WO2002040996A2

WO2002040996A2 - Differential gene expression in specific regions of the brain in neurodegenerative diseases

Info

Publication number: WO2002040996A2
Application number: PCT/US2001/046699
Authority: WO
Inventors: Jung-Joo Hwang; David K. Becker; Robert E. Davis
Original assignee: Mitokor
Priority date: 2000-11-08
Filing date: 2001-11-08
Publication date: 2002-05-23
Also published as: AU2002239531A1; WO2002040996A9; WO2002040996A8; WO2002040996A3

Abstract

The present invention provides methods and compositions for screening assays for compounds that alter mitochondrial function, and in particular that alter apoptosis, based on the observations that expression of the transcription factor FREAC-2 is up-regulated in Alzheimer's disease and that induction of FREAC-2 expression in a cell results in apoptosis. The invention thus provides methods for identifying agents that alter mitochondrial function by determining the activity of reducing or enhancing apoptosis that follows the induced expression of FREAC-2, useful, for example, in treatment of neurodegenerative diseases (e.g., Alzheimer's) and/or of diseases of inappropriate cell proliferation or survival (e.g., cancer). The present invention also includes methods of identifying pharmaceutical/ molecular targets for compounds that alter mitochondrial function (e.g., by altering apoptosis).

Description

DIFFERENTIAL GENE EXPRESSION IN SPECIFIC REGIONS OF THE BRAIN IN

NEURODEGENERATIVE DISEASES

FIELD OF THE INVENTION The present invention generally relates generally to the fields of molecular biology, neurobiology, neurodegenerative diseases and the diagnosis and treatment thereof.

BACKGROUND OF THE INVENTION

Neurodegenerative diseases afflict humans with a variety of debilitating effects, such as memory loss, loss of musculoskeletal and fine motor control or coma. It has been recognized that some neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), diffuse Lewy body disease (DLB), vascular dementia, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS) myoclonic epilepsy lactic acidosis and stroke (MELAS) and myoclonic epilepsy ragged red fiber syndrome (MERRF) are or may be related to mitochondrial defects.

Parkinson's disease (PD) is a progressive, neurodegenerative disorder associated with altered mitochondrial function and characterized by the loss and/or atrophy of dopamine-containing neurons in the pars compacta of the substantia nigra of the brain. Like Alzheimer's Disease (AD), PD also afflicts the elderly. It is characterized by bradykinesia (slow movement), rigidity and a resting tremor. Although L-Dopa treatment reduces tremors in most patients for a while, ultimately the tremors become more and more uncontrollable, making it difficult or impossible for patients to even feed themselves or meet their own basic hygiene needs.

It has been shown that the neurotoxin l-methyl-4-phenyl-l,2,3,6- tetiahydropyridine (MPTP) induces parkinsonism in animals and man at least in part through its effects on mitochondria. MPTP is converted to its active metabolite, MPP+, in dopamine neurons; it then becomes concentrated in the mitochondria. The MPP+ then selectively inhibits the mitochondrial enzyme NADH:ubiquinone oxidoreductase ("Complex I"), leading to the increased production of free radicals, reduced production of adenosine triphosphate, and ultimately, the death of affected dopamine neurons.

Mitochondrial Complex I is composed of 40-50 subunits; most are encoded by the nuclear genome and seven by the mitochondrial genome. Since parkinsonism may be induced by exposure to mitochondrial toxins that affect Complex I activity, it appears likely that defects in Complex I proteins may contribute to the pathogenesis of PD by causing a similar biochemical deficiency in Complex I activity. Indeed, defects in mitochondrial Complex I activity have been reported in the blood and brain of PD patients (Parker et al., Am. J. Neurol. 26:119-723, 1989). Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is characterized by loss and/or atrophy of neurons in discrete regions of the brain, and that is accompanied by extracellular deposits of β-amyloid and the intracellular accumulation of neurofibrillary tangles. It is a uniquely human disease, affecting over 13 million people worldwide. It is also a uniquely tragic disease. Many individuals who have lived normal, productive lives are slowly stricken with AD as they grow older, and the disease gradually robs them of their memory and other mental faculties. Eventually, they cease to recognize family and loved ones, and they often require continuous care until their eventual death.

There is evidence that defects in oxidative phosphorylation within the mitochondria are at least a partial cause of sporadic AD. The enzyme cytochrome c oxidase (COX), which makes up part of the mitochondrial electron transport chain (ETC), is present in normal amounts in AD patients; however, the catalytic activity of this enzyme in AD patients and in the brains of AD patients at autopsy has been found to be abnormally low. This suggests that the COX in AD patients is defective, leading to decreased catalytic activity that in some fashion causes or contributes to the symptoms that are characteristic of AD.

Focal defects in energy metabolism in the mitochondria, with accompanying increases in oxidative stress, may be associated with AD. It is well- established that energy metabolism is impaired in AD brain (Palmer et al., Brain Res. 645:338-42, 1994; Pappolla et al., Am. J. Pathol. 140:621-28, 1992; Jeandel et al., Gerontol. 35:275, 1989; Balazs et al, Neurochem. Res. 19:1131-31, 1994; Mecocci et al., Ann. Neurol. 3(5:747-751, 1994; Gsell et al., J. Neurochem. 64:1216-23, 1995). For example, regionally specific deficits in energy metabolism in AD brains have been reported in a number of positron emission tomography studies (Kuhl, et al., J. Cereh. Blood Flow Metab. 7:S406, 1987; Grady, et al., J. Clin. Exp. Neuropsychol. 10:576-96, 1988; Haxby et al., Arch. Neurol. 47:753-60, 1990; Azari et al., J. Cereh. Blood Flow Metab. 13:438-47, 1993). Metabolic defects in the temporoparietal neocortex of AD patients apparently presage cognitive decline by several years. Skin fibroblasts from AD patients display decreased glucose utilization and increased oxidation of glucose, leading to the formation of glycosylation end products (Yan et al., Proc. Nat. Acad. Sci. USA 91:1181-91, 1994). Cortical tissue from postmortem AD brain shows decreased activity of the mitochondrial enzymes pyruvate dehydrogenase (Sheu et al., Ann. Neurol. 17:444-49, 1985) and α-ketoglutarate dehydrogenase (Mastrogiacomo et al., J. Neurochem. 6^':2007-14, 1994), which are both key enzymes in energy metabolism. Functional magnetic resonance spectroscopy studies have shown increased levels of inorganic phosphate relative to phosphocreatine in AD brain, suggesting an accumulation of precursors that arises from decreased ATP production by mitochondria (Pettegrew et al., Neurobiol. of Aging 15:117-32, 1994; Pettigrew et al., Neurobiol. of Aging 16:913-75, 1995). In addition, the levels of pyruvate, but not of glucose or lactate, are reported to be increased in the cerebrospinal fluid of AD patients, consistent with defects in cerebral mitochondrial electron transport chain (ETC) activity (Parnetti et al., Neurosci. Lett. 99:231-33, 1995).

Signs of oxidative injury are prominent features of AD pathology and, as noted above, reactive oxygen species (ROS) are critical mediators of neuronal degeneration. Indeed, studies at autopsy show that markers of protein, DNA and lipid peroxidation are increased in AD brain (Palmer et al., Brain Res. 645:338-42, 1994; Pappolla et al., Am. J. Pathol. 140:621-28, 1992; Jeandel et al., Gerontol. 35:275-82, 1989; Balazs et al., Arch. Neurol. 4:864, 1994; Mecocci et al., Ann. Neurol. 36:747-51, 1994; Smith et al., Proc. Nat. Acad. Sci. USA 88:10540-43, 1991). In hippocampal tissue from AD but not from controls, carbonyl formation indicative of protein oxidation is increased in neuronal cytoplasm, and nuclei of neurons and glia (Smith et al., Nature 382:120-21, 1996). Neurofibrillary tangles also appear to be prominent sites of protein oxidation (Schweers et al., Proc. Nat. Acad. Sci. USA 92:8463, 1995; Blass et al., Arch. Neurol. 4:864, 1990). Under stressed and non-stressed conditions incubation ^" of cortical tissue from AD brains taken at autopsy demonstrate increased free radical production relative to non-AD controls. In addition, the activities of critical antioxidant enzymes, particularly catalase, are reduced in AD (Gsell et al., J. Neurochem.

64:1216-23, 1995), suggesting that the AD brain is vulnerable to increased ROS production. Thus, oxidative stress may contribute significantly to the pathology of mitochondria associated diseases such as AD, where mitochondrial dysfunction and/or elevated ROS may be present.

One hallmark pathology of AD is the death of selected neuronal populations in discrete regions of the brain. Cell death in AD is presumed to be apoptotic because signs of programmed cell death (PCD) are seen and indicators of active gliosis and necrosis are not found. (Smale et al., Exp. Neurolog. 133:225-230, 1995; Cotman et al., Molec. Neurobiol. 10:19-45, 1995.) The consequences of cell death in AD, neuronal and synaptic loss, are closely associated with the clinical diagnosis of AD and are highly correlated with the degree of dementia in AD (DeKosky et al, Ann. Neurology 27:451-464, 1990). Mitochondrial dysfunction is thought to be critical in the cascade of events leading to apoptosis in various cell types (Kroemer et al., FASEB J. 9:1211-87, 1995), and may be a cause of apoptotic cell death in neurons of the AD brain. Altered mitochondrial physiology may be among the earliest events in PCD (Zamzami et al., J. Exp. Med. 182:361-77, 1995; Zamzami et al., J. Exp. Med. 181:1661-12, 1995) and elevated reactive oxygen species (ROS) levels that result from such altered mitochondrial function may initiate the apoptotic cascade (Ausserer et al, Mol. Cell. Biol. i :5032-42, 1994). hi several cell types, including neurons, reduction in the mitochondrial membrane potential (ΔΨm) precedes the nuclear DNA degradation that accompanies apoptosis. In cell-free systems, mitochondrial, but not nuclear, enriched fractions are capable of inducing nuclear apoptosis (Newmeyer et al., Cell 70:353-64, 1994). Perturbation of mitochondrial respiratory activity leading to altered cellular metabolic states, such as elevated intracellular ROS, may occur in mitochondria associated diseases and may further induce pathogenetic events via apoptotic mechanisms. ^* " Thus, in addition to their role in energy production in growing cells, mitochondria (or, at least, mitochondrial components) participate in apoptosis (Newmeyer et al, 1994, Cell 79:353-364; Liu et al, 1996, Cell 86:147-157). Apoptosis is apparently also required for, inter alia, normal development of the nervous system and proper functioning of the immune system. Moreover, some disease states are ^"thought to be associated with inappropriate cell proliferation and/or cell survival, for example, either insufficient (e.g., cancer, autoimmune diseases) or excessive (e.g., stroke damage, AD-associated neurodegeneration) levels of apoptosis. For general reviews of apoptosis, and the role of mitochondria therein, see Green and Reed (1998, Science 257:1309-1312), Green (1998, Cell 94:695-698) and Kramer (1997, Nature Medicine 3:614-620). Hence, agents that effect apoptotic events, including those associated with mitochondrial components, might have a variety of palliative, prophylactic and therapeutic uses.

Oxidatively stressed mitochondria may release a pre-formed soluble factor that can induce chromosomal condensation, an event preceding apoptosis (Marchetti et al., Cancer Res. 56:2033-38, 1996). In addition, members of the Bcl-2 family of anti-apoptosis gene products are located within the outer mitochondrial membrane (Monaghan et al., J. Histochem. Cytochem. 40:1819-25, 1992) and these proteins appear to protect membranes from oxidative stress (Korsmeyer et al, Biochim. Biophys. Act. 1271:63, 1995). Localization of Bcl-2 to this membrane appears to be indispensable for modulation of apoptosis (Nguyen et al., J. Biol. Chem. 269:16521-24, 1994). Thus, changes in mitochondrial physiology may be important mediators of apoptosis. To the extent that apoptotic cell death is a prominent feature of neuronal loss in AD, mitochondrial dysfunction may be critical to the progression of this disease and may also be a contributing factor in other mitochondria associated diseases. Regardless of whether a defect underlying a disease associated with altered mitochondrial function may have mitochondrial or extramitochondrial origins, and regardless of whether a defect underlying altered mitochondrial function has been identified, the present invention provides methods that are useful for determining the risk or presence of diseases associated with such altered mitochondrial function, and for identifying agents that are suitable for treating such diseases.

Diffuse Lewy body disease (DLB), or Lewy body dementia, is a degenerative disorder of the central nervous system (CNS) that typically presents in older patients, initially as psychosis or progressively deteriorating dementia that may precede tremors, rigidity of movement or other manifestations of parkinsonism. Pathology of DLB reveals diffuse distributions of intracytoplasmic neuronal inclusions known as Lewy bodies, particularly in the nuclei of brainstem, basal forebrain and hypothalamic neurons. DLB may also be accompanied by one or more of myoclonus, dysphagia, orthostatic hypotension and involuntary movements of the skeletal muscles. Vascular dementia, or "multi-infarct", refers to a variety of disorders characterized by progressively deteriorating cognitive abilities that results from multiple infarction events in the cerebral vasculature. Impaired memory and intellectual ability are typically accompanied by focal neurologic signs in vascular dementia.

Multiple sclerosis is a chronic disease in humans that is related to central nervous system demyelination, occurring either as a succession of phases characterized by alternating remission and exacerbation ("relapsing/remitting" disease), or as a steadily progressing disease, often leading to paralysis. The anatomopathological features of this disease include well-delimited patches of demyelination in the white matter of the brain and spinal cord. It is believed that inflammatory, genetic, environmental and etiological factors contribute to the pathogenesis of multiple sclerosis.

Amyotrophic lateral sclerosis (ALS) is a commonly diagnosed progressive motor neuron disease. The disease is characterized by degeneration of motor neurons in the cortex, brainstem and spinal cord. The onset of the disease is between about the third and sixth decade and is uniformly fatal. The cause of ALS is unknown, and is diagnosed when symptoms, such as asymmetric limb weakness, localized fasciculation of the limbs or spasticity in the legs are noted.

Myoclonic epilepsy lactic acidosis and stroke (MELAS) is a disease characterized by stroke-like episodes and lactic acidosis. The stroke-like episodes can be precipitated by metabolic stress. The disease results in neurological pathologies including multiple infarction-like lesions with various degrees of generalized cerebral and cerebellar atrophy that are not related to the vascular territories of the central nervous system.

Myoclonic epilepsy ragged red fiber syndrome (MERRF) is characterized by sensor and motor skill dysfunction, lactic acidosis, encephalopathy, stroke-like episodes, seizures and muscle weakness. The disease results in microscopic degeneration of central nervous system tissues with loss of nerve cells.

Forkhead Related Activator-2 (FREAC-2) is a member of the family of "winged helix" or "forkhead" transcription factors (for a review, see Kaufmann and Knochel, Mechanisms of Development 57:3-20, 1996). Previously published reports suggested that expression of FREAC-2 in the adult is limited to lung and placenta; accordingly, the expression of FREAC-2 in AD brains as disclosed in WO 00/70099 may be an abnormal process. FREAC-2 binds to DNA as a monomer and plays regulatory roles in embryogenesis (Aitola et al, Dev. Dyn. 218:136-49, 2000) and pattern formation in multicellular organisms, in tumorigenesis and in the maintenance of differentiated cells.

FREAC-2 was initially cloned from a human cDNA library by Pierrou et al. (EMBO J. 13:5002, 1994). There are various described activities of FREAC-2, including DNA binding (Hellqvist et al, J. Biol. Chem. 271:4482-4490, 1996) and transactivation of gene expression. It has also been reported that FREAC-2 binds to a consensus DNA sequence, AACGTAAACAA (Pierrou et al, Anal. Biochem. 229:99- 105, 1995; Pierrou et al, EMBO J. 13:5002-5012, 1994), and that a number of genes expressed in the adult lung contain binding sites for FREAC-2, including genes for surfactant apoproteins (Hellqvist et al, J. Biol. Chem. 271:4482-4490, 1996). An amino acid sequence identified in the FREAC-2 polypeptide is associated with DNA binding activity and is similar to DNA binding motifs found in other forkhead proteins (Clark et al, Nature 364:412-420, 1993).

Additional polypeptide sequences in FREAC-2 have been identified that appear to mediate transcriptional activation by a mechanism distinct from DNA binding. Thus far, two such independent transcription activation domains have been identified, referred to as AD1 and AD2. The carboxy-terminal 23 amino acids of FREAC-2 comprise the ADl domain, while the AD2 transactivation domain is less well defined, being distributed over approximately 200 non-contiguous amino acids within the FREAC-2 polypeptide. FREAC-2 has also been shown to interact with cellular factors comprising the basal transcriptional machinery of the cell (Hellqvist et al, J. Biol. Chem. 273(36):23335-23343, 1998). hi particular, two general transcription factors have been shown to bind the carboxy-terminal domain of FREAC-2; they are TBP and TFIIB. FREAC-2 localization to the cell nucleus appears to be a necessary prerequisite for its DNA binding and transcriptional activation activities, and a bipartite nuclear localization sequence comprising 16 carboxy-terminal amino acids (residues

151-166) and 38 internally located amino acids (residues 115-152) promotes nuclear localization of FREAC-2, with both domains required for complete nuclear localization.

Human contactin protein, and its homologs from mouse (F3 protein) and chicken (Fl l protein), are cell surface adhesion proteins that are involved in cell attachment to substrate. Contactin comprises an Ig-like domain and multiple fibronectin Ill-like domains (Brummendorf et al., J. Neurochem. 61:1207-1219 (1993)). Unlike many cell adhesion molecules, contactin is not a transmembrane protein, but instead is anchored to cell surfaces via linkage to glycosylphosphatidylinositol (GPI) in the plasma membrane outer leaflet (Id.). In human tissues, relatively high levels of a major contactin mRNA (6.5 kb) are expressed in adult brain along with three minor transcripts (9.7 kb, 4.4 kb and 3.4 kb), whereas low levels of expression of multiple forms of contactin mRNA are found in the adult lung, pancreas, kidney and skeletal muscles (6.8 kb and 6.0 kb) (Reid et al., Mol. Brain Res. 21:1-8 (1994)). High levels of expression of the multiple forms of contactin mRNA are found in neuroblastoma and retinoblastoma cell lines (6.8 kb, 6.0 kb and 4.2 kb) (Id.). The expression of contactin in developing neural tissue is complex, transient, and temporally regulated. Contactin is believed to have a role in neurite outgrowth, perhaps by binding to the cell recognition molecule Ng-CAM and/or by interacting with the extracellular matrix glycoprotein restrictin (Faivre-Sarrailh et al., J. Neurosci. 12:257-267 (1992), Brummendorf et al., Neuron 10:711-7272 (1993)). Adult neural stem cells can give rise to hematopoietic cells, including cells of the myeloid and lymphoid lineages (Bjornson et al., Science 283:534-537 (1999)); thus, contactin mRNA, contactin DNA or contactin protein may be detected in blood.

Identification of the underlying causes of neurodegenerative diseases has often been elusive, as has been the development of reliable diagnosis, prognosis and treatment of such diseases. There clearly exists a need to provide improved methods and compositions for treating these neurodegenerative diseases. The present invention satisfies these needs by providing the first recognition of a role for differentially expressed gene products, such as FREAC-2 and contactin, in neurodegenerative diseases, and further provides other related advantages.

SUMMARY OF THE INVENTION

The present invention is directed in part to exploitation of the relationship between FREAC-2 expression in cells and the onset of apoptosis, in particular, as pertains to neurodegenerative diseases and to disorders associated with cell proliferation and cell survival such as cancer. More specifically, according to the present invention there is provided a correlation between cellular expression levels of FREAC-2 and the induction of altered (e.g., increased or decreased in a statistically significant manner) mitochondrial function, notably apoptosis. In one aspect, the present invention therefore provides a method of identifying an agent that alters a mitochondrial function, comprising inducing FREAC-2 expression in a host cell in the presence and absence of a candidate agent, wherein the host cell comprises a recombinant expression construct comprising a regulated promoter operably linked to a polynucleotide encoding a FREAC-2 polypeptide, a fragment or variant thereof; and comparing a level of apoptosis in the host cell in the absence of the agent to the level of apoptosis in the presence of the agent, whereby an agent that alters the level of apoptosis indicates an agent that alters mitochondrial function. In certain further embodiments, induction of FREAC-2 expression is performed in vitro, and in - certain other further embodiments, induction of FREAC-2 expression is performed in vivo, m one embodiment the host cell is a neuronal cell. In another embodiment, the host cell is of a cell type that is associated with a neurodegenerative disease that is Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, MELAS or MERRF. In other embodiments the host cell is of a cell type that is associated with a neurodegenerative disease that is Alzheimer's disease, multiple sclerosis, or a disease of inappropriate cell proliferation such as cancer.

In another embodiment, the present invention provides a method of identifying a molecular target of an agent that alters a mitochondrial function, comprising (a) contacting an agent identified according to the method of claim 1 with a biological sample under conditions and for a time sufficient to permit formation of a complex comprising the agent and at least one molecule with which the agent specifically interacts, wherein the molecule is derived from the sample; and (b) isolating the complex, and therefrom identifying a molecular target of the agent.

In another embodiment, the present invention provides a method of identifying a molecular target for therapeutic intervention for treating a neurodegenerative disease or a proliferative disorder, comprising (a) comparing (i) at least one expression level of at least one gene product in a first biological sample comprising a host cell in which expression of a FREAC-2 polypeptide or a variant or fragment thereof has been induced, wherein the host cell comprises a recombinant expression construct comprising a regulated promoter operably linked to a polynucleotide encoding a FREAC-2 polypeptide, or a variant or fragment thereof, to (ii) at least one expression level of the gene product in a second biological sample comprising a host cell in which expression of a FREAC-2 polypeptide or a variant or fragment thereof has not been induced; and (b) detecting an altered expression level of at least one gene product in the first sample relative to the expression level of the gene product in the second biological sample, and therefrom identifying a molecular target for therapeutic intervention.

According to another embodiment of the invention, there is provided a method of treating a neurodegenerative disease or a disease associated with inappropriate cell proliferation or cell survival in a subject, comprising administering to a subject in need thereof a recombinant expression construct comprising a promoter operably linked to a polynucleotide encoding a FREAC-2 polypeptide or a variant or fragment thereof, wherein expression of the FREAC-2 polypeptide or a variant or fragment thereof results in altered mitochondrial function. In a further embodiment the promoter is a regulated promoter. In certain other further embodiments of the above described methods, the altered mitochondrial function is apoptosis.

In certain other embodiments, the invention provides a method of treating a neurodegenerative disease or a disease associated with inappropriate cell proliferation or cell survival in a subject, comprising administering to a subject in need thereof a recombinant expression construct comprising a promoter operably linked to a FREAC-2 polynucleotide that is in reverse orientation such that an antisense nucleic acid or variant or fragment thereof is expressed, wherein expression of the antisense nucleic acid or variant or fragment thereof alters a neurodegenerative disease or a disease associated with inappropriate cell proliferation or cell survival. In certain further embodiments of the just-described methods, the disease associated with inappropriate cell proliferation Or cell survival is cancer.

Other embodiments of the invention relate to differential gene expression in neurodegenerative diseases, exemplified in the present disclosure with reference to nucleic acids encoding contactin and/or to contactin polypeptides, although the invention is not intended to be so limited where contactin is a representative example, used for illustrative purposes, of any of a number of differentially expressed genes and gene products that exhibit altered (i.e., increased or decreased in a statistically significant manner relative to normal, disease-free controls) expression levels, as disclosed herein, including in the Examples, the Drawings and in the Sequence Listing. h certain related embodiments, the invention relates to another exemplary gene, that encoding FREAC-2, which exhibits differential expression in a neurodegenerative disease.

These and other aspects of the present invention will become evident upon reference to the following detailed description. In addition, various references are set forth herein which describe in more detail certain aspects of this invention, and are therefore incorporated by reference in their entireties.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows western blot determination of tetracycline (Tet) induced expression of FREAC-2 protein by stably transfected SH-S Y5 Y/tef¹¹ cells.

Figure 2 shows caspase 3 activity determined in vector control- or FREAC-2-transfected, tetracycline induced SY5Y cells in the absence or presence of thapsigargin and ethacrynic acid (TE) treatment.

Figure 3 shows caspase 3 activity determined in vector control- or FREAC-2-transfected, tetracycline induced SY5Y cells in the absence or presence of staurosporine (St) treatment.

Figure 4 shows cell viability determined in vector control- or FREAC-2- transfected, tetracycline induced SY5Y cells.

Figure 5 shows determination of F-actin distribution by fluorescence microscopy in vector control- or FREAC-2-transfected, tetracycline induced SY5Y cells.

Figure 6 shows determination of alpha-tubulin distribution by immunofluorescence microscopy in vector control- or FREAC-2-transfected, tetracycline induced SY5Y cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed in pertinent part to the surprising observation that expression by a host cell of the transcription factor FREAC-2, which as disclosed herein and in WO 00/70099 exhibits increased expression levels in central nervous tissue derived from subjects having Alzheimer's disease (AD), promotes the onset of apoptosis in such a host cell. As discussed herein, in view of the role played by mitochondria in the process of apoptosis, the present invention thus relates to the unexpected participation of a FREAC-2 gene product, heretofore believed to function as a nuclearly localizing transcription factor, in the regulation of a mitochondrial function.

As also discussed herein, the present invention also therefore relates to molecular mechanisms of neurodegenerative diseases and of diseases associated with inappropriate cell proliferation (e.g., cancer or other conditions pertaining to cell survival), given the relationship of apoptosis to such disease states.

Accordingly, and as described in greater detail below, certain

\ embodiments of the invention provide a method for identifying an agent that alters a mitochondrial function, by inducing recombinant FREAC-2 expression in a host cell and screening for an agent capable of altering (e.g., increasing or decreasing in a statistically significant manner relative to a control where the agent is absent) the level of apoptosis that results from such induced FREAC-2 expression. Although not intended to be so limited, in certain preferred embodiments the method is performed in vitro using conventional equipment and procedures, and in certain preferred embodiments the host cell may be of a cell type that represents a disease-relevant model for a neurodegenerative disease (e.g., a cultured neuron or a neuronal cell line, such as a neuroblastoma cell line for AD; a neuronal cell, an astrocytic cell, a glial cell, a CNS endothelial cell or an inflammatory cell such as a lymphocyte, leukocyte or other inflammatory cell for multiple sclerosis; and so forth for affected cell types in other neurodegenerative diseases as provided herein) or for a disease associated with inappropriate cell proliferation (e.g., a transformed, neoplastic, malignant, metastatic or other proliferating cell of an appropriate lineage for the various particular types of cancers).

The present invention also provides advantages associated with methods for identifying a molecular target of an agent identified according to the above described method. For example, and without wishing to be bound by theory, insofar as the present disclosure provides genes and/or gene products that, along with FREAC-2, exhibit altered (e.g., increased or decreased in a statistically significant manner relative to a disease-free control) expression levels in a neurodegenerative disease, and further provides the observation that induction of FREAC-2 expression results in apoptosis, it is contemplated that one or more genes and or gene products may exhibit an altered expression level as a direct or indirect consequence of induced FREAC-2 expression. Also according to non-limiting theory, where the instant disclosure relates to regulation of apoptosis through FREAC-2 expression, the present invention contemplates methods of treating disorders such as neurodegenerative diseases or diseases associated with inappropriate cell proliferation or cell survival (e.g., cancer, autoimmune disease) by administering compositions that may promote or inhibit entry of a cell into an apoptotic pathway, depending on whether preservation of a cell (e.g., of a neuron in neurodegenerative disease) or ablation of a cell (e.g., of a malignant cell in cancer) may be desired. The present invention is also directed in part to the unexpected finding that the GPI-linked, neuronal cell associated recognition molecule contactin is associated with neurodegenerative diseases. In certain embodiments neurodegenerative disease may be correlated with altered (e.g., increased or decreased in a statistically significant manner relative to disease-free controls) expression levels of a gene encoding contactin, or of one or more other genes described herein where, as discussed above, disclosure relating to contactin in the present application is intended to be a representative example of, and applicable to any of the other differentially expressed genes and/or gene products described herein. For example, as provided herein altered levels of contactin expression may be observed as increases or decreases in the amount of contactin protein in a sample relative to a control sample. As another example, altered levels of contactin may be detected as altered amounts of contactin mRNA, as also described in greater detail below.

The invention thus is directed in part to a method of detecting the risk for having or presence of a neurodegenerative disease in a subject, by comparing the level of contactin expression, such as the amount of contactin protein or contactin mRNA, in a test sample to the amount of contactin protein or mRNA in a control sample from a second subject known to be free of a risk for having or presence of the neurodegenerative disease. The invention also relates to a method of correlating contactin expression with the suitability of an agent for treating a neurodegenerative disease in at least one subject, thereby providing a method for identifying those patients having a neurodegenerative disease who are likely to respond to a particular treatment.

The invention is also directed in part to an oligonucleotide primer capable of specifically amplifying DNA or RNA encoding contactin or a nucleic acid sequence complementary thereto. Such primers may be nucleic acid molecules that can function as PCR primers for the detection of contactin mRNA or contactin DNA in a sample. Such nucleic acid molecules can also be labeled, and may be provided in a kit.

In view of the surprising relationship between contactin and neurodegenerative disease, the present invention further provides compositions and methods for drug screening assays, including the use of cybrid cells, to identify agents that may be useful for the treatment of neurodegenerative diseases, for example when formulated into pharmaceutical compositions as provided herein. Similarly, the present disclosure provides methods for identifying molecular targets for pharmaceutical agents that alter contactin expression levels, as well as related therapeutic methods.

Neurodegenerative diseases to which the present invention may be applied include, but are not limited to, Alzheimer's disease (AD), multiple sclerosis (MS), Parkinson's disease (PD), diffuse Lewy body disease (DLB), vascular dementia and the like, and other neurodegenerative diseases.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, chemistry, microbiology, molecular biology, cell science and cell culture described below are well known and commonly employed in the art. Conventional methods are used for these procedures, such as those provided in the art and various general references (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)). Where a term is provided in the singular, the inventors also contemplate the plural of that term. The nomenclature used herein and the laboratory procedures described below are those well known and commonly employed in the art. As employed throughout the disclosure, the following terms, unless otherwise indicated, shall be understood to have - the following meanings:

"Membrane permeant derivative" refers to a chemical derivative of a compound that increases membrane permeability of the compound. These derivatives are made better able to cross cell membranes because hydrophilic groups are masked to provide more hydrophobic derivatives. Also, the making groups can be designed to be cleaved from the compound within a cell to make the compound more hydrophilic once within the cell. Because the substrate is more hydrophilic than the membrane permeant derivative, it preferentially localizes v thin the cell (U.S. Patent No. 5,741,657 to Tsien et al., issued April 21, 1998).

"Isolated" polynucleotide refers to a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, which by virtue of its origin, the isolated polynucleotide (1) is not associated with the cell in which the isolated polynucleotide is found in nature, or (2) is operably linked to a polynucleotide that it is not linked to in nature. The isolated polynucleotide can optionally be linked to promoters, enhancers, or other regulatory sequences. "Isolated" protein refers to a protein of cDNA, recombinant RNA, or synthetic origin, or some combination thereof, which by virtue of its origin the isolated protein (1) is not associated with proteins normally found within nature, or (2) is isolated from the cell in which it normally occurs, or (3) is isolated free of other proteins from the same cellular source, for example, free of cellular proteins), or (4) is expressed by a cell from a different species, or (5) does not occur in nature.

"Polypeptide" is used herein as a generic term to refer to native protein, fragments, or analogs of a polypeptide sequence.

A gene product may be an RNA molecule or other polynucleotide transcript of a polypeptide-encoding and or non-coding region of a gene, or a protein or polypeptide that results from translation of such a transcript. Of particular interest are those genes and gene products that are differentially expressed in a disease state (t^'.e., differentially expressed in cells from a diseased organism relative to cells from an undiseased, control organism of the same species), in manipulated (e.g., recombinantly transfected and/or induced) cells versus non-manipulated (e.g., non-transfected or uninduced, wildtype) cells, or in a species-specific manner (i.e., differentially expressed in cells from one species relative to cells from a second species). Thus, for example, an "RNA of interest," a "gene of interest" and a "protein of interest" refer to, respectively, a RNA, gene and protein that are differentially expressed with regard to a disease state, in manipulated cells or in a species-specific manner. As one example of a gene of interest that does not directly encode a mitochondrial gene product, a nucleic acid of interest may be an antisense regulator of a mitochondrial gene product (Shayiq, J. Biol. Chem. 272:4050-4057 (1997)). "RNAs of interest" include RNA molecules that are not mRNA molecules but are themselves gene products, for example, ribosomal RNA (rRNA) molecules, transfer RNA (tRNA) molecules, ribozymes, RNA molecules that form part of a nucleoprotein complex, and antisense transcripts.

As regards genes and gene products that. are differentially expressed in a disease or disorder, "mitochondria associated disorders," i.e., diseases associated or thought to be associated with altered mitochondrial function and/or mitochondrial mutations, are of particular interest. Mitochondria associated disorders may include without limitation AD, PD, auto-immune diseases, diabetes mellitus, MELAS, MERFF, arthritis, NARP (Neuropathy; Ataxia; Retinitis Pigmentosa); MNGIE (Myopathy and external ophthalmoplegia; Neuropathy; Gastro- ntestinal; Encephalopathy), LHON (Leber's; Hereditary; Optic; Neuropathy), Kearns-Sayre disease; Pearson's Syndrome; PEO (Progressive External Ophthalmoplegia); congenital muscular dystrophy with mitochondrial structural abnormalities; Wolfram syndrome (DIDMOAD; Diabetes Insipidus, Diabetes Mellitus, Optic Atrophy, Deafness), Leigh's Syndrome, fatal infantile myopathy with severe mtDNA depletion, benign "later-onset" myopathy with moderate reduction in mtDNA; dystonia; schizophrenia; mitochondrial encephalopathy, lactic acidosis, and stroke (MELAS); mitochondrial diabetes and deafness (MIDD); myoclonic epilepsy ragged red fiber syndrome (MERFF); and hyperproliferative disorders, such as cancer, tumors and psoriasis.

"Active fragment" refers to a fragment of a parent molecule, such as an organic molecule, nucleic acid molecule, or protein or polypeptide, or combinations thereof, that retains at least one activity of the parent molecule.

"Naturally occurring" refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism, including viruses, that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring. "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence operably linked to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. "Control sequences" refer to polynucleotide sequences that effect the expression of coding and non-coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal biding site, and transcription termination sequences; in eukaryotes, generally, such control sequences include promoters and transcription termination sequences. The term control sequences is intended to include components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

"Polynucleotide" refers to a polymeric form of nucleotides of a least ten bases in length, either ribonucleotides or deoxynucleotides or a modified from of either type of nucleotide. The term includes single and double stranded forms of DNA or RNA.

"Genomic polynucleotide" refers to a portion of the genome. "Active genomic polynucleotide" or active portion of a genome" refer to regions of a genome that can be up regulated, down regulated or both, either directly or indirectly, by a biological process.

"Directly" in the context of a biological process or processes, refers to direct causation of a process that does not require intermediate steps, usually caused by one molecule contacting or binding to another molecule (the same type or different type of molecule). For example, molecule A contacts molecule B, which causes molecule B to exert effect X that is part of a biological process.

"Indirectly" in the context of a biological process or precesses, refers to indirect causation that requires intermediate steps, usually caused by two or more direct steps. For example, molecule A contacts molecule B to exert effect X which in turn causes effect Y.

"Sequence homology" refers to the proportion of base matches between two nucleic acid sequences or the proportion of amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage, for example

50%, the percentage denotes the proportion of matches of the length of sequences from a desired sequence that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; gap lengths of 15 bases or less are usually used, 6 bases or less are preferred with 2 bases or less more preferred. When using oligonuleotides as probes or treatments, the sequence homology between the target nucleic acid and the oligonucleotide sequence is generally not less than 17 target base matches out of 20 possible oligonucleotide base pair matches (85%); preferably not less than 9 matches out of 10 possible base pair matches (90%), and most preferably not less than 19 matches out of 20 possible base pair matches (95%). "Selectively hybridize" refers to detectably and specifically bind.

Polynucleotides, oligonucleotides and fragments thereof selectively hybridize to target nucleic acid strands, under hybridization and wash conditions that minimize appreciable amounts of detectable binding to nonspecific nucleic acids. High stringency conditions can be used to achieve selective hybridization conditions as known in the art. Generally, the nucleic acid sequence homology between the polynucleotides, oligonucleotides, and fragments thereof and a nucleic acid sequence of interest will be at least 30%, and more typically and preferably of at least 40%, 50%, 60%, 70%, 80% or 90%.

Hybridization and washing conditions are typically performed at high stringency according to conventional hybridization procedures. Positive clones are isolated and sequenced. For example, a full length polynucleotide sequence can be labeled and used as a hybridization probe to isolate genomic clones from an appropriate target library as they are known in the art. Typical hybridization conditions and methods for screening plaque lifts and other purposes are known in the art (Benton and Davis, Science 196: 180 (1978); Sambrook et al, supra, (1989)).

Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less are preferred with 2 or less being more preferred. Alternatively and preferably, two protein sequences (or polypeptide sequences derived from them of at least 30 amino acids in length) are homologous, as this term is used herein, if they have an alignment score of at least 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater (Dayhoff, in Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, volume 5, pp. 101-110 (1972) and Supplement 2, pp. 1-10). The two sequences or parts thereof are more preferably homologous if their amino acids are greater than or equal to 30% identical when optimally aligned using the ALIGN program. "Corresponds to" refers to a polynucleotide sequence is homologous (for example is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to all or a portion of a reference polypeptide sequence. In contradistinction, the term "complementary to" is u sed herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For illustration, the nucleotide sequence TATAC corresponds to a reference sequence TATAC and is complementary to a reference sequence GTATA.

The following terms are used to describe the sequence relationships between two or more polynucleotides: "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity," and "substantial identity." A reference sequence is a defined sequence used as a basis for a sequence comparison; a reference sequence can be a subset of a larger sequence, for example, as a segment of a full length cDNA or gene sequence given in a sequence listing, or may comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length. Since two polynucleotides can each (1) comprise a sequence (for example a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A comparison window, as used herein, refers to a conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window can comprise additions and deletions (for example, gaps) of 20 percent or less as compared to the reference sequence (which would not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window can be conducted by the local homology algorithm (Smith and Waterman, Adv. Appl. Math., 2:482 (1981)), by the homology alignment algorithm (Needleman and Wunsch, J. Mol. Bio., 48:443 (1970)), by the search for similarity method (Pearson and Lipman, Proc. Natl. Acid. Sci. U.S.A. 85:2444 (1988)), by the computerized implementations of these algorithms such as GAP, BESTFIT, FASTA and TFASTA (Wisconsin Genetics Software Page Release 7.0, Genetics Computer Group, Madison, WI), or by inspection. Preferably, the best alignment (for example, the result having the highest percentage of homology over the comparison window) generated by the various methods is selected.

"Sequence identity" means that two polynucleotide sequences are identical (for example, on a nucleotide-by-nucleotide basis) over the window of " comparison.

"Percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (for example, the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

"Substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 30 percent sequence identity, preferably at least 50 to 60 percent sequence identity, more usually at least 60 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 25 to 50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence that may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.

"Substantial identity" as applied to polypeptides herein means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 30 percent sequence identity, preferably at least 40 percent sequence identity, and more preferably at least 50 percent sequence identity, and most preferably at lest 60 percent sequence identity. Preferably, residue positions, which are not identical, differ by conservative amino acid substitutions.

"Conservative amino acid substitutions" refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine and tryptophan; a group of amino acids having basic side chains is lysine, arginine and histidine; and a group of amino acids having sulfur-containing side chan is cysteine and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine; phenylalanine-tyrosine; lysine-arginine; alanine-valine; glutamic-aspartic; and asparagine-glutamine.

"Modulation" refers to the capacity to either enhance or inhibit a functional property of a biological activity or process, for example, enzyme activity or receptor binding. Such enhancement or inhibition may be contingent on the occurrence of a specific event, such as activation of a signal transduction pathway and/or may be manifest only in particular cell types.

"Modulator" refers to a chemical (naturally occurring or non-naturally occurring), such as a biological macromolecule (for example, nucleic acid, protein, non- peptide or organic molecule) or an extract made from biological materials, such as prokaryotes, bacteria, eukaryotes, plants, fungi, multicellular organisms or animals, invertebrates, vertebrates, mammals and humans, including, where appropriate, extracts of: whole organisms or portions of organisms, cells, organs, tissues, fluids, whole cultures or portions of cultures, or environmental samples or portions thereof. Modulators are typically evaluated for potential activity as inhibitors or activators (directly or indirectly) of a biological process or processes (for example, agonist, partial antagonist, partial agonist, antagonist, antineoplastic, cytotoxic, inhibitors of neoplastic transformation or cell proliferation, cell proliferation promoting agents, antiviral agents, antimicrobial agents, antibacterial agents, antibiotics, and the like) by inclusion in assays described herein. The activity of a modulator may be known, unknown or partially known.

"Test compound" refers to a chemical, compound, composition or extract to be tested by at least one method of the present invention to be a putative modulator. Test compounds can be candidate agents and may include small molecules, such as small molecules, drugs, proteins or peptides or active fragments thereof, such as antibodies, nucleic acid molecules such as DNA, RNA or combinations thereof, antisense molecules or ribozymes, or other organic or inorganic molecules, such as lipids, carboydrates, or any combinations thereof. Test compounds that include nucleic acid molecules can be provided in a vector, such as a viral vector, such as a retrovirus, adenovirus or adeno-associated virus, a liposome, a plasmid or with a lipofection agent. Test compounds, once identified, can be agonists, antagonists, partial agonists or inverse agonists of a target. A test compound is usually not known to bind to the target of interest. "Control test compound" refers to a compound known to bind to the target (for example, a known agonist, antagonist, partial agonist or inverse agonist). Test compounds do not typically include a compound added to a mixture as a control condition that alters the function of the target to determine signal specificity in an assay. Such control compounds or conditions include chemicals that (1) non-specifically or substantially disrupt protein structure (for example denaturing agents such as urea or guandium, sulfhydryl reagents such as dithiotritol and beta-mercaptoethanol), (2) generally inhibit cell metabolism (for example mitochondrial uncouples) and (3) non- specifically disrupt electrostatic or hydrophobic interactions of a protein (for example, high salt concentrations or detergents at concentrations sufficient to non-specifically disrupt hydrophobic or electrostatic interactions). The term test compound also does not typically include compounds known to be unsuitable for a therapeutic use for a particular indication due to toxicity of the subject. Usually, various predetermined concentrations of test compounds are used for determining their activity. If the molecular weight of a test chemical is known, the following ranges of concentrations can be used: between about 0.001 micromolar and about 10 millimolar, preferably between about 0.01 micromolar and about 1 millimolar, more preferably between about 0.1 micromolar and about 100 micromolar. When extracts are uses a test compounds, the concentration of test chemical used can be expressed on a weight to volume basis. Under these circumstances, the following ranges of concentrations can be used: between about 0.001 micrograms/ml and about 1 milligram ml, preferably between about 0.01 micrograms/ml and about 100 micrograms/ml, and more preferably between about 0.1 micrograms/ml and about 10 micrograms/ml. A candidate agent for use according to the present invention may be any composition of matter that is suspected of altering a mitochondrial function as provided herein, which according to preferred embodiments pertains to apoptosis, for example in a cell-based assay as described herein. Detectable alteration of a level of apoptosis typically refers to a statistically significant alteration (e.g., increase or decrease) of the level detected in the presence of the agent relative to that detected in the absence of the agent.

Preferably the candidate agent is provided in soluble form. Without wishing to be bound by theory, a candidate agent may directly alter the activity of a mitochondrial molecular component that regulates mitochondrial properties such as mitochondrial membrane potential, mitochondrial membrane permeability or mitochondrial ion transport channels, such as a calcium channel or uniporter (e.g., by physical contact with the calcium channel), or may do so indirectly (e.g., by interaction with one or more additional molecular components such as mitochondrial molecular components present in a sample, where such additional components alter mitochondrial apoptosis regulatory activity in response to contact with the agent). Typically, and in preferred embodiments such as for high throughput screening, candidate agents are provided as "libraries" or collections of compounds, compositions or molecules. Such molecules typically include compounds known in the art as "small molecules" and having molecular weights less than 10⁵ daltons, preferably less than 10⁴ daltons and still more preferably less than 10 daltons.

For example, members of a library of test compounds can be administered to a plurality of samples in each of a plurality of reaction vessels in a high throughput screening array as provided herein, each containing at least one cell containing cytosol, a mitochondrion and a recombinant expression construct comprising a regulated promoter operably linked to a polynucleotide encoding a FREAC-2 polypeptide, a fragment or variant thereof, as provided herein. The samples are contacted with an inducer of FREAC-2 expression and then assayed for a level of apoptosis using a detectable signal according to known methodologies as described herein, and the signal generated from each sample in the presence of the candidate agent is compared to the signal generated in the absence of the agent. Compounds so identified as capable of influencing mitochondrial function (e.g., apoptosis) are valuable for therapeutic and/or diagnostic purposes, since they permit treatment and/or detection of diseases associated with altered mitochondrial function. Such compounds are also valuable in research directed to molecular signaling mechanisms that involve apoptosis. Candidate agents further may be provided as members of a combinatorial library, which preferably includes synthetic agents prepared according to a plurality of predetermined chemical reactions performed in a plurality of reaction vessels. For example, various starting compounds may be prepared employing one or more of solid- phase synthesis, recorded random mix methodologies and recorded reaction split techniques that permit a given constituent to traceably undergo a plurality of permutations and/or combinations of reaction conditions. The resulting products comprise a library that can be screened followed by iterative selection and synthesis procedures, such as a synthetic combinatorial library of peptides (see e.g., PCT/US91/08694 and PCT/US91/04666) or other compositions that may include small molecules as provided herein (see e.g., PCT/US94/08542, EP 0774464, U.S. 5,798,035, U.S. 5,789,172, U.S. 5,751,629). Those having ordinary skill in the art will appreciate that a diverse assortment of such libraries may be prepared according to established procedures, and tested using a biological sample according to the present disclosure. An agent so identified as one that alters (e.g., increases or decreases) mitochondrial function (e.g., apoptosis) is preferably part of a pharmaceutical composition when used in the methods of the present invention. The pharmaceutical composition will include at least one of a pharmaceutically acceptable carrier, diluent or excipient, in addition to one or more selected agent that alters mitochondrial function and, optionally, other components.

"Target" refers to a biochemical entity involved in a biological process. Targets are typically proteins that play a useful role in the physiology or biology of an organism. A therapeutic composition or compound typically binds to a target to alter or modulate its function. As used herein, targets can include, but not be limited to, cell surface receptors, G-proteins, G-protein coupled receptors, kinases, phosphatases, ion channels, lipases, phosholipases, nuclear receptors, intracellular structures, tubules, tubulin, and the like.

"Label" or "labeled" refers to incorporation of a detectable marker, for example by incorporation of a radiolabled compound or attachment to a polypeptide of moieties such as biotin that can be detected by the binding of a section moiety, such as marked avidin. Various methods of labeling polypeptide, nucleic acids, carbohydrates, and other biological or organic molecules are known in the art. Such labels can have a variety of readouts, such as radioactivity, fluorescence, color, chemiluminescence or other readouts known in the art or later developed. The readouts can be based on enzymatic activity, such as beta-galactosidase, beta-lactamase, horseradish peroxidase, alkaline phosphatase, luciferase; radioisotopes such as H, C, S, I or I; fluorescent proteins, such as green fluorescent proteins; or other fluorescent labels, such as FITC, rhodamine, and lanthanides. Where appropriate, these labels can be the product of the expression of reporter genes, as that term is understood in the art. Examples of reporter genes are beta-lactamase (U.S. Patent No. 5,741,657 to Tsien et al., issued April 21, 1998) and green fluorescent protein (U.S. Patent No. 5,777,079 to Tsien et al, issued July 7, 1998; U.S. Patent No. 5,804,387 to Cormack et al., issued September 8, 1998).

"Substantially pure" refers to an object species or activity that is the predominant species or activity present (for example on a molar basis it is more abundant than any other individual species or activities in the composition) and preferably a substantially purified fraction is a composition wherein the object species or activity comprises at least about 50 percent (on a molar, weight or activity basis) of all macromolecules or activities present. Generally , as substantially pure composition will comprise more than about 80 percent of all macromolecular species or activities present in a composition, more preferably more than about 85%, 90%, 95% and 99%. Most preferably, the object species or activity is purified to essential homogeneity, wherein contaminant species or activities cannot be detected by conventional detection methods) wherein the composition consists essentially of a single macromolecular species or activity. The inventors recognize that an activity may be caused, directly or indirectly, by a single species or a plurality of species within a composition, particularly with extracts.

"Pharmaceutical agent or drug" refers to a chemical, composition or activity capable of inducing a desired therapeutic effect when property administered by an appropriate dose, regime, route of administration, time and delivery modality.

"Pharmaceutically effective amount" refers to an appropriate dose, regime, route of administration, time and delivery modality associated with the delivery of an amount of a compound or composition to cause a desired effect. Such pharmaceutically effective amount can be determined using methods described herein or byihe United States Food and Drug Administration (USFDA).

"Sample" means any biological sample, preferably derived from a test animal, such as a mouse, rat, rabbit or monkey, or a patient, such as a human. Samples can be from any tissue or fluid, such as neural tissues, central nervous tissues, internal organs such as pancreas, liver, lung, kidney, muscle, skeletal muscle, urine, feces, blood, fluids from body cavities or the central nervous system, or samples from various body cavities such as the mouth or nose. Samples derived from urine and feces contain cells of the immunological, urinary or digestive tract and can be a rich source of sample. Such samples can be obtained using methods known in the art, such as biopsies, aspirations, scrapings or simple collection. A sample can be taken from a test animal or patient that is either living or dead. Thus, for example, a biological sample may be derived from a normal (i.e., healthy) individual or from an individual having a disease associated with altered mitochondrial function. Biological samples may be derived by obtaining a blood sample, biopsy specimen, tissue explant, organ culture or any other tissue or cell preparation from a subject or a biological source. The subject or biological source may be a biological organism such as a human or non-human animal, a-prokaryote or a eukaryote, a plant, a unicellular organism or a multicellular organism.

According to certain preferred embodiments of the present invention, the subject or biological source may also be a host cell, such as a primary cell culture or culture adapted cell line, including but not limited to genetically engineered cell lines that may contain chromosomally integrated or episomal recombinant nucleic acid sequences (including but not limited to a recombinant expression construct comprising a regulated promoter operably linked to a polynucleotide encoding all or a portion of a FREAC-2 polypeptide or a variant thereof) differentiated or differentiatable cell lines, transformed cell lines and the like. In certain embodiments, for example, a biological sample cell may be transfected with a gene encoding and expressing a biological receptor of interest, which may be a receptor having a known ligand (e.g., a cytokine, hormone or growth factor) or which may be an "orphaned" receptor for which no ligand is known. Further to such embodiments, one or more known ligands or other compounds suspected of being able to interact with the receptor of interest may be optionally included in the subject invention method, for example, a cytokine, hormone, growth factor, antibody, neurotransmitter, receptor activator, receptor inhibitor, ion channel modulator, ion pump modulator, irritant, drug, toxin or any other compound known to have, or suspected of having, a biologically relevant activity.

The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Representative examples of appropriate host cells for use according to the present invention include, but need not be limited to, bacterial cells, such as E. coli, Streptomyces, Salmonella tvphimurium; fungal cells, such as yeast; insect cells, such as Drosophila S2, Trichoplusia ni (PharMingen, San Diego, CA) and Spodoptera Sf9; animal cells, such as CHO, COS or 293 cells; adenoviruses; plant cells, or any suitable cell already adapted to in vitro propagation or so established de novo. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

Various mammalian cell culture systems can also be employed to express recombinant protein, such as induced FREAC-2. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences, for example as described herein regarding the preparation of ANT expression constructs. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements. Introduction of the construct into the host cell can be effected by a variety of methods with which those skilled in the art will be familiar, including but not limited to, for example, calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis et al., 1986 Basic Methods in Molecular Biology).

The present invention also relates to vectors and to constructs that include nucleic acids of the present invention, and in particular to "recombinant expression constructs" that include any nucleic acids encoding FREAC-2 polypeptides or fragments or variants thereof, or encoding other gene products identified according to the invention and as provided herein; and to host cells which are genetically engineered with such vectors and/or constructs by recombinant techniques. Recombinant FREAC- 2 proteins (or other differentially expressed proteins as described herein) can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from DNA constructs prepared using coding regions and, optionally, non-coding regions of differentially expressed polynucleotides identified according to the methods of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989).

Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product. Useful expression constructs for bacterial use are constructed by inserting into an expression vector a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The construct may comprise one or more phenotypic selectable marker and an origin of replication to ensure maintenance of the vector construct and, if desirable, to provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice. Any other plasmid or vector may be used as long as they are replicable and viable in the host. As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GΕM1 (Promega Biotec, Madison, WI, USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter, if it is a regulated promoter as provided herein, is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents; such methods are well know to those skilled in the art. Thus, for example, the nucleic acids of the invention as provided herein may be included in any one of a variety of expression vector constructs as a recombinant expression construct for expressing a FREAC-2 or a FREAC-2-induced polypeptide, or another polypeptide. Such vectors and constructs include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA, such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used for preparation of a recombinant expression construct as long as it is replicable and viable in the host. The appropriate DNA sequence(s) may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described, for example, in Ausubel et al. (1993 Current Protocols in Molecular Biology, Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., Boston, MA); Sambrook et al. (1989 Molecular Cloning, Second Ed., Cold Spring Harbor Laboratory, Plainview, NY); Maniatis et al. (1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, NY); and elsewhere.

The DNA sequence in the expression vector is operatively linked to at least one appropriate expression control sequences (e.g., a promoter or a regulated promoter) to direct mRNA synthesis. Representative examples of such expression control sequences include LTR or SV40 promoter, the E. coli lac or trp, the phage lambda P promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda PR, P and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art, and preparation of certain particularly preferred recombinant expression constructs comprising at least one promoter or regulated promoter operably linked to a nucleic acid - encoding a FREAC-2 polypeptide is described herein.

In certain preferred embodiments the expression control sequence is a "regulated promoter", which may be a promoter as provided herein and" may also be a repressor binding site, an activator binding site or any other regulatory sequence that controls expression of a nucleic acid sequence as provided herein. In certain particularly preferred embodiments the regulated promoter is a tightly regulated promoter that is specifically inducible and that permits little or no transcription of nucleic acid sequences under its control in the absence of an induction signal, as is known to those familiar with the art and described, for example, in Guzman et al. (1995 J. Bacteriol 177:4121), Carra et al. (1993 EMBOJ. 12:35), Mayer (1995 Gene 163:41), Haldimann et al. (1998 J. Bacteriol. 180:1277), Lutz et al. (1997 Nuc. Ac. Res. 25:1203), Allgood et al. (1997 Curr. Opin. Biotechnol. 8:474) and Makrides (1996 Microbiol. Rev. 60:512), all of which are hereby incorporated by reference. In other preferred embodiments of the invention a regulated promoter is present that is inducible but that may not be tightly regulated. In certain other preferred embodiments a promoter is present in the recombinant expression construct of the invention that is not a regulated promoter; such a promoter may include, for example, a constitutive promoter such as an insect polyhedrin promoter as described in the Examples or a yeast phosphoglycerate kinase promoter (see, e.g., Giraud et al., 1998 J. Mol. Biol. 281:409). The expression construct also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.

Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are exacting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

As noted above, in certain embodiments the vector may be a viral vector such as a retroviral vector. For example, retioviruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retioviruses such as Rous Sarcoma Virus, Harvey Sarcoma virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus. ~ \~^~' The viral vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques 7:980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and β- actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvo virus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein, and may be from among either regulated promoters or promoters as described above. ^" The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ψ-2, ψ-AM, PA12, T19- 14X, VT-19-17-H2, ψCRE, ψCRTP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy, 7:5-14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO₄ precipitation, hi one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host. The producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding, for example, the FREAC-2 or contactin or other polypeptides or fusion proteins. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the desired polypeptide or fusion protein. Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells. As another example of an embodiment of the invention in which a viral vector is used to prepare the recombinant expression construct, in one preferred embodiment, host cells transduced by a recombinant viral construct directing the expression of desired (e.g., FREAC-2 or FREAC-2-induced) polypeptides or fusion proteins may produce viral particles containing expressed polypeptides or fusion proteins that are derived from portions of a host cell membrane incorporated by the viral particles during viral budding. In another preferred embodiment, polypeptide encoding nucleic acid sequences are cloned into a baculovirus shuttle vector, which is then recombined with a baculovirus to generate a recombinant baculovirus expression construct that is used to infect, for example, Sf9 or Trichoplusia ni (PharMingen, Inc., San Diego, CA) host cells, as described in Baculovirus Expression Protocols, Methods in Molecular Biology Vol. 39, Christopher D. Richardson, Editor, Human Press, Totowa, NJ, 1995; Piwnica- Worms, "Expression of Proteins in Insect Cells Using Baculoviral Vectors," Section II in Chapter 16 in: Short Protocols in Molecular Biology, 2^nd Ed., Ausubel et al, eds., John Wiley & Sons, New York, New York, 1992, pages 16-32 to 16-48.

"Ribozyme" means enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence- specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Within the scope of the invention are engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of RNA encoding contactin. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target RNA target for ribozyme cleavage sites which include the sequences GUA, GUU and GUC. Once identified, short RNA sequences between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets can also be evaluate by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. "Contactin" refers to contactin mRNA, contactin DNA and contactin protein. "Contactin protein" means a protein that exhibits at least one activity of at least one human contactin, F3 or FI 1. "Contactin mRNA" is a mRNA molecule that encodes a contactin protein. Preferably, the contactin mRNA is derived from nuclear DNA, but it can be derived from mitochondrial DNA "Contactin DNA" is a DNA molecule that encodes a contactin protein. Preferably a contactin DNA is a nuclear DNA, but it can be a mitochondrial DNA.

Other technical terms used herein have their ordinary meaning in the art that they are used, as exemplified by a variety of technical dictionaries, such as the McGraw-Hill Dictionary of Chemical Terms and the Stedman's Medical Dictionary.

As noted above, the present invention recognizes that contactin expression in cells is related to neurodegenerative diseases. As a non-limiting introduction to the breadth of the present invention, the present invention includes several general and useful aspects, including (i) nucleic acid molecules that can function as PCR primers for the detection of contactin mRNA or contactin DNA in a sample; (ii) methods for detecting the presence of a neurodegenerative disease such as multiple sclerosis, including providing a sample from a patient and measuring the amount of contactin protein, contactin DNA or contactin mRNA expressed in the sample; (iii) methods for identifying which patients having a neurodegenerative disease are likely to respond to a treatment for a neurodegenerative disease; (iv) cells and cell lines, including cybrid cell lines, that express a normal, increased or decreased amount of contactin as compared to a control cell; (v) methods for screening compounds for the ability to alter (e.g., increase or decrease) the expression of contactin and compositions or compounds, including pharmaceutical compositions, identified by these methods, that reduce or enhance contactin expression; (vi) methods of treating a neurodegenerative disease using the compositions or compounds identified by the methods of the present invention; and (vii) methods for identifying pharmaceutical targets for compounds that alter or modulate the amount of contactin DNA, contactin mRNA or contactin protein in a cell and targets identified by methods of the present invention.

These aspects of the invention, as well as others described herein, can be achieved by using the methods, articles of manufacture and compositions of matter described herein. To gain a full appreciation of the scope of the present invention, it will be further recognized that various aspects of the present invention can be combined to make desirable embodiments of the invention.

Nucleic Acid Molecules

The present invention includes nucleic acid molecules that can function as PCR primers for the detection of contactin mRNA or contactin DNA in a sample. Such nucleic acid molecules can be detectably labeled and can be provided in a kit.

Human contactin protein, and its homologs from mouse (F3 protein) and chicken (Fll protein), is a cell surface protein that is related to cell attachment to substrate. Contactin comprises a Ig-like domain and multiple fibronectin Hi-like domains. Rather than being a transmembrane protein, contactin attaches to glycosylphosphatidylinositol (GPI) on the outer membrane of the cell surface. Relatively high levels of a contactin mRNA (6.5 kb) are expressed in adult brain with three minor transcripts (9J kb, 4.4 kb and 3.4 kb), whereas low levels of expression of multiple forms of contactin mRNA are found in the adult lung, pancreas, kidney and skeletal muscles (6.8 kb and 6.0 kb). High levels of expression of the multiple forms of contactin mRNA are found in the cancerous cells neuroblastoma and retinoblastoma (6.8 kb, 6.0 kb and 4.2 kb). The expression of contactin in developing neural tissue is complex and transient, and is believed to have a role in neurite outgrowth, binding to the cell recognition molecule Ng-CAM and interaction with the extracellular matrix glycoprotein restrictin. Adult neural stem cells can give rise to hematopoietic cells, - myeloid cells and lymphoid cells (Bjorson et al., Science 283:534 etc. (1998)). Thus, contactin mRNA, contactin DNA or contactin protein may be detected in blood.

A variety of human contactin mRNA sequences have been reported. Reid and Hemperly report a human contactin mRNA having 3360 nucleotides, accession number Z21488 (Reid and Hemperly, Bran Res. 21:1-8 (1994)). Berglund and Ranscht report an mRNA encoding human contactin 1 precursor having 3314 nucleotides, accession number U07819 (Berglund and Ranscht, Genomics 21:571-582 (1994)). Berglund and Ranscht report an mRNA encoding human contactin 2 precursor having 3335 nucleotides, accession number U07820 (Berglund and Ranscht, Genomics 21 :571-582 (1994)). Watanabe et al. report a mRNA encoding bovine F3/F11/contactin having 3412 nucleotides (Watanabe et al., Gene 160:245-248 (1995)). Hosoya et al. report a mRNA encoding rat F3 having 3214 nucleotides (Hosoya et al., Neurosci, Lett. 186:2-3 (1995)). Portions of the sequences of Neuro-1, human contactin, mouse F3 and chicken Fll have been compared (Reid et al., Mol. Brain Res. 21:1-8 (1994)). Also, PCR primers for mouse F3 have been reported (Reid et al., Mol. Brain Res. 21:1-8 (1994)). The functional domains of chicken Fll have been mapped using deletions of various regions of chicken Fll (Brummendorf et al., Neuron 10:711-727 (1993)). These functional domains have been compared to surface molecules having similar functions (Brummendorf and Rathjen , J. Neurochem. 61:1207-1219 (1993)).

The inventors contemplate that there are allelic variants of the various contactins which may have sequences different from those reported in the literature. Allelic variants may have different sequences from the reported sequences, such as those that arise naturally from deletions, insertions or substitutions. Allelic variants may have different structures or different functions from the contactins reported in the literature. Such allelic variants are considered by the inventors to encode contactin. In addition to allelic variants, the inventors contemplate altered nucleic acid sequenced that encode a contactin including deletions, insertions or substitutions of different nucleotides resulting in a polynucleotide that encodes the same, a functionally equivalent contactin or a contactin that retains at least one activity of the parent contactin. Such altered nucleic acid sequences can be made using established methods in the art, such as site directed mutagenesis or random mutagenesis as they are known in the art. Proteins encoded by such altered nucleic acid sequences may exhibit equivalent, similar or different structures or activities as compared to the parent molecule. Such altered nucleic acid sequences are considered by the inventors to encode contactin.

The present invention includes nucleic acid molecules that are useful as primers for use in PCR amplification procedures specific for the amplification of at least one contactin mRNA or contactin DNA, particularly in samples derived from humans (see, U.S. Patent No. 4,683,195; U.S. Patent No. 4,965,188; and Innis et al., PCR Strategies, Academic Press, San Diego (1995) for PCR procedures). Such PCR amplification methods are known in the art and include primer extension PCR, real time PCR, reverse transcriptase PCR, (Freeman et al., BioTechniques 26:112-125 (1999)) inverse PCR (Triglia et al., Nucleic Acids Res. 16:8186 (1988)), capture PCR (Lagerstrom et al., PCR Methods Applic. 1:111-119 (1991)), differential primer extensions (WO 96/30545 to Fahy and Ghosh, published October 3, 1996) and other PCR amplification methods known in the art or later developed (see, Innis et al., PCR Strategies, Academic Press, San Diego (1995)).

In operation, PCR methods generally use primer molecules that are usually chemically synthesized, but they may be generated enzymatically or produced from a recombinant course. PCR primers generally comprise two nucleotide sequences, one with sense orientation (5' -> 3') and one with antisense (3* -> 5'), employed under preferred conditions for identification of a specific gene or condition. The same PCR primers, nested sets of oligomers or a degenerate pool of oligomers can be employed under less stringent conditions for detection and/or quantitation of closely related DNA or RNA sequences. Additionally, methods that can be used to quantitate the expression of a particular molecule include radiolabeling (Melby et al., J. Immunol Methods 159:235- 244 (1993)) or biotinylating (Duplaa et al., Anal. Biochem. 229-236 (1993)) nucleotides, coamplification of a control nucleic acid, and standard curves onto which the experimental results are interpolated. Quantitation of multiple samples can be sped up by running the assay in an ELISA format where the nucleic acid molecule of interest is presented in various dilutions and a spectrophotometric or colorimetric responses gives rapid quantitation. Colorimetric intercalating dyes can be used in such quantitative methods, as is described in the Examples and as are known in the art (Freeman et al., BioTechniques, 26:112-125 (1999); and Spiess et al., BioTechniques 26:46-50 (1999)).

These nucleic acid molecules can also be used individually or in combination as probes to identify contactin mRNA or DNA molecules in a sample. These nucleic acid molecules include .

5'-TCAGTAAGGTCTGGTTCACGCTAT-3* (SEQ ID NO:l),

5'-TCCCGTCACTGTAGATTCATTTGA-3' (SEQ ID NO:2),

5*-CCCCAAGTCTTCTCGGCTTA-3* (SEQ ID NO:3),

5'-CAACACATTCAGAATTCCAAGTAGACA-3' (SEQ ID NO:4),

5'-TCCCCAAGTCTTCTCGGCTTA-3' (SEQ ID NO:5),

5'-CCCATCCCAGCTCAGAAGAC-3' (SEQ ID NO:6), and

5'-GCCGCAGAAATTGGAAGG-3' (SEQ ID NO:7).

SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:6 are forward primers. SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:7 are reverse primers.

The invention includes primers that have substantial identity to the nucleic acid molecules of SEQ ID NO:l, SEQ JJD NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 or portions thereof or antisense versions thereof, so long as they are capable of specifically amplifying contactin RNA or DNA in a sample.

Other primers can be utilized in such PCR procedures, so long as they are specific for the amplification of at least one human contactin mRNA or contactin DNA in a sample, particularly in samples derived from humans. Such PCR primers can be selected by identifying stretches of at least one contactin DNA or RNA that are unique to at least one contactin DNA or mRNA using sequence comparing algorithms, such as Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). The specificity of such primers to amplify at least one contactin mRNA or contactin DNA can be determined using the methods of the present invention as exemplified by the Examples. The invention includes primers that have substantial identity to such identified nucleic acid molecules or portions thereof, so long as they are capable of specifically amplifying at least one contactin mRNA or contactin DNA in a sample. The present invention also includes antisense versions of these nucleic acid molecules. Such antisense molecules are useful as probes to detect DNA or RNA encoding contactin in a sample. Such antisense sequences can be determined by deducing the antisense sequence from a given sequence. Such antisense molecules can be either DNA or RNA, and the skilled artisan would of course appreciate the different base pairs used in the coding scheme for these different nucleic acid molecules. These antisense molecules can be used to modulate gene expression of the targeted sequence. Preferably, such antisense molecules target the transcription initiation site of a gene to prevent transcription, or a mRNA molecule, to present translation or binding of ribosomes to the mRNA. Inhibition of the transcription or translation of a gene can also be accomplished using "triple helix" base-pairing methodologies, which comprises the ability of double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA were reviewed by Gee et al. (In: Huber and Carr, Molecular and Immunologic Approaches, Futura Publishing Co., N.Y. (1994)). The nucleic acid molecules of the present invention can be made by a variety of methods known in the art. For example, nucleic acid molecules can be made using synthetic procedures or molecular biology techniques known in the art (see, Sambrook et al., supra). The length of the nucleic acid molecules of the present invention can be readily chosen by one skilled in the art depending on the particular purpose that the nucleic acid molecule is to be used for. For PCR primers, the length of the nucleic acid molecule is preferably between about 10 nucleotides and about 50 nucleotides in length, more preferably between about 12 nucleotides and about 30 nucleotides in length, and most preferably between about 15 nucleotides and about 20 nucleotides in length. For probes, the length of the nucleic acid molecule is preferably between about 20 nucleotides and about 1,000 nucleotides in length, more preferably between about 100 nucleotides in length and about 500 nucleotides in length, and most preferably between about 200 nucleotides and about 400 nucleotides in length. The nucleic acid molecules of the present invention can be linked to a detectable label to form a labeled nucleic acid molecule. Such labeled nucleic acid molecules can be made using methods known in the art. Such labeled nucleic acid molecules are useful alone or in combination as probes to detect contactin DNA or contactin mRNA in a sample using established nucleic acid hybridization methods, such as solid-phase hybridizations or in situ hybridizations. Such labeled probes can be used in PCR procedures that utilize labeled primers, included multiple labeled primers, such as fluorescence resonance energy transfer (FRET) based amplification procedures as they are known in the art.

The labeled or unlabeled nucleic acids of the present invention can be provided separately or in combination in a kit to practice at least one method of the present invention. The nucleic acid molecules can be provided in a single or separate container, along with other reagents, buffers or materials to be used in practicing at least one method of the present invention. The kit can be provided in a container, such as a packaging container, that can optionally include instructions for performing at least one method of the present invention. The instructions can be provided in any language or format, preferably in a language and format directed towards a target end-user such that the end user can practice at least one method of the present invention.

Methods for Detecting the Presence of a Neurodegenerative Disease

The present invention also includes methods for detecting the presence of a neurodegenerative disease such as multiple sclerosis, including providing a sample from a patient and measuring the amount of contactin protein, contactin DNA or contactin mRNA expressed in the sample.

Samples for use in this method can be taken from any tissue, organ or fluid from the patient. Such samples can be obtained iising methods known in the art, such as biopsies, aspirations or scrapings. Preferred samples include samples from the nervous system, including the central nervous system, pancreas, lung, kidney, blood, mouth, nasal passages, urine, feces and skeletal muscle. Preferably, the sample is derived at least in part from the central nervous system of the patient. The samples can used as they are taken from the patient, or be processed by, for example, thin section preparation, homogenization, or for the culturing of cells within the sample using established methods. Once samples are prepared for a particular detection method, the amount of contactin mRNA, contactin DNA or contactin protein in a sample can be determined.

Contactin mRNA in a sample can be measured using a variety of established methods, such as PCR methods as they are known in the art. Such PCR methods utilize appropriate nucleic acid molecules of the present invention as they are discussed in the previous section and in the Examples. Contactin mRNA can also be measured using hybridization methods, such as blot analysis, such as Northern Blot analysis or slot/dot blot analysis, and in situ hybridization as such methods are known in the art

Contactin DNA can be measured using established methods, such as hybridization methods known in the art. Such hybridization methods would utilize appropriate nucleic acid molecules of the present invention as they are discussed in the previous section. Contactin DNA can also be measured using hybridization methods, such as blot analysis, such as Southern Blot analysis or slot/dot blot analysis, or in situ hybridization as such methods are known in the art. Contactin DNA can also be determined in chromosomal or cellular preparation using fluorescence in situ hybridization (FISH) methods as they are known in the art using the nucleic acid molecules of the present invention (Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, N.Y. (1988)).

Contactin protein in a sample can be determined using a variety of methods. For example, immunological methods, such as ELISA, Western Blot analysis, or immunocytochemical analysis can be utilized. Generally, these methods use a primary antibody that is specific for contactin. Such antibodies are known in the art, or cairbe made using established methods known in the art (Faivre-Sarrailh et al., J. Nurosci., 12:257-267 (1992); Brummendorf et al., Neuron 10:711-727 (1993); and Pesheva et al., Neuron 10:69-82 (1993)). This primary antibody can be attached to a detectable label so that the binding of the primary antibody to contactin in a sample can be detected. Alternatively, the primary antibody is not attached to a detectable label. In this instance, a secondary antibody that specifically binds with the primary antibody, preferably at the Fc region of the primary antibody, is used. The secondary antibody is attached to a detectable label so that the binding of the primary antibody to contactin is detected.

The amount of contactin mRNA, contactin DNA or contactin protein in a sample is then preferably compared to the amount of contactin mRNA, contactin DNA or contactin protein in a control sample. Appropriate control samples are readily chosen by the skilled artisan. For example, appropriate control samples include samples taken from normal patents that do not have a particular disease state or who are known to express a normal amount of contactin. Such a control would be derived from the same tissue, organ or fluid that the test sample was derived from so that a valid comparison can be made. The amount of contactin mRNA, contactin DNA or contactin protein in a control sample can be provided in the form of a chart or other documentation or database such that control samples need not be processed for all assays. Differences between the amount of contactin mRNA, DNA or protein in the sample that are statistically different from the amount of contactin mRNA, DNA or protein in the control sample is indicative of a neurodegenerative disease, particularly from the group consisting of amyotiOphic lateral sclerosis, multiple sclerosis, MELAS and MERRF.

The method of the present invention can also comprise measuring the amount of control mRNA, control DNA or control protein in the sample, wherein the control mRNA or control DNA does not encode contactin and the control protein is not contactin. Preferably, the control mRNA, control DNA or control protein relates to a protein that is constitutively, constantly or highly expressed, such as actin, ribosomal RNA or GAPDH. In this instance, the control is an internal control such that, the amount of contactin mRNA, contactin DNA or contactin protein and the amount of control mRNA, control DNA or control protein are detected in the same sample. Control mRNA, control DNA or control protein can be detected using the methods described herein for detection of contactin mRNA, contactin DNA or contactin protein. The amount of control mRNA, control DNA or control protein are preferably compared to the amount of contactin mRNA, contactin DNA or contactin protein in the sample. Preferably, the ratio of contactin / control mRNA, DNA or protein is determined and compared to the normal values obtained from samples derived from normal samples. Differences in the ratio of control mRNA, DNA or protein that are statistically different from normal values is indicative of a neurodegenerative disease, particularly from the group consisting of amyotrophic lateral sclerosis, multiple sclerosis, MELAS and MERRF. hi certain other preferred embodiments, nucleic acid molecules having specific nucleotide sequences may be efficiently detected, screened and/or quantified by high throughput hybridization methodologies directed to independently probing a plurality of distinct DNAs, or a plurality of distinct oligonucleotide primers as provided herein, that have been immobilized as nucleic acid arrays on a solid phase support. Typically, the solid support may be silica, quartz or glass, or any other material on which nucleic acid may be immobilized in a manner that permits appropriate hybridization, washing and detection steps as known in the art and as provided herein. In preferred embodiments, solid-phase nucleic acid arrays are precisely spatially addressed, as described, for example, U.S. Patent 5,800,992 (see also, e.g., WO 95/21944; Schena et al., 1995 Science 270:467-470, 1995; Pease et al., 1994 Proc. Nat. Acad. Sci. USA 91:5022; Lipshutz et al., 1995 Biotechniques 19: 442-447).

Detection of hybridized (e.g., duplexed) nucleic acids on the nucleic acid array may be achieved according to any known procedure, for example, by spectrometry or potentiometry (e.g., MALDI-MS). Within certain preferred embodiments the array contains oligonucleotides that are less than 5500 nt, in other preferred embodiments less

' than 500 nt, in other preferred embodiments less than 100 nt, and in other preferred embodiments less than 50 nt in length. For high throughput screening of nucleic acid arrays, the format is preferably amenable to automation. It is preferred, for example, that an automated apparatus for use according to high throughput screening embodiments of the present invention is under the control of a computer or other programmable controller. The controller can continuously monitor the results of each step of the nucleic acid deposition, washing, hybridization, detection and related processes, and can automatically alter the testing paradigm in response to those results.

Methods for Identifying Which Patients Having a Neurodegenerative Disease are Likely to Respond to a Treatment for a Neurodegenerative Disease

The present invention includes methods for identifying which patients having a neurodegenerative disease are likely to respond to a treatment for a neurodegenerative disease.Such methods include: providing samples from a group of patients having said neurodegenerative disease; measuring the amount of contactin protein, contactin mRNA or contactin DNA present in the samples; providing said treatment to said patients; measuring the degree, frequency, rate or extent of responses of said patients to said treatment; and determining if a correlation exists between the amount of contactin protein, contactin DNA or contactin mRNA present in said samples and the degree, frequency, rate or extent of said responses. Wherein: 1) if said correlation is a positive correlation, the presence of said positive correlation indicates that the patients providing samples having an increased amount of contactin protein, contactin DNA or contactin mRNA are likely to respond to said treatment; or 2) if said correlation is a negative correlation, the presence of the negative correlation indicates that the patients providing samples having a decreased amount of contactin protein, contactin DNA or contactin mRNA are likely to respond to said treatment.

In the practice of this method, samples are provided from a group of patients that have been diagnosed as having a neurodegenerative disease, such as multiple sclerosis , amyotrophic lateral sclerosis, MELAS or MERRF. The sample can be from any tissue, organ or fluid, but is preferably derived at least in part from neurological tissue, preferably central nervous tissue. The group of patients is preferably greater than one, more preferably at least four, and most preferably at least nine. The amount of contactin mRNA, contactin DNA or contactin protein is measured in these samples using methods known in the art or described herein.

The patients are then provided a treatment that may regress, alleviate, reduce the severity, reduce the progress or cure the neurodegenerative disease. The treatment can be any treatment, including those that administer or do not administer a compound or composition. For example, the treatment can include the use of companion animals or humans, massage, humor or other treatment that does not include the purposeful administration of a compound or composition to the patent by any route of administration. The treatment can also include administering a compound or composition to the patient, including tradition or non-traditional medicines or treatments, such as herbal remedies or aroma therapy. The response of the patients are monitored using criteria and endpoints that are related to or established for the neurodegenerative disease. For example, the degree, frequency, rate or extent of the patients' response to the, treatment can be measured using methods and endpoints established for the neurodegenerative disease. Should a patient die during the treatment, samples can be taken from the cadaver and death be recorded as the progress of the disease.

Samples are taken from the patents at least one time during the course of the treatment. Preferably, the samples are from the same tissue, organ or fluid that the original sample was taken from. The amount of contactin mRNA, contactin DNA or contactin protein is measured in these samples. The response of the patients are correlated to the change in the amount of contactin mRNA, contactin DNA or contactin protein in the samples from the patients. A positive correlation exists if the presence of the correlation indicates that patients providing samples having an increased amount of contactin mRNA, contactin DNA or contactin protein are likely to respond to the treatment. A negative correlation exists if the presence of the correlation indicates that patients providing samples having a decreased amount of contactin mRNA, contactin DNA or contactin protein are likely to respond to the treatment.

For both positive and negative correlations, the response can be desirable or undesirable. A desirable response is a response that regresses, alleviates, reduces the severity, reduces the progress or cures the neurodegenerative disease. An undesirable response is any response that is not a desirable response.

Cells That Exhibit an Increased or Decreased Amount of Contactin

The present invention also includes compositions of matter that include a cell or a cell in culture that exhibit an increased or decreased amount of contactin, such as contactin mRNA, contactin DNA or contactin protein, as compared to a control cell.

The cell can be part of a tissue, organ or fluid, or a portion thereof. The cell can also be cultured in vitro.

The cells of the present invention can be derived from a sample taken from a test animal or patient, such as a human patient. Such cells can be part of a primary cell culture or a continuous cell line. Preferably, the cells of the present invention are a clonal population. Primary cell cultures can be obtained using methods known in the art. Continuous cell lines can be made by repeated passage of a population of cells in culture until a continuous cell line is obtained. Alternatively, a primary cell can be made into a continuous cell line by immortalizing the cell line using methods known in the art. For example, a primary cell can be fused with an immortal cell line using polyethylene glycol or an electric charge. Alternatively, a primary cell can be infected with a transforming virus, such as a retrovirus, to make the cell line immortal. A cell can also be engineered using methods known in the art so that the cell exhibits an increased or decreased amount of contactin mRNA, contactin DNA or contactin protein. For example, cells that exhibit an increased amount of contactin mRNA, contactin DNA or contactin protein can be made by transfecting a cell line with - nucleic acid molecules encoding a contactin protein. The nucleic acid molecule can be provided in a vector, and can be operably linked to control sequences, such as CMV promoters or LTR elements, such that relatively high levels of contactiri are expressed in the cell. The nucleic acid molecule encoding a contactin can also be provided in a vector that is operably linked to a nucleic acid sequences that promote homologous recombination to known regions of the genome such* that the nucleic acid molecule encoding a contactin can be expressed under the control of an endogenous promoter (see, WO 94/24301 to Smith et al., published October 27, 1994). The nucleic acid molecule encoding a contactin can also be provided in a vector that is not operably linked to a control sequences or nucleic acid sequences that promote homologous recombination such that the nucleic acid molecule encoding a contactin is randomly integrated into the genome of the cell (see, WO 98/13353 to Whitney, published April 2, 1998). The increased amount of contactin mRNA, contactin DNA or contactin protein in these cells can be confirmed using the methods of the present invention

Cells that exhibit a decreased amount of contactin can be made by transfecting cell lines with nucleic acid molecules that encode antisense molecules to contactin DNA or contactin mRNA or that encode ribozymes that can degrade contactin mRNA using methods known in the art. The decreased amount of contactin mRNA, contactin DNA or contactin protein in these cells can be confirmed using the methods of the present invention. A cell that exhibits an increased or decreased amount of contactin mRNA, contactin DNA or contactin protein can be used to make a cybrid cell using methods known in the art (see, WO 95/26973 to Herrnstadt et al., published October 12, 1995). Briefly, a cell is made essentially devoid of mitochondria by exposure to ethidium bromide. These cells are then fused with platelets from a patient, such as a patent with a neurodegenerative disorder or a normal patient, which comprise mitochondria. The fused cell lines contain the nuclear elements of the cell and the mitochondria, including the mitochondrial DNA, of the platelets. The amount of contactin mRNA, contactin DNA or contactin in these cybrids can be measured using the methods of the present invention and can be compared to the cell prior to the fusion with platelets.

Furthermore, cybrids that exhibit an increased or decreased amount of contactin mRNA, contactin DNA or contactin protein can be made. A cell is exposed to ethidium bromide to make the cell essentially devoid of mitochondria. The cell essentially devoid of mitochondria (such as human SH-SY5 Y neuroblastoma cell line) is^fused with platelets from a normal patient or a patient with a neurodegenerative disorder. The amount of contactin mRNA, contactin DNA or contactin protein in these cybrids can be measured using the methods of the present invention and compared to the amount of contactin mRNA, contactin DNA or contactin protein expressed in the parent cells to identify cybrid cell lines that exhibit an increased or decreased amount of contactin mRNA, contactin DNA or contactin protein.

As provided herein, the present invention relates in part to at least one differentially expressed nucleic acid molecule that is associated with Alzheimer's disease. For example, such a nucleic acid molecule may encode a contactin polypeptide, although the contemplated invention need not be so limited and may be directed to any of the AD differentially expressed genes described in greater detail below. In certain embodiments, a differentially expressed nucleic acid molecule that is associated with AD may directly or indirectly alter (e.g., increase or decrease in a statistically significant manner relative to an appropriate control) a mitochondrial function, for example, mitochondrial calcium regulation and/or apoptosis. As described herein, the present invention provides the surprising observation that FREAC-2, a known transcription factor, is such a differentially expressed gene product associated with AD that alters apoptosis.

Whereas mitochondria-mediated apoptosis may therefore contribute to the pathogenesis of degenerative diseases (e.g., neurodegenerative diseases such as AD), the present invention also contemplates, according to non-limiting theory, that disorders associated with inappropriate cell proliferation or cell survival, such as cancer, involve the unregulated and undesirable growth (hyperproliferation) of cells that have somehow escaped a mechanism that normally triggers apoptosis in such cells. The present invention therefore, according to certain embodiments, relates to a method for treating not only neurodegenerative diseases, but also for treating a disease associated with inappropriate cell proliferation or cell survival, in view of the instant disclosure that induced FREAC-2 expression in a cell results in that cell becoming apoptotic.

By way of background, enhanced expression of the anti-apoptotic protein Bcl-2 and its homologues is involved in the pathogenesis of numerous human cancers. Bcl-2 acts by inhibiting programmed cell death, and overexpression of Bcl-2 and the related gene product known as Bcl-xL block mitochondrial release of cytochrome c from mitochondria and the activation of caspase 3 (Yang et al, Science 275:1129-1132, 1997; Kluck et al, Science 275:1132-1136, 1997; Kharbanda et al., Proc. Natl. Acad. Sci. USA 94:6939-6942, 1997). Bcl-2 also binds to several proteins that are involved in death regulation (Reed, Nature 387:773-779, 1997). Overexpression of Bcl-2 and Bcl- xL protect against the mitochondrial dysfunction preceding nuclear apoptosis that is induced by chemotherapeutic agents, h addition, acquired multi-drug resistance to cytotoxic drugs is associated with inhibition of cytochrome c release that is dependent on overexpression of Bcl-xL (Kojima et al., J. Biol. Chem. 273: 16647-16650, 1998). Given that mitochondria have been implicated in apoptosis, it is expected according to non-limiting theory that agents which interact with mitochondrial components will effect a cell's capacity to undergo apoptosis. Thus, agents that induce or promote apoptosis in hyperproliferative cells are expected to be useful in treating such diseases associated with inappropriate cell proliferation or cell survival. Another embodiment of the invention therefore relates to the identification of compounds that increase the degree or enhance the rate of apoptosis in hyperproliferative cells present in such diseases and disorders associated with inappropriate cell proliferation or cell survival, for example, cancer and psoriasis (note that, for the purposes of the disclosure, the term "hyperproliferative disease or disorder" specifically excludes pregnancy). Because oncogenic changes render certain tumors more susceptible to apoptosis (Evan and Littlewood, Science 257:1317,1998), such agents are expected to be useful for treating such hyperproliferative diseases or disorders. As disclosed herein, induction of apoptosis following induction of FREAC-2 expression provides compositions and methods for the treatment of such diseases and offers related advantages. In a related embodiment, a biological sample from a patient having or suspected of having a hyperproliferative disease or disorder may be evaluated for susceptibility to such agents using the methods of the invention.

Thus, and as noted above, induced FREAC-2 expression and mitochondrial regulation are believed to provide an induction signal for cellular apoptosis (programmed cell death), which may be determined according to any of a number of apoptosis assay methodologies as known in the art and as provided herein. FREAC-2 expression may be induced in vitro (e.g., in a host cell transfected with a recombinant expression construct as provided herein) or in vivo (e.g., via established transgenic methodologies or via a gene therapy vector comprising a recombinant expression construct as provided herein). In one such apoptosis assay, cells that are suspected of undergoing apoptosis may be examined for morphological, permeability or other changes that are indicative of an apoptotic state. For example by way of illustration and not limitation, apoptosis in many cell types may cause altered morphological appearance such as plasma membrane blebbing, cell shape change, loss of substrate adhesion properties or other morphological changes that can be readily detected by those skilled in the art using light microscopy. As another example, cells undergoing apoptosis may exhibit fragmentation and disintegration of chromosomes, which may be apparent by microscopy and/or through the use of DNA specific or chromatin specific dyes that are known in the art, including fluorescent dyes. Such cells may also exhibit altered plasma membrane permeability properties as may be readily detected through the use of vital dyes (e.g., propidium iodide, trypan blue) or by the detection of lactate dehydrogenase leakage into the extracellular milieu. These and other means for detecting apoptotic cells by morphologic criteria, altered plasma membrane permeability and related changes will be apparent to those familiar with the art. In another example of an apoptosis assay, translocation of cell membrane phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane is detected by measuring outer leaflet binding by the PS-specific protein annexin. (Martin et al., J. Exp. Med. 182:1545, 1995; Fadok et al., J. Immunol. 148:2207, 1992.) In still another apoptosis assay, induction of specific protease activity in a family of apoptosis- activated proteases known as the caspases is measured, for example by determination of caspase-mediated cleavage of specifically recognized protein substrates. These substrates may include, for instance, poly-(ADP-ribose) polymerase (PARP) or other naturally occurring or synthetic peptides and proteins cleaved by caspases that are known in the art (.see, e.g., EUerby et al., 1997 J. Neurosci. 77:6165). The synthetic peptide Z-Tyr-Val-AIa-Asp-AFC (SEQ ID NO:_), wherein "Z" indicates a benzoyl carbonyl moiety and AFC indicates 7-amino-4-trifluoromethylcoumarin (Kluck et al., 1997 Science 275:1132; Nicholson et al., 1995 Nature 376:37), is one such substrate. Other substrates include nuclear proteins such as Ul-70 kDa and DNA-PKcs (Rosen and Casciola-Rosen, 1997 J. Cell. Biochem. 64:50; Cohen, 1997 Biochem. J. 326:1).

As described above, the mitochondrial inner membrane may exhibit highly selective and regulated permeability for many small molecules, including certain cations, but is impermeable to large (>~10 kDa) molecules. (See, e.g., Quinn, 1976 The Molecular Biology of Cell Membranes, University Park Press, Baltimore, Maryland). Thus, in another example of an apoptosis assay, detection of the mitochondrial protein cytochrome c that has leaked out of mitochondria in apoptotic cells may provide an apoptosis indicator that can be readily determined. (Liu et al., Cell 86:147, 1996) Such detection of cytochrome c may be performed spectrophotometrically, immunochemically or by other well established methods for determining the presence of a specific protein.

Release of cytochrome c from cells exposed to apoptotic stimuli (e.g., an apoptogen, including a calcium cation ionophore such as ionomycin, or by transfection with a nucleic acid sequence that provides an apoptotic signal) can be followed by a variety of immunological methods. Matrix-assisted laser desorption ionization time-of- flight (MALDI-TOF) mass spectrometry coupled with affinity capture is particularly suitable for such analysis since apo-cytochrome c and holo-cytochrome c can be distinguished on the basis of their unique molecular weights. For example, the Surface- Enhanced Laser Desorption/Ionization (SELDI™) system (Ciphergen, Palo Alto, California) may be utilized to follow the inhibition by mitochondria protecting agents of ^• cytochrome c release from mitochondria in ionomycin treated cells. In this approach, a cytochrome c specific antibody immobilized on a solid support is used to capture released cytochrome c present in a soluble cell extract. The captured protein is then encased in a matrix of an energy absorption molecule (EAM) and is desorbed from the solid support surface using pulsed laser excitation. The molecular mass of the protein is determined by its time of flight to the detector of the SELDI™ mass spectrometer.

Under certain conditions, apoptosis may be induced by exposing a biological sample to compositions referred to as "apoptogens" that induce programmed cell death, or "apoptosis". A variety of apoptogens are known to those familiar with the art (see, e.g., Green et al., 1998 Science 281:1309 and references cited therein) and may include by way of illustration and not limitation: tumor necrosis factor-alpha (TNF- ); Fas ligand; glutamate; N-methyl-D-aspartate (NMDA); interleukin-3 (IL-3); herbimycin A (Mancini et al, 1997 J. Cell. Biol. 138:449-469); paraquat (Costantini et al., 1995 Toxicology 99:1-2); ethylene glycols; protein kinase inhibitors, such as staurosporine, calphostin C, caffeic acid phenethyl ester, chelerythrine chloride, genistein; l-(5- isoquinolinesulfonyl)-2-methylpiperazine; N-[2-(( -bromocinnamyl)amino)ethyl]-5-5- isoquinolinesulfonamide; KN-93; quercitin; d-ervtbro-sphingosine derivatives, for example, ceramide; UV irradiation; ionophores such as ionomycin and valinomycin; MAP kinase inducers such as anisomycin, anandamine; cell cycle blockers such as aphidicolin, colcemid, 5-fluorouracil, homoharringtonine; acetylcholinesterase inhibitors such as berberine; anti-estrogens such as, tamoxifen; pro-oxidants, such as tert-butyl peroxide, hydrogen peroxide; free radicals such as nitric oxide; inorganic metal ions, such as cadmium; DNA synthesis inhibitors, including, for example, actinomycin D and also including DNA topoisomerase inhibitors, for example, etoposide; DNA intercalators such as doxorubicin, bleomycin sulfate, hydroxyurea, methotiexate, mitomycin C, camptothecin, daunorubicin; protein synthesis inhibitors such as cycloheximide, puromycin, rapamycin; agents that affect microtubulin formation or stability, for example, vinblastine, vincristine, colchicine, 4- hydroxyphenylretinamide, paclitaxel; Bad protein, Bid protein and Bax protein (see, e.g., Jurgenmeier et al., 1998 Proc. Nat. Acad. Sci. USA 95:4997-5002 and references cited therein); calcium and inorganic phosphate (Kroemer et al., 1998 Ann. Rev. Physiol. 60:619).

Thapsigargin is a Ca²⁺ uptake inhibitor of the endoplasmic reticulum (ER), thereby influencing mitochondrial regulation of intracellular calcium homeostasis; this compound is commercially available (Calbiochem, San Diego, CA). Other agents thafrelease Ca²⁺ from extramitochondrial reservoirs, and/or prevent the uptake of Ca²⁺ into such extramitochondrial reservoirs, include without limitation inositol-1,4,5- triphosphate (Streb et al., Nβtwre 306:67-69, 1983; Berridge et al., FASEB J. 2:3074- 3082, 1988), okadaic acid (Hepworth et al., Cell Calcium 21:461-467, 1997), caffeine, and dantrolene (Horton et al., J. Surg. Res. 87:39-50 (1999)). Other agents that influence mitochondrial function are as follows:

Ruthenium Red is a cytological stain that inhibits the Ca²⁺ uniporter and therefore uptake of Ca²⁺ into mitochondria (Reed and Bygrave, Bioch. J, 140:143-155, 1974). It also blocks release of Ca from the sarcoplasmic reticulum (Antonius et al, Biochem. Biophys. Acta 816:9-17, 1985; Chiesi et al, Biiochem. Biophys. Res. Commun. 154:1-8, 1988) and the sequestering ability of the endoplasmic reticulum (Hurley, Am. J. Physiol. 23:621-627, 1988). Ru 360 (Calbiochem, San Diego, CA; 557440) is the dinuclear ruthenium amid portion of Ruthenium Red, proposed to be responsible for the inhibition effects of Ruthenium Red (Ying et al., Biochemistry, 30:4949-4952, 1991; Emerson et al, J. Am. Chem. Soc. 115:11799-11805, 1993). _ FCCP (carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone; Sigma) is a potent uncoupler of oxidative phosphorylation in mitochondria (Heytler et al., Biophys. Res. Commun. 7:272, 1962; Biochem. J. 195:583, 1981). Other non-limiting examples of respiratory uncouplers include carbonyl cyanide m-chlorophenyl- hydrazone (Sigma) (Heytler et al., Biophys. Res. Commun. 7:272-etc, 1962), and those described by Heytler in, e.g., Methods of Enzymology 55:462, 1979, and Pharmacol.

Ther. 10:461-472, 1980, both of which are hereby incorporated by reference.

Rotenone (Sigma) is an inhibitor of mitochondrial electron transport

(Fukami et al., Science 155:713-716, 1967). Other non-limiting examples of inhibitors of mitochondrial ETC include cyanide, amytal and antimycin.

Oligomycin (Sigma) is an inhibitor of mitochondrial ATPase (Nagamune et al., Biochim. Biophys. Acta 1141:231-231 , 1993). The combination of oligomycin and rotenone, or rotenone alone, can be used as a positive control to evaluate the effects of greatly reducing or essentially elimination of the membrane potential. Ethacrynic acid (2,3-dichloro-4-(2methylene-butryl)phenoxylacetic acid; Sigma) removes the ability of cells to tolerate oxidative stress. Ethacrynic acid inhibits gluatathione S-transferase and thus depletes cells of glutathione (Shen et al., Biochem.

Pharmacol. 50:1233-1238, 1995). Experiments suggest that mitochondrially localized glutathione has a critical role in the maintenance of mitochondrial function (Seyfried et al., Neurosci. Lett. 264: 1 -4, 1999).

The person having ordinary skill in the art will readily appreciate that there may be other suitable techniques for quantifying apoptosis, and such techniques for purposes of determining the effects of mitochondria protecting agents on the induction and kinetics of apoptosis are within the scope of the assays disclosed here.

Methods for Screening Compounds for the Activity of Reducing or Enhancing the Expression of Contactin

The present invention includes methods of screening test compounds for the activity of reducing or enhancing the expression of contactin, including providing at least one cell, contacting said at least one cell with at least one test compound; and measuring the change in contactin mRNA, contactin DNA or contactin protein in the at least one cell. The present invention also includes compositions, including pharmaceutical compositions, that include at least one test compound identified by these methods. In operation of this methods, a cell of the present invention is contacted with at least one test compound. The change in the amount of contactin mRNA, contactin DNA or contactin protein in the cell is then measured using at least one method of the present invention. Preferably, the amount of contactin mRNA, contactin DNA or contactin protein exhibited by the cell is known prior to the cell being contacted with a test compound, but that need not be the case. Compounds that increase or decrease the amount of contactin mRNA, contactin DNA or contactin protein in a cell are presumptive therapeutic agents to increase or decrease contactin mRNA, contactin DNA or contactin protein in a cell. Test compounds that increase the amount of contactin mRNA, contactin

DNA or contactin protein in a cell have presumptive therapeutic activity of decreasing cell mobility, enhancing the binding of cells to Ng-CAM, increasing interaction of cells with the extracellular matrix glycoprotein restrictin, and promoting neurite outgrowth. Test compounds that decrease the amount of contactin mRNA, contactin DNA or contactin protein in a cell have presumptive therapeutic activity of treating a neurodegenerative disease such as multiple sclerosis, lateral sclerosis, MELAS or MERRF, increase cell mobility, decreasing the binding of cells to Ng-CAM, decreasing interaction of cells with the extracellular matrix glycoprotein restrictin, and repressing neurite outgrowth. The identified test compounds can be evaluated using the methods set forth herein.

Pharmacology and toxicity of test compounds

The structure of a test compound can be determined or confirmed by methods known in the art, such as mass spectroscopy. For test compounds stored for extended periods of time under a variety of conditions, the structure, activity and potency thereof can be confirmed.

Identified test compounds can be evaluated for a particular activity using are-recognized methods and those disclosed herein. For example, if an identified test compound is found to have anticancer cell activity in vitro, then the test compound would have presumptive pharmacological properties as a chemotherapeutic to treat cancer. Such nexuses are known in the art for several disease states, and more are expected to be discovered over time. Based on such nexuses, appropriate confirmatory in vitro and in vivo models of pharmacological activity, and toxicology, and be selected and performed. The methods described herein can also be used to assess pharmacological selectivity and specificity, and toxicity. Identified test compounds can be evaluated for toxicological effects using known methods (see, Lu, Basic Toxicology, Fundamentals, Target Organs, and Risk Assessment, Hemisphere Publishing Corp., Washington (1985); U.S. Patent Nos; 5,196,313 to Culbreth (issued March 23, 1993) and 5,567,952 to Benet (issued October 22, 1996)). For example, toxicology of a test compound can be established by determining in vitro toxicity towards a cell line, such as a mammalian, for example human, cell line. Test compounds can be treated with, for example, tissue extracts, such as preparations of liver, such as microsomal preparations, to determine increased or decreased toxicological properties of the test compound after being metabolized by a whole organism. The results of these types of studies are predictive of toxicological properties of chemical s in animals, such as mammals, including humans.

Alternatively, or in addition to these in vitro studies, the toxicological properties of a test compound in an animal model, such as mice, rats, rabbits, dogs or monkeys, can be determined using established methods (see, Lu, supra (1985); and Creasey, Drug Disposition in Humans, The Basis of Clinical Pharmacology, Oxford University Press, Oxford (1979)). Depending on the toxicity, target organ, tissue, locus and presumptive mechanism of the test compound, the skilled artisan would not be burdened to determine appropriate doses, LD₅o values, routes of administration and regimes that would be appropriate to determine the toxicological properties of the test compound. In addition to animal models, human clinical trials can be performed following established procedures, such as those set forth by the United States Food and Drug Administration (USFDA) or equivalents of other governments. These toxicity studies provide the basis for determining the efficacy of a test compound in vivo.

Efficacy of test compounds

Efficacy of a test compound can be established using several art recognized methods, such as in vitro methods, animal models or human clinical trials (see, Creasey, supra (1979)). Recognized in vitro models exist for several diseases or conditions. For example, the ability of a test compound to extend the life-span of HTV- infected cells in vitro is recognized as an acceptable model to identify chemicals expected to be efficacious to treat HIV infection or AIDS (see, Daluge et al., Antimicro. - Agents Chemother. 41:1082-1093 (1995)). Furthermore, the ability of cyclosporin A (CsA) to prevent proliferation of T-cells in vitro has been established as an acceptable model to identify chemicals expected to be efficacious as immunosuppressants (see, Suthanthiran et al., supra (1996)). For nearly every class of therapeutic, disease or condition, an acceptable in vitro or animal model is available. The skilled artisan is armed with a wide variety of such models as they are available in the literature or from the USFDA or the National Institutes of Health (NIH). h addition, these in vitro methods can use tissue extracts, such as preparations of liver, such as microsomal preparations, to provide a reliable indication of the effects of metabolism on a test compound. Similarly, acceptable animal models can be used to establish efficacy of test compounds to treat various diseases or conditions. For example, the rabbit knee is an accepted model for testing agents for efficacy in treating arthritis (see, Shaw and Lacy, J. Bone Joint Surg. (Br.) 55:197-205 (1973)). Hydrocortisone, which is approved for use in humans to treat arthritis, is efficacious in this model which confirms the validity of this model (see, McDonough, Phys. Ther. 62:835-839 (1982)). When choosing an appropriate model to determine efficacy of test compounds, the skilled artisan can be guided by the state of the art, the USFDA or the NIH to choose an appropriate model, doses and route of administration, regime and endpoint and as such would not be unduly burdened. In addition to animal models, human clinical trials can be used to determine the efficacy of test compounds. The USFDA, or equivalent governmental agencies, have established procedures for such studies.

Selectivity of test compounds

The in vitro and in vivo methods described above also establish the selectivity of a candidate modulator. It is recognized that chemicals can modulate a wide variety of biological processes or be selective. Panels of cells as they are known in the art can be used to determine the specificity of the a test compound (WO 98/13353 to Whitney et al., published April 2, 1998). Selectivity is evident, for example, in the field of chemotherapy, where the selectivity of a chemical to be toxic towards cancerous cells, but not towards non-cancerous cells, is obviously desirable. Selective modulators are -preferable because they have fewer side effects in the clinical setting. The selectivity of a test compound can be established in vitro by testing the toxicity and effect of a test compound can be established in vitro by testing the toxicity and effect of a test compound on a plurality of cell lines that exhibit a variety of cellular pathways and sensitivities. The data obtained form these in vitro toxicity studies can be extended ^'to animal model studies, including human clinical trials, to determine toxicity, efficacy and selectivity of a test compound.

The selectivity, specificity and toxicology, as well as the general pharmacology, of a test compound can be often improved by generating additional test compounds based on the structure/property relationship of a test compound originally identified as having activity. Test compounds can be modified to improve various properties, such as affinity, life-time in blood, toxicology, specificity and membrane permeability. Such refined test compounds can be subjected to additional assays as they are known in the art or described herein. Methods for generating and analyzing such compounds or compositions are known in the art, such as U.S. Patent No. 5,574,656 to Agrafiotis et al.

Pharmaceutical compositions

The present invention also encompasses a test compound in a pharmaceutical composition comprising a pharmaceutically acceptable carrier prepared for storage and preferably subsequent administration, which have a pharmaceutically effective amount of the test compound in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co., (A.R. Gennaro edit. (1985)). Preservatives, stabilizers, dyes and even flavoring agents can be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid can be added as preservatives. In addition, antioxidants and suspending agents can be used.

The test compounds of the present invention can be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions, suspensions or injectable administration; and the like. Injectables can be prepared in conventional forms either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride and the like, hi addition, if desired, the injectable pharmaceutical compositions can contain minor amounts of nontoxic auxiliary substances, such as wetting agents, pH buffering agents and the like. If desired, absorption enhancing preparation, such as liposomes, can be used.

The pharmaceutically effective amount of a test compound required as a dose will depend on the route of administration, the type of animal or patient being treated, and the physical characteristics of the specific animal under consideration. The dose can be tailored to achieve a desired effect, but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize. In practicing the methods of the present invention, the pharmaceutical compositions can be used alone or in combination with one another, or in combination with other therapeutic or diagnostic agents. These products can be utilized in vivo, preferably in a mammalian patient, preferably in a human, or in vitro. In employing them in vivo, the pharmaceutical compositions can be administered to the patient in a variety of ways, including parenterally, intravenously, subcutaneously, intramuscularly, colonically, rectally, nasally or intraperiotoneally, employing a variety of dosage forms. Such methods can also be used in testing the activity of test compounds in vivo.

As will be readily apparent to one skilled in the art, the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight and type of patient being treated, the particular pharmaceutical composition employed, and the specific use for which the pharmaceutical composition is employed. The determination of effective dosage levels, that is the dose levels necessary to achieve the desired result, can be accomplished by one skilled in the art using routine methods as discussed above, and can be guided by agencies such as the USFDA or NTH. Typically, human clinical applications of products are commenced at lower dosage levels, with dosage level being increased until the desired effect is achieved. Alternatively, acceptable in vitro studies can be used to establish useful doses and routes of administration of the test compounds.

In non-human animal studies, applications of the pharmaceutical compositions are commenced at higher dose levels, with the dosage being decreased until the desired effect is no longer achieved or adverse side effects are reduced of disappear. The dosage for the test compounds of the present invention can range broadly depending upon the desired affects, the therapeutic indication, route of administration and purity and activity of the test compound. Typically, dosages can be between about 1 ng kg and about 10 ng/kg, preferably between about 10 ng/kg and about 1 mg/kg, more preferably between about 100 ng/kg and about 100 micrograms/kg, and most preferably between about 1 microgram/kg and about 10 micrograms/kg.

The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see, Fingle et al., in The Pharmacological Basis of Therapeutics (1975)). It should be noted that the attending physician would know how to and when to teiminate, interrupt or adjust administration due to toxicity, organ dysfunction or other adverse effects. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate. The magnitude of an administrated does in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight and response of the individual patient, including those for veterinary applications. Depending on the specific conditions being treated, such pharmaceutical compositions can be formulated and administered systemically or locally. Techniques for formation and administration can be found in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA (1990). Suitable routes of administration can include oral, rectal, transdermal, otic, ocular, vaginal, transmucosal or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.

For injection, the pharmaceutical compositions of the present invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution or physiological saline buffer. For such transmucosal administration, penetrans appropriate to the barrier to be permeated are used in the formulation. Such penetrans are generally known in the art. Use of pharmaceutically acceptable carriers to formulate the pharmaceutical compositions herein disclosed for the practice of the invention into dosages suitable for systemic administration is within the scope of the invention. With proper choice of carrier and suitable manufacturing practice, the compositions of the present invention, in particular, those formulation as solutions, can be administered parenterally, such as by intravenous injection. The pharmaceutical compositions can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administrations. Such carriers enable the test compounds of the invention to be formulated as tables, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.

Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. Substantially all molecules present in an aqueous solution at the time of liposome formation are incorporated into or within the liposomes thus formed. The liposomal contents are both protected from the external micro-environment and, because liposomes fuse will cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules can be directly administered intracellularly.

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amount of a pharmaceutical composition is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. In addition to the active ingredients, these pharmaceutical compositions can contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active chemicals into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tables, dragees, capsules or solutions. The pharmaceutical compositions of the present invention can be manufactured in a manner that is itself known, for example by means of conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical formulations for parenteral administration include aqueous solutions of active chemicals in water-soluble form.

Additionally, suspensions of the active chemicals may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides or liposomes. Aqueous injection suspensions may contain substances what increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the chemicals to allow for the preparation of highly concentrated solutions. Pharmaceutical compositions for oral use can be obtained by combining the active chemicals with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tables or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone. If desired, disintegrating agents can be added, such as the cross-linked polyvinyl pyralidone, agar, alginic acid or a salt thereof such as sodium alginate. Dragee cores can be provided with suitable coatings. Dyes or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active doses.

The test compounds of the present invention, and pharmaceutical compositions that include such test compounds are useful for treating a variety of ailments in a patient, including a human. As set forth in the Examples, the test "compounds of the present invention have antibacterial, antimicrobial, antiviral, anticancer cell, antitumor and cytotoxic activity. A patient in need of such treatment can be provided a test compound of the present invention, preferably in a pharmacological composition in an effective amount to reduce the number or growth rate of bacteria, microbes, cancer cells or tumor cells in said patent, or to reduce the infectivity of viruses in said patient. The amount, dosage, route of administration, regime and endpoint can all be determined using the procedures described herein or by appropriate government agencies, such as the United Stated Food and Drug Administration.

Methods of Treating a Neurodegenerative Disease or a Proliferative Disorder

According to the present invention and as described above, it may be desirable in certain clinical contexts (e.g., proliferative disorders) to induce apoptosis in certain cells, while in certain other clinical contexts it may be desirable to reduce or inhibit apoptosis in certain cells (e.g., neurodegenerative disease). As will be appreciated by those having familiarity with the art, the subject invention disclosure that induction of FREAC-2 expression in a cell results in the onset of apoptosis in such a cell permits manipulation of the apoptotic state in one or more desired cells by inducing FREAC-2 expression, for example through the use of a recombinant expression construct directing FREAC-2 expression, or by impairing FREAC-2 expression, for e ample by administration of a recombinant expression construct directing the expression of a FREAC-2-specific antisense or ribozyme transcript, or through the use of an agent that enhances or inhibits FREAC-2-induced apoptosis and that is identified according to the methods disclosed and claimed herein. Details with regard to preparation, targeting, administration and monitoring the effects of recombinant expression constructs directed to regulating FREAC-2 expression will vary according to specific applications and are within the knowledge of the art based on the present disclosure.

The present invention also includes methods of treating a neurodegenerative disease using the compositions or compounds identified by the methods of the present invention. As set forth herein, compounds that increase or decrease the amount of contactin mRNA, contactin DNA or contactin protein in a cell have presumptive therapeutic activities. These therapeutic activities can be confirmed using the methods of the present invention. Preferably, this aspect of the invention includes administering an effective amount of a pharmaceutical composition of the present invention by a route of administration sufficient to provide a treatment that is remedial, therapeutic, palliative, rehabilitative, preventative, impeditive or prophylactic in nature using appropriate endpoints for such treatments.

Appropriate end-points or parameters for the treatment of multiple sclerosis include: a reduction in the progression of the disorder or the frequency or occurrence of the symptoms characteristic of the disorder, such as, for example, cognitive impairment, optic neritis, diplopia, vertigo, weakness, tremor, spasticity, hystagmus, ataxia, visual loss, piplopia, fatigue, sphincter dysfunction, gait impairment, or the amount of immunoglobulins in the cerebral spinal fluid. Appropriate end-points or parameters for the treatment of lateral sclerosis include: a reduction in the progression of the disorder or the frequency or occurrence of the symptoms characteristic of the disorder, such as, for example, the loss of function of upper or lower neurons resulting in progressive skeletal muscular wasting or weakness. Appropriate end-points or parameters for the treatment of MELAS include: a reduction in the progression of the disorder or the frequency or occurrence of the symptoms characteristic of the disorder, such as, for example, focal or generalized seizures, dementia, headaches, vomiting, hemianopsia, cortical blindness, hearing loss, elevated serum lactate or encephalopathy. Appropriate end-points or parameters for the treatment of MERRF include: a reduction in the progression of the disorder or the frequency or occurrence of the symptoms characteristic of the disorder, such as, for example, seizures, ataxia, lactic acidosis, dysarthia, optic atrophy, hearing loss, dementia, nystagmus, spasticity, muscular weakness or increased serum pyruvate

Another aspect of the present invention is a method of treatment for a neurodegenerative disease, including: providing a sample from a patient having said neurodegenerative disease; measuring the amount of contactin protein, contactin DNA or contactin mRNA present in the sample; and administering to the patient an effective amount of a composition of the present invention, wherein a positive correlation exists between the amount of contactin protein or contactin mRNA present in a sample from a patient and the efficacy of said compound. This aspect of the present invention uses the methods described herein to obtain samples from a patient having a neurodegenerative disease and measuring the amount of contactin mRNA, contactin DNA or contactin protein in the sample. If the sample exhibits an increased or decreased level of contactin mRNA, contactin DNA or contactin protein, an appropriate composition, such as a pharmaceutical composition of the present invention, is administered to the patient to modulate the amount of contactin mRNA, contactin DNA or contactin protein towards normal levels. The pharmaceutical composition can be administered in an effective amount by an appropriate route of administration using an appropriate regime. The effectiveness of such treatment can be measured using end-points or parameters appropriate for the neurodegenerative disease being treated. Preferably, such treatments are remedial, therapeutic, palliative, rehabilitative, preventative, impeditive or prophylactic in nature.

Methods for Identifying Pharmaceutical Targets and Pharmaceutical Targets Identified by Such Methods

The present invention includes methods for identifying pharmaceutical targets for compounds that alter mitochondrial function (e.g., that alter FREAC-2- induced apoptosis), or that interact with a gene product that is differentially expressed as a result of induced FREAC-2 expression, or that modulate contactin expression or activity; the invention thus also relates to targets identified by methods of the present invention.

Methods for identifying a target (e.g., a pharmaceutical target such as a target for therapeutic intervention in a neurodegenerative disease, in a disease associated with inappropriate cell proliferation or cell survival, or in a disease associated with altered mitochondrial function) include providing a compound that modulates expression or activity of a particular gene product of interest such as FREAC-2 or a FREAC-2 induced gene product or contactin, such as the amount of mRNA, DNA or protein for the gene product of interest in a cell; and identifying the cellular component(s) that binds to the compound to form a molecular complex, preferably through a specific interaction.

As used herein, biological samples are comprised of "biomolecular components" such as cellular or organellar (e.g., mitochondrial) molecular components, which may be a protein, polypeptide, peptide, amino acid, or derivative thereof; a lipid, fatty acid or the like, or derivative thereof; a carbohydrate, saccharide or the like or derivative thereof, a nucleic acid, nucleotide, nucleoside, purine, pyrimidine or related molecule, or derivative thereof, or the like; or another biological molecule that is a constituent of a sample. Isolation and, optionally, identification and/or characterization of a biomolecular component with which an agent that affects FREAC-2-induced apoptosis interacts, or which is a product of a gene that is differentially expressed in a host cell induced to express FREAC-2, may also be desirable and are within the scope of the invention. Once an agent is shown to alter a mitochondrial activity such as FREAC-2-induced apoptosis, for example, those having ordinary skill in the art will be familiar with a variety of approaches that may be routinely employed to isolate the molecular species specifically recognized by such an agent and involved in regulation of mitochondrial function (in this example, apoptosis), where to "isolate" as used herein refers to separation of such molecular species from the natural biological environment.

Techniques for isolating such a biomolecular component may include any biological and/or biochemical methods useful for separating the component from its biological source, and subsequent characterization may be performed according to standard biochemical and molecular biology procedures. Those familiar with the art will be able to select an appropriate method depending on the biological starting material and other factors. Such methods may include, but need not be limited to, radiolabeling or otherwise detectably labeling biomolecular components (e.g., cellular and mitochondrial molecular components) in a biological sample, cell fractionation, density sedimentation, differential extraction, salt precipitation, ultrafiltration, gel filtration, ion-exchange chromatography, partition chromatography, hydrophobic chromatography, electrophoresis, affinity techniques or any other suitable separation method that can be adapted for use with the agent with which the biomolecular component interacts. Antibodies to partially purified components may be developed according to methods known in the art and may be used to detect and/or to isolate such components. Any biological sample as provided herein may be a suitable source of biological starting material. For example, and in certain preferred embodiments including methods for identifying a FREAC-2-induced polypeptide in a biological sample with which an agent identified according to the subject invention method specifically interact (e.g., through a binding interaction that results in the formation of a molecular complex), cell or tissue lysates, homogenates, extracts, suspensions, fractions or the like, or other preparations containing partially or fully purified biomolecular components may be useful. Isolated preparations of biomolecular components may be present in membrane vesicles such as uni- or multilamellar membrane vesicles, or reconstituted into naturally derived or synthetic liposomes or proteoliposomes or similar membrane-bounded compartments, or the like, according to generally accepted methodologies (e.g., Jezek et al., 1990 J. Biol. Chem. 265:10522-10526).

Affinity techniques are particularly useful in the context of isolating FREAC-2-induced biomolecular components that are proteins or polypeptides which participate in specific binding interactions with an agent identified according to the presently disclosed methods, for example through the formation of an affinity complex that can be separated from non-binding components according to standard methodologies. Such approaches may include any method that exploits a specific binding interaction to effect a separation. For example, useful affinity techniques include immunological techniques for isolating and/or detecting a protein or polypeptide antigen, which techniques rely on specific binding interaction between - antibody combining sites for antigen and antigenic determinants present on the factor. Binding of an antibody or other affinity reagent to an antigen is "specific" where the binding interaction involves a Ka of greater than or equal to about 10⁴ M"¹, preferably of greater than or equal to about 10⁵ M"¹, more preferably of greater than or equal to about 10⁶ M"¹ and still more preferably of greater than or equal to about 10⁷ M"¹ . Affinities of binding partners or antibodies can ^kbe readily determined using conventional techniques, for example those described by Scatchard et al., Ann. NY. Acad. Sci. 51:660 (1949).

Immunological techniques include, but need not be limited to, immunoaffinity chromatography, immunoprecipitation, solid phase immunoadsorption or other immunoaffinity methods. For these and other useful affinity techniques, see, for example, Scopes, R.K., Protein Purification: Principles and Practice, 1987, Springer- Verlag, NY; Weir, D.M., Handbook of Experimental Immunology, 1986, Blackwell Scientific, Boston; and Hermanson, G.T. et al., Immobilized Affinity Ligand Techniques, 1992, Academic Press, Inc., California; which are hereby incorporated by reference in their entireties, for details regarding techniques for isolating and characterizing complexes, including affinity techniques.

Additional approaches presented here are described with respect to contactin as an examplar of a differentially expressed gene product in a neurodegenerative disease, but as noted above, it is to be understood that the description which follows of further methods for identifying molecular targets such as pharmaceutical targets or molecular targets for therapeutic intervention is also applicable to FREAC-2, to FREAC-2-induced gene products and to other differentially expressed gene products. In practice, the method includes providing a cell, such as a cell of the present invention, that expresses an increased, decreased or normal amount of contactin mRNA, contactin DNA or contactin protein. Alternatively, an extract of the cell can be provided. The cell or extract thereof is then contacted with a compound that modulates the amount of contactin mRNA, contactin DNA or contactin protein in a sample. The binding of the compound to a cellular component is then detected. The cellular component that binds with the compound is a presumptive therapeutic target for the treatment of neurodegenerative diseases, such as multiple sclerosis, lateral sclerosis, MELAS or MERRF.

The binding of compound to a cellular component can be detected using a variety of methods. For example, the compound can be attached to a detectable label such that the location of the compound can be monitored in a cell. The compound- cellular component can then be isolated using affinity chromatography or immunoprecipitation methods that use antibodies specific for the compound or label. Preferably, gel-shift assays can be used to detect compound-cellular component binding. For example, a compound attached to a detectable label has a certain mobility though a gel in response to an electrical current due to a variety of parameters, including the molecular weight of the compound attached to a detectable label. The binding of compound to cellular component changes the mobility of the resulting compound- cellular component complex through the gel due to the increased molecular weight of the complex relative to the cellular component and compound alone. The regions of the gel containing the compound-cellular component can be collected, and the cellular component identified using methods known in the art, such as reactivity with antibodies, molecular weight, localization of the cellular component in a cell, and activity of the cellular component.

In addition, the ability of a compound to modulate signal transduction pathways can be determined. The ability of a compound to modulate an identified signal transduction pathways identifies such signal transduction pathway as a therapeutic target. A variety of cells that comprise reporter genes that report an increased or decreased activity of a signal transduction pathway in response to a compound are known in the art. Such cells can also be made using methods known in the art (see, WO 98/13353 to Whitney, published April 2, 1999; U.S. Patent No. 5,298,429 to Evans et al., issued March 29, 1994; and Skarnes et al., Genes and Development 6:903-918 (1992)). Compounds of the present invention can be contacted with such cells and the expression of the reporter gene monitored to identify signal transduction pathways modulated by the compound. Such identified signal transduction pathways are themselves pharmaceutical targets, as are the individual components of the identified signal transduction pathway.

The following examples are offered by way of illustration, and not by way of limitation.

EXAMPLES

EXAMPLE 1 AMPLIFICATION OF CONTACTIN MRNA This example establishes that the methods used to detect contactin mRNA are specific for contactin mRNA. Preparations of cDNA having nucleic acids encoding contactin were prepared from total RNA from human brain tissue purchased from Clontech (Palo Alto, CA; catalogue No: 64020-1). cDNA preparations were made using Superscript II kits from GIBCO using oligo dT as a primer.

Contactin nucleic acids in samples was measured by Real-Time PCR amplification methods and SYBR® Green detection system using the primers:

5*-TCAGTAAGGTCTGGTTCACGCTAT-3' (SEQ ID NO:l),

5'-TCCCGTCACTGTAGATTCATTTGA-3' (SEQ ID NO:2),

5*-CCCCAAGTCTTCTCGGCTTA-3* (SEQ ID NO:3),

5'-CAACACATTCAGAATTCCAAGTAGACA-3' (SEQ ID NO:4),

5*-TCCCCAAGTCTTCTCGGCTTA-3' (SEQ ID NO:5),

5'-CCCATCCCAGCTCAGAAGAC-3' (SEQ ID NO:6), and 5'-GCCGCAGAAATTGGAAGG-3' (SEQ ID NO:7).

SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:6 are forward primers. SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:7 are reverse primers. Preferred primer pairs were SEQ ID NO:6 and SEQ ID NO:7; SEQ ID NO:l and SEQ ID NO:2; SEQ ID NO:5 and SEQ ID NO:4; and SEQ ID NO:3 and SEQ ID NO:4.

The PCR amplification products were separated using 4% agarose gels that included appropriate molecular weight markers. The gels were stained with ethidium bromide and all showed a single band of the correct predicted size for contactin-derived PCR products. Thus, these results establish that the primer pairs specifically amplify contactin mRNA.

EXAMPLE 2 OPTIMIZATION OF QUANTITATIVE REAL TIME PCR

The use of real time PCR to quantitate levels of specific nucleic acids has been described in the art (see Freeman et al., BioTechniques 26:112-125 (1999), for a recent review as well as Heid et al., Genome Research 6:986-994 (1996); Gibson et al, Genome Research 6:995-1001 (1996), U.S. Patent No., 4,683,202; U.S. Patent No. 4,683,195; U.S. Patent No. 4,965,188; U.S. Patent No. 5,035,996; SYBR® Green PCR and RT-PCR Reagents, Protocol, Applied Biosystems, 1998; and Spiess et al., BioTechniques 26:46-50 (1990, all references being hereby incorporated by reference). For ease of understanding, a brief explanation of quantitative real time PCR (Q- RTPCR) follows. Until recently, the traditional means of measuring the products of a specific PCR reaction was the "end-point" method of analysis, in which the reaction products are measured and quantitated after the amplification reactions are completed. In contrast, "real-time" PCR monitors amplification reactions in the thermal cycler as they progress. Q-RTPCR provides for improved quantification, because quantification is achieved most accurately during the linear range of amplification, and more information about the amplification reactions is obtained for each cycle. For example, the normalized (i.e., to a passive reference dye that does not bind DNA) fluorescence intensity ("ΔR_n"), which indicates the magnitude of the signal generated by a given set of PCR conditions, can be measured during each cycle. From such data, the cycle at which a statistically significant increase in ΔR_n is first detected can be determined. The "threshold cycle" or "Or value" is determined at one log above the signal first detected and provides a quantitative measure of the amount of the input nucleic acid template of interest present in the original sample.

In order to optimize the PCR reactions for use with the SYBR® Green detection system, which does not discriminate between different amplification products, different concentrations of different primers were used to normalize the rates of PCR product amplification. The results are shown in Table 1.

Preferred primer pairs and concentrations reflect those instances where the average CT is relatively low (i.e., sensitivity is high) and the average ΔR_n is relatively high (i.e., the range of magnitude of signal is large). Based on these criteria, preferred primer pairs and concentrations include:

SEQ ID NO:3 (900 nM) and SEQ ID NO:4 (900 nM),

SEQ ID NO:l (300 nM) and SEQ ID NO:2 (300 nM),

SEQ ID NO:l (900 nM) and SEQ ID NO:2 (900 nM), and

SEQ ID NO:6 (300 nM) and SEQ ID NO:7 (900 nM).

hi order to correct for sample-to-sample variation, an internal RNA normalizer is used in Q-RTPCR. The RNA normalizer may be an endogenous RNA species such as, e.g., an mRNA encoding a constitutively-expressed protein like actin or glyceraldehyde-3 -phosphate dehydrogenase (GAPDH), or a ribosomal RNA such as 18S or 28 S rRNA; RNA molecules produced in vitro may also be used as normalizers. Results of Q-RTPCR analyses are thus often expressed as relative amounts.

For example, when the normalizer is actin and the nucleic acid that is being quantitated is a nucleic acid encoding contactin, the relative amount of contactin RNA is determined as compared to the normalizer actin according to standard curves created for both gene sequences for each RNA sample. Standard curves were typically prepared using between about three and four different amounts of input RNA in triplicate reactions. Standard curves are plotted as log input concentration (x axis) - versus C_t (y axis, also log scale). For each standard curve, the slope (m) and the y- intercept (b) are calculated using standard analysis software.

The log input amount for the normalizer (nN) is calculated for a given C_T (Cτ°). For example, when Cχ° = 20,

(20 -b_N) nN= m„

For a specific target (T) sequence of interest, Cτ^T (the C_T required to reach a log input amount equal to nN) is determined by the formula

C_τ ^τ = (m_τ x nN)+ b_T

The normalized target C_t (normalized Q^τ) is calculated according to the formula:

normalized C_τ ^τ = C_τ ^τ - C_τ°

In the present example, the efficiency of these PCR reactions was compared to the efficiencies for control reactions using primer pairs for actin. Standard curves for determining changes in Ct for these reactions were determined by plotting the concentration of primers used vs. the signal from the Taqman SYBR® Green detection system for contactin as compared to the results obtained for actin (Table 2). The slope of the contactin plots were subtracted from the results for the actin plots. An absolute difference of less than about 0.1 is considered a preferable assay. The contactin primer pair SEQ ID NO:l and SEQ ID NO:2 were preferred using actin primers as a control. The preferred actin primer pairs were 5'-CTGGAACGGTGAAGGTGACA-3' (SEQ ID NO:8) forward primer and 5*-CGGCCACATTGTGAACTTTG-3' (SEQ ID NO:9) reverse primer. These conditions resulted in an absolute difference of 0.063.

TABLE 1

- - NORMALIZATION OF PCR AMPLIFICATION RATES USING CONTACTIN PRΓMERS

TABLE 2 COMPARISON OF EFFICIENCIES OF PCR REACTIONS FOR CONTACTIN AND ACTIN

EXAMPLE 3 EXPRESSION OF CONTACTIN MRNA IN NEUROLOGICAL SAMPLES

Human brain samples were obtained from the occipital pole post mortem as soon as possible after death. Samples were obtained from a patient diagnosed with multiple sclerosis (MS) and a control patient. The biological samples were frozen and stored at -80°C, and shipped on dry ice to analysis facilities.

Total RNA was isolated from the biological samples described in Example 2 using the TRIzol® Reagent supplied by Life Technologies™ following the manufacturer's instructions (one version of the TRIzol® Reagent is described in U.S. Patent No. 5,346,994). Briefly, tissue samples were homogenized in TRIzol® Reagent (1 mL of TRIzol® Reagent per 50 mg to 100 mg of tissue). The homogenized samples were incubated at about 15°C to 30°C for about five minutes. Chloroform (0.2 ml per ml of TRIzol® Reagent used in the initial homogenization) were added and the mixture shaken vigorously for fifteen seconds, incubated at about 15°C to 30°C for about two or three minutes, and centrifuged at about 12,000 x g for fifteen minutes at about 2°C to 8°C. The aqueous phase was transferred to a fresh container and the RNA therein was precipitated by mixing with 0.5 ml of isopropyl alcohol per 1 ml of TRIzol® Reagent used in the initial homogenization. The mixture was incubated at about 15°C to 30°C for about ten minutes and centrifuged at about 12,000 x g for ten minutes at about 2°C to 8°C. The supernatant was removed, and the resulting pellet was washed once with 75% ethanol and recollected by centrifugation at about 12,000 x g for five minutes at about 2°C to 8°C. The resulting pellet was collected and dried.

The contactin mRNA and actin mRNA in the pellets were amplified and quantitated as described in Examples 1 and 2. The percent expression of contactin mRNA was calculated as [(contactin mRNA / actin mRNA) x 100]. For the control sample, the percent expression of contactin mRNA was 22.84. For the multiple sclerosis sample, the percent expression of contactin mRNA was 95.22. These results establish that samples of neurological tissues from patients being diagnosed with MS have elevated levels of contactin mRNA.

EXAMPLE 4 ASSAYS OF CONTACTIN PROTEIN AND MRNAS ΓN BIOLOGICAL SAMPLES

Biological samples comprising material from central nervous system tissues, lung tissues, kidney tissues, skeletal muscle, epithelium, blood or amniotic fluid are prepared according to known methods from patients diagnosed as, or suspected of, having MS, ALS, MELAS, MERRF and/or other neurological disorders. Control samples are taken from individuals, preferably from age- and gender-matched or genetically related individuals, who are apparently free of such neurological disorders. Biological samples may be from living or dead individuals. The biological samples are evaluated for levels of contactin proteins and/or contacting-encoding mRNAs.

To detect and quantitate levels of contactin proteins, biological samples are stained using immunohistochemical methods, or evaluated by immunoassay, using an antibody specific for contactin. Such antibodies include, for example, the monoclonal antibody Neuro-1 (Reid et al., Molecular Brain Research 21:1-8 (1994)) and those described by Brummendorf et al. (Neuron 10:711-727, (1993)). The methods described herein in the Detailed Description and Examples 1 through 3 are used to detect and quantitate levels of contactin-encoding mRNA,

Consistent with the results obtained in Example 3, the amount of contactin in the samples from patients with MS, ALS, MELAS, MERRF and/or other neurological disorders are altered relative to the amount of contactin in the control samples.

EXAMPLE 5 SCREENING OF TEST COMPOUNDS FOR INCREASING OR DECREASING EXPRESSION OF CONTACTIN MRNA OR CONTACTIN PROTEINS Cells that express normal, increased and decreased amounts of contactin mRNA or contactin protein are provided. Such cells may contain, for example, an expression construct that comprises a nucleic acid encoding a contactin protein (Reid et al., Molecular Brain Research 21:1-8 (1994), Ogawa et al., Neurosci. Lett. 218:173-176 (1996)). When the contactin-encoding nucleic acid is transcribed in the "sense" orientation, increased amounts of the contactin mRNA and protein are expected to result. In contrast, when the contactin-encoding nucleic acid is transcribed in the "antisense" orientation, decreased amounts of the contactin mRNA and protein are expected to result.

These cells are contacted with the individual members of a library of test compounds known to have pharmaceutical activity. The amount of contactin mRNA and contactin protein are measured in such cells before and after being contacted with the test compounds using the methods of the present invention. Test compounds that increase or decrease the amount of contactin mRNA or contactin protein are identified and are presumptive therapeutic agents to increase or decrease the amount of contactin mRNA or contactin protein in a patient. EXAMPLE 6

DIFFERENTIAL GENE EXPRESSION IN AD BRAIN SAMPLES

Human brain tissue from the occipital pole of individuals diagnosed with - Alzheimer's disease (AD samples), with MS or from control individuals (control or "C" samples) was promptly obtained post mortem and stored frozen at -80°C. Total poly

A+ RNA was isolated from frozen brain tissue after thawing and extracting the tissue according to standard protocols. Poly (A)+ RNAs were prepared from 3 different regions of the brain from autopsies of 5 normal (control) human and 7 Alzheimer's diseased patients. Preparations of radiolabeled cDNAs derived from the poly (A)+ RNAs were prepared using reactions of the Superscript II kit, using oligo-dT as a primer, essentially according to the manufacturer's (Life Technologies) protocol in order to produce a set of labeled probes. The labeled probes were hybridized with Genome Systems, Inc. (St. Louis, MO) GDA 1.3 (Gene Discovery Array) membranes. (The choice of assay systems for gene discovery can be varied, for example, to include filter systems from Genome Systems, Inc. (GDA), Research Genetics, Inc. or Clontech Laboratories, Inc. (Palo Alto, CA), or high-density microarrays such as Affymetiix GeneChip and Genome Systems/Incyte/Synteni (GEM). For example, the GeneChip containing 40,000 oligonucleotides representing 40,000 human genes (35,000 ESTs and -6,000 full-length cDNAs) from Affymetrix, Inc. or the GEM chip containing 7,000 cDNAs representing 7,000 ESTs with -4,000 known human genes from GenomeSystems Inc., may optionally be screened as high throughput microarrays.)

The probed membranes were washed and exposed to Phosphorimager screens in order to produce a series of digital image files showing the intensities of hybridization of the probes to individual positions on the membrane. According to the manufacturer (Genome Systems, Inc., St. Louis, MO), each individual position on the filter has a DNA sequence derived from a specific known gene. The manufacturer's identification of the gene from which the DNA sequence present at a given position is derived is generally accurate but, in a few instances described herein, some variation was seen.

The image files containing 18,000 differential brain gene expression profile of normal and Alzheimer's patients were initially processed (at Genome Systems, Inc.) for background normalization and average values. These results were used to rank the degree of labeled probe hybridization at each position, both in terms of genetic sequences that are present in greater amounts in AD patients as compared to control samples (i.e., "up-regulated" genes in AD), as well those present in lower amounts in AD patients as compared to control samples (i.e., "down-regulated" genes in AD).

The ranked data were further analyzed as described herein using, ter alia, algorithms to analyze gene clusters that are altered in the diseased brains with special regard to changed gene expression profiles in several affected regions of the brains. Such algorithms are disclosed in U.S. Patent Application Serial No. 09/397,380, filed September 15, 1999, the contents of which are hereby incorporated by reference. The results are shown in Table 3 and summarized in Table 4A. Twenty-eight genes were down-regulated greater than 5-fold, and 38 genes were up-regulated greater than 5- fold, in Alzheimer's diseased (AD) brains compared to normal, control (C) brains.

Table 4A Differential Gene Expression in Neurodegenerative Diseased Brain

Table 4B Nucleotide Sequences of Oligonucleotide Primers and Probes for Studies of Gene Expression in AD Brain

Table 5 Relative VDACl mRNA Levels [ ΔCt value (VDACl-Actin)] in Human AD Brain and Other Neurological Diseases

TABLE 3 Down-Regulated and Up-Regulated Genes in Inferior Temporal Cortex ( ITC), Medial Frontal Cortex (MFC) and Occipital Pole (OP ) Regions of Alzheimer's Disease (AD) Brains

[TABLE 3A: "Φ ITC" = DOWN-REGULATED GENES IN ITC REGION OF AD BRAIN]

[TABLE 3B: " ITC" = Up-regulated Genes in ITC region of AD brain]

[Table 3C: "Ψ MFC" = Down-regulated Genes in MFC region of AD brain]

[Table 3D: "φ MFC" = Up-regulated Genes in MFC region of AD brain]

[TABLE 3E: "Ψ OP" = DOWN-REGULATED GENES IN OP REGION OF AD BRAIN]

[TABLE 3F: "φ OP" = UP-REGULATED GENES IN OP REGION OF AD BRAIN]

(*) Indicates array positions that apparently did not contain the manufacturer-indicated gene sequence. Accordingly, the identities of the proteins (i.e., gene products) described in these instances is based on the nucleotide sequences of these genes, and not on the manufacturer's description. EXAMPLE 7

QUANTITATIVE REAL-TIME PCR ANALYSIS OF AD DIFFERENTIALLY EXPRESSED GENES

Quantitative real-time PCR (Q-RTPCR) was used to measure the expression of several of the AD differentially expressed genes described in the preceding Example. The Q-RTPCR techniques, as they were applied to contactin sequences, are described in detail in Example 2. In the present Example the same basic techniques were performed essentially as in the preceding Examples, with the following exceptions, as detailed below.

Each pair (one forward, one reverse) of oligonucleotide primers had sequences derived not from contactin but from each AD differentially expressed gene for the Q-RTPCR experiments. Oligonucleotide probes having sequences specifically hybridizable to each of the AD differentially expressed genes were also prepared (see "Table B"). Specific nucleic acids were detected and measured by Q-RTPCR and the ABI PRISM 7700 Sequence Detection System using the primers and SYBR® Green PCR reagents.

In order to correct for sample-to-sample variation, an internal RNA normalizer was used in Q-RTPCR. The RNA normalizer was selected from an endogenous RNA species such as an mRNA encoding a constitutively-expressed protein like actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or a ribosomal RNA such as 18S or 28S rRNA. RNA molecules produced in vitro may also be used as normalizers. Results of Q-RTPCR analyses are thus often expressed as relative amounts. The relative amount of the AD differentially expressed RNA was determined as compared to the normalizer essentially according to protocols and formulae described in the manufacturer's (PE Corp., PE Applied Biosystems Division, Foster City, CA) instructions entitled "User Bulletin #2 (version of December 11, 1997), ABI PRISM 7700 Sequence Detection System, section entitled "Comparative C_τ Method, pages 11-15; some of these formulae are provided in Example 2 above. Human brain samples were promptly obtained from various regions of the brain (e.g., inferior temporal cortex (ITC), medial frontal cortex (MFC), occipital pole (OP), temporal parietal cortex and cerebellum). RNA was isolated from the biological samples essentially according to the methods described in Example 3. In brief, total RNA was isolated from the biological samples using the TRIzol® Reagent supplied by Life Technologies™ essentially following the manufacturer's instructions (one version of the TRIzol® Reagent is described in U.S. Patent No. 5,346,994). Briefly, tissue samples were homogenized in TRIzol® Reagent (2 mL of TRIzol® Reagent per 50 mg to 100 mg of tissue). The homogenized samples were incubated at about 15°C to 30°C for about five minutes. Chloroform (0.2 ml per ml of TRIzol® Reagent used in the initial homogenization) was added and the mixture shaken vigorously for fifteen seconds, incubated at about 15°C to 30°C for about two or three minutes, and centrifuged at about 12,000 x g for fifteen minutes at about 2°C to 8°C. The aqueous phase was transferred to a fresh container and the RNA therein was precipitated by mixing with 0.5 ml of isopropyl alcohol per 1 ml of TRIzol® Reagent used in the initial homogenization. The mixture was incubated at about 15°C to 30°C for about 15 minutes and centrifuged at about 12,000 x g for 15 minutes at about 2°C to 8°C. The supernatant was removed, and the resulting pellet was washed once with 75% ethanol and recollected by centrifugation at about 7,500 x g for five minutes at about 2°C to 8°C. The resulting pellet was collected, dried and resuspended in buffer.

Data showing the expression often AD differentially expressed genes, as determined by Q-RTPCR, are shown in Table 5. The data in Table 5 are expressed as a ratio of the level of expression in AD samples as compared to the level of expression in control (C) brains. A ratio greater than 1.0 indicates that a genetic sequence that is up- regulated in AD brains relative to control brains, whereas a ratio of less than 1.0 indicates that a genetic sequence that is down-regulated in AD brains relative to control brains. EXAMPLE 8

CHARACTERISTICS AND FUNCTIONS OF GENES THAT ARE DIFFERENTIALLY EXPRESSED IN

NEURODEGENERATIVE DISEASES.

Without wishing to be bound by any theory, the above-described genes (and proteins encoded thereby) that are differentially expressed in neurodegenerative disease, have characteristics and functions that have been reported in the scientific literature. Summaries of research to date for each of the differentially expressed genes and the products thereof, where known, are provided below.

Contactin Human contactin protein, which is up-regulated in MS brains as described in the preceding Examples, and its homologs from mouse (F3 protein) and chicken (Fl l protein), are cell surface adhesion proteins that are involved in cell attachment to substrate. Contactin comprises an Ig-like domain and multiple fibronectin Ill-like domains (Brummendorf et al., J. Neurochemistry 61:1207-1219 (1993)). Unlike many cell adhesion molecules, contactin is not a transmembrane protein, but instead is anchored to cell surfaces via linkage to glycosylphosphatidylinositol (GPI) in the plasma membrane outer leaflet (Id.). In human tissues, relatively high levels of a major contactin mRNA (6.5 kb) are expressed in adult brain along with three minor transcripts (9.7 kb, 4.4 kb and 3.4 kb), whereas low levels of expression of multiple forms of contactin mRNA are found in the adult lung, pancreas, kidney and skeletal muscles (6.8 kb and 6.0 kb) (Reid et al, Molecular Brain Research 21:1-8 (1994)). High levels of expression of the multiple forms of contactin mRNA are found in neuroblastoma and retinoblastoma cell lines (6.8 kb, 6.0 kb and 4.2 kb) (Id.). The expression of contactin in developing neural tissue is complex, transient, and temporally regulated. Contactin is believed to have a role in neurite outgrowth, perhaps by binding to the cell recognition molecule Ng-CAM and/or by interacting with the extracellular matrix glycoprotein restrictin (Faivre-Sarrailh et al., J. Neurosci. 12:257-267 (1992), Brummendorf et al., Neuron 10:711-7272 (1993)). Adult neural stem cells can give rise to hematopoietic cells, including cells of the myeloid and lymphoid lineages (Bjornson et al., Science 283:534-537 (1999)); thus, contactin mRNA, contactin DNA or contactin protein may be detected in blood.

COX7c

COX7c, which is down-regulated in AD brains as described in the preceding Examples, corresponds to the Nile subunit of Complex IN (cytochrome c oxidase) of the ETC chain. In mammals, COX7c is a mitochondrial protein that is encoded by nuclear genome, not the mitochondrial genome. A number of previously-described assays for activities and properties of COX7c, and/or subunit IV of the ETC, may serve as a useful screening assays for identifying chemical and biological treatments for AD and other neurodegenerative disorders.

FREAC-2

Forkhead Related Activator-2 (FREAC-2), which is up-regulated in AD brains as described in the preceding Examples, was initially cloned from a human cDΝA library by Pierrou et al. (EMBO J. 13:5002, 1994). FREAC-2 is a member of the family of "winged helix" or "forkhead" transcription factors (for a review, see Kaufmann et al., Mechanisms of Development 57:3-20, 1996). FREAC-2 binds to DΝA as a monomer and plays regulatory roles in embryogenesis, tumorigenesis, and the maintenance of differentiated cells. Published reports suggest that expression of FREAC-2 is limited to lung and placenta; accordingly, the elevated expression of FREAC-2 in AD brains may be an abnormal process that plays a role in the development and/or maintenance of AD. There are various activities of FREAC-2, including without limitation its binding to DΝA (Hellqvist et al., J. Biol. Chem. 271:4482-4490, 1996). APCL

The APCL ("Adenomatous Polypopsis Coli Like") gene, which is up- regulated in AD brains as described in the preceding Examples, encodes a protein of 2023 amino acid residues that is expressed specifically in the brain. APCL has a variety of in vitro and in vivo activities and functions, each of which has been examined to some degree, that might relate to its role in AD and other neurodegenerative disorders.

For example, APCL binds to beta-catenin in vitro, and overexpression of APCL in SW480 cells results in a reduction of the intracellular beta-catenin pool (Nakagawa et al., Cancer Research 58:5176-5181, 1988). Up-regulation of APCL, as seen in AD, is thus expected to reduce intracellular levels of beta-catenin. Beta-catenin levels are reduced, and/or beta-catenin proteins are inappropriately targeted intracellularly, in the brains of AD patients who also have mutations in presenilin-1, a protein that complexes with and stabilizes beta-catenin (Zhang et al., Nature 395:698- 702, 1998; Nishi ura et al., Nature Medicine 5:164-169, 1999). Moreover, reduced beta-catenin signalling increases neuronal vulnerability to apoptosis induced by amyloid-beta protein and oxidative stress (Zhang et al., Nature 395:698-702, 1998).

Members of the Tcf family of transcription factors, in association with beta-catenin, mediate the transactivation of downstream genes involved in neural development (Cho et al., Mechanisms of Development 77:9-18, 1998). Thus, according to non-limiting theory, beta-catenins may be involved in the development or propagation of AD and other neurodegenerative disorders.

LAP

Lysosomal acid phosphatase (LAP) is a tartrate-sensitive enzyme with ubiquitous expression that is up-regulated in AD brains. Neither the physiological substrates nor the functional significance of LAP is known. Mice with a deficiency of LAP generated by targeted disruption of the LAP gene are fertile and develop normally, and microscopic examination of various peripheral organs reveals progredient lysosomal storage in podocytes and tubular epithelial cells of the kidney, with regionally different ultrastructural appearance of the stored material. Within the central nervous system of such mice, lysosomal storage was detected to a regionally different extent in microglia, ependymal cells, and astroglia concomitant with the development of a progressive astrogliosis and microglial activation. Although behavioral and neuromotor analyses were unable to distinguish between control and deficient mice, approximately 7% of the deficient animals developed generalized seizures. (Saftig et al., J. Biol. Chem. 272:18628-18635, 1997).

PAF

Peroxisome Assembly Factor-1 (PAF), which is down-regulated in AD, is required for the normal assembly of peroxisomes in the liver and other organs. Defects in peroxisome assembly have many deleterious effects, including the widespread distribution of iron in all organs that is seen in Zellweger Syndrome and other disorders. Zellweger Syndrome is typically fatal by the age of 6 months, but some initial attempts at evaluating the potential for gene therapy for peroxisome assembly defects have been promising (Young, S.P. and Aisen P.: The Liver and Iron, in The Liver Biology and Pathobiology (I.M. Arias, J.L. Boyer, N. Fausto, W.B. Jakoby & D. Schachter, Eds.) pp. 609, Raven Press, New York, 1994.).

6PTS-1

The 6-pyruvoyl-tetrahydropterin synthase (6PTS-1, a.k.a. PTPS), down- regulated in AD, is the second enzyme in the biosynthetic pathway from GTP to tetrahydrobiopterin (BH4). In turn, BH4 is an essential cofactor of NO synthases and aromatic amino acid hydroxylases, the latter being responsible for, inter alia, monoamine neurotransmitter biosynthesis (Turri et al., Biol. Chem. 379:1441-1447, 1998). Thus, down-regulation of 6PTS-1 may lead to decreased synthesis of certain neurotransmitters in AD and other neurodegenerative disorders

VDAC-1 VDAC-1, down-regulated in AD brains, is one of several voltage- dependent anion channels (VDACs, also known as mitochondrial porins). VDACs are small pore-forming proteins of the mitochondrial outer membrane found in all eukaryotes, and are the binding sites for several cytosolic enzymes, including the isoforms of hexokinase and glycerol kinase. VDACs have also been shown to conduct ATP when in the open state, allowing bound kinases preferential access to mitochondrial ATP and providing a possible mechanism for the regulation of adenine nucleotide flux, (for reviews, see Sampson et al., J. Biol. Chem. 272:18966-18973, 1997; Mannella, J. Bioenerg. Biomembr. 29:6525-6531,1997).

Several VDAC isoforms are found in mammals (Blachly-Dyson et al. Genomics 20:162-167, 1994). In vitro, VDACs conduct a variety of small metabolites and in vivo they serve as a binding site for several cytosolic kinases involved in intermediary metabolism, yet the specific physiologic role of each isoform is unknown. Mouse embryonic stem cells lacking each isoform are viable but exhibit a 30% reduction in oxygen consumption. VDAC-1 and VDAC-2 deficient cells exhibit reduced cytochrome c oxidase activity, whereas VDACS deficient cells have normal activity (Wu et al, Biochim. Biophys. Acta 1452:168-178, 1999).

It has been suggested that the mitochondrial permeability transition (MPT) pore may comprise VDAC molecules (Szabό et al., FEBS Lett. 330:206-10, 1993). In support of this proposal, in vitro experiments have demonstrated that the pro- apoptotic proteins Bax and Bak accelerate the opening of VDAC, whereas the anti- apoptotic protein Bcl-x(L) closes VDAC by binding to it directly. Bax and Bak allow cytochrome c to pass through VDAC out of liposomes, but such passage is prevented by Bcl-x(L). These results indicate that the Bcl-2 family of proteins bind to the VDAC in order to regulate the mitochondrial membrane potential and the release of cytochrome c during apoptosis (Shimizu et al., Nature 399:483-487, 1999).

Brain-Expressed Novel EFHD-Like Protein

A number of EF-hand (EFHD) proteins have been identified, so many so that over 40 distinct subfamilies are recognized (Kawasaki et al., Biometals 11 -.211-295, 1998). The EF-hand motif consists of two alpha helices, "E" and "F", joined by a Ca(2+)-binding loop. EF-hands have been identified in numerous Ca(2+)-binding proteins by similarity of amino acid sequence and confirmed in some crystal structures. Functional EF-hands seem to tend to occur in pairs. To date, the EF-hand homolog family contains more than 160 different Ca(2+)-modulated proteins which have a broad range of functions. Among them, are the calmodulin, the troponin C, the myosin regulatory light chain, the parvalbumin, the S-100 proteins and the calbindins 9- and 28 kDa.

The most striking features of members of the EF-hand family are the ability to bind calcium (Ca2+) with dissociation constants in the micromolar range and the ability to modulate the activity of a number of enzymes. The activity of EHFD proteins is generally thought to be modulated by stimulus-induced increases in cytosolic free Ca2+ (Weinman, J. Biol. Buccale 1:90-98,1991). Except for troponin-C, all subfamilies and unique EF-hand homologs represented in vertebrates can be found in the CNS (Persechini et al., Trends Neurosci. 11:462-461, 1989). The Brain-Expressed Novel EFHD-Like Protein described herein is up-regulated in AD as described in the preceding Examples. Assays of various activities and properties of the Brain-Expressed Novel EFHD-Like Protein, including but not limited to intracellular calcium assays As a non-limiting example, one set intracellular calcium assays that may be used in this application are described, for example, in copending U.S. patent application Serial No. 60/176,384, hereby incorporated by reference.

EXAMPLE 9 QUANTITATIVE REAL-TIME PCR ANALYSIS OF EXPRESSION OF AD DIFFERENTIALLY EXPRESSED GENES IN OTHER NEURODEGENERATIVE DISEASES.

The present invention can be used to identify genes that are differentially expressed in a consistent manner in a variety of neurodegenerative diseases ("shared genes") as well as those that are differentially expressed in one particular neurodegenerative disease but not in others ("disease-specific genes"). The expression patterns in neurodegenerative diseases other than AD of several AD up- and down- regulated genes from the preceding Example were further examined as follows.

Quantitative real-time PCR (Q-RTPCR) was used to measure the expression of these genes in samples from other diseases. Human brain samples were obtained from the various regions (for e.g., inferior temporal cortex (ITC), medial frontal cortex (MFC), occipital pole (OP), temporal parietal cortex and cerebellum) post mortem from decedents as soon as possible after death. Samples were obtained from a patient diagnosed with Alzheimer's Disease (AD), Vascular Dementia (VaD), Diffused Lewy Body Disease (DLB), Parkinsons' Disease (PD) and a control (C) patient. The biological samples were frozen and stored at -80°C, and shipped on dry ice.

Q-RTPCR was performed on the brain samples from decedents having one or more neurodegenerative disorders using primers and probes specific for VDAC- 1, FREAC-2, COX7c, and APCL. The results from these experiments were grouped according to evaluations of the stage and degree of AD in each decedent, i.e., "sAD" denotes severe AD, "mAD" denotes mild AΩ, and "No AD" indicates decedents not having characteristics of AD. It should be noted, however, that decedents in each of these classes of AD may nonetheless have one or more other neurological disorders such as, e.g., PD.

The results of these experiments regarding VDAC-1, FREAC-2, COX7c, and APCL are shown in Tables 6, 7, 8 and 9, respectively. The Q-RTPCR primers and probes were used in the reactions as follows. Each probe was designed to be complementary to a portion of an mRNA of interest (i.e., having an antisense sequence as compared to the sense strand present in mRNA). The portion to which the probe hybridized was located between the two primers used for amplification, that is, within the amplification products. The target mRNA was labeled with a fluorescent dye (a "quencher") that served to quench the signal from a second fluorescent dye attached to the oligonucleotide probe. TAMRA was typically used as the quencher, and a variety of dyes can be used for probe labeling (e.g., 6-FAM, TET, JOE and the like).

Q-RTPCR was performed according to instructions provided by the instrument manufacturer (see http://www.pebiodocs . com/pebiodocs /04303859 . pdf , which is incorporated by reference, in particular pages 11-15 therein . ) Before the polymerase chain reactions were initiated, the probes specifically hybridized to complementary sequence in the mRNA of interest. The fluorescent signal from the probe was suppressed due to the close proximity of the probe's dye to the quencher dye present in the mRNA. Thus, at time = 0, little or no fluorescence from the dye linked to the probe was detected. During PCR, however, the labeled probes were separated from the mRNA to which they hybridize due to the helicase and/or exonuclease activity of the polymerase. In either event, the dye initially present in the probe became physically separated from the quencher molecules as the PCR continued and, as a consequence, the fluorescent signal corresponding to displaced probe molecules increased over time. This signal was monitored over time and reflects the amount of the mRNA of interest present in the sample (i.e., a greater amount of mRNA molecules required more PCR cycles, and/or different concentrations of primers and probes, to separate the quencher dye molecules from the reporter dye molecules. These parameters and measurements were used to determine the amount of an mRNA of interest in a sample.

Table7 Relative FREAC-2 mRNA Levels in Human Brain ["delta"Ct Value (FREAC-2 - Actin)] in Human Brain Samples from AD and

Other Neurolo ical Diseases

o

Table 8 Relative COX7c mRNA Levels in Human Brain ["delta"Ct Value (COX7c - Actin)] in Human Brain Samples from AD and Other

o

-4

Table 9 Relative APCL mRNA Levels in Human Brain ["delta"Ct Value (APCL - Actin)] in Human Brain Samples from AD and

Other Neurological Diseases

o

Table 7, as well as other Tables 6, 8 and 9, evaluates data from above experiments in a variety of ways. In order to understand the various results from the data analysis, it must first be recognized that a lower "delta" Ct value corresponds to higher levels of the mRNA in question. The "delta" Ct values from two regions of the brain thought to be affected in AD are compared in several ways. These analyses of the data use some of the mathematical formulae presented in preceding examples.

To begin, the ratio "2 " represents the difference in expression of

FREAC-2 in a patient's TPC as compared to expression in the cerebellum (e,g, lines 2- 6, second to rightmost column. The rightmost column of the table shows the mean (as well as the standard deviation, SD) of the ratio "2 " from three subgroups of patients. These values are 3.5, 1.8 and 1.6, respectively, for patients having severe AD (sAD), moderate AD (mAD) and patients not having AD (controls).

Because the results described above are ratios, they can be achieved in two different ways. A ratio of 3.5 for the relative levels of FREAC-2 mRNA in the TPC as compared to the cerebellum indicates that "more" FREAC-2 mRNA is present in the TPC. This can be realized in 2 different ways: FREAC-2 might be down- regulated in the cerebellum in AD, and/or FREAC-2 might be up-regulated in the TPC. Comparison of other values in Table D2 to one another helps distinguish these causes for the differential expression of FREAC-2 in AD brains. For example, the mean "delta" Ct for TPC FREAC-2 in each patient subgroup is determined; the values for sAD, mAD and no AD are, respectively, 9.9, 11.2 and 11.6 (see lines 7, 15 and 24 of Table D2). Because a lower "delta" Ct TPC corresponds to a higher level of FREAC-2 mRNA, the value of 9.9 for sAD, as compared to 11.2 and 11.6 for mAD and no AD, indicates that FREAC-2 mRNA is present in greater amounts in AD TPCs, i.e., FREAC-2 is up-regulated in AD TPCs.

Similarly, the mean "delta" Ct for Cerebellum FREAC-2 in each patient subgroup is determined; the values for sAD, mAD and no AD are, respectively, 11.5, 11.9 and 12.1 (see lines 8, 16 and 25 of Table D2). Because a lower "delta" Ct TPC corresponds to a higher level of FREAC-2 mRNA, the value of 11.5 for sAD, as compared to 11.9 and 12.1 for mAD and no AD, indicates that FREAC-2 mRNA is present in greater amounts in cerebellums from sAD patients, i.e., FREAC-2 is up- regulated in AD cerebellums. However, the differences between the mean "delta" Ct's for Cerebellum FREAC-2 are less pronounced than those for the TPC, suggesting that differences in the amounts of FREAC-2 in the various cerebellums is slight. Moreover, an increased level of FREAC-2 in cerebellum requires higher than normal levels of FREAC-2 in the TPC in order to achieve a ratio that indicates "more" FREAC-2 is present in the TPC. Taking all of the data into account, the results suggest that FREAC-

2 is up-regulated in the TPC of sAD patients.

EXAMPLE 10

INDUCTION OF FREAC-2 EXPRESSION INDUCES APOPTOSIS

Construction of pTO/FREAC2 (native FREAC-2 protein) and pTO- E/FREAC2-V5 (C-terminal V5/polvHis-tagged FREAC-2 protein). A full-length cDNA encoding FREAC2 was amplified by PCR using as template DNA either human placenta cDNA (Clontech, Palo Alto, CA) or cDNA reverse transcribed from RNA isolated from AD brain as described above (e.g., Example 6). For PCR, the following primer sets were used.

For pTO-E/FREAC2-V5 (C-terminal polyHis fusion form), the primers were:

5'FREe: 5'— GAGATGACCACCGAGGGCGGGCCG— 3' [SEQ ID NO:_J and

3 'FREe: 5 '— ACGCAGGGCTTAATATCCTGACAGACGCTCTGG— 3 '

[SEQ ID NO:_]. For pTO/FREAC2 (native form of FREAC-2), the primers were:

5'FRE: 5 '— GATCGAAAGCTTGAGATGACCACCGAGGGCGG— 3 '

[SEQ ID NO:_| and

3 'FRE: 5 '— TGACGATCTAGATCACATGACGCAGGGCTTAA— 3 ' . For the polyHis-tagged FREAC-2 construct, amplified FREAC-2 encoding cDNA was subcloned into the pUni/V5His donor vector containing V5/His epitopes (Invitrogen, Carlsbad, CA) and subsequently recombined with the pcDNA4/TO-E (Invitrogen) adapted expression vector according to the protocol from the manufacturer (Invitrogen). This construct was designated pTO/FREAC2-V5. For the native FREAC-2 construct, the amplified full-length FREAC2 encoding cDNA was subcloned into the tetracycline-regulatable mammalian expression vector pcDNA4TO (Invitrogen) according to the supplier's recommendations, and was desginated pTO/FREAC2. N-terminal and C-terminal sequences corresponding cumulatively to 85% of the published 1335 base pair FREAC-2 gene sequence (e.g., Pierrou et al., 1994 EMBO J. 13:5002; Kaufmann et al., 1996 Mechanisms of Development 57:3) was confirmed by nucleotide sequencing on a model ABI3700 sequencer (Applied Biosystems Division of PerkinElmer, Inc., Foster City, CA) and sequence analysis according to the instrument manufacturer's instructions. . Transfection and selection. Transfections were performed using

FuGENE-6™ transfection reagent (Roche Diagnostics Co., Indianapolis, IN) according to the supplier's recommendations. SH-SY5Y/Tet^R cells stably expressing tetracycline repressor protein (Yao et al., 1998 Hum. Gene Therapy 9:1939; Yao et al., 1990 Hum. Gene Therapy 10:419) were generated from SH-SY5Y cells (ATCC, Manassas, VA) using plasmid pcDNA6/TR (Invitrogen, Carlsbad, CA) according to the supplier's instructions. SH-SY5Y/Tet^R cells (6 x 10⁵) were plated on 100 x 15 mm plastic dishes and cultured in Dulbecco's modified Eagle medium with high glucose (4.5 g/liter), supplemented with 25 mM HEPES, penicillin, streptomycin and 10% fetal bovine serum. After 24 hours, cells were transfected with 20 μg per plate of one of the following plasmids: For native FREAC-2, pTO/FREAC2; for His-tagged FREAC-2, pTO/FREAC2-V5; as a transfection control, pcDNA/TO-E/Uni containing an E. coli lacZ encoding sequence (Invitrogen) in the place of the FREAC-2 sequence; as an empty vector control, pcDNA4/TO (Invitrogen). Cells were washed, the medium was replaced 16 hours after transfection, and cells were then incubated for an additional 48 hours. To obtain stably transfected cells, these transfectants were selected for 14 days with 100 μg/ml concentration of Zeocin™ (Invitrogen) until individual antibiotic- resistant cells formed colonies, with fresh media containing Zeocin™ used to replace exhausted media every 3 days. Antibiotic-resistant colonies were pooled and used for further studies described below. Confirmation of successful transfection was achieved by reverse transcription-PCR using the following specific FREAC2 primer pairs: QRTf: 5'— AGAGCTACTTGCACCAGAACGC— 3' [SEQ ID NO:_J

QRTr: 5'— TGGAGTAGAGTGATGCTGGTAACG— 3' [SEQ ID NO:_J

Western blot analysis. SH-SY5Y/tet^R cells transfected with pTO/FREAC2, pTO/FREAC2-V5, pcDNA4/TO-E/Uni-lacZ or pcDNA4/TO-E were used to prepare crude cell lysates for SDS-polyacrylamide gel electrophoresis according to standard procedures (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)). Aliquots (30 μg) of protein from the lysates were electrophoresed on SDS-PAGE (10-20% acrylamide gradient) gels (Novex, San Diego, CA) and electrophoretically blot- transferred to a nitrocellulose sheet (Novex) according to the manufacturer's instructions. The nitrocellulose membrane was blocked overnight at 4°C with 5% bovine serum albumin (BSA, fraction V, US Biochemcal, Cleveland, OH) in lxTBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl), incubated with a rabbit anti-FREAC2 antibody (prepared as described below) or a mouse anti-V5 antibody (Invitrogen), washed with TBS, and incubated with horseradish peroxidase conjugated to either goat anti-rabbit IgG or anti-mouse IgG (Amersham, Arlington Heights, IL) according to the supplier's instructions. After washing with TBS, the blot was developed with ECL™ detection reagent and exposed to Hyperfilm™-ECL™ film (Amersham). Figure 1 shows western immunoblot detection of tetracycline-induced expression of the V5/His-tagged FREAC- 2 fusion protein with the murine V5 antibody (mV5), and detection of tetracycline- induced expression of full-length recombinant FREAC-2 with polyclonal rabbit anti- FREAC-2 antibodies (rFREAC2).

Detection of apoptosis in FREAC-2 transfectants. In 96-well microculture plates, cultured human neuroblastoma SH-SY5Y cells (20,000 cells per well) stably transfected with plasmids pTO/FREAC2 or pTO/FREAC2-V5 prepared as described above, or with control plasmids pcDNA4/TO-E/Uni-lacZ or pcDNA4/TO-E as also described above, were cultured in Dulbecco's modified Eagle medium with high glucose (4.5 g/liter), supplemented with 25 mM HEPES and 10% fetal bovine serum, and were induced by the addition of tetracycline (at a final concentration of 1 μg/ml for indicated time periods) to induce FREAC2, FREAC2-V5 or LacZ expression as described above.

Caspase-3 activity was assessed by diluting the fluorogenic peptide substrate acetyl- Asp-Glu-Val- Asp (SEQ ID NO:_) conjugated to AMC (7-amino-4- methylcoumarin; the synthetic peptide is referred to as DEVD-AMC; CalBiochem- Novabiochem Corp., San Diego, California; see Walker et al., 1994 Cell 75:343, and Thornberry et al., 1992 Nature 356:768) from a DMSO stock solution into culture media to a final concentration of 20 μM for uptake by cells. Substrate cleavage liberating the AMC fluorophore was measured continuously using a Cytofluor™ fluorimetric plate reader (model #2350, Millipore Corp., Bedford, Massachusetts; excitation wavelength = 355 nm; emission wavelength = 460 nm). Data are presented as ΔRFU (relative fluorescence units). Caspase 3 is generally regarded ' as a mitochondrial-specific caspase, whereas caspase 1 is not; accordingly, DEVD-AMC is one preferred substrate for this embodiment of the invention. Figures 2 and 3 show caspase-3 activation, an indicator of apoptosis, following tetracycline induced FREAC2 or FREAC2-V5 expression in stably transfected cells. Significantly increased and sustained apoptosis was apparent in tetracycline induced cells that had been transfected with the pTO/FREAC2 or pTO/FREAC2-V5 plasmids directing expression of FREAC2 and FREAC2-V5, respectively, but not in tetracycline-induced cells transfected with pcDNA4/TO-E/Uni- lacZ and expressing bacterial lacZ protein. Caspase-3 activity induced by 1 μM thapsigargin and 25 μM ethacrynic acid treatment (TE) for 20 hours immediately prior to the caspase assay was determined in transfectants induced with tetracycline for 0-3 days. Caspase activity in TE-treated cells did not appear to be additive with caspase-3 activity elicited by tetracycline-induced FREAC2 or FREAC2-V5 expression in the transfected cells (Figure 2). The effects of the protein kinase inhibitor staurosporine (0.5 μM) on caspase-3 activity in tetracycline-induced FREAC-2, FREAC-2/N5 or LacZ transfectants are shown in Figure 3. Cells were cultured in the presence of tetracycline for the indicated number (0-3) of days, and media were then replaced with fresh medium containing staurosporine for a 2 hour incubation that immediately preceded the caspase-3 assay.

Additionally, the cell viability of FREAC-2, FREAC-2/N5 and LacZ transfectants was measured during the course of tetracycline induction using a fluorescent calcein assay (Molecular Probes, Eugene, OR) according to the supplier's instructions. As shown in Figure 4, there was correlation between a gradual increase in the proportion of non-viable cells and tetracycline-induced protein expression in pTO/FREAC2 and pTO/FREAC2-N5 transfected cells, but not in pcDΝA4/TO-E/Uni- lacZ transfected cells.

FREAC-2 antibodies. Two regions of the FREAC-2 protein amino acid sequence were selected for use as immunogens to elicit rabbit polyclonal antibodies. The amino acid sequence of the FREAC-2 polypeptide immunogen designated FRE-2A was

EFMFEEGSFRRRPRGFRRKC [SEQ ID NO:_|, and the amino acid sequence of the polypeptide immunogen designated FRE-2B was LNFNGISSFHPSASGSYC [SEQ ID NO:_J. FRE-2A and

FRE-2B peptides were separately prepared by conventional procedures using a solid- phase peptide synthesizer, coupled to adjuvant matrices via the C-terminal cysteine residues and injected into rabbits according to immunological procedures provided as part of the standard custom antiserum services by Alpha Diagnostic International, Inc. (San Antonio, TX). Polyclonal antisera were obtained from test bleedings of the rabbits, and affinity purified polyclonal antibodies were prepared using well known procedures following immunoaffinity chromatography of each antiserum on a column containing the peptide used for immunization covalently immobilized on Sepharose- 4B™ (Pharmacia, Piscataway, NJ). Fluorescence staining/ Immunocytochemistry. Stable SH-SY5Y transfectants prepared as described above were treated with tetracycline (at a final concentration of 1 μg/ml for the indicated time periods) to induce FREAC2, FREAC2- N5 or LacZ expression. Cells were then washed twice with PBS and fixed with 3.7% formaldehyde-PBS solution for 15 min. The fixed cells were permeabilized using PBS supplemented with 0.1% Tween 20™, 3% bovine serum albumin, 2% normal goat serum and 0.3% Triton X-100™ for 15 min. For F-actin (e.g., microfϊlaments) staining, the fixed and permeabilized cells were stained for 20 min with rhodamine-phalloidin conjugate (Molecular Probes, Inc., Eugene, OR) according to the supplier's instructions. For staining alpha-tubulin (e.g., microtubules) by immunocytochemistry, the fixed and permeabilized cells were incubated with anti-alpha tubulin primary antibody (Calbiochem, Inc., San Diego, CA) for 1 hour, washed, and incubated with secondary antibodies of appropriate species specificity conjugated to either Alexa488 or rhodamine (Molecular Probes), according to the manufacturer's recommendations. Fluorescence microscopy was performed using a Nikon microscope equipped with fluorescence illumination and digital images were captured using the SimplePCI system (Compix Inc., Cranberry Township, PA). Representative images of cells stained with rhodamine-phalloidin are shown in Figure 5 for tetracycline-treated cells transfected with FREAC-2 (Fig. 5C,D) or control (Fig. 5A,B) expression constructs. Figure 6 shows representative alpha-tubulin staining in tetracycline-induced (Fig. 6C,D) and uninduced (Fig. 6A,B) FREAC-2/V5 transfectants. As shown in Figures 5 and 6, tetracycline-induced FREAC-2 expression appeared to correlate with the presence of shortened cellular processes and a reduction in the prominence of threadlike, tubulin- containing meshwork. All publications, including patent documents and scientific articles, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. The invention is not limited except as by the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A method of identifying an agent that alters a mitochondrial function, comprising: inducing FREAC-2 expression in a host cell in the presence and absence of a candidate agent, wherein the host cell comprises a recombinant expression construct comprising a regulated promoter operably linked to a polynucleotide encoding a FREAC-2 polypeptide, a fragment or variant thereof; and comparing a level of apoptosis in the host cell in the absence of the agent to the level of apoptosis in the presence of the agent, whereby an agent that alters the level of apoptosis indicates an agent that alters mitochondrial function.

2. The method of 1 wherein induction of FREAC-2 expression is performed in vitro.

3. The method of claim 1 wherein induction of FREAC-2 expression is performed in vivo.

4. The method of claim 1 wherein the host cell is a neuronal cell.

5. The method of claim 1 wherein the host cell is of a cell type that is associated with a neurodegenerative disease selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, MELAS and MERRF.

6. The method claim 1 wherein the host cell is of a cell type that is associated with a neurodegenerative disease that is Alzheimer's disease.

7. The method of claim 1 wherein the host cell is of a cell type that is associated with a neurodegenerative disease that is multiple sclerosis.

8. The method of claim 1 wherein the host cell is of a cell type that is associated with a disease of inappropriate cell proliferation.

9. The method of claim 8 wherein the host cell is a cancer cell.

10. A method of identifying a molecular target of an agent that alters a mitochondrial function, comprising:

(a) contacting an agent identified according to the method of claim 1 with a biological sample under conditions and for a time sufficient to permit formation of a complex comprising the agent and at least one molecule with which the agent specifically interacts, wherein the molecule is derived from the sample; and

(b) isolating the complex, and therefrom identifying a molecular target of the agent.

11. A method of identifying a molecular target for therapeutic intervention for treating a neurodegenerative disease or a proliferative disorder, comprising:

(a) comparing

(i) at least one expression level of at least one gene product in a first biological sample comprising a host cell in which expression of a FREAC-2 polypeptide or a variant or fragment thereof has been induced, wherein the host cell comprises a recombinant expression construct comprising a regulated promoter operably linked to a polynucleotide encoding a FREAC-2 polypeptide, or a variant or fragment thereof, to

(ii) at least one expression level of the gene product in a second biological sample comprising a host cell in which expression of a FREAC-2 polypeptide or a variant or fragment thereof has not been induced; and (b) detecting an altered expression level of at least one gene product in the first sample relative to the expression level of the gene product in the second biological sample, and therefrom identifying a molecular target for therapeutic intervention.

12. A method of treating a neurodegenerative disease or a disease associated with inappropriate cell proliferation or cell survival in a subject, comprising: administering to a subject in need thereof a recombinant expression construct comprising a promoter operably linked to a polynucleotide encoding a FREAC-2 polypeptide or a variant or fragment thereof, wherein expression of the FREAC-2 polypeptide or a variant or fragment thereof results in altered mitochondrial function.

13. A method according to claim 12 wherein the promoter is a regulated promoter.

14. The method of either one of claims 12 or 13 wherein the altered mitochondrial function is apoptosis.

15. A method of treating a neurodegenerative disease or a disease associated with inappropriate cell proliferation or cell survival in a subject, comprising: administering to a subject in need thereof a recombinant expression construct comprising a promoter operably linked to a FREAC-2 polynucleotide that is in reverse orientation such that an antisense nucleic acid or variant or fragment thereof is expressed, wherein expression of the antisense nucleic acid or variant or fragment thereof alters a neurodegenerative disease or a disease associated with inappropriate cell proliferation or cell survival.

16. The method of any one of claims 12-15 wherein the disease associated with inappropriate cell proliferation or cell survival is cancer.