CN103816540B - The material for reducing the combination that β suppresses albumen 1 and the albumen of APH 1 is preparing the application in preventing and treating nerve degenerative diseases medicine - Google Patents

Abstract

The present invention relates to the material for reducing the combination that β suppresses albumen 1 and the albumen of APH 1 to prepare the application in preventing and treating nerve degenerative diseases medicine.The invention firstly discloses β Arrestin1 to be interacted with a necessary component APH 1 of gamma secretase, so as to influence the activity of gamma secretase.The invention also discloses APH 1AL and the APH 1B of a kind of separation protein fragments, the protein fragments are both that β suppresses albumen 1 and the target spot of gamma secretase related component interaction, it can be used for disturbing β suppression albumen 1 and APH 1 to interact again, so as to suppress the nerve degenerative diseases of the generation of amyloid and correlation.The invention also discloses the method for the medicine of screening preventing and treating nerve degenerative diseases.

Description

The material for reducing the combination of beta-protein inhibitor 1 and APH-1 albumen is preparing preventing and treating nerve Application in degenerative disease medicine

Technical field

The invention belongs to field of biological pharmacy, relates more specifically to a kind of related target spot of new nerve degenerative diseases, And the inhibitor based on the target spot, and medicaments sifting model and method based on the target spot.

Background technology

Alzheimer's disease is a kind of central nervous system regression based on progressive cognitive disorder and memory infringement Property disease.Patients with clinical manifestations is cognition (calculating, judgement, comprehensive etc.) dysfunction, failure of memory, abnormality of personality etc..Ah The amyloid plaque and neurofilaments that the key pathological feature of the silent disease in Wurz sea is formed for the big intracerebral of patient tangle.Starch Sample albuminous plasue is the characteristic pathological change of alzheimer's disease, mainly by intracellular abnormal a large amount of caused amyloid eggs - β (A β) albumen is formed in extracellular accumulation in vain.

The cause of disease of alzheimer's disease is still not clear, and has multiple theories to attempt to explain its pathogenesis.Hardy and Selkoe " the A beta hypothesises " proposed is the theory being widely accepted at present.The theory thinks, makees for a long time in the h and E factor of complexity Under, nerve cell largely produces A β singularly, and accumulation forms oligomer and amyloid plaque, A β (the especially A of oligomerization β) by a series of cascade reactions, (including radical reaction, Mitochondrial oxidative damage and inflammatory reaction etc. are directly or indirectly made For neuron and spongiocyte), cause synaptic function exception and neure damage, and cause microglia and star-like glue Cell plastid activates, the formation that accelerans filament tangles, and causes cognitive disorder after long term.Recent numerous studies provide Many evidences support " A beta hypothesises ", it is shown that central roles of the A β in alzheimer's disease pathogenesis.Therefore, study A β are produced and modulated mechanism is to understand the important foundation of alzheimer's disease pathogenesis.

A β by I type transmembrane proteins amyloid precusor protein (Amyloid Precursor Protein, APP) by β-point The protein digestion effect for secreting enzyme and gamma-secretase mediation produces：Beta-secretase and gamma-secretase are respectively acting on APP A β The aminoterminal and c-terminus of domain, order shearing produce complete A β.APP can also be tied by alpha-secretase enzyme shear action in A β In structure domain, incomplete A β peptide section is resulted in.

A β main generation mechanism is：APP forms two segments, sAPP β and C99, C99 by beta-secretase shear action A β are produced by the shear action of gamma-secretase.Amyloid precursor protein also can be sheared to form sAPP α by alpha-secretase enzyme With two segments of C83, but A β can not be produced.A β are secreted into extracellular rear formation oligomer, and further precipitation forms shallow lake Powder sample albuminous plasue.

Gamma-secretase is vital in the aborning effects of A β, and it is in the digestion effect inside APP in different loci The species of A β caused by determining is (predominantly respectively by the A β of 40 and 42 Amino acid profiles₄₀With A β₄₂).In recent years research hair Existing gamma-secretase is a kind of complicated protein complex, by Presenilin (Presenilin), Nicastrin, APH-1 Four kinds of transmembrane proteins such as (anterior pharynx defective 1) and PEN-2 (presenilin enhancer 2) Assembling is formed, and they are the required components for forming the gamma-secretase with enzymatic activity.Presenilin is gamma-secretase complex Protease catalytic subunit, it is protease that it, which is located at the 257th of the 6th and the 7th transmembrane region and the 385th asparagicacid residue, Avtive spot.More than 100 kinds of Presenilin missense mutation in genotype alzheimer's disease people be present.These mutation change γ- The activity of secretase, make it easier to produce the bigger A β 42 of neurotoxicity.Nicastrin is the function subunit for identifying substrate, APH-1A is the scaffolding protein of gamma-secretase complex assembling, PEN-2 major function be start gamma-secretase maturation and Activation and stable gamma-secretase complex.Nearest research reports some molecules, such as TMP21 and Rer1p, be present in γ- In secretion combined enzyme agent and its activity can be regulated and controled, they are probably the optional component of gamma-secretase.

Suppressing albumen (Arrestin) gene family has four members, is included in the widely distributed β-suppression of each organ-tissue Albumen 1 (β-Arrestin1) and beta-protein inhibitor 2 (β-Arrestin2), and the rod expressed in vision system Arrestin and cone Arrestin.β-Arrestin are a long-chain molecule under quiescent condition, by two different structures The hinge arrangement that domain (amino terminal domain and carboxy-terminal domain) forms via 12 amino acid is formed by connecting.The two structures There is the polarity a being buried kernel in domain because intermolecular active force keeps independent mutually between which.Although β- Arrestin and different protein-interactings, but not yet find to have in β-Arrestin so far clearly occur protein- The specific domain of protein interaction.β-Arrestin specifically identify phosphorylated protein, and it is to phosphorylation form Effective object affinity is far above unphosphorylated form.

β-Arrestin are found as the termination factor of g protein coupled receptor signal.β-Arrestin lead to Cross to be combined with the acceptor of activation and cause receptor desensitization and endocytosis, so as to terminate the signal of G-protein.When activator continuous action When, acceptor can soon weaken to the reactivity of activator with the time, here it is the desensitization of acceptor, embody cell pair Accurately regulate and control on time and intensity in receptor signal.The desensitization effect of acceptor is made up of series reaction, works as activator After activated receptor, g protein coupled receptor kinases is also activated and after being phosphorylated structure occurs for phosphorylated-activated acceptor, acceptor As changing, and β-Arrestin affinity increase, promote β-Arrestin from cytoplasm quick indexing to cell membrane and with Acceptor is combined closely, blocked acceptor and G-protein further combined with the signal of acceptor is terminated.

Regulation and control receptor desensitization effect is the classical functions of β-Arrestin.Later discovery shows that β-Arrestin pass through knot Various signaling molecules are closed, may also participate in other signal paths.β-Arrestin can promote receptors signal transduction, swash Under the conditions of dynamic agent is existing, c-Src is recruited and is arrived β by β-Arrestin₂On AR, MAPK activation is promoted.This recruitment effect The regulation and control to a variety of physiological activities are result in, the receptor-mediated anti-apoptotic function of the exocytosis, NK1 such as neutrophil cell and interior Skin peptide stimulates the indexing of GLUT.β-Arrestin can regulate and control MAPK activation.Due in MAPK cascade pathways In, the kinases of a upstream can activate many downstream kinases, and organism just needs the specific MAPK signal pathways of tissue, to protect Demonstrate,prove specificity, repeatability and the high efficiency of signal transduction.Phase interactions of the β-Arrestin as scaffolding protein mediation respective kinase With, ensure that they form complete signal transduction complex, while this complex is in intracellular specific region, from And be isolated with phosphate, avoid kinase-dead.

In addition to combining traditional g protein coupled receptor, β-Arrestin can also combine other types of acceptor, and Its endocytosis and transhipment effect are influenceed, such as Frizzled, Smoothened, Notch, type III transforming growth factor β receptor (type III transforming growth factor- β receptor), LDL receptor (low- densitylipoprotein receptor)、Na⁺/H⁺Exchange transhipment (Na⁺/H⁺exchanger 5transporter)。

β-Arrestin regionals in central nervous system have expression.In big intracerebral, β-Arrestin1 mRNA Horizontal higher than β-Arrestin2 2-3 times, β-Arrestin1 protein level is even more higher by 10-20 times than β-Arrestin2. Studies have shown that β-Arrestin2 participate in the behavior of regulation and control opiate addiction and dopamine dependence in treatment.But β-Arrestin1 are in maincenter Effect in the physiology and pathology of nervous system is simultaneously indefinite.

In view of important function of the β-Arrestin in adjustment signal path, studying its effect in disease helps to take off Show pathogenesis, gain more insight into the pathogenic factor of disease.

The content of the invention

In the first aspect of the present invention, there is provided a kind of material for the combination for reducing beta-protein inhibitor 1 and APH-1 albumen Purposes, for the composition for preparing prevention, alleviating or treat nerve degenerative diseases.

In another preference, described nerve degenerative diseases are the god characterized by intracerebral forms amyloid plaque Through degenerative disease.

In another preference, described nerve degenerative diseases are alzheimer's disease or parkinsonism.

In another preference, the combination of described reduction beta-protein inhibitor 1 and APH-1 albumen is selected from：Reduce β-suppression The expression of albumen 1 or the function of suppressing beta-protein inhibitor 1；Reduce the expression of APH-1 albumen or suppress the work(of APH-1 albumen Energy；Interference or the combination for suppressing beta-protein inhibitor 1 and APH-1 albumen；Or weaken the phase of beta-protein inhibitor 1 and APH-1 albumen Interaction.

In another preference, described APH-1 albumen is selected from APH-1AL albumen or APH-1B albumen.

In another preference, the material of the combination for reducing beta-protein inhibitor 1 and APH-1 albumen is selected from：(a) contain SEQ ID NO:The protein fragments of 246-265 amino acids sequence in 33；(b) SEQ ID NO are contained:235-265 in 33 The protein fragments of amino acids sequence；(c) SEQ ID NO are contained:The protein fragments of 234-257 amino acids sequence in 34； (d) by the amino acid sequence of protein fragments any (a)-(c), by one or more, (such as 1-5, more preferably 1-3 is individual, more preferably Ground 1-2) amino acid residue substitution, missing or addition and formed, and the polypeptide with (a)-(c) protein fragments functions； Also, described protein fragments do not include the albumen with total length APH-1AL albumen or APH-1B amino acid sequences.

In another preference, the material of the combination for reducing beta-protein inhibitor 1 and APH-1 albumen is selected from：(i) amino Acid sequence such as SEQ ID NO:The protein fragments of 246-265 positions in 33；(ii) amino acid sequence such as SEQ ID NO:In 33 The protein fragments of 235-265 positions；Or (iii) amino acid sequence such as SEQ ID NO:The protein fragments of 234-257 positions in 34.

In another preference, the material of the described activity of suppression beta-protein inhibitor 1 is specific binding beta-protein inhibitor 1 antibody, or the small disturbing molecule that specificity interference beta-protein inhibitor 1 is expressed, or the carrier or virus of the small disturbing molecule of expression (such as slow virus).

In another preference, described small disturbing molecule has SEQ ID NO:Sequence shown in 23.

In another aspect of the present invention, there is provided a kind of protein fragments of the beta-protein inhibitor 1 of separation, described albumen Fragment is selected from：(1) SEQ ID NO are contained:The protein fragments of 241-360 amino acids sequence in 1；(2) SEQ ID are contained NO:The protein fragments of 181-418 amino acids sequence in 1；Or (3) contain SEQ ID NO:241-418 amino acids in 1 The protein fragments of sequence.Also, described protein fragments do not include having SEQ ID NO:The albumen of 1 full length amino acid sequence.

In another aspect of the present invention, there is provided the purposes of a kind of beta-protein inhibitor 1 or its protein fragments, for preparing Or screening specificity suppresses beta-protein inhibitor 1 or the material of its protein fragments, described material is prevention or treatment neurological The potential material of property disease.

In another aspect of the present invention, there is provided a kind of APH-1 protein fragments of separation, be selected from：(a) SEQID is contained NO:The protein fragments of 246-265 amino acids sequence in 33；(b) SEQ ID NO are contained:235-265 amino acids in 33 The protein fragments of sequence；(c) SEQ ID NO are contained:The protein fragments of 234-257 amino acids sequence in 34；(d) by (a)- (c) amino acid sequence of any protein fragments is by one or more (such as 1-5, more preferably 1-3, more preferably 1-2) Substitution, missing or the addition of amino acid residue and formed, and with (a)-(c) protein fragments functions polypeptide；It is also, described Protein fragments do not include the albumen with total length APH-1AL albumen or APH-1B amino acid sequences.

In another preference, described APH-1 protein fragments are selected from：(i) amino acid sequence such as SEQ ID NO:In 33 The protein fragments of 246-265 positions；(ii) amino acid sequence such as SEQ ID NO:The protein fragments of 235-265 positions in 33；Or (iii) amino acid sequence such as SEQ ID NO:The protein fragments of 234-257 positions in 34.

In another preference, the N-terminal or C-terminal of described APH-1 protein fragments also include trans-activator (TAT- tag)；It is preferred that described trans-activator has SEQ ID NO:Amino acid sequence shown in 35.

In another aspect of this invention, there is provided a kind of polynucleotides of separation, appoint before described polynucleotide encoding Protein fragments described in one.

In another aspect of this invention, there is provided a kind of to screen prevention, alleviate or treat the potential of nerve degenerative diseases The method of material, methods described include：

Using beta-protein inhibitor 1 or its protein fragments (above-mentioned protein fragments preferably of the invention) as the target spot of screening, choosing Go out the material that specificity suppresses the target spot, described material is prevention, alleviation or the potential material for treating nerve degenerative diseases.

In an embodiment of the invention, described method includes：

(1) candidate substances are contacted with the system of expression beta-protein inhibitor 1 or its protein fragments；

(2) influence of the candidate substances to beta-protein inhibitor 1 or its protein fragments is detected；

If the candidate substances suppress beta-protein inhibitor 1 or expression or the activity of its protein fragments, show the candidate Matter is prevention, alleviation or the potential material for treating nerve degenerative diseases.

In another preference, in described method, step (1) includes：In test group, candidate substances are added to table Up in the system of beta-protein inhibitor 1 or its protein fragments；And/or step (2) includes：Detect beta-inhibited protein in the system of test group Expression or the activity of white 1 or its protein fragments, and compared with control group, wherein described control group is not add the candidate The expression beta-protein inhibitor 1 of matter or the system of its protein fragments；If the table of beta-protein inhibitor 1 or its protein fragments in test group Reach or activity is statistically less than (preferably significantly lower than, such as low more than 20%, preferably low more than 50%；More preferably low 80% with On) control group, it is prevention, alleviation or the potential material for treating nerve degenerative diseases to indicate that the candidate substances.It is preferred that institute The candidate substances stated include but is not limited to：For beta-protein inhibitor 1 or the disturbing molecule of its upstream or downstream protein design, core Sour mortifier, binding molecule (such as antibody or part), micromolecular compound.

In another aspect of the present invention, there is provided a kind of protein complexes, the complex include：Beta-protein inhibitor 1 Or its protein fragments, the protein fragments include SEQ ID NO:241-360 amino acids sequence in 1；With APH-1 albumen APH-1 protein fragments as described in (such as APH-1AL albumen or APH-1B albumen) or claim 9.

In another aspect of the present invention, there is provided a kind of to screen prevention, alleviate or treat the latent of nerve degenerative diseases In the method for material, methods described includes：

Target spot using described protein complexes as screening, select specificity and suppress the thing that the protein complexes are formed Matter, described material are prevention, alleviation or the potential material for treating nerve degenerative diseases.

In yet another embodiment of the present invention, described method includes：(1) by candidate substances with containing egg of the present invention The system contact of white complex；(2) influence of the candidate substances to the protein complexes is detected；If the candidate substances suppress institute The protein-interacting (such as be combineding with each other) stated in protein complexes weakens, then shows that the candidate substances are preventions, alleviate or control Treat the potential material of nerve degenerative diseases.

In another preference, in described method, step (1) includes：In test group, candidate substances are added to institute State in the system containing protein complexes；And/or step (2) includes：Albumen in protein complexes in the system of detection test group Interaction situation, and compared with control group, wherein described control group be do not add the candidate substances containing described The system of protein complexes；If in test group in protein complexes the interaction of albumen be statistically weaker than it is (preferably aobvious Work is weaker than, such as weak more than 20%, preferably weak more than 50%；More preferably weak more than 80%) control group, indicate that the candidate substances are Prevention, the potential material for alleviating or treating nerve degenerative diseases.It is preferred that described candidate substances include but is not limited to： For the protein fragments of any protein design, disturbing molecule, nucleic acid inhibitor, binding molecule in the protein complexes (as resisted Body or part), micromolecular compound etc..

In another aspect of the present invention, there is provided a kind of to screen prevention, alleviate or treat the latent of nerve degenerative diseases In the method for material, methods described includes：(1) by candidate substances with containing (as expressed) beta-protein inhibitor 1 or its protein fragments Contacted with the system of APH-1 albumen or its protein fragments；(2) detect candidate substances to beta-protein inhibitor 1 or its protein fragments with The influence of the interaction of APH-1 albumen or its protein fragments；If the candidate substances suppress beta-protein inhibitor 1 or its albumen flakes Section and the interaction of APH-1 albumen or its protein fragments, then it is prevention, alleviation or treatment neurological to show the candidate substances The potential material of property disease.It is preferred that in described method, step (1) includes：In test group, candidate substances are added to It is described containing (as express) beta-protein inhibitor 1 or its protein fragments with the system of APH-1 albumen or its protein fragments；And/or Step (2) includes：Detect beta-protein inhibitor 1 or its protein fragments and APH-1 albumen or its protein fragments in the system of test group Interaction situation, and compared with control group, wherein described control group be do not add the candidate substances contain β-suppression The system of albumen 1 or its protein fragments processed and APH-1 albumen or its protein fragments；If in test group beta-protein inhibitor 1 or its The interaction of protein fragments and APH-1 albumen or its protein fragments, which is statistically weaker than, (to be preferably significantly smaller than, such as weak 20% More than, preferably weak more than 50%；More preferably weak more than 80%) control group, it is prevention, alleviation or treatment to indicate that the candidate substances The potential material of nerve degenerative diseases.It is preferred that described candidate substances include but is not limited to：For the beta-inhibited protein White 1 or the protein fragments of APH-1 protein designs, disturbing molecule, nucleic acid inhibitor, binding molecule (such as antibody or part), small point Sub- compound etc..More preferably, candidate substances are detected to beta-protein inhibitor 1 or its protein fragments and APH-1 albumen or its albumen flakes The influence of the interaction of section includes：Detect the phase of beta-protein inhibitor 1 or its protein fragments and APH-1 albumen or its protein fragments Mutually combine, if they be combined with each other statistically weaken (be preferably significantly smaller than, such as weak more than 20%, preferably weak 50% with On；More preferably weak more than 80%), it is prevention, alleviation or the potential thing for treating nerve degenerative diseases to indicate that the candidate substances Matter；Or the action time of detection beta-protein inhibitor 1 or its protein fragments and APH-1 albumen or its protein fragments, if their work Statistically reduced with the time and (preferably substantially reduce, such as reduce more than 20%, preferably reduce more than 50%；More preferably reduce More than 80%) it is prevention, alleviation or the potential material for treating nerve degenerative diseases, to indicate that the candidate substances；Or detection β-suppression The effective binding energy power of albumen 1 or its protein fragments processed and APH-1 albumen or its protein fragments, if they are effectively incorporated in system Meter is learned to reduce and (preferably substantially reduces, such as reduce more than 20%, preferably reduce more than 50%；More preferably reduce more than 80%), just It is prevention, alleviation or the potential material for treating nerve degenerative diseases to show the candidate substances.More preferably, candidate substances pair are detected Beta-protein inhibitor 1 or its protein fragments and the influence of the interaction of APH-1 albumen or its protein fragments include：Detect β-suppression The quantity for the complex that albumen 1 or its protein fragments are formed with APH-1 albumen or its protein fragments, if the number of described complex Amount, which is statistically reduced, (preferably to be substantially reduced, such as reduces more than 20%, preferably reduce more than 50%；More preferably reduce 80% More than), it is prevention, alleviation or the potential material for treating nerve degenerative diseases to indicate that the candidate substances.More preferably, detection is waited Material is selected to include beta-protein inhibitor 1 or its protein fragments and the influence of the interaction of APH-1 albumen or its protein fragments：Inspection The quantity or proteinase activity of gamma-secretase complex are surveyed, if the quantity of described complex or proteinase activity are in statistics Upper reduce (preferably substantially reduces, such as reduces more than 20%, preferably reduce more than 50%；More preferably reduce more than 80%), indicate that The candidate substances are prevention, alleviation or the potential material for treating nerve degenerative diseases.

In another preference, described method also includes：Further cell experiment is carried out to the potential material of acquisition And/or animal experiment, it is useful for preventing or treating nerve degenerative diseases further to select and determine from candidate substances Material.

In another preference, described system is selected from：Cell system, subcellular fraction system, solution system, organizational framework, Organ systems or animal system.

The other side of the present invention is apparent to those skilled in the art due to this disclosure 's.

Brief description of the drawings

β-Arrestin expression in Fig. 1 alzheimer's diseases people tissue.Braak0- is detected with Western hybrid experiments The tissue of patient sample of VI phases.With anti-β-Arrestin antibody As 2CT and rabbit-anti Actin antibody detect respectively β-Arrestin and Actin expression, compareed using actin (Actin) as applied sample amount.Wherein, β arr1 represent β-Arrestin1；β arr2 tables Show β-Arrestin2.

β-Arrestin1 expression in Fig. 2 alzheimer's diseases people tissue.Detected with Western hybrid experiments The tissue of patient sample of Braak0-VI phases.With anti-β-Arrestin antibody As 2CT and rabbit-anti Actin antibody detect respectively β- Arrestin1 and Actin expression, β-Arrestin1 and Actin signal value is measured and counts respectively, using Actin as upper Sample amount compares.Illustrate each round dot and represent a sample, strigula represents the cell mean.**P<0.01, * * * P<0.001.

β-Arrestin2 expression in Fig. 3 alzheimer's diseases people tissue.Detected with Western hybrid experiments The tissue of patient sample of Braak0-VI phases.Detect β-Arrestin2 and Actin table respectively with A2CT and rabbit-anti Actin antibody Reach, measure and count β-Arrestin2 and Actin signal value respectively, compareed using Actin as applied sample amount.Illustrate each circle Point represents a sample, and strigula represents the cell mean.**P<0.01.

β-Arrestin1 expression in Fig. 4 alzheimer's diseases people tissue.Genetic chip (left figure) is used respectively and is quantified β-Arrestin1 mRNA expressions in the tissue of patient sample of PCR experiment (right figure) detection Braak 0-VI phases.Diagram is every One round dot represents a sample, and strigula represents the cell mean.

β-Arrestin1 distributions in Fig. 5 immunohistochemical assay detection human cerebral tissue.With A1CT antibody stainings β-Arrestin1s of the β-Arrestin1 in Healthy People (ND) and alzheimer's disease people (AD) cortical tissue is detected in nerve Distribution in first (being represented with long arrow) and star spongiocyte (being represented with short arrow).

β-Arrestin expression in Fig. 6 .TgCRND8 mouse tissues.With Western hybrid experiments detection wild type (WT) With TgCRND8 mouse tissue samples.Detect β-Arrestin and Actin expression respectively with A2CT and rabbit-anti Actin antibody, point β-Arrestin1, β-Arrestin2 and Actin signal value and Ce Liang not be counted, is compareed using Actin as applied sample amount.*P< 0.05, * * * P<0.001.

β-Arrestin expression in Fig. 7 .APP/PS1 mouse tissues.With Western hybrid experiments detection wild type (WT) With APP/PS1 mouse tissue samples.Detect β-Arrestin and Actin expression respectively with A2CT and rabbit-anti Actin antibody, point β-Arrestin1, β-Arrestin2 and Actin signal value and Ce Liang not be counted, is compareed using Actin as applied sample amount.**P< 0.01。

β-Arrestin1 expression in Fig. 8 immunohistochemical assay detection mouse brain tissue.Contaminated with A1CT antibody Color detects β-Arrestin1 in wild type, TgCRND8 and β-Arrestin1 gene knockouts (KO) mouse cortex and hippocampal tissue In β-Arrestin1 expression.

A β in Fig. 9 β-Arrestin1 knock out mice tissues are horizontal.Detected with enzyme-linked immunosorbent assay wild A β in type (WT) and β-Arrestin1 gene knockouts (KO) mouse cortex (Cx) and hippocampus (Hp) tissue sample₄₀With A β₄₂Water It is flat.β-Arrestin1 gene knockouts cause the A β in cortex and hippocampal tissue₄₀With A β₄₂Level declines.n=4-5；*P< 0.05, * * P<0.01.

APP expression in Figure 10 β-Arrestin1 knock out mice tissues.Detected with Western hybrid experiments wild In type (WT) and β-Arrestin1 gene knockouts (KO) hippocampus of mice (Hp) tissue sample total length APP, APP-CTF α (C83) and APP-CTF β (C99) are expressed, and are compareed by applied sample amount of Actin.Left figure is representational Western hybrid experiments result.Right figure Statistical chart is measured for total length APP, APP-CTF α and APP-CTF β Western hybridization signals.n=3-5；*P<0.05.

Secretase proteinase activity in Figure 11 β-Arrestin1 knock out mice tissues.It is real with fluorescent substrate activity Test α in detection wild type (WT) and β-Arrestin1 gene knockouts (KO) hippocampus of mice (Hp) tissue sample-, β-and gamma-secretase The proteinase activity of enzyme.n=4；**P<0.01.

β-Arrestin the tables of Figure 12 Primary cultured neurons (Neuron) and Neuro-2a neuroblastoma cells Reach.Primary cultured neurons and Neuro-2a neuroblastoma cells transfection control (NS) and β-Arrestin1 (β arr1) are small RNA interfering (left figure), or the wild-type beta-Arrestin1 (right figure) of transfection HA marks, with the detection of Western hybrid experiments Source (A2CT antibody) or the β-Arrestin of external source (anti-HA antibody) expression.

Figure 13 suppress β-Arrestin1 expression and lower A β levels.Primary cultured neurons (left figure) and Neuro-2a nerves Blastoma cell (right figure) transfection control (NS) and β-Arrestin1 (β arr1) siRNA, it is real with Enzyme-linked Immunosorbent Assay The A β tested in detection cell culture medium₄₀With A β₄₂It is horizontal.Data are 3 experimental results, are expressed as mean+/-standard error.*P <0.05, * * P<0.01.

It is horizontal that Figure 14 are overexpressed β-Arrestin1 up-regulated expression A β.Primary cultured neurons (left figure) and Neuro-2a god Through blastoma cell (right figure) transfection control (Con) and β-Arrestin1 (β arr1) plasmid, enzyme-linked immunosorbent assay is used Detect the A β in cell culture medium₄₀With A β₄₂It is horizontal.Data are 3 experimental results, are expressed as mean+/-standard error.*P< 0.05, * * P<0.01, * * * P<0.001.

Figure 15 suppress β-Arrestin1 expression and lower gamma-secretase activity.Primary cultured neurons (left figure) and Neuro-2a neuroblastoma cells (right figure) transfection control (NS) and β-Arrestin1 (β arr1) siRNA, with point Secrete the secretase proteinase activity of enzyme fluorescent substrate activity experiment detection cell.Data are 3 experimental results, are expressed as average value ± standard error.*P<0.05, * * P<0.01.

Figure 16 are overexpressed β-Arrestin1 up-regulated expressions gamma-secretase activity.

Primary cultured neurons (left figure) and Neuro-2a neuroblastoma cells (right figure) transfection control (Con) and β- Arrestin1 (β arr1) plasmid, the gamma-secretase protease activity of detection cell is tested with gamma-secretase fluorescent substrate activity Property.Data are 3 experimental results, are expressed as mean+/-standard error.*P<0.05, * * P<0.01, * * * P<0.001.

Figure 17, which suppress endocytosis transporting pathway, does not influence β-Arrestin1 up-regulation gamma-secretase activity.Neuro-2a nerves Blastoma cell transfecting multiple cloning sites carry the expression plasmid of the coded sequence of Rab5S34N and Rab7T22N albumen, use The gamma-secretase proteinase activity of gamma-secretase fluorescent substrate activity experiment detection cell.Data are 3 experimental results, are represented For mean+/-standard error.

Figure 18 β-Arrestin1 combine APH-1AL.The β of HEK293T cells difference cotransfection FLAG couplings- PS1-NTF, PS1-CTF, Nicastrin (NCT), APH-1AL or the PEN-2 that Arrestin1 (β arr1) and HA is coupled.Transfection Cell lysis after 48 hours, with the Ago-Gel immunoprecipitation β-Arrestin1 for being coupled anti-FLAG antibody.Western hybridizes Experiment shows that APH-1AL is co-precipitated, and PS1-NTF, PS1-CTF, Nicastrin and PEN-2 are not co-precipitated.

The common locations of Figure 19 β-Arrestin1 and APH-1AL in the cell.HEK293T cell cotransfections FLAG couplings The APH-1AL of β-Arrestin1 (β arr1) and HA couplings, the anti-HA antibody being coupled with anti-the FLAG antibody and FITC of Cy3 couplings Dyeing, with confocal laser scanning microscope cell sample and obtains image.

Figure 20 β-Arrestin1 do not combine APP and BACE1.The β of HEK293T cells difference cotransfection FLAG couplings- APH-1AL, APP or BACE1 of Arrestin1 (β arr1) and HA couplings.Cell lysis after transfecting 48 hours, with the anti-HA of coupling Ago-Gel immunoprecipitation APH-1AL, APP and BACE1 of antibody.Western hybrid experiments show that APH-1AL is coprecipitated Form sediment, APP and BACE1 are not co-precipitated.

Figure 21 activation β 2AR do not influence β-Arrestin1 and combine APH-1AL.HEK293T cell cotransfections β 2AR, The APH-1AL of β-Arrestin1 (β arr1) and the HA coupling of FLAG couplings, is stimulated with 10 μM of isoproterenol (ISO) Co-immunoprecipitation experiment is carried out after different time.

Figure 22 β-Arrestin1 do not combine APH-1AS.The β of HEK293T cells difference cotransfection HA couplings- APH-1AL, APH-1AS or APH-1B of Arrestin1 (β arr1) and FLAG couplings.Cell lysis after transfecting 48 hours, with idol Join the Ago-Gel immunoprecipitation β-Arrestin1 of anti-HA antibody.Western hybrid experiments show APH-1AL and APH-1B It is co-precipitated, APH-1AS is not co-precipitated.

The endogenous expression β-Arrestin1 of Figure 23 combine APH-1AL.With anti-β-Arrestin1 after the cracking of HEK293T cells Antibody A 1 CT immunoprecipitation β-Arrestin1.Western hybrid experiments show that the β-Arrestin1 of endogenous expression are co-precipitated. The CT20 of 50 μM of incubation has then competed β-Arrestin1 and APH-1AL co-precipitation altogether.

The proteinase activity of gamma-secretase containing APH-1AL and complex proteins water in Figure 24 human cerebral tissue It is flat.Healthy People (ND) and patient (AD) brain cortical tissue carry out co-immunoprecipitation experiment with anti-APH-1AL antibody A 1s .2.Incubate altogether Educate 50 μM of CT20 and then compete A1.2 for APH-1AL immunoprecipitations.It is heavy with the experiment detection of gamma-secretase fluorescent substrate activity The gamma-secretase proteinase activity of shallow lake compound.(n=10)；***P<0.001.

Figure 25 show gamma-secretase component molecular (the N1 expression normal persons 1 of sediment composite with Western hybrid experiments Number, N2 represent normal person No. 2, A1 represent patient AD No. 1, A2 represent patient AD No. 2).

It is horizontal that Figure 26 activation β 2AR do not influence the gamma-secretase complex proteins containing APH-1AL.HEK293T cells Entered with after 10 μM of isoproterenols (Isoproterenol, ISO) stimulation different time with anti-APH-1AL antibody A 1s .2 Row co-immunoprecipitation experiment.

Figure 27 β-Arrestin1 mutation construction schematic diagrames, the gamma-secretase activity and APH-1AL knots of these mutant Conjunction property.

The co-immunoprecipitation of Figure 28 β-Arrestin1 mutant and APH-1AL.HEK293T cells cotransfection is marked with HA β-Arrestin1 the mutant of note and with FLAG mark APH-1AL plasmids, with co-immunoprecipitation experiment analyze β- The interaction of Arrestin1 mutant and APH-1AL.

Effect of Figure 29 β-Arrestin1 mutant to gamma-secretase activity.Neuro-2a neuroblastoma cells The expression plasmid that multiple cloning sites carry protein mutant coded sequence as shown in the figure is transfected, is lived with gamma-secretase fluorogenic substrate Property experiment detection cell gamma-secretase proteinase activity.Data are 3 experimental results, are expressed as average value ± standard error Difference.*P<0.01.

Figure 30 glycerine gradients separate the gamma-secretase complex of β-Arrestin1 knock out mice hippocampus.Wild type (WT) and the hippocampal tissue extract of β-Arrestin1 gene knockouts (KO) mouse is separated into 20 respectively through 10-30% glycerine gradients Individual component, with Western hybrid experiment (above) detect each component PS1-NTF, PS1-CTF, Nicastrin (NCT), APH-1A, PEN-2, β-Arrestin and APP expression.

Under gamma-secretase component molecular expression in Figure 31 β-Arrestin1 knock out mice hippocampal tissues Adjust.The hippocampal tissue extract of wild type (WT) and β-Arrestin1 gene knockouts (KO) mouse is examined with Western hybrid experiments Survey PS1-NTF, Nicastrin (NCT), APH-1AL, PEN-2 and Actin expression.After analytic statistics calculate PS1-NTF, Nicastrin (NCT), APH-1AL and PEN-2 signal relative to Actin signals ratio, as its relative expression quantity.(n= 8)；*P<0.05, * * * P<0.001.

Figure 32 glycerine gradients separate the gamma-secretase complex of β-Arrestin1 knock out mice hippocampus.Wild type (WT) and the hippocampal tissue extract of β-Arrestin1 gene knockouts (KO) mouse is separated into 20 respectively through 10-30% glycerine gradients Individual component, the gamma-secretase proteinase activity for detecting each component is tested with gamma-secretase fluorescent substrate activity.Insert small Figure is that the gamma-secretase ratio in the 8th component is lived.(n=3)；*P<0.05, * * P<0.01.

Figure 33 glycerine gradients separate the gamma-secretase complex of β-Arrestin1 knock out mice hippocampus.Wild type (WT) and the hippocampal tissue extract of β-Arrestin1 gene knockouts (KO) mouse is separated into 20 respectively through 10-30% glycerine gradients Individual component, it is miscellaneous with the gamma-secretase containing APH-1AL, Western in anti-No. 8 component of APH-1AL antibody A 1s .2 immunoprecipitations Experiment is handed over to show the gamma-secretase component molecular of sediment composite.

Figure 34 glycerine gradients separate the gamma-secretase complex of β-Arrestin1 knock out mice hippocampus.Wild type (WT) and the hippocampal tissue extract of β-Arrestin1 gene knockouts (KO) mouse is separated into 20 respectively through 10-30% glycerine gradients Individual component, with the gamma-secretase containing APH-1AL, gamma-secretase in anti-No. 8 component of APH-1AL antibody A 1s .2 immunoprecipitations The gamma-secretase proteinase activity of fluorescent substrate activity experiment detection sediment composite.(n=3)；*P<0.05.

Figure 35 glycerine gradients separate the gamma-secretase complex of Neuro-2a neuroblastoma cells.Transfect respectively The Neuro-2a neuroblastoma cell extracts of β-gal, wild-type beta-Arrestin1 or 1-240 mutant plasmids are through 10- 30% glycerine gradient is separated into 20 components respectively, and PS1-NTF, PS1- of each component are detected with Western hybrid experiments CTF, Nicastrin (NCT), APH-1A and β-Arrestin expression.

Figure 36 glycerine gradients separate the gamma-secretase complex of Neuro-2a neuroblastoma cells.Transfect respectively The Neuro-2a neuroblastoma cell extracts of β-gal, wild-type beta-Arrestin1 or 1-240 mutant plasmids are through 10- 30% glycerine gradient is separated into 20 components respectively, with gamma-secretase fluorescent substrate activity test detect each component γ- Secretase proteinase activity.It is that the gamma-secretase ratio in the 8th component is lived to insert small figure.**P<0.01.

Figure 37 β-Arrestin1 influence the assembling of gamma-secretase complex.Control and β-Arrestin1 have been transfected respectively The wild type (WT) and presenilin gene of siRNA knock out the anti-APH-1AL antibody of (KO) mice embryonic epithelial cell (A1.2) immunoprecipitation gamma-secretase complex.Western hybrid experiments detection immunoprecipitation product in β-Arrestin, Nicastrin, PS1 (PS1-NTF), PEN-2 and APH-1AL.

Figure 38 suppress the amyloid plaque that endogenous β-Arrestin1 expression reduces TgCRND8 mouse.Bilateral hippocampus point The slow virus (Lenti-siArrb1) of expression LacZ (Lenti-siLacZ) and β-Arrestin1 siRNAs has not been injected The brain sections image of TgCRND8 mouse.By observing the GFP fluoroscopic examinations slow virus of slow virus expression in mouse bilateral hippocampus Expression position and intensity in tissue.Mouse pair is detected by immunohistochemical assay with anti-β-Arrestin1 antibody A 1s CT Endogenous β-Arrestin1 in the hippocampal tissue of side.Mouse bilateral is detected by immunohistochemical assay with anti-amyloid beta antibodies 6E10 Amyloid plaques in hippocampus and cortical tissue.Length representative 400 shown in scale μm.

Figure 39 suppress the amyloid plaque that endogenous β-Arrestin1 expression reduces TgCRND8 mouse.Pass through software point The brain sections image of TgCRND8 mouse is analysed, counts and calculates shared by the amyloid plaque in bilateral hippocampus and cortical tissue The percentage of image area.(n=10)；**P<0.01.

It is horizontal that Figure 40 suppress the A β that endogenous β-Arrestin1 expression reduces TgCRND8 mouse.Noted respectively from bilateral hippocampus Penetrate in the cerebral tissue of the TgCRND8 mouse of the slow virus of expression LacZ and β-Arrestin1 siRNAs and isolated hippocampus (left figure) and cortex (right figure) tissue, protein is extracted with surfactant (SDS) and formic acid (FA), it is real with Enzyme-linked Immunosorbent Assay The A β tested in detection protein extract₄₀With A β₄₂It is horizontal.Measured value is expressed as μ g/g tissue wets.(n=10)；*P< 0.05, * * * P<0.001.

Figure 41, which suppress endogenous β-Arrestin1 expression, reduces the gamma-secretase activity of TgCRND8 mouse.From bilateral hippocampus Inject and separated in the cerebral tissue of the TgCRND8 mouse of the slow virus of expression LacZ and β-Arrestin1 siRNAs respectively Go out hippocampus (Hp) and cortex (Cx) tissue, with gamma-secretase fluorescent substrate activity test detection protein extract in γ- Secretase activity.(n=5)；**P<0.01.

Figure 42 are overexpressed the amyloid plaque of β-Arrestin1 expression increase TgCRND8 mouse.Hippocampus is injected respectively The brain sections image of the TgCRND8 mouse of expression control, wild-type beta-Arrestin1 and 1-240 mutant slow virus.It is logical Cross expression position and intensity of the GFP fluoroscopic examination slow virus of observation slow virus expression in Hippocampus of Mice.Resisted with anti-A β Body 6E10 detects the amyloid plaques in hippocampus of mice and cortical tissue by immunohistochemical assay.Length generation shown in scale 400 μm of table.

Figure 43 are overexpressed the amyloid plaque of β-Arrestin1 expression increase TgCRND8 mouse.Pass through software analysis The brain sections image of TgCRND8 mouse, count and calculate the shared figure of amyloid plaque in bilateral hippocampus and cortical tissue The percentage of image planes product.(n=6)；**P<0.01.

The A β that Figure 44 are overexpressed β-Arrestin1 expression increase TgCRND8 mouse are horizontal.Table has been injected respectively from hippocampus Separate and go to sea up in the cerebral tissue of the TgCRND8 mouse of control, wild-type beta-Arrestin1 and 1-240 mutant slow virus Horse (left figure) and cortex (right figure) tissue, protein is extracted with surfactant (SDS) and formic acid (FA), uses Enzyme-linked Immunosorbent Assay A β in experiment detection protein extract₄₀With A β₄₂It is horizontal.Measured value is expressed as μ g/g tissue wets.(n=6)；*P< 0.05, * * P<0.01.

Figure 45, which suppress endogenous β-Arrestin1 expression, reduces the gamma-secretase activity of TgCRND8 mouse.Distinguish from hippocampus Inject and separated in the cerebral tissue of the TgCRND8 mouse of control, wild-type beta-Arrestin1 and 1-240 mutant slow virus Go out hippocampus (Hp) and cortex (Cx) tissue, with gamma-secretase fluorescent substrate activity test detection protein extract in γ-point Secrete enzymatic activity.(n=6)；***P<0.001.

Figure 46 β-arrestin1 knock out the influence to APP/PS1 mouse cognition and memories.Experiment is sought using novelty to grind Study carefully β arr+ /+, β arr-/-, preference-score of β arr+ /+APP/PS1, β arr-/- APP/PS1 mouse to same object (Preference score) and the cognitive index (Recognition Index) to different objects.And utilize One- WayANOVA combination post hoc tests carry out post-hoc tests analysis cognitive index, P<0.0001(βarr+/+vs.βarr+/+ APP/PS1,P<0.001；βarr+/+APP/PS1vs.βarr-/-APP/PS1,P<0.001).

Figure 47 β-arrestin1 knock out the performance for improving APP/PS1 mouse in invisible platform training.In Morris In the invisible platform training of water maze laboratory, counted after recording the time that every mouse finds platform every time daily.Two- Way ANOVA analyses combine Tukey post hoc tests and carry out post-hoc tests, show that β-arrestin1 are knocked out and significantly lower APP/PS1 mouse found in invisible platform training platform time (n=7-9, β arr1-/- APP/PS1vs. β arr1+ /+ APP/PS1 mouse, P=0.0145).

Figure 48 β-arrestin1 knock out the performance for improving APP/PS1 mouse in probe trial.In Morris water fans In the experiment probe trial of palace, record exploration track and time of the every mouse in pond and counted.One-wayANOVA The time searched with reference to post hoc tests progress post-hoc tests analyses in target quadrant, P=0.0151 (β arr1+ /+small Mouse vs. β arr1+ /+APP/PS1 mouse, P<0.05；β arr1+ /+APP/PS1 mouse vs. β arr1-/- APP/PS1 mouse, P< 0.05)。

Swimming distance and swimming rate of Figure 49 mouse in Morris water maze laboratory probe trial.

Figure 50 β-arrestin1 knock out the amyloid-beta precipitation spot substantially reduced in APP/PS1 Mice brain tissues Block.Heart is carried out after three kinds of mouse genotypes (β arr+ /+, β arr+ /+APP/PS1, β arr-/- APP/PS1) anesthesia of diagram Perfusion, take cerebral hemisphere carry out PFA fix and sucrose dehydration after carry out frozen section.Brain frost is cut with anti-A β antibody Amyloid-beta precipitation patch in piece is dyed, and image is obtained by ZEISS OBSERVER Z1 microscopes, uses Image- Pro Plus 5.1 count the pixel of positive signal patch.(n=4-5, * P<0.05).

Figure 51 β-arrestin1, which are knocked out, significantly reduces in APP/PS1 mouse brains the solubility of hippocampus and cortical area and not Soluble A β 40 and the contents of A β 42.Diagram three kinds of mouse genotypes (β arr+ /+, β arr+ /+APP/PS1, β arr-/- APP/PS1 cardiac perfusion is carried out after) anaesthetizing, takes cerebral hemisphere to peel off hippocampus and cortical area, soluble A β is extracted with SDS, uses first Acid extraction insolubility A β, carry out enzyme-linked immunosorbent assay (ELISA) experiment, detect the A β 40 and A β 42 in Mice brain tissues Content.(n=4-5, * P<0.05, * * P<0.01).

Figure 52 β-arrestin1, which are knocked out, significantly reduces gamma-secretase activity in APP/PS1 Mice brain tissues.The two of diagram Cardiac perfusion is carried out after kind mouse genotypes (β arr+ /+APP/PS1, β arr-/- APP/PS1) anesthesia, takes cerebral hemisphere to peel off Hippocampus, with fluorogenic substrate enzyme activity test detection α-, β-and gamma-secretase activity.(n=4-5, * * * P<0.001).

Figure 53 β-arrestin1 knock out the formation for reducing gamma-secretase holoenzyme complex in APP/PS1 Mice brain tissues. Cardiac perfusion is carried out after two kinds of mouse genotypes (β arr+ /+APP/PS1, β arr-/- APP/PS1) anesthesia of diagram, takes brain Hemisphere peels off hippocampus, and the formation of gamma-secretase holoenzyme complex is detected with BN-PAGE.

The small peptide schematic diagram that Figure 54 synthesize according to APH-1AL and APH-1B intracellular ring and carboxyl terminal sequence.APH-1 It is seven transmembrane proteins, includes 3 extracellular rings, 3 intracellular rings and 1 carboxyl terminal.Because β-arrestin1 are endochylemas Albumen, therefore β-arrestin1 can be only combined to APH-1 these intracellular parts, so intracellular ring and carboxylic according to APH-1AL Base end has synthesized AL1, AL3, AL5 and ACT；According to APH-1B intracellular ring and carboxyl terminal synthesized BL1, BL3, BL5 and BCT。

The short peptide sequence that Figure 55 synthesize according to APH-1AL and APH-1B intracellular ring and carboxyl terminal sequence.RED sector For the sequence of corresponding intracellular ring and carboxyl terminal, wherein underlined in red part is shorter intracellular ring, it is entered during synthesis Gone three repeat；TAT-tag and FITC fluorescence labelings are added in small peptide N-terminal to be easy to detect whether to be efficiently entering cell.

Figure 56 .FITC-TAT-tagged small peptides are efficiently entering cell.HEK293 cells are small with 10 μM of ACT small peptides processing 40 Shi Hou, washed with fresh culture and once change culture medium afterwards.Living cells image is by ZEISS OBSERVERZ1 microscope (Carl Zeiss) is equipped with AxioCamMR3 digital camera and AxioVisionsoftware (Carl Zeiss) and obtained .As a result show, FITC-TAT-tagged small peptides ACT can be efficiently entering cell.

Interaction of Figure 57 .FITC-TAT-tagged small peptides to β-arrestin1 and APH-1AL or APH-1B Influence.Left figure：HEK293T cell cotransfection HA- β-arrestin1 and Flag-APH-1AL, calcium phosphate method transfect 8 hours after more 10 μM of corresponding FITC-TAT-tagged small peptides are added when changing culture medium, transfection is used after 48 hours (after i.e. small peptide is handled 40 hours) Lysate cell lysis containing 1%TritonX-100, carry out co-immunoprecipitation and immunoblot experiment；Right figure：HEK293T cells Cotransfection HA- β-arrestin1 and Flag-APH-1B, other same left figures of processing.As a result show, only ACT, BCT small peptide can be with Suppress β-arrestin1 and APH-1AL and APH-1B interaction, other intracellular ring small peptides are interacted to it without notable Influence.

Figure 58 .FITC-TAT-tagged small peptides are on influence caused by A β.FITC-TAT-tagged with 1 μM and 10 μM is short Peptide handled APPswe HEK293 stable cell strains after 40 hours respectively, collects cell culture medium, uses enzyme-linked immunosorbent assay (ELISA) total A β total therein are determined.Data are experimental result three times, are expressed as mean+/-standard error.***P< 0.001。

Figure 59 .FITC-TAT-tagged small peptides to α-, β-and gamma-secretase activity influence.In Figure 25 APPsweHEK293 surely turns cell after FITC-TAT-tagged small peptides are handled 40 hours, cell is collected, with fluorogenic substrate enzyme activity Experiment detection cell α-, β-and gamma-secretase activity.As a result show, only ACT, BCT small peptide can significantly reduce gamma-secretase Enzymatic activity, and not having an impact to α-or beta-secretase activity, other intracellular ring small peptides to α-, β-and gamma-secretase activity all Do not influence.Data are experimental result three times, are expressed as mean+/-standard error.*P<0.05.

The influence that Figure 60 .ACT, BCT small peptides assemble to gamma-secretase holoenzyme complex.With 10 μM of FITC-TAT- Tagged small peptides (AL5, ACT, BCT) handled HEK293 cells after 40 hours respectively, collected cell, and extraction cell membrane carries out BN- PAGE detects the formation of gamma-secretase holoenzyme complex.Mock is cell culture medium control group.As a result show, ACT, BCT small peptide The assembling of gamma-secretase holoenzyme complex can effectively be suppressed, and control group small peptide AL5 can not then suppress its assembling.

The influence that Figure 61 .ACT, BCT small peptides assemble to complex before NCT/APH-1.The double knock-out mice embryos of PS1/2 are into fibre Cell transfecting β-arrestin1 plasmids are tieed up, after Fugene HD are transfected 8 hours, update culture medium and the FITC-TAT- with 10 μM Tagged small peptides (AL5, ACT, BCT) handled the double knock-out mice embryo fibroblasts of PS1/2 after 40 hours respectively, collected thin Born of the same parents, extraction cell membrane carry out the formation of complex before BN-PAGE detections NCT/APH-1.Mock is cell culture medium control group.Knot Fruit shows that complex assembles before ACT, BCT small peptide can effectively suppress NCT/APH-1, and control group small peptide AL5 can not then suppress It is assembled.

Figure 62 reappear β-arrestin1 and APH-1AL interaction using Split-TEV experiments.By β- Arrestin1 and APH-1AL nitrogen ends with TEV enzymes respectively（NTEV）With the carboxyl terminal of TEV enzymes（CTEV）Coupling, β- Arrestin1 and APH-1AL, which interacts, make it that NTEV and CTEV is close and plays complete TEV enzymatic activitys and starts downstream report Gene signal.

Embodiment

A β of the present inventor by extensive research, first the rising increase cell of discovery β-Arrestin1 expressions Protein level, so as to accelerate disease process；And it also found that β-Arrestin1 can be with a necessary component of gamma-secretase APH-1 (APH-1AL or APH-1B) interacts, so as to influence the activity of gamma-secretase.Therefore, can be with β-Arrestin1 The medicine of prevention or treatment nerve degenerative diseases is screened as target spot with APH-1 complex.

As used herein, " separation " it is (former if crude to refer to that material is separated from its primal environment Beginning environment is natural surroundings).If the polynucleotide under the native state in active somatic cell and polypeptide are not isolate and purify , but same polynucleotide or polypeptide such as from native state with being separated in other existing materials, then to isolate and purify 's.

As used herein, described " nerve degenerative diseases " refer to that a kind of be metabolized with amyloid precusor protein produces cell The related nerve degenerative diseases of toxic fragment (such as A β 40, A β 42), namely characterized by intracerebral forms amyloid plaque Nerve degenerative diseases, such as Alzheimer's disease (also known as senile dementia).

As used herein, described " containing ", " having " or " comprising " include "comprising", " mainly by ... form ", " substantially by ... form " and " by ... form "；" mainly by ... form ", " substantially by ... form " and " by ... form " belong to the subordinate concept of " containing ", " having " or " comprising ".

β-Arrestin1 or its protein fragments

In the research process to Alzheimer's disease, the inventors discovered that β-Arrestin1 suffer from Alzheimer's disease The high expression of selectivity in the brain tissue of person and model mice.

β-Arrestin1 the albumen of described β-Arrestin1 albumen including total length or its bioactive fragment (or be Protein fragments or active fragment).For example, the amino acid sequence of described β-Arrestin1 albumen can be with SEQ ID NO:1 institute The sequence shown is substantially the same.The β formed by the substitution of one or more amino acid residues, missing or addition- The amino acid sequence of Arrestin1 albumen is also included in the present invention.β-Arrestin1 albumen or its bioactive fragment include The alternative sequence of a part of conserved amino acid, the sequence through amino acid substitution have no effect on its activity or remain its part Activity.Appropriate amino acid of replacing is technology well known in the art, and the technology can be easily carried out, and be ensured not Change the bioactivity of gained molecule.These technologies recognize those skilled in the art, in general, in a kind of the inessential of polypeptide Area change single amino acids will not substantially change bioactivity.See the Molecular Biology of such as Watson The Gene, fourth edition, 1987, The Benjamin/Cummings Pub.Co.P224.Any β-Arrestin1 albumen Bioactive fragment can be applied in the present invention.Herein, the bioactive fragment of β-Arrestin1 albumen is meant that Refer to and be used as a kind of polypeptide, it still can keep all or part of function of the β-Arrestin1 albumen of total length.Under normal circumstances, Described bioactive fragment at least keeps the activity of 50% total length β-Arrestin1 albumen.Under still more preferential conditions, institute 60%, 70%, 80%, 90%, 95%, 99% or 100% activity of total length β-Arrestin1 albumen can be kept by stating active fragment.

As the preferred embodiment of the present invention, the fragment of described β-Arrestin1 albumen is to include SEQ ID NO:In 1 The albumen of 241-360 amino acids sequences.Empirical tests, the above-mentioned bioactive fragment of β-Arrestin1 albumen remain total length The bioactivity of β-Arrestin1 albumen.

Present invention includes the nucleic acid for the separation for encoding the β-Arrestin1 protein fragments or its complementation Chain.The DNA sequence dna of β-Arrestin1 protein fragments is encoded, can be artificial synthesized with complete sequence, it is also possible to which the method for PCR amplifications obtains .After the DNA sequence dna of bioactive fragment of the described β-Arrestin1 albumen of coding is obtained, it is suitable to be connected into Expression vector, then be transferred to suitable host cell.

APH-1 or its protein fragments

Components of the APH-1 as gamma-secretase, it is be combined with each other with β-Arrestin1 albumen, influences the work of gamma-secretase Property, promote A β generation.Therefore, APH-1 (including APH-1AL or APH-1B) can be as the target spot of drug screening.

The APH-1 albumen of described APH-1 albumen including total length or its bioactive fragment (or for protein fragments or Active fragment).For example, the amino acid sequence of APH-1AL albumen can be with SEQ ID NO:Sequence shown in 33 is substantially the same； The amino acid sequence of APH-1B albumen can be with SEQ ID NO:Sequence shown in 34 is substantially the same.By one or more ammonia Substitution, missing or the addition of base acid residue and the amino acid sequence of APH-1 albumen that is formed are also included in the present invention.

It is preferred that the APH-1AL albumen or APH-1B fragment are selected from：(i) SEQ ID NO are included:246- in 33 The protein fragments of 265 amino acids sequences；(ii) SEQ ID NO are included:The albumen flakes of 235-265 amino acids sequence in 33 Section；Or (iii) includes SEQ ID NO:The protein fragments of 234-257 amino acids sequence in 34；Also, described albumen flakes Section does not include the albumen with total length APH-1AL albumen or APH-1B amino acid sequences.

The inventors discovered that APH-1AL albumen or APH-1B fragment can by it is competitive with beta-protein inhibitor 1 or its Fragment combines, and carrys out tissue APH-1 and β-Arrestin1 albumen and be combined with each other.By the substitution of one or more amino acid residues, The derived protein of missing or (i) or (ii) or (iii) protein fragments for adding and being formed is also included in the present invention, as long as its Foregoing competitive inhibition can be played.In general, single amino acids base is changed in a kind of unwanted regions of polypeptide It will not change bioactivity on this.Further, it is possible to keep the 60% of (i) or (ii) protein fragments, 70%, 80%, 90%, 95%, 99%, Or 100% active albumen is also included in the present invention.

Present invention includes coding (i) or (ii) or (iii) protein fragments or its derived protein separation nucleic acid, It can be its complementary strand.The DNA sequence dna of (i) or (ii) or (iii) protein fragments is encoded, can be artificial synthesized with complete sequence, also may be used Obtained with the method for PCR amplifications.After (i) or (ii) or (iii) protein fragments DNA sequence dna is encoded, connected Enter suitable expression vector, then be transferred to suitable host cell.

Present invention includes include (i) described in coding or (ii) or the nucleic acid of (iii) protein fragments or its derived protein The carrier of molecule.Described carrier can also include the expression regulation sequence being connected with the series of operations of the nucleic acid molecules, with It is easy to the expression of albumen.Also, contain the nucleic acid sequence of (i) or (ii) or (iii) protein fragments or its derived protein described in coding The recombinant cell of row is also included in the present invention.

Suppress material of β-Arrestin1 and APH-1 interactions and application thereof

New discovery based on the present invention, there is provided one kind suppresses β-Arrestin1 and APH-1 (APH-1AL or APH-1B) The purposes of the material of interaction, for preparing the composition of prevention or treatment nerve degenerative diseases.

The material that described suppression β-Arrestin1 and APH-1 interacts includes：β-Arrestin1 inhibitor, under Adjustment, retarding agent, blocking agent etc.；Any activity for reducing β-Arrestin1 albumen, reduce the steady of β-Arrestin1 albumen It is qualitative, suppress β-Arrestin1 albumen expression, reduce β-Arrestin1 albumen effective acting times, suppress β- The secretion of Arrestin1 albumen or the material of suppression β-Arrestin1 transcription and translation.Described suppression β-Arrestin1 Also include with the material of APH-1 interactions：APH-1 inhibitor, lower adjustment, retarding agent, blocking agent etc.；It is any to reduce The activity of APH-1 albumen, the stability for reducing APH-1 albumen, the expression for suppressing APH-1 albumen, reduction APH-1 albumen are effectively made With the material of the transcription and translation of time, the secretion of suppression APH-1 albumen or suppression APH-1.Described suppression β- The material that Arrestin1 and APH-1 interacts also includes：Pass through the part or all of sequence with β-Arrestin1 or APH-1 Competitive binding and reduce material that both be combined with each other (for example, by with APH-1 protein bindings come reduce APH-1 albumen with The material that beta-protein inhibitor 1 combines；Or reduce APH-1 albumen and the knot of beta-protein inhibitor 1 by being combined with beta-protein inhibitor 1 The material of conjunction).Above-mentioned substance is used equally for the present invention, as preventing and treating the effective material of Alzheimer's disease

As the preferred embodiment of the present invention, the material of the described activity of suppression beta-protein inhibitor 1 is specific binding β-suppression The antibody of albumen 1 processed, or the small disturbing molecule that specificity interference beta-protein inhibitor 1 is expressed, or the expression small disturbing molecule Carrier or virus (such as slow virus).For example, described small disturbing molecule has such as SEQ ID NO:23 sequence, it has good Interference β-Arrestin1 expression effect.

As the preferred embodiment of the present invention, the active material of described suppressions beta-protein inhibitor 1 be APH-1AL albumen or APH-1B fragment, it is combined with beta-protein inhibitor 1 or its fragment by competitiveness so that beta-protein inhibitor 1 or its fragment and The probability that APH-1AL albumen or APH-1B are combined declines.Preferably, APH-1AL albumen or APH-1B fragment are selected from (i) Include SEQ ID NO:The protein fragments of 246-265 amino acids sequence in 33；(ii) SEQ ID NO are included:In 33 The protein fragments of 235-265 amino acids sequences；Or (iii) includes SEQID NO:234-257 amino acids sequence in 34 Protein fragments.

It is preferred that (i) or (ii) or (iii) protein fragments or its derived protein are also combined with entering cell guiding thing, with It is easy to it to be easier into playing a role into the cell, described " entering cell guiding thing (such as cell-penetrating peptide) " refers to that one kind has The fusion protein of the polypeptide of cell membrane penetration, its own or itself and other albumen can be entered into the cell by cell membrane. It is described enter cell guiding thing for example including：Trans-activator (TAT), Penetratin, the peptide based on signal sequence, PVEC, Transportan, Amphiphilic model peptide and Arg9 etc..

As the preferred embodiment of the present invention, it is described enter cell guiding thing be trans-activator (Transactivator, TAT).The inventors discovered that after TAT protein fusion (i) or (ii) or (iii) protein fragments, external source (i) or (ii) can be made Or (iii) protein fragments or its derived protein more enter into the cell, so as to play competitive inhibition.It is preferred that institute The TAT-tag stated has SEQ ID NO:Amino acid sequence shown in 35.

Screening technique

, can base after the interaction for knowing β-Arrestin1 and APH-1 and the correlation of nerve degenerative diseases Suppress both combinations to screen in the new discovery, and then reduce material caused by A β, described material is for preventing or treating god It is useful through degenerative disease.

Therefore, the invention provides a kind of method for the potential material for screening prevention or treatment nerve degenerative diseases, institute The method of stating includes：Using β-Arrestin1 or its protein fragments as the target spot of screening, the thing that specificity suppresses the target spot is selected Matter, described material are prevention or the potential material for treating nerve degenerative diseases.

As the preferred embodiment of the present invention, described method includes：(1) by candidate substances with expression β-Arrestin1 or The system contact of its protein fragments；(2) influence of the candidate substances to β-Arrestin1 or its protein fragments is detected；If the time Material is selected to suppress β-Arrestin1 or expression or the activity of its protein fragments, then it is prevention or treatment god to show the candidate substances Potential material through degenerative disease.It is highly preferred that when being screened, in order to be more easily observable β-Arrestin1 table Reach or the change of activity, also settable control group, described control group can be do not add the expression β of the candidate substances- Arrestin1 or its protein fragments system.

As the preferred mode of the present invention, with β-Arrestin1 protein fragments, particularly to contain SEQ ID NO:In 1 the protein fragments of 241-360 amino acids sequence as screening target spot, so as to screen specific effect in Material of the site without acting on other sites on β-Arrestin1 albumen, the material on β-Arrestin1 albumen for removing Other regions beyond APH-1AL calmodulin binding domain CaMs have minor impact, namely do not influence the other of β-Arrestin1 albumen Biological function.

Specific region is as target spot using on albumen, come screening effect in the method for the material in the region be those skilled in the art Known, these methods are used equally for the present invention.Described candidate substances can be selected from：Peptide, pdef polypeptide, peptidomimetic, non-peptideization Compound, carbohydrate, fat, antibody or antibody fragment, part, organic molecule, inorganic molecules and nucleotide sequence etc..According to The species of material to be screened, those skilled in the art are clear how the applicable screening technique of selection.

By large-scale drug screening, a kind of specific effect can be obtained in β-Arrestin1, be particularly acting on The potential material of the upper 241-360 positions protein domains of β-Arrestin1.The potential material that these preliminary screenings go out may make up one Screen storehouse, in order to people may finally on the basis of further checking, therefrom filter out can for regulation β- Arrestin1 expression and active actually useful material.

In view of the inventors discovered that β-Arrestin1 and APH-1 (particularly APH-1AL or APH-1B) interacts, originally Invention additionally provides a kind of protein complexes, and the complex includes：β-Arrestin1 or its protein fragments, the albumen flakes Section includes SEQ ID NO:241-360 amino acids sequence in 1；With APH-1 or its fragment.Preferred side as the present invention Formula, described APH-1AL amino acid sequence can be with the sequence substantially phase shown in GenBank accession number NP 001071096 Together.Described APH-1AL bioactive fragment (such as its carboxyl-terminal fragment) or derivative can also be used for the present invention.

After β-Arrestin1 and APH-1 interaction is known, the new discovery can be based on screen suppression β- Arrestin1 and APH-1 (particularly APH-1AL or APH-1B) interaction, so as to influence the formation of gamma-secretase, and then Material caused by A β is reduced, described material is useful for preventing or treating nerve degenerative diseases.

Therefore, the invention provides a kind of side for the potential material for screening prevention, alleviation or treatment nerve degenerative diseases Method, methods described include：Target spot using described protein complexes as screening, select specificity and suppress the protein complexes The material of formation, described material are prevention, alleviation or the potential material for treating nerve degenerative diseases.

As the preferred embodiment of the present invention, described method includes：(1) it is candidate substances are compound with containing described albumen The system contact of body；(2) influence of the candidate substances to the protein complexes is detected；If the candidate substances suppress the albumen β-Arrestin1 or its protein fragments and the interaction (such as be combineding with each other) of APH-1 or its fragment, then show this in complex Candidate substances are prevention or the potential material for treating nerve degenerative diseases.

Therefore, the invention provides a kind of side for the potential material for screening prevention, alleviation or treatment nerve degenerative diseases Method, methods described include：(1) by candidate substances with containing (as expressed) beta-protein inhibitor 1 or its protein fragments and APH-1 albumen Or the system contact of its protein fragments；(2) detect candidate substances to beta-protein inhibitor 1 or its protein fragments and APH-1 albumen or The influence of the interaction of its protein fragments；If the candidate substances suppress beta-protein inhibitor 1 or its protein fragments and APH-1 eggs White or its protein fragments interactions, then it is the latent of prevention, alleviation or treatment nerve degenerative diseases to show the candidate substances In material.Phase of the material to beta-protein inhibitor 1 or its protein fragments and APH-1 albumen or its protein fragments is selected in described detection Interaction can be observed in many-side, for example, observation beta-protein inhibitor 1 or its protein fragments and APH-1 albumen or its egg The be combineding with each other of white tiles section, action time length, the ratio effectively combined etc..

Under a kind of filtering mode, by simulating the β-Arrestin1 or its protein fragments, APH-1 or its albumen flakes The three-dimensional structure of section carries out drug screening.The three-dimensional structure of a large amount of protein is by X ray crystal diffraction technology and more at present The technologies such as dimension nuclear magnetic resonance (NMR) are parsed and obtained, and favourable bar is provided to study the molecular recognition of protein-ligand complexes Part.

There are many alternative screening storehouses (such as micromolecular compound screening storehouse) at present so that high flux screening turns into can Energy.Can by Computer-Aided Drug Design, by analyzing the structural property of drug targets, i.e. protein molecular functional site, if Count out specific chemical small molecule, each several part molecular structure and physicochemical property for making its molecule match with target, with reach with Combination and the purpose that plays a role, these technologies are known to those skilled in the art.

In the present invention, a variety of abilities can be used by detecting albumen and the power of interphase interaction and the interaction of albumen Technology known to field technique personnel, such as GST sedimentation techniques (GST-Pull Down), display technique of bacteriophage, yeast two-hybrid System or Immunoprecipitation.Wherein, yeast two-hybrid is a kind of extensive quick research protein-protein interphase interaction Method, there is the advantages of simple, stable.In yeast cells, a kind of bait protein merged with DNA- domains with it is a kind of with The prey protein interaction of activation domain fusion.The molecular pairs of interaction are incorporated into single-minded sequence pattern, so as to activate The transcription of two independent reporters.In a kind of preferred embodiment of the present invention, albumen is verified using immunoprecipitate Specificity interaction between albumen.The principle of described Immunoprecipitation is：Keeping protein-protein phase interaction Under conditions of, harvest and cell lysis, the specifically immunoprecipitation destination protein from cell extract, then pass through electrophoresis The methods of separating immune sediment.The co-precipitation of albumen with known features, the antibody for resisting the albumen can be used, by such as Western blot detects.In addition, if cell had carried out mark with label before cracking, can by radiation from Development or other immunological techniques observe the albumen of co-precipitation.

The present invention's is another it is preferable that reappearing β-arrestin1 using Split-TEV Reporter Systems quantification With APH-1AL interaction.By β-arrestin1 and the APH-1AL nitrogen end with TEV enzymes respectively（NTEV）With TEV enzymes Carboxyl terminal（CTEV）Coupling, β-arrestin1 and APH-1AL, which interacts, make it that NTEV and CTEV is close and it is complete to play TEV enzymatic activitys simultaneously start downstream reporter gene signal.This Reporter System be used directly for disturb β-arrestin1 with The high flux screening of the micromolecular compound of APH-1AL interactions.

Preferred embodiment as the present invention, it is also necessary to the potential material of acquisition is carried out further cell experiment and/or Animal experiment, further to select and determine the thing useful for preventing or treating nerve degenerative diseases from these materials Matter.

Described " system of expression β-Arrestin1 or its protein fragments " or " body containing described protein complexes System " can be selected from：Cell system, subcellular fraction system, solution system, organizational framework, organ systems or animal system.In cellular water On flat, it can be screened by way of building cell model.The structure of relevant cell model is that those skilled in the art are ripe Know.For example, can be by the way that the ceneme of β-Arrestin1 or its protein fragments (such as expression vector) be transferred into host cell It is interior, it is allowed to stably express β-Arrestin1 or its protein fragments.The selection of ceneme and host cell is this area Technology known to personnel.Described host cell is, for example, (but not limited to)：HEK293 cells.It is of course also possible to endogenous table Model up to the cell of β-Arrestin1 or its protein fragments as screening, the cell is, for example, (but not limited to)：Neuron Cell.

With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention Rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to conventional strip Part such as Sambrook et al., molecular cloning：Lab guide (New York：Cold Spring HarborLaboratory Press, 2002 with reference to) described in condition, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise Percentage and number are calculated by weight.

I. material and method

1. material

Cell line and original cuiture Mouse Neuron

HEK293T human embryonic kidney cells：Purchased from ATCC.

Neuro-2a mouse neuroblastoma cell lines：Purchased from ATCC.

Wild type and Presenilin knock out (Psen1^-/-Psen2^-/-) MEC system：Obtained from Billy When Katholieke Universiteit Leuven (being provided by Bart De Strooper and Paul Saftig).

Primary mouse neuron：C57/BL6 mouse (P0 or P1) cerebral cortex is derived from, in vitro culture.

Alzheimer's disease human cerebral tissue's sample

Frontal cortex grey matter tissue sample is obtained from Netherlands Brain Bank in patient.Every Braak sample sets Containing 7 samples, and ensure the sex of each group sample, age (average 77 one full year of life, scope is 52-93 one full year of life), postmortem interval Time (average 6 hours, scope is 3-10 hours), cerebrospinal fluid pH value (mean ph value 6.7, scope 6.3-7.4) and ApoE bases Because type is consistent.

Experimental animal

β-Arrestin1 knock out mice, C57BL/6 inbred strais：Obtained from U.S. Duke University MedicalCenter (being provided by Robert Lefkowitz) [referring to Conner, D.A.et al. β- Arrestin1knockout miceappear normal but demonstrate altered cardiac responses to β-adrenergic stimulation.Circ.Res.81(6),1021-1026(1997)].Hybrid mice is selfed, son For mouse after genotype identification, homozygote is used to test.

1026(1997)].Hybrid mice is selfed, and after genotype identification, homozygote is used to test generation mice.

].Hybrid mice is selfed, and after genotype identification, homozygote is used to test generation mice.

For mouse after genotype identification, homozygote is used to test.

Homozygote is used to test.

TgCRND8 transgenic models mouse, C3H/C57BL6 outbreeding systems：Express APP K670N, M671L, V717F mutation Body, obtained from Canadian University of Alberta (being provided by David Westaway) [referring to Chishti, M.A.et al.Early-onset amyloid deposition and cognitive deficits in transgenicmice expressing a double mutant form of amyloid precursor protein 695.J.Biol.Chem.276(24),21562-21570(2001)].TgCRND8 transgenic mices are handed over B6C3/F1 instruments mouse With breeding filial generation, after genotype identification, transgenic mice is positive to be used to test generation mice.

APP/PS1 transgenic models mouse, C3H/C57BL6 outbreeding systems：Express APP K670N, M671L mutant and PS1 Δ E9 mutant double transgenics, purchased from Jackson Laboratory [referring to Jankowsky, J.L.etal.Mutant presenilins specifically elevate the levels of the 42 residue β- amyloidpeptide in vivo:evidence for augmentation of a 42-specific γ- secretase.Hum.Mol.Genet.13(2),159-170(2004)].APP/PS1 transgenic mices and B6C3/F1 instrument mouse Mating breeding filial generation, after genotype identification, transgenic mice is positive to be used to test generation mice.

Plasmid

The mouse source β-Arrestin1 sequences of small interfering RNAs being implemented in pBluescript-SKII carriers is as follows：5’- GGGAAGCTCAAGCATGAGGACA-3’(SEQ ID NO:23) it is as follows, to compare sequences of small interfering RNAs：5’- GGCCGCAAAGACCTTGTCCTTA-3’(SEQ ID NO:24).Plasmid construction method is as follows：Chemical synthesis carries small interference RNA justice and antisense complementarity single stranded DNA (both ends carry BamHI and HindIII restriction enzyme sites).Complementary single-stranded dna is denatured After annealing forms double-stranded DNA segment, and the pBluescript-SKII carriers linearized with BamHI/HindIII double digestions (Invitrogen) connect to be built into the carrier.

Mouse source β-Arrestin1 siRNAs and control siRNA (slow virus FG12 carriers) construction method are as follows： By the siRNA on pBluescript-SKII carriers and U6 promoters XhoI/XbaI double digestions, slow virus is cloned into FG12 carriers are [referring to Qin, X.F. etc., Inhibiting HIV-1 infection in human T cellsby lentiviral-mediated delivery of small interfering RNA against CCR5.Proc.Natl.Acad.Sci.USA 100(1),183-188(2003)]。

Wild type FLAG- β-Arrestin1 plasmids and HA- β-Arrestin1 plasmid construction methods are as follows：β- Arrestin1 complete encoding sequences are obtained by reverse transcription PCR from HEK293T cell RNAs, pass through EcoRV/XhoI digestions position Point is built into pcDNA3 carriers (Invitrogen).

HA- β-Arrestin1 mutant 1-180,181-418,1-240,241-360,1-360 and 241-418 are using wild Raw type HA- β-Arrestin1 plasmids (wild type HA- β-Arrestin are obtained by reverse transcription PCR) are template, with appropriate primer It is inserted into build in the BamHI/XhoI sites of pcDNA3 carriers after amplification respective segments and obtains.

FLAG-APH-1AL plasmids, FLAG-APH-1AS plasmids and HA-APH-1AL plasmids are obtained from German Ludwig Maximilians University (are provided) by Harald Steiner.

HA-PS1-NTF plasmids and HA-PS1-CTF plasmids are constructed as below：Using HEK293 cell total rnas as template, by inverse Transcription-PCR is expanded, with 5 '-CGC, GGA, TCC, ATG, ACA, GAG, TTA, CCT, GCA, CC-3 ' (SEQ ID NO:25) and 5’-ATC,GCT,CGA,GCT,ATG,TTG,AGG,AGT,AAA,TGA,GAG-3’(SEQ ID NO:26) obtained for primer amplification PS1-NTF coded sequences；With 5 '-CGC, GGA, TCC, ATG, GTG, TGG, TTG, GTG, AAT, ATG-3 ' (SEQ ID NO:27) With 5 '-TCG, CTC, GAG, CTA, GAT, ATA, AAA, TTG, ATG, GA-3 ' (SEQ ID NO:28) obtained for primer amplification PS1-CTF coded sequences.PS1-NTF and PS1-CTF coded sequences are cloned into respectively by BamHI/XhoI double enzyme sites Multiple cloning sites N-terminal and C-terminal are respectively provided with the pcDNA3 carriers of HA epitope coded sequences；

HA-Nicastrin plasmids：Obtained from U.S. University of Pennsylvania School of Medicine (is provided) by Robert Doms.

HA-PEN-2 plasmids, (provided obtained from U.S. University of Chicago by Sangram Sisodia).

HA-APP plasmids are constructed as below：Using HEK293 cell total rnas as template, with 5 '-AGC, GAT, ATC, GAT, GCT, GCC, CGG, TTT, GGC-3 ' (SEQ ID NO:And 5 '-ATG, CTC, TAG, ATT, AGG, CGT, AGT, CGG, GGA, CGT, 29) CGT,AGG,GGT,AGT,TCT,GCA,TCT,GCT,CAA,AG-3’(SEQ ID NO:30) expanded for primer by reverse transcription-pcr Increase and obtain APP coded sequences, and introduced at its 3 ' end with HA epitopes by design of primers in order to detect.Pass through APP coded sequences are cloned into pcDNA3 carriers by EcoRV/XbaI double enzyme sites, sport " Swedish " mutant (K670M/N671L)。

HA-BACE1 plasmid construction methods are as follows：Using HEK293 cell total rnas as template, with 5 '-CGC, GGA, TCC, ATG,GCC,CAA,GCC,CTG,CCC,TG-3’(SEQ ID NO:31) and 5 '-ATC, GC T, CGA, GTC, ACT, TCA, GCA, GGG, AGA, TGT-3 ' (SEQ ID NO:32) expanded for primer by reverse transcription-pcr and obtain BAC E1 coded sequences.Pass through BACE1 coded sequences are cloned into multiple cloning sites C-terminal and carry HA epitope coded sequences by BanHI/XhoI double enzyme sites PcDNA3 carriers.

Each plasmid can conventional method conversion relevant cell so that the albumen that recombinant cell expression is related.

Antibody

Rabbit-anti PS1-NTF antibody：Purchased from Calbiochem companies.Rabbit-anti PS1-CTF antibody：It is public purchased from Calbiochem Department.The anti-A β monoclonal antibodies 6E10 of mouse：Purchased from Chemicon companies.Rabbit-anti Flag antibody：Purchased from Sigma companies.Rabbit-anti HA resists Body：Purchased from Sigma companies.Rabbit-anti Nicastrin antibody：Purchased from Sigma companies.Rabbit-anti APH-1A antibody：It is public purchased from Abcam Department.Rabbit-anti PEN-2 antibody：Purchased from Sigma companies.Rabbit-anti APP antibody：Purchased from Sigma companies.Rabbit-anti Actin antibody：It is purchased from Sigma companies.Rabbit-anti β-Arrestin antibody A 1s CT and A2CT：Obtained from U.S. Duke University Medical Center (being provided by Robert Lefkowitz).The anti-APH-1AL antibody of mouse：BALB/c mouse is immunized by conventional method to obtain, antigen To be coupled KLH synthesis small peptide RRQEDSRVMVYSALRIPPED (SEQ ID NO:22).

Other reagents

Enzyme-linked immunosorbent assay kit：Detect people source A β₄₀With A β₄₂It is purchased from enzyme-linked immunosorbent assay kit Biosource companies.

Alpha-secretase enzyme, beta-secretase and gamma-secretase fluorogenic substrate are purchased from Calbiochem companies.

Vectastain Elite ABC immunohistochemical assay kit is public purchased from Vector Laboratories Department.

[35S]-methionine is purchased from GE Healthcare companies.

TNT in-vitro transcriptions/translation kits are purchased from Promega companies.

Plasmid extraction kit is purchased from Qiagen companies.

FuGENE HD transfection reagents are purchased from Roche companies.

2. method

Western hybrid experiments detect the β-Arrestin protein levels of patient and mouse tissue sample

Mouse tissue sample is derived from the wild type and transgenosis TgCRND8 and APP/PS1 hippocampus of mice of brood identical sex Tissue.Patient and mouse tissue sample (contain 5mM Tris-HCl (pH 7.4), 5mM with protein extract buffer respectively EDTA, 5mM EGTA, 1%SDS and protease inhibitors) cracking, determine protein concentration.Equal protein quality sample Western Hybrid experiment detects β-Arrestin, obtains Western hybrid experiments image with Odyssey infrared imaging systems and carries out gray scale Analysis.β-Arrestin opposing proteins level is by β-Arrestin signal intensity to Actin signal intensity rate Average value normalization calculating is got.

β-the Arrestin1mRNA of genetic chip and quantitative PCR experiment detection tissue of patient sample are horizontal

Take 50mg to freeze tissue of patient sample, extracted RNA according to former report methods described, detect RNA mass, carry out RNA reverse transcription reactions and quantitative PCR experiment.Hybrid experiment is carried out using Agilent mankind's full-length genome chip, uses R/ BioConductor limma bags processing microarray data.Quantitative PCR experiment then chooses expression not by alzheimer The gene that disease influences is as reference gene.The primer sequence of β-Arrestin and reference gene is：

ARRB1：5’-CGA GAC GCC AGT AGA TAC CA-3’(SEQ ID NO:2), 5 '-TCA TCCTTC ATG CCT TTC AG-3’(SEQ ID NO:3)；

ARRB2,5 '-ACA CTG GAC CCT CTC TTG CT-3 ' (SEQ ID NO:4), 5 '-TCC CTCCTG TCA CTC CTC TT-3’(SEQ ID NO:5)；

DHX16,5 '-TTG ACT CGG AGT GGC TAC CG-3 ' (SEQ ID NO:6), 5 '-AGC GTGGCT GTT GCT CAA AGA-3’(SEQ ID NO:7)；

DNTTIP2,5 '-GGC TTC CCC AAG TAC TTC CA-3 ' (SEQ ID NO:8), 5 '-AGC CAGCAG TTC TTC CAC AA-3’(SEQ ID NO:9)；

KLHDC5,5 '-AGG ATG CGT GGA ATT TTG TG-3 ' (SEQ ID NO:10), 5 '-TTCATG TTC CGG TCT CTG GT-3’(SEQ ID NO:11)；

TM9SF4,5 '-CCA GCT GTG TGC AGA GGA TT-3 ' (SEQ ID NO:12), 5 '-TCCACG ATG TCC AGC TTG TT-3’(SEQ ID NO:13)；

SNW1,5 '-CTT CTC CCA CCT TTC AGC AT-3 ' (SEQ ID NO:14), 5 '-CTC ACTGTG GTT GGG GTA ACT-3’(SEQ ID NO:15)；

AURKAIP1,5 '-GCC TCA AAT TCA GTG CAA AA-3 ' (SEQ ID NO:16), 5 '-CTCGAA CTT GAT CTG CTT GC-3’(SEQ ID NO:17)；

ISOC2,5 '-TGA GAC AGA GTG GTG CCT TC-3 ' (SEQ ID NO:18), 5 '-TTC TGGATC TCC TTG AAC TGG-3’(SEQ ID NO:19)。

Cell culture and transfection

Cell culture is in 37 DEG C of cell culture incubators containing 5% carbon dioxide.Neuro-2a neuroblastoma cells are trained Support in the MEM culture mediums containing 10% hyclone；The mice embryonic that HEK293T cells, wild type and presenilin gene knock out Fibroblast is incubated in the DMEM culture mediums containing 10% hyclone.Cell is inoculated with into culture dish for 16 hours before transfection It is interior.HEK293T cells and Neuro-2a neuroblastoma cells are transfected using calcium phosphate method, and training is discarded after transfecting 6-8 hours Base is supported, is replaced by fresh culture.MEC system is transfected using FuGENE HD.Primary mouse neuron is trained Support in the Neurobasal culture mediums containing 2%B27, with Amaxa Nucleofector system transfections, culture is used to test after 7 days. All experiments are to transfect beta galactosidase (β-gal) or green fluorescent protein (GFP) plasmid as control.

The packaging purifying of slow virus and stereotaxical injection

Recombinant slow virus particle is obtained by transfecting HEK293T cells.Virus core plasmid FG12 is obtained from the U.S. Calfornia Institute of Technology (being provided by David Baltimore), packaging plasmid pRSVREV are obtained from U.S. Calfornia Institute of Technology (being provided by David Baltimore) and pMDLg/pRRE are obtained from U.S. Calfornia Institute of Technology (being provided by David Baltimore) and expression plasmid PHCMVG passes through phosphorus obtained from U.S. Calfornia Institute of Technology (being provided by David Baltimore) Sour calcium method cotransfection enters HEK293T cells.Collect cell culture medium within the 2nd and the 3rd day after transfection, after 2500rpm is centrifuged 10 minutes, With 0.45 μm of aperture membrane filtration, then 28,000rpm ultracentrifugations are after 2 hours, supernatant discarding.Appropriate physiology is added to precipitation Salt solution, 4 DEG C stand overnight dissolving precipitation, be stored in -80 DEG C it is standby.

TgCRND8 mouse bilateral hippocampus stereotaxical injection slow virus.It is placed in after mouse deep anaesthesia on stereotaxic instrument Performed the operation, according to following setting coordinate injection site：Front and rear -2.0 millimeters；Inside and outside ± 1.5 millimeters；Carry on the back -2.0 millimeters of abdomen.It is double Side hippocampus injects β-Arrestin1 siRNAs slow virus and the μ L of LacZ siRNAs slow virus 2 respectively, and injection speed is 1 μ L/ minutes.After performing the operation 4 weeks, cardiac perfusion physiological saline puts to death mouse, and the paraformaldehyde of perfusion 4% is fixed, and quickly removes cerebral tissue It is soaked in 4% paraformaldehyde to continue to fix 2 hours, 30% sucrose solution dewater treatment, makes the coronal frozen section of 30 μ m thicks.With The GFP fluorescence of fluorescence microscope slow virus expression is to detect slow virus expression.

Enzyme-linked immunosorbent assay detection A β are horizontal

Neuro-2a Human Neuroblastoma Cell Lines and the culture medium of Primary cultured neurons culture 12 hours are collected respectively, Method is provided with BNT77/BA27 and BNT77/BC05 enzyme-linked immunosorbent assays kit (being purchased from Wako) according to manufacturer to detect Contained mouse source A β in culture medium₄₀With A β₄₂It is horizontal.

The cortex and hippocampal tissue of β-Arrestin1 knock out mice are homogenized with 0.4% diethylamine/100mM NaCl, 100,000g centrifugations 1 hour, supernatant adds 1/10 volume 0.5M Tris-HCl (pH 6.8) and neutralized, with BNT77/BA27 and BNT77/BC05 enzyme-linked immunosorbent assays kit provides method according to manufacturer and carries out mouse source A contained in experiment detection supernatant β₄₀With A β₄₂It is horizontal.

Cardiac perfusion physiological saline puts to death TgCRND8 mouse, opens cranium and separates cerebral tissue, immediately with liquid nitrogen flash freezer, protects It is stored in -80 DEG C.2%SDS solution is added to cerebral tissue before experiment, is placed in thaw at RT, then ultrasonic disruption tissue.Treat group Centrifuged 1 hour in 4 DEG C with 10,000g after casting off complete crush.Supernatant is SDS soluble protein components.70% is added in precipitation Formic acid solution, centrifuged again after precipitation is resuspended, supernatant is formic acid soluble protein component, adds the neutralization buffer of 20 times of volumes Liquid (Tris containing 1M and 0.5M Na₂HPO₄).Employment source A β after SDS and formic acid component dilute respectively₄₀With A β₄₂Enzyme-linked Immunosorbent Assay Experiment reagent box provides method according to manufacturer and tested.

Secretase fluorescent substrate activity detects

Cell or tissue is split with lysis buffer (containing 5mM Tris-HCl (pH 7.4), 5mM EDTA and 5mMEGTA) Solution, insulin needle aspirate smudge cells repeatedly.Protein concentration is determined with Coomassie brilliant blue method, takes a certain amount of component through 12, 000g is centrifuged 30 minutes in 4 DEG C, supernatant discarding.

Such as it is used to detect gamma-secretase activity, precipitation (contains 0.25%CHAPSO, 50mMTris-HCl with reaction buffer (pH 6.8), 2mM EDTA and 8 μM of gamma-secretase fluorogenic substrates) be resuspended after, reacted 2 hours in 37 DEG C of water-baths.Reaction production Thing fluorescence spectrophotometer measurement fluorescent value, setting excitation wavelength λ_ex=355nm, wavelength of transmitted light λ_em=440nm。

Such as be used to detect alpha-secretase activity, precipitation with reaction buffer (containing 100mM sodium citrates, (pH 4.0) and 20 μM of alpha-secretase enzyme fluorogenic substrates) be resuspended after, reacted 1 hour in 37 DEG C of water-baths.Measure reaction product fluorescent value setting ginseng Number：λ_ex=325nm, λ_em=393nm。

Such as it is used to detect beta-secretase activity, precipitation (contains 50mM sodium acetates (pH 4.5) and 10 μM with reaction buffer Beta-secretase fluorogenic substrate) be resuspended after, reacted 30 minutes in 37 DEG C of water-baths.Measure reaction product fluorescent value setup parameter：λ_ex= 320nm, λ_em=420nm。

Settle (Pull-down) experiment

[³⁵S] methionine mark β-Arrestin1 transcribed with TNT/translation kits provide method according to manufacturer and close Into.PS1-NTF, PS1-CTF, Nicastrin, APH-1AL and PEN-2 of coupling HA epitopes are expressed in HEK293T cells, are used It is coupled anti-HA antibody-agaroses gel immunoprecipitation, and [³⁵S] methionine mark β-Arrestin1 in combination buffer (50mM HEPES (pH 7.5), 150mM NaCl, 0.5mg/mL bovine serum albumins, 1%Triton X-100,5mM EDTA, 10% Glycerine and protease inhibitors) in be incubated altogether.With reference to the β-Arrestin1 sds polyacrylamide electrophoresis point of Ago-Gel From autoradiograph detection.

Immunoprecipitation method

Cell culture medium is discarded, after washing twice of cell with the PBS of precooling, addition lysis buffer (contains into cell 50mM HEPES (pH 7.5), 150mM NaCl, 5mM EDTA, 10% glycerine, 1%Triton X-100 or 1%CHAPSO and albumen Enzyme inhibitor).Cell is collected to crack 1 hour after 4 DEG C.After cell cracks, centrifuged 15 minutes in 4 DEG C with 12,000g.Take Supernatant, add be coupled anti-Flag antibody or anti-HA antibody-agaroses gel (endogenous APH-1AL with mouse resisting anteserum A1.2 and Protein G-Sepharose gel precipitations), it is incubated 4-6 hours in 4 DEG C.Then agarose is collected by centrifugation in 4 DEG C with 2,000g Gel, washed with lysis buffer after 3 times and add 30 μ LSDS-PAGE sample-loading buffers elution protein.With Western hybridization sides Method detects immunoprecipitation product.

TgCRND8 murine genes type is identified

According to standard method extraction rat-tail DNA.The rat-tail tip of clip about 0.5cm length, adds 0.5mL lysates and (contains 4M Urea, 10mM EDTA, 0.1M Tris HCl (pH 8.0), 0.2M NaCl, 0.5% Sarkosyl and 1mg/mL protease K), 56 DEG C of water-baths are placed in until rat-tail is completely dissolved.Then add 0.7mL phenol chloroform and mix, centrifuged in 4 DEG C with 9000rpm 15 minutes.Supernatant is taken, adds isometric isopropanol and mixing, 15 minutes precipitation DNA are centrifuged with 9000rpm in 4 DEG C.Abandon Clearly, precipitation DNA is washed with 70% ethanol.Concentration is determined after dissolving DNA.

PCR identifies that app gene primer is：DW229：TGTCCAAGATGCAGCAGAACGGCTACGAAAA(SEQ ID NO: 20)；DW191：GGCCGCGGAGAAATGAAGAAACGCCAAGCGCCGTGACT(SEQ ID NO:21).

PCR reaction systems：10 × PCR buffer solutions 2 μ L, 25mM MgCl₂The μ L of 1.6 μ L, 2mM dNTP 2, DNA profiling 100ng, 10 μM of DW2290.5 μ L, 10 μM of DW1910.5 μ L, the μ L of 50% glycerine 4, Taq enzyme 3-5 units, adds water to supply 20 μ L. PCR reaction conditions：94 DEG C of denaturations 3 minutes；94 DEG C are denatured 30 seconds, 60 DEG C of renaturation 30 seconds, and 72 DEG C expand 90 seconds, circulation 35 It is secondary.

Immunohistochemical assay

Mouse brain coronal section provides method according to manufacturer with Vectastain Elite ABC kits and tested Detect β-Arrestin1 and amyloid plaque.A1CT and 6E10 antibody tests β-Arrestin1 and amyloid egg are used respectively Hickie.Staining reaction positive signal the area software analysis of Image-Pro Plus 5.1 and statistics in image.Every mouse 3-5 section statistics are taken, its average value is as the mouse measured value.

Novelty seeks experiment

All mouse are by random number and upset order, tested with double-blind study.Experiment carried out in sound proof box, with The clear plexiglass box of 25 centimetres of 25 cm x, 25 cm x is placed in the standby sound proof box of video camera.First day, own Mouse is transferred to Behaviors survey room to adapt to living environment；Second day, mouse is placed sequentially in transparent organic In Yurisangja 10 minutes to adapt to experimental situation；Third and fourth day, placement two is just the same in clear plexiglass box Object at diagonal, apart from about 5 centimetres of box edge, every mouse is sequentially placed into and is wherein allowed to explore 20 minutes；The Five days, two duplicate objects (identical with third and fourth day) are first placed in clear plexiglass box, allow every mouse Carry out the training of 10 minutes, and with its heuristic process of camera record；One of object is replaced as to brand-new thing after 1 hour Body, mouse is placed in one again, is allowed to explore 10 minutes, and with its heuristic process of camera record.Every mouse is put into Before bright plexiglass box, box bottom surface and object will be cleaned with 70% isopropanol, to remove the smell of previous mouse residual And excrement etc..After experiment terminates, mouse in stopwatch analytic statistics video and each object interactive time are utilized.

Morris water mazes

All mouse are by random number and upset order, tested with double-blind study.Water maze is by 1.2 meters of diameter Pond is made, wherein filling up white milk, temperature is controlled at 22 ± 0.5 DEG C all the time, and water maze surrounding is laid various with obvious The position prompting mark of difference, including：Red triangle, white circle and black bars.Carry out within 1st day to 10 days invisible flat Platform is trained, and platform is the white cylinder of 12 centimetres of diameter, is fixed on the centimeters of underwater about 1.5 of target quadrant center, no Work changes.Mouse is put into water from quadrant separator bar at water tank wall, is allowed to freely explore 1 minute, is not climbed in 1 minute such as Upper mounting plate, then mouse is directed on platform, then allows it to be stopped on platform 30 seconds, taken out, dry mouse hair, put back in cage. Training 4 times daily of every mouse, it is four quadrant separator bars (random) that training, which is put into position, every time, and platform immobilizes, every time Training interval 40-60 minutes.Therebetween using video camera and the movement locus of corresponding software records mouse, and record every mouse The time of platform, the note that oneself can not be looked for 60 seconds are reached every time.At the 4th day, the 7th day, the 11st day respectively when surveyed Examination, now takes platform away, and mouse is put into water from quadrant separator bar at water tank wall, and placement order is north, west, east, south, is appointed It is freely explored 1 minute, records the movement locus of mouse, and taking-up is dried mouse hair, put back in cage.All data finally by Ethovision XJ6 software are counted and analyzed.

Blue Native PAGE (SDS-PAGE) are tested

Conventionally extract membrane pellet.In 25,000g after 4 DEG C centrifuge 1 hour, its supernatant is taken, is used in combination SDS-PAGE immune-blotting methods β arr1WT therein or β arr18M and actin, to make BN-PAGE applied sample amount pair According to.And its film precipitation is then with the buffer solution containing DDM (N-dodecyl- β-d-maltoside, J424, Amresco) detergent (for gamma-secretase holoenzyme complex：0.2%DDM；For complex before NCT/APH1：0.5% DDM；50mM sodium Chloride, 50mM imidazole, 2mM 6-Aminohexanoic acid, 1mM EDTA, pH 7.0) it is resuspended, ice bath 30 Minute.25,000g after 4 DEG C centrifuge 15 minutes, take its supernatant, and every 40 μ l supernatants add the Coomassie brilliant G-250s of 5 μ l 5% and 5 The glycerine of μ l 50%.BN-PAGE each swimming lane applied sample amount is the film extract of 120 μ g holoproteins.BN-PAGE uses Native Makers (MW-GF-1000, Sigma) is used as Protein Marker product, and it includes 29KD, 66KD, 150KD, 220KD, The albumen of 430KD, 669KD 6 kinds of molecular size ranges, they carry out parallel processing with sample upon mixing, and are loaded to glue two The swimming lane of side.From 5%-15% gradient glue, with the Ruby vertical electrophoresis system of SE 600 (Amersham) under the conditions of 4 DEG C of 80V by all samples electrophoresis to glue is concentrated, then by voltage rise to 160V continue electrophoresis about 2 Hour is located to sample leading edge at 1/3rd of monoblock separation gel, changes inside groove electrophoresis liquid only to contain 1/10th coomassies Light blue G-250 buffer solution (relative to interior tank liquor before).After electrophoresis terminates, colloid is peeled off, with containing 0.1%SDS, 25mM Tris, 192mM glycine, 20% methanol buffer solution were in incubation at room temperature 10 minutes, then by albumen transferring film to PVDF On film, next step immunoblot experiment is carried out.

Glycerine gradient protein isolate matter complex

Hippocampus of Mice or Neuro-2a neuroblastoma cells homogenate buffer (5mM HEPES (pH7.4), 1mM EDTA, 0.25M sucrose and protease inhibitors) in be homogenized, 3,000g centrifugation 5 minutes, discard precipitation.Take supernatant, 100, After 000g is centrifuged 1 hour, by precipitation buffer solution (50mM Tris-HCl (pH 7.5), 2mMEDTA, 150mM NaCl, 1% CHAPSO and protease inhibitors) it is resuspended, 100,000g are centrifuged 30 minutes again, and gained supernatant is tested for glycerine gradient. The linear glycerine gradients of 10-30% contain 25mM HEPES (pH 7.2), 150mM NaCl and 0.5%CHAPSO, in 150,000g from After the heart 24 hours, gradient is divided into 20 components and is used for Western hybrid experiments and the experiment of gamma-secretase fluorogenic substrate.

Data statistic analysis

Experimental data is expressed as mean+/-standard error.β-Arrestin the expressions of tissue of patient sample use Between ANOVA analysis groups after significant difference, with the significant difference between two groups of Student-Newman-Keuls check analyses. The experiment of TgCRND8 mouse bilateral hippocampus infection slow virus uses paired t check analyses data.Other experiment the data obtaineds are then adopted With t check analysis significant differences.P<Think there is significant difference between group when 0.05.

II. embodiment

β-Arrestin the expressions of the alzheimer's disease people of embodiment 1. and transgenic models mouse rise

First, in order to study whether β-Arrestin change in alzheimer's disease, the present inventor have detected disease β-Arrestin the expressions of people's brain samples.Patient samples are by stages square according to the Braak dependent on neuropathology index Method is evaluated, and 7 groups of (being followed successively by Braak 0, I, II, III, IV and VI phase) [Braak, H.＆ are grouped into according to lesion degree Braak,E.Neuropathological stageing of Alzheimer-related changes.Acta.Neuropathol.82(4),239-259(1991)].As depicted in figs. 1 and 2, with Western hybridization sides Method detection β-Arrestin expression, finds β-Arrestin1 protein levels and Braak is proportionate (r=0.725, P by stages< 0.001), and β-Arrestin1 expression occurs in the 6th and the 7th group of (i.e. Braak V and VI phases) sample on obvious Rise (Braak V phases, P=0.008；Braak VI phases, P<0.001).As shown in figures 1 and 3, β-Arrestin2 expression Also it is proportionate by stages (r=0.508, P with Braak<0.001), and β-Arrestin2 expression in the Braak VI phases Occurs obvious rising (P=0.003) in sample.

The present inventor have detected the mRNA level in-site of β-Arrestin1 in tissue of patient by method for gene chip, find β- Arrestin1 mRNA level in-site and Braak is proportionate (r=0.584, P by stages<0.001), and in the 7th group of (i.e. Braak The VI phases) occur in sample obvious rising (P=0.048) (Fig. 4).The present inventor also demonstrated with quantitative PCR experiment β- The positive correlation (r=0.442, P=0.004) (Fig. 4) of Arrestin1 mRNA level in-site and Braak by stages.

The present inventor have detected the distribution situation of β-Arrestin1 in tissue by immunohistochemical method, hair Existing β-Arrestin1 are distributed in neuron and star spongiocyte (Fig. 5) in Healthy People and tissue of patient.

The present inventor is in alzheimer's disease transgenic models mouse it has also been found that the phenomenon that β-Arrestin expression rises. TgCRND8 mouse are the transgenic mices of APP " Swedish " and " Indiana " double-mutant, with cognition dysfunction and greatly The amyloid plaque lesion of intracerebral.The hippocampal tissue sample discovery of detection TgCRND8 mouse, β-Arrestin1 and β- Arrestin2 protein level substantially rises (β-Arrestin1, P<0.001；β-Arrestin2, P=0.049) (Fig. 6).

In order to examine whether this phenomenon is only limitted to a kind of this animal model of TgCRND8, the present inventor have detected APP/ again β-Arrestin expression in the hippocampal tissue sample of PS1 mouse.Experiment finds that β-Arrestin1 protein level exists Substantially rise (P=0.006) in APP/PS1 mouse, but β-Arrestin2 protein level does not change (Fig. 7).

With immunohistochemical assay distributions of the detection β-Arrestin1 in mouse brain tissue, it was also found that with it is wild Type mouse is compared, and β-Arrestin1 expressions in the cortex of TgCRN8 mouse and hippocampal tissue substantially rise (Fig. 8).

Experiment display, the protein expression level that β-Arrestin are organized in patient and model mice brain are obvious above Rise, and rising changes of the β-Arrestin1 compared to β-Arrestin2 is more notable, prompt β-Arrestin may and A Er Zi Haimo diseases have certain association, and β-Arrestin1 regulation and control A β are produced and gamma-secretase activity.

In order to disclose possibility effects of the β-Arrestin in alzheimer's disease, the present inventor studies β-Arrestin energy No regulation and control A β are horizontal.As shown in figure 9, detect β-Arrestin1 knock out mice cortexes respectively with enzyme-linked immunosorbent assay With the A β of the endogenous expression of hippocampal tissue₄₀With A β₄₂, find the A β in the two brain areas₄₀With A β₄₂Level is all decreased obviously.Cortex and Hippocampus is the main brain area influenceed by alzheimer's disease, expression shadow of the A β levels in the two brain areas by β-Arrestin1 Ring, prompt β-Arrestin1 to participate in regulation and control A β horizontal.

Western hybrid experiments detect APP and its piece and had no progeny discovery, the APP- in β-Arrestin1 knock out mice CTF α and APP-CTF β are horizontal to be risen, and total length APP levels do not change (Figure 10).APP-CTF α and APP-CTF β is respectively The small pieces that APP is formed after alpha-secretase enzyme and beta-secretase shearing, and the substrate of gamma-secretase.This result prompting β- The A β levels of Arrestin1 knock out mice change be influenceed by secretase caused by, rather than because APP expression Caused by level change.

The present inventor further tests the secretase proteinase activity in detection A β the way of production with fluorescent substrate activity.It is real Issue after examination and approval now, gamma-secretase activity substantially reduces in the hippocampal tissue of β-Arrestin1 knock out mice, but alpha-secretase The activity of enzyme and beta-secretase does not all change (Figure 11).This result is consistent with Figure 10 experimental result, illustrate β- Arrestin1 gene knockouts cause gamma-secretase activity to reduce, and cause substrate accumulation, in APP-CTF α and APP-CTF β levels Rise.

Cultivate whether β-Arrestin1 in cell can influence A β levels and gamma-secretase activity in vitro to examine, Tested respectively with Primary cultured neurons and Neuro-2a neuroblastoma cells.Primary cultured neurons and Neuro- β-the Arrestin expression such as Figure 12 of 2a neuroblastoma cells.

As shown in figure 13, with siRNA suppress primary neuron and the endogenous β of Neuro-2a neuroblastoma cells- Arrestin1 expression, the A β of cell₄₀With A β₄₂Level is all decreased obviously.Opposite, in primary neuron and Neuro-2a god After being overexpressed β-Arrestin1 in blastoma cell, the A β of cell₄₀With A β₄₂It is horizontal all substantially to rise (Figure 14).

Find, use after detecting the gamma-secretase activity of primary neuron and Neuro-2a neuroblastoma cells respectively The expression that siRNA suppresses cellular endogenous β-Arrestin1 causes gamma-secretase activity decrease (Figure 15).Opposite, in original After β-Arrestin1 are overexpressed in neuron and Neuro-2a neuroblastoma cells, the gamma-secretase activity of cell is all It is obvious to rise (Figure 16).

Above experimental result provides positive evidence, and β-Arrestin1 are by changing gamma-secretase activity regulation A β for display It is horizontal.

β-the Arrestin1 of embodiment 2. regulate and control gamma-secretase activity by combining APH-1AL

The present inventor's previous studies find that β 2AR can be by mediating gamma-secretase to increase into endocytic pathway after being activated Its strong proteinase activity.In order to examine whether endocytosis take part in regulating and controlling effects of the β-Arrestin1 for gamma-secretase, The present inventor with regulation and control endocytosis transitional vesicle forming process Rab5 and Rab7 dominant negative mutant (Rab5S34N and Rab7T22N endocytosis transport process) is suppressed, and it was found that Rab5S34N and Rab7T22N can not suppress β-Arrestin1 enhancings Gamma-secretase it is active (Figure 17).Make thus be excluded that endocytosis participates in regulation and control of the β-Arrestin1 for gamma-secretase Possibility.

β-Arrestin1 can be led to by combining cell surface receptor and intracellular signaling pathways molecular regulation unlike signal Road.The present inventor speculates that protein-protein interaction may take part in regulation and control of the β-Arrestin1 for gamma-secretase Effect.In order to examine this supposition, the present inventor detects the interaction of β-Arrestin1 and gamma-secretase component first.

In-vitro transcription/translation synthesis [³⁵S] mark β-Arrestin1, and PS1-NTF, PS1- of cell expression and purification CTF, Nicastrin, APH-1AL and PEN-2 are incubated altogether respectively, by Pull-down test detection β-Arrestin1 and γ- Interaction between secretase component.Autoradiograph shows that β-Arrestin1 combine APH-1AL, but does not combine PS1- NTF, PS1-CTF, nicastin or PEN-2.

With co-immunoprecipitation experiment checking β-Arrestin1 and APH-1AL intracellular interaction.As shown in figure 18, β-Arrestin1 only combine APH-1AL, but do not combine other components of gamma-secretase, as PS1-NTF, PS1-CTF, Nicastrin and PEN-2.Due to the interaction that Triton X-100 can be destroyed between each component completely, will be completely dissociated γ- Secretase, therefore β-Arrestin1 and APH-1AL interaction is special, and independent of the other of gamma-secretase Component.

The present inventor has found β-Arrestin1 and APH-1AL in cell by laser co-focusing microscopic fluorescence Coloration experiment The interior common location for forming spot distribution, and continuous vesica shape common location distribution (figure is formed in the kytoplasm of nucleus periphery 19)。

The present inventors have additionally discovered that β-Arrestin1 do not combine APP or beta-secretase (BACE1).As shown in figure 20, by exempting from Epidemic disease co-precipitation experiment finds that the APP and BACE1 being overexpressed in cell are not co-precipitated with β-Arrestin1.This result Further display, β-Arrestin1 specifically act on the gamma-secretase in A β the way of production, to another protease β-point Secrete enzyme and its substrate A PP does not interact.

In order to examine the interaction generation dynamic effects whether receptor signal can be to β-Arrestin1 and APH-1AL, The present inventor detects the effect of β-Arrestin1 and APH-1AL interactions after β 2AR are activated by co-immunoprecipitation experiment. The inventors discovered that after β 2AR activator isoproterenol (ISO) is stimulated, β-Arrestin1 and APH-1AL co-precipitation Significant change (Figure 21) does not occur.

APH-1AS is APH-1AL c-terminus variant, is produced by mRNA variable sheer.APH-1B is then APH-1A homologous protein [Francis, R.et al.aph-1and pen-2are required for Notch pathway signaling,γ-secretase cleavage of β APP,and presenilin protein accumulation.Dev.Cell3(1),85-97(2002)].As shown in figure 22, co-immunoprecipitation experiment shows APH-1AS not With reference to β-Arrestin1, the c-terminus for prompting APH-1AL is probably with reference to necessary to β-Arrestin1.

With β-Arrestin1, the Western hybridization in anti-β-Arrestin1 antibody A 1s CT immunoprecipitation HEK293T cells Experiment shows that the APH-1AL of endogenous expression is co-precipitated (Figure 23), and this co-precipitation can be by CT20 (APH-1AL carboxyl terminals The peptide fragment that 20 amino acid residues are formed) competition, illustrate that the β-Arrestin1 and APH-1AL of endogenous expression interact, And this interact is to rely on APH-1AL c-terminus.

The present inventor immunospecifically precipitates the gamma-secretase containing APH-1AL with anti-APH-1AL antibody A 1 .2, It was found that its complex proteins is horizontal and proteinase activity rises about 40% (Figure 24-25) in tissue of patient.An other side Face, the inventors discovered that β 2AR have no effect on the horizontal (figure of the gamma-secretase complex proteins containing APH-1AL after being activated 26) activity of β-Arrestin1 and β 2AR by completely different mechanism regulating gamma-secretase, is illustrated.

In order to analyze region necessary to β-Arrestin1 and APH-1AL are combined, the present inventor construct a series of β- Arrestin1 mutant (Figure 27), they are cloned into after carrier and converts HEK293T cells, carried out after cell lysis immune common Precipitation.Co-immunoprecipitation experiment shows that the mutant (1-180 and 1-240) of β-Arrestin1 carboxy-terminal domains missing can not With reference to APH-1AL (Figure 28).Moreover, 1-180 and 1-240 can not strengthen gamma-secretase activity (Figure 29).However, missing carboxylic β-Arrestin1 mutant the 1-360 of base end can combine APH-1AL and enhancing gamma-secretase activity.On the other hand, β- Arrestin1 amino terminal domain missing mutant (181-418 and 241-418) can combine APH-1AL and enhancing γ- Secretase activity.β-Arrestin1 241-360 amino acid residues form segment can combine APH-1AL and enhancing γ-point Secrete enzymatic activity.In summary, β-Arrestin1 regulate and control gamma-secretase activity be to rely on its 241-360 amino acid residue and APH-1AL interaction.

β-the Arrestin1 of embodiment 3. regulation and control gamma-secretase complex assemblings

APH-1AL is that the scaffolding protein in forming process is assembled in gamma-secretase complex, and it is for forming stable and having The gamma-secretase polyprotein macromolecule complex of activity is required [Niimura, M.et al.Aph-1contributes to the stabilization and trafficking of the γ-secretase complex throughmechanisms involving intermolecular and intramolecular interactions.J.Biol.Chem.280(13),12967-12975(2005)].APH-1AL is lacked or mutation can be led Cause gamma-secretase can not assemble and activate [Lee, S.F.et al.A conserved GXXXG motif in APH-1 is critical for assembly andactivity of the γ-secretase complex.J.Biol.Chem.279 (6),4144-4152(2004)].In order to examine whether β-Arrestin1 have impact on assembling and the activation of gamma-secretase, The present inventor is tested using glycerol gradient centrifugation and gamma-secretase complex is separated from Hippocampus of Mice extract, is used Western hybrid experiments analyze the protein complex content in each molecular weight component, real with gamma-secretase fluorescent substrate activity Test the gamma-secretase activity detected in each molecular weight component.As shown in figure 30, Western hybrid experiments detection glycerine gradient from Being found after heart separation component, β-Arrestin1 are distributed mainly in the separation component (66-150kDa) of low molecule amount, and low point Son amount protein complex (APH-1AL of about 150kDa molecular weight and immature Nicastrin dimeric complexes) is high Molecular weight protein matter complex (PS1 of more than 200-669kDa molecular weight, ripe Nicastrin, APH-1A and PEN-2 tetra- Poly- complex) cloth is not divided into significantly.It is worth noting that, molecular weight is 150kDa and 200-443kDa gamma-secretase Level distribution of the protein complex in β-Arrestin1 knock out mice hippocampal tissues compares wild-type mice hippocampus group Knit and be all decreased obviously.Consistent with this result, experiment shows that gamma-secretase is each in β-Arrestin1 knock out mice tissues The protein level of individual component molecular have dropped 20-40%, and reflecting these molecules can not assemble to form gamma-secretase complex The degradation (Figure 31) occurred afterwards.However, in β-Arrestin1 gene knockouts and wild-type mice hippocampal tissue, APP is each The no notable difference of distribution in molecular weight component.Detection proteinase activity is tested with gamma-secretase fluorescent substrate activity to find, Gamma-secretase complex with proteinase activity is mainly enriched in the 8th component, and its molecular weight is about 240kDa.Moreover, β- Gamma-secretase protein content in 8th component of Arrestin1 knock out mice tissues is also decreased obviously.Detect the 8th group Gamma-secretase protease in point compares (digestion activity of unit content protease) living and found afterwards, β-Arrestin1 gene knockouts The ratio for having no effect on gamma-secretase is lived, and illustrate the catalytic capability of gamma-secretase is not influenceed (Figure 32) by β-Arrestin1.

The present inventor analyzes β-Arrestin1 gene knockouts and wild-type mice hippocampal tissue with co-immunoprecipitation experiment In gamma-secretase complex it is horizontal, it is found that β-Arrestin1 knock out mice tissue neutralizes APH-1AL co-precipitation PS1, Nicastrin and PEN-2 level are decreased obviously, and are illustrated that gamma-secretase complex is horizontal and are occurred to decline (Figure 33), and The proteinase activity of gamma-secretase complex is also decreased obviously (Figure 34).

The present inventor is with the Neuro- for having transfected β-gal, wild-type beta-Arrestin1 or 1-240 mutant plasmids respectively 2a neuroblastoma cells have carried out similar glycerine gradient protein isolate matter complex experiment (Figure 35).Experimental result shows Show, overexpression β-Arrestin1 add the APH-1AL/Nicastrin complexs that molecular weight is about 150kDa and molecular weight is The expression of 200-443kDa complete gamma-secretase complex.However, it is overexpressed missing APH-1AL calmodulin binding domain CaMs 1-240 mutant does not have obvious effect to the expression of gamma-secretase complex.Because 1-240 mutant can not combine APH-1AL or enhancing gamma-secretase activity, this phenomenon are consistent with aforementioned result.Moreover, β-Arrestin1 are not overexpressed it also not The gamma-secretase in the 8th component is influenceed than (Figure 36) living.

Above experimental result prompt, β-Arrestin1 by influence gamma-secretase complex assembling process regulate and control γ- Secretase activity.

In order to verify conclusions, the present inventor's immunoprecipitation gamma-secretase complex from cell is analyzed (figure 37).The assembling process of gamma-secretase complex is formed from the starting of two poly- intermediates of APH-1/Nicastrin, then in conjunction with Presenilin/PEN-2 complexs form complete tetramer complex with reference to Presenilin and PEN-2 successively.Missing is early Old albumen can not be ripe by the Nicastrin caused in cell, and PEN-2 expression declines, APH-1/Nicastrin dimerization intermediates Accumulation, and complete gamma-secretase tetramer complex and proteinase activity can not be formed.Therefore, Presenilin is lacked Cell is the highly useful instrument for studying the assembling of gamma-secretase complex.The present inventor is with anti-APH-1AL antibody A 1s .2 from open country The immunoprecipitation APH-1AL and gamma-secretase of a large amount of endogenous expression in the CHAPSO extracts of raw type mice embryonic epithelial cell Enzyme other component PS1, Nicastrin and PEN-2.CHAPSO can keep the integrality of gamma-secretase complex, and Triton X-100 can then destroy gamma-secretase complex completely.Therefore, can only be from wild type mouse embryos epithelial cell with A1.2 Immunoprecipitation APH-1AL in Triton X-100 extracts, but PS1, Nicastrin or PEN-2 can not be co-precipitated.Interesting It is, by the β-Arrestin1 levels of co-immunoprecipitation more than obtaining in Triton X-100 extracts in CHAPSO extracts It is low, it is shown that β-Arrestin1 are easier to combine the APH-1AL for not forming complex.

The present inventors have additionally discovered that the mice embryonic epithelial cell Triton X-100 extractions that wild type and Presenilin knock out β-the Arrestin1 of co-immunoprecipitation are on close level in thing, show β-Arrestin1 and APH-1AL interaction independent of In Presenilin.If suppressing β-Arrestin1 expression with siRNA, wild-type cell CHAPSO is significantly reduced The level of the gamma-secretase component of immunoprecipitation in extract.This result provide evidence support β-Arrestin1 regulation and control γ- Secrete the conclusion of combined enzyme agent expression.If suppress the β-Arrestin1 of Presenilin knockout cell with siRNA Expression, the then Nicastrin for significantly reducing the co-precipitation of CHAPSO extracts are horizontal, it is shown that β-Arrestin1 participate in adjusting Control the forming process of two poly- intermediates of APH-1/Nicastrin.

Above experimental result shows β-Arrestin1 by influenceing to form APH-1AL/Nicastrin with reference to APH-1AL The process of intermediate complexes and complete gamma-secretase complex, so as to regulate and control the proteinase activity of gamma-secretase.

The expression that embodiment 4. changes β-Arrestin1 influences alzheimer's disease pathological change

Because it have been observed that the A β horizontal down-regulations in β-Arrestin1 knock out mice cerebral cortexes and hippocampal tissue, table Bright suppression β-Arrestin1 expression is likely to decrease A β generation and accumulation, and the expression for prompting to suppress β-Arrestin1 has Potential clinical value.In order to study this possibility, the present inventor enters action using transgene mouse model TgCRND8 Thing is tested, and the β in slow virus reduction Hippocampus of Mice by injecting expression β-Arrestin1 siRNAs- Arrestin1 expressions.The slow virus of injection expression LacZ siRNAs is as control in the hippocampal tissue of offside.Such as Shown in Figure 38, the GFP of slow virus expression shows expression region and level of the slow virus in hippocampus, expression β-Arrestin1 It is suitable with the slow virus expression of LacZ siRNAs, the main infection dentate fascia area neuron of hippocampus, it is distributed in cell space On dendron, also there is certain distribution in aixs cylinder.With anti-β-Arrestin1 antibody carry out immunohistochemical assay detection β- Arrestin1 expression (Figure 38), it is found that β-Arrestin1 siRNAs significantly inhibit β-Arrestin1 expression. With anti-amyloid beta antibodies 6E10 carry out immunohistochemical assay detection amyloid plaque (Figure 38), find injected expression β- In the side hippocampal tissue of the slow virus of Arrestin1 siRNAs, amyloid plaque significantly reduces.It is slow in no injection In other brain areas (such as cortex) of virus, the amyloid plaque in bilateral tissue does not have notable difference.Analysis image simultaneously counts After confirm the above find, suppressing the β-Arrestin1 expression deletions of hippocampus reduces the quantity of amyloid plaque, as right According to bi-cortical tissue in amyloid plaque quantity there is no difference (Figure 39).

In addition, the A β during the TgCRND8 mouse brain tissues of slow virus were injected by enzyme-linked immunosorbent assay measurement₄₀ With A β₄₂Found after level, the A β in the hippocampal tissue for the slow virus for having infected expression β-Arrestin1 siRNAs₄₀With A β₄₂ Level is all decreased obviously.As control, not by the A β in the cortical tissue of slow-virus infection₄₀With A β₄₂It is horizontal then there is no difference (Figure 40).

In addition, sent out after testing the gamma-secretase activity in detection TgCRND8 mouse brain tissues by fluorescent substrate activity Existing, in the side hippocampal tissue of slow virus of expression β-Arrestin1 siRNAs has been infected, gamma-secretase activity is obvious Decline.As control, by the bi-cortical tissue of slow-virus infection, gamma-secretase activity is not then without difference (figure 41)。

In order to verify the molecular mechanism of β-Arrestin1 regulation and control gamma-secretase activity in TgCRND8 mouse models, this Inventor is changed in Hippocampus of Mice by injecting expression wild-type beta-Arrestin1 and 1-240 mutant slow virus β-Arrestin1 expressions.As shown in figure 42, the GFP of slow virus expression shows expression region of the slow virus in hippocampus And level, control, wild-type beta-Arrestin1 are with the slow virus expression of 1-240 mutant suitable, main infection hippocampus Dentate fascia area neuron, be distributed on cell space and dendron, also there is certain distribution in aixs cylinder.Carried out with anti-amyloid beta antibodies 6E10 Immunohistochemical assay detection amyloid plaque (Figure 42), finds injecting the slow of expression wild-type beta-Arrestin1 In the side hippocampal tissue of virus, amyloid plaque substantially increases, and is injecting the slow virus of expression 1-240 mutant In the hippocampal tissue of side, amyloid plaque does not have significant change.In other brain areas (such as cortex) of no injecting lentivirus, Amyloid plaque in tissue does not have notable difference (Figure 42).Confirm that the above is found after analysis image and statistics, increase sea β-Arrestin1 the expression of horse adds the quantity of amyloid plaque really, as the amyloid in the cortical tissue of control Albuminous plasue quantity does not have difference (Figure 43).

In addition, the A β during the TgCRND8 mouse brain tissues of slow virus were injected by enzyme-linked immunosorbent assay measurement₄₀ With A β₄₂Found after level, the A β in the hippocampal tissue for the slow virus for having infected expression wild-type beta-Arrestin1₄₀With A β₄₂Water Flat all obvious increase, and infected the A β in the hippocampal tissue of the slow virus of expression 1-240 mutant₄₀With A β₄₂Level does not all have Change.As control, not by the A β in the cortical tissue of slow-virus infection₄₀With A β₄₂It is horizontal then there is no difference (Figure 44).

In addition, sent out after testing the gamma-secretase activity in detection TgCRND8 mouse brain tissues by fluorescent substrate activity It is existing, in the expression wild-type beta-Arrestin1 hippocampal tissue of slow virus has been infected, the obvious increase of gamma-secretase activity, and The gamma-secretase activity infected in the hippocampal tissue of the slow virus of expression 1-240 mutant does not change.As control, Not by the bi-cortical tissue of slow-virus infection, gamma-secretase activity does not have difference (Figure 45) then.

Embodiment 5. knocks out β-arrestin1 in AD model mices can effectively alleviate its learning cognition memory disorders

In order to which the influence under the conditions of verifying β-arrestin1 in vivo to AD Development process, the present inventor are small by AD models Mouse APP/PS1 transgenic mices are hybridized with β-arrestin1 knock-out mices, and obtain the APP/ of β-arrestin1 knockouts PS1 mouse.All Mouse Ages do not have significant difference, are evenly distributed on 7-9 months.APP/PS1 mouse can show year The amyloid-beta precipitation patch and learning cognition memory impairment of age correlation.Then, the present inventor visits first with novelty It is realistic to test the influence that have detected β-arrestin1 knockouts to mouse cognition and memory.As shown in Figure 46 left figures, four kinds of mouse genotypes (β arr+ /+, β arr-/-, β arr+ /+APP/PS1, β arr-/- APP/PS1) in the training stage, during in face of two same objects, Preference (respectively accounting for about 50% with two object interaction times) is not shown, shows to be familiar with two same objects on an equal basis；1 Hour after, one of object is replaced with into new object, find non-APP/PS1 transgenic mices (β arr+ /+, β arr-/-) it is right New object shows bigger Preference (reaching 70-80% with new object average interaction time), and β-arrestin1 are at non-turn Knockout in DNA murine can't have an impact to this, this (Figure 46 right figures) consistent with report before；And β arr+ /+APP/ PS1 mouse do not show Preference to new object, β-arrestin1 knock out after β arr-/- APP/PS1 mouse then with newly Object average interaction time reaches 70-80%, and One-way ANOVA are carried out to each group cognitive index (Recognition Index) Its cognition and memory defect of analysis shows is relieved (Figure 46 right figures).

The shadow to mouse study and spatial memory is further knocked out using Morris water maze laboratories detection β-arrestin1 Ring.As shown in figure 47, shown in invisible platform phase, β arr+ /+APP/PS1 mouse than wild type littermates (β arr + /+) (reach the time of platform increases lower learning ability, P<0.001), and β arr-/- APP/PS1 mouse reach platform Time significantly reduces (P=0.0145) compared to β arr+ /+APP/PS1 mouse, shows that its learning ability is significantly improved.

After invisible platform training terminates, i.e., probe trial are carried out within the 11st day to detect the spatial memory energy of mouse Power.As a result find, compared with β arr+ /+APP/PS1 mouse, β arr-/- APP/PS1 mouse are (i.e. invisible flat in target quadrant Quadrant where platform when platform is trained) in spent the more time to search platform, show the spatial memories of β arr-/- APP/PS1 mouse Ability is significantly improved (Figure 48).It is as shown in figure 42 that typical case of each group mouse in probe trial searches track.Separately Outside, APP/PS1 transgenosis and β-arrestin1 knockout have no effect on swimming distance of the mouse in water maze and swimming speed Degree, shows not influence its locomitivity (Figure 49).

In summary, β-arrestin1 are knocked out in APP/PS1 mouse can significantly improve its learning cognition and space note Recall ability, and its general motor ability can't be influenceed.

After Behaviors survey has been carried out, all mouse are condemned to death and carry out the related biochemical analysis of follow-up AD.Such as Figure 50 It is shown, typical amyloid-beta precipitation patch is have accumulated in APP/PS1 Mice brain tissues, and after β-arrestin1 are knocked out APP/PS1 Mice brain tissues in amyloid-beta precipitation patch substantially reduce.Utilize enzyme-linked immunosorbent assay (ELISA) The A β 40 and the contents of A β 42 in Mice brain tissues are detected, is as a result shown, in the APP/PS1 mouse brains after β-arrestin1 knockouts A β 40 and A β 42 content of the solubility and insolubility of hippocampus and cortical area all significantly reduce (Figure 51).

Using fluorogenic substrate enzyme activity test α in the APP/PS1 Mice brain tissues that detection β-arrestin1 knock out-, β-and The activity of gamma-secretase, it is found that after β-arrestin1 are knocked out, gamma-secretase activity significantly reduces, and α-and beta-secretase enzyme activity Property does not have significant changes (Figure 52).

It is each due to finding that β-arrestin1 can't influence gamma-secretase in β-arrestin1 knock-out mice brain tissues The expression quantity (Figure 10) of individual component.Therefore, Blue Native-Polyacrylamide GelElectrophoresis are utilized (BN-PAGE), the present inventor have detected the influence that β-arrestin1 assemble to gamma-secretase complex.In BN-PAGE, into Ripe gamma-secretase holoenzyme complex is about the band in 440KD or so.β-arrestin1 are have detected using BN-PAGE to strike After removing, the assembling of gamma-secretase in APP/PS1 Mice brain tissues.It was found that compared with β arr+ /+APP/PS1 mouse, β arr-/- The formation of gamma-secretase holoenzyme complex reduces (Figure 53) in APP/PS1 Mice brain tissues.It is worth noting that, β- Arrestin1 knockout does not destroy the formation of gamma-secretase completely, implies that β-arrestin1 are only combined and be have adjusted one Complex before partial NCT/APH-1, and can in vivo under the conditions of regulate and control gamma-secretase activity and holoenzyme complex group Dress.

Embodiment 6., which intervenes β-arrestin1/APH-1 interactions, can reduce A β generations and gamma-secretase activity

According to APH-1AL and APH-1B intracellular ring and carboxyl terminal sequence, the present inventor has synthesized 8 corresponding small peptides (Figure 54), its sequence are as shown in figure 50.All small peptides all use FITC fluorescence labelings, and the TAT- of 11 amino acid in connection Tag, TAT-tag have been reported can help small peptide to enter cell through cell membrane, and cell is being marked by FITC-TAT tag After small peptide processing, the CDCC (Figure 56) that small peptide has is not observed.

With the treated cell for expressing β-arrestin1 and APH-1AL or APH-1B of FITC-TAT-tagged small peptides Afterwards, the interaction for only having ACT and BCT small peptides to suppress β-arrestin1 and APH-1AL (APH1-AL) simultaneously is found, and The small peptide of other intracellular rings can not then influence it and interact (Figure 57 left figures)；In β-arrestin1 and APH-1B (APH1- B in interaction), also observed identical result (Figure 57 right figures), imply β-arrestin1 and APH-1AL or APH-1B interaction is to rely on APH-1AL and APH-1B carboxyl terminal, and this is not tied with β-arrestin1 in Figure 12 The result for closing APH1-AS is consistent.Interacted in addition, BCT small peptides can suppress β-arrestin1 and APH-1AL, ACT can Interacted with suppressing β-arrestin1 and APH-1B, and β-arrestin18M point mutation bodies do not combine APH-1AL simultaneously And APH-1B, show that β-arrestin1 may be interacted by itself same structure domain with APH-1AL and APH-1B.

In order to detect, FITC-TAT-tagged small peptides produce to A β and the influence of gamma-secretase activity, the present inventor use FITC-TAT-tagged small peptides have handled APPswe HEK293 cells, and (HEK293 of expression APP swedish mutant surely turns Cell line；After transfecting APP swedish mutant by HEK293 cell lines, screen to obtain using puromycin puromycin), It was found that only ACT, BCT small peptide can significantly reduce A β generations (Figure 58).Tested by fluorogenic substrate enzyme activity, discovery only ACT, BCT small peptides can significantly reduce gamma-secretase activity, and not have an impact (Figure 59) to α-or beta-secretase activity, other born of the same parents Inner ring small peptide on α-, β-and gamma-secretase activity all do not influence.

In order to further confirm above-mentioned conclusion, the present inventor, which have detected, can suppress β-arrestin1/APH-1 phase interactions Adjustment effect of ACT, BCT small peptide to complex before gamma-secretase holoenzyme complex and NCT/APH-1.As shown in figure 60, After HEK293 cells are handled by ACT, BCT small peptide, relative to control group (culture medium and AL5 small peptides are handled), gamma-secretase holoenzyme Complex, which is formed, to be reduced；β-arrestin1 double knock-out mice the embryo fibroblasts of PS1/2 have been overexpressed, it is short by ACT, BCT After peptide processing, formed relative to complex before control group (culture medium and AL5 small peptides are handled) NCT/APH-1 and reduce (Figure 61), and And ACT, BCT small peptide can effectively suppress the common migration of complex before β-arrestin1 and NCT/APH-1.In summary, β- Regulations of the arrestin1 to gamma-secretase activity and holoenzyme assembling be by adjust the assembling of complex before its NCT/APH-1 come Realize, and be to rely on β-arrestin1 and APH-1 interaction.

The sedimentation of embodiment 7. screens medicine

The method of reference as described in example 2 above, in-vitro transcription/translation synthesis [³⁵S] mark β-Arrestin1, and carefully The APH-1AL of cellular expression purifying is incubated altogether, forms the system of β-Arrestin1 and APH-1AL interactions.Following handle is set Group：

Control group：The system of β-Arrestin1 and the APH-1AL interaction of candidate substances is not given, is establishing system Handled immediately with candidate substances afterwards；With

Test group：Give the system of β-Arrestin1 and the APH-1AL interaction of candidate substances.

Tested by Pull-down in detection test group and control group between β-Arrestin1 and gamma-secretase component Interaction, APH-1AL situation is combined using autoradiography observation β-Arrestin1.If relative to control group, test group Middle β-Arrestin1 and APH-1AL interactions are having decrease statistically significantly, then illustrate that candidate substances are preventions or controlled Treat the potential material of Alzheimer's disease.

The co-immunoprecipitation method of embodiment 8. screens medicine

With reference to method as described in example 2 above, the β-Arrestin1 that cotransfection FLAG is coupled in HEK293T cells (β arr1) and HA are coupled APH-1A, form the system of β-Arrestin1 and APH-1AL interactions.Following treatment group is set：

Control group：The system of β-Arrestin1 and the APH-1AL interaction of candidate substances is not given, is establishing system Handled immediately with candidate substances afterwards；With

Test group：Give the system of β-Arrestin1 and the APH-1AL interaction of candidate substances.

Cell lysis after transfecting 48 hours, with the Ago-Gel immunoprecipitation β-Arrestin1 for being coupled anti-FLAG antibody. The situation that Western hybrid experiments observation APH-1AL is co-precipitated.If relative to control group, in test group β-Arrestin1 with APH-1AL interactions are having decrease statistically significantly, then it is prevention or treatment Alzheimer's disease to illustrate candidate substances Potential material.

The drug screening of the micromolecular compound of embodiment 9.

Using X ray crystal diffraction technology, according to SEQ ID NO:241-360 amino acids sequences Design is simulated in 1 Protein three-dimensional structure, using high flux virtual screening technology, using the compound in SPECS compound databases as candidate Matter.By preliminary screening and SEQ ID NO:The albumen of 241-360 amino acids sequence has the chemical combination that specific binding acts in 1 Thing is sorted out, and as a result obtains some and thinks potential effective candidate compound through primary dcreening operation.

β-arrestin1 and APH-1AL interaction is reappeared using Split-TEV Reporter Systems quantification.Will β-arrestin1 and APH-1AL nitrogen ends with TEV enzymes respectively（NTEV）With the carboxyl terminal of TEV enzymes（CTEV）Coupling, β- Arrestin1 and APH-1AL, which interacts, make it that NTEV and CTEV is close and plays complete TEV enzymatic activitys and starts downstream report Gene signal, as a result such as Figure 62.This Reporter System is used directly for disturbing β-arrestin1 and APH-1AL mutual The high flux screening of the micromolecular compound of effect.

Discuss

The present invention is found that expressions of the β-Arrestin in alzheimer's disease human cerebral tissue rises first, and And also occurs similar phenomenon in transgene mouse model.However, β-Arrestin are only changing with serious pathological Occur expression rising in tissue of patient sample, prompt β-Arrestin expression to occur not produce in early days in disease Obvious change, but influenceed to rise by X factor after alzheimer's disease development enters the later stage course of disease.Enter The research of one step finds that increase β-Arrestin1 expression can increase the A β levels of cell.Speculate accordingly, in A Er In the later stage process of Zi Haimo diseases, it is possible to create a kind of feedback mechanism by β-Arrestin1 mediations participates in regulation and control A Erzi The development of the silent disease in sea, is risen increase A β generation by β-Arrestin1 expressions caused by alzheimer's disease, accelerates disease The development process of disease.

The present inventor further study β-Arrestin1 by strengthening phenomenon caused by gamma-secretase activity increase A β And its molecular mechanism.It was found that β-Arrestin1 regulation and control gamma-secretase activity be to rely on APH-1AL combine after influence γ-point Secrete the realization of combined enzyme agent assembling process.

Gamma-secretase complex is made up of Presenilin, Nicastrin, APH-1 and PEN-2.Presenlin has egg White enzymatic activity, it is main research object.Nicastrin is the substrate identification component of gamma-secretase.APH-1 γ- Scaffolding protein effect is played in secretion combined enzyme agent assembling process.PEN-2 starts the compound body maturation of gamma-secretase and the work(of activation Can, and Presenilin aminoterminal/c-terminus dimer in stable gamma-secretase.

Recent research is found that many Presenilins/gamma-secretase conjugated protein, some of which molecule, such as CD147, phospholipase D1 and TMP21, be also considered to be gamma-secretase optional component and negative regulation γ-point Secrete enzymatic activity.Recently, it has been found that the protein of endoplasmic reticulum 1 (Rer1p) by with APH-1 competition bindings Nicastrin and negative regulation gamma-secretase activity.But the APH-1 conjugated proteins having now been found that are considerably less, this respect Research or a tera incognita.

The inventors discovered that β-Arrestin1 are APH-1 conjugated proteins.But β-Arrestin1 do not combine it The required component of its three kinds of gamma-secretase, is also not present in gamma-secretase complex.Therefore, β-Arrestin1 are not The component of gamma-secretase.β-Arrestin1 accelerate gamma-secretase by strengthening the formation of APH-1AL/Nicastrin intermediates Combined enzyme agent assembles.This novel regulatory mechanism has been deepened the present inventor and answered for this complicated protein of gamma-secretase Fit understanding, for study provided in a manner of the signal path molecular regulation gamma-secretase that β-Arrestin1 are representative it is new Point of penetration.

β is found that in the work of the present inventor in the past₂AR is by strengthening phenomenon caused by gamma-secretase activity increase A β. And it was found that this phenomenon depends on β caused by activator₂AR endocytosis and γ-secretas are to late period endosome/lysosome Transhipment.β₂AR recruits β-Arrestin, β-Arrestin after being activated by activator and further recruits endocytosis related protein, shape Into acceptor endocytic mechanism.Knock out or mutation β-Arrestin can suppress acceptor endocytosis, cause β₂AR can not activate gamma-secretase Enzyme.The studies have shown that β of the present inventor₂AR influences gamma-secretase by combining Presenilin 1.Because β-Arrestin1 are not tied Presenilin 1 is closed, so being not involved in mediating β₂AR and gamma-secretase direct interaction.β-Arrestin1 mutant 181- 418th, 241-418 and 241-360 deletion recipients binding structural domain can not combine β₂AR, but still be able to combine APH-1AL and increasing Strong gamma-secretase activity.β-Arrestin1 mutant the 1-360 and 241-360 for lacking carboxyl terminal can not be related with reference to endocytosis Protein clathrin and adaptor protein 2, they still are able to combine APH-1AL and strengthen gamma-secretase activity.This One result shows that β-Arrestin1 can not possibly utilize the mechanism of g protein coupled receptor by recruiting endocytosis related protein Directly guiding gamma-secretase enters endocytic pathway so as to cause gamma-secretase to be transported into endosome/lysosome system.

To sum up, it can be found that g protein coupled receptor and β-Arrestin1 present two kinds of completely different regulation and control γ-point The mode of enzyme is secreted, also show complicated and fine modulated mechanism caused by the gamma-secretase complexity of its own.By with The interaction of gamma-secretase and common endocytic mechanism, it is small in the cell that the g protein coupled receptor of activation changes gamma-secretase Distribution on bubble, the proteinase activity of gamma-secretase is influenceed with a kind of quick and journey in short-term the mode of action.On the other hand, β- Arrestin1 is then by directly changing the assembling process of gamma-secretase complex and the level of active gamma-secretase, with one kind Slowly the mode of action with long time-histories influences the proteinase activity of gamma-secretase.Both modes are present in into the cell simultaneously, can The different response modes that neuron can be reflected when being stimulated by different extracellular signals.The current Preliminary Study of the present inventor is taken off Show that g protein coupled receptor and β-Arrestin1 are acted in alzheimer's disease pathogenesis, it is shown that be coupled G-protein Acceptor and β-Arrestin1 develop into the possibility of alzheimer's disease treatment novel targets.

All it is incorporated as referring in this application in all documents that the present invention refers to, it is independent just as each document It is incorporated as to make various changes or modifications the present invention with reference to member, these equivalent form of values equally fall within right appended by the application Claim limited range.

Claims (7)

1. a kind of purposes of the material for the combination for reducing beta-protein inhibitor 1 and APH-1 albumen, prevents, alleviates or controls for preparing Treat the composition of nerve degenerative diseases；The material of the combination for reducing beta-protein inhibitor 1 and APH-1 albumen is selected from：(i) ammonia Base acid sequence such as SEQ ID NO:The protein fragments of 235-265 positions in 33；Or (ii) amino acid sequence such as SEQ ID NO:34 In 234-257 positions protein fragments；

Described nerve degenerative diseases are alzheimer's diseases；Described APH-1 albumen is APH-1AL albumen.

2. purposes as claimed in claim 1, it is characterised in that the described combination for reducing beta-protein inhibitor 1 and APH-1 albumen It is the interaction for weakening beta-protein inhibitor 1 and APH-1 albumen.

3. a kind of protein fragments of separation, are selected from：

(a)SEQ ID NO:The protein fragments of 235-265 amino acids sequence in 33；Or

(b)SEQ ID NO:The protein fragments of 234-257 amino acids sequence in 34.

4. protein fragments as claimed in claim 3, it is characterised in that its N-terminal or C-terminal also include trans-activator.

5. protein fragments as claimed in claim 4, it is characterised in that described trans-activator has SEQ ID NO:35 Shown amino acid sequence.

A kind of 6. polynucleotides of separation, it is characterised in that any described eggs of described polynucleotide encoding claim 3-5 White tiles section.

7. a kind of screen prevention, alleviate or the method for the potential material for the treatment of nerve degenerative diseases, described method is not to examine For the purpose of disconnected or treatment, methods described includes：

(1) candidate substances are contacted with containing beta-protein inhibitor 1 with the system of APH-1 albumen；

(2) influence of combination of the candidate substances to beta-protein inhibitor 1 and APH-1 albumen is detected, and compared with control group, wherein institute The control group stated is the system containing beta-protein inhibitor 1 Yu APH-1 albumen for not adding the candidate substances；

If the candidate substances suppress the combination of beta-protein inhibitor 1 and APH-1 albumen, show that the candidate substances are preventions, delayed The potential material of solution or treatment nerve degenerative diseases；

Wherein described nerve degenerative diseases are alzheimer's diseases；Described APH-1 albumen is APH-1AL albumen.