Abstract

Introduction

The first novel paired helical filament (PHF)-tau targeting positron emission tomography (PET) tracers, such as [F-18]-AV-1451 (previously known as T807), have been demonstrated to detect tangle pathology in vivo,¹ opening an exciting opportunity of using them as a potential surrogate marker to measure tau-bearing neurofibrillary lesions in the brain by in vivo neuroimaging. An anticipated barrier to progress in the field of in vivo tau imaging is the need for tracers with high binding selectivity for tau lesions relative to β-amyloid (Aβ) plaques and other amyloid-like proteins with a β-pleated sheet conformation. A recent study suggested that PET tracer [F-18]-AV-1451 is highly selective for tau over Aβ with negligible off-target binding.² Xia and colleagues suggested that the autoradiography pattern of [F-18]-AV-1451 correlates better with PHF-tau immunoreactivity than with Aβ immunostaining.¹ Emerging data from in vivo human [F-18]-AV-1451 PET scans showed significantly higher AV-1451 retention across frontal, parietal and temporal cortices and the hippocampus in Alzheimer’s (AD) patients when compared to individuals with mild cognitive impairment (MCI) and cognitively normal control subjects.³ A comprehensive postmortem validation of this novel PET tracer is critical for determining its usefulness for antemortem diagnosis and staging of AD and other tauopathies, and to understand exactly what the retention of [F-18]-AV-1451 in PET studies means in terms of neuropathological substrate.

The presence of brain aggregates of abnormally hyperphosphorylated tau in the form of filaments and neurofibrillary tangles (NFT) is a defining pathological hallmark of AD and other tauopathies, such as some types of frontotemporal lobe degeneration (FTLD)-tau including Pick’s disease (PiD),⁴ progressive supranuclear palsy (PSP)⁵ and corticobasal degeneration (CBD).⁶ While these various disorders differ in terms of the regional distribution of tau deposits in the brain, there are other important differences as well which are potentially associated with altered ability to be identified by [F-18]-AV-1451. In these disorders, tau aggregates differ in their morphology: NFT, neuropil threads and dystrophic neurites associated with neuritic plaques in AD; Pick bodies in PiD; globose tangles, coiled bodies and tufted astrocytes in PSP; and astrocytic plaques, coiled bodies and neuropil threads in CBD. In addition, these diseases differ according the tau isoform composition of the aggregates: a mixture of three (3R) and four repeat (4R) tau isoforms in AD; 3R in PiD; and 4R in both PSP and CBD. Finally, the ultrastructural characteristics of tau filaments differ across diseases: PHF in AD versus mainly straight filaments in PiD, PSP and CBD.^7–9

Because AV-1451 was initially screened to specifically detect tau in the form of PHF,¹ we predicted that this tracer would exhibit high binding affinity and selectivity for PHF-tau lesions relative to non-PHF-tau lesions, Aβ deposits and α-synuclein and transactive response DNA binding protein-43 (TDP-43) aggregates. To validate the site/s of AV-1451 binding and determine whether there is off-target binding, we carefully examined the regional and substrate-specific autoradiograhic patterns of [F-18]-AV-1451 by using legacy postmortem brain, retina and skin tissue samples. Cases with a definite pathological diagnosis of AD, FTLD-tau (PiD, PSP, CBD), FTLD-TDP-43, CAA, synucleinopathies (dementia with Lewy bodies (DLB) and multiple system atrophy (MSA)), metastatic melanoma, brain hemorrhages, superficial siderosis and controls free of pathology were included in this study. In vitro binding levels of [H-3]-AV-1451 using homogenates prepared from the same brain tissue material were also analyzed. Our results suggest that AV-1451 holds promise as a surrogate marker for the detection of brain PHF tau-bearing neurofibrillary lesions by in vivo imaging in the setting of some off target non-neurodegenerative lesion binding as well.

Methods

Results

A summary of the demographic characteristics of the postmortem brain samples studied is shown in Table 1.

[F-18]-AV-1451 phosphor screen autoradiography

Phosphor screen autoradiography experiments using [F-18]-AV-1451 (aliquots from material prepared from in vivo imaging on the same day) revealed strong binding in the hippocampal formation and EC, frontal, temporal, parietal and occipital cortices from brain slices containing NFT in AD cases (Figure 1A). Of note, binding was almost completely blocked after incubating the slides with 1μM unlabeled AV-1451, demonstrating the specificity of the signal. No signal was detected in the white matter or other non-tangle containing regions in AD cases, with the exception of the substantia nigra (Figure 2E), as discussed further below. Slides from control brains free of neurofibrillary pathology showed no AV-1451 signal in any of the cases analyzed (Figure 1B), except for the substantia nigra (Figure 2E) as in AD and the rest of the cases analyzed. No AV-1451 binding was detected in the cerebellum (Figure 1A), a region that is typically used in neuroimaging studies as a reference region devoid of specific binding, or in the basal ganglia (Figure 1B), in any of the cases studied. We also noticed the absence of detectable AV-1451 binding in brain slices containing non-PHF tau lesions from PiD (Figure 1C), PSP (Figure 1D) and CBD (Figure 1E) cases when compared to control brains. Of note, incidental AV-1451 binding to age-related PHF-tangles in the entorhinal cortex was noted in these non-AD cases with NFT Braak stage II, and served as an internal positive control as it is shown in the PSP case illustrated in Figure 1D. Similarly, we could not detect labeling in the brains of aged rTg4510 mice that contain innumerable P301L (4R) tau neurofibrillary lesions (Figure 1F). The absence of detectable labeling of tau lesions in the mouse model and in the non-AD tauopathies, which share the feature of primarily straight filaments of tau rather than PHF, suggests that AV-1451 has a higher affinity and specificity for PHF-tau. Importantly, brain slices from the D23N Iowa APP mutation carrier¹² displaying very severe CAA pathology completely lacked [F-18]-AV-1451 autoradiographic signal (Figure 2A) and were indistinguishable from control brain slices. This favors the idea that AV-1451 binds with much stronger affinity and selectivity to PHF-tau containing slices than Aβ lesion-containing ones, as it had been suggested in the previous study by Xia and colleagues.¹ Brain slices containing TDP-43 inclusions in FTLD-TDP-43 cases (Figure 2B) and α-synuclein lesions in DLB (Figure 2C) and MSA (Figure 2D) cases, respectively, also lacked detectable AV-1451 binding on phosphor screen autoradiography in all regions studied, with the exception of the substantia nigra (Figure 2E), as noted in all other cases analyzed including controls, and of the incidental age-related PHF-tangle pathology confined to the entorhinal cortex and limbic regions in cases with NFT Braak stages II–III. Of note, this universal and strong binding of [F-18]-AV-1451 to midbrain slices containing substantia nigra, regardless of the presence of underlying tau aggregates, was blocked almost completely when incubating the slides with 1μM unlabeled AV-1451 (Figure 2E), strongly pointing to the substantia nigra as a brain region where AV-1451 has a robust off-target binding. In addition to the substantia nigra, and to a lesser extent, some [F-18]-AV-1451 binding was also noticed in brain slices containing parenchymal and subarachnoidal hemorrhages (Figure 4F–G), suggesting the possibility of some AV-1451 binding also to this type of lesion. No labeling could be detected however in a case with superficial siderosis with iron deposition in the setting of chronic vascular leakage of red blood cells (Figure 4E).

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Figure 1

[F-18]-AV-1451 phosphor screen images of brain slices from an AD case (A), a control (B), a PiD case (C), a PSP case (D), a CBD case (E) and a rTg4510 mouse brain (F). A strong [F-18]-AV-1451 binding was observed in cortical regions containing tangles and PHF-neurites from AD brains; the signal was blocked by adding unlabeled AV-1451 (A) and no [F-18]-AV-1451 signal was detected in the cerebellum (A). Slices from a control case free of pathology did not show detectable [F-18]-AV-1451 binding (B). [F-18]-AV-1451 binding was not detectable either in non-PHF tau-containing slices from a PiD case (C), a PSP case (D), a CBD case (E) and an rTg4510 mouse brain (F). Note that [F-18]-AV-1451 binding was confined to incidental age-related PHF-tangle pathology in superficial layers of EC in a PSP case (asterisk, D). Abbreviations: AD = Alzheimer’s disease; CBD = corticobasal degeneration, CTL = control, EC = entorhinal cortex, HPC = hippocampus, NFT = neurofibrillary tangles; PiD = Pick’s disease, PHF = paired helical filaments, PSP = progressive supranuclear palsy. Scale bar= 1 cm.

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Figure 2

[F-18]-AV-1451 phosphor screen images of brain slices from a CAA carrier of the D23N Iowa APP mutation (A), a FTLD-TDP-43 case (B), a DLB case (C), a MSA case (D) and midbrain containing slices from control, AD, PSP and DLB cases (E). No [F-18]-AV-1451 binding was detected in slices containing CAA lesions (A), TDP-43 inclusions (B), Lewy bodies (C) and glial α-synuclein inclusions (D). Strong [F-18]-AV-1451 signal was observed in the region corresponding to the substantia nigra in all cases studied regardless their pathological diagnosis. (E). Abbreviations: AD = Alzheimer’s disease, APP = amyloid precursor protein, CAA = cerebral amyloid angiopathy, CTL = control, DLB = dementia with Lewy bodies, FTLD = frontotemporal lobar degeneration, MSA = multiple system atrophy, PSP = progressive supranuclear palsy, TDP-43 = TAR DNA binding protein 43. Scale bar= 1 cm.

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Figure 4

[F-18]-AV-1451 phosphor screen and nuclear emulsion autoradiography images of slices containing substantia nigra in a PSP case (A), skin (B) and retinal pigment epithelium (C) in an AD case, metastatic melanoma (D), cerebellum in a superficial siderosis case (E), and parenchymal (F) and subarachnoid (G) hemorrhagic lesions. [F-18]-AV-1451 phosphor screen autoradiography images are displayed in A–E, left and middle panels, and F–G. [F-18]-AV-1451 nuclear emulsion autoradiography images are displayed in A–E, right panel. Strong [F-18]-AV-1451 binding was observed in neuromelanin-containing neurons of the substantia nigra (A), skin melanocytes (B), neuromelanin containing granules in the retinal pigment epithelium (C) and malignant melanocytes from a metastatic melanoma (D). No detectable [F-18]-AV-1451 binding was observed in association with hemosiderin deposits in a superficial siderosis case (E). Mild [F-18]-AV-1451 binding was noticed in association with intraparenchymal (asterisk, F) and subarachnoid (asterisk, G) hemorrhagic lesions. Abbreviations: AD = Alzheimer’s disease, PSP = progressive supranuclear palsy. Scale bar: 1cm (phosphor screen autoradiographic images), 50 μm (A, C, D right panels), 25 μm (B, E, right panels).

[F-18]-AV-1451 nuclear emulsion autoradiography

In order to further confirm the above observations and to obtain cellular resolution, we dipped adjacent brain slices to those used in phosphor screen autoradiography in a photographic nuclear emulsion. When slices are developed, silver grains struck by positrons emitted during [F-18] nuclear decay are visualized allowing the identification of [F-18]-AV-1451 labeled lesions below the silver grains of the emulsion by optical microscopy.

Using this method, we confirmed that tissue sections from AD cases containing hippocampal formation, EC, and frontal, parietal, temporal and occipital cortices displayed a strong and selective concentration of silver grains, reflecting underlying [F-18]-AV-1451 binding in the grey matter with a negligible presence of silver grains in the white matter (Figure 3A left panel). The distribution of silver grains in those areas closely mirrored the pattern of PHF-1 immunostaining reactivity on adjacent slices with the highest amount of silver grains being observed in cortical layers III and V, as one would expect based on the well known robust density of tangle lesions in those cortical layers in AD^{23, 24} (Figure 3A middle panel), rather than the more scattered plaque distribution pattern revealed by Aβ immunostaining (Figure 3A right panel). [F-18]-AV-1451 nuclear emulsion autoradiography followed by immunostaining with PHF-1 or Aβ antibodies on the same brain slices was also performed. This further confirmed that [F-18]-AV-1451 labeled lesions below the silver grains of the emulsion were PHF-tau containing lesions, including classic NFT (Figure 3B left panel), both intra and extraneuronal e.g. “ghost tangles” (Figure 3B middle panel), and PHF-tau containing neurites (Figure 3B right panel), but not Aβ plaques (Figure 3C) or amyloid-laden vessels (Figure 3D).

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Figure 3

[F-18]-AV-1451 nuclear emulsion autoradiography photomicrographs of brain slices from an AD case (A–C), a CAA carrier of the D23N Iowa APP mutation (D), a rTg4510 mouse (E), a PiD case (F), a PSP case (G) a CBD case (H), a DLB case (I), a MSA case (J) and a FTDL-TDP-43 case (K). Combined [F-18]-AV-1451 nuclear emulsion autoradiography followed by immunostaining with appropriate antibodies (B right and left panels, C–K) or H&E counter staining (B middle panel). The primary antibodies used were PHF-1 (kind gift of Dr. Peter Davies, A middle panel, B left and right panels, and E–H), anti-Aβ (A right panel, C and D), anti α-synuclein (I and J) and anti-TDP-43 (K). [F-18]-AV-1451 nuclear emulsion autoradiography showed a strong cortical accumulation of silver grains in cortical layers III and V in AD brains (A, left panel) mirroring the pattern on adjacent slices of tau PHF-1 immunostaining (A, middle panel) but not the Aβ immunostaining (A, right panel). Accumulation of silver grains from the nuclear emulsion colocalized with PHF-1 stained tangles (solid arrows, B, left panel), both intraneuronal (solid arrows) and extraneuronal (black arrowheads, B middle panel) and PHF-tau containing neurites surrounding plaques in AD brains (empty arrows, B, right panel). No detectable accumulations of silver grains were observed in association with diffuse plaques in AD (asterisk, C), amyloid-containing vessels in CAA (empty arrows, D), tangles in Tg4510 mouse brains (empty arrows, E), Pick bodies in a PiD case (empty arrows, F), astrocytic plaques in a PSP case (empty arrows, G) or coiled bodies and globose tangles in a CBD case (empty arrows, H). No silver grains were detected in association with Lewy bodies in a DLB case (empty arrows, I), α-synuclein inclusions in a MSA case (empty arrows, J) or TDP-43 inclusions in a FTLD-TDP-43 case (empty arrows, K). Abbreviations: Aβ = amyloid-β, AD = Alzheimer’s disease; APP = amyloid precursor protein, CAA = cerebral amyloid angiopathy, CBD = corticobasal degeneration, DLB = dementia with Lewy bodies, FTLD = frontotemporal lobar degeneration, H&E = hematoxylin and eosin, MSA = multiple system atrophy, PHF = paired helical filament, PiD = Pick’s disease, PSP = progressive supranuclear palsy, TDP-43 = TAR DNA binding protein 43. Scale bar = 2 mm (A), 20 μm (B), 50 μm (C–K).

In agreement with the results obtained by phosphor screen autoradiography, brain slices from control brains free of neurofibrillary pathology did not show detectable accumulations of silver grains in any of the cortical brain regions examined (data not shown). Similarly, a negligible number of silver grains were observed colocalizing with tau containing lesions in rTg4510 mouse brains (Figure 3E) and in PiD (Figure 3F), PSP (Figure 3G) and CBD (Figure 3H) cases, further strengthening the relatively higher affinity and selectivity of AV-1451 for tau lesions primarily composed of PHF (as seen in AD) compared to those mainly made of straight filaments of tau in non-AD tauopathies and rTg4510 mice. In addition, no silver grains were observed colocalizing with α-synuclein (Figure 3I–J) or TDP-43 containing inclusions (Figure 3K).

Importantly, neuromelanin-containing neurons in the substantia nigra pars compact a (Figure 4A), melanin-containing retinal pigment epithelium (RPE) cells (Figure 4C), as well as leptomeningeal melanocytes (not shown), skin melanocytes (Figure 4B) and malignant melanocytes in a metastatic melanoma (Figure 4D) consistently demonstrated robust concentration of silver grains independent of any tau aggregates. This identified a prominent off-target binding activity of AV-1451 to both neuromelanin and melanin-containing cells. To a lesser extent, a slight AV-1451 signal was also seen in association with acute and subacute parenchymal (Figure 4F) and subarachnoid (Figure 4G) hemorrhages, pointing to some additional [F-18]-AV-1451 off-target binding in these lesions. Interestingly, no [F-18]-AV-1451 signal was detected in the superficial siderosis case containing multiple hemosiderin deposits (Figure 4E) suggesting that the age of the hemorrhages (e.g. acute/subacute vs chronic) could be a factor in the observed variable range of tracer’s binding to these lesions.

Discussion

To understand exactly what [F-18]-AV-1451 PET positivity means in terms of neuropathological substrate, we have carefully characterized the autoradiographic and in vitro binding patterns of this novel tracer in postmortem tissue samples representing a broad spectrum of neurodegenerative pathologies. Our observations, derived from combined [F-18]-AV-1451 sensitive autoradiography and immunohistochemistry and from [H-3]-AV-1451 in vitro binding assays, suggest that AV-1451 strongly binds to lesions primarily made of tau in the form of PHF in tissue samples from brains with AD (intra- and extraneuronal tangles and PHF-tau containing neurites), but does not bind to a significant extent to neuronal and glial inclusions mainly composed of straight tau filaments in tissue samples from brains with non-AD tauopathy lesions or to Aβ, α-synuclein or TDP-43-containing lesions. Importantly, a pronounced off-target binding of AV-1451 to neuromelanin- and melanin-containing cells and, to a lesser extent, to acute and subacute brain hemorrhagic lesions, was identified in this study suggesting that these substrates represent non-specific binding sites of this tracer, and this should be taken carefully into account when interpreting the AV-1451 retention patterns observed by in vivo neuroimaging.

The PET tracer AV-1451 originally came out of compound library screens that used human brain slices laden with PHF-tau tangles as the binding target.¹ Early studies suggested that this compound had more than 29-fold higher affinity for PHF-tau over Aβ, with non-detectable binding to off-target proteins.² The first emerging data from in vivo human [F-18]-AV-1451 PET scans pointed to a significantly higher AV-1451 retention the brain of patients with AD when compared to individuals with MCI and cognitively normal control subjects.³ In agreement with these observations, our recent in vivo studies based on [F-18]-AV-1451 PET and amyloid-β PET using Pittsburgh Compound B (PiB) in older clinically normal individuals and symptomatic patients with MCI or mild AD dementia have also shown high cortical [F-18]-AV-1451 retention in patients with MCI and AD dementia compared to clinically normal controls, suggesting that AV-1451 could have value as a biomarker that reflects tau pathology in AD brains (Johnson K, Becker A, Sepulcre J et al. Initial experience with F18 T807-Tau PET imaging along the spectrum of normal aging and Alzheimer’s disease dementia. Presented in: Human Amyloid Imaging. Miami, Florida, 2014).

This compound is now quickly making its way into use in clinical research including secondary prevention trials in AD like the A4 trial.²⁵ Thus, a careful study to validate the sensitivity and specificity of this novel tracer, and to exactly define the underlying pathological burden responsible for in vivo [F-18]-AV-1451 PET retention is particularly important at this time. Based on the observations above, we predicted that AV-1451 would bind with high affinity and specificity to tau assemblies in the form of tangles of PHF and would show little or no binding affinity to straight filaments lesions or to other abnormal protein aggregates primarily made of Aβ, α-synuclein or TDP-43. To unequivocally confirm or disprove this hypothesis we used three different methods ([F-18]-AV-1451 phosphor screen autoradiography, [F-18]-AV-1451 nuclear emulsion autoradiography and [H-3]-AV-1451 in vitro binding assays) applied to the study of a series of autopsy samples from individuals with a definitive diagnosis of AD, CAA, PiD, PSP, CBD, FTLD-TDP-43, DLB, MSA and control brains free of neurodegenerative pathology.

Our results from phosphor screen autoradiography and nuclear photographic emulsion, both using [F-18]-AV-1451, confirmed that while AV-1451 avidly bound to PHF-tangle containing slices from AD brains, it failed to label CAA, α-synuclein and TDP-43 lesion-containing slices as well as slices from control brains free of pathology. Importantly, [F-18]-AV-1451 also failed to label tau-containing lesions in slices from non-AD tauopathy brains and from rTg4510 mice. Thus, our data suggest that this tracer has greater selectivity for PHF-tau over tau aggregates that are primarily made of straight filaments in non-AD tauopathies. The unequivocally positive labeling in these autoradiographic experiments of incidental age-related PHF-tangles in the entorhinal cortex (NFT Braak stage II) of human non-AD brains, served as an optimal internal positive control. The regional and laminar autoradiographic patterns of distribution of [F-18]-AV-1451, as revealed by the combination of autoradiography using a fine grain nuclear emulsion and immunohistochemistry, closely matched those of classic PHF-tangles in AD brains^{23, 24} demonstrating very low nonspecific binding to the whiter matter and a strong labeling in particular of cortical layers III and V. We confirmed that [F-18]-AV-1451-labeled lesions below the silver grains of the emulsion were PHF-tau containing lesions, including neurofibrillary tangles, both intra and extraneuronal e.g. “ghost tangles” and PHF-tau containing neurites decorating the periphery of dense cored plaques, further pointing to these lesions as the main pathological substrate of [F-18]-AV-1451 signal detected by in vivo neuroimaging. The microscopic examination of diffuse plaques, CAA, non-PHF tau lesions, α-synuclein and TDP-43 aggregates confirmed the absence of detectable accumulation of silver grains of the photographic emulsion associated with those lesions, favoring the relative specificity of AV-1451 for PHF-tau aggregates over β-amyloid plaques and other abnormal protein brain deposits with a β-pleated sheet conformation. In agreement with these autoradiographic findings, our [H-3]-AV-1451 in vitro binding assay showed higher levels of binding in PHF-tangle containing regions in AD brains when compared to non-AD tauopathy brains, CAA, α-synuclein, TDP-43 lesion-containing brains and controls, although the magnitude of the difference detected by this method seemed substantially less robust than that observed by autoradiography. As discussed further below, we believe that nonspecific binding of AV-1451 to sample components such as hemoglobin and the absence of an ethanol washing step in this assay could contribute, at least in part, to explain the background noise and the relatively modest signal-to-noise ratio of the [H-3]-AV-1451 in vitro binding assay used here.

Intriguingly, our recent in vivo studies showed particularly high levels of AV-1451 retention in the inferior temporal cortex and parieto-occipital association cortical areas in MCI and AD patients, and relatively lower AV-1451 retention in medial temporal lobe structures like the hippocampal formation or the entorhinal cortex (Johnson K, Becker A, Sepulcre J et al. Initial experience with F18 T807-Tau PET imaging along the spectrum of normal aging and Alzheimer’s disease dementia. Presented in: Human Amyloid Imaging. Miami, Florida, 2014). It has long been known, from pathological studies of AD brains, that while PHF-tangle densities in AD are highest in the medial temporal lobe, the load of neuritic plaques, and thus of PHF-tau containing neurites, is in fact relatively low in those brain regions with the highest density of these lesions as well as neuropil threads being consistently found in temporal, parietal and occipital association cortices in AD.²⁴ Thus, our finding that AV-1451 binds to PHF-tau containing neurites in addition to classic PHF-tangles, may provide an explanation for the regional pattern of retention noticed in vivo in MCI and AD patients.

Another intriguing but frequent finding emerging from early [F-18]-AV-1451 PET imaging studies is the abnormally high binding of this tracer observed in the midbrain, the basal ganglia and the eyes, in a significant proportion of individuals with diverse clinical diagnoses and across the entire spectrum of cognition from demented to cognitively normal (Keith Johnson personal communication). The results of our nuclear emulsion autoradiography studies provide an explanation for these in vivo findings and demonstrate that AV-1451 not only binds to PHF-tau aggregates but also exhibits some off-target binding, in particular to neuromelanin-containing cells including pigmented neurons in the substantia nigra (regardless of the presence or absence of nigral tau pathology). Even though it is well known that neuromelanin and melanin are distinct, during the course of our experiments, we noticed, along with off-target binding to neuromelanin containing cells, an incidental strong binding of AV-1451 to extracutaneous melanocytes located in the leptomeninges. This observation prompted us to examine melanin-containing retinal pigment epithelium cells, skin melanocytes and lesions containing malignant melanocytes where we also observed prominent off-target binding. Our data also suggest that AV-1451 exhibits some weaker off-target binding to parenchymal and subarachnoid hemorrhages, and this could be particularly relevant for the correct interpretation of AV-1451 in vivo neuroimaging in patients with brain hemorrhagic lesions. Intriguingly, even though high in vivo retention of AV-1451 in the basal ganglia has been noticed in approximately 30–50% of individuals across ages and clinical diagnoses (Keith Johnson personal communication), no detectable [F-18]-AV-1451 binding to basal ganglia was observed by autoradiography in any of the cases studied here, regardless of the presence of robust amounts of tau aggregates in some of the non-AD tauopathy cases. This suggests that either the in vivo tracer uptake in this brain region may represent non-specific binding influenced by biological or technical factors other than underlying tau pathology or that the ethanol washing steps used in the autoradiographic studies removed [F-18]-AV-1451 labeling from the basal ganglia samples.

We also conducted exploratory studies with two AV-1451 fluorescent analogues, T726 and T557 (kindly provided by Avid Radiopharmaceuticals, Inc.), that share a similar but not identical structure to the parent compound AV-1451 which is not fluorescent itself.² Of note, histological staining with these fluorescent compounds required concentrations 3–5 orders of magnitude higher than AV-1451 in autoradiography due to their low signal intensity. Not unexpectedly, these fluorescent analogues not only labeled tangles and PHF-containing neurites, but also non-PHF tau lesions, amyloid plaque cores and amyloid-laden vessels (data not shown). The labeling of non-PHF tau and amyloid-containing lesions by AV-1451 fluorescent analogues is most likely due to a concentration-dependent specificity that is lost at the very high concentrations necessary for fluorescence studies. The results obtained from autoradiography experiments using the actual parent compound [F-18]-AV-1451 at similar concentrations used in vivo for PET studies, as described above, support this notion. Even though we cannot completely rule out the possibility of a weak in vivo binding of this tracer to non-tangle tau lesions or even non-tau aggregates, in this study we have made a very careful selection of non-AD tauopathy cases with particularly robust non-PHF tau pathology, and of Lewy body and amyloid angiopathy cases that virtually lacked any tau pathology, other than incidental age-related tangles confined to the entorhinal cortex and limbic regions, to be able to draw the most accurate conclusions from our autoradiography and in vitro binding assays.

In conclusion, our results suggest that AV-1451 holds promise as a surrogate marker for the in vivo detection of tau pathology in the form of tangles and PHF-containing neurites in AD brains but also provide evidence of the relatively lower affinity of this tracer for tau lesions predominantly consisting of straight filaments in non-AD tauopathy cases, and of the existence of AV-1451 off-target binding to neuromelanin- and melanin-containing cells and to brain hemorraghic lesions. All together these data give important clues for the interpretation of the patterns of [F-18]-AV-1451 retention observed by in vivo neuroimaging. Future radiological-pathological studies on postmortem material from individuals scanned while alive should provide additional information for the reliable interpretation of change of the PHF-tau pathology burden in AD and its relationship to clinical progression of the disease, and the potential use of [F-18]-AV-1451 as an endpoint when testing therapies aimed at decreasing tangle burden or halting tau propagation across neural circuits in AD.

Acknowledgments

Footnotes

References