Autoradiography validation of novel tau PET tracer [F-18]-MK-6240 on human postmortem brain tissue

Abstract

[F-18]-MK-6240, a novel tau positron emission tomography (PET) tracer recently discovered for the in vivo detection of neurofibrillary tangles, has the potential to improve diagnostic accuracy in the detection of Alzheimer disease. We have examined regional and substrate-specific binding patterns as well as possible off-target binding of this tracer on human brain tissue to advance towards its validation. We applied [F-18]-MK-6240 phosphor screen and high resolution autoradiography to postmortem samples from patients with a definite pathological diagnosis of Alzheimer disease, frontotemporal lobar degeneration-tau (Pick's disease, progressive supranuclear palsy and corticobasal degeneration), chronic traumatic encephalopathy, frontotemporal lobar degeneration-Tar DNA-binding protein 43 (TDP-43), dementia with Lewy bodies, cerebral amyloid angiopathy and elderly controls free of pathologic changes of neurodegenerative disease. We also directly compared the binding properties of [F-18]-MK-6240 and [F-18]-AV-1451 in human tissue, and examined potential nonspecific binding of both tau tracers to monoamine oxidases (MAO) by using autoradiography in the presence of selective monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B) inhibitors. Our data indicate that MK-6240 strongly binds to neurofibrillary tangles in Alzheimer disease but does not seem to bind to a significant extent to tau aggregates in non-Alzheimer tauopathies, suggesting that it may have a limited utility for the in vivo detection of these pathologies. There is no evidence of binding to lesions containing β-amyloid, α-synuclein or TDP-43. In addition, we identified MK-6240 strong off-target binding to neuromelanin and melanin-containing cells, and some weaker binding to areas of hemorrhage. These binding patterns are nearly identical to those previously reported by our group and others for [F-18]-AV-1451. Of note, [F-18]-MK-6240 and [F-18]-AV-1451 autoradiographic binding signals were only weakly displaced by competing concentrations of selective MAO-B inhibitor deprenyl but not by MAO-A inhibitor clorgyline, suggesting that MAO enzymes do not appear to be a significant binding target of any of these two tracers. Together these novel findings provide relevant insights for the correct interpretation of in vivo [F-18]-MK-6240 PET imaging.

Fig. 1

[F-18]-MK-6240 phosphor screen images of brain slices from AD (#5, #7, #9, #16) (a), control (#1, #2) (b), CTE (#32, #33) (c), P301L mutation carrier (#21) (d), PSP (#25) (e), and PiD (#20) (f) cases. A strong [F-18]-MK-6240 binding was observed in cortical regions containing tangles from AD brains. No signal was detected in basal ganglia, a region free of tangles. The signal was blocked by adding unlabeled MK-6240. Slices from a control case free of pathology did not show detectable [F-18]-MK-6240 binding (b). [F-18]-MK-6240 binding was not detectable either in non-PHF tau-containing slices from CTE (c), P301L mutation carrier (d), PSP (e) and PiD (f) cases. Abbreviations: AD = Alzheimer’s disease; CTE = chronic traumatic encephalopathy; PSP = progressive supranuclear palsy; PiD = Pick’s disease. Scale bar = 1 cm

Fig. 2

[F-18]-MK-6240 phosphor screen images of brain slices from a CAA carrier of the D23N Iowa APP mutation (#17) (a), FTLD TDP-43 (#40, #41) (b), LBD (#36) (c), and MSA (#37) (d) cases. No [F-18]-MK-6240 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]-MK-6240 signal was observed in the region corresponding to the substantia nigra (off-target) in all cases studied regardless of their pathological diagnosis (c). Abbreviations: APP = amyloid precursor protein; CAA = cerebral amyloid angiopathy; TDP-43 = TAR DNA binding protein 43; DLB = dementia with Lewy bodies; MSA = multiple system atrophy. Scale bar = 1 cm

Fig. 3

[F-18]-MK-6240 phosphor screen and high-resolution autoradiography images of slices containing substantia nigra in a control case (#2) (a), retinal pigment epithelium (b) in an AD case (#11), metastatic melanoma (#18) (c), parenchymal hemorrhagic lesions (#43) (d) and extracutaneous meningeal melanocytes in the cerebellum of an AD case (#8) (e). [F-18]-MK-6240 phosphor screen autoradiography images are displayed in (a-e), left and middle panels. [F-18]-MK-6240 high resolution autoradiography images are displayed in (a-e), right panel. Strong [F-18]-MK-6240 binding was observed in neuromelanin-containing neurons of the substantia nigra (a), melanin containing granules in the retinal pigment epithelium (b), malignant melanocytes from a metastatic melanoma (c), and extracutaneous meningeal melanocytes (e). [F-18]-MK-6240 binding was noticed in association with intraparenchymal hemorrhagic lesions (d). Scale bars = 1 cm (a-e left and middle panels: phosphor screen autoradiography) and 50 μm (a-e right panels; high resolution nuclear emulsion autoradiography)

Fig. 4

Head-to-head comparison of [F-18]-MK-6240 and [F-18]-AV-1451 phosphor screen autoradiographic binding patterns in adjacent section obtained from the same tissue material containing entorhinal cortex (#13) (a) and superior temporal sulcus (#13) (b) from AD cases. Both tracers exhibited comparable strong binding in regions containing tangles; MK-6240 signal was blocked by adding 500 nM unlabeled AV-1451 and AV-1451 signal was almost completely blocked by adding 500 nM unlabeled MK-6240. Scale bar = 1 cm

Fig. 5

Phosphor screen autoradiography experiments in slices containing entorhinal and temporal cortices from AD cases using competing concentrations of 1 μM clorgyline (a selective MAO-A inhibitor) and deprenyl (MAO-B inhibitor). [F-18]-MK-6240 and [F-18]-AV-1451 binding signals are only weakly displaced (by about 20%) with 1 μM deprenyl (a selective MAO-B inhibitor). When a competing concentration of 1 μM clorgyline (a selective MAO-A inhibitor) was added to the blocking solution, neither [F-18]-MK-6240 nor [F-18]-AV-1451 autoradiographic signal displacement could be detected. Scale bar = 1 cm

Fig. 6

[F-18]-AV-1451 and [F-18]-MK-6240 phosphor screen and high resolution autoradiography photomicrographs of brain slices containing occipital (#13) (a), temporal (#9) (b) and entorhinal (#14) (c) cortices from AD cases (left panels). Middle and right panels (a and b) show immunostaining of adjacent sections with PHF-1 antibody (kind gift of Dr. Peter Davies) and anti-Aβ antibody (1:500, mouse, clone 6F/3D, Dako), respectively. [F-18]-AV-1451 (left panels a and c) and [F-18]-MK-6240 (left panel b and right panel c) high resolution nuclear emulsion autoradiography showed a strong cortical accumulation of silver grains in temporal cortical layers III and V (a and b), and in layers II and IV of the entorhinal cortex (c) in AD brains mirroring the laminar pattern of tangles on adjacent slices as revealed by PHF-1 immunostaining (middle panels) rather than the more scattered plaque distribution pattern revealed by Aβ immunostaining (right panels). Abbreviations: AD = Alzheimer’s disease; IHC = immunohistochemistry. Scale bars = 200 μm (a and b) 50 μm (c)

Fig. 7

Photomicrographs showing combined [F-18]-MK-6240 high-resolution nuclear emulsion autoradiography followed by immunostaining with appropriate antibodies of brain slices containing frontal and temporal cortices from AD (#10, #16) (a-c), CAA (#17) (d) CTE (#33) (e), CBD (#23) (f), PSP (#26) (g), PiD (#20) (h), LBD (#34) (i) and FTLD TDP-43 (#39) (j). Accumulation of silver grains from the nuclear emulsion colocalized with PHF-1 stained tangles and PHF-tau containing dystrophic neurites around plaques in AD. No detectable accumulations of silver grains were observed in association with Aβ plaques themselves or amyloid-containing vessels in CAA, tau aggregates in CTE, coiled bodies and globose tangles in CBD, astrocytic plaques in PSP, Pick bodies in PiD, Lewy bodies in LBD or TDP-43 inclusions in FTLD TDP-43. Abbreviations: AD = Alzheimer’s disease; CAA = cerebral amyloid angiopathy; CTE = chronic traumatic encephalopathy; CBD = corticobasal degeneration PSP = progressive supranuclear palsy; PiD = Pick’s disease; LBD = Lewy body disease; FTLD TDP-43 = frontotemporal lobar degeneration with TDP-43 inclusions. Scale bar = 50 μm