Amyloid-Beta Radiotracer [18F]BF-227 Does Not Bind to Cytoplasmic Glial Inclusions of Postmortem Multiple System Atrophy Brain Tissue

Abstract

The accumulation of aggregated alpha-synuclein (α-syn) in multiple brain regions is a neuropathological hallmark of synucleinopathies. Multiple system atrophy (MSA) is a synucleinopathy characterized by the predominant cerebral accumulation of aggregated α-syn as cytoplasmic glial inclusions (CGI). A premortem diagnosis tool would improve early diagnosis and help monitoring disease progression and therapeutic efficacy. One Positron Emission Tomography (PET) study suggested [¹¹C]BF-227 as a promising radiotracer for monitoring intracellular α-syn deposition in MSA patients. We sought to confirm the binding of this radiotracer to α-syn using state-of-the-art autoradiography. Medulla sections were obtained from 9 MSA patients and 9 controls (London Neurodegenerative Diseases Brain Bank). [¹⁸F]BF-227, chemically identical to [¹¹C]BF-227, was used at nanomolar concentrations to perform in vitro autoradiography assays. Autoradiograms were superimposed on fluorescent staining from the conformational anti-α-syn antibody 5G4 and quantified after immunofluorescence-driven definition of regions of interest. Autoradiography showed no specific signals in MSA patients in comparison to controls despite widespread pathology detected by immunofluorescence. Autoradiography does not support a significant binding of [¹⁸F]BF-227 to CGI at concentrations typically achieved in PET experiments.

Figure 1

Two-step methodology for autoradiography assays on MSA patients and CTL subjects. Qualitative experiments to optimize washing conditions were first performed (a), followed by quantitative evaluation of total and nondisplaceable binding of [¹⁸F]BF-227 (b).

Figure 2

Representative autoradiography images obtained from (a) an MSA patient, (b) a CTL subject, and (c) a patient with Alzheimer's disease used as positive control. Three consecutive medulla sections were incubated with [¹⁸F]BF-227 and washed with various ethanol concentrations (50-65-80% from left to right). Corresponding immunofluorescence against pathological α-syn (in red, red arrow) did not match the autoradiography signals (in white) detected after washes with 50 and 65% ethanol (a, b). In contrast, amyloid plaques in the Alzheimer's patient were best detected (white arrow) after washing the slides with 80% ethanol.

Figure 3

Quantification of total and nondisplaceable [¹⁸F]BF-227 binding. (a) Regions of interest, with (MSA⁺, in red) and without (MSA⁻, in white) aggregated alpha-synuclein staining were defined according to immunofluorescence ((A) MSA patient and (D) CTL subject). Corresponding total and nondisplaceable binding were extracted from MSA patients (B and C) and CTL subjects (E and F). (b) Computed displaceable [¹⁸F]BF-227 binding in MSA⁺, MSA⁻, and CTL regions of interest, plotted as individual points (for each MSA patient or CTL subject) and mean ± standard deviation. Differences between the three groups were not significant.