[18F]Flotaza for Aß Plaque Diagnostic Imaging: Evaluation in Postmortem Human Alzheimer's Disease Brain Hippocampus and PET/CT Imaging in 5xFAD Transgenic Mice.

The diagnostic value of imaging Aß plaques in Alzheimer's disease (AD) has accelerated the development of fluorine-18 labeled radiotracers with a longer half-life for easier translation to clinical use. We have developed [18F]flotaza, which shows high binding to Aß plaques in postmortem human AD brain slices with low white matter binding. We report the binding of [18F]flotaza in postmortem AD hippocampus compared to cognitively normal (CN) brains and the evaluation of [18F]flotaza in transgenic 5xFAD mice expressing Aß plaques. [18F]Flotaza binding was assessed in well-characterized human postmortem brain tissue sections consisting of HP CA1-subiculum (HP CA1-SUB) regions in AD (n = 28; 13 male and 15 female) and CN subjects (n = 32; 16 male and 16 female). Adjacent slices were immunostained with anti-Aß and analyzed using QuPath. In vitro and in vivo [18F]flotaza PET/CT studies were carried out in 5xFAD mice. Post-mortem human brain slices from all AD subjects were positively IHC stained with anti-Aß. High [18F]flotaza binding was measured in the HP CA1-SUB grey matter (GM) regions compared to white matter (WM) of AD subjects with GM/WM > 100 in some subjects. The majority of CN subjects had no decipherable binding. Male AD exhibited greater WM than AD females (AD WM♂/WM♀ > 5; p < 0.001) but no difference amongst CN WM. In vitro studies in 5xFAD mice brain slices exhibited high binding [18F]flotaza ratios (>50 versus cerebellum) in the cortex, HP, and thalamus. In vivo, PET [18F]flotaza exhibited binding to Aß plaques in 5xFAD mice with SUVR~1.4. [18F]Flotaza is a new Aß plaque PET imaging agent that exhibited high binding to Aß plaques in postmortem human AD. Along with the promising results in 5xFAD mice, the translation of [18F]flotaza to human PET studies may be worthwhile.

Comparison of Monoamine Oxidase-A, Aß Plaques, Tau, and Translocator Protein Levels in Postmortem Human Alzheimer's Disease Brain.

Increased monoamine oxidase-A (MAO-A) activity in Alzheimer's disease (AD) may be detrimental to the point of neurodegeneration. To assess MAO-A activity in AD, we compared four biomarkers, Aß plaques, tau, translocator protein (TSPO), and MAO-A in postmortem AD. Radiotracers were [18F]FAZIN3 for MAO-A, [18F]flotaza and [125I]IBETA for Aß plaques, [124/125I]IPPI for tau, and [18F]FEPPA for TSPO imaging. Brain sections of the anterior cingulate (AC; gray matter GM) and corpus callosum (CC; white matter WM) from cognitively normal control (CN, n = 6) and AD (n = 6) subjects were imaged using autoradiography and immunostaining. Using competition with clorgyline and (R)-deprenyl, the binding of [18F]FAZIN3 was confirmed to be selective to MAO-A levels in the AD brain sections. Increases in MAO-A, Aß plaque, tau, and TSPO activity were found in the AD brains compared to the control brains. The [18F]FAZIN3 ratio in AD GM versus CN GM was 2.80, suggesting a 180% increase in MAO-A activity. Using GM-to-WM ratios of AD versus CN, a >50% increase in MAO-A activity was observed (AD/CN = 1.58). Linear positive correlations of [18F]FAZIN3 with [18F]flotaza, [125I]IBETA, and [125I]IPPI were measured and suggested an increase in MAO-A activity with increases in Aß plaques and tau activity. Our results support the finding that MAO-A activity is elevated in the anterior cingulate cortex in AD and thus may provide a new biomarker for AD in this brain region.

Measurement of Aß Amyloid Plaques and Tau Protein in Postmortem Human Alzheimer's Disease Brain by Autoradiography Using [18F]Flotaza, [125I]IBETA, [124/125I]IPPI and Immunohistochemistry Analysis Using QuPath.

High-resolution scans of immunohistochemical (IHC) stains of Alzheimer's disease (AD) brain slices and radioligand autoradiography both provide information about the distribution of Aß plaques and Tau, the two common proteinopathies in AD. Accurate assessment of the amount and regional location of Aß plaques and Tau is essential to understand the progression of AD pathology. Our goal was to develop a quantitative method for the analysis of IHC-autoradiography images. Postmortem anterior cingulate (AC) and corpus callosum (CC) from AD and control (CN) subjects were IHC stained with anti-Aß for Aß plaques and autoradiography with [18F]flotaza and [125I]IBETA for Aß plaques. For Tau, [124I]IPPI, a new radiotracer, was synthesized and evaluated in the AD brain. For Tau imaging, brain slices were IHC stained with anti-Tau and autoradiography using [125I]IPPI and [124I]IPPI. Annotations for Aß plaques and Tau using QuPath for training and pixel classifiers were generated to measure the percent of the area of Aß plaques and Tau in each slice. The binding of [124I]IPPI was observed in all AD brains with an AC/CC ratio > 10. Selectivity to Tau was shown by blocking [124I]IPPI with MK-6240. Percent positivity for Aß plaques was 4-15%, and for Tau, it was 1.3 to 35%. All IHC Aß plaque-positive subjects showed [18F]flotaza and [125I]IBETA binding with a positive linear correlation (r2 > 0.45). Tau-positive subjects showed [124/125I]IPPI binding with a stronger positive linear correlation (r2 > 0.80). This quantitative IHC-autoradiography approach provides an accurate measurement of Aß plaques and Tau within and across subjects.

Abnormal [18 F]NIFENE binding in transgenic 5xFAD mouse model of Alzheimer's disease: In vivo PET/CT imaging studies of α4ß2* nicotinic acetylcholinergic receptors and in vitro correlations with Aß plaques.

Since cholinergic dysfunction has been implicated in Alzheimer's disease (AD), the effects of Aß plaques on nicotinic acetylcholine receptors (nAChRs) α4ß2* subtype were studied using the transgenic 5xFAD mouse model of AD. Using the PET radiotracer [18 F]nifene for α4ß2* nAChRs, in vitro autoradiography and in vivo PET/CT studies in 5xFAD mice were carried out and compared with wild-type (C57BL/6) mice. Ratios of [18 F]nifene binding in brain regions versus cerebellum (CB) in 5xFAD mice brains were for thalamus (TH) = 17, hippocampus-subiculum = 7, frontal cortex (FC) = 5.5, and striatum = 4.7. [125 I]IBETA and immunohistochemistry (IHC) in 5xFAD brain slices confirmed Aß plaques. Nicotine and acetylcholine displaced [18 F]nifene in 5xFAD mice (IC50 nicotine = 31-73 nM; ACh = 38-83 nM) and C57BL/6 (IC50 nicotine = 16-18 nM; ACh = 34-55 nM). Average [18 F]nifene SUVR (CB as reference) in 5xFAD mice was significantly higher in FC = 3.04 compared to C57BL/6 mice FC = 1.92 (p = .001), whereas TH difference between 5xFAD mice (SUVR = 2.58) and C57BL/6 mice (SUVR = 2.38) was not significant. Nicotine-induced dissociation half life (t1/2 ) of [18 F]nifene for TH were 37 min for 5xFAD mice and 26 min for C57BL/6 mice. Dissociation half life  for FC in C57BL/6 mice was 77 min , while no dissociation of [18 F]nifene occurred in the medial prefrontal cortex (mFC) of 5xFAD mice. Coregistration of [18 F]nifene PET with MR suggested that the mPFC, and anterior cingulate (AC) regions exhibited high uptake in 5xFAD mice compared to C57BL/6 mice. Ex vivo [18 F]nifene and in vitro [125 I]IBETA Aß plaque autoradiography after in vivo PET/CT scan of 5xFAD mouse brain were moderately correlated (r2 = 0.68). In conclusion, 5xFAD mice showed increased non-displaceable [18 F]nifene binding in mPFC.

Elevated Monoamine Oxidase-A in Anterior Cingulate of Post-Mortem Human Parkinson's Disease: A Potential Surrogate Biomarker for Lewy Bodies?

Lewy bodies (LB) play a neuropathological role in Parkinson's disease (PD). Our goal was to evaluate LB using anti-ubiquitin immunohistochemistry (UIHC) and find correlations with monoamine oxidase-A (MAO-A) using imaging agent, [18F]FAZIN3. Human post-mortem anterior cingulate (AC) and corpus callosum (CC) from control subjects (CN), n = 6; age 81-90 LB = 0 and PD, n = 6, age 77-89, LB = III-IV were sectioned (10 µm slices). Brain slices were immunostained with anti-ubiquitin for LB (UIHC) and analyzed using QuPath for percent anti-ubiquitin per unit area (µm2). Adjacent brain slices were incubated with [18F]FAZIN3 and cortical layers I-III, IV-VI and CC (white matter) regions were quantified for the binding of [18F]FAZIN3. UIHC was correlated with [18F]FAZIN3 binding. All PD brains were positively UIHC stained and confirmed presence of LB. Outer cortical layers (I-III) of PD AC had 21% UIHC while inner layers (IV-VI) had >75% UIHC. In the CN brains LB were absent (<1% UIHC). Increased [18F]FAZIN3 binding to MAO-A in AC was observed in all PD subjects. [18F]FAZIN3 ratio in PD was AC/CC = 3.57 while in CN subjects it was AC/CC = 2.24. Increases in UIHC µm2 correlated with [18F]FAZIN3 binding to MAO-A in DLU/mm2. Increased [18F]FAZIN3 binding to MAO-A in PD is a potential novel "hot spot" PET imaging approach.