In-vivo Imaging of Amyloid Deposited in Alzheimer’s Disease Model Mouse Brain with a 123I-labeled Ligand and Analysis for Binding Characteristic in Postmortem Human Brain

Background/Purpose: Formation and accumulation of senile plaques is usually accompanied with occurrence of neuroinflammation, characterized by microglial activation, in Alzheimer’s disease (AD). Non-invasively visualization of these two neuropathological events by PET/SPECT technology with radiolabeled ligands for fibrillar amyloid peptide (A) and 18kDa-translocator protein (TSPO), a biomarker for activated microglia, has important significance for the early diagnosis and medical intervention for AD. Some studies with combination of amyloid and TSPO imaging, however, failed to find close association between A deposition and neuroinflammation, possibly due to lack in preferential binding of used radioligands to toxic (neuritic plaques) or non-toxic (diffuse plaques) A components, which usually are intermingled in the same brain regions, and therefore lead to difficulty in development of high selective ligand. The previous studies also demonstrated unique super-diffuse A, sometimes being called “lake-like” A, with obviously different feature compared with diffuse/neuritic plaques, exclusively deposited in the parvopyramidal layer of the presubiculum. In the present study, we have performed in vivo imaging with a newly developed 123I-labeled ligand 123I-DRM106 for detecting amyloid deposited in AD model mouse brain and compared preferential binding of this radioligand with other widely-used radioligands including Pittsburgh compound B (PiB), AZD2186 and DRK092. Materials & Methods: Animal: A transgenic (Tg) mouse line (Tg2576) which overexpress a mutant form of amyloid precursor protein and age-matched non-Tg littermates (29 months)PET/SPECT scan: PET imaging with 11C-PiB was performed by microPET Focus 220 animal scanner (Siemens Medical Solutions, Malvern, PA). Experimental animals were anesthetized with 1.5% (v/v) isoflurane, and a 30-G needle connected to a 1-ml polypropylene syringe via polyethylene tubing was inserted into the tail vein. 11C-PiB (30.0 ± 6.8 MBq) was intravenously injected, and a 60-min list-mode emission scan was carried out immediately. Average images from 30 to 60 min were reconstructed by maximum a posteriori reconstruction, and dynamic images were reconstructed with filtered back-projection using a 0.5-mm Hanning filter. ROIs were placed on the neocortex (CT), hippocampus (Hip) and cerebellum (CB) using PMOD® image analysis software (PMOD Group, Zurich, Switzerland) with reference to an MRI template generated. We quantified the binding potential for 11C-PiB in the CT/Hip on the basis of CB data as references. SPECT imaging was performed one month later on the same mice. The injection solution of 123I-DRM106 (340-350 MBq/800 μL saline) was injected via tail vein with an initial bolus injection (200 μL) followed by constant infusion injection (600 μL over 15 min). All SPECT scans were carried out using an integrated small-animal PET/SPECT/CT system InveonTM platform (Siemens Medical Solutions USA, Knoxville, TN). In vitro autoradiography: Postmortem human brains were obtained from the Center for Neurodegenerative Disease Research at the University of Pennsylvania Perelman School of Medicine. The brains were fixed in 10% neutral buffered formalin, embedded in paraffin blocks, and then cut into 10-μm-thick sections. Deparaffinized brain sections were preincubated with PBS for 30 min, followed by treatment with radioligands (5 mM for 11C-PiB, 3H-AZD2184, 125I-DRM106, and 125I-DRK092) in 50 mM Tris-HCl buffer containing 5% (for 11C-PiB and 3H-AZD2184) or 20% (for 125I-DRM106 and 125I-DRK092) ethanol at room temperature for 1 h in the presence or absence of unlabeled DRM106 (10 μM). The sections were then rinsed with ice-cold wash buffer (50 mM Tris-HCl buffer containing the same concentration of ethanol as the incubation buffer) for 2 min twice, and dipped into distilled water for 10 sec. After warmly blow-dried and attached to an imaging plate (BAS-MS2025; GE Healthcare) for contact periods optimized for each radioligand, radiolabeling was detected by scanning the imaging plate using the BAS-5000 system (FUJIFILM).Immunohistochemisty/histochemistry: An anti-A antibody (6E10) and an anti-TSPO antibody (NP157) was used. The deparaffinized brain sections used for TSPO immunostaining were initially autoclaved in citric buffer, and those for A immunostaining were pretreated with formic acid for antigen recovery, followed by a standard immunohistochemical procedure. All stained sections were examined by an all-in-one microscope/digital camera (BZ-9000; Keyence Japan), and captured photomicrographs were semi-automatically tiled and merged into a large high-resolution image containing the regions of interest. Results: In vivo imaging with either 123I-DRM106 or 11C-PiB showed significant higher binding potential in forebrain area of Tg mouse (n = 5) compared to age-matched non-Tg mouse (n = 3) with cerebellum as reference tissue (11C-PiB; **, p<0.01, 123I-DRM106; *, p<0.05, Tg vs non Tg, ttest) and there was excellent correlation (pearson correlation coefficient R = 0.98, *p < 0.01) between 11C-PiB and 123I-DRM106 binding. Autoradiographic images of 11C-PiB, 3H-AZD2184 and 125I-DRK092 showed overt binding to presubicular A deposits, while 125I-DRM106 barely bound to these aggregates, despite its strong binding in the hippocampal CA1 sector (binding ratio of presubiculum to CA1, 125I-DRM106 vs 11C-PiB; 0.64 ± 0.06 vs 2.62 ± 0.50, ***p < 0.001, ttest). Unlike neuritic plaques in the CA1, A lesions in the presubiculum were not accompanied by inflammatory gliosis enriched with TSPO. \nConclusions: The present study demonstrated high potential of 123I-DRM106 for amyloid imaging in preclinical and clinical application. Presubicular A deposits has fibrillar structure and is PET-measurable for the first time. Regionally separated deposition of these A enable classification of radioligand into two groups, based on their preferential binding to these subtypes of A deposits. Accumulation of high affinity ligands exemplified by 11C-PiB, 3H-AZD2184, and 125I-DRK092 showed lack in spatial consistency with induced TSPO expression, supporting published results of in vivo imaging. Meanwhile, brain regions labeled by 125I-DRM106 with low affinity for presubicular A deposits was spatially consistent with induced TSPO expression, suggesting that the combination of TSPO imaging and amyloid imaging with those low affinity ligands for presubicular A deposits might be able to provide additional evidence for relationship between Adeposition and neuroinflammation.The Second Global Conference of Chinese Professionals i