We performed immunohistochemical analyses on adjacent brain sections of individuals with or without AD using three BIN1 antibodies (pAb BSH3, mAb 2F11, and mAb 99D) and markers of AD pathology. Figures A and B show the distribution and density of senile plaques and neurofibrillary tangles in the entorhinal cortex of a patient with AD [immunostained using antibodies against Aβ (mAb 4G8) and Tau (Tau-2)], in relation to BIN1 cellular distribution [immunostained using pAb BSH3 and mAb 2F11]. As described in a recent study, we observed widespread BIN1 immunoreactivity in oligodendrocytes and processes throughout the neuropil, and more intense staining of the white matter. However, the pattern of BIN1 immunoreactivity did not fit the profiles of Tau immunostaining (Figures A and B). Whereas the Tau-2 antibody labeled a number of tangles and neuropil threads in the entorhinal cortex, BIN1 antibodies labeled profuse punctate structures and cell soma that had no resemblance to tangles (Figure C). At closer inspection, only weak BIN1 immunoreactivity was found to be associated with the cytoplasm of neurons (red arrows in Figure C). Robust BIN1 immunoreactivity was found in oligodendrocytes (yellow arrows in Figure C), which are smaller than neurons and can be labeled by antibodies against the oligodendrocyte marker TPPP/p25 (Figure D). Thus, BIN1 immunoreactivity is not associated with neurofibrillary tangles or neuropil threads.
We also examined BIN1 distribution in relation to senile plaques in the brains of patients with AD. Similar to what was recently reported in control brains, immunohistochemical analyses of advanced AD brain tissue revealed a strong BIN1 immunoreactivity in oligodendrocytes and processes throughout the neuropil. However, in areas with a high senile plaque density, we observed an absence of BIN1 immunoreactivity in the areas that corresponded to amyloid deposits (Figure B). This finding is consistent with a recently published report.
To explore BIN1 cellular expression in the context of inflammatory response to AD pathology, we performed immunostaining of adjacent sections with antibodies against BIN1 and cellular markers of microglia (Iba1), activated microglia/macrophages (CD68), and astrocytes (GFAP). A comparison of Iba1 immunostaining with that of BIN1 revealed that the overall pattern of BIN1 immunoreactivity did not fit the overall distribution profiles of microglia, activated microglia/macrophages, and astrocytes, suggesting that these reactive cell types in pathological AD brain do not express BIN1 (Figure B, data not shown). Inspection at higher magnification revealed clusters of Iba1-positive microglia with readily discernible ramified processes near senile plaques. However, BIN1 immunoreactivity was not associated with cells that resembled Iba1-positive microglia (Figure D). This finding is consistent with a recent report showing little evidence for microglial expression of BIN1 in the human (non-AD) and mouse brain. Moreover, a comparison of the morphology of cells positive for CD68 and GFAP revealed no similarity with BIN1 immunoreactivity, suggesting the lack of BIN1 expression in reactive microglia, macrophages, and astrocytes (Figure D). These results suggest that BIN1 is unlikely to be involved in the inflammatory processes associated with pathogenesis in AD. Our findings that BIN1 protein is not detected in ramified microglia, reactive microglia, and macrophages are notable because BIN1 mRNA expression in microglia and macrophages acutely isolated from mouse and human brain are reported in RNA-seq transcriptome databases.
In order to confirm the above findings, we performed immunofluorescence staining of BIN1 and Iba1 along with Thioflavin S staining. We observed a clearance of BIN1 within the area of neurofibrillary tangles stained by Thioflavin S (Figure E). A number of Iba-1 positive microglia were found near the tangles, but they were negative for BIN1 immunostaining. These results from immunofluorescence labeling are in agreement with the observations made by immunohistochemical staining of serial sections described above.
Two studies have previously suggested a link between BIN1 expression and neurofibrillary tangle pathology in AD. The findings that Tau can bind to BIN1 in vitro and can co-immunoprecipitate with the brain-specific BIN1 isoform 1 support this notion. In contrast, a recent study demonstrated an inverse correlation between BIN1 levels and propagation of Tau pathology. Earlier studies on the localization of BIN1 immunoreactivity and neurofibrillary tangles in AD brain described discordant findings. However, a detailed immunohistochemical analysis of a large set of brain tissue at different stages of AD progression demonstrated a lack of correlation between tangles and BIN1 immunoreactivity in neurons and even a negative correlation between neurofibrillary tangle pathology and BIN1 immunoreactivity in the neuropil. Our results presented here are in accordance with this later report as we found a lack of overlap between neurofibrillary tangles and BIN1 immunoreactivity by immunofluorescence analysis. Our negative finding is not due to the complexity of BIN1 alternate splicing, as four different BIN1 antibodies, including two that are capable of reacting to all BIN1 isoforms, failed to stain neurofibrillary tangles in our study (Figure C, data not shown). Finally, it is notable that the levels of brain-specific BIN isoform 1, which was found to be specifically associated with Tau in AD brain, is significantly decreased in AD brain. Thus, additional studies are needed to fully understand the significance of the positive correlation between Tau pathology and the levels of the ubiquitous BIN1 isoform 9, which in the brain appears to be mainly expressed by oligodendrocytes and elucidates how BIN1 mechanistically participates as a risk factor in late-onset AD.