Predominant expression of Alzheimer’s disease-associated BIN1 in mature oligodendrocytes and localization to white matter tracts
- Pierre De Rossi†1,
- Virginie Buggia-Prévot†1,
- Benjamin L. L. Clayton2,
- Jared B. Vasquez3,
- Carson van Sanford3,
- Robert J. Andrew1,
- Ruben Lesnick1,
- Alexandra Botté1,
- Carole Deyts1,
- Someya Salem1,
- Eshaan Rao1,
- Richard C. Rice1,
- Angèle Parent1,
- Satyabrata Kar4,
- Brian Popko2,
- Peter Pytel5,
- Steven Estus3 and
- Gopal Thinakaran1, 2, 5Email authorView ORCID ID profile
© The Author(s). 2016
Received: 28 May 2016
Accepted: 27 July 2016
Published: 3 August 2016
Genome-wide association studies have identified BIN1 within the second most significant susceptibility locus in late-onset Alzheimer’s disease (AD). BIN1 undergoes complex alternative splicing to generate multiple isoforms with diverse functions in multiple cellular processes including endocytosis and membrane remodeling. An increase in BIN1 expression in AD and an interaction between BIN1 and Tau have been reported. However, disparate descriptions of BIN1 expression and localization in the brain previously reported in the literature and the lack of clarity on brain BIN1 isoforms present formidable challenges to our understanding of how genetic variants in BIN1 increase the risk for AD.
In this study, we analyzed BIN1 mRNA and protein levels in human brain samples from individuals with or without AD. In addition, we characterized the BIN1 expression and isoform diversity in human and rodent tissue by immunohistochemistry and immunoblotting using a panel of BIN1 antibodies.
Here, we report on BIN1 isoform diversity in the human brain and document alterations in the levels of select BIN1 isoforms in individuals with AD. In addition, we report striking BIN1 localization to white matter tracts in rodent and the human brain, and document that the large majority of BIN1 is expressed in mature oligodendrocytes whereas neuronal BIN1 represents a minor fraction. This predominant non-neuronal BIN1 localization contrasts with the strict neuronal expression and presynaptic localization of the BIN1 paralog, Amphiphysin 1. We also observe upregulation of BIN1 at the onset of postnatal myelination in the brain and during differentiation of cultured oligodendrocytes. Finally, we document that the loss of BIN1 significantly correlates with the extent of demyelination in multiple sclerosis lesions.
Our study provides new insights into the brain distribution and cellular expression of an important risk factor associated with late-onset AD. We propose that efforts to define how genetic variants in BIN1 elevate the risk for AD would behoove to consider BIN1 function in the context of its main expression in mature oligodendrocytes and the potential for a role of BIN1 in the membrane remodeling that accompanies the process of myelination.
The Bridging INtegrator-1 (BIN1; also referred to as amphiphysin II and SH3P9) is a member of the Bin/Amphiphysin/Rvs (BAR) family of adaptor proteins that regulates membrane dynamics in a variety of cellular functions . BIN1 was first identified as a protein that interacted with the N-terminus of the MYC oncoprotein , and cloned based on sequence similarity to Amphiphysin 1, a protein predominantly expressed in the brain [3–8]. Based on sequence similarity to Amphiphysin 1, it has been proposed that BIN1 might function in synaptic vesicle endocytosis . However, neuronal cultures prepared from Bin1 −/− mice failed to reveal a deficiency in basal synaptic vesicle uptake , raising the possibility that BIN1 and Amphiphysin 1 may have divergent functions in the brain. Unlike its close paralog Amphiphysin 1, BIN1 is broadly expressed in several tissues, with the highest expression in skeletal muscle [2, 4, 6, 11]. Alternate splicing of the 20 exons in BIN1 gene generates at least 10 transcripts encoding ubiquitous and tissue-specific isoforms, which differ not only in their tissue distribution but also in subcellular localization and cellular function [2, 4–6, 12, 13].
All isoforms of BIN1 contain an N-terminal BAR domain, which functions in sensing and generating membrane curvature and is critical for BIN1 function in membrane tubulation and budding/vesicle formation. A C-terminal SH3 domain, also found in all isoforms, mediates BIN1 interaction with proteins involved in endocytosis, such as dynamin, synaptojanin, and endophilin [1, 4–6, 14]. The CLathrin-Associated Protein binding region (CLAP domain), encoded by alternatively spliced exons present only in the neuronal BIN1 isoforms, is important for BIN1’s function in clathrin-mediated endocytosis and synaptic vesicle recycling [1, 9]. Crystal structure and functional studies predicted that the BIN1 CLAP domain is critical to recruiting dynamin and regulate its self-assembly at the clathrin-coated pit . Moreover, the residues encoded by the alternatively spliced exon 7 within the BAR domain promotes BIN1 interaction with dynamin 2 [16, 17]. In addition to proteins involved in endocytosis, the BIN1 SH3 domain can interact with a number of other proteins including the tyrosine kinase c-Abl, the actin nucleation-promoting factor N-WASP, actin, myosin, and Tau, suggesting the involvement of the cytoskeleton in BIN1 function [18–21].
Very little is known about BIN1 expression in the brain. The identification of polymorphisms upstream of BIN1 that act as a major genetic risk factor for late-onset AD has stimulated an interest in understanding BIN1’s role in the central nervous system [22–24]. It is notable that BIN1 is ranked in the AlzGene database as the second most statistically significant AD susceptibility gene based on a meta-analysis . In vitro assays and analysis of mutant flies lacking the expression of the Drosophila BIN1 ortholog Amphiphysin have suggested that BIN1 modifies AD risk through Tau pathology  or β-amyloid production  and studies have reported altered expression of BIN1 in Alzheimer Disease, albeit with discrepancies [26, 28–31]. Nevertheless, since functional BIN1 variants are yet to be identified, the precise link between BIN1 and the susceptibility to AD remains elusive. A survey of the published literature reveals conflicting information on the cellular and subcellular localization of BIN1 in the brain. Previous studies have described the presence of BIN1 in apical dendrites, initial axon segments, along the axons, nerve terminals, or the nodes of Ranvier [4, 26, 30]. Although it has been generally assumed that BIN1 is mainly expressed in neurons, Bin1 was found among the top 50 highly expressed genes in cultured oligodendrocytes by genomic analysis . A precise description of its expression and localization in the nervous system is a prerequisite for understanding BIN1 function and dysfunction in the brain. Here, we report the prominent non-neuronal expression of BIN1 in mouse and the human brain, which contrasts with neuronal expression of Amphiphysin 1. We detail BIN1 expression in mature oligodendrocytes and its enrichment in the white matter and characterize BIN1 isoform diversity in the gray and white matter of individuals with and without AD. The upregulation of BIN1 during in vitro oligodendrocyte maturation and the period of myelination in vivo, and the loss of BIN1 within brain lesions of patients with multiple sclerosis are all consistent and point to a potential role for BIN1 in mature oligodendrocytes in the brain.
Human autopsy tissue
List of human samples used for immunohistochemistry and immunoblot analysisa
AD; Braak 5
AD; Braak 5
AD Braak 5
AD Braak 5
AD Braak 5
AD Braak 2-3
AD Braak 5
Greater than 95% pure cultures of rat oligodendrocyte progenitor cells (OPCs) were isolated from cortices of 6–7 day old pups as described by sequential immunopanning on dishes coated with antibodies against Ran-2, GalC, and O4 . Ran-2−, GalC−, O4+ OPCs were cultured in medium supplemented with the mitogen PDGFα (Peprotech). Mature oligodendrocytes were generated by differentiating OPCs in medium containing the hormone 3-3’-5-Triiodo-L-thyronine (Sigma). Primary rat hippocampal neurons and astrocytes were cultured as described .
Analysis of gene expression
The expression levels of BIN1 isoforms that lack exon 7 (Delta-7-BIN1 (D7-BIN1) or include exon 7 (BIN1 + Ex7) were determined by quantitative polymerase chain reactions (qPCR). The primers for BIN1 + Ex7 were: exon 5 sense 5’-CCTGCTGTGGATGGATTACC-3’ and exon 7 antisense 5’-GCTTTCTCAAGCAGCGAGAC-3’. D7-BIN1 reactions used the same exon 5 sense primer and a junctional antisense primer, 5’-GCTTTCTCAAGCAGCGAGAC-3’ , corresponding to the last 6 nucleotides of exon 6 and the first 11 nucleotides of exon 8. Each 20 μL reaction mixture contained 20 ng of cDNA, 1 μM of each primer, and 2X SYBR Green (Quanta Biosciences) and was subjected to a PCR profile of 40 cycles at 95 °C for 15 sec, 60 °C for 30 sec, and 72 °C for 20 sec. A melting curve was performed after each qPCR run to ensure specific amplification. The BIN1 isoform copy number in samples was determined by using a standard curve generated in parallel. Copy numbers were normalized to the geometric mean of reference genes EIF4H and RPL32. Levels of cell type-specific genes were determined similarly. Primer sequences for microglial and endothelial cells were reported previously [41, 42]. Additional primer sequences were as follows: SYP (synaptophysin), 5’-AGGGAACACATGCAAGGAG-3’ and 5’-CCTTAAACACGAACCACAGG-3’; MBP, 5’-AAGGCCAGAGACCAGGATTT-3’, and 5’-TAGCCATGGGTGATCCAGA-3’; GFAP 5’-AAGAGATCCGCACGCAGTAT-3’ and 5’-GTAGTCGTTGGCTTCGTGCT-3’. Assays were repeated at least two times. The extent that BIN1 mRNA levels were associated with AD-status or cell type-specific gene expression was determined by using a general linear model (SPSS v.19).
BIN1 alternate splicing in RNA isolated from human anterior cingulate samples was determined by PCR. Assays included an exon 12 sense primer 5’-GAGTCAACCACGAGCCAGAG-3’ and exon 18 antisense primer 5’-GAAGGTCTCCACCACGACAG-3’. Each 20 μL reaction mixture contained 20 ng of cDNA, 1 μM of each primer, and Platinum Taq (Life Technologies) and was subjected to a PCR profile of 30 cycles at 95 °C for 15 sec, 59 °C for 30 sec, and 72 °C for 20 sec. Assays were performed in triplicate and PCR amplicons were excised from gels and sequenced.
Immunofluorescence labeling of transfected cells
HEK293 cells cultured on glass coverslips were transiently transfected with an expression plasmid that encodes FLAG-tagged human BIN1 isoform 7 (lacking exons 7, 11, 13–15) [generously provided by Dr. Zhou, Merck Research Laboratories]. The following day, the cells were fixed, permeabilized, and stained with mAb against FLAG or BIN1 antibodies as described .
Human brain immunohistochemistry
5 μm-thick paraffin embedded brain sections from individuals with and without multiple sclerosis were rehydrated, treated with antigen retrieval buffer (DAKO) in a steamer for 20 min, followed by a 5 min incubation with 3% H2O2 and permeabilized with PBST (phosphate-buffered saline containing 0.025% Triton-X 100). Sections were blocked with 10% normal rabbit serum-PBST for 1 h and then incubated with the indicated primary antibodies (Additional file 1: Table S1) for 1 h followed by species-specific secondary antibodies. The antigen-antibody reactions were detected by Envision + kit (Dako) or Vectastain Elite-kit (Vector laboratories). Nuclei were counterstained using hematoxylin. The slides were scanned using CRi Pannoramic Scan Whole Slide Scanner and analyzed by Pannoramic Viewer (3DHISTECH).
Immunofluorescence labeling and analysis
Wild-type C57/BL6 mice (between 1-month and 5-month-old) or adult rats were fixed by perfusion with 4% paraformaldehyde containing 4% sucrose. Immunofluorescence labeling was performed on 40 μm-thick sections. Floating brain sections were subjected to epitope retrieval by incubation in 10 mM trisodium citrate pH 6 and 0.05% Tween20 for 30 min at 90°C prior to blocking. The sections were sequentially incubated with the indicated primary antibodies (Additional file 1: Table S1) and Alexa Fluor-conjugated secondary antibodies (Molecular Probes) diluted in Tris-buffered saline containing 1% BSA and 0.25% Triton-X 100, for 48 h and 2 h, respectively. Nuclei were labeled using Hoechst stain (Molecular Probes) before mounting the sections on slides. Confocal images were acquired on a Leica SP5 II STED-CW Superresolution Laser Scanning Confocal microscope and analyzed by ImageJ .
Frontal cortex and deep frontal white matter regions were weighed and homogenized (20% weight/volume) in ice-cold lysis buffer [150 mM NaCl, 50 mM Tris–HCl pH 7.4, 0.5% NP-40, 0.5% sodium deoxycholate, 5 mM EDTA, 0.25 mM PMSF and protease inhibitor cocktail (Sigma)]. An equal volume of lysis buffer containing 2% SDS was then added to each sample and briefly sonicated. Cultured cells were lysed in 150 mM NaCl, 50 mM Tris pH 7.5, 1 mM EDTA, 1% Triton-X 100, 0.5% sodium deoxycholate, 0.1% SDS. Aliquots of lysates were fractionated by 10% or 4-20% polyacrylamide gel electrophoresis. Primary antibodies used for Western blot analysis are listed in Additional file 1: Table S1. After incubation with infrared dye-conjugated secondary antibodies, signals were recorded by the Odyssey infrared imaging system (LI-COR Biosciences) and quantified using ImageJ .
Differential expression of BIN1 isoforms in the gray and white matter
To investigate this question, the expression of BIN1 + Ex7 and D7-BIN1 was compared to that of genes with cell-type specific expression. Inspection of the results suggested that BIN1 + Ex7 expression was associated positively with synaptophysin expression (neuronal marker) but not with the markers of oligodendrocytes, astrocytes, microglia, or endothelial cells (Fig. 1c and d, Additional file 2: Figure S1A-C). This association was confirmed statistically by evaluating with a general linear model that included BIN1 + Ex7 as the dependent variable and synaptophysin and AD as independent variables. This produced a moderately significant model (adjusted R2 = 0.264). BIN1 + Ex7 was significantly associated with synaptophysin (p = 1.07X10−4) and modestly with AD (p = 0.046, note the limited power of 0.517) (Fig. 1c and Additional file 3: Table S2). If the model was expanded to include the AD-associated BIN1 single-nucleotide polymorphism rs7561528 , the BIN1 + Ex7 association with synaptophysin was unchanged. However, rs7561528 was not associated with BIN1 + Ex7 expression (p = 0.3) and including the single-nucleotide polymorphism in the model pushed AD from significance (p = 0.068) (data not shown). The increase in BIN1 + Ex7 expression in AD appears to be a fairly modest 18% (Additional file 4: Table S3). These results are in accordance with the findings reported by Chapuis et al. where the data were normalized to either actin or an apparent microglia gene, GUSB .
In contrast to BIN1 + Ex7, inspection of the graphs of the D7-BIN1 isoform relative to cell-specific mRNAs suggested that the D7-BIN1 isoform was associated positively with myelin basic protein (MBP), a marker for mature oligodendrocytes and the white matter (Fig. 1e and f) but not with other cell types (Additional 2: Figure S1D–F). We confirmed this notion by qPCR analysis of RNA isolated from gray matter and white matter and found that ~90% of BIN1 in the white matter corresponds to D7-BIN1 isoforms (Fig. 1b). Using a linear model that included MBP and AD status, we found a significant model (R2 = 0.553) wherein D7-BIN1 was significantly associated with MBP (p = 2.97X10−11). However, we observed no significant association between AD and D7-BIN1 expression (p = 0.4) (Additional file 5: Table S4). Additional analyses did not find a significant association between D7-BIN1 and the AD-associated single-nucleotide polymorphism (p = 0.6). These results reveal that the association between BIN1 expression and AD status is more complicated than previously understood due to the expression of alternatively spliced BIN1 transcripts, and raise the possibility that BIN1 expression may not be limited to neurons.
BIN1 is enriched in the human brain white matter
All three BIN1 antibodies revealed significantly higher BIN1 levels in the white matter (Fig. 2c and d). Quantification revealed that in comparison to the gray matter, the white matter overall contains ~3-fold higher levels of BIN1 (normalized to actin) (p <0.0001 by ANOVA) (Fig. 2d). The predominant white matter BIN1 isoforms migrated at ~65-75 kDa and were uniformly detectible by all three antibodies. This observation suggested that the white matter BIN1 isoforms, for the most part, lacked exon 7-encoded residues and contained exon 17-encoded residues. In contrast to its selective expression in the gray matter, BIN1:H isoform was conspicuously absent in the white matter. Our detection of lower molecular weight ~65-75 kDa BIN1 isoforms (henceforth referred as BIN1:L) in the brain are consistent with the size of the non-neuronal and ubiquitous BIN1 isoforms lacking the intact CLAP domain (encoded by exons 13–16). To confirm this notion, we performed RT-PCR analysis of mRNA isolated from human brain using forward and reverse primers within the exons 12 and 18, respectively. By sequence analysis of the PCR products, we identified multiple BIN1 isoforms generated by variable alternate splicing of BIN1 exons 13–16 (Additional file 7: Figure S3 and Fig. 2e). Finally, for comparison with BIN1 expression, we also assessed the levels of Amphiphysin 1, the brain-specific BIN1 homolog [3, 5, 48]. We found that Amphiphysin 1 is enriched in the gray matter, consistent with previous studies describing Amphiphysin 1 as the neuron-specific homolog of BIN1 (Fig. 2c). This finding supports the idea that Amphiphysin 1 and BIN1:H isoform differed in their cell type expression when compared to the BIN1:L isoforms.
Alteration in the levels of BIN1 isoforms in AD
We considered the possibility that the apparent AD-associated decrease in BIN1:H isoform could be the result of an overall neuronal loss in the gray matter. Therefore, we simultaneously quantified the levels of Amphiphysin 1 in the samples and normalized the data to the levels of actin. The results show significant differences in Amphiphysin 1 levels between controls and AD samples (Fig. 3a and b). Similar to BIN1:H isoform, the levels of Amphiphysin1 were markedly lower in the gray matter AD samples in comparison with the controls, with a high degree of correlation (Pearson r 0.934; p = 0.02). To extend this observation, we performed a series of immunoblots to quantify the levels of markers of neurons (CamKII, PSD-95, and Class III β-tubulin), oligodendrocytes (MBP, ASPA), astrocytes (glial fibrillary acidic protein [GFAP]), and microglia/macrophages (CD45). We calculated the relative difference in the levels of each marker protein in AD versus control samples, and then performed a Pearson correlation matrix analysis to assess the degree of relationship between the levels of BIN1 isoforms with the cell-type markers (Fig. 3d). The results show a significant correlation between BIN1:H and Amphiphysin 1 with neuronal markers in their relative expression level differences between controls and AD cases. In contrast, BIN1:L isoforms showed an inverse correlation with neuronal markers. Moreover, AD-associated changes in the levels of BIN1:L isoforms in the gray matter showed a high degree of correlation with the oligodendrocyte marker ASPA (Pearson r 0.897; p < 0.05). Significant correlation was also observed between the levels of BIN1:L isoforms and GFAP in the white matter (Pearson r 0.936; p < 0.02). Taken together, the results of qPCR and quantitative immunoblot analyses suggest neuronal and non-neuronal expression of BIN1, and reveal that the expression of BIN1 isoforms is differentially affected in brains of patients with AD.
Predominant white matter localization and oligodendrocyte expression of BIN1 in the human brain
Oligodendrocytes are responsible for axon myelination in the brain. Based on the intense white matter immunolabeling, we predicted that BIN1 is predominantly expressed in oligodendrocytes. In order to formally determine the identity of BIN1-positive cells in the human brain, we performed labeling of adjacent sections with multiple BIN1 antibodies and specific markers for mature oligodendrocytes, astrocytes, and microglia. Immunolabeling with an antibody against tubulin polymerisation-promoting protein p25 (TPPP), a marker for mature oligodendrocytes, revealed a cellular pattern of immunolabeling very similar to that of BIN1 antibodies in all brain regions studied. For example, TPPP stained nearly all the cells in the cerebral peduncle, which contains a large number of myelinating oligodendrocytes whose processes ensheath the axons that project to the cerebellum and the spinal cord (Fig. 4d). Adjacent sections stained with three BIN1 antibodies revealed intense labeling of the fiber tracts and labeling of the oligodendrocyte cell bodies within the cerebral peduncle. In contrast, BIN1 immunolabeling of oligodendrocytes and their fine processes in the CA1 region, as well as the intense immunolabeling of fiber tracks in the alveus, were strikingly distinct from the evenly dispersed Ionized calcium-binding adapter molecule 1 (Iba1) and GFAP immunolabeling of microglia and astrocytes, respectively (Fig. 4e). The predominant oligodendrocyte cell-type immunolabeling of BIN1 is also discernable based on a comparison of the structure of the stained population of cells, each with their distinctive cellular morphology (Fig. 4f). In order to determine more precisely the localization of BIN1 in myelinated axons, we performed two-color immunostaining with antibodies against BIN1 and Caspr (Contactin-associated protein), an axonal membrane protein. In comparison to Caspr, which becomes concentrated in the paranodal junctions of myelinated axons , BIN1 displayed patchy distribution along short segments of interdigitating processes without an apparent concentration in the nodes or the paranodal area (Fig. 4g). Moreover, we also observed annular immunolabeling of BIN1, which likely represent cross-sections of traversing myelinated axons. Together, these results show predominant oligodendrocyte expression and white matter localization of BIN1 in the human brain.
Cell-type specific expression and complementary localization of BIN1 and Amphiphysin 1
The findings detailed above were surprising because it is generally assumed, and in a few papers described, that BIN1 is expressed in neurons [4, 26, 30]. In order to carefully assess the distribution of BIN1 in the human brain, we performed immunohistochemical analysis of BIN1 and compared it to the distribution of Amphiphysin 1. Strikingly, adjacent sections stained with antibodies against Amphiphysin 1 and BIN1 revealed a near complementary pattern of expression in the cortex, hippocampal CA fields, hilus, subiculum, fimbria, caudate nucleus, lateral geniculate nucleus, and cerebral peduncle (Fig. 5a). Amphiphysin 1 was widely expressed in neurons throughout the cortex and hippocampus. Notably, Amphiphysin 1 immunolabeling was apparent in neuronal soma and was also intense in the neuropil (Fig. 5b and c). In comparison, we observed little, if any, neuronal or neuropil BIN1 immunolabeling in the cortex or hippocampus (Fig. 5b and c). On the contrary, BIN1 immunolabeling showed a strong signal in the white matter. Interestingly, whereas Amphiphysin 1 antibody labeled the mossy fiber terminal field in the hippocampus, these unmyelinated axons of the dentate gyrus granule cells are not labeled by the BIN1 antibody (Fig. 5c). Similarly, the Amphiphysin 1 antibody labeled the terminal zones of the associational and commissural pathways, as well as the perforant pathway in the molecular layer of the dentate gyrus, whereas BIN1 immunolabeling in this area was restricted to a few ramified cells and their processes (Fig. 5c). Thus, immunohistochemical staining and immunoblot analysis with independent antibodies support the assertion that BIN1 is enriched in the human brain white matter and shows little overlap with the expression and neuronal localization of its close homolog Amphiphysin 1.
BIN1 expression in mature oligodendrocytes and localization in the white matter tracts in rodent brain
Finally, BIN1-positive cells in the cortex and corpus callosum were positive for ASPA, TPPP, and CC1 epitope, markers for mature oligodendrocytes (Fig. 7e and f, and data not shown). Immunolabeling using an antibody against neural/glial antigen 2 (NG2) showed little or no expression of BIN1 in oligodendrocyte progenitor cells (Fig. 7g and h). Similarly, BIN1 was not expressed in microglia or astrocytes (Fig. 7i and j). BIN1 was not detected at appreciable levels in dendrites (identified by microtubule-associated protein 2 [MAP2] labeling) or presynaptic sites (synapsin 1 labeling) in the cortex (data not shown). As expected from BIN1 localization in myelinating oligodendrocytes, BIN1 immunolabeling showed overlap with the axonal marker phospho-neurofilament (data not shown). Together, the results described above are consistent with high-level BIN1 expression in mature oligodendrocytes and their processes in the brain.
Upregulation of BIN1 during developmental myelination and oligodendrocyte maturation
Loss of BIN1 immunolabeling in demyelinated plaques of patients with multiple sclerosis
In this paper, we document prominent BIN1 expression in mature oligodendrocytes and its localization within the white matter in the brain, which strikingly contrasts with neuronal and presynaptic localization of the BIN1 paralog, Amphiphysin 1. Our study reveals the upregulation of BIN1 expression during in vivo myelination as well as oligodendrocyte maturation in vitro. Moreover, we reveal the diversity of brain BIN1 isoforms and report on the alteration of select BIN1 isoform expression in patients with AD. Finally, we show that the loss of BIN1 immunoreactivity closely parallels myelin loss within brain lesions of individuals with multiple sclerosis in a pilot study. Altogether, our findings bring clarity to the issue of BIN1 expression and isoform diversity in the brain and provide novel insights on a possible non-neuronal role for BIN1, which has been genetically characterized as the second most significant risk factor for late-onset AD.
Predominant oligodendrocyte expression of BIN1
Summary of BIN1 expression in the brain analysed using cellular markers
NeuN, Amph 1, Calbindin, Parvalbumin
ASPA, TPP25, CNPase, CC1
Previous studies using the same antibodies employed in our analysis identified similar immunolabeling in the human brain as reported here, but attributed the labeling to neuronal soma or nucleus without comparing BIN1 immunolabeling with markers for neurons or oligodendrocytes [13, 26, 30]. Our results clearly show that, unlike Amphiphysin 1, the large majority of BIN1 immunoreactivity is not associated with neurons in the human brain (Fig. 5). In addition, it is clear that very little overlap exists between the cellular immunolabeling of NeuN and BIN1 throughout the rodent brain. Mature oligodendrocyte expression of BIN1 was established in our experiments using three commonly used cellular markers of oligodendrocytes. While our results are in agreement with a previous study reporting presynaptic localization of Amphiphysin 1 resembling that of synaptophysin in the cerebellum , they reveal that BIN1 localization markedly differs from that of synaptophysin. Specifically, BIN1 is highly enriched in white matter tracts in the cerebellum where it overlaps with MBP; in contrast synaptophysin and Amphiphysin 1 do not localize to the white matter (Figs. 5 and 6).
Although BIN1 mRNA expression in microglia acutely-isolated from mouse brain was observed in RNA-seq experiments , our results show little evidence in support of microglial localization of BIN1 at the protein level in the human or mouse brain. Similar to our findings, no overlap in the cellular expression of BIN1 and Iba1 was observed in the hippocampus of patients with AD . Furthermore, our study reveals BIN1 immunolabeling of morphologically distinct cell types from those stained by Iba1, CD45, and CD68, arguing against detectable levels of BIN1 expression in mouse or human brain microglia or macrophages in situ (Figs. 3, 6, 8, and Additional file 10: Figure S6). These findings are summarized in Table 2.
The significance of BIN1 expression in oligodendrocytes
High-level BIN1 protein expression in mature oligodendrocytes and the white matter in our studies are in agreement with the Barres lab’s identification of Bin1 among the top 50 highly expressed genes in cultured oligodendrocytes in a microarray study , and proteomic identification of BIN1 in human and mouse brain myelin preparations . Our results show an increase of BIN1 levels both during oligodendrocyte maturation in vitro and critical period of myelination in vivo. Upregulation of BIN1 during synchronous differentiation of cultured oligodendrocytes is consistent with earlier microarray and recent RNAseq datasets [32, 45]. Similarly, a significant decrease in BIN1 expression was observed in microarray analysis of multiple sclerosis brain lesions in comparison with control brain samples (GEO DataSet GDS4218) . In agreement, our analysis of brain tissue from patients with multiple sclerosis shows a close correlation between BIN1 levels and the intensity of myelin labeling across the different types of lesions. Notably, BIN1 labeling within the shadow plaques indicates possible reemergence of BIN1 expression during remyelination. Thus, BIN1 upregulation during oligodendrocyte maturation and its localization to myelinating oligodendrocytes have potential implications not only for normal physiology in the healthy brain but also for myelin repair under pathological conditions. OPCs remain abundant in the adult brain as lineage-restricted cells, which retain their capacity to proliferate and mature into myelinating oligodendrocytes as needed in response to demyelination resulting from injury or disease conditions. The lack of BIN1 expression in NG2-positive OPCs and the coordinate upregulation of BIN1 along with myelin-specific proteins suggest a possible role for BIN1 in membrane remodeling that accompanies the transition from the progenitors into myelinating oligodendrocytes with long arborized processes.
BIN1 isoform diversity in the brain
We report two important insights based on the results of our immunohistochemistry and immunoblot analyses. First, BIN1 levels are enriched in the human white matter in comparison to the gray matter. This finding is in agreement with the comparison of BIN1 transcript levels between the human white matter and the gray matter reported from independent microarray analyses [50, 52, 53]. Second, the isoform distribution of BIN1 between the gray matter and white matter differs significantly, as revealed by different BIN1 antibodies. The ~90 kDa BIN1 isoform is expressed only in the gray matter at lower levels, whereas the ~65-75 kDa BIN1 isoforms are the predominant forms in the gray matter and white matter. Expression of seven brain-specific and two ubiquitous BIN1 isoforms have been reported previously [4, 6, 7]. Consistent with the previous characterization that the neuronal BIN1 isoform 1 contains the intact CLAP domain responsible for binding to clathrin, we clearly see an enrichment of the ~90 kDa BIN1 (the largest BIN1 isoform in the brain) in the gray matter. However, it is somewhat surprising that the BIN1 isoform 1 is not the predominant BIN1 species in the human brain. Our immunoblot analysis of controls and AD patients revealed that the majority of BIN1 isoforms expressed in the brain corresponded to ~65-75 kDa, the reported size of the ubiquitous BIN1 isoforms, raising the possibility that they may lack the CLAP domain.
By direct sequencing of the human brain RT-PCR products, we have confirmed variable alternate splicing of BIN1 exons 13–16, which encodes the CLAP domain (Additional file 7: Figure S3). Thus, the results from immunoblot and RT-PCR analysis show that several human brain BIN1 isoforms lack the CLAP domain, entirely or at least partially. Our findings are similar to the previously reported RT-PCR analysis of BIN1 isoform composition in rat brain and peripheral tissue . Since the CLAP domain is required for BIN1 interaction with clathrin, these observations raise the possibility that large proportion of BIN1 expressed in the brain may not participate in clathrin-mediated endocytosis . Evidence from the analysis of Drosophila Amphiphysin, encoded by the unique Amphiphysin gene (and thought to be a BIN1 ortholog), show that absence of the CLAP domain results in no detectable defects in synaptic vesicle endocytosis in mutant flies [55–57]. Thus, the precise cellular functions of the major BIN1 isoforms in the brain remain to be established.
BIN1 expression in the brains of patients with AD
The functional connection between BIN1 and the susceptibility to AD remains to be elucidated. Initial attempts to determine whether BIN1 expression might be altered in AD have resulted in discordant findings. While two studies have reported significantly increased BIN1 transcripts levels in brains of individuals with AD [26, 31], another found a positive correlation between BIN1 expression levels and later age at onset of the disease . Apparent disagreements were also reported by immunoblot analysis of BIN1 expression in individuals with AD. Whereas, a significant decrease in BIN1 levels was observed by immunoblot analysis in one study , an increase in BIN1 levels was reported in another study . A comparison of the findings from these two reports with the results of our study offers some clarity to this apparent discrepancy. Uniformly, all three studies report a decrease of the ~90 kDa BIN1:H isoform and an increase of the ~65-75 kDa BIN1:L isoforms (Fig. 3). Further, we show that the decrease of the BIN1:H isoform correlated with a decrease in Amphiphysin 1 and neuronal markers, concurrent with the loss of neurons observed in AD. While BIN1:L expression highly correlated with oligodendrocyte markers, as expected from our immunostaining analysis, the correlation with GFAP or CD45 could also suggest an increase in BIN1:L expression in parallel with activation of astrocytes and microglia as well as infiltration of macrophages during pathogenesis. While this manuscript was under review, Adams and colleagues reported BIN1 expression in white matter and oligodendrocytes in the brain but not in neurons in healthy controls , consistent with our results. Interestingly, their study revealed weak BIN1 immunoreactivity in a subset of neurons in patients with AD. The findings described here complement the report by Adams et al. by identifying selective alterations of BIN1 isoforms in the gray matter of patients with AD.
A potential function for BIN1 in AD pathogenesis
There is substantial interest in understanding the functional connection between BIN1 and AD pathogenesis. In an earlier study, RNAi-mediated silencing of BIN1 expression in HeLa cells resulted in a 1.5- and 2-fold increase in Aβ40 and Aβ42 secretion, respectively . It is tempting to speculate that this effect could have resulted from alterations in proteolytic processing of APP in endosomes in cells lacking BIN1 expression. However, the lack of a significant change in sAPPβ production argues against a simple model whereby the loss of BIN1 had an effect on endocytic trafficking of APP or BACE1, thus promoting amyloidogenic processing of APP. Moreover, the loss of BIN1 function neither impaired endocytic transferrin uptake in Bin1−/− fibroblasts nor phagocytic internalization of zymosan . Thus, it is unlikely that RNAi knockdown of BIN1 introduced endocytic defects that altered Aβ generation. Interestingly, partial or total loss of Amphiphysin expression attenuated the rough eye phenotype induced by the overexpression of human Tau but not Aβ42 in Drosophila eye. A previous study showed an interaction between fly Amphiphysin and human Tau in a fly model of AD Tau pathology . Whether this functional interaction between BIN1 and Tau enhances AD risk in mammalian brain awaits experimental confirmation. In this regard, it is worth noting that Tau is expressed in mature oligodendrocytes and Tau recruitment to the sites of process outgrowth is a critical step in the initiation of myelination [60, 61]. Whether a functional interaction between BIN1 and Tau in oligodendrocytes has physiological and/or pathological implications in myelination is a topic worth investigating in the future.
AD pathology in the white matter includes the loss of myelin and a decrease in oligodendrocyte proteins [62–68]. The exposure of oligodendrocytes to Aβ induces apoptotic cell death  and studies in AD transgenic mouse models show defects in myelin integrity [70, 71]. Moreover, analysis of human post-mortem brain tissue suggests an age-related reduction in myelin repair capacity associated with AD pathogenesis . Therefore, insights into BIN1’s function in oligodendrocytes will advance our understanding of its involvement as an AD risk factor.
BIN1 is the most significant late-onset AD susceptibility locus identified via the genome-wide association studies. Multiple BIN1 isoforms are expressed in the human brain and there are significant changes in the levels of select BIN1 isoforms in the brains of individuals with late-onset AD. BIN1 is predominantly expressed by the myelinating cells in the brain and its expression is upregulated during myelin formation in vitro and in vivo. BIN is enriched in the white matter in the human and rodent brain and its diminution correlates with the extent of demyelination within subcortical plaques of patients with Multiple Sclerosis. Our results suggest a potential role for BIN1 in mature oligodendrocytes in the brain.
ASPA, aspartoacylase; BAR, Bin/Amphiphysin/Rvs; BIN1, Bridging Integrator 1; Caspr, contactin-associated protein; CC1, crenarchaeal chromatin protein 1; cDNA, complementary DNA; CLAP, CLathrin-Associated Protein binding region; CNPase, 2’,3’-Cyclic-Nucleotide 3’-Phosphodiesterase; GFAP, glial fibrillary acidic protein; Iba1, ionized calcium-binding adapter molecule 1; LOAD, late-onset alzheimer’s disease; mAb, monoclonal antibody; MAP2, microtubule-associated protein 2; MBP, myelin basic protein; NG2, neural/glial antigen 2; OPC, oligodendrocyte progenitor cell; pAb, polyclonal antibody; PSD95, post-synaptic density protein 95; qPCR, quantitative polymerase chain reaction; RT, reverse transcription; TPPP/p25, tubulin polymerisation-promoting protein p25
We thank the University of Kentucky AD Center Neuropathology Core for providing the human brain specimens used for RNA analysis. Postmortem tissue specimens for biochemical analysis were provided by NIH Neurobiobank through the Human Brain and Spinal Fluid Resource Center, VA West Los Angeles Healthcare Center, which is sponsored by NINDS/NIMH, National Multiple Sclerosis Society, and the Department of Veterans Administration. We thank the Histology Core Facility at the University of Chicago Human Tissue Resource Center for immunostaining of human tissue. We thank Dr. Manuel F. Utset for helpful suggestions during the course of this study.
This study was supported by Cure Alzheimer’s Fund (GT), National Multiple Sclerosis Society and Illinois Department of Public Health Multiple Sclerosis Research Fund Grant (GT), BrightFocus Foundation (SE), and National Institutes of Health grants AG019070 (GT), AG045775 (SE), and NS034939 (PB). P.D.R. was supported by an AD Research fellowship from the Illinois Department of Public Health and V.B.P was supported by a postdoctoral fellowship from BrightFocus Foundation. C.V.S. was supported by the National Center for Advancing Translational Sciences grant UL1TR000117. The authors acknowledge the University of Kentucky Alzheimer’s Center (supported by P30-AG028383) for human tissue samples. Confocal imaging was performed at the Integrated Microscopy Core Facility at the University of Chicago (supported by S10OD010649). The use of University of Chicago’s Core Facilities was supported by the National Center For Advancing Translational Sciences Award 5 UL1 TR 000430–09.
Availability of data and materials
Supplemental files are made available online along with the manuscript.
PDR, VBP, SE, and GT designed experiments and analyzed the data with the help of SK, BP, and PP. JBV, CVS, and SE performed human brain mRNA expression analysis. PDR, VBP, RL, AB, SS, RCR, and SK performed immunolabeling and PDR, VBP, SK, PP, and GT analyzed the data. PDR, RJA, ER, and CD performed immunoblotting and analyzed the data. BLLC, PDR, and BP characterized BIN1 expression in cultured oligodendrocytes and analyzed the data. PDR, VBP, and GT performed confocal imaging. PDR, VBP, SE, and GT wrote the manuscript. GT conceived of the study, coordinated data analysis, and prepared the final manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
All authors consent for the publication of this study.
Ethics approval and consent to participate
All research involving the animals have been approved by the University of Chicago Animal Care and Use Committee. Human subjects consent to participate is not applicable.
Pierre De Rossi and Virginie Buggia-Prevot are co-first authors.
VBP’s Present Address: The Neurodegeneration Consortium, The University of Texas MD Anderson Cancer Center, Houston, TX 77054, U.S.A.
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