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Bridging integrator 1 fragment accelerates tau aggregation and propagation by enhancing clathrin-mediated endocytosis in mice [1]

['Xingyu Zhang', 'Department Of Neurology', 'Renmin Hospital Of Wuhan University', 'Wuhan', 'Li Zou', 'Zhongnan Hospital Of Wuhan University', 'Li Tang', 'Min Xiong', 'Xiao-Xin Yan', 'Department Of Anatomy']

Date: 2024-01

The bridging integrator 1 (BIN1) gene is an important risk locus for late-onset Alzheimer’s disease (AD). BIN1 protein has been reported to mediate tau pathology, but the underlying molecular mechanisms remain elusive. Here, we show that neuronal BIN1 is cleaved by the cysteine protease legumain at residues N277 and N288. The legumain-generated BIN1 (1–277) fragment is detected in brain tissues from AD patients and tau P301S transgenic mice. This fragment interacts with tau and accelerates its aggregation. Furthermore, the BIN1 (1–277) fragment promotes the propagation of tau aggregates by enhancing clathrin-mediated endocytosis (CME). Overexpression of the BIN1 (1–277) fragment in tau P301S mice facilitates the propagation of tau pathology, inducing cognitive deficits, while overexpression of mutant BIN1 that blocks its cleavage by legumain halts tau propagation. Furthermore, blocking the cleavage of endogenous BIN1 using the CRISPR/Cas9 gene-editing tool ameliorates tau pathology and behavioral deficits. Our results demonstrate that the legumain-mediated cleavage of BIN1 plays a key role in the progression of tau pathology. Inhibition of legumain-mediated BIN1 cleavage may be a promising therapeutic strategy for treating AD.

Funding: This work was supported by the National Key Research and Development Program of China (No. 2019YFE0115900, to Z.Z.), the National Natural Science Foundation of China (No. 82271447 and 81822016, to Z.Z., No. 82301356 to X.Z., https://www.nsfc.gov.cn/english/site_1/index.html ), the Innovative Research Groups of Hubei Province (2022CFA026, to Z.Z., https://kjt.hubei.gov.cn/ ), the “New 20 Terms of Universities in Jinan” grant (No. 202228022, to Z.Z., https://jnsti.jinan.gov.cn/index.html ), the China Postdoctoral Science Foundation (No. 2021M702523, to X.Z., https://www.chinapostdoctor.org.cn ), and the Fundamental Research Funds for the Center University (No. 413000659 to X.Z., http://www.moe.gov.cn/ ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Legumain is an endolysosomal cysteine protease that cleaves its substrates after asparagine (N) residues [ 15 , 16 ]. As has been shown before, legumain is activated in the human AD brain [ 17 ]. Legumain cleaves tau and amyloid precursor protein (APP), mediating the formation of neurofibrillary pathology and the generation of amyloid-β [ 17 , 18 ]. These studies suggest that legumain plays an important role in AD. Here, we further investigated whether legumain is involved in BIN1-mediated tau pathology. We show that legumain cleaves BIN1 at the N277 and N288 residues, with N277 being the major cleavage site in the brains of AD patients. The legumain-generated BIN1 (1–277) fragment promotes the uptake and propagation of tau aggregates by enhancing CME. Moreover, BIN1 (1–277) interacts with tau and accelerates the de novo assembly of tau fibrils. Overexpression of BIN1 (1–277) in tau P301S mice facilitates the propagation of tau pathology and induces behavioral defects. Blockage of legumain-mediated cleavage of BIN1 ameliorates the pathological and behavioral deficits in tau P301S mice.

Bridging integrator 1 (BIN1), a ubiquitously expressed protein, plays a pivotal role in multiple cellular processes, including endocytosis and trafficking, membrane recycling, cell cycle progression, apoptosis, and cytoskeleton regulation [ 8 , 9 ]. At least 10 isoforms of BIN1 are expressed in different tissues, with the longest isoform (isoform 1) being specifically expressed in the brain. Isoform 1 contains a CLAP domain that interacts with clathrin and adaptor protein 2 (AP2), thus playing a role in clathrin-mediated endocytosis (CME) [ 10 , 11 ]. Genome-wide association studies (GWAS) identified BIN1 as the second most significant genetic risk locus for sporadic AD [ 12 , 13 ]. The expression of brain-specific BIN1 is decreased in the brain tissue of patients with AD, while that of shorter BIN1 isoforms is increased. The expression of BIN1 correlates with neurofibrillary tangle pathology [ 14 ]. Furthermore, brain-specific BIN1 was found to alleviate tau pathology, whereas the down-regulation of BIN1 enhanced the propagation of tau pathology [ 9 ]. However, the exact role of BIN1 in the onset and progression of AD has not been elucidated.

Alzheimer’s disease (AD) is the most common progressive neurodegenerative disorder. Pathologically, AD is characterized by the deposition of extracellular amyloid-β plaques and intraneuronal neurofibrillary tangles composed of aggregated tau. The extent of tau aggregation correlates better with the severity of neurodegeneration and cognitive impairment than amyloid deposits [ 1 ]. During the progression of AD, tau pathology usually starts in subcortical nuclei such as the locus coeruleus and then spreads to limbic regions, including the subiculum, hippocampal cornu ammonis (CA), and amygdala, before eventually progressing to the neocortex [ 2 ]. Converging evidence suggests that tau propagation may occur via a “prion-like” transmission mode [ 3 – 5 ], by which the aggregated tau induces the soluble tau monomers to form aggregates of the same conformation, initiating a self-amplifying cascade. Injection of human AD brain extracts containing tau aggregates into a mouse brain induces tau pathology, which spreads from the injection site to the brain regions anatomically connected to the injection site [ 3 , 4 ]. In vitro studies showed that extracellular tau aggregates can be taken up by cultured cells and “seed” the aggregation of soluble tau. Furthermore, pathological tau aggregates transfer between cells in a way similar to the propagation of prion protein [ 5 – 7 ]. However, the mechanisms underlying the propagation of tau pathology have yet to be elucidated.

Results

The BIN1 (1–277) fragment accelerates the uptake of tau aggregates Since BIN1 is an endocytosis-related protein, we further tested whether the legumain-generated BIN1 fragments influence the uptake of pathological tau in primary cultured neurons. The purity of the neurons was confirmed by immunostaining with the neuronal marker MAP2, astrocyte marker GFAP, and microglial marker Iba1 (S4A Fig). The neurons were infected with adeno-associated virus (AAV) encoding GFP-tagged BIN1, BIN1 (1–277), and BIN1 (278–594), respectively. GFP signals were detected only in neurons that stained positive for MAP2 (S4A Fig). The cell viability of neurons expressing full-length BIN1 or its fragments was similar (S4B Fig). The neurons were then exposed to tau RD fibrils. The uptake of tau RD fibrils was significantly enhanced in the presence of the BIN1 (1–277) fragment (Fig 3A and 3B). To investigate whether BIN1 fragments alter the degradation rate of tau, neurons were exposed to K18 fibrils for 30 min and washed with PBS to eliminate free fibrils. The K18 signals in neurons were observed at different time points after washing. There was no difference in degradation rates among the 4 groups (S5A and S5B Fig), indicating that increased signals in BIN1 (1–277) were not due to impairment of the degradation system. PPT PowerPoint slide

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TIFF original image Download: Fig 3. BIN1 (1–277) promotes the propagation of tau pathology. (a, b) Representative immunofluorescence images showing the uptake of K18 fibrils by neurons expressing EGFP, BIN1, BIN1 (1–277), and BIN1 (278–594). Neurons treated with dynasore were used as negative control. Scale bar, 5 μm. Quantification of K18 fibril uptake (b). The uptake of K18 fibrils was calculated as the mean fluorescence density of 60 cells under each condition. The fluorescence density was normalized to the Vector+DMSO group (mean ± SEM n = 60 cells per group; ***P < 0.001). (c) Transmission of tau aggregates from the donor cells to COS-7 cells transfected with HA-vector, BIN1, BIN1 (1–277), and BIN1 (278–594). The right panel shows the three-dimensional image of cells transfected with HA-BIN1 (1–277). Scale bar, 20 μm. (d) Quantification of the percentage of cells containing aggregates in cells expressing HA-vector, BIN1, BIN1 (1–277), or BIN1 (278–594). Data represent mean ± SEM of 4 independent experiments. *P < 0.05, **P < 0.01. Source data can be found in S1 Data. BIN1, bridging integrator 1; DMSO, dimethyl sulfoxide; EGFP, enhanced green fluorescent protein; FL, full-length; HA, human influenza hemagglutinin; YFP, yellow fluorescent protein. https://doi.org/10.1371/journal.pbio.3002470.g003 We further detected the cell-to-cell propagation of tau aggregates in a coculture system. COS-7 cells were transfected with HA-BIN1, HA-BIN1 (1–277), or HA-BIN1 (278–594) and cocultured with HEK293 cells that consistently contain tau inclusions [19]. The cells expressing BIN1 (1–277) exhibited more inclusions translated from the donor cells than the other groups, which was confirmed by the three-dimensional image rendered from the Z-stack (Fig 3C and 3D). Overall, these results demonstrate that BIN1 (1–277) promotes the uptake of tau fibrils and enhances the cell-to-cell transmission of tau pathology.

The BIN1 (1–277) fragment promotes CME BIN1 interacts with clathrin, AP2, and dynamin and regulates CME, a process involved in the uptake of tau fibrils in AD [20,21]. We investigated whether the legumain-mediated fragmentation of BIN1 regulates the CME process using transferrin uptake assay in primary neurons (Fig 4A). Interestingly, overexpression of EGFP-BIN1 (1–277) enhanced transferrin uptake (Fig 4A and 4B). The uptake of transferrin was blocked by the endocytosis inhibitor dynasore in neurons expressing BIN (1–277) (Fig 4C and 4D). The uptake of tau fibrils was also inhibited by dynasore (Fig 3A). We then tested the uptake of FM 4–64 dye (FM) by neurons and found that BIN1 (1–277) dramatically enhanced the uptake of the FM 4–64 dye, while KCl-induced loss of fluorescence was not influenced by BIN1 fragments (Fig 4E–4G). These results indicate that the BIN1 (1–277) fragment promotes CME and the uptake of tau fibrils. PPT PowerPoint slide

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TIFF original image Download: Fig 4. BIN1 (1–277) enhances CME. (a) Transferrin uptake assay of neurons expressing EGFP, BIN1, BIN1 (1–277), and BIN1 (278–594). Scale bar, 5 μm. (b) Quantification of transferrin uptake in (a). The uptake of transferrin was calculated as the mean fluorescence density of 30 cells under each condition. The fluorescence density was normalized to the GFP-Vector group (mean ± SEM n = 30 cells; *P < 0.05, ***P < 0.001). (c, d) Transferrin uptake assay of neurons expressing BIN1 (1–277) in the presence or absence of dynamin inhibitor dynasore. Scale bar, 5 μm. (d) Quantification of transferrin uptake in (c). Mean ± SEM n = 30 cells; ***P < 0.001. (e) FM 4–64 uptake assay in neurons expressing EGFP-vector, BIN1, BIN1 (1–277), and BIN1 (278–594). Scale bar, 5 μm. (f) FM 4–64 dye after KCl-induced loss of fluorescence in neurons expressing EGFP-vector, BIN1, BIN1 (1–277), and BIN1 (278–594). Scale bar, 5 μm. (g) Quantification of FM 4–64 uptake. FM 4–64 labeling was calculated as the integral fluorescence intensity of 30 boutons under each condition. The fluorescence density was normalized to the control group (mean ± SEM n = 30 cells; *P < 0.05, **P < 0.01. ***P < 0.001). (h) Immunofluorescence showing the distribution of dynamin in neurons expressing EGFP, EGFP-BIN1, EGFP-BIN1 (1–277), and EGFP-BIN1 (278–594). Scale bar, 5 μm. (i, j) Quantification of the number and size of the dynamin puncta (mean ± SEM n = 30 cells; *P < 0.05). (k) Immunofluorescence showing the expression of Rab5 in neurons expressing EGFP, EGFP-BIN1, EGFP-BIN1 (1–277), and EGFP-BIN1 (278–594). Scale bar, 5 μm. (l, m) Quantification of the number and size of the Rab5 puncta (mean ± SEM n = 30; *P < 0.05, ***P < 0.001). Source data can be found in S1 Data. BIN1, bridging integrator 1; CME, clathrin-mediated endocytosis; DMSO, dimethyl sulfoxide; EGFP, enhanced green fluorescent protein; FL, full-length; FM, FM 4-64 dye; GFP, green fluorescent protein. https://doi.org/10.1371/journal.pbio.3002470.g004 Considering that BIN1 interacts with dynamin and sequesters dynamin to inhibit CME [22], we assessed whether legumain-fragmented BIN (1–277) plays a different role in this process. Consistent with the previous report [22], full-length BIN1 increased the dynamin puncta size, but BIN1 (1–277) did not affect the size of dynamin puncta (Fig 4H–4J). In addition, the Glutathione S-Transferase (GST) pull-down assay demonstrated that BIN1 (1–277) failed to interact with dynamin as full-length BIN1 did (S5C Fig). These results indicate that BIN1 (1–277) loses the physiological function of full-length BIN1 to sequester dynamin. We further tested the expression of the early endosome marker Rab5. Overexpression of full-length BIN1 resulted in a decreased number and smaller size of Rab5-positive endosomes, while BIN1 (1–277) increased the number and size of Rab5 puncta relative to full-length BIN1 (Fig 4K–4M). These results imply that the BIN1 (1–277) fragment abolishes the inhibitory effect of full-length BIN1 on endocytosis.

BIN1 (1–277) interacts with tau and promotes tau aggregation To determine whether BIN1(1–277) directly interacts with tau and regulates its aggregation, brain sections from tau P301S transgenic mice and age-matched control mice were stained with phospho-tau (p-tau) and BIN1 (1–277) antibodies, revealing that BIN1 (1–277) colocalized with p-tau (Fig 5A). Furthermore, a pull-down assay found that BIN1 (1–277) interacts with fibrils (F) formed by the tau repeat domain (RD, K18) but not K18 monomers (M) or oligomers (O). We further overexpressed GFP-Vector, GFP-BIN1 full-length (FL), GFP-BIN1 (1–277), or GFP-BIN1 (278–594) in HEK293 cells or primary neurons derived from the tau P301S mouse brain. We found that GFP-BIN1 (1–277) aggregated into inclusions after transduction of the K18 fibrils (Fig 5C and 5D). These results suggest that K18 fibrils may initiate the assembly of BIN1 (1–277). Furthermore, we explored the effect of BIN1 (1–277) on the aggregation of tau using HEK293 cells stably expressing the GPF-tagged tau RD as reporter cells, as intracellular inclusions are formed when reporter cells are transduced with tau K18 fibrils [19]. We found that aggregates formed after expressing HA-BIN1 (1–277), and the aggregates of tau RD colocalized with HA-BIN1 (1–277), indicating that BIN1 (1–277) and tau may assemble together (Fig 5E and 5F). PPT PowerPoint slide

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TIFF original image Download: Fig 5. BIN1 (1–277) binds tau and facilitates its assembly. (a) Colocalization of AT8 and BIN1 (1–277) in the cortex and hippocampus of tau P301S mice. Scale bar, 20 μm. (b) His pull-down assay showing the interaction between BIN1 (1–277) and K18 fibrils. (c) BIN1 (1–277) enhances the seeding activity of K18 fibrils in clone 1 cells (upper panel) and primary neurons from tau P301S mice (lower panel). Scale bar, 20 μm. (d) The percentage of cells with inclusions in clone 1 cells (left panel) and primary neurons from tau P301S mice (right panel) (mean ± SEM n = 5 independent experiments. ***P < 0.001). (e, f) The Clone1 cells expressing HA-Vector, HA-BIN1 FL, HA-BIN1 (1–277), or HA-BIN1 (278–594) were transduced with K18 fibrils (1 μM) for 24 h. Scale bar, 20 μm. The bar graph shows the percentage of cells with inclusions (mean ± SEM n = 5 independent experiments. ***P < 0.001). (g) ThS assay showing the assembly kinetics of K18 in the presence of the BIN1 N277 peptide (265–277), BIN1 N288 peptide (265–287), and BIN1 spanning peptide (282–294), respectively. (h) Electron micrographs of the fibrils formed by incubating K18 for 12 h in the presence or absence of BIN1 N277 peptide. Scale bar, 120 nm. Source data can be found in S1 Data and S1 Raw Images. a.u., arbitrary unit; BIN1, bridging integrator 1; F, fibrils; FL, full-length; GFP, green fluorescent protein; HA, human influenza hemagglutinin; M, monomers; O, oligomers; WB, western blot. https://doi.org/10.1371/journal.pbio.3002470.g005 To confirm the effect of BIN1 (1–277) on the aggregation of tau in vitro, we recorded the kinetics of amyloid assembly by tau-K18 in the presence or absence of the BIN1 N277 peptide (265–277 aa). The thioflavin S (ThS) fluorescence assay found that the BIN1 N277 peptide dramatically promoted the aggregation of tau-K18, with shorter lag times, steeper elongation phases, and higher final signals than tau-K18 alone, while the BIN1 spanning peptide (282–294 aa) and BIN1 N288 peptide (265–288 aa) showed no effect on the kinetics of K18 aggregation (Fig 5G). Under electron microscopy, the mixed fibrils consisting of K18 and N277 peptide were longer than the K18 fibrils and were highly ordered with a paired arrangement (Fig 5H), indicating that the BIN1 N277 peptide accelerates tau assembly and generates ordered paired helical structures. Co-sedimentation analysis detected the presence of both tau and BIN1 N277 peptide in the pellet fractions (S6A and S6B Fig). We further tested the seeding activity of K18 fibrils formed in the presence or absence of the BIN1 N277 peptide. When transduced into reporter cells, the BIN1 (1–277)-K18 fibrils induced more inclusions than the K18 fibrils formed in the absence of the BIN1 N277 peptide (S6C Fig). Overall, these results indicate that the BIN1 (1–277) fragment interacts with tau and accelerates its aggregation.

Overexpression of uncleavable BIN1 ameliorates tau pathology in vivo To verify the role of legumain-mediated BIN1 fragmentation in tau propagation, we injected AAVs encoding wild-type BIN1 or N277A/N288A mutant BIN1 that cannot be cleaved by legumain, together with K18 fibrils into the DG area of 2-month-old tau P301S mice. One month later, the severity of tau pathology was similar in both groups (S11A Fig). However, 2 months after injection, tau pathology was observed in the ipsilateral CA1 area in mice expressing wild-type BIN1 but not in mice expressing the uncleavable BIN1 (S11B Fig). Six months after injection, the ipsilateral DG, CA3, and CA1 areas in mice overexpressing wild-type BIN1 displayed more tau pathology than those in mice expressing the uncleavable BIN1 (Figs 7A and S12). PPT PowerPoint slide

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TIFF original image Download: Fig 7. Uncleavable BIN1 inhibits the propagation of tau pathology. (a) AT8 immunostaining of the DG, CA3, and CA1 areas of the HP in tau P301S mice 6 months after the injection of a mixture of K18 fibrils and AAVs encoding wild-type or N277A/N288A mutant BIN1. Scale bar of the whole HP, 280 μm; scale bar of DG, CA3, and CA1, 140 μm. (b, c) Morris water maze analysis of the distance traveled to the platform (b) and the distance traveled to the platform on day 7 (c) (mean ± SEM; n = 6–10 mice per group; *P < 0.05, Student t test). (d) Probe trial of the Morris water maze test analyzed as time spent in the target quadrant versus the average of time spent in other quadrants (mean ± SEM; n = 6–10 mice per group; **P < 0.01, Student t test). (e) Swim speed of mice injected with AAVs encoding EGFP, EGFP-BIN1 FL, and EGFP-BIN1 N277A/N288A (mean ± SEM; n = 6–10 mice per group, Student t test). Source data can be found in S1 Data. AAV, adeno-associated virus; BIN1, bridging integrator 1; DG, dentate gyrus; EGFP, enhanced green fluorescent protein; FL, full-length; HP, hippocampus. https://doi.org/10.1371/journal.pbio.3002470.g007 Electron microscopy and Golgi staining indicated that mice injected with uncleavable BIN1 showed a higher density of hippocampal synapses and dendritic spines than mice injected with uncleavable BIN1 (S12A–S12D Fig). In addition, in the water maze test, mice overexpressing uncleavable BIN1 traveled less distance to find the platform during the training phase than mice expressing wild-type BIN1 (Fig 7B and 7C). In the probe trial, the mice expressing mutant BIN1 spent more time in the target quadrant (Fig 7D). The swimming speeds of all mice were comparable (Fig 7E). These results indicate that blocking the cleavage of BIN1 by legumain alleviates the spreading of tau pathology in a mouse model of tauopathy.

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