The Alzheimer’s disease risk gene BIN1 regulates activity-dependent gene expression in human-induced glutamatergic neurons

doi:10.21203/rs.3.rs-3017048/v1

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The Alzheimer’s disease risk gene BIN1 regulates activity-dependent gene expression in human-induced glutamatergic neurons

https://doi.org/10.21203/rs.3.rs-3017048/v1

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Bridging Integrator 1 (BIN1) is the second most important Alzheimer’s disease (AD) risk gene, but its physiological roles in neurons and its contribution to brain pathology remain largely elusive. In this work, we show that BIN1 plays a critical role in the regulation of calcium homeostasis, electrical activity, and gene expression of glutamatergic neurons. Using single-cell RNA-sequencing of cerebral organoids generated from isogenic BIN1 wild-type (WT), heterozygous (HET) and homozygous knockout (KO) human-induced pluripotent stem cells (hiPSCs), we show that BIN1 is mainly expressed by oligodendrocytes and glutamatergic neurons, like in the human brain. Both HET and KO cerebral organoids show specific transcriptional alterations, mainly associated with ion transport and synapses in glutamatergic neurons. We then demonstrate that BIN1 cell-autonomously regulates gene expression in glutamatergic neurons by using a novel protocol to generate pure culture of human-derived induced neurons (hiNs). Using this system, we also show that BIN1 plays a key role in the regulation of neuronal calcium transients and electrical activity via its interaction with the L-type voltage-gated calcium channel Cav1.2. BIN1 KO hiNs show reduced activity-dependent internalization and higher Cav1.2 expression compared to WT hiNs. Pharmacological blocking of this channel with clinically relevant doses of nifedipine, a calcium channel blocker, partly rescues neuronal electrical and gene expression alterations in BIN1 KO glutamatergic neurons. Further, we show that transcriptional alterations in BIN1 KO hiNs affecting biological processes related to calcium homeostasis are also present in glutamatergic neurons of the human brain at late stages of AD pathology. Together, these findings suggest that BIN1-dependent alterations in neuronal properties could contribute to AD pathophysiology and that treatment with low doses of clinically approved calcium blockers should be considered as an option to dampen disease onset and progression.

Biological sciences/Neuroscience

Biological sciences/Stem cells

Biological sciences/Cell biology

The Bridging Integrator 1 (BIN1) is the second most associated genetic determinant with the risk of late-onset Alzheimer's disease (LOAD), after the Apolipoprotein E (APOE) gene [1–4]. In the adult human brain, BIN1 is mainly expressed by oligodendrocytes, microglial cells and glutamatergic neurons [5–7] and its expression is reduced in AD patients compared to healthy individuals [7, 8]. The consequences of this reduced BIN1 expression to neuronal and glial cells, as well as the mechanisms by which it contributes to AD pathogenesis remain poorly understood.

BIN1 has disputably been associated with amyloidopathy and tauopathy, two pathological hallmarks of AD [9]. Reduced BIN1 expression results in a higher amyloid precursor protein (APP) processing towards the production of amyloid-beta (Aβ) peptides in Neuroblastoma Neuro2a cells [10, 11]. However, we previously showed that BIN1 knockout (KO) does not increase the concentrations of Aβ peptides in hiPSC-derived neurons (human-induced neurons or hiNs) despite impairing endocytic trafficking [12]. Likewise, reduced Bin1 expression in the mouse brain does not affect the production of endogenous Aβ peptides [13]. Concerning Tau pathology, decreased expression of the BIN1 ortholog Amph suppress Tau-mediated neurotoxicity in Drosophila Melanogaster [14]. In contrast, reduced BIN1 expression results in higher Tau aggregation and propagation in primary rat hippocampal neurons [15]. In humans, higher concentrations of phosphorylated Tau are observed in the cerebrospinal fluid of patients with AD and are significantly correlated with genetic variants within the BIN1 locus [16].

More recently, BIN1 has also been associated with the regulation of synaptic transmission and neuronal electrical activity in animal models. Conditional deletion of Bin1 in neurons of the adult mice hippocampus leads to altered frequency of mini excitatory post-synaptic currents (mEPSC), likely due to an impaired presynaptic release probability and slower depletion of neurotransmitters [6]. Knockdown of BIN1 in embryonic rat primary cortical neurons also affects the glutamate AMPA receptor trafficking in the post-synaptic compartment, leading to alterations in the amplitude of mEPSC [17]. Lastly, overexpression of a BIN1-mKate2 fusion protein increases the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in embryonic rat hippocampal cultures, seemingly by affecting the localization of L-type voltage gated calcium channels (LVGCC) in the membrane through a Tau-dependent interaction [18].

Despite these advances, no consensus has been reached on the roles of BIN1 in AD pathogenesis and even its physiological functions in human brain cells remain mostly unknown. In this work, we tackled this important question by generating and characterizing human neural cells derived from isogenic BIN1 wild type (WT), heterozygous (HET) and homozygous knockout (KO) hiPSC lines. We first characterized the transcriptional profile of human neural cells grown in three-dimensional cerebral organoids for more than 6 months and show that reduced BIN1 expression affects mainly glutamatergic neurons. Next, we generated pure BIN1 WT and KO hiNs cultures and show that BIN1 cell-autonomously regulates electrical activity and gene expression of glutamatergic neurons via the interaction with the regulation of the LVGCC Cav_1.2. Pharmacological blockage of this channel with nifedipine partly rescues electrical and gene expression alterations in BIN1 KO hiNs. Our findings suggest that BIN1 is a key regulator of calcium homeostasis in glutamatergic neurons and that repurposing the use of clinically approved calcium channel blockers could be a promising strategy to treat AD.

hiPSC lines and neural differentiation

Isogenic hiPSCs (ASE 9109, Applied StemCell Inc. CA, USA) modified for BIN1 in exon 3 were generated by CRISPR/Cas9. Homozygous null mutants for BIN1 had a 5 bp deletion on one allele and an 8 bp deletion on the other allele. Heterozygous for BIN1 had a 1bp insertion on one allele. All hiPSCs, and all subsequent human induced neural progenitor cells (hiNPCs), hiNs, human induced astrocytes (hiAs), and cerebral organoids derived thereof, were maintained in media from Stemcell Technologies. Maintenance of cell cultures and organoids were done in adherence with manufacturer’s protocols which can be found on the webpage of Stemcell Technologies. To generate pure neuronal hiNs culture, we expressed Ascl1 in hiNPCs.

Electrophysiological recordings and analyses

ASCL1-hiNs were cultured in microfluidic devices bound to multi-electrode arrays (256MEA100/30iR-ITO, Multi-Channel Systems, Germany) and extracellular action potentials were recorded in 5 different cultures for both genotypes at 2, 3, 4 and 6 weeks of differentiation using the MEA2100-256-System (Multi-Channel Systems). For rescue experiments, ASCL1-hiNs were cultured on MEA 96-well plates (CytoView MEA 96, Axion Biosystems, USA) and extracellular action potentials were recorded in 3 independent cultures for either genotype in the presence of 50nM nifedipine (Tocris Bioscience) or vehicle using the MaestroPro (Axion Biosystems, Inc, USA). Channels containing detected waveforms were processed offline for spike waveform separation and classification using Offline Sorter v3 (Plexon, USA).

Single-nucleus RNA-sequencing (snRNAseq)

Nuclei isolation and Hash Tag Oligonucleotide (HTO) libraries preparation were performed as previously described (Lambert et al., 2022).

Statistical analyses

Statistical analyses were performed using GraphPad Prism version 8.0.0 (GraphPad Software, San Diego, California USA, www.graphpad.com) and R 4.2.0 (R Core Team, 2022, https://cran.r-project.org/bin/windows/base/old/4.2.0/). Bar plots show mean ± SD and individual values. Box plots show 1-99 percentile. Sample sizes, statistical tests and p values are indicated in Figure legends.

BIN1 HET and KO cerebral organoids show transcriptional alterations associated with neuronal functional properties

Cerebral organoids (COs) faithfully recapitulate fundamental aspects of the three-dimensional organization of the human brain, including the molecular specification of different neural cell types/subtypes and the generation of complex electrical activity patterns [20, 21]. To investigate the potential role of BIN1 in human neural cells, we generated and characterized COs using isogenic BIN1 wild type (WT), heterozygous (HET) and KO hiPSCs (Figure 1A). After 6.5 months of culture, COs were composed of all the major neural cell types identified by the expression of MAP2, GFAP and NESTIN and we did not observe any gross difference in size or morphology of COs among the three genotypes (Supplementary Figure1A). Western blot analyses confirmed the reduction and absence of BIN1 protein in BIN1 HET and KO COs, respectively (Supplementary Figure 1B). We have then employed single-nucleus RNA sequencing (snRNA-seq) to further characterize individual cell types/subtypes and investigate possible gene expression alterations associated with reduced BIN1 expression. COs (n=4 from each genotype) were divided into two halves that were independently processed for western blotting or snRNA-seq. We observed similar expression of general neuronal and glial proteins in these COs (Supplementary Figure 1C), suggesting a low degree of heterogeneity in these samples. Nevertheless, to further reduce potential batch effects, we pooled COs into a single multiplexed library using Cell Hashing (Stoeckius et al., 2018). After sequencing, quality control and demultiplexing, we recovered 4398 singlets that could be grouped into 7 major cell clusters based on the expression of cell type markers SLC1A3 (GLAST), GFAP and TNC (astrocyte); SNAP25, DCX, MAPT (pan-neuronal); SLC17A7 and SLC17A6 (glutamatergic neurons); DLX1, GAD1 and GAD2 (GABAergic neurons); HES6, CCND2 and CDK6 (NPCs); ITGA8 (choroid plexus); and CLIC6 (pigmented epithelium) (Figure1B-C). BIN1 expression in COs was mainly detected in glutamatergic neurons and oligodendrocytes (Figure1C), similar to the profile described for the human brain [7] - except for brain microglial cells that express BIN1 but are not present in COs. We also observed a reduction and enlargement, respectively, in the proportions of glutamatergic neurons and astrocytes in BIN1 KO compared to WT (Figure1D; ****p<0.0001; Chi-square test).

To identify possible differentially expressed genes (DEGs) in BIN1 KO or HET compared to WT cells, we performed Wilcoxon test for each major cell type/subtype identified in COs. Consistent with the predominant expression of BIN1 in glutamatergic neurons (Figure 1D), we identified a high number of DEGs in this cell type both in BIN1 HET (76 genes) and KO (124 genes) compared to WT genotype (Figure1E; SupplementaryFigure1D). In astrocytes, we also detected 75 DEGs in BIN1 KO, but only 6 DEGs in BIN1 HET compared to WT (Figure 1E; Supplementary Figure 1D). For all other cell types, we observed a maximum of 1-4 DEGs in the comparison between KO vs WT or HET vs WT (Supplementary Table 1). These observations suggest that both BIN1 null (KO) and partial deletion (HET) affect similar biological process in glutamatergic neurons in a dose-dependent manner. Accordingly, similar GO terms were enriched for DEGs identified in BIN1 KO or HET glutamatergic neurons, including several terms associated with synaptic transmission (Figure 1F). For BIN1 KO glutamatergic neurons, we also identified GO terms associated with ion channel complex and calcium ion binding (Supplementary Figure 1E; Supplementary Table 2), further suggesting that BIN1 depletion leads to specific transcriptional changes associated with functional properties of this neuronal subtype.

Altered expression of activity-related genes in BIN1 KO and HET COs

Neuronal firing patterns (such as tonic and burst firing) play a key role on the transcriptional regulation of a particular set of genes designated activity-related genes (ARGs) [22]. While neurons stimulated with brief patterns of electrical activity transcribe rapid primary response genes (rPRGs) or early response genes (ERGs), those stimulated with sustained patterns of electrical activity express delayed primary response genes (dPRGs), secondary response genes (SRGs) or late response genes (LRGs) (Figure 2A)[23, 24]. Using Cell-ID [25], we quantified the enrichment for ARGs signatures (Supplementary Table 3) in COs at single-cell resolution as an indirect read-out of neuronal electrical activity patterns in this model. We first confirmed that ARG signatures were predominantly enriched in neurons (Figure 2B). Then, we quantified the proportion of glutamatergic or GABAergic neurons significantly enriched for such specific response gene signatures (p_adj<0.05; hypergeometric test). We observed a significantly higher proportion of glutamatergic neurons enriched for dPRGs and LRGs both in BIN1 HET and KO, as well as SRGs in BIN1 KO compared to WT glutamatergic neurons, whereas the proportion of glutamatergic neurons enriched for rPRGs and ERGs was reduced in BIN1 HET and KO (Figure2 C). In sharp contrast, the only difference observed in GABAergic neurons was a reduction in the proportion of cells enriched for SRGs (Supplementary Figure 2). These results suggest that reduced BIN1 expression in glutamatergic neurons triggers neuronal firing patterns towards sustained activity leading to a higher expression of late-response ARGs.

Lower numbers of synaptic puncta in BIN1 HET and KO COs compared to WT

Transcriptional alterations in BIN1 HET and KO glutamatergic neurons are also suggestive of synaptic dysfunction, which is an early hallmark of AD pathology [26]. We thus sought to determine whether BIN1 depletion could affect synaptic connectivity in COs. Using immunohistochemistry to detect the expression of the pre-synaptic protein Synaptohysin-1 (SYP) and post-synaptic protein HOMER1, we were able to quantify the frequency of putative synaptic contacts (% SYP assigned) in COs (see methods). We observed a significant reduction in the % of SYP assigned both in BIN1 HET and KO compared to WT, mainly due to a reduction in the number of post-synaptic spots expressing HOMER1 (Figure 2D-G).

Next, we investigated whether APP processing and Tau phosphorylation, which have been previously associated with BIN1 and are known to modulate neuronal electrical activity [10, 15, 27], could also be altered in BIN1 HET and KO COs. To this end, we measured the intracellular levels of full-length APP and APP β-CTF (as a readout of amyloidogenic APP processing), total and phosphorylated TAU proteins by western blotting. Besides a trend for reduced TAU expression, likely explained by the reduced proportion of neurons (Figure 1), we did not detect any significant differences in the intracellular levels of APP, APP β-CTF, TAU or phospho-TAU (Ser202, Thr205) in BIN1 HET and KO compared to WT COs (Supplementary Figure 3). Altogether, these results suggest that BIN1 depletion could alter neuronal functional properties without significantly affecting APP or Tau metabolism in COs.

Cell-autonomous role of BIN1 in the regulation of neuronal gene expression

Our results in COs suggest that reduced BIN1 expression affects mainly glutamatergic neurons. However, at least in BIN1 KO COs, we cannot completely rule out an effect of BIN1 deletion in astrocytes that could indirectly impact glutamatergic neurons. Therefore, to unambiguously probe the cell-autonomous effect of BIN1 deletion on the electrical activity and gene expression of human glutamatergic neurons, we generated BIN1 WT or KO pure neuronal cultures by direct lineage-reprogramming of human NPCs (hNPCs) using doxycycline-inducible expression of ASCL1 (Figure 3A; see online methods). After validation of the efficient lineage-reprogramming of hNPCs into highly pure neurons (hereafter ASCL1-hiNs) (Figure 3B), we added exogenous human cerebral cortex astrocytes to trophically support functional neuronal maturation and synaptic connectivity [28]. Using snRNA-seq after 4 weeks of differentiation we identified 5583 cells (n=5 independent culture batches) clustered into two main glutamatergic neuron (GluNeu-I and II), one GABAergic neuron (GABANeu), one immature/unspecified neuron (UnspNeu), two astrocyte (Astro-I and II) and 1 proliferative NPC groups (Figure 3C-D). Sample-level differential gene expression analysis using DESeq2 [29], revealed 99 DEGs (|log2FC| >0.25 and FDR <0.05) in BIN1 KO GluNeu-II compared to WT, but only 2 in GluNeu-I and 1 in immature neurons (Figure 3E; Supplementary Table 4). As observed in COs (Figure 1H-I), GO term enrichment analysis revealed a significant enrichment for terms associated with synaptic transmission, ion channel activity and calcium signaling pathways (Figure 3F; Supplementary Table 5). The percentage of GluNeu-II enriched for late-response ARGs was slightly greater in BIN1 KO compared to WT, but without statistical differences (Figure 3G-H). Exogenously added human astrocytes co-cultured with BIN1 WT and KO hiNs also showed a low number of DEGs (11 in Astro-I; Supplementary Table 4), likely reflecting an astrocyte reaction to primary changes in hiNs in response to BIN1 deletion.

BIN1 KO leads to alteration in the electrical activity pattern of ASCL1-hiNs

The transcriptional changes observed in our 2D and 3D models could suggest that reduced BIN1 expression is associated with altered electrical properties of glutamatergic neurons. To directly address this possibility, we used multi-electrode arrays (MEA) to record and quantify multi-unit activity (MUA) in ASCL1-hiNs. As previously described in spontaneously differentiated hiPSC-derived neuronal cultures [30], ASCL1-hiNs cells exhibited a diverse range of spontaneous activity patterns, including regular discharges, population bursts and period activity (Supplementary Figure 4A). In this respect, we found a conspicuous change in the temporal organization of MUA after BIN1 deletion (Supplementary Figure 4A), mainly characterized by a greater number of spike bursts at 4 weeks (Supplementary Figure 4D). These alterations may result from changes at the single cell or the population level (different number of neurons contributing to each electrode, for example). To disentangle these possibilities, we used waveform-based spike sorting to examine the functional consequences of BIN1 deletion at the single neuronal level (Figure 4A). We identified a similar number of single units per recording electrode between genotypes (WT: 4.92±2.34; KO: 5.27±2.45), indicating that BIN1 deletion does not affect the density of active neurons within culture. However, we observed reduced single-unit activity (SUA) frequency (Figure 4B) and higher SUA amplitude (Figure 4C) in BIN1 KO compared to WT ASCL1-hiNs. Interestingly, we could not detect significant changes in the number of bursts per neuron (WT: 11.01±6.71; KO: 10.36±8.59), although both the burst duration and the number of spikes within a burst were significantly lower in BIN1 KO compared to WT ASCL1-hiNs (Figure 4D-E). We also observed a prominent temporal disorganization of BIN1 KO hiNs activity by computing the array-wide spike detection rate (ASDR, Figure 4G), which reveals the strength of the synchronized population activity, and the autocorrelograms of SUAs (Figure 4H-I), which allows the apprehension of synchronized periodicity. These analyses revealed that most spikes of BIN1 WT neurons were organized in bursts occurring at periodic intervals of about 8-10 s, whereas the spikes of BIN1 KO neurons were randomly distributed, leading to a higher percentage of spikes occurring outside of bursts compared to WT neurons (Figure 4J).

Altered electrical activity of BIN1 KO ASCL1-hiNs is associated with normal synapse numbers and altered TAU phosphorylation

These changes in neural network activity observed in BIN1 KO hiNs could be explained, among other things, by a reduced synaptic connectivity, as observed in our long-term COs cultures (Figure 2). To test this possibility, we first quantified the number of synaptic contacts in BIN1 WT and KO ASCL1-hiNs cultures. In contrast with COs, we did not detect any significant differences in the number of putative synaptic contacts (% SYP assigned) in BIN1 KO compared to WT ASCL1-hiNs, neither after 4 nor 6 weeks of differentiation (Figure 5A-D). Next, we quantified the number and activity of glutamatergic synapses by using real-time imaging of ASCL1-hiNs expressing the glutamate sensor iGLUSnFr [31]. In accordance with our observations based on immunocytochemistry, we did not detect differences neither in the number of glutamatergic synapses (active spots) nor in the frequency of events (change in fluorescence levels in active spots) in BIN1 KO compared to WT ASCL1-hiNs (Supplementary Figure 5; Supplementary Movies 1 and 2), indicating that changes in neuronal activity observed in our cultures are not related to changes in synaptic transmission.

Taking advantage of this culture system comprising enriched neuronal populations, we also sought to confirm whether BIN1 deletion could be associated with changes in APP processing or Tau phosphorylation. To this end, we measured the extracellular levels of amyloid-beta (Aβ) peptides, as well as the intracellular levels of full-length APP and APP β-CTF, total and phosphorylated TAU proteins in ASCL1-hiNs cultures. Like COs, we did not detect any significant differences neither in the extracellular levels of Aβ1-x or Aβ1-42, nor in the intracellular levels of APP or APP β-CTF in BIN1 KO compared to WT ASCL1-hiNs (Supplementary Figure 6). However, in contrast with our observations in COs, we observed significantly higher levels of phospho-TAU (Ser202, Thr205) relative to β-ACTIN and total TAU in in BIN1 KO compared to WT ASCL1-hiNs (Figure 5E-F). Together, these observations may suggest that BIN1 deletion primarily impairs neuronal intrinsic properties regulating electrical activity and Tau phosphorylation prior to detectable changes in synaptic communication (observed only in long-term cultures) and independently of alterations in APP processing.

BIN1 regulates neuronal Ca²⁺ dynamics through LVGCCs

In neurons, electrical activity is always accompanied by an influx of Ca2+ ions, which play a fundamental role in the regulation of neuronal firing and activity-dependent gene transcription [32]. We therefore postulated that reduced BIN1 expression in human glutamatergic neurons could affect Ca2+ dynamics, as previously suggested for cardiomyocytes [33]. To directly test this possibility, we first studied Ca²⁺ dynamics in BIN1 WT and KO ASCL1-hiNs using real-time calcium imaging experiments. We observed spontaneous synchronous calcium transients among adjacent cells both in BIN1 WT and KO ASCL1-hiNs cultures (Supplementary Movies 3 and 4). By quantifying calcium spike transients (> 2 standard deviations above the noise level) we showed a significantly higher frequency of Ca²⁺ transients in BIN1 KO compared to WT ASCL1-hiNs (Figure 6A,B and D). Moreover, the dynamics of individual Ca²⁺ transients in BIN1 KO were qualitatively different from WT ASCL1-hiNs (Figure 6C). These differences could be quantitatively measured by a longer time to reach the maximum intracellular Ca²⁺levels and to recover baseline levels (Figure 6E-F).

In human heart failure, BIN1 expression is reduced, leading to an impairment in Cav1.2 trafficking, calcium transients, and contractility (Hong et al., 2012). Thus, we sought to determine if BIN1 could also interact and regulate LVGCC expression in human neurons. To that, we performed proximity ligation assay (PLA) to probe a possible interaction between BIN1 and Cav1_.2or Cav_1.3, the two LVGCCs expressed in ASCL1-hiNs (Supplementary Figure 7). We observed a widespread BIN1-Cav_1.2 PLA signal (Figure 6G) and, to a lesser extent, a BIN1-Cav_1.3 PLA signals in neurons (Supplementary Figure 7). Next, we quantified neuronal LVGCC protein by western blotting and observed higher total Cav_1.2 expression in BIN1 KO compared to WT ASCL1-hiNs (Figure 6H-I). Protein expression of neither Cav_1.3, nor the members of the Cav₂ family (Cav_2.1, Cav_2.2 and Cav_2.3) were altered in the same cultures (Supplementary Figure 7), suggesting a specific regulation of Cav_1.2expression by BIN1.

Notably, LVGCCs are key regulators of neuronal firing [30] and activity-dependent internalization of these channels is a key mechanism in firing homeostasis [34]. We thus set out to investigate whether BIN1 deletion could impair this mechanism in human neurons. We stimulated ASCL1-hiNs with KCl 65nM for 30 min to induce neuronal depolarization and collected total and endosomal proteins for analysis. We confirmed a higher global level of Cav_1.2 in BIN1 KO compared to WT ASCL1-hiNs that was independent of KCl treatment (Figure 6J). However, Cav_1.2 expression in the endosomal fraction was 50% higher after KCl treatment in BIN1 WT, whereas this rise was only of 10% in BIN1 KO ASCL1-hiNs (Figure6K-L). This effect was specific for Cav_1.2since both early endosome antigen 1 (EEA1) and Cav_1.3 expression rose in both BIN1 WT and KO ASCL1-hiNs at similar levels after KCl treatment (Figure 6K-L). Altogether, these results indicate that BIN1 regulates activity-dependent internalization and expression of Cav_1.2 in human neurons.

Treatment with the calcium channel blocker nifedipine partly rescues electrical and gene expression alterations in BIN1 KO ASCL1-hiNs

To investigate whether the network dysfunctions observed in BIN1 KO ASCL1-hiNs may be related to the higher expression of Cav_1.2, we treated these cells with a physiologically relevant concentration (50nM) of the Cav_1.2 blocker nifedipine [35]) for 2 weeks and recorded neuronal activity using MEA electrophysiology. We observed a partial recovery of the oscillatory pattern of neuronal electrical activity observed in WT cells (Figure 6M). Interestingly, the percentage of spikes outside bursts was not affected by nifedipine treatment in BIN1 WT but was significantly lower in BIN1 KO ASCL1-hiNs (Figure 6N), indicating a partial recovery of burst organization. To note, no difference in firing rates was observed whatever the models and conditions (Figure 6O). After 2 weeks of nifedipine treatment (4 weeks of differentiation), we also performed snRNA-seq experiments and recovered a total of 1537 cells (n= 2 independent culture batches), which were mapped into the 7 clusters described before (Figure 3; Supplementary Figure 8). Using Wilcoxon test, we found that nifedipine treatment down-regulated several genes in BIN1 KO ASCL1-hiNs, especially in the GluNeu-II population (Supplementary Table 6). Comparison of GO term enrichments between nifedipine-treated and untreated BIN1 KO vs WT GluNeu-II population revealed a consistent reduction of the enrichment for several terms associated with ion channel activity and synapse transmission in nifedipine-treated BIN1 KO cells (Figure 6P; Supplementary Table 7). Altogether, these data support the view that BIN1 contributes to the regulation of electrical activity and gene expression through the regulation of Cav_1.2 expression/localization in human neurons.

1.1. Molecular alterations in BIN1 KO organoids and ASCL-hiNshiNs are also present in glutamatergic neurons of AD patients

We finally sought to evaluate whether molecular alterations in our neural models may recapitulate some of those observed in the brain of AD cases. For this purpose, we used a publicly available snRNA-seq dataset generated from the entorhinal cortex (EC) and superior frontal gyrus (SFG) of AD patients at different Braak stages [36]. We first observed a progressive and significant decrease in BIN1 mRNA levels in glutamatergic neurons (Supplementary Figure 9A), suggesting that reduced BIN1 expression in this cell type may be a common feature occurring in the AD pathology progression. We then compared DEGs identified in BIN1 KO glutamatergic neurons (either from COs or ASCL1-hiNs) with those identified in the same cell subtype of AD brains (Supplementary Table 8). Remarkably, DEGs identified in BIN1 KO glutamatergic neurons (either from COs or ASCL1-hiNs) showed a statistically significant overlap with DEGs detected in this cell population in AD brains at different Braak stages (Supplementary Figure 9B). In astrocytes, however, a similar significant overlap could only be observed between COs and AD brains. GO analysis based on DEG overlap between BIN1 KO ASCL1-hiNs and AD brain glutamatergic neurons indicated significant enrichment for pathways associated with glutamate receptor activity and gated channel activity (Supplementary Figure 9C; Supplementary Table 7). Similarly, DEG overlap between BIN1 KO COs and AD brain glutamatergic neurons was significantly enriched for genes associated with glutamate receptor activity, gated channel activity and calcium ion binding (Supplementary Figure 9D; Supplementary Table 7). No significant enrichment was observed for DEG overlap between BIN1 KO COs and AD brain astrocytes (data not shown).

In this work, we show that the AD genetic risk factor BIN1, plays a critical role in the regulation of neuronal firing homeostasis and gene expression in glutamatergic neurons. Complete deletion of BIN1 gene in these neurons is sufficient to alter the expression of the LVGCC Cav_1.2, leading to altered calcium homeostasis and neural network dysfunctions in human neurons in vitro. These functional changes are correlated with changes in the expression of genes involved in synaptic transmission and ion transport across the membrane, as well as elevated Tau phosphorylation. In long-term neuronal cultures using COs, we show that reduced BIN1 expression is associated with fewer synapses and specific gene expression alterations in glutamatergic neurons associated with activity-dependent transcription. Notably, reduced BIN1 expression in human-induced glutamatergic neurons is sufficient to elicit gene expression alterations that are also present in AD and converge to biological processes related with calcium homeostasis. Together, our findings support the view that altered BIN1 expression in glutamatergic neurons may contribute to AD pathophysiology by dysregulating neuronal firing homeostasis via LVGCCs.

Neuronal network dysfunctions are observed in AD patients at early stages of the disease and precede or coincide with cognitive decline [37–39]. Under physiological conditions, neuronal networks can maintain optimal output through regulation of synaptic plasticity and firing rate [40]. Our results suggest that normal levels of BIN1 expression in glutamatergic neurons are fundamental to regulating neuronal firing rate homeostasis. Accordingly, BIN1 deletion in hiNs is sufficient to dysregulate network oscillations even without impacting the number of functional synaptic contacts, suggesting that the desynchronization observed in BIN1 KO hiNs circuits are a consequence of disordered homeostatic controls of neuronal activity. At later time-points (COs), glutamatergic neurons show both gene expression alterations indicative of altered electrical activity and reduced synaptic densities, which could indicate a synaptic down-scaling in response to earlier augmented electrical activity (Dörrbaum et al., 2020).

One key mechanism controlling neuronal spiking activity is the regulation of Ca²⁺ homeostasis [30, 32, 41]. Boosted neuronal electrical activity induces the turnover of LVGCCs from the plasma membrane through endocytosis [34] and regulates the transcription of genes encoding for calcium-binding proteins and calcium-mediated signaling [42], mechanisms aiming to restore local Ca²⁺ signaling cascades and protect cells against aberrant Ca²⁺ influx. We show that BIN1 interacts with Cav_1.2 in hiNs, similar to previous findings in cardiac T tubules [33] and provide evidence supporting a novel role for BIN1 in the regulation of activity-dependent internalization of Cav_1.2 in human neurons, thus linking the known role of BIN1 in endocytosis [12] to firing homeostasis in human neurons via the LVGCC. These results confirm in human neurons the interaction between endogenous neuronal BIN1 and Cav_1.2 as previously suggested in mouse hippocampal neurons, overexpressing a Bin1-mKate fused protein [18].

Loss of Ca²⁺ homeostasis is an important feature of many neurological diseases and has been extensively described in AD [43, 44]. Interestingly, DEGs identified in BIN1 KO glutamatergic neurons in our different cell culture models are enriched for calcium-related biological processes. This is also observed for DEGs detected in glutamatergic neurons of the AD brain at late stages of pathology when the expression levels of BIN1 in those cells is also decreased. Thus, it is plausible to speculate that reduced expression of BIN1 in glutamatergic neurons may contribute to the breakdown of Ca²⁺ homeostasis in the AD brain, potentially contributing to neuronal circuit dysfunctions. Consistent with this hypothesis, we have previously shown a significant reduction in the expression of the transcript encoding for the neuron-specific BIN1 isoform 1 in bulk RNA-sequencing data from a large number of AD patients [7] and we show in this work that BIN1 expression is reduced in glutamatergic neurons of AD brains at late Braak stages. Moreover, it has been recently shown that Bin1 conditional knockout in neurons and glial cells of the mouse forebrain is sufficient to elicit gene expression changes associated with calcium-dependent mechanisms (Ponnusamy et al., 2022), further supporting the interpretation that BIN1 plays an important role in cellular processes involved in Ca²⁺ homeostasis in the brain.

Lastly, we show that treatment with the clinically approved calcium channel blocker nifedipine for only 2 weeks is sufficient to partly recover electrical and gene expression alterations in BIN1 KO hiNs. These findings further support our interpretation that changes in gene expression and electrical activity observed in BIN1 HET and KO hiNs are a direct consequence of reduced BIN1 expression in glutamatergic neurons and not a possible artifact of CRISPR/Cas9 gene-editing in our hiPSC lines. Moreover, together with our observations of reduced BIN1 expression and transcriptional alterations affecting biological processes related to calcium homeostasis in human-induced glutamatergic neurons of the human brain at late stages of AD pathology, our data strongly support a link between BIN1 and calcium homeostasis.

Thus, it is plausible to speculate that BIN1 depletion in glutamatergic neurons primarily undermines Ca²⁺ homeostasis, leading to changes in neuronal electrical activity. In a later stage, gene expression and circuit-level alterations such as synapse loss would occur, likely because of altered neuronal electrical activity. A corollary to this model would be that early treatments aiming to restore Ca²⁺ homeostasis and neuronal electrical activity may have a beneficial impact in AD onset and progression. Supporting this notion, both a Mendelian randomization and a retrospective population-based cohort study found evidence suggesting that treatment with Ca²⁺ channel blockers in human patients are associated with a reduced risk of AD [45, 46]. In the future, it would be interesting to study the impact of these drugs for AD onset/progress as a function of genetic variants in the BIN1 locus.

An important limitation of our work is the absence of microglial cells in our models, hampering the study of BIN1 roles in this cell type of high relevance to AD pathology [47]. However, our findings provide fundamental information about the molecular and cellular processes impacted by reduced BIN1 expression in human neurons, which will require further confirmation in the brain of patients carrying AD-related BIN1 genetic variants. Moreover, we studied the impact of heterozygous and knockout mutations, which do not necessarily represent the consequences of AD-related BIN1 genetic variants in gene expression. Nevertheless, it is tempting to speculate that slight changes in BIN1 expression provoked by those variants could progressively deteriorate neuronal functions in the human brain, contributing to AD pathogenesis in the elderly brain.

Acknowledgements

The authors thank the BICeL platform of the Institut Biologie de Lille and the Vect’UB viral platform (INSERM US 005 – CNRS 3427 – TBMCore, Université de Bordeaux, France). The authors thank Karine Blary at the IEMN Lille for the microfabrication work. The Maestro Pro multiwell microelectrode array was acquired with the “Prix Claude Pompidou pour la Recherche sur l’Alzheimer (2021)” to MRC.

Conflicts

The authors have no conflicts of interest to declare.

Authors' contributions

Conceptualization, M.R.C.; Methodology, M.R.C., F.D., D.K., C.M.Q.; Investigation, M.R.C., O.S., B.S.L., A.R.M.F., J.G., A.C., A.P., D.S.W., F.D., K.G., D.K., A.C.V., C.M.Q.; Writing - Original Draft, M.R.C.; Writing - Reviews & Editing, M.R.C., F.D., J.C.L., C.M.Q., D.K.; Figures preparation: M.R.C., F.D., A.P., B.S.L., A.R.M.F., D.K. Supervision, M.R.C., F.D., J.C.L., D.K.; Funding Acquisition, M.R.C., J.C.L., F.D, D.K., P.A., A.B. All authors have read and approved the manuscript.

Funding Sources

This work was co-funded by the European Union under the European Regional Development Fund (ERDF) and by the Hauts de France Regional Council (contract no.18006176), the MEL (contract_18006176), and the French State (contract no. 2018-3-CTRL_IPL_Phase2). This work was partly supported by the French RENATECH network (P-18-02737), Fondation pour la recherche médicale (ALZ201912009628, ALZ201906008477), PTR-MIAD, Fondation Recherche Alzheimer and by the Sanofi i-Awards Europe 2019. This work was also funded by the Lille Métropole Communauté Urbaine and the French government’s LABEX DISTALZ program (Development of innovative strategies for a transdisciplinary approach to Alzheimer’s disease). The UMR 8199 LIGAN-PM Genomics platform (Lille, France) belongs to the 'Federation de Recherche' 3508 Labex EGID (European Genomics Institute for Diabetes; ANR-10-LABX-46) and was supported by the ANR Equipex 2010 session (ANR-10-EQPX-07-01; 'LIGAN-PM'). The LIGAN-PM Genomics platform (Lille, France) is also supported by the FEDER and the Region Nord-Pas-de-Calais-Picardie and is a member of the “France Génomique” consortium (ANR-10-INBS-009).

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The Alzheimer’s disease risk gene BIN1 regulates activity-dependent gene expression in human-induced glutamatergic neurons

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