Chromatin remodeling factor BAF155 coordinates oligodendroglial-neuronal communications linked to regional myelination and autism-like behavioral deficits in mice

Domain: biology
Published: 2025-12-21
Editorial outcome: major_revision

Abstract
Autism spectrum disorders (ASD) are neurodevelopmental disorders associated with synaptic deficits. Oligodendrocyte precursor cells (OPCs) are the only type of glial cells that establish direct synaptic connections with neurons within the central nervous system (CNS). However, the mechanism that results in the delicate construction of OPC-neuron synaptic connections remain poorly understood. Here we show in a mouse model that BAF155, a chromatin remodeling factor, is highly expressed in committed OPCs. BAF155 influences the OPC differentiation and myelination by coordinating the expression of multiple synapse-related genes that mediate OPC-neuron synaptic communication. The varying chromatin regulatory roles of BAF155 across brain regions give rise to local myelin deficits, contributing to the diverse clinical manifestations observed in individuals with ASD. Collectively, these results deepen our insight into OPC-neuron interactions under pathophysiological conditions and uncover a mechanism that integrates synaptic and ASD susceptibility genes, implying that abnormal OPC-neuron synaptogenesis could be an early instigator of ASD.

Introduction
Autism spectrum disorders (ASD) is one of the most highly heritable (~80%) neurodevelopmental disorders, affecting ∼1% of children worldwide1. It is characterized by abnormal development of the brain2,3, resulting in a wide range of psychiatric and neurological symptomology, generally affecting social interactions, and showing restricted and repetitive behaviors4,5. At present, the synaptic defect theory is considered the most comprehensive framework in ASD research, as it directly links impaired neuronal connections with excitatory/inhibitory (E/I) imbalances2,6–12; however, the underlying pathogenetic mechanisms remain largely obscure.

Recent studies have expanded the understanding of oligodendrocyte precursor cells (OPCs) by demonstrating their interactions with multiple cell types in the central nervous system (CNS)13. In a physiological context of OPC–neuron interaction, OPCs are unique among glial cells as they express multiple synapse-related genes and form direct synaptic connections with neurons14–16, allowing them to receive neuronal inputs, then adjusting their behavior accordingly17, and even play an active role in synaptic pruning and remodeling18–20. In pathological conditions, OPCs impede synaptogenesis in hippocampal neuronal networks associated with schizophrenia21, and facilitate the release of GABA through synaptic complexes with hippocampal interneurons, thereby contributing to anxiety onset22. Nonetheless, the primary molecular mechanism that integrates the expressions of multiple synapse genes in OPCs, resulting in the delicate construction of OPC–neuron synaptic connections in ASD conditions, remains poorly understood.

Hence, as part of our strategy, we investigate the regulation of synaptic connections through the lens of autism susceptibility genes. Recently, BAF155, a scaffolding subunit of the BRG1/BRM-associated factor (BAF) complex, was identified as a core member of the SWI/SNF ATP-dependent chromatin remodeling system23, playing a crucial role in gene expression during embryonic corticogenesis24–26, and has also been linked to ASD27–30. However, it is still largely unknown whether the Baf155 gene (also known as Smarcc1 in mice) influences the synaptic communication between OPC and neuron, and how it may contribute to behavioral abnormalities in diseases.

In the current study, we generated a transgenic mouse strain by removing the BAF155 exon 4 from neural stem cells, and found that this caused robust hypomyelination. We used this loxP transgenic mouse to create the conditional knockout of Baf155 specifically in oligodendroglia, which reproduced multiple autistic behaviors. We provide mechanistic insight into how BAF155 instructs myelination in different brain regions by modifying OPC–neuron communications. Our findings identify an essential chromatin remodeling factor driving OPC–neuron synaptic interactions, and reveal mechanisms of diverse manifestations of ASD, thus highlighting oligodendroglia-oriented interventions as a potential therapeutic strategy.

Results
BAF155 in the CNS regulates oligodendroglial differentiation and myelination
Baf155 mRNA is highly expressed in various cell types in the CNS, including neurons and OPCs, as illustrated in the UMAP plot from the mouse whole-brain transcriptomic cell type atlas31 (Fig. S1A). To explore the function of Baf155, we generated a Baf155fl/fl transgenic mouse, in which the loxP fragments were inserted into the introns downstream of the ATG-containing exon (Fig. S1B). By crossing with NestinCre mice, Baf155 exon 4 was conditionally deleted, which led to a protein reading frameshift in neural stem cells. In this mouse strain, the fl/fl homozygotes were lethal. In order to modify neural cells in a more controlled way, we crossed the Baf155fl/fl transgenic mice with NestinCreERT2 mice, thereby reducing BAF155 protein levels in neural stem cell-derived neurons, astrocytes, and oligodendroglia, but not in microglia (Fig. S1C, S1D). These fl/+ heterozygotes exhibited significant defects in white matter development, as reflected by the reduced myelin-basic protein (MBP)-positive areas (representing the myelin-like structures) (Fig. S1E), along with decreased densities and proportions of PDGFRα-positive OPCs and CC1-positive mature oligodendrocytes (OLs) in medial prefrontal cortex (mPFC); conversely, the ratio of InsPR-type II (IP R-II)/OLIG2-positive committed OPCs was significantly increased (Fig. S1F), indicating a block in the transition from committed OPCs to mature OLs. In addition, other glial cell types and neurons were unaffected (Fig. S1G, S1H). Therefore, our results suggest a major role of BAF155 in the oligodendroglial lineage.

We then explored the expression of BAF155 in oligodendroglia at different developmental stages. A Pdgfrα-EGFP mouse strain32 was employed to identify PDGFRα-expressing OPCs and track OPC differentiation. In addition, the IPR-II antibody was used to label committed OPCs33, whereas CC1 was used to identify differentiated OLs. This multiple staining showed that BAF155 was significantly upregulated in IPR-II and PDGFRα-EGFP double-positive committed OPCs, compared to PDGFRα-EGFP-positive but IPR-II-negative non-committed OPCs at an earlier developmental stage. Additionally, BAF155 was significantly downregulated in CC1-positive IPR-II-negative mature OLs (Fig. 1A). This expression pattern was confirmed in vitro. qPCR conducted on purified OPC cultures revealed that the highest level of Baf155 mRNA was detected in the committed OPCs (1 day in mitogen-free medium, which switch OPCs to their committed stage), as reflected by the parallel expression of Gpr17 (indicated by the red dotted line), which has also been reported to be highly expressed in the committed OPCs34 (Fig. 1B).

To investigate the role of BAF155 in OPC development, Baf155fl/fl transgenic mice were crossed with the OPC-specific PdgfrαCreER mice (Fig. S2A), allowing Baf155 knockout before the early differentiation stage at postnatal day 4 (P4, Fig. S2B, S2C). The loss of BAF155 in OPCs resulted in long-lasting defects of oligodendroglial differentiation and myelination, as shown by the reduced numbers of CC1/myelin-associated glycoprotein (MAG)-positive OLs in the mPFC (Fig. S2D, S2E), and persistent decreases of MBP-positive areas in mPFC from P14 to 8-week old (Fig. 1C). Impaired myelination was also confirmed by electron microscopy, showing the reduced number of myelinated axons and increased G-ratios (G-ratio is defined as the ratio of an axon’s diameter to the overall diameter of its myelinated fiber. It serves as an essential metric for evaluating myelin integrity and function; a lower G-ratio value reflects thicker myelin sheath and a greater degree of myelination) in Baf155 knockout mice at both P14 and 8 weeks of age (Fig. 1D, E). Fluorescence microscopy further revealed that the MBP-positive areas on parvalbumin (PV)-positive axons of interneurons, assessed through MBP/PV co-staining, decreased significantly when compared to WT controls. Specifically, there was an 86.86±11.71% reduction at P14, and a 28.68±3.15% reduction at 8 weeks of age (Fig. 1F, G). Loss of BAF155, however, did not affect SMI32-positive axons (Fig. 1F, G), the numbers of OLIG2-positive and PDGFRα-positive oligodendroglial lineage cells (Fig. S2F, S2G), or the proliferation of OPCs (Fig. S2G). These findings indicate that BAF155 predominantly operates during the early oligodendroglial differentiation and myelination.

Loss of BAF155 in committed OPCs induces ASD-like phenotypes
To further determine whether the Baf155 knockout-induced hypomyelination triggers functional outcomes, we first recorded spontaneous postsynaptic currents in both mPFC (Fig. 2A) and hippocampus (Fig. 2B), both regions being implicated in ASD2. Our results showed that loss of BAF155 in OPCs resulted in a significant decrease in the frequency of spontaneous excitatory postsynaptic currents (sEPSC), as well as spontaneous inhibitory postsynaptic currents (sIPSC) in pyramidal neurons in the mPFC and hippocampal CA1 region. In contrast, the amplitudes of postsynaptic currents were unaffected (Fig. 2A, B). Additionally, we recorded miniature synaptic events in the same regions and found that the frequency and amplitude of mEPSCs and mIPSCs remained unchanged in BAF155 knockout mice (Fig. 2C, D). The absence of differences in mEPSC frequency and amplitude suggests that synaptic density appears to be intact; while the action potential-dependent transmission seems to be affected, as indicated by the sEPSC data, which suggests that myelination deficits impair action potential-dependent synaptic transmission.

To investigate which type(s) of behavioral defect is triggered by the loss of BAF155 in OPCs, we first performed the three-chamber test and the self-grooming test, both of which are key behavioral tests associated with ASD. In the first phase of the three-chamber test (social preference test), Baf155 knockout mice showed a reduced proportion of sniffing time with the chamber containing another mouse (representing the social stimulus) compared to the empty chamber (representing the non-social stimulus). However, in the second phase of the three-chamber test (social novelty preference test), we found no significant difference in the proportion of sniffing time towards the chamber with a stranger mouse (representing the novel social stimulus) versus a familiar mouse (representing the familiar social stimulus) (Fig. 2E). Thus, ASD-like social preference in Baf155 knockout mice was significantly impaired. In the self-grooming test, Baf155 knockout mice displayed an increase in repetitive behaviors, suggesting ASD-like restricted and stereotyped movements (Fig. 2F). Subsequently, we performed several cognitive and mental-related behavioral tests. The novel object recognition test showed no differences, suggesting that the loss of BAF155 in OPCs does not affect the memory abilities (Fig. 2G). In the open field test, knockout mice covered less distance in the center of the open field chamber, although there was no difference in the total distance covered (Fig. 2H). In the elevated plus maze test, knockout mice displayed a decrease in the distance traveled in the open arms (Fig. 2I). These changes imply an anxiety-related behavior, which often presents in ASD patients35. Finally, there was no difference in the forced swimming test and tail suspension test between knockout and control mice, suggesting an absence of depression-like behaviors (Fig. 2J, K).

Thus, specific elimination of BAF155 from committed OPCs reproduces several pathophysiological changes seen in ASD, encompassing alterations in neuronal activity and a spectrum of behavioral outcomes.

Loss of BAF155 impairs OPC differentiation and myelin formation
To further investigate the role of BAF155 in regulating oligodendroglial differentiation and myelination, we assessed the characteristics of hypomyelination across various brain regions related to ASD-like behavioral outcomes, as well as in different experimental settings. As shown in our results, the loss of BAF155 in OPCs caused varying reductions of MBP-positive areas across ASD-related brain regions, including mPFC, hippocampus, corpus callosum, cerebellum, and striatum (Figs. 3A, S3A, S3E). Notably, the mPFC exhibited the most significant impact (Figs. 3A, S3A, S3E). To determine whether the reductions in MBP staining are caused by a loss of oligodendroglial lineage cells or by impaired differentiation of OPCs, we also evaluated the staining of CC1, OLIG2, and PDGFRα in various brain regions (Fig. S3B–H) and showed a significant reduction in CC1-positive mature OLs, but no differences in OLIG2+ or PDGFRα+ cells (Fig. S3B–H), suggesting that Baf155 knockout impairs the OPC differentiation and myelination.

Remyelination is thought to resemble developmental myelination36, though some studies have found exceptions to this recapitulation hypothesis37,38. To investigate the role of BAF155 in remyelination, we employed a model of lysolecithin-induced demyelination in adult mice (Fig. S4A). In this model, the loss of BAF155 did not impact the number of OLIG2-positive oligodendroglial lineage cells within the demyelinating lesions. However, it significantly impaired the differentiation of OPCs, as evidenced by the reduced number of MAG-positive OLs (Figs. 3B and S4B), and it further hindered the remyelination of axons at 14 days post-lesion (Fig. 3C).

To determine whether BAF155 also operates in the OLs, we crossed Baf155fl/fl mice with mature OL-specific PlpCreERT mice to specifically delete BAF155 at the late stage of OPC differentiation (Fig. S4C). Expression of MBP, numbers of OLIG2-positive oligodendroglial lineage cells, and CC1/MAG-positive mature OLs were unchanged (Figs. 3D, E, S4C, and S4D), demonstrating that depleting BAF155 in OLs does not induce obvious defects in oligodendroglial differentiation and myelination, and providing further confirmation that Baf155 acts only during early oligodendroglial development, significantly impacting OPC differentiation and myelination.

Intriguingly, when we compared the differentiation capacities of primary OPCs isolated from the cortex of both wild-type and Baf155 knockout mice, we found that these OPCs showed no difference in their ability to differentiate into MBP-expressing OLs in culture conditions (Fig. 3F). It implies a non-cell autonomous effect involved in BAF155-mediated oligodendroglial development.

Together, these findings raise important questions about the underlying mechanisms through which (1) BAF155 regulates oligodendroglial development in vivo but not in vitro and (2) BAF155 regulates myelination in specific brain regions.

BAF155 targets multiple synaptic genes in committed OPCs
To investigate the underlying mechanism of BAF155 regulating OPC development, we performed chromatin immunoprecipitation sequencing (ChIP-seq) analysis of purified committed OPCs in vitro to identify the potential chromatin remodeling-targeted genes that may trigger autistic symptoms. Our results identified 5226-binding sites of BAF155 located within 3219 genes during the early developmental stage (1 day in mitogen-free medium, which switch OPCs to their committed stage; see Fig. 2B). At the late developmental stage, which corresponds to 5 days in mitogen-free medium, we identified 3810 binding sites located within 2857 genes (Fig. 4A). Among these genes, 928 genes appeared in both early and late developmental stages (Fig. 4A), and the higher BAF155-binding peaks were localized around the transcription start sites (TSS) (Fig. 4B). In addition, most of these binding peaks were found in evolutionarily conserved intergenic regions or introns (Fig. 4C), suggesting that BAF155 mainly targets regulatory regions within the oligodendroglial lineage. Closer examination of these genes and gene ontology (GO) enrichment analysis revealed enrichment in synapse-related genes, including both presynaptic and postsynaptic compartments, but not in myelin-related genes. This enrichment was particularly significant at the early developmental stage (Fig. 4D). Supporting evidence consistently indicated a marked decrease in synapse-related genes during the late developmental stage when compared to the genes that were enriched in the early developmental stage (Fig. 4E). Thus, binding of BAF155 to synapse-associated genes is more pronounced during the early OPC differentiation.

Moreover, we performed a cross-analysis with previously reported ASD susceptibility genes39–43. Our results revealed that a substantial number (198 genes) of BAF155-binding genes are synapse-associated, including γ-aminobutyric acid type A receptor subunit γ3 (Gabrg3 is a member of the GABA-A receptor gene), as well as Gabrg2, Gria2, Grm7, and Grik2, which encode subunits of neurotransmitter receptors. Additionally, 17 of these BAF155-binding genes were previously implicated as ASD susceptibility genes, and three of them, including Gabrg3, Adnp (activity-dependent neuroprotective protein), and Nuclear factor I A (Nfia), were identified as both synapse-associated and ASD susceptibility genes. Among these genes, Gabrg3 emerged as the most prominent synapse-associated ASD susceptibility gene targeted by BAF155 (Fig. 4F). Thus, BAF155-targeted synaptic genes in committed OPCs may play an as-yet-undiscovered role in ASD. To further determine this possibility, we evaluated the fold enrichment of BAF155 on these genes. Our results showed that the enrichments of BAF155 on Gabrg3, Nfia were significantly higher than other candidate synaptic genes Gria2, Grm7, Adnp, Gabrg2, and Grik2, particularly in the committed OPC stage (Fig. 4G). In addition, significant enrichments of BAF155 on Gabrg3 and Nfia gene were also detected when compared to the input group in the genome browser visualization, and it is notable that binding sites of BAF155 on Gabrg3 and Nfia genes were also enriched for H3K27Ac (Histone H3 Lysine 27 acetylation, locates at promoters and enhancers and is strongly correlated with active transcription) but not H3K4me3 (Histone H3 Lysine 4 trimethylation, locates around TSS of active genes)44–46, again suggesting that BAF155 may target enhancer regions to modulate gene expressions (Fig. 4H, Supplementary Data 1). ChIP-qPCR analysis confirmed significantly increased enrichments of BAF155 protein on Gabrg3 and Nfia genes (Fig. 4I).

These findings indicate that BAF155 predominantly functions during early oligodendroglial development by selectively binding to synaptic genes. This process may be involved in the establishment of OPC–neuron synaptic connections. Since these OPC–neuron synaptic connections are crucial for OPC differentiation and myelination in the CNS14,17,47, we propose that the deficits in differentiation and myelination after BAF155 depletion stem from a lack of neuronal signaling inputs to OPCs. In vitro, the absence of neuronal signaling resulted in no significant changes in OPC differentiation or MBP expression.

BAF155 is required for OPC–neuron synaptic connection
To determine the regulatory role of BAF155 in synaptic connections in OPCs, we measured mRNA and protein levels of these ASD and synapse-associated genes in acutely isolated OPCs and tissue slices from PdgfrαCreER;Baf155fl/fl mouse brains. The qPCR results showed reduced mRNA levels of Gabrg3 and Gabrg2, as well as Gria2 in OPCs with Baf155 deletion (Fig. 5A). Specifically, the numbers of GABRG3-labeled synaptic elements on the surface of OPCs were significantly decreased by 30.54±6.55% in the mPFC, as well as in the hippocampus and corpus callosum (Figs. 5B and S5A).

To investigate whether these BAF155-targeted synaptic genes regulate synaptic connections between OPCs and neurons, the synaptic-like structures on OPCs were examined by super-resolution fluorescence microscopy and immunogold electron microscopy. Both pre- and postsynaptic elements were observed in close association with the OPC cell body and main processes (Figs. 5C, and S5B). We found a significant decrease in postsynaptic elements of OPCs in the brain of PdgfrαCreER;Baf155fl/fl mice, as reflected by the reduced numbers of Homer1- (an excitatory postsynaptic marker) and Gephyrin- (an inhibitory postsynaptic marker) positive puncta on the PDGFRα-positive OPCs (Figs. 5C, D and S5B). Numbers of vGAT- (a GABAergic presynaptic element marker) and vGLUT1- (a glutamatergic presynaptic marker) positive puncta, closely associated with OPCs, were also significantly declined (Figs. 5C, D and S5B). Furthermore, the immunogold electron microscopy revealed that the length of postsynaptic density in neuron–OPC synapse was significantly reduced by Baf155 knockout in OPCs (Fig. 5E), confirming that BAF155 regulates OPC–neuron synaptic transmission.

To examine the functionality of OPC–neuron synaptic transmission in Baf155 deletion mice, we performed calcium imaging in acute brain slices. When monitoring intracellular calcium dynamics in PDGFRα-positive OPCs, we found three distinct patterns of calcium signals in OPCs. As shown by the ΔF/F0 traces, these three types included: (i) ‘flat’ Ca2+ signaling, (ii) spontaneous ‘oscillatory’- Ca2+ signaling with peaks, and (iii) spontaneous high ‘plateau’ transients (Fig. S5C). In Baf155-deficient mice, fewer OPCs exhibited oscillatory and plateau patterns; whereas flat Ca2+ responses dominated (Figs. 5F and S5D). Moreover, we determined the characteristics of calcium signaling in OPCs. The number of oscillatory peaks per minute was significantly decreased in Baf155-deleted OPCs (Fig. 5G and H). We also found that Ca2+ transients had greater amplitude in OPCs with Baf155 deletion (Fig. 5I). This pattern of Ca2+ dynamics was documented previously as indicative of oligodendroglial processes that precede retractions from axons48,49, though the duration of these Ca2+ events was comparable to that observed in WT OPCs (Fig. S5D). The number of higher frequencies of Ca2+ transients diminished in OPCs lacking BAF155 (Fig. 5J). This specific pattern of Ca2+ transients is suggested to play a role in the rapid myelin growing48,49. These findings indicate that OPCs deficient in BAF155 exhibit abnormal Ca2+ dynamics, which may stem from a weakened synaptic transmission between OPCs and neurons.

To functionally verify the presence and activity of the OPC-neuron synaptic connections, we performed whole-cell patch-clamp recordings on YFP+ OPCs from healthy control (PdgfrαCreER;Rosa-YFP) and Baf155-deficient mice (PdgfrαCreER;Baf155fl/fl;Rosa-YFP) (Fig. S5E). We measured the currents mediated by two major ionotropic glutamate receptors specifically: α‑amino‑3‑hydroxy‑5‑methyl‑4‑isoxazole‑propionic acid receptor-evoked EPSCs (AMPAR-eEPSC, recorded at −70 mV) and N‑Methyl‑D‑aspartate receptor-evoked EPSCs (NMDAR-eEPSC, recorded at +40 mV), under a fixed stimulus intensity (Fig. S5F). Subsequently, we calculated the AMPA/NMDA current ratio, which serves as a critical and widely accepted measure for assessing synaptic maturation and strength50,51. Our result showed a potential reduction in this ratio in Baf155-deficient OPCs, suggesting that again, deletion of Baf155 in OPCs disrupts the synaptic communication between OPCs and neurons (Fig. S5G).

Together, these findings demonstrate that BAF155 plays a critical role in the development of oligodendroglia by facilitating synaptic communication between OPCs and neurons.

BAF155 modulates heterogeneous responses of OPCs to neuronal inputs in distinct brain regions
To further investigate why loss of BAF155 in vivo induces local hypomyelination, we assessed the chromatin regulatory role of BAF155 to its specific synaptic gene targets in the mPFC and hippocampus using ATAC-qPCR (the assay for transposase-accessible chromatin) and ChIP-qPCR. Our results showed significantly higher chromatin accessibility levels of Gabrg3, Adnp and Nfia in hippocampal OPCs than in cortical OPCs (Fig. 6A); meanwhile, the binding of BAF155 to these target genes in hippocampus were greater than those observed in the mPFC, implying that BAF155 exerts a stronger regulation on these target genes in hippocampal OPCs (Fig. 6B). We also conducted qPCR analysis, which confirmed that the expression levels of BAF155-targeted synaptic genes were elevated in the hippocampus compared to the mPFC (Fig. 6C). Furthermore, we assessed the differentially expressed genes in acutely isolated OPCs from mPFC and hippocampus of Baf155 knockout mice and their non-Cre littermates by RNA-seq (Fig. 6D). Differential gene expression analysis in each brain region revealed unique gene expression changes and enriched molecular functions specific to the mPFC and hippocampus (Fig. 6E). GO enrichment analysis showed that Baf155-deletion upregulated extracellular matrix organization and downregulated steroid metabolic processes specifically in the mPFC; while upregulated cell morphogenesis and downregulated cell activation regulation specifically in hippocampus (Fig. S6A, S6B). The upregulated differentially expressed genes (DEGs) in Baf155 knockout mice were significantly enriched in multiple biological processes, including regulation of nervous system development and regulation of neuronal synaptic plasticity. The downregulated DEGs were associated with dopaminergic neurogenesis, axon guidance and synapse pruning (Fig. 6F). Among all identified DEGs, 173 out of 1315 DEGs were affected in both mPFC and hippocampus of Baf155 knockout mice, 592 out of 1315 were uniquely affected in the mPFC and 377 out of 1315 in the hippocampus (Fig. 6F). As shown in the chord plot (Fig. 6G), Egr1 (Early growth response 1), Esr1 (Estrogen Receptor 1 Gene) and Drd1 (Dopamine D1 receptor), all highly associated with ASD52–54, were significantly upregulated in both mPFC and hippocampus. However, Slc17a7 (Solute carrier family 17 member a7), which codes for VGLUT1, a protein responsible for glutamate accumulation into synaptic vesicles55, and Neurod1 (Neuronal differentiation 1), which is highly associated with neurogenesis56, were significantly downregulated in the OPCs from the mPFC region but not in those from the hippocampal region. In contrast, Lgals3 (Galectin 3), which is essential for OPC differentiation and myelin integrity57, was specifically downregulated in the hippocampal region.

In line with that, we analyzed the variability of OPC responses to neurotransmitters in various brain regions. The RNA sequencing analysis showed that OPCs from mPFC and hippocampus did not respond in the same manner to the same neurotransmitter GABA (Figs. 6H, S6C, and S6D). When exposed to GABA, Slc7a11 (Solute carrier family 7 member 11) and Odc1 (Ornithine decarboxylase 1), which are highly associated with neurodevelopmental disorders and ASD58,59, were significantly upregulated in the OPCs from the mPFC region but not those from the hippocampus. Furthermore, Lss (Lanosterol synthase) and Dhcr7 (7-Dehydrocholesterol reductase), both highly implicated in ASD, mental disorders, and myelination60,61, were selectively upregulated in the OPCs from the hippocampus but downregulated in the OPCs from mPFC. We also compared the DEGs in OPCs exposed to either GABA or glutamate. As shown by the GO-enrichment analysis, genes related to axonogenesis, dendrite development, synapse development, and cell junction assembly (downregulated) were also significantly different in OPCs between the two groups (Fig. S6E, S6F). As highlighted in the chord plot (Fig. 6I), Snca (Alpha-synuclein), highly associated with oligodendrocyte development, myelin formation and mental disorder62, was upregulated in response to GABA, but not the glutamate; whereas Igfbp5 (Insulin Like Growth Factor Binding Protein 5), Vegfa (Vascular endothelial growth factor A), Yap1 (Yes-associated protein-1), Clic1 (Chloride intracellular channel 1), genes also associated with oligodendrocyte development and myelin formation63–65, were upregulated in response to glutamate, but not the GABA. We also compared heterogeneous responses of OPCs derived from various regions to different neurotransmitters, and indicated that the majority of genes exhibited variability under distinct conditions (Fig. 6J).

Collectively, our findings suggest that BAF155 exhibits heterogeneity in OPCs across various brain regions, influencing their responsiveness to neuronal activity and triggering specific gene set expressions in OPCs.

Methods
All animal studies were performed under the guidelines of the laboratory animal welfare and ethics committee of the Third Military Medical University (AMUWEC20223048). All mice were housed in a temperature- and humidity-controlled environment with free access to standard chow and water and on a 12h/12h light/dark cycle.

Mice
Our study examined male and female animals, and similar findings are reported for both sexes.

PdgfrαCreER and PlpCreERT. PdgfrαCreER mice were acquired from Dr. Stephen Fancy at the University of California, San Francisco101. PlpCreERT mice (JAX lab, Catalog #005975) were purchased from The Jackson Laboratory (USA).

Baf155fl/fl. Baf155-flox mice were generated by inserting a loxP site on both sides of exon 4 of the Baf155 gene using CRISPR-Cas9 technology by Biocytogen Pharmaceuticals (Beijing) Co., Ltd (China). The mice were then crossed with NestinCreERT2 (JAX lab, Catalog #016261), PdgfrαCreER, and PlpCreERT mice to generate PdgfrαCreER;Baf155fl/fl and PlpCreERT;Baf155fl/fl conditional knockout mice.

PdgfrαCreER; Rosa-YFP. Rosa-YFP reporter mice (JAX lab, Catalog #006148) were acquired from Dr. Stephen Fancy at the University of California, San Francisco102. These mice were crossed with the PdgfrαCreER and Baf155-flox mice to specifically label the recombined OPCs.

NestinCreERT2. NestinCreERT2 mice (Catalog #016261) were crossed with Baf155fl/fl mice to generate NestinCreERT2:Baf155fl/+ mice.

Mice were administered tamoxifen (10 mg/kg/day, gavage) for 5 consecutive days from postnatal (P) 4 to P8 to induce cre-mediated reorganization. To ensure comparability between experimental and control mice, all genotypes of mice were given tamoxifen.

Primary OPC cultures
Rat OPC culture. Primary rat OPCs were cultured using our previous publication103. Briefly, cells from P0-3 rat brains were seeded in Dulbecco’s modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS) until confluent. OPCs were detached by the addition of 0.04% EDTA in PBS and repetitive rocking. Then, OPC proliferation medium was used for 2 days to culture the OPCs on a PDL-coated surface. PDGF-AA (10 ng/ml, Peprotech, 100-13A) was removed from the culture medium when OPC differentiation was induced.

Mouse OPC culture. Mouse OPCs were isolated from P10 mice using a modified immunopanning technique based on a previously published method104. Briefly, either specific regions or the entire mouse brain were homogenized and digested with 1 mg/mL papain and DNase I at 37°C for 1 h, with the digestion terminated by adding ovomucoid to inhibit the enzymatic activity. After trituration, the resulting cell suspension was incubated with primary PDGFRα antibody (Abcam, Cat# ab96569), diluted in the panning buffer, for 30 min at room temperature to bind OPCs specifically. The cell suspension was then transferred into a pre-coated secondary antibody dish, and incubated for another 30 min to allow the OPCs to adhere selectively. Then, the panning buffer was used to wash away the non-adherent cells, and the adherent OPCs were released from the panning dish using 0.05% trypsin and cultured in poly-D-lysine-coated 10-cm dishes or 24-well plates with coverslips for further applications.

Behavior tests
Behavioral tests were performed on 6–8-week-old male mice. All mice were acclimatized to the behavioral test apparatus with free access to food and water, and housed on a 12h/1h light/dark cycle. Mice were handled daily for 5 days before the test. The apparatus was wiped with 80% alcohol between each trial. All tests were conducted between 9 a.m. and 6 p.m. The experimenters were blinded to the grouping. VisuTrack Animal Behavior Analysis Software (Shanghai XinRuan) was used for data collection and analysis.

The three-chamber test. The three-chamber test was used to assess the sociability and social novelty preference. The apparatus consisted of three chambers: the center, the left, and the right chambers. The test mice were first placed in the center chamber and allowed to explore the three chambers freely for 10 min to familiarize themselves with the environment. In the first phase (sociability test, indicated as Stranger1-Object, S1-O, in figures), an unfamiliar mouse of the same age and sex was randomly placed in the cage located in either the left or right chamber, and the baffles of the two chambers were opened to allow the test mouse to explore freely. The sociability was assessed by recording the sniffing time of the test mouse at the caged mouse (Stranger1) and the empty cage (Object). In the second phase (social novelty preference test, indicated as Stranger1–Stranger2, S1–S2 in figures), a new unfamiliar mouse was placed in the vacant cage on the other side, and the sniffing time on the stranger2 versus the stranger1 was recorded.

The self-grooming test. The self-grooming test was used to assay repetitive behaviors in mice. Test mice were placed in a transparent chamber and allowed to freely explore and familiarize themselves with the environment for 10 min before being recorded for another 10 min. The cumulative time of self-grooming of the mice was calculated.

Novel object recognition test. A novel object recognition test was used to assess short-term memory function. Two days before the experiment, mice were placed in the chamber (25×25×40 cm) for 10 min each day to acclimatize to the test environment. During the trial, two identical objects were placed in the chamber for the mouse to explore for 5 min. After two hours, one of the objects was replaced with a new one with a different shape and color for the mouse to explore for 5 min. The times spent on the new versus the old object were recorded.

Open field test. The open-field test was used to assess the motor ability, as well as anxiety-like behavior, of mice. Test mice were placed in the center of the arena (50×50 cm) and allowed to explore freely for 15 min. The total distance traveled by the mice, and the time and distance traveled in the central area, were recorded. The time or distance traveled in the center area reflects anxiety-like behavior. Data collection and analysis were performed with the VisuTrack Animal Behavior Analysis Software.

Elevated plus maze. The elevated plus maze test was used to assess anxiety-like behavior in mice. The mouse was placed in the center of the crossing and faced the open arms. Mice were allowed to explore for 10 min. Anxiety-like behavior was assessed by comparing the time or distance traveled in the closed arms.

Tail suspension test. The tail suspension test was used to measure depression-associated behavior. The mouse tail was secured to the top of the test chamber (55×15×11.5 cm) with adhesive tape to keep the head approximately 25 cm from the bottom of the chamber for 10 min. The time of immobility was quantified.

Forced swimming test. The forced swimming test was used to assess depression-associated behavior in mice. The water depth in the arena (10×25 cm) was 15 cm, and the water temperature was 25°C. The mouse was placed in the arena for 6 min and the time of fatigue was recorded.

Lysolecithin-induced demyelinating mouse model
A mouse model of lysolecithin-induced demyelinating has been described in our previous papers105,106. Briefly, for the demyelination mouse model, tamoxifen was administered continuously from P50 to P54. At P56, mice were anesthetized by isoflurane (2–3%); after exposing the skull, 1.5 μl 1% lysolecithin (Sigma-Aldrich, L0906) was injected into the corpus callosum (1.04 mm lateral and 1.0 mm posterior to the bregma, depth: −1.62 mm). Two weeks later, brain tissue was harvested for immunofluorescence staining and in situ hybridization.

Real time-PCR
Total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, Cat# 74134). The PrimeScript RT Reagent Kit (Takara) was used for reverse transcription. The Accurate 96 Real Time PCR System (DLAB) and FastStart Universal SYBR Green Master Mix (Roche, 04913850001) were used for the qPCR experiment. All primer sequences are presented in Supplementary Data 2.

In situ hybridization
In situ hybridization was performed as previously described105. Brains were fixed with 4% PFA and cryoprotected in 30% sucrose with 0.1% diethyl pyrocarbonate before sectioning at 20 μm. The brain sections were incubated with digoxigenin (DIG)-labeled antisense MAG probe at 65°C overnight, followed by the incubation with anti-DIG-AP Fab fragments antibody (1:1000, Sigma-Aldrich, 11093274910) at 4°C overnight and NBT/BCIP alkaline phosphatase combination (Sigma-Aldrich, 11681451001) for 4 h at 37°C or overnight at room temperature. The cells expressing the genes of interest displayed dark purple staining.

Immunohistochemistry
Mice were intracardially perfused with 4% PFA, and brains were removed and fixed in 4% PFA overnight at 4°C. The brains were cryoprotected with 30% sucrose for 3 days before being cryosectioned at 20 μm. Sections were blocked with 5% bovine serum albumin (BSA) with 0.25% Triton-X 100 for 2 h at room temperature before being incubated with primary antibodies overnight at 4°C, followed by secondary antibodies for one hour at room temperature. The primary antibodies include: Rat anti-MBP (1:500, Millipore, MAB395); Mouse anti-CC1 (1:500, Calbiochem, OP80); Guinea anti-vGlut1 (1:2000, Synaptic Systems, 135304); Mouse anti-vGAT (1:1000, Synaptic Systems, 131011); Rabbit anti-Homer1 (1:1000, Synaptic Systems, 160003); Mouse anti-Synapsin-1 (1:1000, Cell Signaling Technology, 5297); Goat anti-PDGFRα (1:500, R&D, AF1062); Mouse anti-Olig2 (1:500, Millipore, MABN50); Rabbit anti-Olig2 (1:500, Millipore, AB9610); Rabbit anti-Ki67 (1:1000, Thermo, MA514520); Goat anti-GFP (1:500, Abcam, ab5450). The Olympus VS200 Research Slide Scanner (Olympus) and Ixplore SpinSR confocal microscope (Olympus) were used for imaging. Fluorescent images were analyzed using the CellSens (Olympus) and ImageJ (NIH, USA).

Electrophysiology
The N-methyl-D-glucamine (NMDG) protective recovery method was used to prepare brain slices107. NMDG-HEPES aCSF contains (in mM): 92 NMDG, 2.5 KCl, 1.25 NaHPO4, 30 NaHCO3, 20 HEPES, 25 glucose, 2 thiourea, 5 Na-ascorbate, 3 Na-pyruvate, 0.5 CaCl2·2H2O, and 10 MgSO4·7H2O. HEPES with aCSF contains (in mM): 92 NaCl, 2.5 KCl, 1.25 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 2 thiourea, 5 Na-ascorbate, 3 Na-pyruvate, 2 CaCl2·2H2O, and 2 MgSO4·7H2O. For spontaneous excitatory postsynaptic currents (sEPSCs) and inhibitory postsynaptic currents (sIPSCs) recording, whole-cell patch-clamp recordings were performed using borosilicate glass pipettes. sEPSCs were recorded at a holding potential of −70 mV, and slPSCs were recorded at a holding potential of +10 mV in regular ACSF. As described previously108, for isolating miniature excitatory postsynaptic currents (mEPSCs), we continuously infused sodium channel blocker tetrodotoxin (TTX, 1 µM; MLC, MBZ10175) into the ACSF. For recording miniature inhibitory postsynaptic currents (mIPSCs), we used a special pipette solution containing CsCl and administered the AMPA receptor antagonist NBQX (10 µM; MCE, HY-15068) and NMDA receptor antagonist D-AP5 (50 µM; MCE, HY-100714A) or TTX (1 µM)+NBQX (10 µM)+D-AP5 (50 µM) into the ACSF throughout the recording. We clamped membrane potentials at −60 mV to record both mEPSCs and mIPSCs for at least 15 min. For voltage-clamp recordings on OPCs15, an individual slice was transferred to the recording chamber with the external ACSF containing (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 10 glucose, 2 CaCl2·2H2O, and 1 MgSO4·7H2O. The patch pipette was injected with internal solution (in mM): 135 Cs methanesulfonate, 8 NaCl, 10 HEPES, 2 Mg ATP, 0.3 Na GTP, 0.1 Spermine, 7 phosphocreatine, and 0.3 EGTA. AMPAR-mediated EPSCs were recorded at −70 mV, NMDAR-mediated EPSCs were recorded for 100 ms at +40 mV. As a stimulation electrode, the electrode was inserted into the stratum radiatum of the CA1 area. A MultiClamp 700B amplifier and pCLAMP10 software were used for electrophysiology (Axon Instruments). Mini analysis and Clampfit software were used for data analysis. The AMPA/NMDA ratio was calculated as described previously50,51.

Immunogold labeling
Mice were perfused intracardially with PBS followed by pre-cold 4% PFA, and the brains were processed by Vibratome (submerged in 0.1 M PBS) to obtain 50 µm free-floating sections. Sections were postfixed with 4% PFA with 0.1% glutaraldehyde in 0.1 M PB for 2 h, washed with 0.1 M PB for 10 min thrice. The residual fixatives were then quenched with 50 mM glycine in 0.1 M PB for 30 min. Sections were washed with 0.1 M PB for 10 min, permeabilized in 0.05% Triton-X 100: PB for 15 min, washed again with 0.1 M PB for 15 min, and incubated in blocking buffer (0.1 M PBS with 0.1% BSA-CTM) for 1.5 h. Sections were then incubated with primary antibody (Rabbit-anti-NG2 (1:200, Millipore, AB5320)) at 4°C overnight, washed with blocking buffer 6×10 min, and incubated with 1.4-nm gold-conjugated secondary antibody (Nanoprobes) overnight. Sections were then washed with blocking buffer 6×10 min, 0.1 M PB 3×10 min, and postfixed in 2.5% glutaraldehyde for 4 h, followed by washing with 0.1 M PB 3×10 min, ddH2O 6×5 min, sodium citrate (pH 7.0) 3×5 min, before being subjected to silver enhancement with the HQ Silver Kit (Nanoprobes). Immunolabelled sections were rinsed in ddH2O 6×10 min, post-fixed with 1% osmium tetroxide in PB for 1 h, and then incubated in 2% uranyl acetate in ddH2O for 40 min in the dark. Sections were dehydrated in graded ethanol, then acetone series, and finally flat-embedded in Epon 812. After polymerization, flat-embedded sections were examined under a light microscope. Serial ultrathin (~70–90 nm) sections were cut with an ultramicrotome (Leica EM UC6, Germany) using a diamond knife (Diatome) and mounted on formvar-coated mesh grids (6–8 sections per grid). They were observed under a Tecnai G2 Spirit 120 kV transmission electron microscope at the Center of Cryo-Electron Microscopy, Zhejiang University.

Calcium imaging
As described previously109, isolated OPCs were incubated with calcium probe Rhod-5 at 37°C in the dark for 30 min. Confocal images were captured by an Ixplore SpinSR confocal microscope (Olympus) at 200 ms per frame and analyzed by the ImageJ software.

Gene expression analysis from online mouse scRNA-seq platform
The Mouse Whole-Brain Transcriptomic Cell Type Atlas data were accessed via the Allen Institute for Brain Science (https://portal.brain-map.org)31. The dataset comprises scRNA-seq data from approximately 4 million cells, covering diverse cell types across the entire mouse brain. To examine the gene expression of the target genes Baf155, Gabrg3, and Gabr subtypes, we used the portal’s search function and interactive features to visualize the transcriptomic data. The platform provides UMAP plots, heatmaps showing the expression of Baf155 and Gabrg3 across different cell types. Ridgeline plots were generated with R “ggridges” package with the data acquired from The Mouse Whole-Brain Transcriptomic Cell Type Atlas.

Assay for transposase-accessible chromatin (ATAC assay)
A chromatin accessibility kit was purchased from Abcam (ab185901), and the assay was performed according to the manufacturer’s instructions110. Briefly, OPCs were isolated from the mouse hippocampus and mPFC by immunopanning. Then, OPCs were incubated on ice, vortexed, and centrifuged (5000×g, 5 min). After cell lysis, the OPC chromatin was treated with a nuclease mix, and OPC DNA was purified (250 µL binding buffer, two washes with 200 µL wash buffer, elution with 20 µL elution buffer), and subjected to qPCR for region-specific analysis of chromatin accessibility. All primer sequences are presented in Supplementary Data 2.

Chromatin immunoprecipitation-sequencing (ChIP-seq) and qPCR
ChIP-seq technology is used to determine BAF155-binding sites as described previously111. In cells, protein–DNA complexes were immobilized using formaldehyde at a final concentration of 1% to preserve the protein–DNA interactions. The crosslinking process was then terminated by adding glycine, typically at a concentration of 0.125 M. Next, cell membranes were disrupted with lysis buffer to release the nuclear material. The chromatin was sheared into small fragments, usually ranging from 200 to 1000 base pairs, through ultrasonic fragmentation. This was followed by enriching the protein–DNA complexes using the BAF155 antibody (Cell Signaling Technology, catalog number 11956). The BAF155 antibody was bound to Protein A+G magnetic beads (Magna ChIP™, catalog number 16-663), and the sonicated samples were incubated overnight with the antibody-bound beads. After incubation, unbound material was removed, and DNA fragments were released from the immunoprecipitation complexes. The purified DNA underwent end repair and splice addition, followed by amplification using PCR to generate a sequencing library. Once the library was tested and validated, it was sequenced using the Illumina HiSeq platform. Following ChIP-seq sequencing, raw reads were obtained and filtered to remove junctions. The data were decontaminated and aligned to the reference genome. Finally, high-quality-mapped reads with a mean Phred quality score (MPAQ) of 30 or greater were used for subsequent analysis. ChIP-qPCR was adopted to quantify the presence and abundance of protein–DNA complexes at specific DNA sequences112. Following similar procedures, the protein–DNA complexes were collected, and the DNA was released by reversing the cross-links through heating, typically overnight at 65°C. Afterward, the DNA samples were purified and analyzed using qPCR. All primer sequences are presented in Supplementary Data 2.

RNA-sequencing and analysis
OPCs were isolated by immunopanning, and RNA was extracted from the isolated cells by Trizol (Thermo, Cat. No. 15596026) according to the manufacturer’s protocol. RNA-seq was performed by the Beijing Genomics Institute (BGI). Raw RNA sequencing data in FASTQ format were initially processed using quality control steps. Adapter sequences and low-quality reads were removed using tools such as Trim Galore to ensure the integrity of the downstream analysis. The clean data were then aligned to GRCm38 for genome reference. Normalization of gene expression was performed by transferring the read counts to FPKM values. Gene expression levels were quantified, and differential expression analysis was performed using DESeq2. Genes with adjusted p-values below 0.05 were considered statistically significant. Data visualization and downstream analysis were conducted using R, with functional enrichment analysis performed using the ClusterProfiler package.

Statistics
Quantification of cell numbers. The number of immunostaining-positive cells was manually counted and normalized to the area of the region of interest. Only cells with visible cell bodies were included.

Quantification of fluorescence intensity or positive area. Quantification of fluorescence intensity or the fluorescence-positive area was performed using the Fiji software, where a threshold was set to distinguish the positive fluorescence signals, followed by the measurement of fluorescence intensity and the positive area percentage within the region of interest. The same threshold was set for different experiment groups in the same batch. For the analysis of representative images of MBP/PDGFRα staining in OPC cultures, the fluorescence intensities were normalized for the total DAPI nuclear area value.

Quantification of synaptic element immunostaining in OPC. The analysis was performed by Imaris 9.0 according to a previous publication113. Briefly, the OPC surface was reconstructed based on the PDGFRα signal, which was then used to mask the synaptic element staining. The surface rendering was performed on the masked synaptic element signal, and the number of object was quantified.

The GraphPad Prism program 9.0 (GraphPad program, San Diego, CA, USA) was used to establish statistical significance between groups. The data were reported as means ± standard error of the mean (SEM). The significance between the two experimental groups was ascertained using the unpaired t-test. All statistical tests were two-tailed. p-values were regarded as statistically significant if they were <0.05. Significant statistical results are indicated as: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data distribution was assumed to be normal, but this was not formally tested. Although sample sizes were not predetermined using statistical techniques, our numbers are comparable to those reported in previous studies. Each experiment has been conducted at least three times.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Wang et al, present a very interesting paper highlighting the role of BAF155, a chromatin\nremodeling factor, in the di;erentiation and myelination of oligodendrocyte progenitor cells\nby integrating the expression of several genes related to OPC-neuronal synaptic regulation.\nThe authors discuss the e;ects of BAF155 KO at the NPC, OPC and OL level with intriguing\nresults. A major concern is how well does BAF155 homozygous KO model heterozygous\nmutations in the ASD patient population.\n1. BAF155 in the CNS regulates OL di;erentiation and myelination\n1. In the NPC het, are the number of Olig2+ cells di;erent between genotypes?\nIs the reduction of mature OLs due to reduced expression of MBP or CC1 at\nthe mature stage or a reduction in the total number of OLs? How about the\nPDGFRa+ cells. Could it be an issue with the progression of the OPCs into\nthe committed and mature stages or is it an overall reduction in the number\nof progenitors?\n2. Related to Fig 1 and Fig S2\n1. Why is there a decrease in PV interneurons? Are the cells dying or are the\nnumber of interneuron progenitors low and, hence, fewer cells?\n3. IF panels need to be brightened, especially the DAPI channel (e.g. Fig1F, Fig3A).\nSingle panels/channel (preferentially in greyscale to improve contrast) should be\nadded to help with the visualization of the data (eg. Fig 1F, 3F).\n4. Are the fluorescence intensities normalized to Dapi counts or Dapi area (as done on\nFig 3F quantification)?\n5. Figure 3\n1. Fig 3A\n1. If possible, please include a full brain image (low magnification or\nstitched)\n2. Olig2 staining may be helpful to evaluate if the MBP di;erences are\ndue to OL loss or reduced expression of the protein.\n2. Fig 3D and 3E: similar comment. Co-staining with Olig2 may be helpful for\nthe quantification.\n3. The lysolecithin-induced demyelination experiments are a bit confusing.\nHow do we know that the injection produced a lesion? A control experiment\ndemonstrating a demyelination lesion was induced is necessary.\n6. Figure 2\n1. The results section states miniature EPSCs were recorded but the figure and\nfigure legend state spontaneous EPSCs. Which is correct? Based on\nfrequency and amplitudes I am guessing these are sEPSCs and sIPSCs. What\nis causing the reduced frequency of spontaneous events in pyramidal\nneurons? Supp Figure 1E,F suggest that axons density and neuron numbers\nare not altered. Presumably spontaneous network activity is reduced due to\nreduced myelination of axons? It would be good to demonstrate this by\nrecording miniature synaptic events, as the TTX would block network activity,\npresumably mEPSC and mIPSC frequency and amplitude would be\nunchanged in BAF155 mutants?\n2. How many neurons were recorded in each animal? The figure legends states\neach data point is a mouse.\n7. Figure 4\n1. For ChIP-seq experiments, how was the antibody validated for its specificity\nfor BAF155? In supp Figure 2, the BAF155 antibody is producing a signal in\nBAF155fl/fl mouse x PDGFRa-CreER. This could be explained by ine;icient\nrecombination, but could also be due to non-specific BAF155 antibody.\n8. Supplemental Figure 1\n1. The e;ect of BAF155 heterozygous mutation is quite small, and the majority\nof the experiments were performed using homozygous KO, which severely\nlimits the relevance of this work in relation to ASD. Patients with BAF155\nmutation will only have heterozygous mutations.\n9. Supplemental Figure 2\n1. In Supp figure 2C and 2D, there is a consistent reduction in CC1+ and MAG+\nstaining in the right quadrant of the images. What is the exact region of the\nmPFC that is imaged and is this imaging region consistent between animals?\n10. Where all genotypes of mice given tamoxifen?\n11. There is a growing body of evidence from several ASD mouse models related to\nmyelination defects in ASD. A discussion of this literature in the introduction and/or\ndiscussion should be included.\nMinor concerns:\n• Abstract, line 54: eliminate the period in the middle of the sentence.\n• In the results section describing Figure 3F – instead of “in vivo specific regulatory\nmechanism”, maybe refer to this as a non-cell autonomous e;ect.\n• Figure 5G – a split y-axis may help to display the data points better.\n• Y-axis labels are missing space in “fold change”.\n• Pg 20, what is meant by “most of these susceptible genes do not reproduce ASD\nphenotype”.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "Summary:\nIn this manuscript from Wang et al, the authors investigate the role of the chromatin remodeler BAF155 in oligodendroglial\nfunction. They generate a new conditional loss-of-function mouse line, BAF155fl/fl mice, then cross this line to\nNestinCreERT2 to hit most brain cells, and PDGFRaCreERT2 to remove BAF155 from OPCs specifically. Analysis of these\nmice showed reduced numbers of mature oligodendrocytes and myelin production (i.e. hypomyelination) especially in white\nmatter as well as mPFC and hippocampus. At the physiological level, loss of OPC-expressed BAF155 decreased excitatory\nand inhibitory miniatures onto pyramidal neurons in mPFC and hippocampus. Behaviorally, removing BAF155 from OPCs\nimpairs social behaviors, and increases repetitive and anxiety-like behaviors consistent with symptoms of ASD in humans.\nWhen the BAF155fl/fl; PDGFRaCreERT2 mouse line was subjected to a demylination model, OPCs without BAF155 were\nless capable of differentiating. Chip-seq and DEG analysis of purified committed OPCs revealed gene targets of BAF155\nrelated to synaptic function. They then suggest, based upon structural experiments and Ca2+ imaging, that neuron-OPC\nsynapses are disrupted by loss of BAF155.\nOverall, this is a well-written study with high quality, well-organized, and clear figures. I think the topic of how neurons and\nOPCs communicate in the brain is highly relevant and timely, and the connection with autism increases interest in the work. I\nthink the paper is a particularly good fit for Nature Communications based upon its strength. I only have two main\nsuggestions, as you will see below, but I feel the suggested experiments are really important to address prior to publication,\nespecially providing more extensive evidence that the newly generated mouse line behaves as expected.\nMajor comments:\n1. This appears to be the first reported publication of this new BAF155fl/fl mouse line. However, perhaps I missed it, but I\ncannot find in the paper the rigorous validation of the line. How do we know that BAF155 is actually disrupted in these mice?\nOnly exon 4 was floxed, so a truncated protein could still exist. To trust in any of these results, a supplemental figure needs\nto be added validating (1) normal expression of BAF155 in BAF155fl/fl mice versus wildtype mice of the same background\nstrain when Cre or tamoxifen is absent, and (2) the efficient loss of BAF155 protein from most brain cells in the\nNestinCreERT2 line and from OPCs specifically in the PDGFRACreERT2 line. These are necessary experiments to\ninterpret the findings of the study.\n2. In several places, including lines 354-356, the authors indicate that BAF155 disrupts neuron-OPC synaptic connectivity.\nTo support this conclusion, the authors should assess functional synaptic connectivity between neurons and OPCs using\nacute slice electrophysiology. In the absence of such an analysis, the claim that BAF155 loss dysregulates neuron-OPC\nsynapses is not fully supported by the data.\nMinor comments:\n1. A lot of speculation around gene expression results, this should be limited or moved to the discussion.", "reviewer_4": "The study entitled \" Chromatin remodeling factor BAF155 coordinates oligodendroglial-neuronal communications linked to\nregional myelination and autism-like behavioral deficits \" by Wang et al. investigates the role of BAF155 in OPCs in the\ncontext of ASD. They use mouse models to delete BAF155 in OPCs in mice to show myelination defects, altered synaptic\nactivity and behaviour, and they correlate these features to altered expression of synaptic genes in OPCs. This is overall\nvery interesting and relevant findings, deserving publication in Nat. comm.\nI have several comments that might help to make even better the manuscript.\nMajor comments:\n1.) In the graphical abstract as well as in regard to Figure 6 the authors claimed to have studied chromatin accessibility. This\nis not correct, since BAF155 ChIP signals could correlate with altered accessibility but do not show it. For a mechanistic\nexplanation of the altered transcription, indeed an ATAC survey or nucleosome positioning of/in the BAF155 deleted OPCs\nmight be of relevance. Correlation to the observed changes in gene expression would be of benefit in addition. Please\ncorrect also the first sentence referencing to Fig. 6A for the above mentioned reason, that BAF presence it not\nnecessarily correlated to accessibility changes.\n2.) The authors claim that the altered expression of synaptic proteins upon BAF155 deletion leads to impaired OPC-neuron\ntransmission. However, they only show altered Ca-transients in OPCs, or synaptic responses in pyramidal neurons\nindependent from each other. It would be nice to see stimulation of neurons and record altered response in OPCs, which\nwould indeed confirm altered synaptic transmission or communication, as written by the authors. Without this experiments\nthere is the correlation between the events, but not the transmission per se. As the authors identified specific receptors on\nthe OPC side, the relevance of synaptic transmission through these specific receptors can be strengthened by applying\nselective inhibitors to the WT condition.\n3.) How is the connection between altered chromatin in OPCs, myelination defects in axons, altered Ca transients in OPCs,\nand altered synaptic protein expression in OPCs? Among the target genes, are there as well myelin affecting genes as a\nmissing functional link? How do the authors explain and validate experimentally the different correlations they observe?\n4.) On page 11 the authors start the paragraph with \" To investigate why loss of BAF155 impairs OPC differentiation in vivo\nbut not in vitro,...\" however, the answer to this question is not given by the experimentation. In addition to this, it is not clear\nwhether the ChIP-seq was done on in vivo cells (as suggested by using purified committed progenitors) or on in vitro\ncultured cells (as suggested by using medium at day 1 and 5). Please clarify - and resolve the difference between native, i.e.\nin vivo and cultured cells.\n5.) \" The enrichment analysis of these shared genes such as Ank3 (Ankyrin-3 61), Chl1 (Close Homolog of L1 62),\nMap1b (Microtubule-associated protein 1b 63), and Mbp 64, indicated their substantial participation in essential biological\nprocesses, including neuron projection development, developmental growth, and axon development (Figure 6I).\" As the\nauthors claim the substantial participation in essential biological processes, they should integrate these genes in their\ncharacterisation to establish the missing functional links between their observations. As a proof of principle that these are\nindeed essential components linking BAF, neurotransmitter signaling and neuronal development, one of them might be used\nto establish the mechanistic link between OPC BAF functions, impacting myelination, and neuronal function/development.\nMinor comments:\nAbstract:\n1. Through the shortening of information in the Abstract, some statements become unprecise:\n\"...BAF155, a newly identified chromatin remodeling factor linked to autism susceptibility, is primarily expressed in\ncommitted OPCs.\" BAF155 is not newly identified per se - it is perhaps novel in its implication in autism. Further, BAF155 is\na member of ncBAF and nBAF; it has been shown to be active in cortical progenitors during embryonic development\n(10.1016/j.isci.2018.05.014). Therefore the claim that BAF155 is primarily expressed in OPCs seems superficial. The\nauthors phrase much better in the introduction - here, it is formulated in a misleading way.\nFurther, the authors used contribute/contributing in the same sentence and inserted a \".\" instead of \",\" in the last sentence.\nResults:\n2. \"...stage (Figure 4G). In addition, an extensive and significant enrichments of...\" please correct\n3.\" the reduced number of myelinated axons and increased G-ratios in Baf155 knockout mice at both P14 and 8 weeks of\nage (Figure 1D).\" Please explain G-ratios for a broader audience.\n4. The authors observed differences for GABA and Glutamatergic neurons (Fig. 1E, 6G, 6H). Please explain these\ndifferences in regard to potential neuronal functions and correlate to the electrophysiological findings.\n5. \"neither it affected proliferation OPCs (Figure S2F).\" Please correct\n6. In regard to Figures 3 and 6 the authors observed differences in mPFC and hippocampus. If the myelination defect is\nhigher in mPFC compared to the hippocampus, but responses on the targets is higher in hippocampus, it seems that the\nprocesses are decoupled? Maybe the authors can explain this?\n7. The origin of several data sets is not clear and need to be referenced by giving the respective numbers of their deposition\nto publicly accessible databases:\n• RNAseq upon GABA and Glutamate\n• Chip-seq H3K27Ac and H3K4me3\n• Chip-seq of BAF155\nPlease give the original data source and make sure these are publicly available to the readership.\n8.\"... which result in regional myelin development and contribute to the diverse onset of symptoms associated with ASD.\"\nDo the authors refer to local defects in myelination?\n9. Figure legends, Figures:\nPlease give n numbers for all experiments, even if individual data points are given.\nIt is a very good way to indicate the p-values in the graphs as done for S1D - why not doing it consistently for all?\nFigure 4: 1d and 5d brains is not understandable; please describe what is used as material correctly. Also give n-numbers\nand statistics for 4I.\nDiscussion:\nMaybe the authors could elaborate on how the communication between OPC receiving inputs from neurons can be related\nto abnormal neuronal functions in more detail and by citing the relevant literature regarding the transmitters involved?\nWhat are these transmitters, and how do the authors integrate their findings of interneurons being heavily impacted, and\nOPCs responding differently to GABA or Glutamate?\nMethods:\nFor the demyelinating mouse model, please precise which age and how long the mice were exposed."}, "author_response": "Response: We sincerely appreciate this comment. To address it, we performed\nimmunostaining experiments to clarify these issues. In addition to OLIG2 staining, we\nalso performed the co-staining for PDGFRα/OLIG2, IPR-II/OLIG2 and CC1/OLIG2 in the\n3\nmPFC of NestinCreERT2;Baf155fl/+ mice. Our results confirmed that the number of OLIG2+ cells\nwas decreased in NestinCreERT2;Baf155fl/+ mice, as well as the numbers of PDGFRα+ OPCs\nand CC1+ OLs. These results suggest that a reduction of total number of OLs, and an\noverall decrease in oligodendroglial population. We have added this data in the New\nFigure S1F, and a statement in the limitations of Discussion section that further studies\nare still needed to elucidate the mechanism underlying the overall reduction of\noligodendroglial lineage in NestinCreERT2;Baf155fl/+ mice.\nTo further determine whether the reduction of total number of OLs due to reduced total\nnumber of oligodendroglial lineage or a reduction in the oligodendroglial differentiation,\nwe quantified the proportion of OPCs by measuring the ratio of PDGFRα/OLIG2 co-\nstaining, the proportion of committed OPCs by IPR-II/OLIG2 co-staining, and the\n3\nproportion of mature OL by CC1/OLIG2 co-staining. Both the OPC and OL ratios were\ndecreased in NestinCreERT2;Baf155fl/+ mice, consistent with the decreased OPC an OL\nnumbers. However, the ratio of committed OPCs significantly increased, indicating a\nblock in the transition from committed OPCs to mature OLs. This finding supports our\nhypothesis that BAF155 is essential for OPC-neuron synaptic connections, which are\ncritical for subsequent differentiation and myelination. We have added this data in Result\nsection, and highlighted in yellow.\nRegarding the question “Is the reduction of mature OLs due to reduced expression of\nMBP or CC1 at the mature stage or a reduction in the total number of OLs”, MBP and\nCC1 are widely used as the mature markers for OLs; without their expressions,\noligodendroglial lineage cells can’t be classified as mature OLs. As shown in the New\nFigure S1F, the mature OL ratio decreases while the committed OPC ratio increases,\nimplying again that BAF155-KO impairs the differentiation of OPCs.\n2. Related to Fig 1 and Fig S2\nWhy is there a decrease in PV interneurons? Are the cells dying or are the number of\ninterneuron progenitors low and, hence, fewer cells?\nResponse: We are sorry for the lack of clarity in our manuscript. In our study, we found\na significant decrease of MBP positive area on PV positive axons by MBP/PV co-staining,\nwhereas no difference was found in PV interneuron numbers or SMI32 positive axons.\nWe have revised the description of this part to make it more accurate: “Fluorescence\nmicroscopy revealed that the MBP-positive areas on parvalbumin (PV)-positive axons of\ninterneurons, assessed through MBP/PV co-staining, decreased significantly when\ncompared to WT controls. Specifically, there was an 86.86±11.71% reduction at P14 and\na 28.68±3.15% reduction at 8 weeks of age (New Figure 1F, G). Loss of BAF155, however,\ndid not affect the SMI32 positive axons (New Figure 1F, G), the numbers of OLIG2-\npositive and PDGFRα-positive oligodendroglial lineage cells (Figure S2F, S2G), or the\nproliferation of OPCs (Figure S2G)”.\n3. IF panels need to be brightened, especially the DAPI channel (e.g. Fig1F, Fig3A). Single\npanels/channel (preferentially in greyscale to improve contrast) should be added to help\nwith the visualization of the data (eg. Fig 1F, 3F).\nResponse: We thank the reviewer for this comment. Based on the reviewer’s suggestion,\nwe have brightened the DAPI channel in New Figure 1F and 3A. Additionally, we have\nreplaced single channel with greyscale to enhance the contrast in New Figure 1F and 3F.\n4. Are the fluorescence intensities normalized to Dapi counts or Dapi area (as done on\nFig 3F quantification)?\nResponse: We thank the reviewer for this comment. We have added this information in\nthe Statistics of Method section and the Figure legend of Fig3F: “For the analysis of\nrepresentative images of MBP/PDGFRα staining in OPC cultures, the fluorescence\nintensities were normalized for the total DAPI nuclear area value”. Because the size of\nDAPI-labelled nuclei is generally consistent, this quantity should represent and correlate\nwith changes in DAPI area (see below reference).\nBolognesi MM, Dall'Olio L, et al. Seeing or believing in hyperplexed spatial proteomics via antibodies:\nNew and old biases for an image-based technology. Biol Imaging. 2024 Oct 23;4:e10. doi:\n10.1017/S2633903X24000138. eCollection 2024.\n5. Figure 3\n1. Fig 3A\nIf possible, please include a full brain image (low magnification or stitched)\n2. Olig2 staining may be helpful to evaluate if the MBP differences are due to OL loss or\nreduced expression of the protein.\nFig 3D and 3E: similar comment. Co-staining with Olig2 may be helpful for\nthe quantification.\n3. The lysolecithin-induced demyelination experiments are a bit confusing. How do we\nknow that the injection produced a lesion? A control experiment demonstrating a\ndemyelination lesion was induced is necessary.\nResponse: We thank the reviewer for these valuable comments. Based on the reviewer’s\nsuggestion, we have added the low magnification images of MBP staining of different\nregions in our New Figure S3A, S3E.\nTo evaluate if the MBP differences are due to OL loss or reduced expression of myelin\nprotein, we also have added the extra data of CC1/OLIG2/PDGFRα staining of different\nregions in New Figure S3B-S3D, S3F-S3H. These new results indicated a significant\nreduction in CC1-positive mature OLs, but no differences in OLIG2+ or PDGFRα+ cells,\nsuggesting that the MBP difference of different regions was attributable to the reduction\nof CC1+ mature OLs rather than the reductions of OLIG2+ or PDGFRα+ cells.\nFor the PlpCreERT;Baf155fl/fl mice, we also have provided the extra data of CC1/OLIG2 co-\nstaining, and MAG in situ hybridization in New Figure S4C, S4D, confirming that\ndepleting BAF155 in OLs does not induce obvious defects in oligodendroglial\ndifferentiation and myelination.\nIn addition, for the lysolecithin-induced demyelination experiments, we have included\nthe results of MBP and DAPI staining to define the lesion areas in mice, as shown in New\nFigure S4A, S4B. This model has been widely used in previous studies both in our lab\nand by other researchers (see below references).\nNiu J, Tsai HH, Hoi KK, et al. Aberrant oligodendroglial-vascular interactions disrupt the blood-brain\nbarrier, triggering CNS inflammation. Nat Neurosci. 2019;22(5):709-718.\nNiu J, Yu G, Wang X, et al. Oligodendroglial ring finger protein Rnf43 is an essential injury-specific\nregulator of oligodendrocyte maturation. Neuron. 2021;109(19):3104-3118.e6.\nFancy SP, Harrington EP, et al. Axin2 as regulatory and therapeutic target in newborn brain injury and\nremyelination. Nature Neuroscience. 2011;14(8):1009-16.\nArnett HA, Fancy SP, et al. bHLH transcription factor Olig1 is required to repair demyelinated lesions\nin the CNS. Science. 2004;306(5704):2111-5.\n6. Figure 2\n1. The results section states miniature EPSCs were recorded but the figure and\nfigure legend state spontaneous EPSCs. Which is correct? Based on frequency and amplitudes I\nam guessing these are sEPSCs and sIPSCs. What is causing the reduced frequency of\nspontaneous events in pyramidal neurons? Supp Figure 1E,F suggest that axons density and neuron numbers\nare not altered. Presumably spontaneous network activity is reduced due to\nreduced myelination of axons? It would be good to demonstrate this by\nrecording miniature synaptic events, as the TTX would block network activity, presumably\nmEPSC and mIPSC frequency and amplitude would be unchanged in BAF155 mutants?\n2. How many neurons were recorded in each animal? The figure legends states each data\npoint is a mouse.\nResponse: We thank the reviewer for his/her careful reading. “Miniature excitatory\npostsynaptic currents (mEPSC) and miniature inhibitory postsynaptic currents (mIPSC)”\nshown in the results section should be “spontaneous EPSCs and IPSCs”, which we have\ncorrected. In addition, we performed electrophysiological experiment as suggested. We\nrecorded miniature synaptic currents in the same regions. For isolating mEPSCs, we\ncontinuously infused sodium channel blocker tetrodotoxin (TTX, 1 mM) into the ACSF.\nFor recording mIPSCs, we used a special pipette solution containing CsCl and\nadministrate the AMPA receptor antagonist BNQX (10 mM) and NMDA receptor\nantagonist AP-5 (50 mM) or TTX (1 mM) + BNQX (10 mM) + AP-5 (50 mM) into the ACSF\nthroughout the recording. Our results showed that the frequency and amplitude of\nmEPSCs and mIPSCs remained unchanged in BAF155 knockout mice (New Figure 2C, D),\nsuggesting that the reduction in spontaneous network activity is attributable to decreased\naxonal myelination. We have added this new result in our Result and Method sections.\nIn the figure legend of Fig2A-B, each data point represents a single neuron cell. We are\nsorry for this mistake in our description. We apologize for this error in our description.\nThis has been corrected in the current version of our manuscript.\n7. Figure 4\n1. For ChIP-seq experiments, how was the antibody validated for its specificity for BAF155?\nIn supp Figure 2, the BAF155 antibody is producing a signal in BAF155fl/fl mouse x\nPDGFRa-CreER. This could be explained by inefficient recombination, but could also be\ndue to non-specific BAF155 antibody.\nResponse: We thank the reviewer for this comment. For ChIP-seq experiments, we\nselected the antibody from CST (catalog number 11956), which is widely used in ChIP\nassays and has been validated for specificity (see below references). We also confirmed\nits specificity for BAF155 by showing the markedly reduced BAF155 staining observed in\ntissues from BAF155-KO mice. The residual BAF155 signal detected in PDGFRα\nCreER; BAF155fl/fl mice is likely attributable to incomplete recombination.\nMondal S, Ramanathan M, Miao W, et al. PROBER identifies proteins associated with programmable\nsequence-specific DNA in living cells. Nat Methods. 2022;19(8):959-968.\nZhang HF, Hughes CS, Li W, et al. Proteomic Screens for Suppressors of Anoikis Identify IL1RAP as a\nPromising Surface Target in Ewing Sarcoma. Cancer Discov. 2021;11(11):2884-2903.\nHu A, Chen G, Bao B, et al. Therapeutic targeting of CNBP phase separation inhibits ribosome\nbiogenesis and neuroblastoma progression via modulating SWI/SNF complex activity. Clin Transl\nMed. 2023;13(4):e1235.\nHarlow ML, Chasse MH, Boguslawski EA, et al. Trabectedin Inhibits EWS-FLI1 and Evicts SWI/SNF\nfrom Chromatin in a Schedule-dependent Manner. Clin Cancer Res. 2019;25(11):3417-3429.\nWu J, Krchma K, Lee HJ, et al. Requisite Chromatin Remodeling for Myeloid and Erythroid Lineage\nDifferentiation from Erythromyeloid Progenitors. Cell Rep. 2020;33(7):108395.\n8. Supplemental Figure 1\n1. The effect of BAF155 heterozygous mutation is quite small, and the majority\nof the experiments were performed using homozygous KO, which severely limits the relevance\nof this work in relation to ASD. Patients with BAF155 mutation will only have heterozygous\nmutations.\nResponse: We thank the reviewer for raising an important point about the translational\nrelevance of our work. We acknowledge that the heterozygous BAF155 mutation\nproduces a modest yet statistically significant effect, whereas the most of our mechanistic\nresults were from homozygous knockout mice. Most ASD-associated genes (such as\nCHD8, PTEN, see below references) exhibit only mild or subclinical neurodevelopmental\nalterations when heterozygously deleted. For instance, CHD8⁺/⁻ mice display modest\nbrain overgrowth and subtle behavioral changes; whereas PTEN⁺/⁻ mice show\nwhite-matter abnormalities with relatively limited behavioral deficits. These examples\nillustrate that heterozygous mutations typically produce fine-scale cellular or molecular\nperturbations rather than dramatic phenotypes. In our NestinCreERT2;Baf155fl/+ mice, the\nMBP-positive area and the number of CC1⁺ mature OLs were reduced, while the\nproportion of IPR-II/OLIG2-positive committed OPCs was significantly increased,\n3\nindicating a block in differentiation. To more definitively demonstrate the relevance of\nBAF155 deficiency in oligodendroglial lineage to ASD-related phenotypes, we employed\nhomozygous knockout mice, a strategy consistent with previous studies of ASD risk genes.\nWe believe our work provides novel evidence linking BAF155 to ASD pathology.\nWe also acknowledge that the use of homozygous mutants limits the direct translatability\nof our findings to human patients, who typically carry heterozygous mutations. In the\nfuture studies, we will incorporate temporally and spatially restricted knockouts,\ngenotype-phenotype correlation with patient data, and multi-omics analyses to further\nbridge the gap between mouse models and the human conditions. We have\nacknowledged this limitation in our Discussion section.\nShi X, Lu C, Corman A, et al. Heterozygous deletion of the autism-associated gene CHD8 impairs\nsynaptic function through widespread changes in gene expression and chromatin compaction. Am J\nHum Genet. 2023;110(10):1750-1768.\nBusch RM, Srivastava S, Hogue O, et al. Neurobehavioral phenotype of autism spectrum disorder\nassociated with germline heterozygous mutations in PTEN. Transl Psychiatry. 2019;9(1):253.\n9. Supplemental Figure 2\n1. In Supp figure 2C and 2D, there is a consistent reduction in CC1+ and MAG+\nstaining in the right quadrant of the images. What is the exact region of the mPFC that is imaged\nand is this imaging region consistent between animals?\nResponse: Thanks for the reviewer for the careful reading. These CC1 and MAG staining\nregions are consistent across animals. This right quadrant which shows a consistent\nreduction in CC1+ and MAG+ staining is the are nearer the cortex. Because cortical\ndevelopment occurs later than that of the CC, fewer CC1⁺/MAG⁺ cells are detected\nthere. Now, we have added the detailed location information to the figure legend. In\naddition, we have added the low magnification images of CC1 and MAG staining in our\nNew Figure S2D, S2E.\n10. Where all genotypes of mice given tamoxifen?\nResponse: We are sorry for the lack of clarity in our manuscript. To ensure comparability\nbetween experimental and control mice, all genotypes of mice were given tamoxifen. We\nhave provided this detail in Method section, and highlighted in yellow.\n11. There is a growing body of evidence from several ASD mouse models related to\nmyelination defects in ASD. A discussion of this literature in the introduction and/or\ndiscussion should be included.\nResponse: We sincerely appreciate the valuable comment, and agree with the reviewer.\nRecently, a growing body of evidence from postmortem and animal studies indicate that\nwhite matter, particularly myelin, undergoes alteration at different stages of development\nin individuals with autism. Given the well-established roles of myelin in facilitating nerve\nimpulse conduction, promoting synaptogenesis and fine-tuning intracortical network, it\nemerges as a potential key player in ASD pathology. However, the certain contribution of\naltered myelin to the diverse symptomology seen in ASD remained unclear. In this study,\nwe successfully induced ASD-like pathology in an OPC-specific knockout mouse model,\nand revealed that BAF155 regulates nearly two hundred synaptic genes, which is\nconsistent with the notion that OPC-neuron synaptic contacts contribute to psychiatric\ndisorders. Among these synaptic genes, such as Gabrg3, the top listed synaptic gene\nenriched in OPCs, is associated with altered myelination, accounting for ~10-20% ASD\ncases. Our findings suggest a complex relationship between all three classical pathways\nof ASD pathogenesis: chromatin remodeling, synaptic formation, and neural projection.\nIn this context, the primary chromatin remodeling mechanism for regulating OPC-neuron\nsynaptic connection significantly impacts myelin-supported neural functions, thereby\ntriggers the onset of autistic symptoms. We have discussed this point in our manuscript,\nand highlighted in yellow.\nMinor concerns:\n• Abstract, line 54: eliminate the period in the middle of the sentence.\n• In the results section describing Figure 3F – instead of “in vivo specific regulatory\nmechanism”, maybe refer to this as a non-cell autonomous effect.\nResponse: We thank the reviewer for this valuable comment. As suggested by the\nreviewer, we have eliminated the period in the middle of the sentence in the line 54 of\nAbstract, and corrected the “in vivo specific regulatory mechanism” into “a non-cell\nautonomous effect”.\n• Figure 5G – a split y-axis may help to display the data points better.\n• Y-axis labels are missing space in “fold change”.\nResponse: Thanks for the reviewer for the careful reading. We have changed these\nfigures accordingly.\n• Pg 20, what is meant by “most of these susceptible genes do not reproduce ASD\nphenotype”.\nResponse: We thank the reviewer for this valuable comment. To make our description\nmore accurate, we have re-written this part: “most of these susceptible genes when\nexamined individually, fail to recapitulate ASD-like phenotypes in animal models, and\nmany of their underlying mechanisms remain unknown”. As another reviewer suggested,\nwe moved this part to the Discussion section, and highlighted it in yellow.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse: We thank the reviewer for his/her help and support.\nReviewer #3 (Remarks to the Author):\nSummary:\nIn this manuscript from Wang et al, the authors investigate the role of the chromatin\nremodeler BAF155 in oligodendroglial function. They generate a new conditional loss-\nof-function mouse line, BAF155fl/fl mice, then cross this line to NestinCreERT2 to hit most\nbrain cells, and PDGFRaCreERT2 to remove BAF155 from OPCs specifically. Analysis of\nthese mice showed reduced numbers of mature oligodendrocytes and myelin production\n(i.e. hypomyelination) especially in white matter as well as mPFC and hippocampus. At\nthe physiological level, loss of OPC-expressed BAF155 decreased excitatory and\ninhibitory miniatures onto pyramidal neurons in mPFC and hippocampus. Behaviorally,\nremoving BAF155 from OPCs impairs social behaviors, and increases repetitive and\nanxiety-like behaviors consistent with symptoms of ASD in humans. When the BAF155fl/fl;\nPDGFRaCreERT2 mouse line was subjected to a demyelination model, OPCs without\nBAF155 were less capable of differentiating. Chip-seq and DEG analysis of purified\ncommitted OPCs revealed gene targets of BAF155 related to synaptic function. They then\nsuggest, based upon structural experiments and Ca2+ imaging, that neuron-OPC\nsynapses are disrupted by loss of BAF155.\nOverall, this is a well-written study with high quality, well-organized, and clear figures. I\nthink the topic of how neurons and OPCs communicate in the brain is highly relevant and\ntimely, and the connection with autism increases interest in the work. I think the paper is\na particularly good fit for Nature Communications based upon its strength. I only have\ntwo main suggestions, as you will see below, but I feel the suggested experiments are\nreally important to address prior to publication, especially providing more extensive\nevidence that the newly generated mouse line behaves as expected.\nMajor comments:\n1. This appears to be the first reported publication of this new BAF155fl/fl mouse line.\nHowever, perhaps I missed it, but I cannot find in the paper the rigorous validation of the\nline. How do we know that BAF155 is actually disrupted in these mice? Only exon 4 was\nfloxed, so a truncated protein could still exist. To trust in any of these results, a\nsupplemental figure needs to be added validating (1) normal expression of BAF155 in\nBAF155fl/fl mice versus wildtype mice of the same background strain when Cre or\ntamoxifen is absent, and (2) the efficient loss of BAF155 protein from most brain cells in\nthe NestinCreERT2 line and from OPCs specifically in the PDGFRACreERT2 line. These are\nnecessary experiments to interpret the findings of the study.\nResponse: We thank the reviewer for this valuable comment. Based on the reviewer’s\nsuggestion, we have performed the staining of BAF155 in four mouse groups (WT without\ntamoxifen vs. Baf155fl/fl without tamoxifen vs. PDGFRαCreER;Baf155fl/fl without tamoxifen vs.\nPDGFRαCreER;Baf155fl/fl with tamoxifen) to validate the newly generated Baf155fl/fl mouse line.\nAs shown in our New Figure S2B, BAF155 protein level was significantly decreased in\nPDGFRαCreER;Baf155fl/fl with tamoxifen group, when compared to other three groups.\nMoreover, there were no differences among WT without tamoxifen, Baf155fl/fl without\ntamoxifen and PDGFRαCreER;Baf155fl/fl without tamoxifen groups. These quantifications\nvalidated our new Baf155fl/fl mouse line, and confirmed that tamoxifen administration can\nreduce the BAF155 protein level in OPCs. Additionally, as shown in the New Figure S2C,\nthe BAF155/PDGFRα co-staining in Baf155fl/fl with tamoxifen and PDGFRαCreER;Baf155fl/fl\nwith tamoxifen groups validated the efficient BAF155 depletion from OPCs.\nFurthermore, to validate the efficient BAF155 depletion from brain cells in the NestinCreERT2\nline, and from OPCs specifically in the PDGFRαCreERT2 line, we have performed co-staining\nof BAF155 with cell type markers: NeuN, Aldh1L1, OLIG2, PDGFRα for brain cells in\nknockout mice. Our results showed reduced BAF155 protein levels in neural\nstem-cell-derived neurons, astrocytes, and oligodendroglia, but not in microglia (New\nFigure S1C, S1D). We have added these results in our manuscript, and highlighted in\nyellow.\n2. In several places, including lines 354-356, the authors indicate that BAF155 disrupts\nneuron-OPC synaptic connectivity. To support this conclusion, the authors should assess\nfunctional synaptic connectivity between neurons and OPCs using acute slice\nelectrophysiology. In the absence of such an analysis, the claim that BAF155 loss\ndysregulates neuron-OPC synapses is not fully supported by the data.\nResponse: We thank the reviewer for this valuable comment. As suggested by the\nreviewer, we performed whole-cell patch-clamp recordings on YFP+ OPCs from healthy\ncontrol and BAF155 deficient mice (PdgfrαCreER;Rosa-YFP vs. PdgfrαCreER;Baf155fl/fl;Rosa-YFP)\nto assess the neuron-OPC synaptic connectivity (New Figure S5E). For voltage-clamp\nrecordings on OPCs, individual slice was transferred to recording chamber with the\nexternal ACSF contains (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 10\nglucose, 2 CaCl2∙2H2O, and 1 MgSO4∙7H2O. The patch pipette was injected with internal\nsolution (in mM): 135 Csmethanesulfonate, 8 NaCl, 10 HEPES, 2 Mg2ATP, 0.3 Na3GTP,\n0.1 Spermine, 7 phosphocreatine and 0.3 EGTA. AMPAR-mediated EPSCs were recorded\nat -70 mV, NMDAR-mediated EPSCs were recorded at +40 mV. As stimulation electrode,\nelectrode was inserted in stratum radiatum of CA1 area under a fixed stimulus intensity\n(New Figure S5F). Then, the AMPA/NMDA current ratio, a critical and widely accepted\nmetric for assessing synaptic maturation and functional strength, was calculated as\ndescribed previously (see below reference). Because of the technical difficulty of OPC\npatch-clamp, we were only able to record three OPCs in each group under identical\nstimulus conditions. However, as shown in our New Figure S5G, the\nAMPA/NMDA current ratio potentially reduced in Baf155-deficient OPCs compared with\ncontrol OPCs, implying again that BAF155 deletion in OPCs impairs the synaptic\ncommunication between OPCs and neurons. We also have included this new result in our\nResult and Method sections, and have acknowledged this limitation of the OPC\npatch-clamp in our Discussion section.\nMinor comments:\n1. A lot of speculation around gene expression results, this should be limited or moved\nto the discussion.\nResponse: We thank the reviewer for this suggestion. We have refined this section and\nmoved these statements into our Discission section.\nReviewer #4 (Remarks to the Author):\nThe study entitled \" Chromatin remodeling factor BAF155 coordinates oligodendroglial-\nneuronal communications linked to regional myelination and autism-like behavioral\ndeficits \" by Wang et al. investigates the role of BAF155 in OPCs in the context of ASD.\nThey use mouse models to delete BAF155 in OPCs in mice to show myelination defects,\naltered synaptic activity and behaviour, and they correlate these features to altered\nexpression of synaptic genes in OPCs. This is overall very interesting and relevant findings,\ndeserving publication in Nat. comm.\nI have several comments that might help to make even better the manuscript.\nMajor comments:\n1.) In the graphical abstract as well as in regard to Figure 6 the authors claimed to have\nstudied chromatin accessibility. This is not correct, since BAF155 ChIP signals could\ncorrelate with altered accessibility but do not show it. For a mechanistic explanation of\nthe altered transcription, indeed an ATAC survey or nucleosome positioning of/in the\nBAF155 deleted OPCs might be of relevance. Correlation to the observed changes in\ngene expression would be of benefit in addition. Please correct also the first sentence\nreferencing to Fig. 6A for the above mentioned reason, that BAF presence it not\nnecessarily correlated to accessibility changes.\nResponse: We feel extremely grateful for the professional reviewing on our work. Based\non the reviewer’s suggestion, we have revised the manuscript to correct the sentence\nregarding chromatin accessibility. In addition, we performed the ATAC assay (the assay\nfor transposase-accessible chromatin) on the OPCs acutely isolated from mPFC and\nhippocampus. This was done to elucidate the role of BAF155 in the transcription of\nGabrg3, Adnp and Nfia through regulating chromatin accessibility. As shown in our New\nFigure 6A, the result of ATAC assay revealed significantly higher chromatin accessibility\nlevels of Gabrg3, Adnp, and Nfia in hippocampal OPCs when compared to cortical OPCs.\nTherefore, we suggest that BAF155 exerts a stronger regulation on these target genes in\nthe hippocampus. We have added this ATAC assay in our Result and Method sections,\nand highlighted this information in yellow.\n2.) The authors claim that the altered expression of synaptic proteins upon BAF155\ndeletion leads to impaired OPC-neuron transmission. However, they only show altered\nCa-transients in OPCs, or synaptic responses in pyramidal neurons independent from\neach other. It would be nice to see stimulation of neurons and record altered response in\nOPCs, which would indeed confirm altered synaptic transmission or communication, as\nwritten by the authors. Without this experiments there is the correlation between the\nevents, but not the transmission per se. As the authors identified specific receptors on\nthe OPC side, the relevance of synaptic transmission through these specific receptors can\nbe strengthened by applying selective inhibitors to the WT condition.\nResponse: We thank the reviewer for this valuable comment. We think this is an excellent\nsuggestion. In response, we performed whole-cell patch-clamp recordings on YFP+\nOPCs from healthy control and Baf155 deficient mice (PdgfrαCreER;Rosa-YFP vs.\nPdgfrαCreER;Baf155fl/fl;Rosa-YFP) to assess the neuron-OPC synaptic connectivity (New\nFigure S5E). For voltage-clamp recordings on OPCs, individual slice was transferred to\nrecording chamber with the external ACSF contains (in mM): 125 NaCl, 2.5 KCl, 1.25\nNaH2PO4, 26 NaHCO3, 10 glucose, 2 CaCl2∙2H2O, and 1 MgSO4∙7H2O. The patch\npipette was injected with internal solution (in mM): 135 Csmethanesulfonate, 8 NaCl, 10\nHEPES, 2 Mg2ATP, 0.3 Na3GTP, 0.1 Spermine, 7 phosphocreatine and 0.3 EGTA. AMPAR-\nmediated EPSCs were recorded at -70 mV, NMDAR-mediated EPSCs were recorded at\n+40 mV. As stimulation electrode, electrode was inserted in stratum radiatum of CA1 area\nunder a fixed stimulus intensity (New Figure S5F). Then, the AMPA/NMDA current ratio,\na critical and widely accepted metric for assessing synaptic maturation and functional\nstrength, was calculated as described previously (see below reference). Because of the\ntechnical difficulty of OPC patch-clamp, we were only able to record three OPCs in each\ngroup under identical stimulus condition. However, as shown in our New Figure S5G, the\nAMPA/NMDA current ratio was potentially reduced in Baf155-deficient OPCs compared\nwith control OPCs, suggesting that BAF155 deletion in OPCs impairs the synaptic\ncommunication between OPCs and neurons. We have added this new result in our Result\nand Method sections, and we also have acknowledged this limitation of the OPC patch\nclamp technique in Discussion section.\nAlthough we have identified serval potential receptors on the OPC side, GO enrichment\nanalysis revealed that enrichments in synapse-related genes, not only including both\npresynaptic and postsynaptic compartments, but also including both GABA receptor\ngenes, such as Gabrg3, Gabrg2, and glutamate receptor genes, such as Gria2, Grm7, Grik2.\nThese neurotransmitter receptors play a crucial role in various developmental processes\nand myelination, which is significantly more complex than the primary focus of this study,\nwhich is to establish a connection between BAF155 in oligodendroglia and ASD\npathogenesis. Nonetheless, we believe it would be beneficial for future studies to\nmanipulate one of these receptors on OPCs (We have another ongoing project focusing\non GABRG3 on OPCs right now). This could help demonstrate the role of neuronal activity\ninputs to OPCs, as well as in OPC-related physiology and pathophysiology. We have\nacknowledged these limitations in our Discussion section, and highlighted in yellow.\n3.) How is the connection between altered chromatin in OPCs, myelination defects in\naxons, altered Ca transients in OPCs, and altered synaptic protein expression in OPCs?\nAmong the target genes, are there as well myelin affecting genes as a\nmissing functional link? How do the authors explain and validate experimentally the\ndifferent correlations they observe?\nResponse: We sincerely appreciate the valuable comment. Recently, a growing body of\nevidence from postmortem and animal studies indicate that white matter, particularly\nmyelin, undergoes alteration at different stages of development in individuals with autism.\nGiven the well-established roles of myelin in facilitating nerve impulse conduction,\npromoting synaptogenesis and fine-tuning intracortical network, it emerges as a\npotential key player in ASD pathology. However, the certain contribution of altered myelin\nto the diverse symptomology seen in ASD remained unclear. In this study, we successfully\ninduced ASD-like pathology in an OPC-specific knockout mouse model, and revealed\nthat BAF155 regulates nearly two hundred synaptic genes, which is consistent with the\nnotion that OPC-neuron synaptic contacts contribute to psychiatric disorders. Among\nthese synaptic genes, such as Gabrg3, the top listed synaptic gene enriched in OPCs, is\nassociated with altered myelination, accounting for ~10-20% ASD cases (see below\nreference).\nTo examine the function of OPC-neuron synapses, Ca2+ imaging was used to reflect the\nOPC activity. Meanwhile, the Ca2+ dynamics has been documented previously as indicative\nof oligodendroglial processes that precede retractions from axons (see below reference).\nSpecific patterns of Ca2+ transients play different roles in myelin growing. These findings\nindicate that OPCs deficient in BAF155 exhibit abnormal Ca2+ dynamics, which may stem\nfrom a weakened synaptic transmission between OPCs and neurons. In the present study,\nwe found that the number of oscillatory peaks per minute were significantly decreased in\nBaf155-deleted OPCs (Figure 5G-H), Ca2+ transients had greater amplitude in OPCs with\nBaf155 deletion (Figure 5I), and the number of higher frequencies Ca2+ transients,\ndiminished in OPCs lacking BAF155 (Figure 5J). Hence, our results link the OPC-neuron\nsynapse with OPC Ca2+ dynamics and myelination.\nIn present study, our findings may suggest a complex relationship between all three\nclassical pathways of ASD pathogenesis: chromatin remodeling, synaptic formation, and\nneural projection. In this context, the primary chromatin remodeling mechanism for\nregulating OPC-neuron synaptic connection significantly impacts myelin-supported\nneural functions, thereby triggers the onset of autistic symptoms. Right now, we have\ndiscussed this point in our manuscript.\nStefansson, H., Meyer-Lindenberg, A., Steinberg, S., Magnusdottir, B., Morgen, K., Arnarsdottir, S.,\nBjornsdottir, G., Walters, G.B., Jonsdottir, G.A., Doyle, O.M., et al. (2014). CNVs conferring risk of autism\nor schizophrenia affect cognition in controls. Nature 505, 361-366. 10.1038/nature12818.\nKrasnow, A.M., Ford, M.C., Valdivia, L.E., Wilson, S.W., and Attwell, D. (2018). Regulation of developing\nmyelin sheath elongation by oligodendrocyte calcium transients in vivo. Nat Neurosci 21, 24-28.\n10.1038/s41593-017-0031-y.\nBaraban, M., Koudelka, S., and Lyons, D.A. (2018). Ca (2+) activity signatures of myelin sheath\nformation and growth in vivo. Nat Neurosci 21, 19-23. 10.1038/s41593-017-0040-x.\n4.) On page 11 the authors start the paragraph with \" To investigate why loss of BAF155\nimpairs OPC differentiation in vivo but not in vitro,...\" however, the answer to this question\nis not given by the experimentation. In addition to this, it is not clear whether the ChIP-\nseq was done on in vivo cells (as suggested by using purified committed progenitors) or\non in vitro cultured cells (as suggested by using medium at day 1 and 5). Please clarify -\nand resolve the difference between native, i.e. in vivo and cultured cells.\nResponse: We appreciate this insightful comment. In our study, the ChIP-seq\nexperiments were performed using in vitro cultured OPCs, because isolating sufficient\nnumbers of committed OPCs from brain tissue was too difficult and infeasible in\ntechnique. Nevertheless, we don’t think that the use of cultured cells compromises our\npurpose of investigating the underlying mechanism of BAF155 regulating OPC\ndevelopment. In the present study, our data showed that BAF155 depletion impairs OPC\ndifferentiation in vivo, whereas the same depletion has no effect on differentiation in vitro.\nGiven BAF155 is required for establishing OPC-neuron synaptic connection, and this\nconnection is essential for OPC differentiation and myelination in the CNS (this conclusion\nis supported both by our findings and by previous studies from other labs, see references).\nWe therefore propose that the differentiation and myelin defects observed after BAF155\nloss arises from the absence of neuronal signaling inputs to OPCs. In the in vitro\nexperiment, although the chromatin remodeling system was affected by BAF155\ndepletion, and synaptic genes changed in cultured committed OPCs (shown in Figure 4),\nthere was no neuronal signaling; consequently, OPC differentiation was not altered.\nThis is why we observed no differences in OPC differentiation, or MBP expression in vitro.\nIn fact, we think this finding supports the hypothesis that BAF155 plays a critical role in\noligodendroglia development and ASD pathogenesis. Right now, we have refined the\nrelated texts in our manuscript, and highlighted in yellow.\nSynapses shape oligodendrocyte precursor cell development and predict myelination location. Nat\nNeurosci. 2024;27(2):217-218.\nMoura DMS, Brennan EJ, Brock R, Cocas LA. Neuron to Oligodendrocyte Precursor Cell Synapses:\nProtagonists in Oligodendrocyte Development and Myelination, and Targets for Therapeutics. Front\nNeurosci. 2022;15:779125.\n5.) \" The enrichment analysis of these shared genes such as Ank3 (Ankyrin-3 61), Chl1\n(Close Homolog of L1 62), Map1b (Microtubule-associated protein 1b 63), and Mbp 64,\nindicated their substantial participation in essential biological processes, including neuron\nprojection development, developmental growth, and axon development (Figure 6I).\" As\nthe authors claim the substantial participation in essential biological processes, they\nshould integrate these genes in their characterization to establish the missing functional\nlinks between their observations. As a proof of principle that these are indeed essential\ncomponents linking BAF, neurotransmitter signaling and neuronal development, one of\nthem might be used to establish the mechanistic link between OPC BAF functions,\nimpacting myelination, and neuronal function/development.\nResponse: We appreciate this insightful comment. We have to acknowledge that the\npresent study is not able to address all of these intriguing questions. So far, the genes\nidentified in our work are linked with oligodendroglial and neuronal development, as well\nas the pathogenesis of ASD. However, proving that they are essential mediators linking\nBAF155, neurotransmitter signaling, and neuronal development is far more intricate than\nthe primary focus of this study, which is to establish a connection between BAF155 in\noligodendroglia and ASD pathogenesis. Nonetheless, we believe this line of inquiry is\nworthwhile for future studies. We have acknowledged this limitation in our Discussion\nsection.\nMinor comments:\nAbstract:\n1. Through the shortening of information in the Abstract, some statements become\nunprecise:\n\"...BAF155, a newly identified chromatin remodeling factor linked to autism susceptibility,\nis primarily expressed in committed OPCs.\" BAF155 is not newly identified per se - it is\nperhaps novel in its implication in autism. Further, BAF155 is a member of ncBAF and\nnBAF; it has been shown to be active in cortical progenitors during embryonic\ndevelopment (10.1016/j.isci.2018.05.014). Therefore the claim that BAF155 is primarily\nexpressed in OPCs seems superficial. The authors phrase much better in the introduction\n- here, it is formulated in a misleading way.\nFurther, the authors used contribute/contributing in the same sentence and inserted a \".\"\ninstead of \",\" in the last sentence.\nResponse: We thank the reviewer for careful reading. We have addressed these issues\naccordingly and refined our Abstract.\nResults:\n2. \"...stage (Figure 4G). In addition, an extensive and significant enrichments of...\" please\ncorrect\nResponse: We thank the reviewer for careful reading. We have corrected this mistake.\n3.\" the reduced number of myelinated axons and increased G-ratios in Baf155 knockout\nmice at both P14 and 8 weeks of age (Figure 1D).\" Please explain G-ratios for a broader\naudience.\nResponse: We thank the reviewer for this suggestion. We have provided more detailed\ninformation of G-ratio in the result of Figure 1D: “G-ratio is defined as the ratio of an\naxon’s diameter to the overall diameter of its myelinated fiber. It serves as a key metric\nfor evaluating myelin integrity and function; lower G-ratio value reflects thicker myelin\nsheath and a greater degree of myelination”.\n4. The authors observed differences for GABA and Glutamatergic neurons (Fig. 1E, 6G,\n6H). Please explain these differences in regard to potential neuronal functions and\ncorrelate to the electrophysiological findings.\nResponse: We appreciate the valuable comment. As shown in Figure 1, PV+ neurons are\na class of fast-spiking GABAergic interneurons that provide inhibitory control in the brain.\nThe significant hypomyelination of PV+ axons in BAF155-deficient mice is likely to\ndesynchronize neural networks. This phenomenon is a well-established\npathophysiological feature of ASD and schizophrenia, where dysfunction of PV+\ninterneurons is a common theme. In Figure 6, OPCs exposed to GABA versus glutamate\nexhibit different gene expression profiles. Serval key genes involved in oligodendroglial\ndevelopment and myelination are differentially regulated. Besides, our results also\nindicate that OPCs express both GABA and glutamate receptors, as shown in Figure 4,\nand 5. These findings suggest that neuronal activity, mediated by the release of GABA\nand glutamate, provides instructional cues to OPCs. The distinct genetic programs\ninduced by these two primary neurotransmitters imply a sophisticated signaling “code”.\nVarious patterns of signaling “code” could regulate OPCs to differentiate or myelinate in\nways that are specifically aligned with the needs of the local circuit. This notion is also\nsupported by previous studies showing that glutamate promotes OPC migration, and at\nlater stages, drives differentiation and myelination; GABA-induced GABAA activation\ninhibits OPC proliferation and reduces myelin thickness, while GABAB activation\nstimulates OPC proliferation and migration (see below reference). The chromatin\nremodeling factor BAF155 is a key regulator that enables OPCs to accurately interpret\nthese neuronal signals. Its deletion disrupts this signaling “code”, leading to inappropriate\ntranscriptional responses. The downregulation of genes associated with synaptic\ndevelopment observed in the DEGs, and histological experiments, as shown in Figure 4,\n5 and 6, suggests that the ability of OPCs to support neuronal development and maintain\nsynaptic connections is disrupted.\nOur new electrophysiological results presented in New Figure S5E-G, show a potentially\nreduced AMPA/NMDA current ratio in BAF155-deficient OPCs, suggesting impaired\nsynaptic transmission from neurons to OPCs. Although we were only able to record from\nthree OPCs in each group under identical stimulus conditions due to the technical\ndifficulties associated with patch-clamping OPCs, this electrophysiological deficit\nprovides a functional link between the molecular changes represented by the DEGs in\nFigure 6 and the structural changes, as reflected by myelin deficits in Figure 1, and the\nOPC-neuron synaptic deficits in Figure 5. If OPCs can’t properly sense neuronal activity\ndue to faulty synapses, they will be unable to execute their developmental program\nappropriately, including the precise timing and targeting of myelination. We have added\nthis statement in Discussion section and highlighted it in yellow.\nAs mentioned above in the point 2, although we have identified potential\nneurotransmitter receptors on the OPC side, these neurotransmitter receptors play a\ncomplex role in various developmental processes and myelination, which is beyond the\nprimary focus of this study. However, we do believe it is a very important comment. Our\nfuture study will manipulate specific receptors, such as GABRG3 on OPCs This will be a\ncrucial next step to directly test the causal chain, and further clarify the role of neuronal\nactivity in guiding myelination through BAF155 in OPCs. We have discussed this point in\nour Discussion section, acknowledged these limitations, and highlighted them in yellow.\nBraaker, P.N., Mi, X., Soong, D., Bin, J.M., Marshall-Phelps, K., Bradley, S., Benito-Kwiecinski, S., Meng,\nJ., Arafa, D., Richmond, C., et al. (2025). Activity-driven myelin sheath growth is mediated by mGluR5.\nNat Neurosci 28, 1213-1225. 10.1038/s41593-025-01956-9.\nChen, T.J., Kula, B., Nagy, B., Barzan, R., Gall, A., Ehrlich, I., and Kukley, M. (2018). In Vivo Regulation\nof Oligodendrocyte Precursor Cell Proliferation and Differentiation by the AMPA-Receptor Subunit\nGluA2. Cell Rep 25, 852-861.e857. 10.1016/j.celrep.2018.09.066.\nHamilton, N.B., Clarke, L.E., Arancibia-Carcamo, I.L., Kougioumtzidou, E., Matthey, M., Káradóttir, R.,\nWhiteley, L., Bergersen, L.H., Richardson, W.D., and Attwell, D. (2017). Endogenous GABA controls\noligodendrocyte lineage cell number, myelination, and CNS internode length. Glia 65, 309-321.\n10.1002/glia.23093.\nLuyt, K., Slade, T.P., Dorward, J.J., Durant, C.F., Wu, Y., Shigemoto, R., Mundell, S.J., Váradi, A., and\nMolnár, E. (2007). Developing oligodendrocytes express functional GABA(B) receptors that stimulate\ncell proliferation and migration. J Neurochem 100, 822-840. 10.1111/j.1471-4159.2006.04255.x.\n5. \"neither it affected proliferation OPCs (Figure S2F).\" Please correct\nResponse: We thank the reviewer for careful reading. We have addressed this issue\naccordingly.\n6. In regard to Figures 3 and 6 the authors observed differences in mPFC and\nhippocampus. If the myelination defect is higher in mPFC compared to the hippocampus,\nbut responses on the targets is higher in hippocampus, it seems that the processes are\ndecoupled? Maybe the authors can explain this?\nResponse: We thank the reviewer for careful reading and insightful comment. We think\nthere are three main reasons may underlie the regional differences we observed. First,\nthe timing and spatial pattern of myelination differ across brain regions. In mice,\nhippocampal myelination begins earlier. MBP expression can be detected around P8;\nwhereas the mPFC shows myelin formation around P10. This developmental lag has been\nreported previously (see reference). While mice complete their myelination within a few\nweeks, human myelination extends over several years and continues into early adulthood,\nespecially in the prefrontal cortex (see reference). Consequently, in our case, the\nhippocampus, which may develop earlier, and undergo a longer period of myelin\nrefinement; therefore, the myelination deficit in hippocampus tends to diminish over time,\nand the myelination deficit in mPFC is larger. Our data shown in Figure 1C also reflects\nthis trend: the myelin deficit at 8 weeks is markedly smaller than that observed at P14.\nSecond, as shown in our New Figure 6A-C, BAF155 exhibits higher chromatin\naccessibility at Gabrg3, Adnp and Nfia loci in the hippocampus than in the mPFC,\nsuggesting that region-specific chromatin accessibility of BAF155 may also contribute to\nregional myelin differences. Moreover, the residual BAF155 signal is still detectable in\nPDGFRαCreER; BAF155fl/fl mice. Hence, we suggest that BAF155 more strongly regulates\nthese target genes in the hippocampus, potentially buffering or compensating for the\nimpact of BAF155 loss more effectively, resulting in a milder myelin phenotype compared\nwith the mPFC.\nThird, given the myelin formation is highly adaptive progress in the CNS, and the number\nof myelin segments that each mature OLs can form is different (see reference), along with\nthe variations among different animals, these factors contribute to the different myelin\nphenotypes observed in different brain regions.\nOzarkar SS, Patel RKR, Vulli T, Friar CA, Burette AC, Philpot BD. Regional analysis of myelin basic\nprotein across postnatal brain development of C57BL/6J mice. Front Neuroanat. 2025;19:1535745.\nSemple BD, Blomgren K, Gimlin K, Ferriero DM, Noble-Haeusslein LJ. Brain development in rodents\nand humans: Identifying benchmarks of maturation and vulnerability to injury across species. Prog\nNeurobiol. 2013;106-107:1-16.\nZeiss CJ. Comparative Milestones in Rodent and Human Postnatal Central Nervous System\nDevelopment. Toxicol Pathol. 2021;49(8):1368-1373.\nBenamer N, Vidal M, Balia M, Angulo MC. Myelination of parvalbumin interneurons shapes the\nfunction of cortical sensory inhibitory circuits. Nat Commun. 2020;11(1):5151.\n7. The origin of several data sets is not clear and need to be referenced by giving the\nrespective numbers of their deposition to publicly accessible databases:\n• RNAseq upon GABA and Glutamate\n• Chip-seq H3K27Ac and H3K4me3\n• Chip-seq of BAF155\nPlease give the original data source and make sure these are publicly available to the\nreadership.\nResponse: We thank the reviewer for careful reading. RNAseq upon GABA and Glutamate\nand ChIP-seq data of BAF155 in this manuscript have been deposited in the NCBI GEO\ndatabase under the accession number GSE282099 (reviewer token: yfaliyyijhkhdeb) and\nGSE282098 (reviewer token: wzibcscgdviptqp), respectively. Chip-seq H3K27Ac and\nH3K4me3 data was from GSM1040161, GSM2225634 and GSM1040162 (see below\nreference). We have added this information in our manuscript. These data sets are\npublicly available as of the date of publication.\nOu Z, Sun Y, Lin L, et al. Olig2-Targeted G-Protein-Coupled Receptor Gpr17 Regulates\nOligodendrocyte Survival in Response to Lysolecithin-Induced Demyelination. J Neurosci.\n2016;36(41):10560-10573.\n8.\"... which result in regional myelin development and contribute to the diverse onset of\nsymptoms associated with ASD.\" Do the authors refer to local defects in myelination?\nResponse: Yes. We have refined this word accordingly.\n9. Figure legends, Figures:\nPlease give n numbers for all experiments, even if individual data points are given.\nIt is a very good way to indicate the p-values in the graphs as done for S1D - why not\ndoing it consistently for all?\nFigure 4: 1d and 5d brains is not understandable; please describe what is used as material\ncorrectly. Also give n-numbers and statistics for 4I.\nResponse: We thank the reviewer for this suggestion. We have provided N numbers and\np-values for all figures.\nDiscussion:\nMaybe the authors could elaborate on how the communication between OPC receiving\ninputs from neurons can be related to abnormal neuronal functions in more detail and\nby citing the relevant literature regarding the transmitters involved?\nWhat are these transmitters, and how do the authors integrate their findings of\ninterneurons being heavily impacted, and OPCs responding differently to GABA or\nGlutamate?\nResponse: We appreciate the reviewer’s suggestion, and have expanded the Discussion\nsection about how neuronal inputs to OPCs may link BAF155 deficiency to abnormal\nneuronal function. OPCs are the only glial cells known to form direct synapses with\nneurons. Glutamatergic (AMPA/NMDA) and GABAergic (GABA /GABA ) receptors are\nA B\nexpressed on OPC membranes, allowing them to sense activity-dependent release of the\ntwo principal CNS neurotransmitters. Glutamate promotes OPC migration, and in later\nstages, drives differentiation and myelination; excessive activation also can transiently\ninhibit proliferation. GABA induced GABA activation inhibits OPC proliferation and\nA\nreduces myelin thickness; while GABA activation stimulates OPC proliferation and\nB\nmigration. ATP and adenosine regulate intracellular Ca2+, influencing OPC migration and\ndifferentiation. Other modulators, such as acetylcholine, serotonin, contribute to\nregion-specific OPC responses during early development. Overall, we suggest that a\nnetwork effect of neuronal activity on OPCs. If OPCs can’t properly sense neuronal activity\ndue to faulty synapses, they will be unable to execute their developmental program\nappropriately, including the precise timing and targeting of myelination. Besides, OPC-\nrelated physiology and pathophysiology, may depend on the balance of excitatory and\ninhibitory inputs via the specific receptors on OPCs. We have discussed this point in our\nmanuscript, and highlighted in yellow.\nMethods:\nFor the demyelinating mouse model, please precise which age and how long the mice\nwere exposed.\nResponse: We thank the reviewer for this comment. As suggested, we have added more\ndetailed information about the demyelinating mouse model in the Method section and\nhighlighted in yellow: “Briefly, for the demyelination mouse model, tamoxifen was\nadministered continuously from P50 to P 54. At P 56, mice were anesthetized by\nisoflurane (2-3%); after exposing the skull, 1.5μl 1% lysolecithin (Sigma-Aldrich, L0906) was\ninjected into the corpus callosum (1.04 mm lateral and 1.0 mm posterior to the bregma,\ndepth: -1.62 mm). Two weeks later, brain tissue was harvested for immunofluorescence\nstaining and in situ hybridization”."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have adequately addressed our concerns, except for a few minor edits requested below. It is our opinion the\nmanuscript is suitable for publication.\n1. Figure legend for supplementary figure one is missing the word supplementary in the legend title.\n2. Concentrations for TTX, BNQX and AP-5 should be in micromolar not millimolar.\n3. Never heard of BNQX, I have heard of CNQX and DNQX.\n4. Pg. 9, a better explanation for the differing results between sEPSC and mEPSC data would be helpful. It currently states\n\"suggesting that the reduction in spontaneous network activity is attributable to decreased axonal myelination\"\nshould also include statements regarding that the no difference in mEPSC frequency and amplitude suggests that synaptic density\nappears to be intact, and only AP-dependent transmission is affected based on sEPSC data, which indicates mylenation\ndefects may be effection AP-dependent synaptic transmission.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.", "reviewer_3": "The authors have addressed my concerns. The paper is now suitable for publication in Nat Comms", "reviewer_4": "The authors addressed my comments to enhance their manuscript. There is few things left, that could be added and\ncorrected in the manuscript.\n1. Lines 235-236: the authors write about potential enhancer regions. As the H3K27ac data are available and used in the\nmanuscript, I suggest to map the BAF155 binding sites and H3K27ac enrichment and to strengthen the statement, that\n\"enhancers\" are bound by BAF155. Presently, without this information, it is more suitable to write about regulatory regions,\nas intragenic regions are not per se enhancers.\n2. Line 260: instead of \"specific for H3K27Ac\" it would be more suitable to write \"enriched for\".\n3. Line 278: correct \"labled\"\n4. Line 321: correct \"play\"\n5. Line 426: change \"triggers\" to \"triggering\"\n6. Line 427: change to \" in the present study,\"\n7. Line 430: change \"response\" to \"respond\"\n8. Line 442: change to \"provides an instructional\"\n9. Line 447: change to \"sensing the neuronal\"\n10. Line 451: change \"oligodendroglial lineage\" to \"oligodendrocytes\"\n11. Line 460: change to \"and humans\""}, "author_response": "Response: We thank the reviewer for the careful reading. We have refined the figure\nlegend for supplementary figures one accordingly.\n2. Concentrations for TTX, BNQX and AP-5 should be in micromolar not millimolar.\n3. Never heard of BNQX, I have heard of CNQX and DNQX.\nResponse: We thank the reviewer for the careful reading. The concentration of TTX,\nNBQX and AP-5 is indeed micromolar rather than millimolar, and we have added the\ninformation in the Method section.\nWe sincerely apologize for mistakenly writing NBQX as BNQX. We have already\ncorrected it.\n4. Pg. 9, a better explanation for the differing results between sEPSC and mEPSC data\nwould be helpful. It currently states \"suggesting that the reduction in spontaneous\nnetwork activity is attributable to decreased axonal myelination\" should also\ninclude statements regarding that the no difference in mEPSC frequency and amplitude\nsuggests that synaptic density appears to be intact, and only AP-dependent\ntransmission is affected based on sEPSC data, which indicates mylenation\ndefects may be effection AP-dependent synaptic transmission.\nResponse: We thank the reviewer for this valuable comment and suggestion. We have\nrefined the text accordingly.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nResponse: We thank the reviewer for his/her help and support.\nReviewer #3 (Remarks to the Author):\nThe authors have addressed my concerns. The paper is now suitable for publication in\nNat Comms\nResponse: We thank the reviewer for his/her help and support.\nReviewer #4 (Remarks to the Author):\nThe authors addressed my comments to enhance their manuscript. There is few things\nleft, that could be added and corrected in the manuscript.\n1. Lines 235-236: the authors write about potential enhancer regions. As the H3K27ac\ndata are available and used in the manuscript, I suggest to map the BAF155 binding\nsites and H3K27ac enrichment and to strengthen the statement, that \"enhancers\" are\nbound by BAF155. Presently, without this information, it is more suitable to write about\nregulatory regions, as intragenic regions are not per se enhancers.\nResponse: We thank the reviewer for this valuable comment and suggestion. We have\nrefined the text accordingly.\n2. Line 260: instead of \"specific for H3K27Ac\" it would be more suitable to write\n\"enriched for\".\n3. Line 278: correct \"labled\"\n4. Line 321: correct \"play\"\n5. Line 426: change \"triggers\" to \"triggering\"\n6. Line 427: change to \" in the present study,\"\n7. Line 430: change \"response\" to \"respond\"\n8. Line 442: change to \"provides an instructional\"\n9. Line 447: change to \"sensing the neuronal\"\n10. Line 451: change \"oligodendroglial lineage\" to \"oligodendrocytes\"\n11. Line 460: change to \"and humans\"\nResponse: We sincerely appreciate the reviewer for the careful reading. We have\naddressed these issues accordingly and refined our manuscript."}]

Decision letter