ANKS1B encoded AIDA-1 regulates social behaviors by controlling oligodendrocyte function

Cho, Chang Hoon; Deyneko, Ilana Vasilisa; Cordova-Martinez, Dylann; Vazquez, Juan; Maguire, Anne S.; Diaz, Jenny R.; Carbonell, Abigail U.; Tindi, Jaafar O.; Cui, Min-Hui; Fleysher, Roman; Molholm, Sophie; Lipton, Michael L.; Branch, Craig A.; Hodgson, Louis; Jordan, Bryen A.

doi:10.1038/s41467-023-43438-1

Download PDF

Article
Open access
Published: 21 December 2023

ANKS1B encoded AIDA-1 regulates social behaviors by controlling oligodendrocyte function

Chang Hoon Cho¹^na1^nAff8,
Ilana Vasilisa Deyneko¹^na1,
Dylann Cordova-Martinez¹,
Juan Vazquez¹,
Anne S. Maguire ORCID: orcid.org/0000-0002-3037-9125¹,
Jenny R. Diaz¹,
Abigail U. Carbonell¹,
Jaafar O. Tindi¹,
Min-Hui Cui^2,3,
Roman Fleysher^2,3,
Sophie Molholm^1,4,5,
Michael L. Lipton^1,2,3,5,
Craig A. Branch^2,3,
Louis Hodgson ORCID: orcid.org/0000-0001-9188-7580^6,7 &
…
Bryen A. Jordan ORCID: orcid.org/0000-0002-4967-444X^1,5

Nature Communications volume 14, Article number: 8499 (2023) Cite this article

1924 Accesses
1 Altmetric
Metrics details

Subjects

Abstract

Heterozygous deletions in the ANKS1B gene cause ANKS1B neurodevelopmental syndrome (ANDS), a rare genetic disease characterized by autism spectrum disorder (ASD), attention deficit/hyperactivity disorder, and speech and motor deficits. The ANKS1B gene encodes for AIDA-1, a protein that is enriched at neuronal synapses and regulates synaptic plasticity. Here we report an unexpected role for oligodendroglial deficits in ANDS pathophysiology. We show that Anks1b-deficient mouse models display deficits in oligodendrocyte maturation, myelination, and Rac1 function, and recapitulate white matter abnormalities observed in ANDS patients. Selective loss of Anks1b from the oligodendrocyte lineage, but not from neuronal populations, leads to deficits in social preference and sensory reactivity previously observed in a brain-wide Anks1b haploinsufficiency model. Furthermore, we find that clemastine, an antihistamine shown to increase oligodendrocyte precursor cell maturation and central nervous system myelination, rescues deficits in social preference in 7-month-old Anks1b-deficient mice. Our work shows that deficits in social behaviors present in ANDS may originate from abnormal Rac1 activity within oligodendrocytes.

PKBβ/AKT2 deficiency impacts brain mTOR signaling, prefrontal cortical physiology, hippocampal plasticity and select murine behaviors

Article 16 December 2020

Targeted Tshz3 deletion in corticostriatal circuit components segregates core autistic behaviors

Article Open access 15 March 2022

Corticostriatal dysfunction and social interaction deficits in mice lacking the cystine/glutamate antiporter

Article Open access 04 May 2020

Introduction

Despite evidence for a large genetic contribution to neurodevelopmental disorders (NDDs), defining causal pathways is difficult because individual mutations typically exert only mild effects and the risk for disease increases with multiple mutations¹. NDDs associated with copy number variations (CNVs) or other mutations predicted to have major effects on protein function², especially those associated with CNVs affecting single genes (monogenic), are critically important for research because they can establish a direct link between one gene and a set of cellular and behavioral phenotypes. We recently identified individuals around the world with heterozygous and monogenic deletions in the ANKS1B gene that cause haploinsufficiency³, confirming a previously uncharacterized link between ANKS1B haploinsufficiency and a novel neurodevelopmental syndrome we term ANKS1B Neurodevelopmental Syndrome (ANDS)³. Patients with ANDS exhibit a range of NDDs, including attention-deficit/hyperactivity disorder (ADHD), motor impairments, speech apraxia and autism spectrum disorder (ASD), the latter of which is present in >60% of patients. The ANKS1B gene encodes for the protein AIDA-1, which is highly expressed in excitatory neurons of the central nervous system (CNS) and is one of the most abundant proteins at neuronal synapses^4,5. AIDA-1 is localized at postsynaptic densities (PSDs), where it binds to N-methyl-D-aspartate receptors (NMDARs) and the synaptic scaffolding protein PSD95⁶. Excitatory neuron-specific (Camk2a-Cre) Anks1b knockout mice show reduced synaptic expression of the NMDAR subunit GluN2B and impaired hippocampal NMDAR-dependent synaptic plasticity⁷, and CNS-wide (Nestin-Cre) Anks1b heterozygous mice display behavioral deficits across domains relevant to clinical features of ANDS³. Based on these findings, we previously postulated that neuronal dysfunctions and synaptopathies are involved in the pathogenesis of ANDS³.

Despite a historical shortage on research into the role of white matter in brain diseases, recent literature has described myelin deficits early in the pathological cascade of prevalent disorders such as Alzheimer disease, Parkinson disease, and schizophrenia. In the process of investigating myelination in normal and abnormal CNS physiology, recent studies on oligodendrocytes have triggered rapid shifts in our understanding of this cell lineage. Oligodendrocyte precursor cells (OPCs) represent 5–10% of the glial population in the CNS and are increasingly recognized as active participants in normal and abnormal brain functions^{8,9,10,11,12,13}. Oligodendrocytes are primarily involved in myelination of CNS axons, which emerging research reveals is dynamically regulated by sensory, motor, and cognitive activity in both humans and rodents throughout the lifespan^{14,15,16,17,18}. There is also growing appreciation for the role of OPCs in the pathogenesis of neurodegenerative disorders including Alzheimer disease^19,20 and Parkinson disease²¹. OPCs have been implicated in ASD because altered white matter integrity^{22,23,24,25,26} and reduced axonal conduction velocity^27,28 have been extensively documented in a subset of patients with this disorder. A recent study showed that haploinsufficiency of Chd8, a gene robustly linked to ASD etiology, leads to oligodendrocyte dysfunction and autism-related behaviors²⁹. Recent large-scale single-cell transcriptomic initiatives suggest that AIDA-1 is expressed in oligodendrocytes and OPCs^30,31,32,33, as well as in newborn OPCs linked to lesions in multiple sclerosis (MS)³⁴. Here, we test the hypothesis that oligodendrocyte dysfunction contributes to ANDS etiology.

Consistent with this speculation, we previously reported that magnetic resonance images (MRIs) from ~40% of ANDS patients reveal thinning or other structural anomalies of the corpus callosum, which is highly myelinated³. Here we reveal additional profound white matter abnormalities in ANDS patients that are also modeled by mice with Anks1b haploinsufficiency. Furthermore, we show that selective Anks1b ablation from oligodendrocytes is sufficient to reproduce both myelination deficits as well as behavioral correlates of ASD found in a brain-wide (Nestin-Cre) Anks1b haploinsufficiency model³. Surprisingly, these behaviors were not recapitulated by selective ablation of Anks1b from neuronal populations that highly express this gene. Moreover, we found that social deficits were rescued by treatment with clemastine fumarate, an FDA approved antihistamine used for seasonal allergies that was recently and unexpectedly found to increase myelination in a high-throughput screen for novel MS therapeutics³⁵.

Our previous proteomic and functional analyses reveal that AIDA-1 may be linked to small Rho family small GTPases, particularly to Rac1 function³⁶. Rho GTPases are crucial for cytoskeletal dynamics³⁷, migration³⁸, and signal transduction³⁹, and have been shown to regulate the cellular morphology, maturation, and migration of oligodendrocyte lineage cells^{40,41,42,43,44}. Additionally, dysregulation of these GTPases is linked to neurodevelopmental (ASD, Rett syndrome), neurodegenerative (Alzheimer, Parkinson), and psychiatric disorders^45,46,47. Rac1 in particular has garnered significant attention due to its role in various neurodevelopmental^48,49 and neurodegenerative diseases⁵⁰ as well as its critical role in supporting oligodendrocyte functions including OPC migration, differentiation, and the formation of myelin sheaths around axons^40,41,51,52. Dysregulation of Rac1 can therefore lead to faulty myelination and contribute to demyelinating disorders like MS⁵³. Using FRET-based G-protein sensors, we confirm that loss of AIDA-1 affects Rac1 activation in primary cultured oligodendrocytes isolated from the Anks1b-deficient mice. We also show that that in vitro activation of Rac1 (as well as treatment with clemastine) rescues maturation deficits observed in these cells. Overall, our results suggest that the social and sensory behavioral correlates of ASD present in mouse models of ANDS originate from oligodendrocyte dysfunction.

Results

Anks1b haploinsufficiency impairs structural integrity of white matter

Several patients with ANDS had undergone brain MRI during prior clinical evaluation³. Radiologist impressions frequently cited abnormalities in the corpus callosum, among other findings in white matter (Fig. 1a). To substantiate the clinical observations, two ANDS patients underwent detailed Diffusion Tensor Imaging (3 Tesla-DTI) at the Gruss Magnetic Resonance Research Center at Albert Einstein College of Medicine to quantitatively assess integrity of white matter microstructure using Fractional Anisotropy (FA) (Fig. 1b, c). Patient FA values were compared to those of 101 neurotypical individuals previously collected as part of the Einstein Lifespan Study⁵⁴ (Fig. 1b–d, Supplementary Fig. 1). In both patients, we found multiple brain regions displaying FA values significantly (p < 0.05) below and above the average of the control group (Fig. 1d). These results reveal profound alterations in white matter microstructure throughout the brain in individuals with ANDS.

**Fig. 1: *Anks1b* haploinsufficiency leads to structural abnormalities in corpus callosum.**

We had previously shown that CNS-wide (Nestin-Cre) Anks1b heterozygous mice (Nestin-Het mice) display behavioral correlates of patient phenotypes including hyperactivity, abnormal approach-avoidance behaviors, sensory hyperreactivity, impaired sensorimotor gating and fine motor coordination, and reduced social preference³. We performed volumetric analysis on T2-weighted MRI data to detect gross anatomical abnormalities and carried out DTI (Fig. 1e). Testing was performed on Nestin-Het and wildtype (WT) littermates for in vivo analyses (n = 5 per genotype), followed by ex vivo imaging of fixed brains of these and an additional set of mice (n = 10 per genotype). Volumetric analyses of ex-vivo brains revealed no statistically significant differences in overall brain volume between genotypes (Fig. 1f). However, Nestin-Het mice had a significant decrease in the volume of the corpus callosum (Fig. 1g), as well as in other highly myelinated brain structures including the optic tract (Supplementary Data 1). Moreover, in vivo DTI analyses revealed a significant decrease in FA of callosal (Fig. 1h) and other tracts (Supplementary Data 1), suggesting deficient white matter integrity and deficits in myelinated axons. These results show that mice with Anks1b haploinsufficiency model the white matter abnormalities found in patients.

Anks1b haploinsufficiency results in impaired myelination and reduced oligodendrocyte abundance

To explore the white matter abnormalities observed in Nestin-Het mice, we performed histological and immunocytochemical analyses. Nissl staining confirmed that Nestin-Het mice have a thinner corpus callosum throughout the rostrocaudal axis (Fig. 2a), as well as larger lateral ventricles (LV) compared to WT littermates, although their dimensions were highly variable among animals (Supplementary Fig. 2a). Comprehensive analyses of other structures also revealed significant changes in the CA1/2 region of the hippocampus as well as in cerebellar lobule VI (Supplementary Fig. 2a, b), an area increasingly correlated with ASDs⁵⁵. Since the decrease in callosal volume suggested impaired myelination, we stained brain slices with Luxol Fast Blue (LFB), a copper phthalocyanine dye that binds to lipoproteins of the myelin sheath. Both male and female Nestin-Het mice had decreased LFB staining intensity compared to WT littermates in the corpus callosum (Fig. 2b) as well as in the cerebellum (Supplementary Fig. 2c). To directly measure myelination, we analyzed transmission electron microscopy (TEM) images of the corpus callosum in the sagittal plane. Quantification revealed a significant increase in g-ratio (signifying decreased myelination) in Nestin-Het mice in the most common axon diameters (0.3–1.7 µm) (Fig. 2c, d). A decrease in immunostaining for myelin basic protein (MBP) in the corpus callosum (Fig. 2e) and cerebral cortex (Supplementary Fig. 2d) corroborated our TEM findings. We also observed fewer Olig2-positive oligodendrocytes in the corpus callosum (Fig. 2f). These results show that Anks1b haploinsufficiency reduces myelin sheath integrity and MBP expression, possibly by reducing the numbers of oligodendrocytes.

**Fig. 2: Abnormal myelin sheath integrity and fewer oligodendrocytes in an *Anks1b* haploinsufficiency mouse model.**

Anks1b haploinsufficiency impairs oligodendrocyte maturation and migration

To explore the mechanisms underlying these findings, we tracked the birth and maturation of oligodendrocyte lineage cells in Nestin-Het and WT mice. We injected bioconjugatable EdU intraperitoneally (IP) into pregnant mice at gestational age E18, and into mice 2, 6, and 12 months of age and tracked nucleotide incorporation over 4 weeks using markers of oligodendrocyte maturation to identify oligonucleotide lineage cells¹¹ (Fig. 3a). The cell surface tyrosine receptor kinase PDGFRa (platelet-derived growth factor receptor A) was used as a marker of OPCs and immature oligodendrocytes, and CC1 antibodies (targeting the RNA binding protein Quaking7⁵⁶) were used to mark mature oligodendrocytes. Mice were sacrificed 4 weeks after EdU injections, fixed brain tissue was cut in coronal slices, and EdU-positive newborn cells were labeled with reactive fluorophores. EdU-labeled mice at 3 weeks of age were obtained by injecting pregnant female mice at E18. Quantitation of labeled cells revealed significantly fewer newborn cells overall in the corpus callosum of Nestin-Het mice (Supplementary Fig. 3a), and significantly fewer mature oligodendrocytes (CC1-positive) derived from newborn cells throughout all time points tested (Fig. 3b). We also found a significant increase in OPCs and immature oligodendrocytes (PDGFRa-positive) in mice at 3 weeks and 6 months of age (Fig. 3c). However, there were no changes overall in the total number of proliferating cells as measured by BrdU-positive cells in neurogenic subventricular or subgranular zones (Supplementary Fig. 3a, b). These results provide evidence for impaired maturation.

**Fig. 3: Impaired oligodendrocyte maturation and motility in *Anks1b* Nestin-Het mice.**

Because oligodendrocyte migration is important for white matter integrity, we performed a remyelination experiment⁵⁷ by injecting lysophosphatidylcholine (LPC) into the corpus callosum to generate a myelin lesion, and tracked OPCs in Nestin-Het and WT mice. Mice were sacrificed at 7 days after injection to document the extent of the myelin lesion, at 10 days after injection to measure the initial stages of remyelination, and at 14 days after injection when complete remyelination would be expected (Fig. 3d, e). Newborn oligodendrocyte-lineage cells were tracked using EdU as described above, and both myelin deposition and OPC numbers were assessed. As expected, newborn oligodendrocytes accumulated at lesioned areas in WT mice (Supplementary Fig. 3c, d), and then dispersed throughout the corpus callosum (Supplementary Fig. 3e), validating the assay and timeline selected. However, we found significantly fewer newborn cells in demyelinated areas in Nestin-Het mice (Fig. 3f) compared to WT littermates. Moreover, significantly fewer of these cells were of oligodendrocyte lineage, as indicated by co-labeling with Olig2 (Fig. 3g). Interestingly, we did not observe a difference in the extent of MBP deposition at lesioned areas between genotypes (Supplementary Fig. 3f). These effects were observed during early remyelination (10 days post lesion) and persisted 2 weeks post lesion, suggesting that AIDA-1 plays an important role in the birth, maturation, and/or the migration of OPCs into demyelinated areas.

AIDA-1 is expressed in oligodendrocytes

Deficits in myelination and oligodendrocyte numbers were unexpected given that Anks1b-encoded AIDA-1 is one of the most abundant proteins at neuronal synapses^4,5 and has no published role in CNS myelination. To explore whether cell-autonomous effects played a role in the observed phenotypes, we searched through single-cell transcriptomic databases and found that Anks1b is selectively expressed in the CNS not only in neurons, but also oligodendrocytes and OPCs^33,58,59 (Fig. 4a, Supplementary Fig. 4). This is consistent with recent studies suggesting the expression of AIDA-1 in these cells^30,31,32,33. Immunocytochemistry using a knockdown-verified antibody against AIDA-1^3,60 in rat neuron-glia primary cultures shows that AIDA-1 is present in punctae throughout the cell body of oligodendrocytes (Fig. 4b). Fluorescent in situ hybridization (FISH) and RT-PCR confirms the presence of different Anks1b transcripts in these cells, as well as corpus callosum in mouse acute brain sagittal sections (Fig. 4c, Supplementary Fig. 5a, b). Western blots of mouse primary cell cultures revealed several AIDA-1 isoforms in oligodendrocytes, and at higher relative levels compared to neurons (Fig. 4d). No expression was observed in astrocytes (Fig. 4d), which corroborates diverse single-cell transcriptomic databases (Supplementary Fig. 4). Taken together, these results confirm that AIDA-1 is expressed in oligodendrocytes and OPCs.

**Fig. 4: AIDA-1 is expressed in oligodendrocyte lineage cells.**

Loss of Anks1b from oligodendrocyte lineage cells results in oligodendrocyte dysfunction

To test the oligodendrocyte-specific contribution to the phenotypes observed, we conditionally deleted Anks1b from these cells using Olig2-Cre mice. Previously described Anks1b floxed mice⁷ were crossed to Olig2-Cre mice (tm1.1(Cre), Jackson labs) on a similar C57BL/6J background. Oligodendrocyte-specific Anks1b heterozygous (Olig2-Het) and homozygous (Olig2-KO) oligodendrocyte-specific Anks1b knockout mice were viable, although knockout mice were born at rates below expected Mendelian ratios and were smaller than their WT counterparts (Fig. 5a). To confirm the specificity of Cre recombinase activity, we crossed Olig2-Cre mice to Rosa26-GFP reporter mice, and harvested brains were clarified using CUBIC⁶¹ and imaged by light sheet microscopy (Fig. 5b). As would be expected for oligodendrocytes, GFP-expressing cells appeared tiled, small and with fine cloud-like processes (Fig. 5b, Supplementary Fig. 6a), and overwhelmingly expressed in corpus callosum where they co-localized with MBP (Fig. 5c). These results corroborate the extensive use of Olig2-Cre lines to target oligodendrocytes^{62,63,64,65,66}, as well as studies that show that Olig2 expression is highly specific and universal for oligodendrocyte lineage cells⁶⁷.

**Fig. 5: Generation and characterization of *Anks1b Olig2*-Cre transgenic mice.**

As in Anks1b Nestin-Het mice, imaging experiments show that Olig2-Het mice exhibit fewer oligodendrocytes in the corpus callosum (Fig. 5d), and Western blots confirm reduced MBP and decreased oligodendrocyte makers Olig2 and Sox10 overall (Fig. 5e, f). TEM imaging confirms a decrease in myelination of callosal axons as measured using g-ratios (Fig. 5g, h). To validate the haploinsufficiency of Anks1b in this model, we performed Western blots on lysates from brain tissues and primary cell cultures. Whole brain tissue lysates from WT and Olig2-Het mice revealed no discernible changes in AIDA-1 expression (Fig. 5i, j). Because heterozygosity in a small subset of cells would not be detectable by Western blot, this result is consistent with specific cre-recombination in OPCs and oligodendrocytes, which represent only 5–10% of all brain cells. However, AIDA-1 expression was substantially reduced in cultured primary oligodendrocytes, but not in neuronal cultures grown from Olig2-Het mice⁶⁸ (Fig. 5i, j). Together, these results validate the specificity of the Olig2-Cre driver for oligodendrocytes and suggest an oligodendrocyte-autonomous role for AIDA-1 in the phenotypes observed.

Oligodendrocytes deficient in Anks1b display Rac1-based functional deficits

Western blots (Fig. 6a) and imaging analyses (Fig. 6j, k) show that oligodendrocytes from Olig2-Het mice have deficits in the expression of MBP, a marker of mature oligodendrocytes. Similar results were also observed in rat oligodendrocytes transduced with previously validated AIDA-1 specific shRNAs^7,60 (Fig. 6b, c; Supplementary Fig. 10c, d), suggesting that acute AIDA-1 knockdown can also lead to deficits in OPC maturation. Moreover, these results corroborate the assertion that myelination phenotypes observed in Anks1b-deficient mice originate from oligodendrocyte-autonomous deficits. To glean insights into the molecular mechanism linking AIDA-1 to cellular and behavioral phenotypes, we reanalyzed our published AIDA-1 interactome³ and proteomic analyses of synaptosomes isolated from Anks1b Nestin-Het mice³⁶. We reasoned that proteins that: 1) bind to AIDA-1 and 2) are altered by its absence, would be strong candidates for functional links. A weighted interaction score based on co-immunoprecipitation data from several AIDA-1 antibodies normalized to a non-specific control revealed many small Rho GTPases and their regulators, as well as key proteins involved in myelination or oligodendrocyte function (Fig. 6d, Supplementary Fig. 7a). We then compared these proteins to our previous synaptic proteomic dataset, which identified proteins that were up- or down-regulated at hippocampal synapses in Anks1b Nestin-Het mice (Fig. 6e, Supplementary Fig. 7b, c, Supplementary Data 2). An unbiased dataset of proteins that showed at least a 10% change compared to their respective controls in both proteomic studies were then included in a StringDB network model to identify functional clusters (Fig. 6e, f). Analyses revealed that by far the most enriched protein domains amongst this group were associated with GTPase catalytic activity (Fig. 6f). “Component” and “Function” Gene Ontology (GO) annotations similarly point to an enrichment for GTP binding as well as links to the myelin sheath (Fig. 6f). The prominence of Rac1 and associated regulators in these analyses confirmed our previous research pointing to Rac1 dysfunction in mouse models of ASD³⁶.

**Fig. 6: Deficits in Rac1 activity underlie abnormal oligodendrocyte function.**

To directly test the effects of AIDA-1 on Rac1 function, we used a cutting-edge Fluorescence Resonance Energy Transfer (FRET)-based Rac1 biosensor to measure Rac1 activity^69,70. To perform live imaging of primary oligodendrocytes (Fig. 6g), OPCs from Olig2-Het and WT mice were grown as described and then transduced with viruses to express the Rac1 biosensor. Two days after transduction, oligodendrocytes were imaged for 16 minutes in the presence of a Rac1 stimulator (CN04, Cytoskeleton, inc; IGF-1) to increase basal activity (Fig. 6h, i). While stimulation showed minimal effects on the overall FRET signal in this time frame, cells derived from Olig2-Het mice showed a marked increase in Rac1 activity compared to cells from WT mice (Fig. 6i). Similar experiments performed in rat oligodendrocytes transduced with AIDA-1 shRNAs also yielded significant differences in Rac1 activity, but curiously in the opposite direction from mouse cells (Supplementary Fig. 10a, b). To test whether abnormal Rac1 activity was linked to the maturation deficits observed in cells, we treated Olig2-Het oligodendrocytes for 5 days with Rac1 activator and found that this rescued the expression of MBP (Fig. 6j, k). These results corroborate the hypothesis that abnormal Rac1 activity underlies the myelination phenotypes observed in Olig2-Het mice.

Loss of Anks1b from oligodendrocyte lineage cells results in behavioral correlates of ASD

To test the contribution of oligodendrocyte dysfunction to ANDS, we carried out behavioral assays in Olig2-Cre Anks1b conditional knockout mice across domains relevant to clinical features of this genetic disease. As in the Nestin-Het mice, no deficits were observed in novel object placement (Fig. 7a), a test of hippocampus-dependent learning and perseverative behaviors. Because we had found reduced anxiety-like behavior in Nestin-Het mice³, we examined the behavior of Olig2-Cre transgenic mice on the elevated plus maze. Results were mixed as Olig2-Het and Olig2-KO mice spent more time in the open arms of the elevated plus maze (decreased anxiety) (Fig. 7b) but covered less distance in the open arms (increased anxiety) (Supplementary Fig. 8d). There were no differences in center exploration in the open-field test or in the forced swim test, two other measures of anxiety-like behaviors (Supplementary Fig. 8b, c). Hyperactivity present in Nestin-Het mice³ was not observed in Olig2-Cre transgenic mice, as these showed no changes in locomotor activity in the open field (Supplementary Fig. 8a, c).

**Fig. 7: Mice with oligodendrocyte specific *Anks1b* deletion display behavioral deficits associated with ASD.**

Autism is a prominent feature of ANDS and Nestin-Het mice have deficits in sensory reactivity and impaired social behaviors³. Like Nestin-Het mice, Olig2-Het and Olig2-KO mice of both sexes show significantly increased sensory hyperreactivity as demonstrated by increased startle to sounds at 100 db and 110 db, but not at 90db compared to WT littermates (Fig. 7c). Unlike Nestin-Het mice, Olig2-Cre transgenic mice had no impairment in sensorimotor gating as tested using pre-pulse inhibition (Supplementary Fig. 8e). Like Nestin-Het mice, Olig2 mice showed no differences in stereotypic and perseverative behaviors such as grooming (Supplementary Fig. 8a). Surprisingly, we found that both Olig2-Het and Olig2-KO mice displayed significantly decreased social preference in the three-chamber assay (Fig. 7d). For comparison, we also analyzed mice with homozygous Anks1b deletion specifically from forebrain excitatory neurons, using Camk2a-Cre (Camk2a-KO; generated previously⁷), or from cerebellar Purkinje neurons using L7-Cre (L7-KO). These neuronal subtypes display high Anks1b expression as observed in the Allen Brain Atlas (Supplementary Fig. 8g). Notably, only Olig2-Het and Olig2-KO mice displayed deficits in social preference similar to Nestin-Het mice (Fig. 7d), showing that social deficits in a mouse model of ANDS can originate from targeted oligodendrocyte disruption. A characterization of Camk2a-KO mice (Supplementary Fig. 9a) revealed that they did not exhibit any changes in the area of the corpus callosum compared to WT controls (Supplementary Fig. 9b), or changes in overall MBP expression (Supplementary Fig. 9c), supporting a correlation between deficits in social behaviors and white matter abnormalities. Overall, no significant sex differences were observed in any behaviors evaluated. Statistical analyses for the Olig2-Het (n = 82), Olig2-KO (n = 13), and WT (n = 53) mice demonstrate robust results with high power (beta > 0.85) for all tests where results were significant and beta >0.95 for social preference tests.

Clemastine rescues social deficits in mice lacking Anks1b in oligodendrocytes

To test whether the social impairments were caused by the myelination deficits, we sought compounds shown to induce myelination. Conveniently, we found that clemastine fumarate is a well-tolerated, FDA approved, and blood brain barrier permeable compound shown to increase myelination specifically by increasing OPC maturation^{17,35,71,72,73}. We first tested this compound in primary oligodendrocytes and found that long-term treatment (5 days, 1 µM clemastine) significantly increased MBP expression in cells derived from Olig2-Het mice (Fig. 8a, b), as well as in rat oligodendrocytes transduced with shRNAs to knock down AIDA-1 (Supplementary Fig. 10f). We then took Olig2-Het and WT mice aged 6–7 months that had been previously tested for social preference (at 3–4 months of age) and injected them with clemastine (10 mg/kg) or vehicle daily for 14 days IP and allowed them to recover for 7 days (Fig. 8c). Clemastine had no effects on overall locomotor activity (Fig. 8d). Notably, paired results show that clemastine treatment significantly rescued social preference in mice that had previously exhibited no social preference (Fig. 8e). Drug effects were largest for mice that initially displayed the poorest scores. No effects were observed for Olig2-Het mice treated with vehicle (Fig. 8f), or WT controls treated with either clemastine or vehicle (Fig. 8g), showing that improved social preference was neither a consequence of behavioral retesting, nor of vehicle injections. Following behavioral testing, a randomized subset of the mice was euthanized (Olig2-Het clemastine-treated n = 3, Olig2-Het vehicle-treated n = 3), and dissected brains were fixed and prepared for TEM analyses (Fig. 8h). We found that clemastine treatment resulted in a significant decrease in g-ratio across most callosal axon diameters, which indicates a substantial increase in myelination (Fig. 8i). These results show that OPC maturation and induction of new myelin formation through clemastine treatment can improve social behaviors even in 6–7-month-old mice that are significantly beyond critical periods for myelination. Overall, these results strongly support the assertion that behavioral deficits in Olig2-Het mice are caused by deficits in oligodendrocyte function and myelination.

**Fig. 8: Clemastine rescues deficits in social preference in *Anks1b* Olig2-Het mice.**

Discussion

Here we reveal an unexpected role for ANKS1B in oligodendrocyte maturation and function, and a potential role for oligodendrocytes in sociability and NDDs. We show that ANDS patients display profound white matter abnormalities, and that CNS-wide (Nestin-Het) mouse models reveal similar white matter aberrations (Fig. 1), as well as decreases in oligodendrocyte abundance, maturation, and myelination in the corpus callosum (Figs. 2 and 3). The Anks1b-encoded protein AIDA-1 was previously reported to be neuron-specific^{3,4,6,7,60,74} and expressed mainly at PSDs in dendritic spines^4,5,6,60. Here we show that AIDA-1 is also expressed in oligodendrocyte lineage cells (Fig. 4). While expression of Anks1b in neurons and oligodendrocyte lineage cells points to multifactorial dysfunctions in disease etiology, selective Anks1b deletion from oligodendrocytes recapitulates oligodendrocyte phenotypes (Fig. 5) as well as the social and sensory correlates of ASD observed in Nestin-Het mice (Fig. 6). Notably, these effects were not observed in transgenic mice targeting neuronal populations that highly express Anks1b. Overall, our results suggest that oligodendrocyte-autonomous Anks1b haploinsufficiency mediates the deficits in white matter microstructure, oligodendrocyte abundance and maturation, and social behaviors observed in Anks1b-deficient mice.

We present a potential mechanism linking AIDA-1 to oligodendrocyte function by regulating Rac1 activity. We previously found that AIDA-1 interacts with Rho family GTPases and associated regulatory proteins^3,36. Signaling pathways mediated by Rho Family GTPases are known to play pivotal roles in orchestrating the dynamics of the actin cytoskeleton. These signaling mechanisms have also emerged as crucial regulators of morphology, maturation, and migratory capacities of oligodendrocyte lineage cells^{40,41,42,43,44}. Disruptions in these finely tuned processes might contribute to the phenotypes associated with oligodendrocytes that we observed in mouse models of ANDS. This suggests that the syndromic phenotypes we see caused by loss of Anks1b in oligodendrocytes may be caused by impaired interactions of AIDA-1 via Rac1 or Rac1-mediated processes. For example, p21-activated kinases (Pak1, Pak2, and Pak3) are highlighted in our proteomic and bioinformatic analyses (Fig. 6d–f). These processes are essential for proper oligodendrocyte development and differentiation, with Pak1 promoting oligodendrocyte morphogenesis and myelination, Pak2 involved in proliferation and survival, and Pak3 involved in OPC differentiation^{51,75,76,77,78,79}. Disruption of Rac1 and Pak1, 2 or 3 activities has been linked to various disorders, including ASD^78,80. Other potential mechanistic pathways of interest include RhoA-Rock2, DCC, and Srgap2 (Fig. 6d–f). Our results show that AIDA-1 regulates Rac1 activity in mouse oligodendrocytes (Fig. 6). Curiously, Rac1 activity in rat cells transduced with AIDA-1 shRNA was also significantly altered, but in the opposite direction (Supplementary Fig. 10a, b). The different results in mouse and rat cells could be due to species differences, developmental compensation in the Olig2-Het mice, or significant differences reported between the two types of cells⁸¹. Alternatively, these results could point to a dose-dependent effect on complex Rac1 functional dynamics, given that the amount of AIDA-1 knockdown observed in rat cells was small (Supplementary Fig. 10e) compared to the reduction observed in Olig2-Het mouse cells (Fig. 5i, j). In both cases, we were only able to reliably record Rac1 activity for 12 min post-stimulation because primary oligodendrocytes are highly sensitive and do not survive long in the live-cell FRET recording conditions. This means that we may have missed the complex temporal patterns displayed by RhoA family GTPases. We also report that long-term stimulation of Rac1 was able to rescue the expression of MBP in cells in culture (Fig. 6j, k). Taken together, these results suggest that the regulation of Rac1 is part of the mechanism underlying the interaction of AIDA-1 in oligodendrocyte and myelination processes, and that disruption of this mechanism is related to prominent phenotypes seen in ANDS patients.

It is worth noting that Anks1b (as EB1) was originally identified as a transcript upregulated in pre-B myeloid leukemia cells⁸², that it is ectopically expressed in transformed cell lines^82,83, and has been linked to diverse cancers in GWAS studies^84,85. While this research suggests a potential function in contact inhibition or cellular division, we did not detect any effects on overall cell birth in neurogenic zones of the CNS (Supplementary Fig. 3b). Despite the deficits in OPC maturation and significant reduction in the number of newborn oligodendrocytes in acutely demyelinated areas of Nestin-Het mice, we found that MBP was deposited at similar rates in lesioned areas compared to WT controls (Fig. 3). These results are consistent with recent findings showing that mature oligodendrocytes, and not OPC turnover, contribute to the regulation of myelination⁸⁶. Another potential explanation for our findings is that our time points were insufficient to capture the contribution of newborn cells to remyelination. Because OPCs require at least two weeks (the time of our remyelinating assays) to differentiate into myelinating oligodendrocytes^17,18 it is possible that our assay quantified MBP deposited by pre-existing mature or immature oligodendrocytes present or near lesioned areas. Yet another explanation is that oligodendrocytes are highly heterogeneous³⁰ and Anks1b may not be expressed in cells that participate in acute remyelinating processes.

Studies of ASD etiology have implicated diverse convergent cellular pathways, including those regulating synaptic function^{87,88,89,90,91,92,93,94,95,96,97,98,99,100}. Databases for ASD-linked genes such as SFARI Gene (Simons Foundation) are enriched in synaptic components, and mouse models for syndromic ASD are primarily associated with synaptopathies arising from mutations in highly synaptic proteins such as SHANKs, neurexins, neuroligins, CNTNAP2, and SynGAP1^95,101,102. Given that Anks1b-encoded AIDA-1 is one of the most abundant proteins at neuronal excitatory synapses⁶⁰ and regulates NMDAR function⁷, we expected that neuron-specific Anks1b deletion would drive ASD-related behavioral deficits. We focused on forebrain excitatory neurons (Camk2a-Cre) and cerebellar Purkinje cells (L7-Cre) because these cells most prominently express Anks1b, and because previous studies have linked these cell populations to other mouse synaptopathy models of ASD^103,104. Surprisingly, only Olig2-Cre driven Anks1b conditional mutant mice had social deficits, suggesting a significant contribution of oligodendrocyte dysfunction to autism-like features in ANDS. Clearly, our experiments do not rule out other neuronal populations, early developmental processes in neurons, or interactions between oligodendrocytes and neurons in which AIDA-1 may participate. Moreover, while Olig2-Cre mice have been used extensively for the study of oligodendrocyte function^{62,63,64,65,66}, Olig2 expression has been reported in interneuron subpopulations early in development¹⁰⁵ as well as in cerebellar Purkinje cells¹⁰⁶. However, we saw no changes in established markers of interneurons (PV, SST) or cerebellar Purkinje cells (calbindin) throughout the brain (Supplementary Fig. 6b) and no changes in cerebellar Purkinje cell numbers in Nestin-Het mice (Supplementary Fig. 6c). Additionally, Purkinje-specific Anks1b knockout mice (L7-KO) exhibit no deficits in social preference (Fig. 7d), diminishing concerns that confounding expression of Olig2 in these cells play a role in the social deficits observed. However, we recognize that a limitation of our work is that we cannot fully rule out the influence of a small subset of interneurons on behavioral deficits in the Olig2-Het mice.

The important role for AIDA-1 at neuronal synapses^3,6,7,60 raises the possibility for a similar role in oligodendrocyte lineage cells. Seminal findings show that OPCs can also form functional synapses with neurons in the hippocampus^{107,108,109,110,111,112}, and that these synapses display properties similar to canonical neuronal synapses¹¹³. OPCs express components of PSDs^33,114, and ultrastructural images show PSD-like structures in OPCs and synaptic vesicles in presynaptic neurons^107,115,116. Indeed, OPCs functionally express the full gamut of ionotropic and metabotropic neurotransmitter receptors, including AMPA, kainate, and NMDA receptors^109,110. Well-established findings show that neuronal activity regulates oligodendrocyte proliferation, maturation, and myelination^{117,118,119,120,121}. Similar to what has been observed in neurons⁶, AIDA-1 may relay fast synaptic information from neuron-OPC synapses into the nucleus to regulate protein synthesis or other genetic signals necessary for OPC maturation. Punctate staining of AIDA-1 throughout cell bodies of oligodendrocytes (Fig. 4) may mark neuron-OPC synaptic junctions. Thus, an additional synaptopathy may underlie ANKS1B syndrome, one of unique neuron-OPC synapses as well as of canonical neuron-neuron synapses. Additional experiments will be required to elucidate how AIDA-1 regulates oligodendrocyte maturation and function.

Our comprehensive behavioral assessment of Olig2-Cre driven Anks1b conditional knockout mice reveals that some ASD-related behavioral phenotypes including social preference can originate from oligodendrocyte dysregulation. Together with a recent report of oligodendrocyte dysfunction in a rodent Chd8 transgenic rodent model of autism²⁹, our results highlight an underappreciated role for these cells in NDDs. How these cells contribute to complex behaviors such as sociability is unclear. While tantalizing research suggests that oligodendrocytes can fire action potentials^110,122 and establish functionally connected circuits throughout the brain¹²³, any effects on myelination will clearly impact neuronal circuit activity. Changing myelinated axon structure by altering internode number and length, and myelin thickness and stability, will regulate conduction velocities to alter neural networks and circuit plasticity^{117,124,125,126}. Two recent studies on memory consolidation showed that inhibiting active myelination through pharmacological or genetic manipulation blocks recall of spatial learning in both water maze and conditioned fear assays^17,18, and chronic administration of clemastine preserved the retrieval of remote fear memory¹⁷. While our results suggest that Ansk1b broadly regulates myelination by affecting oligodendrocyte maturation, more detailed analyses may reveal preferential targeting of specific circuits involved in regulating social behaviors. Targeted Anks1b expression in a specific oligodendrocyte subtype may provide the basis for selective regulation of neural circuits.

The few available therapies for NDDs include behavioral therapy, physical rehabilitation, and symptoms-based pharmacotherapy, but specific and effective treatments are lacking due to a poor understanding of disease mechanisms¹²⁷. We found that clemastine fumarate rescues the impaired social preference in Olig2-Het mice. Recent high-throughput screens identified clemastine fumarate as possible therapeutic compounds for multiple sclerosis^35,128. Clemastine is a blood brain barrier permeable compound approved by the FDA to treat seasonal allergies. It binds to the histamine receptor 1 but promotes OPC maturation and myelination by stimulating M1 acetylcholine receptors¹²⁸. Clemastine has shown promising therapeutic potential by promoting myelination in hypoxia, mouse models of demyelination, and neurodegenerative disorders^{71,72,128,129}. It has recently been shown to relieve the severity of clinical symptoms from inflammatory demyelination^73,130 by increasing OPC maturation and myelination^{17,35,71,72,73}, as well as rescue deficits in memory consolidation due to abnormal myelination¹⁷. To our knowledge, our study represents the first use of clemastine to rescue phenotypes in an animal model of ASD.

Our research supports the notion that impaired myelination contributes to the pathogenesis of NDDs and directly implicates dysfunction of oligodendrocyte lineage cells in disease. Recent studies^{26,131,132,133,134,135,136} corroborate long-standing evidence of deficits in white matter organization in children and adults with ASD^22,23,24,25. In addition, analyses of differentially expressed genes in several mouse models of syndromic ASD highlighted dysregulation of oligodendrocyte function¹³⁶. Although the developmental component of genetic diseases like ANDS has previously given little hope for therapeutic intervention in adults¹³⁷, new findings, including ours, indicate that at least some behavioral correlates of NDDs are reversible well into adulthood in animal models. This may be especially true for phenotypes caused by deficits in myelination, given that myelination is a dynamic process that remains plastic throughout the lifespan. In a SHANK3 model of ASD, morphological, functional, and behavioral anomalies such as self-injurious grooming and social interaction deficits (but not anxiety or motor deficits) are reversible in adult mice¹²⁸. Here we find that deficits in social preferences can be ameliorated even in 7-month-old mice, an age that is significantly far into adulthood in these animals. Our results suggest that interventions targeting myelination may represent a potential therapy for ASD-related phenotypes in ANDS, and perhaps for other syndromic ASDs that display impaired myelination.

Methods

Reagents

Antibodies, reagents, mouse lines, and genotyping primers are detailed in Supplementary Data 3.

Brain imaging

Human subjects and clinical data

All procedures were performed under the ethical approval by the Institutional Review Board (IRB) at AECOM and are described in IRB protocol #2011-320. Written consent was obtained from all participants prior to MRI and DTI analyses. The two affected individuals are men, and the neurotypical group was comprised of 50 men and 51 women.

Patient MRI scans

Imaging data was collected using a 3.0 T Philips Achieva TX scanner (Philips Medical Systems, Best, The Netherlands) with a 32-channel head coil. Structural 3D T1-weighted (T1W) magnetization-prepared rapid acquisition of gradient echo imaging was performed with TR/TE/TI = 8.5/3.9/900 ms, α = 8°, SENSE factor 2.0/2.6 in SI/RL directions, 1 mm³ isotropic resolution, 240 × 240 × 220 matrix. Diffusion tensor imaging (DTI) data was acquired using 2D single-shot spin echo EPI with 32 diffusion-weighting directions at b = 800 s/mm² and TE = 56 ms, TR = 7.6 s, SENSE factor = 2.9, 2 mm³ isotropic resolution, 128 × 128 matrix, 70 slices. An auxiliary field map scan was acquired using FOV = 256 mm, 4.0 mm³ isotropic resolution, TR = 26 ms, TE/DTE = 2.5/2.3 ms, α = 26°. The field map is used to correct susceptibility-induced distortions in echo-planar (diffusion) scan.

MRI data analysis

Most of the image analysis steps, correction of DTI data for motion, eddy currents, EPI distortion as well as generation of map of fractional anisotropy and its rigid body registration to subject’s T1W image, were performed using FMRIB-FSL package¹³⁸ as previously described¹³⁹. Normative data from the Einstein Lifespan Study was pre-processed in a similar way and registered to the subject’s T1W image using non-linear ANTs registration algorithm¹⁴⁰ for voxel-wise comparison using EZ-Map¹⁴¹. This subject-based method was shown to outperform template-based approach⁵⁴.

In vivo mouse brain MRI

All MRI data were acquired in a 9.4 T Varian Direct Drive system (Agilent Technologies, Santa Clara, CA, USA) at the Einstein Gruss Magnetic Resonance Research Center. A 14-mm diameter receive-only surface RF coil (Doty Scientific, Columbia, SC, USA) along with a 7-cm ID ¹H transmit and receive body coil (M2M Imaging, Cleveland, OH, USA) were used. Anesthesia was achieved with ~1.25% isoflurane mixed with room air. Respiration was monitored with a pressure pad (SA Instruments, Stony Brook, NY, USA). Rectal temperature was maintained at ~38 C using warm air with feedback from a rectally placed thermocouple (SA Instruments). T2-weighted images were acquired using fast spin echo (fse) sequence covering the whole brain with the following parameters: TR = 3000 ms, ESP = 8.8 ms, segments = 48, ETL = 4, kzero = 3, effective TE = 26.4 ms, NSA = 4, data matrix = 192 × 192 (zero filled to 256 × 256), FOV = 25 mm², slices = 60, thickness = 0.3 mm, gap = 0 mm. Diffusion tensor image (DTI) was acquired with the following parameters: TR = 3000 ms, TE = 19.62 ms, kzero = 6, shots = 8, number of signals averaged (NSA) = 1, slices = 24, slice thickness = 0.5 mm, gap = 0, field of view (FOV) = 25 mm², data matrix = 128 × 128 (zero-filled to 256 × 256), b-value = 825.9 s/mm², G = 0.45 T/m, d = 2.56 ms, D = 8 ms, 42 directions plus 6 unweighted images, acquisition time = 40 min 18 s. 3D multiecho gradient echo (MGRE) images from which T2* mapping was generated were acquired with the following parameters: TR = 90 ms, 20 echoes with first echo at 1.78 ms, time between echoes 2.59 ms, flip angle 17, resolution = 125 × 125 × 125 µm³, 0 with outer volume suppression.

Ex vivo mouse brain MRI

Fixed mouse brains were positioned in a plastic tube filled with Fomblin Y (perfluoropolyether; Sigma-Aldrich Co., St. Louis, MO). Imaging was performed on the same system and coils as described above. Parameters for fse sequence were as following: TR = 5000 ms, ESP = 17.54 ms, segments = 64, ETL = 4, kzero = 3, effective TE = 52.61 ms, NSA = 16, data matrix = 256 × 256, FOV = 12.8 mm², slices = 50, thickness = 0.3 mm, gap = 0 mm. DTI (30 orthogonal directions¹⁴² was accomplished using b-values of 939 s/mm², a 128 × 128 matrix and FOV of 12.8 mm², and 30 slices (0.3-mm thickness and no gap).

MRI data analysis

The Dorr mouse brain atlas¹⁴³ was registered to T2-weighted images covering the whole brain using ANTs¹⁴⁰ registration software. Maps of diffusion metrics including fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD) were generated using the FMRIB FSL diffusion toolbox¹³⁸. Volumes of segmented regions¹⁴³ and average diffusion metrics over the segmented regions were then calculated.

Anks1b mouse models

All protocols were approved by Albert Einstein College of Medicine’s Institutional Animal Care and Use Committee (IACUC) in accordance with National Institutes of Health guidelines. Mice were maintained in a pathogen-free animal room under a 12-h light/dark cycle. Food and water ad libitum were available prior to the beginning of all experiments. Both sexes were used in biochemical, morphological, and behavioral studies. Anks1b floxed mice were generated as previously described⁷. All mice used for conditional knockdown are described in Supplementary Data 3. Anks1b Olig2-Het and Olig2-KO mice were generated by crossing previously described Anks1b floxed mice⁷ with Olig2-Cre mice from Jackson Laboratories. Wildtype animals (WT) used throughout refer to one of these three lines: 1) animals with the Cre transgene and without the floxed Anks1b allele, 2) mice expressing the floxed allele without the Cre transgene, or 3) mice expressing neither transgene nor floxed allele.

Tissue preparation, histology, clearing, immunofluorescence

Animals were perfused with PBS followed by 4% paraformaldehyde (PFA). Brains were harvested and post-fixation was performed in 4% PFA at 4 °C for 24 h. Fixed brains were then equilibrated in 30% sucrose for 2 days. Entire brain sections (coronal 40 µm thickness) running anterior to posterior were collected using a microtome in serial order for histological analysis and immunolabeling analyses.

Histology

Nissl staining was performed using Cresyl Violet dye to measure the surface area of each region of interest. Briefly, serial coronal sections were permeabilized with a 1:1 ratio of cold (−20 °C) acetone and methanol for 10 min, then stained with 0.1% Cresyl Violet containing 0.3% acetic acid. Brain sections were then dehydrated with 100% ethanol and mounted with Permount (Fisher).

Luxol Fast Blue staining

Luxol Fast Blue staining was performed to measure the density of myelinated regions. Brain sections were mounted on slides and placed into a 1:1 ethanol:chloroform solution for 6 h. Sections were then incubated in a 0.1% Luxol Fast Blue solution dissolved in 95% ethanol, solvent blue 38 (Millipore Sigma), and 0.5% acetic acid, in a 56 °C incubator overnight. The following day, sections were rinsed of excess stain with 95% ethanol, and differentiated in a 0.1% lithium carbonate solution for 1 min. Brain sections were then dehydrated with 100% ethanol followed by xylene for 5 min. Permount mounting medium was used for microscopy.

Immunofluorescence

All brain sections were stored in antifreeze solution (30% ethylene glycol, 30% sucrose in 30% pH 7.4 PBS) at −20 °C prior to immunolabeling. By using the free-floating method, sections were washed with TBS-T (0.1% Tween-20 buffer), and primary antibodies were incubated in blocking solution (0.1% Triton X-100 in SuperBlock TBS buffer, Thermo) at 4 °C overnight. The following day, sections were washed with TBS-T and incubated with secondary fluorescent antibodies (Jackson ImmunoResearch) for 2 h at room temperature. Sections were then washed with TBS-T and mounted onto coverslips using Mowiol (2.5% PVA/DABCO) for imaging.

Imaging

Nissl and LFB Images were acquired on a Zeiss AxioScan Z1 slide scanner with a 5× objective. Immunofluorescence images were acquired on an LSM 880 airy confocal system with 20X and 40X objectives using a tile scanning configuration.

Analysis

For Nissl analysis, areas were quantified using ZEN software (blue edition). For LFB analysis, ROIs were quantified using Image J software (inverted image). Stereological quantifications of corpus callosum (genu and body) were performed for immunolabeling analysis by using ZEN software.

CUBIC tissue clearing and LSFM

Tissues were cleared using CUBIC reagents (TCI, T3740, T3741) as previously described^61,144. Mice were perfused and brains were harvested and postfixed as described above. On day 2, brains were washed with PBS for 2 h at RT and then immersed in 50% CUBIC-L. On day 3, the solution was replaced with 100% CUBIC-L. On day 5, brains were washed with PBS for 3 h, immersed in 50% CUBIC-R, and changed to 100% CUBIC-R on day 6. On day 8, brains were transferred to CUBIC mounting solution (TCI, RI 1.520, M3294). Images were acquired on a Light Sheet Fluorescence Microscopy (LSFM) at Zeiss Microscopy (Thornwood, NY).

Transmission electron microscopy sample preparation and processing

Brain tissue samples were collected from the genu region of the corpus callosum of mice. Specifically, samples were sourced from 3-month-old mice for the Nestin and Olig2 groups, and from 7-month-old mice for the clemastine treatment group. The mice were perfused with a 4% PFA solution. Following perfusion, the tissue samples were fixed for 1 h at room temperature using a solution comprising 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer then incubated overnight at 4 °C. The samples underwent postfixation with 1% osmium tetroxide, followed by 2% uranyl acetate. Dehydration was performed using a graded series of ethanol, transitioning from 30% to 100%. The samples were embedded in LX112 resin (LADD Research Industries, Burlington VT). Ultrathin sections were prepared using a Leica Ultracut UC7, stained sequentially with uranyl acetate and lead citrate, and subsequently visualized on a JEOL 1400 Plus transmission electron microscope operated at 120 kV (Albert Einstein College of Medicine Analytical Imaging Facility; SIG # 1S10OD016214-01A1). Digital TEM images were originally acquired as.dm4 files and then converted to 8-bit TIFF files using ImageJ software for compatibility and further processing. The images were then analyzed using hand tracing and photoshop measurement tools. For each mouse, 100 axons were analyzed, and processing was completed while blinded to mouse identity. The Olig2 group was processed with a resolution of 0.00539 um per pixel, while both Nestin and clemastine groups were processed at a resolution of 0.00258 um per pixel. Axons were classified in groups based on their axonal diameter (grouped in microns as 0.1 to 0.29, to 0.4, to 0.51, to 0.66, to 1.7, >1.7).

Fluorescent In situ hybridization (FISH)

FISH was performed according to the procedure outlined in the StellarisⓇFISH kit (Biosearch Technologies, Inc., Petaluma, CA)¹⁴⁵. Custom StellarisⓇFISH Probes were designed against the rat/mouse Anks1b gene using the StellarisⓇFISH Probe Designer, targeting the 3’ region of the Anks1b gene to create a specific probe for the larger ankyrin-repeat containing splice variant (AIDA-1B), and targeting the 5’ region to create probes for both smaller AIDA-1D and larger AIDA-1B variants. Brain sections were hybridized with FAM-AIDA-1B and CAL Fluor Red 590-AIDA-1D probes. Fixation, pre-hybridization, and hybridization were performed following the manufacturer’s protocol.

Cell culture

Oligodendrocytes were grown on 18 mm round glass coverslips. Cells were fixed and permeabilized with 70% ethanol for 1 h at 4 °C. Coverslips were washed with Wash buffer A (Stellaris, Biosearch Technologies) for 5 min at room temperature. Cells were then incubated with hybridization buffer containing Anks1b probes for 6 hr at 37 °C. Then, coverslips were washed with Wash Buffer A and incubated in the dark at 37 °C for 30 min. After Wash Buffer A was aspirated, cells were stained with DAPI, incubated in the dark at 37 °C for 30 min, and mounted on slides for imaging.

Fixed tissue

Sagittal brain sections (5 µm thick) were mounted on slides and fixed with cold electron microscopy grade PFA in PBS for 15 min at RT. They were washed with PBS for 5 min, dipped in nuclease-free water, and dipped in TEA buffer (13.3 mL Triethanolamine, up to 100 mL Nuclease-free water, pH 8.0). Slides were immersed in TEA with acetic anhydride for 10 min with stirring, immersed in in 2x SSC for 3 min, washed sequentially in 70%/95%/100% ethanol for 3 min each, fixed with chloroform for 3 min, washed in 100% and 95% of ethanol for 3 min each, then dried for 90 min at RT. Hybridization Buffer containing Anks1b probes was dispensed for 12 h at 37 °C. Slides were immersed in Wash Buffer A and incubated in the dark at 37 °C for 30 min. After Wash Buffer A was aspirated, slides were stained with DAPI and incubated in the dark at 37 °C for 30 min. Slides were immersed in Wash Buffer B for 3 min and immersed sequentially in 50%/85%/100% ethanol for 3 min each. After air drying for 10 min, Prolong Gold Antifade Mounting solution was added and sections were coverslipped for imaging.

Neuron and oligodendrocyte cultures

Primary cortical and hippocampal cultures were prepared from Sprague-Dawley rat embryos at E18 as previously described¹⁴⁶. For mixed cultures containing neurons and oligodendrocytes, primary cells plated in standard media were switched either to Neurobasal + B27 + 2.5% FBS or Neurobasal and MYM media as described¹⁴⁷. For enriched oligodendrocyte cultures, brain tissues from mouse (postnatal days 1–5) or rat (Embryonic E20-E22; Postnatal P1-P5) pups were obtained. After decapitation, brains were extracted from skulls, and the cortex and hippocampi were dissected in aseptic conditions. Cerebral cortices were stripped of dura matter in Leibovitz’s L-15 Medium supplemented with glucose 6 g/mL (dissection media) and incubated in 1X Trypsin (Gibco; 15–25 min at 37 °C). Tissues were washed of trypsin using dissection media and finally incubated in OPC base media composed of DMEM F12 with glucose 6 g/L, glutamax 2 mM penicillin and streptomycin 50 mg/mL, Neomycin 100 mg/mL, and 1X B27 (Gibco). Cells were then triturated by mechanical means which typically involved passing tissues sequentially through 1 ml, 200 ul and 20 ul tips or using a fire-polished glass pipette. For experiments with mouse OPCs, cortices from each pup were processed separately and incubated in media on ice while PCR genotyping experiments were performed. For rat cells, all cortices were combined and processed together. Following dissociation and genotyping (if necessary), cells were plated and grown on wells/coverslips coated with poly-L-lysine (Sigma-Aldrich, St. Louis, MO, United States) in OPC media supplemented with PDGF-AA 30 mg/mL and FGF 10 mg/mL (growth media). Cells were fed by replacing 1/3 of media typically every 3–4 days. For maturation, which typically occurred 6–10 days following OPC expansion and once sufficient growth was observed, the growth media was replaced by OPC media supplemented with T3 0.75 ng/ml and followed for 5–7 days. Where appropriate, cells were infected with lentivirally delivered shRNAs described previously⁷.

Western blot

For western blot, the cells were lysed with LDS (Lithium dodecyl sulfate) buffer containing 150 mM tris HCl, 2% LDS, 10% glycerol, 0.51 mM EDTA, 0.22 mM Orange G at pH 8.5 and disaggregated using a syringe with a 28 G ½ needle. The samples were run on precast 4–12% Criterion gels (Bio-Rad Laboratories, Inc., Hercules, CA, United States). After electrophoresis, the gels were transferred onto nitrocellulose membranes for 1 h. The membranes were blocked with 5% dry milk diluted in Tris-buffered saline (TBS) buffer with 0.1% tween 20. Membranes were incubated for 1 h at room temperature with primary antibodies described in Supplementary Data 3. Proteins were visualized with the incubation of fluorescent secondary antibodies and imaged using the Odyssey CLX imaging system (LI-COR Biosciences, Nebraska).

Oligodendrocyte maturation assay with EdU-labeling

Mice at different developmental stages were injected IP with Click-iTTM EdU (50 mg/kg, Invitrogen) five times a day (2 h intervals) and sacrificed 4 weeks later as described¹⁴⁸. For the 3-week-old time point, EdU was injected in pregnant females at E18.5, and pups were sacrificed at P21. In adult (2 and 6 months old), and aged (12 months old) mice, EdU was injected at 2, 6, and 12 months old, and mice were sacrificed 4 weeks later at 3, 7, and 13 months old, respectively. Coronal sections (40 µm thickness) were prepared and processed for immunofluorescence. Images were acquired on a Zeiss LSM 880 airy confocal system with 20× and 40× objectives using a tile scanning configuration. Stereological quantification of EdU+ cells (newborn cells that had migrated into the corpus callosum), EdU+/PDGF1a+ cells (OPCs and immature oligodendrocytes), and EdU+/CC1+ cells (mature oligodendrocytes) were quantified within the corpus callosum area using Zen software.

Demyelination and remyelination assays

Mice at 6–8 weeks of age were anesthetized with 5% isoflurane and maintained at 1–2% isoflurane on a stereotaxic apparatus (Stoelting). Mice were given flunixin to manage pain and moisturizing eye ointment was added at the beginning and end of surgery. LPC (Lysophosphatidylcholine, 62963, Sigma, 1% LPC in 0.9% NaCl) was stereotactically injected into the corpus callosum at 1 site (2 µL per site) at the following coordinates (AP: +1, ML: +1.5, DV: −1.8 mm). On the following day, EdU (50 mg/kg) was injected via IP route 3 times (at 2 h intervals) for 1 day to track the population of newborn cells in the corpus callosum during the de-, and re-myelination phases^116,149. Mice were sacrificed at 7 days post injection (dpi) (D, demyelination), 10 dpi (RI, remyelination initiation), and 14 dpi (R, remyelination). Focal demyelinating region was validated with LFB staining and MBP staining. Images were acquired on a Zeiss LSM 880 airy confocal system with 20X and 40X objectives using a tile scanning configuration. Stereological quantification of EdU+ cells (newborn cells migrated into the demyelinated region of the corpus callosum) was quantified using Zen software.

FRET live imaging of Rac1 activation

OPCs, obtained as described above, were plated at a cell density of 2 × 10⁵ on 25 mm glass coverslips. For live-cell imaging, artificial cerebral spinal fluid (ACSF) containing (in mM) 124 NaCl, 26 NaHCO₃, 10 glucose, 2.5 KCl, 1 NaH₂PO₄, 2.5 CaCl₂, and 1.3 MgSO₄ was used. Cells were imaged for a total of 16 minutes, with Rac1 stimulator (CN04 at 40% recommended, Cytoskeleton, inc; IGF-1 at 1000x) added after 4 min. Image acquisitions were performed through a 40× magnification objective lens (UIS 40× 1.30 NA; Olympus) using a custom microscope³⁷. This microscope was capable of simultaneous acquisition of FRET and donor mCerulean emissions through two PrimeBSI-Express cameras (Photometrics, Tucson, AZ, USA) that are mounted via a 4-way optical beam splitter (Cairn Research, Kent, UK) and containing a T505LPXR mirror, ET480/40M for mCerulean emission, and ET535/30M for mVenus-FRET emission (Chroma Technology Corp, Bellows Falls, VT, USA). The relative intensities between the two channels were balanced by the inclusion of a neutral density filter (ND0.2 in FRET channel) to ensure that the range of brightness in both mCerulean and FRET channels were similar, to maximize the signal to noise ratio. Cells were illuminated with a 100 W Hg arc lamp through a neutral density filter to attenuate the light as needed and then through an ET436/20X (Chroma Technology) bandpass filter for mCerulean excitation. The main fluorescence turret of the microscope contained a custom T-cfp/yfp/cy5pc-WF dichroic mirror (Chroma Technology). The mCherry fluorescence (a marker of rat cells expressing AIDA-1 shRNAs) was acquired through a PrimeBSI camera (Photometrics) also attached on the third port of the optical beamsplitter (Cairn Research) via the T560LPXRXT mirror (Chroma Technology) and FF628/32 emission filter (Semrock). MetaMorph software ver. 7.10.5 (Molecular Devices) was used to control the microscope, motion control devices, and image acquisition. Metamorph and MatLab (ver 2011a; Mathworks, Natick, MA, USA) software were used to perform image processing and data analyses, as previously described^150,151,152. Ratiometric image processing included flatfield correction, background subtraction, image registration, and ratio calculations¹⁵³. In brief, flatfield correction involved the acquisition of cell-free fields of view with the same exposure and field illumination conditions as the foreground image sets (shading images). The foreground images were then divided by the shading images to obtain flatfield-corrected images. A small region of interest in the background (cell-free) area was selected in the flatfield-corrected foreground image sets, and the mean gray value from such a region was subtracted from the whole field of view, calculated, and processed at each time point to obtain the background-subtracted image sets. The background-subtracted image sets were then subjected to an affine transformation based on a priori calibration to account for misalignments between the three cameras used for the simultaneous imaging of the FRET and mCerulean channels, plus the mCherry channel. After the transformation, a linear X–Y registration was performed on the resulting image sets, before ratio calculations, in which the FRET image set was divided by the mCerulean channel image set. For the imaging of biosensors, we adjusted the intensity of excitation light and the camera acquisition time duration by targeting to fill approximately 50% of the total digitization range of the sCMOS device circuitry, to maximize the dynamic range, using excitation light intensities of 0.4–1.0 mW at the specimen plane.

Animal behavioral assays

All experiments were approved by and complied with the Albert Einstein College of Medicine IACUC. All mice were housed and handled at Einstein and behavioral phenotyping was performed as 7 independent cohorts in the Animal Behavior Core under the supervision of Dr. Maria Gulinello. All behavioral testing was performed as previously reported³. In the Behavioral Spectrometer, mice were recorded for 9 min (3 min, 3×) in an open field with a center area of 18.0 × 18.0 cm; validity and specificity of the system in identifying ASD-related behaviors has been previously described¹⁵⁴. Elevated plus maze (5 min), forced swim test for 9 min (3 min training, 4 min test in 25 °C water bath), and three-chamber test (5 min with ovariectomized C57BL/6J mouse, Jax) were performed using standard procedures¹⁵⁵. Social preference was calculated using the following formula: social preference = (social sniffing time)/(social sniffing time + object sniffing time) x 100%. Acoustic startle reflex and prepulse inhibition of the startle response were assayed¹⁵⁶ in a single session for each mouse using randomized, interleaved trials (5 each) for acoustic startle response (115 dB) and prepulse inhibition (PPI). Short-delay PPI trials were conducted with an 81-dB prepulse 40 ms before the 115-dB target stimulus, while long-delay PPI was tested with a 200-ms inter-stimulus interval. Balance beam assay for motor coordination was performed as described¹⁵⁷. Mice were started at the center of a wood-circular beam (Diameters: 1.2 cm, 1.5 cm, 1.8 cm) after pre-training. The novel object placement test was performed¹⁵⁸ using a 5-min training phase, 4-min testing phase, and short (40 min) retention intervals. During the testing phase, preference for the new placement was calculated using the following formula: preference for new = (time exploring new placement)/(time exploring new placement + time exploring old placement) x 100%. All behavioral testing in adult mice aged 3–4 months was performed with experimenter blind to genotype. Feeding, mixed-genotype group housing, light-dark cycles, and time of testing were controlled across all cohorts¹⁵⁹. Testing chambers were sanitized with 70% ethanol between trials. Mouse movement was recorded by a video tracking software (Viewer) and analyzed by off-line video tracking software (EthoVision XT 14). Sample sizes were estimated in JMP (version 16 and 17, SAS) to be of sufficient power to show effects independently in either sex if main effect of genotype-sex interaction (a = 0.05) were detected in 2-way ANOVA. For all behavioral tests, post hoc t test (Tukey) was performed for tests in which genotype was a significant main effect in 2-way ANOVA. For the three-chamber test, Likelihood Ratio effect tests were additionally performed for passing (>50% social preference) or failing (<50% social preference). Post hoc contingency testing included chi-squared analyses and Fisher’s exact test. Since no main effect of sex or genotype-sex interaction was observed in any behavioral test, males and females of each genotype were combined for all post hoc testing¹⁵⁹. Power analysis and least significant number (LSN) were calculated in JMP for post hoc t tests on significant effects from 2-way ANOVA analyses. 2-way ANOVAs were performed to detect main effects and interactions of genotype, and sex.

Clemastine rescue experiments

For treatment with clemastine fumarate (Cat #:1453; Tocris), the compound was solubilized at 1 mg/ml in a PBS solution containing 10% DMSO and filter sterilized (0.22 um). Clemastine was administered IP at a dose of 10 mg/kg (~270 uL per animal) daily at noon for 2 weeks, alternating the location of injection each day (left or right peritoneum), as has been previously described^17,72,160. Controls were injected with the same volume of 10% DMSO/PBS (vehicle). All injections were performed blind to mouse genotype. All injected animals were 7–8 months of age and group-housed according to the experimental group. After the injection period, animals were allowed to recover for 7 days prior to behavioral testing. Social preference and behavioral spectrometer analyses were performed as described above. All mice had been previously tested in these assays at 3–4 months of age.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All raw data and the datasets generated during and/or analyzed during the current study are available from the corresponding author on request. Source data are provided as a Source Data file. Source data are provided with this paper.

References

Anney, R. et al. Individual common variants exert weak effects on the risk for autism spectrum disorders. Hum. Mol. Genet. 21, 4781–92 (2012).
Article CAS PubMed PubMed Central Google Scholar
Barrett, T. et al. NCBI GEO: archive for functional genomics data sets–update. Nucleic Acids Res. 41, D991–5 (2013).
Article CAS PubMed Google Scholar
Carbonell, A. U. et al. Haploinsufficiency in the ANKS1B gene encoding AIDA-1 leads to a neurodevelopmental syndrome. Nat. Commun. 10, 3529 (2019).
Article ADS PubMed PubMed Central Google Scholar
Jordan, B. A. et al. Identification and verification of novel rodent postsynaptic density proteins. Mol. Cell Proteomics 3, 857–71 (2004).
Article CAS PubMed Google Scholar
Lowenthal, M. S., Markey, S. P. & Dosemeci, A. Quantitative mass spectrometry measurements reveal stoichiometry of principal postsynaptic density proteins. J. Proteome Res. 14, 2528–38 (2015).
Article CAS PubMed PubMed Central Google Scholar
Jordan, B. A. et al. Activity-dependent AIDA-1 nuclear signaling regulates nucleolar numbers and protein synthesis in neurons. Nat. Neurosci. 10, 427–35 (2007).
Article CAS PubMed Google Scholar
Tindi, J. O. et al. ANKS1B gene product AIDA-1 controls hippocampal synaptic transmission by regulating GluN2B subunit localization. J. Neurosci. 35, 8986–96 (2015).
Article CAS PubMed PubMed Central Google Scholar
Akay, L. A., Effenberger, A. H. & Tsai, L. H. Cell of all trades: oligodendrocyte precursor cells in synaptic, vascular, and immune function. Genes Dev. 35, 180–198 (2021).
Article CAS PubMed PubMed Central Google Scholar
Bonetto, G. et al. Unraveling myelin plasticity. Front. Cell Neurosci. 14, 156 (2020).
Article CAS PubMed PubMed Central Google Scholar
Clayton, B. L. L. & Tesar, P. J. Oligodendrocyte progenitor cell fate and function in development and disease. Curr. Opin. Cell Biol. 73, 35–40 (2021).
Article CAS PubMed PubMed Central Google Scholar
Kuhn, S. et al. Oligodendrocytes in development, myelin generation and beyond. Cells 8, 1424 (2019).
Article CAS PubMed PubMed Central Google Scholar
Monje, M. & Karadottir, R. T. The bright and the dark side of myelin plasticity: neuron-glial interactions in health and disease. Semin. Cell Dev. Biol. 116, 10–15 (2021).
Article PubMed Google Scholar
Paez, P. M. & Lyons, D. A. Calcium signaling in the oligodendrocyte lineage: regulators and consequences. Annu. Rev. Neurosci. 43, 163–186 (2020).
Article CAS PubMed Google Scholar
Liu, J. et al. Impaired adult myelination in the prefrontal cortex of socially isolated mice. Nat. Neurosci. 15, 1621–3 (2012).
Article CAS PubMed PubMed Central Google Scholar
Makinodan, M. et al. A critical period for social experience-dependent oligodendrocyte maturation and myelination. Science 337, 1357–60 (2012).
Article ADS CAS PubMed PubMed Central Google Scholar
Scholz, J. et al. Training induces changes in white-matter architecture. Nat. Neurosci. 12, 1370–1 (2009).
Article CAS PubMed PubMed Central Google Scholar
Pan, S. et al. Preservation of a remote fear memory requires new myelin formation. Nat. Neurosci. 23, 487–499 (2020).
Article CAS PubMed PubMed Central Google Scholar
Steadman, P. E. et al. Disruption of oligodendrogenesis impairs memory consolidation in adult mice. Neuron 105, 150–164 e6 (2020).
Article CAS PubMed Google Scholar
Butt, A. M., De La Rocha, I. C. & Rivera, A. Oligodendroglial cells in Alzheimer’s disease. Adv. Exp. Med. Biol. 1175, 325–333 (2019).
Article CAS PubMed Google Scholar
Hoy, A. R. et al. Microstructural white matter alterations in preclinical Alzheimer’s disease detected using free water elimination diffusion tensor imaging. PLoS One 12, e0173982 (2017).
Article PubMed PubMed Central Google Scholar
Agarwal, D. et al. A single-cell atlas of the human substantia nigra reveals cell-specific pathways associated with neurological disorders. Nat. Commun. 11, 4183 (2020).
Article ADS PubMed PubMed Central Google Scholar
Aoki, Y. et al. Association of white matter structure with autism spectrum disorder and attention-deficit/hyperactivity disorder. JAMA Psychiatry 74, 1120–1128 (2017).
Article PubMed PubMed Central Google Scholar
Deoni, S. C. & Kolind, S. H. Investigating the stability of mcDESPOT myelin water fraction values derived using a stochastic region contraction approach. Magn. Reson. Med. 73, 161–9 (2015).
Article CAS PubMed Google Scholar
Sakurai, T. et al. Slc25a12 disruption alters myelination and neurofilaments: a model for a hypomyelination syndrome and childhood neurodevelopmental disorders. Biol. Psychiatry 67, 887–94 (2010).
Article CAS PubMed Google Scholar
Whittall, K. P. et al. In vivo measurement of T2 distributions and water contents in normal human brain. Magn. Reson. Med. 37, 34–43 (1997).
Article CAS PubMed Google Scholar
Zikopoulos, B. & Barbas, H. Changes in prefrontal axons may disrupt the network in autism. J. Neurosci. 30, 14595–609 (2010).
Article CAS PubMed PubMed Central Google Scholar
Roberts, T. P. et al. MEG detection of delayed auditory evoked responses in autism spectrum disorders: towards an imaging biomarker for autism. Autism. Res. 3, 8–18 (2010).
Article ADS PubMed PubMed Central Google Scholar
Sokhadze, E. et al. Event-related potential study of novelty processing abnormalities in autism. Appl. Psychophysiol. Biofeedback 34, 37–51 (2009).
Article PubMed Google Scholar
Kawamura, A. et al. Oligodendrocyte dysfunction due to Chd8 mutation gives rise to behavioral deficits in mice. Hum. Mol. Genet. 29, 1274–1291 (2020).
Article CAS PubMed Google Scholar
Marques, S. et al. Oligodendrocyte heterogeneity in the mouse juvenile and adult central nervous system. Science 352, 1326–1329 (2016).
Article ADS CAS PubMed PubMed Central Google Scholar
Regev, A., et al. The human cell atlas. Elife, 6, eLife.27041 (2017).
Article Google Scholar
Sharma, K. et al. Cell type- and brain region-resolved mouse brain proteome. Nat. Neurosci. 18, 1819–31 (2015).
Article CAS PubMed PubMed Central Google Scholar
Tabula Muris, C. et al. Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris. Nature 562, 367–372 (2018).
Article ADS Google Scholar
Voskuhl, R. R. et al. Gene expression in oligodendrocytes during remyelination reveals cholesterol homeostasis as a therapeutic target in multiple sclerosis. Proc. Natl Acad. Sci. USA 116, 10130–10139 (2019).
Article ADS CAS PubMed PubMed Central Google Scholar
Mei, F. et al. Micropillar arrays as a high-throughput screening platform for therapeutics in multiple sclerosis. Nat. Med. 20, 954–960 (2014).
Article CAS PubMed PubMed Central Google Scholar
Carbonell, A. U. et al. Comparing synaptic proteomes across five mouse models for autism reveals converging molecular similarities including deficits in oxidative phosphorylation and Rho GTPase signaling. Front. Aging Neurosci. 15, 1152562 (2023).
Article CAS PubMed PubMed Central Google Scholar
Spiering, D. & Hodgson, L. Dynamics of the Rho-family small GTPases in actin regulation and motility. Cell Adh Migr. 5, 170–180 (2011).
Article PubMed PubMed Central Google Scholar
Ridley, A. J. Rho GTPases and cell migration. J. Cell Sci. 114, 2713–22 (2001).
Article CAS PubMed Google Scholar
Mosaddeghzadeh, N. & Ahmadian, M. R. The RHO family GTPases: mechanisms of regulation and signaling. Cells 10, 1831 (2021).
Article CAS PubMed PubMed Central Google Scholar
Biname, F. et al. NG2 regulates directional migration of oligodendrocyte precursor cells via Rho GTPases and polarity complex proteins. J. Neurosci. 33, 10858–74 (2013).
Article CAS PubMed PubMed Central Google Scholar
Liang, X., Draghi, N. A. & Resh, M. D. Signaling from integrins to Fyn to Rho family GTPases regulates morphologic differentiation of oligodendrocytes. J. Neurosci. 24, 7140–9 (2004).
Article CAS PubMed PubMed Central Google Scholar
Liu, X. et al. Slit2 regulates the dispersal of oligodendrocyte precursor cells via Fyn/RhoA signaling. J. Biol. Chem. 287, 17503–17516 (2012).
Article CAS PubMed PubMed Central Google Scholar
Pedraza, C. E. et al. Induction of oligodendrocyte differentiation and in vitro myelination by inhibition of rho-associated kinase. ASN Neuro 6, 1759091414538134 (2014).
Article PubMed PubMed Central Google Scholar
Rajasekharan, S. et al. A central role for RhoA during oligodendroglial maturation in the switch from netrin-1-mediated chemorepulsion to process elaboration. J. Neurochem. 113, 1589–97 (2010).
Article CAS PubMed Google Scholar
Antoine-Bertrand, J., Villemure, J. F. & Lamarche-Vane, N. Implication of rho GTPases in neurodegenerative diseases. Curr. Drug Targets 12, 1202–15 (2011).
Article CAS PubMed Google Scholar
DeGeer, J. & Lamarche-Vane, N. Rho GTPases in neurodegeneration diseases. Exp. Cell Res. 319, 2384–94 (2013).
Article CAS PubMed Google Scholar
Huang, G. H. et al. Rho GTPase-activating proteins: regulators of Rho GTPase activity in neuronal development and CNS diseases. Mol. Cell Neurosci. 80, 18–31 (2017).
Article CAS PubMed Google Scholar
Wang, X. et al. Stress-sensitive protein Rac1 and its involvement in neurodevelopmental disorders. Neural Plast 2020, 8894372 (2020).
Article ADS PubMed PubMed Central Google Scholar
Guo, D. et al. Autism-like social deficit generated by Dock4 deficiency is rescued by restoration of Rac1 activity and NMDA receptor function. Mol. Psychiatry 26, 1505–1519 (2021).
Article CAS PubMed Google Scholar
Marei, H. & Malliri, A. Rac1 in human diseases: the therapeutic potential of targeting Rac1 signaling regulatory mechanisms. Small GTPases 8, 139–163 (2017).
Article CAS PubMed Google Scholar
Thurnherr, T. et al. Cdc42 and Rac1 signaling are both required for and act synergistically in the correct formation of myelin sheaths in the CNS. J. Neurosci. 26, 10110–9 (2006).
Article CAS PubMed PubMed Central Google Scholar
Xiao, L. et al. NMDA receptor couples Rac1-GEF Tiam1 to direct oligodendrocyte precursor cell migration. Glia 61, 2078–99 (2013).
Article PubMed Google Scholar
Kurdi, A. T. et al. Tiam1/Rac1 complex controls Il17a transcription and autoimmunity. Nat. Commun. 7, 13048 (2016).
Article ADS MathSciNet CAS PubMed PubMed Central Google Scholar
Suri, A. K., Fleysher, R. & Lipton, M. L. Subject based registration for individualized analysis of diffusion tensor MRI. PLoS One 10, e0142288 (2015).
Article PubMed PubMed Central Google Scholar
Crucitti, J. et al. Are vermal lobules VI-VII smaller in autism spectrum disorder? Cerebellum 19, 617–628 (2020).
Article PubMed Google Scholar
Bin, J. M., Harris, S. N. & Kennedy, T. E. The oligodendrocyte-specific antibody ‘CC1’ binds Quaking 7. J. Neurochem. 139, 181–186 (2016).
Article CAS PubMed Google Scholar
Kotter, M. R. et al. Myelin impairs CNS remyelination by inhibiting oligodendrocyte precursor cell differentiation. J. Neurosci. 26, 328–32 (2006).
Article CAS PubMed PubMed Central Google Scholar
Yao, Z. et al. A taxonomy of transcriptomic cell types across the isocortex and hippocampal formation. Cell 184, 3222–3241 e26 (2021).
Article CAS PubMed PubMed Central Google Scholar
Zeisel, A. et al. Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq. Science 347, 1138–42 (2015).
Article ADS CAS PubMed Google Scholar
Jacob, A. L., Jordan, B. A. & Weinberg, R. J. Organization of amyloid-beta protein precursor intracellular domain-associated protein-1 in the rat brain. J. Comp. Neurol. 518, 3221–36 (2010).
Article CAS PubMed PubMed Central Google Scholar
Susaki, E. A. et al. Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. Nat. Protoc. 10, 1709–27 (2015).
Article CAS PubMed Google Scholar
Grier, M. D. et al. Loss of mTORC2 signaling in oligodendrocyte precursor cells delays myelination. PLoS One 12, e0188417 (2017).
Article PubMed PubMed Central Google Scholar
Harrington, E. P. et al. Oligodendrocyte PTEN is required for myelin and axonal integrity, not remyelination. Ann. Neurol. 68, 703–16 (2010).
Article CAS PubMed PubMed Central Google Scholar
Maire, C. L. et al. Pten loss in Olig2 expressing neural progenitor cells and oligodendrocytes leads to interneuron dysplasia and leukodystrophy. Stem Cells 32, 313–26 (2014).
Article ADS CAS PubMed Google Scholar
Sun, L. O. et al. Spatiotemporal control of CNS myelination by oligodendrocyte programmed cell death through the TFEB-PUMA axis. Cell 175, 1811–1826 e21 (2018).
Article CAS PubMed PubMed Central Google Scholar
Xu, H. et al. m(6)A mRNA methylation is essential for oligodendrocyte maturation and CNS myelination. Neuron 105, 293–309 e5 (2020).
Article CAS PubMed Google Scholar
Miyauchi, E. et al. Identification of blood biomarkers in glioblastoma by SWATH mass spectrometry and quantitative targeted absolute proteomics. PLoS One 13, e0193799 (2018).
Article PubMed PubMed Central Google Scholar
Yoshida, A. et al. Establishment of a simple one-step method for oligodendrocyte progenitor cell preparation from rodent brains. J. Neurosci. Methods 342, 108798 (2020).
Article CAS PubMed Google Scholar
Bhalla, R. M. et al. Multiplex imaging of Rho GTPase activities in living cells. Methods Mol. Biol. 2350, 43–68 (2021).
Article CAS PubMed PubMed Central Google Scholar
Shcherbakova, D. M. et al. Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET. Nat. Chem. Biol. 14, 591–600 (2018).
Article CAS PubMed PubMed Central Google Scholar
Cree, B. A. C. et al. Clemastine rescues myelination defects and promotes functional recovery in hypoxic brain injury. Brain 141, 85–98 (2018).
Article PubMed Google Scholar
Li, Z. et al. Clemastine rescues behavioral changes and enhances remyelination in the cuprizone mouse model of demyelination. Neurosci. Bull. 31, 617–25 (2015).
Article CAS PubMed PubMed Central Google Scholar
Liu, J. et al. Clemastine enhances myelination in the prefrontal cortex and rescues behavioral changes in socially isolated mice. J. Neurosci. 36, 957–62 (2016).
Article CAS PubMed PubMed Central Google Scholar
McClay, J. L. et al. Genome-wide pharmacogenomic analysis of response to treatment with antipsychotics. Mol. Psychiatry 16, 76–85 (2011).
Article CAS PubMed Google Scholar
Hoppe, A. D. & Swanson, J. A. Cdc42, Rac1, and Rac2 display distinct patterns of activation during phagocytosis. Mol. Biol. Cell 15, 3509–19 (2004).
Article CAS PubMed PubMed Central Google Scholar
Stankiewicz, T. R. & Linseman, D. A. Rho family GTPases: key players in neuronal development, neuronal survival, and neurodegeneration. Front. Cell Neurosci. 8, 314 (2014).
Article PubMed PubMed Central Google Scholar
Niederost, B. et al. Nogo-A and myelin-associated glycoprotein mediate neurite growth inhibition by antagonistic regulation of RhoA and Rac1. J. Neurosci. 22, 10368–76 (2002).
Article CAS PubMed PubMed Central Google Scholar
Maglorius Renkilaraj, M. R. L. et al. The intellectual disability protein PAK3 regulates oligodendrocyte precursor cell differentiation. Neurobiol. Dis. 98, 137–148 (2017).
Article CAS PubMed Google Scholar
Brown, T. L. et al. PAK1 positively regulates oligodendrocyte morphology and myelination. J. Neurosci. 41, 1864–1877 (2021).
Article CAS PubMed PubMed Central Google Scholar
Wang, Y. et al. PAK2 haploinsufficiency results in synaptic cytoskeleton impairment and autism-related behavior. Cell Rep. 24, 2029–2041 (2018).
Article PubMed Google Scholar
Horiuchi, M. et al. Differing in vitro survival dependency of mouse and rat NG2+ oligodendroglial progenitor cells. J. Neurosci. Res. 88, 957–70 (2010).
Article CAS PubMed PubMed Central Google Scholar
Fu, X. et al. EB-1, a tyrosine kinase signal transduction gene, is transcriptionally activated in the t(1;19) subset of pre-B ALL, which express oncoprotein E2a-Pbx1. Oncogene 18, 4920–9 (1999).
Article CAS PubMed Google Scholar
Xu, H. & Hebert, M. D. A novel EB-1/AIDA-1 isoform, AIDA-1c, interacts with the Cajal body protein coilin. BMC Cell Biol. 6, 23 (2005).
Article PubMed PubMed Central Google Scholar
Eckel-Passow, J. E. et al. ANKS1B is a smoking-related molecular alteration in clear cell renal cell carcinoma. BMC Urol. 14, 14 (2014).
Article PubMed PubMed Central Google Scholar
Zeng, K. et al. The pro-metastasis effect of circANKS1B in breast cancer. Mol. Cancer 17, 160 (2018).
Article CAS PubMed PubMed Central Google Scholar
Yeung, M. S. et al. Dynamics of oligodendrocyte generation and myelination in the human brain. Cell 159, 766–74 (2014).
Article CAS PubMed Google Scholar
Brett, M. et al. Massively parallel sequencing of patients with intellectual disability, congenital anomalies and/or autism spectrum disorders with a targeted gene panel. PLoS One 9, e93409 (2014).
Article ADS PubMed PubMed Central Google Scholar
Bucan, M. et al. Genome-wide analyses of exonic copy number variants in a family-based study point to novel autism susceptibility genes. PLoS Genet. 5, e1000536 (2009).
Article PubMed PubMed Central Google Scholar
Buxbaum, J. D. et al. The Autism Simplex Collection: an international, expertly phenotyped autism sample for genetic and phenotypic analyses. Mol. Autism. 5, 34 (2014).
Article PubMed PubMed Central Google Scholar
Buxbaum, J. D. et al. Optimizing the phenotyping of rodent ASD models: enrichment analysis of mouse and human neurobiological phenotypes associated with high-risk autism genes identifies morphological, electrophysiological, neurological, and behavioral features. Mol. Autism. 3, 1 (2012).
Article CAS PubMed PubMed Central Google Scholar
Cukier, H. N. et al. Exome sequencing of extended families with autism reveals genes shared across neurodevelopmental and neuropsychiatric disorders. Mol. Autism. 5, 1 (2014).
Article PubMed PubMed Central Google Scholar
Glessner, J. T. et al. Autism genome-wide copy number variation reveals ubiquitin and neuronal genes. Nature 459, 569–73 (2009).
Article ADS CAS PubMed PubMed Central Google Scholar
Hussman, J. P. et al. A noise-reduction GWAS analysis implicates altered regulation of neurite outgrowth and guidance in autism. Mol. Autism. 2, 1 (2011).
Article PubMed PubMed Central Google Scholar
Iossifov, I. et al. The contribution of de novo coding mutations to autism spectrum disorder. Nature 515, 216–21 (2014).
Article ADS CAS PubMed PubMed Central Google Scholar
Pinto, D. et al. Convergence of genes and cellular pathways dysregulated in autism spectrum disorders. Am. J. Hum. Genet 94, 677–94 (2014).
Article CAS PubMed PubMed Central Google Scholar
Ronemus, M. et al. The role of de novo mutations in the genetics of autism spectrum disorders. Nat. Rev. Genet 15, 133–41 (2014).
Article CAS PubMed Google Scholar
Sanders, S. J. et al. De novo mutations revealed by whole-exome sequencing are strongly associated with autism. Nature 485, 237–41 (2012).
Article ADS CAS PubMed PubMed Central Google Scholar
Toma, C. et al. Exome sequencing in multiplex autism families suggests a major role for heterozygous truncating mutations. Mol. Psychiatry 19, 784–90 (2014).
Article CAS PubMed Google Scholar
Yu, T. W. et al. Using whole-exome sequencing to identify inherited causes of autism. Neuron 77, 259–73 (2013).
Article CAS PubMed PubMed Central Google Scholar
Yuen, R. K. et al. Whole-genome sequencing of quartet families with autism spectrum disorder. Nat. Med. 21, 185–91 (2015).
Article CAS PubMed Google Scholar
Ramaswami, G. & Geschwind, D. H. Genetics of autism spectrum disorder. Handb. Clin. Neurol. 147, 321–329 (2018).
Article PubMed Google Scholar
Yoo, H. Genetics of autism spectrum disorder: current status and possible clinical applications. Exp. Neurobiol. 24, 257–72 (2015).
Article MathSciNet PubMed PubMed Central Google Scholar
Kim, R. et al. Cell-type-specific shank2 deletion in mice leads to differential synaptic and behavioral phenotypes. J. Neurosci. 38, 4076–4092 (2018).
Article CAS PubMed PubMed Central Google Scholar
Tsai, P. T. et al. Autistic-like behaviour and cerebellar dysfunction in Purkinje cell Tsc1 mutant mice. Nature 488, 647–51 (2012).
Article ADS CAS PubMed PubMed Central Google Scholar
Miyoshi, G. et al. Physiologically distinct temporal cohorts of cortical interneurons arise from telencephalic Olig2-expressing precursors. J. Neurosci. 27, 7786–98 (2007).
Article CAS PubMed PubMed Central Google Scholar
Ju, J. et al. Olig2 regulates Purkinje cell generation in the early developing mouse cerebellum. Sci. Rep. 6, 30711 (2016).
Article ADS CAS PubMed PubMed Central Google Scholar
Bergles, D. E. et al. Glutamatergic synapses on oligodendrocyte precursor cells in the hippocampus. Nature 405, 187–91 (2000).
Article ADS CAS PubMed Google Scholar
Gautier, H. O. et al. Neuronal activity regulates remyelination via glutamate signalling to oligodendrocyte progenitors. Nat. Commun. 6, 8518 (2015).
Article ADS CAS PubMed Google Scholar
Karadottir, R. et al. NMDA receptors are expressed in oligodendrocytes and activated in ischaemia. Nature 438, 1162–6 (2005).
Article ADS CAS PubMed PubMed Central Google Scholar
Karadottir, R. et al. Spiking and nonspiking classes of oligodendrocyte precursor glia in CNS white matter. Nat. Neurosci. 11, 450–6 (2008).
Article CAS PubMed PubMed Central Google Scholar
Lundgaard, I. et al. Neuregulin and BDNF induce a switch to NMDA receptor-dependent myelination by oligodendrocytes. PLoS Biol. 11, e1001743 (2013).
Article PubMed PubMed Central Google Scholar
Ziskin, J. L. et al. Vesicular release of glutamate from unmyelinated axons in white matter. Nat. Neurosci. 10, 321–30 (2007).
Article CAS PubMed PubMed Central Google Scholar
Ge, W. P. et al. Long-term potentiation of neuron-glia synapses mediated by Ca2 + -permeable AMPA receptors. Science 312, 1533–7 (2006).
Article ADS CAS PubMed Google Scholar
Hughes, A. N. & Appel, B. Oligodendrocytes express synaptic proteins that modulate myelin sheath formation. Nat. Commun. 10, 4125 (2019).
Article ADS PubMed PubMed Central Google Scholar
Haberlandt, C. et al. Gray matter NG2 cells display multiple Ca2+-signaling pathways and highly motile processes. PLoS One 6, e17575 (2011).
Article ADS CAS PubMed PubMed Central Google Scholar
Sahel, A. et al. Alteration of synaptic connectivity of oligodendrocyte precursor cells following demyelination. Front. Cell Neurosci. 9, 77 (2015).
Article PubMed PubMed Central Google Scholar
Gibson, E. M. et al. Neuronal activity promotes oligodendrogenesis and adaptive myelination in the mammalian brain. Science 344, 1252304 (2014).
Article PubMed PubMed Central Google Scholar
Mitew, S. et al. Pharmacogenetic stimulation of neuronal activity increases myelination in an axon-specific manner. Nat. Commun. 9, 306 (2018).
Article ADS PubMed PubMed Central Google Scholar
Demerens, C. et al. Induction of myelination in the central nervous system by electrical activity. Proc. Natl Acad. Sci. USA 93, 9887–92 (1996).
Article ADS CAS PubMed PubMed Central Google Scholar
Mensch, S. et al. Synaptic vesicle release regulates myelin sheath number of individual oligodendrocytes in vivo. Nat. Neurosci. 18, 628–30 (2015).
Article CAS PubMed PubMed Central Google Scholar
Tauber, H., Waehneldt, T. V. & Neuhoff, V. Myelination in rabbit optic nerves is accelerated by artificial eye opening. Neurosci. Lett. 16, 235–8 (1980).
Article CAS PubMed Google Scholar
Ge, W. P. et al. Dividing glial cells maintain differentiated properties including complex morphology and functional synapses. Proc. Natl Acad. Sci. USA 106, 328–33 (2009).
Article ADS CAS PubMed Google Scholar
Mount, C. W. et al. Monosynaptic tracing maps brain-wide afferent oligodendrocyte precursor cell connectivity. Elife 8, e49291 (2019).
Article PubMed PubMed Central Google Scholar
McKenzie, I. A. et al. Motor skill learning requires active central myelination. Science 346, 318–22 (2014).
Article ADS CAS PubMed PubMed Central Google Scholar
Pajevic, S., Basser, P. J. & Fields, R. D. Role of myelin plasticity in oscillations and synchrony of neuronal activity. Neuroscience 276, 135–47 (2014).
Article CAS PubMed Google Scholar
Sampaio-Baptista, C. et al. Motor skill learning induces changes in white matter microstructure and myelination. J. Neurosci. 33, 19499–503 (2013).
Article CAS PubMed PubMed Central Google Scholar
Homberg, J. R. et al. Improving treatment of neurodevelopmental disorders: recommendations based on preclinical studies. Expert. Opin. Drug Discov. 11, 11–25 (2016).
Article CAS PubMed Google Scholar
Mei, F. et al. Accelerated remyelination during inflammatory demyelination prevents axonal loss and improves functional recovery. Elife 5, e18246 (2016).
Article PubMed PubMed Central Google Scholar
Chen, B. S. et al. NMDA receptor-dependent regulation of dendritic spine morphology by SAP102 splice variants. J. Neurosci. 31, 89–96 (2011).
Article PubMed PubMed Central Google Scholar
Green, A. J. et al. Clemastine fumarate as a remyelinating therapy for multiple sclerosis (ReBUILD): a randomised, controlled, double-blind, crossover trial. Lancet 390, 2481–2489 (2017).
Article CAS PubMed Google Scholar
Andrews, D. S. et al. A longitudinal study of white matter development in relation to changes in autism severity across early childhood. Biol. Psychiatry 89, 424–432 (2021).
Article PubMed Google Scholar
Dimond, D. et al. Reduced white matter fiber density in autism spectrum disorder. Cereb. Cortex 29, 1778–1788 (2019).
Article PubMed PubMed Central Google Scholar
Ismail, M. M. et al. Studying autism spectrum disorder with structural and diffusion magnetic resonance imaging: a survey. Front. Hum. Neurosci. 10, 211 (2016).
Article PubMed PubMed Central Google Scholar
Ohta, H. et al. White matter alterations in autism spectrum disorder and attention-deficit/hyperactivity disorder in relation to sensory profile. Mol. Autism. 11, 77 (2020).
Article CAS PubMed PubMed Central Google Scholar
Pagnozzi, A. M. et al. A systematic review of structural MRI biomarkers in autism spectrum disorder: a machine learning perspective. Int. J. Dev. Neurosci. 71, 68–82 (2018).
Article PubMed Google Scholar
Phan, B. N. et al. A myelin-related transcriptomic profile is shared by Pitt-Hopkins syndrome models and human autism spectrum disorder. Nat. Neurosci. 23, 375–385 (2020).
Article CAS PubMed PubMed Central Google Scholar
Lord, C. et al. Autism spectrum disorders. Neuron 28, 355–63 (2000).
Article CAS PubMed Google Scholar
Smith, S. M. et al. Advances in functional and structural MR image analysis and implementation as FSL. Neuroimage 23, S208–19 (2004).
Article PubMed Google Scholar
Fleysher, R. et al. White matter structural integrity and transcranial Doppler blood flow pulsatility in normal aging. Magn. Reson. Imaging 47, 97–102 (2018).
Article PubMed Google Scholar
Avants, B. B. et al. A reproducible evaluation of ANTs similarity metric performance in brain image registration. Neuroimage 54, 2033–44 (2011).
Article PubMed Google Scholar
Lipton, M. L. et al. Robust detection of traumatic axonal injury in individual mild traumatic brain injury patients: intersubject variation, change over time and bidirectional changes in anisotropy. Brain Imaging Behav. 6, 329–42 (2012).
Article PubMed Google Scholar
Jones, D. K. et al. Characterization of white matter damage in ischemic leukoaraiosis with diffusion tensor MRI. Stroke 30, 393–7 (1999).
Article CAS PubMed Google Scholar
Dorr, A. E. et al. High resolution three-dimensional brain atlas using an average magnetic resonance image of 40 adult C57Bl/6J mice. Neuroimage 42, 60–9 (2008).
Article CAS PubMed Google Scholar
Nojima, S. et al. CUBIC pathology: three-dimensional imaging for pathological diagnosis. Sci Rep 7, 9269 (2017).
Article ADS PubMed PubMed Central Google Scholar
Raj, A. et al. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods 5, 877–9 (2008).
Article CAS PubMed PubMed Central Google Scholar
Osten, P. et al. Mutagenesis reveals a role for ABP/GRIP binding to GluR2 in synaptic surface accumulation of the AMPA receptor. Neuron 27, 313–25 (2000).
Article CAS PubMed Google Scholar
Lariosa-Willingham, K. D. et al. Development of a central nervous system axonal myelination assay for high throughput screening. BMC Neurosci. 17, 16 (2016).
Article PubMed PubMed Central Google Scholar
Sherafat, A. et al. Microglial neuropilin-1 promotes oligodendrocyte expansion during development and remyelination by trans-activating platelet-derived growth factor receptor. Nat. Commun. 12, 2265 (2021).
Article ADS CAS PubMed PubMed Central Google Scholar
Jablonska, B. et al. Chordin-induced lineage plasticity of adult SVZ neuroblasts after demyelination. Nat. Neurosci. 13, 541–550 (2010).
Article CAS PubMed PubMed Central Google Scholar
Bravo-Cordero, J. J. et al. Live cell imaging of RhoGTPase biosensors in tumor cells. Methods Mol. Biol. 1046, 359–70 (2013).
Article PubMed PubMed Central Google Scholar
Donnelly, S. K. et al. Rac3 regulates breast cancer invasion and metastasis by controlling adhesion and matrix degradation. J. Cell Biol. 216, 4331–4349 (2017).
Article CAS PubMed PubMed Central Google Scholar
Moshfegh, Y. et al. A Trio-Rac1-Pak1 signalling axis drives invadopodia disassembly. Nat. Cell Biol. 16, 574–86 (2014).
Article CAS PubMed PubMed Central Google Scholar
Spiering, D. et al. Quantitative ratiometric imaging of FRET-biosensors in living cells. Methods Cell Biol. 114, 593–609 (2013).
Article PubMed PubMed Central Google Scholar
Brodkin, J. et al. Validation and implementation of a novel high-throughput behavioral phenotyping instrument for mice. J. Neurosci. Methods 224, 48–57 (2014).
Article PubMed Google Scholar
Lopez-Granero, C. et al. BXD recombinant inbred strains participate in social preference, anxiety and depression behaviors along sex-differences in cytokines and tactile allodynia. Psychoneuroendocrinology 80, 92–98 (2017).
Article CAS PubMed PubMed Central Google Scholar
Gulinello, M., Orman, R. & Smith, S. S. Sex differences in anxiety, sensorimotor gating and expression of the alpha4 subunit of the GABAA receptor in the amygdala after progesterone withdrawal. Eur. J. Neurosci. 17, 641–8 (2003).
Article CAS PubMed PubMed Central Google Scholar
Molero, A. E. et al. Selective expression of mutant huntingtin during development recapitulates characteristic features of Huntington’s disease. Proc. Natl Acad. Sci. USA 113, 5736–41 (2016).
Article ADS CAS PubMed PubMed Central Google Scholar
Li, Y. et al. Systemic methotrexate induces spatial memory deficits and depletes cerebrospinal fluid folate in rats. Pharmacol. Biochem. Behav. 94, 454–63 (2010).
Article CAS PubMed Google Scholar
Gulinello, M. et al. Rigor and reproducibility in rodent behavioral research. Neurobiol. Learn Mem. 165, 106780 (2019).
Article PubMed Google Scholar
Lee, J. I. et al. Clemastine improves electrophysiologic and histomorphometric changes through promoting myelin repair in a murine model of compression neuropathy. Sci. Rep. 11, 20886 (2021).
Article ADS CAS PubMed PubMed Central Google Scholar

Download references

Acknowledgements

Supported by NIH R01NS118820, NIH R56MH115201, NIH 1UL1TR002556-01 and Simons AR-PI-0000251 to B.A.J., NIH T32HD098067 to A.M., NIH T32GM007288 to A.U.C., R35GM136226 to L.H., R01NS123374 and R01NS082432 to M.L.L. and SIG #1S10OD016214 and P30CA01330 to the Analytical Imaging Facility at Albert Einstein College of Medicine. Significant support for this work came from the Rose F. Kennedy Intellectual and Developmental Disabilities Research Center (IDDRC), which is funded through a center grant from the Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD U54HD090260). We thank Xheni Nishku, Leslie Cummins, and Joseph Churaman in the Analytical Imaging Facility at the Albert Einstein College of Medicine. We thank the participating families for their time and contributions to this work.

Author information

Chang Hoon Cho
Present address: Human Pathobiology and OMNI Reverse Translation, Genentech, Inc., San Francisco, CA, USA
These authors contributed equally: Chang Hoon Cho, Ilana Vasilisa Deyneko.

Authors and Affiliations

Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY, USA
Chang Hoon Cho, Ilana Vasilisa Deyneko, Dylann Cordova-Martinez, Juan Vazquez, Anne S. Maguire, Jenny R. Diaz, Abigail U. Carbonell, Jaafar O. Tindi, Sophie Molholm, Michael L. Lipton & Bryen A. Jordan
Department of Radiology, Albert Einstein College of Medicine, Bronx, NY, USA
Min-Hui Cui, Roman Fleysher, Michael L. Lipton & Craig A. Branch
Gruss Magnetic Resonance Research Center, Albert Einstein College of Medicine, Bronx, NY, USA
Min-Hui Cui, Roman Fleysher, Michael L. Lipton & Craig A. Branch
Department of Pediatrics, Albert Einstein College of Medicine, Bronx, NY, USA
Sophie Molholm
Department of Psychiatry and Behavioral Sciences, Albert Einstein College of Medicine, Bronx, NY, USA
Sophie Molholm, Michael L. Lipton & Bryen A. Jordan
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, USA
Louis Hodgson
Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY, USA
Louis Hodgson

Authors

Chang Hoon Cho
View author publications
You can also search for this author in PubMed Google Scholar
Ilana Vasilisa Deyneko
View author publications
You can also search for this author in PubMed Google Scholar
Dylann Cordova-Martinez
View author publications
You can also search for this author in PubMed Google Scholar
Juan Vazquez
View author publications
You can also search for this author in PubMed Google Scholar
Anne S. Maguire
View author publications
You can also search for this author in PubMed Google Scholar
Jenny R. Diaz
View author publications
You can also search for this author in PubMed Google Scholar
Abigail U. Carbonell
View author publications
You can also search for this author in PubMed Google Scholar
Jaafar O. Tindi
View author publications
You can also search for this author in PubMed Google Scholar
Min-Hui Cui
View author publications
You can also search for this author in PubMed Google Scholar
Roman Fleysher
View author publications
You can also search for this author in PubMed Google Scholar
Sophie Molholm
View author publications
You can also search for this author in PubMed Google Scholar
Michael L. Lipton
View author publications
You can also search for this author in PubMed Google Scholar
Craig A. Branch
View author publications
You can also search for this author in PubMed Google Scholar
Louis Hodgson
View author publications
You can also search for this author in PubMed Google Scholar
Bryen A. Jordan
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

C.H.C. developed the idea that AIDA-1 regulates oligodendrocyte function. C.H.C. performed all histology and immunofluorescence to measure myelin and OPC development in tissue, as well as behavioral analyses of Olig2 Het mice, which were created and maintained by C.H.C. and I.V.D. I.V.D. assisted in histology and immunofluorescence assays and developed the idea that AIDA-1 regulates Rac1. I.V.D. performed all proteomic analyses and Rac1 activity measurements together with L.H. I.V.D. and D.C.M. performed all EM experiments and quantitation in diverse Anks1b mouse models. D.C.M., J.V. and B.A.J. performed clemastine rescue assays. J.R.D., A.S.M. and B.A.J. performed in vitro assays in primary oligodendrocyte cultures. A.U.C. and J.O.T. performed behavioral analyses on CaMKII and L7 Anks1b mouse models. R.F., M.H.C., J.V., C.A.B. and M.L.L. performed and analyzed MRI and DTI experiments on ANDS patients and mouse models. S.M. was involved in the recruitment of patients and MRI analyses. C.H.C., I.V.D. and B.A.J. wrote the manuscript.

Corresponding author

Correspondence to Bryen A. Jordan.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Communications thanks Hideki Hida, Masaaki Nishiyama and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Peer Review File

Description of Additional Supplementary Files

Supplementary Data 1

Supplementary Data 2

Supplementary Data 3

Reporting Summary

Source data

Source Data

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Cho, C.H., Deyneko, I.V., Cordova-Martinez, D. et al. ANKS1B encoded AIDA-1 regulates social behaviors by controlling oligodendrocyte function. Nat Commun 14, 8499 (2023). https://doi.org/10.1038/s41467-023-43438-1

Download citation

Received: 08 April 2022
Accepted: 09 November 2023
Published: 21 December 2023
DOI: https://doi.org/10.1038/s41467-023-43438-1

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Subjects

Abstract

Similar content being viewed by others

Introduction

Results

Anks1b haploinsufficiency impairs structural integrity of white matter

Anks1b haploinsufficiency results in impaired myelination and reduced oligodendrocyte abundance

Anks1b haploinsufficiency impairs oligodendrocyte maturation and migration

AIDA-1 is expressed in oligodendrocytes

Loss of Anks1b from oligodendrocyte lineage cells results in oligodendrocyte dysfunction

Oligodendrocytes deficient in Anks1b display Rac1-based functional deficits

Loss of Anks1b from oligodendrocyte lineage cells results in behavioral correlates of ASD

Clemastine rescues social deficits in mice lacking Anks1b in oligodendrocytes

Discussion

Methods

Reagents

Brain imaging

Human subjects and clinical data

Patient MRI scans

MRI data analysis

In vivo mouse brain MRI

Ex vivo mouse brain MRI

MRI data analysis

Anks1b mouse models

Tissue preparation, histology, clearing, immunofluorescence

Histology

Luxol Fast Blue staining

Immunofluorescence

Imaging

Analysis

CUBIC tissue clearing and LSFM

Transmission electron microscopy sample preparation and processing

Fluorescent In situ hybridization (FISH)

Cell culture

Fixed tissue

Neuron and oligodendrocyte cultures

Western blot

Oligodendrocyte maturation assay with EdU-labeling

Demyelination and remyelination assays

FRET live imaging of Rac1 activation

Animal behavioral assays

Clemastine rescue experiments

Reporting summary

Data availability

References

Acknowledgements

Author information

Authors and Affiliations

Contributions

Corresponding author

Ethics declarations

Competing interests

Peer review

Peer review information

Additional information

Supplementary information

Source data

Rights and permissions

About this article

Cite this article

Share this article

Comments

Search

Quick links