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The X-linked intellectual disability gene CUL4B is critical for memory and synaptic function

Acta Neuropathologica Communications volume 12, Article number: 188 (2024) Cite this article

Abstract

Cullin 4B (CUL4B) is the scaffold protein in the CUL4B-RING E3 ubiquitin ligase (CRL4B) complex. Loss-of-function mutations in the human CUL4B gene lead to syndromic X-linked intellectual disability (XLID). Till now, the mechanism of intellectual disability caused by CUL4B mutation still needs to be elucidated. In this study, we used single-nucleus RNA sequencing (snRNA-seq) to investigate the impact of CUL4B deficiency on the transcriptional programs of diverse cell types. The results revealed that depletion of CUL4B resulted in impaired intercellular communication and elicited cell type-specific transcriptional changes relevant to synapse dysfunction. Golgi-Cox staining of brain slices and immunostaining of in vitro cultured neurons revealed remarkable synapse loss in CUL4B-deficient mice. Ultrastructural analysis via transmission electron microscopy (TEM) showed that the width of the synaptic cleft was significantly greater in CUL4B-deficient mice. Electrophysiological experiments found a decrease in the amplitude of AMPA receptor-mediated EPSCs in the hippocampal CA1 pyramidal neurons of CUL4B-deficient mice. These results indicate that depletion of CUL4B in mice results in morphological and functional abnormalities in synapses. Furthermore, behavioral tests revealed that depletion of CUL4B in the mouse nervous system results in impaired spatial learning and memory. Taken together, the findings of this study reveal the pathogenesis of neurological disorders associated with CUL4B mutations and promote the identification of therapeutic targets that can halt synaptic abnormalities and preserve memory in individuals.

Introduction

Intellectual disability (ID) is a class of neurodevelopmental disorder with extreme clinical and genetic heterogeneity, with an estimated prevalence of 2–3% worldwide [1]. X-linked intellectual disability (XLID) accounts for 5–10% of all intellectual disability in males [2]. Loss-of-function mutations in the human CUL4B gene lead to syndromic X-linked intellectual disability [3, 4]. Patients with CUL4B mutations exhibit severe intellectual disability associated with seizures, tremors, speech delays, aggressive outbursts, short stature, central obesity, etc [3,4,5,6].

Cullin 4B (CUL4B) is a member of the Cullin family and functions as a scaffold protein in the CUL4B-RING E3 ligase (CRL4B) complex [7, 8]. The CRL4B complex catalyzes the polyubiquitination of specific substrates for ubiquitin-dependent proteasomal degradation. In addition, the CRL4B complex can catalyze the monoubiquitination of H2AK119 and coordinate with transcriptional repression complexes to repress gene transcription, such as coordinate with the PRC2 complex to promote H3K27 trimethylation (H3K27me3) [9], coordinate with the SUV39H1/HP1/DNMT3A complex to promote H3K9 trimethylation (H3K9me3) and DNA methylation [10], and coordinate with the SIN3A-HDAC complex to promote the deacetylation of H3 and H4 [11]. CRL4B complexes participate in the regulation of diverse physiological and developmental processes, including embryogenesis [12, 13], spermatogenesis [14, 15], adipogenesis [16], and tumor progression [9, 17].

Deletion of Cul4b in mouse epiblasts led to impaired spatial learning and memory, potentially mimicking intellectual disability in human patients with CUL4B mutations [18]. In addition, CUL4B has been intensively reported to regulate neurite morphogenesis during neurodevelopment [19], the abundance of GFAP-positive cells [20], and human cortical neurogenesis [21]. Despite these advances, the mechanism of CUL4B mutation-associated intellectual disability still needs to be elucidated.

Single-cell transcriptomics enables the precise measurement of gene expression in single cells, providing high resolution for understanding the complexity and heterogeneity of cell types in the brain [22,23,24]. This precision is crucial for understanding the molecular mechanism of mental diseases, which often involve cell type-specific changes in gene expression. For example, in disorders such as Down syndrome [25] or epilepsy [26], single-nucleus RNA sequencing (snRNA-seq) can reveal alterations in specific neuronal subtypes or glial cells that contribute to disease pathology, providing insights that bulk RNA-seq cannot achieve. In this study, using snRNA-seq analysis, we found that depletion of CUL4B resulted in impaired intercellular communication and elicited cell type-specific transcriptional changes toward defects in axons, dendrites and synapses. Furthermore, Golgi-Cox staining, immunostaining, ultrastructural analysis and electrophysiological experiments revealed that depletion of CUL4B in mice results in morphological and functional abnormalities in synapses. Depletion of CUL4B in the mouse nervous system also results in impaired spatial learning and memory. Our findings could provide a blueprint for investigating the molecular and cellular processes of intellectual disability caused by mutations in the CUL4B gene.

Results

Single-nucleus RNA sequencing of the mouse hippocampus

To investigate the mechanism underlying the intellectual disability associated with CUL4B loss-of-function mutations, we generated nervous system-specific Cul4b knockout mice (Nestin-Cre+/−;Cul4bflox/Y mice, designated CKO) and their littermate controls (Cul4bflox/Y mice, designated CON). Western blotting showed that the expression of CUL4B was undetectable in the brains of CKO mice but was comparable to that in other tissues of CON mice (Additional file 1: Fig. S1a). Moreover, CUL4B was undetectable in the hippocampus, cortex, olfactory bulbs and cerebellum of CKO mice (Additional file 1: Fig. S1b). These results indicated that CUL4B was specifically deleted in the nervous system of CKO mice.

As CUL4B plays important roles in transcriptional repression by catalyzing the monoubiquitination of H2AK119 and epigenetic modifications, we performed snRNA-seq to investigate the impact of CUL4B deficiency on the transcriptional programs of diverse cell types. Hippocampal samples from three 8-week-old male mice of each genotype were collected. After quality filtering (Additional file 1: Fig. S2a-c), a total of 22,994 nuclei were obtained for analysis (11,270 from CKO and 11,724 from CON mice). To classify major cell types, we clustered all cells jointly across each genotype in Uniform Manifold Approximation and Projection (UMAP) space, which revealed 29 distinct clusters (Additional file 1: Fig. S2d). Importantly, all major clusters were present in each genotype and showed no significant alterations (Additional file 1: Fig. S2e). Eight major cell types were annotated by known marker genes: mature granule cells (mGC, marked by Prox1, Lct, and Gsg1l), immature granule cells (imGC, marked by Prox1 but lacking Calb1 expression), catecholaminergic neurons (CA neurons, marked by Mpped1 and Dkk3), GABAergic neurons (GABA neurons, marked by Gad1), astrocytes (marked by Aqp4, Gfap, and S1pr1), oligodendrocytes (marked by Sox10, Mobp, and Mog), oligodendrocyte progenitor cells (marked by Pdgfra), and microglia (marked by Ptprc, C1qb, and Inpp5d) (Fig. 1a; Additional file 2: Table S1). Less abundant cell types, such as vascular and leptomeningeal cells (marked by Col1a2), were also observed. Any remaining cells that did not fall into these categories were labeled “Unsigned.” UMAP plots (Fig. 1b) and violin plots (Fig. 1c) demonstrated the marker genes central to the cell types. Additionally, Gene Set Variation Analysis (GSVA) revealed that the top expressed genes for each cluster were enriched in hallmark terms associated with their cell type, further validating the identification of each cluster (Fig. 1d). To investigate whether the cellular composition was altered in the CKO hippocampus, we detected the abundance of each cell type in each genotype. The results demonstrated that the cellular composition was largely changed in the CKO hippocampus. The percentage of neurons was significantly greater in the CKO (80.66%) hippocampus than in the control hippocampus (74.55%) (Fig. 1e). The changes in the cell-type ratios suggest that the biological functions of these cell types may be affected.

Fig. 1
figure 1

Single-nucleus RNA sequencing analysis of the mouse hippocampus. (a) UMAP plot of all the nuclei used for analysis. Clustering analysis revealed 9 broad categories of cell type identity, including astrocytes (ASC), microglia (MIC), cornu ammonis neurons (CA neurons), GABAergic neurons (GABA neurons), immature dentate granule cells (imGC), mature dentate granule cells (mGC), oligodendrocytes (ODC), oligodendrocyte progenitor cells (OPC), vascular and leptomeningeal cells (VLMC) and unsigned cells. A total of 22,994 cells were obtained from the hippocampi of 3 CKO (11,270 cells) and 3 control mice (11,724 cells) at 8 weeks of age. (b) UMAP plots of all nuclei labeled for the expression of marker genes for each cell type (Slc17a7, excitatory neurons; Prox1, mGC and imGC; Dkk3, CA neurons; Gad1, GABA neurons; Sox10, ODC; Pdgfra, OPC; Aqp4, ASC; Ptprc, MIC; Col1a2, VLMC). The color scale indicates the level of gene expression. (c) Violin plots showing the expression levels of known cell type-enriched marker genes across different cell populations. (d) Heatmap showing GSVA for hallmark terms from the Molecular Signatures Database (MSigDB). The color indicates the normalized enrichment score. (e) Percentages of major cell types in the control and CKO hippocampi

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Depletion of CUL4B resulted in impaired intercellular communication

To explore the interactions among different cell types in the hippocampus of control and CKO mice, we employed the CellChat tool for a detailed quantitative analysis of the intercellular communication network. The results demonstrated that the total number of inferred interactions was reduced in the CKO hippocampus (Fig. 2a). The number of interactions between both neuron–neuron and neuron–glia cells was reduced in the CKO hippocampus (Fig. 2b-c). The interaction strength was also reduced in the CKO hippocampus (Fig. 2d-f). To understand how the interactions are altered with CUL4B deficiency, we utilized CellChat to detect significant ligand–receptor pairs among the 8 cell clusters. We found that the loss of 6 signaling pathways, including the EGF, COLLAGEN, TGFβ, THBS, VISFATIN and TENASCIN pathways, was a theme across the cell types in the CKO mice (Fig. 2g-h). The loss of these cell signaling pathways may represent a mechanism for alterations in synaptogenesis in the CUL4B-deficient hippocampus.

Fig. 2
figure 2

Analysis of intercellular communication in the control and CKO hippocampi via CellChat. (a) The total number of inferred interactions in the control and CKO hippocampi. (b) The absolute number of interactions between different cell types in the control and CKO hippocampi. The interaction directions are indicated by arrows whose thickness is correlated with the interaction frequency. (c) Heatmap showing the differential numbers of interactions in each cluster. The Y-axis represents the sender, whereas the X-axis represents the receptor. The blue color indicates a decrease in CKO, and the red color indicates an increase. (d) The total interaction strength in the control and CKO hippocampi. (e) The interaction strength between different cell types in the control and CKO hippocampi. The arrows indicate the direction of the interaction, and their thickness symbolizes the relative strength of the interaction. (f) Heatmap showing the differential interaction strength in each cluster. The Y-axis represents the sender, whereas the X-axis represents the receptor. The blue color indicates a decrease in CKO, and the red color indicates an increase. (g-h) Aggregated view of incoming (g) and outgoing (h) signaling patterns in ligand–receptor pathways in the control and CKO hippocampi. The incoming signaling patterns indicate the cell types expressing the respective receptors, whereas the outgoing signaling patterns illustrate the origin of pathway interaction ligands by cell type. The height of the top-colored bar indicates the total interaction strength of each cell type. The color shows the relative strength of each pathway across all cell types

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Depletion of CUL4B elicits cell type-specific transcriptional changes toward defects in axons, dendrites and synapses

We next investigated the changes in gene expression associated with CUL4B deficiency in the major cell types of the hippocampus. To gain a global understanding of how gene expression is altered in the CKO hippocampus, we first performed differential expression analysis on all cells via model-based analysis of single-cell transcriptomics (MAST). We identified 7639 differentially expressed genes (DEGs) between the CKO and control hippocampi (Fig. 3a; Additional file 3: Table S2). Next, we investigated the impact of CUL4B deficiency on gene expression in each major cell type. Neurons, including mGC, CA and GABA neurons, presented the greatest number of DEGs (Fig. 3b; Additional file 4: Table S3). Additionally, the coefficient of variation (CV) analysis of the major cell types revealed a significant difference between the CKO and control hippocampi in CA neurons, GABA neurons, astrocytes, microglia and oligodendrocytes (Fig. 3c). Specifically, the coefficients of variation of CA neurons and GABA neurons were significantly increased in the CKO hippocampus, suggesting that variability in gene expression increases in these two cell types of the CKO hippocampus, which could contribute to cellular dysfunction. To investigate the cellular processes that are altered with CUL4B deficiency in different cell types in the hippocampus, we performed Gene Set Enrichment Analysis (GSEA) on the Biological Process (BP) gene set. The results revealed that CUL4B deficiency led to enrichment of biological processes related to ribosome biogenesis, the respiratory chain, and rRNA metabolism among CA neurons, GABA neurons and mGC but not in glial cells (Additional file 1: Fig. S3; Additional file 5: Table S4). Conversely, CUL4B deficiency led to under-enrichment (negative normalized enrichment score) of biological processes related to cognition, axon development, dendrite development and synapse organization among almost all cell types, which are crucial processes for proper neuron function (Fig. 3d-k; Additional file 5: Table S4).

Fig. 3
figure 3

Cell type-specific transcriptional changes in CON and CKO hippocampi derived from snRNA-seq. (a) Volcano plot showing the log2 fold change (FC) and -log10P value for all genes expressed in the hippocampi of control and CKO mice (log2FC > 0 represents upregulation in the CKO hippocampus, and log2FC < 0 indicates downregulation in the CKO hippocampus compared with the control hippocampus). The top DEGs are highlighted in red (|log2FC| > 0.2, P < 0.05). MAST analysis with random effects for sample of origin and sequencing depth, with Benjamin adjustment of P values. (b) UpSet plot showing DEGs in each cell type. (c) Coefficient of variation (c.v.) analysis for each cell type (two-sided Wilcoxon test with Bonferroni correction, * Padj<0.05, ** Padj<0.01, **** Padj<0.0001; ns, not significant). The box indicates the range from the 25th to 75th percentiles, with whiskers extending to 1.5 times the interquartile range. The outliers are plotted separately, and the center indicates the median value. (d-k) Dot plot showing GSEA for biological process (BP) terms under-enriched in CA neurons (d), GABA neurons (e), mGC (f), imGC (g), astrocytes (h), microglia (i), oligodendrocytes (j) and oligodendrocyte progenitor cells (k). NES, normalized enrichment score (the negative number represents down-regulation). The dot size indicates the gene ratio, whereas the color indicates the adjusted P value

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For validation, bulk RNA sequencing (RNA-seq) from 6 control and 7 CKO hippocampi was performed, and the snRNA-seq results were cross-checked with bulk RNA-seq data. The results of the GO enrichment analyses revealed that the DEGs were related mainly to neuron and synapse development (Additional file 1: Fig. S4; Additional files 6–7: Tables S5-6). Overall, the above results suggest that CUL4B deficiency may affect axon development, dendrite development and synapses and eventually affect the functions of neurons.

Neuronal subcluster-specific transcriptional changes toward defects in axons, dendrites and synapses in CUL4B-deficient mice

To conduct a more detailed analysis of the transcriptional programs of diverse sub-types of neurons, we subdivided the neuronal cells into 33 sub-clusters (Fig. 4a). Each sub-cluster was named on the basis of the top 2 expressed genes, and the clusters were classified into GABA, CA, and GC neurons, with GABA neurons defined by Gad1, GC neurons defined by Prox1, and CA neurons defined by Slc8a1 (Fig. 4b; Additional file 8: Table S7). Next, we investigated the effect of CUL4B deficiency on gene expression in each sub-cluster. The greatest numbers of DEGs were shown in sub-clusters 2, 13, 19–24, 26–28, and 32 (Fig. 4c; Additional file 9: Table S8). We matched the DEGs in 33 cell populations against the GWAS database and found that the DEGs were related to learning and memory. Intelligence and cognitive function were related mainly to clusters 1, 2, 7, 13, 20–25, 27 and 28 (Fig. 4d; Additional file 10: Table S9). The results of Gene Set Enrichment Analysis (GSEA) of each sub-cluster on the Biological Process (BP) gene set indicated that CUL4B deficiency let to under-enrichment of biological processes involving synapses, dendritic spines and axons (Fig. 4e; Additional file 11: Table S10). These results also support that CUL4B deficiency may affect dendrites, axons and synapses.

Fig. 4
figure 4

Neuronal subcluster-specific transcriptional change analysis. (a) UMAP plot showing that neurons were subdivided into 33 sub-clusters. (b) Dot plot showing the expression levels of typical neuron markers in each sub-cluster, which was named the top 2 expressed genes. (c) Strip plot showing DEGs in each neuronal sub-cluster. Significant genes are in color, and nonsignificant genes are in gray. (d) UP: Bubble chart showing the enrichment of human GWAS signals in each neuronal sub-cluster. Association analysis was performed via MAGMA. The color corresponds to the log10(P) for each association test. The size corresponds to the effect size (Beta). Below: Bar plot showing the relative percentage of each sub-cluster’s cell number in the control and CKO groups. (e) Dot plot showing GSEA for Biological Process (BP) terms in each neuronal sub-cluster. The dot size indicates the -log10(adjusted P value), whereas the color indicates the normalized enrichment score (NES)

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CUL4B-deficient mice exhibit synaptic dysfunction

To validate the synaptic dysfunction suggested by snRNA-seq, we first tested whether CUL4B deficiency may result in synapse loss. Golgi-Cox staining revealed that the dendritic spine density in the hippocampus was significantly reduced in CKO mice, suggesting remarkable synapse loss in CKO mice (Fig. 5a-b). For the analysis of spine subtypes, fewer mushroom spines and more stubby spines were noted in CKO mice than in CON mice (Fig. 5c). We subsequently examined the synaptic density of neurons cultured in vitro by immunostaining for synapse-associated proteins. As shown in Fig. 5d, the density of synapses positive for Synapsin1 (an excitatory presynaptic membrane marker) (Fig. 5e) or PSD95 (an excitatory postsynaptic membrane marker) (Fig. 5f) was lower in Cul4b-deficient neurons than in control neurons. The colocalization of Synapsin1 with PSD95, which represents functional excitatory synapses, was also reduced in Cul4b-deficient neurons (Fig. 5g). Similarly, as shown in Fig. 5h, the density of synapses positive for GAD67 (an inhibitory presynaptic membrane marker) (Fig. 5i) or Gephyrin (an inhibitory postsynaptic membrane marker) (Fig. 5j) was lower in Cul4b-deficient neurons than in control neurons. The colocalization of GAD67 with Gephyrin, which represents functional inhibitory synapses, was also reduced in Cul4b-deficient neurons (Fig. 5k). These data indicate that CUL4B plays an important role in the regulation of synaptic integrity.

Fig. 5
figure 5

CUL4B-deficient mice exhibit synapse loss. (a) Golgi-Cox staining for the measurement of synapse density in pyramidal neurons in the hippocampus. Scale bar, 1 μm. (b) Spine density on basal dendrites per 10 μm (N = 50 dendrites from 3 CON mice and 45 dendrites from 3 CKO mice). (c) Percentages of different subtypes, mushroom (M), stubby (S), thin (T) and filiform pseudopodia (F) spines in CON and CKO mice. (N = 50 dendrites from 3 CON mice and 45 dendrites from 3 CKO mice). (d) Synapsin1 and PSD95 immunostaining of DIV14 neurons from CKO and control mice. Scale bars: 100 μm (upper) or 20 μm (lower). (e-g) Statistical analysis of Synapsin1+ (e), PSD95+ (f), and PSD95+/Synapsin1+ synapse density (g) (N = 29 cells from 3 mice). (h) GAD67 and Gephyrin immunostaining of DIV14 neurons from CKO and littermate control mice. Scale bars: 100 μm (upper) or 20 μm (lower). (i-k) Statistical analysis of GAD67+ (i), Gephyrin+ (j), and GAD67+/Gephyrin+ synapse density (k) (N = 12 cells from 3 mice). * P < 0.05; *** P < 0.001; **** P < 0.0001; ns: not significant

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Next, we investigated the effect of CUL4B deficiency on synaptic structure via transmission electron microscopy (TEM) (Fig. 6a). Compared with those in control mice, there were no significant differences in the length of the active zone (Fig. 6b), the curvature of the synaptic interface (Fig. 6c) or the thickness of the postsynaptic density (PSD) (Fig. 6d) in the CA1 region of the hippocampus in CKO mice, but the width of the synaptic cleft was significantly greater in CKO mice (Fig. 6e), suggesting a reduction in signal transduction. The difference in the synaptic cleft mainly occurred in asymmetric synapses (Fig. 6f) but not in symmetric synapses (Fig. 6g). Moreover, the number of synaptic vesicles was lower in the CKO mice than in the CON mice (Fig. 6h). Next, we performed whole-cell voltage–clamp recordings of hippocampal pyramidal neurons freshly prepared from 8-week-old mice. We recorded evoked AMPA receptor-mediated EPSCs (AMPAR-EPSCs) at a holding potential of − 60 mV, which respond to incremental stimulus intensities applied at Schaffer collaterals. As the stimulus intensity increased, the AMPAR EPSC amplitude of CA1 pyramidal neurons in CKO hippocampal slices tended to decrease (Fig. 6i-k). The above results concerning activity-dependent synaptic responses suggested that basic excitatory synaptic transmission from CA3 to CA1 pyramidal neurons was decreased. Taken together, these results indicate that depletion of CUL4B in mice results in morphological and functional abnormalities in synapses.

Fig. 6
figure 6

CUL4B-deficient mice exhibit morphological and functional abnormalities in synapses. (a) Representative TEM images of the synaptic structure in the CA1 region of the hippocampus in control and CKO mice. (b-e) Quantification of the length of the active zones (b), the curvature of the synaptic interface (c), the thickness of the postsynaptic density (PSD) (d) and the width of the synaptic cleft (e) (N = 36 synapses from 4 CON mice and 42 synapses from 4 CKO mice). (f) Quantification of the width of the synaptic cleft in asymmetric synapses (N = 24 synapses from 4 CON mice and 27 synapses from 4 CKO mice). (g) Quantification of the width of the synaptic cleft in symmetric synapses (N = 12 synapses from 4 CON mice and 15 synapses from 4 CKO mice). (h) Quantification of the number of synaptic vesicles per synaptosome (N = 36 synapses from 4 CON mice and 42 synapses from 4 CKO mice). (i) Representative traces showing evoked AMPAR EPSCs in the CA1 pyramidal neurons of control and CKO mice. (j) Analysis of the input–output (I–O) relationship between AMPAR EPSCs and incremental stimulation intensities (in µA) (N = 12 cells from 4 mice). (k) Input/output slope of each cell (N = 12 cells from 4 mice). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant

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Depletion of CUL4B in the nervous system results in impaired spatial learning and memory

We then evaluated the effects of CUL4B depletion on spatial learning and memory via the Morris water maze. During the 5-day training period, the escape latency of each mouse from the edge of the pool to the submerged hidden platform was recorded. The escape latency of control and CKO mice decreased over time, indicating progressive learning effects. However, the escape latency of CKO mice was significantly longer than that of control mice, indicating impaired spatial learning ability in CKO mice (Fig. 7a). To evaluate spatial memory, the original submerged platform was removed on day 6, and the mice were subjected to a probe trial. Compared with control mice, CKO mice spent less time in the target quadrant, in which the platform was originally placed (Fig. 7b-c), and crossed the original platform cycle less frequently (Fig. 7d), indicating that the spatial memory of CKO mice was deficient. Both CKO and control mice had comparable swimming velocities (Fig. 7e) and swimming distances (Fig. 7f), indicating that the impaired spatial learning and memory in CKO mice were not due to altered motor function. Next, the Y-maze was used to evaluate the spatial working memory of the mice (Fig. 7g). There was no difference in the number of total entries between the CKO and control mice (Fig. 7h). However, compared with control mice, CKO mice presented a notable decrease in the alternation ratio (Fig. 7i), indicating impaired spatial working memory in CKO mice. These results suggest that depletion of CUL4B in the nervous system results in impaired spatial learning and memory.

Fig. 7
figure 7

Depletion of CUL4B in the nervous system results in impaired spatial learning and memory. (a) Escape latency (time spent prior to finding the platform) was measured during 5 consecutive days of training in the Morris water maze test (N = 10 mice). (b) Representative traces of the real path of the probe trail in the Morris water maze test. (c) The movement time spent in the target quadrant, in which the platform was originally placed, in the probe trial (N = 10 mice). (d) The number of times the mice crossed the circle where the platform was originally placed (N = 10 mice). (e) Swimming velocity of the mice in the Morris water maze test (N = 10 mice). (f) Swimming distance of the mice in the Morris water maze test (N = 10 mice). (g) Activity trajectories of the mice in the Y maze monitored by an infrared video recorder within 8 min. (h) The number of total entries of the mice into the arms of the Y maze (N = 8 mice for CON and 12 mice for CKO). (i) The percentage of alternation ratio of the mice that traveled in the Y maze (N = 8 mice for CON, and 12 mice for CKO). * P < 0.05; ** P < 0.01; **** P < 0.0001; ns, not significant

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On the other hand, the anxiety and depression-related behaviors of CKO mice were evaluated via the open field test, elevated zero maze, and tail suspension test. The open field test (Additional file 1: Fig. S5a) revealed that the total distance traveled (Additional file 1: Fig. S5b) and the maximum speed (Additional file 1: Fig. S5c) of CKO mice were comparable to those of littermate controls, suggesting that CKO mice exhibited normal locomotor activity. The two groups showed no marked differences in the territories (center vs. periphery) they explored (Additional file 1: Fig. S5d-i), suggesting that CKO mice did not experience increased anxiety or depression. The elevated zero maze test revealed that there were no differences in total distance traveled, open arm duration or number of open arm entries between CKO and control mice (Additional file 1: Fig. S5j-m). The tail suspension test also revealed similar struggling time and immobility time between the two genotypes (Additional file 1: Fig. S5n-p). These results indicate that depletion of CUL4B in the nervous system does not result in anxiety or depression in mice.

Discussion

In this study, we utilized multiple methods, including snRNA-seq, morphology, electrophysiology and behavior tests, to investigate the possible mechanism of intellectual disability caused by CUL4B mutation. We demonstrated the important role of CUL4B in synaptic function and memory, providing a potential drug target for the treatment of synaptic dysfunction and memory loss in individuals with intellectual disability.

First, we performed comprehensive single-nucleus transcriptional analyses of the hippocampus in mice, substantially extending previous observations and providing important insights into CUL4B function. SnRNA-seq is currently the optimal method for single-cell transcriptomic profiling of the diversity of cell types in the adult mammalian brain [27]. The results of snRNA-seq analysis revealed that depletion of CUL4B resulted in impaired intercellular communication. CKO mice lose some important communication pathways, which may contribute to impaired spatial memory and defects in synapses. For example, TGF-β1 is a crucial regulator of brain homeostasis, memory formation and neuronal plasticity [28]. The increase in TGF-β1 in the prefrontal cortex may be involved in memory consolidation [29]. Increasing the level of TGF-β1 in the hippocampal CA1 region can also increase synaptic stability by increasing the distribution of AMPA [30]. Loss of TGF-β2 could lead to impairment of central synapse function [31]. The TGFβ interaction pathway is significantly absent in the mGC and CA neurons of Cul4b CKO mice, which may lead to synaptic defects and make it difficult to switch from short-term memory to long-term memory. THBS1 is an essential astrocyte-derived synaptogenesis-promoting factor [32], that regulates synaptic plasticity and inhibits microglial activation [33]. VISFATIN can reduce hippocampal CA1 cell death [34] and is necessary for synaptic integrity and development [35]. Tenascin C is a key molecule in the DG and CA regions that maintains neuronal plasticity and can promote the formation of Perineural networks (PNNs) and the synaptogenesis of excitatory and inhibitory synapses [36]. Tenascin-R is expressed by oligodendrocytes and small interneurons in the hippocampus and is a mediator of neuron-glia interactions, contributing to the formation and maintenance of synapses during development of the hippocampus [37]. Loss of Tenascin-R impairs cognition, synaptic plasticity and motor ability in mice [38]. Compared with those in CON mice, neurons in the hippocampus of Cul4b CKO mice (including mGC, CA and GABA) lack THBS, VISFATIN and TENASCIN interaction pathways, which may severely affect the number, plasticity and function of synapses during development and subsequently cause mental retardation and other phenotypes.

The results of snRNA-seq analysis also revealed that depletion of CUL4B resulted in elicited cell type-specific transcriptional changes toward defects in axons, dendrites and synapses. Considering the important role of CUL4B in transcriptional repression, the altered transcriptional profile may be directly or indirectly regulated by CUL4B or the result of compensatory mechanisms. Future investigations will focus on exploring the target gene of CUL4B, which plays important roles in memory and synaptic function. For example, PISD functions in phospholipid metabolism, and it is a key molecule related to both global developmental delay and intellectual disability [39]. In liberfarb patients, loss of PISD function leads to intellectual disability [40]. In another model of mental retardation, the expression of PISD was significantly up-regulated, and the mice presented significantly impaired spatial memory, accompanied by abnormal dendritic spine morphology [41]. As CUL4B plays important roles in both protein degradation and transcriptional inhibition, the altered gene expression profile may partially explain the phenotypes. The protein degradation substrates of CUL4B may also play important roles in these processes.

The results of follow-up experiments further confirmed the above findings of synaptic dysfunction, including decreased dendritic spine density, increased width of the synaptic cleft and a decrease in AMPAR EPSC amplitude in CKO mice. We showed that the dendritic spine density in the hippocampus was significantly reduced in CKO mice, and fewer mushroom spines and more stubby spines were noted in CKO mice. The synaptic density reflects the degree of neurotransmission at synapses [42]. Dendritic spines are the most important postsynaptic compartment of excitatory neurons in the brain and are essential for synaptic transmission and plasticity [43,44,45]. Dendritic spine abnormalities may lead to synapse loss and affect synaptic function. Stubby spines are thought to represent an immature type, as they tend to disappear during development [46, 47], whereas mushroom spines are responsible for information transmission and learning in adults [44, 48]. Therefore, the lower percentage of mushroom spines and higher percentage of stubby spines may be related to decreased synaptic transmission.

Moreover, the TEM results revealed synaptic ultrastructure alterations, including an increase in the width of the synaptic cleft. Synaptic cleft widening is strongly associated with a reduction in signal transduction [49]. The difference in the synaptic cleft mainly occurs in asymmetric synapses but not in symmetric synapses, which is consistent with the decreased AMPAR EPSC amplitude of CA1 pyramidal neurons. Indeed, the decrease in neurotransmission at synapses was confirmed by electrophysiological experiments. In individuals with intellectual disability, behavioral abnormalities such as learning and memory defects are usually associated with alterations in synaptic transmission and abnormal spine density [50, 51]. Defects in spines are believed to lead to impaired synaptic maturation, which serves as pivotal signal processing units essential for higher brain function. Consequently, this may contribute to the cognitive deficits observed in individuals suffering from this debilitating condition [52].

Patients with CUL4B mutations exhibit severe intellectual disability [3,4,5,6]. Consistent with patients with CUL4B mutations, CKO mice presented impaired spatial learning and memory in the Morris water maze and Y maze. These phenotypes resemble those of a mouse model in which Cul4b was knocked out in epiblasts [18]. Nonetheless, the specific deletion of Cul4b in the nervous system described in this report may exclude systemic effects from other tissues. Our findings suggest that CUL4B may influence learning and memory by modulating synaptic structure and function, underscoring the pivotal role of synapses and spines in the pathophysiological mechanism underlying intellectual disability.

Conclusions

In summary, our study demonstrated that the X-linked intellectual disability gene CUL4B is critical for memory and synaptic function. The findings of this study reveal the pathogenesis of neurological disorders associated with CUL4B mutations and promote the identification of therapeutic targets that can halt synaptic abnormalities and preserve memory in individuals.

Materials and methods

Animals

Cul4b floxed mice were generated at the Model Animal Research Center of Nanjing University as previously reported [13]. To produce conditional knockout mice in which the Cul4b gene was specifically deleted in brain tissue, Cul4bflox/flox female mice were crossed with Nestin-Cre transgenic male mice (The Jackson Laboratory, 003771), in which Cre recombinase was under the control of the promoter and enhancer of rat nestin, which is expressed primarily in the nervous system [53]. These crosses are expected to yield conditional knockout male mice (Nestin-Cre+/−;Cul4bflox/Y, referred to as CKO), littermate control male mice (Nestin-Cre−/−;Cul4bflox/Y, referred to as CON), heterozygous female mice (Nestin-Cre+/−;Cul4bflox/+) and control female mice (Nestin-Cre−/−;Cul4bflox/+) at a ratio of 1:1:1:1. All the mice were on the C57BL/6J background. All the mice were housed in a specific-pathogen-free facility at 22 °C to 24 °C and 40–70% humidity with 15 times/h ventilation, noise less than or equal to 60 dB, and 12/12 h light–dark cycles. All animal care and experiments were approved by the Animal Care and Use Committee of Shandong University School of Basic Medical Sciences (No. ECSBMSSDU2023-2-27).

Genotyping

Genomic DNA was extracted from tails and used for genotyping via PCR analysis. For the genotyping of Cul4b floxed mice, the primers 5’-ACAGGTATTTGCCAGTGCTGTC-3’ and 5’-TTCTGTTACCTTCCTACCGAGAG-3’ were used to amplify the Cul4b floxed allele (501 bp) and the wild-type allele (383 bp). For the genotyping of Nestin-Cre transgenic mice, the primers 5′-CCCGCAGAACCTGAAGATG-3′ and 5′-GACCCGGCAAAACAGGTAG-3′ were used for detection of the Cre allele (534 bp).

Single-nucleus RNA sequencing

To reduce noise stemming from individual differences, three whole hippocampi were pooled into each biological replicate, with one replicate for the CON and CKO conditions. Nuclei were extracted via the BOAOJINGDIAN Nuclei Extraction Kit according to the manufacturer’s instructions with the following modifications: for each sample, the hippocampus was dissected from the animal and rinsed in cold PBS buffer. Fresh tissue was homogenized in a Dounce homogenizer in lysis buffer with 1 mM DTT and 1 U/µL RNase inhibitor and incubated on ice for 5 min. The suspension was filtered through a 40 μm filter to remove debris and centrifuged at 500 × g at 4 °C for 5 min. After the nuclei were resuspended in 300 µL of lysis buffer and 300 µL of RB buffer in a 2 mL tube, the mixture was centrifuged with a density gradient to separate the nuclei from the cell debris. The intermediate layer was collected and washed with RB buffer. Finally, the nuclei were counted via a hemocytometer, and 10,000 cells per sample were loaded onto the Chromium Single Cell 3′ Chip (10x Genomics) and processed with the Chromium Controller (10x Genomics). CON and CKO samples were prepared via the Chromium Single Cell 3′ Library & Gel Bead Kit V3.1 according to the manufacturer’s instructions. The samples were sequenced at GENEWIZ Inc. on an Illumina HiSeq platform, with a target of more than 500 million reads per sample. The sequencing work was conducted by the BOAOJINGDIAN company.

Quality control, data processing and analysis

We performed sequence alignment to the mm10 genome (2020) via CellRanger software (cellranger/6.0.0) from 10x Genomics. The resulting feature–barcode matrices were processed via R (version 4.1.0), excluding any nuclei expressing fewer than 200 or more than 5000 genes, and excluding any gene expressed in fewer than three nuclei. Filtering and visualization were performed via Seurat (4.3.0) [54]. Similarly, nuclei with greater than 10% mitochondrial mapping were removed, resulting in 11,270 nuclei in the CKO condition and 11,724 nuclei in the CON condition. The datasets were integrated via the IntegrateData function on 2000 variable features. The number of nuclei, unique molecular identifiers, and unique genes per sample are reported in Supplementary Fig. 2. We identified and annotated the major cell types of the mouse brain by interrogating the expression patterns of known marker genes. Differential expression analysis was performed via MAST (1.18.0) [55], with random effects for sequencing depth and sample origin. Genes were considered significant if the adjusted p value was less than 0.05 and if the log2 fold change was greater than 0.25 or less than − 0.25. A volcano plot was generated with EnhancedVolcano (https://github.com/kevinblighe/EnhancedVolcano), and an UpSet plot was generated with UpSetR [56]. Other bar or dot plots were visualized via ggplot2 [56].

Gene set enrichment analysis

Gene Set Enrichment Analysis (GSEA) was performed via the fgsea package (1.18.0) [57] via the Hallmark gene set list, GO biological process (BP) gene set list and KEGG gene set list from MSigDB (version 7.2.) [58]. For each cluster, genes were ranked by log2 fold change, and the analysis was performed via the fgseaMultilevel command with default settings and a seed set at 1000.

Cell–to–cell ligand–receptor interaction analysis

The “CellChat” package was used to predict and visualize biologically significant intercellular communication [59] and was run with default parameters. Briefly, a CellChat object was made via an annotated Seurat object via the createCellChat function. identifyOverExpressedInteractions was applied to conduct a differential interaction analysis of the ligand and receptor pairs, and the computeCommunProb function was applied to infer the communication probability. Intercellular communications of each cell signaling pathway were predicted with the computeCommunProbPathway function.

GWAS data and enrichment

The mouse gene symbols were converted to the corresponding human gene symbols via the biomaRt (v2.38.0) [59] package for comparison with the human dataset. We downloaded summary statistics of GWAS on cognitive traits and nonbrain disorders from the Psychiatric Genomics Consortium and the GIANT Consortium. We used MAGMA (v1.10) [60, 61] for genome-wide gene-based association analysis. We used the 19,427 protein-coding genes from human GENCODE v19 as a background for the gene-based association analysis. SNPs were selected within exonic, intronic, and UTR regions as well as within 10 kb up/downstream of the protein-coding gene. Gene-based association tests were performed using linkage disequilibrium between SNPs. The beta value (effect size) from a linkage disequilibrium model was calculated. Benjamin–Hochberg correction was applied, and significant enrichment was reported with a cutoff of P < 0.05. We used GWAS acronyms for the figures (AD [62] = Alzheimer’s disease, ADHD [62] = attention deficit hyperactivity disorder, ANX [63] = Anx disorders, ASD [64] = autism spectrum disorders, BMI [65] = body mass index, ED [66] = Anorexia nervosa, fEpilepsy [67] = focal epilepsy, fHS [67] = focal epilepsy with hippocampal sclerosis, gEpilepsy [67] = genetic generalized epilepsy, MDD [67] = major depressive disorder, tIntelligence [67] = intelligence, tCognFunc [68] = cognitive functions, PTSD [69] = posttraumatic stress disorder).

Bulk RNA-seq analysis of differentially expressed genes

The hippocampi of 6 CON mice and 7 CKO mice were dissected separately, and the lysates were used for RNA extraction and cDNA library construction. The sequencing was conducted by BGI Technology Company via the DNBseq platform. SOAPnuke [70], STAR [71] and DESeq2 [72] were used for data quality control, alignment and differentially expressed gene analysis across samples. Genes with a log2 fold change in expression of at least ± 0.58 and a p value < 0.05 were considered differentially expressed.

Functional enrichment analysis

WebGestaltR [73] was used to perform functional enrichment analysis of upregulated and downregulated genes in the CKO vs. CON comparison, resulting in lists of significantly enriched terms (adjusted p < 0.05 with Benjamin–Hochberg correction). We checked the gene set databases GO, Biological Process and KEGG, and visualized the 10 related terms in the bar plot.

Golgi-cox staining

The animals were anesthetized with tribromoethanol and decapitated, and the mouse brain tissue was directly placed in Golgi stain fixing solution. We replaced the Golgi dye solution, completely immersed the tissue blocks, and placed them in a cool and ventilated place at 26℃ away from light for 14 days (after soaking for 48 h, the new dye solution was changed once, and then the new dye solution was changed every 3 days). After the samples were dyed, the tissue treatment solution was changed for 1 h, and then the tissue treatment solution was changed once at 4℃ in the dark. Slices (60 μm thick) were cut on a microslicer (VT-1000 S, Leica) in the tissue treatment solution, followed by 30 min of Golgi developer solution.

Neuronal culture and immunostaining

The forebrains of E18.5 embryos were dissected and dissociated via TrypLE (Invitrogen) digestion and pipetting. The samples were subsequently resuspended in neurobasal medium supplemented with L-glutamate and B-27 (Invitrogen). The cells were plated at 30,000 cells per well in 24-well plates with cover glasses and cultured at 37 °C with 5% CO2. Twenty-four-well plates and cover glasses were coated with poly-L-lysine before use. Double-staining analysis of colocalization was conducted on DIV14 neurons. The primary antibodies used included rabbit anti-Synapsin-1 (CST, 1:200), mouse anti-PSD95 (Synaptic Systems, 1:50), mouse anti-GAD67 (Synaptic Systems, 1:400) and rabbit anti-Gephyrin (Synaptic Systems, 1:500) antibodies. The secondary antibodies used included goat anti-mouse (or rabbit) TRITC or FITC (Jackson ImmunoResearch; 1:200). Negative controls were obtained by substituting the primary antibody with normal serum. Images were acquired on a Zeiss LSM780 laser-scanning confocal microscope. The number of positive synapses within 100 μm of the dendritic length from the soma was counted. ImageJ was used to quantify the number of colocalized Synapsin-1/PSD95 or GAD67/Gephyrin puncta and the synapse density of colocalized puncta.

Electron microscopic imaging

Anesthetized mice were perfused with saline and 4% PFA in PBS buffer containing 0.075% glutaraldehyde. The brains were quickly removed, and coronal Sect. (1 mm) were cut within 1–3 min by using a precooled mouse brain matrix. The brain fields of interest were subsequently extracted via a punch (1 mm in diameter). These punched tissues were immersed in 2.5% glutaraldehyde for 2 h postfixation at 4oC, followed by washing three times with 0.1 M phosphate buffer. The 2.5% glutaraldehyde was prepared by mixing 9 ml of 0.1 M phosphate buffer and 1 ml of 25% glutaraldehyde. The tissues were fixed with 1% OsO4 in 0.1 M phosphate buffer (pH 7.4) in the dark for 2 h at room temperature and then washed three times with 0.1 M phosphate buffer. Dehydrate, resin penetration and embedding were performed, and the samples were then moved into a 60℃ oven to polymerize for more than 48 h. The resin blocks were sectioned into 1.5 μm slices via a semithin microtome, subsequently stained with toluidine blue, and positioned under a light microscope for positioning. After ultrathin sectioning, the samples were stained in the dark for 8 min, and the cuprum grids were observed under a transmission electron microscopy.

Electrophysiology

Electrophysiological experiments were carried out on 8-week-old male CON and CKO mice [74]. Animals were anesthetized with tribromoethanol and decapitated, and hippocampal slices (300 μm thick) were cut on a microslicer (VT-1000 S, Leica) in ice-cold dissection solution (213 mM sucrose, 10 mM glucose, 3 mM KCl, 0.5 mM CaCl2, 5 mM MgCl2, 26 mM NaHCO3 and 1 mM NaH2PO4, pH 7.4). Following sectioning, the slices were kept at room temperature for > 1 h before being used for experiments in artificial cerebrospinal fluid (ACSF) solution (10 mM glucose, 125 mM NaCl, 5 mM KCl, 2.6 mM CaCl2, 1.3 mM MgCl2, 26 mM NaHCO3 and 1.2 mM NaH2PO4, pH 7.4).

For the voltage–clamp configuration, glass pipettes were filled with a solution (4 mM NaCl, 1 mM MgCl2, 10 mM CsCl, 130 mM Cs-methanesulfonate, 12 mM phosphocreatine, 10 mM HEPES, 5 mM Mg–ATP, 0.5 mM Na2–GTP, 5 mM EGTA, pH 7.2–7.3, approximately 265–270 mOsm) for sEPSC recording [75]. Evoked EPSCs (AMPAR-EPSCs) were recorded in the presence of 50 µM D-AP-5 and 100 µM picrotoxin. A concentric bipolar electrode (CBBEB75, Frederick Haer) triggered with an impulse isolator (A-M system 2100) was placed 100 μm away from the recording neuron in the CA1 strata radiatus in the same plane. To measure the input–output curves for pyramidal neurons, a sequential increase in stimulus strength from 10 µA to 100 µA was applied. Each stimulus was repeated 3 times at a stimulus interval of 15 s, and the average response under the stimulus strength was subjected to statistical analysis. All data sampling was set at 10 kHz for voltage-clamp. The data were analyzed with pClamp10.4 (Axon instruments).

Behavioral tests

Two-month-old mice were used in the behavioral tests. The behavioral tests were performed sequentially on the same set of mice in the following sequence: open field test (1 day), Y maze (1 day), elevated zero maze (1 day), and tail suspension test (1 day). The Morris water maze (6 days) was performed on another set of mice. The mice were housed under an inverted light/night cycle. To reduce circadian effects, behavioral tests were performed during the same time interval each day as much as possible.

Morris water maze

The Morris water maze was used to assess hippocampus-dependent spatial long-term learning and memory. The water maze consisted of a circular tank (diameter = 1.25 m; height = 70 cm) that was filled with tepid water (23 ± 1 °C) that was made opaque by the addition of powdered milk. The circular tank was divided into four equal quadrants, each with a spatial cue on the tank wall. A white escape platform (diameter = 10 cm, height = 10 cm) was located 1 cm below the water. First, the mice were trained for 5 consecutive days and underwent four training trials per day, for a total of 20 training trials, each starting at the same time of day. In each training trial, a mouse was placed in one of the quadrants and allowed to search for the hidden platform for 1 min. If the mouse did not find the platform within 1 min, the experimenter led the animal to it. After the platform was located, the mouse was left on it for 15 s to memorize the spatial cues. After that, the mouse was placed in a cage to rest before the next trial. Throughout the experiment, the platform remained at its original position. To assess learning ability, the latency to find the platform in each trial was recorded. Finally, on day 6, a probe trial was administered: the platform was removed, the mouse was placed in the opposite quadrant, and the time spent in each quadrant within 1 min was measured. TopScan video tracking software (V.3.0, CleverSys Inc., USA) was used to record and analyze the data.

Y-maze

The apparatus consisted of three enclosed arms (L×W×H: 30 × 6 × 15 cm). The mice were placed in a random arm of the maze and allowed to explore it for 8 min with all three arms open. Trials were recorded via an overhead camera, and video files were scored manually by an observer who was blinded to the genotype. One alternation was counted when the mice visited the three different arms consecutively. Immediate re-entries were discounted. The percentage of alternations (i.e., the number of alternations divided by the total possible alternations and multiplied by 100) was used as a measure of spatial working memory and executive function. Once a test was completed, we carefully removed the mouse from the maze and proceeded to clean the maze using 30% ethanol followed by distilled water. SMART video tracking software (V.3.0, Panlab, USA) was used to record and analyze the data.

Open field test

The open field test was used to assess locomotion and anxiety-like behavior in the mice. The open field test was performed in a plastic apparatus (40 cm height × 40 cm width × 40 cm depth) with a 20 cm× 20 cm central zone. The total distance and maximum speed moved and the numbers of entries into the peripheral zone and central zone were recorded over a 10-minute test. The 30% ethanol was sprayed to clean the apparatus between each test to avoid odor and waste left by the last mouse. SMART video tracking software (V.3.0, Panlab, USA) was used to record and analyze the data.

Elevated zero maze

The mice were introduced into a circular elevated maze (50 cm diameter, 5 cm track width) with 30% enclosed (15 cm wall height) and 70% open arenas. The mice were allowed to freely explore the novel environment for 8 min. To prevent olfactory cue bias, the maze apparatus was thoroughly washed with 30% alcohol following each test. SMART video tracking software (V.3.0, Panlab, USA) was used to record and analyze the data.

Tail suspension test

The tail suspension test was performed in a white plastic chamber (30 cm height × 20 cm width × 20 cm depth). Each mouse was suspended from its tail tip with adhesive tape in a head-down position for 6 min. Immobility time was defined as the cessation of any movements of the limbs or the trunk. To avoid bias affected by the stress response, each mouse was allowed to adapt for 2 min after being suspended, and only the remaining 4 min were recorded and analyzed. SMART video tracking software (V.3.0, Panlab, USA) was used to record and analyze the data.

Immunoblotting

Hippocampal samples from 7- to 8-week-old mice were homogenized and lysed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, and protease inhibitor, pH 7.5). The supernatants were collected after centrifugation at 13,000 ×g and 4 °C for 30 min. The protein concentration was estimated via BCA assays (Vazemy). Then, the proteins were mixed with 5×SDS‒PAGE loading buffer (New Cell & Molecular Biotech) and boiled at 99 °C for 10 min. The samples were resolved via 7.5% SDS‒PAGE, followed by electrotransfer to polyvinylidene difluoride (PVDF) membranes (Millipore). For immunoblotting, blots were probed with antibodies against CUL4B (1:1000; Sigma; Cat# C9995; RRID: AB_1840781), beta-actin (1:1000; Proteintech; Cat# 66009-1-Ig; RRID: AB_2687938) and detected via peroxidase AffiniPure goat anti-rabbit IgG (H + L) (1:5000; Jackson ImmunoResearch; Cat# 111-035-003; RRID: AB_2313567) and peroxidase AffiniPure goat anti-mouse IgG (H + L) (1:5000; Jackson ImmunoResearch; Cat# 115-035-003; RRID: AB_10015289).

Statistical analysis

All the data are presented as the mean ± SEM. Statistical analysis was performed via GraphPad Prism 8.0. Statistical significance was determined via an unpaired two-tailed t test for two-sample comparisons or two-way ANOVA to examine the effect of two factors on a dependent variable. A P value < 0.05 was considered significant. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant.

Data availability

snRNA-seq data that support the findings of this study have been deposited in the China National Center for Bioinformation with the primary accession https://ngdc.cncb.ac.cn/omix/preview/p8qpZmh2; Bulk RNA-seq data that support the findings of this study have been deposited in the China National Center for Bioinformation with the primary accession https://ngdc.cncb.ac.cn/omix/preview/h90DuNwN.

Abbreviations

CRL4B:

CUL4B-RING E3 ubiquitin ligase

XLID:

X-Linked Intellectual Disability

snRNA-seq:

single-nucleus RNA sequencing

TEM:

Transmission Electron Microscopy

ID:

Intellectual Disability

UMAP:

Uniform Manifold Approximation and Projection

mGC:

mature Granule Cells

imGC:

immature Granule Cells

GSVA:

Gene Set Variation Analysis

MAST:

Model-Based Analysis of Single-Cell Transcriptomics

DEGs:

Differentially Expressed Genes

CV:

Coefficient of Variation

GSEA:

Gene Set Enrichment Analysis

BP:

Biological Process

RNA-seq:

RNA sequencing

ASC:

Astrocytes

MIC:

Microglia

GABA neurons:

GABAergic neurons

ODC:

Oligodendrocytes

OPC:

Oligodendrocyte Progenitor Cells

VLMC:

Vascular and Leptomeningeal Cells

AD:

Alzheimer’s Disease

ADHD:

Attention Deficit Hyperactivity Disorder

ANX:

Anx disorders

ASD:

Autism Spectrum Disorders

BMI:

Body Mass Index

ED:

Anorexia Nervosa

fEpilepsy:

Focal Epilepsy

fHS:

Focal Epilepsy with Hippocampal Sclerosis

gEpilepsy:

genetic Generalized Epilepsy

MDD:

Major Depressive Disorder

tIntelligence:

Intelligence

tCognFunc:

Cognitive Functions

PTSD:

Posttraumatic Stress Disorder

ACSF:

Artificial Cerebrospinal Fluid

MSigDB:

Molecular Signatures Database

FC:

Fold Change

NES:

Normalized Enrichment Score

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Acknowledgements

We thank the Translational Medicine Core Facility and the School of Basic Medical Sciences Core Facility of Shandong University for consultation and instrument availability.

Funding

This work was supported by grants from the Shandong Provincial Natural Science Foundation (ZR2022QH343) and the National Natural Science Foundation of China (32370652, 82171851, 31970559).

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Authors and Affiliations

  1. The Key Laboratory of Experimental Teratology of the Ministry of Education and Department of Genetics, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, China

    Wei Jiang, Jian Zhang, Molin Wang, Yongxin Zou, Qiao Liu, Yu Song, Yaoqin Gong & Baichun Jiang

  2. The Key Laboratory of Experimental Teratology of the Ministry of Education, Department of Histology and Embryology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, China

    Gongping Sun

  3. Medical Morphology Teaching Laboratory, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, China

    Fan Zhang

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  1. Wei Jiang
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Contributions

Y.G., B.J. and F.Z. conceived the study concept and design; W.J. performed most of the experiments; F.Z. analyzed the snRNA-Seq data; J.Z. performed the immunostaining experiments; M.W., Y.Z., Q.L., Y.S. and G.S. participated in the acquisition, analysis, and interpretation of the data; F.Z. and B.J. wrote and revised the manuscript.

Corresponding authors

Correspondence to Fan Zhang or Baichun Jiang.

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Ethics approval and consent to participate

All animal care and experiments were approved by the Animal Care and Use Committee of Shandong University School of Basic Medical Sciences (No. ECSBMSSDU2023-2-27).

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Not applicable.

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The authors declare no competing interests.

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Jiang, W., Zhang, J., Wang, M. et al. The X-linked intellectual disability gene CUL4B is critical for memory and synaptic function. acta neuropathol commun 12, 188 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s40478-024-01903-y

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Acta Neuropathologica Communications

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