NEK4 modulates circadian fluctuations of emotional behaviors and synaptogenesis in male mice.

GWASs have linked the 3p21.1 locus, which is associated with the expression levels of NEK4, to bipolar disorder. Here, we use integrative analyses of GWAS statistics and eQTL annotations to establish that elevated NEK4 expression in the hippocampus is associated with an increased risk of bipolar disorder. To further study this association, we generate transgenic male mice that conditionally overexpress NEK4 in the pyramidal neurons of the adult forebrain, or use AAV to overexpress NEK4 in the dorsal hippocampus. Compared to the control mice, male mice of both strains exhibit a shift from a diurnal anxiety state to a nocturnal normal or anxiolytic-like state. Overexpression of NEK4 also affects the circadian fluctuations in dendritic spine morphology and synaptic structure. Furthermore, we show that treatment with lithium ameliorates the effects of NEK4 overexpression in male mice. We then perform phosphoproteomic analyses to demonstrate that the diurnal and nocturnal phosphoproteomic profiles of male control and NEK4 overexpressing mice are different. These results suggest that male mice with different NEK4 expression levels may recapitulate some of the core features observed in patients with bipolar disorder, indicating that interruption of the homeostatic dynamics of synapses may underlie the emotional swings in bipolar disorder.

Genomic landscape and functional characterization of structural variations in schizophrenia and bipolar disorder.

Multiple types of variations have been postulated to confer risk of schizophrenia and bipolar disorder, but majority of present GWAS solely focused on SNPs or small indels, and the impacts of structural variations (SVs) remain less understood. Nevertheless, accumulating evidence suggest that SVs may explain the association signals in certain GWAS hits. Here, we conducted pairwise linkage disequilibrium (LD) analyses of SNPs and SVs in populations from 1000 Genomes Project. Among the 299 psychiatric GWAS loci, 1213 SVs showed an LD of r2 > 0.1 with GWAS risk SNPs, and 66 of them were in moderate to strong LD (r2 > 0.6) with at least one GWAS risk SNP. Nine SVs were subject to further explorative analyses, including eQTL analysis in DLPFC, luciferase reporter gene assays, CRISPR/Cas9-mediated genome deletion and RT-qPCR. These assays highlighted several functional SVs showing regulatory effects on transcriptional activities, and some risk genes (e.g., BORCS7, GNL3) affected by the SVs were also annotated. Finally, mice overexpressing Borcs7 in the mPFC exhibited schizophrenia-like behaviors, such as abnormal prepulse inhibition and social dysfunction. These data suggest that SNPs association signals at GWAS loci might be driven by SVs, highlighting the necessities of considering such variants in future.

Efficacy of Fire-Needle Therapy in Improving Neurological Function Following Cerebral Infarction and Its Effect on Intestinal Flora Metabolites.

Objective: This study was to investigate the mechanism of action and clinical efficacy of fire-needle therapy in improving neurological function in patients with acute cerebral infarction (identified as a wind-phlegm-blood stasis syndrome in traditional Chinese medicine). Methods: We included patients diagnosed with acute cerebral infarction (wind-phlegm-blood stasis syndrome) admitted to the Encephalopathy and Acupuncture Center of the Second Affiliated Hospital of Tianjin University of Chinese Medicine. We randomly allocated them into the treatment and control groups, with 45 cases in each group. Acupuncture treatments that focused on regulating the mind and dredging the collaterals were used in the control group, while the treatment group additionally received fire-needle therapy. Our indicators included the National Institutes of Health Stroke Scale (NIHSS) scores, the Fugl-Meyer Assessment (FMA) scale, peripheral blood tumor necrosis factor-α (TNF-α), interleukin-17 (IL-17), hypersensitivity C-reactive protein (hs-CRP), and intestinal metabolites short-chain fatty acids (SCFAs). We measured these indicators before treatment and 14 days after treatment. Results: The post-treatment NIHSS scores of the two groups were significantly reduced (P < 0.05), and the treatment group showed a more significant decline in the score when compared to the control group (P < 0.05). The treatment group showing significant improvement in the domains of reflex activity, mobility, cooperative movement, and finger movement (P < 0.05). Both groups showed a significant decrease in the IL-17 and hs-CRP levels (P < 0.05), with the treatment group demonstrating a significant declining trend when compared to the control group (P < 0.05). The levels of acetic acid, propionic acid, butyric acid, and valeric acid all increased significantly in the two groups (P < 0.05), with acetic acid and butyric acid increasing significantly in the treatment group when compared to the control group (P < 0.05). Clinical efficacy rate: 78.6% of patients in the treatment group had an excellent rate, whereas it was 30.0% in the control group, and the difference was statistically significant (P < 0.001). Conclusion: Fire-needle therapy was effective in upregulating the SCFA content in patients with acute cerebral infarction (wind-phlegm-blood stasis syndrome), inhibiting the level of the inflammatory response, and improving the recovery of neurological functions. Clinical registration number: Registration website link: https://www.chictr.org.cn. Registration date: 2022/9/27. Registration number: ChiCTR2200064122.

Shared genetic architecture and causal relationship between sleep behaviors and lifespan.

Poor sleep health is associated with a wide array of increased risk for cardiovascular, metabolic and mental health problems as well as all-cause mortality in observational studies, suggesting potential links between sleep health and lifespan. However, it has yet to be determined whether sleep health is genetically or/and causally associated with lifespan. In this study, we firstly studied the genome-wide genetic association between four sleep behaviors (short sleep duration, long sleep duration, insomnia, and sleep chronotype) and lifespan using GWAS summary statistics, and both sleep duration time and insomnia were negatively correlated with lifespan. Then, two-sample Mendelian randomization (MR) and multivariable MR analyses were applied to explore the causal effects between sleep behaviors and lifespan. We found that genetically predicted short sleep duration was causally and negatively associated with lifespan in univariable and multivariable MR analyses, and this effect was partially mediated by coronary artery disease (CAD), type 2 diabetes (T2D) and depression. In contrast, we found that insomnia had no causal effects on lifespan. Our results further confirmed the negative effects of short sleep duration on lifespan and suggested that extension of sleep may benefit the physical health of individuals with sleep loss. Further attention should be given to such public health issues.

Reduced Vrk2 expression is associated with higher risk of depression in humans and mediates depressive-like behaviors in mice.

BACKGROUND: Genome-wide association studies (GWAS) have reported single-nucleotide polymorphisms (SNPs) in the VRK serine/threonine kinase 2 gene (VRK2) showing genome-wide significant associations with major depression, but the regulation effect of the risk SNPs on VRK2 as well as their roles in the illness are yet to be elucidated. METHODS: Based on the summary statistics of major depression GWAS, we conducted population genetic analyses, epigenome bioinformatics analyses, dual luciferase reporter assays, and expression quantitative trait loci (eQTL) analyses to identify the functional SNPs regulating VRK2; we also carried out behavioral assessments, dendritic spine morphological analyses, and phosphorylated 4D-label-free quantitative proteomics analyses in mice with Vrk2 repression. RESULTS: We identified a SNP rs2678907 located in the 5' upstream of VRK2 gene exhibiting large spatial overlap with enhancer regulatory marks in human neural cells and brain tissues. Using luciferase reporter gene assays and eQTL analyses, the depression risk allele of rs2678907 decreased enhancer activities and predicted lower VRK2 mRNA expression, which is consistent with the observations of reduced VRK2 level in the patients with major depression compared with controls. Notably, Vrk2-/- mice exhibited depressive-like behaviors compared to Vrk2+/+ mice and specifically repressing Vrk2 in the ventral hippocampus using adeno-associated virus (AAV) lead to consistent and even stronger depressive-like behaviors in mice. Compared with Vrk2+/+ mice, the density of mushroom and thin spines in the ventral hippocampus was significantly altered in Vrk2-/- mice, which is in line with the phosphoproteomic analyses showing dysregulated synapse-associated proteins and pathways in Vrk2-/- mice. CONCLUSIONS: Vrk2 deficiency mice showed behavioral abnormalities that mimic human depressive phenotypes, which may serve as a useful murine model for studying the pathophysiology of depression.

Identification of a psychiatric risk gene NISCH at 3p21.1 GWAS locus mediating dendritic spine morphogenesis and cognitive function.

BACKGROUND: Schizophrenia and bipolar disorder (BD) are believed to share clinical symptoms, genetic risk, etiological factors, and pathogenic mechanisms. We previously reported that single nucleotide polymorphisms spanning chromosome 3p21.1 showed significant associations with both schizophrenia and BD, and a risk SNP rs2251219 was in linkage disequilibrium with a human specific Alu polymorphism rs71052682, which showed enhancer effects on transcriptional activities using luciferase reporter assays in U251 and U87MG cells. METHODS: CRISPR/Cas9-directed genome editing, real-time quantitative PCR, and public Hi-C data were utilized to investigate the correlation between the Alu polymorphism rs71052682 and NISCH. Primary neuronal culture, immunofluorescence staining, co-immunoprecipitation, lentiviral vector production, intracranial stereotaxic injection, behavioral assessment, and drug treatment were used to examine the physiological impacts of Nischarin (encoded by NISCH). RESULTS: Deleting the Alu sequence in U251 and U87MG cells reduced mRNA expression of NISCH, the gene locates 180 kb from rs71052682, and Hi-C data in brain tissues confirmed the extensive chromatin contacts. These data suggested that the genetic risk of schizophrenia and BD predicted elevated NISCH expression, which was also consistent with the observed higher NISCH mRNA levels in the brain tissues from psychiatric patients compared with controls. We then found that overexpression of NISCH resulted in a significantly decreased density of mushroom dendritic spines with a simultaneously increased density of thin dendritic spines in primary cultured neurons. Intriguingly, elevated expression of this gene in mice also led to impaired spatial working memory in the Y-maze. Given that Nischarin is the target of anti-hypertensive agents clonidine and tizanidine, which have shown therapeutic effects in patients with schizophrenia and patients with BD in preliminary clinical trials, we demonstrated that treatment with those antihypertensive drugs could reduce NISCH mRNA expression and rescue the impaired working memory in mice. CONCLUSIONS: We identify a psychiatric risk gene NISCH at 3p21.1 GWAS locus influencing dendritic spine morphogenesis and cognitive function, and Nischarin may have potentials for future therapeutic development.

Genetic associations between bipolar disorder and brain structural phenotypes.

Patients with bipolar disorder (BD) and their first-degree relatives exhibit alterations in brain volume and cortical structure, whereas the underlying genetic mechanisms remain unclear. In this study, based on the published genome-wide association studies (GWAS), the extent of polygenic overlap between BD and 15 brain structural phenotypes was investigated using linkage disequilibrium score regression and MiXeR tool, and the shared genomic loci were discovered by conjunctional false discovery rate (conjFDR) and expression quantitative trait loci (eQTL) analyses. MiXeR estimated the overall measure of polygenic overlap between BD and brain structural phenotypes as 4-53% on a 0-100% scale (as quantified by the Dice coefficient). Subsequent conjFDR analyses identified 54 independent loci (71 risk single-nucleotide polymorphisms) jointly associated with BD and brain structural phenotypes with a conjFDR < 0.05, among which 33 were novel that had not been reported in the previous BD GWAS. Follow-up eQTL analyses in respective brain regions both confirmed well-known risk genes (e.g. CACNA1C, NEK4, GNL3, MAPK3) and discovered novel risk genes (e.g. LIMK2 and CAMK2N2). This study indicates a substantial shared genetic basis between BD and brain structural phenotypes, and provides novel insights into the developmental origin of BD and related biological mechanisms.

Genetic Insights of Schizophrenia via Single Cell RNA-Sequencing Analyses.

BACKGROUND: Schizophrenia is a complex and heterogeneous disorder involving multiple regions and types of cells in the brain. Despite rapid progress made by genome-wide association studies (GWAS) of schizophrenia, the mechanisms of the illness underlying the GWAS significant loci remain less clear. STUDY DESIGN: We investigated schizophrenia risk genes using summary-data-based Mendelian randomization based on single-cell sequencing data, and explored the types of brain cells involved in schizophrenia through the expression weighted cell-type enrichment analysis. RESULTS: We identified 54 schizophrenia risk genes (two-thirds of these genes were not identified using sequencing data of bulk tissues) using single-cell RNA-sequencing data. Further cell type enrichment analysis showed that schizophrenia risk genes were highly expressed in excitatory neurons and caudal ganglionic eminence interneurons, suggesting putative roles of these cells in the pathogenesis of schizophrenia. We also found that these risk genes identified using single-cell sequencing results could form a large protein-protein interaction network with genes affected by disease-causing rare variants. CONCLUSIONS: Through integrative analyses using expression data at single-cell levels, we identified 54 risk genes associated with schizophrenia. Notably, many of these genes were only identified using single-cell RNA-sequencing data, and their altered expression levels in particular types of cells, rather than in the bulk tissues, were related to the increased risk of schizophrenia. Our results provide novel insight into the biological mechanisms of schizophrenia, and future single-cell studies are necessary to further facilitate the understanding of the disorder.

Bidirectional genetic overlap between bipolar disorder and intelligence.

BACKGROUND: Bipolar disorder (BD) is a highly heritable psychiatric illness exhibiting substantial correlation with intelligence. METHODS: To investigate the shared genetic signatures between BD and intelligence, we utilized the summary statistics from genome-wide association studies (GWAS) to conduct the bivariate causal mixture model (MiXeR) and conjunctional false discovery rate (conjFDR) analyses. Subsequent expression quantitative trait loci (eQTL) mapping in human brain and enrichment analyses were also performed. RESULTS: Analysis with MiXeR suggested that approximately 10.3K variants could influence intelligence, among which 7.6K variants were correlated with the risk of BD (Dice: 0.80), and 47% of these variants predicted BD risk and intelligence in consistent allelic directions. The conjFDR analysis identified 37 distinct genomic loci that were jointly associated with BD and intelligence with a conjFDR < 0.01, and 16 loci (43%) had the same directions of allelic effects in both phenotypes. Brain eQTL analyses found that genes affected by the "concordant loci" were distinct from those modulated by the "discordant loci". Enrichment analyses suggested that genes related to the "concordant loci" were significantly enriched in pathways/phenotypes related with synapses and sleep quality, whereas genes associated with the "discordant loci" were enriched in pathways related to cell adhesion, calcium ion binding, and abnormal emotional phenotypes. CONCLUSIONS: We confirmed the polygenic overlap with mixed directions of allelic effects between BD and intelligence and identified multiple genomic loci and risk genes. This study provides hints for the mesoscopic phenotypes of BD and relevant biological mechanisms, promoting the knowledge of the genetic and phenotypic heterogeneity of BD. The essential value of leveraging intelligence in BD investigations is also highlighted.

Genetic similarities and differences among distinct definitions of depression.

Depression is a common and complex psychiatric illness with considerable heritability. Genome-wide association studies (GWAS) have been conducted among different definitions of depression based on different diagnostic criteria. However, the heritability explained by different depression GWAS and the identified loci varied widely. To understand the genetic architectures of different definitions of depression, we conducted a series of genetic analyses including linkage disequilibrium score regression (LDSC), Mendelian randomization, and polygenic overlap quantification and identification. Different definitions of depression and other common psychiatric traits were included in this analysis. We found that although genetic correlations between different definitions of depression were relatively high, they showed substantially different genetic correlation and causality with other psychiatric traits. Using bivariate causal mixture mode (MiXeR) and conjunctional false discovery rate (conjFDR) approach, we observed both shared and unique risk loci across different definitions of depression. Further functional mapping with expression quantitative trait loci (eQTL) information from multiple brain tissues and single cell types indicated distinct genes underlying different definitions of depression, and pathways associated with synapses were significantly enriched in the illness. Our study showed that the genetic architectures of different definitions of depression were distinct and genetic studies of depression should be conducted more cautious.

Revisiting tandem repeats in psychiatric disorders from perspectives of genetics, physiology, and brain evolution.

Genome-wide association studies (GWASs) have revealed substantial genetic components comprised of single nucleotide polymorphisms (SNPs) in the heritable risk of psychiatric disorders. However, genetic risk factors not covered by GWAS also play pivotal roles in these illnesses. Tandem repeats, which are likely functional but frequently overlooked by GWAS, may account for an important proportion in the "missing heritability" of psychiatric disorders. Despite difficulties in characterizing and quantifying tandem repeats in the genome, studies have been carried out in an attempt to describe impact of tandem repeats on gene regulation and human phenotypes. In this review, we have introduced recent research progress regarding the genomic distribution and regulatory mechanisms of tandem repeats. We have also summarized the current knowledge of the genetic architecture and biological underpinnings of psychiatric disorders brought by studies of tandem repeats. These findings suggest that tandem repeats, in candidate psychiatric risk genes or in different levels of linkage disequilibrium (LD) with psychiatric GWAS SNPs and haplotypes, may modulate biological phenotypes related to psychiatric disorders (e.g., cognitive function and brain physiology) through regulating alternative splicing, promoter activity, enhancer activity and so on. In addition, many tandem repeats undergo tight natural selection in the human lineage, and likely exert crucial roles in human brain evolution. Taken together, the putative roles of tandem repeats in the pathogenesis of psychiatric disorders is strongly implicated, and using examples from previous literatures, we wish to call for further attention to tandem repeats in the post-GWAS era of psychiatric disorders.

An alternative splicing hypothesis for neuropathology of schizophrenia: evidence from studies on historical candidate genes and multi-omics data.

Alternative splicing of schizophrenia risk genes, such as DRD2, GRM3, and DISC1, has been extensively described. Nevertheless, the alternative splicing characteristics of the growing number of schizophrenia risk genes identified through genetic analyses remain relatively opaque. Recently, transcriptomic analyses in human brains based on short-read RNA-sequencing have discovered many "local splicing" events (e.g., exon skipping junctions) associated with genetic risk of schizophrenia, and further molecular characterizations have identified novel spliced isoforms, such as AS3MTd2d3 and ZNF804AE3E4. In addition, long-read sequencing analyses of schizophrenia risk genes (e.g., CACNA1C and NRXN1) have revealed multiple previously unannotated brain-abundant isoforms with therapeutic potentials, and functional analyses of KCNH2-3.1 and Ube3a1 have provided examples for investigating such spliced isoforms in vitro and in vivo. These findings suggest that alternative splicing may be an essential molecular mechanism underlying genetic risk of schizophrenia, however, the incomplete annotations of human brain transcriptomes might have limited our understanding of schizophrenia pathogenesis, and further efforts to elucidate these transcriptional characteristics are urgently needed to gain insights into the illness-correlated brain physiology and pathology as well as to translate genetic discoveries into novel therapeutic targets.

Identification of a Risk Locus at 7p22.3 for Schizophrenia and Bipolar Disorder in East Asian Populations.

Background: Shared psychopathological features and mechanisms have been observed between schizophrenia (SZ) and bipolar disorder (BD), but their common risk genes and full genetic architectures remain to be fully characterized. The genome-wide association study (GWAS) datasets offer the opportunity to explore this scientific question using combined genetic data from enormous samples, ultimately allowing a better understanding of the onset and development of these illnesses. Methods: We have herein performed a genome-wide meta-analysis in two GWAS datasets of SZ and BD respectively (24,600 cases and 40,012 controls in total, discovery sample), followed by replication analyses in an independent sample of 4,918 SZ cases and 5,506 controls of Han Chinese origin (replication sample). The risk SNPs were then explored for their correlations with mRNA expression of nearby genes in multiple expression quantitative trait loci (eQTL) datasets. Results: The single nucleotide polymorphisms (SNPs) rs1637749 and rs3800908 at 7p22.3 region were significant in both discovery and replication samples, and exhibited genome-wide significant associations when combining all East Asian SZ and BD samples (29,518 cases and 45,518 controls). The risk SNPs were also significant in GWAS of SZ and BD among Europeans. Both risk SNPs significantly predicted lower expression of MRM2 in the whole blood and brain samples in multiple datasets, which was consistent with its reduced mRNA level in the brains of SZ patients compared with normal controls. The risk SNPs were also associated with MAD1L1 expression in the whole blood sample. Discussion: We have identified a novel genome-wide risk locus associated with SZ and BD in East Asians, adding further support for the putative common genetic risk of the two illnesses. Our study also highlights the necessity and importance of mining public datasets to explore risk genes for complex psychiatric diseases.

Independent replications and integrative analyses confirm TRANK1 as a susceptibility gene for bipolar disorder.

Genetic analyses for bipolar disorder (BD) have achieved prominent success in Europeans in recent years, whereas its genetic basis in other populations remains relatively less understood. We herein report that the leading risk locus for BD in European genome-wide association studies (GWAS), the single-nucleotide polymorphism (SNP) rs9834970 near TRANK1 at 3p22 region, is also genome-wide significantly associated with BD in a meta-analysis of four independent East Asian samples including 5748 cases and 65,361 controls (p = 2.27 × 10-8, odds ratio = 1.136). Expression quantitative trait loci (eQTL) analyses and summary data-based Mendelian randomization (SMR) analyses in multiple human brain samples suggest that lower TRANK1 mRNA expression is a principal BD risk factor explaining its genetic risk signals at 3p22. We also identified another SNP rs4789 in the 3' untranslated region (3'UTR) of TRANK1 showing stronger eQTL associations as well as genome-wide significant association with BD. Despite the relatively unclear neuronal function of TRANK1, our mRNA expression analyses in the human brains and in rat primary cortical neurons reveal that genes highly correlated with TRANK1 are significantly enriched in the biological processes related to dendritic spine, synaptic plasticity, axon guidance and circadian entrainment, and are also more likely to exhibit strong associations in psychiatric GWAS (e.g., the CACNA1C gene). Overall, our results support that TRANK1 is a potential BD risk gene. Further studies elucidating its roles in this illness are needed.

Novel Risk Loci Associated With Genetic Risk for Bipolar Disorder Among Han Chinese Individuals: A Genome-Wide Association Study and Meta-analysis.

Importance: The genetic basis of bipolar disorder (BD) in Han Chinese individuals is not fully understood. Objective: To explore the genetic basis of BD in the Han Chinese population. Design, Setting, and Participants: A genome-wide association study (GWAS), followed by independent replication, was conducted to identify BD risk loci in Han Chinese individuals. Individuals with BD were diagnosed based on DSM-IV criteria and had no history of schizophrenia, mental retardation, or substance dependence; individuals without any personal or family history of mental illnesses, including BD, were included as control participants. In total, discovery samples from 1822 patients and 4650 control participants passed quality control for the GWAS analysis. Replication analyses of samples from 958 patients and 2050 control participants were conducted. Summary statistics from the European Psychiatric Genomics Consortium 2 (PGC2) BD GWAS (20 352 cases and 31 358 controls) were used for the trans-ancestry genetic correlation analysis, polygenetic risk score analysis, and meta-analysis to compare BD genetic risk between Han Chinese and European individuals. The study was performed in February 2020. Main Outcomes and Measures: Single-nucleotide variations with P < 5.00 × 10-8 were considered to show genome-wide significance of statistical association. Results: The Han Chinese discovery GWAS sample included 1822 cases (mean [SD] age, 35.43 [14.12] years; 838 [46%] male) and 4650 controls (mean [SD] age, 27.48 [5.97] years; 2465 [53%] male), and the replication sample included 958 cases (mean [SD] age, 37.82 [15.54] years; 412 [43%] male) and 2050 controls (mean [SD] age, 27.50 [6.00] years; 1189 [58%] male). A novel BD risk locus in Han Chinese individuals was found near the gene encoding transmembrane protein 108 (TMEM108, rs9863544; P = 2.49 × 10-8; odds ratio [OR], 0.650; 95% CI, 0.559-0.756), which is required for dendritic spine development and glutamatergic transmission in the dentate gyrus. Trans-ancestry genetic correlation estimation (ρge = 0.652, SE = 0.106; P = 7.30 × 10-10) and polygenetic risk score analyses (maximum liability-scaled Nagelkerke pseudo R2 = 1.27%; P = 1.30 × 10-19) showed evidence of shared BD genetic risk between Han Chinese and European populations, and meta-analysis identified 2 new GWAS risk loci near VRK2 (rs41335055; P = 4.98 × 10-9; OR, 0.849; 95% CI, 0.804-0.897) and RHEBL1 (rs7969091; P = 3.12 × 10-8; OR, 0.932; 95% CI, 0.909-0.956). Conclusions and Relevance: This GWAS study identified several loci and genes involved in the heritable risk of BD, providing insights into its genetic architecture and biological basis.