Stimulating Wnt signaling reveals context-dependent genetic effects on gene regulation in primary human neural progenitors.

Gene regulatory effects have been difficult to detect at many non-coding loci associated with brain-related traits, likely because some genetic variants have distinct functions in specific contexts. To explore context-dependent gene regulation, we measured chromatin accessibility and gene expression after activation of the canonical Wnt pathway in primary human neural progenitors (n = 82 donors). We found that TCF/LEF motifs and brain-structure-associated and neuropsychiatric-disorder-associated variants were enriched within Wnt-responsive regulatory elements. Genetically influenced regulatory elements were enriched in genomic regions under positive selection along the human lineage. Wnt pathway stimulation increased detection of genetically influenced regulatory elements/genes by 66%/53% and enabled identification of 397 regulatory elements primed to regulate gene expression. Stimulation also increased identification of shared genetic effects on molecular and complex brain traits by up to 70%, suggesting that genetic variant function during neurodevelopmental patterning can lead to differences in adult brain and behavioral traits.

Genetics of cell-type-specific post-transcriptional gene regulation during human neurogenesis.

The function of some genetic variants associated with brain-relevant traits has been explained through colocalization with expression quantitative trait loci (eQTL) conducted in bulk postmortem adult brain tissue. However, many brain-trait associated loci have unknown cellular or molecular function. These genetic variants may exert context-specific function on different molecular phenotypes including post-transcriptional changes. Here, we identified genetic regulation of RNA editing and alternative polyadenylation (APA) within a cell-type-specific population of human neural progenitors and neurons. More RNA editing and isoforms utilizing longer polyadenylation sequences were observed in neurons, likely due to higher expression of genes encoding the proteins mediating these post-transcriptional events. We also detected hundreds of cell-type-specific editing quantitative trait loci (edQTLs) and alternative polyadenylation QTLs (apaQTLs). We found colocalizations of a neuron edQTL in CCDC88A with educational attainment and a progenitor apaQTL in EP300 with schizophrenia, suggesting that genetically mediated post-transcriptional regulation during brain development leads to differences in brain function.

Cross-site reproducibility of human cortical organoids reveals consistent cell type composition and architecture.

While guided human cortical organoid (hCO) protocols reproducibly generate cortical cell types at one site, variability in hCO phenotypes across sites using a harmonized protocol has not yet been evaluated. To determine the cross-site reproducibility of hCO differentiation, three independent research groups assayed hCOs in multiple differentiation replicates from one induced pluripotent stem cell (iPSC) line using a harmonized miniaturized spinning bioreactor protocol across 3 months. hCOs were mostly cortical progenitor and neuronal cell types in reproducible proportions that were consistently organized in cortical wall-like buds. Cross-site differences were detected in hCO size and expression of metabolism and cellular stress genes. Variability in hCO phenotypes correlated with stem cell gene expression prior to differentiation and technical factors associated with seeding, suggesting iPSC quality and treatment are important for differentiation outcomes. Cross-site reproducibility of hCO cell type proportions and organization encourages future prospective meta-analytic studies modeling neurodevelopmental disorders in hCOs.

Eye-brain connections revealed by multimodal retinal and brain imaging genetics.

The retina, an anatomical extension of the brain, forms physiological connections with the visual cortex of the brain. Although retinal structures offer a unique opportunity to assess brain disorders, their relationship to brain structure and function is not well understood. In this study, we conducted a systematic cross-organ genetic architecture analysis of eye-brain connections using retinal and brain imaging endophenotypes. We identified novel phenotypic and genetic links between retinal imaging biomarkers and brain structure and function measures from multimodal magnetic resonance imaging (MRI), with many associations involving the primary visual cortex and visual pathways. Retinal imaging biomarkers shared genetic influences with brain diseases and complex traits in 65 genomic regions, with 18 showing genetic overlap with brain MRI traits. Mendelian randomization suggests bidirectional genetic causal links between retinal structures and neurological and neuropsychiatric disorders, such as Alzheimer's disease. Overall, our findings reveal the genetic basis for eye-brain connections, suggesting that retinal images can help uncover genetic risk factors for brain disorders and disease-related changes in intracranial structure and function.

Cross-ancestry atlas of gene, isoform, and splicing regulation in the developing human brain.

Neuropsychiatric genome-wide association studies (GWASs), including those for autism spectrum disorder and schizophrenia, show strong enrichment for regulatory elements in the developing brain. However, prioritizing risk genes and mechanisms is challenging without a unified regulatory atlas. Across 672 diverse developing human brains, we identified 15,752 genes harboring gene, isoform, and/or splicing quantitative trait loci, mapping 3739 to cellular contexts. Gene expression heritability drops during development, likely reflecting both increasing cellular heterogeneity and the intrinsic properties of neuronal maturation. Isoform-level regulation, particularly in the second trimester, mediated the largest proportion of GWAS heritability. Through colocalization, we prioritized mechanisms for about 60% of GWAS loci across five disorders, exceeding adult brain findings. Finally, we contextualized results within gene and isoform coexpression networks, revealing the comprehensive landscape of transcriptome regulation in development and disease.

Rigor and reproducibility in human brain organoid research: Where we are and where we need to go.

Human brain organoid models have emerged as a promising tool for studying human brain development and function. These models preserve human genetics and recapitulate some aspects of human brain development, while facilitating manipulation in an in vitro setting. Despite their potential to transform biology and medicine, concerns persist about their fidelity. To fully harness their potential, it is imperative to establish reliable analytic methods, ensuring rigor and reproducibility. Here, we review current analytical platforms used to characterize human forebrain cortical organoids, highlight challenges, and propose recommendations for future studies to achieve greater precision and uniformity across laboratories.

Mapping of neuronal and glial primary cilia contactome and connectome in the human cerebral cortex.

Primary cilia act as antenna receivers of environmental signals and enable effective neuronal or glial responses. Disruption of their function is associated with circuit disorders. To understand the signals these cilia receive, we comprehensively mapped cilia's contacts within the human cortical connectome using serial-section EM reconstruction of a 1 mm3 cortical volume, spanning the entire cortical thickness. We mapped the "contactome" of cilia emerging from neurons and astrocytes in every cortical layer. Depending on the layer and cell type, cilia make distinct patterns of contact. Primary cilia display cell-type- and layer-specific variations in size, shape, and microtubule axoneme core, which may affect their signaling competencies. Neuronal cilia are intrinsic components of a subset of cortical synapses and thus a part of the connectome. This diversity in the structure, contactome, and connectome of primary cilia endows each neuron or glial cell with a unique barcode of access to the surrounding neural circuitry.

Systematic investigation of allelic regulatory activity of schizophrenia-associated common variants.

Genome-wide association studies (GWASs) have successfully identified 145 genomic regions that contribute to schizophrenia risk, but linkage disequilibrium makes it challenging to discern causal variants. We performed a massively parallel reporter assay (MPRA) on 5,173 fine-mapped schizophrenia GWAS variants in primary human neural progenitors and identified 439 variants with allelic regulatory effects (MPRA-positive variants). Transcription factor binding had modest predictive power, while fine-map posterior probability, enhancer overlap, and evolutionary conservation failed to predict MPRA-positive variants. Furthermore, 64% of MPRA-positive variants did not exhibit expressive quantitative trait loci signature, suggesting that MPRA could identify yet unexplored variants with regulatory potentials. To predict the combinatorial effect of MPRA-positive variants on gene regulation, we propose an accessibility-by-contact model that combines MPRA-measured allelic activity with neuronal chromatin architecture.

The pivotal role of the X-chromosome in the genetic architecture of the human brain.

Genes on the X-chromosome are extensively expressed in the human brain, resulting in substantial influences on brain development, intellectual disability, and other brain-related disorders. To comprehensively investigate the X-chromosome's impact on the cerebral cortex, white matter tract microstructures, and intrinsic and extrinsic brain functions, we examined 2,822 complex brain imaging traits obtained from n=34,000 subjects in the UK Biobank. We unveiled potential autosome-X-chromosome interaction, while proposing an atlas of dosage compensation (DC) for each set of traits. We observed a pronounced X-chromosome impact on the corticospinal tract and the functional amplitude and connectivity of visual networks. In association studies, we identified 50 genome-wide significant trait-locus pairs enriched in Xq28, 22 of which replicated in independent datasets (n=4,900). Notably, 13 newly identified pairs were in the X-chromosome's non-pseudo-autosomal regions (NPR). The volume of the right ventral diencephalon shared genetic architecture with schizophrenia and educational attainment in a locus indexed by rs2361468 (located ~3kb upstream of PJA1, a conserved and ubiquitously expressed gene implicated in multiple psychiatric disorders). No significant associations were identified in the pseudo-autosomal regions (PAR) or the Y-chromosome. Finally, we explored sex-specific associations on the X-chromosome and compared differing genetic effects between sexes. We found much more associations can be identified in males (33 versus 9) given a similar sample size. In conclusion, our research provides invaluable insights into the X-chromosome's role in the human brain, contributing to the observed sex differences in brain structure and function.

Genetics of cell-type-specific post-transcriptional gene regulation during human neurogenesis.

The function of some genetic variants associated with brain-relevant traits has been explained through colocalization with expression quantitative trait loci (eQTL) conducted in bulk post-mortem adult brain tissue. However, many brain-trait associated loci have unknown cellular or molecular function. These genetic variants may exert context-specific function on different molecular phenotypes including post-transcriptional changes. Here, we identified genetic regulation of RNA-editing and alternative polyadenylation (APA), within a cell-type-specific population of human neural progenitors and neurons. More RNA-editing and isoforms utilizing longer polyadenylation sequences were observed in neurons, likely due to higher expression of genes encoding the proteins mediating these post-transcriptional events. We also detected hundreds of cell-type-specific editing quantitative trait loci (edQTLs) and alternative polyadenylation QTLs (apaQTLs). We found colocalizations of a neuron edQTL in CCDC88A with educational attainment and a progenitor apaQTL in EP300 with schizophrenia, suggesting genetically mediated post-transcriptional regulation during brain development lead to differences in brain function.

Cross-site reproducibility of human cortical organoids reveals consistent cell type composition and architecture.

Background: Reproducibility of human cortical organoid (hCO) phenotypes remains a concern for modeling neurodevelopmental disorders. While guided hCO protocols reproducibly generate cortical cell types in multiple cell lines at one site, variability across sites using a harmonized protocol has not yet been evaluated. We present an hCO cross-site reproducibility study examining multiple phenotypes. Methods: Three independent research groups generated hCOs from one induced pluripotent stem cell (iPSC) line using a harmonized miniaturized spinning bioreactor protocol. scRNA-seq, 3D fluorescent imaging, phase contrast imaging, qPCR, and flow cytometry were used to characterize the 3 month differentiations across sites. Results: In all sites, hCOs were mostly cortical progenitor and neuronal cell types in reproducible proportions with moderate to high fidelity to the in vivo brain that were consistently organized in cortical wall-like buds. Cross-site differences were detected in hCO size and morphology. Differential gene expression showed differences in metabolism and cellular stress across sites. Although iPSC culture conditions were consistent and iPSCs remained undifferentiated, primed stem cell marker expression prior to differentiation correlated with cell type proportions in hCOs. Conclusions: We identified hCO phenotypes that are reproducible across sites using a harmonized differentiation protocol. Previously described limitations of hCO models were also reproduced including off-target differentiations, necrotic cores, and cellular stress. Improving our understanding of how stem cell states influence early hCO cell types may increase reliability of hCO differentiations. Cross-site reproducibility of hCO cell type proportions and organization lays the foundation for future collaborative prospective meta-analytic studies modeling neurodevelopmental disorders in hCOs.

Donor perspectives on informed consent and use of biospecimens for brain organoid research.

Debates about the ethics of human brain organoids have proceeded without the input of individuals whose brains are being modeled. Interviews with donors of biospecimens for brain organoid research revealed overall enthusiasm for brain organoids as a tool for biomedical discovery, alongside a desire for ongoing engagement with research teams to learn the results of the research, to allow transfer of decision-making authority over time, and to ensure ethical boundaries are not crossed. Future work is needed to determine the most feasible and resource-efficient way to longitudinally engage donors participating in brain organoid research.

Heart-brain connections: Phenotypic and genetic insights from magnetic resonance images.

Cardiovascular health interacts with cognitive and mental health in complex ways, yet little is known about the phenotypic and genetic links of heart-brain systems. We quantified heart-brain connections using multiorgan magnetic resonance imaging (MRI) data from more than 40,000 subjects. Heart MRI traits displayed numerous association patterns with brain gray matter morphometry, white matter microstructure, and functional networks. We identified 80 associated genomic loci (P < 6.09 × 10-10) for heart MRI traits, which shared genetic influences with cardiovascular and brain diseases. Genetic correlations were observed between heart MRI traits and brain-related traits and disorders. Mendelian randomization suggests that heart conditions may causally contribute to brain disorders. Our results advance a multiorgan perspective on human health by revealing heart-brain connections and shared genetic influences.

Inferring cell-type-specific causal gene regulatory networks during human neurogenesis.

BACKGROUND: Genetic variation influences both chromatin accessibility, assessed in chromatin accessibility quantitative trait loci (caQTL) studies, and gene expression, assessed in expression QTL (eQTL) studies. Genetic variants can impact either nearby genes (cis-eQTLs) or distal genes (trans-eQTLs). Colocalization between caQTL and eQTL, or cis- and trans-eQTLs suggests that they share causal variants. However, pairwise colocalization between these molecular QTLs does not guarantee a causal relationship. Mediation analysis can be applied to assess the evidence supporting causality versus independence between molecular QTLs. Given that the function of QTLs can be cell-type-specific, we performed mediation analyses to find epigenetic and distal regulatory causal pathways for genes within two major cell types of the developing human cortex, progenitors and neurons. RESULTS: We find that the expression of 168 and 38 genes is mediated by chromatin accessibility in progenitors and neurons, respectively. We also find that the expression of 11 and 12 downstream genes is mediated by upstream genes in progenitors and neurons. Moreover, we discover that a genetic locus associated with inter-individual differences in brain structure shows evidence for mediation of SLC26A7 through chromatin accessibility, identifying molecular mechanisms of a common variant association to a brain trait. CONCLUSIONS: In this study, we identify cell-type-specific causal gene regulatory networks whereby the impacts of variants on gene expression were mediated by chromatin accessibility or distal gene expression. Identification of these causal paths will enable identifying and prioritizing actionable regulatory targets perturbing these key processes during neurodevelopment.

Cross-ancestry, cell-type-informed atlas of gene, isoform, and splicing regulation in the developing human brain.

Genomic regulatory elements active in the developing human brain are notably enriched in genetic risk for neuropsychiatric disorders, including autism spectrum disorder (ASD), schizophrenia, and bipolar disorder. However, prioritizing the specific risk genes and candidate molecular mechanisms underlying these genetic enrichments has been hindered by the lack of a single unified large-scale gene regulatory atlas of human brain development. Here, we uniformly process and systematically characterize gene, isoform, and splicing quantitative trait loci (xQTLs) in 672 fetal brain samples from unique subjects across multiple ancestral populations. We identify 15,752 genes harboring a significant xQTL and map 3,739 eQTLs to a specific cellular context. We observe a striking drop in gene expression and splicing heritability as the human brain develops. Isoform-level regulation, particularly in the second trimester, mediates the greatest proportion of heritability across multiple psychiatric GWAS, compared with eQTLs. Via colocalization and TWAS, we prioritize biological mechanisms for ~60% of GWAS loci across five neuropsychiatric disorders, nearly two-fold that observed in the adult brain. Finally, we build a comprehensive set of developmentally regulated gene and isoform co-expression networks capturing unique genetic enrichments across disorders. Together, this work provides a comprehensive view of genetic regulation across human brain development as well as the stage-and cell type-informed mechanistic underpinnings of neuropsychiatric disorders.

Wnt activity reveals context-specific genetic effects on gene regulation in neural progenitors.

Gene regulatory effects in bulk-post mortem brain tissues are undetected at many non-coding brain trait-associated loci. We hypothesized that context-specific genetic variant function during stimulation of a developmental signaling pathway would explain additional regulatory mechanisms. We measured chromatin accessibility and gene expression following activation of the canonical Wnt pathway in primary human neural progenitors from 82 donors. TCF/LEF motifs, brain structure-, and neuropsychiatric disorder-associated variants were enriched within Wnt-responsive regulatory elements (REs). Genetically influenced REs were enriched in genomic regions under positive selection along the human lineage. Stimulation of the Wnt pathway increased the detection of genetically influenced REs/genes by 66.2%/52.7%, and led to the identification of 397 REs primed for effects on gene expression. Context-specific molecular quantitative trait loci increased brain-trait colocalizations by up to 70%, suggesting that genetic variant effects during early neurodevelopmental patterning lead to differences in adult brain and behavioral traits.

Eye-brain connections revealed by multimodal retinal and brain imaging genetics in the UK Biobank.

As an anatomical extension of the brain, the retina of the eye is synaptically connected to the visual cortex, establishing physiological connections between the eye and the brain. Despite the unique opportunity retinal structures offer for assessing brain disorders, less is known about their relationship to brain structure and function. Here we present a systematic cross-organ genetic architecture analysis of eye-brain connections using retina and brain imaging endophenotypes. Novel phenotypic and genetic links were identified between retinal imaging biomarkers and brain structure and function measures derived from multimodal magnetic resonance imaging (MRI), many of which were involved in the visual pathways, including the primary visual cortex. In 65 genomic regions, retinal imaging biomarkers shared genetic influences with brain diseases and complex traits, 18 showing more genetic overlaps with brain MRI traits. Mendelian randomization suggests that retinal structures have bidirectional genetic causal links with neurological and neuropsychiatric disorders, such as Alzheimer's disease. Overall, cross-organ imaging genetics reveals a genetic basis for eye-brain connections, suggesting that the retinal images can elucidate genetic risk factors for brain disorders and disease-related changes in intracranial structure and function.

MicroRNA-eQTLs in the developing human neocortex link miR-4707-3p expression to brain size.

Expression quantitative trait loci (eQTL) data have proven important for linking non-coding loci to protein-coding genes. But eQTL studies rarely measure microRNAs (miRNAs), small non-coding RNAs known to play a role in human brain development and neurogenesis. Here, we performed small-RNA sequencing across 212 mid-gestation human neocortical tissue samples, measured 907 expressed miRNAs, discovering 111 of which were novel, and identified 85 local-miRNA-eQTLs. Colocalization of miRNA-eQTLs with GWAS summary statistics yielded one robust colocalization of miR-4707-3p expression with educational attainment and brain size phenotypes, where the miRNA expression increasing allele was associated with decreased brain size. Exogenous expression of miR-4707-3p in primary human neural progenitor cells decreased expression of predicted targets and increased cell proliferation, indicating miR-4707-3p modulates progenitor gene regulation and cell fate decisions. Integrating miRNA-eQTLs with existing GWAS yielded evidence of a miRNA that may influence human brain size and function via modulation of neocortical brain development.

Genetic variations within human gained enhancer elements affect human brain sulcal morphology.

The expansion of the cerebral cortex is one of the most distinctive changes in the evolution of the human brain. Cortical expansion and related increases in cortical folding may have contributed to emergence of our capacities for high-order cognitive abilities. Molecular analysis of humans, archaic hominins, and non-human primates has allowed identification of chromosomal regions showing evolutionary changes at different points of our phylogenetic history. In this study, we assessed the contributions of genomic annotations spanning 30 million years to human sulcal morphology measured via MRI in more than 18,000 participants from the UK Biobank. We found that variation within brain-expressed human gained enhancers, regulatory genetic elements that emerged since our last common ancestor with Old World monkeys, explained more trait heritability than expected for the left and right calloso-marginal posterior fissures and the right central sulcus. Intriguingly, these are sulci that have been previously linked to the evolution of locomotion in primates and later on bipedalism in our hominin ancestors.

Cellular Genome-wide Association Study Identifies Common Genetic Variation Influencing Lithium-Induced Neural Progenitor Proliferation.

BACKGROUND: Bipolar disorder is a highly heritable neuropsychiatric condition affecting more than 1% of the human population. Lithium salts are commonly prescribed as a mood stabilizer for individuals with bipolar disorder. Lithium is clinically effective in approximately half of treated individuals, and their genetic backgrounds are known to influence treatment outcomes. While the mechanism of lithium's therapeutic action is unclear, it stimulates adult neural progenitor cell proliferation, similar to some antidepressant drugs. METHODS: To identify common genetic variants that modulate lithium-induced proliferation, we conducted an EdU incorporation assay in a library of 80 genotyped human neural progenitor cells treated with lithium. These data were used to perform a genome-wide association study to identify common genetic variants that influence lithium-induced neural progenitor cell proliferation. We manipulated the expression of a putatively causal gene using CRISPRi/a (clustered regularly interspaced short palindromic repeats interference/activation) constructs to experimentally verify lithium-induced proliferation effects. RESULTS: We identified a locus on chr3p21.1 associated with lithium-induced proliferation. This locus is also associated with bipolar disorder risk, schizophrenia risk, and interindividual differences in intelligence. We identified a single gene, GNL3, whose expression temporally increased in an allele-specific fashion following lithium treatment. Experimentally increasing the expression of GNL3 led to increased proliferation under baseline conditions, while experimentally decreasing GNL3 expression suppressed lithium-induced proliferation. CONCLUSIONS: Our experiments reveal that common genetic variation modulates lithium-induced neural progenitor proliferation and that GNL3 expression is necessary for the full proliferation-stimulating effects of lithium. These results suggest that performing genome-wide associations in genetically diverse human cell lines is a useful approach to discover context-specific pharmacogenomic effects.