The association of GNB5 with Alzheimer disease revealed by genomic analysis restricted to variants impacting gene function.

Disease-associated variants identified from genome-wide association studies (GWASs) frequently map to non-coding areas of the genome such as introns and intergenic regions. An exclusive reliance on gene-agnostic methods of genomic investigation could limit the identification of relevant genes associated with polygenic diseases such as Alzheimer disease (AD). To overcome such potential restriction, we developed a gene-constrained analytical method that considers only moderate- and high-risk variants that affect gene coding sequences. We report here the application of this approach to publicly available datasets containing 181,388 individuals without and with AD and the resulting identification of 660 genes potentially linked to the higher AD prevalence among Africans/African Americans. By integration with transcriptome analysis of 23 brain regions from 2,728 AD case-control samples, we concentrated on nine genes that potentially enhance the risk of AD: AACS, GNB5, GNS, HIPK3, MED13, SHC2, SLC22A5, VPS35, and ZNF398. GNB5, the fifth member of the heterotrimeric G protein beta family encoding Gß5, is primarily expressed in neurons and is essential for normal neuronal development in mouse brain. Homozygous or compound heterozygous loss of function of GNB5 in humans has previously been associated with a syndrome of developmental delay, cognitive impairment, and cardiac arrhythmia. In validation experiments, we confirmed that Gnb5 heterozygosity enhanced the formation of both amyloid plaques and neurofibrillary tangles in the brains of AD model mice. These results suggest that gene-constrained analysis can complement the power of GWASs in the identification of AD-associated genes and may be more broadly applicable to other polygenic diseases.

Loss of Ezh2 in the medial ganglionic eminence alters interneuron fate, cell morphology and gene expression profiles.

Introduction: Enhancer of zeste homolog 2 (Ezh2) is responsible for trimethylation of histone 3 at lysine 27 (H3K27me3), resulting in repression of gene expression. Here, we explore the role of Ezh2 in forebrain GABAergic interneuron development. Methods: We removed Ezh2 in the MGE by generating Nkx2-1Cre;Ezh2 conditional knockout mice. We then characterized changes in MGE-derived interneuron fate and electrophysiological properties in juvenile mice, as well as alterations in gene expression, chromatin accessibility and histone modifications in the MGE. Results: Loss of Ezh2 increases somatostatin-expressing (SST+) and decreases parvalbumin-expressing (PV+) interneurons in the forebrain. We observe fewer MGE-derived interneurons in the first postnatal week, indicating reduced interneuron production. Intrinsic electrophysiological properties in SST+ and PV+ interneurons are normal, but PV+ interneurons display increased axonal complexity in Ezh2 mutant mice. Single nuclei multiome analysis revealed differential gene expression patterns in the embryonic MGE that are predictive of these cell fate changes. Lastly, CUT&Tag analysis revealed that some genomic loci are particularly resistant or susceptible to shifts in H3K27me3 levels in the absence of Ezh2, indicating differential selectivity to epigenetic perturbation. Discussion: Thus, loss of Ezh2 in the MGE alters interneuron fate, morphology, and gene expression and regulation. These findings have important implications for both normal development and potentially in disease etiologies.

Enhancer-promoter interactions can bypass CTCF-mediated boundaries and contribute to phenotypic robustness.

How enhancers activate their distal target promoters remains incompletely understood. Here we dissect how CTCF-mediated loops facilitate and restrict such regulatory interactions. Using an allelic series of mouse mutants, we show that CTCF is neither required for the interaction of the Sox2 gene with distal enhancers, nor for its expression. Insertion of various combinations of CTCF motifs, between Sox2 and its distal enhancers, generated boundaries with varying degrees of insulation that directly correlated with reduced transcriptional output. However, in both epiblast and neural tissues, enhancer contacts and transcriptional induction could not be fully abolished, and insertions failed to disrupt implantation and neurogenesis. In contrast, Sox2 expression was undetectable in the anterior foregut of mutants carrying the strongest boundaries, and these animals fully phenocopied loss of SOX2 in this tissue. We propose that enhancer clusters with a high density of regulatory activity can better overcome physical barriers to maintain faithful gene expression and phenotypic robustness.

Generation of single-cell and single-nuclei suspensions from embryonic and adult mouse brains.

Efficient protocols to generate single-cell and single-nuclei suspensions are critical for the burgeoning field of single-cell/single-nuclei sequencing. Here we describe procedures to generate single-cell and single-nuclei suspensions from embryonic and adult mouse brains. This protocol can be modified for any brain region and/or neural cell type. For complete details on the use and execution of this protocol, please refer to Lee et al. (2022),1 Rhodes et al. (2022),2 Mahadevan et al. (2021),3 Ekins et al. (2020),4 and Wester et al. (2019).5.

An epigenome atlas of neural progenitors within the embryonic mouse forebrain.

A comprehensive characterization of epigenomic organization in the embryonic mouse forebrain will enhance our understanding of neurodevelopment and provide insight into mechanisms of neurological disease. Here we collected single-cell chromatin accessibility profiles from four distinct neurogenic regions of the embryonic mouse forebrain using single nuclei ATAC-Seq (snATAC-Seq). We identified thousands of differentially accessible peaks, many restricted to distinct progenitor cell types or brain regions. We integrated snATAC-Seq and single cell transcriptome data to characterize changes of chromatin accessibility at enhancers and promoters with associated transcript abundance. Multi-modal integration of histone modifications (CUT&Tag and CUT&RUN), promoter-enhancer interactions (Capture-C) and high-order chromatin structure (Hi-C) extended these initial observations. This dataset reveals a diverse chromatin landscape with region-specific regulatory mechanisms and genomic interactions in distinct neurogenic regions of the embryonic mouse brain and represents an extensive public resource of a 'ground truth' epigenomic landscape at this critical stage of neurogenesis.

Transcriptional heterogeneity of ventricular zone cells in the ganglionic eminences of the mouse forebrain.

The ventricular zone (VZ) of the nervous system contains radial glia cells that were originally considered relatively homogenous in their gene expression, but a detailed characterization of transcriptional diversity in these VZ cells has not been reported. Here, we performed single-cell RNA sequencing to characterize transcriptional heterogeneity of neural progenitors within the VZ and subventricular zone (SVZ) of the ganglionic eminences (GEs), the source of all forebrain GABAergic neurons. By using a transgenic mouse line to enrich for VZ cells, we characterize significant transcriptional heterogeneity, both between GEs and within spatial subdomains of specific GEs. Additionally, we observe differential gene expression between E12.5 and E14.5 VZ cells, which could provide insights into temporal changes in cell fate. Together, our results reveal a previously unknown spatial and temporal genetic diversity of VZ cells in the ventral forebrain that will aid our understanding of initial fate decisions in the forebrain.

NMDARs Drive the Expression of Neuropsychiatric Disorder Risk Genes Within GABAergic Interneuron Subtypes in the Juvenile Brain.

Medial ganglionic eminence (MGE)-derived parvalbumin (PV)+, somatostatin (SST)+and Neurogliaform (NGFC)-type cortical and hippocampal interneurons, have distinct molecular, anatomical, and physiological properties. However, the molecular mechanisms regulating their maturation remain poorly understood. Here, via single-cell transcriptomics, we show that the obligate NMDA-type glutamate receptor (NMDAR) subunit gene Grin1 mediates transcriptional regulation of gene expression in specific subtypes of MGE-derived interneurons, leading to altered subtype abundances. Notably, MGE-specific early developmental Grin1 loss results in a broad downregulation of diverse transcriptional, synaptogenic and membrane excitability regulatory programs in the juvenile brain. These widespread gene expression abnormalities mirror aberrations that are typically associated with neurodevelopmental disorders. Our study hence provides a road map for the systematic examination of NMDAR signaling in interneuron subtypes, revealing potential MGE-specific genetic targets that could instruct future therapies of psychiatric disorders.

Single nucleus RNA-sequencing defines unexpected diversity of cholinergic neuron types in the adult mouse spinal cord.

In vertebrates, motor control relies on cholinergic neurons in the spinal cord that have been extensively studied over the past hundred years, yet the full heterogeneity of these neurons and their different functional roles in the adult remain to be defined. Here, we develop a targeted single nuclear RNA sequencing approach and use it to identify an array of cholinergic interneurons, visceral and skeletal motor neurons. Our data expose markers for distinguishing these classes of cholinergic neurons and their rich diversity. Specifically, visceral motor neurons, which provide autonomic control, can be divided into more than a dozen transcriptomic classes with anatomically restricted localization along the spinal cord. The complexity of the skeletal motor neurons is also reflected in our analysis with alpha, gamma, and a third subtype, possibly corresponding to the elusive beta motor neurons, clearly distinguished. In combination, our data provide a comprehensive transcriptomic description of this important population of neurons that control many aspects of physiology and movement and encompass the cellular substrates for debilitating degenerative disorders.

Temporal Dynamics and Neuronal Specificity of Grin3a Expression in the Mouse Forebrain.

GluN3A subunits endow N-Methyl-D-Aspartate receptors (NMDARs) with unique biophysical, trafficking, and signaling properties. GluN3A-NMDARs are typically expressed during postnatal development, when they are thought to gate the refinement of neural circuits by inhibiting synapse maturation, and stabilization. Recent work suggests that GluN3A also operates in adult brains to control a variety of behaviors, yet a full spatiotemporal characterization of GluN3A expression is lacking. Here, we conducted a systematic analysis of Grin3a (gene encoding mouse GluN3A) mRNA expression in the mouse brain by combining high-sensitivity colorimetric and fluorescence in situ hybridization with labeling for neuronal subtypes. We find that, while Grin3a mRNA expression peaks postnatally, significant levels are retained into adulthood in specific brain regions such as the amygdala, medial habenula, association cortices, and high-order thalamic nuclei. The time-course of emergence and down-regulation of Grin3a expression varies across brain region, cortical layer of residence, and sensory modality, in a pattern that correlates with previously reported hierarchical gradients of brain maturation and functional specialization. Grin3a is expressed in both excitatory and inhibitory neurons, with strong mRNA levels being a distinguishing feature of somatostatin interneurons. Our study provides a comprehensive map of Grin3a distribution across the murine lifespan and paves the way for dissecting the diverse functions of GluN3A in health and disease.

Emergence of non-canonical parvalbumin-containing interneurons in hippocampus of a murine model of type I lissencephaly.

Type I lissencephaly is a neuronal migration disorder caused by haploinsuffiency of the PAFAH1B1 (mouse: Pafah1b1) gene and is characterized by brain malformation, developmental delays, and epilepsy. Here, we investigate the impact of Pafah1b1 mutation on the cellular migration, morphophysiology, microcircuitry, and transcriptomics of mouse hippocampal CA1 parvalbumin-containing inhibitory interneurons (PV+INTs). We find that WT PV+INTs consist of two physiological subtypes (80% fast-spiking (FS), 20% non-fast-spiking (NFS)) and four morphological subtypes. We find that cell-autonomous mutations within interneurons disrupts morphophysiological development of PV+INTs and results in the emergence of a non-canonical 'intermediate spiking (IS)' subset of PV+INTs. We also find that now dominant IS/NFS cells are prone to entering depolarization block, causing them to temporarily lose the ability to initiate action potentials and control network excitation, potentially promoting seizures. Finally, single-cell nuclear RNAsequencing of PV+INTs revealed several misregulated genes related to morphogenesis, cellular excitability, and synapse formation.

Non-canonical Wnt Signaling through Ryk Regulates the Generation of Somatostatin- and Parvalbumin-Expressing Cortical Interneurons.

GABAergic interneurons have many important functions in cortical circuitry, a reflection of their cell diversity. The developmental origins of this diversity are poorly understood. Here, we identify rostral-caudal regionality in Wnt exposure within the interneuron progenitor zone delineating the specification of the two main interneuron subclasses. Caudally situated medial ganglionic eminence (MGE) progenitors receive high levels of Wnt signaling and give rise to somatostatin (SST)-expressing cortical interneurons. By contrast, parvalbumin (PV)-expressing basket cells originate mostly from the rostral MGE, where Wnt signaling is attenuated. Interestingly, rather than canonical signaling through ß-catenin, signaling via the non-canonical Wnt receptor Ryk regulates interneuron cell-fate specification in vivo and in vitro. Indeed, gain of function of Ryk intracellular domain signaling regulates SST and PV fate in a dose-dependent manner, suggesting that Ryk signaling acts in a graded fashion. These data reveal an important role for non-canonical Wnt-Ryk signaling in establishing the correct ratios of cortical interneuron subtypes.

Neocortical Projection Neurons Instruct Inhibitory Interneuron Circuit Development in a Lineage-Dependent Manner.

Neocortical circuits consist of stereotypical motifs that must self-assemble during development. Recent evidence suggests that the subtype identity of both excitatory projection neurons (PNs) and inhibitory interneurons (INs) is important for this process. We knocked out the transcription factor Satb2 in PNs to induce those of the intratelencephalic (IT) type to adopt a pyramidal tract (PT)-type identity. Loss of IT-type PNs selectively disrupted the lamination and circuit integration of INs derived from the caudal ganglionic eminence (CGE). Strikingly, reprogrammed PNs demonstrated reduced synaptic targeting of CGE-derived INs relative to controls. In control mice, IT-type PNs targeted neighboring CGE INs, while PT-type PNs did not in deep layers, confirming this lineage-dependent motif. Finally, single-cell RNA sequencing revealed that major CGE IN subtypes were conserved after loss of IT PNs, but with differential transcription of synaptic proteins and signaling molecules. Thus, IT-type PNs influence CGE-derived INs in a non-cell-autonomous manner during cortical development.

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains.

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the roles of intrinsic vs. extrinsic mechanisms that regulate this differentiation process is a conundrum for all developmental neurobiologists. This issue is magnified for GABAergic interneurons, an incredibly heterogeneous cell population that is born from transient embryonic structures and undergo a protracted migratory phase to disperse throughout the telencephalon. To explore how different brain environments affect interneuron fate and maturation, we developed a protocol for harvesting fluorescently labeled immature interneuron precursors from specific brain regions in newborn mice (P0-P2). At this age, interneuron migration is nearly complete and these cells are residing in their final resting environments with relatively little synaptic integration. Following collection of single cell solutions via flow cytometry, these interneuron precursors are transplanted into P0-P2 wildtype postnatal pups. By performing both homotopic (e.g., cortex-to-cortex) or heterotopic (e.g., cortex-to-hippocampus) transplantations, one can assess how challenging immature interneurons in new brain environments affects their fate, maturation, and circuit integration. Brains can be harvested in adult mice and assayed with a wide variety of posthoc analysis on grafted cells, including immunohistochemical, electrophysiological and transcriptional profiling. This general approach provides investigators with a strategy to assay how distinct brain environments can influence numerous aspects of neuron development and identify if specific neuronal characteristics are primarily driven by hardwired genetic programs or environmental cues.

Stranger in a Strange Land: Using Heterotopic Transplantations to Study Nature vs Nurture in Brain Development.

The mammalian brain develops from a simple sheet of neuroepithelial cells into an incredibly complex structure containing billions of neurons with trillions of synapses. Understanding how intrinsic genetic programs interact with environmental cues to generate neuronal diversity and proper connectivity is one of the most daunting challenges in developmental biology. We recently explored this issue in forebrain GABAergic inhibitory interneurons, an extremely diverse population of neurons that are classified into distinct subtypes based on morphology, neurochemical markers, and electrophysiological properties. Immature interneurons were harvested from one brain region and transplanted into a different region, allowing us to assess how challenging cells in a new environment affected their fate. Do these grafted cells adopt characteristics of the host environment or retain features from the donor environment? We found that the proportion of interneuron subgroups is determined by the host region, but some interneuron subtypes maintain features attributable to the donor environment. In this commentary, I expound on potential mechanisms that could underlie these observations and explore the implications of these findings in a greater context of developmental neuroscience.

Heterotopic Transplantations Reveal Environmental Influences on Interneuron Diversity and Maturation.

During embryogenesis, neural progenitors in the ganglionic eminences give rise to diverse GABAergic interneuron subtypes that populate all forebrain regions. The extent to which these cells are genetically predefined or determined by postmigratory environmental cues remains unknown. To address this question, we performed homo- and heterotopic transplantation of early postnatal MGE-derived cortical and hippocampal interneurons. Grafted cells migrated, and displayed neurochemical, electrophysiological, morphological, and neurochemical profiles similar to endogenous interneurons. Our results indicate that the host environment regulates the proportion of interneuron classes in the brain region. However, some specific interneuron subtypes retain characteristics representative of their donor brain regions.

Apical versus Basal Neurogenesis Directs Cortical Interneuron Subclass Fate.

Fate determination in the mammalian telencephalon, with its diversity of neuronal subtypes and relevance to neuropsychiatric disease, remains a critical area of study in neuroscience. Most studies investigating this topic focus on the diversity of neural progenitors within spatial and temporal domains along the lateral ventricles. Often overlooked is whether the location of neurogenesis within a fate-restricted domain is associated with, or instructive for, distinct neuronal fates. Here, we use in vivo fate mapping and the manipulation of neurogenic location to demonstrate that apical versus basal neurogenesis influences the fate determination of major subgroups of cortical interneurons derived from the subcortical telencephalon. Somatostatin-expressing interneurons arise mainly from apical divisions along the ventricular surface, whereas parvalbumin-expressing interneurons originate predominantly from basal divisions in the subventricular zone. As manipulations that shift neurogenic location alter interneuron subclass fate, these results add an additional dimension to the spatial-temporal determinants of neuronal fate determination.

Duration of culture and sonic hedgehog signaling differentially specify PV versus SST cortical interneuron fates from embryonic stem cells.

Medial ganglionic eminence (MGE)-derived GABAergic cortical interneurons (cINs) consist of multiple subtypes that are involved in many cortical functions. They also have a remarkable capacity to migrate, survive and integrate into cortical circuitry after transplantation into postnatal cortex. These features have engendered considerable interest in generating distinct subgroups of interneurons from pluripotent stem cells (PSCs) for the study of interneuron fate and function, and for the development of cell-based therapies. Although advances have been made, the capacity to generate highly enriched pools of subgroup fate-committed interneuron progenitors from PSCs has remained elusive. Previous studies have suggested that the two main MGE-derived interneuron subgroups--those expressing somatostatin (SST) and those expressing parvalbumin (PV)--are specified in the MGE from Nkx2.1-expressing progenitors at higher or lower levels of sonic hedgehog (Shh) signaling, respectively. To further explore the role of Shh and other factors in cIN fate determination, we generated a reporter line such that Nkx2.1-expressing progenitors express mCherry and postmitotic Lhx6-expressing MGE-derived interneurons express GFP. Manipulations of Shh exposure and time in culture influenced the subgroup fates of ESC-derived interneurons. Exposure to higher Shh levels, and collecting GFP-expressing precursors at 12 days in culture, resulted in the strongest enrichment for SST interneurons over those expressing PV, whereas the strongest enrichment for PV interneurons was produced by lower Shh and by collecting mCherry-expressing cells after 17 days in culture. These findings confirm that fate determination of cIN subgroups is crucially influenced by Shh signaling, and provide a system for the further study of interneuron fate and function.

Enhanced derivation of mouse ESC-derived cortical interneurons by expression of Nkx2.1.

Forebrain GABAergic interneurons are divided into subgroups based on their neurochemical markers, connectivity and physiological properties. Abnormal interneuron function is implicated in the pathobiology of neurological disorders such as schizophrenia, autism, and epilepsy. Studies on interneuron development and their role in disease would benefit from an efficient mechanism for the production and selection of specific interneuron subgroups. In this study, we engineered a mouse embryonic stem cell (mESC) line for doxycycline-inducible expression of Nkx2.1, a required transcription factor for cortical interneurons derived from the medial ganglionic eminence (MGE). This mESC line was modified to express GFP in Lhx6(+) cells, a marker of newly postmitotic and mature MGE-derived cortical interneurons. The addition of doxycycline to differentiating ESCs efficiently induced Nkx2.1 protein and increased the production of GFP(+) cells. Transplantation of GFP(+) putative interneuron precursors resulted in migratory, morphological, and neurochemical features consistent with cortical interneuron fates. To test the hypothesis that Sonic hedgehog (Shh) primarily influences cortical interneuron fate determination through the induction of Nkx2.1, ESCs were grown with doxycycline and the Shh antagonist cyclopamine. We found induced Nkx2.1 renders Shh signaling dispensable for the generation of MGE-derived interneurons. These results demonstrate that inducible expression of fate determining genes in embryonic stem cells can be used to study fate determination of the developing forebrain.

Losing your inhibition: linking cortical GABAergic interneurons to schizophrenia.

GABAergic interneurons of the cerebral cortex (cINs) play crucial roles in many aspects of cortical function. The diverse types of cINs are classified into subgroups according to their morphology, intrinsic physiology, neurochemical markers and synaptic targeting. Recent advances in mouse genetics, imaging and electrophysiology techniques have greatly advanced our efforts to understand the role of normal cIN function and its dysfunction in neuropsychiatric disorders. In schizophrenia (SCZ), a wealth of data suggests that cIN function is perturbed, and that interneuron dysfunction may underlie key symptoms of the disease. In this review, we discuss the link between cINs and SCZ, focusing on the evidence for GABAergic signaling deficits from both SCZ patients and mouse models.

Optic chiasm presentation of Semaphorin6D in the context of Plexin-A1 and Nr-CAM promotes retinal axon midline crossing.

At the optic chiasm, retinal ganglion cells (RGCs) project ipsi- or contralaterally to establish the circuitry for binocular vision. Ipsilateral guidance programs have been characterized, but contralateral guidance programs are not well understood. Here, we identify a tripartite molecular system for contralateral RGC projections: Semaphorin6D (Sema6D) and Nr-CAM are expressed on midline radial glia and Plexin-A1 on chiasm neurons, and Plexin-A1 and Nr-CAM are also expressed on contralateral RGCs. Sema6D is repulsive to contralateral RGCs, but Sema6D in combination with Nr-CAM and Plexin-A1 converts repulsion to growth promotion. Nr-CAM functions as a receptor for Sema6D. Sema6D, Plexin-A1, and Nr-CAM are all required for efficient RGC decussation at the optic chiasm. These findings suggest a mechanism by which a complex of Sema6D, Nr-CAM, and Plexin-A1 at the chiasm midline alters the sign of Sema6D and signals Nr-CAM/Plexin-A1 receptors on RGCs to implement the contralateral RGC projection.