Schizophrenia (SCZ) is a common psychiatric disorder characterized by psychosis, emotional withdrawal, and cognitive deficits. Most SCZ risk variants reside in non-coding regions of the genome and are thought to influence disease risk by modulating gene regulation. However, the target genes, biological pathways, and cell types through which these variants exert their effects remain poorly understood. To address this gap, we employed in vivo CRISPR droplet sequencing (CROP-seq) in the postnatal mouse neocortex. We perturbed 12 SCZ risk genes previously linked to functionally validated risk variants, followed by single-cell RNA sequencing. We identified 3,031 differentially expressed genes (DEGs) that recapitulate transcriptional alterations observed in postmortem SCZ brains. Integrative analysis using DEG clustering, factor analysis, and gene regulatory network inference uncovered convergent gene programs with distinct biological functions and cell type specificity. Notably, ciliary transcriptional programs consistently emerged across analytical frameworks. The primary cilium is a neurocircuit modulating signaling organelle in neurons and glia that remains understudied in SCZ. Perturbation of key contributors to the ciliary transcriptional programs led to significant alterations in ciliary structure, suggesting that SCZ genetic risk factors may influence how brain cells sense and transduce extracellular signals through synapse-independent mechanisms. Together, this study provides the first in vivo characterization of the functional consequence of common variant architecture in SCZ and implicates ciliary dysfunction as a convergent downstream mechanism.

Mutations in the chromatin remodeler CHD8 are associated with autism and macrocephaly. While mouse models of Chd8 haploinsufficiency recapitulate brain overgrowth, the specific cellular mechanisms and developmental timing that lead to these anatomical abnormalities remain poorly understood. Here, we conducted 3D imaging of Chd8 V986*/+ mouse brains using magnetic resonance imaging followed by tissue clearing and cellular resolution light-sheet microscopy across embryonic and postnatal developmental stages. We found that brain overgrowth occurs postnatally, driven by an expansion of oligodendrocytes and microglia. Unexpectedly, we identified prevalent molecular layer heterotopias within the frontal cortex of Chd8 V986*/+ mice appearing during embryonic development and persisting throughout life. Molecular layer heterotopias were previously identified in post-mortem brains from individuals with autism and other neurodevelopmental disorders, suggesting functional significance in human patients.

Angelman syndrome is a neurodevelopmental disorder caused by loss of the maternal UBE3A allele, the sole source of UBE3A in mature neurons owing to epigenetic silencing of the paternal allele. Although emerging therapies are being developed to restore UBE3A expression by activating the dormant paternal UBE3A allele, existing mouse models for such preclinical studies have limited throughput and utility, creating bottlenecks for both in vitro therapeutic screening and in vivo characterization. To address this, we developed the Ube3a-INSG dual-reporter knockin mouse, in which an IRES-Nanoluciferase-T2A-Sun1-sfGFP (INSG) cassette was inserted downstream of the endogenous Ube3a stop codon. The INSG model preserves UBE3A protein levels and function while enabling 2 complementary allele-specific readouts: Sun1-sfGFP and Nanoluciferase. We show that Sun1-sfGFP, a nuclear envelope-localized reporter, enables single-cell fluorescence analysis, whole-brain light-sheet imaging, and nuclear quantification by flow cytometry. Further, Nanoluciferase supports high-throughput luminescence assays for sensitive pharmacological profiling in cultured neurons and noninvasive in vivo bioluminescence imaging for pharmacodynamic assessment. By combining scalable screening, cellular analysis, and real-time in vivo monitoring in a single model, the Ube3a-INSG dual-reporter mouse provides a powerful platform to accelerate therapeutic development centered on UBE3A.

Loss- and gain-of-function (LOF and GOF) mutations in the E3 ubiquitin ligase UBE3A cause distinct neurodevelopmental disorders. The AZUL domain of UBE3A binds with low nanomolar affinity to a 45 amino acid (aa) region of PSMD4. We fused this 45-aa sequence to Firefly luciferase to generate a luminescence-based biosensor, Firefly-45aa, that acts as an artificial UBE3A substrate and exhibits exceptional sensitivity in measuring UBE3A activity. Testing UBE3A variants using Firefly-45aa in HEK293T cells revealed distinct biosensor activity profiles, enabling the classification of LOF and GOF mutations based on maximum activity, inhibitory concentration-50, and activity per unit of protein. Some strong LOF mutations had dominant negative activity. In addition, Firefly-45aa can be used to quantify endogenous UBE3A activity in primary cells from Angelman syndrome and GOF mouse models. This biosensor reveals a mutational spectrum of UBE3A enzyme activity and is a sensitive tool for functional characterization and therapeutic development in UBE3A-related disorders.

Environmental exposures can impact brain development and contribute to neurodevelopmental disorder risk. In this study, we leveraged insights from in vitro high-throughput screening studies that examined the developmental toxicity of environmental chemicals to select 47 chemicals for in vivo testing as complex random mixtures in pregnant female mice. Our objectives were to identify mixtures that impact key neurodevelopmental endpoints-embryonic body, brain, and placenta weight-and subsequently to use mass spectrometry to ascertain which chemicals from each active mixture entered the developing brain following gestational exposure. We identified 3 chemicals that entered the embryonic brain and reduced embryonic brain weight: Perfluorooctanoic acid (PFOA), fenpyroximate, and 4-tert-octylphenol. Given its effect on embryonic brain weight and its widespread presence in environmental samples, we selected PFOA for further study using single-nuclei RNA sequencing. We found that PFOA altered neural progenitor cell proliferation and neuronal differentiation in the developing mouse cerebral cortex. Furthermore, we found that gestational exposure to PFOA disrupted neurodevelopment by altering the cell cycle in neural progenitor cells of males and females. In conclusion, we identified environmental chemicals that impact neurodevelopmental processes in vivo and found that single-nuclei RNA sequencing can provide new insights into the cellular mechanisms of neurotoxicity.

Cas9 must be localized to the nucleus to access the genome of mammalian cells. For most proteins, adding a single nuclear localization signal (NLS) is sufficient to promote nuclear entry. However, Cas9 nuclear entry appears to be inefficient as multiple NLSs are typically added to Cas9. Here, we found that three different Cas9 variants interact with the ribosome in HEK293T cells, and that this interaction is RNA mediated. Following immunoprecipitation-mass spectrometry of cytoplasmic-localized Cas9-0NLS and nuclear-localized Cas9-4NLS constructs, we identified novel Cas9 interactors in postmitotic neurons, including KEAP1 and additional ribosomal subunits, the latter were enriched in Cas9-0NLS samples. Collectively, our results suggest that Cas9 is sequestered in the cytoplasm of mammalian cells, in part, via interaction with the ribosome. Increasing the number of NLSs on Cas9 and/or increasing the amount of cytoplasmic guide RNA has the potential to outcompete ribosomal RNA binding and promote efficient nuclear localization of CRISPR-Cas9 variants.

The E3 ubiquitin ligase Ube3a is biallelically expressed in neural progenitors and glial cells, suggesting that UBE3A gain-of-function mutations might cause neurodevelopmental disorders irrespective of parent of origin. Here, we engineered a mouse line that harbors an autism-linked UBE3AT485A (T503A in mouse) gain-of-function mutation and evaluated phenotypes in animals that inherited the mutant allele paternally, maternally, or from both parents. We find that paternally and maternally expressed UBE3AT503A results in elevated UBE3A activity in neural progenitors and glial cells. Expression of UBE3AT503A from the maternal allele, but not the paternal one, leads to a persistent elevation of UBE3A activity in neurons. Mutant mice display behavioral phenotypes that differ by parent of origin. Expression of UBE3AT503A, irrespective of its parent of origin, promotes transient embryonic expansion of Zcchc12 lineage interneurons. Phenotypes of Ube3aT503A mice are distinct from Angelman syndrome model mice. Our study has clinical implications for a growing number of disease-linked UBE3A gain-of-function mutations.

Adult twin neuroimaging studies have revealed that cortical thickness (CT) and surface area (SA) are differentially influenced by genetic information, leading to their spatially distinct genetic patterning and topography. However, the postnatal origins of the genetic topography of CT and SA remain unclear, given the dramatic cortical development from neonates to adults. To fill this critical gap, this study unprecedentedly explored how genetic information differentially regulates the spatial topography of CT and SA in the neonatal brain by leveraging brain magnetic resonance (MR) images from 202 twin neonates with minimal influence by the complicated postnatal environmental factors. We capitalized on infant-dedicated computational tools and a data-driven spectral clustering method to parcellate the cerebral cortex into a set of distinct regions purely according to the genetic correlation of cortical vertices in terms of CT and SA, respectively, and accordingly created the first genetically informed cortical parcellation maps of neonatal brains. Both genetic parcellation maps exhibit bilaterally symmetric and hierarchical patterns, but distinct spatial layouts. For CT, regions with closer genetic relationships demonstrate an anterior-posterior (A-P) division, while for SA, regions with greater genetic proximity are typically within the same lobe. Certain genetically informed regions exhibit strong similarities between neonates and adults, with the most striking similarities in the medial surface in terms of SA, despite their overall substantial differences in genetic parcellation maps. These results greatly advance our understanding of the development of genetic influences on the spatial patterning of cortical morphology. SIGNIFICANCE STATEMENT Genetic influences on cortical thickness (CT) and surface area (SA) are complex and could evolve throughout the lifespan. However, studies revealing distinct genetic topography of CT and SA have been limited to adults. Using brain structural magnetic resonance (MR) images of twins, we unprecedentedly discovered the distinct genetically-informed parcellation maps of CT and SA in neonatal brains, respectively. Each genetic parcellation map comprises a distinct spatial layout of cortical regions, where vertices within the same region share high genetic correlation. These genetic parcellation maps of CT and SA of neonates largely differ from those of adults, despite their highly remarkable similarities in the medial cortex of SA. These discoveries provide important insights into the genetic organization of the early cerebral cortex development.

Brain subcortical structures are paramount in many cognitive functions and their aberrations during infancy are predisposed to various neurodevelopmental and neuropsychiatric disorders, making it highly essential to characterize the early subcortical normative growth patterns. This study investigates the volumetric development and surface area expansion of six subcortical structures and their associations with Mullen scales of early learning by leveraging 513 high-resolution longitudinal MRI scans within the first two postnatal years. Results show that (1) each subcortical structure (except for the amygdala with an approximately linear increase) undergoes rapid nonlinear volumetric growth after birth, which slows down at a structure-specific age with bilaterally similar developmental patterns; (2) Subcortical local area expansion reveals structure-specific and spatiotemporally heterogeneous patterns; (3) Positive associations between thalamus and both receptive and expressive languages and between caudate and putamen and fine motor are revealed. This study advances our understanding of the dynamic early subcortical developmental patterns.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is caused by mutations in SACS, which manifest as a childhood-onset cerebellar ataxia. Cellular ARSACS phenotypes include mitochondrial dysfunction, intermediate filament (IF) disorganization, and loss of Purkinje neurons. It is unclear how the loss of SACS causes these deficits, or why they manifest as cerebellar ataxia. We employed a multi-omics approach to characterize molecular and cellular deficiencies in SACS knockout (KO) cells. We identified alterations in microtubule structure and dynamics, protein trafficking, and mislocalization of synaptic and focal adhesion proteins. Targeting PTEN, a negative regulator of focal adhesions, rescued several cellular phenotypes in SACS KO cells. We found sacsin interacts with proteins implicated in vesicle transport, including HSP proteins, and interactions between structural and cell adhesion proteins were diminished in SACS KO cells. In all, this study suggests that trafficking and localization of synaptic adhesion proteins is a causal molecular deficiency in ARSACS.

Tissue-clearing methods allow every cell in the mouse brain to be imaged without physical sectioning. However, the computational tools currently available for cell quantification in cleared tissue images have been limited to counting sparse cell populations in stereotypical mice. Here, we introduce NuMorph, a group of analysis tools to quantify all nuclei and nuclear markers within the mouse cortex after clearing and imaging by light-sheet microscopy. We apply NuMorph to investigate two distinct mouse models: a Topoisomerase 1 (Top1) model with severe neurodegenerative deficits and a Neurofibromin 1 (Nf1) model with a more subtle brain overgrowth phenotype. In each case, we identify differential effects of gene deletion on individual cell-type counts and distribution across cortical regions that manifest as alterations of gross brain morphology. These results underline the value of whole-brain imaging approaches, and the tools are widely applicable for studying brain structure phenotypes at cellular resolution.

Appropriate axonal growth and connectivity are essential for functional wiring of the brain. Joubert syndrome-related disorders (JSRD), a group of ciliopathies in which mutations disrupt primary cilia function, are characterized by axonal tract malformations. However, little is known about how cilia-driven signaling regulates axonal growth and connectivity. We demonstrate that the deletion of related JSRD genes, Arl13b and Inpp5e, in projection neurons leads to de-fasciculated and misoriented axonal tracts. Arl13b deletion disrupts the function of its downstream effector, Inpp5e, and deregulates ciliary-PI3K/AKT signaling. Chemogenetic activation of ciliary GPCR signaling and cilia-specific optogenetic modulation of downstream second messenger cascades (PI3K, AKT, and AC3) commonly regulated by ciliary signaling receptors induce rapid changes in axonal dynamics. Further, Arl13b deletion leads to changes in transcriptional landscape associated with dysregulated PI3K/AKT signaling. These data suggest that ciliary signaling acts to modulate axonal connectivity and that impaired primary cilia signaling underlies axonal tract defects in JSRD.

RASopathies are a family of related syndromes caused by mutations in regulators of the RAS/Extracellular Regulated Kinase 1/2 (ERK1/2) signaling cascade that often result in neurological deficits. RASopathy mutations in upstream regulatory components, such as NF1, PTPN11/SHP2, and RAS have been well-characterized, but mutation-specific differences in the pathogenesis of nervous system abnormalities remain poorly understood, especially those involving mutations downstream of RAS. Here, we assessed cellular and behavioral phenotypes in mice expressing a Raf1L613V gain-of-function mutation associated with the RASopathy, Noonan Syndrome. We report that Raf1L613V/wt mutants do not exhibit a significantly altered number of excitatory or inhibitory neurons in the cortex. However, we observed a significant increase in the number of specific glial subtypes in the forebrain. The density of GFAP+ astrocytes was significantly increased in the adult Raf1L613V/wt cortex and hippocampus relative to controls. OLIG2+ oligodendrocyte progenitor cells were also increased in number in mutant cortices, but we detected no significant change in myelination. Behavioral analyses revealed no significant changes in voluntary locomotor activity, anxiety-like behavior, or sociability. Surprisingly, Raf1L613V/wt mice performed better than controls in select aspects of the water radial-arm maze, Morris water maze, and cued fear conditioning tasks. Overall, these data show that increased astrocyte and oligodendrocyte progenitor cell (OPC) density in the cortex coincides with enhanced cognition in Raf1L613V/wt mutants and further highlight the distinct effects of RASopathy mutations on nervous system development and function.

The development of the mammalian cerebral cortex depends on careful orchestration of proliferation, maturation, and migration events, ultimately giving rise to a wide variety of neuronal and non-neuronal cell types. To better understand cellular and molecular processes that unfold during late corticogenesis, we perform single-cell RNA-seq on the mouse cerebral cortex at a progenitor driven phase (embryonic day 14.5) and at birth—after neurons from all six cortical layers are born. We identify numerous classes of neurons, progenitors, and glia, their proliferative, migratory, and activation states, and their relatedness within and across age. Using the cell-type-specific expression patterns of genes mutated in neurological and psychiatric diseases, we identify putative disease subtypes that associate with clinical phenotypes. Our study reveals the cellular template of a complex neurodevelopmental process, and provides a window into the cellular origins of brain diseases.

Seizures are bursts of excessive synchronized neuronal activity, suggesting that mechanisms controlling brain excitability are compromised. The voltage-gated potassium channel Kv4.2, a major mediator of hyperpolarizing A-type currents in the brain, is a crucial regulator of neuronal excitability. Kv4.2 expression levels are reduced following seizures and in epilepsy, but the underlying mechanisms remain unclear. Here, we report that Kv4.2 mRNA is recruited to the RNA-induced silencing complex shortly after status epilepticus in mice and after kainic acid treatment of hippocampal neurons, coincident with reduction of Kv4.2 protein. We show that the microRNA miR-324-5p inhibits Kv4.2 protein expression and that antagonizing miR-324-5p is neuroprotective and seizure suppressive. MiR-324-5p inhibition also blocks kainic-acid-induced reduction of Kv4.2 protein in vitro and in vivo and delays kainic-acid-induced seizure onset in wild-type but not in Kcnd2 knockout mice. These results reveal an important role for miR-324-5p-mediated silencing of Kv4.2 in seizure onset.

Aberrant signaling through the Raf/MEK/ERK (ERK/MAPK) pathway causes pathology in a family of neurodevelopmental disorders known as 'RASopathies' and is implicated in autism pathogenesis. Here, we have determined the functions of ERK/MAPK signaling in developing neocortical excitatory neurons. Our data reveal a critical requirement for ERK/MAPK signaling in the morphological development and survival of large Ctip2+ neurons in layer 5. Loss of Map2k1/2 (Mek1/2) led to deficits in corticospinal tract formation and subsequent corticospinal neuron apoptosis. ERK/MAPK hyperactivation also led to reduced corticospinal axon elongation, but was associated with enhanced arborization. ERK/MAPK signaling was dispensable for axonal outgrowth of layer 2/3 callosal neurons. However, Map2k1/2 deletion led to reduced expression of Arc and enhanced intrinsic excitability in both layers 2/3 and 5, in addition to imbalanced synaptic excitation and inhibition. These data demonstrate selective requirements for ERK/MAPK signaling in layer 5 circuit development and general effects on cortical pyramidal neuron excitability.

The cellular pathology of a complex neurodevelopmental disorder is teased apart

Posttranscriptional regulation of gene expression by mRNA-binding proteins is critical for neuronal development and function. hnRNP-Q1 is an mRNA-binding protein that regulates mRNA processing events, including translational repression. hnRNP-Q1 is highly expressed in brain tissue, suggesting a function in regulating genes critical for neuronal development. In this study, we have identified Growth-associated protein 43 (Gap-43) mRNA as a novel target of hnRNP-Q1 and have demonstrated that hnRNP-Q1 represses Gap-43 mRNA translation and consequently GAP-43 function. GAP-43 is a neuronal protein that regulates actin dynamics in growth cones and facilitates axonal growth. Previous studies have identified factors that regulate Gap-43 mRNA stability and localization, but it remains unclear whether Gap-43 mRNA translation is also regulated. Our results reveal that hnRNP-Q1 knockdown increased nascent axon length, total neurite length, and neurite number in mouse embryonic cortical neurons and enhanced Neuro2a cell process extension; these phenotypes were rescued by GAP-43 knockdown. Additionally, we have identified a G-quadruplex structure in the 5' untranslated region of Gap-43 mRNA that directly interacts with hnRNP-Q1 as a means to inhibit Gap-43 mRNA translation. Therefore hnRNP-Q1-mediated repression of Gap-43 mRNA translation provides an additional mechanism for regulating GAP-43 expression and function and may be critical for neuronal development.

The role of higher‐order thalamic structures in sensory processing remains poorly understood. Here, we used the ferret (Mustela putorius furo) as a novel model species for the study of the lateral posterior (LP)–pulvinar complex and its structural and functional connectivity with area 17 [primary visual cortex (V1)]. We found reciprocal anatomical connections between the lateral part of the LP nucleus of the LP–pulvinar complex (LPl) and V1. In order to investigate the role of this feedback loop between LPl and V1 in shaping network activity, we determined the functional interactions between LPl and the supragranular, granular and infragranular layers of V1 by recording multiunit activity and local field potentials. Coherence was strongest between LPl and the supragranular V1, with the most distinct peaks in the delta and alpha frequency bands. Inter‐area interaction measured by spike‐phase coupling identified the delta frequency band being dominated by the infragranular V1 and multiple frequency bands that were most pronounced in the supragranular V1. This inter‐area coupling was differentially modulated by full‐field synthetic and naturalistic visual stimulation. We also found that visual responses in LPl were distinct from those in V1 in terms of their reliability. Together, our data support a model of multiple communication channels between LPl and the layers of V1 that are enabled by oscillations in different frequency bands. This demonstration of anatomical and functional connectivity between LPl and V1 in ferrets provides a roadmap for studying the interaction dynamics during behaviour, and a template for identifying the activity dynamics of other thalamo‐cortical feedback loops.