ARX regulates cortical interneuron differentiation and migration.

Mutations in aristaless-related homeobox ( ARX ) are associated with neurodevelopmental disorders including developmental epilepsies, intellectual disabilities, and autism spectrum disorders, with or without brain malformations. Aspects of these disorders have been linked to abnormal cortical interneuron (cIN) development and function. To further understand ARX's role in cIN development, multiple Arx mutant mouse lines were interrogated. We found that ARX is critical for controlling cIN numbers and distribution, especially, in the developing marginal zone (MZ). Single cell transcriptomics and ChIP-seq, combined with functional studies, revealed ARX directly or indirectly regulates genes involved in proliferation and the cell cycle (e.g., Bub3 , Cspr3 ), fate specification (e.g., Nkx2.1 , Maf , Mef2c ), and migration (e.g., Nkx2.1 , Lmo1 , Cxcr4 , Nrg1 , ErbB4 ). Our data suggest that the MZ stream defects primarily result from disordered cell-cell communication. Together our findings provide new insights into the mechanisms underlying cIN development and migration and how they are disrupted in several disorders.

The spectrum of movement disorders in young children with ARX-related epilepsy-dyskinesia syndrome.

Children with developmental and epileptic encephalopathies often present with co-occurring dyskinesias. Pathogenic variants in ARX cause a pleomorphic syndrome that includes infantile epilepsy with a variety of movement disorders ranging from focal hand dystonia to generalized dystonia with frequent status dystonicus. In this report, we present three patients with severe movement disorders as part of ARX-associated epilepsy-dyskinesia syndrome, including a patient with a novel pathogenic missense variant (p.R371G). These cases illustrate diagnostic and management challenges of ARX-related disorder and shed light on broader challenges concerning epilepsy-dyskinesia syndromes.

Shaping the brain: The emergence of cortical structure and folding.

The cerebral cortex-the brain's covering and largest region-has increased in size and complexity in humans and supports higher cognitive functions such as language and abstract thinking. There is a growing understanding of the human cerebral cortex, including the diversity and number of cell types that it contains, as well as of the developmental mechanisms that shape cortical structure and organization. In this review, we discuss recent progress in our understanding of molecular and cellular processes, as well as mechanical forces, that regulate the folding of the cerebral cortex. Advances in human genetics, coupled with experimental modeling in gyrencephalic species, have provided insights into the central role of cortical progenitors in the gyrification and evolutionary expansion of the cerebral cortex. These studies are essential for understanding the emergence of structural and functional organization during cortical development and the pathogenesis of neurodevelopmental disorders associated with cortical malformations.

Cell lineage analysis with somatic mutations reveals late divergence of neuronal cell types and cortical areas in human cerebral cortex.

The mammalian cerebral cortex shows functional specialization into regions with distinct neuronal compositions, most strikingly in the human brain, but little is known in about how cellular lineages shape cortical regional variation and neuronal cell types during development. Here, we use somatic single nucleotide variants (sSNVs) to map lineages of neuronal sub-types and cortical regions. Early-occurring sSNVs rarely respect Brodmann area (BA) borders, while late-occurring sSNVs mark neuron-generating clones with modest regional restriction, though descendants often dispersed into neighboring BAs. Nevertheless, in visual cortex, BA17 contains 30-70% more sSNVs compared to the neighboring BA18, with clones across the BA17/18 border distributed asymmetrically and thus displaying different cortex-wide dispersion patterns. Moreover, we find that excitatory neuron-generating clones with modest regional restriction consistently share low-mosaic sSNVs with some inhibitory neurons, suggesting significant co-generation of excitatory and some inhibitory neurons in the dorsal cortex. Our analysis reveals human-specific cortical cell lineage patterns, with both regional inhomogeneities in progenitor proliferation and late divergence of excitatory/inhibitory lineages.

Exome Sequencing and the Identification of New Genes and Shared Mechanisms in Polymicrogyria.

Importance: Polymicrogyria is the most commonly diagnosed cortical malformation and is associated with neurodevelopmental sequelae including epilepsy, motor abnormalities, and cognitive deficits. Polymicrogyria frequently co-occurs with other brain malformations or as part of syndromic diseases. Past studies of polymicrogyria have defined heterogeneous genetic and nongenetic causes but have explained only a small fraction of cases. Objective: To survey germline genetic causes of polymicrogyria in a large cohort and to consider novel polymicrogyria gene associations. Design, Setting, and Participants: This genetic association study analyzed panel sequencing and exome sequencing of accrued DNA samples from a retrospective cohort of families with members with polymicrogyria. Samples were accrued over more than 20 years (1994 to 2020), and sequencing occurred in 2 stages: panel sequencing (June 2015 to January 2016) and whole-exome sequencing (September 2019 to March 2020). Individuals seen at multiple clinical sites for neurological complaints found to have polymicrogyria on neuroimaging, then referred to the research team by evaluating clinicians, were included in the study. Targeted next-generation sequencing and/or exome sequencing were performed on probands (and available parents and siblings) from 284 families with individuals who had isolated polymicrogyria or polymicrogyria as part of a clinical syndrome and no genetic diagnosis at time of referral from clinic, with sequencing from 275 families passing quality control. Main Outcomes and Measures: The number of families in whom genetic sequencing yielded a molecular diagnosis that explained the polymicrogyria in the family. Secondarily, the relative frequency of different genetic causes of polymicrogyria and whether specific genetic causes were associated with co-occurring head size changes were also analyzed. Results: In 32.7% (90 of 275) of polymicrogyria-affected families, genetic variants were identified that provided satisfactory molecular explanations. Known genes most frequently implicated by polymicrogyria-associated variants in this cohort were PIK3R2, TUBB2B, COL4A1, and SCN3A. Six candidate novel polymicrogyria genes were identified or confirmed: de novo missense variants in PANX1, QRICH1, and SCN2A and compound heterozygous variants in TMEM161B, KIF26A, and MAN2C1, each with consistent genotype-phenotype relationships in multiple families. Conclusions and Relevance: This study's findings reveal a higher than previously recognized rate of identifiable genetic causes, specifically of channelopathies, in individuals with polymicrogyria and support the utility of exome sequencing for families affected with polymicrogyria.

Glial dysregulation in the human brain in fragile X-associated tremor/ataxia syndrome.

Short trinucleotide expansions at the FMR1 locus are associated with the late-onset condition fragile X-associated tremor/ataxia syndrome (FXTAS), which shows very different clinical and pathological features from fragile X syndrome (associated with longer expansions), with no clear molecular explanation for these marked differences. One prevailing theory posits that the shorter, premutation expansion uniquely causes extreme neurotoxic increases in FMR1 mRNA (i.e., four to eightfold increases), but evidence to support this hypothesis is largely derived from analysis of peripheral blood. We applied single-nucleus RNA sequencing to postmortem frontal cortex and cerebellum from 7 individuals with premutation and matched controls (n = 6) to assess cell type-specific molecular neuropathology. We found only modest upregulation (~1.3-fold) of FMR1 in some glial populations associated with premutation expansions. In premutation cases, we also identified decreased astrocyte proportions in the cortex. Differential expression and gene ontology analysis demonstrated altered neuroregulatory roles of glia. Using network analyses, we identified cell type-specific and region-specific patterns of FMR1 protein target gene dysregulation unique to premutation cases, with notable network dysregulation in the cortical oligodendrocyte lineage. We used pseudotime trajectory analysis to determine how oligodendrocyte development was altered and identified differences in early gene expression in oligodendrocyte trajectories in premutation cases specifically, implicating early cortical glial developmental perturbations. These findings challenge dogma regarding extremely elevated FMR1 increases in FXTAS and implicate glial dysregulation as a critical facet of premutation pathophysiology, representing potential unique therapeutic targets directly derived from the human condition.

TMEM161B regulates cerebral cortical gyration, Sonic Hedgehog signaling, and ciliary structure in the developing central nervous system.

Sonic hedgehog signaling regulates processes of embryonic development across multiple tissues, yet factors regulating context-specific Shh signaling remain poorly understood. Exome sequencing of families with polymicrogyria (disordered cortical folding) revealed multiple individuals with biallelic deleterious variants in TMEM161B, which encodes a multi-pass transmembrane protein of unknown function. Tmem161b null mice demonstrated holoprosencephaly, craniofacial midline defects, eye defects, and spinal cord patterning changes consistent with impaired Shh signaling, but were without limb defects, suggesting a CNS-specific role of Tmem161b. Tmem161b depletion impaired the response to Smoothened activation in vitro and disrupted cortical histogenesis in vivo in both mouse and ferret models, including leading to abnormal gyration in the ferret model. Tmem161b localizes non-exclusively to the primary cilium, and scanning electron microscopy revealed shortened, dysmorphic, and ballooned ventricular zone cilia in the Tmem161b null mouse, suggesting that the Shh-related phenotypes may reflect ciliary dysfunction. Our data identify TMEM161B as a regulator of cerebral cortical gyration, as involved in primary ciliary structure, as a regulator of Shh signaling, and further implicate Shh signaling in human gyral development.

Loss of non-motor kinesin KIF26A causes congenital brain malformations via dysregulated neuronal migration and axonal growth as well as apoptosis.

Kinesins are canonical molecular motors but can also function as modulators of intracellular signaling. KIF26A, an unconventional kinesin that lacks motor activity, inhibits growth-factor-receptor-bound protein 2 (GRB2)- and focal adhesion kinase (FAK)-dependent signal transduction, but its functions in the brain have not been characterized. We report a patient cohort with biallelic loss-of-function variants in KIF26A, exhibiting a spectrum of congenital brain malformations. In the developing brain, KIF26A is preferentially expressed during early- and mid-gestation in excitatory neurons. Combining mice and human iPSC-derived organoid models, we discovered that loss of KIF26A causes excitatory neuron-specific defects in radial migration, localization, dendritic and axonal growth, and apoptosis, offering a convincing explanation of the disease etiology in patients. Single-cell RNA sequencing in KIF26A knockout organoids revealed transcriptional changes in MAPK, MYC, and E2F pathways. Our findings illustrate the pathogenesis of KIF26A loss-of-function variants and identify the surprising versatility of this non-motor kinesin.

Impaired catabolism of free oligosaccharides due to MAN2C1 variants causes a neurodevelopmental disorder.

Free oligosaccharides (fOSs) are soluble oligosaccharide species generated during N-glycosylation of proteins. Although little is known about fOS metabolism, the recent identification of NGLY1 deficiency, a congenital disorder of deglycosylation (CDDG) caused by loss of function of an enzyme involved in fOS metabolism, has elicited increased interest in fOS processing. The catabolism of fOSs has been linked to the activity of a specific cytosolic mannosidase, MAN2C1, which cleaves α1,2-, α1,3-, and α1,6-mannose residues. In this study, we report the clinical, biochemical, and molecular features of six individuals, including two fetuses, with bi-allelic pathogenic variants in MAN2C1; the individuals are from four different families. These individuals exhibit dysmorphic facial features, congenital anomalies such as tongue hamartoma, variable degrees of intellectual disability, and brain anomalies including polymicrogyria, interhemispheric cysts, hypothalamic hamartoma, callosal anomalies, and hypoplasia of brainstem and cerebellar vermis. Complementation experiments with isogenic MAN2C1-KO HAP1 cells confirm the pathogenicity of three of the identified MAN2C1 variants. We further demonstrate that MAN2C1 variants lead to accumulation and delay in the processing of fOSs in proband-derived cells. These results emphasize the involvement of MAN2C1 in human neurodevelopmental disease and the importance of fOS catabolism.

Systematic Analysis of Brain MRI Findings in Adaptor Protein Complex 4-Associated Hereditary Spastic Paraplegia.

BACKGROUND AND OBJECTIVES: AP-4-associated hereditary spastic paraplegia (AP-4-HSP: SPG47, SPG50, SPG51, SPG52) is an emerging cause of childhood-onset hereditary spastic paraplegia and mimic of cerebral palsy. This study aims to define the spectrum of brain MRI findings in AP-4-HSP and to investigate radioclinical correlations. METHODS: We performed a systematic qualitative and quantitative analysis of 107 brain MRI studies from 76 individuals with genetically confirmed AP-4-HSP and correlation with clinical findings including surrogates of disease severity. RESULTS: We define AP-4-HSP as a disorder of gray and white matter and demonstrate that abnormal myelination is common and that metrics of reduced white matter volume correlate with severity of motor symptoms. We identify a common diagnostic imaging signature consisting of (1) a thin splenium of the corpus callosum, (2) an absent or thin anterior commissure, (3) characteristic signal abnormalities of the forceps minor ("ears of the grizzly sign"), and (4) periventricular white matter abnormalities. The presence of 2 or more of these findings has a sensitivity of ∼99% for detecting AP-4-HSP; the combination of all 4 is found in ∼45% of cases. Compared to other HSPs with a thin corpus callosum, the absent anterior commissure appears to be specific to AP-4-HSP. Our analysis identified a subset of patients with polymicrogyria, underscoring the role of AP-4 in early brain development. These patients displayed a higher prevalence of seizures and status epilepticus, many at a young age. DISCUSSION: Our findings define the MRI spectrum of AP-4-HSP, providing opportunities for early diagnosis, identification of individuals at risk for complications, and a window into the role of the AP-4 complex in brain development and neurodegeneration.

Early role for a Na+,K+-ATPase (ATP1A3) in brain development.

Osmotic equilibrium and membrane potential in animal cells depend on concentration gradients of sodium (Na+) and potassium (K+) ions across the plasma membrane, a function catalyzed by the Na+,K+-ATPase α-subunit. Here, we describe ATP1A3 variants encoding dysfunctional α3-subunits in children affected by polymicrogyria, a developmental malformation of the cerebral cortex characterized by abnormal folding and laminar organization. To gain cell-biological insights into the spatiotemporal dynamics of prenatal ATP1A3 expression, we built an ATP1A3 transcriptional atlas of fetal cortical development using mRNA in situ hybridization and transcriptomic profiling of ∼125,000 individual cells with single-cell RNA sequencing (Drop-seq) from 11 areas of the midgestational human neocortex. We found that fetal expression of ATP1A3 is most abundant to a subset of excitatory neurons carrying transcriptional signatures of the developing subplate, yet also maintains expression in nonneuronal cell populations. Moving forward a year in human development, we profiled ∼52,000 nuclei from four areas of an infant neocortex and show that ATP1A3 expression persists throughout early postnatal development, most predominantly in inhibitory neurons, including parvalbumin interneurons in the frontal cortex. Finally, we discovered the heteromeric Na+,K+-ATPase pump complex may form nonredundant cell-type-specific α-ß isoform combinations, including α3-ß1 in excitatory neurons and α3-ß2 in inhibitory neurons. Together, the developmental malformation phenotype of affected individuals and single-cell ATP1A3 expression patterns point to a key role for α3 in human cortex development, as well as a cell-type basis for pre- and postnatal ATP1A3-associated diseases.

Parallel RNA and DNA analysis after deep sequencing (PRDD-seq) reveals cell type-specific lineage patterns in human brain.

Elucidating the lineage relationships among different cell types is key to understanding human brain development. Here we developed parallel RNA and DNA analysis after deep sequencing (PRDD-seq), which combines RNA analysis of neuronal cell types with analysis of nested spontaneous DNA somatic mutations as cell lineage markers, identified from joint analysis of single-cell and bulk DNA sequencing by single-cell MosaicHunter (scMH). PRDD-seq enables simultaneous reconstruction of neuronal cell type, cell lineage, and sequential neuronal formation ("birthdate") in postmortem human cerebral cortex. Analysis of two human brains showed remarkable quantitative details that relate mutation mosaic frequency to clonal patterns, confirming an early divergence of precursors for excitatory and inhibitory neurons, and an "inside-out" layer formation of excitatory neurons as seen in other species. In addition our analysis allows an estimate of excitatory neuron-restricted precursors (about 10) that generate the excitatory neurons within a cortical column. Inhibitory neurons showed complex, subtype-specific patterns of neurogenesis, including some patterns of development conserved relative to mouse, but also some aspects of primate cortical interneuron development not seen in mouse. PRDD-seq can be broadly applied to characterize cell identity and lineage from diverse archival samples with single-cell resolution and in potentially any developmental or disease condition.

The trajectory of gait development in mice.

OBJECTIVE: Gait irregularities are prevalent in neurodevelopmental disorders (NDDs). However, there is a paucity of information on gait phenotypes in NDD experimental models. This is in part due to the lack of understanding of the normal developmental trajectory of gait maturation in the mouse. MATERIALS AND METHODS: Using the DigiGait system, we have developed a quantitative, standardized, and reproducible assay of developmental gait metrics in commonly used mouse strains that can be added to the battery of mouse model phenotyping. With this assay, we characterized the trajectory of gait in the developing C57BL/6J and FVB/AntJ mouse lines. RESULTS: In both lines, a mature stride consisted of 40% swing and 60% stance in the forelimbs, which mirrors the mature human stride. In C57BL/6J mice, developmental trajectories were observed for stance width, paw overlap distance, braking and propulsion time, rate of stance loading, peak paw area, and metrics of intraindividual variability. In FVB/AntJ mice, developmental trajectories were observed for percent shared stance, paw overlap distance, rate of stance loading, and peak paw area, although in different directions than C57 mice. By accounting for the impact of body length on stride measurements, we demonstrate the importance of considering body length when interpreting gait metrics. CONCLUSION: Overall, our results show that aspects of mouse gait development parallel a timeline of normal human gait development, such as the percent of stride that is stance phase and swing phase. This study may be used as a standard reference for developmental gait phenotyping of murine models, such as models of neurodevelopmental disease.