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While large trinucleotide repeat expansions at the FMR1 locus cause Fragile X Syndrome (FXS), smaller “premutations” are associated with the late-onset condition Fragile X-associated tremor/ataxia syndrome (FXTAS), which shows very different clinical and pathological features, with no clear molecular explanation for these marked differences. One prevailing theory posits that the premutation uniquely causes neurotoxic increases in FMR1 mRNA (i.e., 4-8-fold increases), but evidence to support this hypothesis is largely derived from analysis of peripheral blood. We applied single- nucleus RNA-sequencing to post-mortem frontal cortex and cerebellum from 9 individuals with Fragile X mutations as well as age and sex matched controls (n=6) to assess cell-type specific molecular neuropathology. We found robust reduction of FMR1 mRNA in FXS as expected, with modest but significant upregulation (∼1.3 fold) of FMR1 in glial clusters associated with premutation expansions. In premutation cases we identified alterations in glia number in cortex and cerebellum. Differential expression analysis demonstrated altered cortical oligodendrocyte development, while gene ontology analysis revealed alterations in neuroregulatory roles of glia, such as glial modulation of neurotransmission and synaptic structure. We identified significant enrichment of known FMR1 protein target genes in differentially expressed gene lists in FXS as well as the premutation, suggesting FMR1 protein target pathways may represent a shared source of dysfunction in both conditions despite opposite FMR1 mRNA changes. These findings challenge existing dogma regarding FXTAS and implicate glial dysregulation as a critical facet of premutation pathophysiology, representing novel therapeutic targets directly derived from the human condition.

International audience; Free oligosaccharides (fOSs) are soluble oligosaccharide species generated during N-glycosylation of proteins. Although little is knownabout fOS metabolism, the recent identification of NGLY1 deficiency, a congenital disorder of deglycosylation (CDDG) caused by loss offunction of an enzyme involved in fOS metabolism, has elicited increased interest in fOS processing. The catabolism of fOSs has beenlinked to the activity of a specific cytosolic mannosidase, MAN2C1, which cleaves a1,2-, a1,3-, and a1,6-mannose residues. In this study,we report the clinical, biochemical, and molecular features of six individuals, including two fetuses, with bi-allelic pathogenic variants inMAN2C1; the individuals are from four different families. These individuals exhibit dysmorphic facial features, congenital anomaliessuch as tongue hamartoma, variable degrees of intellectual disability, and brain anomalies including polymicrogyria, interhemisphericcysts, hypothalamic hamartoma, callosal anomalies, and hypoplasia of brainstem and cerebellar vermis. Complementation experimentswith isogenic MAN2C1-KO HAP1 cells confirm the pathogenicity of three of the identified MAN2C1 variants. We further demonstratethat MAN2C1 variants lead to accumulation and delay in the processing of fOSs in proband-derived cells. These results emphasize theinvolvement of MAN2C1 in human neurodevelopmental disease and the importance of fOS catabolism.

Significance By evaluating children with a malformed cerebral cortex, we identified an ATPase pump (ATP1A3) with an early role in brain development. The ATP1A3 pump maintains the physiological concentration of sodium and potassium ions in cells, a process critical for osmotic equilibrium and membrane potential across several developing cell populations. We employed single-cell sequencing approaches to identify key enrichments for ATP1A3 expression during human cortex development. Unravelling this early cell-type–specific pathophysiology in the developing brain offers a potential basis for the treatment of ATP1A3-related diseases affecting prenatal and early childhood 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-β isoform combinations, including α3-β1 in excitatory neurons and α3-β2 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.

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-β isoform combinations, including α3-β1 in excitatory neurons and α3-β2 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.

Background and ObjectivesAP-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.MethodsWe 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.ResultsWe 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.DiscussionOur 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.

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 that is catalyzed by the Na,K-ATPase alpha subunit. In vertebrates, four paralogous genes, ATP1A1-4, encode distinct alpha subunit isoforms (a1-a4), three of which (a1, a2, a3) are expressed in the brain, and two (a1, a3) predominantly in neurons. The a3 isoform, encoded by ATP1A3, is critical to neuronal physiology, and a growing spectrum of neurological diseases are associated with ATP1A3 pathogenic variants, with ages of onset ranging from early childhood to adulthood. Here, we describe ATP1A3 variants encoding dysfunctional a3 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 established a transcriptional atlas of ATP1A3 expression during cortical development using mRNA in situ hybridization and transcriptomic profiling of ~125,000 individual cells with single-cell RNA sequencing (Drop-Seq) from various areas of the midgestational human neocortex. We find that fetal expression of ATP1A3 is restricted to a subset of excitatory neurons carrying transcriptional signatures of neuronal activity and maturation characteristic of the developing subplate. Furthermore, by performing Drop-Seq on ~52,000 nuclei from four different areas of an infant human neocortex, we show that ATP1A3 expression persists throughout early postnatal development, not only within excitatory neurons across all cortical layers, but also and more predominantly in inhibitory neurons, with specific enrichment in fast-spiking basket cells. In addition, we show that ATP1A3 expression, both in fetal and postnatal neurons, tends to be higher in frontal cortical areas than in occipital areas, in a pattern consistent with the rostro-caudal maturation gradient of the human neocortex. Finally, we discover distinct co-expression patterns linking catalytic α subunit isoforms (ATP1A1,2,3) and auxiliary isoforms (ATP1B1,2,3), suggesting the ATPase pump may form non-redundant, cell-type specific α-β combinations. Together, the importance of the developmental phenotypes and dynamic expression patterns of ATP1A3 point to a key role for a3 in the development and function of human cortex.

Elucidating the lineage relationships among different cell types is key to understanding human brain development. Here we developedParallelRNA andDNA analysis afterDeep-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 the first-ever 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 the first 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.Significance StatementStem cells and progenitors undergo a series of cell divisions to generate the neurons of the brain, and understanding this sequence is critical to studying the mechanisms that control cell division and migration in developing brain. Mutations that occur as cells divide are known as the basis of cancer, but have more recently been shown to occur with normal cell divisions, creating a permanent, forensic map of the clonal patterns that define the brain. Here we develop new technology to analyze both DNA mutations and RNA gene expression patterns in single cells from human postmortem brain, allowing us to define clonal patterns among different types of human brain neurons, gaining the first direct insight into how they form.

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., To fill the gap in our understanding of the normal developmental trajectory of gait in the mouse, we characterized gait in two mouse strains across development. Our results revealed metrics that best reflect gait maturation in both strains and underscore the importance of considering body length when interpreting gait metrics. Overall, our results show that aspects of mouse gait development parallel a timeline of normal human gait development.

Visual Abstract, Serotonergic dysregulation is implicated in numerous psychiatric disorders. Serotonin plays widespread trophic roles during neurodevelopment; thus perturbations to this system during development may increase risk for neurodevelopmental disorders. Epidemiological studies have examined association between selective serotonin reuptake inhibitor (SSRI) treatment during pregnancy and increased autism spectrum disorder (ASD) risk in offspring. It is unclear from these studies whether ASD susceptibility is purely related to maternal psychiatric diagnosis, or if treatment poses additional risk. We sought to determine whether maternal SSRI treatment alone or in combination with genetically vulnerable background was sufficient to induce offspring behavior disruptions relevant to ASD. We exposed C57BL/6J or Celf6 +/- mouse dams to fluoxetine (FLX) during different periods of gestation and lactation and characterized offspring on tasks assessing social communicative interaction and repetitive behavior patterns including sensory sensitivities. We demonstrate robust reductions in pup ultrasonic vocalizations (USVs) and alterations in social hierarchy behaviors, as well as perseverative behaviors and tactile hypersensitivity. Celf6 mutant mice demonstrate social communicative deficits and perseverative behaviors, without further interaction with FLX. FLX re-exposure in adulthood ameliorates the tactile hypersensitivity yet exacerbates the dominance phenotype. This suggests acute deficiencies in serotonin levels likely underlie the abnormal responses to sensory stimuli, while the social alterations are instead due to altered development of social circuits. These findings indicate maternal FLX treatment, independent of maternal stress, can induce behavioral disruptions in mammalian offspring, thus contributing to our understanding of the developmental role of the serotonin system and the possible risks to offspring of SSRI treatment during pregnancy.

SummarySerotonergic dysregulation is implicated in psychiatric disorders, including autism spectrum disorders (ASD). Epidemiological studies suggest selective serotonin reuptake inhibitor (SSRI) treatment during pregnancy may increase ASD risk in offspring, however it is unclear from these studies whether ASD susceptibility is related to the maternal diagnosis or if treatment poses additional risk. Here, we exposed mouse dams to fluoxetine and characterized the offspring to isolate possible effects of SSRI exposure on ASD-relevant behaviors. We demonstrate social communication and interaction deficits and repetitive behaviors, with corresponding dendritic morphology changes in pertinent brain regions. Also, using a novel application of optical intrinsic signal imaging, we show altered stimulus-evoked cortical response and region-specific decreases in functional connectivity. These findings indicate drug exposure alone is sufficient to induce long-term behavioral, cellular, and hemodynamic-response disruptions in offspring, thus contributing to our understanding of ASD pathogenesis, risk and mechanism, as well as the developmental role of serotonin.

This chapter takes a look at stereotypes at the individual relationship level and proposes the tool of storytelling as a method for people to move beyond stereotypes. Stories of personal experiences have power because of their capacity to evoke empathy, to force perspective, and to build trust in relationships. Thus, storytelling represents a tool for individuals to use in personal development surrounding issues of identity, particularly race. Stories oppose stereotypes by providing context to people’s actions, mannerisms, and choices. If the sin of stereotyping is oversimplification, storytelling allows us to describe experiences with the feeling, personal commentary, and context necessary to help people overcome implicit and explicit biases.

While Washington University in St. Louis (the University) has enjoyed success in recruiting higher numbers of low-income students, it has not achieved comparable results in ensuring an equally successful college experience for this underrepresented group. Data shows that low-income students, as compared to their financially unaided peers, have an inequitable undergraduate experience at Washington University that includes performance gaps in STEM-intensive curricula and less-robust co-curricular experiences. This chapter presents the outcomes of a report entitled “Honoring Our Investment” that focused on how, from an institutional perspective, to best support the academic and co-curricular success of low-income students at Washington University. The Undergraduate Representatives to the Board of Trustees of the 2015–2016 academic year wrote this report and compiled information, research, and data about the low-income student experience as part of a dialogue focused on improvement. Additionally, this chapter recommends changes for improving outcomes.

After a person chooses between two items, preference for the chosen item will increase and preference for the unchosen item will decrease because of the choice made. In other words, we tend to justify or rationalize our past behavior by changing our attitude. This phenomenon of choice-induced preference change has been traditionally explained by cognitive dissonance theory. Choosing something that is disliked or not choosing something that is liked are both cognitively inconsistent and, to reduce this inconsistency, people tend to change their subsequently stated preference in accordance with their past choices. Previously, human neuroimaging studies identified posterior medial frontal cortex (pMFC) as a key brain region involved in cognitive dissonance. However, it remains unknown whether the pMFC plays a causal role in inducing preference change after cognitive dissonance. Here, we demonstrate that 25 min, 1 Hz repetitive transcranial magnetic stimulation applied over the pMFC significantly reduces choice-induced preference change compared with sham stimulation or control stimulation over a different brain region, demonstrating a causal role for the pMFC.