Background: The M105I point mutation in α-SNAP (Soluble N-ethylmaleimide-sensitive factor attachment protein-alpha) leads in mice to a complex phenotype known as hyh (hydrocephalus with hop gait), characterized by cortical malformation and hydrocephalus, among other neuropathological features. Studies performed by our laboratory and others support that the hyh phenotype is triggered by a primary alteration in embryonic neural stem/progenitor cells (NSPCs) that leads to a disruption of the ventricular and subventricular zones (VZ/SVZ) during the neurogenic period. Besides the canonical role of α-SNAP in SNARE-mediated intracellular membrane fusion dynamics, it also negatively modulates AMP-activated protein kinase (AMPK) activity. AMPK is a conserved metabolic sensor associated with the proliferation/differentiation balance in NSPCs. Methods: Brain samples from hyh mutant mice (hydrocephalus with hop gait) (B6C3Fe-a/a-Napahyh/J) were analyzed by light microscopy, immunofluorescence, and Western blot at different developmental stages. In addition, NSPCs derived from WT and hyh mutant mice were cultured as neurospheres for in vitro characterization and pharmacological assays. BrdU labeling was used to assess proliferative activity in situ and in vitro. Pharmacological modulation of AMPK was performed using Compound C (AMPK inhibitor) and AICAR (AMPK activator). Results: α-SNAP was preferentially expressed in the brain, showing variations in the levels of α-SNAP protein in different brain regions and developmental stages. NSPCs from hyh mice (hyh-NSPCs) displayed reduced levels of α-SNAP and increased levels of phosphorylated AMPKα (pAMPKαThr172), which were associated with a reduction in their proliferative activity and a preferential commitment with the neuronal lineage. Interestingly, pharmacological inhibition of AMPK in hyh-NSPCs increased proliferative activity and completely abolished the increased generation of neurons. Conversely, AICAR-mediated activation of AMPK in WT-NSPCs reduced proliferation and boosted neuronal differentiation. Discussion: Our findings support that α-SNAP regulates AMPK signaling in NSPCs, further modulating their neurogenic capacity. The naturally occurring M105I mutation of α-SNAP provokes an AMPK overactivation in NSPCs, thus connecting the α-SNAP/AMPK axis with the etiopathogenesis and neuropathology of the hyh phenotype.

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder that occurs in early childhood and can persist to adulthood. It can affect many aspects of a patient\'s daily life, so it is necessary to explore the mechanism and pathological alterations. For this purpose, we applied induced pluripotent stem cell (iPSC)-derived telencephalon organoids to recapitulate the alterations occurring in the early cerebral cortex of ADHD patients. We found that telencephalon organoids of ADHD showed less growth of layer structures than control-derived organoids. On day 35 of differentiation, the thinner cortex layer structures of ADHD-derived organoids contained more neurons than those of control-derived organoids. Furthermore, ADHD-derived organoids showed a decrease in cell proliferation during development from day 35 to 56. On day 56 of differentiation, there was a significant difference in the proportion of symmetric and asymmetric cell division between the ADHD and control groups. In addition, we observed increased cell apoptosis in ADHD during early development. These results show alterations in the characteristics of neural stem cells and the formation of layer structures, which might indicate key roles in the pathogenesis of ADHD. Our organoids exhibit the cortical developmental alterations observed in neuroimaging studies, providing an experimental foundation for understanding the pathological mechanisms of ADHD.

Thyroid hormone (TH) action controls brain development in a spatiotemporal manner. Previously, we demonstrated that perinatal hypothyroidism led to formation of a periventricular heterotopia in developing rats. This heterotopia occurs in the posterior telencephalon, and its formation was preceded by loss of radial glia cell polarity. As radial glia mediate cell migration and originate in a progenitor cell niche called the ventricular zone (VZ), we hypothesized that TH action may control cell signaling in this region. Here we addressed this hypothesis by employing laser capture microdissection and RNA-Seq to evaluate the VZ during a known period of TH sensitivity. Pregnant rats were exposed to a low dose of propylthiouracil (PTU, 0.0003%) through the drinking water during pregnancy and lactation. Dam and pup THs were quantified postnatally and RNA-Seq of the VZ performed in neonates. The PTU exposure resulted in a modest increase in maternal thyroid stimulating hormone and reduced thyroxine (T4). Exposed neonates exhibited hypothyroidism and T4 and triiodothyronine (T3) were also reduced in the telencephalon. RNA-Seq identified 358 differentially expressed genes in microdissected VZ cells of hypothyroid neonates as compared to controls (q-values ≤0.05). Pathway analyses showed processes like maintenance of the extracellular matrix and cytoskeleton, cell adhesion, and cell migration were significantly affected by hypothyroidism. Immunofluorescence also demonstrated that collagen IV, F-actin, radial glia, and adhesion proteins were reduced in the VZ. Immunohistochemistry of integrin αvβ3 and isoforms of both thyroid receptors (TRα/TRβ) showed highly overlapping expression patterns, including enrichment in the VZ. Taken together, our results show that TH action targets multiple components of cell junctions in the VZ, and this may be mediated by both genomic and nongenomic mechanisms. Surprisingly, this work also suggests that the blood-brain and blood-cerebrospinal fluid barriers may also be affected in hypothyroid newborns.

Mfge8, a secreted glycoprotein, is a key molecule that mediates the phagocytosis of apoptotic cells. Previous research reported that Mfge8 is critical for the proliferation and differentiation of radial glial cells (RGCs) in the dentate gyrus of adult mice. The treatment of Mfge8 is also beneficial for the repair of central nervous system (CNS) injury after cerebral ischemia. This study aimed to investigate whether the expression of mfge8a in zebrafish embryos was associated with the development of CNS and larval behavior. We found that zebrafish mfge8a was initially expressed at 48 hpf, and its expression was gradually increased in the ventricular zone. Knocking down mfge8a with antisense morpholino oligonucleotides impaired both spontaneous and photoinduced swimming locomotion in the behavioral tests. The neurogenesis analysis in telencephalon showed that mfge8a morphants excessively promoted neural differentiation over self-renewal after RGCs division, and consequently depleted proliferative RGC population during early neurogenesis. Furthermore, downregulation of mfge8a was shown to alter the expression patterns of genes associated with Notch signaling pathway. Our results demonstrated that mfge8a is involved in the maintenance of the progenitor identity of RGCs in embryonic zebrafish brain through regulating Notch signaling pathway, thereby contributing to consistent neurogenesis and locomotor development.

The cerebellum is a key player in many brain functions and a major topic of neuroscience research. However, the cerebellar nuclei (CN), the main output structures of the cerebellum, are often overlooked. This neglect is because research on the cerebellum typically focuses on the cortex and tends to treat the CN as relatively simple output nuclei conveying an inverted signal from the cerebellar cortex to the rest of the brain. In this review, by adopting a nucleocentric perspective we aim to rectify this impression. First, we describe CN anatomy and modularity and comprehensively integrate CN architecture with its highly organized but complex afferent and efferent connectivity. This is followed by a novel classification of the specific neuronal classes the CN comprise and speculate on the implications of CN structure and physiology for our understanding of adult cerebellar function. Based on this thorough review of the adult literature we provide a comprehensive overview of CN embryonic development and, by comparing cerebellar structures in various chordate clades, propose an interpretation of CN evolution. Despite their critical importance in cerebellar function, from a clinical perspective intriguingly few, if any, neurological disorders appear to primarily affect the CN. To highlight this curious anomaly, and encourage future nucleocentric interpretations, we build on our review to provide a brief overview of the various syndromes in which the CN are currently implicated. Finally, we summarize the specific perspectives that a nucleocentric view of the cerebellum brings, move major outstanding issues in CN biology to the limelight, and provide a roadmap to the key questions that need to be answered in order to create a comprehensive integrated model of CN structure, function, development, and evolution.

Heterozygous loss-of-function variants of FOXP4 are associated with neurodevelopmental disorders (NDDs) that exhibit delayed speech development, intellectual disability, and congenital abnormalities. The etiology of NDDs is unclear. Here we found that FOXP4 and N-cadherin are expressed in the nuclei and apical end-feet of radial glial cells (RGCs), respectively, in the mouse neocortex during early gestation. Knockdown or dominant-negative inhibition of Foxp4 abolishes the apical condensation of N-cadherin in RGCs and the integrity of neuroepithelium in the ventricular zone (VZ). Inhibition of Foxp4 leads to impeded radial migration of cortical neurons and ectopic neurogenesis from the proliferating VZ. The ectopic differentiation and deficient migration disappear when N-cadherin is over-expressed in RGCs. The data indicate that Foxp4 is essential for N-cadherin-based adherens junctions, the loss of which leads to periventricular heterotopias. We hypothesize that FOXP4 variant-associated NDDs may be caused by disruption of the adherens junctions and malformation of the cerebral cortex.

Interactions between angiogenesis and neurogenesis regulate embryonic brain development. However, a comprehensive understanding of the stages of vascular cell maturation is lacking, especially in the prenatal human brain. Using fluorescence-activated cell sorting, single-cell transcriptomics, and histological and ultrastructural analyses, we show that an ensemble of endothelial and mural cell subtypes tile the brain vasculature during the second trimester. These vascular cells follow distinct developmental trajectories and utilize diverse signaling mechanisms, including collagen, laminin, and midkine, to facilitate cell-cell communication and maturation. Interestingly, our results reveal that tip cells, a subtype of endothelial cells, are highly enriched near the ventricular zone, the site of active neurogenesis. Consistent with these observations, prenatal vascular cells transplanted into cortical organoids exhibit restricted lineage potential that favors tip cells, promotes neurogenesis, and reduces cellular stress. Together, our results uncover important mechanisms into vascular maturation during this critical period of human brain development.

The fluctuation of intracellular calcium concentration ([Ca2+]i) is known to be involved in various processes in the development of central nervous system, such as the proliferation of neural progenitor cells (NPCs), migration of intermediate progenitor cells (IPCs) from the ventricular zone (VZ) to the subventricular zone (SVZ), and migration of immature neurons from the SVZ to cortical plate. However, the roles of [Ca2+]i fluctuation in NPC development, especially in the differentiation of the self-renewing NPCs into neuron-generating NPCs and immature neurons have not been elucidated. Using calcium imaging of acute cortical slices and cells isolated from mouse embryonic cortex, we examined temporal changes in the pattern of [Ca2+]i fluctuations in VZ cells from E12 to E16. We observed intracellular Ca2+ levels in Pax6-positive self-renewing NPCs decreased with their neural differentiation. In E11, Pax6-positive NPCs and Tuj1-positive immature neurons exhibited characteristic [Ca2+]i fluctuations; few Pax6-positive NPCs exhibited [Ca2+]i transient, but many Tuj1-positive immature neurons did, suggesting that the change in pattern of [Ca2+]i fluctuation correlate to their differentiation. The [Ca2+]i fluctuation during NPCs development was mostly mediated by the T-type calcium channel and blockage of T-type calcium channel in neurosphere cultures increased the number of spheres and inhibited neuronal differentiation. Consistent with this finding, knockdown of Cav3.1 by RNAi in vivo maintained Pax6-positive cells as self-renewing NPCs, and simultaneously suppressing their neuronal differentiation of NPCs into Tbr1-positive immature neurons. These results reveal that [Ca2+]i fluctuation mediated by Cav3.1 is required for the neural differentiation of Pax6-positive self-renewing NPCs.

Developmental abnormalities of the cerebellum are among the most recognized structural brain malformations in human prenatal imaging. Yet reliable information regarding their cause in humans is sparse, and few outcome studies are available to inform prognosis. We know very little about human cerebellar development, in stark contrast to the wealth of knowledge from decades of research on cerebellar developmental biology of model organisms, especially mice. Recent studies show that multiple aspects of human cerebellar development significantly differ from mice and even rhesus macaques, a nonhuman primate. These discoveries challenge many current mouse-centric models of normal human cerebellar development and models regarding the pathogenesis of several neurodevelopmental phenotypes affecting the cerebellum, including Dandy-Walker malformation and medulloblastoma. Since we cannot model what we do not know, additional normative and pathological human developmental data are essential, and new models are needed.

The study of cerebellar development has been at the forefront of neuroscience since the pioneering work of Wilhelm His Sr., Santiago Ramón y Cajal and many others since the 19th century. They laid the foundation to identify the circuitry of the cerebellum, already revealing its stereotypic three-layered cortex and discerning several of its neuronal components. Their work was fundamental in the acceptance of the neuron doctrine, which acknowledges the key role of individual neurons in forming the basic units of the nervous system. Increasing evidence shows that the cerebellum performs a variety of homeostatic and higher order neuronal functions beyond the mere control of motor behaviour. Over the last three decades, many studies have revealed the molecular machinery that regulates distinct aspects of cerebellar development, from the establishment of a cerebellar anlage in the posterior brain to the identification of cerebellar neuron diversity at the single cell level. In this review, we focus on summarizing our current knowledge on early cerebellar development with a particular emphasis on the molecular determinants that secure neuron specification and contribute to the diversity of cerebellar neurons.