Focal cortical dysplasia (FCD) is a developmental abnormality of cortex commonly linked with drug-resistant seizures. Altered GABAergic activity is a key contributor to interictal discharges in FCD. In FCD, GABAA receptor associated epileptogenicity is dependent upon the age at seizure onset, as differential epileptogenic networks are observed in early and late onset FCD patients. But the contribution of GABAA receptor alteration to epileptogenic networks during development is unclear. We hypothesize that GABAergic signaling in FCD undergoes age-dependent molecular alterations, contributing to the development of distinct epileptogenic networks. In this study, we investigated age-dependent changes in GABA neurotransmitter levels, GABAA receptor α subunit expression, and GABAA receptor-mediated synaptic activity using the BCNU-rat model of FCD. GABA levels, mRNA, and protein expression of GABAA receptor α subunits were determined by HPLC, qPCR and western blot and spontaneous GABAergic activity from pyramidal neurons was recorded using whole cell patch-clamp technique. At postnatal days (P) 12 and 21, reduced expression of α1, 2 and 4 subunits were observed in FCD rats compared to control. Consistent with this, decreased amplitude and frequency of GABAergic events were observed in FCD rats. In contrast, at P30 and P65, decreased GABA levels, without changes in receptor expression, were observed in FCD rats. Consistently, reduction in the frequency of GABAergic events was observed in FCD rats compared to the control. Furthermore, treatment with tetrodotoxin (TTX) revealed that the observed alterations in GABAergic activity were predominantly action potential (AP)-dependent. Our findings indicate that distinct epileptogenic networks exist in FCD during early and late developmental stages. These networks are driven primarily by altered GABAergic activity, with early age changes linked to aberrant GABAA receptor configurations and late age changes associated with abnormal GABA levels.

MicroRNA (miRNA) clusters, defined as genomically co-localized miRNAs regulated by a shared promoter and processed from polycistronic transcripts, exhibit synergistic regulatory roles in developmental processes. Among these, the evolutionarily conserved miR-125/let-7 cluster has been identified as a key regulator of neural stem cell (NSC) dynamics. In this study, we used Dicer conditional knockout (cKO) mice to confirm the essential role of miRNAs in mouse neocortical layer formation. The miR-125/let-7 cluster is co-expressed in mice and shows significant enrichment in upper-layer (UL) neurons. Using in utero electroporation (IUE), we found that miR-125b or let-7b overexpression partially rescues cortical phenotypes in Dicer-deficient mice, restoring proper UL organization but failing to rescue laminar fate defects in deep-layer cortical neurons. Our findings demonstrate that the miR-125b/let-7b exhibits a specialized function in regulating UL neuronal fate specification in mice and promotes the differentiation of NSC. Notably, miR-125b and let-7b exhibit both overlapping and distinct regulatory functions. Collectively, these results underscore the cooperative mechanisms by which miRNA clusters orchestrate cortical development.

Hippocampal neural stem/progenitor cells (HNPCs) in the hippocampus can differentiate into neurons and astrocytes, and are regulated by complex signaling pathways, such as the NOTCH1-signal transducer and activator of transcription 3 (STAT3) axis, which are crucial for cell fate determination. However, the exact molecular mechanism underlying HNPC differentiation remains unclear. This study investigated the role of NOTCH1-STAT3 pathway in HNPC differentiation into neuronal and astrocytic lineages during embryonic development. Mouse HNPCs were cultured with basic fibroblast and epidermal growth factors to promote proliferation. Differentiation was assessed using western blotting, immunofluorescence, RNA sequencing (RNA-seq), and reverse transcription-quantitative PCR to analyze gene expression. The roles of NOTCH1 and STAT3 in cell fate were assessed using their respective inhibitors, DAPT and Stattic, respectively. Immunoprecipitation was performed to investigate the interactions between NOTCH1 and STAT3. Proliferative conditions induced a shift from neurogenesis to astrocytic differentiation in HNPCs, as demonstrated by the increased GFAP and decreased TUJ1 levels. RNA-seq and gene ontology analyses revealed the upregulation of astrocyte-related genes and suppression of neurogenesis. NOTCH1 signaling promoted astrocytic differentiation through elevated DLL1 expression. Additionally, the inhibition of STAT3 or NOTCH1 reduced GFAP expression, whereas NOTCH1 knockdown reduced STAT3 activation, suggesting that NOTCH1 regulates astrocytic fate via STAT3 in proliferating HNPCs. These findings reveal the regulatory mechanisms of neural differentiation, emphasizing the critical role of the NOTCH1-STAT3 signaling axis in astrocytic differentiation of HNPCs, thereby enhancing our understanding of the molecular basis of neural cell fate decisions during brain development.

Neurodevelopmental studies employing animal models encounter challenges due to interspecies differences and ethical concerns. Maturing neurons of human origin, undergoing several developmental stages, present a powerful alternative. In this study, human embryonic stem cell (H9 cell line) was differentiated into neural stem cells and subsequently matured into neurons over 30 days. Ion AmpliSeq™ was used for transcriptomic characterization of human stem cell-derived neurons at multiple time points. Data analysis revealed a progressive increase of markers associated with neuronal development and astrocyte markers, indicating the establishment of a co-culture accommodating both glial and neurons. Transcriptomic and pathway enrichment analysis also revealed a more pronounced GABAergic phenotype in the neurons, signifying their specialization toward this cell type. The findings confirm the robustness of these cells across different passages and demonstrate detailed progression through stages of development. The model is intended for neurodevelopmental applications and can be adapted to investigate how genetic modifications or exposure to chemicals, pharmaceuticals, and other environmental factors influence neurons and glial maturation.

Adult stem cells residing in the body's tissues are responsible for the regeneration and replacement of old cells by new ones, thanks to their ability to differentiate. Scientific research increasingly focuses on the regeneration processes associated with these cells and the ability to modulate the microenvironment in which they are located. The modulatory effect can occur through direct interactions of stem cells with other cells or through their paracrine activity by releasing biologically active substances. For the nervous system, neural stem/progenitor cells are located in the subgranular zone in the hippocampal dentate gyrus and the subventricular zone around the lateral ventricles. This type of cell, in addition to giving rise to new neurons depending on the physiological state of the body, is also involved in the modulation of the niche in which they are found. This process plays a particular role in inflammation associated with many neurodegenerative diseases, which is connected with increased activity of the immune system cells. In this review article, we wanted to present the biologically active factors found in the neural stem/progenitor cells' secretome, which are key factors that can contribute physiologically to the silencing of inflammatory processes.

The development of the cerebral cortex is a stepwise process that involves numerous cell types and signaling pathways to achieve the functional assembly of neural circuits. Our understanding of this process is primarily rooted in findings from studying transgenic knockout models, which reveal coordinated molecular actions, particularly transcription factor cascades critical for cell type acquisition and maintenance in a context-dependent manner. Further resolving their cell type specificity necessitates the use of high-throughput, high-content methodologies. Over the past decade, the emerging single-cell genomics and in vivo CRISPR tools have provided new approaches to study neurodevelopment with elevated scale and resolution. In this review, we discussed efforts to study mouse cortical cell fate determination using single-cell genomics methods. Additionally, we explored recent studies combining programmable gene editing with single-cell phenotypic assays to investigate the function of transcription factors in perinatal cortical development, delineating cell-type specific, functional cytoarchitecture of the developing brain.

The adult mammalian brain exhibits remarkable forms of neural plasticity, enabling it to adapt and reorganize in response to internal and external stimuli. These plastic mechanisms include cytogenesis, the capacity of producing new neuronal and glial cells in restricted brain regions through processes known as neuro- and gliogenesis, respectively. Although many advances have been made in understanding adult brain plastic processes associated with cell genesis, as well as its functional and behavioral implications, most of the evidence is focused on neuronal cells. Even though astrocytes play a critical role in maintaining a neurochemical and electrophysiological homeostasis in the brain and provide a pivotal support to neuronal activity, the molecular mechanisms underlying the formation and functional integration of newly formed astroglial cells are poorly understood. However, some studies have provided key insights into the molecular mechanisms driving the generation of adult neural stem cell (NSC)-derived astrocytes, focusing on the dentate gyrus of the hippocampal cytogenic niche. Recent work has demonstrated that intrinsic and extrinsic factors can modulate astrogliogenesis. In the context of neuropathogenesis, this mechanism may be compromised in the hippocampus, contributing to functional and behavioral impairments. Here, we review the mechanisms underlying NSC-derived hippocampal astrogliogenesis, examining current perspectives on how adult-born astrocytes develop in the adult brain, their functional relevance, and the intricate regulation of the astrogliogenic process.

Disruptor of Telomeric Silencing 1-Like (DOT1L) is the sole methyltransferase in mammals responsible for catalyzing the mono-, di-, and trimethylation of histone H3 at lysine 79 (H3K79), a modification crucial for various cellular processes, including gene transcription, cell cycle regulation, DNA repair, and development. Recent studies have increasingly linked DOT1L to the nervous system, where it plays a vital role in neurodevelopment and neuronal function. It has been shown to regulate the proliferation and differentiation of neural progenitor cells, promote neuronal maturation, and influence synaptic function, all of which are essential for proper neural circuit formation and brain function. Moreover, dysregulation of DOT1L has been associated with several neurological disorders, highlighting its potential role in disease pathology. Abnormal expression or activity of DOT1L has been implicated in cognitive deficits and neurodegenerative diseases, underscoring the enzyme's significance in both the development and maintenance of the nervous system. This review synthesizes recent findings on DOT1L's role in the nervous system, emphasizing its importance in neurodevelopment and exploring its potential as a therapeutic target for treating neurological disorders.

Single-cell chromatin accessibility sequencing (scATAC-seq) reconstructs developmental trajectory by phenotypic similarity. However, inferring the exact developmental trajectory is challenging. Previous studies showed age-associated DNA methylation (DNAm) changes in specific genomic regions, termed clock-like differential methylation loci (ClockDML). Age-associated DNAm could either result from or result in chromatin accessibility changes at ClockDML. As cells undergo mitosis, the heterogeneity of chromatin accessibility on clock-like loci is reduced, providing a measure of mitotic age. In this study, we developed a method, called EpiTrace, that counts the fraction of opened clock-like loci from scATAC-seq data to determine cell age and perform lineage tracing in various cell lineages and animal species. It shows concordance with known developmental hierarchies, correlates well with DNAm-based clocks and is complementary with mutation-based lineage tracing, RNA velocity and stemness predictions. Applying EpiTrace to scATAC-seq data reveals biological insights with clinically relevant implications, ranging from hematopoiesis, organ development, tumor biology and immunity to cortical gyrification.

The subventricular zone (SVZ) is a region surrounding the lateral ventricles that contains neural stem cells and neural progenitor cells, which can proliferate and differentiate into various neural and glial cells. SVZ cells play important roles in neurological diseases like neurodegeneration, neural injury, and glioblastoma multiforme. Investigating the anatomy, structure, composition, physiology, disease associations, and related mechanisms of SVZ is significant for neural stem cell therapy and treatment/prevention of neurological disorders. However, challenges remain regarding the mechanisms regulating SVZ cell proliferation, differentiation, and migration, delivering cells to damaged areas, and immune responses. In-depth studies of SVZ functions and related therapeutic developments may provide new insights and approaches for treating brain injuries and degenerative diseases, as well as a scientific basis for neural stem cell therapy. This review summarizes research findings on SVZ and neurological diseases to provide references for relevant therapies.

Rationale: As a highly plastic population, parenchymal astrocytes have demonstrated the capacity to become activated and recapitulate neurogenic potential in response to traumatic central nervous system (CNS) injuries, representing a latent reservoir for neuronal regeneration in non-neurogenic brain regions. However, the extrinsic and intrinsic factors regulating this process remain poorly characterized. Elucidating these molecular mechanisms is crucial to harnessing the regenerative potential of reactive astrocytes in CNS repair. Methods: A multidisciplinary approach combining immunostaining, western blotting, RNA interference (RNAi), gene knock out and fate-mapping was used to investigate the role of topoisomerase IIα (TOP2a) in regulation of the stemness response in reactive astrocytes to traumatic brain injury (TBI). Results: Both in vitro and in vivo analyses demonstrated that TBI induces a stem cell-like response in reactive astrocytes concomitant with TOP2a upregulation. Pharmacological inhibition or genetic deletion of TOP2a significantly attenuated this stemness response. Neurosphere culture assay indicates that TOP2a might act as a downstream factor of Sonic Hedgehog (SHH) signaling to mediate the acquisition of stem cell potential. Conclusions: This study identifies TOP2a as a pivotal intrinsic regulator of astrocytic stem cell potential in the injured brain, which will advance our understanding of the molecular underpinnings of the stem cell response and its therapeutic application in neural regeneration.

In restricted regions of the rodent brain, neurogenesis persists throughout life, hinting that perhaps similar phenomena may exist in humans. Neural stem cells (NSCs) that reside within the ventricular-subventricular zone (V-SVZ) continually produce functional cells, including neurons that integrate into the olfactory bulb circuitry. The ability to achieve this feat is based on genetically encoded transcriptional programs that are controlled by environmentally regulated post-transcriptional signaling pathways. One such pathway that molds V-SVZ neurogenesis is the mTOR pathway. This pathway integrates nutrient sufficiency with growth factor signaling to control distinct steps of neurogenesis. Alterations in mTOR pathway signaling occur in numerous neurodevelopmental disorders. Here, we provide a narrative review for the role of the mTOR pathway in this process and discuss the use of this region to study the mTOR pathway in both health and disease.

Absence of the structural protein, dystrophin, results in the neuromuscular disorder Duchenne Muscular Dystrophy (DMD). In addition to progressive skeletal muscle dysfunction, this multisystemic disorder can also result in cognitive deficits and behavioural changes that are likely to be consequences of dystrophin loss from central neurons and astrocytes. Dystrophin-deficient mdx mice exhibit decreases in grey matter volume in the hippocampus, the brain region that encodes and consolidates memories, and this is exacerbated with ageing. To understand changes in cellular composition that might underpin these age-related developments, we have compared neurogenesis and the prevalence of immunofluorescently identified newly born and mature neurons, astrocytes and microglia in the dentate gyrus of mdx and wild-type mice at 2, 4, 8 and 16 months of age. The number of adult-born neurons was suppressed in the dentate gyrus subgranular zone of 2-month-old mdx mice. However, the numbers of granule cells and GABAA receptor, alpha 1-expressing cells were similar in wild-type and mdx mice at all ages. Strikingly, the numbers of astrocytes, particularly in the dentate gyrus molecular layer, were suppressed in mdx mice at all time points. Thus, dystrophin loss was associated with reduced hippocampal neurogenesis in early life but did not impact the prevalence of mature neurons across the lifespan of mdx mice. In contrast, normal age-related dentate gyrus astrocyte proliferation was suppressed in dystrophic mice. Astrocytes are the most abundant cell type in the brain and are crucial in supporting neuronal function, such that loss of these cells is likely to contribute to hippocampal dysfunction reported in mdx mice.

The IKKβ signaling pathway regulates NF-κB, influencing inflammation and cell survival in the brain. Radial glia cells are crucial for hippocampal neurogenesis and cognition. However, the role and mechanisms of IKKβ in modulating radial glia behavior and its impact on memory and neurogenesis remain unclear. Further studies are needed to understand how alterations in this pathway affect hippocampal function.

The role of IKKβ in memory and hippocampal neurogenesis was examined using GFAP-CreERT2/IKKβflox/flox mice with IKKβ knockdown in radial glia cells. IKKβ expression, NSC proliferation, and differentiation were assessed by immunohistochemistry. NF-κB and β-catenin interactions were evaluated by immunoprecipitation. Cultured adult hippocampal NSCs, with IKKβ or β-catenin shRNA transfection, were analyzed by flow cytometry and western blot to examine stem cell characteristics, NF-κB signaling, cell cycle, and β-catenin pathways.

Our results showed IKKβ cKD increased exploratory activity in the open-field and hyperactivity in the Y-maze, as well as enhanced spatial memory in the object location and Morris water maze tests. It also promoted adult hippocampal NSC proliferation by upregulating positive and inhibiting negative cell cycle regulators. Neuronal differentiation was enhanced, affecting β-catenin signaling and NeuroD1 expression. Additionally, IKKβ cKD promoted NSC survival, as shown by decreased cleaved caspase-3 and reduced Bax and cytochrome c in the hippocampus.

These findings suggest that in hippocampal NSCs, IKKβ inhibits locomotion, cognitive function, and adult hippocampal neurogenesis by suppressing the β-catenin signaling, highlighting its key role in decreasing hippocampal neurogenesis and cognitive function through NF-κB signaling in adult NSCs.

Haploinsufficiency of CHD7 (Chromo-Helicase-DNA binding protein 7) causes a severe congenital disease CHARGE syndrome. Brain anomaly such as microcephaly and olfactory bulb agenesis seen in CHARGE patients have not been mimicked in previous animal models. Here, we uncover an indispensable function of CHD7 in the neuroepithelium (NE) but not in the neural stem cells (NSCs) after NE transition. Loss of Chd7 in mouse NE resulted in CHARGE-like brain anomalies due to reduced proliferation and differentiation of neural stem and progenitor cells, which were recapitulated in CHD7 KO human forebrain organoids. Mechanistically, we find that CHD7 activates neural transcription factors by removing the repressive histone mark H3K27me3 and promoting chromatin accessibility. Importantly, neurodevelopmental defects caused by CHD7 loss in human brain organoids and mice were ameliorated by the inhibition of H3K27me3 methyltransferase EZH2. Altogether, by implementing appropriate experimental models, we uncover the pathogenesis of CHD7-associated neurodevelopmental diseases, and identify a potential therapeutic opportunity for CHARGE syndrome.

We determined the roles of two coevolved and coexpressed human-specific genes, NBPF14 and NOTCH2NLB, on the abundance of the cortical progenitors that underlie the evolutionary expansion of the neocortex, the seat of higher cognitive abilities in humans. Using automated microinjection into apical progenitors (APs) of embryonic mouse neocortex and electroporation of APs in chimpanzee cerebral organoids, we show that NBPF14 promotes the delamination of AP progeny, by promoting oblique cleavage plane orientation during AP division, leading to increased abundance of the key basal progenitor type, basal radial glia. In contrast, NOTCH2NLB promotes AP proliferation, leading to expansion of the AP pool. When expressed together, NBPF14 and NOTCH2NLB exert coordinated effects, resulting in expansion of basal progenitors while maintaining self-renewal of APs. Hence, these two human-specific genes orchestrate the behavior of APs, and the lineages of their progeny, in a manner essential for the evolutionary expansion of the human neocortex.

Neurogenesis and gliogenesis continue in the ventricular-subventricular zone (V-SVZ) of the adult rodent brain. V-SVZ astroglial cells with apical contact with the ventricle (B1 cells) function as neural stem cells (NSCs). B1 cells sharply decline during early postnatal life; in contrast, neurogenesis decreases at a slower rate. Here, we show that a second population of astroglia (B2 cells) that do not contact the ventricle also function as NSCs in the adult mouse brain. B2 cell numbers increase postnatally, are sustained in adults, and decrease with aging. We reveal the transcriptomic profile of B1 and B2 cells and show that, like B1 cells, B2 cells can be quiescent or activated. Transplantation and lineage tracing of B2 cells demonstrate their function as primary progenitors for adult neurogenesis. This study reveals that NSC function is progressively relayed from B1 to B2 progenitors helping explain how neurogenesis is maintained into adult life.

Female Alzheimer's disease (AD) patients display greater cognitive deficits and worse AD pathology as compared to male AD patients. In this study, we found that conditional knockout (cKO) of Atg5 in female microglia failed to obtain disease-associated microglia (DAM) gene signatures in familiar AD mouse model (5xFAD). Next, we analyzed the maintenance and neurogenesis of neural stem cells (NSCs) in the hippocampus and subventricular zone (SVZ) from 5xFAD mice with Atg5 cKO. Our data indicated that Atg5 cKO reduced the NSC number in hippocampus of female but not male 5xFAD mice. However, in the SVZ, Atg5 cKO only impaired NSCs in male 5xFAD mice. Interestingly, female 5xFAD;Fip200 cKO mice and 5xFAD;Atg14 cKO mice did not show NSC defects. These autophagy genes cKO 5xFAD mice exhibited a higher neurogenesis activity in their SVZ. Together, our data indicate a sex-specific role for microglial Atg5 in postnatal neurogenesis in AD mice.

Oxygen plays a critical role in early neural development in brains, particularly before establishment of complete vasculature; however, it has seldom been investigated due to technical limitations. This study uses an in vitro human cerebral organoid model with multiomic analysis, integrating advanced microscopies and single-cell RNA sequencing, to monitor tissue oxygen tension during neural development. Results reveal a key period between weeks 4 and 6 with elevated intra-organoid oxygen tension, altered energy homeostasis, and rapid neurogenesis within the organoids. The timed oxygen tension elevation can be suppressed by hypoxia treatment or silencing of neuroglobin gene. This study provides insights into the role of oxygen in early neurogenesis from functional, genotypic, phenotypic, and proteomic aspects. These findings highlight the significance of the timed tissue oxygen tension elevation in neurogenesis and provide insights into the role of neuroglobin in neural development, with potential implications for understanding neurodegenerative diseases and therapeutic strategies.

Adult neurogenesis is a unique cellular process of the ongoing generation of new neurons throughout life, which primarily occurs in the subgranular zone (SGZ) of the dentate gyrus (DG) and the subventricular zone (SVZ) of the lateral ventricle. In the adult DG, newly generated granule cells from neural stem cells (NSCs) integrate into existing neural circuits, significantly contributing to cognitive functions, particularly learning and memory. Recently, more and more studies have shown that rather than being a homogeneous population of identical cells, adult NSCs are composed of multiple subpopulations that differ in their morphology and function. In this study, we provide an overview of the origin, regional characteristics, prototypical morphology, and molecular factors that contribute to NSC heterogeneity. In particular, we discuss the molecular mechanisms underlying the balance between activation and quiescence of NSCs. In summary, this review highlights that deciphering NSC heterogeneity in the adult brain is a challenging but critical step in advancing our understanding of tissue-specific stem cells and the process of neurogenesis in the adult brain.

The cerebral cortex comprises diverse types of glutamatergic projection neurons (PNs) generated from radial glial progenitors (RGs) through either direct neurogenesis (dNG) or indirect neurogenesis (iNG) via intermediate progenitors (IPs). A foundational concept in corticogenesis is the "inside-out" model, whereby successive generations of PNs sequentially migrate first to deep and then progressively to more superficial layers. However, its biological significance remains unclear, and the role of iNG in this process is unknown. Using genetic strategies linking PN birth dating to projection mapping in mice, we found that the laminar deployment of IP-derived PNs substantially deviates from a stringent inside-out rule: PNs destined to non-consecutive layers are generated at the same time, and different PN types of the same layer are generated at non-contiguous times. The overarching scheme of iNG is the sequential specification and precise laminar deployment of projection-defined PN types, which may contribute to the orderly assembly of cortical output channels and processing streams.

During cortical development, radial glial cells (neural stem cells) initially are neurogenic, generating intermediate progenitor cells that exclusively produce glutamatergic pyramidal neurons. Next, radial glial cells generate tripotential intermediate progenitor cells (Tri-IPCs) that give rise to cortical astrocytes and oligodendrocytes, and olfactory bulb interneurons. The molecular mechanisms underlying the transition from cortical neurogenesis to gliogenesis, and the subsequent fate determination of cortical astrocytes, oligodendrocytes, and olfactory bulb interneurons, remain unclear. Here, we report that extracellular signal-regulated kinase (ERK) signaling plays a fundamental role in promoting cortical gliogenesis and the generation of Tri-IPCs. Additionally, sonic hedgehog-smoothened-glioma-associated oncogene homolog (SHH-SMO-GLI) activator signaling has an auxiliary function to ERK during these processes. We further demonstrate that, from Tri-IPCs, NOTCH signaling is crucial for the fate determination of astrocytes, while ERK signaling plays a prominent role in oligodendrocyte fate specification, and SHH signaling is required for the fate determination of olfactory bulb interneurons. We provide evidence suggesting that this mechanism is conserved in both mice and humans. Finally, we propose a unifying principle of mammalian cortical gliogenesis.

Regulated cell death is integral to sculpting the developing brain, yet the relative contributions of extrinsic apoptosis and necroptosis remain unclear. Here, we leverage single-cell mass cytometry (CyTOF) to characterize the cellular landscape of the mouse telencephalon in wild-type (WT), RIPK3 knockout (RIPK3 KO), and RIPK3/Caspase-8 double knockout (DKO) mice. Strikingly, combined deletion of RIPK3 and Caspase-8 leads to a 12.6% increase in total cell count, challenging the prevailing notion that intrinsic apoptosis exclusively governs developmental cell elimination. Detailed subpopulation analysis reveals that DKO mice display selective enrichment of Tbr2⁺ intermediate progenitors and endothelial cells, underscoring distinct, cell type-specific roles for extrinsic apoptotic and necroptotic pathways. These findings provide a revised framework for understanding the coordinated regulation of cell number during telencephalic development and suggest potential mechanistic links to neurodevelopmental disorders characterized by aberrant cell death.

Human's robust cognitive abilities, including creativity and language, are made possible, at least in large part, by evolutionary changes made to the cerebral cortex. This paper reviews the biology and evolution of mammalian cortical radial glial cells (primary neural stem cells) and introduces the concept that a genetically step wise process, based on a core molecular pathway already in use, is the evolutionary process that has molded cortical neurogenesis. The core mechanism, which has been identified in our recent studies, is the extracellular signal-regulated kinase (ERK)-bone morphogenic protein 7 (BMP7)-GLI3 repressor form (GLI3R)-sonic hedgehog (SHH) positive feedback loop. Additionally, I propose that the molecular basis for cortical evolutionary dwarfism, exemplified by the lissencephalic mouse which originated from a larger gyrencephalic ancestor, is an increase in SHH signaling in radial glia, that antagonizes ERK-BMP7 signaling. Finally, I propose that: (1) SHH signaling is not a key regulator of primate cortical expansion and folding; (2) human cortical radial glial cells do not generate neocortical interneurons; (3) human-specific genes may not be essential for most cortical expansion. I hope this review assists colleagues in the field, guiding research to address gaps in our understanding of cortical development and evolution.

The phenotypic and functional states of cells are modulated by a complex interactive molecular hierarchy of multiple omics layers, involving the genome, epigenome, transcriptome, proteome and metabolome. Spatial omics approaches have enabled the study of these layers in tissue context but are often limited to one or two modalities, offering an incomplete view of cellular identity. Here we present spatial-Mux-seq, a multimodal spatial technology that allows simultaneous profiling of five different modalities: two histone modifications, chromatin accessibility, whole transcriptome and a panel of proteins at tissue scale and cellular level in a spatially resolved manner. We applied this technology to mouse embryos and mouse brains, generating detailed multimodal tissue maps that identified more cell types and states compared to unimodal data. This analysis uncovered spatiotemporal relationships among histone modifications, chromatin accessibility, gene expression and protein levels during neuron differentiation, and revealed a radial glia niche with spatially dynamic epigenetic signals. Collectively, the spatial multi-omics approach heralds a new era for characterizing tissue and cellular heterogeneity that single-modality studies alone could not reveal.

Proper formation of the complex neurovascular unit (NVU) along with the blood-brain barrier is critical for building and sustaining a healthy, functioning central nervous system. The RNA binding protein argonaute2 (Ago2) mediates microRNA (miRNA)-mediated gene silencing, which is critical for many facets of brain development, including NVU development. Here, we found that Ago2 in glutamatergic neurons was critical for NVU formation in the developing cortices of mice. Glutamatergic neuron-specific loss of Ago2 diminished synaptic formation, neuronal-to-endothelial cell contacts, and morphogenesis of the brain vasculature, ultimately compromising the integrity of the blood-brain barrier. Ago2 facilitated miRNA targeting of phosphatase and tensin homolog (Pten) mRNA, which encodes a phosphatase that modulates reelin-dependent phosphatidylinositol 3-kinase (PI3K)-Akt signaling within the glutamatergic subpopulation. Conditionally deleting Pten in Ago2-deficient neurons restored Akt2 phosphorylation as well as postnatal development and survival. Several mutations in AGO2 impair small RNA silencing and are associated with Lessel-Kreienkamp syndrome, a neurodevelopmental disorder. When expressed in a neuronal cell line, these human AGO2 loss-of-function variants failed to suppress PTEN, resulting in attenuated PI3K-Akt signaling, further indicating that dysregulation of Ago2 function may contribute to both impaired development and neurological disorders. Together, these results identify Ago2 as central to the engagement of neurons with blood vessels in the developing brain.

RNA interference (RNAi) pathways are crucial for regulating viral infections in both animals and plants, acting as defense mechanisms that limit pathogen replication. This has led to the evolution of viral suppressors of RNA silencing (VSRs) across various plant and insect viruses, with potential analogs in arthropod-borne human pathogens. However, while functionally similar, VSRs often lack genetic conservation due to convergent evolution. Research on VSRs typically involves analyzing individual proteins expressed in host cells with secondary reporter constructs, but the lack of a standardized system can lead to inconsistent findings. Our study examined how genomic insertion sites affect VSR activity using a transgenic Drosophila melanogaster reporter system. We integrated the VSR protein DCV-1A into three different attP sites and assessed silencing. The results showed significant variation in VSR activity across loci due to position effects. However, by flanking the transgenes with gypsy retrovirus insulators, we achieved consistent high-level silencing across all sites. These findings suggest the potential for establishing a standardized reporter system in Drosophila , facilitating the identification, study and comparison of VSR proteins. However, our results also highlight the limitations of using isolated proteins in reporter systems, emphasizing the need for a comprehensive holistic approach to definitively determine VSR functions.

Myelination is fundamental to brain function, enabling rapid neural communication and supporting neuroplasticity throughout the lifespan. While hierarchical patterns of myelin maturation across the cortical surface are well-documented in humans, it remains unclear which features reflect evolutionarily conserved developmental processes versus human-characteristic adaptations. Moreover, the laminar development of myelin across the primate cortical surface, which shapes hierarchies and supports functions ranging from sensory integration to network communication, has been largely unexplored. Using neuroimaging to measure the T1-weighted/T2-weighted ratio in tissue contrast as a proxy for myelin content, we systematically compared depth-dependent trajectories of myelination across the cortical surface in humans and macaques. We identified a conserved "inside-out" pattern, with deeper layers exhibiting steeper increases in myelination and earlier plateaus than superficial layers. This depth-dependent organization followed a hierarchical gradient across the cortical surface, progressing from early maturation in sensorimotor regions to prolonged development in association areas. Humans exhibited a markedly extended timeline of myelination across both cortical regions and depths compared to macaques, allowing for prolonged postnatal plasticity across the entire cortical hierarchy - from sensory and motor processing to higher-order association networks. This extended potential for plasticity may facilitate the shaping of cortical circuits through postnatal experience in ways that support human-characteristic perceptual and cognitive capabilities.

The digestive tract represents the most complex interface of an organism with its biotope. Food may be contaminated by pathogens and toxicants while an abundant and complex microbiota strives in the gut lumen. The organism must defend itself against potentially noxious biotic or abiotic stresses while preserving its microbiota, provided it plays a beneficial role. The presence of intestinal viruses adds another layer of complexity. Starting from a differential sensitivity of two lines from the same Drosophila wild-type strain to ingested Pseudomonas aeruginosa, we report here that the presence of Nora virus in the gut epithelium promotes the sensitivity to this bacterial pathogen as well as to an ingested oxidizing xenobiotic. The genotype, age, nature of the ingested food and to a limited extent the microbiota are relevant parameters that influence the effects of Nora virus on host fitness. Mechanistically, we detect the initial presence of viral proteins essentially in progenitor cells. Upon stress such as infection, exposure to xenobiotics, aging or feeding on a rich-food diet, the virus is then detected in enterocytes, which correlates with a disruption of the intestinal barrier function in aged flies. Finally, we show that the virus proliferates only when ISCs are induced to divide and that blocking either enterocyte apoptosis or JAK/STAT-driven ISC division leads to a drastically reduced Nora virus titer. In conclusion, it is important to check that experimental strains are devoid of intestinal viruses when monitoring survival/life span of fly lines or when investigating the homeostasis of the intestinal epithelium as these viruses can constitute significant confounding factors.

Yin Yang 1 is a ubiquitously expressed transcription factor that has been extensively studied given its particular dual transcriptional regulation. Yin Yang 1 is involved in various cellular processes like cell cycle progression, cell differentiation, DNA repair, cell survival and apoptosis among others. Its malfunction or alteration leads to disease and even to malignant transformation. This transcription factor is essential for the proper central nervous system development and function. The activity of Yin Yang 1 depends on its interacting partners, promoter environment and chromatin structure, however, its mechanistic activity is not completely understood. In this review, we briefly discuss the Yin Yang 1 structure, post-translational modifications, interactions, mechanistic functions and its participation in neurodevelopment. We also discuss its expression and critical involvement in the physiology and physiopathology of glial cells, summarizing the contribution of Yin Yang 1 on different aspects of cellular function.

Tuberous sclerosis complex is a genetic disorder caused by mutations in the TSC1 or TSC2 genes, affecting multiple systems. These genes produce proteins that regulate mTORC1 activity, essential for cell function and metabolism. While mTOR inhibitors have advanced treatment, maintaining long-term therapeutic success is still challenging. For over 20 years, significant progress has linked TSC1 or TSC2 gene mutations in stem cells to tuberous sclerosis complex symptoms.

A comprehensive review was conducted using databases like Web of Science, Google Scholar, PubMed, and Science Direct, with search terms such as "tuberous sclerosis complex," "TSC1," "TSC2," "stem cell," "proliferation," and "differentiation." Relevant literature was thoroughly analyzed and summarized to present an updated analysis of the TSC1-TSC2 complex's role in stem cell fate determination and its implications for tuberous sclerosis complex.

The TSC1-TSC2 complex plays a crucial role in various stem cells, such as neural, germline, nephron progenitor, intestinal, hematopoietic, and mesenchymal stem/stromal cells, primarily through the mTOR signaling pathway.

This review aims shed light on the role of the TSC1-TSC2 complex in stem cell fate, its impact on health and disease, and potential new treatments for tuberous sclerosis complex.

Food-derived nucleic acids exhibit various biological activities and may act as nutrients. Oral ingestion of the nucleic acid fraction (NAF) of salmon milt extract hydrolysates enhances cognitive function in mice, although their active ingredients have not yet been identified, and detailed mechanisms of action are unknown.

To identify active ingredients enhancing cognitive function contained in the NAF and its possible underlying mechanism.

Because the NAF is rich in trinucleotides, proliferative effects of all 64 types of trideoxyribonucleotides were examined in primary cultured neural stem cells (pcNSCs). The active trideoxyribonucleotide was administered intrahippocampally (5 μg) in 7-wk-old male Institute of Cancer Research (ICR) mice (n = 4-6) or orally (1 mg) 3 times a week for 2 wk in 6-wk-old male ICR mice (n = 5), followed by evaluating neurogenesis and cognitive function by immunohistochemical analysis and spatial recognition test, respectively. The mechanism of action was examined by proteomic analysis of trideoxyribonucleotide-treated pcNSCs using the software DAVID, followed by western blot analysis in pcNSCs and hippocampus.

Among all trideoxyribonucleotides, only trideoxycytidine diphosphate (CCC) significantly promoted NSC proliferation (P < 0.05), whereas exposure to putative metabolites of CCC did not affect the proliferation. Intrahippocampal or oral CCC administration in mice increased doublecortin-positive cells in hippocampus (P < 0.05) and enhanced spatial memory (P < 0.05). Proteomic analysis revealed significant alterations in expression of PI3K-Akt signaling-related proteins, including eEF1A2 (P < 0.05). Intrahippocampal CCC administration significantly increased the Akt phosphorylation (p-Akt/Akt) (P < 0.05), which was abolished with the PI3K-Akt inhibitor, LY294002 (P < 0.05). CCC exposure increased p-Akt/Akt (P < 0.05) in pcNSCs, whereas LY294002 or small interfering RNA for eef1a2 suppressed CCC-induced increase in p-Akt/Akt (P < 0.05) and cell proliferation (P < 0.05).

A simple food-derived structural molecule CCC promotes NSC proliferation through eEF1A2/PI3K-Akt signaling pathway, thereby enhancing neurogenesis.

Heterozygous mutations in the histone lysine acetyltransferase gene KAT6B (MYST4/MORF/QKF) underlie neurodevelopmental disorders, but the mechanistic roles of KAT6B remain poorly understood. Here, we show that loss of KAT6B in embryonic neural stem and progenitor cells (NSPCs) impaired cell proliferation, neuronal differentiation, and neurite outgrowth. Mechanistically, loss of KAT6B resulted in reduced acetylation at histone H3 lysine 9 and reduced expression of key nervous system development genes in NSPCs and the developing cortex, including the SOX gene family, in particular Sox2, which is a key driver of neural progenitor proliferation, multipotency and brain development. In the fetal cortex, KAT6B occupied the Sox2 locus. Loss of KAT6B caused a reduction in Sox2 promoter activity in NSPCs. Sox2 overexpression partially rescued the proliferative defect of Kat6b -/- NSPCs. Collectively, these results elucidate molecular requirements for KAT6B in brain development and identify key KAT6B targets in neural precursor cells and the developing brain.

The importance of complex systems has become increasingly evident in recent years. The nervous system is one such example, with neural networks sitting at the intersection of complex networks and biology. A particularly exciting feature is the resilience of complex systems. For example, the ability of the nervous system to perform even in the face of challenges that include neuronal loss, neuroinflammation, protein accumulation, axonal disruptions, and metabolic stress is an intriguing and exciting line of investigation. In neurodegenerative diseases, neural network resilience is responsible for the time between the earliest disease-linked changes and clinical symptom onset and disease diagnosis. In this way, connectivity resilience of neurons within the complex network of cells that make up the nervous system has significant implications. This review provides an overview of relevant concepts related to complex systems with a focus on the connectivity of the nervous system. It discusses the development of the neural network and how a delicate balance determines how this complex system responds to injury, with examples illustrating maladaptive plasticity. The review then addresses the implications of these concepts, methods to understand brain connectivity and neural networks, and recent research efforts aimed at understanding neurodegeneration from this perspective. This study aims to provide foundational knowledge and an overview of current research directions in this evolving and exciting area of neuroscience.

The TEAD family of transcription factors are best known as the DNA-binding factor in the Hippo pathway, where they act by interacting with transcriptional coactivators YAP and TAZ (YAP/TAZ). Despite the importance of the Hippo pathway, the in vivo functions of TEAD in mammals have not been well established. By comparing mouse mutants lacking TEAD1 and TEAD2 (TEAD1/2) to those lacking YAP/TAZ, we found that TEAD1/2 have both YAP/TAZ-dependent and -independent functions during ventral telencephalon development. TEAD1/2 loss and YAP/TAZ loss similarly disrupt neuroepithelial apical junctions. However, the impacts of their losses on progenitor lineage progression are essentially opposite: Whereas YAP/TAZ loss depletes early progenitors and increases later progenitors-consistent with their established function in promoting progenitor self-renewal and proliferation, TEAD1/2 loss expands early progenitors and reduces late progenitors, indicating that TEAD1/2 promote lineage progression. We further show that TEAD1/2 promote neural progenitor lineage progression by, at least in part, inhibiting Notch signaling and by cooperating with Insulinoma-associated 1 (INSM1). Orthologs of TEAD and INSM1 have been shown to cooperatively regulate neuronal cell fate decisions in worms and flies. Our study reveals a remarkable evolutionary conservation of the function of this transcription factor complex during metazoan neural development.

Ischemic stroke is a significant global health crisis, frequently resulting in disability or death, with limited therapeutic interventions available. Although various intrinsic reparative processes are initiated within the ischemic brain, these mechanisms are often insufficient to restore neuronal functionality. This has led to intensive investigation into the use of exogenous stem cells as a potential therapeutic option. This comprehensive review outlines the ontogeny and mechanisms of activation of endogenous neural stem cells within the adult brain following ischemic events, with focus on the impact of stem cell-based therapies on neural stem cells. Exogenous stem cells have been shown to enhance the proliferation of endogenous neural stem cells via direct cell-to-cell contact and through the secretion of growth factors and exosomes. Additionally, implanted stem cells may recruit host stem cells from their niches to the infarct area by establishing so-called "biobridges." Furthermore, xenogeneic and allogeneic stem cells can modify the microenvironment of the infarcted brain tissue through immunomodulatory and angiogenic effects, thereby supporting endogenous neuroregeneration. Given the convergence of regulatory pathways between exogenous and endogenous stem cells and the necessity for a supportive microenvironment, we discuss three strategies to simultaneously enhance the therapeutic efficacy of both cell types. These approaches include: (1) co-administration of various growth factors and pharmacological agents alongside stem cell transplantation to reduce stem cell apoptosis; (2) synergistic administration of stem cells and their exosomes to amplify paracrine effects; and (3) integration of stem cells within hydrogels, which provide a protective scaffold for the implanted cells while facilitating the regeneration of neural tissue and the reconstitution of neural circuits. This comprehensive review highlights the interactions and shared regulatory mechanisms between endogenous neural stem cells and exogenously implanted stem cells and may offer new insights for improving the efficacy of stem cell-based therapies in the treatment of ischemic stroke.

In mice engineered to express enhanced green fluorescent protein (eGFP) under the control of the entire glutamate transporter 1 (GLT1) gene, eGFP is found in all 'adult' cortical astrocytes. However, when 8.3 kilobases of the human GLT1/EAAT2 promoter is used to control expression of tdTomato (tdT), tdT is only found in a subpopulation of these eGFP-expressing astrocytes. The eGFP mice have been used to define mechanisms of transcriptional regulation using astrocytes cultured from cortex of 1-3 day old mice. Using the same cultures, we were never able to induce tdT+ expression. We hypothesized that these cells might not have migrated into the cortex by this age. In this study, we characterized the ontogeny of tdT+ cells, performed single-cell RNA sequencing (scRNA-seq), and tracked their migration in organotypic slice cultures. At postnatal day (PND) 1, tdT+ cells were observed in the subventricular zone and striatum but not in the cortex, and they did not express eGFP. At PND7, tdT+ cells begin to appear in the cortex with their numbers increasing with age. At PND1, scRNA-seq demonstrates that the tdT+ cells are molecularly heterogeneous, with a subpopulation expressing astrocytic markers, subsequently validated with immunofluorescence. In organotypic slices, tdT+ cells migrate into the cortex, and after 7 days they express GLT1, NF1A, and eGFP. An ionotropic glutamate receptor (iGluR) antagonist reduced by 50% the distance tdT+ cells migrate from the subventricular zone into the cortex. The pan-glutamate transport inhibitor, TFB-TBOA, increased, by sixfold, the number of tdT+ cells in the cortex. In conclusion, although tdT is expressed by non-glial cells at PND1, it is also expressed by glial progenitors that migrate into the cortex postnatally. Using this fluorescent labeling, we provide novel evidence that glutamate signaling contributes to the control of glial precursor migration.

The deubiquitinating enzyme BAP1, the catalytic subunit of the PR-DUB complex, is implicated in several cancers, in the familial cancer syndrome BAP1 Tumor Predisposition Syndrome, and in the neurodevelopmental disorder Küry -Isidor syndrome. In Drosophila, there are numerous reports in the literature describing developmental patterning phenotypes for several chromatin regulators including the discovery of Polycomb itself, but corresponding adult morphological phenotypes caused by developmental dysregulation of Drosophila BAP1 ortholog calypso ( caly ) are less well-described. We report here that knockdown of caly in the eye and wing produce concomitant chromatin dysregulation phenotypes. RNAi to caly in the early eye reduces survival and leads to changes in eye size and shape including eye outgrowths, some of which resemble homeotic transformations whereas others resemble tumor-like outgrowths seen in other fly cancer models. Mosaic eyes containing caly loss-of-function tissue phenocopy caly RNAi. Knocking down caly across the wing disrupts wing shape and patterning including effects on wing vein pattern. This phenotypic characterization reinforces the growing body of literature detailing developmental mis-patterning driven by chromatin dysregulation and serves as a baseline for future mechanistic studies to understand the role of BAP1 in development and disease.

PR-DUB catalytic subunit deubiquitinating enzyme BAP1 plays an important role in tumor suppression and chromatin regulation. Whereas many chromatin regulators are well-characterized for their roles in patterning, the mis-patterning phenotypes in adult structure for dysregulating BAP1 ortholog calypso ( caly ) in development are less well described. We report mis-patterned adult eye and wing phenotypes caused by caly RNAi in the developing eye and wing respectively.

The development of the human neocortex is highly dynamic, involving complex cellular trajectories controlled by gene regulation1. Here we collected paired single-nucleus chromatin accessibility and transcriptome data from 38 human neocortical samples encompassing both the prefrontal cortex and the primary visual cortex. These samples span five main developmental stages, ranging from the first trimester to adolescence. In parallel, we performed spatial transcriptomic analysis on a subset of the samples to illustrate spatial organization and intercellular communication. This atlas enables us to catalogue cell-type-specific, age-specific and area-specific gene regulatory networks underlying neural differentiation. Moreover, combining single-cell profiling, progenitor purification and lineage-tracing experiments, we have untangled the complex lineage relationships among progenitor subtypes during the neurogenesis-to-gliogenesis transition. We identified a tripotential intermediate progenitor subtype-tripotential intermediate progenitor cells (Tri-IPCs)-that is responsible for the local production of GABAergic neurons, oligodendrocyte precursor cells and astrocytes. Notably, most glioblastoma cells resemble Tri-IPCs at the transcriptomic level, suggesting that cancer cells hijack developmental processes to enhance growth and heterogeneity. Furthermore, by integrating our atlas data with large-scale genome-wide association study data, we created a disease-risk map highlighting enriched risk associated with autism spectrum disorder in second-trimester intratelencephalic neurons. Our study sheds light on the molecular and cellular dynamics of the developing human neocortex.

The peripheral nervous system (PNS) includes all nerves outside the brain and spinal cord, comprising various cells like neurons and glial cells, such as schwann and satellite cells. The PNS is increasingly recognized for its bidirectional interactions with tumors, exhibiting both pro- and anti-tumor effects. Our review delves into the complex mechanisms underlying these interactions, highlighting recent findings that challenge the conventional understanding of PNS's role in tumorigenesis. We emphasize the contradictory results in the literature and propose novel perspectives on how these discrepancies can be resolved. By focusing on the PNS's influence on tumor initiation, progression, and microenvironment remodeling, we provide a comprehensive analysis that goes beyond the structural description of the PNS. Our review suggests that a deeper comprehension of the PNS-tumor crosstalk is pivotal for developing targeted anticancer strategies. We conclude by emphasizing the need for future research to unravel the intricate dynamics of the PNS in cancer, which may lead to innovative diagnostic tools and therapeutic approaches.

Neurodevelopmental disorders (NDD) encompass a range of conditions marked by abnormal brain development in conjunction with impaired cognitive, emotional and behavioural functions. Transgenic animal models, mainly rodents, traditionally served as key tools for deciphering the molecular mechanisms driving NDD physiopathology and significantly contributed to the development of pharmacological interventions aimed at treating these disorders. However, the efficacy of these treatments in humans has proven to be limited, due in part to the intrinsic constraint of animal models to recapitulate the complex development and structure of the human brain but also to the phenotypic heterogeneity found between affected individuals. Significant advancements in the field of induced pluripotent stem cells (iPSCs) offer a promising avenue for overcoming these challenges. Indeed, the development of advanced differentiation protocols for generating iPSC-derived brain organoids gives an unprecedented opportunity to explore human neurodevelopment. This review provides an overview of how 3D brain organoids have been used to investigate various NDD (i.e. Fragile X syndrome, Rett syndrome, Angelman syndrome, microlissencephaly, Prader-Willi syndrome, Timothy syndrome, tuberous sclerosis syndrome) and elucidate their pathophysiology. We also discuss the benefits and limitations of employing such innovative 3D models compared to animal models and 2D cell culture systems in the realm of personalized medicine.

Amniotic fluid is a complex and dynamic biological matrix that surrounds the fetus during the pregnancy. From this fluid, is possible to isolate various cell types with particular interest directed towards stem cells (AF-SCs). These cells are highly appealing due to their numerous potential applications in the field of regenerative medicine for tissues and organs as well as for treating conditions such as traumatic or ischemic injuries to the nervous system, myocardial infarction, or cancer. AF-SCs, when subcultured in the presence of basic Fibroblast Growth Factor (bFGF), have been shown to survive and migrate when transplanted into the striatum of the rat brain, exhibiting behavior characteristics of neuronal/glial progenitor cells. In this work, we performed an electrophysiological characterization to ascertain the propensity of AF-SCs to differentiate into glial and neuronal cells by bFGF. By using patch clamp technique we characterized a fibroblast-like morphology that display a barium-sensitive inward-rectifying potassium current (Kir) and calcium-activated potassium currents (KCa). The electrophysiological and calcium dynamics of histamine, a marker of undifferentiated neural progenitors, was further studied. Histamine promoted intracellular calcium increase by Fura-2 recording and calcium-activated potassium current activation with a similar temporal profile in AF-SC. The data presented in this paper ultimately confirm the expression in AF-SCs of the Kir and KCa currents, also showing regulation by endogenous stimuli such as histamine for the latter.