Radial Glia Research Articles

CCRG-02. DEVELOPMENTALLY PARTITIONED PROLIFERATIVE COMPARTMENTS IN GLIOBLASTOMA

Abstract It is now well established from single-cell RNA-seq studies that glioblastoma (GBM) tumors are complex, maligned neuro-developmental ecosystems harboring diverse tumor cell types. Neoplastic cells can resemble astrocytes, neural progenitors, oligodendrocyte progenitor cells, mesenchymal cells, and radial glial cells that contribute to tumor growth and homeostasis in specific ways. However, GBM single-cell data sets have failed to produce general models for transitions in and out of specific developmental and proliferative states in tumors. One reason is that human GBM tumors do not neatly resolve into developmental hierarchies. Here we focused on modeling GBM tumor cellular heterogeneity by defining "proliferative compartments" in single-cell transcriptomic data derived from primary tumors and early passage tumorsphere cultures. Previously, we observed that each tumor cell entering S-phase has a unique developmental signature that can be leveraged to define broader partitioned proliferative compartments (PPCs) with distinct developmental gene expression and genomic alteration patterns. Thus, we extracted the S-phase cells from a tumor, defined the proliferative compartments in that tumor using de novo clustering, determined the marker genes for each compartment, and defined the broader PPCs across tumors by de novo clustering based on similarity in marker genes. From a cohort of six tumors we observed eight broader PPCs and found that tumors can contain as many as five PPCs or as few as two. Because tumor growth and recurrence both require cell proliferation, we propose that patient-specific PPCs represent the engines of GBM progression.

STEM-17. IMPLICATING ASTROCYTE PROGENITOR SIGNATURES FROM THE DEVELOPING HUMAN SUBVENTRICULAR ZONE IN GLIOBLASTOMA PROGRESSION AND RECURRENCE

Abstract Although astrocyte-like progenitors in the adult subventricular zone (SVZ) have been put forward as the glioblastoma (GBM) cell of origin, the mechanisms that trigger astrogenesis in the developing SVZ are poorly understood. We previously reconstructed astrocyte lineages using computational tools and identified PDGFRβ+ astrogenesis-committed radial glia (RG-A) and EGFR+ gliogenic intermediate progenitor cells (gIPCs). We have, however, yet to identify the transcription factors (TFs) that drive the differentiation of these progenitors in the pre- and postnatal SVZ. As malignant cells are known to co-opt neurodevelopmental pathways, the exploitation of TF driver vulnerabilities in malignant cells could bring novel therapeutic strategies to hinder GBM progression or recurrence. Here, we integrated single nucleus RNA- (snRNA-seq) and ATAC-sequencing (snATAC-seq) datasets from 1) surgically resected GBMs (n = 5), 2) the subventricular zone and neocortex from non-pathological adult cortices (n = 3) and 3) the corresponding germinal matrix and cortical plate of non-pathological cortices ranging from 17 to 41 gestational weeks (n = 17) and 21 to 301 postnatal days in age (n = 9). To prioritize candidate drivers of astrogenesis computationally, we correlated motif enrichment with gene expression over pseudotime in gIPC and RG-A lineages. Subsequently, we projected transcriptional signatures from non-pathological neocortices onto GBMs. Interestingly, we observed high proportions of tumor cells with preferentially enriched prenatal gIPC-like signature in EGFR amplified GBMs and with preferentially enriched postnatal RG-A-like signature in GBMs with PFGFRα or FGFR3 amplification. As tumor recurrence is associated with a mesenchymal-like switch, we were intrigued to find that RG-A-like cells exhibited a mesenchymal-like phenotype in a recurrent GBM. Currently, we are developing an in vitro assay to manipulate the expression of astrogenic drivers and force the differentiation of patient-derived, primary GBM cells into astrocytes; a strategy that we will later apply in patient-derived xenograft models.

CCRG-03. KAT5 REGULATES DEVELOPMENTAL TRANSITIONS AND CELLULAR HETEROGENEITY IN AN IN VIVO MODEL OF GLIOBLASTOMA

Abstract In solid tumors, G0-like states are likely critical for maintaining developmental hierarchies and cellular heterogeneity and promoting tumor growth/recurrence, yet little is known about tumor G0 states or regulation of their ingress/egress. To discover G0-like states and their regulators for glioblastoma (GBM), we previously performed a genome-wide CRISPR-Cas9 screen in patient-derived GBM stem-like cells (GSCs) for genes that trap cells in G0 when inhibited. We identify the protein acetyltransferase KAT5 as a key regulator of GBM G0 ingress/egress. Here, we show in an in vivo GSC-derived orthotopic xenograft model that KAT5 regulates transcriptional, epigenetic, and proliferative heterogeneity impacting transitions into G0-like states. We show that KAT5 activity suppresses the emergence of non-dividing subpopulations with oligodendrocyte progenitor and radial glial cell characteristics. With regard to chromatin regulation, we show that loss of KAT5 activity alters sites of mixed epigenetic valency (which have both activating and repressive marks in bulk tumor populations) to promote emergence of OPC/RG G0-like populations in tumors. Our data reveal KAT5-associated transcriptional and epigenetic changes in tumors, which indicate that KAT5 activity biases cell state distribution away from OPC and RG-like subpopulations. In addition, loss of KAT5 resulted in a less invasive phenotype and prolonged survival of mice harboring orthotopic xenografts.

TMIC-05. UNVEILING THE IMPACT OF PTN-PTPRZ1 SIGNALING ON GLIOBLASTOMA PROGRESSION THROUGH TUMOR MICROENVIRONMENT COMMUNICATION

Abstract Glioblastoma (GBM) is the most lethal primary brain tumor with high rate of recurrence. GBM’s high inter- and intra-heterogeneity present a huge challenge to both disease modeling and treatment. Recent progress in the field has highlighted the role of signals associated with the microenvironment factor in driving tumor progression, suggesting that more detailed characterizations of the GBM microenvironment may provide novel therapeutic targets. In this study, we used a novel stem cell derived cortical organoid transplantation model, where we transplanted patient IDH wild type GBM cells onto organoids and provided multiple culture conditions to explore the impact of the environment on tumor cell identity. After several weeks of culture, single cell RNA-sequencing of tumor and non-neoplastic organoid cells was used to profile molecular features, demonstrating not only cellular composition of GBM and its surrounding normal brain cell, but also the bidirectional communication between the two. We observed functional heterogeneity where individual tumors responded differently to environmental perturbations. However, the most significant cell-cell interaction between tumors and the microenvironment was found to be PTN-PTPRZ1, which is well-maintained across all transplantation models we tested, as well as in re-analysis of published patient data that included tumor and normal cells. Encouraged by our previous works as well as others, which have revealed PTPRZ1 as an outer radial glia marker gene driving tumor invasion, we further verified PTN/PTPRZ1 expression not only in our organoid transplantation models but also on FFPE sections from GBM patients and mouse xenograft samples. These data suggest that this signaling axis promotes tumor progression through tumor – microenvironmental crosstalk, and using the organoid model we show a functional role for this communication. Together, this study presents a GBM organoid transplantation modeling system, which we leverage to detect a promising therapeutically target existing between GBM and its microenvironment.

Non-synaptic function of the autism spectrum disorder-associated gene SYNGAP1 in cortical neurogenesis.

Genes involved in synaptic function are enriched among those with autism spectrum disorder (ASD)-associated rare genetic variants. Dysregulated cortical neurogenesis has been implicated as a convergent mechanism in ASD pathophysiology, yet it remains unknown how 'synaptic' ASD risk genes contribute to these phenotypes, which arise before synaptogenesis. Here, we show that the synaptic Ras GTPase-activating (RASGAP) protein 1 (SYNGAP1, a top ASD risk gene) is expressed within the apical domain of human radial glia cells (hRGCs). In a human cortical organoid model of SYNGAP1 haploinsufficiency, we find dysregulated cytoskeletal dynamics that impair the scaffolding and division plane of hRGCs, resulting in disrupted lamination and accelerated maturation of cortical projection neurons. Additionally, we confirmed an imbalance in the ratio of progenitors to neurons in a mouse model of Syngap1 haploinsufficiency. Thus, SYNGAP1-related brain disorders may arise through non-synaptic mechanisms, highlighting the need to study genes associated with neurodevelopmental disorders (NDDs) in diverse human cell types and developmental stages.

Temporal-spatial Generation of Astrocytes in the Developing Diencephalon.

Astrocytes are the largest glial population in the mammalian brain. However, we have a minimal understanding of astrocyte development, especially fate specification in different regions of the brain. Through lineage tracing of the progenitors of the third ventricle (3V) wall via in-utero electroporation in the embryonic mouse brain, we show the fate specification and migration pattern of astrocytes derived from radial glia along the 3V wall. Unexpectedly, radial glia located in different regions along the 3V wall of the diencephalon produce distinct cell types: radial glia in the upper region produce astrocytes and those in the lower region produce neurons in the diencephalon. With genetic fate mapping analysis, we reveal that the first population of astrocytes appears along the zona incerta in the diencephalon. Astrogenesis occurs at an early time point in the dorsal region relative to that in the ventral region of the developing diencephalon. With transcriptomic analysis of the region-specific 3V wall and lateral ventricle (LV) wall, we identified cohorts of differentially-expressed genes in the dorsal 3V wall compared to the ventral 3V wall and LV wall that may regulate astrogenesis in the dorsal diencephalon. Together, these results demonstrate that the generation of astrocytes shows a spatiotemporal pattern in the developing mouse diencephalon.

Estrogenic regulation of claudin 5 and tight junction protein 1 gene expression in zebrafish: A role on blood-brain barrier?

The blood-brain barrier (BBB) is a physical interface between the blood and the brain parenchyma, playing key roles in brain homeostasis. In mammals, the BBB is established thanks to tight junctions between cerebral endothelial cells, involving claudin, occludin, and zonula occludens proteins. Estrogens have been documented to modulate BBB permeability. Interestingly, in the brain of zebrafish, the estrogen-synthesizing activity is strong due to the high expression of Aromatase B protein, encoded by the cyp19a1b gene, in radial glial cells (neural stem cells). Given the roles of estrogens in BBB function, we investigated their impact on the expression of genes involved in BBB tight junctions. We treated zebrafish embryos and adult males with 17β-estradiol and observed an increased cerebral expression of tight junction and claudin 5 genes in adult males only. In females, treatment with the nuclear estrogen receptor antagonist (ICI182,780 ) had no impact. Interestingly, telencephalic injuries performed in males decreased tight junction gene expression that was partially reversed with 17β-estradiol. This was further confirmed by extravasation experiments of Evans blue showing that estrogenic treatment limits BBB leakage. We also highlighted the intimate links between endothelial cells and neural stem cells, suggesting that cholesterol and peripheral steroids could be taken up by endothelial cells and used as precursors for estrogen synthesis by neural stem cells. Together, our results show that zebrafish provides an alternative model to further investigate the role of steroids on the expression of genes involved in BBB integrity, both in constitutive and regenerative physiological conditions. The link we described between capillaries endothelial cells and steroidogenic neural cells encourages the use of this model in understanding the mechanisms by which peripheral steroids get into neural tissue and modulate neurogenic activity.

Echinoderm radial glia in adult cell renewal, indeterminate growth, and regeneration.

Echinoderms are a phylum of marine deterostomes with a range of interesting biological features. One remarkable ability is their impressive capacity to regenerate most of their adult tissues, including the central nervous system (CNS). The research community has accumulated data that demonstrates that, in spite of the pentaradial adult body plan, echinoderms share deep similarities with their bilateral sister taxa such as hemichordates and chordates. Some of the new data reveal the complexity of the nervous system in echinoderms. In terms of the cellular architecture, one of the traits that is shared between the CNS of echinoderms and chordates is the presence of radial glia. In chordates, these cells act as the main progenitor population in CNS development. In mammals, radial glia are spent in embryogenesis and are no longer present in adults, being replaced with other neural cell types. In non-mammalian chordates, they are still detected in the mature CNS along with other types of glia. In echinoderms, radial glia also persist into the adulthood, but unlike in chordates, it is the only known glial cell type that is present in the fully developed CNS. The echinoderm radial glia is a multifunctional cell type. Radial glia forms the supporting scaffold of the neuroepithelium, exhibits secretory activity, clears up dying or damaged cells by phagocytosis, and, most importantly, acts as a major progenitor cell population. The latter function is critical for the outstanding developmental plasticity of the adult echinoderm CNS, including physiological cell turnover, indeterminate growth, and a remarkable capacity to regenerate major parts following autotomy or traumatic injury. In this review we summarize the current knowledge on the organization and function of the echinoderm radial glia, with a focus on the role of this cell type in adult neurogenesis.

The Reissner fiber under tension in vivo shows dynamic interaction with ciliated cells contacting the cerebrospinal fluid.

The Reissner fiber (RF) is an acellular thread positioned in the midline of the central canal that aggregates thanks to the beating of numerous cilia from ependymal radial glial cells (ERGs) generating flow in the central canal of the spinal cord. RF together with cerebrospinal fluid (CSF)-contacting neurons (CSF-cNs) form an axial sensory system detecting curvature. How RF, CSF-cNs and the multitude of motile cilia from ERGs interact in vivo appears critical for maintenance of RF and sensory functions of CSF-cNs to keep a straight body axis, but is not well-understood. Using in vivo imaging in larval zebrafish, we show that RF is under tension and resonates dorsoventrally. Focal RF ablations trigger retraction and relaxation of the fiber's cut ends, with larger retraction speeds for rostral ablations. We built a mechanical model that estimates RF stress diffusion coefficient D at 5 mm2/s and reveals that tension builds up rostrally along the fiber. After RF ablation, spontaneous CSF-cN activity decreased and ciliary motility changed, suggesting physical interactions between RF and cilia projecting into the central canal. We observed that motile cilia were caudally-tilted and frequently interacted with RF. We propose that the numerous ependymal motile monocilia contribute to RF's heterogenous tension via weak interactions. Our work demonstrates that under tension, the Reissner fiber dynamically interacts with motile cilia generating CSF flow and spinal sensory neurons.

Insulin signaling promotes neurogenesis in the brain of adult zebrafish.

Insulin is a peptide hormone that plays a central role in the regulation of circulating blood glucose in vertebrates, including zebrafish. Increasing evidence has demonstrated the important role of insulin in many brain functions. In zebrafish, two insulin receptor genes (insra and insrb) have been identified. However, their biodistribution in the adult brain as well as their cell-specific expression pattern has not been well described. Using gene expression analysis, in situ hybridization and transgenic fish, we confirmed the expression of insra, insrb, and irs1 (insulin receptor substrate 1, the downstream effector of insulin receptor) in the brain of adult zebrafish and characterized their specific expression in neurons and neural stem cells (radial glia). After demonstrating that intracerebroventricular (ICV) injection resulted in the diffusion of the injected solution within the ventricular system, we analyzed the effect of insulin ICV injection on neurogenesis. We showed that insulin promotes ventricular cell proliferation 24h postinjection. This neurogenic effect appeared to be independent of neuroinflammatory processes. Also, after a mechanical telencephalic stab-wound injury, we highlighted the overexpression of irs1 gene 5 days postlesion notably in the ventricular zone where radial glial cells (RGCs) are localized, suggesting key roles of insulin signaling in regenerative processes. Finally, our results reinforced the expression of insulin-related proteins in the brain of adult zebrafish, highlighting the potential role of insulin signaling on neurogenesis.

P02.21.A A BIOBANK OF IDH1-MUTANT CELL LINES FOR STUDYING TUMOR HETEROGENEITY, PROGRESSION AND TARGET IDENTIFICATION

Abstract BACKGROUND Diffuse low grade gliomas are grade II brain tumors mainly affecting young patients. Most of these tumors have a mutation in the IDH1 gene (isocitrate dehydrogenase 1) gene causing differentiation defects and cell accumulation in the brain. These tumors are classified as astrocytomas or oligodendrogliomas and often progress into high grade gliomas (i.e IDH1-mutant grade IV astrocytomas and grade III oligodendrogliomas). We and others have shown that IDH1-mutant diffuse low grade gliomas contain 3 types of tumoral cells, resembling astrocytes, oligodendrocytes and neural progenitors, the formation of which is regulated by Notch signaling (Augustus et al, 2021, Cells, Suvà et al, 2020, Cancer Cell). There is a significant lack of cellular tools to study these tumors in vitro, hindering the development of new therapies. We aimed to address this by creating a fully annotated biobank of IDH1-mutant cell lines derived from different grades of tumors and carrying various rare mutations. MATERIAL AND METHODS We derived and collected 8 IDH1-mutant cell lines (4 astrocytomas grade II/III/IV and 4 grade III oligodendrogliomas). These cell lines were characterized using bulk RNA sequencing under different culture conditions, single-cell RNA sequencing to determine their cellular heterogeneity, and immunofluorescence and western blotting. RESULTS Our findings show that these IDH1-mutant cell lines exhibit different growth rates and morphologies and are phenotypically related to brain radial glial cells, oligodendrocyte progenitor cells, or more mature oligodendrocytes. Some of these cell lines are multipotent and contain astrocyte-like and oligodendrocyte-like cells. Our current work aims to purify different cell subpopulations to study their properties and plasticity. Additionally, we are analyzing pathways and differentially expressed genes involved in IDH1-mutant cell quiescence and malignant progression. CONCLUSION In summary, we present a novel and deeply-annotated biobank of 8 IDH1-mutant cell lines derived from different grades of gliomas, providing an invaluable resource to study the cellular and molecular heterogeneity of IDH1-mutant gliomas, their progression to high-grade gliomas, and the identification of new therapeutic targets. We anticipate that our biobank may facilitate the development of personalized medicine for patients with IDH1-mutant gliomas and accelerate the discovery of new therapeutic strategies.

P16-10: Refinement of an in vitro testing battery for developmental neurotoxicity assessment by integration of radial glia- and astrocyte-specific endpoints

P16-10: Refinement of an in vitro testing battery for developmental neurotoxicity assessment by integration of radial glia- and astrocyte-specific endpoints

4E-BP1 expression in embryonic postmitotic neurons mitigates mTORC1-induced cortical malformations and behavioral seizure severity but does not prevent epilepsy in mice.

Hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) pathway during neurodevelopment leads to focal cortical malformations associated with intractable seizures. Recent evidence suggests that dysregulated cap-dependent translation downstream of mTORC1 contributes to cytoarchitectural abnormalities and seizure activity. Here, we examined whether reducing cap-dependent translation by expressing a constitutively active form of the translational repressor, 4E-BP1, downstream of mTORC1 would prevent the development of cortical malformations and seizures. 4E-BP1CA was expressed embryonically either in radial glia (neural progenitor cells) that generate cortical layer 2/3 pyramidal neurons or in migrating neurons destined to layer 2/3 using a conditional expression system. In both conditions, 4E-BP1CA expression reduced mTORC1-induced neuronal hypertrophy and alleviated cortical mislamination, but a subset of ectopic neurons persisted in the deep layers and the white matter. Despite the above improvements, 4E-BP1CA expression in radial glia had no effects on seizure frequency and further exacerbated behavioral seizure severity associated with mTORC1 hyperactivation. In contrast, conditional 4E-BP1CA expression in migratory neurons mitigated the severity of behavioral seizures but the seizure frequency remained unchanged. These findings advise against targeting 4E-BPs by 4E-BP1CA expression during embryonic development for seizure prevention and suggest the presence of a development-dependent role for 4E-BPs in mTORC1-induced epilepsy.

Therapeutic hypothermia modulates the neurogenic response of the newborn piglet subventricular zone after hypoxia-ischemia

BackgroundNeuroprotection combined with neuroregeneration may be critical for optimizing functional recovery in neonatal encephalopathy. To investigate the neurogenic response to hypoxia-ischemia (HI) followed by normothermia (38.5 °C) or three different hypothermic temperatures (35, 33.5, or 30 °C) in the subventricular zone (SVZ) of the neonatal piglet.MethodsFollowing transient cerebral HI and resuscitation, 28 newborn piglets were randomized to: normothermia or whole-body cooling to 35 °C, 33.5 °C, or 30 °C during 2–26 h (all n = 7). At 48 h, piglets were euthanized and SVZ obtained to evaluate its cellularity, pattern of cell death, radial glia length, doublecortin (DCX, neuroblasts) expression, and Ki67 (cell proliferation) and Ki67/Sox2 (neural stem/progenitor dividing) cell counts.ResultsNormothermic piglets showed lower total (Ki67+) and neural stem/progenitor dividing (Ki67+Sox2+) cell counts when compared to hypothermic groups. Cooling to 33.5 °C obtained the highest values of SVZ cellularity, radial glia length processes, neuroblast chains area and DCX immunohistochemistry. Cooling to 30 °C, however, revealed decreased cellularity in the lateral SVZ and shorter radial glia processes when compared with 33.5 °C.ConclusionsIn a neonatal piglet model, hypothermia to 33.5 °C modulates the neurogenic response of the SVZ after HI, highlighting the potential beneficial effect of hypothermia to 33.5 °C on endogenous neurogenesis and the detrimental effect of overcooling beyond this threshold.ImpactNeuroprotection combined with neuroregeneration may be critical for optimizing functional recovery in neonatal encephalopathy.Hypothermia may modulate neurogenesis in the subventricular zone (SVZ) of the neonatal hypoxic-ischemic piglet.Cooling to 33.5 °C obtained the highest values of SVZ cellularity, radial glia length processes, neuroblast chains area and doublecortin immunohistochemistry; cooling to 30 °C, however, revealed decreased cellularity and shorter radial glia processes.In a neonatal piglet model, therapeutic hypothermia (33.5 °C) modulates the neurogenic response of the SVZ after hypoxia-ischemia, highlighting also the detrimental effect of overcooling beyond this threshold.

FOXP1 orchestrates neurogenesis in human cortical basal radial glial cells.

During cortical development, human basal radial glial cells (bRGCs) are highly capable of sustained self-renewal and neurogenesis. Selective pressures on this cell type may have contributed to the evolution of the human neocortex, leading to an increase in cortical size. bRGCs have enriched expression for Forkhead Box P1 (FOXP1), a transcription factor implicated in neurodevelopmental disorders (NDDs) such as autism spectrum disorder. However, the cell type-specific roles of FOXP1 in bRGCs during cortical development remain unexplored. Here, we examine the requirement for FOXP1 gene expression regulation underlying the production of bRGCs using human brain organoids. We examine a developmental time point when FOXP1 expression is highest in the cortical progenitors, and the bRGCs, in particular, begin to actively produce neurons. With the loss of FOXP1, we show a reduction in the number of bRGCs, as well as reduced proliferation and differentiation of the remaining bRGCs, all of which lead to reduced numbers of excitatory cortical neurons over time. Using single-nuclei RNA sequencing and cell trajectory analysis, we uncover a role for FOXP1 in directing cortical progenitor proliferation and differentiation by regulating key signaling pathways related to neurogenesis and NDDs. Together, these results demonstrate that FOXP1 regulates human-specific features in early cortical development.

A defined roadmap of radial glia and astrocyte differentiation from human pluripotent stem cells.

Human gliogenesis remains poorly understood, and derivation of astrocytes from human pluripotent stem cells (hPSCs) is inefficient and cumbersome. Here, we report controlled glial differentiation from hPSCs that bypasses neurogenesis, which otherwise precedes astrogliogenesis during brain development and invitro differentiation. hPSCs were first differentiated into radial glial cells (RGCs) resembling resident RGCs of the fetal telencephalon, and modulation of specific cell signaling pathways resulted in direct and stepwise induction of key astroglial markers (NFIA, NFIB, SOX9, CD44, S100B, glial fibrillary acidic protein [GFAP]). Transcriptomic and genome-wide epigenetic mapping and single-cell analysis confirmed RGC-to-astrocyte differentiation, obviating neurogenesis and the gliogenic switch. Detailed molecular and cellular characterization experiments uncovered new mechanisms and markers for human RGCs and astrocytes. In summary, establishment of a glia-exclusive neural lineage progression model serves as a unique serum-free platform of manufacturing large numbers of RGCs and astrocytes for neuroscience, disease modeling (e.g., Alexander disease), and regenerative medicine.

Potential effects of brain lipid binding protein in the pathogenesis of amyotrophic lateral sclerosis.

Current studies suggest that the abnormal alteration of brain lipid binding protein (BLBP) might participate in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, the detailed understanding of ALS pathogenesis been yet to be elucidated. Therefore, this research intended to explore the potential effects of BLBP in ALS. The observation and analysis of BLBP-altered features in various anatomical areas and different spinal segments was conducted at the pre-onset, onset, and progression stages of Tg(SOD1*G93A)1Gur (TG) mice and the same periods of age-matched SOD1 wild-type (WT) mice by fluorescence immunohistochemistry and western blotting. BLBP-positive cells were comprehensively distributed in various spinal anatomical areas, especially in both the anterior and posterior horn, around the central canal and in anterior, lateral, and posterior funiculi. Overall, BLBP expression tended to increase from the pre-onset to the onset to the progression stages of the same periods of age-matched WT mice. Furthermore, in TG mice, BLBP expression in the entire spinal cord significantly increased from onset to the progression stage. BLBP was expressed in neurons, astrocytes, and radial glial cells, and at the early and late stages of neural precursor cells (NPCs) and was predominantly distributed outside the cell nucleus. The increase of BLBP-positive cells was closely related to neural cell reduction in TG mice. The distribution and increased expression of BLBP among the cervical, thoracic, and lumbar segments of the spinal cord might participate in the development of ALS and exert potential effects in the pathogenesis of ALS by regulating NPCs.

Mutation of brain aromatase disrupts spawning behavior and reproductive health in female zebrafish.

Aromatase (Cyp19a1) is the steroidogenic enzyme that converts androgens into bioactive estrogens, and hence is in a pivotal position to mediate reproduction and sexual behavior. In teleosts, there are two aromatase paralogs: cyp19a1a that is highly expressed in granulosa and Leydig cells in the gonads with critical function in sexual differentiation of the ovary, and cyp19a1b that is highly expressed in radial glial cells in the brain with unknown roles in reproduction. Cyp19a1 -/- mutant zebrafish lines were used to investigate the importance of the cyp19a1 paralogs for spawning behavior and offspring survival and early development. Mutation of cyp19a1b was found to increase the latency to the first oviposition in females. Mutation of cyp19a1b in females also increased the number of eggs spawned; however, significantly more progeny died during early development resulting in no net increase in female fecundity. This finding suggests a higher metabolic cost of reproduction in cyp19a1b -/- mutant females. In males, the combined mutation of both cyp19a1 paralogs resulted in significantly lower progeny survival rates, indicating a critical function of cyp19a1 during early larval development. These data establish the specific importance of cyp19a1b for female spawning behavior and the importance of the cyp19a1 paralogs for early larval survival.

Ccdc85c-Par3 condensates couple cell polarity with Notch to control neural progenitor proliferation

Polarity proteins regulate the proliferation and differentiation of neural progenitors to generate neurons during brain development through multiple signaling pathways. However, how cell polarity couples the signaling pathways remains unclear. Here, we show that coiled-coil domain-containing protein 85c (Ccdc85c) interacts with the polarity protein Par3 to regulate the proliferation of radial glial cells (RGCs) via phase separation coupled to percolation (PSCP). We find that the interaction with Ccdc85c relieves the intramolecular auto-inhibitionof Par3, which leads to PSCP of Par3. Downregulation of Ccdc85c causes RGC differentiation. Importantly, the open conformation of Par3 facilitates the recruitment of the Notch regulator Numb to the Par3 condensates, which might prevent the attenuation of Notch activity to maintain RGC proliferation. Furthermore, ectopic activation of Notch signaling rescues RGC proliferation defects caused by the downregulation of Ccdc85c. These results suggest that Ccdc85c-mediated PSCP of Par3 regulates Notch signaling to control RGC proliferation during brain development.

Role of vitamin C and SVCT2 in neurogenesis.

Different studies have established the fundamental role of vitamin C in proliferation, differentiation, and neurogenesis in embryonic and adult brains, as well as in in vitro cell models. To fulfill these functions, the cells of the nervous system regulate the expression and sorting of sodium-dependent vitamin C transporter 2 (SVCT2), as well as the recycling of vitamin C between ascorbic acid (AA) and dehydroascorbic acid (DHA) via a bystander effect. SVCT2 is a transporter preferentially expressed in neurons and in neural precursor cells. In developmental stages, it is concentrated in the apical region of the radial glia, and in adult life, it is expressed preferentially in motor neurons of the cerebral cortex, starting on postnatal day 1. In neurogenic niches, SVCT2 is preferentially expressed in precursors with intermediate proliferation, where a scorbutic condition reduces neuronal differentiation. Vitamin C is a potent epigenetic regulator in stem cells; thus, it can induce the demethylation of DNA and histone H3K27m3 in the promoter region of genes involved in neurogenesis and differentiation, an effect mediated by Tet1 and Jmjd3 demethylases, respectively. In parallel, it has been shown that vitamin C induces the expression of stem cell-specific microRNA, including the Dlk1-Dio3 imprinting region and miR-143, which promotes stem cell self-renewal and suppresses de novo expression of the methyltransferase gene Dnmt3a. The epigenetic action of vitamin C has also been evaluated during gene reprogramming of human fibroblasts to induced pluripotent cells, where it has been shown that vitamin C substantially improves the efficiency and quality of reprogrammed cells. Thus, for a proper effect of vitamin C on neurogenesis and differentiation, its function as an enzymatic cofactor, modulator of gene expression and antioxidant is essential, as is proper recycling from DHA to AA by various supporting cells in the CNS.