Mapping the cellular expression patterns of vascular endothelial growth factor aa and bb genes and their receptors in the adult zebrafish brain during constitutive and regenerative neurogenesis

SummaryThe consequences of DNA damage generation in mammalian somatic stem cells, including neural stem cells (NSCs), are poorly understood despite their potential relevance for tissue homeostasis. Here, we show that, following ionizing radiation-induced DNA damage, NSCs enter irreversible proliferative arrest with features of cellular senescence. This is characterized by increased cytokine secretion, loss of stem cell markers, and astrocytic differentiation. We demonstrate that BMP2 is necessary to induce expression of the astrocyte marker GFAP in irradiated NSCs via a noncanonical signaling pathway engaging JAK-STAT. This is promoted by ATM and antagonized by p53. Using a SOX2-Cre reporter mouse model for cell-lineage tracing, we demonstrate irradiation-induced NSC differentiation in vivo. Furthermore, glioblastoma assays reveal that irradiation therapy affects the tumorigenic potential of cancer stem cells by ablating self-renewal and inducing astroglial differentiation.

Vascular endothelial growth factor receptor 3 controls neural stem cell activation in mice and humans.

High density lipoproteins (HDLs) display pleiotropic functions such as anti-inflammatory, antioxidant, anti-protease, and anti-apoptotic properties. These effects are mediated by four main receptors: SCARB1 (SR-BI), ABCA1, ABCG1, and CD36. Recently, HDLs have emerged for their potential involvement in brain functions, considering their epidemiological links with cognition, depression, and brain plasticity. However, their role in the brain is not well understood. Given that the zebrafish is a well-recognized model for studying brain plasticity, metabolic disorders, and apolipoproteins, it could represent a good model for investigating the role of HDLs in brain homeostasis. By analyzing RNA sequencing data sets and performing in situ hybridization, we demonstrated the wide expression of scarb1, abca1a, abca1b, abcg1, and cd36 in the brain of adult zebrafish. Scarb1 gene expression was detected in neural stem cells (NSCs), suggesting a possible role of HDLs in NSC activity. Accordingly, intracerebroventricular injection of HDLs leads to their uptake by NSCs without modulating their proliferation. Next, we studied the biodistribution of HDLs in the zebrafish body. In homeostatic conditions, intraperitoneal injection of HDLs led to their accumulation in the liver, kidneys, and cerebral endothelial cells in zebrafish, similar to that observed in mice. After telencephalic injury, HDLs were diffused within the damaged parenchyma and were taken up by ventricular cells, including NSCs. However, they failed to modulate the recruitment of microglia cells at the injury site and the injury-induced proliferation of NSCs. In conclusion, our results clearly show a functional HDL uptake process involving several receptors that may impact brain homeostasis and suggest the use of HDLs as delivery vectors to target NSCs for drug delivery to boost their neurogenic activity.

Adult Neural Stem Cells Bridge Their Niche

Failure of neural stem cell (NSC) activity and subsequently neurogenesis during brain development has been linked to cognitive impairment and intellectual disability. However, it remains unclear if changes in metabolism, recently discovered as a key regulator of somatic stem cell activity, contribute to altered neurogenesis and cognitive deficits in humans. To investigate a link between NSC-associated lipid metabolism and cognition we generated mice and human embryonic stem cells (hESCs) mimicking a variant in fatty acid synthase (FASN; R1819W), a metabolic regulator of rodent NSC activity recently identified in humans with intellectual disability. Strikingly, mice homozygous for the FASN R1812W variant have impaired hippocampal NSC activity associated with cognitive impairment due to presumed toxic accumulation of lipids in NSCs and subsequent lipogenic ER stress. Moreover, human NSCs (hNSCs) homozygous for the FASN R1819W variant show reduced rates of proliferation in 2D cultures and 3D forebrain regionalized organoids, revealing that the functional significance of lipid metabolism for NSC activity is conserved between rodents and humans. By taking a comprehensive disease modeling approach, using mouse and human tissue genome engineering, our data provide the first genetic evidence for a link between altered lipid metabolism, NSC activity and brain function in humans.

Microglia are macrophage-like cells exerting determinant roles in neuroinflammatory and oxidative stress processes during brain regeneration. We used zebrafish as a model of brain plasticity and repair. First, by performing L-plastin (Lcp1) immunohistochemistry and using transgenic Tg(mpeg1.1:GFP) or Tg(mpeg1.1:mCherry) fish, we analyzed the distribution of microglia/immune cells in the whole brain. Specific regional differences were evidenced in terms of microglia/immune cell density and morphology (elongated, branched, highly branched, and amoeboid). Taking advantage of Tg(fli:GFP) and Tg(GFAP::GFP) enabling the detection of endothelial cells and neural stem cells (NSCs), we highlighted the association of elongated microglia/immune cells with blood vessels and rounded/amoeboid microglia with NSCs. Second, after telencephalic injury, we showed that L-plastin cells were still abundantly present at 5 days post-lesion (dpl) and were associated with regenerative neurogenesis. Finally, RNA-sequencing analysis from injured telencephalon (5 dpl) confirmed the upregulation of microglia/immune cell markers and highlighted a significant increase of genes involved in oxidative stress (nox2, nrf2a, and gsr). The analysis of antioxidant activities at 5 dpl also revealed an upregulation of superoxide dismutase and persistent H2 O2 generation in the injured telencephalon. Also, microglia/immune cells were shown to be a source of oxidative stress at 5 dpl. Overall, our data provide a better characterization of microglia/immune cell distribution in the healthy zebrafish brain, highlighting some evolutionarily conserved features with mammals. They also emphasize that 5 days after injury, microglia/immune cells are still activated and are associated to a persistent redox imbalance. Together, these data raise the question of the role of oxidative stress in regenerative neurogenesis in zebrafish.

Neural stem cell quiescence and activation dynamics are regulated by feedback input from their progeny under homeostatic and regenerative conditions.

Multipotent precursors in the anterior and hippocampal subventricular zone display similar transcription factor signatures but their proliferation and maintenance are differentially regulated

Adult neural stem cell activation in mice is regulated by the day/night cycle and intracellular calcium dynamics

Adult mammalian brain, including humans, has rather limited addition of new neurons and poor regenerative capacity. In contrast, neural stem cells (NSC) with glial identity and neurogenesis are highly abundant throughout the adult zebrafish brain. Importantly, the activation of NSC and production of new neurons in response to injuries lead to the brain regeneration in zebrafish brain. Therefore, understanding of the molecular pathways regulating NSC behavior in response to injury is crucial in order to set the basis for experimental modification of these pathways in glial cells after injury in the mammalian brain and to elicit neuronal regeneration. Here, we describe the procedure that we successfully used to prospectively isolate NSCs from adult zebrafish telencephalon, extract RNA, and prepare cDNA libraries for next generation sequencing (NGS) and full transcriptome analysis as the first step toward understanding regulatory mechanisms leading to restorative neurogenesis in zebrafish. Moreover, we describe an alternative approach to analyze antigenic properties of NSC in the adult zebrafish brain using intracellular fluorescence activated cell sorting (FACS). We employ this method to analyze the number of proliferating NSCs positive for proliferating cell nuclear antigen (PCNA) in the prospectively isolated population of stem cells.

The future belongs to those who prepare for it today

Secreted Frizzled-Related Protein 3 Regulates Activity-Dependent Adult Hippocampal Neurogenesis

Home at Last: Neural Stem Cell Niches Defined

The central nervous system of adult zebrafish displays an extraordinary neurogenic and regenerative capacity. In the zebrafish adult brain, this regenerative capacity relies on neural stem cells (NSCs) and the careful management of the NSC pool. However, the mechanisms controlling NSC pool maintenance are not yet fully understood. Recently, Bone Morphogenetic Proteins (BMPs) and their downstream effector Id1 (Inhibitor of differentiation 1) were suggested to act as key players in NSC maintenance under constitutive and regenerative conditions. Here, we further investigated the role of BMP/Id1 signaling in these processes, using different genetic and pharmacological approaches. Our data show that BMPs are mainly expressed by neurons in the adult telencephalon, while id1 is expressed in NSCs, suggesting a neuron-NSC communication via the BMP/Id1 signaling axis. Furthermore, manipulation of BMP signaling by conditionally inducing or repressing BMP signaling via heat-shock, lead to an increase or a decrease of id1 expression in the NSCs, respectively. Induction of id1 was followed by an increase in the number of quiescent NSCs, while knocking down id1 expression caused an increase in NSC proliferation. In agreement, genetic ablation of id1 function lead to increased proliferation of NSCs, followed by depletion of the stem cell pool with concomitant failure to heal injuries in repeatedly injured mutant telencephala. Moreover, pharmacological inhibition of BMP and Notch signaling suggests that the two signaling systems cooperate and converge onto the transcriptional regulator her4.1. Interestingly, brain injury lead to a depletion of NSCs in animals lacking BMP/Id1 signaling despite an intact Notch pathway. Taken together, our data demonstrate how neurons feedback on NSC proliferation and that BMP1/Id1 signaling acts as a safeguard of the NSC pool under regenerative conditions.

The activation of neural stem cells (NSCs) residing in the subventricular zone (SVZ) and dentate gyrus (DG) has been shown to promote the restoration of damaged brain tissues. Ginsenoside Rb3 (Rb3) is a bioactive substance known for its pharmacological properties in treating neurological disorders. This study investigated the effects of Rb3 on neural regeneration following ischaemic stroke (IS) and the underlying mechanisms involved. Male C57BL/6 mice were utilized and were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R). Post-ischemia, Rb3 was administered through intraperitoneal (i.p.) injection for either 7 or 28 days. The promotion of Rb3 on regenerative neurogenesis was detected by immunofluorescence staining. NSCs were pretreated with different concentrations of Rb3 for 24 h before oxygen-glucose deprivation/reoxygenation (OGD/R) exposure. Afterward, immunofluorescence staining and flow cytometry were used to detect the migration and proliferation of Rb3 in OGD/R-induced NSCs. Furthermore, Adeno-associated virus (AAV) transduction experiments, siRNA transfection experiments, gene knockout experiments, targeted metabolomics analysis, molecular dynamics simulation, cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) assays were used to explore the promotion and mechanism of Rb3 on regenerative neurogenesis following IS. Rb3 promoted Opa1-mediated NSCs migration and proliferation. Knockdown of Opa1 blunted the above-promoting effects of Rb3 in both the brains of ischemia-reperfusion (I/R)-treated mice and OGD/R-treated NSCs. Mechanistically, targeted metabolomics, molecular dynamics, molecular docking, CETAS, and DARTS experiments showed that Rb3 promoted Opa1-mediated neural regeneration required the activation of Ido1 and that Ido1 served as a direct target of Rb3 to repair I/R injury. Moreover, studies in siRNA-mediated knockdown and KO mice revealed that inhibition of Ido1 attenuated the enhancing effect of Rb3 on mitochondrial fusion. Our study provides novel evidence that Rb3 promotes neurogenesis through an Ido1/Opa1-mediated pathway involving the interaction between Rb3 and Ido1, leading to improved long-term neurological function. These results indicate that Rb3 or other mitochondrial fusion promoters could be a potential neurorestorative strategy for regenerative neurogenesis following IS.