Glial ferritin maintains neural stem cells via transporting iron required for self-renewal in Drosophila.

Stem cell niche is critical for regulating the behavior of stem cells. Drosophila neural stem cells (Neuroblasts, NBs) are encased by glial niche cells closely, but it still remains unclear whether glial niche cells can regulate the self-renewal and differentiation of NBs. Here, we show that ferritin produced by glia, cooperates with Zip13 to transport iron into NBs for the energy production, which is essential to the self-renewal and proliferation of NBs. The knockdown of glial ferritin encoding genes causes energy shortage in NBs via downregulating aconitase activity and NAD+ level, which leads to the low proliferation and premature differentiation of NBs mediated by Prospero entering nuclei. More importantly, ferritin is a potential target for tumor suppression. In addition, the level of glial ferritin production is affected by the status of NBs, establishing a bicellular iron homeostasis. In this study, we demonstrate that glial cells are indispensable to maintain the self-renewal of NBs, unveiling a novel role of the NB glial niche during brain development.

Iron is an essential nutrient for almost all living organisms. For example, iron contributes to the replication of DNA, the generation of energy inside cells, and the transport of oxygen around the body. Iron deficiency is the most common of all nutrient deficiencies, affecting over 40% of children worldwide. This can lead to anemia and also impair how the brain and nervous system develop, potentially resulting in long-lasting cognitive damage, even after the deficiency has been treated. It is poorly understood how iron contributes to the development of the brain and nervous system. In particular, whether and how it supports nerve stem cells (or NSCs for short) which give rise to the various neural types in the mature brain. To investigate, Ma et al. experimentally reduced the levels of ferritin (a protein which stores iron) in the developing brains of fruit fly larvae. This reduction in ferritin led to lower numbers of NSCs and a smaller brain. Unexpectedly, this effect was largest when ferritin levels were reduced in glial cells which support and send signals to NSCs, rather than in the stem cells themselves. Ma et al. then used fluorescence microscopy to confirm that glial cells make and contain a lot of ferritin which can be transported to NSCs. Adding iron supplements to the diet of flies lacking ferritin did not lead to normal numbers of stem cells in the brains of the developing fruit flies, whereas adding compounds that reduce the amount of iron led to lower numbers of stem cells. Together, this suggests that ferritin transports iron from glial cells to the NSCs. Without ferritin and iron, the NSCs could not produce enough energy to divide and make new stem cells. This caused the NSCs to lose the characteristics of stem cells and prematurely turn into other types of neurons or glial cells. Together, these findings show that when iron cannot move from glial cells to NSCs this leads to defects in brain development. Future experiments will have to test whether a similar transport of iron from supporting cells to NSCs also occurs in the developing brains of mammals, and whether this mechanism applies to stem cells in other parts of the body.

Secretion of GPNMB from Neural Stem Cells Induced by ET-1 Contributes to Angiogenesis after Spinal Cord Injury.

BACKGROUND: The feeble plasticity of spinal cord microvascular endothelial cells (SCMECs) after trauma is one of the major causes of spinal cord injury (SCI). Neural stem cells (NSCs) play an important role in nerve repair. Glycoprotein nonmetastatic B (GPNMB) has neuroprotective effects and can be stimulated by endothelin 1 (ET-1), and its expression is upregulated in SCI. Here, we aim to investigate whether elevated ET-1 levels stimulate NSCs to secrete GPNMB, thereby further promoting angiogenesis. METHODS: Mouse SCMECs and NSCs were isolated, cultured, and identified by flow cytometry and immunofluorescence staining. NSCs were treated with ET-1, while SCMECs were cocultured with NSCs, followed by treatment with ET-1. NCS and SCMEC viability were evaluated using cell counting kit 8 (CCK-8) assay, while cell proliferation, migration, invasion, and angiogenesis were examined using 5'-Ethynyl-2'-Deoxyuridine (EdU) staining, wound healing assay, Transwell assay, and tube formation assay. GPNMB expression in NCSs and SCMECs was quantified by western blot assay, quantitative Real-Time polymerase chain reaction (qRT-PCR), or enzyme-linked immunosorbent assay (ELISA). RESULTS: Mouse SCMECs and NSCs were successfully isolated and cultured. ET-1 promoted NSC viability and proliferation and upregulated GPNMB expression. NSCs and ET-1-treated NSCs promoted the viability, migration, invasion, angiogenesis, and GPNMB expression in SCMECs compared with control group cells, while GPNMB antibody reversed the above effects of ET-1 on the SCMECs. CONCLUSION: ET-1 promotes SCMEC migration and invasion, along with angiogenesis, by enhancing NSC-mediated GPNMB secretion, so ET-1 may be a novel therapeutic target for SCI.

Comparison of Neurogenesis Promotion Effects between Cinnamoylquinic Acids in Neural Stem Cells from Adult Mice Brains.

Caffeoylquinic acids (CQAs) and feruloylquinic acids (FQAs), as cinnamoylquinic acids, have neurogenesis promotion effects. We studied for the first time the neurogenesis-enhancing effect of 3,4,5-tri-feruloylquinic acid (TFQA) compared to 3,4,5-tri-caffeoylquinic acid (TCQA), which has a similar structure, and explored their different cellular and molecular mechanisms in neural stem cells (NSCs) of mice brains. After 2 weeks of incubation, we first assessed the number and size of NSCs in TCQA and TFQA treatments. In NSCs treated for TCQA and TFQA, the NSC proliferation gene expression as well as neuronal and glial cell differentiation gene expressions improved. In the microarray assay, the erythroblastic oncogene B (ErbB) signaling pathway, as the common signaling of TCQA and TFQA treatments, was focused on and discussed. In our study, TCQA and TFQA treatments in NSCs showed a significant performance on improving synapse growth and neurogenesis compared with no treatment of NSCs. The two treatments in NSCs also had a significant activation of the ErbB signaling pathway, protein kinase B (AKT), and mitogen-activated protein kinase (MAPK) kinases. In particular, the TCQA-expressed proliferation gene myelocytomatosis oncogene (Myc) had the greatest connections significantly. TFQA treatment remarkably regulated the differentiation gene jun proto-oncogene (Jun), which was the gene with greatest direct relations, while Myc was also induced in TFQA treatment. In the overall quantitative real-time polymerase chain reaction (PCR) assay, TFQA had more outstanding neural proliferation and differentiation capabilities than TCQA in NSCs. Our study suggests that TFQA has greater therapeutic potential in neurogenesis promotion and neurodegenerative diseases compared with TCQA.

Dendritic morphological development of traumatic brain injury-induced new neurons in the dentate gyrus is important for post-injury cognitive recovery and is regulated by Notch1.

Traumatic brain injury (TBI) is a prevalent problem with survivors suffering from chronic cognitive impairments. Following TBI there is a series of neuropathological changes including neurogenesis. It is well established that neurogenesis in the dentate gyrus (DG) of the hippocampus is important for hippocampal dependent learning and memory functions. Following TBI, injury-enhanced hippocampal neurogenesis is believed to contribute to post-injury cognitive recovery. Behavioral function is connected to synaptic plasticity and neuronal dendritic branching is critical for successful synapse formation. To ascertain the functional contribution of injury-induced DG new neurons in post-TBI cognitive recovery, it is necessary to study their dendritic morphological development and the molecular mechanisms controlling this process. Utilizing transgenic mice with tamoxifen-induced GFP expression and Notch1 knock-out in nestin+ neural stem cells, this study examined dendritic morphology, the role of Notch1 in regulating dendritic complexity of injury-induced DG new neurons, and their association to post-TBI cognitive recovery. We found that at 8 weeks after a moderate TBI, injury-induced DG new neurons in the injured control mice displayed a similar dendritic morphology as the cells in non-injured mice accompanied with cognitive recovery. In comparison, in Notch1 conditional knock-out mice, DG new neurons in the injured mice had a significant reduction in dendritic morphological development including dendritic arbors, volume span, and number of branches in comparison to the cells in non-injured mice concomitant with persistent cognitive dysfunction. The results of this study confirm the importance of post-injury generated new neurons in cognitive recovery following TBI and the role of Notch1 in regulating their maturation process.

Investigation of Persistent Photoconductivity of Gallium Nitride Semiconductor and Differentiation of Primary Neural Stem Cells.

A gallium nitride (GaN) semiconductor is one of the most promising materials integrated into biomedical devices to play the roles of connecting, monitoring, and manipulating the activity of biological components, due to its excellent photoelectric properties, chemical stability, and biocompatibility. In this work, it was found that the photogenerated free charge carriers of the GaN substrate, as an exogenous stimulus, served to promote neural stem cells (NSCs) to differentiate into neurons. This was observed through the systematic investigation of the effect of the persistent photoconductivity (PPC) of GaN on the differentiation of primary NSCs from the embryonic rat cerebral cortex. NSCs were directly cultured on the GaN surface with and without ultraviolet (UV) irradiation, with a control sample consisting of tissue culture polystyrene (TCPS) in the presence of fetal bovine serum (FBS) medium. Through optical microscopy, the morphology showed a greater number of neurons with the branching structures of axons and dendrites on GaN with UV irradiation. The immunocytochemical results demonstrated that GaN with UV irradiation could promote the NSCs to differentiate into neurons. Western blot analysis showed that GaN with UV irradiation significantly upregulated the expression of two neuron-related markers, ßIII-tubulin (Tuj-1) and microtubule-associated protein 2 (MAP-2), suggesting that neurite formation and the proliferation of NSCs during differentiation were enhanced by GaN with UV irradiation. Finally, the results of the Kelvin probe force microscope (KPFM) experiments showed that the NSCs cultured on GaN with UV irradiation displayed about 50 mV higher potential than those cultured on GaN without irradiation. The increase in cell membrane potential may have been due to the larger number of photogenerated free charges on the GaN surface with UV irradiation. These results could benefit topical research and the application of GaN as a biomedical material integrated into neural interface systems or other bioelectronic devices.

Cocaine amphetamine-regulated transcription peptide inhibits apoptosis in oxygen-glucose deprived neural stem cells.

Background: Apoptosis has been recognized as a critical pathophysiological process during cerebral ischemia. The neuroprotective effect of CART on ischemic brain injury is determined. However, there is little research on the protective effect of CART on neural stem cells (NSCs). Methods: Primary cultured rat NSCs were utilized as the research subject. In vitro oxygen glucose deprivation (OGD) treatment was employed, and NSCs were extracted from SD pregnant rats following previous experimental protocols and identified through cell immunofluorescence staining. The appropriate concentration of CART affecting OGD NSCs was initially screened using Cell Counting Kit-8 (CCK-8) and Lactate Dehydrogenase (LDH) assays. EdU staining and Western blotting (WB) techniques were employed to assess the impact of the suitable CART concentration on the proliferation and apoptosis of OGD NSCs. Finally, Western blot analysis was conducted to investigate the cAMP-response element binding protein (CREB) pathway and expression levels of related proteins after KG-501 treatment in order to elucidate the mechanism underlying apoptosis and proliferation regulation in OGD NSCs. Results: CCK-8 and LDH assays indicated that a concentration of 0.8 nM CART may be the optimal concentration for modulating the proliferation of OGD NSCs. Subsequently, cellular immunofluorescence and EdU detection experiments further confirmed the findings obtained from CCK-8 analysis. Western blot analysis of apoptosis-related protein expression also demonstrated that an appropriate concentration of CART could suppress the apoptosis of OGD NSCs. Finally, Western blotting was conducted to examine the CREB pathway and related protein expression after treatment with KG-501, revealing that an appropriate concentration of CART regulated both apoptosis and proliferation in OGD NSCs through CREB signaling. Conclusion: The concentration of CART at 0.8 nM may be deemed appropriate for inhibiting apoptosis and promoting proliferation in OGD NSCs in vitro. The mechanism maybe through activating the CREB pathway.

bHLH family proteins control the timing and completion of transition from neuroepithelial cells into neural stem cells.

The number of neural stem cells reflects the total number of neurons in the mature brain. As neural stem cells arise from neuroepithelial cells, the neuroepithelial cell population must be expanded to secure a sufficient number of neural stem cells. However, molecular mechanisms that regulate timely differentiation from neuroepithelial to neural stem cells are largely unclear. Here, we show that TCF4/Daughterless is a key factor that determines the timing of the differentiation in Drosophila. The neuroepithelial cells initiated but never completed the differentiation in the absence of TCF4/Daughterless. We also found that TCF4/Daughterless binds to the Notch locus, suggesting that Notch is one of its downstream candidate genes. Consistently, Notch expression was ectopically induced in the absence of TCF4/Daughterless. Furthermore, ectopic activation of Notch signaling phenocopied loss of TCF4/Daughterless. Our findings demonstrate that TCF4/Daughterless directly inactivates Notch signaling pathway, resulting in completion of the differentiation from neuroepithelial cells into neural stem cells with optimal timing. Thus, the present results suggest that TCF4/Daughterless is essential for determining whether to move to the next state or stay in the current state in differentiating neuroepithelial cells.

Vitamin D Deficiency Does Not Affect Cognition and Neurogenesis in Adult C57Bl/6 Mice.

Vitamin D deficiency is a global problem. Vitamin D, the vitamin D receptor, and its enzymes are found throughout neuronal, ependymal, and glial cells in the brain and are implicated in certain processes and mechanisms in the brain. To investigate the processes affected by vitamin D deficiency in adults, we studied vitamin D deficient, control, and supplemented diets over 6 weeks in male and female C57Bl/6 mice. The effect of the vitamin D diets on proliferation in the neurogenic niches, changes in glial cells, as well as on memory, locomotion, and anxiety-like behavior, was investigated. Six weeks on a deficient diet was adequate time to reach deficiency. However, vitamin D deficiency and supplementation did not affect proliferation, neurogenesis, or astrocyte changes, and this was reflected on behavioral measures. Supplementation only affected microglia in the dentate gyrus of female mice. Indicating that vitamin D deficiency and supplementation do not affect these processes over a 6-week period.

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.

The complex interplay between vascular signaling and neurogenesis in the adult brain remains a subject of intense research. By exploiting the unique advantages of the zebrafish model, in particular the persistent activity of neural stem cells (NSCs) and the remarkable ability to repair brain lesions, we investigated the links between NSCs and cerebral blood vessels. In this study, we first examined the gene expression profiles of vascular endothelial growth factors aa and bb (vegfaa and vegfbb), under physiological and regenerative conditions. Employing fluorescence in situ hybridization combined with immunostaining and histology techniques, we demonstrated the widespread expression of vegfaa and vegfbb across the brain, and showed their presence in neurons, microglia/immune cells, endothelial cells and NSCs. At 1 day post-lesion (dpl), both vegfaa and vegfbb were up-regulated in neurons and microglia/peripheral immune cells (macrophages). Analysis of vegf receptors (vegfr) revealed high expression throughout the brain under homeostatic conditions, with vegfr predominantly expressed in neurons and NSCs and to a lower extent in microglia/immune cells and endothelial cells. These findings were further validated by Vegfr3 and Vegfr4 immunostainings, which showed significant expression in neurogenic radial glial cells.Following brain lesion (1 dpl), while vegfr gene expression remained stable, vegfr transcripts were detected in proliferative cells within the injured parenchyma. Collectively, our results provide a first overview of Vegf/Vegfr signaling in the brain and suggest important roles for Vegf in neurogenesis and regenerative processes.

DVL/GSK3/ISL1 pathway signaling: unraveling the mechanism of SIRT3 in neurogenesis and AD therapy.

BACKGROUND: The established association between Alzheimer's disease (AD) and compromised neural regeneration is well-documented. In addition to the mitigation of apoptosis in neural stem cells (NSCs), the induction of neurogenesis has been proposed as a promising therapeutic strategy for AD. Our previous research has demonstrated the effective inhibition of NSC injury induced by microglial activation through the repression of oxidative stress and mitochondrial dysfunction by Sirtuin 3 (SIRT3). Nonetheless, the precise role of SIRT3 in neurogenesis remains incompletely understood. METHODS: In vivo, SIRT3 overexpression adenovirus was firstly injected by brain stereotaxic localization to affect the hippocampal SIRT3 expression in APP/PS1 mice, and then behavioral experiments were performed to investigate the cognitive improvement of SIRT3 in APP/PS1 mice, as well as neurogenic changes in hippocampal region by immunohistochemistry and immunofluorescence. In vitro, under the transwell co-culture condition of microglia and neural stem cells, the mechanism of SIRT3 improving neurogenesis of neural stem cells through DVL/GSK3/ISL1 axis was investigated by immunoblotting, immunofluorescence and other experimental methods. RESULTS: Our findings indicate that the overexpression of SIRT3 in APP/PS1 mice led to enhanced cognitive function and increased neurogenesis. Additionally, SIRT3 was observed to promote the differentiation of NSCs into neurons during retinoic acid (RA)-induced NSC differentiation in vitro, suggesting a potential role in neurogenesis. Furthermore, we observed the activation of the Wnt/ß-catenin signaling pathway during this process, with Glycogen Synthase Kinase-3a (GSK3a) primarily governing NSC proliferation and GSK3ß predominantly regulating NSC differentiation. Moreover, the outcomes of our study demonstrate that SIRT3 exerts a protective effect against microglia-induced apoptosis in neural stem cells through its interaction with DVLs. CONCLUSIONS: Our results show that SIRT3 overexpressing APP/PS1 mice have improved cognition and neurogenesis, as well as improved neurogenesis of NSC in microglia and NSC transwell co-culture conditions through the DVL/GSK3/ISL1 axis.