Extracellular vesicle-induced differentiation of neural stem progenitor cells

Neural stem progenitor cells (NSPCs) from E13.5 mouse embryos can be maintained in culture under proliferating conditions. Upon growth-factor removal, they may differentiate toward either neuronal or glial phenotypes or both. Exosomes are small extracellular vesicles that are part of the cell secretome; they may contain and deliver both proteins and genetic material and thus play a role in cell–cell communication, guide axonal growth, modulate synaptic activity and regulate peripheral nerve regeneration. In this work, we were interested in determining whether NSPCs and their progeny can produce and secrete extracellular vesicles (EVs) and if their content can affect cell differentiation. Our results indicate that cultured NSPCs produce and secrete EVs both under proliferating conditions and after differentiation. Treatment of proliferating NSPCs with EVs derived from differentiated NSPCs triggers cell differentiation in a dose-dependent manner, as demonstrated by glial-and neuronal-marker expression

Polymerogenic neuroserpin causes mitochondrial alterations and activates NFκB but not the UPR in a neuronal model of neurodegeneration FENIB

The neurodegenerative condition FENIB (familiar encephalopathy with neuroserpin inclusion bodies) is caused by heterozygous expression of polymerogenic mutant neuroserpin (NS), with polymer deposition within the endoplasmic reticulum (ER) of neurons. We generated transgenic neural progenitor cells (NPCs) from mouse fetal cerebral cortex stably expressing either the control protein GFP or human wild type, polymerogenic G392E or truncated (delta) NS. This cellular model makes it possible to study the toxicity of polymerogenic NS in the appropriated cell type by in vitro differentiation to neurons. Our previous work showed that expression of G392E NS in differentiated NPCs induced an adaptive response through the upregulation of several genes involved in the defence against oxidative stress, and that pharmacological reduction of the antioxidant defences by drug treatments rendered G392E NS neurons more susceptible to apoptosis than control neurons. In this study, we assessed mitochondrial distribution and found a higher percentage of perinuclear localisation in G392E NS neurons, particularly in those containing polymers, a phenotype that was enhanced by glutathione chelation and rescued by antioxidant molecules. Mitochondrial membrane potential and contact sites between mitochondria and the ER were reduced in neurons expressing the G392E mutation. These alterations were associated with a pattern of ER stress that involved the ER overload response but not the unfolded protein response. Our results suggest that intracellular accumulation of NS polymers affects the interaction between the ER and mitochondria, causing mitochondrial alterations that contribute to the neuronal degeneration seen in FENIB patients

Molecular profiling of aged neural progenitors identifies Dbx2 as a candidate regulator of age-associated neurogenic decline.

Adult neurogenesis declines with aging due to the depletion and functional impairment of neural stem/progenitor cells (NSPCs). An improved understanding of the underlying mechanisms that drive age-associated neurogenic deficiency could lead to the development of strategies to alleviate cognitive impairment and facilitate neuroregeneration. An essential step towards this aim is to investigate the molecular changes that occur in NSPC aging on a genomewide scale. In this study, we compare the transcriptional, histone methylation and DNA methylation signatures of NSPCs derived from the subventricular zone (SVZ) of young adult (3 months old) and aged (18 months old) mice. Surprisingly, the transcriptional and epigenomic profiles of SVZ-derived NSPCs are largely unchanged in aged cells. Despite the global similarities, we detect robust age-dependent changes at several hundred genes and regulatory elements, thereby identifying putative regulators of neurogenic decline. Within this list, the homeobox gene Dbx2 is upregulated in vitro and in vivo, and its promoter region has altered histone and DNA methylation levels, in aged NSPCs. Using functional in vitro assays, we show that elevated Dbx2 expression in young adult NSPCs promotes age-related phenotypes, including the reduced proliferation of NSPC cultures and the altered transcript levels of age-associated regulators of NSPC proliferation and differentiation. Depleting Dbx2 in aged NSPCs caused the reverse gene expression changes. Taken together, these results provide new insights into the molecular programmes that are affected during mouse NSPC aging, and uncover a new functional role for Dbx2 in promoting age-related neurogenic decline.This work was supported by grants to P.J.R.-G. from the 6 Wellcome Trust (WT093736) and the BBSRC (BB/P013406/1, BB/M022285/1), by funding from 7 Sapienza University of Rome (G.L, S.B., E.C) and by a grant from the Spanish Ministry of 8 Economy to P.B. (BFU2016-75412-R, co-financed by FEDER). The Babraham Institute Biological 9 Services Unit is supported by Campus Capability Grant funding from the BBSRC

X-ray irradiated cultures of mouse cortical neural stem/progenitor cells recover cell viability and proliferation with dose-dependent kinetics

Exposure of the developing or adult brain to ionizing radiation (IR) can cause cognitive impairment and/ or brain cancer, by targeting neural stem/progenitor cells (NSPCs). IR effects on NSPCs include transient cell cycle arrest, permanent cell cycle exit/differentiation, or cell death, depending on the experimental conditions. In vivo studies suggest that brain age influences NSPC response to IR, but whether this is due to intrinsic NSPC changes or to niche environment modifications remains unclear. Here, we describe the dose-dependent, time-dependent effects of X-ray IR in NSPC cultures derived from the mouse foetal cerebral cortex. We show that, although cortical NSPCs are resistant to low/moderate IR doses, high level IR exposure causes cell death, accumulation of DNA double-strand breaks, activation of p53- related molecular pathways and cell cycle alterations. Irradiated NSPC cultures transiently upregulate differentiation markers, but recover control levels of proliferation, viability and gene expression in the second week post-irradiation. These results are consistent with previously described in vivo effects of IR in the developing mouse cortex, and distinct from those observed in adult NSPC niches or in vitro adult NSPC cultures, suggesting that intrinsic differences in NSPCs of different origins might determine, at least in part, their response to IR

Nitric oxide from inflammatory origin impairs neural stem cell proliferation by inhibiting epidermal growth factor receptor signaling

Neuroinflammation is characterized by activation of microglial cells, followed by production of nitric oxide (NO), which may have different outcomes on neurogenesis, favoring or inhibiting this process. In the present study, we investigated how the inflammatory mediator NO can affect proliferation of neural stem cells (NSCs), and explored possible mechanisms underlying this effect. We investigated which mechanisms are involved in the regulation of NSC proliferation following treatment with an inflammatory stimulus (lipopolysaccharide plus IFN-gamma), using a culture system of subventricular zone (SVZ)-derived NSCs mixed with microglia cells obtained from wild-type mice (iNOS(+/+)) or from iNOS knockout mice (iNOS(-/-)). We found an impairment of NSC cell proliferation in iNOS(+/+) mixed cultures, which was not observed in iNOS(-/-) mixed cultures. Furthermore, the increased release of NO by activated iNOS(+/+) microglial cells decreased the activation of the ERK/MAPK signaling pathway, which was concomitant with an enhanced nitration of the EGF receptor. Preventing nitrogen reactive species formation with MnTBAP, a scavenger of peroxynitrite (ONOO-), or using the ONOO- degradation catalyst FeTMPyP cell proliferation and ERK signaling were restored to basal levels in iNOS(+/+) mixed cultures. Moreover, exposure to the NO donor NOC-18 (100 mu M), for 48 h, inhibited SVZ-derived NSC proliferation. Regarding the antiproliferative effect of NO, we found that NOC-18 caused the impairment of signaling through the ERK/MAPK pathway, which may be related to increased nitration of the EGF receptor in NSC. Using MnTBAP nitration was prevented, maintaining ERK signaling, rescuing NSC proliferation. We show that NO from inflammatory origin leads to a decreased function of the EGF receptor, which compromised proliferation of NSC. We also demonstrated that NO-mediated nitration of the EGF receptor caused a decrease in its phosphorylation, thus preventing regular proliferation signaling through the ERK/MAPK pathway.Foundation for Science and Technology, (FCT, Portugal); COMPETE; FEDER [PEst-C/SAU/LA0001/2013-2014, PEst-OE/EQB/LA0023/2013-2014, PTDC/SAU-NEU/102612/2008, PTDC/NEU-OSD/0473/2012]; FCT, Portugal [SERH/BPD/78901/2011, SERH/BD/38127/2007, SFRH/BD/77903/2011, SFRH/BD/79308/2011]info:eu-repo/semantics/publishedVersio

Abrogated Inflammatory Response Promotes Neurogenesis in a Murine Model of Japanese Encephalitis

Japanese encephalitis virus (JEV) induces neuroinflammation with typical features of viral encephalitis, including inflammatory cell infiltration, activation of microglia, and neuronal degeneration. The detrimental effects of inflammation on neurogenesis have been reported in various models of acute and chronic inflammation. We investigated whether JEV-induced inflammation has similar adverse effects on neurogenesis and whether those effects can be reversed using an anti-inflammatory compound minocycline.Here, using in vitro studies and mouse models, we observed that an acute inflammatory milieu is created in the subventricular neurogenic niche following Japanese encephalitis (JE) and a resultant impairment in neurogenesis occurs, which can be reversed with minocycline treatment. Immunohistological studies showed that proliferating cells were replenished and the population of migrating neuroblasts was restored in the niche following minocycline treatment. In vitro, we checked for the efficacy of minocycline as an anti-inflammatory compound and cytokine bead array showed that production of cyto/chemokines decreased in JEV-activated BV2 cells. Furthermore, mouse neurospheres grown in the conditioned media from JEV-activated microglia exhibit arrest in both proliferation and differentiation of the spheres compared to conditioned media from control microglia. These effects were completely reversed when conditioned media from JEV-activated and minocycline treated microglia was used.This study provides conclusive evidence that JEV-activated microglia and the resultant inflammatory molecules are anti-proliferative and anti-neurogenic for NSPCs growth and development, and therefore contribute to the viral neuropathogenesis. The role of minocycline in restoring neurogenesis may implicate enhanced neuronal repair and attenuation of the neuropsychiatric sequelae in JE survivors

HIV-1-Infected and Immune-Activated Macrophages Induce Astrocytic Differentiation of Human Cortical Neural Progenitor Cells via the STAT3 Pathway

Diminished adult neurogenesis is considered a potential mechanism in the pathogenesis of HIV-1-associated dementia (HAD). In HAD, HIV-1-infected and immune-activated brain mononuclear phagocytes (MP; perivascular macrophages and microglia) drive central nervous system (CNS) inflammation and may alter normal neurogenesis. We previously demonstrated HIV-1-infected and lipopolysaccharide (LPS) activated monocyte-derived macrophages (MDM) inhibit human neural progenitor cell (NPC) neurogenesis, while enhancing astrogliogenesis through the secretion of the inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), in vitro and in vivo. Here we further test the hypothesis that HIV-1-infected/activated MDM promote NPC astrogliogenesis via activation of the transcription factor signal transducer and activator of transcription 3 (STAT3), a critical factor for astrogliogenesis. Our results show that LPS-activated MDM-conditioned medium (LPS-MCM) and HIV-infected/LPS-activated MDM-conditioned medium (LPS+HIV-MCM) induced Janus kinase 1 (Jak1) and STAT3 activation. Induction of the Jak-STAT3 activation correlated with increased glia fibrillary acidic protein (GFAP) expression, demonstrating an induction of astrogliogenesis. Moreover, STAT3-targeting siRNA (siSTAT3) decreased MCM-induced STAT3 activation and NPC astrogliogenesis. Furthermore, inflammatory cytokines (including IL-6, IL-1β and TNF-α) produced by LPS-activated and/or HIV-1-infected MDM may contribute to MCM-induced STAT3 activation and astrocytic differentiation. These observations were confirmed in severe combined immunodeficient (SCID) mice with HIV-1 encephalitis (HIVE). In HIVE mice, siRNA control (without target sequence, sicon) pre-transfected NPCs injected with HIV-1-infected MDM showed more astrocytic differentiation and less neuronal differentiation of NPCs as compared to NPC injection alone. siSTAT3 abrogated HIV-1-infected MDM-induced astrogliogenesis of injected NPCs. Collectively, these observations demonstrate that HIV-1-infected/activated MDM induces NPC astrogliogenesis through the STAT3 pathway. This study generates important data elucidating the role of brain inflammation in neurogenesis and may provide insight into new therapeutic strategies for HAD

Clonal human fetal ventral mesencephalic dopaminergic neuron precursors for cell therapy research

A major challenge for further development of drug screening procedures, cell replacement therapies and developmental studies is the identification of expandable human stem cells able to generate the cell types needed. We have previously reported the generation of an immortalized polyclonal neural stem cell (NSC) line derived from the human fetal ventral mesencephalon (hVM1). This line has been biochemically, genetically, immunocytochemically and electrophysiologically characterized to document its usefulness as a model system for the generation of A9 dopaminergic neurons (DAn). Long-term in vivo transplantation studies in parkinsonian rats showed that the grafts do not mature evenly. We reasoned that diverse clones in the hVM1 line might have different abilities to differentiate. In the present study, we have analyzed 9 hVM1 clones selected on the basis of their TH generation potential and, based on the number of v-myc copies, v-myc down-regulation after in vitro differentiation, in vivo cell cycle exit, TH+ neuron generation and expression of a neuronal mature marker (hNSE), we selected two clones for further in vivo PD cell replacement studies. The conclusion is that homogeneity and clonality of characterized NSCs allow transplantation of cells with controlled properties, which should help in the design of long-term in vivo experimentsThis work was supported by grants from the Spanish Ministry of Economy and Competitiveness (formerly Science and Innovation; PLE2009-0101, SAF2010-17167), Comunidad Autónoma Madrid (S2011-BMD-2336), Instituto Salud Carlos III (RETICS TerCel, RD06/0010/0009) and European Union (Excell, NMP4-SL-2008-214706). This work was also supported by an institutional grant from Foundation Ramón Areces to the Center of Molecular Biology Severo Ocho