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

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

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

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

Glial phenotype induction of neural stem progenitor cells using astrocyte-derived exosomes

Exosomes, small vescicles with a lipid bilayer, are released from many cell types and are part of the cell secretrome that can play a role in intercellular communication. They originate from cytoplasmic multivescicular bodies, fuse with the plasma membrane and release their content of lipids, proteins and RNAs in the extracellular space or in target cells. A growing body of evidence suggests that exosomes contribute to many aspects of healthy and pathological cells, and they may influence the homeostasis of target cells. With the aim of understanding the role of exosomes during tissue development, we investigated the role of these extracellular vesicles in neural progenitor stem cells (NSPCs) commitment. By using NSPCs from embryonic mouse spinal cord, we demostrate that neuronal and/or astrocytes induction of differentiation may be modulated by exosomes obtained from astrocytes. Treatment of proliferating NSPCs with exosomes, produced and released by differentiated cells, triggers cell differentiation toward astrocytic phenotype, as demostrated by glial and neuronal marker expression. The same effect can also be observed upon growth factor withdrawal, a condition in which NSPCs may autonomously differentiate in a mixed population of neurons and glial cells. We also show that the effect of exosomes treatment is dose-dependent

Regulation of adult mouse SVZ-derived neural stem cells properties by the transcriptional factor REST

Neural stem cell (NSC) properties are finely regulated by changes in their transcriptional program. A compelling factor involved in neuronal differentiation is the RE1 silencing transcription factor (REST). REST is a multifunction transcriptional regulator that represses or silences many genes in both neural and non-neural cells by largely unclear mechanisms that seem to depend upon developmental stage and cell type (Johnson R et al, 2008). REST is expressed at high levels in embryonic stem (ES) cells and declines during ES conversion into the neural cell lineage and fetal NSC differentiation into neurons. Recent studies highlight the crucial role of REST in regulating adult neurogenesis (Gao Z et al, 2011). We have explored how REST regulates the properties of NSCs derived from the mouse adult subventricular zone. We demonstrated that REST is expressed in different clonally derived aNSC lines and that it binds to several well-known target genes, repressing their expression. RNA interference-mediated knockdown of REST in a transiently or stable transfected clonal cell line (aNSC1) promoted cell differentiation into neurons and glia, despite the presence of growth factors in the culture medium. Interestingly REST silencing did not modify the neurogenic and gliogenic potential of aNSC1 induced to differentiate by growth factors withdrawal. These data suggest that REST knock down is sufficient to induce NSC differentiation but it does not affect their lineage-specific differentiation program. Consistently, knockdown of REST in primary neurospheres impaired their growth and clonogenic potential, as evidenced in proliferation- and colony-forming assays. We are currently performing transcriptome and ChIP Seq analyses to identify genes regulated by REST and possibly involved in NSC maintenance. Comparison with ES cell-derived NSC genome and transcriptome-wide analyses will help to define difference in Rest binding and/or regulative capability between embryonic and adult NSC