A Novel and Efficient Gene Transfer Strategy Reduces Glial Reactivity and Improves Neuronal Survival and Axonal Growth In Vitro

Background: The lack of axonal regeneration in the central nervous system is attributed among other factors to the formation of a glial scar. This cellular structure is mainly composed of reactive astrocytes that overexpress two intermediate filament proteins, the glial fibrillary acidic protein (GFAP) and vimentin. Indeed, in vitro, astrocytes lacking GFAP or both GFAP and vimentin were shown to be the substrate for increased neuronal plasticity. Moreover, double knockout mice lacking both GFAP and vimentin presented lower levels of glial reactivity in vivo, significant axonal regrowth and improved functional recovery in comparison with wild-type mice after spinal cord hemisection. From these results, our objective was to develop a novel therapeutic strategy for axonal regeneration, based on the targeted suppression of astroglial reactivity and scarring by lentiviral-mediated RNA-interference (RNAi). Methods and Findings: In this study, we constructed two lentiviral vectors, Lv-shGFAP and Lv-shVIM, which allow efficient and stable RNAi-mediated silencing of endogenous GFAP or vimentin in vitro. In cultured cortical and spinal reactive astrocytes, the use of these vectors resulted in a specific, stable and highly significant decrease in the corresponding protein levels. In a second model -scratched primary cultured astrocytes- Lv-shGFAP, alone or associated with Lv-shVIM, decreased astrocytic reactivity and glial scarring. Finally, in a heterotopic coculture model, cortical neurons displayed higher survival rates and increased neurite growth when cultured with astrocytes in which GFAP and vimentin had been invalidated by lentiviral-mediated RNAi. Conclusions: Lentiviral-mediated knockdown of GFAP and vimentin in astrocytes show that GFAP is a key target for modulating reactive gliosis and monitoring neuron/glia interactions. Thus, manipulation of reactive astrocytes with the Lv-shGFAP vector constitutes a promising therapeutic strategy for increasing glial permissiveness and permitting axonal regeneration after central nervous system lesions. Copyright: © 2009 Desclaux et al.This work was supported by the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Sante et de la Recherche Medicale (INSERM), the Universite Pierre et Marie Curie - Paris 6 (UPMC), the Universite de Montpellier 2, Verticale, Demain Debout, and the Institut de Recherche sur la Moelle epiniere et l’Encephale (IRME).Peer Reviewe

Isolation of mineralizing Nestin+ Nkx6.1+ vascular muscular cells from the adult human spinal cord

<p>Abstract</p> <p>Background</p> <p>The adult central nervous system (CNS) contains different populations of immature cells that could possibly be used to repair brain and spinal cord lesions. The diversity and the properties of these cells in the human adult CNS remain to be fully explored. We previously isolated Nestin⁺Sox2⁺neural multipotential cells from the adult human spinal cord using the neurosphere method (i.e. non adherent conditions and defined medium).</p> <p>Results</p> <p>Here we report the isolation and long term propagation of another population of Nestin⁺cells from this tissue using adherent culture conditions and serum. QPCR and immunofluorescence indicated that these cells had mesenchymal features as evidenced by the expression of Snai2 and Twist1 and lack of expression of neural markers such as Sox2, Olig2 or GFAP. Indeed, these cells expressed markers typical of smooth muscle vascular cells such as Calponin, Caldesmone and Acta2 (Smooth muscle actin). These cells could not differentiate into chondrocytes, adipocytes, neuronal and glial cells, however they readily mineralized when placed in osteogenic conditions. Further characterization allowed us to identify the Nkx6.1 transcription factor as a marker for these cells. Nkx6.1 was expressed in vivo by CNS vascular muscular cells located in the parenchyma and the meninges.</p> <p>Conclusion</p> <p>Smooth muscle cells expressing Nestin and Nkx6.1 is the main cell population derived from culturing human spinal cord cells in adherent conditions with serum. Mineralization of these cells in vitro could represent a valuable model for studying calcifications of CNS vessels which are observed in pathological situations or as part of the normal aging. In addition, long term propagation of these cells will allow the study of their interaction with other CNS cells and their implication in scar formation during spinal cord injury.</p

Asymmetric Distribution of GFAP in Glioma Multipotent Cells

International audienceAsymmetric division (AD) is a fundamental mechanism whereby unequal inheritance of various cellular compounds during mitosis generates unequal fate in the two daughter cells. Unequal repartitions of transcription factors, receptors as well as mRNA have been abundantly described in AD. In contrast, the involvement of intermediate filaments in this process is still largely unknown. AD occurs in stem cells during development but was also recently observed in cancer stem cells. Here, we demonstrate the asymmetric distribution of the main astrocytic intermediate filament, namely the glial fibrillary acid protein (GFAP), in mitotic glioma multipotent cells isolated from glioblastoma (GBM), the most frequent type of brain tumor. Unequal mitotic repartition of GFAP was also observed in mice non-tumoral neural stem cells indicating that this process occurs across species and is not restricted to cancerous cells. Immunofluorescence and videomicroscopy were used to capture these rare and transient events. Considering the role of intermediate filaments in cytoplasm organization and cell signaling, we propose that asymmetric distribution of GFAP could possibly participate in the regulation of normal and cancerous neural stem cell fate

Brca1 is expressed in human microglia and is dysregulated in human and animal model of ALS

International audienceBackground : There is growing evidence that microglia are key players in the pathological process of amyotrophiclateral sclerosis (ALS). It is suggested that microglia have a dual role in motoneurone degeneration through therelease of both neuroprotective and neurotoxic factors.Results : To identify candidate genes that may be involved in ALS pathology we have analysed at early symptomaticage (P90), the molecular signature of microglia from the lumbar region of the spinal cord of hSOD1G93Amice, the mostwidely used animal model of ALS. We first identified unique hSOD1G93Amicroglia transcriptomic profile that, in additionto more classical processes such as chemotaxis and immune response, pointed toward the potential involvement of thetumour suppressor gene breast cancer susceptibility gene 1(Brca1). Secondly, comparison with our previous data onhSOD1G93Amotoneurone gene profile substantiated the putative contribution of Brca1 in ALS. Finally, we establishedthat Brca1 protein is specifically expressed in human spinal microglia and is up-regulated in ALS patients.Conclusions : Overall, our data provide new insights into the pathogenic concept of a non-cell-autonomous disease andthe involvement of microglia in ALS. Importantly, the identification of Brca1 as a novel microglial marker and as possiblecontributor in both human and animal model of ALS may represent a valid therapeutic target. Moreover, our data pointstoward novel research strategies such as investigating the role of oncogenic proteins in neurodegenerative diseases

Notch1 Stimulation Induces a Vascularization Switch With Pericyte-Like Cell Differentiation of Glioblastoma Stem Cells

International audienceGlioblastoma multiforms (GBMs) are highly vascularized brain tumors containing a subpopulation of multipotent cancer stem cells. These cells closely interact with endothelial cells in neurovascular niches. In this study, we have uncovered a close link between the Notch1 pathway and the tumoral vascularization process of GBM stem cells. We observed that although the Notch1 receptor was activated, the typical target proteins (HES5, HEY1, and HEY2) were not or barely expressed in two explored GBM stem cell cultures. Notch1 signaling activation by expression of the intracellular form (NICD) in these cells was found to reduce their growth rate and migration, which was accompanied by the sharp reduction in neural stem cell transcription factor expression (ASCL1, OLIG2, and SOX2), while HEY1/2, KLF9, and SNAI2 transcription factors were upregulated. Expression of OLIG2 and growth were restored after termination of Notch1 stimulation. Remarkably, NICD expression induced the expression of pericyte cell markers (NG2, PDGFRβ, and α-smooth muscle actin [αSMA]) in GBM stem cells. This was paralleled with the induction of several angiogenesis-related factors most notably cytokines (heparin binding epidermal growth factor [HB-EGF], IL8, and PLGF), matrix metalloproteinases (MMP9), and adhesion proteins (vascular cell adhesion molecule 1 [VCAM1], intercellular adhesion molecule 1 [ICAM1], and integrin alpha 9 [ITGA9]). In xenotransplantation experiments, contrasting with the infiltrative and poorly vascularized tumors obtained with control GBM stem cells, Notch1 stimulation resulted in poorly disseminating but highly vascularized grafts containing large vessels with lumen. Notch1-stimulated GBM cells expressed pericyte cell markers and closely associated with endothelial cells. These results reveal an important role for the Notch1 pathway in regulating GBM stem cell plasticity and angiogenic properties

Secreted α-Klotho maintains cartilage tissue homeostasis by repressing NOS2 and ZIP8-MMP13 catabolic axis.

International audienceProgressive loss of tissue homeostasis is a hallmark of numerous age-related pathologies, including osteoarthritis (OA). Accumulation of senescent chondrocytes in joints contributes to the age-dependent cartilage loss of functions through the production of hypertrophy-associated catabolic matrix-remodeling enzymes and pro-inflammatory cytokines. Here, we evaluated the effects of the secreted variant of the anti-aging hormone α-Klotho on cartilage homeostasis during both cartilage formation and OA development. First, we found that α-Klotho expression was detected during mouse limb development, and transiently expressed during in vitro chondrogenic differentiation of bone marrow-derived mesenchymal stem cells. Genome-wide gene array analysis of chondrocytes from OA patients revealed that incubation with recombinant secreted α-Klotho repressed expression of the NOS2 and ZIP8/MMP13 catabolic remodeling axis. Accordingly, α-Klotho expression was reduced in chronically IL1β-treated chondrocytes and in cartilage of an OA mouse model. Finally, in vivo intra-articular secreted α-Kotho gene transfer delays cartilage degradation in the OA mouse model. Altogether, our results reveal a new tissue homeostatic function for this anti-aging hormone in protecting against OA onset and progression

Symmetric and Asymmetric distribution of GFAP in mGb4 and mNSC observed by IF.

<p><b>(A)</b> Examples of symmetric and asymmetric distribution of GFAP during mitosis in mGb4 cells detected using a rabbit polyclonal antibody (Z0034, upper panel), mouse monoclonal antibody (G3893, middle panel) or chicken polyclonal antibody (AB4674, lower panel). <b>(B)</b> Examples of asymmetric and symmetric distribution of GFAP in mitotic cells stained for spindle apparatus (β-tubulin in red). <b>(C)</b> Examples of symmetric and asymmetric distribution of GFAP in mitotic spinal cord neural stem cells. Quantifications are presented on right-hand panels. n = number of observed mitotic cells (late anaphase or telophase) of at least three independent experiments. The Percent deviation in staining between two cibling cells is displayed in the bottom right corner of images. Scale bars = 10μm.</p

Using IPA software and analysis of the literature, the genes deregulated by either MK801 or GK11 treatment were each allocated one main biological function.

<p>Each bar represents the number of genes (expressed as the percentage of the total number of deregulated genes after each treatment) assigned to each specific biological function. Although the functions of a significant number of genes affected by the NMDAR antagonists are unknown, the most affected biofunction corresponds to the inflammatory and immune response regulators.</p