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

Diabetes-induced mechanical hyperalgesia involves spinal mitogen-activated protein kinase activation in neurons and microglia via N-methyl-D-aspartate-dependent mechanisms

ABSTRACT Molecular mechanisms underlying diabetes-induced painful neuropathy are poorly understood. We have demonstrated, in rats with streptozotocin-induced diabetes, that mechanical hyperalgesia, a common symptom of diabetic neuropathy, was correlated with an early increase in extracellular signal-regulated protein kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) phosphorylation in the spinal cord and dorsal root ganglion at 3 weeks after induction of diabetes. This change was specific to hyperalgesia because nonhyperalgesic rats failed to have such an increase. Immunoblot analysis showed no variation of protein levels, suggesting a post-translational regulation of the corresponding kinases. In diabetic hyperalgesic rats, immunocytochemistry revealed that all phosphorylated mitogen-activated protein kinases (MAPKs) colocalized with both the neuronal (NeuN) and microglial (OX42) cell-specific markers but not with the astrocyte marker [glial fibrillary acidic protein (GFAP)] in the superficial dorsal horn-laminae of the spinal cord. In these same rats, a 7-day administration [5 g/rat/day, intrathecal (i.t.)] of 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580), and anthra(1,9-cd)pyrazol-6(2H)-one (SP600125), which inhibited MAPK kinase, p38, and JNK, respectively, suppressed mechanical hyperalgesia, and decreased phosphorylation of the kinases. To characterize the cellular events upstream of MAPKs, we have examined the role of the NMDA receptor known to be implicated in pain hypersensitivity. The prolonged blockade of this receptor during 7 days by (5R,10S)-(ϩ)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]-cyclohepten-5-10-imine hydrogen maleate (MK801; 5 g/rat/day, i.t.), a noncompetitive NMDA receptor antagonist, reversed hyperalgesia developed by diabetic rats and blocked phosphorylation of all MAPKs. These results demonstrate for the first time that NMDA receptor-dependent phosphorylation of MAPKs in spinal cord neurons and microglia contribute to the establishment and longterm maintenance of painful diabetic hyperalgesia and that these kinases represent potential targets for pain therapy. Sensitive peripheral neuropathies represent a common and debilitating complication of diabetes (types 1 and 2) and affect an increasing proportion of diabetic patients as the disease progresses. Even though antidepressant and antiepileptic agents have been shown to be partially effective, clinical studies have reported the difficulty of managing pain caused by these neuropathies The mitogen-activated protein kinase (MAPK) cascade is a family of serine/threonine kinases that are activated by dual phosphorylation on threonine and tyrosine residues. The Article, publication date, and citation information can be found a

Grafted Human Embryonic Progenitors Expressing Neurogenin-2 Stimulate Axonal Sprouting and Improve Motor Recovery after Severe Spinal Cord Injury

7 p.Background: Spinal cord injury (SCI) is a widely spread pathology with currently no effective treatment for any symptom. Regenerative medicine through cell transplantation is a very attractive strategy and may be used in different non-exclusive ways to promote functional recovery. We investigated functional and structural outcomes after grafting human embryonic neural progenitors (hENPs) in spinal cord-lesioned rats.Methods and Principal Findings: With the objective of translation to clinics we have chosen a paradigm of delayed grafting, i.e., one week after lesion, in a severe model of spinal cord compression in adult rats. hENPs were either naive or engineered to express Neurogenin 2 (Ngn2). Moreover, we have compared integrating and non-integrating lentiviral vectors, since the latter present reduced risks of insertional mutagenesis. We show that transplantation of hENPs transduced to express Ngn2 fully restore weight support and improve functional motor recovery after severe spinal cord compression at thoracic level. This was correlated with partial restoration of serotonin innervations at lumbar level, and translocation of 5HT1A receptors to the plasma membrane of motoneurons. Since hENPs were not detectable 4 weeks after grafting, transitory expression of Ngn2 appears sufficient to achieve motor recovery and to permit axonal regeneration. Importantly, we also demonstrate that transplantation of naive hENPs is detrimental to functional recovery.Conclusions and Significance: Transplantation and short-term survival of Ngn2-expressing hENPs restore weight support after SCI and partially restore serotonin fibers density and 5HT1A receptor pattern caudal to the lesion. Moreover, grafting of naive-hENPs was found to worsen the outcome versus injured only animals, thus pointing to the possible detrimental effect of stem cell-based therapy per se in SCI. This is of major importance given the increasing number of clinical trials involving cell grafting developed for SCI patients.This study was supported by the European Union FP6 "RESCUE" STREP; the "Institut pour la Recherche sur la Moelle Epiniere"; the "Academie de Medecine"; the "Societe Francaise de Neurochirurgie"; "Verticale" and the "Association Demain Debout Aquitaine". The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Socializing One Health: an innovative strategy to investigate social and behavioral risks of emerging viral threats

In an effort to strengthen global capacity to prevent, detect, and control infectious diseases in animals and people, the United States Agency for International Development’s (USAID) Emerging Pandemic Threats (EPT) PREDICT project funded development of regional, national, and local One Health capacities for early disease detection, rapid response, disease control, and risk reduction. From the outset, the EPT approach was inclusive of social science research methods designed to understand the contexts and behaviors of communities living and working at human-animal-environment interfaces considered high-risk for virus emergence. Using qualitative and quantitative approaches, PREDICT behavioral research aimed to identify and assess a range of socio-cultural behaviors that could be influential in zoonotic disease emergence, amplification, and transmission. This broad approach to behavioral risk characterization enabled us to identify and characterize human activities that could be linked to the transmission dynamics of new and emerging viruses. This paper provides a discussion of implementation of a social science approach within a zoonotic surveillance framework. We conducted in-depth ethnographic interviews and focus groups to better understand the individual- and community-level knowledge, attitudes, and practices that potentially put participants at risk for zoonotic disease transmission from the animals they live and work with, across 6 interface domains. When we asked highly-exposed individuals (ie. bushmeat hunters, wildlife or guano farmers) about the risk they perceived in their occupational activities, most did not perceive it to be risky, whether because it was normalized by years (or generations) of doing such an activity, or due to lack of information about potential risks. Integrating the social sciences allows investigations of the specific human activities that are hypothesized to drive disease emergence, amplification, and transmission, in order to better substantiate behavioral disease drivers, along with the social dimensions of infection and transmission dynamics. Understanding these dynamics is critical to achieving health security--the protection from threats to health-- which requires investments in both collective and individual health security. Involving behavioral sciences into zoonotic disease surveillance allowed us to push toward fuller community integration and engagement and toward dialogue and implementation of recommendations for disease prevention and improved health security

Synaptogenesis : facts and prospects

Dans la première moitié du XXe siècle, deux théories sopposaient concernant la constitution du système nerveux. Nous savons maintenant qu'il est bien formé de cellules et de neurones dont la connexion s'effectue grâce aux synapses. Santiago Ramon y Cajal a inventé ce terme de synapse aux environs de 1930. Partisan de cette théorie neuronale du système nerveux, il s'opposait ainsi aux partisans de la théorie réticulaire. Après ce préambule historique, l'A. sintéresse au processus de genèse des synapses durant la constitution de l'embryon

Mise au point d un modèle de compression médullaire chez la souris (étude comportementale et histopathologique)

Malgré des efforts considérables effectués au cours des deux dernières décennies, il n'existe jusqu'à présent aucune approche thérapeutique efficace dans les lésions de la moelle épinière (SCI). Ceci est attribuable, du moins en partie, aux difficultés rencontrées pour l'élaboration de modèles animaux prédictifs précis. Des études antérieures ont identifié deux conditions pertinentes à ces modèles. D'abord, on note l'intérêt de valider des modèles de SCI de souris à partir des données provenant de modèles chez le rat. Ceci permet d'avoir accès à un large répertoire de modèles transgéniques afin de valider les hypothèses physiopathologiques. L'autre élément est l'exploration des conséquences de chaque mécanisme de traumatisme de la moelle. Ceci requiert la modélisation des différents aspects étiologiques de la lésion (c'est-à-dire l'ischémie, la contusion et la compression). Ainsi, nous avons conçu un nouveau modèle de compression de la moelle épinière de souris en utilisant un dispositif olivaire que nous décrivons. Nous rapportons les premiers résultats fonctionnels moteurs et histologiques pour 3 différents calibres d'olive et différentes durées de compressions. Nous montrons que le modèle est calibré et reproductible, avec un retentissement fonctionnel et une extension lésionnelle, tous les deux corrélés à la gravité de la compression. Pour étayer la caractérisation de ce modèle, nous l'avons validé pharmacologiquement par un antagoniste du récepteur NMDA, la gacyclidine, et démontré l'implication de l'excitotoxicité dans mécanisme lésionnel. Nous avons donc la preuve que ce modèle de compression chez la souris est reproductible, bien caractérisé et prévisible. Son caractère non magnétique et contrôlable à distance permettra en outre l'analyse de la SCI en temps réel par IRM, donnant un aperçu de la caractérisation spatiale et temporelle de l'évolution de la lésion. En outre, il ouvre la voie à des études in vivo de SCI chez en animaux génétiquement modifiés.Despite considerable efforts over the last two decades there is so far no efficient therapeutic approach in spinal cord injuries (SCI). This may be attributable, at least in part, to difficulties encountered for elaborating predictive and accurate experimental animal models of this pathology. Previous studies have identified two relevant conditions of such model. First, the comparison of data arising from rat models by developing mice models that permits to have access to a large repertory of transgenic models, thus allowing to probe precise pathophysiological hypotheses. Second, the exploration of the consequences of each mechanism of the spinal trauma requires modeling the different etiologic aspects of the injury (i.e. ischemia, mechanic contusion and compression). In order to fulfill these two conditions we have devised a new model of compression injury of the mouse spinal cord using a thread-driven olive-shaped compressive device. We havecharacterized early motor, sensory and histological outcomes using three olive diameters anddifferent compression duration. We show a gradual and reproducible functional severity thatcorrelates with lesion extension. To further substantiate the characterization of this model, wedispensed a non-competitive NMDA antagonist and demonstrated the involvement ofexcitotoxicity in this model. We thus demonstrate that spinal olive-compression injury in the mouse is a reproducible, well-characterized and predictable model to analyse early event following SCI. The nonmagnetic and remote controlled design of this model will allow further real-time MRI studies that will give insights in the characterization of spatial and temporal evolution of SCI. Moreover it paves the way toward in vivo studies of functional and histological outcomes following SCI in genetically engineered animals.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF