Adult neural stem cells in neuroinflammation

University dissertation from Stockholm : Karolinska Institutet, Department of Clinical Neuroscience

Abstract: Adult neural stem cells (NSC) can be isolated from the subventricular zone (SVZ) of the lateral ventricles, the subgranular zone (SGZ) of the hippocampus and the central canal of the spinal cord. These cells are thought to have regenerative potential and pose important therapeutic possibilities in neuroinflammatory conditions such as Multiple Sclerosis (MS). The aim of this thesis was to investigate the function of these cells during neuroinflammation. To that end we employed both in vitro primary cultures of NSC and the experimental autoimmune encephalomyelitis (EAE) model. I. NSC generate neurons in demyelinated spinal cord lesions. In order to monitor NSC behaviour in EAE we labelled the endogenous NSC by injecting a lipophilic dye, DiI in the ventricle system of Dark Agouti rats. These rats were immunized to develop EAE and injected with BrdU to identify proliferating cells. We report that NSC proliferated and migrated to demyelinated lesions in the spinal cord of EAE diseased rats, where some of the cells started to express betaIIItubulin or NeuN. Ultimately, these NSC-derived neuronal-like cells could generate overshooting action potentials. II. Nitric oxide (NO) suppresses NSC-derived neurogenesis. In this study we used NSC cultures to analyse the effects of nitric oxide (NO) on NSC proliferation and differentiation. NO is produced within EAE and MS lesions and has been correlated with disease exacerbation in MS. We report that exposure of NSC to pathophysiological concentrations of NO diverted their differentiation potential from neurogenesis towards astrogliogenesis. Using immunocytochemistry we could demonstrate a lower percentage of betaIIItubulin-IR neurons but a higher percentage of O4-IR oligodendrocytes in NO-exposed cultures. The higher rate of gliogenesis in these cultures was also confirmed by western blotting for the astrocyte-specific protein GFAP and activated STAT1, a transcription factor involved in gliogenic differentiation. Moreover, the pro-neurogenic determinant neurogenin-2 was down-regulated subsequent to NO exposure, constituting a potential mechanism for the NO-mediated down-regulation of neurogenesis. III. High Mobility Group Box Protein 1 (HMGB1) expression correlates with inflammation in MS and EAE. In the third study we focused on the expression of the cytokine HMGB1 and its receptors RAGE, TLR-2 and TLR-4 in MS and EAE. Our interest in HMGB1 is related both to its cytokine function in inflammatory diseases and to its possible involvement in cell migration and differentiation. We detected cytoplasmic translocation of HMGB1, indicative of active release, in microglia and macrophages located in MS lesions. Moreover, the expression of HMGB1 and its receptors in immune cells isolated from the cerebrospinal fluid (CSF) of MS patients was significantly higher compared to cells from CSF of control patients. In EAE, an increased transcript level of HMGB1 correlated with higher disease severity. Finally, microglia cells could translocate HMGB1 to the cytoplasm, implying their ability to actively release this protein and indicating their potential contribution to inflammation. IV. TLR-2 and TLR-4 agonists induce TNFalpha release from NSC. TLR-2 and TLR-4 recognise bacterial moieties and can also ligate HMGB1. TLR activation in innate immune cells leads to release of inflammatory agents aimed at clearing invading pathogens. The connection between TLR and NSC originates from the Drosophila ortholog, Toll, which participates in neuronal patterning. Immunocytochemical investigations of primary NSC cultures revealed the presence of both TLR-2 and TLR-4 on these cells. Moreover, the expression of these receptors was differentially regulated by inflammatory conditions and cytokines. Agonist-induced TLR activation was not involved in differentiation or proliferation of NSC. Activation of these receptors prompted NSC to express the pro-inflammatory cytokine TNFalpha at both mRNA and protein levels. In conclusion, we demonstrated that inflammatory conditions can both promote and inhibit the ectodermal differentiation capacity of NSC, but also to yield them unexpected immune features.

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