Proliferation, differentiation, and migration of endogenous adult neural stem cells and their progeny in vivo.

摘要

The adult CNS contains pluripotent, self-renewing stem cells which are capable of generating neurons, astrocytes, and oligodendrocytes. The factors controlling the proliferation, differentiation, and migration of neural stem cells and their progeny are currently the focus of intense study. This thesis is composed of three main projects. The first project assessed the role of the cytokine, ciliary neurotrophic factor (CNTF), on the proliferation and differentiation of neural stem cells in two major neurogenic regions of the forebrain, the subventricular zone (SVZ) and the dentate gyrus of the hippocampal formation. Injection of CNTF in adult C57BL/6 mouse forebrain induced proliferation in both neurogenic regions as assessed by bromodeoxyuridine (BrdU) incorporation. In the dentate gyrus, CNTF enhanced neuronal differentiation, and migration into the granule cell layer. Intraventricular injection of neutralizing anti-CNTF antibodies reduced proliferation. CNTF and anti-CNTF slightly decreased and increased, respectively, the number of apoptotic cells in the neurogenic regions. These results suggest that endogenous CNTF regulates adult neurogenesis by increasing proliferation and/or survival of newly-formed neurons. CNTFRα was most clearly present in astrocytes in neurogenic regions, and therefore the effects reported here could be indirect via neighbouring astroglia. The restricted expression of CNTF in the nervous system makes it a potential endogenous target for therapeutic cell replacement strategies.; New neuroblasts are constantly generated in the adult mammalian SVZ and migrate via a “rostral migratory stream” (RMS) to the olfactory bulb, where they differentiate into functional neurons. Little is known about molecules involved in the directed nature of this migration. The second major component of this thesis investigated the role of the α6β1 integrin, and its ligand, laminin, in controlling guidance of migrating neuroblasts. Immuno-staining for both α6β1 integrin and laminin was present within the RMS. Inhibition of the endogenous α6 or β1 integrin subunits with locally injected antibodies disrupted the cohesive nature of the RMS. Local infusion of a 15 a.a. peptide, representing the E8 domain of the laminin α chain, and which is recognized by the α6β1 integrin, redirected the neuroblasts away from the RMS into the neostriatum. Injection of a narrow tract of intact laminin also drew the neuroblasts away from the RMS, but in a more restricted localization. These results establish a critical role for integrins and laminin in adult SVZ-derived neuroblast migration.; The third component of this thesis examined retrograde cell tracing techniques in models of Parkinson's disease. Six sites of Dil injection labeled the substantia nigra more broadly and effectively than did 2 injection sites. Two injections of Fluorogold labeled fewer neurons, but their morphology was clearer. After injection of the neurotoxin 6-OHDA, neuronal survival was greater with 6 sites of Dil than with 2, but survival within the middle region was lower. Survival after 6-OHDA or axotomy was similar with Dil or Fluorogold. These results suggest that, because of a complex projection pattern of the nigrostriatal neurons, detailed quantification of neuronal survival should rely on extensive labeling.