Neurorestorative strategies involving neurogenesis, neuronal precursors and stem cells in animal models of Parkinson's disease

Abstract: The general aims of the thesis is to provide evidence for neurogenesis in the adult substantia nigra pars compacta (SNpc), where the dopamineproducing neurons lost in Parkinson's disease (PD) reside; to optimize methods used for detecting newborn nerve cells in adult brain regions with a low rate of neurogenesis; to explore the mechanism of nerve cell death in animal models of parkinsonism and early degenerative and restorative changes in nigral cell populations; and to develop a therapeutic approach that could translate into a future restorative disease-modifying strategy in PD. Paper I. We provide evidence for the generation of dopaminergic projection neurons in adult SNpc, and estimate the low rate of turnover in this brain region. We administered the thymidine-analogue 5-bromodeoxyuridine (BrdU) or [3H]-thymidine using various regimen strategies to establish the generation of new neurons in SNpc in the confocal light and electron microscope. To trace the origin of the newly generated cells from stem cells lining the cerebroventricular system, we labeled ependymal cells with 1,1´-dioctadecyl-6,6´-di-(4-sulfophenyl)-3,3,3´,3´-tetramethylindocarbocyanine (DiI) or rhodamine-conjugated latex beads. The results obtained in this work show that (1) the size of the nigral nerve cell population remains constant during a large part of the mouse life span; (2) neurogenesis occurs in the substantia nigra and the BrdU-labeled neurons do not represent DNA repair; (3) the newly generated neurons project to the striatum and integrate into synaptic circuits; (4) there is increased neurogenesis after a partial 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced lesion. Paper II. Methodological differences may explain discrepancies between research reports on adult mammalian neurogenesis in the brain outside the widely accepted neurogenic regions, i.e. hippocampus and olfactory bulb/subventricular zone. We describe a method to dissolve and administer BrdU at high concentrations (150 mg/mL) into the adult mouse right cerebral ventricle to demonstrate neuronal incorporation of this thymidine analogue in CNS regions with a low rate of neurogenesis. The dosage regimen, duration and survival time of the mouse after the end of the nucleotide analogue administration all are critical, since e.g. exposure to low doses (paper I) did not result in a robust neuronal label. Techniques to optimize BrdU detection include tissue denaturation to fully expose the incorporated nuclear thymidine analogue for immunohistochemical staining. On the other hand, strategies to protect other tissue antigens against deterioration by this denaturation (HCl and pepsin) are necessary. Nigral neuronal incorporation of another proliferative marker, [3H]-thymidine confirmed the presence of neurogenesis in adult mammalian substantia nigra under physiological conditions. Paper III. To understand early neurodegenerative and neurorestorative post-lesion events in SNpc after a single dose of MPTP, we analyzed cell death by apoptosis as well as parallel dynamic changes in nigral populations of neurons, glia and progenitor cells. At the time of the peak of the MPTP-induced apoptosis, condensed neuronal nuclei containing fragmented DNA and activated caspases in the cytoplasm was found in nigral neurons, but neither in glial nor progenitor cells. Ultrastructural analysis confirmed the neuronal phenotype of cells with apoptotic morphologies. Moreover, the dynamic changes during the first 7 days after a single MPTP-induced lesion indicate that neuronal apoptosis is slow, lasting over several days, and that parallel changes in neuronal and glial progenitor populations occur, possibly representing both neurodegenerative and neurorestorative events. Stereological cell counts of nigral neurons using antibodies against tyrosine hydroxylase and neuronal-specific nuclear protein combined with Nissl staining all provide evidence that neurons are lost by a single dose of MPTP, in contrast to earlier reports indicating that neurons do not die in this lesion model, but merely lose expression of the rate limiting enzyme for dopamine synthesis. Paper IV. Intracerebroventricular administration of platelet-derived growth factor (PDGF)-BB for two weeks in parkinsonian animal models result in long-lasting dopaminergic restoration and functional recovery. We used several animal models, all inducing partial lesions in the nigrostriatal dopamine system, including mice and monkeys lesioned with MPTP and rats with a 6-hydroxydopamine (6OHDA)-injection in the right medial forebrain bundle. PDGF-BB promoted proliferation of neural progenitor cells in the subventricular zone in all animal species used. When the proliferation inhibitor cytosine-D-arabinofuranoside was coadministered with PDGF-BB in the 6-OHDA lesioned rats for two weeks, both structural and behavioral restoration was blocked. A link between the anti-parkinsonian effect of PDGF-BB and proliferation points to a new strategy in the search for disease-modifying agents that could lead to the development of new therapies against PD.