Neuroprotection and Neuronal Differentiation Studies Using Substantia Nigra Dopaminergic Cells Derived from Transgenic Mouse Embryos

Abstract

The major pathological lesion of Parkinson's disease (PD) is the selective cell death of dopaminergic (DA) neurons in substantia nigra (SN). Although the initial cause and subsequent molecular signaling mechanisms leading to DA cell death underlying the PD process remain elusive, brain-derived neurotrophic factor (BDNF) is thought to exert neuroprotective as well as neurotrophic roles for the survival and differentiation of DA neurons in SN. Addressing molecular mechanisms of BDNF action in both primary embryonic mesencephalic cultures and in vivo animal models has been technically difficult because DA neurons in SN are relatively rare and present with many heterogeneous cell populations in midbrain. We have developed and characterized a DA neuronal cell line of embryonic SN origin that is more accessible to molecular analysis and can be used as an in vitro model system for studying SN DA neurons. A clonal SN DA neuronal progenitor cell line SN4741, arrested at an early DA developmental stage, was established from transgenic mouse embryos containing the targeted expression of the thermolabile SV40Tag in SN DA neurons. The phenotypic and morphological differentiation of the SN4741 cells could be manipulated by environmental cues in vitro. Exogenous BDNF treatment produced significant neuroprotection against 1-methyl-4-phenylpyridinium, glutamate, and nitric oxide-induced neurotoxicity in the SN4741 cells. Simultaneous phosphorylation of receptor tyrosine kinase B accompanied the neuroprotection. This SN DA neuronal cell line provides a unique model system to circumvent the limitations associated with primary mesencephalic cultures for the elucidation of molecular mechanisms of BDNF action on DA neurons of the SN. Key words: neuroprotection; BDNF; substantia nigra; dopaminergic neuron; Parkinson's disease; transgenic mice; neuronal differentiation; conditional immortalizationThe neuropathological symptoms of Parkinson's disease (PD) result from the greater than normal selective degeneration of substantia nigra (SN) dopaminergic (DA) neurons during aging. In PD the initial cause or causes and molecular mechanisms leading to the DA cell death are unknown. Treatment to prevent DA cell loss in PD is not available. Because of the paucity of DA neurons and the presence of numerous other cell populations in SN, it has not been possible to definitively address the molecular mechanisms of both DA neuronal survival and differentiation. Thus, it is crucial to acquire an abundant source of homogeneous DA neurons in vitro for the molecular dissection of SN DA neurons. To generate a consistent and abundant source of SN DA neurons in vitro, several approaches, using developmental, anatomical, somatic, or genetic manipulations, have been investigated. First, Hynes et al. (1995) tried to recapitulate the ontogeny of SN DA neuronal differentiation in explant cultures. Sonic hedgehog can induce effectively DA neurons in rat embryonic forebrain /midbrain explant. But, to practically manipulate the DA neuronal differentiation in vitro...

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