Oligodendrocytes and motoneurons, two cholinergic cell types derived from multipotent spinal neuroepithelial precursor cells

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MacDonald, Stephen Christopher
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Cholinergic neurons have been implicated in various neural behaviours including motor control, autonomic function and propriospinal communication. Neuroepithelial precursors have recently emerged in the field of regenerative neurobiology and have been shown to proliferate in vitro under the influence of mitogenic growth factors. Upon removal of these mitogens, the precursor cells differentiate down neuronal and glial lineage pathways. Neural precursors offer new avenues in the study of cholinergic developmental biology and cell replacement therapies. In pursuing cholinergic neurons derived from neuroepithelial precursors, two discoveries were made that are described in this thesis. The first study reports that some ChAT expressing cells in differentiated neurosphere cultur s express oligodendrocyte and not neuron markers. Neurospheres were cultured in the presence of trophic factors and processed immunohistochemically for oligodendroglial and cholinergic markers. Almost all ChAT expressing cells in treated cultures expressed oligodendrocyte markers and displayed dramatic growth responses to the cytokine CNTF. Treatment with the cholinergic antagonist atropine during differentiation significantly decreased the amount of oligodendroglial differentiation, indicating a role for acetylcholine in oligodendrocyte differentiation. In differentiated neuroepithelial precursor cultures, a small fraction of ChAT expressing cells differentiated not into oligodendrocytes, but neurons expressing MAP-2. The multipolar morphology and large size of these cells could suggest a motoneuron identity. Further immunostaining revealed cells expressing the motoneuron markers Islet-1 and REG2. When co-cultured with dissociated skeletal myocytes, functional neuromuscular junctions were established as indicated by immunohistochemistry, drug perfusion and electrophysiology. After transplantation into the transected sciatic nerve, precursors differentiated into neurons expressing ChAT and the _-1c calcium channel subunit found on spinal motoneurons. Precursor-derived neurons also projected axons towards the distal musculature and formed cholinergic terminals. These results suggest that functional motoneurons can differentiate from proliferative neural precursors. Together these two projects demonstrate that cholinergic cell types differentiate from neuroepithelial precursors of a common origin. These findings support the hypothesis that functional motoneurons can differentiate from proliferative precursors and serendipitously demonstrate a traditional neurotransmitter synthesis enzyme being expressed in developing oligodendrocytes. This thesis also contains the most comprehensive study to date characterizing neural precursor-derived motoneurons which has relevance to cell replacement strategiesfor neurodegenerative conditions such as Amytrophic Lateral Sclerosis (ALS).