Amyotrophic lateral sclerosis (ALS) results in progressive paralysis and eventual death. Its cause is unknown and there is no effective treatment despite many attempted experimental therapies. The wobbler mouse is the most studied animal model of human motor neuron disease and its pathology closely resembles that seen in ALS. This proposal will examine mechanisms underlying the degenerative changes of spinal cord motor neurons in a genetic mouse model (wobbler) of ALS and their prevention with specific neurotrophic factors. This will be done using immunocytochemical, biochemical and molecular biology techniques to: l) identify early changes of cell degeneration as revealed by specific markers (stress proteins: Grp78, Hsp7O ubiquitin; and phosphorylated neurofilament) both in vivo and in vitro, 2) examine the effects of neurotrophic factors, CNTF and BDNF on degenerating wobbler motor neurons in vitro.Two hypotheses to be tested include: 1) the wobbler mouse model of motor neuron degeneration mimics, at least in part, what is occurring in ALS; 2) the markers chosen to reveal the onset of this degeneration are good indicators of neuronal pathology.
Specific Aim 1 will determine when the first evidence of wobbler mouse spinal cord motor neuron degeneration appears in in vivo. This information will help us establish the temporal significance of other pathological changes which may reveal underlying mechanisms. Because the degeneration begins when animals are still presymptomatic (based on previous data), PCR- based wobbler gene linkage analysis will be used to identify affected animals. The changes in markers of motor neuron degeneration in vivo will provide the basis for subsequent studies.
Specific Aim 2 is to develop an organotypic explant culture of 7 day-old wobbler cervical and lumbar spinal cord slices to allow more direct observation and manipulation than is possible in vivo. Markers of cholinergic neurons and motor neuron degeneration used in Specific Aim will characterize the pathology of these cells in vitro for comparison with the in vivo changes.
Specific Aim 3 will examine whether CNTF and BDNF can prevent degeneration of wobbler spinal cord motor neurons in vitro as monitored by markers used in specific aim 2. Two related outcomes expected as a result of this proposal are: l) a solid understanding and extensive practical experience with defined immunocytochemical, biochemical, molecular biology and tissue culture techniques, 2) a better understanding of the mechanisms of wobbler mouse motor neuron degeneration in vivo and in vitro and its prevention by neurotrophic factors (e.g. CNTF, BDNF). The information obtained from this study may provide insights into the pathogenesis and treatment of neuronal degeneration in ALS.
| Mitsumoto, H; Klinkosz, B; Pioro, E P et al. (2001) Effects of cardiotrophin-1 (CT-1) in a mouse motor neuron disease. Muscle Nerve 24:769-77 |
| Tsuzaka, K; Ishiyama, T; Pioro, E P et al. (2001) Role of brain-derived neurotrophic factor in wobbler mouse motor neuron disease. Muscle Nerve 24:474-80 |
| Pioro, E P; Majors, A W; Mitsumoto, H et al. (1999) 1H-MRS evidence of neurodegeneration and excess glutamate + glutamine in ALS medulla. Neurology 53:71-9 |
| Pioro, E P; Wang, Y; Moore, J K et al. (1998) Neuronal pathology in the wobbler mouse brain revealed by in vivo proton magnetic resonance spectroscopy and immunocytochemistry. Neuroreport 9:3041-6 |
