The overall goal of our laboratory is to understand the mechanisms of selective vulnerability of motor neurons in amyotrophic lateral sclerosis (ALS) and to search for specific neuroprotective strategies applicable to motor neuron degenerations. The specific goal of this proposal is to determine whether (and by what mechanisms) recently engineered recombinant neurotrophic factors can be protective by (1) preserving the phenotype of relatively mature motor neurons, and (2) protecting them from experimentally induced chronic degeneration. Inevitably, much of the previous work identifying the role of neurotrophic factors in motor neuron survival has focused on models of the developing nervous system in which embryonic motor neurons undergo programmed cell death and neonatal motor neurons succumb to axonal injury. Few studies have actually considered the role of neurotrophic factors in more mature postnatal motor neurons, when the processes of naturally occurring or axotomy-induced motor neuron death are no longer applicable. Several such factors (CNTF, BDNF, IGF-I) have already been rushed into clinical trials (some would say prematurely) for neurodegenerative diseases. This work will provide important preclinical data on the use of these factors as therapy in motor neuron diseases. A unique organotypic culture system has been developed in our laboratory for postnatal rat spinal cord. Because this preparation combines the advantages of long-term survival of stable motor neurons in culture with partially preserved synaptic connections, it is an ideal system in which to test whether neurotrophic factors can have constitutive effects on relatively mature motor neurons or provide protection against motor neuron degeneration. It simultaneously provides a way of determining any inherent toxicity of these growth factors, particularly those with cytokine-like activity. The specific localization of choline acetyltransferase activity (ChAT) to large motor neurons in the ventral lumbar cord allows techniques for systematically assessing motor neuron viability. These include biochemical assay of ChAT activity and immunocytochemical staining of ChAT for morphologic analysis and cell counts. We also use organotypic spinal cord cultures to provide two novel models of chronic motor neuron degeneration due to either glutamate-mediated or oxygen radical-induced injury - both postulated mechanisms of neurotoxicity in ALS. Long term incubation of spinal cord in the presence of threohydroxyaspartate, a potent glutamate transport inhibitor, reproduces relatively selective chronic motor neuron degeneration. Incubation of spinal cord under conditions of chronic SOD-1 inhibition, using antisense oligodeoxynucleotides or diethyldithiocarbamate (a metal chelator agent), results in apoptotic neuronal degeneration, including motor neurons, morphologically distinct from excitotoxin-induced vacuolar degeneration. In these models, the neuroprotectant effects of the neurotrophic factors on motor neurons will be studied, providing further insight into the mechanisms of neurotrophic actions and potential pharmacologic strategies for therapies of motor neuron diseases such as ALS.
| Bilak, M M; Corse, A M; Bilak, S R et al. (1999) Pigment epithelium-derived factor (PEDF) protects motor neurons from chronic glutamate-mediated neurodegeneration. J Neuropathol Exp Neurol 58:719-28 |
| Corse, A M; Bilak, M M; Bilak, S R et al. (1999) Preclinical testing of neuroprotective neurotrophic factors in a model of chronic motor neuron degeneration. Neurobiol Dis 6:335-46 |
