The neuronal cytoskeleton consists of three types of interlinked cytoskeletal elements, the MTs (MTs), the microfilaments and the neuronal intermediate filaments (IFs). Neurofilamentous swellings are generally observed in motor neuron diseases, such as amyotrophic lateral sclerosis (ALS). In both sporadic and familial ALS, neurofilamentous accumulations are observed in the cell bodies and proximal axons of motor neurons. Axonal transport is likely to be disrupted in diseases where neurofilamentous swellings occur. During the past grant period, we have shown that transport of neurofilaments occurs by a series of rapid transport episodes interrupted by frequent pauses. It has been suggested therefore that MT based motors participate in neurofilament transport. We hypothesize that defects in microtubule dependant transport of neurofilaments lead to the abnormal accumulations of neurofilaments that typify a number of neurodegenerative diseases, including ALS. Recently dominant mutations in the NFL gene have been described as the primary cause of an axonal form of Charcot-Marie Tooth (CMT2) disease. We have made the mutations in both human and rat NFL cDNAs and performed cell transfections studies. We found that these mutant NFL proteins have abnormal assembly properties. Our preliminary data also indicate that they may also have abnormal transport properties. We will therefore focus this proposal on the two published NFL mutations associated with CMT2, P8R and Q333P, the former in the head region of NFL and the latter in the rod region. We will characterize the assembly of the mutant NFL in more detail, study the transport of the mutant neurofilaments and generate transgenic mice that express these mutant NFL proteins. There are currently no model systems for the CMT2E disease. We hope that these transgenic models will give us a better indication of how the disease progresses and they should also give us the best system to study the downstream effects of the mutations.
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