Cytoplasmic accumulations of neurofilaments are a hallmark pathological feature of a number of human neurodegenerative diseases, most notably amyotrophic lateral sclerosis. These neurofilamentous accumulations are thought to be caused by changes in the mechanisms of slow axonal transport, which move cytoskeletal and cytosolic proteins along axons from their site of synthesis in the nerve cell body. We have recently observed the slow axonal transport of neurofilament protein in cultured nerve cells. The proteins move in the form of filamentous structures that may represent single neurofilament polymers. Contrary to the widely held view that slow axonal transport is a slow, synchronous and exclusively anterograde movement, we found that the filaments actually move at very fast rates, approaching the rate of fast axonal transport, and that the movements are also infrequent, bi-directional and highly asynchronous. Based on these observations, we have proposed a new model for slow axonal transport in which the actual rate of movement is fast, but the overall rate is slow because the rapid movements are interrupted by prolonged pauses. In this application, we propose to use live-cell fluorescence imaging strategies to test specific aspects of this hypothesis.
In Aim 1 we will test the hypothesis that the moving filaments represent single neurofilament polymers. We expect that these experiments will also reveal the tracks along which the filaments move.
In Aim 2 we will test the hypothesis that moving and stationary filaments differ in their phosphorylation state at specific epitopes and that they differ in their association with specific microtubule motor proteins.
In Aim 3 we will test the hypothesis that rapidly moving filaments are delivered to the tip of growing axons in sufficient quantity to support the elaboration of the axonal neurofilament array during axon growth. We will also test the hypothesis that the growth cone is a site of frequent reversals in the direction of filament movement and that the frequency and/or directionality of filament movements in the distal axon is regulated in response to the rate of axon growth. The long-term goal of our research is to determine the mechanism and regulation of neurofilament protein transport along axons and the mechanisms that lead to the accumulation of neurofilaments in neurofilamentous neuropathies. ? ?
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