Neurofilaments are thought to play a central role in the etiology of a number of human neurodegenerative diseases, most notably amyotrophic lateral sclerosis. These disorders are characterized by massive accumulations of neurofilaments in the axons of affected neurons, leading to axonal degeneration. The accumulation of neurofilaments in these diseases is 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 cell body. However, these mechanisms are poorly understood and controversial. The principal issue concerns the site of assembly of cytoskeletal proteins and the form in which they move. The polymer transport hypothesis proposes that the cell body and proximal axons are principal sites of assembly of cytoskeletal proteins and the form in which they move. The polymer transport hypothesis proposes that the cell body and proximal axon are principal sites of assembly and that cytoskeletal proteins are transported in the form of moving polymers. In contrast, the cytoskeletal proteins are transported in the form of subunits of oligomers that assemble locally along the axon and the axon tip. To test these hypotheses, the assembly and axonal transport of neurofilament proteins will be investigated in cultured neurons, which are advantageous because of their accessibility to direct observation and experimentation. The proposed experiments will address two specific aims.
For Specific Aim 1, immunofluorescence and immunoelectron microscopy will be combined with quantitative digital image analysis to determine the sites of assembly of biotinylated and endogenous neurofilament proteins in neurons.
For Specific Aim 2, novel strategies that include constriction that includes constriction of axons will be combined with direct observation of fluorescent neurofilament proteins in living cells to determine the form in which neurofilament proteins are transported. The long-term goal of this research is to determine the mechanism by which neurofilament proteins move in axons and the mechanisms that lead to the accumulation of neurofilaments in certain neurodegenerative diseases. By testing specific hypotheses on the assembly and axonal transport of neurofilaments, the studies proposed here represent an important step toward this goal.
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