Axnoal Transport and Cytoskeletal Incorporation of Neurofilaments

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Tom Shea99-05123NON-TECHNICAL SUMMARYNeurons (the "thinking" cells of the brain) interconnect with each other by long extensions ("axons") that, like telephone wires, transmit the signals that allow us to think, feel and move. Once neurons have connected in this manner, their overall structure remains largely unchanged throughout an entire lifetime. These axons are, however, as alive, must be maintained, and are even more prone to stress than most of our cells. Their shape and ability to remain connected to the next neuron is derived from fibrous protein assemblies that are deposited along their length. This array, collectively referred to as the "cytoskeleton", includes "neurofilaments" and "microtubules". A major complicating factor is that the axon cannot manufacture any required proteins. Rather, everything in the axon, including the fibers themselves, is first synthesized in the cell body, and subsequently passed along the axon by a process referred to as "axonal transport." Certain proteins function as motors and carry other proteins and nutrients along the axon using microtubules essentially as "railroad tracks." The motor(s) that mediate axonal transport of neurofilaments are not known. Since neurofilaments are thought to provide structural support to the mature axon, elucidation of their transport mechanism is of great interest both for understanding neuronal development and for considerations of regeneration. Studies suggest that neurofilaments undergo transport as cargo of the motor protein "kinesin", and that they are also transported along microtubule tracks. The proposed studies will examine the nature and extent of interaction of neurofilaments with this motor protein, including determining which of the 3-neurofilament subunits, and moreover which region of the subunit(s), mediates this interaction. Once in the axon, neurofilaments undergo a series of metabolic modifications ("phosphorylation") which promote neurofilament-neurofilament associations, and, in doing so, form the latticework of the cytoskeleton. It will also be determined whether or not such neurofilament-neurofilament associations compete with neurofilament-motor associations. These analyses will be carried out by expressing foreign genes in cultured neurons and in mice that have been genetically engineered to lack one or more neurofilament subunits. These latter analyses will also provide information as to how precocious neurofilament phosphorylation, by fostering prematuredissociation from their transport motor, could contribute to their aberrant accumulation in certain motor neuron disorders.