Axons and nerve terminals are unique subcellular structures of the neuron that play a critical role in the development and maintenance of neural connectivity. One of the central tenets in neuroscience is that the protein constituents of these distal neuronal compartments are synthesized in the nerve cell body and subsequently transported to their ultimate sites of function. Hence, the structure and function of these highly specialized distal domains of the neuron are totally dependent on slow anterograde axoplasmic transport. Although the majority of neuronal mRNAs are indeed transcribed and translated in the neuronal cell soma, recent findings have established the presence of a diverse population of mRNAs in the distal structural/functional domains of the neuron to include the axon and presynaptic nerve terminal. It has also become well established that proteins synthesized from these mRNAs play a key role in the development of the neuron and the function of the axon and nerve terminal, including navigation of the axonal growth cone, synthesis of membrane receptors employed as axon guidance molecules, axon transport, synapse formation, and in activity-dependent synaptic plasticity. In previous studies, we reported that several nuclear-encoded mitochondrial mRNAs were present in the axon and the presynaptic nerve terminal and that approximately 25% of the total protein synthesized locally in the nerve terminal was destined for the mitochondria. Based upon these findings, we hypothesized that the local protein synthetic system plays a critical role in the maintenance of the mitochondrial population and ultimately, the function of the axon and presynaptic nerve terminal. Currently, we are testing this working hypothesis using rat primary sympathetic neurons cultured in multicompartment Campenot chambers. Results of these studies established that acute inhibition of the local protein synthetic system significantly diminished the membrane potential of mitochondria, and also reduced the ability of mitochondria to maintain basal levels of axonal ATP and restore levels of axonal ATP after KCl-induced depolarization. Moreover, the inhibition of local protein synthesis for more than six hours significantly reduced the viability of the axons, as judged by the stucture's ability to thrive in the cell culture system. Most recently, we have discovered that Cytochrome c oxidase IV mRNA is present in the axon. This protein is a rate-limiting factor in the mitochondrion's ability to generate ATP in the cell. This past year, we also completed an expression array analysis to annotate the composition of the microRNA population present in the axons of sympathetic neurons. These RNAs belong to a family of small noncoding RNA molecules that regulate the posttranscriptional expression of genes that are involved in various fundamental biological processes such as cell growth and differentiation. Among the 130 different microRNAs present in the axon, four were found to be highly abundant in the axon as compared to their levels in the parental cell bodies. Moreover, a number of microRNAs encoded by a common primary nuclear transcript were differentially expressed in the axon, a finding that suggests that there is a differential and/or selective transport of microRNAs into the axonal compartment of the neuron. Surprisingly, the expression of COXIV in the axon is being regulated by a brain-specific microRNA which binds to a signal sequence present in a putative hair-pin loop structure located in the 3'untranslated region (3'UTR) of the mRNA. Modulation of axonal COXIV expression by this microRNA moiety has marked effects on the axon's metabolic activity and its capacity to generate energy. The silencing of the local expression of COXIV also has profound effects on the basal levels of reactive oxygen species (ROS) in the axon, as well as on the growth of the axon itself. Most recently, we have identified a second hair-pin loop structure comprised of a 38 nucleotide sequence in the 3'UTR of COXIV mRNA that directs the trafficking of this mRNA to the axon (""""""""zip-code""""""""). Deletion of this sequence blocks the movement of COXIV mRNA to the axon and markedly reduces COXIV levels, diminishing the metabolic activity and function of the axon. Taken together, these results indicate that the local protein synthetic system plays a key role in the regulation of mitochondrial activity and the growth and maintanence of the axon. We hope that this line of investigation will augment our understanding of the molecular mechanisms that underlie neuronal development, regeneration, and plasticity and generate new avenues of research into the pathophysiology of mental illness.
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