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, it is now well- established that there exists 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- accepted 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 the surprising finding that several nuclear-encoded mitochondrial mRNAs were present in the axon 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 played 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 multi-compartment 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 prolonged neural activity (e.g., stress). Moreover, the inhibition of local protein synthesis for more than six hours significantly reduced the viability of the axon, as judged by the structure's ability to grow and thrive in the cell culture system. Most recently, we have discovered that Cytochrome c oxidase IV (COXIV) and ATP synthase mRNAs are present in the axon. These proteins are key components of Complexes IV and V of the oxidative phosphorylation chain and are rate-limiting factors in the mitochondrion's ability to generate ATP in the cell. Inhibition of the local synthesis of these proteins results in marked decrements in axon respiration, elevation in axon levels of reactive oxygen species (ROS), and attenuation of the growth of the axon. Interestingly, the deleterious effects on axonal metabolism derived from the inhibition of the local synthesis of these two proteins were additive, suggesting that ATP generation in the axon was controlled by the local translation of multiple nuclear-encoded mitochondrial mRNAs. These new findings are now being prepared for publication. During this past year, we also completed a structural/functional analysis of the 3'untranslated region (3'UTR) of the COXIV mRNA. Our results revealed that the region contained three putative regulatory domains which comprised three hairpin-loop structures. Findings derived from a deletion mutation analysis of the 3'UTR revealed that the first putative regulatory stem-loop structure contained sequences that controlled the trafficking of this mRNA to the axon (i.e., the """"""""zip-code"""""""" element). The second and third regulatory domains identified by the secondary structural analysis contained target sequences for two different brain-specific microRNAs. These RNAs comprise 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. Results of a microarray expression analysis indicated that there were approximately 130 different microRNAs present in the axon, several of which were highly abundant and appeared selectively transported into and/or were being preferentially stabilized this subcellular compartment of the neuron. Surprisingly, the expression of COXIV in the axon is being regulated by two different brain-specific microRNAs which bind to their respective signal sequences present in the second and third hair-pin loop structures located in the 3'untranslated region (3'UTR) of the mRNA. Modulation of axonal COXIV expression by these microRNAs has marked effects on the axon's metabolic activity and its capacity to generate energy. Most recently, we have identified a second nuclear-encoded mitochondrial mRNA which is regulated by these two microRNAs and also contains the """"""""zip-code"""""""" element in its 3'UTR. This finding is important because it raises the possibility that the trafficking and translation of several of the mRNAs that encode key proteins in the oxidative phosphorylation chain present in mitochondria are being co-ordinately regulated in the axon. Last, we have discovered that mRNAs encoding two translation initiation factors are present in the axon. The results generated from this portion of our investigation establish that the local expression of these factors regulate the activity of the intra-axonal protein synthetic system. The inhibition of local expression of these key proteins has a profound inhibitory effect on axon growth, as well as the long-term viability of the axon. A manuscript describing these recent results is now in preparation. 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 maintenance of the axon. We anticipate 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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