Although once highly controversial, it is now well-accepted that there exists a diverse population of mRNAs and non-coding microRNAs in the distal structural/functional domains of the neuron to include the dendrite, axon, and presynaptic nerve terminal. It has also become well-established that proteins synthesized from these mRNA templates play a key role in the development and long-term viability of the axon. The findings derived from this new area of research have recently been reviewed (Scott et al., 2015). In 2015, we continued our investigation into the composition and function of this heterogeneous axonal mRNA populations. First,in regard to this past years research activities, we reported that the mRNAs encoding the key enzymes comprising the catecholamine neurotransmitter biosynthetic pathway were present in the axons and presynaptic nerve terminals of sympathetic neurons and were indeed locally translated. These findings are at odds with the widely held belief that these enzymes are synthesized solely in the cell body of the neuron and are later transported to their ultimate sites of function. Based, in part, on our preliminary findings, we now postulate that the synthesis of the proteins mediating the synthesis of this family of neurotransmitters is regulated locally and that the dysregulation of this regulatory network might play an important role in the pathophysiology of both developmental neuropsychiatric disorders and neurodegenerative disease. The findings, derived from this exciting aspect of the Section's research program, have recently been published (Gervasi et al., 2016). Second, we continue our studies on the regulation of local mitochondrial activity. During the past year, using a microarray analysis conducted in collaboration with the National Human Genome Research Institute (NHGRI) Microarray Core Facility (Dr. Elkahloun, Director), quantitative PCR methodology, and in situ hybridization histochemistry, we reported that there were over 100 nuclear-encoded mitochondrial mRNAs present in the axon, and that the function of this organelle, as well as the growth of the axon, per se, are dependent on thelocal translational activity of these mRNA(Aschrafi et al., 2016). Interestly, over half of these nuclear-encoded mitochondrial mRNAs were present in the axon at levels two-fold greater than in the parental cell bodies. This observation suggests that a subset of the nuclear-encoded mitochondrial mRNA population was being selectively transported into this distal subcellular compartment of the neuron. In addition,the levels of several of these mRNAs is modulated by a family of small (approximately 20-25 nucleotides), non-coding RNAs called microRNAs (miRNAs. Interestingly, the precursors of a few of these miRNAs are selectively transported to the axon and are associated (i.e., co-localized) with the mitochondria itself. Based upon these findings, it is hypothesized that these precursor-miRNAs serve as a reservoir in the activity-dependent regulation of the levels of the mature, biologically functional forms of the molecule and are situated in the axon in the form of stable ribonucleoprotein particles that are juxtaposed to the organelle itself. These new observations have recently been published (Vargas et al., 2016). This past year, we completed our collaborative investigations into the function of miRNAs in the axon. This international collaborative research effort has proven rather fruitful. For example, we discovered that miRNA-338 regulated the expression of several axon guidance genes which markedly affected the migration and differentiation of cortical neurons during development (Kos et al., 2015). Moreover, we have just completed two pilot studies on the involvement of local miRNA expression in the pathobiology of animal models of neuropsychiatric disorders (Olde Loohius et al., 2015; Olde Loohius et al., 2016). Last, we initiated an exciting new series of proteomic investigations into the molecular mechanism(s) regulating the trafficking of RNA to the axon. This work is being conducted in collaboration with the NINDS/NIMH Proteomics Core (J. Kowalak, investigator). These RNAs appear to be transported to the axon in association with a large number of proteins (approx. 80-100) which form a RNA-protein trafficking granule/complex. Our initial findings describing the experimental paradigm, as well as the components of the COX IV mRNA trafficking granule are in press (Kar et al., 2016). In the future, we plan to extend these studies to include the axonal trafficking of the mRNAs encoding additional nuclear-encoded mitochondrial mRNAs (e.g., ATP Synthase), the mRNAs encoding the catecholamine biosynthetic enzymes (e.g., tyrosine hydroxylase and dopamine beta hydroxylase), as well as precursor miRNAs.(see for example, Vargas et al., 2016) Moreover, we plan to create a series of transgenic animal lines designed to disrupt the normal trafficking of these RNAs to CNS axons in both developing and adult animals. These studies will include assessments of the effects of transgene expression on brain development and animal behavior.
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