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. In 2015, we continued our investigation into the composition and function of these heterogeneous axonal RNA populations. This past year also represented a significant transitional period for the Section on Neurobiology. For example, during the past fiscal year, the Section relocated its research facility from Building 10, which it had occupied for the past several years, to Building 49. The extensive period of time spent in the preparation for the move greatly facilitated the relocation as the laboratory was fully functional within ten days after the move. In addition, in November 2013, the Section Chief, Dr. B.B. Kaplan, stepped-down from his administrative post as Director of Fellowship Training, a position that he held for the past eighteen years, to commit 100% of his time and effort to research and management of the Section's research program. In regard to this past years research activities, members of the Section 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 in the cell body of the neuron and are later transported to their ultimate sites of function. Based, in part, on these 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 a key role in the pathophysiology of both developmental neuropsychiatric disorders and neurodegenerative disease. The findings, derived from this aspect of the Sections research program, have been submitted for publication (see Gervasi et al., 2015). 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) and quantitative PCR methodology, we discovered that there are over 350 nuclear-encoded mitochondrial mRNAs present in the axon, and that the function of this organelle, as well as the growth of the axon are dependent on the local translational activity of these mRNAs. In addition, the local translational activity of several of these mRNAs is modulated by a family of small (approximately 20-25 nucleotides), non-coding RNAs called microRNAs (e.g., Cytochrome c-Oxidase IV and ATP synthase). Interestingly, the precursors of these miRNAs are selectively transported to the axon and 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 were recently submitted for publication (Kar et al., 2015; Vargas et al., 2015). This past year, we have also broadened our investigations into the function of miRNAs in the axon through an international collaboration. This newly established collaborative research effort has already begun to bear fruit. For example, we have discovered that miRNA-338 regulates the expression of several axon guidance genes which markedly affect the migration and differentiation of cortical neurons during development (Kos et al., 2015). Moreover, we have just completed a pilot study on the involvement of local miRNA expression in neuropsychiatric disorders using a rat model of autism (Olde Loohius et al., 2015). Last, in 2015, 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., 2015). 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. Moreover, we plan to create a series of transgenic animal lines designed to disrupt the normal trafficking of mRNAs to CNS axons in both developing and adult animals.
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