Although once highly controversial, it is now well- established that there exists a diverse population of mRNAs and non-coding microRNAs in the distal structural/functional domains of the neuron which include the dendrite, axon, and presynaptic nerve terminal. It has also become well- accepted 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 investigation have recently been reviewed (Scott et al., 2015). In regard to this past years research activities, members of the Section were committed to the conduct of two major projects. The progress effected in each of these projects is summarized, briefly, below: A third project which continued our investigation of axonal microRNA was conducted in the form of an international collaboration and employed only minimal laboratory personnel and resources . Project #1. Regulation of the axonal trafficking of RNA. This past year, the protein constituents of the axonal trafficking granule associated with Cytochrome c-oxidase (Cox IV) mRNA, a messenger that codes for a key component of the oxidative phosphorylation chain, have been tentatively identified by a combination of RNA-affinity purification and mass spectroscopy. This work was conducted in collaboration with Dr. J.A. Kowalak, a staff scientist in the new NIMH/NINDS Mass Spectroscopy Core Facility. Previously the local translation of two of these nuclear-encoded mitochondrial mRNAs was found to play a key role in the regulation of local energy metabolism, modulating the synthesis of ATP and ultimately the production of harmful reactive oxygen species (ROS) (reviewed in Scott et al., 2015; Gale et al., 2018). Dysregulation of the local translation of this mRNA resulted in reductions in the levels of ATP in the axon and an elevation in the production of ROS, two factors which negatively affected the growth and health of the axon. Surprisingly, there are approximately 30-50 different proteins that associate with the RNA sequences that regulate the transport of this mRNA to the axon (i.e., zipcode), a finding that suggests that the zipcode serves as a nucleation site for the formation of an RNA-protein trafficking complex ( see Kar et al., 2017). In addition, the proteins associated with the zipcode, regulating the trafficking of TH mRNA to the axon were identified. The protein composition of the TH mRNA trafficking complex are now being prepared for publication (Aschrafi et al., Manuscript in preparation). Project # 2. MicroRNAs present in the axon Previously, we had reported, that in addition to a highly diverse population of mRNAs, that the axon contained over 100 different small, noncoding RNAs One of these miRNAs, miRNA338, was found to coordinately regulate the local expression of several nuclear-encoded mitochondrial mRNAs that coded for key components of the oxidative phosphorylation chain and regulated local energy metabolism (e,g., Cytochrome-c oxidase IV and ATP synthase mRNAs).. Interestingly, the precursor of the mature, functional form of miRNA 338 was observed to be associated with the organelle, itself. This finding suggested that this precursor miRNA was bound to the mitochondria and served as a reservoir in the activity-dependent activation of the mature miRNA 338 (Vargas et al., 2016). The results of affinity purification and mass spectroscopy experiments indicated that the precursor miRNA338 associated with a large number of proteins that regulated its transport to the axon, as well as its binding to the mitochondria (Vargas et al., 2016). The results of gene ontology analysis indicated that these proteins included mitochondrial, cytoskeletal, and axonal motor proteins, as well as a number of well-known RNA-binding proteins. Additionally, Members of our international collaborative research team have found that miRNA338 also regulates the expression of several axon guidance genes that markedly affect the migration and differentiation of cortical neurons during development. Project #3. Local Regulation of Neurotransmitter Synthesis. This year members of the laboratory reported that mRNAs encoding the enzymes that comprise the catecholamine biosynthetic pathway were present in the axon of primary sympathetic neurons and were being actively translated. (Gervasi et al., 2016; Aschrafi et al., 2017). Importantly the introduction of the mRNA encoding tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, directly into the axon by transfection, markedly increased the synthesis of the catecholamine neurotransmitters, dopamine and norepinephrine. Stimulation of these sympathetic neurons resulted in significant increases in the release of these two neurotransmitters into the cell culture media (Aschrafi et al., 2017). Conversely, removal of the zipcode from the 3UTR of the TH mRNA, by gene editing techniques, blocked the transport of the mRNA to the axon and significantly diminished the local synthesis of protein, as well as decreased the axonal levels and release of DA and NE. This year it was also demonstrated that Angiotensin II ( Ang II) augmented the axonal trafficking of TH and DBH mRNAs and enhanced the synthesis of DA and NE. These effects were abolished by the pretreatment of the neurons with an Angiotensin II antagonist. Surprisingly, the affinity purification of RNP trafficking granules, using the TH zipcode, resulted in the enrichment of the relative abundance of both the TH and DBH mRNAs, suggesting that both of these mRNAs were being transported to the axon together, perhaps in the same RNP granule. Taken together, these results point to a novel mechanism by which Ang II participates in the regulation of the axonal synthesis of NE by modulating the local expression of TH and DBH, two enzymes involved in the catecholamine biosynthetic pathway.
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