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 which 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 regard to this past years research activities, members of the Section were committed to the conduct of three independent projects. The progress effected in each of these projects is summarized, briefly, below: Project #1. Nuclear-encoded Mitochondrial mRNAs Present in the Axon. This past year, the components of the axonal nuclear-encoded mitochondrial mRNAs were delineated using a microarray analysis. This experiment was conducted in collaboration with Dr. Abdel Elkahoun, Director of the Microarray Core Facility (NHGRI). Results of this experiment were surprising, indicating that there were approximately 130 different nuclear-encoded mitochondrial mRNAs present in the axon (Aschrafi et al., 2016). Interestingly, the levels of several of these mRNAs far exceeded their corresponding levels in their parental cell bodies. This observation raises the possibility that some of these mRNAs were being selectively transported to this distal structural/functional domain of the neuron. . 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). 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 (for review, see Gale et al., 2017). 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 identified by a combination of RNA-affinity purification and mass spectroscopy. Surprisingly, there are approximately 50-60 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 (Kar et al., 2017). 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 (for review, see Kaplan et al., 2013). 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). Members of our international collaborative research team have found that miRNA338 also regulates the expression of several axon guidance genes that markedly effect the migration and differentiation of cortical neurons during development. 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. 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 and were being actively translated locally in sympathetic neurons (Gervasi et al., 2016; Aschrafi et al., 2017). The introduction of the mRNA encoding tyrosine hydroxylase, 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).
Showing the most recent 10 out of 22 publications
