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 are 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. 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 activity of the local mitochondrial population and ultimately, the function of the axon and presynaptic nerve terminal. Most recently, we have reported that Cytochrome c oxidase IV (COXIV) and ATP synthase mRNAs are present in the axon. These mRNAs encode proteins that are key components of the oxidative phosphorylation chain and are rate-limiting factors in the mitochondrions ability to generate ATP. During the past year, we completed studies on the regulation of the local expression of these two nuclear-encoded mitochondrial mRNAs by a brain-specific micro RNA-338. Micro RNAs are small, non-coding RNA molecules that regulate the post-transcriptional expression of a large number of gene products. The results of these experiments established that the miRNA-338 binding sites located in the 3UTR of COXIV and ATP synthase mRNAs were biologically active and that the co-ordinate regulation of the levels of these nuclear-encoded mitochondrial mRNAs in the axon had a profound effect on the axons metabolic activity and its capacity to generate energy. In addition, the down-regulation of the expression of these mRNAs also had significant inhibitory effects on the growth of the axon and neuronal differentiation. Our work on the composition and function of the axonal miRNA population was recently reviewed (Kaplan et al., 2013;see below). This past year, we also discovered that mRNAs encoding two translation initiation factors were also 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 new findings was recently published (Kar et al., 2013). Within the context of this study, we were successful in establishing a transgenic mouse line that over-expressed the COXIV and ATP synthase mRNA zip-code. This 38-nt hair-pin loop structure present in the 3UTR of these mRNAs regulates their trafficking to the axon. The findings of our previous in vitro experiments indicate that over-expression of these sequences inhibits the normal transport of the endogenous mRNAs to the axon and disrupts the activity of the local mitochondria. Our newly created transgenic animals over-express the zip-code specifically in neurons located in the deep layers of the cerebral cortex. The over-expression of the zip-code in these neurons results in significant increases in ROS production and the manifestation of anxiety-like behavior in the transgenic mice. A manuscript describing these new findings has been submitted for publication. (Kar et all., 2013;see below) Last, we continue to conduct our exploratory, high-risk investigation designed to test the working hypothesis that the synthesis and release/reuptake of the catecholamine neurotransmitters are regulated locally in the axon and presynaptic nerve terminal. In this regard, our preliminary data indicate that the mRNAs encoding all the enzymes of the catecholamine biosynthesis pathway are present in the axons of sympathetic neurons, and are locally translated. Additionally, the levels of these mRNAs in the polysome fraction could be induced 5-to 8-fold by elevating the levels of cyclic AMP in the axon, a finding that suggests that the intra-axonal synthesis of these enzymes can be modulated by synaptic activity. These provocative results are in contrast to the commonly held belief (e.g., central dogma) that the catecholamine biosynthesis enzymes are synthesized in the cell body and are transported to their sites of function (i.e., nerve terminal) by slow anterograde axonal transport, and may ultimately lead to a paradigm shift in the manner in which we envision the regulation of neurotransmitter synthesis and release. Taken together, these results indicate that the local protein synthetic system plays a key role in the regulation of mitochondrial activity and axon growth, as well as neurotransmitter metabolism. 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. Kaplan BB, Kar AN, Gioio AE, Aschrafi A. (2013). MicroRNAs in the axon and presynaptic nerve terminal. Fronteirs Cell Neurosci. In press Kar AN, Sun C-Y, Reichard K, Pickel J, Nakazawa K, Gervasi NM, Gioio AE, Kaplan BB (2013). Dysregulation of the axonal trafficking of nuclear-encoded mitochondrial mRNAs alters axonal growth and mouse behavior. Dev. Neurobio. In revision.

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U.S. National Institute of Mental Health
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Aschrafi, Armaz; Kar, Amar N; Gale, Jenna R et al. (2016) A heterogeneous population of nuclear-encoded mitochondrial mRNAs is present in the axons of primary sympathetic neurons. Mitochondrion 30:18-23
Kos, Aron; Wanke, Kai A; Gioio, Anthony et al. (2016) Monitoring mRNA Translation in Neuronal Processes Using Fluorescent Non-Canonical Amino Acid Tagging. J Histochem Cytochem 64:323-33
Vargas, Jose Norberto S; Kar, Amar N; Kowalak, Jeffrey A et al. (2016) Axonal localization and mitochondrial association of precursor microRNA 338. Cell Mol Life Sci 73:4327-4340
Kos, Aron; Klein-Gunnewiek, Teun; Meinhardt, Julia et al. (2016) MicroRNA-338 Attenuates Cortical Neuronal Outgrowth by Modulating the Expression of Axon Guidance Genes. Mol Neurobiol :
Kos, Aron; Olde Loohuis, Nikkie; Meinhardt, Julia et al. (2016) MicroRNA-181 promotes synaptogenesis and attenuates axonal outgrowth in cortical neurons. Cell Mol Life Sci 73:3555-67
Gervasi, Noreen M; Scott, Shane S; Aschrafi, Armaz et al. (2016) The local expression and trafficking of tyrosine hydroxylase mRNA in the axons of sympathetic neurons. RNA 22:883-95
Aschrafi, Armaz; Verheijen, Jan M; Gordebeke, Peter M et al. (2016) MicroRNA-326 acts as a molecular switch in the regulation of midbrain urocortin 1 expression. J Psychiatry Neurosci 41:342-53
Olde Loohuis, N F M; Kole, K; Glennon, J C et al. (2015) Elevated microRNA-181c and microRNA-30d levels in the enlarged amygdala of the valproic acid rat model of autism. Neurobiol Dis 80:42-53
Kar, Amar N; Sun, Ching-Yu; Reichard, Kathryn et al. (2014) Dysregulation of the axonal trafficking of nuclear-encoded mitochondrial mRNA alters neuronal mitochondrial activity and mouse behavior. Dev Neurobiol 74:333-50
Kar, Amar N; Macgibeny, Margaret A; Gervasi, Noreen M et al. (2013) Intra-axonal Synthesis of Eukaryotic Translation Initiation Factors Regulates Local Protein Synthesis and Axon Growth in Rat Sympathetic Neurons. J Neurosci 33:7165-74

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