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, including navigation of the axonal growth cone, synthesis of membrane receptors employed as axon guidance molecules, synapse formation, and in activity-dependent synaptic plasticity. 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 mitochondrial population and ultimately, the function of the axon and presynaptic nerve terminal. Currently, we are testing this working hypothesis using rat primary sympathetic neurons cultured in multi-compartment Campenot chambers. Results of these studies established that acute inhibition of the local protein synthetic system significantly diminished the membrane potential of mitochondria, and also reduced the ability of mitochondria to maintain basal levels of axonal ATP and restore levels of axonal ATP after prolonged neural activity (e.g., stress). Moreover, the inhibition of local protein synthesis for more than six hours significantly reduced the viability of the axon, as judged by the structure's ability to grow and thrive in the cell culture system. Most recently, we have reported that Cytochrome c oxidase IV (COXIV) and ATP synthase mRNAs are present in the axon. These proteins are key components of Complexes IV and V of the oxidative phosphorylation chain and are rate-limiting factors in the mitochondrion's ability to generate ATP in the cell. Inhibition of the local synthesis of these proteins results in marked decrements in axon respiration, elevation in axon levels of reactive oxygen species (ROS), and attenuation of the growth of the axon. Interestingly, the deleterious effects on axonal metabolism derived from the inhibition of the local synthesis of these two proteins were additive, suggesting that ATP generation in the axon was controlled by the local translation of multiple nuclear-encoded mitochondrial mRNAs. These new findings have recently been published (Natera et al., 2012;Aschrafi et al., 2012). During this past year, we also completed a structural/functional analysis of the 3'untranslated region (3'UTR) of the COXIV mRNA. Our results revealed that the region contained three putative regulatory domains which comprised three hairpin-loop structures. Findings derived from a deletion mutation analysis of the 3'UTR revealed that two of these stem-loop structure contained target sequences for two brain-specific microRNAs. These RNAs comprise a family of small noncoding RNA molecules that regulate the posttranscriptional expression of genes that are involved in various fundamental biological processes, such as cell growth and differentiation. Surprisingly, the expression of COXIV in the axon is being regulated by two different brain-specific microRNAs which bind to their respective signal sequences present in the second and third hair-pin loop structures located in the 3'untranslated region (3'UTR) of the mRNA. Modulation of axonal COXIV expression by these microRNAs has marked effects on the axon's metabolic activity and its capacity to generate energy. Most recently, we have identified a second nuclear-encoded mitochondrial mRNA which is regulated by these two microRNAs. This finding is important because it raises the possibility that the translation of several of the mRNAs that encode key proteins in the oxidative phosphorylation chain present in mitochondria are being co-ordinately regulated in the axon. This past year, we also discovered that mRNAs encoding two translation initiation factors are 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 exciting new findings is now under review (Kar et al., J. Neurosci., in revision). Last, we have observed that the mRNAs encoding several of the key enzymes comprising the catecholamine neurotransmitter biosynthetic pathway are present in the axon of sympathetic neurons. Using a newly established metabolic labeling paradigm, and immunopurification we have been able to establish that these enzymes are locally synthesized in the axon: This surprising finding is in contrast to the commonly held belief (e.g., central dogma) that the catecholamine biosynthetic enzymes are synthesized in the cell body and are transported to their ultimate sites of function (i.e., the nerve terminal). These provocative new observations were recently presented at the Tenth International Catecholamine Symposium held in Asilomar, CA. Taken together, the results obtained over the past year indicate that the local protein synthetic system plays a key role in the regulation of mitochondrial activity, neurotransmitter biosynthesis, as well as the growth and maintenance of the axon. 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.

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Project End
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Budget End
Support Year
15
Fiscal Year
2012
Total Cost
$844,318
Indirect Cost
Name
U.S. National Institute of Mental Health
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Olde Loohuis, Nikkie F M; Martens, Gerard J M; van Bokhoven, Hans et al. (2017) Altered expression of circadian rhythm and extracellular matrix genes in the medial prefrontal cortex of a valproic acid rat model of autism. Prog Neuropsychopharmacol Biol Psychiatry 77:128-132
Kar, Amar N; Vargas, Jose Norberto S; Chen, Cai-Yun et al. (2017) Molecular determinants of cytochrome C oxidase IV mRNA axonal trafficking. Mol Cell Neurosci 80:32-43
Olde Loohuis, Nikkie F M; Nadif Kasri, Nael; Glennon, Jeffrey C et al. (2017) The schizophrenia risk gene MIR137 acts as a hippocampal gene network node orchestrating the expression of genes relevant to nervous system development and function. Prog Neuropsychopharmacol Biol Psychiatry 73:109-118
Aschrafi, Armaz; Gioio, Anthony E; Dong, Lijin et al. (2017) Disruption of the Axonal Trafficking of Tyrosine Hydroxylase mRNA Impairs Catecholamine Biosynthesis in the Axons of Sympathetic Neurons. eNeuro 4:
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Kos, Aron; de Mooij-Malsen, Annetrude J; van Bokhoven, Hans et al. (2017) MicroRNA-338 modulates cortical neuronal placement and polarity. RNA Biol 14:905-913
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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; 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
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

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