Angiotensin II mediates the axonal trafficking of tyrosine hydroxylase and dopamine β-hydroxylase mRNAs and enhances norepinephrine synthesis in primary sympathetic neurons.

In the sympatho-adrenal system, angiotensin II (Ang II) acts as a key neuromodulatory component. At sympathetic nerve terminals, Ang II influences sympathetic transmission by enhancing norepinephrine (NE) synthesis, facilitating NE release and inhibiting NE uptake. Previously, it was demonstrated that tyrosine hydroxylase (TH) mRNA is trafficked to the distal axons of primary superior cervical ganglia (SCG) neurons, directed by a cis-acting regulatory element (i.

Disruption of the Axonal Trafficking of Tyrosine Hydroxylase mRNA Impairs Catecholamine Biosynthesis in the Axons of Sympathetic Neurons.

Tyrosine hydroxylase (TH) is the enzyme that catalyzes the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. In a previous communication, evidence was provided that TH mRNA is trafficked to the axon, where it is locally translated. In addition, a 50-bp sequence element in the 3'untranslated region (3'UTR) of TH mRNA was identified that directs TH mRNA to distal axons (i.

Nuclear-Encoded Mitochondrial mRNAs: A Powerful Force in Axonal Growth and Development.

Axons, their growth cones, and synaptic nerve terminals are neuronal subcompartments that have high energetic needs. As such, they are enriched in mitochondria, which supply the ATP necessary to meet these demands. To date, a heterogeneous population of nuclear-encoded mitochondrial mRNAs has been identified in distal axons and growth cones.

Molecular determinants of cytochrome C oxidase IV mRNA axonal trafficking.

In previous studies, we identified a putative 38-nucleotide stem-loop structure (zipcode) in the 3' untranslated region of the cytochrome c oxidase subunit IV (COXIV) mRNA that was necessary and sufficient for the axonal localization of the message in primary superior cervical ganglion (SCG) neurons. However, little is known about the proteins that interact with the COXIV-zipcode and regulate the axonal trafficking and local translation of the COXIV message. To identify proteins involved in the axonal transport of the COXIV mRNA, we used the biotinylated 38-nucleotide COXIV RNA zipcode as bait in the affinity purification of COXIV zipcode binding proteins.

A heterogeneous population of nuclear-encoded mitochondrial mRNAs is present in the axons of primary sympathetic neurons.

Mitochondria are enriched in subcellular regions of high energy consumption, such as axons and pre-synaptic nerve endings. Accumulating evidence suggests that mitochondrial maintenance in these distal structural/functional domains of the neuron depends on the "in-situ" translation of nuclear-encoded mitochondrial mRNAs. In support of this notion, we recently provided evidence for the axonal targeting of several nuclear-encoded mRNAs, such as cytochrome c oxidase, subunit 4 (COXIV) and ATP synthase, H+ transporting and mitochondrial Fo complex, subunit C1 (ATP5G1).

Axonal localization and mitochondrial association of precursor microRNA 338.

MicroRNAs (miRNAs) selectively localize to subcompartments of the neuron, such as dendrites, axons, and presynaptic terminals, where they regulate the local protein synthesis of their putative target genes. In addition to mature miRNAs, precursor miRNAs (pre-miRNAs) have also been shown to localize to somatodendritic and axonal compartments. miRNA-338 (miR-338) regulates the local expression of several nuclear-encoded mitochondrial mRNAs within axons of sympathetic neurons.

The local expression and trafficking of tyrosine hydroxylase mRNA in the axons of sympathetic neurons.

Synthesis and regulation of catecholamine neurotransmitters in the central nervous system are implicated in the pathogenesis of a number of neuropsychiatric disorders. To identify factors that regulate the presynaptic synthesis of catecholamines, we tested the hypothesis that the rate-limiting enzyme of the catecholamine biosynthetic pathway, tyrosine hydroxylase (TH), is locally synthesized in axons and presynaptic nerve terminals of noradrenergic neurons. To isolate pure axonal mRNA and protein, rat superior cervical ganglion sympathetic neurons were cultured in compartmentalized Campenot chambers.

Monitoring mRNA Translation in Neuronal Processes Using Fluorescent Non-Canonical Amino Acid Tagging.

A steady accumulation of experimental data argues that protein synthesis in neurons is not merely restricted to the somatic compartment, but also occurs in several discrete cellular micro-domains. Local protein synthesis is critical for the establishment of synaptic plasticity in mature dendrites and in directing the growth cones of immature axons, and has been associated with cognitive impairment in mice and humans. Although in recent years a number of important mechanisms governing this process have been described, it remains technically challenging to precisely monitor local protein synthesis in individual neuronal cell parts independent from the soma.

Dysregulation of the axonal trafficking of nuclear-encoded mitochondrial mRNA alters neuronal mitochondrial activity and mouse behavior.

Local translation of nuclear-encoded mitochondrial mRNAs is essential for mitochondrial activity, yet there is little insight into the role that axonal trafficking of these transcripts play in neuronal function and behavior. Previously, we identified a 38 nucleotide stem-loop structure (zipcode) in the 3' untranslated region of the Cytochrome C oxidase IV (COXIV) mRNA that directs the transport of a reporter mRNA to the axon of superior cervical ganglion neurons (SCG). Overexpression of a chimeric reporter mRNA with the COXIV zipcode competed with the axonal trafficking of endogenous COXIV mRNA, and led to attenuated axon growth in SCG neurons.

MicroRNAs in the axon and presynaptic nerve terminal.

The distal structural/functional domains of the neuron, to include the axon and presynaptic nerve terminal, contain a large, heterogeneous population of mRNAs and an active protein synthetic system. These local components of the genetic expression machinery play a critical role in the development, function, and long-term viability of the neuron. In addition to the local mRNA populations these presynaptic domains contain a significant number of non-coding RNAs that regulate gene expression post-transcriptionally.

Intra-axonal synthesis of eukaryotic translation initiation factors regulates local protein synthesis and axon growth in rat sympathetic neurons.

Axonal protein synthesis is a complex process involving selective mRNA localization and translational regulation. In this study, using in situ hybridization and metabolic labeling, we show that the mRNAs encoding eukaryotic translation initiation factors eIF2B2 and eIF4G2 are present in the axons of rat sympathetic neurons and are locally translated. We also report that a noncoding microRNA, miR16, modulates the axonal expression of eIF2B2 and eIF4G2.

MicroRNA-338 regulates the axonal expression of multiple nuclear-encoded mitochondrial mRNAs encoding subunits of the oxidative phosphorylation machinery.

MicroRNAs (miRNAs) constitute a novel class of small, non-coding RNAs that act as post-transcriptional regulators of gene expression. Remarkably, it has been shown that these small molecules can coordinately regulate multiple genes coding for proteins with related cellular functions. Previously, we reported that brain-specific miR-338 modulates the axonal expression of cytochrome c oxidase IV (COXIV), a nuclear-encoded mitochondrial protein that plays a key role in oxidative phosphorylation and axonal function.

Local translation of ATP synthase subunit 9 mRNA alters ATP levels and the production of ROS in the axon.

To date, it has been demonstrated that axonal mRNA populations contain a large number of nuclear-encoded mRNAs for mitochondrial proteins. Here, we report that the mRNA encoding ATP synthase subunit 9 (ATP5G1), a key component of Complex V of the oxidative phosphorylation chain, is present in the axons of rat primary sympathetic neurons, as judged by in situ hybridization and qRT-PCR methodology. Results of metabolic labeling studies establish that this nuclear-encoded mRNA is translated in the axon.

Identification and quantitative analyses of microRNAs located in the distal axons of sympathetic neurons.

microRNAs (miRNAs) constitute a novel class of small, noncoding RNAs that act as negative post-transcriptional regulators of gene expression. Although the nervous system is a prominent site of miRNA expression, little is known about the spatial expression profiles of miRNAs in neurons. Here, we employed compartmentalized Campenot cell culture chambers to obtain a pure axonal RNA fraction of superior cervical ganglia (SCG) neurons, and determined the miRNA expression levels in these subcellular structural domains by microarray analysis and by real-time reverse-transcription polymerase chain reaction.

Regulation of axonal trafficking of cytochrome c oxidase IV mRNA.

Trafficking and local translation of axonal mRNAs play a critical role in the development and function of this neuronal subcellular structural domain. In this report, we studied cytochrome c oxidase subunit IV (COXIV) mRNA trafficking into distal axons of primary superior cervical ganglia (SCG) neurons, and provided evidence that axonal trafficking and mitochondrial association of the mRNA are mediated by an element located in a 38bp-long, hairpin-loop forming region within the 3'UTR of the transcript. Our results also suggest that suppression of local translation of COXIV mRNA results in significant attenuation of axonal elongation.

Axonal protein synthesis and the regulation of local mitochondrial function.

Axons and presynaptic nerve terminals of both invertebrate and mammalian SCG neurons contain a heterogeneous population of nuclear-encoded mitochondrial mRNAs and a local cytosolic protein synthetic system. Nearly one quarter of the total protein synthesized in these structural/functional domains of the neuron is destined for mitochondria. Acute inhibition of axonal protein synthesis markedly reduces the functional activity of mitochondria.

MicroRNA-338 regulates local cytochrome c oxidase IV mRNA levels and oxidative phosphorylation in the axons of sympathetic neurons.

MicroRNAs (miRs) are evolutionarily conserved, noncoding RNA molecules of approximately 21 nt that regulate the expression of genes that are involved in various biological processes, such as cell proliferation and differentiation. Previously, we reported the presence of a heterogeneous population of mRNAs present in the axons and nerve terminals of primary sympathetic neurons to include the nuclear-encoded mitochondrial mRNA coding for COXIV. Sequence analysis of the 3'UTR of this mRNA revealed the presence of a putative binding site for miR-338, a brain-specific microRNA.

Axon viability and mitochondrial function are dependent on local protein synthesis in sympathetic neurons.

(1) Axons contain numerous mRNAs and a local protein synthetic system that can be regulated independently of the cell body. (2) In this study, cultured primary sympathetic neurons were employed, to assess the effect of local protein synthesis blockade on axon viability and mitochondrial function. (3) Inhibition of local protein synthesis reduced newly synthesized axonal proteins by 65% and resulted in axon retraction after 6 h.

Nerve terminals of squid photoreceptor neurons contain a heterogeneous population of mRNAs and translate a transfected reporter mRNA.

It is now well established that the distal structural/functional domains of the neuron contain 2a diverse population of mRNAs that program the local synthesis of protein. However, there is still a paucity of information on the composition and function of these mRNA populations in the adult nervous system. To generate empirically, hypotheses regarding the function of the local protein synthetic system, we have compared the mRNAs present in the squid giant axon and its parental cell bodies using differential mRNA display as an unbiased screen.

Subcellular compartmentation of neuronal protein synthesis: new insights into the biology of the neuron.

During the past few years, it has become well established that the distal structural/functional domains of the neuron contain numerous mRNAs. However, there is a paucity of information on the composition and function of these unique mRNA populations. In this article, we review recent evidence to support the hypothesis that protein synthesis occurs in multiple subcellular compartments in the neuron, to include the axon and presynaptic nerve terminal.

Identification of a novel membrane-associated protein expressed in neurons of the squid and rodent nervous systems.

In a previous communication, we reported the isolation of a novel cDNA clone (pA6) from a library constructed from squid axonal mRNAs. The partial cDNA clone contained a unique open reading frame that encoded 84 amino acids and was complementary to a moderately abundant mRNA approximately 550-600 nucleotides in length [Chun et al., J.

Protein synthesis in synaptosomes: a proteomics analysis.

A proteomics approach was used to identify the translation products of a unique synaptic model system, squid optic lobe synaptosomes. Unlike its vertebrate counterparts, this preparation is largely free of perikaryal cell fragments and consists predominantly of pre-synaptic terminals derived from retinal photoreceptor neurones. We metabolically labelled synaptosomes with [(35)S] methionine and applied two-dimensional gel electrophoresis to resolve newly synthesized proteins at high resolution.

Local synthesis of nuclear-encoded mitochondrial proteins in the presynaptic nerve terminal.

One of the central tenets in neuroscience has been that the protein constituents of distal compartments of the neuron (e.g., the axon and nerve terminal) are synthesized in the nerve cell body and are subsequently transported to their ultimate sites of function.

An association between a microsatellite polymorphism at the DRD5 gene and the liability to substance abuse: pilot study.

We have conducted a population-based association study of substance abuse and a microsatellite at the dopamine D5 receptor locus (DRD5) in a sample of European-American males and females with substance dependence (SA) or without any psychiatric disorder. Overrepresentation of the most frequent allele (148 bp) was found in males in the SA group (OR = 2.2, P = .

Molecular cloning and characterization of a novel mRNA present in the squid giant axon.

Previously, we reported the presence of a heterogeneous population of mRNAs in the squid giant axon. The construction of a cDNA library to this mRNA population has facilitated the identification of several of the constituent mRNAs which encode several cytoskeletal and motor proteins as well as enolase, a glycolytic enzyme. In this communication, we report the isolation of a novel mRNA species (pA6) from the axonal cDNA library.