The functional organization of axonal mRNA transport and translation.

Axons extend for tremendously long distances from the neuronal soma and make use of localized mRNA translation to rapidly respond to different extracellular stimuli and physiological states. The locally synthesized proteins support many different functions in both developing and mature axons, raising questions about the mechanisms by which local translation is organized to ensure the appropriate responses to specific stimuli. Publications over the past few years have uncovered new mechanisms for regulating the axonal transport and localized translation of mRNAs, with several of these pathways converging on the regulation of cohorts of functionally related mRNAs - known as RNA regulons - that drive axon growth, axon guidance, injury responses, axon survival and even axonal mitochondrial function. Recent advances point to these different regulatory pathways as organizing platforms that allow the axon's proteome to be modulated to meet its physiological needs.

Intra-axonal translation of Khsrp mRNA slows axon regeneration by destabilizing localized mRNAs.

Axonally synthesized proteins support nerve regeneration through retrograde signaling and local growth mechanisms. RNA binding proteins (RBP) are needed for this and other aspects of post-transcriptional regulation of neuronal mRNAs, but only a limited number of axonal RBPs are known. We used targeted proteomics to profile RBPs in peripheral nerve axons. We detected 76 proteins with reported RNA binding activity in axoplasm, and levels of several change with axon injury and regeneration. RBPs with altered levels include KHSRP that decreases neurite outgrowth in developing CNS neurons. Axonal KHSRP levels rapidly increase after injury remaining elevated up to 28 days post axotomy. Khsrp mRNA localizes into axons and the rapid increase in axonal KHSRP is through local translation of Khsrp mRNA in axons. KHSRP can bind to mRNAs with 3'UTR AU-rich elements and targets those transcripts to the cytoplasmic exosome for degradation. KHSRP knockout mice show increased axonal levels of KHSRP target mRNAs, Gap43, Snap25, and Fubp1, following sciatic nerve injury and these mice show accelerated nerve regeneration in vivo. Together, our data indicate that axonal translation of the RNA binding protein Khsrp mRNA following nerve injury serves to promote decay of other axonal mRNAs and slow axon regeneration.

Selective axonal translation of the mRNA isoform encoding prenylated Cdc42 supports axon growth.

The small Rho-family GTPase Cdc42 has long been known to have a role in cell motility and axon growth. The eukaryotic Ccd42 gene is alternatively spliced to generate mRNAs with two different 3' untranslated regions (UTRs) that encode proteins with distinct C-termini. The C-termini of these Cdc42 proteins include CaaX and CCaX motifs for post-translational prenylation and palmitoylation, respectively. Palmitoyl-Cdc42 protein was previously shown to contribute to dendrite maturation, while the prenyl-Cdc42 protein contributes to axon specification and its mRNA was detected in neurites. Here, we show that the mRNA encoding prenyl-Cdc42 isoform preferentially localizes into PNS axons and this localization selectively increases in vivo during peripheral nervous system (PNS) axon regeneration. Functional studies indicate that prenyl-Cdc42 increases axon length in a manner that requires axonal targeting of its mRNA, which, in turn, needs an intact C-terminal CaaX motif that can drive prenylation of the encoded protein. In contrast, palmitoyl-Cdc42 has no effect on axon growth but selectively increases dendrite length. Together, these data show that alternative splicing of the Cdc42 gene product generates an axon growth promoting, locally synthesized prenyl-Cdc42 protein. This article has an associated First Person interview with one of the co-first authors of the paper.

hnRNPs Interacting with mRNA Localization Motifs Define Axonal RNA Regulons.

mRNA translation in axons enables neurons to introduce new proteins at sites distant from their cell body. mRNA-protein interactions drive this post-transcriptional regulation, yet knowledge of RNA binding proteins (RBP) in axons is limited. Here we used proteomics to identify RBPs interacting with the axonal localizing motifs of Nrn1, Hmgb1, Actb, and Gap43 mRNAs, revealing many novel RBPs in axons. Interestingly, no RBP is shared between all four RNA motifs, suggesting graded and overlapping specificities of RBP-mRNA pairings. A systematic assessment of axonal mRNAs interacting with hnRNP H1, hnRNP F, and hnRNP K, proteins that bound with high specificity to Nrn1 and Hmgb1, revealed that axonal mRNAs segregate into axon growth-associated RNA regulons based on hnRNP interactions. Axotomy increases axonal transport of hnRNPs H1, F, and K, depletion of these hnRNPs decreases axon growth and reduces axonal mRNA levels and axonal protein synthesis. Thus, subcellular hnRNP-interacting RNA regulons support neuronal growth and regeneration.

Axonal localization of neuritin/CPG15 mRNA is limited by competition for HuD binding.

HuD protein (also known as ELAVL4) has been shown to stabilize mRNAs with AU-rich elements (ARE) in their 3' untranslated regions (UTRs), including Gap43, which has been linked to axon growth. HuD also binds to neuritin (Nrn1) mRNA, whose 3'UTR contains ARE sequences. Although the Nrn1 3'UTR has been shown to mediate its axonal localization in embryonic hippocampal neurons, it is not active in adult dorsal root ganglion (DRG) neurons. Here, we asked why the 3'UTR is not sufficient to mediate the axonal localization of Nrn1 mRNA in DRG neurons. HuD overexpression increases the ability of the Nrn1 3'UTR to mediate axonal localizing in DRG neurons. HuD binds directly to the Nrn1 ARE with about a two-fold higher affinity than to the Gap43 ARE. Although the Nrn1 ARE can displace the Gap43 ARE from HuD binding, HuD binds to the full 3'UTR of Gap43 with higher affinity, such that higher levels of Nrn1 are needed to displace the Gap43 3'UTR. The Nrn1 3'UTR can mediate a higher level of axonal localization when endogenous Gap43 is depleted from DRG neurons. Taken together, our data indicate that endogenous Nrn1 and Gap43 mRNAs compete for binding to HuD for their axonal localization and activity of the Nrn1 3'UTR.

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. qRT-PCR and RNA in situ hybridization analyses showed that TH mRNA is present in distal axons. Colocalization experiments with nerve terminal marker proteins suggested that both TH mRNA and protein localize in regions of the axon that resemble nerve terminals (i.e., synaptic boutons). Analysis of polysome-bound RNA showed that TH mRNA is present in polysomes isolated from distal axons. Metabolic labeling of axonally synthesized proteins labeled with the methionine analog, L-azidohomoalanine, showed that TH is locally synthesized in axons. Moreover, the local transfection and translation of exogenous TH mRNA into distal axons facilitated axonal dopamine synthesis. Finally, using chimeric td-Tomato-tagged constructs, we identified a sequence element within the TH 3'UTR that is required for the axonal localization of the reporter mRNA. Taken together, our results provide the first direct evidence that TH mRNA is trafficked to the axon and that the mRNA is locally translated. These findings raise the interesting possibility that the biosynthesis of the catecholamine neurotransmitters is locally regulated in the axon and/or presynaptic nerve terminal.

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. Gel-shift assays of the biotinylated COXIV zipcode indicated that the putative stem-loop structure functions as a nucleation site for the formation of ribonucleoprotein complexes. Mass spectrometric analysis of the COXIV zipcode ribonucleoprotein complex led to the identification of a large number RNA binding proteins, including fused in sarcoma/translated in liposarcoma (FUS/TLS), and Y-box protein 1 (YB-1). Validation experiments, using western analyses, confirmed the presence of the candidate proteins in the COXIV zipcode affinity purified complexes obtained from SCG axons. Immunohistochemical studies show that FUS, and YB-1 are present in SCG axons. Importantly, RNA immunoprecipitation studies show that FUS, and YB-1 interact with endogenous axonal COXIV transcripts. siRNA-mediated downregulation of the candidate proteins FUS and YB-1 expression in the cell-bodies diminishes the levels of COXIV mRNA in the axon, suggesting functional roles for these proteins in the axonal trafficking of COXIV mRNA.

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. Previous work has shown that precursor miR-338 (pre-miR-338) introduced into the axon can locally be processed into mature miR-338, where it can regulate local ATP synthesis. However, the mechanisms underlying the localization of pre-miRNAs to the axonal compartment remain unknown. In this study, we investigated the axonal localization of pre-miR-338. Using proteomic and biochemical approaches, we provide evidence for the localization of pre-miR-338 to distal neuronal compartments and identify several constituents of the pre-miR-338 ribonucleoprotein complex. Furthermore, we found that pre-miR-338 is associated with the mitochondria in axons of superior cervical ganglion (SCG) neurons. The maintenance of mitochondrial function within axons requires the precise spatiotemporal synthesis of nuclear-encoded mRNAs, some of which are regulated by miR-338. Therefore, the association of pre-miR-338 with axonal mitochondria could serve as a reservoir of mature, biologically active miRNAs, which could coordinate the intra-axonal expression of multiple nuclear-encoded mitochondrial mRNAs.

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. Transfection of axons with precursor miR16 and anti-miR16 showed that local miR16 levels modulated axonal eIF2B2 and eIF4G2 mRNA and protein levels, as well as axon outgrowth. siRNA-mediated knock-down of axonal eIF2B2 and eIF4G2 mRNA also resulted in a significant decrease in axonal eIF2B2 and eIF4G2 protein. Moreover, results of metabolic labeling studies showed that downregulation of axonal eIF2B2 and eIF4G2 expression also inhibited local protein synthesis and axon growth. Together, these data provide evidence that miR16 mediates axonal growth, at least in part, by regulating the local protein synthesis of eukaryotic translation initiation factors eIF2B2 and eIF4G2 in the axon.

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. Here, we report that ATP synthase (ATP5G1), like COXIV mRNA, contains a putative miR-338 binding site, and that modulation of miR-338 levels in the axon results in alterations in both COXIV and ATP5G1 expression. Importantly, miR-338 modulation of local COXIV and ATP5G1 expression has a marked effect on axonal ROS levels, as well as axonal growth. These findings point to a mechanism by which miR-338 modulates local energy metabolism through the coordinate regulation of the expression of multiple nuclear-encoded mitochondrial mRNAs in the axon.

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. The siRNA-mediated knock-down of axonal ATP5G1 mRNA resulted in a significant reduction of axonal ATP5G1 protein and ATP levels. Silencing of local ATP5G1 expression enhanced the production of local reactive oxygen species (ROS). Importantly, reduction in the levels of ATP5G1 expression resulted in a marked attenuation in the rate of elongation of the axon. Exposure of the distal axons to nordihydroguaiaretic acid (NDGA), a ROS scavenger, mitigated the reduction in the rate of axon elongation observed after knock-down of ATP5G1. Taken together, these data call attention to the key regulatory role that local translation of nuclear-encoded mitochondrial mRNAs plays in energy metabolism and growth of the axon.

MicroRNAs 21 and 199a-3p Regulate Axon Growth Potential through Modulation of Pten and mTor mRNAs.

Increased mTOR activity has been shown to enhance regeneration of injured axons by increasing neuronal protein synthesis, while PTEN signaling can block mTOR activity to attenuate protein synthesis. MicroRNAs (miRs) have been implicated in regulation of PTEN and mTOR expression, and previous work in spinal cord showed an increase in miR-199a-3p after spinal cord injury (SCI) and increase in miR-21 in SCI animals that had undergone exercise. Pten mRNA is a target for miR-21 and miR-199a-3p is predicted to target mTor mRNA. Here, we show that miR-21 and miR-199a-3p are expressed in adult dorsal root ganglion (DRG) neurons, and we used culture preparations to test functions of the rat miRs in adult DRG and embryonic cortical neurons. miR-21 increases and miR-199a-3p decreases in DRG neurons after in vivo axotomy. In both the adult DRG and embryonic cortical neurons, miR-21 promotes and miR-199a-3p attenuates neurite growth. miR-21 directly bound to Pten mRNA and miR-21 overexpression decreased Pten mRNA levels. Conversely, miR-199a-3p directly bound to mTor mRNA and miR-199a-3p overexpression decreased mTor mRNA levels. Overexpressing miR-21 increased both overall and intra-axonal protein synthesis in cultured DRGs, while miR-199a-3p overexpression decreased this protein synthesis. The axon growth phenotypes seen with miR-21 and miR-199a-3p overexpression were reversed by co-transfecting PTEN and mTOR cDNA expression constructs with the predicted 3' untranslated region (UTR) miR target sequences deleted. Taken together, these studies indicate that injury-induced alterations in miR-21 and miR-199a-3p expression can alter axon growth capacity by changing overall and intra-axonal protein synthesis through regulation of the PTEN/mTOR pathway.

Intra-axonal mechanisms driving axon regeneration.

Traumatic injury to the peripheral and central nervous systems very often causes axotomy, where an axon loses connections with its target resulting in loss of function. The axon segments distal to the injury site lose connection with the cell body and degenerate. Axotomized neurons in the periphery can spontaneously mount a regenerative response and reconnect to their denervated target tissues, though this is rarely complete in humans. In contrast, spontaneous regeneration rarely occurs after axotomy in the spinal cord and brain. Here, we concentrate on the mechanisms underlying this spontaneous regeneration in the peripheral nervous system, focusing on events initiated from the axon that support regenerative growth. We contrast this with what is known for axonal injury responses in the central nervous system. Considering the neuropathy focus of this special issue, we further draw parallels and distinctions between the injury-response mechanisms that initiate regenerative gene expression programs and those that are known to trigger axon degeneration.

A Ca2+-Dependent Switch Activates Axonal Casein Kinase 2α Translation and Drives G3BP1 Granule Disassembly for Axon Regeneration.

The main limitation on axon regeneration in the peripheral nervous system (PNS) is the slow rate of regrowth. We recently reported that nerve regeneration can be accelerated by axonal G3BP1 granule disassembly, releasing axonal mRNAs for local translation to support axon growth. Here, we show that G3BP1 phosphorylation by casein kinase 2α (CK2α) triggers G3BP1 granule disassembly in injured axons. CK2α activity is temporally and spatially regulated by local translation of Csnk2a1 mRNA in axons after injury, but this requires local translation of mTor mRNA and buffering of the elevated axonal Ca2+ that occurs after axotomy. CK2α's appearance in axons after PNS nerve injury correlates with disassembly of axonal G3BP1 granules as well as increased phospho-G3BP1 and axon growth, although depletion of Csnk2a1 mRNA from PNS axons decreases regeneration and increases G3BP1 granules. Phosphomimetic G3BP1 shows remarkably decreased RNA binding in dorsal root ganglion (DRG) neurons compared with wild-type and non-phosphorylatable G3BP1; combined with other studies, this suggests that CK2α-dependent G3BP1 phosphorylation on Ser 149 after axotomy releases axonal mRNAs for translation. Translation of axonal mRNAs encoding some injury-associated proteins is known to be increased with Ca2+ elevations, and using a dual fluorescence recovery after photobleaching (FRAP) reporter assay for axonal translation, we see that translational specificity switches from injury-associated protein mRNA translation to CK2α translation with endoplasmic reticulum (ER) Ca2+ release versus cytoplasmic Ca2+ chelation. Our results point to axoplasmic Ca2+ concentrations as a determinant for the temporal specificity of sequential translational activation of different axonal mRNAs as severed axons transition from injury to regenerative growth.

Dysbiosis-Associated Enteric Glial Cell Immune-Activation and Redox Imbalance Modulate Tight Junction Protein Expression in Gulf War Illness Pathology.

About 14% of veterans who suffer from Gulf war illness (GWI) complain of some form of gastrointestinal disorder but with no significant markers of clinical pathology. Our previous studies have shown that exposure to GW chemicals resulted in altered microbiome which was associated with damage associated molecular pattern (DAMP) release followed by neuro and gastrointestinal inflammation with loss of gut barrier integrity. Enteric glial cells (EGC) are emerging as important regulators of the gastrointestinal tract and have been observed to change to a reactive phenotype in several functional gastrointestinal disorders such as IBS and IBD. This study is aimed at investigating the role of dysbiosis associated EGC immune-activation and redox instability in contributing to observed gastrointestinal barrier integrity loss in GWI via altered tight junction protein expression. Using a mouse model of GWI and in vitro studies with cultured EGC and use of antibiotics to ensure gut decontamination we show that exposure to GW chemicals caused dysbiosis associated change in EGCs. EGCs changed to a reactive phenotype characterized by activation of TLR4-S100ß/RAGE-iNOS pathway causing release of nitric oxide and activation of NOX2 since gut sterility with antibiotics prevented this change. The resulting peroxynitrite generation led to increased oxidative stress that triggered inflammation as shown by increased NLRP-3 inflammasome activation and increased cell death. Activated EGCs in vivo and in vitro were associated with decrease in tight junction protein occludin and selective water channel aquaporin-3 with a concomitant increase in Claudin-2. The tight junction protein levels were restored following a parallel treatment of GWI mice with a TLR4 inhibitor SsnB and butyric acid that are known to decrease the immunoactivation of EGCs. Our study demonstrates that immune-redox mechanisms in EGC are important players in the pathology in GWI and may be possible therapeutic targets for improving outcomes in GWI symptom persistence.

Deacetylation of Miro1 by HDAC6 blocks mitochondrial transport and mediates axon growth inhibition.

Inhibition of histone deacetylase 6 (HDAC6) was shown to support axon growth on the nonpermissive substrates myelin-associated glycoprotein (MAG) and chondroitin sulfate proteoglycans (CSPGs). Though HDAC6 deacetylates α-tubulin, we find that another HDAC6 substrate contributes to this axon growth failure. HDAC6 is known to impact transport of mitochondria, and we show that mitochondria accumulate in distal axons after HDAC6 inhibition. Miro and Milton proteins link mitochondria to motor proteins for axon transport. Exposing neurons to MAG and CSPGs decreases acetylation of Miro1 on Lysine 105 (K105) and decreases axonal mitochondrial transport. HDAC6 inhibition increases acetylated Miro1 in axons, and acetyl-mimetic Miro1 K105Q prevents CSPG-dependent decreases in mitochondrial transport and axon growth. MAG- and CSPG-dependent deacetylation of Miro1 requires RhoA/ROCK activation and downstream intracellular Ca2+ increase, and Miro1 K105Q prevents the decrease in axonal mitochondria seen with activated RhoA and elevated Ca2+ These data point to HDAC6-dependent deacetylation of Miro1 as a mediator of axon growth inhibition through decreased mitochondrial transport.

Expanding Axonal Transcriptome Brings New Functions for Axonally Synthesized Proteins in Health and Disease.

Intra-axonal protein synthesis has been shown to play critical roles in both development and repair of axons. Axons provide long-range connectivity in the nervous system, and disruption of their function and/or structure is seen in several neurological diseases and disorders. Axonally synthesized proteins or losses in axonally synthesized proteins contribute to neurodegenerative diseases, neuropathic pain, viral transport, and survival of axons. Increasing sensitivity of RNA detection and quantitation coupled with methods to isolate axons to purity has shown that a surprisingly complex transcriptome exists in axons. This extends across different species, neuronal populations, and physiological conditions. These studies have helped define the repertoire of neuronal mRNAs that can localize into axons and imply previously unrecognized functions for local translation in neurons. Here, we review the current state of transcriptomics studies of isolated axons, contrast axonal mRNA profiles between different neuronal types and growth states, and discuss how mRNA transport into and translation within axons contribute to neurological disorders.

Axonal G3BP1 stress granule protein limits axonal mRNA translation and nerve regeneration.

Critical functions of intra-axonally synthesized proteins are thought to depend on regulated recruitment of mRNA from storage depots in axons. Here we show that axotomy of mammalian neurons induces translation of stored axonal mRNAs via regulation of the stress granule protein G3BP1, to support regeneration of peripheral nerves. G3BP1 aggregates within peripheral nerve axons in stress granule-like structures that decrease during regeneration, with a commensurate increase in phosphorylated G3BP1. Colocalization of G3BP1 with axonal mRNAs is also correlated with the growth state of the neuron. Disrupting G3BP functions by overexpressing a dominant-negative protein activates intra-axonal mRNA translation, increases axon growth in cultured neurons, disassembles axonal stress granule-like structures, and accelerates rat nerve regeneration in vivo.

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). Furthermore, we showed that axonal trafficking and local translation of these mRNAs plays a critical role in the generation of axonal ATP. Using a global gene expression analysis, this study identified a highly diverse population of nuclear-encoded mRNAs that were enriched in the axon and presynaptic nerve terminals. Among this population of mRNAs, fifty seven were found to be at least two-fold more abundant in distal axons, as compared with the parental cell bodies. Gene ontology analysis of the nuclear-encoded mitochondrial mRNAs suggested functions for these gene products in molecular and biological processes, including but not limited to oxidoreductase and electron carrier activity and proton transport. Based on these results, we postulate that local translation of nuclear-encoded mitochondrial mRNAs present in the axons may play an essential role in local energy production and maintenance of mitochondrial function.

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. Here, we show that exogenous expression of the COXIV zipcode in cultured SCG neurons also results in the reduction of local ATP levels and increases levels of reactive oxygen species (ROS) in the axon. We took advantage of this "competition" phenotype to investigate the in vivo significance of axonal transport of COXIV mRNA. Toward this end, we generated transgenic mice expressing a fluorescent reporter fused to COXIV zipcode under a forebrain-specific promoter. Immunohistological analyses and RT-PCR analyses of RNA from the transgenic mouse brain showed expression of the reporter in the deep layer neurons in the pre-frontal and frontal cortex. Consistent with the in vitro studies, we observed increased ROS levels in neurons of these transgenic animals. A battery of behavioral tests on transgenic mice expressing the COXIV zipcode revealed an "anxiety-like" behavioral phenotype, suggesting an important role for axonal trafficking of nuclear-encoded mitochondrial mRNAs in neuronal physiology and animal behavior.