Axonally synthesized ß-actin and GAP-43 proteins support distinct modes of axonal growth.

Increasing evidence points to the importance of local protein synthesis for axonal growth and responses to axotomy, yet there is little insight into the functions of individual locally synthesized proteins. We recently showed that expression of a reporter mRNA with the axonally localizing ß-actin mRNA 3'UTR competes with endogenous ß-actin and GAP-43 mRNAs for binding to ZBP1 and axonal localization in adult sensory neurons (Donnelly et al., 2011). Here, we show that the 3'UTR of GAP-43 mRNA can deplete axons of endogenous ß-actin mRNA. We took advantage of this 3'UTR competition to address the functions of axonally synthesized ß-actin and GAP-43 proteins. In cultured rat neurons, increasing axonal synthesis of ß-actin protein while decreasing axonal synthesis of GAP-43 protein resulted in short highly branched axons. Decreasing axonal synthesis of ß-actin protein while increasing axonal synthesis of GAP-43 protein resulted in long axons with few branches. siRNA-mediated depletion of overall GAP-43 mRNA from dorsal root ganglia (DRGs) decreased the length of axons, while overall depletion of ß-actin mRNA from DRGs decreased the number of axon branches. These deficits in axon growth could be rescued by transfecting with siRNA-resistant constructs encoding ß-actin or GAP-43 proteins, but only if the mRNAs were targeted for axonal transport. Finally, in ovo electroporation of axonally targeted GAP-43 mRNA increased length and axonally targeted ß-actin mRNA increased branching of sensory axons growing into the chick spinal cord. These studies indicate that axonal translation of ß-actin mRNA supports axon branching and axonal translation of GAP-43 mRNA supports elongating growth.

Nerve growth factor-induced formation of axonal filopodia and collateral branches involves the intra-axonal synthesis of regulators of the actin-nucleating Arp2/3 complex.

Nerve growth factor (NGF) induces collateral branching along sensory axons by promoting the formation of axonal filopodia dependent on the actin-nucleating Arp2/3 complex. This study shows that chicken embryonic sensory axons contain mRNAs for the actin-nucleating Arp2/3 complex activator WAVE1 and the complex stabilizer cortactin. NGF increases the axonal levels of WAVE1 and cortactin through localized protein synthesis even in axons isolated from the cell body. Inhibition of protein synthesis in severed axons impairs NGF-induced branching, the formation of axonal filopodia, and the initiation of Arp2/3-dependent axonal actin patches, which serve as precursors to the emergence of filopodia. Overexpression of WAVE1 or cortactin in axons not treated with NGF increased the rate of actin patch formation and the frequency of the emergence of filopodia from actin patches, respectively. Antisense inhibition of cortactin mRNA translation in isolated axons blocked NGF-induced filopodia. NGF also activated the Rac1 GTPase, which drives WAVE1 activity, in a protein synthesis-independent manner. Similarly, inhibition of protein synthesis did not impair the effects of NGF on the axonal microtubule cytoskeleton during branching. The effects of NGF on Rac1 activity and increases in axonal levels of WAVE1 and cortactin were both dependent on phosphoinositide 3-kinase (PI3K) signaling. Collectively, the data indicate that NGF promotes sensory axon branching through regulation of the actin cytoskeleton using both canonical signaling mechanisms and intra-axonal protein synthesis downstream of PI3K signaling. Finally, we present experimental evidence of axonal mRNA translation in sensory axons in the living embryonic spinal cord.

Lysophosphatidic acid differentially regulates axonal mRNA translation through 5'UTR elements.

Sensory neurons transport a complex population of mRNAs into their axons, including many encoding ER chaperone proteins. Transport of the mRNA encoding the ER chaperone protein calreticulin is regulated through 3'UTR elements. In other cellular systems, translation of chaperone protein mRNAs can be regulated by ER stress. Here, we have asked if the translation of axonal calreticulin mRNA is regulated in a different manner than its transport into axons. Treatment with lysophosphatidic acid, which is known to trigger axon retraction and stimulate ER Ca(2+) release, caused a translation-dependent increase in axonal calreticulin protein levels. RNA sequences in the 5'UTR of calreticulin confer this translational control through a mechanism that requires an inactivating phosphorylation of eIF2α. In contrast to calreticulin, these signaling events do not activate axonal translation through ß-actin's 5'UTR. Together, these data indicate that stimulation of ER stress can regulate specificity of localized mRNA translation through 5'UTR elements.

A novel role for Gemin5 in mRNA translation.

In eukaryotic cells translation initiation occurs through two alternative mechanisms, a cap-dependent operating in the majority of mRNAs, and a 5'-end-independent driven by internal ribosome entry site (IRES) elements, specific for a subset of mRNAs. IRES elements recruit the translation machinery to an internal position in the mRNA through a mechanism involving the IRES structure and several trans-acting factors. Here, we identified Gemin5 protein bound to the foot-and-mouth disease virus (FMDV) and hepatitis C virus (HCV) IRES using two independent approaches, riboproteomic analysis and immunoprecipitation of photocrosslinked factors. Functional analysis performed in Gemin5 shRNA-depleted cells, or in in vitro translation reactions, revealed an unanticipated role of Gemin5 in translation control as a down-regulator of cap-dependent and IRES-driven translation initiation. Consistent with this, pull-down assays showed that Gemin5 forms part of two distinct complexes, a specific IRES-ribonucleoprotein complex and an IRES-independent protein complex containing eIF4E. Thus, beyond its role in snRNPs biogenesis, Gemin5 also functions as a modulator of translation activity.

Axotomy Induces Drp1-Dependent Fragmentation of Axonal Mitochondria.

It is well established that CNS axons fail to regenerate, undergo retrograde dieback, and form dystrophic growth cones due to both intrinsic and extrinsic factors. We sought to investigate the role of axonal mitochondria in the axonal response to injury. A viral vector (AAV) containing a mitochondrially targeted fluorescent protein (mitoDsRed) as well as fluorescently tagged LC3 (GFP-LC3), an autophagosomal marker, was injected into the primary motor cortex, to label the corticospinal tract (CST), of adult rats. The axons of the CST were then injured by dorsal column lesion at C4-C5. We found that mitochondria in injured CST axons near the injury site are fragmented and fragmentation of mitochondria persists for 2 weeks before returning to pre-injury lengths. Fragmented mitochondria have consistently been shown to be dysfunctional and detrimental to cellular health. Inhibition of Drp1, the GTPase responsible for mitochondrial fission, using a specific pharmacological inhibitor (mDivi-1) blocked fragmentation. Additionally, it was determined that there is increased mitophagy in CST axons following Spinal cord injury (SCI) based on increased colocalization of mitochondria and LC3. In vitro models revealed that mitochondrial divalent ion uptake is necessary for injury-induced mitochondrial fission, as inhibiting the mitochondrial calcium uniporter (MCU) using RU360 prevented injury-induced fission. This phenomenon was also observed in vivo. These studies indicate that following the injury, both in vivo and in vitro, axonal mitochondria undergo increased fission, which may contribute to the lack of regeneration seen in CNS neurons.

Insights into the biology of IRES elements through riboproteomic approaches.

Translation initiation is a highly regulated process that exerts a strong influence on the posttranscriptional control of gene expression. Two alternative mechanisms govern translation initiation in eukaryotic mRNAs, the cap-dependent initiation mechanism operating in most mRNAs, and the internal ribosome entry site (IRES)-dependent mechanism, first discovered in picornaviruses. IRES elements are highly structured RNA sequences that, in most instances, require specific proteins for recruitment of the translation machinery. Some of these proteins are eukaryotic initiation factors. In addition, RNA-binding proteins (RBPs) play a key role in internal initiation control. RBPs are pivotal regulators of gene expression in response to numerous stresses, including virus infection. This review discusses recent advances on riboproteomic approaches to identify IRES transacting factors (ITAFs) and the relationship between RNA-protein interaction and IRES activity, highlighting the most relevant features on picornavirus and hepatitis C virus IRESs.

Mechanism and role of the intra-axonal Calreticulin translation in response to axonal injury.

Following injury, sensory axons locally translate mRNAs that encode proteins needed for the response to injury, locally and through retrograde signaling, and for regeneration. In this study, we addressed the mechanism and role of axotomy-induced intra-axonal translation of the ER chaperone Calreticulin. In vivo peripheral nerve injury increased Calreticulin levels in sensory axons. Using an in vitro model system of sensory neurons amenable to mechanistic dissection we provide evidence that axotomy induces local translation of Calreticulin through PERK (protein kinase RNA-like endoplasmic reticulum kinase) mediated phosphorylation of eIF2α by a mechanism that requires both 5' and 3'UTRs (untranslated regions) elements in Calreticulin mRNA. ShRNA mediated depletion of Calreticulin or inhibition of PERK signaling increased axon retraction following axotomy. In contrast, expression of axonally targeted, but not somatically restricted, Calreticulin mRNA decreased retraction and promoted axon regeneration following axotomy in vitro. Collectively, these data indicate that the intra-axonal translation of Calreticulin in response to axotomy serves to minimize the ensuing retraction, and overexpression of axonally targeted Calreticulin mRNA promotes axon regeneration.

Neurotrophins induce fission of mitochondria along embryonic sensory axons.

Neurotrophins are growth factors that have a multitude of roles in the nervous system. We report that neurotrophins induce the fission of mitochondria along embryonic chick sensory axons driven by combined PI3K and Mek-Erk signaling. Following an initial burst of fission, a new steady state of neurotrophin-dependent mitochondria length is established. Mek-Erk controls the activity of the fission mediator Drp1 GTPase, while PI3K may contribute to the actin-dependent aspect of fission. Drp1-mediated fission is required for nerve growth factor (NGF)-induced collateral branching in vitro and expression of dominant negative Drp1 impairs the branching of axons in the developing spinal cord in vivo. Fission is also required for NGF-induced mitochondria-dependent intra-axonal translation of the actin regulatory protein cortactin, a previously determined component of NGF-induced branching. Collectively, these observations unveil a novel biological function of neurotrophins; the regulation of mitochondrial fission and steady state mitochondrial length and density in axons.

Riboproteomic analysis of polypeptides interacting with the internal ribosome-entry site element of foot-and-mouth disease viral RNA.

Initiation of translation driven by internal ribosome entry site (IRES) elements depends upon the structural organization of this mRNA region. Besides translation initiation factors (eIFs), auxiliary proteins can also affect IRES activity. With the aim to identify proteins interacting with two unrelated IRESs present in the genome of foot-and-mouth disease virus (FMDV) and hepatitis C virus (HCV) we have used a proteomic approach. This procedure allowed the identification of 21 RNA-binding proteins interacting with discrete regions of the FMDV IRES, domains 3 and 5, and 16 interacting with domain III of the HCV IRES. In support of the binding specificity, the factors interacting with domain 3 differed from those interacting with domain 5, and included three poly(rC)-binding protein (PCBP) members, besides proliferation-associated 2G4 (PA2G4) and deleted-azoospermia 1 (DAZ1) protein. Around 71% of the identified factors associated with the FMDV IRES differ from those interacting with the HCV IRES. The group of proteins interacting with the FMDV or the HCV IRES includes eIF4B and 5 subunits of eIF3, respectively, known to interact with each of these RNAs, validating the results of this approach. According to the function of the identified proteins, 55% are involved in translation control, whereas 35% play a role in different aspects of RNA lifespan. Compilation of factors preferentially associated with FMDV or HCV IRES provides a basis for examining the strategies used by IRESs to recruit the translation machinery.

Actin filament-microtubule interactions in axon initiation and branching.

Neurons begin life as spherical cells. A major hallmark of neuronal development is the formation of elongating processes from the cell body which subsequently differentiate into dendrites and the axon. The formation and later development of neuronal processes is achieved through the concerted organization of actin filaments and microtubules. Here, we review the literature regarding recent advances in the understanding of cytoskeletal interactions in neurons focusing on the initiation of processes from neuronal cell bodies and the collateral branching of axons. The complex crosstalk between cytoskeletal elements is mediated by a cohort of proteins that either bind both cytoskeletal systems or allow one to regulate the other. Recent studies have highlighted the importance of microtubule plus-tip proteins in the regulation of the dynamics and organization of actin filaments, while also providing a mechanism for the subcellular capture and guidance of microtubule tips by actin filaments. Although the understanding of cytoskeletal crosstalk and interactions in neuronal morphogenesis has advanced significantly in recent years the appreciation of the neuron as an integrated cytoskeletal system remains a frontier.

Specific interference between two unrelated internal ribosome entry site elements impairs translation efficiency.

Internal ribosome entry site (IRES) elements allow simultaneous synthesis of multiple proteins in eukaryotic cells. Here, two unrelated IRESs that perform efficiently in bicistronic constructs, the picornavirus foot-and-mouth disease virus (FMDV) and the cellular immunoglobulin heavy chain binding protein (BiP) IRES, were used to generate a tricistronic vector. Functional analysis of the tricistronic RNA evidenced that the efficiency of protein synthesis under the control of BiP IRES was lower than that of the FMDV IRES, relative to the efficiency measured in bicistronic vectors. A specific competition between these elements was verified using two separate mono- or bicistronic constructs in vivo and in vitro. In contrast, no interference was detected with the hepatitis C virus (HCV) IRES. The interference effect of FMDV IRES was observed in cis and trans, in support of competition for common transacting factors different than those used in cap- and HCV-dependent initiation.

Localized IRES-dependent translation of ER chaperone protein mRNA in sensory axons.

Transport of neuronal mRNAs into distal nerve terminals and growth cones allows axonal processes to generate proteins autonomous from the cell body. While the mechanisms for targeting mRNAs for transport into axons has received much attention, how specificity is provided to the localized translational apparatus remains largely unknown. In other cellular systems, protein synthesis can be regulated by both cap-dependent and cap-independent mechanisms. The possibility that these mechanisms are used by axons has not been tested. Here, we have used expression constructs encoding axonally targeted bicistronic reporter mRNAs to determine if sensory axons can translate mRNAs through cap-independent mechanisms. Our data show that the well-defined IRES element of encephalomyocarditis virus (EMCV) can drive internal translational initiation of a bicistronic reporter mRNA in distal DRG axons. To test the potential for cap-independent translation of cellular mRNAs, we asked if calreticulin or grp78/BiP mRNA 5'UTRs might have IRES activity in axons. Only grp78/BiP mRNA 5'UTR showed clear IRES activity in axons when placed between the open reading frames of diffusion limited fluorescent reporters. Indeed, calreticulin's 5'UTR provided an excellent control for potential read through by ribosomes, since there was no evidence of internal initiation when this UTR was placed between reporter ORFs in a bicistronic mRNA. This study shows that axons have the capacity to translate through internal ribosome entry sites, but a simple binary choice between cap-dependent and cap-independent translation cannot explain the specificity for translation of individual mRNAs in distal axons.

Group I p21-activated kinases: emerging roles in immune function and viral pathogenesis.

Group I p21-activated kinases are a highly conserved three-member family of serine/threonine kinases that act as key effectors for the small GTPases Cdc42 and Rac. In man, these enzymes have been implicated in a wide range of biological processes and are beginning to draw the attention of the pharmaceutical industry as potential therapeutic targets in cancer and in inflammatory processes. In this review, we summarize basic properties of group I Paks and discuss recently uncovered roles for these kinases in immune function and in viral infection.

New insights into internal ribosome entry site elements relevant for viral gene expression.

A distinctive feature of positive-strand RNA viruses is the presence of high-order structural elements at the untranslated regions (UTR) of the genome that are essential for viral RNA replication. The RNA of all members of the family Picornaviridae initiate translation internally, via an internal ribosome entry site (IRES) element present in the 5' UTR. IRES elements consist of cis-acting RNA structures that usually require specific RNA-binding proteins for translational machinery recruitment. This specialized mechanism of translation initiation is shared with other viral RNAs, e.g. from hepatitis C virus and pestivirus, and represents an alternative to the cap-dependent mechanism. In cells infected with many picornaviruses, proteolysis or changes in phosphorylation of key host factors induces shut off of cellular protein synthesis. This event occurs simultaneously with the synthesis of viral gene products since IRES activity is resistant to the modifications of the host factors. Viral gene expression and RNA replication in positive-strand viruses is further stimulated by viral RNA circularization, involving direct RNA-RNA contacts between the 5' and 3' ends as well as RNA-binding protein bridges. In this review, we discuss novel insights into the mechanisms that control picornavirus gene expression and compare them to those operating in other positive-strand RNA viruses.