Jeffery L. Twiss - US grants

The neurotrophins play a vital role in neuronal differentiation and maintenance. Though the low and high affinity neurotrophin receptors (p75NGFR and Trk proteins, respectively) have been isolated, their specific functions remain unclear. They hypothesis that p75NGFR and TrkA interact to form the high affinity NGF receptor holds true in some cell types, while others, only TrkA is required, challenging the proposed role of p75NGFR. The intracellular environment progressively changes during neuronal differentiation; though presumptive, the functional interactions of p75NGFR and TrkA may change dependent upon alterations in the cellular milieu. That is, these proteins may interact as a receptor complex at one stage of differentiation while showing convergent, parallel or even divergent actions in response to NGF at another. In this proposal, the expression of these receptors will be specifically inhibited by stably transfecting PC12 cells with vectors encoding antisense p75NGFR or trkA cRNA whose expression is controlled by inducible promoters. This will allow the dissociation of the receptor molecules in an appropriate intracellular environment to adequately address their individual, temporal functions. Since each receptor mRNA is further induced in response to NGF, it will be essential to investigate the role of these receptors in later stages of neuronal differentiation. In addition to acting as a differentiative factor, neurotrophins are also necessary for the survival of specific neuronal populations. Though NGF causes rapid transcriptional induction generating nascent proteins necessary for differentiation, the hormone also has effects upon mRNA survival. This aspect of NGF's actions likely represents a function of neurotrophin-dependent phenotypic maintenance. PC12 cells continually express and accumulate mRNAs necessary for transcriptional-independent neuritic reextension in the event of injury. Those mRNAs whose stability is increased by NGF will be identified and characterized. Since transcriptional activation represents a distinct intracellular mechanism from mRNA degradation, it will be essential to elucidate the signal transduction cascade involved. While much current work addresses the molecular mechanisms of rapid mRNA degradation, that of enhanced mRNA survival has yet to be adequately studied. The neurotrophins also function in neuronal regeneration and degeneration. The understanding of their mechanisms of action will provide invaluable insight to the possible prevention and treatment of peripheral neuropathies and neurodegenerative disorders. Thus, it will be essential to extend the observations of mRNA stability to in vivo models of regeneration and aging.

The neurotrophins play a vital role in neuronal differentiation and maintenance. Though the low and high affinity neurotrophin receptors (p75NGFR and Trk proteins, respectively) have been isolated, their specific functions remain unclear. They hypothesis that p75NGFR and TrkA interact to form the high affinity NGF receptor holds true in some cell types, while others, only TrkA is required, challenging the proposed role of p75NGFR. The intracellular environment progressively changes during neuronal differentiation; though presumptive, the functional interactions of p75NGFR and TrkA may change dependent upon alterations in the cellular milieu. That is, these proteins may interact as a receptor complex at one stage of differentiation while showing convergent, parallel or even divergent actions in response to NGF at another. In this proposal, the expression of these receptors will be specifically inhibited by stably transfecting PC12 cells with vectors encoding antisense p75NGFR or trkA cRNA whose expression is controlled by inducible promoters. This will allow the dissociation of the receptor molecules in an appropriate intracellular environment to adequately address their individual, temporal functions. Since each receptor mRNA is further induced in response to NGF, it will be essential to investigate the role of these receptors in later stages of neuronal differentiation. In addition to acting as a differentiative factor, neurotrophins are also necessary for the survival of specific neuronal populations. Though NGF causes rapid transcriptional induction generating nascent proteins necessary for differentiation, the hormone also has effects upon mRNA survival. This aspect of NGF's actions likely represents a function of neurotrophin-dependent phenotypic maintenance. PC12 cells continually express and accumulate mRNAs necessary for transcriptional-independent neuritic reextension in the event of injury. Those mRNAs whose stability is increased by NGF will be identified and characterized. Since transcriptional activation represents a distinct intracellular mechanism from mRNA degradation, it will be essential to elucidate the signal transduction cascade involved. While much current work addresses the molecular mechanisms of rapid mRNA degradation, that of enhanced mRNA survival has yet to be adequately studied. The neurotrophins also function in neuronal regeneration and degeneration. The understanding of their mechanisms of action will provide invaluable insight to the possible prevention and treatment of peripheral neuropathies and neurodegenerative disorders. Thus, it will be essential to extend the observations of mRNA stability to in vivo models of regeneration and aging.

DESCRIPTION (Applicant's abstract): Decreased neurotrophic support has been postulated as a cause of diabetic peripheral neuropathy and exogenous neurotrophic factors have shown some promise for treatment of this diabetic complication. Levels of some neurotrophic factors are decreased in diabetic animals. There is also reason to believe that responsiveness to neurotrophic. factors is altered in diabetes mellitus. The objective of this proposal is to address the role of neurotrophic responsiveness in the pathogenesis of diabetic peripheral neuropathy. In Specific aim I, we will directly test the intracellular responsiveness of sensory neurons from diabetic rodents to exogenous and endogenous neurotrophins. Application of exogenous NGF and BDNF will show whether the diabetic sensory neurons can initiate appropriate intracellular signaling mechanisms in response to these neurotrophins. Autocrine/paracrine-produced BDNF supports survival of adult sensory neurons in vitro. Curiously, BDNF mRNA is actually increased in sensory ganglia of diabetic animals, raising the question of whether these neurons can respond to BDNF. Responsiveness of these sensory neurons to endogenous BDNF will be determined by selectively inhibiting the BDNF receptor (TrkB) or competitively removing BDNF from the culture medium. The loss of activity of TrkB and downstream signaling pathways (Ras-Erk and P13K-Akt) will provide a measure of the capacity of diabetic sensory neurons to maintain intracellular signaling mechanisms in response to neurotrophins. In Specific Aim II, we will address the molecular responsiveness of diabetic sensory neurons to the endogenous neurotrophic factors that are increased after nerve injury. Altered neurotrophic factor responsiveness both before and after nerve injury may indeed account for the aborted nerve regeneration seen in diabetic animals. We will use conditioning crush lesions to determine if diabetic sensory neurons can i) generate a population of mRNAs needed for axonal regeneration after nerve injury, and ii) regulate the translation of these mRNAs to rapidly extend axons in vitro.

DESCRIPTION: (Verbatim from the Applicant's Abstract) The proposal focuses upon the role of mRNA translational control during nerve regeneration. It has become increasingly clear that the translation of mRNA into its protein product is a regulated and, often times, specific process. Additionally, localization of mRNAs to particular regions of the cell provides a means to spatially regulate mRNA translation. There are examples for both temporal and spatial regulation of neuronal protein synthesis. Protein synthesis within dendrites has recieved much attention in recent years, but only immature neurons were thought to be capable of intra-axonal protein synthesis. Preliminary data from our laboratory indicate that protein synthesis occurs directly within regenerating axons of adult sensory neurons. Moreover, the rapid axonal regeneration from conditioned sensory neurons occurs by translational regulation of existing neuronal mRNAs. Taken together, these findings suggest that temporal and spatial regulation of protein synthesis occurs during axonal regeneration from adult neurons. In this proposal we will address the role of temporal and spatial regulation of protein synthesis during axonal regeneration. We have two specific aims. In the first aim, we will take two different approaches to determine what mRNAs are translationally regulated in the conditioned sensory neuron cultures. First, we will focus upon the translational regulation of mRNAs for cytoskeletal and growth-associated proteins in the conditioned neurons. We reason that the neuron will need more of these proteins to extend axons. Second, we will use polysomal RNAs from the conditioned and naive DRG neurons to probe cDNA arrays. In this second aim, we will use a tissue culture model developed by Torre and Steward (1992) to specifically clone intra-axonal mRNAs from conditioned sensory neurons. We will identify these intra-axonal mRNAs and prove that they are translated directly within axons.

DESCRIPTION (provided by applicant): The functional component of the nervous system, neurons, are the largest cells in the body. Animals have somehow evolved mechanisms to maintain these huge cells. While most neuronal proteins are provided to distant cellular regions (i.e., dendritic and axonal compartments) by transport from the cell body, it has become clear in recent years that some proteins are produced locally. Localized protein synthesis has been best characterized in dendrites where activity appears to regulate local post-synaptic protein synthesis. Several lines of evidence indicate that translation also occurs in the axonal compartment. While this has been best documented in invertebrate species, several laboratories have reported that vertebrate neurons can locally synthesize proteins in developing axons. We have recently shown that regenerating axons of adult rat neurons locally synthesize proteins during regeneration. In both the developing and regenerating neurons, intra-axonal translation contributes to the structure of the growing axon by providing a locally renewable source of cytoskeleton. Most studies of localized neuronal protein synthesis have been restricted to cultured neurons. In situ hybridization and immunofluorescence have been used to confirm that axons contain RNA and translation machinery in vivo. However, there are no other methods to visualize RNA transport in real tissues. In situ hybridization suffers from low resolution and provides no clue of where the mRNA is utilized. The objective of this proposal is to develop a mouse model in which we can visualize RNA trafficking and local protein synthesis in vivo. Here we will generate transgenic mice where localization of a reporter is driven by a well-characterized cis-element of beta-actin mRNA. Beta-actin is the most abundant mRNA that we have identified in regenerating axons and it also extends into developing axons. We will initially determine the elements necessary for localization of beta-actin mRNA in cultured rat and mouse sensory neurons. We will then use this information to generate mice whose neurons express chimeric mRNAs with the axonal-localization motif. If successful, these animals will allow us to analyze RNA trafficking and local protein synthesis in vivo. Furthermore, we will be able to determine if local protein synthesis occurs in axons of CNS neurons that we cannot culture from adult animals (e.g., spinal motor neurons) and whether we can utilize exogenous agents to increase local axonal protein synthesis for expediting axonal regeneration.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Subcellular localization and translation of mRNAs provides cells with a locally renewable source of proteins to autonomously respond to extracellular stimuli. For neurons, this is critical since the cytoplasm and membranes of neurons can extend for several hundred times the dimensions of the cell body. Work in developing neurons has shown that localized protein synthesis plays a role in axonal pathfinding, provides structural protection to the axon, and triggers anterograde and retrograde axonal transport. Studies from the PI's group have shown that axonal protein synthesis is triggered by injury in adult neurons and that regenerating axons show particularly robust intra-axonal protein synthesis. This localized protein synthesis represents a mechanism that could be modulated to facilitate the regenerative capacity of axons in the adult nervous system. Despite the obvious functional significance of and newly increased interests in axonal protein synthesis, we know of excessively few mRNAs whose local translation is regulated by extracellular stimuli. Our preliminary studies indicate that adult axons have the potential to synthesize a complex population of more than 200 different proteins. We hypothesize that axonal stimulation alters localized protein synthesis through both directing the transport of particular mRNAs into the axonal compartment and locally controlling the activity of the axonal translational machinery. The objective of this BioAMS project is to use the high sensitivity of AMS for quantifying how protein synthesis is regulated in regenerating axons.

It has become accepted that the axonal compartment can autonomously synthesize proteins. This local translation provides the axon with a renewable source of proteins to respond to extracellular stimuli. Studies from the Pi's group have shown that axonal protein synthesis is triggered by neural injury and that particularly robust protein synthesis occurs in regenerating axons. This localized protein synthesis represents a mechanism that can likely be harnessed to facilitate the regeneration of axons in the adult nervous system. Despite the obvious functional significance and newly increased interests in axonal protein synthesis, we little understanding of how this process is regulated. Our preliminary studies indicate that adult axons have the potential to synthesize a complex population of more than 200 different proteins;there is clearly some specificity to choose which proteins are generated when and where. The objective of this proposal is to determine how this axonal protein synthesis is regulated. We hypothesize that axonal stimulation alters localized protein synthesis through both directing the transport of particular mRNAs into the axonal compartment and locally controlling the activity of the axonaltranslational machinery. In Aim 1, we will determine how axonal guidance cues and growth promoting stimuli modify specificity of axonal protein synthesis. The contributions of axonal transport vs. localized synthesis will be determined for each axonally synthesized protein identified. The physiological consequences of localized synthesis of these proteins in axonal growth will be addressed. In Aim 2 we will ask how the axon controls synthesis of organelle and membrane proteins and the functional relevance of these pathways to nerve regeneration. Nerve regeneration is abysmally slow and rarely successful. It has recently been recognized that injured nerve processes are capable of generating their own proteins. Our studies indicate that this may be used to enhance recovery after injury of the nervous system. The objective of this grant application is to determine how local protein synthesis in nerve processes is regulated by extracellular stimuli.

[unreadable] DESCRIPTION (provided by applicant): It has become accepted that the axonal compartment can autonomously synthesize proteins. This local translation provides the axon with a renewable source of proteins to respond to extracellular stimuli. Studies from the Pi's group have shown that axonal protein synthesis is triggered by neural injury and that particularly robust protein synthesis occurs in regenerating axons. This localized protein synthesis represents a mechanism that can likely be harnessed to facilitate the regeneration of axons in the adult nervous system. Despite the obvious functional significance and newly increased interests in axonal protein synthesis, we little understanding of how this process is regulated. Our preliminary studies indicate that adult axons have the potential to synthesize a complex population of more than 200 different proteins; there is clearly some specificity to choose which proteins are generated when and where. The objective of this proposal is to determine how this axonal protein synthesis is regulated. We hypothesize that axonal stimulation alters localized protein synthesis through both directing the transport of particular mRNAs into the axonal compartment and locally controlling the activity of the axonal translational machinery. In Aim 1, we will determine how axonal guidance cues and growth promoting stimuli modify specificity of axonal protein synthesis. The contributions of axonal transport vs. localized synthesis will be determined for each axonally synthesized protein identified. The physiological consequences of localized synthesis of these proteins in axonal growth will be addressed. In Aim 2 we will ask how the axon controls synthesis of organelle and membrane proteins and the functional relevance of these pathways to nerve regeneration. Nerve regeneration is abysmally slow and rarely successful. It has recently been recognized that injured nerve processes are capable of generating their own proteins. Our studies indicate that this may be used to enhance recovery after injury of the nervous system. The objective of this grant application is to determine how local protein synthesis in nerve processes is regulated by extracellular stimuli. [unreadable] [unreadable] [unreadable]

Localization of mRNAs with subsequent translation into new proteins provides a means to geographically regulate neuronal protein composition in distinct subcellular regions. In axons, this localized protein synthesis is needed for growth cone guidance and regeneration. Although mature axons show little evidence for localized protein synthesis, injury appears to increase the neuron's ability to locally generate new axonal proteins. We suspect that injury invokes a fundamental change in how the neuron targets mRNAs into and translates mRNAs within axons. Locally synthesizing proteins at sites distal from the neuronal perikaryon requires a coordinated effort to package and target mRNAs and translational machinery for delivery to the correct locale. In large part, this targeting is accomplished through specific RNA-protein interactions. For axonal mRNA transport and translation, exceptionally little is known of which mRNA elements are needed for this RNA-protein interaction and which RNA binding proteins are targeted into axons. Here we propose to dissect the molecular determinants for targeting mRNAs into axons. In the first aim, we will determine the localizing elements of axonal mRNAs that are regulated at the level of transport, translation, or both using PNS sensory neurons. In the second aim, we will test functionality of these RNA localization elements in CNS neurons that show complete axonal-dendritic polarization. Finally, we will generate and characterize viral reporter constructs that will enable us to address RNA localization mechanisms in vivo.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The objective of this project is to determine how the distal processes of nerve cells regulate their protein levels after injury. These 'axonal processes'are needed for long-range communication in the brain and spinal cord. If these processes are disrupted, communication ceases and function of the brain and spinal cord is lost. Improving regeneration of injured axonal processes will restore function to the brain and spinal column. Several lines of evidence indicate that distal axonal processes can autonomously regulate levels of proteins needed for regeneration through modulating synthesis and degradation of these proteins locally. Until recently we have had no means to dissect the proteome of regenerating axons, since the materials available for study are exceptionally limiting and most often contaminated with other cellular constituents. We will take advantage of an axonal preparation that our laboratory has developed and the high sensitivity proteomics applications of the UCSF Mass Spectrometry Facility to determine how the precursors of axonally synthesized proteins are targeted for transport into axons and what becomes of the protein products encoded by these precursors. We will use affinity purification of mRNA: protein complexes to identify the proteins needed for transport of the mRNA precursors into axons. Integrating these data with ongoing axonal mRNA profiling from our laboratory will provide a systematic view of protein dynamics of distal axons. Ultimately, these studies will provide us with a unique perspective of axonal biology that has not been feasible until now and should lead to novel strategies for accelerating regeneration after traumatic injury of the brain and spinal cord.

DESCRIPTION (provided by applicant): This R13 application requests funds to increase participation by US scientists in an EMBO workshop series entitled Systems Dynamics of Intracellular Communication ('Spatial 2011'). Information transfer is a central aspect of biology, and signaling within and between cells is a fundamental aspect of the functioning of multicellular organisms. Signaling over long distances between cells requires specific and regulated mechanisms to transport signaling molecules and generate functional gradients to control processes such as chemotaxis, axon guidance, developmental differentiation and others. In recent years it has become increasingly clear that equally sophisticated and diverse mechanisms are required for information transfer and signaling within cells. Diffusion alone is not sufficient to account for effective transfer of different signals from cell surface to the nucleus against unfavorable enzymatic activities. Long distance communication within cells requires protected or facilitated mechanisms of signal propagation over any intracellular distance of more than a few micrometers. Different communities of researchers are interested in spatial aspects of propagation of intracellular signals and information within cells, including theoreticians, cell biologists, and neurobiologists. Information transfer within large and morphologically complex cells (such as neurons and other polarized eukaryotic cells) requires specialized mechanisms to facilitate propagation and translocation of signals. Spatial 2011 will provide a unique platform for communication and interaction between the experimentalists and theoreticians involved in these diverse research communities. The 2007 and 2009 Spatial meetings (held in Israel) saw participation from relatively few US scientists. We have relocated the venue to central Europe to specifically facilitate US participation and are seeking NIH support to support US representation by young faculty, fellows, and students in this growing interdisciplinary field. PUBLIC HEALTH RELEVANCE: This application seeks funds to support US scientists'participation in a unique interdisciplinary conference focusing on spatial communication in health and disease. Signaling over long distances between cells requires specific and regulated mechanisms to transport signaling molecules and generate functional gradients to control processes such as chemotaxis, axon guidance, developmental differentiation and others. Malfunction of intracellular propagation of survival and other signals in large cells is tightly linked with disease etiology in the nervous and immune systems.

Axons provide long-range communication in the nervous system. Regeneration of axons in the injured spina! cord brings the potential to reconnect the caudal spinal cord to rostra! brain stem and cerebrum and restore sensory and motor function. Significant advances have been made in the field of neura! repair that hold promise for restoring function in spinal cord injury, particularly when interventions can be combined to target multiple repair mechanisms. The studies proposed in this project will explore the intracellular mechanisms underlying improved functional recovery in spinal cord injury interventions, focusing on novel interactions in the axonal compartment. We will test the hypothesis that the microenvironment of the injured spina! cord and interventions aimed at overcoming the inhibitory microenvironment can modulate intra- axonal signaling events that converge on the local protein synthesis machinery and this contributes to axonal growth and maturation. We wil! test this hypothesis with two specific aims that bring together expertise of the principal investigator in axonal growth and intra-axonal signaling with expertise from Project 1 (Houle) in regenerative therapies for spinal cord injury and Project II (Fischer) in progenitor cell therapies for spinal cord injury. The first aim of this project asks if exercise/training regimens that have been shown to improve recovery from spinal cord injury regulate axonal growth potential through post-transcriptional mechanisms. Both overall and intra-axonal translational control mechanisms will be tested using primary neuronal cultures and peripheral nerve grafting into the transected spinal cord. The second aim will ask if precursor cells used for spinal cord injury can directly modulate intra-axonal signaling to regulate the intrinsic growth potential and maturation of axons through axonal mRNA transport and translational control mechanisms. We will integrate these data with Project II to address mRNA translation in host axons as they interact with grafted precursor cells in SCI. The overall objective of these experiments is to uncover mechanisms underlying enhanced axonal growth and signaling that can be used to rationally fine tune future neural repair strategies.

DESCRIPTION (provided by applicant): Although neurons in the peripheral nervous system can spontaneously regenerate injured axons, those in the central nervous system have to be coaxed to regenerate. Both extrinsic inhibitor factors and intrinsically lower growth capacity limi central regeneration. Conditioning neurons by peripheral nerve injury can increase the intrinsic growth capacity of neurons. This increase in intrinsic growth capacity is accompanied by an increase in localized protein synthesis directly within regenerating axons. We have recently shown that intra-axonal translation of ?-actin, GAP-43, and Importin ?1 mRNAs is needed for peripheral nerve regeneration (Donnelly et al., 2011; Perry et al., 2012). Limiting of the delivery of these mRNAs into peripheral axons compromises regeneration. Moreover, increasing delivery of ?-actin and GAP-43 mRNAs into axons increases axonal growth, including axonal growth in the developing spinal cord (Donnelly et al., 2013). Although the extent to which mRNAs can localize into CNS axons after spinal cord injury has not been thoroughly tested, ?-actin's 3'UTR can drive mRNA into central process of sensory neurons after spinal cord injury (Willis et al., 2011). This suggests that local mRNA translation occurs in injured axons in the spinal cord. Over the past funding period, we have defined RNA localization elements for multiple axonal mRNAs encoding regeneration-associated genes like ?-actin. These provide a singularly unique resource of cellular tools to move our studies from in vitro preparations to in vivo neural injury models. The experiments proposed here will take advantage of these tools to determine if modifying the axonal transcriptome can be used to increase the intrinsic growth capacity of adult neurons in peripheral and central nervous systems. The studies in Aims 1 and 2 will tell us if transport into PNS and CNS axons is regulated by injury, the extent to which this compares to PNS axons, and whether axonal protein synthesis can be used to facilitate axonal regeneration in both the PNS and CNS in vivo. The studies in Aim 3 examine whether and how CNS growth promoting and growth inhibiting agents modulate translation in axons uncovering novel genetic and/or pharmacological approaches to alter the axon's translational response to stimuli that impede regeneration.

? DESCRIPTION (provided by applicant): Extension and maturation of axons and dendrites are essential developmental steps that allow the nervous system to function. This development requires precise regulation of gene expression, with coordinated activation and inactivation of gene expression programs associated with growth, maturation, and function of neurons. Growth of neuronal processes or `neurites' must be precisely timed and regulated to generate functional neural circuits. Regulation of gene expression extends beyond transcribing DNA into mRNAs, and it has become increasingly clear that much regulation occurs post-transcriptionally in neurons. Regulatory steps include splicing, subcellular localization, and translational control of mRNAs. Stability of mRNAs plays a critical role in gene expression by modifying the amount of an individual mRNA available as a template for generating new protein over time. Stabilization and destabilization of mRNAs within growing neurites also impacts where new proteins are produced. Despite increased recognition of importance of this mechanism, we have little understanding of how neuronal mRNA stability is regulated. Recent work from the PI's and Co- PI's labs have uncovered a mechanism for modulation of mRNA stability in neurons. The RNA binding proteins KSRP and HuD compete for binding to GAP-43 mRNA. Both these RNA binding proteins are known to have multiple functions, and our data suggest that KSRP and HuD have antagonistic functions. For GAP-43 mRNA, KSRP binding destabilizes the transcript while HuD binding stabilizes the transcript. By initial CLIP analyses, KSRP and HuD can bind to overlapping cohorts of mRNAs and cytoplasmic KSRP appears to provide a governor to limit neurite length by destabilizing mRNAs. These data have led us to hypothesize that competitive interactions of HuD and KSRP with specific cohorts of ARE-containing mRNAs control the temporal and spatial pattern of neuronal protein expression during the initiation and termination of neurite outgrowth through changes in mRNA stability. We will test this hypothesis with three specific aims: 1) Does KSRP destabilize neuronal mRNA cohorts? 2) Do KSRP or HuD interactions alter stability of localized mRNAs? 3) Do KSRP and HuD compete for binding to a shared cohort of mRNAs with antagonistic functions? Completion of these aims will fill a gap in knowledge on mechanisms of neuronal mRNA stability and its contributions to brain development.

Peripheral nerve injuries are common with more than 200,000 new cases reported each year in the United States alone. Only about 10% of these individuals regain much function. Nerve injury significantly impacts long-term quality of life, and most injured individuals seek continued treatments for associated disabilities and pain. The most common explanation for poor functional outcomes is the slow and inefficient process of axon regeneration. Proteins synthesized locally in axons contribute to peripheral nerve regeneration by providing retrograde signals for injury responses and supporting axon regrowth locally. We have shown that mRNAs are stored in PNS axons in RNA-protein aggregates that contain the stress granule protein G3BP1. G3BP1 protein can drive stress granule aggregation, and G3BP1 phosphorylation blocks stress granule assembly. G3BP1 binds to mRNAs in axons and attenuates their translation. We have discovered exogenous agents and endogenous signals that trigger disassembly of axonal G3BP1 aggregates. The exogenous agents specifically increase axonal protein synthesis and accelerate axon growth rates in vitro and in vivo. However, translational regulation of axonal mRNAs has been demonstrated in models of neuropathic pain, and nerve injury patients frequently seek additional treatment after peripheral nerve injury for the development of neuropathic pain. Whether acute disassembly of G3BP1 RNAs within nociceptive neurons will lead to or exacerbate neuropathic pain is unknown and is a key step toward translatability of this type of therapy. This ?Research Supplement to Promote Diversity in Health-Related Research? is designed to bring translational research experience to a post- baccalaureate fellow with mentoring from a team of one junior and two senior investigators. This will prepare the fellow for graduate school and strengthen her applications.

SUMMARY This application asks how localized mRNA stability modifies axonal regeneration capacity, focusing on contributions of the RNA binding protein [RBP] KHSRP. The nervous system makes extensive use of post- transcriptional mechanisms to regulate cellular proteomes in response to extracellular stimuli and physiologic environments during development, function, & in response to axonal injury. Since one mRNA can be translated into protein many times over, how long a given mRNA is available for translation impacts the amount of protein generated from that mRNA. Stability of mRNAs is indeed regulated, with interactions with RBPs stabilizing & destabilizing different mRNAs, as well as interactions with microRNAs targeting some targets for degradation. Translation of mRNAs clearly supports axon regeneration, but we have little knowledge for how stability of axonal mRNAs is locally regulated. We have shown that the RBPs HuD (also called ELAVL4) and KHSRP (also called FUBP2, MARTA1, & ZBP2) compete for binding to neuronal mRNAs with AU-rich elements, where HuD interaction stabilizes and KHSRP interaction destabilizes target mRNAs. At the molecular level, this interaction is impacted by an mRNA?s affinity for binding to HuD or KHSRP. Our work over years 01-05 show that loss of KHSRP increases KHSRP target mRNA levels, causes excessive axonal and dendritic growth, impairs memory consolidation in hippocampus & prefrontal cortex, and increases presynaptic activity in prefrontal cortex and hippocampus. KHSRP is expressed into adulthood, and we surprisingly find that axonal KHSRP levels rapidly increase in peripheral nerves after injury. This increase in axonal KHSRP occurs through intra-axonal translation of its encoding mRNA. Our preliminary data indicate that KHSRP knockout mice show accelerated nerve regeneration pointing to axon-intrinsic functions for KHSRP in regeneration. Based on these observations, we hypothesize that axonal KHSRP controls the rates of axon regeneration through regulation of localized mRNA stability. We will test this hypothesis with the following specific aims: 1) Does KHSRP regulate PNS axon regeneration through a neuron intrinsic mechanism? 2) Does increased axonal KHSRP limit axon regeneration by destabilizing axonal mRNAs encoding regeneration-associated proteins? and 3) Does KHSRP?s protein interactome influence its intra-axonal functions? Completion of the studies here will begin to fill this knowledge gap by focusing on RNA-protein interactions initiated in axons that can affect mRNA survival. This will provide the first subcellular analyses of RBP domain-specific RNA regulons and will bring the first systematic assessment for contributions of RNA survival to peripheral nerve regeneration.