Valeria Cavalli - US grants

DESCRIPTION (provided by applicant): Our previous work suggests that the protein "Sunday Driver" (syd) organizes a damage surveillance system in peripheral neurons. In this proposal, we will further test the role of syd in conveying vesicle packages and signaling molecules along axons from the injury site back to the cell body to initiate a regenerative response. Our long-term goal is to identify the mechanisms that activate the intrinsic growth capacity of neurons following axonal injury. This knowledge will be critical to enable the design of new strategies to enhance nerve regeneration following injury. To uncover how syd and its associated membrane compartment may serve as what we call an "injury signaling platform", our first aim is to determine that axonal injury regulates syd interaction with the molecular motors. We will perform immunoprecipitations and sucrose velocity gradients from injured and control mouse sciatic nerves to determine whether syd phosphorylation triggered by nerve injury increases syd interaction with the retrograde motor complex dynein/dynactin, and decreases its interaction with the anterograde motor kinesin. To reveal the role of the c-Jun-N-terminal kinase (JNK) in this process, we will use known JNK inhibitors. This approach will allow us to determine whether JNK activity regulates syd axonal transport in vivo. To further dissect the role of syd phosphorylation by JNK, we will use a primary neuronal cell culture approach to determine whether deleting JNK consensus phosphorylation sites within syd sequence affects syd interaction with the motors and its subcellular distribution. Our second aim will focus on determining the mechanisms by which syd and the motors are recruited to axonal membrane compartments. We will first employ an EM approach to determine whether syd is associated with retrogradely transported endosomes in vivo. We will then perform immunoprecipitation, subcellular fractionation and immunofluorescence experiments to test whether small GTPases of the Rab family play a role in the recruitment of syd and motors to membrane compartments. Finally our third aim will assess the role of syd in nerve regeneration. An RNAi approach in cultured primary dorsal root ganglia (DRG) neurons will allow us to test syd function in axonal regeneration in vitro. Using a syd conditional knockout strategy, we will determine whether syd is critical for the regenerative capacity of peripheral neurons in vivo. To test whether syd injury-induced retrograde transport underlies the ability of PNS neurons to regenerate, we will determine whether syd phosphorylation and interaction with the molecular motors is differentially regulated following injury to peripheral vs. central axons. Altogether, this proposal will address whether the intracellular transport of vesicles that possess syd on their membranes participate in mechanisms that sense axonal injury and organize regenerative responses. PUBLIC HEALTH RELEVANCE: While peripheral neurons have a remarkable ability to repair themselves after injury, most parts of the adult central nervous system, comprised of the brain and the spinal cord, fail to extend new axons when damaged. Understanding the cellular events that confer such properties onto peripheral neurons will improve our ability to restore nerve function following spinal cord injury and stroke, as well as in cases of neurodegenerative diseases in which axonal pathologies interrupt the cell-body/synapse connection. Our work is centered on dissecting the molecular mechanisms regulating how information from the site of injury in axons is conveyed to the cell body to initiate a repair program in peripheral nerves.

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. While most peripheral nerves have a remarkable ability to repair themselves after injury, the central nervous system does not spontaneously regenerate. The retrograde transport of injury signals is one of the essential components that allow peripheral neurons to regenerate. Yet, the mechanisms regulating their transport are poorly understood. Our goal is to elucidate the molecular mechanisms by which injury signals are conveyed to the cell body to initiate the cascade of events leading to regeneration. Our previous studies implicate the protein "Sunday Driver" (syd) in transmitting information about injury from the axon back to the neuronal cell body. A better understanding of how nerve injury regulates syd association with the molecular motors and syd recruitment to axonal vesicles may define the mechanisms by which neurons initiate the appropriate regenerative response. We will use proteomics to reveal syd phosphorylation pattern and binding partners prior and following sciatic nerve injury. Syd will be immunoprecipitated from injured or non-injured rat sciatic nerves;the immunoprecipitated material will be separated by SDS-PAGE and sypro-stained bands of interest will be cut and analyzed by mass spectrometry. This proposed collaboration with the UCSF Mass Spectrometry Facility will help understand the role of syd in nerve regeneration and define the molecular mechanisms regulating anterograde and retrograde axonal transport in the context of nerve injury. Our studies may guide the development of new strategies to enhance nerve regeneration following injury.

DESCRIPTION (provided by applicant): Acute pain in the wake of peripheral injury is an important, adaptive physiological response. Pain helps to reduce re-injury, thus hastening recovery. Acute pain normally resolves when the injury heals. However, in many cases involving damage to peripheral nerves, acute pain transforms into chronic pain, which persists long after wounds have fully healed, and can be severely debilitating. Injury to peripheral nerves elicits a regenerative response and may also lead to long-term sensitization that contributes to the development of chronic pain. Protein synthesis in sensory neurons is required for both axon regeneration and for the development and maintenance of sensitization and pain following injury. Our goal is to gain new insights into the signaling pathways controlling protein synthesis in response to nerve injury to develop strategies that stimulate neurological recovery without coincidently promoting the development of chronic pain. The evolutionarily conserved mammalian Target Of Rapamycin (mTOR), a master regulator of the protein synthesis machinery, and the extracellular signal-regulated kinase (ERK) pathway are linked to both axon regeneration and to the development of neuropathic pain. Activation of mTOR by conditional deletion of the negative regulator tuberin (TSC2) in sensory neurons is sufficient to sustain regenerative growth, but whether it affects the development of nerve injury-induced pain is not known. ERK1/2 signaling is a major upstream regulator or mTOR activity. Although sensitization and regenerative axon growth are two processes known to require protein synthesis, the role or ERK1/2 signaling in regulating protein synthesis in sensory neurons has not been explored. Furthermore, in the studies performed to date, no discrimination was made regarding which ERK isoform, ERK1 or ERK2, is involved in sensitization and regenerative axon growth. Our preliminary data reveal ERK2 as the critical isoform for nociceptor sensitization and as a negative regulator of axon regeneration. This provocative result challenges the current model of the role of ERK1/2 signaling in axon regeneration. We propose here to identify the overlapping molecular events that regulate axon regeneration and the conversion from acute to chronic pain after nerve injury. Specifically, we will determine if ERK signaling regulates protein synthesis in naive or injured primary sensory neurons. We will determine which ERK isoform affects the regenerative ability of sensory neurons and if this effect is dependent on ERK-mediated regulation of protein synthesis. Finally, we will evaluate the effect of TSC2, ERK1 and ERK2 deletion on the development and maintenance of nerve injury-induced pain. Together, these experiments will test whether TSC2 and ERK signaling converge to mTOR-dependent protein translation to regulate nerve regeneration and the development of injury-induced chronic pain.

DESCRIPTION (provided by applicant): Lack of robust axonal regeneration represents one of the major barriers to recovery of neurological functions following injury to neurons within the central nervous system (CNS). In contrast, neurons in the peripheral nervous system (PNS) have a remarkable ability to regenerate after injury. The extent of axonal regeneration not only depends on the presence or absence of inhibitory cues in the environment, but also on the intrinsic growth capacity of damaged neurons. Indeed, blocking extracellular inhibitory influences alone is not sufficient to allow complete axon regeneration, emphasizing the need for a better understanding of the mechanisms controlling the intrinsic regenerative ability of injured neurons. The mechanisms that govern axon regeneration operate both in the cell body and locally in the axon. The local axonal responses allow injured neurons to signal back to the cell body and to transform their damaged axonal tips into a new growth- cone-like structure, two processes that are essential to initiate regeneration. In pursuing our studies on the response of axons to injury, we recently focused on the microtubule (MT) cytoskeleton. We found that the histone deacetylase HDAC5 is a novel injury-regulated tubulin deacetylase controlling axon regeneration. HDAC5 accumulates and deacetylates tubulin at the tip of injured PNS, but not CNS axons. HDAC5-mediated tubulin deacetylation is essential for PNS neuron's ability to regenerate, but fails to occur in CNS neurons. In addition to tubulin deacetylation, we observed that PNS axon injury also increases tubulin tyrosination. Tubulin acetylation and tyrosination are known to contribute to the dynamics properties of MTs and to MT-dependent axonal transport. However, the signaling pathways elicited by injury, which regulate MT posttranslational modifications and the precise role these modifications play in axon regeneration remain elusive. Here we propose to uncover the mechanisms controlling MT post-translational modifications in injured axons and to establish their specific roles in injured axons. Specifically, we will determine how a tubulin deacetylation gradient is maintained over time to sustain axon regeneration. We will also determine whether tubulin tyrosination initiates the retrograde transport of injury signals to activate a pro- regenerative program. Our long-term goal is to gain new insights into the molecular events that dictate the regenerative response of PNS neurons, and identify potential targets for future therapeutic interventions in the setting of CNS injury.

ABSTRACT Lack of robust axonal regeneration represents a major barrier to functional recovery following injury to neurons within the central nervous system (CNS). In contrast, peripheral neurons can regenerate after injury. Activation of a pro-regenerative growth program in peripheral neurons relies on the expression of regeneration-associated genes (RAGs) that allow for robust axonal re-growth. Although several genes have been identified for their pro-regenerative influence, individual gene based approaches have yielded limited success in axon regeneration, illustrating that manipulation of individual RAGs is unlikely to be sufficient to stimulate robust long-distance axon regeneration in the injured CNS. Therefore, understanding how a large ensemble of RAGs can be simultaneously activated after injury could reveal strategies to initiate the transcriptional pro-regenerative program. Epigenetic regulations, which include modification of the chromatin, affect combinations of multiple genes and hence represent ideal strategies to promote neural repair. Our goal is to gain new insights into the molecular events that regulate chromatin function in response to injury in peripheral neurons, and identify potential targets for future treatment of CNS injuries We previously demonstrated that axon injury elicits an epigenetic switch stimulating the regenerative competence of sensory neurons. Specifically, we discovered that calcium wave back-propagating from the site of axonal injury increases histone acetylation levels, stimulating the regenerative competence of sensory neuron. This work demonstrates a link between axon injury and chromatin remodeling and suggests that a coordinated pro-regenerative program is initiated by changes in the epigenetic landscape. In our recent studies, we identified hypoxia-inducible factor 1? (HIF-1?) as an important factor regulating axon regeneration via epigenetic as well as transcriptional regulatory mechanisms. We found that HIF-1? is required in injured sensory neurons to increase histone acetylation levels, to stimulate the expression of pro- regenerative genes and to promote axon regeneration. In mice breathing repeatedly low oxygen levels for brief periods (i.e., acute intermittent hypoxia, AIH) we observed increased levels of HIF-1? and enhanced axon regeneration in sensory neurons. However, the signaling pathways in normoxic conditions regulating HIF-1? accumulation and the precise mechanisms by which HIF-1? regulates chromatin in injured neurons remain elusive. Here we propose to uncover the molecular mechanisms controlling HIF-1? stability and activity following injury and to establish its specific roles in chromatin remodeling in injured neurons. We will also test if AIH can recapitulate at least in part the epigenetic changes elicited by peripheral axon injury and activate a pro-regenerative program in both peripheral and central neurons. This proposal has the potential to provide further rationale for the improvement of AIH-based treatment strategies for human patients. .

Trophic support and myelination of axons by Schwann cells in the peripheral nervous system (PNS) are essential for normal nerve function. Disruptions to myelin result in many neurological diseases, including Charcot-Marie-Tooth disease and numerous other peripheral neuropathies. Aberrant Schwann cell physiology leads to axon degeneration, demonstrating that glial-derived signals are required for axonal integrity. Non- myelinating Schwann cells in peripheral nerves, known as Remak Schwann cells, surround and ensheath small diameter axons into ?Remak bundles,? and structural defects in Remak bundles were shown to be associated with chronic pain. Schwann cell?axonal interactions are thus essential for proper nerve function, but the extent to which neurons contribute to Remak Schwann cell development is not well understood. In a mouse model of tuberous sclerosis, in which cortical neurons lack Tuberous sclerosis 1 (Tsc1), a negative regulator of the master regulator of protein synthesis, mTOR (mammalian Target Of Rapamycin), a striking delay in myelination was observed. Furthermore, loss of Tsc2, another negative regulator of mTOR, in excitatory neurons affects astrocyte development. These studies indicate that mTOR activation by neuronal deletion of Tsc1 or Tsc2 affects the development of glia, including oligodendrocytes and astrocytes. In agreement with these studies, our preliminary results in the peripheral nervous system indicate that in mice lacking Tsc2 in sensory neurons, Remak bundles are disorganized: the Remak bundles are oddly shaped and possess abnormally large diameter axons as well as fewer axons per bundle. We also noted thicker myelin around some axons and evidence of lost axon-Schwann cell contact. These results indicate that Tsc2 deletion and the resulting activation of mTOR in sensory neurons generates abnormal signals that disrupt Schwann cell development and/or maintenance, with a prominent effect on Remak bundles. Our goal is to understand the molecular mechanisms by which neuronal mTOR signaling impacts Schwann cells and Remak bundle organization. In Aim 1 we will expand and thoroughly define the consequence of Tsc2 deletion in sensory neurons on Schwann cell development and peripheral nerve function. In Aim 2, we will use genetic and next generation sequencing approaches to identify the molecular mechanisms underlying neuronally induced Schwann cell defects. These studies will help elucidate the role of axonally-derived signals in Remak Schwann cell development and may uncover new therapeutic avenues to treat peripheral neuropathy.

ABSTRACT Permanent disabilities following central nervous system (CNS) injuries result from the failure of injured axons to regenerate and re-build functional connections. The poor intrinsic regenerative capacity of adult CNS neurons is a major contributor to the regeneration failure and remains a major problem in neurobiology as well as an unmet medical need. In patients with glaucoma, the axon of retinal ganglion cells? (RGC) projecting into the optic nerve are injured, leading to RGCs death and loss of vision. Some treatment strategies can delay progression of the disease, but vision lost from the disease cannot be restored. Using adult mouse peripheral sensory neurons, which can switch to a robust axon regeneration state upon injury, my laboratory has discovered that injury elicits epigenetic changes that promote axon regeneration. Acetylation of the histone tail, which regulates chromatin dynamics and transcriptional activity, promotes expression of regeneration-associated genes (RAGs) and axon regeneration. We demonstrated that histone deacetylase 5 (HDAC5) and HDAC3 are involved in peripheral axon regeneration. Whereas neurons in the adult peripheral nervous system alter their epigenetic landscape to permit axon regeneration, RGCs may not be able to perform this task. Indeed, HDAC3 was shown to accumulate in the nucleus of injured RGCs, where it suppresses gene expression and promotes RGCs death after optic nerve injury. Consistently, inhibiting HDAC activity was shown to ameliorate RGCs survival and axon regeneration. Our data indicate that expression in RGCs of an HDAC5 mutant (with increased interaction with HDAC3) promotes axon regeneration after optic nerve injury. HDAC5 can regulate transcription by scaffolding the HDAC3 to a specific set of transcriptional targets, and we found that this HDAC5 mutant suppresses the level of SOCS3, an important negative regulator of axon regeneration. Together, these results strongly imply that the epigenetic landscape of RGCs represents a barrier to axon growth and survival after optic nerve damage and that modulating the chromatin can change RGCs? response to injury. We propose here to identify novel epigenetic modifiers that stimulate RGCs survival and regenerative capacity. These experiments may offer novel therapeutic avenues towards treatment of patients with glaucoma or other types of optic neuropathies.

Adult; Afferent Neurons; Attention; Axon; axon growth; axon regeneration; Biology; Brain; Candidate Disease Gene; Cell Differentiation process; Cells; chemotherapy; Coculture Techniques; Complex; Cues; Data; Embryo; Enzymes; Ethers; experimental study; Fatty Acids; Fatty-acid synthase; functional outcomes; Gene Expression; Gene Expression Profile; Generations; Genes; Genetic; Genetic Transcription; Goals; Human; human model; Immunofluorescence Immunologic; Impairment; In Situ Hybridization; in vivo; Inflammation; injured; Injury; innovation; Link; lipid metabolism; Lipids; live cell imaging; mature animal; Molecular; mouse model; Mus; Nerve; nerve injury; Nerve Regeneration; nervous system disorder; Neuraxis; Neuroglia; neuronal cell body; Neurons; novel marker; Pain; painful neuropathy; Palmitic Acids; Pathologic; Peripheral; peripheral nerve regeneration; Peripheral Nerves; Phospholipids; Physiology; Plasmalogens; Play; Process; Property; Recovery of Function; regenerative; Regenerative response; repaired; Research; response; Role; Schwann Cells; sciatic nerve; Sensory; Signal Pathway; Signal Transduction; single-cell RNA sequencing; Spinal Ganglia; Structure; System; Testing; Time; virtual;

ABSTRACT Spinal cord injury (SCI) damages long projecting axons leading to loss of sensory and motor function. Permanent disability results because injured axons in the spinal cord fail to regenerate, leaving them disconnected from their targets. There are currently no therapies to restore mobility and sensation following SCI. Therefore, a better understanding of the cellular and molecular mechanisms that compromise axon regeneration after SCI is needed to develop new strategies to restore function. Sensory dysfunctions and neuropathic pain are often the consequences of SCI. Sensory neurons are located in dorsal root ganglia (DRG) and are pseudo unipolar: in addition to a peripherally projecting axon that receive sensory information, they extend a centrally-projecting axon into the spinal cord that transmit this information to the brain. Whereas injury to the peripheral axon elicits a regenerative response, injury to the central axon fails to do so. Regeneration failure has been attributed in part to the weak regeneration- associated gene (RAGs) response upon injury. However, previous studies have assessed changes in gene expression using whole lumbar DRG after SCI, but only proprioceptors and low threshold mechanoreceptors (LTMRs) ascend the spinal cord and are injured after thoracic or cervical SCI. Furthermore, neurons are outnumbered by glial cells and other cell types within DRG, which may also contribute to the failed regeneration after SCI. Because of this remarkable heterogeneity in cell types, analyses of the neuronal and non-neuronal responses to injury has remained a challenge. Therefore, whether and how sensory neurons and the surrounding cells respond to SCI remains unclear. Based on our preliminary studies, we have now reason to believe that SCI results in gene expression changes in proprioceptors and LTMRs that have little overlap with those of peripheral injury, potentially constituting a roadblock for regeneration. The indirect effects of SCI on the non-neuronal cellular environment in the DRG also remain largely unexplored. Using transcriptional analysis at the single cell resolution, we identified a pro-regenerative role of the glial cells that envelop the neuronal soma, known as satellite glial cells. Our goal is now to use single cell transcriptional approaches to unravel how distinct sensory neuron subtypes and the surrounding non-neuronal cells respond to SCI. These approaches may lead to new therapeutic targets to promote axon regeneration and treat neuropathic pain after SCI.

ABSTRACT Permanent disabilities following central nervous system (CNS) injuries result from the failure of injured axons to re-build functional connections. There are currently no therapies to restore mobility and sensation following spinal cord injury or vision after optic nerve damage. The poor intrinsic regenerative capacity of mature CNS neurons is a major contributor to the regeneration failure and remains a major problem in neurobiology. In contrast, peripheral sensory neurons successfully switch to a regenerative state after axon injury. The long-term goal of my research program is to understand the multicellular mechanisms by which injured sensory neurons activate a pro-regenerative program and identify potential targets for future treatment of CNS injuries. Activation of an axon growth program relies in part on the expression of regeneration-associated genes. Because individual gene based approaches have yielded limited success in axon regeneration, we are focusing on epigenomic regulations, which affect globally, yet specifically a combination of multiple genes. Our goal is to uncover how the epigenetic landscape is re-organized in the context of axon injury to enable axon repair. These studies will incorporate cell-type specific epigenomic analyses to study the transcriptional and chromatin conformation changes elicited by peripheral and central axon injury. Axon regeneration is not cell autonomous and is influenced by the environment at the level of the axon injury site and at the level of the cell soma. We have recently discovered that satellite glial cells, the main type of glial cells in sensory ganglia respond to axon injury and contribute to the repair process. We propose to use powerful combinations of tools to pursue an innovative line of research aimed at dissecting the multicellular mechanisms orchestrating axon regeneration and build upon these findings to improve regeneration in CNS models. To achieve this goal, we will determine the intrinsic neuronal mechanisms controlling axon regeneration, focusing on epigenomics studies. We will elucidate the contribution of the microenvironment surrounding neuronal soma to the axon regeneration process, including satellite glial cells and other non-neuronal cells. To determine if findings made in the mouse model system are predictive of human physiology, we will determine the molecular profile of human cells surrounding sensory neurons. Finally we propose to manipulate novel pathways we discover to improve regeneration in two CNS models, spinal cord injury and optic nerve injury. This proposal will use powerful combinations of tools to pursue an innovative line of research aimed at dissecting the multicellular mechanisms orchestrating axon regeneration and build upon these findings to improve regeneration in CNS models.