Young-Jin Son - US grants

DESCRIPTION (provided by applicant): Intact motor nerve terminals sprout in response to paralysis or partial denervation and re-establish functional synaptic connections with inactive muscle fibers, thus insuring the maintenance of muscle innervation and strength. This remarkable feature of neuromuscular plasticity has been studied intensively, both because it is essential to our understanding of synapse formation and maintenance and because it provides a potential avenue for promoting repair of nerve injuries. However, the mechanisms underlying this type of neural plasticity are poorly understood. We and others have recently shown that the terminal Schwann cells (tSCs) that cap the nerve terminal play a central role in this process, by extending processes that trigger terminals to sprout and that guide these sprouts to inactive muscle fibers. We now propose to identify the molecules that activate quiescent tSCs to extend processes and the molecules that activated tSCs use to elicit sprouting. Aim 1 will test the hypothesis that reduced neurotransmitter release, and subsequent inactivation of muscarinic signaling, activates tSCs. Pharmacological inhibition of this muscarinic signaling in vivo and analyses of knockout mouse lines that are defective in muscarinic signaling will be used. Aim 2 will assess the role of tSCs in mediating growth factor induced sprouting. We will test this by exogenous application of IGFs and CNTF, two growth factors known to elicit sprouting, to determine whether these agents activate tSCs to extend processes. We will then examine CNTF -/- mice, whose nerve terminals apparently do not sprout in response to partial denervation or botulinum toxin, to determine whether their tSCs are defective in their response to normal sprouting stimuli. In Aim 3, we will test the hypothesis that NCAM and N-cadherin, the two cell adhesion molecules (CAMs) upregulated in activated tSCs, elicit sprouting. We will exploit the ballistic transfection technique to overexpress these CAMs selectively in quiescent tSCs in vivo, and examine the incidence of terminal sprouting. Secondly, we will transplant fibroblasts genetically modified to express the CAMs to intact endplates, to determine whether these fibroblasts acquire the ability to induce terminal sprouting. Through this work, we attempt to further define the role of Schwann cells in the reactive sprouting and to gain new insights into the inter- and intracellular signaling associated with terminal sprouting.

DESCRIPTION (provided by applicant): Several different therapeutic approaches have produced significant recovery of motor function in experimental models of spinal cord injury (SCI). These treatments include strategies designed to enhance axon regeneration and strategies targeted towards remyelination, tissue sparing and training the spared circuits. Their potential usefulness at the bedside, however, is critically dependent on nerve-muscle connectivity that not only remains functional but also functions as efficiently as possible. Surprisingly, however, the synaptic responses of muscles to SCI have received little attention, and the neuromuscular junctions (NMJs) caudal to SCI are assumed to remain intact. Our preliminary morphological analyses, however, suggest that NMJs in hindlimb muscles of adult rats paralyzed by SCI may be extremely dysfunctional. Furthermore, these studies imply that adult NMJs may be extraordinarily diverse and specific in their sensitivity to paralysis. In this R21 application, we will use fluorescent transgenic mice, in vivo time-lapse imaging, and combined electrophysiological and morphological analyses to determine (1) if there are multiple subpopulations of NMJs that differ in pre- and postsynaptic sensitivity to the SCI-elicited paralysis, and (2) if physiologically significant loss of nerve-muscle connectivity accompanies morphological instability of NMJs distal to SCI. These results will provide critical data to justify a larger grant application to: explore the molecular mechanisms underlying the unexpected diversity of mature NMJs;comprehensively assess the contribution of NMJ loss to motor deficits associated with SCI;and attempt to promote motor recovery by stabilizing NMJs. The proposed work therefore has the potential to establish a strong foundation for developing novel treatments for spinal cord injured patients. PUBLIC HEALTH RELEVANCE: Several different therapeutic approaches have produced significant recovery of motor function in experimental models of spinal cord injury. These treatments include strategies designed to enhance axon regeneration and strategies targeted towards remyelination, tissue sparing and training the spared circuits. The project seeks to provide a novel basis for motor deficits and recovery following spinal cord injury by identifying nerve-muscle connections, termed neuromuscular junctions, as novel therapeutic targets.

DESCRIPTION (provided by applicant): Primary sensory axons injured by dorsal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. The mechanisms that prevent regeneration at the CNS-PNS interface, the dorsal root entry zone (DREZ), are unknown. The present approaches for overcoming this regeneration failure have had only limited success. Over the past few years, we have pioneered in applying in vivo imaging to directly monitor sensory axons arriving at the DREZ in living mice. These studies lead us to hypothesize that the regeneration failure and the limited success with current interventions might be because regenerating axons undergo rapid and aberrant synaptic differentiation that causes growth to cease prematurely at the DREZ. To test this idea, we will apply advanced techniques, including in vivo imaging, inducible transgenic mice, and targeted electron microscopy. In Aim 1, we will identify postsynaptic mechanisms by testing whether NG2 glia induce presynaptic differentiation and/or growth arrest. In Aim 2, we will identify presynapti mechanisms by testing whether targeting calcium channel alpha2delta subunits and their interaction with thrombospondins will promote regeneration. In Aim 3, we will promote robust regeneration by combining treatment with gabapentin (GBP) and pregabalin (PG), which prevent synaptogenesis, with conventional interventions targeting intrinsic and extrinsic growth barriers, which individually elicit little regeneration. The proposed work has the potential to revise the prevailing explanation for the regeneration failure of primary sensory neurons and may also be applicable to spinal cord injury. In addition, GBP and PG are commonly prescribed anti-neuropathic pain medications already approved by the FDA. Our work therefore can be quickly applied to patients with brachial plexus, lumbosacral plexus and cauda equina injuries, which are common and debilitating and have no effective treatment.

? DESCRIPTION (provided by applicant): Peripheral nerve injuries are common and affect >250,000 individuals annually in the US. Full recovery is rare even after surgical repair; >90% of patients suffer long-term motor and sensory deficits. Functional recovery is poor because peripheral nerves rarely regenerate over long distances. Long-distance regeneration fails largely because Schwann cells lose their ability to support axon regeneration over time after injury. No therapy is currently available to encourage Schwann cells to continue to promote regeneration, and it is not even known whether Schwann cell atrophy can be prevented or reversed. We have recently discovered that induced expression of constitutively active ErbB2 receptor tyrosine kinase (caErbB2) dramatically enhances the regeneration-promoting capability of Schwann cells. Moreover, our preliminary results show that caErbB2 enables chronically denervated, atrophied Schwann cells to enlarge, divide, migrate and extend processes. Notably, we have observed few, if any, effects on innervated Schwann cells that were spared injury, suggesting that therapeutic caErbB2 would have few off-target effects. In this R21 application, we propose to explore further the possibility that caErbB2 reverses Schwann cell atrophy and determine whether it promotes long-distance axon regeneration. We will combine conventional anatomical and functional analyses with advanced techniques such as inducible fluorescent transgenic mice, in vivo imaging and CLARITY, to determine: (1) if induced expression of caErbB2 reactivates atrophied Schwann cells in chronically denervated nerve, and (2) if the Schwann cells reactivated by caErbB2 promote axon regeneration. These proof-of- concept experiments will provide critical data to justify a larger grant application that will investigate the signalin cascades evoked by caErbB2, identify its key effectors, define the therapeutic window of caErbB2 efficacy and develop a therapeutically applicable method of boosting ErbB2 signaling. The proposed work therefore has the potential to establish a strong foundation for developing a potent treatment that enables long- distance nerve regeneration in patients.

Abstract In the vertebrate peripheral nervous system, Schwann cells (SCs) make myelin that insulates large axons and allows rapid conduction of nerve impulses. Myelinating SCs possess the innate ability to demyelinate and transform into repair SCs, which promote axonal regeneration and remyelination after traumatic injury. Demyelination can also occur pathologically, and there are no effective treatments to promote or enhance remyelination after injury or disease. Myelination during development is triggered by activation of several SC membrane-associated proteins, and requires that the transcription factor Krox20 be in the nucleus. However, we know little about how myelination signals move from the membrane to the nucleus during development and even less about the signaling required for myelin maintenance and remyelination. YAP/TAZ are paralogous transcriptional co-activator proteins with diverse cellular functions, known best as potent promoters of cell proliferation. Their activity is regulated by nucleocytoplasmic shuttling: when nuclear, they are transcriptionally active. We recently showed that in SCs, YAP/TAZ are nuclear and required for Krox20 expression, myelin formation and maintenance, suggesting that YAP/TAZ shuttle signals from membrane to nucleus to regulate myelination. These findings lead us to hypothesize that YAP/TAZ are a nexus for multiple signaling pathways that lead to transcriptional activation of Krox20 and myelin genes, and which thereby regulate developmental myelination, myelin maintenance, demyelination and remyelination. To test our hypothesis, we propose the following Aims: 1) Determine if YAP/TAZ mediate demyelination and remyelination; 2) Identify upstream regulators of YAP/TAZ in myelin formation and maintenance; 3) Determine how YAP/TAZ regulate transcription of Krox20 and myelin genes. We will use unique and conventional in vitro and in vivo techniques, including multiple lines of inducible transgenic mice and RiboTag translatome profiling. The proposed study should significantly enhance our understanding of how SCs form, maintain and repair peripheral myelin. It is also likely to provide important new insights into how to prevent demyelination or promote robust remyelination in peripheral nerve diseases.