Gregorio Valdez, Ph.D. - US grants

DESCRIPTION (provided by applicant): Age-related changes in mental function are associated with, and are presumably due to, morphological and molecular alterations in the central and peripheral nervous systems. Most neurobiologists agree that some of these changes involve synapses, but relationships of aging to synaptic alteration are poorly understood. The neuromuscular junction (NMJ) is an ideal system to examine age-associated structural and functional alterations in synapses, and to elucidate their molecular bases. Exciting preliminary results show that aging causes major morphological and molecular changes to the pre- and post-synaptic components of the NMJ. Presynaptically, there are fewer axons, more varicose axons and more axonal sprouting. The postsynaptic sites are often fragmented and partially innervated. Concomitant with these morphological changes, three synaptic adhesion molecules, laminin alpha4, laminin beta2 and agrin, are significantly decreased in aged NMJs. Supporting a role for laminin alpha4 in aging, its deletion causes premature synaptic aging in mice. Surprisingly, old age and the absence of laminin alpha4 do not alter the morphology of extraocular NMJs, a muscle also spared in the neuromuscular disease, amyotrophic lateral sclerosis (ALS). These novel findings suggest that laminin alpha4, laminin aeta2, agrin and their cognate receptors may play a critical role in synaptic aging and neurological disorders. To begin uncovering the role of these molecules in aging synapses, I will first assess when and how these molecules decrease using immunohistochemistry and in situ hybridization. I will also seek age-related alternations in their receptors, such as MuSK, Integrins and P/Q-type calcium channels. To test for a causal role for laminin beta2 and agrin in aging, I will use conditional and heterozygous mice to assess if their absence or substantial decrease causes defects in young adult NMJs. Finally, to probe the relationship of aging to ALS, I will assess the expression of synaptic adhesion molecules in a mouse model of ALS. Together, these studies may allow me to identify molecules that can attenuate or even reverse age-induced deleterious effects on synapses. Consequently, these experiments could provide tools to ameliorate a number of synaptic defects underlying cognitive and spinalmuscular diseases.

Project Summary/Abstract I am a tenure-track assistant professor at the Virginia Tech Carilion Research Institute. The interdisciplinary, interactive, collegial and nurturing atmosphere at the institute is truly conducive for developing a successful research and academic career. To help with my career goals, I have assembled an exceptional team of mentors, with Dr. Michael Friedlander (at VTCRI) serving as a mentor and Dr. Kenneth Fischbeck (at NINDS) and Dr. Michael Fox (at VTCRI) serving as co-mentors. In the short-term, I will obtain further training, acquire new skills and the experience needed to successfully run a team-driven research project and compete for independent sources of funding, including an R01. These experiences will provide me with the foundation necessary to obtain tenure and run a highly successful and well-funded laboratory. In my laboratory, I will seek to discover and manipulate molecules that act to maintain synapses, and thereby prevent the decline of motor skills that occur with normal aging and in blunting the effects of a multitude of age-related neurodegenerative diseases. In this proposal, I hypothesize that maintaining the normal function of the neuromuscular junction (NMJ), a large and experimentally accessible synapse formed between motor neurons and muscles fibers, could be sufficient to slow or prevent the erosion of motor skills caused by aging and amyotrophic lateral sclerosis (ALS). This hypothesis stems from the fact that deleterious changes at the NMJ appears to precede death of motor neurons and atrophy of muscle fibers during the progression of normal aging and ALS. In this regard, I have gathered preliminary data suggesting that three members of the fibroblast growth factor (FGF) signaling pathway, FGF-7/10/22, and a FGF-binding protein (FGFBP1) could be promising candidate molecules for protecting NMJs from insults emanating from normal aging and ALS. In mice, deletion of FGF- 22 results in premature aging of the NMJ. It also delays reinnervation of skeletal muscles after injury. Similarly, a reduction in FGFBP1 delays reinnervation of skeletal muscles. Thus, these results suggest that FGF-22 and FGFBP1 could function to repair the NMJ. In this project, my goal is to investigate the function of these growth factors in aging NMJs and in the initiation and progression of ALS. In addition to the training opportunities, the proposed experiments could lead to new therapeutic targets and approaches for protecting the motor system.

? DESCRIPTION (provided by applicant): Motor function declines with aging resulting in impaired mobility, loss of independence and increased susceptibility to injury and diseases. Many studies have sought to determine the cellular and molecular factors that contribute to aging of the neuromuscular system. Recent findings strongly suggest that structural and molecular alterations at neuromuscular junctions occur before motor neurons and skeletal muscle fibers exhibit obvious age-related pathological changes. Thus, deleterious changes at the neuromuscular junction may trigger the slow but consistent loss of motor function during aging. While it is known that muscle-derived factors are required to maintain and repair the neuromuscular junction, the identity of such factors has remained elusive. To this end, we have taken varied, yet complementary, approaches to identify muscle- derived factors that function to maintain the structural and functional integrity of neuromuscular junctions from the ravages of aging. In this regard, we have gathered preliminary data suggesting that three members of the fibroblast growth factor (FGF) signaling pathway, FGF-7/10/22, and a FGF-binding protein (FGFBP1) are promising candidate molecules for protecting neuromuscular junctions from insults emanating from normal aging, ALS-causing mutations and injury to peripheral nerves. In mice, deletion of FGF-22 results in premature aging of the neuromuscular junction. It also delays reinnervation of skeletal muscles after injury. Similarly, a reduction in FGFBP1 accelerates age-associated changes at neuromuscular junctions and compromises motor function in young adult mice. Importantly, introducing FGFBP1 and FGF-22 into denervated muscles accelerates their reinnervation, further indicating that FGF-22 and FGFBP1 function to repair the neuromuscular junction. We strongly believe that the proposed experiments could lead to new molecular targets for developing therapeutic interventions to protect and repair the neuromuscular junction, and thus slow, prevent or even reverse aging of the motor system.

Project Summary/ Abstract The loss of motor function that occurs with aging is closely associated with adverse health outcomes. To date, the contribution of different components of the motor system to age-related motor deficits remains unknown and despite extensive efforts. In addition, there are no therapeutics that can prevent the cellular and molecular changes that underlie loss of motor function with aging. The purpose of this proposal is to identify the contribution of the neuromuscular system to loss of motor function. We also seek to identify and test factors that function to maintain healthy skeletal muscles and motor neurons in the spinal cord, and thus preserve motor function. Specifically, we will determine whether maintaining normal levels of acetylcholine, genetically and pharmacologically, is sufficient to slow aging of skeletal muscle and motor neurons.

Abstract: Our funded R01 is based on the discovery that FGFBP1 is associated with synapses and progressively decreases with advancing age and in Amyotrophic Lateral Sclerosis (ALS). Specifically, we have discovered that genetic deletion of FGFBP1 accelerates degeneration of the neuromuscular synapse during normal aging and progression of ALS. Since FGF ligands are present and play critical functions in the brain, our findings suggest that FGFBPs, which are master regulators of FGF ligand signaling, may play similar roles in brain synapses harboring AD-inducing factors and with advancing age. Additionally, the increased presence of glycosylated ECM proteins in amyloid plaques and elsewhere due to fibrosis in AD strongly indicates that changes in levels and secretion of FGFBPs would further compromise FGF signals in AD. Thus, there is a strong rationale to posit that FGFBPs may be necessary for FGF ligands to appropriately promote the maintenance, repair, and survival of synapses and neurons most susceptible to AD. In this study, we propose to evaluate the role of FGFBPs in AD in the following two aims: 1) We will determine the expression and distribution of FGFBPs in brain regions affected with AD. 2) We will test the hypothesis that FGFBP1 plays important roles in AD pathogenesis in mouse models of AD and in cultured neurons harboring AD-causing mutant genes.

Project Summary/Abstract The loss of motor function that occurs with aging is closely associated with adverse health outcomes. In recent years, published findings strongly suggest that malfunction and degeneration of the neuromuscular junction (NMJ), the synapse formed between ?-motor neurons and skeletal muscle fibers, contributes to age-related motor dysfunction. As the final output of the somatic motor system, degeneration of the NMJ inevitably results in degeneration of motor axons and atrophy of muscle fibers, thus affecting voluntary movement. Thus, it is critical to identify factors that function to maintain and repair the NMJ. Using an R56 grant provided by NIA, our lab has identified the fibroblast growth factor binding protein 1 (FGFBP1) as a promising candidate factor secreted by muscle fibers to preserve and restore the integrity of NMJs during aging. FGFBP1 functions to chaperone FGF ligands from the extracellular matrix to cognate receptors. In this manner, it enhances FGF signaling. We have found that while FGFBP1 concentrates at NMJs in young adult mice, it progressively decreases during normal aging and in SOD1G93A mice, a mouse model for ALS. Using knockout mice, we observed that FGFBP1 expression is required to slow aging of NMJs and motor deficits during normal aging. Furthermore, FGFBP1 deletion in mice expressing SOD1G93A, a model for amyotrophic lateral sclerosis (ALS), accelerates NMJ degeneration, disease progression and death. These initial discoveries strongly suggest that preventing loss of endogenous of FGFBP1 during aging may be sufficient to slow degeneration of NMJs, and thus preserve motor function. To test this hypothesis, we proposed three specific aims that build on each other. In aim 1, we test the hypothesis that motor deficits in mice deficient for FGFBP1 result from cellular, molecular, and physiological changes at NMJs. In aim 2, we will seek to identify molecular mechanisms that inhibit FGFBP1 expression in aging muscles. In aim 3, we will test the hypothesis that FGFBP1 is sufficient to prevent and reverse age-related changes of NMJs. These aims are designed to uncover the initial changes that precipitate aging of NMs, the molecular factors that result in decreased FGFBP1 expression in aging muscle, and the therapeutic potential of FGFBP1 in preserving NMJs and motor function.

Project Summary To continue to march towards developing an effective cure for ALS, it is imperative to: 1) ascertain the contribution of neurons, other than motor neurons, to the disease: 2) identify the molecular mechanisms that trigger or fail to stop pathophysiological changes in neurons, including motor neurons; and 3) develop culture assays that faithfully and reliably recapitulate pathogenic cellular and molecular changes that occur in vivo. In published findings, we and others have shown that sensory axons and nerve endings in the periphery degenerate early and progressively in SOD1G93A and TDP43A315T mice. These cellular features are also found in motor neurons afflicted with ALS, suggesting that the same molecular mechanisms critical for maintaining and repairing axons and their nerve endings may become dysfunctional in both types of neurons. However, the soma of sensory neurons does not atrophy in mice harboring ALS-causing mutant genes despite the degeneration of their nerve endings, indicating that there are differences in how these two neuronal populations respond to ALS-induced pathogenesis. These similarities and differences between sensory and motor neurons affected with ALS present unique opportunities to uncover shared mechanisms involved in axonal degeneration and activated to maintain the viability of the neuronal soma. In this proposal, we will examine the impact of two ALS-causing mutant genes on sensory neurons in vivo and in culture. In aim 1, we will determine cellular changes altered in sensory neurons expressing ALS-causing mutant genes maintained in culture for weeks. In aim 2, we will test the hypothesis that sensory neurons harboring ALS-causing mutant genes are sensitive to cellular stressors, an experiment designed to recapitulate stressors these and other neurons encounter in vivo. In aim 3, we will determine molecular features shared between sensory neurons expressing different ALS-causing mutant genes. Altogether, this proposal will further our understanding of pathological changes that occur in sensory neurons harboring ALS-causing mutant genes, establish primary sensory neurons as a cellular assay in culture to identify and test therapeutics for ALS and uncover molecular mechanisms altered by expression of both, SOD1G93A and TDP43A315T.