Aaron DiAntonio - US grants

The long-term objective of this proposal is to define the role of the postsynaptic cell in the regulation of synaptic development The general strategy is to combine genetic, molecular, anatomical, and electroph2rsiological analysis in Drosophila to identify mechanisms by which postsynaptic cells shape the developing synapse Recent e_ idence at the Drosophila neuromuscular junction demonstrates that postsynaptic dysfunction leads to a compensatory increase in presynaptic neurotransmitter release This proposal investigates the hypothesis that postsynaptic activity controls a homeostatic signaling mechanism that regulates presynaptic function Such homeostatic mechanisms can regulate synaptic activity during development and may compensate for synapse loss following injury o1 disease This proposal will inx estigate the mechanism of the homeostatic response and characterize molecules that regulate the homeostatic, retrograde signaling pathway Spatial and temporal requirements for homeostatic signaling will be investigated and the presynaptic change underlying homeostatic compensation will be determined Postsynaptic glutamate receptors play a central role in regulating homeostasis A novel glutamate receptor has been identified and mutations in this gene have been isolated This receptor's role in regulating homeostasis will be investigated In addition, a structure/function analysis of glutamate receptors will be undertaken to determine how they regulate homeostasis Finally, two novel mutations have been identified that regulate homeostatic and retrogIade signaling These enhancers will be characterized to elucidate their role in the homeostasis pathway These studies seek to define the mechanisms and molecules that underlie synaptic homeostasis, which is a necessary )rerequisite for understanding the role of homeostasis in development and disease

DESCRIPTION (provided by applicant): Glutamate is the primary excitatory neurotransmitter of the vertebrate CNS and the Drosophila neuromuscular junction (NMJ). The vesicular glutamate transporter (VGLUT) is responsible for filling synaptic vesicles with transmitter at glutamatergic synapses. The glutamate content of a synaptic vesicle is a fundamental parameter controlling the strength of synaptic transmission in the healthy brain, while the misregulated release of vesicular glutamate may contribute to the pathophysiology of such diseases as stroke, ALS, and epilepsy. This proposal will test the hypothesis that the expression, trafficking, and activity of the Drosophila homolog of VGLUT (D VGLUT) regulates the function, plasticity, and development of a glutamatergic synapse by controlling the glutamate content of synaptic vesicles. Our studies will combine genetic, cell biological, and electrophysiological methods to test the function of this important protein. The Drosophila homolog of the vesicular glutamate transporter (DVGLUT) has been identified and mutants have been generated that allow for the manipulation of its expression at the synapse. Biochemical, electrophysiological, and electron microscopic techniques will be employed to investigate the physiological role of DVGLUT in the filling of synaptic vesicles with glutamate (Aim 1). Neurotransmitter not only mediates synaptic transmission, but can also regulate the development of synapses. DVGLUT mutants will be used to manipulate the levels of glutamate in synaptic vesicles and the consequences for synapse formation, glutamate receptor localization, and synaptic maturation will be investigated (Aim 2). Finally, the mechanisms regulating the membrane trafficking of DVGLUT will be determined in order to define the role of DVGLUT trafficking in the regulation of glutamatergic transmission (Aim 3). The results of these experiments will provide novel and fundamental information on the mechanisms by which VGLUT contributes to the development and function of glutamatergic synapses.

DESCRIPTION (provided by applicant): The development and maintenance of neural circuits is essential for the formation and function of the nervous system. Circuit development entails axon guidance, synaptic target selection, synapse formation, and synaptic growth and remodeling in response to developmental and environmental inputs. Disrupting these processes in childhood can lead to neurodevelopmental disorders such as mental retardation and autism. In the adult, plasticity mechanisms inducing circuit remodeling likely allow for the encoding of memories, but also contribute to the development of chronic pain and drug addiction. While circuit plasticity is important, it is also essential that circuits be maintained. Disease, trauma,and neurotoxins, including medicines such as chemotherapeutics as well as drugs of abuse such as alcohol and ecstasy, can all trigger an ill-defined axonal degeneration cascade that leads to axonal loss with profound consequences for neurological function. Inhibiting this degeneration program has tremendous potential for the treatment of a host of devastating neurological disorders including Parkinson's disease, Multiple Sclerosis, and neuropathies, but the critical barrier in the field is the lack of knowledge of the key components of the degeneration pathway. Remarkably, a single molecule, the ubiquitin ligase Phr1/Highwire, is a central regulator of both these developmental and degenerative axonal responses. This application explores the function of the Phr1/Highwire ligase, focusing on the recent finding that it is required for normal axonal degeneration in the mouse. The therapeutic potential of inhibiting Phr1 is tested in vivo, while in vitro neuronal culture experiments explore the mechanism of the Phr1-dependent degeneration program. In addition, new Drosophila mutants have been identified that regulate synaptic development and that interact with highwire and its target the MAPKKK Wallenda/DLK. Powerful genetic techniques will be used to analyze these mutants to define molecular pathways controlled by this ligase, allowing for integration of findings in fly and mouse. This innovative, multi-system approach will generate insights into the mechanisms by which neuronal circuits develop and degenerate, and test the therapeutic potential of targeting this essential, evolutionarily conserved ligase and its interaction partners for the treatment of a host of neurological disorders characterized by axon loss.

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to define molecular mechanisms that control synaptic strength. Synapses are dynamic-once formed, neural circuits evolve by the addition and elimination of synaptic connections and the modification of their strength. Setting and modifying the strength of synapses is important for refining developing circuits and defects in these mechanisms are a likely etiology of neurodevelopmental disorders such as autism and mental retardation. In the mature nervous system, modifying synaptic strength is important for normal processes such as memory formation and pathophysiological events such as the synaptic rearrangements underlying chronic pain or the synaptic loss in neurodegenerative disorders. To define molecular mechanisms that control synaptic strength, we are undertaking a genetic, anatomical, and electrophysiological analysis in Drosophila. Neurotransmitter is released from the presynaptic cell at specialized sites called active zones. Efficient synaptic transmission requires that active zones contain a normal complement of proteins, and that these specialized release sites be apposed to postsynaptic clusters of neurotransmitter receptor. Little is known of the molecular mechanisms that regulate the protein composition of active zones and ensure the alignment of neurotransmitter release and reception machinery. In screens for genes required for such processes, four mutants were identified in which a large proportion of glutamate receptor clusters are apparently unapposed to presynaptic release sites. These mutants will be characterized to uncover molecular mechanisms that form and maintain the active zone/receptor cluster dyad. PUBLIC HEALTH RELEVANCE: This research is relevant to public health because it will improve our understanding of how nerve cells connect and communicate in the brain. If these connections do not form or function properly in a child, it may lead to neurological diseases such as mental retardation, epilepsy, and autism, while in the adult, loss of these connections may contribute to neurodegenerative disorders such as Alzheimer's disease. An understanding of the molecules that control the formation, function, and maintenance of nerve cell connections could aid in the future development of new therapies for devastating neurological diseases.

DESCRIPTION (provided by applicant): Axonal degeneration is a common feature of many neurological diseases including neurodegenerative disorders, hereditary neuropathies, traumatic injury, diabetes, glaucoma, and chemotherapy-induced neurotoxicity. Axonal dysfunction is an early event in many of these disorders, and so axo-protective therapies are a central focus for the development of new treatments for these conditions. Recent studies demonstrate that axonal degeneration is an active and highly regulated process, yet the intrinsic, neuronal mechanism promoting degeneration is poorly understood. Expression of Nmnat is the most potent axo-protective strategy yet identified. The ability of Nmnat to protect axons following a wide range of insults in both mammals and Drosophila indicates that it modulates a fundamental, evolutionarily conserved axonal degeneration pathway. However the identity of this pathway is unknown. We have developed novel, large-scale screening paradigms in both mammalian neurons and Drosophila that will allow us to identify genes required for Nmnat-dependent axonal protection as well as genes that promote axonal degeneration following injury. We propose to explore the potential of these new assays to identify genes that are crucial for the prevention of axonal degeneration and, as such, may have therapeutic potential in neurological disorders where axonopathy is a major contributor. By performing these complementary genetic screens, we hope to identify a larger cohort of genes involved in the evolutionarily conserved axonal degeneration process than would likely be found using any individual screen. It is our expectation that findings derived from these exploratory studies will allow our labs as well as others throughout the world to make rapid progress in understanding the process of axonal loss in disease. PUBLIC HEALTH RELEVANCE: This research is relevant to public health because it will identify components of pathways that promote axonal degeneration following injury and disease. Axonal degeneration is a prominent component of many neurological disorders including neurodegenerative diseases, hereditary neuropathies, trauma, diabetes, glaucoma, and chemotherapy-induced neurotoxicity. Identifying components of the pathways in axons that promote degeneration will provide insights into the fundamental mechanism underlying axonal degeneration as well as potential therapeutic targets for the many neurological diseases characterized by axonal degeneration.

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to define the molecular mechanisms that promote axonal degeneration following injury or disease. Axonal degeneration is a common feature of many neurological diseases. Neuropathies due to axonal degeneration are a hallmark of disorders such as diabetes, glaucoma, and chemotherapy-induced neurotoxicity and axonal loss is an early feature of debilitating neurodegenerative diseases. The great length of many axons makes them particularly vulnerable to mechanical injury, and axonal degeneration following trauma is a major cause of disability. Recent studies demonstrate that axonal degeneration is an active and highly regulated process, yet the intrinsic, neuronal mechanism promoting degeneration is poorly understood. This proposal investigates what causes axons to degenerate, and how this can be prevented. Axonal degeneration is an active process of self-destruction that appears to be naturally primed and waiting for a triggering stimulus that activates the execution phase. It proceeds as a stepwise process that begins with microtubule destabilization, followed by rapid blebbing of the axonal membrane, axonal fragmentation, cytoskeletal degradation and eventual engulfment by glial and/or phagocytic cells. We now demonstrate that the DLK pathway functions in the intrinsic neuronal pathway that promotes axonal degeneration following injury. Identifying and characterizing the function of components of the intrinsic axonal degeneration pathway will provide insights into its mechanism as well as potential therapeutic targets for the many neurological diseases characterized by axonal degeneration. PUBLIC HEALTH RELEVANCE: This research is relevant to public health because it will improve our understanding of the mechanism of axonal degeneration following injury and disease. Axonal degeneration is a prominent component of many neurological disorders including neuropathies associated with trauma, diabetes, glaucoma, chemotherapy-induced neurotoxicity, and neurodegenerative diseases. Identifying components of the pathway in axons that promote degeneration will provide insights into the fundamental mechanism underlying axonal degeneration as well as potential therapeutic targets for the many neurological diseases characterized by axonal degeneration.

DESCRIPTION (provided by applicant): Neurological disease represents a tremendous personal burden to patients and families and financial burden to society. With our rapidly aging population, these burdens are estimated to increase dramatically in the coming decades. Conventional research efforts focus on identifying the distinct etiologies and developing disease-specific treatments for debilitating neurological disorders such as Alzheimer's Disease, stroke, Multiple Sclerosis, glaucoma, and peripheral neuropathy. As an alternative, we are focusing on a shared feature of these disorders-the degeneration of injured axons. We hypothesize that a common, evolutionarily conserved cell biological pathway triggers axonal degeneration, and that inhibiting this pathway will preserve axonal connections and serve as an effective treatment in these and other neurological diseases. To test this hypothesis, we are developing an innovative, high-throughput set of tools for the genome-wide identification and characterization of proteins and pathways involved in axonal degradation using both Drosophila and primary mouse neuronal systems. By focusing on candidates validated in both systems, we anticipate elucidating this critical program. We will identifying a host of proteins, some of which are likely to represent reasonable pharmacological targets that could be modulated in order to block or delay axonal degeneration. If successful, this proposal will stimulate the development of treatments for a wide range of devastating neurological disorders.

DESCRIPTION (provided by applicant): Methods to promote axonal regeneration have tremendous potential to treat the injured and diseased nervous system. This potential is most clear in the injured CNS, such as in spinal cord injury, where there is essentially no axon regeneration. Even in the periphery, increasing the speed and extent of axonal regeneration would provide important therapeutic benefits. In this proposal, we outline experiments to promote axon regeneration by therapeutically invoking the preconditioning response. The molecular basis of preconditioning is poorly understood but its ability to stimulate axonal regeneration after injury and to enhance axonal growth over non-permissive substrates makes it an important target for development of new approaches for treating the damaged nervous system. We have developed a fully in vitro preconditioning assay in primary neurons, which will allow for the first high throughput drug and genetic studies of this process. We plan to identify preconditioning pathways using high-throughput methods adapted from those we developed to explore axonal degeneration that enable rapid screening of compounds and genetic pathways. Using adult DRG neurons we plan to screen libraries of drug compounds and lentivirus open reading frame (ORF) libraries. First, we will optimize and miniaturize the screening assay and image analysis (R21 phase) and then use high-throughput screening and imaging analysis to identify compounds and/or genes that enhance axon re-growth by promoting a 'preconditioning' response (R33 phase). Second, we will develop secondary screens to assay molecular markers of preconditioning, neuronal sub-type-specific preconditioning responses, and a microfluidics based assay for axonal growth on inhibitory substrates (R21 phase). These assays will be used to further characterize 'hits' from the primary screens (R33 phase). Third, we will use a sciatic nerve crush assay to examine the in vivo activity of a few prioritized compounds identified in the screens (R33 phase). Through these experiments we hope to uncover agents and pathways of injury-induced preconditioning that will lead to new methods for stimulating robust axonal regrowth and growth on inhibitory substrates that will potentially lead to new treatments for the damaged nervous system.

Project Summary/Abstract: Axonal degeneration is an early and likely initiating event in some of the most prevalent neurological diseases, including peripheral neuropathies, traumatic brain injury, Parkinson's disease and glaucoma. Although axon loss is central to many neurological disorders, no treatments currently exist that effectively target axonal breakdown. Axon degeneration is a subcellular self-destructive process that is activated by traumatic, metabolic, and neurodegenerative insults. Conceptually this degeneration program is akin to the apoptotic pathway?it is a biochemical pathway that dismantles injured axons in much the same way that the apoptotic pathway orchestrates the programmed death of dysfunctional cells, although the molecular mechanisms are primarily distinct. Others and we discovered that SARM1 is an essential component of the injury-activated axonal degeneration program. Importantly, SARM1 is also required for axon loss in models of neurological disease, including peripheral neuropathies and traumatic brain injury. Hence, agents that block SARM1 activity are exciting therapeutic candidates for axonal preservation in diseases of axon loss. In the prior funding period we made a major conceptual breakthrough, discovering that SARM1 is a NAD+ cleaving enzyme and, hence, a druggable target in the axon degeneration pathway. However, to exploit the full promise of targeting SARM1 we must understand the molecular mechanisms upstream and downstream of SARM1 enzyme activity. Here we will explore the role of SARM1-derived NAD+ metabolites as biomarkers and mediators of axon degeneration. We will also identify the mechanisms that keep SARM1 `off' in a healthy axon and turn SARM1 `on' in a diseased axon. If successful, these studies will define the molecular mechanisms upstream and downstream of SARM1 enzyme activity and identify novel therapeutic targets for the preservation of axons in neurological diseases.

Project Summary/Abstract: Therapy-induced peripheral neuropathy (TIPN) is a very common and often dose-limiting side effect of anti-cancer therapy. Clinically, TIPN is a predominantly sensory peripheral neuropathy characterized by numbness, tingling, and often, neuropathic pain. These symptoms can persist for years after cessation of treatment, and so TIPN can significantly diminish patient's quality-of-life both during and after treatment. Moreover, the development of TIPN often necessitates reducing drug dosage or switching regimens, and therefore limits the effectiveness of anti-cancer therapy. Currently, there are no effective treatments for TIPN. Axon loss is a hallmark of this neuropathy, suggesting that mechanistically distinct chemotherapeutics may feed into a common axonal degeneration program. We have demonstrated that genetic inhibition of SARM1, the central executioner of this core axonal degeneration program, blocks the development of TIPN in a mouse model of vincristine-induced peripheral neuropathy. The SARM1 pathway induces axon loss by triggering depletion of the essential metabolic co-factor NAD. Here we seek to block the development of TIPN by countering this loss of NAD in order to maintain axonal health. We also explore mechanisms to block the activation of SARM1 as novel therapeutic strategies for blocking the development of TIPN. Finally, targeting the SARM1 pathway will be a useful treatment for TIPN if manipulating this pathway does not affect tumor growth or chemotherapeutic efficacy. We will explore this using genetic tumor models. If successful, this project will identify novel treatment strategies for the prevention of TIPN.

Project Summary/Abstract: Therapy-induced peripheral neuropathy (TIPN) is a very common and often dose-limiting side effect of anti-cancer therapy. Clinically TIPN is a predominantly sensory peripheral neuropathy characterized by numbness, tingling, and often, neuropathic pain. These symptoms can persist for years after cessation of treatment, and so TIPN can significantly diminish patient's quality-of-life both during and after treatment. Moreover, the development of TIPN often necessitates reducing drug dosage or switching regimens, and therefore limits the effectiveness of anti-cancer therapy. Currently, there are no effective treatments for TIPN. Axon loss is a hallmark of this neuropathy, suggesting that mechanistically distinct chemotherapeutics may feed into a common axonal degeneration program. We have demonstrated that genetic inhibition of this core axonal degeneration program blocks the development of TIPN in a mouse model of vincristine-induced peripheral neuropathy. Now we seek to identify small molecules that can block this axonal degeneration program that could serve as a) chemical probes for the study of axon degeneration and b) therapeutic lead compounds for the development of new treatments to prevent or treat TIPN and other disorders characterized by axon loss. We have developed an assay to identify such inhibitors, and now propose to optimize this assay for a high-throughput screening format. We will use this high-throughput screening platform to conduct a pilot screen and develop a series of counter screens to eliminate false positives and to assess various parameters of identified compounds to prioritize them for further validation. These assays will also serve to characterize mechanistic features of the hits and cluster them based on chemical properties and mechanism of action for biological assays. Finally, we will test the therapeutic potential of identified compounds by examining their activity in cultured neurons using phenotypic axon degeneration assays. If successful, this project will yield a reliable and efficient high-throughput screening platform and follow-up testing funnel for the identification of novel therapeutic candidates for the development of therapies for TIPN and other disorders of the injured and diseased nervous system.

Motor axon loss is a cardinal symptom of amyotrophic lateral sclerosis (ALS). Axon loss can be driven by a genetically encoded program in which the axon survival factors NMNAT2 and STMN2 inhibit the activity of the axon destruction factor SARM1. Recent data suggest that this program of axon self-destruction may contribute to pathology in ALS. First, aggregation of TDP-43, a hallmark of most ALS cases, results in the selective loss of mRNA encoding functional STMN2, a key axon survival factor. Second, loss of SARM1 suppresses some neurodegenerative phenotypes in a mouse ALS model that expresses pathogenic human TDP-43. Here we investigate the contribution of this axon degeneration pathway to ALS. We have defined the mechanism of action of SARM1, demonstrating that it is the founding member of a new class of NAD-cleaving enzymes. SARM1 enzyme activity is normally held in check via an autoinhibitory domain. Injury- or disease- induced loss of NMNAT2 and STMN2 disinhibits SARM1, leading to rapid NAD+ depletion, metabolic catastrophe, and axon fragmentation. Our structure-function studies of the SARM1 protein have identified mutations with a range of consequences, from constitutively active variants that promote cell death and axon loss, to dominant negative variants that are neuroprotective. These findings imply that human variants may exist that either promote or protect against neurodegeneration, and that understanding the phenotypic consequences of genetic variation requires functional studies of enzyme activity. In support of this hypothesis, we have identified several rare SARM1 variants in ALS patients, but not in controls, that have constitutive NADase activity and promote neuron death and axon loss. These variants also cause motor dysfunction and paralysis when expressed in the mouse CNS, suggesting that activating SARM1 mutations may contribute to ALS pathogenesis. Here we propose to define the function of SARM1 variants from ALS patients, controls, and the general population. These studies will allow us to categorize SARM1 variants as putatively pro-degenerative, neuroprotective, or neutral. In parallel, we will dissect the contribution of variation in components of the programmed axon destruction pathway to ALS phenotypes, alone and in combination with known ALS genetic risk-factors, in motor neurons differentiated from human induced pluripotent stem cells (iPSCs). Finally, we will investigate neurodegeneration in a mouse knock-in model carrying a Sarm1 allele equivalent to a pro- degenerative allele found in ALS patients, alone and in combination with a SOD1 model, based on a specific patient genotype that we identified. We will attempt to suppress ALS phenotypes with SARM1 inhibition via a proven gene therapy approach and with experimental small molecule inhibitors. Results of these studies will establish the relationship between the SARM1-mediated axon destruction program and ALS, and build the foundation to develop axoprotective therapeutics to treat this devastating disease.