Jeffrey Milbrandt, MD, PhD - US grants

The development of the nervous system is dominated by the central consideration that its ability to function properly depends on the accuracy of its spatial organization. The neuronal precursors must attain the correct position within the organism to insure that future axons are able to make synaptic contact with their designated targets. Several mechanisms operate to achieve this goal: the embryonic environment influences both the pattern of cellular migration and neuronal differentiation; diffusible factors and recognition molecules on cell surfaces guide axons to their appropriate targets; and signals are released from target cells that allow neuronal degeneration to occur in response to incongruous neuron-target interactions. Studies of the peripheral nervous system have been invaluable in elucidating many of these developmental processes. The discovery of the well-characterized trophic agent, nerve growth factor (NGF), is one example. NGF is a polypeptide hormone that is crucial for the survival and differentiation of sympathetic neuroblasts and mature sympathetic neurons. Recently, a cell line (PC12) that responds to NGF has been used extensively to study the mechanism of action of this hormone. Experiments with inhibitors of RNA transcription have demonstrated that many NGF-stimulated responses require alterations in the pattern of gene expression. In this proposal, experiments designed to identify, isolate, and characterize the gene products whose synthesis is induced by the actions of NGF will be outlined. (1) cDNA libraries of NGF-treated PC12 cells will be constructed and subsequently screened with probes enriched for NGF-regulated mRNA species. (2) The identity, structure, and function of these gene products will be investigated by determining the nucleotide sequence of the NGF-activated genes. Antibodies directed against the peptides encoded by the NGF-regulated cDNAs will be produced. (3) The temporal and spatial patterns of expression of these gene products in the developing embryo and adult animal will be determined. From these studies, new insight will be gained with regard to the role of NGF in neuronal development. Knowledge in this area may lead to an increased understanding of the defects manifested in familial dysautonomia hereditary sensory neuropathies, and other neurological defects in the newborn.

Nerve growth factor (NGF) is an important modulator of neuronal development and survival. To understand how NGF alters the pattern of gene expression in responsive cells, we have identified several genes that are rapidly induced by NGF in PC12 cells. Each of these three genes, c-fos, NGFI-A and NGFI-B, are rapidly transcribed in response to NGF, phorbol esters (TPA), and calcium ionophores (A23187), and the induction process does not require de novo protein synthesis. The c-fos protein is also a transcriptional activating factor, and NGFI-A and -B are homologous to transcriptional activating factors; therefore, each of them could play a role in controlling the differentiation process. To determine the sequence elements, or enhancers, that control the NGF-mediated induction of these genes, we plan to isolate the 5'flanking regions of each of these genes. The transcription start site will be established, and the nucleotide sequence will be determined from the start site upstream 1 to 2 kb. DNA fragments from each of these regions will be cloned upstream of the chloramphenicol acetyltransferase (CAT) gene, and each construct will be transfected into PC12 cells. In each instance, expression of the CAT gene will be monitored in the presence and absence of NGF to determine whether an NGF responsive element (NRE) resides in the fragment. Deletion mutants constructed from fragments that contain the NREs will be used to further delimit their location. "Trans-acting" factors that bind to the NREs and presumably promote the transcription of these genes will be detected by gel retardation and DNAse I protection experiments. To explore whether these genes are activated by a common mechanism, we will perform a comparison of the NRE sequences from each gene, and carry out competition experiments aimed at determining whether the same proteins bind to NREs located in different genes. Once the nucleotide sequence of the NREs is determined, oligonucleotide affinity chromatography will be exploited to purify these "trans-acting" factors. Elucidating the molecular mechanism by which NGF regulates gene expression will help us understand how NGF influences the survival and differentiation of neural-crest derived cell types.

The Oligonucleotide core facility will provide oligonucleotides to all participants in the program project. They will be synthesized in a timely fashion on an Applied Biosystems model 394 and distributed to the various laboratories. Quality control, rapid synthesis, and a large cost savings are the primary benefits of this core.

DESCRIPTION: The PI is investigating the function of neurturin, a novel neurotrophic factor purified from Chinese hamster ovary cell conditioned media on the basis of its ability to promote the survival of sympathetic neurons in vitro. Subsequent to obtaining partial amino acid sequence of the purified protein, a cDNA encoding neurturin was isolated. Sequence analysis revealed that it is related to a distant member of the TGF-B superfamily, glial cell line-derived neurotrophic factor (GDNF), which promotes survival of midbrain dopaminergic and motor neurons. Comparison of the neurturin and GDNF sequences has revealed several highly conserved domains which may allow us to identify additional neurotrophic factors, just as the discovery of BDNF allowed the identification of NT-3 and NT-4/5. To pursue the biological functions of neuturin, recombinant neurturin will be produced and characterized with regard to its survival promoting activities. To characterize the interaction of neurturin with its receptor, binding and crosslinking studies will be performed. The identification of the neurturin receptor will be approached by first determining whether it is a known or structurally related member of the Ser/Thr kinase receptor family, such as those which interact with other TGF-B family members. If it is not, the receptor will be isolated by ligand-affinity chromatography or, by expression cloning. The effects of neurturin on sympathetic neuron neurotransmitter phenotype will be examined by monitoring expression of genes encoding neuropeptides and enzymes involved in neurotransmitter synthesis. Chimeric mutants generated by swapping domains between neurturin and GDNF will be analyzed to identify the particular neurturin domains required for promoting sympathetic neuronal survival and, potentially, for interacting with its receptor (s). To elucidate the physiological role of neurturin, the phenotypes of transgenic mice either deficient in neurturin, or in which neurturin is ectopically expressed, will be examined. Special attention will be given to the development and function of the nervous system in these mice, both in its normal and injured state. Because of the wide range of activities noted for members of the TGF-B family, we will also perform a global analysis of these animals as neurturin may influence other cell types as well. The studies outlined in this and the accompanying proposal of the IRPG will provide a solid foundation of basic information regarding the biological role of neurturin. As neurotrophic factors are now considered as potential therapeutic agents for neurodegenerative diseases, such as Alzheimer's as well as for more acute conditions, understanding the function of neurturin could have a major impact on the future treatment of these afflictions.

Prostate cancer is a very important health problem in the United States. It will be diagnosed in approximately 350,000 American men and will cause approximately 42,000 deaths in 1998. Genetic mutations are known to cause a variety of human cancers however the molecular genetic defects leading to prostate carcinoma are still unidentified despite the detection of a variety of chromosomal abnormalities in prostate tumors. The most commonly observed abnormality is a deletion at 8p12-22, suggesting that a gene(s) important for prostate carcinogenesis is located within this chromosomal region. Evidence is rapidly accumulating that inappropriate activity of developmental regulators can result in malignant transformation. A recently discovered factor, Nkx3.1, possesses may properties expected of such a regulator: it is a homeodomain protein, a class of transcription factors critically involved in many developmental processes; it is expressed in developing and mature prostate, and its chromosomal location, 8p21, suggests it is lost in many prostate tumors. We therefore plan to investigate whether Nkx3.1 regulates prostate development and whether loss of Nkx3.1 results in differentiation of prostate cells, hence contributing to prostate carcinogenesis. The capacity of Nkx3.1 to drive prostate-specific differentiation in vitro, and its effects on growth, patterns of gene expression, and tumorigenicity of prostate carcinoma cell lines will be investigated. The Nkx3.1 in human prostate tumors will be examined using immunohistochemistry, and microdissected prostate tumor specimens will be used to search for Nkx3.1 mutations and/or evidence of hypermethylation. Specific cell types expressing Nkx3.1 in developing and mature urogenital tract will be identified using immunohistochemistry. Gene targeting using the Cre/lox recombination system will be used to generate mice that suffer sudden loss of Nkx3.1 function in adulthood. These mice will be examined for evidence of prostate hyperplasia and neoplasia, and for functional and morphological alterations. In summary, our work will determine whether loss of Nkx3.1 function is a major cause of prostate cancer.

Neuronal degeneration and death occur during development, during senescence, and as a consequence of pathological events throughout life. Neuronal survival and function are critically influenced by the actions of neurotrophic factors. We are investigating the physiologic roles of the recently identified neurotrophic factors GDNF, Artemin, Neurturin and Persephin. Together, they represent a family of proteins that are a distantly related subgroup (GDNF Family Ligands or GFLs) of the TGFbeta superfamily. GFLs utilizes a receptor complex composed of a signaling component (the Ret tyrosine kinase), and a binding component (a member of a family of recently identified GPI-linked co-receptors (GFRalpha family). Experiments using in vitro cultures have shown that GF ligands support the survival of a variety of CNS (dopaminergic midbrain, spinal cord motor) and PNS neurons (sensory, sympathetic, parasympathetic, enteric). In this proposal, we outline experiments aimed at determining the molecular interactions between GF ligands and the GFRalpha/Ret receptor complexes. The physiologic roles of these ligands and cognate GFRalpha coreceptors will be investigated by studying mice that lack one or more of these components. Through this type of analysis, neuronal populations dependent on these factors for survival as well as maintenance and function will be identified. Further analysis will examine the role of these factors in Schwann cell biology and in regulating Schwann cell-neuronal interactions during development and nerve regeneration. From these studies further understanding concerning the role of GFLs in disease processes, as well as insights into modulating GFL signaling, will become available. This is vital information because these neurotrophic factors influence neurons involved in several neurodegenerative diseases (Parkinson's and ALS), neurons affected in peripheral neuropathies of chronic diseases such as diabetes, and enteric neurons which function poorly in gut motility syndromes that accompany chronic diseases. Since both chronic as well as acute nervous system injuries are characterized by structural damage, disease-induced apoptosis, and neuronal dysfunction, neurotrophic factors, such as the GF ligands, have potential value as therapeutic agents for these conditions.

DESCRIPTION (From the Applicant's Abstract): Inherited neuropathies are among the most common human genetic diseases. These syndromes are characterized by severe motor and sensory deficits secondary to abnormal nerve myelination resulting in significant patient morbidity and mortality. The underlying genetic defects of these neuropathies occur primarily in genes encoding the myelin structural proteins MPZ, PMP-22 and connexin-32. Recently, however, mutations in the transcription factor Egr2 have also been associated with these syndromes. The connection between Egr2 and these syndromes was made after peripheral nerves in Egr2-deficient mice appeared poorly myelinated due to a Schwann cell differentiation arrest at the promyelinating stage. Together, these results strongly suggest that Egr2 is a crucial regulator of a differentiation program, which culminates in the myelinating Schwann cell phenotype. In this proposal, we outline experiments aimed at understanding the molecular mechanisms by which Egr2 regulates the myelination process. Gain-of-function experiments using adenovirus infection of Schwann cells will be utilized to perform Egr2 target gene profiling via microarray screening. Egr2 mutants associated with inherited neuropathies will be characterized in in vitro myelination assays and tested for their ability to activate expression of Egr2 target genes. In addition, one of the neuropathy-associated Egr2 mutations is located in the domain that interacts with the Nab proteins, modulators of Egr2 activity. We will therefore investigate the role of the Nab proteins in regulating myelination. The presence of mutations in the Nabl or Nab2 genes will be sought in patients with inherited neuropathy. Finally, gene targeting will be used to produce mice that harbor neuropathy-associated Egr2 mutations in order to create mouse models of these inherited neuropathies. The peripheral nervous system of these mice will be examined for deficits in Schwann cell differentiation and peripheral nerve myelination. The expression of Egr2-regulated genes will be examined in nerves of these mutant mice. These studies will provide new insight into how mutations in Egr2 lead to peripheral neuropathies, information that may lead to novel therapies for these diseases.

DESCRIPTION (provided by applicant): The GDNF family ligands (GFLs - GDNF, neurturin, artemin and persephin) are a family of neurotrophic factors of critical importance to neurodevelopment, and are potential therapeutic agents for many neurodegenerative diseases from Parkinson's disease to peripheral neuropathy. GFLs signal through a multimolecular complex comprised of RET (a receptor tyrosine kinase) and a specific ligand-binding coreceptor, GFRa1-4. The expression pattern of RET and the different GFRas is critical to mediate responsiveness to a particular GFL and proper neurodevelopment, but the genetic network underlying regulation of these receptors in specific neuronal subtypes remains unknown. GFLs can alter addictive behaviors through their influence on the ventral tegmental area, broadening their relevance to another critical population of midbrain dopaminergic neurons, and opening a new avenue in the treatment of substance addiction. GFLs and their receptors are expressed in the retina, and exogenous GDNF can slow retinal degeneration. Although the critical role GFLs play in peripheral nervous system development, and their promise as therapeutic agents for Parkinson's disease are firmly established, much work remains to be done to characterize the role of GFLs particularly in postnatal central nervous system development and maintenance, and to optimize and broaden their therapeutic potential into different avenues. With these goals in mind, we propose the following specific aims: (1) to identify the genetic network that regulates the expression of GFL receptors in different cell types using bioinformatics analysis of RET and the GFRa loci, together with targeted in vitro and in vivo confirmation, (2) to characterize the importance of RET signaling in midbrain dopaminergic neurons in adult animals, in regards to susceptibility to Parkinsonism-inducing toxins and modulation of addictive behaviors, using conditional removal of RET from dopaminergic neurons in transgenic mice, (3) to characterize the role of Neurturin-RET signaling in retinal development/maintenance using transgenic mice, and to investigate the utility of GFLs in a mouse model of retinal degeneration. Relevance: Neurodegenerative diseases are a tremendous burden to the public, with few effective treatments. Neurotrophic factors such as the GFLs have potential in relieving this burden, and the research in the proposal is aimed at furthering that potential.

Prostate cancer is a very important health problem in the United States. It will be diagnosed in approximately 350,000 American men and will cause approximately 42,000 deaths in 1998. Genetic mutations are known to cause a variety of human cancers however the molecular genetic defects leading to prostate carcinoma are still unidentified despite the detection of a variety of chromosomal abnormalities in prostate tumors. The most commonly observed abnormality is a deletion at 8p12-22, suggesting that a gene(s) important for prostate carcinogenesis is located within this chromosomal region. Evidence is rapidly accumulating that inappropriate activity of developmental regulators can result in malignant transformation. A recently discovered factor, Nkx3.1, possesses may properties expected of such a regulator: it is a homeodomain protein, a class of transcription factors critically involved in many developmental processes; it is expressed in developing and mature prostate, and its chromosomal location, 8p21, suggests it is lost in many prostate tumors. We therefore plan to investigate whether Nkx3.1 regulates prostate development and whether loss of Nkx3.1 results in differentiation of prostate cells, hence contributing to prostate carcinogenesis. The capacity of Nkx3.1 to drive prostate-specific differentiation in vitro, and its effects on growth, patterns of gene expression, and tumorigenicity of prostate carcinoma cell lines will be investigated. The Nkx3.1 in human prostate tumors will be examined using immunohistochemistry, and microdissected prostate tumor specimens will be used to search for Nkx3.1 mutations and/or evidence of hypermethylation. Specific cell types expressing Nkx3.1 in developing and mature urogenital tract will be identified using immunohistochemistry. Gene targeting using the Cre/lox recombination system will be used to generate mice that suffer sudden loss of Nkx3.1 function in adulthood. These mice will be examined for evidence of prostate hyperplasia and neoplasia, and for functional and morphological alterations. In summary, our work will determine whether loss of Nkx3.1 function is a major cause of prostate cancer.

DESCRIPTION (provided by applicant): The GDNF Family of Ligands (GFLs) signal through a receptor complex composed of the Ret tyrosine kinase and a member of the GFRalpha coreceptor family. Studies of mice deficient in various components of the GFL system have revealed that GFL-signaling is important for the development and function of a wide variety of neuronal populations as well as the urogenital system. Deficits in these mutant animals lead to the conclusion that Ret activity supports progenitor cell proliferation, cell migration and axonal projection. Furthermore, both activating and inactivating mutations in Ret result in human disease, highlighting the importance of understanding the signal transduction pathways that normally transmit GFL-mediated signals to their cellular targets. Our central hypothesis is that signals emanating from specific Ret isoforms and through individual Ret tyrosine residues specifically regulate these physiologic processes. To test this hypothesis, we plan to examine Ret signaling in mice expressing specific Ret isoforms, either wild type or those containing mutations that eliminate specific protein interactions. The phenotypes of these mice will be examined for deficits in the nervous system that highlight the contributions of specific domains and tyrosine residues to the physiologic functions of Ret. In vitro models will be used to study the effects of specific Ret mutants and/or alterations in adaptor protein activities to further define the effects of Ret signaling on migration, proliferation and axonal projection. Finally, spermatogenesis has recently been discovered to depend on Ret activity. The availability of Ret mutants that survive the neonatal period will be exploited to explore this newly discovered role of Ret. The implementation of testicular transplantation technology will allow us to examine spermatogenesis in mouse mutants that die during the neonatal period. This technique will be used to examine spermatogenesis in GDNF, GFRalpha1 and Ret-deficient animals, as well as other Ret mutants we generate that might die at birth. Through these studies, new insights into the molecular mechanisms of GFL-mediated signaling will be forthcoming.

DESCRIPTION (provided by applicant): Prostate cancer is the most common non-skin cancer in men and the second-leading cause of death from cancer in the US. As with most human cancers, prostate tumorigenesis requires the sequential accumulation of multiple genetic lesions. The multistep nature of tumorigenesis is widely accepted;however little is known about the initiation steps that lead to pre-malignant alterations. Haploinsufficiency at tumor suppressor loci presents a potential central mechanism for tumor initiation. In the prostate, tumor initiation is often linked to loss-of-heterozygosity at the NKX3.1 locus. In mice, haploid loss ofNkx3.1, which encodes a homeodomain protein, is sufficient to cause prostate epithelial hyperplasia and eventual PIN formation. In both the Myc overexpression and Pten loss-of-function mouse models of prostate cancer, Nkx3.1 expression is lost in the early stages of tumorigenesis. Our central hypothesis is that NKXS.1 serves a 'gatekeeper'function in the prostate and that its loss is a major initiating event in prostate cancer. To test this hypothesis, we plan to investigate whether overexpression of NKX3.1 in the prostate can prevent the development of prostate cancer in several mouse models. We will also attempt to identify effectors of NKX3.1 that would provide a link between it and abnormal proliferation. For this search, we will identify genes that are directly regulated by NKX3.1 through a series of analyses including expression profiling, comparative genomic analysis chromatin immunoprecipitation and reporter gene assays. Finally, we will determine whether the effects of NkxS.1 loss on prostate epithelial cell proliferation are mediated through alterations in mTOR activation. We will also attempt to prevent hyperplasia in Nkx3.1 -deficient mice using mTOR inhibitors. For these studies, we will utilize a combination of techniques, including computational techniques, lentivirus mediated overexpression and knockdown using siRNA, mutagenesis, and functional assays conducted in vitro and using transgenic mice.

[unreadable] DESCRIPTION (provided by applicant): Axonal degeneration often precedes the death of neuronal cell bodies in neurodegenerative diseases, thus inhibiting axonal degeneration represents a potential novel point of intervention in combating these diseases. Axonal degeneration is now regarded as an active, self-destructive program in which the ubiquitin proteosome pathway plays an important role. We have identified a novel E3 ubiquitin ligase, ZNRF1 that is induced in Schwann cells after nerve injury. ZNRF1 is also specifically upregulated in neurons, along with ubiquitin, after axonal damage. ZNRF1 and another related E3 ligase, ZNRF2, are both highly expressed throughout the nervous system during development and in adulthood. Using in vitro assays we have found that ZNRF protein activity is required for normal axonal degeneration. In this proposal, we have outlined experiments to define how ZNRF proteins influence axonal degeneration. First, we will analyze the phenotypes of ZNRF-deficient mice in order to determine physiological roles of these proteins. In particular, the will focus our analysis on the processes of nerve degeneration/regeneration after mechanical and metabolic injury as well as synaptogenesis. We also aim to identify proteins targeted by ZNRF-mediated ubiquitination. A proteomic analysis will be performed to identify ZNRF targets either from brain tissues of mice deficient for ZNRF proteins or from affinity-purified complex containing dominant negative ZNRF mutants and ZNRF targets. These studies will improve our understanding of axonopathy and neuronal degeneration, and provide a basis for developing novel treatments for neurodegenerative diseases [unreadable] [unreadable]

The discovery that Egr2-deficient mice have hypomyelinated peripheral nerves led to the realization that EGR2 is an essential regulator of the Schwann cell myelination program and, moreover, to the identification of EGR2 mutations in patients with inherited neuropathy. The mutant EGR2 in these patients causes myelinopathy by acting as a dominant inhibitor of the normal EGR2-mediated expression of myelin proteins. Further study demonstrated that NAB proteins, which interact with EGR2 and modulate its transcriptional activity, are also necessary for peripheral nerve myelination. Together, these observations have led us to hypothesize that EGR/NAB complexes are the prime regulators of myelination. Expression profiling experiments using cultured Schwann cells, or sciatic nerves from hypomyelinated mouse mutants and developing mice, or distal segments from transected or crushed sciatic nerves has enabled the identification of a 'myelination-associated gene cluster'. The expression of genes in this cluster is tightly correlated with that of myelin proteins, inferring that they are also involved in the myelination process and regulated by the same transcriptional regulators, which we believe include EGR/NAB complexes. Here, we have outlined experiments aimed at identifying the genetic networks that are regulated by these complexes and are functionally important in myelinating Schwann cells. Having identified candidate target genes by expression profiling, comparative genomic analysis and chromatin immunoprecipitation experiments will now be used to identify genomic loci where EGR2/NAB complexes are bound. Our expression profiling analyses also revealed a novel transcript whose expression in Schwann cells is regulated by EGR2/NAB complexes and is concordant with that of myelin proteins, and whose genomic loci is tightly linked with a CMT neuropathy locus. The function of the MP11 protein encoded by this transcript will be investigated. Finally, we will determine whether EGR2 is necessary and sufficient to mediate the axonal signals that direct Schwann cells to activate the myelination program.

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 development of new treatments for neurodegenerative diseases is a major unmet medical need, with the numbers of affected individuals (already tens of millions worldwide) projected to dramatically increase with the aging population. Axonal dysfunction appears to be an early event in many of these disorders, thus it has become a prime focus for the development of new treatments for these conditions. AxD is a self-destructive process that is similar to, but distinct from, apoptosis. Most studies indicate that it is a caspase-independent process as manipulation of the mitochondrial apoptotic machinery or caspase inhibitors fail to block AxD. However, the recent identification of an APP/DR6/Casp6 axon degeneration pathway induced by trophic factor deprivation suggests a link between these two processes. Studies of the wlds mutant mouse led to the discovery that the NAD biosynthetic enzyme Nmnat1 can protect axons from a wide variety of insults, including mechanical trauma, loss of trophic support, and mitochondrial inhibition. Nmnat enzymatic activity is required for the axonal protective effects; however, its mechanism of action remains obscure. In this proposal, we outline experiments aimed at 1) identifying the signaling pathway(s) responsible for axonal surface APP (sAPP) shedding, caspase 6 (Casp6) cleavage, and axonal degeneration (AxD) induced by trophic factor deprivation using Ret knockin Tyr->Phe mutant mice; 2) understanding the mechanism by which Nmnat protects axons after trophic factor deprivation without altering cleaved Casp6 levels via monitoring cleavage of Casp6 targets; 3) investigating the idea that different Nmnat isoforms produce NAD for specific neuronal subcellular compartments important for axonal integrity or that they have additional functions besides NAD production, such as the synthesis of novel metabolites that are important mediators of axonal protection; and 4) testing the therapeutic efficacy of combined neurotrophic factor and axonal protectant treatment in animal models of Parkinson's disease. If the proposed combination therapy shows dramatically increased benefit over single agent therapy in the PD models, this could have a wide-ranging impact as it is likely to be applicable to other neurodegenerative diseases. Overall, we believe that understanding the convergence of growth factor signaling and the common Nmnat-sensitive pathway of axon self-destruction could foster new therapeutic strategies for a wide range of disease and injury conditions.

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. PUBLIC HEALTH RELEVANCE: This research is relevant to public health because it will identify potential therapeutic targets for a host of debilitating neurological disorders. Axonal degeneration is a shared component of many neurological disorders including neurodegenerative diseases like Alzheimer's, Parkinson's and Multiple Sclerosis, and it is also an important aspect of hereditary, chemotherapy-induced and diabetic neuropathies, stroke, and glaucoma. Defining the molecular mechanisms of the axonal self-destructive pathway will identify novel therapeutic candidates for these disorders where morbidity is largely caused by axonal dysfunction.

DESCRIPTION (provided by applicant): Schwann cell transplantation holds great promise for the treatment of spinal cord injuries and some neuropathies. In addition, Schwann cell functions are coming under wider scrutiny due to their potential importance in hematopoiesis. A major bottleneck hindering the progress of Schwann cell-based therapy and Schwann cell functional genomics is the lack of methods to produce large numbers of transplantable cells and the easy perturbation of their genetic network. Recently, it has become possible to reprogram fibroblasts into different cell types by expressing a small number of transcription factors. However, the efficiencies are typically low, and only a few cell types (e.g. neurons, cardiomyocytes, oligodendrocytes) have been produced to date. We propose to overcome these difficulties by creating artificial transcription factors (ATFs) based on the Cas9 protein. Cas9 can be directed to bind specific genomic sequences using guide RNAs, so it will possible to specifically activate hundreds or even thousands of genes. We will use Cas9 ATFs to reprogram fibroblasts into neurons and Schwann cells by activating transcription factors that are specific to these cell types. We anticipate that this approach will substantially improve the efficiencies of existing transdifferentiation protocols (for conversion into neurons), as well as enable transdifferentiatio to previously unobtainable cell types (Schwann cells). Our preliminary experiments suggest our strategy is feasible. We have demonstrated that Cas9 ATFs can achieve potent gene activation (>100 fold), and we have developed computational methods to predict the sets of genes required for transdifferentiation. Our specific aims are as follows: 1) To determine the rules that govern gene activation by Cas9-based artificial transcription factors (ATFs). 2) To develop tunable Cas9 mutant proteins bearing transcriptional activation or repression domains wherein their activity can be controlled by addition of small molecules to enable regulable perturbation of large-scale genetic networks. 3) To transdifferentiate fibroblasts into Schwann cells or their precursors by simultaneously activating the expression of 75-100 transcription factors that are differentially expressed between these two cell types.

DESCRIPTION (provided by applicant): Acquired peripheral neuropathy commonly results from diabetes, chemotherapy and HIV/AIDS, and therefore imposes a considerable, and increasing, health care burden on society. Effective treatment of neuropathy requires an understanding of the interactions between glia and axons in the peripheral nerve. Myelinating Schwann cells have been studied extensively, but less is known about non-myelinating Schwann cells. Because they are associated with unmyelinated sensory fibers that transmit pain, they are likely to participate in the pathology associated with diabetic and other small-fibe neuropathies. Furthermore, Schwann cell-specific mutations in genes that are important for cellular metabolism strongly affect unmyelinated axon stability, revealing an important but little explored link between these cells and peripheral nerve disease. Therefore an understanding of the biology of non-myelinating Schwann cells is an important approach toward developing new treatments for peripheral nerve diseases. We have developed several independent approaches for obtaining gene expression profiles of non-myelinating Schwann cells in order to identify new markers for these cells, and to identify mechanisms whereby their functional impairment in disease, particularly metabolic disease, affects peripheral nerve function. In addition, we have generated transgenic mice that express two proteins, an eGFP-tagged ribosomal protein, and tamoxifen-inducible Cre recombinase, in non-myelinating Schwann cells. The tagged ribosomal protein will permit the isolation and profiling of polyribosome-associated mRNAs in non-myelinating Schwann cells by translating ribosome affinity purification, or TRAP, a powerful method for determining gene expression profiles of specific cell types. The Cre recombinase will permit us to specifically manipulate gene expression in non-myelinating Schwann cells, or ablate them entirely. Such experiments are necessary to dissect the complex interactions between non-myelinating Schwann cells and their associated axons. These new transgenic mouse lines will permit us to establish gene expression signatures of non-myelinating Schwann cells after genetic or environmental perturbation and in models of inherited or acquired PNS disease. In particular we will use these methods to investigate the molecular aspects of how metabolic deficits lead to unmyelinated axon loss and neuropathy. Through the identification and manipulation of genes altered in non-myelinating Schwann cells in health and disease, we hope to gain new insights into the underlying pathology and open up new avenues for treatment of peripheral neuropathies.

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.

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.

Diabetic peripheral neuropathy is an increasingly common disorder that affects up to 25% of diabetic patients. The pathological underpinning of diabetic neuropathy is the loss of axonal integrity and function. In addition to axonal loss, impaired nerve fiber regeneration after injury is commonplace in diabetics and is recapitulated in rodent diabetic models. Axonal injury triggers a dramatic reprogramming of the Schwann cells (SCs) surrounding the damaged axon that culminates in the adoption of a `repair SC' phenotype. The repair SC promotes axon/myelin breakdown and disposal, attracts macrophages, produces neurotrophic factors, and elaborates adhesive molecules. Upon contact with the regenerating axon, it transforms back into a differentiated SC to ensure remyelination or Remak bundle formation. The primary regulator of this repair SC transition is the transcription factor c-Jun, which is rapidly activated after injury in SCs surrounding damaged axons. In Jun-deficient mice, nerve regeneration is impaired. In keeping with the impaired axon regeneration in diabetes, we find that mice with mutations that alter SC metabolism fail to effectively promote nerve regeneration. Most recently, we characterized OGT-SCKO mice that lack SC expression of O-GlcNAc transferase (OGT), the enzyme that catalyzes addition of O-GlcNAc moieties to proteins at Ser and Thr residues. OGT activity is regulated by the flux of glucose through the hexosamine biosynthetic pathway, thus it serves as a sensor that aggregates information regarding glucose metabolism and transmits it into changes in cell physiology. Notably, abnormal O-GlcNAcylation has been implicated in diabetes, cancer, and neurodegenerative diseases. Mice lacking O-GlcNAcylation in SCs develop a tomaculous demyelinating neuropathy. Expression profiling of OGT-SCKO sciatic nerve revealed high expression of many AP-1 targets. Moreover, we find that JUN phosphorylation and transcriptional activity are regulated by O-GlcNAcylation. In keeping with abnormalities in JUN activity, we find that loss of OGT leads to a substantial decrease in regeneration/remyelination, indicating that the SC injury response is modulated by metabolism. These results lead us to hypothesize that poor nerve regeneration in diabetes, and potentially the neuropathy itself, is caused by the impact of abnormal metabolism on the generation, function, and/or cessation of the SC injury response. To pursue this hypothesis we propose three aims: 1) To investigate how metabolism impacts the SC injury response; 2) To investigate the role of O-GlcNAcylation in regulating JUN activity; and 3) To determine the role of AP-1 partners and other regulators in the SC injury response. Through these studies, we hope to show that therapies targeting Schwann cells and their repair functions will be useful in treating neuropathy and traumatic nerve injury.

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.

Project Summary/Abstract: Alzheimer's disease is the most common cause of dementia. Axon degeneration is an important contributor to the functional deficits of Alzheimer's disease (AD) and is observed early in the disease process. Metabolic abnormalities also contribute to AD, a link made apparent by the increased risk of AD in patients with type II diabetes. Metabolism is also a critical regulator of axonal health. We have found that neuronal metabolism, particularly NAD metabolism, plays an important role in maintaining axon integrity and function. Injured axons degenerate via activation of a self-destructive program that is controlled, in part, by the TIR adaptor protein SARM1. Upon injury, the NAD biosynthetic enzyme NMNAT2 is rapidly lost from the axon. At the same time, SARM1 is activated and stimulates a pathway that leads to the rapid degradation of axonal NAD. This disruption in axonal NAD homeostasis, along with calcium and kinase signaling cascades, culminates in the degeneration of the damaged axon. Conversely, overexpression of enzymes in the NAD biosynthetic pathway counteract this program and prevent damaged axons from degenerating. To understand how NAD homeostasis is controlled, we have developed assays to measure a wide range of NAD metabolites and to follow the synthesis and consumption of these molecules in healthy and damaged axons. The hallmark of tauopathy-related neurodegenerative disease like AD is the hyperphosphorylation and aggregation of tau. Abnormal tau conformers act like prion-like seed molecules that can propagate from cell-to- cell. These prion-like tau oligomers are thought to represent the predominant axonal insult in Alzheimer's disease. Interestingly, tau is also post-translationally modified by acetylation and O-GlcNAcylation, and these additions contribute to tau solubility and pathological potential. These modifications are regulated by cellular metabolism, thus providing a direct linkage between metabolism and neurodegeneration. These results lead us to hypothesize that changes in NAD metabolism contribute to AD progression through impaired axon stability due to alterations in tau modification that lead to increased formation of tau prion-like oligomers. To pursue this hypothesis we propose three aims: 1) To determine how alterations in NAD homeostasis lead to axon degeneration; 2) To identify the enzymology responsible for rapid NAD degradation in degenerating axons; and, 3) To establish the molecular association between NAD homeostasis and AD-related tau pathophysiology. Through these studies, we hope to show that therapies targeting NAD metabolism will be useful in treating neurodegenerative diseases like Alzheimer's disease.

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: Peripheral neuropathy is an increasingly common disorder that affects up to 25% of diabetics and 35-40% of elderly individuals. The pathological underpinning of peripheral neuropathy from any etiology is the loss of axonal integrity and function. In diabetics and the aged, the axonal loss is accompanied by impaired nerve fiber regeneration after injury; a state that is recapitulated in rodent models. Axonal injury triggers a dramatic reprogramming of the Schwann cells (SCs) surrounding the damaged axon that culminates in the adoption of a `repair SC' phenotype. The repair SC promotes axon/myelin breakdown and disposal, attracts macrophages, and produces neurotrophic factors. Upon contact with the regenerating axon, it transforms back into a differentiated SC to ensure remyelination or Remak bundle formation. The primary regulator of this transition to the repair SC is the transcription factor JUN, which is rapidly activated after injury in SCs surrounding damaged axons. In Jun-deficient mice, nerve regeneration is impaired. In keeping with the impaired axon regeneration in diabetic and aged animals, we find that mice with mutations that alter SC metabolism fail to effectively promote nerve regeneration. Most recently, we characterized OGT-SCKO mice that lack SC expression of O-GlcNAc transferase (OGT), the enzyme that catalyzes addition of O-GlcNAc moieties to proteins at Ser and Thr residues. OGT activity is regulated by the flux of glucose through the hexosamine biosynthetic pathway, thus it serves as a sensor that aggregates information regarding glucose metabolism and transmits it into changes in cell physiology. Notably, abnormal O-GlcNAcylation has been implicated in diabetes, cancer, and neurodegenerative diseases. Mice lacking O- GlcNAcylation in SCs develop a tomaculous demyelinating neuropathy. Expression profiling of OGT-SCKO sciatic nerve revealed high expression of many AP-1 targets. Moreover, we find that JUN phosphorylation and transcriptional activity are regulated by O-GlcNAcylation. In keeping with abnormalities in JUN activity, we find that loss of OGT leads to a substantial decrease in regeneration/remyelination, indicating that the SC injury response is modulated by metabolism. These results lead us to hypothesize that poor nerve regeneration, and potentially the neuropathy itself, in diabetic and aged individuals is caused by the impact of abnormal metabolism on the generation, function, and/or cessation of the SC injury response via direct effects on JUN activity. To pursue this hypothesis we propose three aims: 1) To investigate how metabolism impacts the SC injury response; 2) To investigate the role of O-GlcNAcylation in regulating JUN activity; and 3) To determine the role of AP-1 partners and other regulators in the SC injury response. Through these studies, we hope to show that therapies targeting Schwann cells and their repair functions will be useful in treating peripheral neuropathy and traumatic nerve injury.

Project Summary Genome engineering and patient-derived pluripotent stem cell technology have dramatically changed our abilities to understand disease pathways. These two technologies are recent additions to our investigative armamentarium but have rapidly permeated all aspects of biomedical science, providing unprecedented power to discover how perturbations of components in specific pathways lead to disease. The Genome Engineering Core C is composed of 11 investigators and all of the necessary equipment, reagents and expertise required to perform the experiments needed to support the research goals of the 3 projects outlined in this PO1 renewal application entitled ?New therapies for liver fibrosis and hyperproliferation in alpha1-AT deficiency (ATD)?. Our facility is housed within the Washington University Genome Engineering and IPSC Center (GEiC) and was established and is overseen by the Department of Genetics. It was created to facilitate the implementation of these powerful new technologies in laboratories at Washington University. All projects proposed within this PO1 renewal application plan to extensively utilize the services provided by the Core. These experiments are largely aimed at the ongoing evaluation of modifying variants in ATD and their potential exploitation for development of new treatments for this disorder. The services to be utilized include the design, construction and validation of genome editing reagents (e.g. gRNAs, donor plasmids, and Cas9 derivatives). The Core also produces modified cell lines using genome editing technologies, including gene knockout, variant introduction, epitope tagging or gene replacement. These cell lines will be used to better understand the biology of ATD and to assess the impact of variants selected for their potential to modify disease progression and facilitate drug development. The Core will also design and generate materials needed for rapid production of animal models harboring selected variants. The Core produces iPSCs from both skin biopsies and the renal tubular epithelial cells present in urine samples. Genome engineering of iPSCs is now routinely performed in the Core to introduce new variants and to convert disease-associated mutant alleles back to wildtype or use as controls. Skin biopsies (fibroblasts) or urine (renal tubular epithelia) will be procured from ATD patients or controls and the cells will be reprogrammed to produce iPSC lines. Genetically modified patient-derived iPSCs will be generated using genome editing techniques. In addition, the personnel in the Core provide assistance for investigators in performing genome engineering, particularly in the development of new techniques and reagents, and for the maintenance and differentiation of iPSC lines.

Research Project, Project Summary Intellectual and developmental disabilities (IDDs) exact a heavy emotional and financial toll on society, affecting an estimated 1 in 6 children in the US. Developing effective therapeutic interventions to treat IDD is a challenging problem because a large number of environmental and genetic risk factors contribute to these diseases. Indeed, IDD-associated genetic variants have been identified in more than 700 genes, but each variant is present in only a small number of patients, and our understanding of how these variants contribute to the disease is limited. This high degree of genetic heterogeneity and lack of mechanistic insights confound efforts to develop effective therapies to treat IDD. If individual mutations can be grouped by shared molecular pathways, then targeting these pathways may be efficacious in large subsets of patients. The overall goal of our proposal is to develop CRANIUM, a platform that will read out the genomic, transcriptional, and neuronal phenotypic signatures of IDD genes to reveal common pathways disrupted by IDD-associated mutations.

PROJECT SUMMARY This supplement will continue to fund our Center for Common Disease Genomics (UM1HG008853) at the McDonnell Genome Institute at Washington University entitled, ?A platform for large-scale discovery in common disease.? In the supplemental period we will continue our multi-ethnic case-control whole genome sequencing (WGS) study focused on mapping novel disease genes and variants underlying risk and protection from early- onset coronary artery disease (EOCAD). Along with EOCAD cases, we aim to select deeply phenotyped controls whenever possible in order to study the genetic basis of quantitative cardiometabolic risk factors. After completing the WGS, we will assemble a joint callset that includes all EOCAD cases and controls from our center to enable association testing between genotypes and disease outcomes. Genotypes for the primary analysis will include testing common (individual) and rare (burden) single nucleotide variants (SNVs), insertion/deletion variants (indels), and structural variants (SVs) across coding and non-coding space. In secondary analyses, we will test for association with quantitative cardiometabolic risk factor traits and will leverage differential patterns of admixture to map causal variants underlying previously mapped disease and trait associated loci. Beyond disease association studies, we will continue to collaborate with consortium members to create genomic resources that will be used by the scientific community such as aggregated site frequencies, imputation resources, and open source analysis methods.

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.