Samuel L. Pfaff - US grants

DESCRIPTION (provided by applicant): This grant is directed at understanding the molecular mechanisms that control the development and connectivity of spinal neurons involved in the control of locomotion. Motor neurons represent one of the key cell types that directly mediate the control of movement and respiration by forming synaptic connections with muscles. Therefore diseases of these cells such as ALS, post polio syndrome, and spinal muscle atrophy are extremely debilitating and frequently lethal. Although tremendous progress has been made in identifying the factors that trigger motor neuron development, regulate gene expression, and mediate synapse formation, our understanding of the molecular mechanisms by which some of these factors act is in many cases rather limited. During development motor neuron subtypes are generated that exhibit distinct cell migration patterns and whose axons display specific preferences for particular nerve pathways. A family of LIM homeodomain (LIM-HD) transcription factors is expressed in unique combinations within individual motor neuron subtypes (LIM code). Moreover, functional experiments have now demonstrated that the combinatorial activity of LIM-HD factors contribute to the specification of distinct types of motor neurons as monitored by examining motor neuron cell migration, axon navigation, and gene profiles. Therefore LIM-HD factors are intimately involved in establishing motor neuron identity and connectivity. This proposal addresses two main deficiencies in our understanding of the LIM-HD factors: how they actually regulate gene expression, and the identity and function of the target genes under their control. In the first aim we plan to address the biochemical basis for the LIM code and to examine how LIM-HD factors act in the context of other regulatory factors such as those that control neurogenesis. In the second aim we will test the function of LIM-only factors to determine whether they negatively-regulate the activity of the LIMHD factors. In the third aim we plan to characterize the molecular basis for NLI's involvement with the LIM-HD factors. And in aim four we will screen for target genes of the LIM-HD factors and characterize their function. Over the long term these studies should contribute to our general understanding of the molecular mechanisms that control neuronal differentiation and provide insight into novel methods for restoring motor function lost due to injury or disease

This research will be done in Mexico as an extension of NIH Grant NS37116. Hindbrain reticulospinal neurons are involved in the modulation of spinal visceral and somatic motor responses such as maintenance of posture and the control of cardiovascular and respiratory functions. Two large groups of reticulospinal neurons have been described in the mammalian brainstem, one in the pons and the other in the medulla. These groups are dissimilar in the way they project into the spinal cord (ipsilaterally in the ventral funiculus and bilaterally in the lateral funiculus, respectively) and in their termination sites (pontine fibers terminating more ventrally in the spinal cord than do the medullary ones). The molecular processes underlying the development of these and other morphologically and physiologically distinguishable reticulospinal neuron subtypes, however, remains to be elucidated. The specific aims of this proposal address two major questions. First, can the different subtypes of reticulospinal neurons be defined on the basis of the transcription factors and neurotransmitters expressed? Second, do these factors functionally predict identity and axonal projection pathway? To answer these questions, fluorescent retrograde labeling will first be used in combination with in situ hybridization and immunostaining to determine the expression of LIM homeodomain and other transcription factors in the subtypes of reticulospinal neurons. A similar strategy will be employed to define their neurotransmitter phenotype. Second, a dominant-negative form of the nuclear LIM interactor will be employed to block LIM homeodomain function and thus to investigate the role these factors play in reticulospinal development. Third, ectopic expression of the transcription factors under study will be attained by in ovo electroporation in chick hindbrain to assess its effect in reticulospinal axon projection. Obtaining the answers to these questions will lead to a better understanding of the molecular determinants of reticulospinal neuron identity and axonal pathfinding. This will, in turn, facilitate the study of the establishment of complex motor circuits. From these efforts, procedures might emerge to restore motor control functions lost by injury or disease.

DESCRIPTION (provided by applicant): Spinal cord regeneration requires injured neurons to survive, re-grow axons, and reconnect with appropriate targets. Since many of these events are related to what must occur during spinal cord development, it may be helpful to use developmental strategies in order to restore locomotor function following spinal cord injury (SCI). Numerous studies have focused on anti-inflammatory steroids and glutamate antagonists, to minimize neuronal death, and inhibitory factors such as myelin-associated glycoprotein (MAG) and Nogo-A, to overcome the inhibition of axon growth; but less research has addressed the problem of reestablishing functional and appropriate synaptic connections. In addition to the obvious need to reestablish appropriate connections to mediate coordinated locomotion, it is important not to promote random growth and connections because maladaptive function could arise resulting in neuropathic pain, for example. Developmental studies have implicated cellular depolarization (activity) as an important process in establishing appropriate locomotor function during fetal development. The formation of spinal locomotor networks, initial synaptogenesis, refinement of connectivity, cell survival, as well as maturation of muscle targets are all thought to depend, at least in part, on activity. Therefore, the overall aim of this pilot project is to define mechanistically how cellular activity affects SCI recovery. The two major goals of this proposal are: (L) to develop a novel genetically-modified mouse line in which the activity of motor neurons can be switched ON and OFF, and (2.) to use genetic and pharmacological approaches in mice recovering from mild contusive SCI to precisely define the role of activity in reestablishing, locomotor function. These studies should define how activity influences SCI recovery, and may provide insight into desirable cellular and molecular targets for pharmacalogical agents that promote the reformation of functional locomotor circuitry following SCI.

Project 2: Eph Signaling in Embryonic Motor Neurons Several recent studies have shown that ephrin-A - EphA "forward" and"reverse" signaling are involved in controlling motor neuron axon navigation during embryonic development. This grant is directed at understanding the mechanisms that control how and where ephrin-A - EphA signaling occurs, in order to understand how spinal locomotor circuitry is formed. Cellular and developmental studies of motor neurons have revealed the inductive interactions that trigger their differentiation, the cellular interactions that control their axonal projections, the trophic interactions that support their survival, and the post synaptic interactions that lead to maturation of their synapses. The signaling pathways that actually guide motor neuron axons to their appropriate targets, however, remain poorly defined. During vertebrate development motor neuron subtypes are generated that exhibit distinct cell migration patterns and specific preferences for axon pathways. In this grant we propose to examine how a well defined family of axon guidance molecules, the EphAs-ephrinAs, are used in sophisticated temporal and spatial ways to control the axonal navigation of multiple classes of motor neurons. Genetic studies indicate that EphA4 is used at multiple choice points for motor neuron pathfinding. This appears to be based on a precise spatial localization of EphA4 protein along the proximo-distal axis of specific motor neuron subtypes. In Aim 1 we will investigate whether this localization is mediated by RNAtransport and selective translation, protein degradation, and/or temporal regulation of transcription. In Aim 2 we will examine the mechanisms that control EphA4 expression in motor neurons by investigating a possible connection between the Lim1 (Lhx1) LIM-HD transcription factor and neuronal activity. In Aim 3 we examine how EphA proteins can function as ligands to reverse signal through ephrin-As expressed by motor neurons, focusing on p75NTR as a possible coreceptor. More generally, these studies should provide a better understanding of how a limited number of guidance molecules can be used in diverse ways to wire the CMS.These findings should help to develop innovative methods for restoring motor function lost due to injury or disease.

DESCRIPTION (provided by applicant): This grant is directed at understanding the molecular mechanisms that control the development and connectivity of motor neurons. Since these cells are needed to control movement and respiration, diseases of motor neurons (e.g. ALS and SMA) are extremely costly and frequently lethal due to the lack of any treatment. The two main goals of this application are to characterize the function and biochemistry of motor neuron transcription factors and to identify the genetic pathways involved in their proper development. Our past studies have shown that LIM-HD factors function in a combinatorial manner to specify individual motor neuron subtypes (LIM code). In this grant we will examine how LIM-HD factors acquire cell type specific activities through functional and genetic interactions with other transcription factors. We will test whether LIM-HD factors have temporally regulated functions that direct the sequential refinement of motor neuron identity and function. Finally, we will use "forward" mouse genetic screens to identify and characterize new genes involved in motor neuron development. The experiments in this grant rely extensively on mouse genetics using transgenic and knockout methods, biochemistry and transcription assays, explant assays and imaging, and ENU-based mutagenesis screens. In aim one we will examine the function of LIM-HD factors Isl1 and Isl2, LMO factor LMO4, and Tbx factor Tbx20 using mouse knockout mutations to define functional interactions between LIM-HD factors and other classes of transcription factors expressed by motor neurons. In aim two we will use biochemical assays to investigate how gene regulation is controlled during motor neuron differentiation. In aim three we will examine whether motor neuron transcription factors Isl1, LMO4, Hb9, and Tbx20 are required for the survival and proper function of post natal motor neurons, since this could shed new light on motor neuron disease pathways. In aim four we will characterize new genetic pathways involved in motor neuron development by characterizing genes identified through an END mutagenesis screen. Our studies should provide novel information about the molecular pathways that operate to control motor neuron specification, axon navigation, circuit formation, and survival in adults.

DESCRIPTION (provided by applicant): This grant proposes to study how embryonic motor neuron axons are guided to their appropriate muscle targets. These cells directly mediate the nervous system's control of respiration and movement. Thus, diseases of motor neurons such as ALS and SMA have devastating consequences for human health and care. Our studies should provide insight into how these cells develop and over the long term contribute to our general understanding of the mechanisms that control neuronal connectivity and circuit formation in the brain. During development, motor neuron subtypes are generated that exhibit distinct cell migration patterns and specific preferences for axon pathways. Although families of transcription factors have been identified in motor neuron subtypes, less is known about the molecular signals that control the connectivity of individual motor neuron subtypes in mammals. Our preliminary studies have implicated a family of receptor tyrosine kinases in axonal navigation, which others have found are proto-oncogenes in non-neuronal tissues. In aim 1 we will characterize how EphA and ephrin-A signaling is used to guide both MMCm and LMCI cells. These studies will help to understand how axon guidance molecules expand their repertoire of functions. In aim 2 we will examine inter-axonal interactions between motor and sensory neurons to understand how proper afferent and efferent pathways develop, focusing on the role of EphAs and ephrin-As present on motor and sensory neuron axons. In aim 3 we will characterize the role of FgfR1 in MMCm motor neuron axon guidance and study FgfR1-EphA4 receptor "cross-talk". This will help to understand how attractive and repulsive guidance cues are integrated by MMCm growth cones. In aim 4 we will identify coreceptors needed for reverse signaling by the ephrin-A GPI-anchored proteins and determine whether other GPI-anchored proteins function as motor axon guidance molecules.

[unreadable] DESCRIPTION (provided by applicant): There is a tremendous need to continue to understand the basic principles of nervous system development more completely so that novel cellular and molecular targets for treating diseases can be identified in order to fuel the pipeline toward translational medicine. The goal of this meeting is to provide a forum to discuss new research that will strengthen the foundation of our knowledge of the basic mechanisms that underlie the development of the nervous system. We aim to achieve this goal by holding an intimate meeting that is organized around scientific presentations and interactive discussions. The Gordon Conference in Neural Development has become a key meeting in the larger field of neuroscience and has been held biannually for over 25 years. It attracts superb speakers, excellent students and postdoctoral fellows, and a wide range of scientists that span different stages in their careers coming from national and international labs. This popular meeting has a long history of being fully subscribed and is organized to promote maximal interactions among the participants. There will be 7 themes covered in the 2008 meeting: genetic control of circuit formation, gene regulation and CNS development, neural progenitors and glia, neuron morphology and [unreadable] growth, stem cells and cell fate, CNS disorders and development, and cell-cell communication/signaling. The talks in each section will share a focus on using multidisciplinary approaches that frequently incorporates the use of molecular, cellular, electrophysiologial, and behavioral techniques in a variety of model systems. This meeting is organized to provide a cost efficient mechanism for exchanging information and discussion of new strategies for approaching research questions. Well run research meetings are a proven mechanism for enhancing science. Support of the 2008 Gordon Conference in Neural Development is relevant to public health in two major [unreadable] ways: 1. findings from studies of nervous system development are likely to provide novel targets for treating diseases and injuries to the CNS, and 2. many CNS disorders arise from developmental defects and discussions among scientists with different backgrounds in developmental neuroscience are likely to generate novel ideas for studying and treating these disorders. [unreadable] [unreadable] [unreadable]

Charcot Marie Tooth 2D (CMT2D) is caused by dominant mutations in the glycine RNA synthetase gene (GlyRS). Despite the ubiquitous requirement for this cytoplasmic enzyme in generating glycine-tRNA for translation, mutations cause selective peripheral neuron axon degeneration leading to motor and sensory deficits. Although CMT2D was initially thought to be caused by inadequate GlyRS enzyme, previous genetic studies indicate that CMT2D is caused by a neomorphic function of GlyRS (i.e. gain-of-function). The mechanism of action by which mutant GlyRS causes CMT2D is not known and therefore is the focus of this grant. Preliminary in vitro and biochemical data from our collaborator Xiang-Lei Yang's lab has unmasked a heretofore unknown biological pathway in which GlyRS is secreted and binds competitively with VEGF to the Nrp1 receptor. Since motor neurons express Nrp1, and VEGF isoform 164 has neurotrophin-activity, they hypothesize that the novel extracellular interaction between mutant GlyRS and Nrp1 underpins CMT2D. The goal in this exploratory R21 grant is to establish whether key aspects of their hypothesis are correct in order to determine if VEGF can be used as a therapeutic agent for CMT2D disease. The four aims are (1) to establish whether GlyRS (normal and mutant protein) is secreted in mice, (2) to characterize the motor deficits in CMT2D mice and determine if GlyRS genetically interacts with VEGF and Nrp1 in vivo, (3) to assay the influence of GlyRS on Nrp1 signaling, and (4) to express VEGF in CMT2D mouse muscles using AAV-VEGF vectors and assay whether the progression of peripheral neuropathy (i.e. axon loss) is slowed or prevented. These studies may lead to a paradigm shift in our understanding of normal GlyRS function as an extracellular ligand in a broad array of biological contexts, but the main goal here is to determine whether mutant GlyRS has acquired a neomorphic activity as a VEGF antagonist of Nrp1 signaling in motor neurons. This grant aims to establish sufficient preliminary data to justify further investigation of VEGF as a feasible and novel treatment for CMT2D.

DESCRIPTION (provided by applicant): The main goal of this proposal is to generate new bioinformatics tools and mouse transgenic reagents for monitoring astrocyte gene expression in the striatum and amygdala. These experiments are designed to: (1) reveal how transplanted human astrocytes react to engraftment, (2) reveal how transplanted astrocytes influence their surroundings in mouse Rett models (MeCP2 mutant) and anxiety models (SERT mutant), and (3) to create novel transgenic animals for characterization of astrocyte heterogeneity and function. The existing tools and methods to address these questions are inadequate, in part because the dynamic range and sensitivity of microarrays is limited, FAC-sorting and immunopanning risk causing cellular changes during the purification, and laser capture requires massive RNA amplification. Two Aims will develop new methods and reagents to address these limitations. In Aim 1 a bioinformatics pipeline will be optimized to identify the origin of transcipts from mouse and human cells. Next, human astrocyte progenitors derived from H9 ES cells or Qthera human fetal glial-restricted precursors (GRPs, Q cells) will be stereotaxically transplanted into the striatum and amygdala of mice and allowed to mature. High content long-read RNA-sequencing and bioinformatics will be used to deconvolute the origin of transcripts from the human-into-mouse transplants. This species-specific gene profiling data should provide new information on how human astrocytes precursors behave in the striatum versus the amygdala. At the same time the response of the striatum and amygdala to the transplanted cells will be monitored. Finally, how astrocytes respond-to and influence the striatum and amygdala of MeCP2 and SERT mutant mice will be examined, using RNA-sequencing. Transplantation of astrocyte precursors is being considered for many clinical trials to treat mental illness, disease and CNS injury. These bioinformatics methods and new data sets should be useful for further characterization of astrocyte function. In Aim 2 novel mouse lines will be generated to enable the selective purification of RNAs from astrocyte-subtypes and lineally related neurons present in heterogeneous brain tissue. Transgenic mice will be created that express the ribosomal affinity tag Rpl22 and the fluorescent calcium reporter GCaMP3 under the control of the astrocyte promoter GFAP or the neuron promoter Synapsin. These reporters will be restricted to glial and neuronal subtypes using an intersectional approach that requires Cre-recombination. These transgenics will be characterized using crosses to progenitor-specific and inducible Cre lines to activate the reporters in subsets of glia. RNAs bound to Rpl22 will be isolated and sequenced to determine whether glia and neurons originating from the same progenitor cells share molecular-genetic features. These new mouse lines should have broad value to the neuroscience community and help to identify molecular features that either bind or distinguish subsets of astroctyes and neurons arising from shared progenitors.

Summary: Project 3 ? Cell Phenotyping: Intrinsic Physiology and Genetic Characteristics The identification of developmental pathways and neuronal subtype markers has made circuit studies of the spinal cord one of the most tractable CNS systems to investigate how neural networks control behaviorally relevant activity. Although a general framework now exists for labeling cardinal interneuron and motor neuron populations within the ventral spinal cord using Cre-mouse lines, it is apparent that each cardinal spinal neuron population is in fact a complex mixture of many heterogeneous cell types when viewed from the perspective of inputs, outputs, firing properties, and molecular-genetic attributes. Despite clear evidence for this heterogeneity, the relationship between each of these cellular properties is very fragmentary. The goal of Project 3 is to interrelate how cell lineage (cardinal neuron identity), connectivity to motor pools, intrinsic firing properties, and molecular genetics define cell types to provide a true definition of cell identity. This interconnected framework of cell features is critical because it will allow modeling to predict how spinal circuitry modulates the control of movement, and it will serve as the basis for genetic experiments that perturb neuronal function in order to test predictions of the model. This U19 Spinal Cord Circuit Team hypothesizes that the heterogeneity among premotor interneurons will scale with the complexity of motor functions mediated by different motor pools. If this hypothesis is correct, muscle groups controlling the wrist will be controlled by a more diverse population of premotor interneurons than the subset controlling the elbow because the degrees of freedom in movement differ between these two joints. There are two main approaches that will be employed to define interneuron heterogeneity: patch clamp electrophysiology in order to define input/output relationships, and single cell sequencing transcriptomics (scRNAseq) to define molecular heterogeneity. These methods will be anchored to connectivity and lineage by recording and sequencing cells that have been Cre-tagged to mark their lineage of origin (i.e. V1, V2a, V2b, V3) and retrograde trans-synaptically labeled with rabies to identify motor pool connectivity. How will the characterization of cell type-specific intrinsic firing patterns and transcriptome be applied to the broader understanding of neuroscience and limb movements in particular? First, each cardinal interneuron group will be divided into many additional subtypes based on their unique combinatorial patterns of gene expression. However, the goal is not to attempt to fractionate the cardinal interneuron groups into as many subpopulations as possible, rather it is to establish a set of molecular landmarks that can be used to reliably identify and genetically perturb subsets of interneurons with known firing patterns and connectivity. It is only with information about cell numbers, connectivity, synaptic strength, firing properties, and ?surgical? molecular tools to perturb neuronal subtype activity can models of cervical spinal circuitry be created and functionally tested to understand how forelimb movements are regulated.

Abstract: The CNS performs extremely complex computations with remarkable efficiency. This is exemplified by the ability to seamlessly execute motor behaviors that necessitate the coordination of multiple muscle groups controlling joints with many degrees of freedom. It is thought that one strategy to simplify motor computations is to adopt a circuit organization that links combinations of motor pools into functional units called ?synergies? or ?primitives?. Thus, the circuit elements that underlie motor synergies are thought to represent the basic building blocks for orchestrating the neural control of routine motor behaviors. Elegant stimulation and recording experiments from labs working with amphibians, rodents, and primates have found evidence for motor synergy circuits within the spinal cord. The major questions addressed in this grant are: (a) what is the underlying cellular and connectivity organization of lumbar spinal motor synergy circuits, (b) what neuronal subtypes comprise these circuits, and (c) what intrinsic and extrinsic factors shape the formation of these circuits? The laboratory has used trans-synaptic neuronal tracing, optogenetics, and molecular screens to identify a heterogenous (Satb1+, Satb2+, Tcfap2b+, Tcf4+) population of interconnected excitatory and inhibitory pre- motor interneurons within lamina V of the lumbar spinal cord. Based on their properties these lamina V cells are generically referred to as motor synergy encoders (MSE). The hypothesize is that the MSE cell network comprises a major computational node for motor control within the spinal cord. These cells receive inputs from the cortex and sensory neurons such as those that relay proprioceptive information. Thus, MSE neurons are well positioned to mediate coordinated muscle activation patterns arising from command centers for volitional movement as well as reflex pathways activated by sensory feedback locally within the spinal cord. The aims of this grant are designed to unravel the wiring and cellular constituents within motor synergy circuits, and to examine how these circuits form during embryonic development and early postnatal life. Aim 1 will create a cellular atlas and connectivity map of MSE neurons. This will define whether the molecular heterogeneity of MSE neurons corresponds to separate motor pool circuit-modules or physiologically-different classes of neurons used for controlling all motor pools. Aim 2 will define the pattern of propriospinal feedback from muscles onto MSE neurons. Here the goal is to establish whether the MSE circuit is based on simple labeled line pathways or has a more complex input-output relationship. Aim 3 will use transcription factor knockouts to determine whether hardwired intrinsic genetic programs establish the MSE circuitry. Aim 4 will test whether the functional MSE network arises from activity dependent feedback from proprioceptive sensory neurons. Taken together, these aims will provide a detailed molecular-cellular understanding of a critical node within the local spinal system for computing and coordinating motor activation patterns. These findings may help target motor circuits using genetics and/or neural activity to facilitate recovery from spinal cord injury.

PROJECT SUMMARY/ABSTRACT Amyotrophic lateral sclerosis (ALS) is a relatively-rare disease that leads to motor neuron degeneration in 1:50,000 people in the US. Since mutations in ~50 different genes have been linked to ALS, there is the daunting possibility that treatments will require the development of many separate therapies. Consequently, identification of the shared pathways and key nodal points affected in multiple forms of sporadic and familial ALS could provide greater impact for the overall ALS community. In this regard much effort is currently focused on pathways relevant to protein-stasis, autophagy and cell stress, since protein aggregates are a common feature of ALS. This proposal takes a complimentary approach by examining the mechanistic role played by an essential motor neuron-specific microRNA (miR-218) that is affected by ALS. Gene expression studies to identify microRNAs dysregulated in sporadic and familial ALS consistently detect downregulation of miR-218. A recent analysis of ALS patients found a cohort with mutations in miR-218, suggesting insufficient miR-218 levels/activity may be a risk factor for the disease. Because many ALS-linked genes affect RNA metabolism and microRNA processing complexes, it is hypothesized that miR-218 activity is downregulated in many types of ALS leading to a pattern of gene dysregulation that fails to sustain motor neurons. Conversely, it is predicted that ectopic miR-218 may restore proper gene expression in motor neurons and counteract ALS. While previous studies have established that miR-218 controls motor neuron connectivity in embryos, the goal of this grant is to identify the gene networks controlled by miR-218 in adult motor neurons using genetics to decrease (aim 1) and elevate (aim 2) miR-218 with precise spatiotemporal control. A floxed- miR-218 allele was created and following Cre-mediated deletion in adult motor neurons it was found that neuromuscular defects arose - indicating miR-218 is a critical regulatory molecule in mature motor neurons. In Aim 1 miR-218 will be conditionally deleted in adult mouse motor neurons and next generation RNA sequencing from single-nuclei will be used to (1a) uncover the miR-218 gene network in adult motor neurons, and (1b) cross- correlate miR-218-regulated genes with dysregulated genes in mouse models of ALS. In Aim 2, miR-218 will be conditionally (ectopically) expressed in mice to (2a) define non-cell-autonomous effects of mir-218, and (2b) model baseline levels of miR-218 in motor neurons that would be tolerable for potential ALS-therapies. These studies will pave the way for future experiments to directly test whether miR-218 can be used to attenuate ALS. To make this possible an independent but complementary R03 was submitted (see complimentary application) to allow others with ALS-models to explore this promising possibility with our miR-218 reagents. This R21 grant is an important step toward understanding the mechanism-of-action of miR-218 and how it might be used to attenuate ALS.

MiR-218 is a motor neuron-specific microRNA conserved across vertebrate evolution that is expressed by both fetal and adult lower motor neurons in humans and mice. There are two alleles of miR-218 in humans and mice, and null alleles of both miR-218-1 and miR-218-2 have been generated. It was found that embryos lacking both alleles of miR-218 develop normally with proper motor neuron numbers, however during the final week of embryonic development their motor neurons degenerate with a pathophysiology that mimics spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Although motor degeneration in ALS occurs in adults rather than at fetal stages as observed in miR-218 mutant mice, several findings provide further indirect support for a connection between ALS and miR-218: (1) Gene expression studies to identify microRNAs dysregulated in sporadic and familial ALS repeatedly detect downregulation of miR-218. (2) A recent analysis of ALS patients found a cohort with mutations in miR-218, suggesting insufficient miR-218 levels or activity may be a risk factor for the disease. (3) Many ALS-linked genes affect RNA metabolism and microRNA processing complexes, including TDP43, FUS and C9orf72, suggesting a plausible mechanism for miR-218 downregulation in ALS. (4) In preliminary studies a floxed-mutation of miR-218 was generated and it was found that deletion in adults leads to neuromuscular pathology. These observations suggest several obvious research possibilities, including tests to: (1) determine whether genetically-lowering miR-218 accelerates ALS-pathology in animal models and (2) whether ectopic miR-218 can reprogram the normal genetic circuits in motor neurons and attenuate ALS degeneration. The goal of this grant is to generate and provide miR-218 reagents to the ALS research community to accelerate the investigation of this promising new candidate for ALS-therapy. In Aim 1, the concentration levels of miR-218 will be defined in an allelic series of mouse lines. It is anticipated that these lines can be used by others to cross to their ALS-models systematically to raise the lower miR-218 levels in vivo. In Aim 2 mouse embryonic stem cell lines (mESC) will be derived from our allelic series of miR-218 lines which is anticipated to have value in culture assays since motor neurons with different miR-218 levels can easily and efficiently be generated from mES lines for investigating the cellular/molecular features of ALS in vitro. The reagents generated in this grant will be made widely available to researchers by depositing our mouse strains in the Jackson Laboratories repository and by maintaining a dedicated stock of mES cell lines for distribution. non- overlapping complementary R21 grant has also been submitted to characterize the genetic networks regulated by miR-218, in order to define mechanistically the motor neuron synaptic and survival modules regulated by this microRNA.