Neil A. Shneider - US grants

In the developing brainstem and spinal cord, distinct classes of motor neurons and ventral interneurons are generated by the graded signaling activity of the secreted protein Sonic Hedgehog (Shh). Shh controls neuronal fate by establishing different progenitor cell populations in the ventral neural tube that are defined by the expression of the transcription factors Pax6 and Nkx2.2. Pax6 functions as a key intermediary in the Shh-mediated control of motor neuron subtype identity. In the caudal hindbrain, elimination of Pax6 expression alters motor neuron subtype identity, transforming hypoglossal to vagal motor neurons. The mechanism by which Pax6 functions to control ventral neuronal fate remains unclear. Shh inhibits the expression of Pax6 in a concentration-dependent manner such that an inverse gradient of Pax6 results from the expression of secreted Shh by ventral midline structures. The specific aims of this proposal include determining whether the graded expression of Pax6 is important for the control of neuronal fate and not simply an insignificant consequence of the requirement for graded Shh signaling. Pax6 is expressed as two alternatively spliced proteins with distinct DNA binding characteristics. The regulation of this alternative splice choice by graded Shh signaling will be investigated as a possible mechanism by which graded Shh signaling generates cellular diversity within the spinal cord. Finally, a PCR-based cloning strategy will be used to differentially screen cDNA libraries constructed from single ventral progenitor cells specific to a particular motor neuron subtype to isolate genes involved in the specification of motor neuron subtype identity. The role of these genes in motor neuron development will be examined by studying the effect of their ectopic expression on motor neuron identity in the developing spinal cord. Through a more complete understanding of the genetic events which control motor neuron identity and early differentiation in the developing central nervous system, these experiments will provide considerable insight into the pathogenesis of amyotrophic lateral sclerosis (ALS) and other motor neuron diseases which result from the selective degeneration of this neuronal population. The manipulation of the developmental programs which control the specification of motor neurons may make possible novel therapeutic strategies to restore this specific subclass of central neurons lost either by traumatic spinal cord injury or progressive neurodegenerative disease.

DESCRIPTION (provided by applicant): Gamma motor neurons (g-MNs) are a functionally and anatomically distinct subclass of neurons found in large number in most motor pools. These "fusimotor" neurons exclusively innervate the intrafusal fibers of the muscle spindle and comprise a parallel motor system that activates muscle spindle fibers independently of extrafusal muscle. The mechanisms that control the specification and differentiation of g-MNs are unknown, and no specific markers have been identified to distinguish g from a motor neurons in early development. We have found that g fusimotor neurons are selectively dependent on target muscle spindle-derived GDNF, and a recent study reports that fusimotor neurons are also selectively dependent on GDNF signaling in embryogenesis. Using this trophic requirement as a functional marker, we propose to perform a differential screen for genes selectively expressed in g-MNs using a mutant mouse in which g- MN precursors are selectively lost in the absence of GDNF signaling. We will also exploit this specific property of g-MNs in cultures of mouse embryonic stem cell-derived motor neurons to profile the pattern of gene expression in g-MNs in vitro. Finally, we will use molecular and size criteria established in our previous work to selectively isolate postnatal g-MNs for molecular analysis. The identification of genes specifically expressed in g-MNs at different stages of development will make possible a molecular genetic approach to study the formation and function of the fusimotor system, including its role in normal motor behaviors and disorders of motor control. PUBLIC HEALTH RELEVANCE: Gamma motor neurons regulate sensory feedback to the central nervous system from the periphery by controlling the sensitivity of stretch receptors in skeletal muscle. Their function is critical for motor control and for our perception of where our limbs are in space. This study will provide a molecular profile of gamma motor neurons, opening the way to future study of this motor system in health and disease.

DESCRIPTION (provided by applicant): Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder in which preferential loss of motor neurons (MNs) results in paralysis and death. Although ALS is largely a sporadic disease, research has focused on heritable forms of the disorder because clinical and pathological evidence suggests common pathogenic mechanisms. Mutations in the gene FUS (or TLS) were recently reported in rare ALS families, and FUS pathology has since been found in sporadic ALS, suggesting that FUS may provide a mechanistic link between familial and sporadic disease. Structural and functional similarities between FUS and TDP-43 - another RNA/DNA-binding protein involved in the pathogenesis of sporadic and familial ALS - have also led to speculation that the molecular pathways regulated by both of these factors are vital to our understanding of common disease mechanisms. We know very little about how mutations in FUS cause motor neuron degeneration. Dominant inheritance of FUS mutations suggests a novel gain of function that is selectively toxic to motor neurons. Alternatively, mutant FUS may act as a dominant negative, inhibiting the normal activity of wild type protein, perhaps by sequestering it in abnormal FUS-positive, cytoplasmic aggregates that are a hallmark of sporadic and familial ALS. If ALS results as a consequence of FUS deficiency, then it is critical to understand more about the normal functions of FUS in the central nervous system, and specifically in the motor circuits affected in the disease. In this project, loss and gain of function strategies are used to explore the role of FUS in normal motor neuron development in animal and cellular models, and to relate that function to mutant FUS-mediated ALS. In vitro studies will take advantage of our ability to generate FUS mutant embryonic stem cell-derived motor neurons in large numbers. In Aim 1, FUS knockout mice will be used to support the hypothesis that motor neuron degeneration in ALS is a consequence of FUS deficiency. We will test the effect of FUS loss on motor neuron differentiation and survival and on the functional development of spinal motor circuits required for normal motor activity. By high-throughput RNA sequencing (RNA Seq), we will explore the normal role of FUS in the regulation of gene expression in the nervous system. In Aim 2, we will use overexpression studies of mutant FUS to characterize the effect on motor neuron survival and function in vivo, and to determine how mutations alter the functional properties of FUS in ALS. RNA Seq analysis will be used to identify molecular pathways involved in the pathogenesis of disease. In Aim 3, we will use motor neurons derived from mouse embryonic stem cells to study cellular and molecular mechanism of FUS-mediated motor neuron degeneration. This project will address fundamental questions about the role of FUS in ALS, and generate novel models of FUS-mediated disease in mice and cultured motor neurons that will be critical tools for future studies of disease mechanism and drug discovery in the ALS research community.

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder in which preferential loss of motor neurons (MNs) results in paralysis and death. Although ALS is largely a sporadic disease, research has focused on heritable forms of the disorder because clinical and pathological evidence suggests common pathogenic mechanisms. Mutations in the gene FUS cause some of the most aggressive early-onset forms of ALS. In a recent study, our lab demonstrated in a mouse model of disease that mutant FUS causes motor neuron degeneration not by a loss-of-function, by a toxic gain-of-function that does not involve an excess of FUS activity. FUS is one of a number of RNA binding proteins ? including TDP-43 and hnRNP A1 ? that have been causally related to ALS. Our recent work ? together with related studies from several labs ? has led to a disease model in which the low complexity (LC) ?prion-like? domain of FUS and related proteins drives its phase transition to an irreversible, neurotoxic assembly. Our data demonstrates that ALS-related mutations in FUS increase the natural tendency of the protein to form these toxic assemblies, which trap and sequester other ribonucleoprotein granule components, including proteins involved in translational control. In this project we will explore the mechanisms of FUS toxicity in a series of transgenic and knock-in mutant mice with which we have modelled key aspect of the FUS-ALS phenotype. In addition, in vitro studies using motor neurons and astrocytes derived from these mouse models will be used to investigate cell autonomous and non-autonomous mechanisms of disease. In Aim 1, we will use a conditional knock-in mouse model to express mutant FUS in MNs or astrocytes, or more broadly in the nervous system to explore the effects of regulated mutant FUS expression on MN survival and function, and on gene expression changes that may underlie MN degeneration in FUS-ALS. We will also analyze mice with ALS-causing mutations in the LC domain of FUS to test the role of this critical domain in the disease. In Aim 2, we will use microfluidics to isolate MN axons and test the idea that FUS-dependent defects in axonal protein synthesis contribute to MN degeneration, and we will pursue our finding that hnRNP U selectively interacts with ALS-mutant FUS by exploring in vivo the functional role of this RNA binding protein in disease progression. Finally in Aim 3, we will apply a combination of single-cell RNA sequencing and topological data analysis to a mixed distribution of in vitro differentiated MNs derived from our FUS knock-in mutant mice. This sophisticated integration of in vivo and in vitro experimental systems, combined with our integrative computational and analytical approach will allow us to elucidate pathways of disease in vulnerable subpopulations of MNs and to identify potential therapeutic targets for the treatment of FUS-ALS and related forms of motor neuron disease.

Project summary Amyotrophic lateral sclerosis (ALS) is a predominantly sporadic condition affecting motor neurons (MNs). Its etiology is unknown but several environmental neurotoxicants have been associated with ALS. Though, none have a clear pathogenic role. Only a few of the previous studies investigating the role of environmental factors in ALS have assessed individual biomarkers of exposure (mostly to persistent pollutants) and none has demonstrated a direct concordance between signaling pathways produced by neurotoxic exposure and those implicated in ALS. So far, the progress in this field is hampered by the lack of CNS-relevant specific biomarkers for monitoring both environmental exposure to neurotoxicants and disease progression. Recently, we screened a series of highly prevalent neurotoxicants associated with ALS in vitro and found that mouse and human MNs exhibit a preferential and dose-dependent vulnerability to the metals manganese (Mn) and arsenic (As), and several organophosphate (OP) and pyrethroid (PT) pesticides (e.g. chlorpyrifos [CPS] and cypermethrin). Also, we found that MNs expressing an incompletely penetrant familial ALS variant of TAR DNA-binding protein 43 (G298S TDP-43) are vulnerable to As, Mn and CPS at very low doses that are innocuous to wild-type MNs. TDP-43 pathology is a hallmark of the large majority of ALS cases, both familial and sporadic, so altogether our data suggest that these toxicants could be environmental modifiers of most of the forms of ALS. Intriguingly, excess metals and pathological proteins such as TDP-43 can both be extruded from cells through a homeostatic mechanism that involves the release of tiny membrane-bound compartments called extracellular vesicles (EVs). EVs are now of particular interest as disease biomarkers in other fields. CNS-derived EVs can be isolated from blood because of their ectopic membrane expression of L1CAM. In this study, we will use biospecimens available in the US ALS National Biorepository and from the Target ALS brain bank to reach the following objectives: 1) we will validate hair as a useful biospecimen in ALS for the measure of poorly investigated non-persistent pollutants like OPs and PTs (200 patients x2; taken at 2 ALS stages); 2) we will examine the use of CNS-L1CAM-EVs isolated from the blood of the same 200 patients (2 ALS stages) as a biomarker of environmental exposure (compare metal levels and profiles), as well as a biomarker of disease progression via the accumulation of TDP-43; 3) our epidemiological and statistical analysis will determine association between environmental toxicants, TDP-43 and ALS progression; 4) we will examine concordance between toxicant-specific exposure extracted from gene-expression profiling in mice exposed to CPS and Mn and patient pathogenic transcriptional signatures obtained from our Target ALS consortium to identify disease pathways with therapeutic potential for ALS. By elucidating molecular mechanisms and biomarkers of environmental ALS, our study will have several far-reaching clinical and therapeutic implications.

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder in which preferential loss of motor neurons (MNs) results in paralysis and death. Although ALS is largely a sporadic disease, research has focused on heritable forms of the disorder because clinical and pathological evidence suggests common pathogenic mechanisms. Mutations in the gene FUS cause some of the most aggressive early-onset forms of ALS. FUS pathology ? and rarely, mutations - are also associated with the related neurodegenerative disorder, frontotemporal dementia (FTD). In a recent study, our lab demonstrated in a mouse model of disease that mutant FUS causes motor neuron degeneration not by a loss-of-function, by a toxic gain-of-function that does not involve an excess of FUS activity. FUS is one of a number of RNA binding proteins ? including TDP-43 and hnRNP A1 ? that have been causally related to ALS and FTD. Recent work has led to a disease model in which the intrinsically disordered ?prion-like? domain of FUS and related proteins drives a phase transition that results in the formation of an irreversible, neurotoxic aggregate. ALS-related mutations in FUS increase the natural tendency of the protein to form these toxic assemblies, which trap and sequester other ribonucleoprotein granule components. In this project we will explore the mechanisms of FUS toxicity in a novel series of knock-in mutant mice that reproduce key aspect of the FUS-ALS phenotype. In addition, in vitro studies using motor neurons and astrocytes derived from these mouse models will be used to investigate cell autonomous and non- autonomous mechanisms of disease. In Aim 1, we will use a conditional knock-in mouse model to express mutant FUS in MNs or astrocytes, or more broadly in the nervous system to explore the effects of temporally and spatially regulated mutant FUS expression on MN survival and function; and we will also explore the relative toxicity of human FUS in a fully humanized mouse model of FUS-ALS. In this highly disease-relevant model of ALS/FTD, we will also test the therapeutic potential of the FUS disaggregase, Kap?2 as a means to slow or stop the onset and progression of MN degeneration. In Aim 2, we will combine sophisticated electrophysiological and behavioral methods to explore the functional consequence of mutant FUS throughout disease progression in the FUS knock-in mouse. Finally in Aim 3, we will apply a combination of single-cell RNA sequencing and topological data analysis to a mixed distribution of in vitro differentiated MNs derived from our FUS knock-in mutant mice. This sophisticated integration of in vivo and in vitro experimental systems, combined with our integrative computational and analytical approach will allow us to elucidate pathways of disease in vulnerable subpopulations of MNs and to identify potential therapeutic targets for the treatment of FUS-ALS and related forms of motor neuron disease.