Justin R. Fallon - US grants

Proper function of the synapse relies on a remarkably precise assemblage of molecular and morphological specializations. However, while much has been learned about the biochemical, structural and physiological features which characterize the mature synapse, much less is known about the mechanisms which direct synapse formation. Agrin is an extracellular matrix protein derived from Torpedo electric organ that organizes acetylcholine receptor (AchR) and acetylcholinesterase (AchE) on cultured myotubes. At the mature neuromuscular junction, immunocytochemical studies have shown that molecules closely related to agrin are highly concentrated in the synaptic basal lamina, a structure known to play a central part in directing synapse regeneration. These results strongly suggest that agrin- related molecules play a role in directing the formation of the regenerating nerve-muscle synapse. The goal of the studies proposed here is to determine how agrin- related molecules might be involved in the differentation of the developing synapse. The experimental plan is to use recently developed monoclonal antibodies to Torpedo agrin to establish the time of appearance and the distribution of agrin-related molecules at the developing chick neuromuscular synapse in vivo. These antibodies will be used in concert with other markers to correlate the localization and expression of agrin-related molecules with other key events in synapse formation, such as the onset of functional innervation and the formation of high density clusters of AchR and AchE. The relationship of the expression and localization of agrin-related molecules to innervation will be investigated in experiments utilizing paralyzed and aneural muscles. These studies should yield important insights into the molecular mechanisms which underlie synapse formation in the developing embryo, and could provide important clues toward developing effective treatments for diseases and trauma which affect the neuromuscular synapse.

Proper function of the synapse relies on a remarkably precise assemblage of molecular and morphological specializations. However, while much has been learned about the biochemical, structural and physiological features which characterize the mature synapse, much less is known about the mechanisms which direct synapse formation. Agrin is an extracellular matrix protein derived from Torpedo electric organ that organizes acetylcholine receptor (AchR) and acetylcholinesterase (AchE) on cultured myotubes. At the mature neuromuscular junction, immunocytochemical studies have shown that molecules closely related to agrin are highly concentrated in the synaptic basal lamina, a structure known to play a central part in directing synapse regeneration. These results strongly suggest that agrin- related molecules play a role in directing the formation of the regenerating nerve-muscle synapse. The goal of the studies proposed here is to determine how agrin- related molecules might be involved in the differentation of the developing synapse. The experimental plan is to use recently developed monoclonal antibodies to Torpedo agrin to establish the time of appearance and the distribution of agrin-related molecules at the developing chick neuromuscular synapse in vivo. These antibodies will be used in concert with other markers to correlate the localization and expression of agrin-related molecules with other key events in synapse formation, such as the onset of functional innervation and the formation of high density clusters of AchR and AchE. The relationship of the expression and localization of agrin-related molecules to innervation will be investigated in experiments utilizing paralyzed and aneural muscles. These studies should yield important insights into the molecular mechanisms which underlie synapse formation in the developing embryo, and could provide important clues toward developing effective treatments for diseases and trauma which affect the neuromuscular synapse.

Proper functioning of the nervous system requires highly ordered and tightly regulated synapse formation. The long-range goal of this project is to elucidate the molecular and cellular mechanisms that underlie postsynaptic differentiation. Agrin is an extracellular matrix protein that induces the clustering of acetylcholine receptors (AChRs) on myotubes in culture. At developing neuromuscular junctions in vivo, agrin is likely to direct the assembly of key elements of the postsynaptic apparatus. Agrin mRNA is alternatively spliced yielding agrin isoforms that have large differences in AChR clustering potency. During the previous funding period, a candidate agrin receptor from Torpedo electric organ postsynaptic membranes was identified and was shown to be a heteromeric complex whose subunits are homologous to alpha- and beta- dystroglycan. Preliminary experiments using alternatively-spliced agrin isoforms have indicated that myotubes and postsynaptic membranes express several classes of agrin receptors, each of which contains alpha-dystroglycan. The proposed experiments are designed to characterize these newly recognized classes of agrin receptors at the cellular, biochemical, and molecular levels. The first goal (Specific Aims #1 and #2) is to analyze agrin- isoform selective receptors at the cellular and biochemical levels. It is hypothesized that the different classes of agrin receptors perform distinct but interrelated roles during synapse development. The experiments in Specific Aim #3, are designed to establish the developmental profile of the expression of these agrin receptor classes, to localize them at synapses in vivo, and to study their regulation during key stages in nerve-muscle synaptogenesis. Further, it is hypothesized that other proteins that associate with the dystroglycans play important roles in mediating agrin's actions. Experiments in Specific Aim #4 are designed to characterize and to clone one such protein, a novel dystroglycan/dystrophin- associated protein discovered in this laboratory. The results of these studies will provide vital insights into the means by which synaptic differentiation proceeds during development and regeneration. In addition, the experiments proposed here focus on the dystroglycans, part of a protein complex that is defective in several muscular dystrophies. Knowledge of the function of this complex, and the factors that mediate its assembly, could provide new keys to understanding and eventually treating these diseases.

Proper development and functioning of the brain relies on the precise and rapid flow of signals between neurons. Synapses play the foremost role in this information exchange. While the physiology and structure of mature synapses are understood in considerable depth, much less is known about the molecular cues mediating synaptic differentiation, maintenance, and turnover. A cardinal event in synapse formation is the marshalling of neurotransmitter receptors to the postsynaptic membrane. Mounting evidence suggests that agrin, a protein that directs postsynaptic differentiation at developing neuromuscular junctions, may also play a role in synaptogenesis in the brain. A full understanding of agrin's function in the brain requires the characterization of its cell surface receptor. In preliminary experiments we have found that agrin binds to cultured neurons and to synaptosomes. In some cells, agrin elicits the redistribution of these binding sites, suggesting that they constitute functional agrin receptors. Further, we have identified two agrin binding polypeptides in brain membranes. In the proposed studies we will characterize neuronal agrin receptor(s) at the cellular, biochemical, and functional levels, with the overall goal of elucidating the molecular events underlying synapse formation among neurons. Using well characterized primary hippocampal cultures, we will determine the time course of expression, the cellular localization, and the functional properties of agrin receptors on differentiating neurons. We will compare the distribution of agrin receptors to that of neurotransmitter receptors and other synaptic specializations. The subcellular localization of agrin receptors will be analyzed at the ultrastructural level. We will also determine whether agrin induces changes in the distribution of its own receptor or of related markers of synaptic differentiation. In parallel with this cell biological approach, we will characterize neural agrin receptors biochemically. Ligand overlay techniques and affinity chromatography methods will be used to identify and to determine the subunit composition of agrin receptors on neurons, and partial amino acid sequence of the purified receptors will be obtained. In addition, we will determine how alternative splicing of agrin may regulate binding to agrin receptors, and investigate the ensuing functional consequences. The results of these experiments will provide fundamental insights into synapse formation, modification, loss, and recovery. Knowledge of these basic mechanisms is crucial to our understanding of normal synaptic function, and forms a bedrock for the development of diagnostic and therapeutic approaches aimed at ameliorating or curing mental illnesses.

This application is for a training program to support an interdisciplinary predoctoral training in the Neurosciences at Brown University, under the Jointly Sponsored NIH Predoctoral Training Program in the Neurosciences. The training program is intended to produce new Ph.D.'s capable of establishing independent research in the interdisciplinary field of neuroscience. The training program will be operated by the Neuroscience Graduate Program, and includes 26 training faculty drawn from Departments including Neuroscience, Cognitive Sciences and Linguistics, Psychology, Physics, Applied Mathematics, and Molecular Pharmacology at Brown University. Funds are requested for 5 years, for 7 predoctoral trainees per year. The research of the training faculty reflects the diversity and high quality of the neuroscience program. The long-standing strength in systems, computational, and behavioral neurobiology has been recently complemented by the addition of several new faculty in the area of cellular and molecular neurobiology. Importantly, these diverse areas are well integrated. Several faculty use behavioral measures in conjunction with physiological experiments and modeling of neural systems, while others work with computational models, bringing behavioral or physiological data to bear on testing the validity of the model. Many of the faculty also shares a common interest in synaptic plasticity. This problem is being attacked at levels ranging from multi-electrode recording in awake behaving animals, to the synaptic basis of ocular dominance column formation, to the cloning novel genes mediating memory consolidation. The program thus offers broad yet well-integrated training in modern neuroscience. This program uses courses, supervised laboratory research, a strong colloquium series, seminars, journal clubs, and retreats to train students from a multidisciplinary perspective. The trainees will also participate in programs specifically designed to provide insight into the responsible conduct of research.

A major problem in neuroscience is understanding how ephemeral episodes of neural activity are transformed into stable changes in synaptic efficacy. The creation of long-lasting synaptic modifications requires new protein synthesis. One source of these newly synthesized polypeptides is the translation of mRNAs that are localized to synapses. Recent work from our laboratories has provided strong evidence that the activation of translationally dormant mRNAs by cytoplasmic polyadenylation is an important mechanism for experience-driven local synthesis. T his polyadenylation is mediated by a sequence-specific RNA binding protein called CPEB (cytoplasmic polyadenylation element binding protein) that is localized at synapses and is a component of postsynaptic density fractions. In this sub-project we will use an integrated approach to determine CPEB protein and mRNA localization and expression in the developing adult brain, and in cultured hippocampal neurons. We will test the role of the CPE (cytoplasmic polyadenylation element) and CPEB in the dendritic targeting of alpha-CaMKII mRNA. We will study the function of CPEB using mice (produced in Core B) with targeted mutations of CPEB in area CA1. The results of these studies will provide vital insights into the means by which long-term synaptic modifications are formed by the healthy brain. Knowledge of these fundamental mechanisms forms the basis for developing diagnostic and therapeutic approaches to the treatment of brain disorders.

The overall goal of this Program is to understand the protein synthesis- dependent synaptic modifications that underlie learning and memory. It has been established that activity stimulates protein synthesis, and that some of these proteins are synthesized from mRNA localized at or near the synapse. The investigators recently described a novel mechanism for such experience-driven local translation wherein translationally dormant synaptic mRNA is polyadenylated through the action of the CPEB (cytoplasmic polyadenylation element binding protein). This discovery provides an unprecedented opportunity to study the role of local mRNA translation in synaptic plasticity, and forms the basis for a coherent, integrated, and broadly-based investigation into the regulation of protein synthesis-dependent synaptic plasticity. Project 1 (Richter) includes the characterization of new CPEB isoforms and a novel targeted CPEB knock-out mouse using Cre-loxP technology; and the identification of additional CPEB-regulated mRNAs. Project 2 (Fallon) includes the localization, at the light and EM level, of CPEB in developing an adult brain, and in cultured hippocampal neurons; the characterization of the intracellular signaling pathways leading from synaptic activation to cytoplasmic polyadenylation; and the regulation of the targeting of CPE- containing mRNAs and of CPEB to dendrites. Project 3 (Bear) will characterize a noel form of protein synthesis-dependent synaptic plasticity (DHPG-LTD); use mice lacking CPEB in the consolidation; and characterize the role of local translation and CPEB in experience- dependent regulation of NMDA receptor subunit expression. There will be two cores, one administrative and the second for the production and maintenance of CPEB knock-out mice, which will be used by all the investigators. These should lead to better understanding of the mechanisms of long-term memory formation. The results could provide insights needed to understand diseases that affect learning and memory, and may be useful in designing therapeutic strategies to combat them.

Dementia is among the most debilitating disorders affecting the elderly. The majority of cases are caused by Alzheimer's disease (AD), which is characterized by senile plaques, neurofibrillary tangles, and loss of synapses. Despite their rapidly increasing prevalence within the aged population, progress in understanding the etiology and pathogenesis of these diseases, and developing therapies for them, has been severely hampered by the lack of suitable animal models. Further, microvascular disease is a common finding in the aged brain and it may precede the development of dementia. The ApoE4 gene is a predisposing factor for various types of vascular pathology as well as an important risk factor for the development of sporadic AD. The autosomal dominant disorder CADASIL, which results from mutations in the Notch3 gene, is a brain microvascular disease causing stroke and dementia and is characterized by thickening of the smooth muscle layer of brain arterioles. Agrin, a synapse organizing molecule in the peripheral nervous system, is expressed in normal brain but its function there remains unknown. In the central nervous system, it is found in neurons as well as within the basement membranes of the capillaries that form the blood brain barrier. Recent studies from two members of the project (Fallon and Stopa) have shown agrin redistributes to the Abeta-containing senile plagues and becomes insoluble in AD brains. These patients also exhibit fragmentation on the agrin-containment basement membranes of the brain microvasculature. In this proposal we will use tissue-specific targeted disruption of the agrin gene as a tool to study agrin function at synapses in normal brain (Aim #1) and in mouse models of AD (Aim #2). We will also examine the status of the microvasculature in mouse models of AD as well as in ApoE4 homozygous animals, particularly with respect to agrin expression in the basement membrane. Finally, we will generate mouse models of CADASIL (Aim #3) by 'knocking-in' mutant Notch3 alleles. We will characterize the microvasculature in these animals for comparison with CADASIL patients. We will also determine the prevalence of dominant Notch3 mutations in patients demonstrating MRI evidence of the CADASIL phenotype.

DESCRIPTION (Adapted from applicant's abstract): This proposal has two overall, interrelated goals. The first is to deepen our understanding of how synapses are formed, shaped, maintained and eliminated. The second is to elucidate how the integrity of the muscle fiber membrane is maintained, with particular regard to muscular dystrophy. Agrin secreted from the nerve terminal induces the formation of nerve-muscle synapses. The agrin signaling receptor MuSK is essential for this induction. However, agrin does not bind MuSK directly and the mechanisms of MuSK activation and localization are unresolved. In the previous funding period we discovered a novel component of Torpedo electric organ postsynaptic membranes, biglycan. Biochemical studies show that this small leucine-rich repeat proteoglycan (SLRP) binds via distinct domains to a a-dystroglycan, the ectodomain of MuSK and to a-and g- sarcoglycan. Both biglycan and its homolog decorin induce MuSK tyrosine phosphorylation when added to cultured myotubes. Moreover, agrin-induced AChR clustering is greatly reduced on myotubes from biglycan null (biglycan-10) mice. Finally, serum creatine kinase levels are markedly elevated in biglycan-10 mice. Together, these observations point to an important role for biglycan and/or decorin in postsynaptic differentiation, and for biglycan in maintaining the integrity of the muscle cell plasma membrane. In the present proposal we will take a combined molecular, biochemical, cell biological, and genetic approach to elucidate the role of biglycan and decorin in synaptic differentiation and in maintaining muscle cell integrity.

disease /disorder model; laboratory mouse

[unreadable] DESCRIPTION (provided by applicant): A fundamental problem in neuroscience is understanding how ephemeral episodes of experience are transformed into stable changes in synaptic architecture and efficacy. The creation of such long-lasting synaptic modifications requires new protein synthesis, which in turn is regulated at both transcriptional and translational levels. Moreover, the transcriptional profile of the neuron is a function of its developmental stage - e.g. critical period - and its history of activation. A major challenge in unraveling the mechanisms of long term plasticity then is to relate both developmental timing and experience-induced neural activity to the regulation of identified molecules that play key roles in synaptic plasticity. Fragile X Syndrome (FXS) offers a portal to the heart of this problem. FXS affects about 1:4000 boys and is caused by a triplet repeat expansion and hypermethylation of the Fmr1 promoter, leading to gene silencing. The protein product of the Fmr1 gene, FMRP, plays a central role in regulating protein synthesis-dependent synaptic plasticity. Our laboratory has established in vivo and cell culture systems for the study of Fmr1 transcription and expression. We find that Fmr1 transcripts are highly abundant in the developing and adult olfactory bulb and are bi-directionally regulated by olfactory experience. Preliminary in vivo and ce|l culture studies have provided evidence for two molecular mechanisms that regulate Fmr1 transcription: the transcription factor AP-2a and the selective, developmentally-regulated epigenetic modification of the Fmr1 gene regulatory regions. In the proposed studies we will use the olfactory system together with genetic and cell culture models to elucidate the molecular logic of Fmr1 gene regulation in the intact CNS. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The pharmacological upregulation of utrophin has been proposed as a therapeutic strategy for treating DMD. Genetic studies show that increasing utrophin expression can compensate for the loss of dystrophin and correct dystrophic pathology in the mdx mouse. However, genetic approaches for utrophin upregulation are not yet feasible in humans. Biglycan is an extracellular protein that is a component of the Dystrophin complex and binds to a-dystroglycan (Bowe et al., 2000), a - and ?- sarcoglycan (Rafii et al., 2006). Recombinant biglycan protein can be delivered to muscle in vivo where it rescues the sarcolemmal expression of intracellular DAPC components in biglycan null mice (Mercado et al., 2006). In the proposed studies we will use mdx mice to test the potential efficacy of biglycan as a therapeutic for DMD. In preliminary experiments we have shown that purified recombinant biglycan protein delivered systemically to mdx mice upregulates utrophin expression and decreases myofiber death and mononuclear infiltration. Moreover, single doses at therapeutically-practical levels are effective for up to three weeks. In the proposed studies we will produce recombinant biglycan and use histopathological markers to determine the optimal dose, frequency and route(s) of administration for its use. Functional studies will then be carried out to determine whether biglycan treatment improves the function of skeletal muscle in mdx mice. Positive data from these studies would form the basis for a rapid translation of this therapeutic approach to clinical trials. Duchenne Muscular Dystrophy (DMD) is the most common form of inherited muscular dystrophy. Currently there are no effective treatments and affected boys are wheelchair bound by age twelve and die by their third decade. In the proposed studies we will perform critical preclinical studies to test a novel therapy for DMD. [unreadable] [unreadable] [unreadable]

This U01 Translational Research in iVluscular Dystrophy will support the deveiopment of recombinant human biglycan (rhBGN) as a therapeutic and to prepare the data package necessary for an IND filing with the FDA. It builds upon work supported by an exploratory R21 proposal to our laboratory where we have demonstrated that systemically-delivered, rhBGN counters the dystrophic phenotype in mdx mice and improves muscle function. Notably, rhBGN is effective when delivered at 3 week intervals and at doses of <10mg/kg. We propose a 4 year development plan to produce and to validate rhBGN material that is compatible with use in clinical trials. This plan will extend from pilot studies with material produced by transient transfection, to PK/PD in the mdx mouse model, to dose optimization with highly characterized material produced from a stable cell line under cGMP conditions. To maximize the efficiency and speed of this process, the work will make use of standardized models and assays as well as collaborations with several expert vendors of biologic development services. We have also enlisted consultants who have extensive expertise in the manufacturing, testing and regulatory issues that are unique to biologies.

DESCRIPTION (provided by applicant): This U01 Translational Research in Muscular Dystrophy will support the development of recombinant human biglycan (rhBGN) as a therapeutic and to prepare the data package necessary for an IND filing with the FDA. It builds upon work supported by an exploratory R21 proposal to our laboratory where we have demonstrated that systemically-delivered, rhBGN counters the dystrophic phenotype in mdx mice and improves muscle function. Notably, rhBGN is effective when delivered at 3 week intervals and at doses of <10mg/kg. We propose a 4 year development plan to produce and to validate rhBGN material that is compatible with use in clinical trials. This plan will extend from pilot studies with material produced by transient transfection, to PK/PD in the mdx mouse model, to dose optimization with highly characterized material produced from a stable cell line under cGMP conditions. To maximize the efficiency and speed of this process, the work will make use of standardized models and assays as well as collaborations with several expert vendors of biologic development services. We have also enlisted consultants who have extensive expertise in the manufacturing, testing and regulatory issues that are unique to biologies. PUBLIC HEALTH RELEVANCE: In these translational studies we will develop a novel protein therapeutic for Duchenne Muscular Dystrophy. The goal is to take our findings in mouse models of this disease and do the work necessary to test this therapy in boys with this devastating disease. If the proposed work is successful, we will be able to move immediately into Phase I clinical trials.

DESCRIPTION (provided by applicant): This U01 Translational Research in Muscular Dystrophy will support the development of recombinant human biglycan (rhBGN) as a therapeutic and to prepare the data package necessary for an IND filing with the FDA. It builds upon work supported by an exploratory R21 proposal to our laboratory where we have demonstrated that systemically-delivered, rhBGN counters the dystrophic phenotype in mdx mice and improves muscle function. Notably, rhBGN is effective when delivered at 3 week intervals and at doses of <10mg/kg. We propose a 4 year development plan to produce and to validate rhBGN material that is compatible with use in clinical trials. This plan will extend from pilot studies with material produced by transient transfection, to PK/PD in the mdx mouse model, to dose optimization with highly characterized material produced from a stable cell line under cGMP conditions. To maximize the efficiency and speed of this process, the work will make use of standardized models and assays as well as collaborations with several expert vendors of biologic development services. We have also enlisted consultants who have extensive expertise in the manufacturing, testing and regulatory issues that are unique to biologies. PUBLIC HEALTH RELEVANCE: In these translational studies we will develop a novel protein therapeutic for Duchenne Muscular Dystrophy. The goal is to take our findings in mouse models of this disease and do the work necessary to test this therapy in boys with this devastating disease. If the proposed work is successful, we will be able to move immediately into Phase I clinical trials.

Project Summary Amyotrophic lateral sclerosis (ALS) and Frontotemporal Dementia FTD are devastating neurodegenerative disorders that lie on a genetic and mechanistic continuum. ALS is a disease of motor neurons that that is almost uniformly lethal within only 3-5 years of diagnosis. FTD is a heterogeneous, rapidly progressing syndrome that is among the top three causes of presenile dementia. About 10% of ALS cases are caused by dominantly transmitted gene defects. SOD1 and FUS mutations cause aggressive motor neuron pathology while TDP43 mutations cause ALS-FTD. Further, wild type FUS and TDP43 are components of abnormal inclusions in many FTD cases, suggesting a mechanistic link between these disorders. Early phenotypes are of particular interest because these could lead to targeted interventions aimed at the root cause of the disorder that could stem the currently inexorable disease progression. Elucidating such early, potentially shared characteristics of these disorders should be greatly aided by: 1) knock-in animal models expressing familial ALS-FTD genes; 2) sensitive, rigorous and objective behavioral phenotyping methods to analyze and compare models generated in different laboratories. In published work the co-PIs applied their first-generation, machine vision-based automated phenotyping method, ACBM ?1.0? (automated continuous behavioral monitoring) to detect and quantify the earliest-observed phenotypes in Tdp43Q331K knock-in mice. This method entails continuous video recording for 5 days to generate >14 million frames/mouse. These videos are then scored by a trained computer vision system. In addition to its sensitivity, objectivity and reproducibility, a major advantage of this method is the ability to acquire and archive video recordings and to analyze the data at sites, including the Cloud, remote from those of acquisition. We will use Google Cloud TPUs supercomputers that have been designed from the ground up to accelerate cutting-edge machine learning workloads, with a special focus on deep learning. We will analyze this data using Bayesian hierarchical spline models that describe the different mouse behaviors along the circadian rhythm. The current proposal has two main goals: 1) Use deep learning to refine and apply a Next Generation ACBM - ?2.0? - that will allow for more sensitive, expansive and robust automated behavioral phenotyping of four novel knock-in models along with the well characterized SOD1G93A transgenic mouse. 2) To establish and validate procedures to enable remote acquisition of video recording data with cloud-based analysis. Our vision is to establish sensitive, robust, objective, and open-source machine vision-based behavioral analysis tools that will be widely available to researchers in the field. Since all the computer-annotated video data is standardized in ACBM 2.0 and will be archived, we envision a searchable ?behavioral database?, that can be freely mined and analyzed. Such tools are critical to accelerate the development of novel and effective therapeutics for ALS-FTD.

Project Summary Adult Hippocampal Neurogenesis (AHN) is critical for normal learning and memory and reduced AHN is an early hallmark of Alzheimer?s Disease (AD). Thus, restoring AHN has emerged as an attractive target for AD therapy. The accumulation of negative signals that degrade the neurogenic niche contributes to the reduction in newborn neurons in AD and aging. BMPs are components of the niche that negatively regulate neurogenesis and their levels are increased in AD in humans and in mouse FAD models. We recently reported that full length MuSK harboring its Ig3 domain, which is necessary for high affinity BMP binding, is a BMP co-receptor that augments and shapes BMP signaling. In preliminary studies we established that MuSK is endogenously expressed in neural stem cells (NSCs). We generated knock-in mice engineered to constitutively express an alternatively spliced form of MuSK lacking the Ig3 domain (??Ig3- MuSK?). The animals are viable, fertile and have a normal life span. NSCs isolated from ?Ig3- MuSK mice show impaired BMP responsiveness. Remarkably, the ?Ig3-MuSK mice exhibit over a two-fold increase in AHN and improved hippocampal-dependent learning. These results suggest that reducing MuSK-BMP activity by modulating MuSK alternative splicing is a potential target for promoting AHN in AD. Importantly, such alternative splicing is expected to be amenable to manipulation by exon-skipping antisense oligonucleotides. The recent success of the ASO Spinraza for Spinal Muscular Atrophy has demonstrated that this class of drugs can be highly effective in the human CNS, with a favorable pharmacokinetic and safety profile. In the proposed experiments we will use mouse FAD models to test whether inhibition of the MuSK- BMP pathway can promote AHN in the plaque-rich and inflammatory ?AD environment?. To more closely model the pathological and therapeutic landscape in humans, we will also use conditional mutants to test whether manipulating the MuSK-BMP pathway after amyloid plaque formation can promote AHN and improve cognition. If successful, this work will form an important part of the rationale and impetus for a pursuing a MuSK-directed ASO therapy for AD.