Gary J. Bassell, Ph.D. - US grants

DESCRIPTION Nerve cells are the quintessential asymmetric cell and differentiate two morphologically and functionally distinct types of processes: axons and dendrites. The development of the nervous system proceeds by the extension of axons over long distances to reach their appropriate target cells. The growth cone is a specialized structure at the termini of neuronal processes that is essential for directed process outgrowth of both axons and dendrites. The ability of the growth cone to reach its correct targets may depend on mRNA transport and local protein synthesis to modulate cytoskeletal composition in response to external signals. In recent years, mechanisms for fast and slow transport systems been elucidated for membrane bound vesicles and cytoskeletal complexes, yet contemporary models for protein sorting into axons and dendrites have not considered mRNA transport as an additional mechanism. Using high resolution in situ hybridization and digital imaging microscopy methods, we have shown that beta actin mRNAs were localized to growth cones of developing dendritic and axonal processes. Beta actin mRNAs were detected in the form of spatially distinct granules that were associated with microtubules. The localization of beta actin mRNAs to neuronal growth cones of cultured neurons is proposed to be dependent on cis-acting sequences within the 3'UTR. Preliminary data suggests that short zip code sequences within the 3'UTR are involved in beta actin mRNA localization within neurons. The hypothesis is that these cis-acting sequences are necessary and sufficient to localize beta actin mRNA and that perturbation of these elements disrupts protein localization within the growth cone and has deleterious effects on process outgrowth. The spatial component of translation will be studied by expression of epitope tagged beta actin constructs. Trans-acting factors are proposed to bind the cis-acting elements and to be involved in beta actin mRNA localization and its association with cytoskeletal filaments. Using ultrastructural and fluorescence in situ hybridization methods, the localization and cytoskeletal association of beta actin mRNA complexes will be determined. The dynamic aspects of RNA transport in processes will be studied by microinjection of fluorescent mRNA sequences in living neurons. Proteins which bind the RNA localization sequences will be isolated, cloned and sequenced. The elucidation of a mechanism to regulate mRNA localization to growth cones will provide insight into the regulation of growth cone structure and mechanisms that may underlie process outgrowth.

Analysis of gene expression within the nervous system is particularly challenging because of the heterogeneity between cell types and the complex distributions patterns of specific mRNAs within single cells. Careful analysis of gene expression within the neurons must consider the fact that protein synthetic machinery is not restricted to the perikarya and that specific mRNAs can be localized to dendrites and subsynaptic locations. It is the contention of this proposal that conventional biochemical methods as well as micro-dissection approaches lack the ability to accurately measure gene expression within dendrites. This logic provides the rationale for this proposal and the need to develop technology capable of analysis of local changes in gene expression which are under regulatory control and vary during normal development, differentiation and disease processes. We have developed high resolution in situ hybridization methods to visualize the localization of mRNA within neurons. Further work is needed to develop methodology which is quantitative, highly sensitive and capable of analysis of changes in gene expression which are cell-type and/or cell- compartment specific. We propose to develop high-throughput technology in multi-color fluorescence in situ hybridization and non-isotopic methods at the ultrastructural level to permit quantitative analysis of gene transcription and localized gene expression within neurons. This proposal addresses four of the six areas of focus outlined in the RFA: (1) high throughput methods for quantifying the expression of multiple genes (2) methods for quantifying multiple spliced or edited variants of a given transcript (3) methods for comparing protein levels to corresponding mRNA levels within a cell (4) techniques for visualizing RNA distribution within cells. This approach has as its goal the assessment of the expression of many genes within a single cell. It relies on technology developed in the laboratory of the Co-Principal Investigator which allows the quantitative interrogation of sites of transcription within cells with a sensitivity sufficient to detect single molecules, or single nascent transcripts. This proposal describes a program of technological innovation based on the technical strengths of collaborations and existing expertise within the institution: that of FISH technology, primary neuronal culture, intracellular localization within neurons, slice cultures, long-term potentiation and in situ hybridization in brain sections.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The active transport of b-actin mRNAs to growth cones of developing neurons may provide a basic mechanism to enrich b-actin protein and influence growth cone motility. Using high resolution fluorescence in situ hybridization and digital imaging microscopy methods, we have shown that b-actin mRNAs were localized to growth cones of developing axonal and dendritic processes in the form of granules that were associated with microtubules. The molecular mechanism of b-actin mRNA localization in these cultured neurons was shown to be dependent on a 54 nt sequence in the 3'UTR, termed the zipcode, which is bound by two mRNA binding proteins, ZBP1 and ZBP2. Live cell imaging of neurons transfected with EGFP-ZBP1 revealed fast, bi-directional movements of granules in processes. This proposal will test the hypothesis that ZBP1 and/or ZBP2 function as adapter molecules between the b-actin zipcode and distinct types of cytoskeletal motors involved in both microtubule and microfilament-dependent movements. Preliminary findings presented in this application document the association of ZBP1 with microtubules. Experiments in Specific Aim 1 will elucidate the roles of ZBP1, ZBP2 and cytoskeletal motors in the transport of b-actin mRNA in processes and growth cones of cultured neurons. High resolution fluorescence microscopy and live cell imaging technology will be developed to visualize the co-transport of b-actin mRNA and ZBPs. Molecular and biochemical methods will be used to characterize the interactions between ZBPs and cytoskeletal motors. We will determine whether disruption of the expression or function of motors, and their interaction with ZBPs, can impair b-actin mRNA localization into processes and growth cones. Experiments in Specific Aim 2 will elucidate the function for ZBP/motor dependent transport of b-actin mRNA in the local synthesis of b-actin and growth cone motility. We will determine whether disruption of ZBPs or interacting motors can inhibit the local synthesis of b-actin within growth cones and impair regulation of growth cone motility and dynamics. The elucidation of molecular mechanisms involved in mRNA transport and their perturbation will provide new insight into protein sorting mechanisms that regulate growth cone motility and process outgrowth during neuronal development. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Elucidating the mechanistic function of Fragile X Mental Retardation Protein (FMRP) in neurons is a critical goal in understanding the basis of Fragile X Syndrome. This proposal will test the hypothesis that FMRP is required for the glutamatergic regulation of mRNA transport in dendrites and its subsynaptic translation. Recent identification of mRNAs that are bound by FMRP now makes it possible to investigate whether these RNA-protein interactions occur in dendrites and at synapses. An inherent difficulty in studying RNA-protein interactions in dendrites has been the lack of suitable high resolution microscopic technology to visualize mRNA transport and identify sites of local translation. A new view of FMRP function is made possible by utilizing novel microscopic and imaging technology to visualize mRNP complexes in live neurons. We have recently shown that FMRP is localized in the form of RNA granules that exhibit dynamic and activity-dependent movements in dendrites and spines. Experiments in Specific Aim 1 will apply high resolution fluorescence in situ hybridization methods and quantitative digital imaging analysis to determine whether specific mRNAs have altered localization and regulation in hippocampal cultures from Fmr1 knockout mice. We will determine whether FMRP binding elements function as zipcodes to localize FMRP target mRNAs using transfection of reporter constructs. Experiments in Specific Aim 2 will use live cell imaging technology to determine whether glutamatergic signaling and synaptic activity regulates the dynamic trafficking of FMRP and bound mRNAs in dendrites and spines. Experiments in Specific Aim 3 will use combined imaging and biochemical methods to elucidate a role for FMRP in the glutamatergic regulation of dendritic and synaptic protein synthesis. These studies will provide new insight into the molecular and cellular basis for altered synaptic plasticity in Fragile X Syndrome.

DESCRIPTION (provided by applicant): Spinal Muscular Atrophy (SMA) is a common inherited neurodegenerative disease caused by mutations or deletions in the SMN1 gene that encodes for the ubiquitous Survival of Motor Neuron Protein (SMN), which is essential for the assembly of spliceosomal snRNP complexes in all cells. A major unanswered question is how the loss of SMN leads to neuronal dysfunction and SMA. Further work is greatly needed to identify functions for SMN in neurons. Immunohistochemical studies have revealed localization of SMN to axons and dendrites from sections of spinal cord. Using high-resolution immunofluorescence of cultured neurons, we have shown that SMN forms granules which colocalize with RNA and ribosomes in growth cones. Using live cell imaging, we showed that EGFP-SMN granules exhibited rapid and bi-directional movements in neuronal processes and growth cones of live neurons. Primary motor neurons cultured from a transgenic mouse model of SMA displayed axonal defects that include loss of beta-actin mRNA from growth cones, suggesting a novel function for SMN in the mechanism of mRNA localization. Our preliminary data indicate that SMN associates with the mRNA binding proteins, ZBP1, ZBP2 and HuD, which are known to be involved in neuronal mRNA localization and stability. We hypothesize that these mRNA binding proteins interact with SMN to facilitate mRNA localization in processes and stabilization within growth cones. Experiments proposed here will further characterize the association, molecular interactions and dynamic regulation between SMN, HuD, ZBPs and associated mRNAs within RNA transport granules. SMN deficient motor neurons will be used to identify specific mRNAs that are altered in their localization and translation. We will investigate whether dysfunction of local mRNA regulation within SMN-deficient neurons contributes to altered growth cone motility. This research will provide new insight into neuronal functions for SMN, whereby possible defects in the assembly, localization and/or translation of mRNP complexes may contribute to the disease process in SMA.

[unreadable] DESCRIPTION (provided by applicant): Recent studies using cell culture models of axon regeneration and a biochemical approach have demonstrated that many mRNAs are targeted and translated within regenerating axons following injury. As recent studies have also shown that the local translation of specific mRNAs directly within the growth cones plays a critical role in axon guidance in vitro, an important question is whether specific mRNAs are targeted and translated within the growth cone of regenerating neurons in vitro. It is also critical to extend the above findings from cell culture into in vivo models for nerve regeneration. Almost nothing is known on role of local protein synthesis in vivo to promote axon regeneration. Future work is also critically needed to identify molecular mechanisms involved in mRNA localization and local protein synthesis during development and regeneration. Continued studies to identify underlying mechanisms and their extrapolation in vivo will provide critical tools and approaches to test the hypothesis that specific mRNA localization and translation is necessary for optimal axon regeneration. We have a long-standing interest in studying the mechanism, regulation and function of mRNA transport and local protein synthesis in cultured neurons. An area of technical expertise has been the use of high resolution fluorescence microscopy and digital imaging methods to visualize the localization and dynamic trafficking of mRNAs in fixed and live neurons respectively. We propose to use this expertise to investigate the role of specific mechanisms in mRNA targeting and local translation in axon regeneration, using established in vitro and in vivo models. Aim-1 will use quantitative fluorescence in situ hybridization methods to define whether [unreadable]-actin mRNAs and others are localized specifically within the growth cone of regenerating axons. Live cell imaging methods will be applied to visualize mRNA transport in the axon and its translation at the growth cone. Knockdown approaches and overexpression approaches will be used to assess whether specific mRNA binding proteins can influence axon regeneration. Aim-2 will involve the use of an established in vivo model to study axon regeneration which will be used, in similar fashion to aim-1, to document the presence of specific mRNAs, mRNA binding proteins and translational components, followed by efforts to manipulate the molecular mechanisms with viral vectors and affect axon regeneration in vivo. The proposed studies will provide new insight into novel mechanisms for axon regeneration which will suggest new targets for therapeutic targets for treatment of nerve injury. Axonal degeneration is a common pathway leading to loss of neural function, as seen in various neurological diseases and after spinal cord and nerve injuries. Therapeutic approaches to enhance axon regeneration are very limited. This proposal will investigate a role for specific mRNA targeting and translation mechanisms in optimal nerve regeneration. We anticipate that the proposed basic research will provide new insight into molecular mechanisms underlying optimal axon regeneration and has important implications for future developments of therapies and treatments for nerve injury. [unreadable] [unreadable]

This proposal seeks to establish the Emory Neuroscience NINDS Core Facilities. Neuroscience research has grown dramatically at Emory, with dozens of new investigators receiving NINDS-funding in recent years. The research, based in more than 10 basic science and clinical departments, pursues key issues ranging from the cellular and molecular basis of neural function and mechanisms of neurological disease, to clinical trials of new therapies in stroke and other common diseases. The Emory Neuroscience NINDS Core Facilities will coordinate core activities for 37 NINDS-funded investigators and their 51 qualifying grants, to provide these investigators and other neuroscientists access to a variety of state-of-the-art technologies and approaches that will enhance collaborative, multidisciplinary research. The facility will leverage generous institutional support for personnel, equipment, space and the new Center for Neurodegenerative Disease, to develop or expand the following shared core facilities: a) Proteomics b) Imaging c) Neuropathology/Histochemistry d) Viral Vector and e) Genomics. As directors, Drs. Allan Levey and Ray Dingledine will provide outstanding administrative support for the Center by a) facilitating, coordinating and monitoring access to the cores;b) assisting with budgeting, reporting, and maintaining fiscal responsibility;and c) providing an environment that fosters collaborative research utilizing cutting edge technologies and multidisciplinary approaches. A steering committee comprised of the Directors of the Center and the leaders of the respective Cores will meet at least once every 6 months to assure fair access, provide oversight of the operations of the cores, and to establish priorities and resolves issues. Neurological disorders are major causes of morbidity and mortality. The cores described in this application will facilitate a broad range of NINDS-sponsored research at Emory that is aimed at improving the understanding of disease, and producing new diagnostic approaches, therapies, and prevention.

Image; Mental Retardation and Developmental Disabilities Research Centers; Neurons

The frag ile X mental re tardation protein (FMRP) binds mRNA and micro RNA, is associated with polyribosomes, and is localized in dendrites and axons. Hence, FMRP is thought to reg ulate the local translation of its mRNA targets as a means to influence neuronal development and plasticity. The lack of FMRP resu lts in dysregulated protein synthesis, wh ich is an underlying pathomechanism for the deficits in synaptic plasticity and mental impairment in frag ile X syndrome. Our long term -goal is to elucidate how FMRP controls mRNA translation and local protein synthesis du ring normal neuronal development and how FMRP deficiency leads to fragile X syndrome, the most common form of inherited mental retardation. A decade of extensive studies have characterized the biochemical interactions between FMRP and its mRNA ligands. In fact, more than 400 mRNAs have been found to associate with FMRP. However, the molecular mechanims by wh ich FMRP controls translat ion of its mRNA targets are st ill poorly understood. Moreover, how dysregulated translation , as a result of FMRP deficiency, may lead to aberrant neuronal development in the frag ile X brain remains unknown. Several lines of evidence, including our previous work, suggest that the mRNA encoding microtubule associated protein 1 B (MAP1 B) is a funct ional target of FMRP, and the lack of FMRP resu lts in dysregulated MAP1 B translation in Fmr1 KO neurons. The goal of th is proposal is to use MAP1 B as a model target of FMRP to delineate molecular mechanisms for FMRP to regulate protein synthesis in response to neuronal activation and the functional importance of FMRP-dependent trans lational regulat ion in neuronal development. Two specific aims are proposed : 1) To del ineate how FMRP-dependent local translation of MAP1 B may control growth cone dynamics in repsonse to an axon gu idance factor and activation of group 1 metabotropic glutamate recepto r;2) To determine whether and how FMRP-mediated translat ional regu lation of MAP1 B governs projections of hippocampal mossy fiber axons during normal development and in epilepsy.

DESCRIPTION (provided by applicant): miRNAs have emerged as a powerful class of conserved noncoding RNAs that regulate gene expression post-transcriptionally and play critical roles in numerous aspects of development. Numerous miRNAs are expressed at high levels in the nervous system, where they regulate neural development and synaptic plasticity. Impaired expression of miRNAs are implicated in several neurological diseases. Recent studies have identified a few miRNAs that are localized to dendrites in vitro, and in one case, shown to regulate dendritic mRNA translation important for spine development. However, the identity of miRNAs localized to dendrites in vivo is unknown. Axonal miRNAs may regulate local protein synthesis underlying axon guidance, however, the identity of specific miRNAs within axons is unknown so far. We hypothesize that the levels of specific miRNAs within dendrites and axons can be regulated by neuronal activity and receptor signaling pathways to influence the regulation of local protein synthesis important for neuronal development. A major limitation of current technology is that it is unable to quantify changes in miRNA expression in neuronal processes. This project will develop and apply new technology that is capable to identify and quantify novel miRNAs within dendrites and axons. In aim-1, we will quantify miRNA localization in dendrites of cultured hippocampal and striatal neurons in response to neuronal activity and activation of neurotrophin, glutamate and dopamine receptors. GFP and luciferase reporters will be used to assess the role of a few candidate miRNA in the regulation of dendritic protein synthesis. In aim-2, we will use similar approaches to identify and assess the role of axonal miRNAs in the regulation of local protein synthesis underlying signaling by axon guidance factors. These studies will advance our understanding of the critical importance played by miRNAs in local protein synthesis underlying synaptic plasticity and axon guidance, which may be altered in disease states, including mental retardation, autism and drug addiction. The quantitative assays developed will be broadly applicable to quantify miRNA expression in models of neurological disease and drug addiction. This research has the potential to lead to the identification of new therapeutic strategies that involve manipulation of miRNAs. PUBLIC HEALTH RELEVANCE: This research will advance our understanding of the critical importance played by miRNAs in local protein synthesis underlying neuronal development, which may be altered in disease states, including drug addiction. The quantitative assays developed will be broadly applicable to quantify miRNA expression in models of neurological disease and drug addiction. This research has the potential to lead to the identification of new therapeutic strategies that involve manipulation of miRNAs.

DESCRIPTION (provided by applicant): A number of neurological and neuropsychiatric disorders, including fragile x syndrome, autism spectrum disorders, and drug addiction, may result from altered regulation of microRNAs. MicroRNAs (miRs) are small, conserved, noncoding RNAs that act in association with the RNA-induced silencing complex (RISC) to regulate gene expression post-transcriptionally. MicroRNAs are hypothesized to play a critical role in activity-regulated mRNA translation that controls neuronal development and synaptic plasticity. Our recent work has discovered a novel molecular mechanism for neurotransmitter-regulated protein synthesis that involves the release of a microRNA induced silencing complex (miRISC) from its target mRNA. This proposal will investigate whether this is used as a general mechanism for microRNAs to modulate activity mediated mRNA translation in neurons. We will identify new microRNAs and target mRNAs that utilize reversibility of the miRISC complex to regulate activity-mediated translation. A key question to be addressed is how different neurotransmitter signaling pathways may differentially affect microRNA/RISC targeting to mRNAs. Lastly, we will assess a role for phosphorylation of the fragile x mental retardation protein, FMRP, as a mechanism to regulate a subset of microRNAs at synapses. This research is expected to uncover new molecular mechanisms for activity mediated gene expression that allows for selective, dynamic and spatiotemporal control. The proposal will fill a critical gap in our understanding by characterization of a unifying molecular mechanism allowing for activity- regulated and sequence specific mRNA translation. Aim 1 will use candidate analysis and microarrays to test the hypothesis that neurotransmitter receptor signaling alters the interactions of microRNA/RISC complexes from target mRNAs as a novel mechanism for activity-regulated mRNA translation. Aim 2 will test the hypothesis that a subset of microRNAs and their regulated targeting to mRNAs by gp1 mGlu signaling is dysregulated in a mouse model of fragile x syndrome. This research has the potential to identify novel microRNAs regulated by neuronal activity, understand a unifying molecular mechanism for activity-regulated translation and help to elucidate the pathophysiology of a neurological disorder at the molecular level. This research is expected to have a broad impact on understanding how the posttranscriptional regulation of gene expression is dynamically controlled by RISC and miRNAs in response to neuronal activity to promote neuronal function, which may be altered in neurodevelopmental, neuropsychiatric disorders and drug addiction. These studies are envisioned to have important implications for future therapeutic strategies to manipulate activity-regulated protein synthesis in the treatment of neurological disorders and drug addiction.

DESCRIPTION (provided by applicant): MiRNAs have emerged as a powerful class of conserved noncoding RNAs that regulate gene expression post- transcriptionally and play critical roles in numerous aspects of neuronal development and synaptic function. Impaired expression and/or function of miRNAs is implicated in several neurological diseases. A major gap is our poor understanding on the localization of specific microRNAs to the axon growth cone, and whether they play important roles to regulate local protein synthesis dependent effects on growth cone motility and guidance. The objectives of this application are to explore the new concept that microRNAs (miRNAs) localized to growth cones provide a regulated molecular mechanism to influence local protein synthesis underlying cue mediated and local protein synthesis dependent axon guidance. We will test the hypothesis that growth cone microRNAs regulate local translation to modulate attractive versus repulsive steering. Quantitative fluorescent in situ hybridization on cultured cortical neurons will be used to assess possible growth cone localization for microRNAs enriched in axonal fractions. We propose approaches to visualize the localization of microRNAs in cultured cortical neurons, to manipulate axonal microRNAs and target mRNAs translation in microfluidic devices, and image fluorescent reporters for local translation and growth cone guidance in live neurons. Aim 1 will identify miRNAs that are localized to axons by profiling microRNAs from axonal fractions of cortical neuron balls and visualization of their localization using fluorescent in situ hybridizatin. Aim 2 will examine the effects of manipulating axonal microRNA levels and function on axon outgrowth, growth cone morphology and steering responses to attractive and repulsive cues. Aim 3 will examine the effects of select candidate microRNAs on axonal protein expression and local protein synthesis in growth cones using immunofluorescence and live cell imaging of fluorescent reporters. The proposed research will advance our limited understanding of growth cone localized miRNAs and elucidate their mechanistic role in local protein synthesis underlying growth cone guidance. This research is expected to motivate studies to investigate the function of axonal microRNAs in the development of the nervous system in vivo, as well as dysfunction of micoRNA regulation leading to neurological diseases. These studies have important implications for future therapeutic strategies to modulate microRNA expression or function to manipulate axonal growth and connectivity in the treatment of neurologic disorders.

DESCRIPTION (provided by applicant): There is a lack of effective therapeutic strategies to treat Fragile X syndrome (FXS), the most frequent inherited intellectual disability, and the primary monogenic cause of autism. A major challenge for the development of therapies for FXS is to understand the molecular mechanisms leading to the defects in receptor-mediated signaling and protein synthesis that may underlie the broad spectrum of neuronal dysfunctions in FXS. FXS is caused by loss of Fragile X Mental Retardation Protein (FMRP), which binds and translationally regulates many different mRNAs. A critical gap is absence of information on FMRP target mRNAs that are functionally relevant to the global dysregulation of neuronal protein synthesis in FXS. The major objective of the proposed research is to provide evidence that (1) dysregulated expression of the phosphoinositide-3 kinase enhancer (PIKE), a confirmed FMRP target mRNA, is a major mediator of impaired neuronal function in FXS, and that (2) reducing PIKE activity is a promising disease-targeted therapeutic strategy for FXS. Recent studies imply that FMRP is a key regulator of neurotransmitter-mediated PI3K signaling by translationally regulating PI3K signaling components: FMRP was suggested to associate with a few mRNAs coding for components of the PI3K signaling complex, and loss of FMRP leads to increased and dysregulated PI3K activity in mice and patient cells. The PI3K enhancer PIKE is a confirmed FMRP target mRNA within the PI3K complex, and expressed excessively in the absence of FMRP. PIKE is of special interest, because PIKE plays a central role in mediating metabotropic glutamate receptor 1 and 5 (mGlu1/5) - induced activation of PI3K downstream signaling and protein synthesis. Dysregulated mGlu1/5-mediated synaptic plasticity is a hallmark of FXS animal models, and negative modulators of mGlu1/5 receptors are currently in clinical trials with FXS patients; however, these therapies do not considerably ameliorate major phenotypes, such as cognition, probably because they are not targeting the underlying mechanism. The central hypothesis of this proposal is that dysregulated PIKE expression is a key pathomechanism of FXS and underlies phenotypes on the molecular, cellular and behavioral level, including dysregulated mGlu1/5 signaling. This hypothesis will be tested in four specific aims following a comprehensive and multidisciplinary strategy that uses two animal models of FXS, a mouse and a fly model, will employ two different rescue strategies targeting PIKE, genetic Pike knockdown and small peptide inhibitors disrupting mGlu1/5-PIKE-PI3K signaling, and will test phenotypes in four major domains, on the molecular, morphological, electrophysiological and behavioral level. Taken together, the proposed research may not only fill a major gap of knowledge by providing critical information about dysregulated PIKE as a potential mechanism underlying impaired signal transduction in FXS, but may also represent a first step towards developing a novel disease-targeted therapy by testing small peptide inhibitors reducing mGlu1/5-PIKE signaling to ameliorate FXS-associated phenotypes.

? DESCRIPTION (provided by applicant): Fragile X syndrome (FXS), caused by the inherited loss of the Fragile X Mental Retardation Protein (FMRP), is the most common form of inherited intellectual disability and the leading monogenetic cause of autism. FMRP is an mRNA binding protein that binds numerous mRNAs and often represses their translation. FMRP is detected at postsynaptic sites within dendritic spines where it is believed to play a role in local protein synthesis involved in synapse development and synaptic plasticity underlying learning and memory. Our long-term objectives are to characterize mechanisms of local protein synthesis in neurons, elucidate their functions in synapse development, and use this knowledge to develop strategies to restore synaptic protein homeostasis in fragile x syndrome and other neurodevelopmental disorders. A critical gap is lack of understanding of the underlying mechanisms of FMRP mediated regulation of local protein synthesis and how dysregulated translation may contribute to impaired synaptic protein homeostasis and dendritic spine development in fragile x syndrome. We hypothesize that FMRP is necessary for the activity dependent regulation of mRNA translation in dendrites and spines, and that loss of FMRP in FXS results in dysregulated translation and altered postsynaptic protein homeostasis leading to impaired synaptic development. To accomplish these goals, we will develop and employ novel fluorescent reporters and imaging assays to visualize and characterize novel mechanisms of FMRP mediated local protein synthesis in live cultured hippocampal neurons. Aim 1 will test the hypothesis that loss of FMRP in a mouse model of FXS results in dysregulation of mRNA translation in dendrites and spines. Aim 2 will test the hypothesis that loss of either FMRP, or mRNA target sequences involved in local protein synthesis, results in dysregulation of protein ubiquitination and homeostasis in dendrites and spines. The characterization of dysregulated protein homeostasis at synapses in fragile x syndrome has broader significance toward elucidation of the shared neurobiology of synaptopathies in autism spectrum disorders. This research will provide methods and rationale to assess other autism disease models and test therapeutic strategies that restore synaptic protein homeostasis.

? DESCRIPTION (provided by applicant): Local protein synthesis within dendritic spines has long been hypothesized to provide a synapse specific mechanism for the control of spine development and synaptic plasticity. An inherent difficulty in testing this hypothesis has been the lack of suitable methods to directly visualize and quantify local translation with sufficient spatil and temporal resolution. While numerous mRNAs have been localized to dendrites by in situ hybridization methods, only a few have been directly shown to be locally translated in dendrites using fluorescent reporters, and technical limitations often preclude visualization of the precise loci where translation occurs and how translational responses are regulated by physiological signals within a morphologic context. Thus, a critical gap is lack of suitable methods to address fundamental questions about the types of mRNAs that are translated in spines, and to ask questions about the underlying regulatory mechanisms and their function in protein sorting and spine development. Related to this major gap in knowledge, a fundamental but as yet unanswered question is whether membrane proteins can be translated in or near spines, and whether mechanisms exist to couple synthesis with trafficking to the cell surface in response to plasticity inducing stimuli. Our goal for the proposed research is to develop and apply a single molecule imaging approach using fluorescent reporters in microfluidic devices to visualize and quantify translational events within dendrites and spines. Aim 1 will investigate the hypothesis that local translation of receptors and channels occurs locally in spines and that mechanisms of synthesis and surface expression are coordinately regulated by plasticity inducing stimuli. Aim 2 will investigate the hypothesis that dendritic microRNAs are necessary for both the activity regulated local translation and surface expression of membrane proteins in response to plasticity inducing stimuli. We anticipate that development and application of this technological approach will uncover novel mechanisms for receptor-mediated regulation of local mRNA translation in dendritic spines that play important roles in protein trafficking and neuronal development. We anticipate that the proposed research will lead to a new perspective on how local translation is under spatiotemporal control by synaptic mechanisms that are likely impaired in neurologic disorders, including epilepsy, fragile x syndrome and schizophrenia.

SUMMARY The primary goals of the ENNCF Imaging core are to provide: (1) access to state-of-the-art light microscopy including confocal and widefield deconvolution microscopy, live cell imaging, multi-photon animal and tissue imaging, total internal reflection fluorescence (TIRF) microscopy, super-resolution Structured Illumination Microscopy (SIM) and image analysis equipment including Imaris suite, deconvolution and emerging technologies; (2) expertise and guidance on use of microscopy equipment, research design, image analysis and services through direct consultation with Core director and core personnel; and (3) an outstanding research environment that provides infrastructure, education and resources to foster collaborations between investigators and cores through organization of workshops, data clubs and user group meetings and maintenance of the Imaging core website with regular updates on recent technologies and data analysis platforms. These objectives implemented by the imaging core have proven to stimulate multidisciplinary research and innovation in basic and translational neuroscience.

Fragile X syndrome (FXS), caused by the inherited loss of the Fragile X Mental Retardation Protein (FMRP), is the most common form of inherited intellectual disability and the leading monogenetic cause of autism. FMRP binds to many target mRNAs encoding proteins that play key roles at the synapse. FMRP has been shown to repress translation of many target mRNAs, and a few mechanisms have been proposed. FMRP interactions with microRNAs have been shown to play a role in translational control but the molecular mechanisms are not well understood. FMRP mediated repression of translation is reversibly regulated and dependent on the phosphorylation status of FMRP. FMRP has also been shown to be ubiquitinated in response to glutamate receptor stimulation, providing a potential mechanism to dynamically remove translational repression. It remains unclear whether any of the above mechanisms occur locally within dendrites to regulate local translation important for protein synthesis dependent synaptic plasticity. It is likely that some or all of these mechanisms are inter-related but critical details are lacking to understand FMRP mediated translational control and its reversibility in response to receptor signaling. A critical gap is lack of a unifying model for FMRP mediated repression and its reversible regulation at the synapse. We hypothesize that FMRP ubiquitination and UPS-mediated degradation in response to receptor stimulation provides a unifying mechanism to remove translational repression and regulate local protein synthesis at the synapse. The specific role of the E3 ligase Cdh1-APC in FMRP mediated regulation of local protein synthesis will be investigated. To elucidate the local functions of these mechanisms within dendrites and spines, we will continue to develop and apply fluorescent reporters and single molecule imaging of live cultured hippocampal neurons. Using dissociated and organotypic slice cultures as model systems, the role of UPS mediated FMRP degradation, as a local translational switch, to regulate spine morphology, synapse development and plasticity will be investigated. We will analyze the role of FMRP mutants that are resistant to ubiquitination or unable to bind Cdh1-APC to modulate or rescue FXS-associated impairments in dendritic spine development, synapse function and plasticity. Aim 1 will test the hypothesis that FMRP dephosphorylation, ubiquitination by Cdh1-APC and UPS- mediated degradation are components of a dynamic molecular switch to regulate local mRNA translation that functions in control of dendritic spine morphology, synapse development and plasticity. Aim 2 will test the hypothesis that FMRP ubiquitination and UPS-mediated degradation provides a mechanism to regulate targeting of RISC/microRNAs. This research is expected to uncover a novel role for Cdh1-APC and FMRP ubiquitination in regulation of microRNAs and local protein synthesis. The development of disease mechanism based therapeutic strategies for FXS will benefit from this in depth understanding of the mechanism and function of FMRP mediated control of local mRNA translation at synapses.  

Project Summary/Abstract 3q29 deletion syndrome is caused by a recurrent typically de novo 1.6 Mb heterozygous deletion and is associated with a range of neuropsychiatric phenotypes, including mild to moderate intellectual disability, autism, anxiety, and a 40-fold increased risk for schizophrenia. Although the 3q29 deletion is rare (~1 in 30,000 births), its high risk for neuropsychiatric phenotypes coupled with its relatively low complexity (22 genes in the deletion interval) make it ideal for molecular dissection. Investigating the neuronal phenotype caused by 3q29 deletion may reveal a core neurodevelopmental process that is disrupted in schizophrenia, autism, and/or intellectual disability. We propose to assess deletion carriers for behavioral traits along 4 dimensions: cognitive ability, anxiety, autism spectrum, and presence of psychosis or prodromal features. We will also collect blood samples from deletion carriers and related controls and bank these materials in the Rutgers University Cell and DNA Repository (RUCDR) for use by the research community. Finally, we will model the human neuronal phenotype using iPSC lines derived from deletion carriers who have psychosis. This will be the first human neuronal model of the 3q29 deletion. Understanding the specific biological processes disrupted by deletion of the 22 genes in this interval may provide a molecular window into key neurodevelopmental processes relevant to neuropsychiatric phenotypes. All phenotypic data, molecular data, and cell lines will be rapidly shared through NDAR, dbGaP (dbgap.ncbi.nlm.nih.gov) and the NIMH Repository and Genomics Resource (nimhgenetics.org).

Expanded GGGGCC (G4C2) hexanucleotide repeats in the C9orf72 gene were recently identified as the most common genetic cause of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD), two neurodegenerative disorders with genetic and pathological overlap. Repeat-RNA-toxicity mediated by sequestering key RNA binding proteins is thought to play key roles in c9ALS/FTD pathogenesis. However, which RNA binding protein(s) might be sequestered by long G4C2 repeat RNAs is still unknown. We propose to identify RNA-protein interactions in c9ALS/FTD using disease-relevant repeat lengths and cell models, and by comparison to interacting-proteins of (TG3C2) repeats, a similar repeat expansion that leads to a clinically disparate disease Spinocerebellar Ataxia type 36 (SCA36). The identified RNA binding protein(s) by the G4C2 repeats will be further validated in c9ALS/FTD patient postmortem brain samples and its functions will be studied in iPS-derived motor neurons and in mice. Results from this proposal will provide new insight into the cellular cascades that cause neurodegeneration in c9ALS/FTD.

Myotonic dystrophy (dystrophia myotonica, DM) is an autosomal dominant genetic disease, with a diagnosed prevalence of 1:8000 people worldwide, that affects multiple tissues of the body, including skeletal muscle, heart, and related to this proposal, the central nervous system (CNS). DM1 is caused by expanded CTG repeats in the 3' UTR of dystrophia myotonica protein kinase (DMPK). There is substantial evidence in mouse DM1 models and human DM1 postmortem tissue to support an RNA-mediated disease mechanism where toxic intranuclear CUG RNA foci sequester Muscleblind (MBNL) RNA binding proteins that normally play crucial roles to regulate various aspects of post-transcriptional gene regulation. A major gap in our understanding is that we do not know which RNA processing defects underlie specific impairments in DM1 brain function. Recent work together with our new findings suggests that missplicing of RNAs encoding synaptic proteins is responsible for CNS dysfunction in DM1. Our central hypothesis is that CNS phenotypes are directly attributed to loss of MBNL mediated RNA processing and that restoration of MBNL activity and/or splicing can restore brain function. Our goal is to gain a thorough understanding of RNA processing-mediated mechanisms of CNS dysfunction in DM1 and use this to develop and rigorously evaluate novel therapeutic strategies. The overall objectives of this proposal are to use both candidate and genome wide approaches, applied to MBNL KO mice and a new AAV9 based neuronal mouse model, compared to RNAseq analysis of human postmortem brain, to evaluate the role of specific splicing events to drive symptoms, and to comprehensively identify changes in missplicing and RNA processing. Aim 1 will characterize how dysregulation of GABRG2, GRIN1, and SNAP25 splicing events is linked to molecular, cellular, and behavioral phenotypes observed in DM1. Aim 2 will develop a new AAV9 based mouse model to elucidate the set of RNA processing events in neurons that cause DM1 phenotypes, through transcriptional profiling and overlap of human DM brains with DM mouse model brains. Aim 3 will assess the extent to which antisense oligonucleotides or MBNL expression can rescue molecular, cellular, physiologic and behavioral phenotypes in DM1 mouse models. These studies will provide new mechanistic insights into how perturbations to specific RNA processing events can lead to CNS symptoms in myotonic dystrophy, and provide a broader comprehensive view of all transcriptome changes occurring in the DM CNS. The proposed work is significant, as no molecular changes have been linked to any phenotypes in the DM CNS. This provides the framework for future therapeutic efforts aimed at correcting CNS defects.

The development and maintenance of neurons and synaptic connections are highly complex processes, in part due to the massive cytoplasmic volume and complex branching morphology of axons and dendrites. As one mechanism, it is well appreciated that RNA localization and local translation are required to precisely regulate protein homeostasis at synapses. Indeed, loss of FMRP in Fragile X Syndrome, or other impairments to RNA localization and local translation at synapses, likely contribute to brain disorders. To better understand RNA localization and local translation in neurons, we must elucidate the RNA cis-elements, RBP trans-factors, and cytoskeletal motors mediating these processes. Although ongoing efforts have demonstrated how RNA binding proteins (RBPs) can regulate local translation at post-synaptic sites, there still exists a major gap in our understanding of how RBPs transport RNAs to regulate synaptic function. Fortunately, recent observations provide clues about fruitful lines of investigation. For example, multiple studies report that distally localized RNAs are enriched for cis-elements targeted by Muscleblind-like (MBNL) proteins. Although these observations suggest that MBNL may be a major player in localizing RNAs to the pre- and post-synapse, we still lack a mechanistic understanding for how MBNL proteins may achieve this task, or what functions depend on MBNL-mediated RNA localization. This line of research has important implications for the neurological disease myotonic dystrophy (dystrophia myotonica, DM), in which MBNLs are depleted by toxic CUG repeats. Therefore, an emerging hypothesis is that RNA localization functions of MBNL are important for proper synapse function, and that mis-localized RNAs might account for some neurological features of DM patients, particularly early in disease. Here, using MBNL depletion and DM-associated models, we propose to identify specific functions for the localization of MBNL targets. Aim 1 will elucidate mechanisms of MBNL-mediated mRNA localization in neurons. We will define the RNA targets that are localized by MBNL in the pre- and post-synapse. We will characterize dynamic properties of motile MBNL RNA granules in live neurons and identify cytoskeletal motors and adaptors associated with these granules. Using genomics, live cell imaging, and biochemical approaches, we will establish mechanisms of how MBNL-interacting RNAs are transported. Aim 2 will define functions conferred by MBNL-dependent RNA localization using models of synapse development and function, and models of myotonic dystrophy. By depleting cytoplasmic MBNL and other proteins required for MBNL-dependent RNA localization, we will assess cellular functions dependent on this process. We will identify specific neuronal functions, such as synaptic vesicle release, that depend on proper localization of mRNAs by MBNL proteins. The impact of this research is to better understand how RNA localization and local translation confers important synaptic functions and how they may go awry in DM. As few RNA binding proteins have been linked to motors, this may evolve into a unifying model for mRNA transport to synapses.

Project Summary Previous work in animal models of fragile X syndrome (FXS) has provided invaluable insight into the normal molecular, cellular, and physiological functions of fragile X mental retardation protein (FMRP); however, an effective treatment remains elusive. Although these failures could be attributed to several factors, it is now apparent that it is imperative that FXS-associated phenotypes, the efficacy of drugs, and rescue strategies characterized in animal models of FXS be validated and/or new phenotypes characterized in human FXS patient- derived, disease-relevant cell types. A critical limitation is lack of an available human FXS patient-derived neural model to investigate the role of FMRP-mediated regulation of protein synthesis and signaling. We have recently developed multiple human iPSC-derived 2D neural and 3D cortical organoid models to investigate the role of FMRP-mediated regulation of protein synthesis and signaling during brain development. The objectives of Project 1 are to use these FXS patient iPSC-derived 2D monolayers as well as 3D cortical and hippocampal organoids to address questions delineated in three specific aims. Aim 1 is to characterize protein synthesis dysregulation and associated molecular, cellular and neurophysiological phenotypes in specific cell types across neural development in human FXS iPSC neural models. Our preliminary data indicate that FXS patient cells have increased protein synthesis rates, increased proliferation and altered migration, resulting in delayed acquisition of cell fate and neuronal differentiation. These early neurodevelopmental defects are anticipated to have consequences on neuronal development and function. Aim 2 is to identify FMRP targets and translationally dysregulated mRNAs during brain development in multiple human FXS iPSC neural models. Using CLIP-seq we have identified FMRP target mRNAs in both human cortical organoids and mouse embryonic cortex at similar developmental stages. Our comparative analyses have revealed three groups of FMRP mRNA targets, human only, mouse only and shared ones. We have also recently used ribosome profiling to identify translationally dysregulated mRNAs, some of which are FMRP targets, in whole cortex in the adult mouse brain. Thus, ribosome profiling will be applied to characterize the translatomes of FXS patients and controls using both isogenic i3Neurons and i3Neurons from multiple patients, as well as from isogenic 3D cortical organoids. For comparison between FXS models, we also will conduct ribosome profiling of FXS mouse embryonic cortex. In Aim 3, we will devise targeted strategies to rescue cellular and synaptic phenotypes in human FXS iPSC neural models. We will manipulate expression of dysregulated FMRP targets using lentivirus-based approaches to rescue FXS- associated cellular and synaptic phenotypes. The outcome of the experiments in this Project, coupled with synergy with the other projects, will uncover novel mechanisms and key drivers of FXS-associated phenotypes in cortical development using our newly generated human iPSC-derived 2D and 3D neural models.