Mustafa Sahin - US grants

The goal of this project is to characterize the signaling pathways that regulate synaptogenesis in the developing brain. Previous studies have demonstrated that synapses differentiate in a series of steps, one of which is the clustering of postsynaptic neurotransmitter receptors. In the neuromuscular junction, clustering of the cholinergic receptors is induced by a proteoglycan (agrin), which activates a receptor tyrosine kinase (MuSK). In the central nervous system, it is not clear what regulates the clustering of postsynaptic receptors such as the NMDA receptors at excitatory synapses. Preliminary data from the Sponsor's laboratory indicate that activation of Eph receptor tyrosine kinases on cultured hippocampal neurons induces the clustering of a NMDA receptor subunit, NR1. Furthermore, Eph receptor ligands (ephrins) induce a biochemical interaction between the Eph and NMDA receptors. This study will examine the protein-protein interactions that mediate ephrin-induced NMDA receptor clustering through three specific aims. First, we will identify the domains of Eph and NR1 receptors that are critical for ephrin-induced receptor clustering. Then, we will investigate the role of tyrosine phosphorylation on the clustering of NMDA receptors by Eph tyrosine kinases. Finally, the cellular mechanisms of NMDA receptor clustering will be examined through two sets of experiments. We will present the ephrin ligands on the cell membrane and determine their effect on the NMDA receptors on neighboring neurons. Mechanisms of synapse formation are of particular interest to pediatric neurologists because aberrant synapse formation is likely to underlie common developmental disorders such as mental retardation and epilepsy. Through intensive training in basic science under the supervision of Dr. Michael Greenberg and clinical practice in pediatric neurology at the Children's Hospital in Boston, the candidate expects to become a physician- scientist equipped with the molecular and cellular tools to study neuronal dysgenesis in children.

DESCRIPTION (provided by applicant): Tuberous sclerosis complex (TSC) is an autosomal dominant disease characterized by the presence of benign tumors, called hamartomas, which can affect virtually every organ system of the body. The hallmark of TSC and the predominant cause of morbidity in this disease are the neurological symptoms, such as epilepsy, autism and mental retardation, which occur in 95% of affected individuals. Over the last decade, significant progress has been made in identifying a function for TSC in regulating protein synthesis and cell size. However, the cause of the neurological symptoms in TSC patients has not yet been resolved. We propose a set of experiments to investigate neuronal connectivity and network formation in mouse models of TSC. These experiments are based on our recent observation that TSC pathway components are expressed in axons of hippocampal neurons at high levels starting with the initial stages of axonal polarization. Furthermore, we find that in mice lacking Tsc2, topographic mapping of connections in the brain are aberrant compared to wild-type mice. These findings have led us to hypothesize that TSC proteins are crucial not only for determining cell size, but also for determining axonal connectivity in the brain. We propose two sets of experiments to elucidate the role played by TSC1/TSC2 proteins in the establishment of axonal polarity and the mediation of axonal pathfinding. By using dissociated cultures, acute brain slices, and brain sections, we will first characterize the role of TSC proteins in the establishment of neuronal polarity. In the second phase of the project, we will characterize the role of TSC proteins in axon pathfinding and growth cone dynamics. Ultimately, our work will determine the role of TSC proteins in neuronal network formation by examining the visual pathways in Tsc-deficient mice - the best-studied and most accessible axon pathway in the central nervous system. The operations of TSC protein complex may be the key to a fuller understanding of the abnormal neural networks of TSC. Our results, thus, may suggest further avenues of exploration to illuminate the complex causes of the devastating neurological symptoms that characterize TSC.

DESCRIPTION (provided by applicant): Autism Spectrum Disorders (ASD) has complex and varying etiologies. A lack of understanding regarding the underlying molecular and cellular mechanisms is a key barrier in finding effective treatments. Single-gene disorders that have a high prevalence of ASD provide unique opportunities to investigate the underlying biology and test treatments for autism. Tuberous Sclerosis Complex (TSC) is a genetic disorder in which approximately 50% of individuals are also affected with ASD. Importantly, TSC can be diagnosed before or at the time of birth, and thus infants with TSC allow us to observe prospectively the natural history of ASD and develop better tools for early detection of autism. On a molecular level, TSC disease manifestations result from the aberrant hyperactivity of mTOR that is caused by mutation in one of two TSC genes. Pharmacologic mTOR inhibition to correct the cellular defects in TSC is no longer a hope for the future, but rather an exciting reality with proven efficacy against various TSC disease manifestations. To determine the potential benefit these agents may have in treating or preventing ASD, it is imperative to identify early markers of autism in infants with TSC, so as to not put young children who will not develop ASD at unnecessary risk. Accumulating clinical and basic science evidence suggests that aberrant white matter connectivity represents a rational candidate as a biomarker in TSC. TSC mouse models demonstrate defects in the specification, guidance, and myelination of axons. More importantly, several groups have reported abnormalities in the normal-appearing white matter of TSC patients that can be identified by MR imaging, and loss of white matter microstructural integrity is associated with neurological and cognitive deficits. Furthermore, there is preliminary data indicating that white matter integrity can be improved by treatment with mTOR inhibitors in both animal models and in TSC patients. Taken together, these findings lead to the hypothesis that longitudinal assessment of white matter integrity and neural connectivity in TSC infants, through advanced MRI and EEG analysis, can be used as an early biomarker of subsequent ASD in this genetic disease. This proposal aims to establish a consortium of five Children's Hospitals that are geographically-distributed throughout the US to recruit TSC patients in the first year of life to test this hypothesis. State of the art imaging with 3Tesla MR scanners, advanced EEG technology, validated neurodevelopmental assessment tools, genetic analysis, and standardized clinical measures through age 36 months will be utilized. The clinical consortium will be supported by a centralized Data Coordinating Center with experience in another rare disease (neurofibro- matosis). Collaboration with Leadership Education in Neurodevelopmental and Related Disabilities (LEND) programs at each center will provide additional interdisciplinary research training and education expertise in ASD and TSC. As a result of the research outlined in this proposal, better understanding of brain connectivity and it relationship to ASD in TSC will pave the way for new interventions for this and related causes of autism.

DESCRIPTION (provided by applicant) Autism spectrum disorder and intellectual disability (ASD/ID) are severe neurodevelopmental conditions with early childhood onset. Advances in genetics have illustrated that ASD/ID represent a spectrum of rare disorders and that mutations in hundreds of genes may result in susceptibility to ASD/ID. This heterogeneity represents significant challenges but at the same time unique opportunities for research in the field of ASD/ID. Many of the genes implicated in ASD/ID appear to converge on a few common pathways, suggesting that there may be a common dysfunction at the cellular or systems level. Deeper understanding of the shared pathophysiology of these diseases may serve as gateways for understanding mechanisms of other causes of ASD/ID and for shared treatment possibilities. Here we focus of three well-established genetic syndromes that are associated with high penetrance for ASD/ID: TSC1/2, PTEN and SHANK3 mutations. Specific aims for TSC are: 1) characterize the developmental phenotype of ASD and ID in a large cohort of pediatric patients with TSC; 2) identify biomarkers using advanced MR imaging; 3) establish infrastructure for the collection and storage of human bio-specimens, including genetic material, from TSC patients and their family members with ASD. Specific aims for PTEN are: 1) determine cross-sectional and longitudinal medical, behavioral, and cognitive differences between PTEN ASD and other groups; 2) identify cognitive, neural systems, and molecular biomarkers specific to PTEN ASD; 3) create and maintain a biorepository and linked phenotypic database for PTEN ASD. Specific aims for SHANK3 are: 1) characterize PMS using standardized medical, behavioral, and cognitive measures and to track the natural history of the syndrome using repeated longitudinal assessments; 2) identify biomarkers using advanced MR imaging; S) identify genetic factors that contribute to diverse phenotypes in patients with PMS. As detailed in the resources sections, this Consortium involves experienced physician researchers from premier academic institutions with strong institutional support, impressive mentors for training of future physician researchers, and long-standing connections to patient advocacy organizations with extensive recruitment networks.

The Developmental Synaptopathies Consortium (DSC) Administrative Unit will facilitate the collaboration and communication of clinical research in the rare diseases of TSC, PTEN and PMS across the ten participating sites. The Administrative Unit will ensure collaboration among Project Leaders, Principal Investigators, and site study staff for the longitudinal research in each of the three identified diseases, the training component, the clinical pilot studies, and the access to information related to the rare diseases for basic and clinical researchers, other health professionals and the public.

Autism spectrum disorder and intellectual disability (ASD/ID) are severe neurodevelopmental conditions with early childhood onset. Advances in genetics have illustrated that ASD/ID represent a spectrum of rare disorders and that mutations in hundreds of genes may result in susceptibility to ASD/ID. This heterogeneity represents significant challenges but at the same time unique opportunities for research in the field of ASD/ID. Many of the genes implicated in ASD/ID appear to converge on a few common pathways, suggesting that there may be a common dysfunction at the cellular or systems level. Deeper understanding of the shared pathophysiology of these diseases may serve as gateways for understanding mechanisms of other causes of ASD/ID and for shared treatment possibilities. Here we focus of three well-established genetic syndromes that are associated with high penetrance for ASD/ID: TSC1/2, PTEN and SHANK3 mutations. Specific aims for TSC are: 1) characterize the developmental phenotype of ASD and ID in a large cohort of pediatric patients with TSC; 2) identify biomarkers using advanced MR imaging; 3) establish infrastructure for the collection and storage of human bio-specimens, including genetic material, from TSC patients and their family members with ASD. Specific aims for PTEN are: 1) determine cross-sectional and longitudinal medical, behavioral, and cognitive differences between PTEN ASD and other groups; 2) identify cognitive, neural systems, and molecular biomarkers specific to PTEN ASD; 3) create and maintain a biorepository and linked phenotypic database for PTEN ASD. Specific aims for SHANK3 are: 1) characterize PMS using standardized medical, behavioral, and cognitive measures and to track the natural history of the syndrome using repeated longitudinal assessments; 2) identify biomarkers using advanced MR imaging; 3) identify genetic factors which contribute to diverse phenotypes in patients with PMS. As detailed in the Resources sections, this Consortium involves experienced physician-researchers from premier academic institutions with strong institutional support, impressive mentors for training of future physician-researchers, and long-standing connections to patient advocacy organizations with extensive recruitment networks.

CLINICAL TRANSLATIONAL CORE (CORE B) ABSTRACT The objectives of the new Clinical Translational Core of our IDDRC are to accelerate the translation of research discoveries into new treatments for neurodevelopmental disorders, through collaboration with basic scientists and clinicians, as well as to train future leaders in translational neuroscience. Resources offered by the Clinical Translational Core include preclinical support through a Human Neuron Core Component, composed of a Cellular Assay Development and Screening Service and a Human Neuron Differentiation Service. Additionally, support for translational work includes resources through a Clinical and Regulatory Affairs Service, a Data Analysis Core Component, Biorepository and preclinical consultation. The core has a proven record of success in designing and launching preclinical and clinical projects for IDDRC investigators.

Project Summary/Abstract: The Translational Post-doctoral Training in Neurodevelopment (TPND) Program at Boston Children's Hospital is designed to provide promising post-doctoral neuroscience investigators (MD, PhD or MD/PhD) with advanced training in essential translational topics ranging from preclinical considerations through implementation of clinical trials for individuals with a range neurodevelopmental disorders. Large numbers of children, adolescents, and adults are affected by neurodevelopmental disorders that begin early in life, are rooted in aberrant brain circuitry, and have profound short- and long-term consequences on critical domains of development, cognition, social interaction, and behavioral regulation; yet most available medical and psychological treatments have had limited impact on the course of neurodevelopmental disorders. We propose to build on the significant strengths of the Translational Neuroscience Center (TNC) and the Laboratories of Cognitive Neuroscience at Boston Children's Hospital and Harvard Medical School to provide trainees with research experiences ranging from pre-clinical and cognitive neuroscience labs through clinical trial involvement in neurodevelopmental disorders. In addition, a key premise of this program is that effective research training in the field of translational neuroscience requires both mentors with expertise in areas across the continuum of translational research with neurodevelopmental disorders and ongoing programs to support the trainees to conduct innovative, high impact translational research. The 17 faculty mentors will involve trainees over a 2 year period (2 new entrants per year) with a range of state of the art methods in translational neurodevelopmental science that reflect core areas of the TNC program including basic science and translational methods as well as application to clinical populations in therapeutic trials for neurodevelopmental disorders. This research experience will be supplemented with both didactic and clinical immersion experiences designed to provide trainees with the skills needed to be successful independent investigators in this critical and emerging field. Ultimately this research experience will yield treatments that have an impact on the field by reducing the burdens and costs of care and costs to society that is now associated with neurodevelopmental disorders. In addition, the TPND will develop models of interdisciplinary research training for promising young scientists that will have a transformative impact on the field.

An e?cient administrative structure is essential to the design and implementation of the Developmental Synaptopathies Consortium (DSC). The overarching goal of the Administrative Core is to create an environment in which scientific research relevant to the goals of the DSC can be effectively and efficiently designed, conducted and disseminated. This Core was very successful in establishing an administrative and management structure for the DSC in the first five years of funding and has worked closely with patient advocacy groups to raise additional funds, recruit subjects and disseminate information with respect to the three rare disorders: TSC, PHTS and PMS. The Core will be staffed by senior physician/scientists as well as a biostatistician, a project manager and a regulatory manager, all of whom are experienced in multi-site clinical research projects. The Administrative Core focuses on facilitating communication, collaboration, planning, data sharing, and scienti?c and ?scal oversight of all research projects for the DSC. The Administrative Core will organize the investigator launch meeting as well as the Executive Advisory Committee, Scholarly Awards Committee, Steering Committee, study staff and neuropsychologist meetings at regular intervals. The Administrative Core will be responsible for the following Speci?c Aims: 1) coordinating clinical research projects across all three rare disorders leveraging the expertise of the neuropsychology and biomarker teams; 2) developing superior core processes (Pilot Feasibility Core and Career Enhancement Core), meeting the needs of investigators and trainees; 3) serving as a liaison to other organizations, including PAGs, the NIH, and the RDRCN Network; 4) disseminating the results of DSC-related research to the patient and investigator communities; 5) promoting a cohesive and integrated consortium in order to maximize the combined efforts of our investigators and resources ? which will be achieved through detailed communication and operational planning; and 6) providing outstanding administrative leadership with respect to managing day- to-day operations and maintaining high-quality, efficient and cost-effective services throughout the DSC.

Tuberous Sclerosis Complex (TSC) is a multisystem genetic disorder affecting several organs. Individuals with TSC suffer from refractory epilepsy, intellectual disabilities and autism spectrum disorder, and the neurological manifestations are often the most disabling for these patients. Central nervous system (CNS) manifestations include disorganized brain connectivity, increased astrogliosis, and presence of immature dysmorphic neurons. Despite the fact that increased mTORC1 activity has been clearly implicated in the brain manifestations of TSC, a critical unmet medical need remains to identify the downstream molecular pathways implicated in the abnormal brain development. Ciliopathies are genetic disorders caused by mutations in genes affecting primary cilia which are sensory cellular antenna with a role in brain homeostasis and development, thought to act as a key regulatory node for sonic hedgehog signaling. Ciliopathies encompass a range of genetic disorders that share ciliary dysfunction and can affect several organs, including the brain. This proposal builds on robust in vitro and in vivo data indicating that certain brain abnormalities of TSC recapitulate the manifestations of ciliopathies with alteration in the Shh signaling pathway. In particular, we found reduced ciliation in Tsc2-knockdown hippocampal neurons, in neuronal-specific Tsc1 and Tsc2 conditional knockout mouse models and in the giant cells of the cortical tubers of TSC patients. Notably, defective ciliogenesis was associated with an altered Shh signaling pathway and presence of immature neurons. To gain insights into the molecular mechanism implicated in defective ciliation, we performed a phenotypic screen in the Tsc2-deficient neurons and identified the heat shock protein hsp90 as a drug target that reverses altered ciliation independently from mTORC1 hyperactivation. Using a high throughput cell-based assay, we uncovered the existence of a therapeutic window for cilia restoration through hsp90 inhibition, without affecting TORC1 activation. These findings enable us to build our central hypothesis that hsp90 is the mTORC1 downstream target responsible for the ciliopathy-like phenotype seen in TSC. Here, we propose to determine the mechanistic basis by which hsp90 inhibition restores cilia in Tsc2 deficient neurons using multiple pharmacological and genetic techniques and by identifying the hsp90 interactome in the TSC1/2 mutant neurons. Presence of immature neuronal properties, astrogliosis, and aberrant regulation of the Shh pathway are some of the neuronal TSC manifestations that will be investigated to establish the functional relevance of restoring cilia. Finally, we will first examine cilia in cortical neurons generated from TSC patient-derived induced pluripotent stem cells (iPSCs) and their isogenic controls. We will perform preclinical pharmacokinetics/pharmacodynamics (PK/PD) assessment of brain penetration and exposure of hsp90 inhibitors in mice. Taken together, the outcome of these experiments will help elucidate the downstream pathways affected by hsp90 in TSC, provide fundamental insights into cilia biology and potentially shed light for other ciliopathies caused by dysfunctional heat shock response.

Project Summary Novel technologies to advance discovery of disease mechanisms and therapeutics Genetic syndromes with increased prevalence of autism spectrum disorder (ASD) and intellectual disability (ID) offer an opportunity to understand the brain pathophysiology that manifests as ASD; this knowledge can suggest potential targeted therapies. It is now clear that the genetics underlying ASD are complicated; with at least several hundred genes conferring small amounts of risk. More recently, studies have turned to genomic sequencing and found several rare variants in individuals with ASD. These studies suggest a high degree of convergence on particular cellular processes and biochemical pathways, suggesting there may be convergence of underlying mechanisms and potential therapeutic strategies. The study of monogenic or syndromic forms of ASD, has been a leading strategy to gain insight into the complex mechanisms of ASD. Fragile X Syndrome (FXS) is one of the most common inherited forms of ASD and ID. Since the Fragile X gene (FMR1) was cloned in 1991, the field has used cellular assays and model organisms to elucidate the functions of the FMR1 protein (FMRP), the consequences of its loss and identify therapeutic targets for FXS and ASD. Other syndromic forms of ASD, such as tuberous sclerosis complex, Rett Syndrome, Angelman Syndrome and others, are being investigated using similar approaches. Recent technological advances in stem-cell derived neurons, single cell sequencing, gene therapy and novel model organisms are setting the stage for transformative advances in therapeutic development for these neurodevelopmental disorders. This conference will bring together leading scientists and clinicians studying FXS, ASD and related neurodevelopmental disorders with the ultimate goal of identifying the mechanisms that underlie the heterogeneous symptomatology associated with these disorders and the best potential pathways for translating this knowledge into clinical trials.

ABSTRACT Our overarching vision of the Boston Children?s Hospital and Harvard Medical School Intellectual and Developmental Disorders Research Center (IDDRC) is to improve the lives of individuals with IDD with timely and efficient translation of scientific research through collaboration among our institutions? exceptional investigators and clinicians in partnership with the external IDD community. To achieve our broad vision, we organize the Center?s research around four clearly defined themes: 1) Discovery of genetic and non-genetic causes of IDD; 2) Determination of the cellular bases of IDDs using advanced imaging and analysis tools; 3) Identification of translational phenotypes in animal models of IDD to validate therapeutics; 4) Accelerated translation of research discoveries into new prevention and treatment strategies for IDDs. The Center currently supports 106 research projects and 68 investigators through the Administrative Core and the four scientific Cores. The Administrative Core is the hub of the Center as it provides both scientific and administrative leadership which promotes synergistic, interdisciplinary interactions that address IDD-related issues at multiple levels, trains the next generation of young investigators and facilitates outreach and dissemination of IDD research to diverse audiences. The Genetic Analysis and Editing Core (GAEC) provides access to the latest technological advances both in genetic analysis and in gene editing. The Cellular Imaging Core (CIC) facilitates the study of cellular and circuit biology through state-of-the-art imaging and image analysis services which enable visualization of fixed tissue, in vitro organ explants and in vivo awake behaving model organisms. The Animal Behavior and Physiology (AB&P) Core provides investigators with access to a wide range of validated technologies and scientific expertise for in vivo rodent behavioral, biochemical and physiological measures in a well-controlled and rigorous preclinical setting. Finally, the Clinical Translational Core (CTC) provides full access for IDDRC PIs to all services required for translation of research discoveries into clinical innovation; this ranges from biosample collection and storage, generation of patient-derived stem cell models of diseases, drug screening platforms on the preclinical side to neurobehavioral and electrophysiological assessment of IDD patients of different age groups as well as statistical and regulatory support of clinical trials. The Cores all interact through shared projects, providing complementary expertise and tools to address unique aspects of central scientific questions, and the Core directors meet to exchange ideas and optimize resource utilization in the monthly Executive Committee meetings. This integrated approach aims to enhance the translational potential of basic research in IDD by putting patients at the center of the drug discovery cycle starting with genetic and molecular screens through clinical trials. The Center engages frequently with patient- advocacy groups and the IDD community in a bi-directional manner to ensure that their needs and concerns steer the Center?s efforts.

CLINICAL TRANSLATIONAL CORE ABSTRACT The objectives of the Clinical Translational Core (CTC) of our IDDRC are to accelerate the translation of research discoveries into new treatments for neurodevelopmental disorders, through collaboration with basic scientists and clinicians, as well as to train future leaders in translational neuroscience. Services offered by the Clinical Translational Core to support translational work include three sub-cores: 1) Patient-based Preclinical Services, comprised of a Biorepository and the induced pluripotent stem cell (iPSC) based Human Neuron Unit, 2) Clinical Phenotyping Services, consisting of the Neurobehavioral Unit and the Neurophysiology Unit, and 3) Clinical Trial and Regulatory Services, containing the Regulatory Unit and Research Participant Registry. The suite of services offered by the CTC enables investigators to pursue an integrated approach to therapeutic discovery for neurodevelopmental disorders, from patient derived samples in preclinical research to biomarker development to natural history and interventional clinical trials. Sub-core 1 - Patient Based Preclinical Services - maintains the regulatory approval to collect patient samples that can be used for primary research or for the generation of iPSC lines and neuronal differentiation within the Human Neuron Unit. Sub- core 2 - Clinical Phenotyping Services - provides expertise and access to high density EEG equipment as well as direct phenotyping through neuropsychological assessment and neurophysiological recording. Sub-core 3 - Clinical Trial and Regulatory Services - provides expert consultation in the regulatory framework supporting clinical research which enables both the services provided within the Core and IDDRC investigators of all backgrounds. To facilitate engagement in clinical research by basic science researchers and young investigators, the sub-core assists in the preparation of regulatory documents, the training of study staff, patient recruitment and data analysis. Patient recruitment is supported by the Research Participant Registry, a collection of over 30,000 individuals willing to participate in clinical research, including patients with a myriad of neurodevelopmental diagnoses, their parents and siblings, and typically developing controls. To ensure that the efforts of the Core are focused in areas that will produce meaningful benefits for the intellectual and developmental disability population, the Core is strongly connected to clinical care centers and patient advocacy groups, both of which advise the activity and development of the group. Through these efforts, the Core has a proven record of success in designing and launching preclinical translational and clinical projects for IDDRC investigators.

ABSTRACT This grant application requests funding for the purchase of a high-density electroencephalography (EEG) system to reside in the Human Neurophysiology Core at Boston Children's Hospital. The EEG system offers state-of- the-art technology that will allow investigators flexibility in experimental design to incorporate event related potentials, magnetic resonance imaging (MRI) co-acquisition, transcranial magnetic stimulation (TMS) application and physiology co-registering into high density EEG collection. The equipment selected has been optimized for EEG biomarker collection in pediatric populations, including infants, young children, children with neurodevelopmental delays and neuropsychiatric symptomatology. Importantly, the equipment will be located within a Core facility staffed with experts in the design, collection and analysis of EEG data enabling users to obtain impactful results related to a broad array of NIMH funded research aims. EEG is being incorporated into an increasing number of research studies due to the capacity of this technique to yield high quality data reflecting neural function and connectivity from sensitive populations. Biomarkers of disease state, prognostics, stratification and treatment response have been identified in a number of neuropsychiatric disorders including autism spectrum disorder, ADHD, psychotic disorders and depression/anxiety. Furthermore, considerable EEG research is being undertaken specifically for therapeutic indications in combination with physiological measures (heart rate, galvanic skin response), TMS and MRI neuro- feedback. The realization of these research goals, however, is dependent on the collection of standardized, reproducible data, which is dependent on high quality equipment with built-in validation capabilities and deep technical expertise. To this end, availability of the state-of-the art equipment outlined here within a core setting makes incorporation of EEG based biomarkers and therapeutic applications possible for all investigators focused on improving mental health outcomes.