Helen S. Bateup - US grants

PROJECT SUMMARY Tuberous Sclerosis Complex (TSC) is a neurodevelopmental disorder caused by mutations in the TSC1 or TSC2 genes. The protein products of TSC1 and 2 form a complex that is a key negative regulator of mTOR signaling. TSC is associated with an array of neurological and psychiatric problems that can include epilepsy, autism spectrum disorder, and intellectual disability. The neurological manifestations of TSC are amongst the most debilitating to patients, yet our knowledge of the neuropathophysiology of TSC is limited. Animal models of TSC have been valuable to address questions of basic biology, revealing alterations in neuronal development, morphology, and synaptic communication. The next major challenge is to translate these findings to humans. This will require defining the consequences of TSC mutations in a human genetic and developmental context. To achieve this we will establish a novel human neuronal model for TSC based on Cas9-mediated gene editing of human embryonic stem cells (hESCs). To this end we have generated an isogenic panel of hESCs with heterozygous, homozygous, and conditional loss of function mutations in the TSC1 or TSC2 genes. These cells will be differentiated into neural progenitors, neurons, and cerebral organoids to model the early stages of human cortical development when TSC-related phenotypes first arise. We will determine how mutations in TSC1 or 2 affect the development and function of human neurons using biochemical, genome-wide profiling, imaging, and electrophysiological approaches. Our findings will answer several key questions related to genotype-phenotype relationships in TSC and the developmental origin of the cortical malformations that are a hallmark of this disorder. In addition, the cell lines we generate will be a valuable resource for the research community to investigate disease mechanisms and test potential therapeutics for TSC and other ?mTOR-opathies? directly in primary human cells.

PROJECT SUMMARY Elucidating the molecular basis of neurological and psychiatric disease is of critical importance for the design of improved therapies. One signaling pathway that has been identified as a hub of dysregulation in several brain disorders is the mTOR pathway. A large body of work has established that altered mTOR signaling affects key aspects of neuronal structure and function including cell morphology, connectivity, synaptic plasticity, and excitability. However, very little is known about the molecules downstream of mTOR responsible for regulating these processes. This information is essential as the effectors of mTOR signaling represent potential drug targets that could provide improved specificity compared with systemic mTOR blockade; a current treatment strategy that is associated with problematic side-effects. A central cellular process regulated by mTOR complex 1 (mTORC1) is protein synthesis. Therefore, identifying the translational targets of mTORC1 is a key first step towards understanding how this pathway controls neuronal function in both normal and disease states. Here we will generate a comprehensive and unbiased assessment of mTORC1's influence on protein synthesis in distinct disease-relevant cell types in the mouse brain. To do this we will genetically and pharmacologically manipulate mTORC1 signaling in vivo and perform translational profiling of defined classes of neurons. We will use a novel approach, called FLEX-TRAP, to express a tagged-ribosomal protein selectively in sub-populations of neurons and use translating ribosome affinity purification (TRAP) to isolate mRNAs that are being actively translated. These mRNAs will be analyzed using next generation sequencing and translational profiles will be compared across conditions and cell types. For this exploratory project we will examine the impact of mTORC1 signaling on the translational profile of four disease-relevant populations of neurons: hippocampal pyramidal cells, direct and indirect pathway striatal projection neurons, and midbrain dopamine neurons. This analysis will provide the first comprehensive and cell type-specific description of mTORC1's impact on mRNA translation in the brain and identify the molecules downstream of mTORC1 responsible for mediating changes in cellular and synaptic function.

Significant progress has been made in identifying genetic factors contributing to autism spectrum disorder (ASD). Although ASD-risk genes have diverse biological functions, many of them are important for controlling the structure and function of synapses. We propose that a core feature of ASD, restricted, repetitive patterns of behavior, is caused by synaptic dysfunction in the basal ganglia, a brain region that controls the selection and learning of appropriate actions. Little is known about the mechanisms by which mutations in genes associated with ASD affect the basal ganglia. To address this, we will determine how disruption of the ASD-risk gene Tsc1 affects the cellular physiology and behavioral output of neurons comprising key basal ganglia circuits. To isolate specific cell types, we will use genetic mouse models in which Tsc1 is selectively deleted from defined cell populations. Our goal in Aim 1 is to investigate how Tsc1 loss affects intrinsic and synaptic excitability in the two classes of striatal projection neurons that initiate the primary output pathways of the basal ganglia. In addition, we will test the idea that synaptic alterations in striatal neurons lead to altered motor behaviors and increased propensity for habit formation. Striatal activity is dynamically regulated by dopamine signaling, which exerts a powerful control over basal ganglia-mediated behaviors. In Aim 2, we will determine how selective deletion of Tsc1 from dopamine neurons affect their physiology and output. Using behavioral experiments, we will test the hypothesis that altered dopamine signaling due to loss of Tsc1 leads to behavioral inflexibility. This strategy represents a key step towards defining the neural basis of ASD, which may ultimately inform the rational design of new therapeutic strategies for this disorder.

PROJECT SUMMARY Tuberous Sclerosis Complex is a neurodevelopmental disorder caused by mutations in the TSC1 or 2 genes that encode negative regulators of mTOR complex 1 signaling. TSC is associated with a high prevalence of autism spectrum disorder (ASD) and other neuropsychiatric conditions, which are debilitating for patients and caregivers. Despite their prevalence in TSC, relatively little is known about the neurobiology of these manifestations including the cell types responsible. We propose that a core aspect of ASD, repetitive, inflexible patterns of behavior, is caused by synaptic changes in the basal ganglia, a brain region responsible for the selection and learning of appropriate actions. Here we will investigate this in the context of TSC by determining how mutations in Tsc1 affect the cellular physiology and behavioral output of neurons comprising key basal ganglia circuits. To isolate specific cell types, we will use genetic mouse models in which Tsc1 is selectively deleted from defined cell populations. The experiments in Aim 1 will determine how Tsc1 loss affects synaptic transmission and plasticity in the two classes of striatal projection neurons that initiate the primary output pathways of the basal ganglia. We will test the idea that increased cortical synaptic drive of direct pathway striatal neurons leads to altered learning and increased propensity for motor habit formation. Striatal activity is dynamically regulated by dopamine signaling, which exerts powerful control over behavior. In Aim 2, we will determine how selective deletion of Tsc1 from dopamine neurons affect their physiology and output. We will test the hypothesis that loss of Tsc1 causes hypofunctional striatal dopamine signaling leading to impaired cognitive flexibility in reversal learning tasks. This strategy represents a key step towards dissecting the cellular and circuit basis of TSC, and may ultimately inform new therapeutic strategies for this and related ASDs.

PROJECT SUMMARY SYNGAP1-related non-syndromic intellectual disability is a neurodevelopmental disorder caused by mutations in the SYNGAP1 gene. SYNGAP1 encodes the protein SynGAP, which is a highly abundant protein in the post-synaptic density of excitatory synapses. At synapses, SynGAP functions to repress downstream NMDAR signaling and AMPAR trafficking through its inhibition of small GTPases. Translocation of SynGAP out of the post-synaptic density is required to allow NMDAR-dependent long- term potentiation (LTP). In the absence of SynGAP, NMDAR-dependent plasticity is unrestrained leading to alterations in synapse strength, spine structure, and plasticity. While the functions of SynGAP have been nearly exclusively studied in the cortex and hippocampus, the striatum also exhibits high levels of SynGAP expression. Striatal projection neurons are GABA-ergic neurons covered in a dense array of dendritic spines that receive excitatory input from multiple cortical areas. SynGAP is therefore positioned to play a key role in gating synaptic transmission and plasticity at corticostriatal synapses. Despite this, SynGAP?s functions in striatal synaptic physiology have not yet been defined. Moreover, several of the major symptoms of SYNGAP1 disorder likely involve striatal pathophysiology including autism spectrum disorder, obsessive-compulsive behavior, motor developmental delay, hyperexcitability, and other behavioral problems. In this exploratory study, we will elucidate the consequences of SynGAP loss on striatal synaptic function and determine whether loss of SynGAP from striatal neurons is sufficient to induce behavioral alterations relevant for SYNGAP1 disorder. Specifically, in Aim 1 we will determine how loss of SynGAP impacts corticostriatal synaptic transmission and plasticity. In addition, we will use advanced imaging approaches to investigate how SynGAP deficiency affects dendritic spine number and morphology. In Aim 2, we will determine whether deletion of Syngap1 from specific striatal cell types is sufficient to alter motor behaviors, habit learning, and cognitive flexibility. We will further test whether restoration of SynGAP expression only in striatal projection neurons is capable of preventing behavioral abnormalities using a genetic rescue strategy. Together, this work will provide an essential starting point for understanding SynGAP?s functions at striatal synapses and identify the striatal cell type(s) most relevant for the manifestations of SYNGAP1-related disorders.

PROJECT SUMMARY Tuberous Sclerosis Complex (TSC) is a multi-system developmental disorder caused by mutations in the TSC1 or TSC2 genes. The protein products of these genes form a complex that is an essential negative regulator of mTORC1 signaling. In the absence of a functional TSC1/2 complex, mTORC1 signaling is deregulated and constitutively active. While the manifestations of TSC can affect several different organ systems, the neurological and psychiatric aspects of the disease are the most burdensome for caregivers and least well understood. These include early-onset epilepsy, varying degrees of intellectual disability, and a high prevalence of autism spectrum disorder and other behavioral conditions. A hallmark pathology of TSC is the presence of cortical tubers, which are focal regions of enlarged, dysplastic neurons and glia in the cortex that form during embryonic development. Cortical tubers can become epileptic foci and in some cases are surgically removed in individuals with intractable seizures. The size and number of cortical tubers is variable between patients and increased cortical tuber load is associated with worse outcomes including more severe epilepsy and cognitive impairment. The goal of this project is to determine the molecular mechanism(s) by which mutations in TSC1 or TSC2 lead to the formation of cortical tuber cells. To do this we will use our recently established human brain organoid models of TSC in which we have engineered loss of function mutations in TSC1 or TSC2. These human brain organoid models robustly reproduce key cellular features of cortical tubers including dysmorphic neurons, reactive astrocytes, and giant/balloon cells. In addition, we have observed a strong bias towards the production of glial-lineage cells at the expense of neurons in TSC brain organoids, which recapitulates observations from patient tuber samples. Here we will define the molecular basis for altered cortical cell development due to TSC1/2 mutations and investigate how the resulting tuber cells impact the function of the surrounding cortical network. In Aim 1 we will explore two potential hypotheses for altered differentiation of TSC1/2 mutant cells in brain organoids: 1) premature activation of astrogenic transcription programs that interfere with normal neurogenesis and/or 2) impaired survival and development of newborn neurons. To test these hypotheses we will use pharmacological, shRNA, and CRISPRi manipulations to test the contribution of candidate pathways. In Aim 2 we will use different strategies to manipulate mTORC1 signaling and specific downstream arms of the pathway to test whether these can prevent or rescue altered cellular development. In Aim 3, we will perform functional analyses to determine how the presence of cortical tuber cells impacts the activity of the surrounding cortical network. Together the results of these aims will generate new insights into the molecular and cellular mechanisms leading to cortical tuber formation and how these cells ultimately impact cortical function.