Stephen J. Haggarty - US grants

DESCRIPTION (provided by applicant): The serine/threonine kinase glycogen synthase kinase-3 beta (GSK-3b) is a known master regulator for multiple cellular pathways that include insulin signaling and glycogen synthesis, neurotrophic factor signaling, and Wnt signaling. Consequently, this enzyme has a critical role in metabolism, transcription, development, and neuronal functions and has been implicated in multiple human disorders including Alzheimer's disease, bipolar disorder, noninsulin-dependent diabetes mellitus, cardiac hypertrophy, and cancer. Precisely how GSK-3b maintains its pathway specificity efficiently at the crossroads of many cellular processes is unclear. This regulation may involve allosteric sites within distinct structural domains of this complex kinase. The majority of the existing chemical inhibitors compete for the ATP-binding site of GSK-3b and inhibit additional kinases, while the two known structural series of ATP-noncompetitive inhibitors have low potency and suboptimal pharmacological properties that limit their use. Small molecules targeting the allosteric sites of GSK-3b could have the potential to provide highly specific GSK-3b inhibitors that may help elucidate GSK-3b function and regulation in distinct cellular pathways. Hence we propose a primary screen and a cascade of secondary biochemical and cellular assays to identify ATP-non competitive, allosteric inhibitors of GSK-3b. The anticipated high specificity of such probes could lead to the selective modulation of distinct cellular pathways dependent on GSK-3b. This probe development plan includes: 1) A primary kinase activity assay that will identify ATP-competitive as well as ATP- noncompetitive inhibitors that affect GSK-3b kinase activity. 2) A secondary kinase activity assay based on time resolved fluorescence (HTRF) detection of ADP that will distinguish allosteric modulators and ATP-competitive inhibitors. 3) Binding-based assays that will more precisely define a compound's mode of action. 4) Importantly, we have established a number of cellular assays that will help differentiate allosteric inhibitors and identify pathway-specific GSK-3b inhibitors. Ultimately, we plan to optimize the pharmacokinetic properties of pathway-specific, allosteric GSK-3b inhibitors, and test them in the established rodent models of mood and memory. PUBLIC HEALTH RELEVANCE: Glycogen synthase kinase-3 beta is a master regulator of multiple cell signaling pathways and is implicated in multiple human disorders. We propose to screen the MLPCN library to identify potent and selective allosteric modulators of GSK-3b. Collaborating with MLPCN and Stanley Center chemists, we will optimize the hit compounds to generate chemical probes to elucidate GSK-3b biology in distinct cellular pathways.

DESCRIPTION (provided by applicant): Bipolar disorder and schizophrenia are significant burdens to patients, families and society. Despite consistent evidence for high heritability (up to 80%), the etiology remains poorly understood. In 2008, several groups including ours observed that copy number variants, and in particular, large deletions each containing many genes accounted for some fraction of disease susceptibility. Several groups also identified a more general excess of rare CNVs in schizophrenia and more recently, common single nucleotide variation has been shown to contribute to a polygenic component that also accounts for a fraction of disease risk. Finally, several statistically compelling specific risk loci have also been reported. With what is proving to be a highly complex genetic landscape, existing approaches that model changes in single or small numbers of genes across development are not well suited to capture all the genetic factors interacting in an individual. Thus, the overall goal of this project is to develop human induced pluripotent stem (iPS) cell-based models of neuropsychiatric disorders that are genetically based. In the R21 Phase, pre-existing publicly available fibroblasts from bipolar disorder and schizophrenia patients along with healthy controls will be used to pilot human iPS cell model methods. Once generated, methods for the differentiation of these iPS cells into neural lineages will be employed to characterize potential between-subject and within-subject differences. Specific signaling pathways previously implicated in psychiatric disease will be characterized using a panel of neurotrophic factors and small-molecule probes of Wnt/GSK-3 signaling to develop automated microscopy- based imaging and pathway-selective reporter gene assays. Upon completion of these studies we will have established a framework for systematic development of a Mental Illness Stem Cell Library (MISCL) that will be expanded in the second R33 Phase to include samples obtained from patients from our ongoing genome-wide association studies (GWAS) of bipolar disorder and schizophrenia. The driving questions in the R33 phase will be to use iPS cells derived from clinical samples that reflect the entire genome of an individual to determine the effects on neurodevelopment of: 1) large multigenic deletions on chromosome 1q21.1, 22q11 or 15q13.1 and 2) multiple polygenes of small effect. Similarly, we will use iPS cells from individuals with bipolar disorder associated ankyrin-G (ANK3) haplotypes to determine the effect on basic ion channel physiology, molecular organization of the axon initial segment, and neuronal polarity. PUBLIC HEALTH RELEVANCE: The purpose of this Project is to use newly developed methods for reprogramming human somatic cells (e.g., fibroblasts) to create induced pluripotent stem (iPS) cells that can be used as genetically accurate models of bipolar disorder and schizophrenia. To do so we will work closely with the Harvard Stem Cell Institute (HSCI) iPS Core facility at Massachusetts General Hospital, which has extensive experience with human iPS cell technologies and have successfully derived iPS lines from adult fibroblasts. Methods for inducing the differentiation of iPS cells into neural and glial cells will be implemented as well as novel methods for phenotyping cells using imaging and plate reader assays.

DESCRIPTION (provided by applicant): Advancing our understanding of neuroplasticity and the development of novel therapeutics based upon this knowledge is critical in order to improve the treatment and prevention of nervous system disorders. Recent molecular, cellular, and behavioral findings have revealed the importance of epigenetic mechanisms that alter chromatin structure in maintaining stable patterns of gene expression and altering neuroplasticity associated with mood and memory formation. However, the dynamic and combinatorial nature of these signaling events has meant that the state of our understanding and ability to manipulate the underlying molecular mechanisms in the nervous system remains limited. To overcome these limitations, the long-term goals of the studies outlined in this proposal are to systematically develop selective, brain-penetrant, small-molecule probes (SMPs) of chromatin-remodeling complexes that affect neural activity-regulated gene transcription. Our overall hypothesis is that by selectively targeting the enzymatic activity of specific members of the histone deacetylase (HDAC) and histone acetyltransferase (HAT) families that it will be possible manipulate the acetylation state of histones in the promoters of certain immediate early genes (IEGs) thereby affecting neural- activity-regulated gene transcription and neuroplasticity. To develop the methods and SMPs necessary to rigorously test this hypothesis the proposed studies will address the following aims. In Aim I, the structure- activity-relationships of two types of SMPs that enhance cAMP response element (CRE)-mediated transcription through affecting the activity of certain HDAC and HAT isoforms will be determined. As a sub-aim, proteomic profiling using affinity probes will be used to determine the components of the chromatin-remodeling complexes targeted by both types of SMPs. In Aim II, a real time, automated microscopy-based imaging assay of cultured neurons from bacterial artificial chromosome (BAC)-transgenic mice expressing a genetically encoded fluorescent reporter of IEG expression, will be developed. As a sub-aim, this assay will be used in combination with immunofluorescent detection of histone-modifications to characterize the effect of manipulating HDAC/HAT-complex activities on IEG expression using SMPs and RNAi-mediated gene silencing. In Aim III, the effect of specific HDAC inhibitors and HAT activators in mouse behavioral tests of hippocampal-dependent memory and depression-like behavior will be determined along with measurements of corresponding changes in brain gene expression patterns and histone acetylation. Significance: We anticipate these multidisciplinary studies will shed new light on molecular mechanisms of neuroplasticity and the relevance of these mechanisms to the development of novel therapeutics for memory and mood disorders. PUBLIC HEALTH RELEVANCE: Advancing our understanding of brain plasticity and the development of novel therapeutics based upon this knowledge is critical in order to improve the treatment and prevention of a myriad of central nervous system disorders. This work will characterize the role that gene expression plains in mediating aspects of brain plasticity relevant to mood and memory disorders using small molecules as probes in biochemical and mouse behavioral studies. These multidisciplinary studies will shed new light on molecular mechanisms of brain plasticity and the relevance of these mechanisms to the development of novel therapeutics for their treatment of memory and mood disorders.

DESCRIPTION (provided by applicant): Schizophrenia is a severe neuropsychiatric disorder with complex etiology that affects approximately 1% of the human population. There exists a critical need to understand the underlying etiology and pathophysiology of the disease in order to advance the development of novel therapeutics, particularly to target cognitive dysfunction. Recent genome-wide association and family-based studies have identified several copy number variants (CNVs) associated with schizophrenia. However, despite the strong genetic risk for schizophrenia associated with these CNVs, the etiologically relevant gene(s) in each of these genomic intervals remain unknown. There is therefore a pressing need for systematic and scalable strategies to gain insight into the function of genes associated with schizophrenia as well as other neuropsychiatric disorders. The overall goal of our current application is test the hypothesis that loss-of-function of specific genes within schizophrenia CNVs leads to a dysfunction in mechanisms regulating neuroplasticity. To test this hypothesis our proposal has 3 aims: 1. We will devise robust, high-throughput, image-based assays of neuroplasticity along with an integrated image analysis framework. 2. We will perform high-throughput, lentiviral short hairpin RNA (shRNA) screens of schizophrenia genes to identify regulators of neuroplasticity. 3. We will validate loss-of-function cellular and synaptic phenotypes of schizophrenia genes by performing more detailed, and higher resolution, morphological and functional analyses of candidate shRNAs using imaging and measures of basal and evoked synaptic transmission in cultured neurons and brain slices. It is our hope that the experiments proposed in this application will contribute to gaining a better understanding of the molecular and cellular mechanisms of neuroplasticity and ultimately help illuminate the etiology and pathophysiology of schizophrenia. This proposal seeks to devise innovative strategies for assessing the function of genes in the nervous system and to define the mechanisms through which neurons adapt to changes during development and in response to external stimuli. Once our experimental aims have been achieved, it is our long-term goal to apply similar strategies to other severe mental disorders in order to advance our understanding of their causes and to contribute to the discovery of new types of medicines for improving mental health.

DESCRIPTION (provided by applicant): Major depressive episodes contribute substantially to morbidity and mortality in bipolar disorder. While multiple medications have demonstrated efficacy for treating these episodes, a majority of patients do not reach complete symptomatic remission or have difficulty tolerating these medications. Studies of the mechanism of action of treatments known to be effective in bipolar may provide new treatment targets. Work by our group and others strongly implicates Wnt/GSK3 signaling in the mechanism of action of lithium, which remains a first-line treatment for bipolar depression as well as prevention of recurrence. We therefore have utilized high-throughput cell-based screening in neuronal cells to identify compounds that showed potential additivity or synergy with lithium in terms of effects on Wnt/GSK3 signaling. Among the active F.D.A.-approved drugs with safety profiles compatible with long-term use, we identified multiple statins that acted synergistically with Wnt3a treatment and show further additivity with lithium treatment, including simvastatin, one of the most potent statins known to be capable of crossing the blood-brain barrier. We have validated 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase as the relevant target. Statins have not been directly examined in the treatment of bipolar disorder. A recent rodent study found evidence that a statin augmented the antidepressant-like effects with fluoxetine. Intriguingly, multiple population-based studies also suggest that statins may be associated with a statistically significant decrease in depressive symptoms, and a decrease in the likelihood of adverse psychiatric outcomes. Another study examining statin treatment of dementia indicated a decrease in depressive symptoms compared to placebo. We now propose to conduct a randomized, double-blind, placebo-controlled, proof-of-concept investigation of simvastatin as add-on treatment to lithium in outpatients with bipolar I disorder in a major depressive episode. In parallel, we will collect fibroblasts and derive induced pluripotent stem cells (iPSCs) and neuronal progenitor (NP) cells. The function of the Wnt signaling pathway in patient-specific iPSC-derived NP cells will then be quantified in cell-based assays, with and without treatment with lithium and simvastatin, to enable examination of the association between Wnt/GSK3 signalling and magnitude of improvement in depressive symptoms. These experiments are expected to serve as a crucial first step in the development of new bipolar pharmacotherapies. By predicting the benefit of an adjunctive therapy for bipolar disorder based upon its effects on Wnt/GSK3 signaling pathway and attempting to correlate these responses using patient-specific neuronal cell models, our proposed study will provide a critical test of the importance of Wnt/GSK3 signaling in regulating neuroplasticity in bipolar disorder and depression. At a minimum, these studies will also provide a well-phenotyped, patient-derived cellular resource for future investigation of lithium response and bipolar disorder that can be applied in future studies toward high-throughput screening for lithium-like drugs.

DESCRIPTION (provided by applicant): Frontotemporal dementia (FTD) is a spectrum of devastating neurodegenerative diseases characterized by progressive impairment in emotional and social behavior, language and executive function. There are no effective treatments at present, although clinical trials are being planned. Some forms of FTD are inherited in an autosomal dominant manner as a result of a mutation in one of several genes that have been identified. Despite this genetic information, we currently understand very little about the pathways altered in human neurons leading to neurodegeneration. To address this gap in our knowledge, the overall goal of the proposed study is to apply state-of-the-art methods for deriving human induced pluripotent cells (iPSCs) from FTD patients to model aspects of the underlying disease mechanisms and to investigate the step-by-step progression and development of pathophysiology in living human neurons cultured in the laboratory. In this exploratory proposal, we aim to perform a preliminary investigation of individuals who carry one of these genetic mutations. We will obtain skin biopsies and transform the fibroblasts into induced pluripotent cells and then into neurons in culture in order to study abnormalities in cellular metabolic pathways that are specific to each individual's genetic mutation and context. The ultimate goal of this research is to generate neuronal cellular models of FTD to provide a platform for the development of high-content, cell-based assays to be used in chemical and genetic screens for modifiers of tau toxicity, and potential therapeutics that can reverse or prevent disease phenotypes.

DESCRIPTION (provided by applicant): Growing evidence points to a critical role for epigenetic mechanisms in diverse aspects of human health and disease, including neurodegenerative and neuropsychiatric disorders. This role includes fundamental cellular processes ranging from neurogenesis to synaptogenesis, with significant implications for the development of novel therapeutics to treat and ideally prevent disease pathophysiology. However, despite dramatic advances in our ability to observe the epigenome and transcriptome, our ability to perturb the epigenome and manipulate transcriptional programs with precise temporal control and spatial resolution remains severely limited due to the pleiotropic effects of most existing pharmacological probes and the lack of suitable genetic tools. To overcome these limitations and enable targeting specific cell types within neurocircuits, we propose an integrated, multidisciplinary approach-spanning synthetic chemistry to neurobiology- combining innovative, and scalable 'chemical optoepigenetic' technologies together with epigenome and transcriptome analysis in human and mouse neurons. Our strategy for neuromodulation exploits photoswitchable compounds with fast thermal relaxation kinetics that possess slow-binding kinetics with their epigenetic targets. Using the family of class I histone deacetylase (HDAC)-containing chromatin-modifying complexes, which our work has demonstrated as key regulators of chromatin-mediated neuroplasticity, to advance the testing of this methodology, the specific aims of the proposed project are to: 1) synthesize, characterize and optimize the physical properties, biochemical potency and selectivity of optoepigenetic probes capable of light-dependent inhibition of the deacetylase activity of neuronal chromatin-modifying complexes containing different class I HDAC isoforms; 2) determine the epigenome and transcriptome changes in cultured human stem cell-derived neurons after precise temporal manipulation of different HDAC complexes; and 3) use the novel optoepigenetic probes to temporally manipulate the epigenome of spatially defined mouse neurons to enhance synaptogenesis and modulate hippocampal circuit function. Overall, by providing significant improvements in spatiotemporal control of HDAC activity in combination with advances in the generation of isoform and complex-selective HDAC inhibitors, we anticipate our approach will limit the pleiotropic effects of currently available small molecule tools. Through selective manipulation of the epigenome in specific regions of neurocircuits, we anticipate being able to significantly improve our understanding of how specific temporal regulation of epigenetic states affects neuroplasticity and to be able to delineate the contribution of epigenetic mechanisms in defined neuronal subtypes within neurocircuits. Importantly, our approach developing chemical optoepigenetic probes is broadly applicable to manipulating epigenetic regulatory mechanisms and could be scaled to enable the assembly of a molecular tool kit for combinatorial optoepigenetic studies. Such tools could have wide applicability in the field of neuroepigenetics and help advance efforts to develop improved therapeutics targeting neuroplasticity.

PROJECT 3 ABSTRACT: Major depressive disorder (MDD) remains one of the most prevalent and costly medical disorders. A third or more of patients may not achieve symptomatic remission despite multiple medication treatments; many other individuals are simply unable or unwilling to initiate prescription pharmacologic or psychosocial treatment. Complementary and alternative medications (CAM) represent an important option for such patients. In addition to understanding which CAM antidepressant strategies are efficacious for depression as well as for stress, the aim of Project 1, it would be highly valuable to understand the mechanisms by which certain CAM treatments exert their therapeutic effect. This understanding could increase the acceptability of current treatments, allow better matching of patients with effective treatments, and facilitate the investigation and development of novel CAM treatments. For standard antidepressant treatments, multiple hypotheses regarding mechanisms of action have been developed. These include (i) promotion of neuroplasticity, (ii) modulation of inflammation, and (iii) promotion of neurogenesis. To date, investigation of these latter hypotheses has been hampered by a lack of direct models of human neurobiology ? and particularly neuropathology - amenable to rapid screening and quantitative functional assessment. That is, it has not been possible to examine whether these hypotheses are supported in neural tissue from patients with the particular disease targeted by these interventions. Progress in stem cell technology and developmental neurobiology allows a novel strategy that forms the focus of Project 3. Dermal fibroblasts from 180 patient participants in the randomized trial of Project One will be reprogrammed (transdifferentiated) to induced neurons (iNs). Transcriptional profiles of these iN's will be compared after exposure to n-3 fatty acids, S- adenosyl L-methionine (SAMe), or vehicle to test whether the two CAMs regulate genes related to neuroplasticity (Aim 1a), and whether degree of modulation of neuroplasticity is associated with treatment efficacy. In parallel, a subset (10 per treatment arm) of patient-derived fibroblasts will be reprogrammed to induced pluripotent stem cells. These cell lines will then be differentiated into neuronal precursors and ultimately to mature neurons. Work by this group and others indicates that it is possible to generate such cells and incorporate them in high-throughput, quantitative functional assays to characterize phenotypes relevant to antidepressant mechanism. Specifically, the hypothesis that n-3 fatty acids and SAMe modulate inflammatory markers on neural-lineage cells (Aim 2a), and promote neurogenesis (Aim 2b), will be tested using validated assays. The hypothesis that these mechanisms are associated with treatment efficacy will also be tested. In addition to examining these primary hypotheses, this project will establish a critical resource for future investigation of CAM compounds, a biobank of 180 fibroblasts and 30 pluripotent stem cells and neuronal precursor cells, all derived from patients with MDD participating in Project 1's placebo-controlled investigations. NARRATIVE Major depressive disorder is a major contributor to morbidity worldwide, and existing treatments fail to yield symptomatic remission in ~1/3 of patients. While Complementary and Alternative Medicine (CAM) compounds are increasingly used to treat depression, and well-accepted by patients, their mechanisms of effect have not been fully characterized. The proposed investigation will test specific hypotheses extending preliminary data using unique patient-derived stem cells and induced neurons, while at the same timing establishing biomarkers, biosignatures, and a biobank to facilitate future CAM studies.

Project Summary The development of isoform-selective histone deacetylase (HDAC) radiotracers will fill critical knowledge gaps that exist in our understanding of HDAC function in the human brain. HDAC6 is primary among the isoforms of HDAC that, to date, have exhibited strong therapeutic potential. Compelling preclinical evidence supports HDAC6 inhibition for the treatment of depression and neurodegenerative diseases and translational tools such as a positron emission tomography radiotracer targeting HDAC6 will allow us to assess the relevance of preclinical findings in the human brain. HDAC6 acts not by removing acetyl groups from histones as its name might suggest, but instead by regulating the acetylation state of several non-histone proteins including ?- tubulin, cortactin, tau, HSP90 and ?-catenin. Inhibitors that selectively engage HDAC6 over the other ten isoforms of HDAC have been developed by our team and by others in the field and provide the foundation for PET radiotracer development. Using known HDAC6 inhibitor scaffolds, we will design and synthesize a library of HDAC6 inhibitors that can be readily labeled with a positron-emitting isotope (carbon-11 or fluorine-18). The library will undergo rigorous physiochemical and biochemical profiling including assays that evaluate HDAC- isoform selectivity and efficacy in cultured human neurons and that predict blood-brain barrier penetration. Compounds prioritized through these assays will be radiolabeled and evaluated in rodents for brain uptake, pharmacokinetics and specific (saturable) binding. Promising in vivo imaging results will be validated using HDAC6 knock-out mouse imaging. Finally to set the stage for translation to human HDAC6 imaging (which our team has accomplished for class-I HDAC imaging), we will evaluate priority compounds in non-human primates to determine regional brain binding using full metabolite-corrected arterial blood input function correction and kinetic modeling. By the end of the grant project period, our team will be poised to measure the density-distribution of HDAC6 in the human brain over the course of natural heterogeneity (e.g. age and sex differences) and in patients with HDAC6-implicated brain disorders.

PROJECT SUMMARY/ABSTRACT Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) represent a spectrum of devastating neurodegenerative diseases characterized by progressive impairment in cognition, emotion, and/or motor function. There are no effective treatments at present, although clinical trials are in process and being planned. Some forms of FTD and ALS are inherited in an autosomal dominant manner as a result of a mutation in one of several genes. The major gene that links familial FTD and ALS is C9orf72, which was identified in 2011 and in which the mutation is an expansion of a hexanucleotide (G4C2) repeat sequence. The mechanism(s) by which this mutation results in neurodegeneration is currently unclear. C9orf72 ALS/FTD is one of over thirty neurodegenerative diseases caused by the expansion of a short repetitive repeat tract (microsatellite repeat). In general, these repeats tend to be unstable, changing in length both across generations and over time in somatic cells, with considerable evidence that changes in somatic repeat length can modify disease phenotype. Notably, the C9orf72 repeat is highly somatically unstable. DNA repair genes play critical roles in determining the instability of microsatellite repeats; most notably, genes encoding mismatch repair proteins modify the instability of several different disease-associated repeats (CAG/CTG, CGG and GAA), indicating a fundamental role for these proteins in regulating the length of diverse types of repeat tracts, and signaling common mechanisms of disease modification. Here, we will carry out studies to explore factors contributing to C9orf72 G4C2 repeat dynamics in mice and in patient-derived neurons, investigating DNA repair genes as well as other candidate genes that control aspects of DNA and chromatin structure that have been implicated in C9orf72 pathogenesis. Using C9orf72 BAC transgenic mice expressing Cas9 we will use adeno-associated viruses (AAVs) to deliver single guide RNAs (sgRNAs) to inactivate genes of interest in the brain and periphery, and will analyze G4C2 repeat length over time in different tissues. As a parallel and complementary approach, we will use CRISPR/Cas9 technology to inactivate genes of interest in patient-derived induced pluripotent stem cells (iPSCs) harboring C9orf72 expansions, and will determine G4C2 repeat length during iPSC passaging and upon differentiation to both cortical and motor neurons. Together, these studies will provide novel insight into factors underlying C9orf72 repeat dynamics and explore mechanisms of disease modification that are common across microsatellite expansion disorders, with the ultimate goal of identifying therapeutics targeting the causative mutation itself. .

Tuberous Sclerosis Complex (TSC) is a monogenic disorder with increased incidence of seizures, intellectual disability (ID), and autism spectrum disorder (ASD). Although significant progress has been achieved in understanding TSC, the ability to fully comprehend TSC as a neurodevelopmental disorder, and the shared molecular mechanisms that may explain the overlapping phenotypes between TSC and ASD is hampered by the lack of suitable human neuronal cell lines as well as challenges in establishing disease-relevant human isogenic cellular models. The capability to reprogram somatic cells into induced pluripotent stem cells (iPSCs), and the recent advances in genome editing technologies provide a timely opportunity to establish genetically matched sets of human iPSC lines that differ exclusively at the disease-causing genetic alteration. Employing CRISPR/Cas9 genome editing, we have recently generated such isogenic iPSC lines from two unrelated TSC patients, with a defined heterozygous inactivating mutation in TSC1 or TSC2, respectively. Further, we have obtained iPSC lines from three additional unrelated TSC2 individuals, which will serve as a validation cohort ensuring robustness and reproducibility of our data. These iPSC lines have allowed us to derive lineages of neural progenitor cells (NPCs), the cell of origin for the CNS manifestations of TSC, and neural crest cells (NCCs), responsible for the non-CNS aspects of TSC. Initial studies carried out with the genetically matched sets of TSC1-NPCs (Het, Null and Corrected-WT) confirm an increase in cell size and activation of mTORC1 in TSC1 Het and Null cells. We observe distinct activation of ERK1/2 signaling and an increase in MNK-eIF4E after treating NPCs with rapamycin. Interestingly, TSC1 Het and Null NPCs when compared with the matched WT control reveal an increase in NPC proliferation as well as neurite number and length, which are early-stage neurodevelopmental phenotypes linked to ASD. As mTORC1, MEK-ERK a as well s MNK-eIF4E signaling regulate translation, we propose to generate comprehensive transcriptome and translatome profiles in TSC1/2 Het, Null and Corrected-WT NPCs and NCCs, which will define the underlying molecular changes upon TSC1/2 loss. Finally, we will undertake an unbiased high-throughput single and combination drug screen in TSC1/2 isogenic sets of NPCs, in collaboration with NIH-NCATS, to identify potential drugs that exert preferential impact on TSC1/2 Het and Null cells. The top single and combination drugs will be independently validated in the Ramesh lab in multiple TSC patient-derived NPC lines. The effects of compounds that show selective bias toward TSC1/2 Het or Null cells will be further tested in secondary assays that will assess their ability to normalize transcriptome and translatome signatures. The use of patient-specific, iPSC-derived NPCs and NCCs as genetically accurate human cellular models for understanding the disease and for drug screening will provide insights into pathophysiology and novel targets for therapeutic development, thus having a direct impact on TSC research as well as patient care, and ultimately will lead to a better understanding of the shared molecular mechanisms between TSC, ASD, and ID.

Haploinsufficiency of the GRN gene encoding Progranulin (PGRN) is the genetic cause for a common form of frontotemporal lobar degeneration (FTLD) giving rise to a distinctive frontotemporal dementia syndrome. It is the second most common form of dementia after Alzheimer's disease and currently no effective cure exists for either form of neurodegeneration. Complete lack of PGRN causes a form of neuronal ceroid lipofuscinosis (NCL), a genetically heterogeneous form of lysosomal storage disease in which the digestion of cellular membranes and glycosphingolipids is impaired, resulting in the accumulation of large misshaped lysosomes especially in neurons. This discovery has shaped our current understanding of GRN haploinsufficiency as a genetically distinct latent form of lysosomal dysfunction that accelerates the `normal' progressively diminishing lysosomal capacity during aging. Lysosomal dysfunction syndromes are thought to induce the production of inflammatory cytokines in part through the reduced generation of physiological lipid ligands for inflammation suppressing nuclear hormone receptors, which further promotes neurodegeneration by increasing microglial activation and synaptophagy. FTLD caused by GRN haploinsufficiency offers a unique therapeutic avenue by increasing gene expression from the remaining functional allele. In theory, doubling of baseline GRN expression should completely negate the risk for this form of FTLD in affected individuals. Our team has developed a comprehensive small molecule discovery strategy that has led to the identification of several chemically and mechanistically distinct classes of GRN transcriptional enhancers. The purpose of this project is to investigate the biochemical and cell biological mechanisms through which these small molecules act on the GRN gene and optimize their specificity and preclinical efficacy. This will be achieved by pursuing three major aims: Aim 1 Prioritize and optimize PGRN enhancers for preclinical development based upon efficacy and functional signatures from integrated ex vivo and in vivo studies; Aim 2 Determine the translational potential of PGRN enhancing compounds by evaluating their ability to normalize the cellular and brain transcriptome and lipidome; and Aim 3 Determine the translational potential of PGRN enhancing compounds by investigating their effects on cellular membrane lipid composition and the lysosomal lipidome and proteome.