Gavin Rumbaugh - US grants

DESCRIPTION (provided by applicant): This project focuses on the molecular mechanisms that are activated by learning and support long-term memory formation in the brain. Many signaling pathways lie downstream of NMDA receptors (NMDARs), and several have been shown to contribute importantly to memory formation. These signaling events are believed to induce structural and functional synaptic modifications. However, the regulation of these signaling pathways is poorly understood. My laboratory believes that understanding the spatial and temporal signals that are engaged during the processes of learning will produce robust targets for the development of drugs that treat disorders of memory. SynGAP, which interacts with NMDARs, can regulate a broad spectrum of signaling pathways that lie downstream of these channels. This protein is unique because it is a core PSD component that also stimulates the dynamic regulation of small G-proteins. Interestingly, SynGAP has been shown to regulate both synapse structure and function, though the signaling pathways that mediate SynGAP-induced synaptic modifications are unknown. Recently, functional mutations in SynGAP were discovered in children with severe mental retardation, and these mutations are thought to cause non-syndromic and non-inherited forms of this disorder. We hypothesize that SynGAP modulates distinct signaling pathways in dendritic spines that maintains synapse structure and constrains AMPAR function. We believe that SynGAP lies downstream of learning-activated NMDARs, which would impart this protein with the unique ability to trigger rapid structural and functional plasticity at synapses. These synaptic changes would support the eventual consolidation of a newly acquired memory, and this hypothesis could explain how mutations in SynGAP cause cognitive impairments associated with mental retardation. To investigate this hypothesis, we propose to employ a novel approach to study AMPAR trafficking and synapse structure. We will combine SynGAP inhibitory peptides with two-photon imaging and electrophysiology to simultaneously assay changes in AMPAR function and corresponding changes to synapse structure. We will then attempt to dissociate signaling pathways that underlie each type of synaptic modification. Finally, we directly investigate the role of SynGAP in acquisition, consolidation and retrieval of hippocampus-dependent memories. This multifaceted approach will explain, at the molecular level, how a synaptic protein that regulates signaling dynamics, such as SynGAP, can support the formation of new memories in animals. Overall, we are optimistic that these studies will provide new insights into the molecular mechanisms of learning and memory, and could lead to potential new treatments for memory disorders and cognitive impairments. PUBLIC HEALTH RELEVANCE: Overall, this Project explores an innovative hypothesis aimed at evaluating and understanding if synaptic signaling pathways underlie the mechanisms of memory formation in the CNS. Understanding signaling pathways that are engaged by learning, such as those controlled by SynGAP, will give us novel insights into how protein function contributes to behavioral changes, and will hopefully lead to new treatment options and avenues for drug development for people with illnesses and conditions that cause memory dysfunction.

DESCRIPTION (Provided by Applicant): This project is aims to understand the cellular and molecular mechanisms that support emotional processing in adults. In this application, the investigators will explore a novel hypothesis that suggests early neonatal experience contributes to the shaping of neuronal circuits that govern emotional responses. It has long been known that interactions between an individual and the environment can contribute to brain development. Therefore, it is not surprising that events early on in childhood can impact one's ability to respond to emotionally relevant environmental cues as an adult. The investigators propose that social experiences in neonates trigger neuronal activity that refine circuits that project from, or innervate directly, various nuclei within the amygdala. They arrived at the hypothesis in response to preliminary studies indicating that an NMDAR-interacting protein, SynGAP, is necessary for proper responses to fear and anxiety in adult mice. Published studies indicate that this protein can regulate signaling pathways downstream of NMDARs. Because these receptors are essential for activity-dependent refinement of cortical areas that control sensory processes, it is conceivable that SynGAP mice express abnormal fear and anxiety because of improper wiring among brain regions that govern emotional processing. Importantly, SynGAP has been implicated as a cause of nonsyndromic mental retardation, supporting the investigators'hypothesis that this protein contributes importantly to brain development. To directly test this idea, they will generate a mouse line that will provide temporal control over SynGAP expression in the nervous system. The investigators will compare fear and anxiety from fully developed adult mice with reduced SynGAP expression to the phenotype of adult mice with SynGAP protein reduced before neonatal development. The outcome of these experiments will definitively determine the role NMDAR-SynGAP signaling serves in the maturation of emotional circuitry. In addition, they will also measure the possibility that reduced SynGAP expression during development promotes abnormal cell death in brain areas that govern emotion. Overall, the investigators are optimistic that these studies will provide initial insight into the molecular and cellular mechanisms that contribute to circuits required for emotional preservation. This application has a high probability of maturing into a more substantial research program targeted at understanding the details of how early childhood experience may shape behavior as adults. RELEVANCE: Overall, this application explores an innovative hypothesis aimed at evaluating the effects of signaling through NMDARs on the postnatal development of circuits governing emotion. Understanding the molecular mechanisms that trigger the maturation of emotional responses may give us novel insights into the causes of childhood psychiatric conditions.

DESCRIPTION (provided by applicant): Synapse dysfunction is emerging as a leading cause of neurodevelopmental disorders and many genes that encode for synapse proteins are mutated in these patients. Several rare mutations that affect dendritic spine structure and function have recently been shown to cause ID, while also increasing the risk for developing ASD or epilepsy. While unique combinations of environment and common genetic mutations likely underlie most cases of ID and ASD, mouse models of rare pathogenic mutations offer excellent experimental systems to search for a common pathobiology underlying these disorders. However, it remains largely unknown how developmental synaptic dysfunction resulting from pathogenic mutations impacts circuit function and behavior. This is a particularly important consideration in ID and ASD because these are disorders first diagnosed in young children. Studies that connect developmental synaptic dysfunction to network and behavioral abnormalities are needed to develop new hypotheses that explain the patho-neurobiology of these disorders. These new hypotheses will guide future therapeutic strategies to treat afflicted patients. In this proposal, we aim to understand how dendritic spine synapses are affected by mutations implicated in ID and ASDs. We will perform studies in an emerging mouse model of ID, which provides us with the experimental flexibility to test the idea that abnormal maturation of dendritic spine synapses in neonatal development are driving circuit-level abnormalities that prevent the emergence of normal cognition and behavior. Specifically, we propose that haploinsufficiency of the SYNGAP1 gene, which has recently been shown to cause a form of sporadic ID, induces an early maturation of dendritic spine synapse in periods of postnatal mouse brain development. The early maturation of these excitatory synapses is expected to directly disrupt E/I balance in nascent neural networks, which then impacts key neurodevelopmental milestones, such as the opening of critical period windows of plasticity and supernumerary cortical spine pruning. Our hypothesis predicts that these types of developmental disruptions prevent the acquisition of cognitive and behavioral skills, which could explain both the early onset and persistent nature of these abnormalities in ID patients. Thus, this project Aims to better understand the pathobiology of ID/ASD by linking genetic mutations that disrupt dendritic spine maturation to systems-level processes known to impact the maturation of cognitive and behavioral modalities. It is expected that knowledge gained from these studies will contribute to novel therapeutic strategies to improve the lives of ID patients.

DESCRIPTION (provided by applicant): Despite an overwhelming need for effective CNS therapeutics, little progress has been made. Improving CNS drug discovery efforts is an urgent goal, as an estimated 1.5 billion people suffer from a CNS-related disease or disorder worldwide. We believe that a major roadblock toward more effective CNS therapeutics is the lack of neuron-based probe discovery platforms cable of supporting HTS-level screening. It seems logical that CNS disease targets should be assayed in neurons instead of cell-lines, though the use of neurons in HTS screening campaigns is uncommon. We argue that development of a flexible and scalable neuron-based assay development platform that is compatible with HTS would facilitate probe development, while also perhaps spurring drug discovery efforts aimed at treating a variety of brain diseases. One of the significant barriers preventing R01-driven investigators from interacting with screening centers is the inability of these centers to miniaturize and scale-up neuron-based assays. We have developed an innovative approach for migrating neuron-based benchtop assays to an HTS-ready platform in order to make HTS more accessible to neurobiologists interested in probe discovery. The Neuroscience Department at Scripps Florida has engaged the Screening Center and the Lead ID group at TSRI in collaborative efforts to overcome this barrier and we have identified ways to alter current procedures so that neuron-based benchtop assays can be miniaturized and automated to produce turnkey assays capable of supporting biological and chemical screens with tens-of-thousands of molecules. We propose that the neuroscience field would benefit tremendously from a system that enabled a migratory route for bench-top neuron-based assays to be miniaturized and then scaled, leading to their use in HTS probe discovery campaigns. Importantly, the system that we have developed will also provide investigators with a toolset to develop novel assays for neuron-based HTS. Our proposal details plans to develop novel reagents, instrumentation and workflows that will demonstrate that a bench-top assay developed in primary neurons can be migrated to an HTS-compatible assay. As a proof of principle, we will migrate a bench-top synaptogenesis assay to this HTS-enabled system. We then propose to take this HTS-ready, neuron-based assay through a screen of ~25,000 compounds. Successful achievement of a screen of this magnitude in primary neurons would demonstrate that primary neurons can be used as a common platform for drug/probe discovery and that our approach could serve as a general assay development system for neuron-based HTS. Thus, the outcome of this project is expected to provide researchers and Screening Centers with an assay development platform capable of supporting HTS-level screens in a neuronal environment. Thus, funding this proposal will result in novel probes to regulate synaptogenesis, but will also generally serve the Neuroscience community as a whole by providing an assay development platform that can be used by anyone interested in scaling up bench-top assays for HTS campaigns.

? DESCRIPTION (provided by applicant): The focus of this revised competitive renewal is to understand the neurobiological substrates that link single gene mutations to cognitive deficits. Currently, there is no effective treatment for the cognitive disruptions that define neurodevelopmental disorders (NDDs). In order to develop therapeutic strategies that improve the lives of affected individuals, it is imperative to understand how the neurobiological substrates that underlie reduced cognition and behavioral adaptations are damaged in these disorders. Our work during the last budget period established Syngap1 Heterozygous KO mice as a robust model that will advance our understanding of how cognitive and behavioral disruptions arise in NDDs. In particular, we established that pathogenic Syngap1 mutations damage the developing brain by altering the maturation rate of forebrain excitatory neurons. We also succeeded in connecting alterations to forebrain pyramidal neuron maturation in Syngap1 mutant mice to reduced cognitive ability. However, it remains unclear how altered neuronal maturation actually degrades cognitive ability. Our studies in this revised proposal are designed to explore the hypothesis that there is a spatial pattern of circuit assembly errors in Syngap1 mutants. We believe that alternated maturation of forebrain pyramidal neurons disrupts the assembly of cortical circuits that underlies cognitive ability, which is hypothesized to be a key neurobiological substrate that connects pathogenic Syngap1 mutations to altered cognition. The impact of our expected results is that the pattern of circuit assembly errors could define the particular cognitive endophenotype displayed by Syngap1 mutant mice, which would provide significant insight into the etiology of reduced cognition due to a single gene disruption. In addition, the patterns of assembly errors in Syngap1 mutants could be used as a comparative benchmark to understand similarities and/or differences in patterns of altered circuitry in other monogenic forms of NDDs. The long-term goal of this proposal is to identify the cells in the brain that are most severely impacted by pathogenic Syngap1 mutations. Once we understand the cellular origins of the disorder, we will have a much better entry point for gaining insight into te molecular perturbations that trigger the systems level dysfunction that directly leads to reduced cognitive ability. At the conclusion of the next requested budget period, we believe we will have reduced this disorder down to a relatively selective pool of neurons that are particularly sensitiv to pathogenic Syngap1 mutations. When this information is combined with the critical period information discovered in the previous budget period, we will have the ideal entry point for assessing how pathogenic Synagp1 mutations disrupt the molecular pathways that control growth and maturation of developing forebrain pyramidal neurons. In the subsequent budget period, we would then begin studying how altered developmental Syngap1 expression in this pool of neurons triggers changes in neuronal maturation.

? DESCRIPTION (provided by applicant): We aim to advance the tools and methodologies for preclinical translation studies in mouse models of intellectual disability (ID) and related disorders, such as autism spectrum disorder and epilepsy. The goal of this project is to optimize a panel of measures that predict the extent of developmental brain damage in an emerging mouse model of ID and then use this panel to test the efficacy of FDA-approved RAS/ERK inhibitors. Diagnostic exome sequencing has identified SYNGAP1/Syngap1 as one of the most commonly disrupted genes in patients with sporadic brain developmental disorders. Our studies in mice that model this monogenic brain disorder demonstrated that life-long cognitive disruptions are caused by isolated damage to developing forebrain glutamatergic neurons. Damage to these neurons disrupts a critical period (CP) of development that drives life-long cognitive and behavioral disruptions. Syngap1 encodes a neuron-specific RasGAP and pathogenic mutations leading to haploinsufficiency enhance Ras/ERK signaling in the brain. However, it is currently unknown if elevated RAS/ERK signaling within forebrain glutamatergic neurons is the primary cause of CP damage that leads to life-long cognitive disability in this mouse model. Based on our past work that identified the core neurobiological defects that underlie this genetic from of ID, we have developed a clear and testable therapeutic hypothesis: that normalizing elevated Ras/ERK signaling in neonatal Syngap1 mutants will protect the CP from damage and thus mitigate the development of persistent cognitive and behavioral disruptions. Therapeutic development in mouse models of ID is expensive, time consuming and has yielded few, if any, translational successes. One possible reason for the lack of translatability in mouse models of ID is the dearth of highly quantifiable surrogate measures of cognitive function. Thus, in order to most effectively assess the efficacy of experimental therapeutics in Syngap1 model mice, we are also proposing to validate several highly quantifiable biomarkers of CP damage. Because abnormal cognition in these mice is caused by damage to a developmental CP, these surrogate measures have the potential to be highly informative with respect to cognitive ability in Syngap1 mice. In addition, these candidate biomarkers of CP damage have a high potential for translatability to human subjects because they can be acquired easily in both mice and humans. Importantly, some of these potential biomarkers are known to give very similar signals in both mice and humans patients with similar Syngap1 loss-of-function mutations. Validation of highly sensitive and translatable biomarkers in Syngap1 mice, combined with efficacy testing of a unique therapeutic hypothesis centered on CP protection, suggests that the work outlined in this proposal could advance the tools and methodologies used to develop experiential therapeutics. These advances could increase the success rate of therapies translated from mouse ID models to corresponding patient populations.

Project Summary The goal of this proposal is to secure funds to support the 1st International SYNGAP1 Conference. MRD5 (MIM#: 612621; www.omim.org/entry/612621) is a recently discovered sporadic form of intellectual disability (ID). This disorder is caused by deleterious de novo mutations in the SYNGAP1 gene, which encodes the synaptic RasGAP, SynGAP. Symptoms of MRD5 include cognitive impairment and severely impaired expressive and receptive language. Epilepsy, ASD and ADHD are common comorbid conditions often described in these patients. Pathogenic mutations in SYNGAP1 are surprisingly common, with the incidence reported as 1-4/10,000 individuals, or approximately 1-2% of all ID cases, making it one of the most common genetic causes of ID. It is crucial in the case of SYNGAP1 to have an international meeting bringing together all relevant stakeholders (i.e. patient families, clinicians and researchers) because so little is understood about this emerging brain disorder. While it is known that SynGAP protein is critical for regulating learning and neural excitability in both humans and mice, it remains unclear how loss of functional SynGAP protein leads to symptoms of the disorder. Furthermore, there is a general lack of awareness of the disease, resulting in delay of diagnosis and there is no centralized location to access medical, research, or patient information. Lastly, patient families have poor access to cutting-edge medical and scientific expertise that will inform their understanding of the disorder. Therefore, we believe that there is an urgent need to hold this meeting. The proposed conference will bring together stakeholders with the primary goal of maximizing scientific resources by building collaborative approaches that are efficient and synergistic, thereby accelerating the identification of effective treatments. We have commitments from the leading clinicians and researchers studying SynGAP biology and the related human disorders. Importantly, the meeting will be run in conjunction with the major MRD5 patient support network, Bridge-the GAP (www.bridgesyngap.org), which will organize the attendance of several families with affected children. The symposium will include sessions related to MRD5 clinical genetics/patient phenotypes, Syngap1 neurobiology, common substrates in neurodevelopmental disorders, epilepsy genetics, translational approaches in RASopathies, and accelerating translation in rare genetic disorders. We anticipate the creation of impactful opportunities for junior investigators, including women and minorities, to participate in scientific exchange and to meet MRD5 patients and their families. Key outcomes expected from this meeting include: (i) networking and establishment of collaborative research and clinical outreach programs; (ii) generation of new ideas on the pathogenesis and possible treatment of MRD5; (iii) expansion of the MRD5 research and clinical community; and (iv) establishment of an international MRD5 research and clinical network to foster fully collaborative, multi-laboratory basic research and to encourage initiation of a patient registry and natural history study in order to advance patient care and treatment.

? DESCRIPTION (provided by applicant): Whole-brain mapping of cellular data in animal models will revolutionize our understanding of neural circuits and behavior. Microscopy hardware has progressed to a point where it is becoming routine to archive labeled whole animal brains. Furthermore, computing power and the software ecosystem are now mature enough that very large datasets can be managed. However, bioinformatics tools specifically designed for whole-brain mapping of cellular signals in animal models are lacking. Our collaborative group has recently created a suite of bioinformatics tools capable of reconstructing and then analyzing whole-brain data sets. However, because of their complexity, these tools can only be used by computer scientists in their current form. Thus, the goal of this project is to optimize a streamlined approach to digitally archive mouse brains so that whole-brain cellular measures can be more easily reconstructed and then analyzed. The results of such experiments are expected to vastly increase understanding of brain function. This will be accomplished by developing a seamless protocol for immunolabeling the mouse brain, followed by digital archiving of the brains, detection of cellular signals and analysis of these signals across the entire mouse brain. As part of developing this protocol, a web-based portal will be created that users around the world will be able to utilize to perform 3D reconstructions and subsequent analysis of their own mouse brain digital sections. Importantly, this system will be amenable to any form of cellular labeling, such as signals arising from any number of fluorescent reporters. In order to optimize this workflow and to test the utility of the web-based portal, mapping of whole brain activity is proposed in a mouse model of autism spectrum disorders and intellectual disability in response to social novelty. These would be the first whole brain cellular activation maps in a model of a developmental brain disorder. It is expected that these maps will advance our understanding of the neurobiology of developmental brain disorders by revealing brain areas that are dysfunctional during abnormal social behaviors. Importantly, the web-based portal will make whole brain mapping of cellular signals more accessible to the neuroscience community. This would enable other labs to map whole brain cellular data in any neuropsychiatric disease model. Collectivity, whole-brain mapping data from numerous distinct disease models by labs throughout the world would be expected to reveal common systems-level substrates of severe brain disorders.

PROJECT SUMMARY    Drug discovery pipelines for neuropsychiatric disorders are dry. One approach to rejuvenating these pipelines  would  be  to  create  assays  based  on  relevant  disease  phenotypes  in  primary  neurons,  something  that  is  currently  lacking.  However,  a  scalable  assay  development  platform  that  is  based  on  bona  fide  neurons,  remains  cost  effective,  and  that  can  support  industrial  level  HTS  does  not  currently  exist.  Over  the  past  five  years, our collaborative group has created a flexible and scalable primary neuron assay development system  that is compatible with industrial-­level HTS. Here, our goal is to optimize these procedures and workflows  to  determine  the  limit  of  scalability  of  neuron-­based  HTS  phenotypic  assays  so  that  they  can  easily  support very large campaigns of >200K compounds.    A  substantial  proportion  of  childhood  brain  disorders  are  caused  by  single  autosomal  dominant  variants  resulting in genetic haploinsufficiency. The rare genetic brain disorders that arise from these variants offer the  greatest potential for discovery of robust therapeutics because the disease mechanism is often straight forward  (i.e. low protein expression). Therefore, a rationale strategy to improve conditions in these patients would be to  treat  them  with  ?magic  bullet?  compounds  that  raise  expression  of  functional  proteins  from  the  remaining  undamaged  allele  (e.g.  ?boosting  compounds?).  De  novo  nonsense  variants  that  cause  SYNGAP1  haploinsufficiency  lead  to  a  genetically-­defined  form  of  intellectual  disability  with  autism  and  epilepsy  (MRD5;?  OMIM#603384)  that  may  explain  up  to  1-­2%  of  all  ID  cases.  The  accepted  cause  of  this  disorder  is  low  functional protein expression in neurons caused most often by truncating SYNGAP1 nonsense variants. As a  means to refine the neuron-­based HTS system, and to advance treatment for ASD-­related disorders, we  are  seeking  to  scale-­up  and  implement  an  assay  for  SynGAP  expression  that  is  compatible  with  industrial-­level robotics. In the first Aim, we will optimize an HTS-­compatible and disease-­relevant SynGAP  expression  assay.  This  assay  is  based  on  mouse  primary  neurons  where  tdTomato  fluorescence  reflects  steady-­state  endogenous  SynGAP  protein  levels.  In  the  second  aim,  we  will  miniaturize  the  SynGAP  expression assay to the 1536-­well format. This miniaturization process would enable an HTS-­scale screen of  this,  or  any  other  related  neuron-­based  phenotypic  assay,  of  up  to  400,000  culture  wells  using  a  standard  screening budget. Finally, we will implement the SynGAP expression assay in a true uHTS environment and  then  validate  lead  compounds  that  emerge  from  a  20K  compound  pilot  screen,  including  a  10K  compound  repurposing  screen  of  known  ?safe  in  human?  compounds.  The  impact  of  this  project  that  we  expect  to  develop procedures that will increase the scale of HTS campaigns in neurons by 10-­fold or more relative to the  current state-­of-­the-­art in academic screening centers. We also expect to validate at least one lead compound  that boosts SynGAP expression, hopefully from the repurposing library.     

Project Summary    A goal of basic mental health research is to understand the molecular, cellular and circuit level substrates that  contribute to neuropsychiatric disorders. The goal of this project is to better understand the principles  underlying circuit dysfunction associated with cognitive and social impairments common to these disorders. A  promising approach to better understand these substrates is to perform in-­depth studies in animal models with  high construct and face validities. De novo pathogenic SYNGAP1 mutations leading to haploinsufficiency  cause one the most common genetically defined and non-­inherited forms of intellectual disability (ID) with  autism spectrum disorder (ASD;? termed MRD5;? OMIN# 612621). Studies supported by the first budget period  identified Syngap1 heterozygous KO mice as an outstanding genetic model of ASD with ID. Using this model,  we discovered a developmental sensitive period of Syngap1 function that promotes the proper function of  cortical networks. The neurobiological studies we published in the last period were significant because they  identified the developmental timing of dendrite and spine maturation selectivity within forebrain excitatory  neurons as a critical substrate that shapes brain function relevant to cognitive and social development.     For this competitive renewal, we will build on our discoveries in the first budget period by studying the key  substrates of circuit dysfunction in the Syngap1 model by probing how this gene regulates cortical sensory  processing relevant to cognition and learning. This approach is significant because sensory impairments are  extremely common in ASD/ID and these impairments influence behavioral adaptations, including learning.  Syngap1 patients express sensory abnormalities related to touch and pain. However, the circuit abnormalities  that underlie sensory dysfunction are unclear. Thus, our approach is innovative because studies will be  performed in the mouse somatosensory cortex, which will enable powerful in vivo experiments that are capable  of directly linking cellular-­ and circuit-­level functional impairments to sensory-­based learning and behavioral  abnormalities. The first Aim will investigate the cellular mechanisms underlying impaired somatosensory cortex  network function caused by pathogenic Syngap1 mutations, with an emphasis on how network-­level E/I  imbalances emerge within cortical circuits that directly encode sensory representations. Research proposed in  the second Aim will determine the cellular mechanisms that contribute to sensory-­driven learning impairments  in Syngap1 mice. The impact of these studies is that they are expected to advance our understanding how  cortical circuit dysfunction leads to behavioral impairments associated with ASD/ID.    

PROJECT SUMMARY    The  overarching  goal  of  this  project  is  to  better  understand  the  links  between  ASD  genetic  risk,  resulting  distributed brain connectivity impairments, and the impact of this on ASD-­relevant behaviors. We will do this by  performing state-­of-­the art in vivo electrophysiology studies in awake-­behaving animals that model a monogenic  form of ASD. This research project is significant because altered brain connectivity is routinely observed  in  ASD  patients,  though  it  remains  unknown  how  brain  connectivity  alterations  cause  abnormal  behaviors relevant to ASD. In the animal model, we will focus on behaviors that optimize active touch. This is  approach  is  valid  because  altered  sensory  function,  including  touch,  is  a  core  manifestation  of  ASD  and  somatomotor brain areas display altered activation in ASD patients. An emerging idea is that altered functioning  of  sensory  systems  directly  impairs  the  functions  of  other  major  neural  domains,  such  as  cognitive  and  social  systems. Active touch arises through rapid adaptions in the dynamics of touch organs in response to physical  contact  with  objects.  This  behavioral  transformation  optimizes  touch-­related  input  into  the  brain  and  is  an  emergent  behavior  resulting  from  sensorimotor  integration  at  various  levels  in  the  nervous  system.  Therefore,  we generally hypothesize that genetic variants that cause ASD disrupt key points of functional connectivity within  the  somatomotor  system,  which  in  turn causes  altered  active touch  behaviors,  leading  to altered  acquisition of  tactile information. This hypothesis is significant because it could define a neural process (i.e. altered distributed  functional  connectivity)  that  explains  how  sensory-­guided  adaptive  behaviors  are  impaired  by  genetic  variants  that cause ASD. Our modeling studies also have the potential to define how altered brain connectivity can disrupt  relevant  behaviors.  We  will  test  this  hypothesis  in  the  first  aim  by  recording  the  flow  of  information  throughout  the major areas of the somato-­motor system in a mouse model for a monogenic form of ASD. The proposed in  vivo  recordings  in  awake-­behaving  animals  will  utilize  state-­of-­art  silicon  neural  probes  that  will  enable  us  to  measure local and long-­range functional connectivity of neurons during distinct behaviors, including during active  touches of objects. These sophisticated measurements will identify circuits that are functionally impaired during  ASD-­relevant  behaviors.  The  second  aim  takes  a  distinct,  but  complementary  approach  by  regionally  and  temporally disrupting expression of the causal ASD gene and then observing the impact of these perturbations  on behaviors that define etiologically-­relevant active touch. We expect to find that proper expression of the ASD  gene  is  required  in  developing  somatomotor  cortical  areas  to  promote  normal  active  touch  behaviors.  The  combined impact of these complementary approaches is that they are expected to define the circuits that cause  abnormal  active  touch-­related  behaviors  in  the  mouse  model.  Thus,  the  proposed  research  is  expected  to  advance our understanding of how major ASD risk genes disrupt the connectivity of neural circuits that underlie  relevant behaviors.       

PROJECT SUMMARY In 2010, an estimated 6 million individuals in the United States abused prescription pain relievers, triggering the current opioid epidemic. Further, an estimated 10-20% of women in the U.S. receive a prescription each year for an opioid, such as oxycodone (OXY), during pregnancy. Collectively, this has resulted in a five-fold increase in prescription drug use among expectant mothers over the last decade, as well as one baby born every 15 minutes in opioid withdrawal, termed neonatal abstinence syndrome (NAS). Despite a rapidly growing population of individuals born with NAS, basic research efforts on the effects of prenatal exposure to opioids on brain development, as well as the lifelong behavior impacts, are poorly defined. Better identifying the effects of prenatal OXY exposure on the development of neural circuits and behavior will allow for future investigations into the underlying mechanisms. The long-term goal is development of novel and innovative strategies to mitigate the lifelong impact and the current focus on OXY specifically is based on the perceived safety due to its FDA-approved status. A broad battery of behavioral tests performed in adult mice exposed to OXY in utero indicates this developmental insult produces behavioral deficits related to impulse control and response to opioids, with sex-specific effects, that are consistent with the limited data available on children exposed to opioids in utero. The medial prefrontal cortex (mPFC) is a core member of the neural circuitry governing these behaviors, often with a link to hypofrontality driven by the striatum and amygdala. Thus, the overarching hypothesis is that prenatal OXY alters the development of long-range inputs to the prefrontal cortex, resulting in behavioral dysregulation. To begin addressing this, unbiased monosynaptic circuit tracing was performed in GAD2-Cre mice to create whole brain maps in both sexes of all direct, long-range inputs to mPFC inhibitory neurons. Consistent with the possibility of hypofrontality, this analysis revealed a marked and selective elevation in structural connectivity to mPFC interneurons (INs) from the basolateral amygdala (BLA) in females exposed to prenatal OXY. This led to the working hypothesis to be addressed here, that prenatal OXY exposure produces BLA-mediated inhibition of the mPFC, resulting in behavioral dysregulation. Completion of the two proposed Aims is expected to produce the following: (1) Unbiased whole brain maps of monosynaptic long-range inputs to excitatory and inhibitory (PV, SST and VIP+) mPFC neurons in the context of prenatal OXY exposure, to determine the source and balance of these inputs. (2) Determination of the BLA?s influence over the mPFC following prenatal OXY exposure, in terms of functional connectivity, using high density silicon probes with optogenetic stimulation and behavior, using chemogenetics. The proposed research is expected to provide a framework for future mechanistic studies aimed at further defining the functional subcircuits, how to best mitigate the consequences of maternal opioid use and assessing the impact of current NAS interventions employed in NICUs, which consists of postnatal opioid replacement therapies.

PROJECT SUMMARY    Drug discovery pipelines for neuropsychiatric disorders are dry. One approach to rejuvenating these  pipelines would be to create assays based on relevant disease phenotypes in primary neurons, something that  is currently lacking. However, a scalable assay development platform that is based on bona fide neurons,  remains cost effective, and that can support industrial level high-­throughput screening (HTS) does not currently  exist. Over the past eight years (spread across different NIH-­sponsored grants), our collaborative group has  created a flexible and scalable primary neuron-­based assay development system that is compatible with  industrial-­level HTS. Our long-­standing goal for this project has been to optimize these procedures and  workflows to support neuron-­based HTS phenotypic assays so that they can support very large  screening campaigns of up to 200K compounds.   We are happy to report that progress over the last budget period has pushed us closer toward this  stated goal. We have invented a state-­of-­the-­art, disease-­modeling assay created in primary neurons that is  designed to discover compounds that reverse the cellular consequences of genetic haploinsufficiency. Indeed,  a substantial proportion of childhood brain disorders are caused by single autosomal dominant variants  resulting in genetic haploinsufficiency. The rare genetic brain disorders that arise from these variants offer  great potential for translation because the disease mechanism is well-­understood (i.e. low protein expression).  Therefore, a rationale precision therapy for treating genetic haploinsufficiency disorders would be to discover  ?magic bullet? compounds that raise expression of functional proteins from the remaining undamaged allele  (e.g. ?boosting compounds?). In this renewal project, we will employ technical innovations that have unlocked  the scalability of primary neurons for phenotypic HTS. As a proof-­of-­principle, we will scale-­up and  implement an assay that reports reversal of low SynGAP expression in neurons caused by genetic  haploinsufficiency of the SYNGAP1/Syngap1 gene. We will miniaturize an HTS-­compatible and disease-­ modeling steady-­state endogenous SynGAP expression assay so that it is compatible with industrial scale HTS  automation. Once implemented, we will then screen up to 200,000 unique substances using a completely  automated version of the neuron-­based SynGAP expression assay. Finally, using a comprehensive multi-­stage  biological validation funnel, we will identify and prioritize the most translatable chemical probes that raise  SynGAP protein expression. The overall impact of this project is that discovery of multiple, validated SynGAP  boosting compounds would provide proof-­of-­principle that our flexible platform is an effective tool for  phenotypic drug discovery for nervous system disorders.        

Summary Autism spectrum disorder (ASD) is a neurodevelopmental disorder with deficits in two core domains: social interaction and communication, and repetitive behaviors or restrictive behaviors. It is diagnosed four times more frequently in boys than in girls. Among a large number of risk loci for ASD, elevated protein synthesis has been recognized as a converging pathological mechanism. ASD is associated with a high percentage of patients with inactivating mutations in genes for several negative translation regulators, such as PTEN, TSC1, TSC2 and FMR1. These mutations increase the availability of eukaryotic translation initiation factor 4E (eIF4E), consequently elevating translation of a selective group of mRNAs. However, it remains unknown in which type of brain cells and how elevated translation leads to dysfunction of neural circuits and subsequently ASD behaviors. We have generated a knock-in mouse strain in which eIF4E is overexpressed from the Rosa26 locus in a Cre-dependent manner. We found that eIF4E overexpression in microglia, but not neurons or astrocytes, led to ASD-like synaptic and behavioral aberrations only in male mice, including increased dendritic spine density, excitation/inhibition imbalance, social interaction impairment, increased repetitive behavior, and selective cognitive deficits. We further found that microglial eIF4E overexpression elevated translation in both sexes but only increased microglial density and size in males. Given critical roles of microglia in synapse development, we posit that elevated synthesis of some proteins alters microglial functions only in male mice, which in turn impairs synapse development and thereby male-biased ASD. We will test this hypothesis in the following three specific aims. Aim 1 is to investigate the molecular mechanism by which elevated protein synthesis alters microglia; Aim 2 is to understand the mechanism underlying the sexual dimorphism of ASD- like phenotypes in MG4E mice; Aim 3 is to determine how microglial alterations impact synapse development by imaging in vivo dynamics of dendritic spines and microglia in control and MG4E mice. This research project will not only provide insights into the pathological mechanism by which mutations in negative translation regulators lead to ASD, but also show microglial dysfunction as a possible etiology of ASD. It may also uncover a mechanism that underlies the strong male bias of ASD, which could guide strategies for innovative therapies of the disorder.

PROJECT SUMMARY  There are no pharmacotherapies for stimulant abuse, including methamphetamine (METH) and relapse rates  are high. Relapse triggered by reminders of drug use is a particular challenge to prevent, as the underlying  memories exert a powerful motivational influence over behavior and represent a lifelong relapse risk factor.  Learning is supported by structural plasticity in dendritic spines, driven by training-­induced actin polymer-­ ization. Memory stability is subsequently achieved by arresting actin dynamics, stabilizing the cytoskeleton. As  a result, memory is impervious to actin depolymerization within minutes of learning. However, prior work in the  lab discovered that the actin cytoskeleton supporting METH memories remains uniquely dynamic in the  amygdala long after training. This enables selective, retrieval-­independent disruption of METH-­associated  memories and drug seeking with a single administration of an actin depolymerizer. Because actin?s critical  roles in the body limit its therapeutic potential, focus shifted to nonmuscle myosin II (NMII), a direct driver of  learning-­stimulated actin polymerization in spines. The effect of NMII inhibition is specific to the amygdala and  METH. Indeed, NMII inhibition has no effect on METH memories when other regions of the drug-­memory  neural circuit are targeted and there is no similar retrieval-­independent effect on memories for fear, food  reward or other drugs of abuse, including opioids. Genetic and pharmacologic targeting of NMII established it  is a viable therapeutic target and an NIH-­funded medication development project for a clinically safe NMII  inhibitor is underway (UH3 NS096833). However, fundamental knowledge needed to understand and further  leverage this specificity is lacking. This will be addressed through the central hypothesis in this new project:  that METH-­associated memories are uniquely supported in the amygdala by NMII, leaving those memories  selectively vulnerable to disruption long after learning, even when other associative learning is introduced. The  focus of this application is two-­fold: (1) The key mechanistic question regarding the specific requirement of  the amgydala, actin-­NMII and METH for selective memory storage disruption will be addressed. For this, the  impact of METH-­related neuromodulators (Aim 1), as well as NMII phosphorylation and interacting partners  (Aim 2) will be studied on NMII-­dependent BLA synaptic actin dynamics and METH-­associated memory, with a  focus on factors that are unique to METH and the BLA. Once identified, the mechanism(s) responsible could  be harnessed to render relapse-­inducing memories for other drugs of abuse vulnerable to disruption. (2)  Because most individuals with METH use disorder use multiple substances, including opioids, it is necessary  to determine the impact of polydrug administration on METH memory susceptibility to NMII inhibition.  Preliminary data indicate that METH confers susceptibility to previously impervious opioid associations.  Technically innovative approaches will be employed throughout the project, spanning from single synapse  manipulations in live tissue slices to memory-­based self-­administration studies.