Grants

NIGMS - National Institute of General Medical Sciences

Project Summary In 1966, Francis Crick described proteins as one of biology's two "great polymer languages". Despite progress, achieving fluency in this language—full understanding and the ability to engineer synthetic proteins for precise control of biological systems—remains a foundational challenge. Synthetic proteins, exemplified by engineered gene editors and chimeric antigen receptors, hold immense potential for medicine. While computational biology has advanced protein science through predictions of structure, stability, and mutation effects on disease, engineering multi-domain synthetic proteins remains difficult due to complex fitness landscapes. This limits our ability to build sophisticated proteins that integrate multiple inputs to control cellular states. Current biophysics- based models struggle with large multidomain proteins, and designs are constrained by cellular contexts, such as folding and trafficking. To address these challenges, my lab developed a Multiplexed Assays of Variant Effect (MAVE) pipeline that combines engineering comprehensive synthetic protein variant libraries, analyzing their phenotypes in diverse cellular contexts, with data-driven models of protein structure and function. While this has enabled better synthetic proteins, including opto- and chemogenetic reagents and cell type specific gene delivery vectors, we need to make MAVE cell context aware to understand how cellular contexts impinge on molecular mechanisms that constrain synthetic protein design. Over the next five years, I will pursue this through: 1) studying how protein molecular traits like folding stability relate to their functions within different cells and tissues, providing insight into cell-type specific variation, 2) assessing multiple phenotypes to uncover structure-function relationships and inform engineering strategies, and 3) exploring diverse systems like ion channels and viral capsids while creating biomedical tools. Through collaborations, these approaches will generate discoveries for independent investigation.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY Microorganisms are frequently challenged by conditions that are suboptimal for growth and reproduction. To persist in such environments, microbes engage in dormancy, a strategy whereby individuals enter a reversible state of reduced metabolic activity. Traditionally, dormancy has been viewed as an adaptation to cope with harsh and fluctuating abiotic conditions, such as resource limitation, extreme temperature, or exposure to antimicrobial agents. However, growing evidence suggests that dormancy plays an important role in modifying host-virus interactions. Although dormancy can provide protection from infection, our findings demonstrate that viruses have evolved strategies to circumvent this metabolic defense. For example, certain viruses hijack the dormancy program of spore-forming bacteria (Bacillus and Clostridia) with the aid of auxiliary genes acquired from their hosts. During this process, viral genomes become entrapped within bacterial spores where they are shielded from the extracellular environment. Upon germination, these non-integrated viruses resume their lytic cycle and exploit the host for replication. Using Bacillus subtilis as a model system, this proposal aims to advance our understanding of host-virus coevolution within the framework of dormancy theory. First, we will test competing models of entrapment using CRISPR gene editing of viruses and quantitative single-cell assays to evaluate the role of auxiliary sporulation genes in lytic suppression and chromosomal segregation. Second, we will investigate the developmental consequences of entrapment based on host-virus expression profiles through single-cell transcriptomics and AI-informed morphological analyses. Third, we will track the fate of infected spores (“virospores”) using a microfluidic platform to quantify the dynamics and consequences of entrapment for host and virus fitness. To synthesize, we will integrate experimental data with mechanistic models to resolve conflicts about virus infection and dormancy decision-making in uncertain environments. The proposed work will advance host-pathogen theory and inform clinical applications, including phage therapies for chronic infections caused by spore-forming bacteria and other microorganisms with quiescent life stages.

NIGMS - National Institute of General Medical Sciences

Cell–cell fusion, a fundamental biological process that is critical for multicellular organism development, has essential roles in conception, placentation, myogenesis, the immune response, and tissue regeneration. Despite the ubiquity of cell–cell fusion, the molecular mechanisms governing gene expression and nuclear function in multinucleate cells remain largely unknown. This research proposal uses the C. elegans epidermal syncytium as a powerful in vivo system to uncover fundamental mechanisms of multinucleate cell formation and function. This innovative, multidisciplinary approach combines single-nucleus genomics, advanced microscopy, and genome engineering to investigate three key scientific aims. First, this work will explore how cell fusion facilitates epidermal specification by examining transcriptional regulation and focusing on the ELT-3 transcription factor and its downstream genes. By tracking target gene expression and ELT-3 localization, the study will determine how cytoplasmic sharing during fusion drives developmental progression. The second aim will investigate fusion's role in synchronizing oscillatory gene control and extracellular matrix organization. Through longitudinal RNA sequencing paired with confocal light and transmission electron microscopy, I will track cuticle gene expression patterns and structural changes in fusion-defective mutants. This approach will reveal how cell fusion coordinates developmental timing and gene regulation across different developmental stages. The third aim will comprehensively examine nuclear heterogeneity within the syncytium. Using single-nucleus RNA sequencing and single-molecule FISH, I will map individual nuclear transcriptomes and connect them to developmental lineages to explore whether nuclei maintain distinct identities or converge to a shared fate. This study will offer unprecedented insights into the transcriptional consequences of cell fusion, in part through innovative technical approaches—including conditional fusion mutants and single-nucleus sequencing. This research will provide a comprehensive understanding of how cytoplasmic sharing and nuclear interactions drive cellular differentiation and tissue development. My findings will have broad implications across biology, illuminating mechanisms underlying developmental processes in syncytial tissues and providing a basis for understanding the implications of fusion defects in human health and disease.

NIAID - National Institute of Allergy and Infectious Diseases

The order Rickettsiales comprises arthropod-associated, obligate intracellular bacteria that are constrained to grow within a eukaryotic host as a result of substantial genome reduction. Their obligate intracellular lifestyle imposes challenges in culturing, genetically manipulating, and imaging these species. As a result, our understanding of fundamental aspects of rickettsial cell biology is limited. The Rickettsiales include a growing number of important human pathogens, however, and a mechanistic understanding of growth and cellular organization would potentiate therapeutic strategies to control rickettsial disease. Within the Rickettsia genus, the Spotted Fever Group (SFG) includes tick-borne human pathogens that cause diseases ranging from mild to life-threatening. Among the SFG bacteria, Rickettsia parkeri causes a relatively mild disease and presents a tractable model for probing the cell biology of this group. Interestingly, despite its reduced genome and lack of obvious morphological polarity, R. parkeri and other Rickettsiales encode putative cell polarity factors, including the polar organizing protein PopZ. We hypothesize that R. parkeri and other Rickettsiales exploit cell polarity to promote their intracellular growth and interactions with a host and that PopZ is important for establishing cell polarity in this group. Here, we propose to identify the suite of polar proteins in R. parkeri and functionally characterize PopZ’s role in growth and host interactions. In Aim 1, we will leverage proximity labeling to comprehensively identify the polar protein repertoire in R. parkeri and will validate putative polar proteins. In Aim 2, will leverage a transposon mutant of popZ to characterize the function of PopZ in fundamental events during R. parkeri growth and interactions with a host cell. Completion of this project will provide foundational knowledge on the mechanisms and function of cell polarity in an important tick-borne, obligate intracellular pathogen that may aid in the design of new antibacterial therapeutic approaches.

NIGMS - National Institute of General Medical Sciences

Project Summary/Abstract Glycosylation – the enzymatic attachment of carbohydrates onto biomolecules – is the most abundant post-translational modification (PTM) of proteins in nature. In mammals, glycosylation influences all aspects of cell biology, including protein quality control and secretion, intracellular signaling, membrane composition and fluidity, cell adhesion and migration, cell-cell communication, and organogenesis. Aberrant glycosylation also underlies a wide range of human diseases, such as developmental defects, obesity and metabolic syndrome, cancer, neurodegeneration, and atherosclerosis. Despite its significance, protein glycosylation remains an under-studied aspect of cell biology, due partly to the unique challenges in characterizing it. Indeed, protein glycosylation is heterogeneous, chemically complex, and created ab initio, without a template molecule, unlike DNA, RNA, or protein biosynthesis. New, interdisciplinary approaches are needed to understand protein glycosylation in physiology and disease. My research program focuses on protein O-glycosylation (i.e., glycans modifying serine and threonine side-chains). In particular, we have a longstanding interest in O-linked β-N-acetylglucosamine (O-GlcNAc), a ubiquitous intracellular PTM in mammals, which decorates thousands of nuclear and cytoplasmic substrates. O- GlcNAc is an essential regulator of myriad aspects of cell physiology and is dysregulated in numerous human diseases, such as cancer, X-linked intellectual disability, and neurodegeneration. However, major aspects of O- GlcNAc signaling are incompletely understood, including the biochemical mechanisms through which O-GlcNAc transduces information. Our work has addressed this challenge by studying O-GlcNAc-mediated protein-protein interactions (PPIs) in fundamental cell biological processes, including intermediate filament structure and dynamics, vesicle trafficking in the early secretory pathway, and regulation of proteostasis through ubiquitin E3 ligase adaptor proteins. We have also systematically identified candidate human O-GlcNAc “reader” proteins that bind this glycan directly and reported the first structures of potential readers with model glycopeptides, illuminating the biophysical basis of these PPIs. Our work is highly interdisciplinary, combining cell biology with structural biology, glycoproteomics, protein biochemistry, and embryonic development. Moreover, as an important complement to conventional techniques, we frequently develop and deploy novel chemical biology approaches to understand O-glycosylation. Building on this foundation, here we propose to study the biochemical mechanisms and downstream functions of O-glycosylation, with a specific focus on O-GlcNAc-mediated PPIs in key cell biological processes. Our work will provide new insight into several pathways dysregulated in human disease and advance our long-term goal of understanding the mechanisms and functions of mammalian O-glycosylation.

NIA - National Institute on Aging

Summary Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and is one of the leading causes of dementia. In addition to memory deficits, Alzheimer’s patients exhibit sleep impairments. Aberrant neuronal circuit activity contributes to the disease etiology and its progression. Anomalies in sleep-dependent brain rhythms, specifically slow oscillations important for consolidation of memories during NREM sleep, have been reported in Alzheimer’s patients. Multiple lines of evidence suggest that disruptions in slow oscillations facilitate Alzheimer’s progression and might contribute to dementia. Thus, aberrant slow wave activity is not simply symptomatic but can be targeted with therapies. Therefore, it is necessary to develop therapeutic strategies targeting restoration of circuit function, such as slow wave activity, to rescue cognitive impairments associated with sleep-dependent memory dysfunction. Stem cell-based therapies are being developed for a number of neurological disorders and could be applicable to Alzheimer’s disease. Alzheimer’s disease is characterized by circuit hyperexcitation at early prodromal stages due to deficits in inhibition, thus disrupting slow brain rhythms, including slow oscillations. Thus, restoration of inhibitory tone through transplantation of inhibitory interneuron progenitors might restore circuit function and slow Alzheimer’s progression. We show that isolation of embryonic mouse MGE-derived interneuron progenitors and their transplantation into an animal model of amyloidosis restores slow wave activity in young mice. We will test the degree to which cell therapy slows neuropathophysiology and rescues sleep as well as memory impairments. Furthermore, to increase translational impact of this work, we will transplant human iPSC-derived interneuron progenitors and determine their role on circuit function and Alzheimer’s progression in a mouse model of amyloidosis. We will implement leading-edge methodology including imaging with voltage-sensors and high-resolution multiphoton microscopy to monitor circuit function as well as optogenetics to control neuronal activity with high temporal precision. Thus, as a result of this work we will evaluate the efficacy of stem cell therapy using mouse and human progenitors for the treatment of Alzheimer’s disease in a mouse model of amyloidosis. This work will provide strong bases for translating cell therapy as a cure to slow AD progression in patients as part of a novel therapeutic approach.

NIA - National Institute on Aging

Project Summary Alzheimer’s Disease (AD) is a progressive neurodegenerative disorder which currently affects nearly 7 million people nationwide. Although the causes of AD are still not completely understood, it is clear that a combination of genetic, environmental, and age-related factors contribute to disease development and progression. However, how these diverse factors coalesce to lead to a pathogenic state remains poorly understood. Moreover, the molecular changes that occur in distinct cell types in the brain in AD and how brain aging affects these changes are not fully known. Of relevance to these critical unanswered questions, recent studies have revealed the importance of RNA regulatory pathways during both brain aging and AD. In particular, chemical modifications to mRNA have emerged as important regulators of gene expression in the brain with links to AD in both human and animal studies. The most abundant internal mRNA modification is adenosine methylation (m6A), which is found in thousands of mRNAs in the brain and which plays critical roles in regulating mRNA processing and expression in cells. Dysregulation of m6A leads to defects in neurodevelopment, learning and memory, and synaptic plasticity, and abnormal expression of m6A writer, reader, and eraser proteins has been linked to AD in both humans and mice. Our recent studies have also shown that some AD-associated mRNAs undergo dynamic methylation in distinct cell types in the brain with age. However, the effects of this dynamic methylation on gene expression during aging are unknown. Moreover, how mRNA methylation is differentially regulated within distinct brain cell types during AD has not been explored. Here, we will address both of these gaps in knowledge by determining how age-dependent differential methylation in the brain influences the expression of AD-associated genes and identifying the m6A reader proteins that mediate m6A function with age (Aim 1). Then, we will determine for the first time how m6A is altered in the AD mouse brain at single-cell resolution (Aim 2). Finally, we will investigate the effects of m6A on gene expression in distinct cell types in the AD mouse brain during disease progression (Aim 3). Altogether, these studies will provide important new insights into the role of m6A in regulating cell type-specific gene expression during brain aging and in AD, and they will provide critical new datasets for the research community.

NIDA - National Institute on Drug Abuse

Methamphetamine (METH) use disorder is chronic and progressive, and currently no therapeutic is approved by the FDA for its treatment. Dopamine (DA) neurons in the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) are necessary for learning associated with drug reinforcers. Our published and preliminary work highlights a role for the peptide transmitter neurotensin in modulation of METH self-administration behavior and plasticity of inhibitory neurotransmission in the SNc/VTA. Neurotensin receptors in the SNc/VTA are localized not only on neurons but also on astrocytes, which are glial cells that can act through local circuits to affect DA neuron excitability and reward-related behavior. We have created floxed neurotensin receptor mouse lines that will enable, for the first time, the determination of the role of specific neurotensin receptors on individual cell types that contribute to METH self-administration and responding to related cues. Filling this knowledge gap is necessary to assess the feasibility of targeting cell type-specific neurotensin signaling and other forms of astrocyte glial modulation as a viable treatment for METH use disorder. The objective of this application is to identify the role of specific neurotensin receptors (NtsR1 and NtsR2), cell types, and circuits responsible for modulating inhibitory input to DA neurons and METH self-administration behavior in mice. Our central hypothesis is that repeated METH self-administration affects VTA circuits through NtsR1 on DA neurons and NtsR2 on astrocytes, which enhances METH intake and responding for METH-related cues. The experiments will combine intravenous METH self-administration in genetically-modified mice, brain slice electrophysiology, and virus-induced expression of Cre recombinase and designer ligand-sensitive proteins to determine the circuits responsible for neurotensin effects in the SNc and VTA. The studies in Aim 1 will elucidate the receptor subtypes and localization that are essential for inhibitory synaptic plasticity using patch clamp electrophysiology in mouse brain slices. The studies in Aim 2 will determine the consequences of METH self-administration, as well as forced abstinence, on neurotensin modulation of DA neurons. Experiments will combine intravenous self-administration of METH in mice with patch clamp electrophysiology, RNAScope, Patch-seq of previously-recorded dopamine neurons, and transcriptomics in midbrain astrocytes. The experiments in Aim 3 will combine chemogenetics and behavior to probe the effects of distinct receptors and neurotensin input on self-administration of METH and responding for METH-related cues after a forced abstinence. The knowledge gleaned from these studies will clarify the role of specific neurotensin receptors by identifying plasticity mechanisms for inhibitory input to DA neurons, and by determining how neurotensin contributes to multiple stages of METH self-administration (acquisition, maintenance, responding for cues after abstinence). The increased understanding of neurotensin and inhibitory circuits in the SNc/VTA could open the door to novel targets for the treatment of stimulant use disorders.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary A typical course of antibiotics for tuberculosis (TB) lasts for six months. Subpopulations of non/slow-replicating Mycobacterium tuberculosis have long been hypothesized to be tolerant to antibiotics and contribute to lengthy treatment. Pathways that are active in quiescent M. tuberculosis are high value targets for drug discovery, particularly extracellular components that are freely accessible to small molecules. Cell envelope recycling is an example of a vulnerable pathway in stressed, non/slow-replicating M. tuberculosis. Recycling of the trehalose component of the outer mycomembrane, which occurs in several in vitro models of stress, promotes M. tuberculosis survival in macrophages and in vivo as well as antibiotic tolerance. While the mycomembrane is a distinctive attribute of the Mycobacteriales cell envelope, these organisms also have a more-conserved peptidoglycan cell wall. Very little is known about cell wall turnover and reuse in the Mycobacteriales. In other bacteria, cell wall recycling genes usually do not contribute to fitness in standard laboratory conditions but can support survival in stationary phase and tolerance to certain cell wall-acting antibiotics. Using bioorthogonal metabolic labeling, the investigators serendipitously discovered that M. tuberculosis, M. smegmatis, and Corynebacterium glutamicum recycle the peptidoglycan sugar N-acetylmuramic acid in a manner that is not explained by their known repertoire of cell wall recycling genes. The central hypothesis of this application is that Mycobacteriales MurNAc recycling occurs via a novel mechanism and promotes survival in stressed, non/slow-replicating M. tuberculosis. To test, the investigators will identify and characterize MurNAc recycling genes in M. tuberculosis and determine the phylogenetic distribution of the pathway within and outside of the Mycobacteriales. Successful execution of the proposed work 1) will reveal fundamental mechanistic insight into cell envelope homeostasis not previously defined in standard model organisms and 2) uncover new vulnerabilities in non/slow-replicating M. tuberculosis, including inner- and mycomembrane transporters with good accessibility to small molecules. The results will also form the basis for future investigation of MurNAc recycling in vivo and as a potential antibiotic target.

NIH

Significance to VA. Disrupted sleep occurs in many conditions common in Veterans, including VA research priorities such as post-traumatic stress disorder (PTSD), substance abuse, major depression and neurodegenerative disorders. Insomnia is associated with an increased risk for suicide and increases the risk for relapse in opioid use disorder. Furthermore, disrupted sleep and abnormal sleep spindles are common in expensive, hard-to-treat conditions such as dementia and schizophrenia. Increasing evidence suggests that sleep spindles are needed for memory consolidation. Deep sleep is vital in clearing the toxic proteins which are increased by traumatic brain injury and cause neurodegeneration. Accordingly, novel strategies to promote deep restorative sleep are needed for a wide variety of disorders affecting Veterans. Cognitive treatments have utility but exert a relatively modest effect and the most widely used existing pharmacological treatments may disrupt deep restorative sleep and be habit-forming. Thus, alternative treatments are needed. Innovation and Impact. Here, in mice we evaluate for the first time whether deep restorative sleep can be enhanced through cell-type specific modulation of an important basal ganglia structure, the globus pallidus, pars externa (GPe). Our data suggest inhibition of GPe neurons which express the calcium-binding protein parvalbumin (PV+) shortens sleep latency and strongly increases the depth and consolidation of sleep. Our novel data show that another group of neurons which express the transcription factor neuronal PAS domain 1 (Npas1+) regulate sleep spindles, important in memory consolidation. Thus, pharmacological or brain stimulation strategies which inhibit GPe PV+ neurons may be a novel strategy to treat insomnia, whereas inhibition of Npas1+ neurons may promote memory consolidation. Our new data in Aim3 identify a safe natural pharmacological agent which opens potassium channels to inhibit GPe PV+ neurons and promote sleep. Specific Aims. Here, experiments focus on two major, non-overlapping neuronal-types of the GPe, PV+ and Npas1+, which make up 50 % and 30 % of GPe neurons, respectively. Aims 1 and 2 will use state-of-the- art neuromodulatory approaches in genetically-modified mice to test the effects of exciting or inhibiting these neurons on sleep. Aims 1 and 2 will also extend our approach to wild-type mice and identify the downstream neuronal targets. In Aim 3 we will test whether we can enhance sleep via systemic or local application of a safe natural compound which preferentially inhibits GPe PV+ neurons by opening potassium channels. Methodology. All aims investigate the effects of GPe manipulations on sleep and cortical electrical oscillations using electroencephalographic and electromyogram recordings in mice. Aim 1 uses chemogenetics to excite or inhibit GPe PV+ or NPas1+ neurons. Chemogenetics allows a prolonged increase or decrease in neuronal activity through the cell-type specific expression of G-protein activated receptors which respond selectively to an otherwise inert drug. Aim 2 will use closed-loop optogenetics to specifically inhibit (Aim2a) or excite (Aim2b) PV+ or Npas1+ during non-rapid-eye-movement sleep. Optogenetics allows fast and precise manipulation by applying light to neurons expressing light-activated ion channels or pumps. Aim 3 will use a pharmacological approach to inhibit GPe PV+ neurons by opening the potassium channels they express. Path to translation/implementation: Potentially the fastest translational application of this research is to use a pharmacological approach, as in Aim3, to enhance sleep by inhibiting GPe PV+ neurons via opening of the potassium channels they express. Several potassium channel openers, including the natural one we test here are safe and approved for use in humans and proposed as therapeutic agents. Another approach could be to modify existing electrical deep brain stimulation protocols or non-invasive stimulation approaches to target GPe to promote healthy sleep and daytime alertness. Ultimately, in the longer-term, we believe that cell- type specific approaches such as optogenetics or chemogenetics could be applied.

U.S. National Science Foundation

Cellular and Biochemical Engineering

NINDS - National Institute of Neurological Disorders and Stroke

PROJECT SUMMARY Oligodendrocytes (OLs), glial cells in the central nervous system, critically contribute to neuronal conduction and health. OLs wrap axons in insulating myelin sheaths – increasing conduction speed – and shuttle metabolites to the local axon to provide necessary energetic support. OLs generate throughout life, deriving from a dedicated pool of OL progenitor cells (OPCs) that tile the brain. Oligodendrogenesis, or the differentiation of OLs from OPCs, is modulated in adulthood by neuronal activity; new OLs and myelin form at increased rates on circuits activated by artificial stimulation or learning and memory tasks. This activity-dependent oligodendrogenesis is consequential for circuit function; across a variety of paradigms, the irreversible genetic blockade of oligodendrogenesis prior to learning results in significantly impaired behavioral performance. Furthering our understanding of the roles adult-born OLs play on neuronal circuits will greatly expand our understanding of neural-glial interactions and assist in determining the extent of functional benefits potentially garnered by therapeutics that induce oligodendrogenesis. This study investigates the cellular biology and circuit interactions of adult-born OLs, using a novel knock-in mouse line enabling the selective labeling and delayed ablation of new OLs generated after a designated time. We first propose to use in vivo two-photon imaging through cranial windows to track and visualize the generation, loss, and replacement dynamics of OLs and myelin in this model, determining the timeline of events occurring after ablation is induced and whether OLs demonstrate cellular age- related resistance to ablation. Data acquired in this aim will provide interesting insight into the cellular and subcellular changes adult-born OLs undergo in response to ablation. To elucidate the functional contribution of activity-generated OLs on neuronal circuitry, we will use a contextual fear conditioning paradigm to induce oligodendrogenesis. Rather than blocking the generation of new OLs after learning, a common model in the field, we will permit OLs to generate and integrate onto neuronal circuitry before ablation and use post-ablation freezing behavior to determine whether the sustained integration of OLs is required for fear memory. The data and knowledge gathered from successful completion of these aims will provide valuable insight into the cellular and circuit dynamics of adult-born OLs.

NIMH - National Institute of Mental Health

Project Summary/Abstract Most sensory stimuli acquire value through learning and experience. While progress has been made in deline- ating the circuits involved in sensory value-guided behaviors, the cellular and circuit mechanisms that underlie how distinct value representations are formed during learning, regulated during behavior, and modified to ac- commodate changes in cue-outcome contingencies remain unclear. Resolving these open questions is a funda- mental challenge in neuroscience and will advance our understanding of how synaptic plasticity induced by reinforcement learning is maintained, yet behaviors remain adaptive. The overarching goal of this proposal is to seek a mechanistic understanding of the circuits underlying value-guided learning and action in the mouse me- dial prefrontal cortex (mPFC), a region essential for the imposition of value on sensory stimuli to guide behavior. Using an olfactory-based appetitive classical trace conditioning task in combination with high-density electro- physiological recordings and optogenetics, I will focus on how functionally distinct yet spatially intermingled pop- ulations of neurons encoding rewarded (conditioned stimuli, CS+) and unrewarded (CS-) odors interact within the local network to drive stable yet flexible reward-seeking behaviors. I will test the hypothesis that the CS+ and CS- populations form a mutually inhibitory circuit that underlies their behaviorally-opposing roles in reward-seek- ing (Aim 1, K99). I will investigate how the existence of a CS- population may represent a circuit mechanism to permit the maintenance of prior reinforcement despite the extinction of learned behavior, thus uncovering a pre- viously unappreciated role for the CS- ensemble in reinforcement learning (Aim 2, K99). In my R00 phase, I will interrogate the mechanisms by which CS+ and CS- representations arise during learning, testing the hypothesis that distinct inputs to the mPFC drive the reinforcement of odors predicting the presence or absence of reward (Aim 3a,b). Further, I will study how the CS+ and CS- representations are updated when cue-outcome contin- gencies are reversed (Aim 3c). Finally, I will model the mPFC circuit to reveal fundamental principles on value- guided learning, which will inform future experiments and also extend the insights that could be gained beyond that which is feasible by experimentation (Aim 3d). This work will have broad implications for various neuropsy- chiatric conditions in which adaptive value-guided learning is disrupted, including addiction and depression. Can- didate and Career Goals. I aim to establish an independent research program investigating the neural basis by which value representations are generated and flexibly regulated to support context-dependent behaviors. I have extensive experience in molecular and systems neuroscience and have developed foundational tools and in- sights for studying value learning. Career Development Plan. I will be trained by my mentors Drs. Richard Axel and Larry Abbott. Dr. Axel is a foremost leader in sensory neuroscience, who will guide me on all aspects of experimental design. Dr. Abbott is a world-renowned theorist who will provide training in the analysis and mod- eling of complex datasets. All mentors will provide career development training for my transition to independence.

National Institutes of Health

Cellular and Molecular Biology of Complex Brain Disorders (R01 Clinical Trial Not Allowed)

National Institutes of Health

Cellular and Molecular Biology of Complex Brain Disorders (R21 Clinical Trial Not Allowed)

NIGMS - National Institute of General Medical Sciences

A branch of the regenerative sciences looks to non-mammalian species with hyper-regenerative abilities for clues to improve human wound healing. However, this approach has struggled to (1) select appropriate model species for meaningful translation to humans and (2) identify specific, actionable molecular targets for manipulating regenerative capabilities. Lizards are the only adult amniotes and closest relatives of humans able to suppress fibrosis and regrow multiple tissue types following appendage amputation. This extraordinary process involves formation of specialized regenerative structures known as blastemas, collections of reprogrammed fibroblasts that differentiate into replacement tissues. Interestingly, not all lizard species are capable of appendage regrowth, distinguishing lizards as the only group of tetrapods to contain both regenerative and non-regenerative species. My lab has committed to studying singular lizard species that have evolved to lack tail regeneration capabilities as natural losses-of-function models toward understanding the fundamental requirements for blastema development. We hypothesized that losses of regenerative capabilities during lizard speciation are due to mutation accumulations within genetic regions responsible for regulating critical aspects of fibrosis suppression and/or blastema establishment. Consequently, we conducted inter-species hybridizations, phylogenomic sequencing, and CRISPR screens to successfully identify mutation signatures significantly associated with loss of blastema formation capabilities. These studies identified three candidate wound healing processes dysregulated in non-regenerative lizard species, and each of the independent yet synergistic projects proposed here investigates the roles of these processes in blastema formation. Project 1 focuses on immunomodulations of pro-inflammatory signals that result in fibrosis suppression. Project 2 investigates pro- regenerative programs that support stem cell survival, proliferation, and activation within wound environments. Project 3 focuses on the epigenetic changes that establish wound site proximodistal patterning essential for proper blastema formation. Successful completion of the proposed projects will meet our short-term goals of characterizing and correcting mutations affecting key wound healing processes in non-regenerative lizard species. The invaluable experience gained during the course of these projects will directly propel my lab toward fulfilling our longer-term goals of inducing nature’s first blastemas in naturally non-regenerative lizard species. My envisioned research program views these endeavors as “steppingstones” for bridging specific gaps in wound healing capabilities between lizards and mammals. A blueprint for supporting lizard-like healing capabilities will be applied to established models of non-regenerating mammalian injuries, such as proximal mouse digit amputations. Ultimately, these lines of research will be applied to forming blastemas in human patients, holding promise to limit painful scarring, support organized tissue growth, facilitate prosthesis attachment, and bring much-needed improvements to patient quality of life following amputation injuries.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ABSTRACT This proposal describes a five-year training plan for the development of an independent research career focused on studying how endothelial immune signaling affects the lung’s response to viral injury. The applicant strives to understand how endothelial cells coordinate immune responses early after influenza infection and how a novel population of injury-associated capillary endothelial cells is regulated at sites of persistent lung injury late after infection. The applicant is a Postdoctoral Fellow of Medicine in the Division of Pulmonary, Allergy and Critical Care at the University of Pennsylvania. He has PhD training in molecular microbiology with a focus on using bioinformatic approaches to understand how populations of cells interact with the adaptive immune system. The goals of this award are for the applicant to gain additional expertise that will help him pursue a career as an independent investigator with a focus on endothelial-immune interactions. The mentor for this award is Dr. Edward Morrisey, an internationally recognized leader in lung regeneration with an outstanding training record. An advisory committee comprising scientists with expertise in domains related to this proposal will support the goals articulated in the training plan. The applicant will benefit from the unreserved support of his institution as well as the unparalleled mentoring, resources, and scientific community at the University of Pennsylvania. The applicant’s preliminary data define how the pulmonary endothelium responds to influenza infection by upregulating genes involved in immune responses. After resolution of the infection, a novel population of capillary endothelial cells is observed adjacent to immune cells in sites of persistent injury, exhibiting increased expression of genes involved in immune signaling. The proposed studies will address the hypothesis that pulmonary endothelial cells coordinate the early antiviral immune response and are maintained in an aberrant inflammatory state late after infection at sites of dysplastic tissue repair. This will be accomplished through experiments in two Specific Aims incorporating mouse genetic knockout models, lineage tracing, immunofluorescence imaging, and spatial transcriptional profiling.The first aim seeks to define the role of two important immune signaling pathways that are expressed in lung endothelial cells and upregulated during inflammation. The second aim investigates the molecular and cellular pathways that give rise to and sustain inflamed capillary endothelial cells late after lung injury. The applicant has established a career development plan that complements his current strengths with additional training in immunology, physiologic measurements of lung disease, and advanced bioinformatics. These training goals will be integrated with professional development activities, additional coursework, and input from his Advisory Committee to help the applicant establish an independent basic science research program focused on endothelial-immune signaling in viral injury and lung regeneration. This work addresses a needed area of investigation and has a high potential for therapeutic impact in patients recovering from lung injury.

NIEHS - National Institute of Environmental Health Sciences

PROJECT SUMMARY Neurodevelopmental disorders such as autism spectrum disorder present a particular challenge due to the complex nature of clinical symptoms and etiology. To understand and dissect human disorders, animal models provide valuable insights that can lead to novel therapeutics and/or drive public health decisions. To facilitate this critical level of comprehension, the mouse model has significantly progressed our understanding of how in utero exposure to common toxins, such as bisphenol A (BPA), can disrupt neural development. Tremendous progress has been made using this model to elucidate the toxin-related pathophysiology. In particular, the mouse model is highly valuable due to the relative evolutionary similarity between rodents and humans. That said, other models, specifically zebrafish (Danio rerio), can complement murine systems because of their unique set of biological attributes and the technologies that have been developed to exploit these biological advantages. Unlike mammalian development, zebrafish development is rapid and external. When seeking to understand neurodevelopmental disorders, these factors provide significant benefits in determining where and when critical periods of dysfunction might occur. Zebrafish also have a relatively reduced nervous system, at least early in development, and are translucent. These factors provide crucial advantages to both identifying aberrant neural circuits and pinpointing cellular and systems-wide perturbations. Perhaps most importantly, the small size and high fecundity of zebrafish facilitate high-throughput screens that can be used to develop novel therapeutics. To further understand how toxins like BPA disrupt neural development, this project seeks to determine the effects of BPA exposure at behavioral and synaptic levels. Behavioral assays used in this project are amenable to high-throughput screens and are mediated by well-annotated and restricted neural circuits. This should expedite investigations at the level of the synapse, another goal of this project. Finally, the project seeks to understand the cellular and molecular mechanisms underlying BPA-mediated pathophysiology and rescue deficits due to this exposure.

NIGMS - National Institute of General Medical Sciences

Project Summary/Abstract: Tissue homeostasis requires the precise control of the development of differentiated cell types from multipotent stem and progenitor cells (SPCs). This balance is regulated by signals originating from multi-tissue niches or SPCs themselves, which affect SPC development. Increasing data supports that these normal developmental processes can be aided or disrupted by inflammatory signal transduction pathways. While prior studies using whole animal knockouts identified the role of inflammatory signaling pathways in HSPC differentiation, open questions remain about the relative contributions of specific cell types to the observed phenotypes and the downstream targets of these pathways. The proposed research program uses the Drosophila melanogaster blood system as a model to address unresolved questions about how multiple cell types found within SPC niches use inflammatory signaling pathways to control the balance between SPCs and differentiated cells during homeostatic development and after injury. As blood SPCs differentiate they make fate choices between alternate paths of development, specifically between distinct intermediate and differentiated cell types, which in turn produce signals to influence the balance between SPC maintenance and differentiation. One goal of the proposed research is to address open questions about how the inflammatory signals activated by injury are propagated through distant niches to control the balance between SPCs and differentiated cells, and how these injury-induced changes influence wound healing. Another goal is to address unresolved questions about how pro-inflammatory signaling pathways influence normal SPC development, specifically which cell types and downstream targets are involved. Tissue damage is a hallmark of many human diseases including atherosclerosis and type 2 diabetes. These inflammatory diseases are also associated with disruption of normal SPC biology in multiple organ systems and increased likelihood of developing diseases that result from SPC dysfunction. Thus, determining the molecular mechanisms required for homeostatic tissue development and understanding how differentiation and SPC biology are changed by tissue damage and inflammatory signaling will provide information that gives insight into disease-related SPC dysfunction.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY / ABSTRACT Endosymbiotic events have driven the evolution of dramatic and consequential biological traits, however most known examples occurred billions of years ago, resulting in deeply integrated associations that mask early steps in the evolution of these associations. “Solar powered, sap sucking” Sacoglossan sea slugs represent a recent and incompletely established endosymbiosis in which stolen, functional chloroplasts are stored in slug tissues. Here, I propose to leverage this unique organism to understand the mechanistic basis of stolen chloroplast (kleptoplast) retention and integration into host cell physiology as a model for understanding endosymbiosis more broadly. To accomplish this, I will: (Aim 1) use organellar proteomics to identify proteins critical for establishment and maintenance of this endosymbiosis (often called “functional kleptoplasty”), (Aim 2) determine the function of those proteins using a combination of pharmacology, patch clamp experiments, and heterologous expression studies, and finally (Aim 3) expand these experiments in a comparative approach using different species of Sacoglossan slugs with different retention abilities. Thus, this study will employ rigorous methodology combining state-of-the-art sequencing technology and multi-omics approaches with physiological approaches, functional assays, and a diverse set of imaging techniques. Further, I will leverage the incredible biodiversity of sea slugs to empower a comparative approach for a comprehensive understanding of a truly unique biological phenomenon: the maintenance of functional photosynthetic chloroplasts within animal tissues. This project will take place at host institution Harvard Medical School (HMS, Cell Biology) and builds on my expertise in transcriptomics, evolutionary biology, and bioinformatics with training in new skills from my primary sponsor, Dr. Corey Allard (HMS, Cell Biology), in electrophysiology, biochemistry, and molecular techniques. I will be aided by an interdisciplinary advisory team composed of my co-sponsor, Dr. Wade Harper (HSM, Cell Biology), an expert in organelle proteomics, Dr. Steven Gygi (HMS, Cell Biology), a world leader in proteomics, and Dr. Amy Lee (HMS, Cell Biology/Dana Farber Cancer Institute), an expert in transcription and RNA biology. Investigation of functional kleptoplasty in Sacoglossan sea slugs will fill a critical gap in our understanding of the evolution of endosymbiosis and the integration of novel organelles into host cells. Completion of these aims will reveal mechanisms used by slugs for sequestration and maintenance of chloroplasts, and thus advance our understanding of the origins and functions of organelles and the fundamental processes that drive the evolution of novel traits. These insights may have broad applications in biotechnology, and could serve as a blueprint to engineer other types of cells to perform photosynthesis, or to take up different foreign cargoes for applications in agriculture, medicine, or even space travel.

NIDCR - National Institute of Dental and Craniofacial Research

PROJECT SUMMARY Temporomandibular joint osteoarthritis (TMJ-OA) is a degenerative joint disease of the jaw and a major driver of temporomandibular disorders (TMDs) that affects approximately 11 million individuals in the United States alone. TMDs are commonly characterized by severe pain and functional defects that lead to TMJ dysfunction and negatively affect the quality-of-life in patients. However, the developmental and pathophysiological mechanisms in TMJ development and disease are poorly understood, thus creating significant barriers to identifying novel non-invasive treatments for TMDs. Here, we aim to examine the cell lineage heterogeneity and molecular mechanisms that contribute to TMJ development and assess how TMJ cell subpopulations are dysregulated in disease. We hypothesize that the fibrous surface layers of the mandibular condyle are composed of unique progenitor cell populations that regulate TMJ development and may contribute to TMJ-OA pathophysiology. Further, we will examine how alterations in the structural organization of the progenitor niche can impact cell lineage plasticity. In Aim 1, we will use single-cell transcriptomic analysis, lineage tracing, and TMJ-OA preclinical modeling to define unique cell subpopulations in the TMJ and their role in TMJ-OA pathogenesis. In Aim 2, we will determine how impaired organization of the fibrous superficial layers of the mandibular condyle alters TMJ progenitor cell differentiation using single-cell multi-omics approaches and investigate the underlying molecular mechanisms that cause TMJ dysfunction.

NEI - National Eye Institute

PROJECT SUMMARY / ABSTRACT Proposed experiments in this multiple-PI application address the retinal functions of MYO7A, which when mutated causes Usher syndrome 1B. Individuals with USH1B are congenitally deaf, have vestibular deficiencies, and develop progressive photoceptor degeneration. Previous studies have focused on the hearing and vestibular phenotypes of USH1B, as these are modeled by the mouse Myo7A knock-out. The knock-out mice also show Retinal Pigment Epithelium (RPE) dysfunction including defects in melanosome motility and localization, as well as impaired phagocytosis of photoreceptor outer segments. It also has a photoreceptor phenotype but does not develop photoreceptor degeneration. Here we provide novel observations that RPE in multiple species show severe cell junction abnormalities. We also show evidence that myo7ab mutations in zebrafish not only have RPE defects, but also develop progressive photoreceptor degeneration. Thus, we define the first experimental model of USH1B to model vision loss. A third important observation is that retinal MYO7A in both mammals and zebrafish express splice variants that affect the MyTH4-FERM domain, a well characterized cargo binding domain of the actin-based motor protein. Leveraging the complimentary expertises of the two PIs, as well as multiple complimentary experimental models (USH1B patient derived RPE cells, Myo7a-mutant mice and pigs, and myo7ab zebrafish mutants), two Aims address the mechanisms underlying cell junction dysfunction, the role of each MYO7A isoform, and the ability of either MyTH4-FERM variant to prevent USH1B pathologies, including photoreceptor loss, when they are re-expressed in relevant retinal cell types. Findings from this project will provide fundamental knowledge of basic cellular functions of MYO7A, and its variants expressed within the retina, including potential cell- autonomous and non-cell-autonomous roles in photoreceptor health. From a clinical relevance perspective, results can shape future gene augmentation therapies, including what are the important MYO7A isoforms and what are the critical cell types to target in order to slow or prevent photoreceptor degeneration.

NIDA - National Institute on Drug Abuse

PROJECT SUMMARY The nucleus accumbens (NAc) is a key node of the brain’s reward circuitry, and perturbations in NAc-mediated reward signaling are highly implicated in substance use disorders. The human NAc has unique neuroanatomical features, which correspond to distinct biological functions. Given the close relationship between brain structure and function, precisely assigning spatial coordinates to transcriptionally-distinct cell populations within the intact cytoarchitectural structure is important to advance our understanding of how NAc cell types mediate reward signaling and control motivated behavior. Towards this goal, we recently generated spatial transcriptomic maps combined with paired single-nucleus RNA sequencing (snRNA-seq) to register molecularly-defined cell types to precise spatial coordinates across the dorsal-ventral and medial-lateral axes of the human NAc. Results from those data suggest existence of topographically organized and molecularly-defined cell types across NAc sub- regions in two-dimensional space. In addition to topographical expression in two-dimensional space, the data also suggest potential changes in the density and localization of these transcriptionally-distinct subtypes across the anterior-posterior axis. This application extends our ongoing studies to generate a three-dimensional cellular resolution spatio-molecular map across the neuroanatomical extent of the human NAc, which will be used to computationally assign molecular identities to NAc cell types across dorsal-ventral, medial-lateral and anterior- posterior axes. These refined annotations will be used to register gene expression changes linked to behavioral phenotypes and genetic risk associated with substance use to spatially-defined NAc cell populations. These data are critical for understanding NAc function and identifying clinical associations with molecularly- and spatially- defined cell populations that can be targeted for prevention and treatment.

NIEHS - National Institute of Environmental Health Sciences

PROJECT SUMMARY Numerous environmental agents (hereafter, genotoxins) induce DNA lesions or create other types of barriers that stall replication forks. This can lead to replication stress, and failure to alleviate this stress and restart stalled forks can cause genome instability. Elevated replication stress and the replication-associated mutations that result from genotoxin-induced fork stalling contribute to aging, inflammation, cancer, and numerous other chronic diseases in humans. This project aims to advance understanding of the cellular responses to replication stress. One crucial aspect of the replication stress response involves replication fork reversal, a process that remod- els both the nascent and parental DNA strands to form a four-way junction structure. Fork reversal is carried out by a family of ATP-dependent translocases. Why multiple enzymes with similar activities are involved in this process is not understood. We showed that one of these translocases, HLTF, prevents a remarkably DNA dam- age-tolerant mode of replication by promoting fork reversal and preventing alternative and potentially error-prone modes of DNA synthesis. The unanticipated resilience of the replication fork to various genotoxins in HLTF’s absence may drive mutagenesis and promote the survival of damaged cells. We will investigate the cellular responses to genotoxic and oxidative damage, focusing on the mechanism of replication fork reversal and the impact of loss of fork reversal on genome stability and cell fitness. By combining molecular, biochemical, genomic and proteomic approaches, as well as state-of-the-art single-molecule ap- proaches, we will address the following broad questions: What are the functions of fork reversal and how does it occur in response to environmentally relevant forms of genotoxic damage? What are the specific functions of a central regulator of fork reversal, HLTF, in the face of oxidative and genotoxic DNA damage? Does loss of HLTF and fork reversal increase mutation (rates) in the context of environmental stressors? Our strategy will initially focus in large part on HLTF, elucidating its unique roles. As the project evolves, we will phase in further studies on other remodelers so as to understand, over the long-term, the overall fork reversal process and the unique contributions of each protein to the replication stress response. The knowledge we gain from this research will ultimately facilitate the development of new strategies that i) alleviate the pathological states observed in the absence of the replication stress response, and ii) prevent cancer cells' ability to tolerate DNA damage and develop drug resistance.