Ut Southwestern Medical Center

NIH Research Projects · FY 2025 · 2024-08

Project Summary Mutations in nuclear envelope proteins (NEPs) cause devastating genetic diseases, known as envelopathies, which primarily affect the heart and skeletal muscle. The LEM domain nuclear envelope protein 2 (LEMD2) is a ubiquitously expressed inner nuclear membrane protein. In vitro studies reported that LEMD2 interacts with DNA-binding proteins, implicating LEMD2 in genome regulation and chromatin organization processes. Importantly, a missense mutation in the LEMD2 coding sequence causes severe cardiomyopathy in humans. In a recent study, we reported that “humanized” mice carrying the Lemd2 c.T38>G human mutation as well as cardiac-specific Lemd2 KO (cKO) mice develop dilated cardiomyopathy (DCM) and die prematurely due to heart failure. Moreover, Lemd2- deficient cardiomyocytes (CMs) display high levels of DNA damage. Despite its association with cardiac phenotypes in both human and mice, the role of LEMD2 in the mammalian heart and the pathological mechanisms responsible for its association with cardiac disease are far from being understood. Based on my preliminary findings, I hypothesize that high levels of DNA damage in LEMD2-mutant CMs cause the development of cardiomyopathy. To test this hypothesis, I will first perform a comprehensive characterization of the DNA damage and DNA damage response (DDR) in both mice and human CMs carrying LEMD2 mutations. The second aim is focused on the mechanistic link between LEMD2 loss-of-function, DNA damage and cardiomyopathy. I will determine if LEMD2 interacts with the chromatin-binding protein BAF, and whether the LEMD2 c.T38>G mutation disrupts this interaction. I will also detect nuclear envelope ruptures, cytosolic DNA leakage, and the activation of the pro-inflammatory pathway cGAS in LEMD2-mutant CMs. The third aim will determine the therapeutic potential of LEMD2 gene therapy for envelopathies characterized by the presence of DNA damage. I will overexpress LEMD2 in CMs and mice carrying mutations in the lamin A gene and assess the extent of rescue at the structural and functional levels. By accomplishing the objectives of this proposal, we will reveal important mechanistic insights regarding LEMD2 functions and its associated cardiomyopathy. With this new knowledge, we hope to ultimately design new therapeutic strategies and preventive methods for genetic cardiomyopathies.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY One in three liver transplant recipients experiences a cardiovascular event within one year of transplant because of immunosuppression medications and a high prevalence of potentially modifiable cardiovascular risk factors, such as high blood pressure. Hypertension is the foremost modifiable risk factor for cardiovascular events in liver transplant recipients (LTRs); however, there is insufficient evidence to guide treatment approaches (e.g., blood pressure target level, medication choice, treatment timing) of hypertension in LTRs that maximize cardiovascular benefit while minimizing potential harms to the liver, kidney, and brain. Intensive blood pressure lowering is indicated in high cardiovascular risk populations and cardiac biomarker levels can aid in matching blood pressure-lowering therapy to those most likely to benefit; however, in LTRs, concerns about the harms and tolerability of intensive blood pressure control limit aggressive blood pressure targets in clinical practice, highlighting a need for a precision medicine approach to hypertension management in LTRs informed by cardiac biomarker levels. To address these knowledge gaps for clinical practice, we will conduct a prospective, multicenter, cohort study of 1,268 LTRs and measure 7-day home blood pressure, cardiac biomarker levels, cardiovascular events, liver graft function, renal function, and cognitive function over two years with outcomes up to five years post-transplant. We will determine the net clinical benefit of blood pressure-lowering on target organs in LTRs (Aim 1), determine whether cardiac biomarker levels can improve stratification of cardiovascular risk among LTRs with elevated blood pressure or hypertension (Aim 2), and identify optimal treatment approaches in hypertension post-liver transplant that lower blood pressure and minimize cardiovascular events (Aim 3). Our central hypothesis is that elevated cardiac biomarker levels can inform optimal treatment approaches for hypertension in LTRs that maximize cardiovascular benefit and minimize harms to the liver, kidney, and brain. We will leverage the existing infrastructure of the Avoiding Cardiovascular Complications in Liver Transplantation through Novel Studies (ACCT NOW) consortium, comprised of multidisciplinary clinicians and researchers across six U.S. transplant centers, whose long-term goal is to establish evidence-based guidelines for cardiovascular disease prevention in liver transplantation. The current aims, when achieved, will (1) define hypertension treatment approaches that are most likely to effectively reduce blood pressure, a key modifiable risk factor for cardiovascular events, without causing harm in LTRs, (2) delineate the role of cardiac biomarkers to guide these treatment decisions, and (3) identify which LTRs are most likely to benefit from intensive blood pressure lowering to reduce cardiovascular risk. These data are essential for design of a future randomized type II hybrid intervention trial that will test biomarker-based hypertension management strategies in liver transplant recipients and support the first evidence-based guidance for managing hypertension after liver transplantation.

NIH Research Projects · FY 2025 · 2024-08

Project Summary Clear cell renal cell carcinoma (ccRCC), which accounts for approximately 85% of all renal cancers, is resistant to a variety of cancer therapies and is highly lethal. von Hippel-Lindau (VHL) is the most important tumor suppressor in renal cancer, its loss leads to hypoxia inducible factor  (HIF, including HIF1α and HIF2α) accumulation. Although HIF2α inhibitor has been developed as a potential therapeutic target in renal cancer, only a small propotion of patients will respond to HIF2α inhibitore treatment, suggesting the importance of identifying additional therapeutic vulnerabilities in VHL-deficient kidney cancer. Methylation of adenosine (m6A) determines the fate of modified messenger RNAs (mRNAs) and has recently been shown to play a key role regulating gen expression critical in cancer development. Although previous research has suggested that m6A may be important in renal cancer, the molecular mechanism behind its role in ccRCC tumorigenesis and aggression is not yet well understood. Our preliminary data suggest that VHL binds to m6A enzymatic complex proteins Mettl3/14 and regulates their interaction. By performing m6A RIP-Seq and RNA-Seq in renal cells with and without VHL, we have identified a set of genes whose expression may be regulated by m6A in a VHL- dependent manner. Among them, Phosphoinositide-3-Kinase Regulatory Subunit 3 (PIK3R3), an important component of PI3K pathway, its mRNA stability is regulated by VHL in an m6A-dependent manner. Functionally, PIK3R3 depletion promotes renal cell growth and tumor growth while its overexpression diminishes ccRCC cell growth on 2-D colony formation, 3-D soft agar growth and tumor growth. We hypothesize that VHL regulates m6A-dependent gene expression, thereby controlling ccRCC tumorigenesis. This is the first study to link VHL with mRNA stability regulation via regulating the Mettl3/14 m6A enzymatic complex, which characterizes the noncanonical function of VHL distinctive from its role as an E3 ubiquitin ligase. In Specific Aim 1, we will determine the molecular mechanism by which VHL regulates m6A modification in kidney cancer. In Specific Aim 2, we will examine the functional role of VHL-m6A signaling in ccRCC tumorigenesis. Specifically, we will examine the therapeutic efficacy or PI3K and AKT inhibitors in kidney cancer xenografts and patient derived xenografts. Successful completion of these aims will provide mechanistic insight into a novel signaling pathway on how VHL regulates gene expression important in ccRCC and set the foundation for therapeutic intervention targeting the VHL-Mettl3/14-m6A signaling axis in ccRCC.

NIH Research Projects · FY 2025 · 2024-08

Project Summary/Abstract Asthma prevalence has increased in recent years with 8.4% of adults diagnosed with asthma. Asthma can be a lifelong disease. These rates increase with age, are 60% higher in women than men and are especially high in African Americans. Thus, adult asthma is a major health concern. Rates of uncontrolled asthma also increase with age and are higher in women and in minorities, especially Hispanics. Texas, where the proposed study will take place, has the third highest rate of uncontrolled asthma; therefore, more effective treatments for adult asthma are needed. Asthma patients with frequent exacerbations despite appropriate treatment pose a particular challenge. New treatments are needed to improve asthma control in this subpopulation. Our pilot data from three preliminary studies suggest that the selective serotonin reuptake inhibitors (SSRIs) citalopram and escitalopram, as compared to placebo, improve asthma control and decrease asthma exacerbations in depressed people with very frequent exacerbations. Effect sizes for exacerbations were much greater than for depression improvement. In addition, baseline depressive symptom severity did not predict asthma improvement. Thus, the asthma effect did not appear to be related to depression improvement. These clinical trial findings were supported by an electronic medical record analysis suggesting patients with asthma, who did not have a depression diagnosis, showed reductions in prednisone use after receiving SSRIs. SSRIs might improve asthma control through effects on subclinical anxiety and depressive symptoms or psychological triggers of asthma or independent of emotions through anti-inflammatory effects. In vitro studies suggest SSRIs reduce the frequency of IL-4- and IL-2-producing cells triggered by CD3 stimulation and potentiate glucocorticoid receptor-mediated gene transcription, suggesting they might enhance the effects of inhaled steroids. SSRIs also decrease IL-6 levels, which are elevated with asthma. In animal models of allergic asthma, SSRIs reduce bronchial inflammation with decreases in macrophages, lymphocytes, neutrophils and eosinophils in bronchoalveolar lavage fluid. A single-site investigator-initiated clinical trial (R61/R33) is proposed to examine escitalopram compared to placebo in adults with asthma requiring frequent steroid bursts but without current major depressive disorder. A milestone-driven R61 start-up phase of less than one year will set the stage for the R33 phase 24-week, randomized, double-blind, placebo-controlled trial of escitalopram in 105 men and women with moderate to severe persistent asthma who, despite treatment with medium-high dose inhaled corticosteroids and long-acting beta agonist therapy, had ≥ 3 asthma exacerbations in the past year. Need for steroid bursts (primary outcome) and asthma control, as well as functioning, quality of life, serum IL-6 levels and exhaled nitric oxide, will be assessed. Effects on mood and anxiety will be explored and their influence on asthma control assessed. An experienced investigative team with a long history of collaboration and expertise in asthma, SSRIs, clinical trials and statistical analysis will conduct the study.

NIH Research Projects · FY 2025 · 2024-08

Canonical Wnt signaling plays a crucial role in several vital processes, including neural tube and retinal vascularization, as well as the maintenance of the blood-brain barrier (BBB) and blood-retina barrier (BRB). Specifically, the Wnt signal triggered by Norrin has been identified as a key regulator of vascular development in the vertebrate retina and is essential for regulating significant blood vessels in the ear. Norrin plays an essential role in the development and maintenance of the retina, which is the light-sensitive tissue at the back of the eye responsible for vision. Mutations or abnormalities in the Norrin gene can lead to various retinal diseases, like Norrie disease which is characterized by abnormal development of the retina and other structures in the eye. When Frizzled receptor 4 (FZD4), the receptor of Norrin, is knocked out in mice, it leads to abnormal vascular development in the retina. TSPAN12 interacts with FZD4 and Norrin and acts as co-receptor that enhances FZD4-meidted Norrin signaling. To ensure the integrity of the BBB and BRB, the involvement of two related Wnt signaling coreceptors, namely LDL receptor-related protein 5 (LRP5) and LRP6, collaborate with Norrin or Wnt to bind to the FZD, initiating the Wnt signaling pathway. Furthermore, disruptions in Norrin/FZD4 signaling in the adult retina and cerebellum result in cell-autonomous changes in the BRB and BBB function, respectively, indicating a necessity for Norrin/FZD4 signaling in the maintenance of barriers and underscores the remarkable adaptability of the mature central nervous system's (CNS) vascular structure. Additionally, the integrity of the BBB relies on the presence of Wnt7 and its co-receptors, RECK and GPR124. The combined action of these signaling molecules and receptors is essential for the proper functioning and maintenance of the BBB and BRB. Our main objective is to understand the molecular mechanisms by which Norrin, TSPAN12, RECK and GPR124 contribute to the ligand specific Wnt/-catenin signaling in retinal and CNS angiogenesis. To accomplish this, we will use recombinant receptor complexes to determine their molecular mechanisms of action by biochemical and biophysical approaches. Through our investigations, we seek to unravel the precise role of Norrin, Wnt7 and their co-receptors in initiating signaling events. The results will have implications for the development of novel therapeutic strategies targeting retinal diseases and the maintenance of barrier functions in the CNS.

NIH Research Projects · FY 2025 · 2024-08

Project Summary This project seeks to examine the role of the exposome as a significant risk factor for Alzheimer’s disease (AD) among people living in different communities. This work will utilize blood samples from the 3,000 participants in the ongoing Health & Aging Brain Study (NIA, U19AG078109). This work can lead to strategies to lower environmental exposures in at-risk populations in order to reduce their risk for developing AD.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Elimination of integrated, replication-competent HIV-1 proviruses from host genomes persisting despite suppressive anti-retroviral therapy (ART) is the major roadblock to a functional cure. Cells harboring these types of proviruses produce marginal levels of viral products thereby becoming refractory to immune surveillance mechanisms. This lack of detection by the immune system, in addition to its increased growth potential, due to homeostatic proliferation and clonal expansion, extend the lifespan of latently infected cells generating a persistent HIV-1 reservoir. There is enormous enthusiasm for the potential of precision therapies targeting the latent reservoir in clinical settings. To achieve this major biomedical goal, the characterization of novel basic regulatory mechanisms can pave the way to devise alternative therapeutic approaches. A large body of work has proposed that HIV-1 latency maintenance and reactivation is regulated by histone modifications. Previous studies mainly focused on the repressive roles of histone modifications involved in latency maintenance and their targeting for latency reversal. However, we have made the unexpected observation of selective H3K9me3 deposition at the provirus 3’-end during latency reactivation. This result was surprising because H3K9me3 is typically seen as a repressive histone modification, which was counterintuitive with the viral latency reversal phenotype. Despite this well-established role, H3K9me3 has also been linked to a pathway of DNA double strand breaks repair. Given these knowledge, two possible models on the function of H3K9me3 at the provirus 3’-end arise. On the viral model, it is possible that H3K9me3 deposition is used by the provirus during latency reactivation to regulate its own gene expression. On the host model, it is possible that H3K9me3 deposition is used by host cells to initiate a process of repair of damaged DNA arising as a consequence of the high levels of transcription accumulated during the exponential phase of latency reactivation. In this exploratory and developmental R21 grant application, we will test these two models using a series of genetic approaches in immortalized and primary models of latency. If successful, our studies will fill a void in our understanding of HIV-1 latency biology by describing the functions of H3K9me3 deposition at the provirus 3’-end and the enzymes implicated. In keeping with NIAID’s mission of ending the HIV-1 epidemic, our long-term objective is to leverage the basic discoveries to devise novel alternative strategies for the selective elimination of inducible HIV-1 proviruses. As such, this fundamental knowledge can be used in future studies beyond the scope of this focused grant application, to therapeutically inhibit select H3K9 methyl transferases to sensitize cells to the natural process of latency reactivation. Collectively, the proposed research will have a sustained impact in the field.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY / ABSTRACT The mechanisms by which mammalian cells sense phosphate availability to maintain homoestasis of this critical nutrient are unknown. Phosphate is indispensable for many biological functions including DNA and RNA synthesis, bone formation, and preservation of energy as high energy phosphates. Consequently, serious complications develop during phosphate deficiency, e. g. rhabdomyolysis, and during phosphate excess, e. g. cardiovascular disease and vascular calcification. Genetic mutations that lead to massive phosphate excess are associated with a premature aging syndrome. A hallmark of vascular calcification is induction of the sodium- coupled phosphate transporter SLC20A1/PiT1, and SLC20A1 is also induced in aggressive cancers. SLC20A1 is 1) widely expressed, 2) provides phosphate for basic cellular functions, and is 3) massively upregulated in phosphate-starved cells. To interrogate the mechanisms underlying SLC20A1 regulation and more broadly phosphate homeostasis and sensing, we performed a set of complementary CRISPR-based genome-wide loss- of-function genetic screens in phosphate-replete and in phosphate-starved cultured mammalian cells. We found that proteostasis plays a crucial role in the regulation of SLC20A1 protein abundance through regulation of protein degradation, recycling, and synthesis. We hypothesize that phosphate starvation is detected by thus far unidentified phosphate sensing machinery, which leads to coordinated regulation of SLC20A1 degradation, recycling, and synthesis resulting in increased SLC20A1 protein abundance. We will examine this hypothesis through three independent yet synergistic projects. In Project 1, we will examine the mechanisms governing SLC20A1 internalization and degradation. Interestingly, loss of one of our candidate negative regulators of SLC20A1 degradation leads to a premature aging syndrome that closely resembles genetic syndromes with phosphate excess. Therefore, we will examine if decreased SLC20A1 internalization and degradation with dysregulated cellular phosphate uptake also leads to a premature aging phenotype. In Project 2, we will examine the mechanisms for SLC20A1 recycling to the plasma membrane, which will also be informative for many other proteins as this process is generally not well understood. In Project 3, we will examine the role of protein synthesis in the regulation of SLC20A1 protein abundance. Each Project is designed to also identify members of the underlying phosphate sensing machinery. Our ultimate goal is identification of novel pharmacological targets for the treatment of hypo- and hyperphosphatemic patients, which makes these studies highly clinically relevant.

NIH Research Projects · FY 2025 · 2024-08

Project Summary Loss of muscle mass, as seen in cancer cachexia, and sarcopenia correlates with heightened mortality rates, making it a global health concern with the aging population. We desperately need advanced molecular understanding to develop effective therapies. The intricate regulation of muscle mass involves several signaling pathways, orchestrating gene expression changes via activation and repression of transcription factors activity. Recent findings from our lab have uncovered the role for the transcription factor Maf in the maturation of fast twitch myofibers by directly activating fast muscle gene expression. Fast twitch myofibers are more affected than slow twitch myofibers in numerous atrophic conditions. Interestingly, Maf expression and activity are repressed during muscle atrophy. Our preliminary data indicate that Maf overexpression can suppress muscle atrophy. We hypothesize that Maf functions as a transcriptional repressor of the atrophic gene program, offering a promising therapeutic avenue. This proposal outlines a comprehensive plan to dissect the functions of Maf during muscle atrophy. To accomplish this, I will employ the following specific Aims: 1. To examine the transcriptional role of Maf in skeletal muscle atrophy. 2. To identify the partners and regulators of Maf activity in skeletal muscle. 3. To investigate the protective role of Maf in cachexia and sarcopenia. The K99 portion of this proposal will be carried out in the lab of the renowned molecular biologist, Dr. Eric Olson. In order to gain research independence through mentored training, I will continue to develop expertise in applying transcriptome profiling, including single nuclei RNAseq, to study how Maf transcriptionally reprograms myofibers (Aims 1). The proposed research also requires that I acquire additional mentoring to evaluate the partners of Maf using proteomic mass spectrometry, and the AAV gene therapy detailed in Aim 2 and 3. The knowledge and skills that I acquire during this time will serve as critical components of the foundation of my own independent lab. In summary, the structured approach detailed in this Pathway to Independence Award application not only promises to advance my growth as an independent researcher but also intends to make significant contributions to neuromuscular research. The anticipated outcomes from the proposed aims have the potential to reshape the understanding of muscle atrophy and lay the groundwork for my scientific journey.

NIH Research Projects · FY 2026 · 2024-08

The development of modern medicines relies on the ability to access compounds that are potent, selective, and ideally curative. To achieve these goals, chemists have sought to prepare evermore complex structures in search of nuanced activity and selectivity profiles, advances dependent on the availability of tools and strategies to synthesize such compounds in an economical and scalable fashion. Complex natural products have traditionally inspired the development of such tools, often acting as key milestones in the advancement of organic synthesis and as vital medicines in their own right. However, at the apex of complexity, their structures continue to test the limits of current capabilities: highly intricate targets are prepared only through enormous effort in terms of man-years and number of synthetic operations, often only in a target-specific manner. Here, we propose to utilize such compounds as vehicles for the discovery of complexity-generating tools and tactics. We outline approaches to several families of natural products built upon two distinct complexity-building strategies: dearomative transformations of readily available aromatic compounds and desymmetrization of simple symmetrical precursors. In the former case, aromatic feedstocks – available as petroleum byproducts or from renewable biomass – represent a versatile synthesis platform given well-established, predictable modes for their functionalization. We seek to deploy these materials in complexity-building dearomative reactions, disrupting their otherwise stable aromatic systems and transforming their flat sp2-scaffolds into sp3-rich compounds containing multiple stereogenic centers. Specifically, we propose to utilize the regiodivergent oxidative coupling of simple bisphenolic compounds to access the structural complexity inherent to the quassinoids, a family of potent anticancer terpenoids. Our synthesis platform will leverage the built-in oxidation of the aromatic systems to encode for much of the sp3-skeletal oxidation in these highly oxidized terpenoids, providing a unique means to access these targets and analogues, and illuminate their biological mode of action. In a symmetry-breaking transformation, we will develop new photochemistry of pyridinium ions to provide convenient access to highly functionalized cyclopentane building blocks, in particular via enantioselective desymmetrization of symmetrical allylic cation intermediates. We will apply these intermediates to the asymmetric synthesis of pactamycin-type aminocyclopentitols, highly potent antiparasitic agents. We will also develop a related approach to two subclasses of the Lycopodium alkaloids, which include central nervous system-active compounds, built upon two sequential photochemical dearomatizations of a simple quinoline. Overall, such discoveries will open up new areas of complex chemical space from simple aromatic or symmetrical precursors, providing natural products and designed analogues for interrogation of biological function. These studies will lead to innovations in dearomatization and desymmetrization chemistry, afford novel chemotypes for disease treatment, and provide tool compounds that may lead to new discoveries in biology.

NIH Research Projects · FY 2025 · 2024-08

Project Summary Dynamic rewiring of gene regulatory networks enables cell fate determination partly through differential gene expression. This process requires spatial organization of dozens of factors at precise genomic loci. Compartmentalization of transcription machinery into condensates—dynamic, mesoscale, local densities of proteins—has emerged as a mechanism coordinating this process. Although the function of condensates has been described in some cases, the mechanisms of their activity—particularly during cell fate determination— remain unclear. Many cell types are characterized by expression of cell-type-specific transcriptional regulators. The MYOCD coactivator is one such regulator for smooth muscle cell lineages. Using MYOCD as a case study, I have found that MYOCD-mediated gene activation and cell fate determination depend on switch-like, condensate formation. These studies also demonstrated the MYOCD trans-activation (TAD) domain is required for condensate formation and function. Surprisingly, substitution of the TAD domain for the intrinsically disordered regions (IDRs) of FUS or CDT1, two unrelated proteins known to form condensates, maintains condensate formation, but only substitution with the FUS-IDR activates gene expression. This indicates not only is self-association of the MYOCD coactivator essential for function, but that condensate formation mediated by distinct IDRs can exhibit distinct functionalities. I hypothesize coactivator condensates—mediated by different IDRs—confer distinct biochemical environments by directing genomic localization of gene expression machinery and selectively compartmentalizing transcriptional machinery into condensates to drive gene activation and cell fate determination. To address this hypothesis, I have developed an interdisciplinary and rigorous approach using various microscopy-, biochemical-, and sequencing-based experiments to model MYOCD-mediated condensate formation, gene activation and cell fate determination.

NIH Research Projects · FY 2024 · 2024-08

Project Summary/Abstract Asthma prevalence has increased in recent years with 8.4% of adults diagnosed with asthma. Asthma can be a lifelong disease. These rates increase with age, are 60% higher in women than men and are especially high in African Americans. Thus, adult asthma is a major health concern. Rates of uncontrolled asthma also increase with age and are higher in women and in minorities, especially Hispanics. Texas, where the proposed study will take place, has the third highest rate of uncontrolled asthma; therefore, more effective treatments for adult asthma are needed. Asthma patients with frequent exacerbations despite appropriate treatment pose a particular challenge. New treatments are needed to improve asthma control in this subpopulation. Our pilot data from three preliminary studies suggest that the selective serotonin reuptake inhibitors (SSRIs) citalopram and escitalopram, as compared to placebo, improve asthma control and decrease asthma exacerbations in depressed people with very frequent exacerbations. Effect sizes for exacerbations were much greater than for depression improvement. In addition, baseline depressive symptom severity did not predict asthma improvement. Thus, the asthma effect did not appear to be related to depression improvement. These clinical trial findings were supported by an electronic medical record analysis suggesting patients with asthma, who did not have a depression diagnosis, showed reductions in prednisone use after receiving SSRIs. SSRIs might improve asthma control through effects on subclinical anxiety and depressive symptoms or psychological triggers of asthma or independent of emotions through anti-inflammatory effects. In vitro studies suggest SSRIs reduce the frequency of IL-4- and IL-2-producing cells triggered by CD3 stimulation and potentiate glucocorticoid receptor-mediated gene transcription, suggesting they might enhance the effects of inhaled steroids. SSRIs also decrease IL-6 levels, which are elevated with asthma. In animal models of allergic asthma, SSRIs reduce bronchial inflammation with decreases in macrophages, lymphocytes, neutrophils and eosinophils in bronchoalveolar lavage fluid. A single-site investigator-initiated clinical trial (R61/R33) is proposed to examine escitalopram compared to placebo in adults with asthma requiring frequent steroid bursts but without current major depressive disorder. A milestone-driven R61 start-up phase of less than one year will set the stage for the R33 phase 24-week, randomized, double-blind, placebo-controlled trial of escitalopram in 105 men and women with moderate to severe persistent asthma who, despite treatment with medium-high dose inhaled corticosteroids and long-acting beta agonist therapy, had ≥ 3 asthma exacerbations in the past year. Need for steroid bursts (primary outcome) and asthma control, as well as functioning, quality of life, serum IL-6 levels and exhaled nitric oxide, will be assessed. Effects on mood and anxiety will be explored and their influence on asthma control assessed. An experienced investigative team with a long history of collaboration and expertise in asthma, SSRIs, clinical trials and statistical analysis will conduct the study.

NIH Research Projects · FY 2024 · 2024-08

Project Summary Compromised tissue integrity is a major cause of debility during aging. Emerging findings suggest critical functions of lysosomes promoting tissue integrity. Our unpublished findings indicate key roles for C. elegans PMK-1, a conserved p38 MAPK member, in promoting lysosome formation and tissue integrity during development and aging. These non-cell autonomous functions signal between germline, epidermis and nerve. Moreover, our data indicate that p38 MAPK acts on distinct targets in a tissue-specific and stage- specific manner to promote lysosome function. It is not known how p38 MAPK signals between tissues to coordinate lysosome function. We hypothesize that PMK-1 promotes lysosome function by signaling a cell non-autonomous network regulating the regeneration and heterogeneity of lysosomes across tissues. Over the next 2 years, the critical goals for this project are to: 1) define the PMK-1 interactome required for lysosome regeneration, 2) reveal tissue and stage-specific functions of PMK-1 in lysosome regeneration and heterogeneity, and 3) delineate tissue-specific contributions to PMK-1 cross-tissue signaling. We have built an array of tools to deeply understand how p38 signaling regulates lysosome regeneration and heterogeneity to promote tissue integrity. Our proposed interdisciplinary studies include proteomics, genetic screens, biochemical analyses, and cell biology approaches to tackle this complex problem of understanding non-cell autonomous p38 MAPK function. The long-term objective of the proposed studies is to understand the molecular and physiological mechanisms of p38 MAPK signaling in tissue integrity during development and aging.

NIH Research Projects · FY 2024 · 2024-07

Project Summary The ability to collect high resolution volumetric datasets of whole organs is arguably most applicable to the neuroscience field. Individual neurons on the micron scale comprise circuits that extend across large brain territories over millimeters apart; thus, perturbations of neuronal circuits in models of neurological, psychiatric or brain injury conditions can become evident only by assessing volumetric datasets spanning the micro-, meso- and macro-scales. We are requesting funds to purchase a TissueVision TissueCyte 1600FC serial two-photon tomography (STPT) microscope to enable high-throughput, high resolution automated imaging of whole, uncleared rodent brains or spinal cord segments by NIMH/NIH supported investigators at UT Southwestern Medical Center in Dallas, TX. STPT is a block face volumetric imaging modality in which two-photon mosaic tile imaging is alternated repeatedly with vibrotome sectioning to obtain full 3D volumes of the entire brain or spinal cord segment. The instrument will be housed in and maintained by the Whole Brain Microscopy Facility (WBMF) and will be available to all investigators on campus as well as external users. This instrument will complement and expand the capacity of two existing STPT systems within the facility (TissueVision TissueCyte 1000 - both 9 years old) which are heavily used (1100 total samples imaged since 2014; projected total of 168 samples for CY23) and lack many features of the updated TissueCyte 1600FC. Our existing TC1000 systems have a single tunable Ti:Sapphire excitation laser and are configured for detection of three emission channels (blue, green and red), which greatly limits their range of detectable fluorophores and multiplexing capability. The new TC1600FC system offers a dual laser line system, four detection channels and the innovative robotic “SlicePlacer” module to automatically mount the sections onto slides after imaging for correlative staining. Our existing TC1000s cannot accommodate imaging of far-red fluorophores, multiplex imaging of fluorophores with widely separated excitation spectra, ratiometric imaging of inherent autofluorescent signals, label free nonlinear microscopy methods such as second and third harmonic generation (SHG and THG), or automatic mounting of sections. The WBMF has a full time computational scientist on staff and robust, custom developed registration and analysis pipelines to accommodate quantification of STPT whole brain and cord datasets. There are no other STPT microscopes available on the UT Southwestern campus or in the state of Texas to our knowledge. The WBMF has a broad user base, currently serving 237 active clients from 98 individual laboratories across 31 basic science and clinical departments with a wide variety of project foci. Since the installation of the current TissueCyte 1000 instruments in 2014, we have supported STPT experiments from 41 individual investigators, the vast majority of which are neuroscience focused. We have 22 active clients using these systems currently, including the 8 major users described in this application. Research projects that will benefit from the use of the proposed equipment are focused on multiple devastating neuropsychiatric and neurological conditions, including autism, schizophrenia, depression, epilepsy, dementia and spinal cord injury.

NIH Research Projects · FY 2026 · 2024-07

PROJECT SUMMARY Experience-driven changes to chromatin architecture help activate gene transcription programs that allow animals to learn and develop adaptive behaviors. Yet the forces that remodel chromatin in response to neuronal activity remain obscure. Torsion from DNA supercoiling injects free energy into the DNA and has the potential to organize chromatin, yet how dynamic supercoiling distributes and acts within the neuronal genome are poorly understood. RNA polymerases (RNAPs) and DNA topoisomerases generate and resolve supercoils, respectively, and their concerted actions affect dynamic supercoiling distribution. However, to what extent supercoils propagate from the sites of RNAP activity, and how chromatin structure affects supercoil distribution remain unexplored. Furthermore, while supercoiling is a ubiquitous feature of DNA topology, how torsional free energy from supercoiling affects chromatin structure and gene activity patterns is also unknown. To address these issues, psoralen crosslinking and sequencing (TMP-seq) was recently performed to map the distribution of underwound (negatively supercoiled) DNA at high resolution in cultured mouse cortical neurons under basal conditions. Additionally, new methods were developed to map genome-wide sites of catalytically engaged topoisomerases (TOP1cc-seq and TOP2Bcc-seq). These studies revealed that supercoils transmit widely (> 200 kb) from the sites of active RNAPs, while topoisomerases only act at selective locations to resolve supercoils. These data indicate that torsional free energy from supercoiling could be widely available to influence chromatin structure. Based on these results, this project will perform TMP-seq at various times following neuronal stimulation and assess how activity-driven supercoiling distributes within the genome. Additionally, TOP1cc-seq and TOP2Bcc-seq will be performed under the same conditions to determine how topoisomerases are utilized to regulate dynamic supercoiling in stimulated neurons. While supercoils generally distribute freely within chromatin, preliminary data indicate that they also accrue at specific nucleosome configurations, particularly at active promoters with broad H3K4me3 distributions and in regions flanking H3K27me3-rich chromatin. Intriguingly, a broadening of H3K4me3 has been observed following neuronal stimulation in vivo, suggesting that specific stimulus-driven epigenetic changes could “constrain” dynamic supercoils. The proposed research will test this idea by assessing how knockdown of WDR5, which mediates H3K4 methylation, and expression of JMJD3, which erases H3K27me3, affect neuronal activity-dependent supercoiling patterns. Finally, topoisomerase inhibitors and locus-specific modulators of DNA supercoiling will be used to perturb supercoiling patterns in stimulated neurons, and chromosome conformation capture (Micro-C), ATAC-seq, and nascent transcription analysis (fastGRO) will performed to test the idea that dynamic supercoiling has a causal role in neuronal activity-driven chromatin reconfiguration. Together, these efforts will provide new insights into how DNA mechanics regulates neuronal functions during development and disease.

NIH Research Projects · FY 2025 · 2024-07

Project Summary/Abstract Cardiovascular disease is the leading cause of mortality in the US and worldwide. Decades of endeavors in studying cardiovascular disease have led to a substantial understanding of underlying mechanisms, but there is still much to be learned. Cardiovascular homeostasis is regulated at both transcriptional and posttranscriptional levels, and an increasing body of evidence suggests that RNA posttranscriptional modifications, such as alternative RNA splicing, play essential roles in regulating cardiac function and disease. A previous study by the applicant (Dr. Peiheng Gan) has revealed that RNA-binding protein with multiple splicing (RBPMS) is crucial for cardiomyocyte proliferation and heart development through modulating alternative RNA splicing. In the new study, the applicant found that RBPMS is required for adult cardiac contractility. The absence of RBPMS impaired cardiomyocyte contractility and impacted the splicing of various sarcomeric genes, which displayed distinct patterns. Cardiac RBPMS expression was decreased in patients with heart failure and doxorubicin-induced heart failure in animal models. Intriguingly, the overexpression of RBPMS showed protective effects on contractility and survival in doxorubicin-treated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Together, these data suggest that RBPMS is a key regulator of adult heart function and heart failure progression. During the K99 phase of this proposal (Aim 1), the applicant will characterize how RBPMS regulates cardiomyocyte contraction through modulating RNA splicing using both mouse models and hiPSC-CMs. In Aim 2, the applicant will leverage cellular and molecular tools to characterize the functional interactions between RBPMS and other cardiac RNA binding proteins (RBPs), including RBM20 and RBFOX1. This study provides the applicant with a unique opportunity to investigate how different RBPs interact to regulate cardiac function and alternative RNA splicing. During the R00 phase (Aim 3), the applicant will explore the therapeutic potential of overexpressing RBPMS in doxorubicin-induced mouse heart failure and determine the molecular mechanisms of RBPMS-mediated protective effects. The completion of the proposed study will provide critical insights into the posttranscriptional regulation of cardiac function by RBPs and create new opportunities for curing cardiovascular disease. The applicant will acquire crucial knowledge and skills by studying RBPs and RNA posttranscriptional modifications during his K99 phase to complement his previous expertise in cardiovascular research. Additionally, the applicant’s career development will be enhanced by the expertise of an exceptional mentoring and advisory committee, as well as the unparalleled resources and ample educational and training opportunities at UT Southwestern Medical Center, a world-class research institution. The outstanding mentoring, unmatched resources, and strong commitment from his department will strengthen the applicant’s candidacy for and transition to an independent tenure-track faculty position.

NIH Research Projects · FY 2025 · 2024-07

7. Project Summary/Abstract The overarching goal of this NGC Center is to leverage expertise in small molecule drug discovery, target identification, and protein and IDR biochemistry to identify and advance small molecules that target transcriptional fusion oncoproteins by 1) targeting the transcriptional machinery that is co-opted by fusion proteins to drive the oncogenic transcriptional programs, and 2) Directly impair IDR function with small molecules. Transcriptional fusion proteins induce oncogenic gene expression programs that drive cancer development and progression. These proteins act as neomorphic transcription factors by recruiting and re-wiring transcriptional regulatory complexes. Rational drug design to directly target transcriptional fusion proteins remains a major challenge because these proteins lack enzymatic activity and do not have obvious pockets amenable for small molecule binding. In addition, the mechanisms by which fusion oncoproteins co-opt transcriptional regulatory complexes are incompletely understood. As such, the development of new therapies to target transcriptional fusion proteins has lagged efforts to develop targeted therapies for mutant oncoproteins involved in cellular signaling. Our team has discovered that the Mediator transcriptional co-activator complex is co-opted to control the oncogenic gene expression programs induced by transcriptional fusion proteins. We have further identified the CDK8 kinase module as a druggable entry point by which to impair Mediator function through a novel trapping mechanism. Our team will probe the biochemical and transcriptional mechanisms through which CDK8/19 inhibitors alter Mediator function and develop CDK8/19 inhibitors that maximize Mediator impairment and study these small molecules in advanced preclinical models of Ewing sarcoma and rhabdomyosarcoma. We further identified that vaccinia-related kinase 1 (VRK1) is a kinase that becomes synthetically lethal following CDK8/19 impairment, and our team will develop novel specific VRK1 inhibitors and evaluate their efficacy in single agent and combination strategies. A central theme has recently emerged that many fusion proteins consist of a DNA binding domain linked to an intrinsically disordered region (IDR). The IDR mediates self-association or phase separation that has been shown to be essential for the oncogenic nature of these fusion proteins. Our team has developed an innovative biochemical method of identifying the key protein regions within IDRs that mediate self-association. We will map the key self-association residues in three fusion protein IDRs and identify small molecules that impair the ability of these regions to drive phase separation.

NIH Research Projects · FY 2025 · 2024-07

Project Summary Human respiratory tissues have a unique cellular architecture and a mucus-based clearance system that, together, constitute an impressive barrier to viruses that use the airway epithelium as their portal of entry. The mucus-based clearance system acts as a physical, innate defense mechanism. Nasal epithelial cells have two classes of protrusions on their surfaces—long motile cilia and short microvilli. Motile cilia are specialized organelles that extend from the cell into the airway to drive mucociliary clearance. However, the mechanisms used by respiratory viruses to breach this barrier and infect the respiratory epithelium are not well understood. Our previous work has highlighted the crucial role of cilia in respiratory virus infection. Using cultured human primary nasal epithelial cells, we have demonstrated that SARS-CoV-2 and RSV specifically attach to motile cilia during infection. Importantly, inhibiting the interaction between SARS-CoV-2 or RSV and cilia can effectively suppress viral infections, indicating the critical role of the virus-cilia interaction. Furthermore, treatment with an inhibitor of ciliary protein trafficking significantly decreased SARS-CoV-2 infection in nasal epithelial cells. These findings underscore the critical role of cilia during viral entry. Based on these findings, we propose specific aims to understand how RSV and SARS-CoV-2 hijack nasal cilia for viral entry using the primary nasal epithelium as a model. By comparing the infection mechanisms of RSV and SARS-CoV-2, we aim to gain a clearer understanding of the differences between these viruses and the significance of nasal cilia in their respective contexts.

NIH Research Projects · FY 2026 · 2024-07

Project Summary/Abstract Lipid signaling is crucial for regulating organismal physiology and metabolic expenditure, and disturbances in lipid homeostasis can deleteriously impact health. Cells tightly control the absorption, synthesis, and breakdown of lipids to accommodate energetic demands and ensure energetic reserves later in life. We recently reported a mechanism by which cells sense metabolic demand resulting from lipid depletion and respond by increasing expression of genes involved in membrane trafficking as well as nutrient absorption and catabolism. This is accomplished by the release of the starvation-responsive nuclear hormone receptor, NHR-49, from endocytic vesicles and its subsequent nuclear translocation and transcriptional activation. Yet the mechanisms and participating co-factors facilitating NHR-49 activation and attenuation remain unclear and are critical for our fundamental understanding of lipid sensing and metabolism. Our examination has uncovered a mechanism in which loss of antagonistic ligand binding releases NHR-49 from endocytic vesicles. Next, NHR- 49 sequentially engages a series of cofactor, which through direct interactions and post-translational modifications, facilitate its nucleocytoplasmic shuttling, transcriptional activation, and proteolytic degradation. Through the proposed five-year research period, we aim to understand how cells activation and attenuation this lipid surveillance response by defining the molecular details underlying the lifecycle of NHR-49 including its nuclear import, intranuclear retention, and transcriptional regulation followed by its phosphorylation, nuclear export and degradation. Our preliminary data has identified a series of co-factors including karopherins involved in its nucleocytoplasmic shuttling, nuclear pore complexes involved in its nuclear retention and transcriptional activation, kinases involved in its nuclear export, and a ubiquitin ligase involved in its proteasomal degradation. Through a more detailed description of NHR-49 co-factor interactions and their associated post-translational modifications, we can gain a better understanding of intracellular lipid sensing and metabolic homeostasis.

NIH Research Projects · FY 2025 · 2024-07

Project Summary Rapidly evolving proteins constitute a significant portion of a bacterial proteome. These proteins diverge substantially from their homologs, frequently change functions and locations of active sites, and therefore are more challenging to study. Fast-evolving proteins are nevertheless as crucial as conserved ones for our fundamental understanding of molecular evolution and biomedical applications: pathogenicity factors in bacterial pathogens typically undergo fast evolution due to arms races between hosts and pathogens. Revolutionary advancements in computational protein science now facilitate the efficient investigation of fast- evolving proteins. Advanced artificial intelligence methods such as AlphaFold have produced accurate models of protein 3D structures, which could revolutionize bioinformatics approaches. Now, instead of relying on sequence-based homology searches, which frequently fail to find relatives of fast-evolving proteins, we can use similarity in 3D structures, which tend to be more conserved evolutionarily. Additionally, methods for predicting protein-protein interactions (PPIs) are nearing the accuracy of high-throughput experiments, offering another approach to gain functional insights to a protein by finding a well-studied interaction partner. The time is ideal to capitalize on these developments and tackle challenging problems that were previously impossible to address. In this proposal, I intend to study fast-evolving proteins using state-of-the-art computational methods, followed by experimental validation. First, I have developed methods to recognize domains from AlphaFold models and assign them to an evolutionary context for functional inference. I plan to enhance these methods using deep learning techniques and develop additional methods to predict functional categories. While existing tools are primarily optimized for conserved protein families with deep sequence alignments and extensive experimental data, my methods will cater specifically to fast-evolving proteins. Subsequently, I will apply these tools and my expertise in comparative genomics and PPI modeling to study the fast-evolving and pathogenicity-associated proteins encoded by the pan-genomes of Vibrio parahaemolyticus (Vpara) strains isolated from human patients. By determining their evolutionary origins, predicting their interacting partners, and inferring their functions, I aim to identify a set of uncharacterized and fast-evolving candidate pathogenicity factors (CPFs). Finally, I will select several promising CPFs and experimentally validate their secretion and interacting partners using medium- throughput experiments. This preliminary functional characterization will be the initial step toward elucidating the mechanisms of these novel PFs and will pave the way for future discoveries in my lab and in the field. This project will allow me to undergo rigorous and multidisciplinary training in cutting-edge computational methods and experimental techniques that can generate large datasets to complement and validate computational studies. I expect to contribute to science by sharing my computational tools and analytical results through a web server and an online database, and by characterizing novel pathogenicity factors in Vpara.

NIH Research Projects · FY 2025 · 2024-07

Project Summary The pathways regulating the development of early-onset glaucoma are not well understood. Despite evidence suggesting that a large proportion of early-onset glaucoma cases may have a genetic basis, known genes only account for about 20% of cases. In fact, only 12 genes have been described so far, for primary forms of this disease, compared to hundreds for retinal dystrophies. In this complex biological system, a Forward Genetics approach is an ideal strategy to ask, without preconceptions, which genes/molecules are important in regulating early-onset glaucoma. Our short-term goal is to identify and characterize gene/protein defects and molecular pathways that lead to early-onset glaucoma. The long-term goal is to leverage our research discoveries to understand this blinding disease, improve screening strategies, and identify novel therapeutic opportunities. We propose that a high-throughput and unbiased strategy provides an ideal approach to discovery of gene/phenotype associations for early-onset glaucoma. In collaboration with Nobel laureate Bruce Beutler, we have been employing a robust state-of-the-science and unbiased Forward Genetics pipeline in which random mutations are generated and mice can be screened for signs of glaucoma. We have already collected retinal images from 6000 mutagenized mice and proposing to use this extensive database to screen for genes that lead to inner retinal thinning. Our approach has significant advantages compared to other existing protocols. Most importantly, ours is the first and only protocol in which all mice have been pre-genotyped at all mutant loci. In addition, the large scale of our system and the large pedigree size will also add to the discovery power. Together, these advantages will allow us to identify and pursue novel gene/phenotype associations related to glaucoma. In our retinal studies we have identified over 45 gene-phenotype associations after covering just 5%-8% of the mouse genome. Of these, 20 genes have not been reported to be associated with the retina. These results attest to the strength of our pipeline. Having an excellent continuous variable parameter to monitor for early-onset glaucoma (ganglion cell complex OCT measurements) also supports the feasibility of our proposal. We will harness the power of CRISPR/Cas9 gene editing, single cell RNA sequencing, and co- immunoprecipitation experiments with highly sensitive mass spectrometry and proteomics analysis, and other techniques to explore the mechanisms of these associations. This proposed research will advance our knowledge of the genetic basis in early-onset glaucoma. We also anticipate that our results will lead to the identification of novel diagnostic and therapeutic avenues.

NIH Research Projects · FY 2025 · 2024-07

PROJECT ABSTRACT Site-one protease (S1P) is a membrane anchored protease in the secretory system and a critical component of cellular signaling pathways including cholesterol biogenesis, the ER stress response, and lysosome biogenesis. S1P begins as an inactive pro-enzyme in the ER and becomes active through autoproteolysis of its inhibitory pro-domains as it folds in the ER and traffics to the Golgi, where it functions as an active enzyme. S1P is best studied in cholesterol metabolism. The spatial control of S1P activity, with the inactive form in the ER and the active form in the Golgi, underpins cholesterol metabolism in human cells. Lipogenic transcription programs that cause the uptake and synthesis of cholesterol are controlled by a family of transcription factor proteins known as Sterol response element binding proteins (SREBP). The SREBP precursors are folded in the ER, where they must be protected from proteolysis by S1P. When cholesterol levels are low, SREBP precursors are transported from the ER to the Golgi, where it they cleaved by S1P to initiate a cascade that results in the liberation of the SREBP transcription factor domain and the upregulation of lipogenesis. Ensuring S1P is active in the Golgi but inactive in the ER is critically important to cells and animals. SPRING (also C12ORF49) is a newly identified co-factor that is critical for the controlled maturation of S1P and understanding their relationship will provide new insights into cholesterol metabolism specifically and protease maturation in the secretory pathway more broadly. In preliminary work, we obtained a high-resolution structure of the soluble S1P-SPRING complex using cryo-electron microscopy (cryo-EM). Structural and biochemical data develop the hypothesis for a proposed mechanism where SPRING matures S1P by competing with and displacing an inhibitory pro-domain. Removing this pro-domain is necessary for S1P to proteolyze external substrates. Experiments with S1P trapped in different maturation stages suggest SPRING binds S1P at an intermediate stage of maturation as S1P traffics from the ER to the Golgi. In this proposal, we will use structural biology, biochemistry, and cellular biology to elucidate how S1P matures in the presence or absence of SPRING and how SPRING controls the enzymatic activity of S1P. In Aim 1, we will obtain cryo-EM reconstructions of S1P in distinct stages of maturation. We will use competition assays to test whether SPRING and the S1P pro-domains compete for binding at the same site of the S1P enzyme. In Aim 2, will use a peptide cleavage assay to determine the substrate specificity of the S1P-SPRING complex and which S1P substrate motifs require SPRING for proteolysis by S1P. In Aim 3, we will determine the functional consequences of disrupting the S1P-SPRING interaction in biochemical and cell-based signaling assays that measure SREBP activity and the ER stress response.

NIH Research Projects · FY 2025 · 2024-07

The inferior olivary nucleus plays a vital role in cerebellar function and motor control, and olivary degeneration is central to the pathophysiology of tremor and ataxia. A striking feature of olivary neurons is that they are susceptible to degeneration both from intrinsic molecular pathology (e.g., in spinocerebellar ataxias) as well as from extrinsic denervation of inhibitory cerebellar input which causes hypertrophic olivary degeneration (HOD). These triggers for olivary degeneration are assumed to reflect distinct cell death mechanisms, but our recent data strongly suggests that these different etiologies utilize shared molecular events key to olivary neuron viability. This proposal will test this novel hypothesis, which has profound implications for inferior olivary neuron survival and function. In a genetically precise mouse model of spinocerebellar ataxia type 1 (SCA1; SCA1- knockin or SCA1-KI mice), we find olivary neuron hypertrophy accompanied by the characteristic increase in dendrite length. Remarkably, these features occur in the absence of loss of inhibitory input. Moreover, we find no olivary pathology in a separate line of SCA1 transgenic mice that exhibit more severe but restricted cerebellar Purkinje neuron degeneration. A significant loss of calbindin positive neurons, characteristic of olivary degeneration, accompanies HOD in SCA1-KI mice. Considered together, these findings challenge current dogma by dissociating HOD and denervation. Preliminary studies using in vivo electrophysiological recordings demonstrate that synaptic (extrinsic) excitation on the olive, the cause of HOD due to inhibitory neuron denervation, is not increased. Additional studies demonstrate that hypertrophic olivary neurons exhibit increased intrinsic excitability of neurons due to potassium (K+) channel loss-of-function. Based on these findings, we hypothesize that this increased intrinsic excitability is the trigger for olivary degeneration in SCA1, and that increased excitability from whatever source (intrinsic or extrinsic) is a shared mechanism for inferior olive degeneration. We further hypothesize that olivary degeneration can therefore be prevented by normalizing membrane excitability. We will test these hypotheses through the following Specific Aims: Aim 1. Define the relationship between membrane excitability and olivary degeneration in SCA1. Aim 2: Define intrinsic and extrinsic contributions to olivary dysfunction in models of SCA1. Aim 3: Determine the basis for increased intrinsic excitability in hypertrophic olivary degeneration in SCA1. Results from these studies are expected to establish that increased membrane excitability is a convergent mechanism for olivary degeneration. These studies would be expected to provide insight into whether neuronal degeneration due to synaptic excitotoxicity may be addressed by reducing intrinsic excitability as a therapeutic strategy.

NIH Research Projects · FY 2025 · 2024-07

Increasing evidence indicates that Autism Spectrum Disorder (ASD) is manifests differently in males and females behaviorally and affects brain function and connectivity differently between sexes. There has been little known of how ASD genes affect the female brain on a cellular and microcircuit level. During the previous grant period we discovered that deletion of the ASD-risk gene, Pten (Phosphatase and tensin homolog deleted on chromosome 10), in neocortical pyramidal neurons (NSEPten KO) resulted in robust hyperexcitability of local neocortical circuits in female, but not male, mice, observed as prolonged, spontaneous persistent activity states (UP states). We also demonstrated that circuit hyperexcitability in NSEPten KO mice is mediated by enhanced signaling of metabotropic glutamate receptor 5 (mGluR5) and estrogen receptor α (ERα) to ERK and de novo protein synthesis selectively in Pten deleted female neurons. Pten deleted Layer 5 cortical neurons have a female-specific increase in mGluR5 levels and mGluR5-driven protein synthesis. In addition, mGluR5-ERα complexes are elevated in female cortex and genetic reduction of ERα in Pten KO cortical neurons rescues circuit excitability, protein synthesis and enhanced neuron size selectively in females. Abnormal timing and hyperexcitability of neocortical circuits in female NSEPten KO mice are associated with deficits in temporal processing of sensory stimuli and social behaviors as well as mGluR5-dependent seizures. Female-specific cortical hyperexcitability and mGluR5-dependent seizures are also observed in a human disease relevant mouse model, germline Pten+/- mice. Our results demonstrate sex-specific dysfunction of developing cortical circuits with loss of function of a high-confidence ASD-risk gene. Importantly, we demonstrate a distinct, female-specific dysfunction of mGluR5- ERα signaling pathways that drive excitability. For the Phase 2 extension of this work, we propose to determine the female-specific, Pten-regulated cellular and molecular alterations in cortical neurons that give rise to hyperexcitability of circuits. In Aim 1, we will determine the sex-specific, mGluR5 and ERα -regulated neuronal and synaptic properties in L5 PTEN KO neurons. In Aim 2 we will determine the sex- specific, PTEN-regulated ribosome- associated transcripts in L5 cortical neurons and their regulation by mGluR5 and ERα. Our results reveal sex-specific and estrus cycle-dependent deficits in sensory-processing in Pten loss of function models. In Aim 3 we will determine if a reduction in mGluR5 or ERα function corrects these sensory processing deficits and in Aim 4, we will examine the role of estrogen and estrus cycle in dysfunction of developing and mature cortical circuits and in vivo resting and sensory-driven EEG phenotypes in Pten loss of function models. Results of these aims are expected to provide knowledge of the sex-specific mechanisms by which ASD-risk genes regulate the development and function of sensory cortical circuits and affect sensory processing.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Mounting evidence supports a central role for chronic inflammation in obesity and metabolic dysfunction, yet our understanding of actual causal links remains limited. We have identified and characterized numerous points of crosstalk between inflammation and the metabolic effects of overnutrition. Activation of the innate immune system triggers the robust production of interferon stimulated genes (ISGs), many of which have potent roles in metabolism. One such gene encodes for the ubiquitin-like modifier ISG15 that is conjugated to target proteins in a reversible process referred to as ISGylation. Our preliminary data show diet-induced obesity increases liver expression of Isg15 and protein ISGylation. Deficiency in liver Isg15 (ISG15LKO) renders mice resistant to diet- induced weight gain, and improves insulin sensitivity and hepatic steatosis. The liver, however, is not a thermogenic organ. Therefore, the impact of liver ISGylation on the regulation of body weight is likely mediated through endocrine signaling to other organs capable of influencing food intake, locomotor activity and/or energy expenditure. To identify ISGylated proteins, we engineered endogenously tagged Isg15 mice, and mass spectrometry proteomics data revealed robust enrichment of proteins involved in peroxisomal and mitochondrial oxidation. The overall goal of this proposal is to interrogate the role of liver ISGylation in regulating organismal energy balance. The central hypothesis is that in the liver, obesity triggers ISGylation of critical oxidative proteins that influence the hepatocyte secretome and promote weight gain, either by increasing food intake or decreasing energy expenditure. This hypothesis will be tested through the following two specific aims. Aim 1 will determine the effects of hepatic ISGylation on energy balance, and Aim 2 will identify liver ISGylation targets responsible for systemic metabolic regulation.