genes

Utilizing integrin-targeted PET imaging and therapeutics to predict and treat radiation-induced pulmonary fibrosis

Project Summary/Abstract. Lung cancer is the leading cause of cancer death in the US, with over 125,000 deaths annually. Radiation therapy (RT) is a critical component of curative lung cancer treatment for many patients. However, radiationinduced pulmonary fibrosis (RIPF) is a common side effect that carries a poor prognosis with limited treatment options. Up to 40% of patients with lung cancer who receive RT may experience RIPF. RIPF is a late effect of RT, typically occurring 3 or more months after treatment. The symptoms of RIPF can include shortness of breath, pleural effusions, decreased lung function, and respiratory failure. Cell surface integrin heterodimers play a key role in the pathogenesis of RIPF. In particular, the integrin αvβ6, which is expressed at a low level in the alveolar epithelium at baseline, is significantly upregulated upon RT damage. The key role of integrin αvβ6 in RIPF is illustrated by studies in which mice lacking integrin αvβ6, or treated with an αvβ6-blocking antibody, do not develop RIPF. Here, we propose to translate this mechanistic understanding of RIPF into novel approaches for monitoring and treating RIPF. We hypothesize that non-invasive αvβ6 PET imaging will be safe and can specifically bind to αvβ6 in patients with RIPF. Additionally, we hypothesize that a novel small-molecule integrin antagonist, IDL2965, can mitigate and treat RIPF in mice. In this project, we are utilizing mice to model RIPF, as mice develop RIPF that mimics human disease. In addition, cellular and in vitro models do not approximate the complex biology leading to the development of RIPF. Our data using [64Cu]Cu-DOTA-αvβ6-BP to detect early RIPF in mice are compelling in both single-fraction high-dose RT and lower dose-larger volume RT models (Lo et. al, IJROBP 2025). However, to progress to clinical trials in patients with cancer, we will obtain data to submit an Investigational New Drug (IND) application to the FDA. Importantly, we propose translating [64Cu]Cu-DOTA-αvβ6-BP PET imaging into patients with lung cancer, allowing us to better identify RIPF and develop a tool to determine the efficacy of IDL-2965 in future clinical studies. The specific aims of the proposal are: (1) Characterize the utility of [64Cu]Cu-DOTA-αvβ6-BP in mice with conventionally fractionated RT and identify circulating biomarkers of RIPF, and determine the in vivo toxicology of [64Cu]Cu-DOTA-αvβ6-BP to prepare and submit an exploratory Investigational New Drug (eIND) application to the FDA, (2) Conduct a first-in-human clinical trial of [64Cu]Cu-DOTA-αvβ6-BP to determine its safety and human dosimetry in patients with evidence of RIPF from computed tomography or in healthy controls, and (3) Determine the effect of integrin antagonism using IDL-2965 on mitigating RIPF in preclinical mouse models. The goals of this proposal are two-fold: (1) demonstrate safety and target specificity for [64Cu]Cu-DOTA-αvβ6-BP so that it can be used in future studies to identify RIPF and evaluate the efficacy of anti-fibrotic therapies, and 2) determine the ability of IDL-2965 to prevent RIPF in preclinical mouse models.

Staphylococcus aureus metabolic requirements during skin colonization

Project Summary Staphylococcus aureus causes 76% of all skin infections, and yet simultaneously this pathogen asymptomatically colonizes the skin of 8-22% of healthy adults. Since the majority of S. aureus disease is the result of autoinfection from the colonizing strain, and invasive infections often originate from the skin, there is an urgent need to understand colonization mechanisms. In colonizing the skin, S. aureus encounters abundant levels of amino acid derivatives like urocanic acid and 5-oxoproline (OP) that contribute to the skin’s “acid mantle” and have reported anti-Staphylococcal properties. The central hypothesis of this project is that amino acid transport and catabolism is a critical feature of S. aureus skin colonization. To model this environment, we developed a skin-like media (SLM) to assess S. aureus physiology on the human skin surface. We determined the S. aureus transcriptional response using RNAseq and performed metabolomics in SLM, both of which demonstrated that amino acid catabolism genes are upregulated and that amino acids are rapidly consumed. These findings indicate that S. aureus has a skin expression program that enables survival and growth in this harsh environment. In Specific Aim 1, we are investigating S. aureus metabolism of serine, the second most abundant amino acid on human skin. We hypothesize that serine transport and catabolism is critical for S. aureus skin colonization. We will assess growth of mutant strains disrupted in serine pathways in the SLM and during mouse skin colonization. With 13C-tracing experiments we will investigate serine flux in S. aureus using metabolomics. We will determine serine transport mechanisms using bioinformatic guided targets and serine analogues. In Specific Aim 2, we will assess S. aureus resistance to toxic skin metabolites. OP is abundant on human skin and is known to be deleterious to bacteria. Our preliminary metabolomics studies indicate that S. aureus metabolizes OP in SLM, and we have identified a putative oxoprolinase (genes SAUSA300_1566-1561) that is upregulated on skin. We hypothesize that the detoxification of OP contributes to S. aureus survival on the skin. We will construct mutants in the 1566-1561 locus and test their contributions to OP metabolism in SLM with growth and metabolomics experiments. We will also investigate OP transport and test mutant strains in our mouse skin colonization model. In Specific Aim 3, we will identify new determinants of S. aureus skin colonization using TnSeq. We have developed an improved TnSeq library preparation and analysis protocol, and in our preliminary studies we performed TnSeq in SLM and in our mouse skin colonization model. We will evaluate pathway hits, such as respiration and fermentation, and aspartate metabolism targets by testing constructed mutants during SLM growth and in the mouse model. Novel hits will be validated with follow-up genetic experiments and 13C-tracing experiments. Collectively, the proposed studies will advance our knowledge of S. aureus colonization and adaptation to the skin environment.

Eosinophils promote persistence and transmission during Bordetella spp. infections

ABSTRACT Despite widespread vaccination, Bordetella spp., the causative agents of whooping cough, continue to circulate globally. Resurgent outbreaks contribute to significant healthcare burdens and costs estimated up to $79 million annually. This persistence and reemergence highlight a critical need for new therapies and prevention methods. Our laboratory investigates bacterial and host drivers that enable Bordetella success, defined as enhanced persistence, reinfection, and transmission. We have identified the Bordetella sigma factor BtrS as a regulator of immunosuppressive pathways that modulate eosinophil function. Leveraging genetically tractable Bordetella strains, advanced murine models, and immunological tools, we are uniquely positioned to dissect how eosinophils contribute to respiratory bacterial infections. Our preliminary data reveal that eosinophils promote Bordetella persistence. Our results also show that the anti-inflammatory cytokine IL1 receptor antagonist (IL1Ra) also contribute to persistence. However, the contribution of eosinophil-derived immunosuppressors remains unclear and will be investigated in Specific Aim 1. Moreover, we have evidence that eosinophils are required for nasal shedding, through mucus enhancement, and paroxysmal coughing, via exacerbation of bronchoconstriction, during Bordetella spp. infection, two key metrics of transmission. The eosinophil-effectors that promote shedding, coughing, and transmission, will be investigated in Specific Aim 2. Based on our data, we hypothesize that eosinophils contribute to Bordetella pathogenesis by (1) promoting persistent infection and (2) enhancing transmission through mucus-driven shedding and cough reflex induction. This proposal will test this hypothesis through two specific aims: Aim 1: Delineate the immunosuppressive role of eosinophils in modulating host responses and enabling Bordetella persistence. Aim 2: Define the mechanisms by which eosinophils facilitate Bordetella spp. transmission. By reframing eosinophils as active modulators of bacterial pathogenesis, this research challenges traditional views of eosinophils as terminal effector cells and positions them as novel targets for therapeutic intervention, that might be applicable to other mucosal pathogens. The outcomes will contribute to our understanding of eosinophil biology in infection and may lead to innovative strategies to halt bacterial persistence and transmission.

Borrelia burgdorferi genotypic diversity, pathogenesis, and host cellular responses

PROJECT SUMMARY Lyme disease is the most common tick-borne illness in the United States, with an estimated 476,000 cases annually, and Pennsylvania (PA) consistently reports one of the highest case numbers nationwide. Borrelia burgdorferi sensu stricto (Bb) is a causative agent of Lyme disease in the US and is transmitted by Ixodes spp. ticks. Bb produces various outer surface proteins (Osp) and other mechanisms to survive in vectors, evade host immune systems, and to propagate infection within a host. Over 35 OspC genotypes have been characterized, which fluctuate in abundance in natural vector and host populations, suggesting host adaptation. While many Lyme-infected patients recover following antibiotic treatment, some may experience neurological symptoms, Lyme neuroborreliosis (LNB), which may be associated with specific genotypes. While previous studies focused on clinical manifestations, pathogenicity, genetic variations, and host immune responses using mouse models or patient samples, the genotype-specific immune responses that contribute to disease progression in humans remain poorly understood. Our central hypothesis is that certain Bb OspC genotypes, maintained in natural populations, are associated with distinct host immune responses that influence disease severity, progression, and persistence. Aim 1 will define the dynamics of OspC genotypes in tick and small mammal populations over time in Western PA to establish a 16-year longitudinal tick study and an 8-year longitudinal small mammal study. Using deep amplicon sequencing, we will quantify genotype diversity, detect low-abundance genotypes, and identify potential host-adapted genotypes. These empirical data will inform a compartmental mathematical model to evaluate OspC genotype prevalence, distribution, and public health risks, including LNB, across space and time. Aim 2 will assess how distinct Bb OspC genotypes affect the host immune landscape and cellular responses using human samples. To determine how Bb genotype contributes to disease phenotype, we will perform immune profiling studies which will include microscopy-based assessment of infected cell cultures, flow cytometric analysis of immune cell phenotypes, and measurement of genotype-specific cytokine, chemokine, and antigen production (sub-Aim2a). We will also employ multi-omics approaches that integrate single cell RNA sequencing with antibody-based protein profiling (scRNA-seq/Ab-seq) to characterize transcriptional and functional changes in immune cell populations exposed to different Bb genotypes (sub-Aim2b). This work is innovative in its integration of long-term ecological data with advanced immune profiling and single cell multi- omics to uncover genotype-specific mechanisms of Bb pathogenicity and human immune response—an approach not previously applied in Lyme disease research. These studies will clarify how specific genotypes influence immune responses and disease severity. Together, the proposed aims will identify critical genetic and immunological mechanisms that drive Bb pathogenicity and human susceptibility, informing the development of improved diagnostics, targeted therapies, and public health interventions to reduce the burden of Lyme disease.

BKCa Channel Contributions to Cerebellar Regulated TSC-Associated Neuropsychiatric Disorders

Project Summary TSC is associated with neurodevelopmental disability including cognitive disability and autism spectrum disorders (ASD) that make up part of TSC associated neuropsychiatric disorders (TAND). The mechanisms for TAND remain poorly understood but studies have increasingly implicated cerebellar dysfunction in the pathogenesis of cognitive and behavioral deficits in both TSC and other neurodevelopmental disorders. A shared feature is cerebellar Purkinje cell (PC) dysfunction. Changes in intrinsic properties of PCs results in both motor and cognitive/ behavioral changes in disease models and in individuals afflicted by these disorders. Mechanistic underpinnings of these altered properties remain unknown, but a significant emerging body of data implicate ion channel dysfunction as the primary etiology of these deficits. The current proposal seeks to delineate the ion channel contribution to PC dysfunction and to TAND-relevant behaviors. In doing so, these studies will produce significant both short- and long-term impact. Short-term: These proposed studies will provide a mechanistic understanding of the contribution of ion channels to the neuronal dysfunction in the cerebellum that has been demonstrated to be causally linked to abnormal TAND-relevant behaviors. In addition, we will target specific ion channels both genetically and pharmacologically to evaluate the benefits of ion channel restoration on both electrophysiological abnormalities but also the TAND-relevant behaviors observed in the model. Long-term: These studies, thus, provide a framework for subsequent clinically-relevant therapeutic development for TAND. First, these studies will uncover the ability for TAND-relevant behaviors to be improved upon targeting ion channel alterations in TSC. These studies will also define molecular targets on which therapeutic development can be targeted, thereby potentially providing a molecular-informed pipeline for therapeutic development. In addition, these studies will utilize clinically-available, FDA-approved pharmacological agents to target ion channel function and investigate the potential therapeutic benefits for these agents for TAND-relevant behaviors. Thus, these studies will address a core gap in knowledge to achieve a better mechanistic understanding of TAND and to develop therapeutic opportunities to address TAND. These studies will not only reveal previously understudied and novel mechanistic underpinnings for these behaviors but will provide pre-clinical insights into the therapeutic utility of clinically-utilized agents for the treatment of TAND-related behaviors, thus potentially providing both immediate and long-term opportunities for the treatment of TAND. Moreover, although these studies focus on TSC, these mechanisms may prove generalizable beyond TSC and provide a shared basis and therapeutic opportunity for other neuropsychiatric/developmental conditions.

Neuroinflammation in Cerebral Small Vessel Disease

Project Summary/Abstract Cerebral small vessel disease (cSVD) is a leading cause of vascular contributions to cognitive impairment and dementia (VCID), which is the 2nd leading cause of dementia and a significant contributor to Alzheimer’s disease (AD). Thus far, the underlying pathogenesis of cSVD is poorly understood. Several lines of evidence, including animal models, postmortem human brain pathology, and systemic inflammatory markers, demonstrated the damaging role of chronic neuroinflammation in cSVD. Direct evidence of neuroinflammation at the tissue level in patients with cSVD is still critically needed. The sphingosine-1-phosphate receptor 1 (S1PR1) regulates neuroinflammation through microglial and astrocyte activation and trafficking and has emerged as a promising target for neuroinflammation. In postmortem brains of patients with cSVD, we observed elevated S1PR1 expression and colocalization of S1PR1 with astrocytes and microglia. A novel 11C-CS1P1 PET radiotracer with high affinity and specificity targeting S1PR1 has been recently developed and validated in animal models and post-mortem human specimens. Under an FDA-approved eIND (IND 146548), we have successfully completed the safety and dosimetry study in healthy participants and performed preliminary studies in patients with cSVD. We found that 11C-CS1P1 PET uptake is significantly associated with WMH lesion burden in patients with cSVD after controlling for age, sex, race, vascular risk factors, and amyloid deposition. We hypothesize that 11C-CS1P1 PET uptake is a tissue-level biomarker of neuroinflammation to provide insight into cSVD severity, progression, and prognosis. We will 1) evaluate the relationship between 11C-CS1P1 PET uptake and cSVD neuroimaging abnormalities and cognitive impairment, 2) evaluate the test-retest repeatability and longitudinal evolution, and 3) determine whether 11C-CS1P1 PET uptake at baseline predict cSVD progression. The successful completion of this study will establish 11C-CS1P1 PET as an neuroinflammation imaging biomarker and investigate the role of neuroinflammation in cSVD pathogenesis and progression. It will lay a foundation for developing future therapies in modulating neuroinflammation.

The role of GPR132 in regulating T cell responses in infection and cancer

PROJECT SUMMARY. CD8 T cells play a critical role in protection from a variety of infectious microorganisms, and pathogen-specific CD8 T cells undergo robust expansion, with an individual T cell clones expanding up to 10,000-fold in a matter of days. After infection is resolved, the majority of these T cells die, leaving a small population of memory cells to provide protective immunity from secondary challenge. T cell expansion and contraction are tightly orchestrated processes that involve a delicate balance between stimulatory and inhibitory signals to ensure proper immune function. Dysregulation of the T cell response can have detrimental effects; too little proliferation and the host fails to mount a successful immune response, while excessive proliferation and persistence of effector T cell populations can lead to tissue damage. This proposal aims to determine the role of the G protein coupled receptor GPR132 in the regulation of CD8 T cell responses during infection and tumorigenesis. GPR132 detects oxidized endogenous and microbial lipids, and this can lead to cell cycle arrest; however, the role of GPR132 in CD8 T cells remains unexplored. Here we identify GPR132 as a critical regulator of CD8 T cell expansion and memory differentiation. Completion of the proposed aims will: 1) uncover the temporal role of GPR132 in regulating T cell accumulation and function during infection and tumorigenesis, 2) examine the abundance of GPR132-activating ligands within the tissue during health and disease, and 3) determine how altering GPR132 ligand availability could be used to enhance/inhibit T cell responses. Overall, these studies will provide fundamental insights into the regulatory mechanisms that dictate the magnitude of T cell responses and how they can be modulated therapeutically, which would allow us to boost responses to pathogens/tumors or inhibit pathogenic responses in the context of autoimmune disease.

Defining Microbial and Host Pathways Driving Asymptomatic C. difficile Colonization Associated with Aging and High-Sugar Diets

SUMMARY Clostridioides difficile infection (CDI) is a leading cause of healthcare-associated diarrhea, with rising incidence in community settings and a growing burden of asymptomatic colonization. Asymptomatic car- riers, particularly among the elderly and individuals consuming high-sugar diets, represent a critical but underexplored reservoir for transmission and disease progression. This proposal introduces novel, anti- biotic-independent mouse models demonstrating that both dietary sugar and aging independently pro- mote asymptomatic C. difficile colonization. We hypothesize that these factors disrupt colonization re- sistance (CR) through distinct but overlapping microbial, metabolic, and immune pathways. In Aim 1, we will define how traditional and emerging dietary sugars alter the gut environment to permit C. difficile colonization using in vitro bioreactors and in vivo models. Aim 2 will identify age-associated changes in microbiota and mucosal immunity that impair CR, using longitudinal studies and fecal micro- biota transfer. Aim 3 will functionally validate C. difficile genes upregulated during asymptomatic carriage using CRISPR-Cas9 mutants in both sugar- and age-induced models. This integrative, multi-omics approach will uncover the mechanisms enabling asymptomatic colonization and identify microbial and host targets for intervention. The findings will inform microbiome-based strat- egies to prevent CDI in vulnerable populations and shift current paradigms in CDI risk assessment and prevention.

Linear diribonucleotides regulation of bacterial physiology and infections

RNA degradation was thought to proceed through endonucleolytic fragmentation, followed by exo- ribonuclease trimming which generate short RNA fragments that are turned over into mononucleotides by oligoribonuclease (Orn). In the last funding period, we published data supporting that only specific enzymes (Orn, NrnA, NrnB, and NrnC) cleave diribonucleotides into monoribonucleotides, and that prokaryotic organisms need to encode at least one diribonuclease to fulfill this specific function. These results support a new perspective on RNA degradation in which the short oligoribonucleotides are processed through a sequence of discrete steps involving distinct enzymes. In addition, linear diribonucleotides appear to be biologically active molecules since we reported that mutants lacking these enzymes accumulate diribonucleotides and have altered cell growth, biofilm formation, motility, and sporulation. Here we present additional preliminary data supporting diribonucleotides as active signaling molecules in the cell including: 1. Specific enzymes act trinucleases to generate diribonucleotides, 2. RNase AM of Pseudomonas aeruginosa ∆orn is a cryptic diribonuclease, 3. Two enzymes in central metabolism are diribonucleotide- binding proteins, and 4. P. aeruginosa ∆orn has virulence defects in an animal model of catheter-associated urinary tract infection. Our past publications and preliminary data provide the scientific premise for our hypothesis that cells generate linear dinucleotides from RNA degradation and linearization of cyclic dinucleotides, which can bind target proteins to alter cell physiology and pathogenesis. To test these aims, we will perform the following specific aims: In Aim 1, we will characterize the generation and degradation of diribonucleotides by characterizing how diribonucleases and triribonucleases bind their respective substrates through molecular biology, biochemistry, and computational docking. In Aim 2, we will identify effects of dinucleotides on bacterial metabolism and physiology by characterizing the binding proteins that specifically interact with linear diribonucleotides. Building on our success of identifying cellular diribonucleotide receptors, we will screen for additional proteins from open reading libraries of P. aeruginosa and Bacillus anthracis. We will exploit the strains available to us that lack all diguanylate cyclases to reveal whether the effect of linear diribonucleotides is independent of c-di-GMP signaling. In Aim 3, we will characterize the effect of expression levels of dinucleases and the effect of dinucleotide accumulation on bacterial physiology and pathogenesis. We will develop mass spectrometry methods to detect di- and triribonucleotides. We will employ existing mutants lacking diribonucleases, including P. aeruginosa ∆orn to study the defects in chronic infection in a murine model of catheter-associated urinary tract infection. Results from these studies will advance our understanding of RNA degradation and open a new area of signaling by linear diribonucleotides with the potential to be applied to novel antibacterial strategies.

Causal mechanisms driving germline predisposition to myeloproliferative disorders

SUMMARY/ABSTRACT Although human genetic studies have indicated a significant hereditary predisposition to myeloproliferative neoplasms (MPNs) the underlying mechanisms driving the genetic risk remains unknown. Our large genome wide association study (GWAS) on MPNs identified several non-coding genetic risk loci associated with disease and implicated modulation of hematopoietic stem cell (HSC) self-renewal by the genetic variants. The long-term goal is to utilize our GWAS results to better understand MPN disease initiation and progression and draw out key unknown MPN predisposition genes. The overall objectives in this application are to elucidate the mechanisms by which MPN risk variants promote disease initiation and progression. The central hypothesis is that common genetic variants increase MPN risk by affecting regulatory elements that influence clonal expansion of HSCs carrying MPN driver mutations. The rationale for this project is that the HSC clones with most prevalent driver mutation found in MPN, JAK2V617F show individual specific growth rates and can develop into MPN or remain as clonal hematopoiesis without any consequences indicating that germline genetic factors influence this process. The central hypothesis will be tested by pursuing two specific aims: 1) To determine the mechanisms by which genetic variation at the GFI1B locus influences MPN predisposition in vivo. 2) To define upstream transcriptional mechanisms disrupted by common genetic variants that predispose to MPN. Under the first aim, a newly generated mouse model will be used to evaluate clonal expansion of JAK2V617F HSCs in the context of a germline Gfi1b enhancer deletion by in vivo competitive transplantation assays. The murine studies will be complemented by an assessment of Gfi1b allele specific clonal expansion in primary human hematopoietic stem and progenitor cells (HSPCs) engineered to carry JAK2V617F mutation. Mechanistically activated mitochondrial respiration will be examined in germline enhancer inactivated JAK2V617F HSPCs in murine models and human patient samples. For the second aim, perturbation of RUNX1 bound cis-regulatory elements by MPN risk variants will be evaluated as a mechanism of clonal expansion in MPN by using lentiviral reporter assays and endogenous CRISPR/Cas9 editing approaches in primary human HSPCs and degron tagged RUNX1 cell lines. A Runx1 haploinsufficiency mouse model will be used to assess global influences of RUNX1 transcriptional network on MPN initiation. Collectively, our proposed studies aim to bridge the gap between inherited genetic variations and the clonal expansion dynamics of MPN stem cells, shedding light on crucial factors influencing disease development. The mouse models proposed in this study provide the in vivo physiological context and functional readouts required to investigate HSC clonal expansion and MPN pathogenesis.

Transcriptional control of activation induced deaminase (AID) function

SUMMARY Somatic hypermutation (SHM) and class switch recombination (CSR) are vital for the generation of high affinity antibodies with appropriate effector function, protection against infection, and vaccine efficiency. They are initiated when the activation induced deaminase (AID) deaminates cytidines in single-stranded DNA in the context of transcription by RNA polymerase 2 (Pol2). Aberrant DNA deamination by AID is an important driver of genetic instability and the development of B cell malignancies. Understanding the factors and mechanisms that coordinate AID-mediated deamination with Pol2 transcription is an important objective in the study of humoral immunity and the central goal of research under this grant. Our preliminary data demonstrate that Pol2 pause factor NELF, Super Elongation Complex (SEC) components MLLT1/3, and the phosphatase module of the Integrator-protein phosphatase complex (INT-PP2A) are required for SHM, with MLLT1/3 but not NELF being required for AID binding to its chromatin targets. Our findings yield a new conceptual framework and model for AID-Pol2 collaboration in which NELF and a balance between kinase and phosphatase activities of SEC and INT-PP2A regulate Pol2 pausing/elongation to generate the critical stalled Pol2 complex on which AID acts. Further, our work has yielded major methodological advances that allow us to overcome obstacles that have stymied progress in the field. In this proposal, we take advantage of these conceptual and technical advances to pursue our central goal through the following two aims: Aim 1: Determine the molecular mechanisms by which NELF and other Pol2 regulatory factors enable AID-Pol2 collaboration and SHM/CSR. It has previously been very difficult to assess the role of cell-essential factors in SHM. By combining our new Rapid Assay for SHM (RASH) cells with degron technology, we will determine the mechanism of action of our newly discovered regulators of SHM using genomic, transcriptomic, and interaction assays that assess Pol2 distribution, phosphorylation, and activity, and the chromatin binding profiles of and interactions between AID and components of NELF, SEC, and INT-PP2A. AID and MLLT1 appear to co-associate in a complex and we will test for a direct interaction between AID and MLLT1/3. Factors will be tested for roles in CSR and validated in human cell line and germinal center B cell models and in mice. Aim 2: Hypothesis testing and deep mechanistic analysis through perturbation of the balance between Pol2 pause/arrest and elongation. We will rigorously test our new model for AID-Pol2 collaboration using degron, reconstitution, mutagenesis, and small molecular inhibitor approaches to perturb the balance between Pol2 pausing and elongation, revealing how altering NELF-Pol2 interactions and the balance between SEC kinase and INT-PP2A phosphatase activities influences SHM efficiencies and AID binding. Together, our proposed studies are significant for the development of new technologies and for understanding mechanisms of antibody gene diversification and causes of genome instability and cancer.

Characterization and functional impact of somatic numtogenesis in the human cortex

Project Summary This project focuses on studying nuclear mitochondrial insertions (numts), which are fragments of mitochondrial DNA that get integrated into the nuclear DNA of human cells. While this process, called numtogenesis, occurs naturally and can be passed down to future generations, it has also been observed to occur somatically in our bodies. Historically the function of numts has been difficult to study because they are repetitive and difficult to map with short read sequencing technologies, but there is emerging evidence that they can influence cell function and play a role in diseases, aging, and even complicate genetic studies. Our recent research discovered numts in the human brain’s cortex, and their presence appeared to be linked with earlier death, suggesting they may play a role in aging. However, due to limitations in the data we used, we could not fully explore the extent or impact of these insertions across different tissues or individuals. This project aims to map and study numts in more detail, especially in the human cortex, to further explore this ongoing transfer of DNA from the mitochondria to the nuclear genome and their potential to impact aging and brain function. We will accomplish this by 1) improving sequencing methods to detect numts, 2) comparing their presence across different tissues, and 3) investigating how they affect gene expression and DNA structure. By the end of the project, we aim to provide a model for how such somatic variation may occur and impact cellular function at the tissue level.

Validating Causality of Disputed Mitochondrial Variants in Inborn Errors of Metabolism

PROJECT SUMMARY Primary mitochondrial disease (PMD) encompasses multi-systemic disorders caused by impaired mitochondrial function. PMDs arise from pathogenic variants in either nuclear genes encoding mitochondrial proteins, or in the mitochondrial DNA (mtDNA) genome. Clinical diagnosis is challenging due to phenotypic heterogeneity, underscoring the importance of genetic diagnosis. ACMG/AMP guidelines provide a well-established framework for interpreting nuclear DNA variants while diagnosing genetic diseases. Their application to mtDNA variants, however, remains challenging due to unique features of mtDNA: maternal inheritance, heteroplasmy, threshold effects, and effect of transfer or ribosomal RNA rather than coding variants. To address these challenges, the ClinGen Mitochondrial Disease Nuclear and Mitochondrial Variant Curation Expert Panel, co-chaired by the Multi-PIs of this study, developed widely adopted ACMG/AMP revised guidelines for mtDNA variant interpretation. Over the past five years, this global expert panel has curated more than 280 mtDNA variant. Because of the lack of functional data of individual mtDNA variants in the literature, 23 previously reported pathogenic (P) variants were classified as Variants of Uncertain Significance (VUS), hindering definitive PMD diagnoses and therapeutic development. This R01 project aims to resolve the pathogenicity of these 23 mtDNA VUS through functional validation, leveraging advanced mtDNA base editing and single-cell genomics in in vitro and in vivo models. In Aim 1, we will create human 143B cell line models for 20 VUS using cutting-edge mtDNA editing techniques, optimized for efficiency and minimal off-target effects. Single-cell genomics (mtscATAC-seq and scRNA-seq) will assess heteroplasmy and genomic changes, while functional assays will evaluate mitochondrial ATP production, oxidative phosphorylation, membrane potential, and redox stress. Aim 2 will develop zebrafish models for 17 conserved VUS, characterizing phenotypic and mitochondrial outcomes to corroborate in vitro findings and PMD patient phenotypes. This study will clarify longstanding uncertainties regarding the pathogenicity of these mtDNA VUSs which were nonetheless reported to be pathogenic with often strong genetic evidence but limited functional data. The study will also establish valuable cell and zebrafish models and provide mechanistic insights of PMDs. The resulting resources will be shared with the scientific community to accelerate research and therapeutic advancements for novel precision medicine approaches for PMDs.

Molecular Mechanism of Immunoglobulin Class Switch Recombination

Antibodies produced by B cells are a critical component of the adaptive immune system in mammals that can respond to and clear a plethora of different pathogens. A key property of B cells is their ability to alter the coding sequence of the immunoglobulin heavy and light chain genes, via VDJ-recombination, somatic hypermutation (SHM) and class switch recombination (CSR). While VDJ-recombination and SHM alter the variable regions of antibodies that directly contact pathogen antigens, CSR changes the constant region of the antibody, which dictates its effector function to optimally respond to the antigen recognized by the antibody. CSR occurs via targeted DNA double strand break (DSB) induction in the switch regions preceding the distinct constant region coding sequences. DSB induction requires active transcription of the switch regions and is initiated by activation-induced cytidine deaminase (AID) induced cytosine deamination (converting cytosine to uracil) within the switch regions. Fusion of the DSBs in the switch regions results in deletion of intervening genomic sequence, completing CSR. Since AID is inherently a mutagenic enzyme that can trigger both point mutations and genomic translocations, its activity has to be tightly controlled, and aberrant AID activity has been directly implicated in the genetic changes that lead to B cell lymphoma formation. Thus, define the molecular mechanism of CSR is critical to understand our adaptive immune system and B cell cancer development, both highly relevant to human health. To study CSR in living B cells, cellular models have been developed to analyze AID function and switch region transcription at the single molecule level. With this new methodology, the critical unanswered question of how AID is specifically recruited to the immunoglobulin heavy chain locus and not other genomic locations will be addressed. In addition, the overall kinetics of CSR will be determined and how transcription controls specific DSB induction in switch regions will be defined. The results of these works will significantly advance our understanding of CSR and provide new insights on how AID contributes to B cell lymphoma formation.

TAR RNA binding to INI1/SMARCB1 and its role in HIV-1 transcription and latency reactivation

Abstract The goal of this application is to study the role of interplay between the components of chromatin remodeling SWI/SNF (BAF complex) and HIV-1 transcription machinery, focusing on the interaction of a BAF component, INI1 (Integrase Interactor 1) with TAR RNA. HIV-1 reservoirs are a mixture of latent cells harboring proviruses silenced at transcriptional level. Cure strategies need a deeper understanding of HIV-1 transcriptional regulation. HIV-1 transcription, initiated by RNA Pol II, pauses producing short TAR transcripts. pTEFb recruitment to TAR by Tat overcomes this transcriptional pause, facilitating elongation. Beyond Tat, the action of chromatin remodeling complexes (CRCs) is required to facilitate elongation. The BAF complexes CBAF and PBAF play distinct roles. While CBAF represses proviral transcription by maintaining nucleosomes in an unfavorable state, PBAF remodels nucleosomes to facilitate elongation. INI1 is a component of both CBAF and PBAF, and its role in transcription is not fully understood. INI1 was identified as a binding partner for HIV-1 integrase (IN) and exerts multifacted roles in virus assembly, production and morphogenesis. INI1 has multiple functional domains. IN binding Rpt1 domain structurally mimics TAR RNA & is necessary for late events. We have made a novel observation that another domain of INI1, the N-terminal Winged Helix DNA binding domain (WHD) specifically binds to TAR RNA and that this interaction is necessary for mediating HIV-1 transcriptional elongation. These exciting results suggest that different functional domains of INI1(Rpt1 and WHD) involved in “TAR RNA mimicry” or “TAR RNA binding” regulate distinct stages of replication. We hypothesize that INI1 WHD domain-TAR interaction is necessary for recruitment of PBAF to HIV-1 LTR for transcriptional elongation and latency reactivation. Disrupting this interaction results in transcriptional repression. We will investigate the role of this novel INI1:TAR RNA interaction in HIV-1 transcription and latency reactivation. This is a multi-PI application involving Drs. Kalpana (HIV-1 virologist), Heng (NMR biophysicist) and Zou (computational biologist/protein-RNA structure). In Aim 1, we will characterize INI1-WHD:TAR interaction in vitro and in vivo via molecular/genetic analyses (Kalpana/Heng). We will employ alanine scanning mutagenesis based on WHD NMR structure to test WHD:TAR interaction. We will use biophysical & biochemical approaches to probe TAR structural elements required for this interaction. In Aim 2, we will employ computational modeling and NMR to determine the structure of INI1- WHD:TAR RNA complex (Zou/Heng). In Aim 3, we will determine the role of INI1:TAR interactions in HIV-1 transcription, latency reactivation and mechanism of action (Kalpana). We will analyze the effect of TAR- Interaction-Defective (TID) INI1 mutants on transcription of LTR-reporters and full-length HIV in INI1-/- cells. Latent cells in which TID-INI1 mutants are knocked in (KI) will be used to assess effect on reactivation via RNA-FISH and qRT-PCR assays. Our studies will establish INI1:TAR interaction as a drug target. Inhibiting this interaction could block latency reactivation promoting deep latency and advancing cure strategies.

Tbx4-Driven Pulmonary Hypertension: Mechanisms and Therapeutic Targets

Project Summary: Heterozygous rare variants in TBX4 are the second most common cause of heritable pulmonary arterial hypertension (PAH). Presentation of this form is commonly in children. Patients with mutations in TBX4 generally have alveolar simplification or hypoplasia in addition to elevated pulmonary vascular resistance. We have developed a set of three tools to help determine the molecular etiology of TBX4-induced PAH; (1) we identified the direct binding targets using a combination of ChIP-seq and RNA-seq; (2) we developed a mouse model with Tbx4 knockout after birth, that substantially phenocopies human disease; (3) we performed single-cell RNA-seq on these mice. By combining these three tools, we can develop a complete model for how loss of a transcription factor leads to the molecular and physiologic changes we see in our mice. The phenotype in mice appears to be dominated by defects in pericytes, resulting in impaired angiogenesis. Pericytes, which strongly express Tbx4, are cells located on the outside of capillaries and precapillary arterioles, and can either stabilize vessels (mesh pericytes), or drive angiogenesis (angiogenic pericytes). The pericytes in Tbx4 mutant mice are heavily skewed towards mesh and away from the angiogenic phenotype. Loss of Tbx4 results in derepression of Tbx4 binding target Rgs5 (10x induction), which directly results in inhibition of Pi3K, and the phenotypic switch in pericytes. We will test this hypothesis through pericyte-specific Tbx4 knockout (Aim 1) and pharmacologic induction of Pi3K in vivo in prevention and rescue models, as well as by siRNA to Rgs5 in precision-cut lung slices from Tbx4 KO mice (Aim 3). We will also test the role of Tbx4 in fibroblasts and smooth muscle using cell-specific knockouts – based on our mouse and single cell data, we expect they contribute somewhat, but primarily through increased stiffness (Aim 2). Finally, we will confirm relevance to human disease through spatial transcriptomics in lung sections explanted from patients with TBX4 mutation or rearrangement (Aim 1), and through determining whether defects in human patient iPSC-derived pericytes can be corrected through Rgs5 or Pi3K interventions (Aim 3). In combination, these aims determine the cellular and molecular mechanisms leading from mutation to physiology with loss of TBX4, and establish therapeutic targets.

Airway Epithelial Defense Mechanisms in Combating STAT3-Deficiency-Related Lung Infections

Airway Epithelial Defense Mechanisms in Combating STAT3-Deficiency-Related Lung Infections Signal transducer and activator of transcription 3 (STAT3) regulates the expression of genes essential for various cellular processes, including survival, proliferation, differentiation, self-renewal, angiogenesis, and immune response. Abnormal and persistent STAT3 activation is detected in diverse human cancers, driving multiple pro- oncogenic functions. Multiple antitumor drug development targets the inhibition of STAT3 to treat various types of cancer. Unfortunately, downregulated STAT3 significantly increases host susceptibility to recurrent infections, especially pneumonia. Additionally, individuals with genetic polymorphisms associated with lower STAT3 expression are more susceptible to severe tuberculosis. Furthermore, patients with autosomal dominant hyper- IgE syndrome (AD-HIES), also known as Job Syndrome, which is caused by de novo STAT3 mutations and substantially decreased STAT3 expression, have a significantly increased susceptibility to bacterial and fungal infections, with high mortality rates and a shortened life span often associated with Pseudomonas aeruginosa infections. Gram-negative bacteria, particularly P. aeruginosa, are opportunistic pathogens that frequently cause hospital-acquired infections. The problems are worsened by the emerging P. aeruginosa with multidrug resistance (MDR), especially in patients with repeated antibiotic treatments, such as Job Syndrome sufferers. Notably, airway epithelial cell-derived proteins play a significant role in the antimicrobial milieu, promoting effective host defense against invading pathogens. One of the most critical STAT3-regulated antimicrobial molecules is bactericidal permeability-increasing protein fold A1 (BPIFA1, also known as SPLUNC1), a multifunctional innate immunity molecule and indispensable host defense protein that is abundantly secreted in the lungs. This application aims to elucidate how STAT3 deficiency impairs host epithelial defense against microbial infections and whether BPIFA1-mediated innate immune responses can sufficiently restore effective antimicrobial protection to prevent pneumonia. The long-term objective is to advance our understanding of the respiratory innate immune response, particularly in relation to epithelial cell-specific antimicrobial defense. We characterized BPIFA1 as an airway lining fluid protein secreted apically in the airway lumen and in primary human airway epithelial cultures. In this study, we hypothesize that mucosal BPIFA1 is an essential antimicrobial protein that plays a critical role in host defense against microbial infections in STAT3-deficiency- associated pneumonia. Our proposed studies will assess innate immunity mechanisms regulating the antimicrobial activity of the airway epithelium in STAT3 deficiency-associated lung infections. By focusing on the crucial epithelial-derived protein product, BPIFA1, our study will provide an alternative treatment for respiratory infections by augmenting native host defense mechanisms in high-risk individuals, including AD-HIES, cancer, and immunocompromised patients.

Role of stress signals in the pathogenesis of pulmonary veno-occlusive disease

PROJECT SUMMARY/ABSTRACT Pulmonary veno-occlusive disease (PVOD) is a subclass of pulmonary hypertension characterized by preferential remodeling of the pulmonary venules and capillaries, and currently, there are no efficacious drug therapies. The clinical presentations and the radiographic findings of PVOD are indistinguishable from PAH, and therefore, it is often misclassified as PAH. However, the application of PAH therapeutics to PVOD patients leads to life-threatening pulmonary edema, thus, there is a critical need for diagnostic methods that accurately differentiate PVOD from PAH. Genetically, PVOD is associated with biallelic loss of function (LOF) mutations in the EIF2AK4 gene encoding GCN2. GCN2 phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF2α), shuts down protein synthesis, and activates the integrated stress response (ISR). However, the molecular mechanisms connecting the loss of GCN2 with pulmonary vascular remodeling are poorly understood. Recent studies find that biallelic EIF2AK4 mutations are identified in ~9% of PAH patients. Conversely, heterozygous mutations in the BMPR2 gene, a leading cause of PAH, have been reported in PVOD patients. These results suggest that (i) PVOD and PAH share some of the pathophysiological mechanisms, and (ii) the presence of EIF2AK4 or BMPR2 mutations does not provide an accurate genetic diagnosis for PVOD. The long-term goal of this proposal is to elucidate the pathophysiological mechanisms involved in remodeling not only pulmonary arterioles but also venules and capillaries and develop those pathways as potential therapies for POVD. It has been observed that cancer patients administered with the chemotherapeutic agent mitomycin-C (MMC) rapidly develop PVOD. Rats administered with MMC develop PVOD-like phenotypes, including right ventricular (RV) hypertrophy, increased RV systolic pressure, and pulmonary vascular lesions in arteries and veins. We found that Rad51, an essential enzyme for double-strand DNA break repair, associates with VE-Cad in the vascular endothelium; however, upon MMC treatment, Rad51 and VE-Cad complex (VRC) were released into the circulation, resulting in increased vascular permeability and reduced barrier integrity. MMC treatment also mediates the depletion of GCN2, which recapitulates the genetic cause of PVOD (LOF EIF2AK4 mutations). Based on these data, this proposal will test the hypothesis that the vascular remodeling in PVOD involves (i) the release of VRC, (ii) the aberrant protein synthesis due to the activation of ISR, and (iii) the mechanism of maladaptive ISR activation. Finally, we will explore the potential application of the circulating VRC as a blood biomarker for PVOD.

Multiplex single-cell chemical genomics to identify small molecule modulators of tumor cell-intrinsic immunogenicity in glioblastoma

PROJECT SUMMARY/ABSTRACT Glioblastoma multiforme is the most common and aggressive primary brain cancer. Despite a multimodal treatment regimen of surgical resection, chemotherapy, radiotherapy, and tumor-treating fields, most patients succumb to the disease within two years of diagnosis. Cancer immunotherapy strategies have emerged as a powerful tool for treating aggressive solid tumors such as melanoma and non-small cell lung cancer. However, current strategies have led to low response rates in glioblastoma, resulting from its low immunogenicity. The proposed research program aims to identify small molecules capable of increasing the immunogenicity of glioblastoma cells, focusing on altering gene expression programs associated with recognition by the immune system and the ability of cytotoxic immune cells to target glioblastoma for destruction. We will use highly multiplex chemical transcriptomic profiling to determine the molecular consequence of exposing glioblastoma neurosphere models to 3,792 small molecules, targeting the majority of cellular activities and clinically relevant drug targets as well as a collection of previously identified immunomodulators. We will then determine how each exposure alters the expression of gene programs associated with tumor cell immunogenicity and response to therapy, including the expression of genes associated with the recognition by the immune system and those associated with immune checkpoints, as well as programs more broadly correlated with resistance to anti-cancer therapies. Chemical hits that meet specific criteria will be subjected to a medicinal chemistry review to further classify compounds by their suitability for treating malignancies in the brain. We will then screen chemical hits to determine their ability to modulate immune-mediated tumor cell killing using tumor- immune cell co-culture. Lastly, we will leverage gene editing and flow cytometry to validate hits based on on- target molecular effects and further refine the mechanism of action by inspecting the ability of drugs to modulate immunogenic programs at the protein level. Our chemical genomics screens aim to provide crucial information regarding the link between pathway activity and immunomodulation in GBM, a critical step to guide future efforts in GBM immunotherapy. More broadly, our study will establish single-cell chemical genomics as a scalable platform for phenotype-based screening for preclinical prioritization of chemical modulators of complex transcriptional phenotypes and provide a framework for hit prioritization, establishment of pipeline robustness and hit validation in the context of single- cell chemical genomics screens.

Effects of Apolipoprotein A4 on Lipid Metabolism via Sympathetic Regulation

Obesity increases the risks and progression of hypertriglyceridemia, metabolic dysfunction- associated steatotic liver disease (MASLD), and cardiovascular diseases. Previous studies demonstrate that a single injection of apolipoprotein A4 (APOA4) elevates sympathetic neural activity and fatty acid β-oxidation in adipose tissues; and consistent infusion of APOA4 in obese mice fed a high-fat diet lowers fat mass, reduces hypertriglyceridemia, elevates brown adipose tissue thermogenesis, and attenuates steatosis and enhances sympathetic neural activity in the liver. This project hypothesizes that APOA4 reduces hypertriglyceridemia by regulating lipid metabolism through sympathetic stimulation in adipose tissues (Specific Aim 1) and sympathetic action in the liver (Specific Aim 2). The role of sympathetic action via the neurotransmitter norepinephrine and adrenergic receptor-mediated pathways will be investigated, and their necessity in APOA4-mediated lipid metabolism will be tested. A strength of this project is the interdisciplinary collaboration between investigators with established successful collaboration and publications. The project will provide physiological, molecular, and neurochemical mechanisms underlying how APOA4 differentially regulates metabolism through sympathetic activation in various types of adipose tissues and the liver in male and female obese mice. Findings would provide impetus to develop unique, novel, targeted therapeutic applications against hypertriglyceridemia and MASLD. Importantly, this project will expose undergraduates and graduate students to meritorious research, provide students with hands-on biomedical research experience, and strengthen research environment at R15 eligible institutions.

Response and defense mechanisms of extraintestinal Escherichia coli to reactive oxygen and chlorine species

Members of the Escherichia coli species are remarkably diverse and comprise commensal, probiotic and pathogenic strains. While some pathogenic E. coli cause intestinal diseases, extraintestinal E. coli (ExPEC) can colonize and infect environments outside the gut. For instance, members of this pathotype can inhabit the urinary tract where they are confronted with a multitude of bactericidal host defense strategies, which requires specialized genetic adaption for survival. ExPEC must defend highly toxic antimicrobials such as hypochlorous acid (HOCl), a potent reactive oxygen and chlorine species (RO/CS) generated during neutrophil-mediated phagocytosis and by enzymes in uroepithelial cells to control bacterial colonization. The increasing rate of ExPEC infections in humans due to changing infection dynamics demonstrate the critical need for a better understanding of ExPEC pathogenesis, which is desperately needed to improve approaches for infection prevention and treatment given the rise in antibiotic resistance spreading among E. coli. Our lab has reported that members of the ExPEC pathotype are more resistant to RCS in vitro and to neutrophil-mediated phagocytosis when compared to non-pathogenic and enteropathogenic E. coli. We identified the defense system responsible for these phenotypes and characterized its regulation during RCS stress: the RcrR regulon consisting of the rcrARB genes is controlled by the RCS-sensing transcriptional repressor RcrR, which reversibly loses its repressor activity upon oxidation by RCS, resulting in de-repression of its downstream targets. Induced expression of rcrB contributes significantly to ExPEC’s increased RCS resistance, however, the precise mechanism of RcrB and the role of RcrA (and potentially other defense players) during RCS stress remain enigmatic. Our long-term goal is to increase the efficacy of existing antimicrobial therapies by purposefully and selectively sensitizing ExPEC to clearance by innate immune cells. The overall objective of this application is a comprehensive analysis of ExPEC’s RCS defense with particular focus on the mechanism of the RcrR regulon. We hypothesize that RcrB directly protects cells from HOCl, while RcrA, another member of the RcrR regulon, mediates evasion from HOCl and invasion into host cells. In Aim 1, we will use phenotypic, biochemical, and imaging approaches to investigate the mechanism by which RcrB contributes to ExPEC’s increased RCS resistance. In Aim 2, we will study the role of RcrA for ExPEC motility, biofilm formation, and host cell invasion. In Aim 3, we will use independent unbiased and targeted approaches, including phenotypic characterization of transposon mutants, to fully comprehend ExPEC-specific responses to and defenses against RCS. Identifying, characterizing and targeting ExPEC-specific defense systems has the potential to increase the body’s own capacity to fight UTIs. Overall, we will involve at least four undergraduate students in our research projects, which we believe will provide an excellent training opportunity for the next generation of scientists.

Molecular strategies for resolving differential regulation of dopamine subpopulations

Project Summary/Abstract Dopamine neurons in the ventral tegmental area (VTA) fire action potentials in complex patterns of tonic and phasic activity in response to environmental stimuli and during behavioral tasks. Transcriptomic, anatomical, and functional studies have established that VTA dopamine neurons can be divided into multiple subpopulations with variable gene expression, projection patterns, and response profiles. We recently completed a transcriptomic study that identified genetic markers for three distinct subpopulations of VTA dopamine neurons, and also found evidence for variability in ion channel gene expression between populations that correlated with differences in activity-dependent gene expression. However, much remains unknown regarding how specific genes encoding ion channels, receptors, transcription factors, or other signaling components contribute to the variability in baseline physiological properties observed across the VTA. Here we propose to combine slice electrophysiology recordings of VTA dopamine neurons with post-hoc single-cell sequencing analysis (i.e. patch-seq), which will allow us to directly correlate gene expression and physiological properties in order to identify candidate genes that may be key drivers of the variability between subpopulations. We also propose to validate and utilize a novel dual-recombinase CRISPR/Cas9 system for targeted gene mutagenesis in intersectional neuronal populations, which will provide a mechanism for testing gene function with unprecedented precision. We will use this approach to test the function of two candidate ion channel genes, the potassium channels Kcnh5 and Kcnh7, previously identified in our transcriptomic study as potential contributors to dopamine neuron action potential firing properties. We hypothesize that these genes are important for enabling rapid action potential firing in highly excitable dopamine neurons found in specific subpopulations. As a whole, with this proposal we aim to generate a valuable dataset linking gene expression in VTA dopamine neurons with physiology and subpopulation identification, as well as develop an intersectional gene mutagenesis strategy that can be used throughout the brain to precisely target neuronal subpopulations to test gene function. With this approach, we hope to facilitate future precision targeting of the dopamine system and dopamine-dependent behaviors.

Transposable element silencing as a regulator of salivary gland immune homeostasis

PROJECT SUMMARY/ABSTRACT Sjogren’s syndrome (SjS) is a chronic autoimmune disorder marked by salivary and lacrimal gland dysfunction, lymphocytic infiltration, and progressive secretory decline. While traditionally viewed as immune cell–driven, emerging evidence suggests that epithelial cells may initiate local inflammation. However, the molecular triggers originating from epithelial cells remain poorly defined. Transposable elements (TEs), including endogenous retroviruses (ERVs) and LINEs, are normally repressed through DNA methylation, histone modifications, and heterochromatin organization. Failure of TE silencing mechanisms due to aging, hormonal changes, or stress results in cytoplasmic dsRNA accumulation, nucleic acid sensor activation, and type I interferon signaling. These TE-derived nucleic acids are increasingly recognized as endogenous triggers of immunological stress that disrupt cellular homeostasis. Our preliminary data show widespread TE derepression and upregulation of interferon-stimulated genes in salivary glands from patients with SjS. To mimic this phenomenon, we will inducibly delete Setdb1, a key histone H3K9 methyltransferase, in defined epithelial compartments of the salivary gland. This will allow us to model compartment-specific TE derepression and assess its impact on both innate immune activation and adaptive immune responses. We will also test how aging and estrogen deficiency disrupt TE repression in basal/ductal versus acinar cells using lineage tracing and epigenomic profiling. Finally, we will evaluate the therapeutic potential of reverse transcriptase inhibitors and chromatin-modifying drugs in attenuating TE-driven inflammation. This exploratory study will uncover how failure of TE silencing contributes to epithelial-driven autoimmunity in SjS and will provide a foundation for future targeted epigenetic manipulations in human tissues and patients.

Mechanisms of age-related inflammatory dysregulation in the pathogenesis of periodontal disease

Periodontal disease is a chronic inflammatory condition that affects the supporting tissues of the dentition. Similar to other chronic inflammatory conditions, the prevalence of periodontal disease increases with age. Dysregulation of the host inflammatory response is central to the pathogenesis of periodontal disease and other age-related diseases. Therefore, an improved understanding of the pathologic mechanisms that contribute to age-related inflammatory dysregulation is needed to better manage periodontal disease in older adults. Towards understanding a mechanism of age-related inflammatory dysregulation in periodontal disease, we will investigate the role of triggering receptor expressed on myeloid cells 2 (TREM2). TREM2 is a potent immunoregulator expressed on macrophages. Signaling through TREM2 downregulates inflammation, in part, through inhibition of inflammatory cytokine expression. Dysregulation of TREM2 has been implicated in chronic inflammatory disease and age-related conditions, such as Alzheimer’s disease, liver disease, and osteoarthritis. However, the role of TREM2 in periodontal disease is understudied. Therefore, we propose to study TREM2 in the pathogenesis of periodontal disease and age-related inflammatory dysregulation. Our preliminary work has demonstrated that TREM2 is critical in macrophage immunoregulatory processes in the periodontium and TREM2 dysregulation contributes to periodontal disease in mice. We have shown that Trem2 is expressed in macrophages isolated form the periodontium in mice. We demonstrated that old mice expressed less Trem2 in the periodontium compared to young, which was associated with local inflammatory dysregulation and increased periodontal disease severity. Interestingly, Trem2 depletion in young mice resulted in increased inflammatory dysregulation and periodontal disease severity, similar to what is observed in old mice. From the preliminary data, we hypothesize that TREM2 modulates macrophage activity in the periodontium and age-related dysregulation of TREM2 drives a pathologic inflammatory response in periodontal disease. In Aim 1, we will demonstrate the extent to which TREM2 modulates inflammation and periodontal disease severity using old, young, and Trem2-/- mouse models of periodontal disease. In Aim 2, we will develop tissue-specific, single cell map of the immune cells in the periodontium and understand the effect of age and Trem2 on immune cell phenotypes and subpopulations. Findings from this proposal will elucidate a novel mechanism in age-related inflammatory dysregulation in the pathogenesis of periodontal disease and further advance our understanding of the role of TREM2 within oral tissues. This proposal was designed to generate a novel body of work that will be used to develop the independent research program of an early stage investigator and to support an R01 proposal to be submitted at the completion of this project period.

Developing a novel technology for studying T cell differentiation in vivo

Summary CRISPR-based genetic screens have revolutionized our understanding of gene functions and molecular mechanisms across various biological processes. In the field of T cell biology, CRISPR screens have played a pivotal role in identifying genes that impact critical aspects, such as T cell development, differentiation, and function. However, traditional screens have struggled to distinguish genes with diverse mechanisms of action, necessitating further investigations. To address this challenge, researchers have harnessed the power of CRISPR screens combined with single-cell sequencing (scCRISPR-seq), enabling the simultaneous assessment of genetic perturbations and high-dimensional phenotypes at the single-cell level. While scCRISPR- seq has predominantly been performed in vitro using immortalized cell lines, its physiological relevance is limited due to oversimplified biological context and disparities compared to primary cells. This limitation highlights the urgent need for large-scale in vivo scCRISPR-seq with primary T cells. However, various challenges have discouraged its widespread adoption. The use of viral vectors for sgRNA delivery compromises physiological relevance, as the in vitro activation conditions fail to faithfully represent the intricate T cell priming process in vivo. Moreover, viral vector components and continuous Cas9 expression can trigger immunogenicity and cytotoxicity, leading to cell depletion and hindering long-term studies. Additionally, current scCRISPR-seq methods face technical limitations, including low editing efficiency and inadequate perturbation identity recovery rates, which impede efficient large-scale in vivo applications. Fortunately, recent advances in ribonucleoprotein complex (RNP) transfection have addressed many of these challenges. This cutting-edge technology enables efficient gene editing in primary T cells without the need for in vitro activation or permanent Cas9 expression. Leveraging the high editing efficiency of RNP transfection, the investigator’s team aims to develop a novel strategy for in vivo T cell CRISPR screens. This innovative approach involves arrayed RNP transfection and co- transfer of T cells that recognize the relevant antigens. Instead of traditional genetic barcodes, the strategy utilizes congenic markers (CD45.1/45.2 and CD90.1/CD90.2) from donor TCR transgenic T cells as "external barcodes." These markers facilitate the recovery of gene perturbation identity at the single-cell level through the application of CITE-seq. Importantly, this RNP-based strategy seamlessly integrates with existing single-cell sequencing protocols, enabling the comprehensive assessment of transcripts, epitopes, and chromatin accessibility simultaneously. To demonstrate the efficacy of this strategy, the team plans to develop two benchmarking approaches: RNP-CET-seq to investigate the role of TCR regulators in T cell exhaustion and RNP-CATE-seq to map the gene regulatory atlas of exhausted CD8 T cells. In summary, the proposed RNP- based scCRISPR-seq strategy overcomes the limitations of current approaches, enabling large-scale, multi- module in vivo genetic screens within a physiologically relevant context across various disease models.

A NOVEL GEMM TO ELUCIDATE THE ROLE OF CHAF1A IN NEUROBLASTOMA DEVELOPMENT

PROJECT SUMMARY: This proposal focuses on the fundamental understanding on how the CHAF1A oncogene drives molecular mechanisms, cellular signaling, and metabolic processes in the oncogenesis of neuroblastoma (NB). NB is an aggressive pediatric cancer, which accounts for 15% of pediatric cancer mortalities. High-risk NB is thought to arise from a small number of recurrent genetic alterations that block the ability of neural crest cells (NCCs) to differentiate. To assess the molecular mechanisms governing NC differentiation, our laboratory has established a definitive role of the epigenetic regulator CHAF1A in blocking NC differentiation and driving NB oncogenesis. In this proposal, we will determine the impact of CHAF1A on NB initiation and progression. To accomplish this goal, we propose to develop a novel CHAF1A-driven genetically-engineered mouse model (GEMM) of NB and test the impact of CHAF1A on NB incidence, histology and metastasis, and the tumor immune microenvironment (TIME). We hypothesize that CHAF1A will increase de novo incidence of NB, reduce mouse survival, and promote a suppressive TIME. By developing a novel GEMM of NB and employing innovative technology (including ATAC-seq, lipidomics, and scRNA-seq), we will: 1- elucidate the role of CHAF1A in NB tumor initiation and progression; and 2- determine the impact of CHAF1A on MYCN-induced oncogenesis. These findings will provide a novel view on the molecular mechanisms driving NB initiation, and will have high clinical implications, informing future differentiation-based interventions for high-risk NBs.

Pathogenic mechanisms of expanded ZFHX3 in SCA4 cerebellar organoids

Spinocerebellar ataxia type 4 (SCA4) is a disabling neurodegenerative disease characterized by progressive cerebellar ataxia, and the causative GGC-repeat expansion in ZFHX3 (ZHFX3-exp) was just discovered this year by our lab and others. Our research aims to understand how ZFHX3-exp causes SCA4 and to identify molecular therapeutic targets that can be quickly advanced into clinical trials. SCA4 is one of the four poly-glycine diseases that share the presence of neuronal intranuclear inclusion (NIIs) as a disease hallmark. In SCA4, NIIs are positive for ZFHX3, p62 and ubiquitin, indicating the loss of proteostasis as a mechanism of neurodegeneration. In addition, ZFHX3 RNA-gain-of-function may also contribute to neurodegeneration. Beyond this, knowledge of the disease mechanisms that underly SCA4 is extremely limited and there are currently no disease-modifying treatments for SCA4 or other polyG/NII diseases. There are no SCA4 mouse models and because of the high GC content in the repeat expansion complicates the production of SCA4 mouse models. We propose a novel approach to characterizing SCA4 Purkinje cell (PC) pathogenesis using human cerebellar organoids. Our approach allows for rapidly advancing the understanding of the pathogenesis and potential treatments of SCA4. Using cerebellar organoids will enable investigation on functional PCs, cerebellar neurodegeneration and the testing of potential therapeutic strategies. In aim 1, we will generate cerebellar organoids from five SCA4 patient-derived iPSC lines, and normal control iPSCs from individuals of the same family. These iPSC lines are already established in our laboratory. In aim 2, we will investigate PC viability, NII protein composition, proteostasis pathways, RNA gain-of-function and cell-type-specific dysregulated pathways by single nucleus RNA sequencing. In addition, we will study potential therapeutic targets by lentiviral knockdown and single nucleus RNA sequencing. SCA4 patient iPSCs express overabundant STAU1 and ATXN2. We will evaluate how lowering the abundance of these proteins modifies the PC molecular phenotype. Together, these experiments will establish a model to greatly enhance the understanding of human PC neurodegeneration, the pathological mechanisms of SCA4 and possible avenues of treatment.

Investigating the role of noncoding RNAs in malaria parasites through targeted Cas13-mediated degradation

Project Summary/Abstract One of the most significant sources of morbidity and mortality throughout large regions of the developing world continues to be malaria caused by infection with mosquito-borne parasites of the genus Plasmodium. The parasite species responsible for the most severe form of the disease is P. falciparum. To avoid antibodies produced by their host and thereby maintain lengthy infections, these parasites undergo a process called antigenic variation by which they can extend an infection for over a year. This results from changes in expression of a protein called PfEMP1, the primary antigenic and virulence determinant expressed on the surface of infected red blood cells. A large, multicopy gene family called var encodes different forms of PfEMP1, and switching expression between var genes enables parasites to evade antibody recognition and destruction by the immune system. The process requires precise and coordinated regulation of transcription of each var gene, however how this is accomplished is unknown. It was recently hypothesized that a family of noncoding RNAs (ncRNAs) plays a key role in controlling the expression of each var gene and in determining the likelihood of activation of any given gene. If correct, this would represent a significant advance in our understanding of how P. falciparum controls antigenic variation and avoids immune clearance. To test this hypothesis, we propose to adapt the CRISPR/Cas13 system of targeted RNA degradation for use in P. falciparum. Similar to the extensively used CRISPR/Cas9 system, CRISPR/Cas13 employes guide RNAs to target a nuclease to a sequence-specific target, however Cas13 targets single stranded RNA rather than DNA. By applying this system to the study of var-related ncRNAs, we will degrade specific ncRNAs and determine the effect on var gene expression. Two classes of ncRNAs previously proposed to regulate var gene expression will be targeted, one called ruf6 and a second encoded by the second exon of all var genes. This will enable us to alter ncRNA expression while leaving the underlying genomic DNA untouched, thereby allowing the unambiguous attribution of any resulting phenotypes to the ncRNAs. Aim 1 will optimize the Cas13 system for P. falciparum by testing different variants of the Cas13 endonuclease for their ability to degrade mRNAs encoding fluorescent reporter proteins. We will determine both the efficiency and sequence specificity of the system. Aim 2 will apply the system to var-associated ncRNAs and quantitatively measure changes in var gene expression and transcriptional switching. If successful, the adaptation of the Cas13 system to P. falciparum will provide the malaria research community with a powerful new tool for manipulating gene expression. In addition, we will gain valuable new insights into how malaria parasites regulate var gene expression, antigenic variation and immune evasion.

Overcoming Treatment Resistance by Targeting Polyploid Breast Cancer Cells with AI assisted Single-Cell Analysis

Therapy resistance remains a formidable challenge in breast cancer treatment, with emerging evidence identifying polyploid giant cancer cells (PGCCs) as key drivers. These cells, arising through whole-genome doubling (WGD) events, exhibit enhanced resistance to therapies, contributing to disease relapse. PGCCs are characterized by enlarged cell and nuclear sizes, increased DNA content, and greater resilience compared to non-PGCCs. Their prevalence escalates with disease progression and therapeutic stress, underscoring their critical role in treatment resistance. As such, we hypothesize that inhibiting polyploid cancer cells can effectively reduce therapeutic resistance. Despite this, effective strategies targeting PGCCs are limited, hindered by the lack of high-throughput methods to assess PGCC viability and abundance. Traditional screening assays lack the sensitivity to detect the elimination of small populations of PGCCs, while current detection methods, such as visual inspection and flow cytometry, are not suited for high-throughput compound screening. Our preliminary work has established a high-throughput single-cell morphological analysis pipeline capable of quantifying PGCCs, and we successfully screened 2,726 compounds for their efficacy on PGCCs. Based on the preliminary success, we aim to further improve its robustness and accuracy under diverse staining and imaging conditions, ensuring consistent performance across multiple labs for widespread use in PGCC/WGD studies, with deep learning to accelerate the discovery of therapeutic strategies targeting PGCCs. In addition to empirical screening, our scRNA-Seq analysis of PGCCs has revealed altered gene expression, particularly in genes associated with FOXM1, a transcription factor critical in cell cycle regulation and linked to poor outcomes in various cancers. PGCCs also show altered ferroptosis regulators and elevated reactive oxygen species (ROS), indicating susceptibility to ferroptosis. Here, we propose two independent and complementary aims. Aim 1: We will develop and validate a robust deep learning–based single-cell morphological analysis pipeline for accurate PGCC/non-PGCC discrimination across variable staining, imaging, and lab settings. The model will be benchmarked on independent datasets from external labs and released as open-source, version-controlled software with full documentation to support reproducibility and broad adoption in PGCC/WGD research. Aim 2: Leveraging our screen of 2,726 FDA-approved compounds and mechanistic studies of FOXM1 and ferroptosis, we will prioritize and validate therapies that eradicate PGCCs and reduce treatment resistance. Using patient- derived cells, 3D spheroids, and syngeneic/xenograft models, we will rigorously assess top candidates as monotherapy and in combination with standard-of-care agents. Successful completion of this project will accelerate PGCC/WGD research, advance therapeutic strategies to overcome breast cancer resistance, and especially deliver benefits to patients with high PGCC burden. Given the prevalence of WGD across solid tumors and its induction by standard therapies, our approach holds broad clinical relevance and translational impact.

A Novel Mitochondrial-Targeted Inhibitor of NLRP3 Inflammasome Activation

PROJECT ABSTRACT Inflammasomes are multiprotein complexes of the innate immune system that assemble upon detecting specific molecular patterns associated with pathogens and cellular damage. Once assembled, activated inflammasomes trigger a cascade of downstream events that culminate in cell death and inflammation. Aberrant activation of the NLRP3 inflammasome contributes to the pathogenesis of numerous inflammatory and degenerative diseases, including gout, atherosclerosis, type 2 diabetes, and Alzheimer’s disease. Despite its central role in innate immunity and inflammation, there are no FDA-approved therapies that directly target the NLRP3 inflammasome. Current strategies rely on biologics that inhibit downstream pro-inflammatory cytokines produced from inflammasome activation, such as interleukin-1β (IL-1β), but do not block upstream inflammasome assembly or pyroptotic cell death, highlighting a critical unmet need for selective small-molecule inhibitors with novel mechanisms of action. To address this gap, we identified a covalent small molecule, Compound-2 (C-2), that robustly inhibits NLRP3 inflammasome activation in murine and human immune cells. C-2 suppresses multiple downstream events triggered by inflammasome activation, including IL-1β secretion and pyroptosis, with no apparent toxicity. Chemoproteomic profiling revealed that C-2 interacts with SLC25A3, a mitochondrial phosphate and copper transporter, suggesting a previously unrecognized regulatory node in inflammasome signaling. This R21 project aims to (1) elucidate the mechanism by which C-2 suppresses NLRP3 activation and (2) define the molecular interaction between C-2 and SLC25A3 and its functional consequences. Our studies will integrate biochemical, cellular, and in vivo approaches to uncover a novel mitochondrial mechanism of inflammasome regulation and validate C-2 as a first-in-class inflammasome inhibitor. Successful completion of this project will lay the foundation for future therapeutic development targeting mitochondrial- inflammasome crosstalk in inflammatory disease.

A dynamic regulatory mechanism controlling bacterial persister formation and resuscitation within biofilms

PROJECT SUMMARY Persisters present a major challenge in clinical infection treatment and recurrent infection management. A continued effort towards a better understanding of the molecular mechanisms of persister formation and resuscitation is needed to provide novel treatment strategies for the control of chronic infections and problems related to persisters. Unlike resistant bacteria, persisters are genetically identical to their susceptible counterparts, and this phenotypic state is inherently transient and shifts in response to environmental conditions. Therefore, it is essential to use an approach tailored to the transient and rare nature of this phenomenon. Pseudomonas aeruginosa (Pa) is an important human pathogen frequently implicated in both acute and chronic infections. Persisters have been identified in both Pa planktonic and biofilm modes of growth, with higher frequencies of persister formation being observed in biofilm, especially in the interior of the mature biofilm structure. In this study, we obtained the first high-resolution single-cell transcriptomes of persister and resuscitated cells isolated directly from the interior of mature biofilms. The results led to the identification of a previously uncharacterized transcriptional regulator that controls persister formation and resuscitation. This regulator, named PriR here, is conserved in Pseudomonas species and has homologs in two critical bacterial pathogens, Acinetobacter baumannii and Enterobacter cloacae. We showed that PriR has a dynamic spatiotemporal gene expression profile, and its expression directly correlates with and causes persister resuscitation. In this application, we propose two specific aims to investigate this novel regulation mechanism of persister formation and resuscitation. Aim 1 will identify the physiological effects of this novel regulatory system on antibiotic tolerance in vitro and in hosts using the Drosophila melanogaster biofilm infection model. Aim 2 will determine its molecular regulatory mechanism via ChIP-seq and RNA-seq, and analyze the putative PriR- controlled genes on persister formation and resuscitation in additional clinically-relevant Pa strains. The insights gained from this proposal will provide crucial new information about the dynamic regulatory mechanism of persister formation and resuscitation. The PriR-controlled resuscitation mechanism could be a promising target for persister eradication approaches by re-sensitizing persister cells to conventional antimicrobials or preventing persister formation. Understanding this novel regulatory system that controls bacterial persister formation and resuscitation could provide new drug targets and/or treatment strategies for persistent infections.

Autoreactive T cells in lupus

The autoimmune disease systemic lupus erythematosus (SLE) is characterized by loss of adaptive immune tolerance in conjunction with innate immune system hyperactivity. Autoantibodies, produced by plasma cells derived from activated B cells, form proinflammatory immune complexes. These immune complexes drive feed forward loops that sustain a systemic inflammatory environment and deposit in tissues leading to potentially fatal organ damage. B cells receive help from T cells to produce antibodies. They also contribute to disease by shaping T cell responses and secreting cytokines. Recent case reports in which SLE patients were treated with anti-CD19 CAR-T cell therapy to deplete B cells highlight the pathogenic role of B cells in lupus and their value as a therapeutic target. However, a better understanding of how autoreactive B cells interact with autoreactive T cells may reveal more targeted points of therapeutic intervention that specifically block autoreactive responses while sparing protective ones. Antigen specific interactions between CD4+ T cells and B cells are required for the development of autoimmune disease in lupus. However, whether these critical interactions occur in germinal centers, where competition for CD4+ T cell help selects high affinity B cells, or in extrafollicular responses, where B cells may avoid peripheral tolerance checkpoints, is unclear. Gene expression profiles and pathways specific to autoreactive CD4+ T cells, and how they are shaped by their interaction with autoreactive B cells, are also ill defined. CD8+ T cells, which recognize antigen presented on MHC Class I, have also been suggested to modulate the fate of autoreactive B cells. They can directly kill autoreactive B cells as a means of tolerance, and a subset of CD8+ T cells has recently been shown to have B cell helper function. Whether and how such interactions between B and CD8+ T cells enhance or suppress the development of lupus is unknown. Here, we will use genetic and in vivo proximity labeling approaches to address these knowledge gaps. In Aim 1, we will test the hypothesis that antigen specific interactions between B and CD8+ T cells promote B cell activation and autoantibody production in lupus. We will prevent B cells, but not other cells, from undergoing cognate interactions with CD8+ T cells via B cell-specific deletion of B2M, a component of the MHC Class I complex, in two lupus models. In Aim 2, will use the uLIPSTIC in vivo proximity system to label all T cells interacting with B cells in lupus models compared to wild type controls. Features specific to these autoreactive T cells will be defined by flow cytometry, scRNA Seq, and scTCR-Seq. These studies will provide valuable molecular and cellular insight into the mutual activation of B and T cells in lupus. They will set the stage for future mechanistic studies defining the role of autoreactive T cell specific genes and pathways and potentially highlight new therapeutic targets specific to autoreactive B/T interactions.

Bacterial ferrous iron sensing via the BqsRS (CarRS) two-component system

Project Summary Pseudomonas aeruginosa (Pa) is an opportunistic and increasingly antibiotic resistant Gram-negative bacterium that is one of the major causes of chronic nosocomial infections in the United States. The colonization of Pa within a host is often linked to the bioavailability of nutrients, such as iron, and Pa has multiple iron acquisition pathways that allow it to adapt readily to the variety of environments it may encounter within a human host. Pa responds to these dynamic environments commonly through the use of two-component signal transduction systems (TCSs) that are important mediators of signal transduction and allow pathogens to detect chemical and/or physical changes in the environment in order to control basic cellular processes. Previous studies have identified a biofilm and quorum sensing TCS known as BqsRS (also known as CarRS) that regulates biofilm formation and decay in Pa through the sensing of extracytoplasmic Fe2+ and Ca2+. Among its targets, the BqsRS TCS is known to regulate rhlAB and rhlC, critical genes for rhamnolipid production and biofilm formation that are also known to be connected to iron homeostasis and antibiotic resistance. Moreover, the deletion of either bqsR or bqsS in PAO1 results in a significant increase in biofilm formation but reduced biofilm dispersion, the latter of which is important for downstream infections. These observations highlight the importance of the BqsRS TCS to Pa virulence, but there is a foundational lack of understanding regarding the structure, the selectivity, and the mechanism of this system. The ultimate goal of this proposal is to generate a mechanistic and functional understanding of BqsRS at atomic, molecular, and organismal levels in order to exploit this system as a means of reducing or stemming the virulence of opportunistic pathogens such as Pa. The objectives of this exploratory grant are to determine the structural and molecular characteristics of BqsRS, to define how these properties govern BqsRS metal selectivity and function, and to examine a new role of the BqsRS system in regulating the Feo system in P. aeruginosa. Ultimately, the accomplishment of this exploratory grant will deliver fundamental mechanistic insight into a critical metal-sensing TCS and lay the groundwork for future studies that may be designed to target this system and its homologs for additional bacterial exploits.

Programming Offspring Metabolism: The Role of Milk Extracellular Vesicles in Fat Development

SUMMARY Obesity is a global health crisis, contributing significantly to the prevalence of metabolic disorders, cardiovascular diseases, and various chronic conditions. A growing body of evidence suggests that maternal obesity during pregnancy and lactation can predispose offspring to obesity and metabolic dysfunction later in life. However, the mechanisms by which maternal obesity programs these adverse outcomes in offspring remain poorly understood. Breast milk is not only a source of essential nutrients but also contains bioactive components, including extracellular vesicles (EVs), which play crucial roles in cellular communication and development. Recent studies have shown that EVs can survive digestion and enter the infant’s circulation, influencing immune and metabolic development. Despite the established link between maternal obesity and altered breast milk composition, no study has investigated the role of milk-derived EVs (mEVs) in programming offspring fat development and metabolism. Understanding this novel pathway could revolutionize our approach to preventing intergenerational transmission of obesity. Our preliminary studies using a mouse model of maternal high-fat diet-induced obesity revealed significant alterations in mEV biogenesis and cargo composition, including changes in specific miRNAs. Oral administration of mEVs from obese dams to neonatal mice increased adiposity and impaired lipid metabolism, indicating that mEVs are crucial in modulating fat development and metabolic pathways in offspring. Several key miRNAs found in mouse mEVs are conserved in human milk EVs, highlighting the potential translational relevance of our findings to human health. We hypothesize that mEVs are critical mediators of maternal obesity’s programming effects on offspring metabolism and adiposity. In specific aim 1, we will use mouse models and advanced molecular techniques (miRNA sequencing, proteomics, and lipidomics) to characterize how maternal obesity affects mEV biogenesis and the composition of their bioactive cargo. We will also evaluate how maternal dietary intake, independent of obesity, influences mEV composition. Specific aim 2 will define the programming effects of mEVs on offspring energy metabolism and obesity. In addition, we will explore whether human milk EVs from lean and obese mothers exert similar programming effects on fat development and metabolism in a mouse model. This R21 application embodies a high-risk, high-reward approach to obesity research. It ventures into uncharted territory by proposing that mEVs are novel regulators of metabolic programming, a concept that has not been explored in prior studies. The potential reward is substantial: discovering a new mechanism by which maternal obesity influences offspring health could fundamentally shift our understanding of early-life metabolic programming and lead to innovative strategies for obesity prevention. If successful, this research could open a new field of study with broad implications for maternal and child health.

2-Deoxyglucose Therapy for Organophosphate Intoxication

Project Summary The main goal of this project is to determine the therapeutic potential of glycolysis inhibition as an adjunct to midazolam therapy in mitigating the long-term neurological effects from acute organophosphate pesticide and nerve agent (OPNA) exposure. Novel countermeasures are desperately needed for effective mitigation of morbidity and long-term effects of OPNAs. A variety of agents targeting glutamate, GABA and oxidative stress have been proposed, but glycolysis inhibitors have not been widely studied in OPNA intoxication. Dysregulated glucose metabolism plays a key role in seizures and neuronal injury following OPNA exposure. 2-Deoxyglucose (2-DG), a selective glycolysis inhibitor, has anticonvulsant and neuroprotection effects and hence can effectively mitigate acute and long-term OPNA neurotoxicity. In this project, we seek to identify the glycolysis inhibition as novel adjunct neuroprotection to midazolam therapy for OPNA exposure, with the goal of identifying 2-DG or related drugs as medical countermeasures. The glycolytic pathway represents a logical target for such intervention because glycolysis controls seizures and neuronal injury by regulating glucose utilization and activity in neurons and astrocytes in the brain. The proposed therapy is based on the hypothesis that acute OPNA neurotoxicity imparts sustained activation of the glycolysis pathway in the brain and therefore, 2- DG and selective glycolysis inhibitors prevents long-term neuronal damage neurological dysfunction. This hypothesis will be tested by using the FDA-approved (2-DG) or clinical-stage glycolytic inhibitors in two distinct OPNA models in rats: (Aim 1) To investigate the protective efficacy of 2-DG and novel glycolysis inhibitors against DFP-induced acute and long-term neuronal damage and neurological dysfunction. (Aim 2) Aim 2 (Year 2). To determine brain penetration, pilot toxicity and pharmacokinetic of 2-DG or other lead drug in naïve and DFP-exposed animals. Test drugs will be evaluated as per the NIH rigor criteria in a dose-related design in male and female rats and behavior/neuropathology will be checked for 3 months post-exposure. 2-DG and test drugs will be given starting 40-min after exposure to ONAs. Three primary outcome measures will be addressed for therapy effectiveness: (i) acute adjunct neuroprotection; (ii) chronic neuroprotectant efficacy; and (iii) prevention of neurological and behavioral deficits. The primary measures of neuroprotection include longitudinal MRI scanning, and extent of neurodegeneration, neuroinflammation, aberrant neurogenesis, and mossy fiber sprouting. Key neurological outcomes include memory deficits, depression, anxiety behavior, and neurological/motor deficits. The outcome of this project will provide “proof-of-efficacy” of a novel glycolytic therapy with FDA-approvable, repurposed drugs with promising potential to limit long-term effects of OPNAs in humans. Thus, the overall impact of the outcome is enormous for civilians, especially in developing a highly effective and safe post-exposure medical countermeasure for chemical nerve agents.

Chromatin-Based Mechanisms Linking Transcriptional Dysregulation to Genome Instability in Neurodevelopmental Disorders.

PROJECT SUMMARY/ABSTRACT Neurons depend on a finely tuned interplay between chromatin regulation and genome maintenance, yet they are acutely vulnerable to DNA damage generated during activity-dependent transcription of long, synaptic genes. Disruption of this balance is increasingly recognized as a driver of neurodevelopmental disorders (NDDs) such as autism spectrum disorder (ASD), intellectual disability, and epilepsy. High-confidence genetic studies converge on regulators of histone H3 lysine 4 (H3K4) methylation, such as the writers ASHIL and Klv1T2C and the eraser KDNISB, as recurrently mutated loci in NTIDs. The overarching goal of this study is to investigate how dysregulated H3K4 methylation compromises genome integrity in human neurons, thereby contributing to the pathogenesis of NDDs. The central, hypothesis is that coordinated II3K4 methylation safeguards neuronal genomes by maintaining an open chromatin architecture that permits the efficient detection and repair of transcription-coupled DNA lesions. The rationale/Or this study is to define the epigenetic control of DNA repair, which will illuminate a shared pathogenic hub across multiple ~I)D-linked genes. During the mentoredK99 phase, I will define how ASHIL, KMT2C, and KDM5B regulate chromatin structure and DNA repair at baseline and during transcriptional stress. Aim-1: I will use isogenic iPSC-derived cortical neurons with patient-relevant mutations or CRrSPRi knockdowns of these regulators, applying an integrated multi-omic pipeline: CUT&Tag and Micro-C to map H3K4 methylation and 3D chromatin topology. Aim-2: I will use Paired-Damage-seq, and CUT&RUN to chart oxidative lesions, repair synthesis, and recruitment of key repair factors; and RNA-seq to relate damage hotspots to altered gene expression. Aims l and 2 will be performed under the guidance of Dr. Lizarraga and Dr. Morrow, experts in the field of neurodevelopmental biology. My advisory team brings unique and complementary skills, enhancing my knowledge in 3D chromatin structure, transcription-coupled repair, gene editing, and multi-omics analysis. I will utilize these skills in the R00 phase (Aim 3), expanding the framework to include additional H3K4 regulators (e.g., LSD1, KMT2A) and broader neural lineages, thereby developing a comprehensive model. This study is innovative in its integration of single-cell D.NA damage mapping with chromatin topology and transcriptional profiling, enabling a direct and mechanistic connection between disrupted H3K4 methylation and genome instability. By uncovering how H3.K4 methylation prevents transcription-coupled genome instability in the developing brain, this research will address a critical gap in our understanding of NDD mechanisms. This award will enable me to launch an independent research program dedicated to determining mechanisms of chromatin-based processes that maintain genome stability in the developing human brain.

Structure-function and mechanistic studies of a specific glycosyltransferase complex in fusion-driven pediatric gliomas

Abstract Glycosylation is a co/post-translational modification involved in cell-matrix interactions, antigen-antibody interactions, tumor invasion, and cell motility. Abnormal glycosylation is a hallmark of cancer, with various glycosylation-related genes linked to glioma prognosis and tumor heterogeneity. Pediatric low-grade gliomas (pLGGs) stand as the most common childhood central nervous system tumor, accounting for 30%-40% of all CNS tumors in children. Despite its relatively low mortality rate, pLGGs are associated with devastating lifelong morbidity. The most common alteration found in 75% of tumors is the KIAA1549:BRAF fusion, causing an aberrant activation of the MAPK/ERK signaling pathway. Current treatments, such as traditional chemotherapies and targeted therapies, have limitations such as resistance, lack of specificity, toxicity and paradoxical activation of the MAPK pathway. This highlights the urgent need for novel therapeutic approaches. Investigations into KIAA1549:BRAF-driven pLGGs identified their dependency on the protein-O-mannosyl transferase (POMT) complex for survival. In contrast, BRAFV600E-mutant cells did not show dependency, suggesting the POMT complex as a vulnerability and promising target in KIAA1549:BRAF-driven pLGGs. Therefore, our goal is to characterize the POMT complex structurally and biochemically and study its roles in KIAA1549:BRAF-driven pLGGs. In this proposal, we aim to 1) determine the high-resolution structures of the complex in its unbound, substrate-bound, and inhibitor-bound forms and 2) elucidate the POMT complex mechanisms in KIAA1549:BRAF-driven pLGGs. We will define the critical functional domains, active sites, interaction interfaces and translational modifications crucial for enzymatic activity using cryo-EM techniques, mutagenesis, and functional studies. To study biological pathways and molecular events modulated by the POMT complex, we will implement global proteomics and transcriptomics analysis in well-characterized disease models. In parallel, we will assess the effect of the POMT complex on the MAPK/ERK signaling pathway. This study will guide the structure-based design of probes and drugs targeting the POMT complex and will unveil glycosylation-mediated oncogenesis in pediatric gliomas. It will aid in the development of new targeted therapies and the identification of new biomarkers for pLGGs harboring the KIAA1549:BRAF fusion. The research will be conducted in the Fischer lab at Dana-Farber Cancer Institute, which provides a collaborative and resource-rich environment. The career development plan includes training in scientific writing, mentoring, and presentation skills, as well as interdisciplinary networking with experts in structural biology and pediatric oncology. The candidate’s career goal is to establish an independent research laboratory focused on developing new therapeutic modalities for pediatric neurooncology. The training provided through this fellowship represents a critical step toward achieving this goal.

Continued HIV Production From Infected Macrophage In People On ART

PROJECT ABSTRACT After a few weeks of antiretroviral therapy (ART), HIV-1 RNA often decays to undetectable levels in blood. The initial decay is typically rapid due to the loss of short-lived, HIV-infected CD4+ T cells, but despite being adherent to ART, some people experience a subsequent period of slower decay and may require months to years to reach virologic suppression. The clinical significance of ‘slow decay’ of HIV-1 RNA after starting ART is currently unknown. Assessing the clinical significance of ‘slow decay virus’ requires identify the mechanisms generating it and exploring whether there is ongoing inflammation and neuronal damage in these people. There are three potential mechanisms that may generate ‘slow decay virus’ and they may have very different clinical implications. (1) Continued HIV-1 replication due to ineffective ART, poor ART adherence or drug- resistance. (2) Alternatively, ART could stop HIV-1 replication, but HIV-1 virions may continue to be produced by HIV-infected CD4+ T cells or (3) macrophage. Virus production without replication that emerges at the time of ART initiation is called primary nonsuppresible viremia (NSV) and is mechanistically distinct from secondary NSV observed in people who were previously suppressed. We recently examined four people who required approximately a year to become suppressed and found that ART stopped HIV-1 replication, but HIV-infected macrophage continued to produce substantial amounts of virus. These preliminary results are consistent with the long-held belief that after starting ART there is a period of rapid viral decay due to loss of HIV-infected CD4+ T cells, but some people have a subsequent period of slower decay due to continued virus production from long- lived, HIV-infected macrophage. The proposed work will expand on these observations and examine the mechanisms generating ‘slow decay virus’ in a much larger cohort of people on ART and explore the clinical implications of having ‘slow decay virus’ after starting ART (i.e. primary NSV). We will use existing, archived, longitudinal blood samples from 99 people in the MACS/WIHS Combined Cohort Study (MWCCS) who did not suppress HIV-1 RNA to undetectable levels by 6 months on ART (i.e. people with ‘slow decay virus’) and samples from 30 people who suppressed virus with typical, rapid kinetics. The proposed experiments will identify the mechanisms generating ‘slow decay virus’ during ART and the clinical implications of ‘slow decay virus’ (Aim 1). In our previous study, we also observed that ‘slow decay virus’ produced by macrophage often had nonsense/frameshift mutations in the HIV-1 vpr gene that may have promoted continued HIV-1 production from macrophage during ART. Specifically, we will explore whether ‘slow decay virus’ populations produced by macrophage have mutations in vpr or other genes that impact macrophage survival and/or HIV-1 production from infected macrophage (Aim 2). We will accomplish these aims using cutting-edge, but highly rigorous approaches. Accomplishing these aims will address clinical concerns about ‘slow decay virus’, the source of ‘slow decay virus’ as well as the role that Vpr plays in HIV-1 persistence and expression in macrophage during ART.

Optimizing gamma-delta T cell receptor-mediated signaling to improve cancer immunotherapy

PROJECT SUMMARY The recent development of T cell-based cancer immunotherapies, including checkpoint blockade (anti-PD-1, anti-CTLA-4 and others) or adoptive cell therapy (ACT) using modified patient T cells, has led to improved patient outcomes for a variety of cancers. However, durable responses are observed in only a fraction of patients. Further progress can be made by studying and targeting different T cell subpopulations, such as the gd T cells which are known to possess antitumor activities. Further, gd T cells are mostly independent of MHC-restriction, unconstrained by neoantigen burden, preferential homing to peripheral tissues and possess unique properties of T cells as well as natural killer cells making them an extremely attractive cancer immunotherapy target. One way of gd T cell activation involves the gd T cell receptor (gdTCR)-CD3 signaling pathway. gd T cell recognition of antigen by the gdTCR and the resulting proximal signaling through surrounding CD3 subunits are key steps of gd T cell activation. Even though the individual components of the gdTCR-CD3 and abTCR-CD3 complexes remain the same except for the TCRs, the complete gdTCR-CD3 complex extracellular structure is unknown. Identification of the specific extracellular interactions between the gdTCR and CD3 subunits could offer precise guidance for the development of immunotherapeutic strategies that modulate gdT cell immunity by targeting signaling through the gdTCR-CD3 complex. Our previous data showed that mutating residues in the constant domain of the abTCR resulted in altered ab T cell cytokine responses. Based on this data, our hypothesis is that gdTCR-CD3 signaling can also be modulated by targeting specific regions of the gdTCR by mutagenesis to improve gd T cell antitumor activities. To test our hypothesis, in Aim 1, we will use a novel photo-crosslinking and computational docking methodology to solve the complete extracellular structure of a gdTCR-CD3 complex. Further, we will use an in silico structure-based TCR design approach to identify gdTCR mutants that enhance signaling. In Aim 2, we will use an in vitro retroviral TCR display method using degenerate primers to create gdTCR mutant libraries at specific gdTCR sites such as Cg helix 3 and connecting peptide (CP) regions. In both instances, identified mutants will be tested for improved functionalities in an MHC-independent gd TCR (G115 Vg9Vd2 TCR) using in vitro cytokine and tumor-killing assays. Overall, the newly identified enhanced gd T cell clones could potentially lead to a new wave of effective cancer immunotherapy strategy by leaning into the largely untapped potential of gd T cells.

Circulating and Mucosal Predictors and Effects of Therapeutic Interleukin-23 Blockade in Crohn's Disease

PROJECT SUMMARY/ABSTRACT Since its discovery 20 years ago, the cytokine interleukin (IL)-23 has increasingly been implicated in the pathogenesis of immune mediated diseases, such as Crohn’s disease (CD). Consequently, four monoclonal antibodies that block IL-23 are currently approved CD therapies, including risankizumab. Although suppression of pathogenic Th17 cells has been widely cited as the mechanism by which IL-23 blockade controls disease, there is a paucity of data to indicate that this is how such therapy works, and a few other immune cell populations expressing the IL-23 receptor could instead be its target. We therefore propose to study how risankizumab affects not only Th17 cells, but also mucosa-associate invariant T (MAIT) cells γδ T cells and (in the colon) type 3 innate lymphoid cells (ILC3s). In addition to quantifying these cells, we will study their gene expression to detect phenotypic differences in treated patients, and in the case of T cells, track their clonal expansion and deletion through their unique T cell receptor sequences. In colon samples, we will use a combination of single cell sequencing of sort-enriched immune cell populations and spatial transcriptomics to characterize cells in situ, at the site of disease, and determine how IL-23 blockade affects their microenvironment in vivo. By contrasting results in patients who do or do not respond therapeutically to IL-23 blockade, we will reveal valuable insights into how this treatment succeeds or fails in CD, in the process identifying predictive biomarkers to guide treatment decisions, and potentially identifying future molecular targets with which to prevent treatment failure.

Spike train structure of cortical transcriptomic populations in vivo

The cortex comprises many neuronal types, which can be distinguished by their transcriptomes: the sets of genes they express. Little is known about the in vivo activity of these cell types, particularly as regards the structure of their spike trains, which might provide clues to cortical circuit function. To address this question, we used Neuropixels electrodes to record layer 5 excitatory populations in mouse V1, then transcriptomically identified the recorded cell types. To do so, we performed a subsequent recording of the same cells using 2-photon (2p) calcium imaging, identifying neurons between the two recording modalities by fingerprinting their responses to a “zebra noise” stimulus and estimating the path of the electrode through the 2p stack with a probabilistic method. We then cut brain slices and performed in situ transcriptomics to localize ~300 genes using coppaFISH3d, a new open source method, and aligned the transcriptomic data to the 2p stack. Analysis of the data is ongoing, and suggests substantial differences in spike time coordination between ET and IT neurons, as well as between transcriptomic subtypes of both these excitatory types.

Cellular Crosstalk in Brain Development, Evolution and Disease

Cellular crosstalk is an essential process during brain development and is influenced by numerous factors, including cell morphology, adhesion, the local extracellular matrix and secreted vesicles. Inspired by mutations associated with neurodevelopmental disorders, we focus on understanding the role of extracellular mechanisms essential for the proper development of the human brain. Therefore, we combine 2D and 3D in vitro human models to better understand the molecular and cellular mechanisms involved in progenitor proliferation and fate, migration and maturation of excitatory and inhibitory neurons during human brain development and tackle the causes of neurodevelopmental disorders.

Expanding mechanisms and therapeutic targets for neurodegenerative disease

A hallmark pathological feature of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the depletion of RNA-binding protein TDP-43 from the nucleus of neurons in the brain and spinal cord. A major function of TDP-43 is as a repressor of cryptic exon inclusion during RNA splicing. By re-analyzing RNA-sequencing datasets from human FTD/ALS brains, we discovered dozens of novel cryptic splicing events in important neuronal genes. Single nucleotide polymorphisms in UNC13A are among the strongest hits associated with FTD and ALS in human genome-wide association studies, but how those variants increase risk for disease is unknown. We discovered that TDP-43 represses a cryptic exon-splicing event in UNC13A. Loss of TDP-43 from the nucleus in human brain, neuronal cell lines and motor neurons derived from induced pluripotent stem cells resulted in the inclusion of a cryptic exon in UNC13A mRNA and reduced UNC13A protein expression. The top variants associated with FTD or ALS risk in humans are located in the intron harboring the cryptic exon, and we show that they increase UNC13A cryptic exon splicing in the face of TDP-43 dysfunction. Together, our data provide a direct functional link between one of the strongest genetic risk factors for FTD and ALS (UNC13A genetic variants), and loss of TDP-43 function. Recent analyses have revealed even further changes in TDP-43 target genes, including widespread changes in alternative polyadenylation, impacting expression of disease-relevant genes (e.g., ELP1, NEFL, and TMEM106B) and providing evidence that alternative polyadenylation is a new facet of TDP-43 pathology.

Rejuvenating the Alzheimer’s brain: Challenges & Opportunities

Pharmacological exploitation of neurotrophins and their receptors to develop novel therapeutic approaches against neurodegenerative diseases and brain trauma

Neurotrophins (NGF, BDNF, NT-3) are endogenous growth factors that exert neuroprotective effects by preventing neuronal death and promoting neurogenesis. They act by binding to their respective high-affinity, pro-survival receptors TrkA, TrkB or TrkC, as well as to p75NTR death receptor. While these molecules have been shown to significantly slow or prevent neurodegeneration, their reduced bioavailability and inability to penetrate the blood-brain-barrier limit their use as potential therapeutics. To bypass these limitations, our research team has developed and patented small-sized, lipophilic compounds which selectively resemble neurotrophins’ effects, presenting preferable pharmacological properties and promoting neuroprotection and repair against neurodegeneration. In addition, the combination of these molecules with 3D cultured human neuronal cells, and their targeted delivery in the brain ventricles through soft robotic systems, could offer novel therapeutic approaches against neurodegenerative diseases and brain trauma.

Rett syndrome, MECP2 and therapeutic strategies

The development of the iPS cell technology has revolutionized our ability to study development and diseases in defined in vitro cell culture systems. The talk will focus on Rett Syndrome and discuss two topics: (i) the use of gene editing as an approach to therapy and (ii) the role of MECP2 in gene expression (i) The mutation of the X-linked MECP2 gene is causative for the disease. In a female patient, every cell has a wt copy that is, however, in 50% of the cells located on the inactive X chromosome. We have used epigenetic gene editing tools to activate the wt MECP2 allele on the inactive X chromosome. (ii) MECP2 is thought to act as repressor of gene expression. I will present data which show that MECP2 binds to Pol II and acts as an activator for thousands of genes. The target genes are significantly enriched for Autism related genes. Our data challenge the established model of MECP2’s role in gene expression and suggest novel therapeutic approaches.

Modeling human brain development and disease: the role of primary cilia

Neurodevelopmental disorders (NDDs) impose a global burden, affecting an increasing number of individuals. While some causative genes have been identified, understanding the human-specific mechanisms involved in these disorders remains limited. Traditional gene-driven approaches for modeling brain diseases have failed to capture the diverse and convergent mechanisms at play. Centrosomes and cilia act as intermediaries between environmental and intrinsic signals, regulating cellular behavior. Mutations or dosage variations disrupting their function have been linked to brain formation deficits, highlighting their importance, yet their precise contributions remain largely unknown. Hence, we aim to investigate whether the centrosome/cilia axis is crucial for brain development and serves as a hub for human-specific mechanisms disrupted in NDDs. Towards this direction, we first demonstrated species-specific and cell-type-specific differences in the cilia-genes expression during mouse and human corticogenesis. Then, to dissect their role, we provoked their ectopic overexpression or silencing in the developing mouse cortex or in human brain organoids. Our findings suggest that cilia genes manipulation alters both the numbers and the position of NPCs and neurons in the developing cortex. Interestingly, primary cilium morphology is disrupted, as we find changes in their length, orientation and number that lead to disruption of the apical belt and altered delamination profiles during development. Our results give insight into the role of primary cilia in human cortical development and address fundamental questions regarding the diversity and convergence of gene function in development and disease manifestation. It has the potential to uncover novel pharmacological targets, facilitate personalized medicine, and improve the lives of individuals affected by NDDs through targeted cilia-based therapies.

How are the epileptogenesis clocks ticking?

The epileptogenesis process is associated with large-scale changes in gene expression, which contribute to the remodelling of brain networks permanently altering excitability. About 80% of the protein coding genes are under the influence of the circadian rhythms. These are 24-hour endogenous rhythms that determine a large number of daily changes in physiology and behavior in our bodies. In the brain, the master clock regulates a large number of pathways that are important during epileptogenesis and established-epilepsy, such as neurotransmission, synaptic homeostasis, inflammation, blood-brain barrier among others. In-depth mapping of the molecular basis of circadian timing in the brain is key for a complete understanding of the cellular and molecular events connecting genes to phenotypes.

The immunopathogenesis of autoimmune seizure disorders

Immune-mediated mechanisms are increasingly recognised as a cause of epilepsy even in the absence of an immune response against a specifical neuronal antigen. In some cases, these autoimmune processes are clearly pathogenic, for example acute seizures in autoimmune encephalitis, whereas in others this is less clear, for example autoimmune-associated epilepsy. Recent research has provided novel insights into the clinical, paraclinical and immunopathogenetic mechanisms in these conditions. I will provide an overview of clinical and paraclinical features of immune-associated seizures. Furthermore, I will describe specific immunopathogenic examples implicating lymphoid follicular autoimmunisation and intrathecal B cells in these conditions. These insights into immunopathogenesis may help to explain the role of current and immunotherapies in these conditions.

The many roles of microglia in the pathogenesis of neurodegeneration

Cellular and genetic mechanisms of cerebral cortex folding

One of the most prominent features of the human brain is the fabulous size of the cerebral cortex and its intricate folding, both of which emerge during development. Over the last few years, work from my lab has shown that specific cellular and genetic mechanisms play central roles in cortex folding, particularly linked to neural stem and progenitor cells. Key mechanisms include high rates of neurogenesis, high abundance of basal Radial Glia Cells (bRGCs), and neuron migration, all of which are intertwined during development. We have also shown that primary cortical folds follow highly stereotyped patterns, defined by a spatial-temporal protomap of gene expression within germinal layers of the developing cortex. I will present recent findings from my laboratory revealing novel cellular and genetic mechanisms that regulate cortex expansion and folding. We have uncovered the contribution of epigenetic regulation to the establishment of the cortex folding protomap, modulating the expression levels of key transcription factors that control progenitor cell proliferation and cortex folding. At the single cell level, we have identified an unprecedented diversity of cortical progenitor cell classes in the ferret and human embryonic cortex. These are differentially enriched in gyrus versus sulcus regions and establish parallel cell lineages, not observed in mouse. Our findings show that genetic and epigenetic mechanisms in gyrencephalic species diversify cortical progenitor cell types and implement parallel cell linages, driving the expansion of neurogenesis and patterning cerebral cortex folds.

Astrocyte reprogramming / activation and brain homeostasis

Astrocytes are multifunctional glial cells, implicated in neurogenesis and synaptogenesis, supporting and fine-tuning neuronal activity and maintaining brain homeostasis by controlling blood-brain barrier permeability. During the last years a number of studies have shown that astrocytes can also be converted into neurons if they force-express neurogenic transcription factors or miRNAs. Direct astrocytic reprogramming to induced-neurons (iNs) is a powerful approach for manipulating cell fate, as it takes advantage of the intrinsic neural stem cell (NSC) potential of brain resident reactive astrocytes. To this end, astrocytic cell fate conversion to iNs has been well-established in vitro and in vivo using combinations of transcription factors (TFs) or chemical cocktails. Challenging the expression of lineage-specific TFs is accompanied by changes in the expression of miRNAs, that post-transcriptionally modulate high numbers of neurogenesis-promoting factors and have therefore been introduced, supplementary or alternatively to TFs, to instruct direct neuronal reprogramming. The neurogenic miRNA miR-124 has been employed in direct reprogramming protocols supplementary to neurogenic TFs and other miRNAs to enhance direct neurogenic conversion by suppressing multiple non-neuronal targets. In our group we aimed to investigate whether miR-124 is sufficient to drive direct reprogramming of astrocytes to induced-neurons (iNs) on its own both in vitro and in vivo and elucidate its independent mechanism of reprogramming action. Our in vitro data indicate that miR-124 is a potent driver of the reprogramming switch of astrocytes towards an immature neuronal fate. Elucidation of the molecular pathways being triggered by miR-124 by RNA-seq analysis revealed that miR-124 is sufficient to instruct reprogramming of cortical astrocytes to immature induced-neurons (iNs) in vitro by down-regulating genes with important regulatory roles in astrocytic function. Among these, the RNA binding protein Zfp36l1, implicated in ARE-mediated mRNA decay, was found to be a direct target of miR-124, that be its turn targets neuronal-specific proteins participating in cortical development, which get de-repressed in miR-124-iNs. Furthermore, miR-124 is potent to guide direct neuronal reprogramming of reactive astrocytes to iNs of cortical identity following cortical trauma, a novel finding confirming its robust reprogramming action within the cortical microenvironment under neuroinflammatory conditions. In parallel to their reprogramming properties, astrocytes also participate in the maintenance of blood-brain barrier integrity, which ensures the physiological functioning of the central nervous system and gets affected contributing to the pathology of several neurodegenerative diseases. To study in real time the dynamic physical interactions of astrocytes with brain vasculature under homeostatic and pathological conditions, we performed 2-photon brain intravital imaging in a mouse model of systemic neuroinflammation, known to trigger astrogliosis and microgliosis and to evoke changes in astrocytic contact with brain vasculature. Our in vivo findings indicate that following neuroinflammation the endfeet of activated perivascular astrocytes lose their close proximity and physiological cross-talk with vasculature, however this event is at compensated by the cross-talk of astrocytes with activated microglia, safeguarding blood vessel coverage and maintenance of blood-brain integrity.

ALBA webinar series - Breaking down the ivory tower: Ep. 4 Maria José Diógenes

With this webinar series, the ALBA Disability & Accessibility Working Group aims to bring down the ivory tower of ableism among the brain research community, one extraordinary neuroscientist at a time. These webinars give a platform to scientists with disabilities across the globe and neuroscience disciplines, while reflecting on how to promote inclusive working environments and accessibility to research. For this 4th episode, Dr. Maria José Diógenes (iMM - ULisboa, PT) will talk about how her personal story changed her professional life: from the pharmacy to the laboratory bench and from ageing to Rett Syndrome.

Gut/Body interactions in health and disease

The adult intestine is a major barrier epithelium and coordinator of multi-organ functions. Stem cells constantly repair the intestinal epithelium by adjusting their proliferation and differentiation to tissue intrinsic as well as micro- and macro-environmental signals. How these signals integrate to control intestinal and whole-body homeostasis is largely unknown. Addressing this gap in knowledge is central to an improved understanding of intestinal pathophysiology and its systemic consequences. Combining Drosophila and mammalian model systems my laboratory has discovered fundamental mechanisms driving intestinal regeneration and tumourigenesis and outlined complex inter-organ signaling regulating health and disease. During my talk, I will discuss inter-related areas of research from my lab, including:1- Interactions between the intestine and its microenvironment influencing intestinal regeneration and tumourigenesis. 2- Long-range signals from the intestine impacting whole-body in health and disease.

Neuroinflammation in Epilepsy: what have we learned from human brain tissue specimens ?

Epileptogenesis is a gradual and dynamic process leading to difficult-to-treat seizures. Several cellular, molecular, and pathophysiologic mechanisms, including the activation of inflammatory processes.  The use of human brain tissue represents a crucial strategy to advance our understanding of the underlying neuropathology and the molecular and cellular basis of epilepsy and related cognitive and behavioral comorbidities,  The mounting evidence obtained during the past decade has emphasized the critical role of inflammation  in the pathophysiological processes implicated in a large spectrum of genetic and acquired forms of  focal epilepsies. Dissecting the cellular and molecular mediators of  the pathological immune responses and their convergent and divergent mechanisms, is a major requisite for delineating their role in the establishment of epileptogenic networks. The role of small regulatory molecules involved in the regulation of  specific pro- and anti-inflammatory pathways  and the crosstalk between neuroinflammation and oxidative stress will be addressed.    The observations supporting the activation of both innate and adaptive immune responses in human focal epilepsy will be discussed and elaborated, highlighting specific inflammatory pathways as potential targets for antiepileptic, disease-modifying therapeutic strategies.

Cellular crosstalk in Neurodevelopmental Disorders

Cellular crosstalk is an essential process during brain development and it is influenced by numerous factors, including the morphology of the cells, their adhesion molecules, the local extracellular matrix and the secreted vesicles. Inspired by mutations associated with neurodevelopmental disorders, we focus on understanding the role of extracellular mechanisms essential for the correct development of the human brain. Hence, we combine the in vivo mouse model and the in vitro human-derived neurons, cerebral organoids, and dorso-ventral assembloids in order to better comprehend the molecular and cellular mechanisms involved in ventral progenitors’ proliferation and fate as well as migration and maturation of inhibitory neurons during human brain development and tackle the causes of neurodevelopmental disorders. We particularly focus on mutations in genes influencing cell-cell contacts, extracellular matrix, and secretion of vesicles and therefore study intrinsic and extrinsic mechanisms contributing to the formation of the brain. Our data reveal an important contribution of cell non-autonomous mechanisms in the development of neurodevelopmental disorders.

Sex hormone regulation of neural gene expression

Gonadal steroid hormones are the principal drivers of sex-variable biology in vertebrates. In the brain, estrogen (17β-estradiol) establishes neural sex differences in many species and modulates mood, behavior, and energy balance in adulthood. To understand the diverse effects of estradiol on the brain, we profiled the genomic binding of estrogen receptor alpha (ERα), providing the first picture of the neural actions of any gonadal hormone receptor. To relate ERα target genes to brain sex differences we assessed gene expression and chromatin accessibility in the posterior bed nucleus of the stria terminalis (BNSTp), a sexually dimorphic node in limbic circuitry that underlies sex-differential social behaviors such as aggression and parenting. In adult animals we observe that levels of ERα are predictive of the extent of sex-variable gene expression, and that these sex differences are a dynamic readout of acute hormonal state. In neonates we find that transient ERα recruitment at birth leads to persistent chromatin opening and male-biased gene expression, demonstrating a true epigenetic mechanism for brain sexual differentiation. Collectively, our findings demonstrate that sex differences in gene expression in the brain are a readout of state-dependent hormone receptor actions, rather than other factors such as sex chromosomes. We anticipate that the ERα targets we have found will contribute to established sex differences in the incidence and etiology of neurological and psychiatric disorders.

Freeze or flee ? New insights from rodent models of autism

Individuals afflicted with certain types of autism spectrum disorder often exhibit impaired cognitive function alongside enhanced emotional symptoms and mood lability. However, current understanding of the pathogenesis of autism and intellectual disabilities is based primarily on studies in the hippocampus and cortex, brain areas involved in cognitive function. But, these disorders are also associated with strong emotional symptoms, which are likely to involve changes in the amygdala and other brain areas. In this talk I will highlight these issues by presenting analyses in rat models of ASD/ID lacking Nlgn3 and Frm1 (causing Fragile X Syndrome). In addition to identifying new circuit and cellular alterations underlying divergent patterns of fear expression, these findings also suggest novel therapeutic strategies.

Regulation of Cerebral Cortex Morphogenesis by Migrating Cells

Epigenetic rewiring in Schinzel-Giedion syndrome

During life, a variety of specialized cells arise to grant the right and timely corrected functions of tissues and organs. Regulation of chromatin in defining specialized genomic regions (e.g. enhancers) plays a key role in developmental transitions from progenitors into cell lineages. These enhancers, properly topologically positioned in 3D space, ultimately guide the transcriptional programs. It is becoming clear that several pathologies converge in differential enhancer usage with respect to physiological situations. However, why some regulatory regions are physiologically preferred, while some others can emerge in certain conditions, including other fate decisions or diseases, remains obscure. Schinzel-Giedion syndrome (SGS) is a rare disease with symptoms such as severe developmental delay, congenital malformations, progressive brain atrophy, intractable seizures, and infantile death. SGS is caused by mutations in the SETBP1 gene that results in its accumulation further leading to the downstream accumulation of SET. The oncoprotein SET has been found as part of the histone chaperone complex INHAT that blocks the activity of histone acetyltransferases suggesting that SGS may (i) represent a natural model of alternative chromatin regulation and (ii) offer chances to study downstream (mal)adaptive mechanisms. I will present our work on the characterization of SGS in appropriate experimental models including iPSC-derived cultures and mouse.

Establishment and aging of the neuronal DNA methylation landscape in the hippocampus

The hippocampus is a brain region with key roles in memory formation, cognitive flexibility and emotional control. Yet hippocampal function is impaired severely during aging and in neurodegenerative diseases, and impairments in hippocampal function underlie age-related cognitive decline. Accumulating evidence suggests that the deterioration of the neuron-specific epigenetic landscape during aging contributes to their progressive, age-related dysfunction. For instance, we have recently shown that aging is associated with pronounced alterations of neuronal DNA methylation patterns in the hippocampus. Because neurons are generated mostly during development with limited replacement in the adult brain, they are particularly long-lived cells and have to maintain their cell-type specific gene expression programs life-long in order to preserve brain function. Understanding the epigenetic mechanisms that underlie the establishment and long-term maintenance of neuron-specific gene expression programs, will help us to comprehend the sources and consequences of their age-related deterioration. In this talk, I will present our recent work that investigated the role of DNA methylation in the establishment of neuronal gene expression programs and neuronal function, using adult neurogenesis in the hippocampus as a model. I will then describe the effects of aging on the DNA methylation landscape in the hippocampus and discuss the malleability of the aging neuronal methylome to lifestyle and environmental stimulation.

Nature over Nurture: Functional neuronal circuits emerge in the absence of developmental activity

During development, the complex neuronal circuitry of the brain arises from limited information contained in the genome. After the genetic code instructs the birth of neurons, the emergence of brain regions, and the formation of axon tracts, it is believed that neuronal activity plays a critical role in shaping circuits for behavior. Current AI technologies are modeled after the same principle: connections in an initial weight matrix are pruned and strengthened by activity-dependent signals until the network can sufficiently generalize a set of inputs into outputs. Here, we challenge these learning-dominated assumptions by quantifying the contribution of neuronal activity to the development of visually guided swimming behavior in larval zebrafish. Intriguingly, dark-rearing zebrafish revealed that visual experience has no effect on the emergence of the optomotor response (OMR). We then raised animals under conditions where neuronal activity was pharmacologically silenced from organogenesis onward using the sodium-channel blocker tricaine. Strikingly, after washout of the anesthetic, animals performed swim bouts and responded to visual stimuli with 75% accuracy in the OMR paradigm. After shorter periods of silenced activity OMR performance stayed above 90% accuracy, calling into question the importance and impact of classical critical periods for visual development. Detailed quantification of the emergence of functional circuit properties by brain-wide imaging experiments confirmed that neuronal circuits came ‘online’ fully tuned and without the requirement for activity-dependent plasticity. Thus, contrary to what you learned on your mother's knee, complex sensory guided behaviors can be wired up innately by activity-independent developmental mechanisms.

Integration of 3D human stem cell models derived from post-mortem tissue and statistical genomics to guide schizophrenia therapeutic development

Schizophrenia is a neuropsychiatric disorder characterized by positive symptoms (such as hallucinations and delusions), negative symptoms (such as avolition and withdrawal) and cognitive dysfunction1. Schizophrenia is highly heritable, and genetic studies are playing a pivotal role in identifying potential biomarkers and causal disease mechanisms with the hope of informing new treatments. Genome-wide association studies (GWAS) identified nearly 270 loci with a high statistical association with schizophrenia risk; however each locus confers only a small increase in risk therefore it is difficult to translate these findings into understanding disease biology that can lead to treatments. Induced pluripotent stem cell (iPSC) models are a tractable system to translate genetic findings and interrogate mechanisms of pathogenesis. Mounting research with patient-derived iPSCs has proposed several neurodevelopmental pathways altered in SCZ, such as neural progenitor cell (NPC) proliferation, imbalanced differentiation of excitatory and inhibitory cortical neurons. However, it is unclear what exactly these iPS models recapitulate, how potential perturbations of early brain development translates into illness in adults and how iPS models that represent fetal stages can be utilized to further drug development efforts to treat adult illness. I will present the largest transcriptome analysis of post-mortem caudate nucleus in schizophrenia where we discovered that decreased presynaptic DRD2 autoregulation is the causal dopamine risk factor for schizophrenia (Benjamin et al, Nature Neuroscience 2022 https://doi.org/10.1038/s41593-022-01182-7). We developed stem cell models from a subset of the postmortem cohort to better understand the molecular underpinnings of human psychiatric disorders (Sawada et al, Stem Cell Research 2020). We established a method for the differentiation of iPS cells into ventral forebrain organoids and performed single cell RNAseq and cellular phenotyping. To our knowledge, this is the first study to evaluate iPSC models of SZ from the same individuals with postmortem tissue. Our study establishes that striatal neurons in the patients with SCZ carry abnormalities that originated during early brain development. Differentiation of inhibitory neurons is accelerated whereas excitatory neuronal development is delayed, implicating an excitation and inhibition (E-I) imbalance during early brain development in SCZ. We found a significant overlap of genes upregulated in the inhibitory neurons in SCZ organoids with upregulated genes in postmortem caudate tissues from patients with SCZ compared with control individuals, including the donors of our iPS cell cohort. Altogether, we demonstrate that ventral forebrain organoids derived from postmortem tissue of individuals with schizophrenia recapitulate perturbed striatal gene expression dynamics of the donors’ brains (Sawada et al, biorxiv 2022 https://doi.org/10.1101/2022.05.26.493589).

Neuron-glial interactions in health and disease: from cognition to cancer

In the central nervous system, neuronal activity is a critical regulator of development and plasticity. Activity-dependent proliferation of healthy glial progenitors, oligodendrocyte precursor cells (OPCs), and the consequent generation of new oligodendrocytes contributes to adaptive myelination. This plasticity of myelin tunes neural circuit function and contributes to healthy cognition. The robust mitogenic effect of neuronal activity on normal oligodendroglial precursor cells, a putative cellular origin for many forms of glioma, suggests that dysregulated or “hijacked” mechanisms of myelin plasticity might similarly promote malignant cell proliferation in this devastating group of brain cancers. Indeed, neuronal activity promotes progression of both high-grade and low-grade glioma subtypes in preclinical models. Crucial mechanisms mediating activity-regulated glioma growth include paracrine secretion of BDNF and the synaptic protein neuroligin-3 (NLGN3). NLGN3 induces multiple oncogenic signaling pathways in the cancer cell, and also promotes glutamatergic synapse formation between neurons and glioma cells. Glioma cells integrate into neural circuits synaptically through neuron-to-glioma synapses, and electrically through potassium-evoked currents that are amplified through gap-junctional coupling between tumor cells This synaptic and electrical integration of glioma into neural circuits is central to tumor progression in preclinical models. Thus, neuron-glial interactions not only modulate neural circuit structure and function in the healthy brain, but paracrine and synaptic neuron-glioma interactions also play important roles in the pathogenesis of glial cancers. The mechanistic parallels between normal and malignant neuron-glial interactions underscores the extent to which mechanisms of neurodevelopment and plasticity are subverted by malignant gliomas, and the importance of understanding the neuroscience of cancer.

Brain mosaicism in epileptogenic cortical malformations

Focal Cortical Dysplasia (FCD) is the most common focal cortical malformation leading to intractable childhood focal epilepsy. In recent years, we and others have shown that FCD type II is caused by mosaic mutations in genes within the PI3K-AKT-mTOR-signaling pathway. Hyperactivation of the mTOR pathway accounts for neuropathological abnormalities and seizure occurrence in FCD. We further showed from human surgical FCDII tissue that epileptiform activity correlates with the density of mutated dysmorphic neurons, supporting their pro-epileptogenic role. The level of mosaicism, as defined by variant allele frequency (VAF) is thought to correlate with the size and regional brain distribution of the lesion such that when a somatic mutation occurs early during the cortical development, the dysplastic area is smaller than if it occurs later. Novel approaches based on the detection of cell-free DNA from the CSF and from trace tissue adherent to SEEG electrodes promise future opportunities for genetic testing during the presurgical evaluation of refractory epilepsy patients or in those that are not eligible for surgery. In utero-based electroporation mouse models allow to express somatic mutation during neurodevelopment and recapitulate most neuropathological and clinical features of FCDII, establishing relevant preclinical mouse models for developing precision medicine strategies.

Microglial efferocytosis: Diving into the Alzheimer's Disease gene pool

Genome-wide association studies and functional genomics studies have linked specific cell types, genes, and pathways to Alzheimer’s disease (AD) risk. In particular, AD risk alleles primarily affect the abundance or structure, and thus the activity, of genes expressed in macrophages, strongly implicating microglia (the brain-resident macrophages) in the etiology of AD. These genes converge on pathways (endocytosis/phagocytosis, cholesterol metabolism, and immune response) with critical roles in core macrophage functions such as efferocytosis. Here, we review these pathways, highlighting relevant genes identified in the latest AD genetics and genomics studies, and describe how they may contribute to AD pathogenesis. Investigating the functional impact of AD-associated variants and genes in microglia is essential for elucidating disease risk mechanisms and developing effective therapeutic approaches." https://doi.org/10.1016/j.neuron.2022.10.015

Indispensable for generating epileptic seizures: where, when, how?

In epilepsy research, a holy grail has been the identification and understanding of the "epileptogenic zone" - operationally defined as the (minimal) area or region of the brain is indispensible for the generation of epileptic seizures. The identification of the epileptogenic zone is particularly important for surgical treatments of focal epilepsy patients, but I will highlight some recent clinical, experimental and theoretical work showing that it is also fundamentally linked with our understanding of epilepsy and seizures. I will conclude with a proposal for an updated understanding of the epileptogenic zone and ictogenesis.

Circuit solutions for programming actions

The hippocampus is one of the few regions in the adult mammalian brain which is endowed with life-long neurogenesis. Despite intense investigation, it remains unclear how neurons newly-generated may retain unique functions that contribute to modulate hippocampal information processing and cognition. In this talk, I will present some recent findings revealing how enhanced forms of plasticity in adult-born neurons underlie the way they become incorporated into pre-existing networks in response to experience.

What shapes the transcriptional identity of a neuron?

Within the vertebrate neocortex and other telencephalic structures, molecularly-defined neurons tend to segregate at first order into GABAergic types and glutamatergic types. Two fundamental questions arise: (1) do non-telencephalic neurons similarly segregate by neurotransmitter status, and (2) do GABAergic (or glutamatergic) types sampled in different structures share many molecular features in common, beyond the few genes directly responsible for neurotransmitter synthesis and release? To address these questions, we used single-nucleus RNA sequencing, analyzing over 2.4 million brain cells sampled from 16 locations in a primate (the common marmoset). Unexpectedly, we find the answer to both is “no”. I will discuss implications for generalizing associations between neurotransmitter utilization and other phenotypes, and share ongoing efforts to map the biodistributions of cell types in the primate brain.

Hidden nature of seizures

How seizures emerge from the abnormal dynamics of neural networks within the epileptogenic tissue remains an enigma. Are seizures random events, or do detectable changes in brain dynamics precede them? Are mechanisms of seizure emergence identical at the onset and later stages of epilepsy? Is the risk of seizure occurrence stable, or does it change over time? A myriad of questions about seizure genesis remains to be answered to understand the core principles governing seizure genesis. The last decade has brought unprecedented insights into the complex nature of seizure emergence. It is now believed that seizure onset represents the product of the interactions between the process of a transition to seizure, long-term fluctuations in seizure susceptibility, epileptogenesis, and disease progression. During the lecture, we will review the latest observations about mechanisms of ictogenesis operating at multiple temporal scales. We will show how the latest observations contribute to the formation of a comprehensive theory of seizure genesis, and challenge the traditional perspectives on ictogenesis. Finally, we will discuss how combining conventional approaches with computational modeling, modern techniques of in vivo imaging, and genetic manipulation open prospects for exploration of yet hidden mechanisms of seizure genesis.

Development and evolution of neuronal connectivity

In most animal species including humans, commissural axons connect neurons on the left and right side of the nervous system. In humans, abnormal axon midline crossing during development causes a whole range of neurological disorders ranging from congenital mirror movements, horizontal gaze palsy, scoliosis or binocular vision deficits. The mechanisms which guide axons across the CNS midline were thought to be evolutionary conserved but our recent results suggesting that they differ across vertebrates.  I will discuss the evolution of visual projection laterality during vertebrate evolution.  In most vertebrates, camera-style eyes contain retinal ganglion cell (RGC) neurons projecting to visual centers on both sides of the brain. However, in fish, RGCs are thought to only innervate the contralateral side. Using 3D imaging and tissue clearing we found that bilateral visual projections exist in non-teleost fishes. We also found that the developmental program specifying visual system laterality differs between fishes and mammals. We are currently using various strategies to discover genes controlling the development of visual projections. I will also present ongoing work using 3D imaging techniques to study the development of the visual system in human embryo.

Chandelier cells shine a light on the emergence of GABAergic circuits in the cortex

GABAergic interneurons are chiefly responsible for controlling the activity of local circuits in the cortex. Chandelier cells (ChCs) are a type of GABAergic interneuron that control the output of hundreds of neighbouring pyramidal cells through axo-axonic synapses which target the axon initial segment (AIS). Despite their importance in modulating circuit activity, our knowledge of the development and function of axo-axonic synapses remains elusive. We have investigated the emergence and plasticity of axo-axonic synapses in layer 2/3 of the somatosensory cortex (S1) and found that ChCs follow what appear to be homeostatic rules when forming synapses with pyramidal neurons. We are currently implementing in vivo techniques to image the process of axo-axonic synapse formation during development and uncover the dynamics of synaptogenesis and pruning at the AIS. In addition, we are using an all-optical approach to both activate and measure the activity of chandelier cells and their postsynaptic partners in the primary visual cortex (V1) and somatosensory cortex (S1) in mice, also during development. We aim to provide a structural and functional description of the emergence and plasticity of a GABAergic synapse type in the cortex.

Epigenome regulation in neocortex expansion and generation of neuronal subtypes

Evolutionarily, the expansion of the human neocortex accounts for many of the unique cognitive abilities of humans. This expansion appears to reflect the increased proliferative potential of basal progenitors (BPs) in mammalian evolution. Further cortical progenitors generate both glutamatergic excitatory neurons (ENs) and GABAergic inhibitory interneurons (INs) in human cortex, whereas they produce exclusively ENs in rodents. The increased proliferative capacity and neuronal subtype generation of cortical progenitors in mammalian evolution may have evolved through epigenetic alterations. However, whether or how the epigenome in cortical progenitors differs between humans and other species is unknown. Here, we report that histone H3 acetylation is a key epigenetic regulation in BP profiling of sorted BPs, we show that H3K9 acetylation is low in murine BPs and high in amplification, neuronal subtype generation and cortical expansion. Through epigenetic profiling of sorted BPs, we show that H3K9 acetylation is low in murine BPs and high in human BPs. Elevated H3K9ac preferentially increases BP proliferation, increasing the size and folding of the normally smooth mouse neocortex. Furthermore, we found that the elevated H3 acetylation activates expression of IN genes in in developing mouse cortex and promote proliferation of IN progenitor-like cells in cortex of Pax6 mutant mouse models. Mechanistically, H3K9ac drives the BP amplification and proliferation of these IN progenitor-like cells by increasing expression of the evolutionarily regulated gene, TRNP1. Our findings demonstrate a previously unknown mechanism that controls neocortex expansion and generation of neuronal subtypes. Keywords: Cortical development, neurogenesis, basal progenitors, cortical size, gyrification, excitatory neuron, inhibitory interneuron, epigenetic profiling, epigenetic regulation, H3 acetylation, H3K9ac, TRNP1, PAX6

Linking GWAS to pharmacological treatments for psychiatric disorders

Genome-wide association studies (GWAS) have identified multiple disease-associated genetic variations across different psychiatric disorders raising the question of how these genetic variants relate to the corresponding pharmacological treatments. In this talk, I will outline our work investigating whether functional information from a range of open bioinformatics datasets such as protein interaction network (PPI), brain eQTL, and gene expression pattern across the brain can uncover the relationship between GWAS-identified genetic variation and the genes targeted by current drugs for psychiatric disorders. Focusing on four psychiatric disorders---ADHD, bipolar disorder, schizophrenia, and major depressive disorder---we assess relationships between the gene targets of drug treatments and GWAS hits and show that while incorporating information derived from functional bioinformatics data, such as the PPI network and spatial gene expression, can reveal links for bipolar disorder, the overall correspondence between treatment targets and GWAS-implicated genes in psychiatric disorders rarely exceeds null expectations. This relatively low degree of correspondence across modalities suggests that the genetic mechanisms driving the risk for psychiatric disorders may be distinct from the pathophysiological mechanisms used for targeting symptom manifestations through pharmacological treatments and that novel approaches for understanding and treating psychiatric disorders may be required.

Role of ASD risk genes on maturation of frontal-sensory cognitive control circuit

At the nexus of genes, aging and environment: Understanding transcriptomic and epigenomic regulation in Parkinson's disease

Parkinson’s Disease (PD), the most common neurodegenerative movement disorder, is based on a complex interplay between genetic predispositions, aging processes, and environmental influences. In order to better understand the gene-environment axis in PD, we pursue a multi-omics approach to comprehensively interrogate genome-wide changes in histone modifications, DNA methylation, and hydroxymethylation, accompanied by transcriptomic profiling in cell and animal models of PD as well as large patient cohorts. Furthermore, we assess the plasticity of epigenomic modifications under influence of environmental factors using longitudinal cohorts of sporadic PD cases as well as mouse models exposed to specific environmental factors. Here, we present gene expression changes in PD mouse models in context of aging as well as environmental enrichment and high-fat diet.

Don't forget the gametes: Neurodevelopmental pathogenesis starts in the sperm and egg

Proper development of the nervous system depends not only on the inherited DNA sequence, but also on proper regulation of gene expression, as controlled in part by epigenetic mechanisms present in the parental gametes. In this presentation an internationally recognized research advocate explains why researchers concerned about the origins of increasingly prevalent neurodevelopmental disorders such as autism and attention deficit hyperactivity disorder should look beyond genetics in probing the origins of dysregulated transcription of brain-related genes. The culprit for a subset of cases, she contends, may lie in the exposure history of the parents, and thus their germ cells. To illustrate how environmentally informed, nongenetic dysfunction may occur, she focuses on the example of parents' histories of exposure to common agents of modern inhalational anesthesia, a highly toxic exposure that in mammalian models has been seen to induce heritable neurodevelopmental abnormality in offspring born of exposed germline.

Cell-type specific genomics and transcriptomics of HIV in the brain

Exploration of genome organization and function in the HIV infected brain is critical to aid in the understanding and development of treatments for HIV-associated neurocognitive disorder (HAND). Here, we applied a multiomic approach, including single nuclei transcriptomics, cell-type specific Hi-C 3D genome mapping, and viral integration site sequencing (IS-seq) to frontal lobe tissue from HIV-infected individuals with encephalitis (HIVE) and without encephalitis (HIV+). We observed reorganization of open/repressive (A/B) compartment structures in HIVE microglia encompassing 6.4% of the genome with enrichment for regions containing interferon (IFN) pathway genes. 3D genome remodeling was associated with transcriptomic reprogramming, including down-regulation of cell adhesion and synapse-related functions and robust activation of IFN signaling and cell migratory pathways, and was recapitulated by IFN-g stimulation of cultured microglial cells. Microglia from HIV+ brains showed, to a lesser extent, similar transcriptional alterations. IS-seq recovered 1,221 integration sites in the brain that were enriched for chromosomal domains newly mobilized into a permissive chromatin environment in HIVE microglia. Viral transcription, which was detected in 0.003% of all nuclei in HIVE brain, occurred in a subset of highly activated microglia that drove differential expression in HIVE. Thus, we observed a dynamic interrelationship of interferon-associated 3D genome and transcriptome remodeling with HIV integration and transcription in the brain.

PET imaging in brain diseases

Talk 1. PET based biomarkers of treatment efficacy in temporal lobe epilepsy A critical aspect of drug development involves identifying robust biomarkers of treatment response for use as surrogate endpoints in clinical trials. However, these biomarkers also have the capacity to inform mechanisms of disease pathogenesis and therapeutic efficacy. In this webinar, Dr Bianca Jupp will report on a series of studies using the GABAA PET ligand, [18F]-Flumazenil, to establish biomarkers of treatment response to a novel therapeutic for temporal lobe epilepsy, identifying affinity at this receptor as a key predictor of treatment outcome. Dr Bianca Jupp is a Research Fellow in the Department of Neuroscience, Monash University and Lead PET/CT Scientist at the Alfred Research Alliance–Monash Biomedical Imaging facility. Her research focuses on neuroimaging and its capacity to inform the neurobiology underlying neurological and neuropsychiatric disorders. Talk 2. The development of a PET radiotracer for reparative microglia Imaging of neuroinflammation is currently hindered by the technical limitations associated with TSPO imaging. In this webinar, Dr Lucy Vivash will discuss the development of PET radiotracers that specifically image reparative microglia through targeting the receptor kinase MerTK. This includes medicinal chemistry design and testing, radiochemistry, and in vitro and in vivo testing of lead tracers. Dr Lucy Vivash is a Research Fellow in the Department of Neuroscience, Monash University. Her research focuses on the preclinical development and clinical translation of novel PET radiotracers for the imaging of neurodegenerative diseases.

Zebrafish models help untangle genetic interactions in motor neuron degeneration

Due to high homology to the human genome and rapid development, zebrafish have been successfully used to model diseases of the neuromuscular system. In this seminar, I will present current advances in modeling genetic causes of Amyotrophic Lateral Sclerosis (ALS), the most common motor neuron degeneration and show how epistatic interaction studies in zebrafish have helped elucidate synergistic effects of major ALS genes and their cellular targets.

Untitled Seminar

G. Quattrocolo: Cajal-Retzius cells in the postnatal hippocampus; F. Garcia-Moreno: Mosaic evolutionary history of brain circuits through the lens of neurogenesis

Malignant synaptic plasticity in pediatric high-grade gliomas

Pediatric high-grade gliomas (pHGG) are a devastating group of diseases that urgently require novel therapeutic options. We have previously demonstrated that pHGGs directly synapse onto neurons and the subsequent tumor cell depolarization, mediated by calcium-permeable AMPA channels, promotes their proliferation. The regulatory mechanisms governing these postsynaptic connections are unknown. Here, we investigated the role of BDNF-TrkB signaling in modulating the plasticity of the malignant synapse. BDNF ligand activation of its canonical receptor, TrkB (which is encoded for by the gene NTRK2), has been shown to be one important modulator of synaptic regulation in the normal setting. Electrophysiological recordings of glioma cell membrane properties, in response to acute neurotransmitter stimulation, demonstrate in an inward current resembling AMPA receptor (AMPAR) mediated excitatory neurotransmission. Extracellular BDNF increases the amplitude of this glutamate-induced tumor cell depolarization and this effect is abrogated in NTRK2 knockout glioma cells. Upon examining tumor cell excitability using in situ calcium imaging, we found that BDNF increases the intensity of glutamate-evoked calcium transients in GCaMP6s expressing glioma cells. Western blot analysis indicates the tumors AMPAR properties are altered downstream of BDNF induced TrkB activation in glioma. Cell membrane protein capture (via biotinylation) and live imaging of pH sensitive GFP-tagged AMPAR subunits demonstrate an increase of calcium permeable channels at the tumors postsynaptic membrane in response to BDNF. We find that BDNF-TrkB signaling promotes neuron-to-glioma synaptogenesis as measured by high-resolution confocal and electron microscopy in culture and tumor xenografts. Our analysis of published pHGG transcriptomic datasets, together with brain slice conditioned medium experiments in culture, indicates the tumor microenvironment as the chief source of BDNF ligand. Disruption of the BDNF-TrkB pathway in patient-derived orthotopic glioma xenograft models, both genetically and pharmacologically, results in an increased overall survival and reduced tumor proliferation rate. These findings suggest that gliomas leverage normal mechanisms of plasticity to modulate the excitatory channels involved in synaptic neurotransmission and they reveal the potential to target the regulatory components of glioma circuit dynamics as a therapeutic strategy for these lethal cancers.

MicroRNAs as targets in the epilepsies: hits, misses and complexes

MicroRNAs are small noncoding RNAs that provide a critical layer of gene expression control. Individual microRNAs variably exert effects across networks of genes via sequence-specific binding to mRNAs, fine-tuning protein levels. This helps coordinate the timing and specification of cell fate transitions during brain development and maintains neural circuit function and plasticity by activity-dependent (re)shaping of synapses and the levels of neurotransmitter components. MicroRNA levels have been found to be altered in tissue from the epileptogenic zone resected from adults with drug-resistant focal epilepsy and this has driven efforts to explore their therapeutic potential, in particular using antisense oligonucleotide (ASOs) inhibitors termed antimirs. Here, we review the molecular mechanisms by which microRNAs control brain excitability and the latest progress towards a microRNA-based treatment for temporal lobe epilepsy. We also look at whether microRNA-based approaches could be used to treat genetic epilepsies, correcting individual genes or dysregulated pathways. Finally, we look at how cells have evolved to maximise the efficiency of the microRNA system via RNA editing, where single base changes is capable of altering the repertoire of genes under the control of a single microRNA. The findings improve our understanding of the molecular landscape of the epileptic brain and may lead to new therapies.

The Synaptome Architecture of the Brain: Lifespan, disease, evolution and behavior

The overall aim of my research is to understand how the organisation of the synapse, with particular reference to the postsynaptic proteome (PSP) of excitatory synapses in the brain, informs the fundamental mechanisms of learning, memory and behaviour and how these mechanisms go awry in neurological dysfunction. The PSP indeed bears a remarkable burden of disease, with components being disrupted in disorders (synaptopathies) including schizophrenia, depression, autism and intellectual disability. Our work has been fundamental in revealing and then characterising the unprecedented complexity (>1000 highly conserved proteins) of the PSP in terms of the subsynaptic architecture of postsynaptic proteins such as PSD95 and how these proteins assemble into complexes and supercomplexes in different neurons and regions of the brain. Characterising the PSPs in multiple species, including human and mouse, has revealed differences in key sets of functionally important proteins, correlates with brain imaging and connectome data, and a differential distribution of disease-relevant proteins and pathways. Such studies have also provided important insight into synapse evolution, establishing that vertebrate behavioural complexity is a product of the evolutionary expansion in synapse proteomes that occurred ~500 million years ago. My lab has identified many mutations causing cognitive impairments in mice before they were found to cause human disorders. Our proteomic studies revealed that >130 brain diseases are caused by mutations affecting postsynaptic proteins. We uncovered mechanisms that explain the polygenic basis and age of onset of schizophrenia, with postsynaptic proteins, including PSD95 supercomplexes, carrying much of the polygenic burden. We discovered the “Genetic Lifespan Calendar”, a genomic programme controlling when genes are regulated. We showed that this could explain how schizophrenia susceptibility genes are timed to exert their effects in young adults. The Genes to Cognition programme is the largest genetic study so far undertaken into the synaptic molecular mechanisms underlying behaviour and physiology. We made important conceptual advances that inform how the repertoire of both innate and learned behaviours is built from unique combinations of postsynaptic proteins that either amplify or attenuate the behavioural response. This constitutes a key advance in understanding how the brain decodes information inherent in patterns of nerve impulses, and provides insight into why the PSP has evolved to be so complex, and consequently why the phenotypes of synaptopathies are so diverse. Our most recent work has opened a new phase, and scale, in understanding synapses with the first synaptome maps of the brain. We have developed next-generation methods (SYNMAP) that enable single-synapse resolution molecular mapping across the whole mouse brain and extensive regions of the human brain, revealing the molecular and morphological features of a billion synapses. This has already uncovered unprecedented spatiotemporal synapse diversity organised into an architecture that correlates with the structural and functional connectomes, and shown how mutations that cause cognitive disorders reorganise these synaptome maps; for example, by detecting vulnerable synapse subtypes and synapse loss in Alzheimer’s disease. This innovative synaptome mapping technology has huge potential to help characterise how the brain changes during normal development, including in specific cell types, and with degeneration, facilitating novel pathways to diagnosis and therapy.

A transcriptomic axis predicts state modulation of cortical interneurons

Transcriptomics has revealed that cortical inhibitory neurons exhibit a great diversity of fine molecular subtypes, but it is not known whether these subtypes have correspondingly diverse activity patterns in the living brain. We show that inhibitory subtypes in primary visual cortex (V1) have diverse correlates with brain state, but that this diversity is organized by a single factor: position along their main axis of transcriptomic variation. We combined in vivo 2-photon calcium imaging of mouse V1 with a novel transcriptomic method to identify mRNAs for 72 selected genes in ex vivo slices. We classified inhibitory neurons imaged in layers 1-3 into a three-level hierarchy of 5 Subclasses, 11 Types, and 35 Subtypes using previously-defined transcriptomic clusters. Responses to visual stimuli differed significantly only across Subclasses, suppressing cells in the Sncg Subclass while driving cells in the other Subclasses. Modulation by brain state differed at all hierarchical levels but could be largely predicted from the first transcriptomic principal component, which also predicted correlations with simultaneously recorded cells. Inhibitory Subtypes that fired more in resting, oscillatory brain states have less axon in layer 1, narrower spikes, lower input resistance and weaker adaptation as determined in vitro and express more inhibitory cholinergic receptors. Subtypes firing more during arousal had the opposite properties. Thus, a simple principle may largely explain how diverse inhibitory V1 Subtypes shape state-dependent cortical processing.

Transcriptional adaptation couples past experience and future sensory responses

Animals traversing different environments encounter both stable background stimuli and novel cues, which are generally thought to be detected by primary sensory neurons and then distinguished by downstream brain circuits. Sensory adaptation is a neural mechanism that filters background by minimizing responses to stable sensory stimuli, and a fundamental feature of sensory systems. Adaptation over relatively fast timescales (milliseconds to minutes) have been reported in many sensory systems. However, adaptation to persistent environmental stimuli over longer timescales (hours to days) have been largely unexplored, even though those timescales are ethologically important since animals typically stay in one environment for hours. I showed that each of the ~1,000 olfactory sensory neuron (OSN) subtypes in the mouse harbors a distinct transcriptome whose content is precisely determined by interactions between its odorant receptor and the environment. This transcriptional variation is systematically organized to support sensory adaptation: expression levels of many genes relevant to transforming odors into spikes continuously vary across OSN subtypes, dynamically adjust to new environments over hours, and accurately predict acute OSN-specific odor responses. The sensory periphery therefore separates salient signals from predictable background via a transcriptional mechanism whose moment-to-moment state reflects the past and constrains the future; these findings suggest a general model in which structured transcriptional variation within a cell type reflects individual experience.

Two lessons from experimental models of generalized absence epilepsy, myelin plasticity dependent epileptogenesis, and circuits of cognitive comorbidities

Epileptogenesis in the developing brain:understanding a moving target

The origins, mechanisms and consequences of epilepsy in the developing brain are incompletely understood. Many developmental epilepsies have a genetic basis and their mechanisms stem from deficits in the function of one or numerous genes. Others, such as those that follow prolonged febrile seizures or severe birth asphyxia in a ‘normal’ brain may depend on the interaction of the insult with the rapidly evolving brain cells and circuits. Yet, how early-life insults may provoke epilepsy is unclear, and requires multiple levels of analysis: behavior, circuits, cells [neurons, glia] and molecules. Here we discuss developmental epileptogenesis, addressing some of its special features: the epilepsy phenotype, the effects insults on the maturation of brain circuits, the role of neuron-glia-neuron communication in cellular and circuit refinement, and how transient epileptogenic insults provoke enduring changes in the structure, connectivity and function of salient neuronal populations. We will highlight resolved questions- and the many unresolved issues that require tackling in 2022 and beyond.

Mapping the Dynamics of the Linear and 3D Genome of Single Cells in the Developing Brain

Three intimately related dimensions of the mammalian genome—linear DNA sequence, gene transcription, and 3D genome architecture—are crucial for the development of nervous systems. Changes in the linear genome (e.g., de novo mutations), transcriptome, and 3D genome structure lead to debilitating neurodevelopmental disorders, such as autism and schizophrenia. However, current technologies and data are severely limited: (1) 3D genome structures of single brain cells have not been solved; (2) little is known about the dynamics of single-cell transcriptome and 3D genome after birth; (3) true de novo mutations are extremely difficult to distinguish from false positives (DNA damage and/or amplification errors). Here, I filled in this longstanding technological and knowledge gap. I recently developed a high-resolution method—diploid chromatin conformation capture (Dip-C)—which resolved the first 3D structure of the human genome, tackling a longstanding problem dating back to the 1880s. Using Dip-C, I obtained the first 3D genome structure of a single brain cell, and created the first transcriptome and 3D genome atlas of the mouse brain during postnatal development. I found that in adults, 3D genome “structure types” delineate all major cell types, with high correlation between chromatin A/B compartments and gene expression. During development, both transcriptome and 3D genome are extensively transformed in the first month of life. In neurons, 3D genome is rewired across scales, correlated with gene expression modules, and independent of sensory experience. Finally, I examined allele-specific structure of imprinted genes, revealing local and chromosome-wide differences. More recently, I expanded my 3D genome atlas to the human and mouse cerebellum—the most consistently affected brain region in autism. I uncovered unique 3D genome rewiring throughout life, providing a structural basis for the cerebellum’s unique mode of development and aging. In addition, to accurately measure de novo mutations in a single cell, I developed a new method—multiplex end-tagging amplification of complementary strands (META-CS), which eliminates nearly all false positives by virtue of DNA complementarity. Using META-CS, I determined the true mutation spectrum of single human brain cells, free from chemical artifacts. Together, my findings uncovered an unknown dimension of neurodevelopment, and open up opportunities for new treatments for autism and other developmental disorders.

Learning binds novel inputs into functional synaptic clusters via spinogenesis

Learning is known to induce the formation of new dendritic spines, but despite decades of effort, the functional properties of new spines in vivo remain unknown. Here, using a combination of longitudinal in vivo 2-photon imaging of the glutamate reporter, iGluSnFR, and correlated electron microscopy (CLEM) of dendritic spines on the apical dendrites of L2/3 excitatory neurons in the motor cortex during motor learning, we describe a framework of new spines' formation, survival, and resulting function. Specifically, our data indicate that the potentiation of a subset of clustered, pre-existing spines showing task-related activity in early sessions of learning creates a micro-environment of plasticity within dendrites, wherein multiple filopodia sample the nearby neuropil, form connections with pre-existing boutons connected to allodendritic spines, and are then selected for survival based on co-activity with nearby task-related spines. Thus, the formation and survival of new spines is determined by the functional micro-environment of dendrites. After formation, new spines show preferential co-activation with nearby task-related spines. This synchronous activity is more specific to movements than activation of the individual spines in isolation, and further, is coincident with movements that are more similar to the learned pattern. Thus, new spines functionally engage with their parent clusters to signal the learned movement. Finally, by reconstructing the axons associated with new spines, we found that they synapse with axons previously unrepresented in these dendritic domains, suggesting that the strong local co-activity structure exhibited by new spines is likely not due to axon sharing. Thus, learning involves the binding of new information streams into functional synaptic clusters to subserve the learned behavior.

BEHAVIORAL AND SPECTRAL EEG BIOMARKERS OF EPILEPTOGENESIS AND PHARMACORESISTANCE IN A LONGITUDINAL PILOCARPINE MODEL OF TEMPORAL LOBE EPILEPSY

FENS Forum 2026

Ago-APP for identifiying a neurogenesis regulating micro-RNA network in fly

Developmental origin of adult neurogenesis: Analysis of the postnatal hipocampal neurogenic niche in Sox5 conditional mutants

The Alzheimer’s amyloid protein APP and G(alpha)o physically and functionally interact to promote neuritogenesis

Alzheimer's disease pathogenesis is influenced by selective brain IL-6 overexpression

Assessment of vascularization and neurogenesis in an iPSC-derived 16p11.2 deletion organoid model

Cannabinoid CB1 receptor gene inactivation in oligodendrocyte precursors disrupts oligodendrogenesis and myelination in mice

The autism risk gene DDX3X in shaping neuronal morphogenesis and synaptogenesis in a sex-specific manner

Behavioral analyses of brain-specific SNORD116 and SNORD115 genes

Bidirectional influence of extremely low frequency electromagnetic field on neurogenesis in healthy brain

Blood-circulating lipids regulate adult hippocampal neurogenesis in the context of anxiety

Brain region‐specific epileptogenesis in a conditional mouse model

Can the brain be taken as a whole when choosing housekeeping genes for studding sex differences?

New Brainbow transgenes for subcellular multicolor barcoding based on quantitative modelling of Cre recombination in lox site arrays

Astrocyte-neuron interplay is critical for Alzheimer's disease pathogenesis and is rescued by TRPA1 channel blockade

Cell-type and brain-region-specific expression of PLPPRs as a molecular code for developmental neuron morphogenesis in the CNS

Cell-type-specific profiling of microRNAs during epileptogenesis: Insights into neurons and microglia microRNA profiles in normal brain function and disease

Cerebellum-postnatal development and behavior ontogenesis in a mouse model of the intellectual disability-associated Coffin-Lowry Syndrome

Characterising Epileptogenesis in a Recurrent Genetic Model of Dravet Syndrome

Chronic maternal SSRI exposure during the prenatal and/or postnatal period induced vocal behavior deficits and affected serotonergic neurogenesis in the mouse offspring

Conditional deletion of Cyclin D2 confirms critical functions in adult hippocampal neurogenesis

A connectome study of postnatal neurogenesis in the olfactory bulb

Contribution of the adipocyte hormone leptin in the pathogenesis of Rett syndrome

Contribution of adult hippocampal neurogenesis to prenatal stress-induced vulnerability to PTSD-like memory

CoQ10 reduces glioblastoma growth and infiltration through proteome remodeling and inhibition of angiogenesis and inflammation

Corticogenesis is impaired in a mouse model of DDX3X syndrome

Craniectomy Camouflages the Mild Fluid Percussion Injury-induced Changes in Neurogenesis, Neuroinflammation, and Behavior

Deciphering the genetics of brain malformation disorders reveals the importance of WD-repeat 47 (WDR47) and WD-repeat 91 (WDR91) genes in mice

Deciphering the role of p75 neurotrophin receptor in adult neurogenesis: a potential pharmacological target against Alzheimer’s Disease

Developmental oligodendrogenesis and myelination : Revisiting canonical and non-canonical Shh signaling

WAT alterations in diabetic mice: its connection and implication in AD pathogenesis

Double-coding: A mechanistic evaluation of neuronal multi-cistronic genes performed on the example of the P/Q-type calcium channel Cav2.1

Dual role of P2X7 receptor during physiological and pathological brain development and dendritogenesis

Effect of a peptide secreted by astrocytes on hippocampal adult neurogenesis

Effects of Chemogenetic Modulation of Hypothalamic Arcuate Nucleus Kisspeptin Neurons on Folliculogenesis in Kiss-Cre Polycystic Ovarian Syndrome Mouse Model

Effects of chronic stress on hippocampal microglia and neurogenesis of mice under social defeat stress

Effects of sequential exposure to physical exercise and cognitive training on hippocampal neurogenesis in mice

Exercising on cannabinoids: a combined effort in regulating postnatal neurogenesis

Experimental arthritis inhibits adult hippocampal neurogenesis in mice

Adult hippocampal neurogenesis signatures in patients with Parkinson’s disease, Dementia with Lewy bodies, and Frontotemporal Dementia