understanding

Delineating the role of TREM2 in chronic pancreatitis

PROJECT SUMMARY Chronic pancreatitis (CP) is a progressive digestive disorder characterized by persistent inflammation, irreversible fibrosis, and acinar cell damage. However, current treatment options remain limited, underscoring the need for effective, targeted therapeutic strategies through a deeper understanding of the disease microenvironment. Macrophages are pivotal players in the CP microenvironment, exhibiting dual roles in inflammation and tissue remodeling. A defining feature of macrophages is their remarkable phenotypic plasticity, enabling them to transition between pro-inflammatory and anti-inflammatory phenotypes. However, the specific macrophage phenotypes contributing to the immune imbalance in CP and their precise mechanisms of action remain poorly understood. TREM2 (Triggering Receptor Expressed on Myeloid cells 2), a transmembrane receptor of the immunoglobulin superfamily, has emerged as a critical modulator of tissue damage responses in multiple disease settings, though its function in CP remains unexplored. Our preliminary single-cell RNA-seq analyses of human CP tissues reveal an enrichment of inflammatory macrophages alongside a marked downregulation of TREM2 compared to non-diseased controls. This reduction in TREM2 correlates with marked increases in pro-inflammatory mediators, such as IL-1β and NF-κB, suggesting that TREM2 in macrophages contributes to maintaining homeostasis and restraining inflammatory signaling. Accordingly, diminished TREM2 expression appears to skew macrophages toward a pathologically hyper-inflammatory state. We hypothesize that loss of TREM2 disrupts the delicate balance among immune cells, fibroblasts, and acinar cells, fueling a self-reinforcing cycle of inflammation and fibrosis that exacerbates pancreatitis. To test this hypothesis, our R01 will leverage integrative single-cell transcriptomics, spatially resolved imaging, transgenic mouse models, functional organoid co-culture assays, and in vivo experiments to elucidate TREM2’s regulatory mechanisms in CP. This research aims to address two key scientific questions: (1) How does TREM2 suppress pro-inflammatory macrophage phenotypes and restrain IL-1β-induced inflammatory signaling? (2) How does the crosstalk among pro-inflammatory macrophages, fibroblasts, and acinar cells exacerbate the local inflammatory environment, leading to further pancreatic damage? Through this study, we aim to establish TREM2 as a pivotal inhibitory checkpoint in the NF-κB/NLRP3/IL-1β axis, preventing unchecked macrophage-driven inflammation, fibroblast activation, and further acinar cell damage. Successful completion of this project will deepen our mechanistic understanding of CP and identify new therapeutic strategies to mitigate fibrotic progression and preserve pancreatic function. Ultimately, these insights may guide the development of immunomodulatory treatments to attenuate CP severity, thereby transforming the clinical management of this devastating disorder.

Exploring in vivo Treg function in T1D through the lens of expanded Tregs

PROJECT SUMMARY/ABSTRACT A critical barrier to optimally treating Type 1 Diabetes (T1D), an autoimmune disease in which the islet beta cells are destroyed by immune cells, is understanding how autoimmunity is regulated in vivo. Several lines of evidence suggest that defective CD4+FOXP3+ regulatory T cells (Treg) likely contribute to the loss of tolerance in T1D. Yet, less is known about how human Treg function in vivo. In the Sanford T-rex study in which adolescents diagnosed with T1D were treated with a single dose of polyclonal autologous in vitro expanded Treg (expTreg), we found that a lower degree of in vitro Treg expansion significantly correlated with better preservation of C- peptide (a biomarker of insulin secretion and beta cell function) a year after treatment. This correlation could not be explained by age, expTreg phenotype or in vitro expTreg suppressive function. However, we did identify an expTreg gene signature that correlated with better C-peptide preservation and this expTreg signature was consistently expressed over time within individuals. Further, lower- and higher- expTreg differed phenotypically and transcriptionally by signatures implicating metabolic, homing and suppressive functions. Together, these data suggest that intrinsic features of an individual’s Treg may contribute to the extent of in vitro Treg expansion. They also suggest that strong activation and expansion can differentially amplify or alter the state of Tregs, leading to changes in homing and function that may impact clinical response. Based on these findings, we hypothesize that Treg proliferative capacity is driven by the activation and metabolic state of Treg resulting in differential in vitro fold expansion, homing potential and in vivo suppressive function that impacts clinical outcome. We will test this hypothesis by leveraging existing primary human samples from both the T-rex clinical trial and the Benaroya Research Institute Registry and Repository that includes individuals with known degree of in vitro Treg expansion and known C-peptide decline. In Aim1, we will identify how activation states of pre- and post- expansion Treg and longitudinal Treg in T-rex participants contribute to proliferative capacity and outcome using cellular, transcriptomic and epigenetic assays. In Aim 2 we will determine how metabolic shifts during Treg in vitro fold expansion alter Treg suppressive function, thereby impacting clinical outcome. In Aim 3, we will compare the in vivo suppressive function of lower- versus higher-expTreg from clinical samples using a xenogeneic graft versus host disease (GvHD) mouse model in addition to assessing in vivo expTreg homing and function using the assays from Aims 1 and 2 and a novel in vitro assay of cell trafficking to pancreatic islets. Successful completion of these aims will reveal mechanisms regulating Treg proliferative capacity and in vivo function that impact clinical outcome. Understanding these mechanisms will guide development of next generation Treg activation and expansion protocols for Treg therapies and help tailor the Treg expansion process to an individual’s baseline Treg signature.

Antibody-guided design of a human astrovirus vaccine

PROJECT SUMMARY Viral diarrheal diseases cause substantial global morbidity and mortality. Diarrheal disease is the second leading cause of childhood mortality in the world, accounting for over 10% of all deaths of children under 5 years old. Gobally, over 1 billion cases of diarrheal diseases occur every year, making prevention of these diseases a public health concern of the highest priority. Human astrovirus (HAstV) infection is a leading cause of viral diarrhea in children and has been shown to cause chronic gastrointestinal disease and fatal neurological disease in immunocompromised patients. There are nearly 4 million cases of HAstV infection each year in the United States alone, and there are no clinically approved HAstV-specific vaccines or therapeutics. Antibody-guided vaccine development leverages a deep understanding of productive antiviral antibody responses in order to design vaccine immunogens that deliberately focus the induced response toward highly conserved epitopes with the goal of reliably inducing broad, durable immunity. Using a cutting-edge monoclonal antibody (mAb) discovery approach based on next-generation antigen barcoding, single cell multi-omics, and sophisticated bioinformatics, we will exhaustively screen the HAstV- specific antibody repertoires of geographically distinct donor cohorts to uncover the structural and immunogenetic features that differentiate broad and potently neutralizing HAstV mAbs. A more complete understanding of these exceptional – and potentially very rare – mAbs will accelerate the development of HAstV vaccines and therapeutics. We have assembled a collaborative, multidisciplinary group of investigators with a long history of productive collaboration and with highly complementary areas of expertise. We expect our work will result in the discovery of thousands of novel anti-HAstV mAbs from cohorts of healthy adult and pediatric participants. Detailed genetic, functional, and structural characterization of these mAbs will reveal conserved sites of viral vulnerability, uncover the precise molecular mechanisms of viral neutralization, and inform our development of a broadly protective HAstV vaccine.

Integrins α4β7 in Leukocyte Rolling in Shear Flow, Firm Adhesion, and Therapy

Abstract. Integrin α4β7 facilitates leukocyte migration to sites of infection and autoimmune disease, making it an important therapeutic target for ulcerative colitis and Crohns disease. However, the currently approved antibody drug vedolizumab targeting α4β7 has limited efficacy. This proposal seeks mechanistic understanding of how α4β7 mediates rolling and firm adhesion of leukocytes during extravasation as well as how therapeutically relevant antibodies modulate α4β7 function to improve drug design. Unlike most integrins, α4β7 mediates rolling adhesion on its ligand MAdCAM. α4β7 can also mediate firm adhesion like α5β1. Integrins typically equilibrate between two low-affinity closed conformations and a high-affinity open conformation. Ligand binding is intimately coordinated with conformational change. During rolling adhesion, receptor-ligand bonds must rapidly form beneath rolling cells as cells are torqued by shear flow onto the substrate. Bonds must also rapidly dissociate at the upstream tethers to the substrate due to hydrodynamic force applied to the cell. To enable their function in rolling adhesion, we hypothesize that α4β7 ligand binding and dissociation and conformational change kinetics are faster than those of other integrins like α5β1 and that α4β7's pathways for conformational change may also differ. We propose that activation of the actin cytoskeleton in the transition from rolling to firm adhesion stabilizes α4β7 in a high-affinity state. Aim 1 will determine high-resolution structures of unliganded α4β7 and its complexes with MAdCAM or medically relevant antibodies using cryo- EM. These structures will reveal how these integrins recognize their ligands, the conformational changes due to ligand binding, and potential structural specializations that enable α4β7 to mediate rolling adhesion. The binding epitopes and conformational specificities of activating antibodies to the β7 subunit will also be defined. The structure of α4β7 bound to vedolizumab will resolve the contention around how it blocks MAdCAM binding. Aim 2 will quantitatively define the mechanisms by which α4β7 mediates both rolling and firm adhesion to improve therapies for inflammatory bowel diseases. Ligand affinity and binding kinetics of α4β7 stabilized in different conformations will be measured as well as single-molecule conformational change rates when bound and unbound to ligand. The effect of mutations that stabilize rolling or firm adhesion will be used to identify parameters important for each adhesion type. The tensile force and bond lifetimes during rolling and firm adhesion will be quantified at the single-molecule level. Together, our studies will enhance our structural, biochemical, and mechanical understanding of α4β7-mediated rolling and firm adhesion and will provide structural and functional information that can be utilized in the development of more effective therapies for inflammatory bowel diseases and multiple myeloma.

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.

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.

Mechanisms of antigen-specific T cell activation in MOGAD

PROJECT SUMMARY / ABSTRACT The overarching goal of this application is to train Dr. Carson E. Moseley, MD, PhD, who is a clinical neurologist and a research immunologist, to become an independent investigator studying and treating neuroimmunologic disorders. Myelin oligodendrocyte glycoprotein (MOG) antibody-associated disease (MOGAD) is a recently described, severe, neuroinflammatory syndrome of the central nervous system (CNS) with no approved therapies. Although MOG-specific antibodies helped define the disease, MOG antibodies alone are not clearly pathogenic and our understanding of MOGAD immunopathology is limited. CD4+ T cells are a dominant lymphocyte population in MOGAD lesions, yet the targets of T cell responses to MOG and how T and B cells interact to drive pathogenic immune response in MOGAD are unknown. This proposal uses a complementary approach of human and mouse immunology along with new technologies in T cell repertoire mapping and genome editing to dissect MOG-specific CD4+ T cell responses in MOGAD. Additionally, it will use new models to investigate how B cells promote pathogenic T cell differentiation and select pathogenic T cell receptors. The proposed training plan involves mentored training, seminars, formal learning, and advising to ensure completion of the proposed research and Dr. Moseley’s career development. He will train at UCSF, which is an outstanding institute for research and environment for physician-scientists. He will receive training in human immunology and CRISPR-based gene editing technologies. He will be mentored by Dr. Scott Zamvil, a leader in identifying antigen-specific T cell responses in neuroimmunologic disorders, and co-mentored by Dr. Alexander Marson, an expert in CRISPR gene editing to understand lymphocyte function. This application will provide Dr. Moseley with the long-term skills needed to become an independent investigator leading efforts to study and treat neuroimmunologic disorders.

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.

TARGETING VAV1 SCAFFOLDING AND ENZYMATIC FUNCTIONS IN MULTIPLE SCLEROSIS VIA BRAIN-PENETRANT MOLECULAR GLUE DEGRADERS

Abstract Multiple Sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) with significant unmet medical needs, as current therapies offer limited efficacy against neurodegeneration and can have considerable side effects. VAV1, a key signaling protein predominantly expressed in hematopoietic cells, plays a crucial role in T and B lymphocyte activation and is genetically and functionally validated as a therapeutic target in MS. This project proposes an innovative approach to target VAV1 through the development of brain-penetrant molecular glue (MG) degraders. Distinct from Proteolysis Targeting Chimeras (PROTACs) that require a high- affinity ligand for the target protein, molecular glues can mediate degradation by engaging specific protein surface features, such as loops, without the necessity of a dedicated binder. These degraders aim to induce the proteasomal degradation of VAV1, thereby ablating both its enzymatic and scaffolding functions, which are implicated in neuroinflammation. The research strategy involves three primary aims: 1) To optimize lead VAV1 molecular glue degraders for enhanced potency, brain penetration, and favorable pharmacokinetic properties using advanced computational modeling and medicinal chemistry. 2) To evaluate the in vivo efficacy of the optimized VAV1 degraders in preclinical mouse models of MS (Experimental Autoimmune Encephalomyelitis - EAE), assessing their ability to ameliorate disease severity, reduce CNS inflammation and demyelination, and engage VAV1 in the CNS. 3) To investigate the Structure-Activity Relationship (SAR) of a novel non-canonical VAV1 degron motif, aiming to expand the understanding of molecular glue-mediated degradation and enable the rational design of degraders for other challenging therapeutic targets. Successful completion of this project is expected to deliver preclinical candidate VAV1 degraders with the potential for a novel, effective, and safer treatment paradigm for MS. Furthermore, the insights gained into non-canonical degron recognition will significantly advance the field of targeted protein degradation, broadening the scope of "undruggable" targets for therapeutic intervention in various diseases.

Mechanisms of Commensal- Specific CD8+ T Cell Differentiation, Restraint and Dysregulation in Intestinal Inflammation

PROJECT SUMMARY Our understanding of immunity largely stems from models of infection with pathogenic microbes. However, the vast majority of microbial-immune encounters occur as a symbiotic relationship with the commensal microbiota. Recently, the contribution of commensal-specific T cells to host physiology has received significant attention. These commensal-specific responses not only control microbiota containment but also promote immune tolerance within the gastrointestinal tract. While commensal-specific CD4+ T cell responses in the lamina propria have dominated models of mucosal immune regulation, these are vastly outnumbered by CD8+ intraepithelial lymphocytes within the epithelium. How CD8+ T cell responses to gut microbiota are primed, differentiate and function under homeostasis has not been addressed. Conversely, aberrant immunity to commensal microbes has been proposed to underlie pathologies of barrier tissues, including inflammatory bowel disease (IBD), where commensal-specific T cells accumulate in blood and intestinal tissues of afflicted patients. A better understanding of the properties and functions of commensal-specific T cell responses is therefore fundamental to studies of tissue immunity in health and disease. Our long term goal is to better understand how commensal-specific T cell responses contribute to barrier tissue homeostasis, and the objective in this application is to investigate the mechanisms regulating induction of commensal-specific CD8+ T cells in homeostasis and how they become dysregulated in IBD. Our rationale for the proposed work is that uncovering these mechanisms has the potential to translate into new therapeutic approaches. Our central hypothesis is that commensal-specific CD8+ T cells develop as functionally restrained intraepithelial lymphocytes (IEL) under homeostasis, but that perturbation of local immune regulation within the intestinal epithelium, in the case of patients with ulcerative colitis, by autoantibody-mediated blockade of integrin avb6 results in aberrant CD8+ effector T cell responses in IBD. Based on strong preliminary data, we will test three specific aims: (1) Determine key antigen-presenting cells (APC) priming SFB-specific CD8⍺β+ IEL. (2) Identify how cell-intrinsic pathways drive differentiation, maintenance and restraint of SFB-specific CD8⍺β+ pIEL. (3) Determine how pathogenic KLRG1+Eomes+ CD8+ T cells arise and contribute to inflammation in murine models of ulcerative colitis Our approach is innovative as it investigates new mechanisms of immunity unique to commensal-specific CD8+ T cell responses. The proposed work is significant because it will establish new insights into the interaction and communication between commensal microbes and immune cells in the gut environment and identify potential targets for therapeutic intervention in conditions of chronic intestinal inflammation.

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.

Targeting disulfidptosis in cancer: mechanisms and preclinical translation

Project Summary Studying regulated cell death is critical for our understanding of cellular homeostasis and tumor suppression. We recently discovered disulfidptosis as a new form of regulated cell death induced by disulfide stress under NADPH-depleting conditions in SLC7A11-high cancer cells. However, in contrast to our deep understanding of other cell death modalities such as apoptosis and ferroptosis, the molecular and metabolic underpinnings of disulfidptosis, along with its therapeutic implications, remain largely unexplored. The objectives of this application are to elucidate the mechanisms underlying disulfidptosis and to therapeutically target this form of cell death in SLC7A11-high cancers. The proposed studies will make extensive use of human cancer cell lines and integrated human cellbased molecular analyses, including metabolomics, proteomics, CRISPR screening, and biochemical studies, to define the metabolic and signaling mechanisms governing disulfidptosis. In addition, select in vivo studies are incorporated in the therapeutic validation components of the project, where tumor growth response, systemic drug exposure and tolerability, tumor microenvironmental influences, and host immune/stromal interactions must be evaluated in an organismal context to ensure translational rigor. Alternative in vitro systems such as organoids may provide useful complementary information on tumor-intrinsic responses, but they cannot fully recapitulate the systemic metabolic stress, pharmacologic exposure, and organism-level therapeutic efficacy required for these studies. It is expected that our proposed studies will reveal novel mechanisms underlying disulfidptosis and identify effective therapies to induce this form of cell death in SLC7A11-high cancers. Our proposal is highly innovative because it focuses on a previously unexplored cell death pathway in cancer therapy. Our proposed studies will have significant impact on both our understanding of the fundamental mechanisms of disulfidptosis and our ability to target this cell death pathway in cancer treatment.

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.

Neural circuits for disinhibition in the cerebellum

ABSTRACT Our long-term goal is to understand how the cerebellum adapts and improves movements in response to motor errors. A critical component of this process is signaling from olivary climbing fibers that, by providing strong excitatory drive onto Purkinje cells, induces long-term synaptic plasticity to instantiate corrective adjustments in motor behavior. However, this signaling process is tightly regulated by molecular layer interneurons (MLIs). By strongly inhibiting Purkinje cells, MLIs oppose climbing fiber-driven excitation and gate the induction of corrective plasticity. Thus, for error-driven climbing fiber-induced plasticity and learning to occur effectively, Purkinje cells must undergo disinhibition through the suppression of MLI-mediated input. Notably, MLI ensembles are composed of several subtypes and have a highly structured interconnectivity and are responsive to convergent climbing fiber inputs, suggesting that climbing fiber synchrony- whose functional significance is poorly understood- can selectively engage MLI networks to alter the state of Purkinje cell inhibition. This engagement may balance inhibition and excitation of Purkinje cells during motor errors, creating a circuit mechanism conducive for the acquisition of adaptive learning. The objective of this proposal is to determine how distinct MLI circuits are organized to modulate Purkinje cell excitability through disinhibition in a context-dependent manner, enabling plasticity and learning in response to motor errors. We will employ functional recordings, circuit-targeted activity manipulations, and behavioral analysis to reveal how error-driven instructive signaling emerges from these circuits. In the first aim, we will use in vivo high-density electrophysiology to map functional interactions among MLIs, climbing fibers, and Purkinje cells in the flocculus during the vestibulo-ocular reflex. We will test whether, during motor errors, climbing fibers synchronize their firing to selectively engage disinhibition of Purkinje cells through MLI subtypes in adapting versus non-adapting contexts. In the second aim, we will combine acute slice recordings and molecular anatomy to define direct versus spillover climbing fiber synapses onto MLI subtypes. We will identify synaptic markers and measure climbing-fiber-evoked currents in MLI subtypes, revealing how structural connectivity supports rapid, subtype-specific circuit engagement. In the third aim, we will determine how long-range inputs to the inferior olive, specifically inhibitory projections from the vestibular nuclei, dynamically tune climbing fiber synchrony in vivo and thereby learning through differential engagement of disinhibitory MLI networks. Using functional recording and optogenetic manipulation during the vestibulo- ocular reflex performance, we will establish causal links between climbing fiber synchrony, MLI network state, and adaptive behavior. By fully understanding the logic of instructive signaling, emergent from cerebellar circuit organization and behavioral engagement, we will advance our knowledge of cerebellum-dependent learning processes and provide broader insights into the neural mechanisms of learning and adaptation more generally.

Biostatistics, Ethics, Data Management, Research Design and Community Engagement(BEDRoC) Core

Biostatistics, Ethics, Data Management, Research Design and Community Engagement (BEDRoC) Core Abstract The Biostatistics, Ethics, Data Management, Research Design and Community Engagement (BEDRoC) Core will promote and support aging with serious illness science for the Center for Aging with Serious Illness (CASI). BEDRoC will provide expertise in statistical design and analysis, research ethics, and community engagement for all components of CASI. The Core's services will support the Research Project Leaders (RPLs) and Pilot Project Leaders (PPLs) and build capacity for the broader Dartmouth Health aging research community to conduct rigorous, impactful research to inform and improve care delivery for older adults with serious illness. BEDRoC includes expertise in mixed methods approaches that feature both quantitative and qualitative research methods to provide a comprehensive understanding of the complex issues related to aging with serious illness, ethical approaches to consent in research trials, multidimensional quality of life measurement, and innovative modeling approaches to studying clinical decision making. BEDRoC faculty have actively collaborated in study planning with each RPL, serving as both mentors and experienced collaborators on the three different projects involving decision aids for patients considering carotid revascularization, a patient-reported outcome-directed referral intervention to improve referral rates to palliative care services, and a pilot trial for a virtual/home-based exercise and a weight management osteoarthritis treatment program in older patients with osteoarthritis and multimorbidity. The BEDRoC Core will further support CASI by establishing an innovative training curriculum with workshops, tutorials, resources, and services, offered locally to RPLs and PPLs and extended to regional and national investigators in the IDeA network. In addition to their primary individual project mentors, each RPL will receive training and guidance from BEDRoC leaders through co-mentoring and RPL-focused works-in-progress sessions. BEDRoC will also provide access to a comprehensive inventory of patient-reported outcomes instruments, which are crucial in geriatric research to provide validated measures of health status, quality of life and functional ability outcomes. BEDRoC will coordinate with the Administrative and Mentoring Core to integrate community advisors in guiding their activities in support of the RPLs. BEDRoC will also enable research collaboration with and within the larger Dartmouth and IDeA investigator communities. The BEDRoC Core will build capacity for aging research and disseminate new resources to RPLs and PPLs, including innovative solutions created through robust community engagement. These services, resources, and solutions will ensure all projects operate in a cohesive, complementary, and collaborative manner to study approaches to improving the health of older patients with serious illness.

Circadian regulation of reperfusion efficacy in acute ischemic stroke

Reperfusion with thrombectomy has changed the clinical landscape for ischemic stroke. Recently, some studies suggest that patients with “large cores” may still benefit from reperfusion. Why? If these “cores” represent dead brain, why should reperfusion help? One logical explanation is that currently used neuroimaging “cores”, do not always identify uniformly dead tissue. Our pilot data suggest that these “cores” include tissue with a wide range of injury, indicated as changes in relative CT Hounsfield Units (rHU). Importantly, circadian mechanisms may be involved. Ischemic tissue with less severe changes in rHU tend to occur in the morning (active phase) when responses to reperfusion are better. In mouse models of stroke, ischemic injury is also less severe when strokes occur during the nighttime (active phase for nocturnal animals). In contrast, more severe ischemic injury during the daytime (inactive phase for mice) is accompanied by dampened vasodilation and CBF response along with increased immunothrombosis and neutrophil extracellular traps (NETosis). Is it possible that understanding these circadian mechanisms may help identify patients who respond best to reperfusion? And is it possible that targeting these circadian mechanisms can help convert non- responders into responders? In this multi-PI project, we use a translational approach (clinical neuroimaging and biomarkers in stroke patients, mouse models of stroke, CT-PET imaging of tissue viability, molecular pharmacology) with three integrated aims that can be pursued in parallel. Aim 1 will use neuroimaging in stroke patients to show that less severe rHU values in reperfusion-responsive “cores” tend to occur in the morning, whereas more severe rHU values in reperfusion-non-responsive “cores” occur later. Aim 2 will use clinical biomarkers to show that more severe rHU “cores” that are not reperfusion-responsive correlate with circadian effects on vasodilation and immunothrombosis. Aim 3 will use mouse stroke models to test whether targeting these circadian mechanisms of vasodilation and immunothrombosis can convert reperfusion-non-responders into reperfusion-responders. Patients cannot choose when they have a stroke. So why should we pay attention to circadian mechanisms? There may be 2 reasons that are addressed by the present project. First, thrombectomy is resource-intensive, and in spite of the very low number-needed-to-treat, only 20% of “large core” patients do well after reperfusion. Our studies may help identify who (when) these responders are. Second, the pathophysiologic mechanisms of cerebral ischemia differ depending on time-of-day. Therefore, understanding and then targeting these circadian mechanisms may allow us to convert reperfusion non-responders into responders.

COCHLEAR SIGNALING MEDIATED BY HENSEN’S CELLS

PROJECT SUMMARY/ABSTRACT The organ of Corti has two types of auditory sensory cells (inner and outer hair cells) surrounded by nearly a dozen different types of supporting cells organized in a very meticulous pattern. Hair cells mediate the mechano-electrical transduction process of the organ of Corti and thus most cochlear auditory research has focused on these sensory cells. In contrast, much less is known about the different types of cochlear supporting cells, even though they likely impact hair cell function. Hensen’s cells are located laterally to the outer hair cell rows and appear to be the only cell type in the cochlear epithelium that expresses TRPA1 channels. These channels are widely known for their role as sensors of tissue damage and inflammation in nociceptive neurons. Not surprisingly, we recently found that Hensen’s cells are main sensors of tissue damage in the cochlear epithelium via the activation of TRPA1 channels (Velez-Ortega et al., Nat Commn, 2023). Additionally, our preliminary data also supports the role of Hensen’s cells in signaling pathways important for the proper innervation of the organ of Corti (aim 1), for the transmission of cochlear damage signals to the brain (aim 2), and for the regulation of hearing sensitivity after acoustic trauma (aim 3). Thus, here we will explore the hypothesis that TRPA1- mediated signaling pathways in the Hensen’s cells are required for the proper innervation and auditory function of the organ of Corti. In Aim 1 we will perform a detailed comparison of the morphology and synapses of afferent cochlear neurons of wild-type and Trpa1-/- mice at several developmental stages (using immunolabeling, confocal microscopy, STED microscopy, and electron microscopy) to assess the role of TRPA1 activity on the postnatal refinement of the cochlear innervation. Aim 2 will evaluate whether the afferent type II spiral ganglion neurons (SGN) can be activated downstream of TRPA1 channel gating in Hensen’s cells by testing responses of neonate and adult type II SGN to TRPA1 agonists (via live-cell time-lapse calcium imaging and patch clamp recordings of type II SGN dendrites). Aim 3 will test the impact of TRPA1 signaling in Hensen’s cells to the operating point of the cochlear transducer (via the recording of cochlear microphonics) and to cochlear tuning (via the recording of ABR tuning curves). This study is significant because it will contribute to our understanding of the cellular (Hensen’s cells plus type II SGN) and molecular (TRPA1 channels) mechanisms of the elusive cochlear nociceptive pathway. In addition, given that the loss of TRPA1 channels does not affect hearing thresholds in mice, we believe that undiagnosed deficits in TRPA1-dependent responses in the human population could represent a hidden susceptibility for cochlear damage after noise exposure or other insults.

Cytoskeletal connectors: Deciphering the fundamental mechanisms of cytoskeletal dynamics and transport

PROJECT SUMMARY The cytoskeleton is a dynamic network of filamentous structures, including microtubules and actin, that regulate essential cellular processes such as cell shape, growth, and signaling. Cytoskeleton also serves as tracks for molecular motors, which transport a variety of cellular cargoes, including organelles, macromolecules, and vesicles. These cargoes are linked to motors by specialized connector proteins. Disruptions in connector proteins are implicated in a range of neurodevelopmental and neurodegenerative diseases, as well as cancers. Despite their importance, these proteins continue to be understudied, primarily due to their perceived role as passive linkers and the technical challenges in working with them. However, recent discoveries suggest that connector proteins may play more active roles, in some cases even have enzymatic functions. This proposal aims to uncover mechanisms of connector protein functions through a detailed investigation of actin-microtubule and motor-cargo interactions. Actin and microtubules are linked by the spectraplakin family of large and evolutionarily conserved proteins, critical for neuronal development and differentiation. Recent discoveries of ATPase domains within these proteins suggest they may haves beyond simply linking cytoskeletal components. One goal of this proposal is to investigate the role of spectraplakin’s ATPase domains via structural, biochemical, and cell biology approaches. Another goal is to explore how dynamic changes in motor-cargo connectors facilitate the transport of diverse cargoes along microtubule tracks. The focus will be on the cytoplasmic dynein-1 (dynein) and the connectors (adaptors) that activate and link dynein to cargo. Dynein is a microtubule minus-end directed motor that plays essential roles in cell division, and transports hundreds of different cellular cargoes. While several motor-cargo connectors have been identified, the regulatory mechanisms enabling cargo transport are not fully understood. We are investigating whether connector proteins work together to activate dynein movement and/or facilitate cargo handoff between different dynein complexes. Using innovative approaches, including time- resolved cryo-EM, complex in-vitro reconstitutions, and live-cell imaging in induced neurons, we are uncovering critical mechanisms that govern cytoskeletal connector proteins, furthering our understanding of how the cytoskeleton regulates essential cellular processes.

Dissecting the role for astrocytes in mediating adverse outcomes of maternal immune activation.

Prenatal infections cause maternal immune activation (MIA), a major risk factor for several neurodevelopmental disorders, including schizophrenia, autism spectrum disorders (ASD), and attention deficit hyperactivity disorder (ADHD). Consequently, elucidating the mechanisms by which MIA alters brain function is critical for understanding the pathophysiology of these disorders and developing effective treatments. While the effects of MIA on neurons and microglia have been extensively studied, the impact of MIA on astrocytes, key regulators of brain physiology and homeostasis, remain unknown that significantly impedes our understanding the mechanisms of MIA-induced neurobehavioral abnormalities. To address this major knowledge gap, we conducted pilot studies that suggest that MIA increases impulsivity-like behaviors and amphetamine-induced hyperactivity and enhances extracellular levels of glutamate (GLU) and dopamine (DA) in the dorsal striatum (DS). MIA also increased pro-inflammatory signatures of astrocytes, including up- regulation of the Nuclear Factor kappa B (NF-κB) pathway and increased GFAP immunoreactivity in DS astrocytes. Collectively, these novel findings support our overarching hypothesis that MIA increases astrocyte reactivity, leading to increased gliotransmission (e.g., GLU), which in turn enhances DS DA release and DA- dependent behaviors. To test this hypothesis, we will leverage the expertise of the research team in molecular, physiological and neurobehavioral approaches and conduct the following Specific Aims: In Aim 1, we will identify the MIA-induced cellular and physiological changes characteristic of astrocyte reactivity. In Aim 2, we will determine the circuit mechanisms by which MIA increases DA signaling. In Aim 3, we will identify the molecular mechanisms whereby reactive astrocytes contribute to MIA-induced cellular and behavioral abnormalities. These studies will enhance the current understanding of the effects of MIA on brain functions and generate new insight into potential treatment strategies for MIA-associated neurodevelopmental disorders.

Circulating extracellular vesicles as functional indicators of maternal mental and physical health in pregnancy and postpartum

Women with high levels of adverse childhood experiences (ACEs) are at significantly greater risk for negative health outcomes in pregnancy and postpartum, including gestational diabetes, PTB, and depressed mood. However, we still lack biomarkers or a sufficient understanding of causal mechanisms. Extracellular vesicles (EVs) are one of the most dynamic and abundant biological signals secreted into maternal circulation, largely produced by the placenta – where levels increase 4-5-fold during pregnancy. Similarly, removal of the placenta at delivery produces a dramatic drop in maternal EV concentration. Across species, we and others have identified significant EV changes during pregnancy associated with homeostatic regulation, including glucose and glucocorticoid levels, supporting key roles for EVs in maternal health. However, longitudinal studies in human pregnancy and postpartum have not been conducted. We know little as to the mechanisms controlling EV secretion or the roles for EVs in maternal pregnancy and postpartum health. Our decade’s long work identified the X-linked gene, O-glycosyltransferase (OGT), in mouse and human placenta as a master gage of the maternal milieu, where OGT regulation of annexin A1 (AA1) is key to EV cargo loading and secretion from the placenta. We recently reported that placental OGT levels positively correlate with maternal EV concentration. How this association may contribute toward postpartum health, including regulating maternal stress physiology and mood in humans is not known. We hypothesize that increased ACEs, similar to stress in preclinical models, are negatively associated with a cell’s ability to secrete EVs important to maintain homeostasis in the face of the challenges of pregnancy and postpartum, producing an increasingly unhealthy state. Therefore, the goals of these proposed studies in both mice and humans are as follows: 1) To identify cellular mechanisms involved in EV secretion important to maternal health outcomes utilizing the placenta as a tool to genetically target OGT in mice and examine maternal homeostatic control related to EV concentration and composition during pregnancy; 2) To examine the functional ability for a dynamic elevation in maternal EV concentration to improve homeostatic regulation in pregnancy and postpartum using chemogenetic activation (DREADDs) of placenta trophoblast cells in pregnancy, and by EV transfer by tail vein injection postpartum; and 3) To examine in women changes in maternal EVs in a longitudinal pregnancy and postpartum study in association with maternal glucose and cortisol changes, we will examine markers of physical (glucose challenge test), HPA stress (hair cortisol & stress- stimulated salivary cortisol) and psychological (Hamilton Rating Scale for Depression, Perceived Stress Scale) health across pregnancy and the postpartum period in 150 healthy women with varying degrees of exposure to ACEs as measured using the ACE Questionnaire (ACE-Q).

Examining the foundations of reading comprehension: a longitudinal study of brain and behavior starting in infancy

SUMMARY Reading comprehension (RC) is one of the most complex skills that we utilize daily and is crucial for functioning in modern society, but despite its significance for academic achievement, employment prospects, and mental health, many children and adults do not exhibit proficient RC abilities. New theoretical models aiming to explain variability in RC suggest a dynamic interplay and co-development among ‘precursor’ foundational and cognitive- linguistic skills, interacting with environmental and socio-ecological factors across the developmental timeline of learning to read. Behavioral and neuroimaging studies in school-age children have demonstrated critical mechanistic support for these multifactorial RC models by identifying the developmental trajectories of precursor skills and further showing that brain areas, tracts, and networks typically underlying language and cognitive skills are also involved in RC. Nevertheless, the precursor skills that support RC start developing in infancy and the brain correlates underlying these precursors begin to develop in utero, which suggests that typical and atypical RC developmental trajectories could diverge long before school age. As such, examining RC development using a multifactorial, longitudinal approach that includes brain and behavior starting in infancy is critical for developing theoretical frameworks that can inform early preventative and intervention strategies. Here, we propose a comprehensive longitudinal study of RC development in which we examine direct and indirect effects on RC from brain, behavioral, familial risk, and environmental data from infancy to adolescence. To achieve this goal, we will combine two existing longitudinal cohorts, one ranging from infancy to late childhood (n = 174) and the other from preschool to early adolescence (n = 137). By applying state-of-the-art pediatric neuroimaging analyses, multiple indicator growth model structural equation models, and an innovative behavior- brain co-development measurement index to this unique, combined dataset, we will be able to identify brain and behavioral measures in infancy that directly and indirectly support subsequent RC development (Aim1). We will further characterize how longitudinal trajectories of behavioral measures as well as brain structure, function, and white matter organization contribute to RC development and how familial risk and environmental factors shape these trajectories (Aim 2). Finally, we will examine how the co-development of brain and behavior, as measured with an innovative co-development index, relates to subsequent RC (Aim 3). If successful, we will contribute the first multifactorial longitudinal model of RC development comprising direct and indirect effects from brain, behavior, brain-behavior co-development, familial risk, and environmental measures beginning in infancy. Understanding RC development using a multifactorial longitudinal lens will be crucial for building theoretical models and developing experimental designs focused on early preventative and intervention approaches long before the start of formal schooling.

Hepatotoxicity of Legacy and Replacement PFAS: Role of BRUCE-Mitochondrial Interactions

Epidemiological studies have shown a strong association between exposure to PFAS (Per- and Poly- fluoroalkyl Substances) and liver toxicity. Particularly, legacy C8-PFAS members, PFOS (perfluorooctane sulfonate) and PFOA (perfluorooctanoic acid), are highly toxic, with PFOS estimated to be approximately 10 times more toxic than PFOA in ecotoxicity models. Consequently, PFAS replacements such as GenX and PFBS are marketed as safe alternatives, although growing evidence indicates that these substitutes also exhibit toxic effects. Lab animal model studies have shown hepatotoxic effects of both legacy and replacement PFAS members, characterized by Metabolic dysfunction-associated steatotic liver disease (MASLD) and its severe form Metabolic dysfunction- associated steatohepatitis (MASH), the two chronic liver diseases affecting an estimated 80-100 million Americans. The broader objective of this project is to understand the underlying mechanisms of PFAS hepatotoxicity in MASLD/MASH. In this context, our initial studies have shown that PFAS exposure of mice downregulates hepatic BRUCE, an autophagy inhibitor, resulting in development of MASLD in WT, and more severe MASLD and even progression to MASH in BRUCE liver-knockdown (BKO) mice. Using primary hepatocytes, we found PFAS-induced BRUCE reduction compromised mitochondrial (mt) functions (respiration, fatty acid oxidation/FAO, and ATP production) and suppressed mitophagy in WT and more so in BKO mice. Pharmacological restoration of mt function in mice prevented PFAS-induced MASLD/MASH. Guided by these compelling preliminary data and scientific premise, we hypothesize that PFAS degradation of BRUCE in hepatocytes induces excessive autophagy (resulting in cytotoxicity) and inhibits mitophagy (resulting in accumulation of damaged mitochondria), leading to release of mtDAMPs to activate inflammation/ fibrosis, thereby facilitating progression from MASLD to MASH. We will test this by three specific aims. Aim 1 (ex vivo) is to determine the human-relevant PFAS doses that modulate BRUCE levels for homeostatic vs cytotoxic autophagy and how BRUCE in turn regulates autophagy. Aim 2 (ex vivo) will investigate BRUCE-driven mitophagy pathway specific to PFAS exposure at human-relevant doses. Aim 3 (ex vivo and in vivo) will involve ex vivo simulation experiments to characterize the role of PFAS-induced, BRUCE-dependent hepatocyte- released mt DAMPs in activation of immune and fibrogenic cells using co-culture assays. Next, we will perform in vivo intervention to validate the role of PFAS-damaged mitochondria in driving MASH progression in mouse models. Furthermore, human relevance of the delineated mechanisms will be ascertained and validated using iPSC-derived human liver organoid system. Impact: This project will advance our understanding of autophagy/mitophagy-centric mechanisms with therapeutic potential in the context of PFAS-induced liver disease MASLD/MASH.

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.

Specific Affinity Requirements for Antibody Somatic Hypermutation

PROJECT SUMMARY Antibodies diversify through two distinct pathways. The first involves the combinatorial assembly of immunoglobulin (Ig) heavy and light chain variable region (V) exons, forming the antigen recognition domains of the B cell receptor (BCR), which is initially expressed as IgM on immature B cells. The second diversification pathway is somatic hypermutation (SHM) of V exons in germinal centers (GCs). In this setting, B cells that acquire mutations enhancing affinity for antigen receive limited cognate T cell help and are selected for clonal expansion, leading to affinity maturation. These primary and secondary diversification systems work together to generate protective antibody responses. The primary, or pre-immune, repertoire provides the foundation for initial antigen recognition. SHM and affinity maturation refine these baseline specificities. While it is well established that SHM improves affinities already present in the primary repertoire, this project explores the hypothesis that SHM can also generate new specificities in B cells that initially lack measurable antigen recognition. This process, termed affinity birth, may enable access to otherwise excluded V gene segments and expand the landscape of antibody evolution. This hypothesis will be tested through two specific aims: (i) To elucidate the extent of SHM-mediated Ig diversification in non-specific or bystander B cells. And, (ii) to define parameters that influence SHM-mediated antibody affinity birth. The significance of this work lies in its potential to reveal previously unappreciated flexibility in the antibody diversification process and to uncover modifiable factors that influence the emergence of new specificities. The proposed studies are innovative in suggesting that B cells possess intrinsic capacity to undergo SHM and selection regardless of their initial antigen specificity. This research may advance understanding of how germinal centers support antibody evolution and inform strategies to design vaccines that anticipate emerging pathogens.

Protective efficacy and immunogenicity of a live attenuated Chlamydia strain

PROJECT SUMMARY The main goal of this project is to rigorously evaluate the immunogenicity and protective efficacy of a mutant, live attenuated Chlamydia trachomatis (CT) vaccine strain in an established nonhuman primate (NHP) model that accurately mimics many aspects of human CT infection. This work is highly significant, as CT is the leading cause of bacterial sexually transmitted infection and an important causative agent of morbidity in women. Although the development of an effective CT vaccine is an urgent medical priority, no approved vaccines exist and it is imperative to pursue new candidates. Historical evidence supports the vaccine efficacy of whole Chlamydia organisms in protecting the reproductive tract from reinfection, primarily using C. muridarum infections in a mouse model. Recent advances in Chlamydia genetic engineering now allow for the development of genetically attenuated strains which can be evaluated as live vaccines in preclinical models. We recently characterized a human-tropic CT mutant with a disruption in garD (CT∆garD); this mutant is sensitive to an intracellular, IFNγ activated defense mechanism and we demonstrated that this strain was attenuated in the female NHP genital tract. In a pilot vaccine efficacy study, we further demonstrated that immunization of macaques with CT∆garD was safe and elicited protection against subsequent challenge with wildtype CT. A unique feature of this strain is that it arrests at an intracellular stage and thus presents a broad array of desirable T and B cell antigens that are broadly conserved across circulating CT strains. We will first generate an improved genetically attenuated CT strain that harbors a clean deletion of garD, and we will subsequently genetically and phenotypically validate its attenuation phenotype. We will then conduct an immunogenicity and efficacy study in female macaques to determine the optimal dosing regimen of live attenuated CT for eliciting protective cellular and humoral immune responses, and also protective efficacy, against challenge with a wild type circulating clinical CT strain. These studies will investigate the potential for a live attenuated human tropic vaccine candidate in a macaque preclinical model and pave the way for greater understanding of immune correlates of protection against CT.

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.

The Pyruvate-Lactate Metabolic Axis in Heart Failure and Recovery

PROJECT SUMMARY/ABSTRACT Heart failure (HF) is a leading cause of mortality worldwide. The metabolism of the failing heart is commonly characterized by increased glucose uptake, glycolytic dependence, and reduced oxidative phosphorylation. We previously demonstrated that blocking glucose oxidation is sufficient to cause hypertrophy and subsequent HF. Additionally, our preliminary data shows that an altered pyruvate-lactate metabolic axis may be pivotal in human HF. Research investigating both the mechanistic regulation and biological roles of the pyruvate-lactate metabolic axis in cardiac metabolism during HF and cardiac recovery is warranted and also has the potential to identify novel druggable pathways to target for future pharmacological approaches. The overall objective of this application is to test the hypothesis that impaired pyruvate oxidation is a cardinal feature of HF in humans and animal models and that myocardial recovery is tightly coupled to normalization of the pyruvate-lactate metabolic axis. We will quantify the pyruvate-lactate metabolic axis in human HF and myocardial recovery (Aim 1). Next, we will determine the essentiality of the pyruvate-lactate metabolic axis for HF and cardiac recovery (Aim 2). Lastly, we will define cell-autonomous mechanisms that regulate the pyruvate-lactate axis in HF and recovery (Aim 3). These experiments will allow us to identify patterns of metabolic alteration in the pyruvate-lactate axis and molecular pathways during HF and myocardial recovery. Understanding the role of pyruvate and lactate metabolism in HF and myocardial recovery is cutting-edge research. Our unique access to human HF myocardium from patients administered stable isotope-labeled glucose or lactate to quantitate pyruvate metabolism in HF and recovery is state-of-the-art and will likely help us reveal new fundamental mechanisms of cardiac metabolism and expedite the successful translation of therapeutics being validated in various models of HF and recovery.

Bridging Local and System-Wide Autoreactive, Extrafollicular B Cell Signatures in a TLR7-Driven Model

Project Summary A substantial body of literature has described the development of autoreactive humoral responses in the context of autoimmune disease and recently discerned an exciting new avenue for investigation. While early work focused on canonical mechanisms of activation through the germinal center (GC) response, recent studies have found GC infrastructure to be dispensable for the onset of chronic autoimmunity. It has become clear that an alternative pathway of B cell activation, the extrafollicular (EF) pathway, can drive the onset of new autoreactivity in multiple human disorders including rheumatoid arthritis and systemic lupus erythematosus (SLE). In comparison to the GC pathway, the EF pathway represents a less stringent method for B cell activation, leads to accelerated antibody-secreting cell (ASC) formation, and thus has a higher propensity for the production of autoreactive B cell effectors and ASCs. Recently, our group has identified a similar skew toward the EF response in the context of severe viral infection, tied to acute tolerance loss, increased disease severity, and complicated recovery from infection. These findings highlight how further study of the EF response is crucial to our understanding of autoimmune induction across multiple areas of disease. Toll-like receptor 7 (TLR7) stimulation has been identified as a key contributor to EF B cell development in SLE, and several studies have now linked TLR7 overstimulation to chronic autoimmune disease. While EF effector B cell populations have now been identified in both murine models and humans, substantial gaps in our knowledge remain to be answered concerning i) the origins of these cells and ii) the system-wide and microenvironmental signaling and organization that drive this differentiation pathway. We propose to address these gaps, here, by utilizing a TLR7 agonist (R848) in a murine model to characterize the autoreactive response within the blood and draining lymph node through innovative high-throughput analytical techniques. Systemic shifts in proteomic signatures and immune cell phenotype will be monitored in the blood throughout the induction of autoreactivity, using novel applications of machine-learning based classification. These signatures will then be connected to developing inflammatory microenvironments identified within the draining lymph node by applying a customized set of software tools to spatial transcriptomic data. This work will deepen our understanding of the immunologic mechanisms by which the EF pathway can lead to “run-away” autoreactive B cell development, with the added potential for identification of early blood-based biomarkers for this developing autoreactivity. The above proposed work will provide an ideal training opportunity for the candidate to develop experience with advanced immunologic laboratory techniques, rigorous bioinformatic analysis, a systems-level view of immunology, and scientific communication. The Woodruff and Sanz Labs are highly experienced within the autoimmune disease space with extensive experience with the required techniques and established routes for clinical collaboration to act on these findings.

Development of an at-home weight-shifting balance game with musical biofeedback for older adults

Reducing fall risk is a dire societal need that requires interventions that over-prepare individuals to perform maneuvers important to daily mobility. Falling is often caused by improper weight shifting, and interventions that focus on developing weight-shifting abilities have shown improvements in clinical balance outcomes, including reduced fall incidence. Interventions that combine challenges to the cognitive and motor systems may be necessary to reduce fall-risk. Our central hypothesis is that leveraging gamification and “musical biofeedback” will improve balance abilities through practicing weight-shifting skills with increased cognitive and physical demands. Musical biofeedback conveys biological sensor data from the participant through specific musical sound parameters in real-time. Of particular interest in the proposal is the applicability to use musical biofeedback to train weight-shifting skills in a musical game. The goal is to develop a wearable sensor system that can be used at-home to practice and develop balance skills, while supporting cognitive engagement and motivation to adhere to exercise goals. To start, we are focusing on older adult end-users who typically have home exercise programs focused on weight-shifting. However, in the future, many other populations can benefit from this technology. In this Trailblazer award, the PI is leveraging her background in studying complex human maneuvers, developing musical biofeedback for older adults, and in algorithm development for mHealth sensors. The transdisciplinary team includes expertise in engineering, gamified rehabilitation technologies, home exercise programs, psychology of aging, and music. In the proposed research, our goals are to evaluate responses to the musical biofeedback game (Aim 1), validate the mHealth sensor system (Aim 2), and phenotype the gameplay behavior of fallers vs. non-fallers (Aim 3), relative to their baseline characteristics (Sub-Aim 3). Our long-term goal is for a variety of people to improve their balance control patterns while supporting and building their self-efficacy. We envision users, including older adults, training with musical biofeedback to safely (and enjoyably) prepare themselves to ambulate in their community – improving and preserving their mobility. The proposed research will pioneer using an emerging clinical technology – musical biofeedback – to train balance during weight-shifting tasks. The proposed research innovates how musical biofeedback, gamification, and focusing on weight-shifting and turns in balance training can be leveraged to challenge cognitive and physical body systems in fall-risk populations. By developing new therapy options and better understanding responses relative to baseline characteristics, this research improves clinical practices to reduce fall risk and deepens our understanding of dynamic balance control. Finally, the results of the proposed research will have translational impacts to help other fall-risk groups.

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.

Impact of environmental toxicants on frontal cortical circuits

Abstract: Human mercury (Hg) exposure has been known for many decades to produce cognitive impairment and mood disorder symptoms. Hg is a global pollutant that poses widespread potential for neurotoxic exposure, earning it a position on the WHO’s list of the top 10 chemicals of major public health concern. However, little is known about the neural mechanisms that lead to neuropsychiatric symptoms from Hg exposure. The objective of this application is to identify specific mechanisms, within the neocortical circuits that control emotion and cognition, that are disrupted by the neurotoxicant, methylmercury (MeHg). The neocortex exhibits especially strong bioaccumulation of Hg, magnifying the risk to these circuits. Therefore, we hypothesize that chronic MeHg exposure leads to persistent circuit dysfunction in prefrontal and insular cortices (mPFC and aIC) – two brain regions critical in control of emotion and cognition. Our recent work showed that mPFC neurons in brain slices are negatively affected by acute MeHg exposure, resulting in hyperexcitability and altered synaptic transmission. Currently, it unknown how these acute effects on synaptic transmission translate to altered neuronal function in vivo. This proposal applies an integrative approach to determine the in vivo effects of MeHg on mPFC and aIC circuits, at the systems neurophysiology, synaptic and molecular levels. We will compare the effects of MeHg exposure on in vivo spiking activity patterns in brain regions of the mPFC-aIC circuit, using multiunit electrophysiological recordings in awake animals. Action potentials will be recorded simultaneously from multiple neurons, distributed across cortical layers, to evaluate effects on spike frequency, temporal patterning and correlation. Using acute brain slices derived from animals chronically treated with MeHg in vivo, electrophysiologically recorded synaptic estimates will be made to compare the effects of MeHg exposure on synaptic transmission and EI-balance within brain regions of the mPFC-aIC circuit. Based on previous evidence, we hypothesize that TDP-43 hyper-phosphorylation and aggregation link MeHg exposure to mPFC and aIC dysfunction. Therefore, immunohistochemistry will be used to measure TDP-43 hyper-phosphorylation and nuclear redistribution from animals treated in vivo +/- MeHg. In addition, tissue will be co-labeled with antibodies for nPAS4, a well-stablished molecular marker of activity, to determine whether TDP-43 hallmarks correlate with MeHg-induced hyper-excitability. The results of our study will substantively improve our mechanistic understanding of how Hg disrupts frontal cortical function and contribute to our understanding of the biological basis of emotional and cognitive sympoms. Identifying specific actions of MeHg at the functional microcircuitry level and cellular/molecular level will help significantly in finding novel targets for therapeutic interventions. If our hypothesis is correct, this will also raise the question of the extent to which chronic low-level environmental mercury exposure contributes to the etiology of fronto-cortical disorders with symptoms that overlap mercury exposure but do not have definitive genetic origins. This is particularly important because fronto-cortical disorders are predominantly sporadic in nature.

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.

Role of Two Medial Prefrontal Long-Range Recurrent Networks in Behavior Initiation and Inhibition

Abstract The medial prefrontal cortex (mPFC) is critical for executive function, yet how its dorsal (dmPFC) and ventral (vmPFC) motor-projecting (MP) neurons coordinate behavioral initiation, inhibition, and cognitive flexibility remains poorly understood. This R21 leverages four translational behavioral paradigms (head-fixed Persistent Licking/Shock-Escape; freely moving FED3-based Reversal Learning/Stop-Signal), high-density neural recordings, circuit manipulations, and Brian2 spiking neural network modeling to test our central hypothesis: dmPFC MP neurons drive action initiation and adaptive switching, while vmPFC MP neurons suppress impulsivity and perseveration. In Aim 1a, we quantify behavior using kinematic analyses (jerk, velocity, z-scored) aligned with human executive dysfunction metrics (Action Latency [AL], Reversal Accuracy [RA], Perseveration Errors [PE], Stop-Signal Reaction Time [SSRT]), combined with optogenetic (stGtACR2/ChR2) and chemogenetic (PSAM/varenicline) perturbations. Aim 1b employs optotagging and population analyses (PCA, SVM, Total Spiking Probability Edges) to decode dmPFC/vmPFC MP dynamics across tasks, resolving specialized versus mixed functional roles. Aim 1c integrates these datasets into Brian2 spiking network models to predict neural-behavioral correlations, validated through cross-validation. Exploratory analyses will link murine kinematic signatures to human stop-signal/reversal learning metrics. By elucidating strain-specific (C57BL/6 vs. CD1) circuit mechanisms and delivering translatable biomarkers (AL, RA, PE, SSRT, kinematics), this work addresses a critical gap in understanding neuropsychiatric disorders like ADHD (impulsivity) and schizophrenia (perseveration). The study’s innovative combination of recurrent neural network theory, FED3-based assays, and New Approach Methodology (NAM)-compliant computational modeling pioneers high-risk, high-reward tools for circuit dissection, fully aligning with NIH’s 2025 priorities.

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.

Characterizing adipocyte heterogeneity in response to metabolic stress

Project Summary Adipose tissue is a central player in metabolism, storing energy healthily under normal conditions but becoming dysfunctional when overloaded. This can lead to the development of metabolic disease, most notably insulin resistance and type 2 diabetes (T2D). Understanding the contribution of adipose tissue to these complications requires knowledge of the individual cell types within adipose tissue and how they respond to different metabolic conditions. My previous work used single nucleus RNA sequencing to profile the cell types in adipose tissue and identified a number of subpopulations of white adipocytes that are differentially associated with clinical characteristics such as body mass index. In this grant, I now aim to better understand how a diverse array of stimuli influences adipocyte development and specification, the role that intra-individual variation plays in the response to these stimuli, and a better understanding of the relationship of adipocyte state to the development of metabolic disease. To do this, I propose using a model in which I can study human adipocyte development and function in mice to perform experiments such as high fat diet and cold exposure that are well-characterized in mice but not in humans. By performing experiments using cells from humans with a range of starting clinical characteristics, I can determine what changes will happen in response to a stimuli in all individuals verses those that only occur in specific populations. The experience that I have in characterizing adipocytes and adipose tissue both at the bench and computationally make me uniquely positioned to answer these questions. Taken together, these studies can test the behavior of adipocyte subpopulations from different people and under different conditions, ultimately leading to a better understanding of how subpopulations develop and, eventually, how we can target these populations to treat metabolic disease.

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.

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.

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.

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.

Consciousness at the edge of chaos

Over the last 20 years, neuroimaging and electrophysiology techniques have become central to understanding the mechanisms that accompany loss and recovery of consciousness. Much of this research is performed in the context of healthy individuals with neurotypical brain dynamics. Yet, a true understanding of how consciousness emerges from the joint action of neurons has to account for how severely pathological brains, often showing phenotypes typical of unconsciousness, can nonetheless generate a subjective viewpoint. In this presentation, I will start from the context of Disorders of Consciousness and will discuss recent work aimed at finding generalizable signatures of consciousness that are reliable across a spectrum of brain electrophysiological phenotypes focusing in particular on the notion of edge-of-chaos criticality.

Competing Rhythms: Understanding and Modulating Auditory Neural Entrainment

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.

How the presynapse forms and functions”

Nervous system function relies on the polarized architecture of neurons, established by directional transport of pre- and postsynaptic cargoes. While delivery of postsynaptic components depends on the secretory pathway, the identity of the membrane compartment(s) that supply presynaptic active zone (AZ) and synaptic vesicle (SV) proteins is largely unknown. I will discuss our recent advances in our understanding of how key components of the presynaptic machinery for neurotransmitter release are transported and assembled focussing on our studies in genome-engineered human induced pluripotent stem cell-derived neurons. Specifically, I will focus on the composition and cell biological identity of the axonal transport vesicles that shuttle key components of neurotransmission to nascent synapses and on machinery for axonal transport and its control by signaling lipids. Our studies identify a crucial mechanism mediating the delivery of SV and active zone proteins to developing synapses and reveal connections to neurological disorders. In the second part of my talk, I will discuss how exocytosis and endocytosis are coupled to maintain presynaptic membrane homeostasis. I will present unpublished data regarding the role of membrane tension in the coupling of exocytosis and endocytosis at synapses. We have identified an endocytic BAR domain protein that is capable of sensing alterations in membrane tension caused by the exocytotic fusion of SVs to initiate compensatory endocytosis to restore plasma membrane area. Interference with this mechanism results in defects in the coupling of presynaptic exocytosis and SV recycling at human synapses.

The Systems Vision Science Summer School & Symposium, August 11 – 22, 2025, Tuebingen, Germany

Applications are invited for our third edition of Systems Vision Science (SVS) summer school since 2023, designed for everyone interested in gaining a systems level understanding of biological vision. We plan a coherent, graduate-level, syllabus on the integration of experimental data with theory and models, featuring lectures, guided exercises and discussion sessions. The summer school will end with a Systems Vision Science symposium on frontier topics on August 20-22, with additional invited and contributed presentations and posters. Call for contributions and participations to the symposium will be sent out spring of 2025. All summer school participants are invited to attend, and welcome to submit contributions to the symposium.

The Systems Vision Science Summer School & Symposium, August 11 – 22, 2025, Tuebingen, Germany

Applications are invited for our third edition of Systems Vision Science (SVS) summer school since 2023, designed for everyone interested in gaining a systems level understanding of biological vision. We plan a coherent, graduate-level, syllabus on the integration of experimental data with theory and models, featuring lectures, guided exercises and discussion sessions. The summer school will end with a Systems Vision Science symposium on frontier topics on August 20-22, with additional invited and contributed presentations and posters. Call for contributions and participations to the symposium will be sent out spring of 2025. All summer school participants are invited to attend, and welcome to submit contributions to the symposium.

The Systems Vision Science Summer School & Symposium, August 11 – 22, 2025, Tuebingen, Germany

Applications are invited for our third edition of Systems Vision Science (SVS) summer school since 2023, designed for everyone interested in gaining a systems level understanding of biological vision. We plan a coherent, graduate-level, syllabus on the integration of experimental data with theory and models, featuring lectures, guided exercises and discussion sessions. The summer school will end with a Systems Vision Science symposium on frontier topics on August 20-22, with additional invited and contributed presentations and posters. Call for contributions and participations to the symposium will be sent out spring of 2025. All summer school participants are invited to attend, and welcome to submit contributions to the symposium.

The Systems Vision Science Summer School & Symposium, August 11 – 22, 2025, Tuebingen, Germany

Applications are invited for our third edition of Systems Vision Science (SVS) summer school since 2023, designed for everyone interested in gaining a systems level understanding of biological vision. We plan a coherent, graduate-level, syllabus on the integration of experimental data with theory and models, featuring lectures, guided exercises and discussion sessions. The summer school will end with a Systems Vision Science symposium on frontier topics on August 20-22, with additional invited and contributed presentations and posters. Call for contributions and participations to the symposium will be sent out spring of 2025. All summer school participants are invited to attend, and welcome to submit contributions to the symposium.

The Systems Vision Science Summer School & Symposium, August 11 – 22, 2025, Tuebingen, Germany

Applications are invited for our third edition of Systems Vision Science (SVS) summer school since 2023, designed for everyone interested in gaining a systems level understanding of biological vision. We plan a coherent, graduate-level, syllabus on the integration of experimental data with theory and models, featuring lectures, guided exercises and discussion sessions. The summer school will end with a Systems Vision Science symposium on frontier topics on August 20-22, with additional invited and contributed presentations and posters. Call for contributions and participations to the symposium will be sent out spring of 2025. All summer school participants are invited to attend, and welcome to submit contributions to the symposium.

The Systems Vision Science Summer School & Symposium, August 11 – 22, 2025, Tuebingen, Germany

Applications are invited for our third edition of Systems Vision Science (SVS) summer school since 2023, designed for everyone interested in gaining a systems level understanding of biological vision. We plan a coherent, graduate-level, syllabus on the integration of experimental data with theory and models, featuring lectures, guided exercises and discussion sessions. The summer school will end with a Systems Vision Science symposium on frontier topics on August 20-22, with additional invited and contributed presentations and posters. Call for contributions and participations to the symposium will be sent out spring of 2025. All summer school participants are invited to attend, and welcome to submit contributions to the symposium.

The Systems Vision Science Summer School & Symposium, August 11 – 22, 2025, Tuebingen, Germany

Applications are invited for our third edition of Systems Vision Science (SVS) summer school since 2023, designed for everyone interested in gaining a systems level understanding of biological vision. We plan a coherent, graduate-level, syllabus on the integration of experimental data with theory and models, featuring lectures, guided exercises and discussion sessions. The summer school will end with a Systems Vision Science symposium on frontier topics on August 20-22, with additional invited and contributed presentations and posters. Call for contributions and participations to the symposium will be sent out spring of 2025. All summer school participants are invited to attend, and welcome to submit contributions to the symposium.

The Systems Vision Science Summer School & Symposium, August 11 – 22, 2025, Tuebingen, Germany

Applications are invited for our third edition of Systems Vision Science (SVS) summer school since 2023, designed for everyone interested in gaining a systems level understanding of biological vision. We plan a coherent, graduate-level, syllabus on the integration of experimental data with theory and models, featuring lectures, guided exercises and discussion sessions. The summer school will end with a Systems Vision Science symposium on frontier topics on August 20-22, with additional invited and contributed presentations and posters. Call for contributions and participations to the symposium will be sent out spring of 2025. All summer school participants are invited to attend, and welcome to submit contributions to the symposium.

The Systems Vision Science Summer School & Symposium, August 11 – 22, 2025, Tuebingen, Germany

Applications are invited for our third edition of Systems Vision Science (SVS) summer school since 2023, designed for everyone interested in gaining a systems level understanding of biological vision. We plan a coherent, graduate-level, syllabus on the integration of experimental data with theory and models, featuring lectures, guided exercises and discussion sessions. The summer school will end with a Systems Vision Science symposium on frontier topics on August 20-22, with additional invited and contributed presentations and posters. Call for contributions and participations to the symposium will be sent out spring of 2025. All summer school participants are invited to attend, and welcome to submit contributions to the symposium.

The Systems Vision Science Summer School & Symposium, August 11 – 22, 2025, Tuebingen, Germany

Applications are invited for our third edition of Systems Vision Science (SVS) summer school since 2023, designed for everyone interested in gaining a systems level understanding of biological vision. We plan a coherent, graduate-level, syllabus on the integration of experimental data with theory and models, featuring lectures, guided exercises and discussion sessions. The summer school will end with a Systems Vision Science symposium on frontier topics on August 20-22, with additional invited and contributed presentations and posters. Call for contributions and participations to the symposium will be sent out spring of 2025. All summer school participants are invited to attend, and welcome to submit contributions to the symposium.

Understanding reward-guided learning using large-scale datasets

Understanding the neural mechanisms of reward-guided learning is a long-standing goal of computational neuroscience. Recent methodological innovations enable us to collect ever larger neural and behavioral datasets. This presents opportunities to achieve greater understanding of learning in the brain at scale, as well as methodological challenges. In the first part of the talk, I will discuss our recent insights into the mechanisms by which zebra finch songbirds learn to sing. Dopamine has been long thought to guide reward-based trial-and-error learning by encoding reward prediction errors. However, it is unknown whether the learning of natural behaviours, such as developmental vocal learning, occurs through dopamine-based reinforcement. Longitudinal recordings of dopamine and bird songs reveal that dopamine activity is indeed consistent with encoding a reward prediction error during naturalistic learning. In the second part of the talk, I will talk about recent work we are doing at DeepMind to develop tools for automatically discovering interpretable models of behavior directly from animal choice data. Our method, dubbed CogFunSearch, uses LLMs within an evolutionary search process in order to "discover" novel models in the form of Python programs that excel at accurately predicting animal behavior during reward-guided learning. The discovered programs reveal novel patterns of learning and choice behavior that update our understanding of how the brain solves reinforcement learning problems.

Neurobiological constraints on learning: bug or feature?

Understanding how brains learn requires bridging evidence across scales—from behaviour and neural circuits to cells, synapses, and molecules. In our work, we use computational modelling and data analysis to explore how the physical properties of neurons and neural circuits constrain learning. These include limits imposed by brain wiring, energy availability, molecular noise, and the 3D structure of dendritic spines. In this talk I will describe one such project testing if wiring motifs from fly brain connectomes can improve performance of reservoir computers, a type of recurrent neural network. The hope is that these insights into brain learning will lead to improved learning algorithms for artificial systems.

Understanding reward-guided learning using large-scale datasets

Understanding the neural mechanisms of reward-guided learning is a long-standing goal of computational neuroscience. Recent methodological innovations enable us to collect ever larger neural and behavioral datasets. This presents opportunities to achieve greater understanding of learning in the brain at scale, as well as methodological challenges. In the first part of the talk, I will discuss our recent insights into the mechanisms by which zebra finch songbirds learn to sing. Dopamine has been long thought to guide reward-based trial-and-error learning by encoding reward prediction errors. However, it is unknown whether the learning of natural behaviours, such as developmental vocal learning, occurs through dopamine-based reinforcement. Longitudinal recordings of dopamine and bird songs reveal that dopamine activity is indeed consistent with encoding a reward prediction error during naturalistic learning. In the second part of the talk, I will talk about recent work we are doing at DeepMind to develop tools for automatically discovering interpretable models of behavior directly from animal choice data. Our method, dubbed CogFunSearch, uses LLMs within an evolutionary search process in order to "discover" novel models in the form of Python programs that excel at accurately predicting animal behavior during reward-guided learning. The discovered programs reveal novel patterns of learning and choice behavior that update our understanding of how the brain solves reinforcement learning problems.

Harnessing Big Data in Neuroscience: From Mapping Brain Connectivity to Predicting Traumatic Brain Injury

Neuroscience is experiencing unprecedented growth in dataset size both within individual brains and across populations. Large-scale, multimodal datasets are transforming our understanding of brain structure and function, creating opportunities to address previously unexplored questions. However, managing this increasing data volume requires new training and technology approaches. Modern data technologies are reshaping neuroscience by enabling researchers to tackle complex questions within a Ph.D. or postdoctoral timeframe. I will discuss cloud-based platforms such as brainlife.io, that provide scalable, reproducible, and accessible computational infrastructure. Modern data technology can democratize neuroscience, accelerate discovery and foster scientific transparency and collaboration. Concrete examples will illustrate how these technologies can be applied to mapping brain connectivity, studying human learning and development, and developing predictive models for traumatic brain injury (TBI). By integrating cloud computing and scalable data-sharing frameworks, neuroscience can become more impactful, inclusive, and data-driven..

Relating circuit dynamics to computation: robustness and dimension-specific computation in cortical dynamics

Neural dynamics represent the hard-to-interpret substrate of circuit computations. Advances in large-scale recordings have highlighted the sheer spatiotemporal complexity of circuit dynamics within and across circuits, portraying in detail the difficulty of interpreting such dynamics and relating it to computation. Indeed, even in extremely simplified experimental conditions, one observes high-dimensional temporal dynamics in the relevant circuits. This complexity can be potentially addressed by the notion that not all changes in population activity have equal meaning, i.e., a small change in the evolution of activity along a particular dimension may have a bigger effect on a given computation than a large change in another. We term such conditions dimension-specific computation. Considering motor preparatory activity in a delayed response task we utilized neural recordings performed simultaneously with optogenetic perturbations to probe circuit dynamics. First, we revealed a remarkable robustness in the detailed evolution of certain dimensions of the population activity, beyond what was thought to be the case experimentally and theoretically. Second, the robust dimension in activity space carries nearly all of the decodable behavioral information whereas other non-robust dimensions contained nearly no decodable information, as if the circuit was setup to make informative dimensions stiff, i.e., resistive to perturbations, leaving uninformative dimensions sloppy, i.e., sensitive to perturbations. Third, we show that this robustness can be achieved by a modular organization of circuitry, whereby modules whose dynamics normally evolve independently can correct each other’s dynamics when an individual module is perturbed, a common design feature in robust systems engineering. Finally, we will recent work extending this framework to understanding the neural dynamics underlying preparation of speech.

Fear learning induces synaptic potentiation between engram neurons in the rat lateral amygdala

Fear learning induces synaptic potentiation between engram neurons in the rat lateral amygdala. This study by Marios Abatis et al. demonstrates how fear conditioning strengthens synaptic connections between engram cells in the lateral amygdala, revealed through optogenetic identification of neuronal ensembles and electrophysiological measurements. The work provides crucial insights into memory formation mechanisms at the synaptic level, with implications for understanding anxiety disorders and developing targeted interventions. Presented by Dr. Kenneth Hayworth, this journal club will explore the paper's methodology linking engram cell reactivation with synaptic plasticity measurements, and discuss implications for memory decoding research.

Impact of High Fat Diet on Central Cardiac Circuits: When The Wanderer is Lost

Cardiac vagal motor drive originates in the brainstem's cardiac vagal motor neurons (CVNs). Despite well-established cardioinhibitory functions in health, our understanding of CVNs in disease is limited. There is a clear connection of cardiovascular regulation with metabolic and energy expenditure systems. Using high fat diet as a model, this talk will explore how metabolic dysfunction impacts the regulation of cardiac tissue through robust inhibition of CVNs. Specifically, it will present an often overlooked modality of inhibition, tonic gamma-aminobuytric acid (GABA) A-type neurotransmission using an array of techniques from single cell patch clamp electrophysiology to transgenic in vivo whole animal physiology. It also will highlight a unique interaction with the delta isoform of protein kinase C to facilitate GABA A-type receptor expression.

What it’s like is all there is: The value of Consciousness

Over the past thirty years or so, cognitive neuroscience has made spectacular progress understanding the biological mechanisms of consciousness. Consciousness science, as this field is now sometimes called, was not only inexistent thirty years ago, but its very name seemed like an oxymoron: how can there be a science of consciousness? And yet, despite this scepticism, we are now equipped with a rich set of sophisticated behavioural paradigms, with an impressive array of techniques making it possible to see the brain in action, and with an ever-growing collection of theories and speculations about the putative biological mechanisms through which information processing becomes conscious. This is all good and fine, even promising, but we also seem to have thrown the baby out with the bathwater, or at least to have forgotten it in the crib: consciousness is not just mechanisms, it’s what it feels like. In other words, while we know thousands of informative studies about access-consciousness, we have little in the way of phenomenal consciousness. But that — what it feels like — is truly what “consciousness” is about. Understanding why it feels like something to be me and nothing (panpsychists notwithstanding) for a stone to be a stone is what the field has always been after. However, while it is relatively easy to study access-consciousness through the contrastive approach applied to reports, it is much less clear how to study phenomenology, its structure and its function. Here, I first overview work on what consciousness does (the "how"). Next, I ask what difference feeling things makes and what function phenomenology might play. I argue that subjective experience has intrinsic value and plays a functional role in everything that we do.

Structural & Functional Neuroplasticity in Children with Hemiplegia

About 30% of children with cerebral palsy have congenital hemiplegia, resulting from periventricular white matter injury, which impairs the use of one hand and disrupts bimanual co-ordination. Congenital hemiplegia has a profound effect on each child's life and, thus, is of great importance to the public health. Changes in brain organization (neuroplasticity) often occur following periventricular white matter injury. These changes vary widely depending on the timing, location, and extent of the injury, as well as the functional system involved. Currently, we have limited knowledge of neuroplasticity in children with congenital hemiplegia. As a result, we provide rehabilitation treatment to these children almost blindly based exclusively on behavioral data. In this talk, I will present recent research evidence of my team on understanding neuroplasticity in children with congenital hemiplegia by using a multimodal neuroimaging approach that combines data from structural and functional neuroimaging methods. I will further present preliminary data regarding functional improvements of upper extremities motor and sensory functions as a result of rehabilitation with a robotic system that involves active participation of the child in a video-game setup. Our research is essential for the development of novel or improved neurological rehabilitation strategies for children with congenital hemiplegia.

Contentopic mapping and object dimensionality - a novel understanding on the organization of object knowledge

Our ability to recognize an object amongst many others is one of the most important features of the human mind. However, object recognition requires tremendous computational effort, as we need to solve a complex and recursive environment with ease and proficiency. This challenging feat is dependent on the implementation of an effective organization of knowledge in the brain. Here I put forth a novel understanding of how object knowledge is organized in the brain, by proposing that the organization of object knowledge follows key object-related dimensions, analogously to how sensory information is organized in the brain. Moreover, I will also put forth that this knowledge is topographically laid out in the cortical surface according to these object-related dimensions that code for different types of representational content – I call this contentopic mapping. I will show a combination of fMRI and behavioral data to support these hypotheses and present a principled way to explore the multidimensionality of object processing.

Rethinking Attention: Dynamic Prioritization

Decades of research on understanding the mechanisms of attentional selection have focused on identifying the units (representations) on which attention operates in order to guide prioritized sensory processing. These attentional units fit neatly to accommodate our understanding of how attention is allocated in a top-down, bottom-up, or historical fashion. In this talk, I will focus on attentional phenomena that are not easily accommodated within current theories of attentional selection – the “attentional platypuses,” as they allude to an observation that within biological taxonomies the platypus does not fit into either mammal or bird categories. Similarly, attentional phenomena that do not fit neatly within current attentional models suggest that current models need to be revised. I list a few instances of the ‘attentional platypuses” and then offer a new approach, the Dynamically Weighted Prioritization, stipulating that multiple factors impinge onto the attentional priority map, each with a corresponding weight. The interaction between factors and their corresponding weights determines the current state of the priority map which subsequently constrains/guides attention allocation. I propose that this new approach should be considered as a supplement to existing models of attention, especially those that emphasize categorical organizations.

Gene regulatory mechanisms of neocortex development and evolution

The neocortex is considered to be the seat of higher cognitive functions in humans. During its evolution, most notably in humans, the neocortex has undergone considerable expansion, which is reflected by an increase in the number of neurons. Neocortical neurons are generated during development by neural stem and progenitor cells. Epigenetic mechanisms play a pivotal role in orchestrating the behaviour of stem cells during development. We are interested in the mechanisms that regulate gene expression in neural stem cells, which have implications for our understanding of neocortex development and evolution, neural stem cell regulation and neurodevelopmental disorders.

LLMs and Human Language Processing

This webinar convened researchers at the intersection of Artificial Intelligence and Neuroscience to investigate how large language models (LLMs) can serve as valuable “model organisms” for understanding human language processing. Presenters showcased evidence that brain recordings (fMRI, MEG, ECoG) acquired while participants read or listened to unconstrained speech can be predicted by representations extracted from state-of-the-art text- and speech-based LLMs. In particular, text-based LLMs tend to align better with higher-level language regions, capturing more semantic aspects, while speech-based LLMs excel at explaining early auditory cortical responses. However, purely low-level features can drive part of these alignments, complicating interpretations. New methods, including perturbation analyses, highlight which linguistic variables matter for each cortical area and time scale. Further, “brain tuning” of LLMs—fine-tuning on measured neural signals—can improve semantic representations and downstream language tasks. Despite open questions about interpretability and exact neural mechanisms, these results demonstrate that LLMs provide a promising framework for probing the computations underlying human language comprehension and production at multiple spatiotemporal scales.

Learning and Memory

This webinar on learning and memory features three experts—Nicolas Brunel, Ashok Litwin-Kumar, and Julijana Gjorgieva—who present theoretical and computational approaches to understanding how neural circuits acquire and store information across different scales. Brunel discusses calcium-based plasticity and how standard “Hebbian-like” plasticity rules inferred from in vitro or in vivo datasets constrain synaptic dynamics, aligning with classical observations (e.g., STDP) and explaining how synaptic connectivity shapes memory. Litwin-Kumar explores insights from the fruit fly connectome, emphasizing how the mushroom body—a key site for associative learning—implements a high-dimensional, random representation of sensory features. Convergent dopaminergic inputs gate plasticity, reflecting a high-dimensional “critic” that refines behavior. Feedback loops within the mushroom body further reveal sophisticated interactions between learning signals and action selection. Gjorgieva examines how activity-dependent plasticity rules shape circuitry from the subcellular (e.g., synaptic clustering on dendrites) to the cortical network level. She demonstrates how spontaneous activity during development, Hebbian competition, and inhibitory-excitatory balance collectively establish connectivity motifs responsible for key computations such as response normalization.

How do we sleep?

There is no consensus on if sleep is for the brain, body or both. But the difference in how we feel following disrupted sleep or having a good night of continuous sleep is striking. Understanding how and why we sleep will likely give insights into many aspects of health. In this talk I will outline our recent work on how the prefrontal cortex can signal to the hypothalamus to regulate sleep preparatory behaviours and sleep itself, and how other brain regions, including the ventral tegmental area, respond to psychosocial stress to induce beneficial sleep. I will also outline our work on examining the function of the glymphatic system, and whether clearance of molecules from the brain is enhanced during sleep or wakefulness.

Understanding the complex behaviors of the ‘simple’ cerebellar circuit

Every movement we make requires us to precisely coordinate muscle activity across our body in space and time. In this talk I will describe our efforts to understand how the brain generates flexible, coordinated movement. We have taken a behavior-centric approach to this problem, starting with the development of quantitative frameworks for mouse locomotion (LocoMouse; Machado et al., eLife 2015, 2020) and locomotor learning, in which mice adapt their locomotor symmetry in response to environmental perturbations (Darmohray et al., Neuron 2019). Combined with genetic circuit dissection, these studies reveal specific, cerebellum-dependent features of these complex, whole-body behaviors. This provides a key entry point for understanding how neural computations within the highly stereotyped cerebellar circuit support the precise coordination of muscle activity in space and time. Finally, I will present recent unpublished data that provide surprising insights into how cerebellar circuits flexibly coordinate whole-body movements in dynamic environments.

Brain-Wide Compositionality and Learning Dynamics in Biological Agents

Biological agents continually reconcile the internal states of their brain circuits with incoming sensory and environmental evidence to evaluate when and how to act. The brains of biological agents, including animals and humans, exploit many evolutionary innovations, chiefly modularity—observable at the level of anatomically-defined brain regions, cortical layers, and cell types among others—that can be repurposed in a compositional manner to endow the animal with a highly flexible behavioral repertoire. Accordingly, their behaviors show their own modularity, yet such behavioral modules seldom correspond directly to traditional notions of modularity in brains. It remains unclear how to link neural and behavioral modularity in a compositional manner. We propose a comprehensive framework—compositional modes—to identify overarching compositionality spanning specialized submodules, such as brain regions. Our framework directly links the behavioral repertoire with distributed patterns of population activity, brain-wide, at multiple concurrent spatial and temporal scales. Using whole-brain recordings of zebrafish brains, we introduce an unsupervised pipeline based on neural network models, constrained by biological data, to reveal highly conserved compositional modes across individuals despite the naturalistic (spontaneous or task-independent) nature of their behaviors. These modes provided a scaffolding for other modes that account for the idiosyncratic behavior of each fish. We then demonstrate experimentally that compositional modes can be manipulated in a consistent manner by behavioral and pharmacological perturbations. Our results demonstrate that even natural behavior in different individuals can be decomposed and understood using a relatively small number of neurobehavioral modules—the compositional modes—and elucidate a compositional neural basis of behavior. This approach aligns with recent progress in understanding how reasoning capabilities and internal representational structures develop over the course of learning or training, offering insights into the modularity and flexibility in artificial and biological agents.

How the brain barriers ensure CNSimmune privilege”

Britta Engelhard’s research is devoted to understanding thefunction of the different brain barriers in regulating CNS immunesurveillance and how their impaired function contributes toneuroinflammatory diseases such as Multiple Sclerosis (MS) orAlzheimer’s disease (AD). Her laboratory combines expertise invascular biology, neuroimmunology and live cell imaging and hasdeveloped sophisticated in vitro and in vivo approaches to studyimmune cell interactions with the brain barriers in health andneuroinflammation.

Cerebrospinal fluid and the meninges : Understanding brain immunology from its borders

Neural mechanisms governing the learning and execution of avoidance behavior

The nervous system orchestrates adaptive behaviors by intricately coordinating responses to internal cues and environmental stimuli. This involves integrating sensory input, managing competing motivational states, and drawing on past experiences to anticipate future outcomes. While traditional models attribute this complexity to interactions between the mesocorticolimbic system and hypothalamic centers, the specific nodes of integration have remained elusive. Recent research, including our own, sheds light on the midline thalamus's overlooked role in this process. We propose that the midline thalamus integrates internal states with memory and emotional signals to guide adaptive behaviors. Our investigations into midline thalamic neuronal circuits have provided crucial insights into the neural mechanisms behind flexibility and adaptability. Understanding these processes is essential for deciphering human behavior and conditions marked by impaired motivation and emotional processing. Our research aims to contribute to this understanding, paving the way for targeted interventions and therapies to address such impairments.

Exploring the cerebral mechanisms of acoustically-challenging speech comprehension - successes, failures and hope

Comprehending speech under acoustically challenging conditions is an everyday task that we can often execute with ease. However, accomplishing this requires the engagement of cognitive resources, such as auditory attention and working memory. The mechanisms that contribute to the robustness of speech comprehension are of substantial interest in the context of hearing mild to moderate hearing impairment, in which affected individuals typically report specific difficulties in understanding speech in background noise. Although hearing aids can help to mitigate this, they do not represent a universal solution, thus, finding alternative interventions is necessary. Given that age-related hearing loss (“presbycusis”) is inevitable, developing new approaches is all the more important in the context of aging populations. Moreover, untreated hearing loss in middle age has been identified as the most significant potentially modifiable predictor of dementia in later life. I will present research that has used a multi-methodological approach (fMRI, EEG, MEG and non-invasive brain stimulation) to try to elucidate the mechanisms that comprise the cognitive “last mile” in speech acousticallychallenging speech comprehension and to find ways to enhance them.

Modelling the fruit fly brain and body

Through recent advances in microscopy, we now have an unprecedented view of the brain and body of the fruit fly Drosophila melanogaster. We now know the connectivity at single neuron resolution across the whole brain. How do we translate these new measurements into a deeper understanding of how the brain processes sensory information and produces behavior? I will describe two computational efforts to model the brain and the body of the fruit fly. First, I will describe a new modeling method which makes highly accurate predictions of neural activity in the fly visual system as measured in the living brain, using only measurements of its connectivity from a dead brain [1], joint work with Jakob Macke. Second, I will describe a whole body physics simulation of the fruit fly which can accurately reproduce its locomotion behaviors, both flight and walking [2], joint work with Google DeepMind.

Characterizing the causal role of large-scale network interactions in supporting complex cognition

Neuroimaging has greatly extended our capacity to study the workings of the human brain. Despite the wealth of knowledge this tool has generated however, there are still critical gaps in our understanding. While tremendous progress has been made in mapping areas of the brain that are specialized for particular stimuli, or cognitive processes, we still know very little about how large-scale interactions between different cortical networks facilitate the integration of information and the execution of complex tasks. Yet even the simplest behavioral tasks are complex, requiring integration over multiple cognitive domains. Our knowledge falls short not only in understanding how this integration takes place, but also in what drives the profound variation in behavior that can be observed on almost every task, even within the typically developing (TD) population. The search for the neural underpinnings of individual differences is important not only philosophically, but also in the service of precision medicine. We approach these questions using a three-pronged approach. First, we create a battery of behavioral tasks from which we can calculate objective measures for different aspects of the behaviors of interest, with sufficient variance across the TD population. Second, using these individual differences in behavior, we identify the neural variance which explains the behavioral variance at the network level. Finally, using covert neurofeedback, we perturb the networks hypothesized to correspond to each of these components, thus directly testing their casual contribution. I will discuss our overall approach, as well as a few of the new directions we are currently pursuing.

Vision Unveiled: Understanding Face Perception in Children Treated for Congenital Blindness

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.

Brain-heart interactions at the edges of consciousness

Various clinical cases have provided evidence linking cardiovascular, neurological, and psychiatric disorders to changes in the brain-heart interaction. Our recent experimental evidence on patients with disorders of consciousness revealed that observing brain-heart interactions helps to detect residual consciousness, even in patients with absence of behavioral signs of consciousness. Those findings support hypotheses suggesting that visceral activity is involved in the neurobiology of consciousness and sum to the existing evidence in healthy participants in which the neural responses to heartbeats reveal perceptual and self-consciousness. Furthermore, the presence of non-linear, complex, and bidirectional communication between brain and heartbeat dynamics can provide further insights into the physiological state of the patient following severe brain injury. These developments on methodologies to analyze brain-heart interactions open new avenues for understanding neural functioning at a large-scale level, uncovering that peripheral bodily activity can influence brain homeostatic processes, cognition, and behavior.

Learning produces a hippocampal cognitive map in the form of an orthogonalized state machine

Cognitive maps confer animals with flexible intelligence by representing spatial, temporal, and abstract relationships that can be used to shape thought, planning, and behavior. Cognitive maps have been observed in the hippocampus, but their algorithmic form and the processes by which they are learned remain obscure. Here, we employed large-scale, longitudinal two-photon calcium imaging to record activity from thousands of neurons in the CA1 region of the hippocampus while mice learned to efficiently collect rewards from two subtly different versions of linear tracks in virtual reality. The results provide a detailed view of the formation of a cognitive map in the hippocampus. Throughout learning, both the animal behavior and hippocampal neural activity progressed through multiple intermediate stages, gradually revealing improved task representation that mirrored improved behavioral efficiency. The learning process led to progressive decorrelations in initially similar hippocampal neural activity within and across tracks, ultimately resulting in orthogonalized representations resembling a state machine capturing the inherent struture of the task. We show that a Hidden Markov Model (HMM) and a biologically plausible recurrent neural network trained using Hebbian learning can both capture core aspects of the learning dynamics and the orthogonalized representational structure in neural activity. In contrast, we show that gradient-based learning of sequence models such as Long Short-Term Memory networks (LSTMs) and Transformers do not naturally produce such orthogonalized representations. We further demonstrate that mice exhibited adaptive behavior in novel task settings, with neural activity reflecting flexible deployment of the state machine. These findings shed light on the mathematical form of cognitive maps, the learning rules that sculpt them, and the algorithms that promote adaptive behavior in animals. The work thus charts a course toward a deeper understanding of biological intelligence and offers insights toward developing more robust learning algorithms in artificial intelligence.

Unifying the mechanisms of hippocampal episodic memory and prefrontal working memory

Remembering events in the past is crucial to intelligent behaviour. Flexible memory retrieval, beyond simple recall, requires a model of how events relate to one another. Two key brain systems are implicated in this process: the hippocampal episodic memory (EM) system and the prefrontal working memory (WM) system. While an understanding of the hippocampal system, from computation to algorithm and representation, is emerging, less is understood about how the prefrontal WM system can give rise to flexible computations beyond simple memory retrieval, and even less is understood about how the two systems relate to each other. Here we develop a mathematical theory relating the algorithms and representations of EM and WM by showing a duality between storing memories in synapses versus neural activity. In doing so, we develop a formal theory of the algorithm and representation of prefrontal WM as structured, and controllable, neural subspaces (termed activity slots). By building models using this formalism, we elucidate the differences, similarities, and trade-offs between the hippocampal and prefrontal algorithms. Lastly, we show that several prefrontal representations in tasks ranging from list learning to cue dependent recall are unified as controllable activity slots. Our results unify frontal and temporal representations of memory, and offer a new basis for understanding the prefrontal representation of WM

From rare Genetic cohorts of Parkinsonism to biomarkers and to understanding broader neurodegenerative disease mechanisms

Using Adversarial Collaboration to Harness Collective Intelligence

There are many mysteries in the universe. One of the most significant, often considered the final frontier in science, is understanding how our subjective experience, or consciousness, emerges from the collective action of neurons in biological systems. While substantial progress has been made over the past decades, a unified and widely accepted explanation of the neural mechanisms underpinning consciousness remains elusive. The field is rife with theories that frequently provide contradictory explanations of the phenomenon. To accelerate progress, we have adopted a new model of science: adversarial collaboration in team science. Our goal is to test theories of consciousness in an adversarial setting. Adversarial collaboration offers a unique way to bolster creativity and rigor in scientific research by merging the expertise of teams with diverse viewpoints. Ideally, we aim to harness collective intelligence, embracing various perspectives, to expedite the uncovering of scientific truths. In this talk, I will highlight the effectiveness (and challenges) of this approach using selected case studies, showcasing its potential to counter biases, challenge traditional viewpoints, and foster innovative thought. Through the joint design of experiments, teams incorporate a competitive aspect, ensuring comprehensive exploration of problems. This method underscores the importance of structured conflict and diversity in propelling scientific advancement and innovation.

Neuromodulation of striatal D1 cells shapes BOLD fluctuations in anatomically connected thalamic and cortical regions

Understanding how macroscale brain dynamics are shaped by microscale mechanisms is crucial in neuroscience. We investigate this relationship in animal models by directly manipulating cellular properties and measuring whole-brain responses using resting-state fMRI. Specifically, we explore the impact of chemogenetically neuromodulating D1 medium spiny neurons in the dorsomedial caudate putamen (CPdm) on BOLD dynamics within a striato-thalamo-cortical circuit in mice. Our findings indicate that CPdm neuromodulation alters BOLD dynamics in thalamic subregions projecting to the dorsomedial striatum, influencing both local and inter-regional connectivity in cortical areas. This study contributes to understanding structure–function relationships in shaping inter-regional communication between subcortical and cortical levels.

Machine learning for reconstructing, understanding and intervening on neural interactions

Bayesian expectation in the perception of the timing of stimulus sequences

In the current virtual journal club Dr Di Luca will present findings from a series of psychophysical investigations where he measured sensitivity and bias in the perception of the timing of stimuli. He will present how improved detection with longer sequences and biases in reporting isochrony can be accounted for by optimal statistical predictions. Among his findings was also that the timing of stimuli that occasionally deviate from a regularly paced sequence is perceptually distorted to appear more regular. Such change depends on whether the context these sequences are presented is also regular. Dr Di Luca will present a Bayesian model for the combination of dynamically updated expectations, in the form of a priori probability, with incoming sensory information. These findings contribute to the understanding of how the brain processes temporal information to shape perceptual experiences.

Piecing together the puzzle of emotional consciousness

Conscious emotional experiences are very rich in their nature, and can encompass anything ranging from the most intense panic when facing immediate threat, to the overwhelming love felt when meeting your newborn. It is then no surprise that capturing all aspects of emotional consciousness, such as intensity, valence, and bodily responses, into one theory has become the topic of much debate. Key questions in the field concern how we can actually measure emotions and which type of experiments can help us distill the neural correlates of emotional consciousness. In this talk I will give a brief overview of theories of emotional consciousness and where they disagree, after which I will dive into the evidence proposed to support these theories. Along the way I will discuss to what extent studying emotional consciousness is ‘special’ and will suggest several tools and experimental contrasts we have at our disposal to further our understanding on this intriguing topic.

Neuronal population interactions between brain areas

Most brain functions involve interactions among multiple, distinct areas or nuclei. Yet our understanding of how populations of neurons in interconnected brain areas communicate is in its infancy. Using a population approach, we found that interactions between early visual cortical areas (V1 and V2) occur through a low-dimensional bottleneck, termed a communication subspace. In this talk, I will focus on the statistical methods we have developed for studying interactions between brain areas. First, I will describe Delayed Latents Across Groups (DLAG), designed to disentangle concurrent, bi-directional (i.e., feedforward and feedback) interactions between areas. Second, I will describe an extension of DLAG applicable to three or more areas, and demonstrate its utility for studying simultaneous Neuropixels recordings in areas V1, V2, and V3. Our results provide a framework for understanding how neuronal population activity is gated and selectively routed across brain areas.

Understanding rat behavior in a complex task via non-deterministic policies

COSYNE 2022

Understanding rat behavior in a complex task via non-deterministic policies

COSYNE 2022

Towards understanding the microcircuit in monkey primary visual cortex in-vivo

COSYNE 2023

Understanding Auditory Cortex with Deep Neural Networks

COSYNE 2023

Understanding network dynamics of compact assemblies of neurons in zebrafish larvae optic tectum during spontaneous activation

COSYNE 2023

Probing Motion-Form Interactions in the Macaque Inferior Temporal Cortex and Artificial Neural Networks for Complex Scene Understanding

COSYNE 2025

Understanding Bi-directional Changes and Rotation in Mitral Cell Population Codes During Repeated Odor Exposure

COSYNE 2025

Understanding the effects of neural perturbations using cell-type dynamical systems

COSYNE 2025

Understanding sensory-motor integration in the PIVC through ensemble analysis

COSYNE 2025

Understanding stochastic decision-making in competitive multi-agent environments

COSYNE 2025

The blood-brain barrier permeability in a preclinical in vivo model of periodontitis and depression: A step forward in the understanding of inflammatory mechanisms

Characterization of neuronal models of glucocerebrosidase deficiency: towards a better understanding of Parkinson's disease

A combined TDP43-Tau cellular model for the understanding of LATE-NC Proteinopathy

The effect of problem contextualization in physics : a neurophysiological approach towards a better understanding of problem solving

How emotional states shape perception: A mechanistic understanding of state dependent auditory processing

Encoding of spatial long term memories in a neural network: understanding fear generalization with miniscope calcium imaging

From neural correlations to population codes and back- understanding a duality in the topological analysis of neural data

Nuclear ATXN3 DUB physiological substrates: towards understanding their role in the context of Machado-Joseph Disease

Souris-City: a multi-environment for understanding the social basis of inter-individual variability and drug vulnerability in mice

A stochastic simulation experiment for a better understanding of uncertainty in self-organizing maps

Studying group behavior in the wild as a new avenue for understanding social brain

Toward a better understanding of ITM2B pathogenicity in a specific retinal dystrophy, and its potential role in mitochondria

Understanding arrhinia and its molecular pathways: where are we now?

Understanding the behavioural and morphological changes in an alpha-synuclein rat model of Parkinson´s disease

Understanding the causes and consequences of individual variation in striatal Oxtr expression in prairie voles: a molecular phenotype relevant to ASD

Understanding the communicative value of ultrasonic calls in male and female laboratory mice: lessons from mouse models of neurodevelopmental disorders

Understanding the emotional consequences of chronic pain: Insight from ACC-LHb pathway

Understanding the impact of early microexon misregulation on zebrafish sleep

Understanding individual differences in reward-guided learning as an efficient adaptation to task uncertainty and computational noise

Understanding how interneurons in the medial prefrontal cortex modulate associative recognition memory

Understanding the interplay between fear anxiety and the stereotypic behavior of grooming in DAT-Cre mice

Understanding the role of VIP+ inhibitory interneurons in the retina

Understanding the role of SYNGAP1 in Parvalbumin-expressing GABAergic circuit development and function

Combining electrophysiology, tissue clearing, and light sheet microscopy for an integrated approach towards brain circuit understanding

FENS Forum 2024

NETSseq enhances the understanding of cerebellar transcriptomic changes in ataxia-telangiectasia

FENS Forum 2024

Neurocognitive profiles of childhood maltreatment subtypes: Understanding the effects of childhood emotional abuse on the adult social brain

FENS Forum 2024

ProB13 and ProD20: Understanding the role of two potential novel retinal amacrine cell types

FENS Forum 2024

Understanding the altered brain metabolism and oxidative stress: Insights into metabolic syndrome and premature aging in a novel obese rodent model

FENS Forum 2024

Understanding CaV2.1 dysfunction in neurological disorders: Insights from novel CRISPR/Cas9 mouse model and iPSC-derived neurons

FENS Forum 2024

Understanding the consequences of prenatal CBD exposure on insular cortex neurons: Sex-specific alterations and the loss of subregional functional differentiation

FENS Forum 2024