Vivo

From B-cell decisions to antibody repertoires

PROJECT SUMMARY/ABSTRACT Vaccine responses are highly variable across the population and not without risk for debilitating side-effects. Antibody-mediated immunity is generated by a Darwinian process to generate B-cells that contain B-cell receptors (BCR) that have high affinity for the pathogen-derived antigen, while also eliminating B-cells that happen to react to self-antigens. This process depends on cell fate decisions such as (i) death vs survival, (ii) entry into a proliferative program, (iii) differentiation into antibody-secreting plasma cells. According to clonal selection theory, B-cell fate decisions are made based on the genetically encoded affinity of the the BCR to the antigen (Signal 1) and the cognate T-cells’ TCR to the antigen peptide (Signal 2). However, single-cell resolution studies have revealed that fate decisions of genetically identical B-cells are remarkably heterogeneous. Our studies of the previous funding period revealed that B-cell epigenetic heterogeneity is in fact dynamically controlled: it is generated during the selection process but remains largely stable during the proliferative burst. This leads to our newly proposed Aim 1 to examine how the dynamic control of epigenetic state variability affects antibody responses. An innovative multi-scale model of Darwinian evolution directs and interprets experimental studies by life cell video microscopy in vitro and in immunization studies in vivo. Our previous studies also found that B-cells are capable of sensing the time gap between signal 1 and 2, suggesting a temporal proofreading mechanism for negative selection. This leads to newly proposed Aim 2 which seeks to identify the regulatory circuits that control the stringency of negative selection, as well as contextual germinal center (GC) cytokines that could be manipulable in vivo. These in silico and in vitro studies are followed by in vivo immunization to extend their physiological relevance. Finally, in Aim 3, we will ask what determines the time-gap of signal1 and signal 2, which occur in the immune- induced structure of the GC. We will develop a new model that simulates B-cell fate decisions as a function of their interactions with antigen-presenting stromal cells and T-cells that may be cognate or non-cognate. Model simulations will be used to interpret spatial transcriptomic data to test different adjuvants and predictions will be tested in in vivo immunization studies. With mouse models of inflammation and aging we will examine how adjuvants alter vaccine efficacy and risk.

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.

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.

Optimization of a novel and effective antiviral agent targeting Zika NS4B

This project focuses on developing novel anti-Zika virus (ZIKV) compounds targeting the NS4B protein, which is crucial for viral replication. ZIKV poses a significant medical challenge due to its potential for severe pathogenic outcomes, such as congenital Zika syndrome and Guillain-Barré Syndrome. Furthermore, its pandemic potential has been increasing with the expansion of carrier mosquito habitats. The project aims to address the urgent need for anti-ZIKV therapeutics that could greatly reduce severity of symptoms and minimize vertical and community transmissions. We have identified a novel small-molecule series with a benzamide scaffold through a cell-based, antiviral ultra-high-throughput screen. This series demonstrates strong potency against ZIKV without measurable cytotoxicity or non-specific antiviral effects, justifying this scaffold as a lead series for further development. Preliminary mechanism-of-action studies, utilizing genetic, biochemical, and virological assays, suggest that this series may inhibit the formation of the ZIKV viral replicase complex by interfering with NS4B. Our goal for this project is to develop a preclinical therapeutic candidate for ZIKV that demonstrates promising therapeutic activity following oral administration in ZIKV-infected mice, at a dosage that shows no clinical toxicity. The project has the following significant and novel objectives: 1) Optimize the benzamide lead for potency and drug-likeness; 2) Develop a lead candidate and a backup compound with optimized pharmacokinetic, pharmacodynamic, and toxicity profiles; 3) Determine the molecular mechanisms of action of the benzamide series using novel structural approaches to assist medicinal chemistry studies; 4) Evaluate the in vivo therapeutic efficacy and safety in mouse models and develop the best therapeutic regime. This project seeks to develop effective antivirals for ZIKV with high retention in the blood and central nervous system (CNS) and high oral bioavailability. The expected successful outcomes will provide significant advancements in ZIKV therapeutics and open new avenues for treating other flavivirus infections

Optimizing CD45-Targeted Astatine-211-Radioimmunotherapy for Malignant and Non-Malignant Blood Disorders

ABSTRACT CD45 is expressed on almost all normal and neoplastic hematopoietic cells but not on non-blood cells and has, therefore, been pursued as a drug target. Initially centered on augmenting conditioning before hematopoietic cell transplantation (HCT) for blood cancers, there is increasing interest in expanding CD45-directed therapies into other settings, with radioimmunotherapy (RIT) being the major therapeutic modality so far. Investigators at our institution pioneered CD45 RIT with b-emitters such as iodine-131 (131I) using the murine monoclonal antibody (mAb), BC8. A phase 3 trial testing 131I-BC8 (131I-apamistamab [Iomab-B]) with allogeneic HCT in older adults with relapsed/refractory acute myeloid leukemia showed improved outcomes over conventional care, validating this approach. More recently, attention has shifted toward a-emitters that deliver substantially higher decay energies over much shorter distances than b-emitters, rendering them more suitable for precise and potent target cell killing. In our work, we focus on astatine-211 (211At) for its ideal half-life and decay without a-emitting daughters. For clinical application, mAbs are conjugated with the bifunctional boron cage molecule, isothiocyantophenethyl-ureido-closo-decaborate(2-) (B10-NCS), to enable stable protein astatination. Three early-phase trials testing 211At-BC8-B10 as augmentation of HCT conditioning for patients with malignant and non-malignant blood disorders are ongoing, with emerging data indicating significant anti-tumor efficacy. Nonetheless, relapses still occur. Other important limitations include marked infusion toxicities and human antimouse antibody (HAMA) responses related to the murine nature of BC8 and dimer formation after 211At labeling of mAb-B10 conjugates with tissue residualization from 211At atom oxidation. The latter may contribute to the risk of liver cell injury, the dose limiting extramedullary toxicity of CD45 RIT. As a first step toward our goal of optimizing CD45 RIT, we have raised new, fully human CD45 mAbs as basis for novel therapeutics. In preliminary in vivo studies in immunodeficient mice, we found some of these mAbs to have greater anti-tumor efficacy than a humanized version of BC8 (HuBC8) we generated as a reference mAb. We will now conduct comparative in vivo CD45+ cell targeting (“biodistribution”) and anti-tumor efficacy studies to select a lead candidate mAb for clinical application and use protein engineering to maximize the selectivity and efficacy of targeted radiation delivery. We will use immunodeficient mice xenotransplanted with human leukemia cells for this purpose as no human approaches are available and in vitro testing is inadequate to measure both the targeting and biologic RIT effects on human leukemia cells. Mice provide the in vivo milieu needed for comprehensive evaluation. Development of improved mAb astatination methodologies to minimize off-target toxicities of 211At-RIT will further increase therapy specificity and reduce toxicity. In parallel, we will conduct genome-scale, unbiased target identification/validation studies to identify partner drugs for rational combination therapies aimed at enhancing the anti-tumor efficacy of 211At-CD45 RIT.

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.

Weak Cell Adhesion is a Prognostic Signature of Invasive Cancer

Project Summary Despite early detection, low-grade and localized breast cancers such as ductal carcinoma in situ (DCIS) can relapse in up to 20% of cases despite standard of care. For DCIS, relapse affects over 12,000 U.S. women annually and has increased 60% in the last 40 years. Current diagnostic assessments including histopathological markers often miss early disseminating cells, lack specificity, or cannot distinguish cancer from non-cancer cells in the stroma. Hence there is an unmet need for cancer diagnostic technologies that employ radically different characterization methods. For example, significant physical differences exist between metastasizing and benign breast cancer cells, owing to metastasizing cells detaching from the primary tumor, migrating through the surrounding stroma, intravasating and extravasating, and ultimately engrafting in distant tissues. We recently demonstrated that cancer cells with weaker adhesion migrate faster and metastasize more frequently in murine breast cancer models than strongly adherent cells. In a small pilot study of human breast tumors, we also observed that the abundance of weakly adherent (WA) cells scales with disease severity; subpopulations from invasive carcinomas were the least adherent. However, a subset of DCIS cases displayed much less adhesion, suggesting that these patients may have a tumor subpopulation that progresses to metastatic disease despite standard-of-care treatment. Weak adhesion is a defining physical characteristic of tumors, but to establish their role in initiation, metastasis, and patient outcomes, we will leverage model systems and our newly patented adhesion technology to answer these fundamental questions of cancer biology and clinical translation. To understand the impact of adhesion on cancer progression, we will evaluate the tumor-initiating potential of WA versus strongly adherent (SA) tumor cells in a murine breast cancer model before confirming how weak adhesion advantages cells to cause secondary disease using bioengineered in vitro models. In dissecting the stages of metastasis where WA cells exhibit advantages, e.g., recapitulating stromal niche, transendothelial migration, and tissue-specific colonization, we will identify mechanisms that enable WA cells to thrive and evaluate therapeutic targets that disrupt these pathways. Finally, we will analyze the adhesion profiles of resected tumors and stroma from 80 breast cancer patients with DCIS or invasive disease. Adhesion data will be correlated with conventional assessment methods and ultimately with patient outcomes, e.g., disease-free and progression-free intervals. We anticipate that the DCIS subpopulation that aligns with the adhesion signature of invasive carcinomas will have shorter intervals and survival time. This integrated study design bridges mouse models, mechanistic bioengineering assays, and human samples to clarify the metastatic potential and prognostic value of WA breast cancer cells. Our use of mouse models in this grant is required to study the interactions among tumor cells, immune cells, vasculature, and stromal tissues that drive tumor formation in vivo. Bioengineered in vitro systems lack the complexity to ask such questions and using injected tumor cells is not possible in humans.

Modulating the Action of Cylindrical Proteases to Eliminate Neisseria Gonorrhea and Chlamydia Trachomatis Infections

Project Summary/Abstract Sexually transmitted bacteria diseases caused by Chlamydia trachomatis (Ctr) and Neisseria gonorrhoeae (NG) are the two most common sexually transmitted bacterial diseases. The infections caused by these pathogens may result in infertility, ectopic pregnancy, blindness, and perinatal mortality. Over 1.70 M cases of chlamydia and 0.65 M cases of drug-resistant gonorrhea are reported yearly in the US. Women with gonorrhea are co- infected with chlamydia in 17.6%–57.9% of cases, while women with chlamydia are co-infected with gonorrhea in 2.1%–17.2% of cases. These infections are treated with broad spectrum antibiotics, which can favor the development of resistance on NG/CTr but also in other bacteria, or damage the microbiota, diminishing its protective function and allowing bacteria and viruses to infect the patient. The Caseinolytic protease (ClpP) proteolytic machinery regulates protein turnover and homeostasis and is key in bacterial growth and development The machinery consists of the proteolytic unit (the ClpP) and its chaperone (ClpX), which transports proteins to be degraded, and it is termed the ClpXP. Our theory is that molecules that inhibit the action of the ClpX chaperone can become efficient antibacterial agents against both pathogens. We have found that the dihydrothiazepines can erradicate both pathogens and prevent the action of the ClpXP complex. Our goal is to advance the dihydrothiazepines as selective agents against Ctr and NG infections. To develop these therapeutic agents, we have envisioned four specific aims. Specific Aim 1. Synthesis and Optimization of the Pharmacophore. Our goal is to use computational models to design dihydrothiazepines molecule that will be synthesized, purified, and characterized using chemical techniques. The molecules will be tested against Ctr and NG and their toxicity against human cells evaluated. Also, we will determine their effect in other bacterial, including those from the microbiota. Specific Aim 2. Assessment of Stability and In Vivo Activity. We will study the stability of the most active molecules under various conditions. Then, we will study the pharmacokinetics, biodistribution , and antibacterial activity against Ctr and NG in mice. Specific Aim 3. Target Validation and Effect. We will study the ability of the compounds to inhibit the activity of ClpX using a luciferase assay and to block protein degradation. We will try grow crystal of the protein and the molecule and will study if the molecules prevent the assembly of the ClpXP system. Finally, we will assess the ability of the bacteria to develop resistance to the molecules.

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

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

The role of endogenous chimeric mRNA encoded GasderminD fusion proteins in immunity

Project Summary: Programmed inflammatory cell death, or pyroptosis, is a crucial innate defense mechanism that protects hosts against infection and orchestrates subsequent immune responses. Central to this process is Gasdermin D (GSDMD), a protein that forms plasma membrane pores upon activation, enabling the release of pro- inflammatory cytokines such as IL-1β and driving cell lysis. Although GSDMD-mediated pyroptosis has been conventionally understood to be controlled mainly at the post-translational level, through proteolytic cleavage by inflammatory caspases, we have discovered compelling evidence that alternative RNA processing may introduce additional, previously unappreciated complexity in GSDMD regulation. Our laboratories have developed and optimized a highly innovative long-read direct RNA sequencing pipeline, which bypasses conventional cDNA synthesis to avoid artifacts and enables unbiased discovery of native chimeric mRNA (chRNA) in mammalian cells. Using this approach, we have uncovered a remarkably diverse repertoire of chRNA species, including over a thousand unique fusions in murine macrophages and more than two thousand in human inflamed tissues. Among the chRNA found in mice, we identified a chRNA joining the effector domain of GSDMD with a novel C-terminal region encoded by Tmem106a, giving rise to the GSDMD:TMEM106A fusion protein. Functional studies demonstrate that GSDMD:TMEM106A is not only produced in response to inflammatory signals in macrophages but is critical for GSDMD-dependent cytokine release and optimal pyroptosis. Genetic loss of GSDMD:TMEM106A in mice results in reduced cytokine secretion and increased susceptibility to bacterial infection, while in vivo delivery of Gsdmd:Tmem106a mRNA is sufficient for protective immunity. Intriguingly, we have also identified a putative human counterpart, GSDMD:S100A6, which is highly inducible in colon biopsies from patients with inflammatory bowel disease. In this application, we propose a comprehensive exploration of this newly defined class of naturally occurring GSDMD fusion proteins. The specific aims are: (1) to elucidate the subcellular localization, protein-protein interactions, and pore-forming function of GSDMD:TMEM106A during canonical and non-canonical inflammasome activation; (2) to determine the transcriptomic, proteomic, and physiological consequences of GSDMD chRNA expression in vivo during infection, sepsis, and inflammatory disease, and to validate and functionally characterize GSDMD:S100A6 in relevant immune and barrier cell populations. Collectively, this work will establish chimeric splicing as a fundamental source of immunoregulatory protein diversity, redefining the landscape of cell death control in the immune system. By revealing new layers of gasdermin regulation and function, our studies have the potential to identify novel therapeutic strategies for infectious, auto-inflammatory, and immune-mediated diseases.

Causal mechanisms driving germline predisposition to myeloproliferative disorders

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

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.

Regulation of neutrophil endoplasmic reticulum stress response by IRE1a

Project Summary/Abstract: The lungs are exposed to pathogens and environmental toxins that trigger stress and cause numerous respiratory diseases. Effective host defenses against lung infection by bacterial pathogens, including methicillin- resistant Staphylococcus aureus (MRSA), rely on innate immune cells including neutrophils, prominent early responders to sites of infection. If host defenses are ineffective, MRSA causes serious lung infection, resulting in severe morbidity and a significant economic burden on healthcare facilities, where it is endemic. MRSA infections have a mortality rate of up to 14% and an estimated $500 million in healthcare costs in the US alone. Increasing resistance to vancomycin, the last resort antibiotic for MRSA infections, underscore the urgent need for innovative treatment approaches. Although directly targeting pathogens with antibiotics has been a successful approach for treating infections, many pathogens, including MRSA, eventually will become resistant to these drugs. As an alternative, immunomodulatory strategies to enhance host defenses, such as those shown to be effective against cancer cells, have the potential for treating drug-resistant pathogen infections. Recently, we showed that the inositol-requiring enzyme 1-α (IRE1α), an endoplasmic reticulum (ER) stress sensor, is required for clearance of MRSA in a murine skin abscess model, where neutrophils are robustly recruited to the site of infection. Further, IRE1α coordinates signaling events upstream of calcium (Ca2+) mobilization, histone citrullination, and production of mitochondrial reactive oxygen species (mitoROS), all of which are important for neutrophil inflammatory responses including the formation of antimicrobial neutrophil extracellular traps (NETs). Because excessive neutrophil activation and NET release can be detrimental to vital organs, it is not clear whether neutrophil IRE1α-mediated stress responses aid or impede the resolution of infection in the lungs. While IRE1α activation has been linked to the development of lung fibrosis through the regulation of alveolar epithelial- to-mesenchymal transition in the context of chronic inflammatory diseases, its role in pulmonary neutrophil defenses is unknown. Thus, there is a gap in our knowledge of how cellular stress responses modulate pulmonary neutrophil defenses and infection outcomes in the lungs. The overarching goal of this proposal is to elucidate the mechanisms by which neutrophil IRE1α signaling influences production of mitoROS and Ca2+ mobilization to drive NET release, injure lungs, and regulate pulmonary host defense against MRSA. We will accomplish the following Aims: (1) Define the molecular mechanisms underlying IRE1α-mediated mitoROS hyperactivation of human and mouse primary neutrophils and excessive NET release, and (2) Elucidate the role of neutrophil IRE1α signaling in excessive NET release, lung injury, and immunity in vivo using a MRSA pneumonia infection mouse model. These studies will yield mechanistic insight into how IRE1α-driven ER stress responses impact pulmonary neutrophil defenses and lung injury revealing potential targets for anti-microbial immunotherapies.

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.

Investigating the nonlinear complex dynamics of the tuft cell-microbiome cross-talk: the impact of feedback loops on immune regulation, microbial modulation and response to tissue insults

Project Abstract Tuft cells (TCs) are specialized chemosensory epithelial cells that are emerging as critical regulators of intestinal homeostasis. Named over 70 years ago based on their distinct morphology, a defined function for TCs was only elucidated in the last decade. TCs in the small intestine sense succinate from helminths to initiate type 2 immune responses that mediate parasite expulsion. Recently, we discovered a novel physiologic function for TCs in the colon, where their role had been considered minimal. Succinate, a key microbial metabolite, is produced by colonic microbiota as both a precursor to other metabolites and a cross-feeding fuel source for pathogens. TCs respond to succinate by secreting interleukin-25 (IL-25), which activates type 2 cytokine- producing lymphocytes (T2Ls), amplifying TC expansion and reinforcing barrier function. We recently demonstrated that this SPB–TC–IL-25–T2L feedback loop is essential for protection against pathogen-induced colitis. Our preliminary data further suggest that TCs actively promote colonization by succinate-producing bacteria (SPBs), establishing positive feedback on TC-supporting microbes, while other epithelial cells such as goblet cells (GCs) and Paneth cells (PCs) may exert complementary or counterbalancing influences. Supported by new modeling insights, we hypothesize that these epithelial–immune–microbiome interactions form coordinated feedback loops that collectively optimize intestinal resilience. These loops may create a dynamic, multi-stable system that flexibly transitions between homeostatic and hyperplastic states, buffering against microbial fluctuations and pathogenic insults while preventing uncontrolled type 2 inflammation. Using a combination of mathematical modeling and experimental validation, we will develop a multi- layered systems framework to explore how epithelial–immune–microbial feedbacks shape resilience or breakdown in clinically relevant models of colonic infection and inflammation. Our three Aims will (1) develop, calibrate, and validate a mathematical model that integrates TCs, GCs, PCs, SPBs, and SCBs; (2) define the immunological circuits governing epithelial–microbiome equilibrium; and (3) determine how epithelial feedbacks regulate microbial community structure and resilience. In line with NIH’s new initiative to prioritize human-based research, our proposal combines computational modeling, human colonic organoids, and complementary mouse models. Organoid experiments will provide human-relevant data for model calibration, while in vivo studies validate systemic predictions, ensuring both rigor and translational relevance while minimizing reliance on animal models. This work will generate interoperable models that integrate epithelial, microbial, and immune networks, providing predictive insight into intestinal outcomes under homeostatic, infectious, and inflammatory conditions and informing therapeutic strategies for microbiome-targeted interventions.

Factors Driving Wear and Implant Failure in Total Shoulder Arthroplasty

Polyethylene (PE) wear and implant-related failure remain leading causes of revision in total shoulder arthroplasty (TSA), a procedure which now surpasses the growth rate of hip and knee arthroplasty. Both anatomic (aTSA) and reverse (rTSA) TSA outcomes are heavily influenced by complex interactions between rotator cuff function, scapular motion, implant design, and patient-specific loading—factors not adequately captured in current preclinical implant testing standards. Emerging evidence suggests that PE wear progression in TSA is highly dependent on shoulder kinematics, joint loading, implant positioning, and individual patient factors. Nonetheless, data on in vivo motion and load profiles remain sparse, and few tools exist to link these profiles to clinically relevant wear patterns or associated periprosthetic inflammatory tissue responses. Accordingly, the primary objective of this project is to develop validated, patient-specific models that predict PE wear in TSA and identify modifiable surgical, design, and rehabilitation targets to improve implant longevity and restore patient mobility. Additionally, we will establish histopathological hallmarks that indicate TSA failure caused by PE wear debris. Our central hypothesis is that specific shoulder kinematics and joint loading drive distinct PE wear patterns in TSA associated with mechanical failure or inflammatory-mediated osteolysis, depending on implant design and positioning. To achieve the overall objective of this work, shoulder motions and muscle excitations across 25 activities of daily living will be collected at pre-op and post-op (>6 months) in both aTSA and rTSA patients, with long-term follow-up of patient-reported outcomes via validated surveys (5 years). Unsupervised machine learning will categorize patients into movement-based phenotypes, which will then inform a multi-scale modeling framework to estimate in vivo shoulder joint loads and implant wear across the varying movement strategies. Predicted wear patterns will be validated using state-of-the-art preclinical wear simulators. Simultaneously, we will quantify how patient, surgical, and implant factors contribute to wear in retrieved TSA components (>400 samples), correlating imaging-based wear patterns with clinical outcomes, patient-reported function, inflammatory tissue responses, and radiographic indications of loosening. For that purpose, we will establish benchmarks of TSA wear rates and introduce a new histopathological approach augmented by infrared spectroscopic imaging. This work is innovative because we are linking patient-specific movement patterns following TSA with multi-scale computational models to predict PE wear, breaking the current approaches of using generic motions and loads in existing testing standards. This work will produce the first integrated, publicly available database of TSA kinematics, joint loading, and PE wear patterns and rates, along with validated computational tools to inform implant design, surgical planning, rehabilitation strategies, and personalized risk assessment. Ultimately, these advances will improve functional outcomes and long-term success for TSA patients and enable better preclinical testing methods and standards.

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.

Validating Causality of Disputed Mitochondrial Variants in Inborn Errors of Metabolism

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

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.

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

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

Tbx4-Driven Pulmonary Hypertension: Mechanisms and Therapeutic Targets

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

Dosing and Deployment Trial: A Home-based Optokinetic Treatment for Ipsilesional Gaze Deviation

Stroke can have devastating consequences including ipsilesional gaze deviation (IGD), which directly impacts mobility and falls. IGD, a hallmark sign of spatial neglect (SN), is a major predictor of poor recovery and can persist after inpatient rehabilitation with targeted treatments. Our preliminary data show that more than half of stroke survivors who have SN at the time of admission to inpatient rehabilitation still have SN at time of discharge, even after treatment. Therefore, because of the challenges of the traditional rehabilitation paradigm we need to bring treatments into the home setting. We plan to examine the feasibility and deployment of Eyemove, an optokinetic stimulation treatment, which induces brain neural plasticity and improves spatial exploration, in turn reducing SN symptoms, including IGD. We hypothesize that by treating IGD, improvements in mobility and fall risk scores will occur, as participants can now interact with the space that was previously “neglected”. Here, we propose to test the following aims with 50 community-dwelling individuals with SN, by identifying the practical dosage associated with mobility improvement: Aim 1 will determine feasibility and acceptability of home deployment of Eyemove. We will collect qualitative information from stroke survivors and their care partners, to determine their pre-treatment and post-treatment perspectives of this home treatment. Aim 2 will determine whether Eyemove in the home is associated with improved mobility-related outcomes (including risk of falls) and to evaluate sufficient dosing. We will randomize participants into either 3 or 5 sessions of a 40-minute treatment given over a week-long intervention period. The primary outcome will be the Mobility Assessment Course and secondary outcomes will be the Stroke Assessment of Fall Risk and the Life Space Assessment. For Aim 1, we expect to learn practical suggestions for home implementation and obtain reports of post-experience enthusiasm and acceptability for specific aspects of the intervention. Our hypotheses for Aim 2 are: 1a-- After controlling for pre-treatment score changes (T2-T1), the intervention (T3) will lead to improved mobility/ fall risk compared to baseline (T1), regardless of treatment group; 1b-- The amount of mobility/ fall risk improvement (T3-T1) in the 3- session and 5-session groups will be different. The expected findings will provide critical insight into the use of Eyemove for spatial neglect remediation. Results from this research will be used to develop a subsequent R01 proposal that uses pragmatic, randomized clinical trial methods to determine the efficacy of Eyemove, in order to provide an effective, accessible treatment to remediate SN at home and improve individuals’ ability to move without spatial bias or risk of falls.

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.

Host-pathogen-microbiome interactions in Mycoplasma genitalium pathology and treatment: experiments in a 3D organotypic cervical epithelium model to strengthen clinical guidelines

ABSTRACT Mycoplasma genitalium (MG) is an emerging sexually transmitted pathogen whose clinical outcomes in women are poorly understood. Unlike other bacterial sexually transmitted infections (STI), the CDC does not recommend MG screening for asymptomatic women because it is unclear how often asymptomatic MG leads to adverse reproductive outcomes like cervicitis, which can lead to further adverse outcomes, including pelvic inflammatory disease, infertility, and ectopic pregnancy. Epidemiologic data on MG and cervicitis are mixed, and mechanistic data primarily come from models that did not faithfully recapitulate in vivo cervical microphysiological conditions. Key elements they lacked are cervical mucus, which mediates host-pathogen interactions, and the cervicovaginal microbiota. The microbiota appears to contribute to MG outcomes, and our preliminary epidemiologic data indicate that MG and bacterial vaginosis (BV) may synergize to promote cervicitis. MG care is further complicated by its ongoing rise in antibiotic resistance. Resistance-guided therapy and novel antibiotics improve treatment outcomes, but these are not available in the US. Recent clinical and in vitro data indicate that metronidazole and tinidazole, two antibiotics that are available in the US and used to treat BV, may hold promise for improving MG treatment outcomes. The overall objective of this R21 is to generate robust experimental data to clarify MG pathology, evaluate potential therapies, and inform more thorough and actionable clinical recommendations. We developed an innovative in vitro 3D organotypic model of the cervical epithelium that is ideally suited for investigating MG pathology, host-MG-microbiota interactions, and potential therapies. The model uses primary human cervical cells and better recapitulates cervical epithelial structure and physiology (including cervical mucus production) than prior 2D models. It also allows for simultaneous STI infection and co- culture of live cervicovaginal microbiota. Using the 3D organotypic cervical epithelium model, we will determine if MG causes microbiota-dependent cervical epithelial damage, a hallmark of cervicitis (Aim 1), and we will test if metronidazole and tinidazole arrest MG infection (Aim 2). In both Aims, we will interrogate the potential mediating role of the microbiota by inoculating models with live representative cervicovaginal microbiota, and we will assess host-MG-microbiota interactions via transcriptomics. We hypothesize that a polymicrobial BV-like microbiota will exacerbate MG-induced cervical epithelial damage, and removal of a polymicrobial BV microbiota will partially mediate metronidazole’s and tinidazole’s anti-MG activity. The proposed Aims have high translational potential and will provide crucial pre-clinical evidence to inform more thorough and actionable MG testing and treatment guidelines and improve reproductive health outcomes. This R21 will generate some of the first experimental data on MG-host and MG-microbiota interactions, which we will use to support an R01 to validate these interactions during in vivo MG infection and identify novel therapeutic targets for MG.

Targeting subtype specification as a driver of PDAC health disparities

PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease that is refractory to current treatment strategies due in part to adaptive mechanisms of chemoresistance. Racial health disparities also confound the treatment and care of these patients. Blacks (people with African genetic ancestry) have significantly higher incidence rates of PDAC and decreased survival times compared to Caucasians (White genetic ancestry) even after socioeconomic status and tumor stages are controlled. Therefore, it is possible different racial groups exhibit unique molecular characteristics in PDAC tumors that contribute to these health disparities. The unique molecular characteristics that distinguish PDAC tumors between racial groups exhibiting disparities have the potential to identify new therapeutic targets. In a previous study, we identified 4 distinct subtypes of PDAC (Metabolic, Progenitor-like, Proliferative, and Inflammatory) that can be distinguished using multivariate analysis of quantitative proteomic data. While these PDAC subtypes are predictive of therapeutic response, this has not yet been analyzed in disparity factor balanced studies. We have examined the proteomes of primary PDAC tumors using quantitative mass spectrometry and identified unique protein signatures for Blacks and Whites. PDAC tumors from Black patients display features consistent with the Inflammatory subtype of PDAC, which is characterized by an inflamed microenvironment expressing complement proteins that can promote resistance to chemotherapy. Therefore, it is possible that race influences subtype and Blacks could preferentially develop the more aggressive and treatment refractory Inflammatory subtype. Strategies are needed to modulate subtype to improve response to chemotherapy. Toward this goal, our proteomic analysis identified polycomb repressor complex 1 (PRC1) protein RNF2 as being upregulated in PDACs from Blacks compared to Whites. We have also discovered that RNF2 regulates mRNA expression of the PDAC subtype specification factor GATA6 and inhibiting RNF2 promotes a molecular shift toward the more chemosensitive Classical subtype of PDAC. Therapeutic targeting can be achieved with Tazemetostat that inhibits the upstream PRC2 to prevent RNF2 binding the GATA6 promoter leading to its increased expression. Additionally, the Inflammatory subtype characterized by innate immune complement protein activation could be targeted with another FDA approved drug, Avacopan, which has not previously been studied in PDAC. Therefore, the Specific Aims of this proposal are designed to: 1) Evaluate the extent to which Tazemetostat treatment impacts chemotherapy-induced subtype plasticity in patient derived organoids; and 2) To determine the extent to which strategies targeting pathways associated with PDAC disparities affect progression and subtype characteristics in vivo. The successful completion of these aims has the potential to be moved quickly into phase I clinical trials since both Tazemetostat and Avacopan are FDA approved drugs. Furthermore, if successful, this project has the potential to mitigate health disparities in PDAC and broadly improve patient outcomes by implementing new precision interventions. The mouse models we propose faithfully recapitulate pancreatic cancer's clinical syndrome, histopathology and molecular properties, including the often-unique features of the stromal and immune responses that constitute the complex desmoplasia of this disease, which cannot be addressed using in vitro model systems

A Novel Mitochondrial-Targeted Inhibitor of NLRP3 Inflammasome Activation

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

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.

Targeted Prodrug Cytokines for Metastatic Breast Cancer Immunotherapy

Project Summary. Our approach directly addresses key limitations in targeting and treating metastatic breast cancer, where we propose the selective activation of modular immune-modulating cytokines within the hypoxic and ROS-active TME for delivery across the BBB, providing the necessary pre-clinical data for future clinical translation. The in vitro and in vivo investigations of this novel immunotherapeutic in immunocompetent models will allow our team to study the interplay between tumor-driven immune activation, cytokine signaling, and anti-tumor immunity in both primary and metastatic sites, and establish a robust groundwork for subsequent clinical validation within the OSUCCC. This proposal addresses two key challenges in developing a novel immunotherapy strategy for breast cancer by answering two hypotheses: (1) can a modular immunotherapy platform with tumor-selective activation of prodrug recombinant cytokines overcome these limitations in drug delivery, and (2) can the development of nanobody-cytokine fusions that can selectively target primary breast cancer tumors and cross the BBB to reach metastatic tumor sites? The first hypothesis focuses on achieving tumor environment-specific activation of prodrug-based recombinant cytokines. Protein cytokines are highly potent, and while others have tried to block their activity using a fused genetic linker to ‘mask’ functionality, no one has yet attempted to use a non-canonical-based chemical strategy to achieve this inhibition. Immune-modulating cytokines will be recombinantly expressed with integrated ncAAs that block cytokine activity until the function is regenerated in the breast cancer TME. Once the cytokine activity is controlled, our second hypothesis will be to achieve selective delivery of the cytokine via fusion to nanobodies. While success has been found in targeting primary tumors in drug and protein delivery, a key challenge remains in reaching secondary metastatic tumors in hard-to-reach sites (i.e., brain). Engineered nanobodies, with affinity for breast cancer tumors and the ability to bind to BBB transcytosis receptors, will enable selective delivery to metastatic breast-to-brain tumors, resulting in tumor- specific activation, immune responses, and improved therapeutic outcomes. This system can significantly improve therapeutic outcomes for patients with mBC by integrating selective activation and delivery mechanisms to reduce off-target effects and enhance tumor-specific immune responses in both primary and secondary metastatic tumor sites. Optimizing drug delivery systems to tune immune responses could offer more effective and less invasive treatment options when compared to traditional and engineered cell-based approaches. Our momentum towards precision medicine and targeted therapies holds significant promise for improving outcomes for mBC patients, and has the potential to serve as a pan-cancer treatment for aggressive metastatic cancers from the following aims: (1) generating a modular platform for tumor-specific activation of prodrug cytokines, (2) evaluating cytokine delivery and anti-cancer immune phenotypes in mBC.

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

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

Neutralizing persistent IFN-I to improve HIV-specific CAR T cell therapy

PROJECT SUMMARY A critical hurdle to further improving the quality of life for people living with HIV (PLWH) is the need to resolve the residual immune activation and inflammation that persists even in those taking effective antiretroviral therapy (ART), which suppresses HIV replication. This unresolved and persistent immune activation is associated with increased type-I interferon (IFN-I) signaling, and increased incidence of comorbidities. Encouragingly, reports demonstrate that blocking IFN-I signaling in animal models of HIV infection can reduce HIV reservoirs and restore T cell immune function. We hypothesize that blocking IFN-I would likewise augment engineered T cell-based therapies against HIV, such as chimeric antigen receptor (CAR) T cells. Our prior work has demonstrated that when engineered to express both the 4-1BB and CD28 costimulatory domains and protected from HIV infection, HIV-specific CD4 ectodomain CAR T cells can reduce acute viremia, prevent CD4+ T cell loss, and reduce viral burden in the tissues of HIV-infected humanized mice. However, the reduction of plasma viral loads was ultimately transient, suggesting that the potency of HIV-specific CAR T cells should be further optimized for clinical translation. Our preliminary data highlights interferon-beta (IFNb) as a key immunosuppressive IFN-I negatively regulating CAR T cell proliferation, and we demonstrate that neutralizing IFNb in vivo enhanced the engraftment and persistence of HIV-specific CAR T cells adoptively transferred into HIV-infected ART- suppressed humanized mice. This proposal will interrogate whether IFNb neutralization augments CAR T cell therapy through 1) identifying the mechanism(s) by which chronic IFNb exposure mediates HIV-specific CAR T cell dysfunction, and 2) determining the effect of neutralizing IFNb on CAR T cell function and persistence in HIV infection in vivo. The proposed aims seek to develop the neutralization of IFNb as a novel immunotherapy approach to maximize the potency of HIV-specific CAR T cells aimed at achieving a functional HIV cure.

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.

Primary cilia protein IFT88 governs smooth muscle phenotype and vascular remodeling

Project Summary/Abstract Cardiovascular disease remains the leading cause of death in the United States, accounting for nearly 1 million deaths in 2022. Vascular diseases such as atherosclerosis, aneurysm, and coronary artery disease are regulated largely by smooth muscle cells (SMCs) residing in the blood vessel wall. The central dogma of vascular SMC biology is that differentiated cells can de-differentiate and give rise to a spectrum of alternative phenotypes promoting invasion, proliferation, fibrosis, and inflammation, but the mechanisms regulating SMC phenotypic transitions are poorly understood. Intraflagellar transport 88 (IFT88) is an essential protein for the formation of primary cilia, centriole-associated plasma membrane organelles that project into the extracellular milieu and regulate cell cycle reentry and responses to stimuli like growth factors and mechanical strain. Non- ciliary functions of IFT88 also include progression of the cell cycle checkpoint and polarized motility, both of which are functionally critical for SMC-mediated vascular remodeling. Little is known about the functional role of the primary cilia in SMCs and the role of the essential cilia protein IFT88 in regulating SMC phenotype. To address this gap in knowledge, my postdoctoral studies focus on the role of IFT88 in the context of intimal hyperplasia (K99). During the independent phase (R00), I will apply these findings to arteriovenous fistula (AVF) maturation, a surgical intervention often required for dialysis individuals with polycystic kidney disease (PKD), an IFT88 loss-of-function disease. I will test my central hypothesis that cilia are key regulators of SMC phenotype in three Specific Aims: 1) determine the role of IFT88-dependent SMC primary cilia in mechanotransduction of extracellular matrix (ECM) stiffness (K99), 2) determine the role of IFT88 in pathological intimal hyperplasia (K99), and 3) test whether SMC IFT88 expression is required for adaptive remodeling of grafted veins following AVF placement (R00). Overall, we propose that IFT88+ ciliated SMC represent an unidentified subclass of the SMC phenotype spectrum that is primarily responsible for vascular remodeling and is an attractive potential target for treatment of vascular diseases. Building on strong existing collaborations, we have formed a research and mentoring team with expertise in SMC pathophysiology, primary cilia biology, mechanobiology, AVF surgery, and PKD to complete the proposed aims. The additional training in cell-ECM interactions (Aim 1, K99), in vivo murine ligation injury and in vivo cilia imaging (Aim 2, K99), and AVF surgery and PKD pathology (Aim 3, R00) will be indispensable for preparing the PI, Dr. O’Brien, for his career as an independent investigator. Completion of the proposed aims will also contribute directly to an understanding of the function of IFT88-dependent primary cilia in SMCs and may likely identify novel therapeutic targets for treatment of vascular diseases.

Targeting the fibrogenic ECM as an alternative approach to treating IPF

Project Abstract Idiopathic pulmonary fibrosis and, more broadly, progressive pulmonary fibrosis are wound healing disorders whose hallmark is unorganized and unchecked extracellular matrix (ECM) deposition leading to scarring/stiffening of the lung interstitium. A highly complex, multicellular process, the generation of scar itself is primarily a function of activated fibroblasts with contributions from multiple subpopulations and non-fibroblastic cells. Myofibroblasts, the contractile cohort of activated fibroblasts, physically perturb (i.e. stretch) the local ECM microenvironment, which we have recently shown triggers site-specific, stretch-dependent conformational changes within the ECM protein fibronectin. We have previously demonstrated that a specific stretch-induced conformational change in the critical receptor binding domain of fibronectin triggers a cellular “integrin switch”, a stark change in the ECM receptors used by cells to engage fibronectin. This integrin switch is sufficient to drive activation of naïve lung fibroblasts, acquisition of mesenchymal characteristics in alveolar epithelial cells, and pathogenic remodeling of vascular structures. In this proposal we hypothesize that fibronectin displays a stretch- dependent conformational change specifically in regions of active lung fibrogenesis and that this conformational change disrupts homeostatic integrin binding dynamics in fibroblasts, leading to their acquisition of a pro-fibrogenic phenotype and transcriptional program. We address this hypothesis in a systematic way through three proposed aims. The first aim focuses on quantifying the presence and spatial localization of the stretch-induced conformational change within a cohort of lung fibrosis patient tissue samples, determining if it represents a consistent marker of active fibrogenic regions and elucidation of critical microenvironmental signatures that further expand our understanding of the impact of fibronectin's integrin switch in driving disease. In the second aim we will begin to unravel the molecular mechanism explaining how the integrin switch that emerges because of the stretch-induced conformational change drives fibroblast activation and fibrogenic gene programs using both idealized in vitro culture systems as well as ex vivo human disease tissue models. Finally, in the third aim we will explore the therapeutic potential of binding and blocking this specific stretch-induced conformation of fibronectin using a promising new and potential antibody drug in both in vivo and ex vivo models of disease.

Spike train structure of cortical transcriptomic populations in vivo

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

Memory Decoding Journal Club: Distinct synaptic plasticity rules operate across dendritic compartments in vivo during learning

Distinct synaptic plasticity rules operate across dendritic compartments in vivo during learning

The Direct Impact Of Amyloid-Beta Oligomers On Neuronal Activity And Neurotransmitter Releases On In Vivo Analysis

Retinal input integration in excitatory and inhibitory neurons in the mouse superior colliculus in vivo

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.

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 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.

In vivo scalable investigation of gene functions in the brain

Epileptic micronetworks and their clinical relevance

A core aspect of clinical epileptology revolves around relating epileptic field potentials to underlying neural sources (e.g. an “epileptogenic focus”). Yet still, how neural population activity relates to epileptic field potentials and ultimately clinical phenomenology, remains far from being understood. After a brief overview on this topic, this seminar will focus on unpublished work, with an emphasis on seizure-related focal spreading depression. The presented results will include hippocampal and neocortical chronic in vivo two-photon population imaging and local field potential recordings of epileptic micronetworks in mice, in the context of viral encephalitis or optogenetic stimulation. The findings are corroborated by invasive depth electrode recordings (macroelectrodes and BF microwires) in epilepsy patients during pre-surgical evaluation. The presented work carries general implications for clinical epileptology, and basic epilepsy research.

Seizure control by electrical stimulation: parameters and mechanisms

Seizure suppression by deep brain stimulation (DBS) applies high frequency stimulation (HFS) to grey matter to block seizures. In this presentation, I will present the results of a different method that employs low frequency stimulation (LFS) (1 to 10Hz) of white matter tracts to prevent seizures. The approach has been shown to be effective in the hippocampus by stimulating the ventral and dorsal hippocampal commissure in both animal and human studies respectively for mesial temporal lobe seizures. A similar stimulation paradigm has been shown to be effective at controlling focal cortical seizures in rats with corpus callosum stimulation. This stimulation targets the axons of the corpus callosum innervating the focal zone at low frequencies (5 to 10Hz) and has been shown to significantly reduce both seizure and spike frequency. The mechanisms of this suppression paradigm have been elucidated with in-vitro studies and involve the activation of two long-lasting inhibitory potentials GABAB and sAHP. LFS mechanisms are similar in both hippocampus and cortical brain slices. Additionally, the results show that LFS does not block seizures but rather decreases the excitability of the tissue to prevent seizures. Three methods of seizure suppression, LFS applied to fiber tracts, HFS applied to focal zone and stimulation of the anterior nucleus of the thalamus (ANT) were compared directly in the same animal in an in-vivo epilepsy model. The results indicate that LFS generated a significantly higher level of suppression, indicating LFS of white matter tract could be a useful addition as a stimulation paradigm for the treatment of epilepsy.

Astrocyte reprogramming / activation and brain homeostasis

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

Cellular crosstalk in Neurodevelopmental Disorders

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

Rodents to Investigate the Neural Basis of Audiovisual Temporal Processing and Perception

To form a coherent perception of the world around us, we are constantly processing and integrating sensory information from multiple modalities. In fact, when auditory and visual stimuli occur within ~100 ms of each other, individuals tend to perceive the stimuli as a single event, even though they occurred separately. In recent years, our lab, and others, have developed rat models of audiovisual temporal perception using behavioural tasks such as temporal order judgments (TOJs) and synchrony judgments (SJs). While these rodent models demonstrate metrics that are consistent with humans (e.g., perceived simultaneity, temporal acuity), we have sought to confirm whether rodents demonstrate the hallmarks of audiovisual temporal perception, such as predictable shifts in their perception based on experience and sensitivity to alterations in neurochemistry. Ultimately, our findings indicate that rats serve as an excellent model to study the neural mechanisms underlying audiovisual temporal perception, which to date remains relativity unknown. Using our validated translational audiovisual behavioural tasks, in combination with optogenetics, neuropharmacology and in vivo electrophysiology, we aim to uncover the mechanisms by which inhibitory neurotransmission and top-down circuits finely control ones’ perception. This research will significantly advance our understanding of the neuronal circuitry underlying audiovisual temporal perception, and will be the first to establish the role of interneurons in regulating the synchronized neural activity that is thought to contribute to the precise binding of audiovisual stimuli.

In vivo direct imaging of neuronal activity at high temporospatial resolution

Advanced noninvasive neuroimaging methods provide valuable information on the brain function, but they have obvious pros and cons in terms of temporal and spatial resolution. Functional magnetic resonance imaging (fMRI) using blood-oxygenation-level-dependent (BOLD) effect provides good spatial resolution in the order of millimeters, but has a poor temporal resolution in the order of seconds due to slow hemodynamic responses to neuronal activation, providing indirect information on neuronal activity. In contrast, electroencephalography (EEG) and magnetoencephalography (MEG) provide excellent temporal resolution in the millisecond range, but spatial information is limited to centimeter scales. Therefore, there has been a longstanding demand for noninvasive brain imaging methods capable of detecting neuronal activity at both high temporal and spatial resolution. In this talk, I will introduce a novel approach that enables Direct Imaging of Neuronal Activity (DIANA) using MRI that can dynamically image neuronal spiking activity in milliseconds precision, achieved by data acquisition scheme of rapid 2D line scan synchronized with periodically applied functional stimuli. DIANA was demonstrated through in vivo mouse brain imaging on a 9.4T animal scanner during electrical whisker-pad stimulation. DIANA with milliseconds temporal resolution had high correlations with neuronal spike activities, which could also be applied in capturing the sequential propagation of neuronal activity along the thalamocortical pathway of brain networks. In terms of the contrast mechanism, DIANA was almost unaffected by hemodynamic responses, but was subject to changes in membrane potential-associated tissue relaxation times such as T2 relaxation time. DIANA is expected to break new ground in brain science by providing an in-depth understanding of the hierarchical functional organization of the brain, including the spatiotemporal dynamics of neural networks.

Mechanisms Underlying the Persistence of Cancer-Related Fatigue

Cancer-related fatigue is a prominent and debilitating side effect of cancer and its treatment. It can develop prior to diagnosis, generally peaks during cancer treatment, and can persist long after treatment completion. Its mechanisms are multifactorial, and its expression is highly variable. Unfortunately, treatment options are limited. Our research uses syngeneic murine models of cancer and cisplatin-based chemotherapy to better understand these mechanisms. Our data indicate that both peripherally and centrally processes may contribute to the developmental of fatigue. These processes include metabolic alterations, mitochondrial dysfunction, pre-cachexia, and inflammation. However, our data has revealed that behavioral fatigue can persist even after the toxicity associated with cancer and its treatment recover. For example, running during cancer treatment attenuates kidney toxicity while also delaying recovery from fatigue-like behavior. Additionally, administration of anesthetics known to disrupt memory consolidation at the time treatment can promote recovery, and treatment-related cues can re-instate fatigue after recovery. Cancer-related fatigue can also promote habitual behavioral patterns, as observed using a devaluation task. We interpret this data to suggest that limit metabolic resources during cancer promote the utilization of habit-based behavioral strategies that serve to maintain fatigue behavior into survivorship. This line of work is exciting as it points us toward novel interventional targets for the treatment of persistent cancer-related fatigue.

PIEZO2 in somatosensory neurons coordinates gastrointestinal transit

The transit of food through the gastrointestinal tract is critical for nutrient absorption and survival, and the gastrointestinal tract has the ability to initiate motility reflexes triggered by luminal distention. This complex function depends on the crosstalk between extrinsic and intrinsic neuronal innervation within the intestine, as well as local specialized enteroendocrine cells. However, the molecular mechanisms and the subset of sensory neurons underlying the initiation and regulation of intestinal motility remain largely unknown. Here, we show that humans lacking PIEZO2 exhibit impaired bowel sensation and motility. Piezo2 in mouse dorsal root but not nodose ganglia is required to sense gut content, and this activity slows down food transit rates in the stomach, small intestine, and colon. Indeed, Piezo2 is directly required to detect colon distension in vivo. Our study unveils the mechanosensory mechanisms that regulate the transit of luminal contents throughout the gut, which is a critical process to ensure proper digestion, nutrient absorption, and waste removal. These findings set the foundation of future work to identify the highly regulated interactions between sensory neurons, enteric neurons and non- neuronal cells that control gastrointestinal motility.

Bridging the gap between artificial models and cortical circuits

Artificial neural networks simplify complex biological circuits into tractable models for computational exploration and experimentation. However, the simplification of artificial models also undermines their applicability to real brain dynamics. Typical efforts to address this mismatch add complexity to increasingly unwieldy models. Here, we take a different approach; by reducing the complexity of a biological cortical culture, we aim to distil the essential factors of neuronal dynamics and plasticity. We leverage recent advances in growing neurons from human induced pluripotent stem cells (hiPSCs) to analyse ex vivo cortical cultures with only two distinct excitatory and inhibitory neuron populations. Over 6 weeks of development, we record from thousands of neurons using high-density microelectrode arrays (HD-MEAs) that allow access to individual neurons and the broader population dynamics. We compare these dynamics to two-population artificial networks of single-compartment neurons with random sparse connections and show that they produce similar dynamics. Specifically, our model captures the firing and bursting statistics of the cultures. Moreover, tightly integrating models and cultures allows us to evaluate the impact of changing architectures over weeks of development, with and without external stimuli. Broadly, the use of simplified cortical cultures enables us to use the repertoire of theoretical neuroscience techniques established over the past decades on artificial network models. Our approach of deriving neural networks from human cells also allows us, for the first time, to directly compare neural dynamics of disease and control. We found that cultures e.g. from epilepsy patients tended to have increasingly more avalanches of synchronous activity over weeks of development, in contrast to the control cultures. Next, we will test possible interventions, in silico and in vitro, in a drive for personalised approaches to medical care. This work starts bridging an important theoretical-experimental neuroscience gap for advancing our understanding of mammalian neuron dynamics.

Hypothalamic episode generators underlying the neural control of fertility

The hypothalamus controls diverse homeostatic functions including fertility. Neural episode generators are required to drive the intermittent pulsatile and surge profiles of reproductive hormone secretion that control gonadal function. Studies in genetic mouse models have been fundamental in defining the neural circuits forming these central pattern generators and the full range of in vitro and in vivo optogenetic and chemogenetic methodologies have enabled investigation into their mechanism of action. The seminar will outline studies defining the hypothalamic “GnRH pulse generator network” and current understanding of its operation to drive pulsatile hormone secretion.

Identifying central mechanisms of glucocorticoid circadian rhythm dysfunction in breast cancer

The circadian release of endogenous glucocorticoids is essential in preparing and synchronizing the body’s daily physiological needs. Disruption in the rhythmic activity of glucocorticoids has been observed in individuals with a variety of cancer types, and blunting of this rhythm has been shown to predict cancer mortality and declines in quality of life. This suggests that a disrupted glucocorticoid rhythm is potentially a shared phenotype across cancers. However, where this phenomenon is driven by the cancer itself, and the causal mechanisms that link glucocorticoid rhythm dysfunction and cancer outcomes remain preliminary at best. The regulation of daily glucocorticoid activity has been well-characterized and is maintained, in part, by the coordinated response of the hypothalamic-pituitary-adrenal (HPA) axis, consisting of the suprachiasmatic nucleus (SCN) and corticotropin-releasing hormone-expressing neurons of the paraventricular nucleus of the hypothalamus (PVNCRH). Consequently, we set out to examine if cancer-induced glucocorticoid dysfunction is regulated by disruptions within these hypothalamic nuclei. In comparison to their tumor-free baseline, mammary tumor-bearing mice exhibited a blunting of glucocorticoid rhythms across multiple timepoints throughout the day, as measured by the overall levels and the slope of fecal corticosterone rhythms, during tumor progression. We further examined how peripheral tumors shape hypothalamic activity within the brain. Serial two-photon tomography for whole-brain cFos imaging suggests a disrupted activation of the PVN in mice with tumors. Additionally, we found GFP labeled CRH+ neurons within the PVN after injection of pseudorabies virus expressing GFP into the tumor, pointing to the PVN as a primary target disrupted by mammary tumors. Preliminary in vivo fiber photometry data show that PVNCRH neurons exhibit enhanced calcium activity during tumor progression, as compared to baseline (no tumor) activity. Taken together, this suggests that there may be an overactive HPA response during tumor progression, which in turn, may result in a subsequent negative feedback on glucocorticoid rhythms. Current studies are examining whether tumor progression modulates SCN calcium activity, how the transcriptional profile of PVNCRH neurons is changed, and test if manipulation of the neurocircuitry surrounding glucocorticoid rhythmicity alters tumor characteristics.

Radiopharmaceutical evaluation of novel bifunctional chelators and bioconjugates for tumour imaging and therapy

Bispidines (3,7-diazabicyclo[3.3.1]nonane) and their derivatives act as bifunctional chelators (BFC), combining the advantages of multidentate macrocyclic and acyclic ligands e.g. high kinetic inertness, rapid radiolabelling under mild conditions. This bicyclic chelator system shows a great diversity in terms of its denticity and type of functional groups, yielding a wide range of multidentate ligands that can bind a variety of different metal ions. In addition, they allow a facile functionalisation of targeting molecules such as peptides, peptidomimetics, and bispeci􀄀c antibodies. Herein, examples of various bispidine complexes labelled with [64Cu]Cu2+, [111In]In3+, [ 177Lu]Lu3+ or [ 225Ac]Ac3+ will be presented which provide a picture of how different substituents in􀄁uence the coordination mode. Target-speci􀄀c radiolabelled bispidine-based conjugates (e.g. peptides, antibody fragments, antibodies) investigated in vivo by positron emission or single-photon emission computed tomography will be presented and discussed in terms of their suitability for nuclear medicine applications.

Hidden nature of seizures

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

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

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

Seeing the world through moving photoreceptors - binocular photomechanical microsaccades give fruit fly hyperacute 3D-vision

To move efficiently, animals must continuously work out their x,y,z positions with respect to real-world objects, and many animals have a pair of eyes to achieve this. How photoreceptors actively sample the eyes’ optical image disparity is not understood because this fundamental information-limiting step has not been investigated in vivo over the eyes’ whole sampling matrix. This integrative multiscale study will advance our current understanding of stereopsis from static image disparity comparison to a morphodynamic active sampling theory. It shows how photomechanical photoreceptor microsaccades enable Drosophila superresolution three-dimensional vision and proposes neural computations for accurately predicting these flies’ depth-perception dynamics, limits, and visual behaviors.

Sharing data from your in vivo studies

Handling data in your in vivo studies

Preregistering your in vivo studies

PET imaging in brain diseases

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

Reprogramming the nociceptive circuit topology reshapes sexual behavior in C. elegans

In sexually reproducing species, males and females respond to environmental sensory cues and transform the input into sexually dimorphic traits. Yet, how sexually dimorphic behavior is encoded in the nervous system is poorly understood. We characterize the sexually dimorphic nociceptive behavior in C. elegans – hermaphrodites present a lower pain threshold than males in response to aversive stimuli, and study the underlying neuronal circuits, which are composed of the same neurons that are wired differently. By imaging receptor expression, calcium responses and glutamate secretion, we show that sensory transduction is similar in the two sexes, and therefore explore how downstream network topology shapes dimorphic behavior. We generated a computational model that replicates the observed dimorphic behavior, and used this model to predict simple network rewirings that would switch the behavior between the sexes. We then showed experimentally, using genetic manipulations, artificial gap junctions, automated tracking and optogenetics, that these subtle changes to male connectivity result in hermaphrodite-like aversive behavior in-vivo, while hermaphrodite behavior was more robust to perturbations. Strikingly, when presented with aversive cues, rewired males were compromised in finding mating partners, suggesting that the network topology that enables efficient avoidance of noxious cues would have a reproductive "cost". To summarize, we present a deconstruction of a sex-shared neural circuit that affects sexual behavior, and how to reprogram it. More broadly, our results are an example of how common neuronal circuits changed their function during evolution by subtle topological rewirings to account for different environmental and sexual needs.

Hebbian Plasticity Supports Predictive Self-Supervised Learning of Disentangled Representations​

Discriminating distinct objects and concepts from sensory stimuli is essential for survival. Our brains accomplish this feat by forming meaningful internal representations in deep sensory networks with plastic synaptic connections. Experience-dependent plasticity presumably exploits temporal contingencies between sensory inputs to build these internal representations. However, the precise mechanisms underlying plasticity remain elusive. We derive a local synaptic plasticity model inspired by self-supervised machine learning techniques that shares a deep conceptual connection to Bienenstock-Cooper-Munro (BCM) theory and is consistent with experimentally observed plasticity rules. We show that our plasticity model yields disentangled object representations in deep neural networks without the need for supervision and implausible negative examples. In response to altered visual experience, our model qualitatively captures neuronal selectivity changes observed in the monkey inferotemporal cortex in-vivo. Our work suggests a plausible learning rule to drive learning in sensory networks while making concrete testable predictions.

Charting the Proteome Landscape of Diverse Synapses In Vivo

A transcriptomic axis predicts state modulation of cortical interneurons

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

Elucidating the mechanism underlying Stress and Caffeine-induced motor dysfunction using a mouse model of Episodic Ataxia Type 2

Episodic Ataxia type 2 (EA2), caused by mutations in the CACNA1A gene, results in a loss-of-function of the P/Q type calcium channel, which leads to baseline ataxia, and attacks of dyskinesia, that can last a few hours to a few days. Attacks are brought on by consumption of caffeine, alcohol, and physical or emotional stress. Interestingly, caffeine and stress are common triggers among other episodic channelopathies, as well as causing tremor or shaking in otherwise healthy adults. The mechanism underlying stress and caffeine induced motor impairment remains poorly understood. Utilizing behavior, and in vivo and in vitro electrophysiology in the tottering mouse, a well characterized mouse model of EA2, or WT mice, we first sought to elucidate the mechanism underlying stress-induced motor impairment. We found stress induces attacks in EA2 though the activation of cerebellar alpha 1 adrenergic receptors by norepinephrine (NE) through casein kinase 2 (CK2) dependent phosphorylation. This decreases SK2 channel activity, causing increased Purkinje cell irregularity and motor impairment. Knocking down or blocking CK2 with an FDA approved drug CX-4945 prevented PC irregularity and stress-induced attacks. We next hypothesized caffeine, which has been shown to increase NE levels, could induce attacks through the same alpha 1 adrenergic mechanism in EA2. We found caffeine increases PC irregularity and induces attacks through the same CK2 pathway. Block of alpha 1 adrenergic receptors, however, failed to prevent caffeine-induced attacks. Caffeine instead induces attacks through the block of cerebellar A1 adenosine receptors. This increases the release of glutamate, which interacts with mGluR1 receptors on PC, resulting in erratic firing and motor attacks. Finally, we show a novel direct interaction between mGluR1 and CK2, and inhibition of mGluR1 prior to initiation of attack, prevents the caffeine-induced increase in phosphorylation. These data elucidate the mechanism underlying stress and caffeine-induced motor impairment. Furthermore, given the success of CX-4945 to prevent stress and caffeine induced attacks, it establishes ground-work for the development of therapeutics for the treatment of caffeine and stress induced attacks in EA2 patients and possibly other episodic channelopathies.

Learning binds novel inputs into functional synaptic clusters via spinogenesis

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

Multi-modal biomarkers improve prediction of memory function in cognitively unimpaired older adults

Identifying biomarkers that predict current and future cognition may improve estimates of Alzheimer’s disease risk among cognitively unimpaired older adults (CU). In vivo measures of amyloid and tau protein burden and task-based functional MRI measures of core memory mechanisms, such as the strength of cortical reinstatement during remembering, have each been linked to individual differences in memory in CU. This study assesses whether combining CSF biomarkers with fMRI indices of cortical reinstatement improves estimation of memory function in CU, assayed using three unique tests of hippocampal-dependent memory. Participants were 158 CU (90F, aged 60-88 years, CDR=0) enrolled in the Stanford Aging and Memory Study (SAMS). Cortical reinstatement was quantified using multivoxel pattern analysis of fMRI data collected during completion of a paired associate cued recall task. Memory was assayed by associative cued recall, a delayed recall composite, and a mnemonic discrimination task that involved discrimination between studied ‘target’ objects, novel ‘foil’ objects, and perceptually similar ‘lure’ objects. CSF Aβ42, Aβ40, and p-tau181 were measured with the automated Lumipulse G system (N=115). Regression analyses examined cross-sectional relationships between memory performance in each task and a) the strength of cortical reinstatement in the Default Network (comprised of posterior medial, medial frontal, and lateral parietal regions) during associative cued recall and b) CSF Aβ42/Aβ40 and p-tau181, controlling for age, sex, and education. For mnemonic discrimination, linear mixed effects models were used to examine the relationship between discrimination (d’) and each predictor as a function of target-lure similarity. Stronger cortical reinstatement was associated with better performance across all three memory assays. Age and higher CSF p-tau181 were each associated with poorer associative memory and a diminished improvement in mnemonic discrimination as target-lure similarity decreased. When combined in a single model, CSF p-tau181 and Default Network reinstatement strength, but not age, explained unique variance in associative memory and mnemonic discrimination performance, outperforming the single-modality models. Combining fMRI measures of core memory functions with protein biomarkers of Alzheimer’s disease significantly improved prediction of individual differences in memory performance in CU. Leveraging multimodal biomarkers may enhance future prediction of risk for cognitive decline.

How evidence synthesis can boost in vivo credibility

As part of the BNA's ongoing Credibility in Neuroscience work, this series of three short webinars will provide neuroscience researchers working in an in vivo setting with tips on how to improve the credibility of their work. Each webinar will be hosted by Emily Sena, member of the BNA's Credibility Advisory Board, with the opportunity for questions.

Embracing variation to boost reproducibility

As part of the BNA's ongoing Credibility in Neuroscience work, this series of three short webinars will provide neuroscience researchers working in an in vivo setting with tips on how to improve the credibility of their work. Each webinar will be hosted by Emily Sena, member of the BNA's Credibility Advisory Board, with the opportunity for questions.

Improving reliability through design and reporting

As part of the BNA's ongoing Credibility in Neuroscience work, this series of three short webinars will provide neuroscience researchers working in an in vivo setting with tips on how to improve the credibility of their work. Each webinar will be hosted by Emily Sena, member of the BNA's Credibility Advisory Board, with the opportunity for questions.

Dissecting the 3D regulatory landscape of the developing cerebral cortex with single-cell epigenomics

Understanding how different epigenetic layers are coordinated to facilitate robust lineage decisions during development is one of the fundamental questions in regulatory genomics. Using single-cell epigenomics coupled with cell-type specific high-throughput mapping of enhancer activity, DNA methylation and the 3D genome landscape in vivo, we dissected how the epigenome is rewired during cortical development. We identified and functionally validated key transcription factors such as Neurog2 which underlie regulatory dynamics and coordinate rewiring across multiple epigenetic layers to ensure robust lineage specification. This work showcases the power of high-throughput integrative genomics to dissect the molecular rules of cell fate decisions in the brain and more broadly, how to apply them to evolution and disease.

Untitled Seminar

Rachel Moore- Microtubules are not required to generate a nascent axon in embryonic spinal neurons in vivo Michael Notaras-TBA Rachel Wong- Circuit assembly in the vertebrate retina

Why is the suprachiasmatic nucleus such a brilliant circadian time-keeper?

Circadian clocks dominate our lives. By creating and distributing an internal representation of 24-hour solar time, they prepare us, and thereby adapt us, to the daily and seasonal world. Jet-lag is an obvious indicator of what can go wrong when such adaptation is disrupted acutely. More seriously, the growing prevalence of rotational shift-work which runs counter to our circadian life, is a significant chronic challenge to health, presenting as increased incidence of systemic conditions such as metabolic and cardiovascular disease. Added to this, circadian and sleep disturbances are a recognised feature of various neurological and psychiatric conditions, and in some cases may contribute to disease progression. The “head ganglion” of the circadian system is the suprachiasmatic nucleus (SCN) of the hypothalamus. It synchronises the, literally, innumerable cellular clocks across the body, to each other and to solar time. Isolated in organotypic slice culture, it can maintain precise, high-amplitude circadian cycles of neural activity, effectively, indefinitely, just as it does in vivo. How is this achieved: how does this clock in a dish work? This presentation will consider SCN time-keeping at the level of molecular feedback loops, neuropeptidergic networks and neuron-astrocyte interactions.

Astrocytes encode complex behaviorally relevant information

While it is generally accepted that neurons control complex behavior and brain computation, the role of non-neuronal cells in this context remains unclear. Astrocytes, glial cells of the central nervous system, exhibit complex forms of chemical excitation, most prominently calcium transients, evoked by local and projection neuron activity. In this talk, I will provide mechanistic links between astrocytes’ spatiotemporally complex activity patterns, neuronal molecular signaling, and behavior. Using a visual detection task, in vivo calcium imaging, robust statistical analyses, and machine learning approaches, my work shows that cortical astrocytes encode the animal's decision, reward, performance level, and sensory properties. Behavioral context and motor activity-related parameters strongly impact astrocyte responses. Error analysis confirms that astrocytes carry behaviorally relevant information, supporting astrocytes' complementary role to neuronal coding beyond their established homeostatic and metabolic roles.

Norepinephrine links astrocytic activity to regulation of cortical state

Cortical state, defined by the synchrony of population-level neuronal activity, is a key determinant of sensory perception. While many arousal-associated neuromodulators—including norepinephrine (NE)—reduce cortical synchrony, how the cortex resynchronizes following NE signaling remains unknown. Using in vivo two-photon imaging and electrophysiology in mouse visual cortex, we describe a critical role for cortical astrocytes in circuit resynchronization. We characterize astrocytes’ sensitive calcium responses to changes in behavioral arousal and NE, identify that astrocyte signaling precedes increases in cortical synchrony, and demonstrate that astrocyte-specific deletion of Adra1A alters arousal-related cortical synchrony. Our findings demonstrate that astrocytic NE signaling acts as a distinct neuromodulatory pathway, regulating cortical state and linking arousal-associated desynchrony to cortical circuit resynchronization.

A nonlinear shot noise model for calcium-based synaptic plasticity

Activity dependent synaptic plasticity is considered to be a primary mechanism underlying learning and memory. Yet it is unclear whether plasticity rules such as STDP measured in vitro apply in vivo. Network models with STDP predict that activity patterns (e.g., place-cell spatial selectivity) should change much faster than observed experimentally. We address this gap by investigating a nonlinear calcium-based plasticity rule fit to experiments done in physiological conditions. In this model, LTP and LTD result from intracellular calcium transients arising almost exclusively from synchronous coactivation of pre- and postsynaptic neurons. We analytically approximate the full distribution of nonlinear calcium transients as a function of pre- and postsynaptic firing rates, and temporal correlations. This analysis directly relates activity statistics that can be measured in vivo to the changes in synaptic efficacy they cause. Our results highlight that both high-firing rates and temporal correlations can lead to significant changes to synaptic efficacy. Using a mean-field theory, we show that the nonlinear plasticity rule, without any fine-tuning, gives a stable, unimodal synaptic weight distribution characterized by many strong synapses which remain stable over long periods of time, consistent with electrophysiological and behavioral studies. Moreover, our theory explains how memories encoded by strong synapses can be preferentially stabilized by the plasticity rule. We confirmed our analytical results in a spiking recurrent network. Interestingly, although most synapses are weak and undergo rapid turnover, the fraction of strong synapses are sufficient for supporting realistic spiking dynamics and serve to maintain the network’s cluster structure. Our results provide a mechanistic understanding of how stable memories may emerge on the behavioral level from an STDP rule measured in physiological conditions. Furthermore, the plasticity rule we investigate is mathematically equivalent to other learning rules which rely on the statistics of coincidences, so we expect that our formalism will be useful to study other learning processes beyond the calcium-based plasticity rule.

NMC4 Short Talk: Two-Photon Imaging of Norepinephrine in the Prefrontal Cortex Shows that Norepinephrine Structures Cell Firing Through Local Release

Norepinephrine (NE) is a neuromodulator that is released from projections of the locus coeruleus via extra-synaptic vesicle exocytosis. Tonic fluctuations in NE are involved in brain states, such as sleep, arousal, and attention. Previously, NE in the PFC was thought to be a homogenous field created by bulk release, but it remains unknown whether phasic (fast, short-term) fluctuations in NE can produce a spatially heterogeneous field, which could then structure cell firing at a fine spatial scale. To understand how spatiotemporal dynamics of norepinephrine (NE) release in the prefrontal cortex affect neuronal firing, we performed a novel in-vivo two-photon imaging experiment in layer ⅔ of the prefrontal cortex using a green fluorescent NE sensor and a red fluorescent Ca2+ sensor, which allowed us to simultaneously observe fine-scale neuronal and NE dynamics in the form of spatially localized fluorescence time series. Using generalized linear modeling, we found that the local NE field differs from the global NE field in transient periods of decorrelation, which are influenced by proximal NE release events. We used optical flow and pattern analysis to show that release and reuptake events can occur at the same location but at different times, and differential recruitment of release and reuptake sites over time is a potential mechanism for creating a heterogeneous NE field. Our generalized linear models predicting cellular dynamics show that the heterogeneous local NE field, and not the global field, drives cell firing dynamics. These results point to the importance of local, small-scale, phasic NE fluctuations for structuring cell firing. Prior research suggests that these phasic NE fluctuations in the PFC may play a role in attentional shifts, orienting to sensory stimuli in the environment, and in the selective gain of priority representations during stress (Mather, Clewett et al. 2016) (Aston-Jones and Bloom 1981).

NMC4 Short Talk: A theory for the population rate of adapting neurons disambiguates mean vs. variance-driven dynamics and explains log-normal response statistics

Recently, the field of computational neuroscience has seen an explosion of the use of trained recurrent network models (RNNs) to model patterns of neural activity. These RNN models are typically characterized by tuned recurrent interactions between rate 'units' whose dynamics are governed by smooth, continuous differential equations. However, the response of biological single neurons is better described by all-or-none events - spikes - that are triggered in response to the processing of their synaptic input by the complex dynamics of their membrane. One line of research has attempted to resolve this discrepancy by linking the average firing probability of a population of simplified spiking neuron models to rate dynamics similar to those used for RNN units. However, challenges remain to account for complex temporal dependencies in the biological single neuron response and for the heterogeneity of synaptic input across the population. Here, we make progress by showing how to derive dynamic rate equations for a population of spiking neurons with multi-timescale adaptation properties - as this was shown to accurately model the response of biological neurons - while they receive independent time-varying inputs, leading to plausible asynchronous activity in the network. The resulting rate equations yield an insightful segregation of the population's response into dynamics that are driven by the mean signal received by the neural population, and dynamics driven by the variance of the input across neurons, with respective timescales that are in agreement with slice experiments. Further, these equations explain how input variability can shape log-normal instantaneous rate distributions across neurons, as observed in vivo. Our results help interpret properties of the neural population response and open the way to investigating whether the more biologically plausible and dynamically complex rate model we derive could provide useful inductive biases if used in an RNN to solve specific tasks.

Untitled Seminar

Leanne Godinho (Germany): Probing the mechanisms underlying cell fate in vivo in the developing retina; Gabriele Ciceri (USA): Directing the timing of maturation in human pluripotent stem cell-derived cortical neurons; Daniel Poppe (Australia): Conserved and divergent features of DNA methylation in embryonic stem cell-derived neurons

Noise-induced properties of active dendrites

Neuronal dendritic trees display a wide range of nonlinear input integrations due to their voltage-dependent active calcium channels. We reveal that in vivo-like fluctuating input enhances nonlinearity substantially in a single dendritic compartment and shifts the input-output relation to exhibiting nonmonotonous or bistable dynamics. In particular, with the slow activation of calcium dynamics, we analyze noise-induced bistability and its timescales. We show bistability induces long-timescale fluctuation that can account for observed dendritic plateau potentials in vivo conditions. In a multicompartmental model neuron with realistic synaptic input, we show that noise-induced bistability persists in a wide range of parameters. Using Fredholm's theory to calculate the spiking rate of multivariable neurons, we discuss how dendritic bistability shifts the spiking dynamics of single neurons and its implications for network phenomena in the processing of in vivo–like fluctuating input.

Keeping axons alive after injury: Inhibiting programmed axon death

Activation of pro-degenerative protein SARM1 in response to diverse physical and disease-relevant injuries triggers programmed axon death. Original studies indicated substantially decreased levels of SARM1 were required for neuroprotection. However, we demonstrate that lowering SARM1 levels by 50% in Sarm1 haploinsufficient mice delays axon degeneration in vivo (after sciatic nerve transection), in vitro (in response to diverse traumatic, neurotoxic, and genetic triggers), and partially prevents neurite outgrowth defects in mice lacking pro-survival factor NMNAT2. We also demonstrate the capacity for Sarm1 antisense oligonucleotides to decrease SARM1 levels by more than 50% which delays or prevents programmed axon degeneration in vitro. Combining Sarm1 haploinsufficiency with antisense oligonucleotides further decreases SARM1 levels and prolongs protection after neurotoxic injuries. These data demonstrate that axon protection occurs in a Sarm1 gene-dose responsive manner and that SARM1 lowering agents have therapeutic potential. Thus, antisense oligonucleotide targeting of Sarm1 is a promising therapeutic strategy against diverse triggers of axon degeneration.

An optimal population code for global motion estimation in local direction-selective cells

Neuronal computations are matched to optimally encode the sensory information that is available and relevant for the animal. However, the physical distribution of sensory information is often shaped by the animal’s own behavior. One prominent example is the encoding of optic flow fields that are generated during self-motion of the animal and will therefore depend on the type of locomotion. How evolution has matched computational resources to the behavioral constraints of an animal is not known. Here we use in vivo two photon imaging to record from a population of >3.500 local-direction selective cells. Our data show that the local direction-selective T4/T5 neurons in Drosophila form a population code that is matched to represent optic flow fields generated during translational and rotational self-motion of the fly. This coding principle for optic flow is reminiscent to the population code of local direction-selective ganglion cells in the mouse retina, where four direction-selective ganglion cells encode four different axes of self-motion encountered during walking (Sabbah et al., 2017). However, in flies we find six different subtypes of T4 and T5 cells that, at the population level, represent six axes of self-motion of the fly. The four uniformly tuned T4/T5 subtypes described previously represent a local snapshot (Maisak et al. 2013). The encoding of six types of optic flow in the fly as compared to four types of optic flow in mice might be matched to the high degrees of freedom encountered during flight. Thus, a population code for optic flow appears to be a general coding principle of visual systems, resulting from convergent evolution, but matching the individual ethological constraints of the animal.

Deriving local synaptic learning rules for efficient representations in networks of spiking neurons

How can neural networks learn to efficiently represent complex and high-dimensional inputs via local plasticity mechanisms? Classical models of representation learning assume that input weights are learned via pairwise Hebbian-like plasticity. Here, we show that pairwise Hebbian-like plasticity only works under specific requirements on neural dynamics and input statistics. To overcome these limitations, we derive from first principles a learning scheme based on voltage-dependent synaptic plasticity rules. Here, inhibition learns to locally balance excitatory input in individual dendritic compartments, and thereby can modulate excitatory synaptic plasticity to learn efficient representations. We demonstrate in simulations that this learning scheme works robustly even for complex, high-dimensional and correlated inputs. It also works in the presence of inhibitory transmission delays, where Hebbian-like plasticity typically fails. Our results draw a direct connection between dendritic excitatory-inhibitory balance and voltage-dependent synaptic plasticity as observed in vivo, and suggest that both are crucial for representation learning.

All-Optical Investigation of Schaller Collateral Synapses in vivo

COSYNE 2023

Multi-modal composition of physiological signals to delineate candidate cell types in-vivo

COSYNE 2023

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

COSYNE 2023

The accumulation of dendritic extracellular Potassium as in vivo model of epilepsy in CA1 pyramidal neurons

COSYNE 2025

In vivo cell-type and brain region classification via multimodal contrastive learning

COSYNE 2025

Anti-GluA3 antibodies in Frontotemporal Dementia: an in vivo approach

Approaching in-vivo BBB permeabilities with engineered blood-brain barrier-on-chip

Autonomic Disbalance During Systemic Inflammation is Associated with Oxidative Stress Changes in Sepsis Survivor Rats

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

Two candidate K-Cl cotransporter 2 (KCC2) enhancers prevent epileptiform activity in vitro and in vivo

Capturing dynamics of inhibitory synaptic connectivity underlying learning using in vivo two-photon optical imaging of hippocampal CA1

Chandelier cells provide diverse inputs to cortical networks in-vivo

Characterisation of in vivo synaptic and neuronal physiology in early and progressed amyloidopathy (APP/PS1 mouse model)

Characterization of hippocampal neuronal activity during the first postnatal week in-vivo

Characterization of a novel Glucocerebrosidase pharmacological chaperone in vitro and in vivo models of alpha synuclein neurotoxicity

Characterization of promoter expression in type 1 and type 2 spiral ganglion neurons in vitro and in vivo

Chronic in vivo alterations of Aeta-alpha peptide levels perturb synaptic plasticity and impact spatial memory

Chronic in vivo electrophysiological recordings of dopamine neurons in a mouse model of the 22q11.2 microdeletion syndrome

Cognitive effects in non-CNS cancer survivors before and 1-year after standard anthracycline-based chemotherapy treatment

Continuous theta burst stimulation decreases striatal dopamine release acutely but not chronically: An in vivo and postmortem study

Depth-resolved fibre photometry for Alzheimer’s disease plaque pathology in vivo

Differential axonal trafficking of Neuropeptide Y-, Lamp1- and Rab7-tagged organelles in vivo

Differential dopamine signalling in ventral and dorsal striatum in vivo

Disruption of hippocampus-based spatial coding in long–sepsis survivors is mediated by HMGB1

Dissecting the role of HCN1 in Developmental and Epileptic Encephalopathy (DEE) by exploiting patient-specific models of cerebral cortex development in vivo

Effects of natural and synthetic catechol-O-methyl transferase inhibitors on two in vivo models of Parkinson’s disease

The emergence of correlated activity in the developing cortex in vivo

Establishment of functional internally tagged sorting receptors of the Vps10p domain receptor family as a novel tool to investigate intracellular neuronal sorting mechanisms in vivo

Examining dendritic plasticity of the barrel cortex layer 4 neurons via in vivo high spatiotemporal-resolution imaging

Exploring the Dynamics of Neuronal Excitability by Optogenetics in ex vivo Cortical Cultures

Focus on the mouse insular cortex: in vivo single-unit recordings during anxiety assays and mapping of the dopaminergic system

Functional studies of neuron-astrocyte interactions in vivo

Further characterisation of the rat model of Tourette-related striatal disinhibition: in vivo electrophysiological and behavioural studies

Glycogen-binding fluorescent probe in astrocytes displays signal translocation in response to metabolic or neuronal activity manipulation in vivo

Hair-thin multimode fiber-based holographic endoscope for deep-brain in-vivo structural and functional imaging of cellular and subcellular structures

Hydrogen Peroxide Diffusivity in vivo in the brain extracellular space supports volume neurotransmission

IFNAR1C291* expression induces neurodegeneration in vivo through impairments in Parkin-mediated mitophagy

Impaired reward processing in chronic stroke survivors

Improved optical recording of in-vivo neuronal activity in 3D cortical neuron populations with patterned excitation of soma-restricted calcium probes

In-vivo dynamical effects of structural white matter disconnections

Bernstein Conference 2024