Vitro

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.

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.

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.

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.

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.

Cartilage targeting exosomes for OA gene therapy and pain treatment

Project Summary Gene therapy has the potential to facilitate targeted expression of therapeutic proteins to promote cartilage regeneration in osteoarthritis (OA). The dense, avascular, aggrecan-glycosaminoglycan rich negatively charged cartilage, however, hinders their transport to reach chondrocytes in effective doses. While viral vector mediated gene delivery has shown promise, concerns over immunogenicity and tumorigenic side-effects persist. To address this, we have developed surface-modified cartilage-targeting MSC exosomes as non-viral carriers for gene therapy. MSC derived exosomes have intrinsic therapeutic potential as they can induce cartilage repair and are non-immunogenic, making them desirable for gene delivery. We have engineered charge-reversed cationic exosomes by anchoring cartilage targeting optimally charged arginine-rich cationic peptide (CPC) motifs into the anionic exosome bilayer (Exo-CPC) by using buffer pH as a charge-reversal switch. Exo-CPC use charge interactions to penetrate through the full thickness of arthritic cartilage (close to tidemark) and deliver the packaged genetic material cargo to chondrocytes residing in the deep tissue layers while native anionic exosomes cannot. They can also bind within the synovial joint, making them effective for OA pain relief gene therapy. Here we will engineer charge-reversed Exo-CPC for delivery of IL-1RA (receptor antagonist of interleukin-1) mRNA and NaV1.8 (voltage gated sodium channel 1.8) inhibitor siRNA to stimulate both disease modifying response and long-term pain relief with a one-time intra-articular dose. IL-1RA mRNA targets are in the chondrocytes and synovium cells; Nav1.8 expressing nerves innervate into synovium and subchondral bone in OA – sites that Exo-CPC can readily target. Aim 1 will engineer cartilage targeting Exo-CPC for delivery of IL- 1RA mRNA and Nav1.8 inhibitor siRNA. Their ability to deliver IL-1RA mRNA to chondrocytes and IL-1RA protein translation efficiency will be evaluated in-vitro. Exo-CPC-Na v1.8’s ability to reduce NaV1.8 bioactivity of sensory nerves will also be evaluated. In Aim 2, their distribution intra-articular (proximity to NaV1.8-positive nerves), extra-articular, and DRG and spinal cord using partial meniscectomy NaV1.8-tdTomato reporter mice OA models will be evaluated. Additionally, their dose dependent reduction on MMP activity, neuronal excitability and pain- related behaviors, and any immunogenicity will be assessed. Aim 3 will use the determined functional doses to study the long-term disease modifying and pain-relief effects of mono and combination therapy with Exo-CPC- IL-1RA and Exo-CPC-Nav1.8 in rescuing injury induced tissue structural damage as well as in reducing pain (weight bearing asymmetry) for up to one month following IA administration in early vs. late stages (intervention at 2 vs 6 weeks) of MMT (medial meniscectomy) induced OA rats. The project paves way for utilizing the intrinsic therapeutic potential of MSC Exosomes as viral-free, non-immunogenic carriers for OA gene therapy by employing cartilage as a drug depot. Cationic exosomes can be used to deliver other OA gene targets, and can be widely used for targeting other negatively charged tissues like meniscus, ligaments, discs, fracture callus etc.

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.

Role of cellular physical interactions in pancreatic cancer progression

Pancreatic cancer, with a 5-year overall survival rate of 13%, has the lowest survival rate of all cancers. The goal of this project is to better understand the biological processes of pancreatic cancer progression and discover their potential as targets for efficient therapies. Pancreatic ductal adenocarcinoma (PDAC) underdoes epithelial architecture changes during its progression. However, the underlying mechanisms for these changes are largely unknown. Interestingly, our recent data demonstrate the recapitulation of the distinct epithelial architectures in the organoid culture of cells derived from the human normal pancreas, primary tumor, and metastatic lesions, thereby developing a unique organoid model for the in vitro studies of PDAC epithelial architecture changes. The primary objective of this project is to understand the regulation of the differential PDAC epithelial architectures as well as their contribution to PDAC progression. Our central hypothesis is that disruption in lumen structure drives PDAC epithelial architecture transition and promotes PDAC progression. We will combine experimental and computational approaches to test our central hypothesis by pursuing the following two specific aims: (Aim 1) define the regulators of PDAC epithelial architecture that drives PDAC progression and (Aim 2) determine the functional consequences of PDAC epithelial architecture on PDAC progression. With the completion of this aims, we expect: (Aim 1) to identify ion and water channels that are important for lumen structure as well as PDAC progression, revealing potential novel targets for therapeutic intervention, and (Aim 2) to uncover YAP’s role in PDAC progression and guide the development of YAP- targeted therapies.

Targeting VIP–VPAC Signaling to Reverse Immune Exclusion and Enhance Immunotherapy Response in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer that is largely unresponsive to chemotherapy and current immune checkpoint blockade drugs, highlighting a critical need for the development of innovative therapeutic strategies. This R01 proposal targets vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide overexpressed in PDAC, which signals through VIP receptors (VPAC) on cancer cells, T cells, and myeloid cells within the tumor microenvironment. Based on our recent success in developing selective and potent VPAC receptor antagonists, we hypothesize that blocking VPAC signaling will reverse immunosuppression in the PDAC TME by reducing immune checkpoint expression, enhancing chemokine-driven infiltration of cytotoxic T cells, and disrupting immunosuppressive interactions between T cells and myeloid cells, ultimately leading to durable anti-cancer immunity. We propose three specific aims to explore the immunosuppressive roles of VPAC signaling in PDAC. Aim 1 will identify the primary sources of VIP in PDAC tumors and characterize the effects of VPAC signaling on immune cell function and phenotype within the tumor microenvironment. Aim 2 will investigate how VPAC signaling influences immune cell migration into tumors by modulating chemokine receptors and directional signaling. Aim 3 will determine how VPAC signaling regulates interactions between T cells and immunosuppressive myeloid cells, particularly tumor-associated macrophages, and the resulting impact on anti-cancer immune responses and immunological memory. Our preliminary findings indicate that combined inhibition of VPAC signaling and PD-1 significantly enhances the regression of PDAC tumors in multiple mouse models, generating lasting protective immunity in cured mice without triggering autoimmune responses. We will use novel methods to pursue our aims, including inducible genetically engineered mouse models (GEMM) of PDAC, long-acting VPAC antagonists engineered with immunoglobulin Fc domains to improve their plasma half-life, and advanced microfluidics technologies to analyze immune cell movement within tumors. Animal experiments will be used to validate the translational potential of observations from in vitro organoids and microfluidic experiments. The GEMM and orthotopic mouse models of PDAC are necessary to provide critical insights into the 3-D structure of the TME and tumor regression in response to our novel immunotherapy. This research will be conducted by a multidisciplinary team with complementary expertise that will clarify the therapeutic potential of VPAC signaling inhibition in PDAC using sophisticated experimental tools and single-cell RNA sequencing. Ultimately, these findings could significantly improve the development of immunotherapeutic strategies for PDAC, potentially enhancing patient outcomes in pancreatic cancer and other malignancies expressing high VIP levels.

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.

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

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

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.

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.

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

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

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.

Breaking Tolerance: Trichloroethylene Provides Survival Signals to Autoreactive CD4s in the Liver

PROJECT SUMMARY The industrial solvent and widespread environmental contaminant, trichloroethylene (TCE) has been linked to autoimmune disease in humans. How TCE impairs tolerance (i.e., unresponsiveness) to self-antigens leading to autoimmunity has not been explored. Autoimmune diseases (ADs) are a class of disorders that affect many different organs and tissues. However, all autoimmune diseases share a feature in common which is the ability of potentially pathogenic autoreactive cells to evade deletion. During early life, peripheral CD4+ cells are primarily comprised of recent thymic emigrants (RTE) which home to the liver. The liver is known to efficiently retain and tolerize self-reactive CD4s to where they are functionally unresponsive to their antigen. Thus, the liver is the first checkpoint in the periphery to filter, retain, and enforce tolerance to autoreactive CD4+ RTEs. The liver is also the site of TCE metabolism. Our Aims are designed to test the hypothesis that TCE, through its metabolite TCAH, delivers costimulatory signals to liver CD4 RTEs via CD28, thereby overriding inhibitory CTLA-4 signaling. This disruption promotes the survival of self-reactive CD4 RTEs by impairing CTLA-4-dependent tolerance mechanisms contributing to the development of ADs. This research will significantly advance the fields of toxicology and autoimmunity, where the origins of environmentally induced AD remain poorly understood. Aim 1 will assess TCE’s effects on RTE migration patterns in real-time in transgenic mice. Aim 2 will investigate TCAH-mediated costimulatory signaling in CD4 RTEs in vitro. Successful completion of these studies will determine how TCE alters key tolerance pathways in the liver resulting in a greater proportion of self-reactive effector memory (EM) peripheral CD4s capable of promoting AD.

Understanding antiretroviral phosphorylation and dephosphorylation using mass spectrometry imaging-based enzyme histochemistry

PROJECT SUMMARY Our overall goal is to understand the mechanistic differences in the activation and deactivation of two widely used first-line antiretroviral drugs: tenofovir (TFV) and emtricitabine (FTC) in colonic tissues. HIV is a global health problem and roughly 1.3 million people became newly infected with HIV globally in 2022. Pre-exposure prophylaxis (PrEP) is an HIV prevention strategy where HIV-negative individuals use antiretrovirals to reduce the risk of HIV infection. Specifically, oral fixed-dose combinations of two antiretrovirals, namely, TFV (TFV; prescribed as TFV disoproxil fumarate or TFV alafenamide prodrugs) and FTC are FDA-approved for HIV PrEP. The pharmacologically active forms of TFV and FTC are TFV-diphosphate (TFV-DP) and FTC-triphosphate (FTC-TP), respectively, and these phosphorylated metabolites are found in cells. Unfortunately, high variability in the responses of TFV and FTC can lead to poor clinical outcomes, including therapeutic failure. However, the molecular mechanisms responsible for the observed variability in TFV and FTC responses are poorly understood. Although the observed variability in TFV and FTC drug responses is likely to be multifactorial, alterations in drug activation and deactivation can contribute to the observed variability in drug responses. Phosphorylation of TFV is known and recent studies suggest that nucleotidases may involve in the dephosphorylation of TFV metabolites. Although the kinases that phosphorylate FTC in peripheral blood mononuclear cells are known, the kinases that are responsible for the phosphorylation of FTC in putative sites of HIV infection such as colonic tissues are yet to be determined. Notably, unprotected receptive anal intercourse has a 20-fold higher risk of HIV transmission than vaginal intercourse. Thus, understanding the biotransformation of TFV and FTC in colonic tissue is important since it is a susceptible tissue to HIV infection. Recently, we have reported the enzymatic activities of nucleotidases toward the pharmacologically active metabolites of TFV and FTC in vitro. However, the mechanistic details of the biotransformation of the above drugs in HIV susceptible tissues such as colonic tissues are yet to be elucidated. Gaining a mechanistic understanding of the biotransformation of TFV and FTC in putative sites of HIV infection is important to improve their therapeutic efficacy. As such, in this application, we propose an innovative mass spectrometry imaging-based interdisciplinary approach to understand the biotransformation of TFV and FTC in the colon. Aim 1 will establish the role of nucleotide kinases and nucleotidases in regulating TFV and FTC metabolites in colonic cells mechanistically. Aim 2 will characterize the region- and cell-type-specific expression patterns, as well as enzymatic activities of nucleotide kinases and nucleotidases in situ. The proposed project will provide novel understandings of TFV and FTC activating and deactivating mechanisms that can be leveraged to optimize the therapeutic efficacy of the above drugs.

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.

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

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

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

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

Multi-modal Micro Electrode Fluidic Array (MEFA) Shells for Brain Organoids

Abstract Brain organoids (BOs) derived from human stem cells bridge the gap between monolayer cell culture studies and animal models, which have well-documented limitations. Monolayer cell culture models fail to accurately replicate the 3D interconnectivity in the brain; animal models, while helpful, are limited due to interspecies differences, with most research focusing on rather phenotypical rather than mechanistic aspects. Concurrent with the advancement of BO models is the urgent need to develop 3D micro instrumentation supporting these organoids to investigate brain development and disease in their accurate physiological environment. Conventional microelectrode arrays (MEAs) used for neuronal cell culture studies are planar, which limits recording access to a small fraction of cells on the bottom side of the organoid. Also, conventional microfluidics is inherently planar, and while recent advances in 3D MEAs and 3D microfluidics have enabled electrical and chemical interrogation in 3D, combining both features with tunability and precision to allow independent and simultaneous control is challenging. Recently, we reported new 3D micro instrumentation in the form of 3D shell MEAs and demonstrated its applicability for electrical recording from BOs. They feature lithographically patterned and chip-integrated electrodes and self-folding polymer shells that can be triggered to wrap around BOs to measure electrical activity from the entire organoid surface. The 3D MEA shell system is modeled on and resembles a miniaturized electroencephalography (EEG) cap; the process used to make them is size-scalable, chip-integrated, and mass- producible. In the research, we aim to develop and validate 3D Micro Electrode Fluidic Array (MEFA) shells with multi-modal electrical recording and biochemical control capabilities, offering high spatiotemporal resolution, tunability, and scalability. Since 3D spatiotemporal patterns of neurochemicals play a critical role in molecular and cellular events of neural development and disease, we propose to apply and validate the MEFA shells in two studies that mimic neurodevelopment and monitor the spatiotemporal effects in neurological disorders and their treatments in vitro. We anticipate that the proposed 3D MEFAs would revolutionize brain sciences by permitting real-time, in-situ studies of electrical and chemical stimulation and interrogation of BOs in a high- throughput manner. The proposed 3D scalable, reproducible, and tunable 3D micro instrumentation for BOs has broad relevance to understanding brain development in utero and the development of anatomically accurate drug and toxicity screening platforms for brain sciences and neurological disorders.

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.

Engineering inducible morphotype switching control in Mycobacterium abscessus for investigating infection outcomes and discovering pathophysiological-targeted treatments

PROJECT SUMMARY Antibiotic-resistant nontuberculous mycobacteria (NTM) infections are rising at a rate of 8% each year and account for ~$1.7 billion in annual U.S. healthcare costs. Mycobacterium abscessus (Mabs), the most common rapidly growing NTM infection, is notoriously nicknamed the “antibiotic nightmare” for its extensive intrinsic and inducible broad-range multidrug resistance to antibiotic countermeasures. As part of its natural infection cycle, Mabs undergoes a morphotypical conversion from smooth to rough, characterized by irreversible genetic changes resulting in the loss of cell envelope glycopeptidolipids (GPLs). This morphotypic conversion is intimately associated with disease progression, ultimately leading to debilitating, refractory Mabs pulmonary disease. Specific stimuli triggering Mabs morphotypical conversion are unknown, thus preventing directed investigations into morphotype-specific immunological responses and the discovery of morphotype-specific therapeutic targets. This project leverages cutting-edge molecular genetic tools, including CRISPR (clustered regularly interspersed short palindromic repeats) interference (CRISPRi) and inducible knockdown control of CRISPRi via the anhydrotetracycline-inducible TetR-regulated promoter-operator system, to create six unique, reversible Mabs smooth to conditional rough morphotype strains. These molecular morphoswitchable strains allow precise investigator-mediated on-off control of Mabs surface GPLs, enabling investigations into Mabs morphological plasticity, unique pathophysiology traits associated with each morphotype, and the complex interplay between Mabs and morphotype-specific immunological responses. In Aim 1, we implement CRISPRi inducible knockdown tunable control of Mabs morphotype switching by targeting six, independent genetic targets directly involved in GPL biosynthesis (mps1, mps2) or transport (mmpS4, mmpL4a, mmpL4b, gap) and validate in vitro morphoswitching. In Aim 2, we establish and confirm Mabs morphoswitching and intracellular growth in infected THP-1 macrophages. Subsequently, we evaluate differential and distinct innate cellular immune responses elicited by Mabs smooth and Mabs conditional rough morphotypes during intracellular infection in human primary monocyte-derived macrophages. Collectively, these studies create a suite of characterized and reversible Mabs smooth and conditional rough morphoswitchable strains with controlled, regulated, and on- demand expression of Mabs surface GPLs. By enabling precisely timed and controlled induction of the Mabs conditional rough morphotype during intracellular growth, we can molecularly dissect and investigate fundamental Mabs host-pathogen interactions and immunological responses that so substantially influence negative clinical outcomes.

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

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.

Cellular Crosstalk in Brain Development, Evolution and Disease

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

Rett syndrome, MECP2 and therapeutic strategies

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

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.

Brain-on-a-Chip: Advanced In Vitro Platforms for Drug Screening and Disease Modeling

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.

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.

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.

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.

A draft connectome for ganglion cell types of the mouse retina

The visual system of the brain is highly parallel in its architecture. This is clearly evident in the outputs of the retina, which arise from neurons called ganglion cells. Work in our lab has shown that mammalian retinas contain more than a dozen distinct types of ganglion cells. Each type appears to filter the retinal image in a unique way and to relay this processed signal to a specific set of targets in the brain. My students and I are working to understand the meaning of this parallel organization through electrophysiological and anatomical studies. We record from light-responsive ganglion cells in vitro using the whole-cell patch method. This allows us to correlate directly the visual response properties, intrinsic electrical behavior, synaptic pharmacology, dendritic morphology and axonal projections of single neurons. Other methods used in the lab include neuroanatomical tracing techniques, single-unit recording and immunohistochemistry. We seek to specify the total number of ganglion cell types, the distinguishing characteristics of each type, and the intraretinal mechanisms (structural, electrical, and synaptic) that shape their stimulus selectivities. Recent work in the lab has identified a bizarre new ganglion cell type that is also a photoreceptor, capable of responding to light even when it is synaptically uncoupled from conventional (rod and cone) photoreceptors. These ganglion cells appear to play a key role in resetting the biological clock. It is just this sort of link, between a specific cell type and a well-defined behavioral or perceptual function, that we seek to establish for the full range of ganglion cell types. My research concerns the structural and functional organization of retinal ganglion cells, the output cells of the retina whose axons make up the optic nerve. Ganglion cells exhibit great diversity both in their morphology and in their responses to light stimuli. On this basis, they are divisible into a large number of types (>15). Each ganglion-cell type appears to send its outputs to a specific set of central visual nuclei. This suggests that ganglion cell heterogeneity has evolved to provide each visual center in the brain with pre-processed representations of the visual scene tailored to its specific functional requirements. Though the outline of this story has been appreciated for some time, it has received little systematic exploration. My laboratory is addressing in parallel three sets of related questions: 1) How many types of ganglion cells are there in a typical mammalian retina and what are their structural and functional characteristics? 2) What combination of synaptic networks and intrinsic membrane properties are responsible for the characteristic light responses of individual types? 3) What do the functional specializations of individual classes contribute to perceptual function or to visually mediated behavior? To pursue these questions, we label retinal ganglion cells by retrograde transport from the brain; analyze in vitro their light responses, intrinsic membrane properties and synaptic pharmacology using the whole-cell patch clamp method; and reveal their morphology with intracellular dyes. Recently, we have discovered a novel ganglion cell in rat retina that is intrinsically photosensitive. These ganglion cells exhibit robust light responses even when all influences from classical photoreceptors (rods and cones) are blocked, either by applying pharmacological agents or by dissociating the ganglion cell from the retina. These photosensitive ganglion cells seem likely to serve as photoreceptors for the photic synchronization of circadian rhythms, the mechanism that allows us to overcome jet lag. They project to the circadian pacemaker of the brain, the suprachiasmatic nucleus of the hypothalamus. Their temporal kinetics, threshold, dynamic range, and spectral tuning all match known properties of the synchronization or "entrainment" mechanism. These photosensitive ganglion cells innervate various other brain targets, such as the midbrain pupillary control center, and apparently contribute to a host of behavioral responses to ambient lighting conditions. These findings help to explain why circadian and pupillary light responses persist in mammals, including humans, with profound disruption of rod and cone function. Ongoing experiments are designed to elucidate the phototransduction mechanism, including the identity of the photopigment and the nature of downstream signaling pathways. In other studies, we seek to provide a more detailed characterization of the photic responsiveness and both morphological and functional evidence concerning possible interactions with conventional rod- and cone-driven retinal circuits. These studies are of potential value in understanding and designing appropriate therapies for jet lag, the negative consequences of shift work, and seasonal affective disorder.

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.

Human stem cell models of Alzheimer’s disease and frontotemporal dementia

The development of human induced pluripotent stem cells (iPSC) and their subsequent differentiation into neurons has provided new opportunities for the generation of physiologically-relevant, in vitro disease models. I will present our work using iPSC to modal familial Alzheimer's Disease (fAD) and Frontotemporal Dementia (FTD). We have investigated the mutation-specific effects of APP and PSEN1 mutations on Abeta generation in neurons generated from individuals with fAD, revealing distinct mechanisms that may contribute to clinical heterogeneity in disease. I will also discuss our work to understand the developmental and pathological changes to tau that occur in iPSC-neurons, particularly the challenges of understanding tau pathology in a developmental system, tau proteostasis and how iPSC-neurons may help us identify early signatures of tau pathology in disease.

2nd In-Vitro 2D & 3D Neuronal Networks Summit

The event is open to everyone interested in Neuroscience, Cell Biology, Drug Discovery, Disease Modeling, and Bio/Neuroengineering! This meeting is a platform bringing scientists from all over the world together and fostering scientific exchange and collaboration.

2nd In-Vitro 2D & 3D Neuronal Networks Summit

The event is open to everyone interested in Neuroscience, Cell Biology, Drug Discovery, Disease Modeling, and Bio/Neuroengineering! This meeting is a platform bringing scientists from all over the world together and fostering scientific exchange and collaboration.

Metabolic spikes: from rogue electrons to Parkinson's

Conventionally, neurons are thought to be cellular units that process synaptic inputs into synaptic spikes. However, it is well known that neurons can also spike spontaneously and display a rich repertoire of firing properties with no apparent functional relevance e.g. in in vitro cortical slice preparations. In this talk, I will propose a hypothesis according to which intrinsic excitability in neurons may be a survival mechanism to minimize toxic byproducts of the cell’s energy metabolism. In neurons, this toxicity can arise when mitochondrial ATP production stalls due to limited ADP. Under these conditions, electrons deviate from the electron transport chain to produce reactive oxygen species, disrupting many cellular processes and challenging cell survival. To mitigate this, neurons may engage in ADP-producing metabolic spikes. I will explore the validity of this hypothesis using computational models that illustrate the implications of synaptic and metabolic spiking, especially in the context of substantia nigra pars compacta dopaminergic neurons and their degeneration in Parkinson's disease.

NaV Long-term Inactivation Regulates Adaptation in Place Cells and Depolarization Block in Dopamine Neurons

In behaving rodents, CA1 pyramidal neurons receive spatially-tuned depolarizing synaptic input while traversing a specific location within an environment called its place. Midbrain dopamine neurons participate in reinforcement learning, and bursts of action potentials riding a depolarizing wave of synaptic input signal rewards and reward expectation. Interestingly, slice electrophysiology in vitro shows that both types of cells exhibit a pronounced reduction in firing rate (adaptation) and even cessation of firing during sustained depolarization. We included a five state Markov model of NaV1.6 (for CA1) and NaV1.2 (for dopamine neurons) respectively, in computational models of these two types of neurons. Our simulations suggest that long-term inactivation of this channel is responsible for the adaptation in CA1 pyramidal neurons, in response to triangular depolarizing current ramps. We also show that the differential contribution of slow inactivation in two subpopulations of midbrain dopamine neurons can account for their different dynamic ranges, as assessed by their responses to similar depolarizing ramps. These results suggest long-term inactivation of the sodium channel is a general mechanism for adaptation.

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.

Dysfunctional synaptic vesicle recycling – links to epilepsy

Accurate and synchronous neurotransmitter release is essential for brain communication and occurs when neurotransmitter-containing synaptic vesicles (SVs) fuse to release their content in response to neuronal activity. Neurotransmission is sustained by the process of SV recycling, which generates SVs locally at the presynapse. Until relatively recently it was believed that most mutations in genes that were essential for SV recycling would be incompatible with life, due to this fundamental role. However, this is not the case, with mutations in essential genes for SV fusion, retrieval and recycling identified in individuals with epilepsy. This seminar will cover our laboratory’s progress in determining how genetic mutations in people with epilepsy translate into presynaptic dysfunction and ultimately into seizure activity. The principal focus of these studies will be in vitro investigations of, 1) the biological role of these gene products and 2) how their dysfunction impacts SV recycling, using live fluorescence imaging of genetically-encoded reporters. The gene products to be discussed in more detail will be the SV protein SV2A, the protein kinase CDKL5 and the translation repressor FMRP.

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.

Improving Communication With the Brain Through Electrode Technologies

Over the past 30 years bionic devices such as cochlear implants and pacemakers, have used a small number of metal electrodes to restore function and monitor activity in patients following disease or injury of excitable tissues. Growing interest in neurotechnologies, facilitated by ventures such as BrainGate, Neuralink and the European Human Brain Project, has increased public awareness of electrotherapeutics and led to both new applications for bioelectronics and a growing demand for less invasive devices with improved performance. Coupled with the rapid miniaturisation of electronic chips, bionic devices are now being developed to diagnose and treat a wide variety of neural and muscular disorders. Of particular interest is the area of high resolution devices that require smaller, more densely packed electrodes. Due to poor integration and communication with body tissue, conventional metallic electrodes cannot meet these size and spatial requirements. We have developed a range of polymer based electronic materials including conductive hydrogels (CHs), conductive elastomers (CEs) and living electrodes (LEs). These technologies provide synergy between low impedance charge transfer, reduced stiffness and an ability to be provide a biologically active interface. A range of electrode approaches are presented spanning wearables, implantables and drug delivery devices. This talk outlines the materials development and characterisation of both in vitro properties and translational in vivo performance. The challenges for translation and commercial uptake of novel technologies will also be discussed.

In vitro bioelectronic models of the gut-brain axis

The human gut microbiome has emerged as a key player in the bidirectional communication of the gut-brain axis, affecting various aspects of homeostasis and pathophysiology. Until recently, the majority of studies that seek to explore the mechanisms underlying the microbiome-gut-brain axis cross-talk relied almost exclusively on animal models, and particularly gnotobiotic mice. Despite the great progress made with these models, various limitations, including ethical considerations and interspecies differences that limit the translatability of data to human systems, pushed researchers to seek for alternatives. Over the past decades, the field of in vitro modelling of tissues has experienced tremendous growth, thanks to advances in 3D cell biology, materials, science and bioengineering, pushing further the borders of our ability to more faithfully emulate the in vivo situation. Organ-on-chip technology and bioengineered tissues have emerged as highly promising alternatives to animal models for a wide range of applications. In this talk I’ll discuss our progress towards generating a complete platform of the human microbiota-gut-brain axis with integrated monitoring and sensing capabilities. Bringing together principles of materials science, tissue engineering, 3D cell biology and bioelectronics, we are building advanced models of the GI and the BBB /NVU, with real-time and label-free monitoring units adapted in the model architecture, towards a robust and more physiologically relevant human in vitro model, aiming to i) elucidate the role of microbiota in the gut-brain axis communication, ii) to study how diet and impaired microbiota profiles affect various (patho-)physiologies, and iii) to test personalised medicine approaches for disease modelling and drug testing.

Human stem cell models of neurodegeneration: complex, relevant and robust

How we can make 3D models more reproducible

Reproducible research using stem cell derived neurons and organoids

Inclusive Basic Research

Methodology for understanding the basic phenomena of life can be done in vitro or in vivo, under tightly-controlled experimental conditions designed to limit variability. However stringent the protocol, these experiments do not occur in a cultural vacuum and they are often subject to the same societal biases as other research disciplines. Many researchers uphold the status quo of biased basic research by not questioning the characteristics of their experimental animals, or the people from whom their tissue samples were collected. This means that our fundamental understanding of life has been built on biased models. This session will explore the ways in which basic life sciences research can be biased and the implications of this. We will discuss practical ways to assess your research design and how to make sure it is representative.

Optogenetic silencing of synaptic transmission with a mosquito rhodopsin

Long-range projections link distant circuits in the brain, allowing efficient transfer of information between regions and synchronization of distributed patterns of neural activity. Understanding the functional roles of defined neuronal projection pathways requires temporally precise manipulation of their activity, and optogenetic tools appear to be an obvious choice for such experiments. However, we and others have previously shown that commonly-used inhibitory optogenetic tools have low efficacy and off-target effects when applied to presynaptic terminals. In my talk, I will present a new solution to this problem: a targeting-enhanced mosquito homologue of the vertebrate encephalopsin (eOPN3), which upon activation can effectively suppress synaptic transmission through the Gi/o signaling pathway. Brief illumination of presynaptic terminals expressing eOPN3 triggers a lasting suppression of synaptic output that recovers spontaneously within minutes in vitro and in vivo. The efficacy of eOPN3 in suppressing presynaptic release opens new avenues for functional interrogation of long-range neuronal circuits in vivo.

Workshop: Spatial Brain Dynamics

Traditionally, the term dynamics means changes in a system evolving over time. However, in the brain action potentials propagate along axons to induce postsynaptic currents with different delays at many sites simultaneously. This fundamental computational mechanism evolves spatially to engage the neuron populations involved in brain functions. To identify and understand the spatial processing in brains, this workshop will focus on the spatial principles of brain dynamics that determine how action potentials and membrane currents propagate in the networks of neurons that brains are made of. We will focus on non-artificial dynamics, which excludes in vitro dynamics, interference, electrical and optogenetic stimulations of brains in vivo. Recent non-artificial studies of spatial brain dynamics can actually explain how sensory, motor and internal brain functions evolve. The purpose of this workshop is to discuss these recent results and identify common principles of spatial brain dynamics.

Workshop: Spatial Brain Dynamics

Traditionally, the term dynamics means changes in a system evolving over time. However, in the brain action potentials propagate along axons to induce postsynaptic currents with different delays at many sites simultaneously. This fundamental computational mechanism evolves spatially to engage the neuron populations involved in brain functions. To identify and understand the spatial processing in brains, this workshop will focus on the spatial principles of brain dynamics that determine how action potentials and membrane currents propagate in the networks of neurons that brains are made of. We will focus on non-artificial dynamics, which excludes in vitro dynamics, interference, electrical and optogenetic stimulations of brains in vivo. Recent non-artificial studies of spatial brain dynamics can actually explain how sensory, motor and internal brain functions evolve. The purpose of this workshop is to discuss these recent results and identify common principles of spatial brain dynamics.

Application of Airy beam light sheet microscopy to examine early neurodevelopmental structures in 3D hiPSC-derived human cortical spheroids

The inability to observe relevant biological processes in vivo significantly restricts human neurodevelopmental research. Advances in appropriate in vitro model systems, including patient-specific human brain organoids and human cortical spheroids (hCSs), offer a pragmatic solution to this issue. In particular, hCSs are an accessible method for generating homogenous organoids of dorsal telencephalic fate, which recapitulate key aspects of human corticogenesis, including the formation of neural rosettes—in vitro correlates of the neural tube. These neurogenic niches give rise to neural progenitors that subsequently differentiate into neurons. Studies differentiating induced pluripotent stem cells (hiPSCs) in 2D have linked atypical formation of neural rosettes with neurodevelopmental disorders such as autism spectrum conditions. Thus far, however, conventional methods of tissue preparation in this field limit the ability to image these structures in three-dimensions within intact hCS or other 3D preparations. To overcome this limitation, we have sought to optimise a methodological approach to process hCSs to maximise the utility of a novel Airy-beam light sheet microscope (ALSM) to acquire high resolution volumetric images of internal structures within hCS representative of early developmental time points.

Workshop: Spatial Brain Dynamics

Traditionally, the term dynamics means changes in a system evolving over time. However, in the brain action potentials propagate along axons to induce postsynaptic currents with different delays at many sites simultaneously. This fundamental computational mechanism evolves spatially to engage the neuron populations involved in brain functions. To identify and understand the spatial processing in brains, this workshop will focus on the spatial principles of brain dynamics that determine how action potentials and membrane currents propagate in the networks of neurons that brains are made of. We will focus on non-artificial dynamics, which excludes in vitro dynamics, interference, electrical and optogenetic stimulations of brains in vivo. Recent non-artificial studies of spatial brain dynamics can actually explain how sensory, motor and internal brain functions evolve. The purpose of this workshop is to discuss these recent results and identify common principles of spatial brain dynamics.

Synchrony and Synaptic Signaling in Cerebellar Circuits

The cerebellum permits a wide range of behaviors that involve sensorimotor integration. We have been investigating how specific cellular and synaptic specializations of cerebellar neurons measured in vitro, give rise to circuit activity in vivo. We have investigated these issues by studying Purkinje neurons as well as the large neurons of the mouse cerebellar nuclei, which form the major excitatory premotor projection from the cerebellum. Large CbN cells have ion channels that favor spontaneous action potential firing and GABAA receptors that generate ultra-fast inhibitory synaptic currents, raising the possibility that these biophysical attributes may permit CbN cells to respond differently to the degree of temporal coherence of their Purkinje cell inputs. In vivo, self-initiated motor programs associated with whisking correlates with asynchronous changes in Purkinje cell simple spiking that are asynchronous across the population. The resulting inhibition converges with mossy fiber excitation to yield little change in CbN cell firing, such that cerebellar output is low or cancelled. In contrast, externally applied sensory stimuli elicits a transient, synchronous inhibition of Purkinje cell simple spiking. During the resulting strong disinhibition of CbN cells, sensory-induced excitation from mossy fibers effectively drives cerebellar outputs that increase the magnitude of reflexive whisking. Purkinje cell synchrony, therefore, may be a key variable contributing to the “positive effort” hypothesized by David Marr in 1969 to be necessary for cerebellar control of movement.

Using human pluripotent stem cells to model obesity in vitro

Obesity and neurodegeneration lead to millions of premature deaths each year and lack broadly effective treatments. Obesity is largely caused by the abnormal function of cell populations in the hypothalamus that regulate appetite. We have developed methods generate human hypothalamic neurons from hPSCs to study how they respond to nutrients and hormones (e.g. leptin) and how disease-associated mutations alter their function. Since human hypothalamic neurons can be produced in large numbers, are functionally responsive, have a human genome that can be readily edited, and are in culture environment that can be readily controlled, there is an unprecedented opportunity to study the genetic and environmental factors underlying obesity. In addition, we are fascinated by the fact that mid-life obesity is a risk factor for dementia later in life, and caloric restriction, exercise, and certain anti-obesity drugs are neuroprotective, suggesting that there are shared mechanisms between obesity and neurodegeneration. Studies of HPSC-derived hypothalamic neurons may help bridge the mechanistic gulf between human genetic data and organismic phenotypes, revealing new therapeutic targets. ​

Learning Neurobiology with electric fish

Electric Gymnotiform fish live in muddy, shallow waters near the shore – hiding in the dense filamentous roots of floating plants such as Eichornia crassipes (“camalote”). They explore their surroundings by using a series of electric pulses that serve as self emitted carrier of electrosensory signals. This propagates at the speed of light through this spongiform habitat and is barely sensed by the lateral line of predators and prey. The emitted field polarizes the surroundings according to the difference in impedance with water which in turn modifies the profile of transcutaneous currents considered as an electrosensory image. Using this system, pulse Gymnotiformes create an electrosensory bubble where an object’s location, impedance, size and other characteristics are discriminated and probably recognized. Although consciousness is still not well-proven, cognitive functions as volition, attention, and path integration have been shown. Here I will summarize different aspects of the electromotor electrosensory loop of pulse Gymnotiforms. First, I will address how objects are polarized with a stereotyped but temporospatially complex electric field, consisting of brief pulses emitted at regular intervals. This relies on complex electric organs quasi periodically activated through an electromotor coordination system by a pacemaker in the medulla. Second, I will deal with the imaging mechanisms of pulse gymnotiform fish and the presence of two regions in the electrosensory field, a rostral region where the field time course is coherent and field vector direction is constant all along the electric organ discharge and a lateral region where the field time course is site specific and field vector direction describes a stereotyped 3D trajectory. Third, I will describe the electrosensory mosaic and their characteristics. Receptor and primary afferents correspond one to one showing subtypes optimally responding to the time course of the self generated pulse with a characteristic train of spikes. While polarized objects at the rostral region project their electric images on the perioral region where electrosensory receptor density, subtypes and central projection are maximal, the image of objects on the side recruit a single type of scattered receptors. Therefore, the rostral mosaic has been likened to an electrosensory fovea and its receptive field referred to as foveal field. The rest of the mosaic and field are referred to as peripheral. Finally, I will describe ongoing work on early processing structures. I will try to generate an integrated view, including anatomical and functional data obtained in vitro, acute experiments, and unitary recordings in freely moving fish. We have recently shown have shown that these fish tract allo-generated fields and the virtual fields generated by nearby objects in the presence of self-generated fields to explore the nearby environment. These data together with the presence of a multimodal receptor mosaic at the cutaneous surface particularly surrounding the mouth and an important role of proprioception in early sensory processing suggests the hypothesis that the active electrosensory system is part of a multimodal haptic sense.

Carnosine negatively modulates pro-oxidant activities of M1 peripheral macrophages and prevents neuroinflammation induced by amyloid-β in microglial cells

Carnosine is a natural dipeptide widely distributed in mammalian tissues and exists at particularly high concentrations in skeletal and cardiac muscles and brain. A growing body of evidence shows that carnosine is involved in many cellular defense mechanisms against oxidative stress, including inhibition of amyloid-β (Aβ) aggregation, modulation of nitric oxide (NO) metabolism, and scavenging both reactive nitrogen and oxygen species. Different types of cells are involved in the innate immune response, with macrophage cells representing those primarily activated, especially under different diseases characterized by oxidative stress and systemic inflammation such as depression and cardiovascular disorders. Microglia, the tissue-resident macrophages of the brain, are emerging as a central player in regulating key pathways in central nervous system inflammation; with specific regard to Alzheimer’s disease (AD) these cells exert a dual role: on one hand promoting the clearance of Aβ via phagocytosis, on the other hand increasing neuroinflammation through the secretion of inflammatory mediators and free radicals. The activity of carnosine was tested in an in vitro model of macrophage activation (M1) (RAW 264.7 cells stimulated with LPS + IFN-γ) and in a well-validated model of Aβ-induced neuroinflammation (BV-2 microglia treated with Aβ oligomers). An ample set of techniques/assays including MTT assay, trypan blue exclusion test, high performance liquid chromatography, high-throughput real-time PCR, western blot, atomic force microscopy, microchip electrophoresis coupled to laser-induced fluorescence, and ELISA aimed to evaluate the antioxidant and anti-inflammatory activities of carnosine was employed. In our experimental model of macrophage activation (M1), therapeutic concentrations of carnosine exerted the following effects: 1) an increased degradation rate of NO into its non-toxic end-products nitrite and nitrate; 2) the amelioration of the macrophage energy state, by restoring nucleoside triphosphates and counterbalancing the changes in ATP/ADP, NAD+/NADH and NADP+/NADPH ratio obtained by LPS + IFN-γ induction; 3) a reduced expression of pro-oxidant enzymes (NADPH oxidase, Cyclooxygenase-2) and of the lipid peroxidation product malondialdehyde; 4) the rescue of antioxidant enzymes expression (Glutathione peroxidase 1, Superoxide dismutase 2, Catalase); 5) an increased synthesis of transforming growth factor-β1 (TGF-β1) combined with the negative modulation of interleukines 1β and 6 (IL-1β and IL-6), and 6) the induction of nuclear factor erythroid-derived 2-like 2 (Nrf2) and heme oxygenase-1 (HO-1). In our experimental model of Aβ-induced neuroinflammation, carnosine: 1) prevented cell death in BV-2 cells challenged with Aβ oligomers; 2) lowered oxidative stress by decreasing the expression of inducible nitric oxide synthase and NADPH oxidase, and the concentrations of nitric oxide and superoxide anion; 3) decreased the secretion of pro-inflammatory cytokines such as IL-1β simultaneously rescuing IL-10 levels and increasing the expression and the release of TGF-β1; 4) prevented Aβ-induced neurodegeneration in primary mixed neuronal cultures challenged with Aβ oligomers and these neuroprotective effects was completely abolished by SB431542, a selective inhibitor of type-1 TGF-β receptor. Overall, our data suggest a novel multimodal mechanism of action of carnosine underlying its protective effects in macrophages and microglia and the therapeutic potential of this dipeptide in counteracting pro-oxidant and pro-inflammatory phenomena observed in different disorders characterized by elevated levels of oxidative stress and inflammation such as depression, cardiovascular disorders, and Alzheimer’s disease.

Physiological importance of phase separation: a case study in synapse formation

Synapse formation during neuronal development is critical to establish neural circuits and a nervous system1. Every presynapse builds a core active zone structure where ion channels are clustered and synaptic vesicles are released2. While the composition of active zones is well characterized2,3, how active zone proteins assemble together and recruit synaptic release machinery during development is not clear. Here, we find core active zone scaffold proteins SYD-2/Liprin-α and ELKS-1 phase separate during an early stage of synapse development, and later mature into a solid structure. We directly test the in vivo function of phase separation with mutants specifically lacking this activity. These mutant SYD-2 and ELKS-1 proteins remain enriched at synapses, but are defective in active zone assembly and synapse function. The defects are rescued with the introduction of a phase separation motif from an unrelated protein. In vitro, we reconstitute the SYD-2 and ELKS-1 liquid phase scaffold and find it is competent to bind and incorporate downstream active zone components. The fluidity of SYD-2 and ELKS-1 condensates is critical for efficient mixing and incorporation of active zone components. These data reveal that a developmental liquid phase of scaffold molecules is essential for synaptic active zone assembly before maturation into a stable final structure.

On the purpose and origin of spontaneous neural activity

Spontaneous firing, observed in many neurons, is often attributed to ion channel or network level noise. Cortical cells during slow wave sleep exhibit transitions between so called Up and Down states. In this sleep state, with limited sensory stimuli, neurons fire in the Up state. Spontaneous firing is also observed in slices of cholinergic interneurons, cerebellar Purkinje cells and even brainstem inspiratory neurons. In such in vitro preparations, where the functional relevance is long lost, neurons continue to display a rich repertoire of firing properties. It is perplexing that these neurons, instead of saving their energy during information downtime and functional irrelevance, are eager to fire. We propose that spontaneous firing is not a chance event but instead, a vital activity for the well-being of a neuron. We postulate that neurons, in anticipation of synaptic inputs, keep their ATP levels at maximum. As recovery from inputs requires most of the energy resources, neurons are ATP surplus and ADP scarce during synaptic quiescence. With ADP as the rate-limiting step, ATP production stalls in the mitochondria when ADP is low. This leads to toxic Reactive Oxygen Species (ROS) formation, which are known to disrupt many cellular processes. We hypothesize that spontaneous firing occurs at these conditions - as a release valve to spend energy and to restore ATP production, shielding the neuron against ROS. By linking a mitochondrial metabolism model to a conductance-based neuron model, we show that spontaneous firing depends on baseline ATP usage and on ATP-cost-per-spike. From our model, emerges a mitochondrial mediated homeostatic mechanism that provides a recipe for different firing patterns. Our findings, though mostly affecting intracellular dynamics, may have large knock-on effects on the nature of neural coding. Hitherto it has been thought that the neural code is optimised for energy minimisation, but this may be true only when neurons do not experience synaptic quiescence.

Mechanisms of pathogenesis in the tauopathies

The distribution of pathological tau in the brain of patients with AD is highly predicable, and as disease worsens, it spreads transynaptically from initial regions of vulnerability. The reason why only some neurons are vulnerable to the accumulation and propagation of pathological forms of tau, and the mechanisms by which tauopathy spreads through the brain are not well understood. Using a combination of immunohistochemistry and computational analysis we have examined pathway differences between vulnerable and resistant neurons. How tau spreads across a synapse has been examined in vitro using different model systems. Our data show that dysregulation of tau homeostasis determines the cellular and regional vulnerability of specific neurons to tau pathology (H. Fu et al. 2019. Nat. Neuro. 22 (1):47-56) and that deficits in tau homeostasis can exacerbate tau accumulation and propagation. Aging appears to impact similar neuronal populations. Mechanisms and consequences of abnormal tau accumulation within neurons, its transfer between cells, pathology propagation and therapeutic opportunities will be discussed.

Functional characterization of human iPSC-derived neurons at single-cell resolution

Recent developments in induced pluripotent stem cell (iPSC) technology have enabled easier access to human cells in vitro. With increasing availability of human iPSC-derived neurons, both healthy and disease cell lines, screening compounds for neurodegenerative diseases on human cells can potentially be performed in the earlier stages of drug discovery. To accelerate the functional characterization of iPSC-derived neurons and the effect of compounds, reproducible and relevant results are necessary. In this webinar, the speakers will: Introduce high-resolution functional imaging of human iPSC-derived neurons Showcase how to extract functional features of hundreds of cells in a cell culture sample label-free Discuss electrophysiological parameters for characterizing the differences among several human neuronal cell lines

INTEGRATING GENETICS, PROTEOMICS, AND <EM>IN VITRO</EM> MODELING TO ELUCIDATE <EM>SLCO1A2</EM>-MEDIATED CEREBROSPINAL FLUID CLEARANCE IN IDIOPATHIC NORMAL PRESSURE HYDROCEPHALUS

FENS Forum 2026

Towards using small topologically constrained networks in-vitro in combination with in-silico models

COSYNE 2022

NIR Laser Photobiomodulation as promising therapy for Brain Hypoxia/Ischemia: in vitro study in organotypic hippocampal slices

Aberrant inflammatory activity of microglia influences neuronal connectivity and activity in schizophrenia in vitro

AQP4 expression level and aggregation state affect astrocyte migration in an in vitro model of reactive gliosis

Behavioural and in vitro studies on processing of bistable stimuli in the mouse visual system

Cholinergic induction of synchronous oscillation in the slug neuronal network in vitro

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

Cannabinoids and cerebral ischemia: experimental studies in an in vitro model

The cell-type specific contribution of EHMT1 to the excitatory/inhibitory balance in in vitro human neuronal networks

Characterisation of a novel NOX2 inhibitor, GSK2795039, using in vitro models of microglial activation

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

Chloride dynamics in specific neuronal subtypes during epileptiform activity in vitro

Bioengineered cortical neuronal network (BIOCONNET): A stem-cell derived neuronal array with defined circuitry architecture in vitro

Development and characterization of an in vitro model of SSADH deficiency using patient IPSC-derived neurons to support unbiased screening of novel therapeutic approaches to treatment

Development of functional in vitro model in Dravet syndrome patient hiPSC-derived cortical neurons

Dimensional Causality analysis method on evoked epileptic activity in vitro

Directly reprogrammed medium spiny neurons for studying pathology and synaptic dysfunction in Huntington's disease in vitro model

Dissecting the link between glial cell shape and function in vitro using human induced pluripotent stem cell-derived astrocytes

The effects of the SOD mimetic, MnTMPyP, on synaptic signalling and viability in an in vitro rat OGD model

Electric axon guidance in embryonic chick retina: molecular mechanism and in vitro optic nerve formation

Empagliflozin effect on the central nervous system: an in vitro study on primary neuronal cell cultures

Engineering a stretchable nerve-on-chip platform to study the changes in nerve conduction under tension in vitro

Ethanol-induced miRNA 137 and 501-3p modulate AMPA neurotransmission in developing hippocampal slices in vitro

Functional effects of human LGI1 autoantibodies on CA3 pyramidal neurons: a species-specific in vitro study in human hippocampal slice cultures

Galectin-3 role in the interaction of microglia and Amyloid-Beta fibrils in vitro

Identification of pro-angiogenic factors for in vitro vascularization of hiPSC-derived brain organoids

The interplay between cell shape/size and function in vitro: Investigating the effect of axonal length on human spinal motor neurons

Ιnvestigation of ependymal cells in the mouse and human Subependymal Zone: in vivo assessment and in vitro cultures

Towards using small topologically constrained networks in-vitro in combination with in-silico models

COSYNE 2022

Loss of GBA activity exacerbate the toxicity of alpha-synuclein oligomers and protofibrils in an in vitro model of Parkinson’s disease

Magnetic field in the extreme low frequency band protects neuronal cells from oxygen-glucose deprivation in-vitro

Measuring pERK (phosphorylated extracellular-signal regulated kinase) activity in acute in-vitro slices of the rat olfactory bulb

A microfluidic based in vitro model to reconstruct the corticostriatal synapse in the study of Huntington’s disease

Mitochondrial morphology and dynamic abnormalities in an in vitro model of Rett syndrome

Modulation of anandamide tone as an effective strategy for in vitro and in vivo stimulation of autophagy in Alzheimer's disease

Modulation of in vitro cortical networks by mechanical perturbation of individual neurons

Multidirectional propagation of SPW-R complexes in the rat hippocampus, in vitro

Disrupting periodic neuronal synchrony with closed-loop stimulation in vitro

COSYNE 2022