therapeutic strategies

Delineating the role of TREM2 in chronic pancreatitis

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

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

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

The Role of the Intestinal Microbiota in Sepsis Mortality

Project Summary/Abstract Sepsis is a life-threatening condition characterized by a dysregulated host response to infection that can cause multi-organ damage and death. As the leading cause of in-hospital mortality, sepsis mortality rates reach up to 50%, and account for approximately 270,000 deaths and $38 billion annually in health care costs in the United States. Notably, patients with similar medical backgrounds can have vastly different sepsis outcomes— some survive with medical treatment while others die. The reasons for this dichotomy are unknown but is seen across all forms of bacterial bloodstream infections, is not specific to any strain-level differences in the infecting pathogen and cannot be explained by human genetic differences. Human microbiota studies suggest that gut microbial dysbiosis is associated with sepsis mortality and that these alterations influence gut barrier breakdown, leading to gram-negative bacteremia—one of the most common causes of sepsis and mortality. However, there are a lack of studies that investigate the causal role of the intestinal microbiota in sepsis mortality. This K08 proposal will elucidate the role of the intestinal microbiota in sepsis mortality. Utilizing the well- established murine model of sepsis by intraperitoneal injection of lipopolysaccharide (LPS), we combine microbiota taxonomic sequencing and metagenomics, advanced bioinformatic techniques and prediction modeling, with knowledge of mucosal immunity and germ-free mouse systems to characterize the microbiota features and members that correlate with, predict, and cause sepsis mortality. This proposal is organized into two specific aims: (1) identify baseline stool microbial features associated with and predictive of sepsis outcomes and (2) determine how colonization with immunostimulatory microbes heightens sepsis mortality. In this work, I will holistically characterize the host immunologic and microbiota features that are associated with and predictive of mortality and experimentally identify microbes and microbial pathways that cause death in our model. These findings will reveal new microbial and host biomarkers of sepsis mortality and identify novel targets for sepsis prevention and treatment to reduce the overall mortality rate of this deadly disease. My long-term goal is to become an independent physician-scientist who integrates cutting-edge computational methods with experimental biology to identify predictive biomarkers of disease onset and outcomes, investigate how they influence disease processes, and develop novel therapeutic and preventive strategies to improve patient care. This proposal details specific research aims and a structured career development and training plan that will allow me to acquire focused, in-depth and multidisciplinary training under the guidance of an internationally recognized team of experts in clinical infectious diseases, host-microbiota interactions, immunology, immunometabolism, and computational biology. The knowledge generated will address the fundamental role of the microbiota in sepsis outcomes and inform future preventative and therapeutic strategies that will lower the sepsis mortality rate worldwide.

Targeting the Molecular Crosstalk Between EZHIP and PRC2 in PFA Ependymoma

Project Summary: PFA ependymoma is a rare and aggressive pediatric brain tumor with a poorly understood molecular mechanism. Unlike many cancers, PFA ependymoma exhibits very few genetic alterations. Instead, it is thought to be driven primarily by epigenetic dysregulation. A key player in this disease is the EZH1/2 inhibitory protein EZHIP, which is normally expressed only in germ cells. EZHIP is aberrantly expressed in PFA ependymoma, where it disrupts the function of Polycomb Repressive Complex 2 (PRC2), a master epigenetic regulator of developmental gene repression through deposition of the trimethylated histone H3 lysine 27 (H3K27me3) repressive histone mark. EZHIP-mediated dysregulation of PRC2 involves both enzymatic inhibition and physical stalling of PRC2 on CpG island (CGI) chromatin, leading to a global loss of H3K27me3 levels, an epigenetic hallmark of PFA ependymoma. PRC2 itself is a highly dynamic and intricate complex that assembles into two functional variants, PRC2.1 and PRC2.2. These two variants share a core composed of the catalytic subunits EZH1/2, along with EED, SUZ12, and RBBP4/7, and differ by incorporating distinct accessory subunits. PRC2.1 includes PHF1/MTF2/PHF19, EPOP, and PALI1/2, while PRC2.2 features AEBP2 and JARID2. Our preliminary data reveal intriguing molecular crosstalk between EZHIP and multiple PRC2 components, suggesting potential competitive or cooperative interplay. The ability of EZHIP to inhibit PRC2 partly stems from its mimicry of the oncohistone H3K27M, which harbors a lysine-to-methionine mutation that causes diffuse midline glioma, another devastating brain tumor in children, where PRC2 activity is also globally suppressed. However, the precise, EZHIP-specific mechanisms behind PRC2 dysregulation in PFA ependymoma remain largely unexplored. Our work aims to uncover these elusive mechanisms using a powerful combination of structural biology, biochemistry, and genomics approaches. Ultimately, we aim to identify therapeutic strategies that disrupt the pathogenic EZHIP–PRC2 crosstalk and restore the normal H3K27me3 epigenetic landscape. Specifically, in Aim 1, we will determine the structural and biochemical mechanisms underlying the enzymatic inhibition of the PRC2 core complex by EZHIP. In Aim 2, we will elucidate the molecular basis of EZHIP-mediated stalling of PRC2 on CGI chromatin, involving PRC2 functional variants. In Aim 3, we will explore an exciting mechanism-based therapeutic strategy to overcome PRC2 enzymatic inhibition and chromatin stalling induced by EZHIP.

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.

Structural and functional characterization of autoimmune antibodies against NMDAR

Project Summary. The goal of this project is to understand the origins and molecular mechanisms underlying the anti-cancer autoimmune response against the N-methyl-D-aspartate receptor (NMDAR) and its correlation with anti-N-methyl-D-aspartate receptor autoimmune encephalitis (NMDARAE). While anti-cancer immune responses can promote tumor elimination, they may also lead to the production of self-reactive antibodies that trigger autoimmune diseases. NMDARAE is the most common form of immune-mediated encephalitis, which results in prominent neuropsychiatric symptoms, including seizures, psychosis, and memory deficits. NMDARs belong to a family of ligand-gated ion channels expressed exclusively in the central nervous system. They are involved in various aspects of brain development and function, including learning and memory. They respond to the neurotransmitter glutamate and a co-agonist, glycine or D-serine, to mediate excitatory neurotransmission, which plays a central role in synaptic plasticity. NMDARAE is associated with ovarian teratomas, where aberrant NMDAR expression is believed to trigger an autoimmune response. In NMDARAE, anti-NMDAR antibodies, as well as B cells and antibody-secreting cells, cross the blood-brain barrier via unknown mechanisms, resulting in the presence of anti-NMDAR antibodies at high titers within the brain and cerebrospinal fluid (CSF). These antibodies target NMDARs, modulating their function and contributing to disease pathology. Emerging evidence, supported by our preliminary data, suggests that NMDARs are also expressed in triple-negative breast cancer (TNBC), extending the relevance of anti-NMDAR autoimmunity beyond ovarian teratomas. In our TNBC mouse model, which ectopically expresses NMDARs (TNBC-NMDAR), we observed the onset of anti-NMDAR autoimmunity, where the produced antibodies cause both anti-tumor activity and symptoms such as lowered seizure threshold, mirroring key features of NMDARAE. Here, we will establish this TNBC mouse model as we develop molecular methods to characterize it. Aim 1 will focus on establishing and characterizing the TNBC- NMDAR mouse model. We will develop a detection method utilizing the intact tetrameric NMDAR channel proteins and a method to isolate B cells expressing B cell receptors against NMDAR from biological samples by using fluorescently labeled intact NMDAR proteins, followed by single-cell RNA sequencing. Aim 2 will utilize single-particle cryo-electron microscopy (cryo-EM) to investigate the interactions between NMDAR and the cloned antibodies, providing insights into epitope recognition, NMDAR subtype specificity, and conformational changes induced by antibody binding. Aim 3 will assess the impact of the cloned antibodies on NMDAR channel activity using electrophysiology. We will also assess anti-tumor activity and NMDARAE onset by each antibody clone. Together, the proposed research will gain insights into the link between anti-cancer anti-NMDAR autoimmunity and NMDARAE. It will also elucidate which functional properties of the cloned antibodies promote anti-tumor activity while contributing to NMDARAE, thereby informing potential therapeutic strategies.

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

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

Uncovering genetic determinants of carbapenem resistance in Klebsiella pneumoniae

Carbapenem-resistant Klebsiella pneumoniae represents an urgent global health threat due to its increasing prevalence and high mortality rates, necessitating a comprehensive understanding of its resistance mechanisms. While key resistance mechanisms and their genetic determinants are known, such as beta- lactamases and porin mutations, the cause of resistance in many strains remains elusive. Moreover, other strains that carry known genetic carbapenem-resistance factors have been found to still be susceptible to carbapenems for unclear reasons. Further, strains can carry genetic elements which, while not conferring resistance directly, can promote resistance indirectly by accelerating its acquisition, such as through mutations in DNA repair systems or mobile genetic elements. To address these knowledge gaps, we propose a genome-wide association study (GWAS), with the aim of maximizing the discovery of gene variants associated with meropenem resistance, with experimental validation of candidates to identify true causal variants. We will overcome limitations of prior studies in the following ways: 1) We have compiled an expanded data set of publicly available K. pneumoniae genomes from strains isolated across a wide distribution of countries, with in hand access to >100 isolates upon which experimental validation studies will be performed. 2) We will perform comprehensive capture of genetic variants by employing a reference-free GWAS, utilizing unitigs, stretches of DNA sequence that represent the entire spectrum of genetic variation. 3) We will enhance statistical power to detect genetic variants with even subtle effects on resistance by using a quantitative, continuous minimum inhibitory concentration (MIC) phenotype to meropenem rather than a binary designation of resistant or susceptible. 4) We will reduce the number of false positives arising from correlation, or linkage disequilibrium (LD), with known carbapenemase and other known resistance factors by performing a conditional GWAS, where known factors are included as covariates. 5) We will further mitigate confounding effects due to population structure and LD, which cause non-random relationships between variants, by utilizing a pangenome-wide regression with an elastic net penalty. 6) Crucially, we will functionally validate our findings, which will include genetic variants associated with increased resistance, whether through direct or indirect mechanisms, as well as those that may restore susceptibility in strains already possessing known resistance factors. We will bridge the gap between GWAS findings and functional validation by leveraging our high-throughput experimental capabilities. This integrated approach promises to uncover novel mechanisms of carbapenem resistance, its acquisition, and susceptibility in K. pneumoniae, with the potential to inform the development of future diagnostics or therapeutic strategies.

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.

Pathogenic mechanisms of expanded ZFHX3 in SCA4 cerebellar organoids

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

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

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

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.

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

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

Freeze or flee ? New insights from rodent models of autism

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

Therapeutic Strategies for Autism: Targeting Three Levels of the Central Dogma of Molecular Biology with a Focus on SYNGAP1

Targeting thalamic circuits rescues motor and mood deficits in PD mice

Although bradykinesia, tremor, and rigidity are hallmark motor defects in Parkinson’s disease (PD) patients, they also experience motor learning impairments and non-motor symptoms such as depression. The neural basis for these different PD symptoms are not well understood. While current treatments are effective for locomotion deficits in PD, therapeutic strategies targeting motor learning deficits and non-motor symptoms are lacking. We found that distinct parafascicular (PF) thalamic subpopulations project to caudate putamen (CPu), subthalamic nucleus (STN), and nucleus accumbens (NAc). While PF-->CPu and PF-->STN circuits are critical for locomotion and motor learning respectively, inhibition of the PF-->NAc circuit induced a depression-like state. While chemogenetically manipulating CPu-projecting PF neurons led to a long-term restoration of locomotion, optogenetic long-term potentiation at PF-->STN synapses restored motor learning behavior in PD model mice. Furthermore, activation of NAc-projecting PF neurons rescued depression-like PD phenotypes. Importantly, we identified nicotinic acetylcholine receptors capable of modulating PF circuits to rescue different PD phenotypes. Thus, targeting PF thalamic circuits may be an effective strategy for treating motor and non-motor deficits in PD.

The glymphatic system in motor neurone disease

Neurodegenerative diseases are chronic and inexorable conditions characterised by the presence of insoluble aggregates of abnormally ubiquinated and phosphorylated proteins. Recent evidence also suggests that protein misfolding can propagate throughout the body in a prion-like fashion via the interstitial or cerebrospinal fluids (CSF). As protein aggregation occurs well before the onset of brain damage and symptoms, new biomarkers sensitive to early pathology, together with therapeutic strategies that include eliminating seed proteins and blocking cell-to-cell spread, are of vital importance. The glymphatic system, which facilitates the continuous exchange of CSF and interstitial fluid to clear the brain of waste, presents as a potential biomarker of disease severity, therapeutic target, and drug delivery system. In this webinar, Associate Professor David Wright from the Department of Neuroscience, Monash University, will outline recent advances in using MRI to investigate the glymphatic system. He will also present some of his lab’s recent work investigating glymphatic clearance in preclinical models of motor neurone disease. Associate Professor David Wright is an NHMRC Emerging Leadership Fellow and the Director of Preclinical Imaging in the Department of Neuroscience, Monash University and the Alfred Research Alliance, Alfred Health. His research encompasses the development, application and analysis of advanced magnetic resonance imaging techniques for the study of disease, with a particular emphasis on neurodegenerative disorders. Although less than three years post PhD, he has published over 60 peer-reviewed journal articles in leading neuroscience journals such as Nature Medicine, Brain, and Cerebral Cortex.

Mutation targeted gene therapy approaches to alter rod degeneration and retain cones

My research uses electrophysiological techniques to evaluate normal retinal function, dysfunction caused by blinding retinal diseases and the restoration of function using a variety of therapeutic strategies. We can use our understanding or normal retinal function and disease-related changes to construct optimal therapeutic strategies and evaluate how they ameliorate the effects of disease. Retinitis pigmentosa (RP) is a family of blinding eye diseases caused by photoreceptor degeneration. The absence of the cells that for this primary signal leads to blindness. My interest in RP involves the evaluation of therapies to restore vision: replacing degenerated photoreceptors either with: (1) new stem or other embryonic cells, manipulated to become photoreceptors or (2) prosthetics devices that replace the photoreceptor signal with an electronic signal to light. Glaucoma is caused by increased intraocular pressure and leads to ganglion cell death, which eliminates the link between the retinal output and central visual processing. We are parsing out of the effects of increased intraocular pressure and aging on ganglion cells. Congenital Stationary Night Blindness (CSNB) is a family of diseases in which signaling is eliminated between rod photoreceptors and their postsynaptic targets, rod bipolar cells. This deafferents the retinal circuit that is responsible for vision under dim lighting. My interest in CSNB involves understanding the basic interplay between excitation and inhibition in the retinal circuit and its normal development. Because of the targeted nature of this disease, we are hopeful that a gene therapy approach can be developed to restore night vision. My work utilizes rodent disease models whose mutations mimic those found in human patients. While molecular manipulation of rodents is a fairly common approach, we have recently developed a mutant NIH miniature swine model of a common form of autosomal dominant RP (Pro23His rhodopsin mutation) in collaboration with the National Swine Resource Research Center at University of Missouri. More genetically modified mini-swine models are in the pipeline to examine other retinal diseases.

Neuron-glia interactions in synapse degeneration in Alzheimer's disease

Tara Spires-Jones’ research focuses on the mechanisms and reversibility of neurodegeneration in Alzheimer’s disease, other degenerative brain diseases, and ageing.  The objective of her research group is to understand why synapses and neurons become dysfunctional and die in these diseases in order to develop effective therapeutic strategies. Her work has shown that soluble forms of the pathological proteins amyloid beta and tau contribute to synapse degeneration, and that lowering levels of these proteins can prevent and reverse phenotypes in model systems. Further, she has pioneered high-resolution imaging techniques in human post-mortem brain and found evidence that these proteins accumulate in synapses in human disease.

Beyond energy - an unconventional role of mitochondria in cone photoreceptors

The long-term goal of my research is to study the mammalian retina as a model for the central nervous system (CNS) -- to understand how it functions in physiological conditions, how it is formed, how it breaks down in pathological conditions, and how it can be repaired. I have focused on two research themes: 1) Photoreceptor structure, synapse, circuits, and development, 2) Hibernation and metabolic adaptations in the retina and beyond. As the first neuron of the visual system, photoreceptors are vital for photoreception and transmission of visual signals. I am particularly interested in cone photoreceptors, as they mediate our daylight vision with high resolution color information. Diseases affecting cone photoreceptors compromise visual functions in the central macular area of the human retina and are thus most detrimental to our vision. However, because cones are much less abundant compared to rods in most mammals, they are less well studied. We have used the ground squirrel (GS) as a model system to study cone vision, taking advantage of their unique cone-dominant retina. In particular, we have focused on short-wavelength sensitive cones (S-cones), which are not only essential for color vision, but are also an important origin of signals for biological rhythm, mood and cognitive functions, and the growth of the eye during development. We are studying critical cone synaptic structures – synaptic ribbons, the synaptic connections of S-cones, and the development of S-cones with regard to their specific connections. These works will provide knowledge of normal retinal development and function, which can also be extended to the rest of CNS; for example, the mechanisms of synaptic targeting during development. In addition, such knowledge will benefit the development of optimal therapeutic strategies for regeneration and repair in cases of retinal degenerative disease. Many neurodegenerative diseases, including retinal diseases, are rooted in metabolic stress in neurons and/or glial cells. Using the same GS model, we aim to learn from this hibernating mammal, which possesses an amazing capability to adapt to the extreme metabolic conditions during hibernation. By exploring the mechanisms of such adaptation, we hope to discover novel therapeutic tactics for neurodegenerative diseases.

CRISPR-based functional genomics in iPSC-based models of brain disease

Human genes associated with brain-related diseases are being discovered at an accelerating pace. A major challenge is an identification of the mechanisms through which these genes act, and of potential therapeutic strategies. To elucidate such mechanisms in human cells, we established a CRISPR-based platform for genetic screening in human iPSC-derived neurons, astrocytes and microglia. Our approach relies on CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa), in which a catalytically dead version of the bacterial Cas9 protein recruits transcriptional repressors or activators, respectively, to endogenous genes to control their expression, as directed by a small guide RNA (sgRNA). Complex libraries of sgRNAs enable us to conduct genome-wide or focused loss-of-function and gain-of-function screens. Such screens uncover molecular players for phenotypes based on survival, stress resistance, fluorescent phenotypes, high-content imaging and single-cell RNA-Seq. To uncover disease mechanisms and therapeutic targets, we are conducting genetic modifier screens for disease-relevant cellular phenotypes in patient-derived neurons and glia with familial mutations and isogenic controls. In a genome-wide screen, we have uncovered genes that modulate the formation of disease-associated aggregates of tau in neurons with a tauopathy-linked mutation (MAPT V337M). CRISPRi/a can also be used to model and functionally evaluate disease-associated changes in gene expression, such as those caused by eQTLs, haploinsufficiency, or disease states of brain cells. We will discuss an application to Alzheimer’s Disease-associated genes in microglia.

Discovering new therapeutic strategies in stroke through APC/C-Cdh1-ROCK2 signalling pathway

Exploring patient-specific mechanisms in SYNGAP1-related pathology to develop targeted therapeutic strategies

FENS Forum 2024

CD9 protein in glioblastoma: Implications for progression and therapeutic strategies

FENS Forum 2024