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Targeting disulfidptosis in cancer: mechanisms and preclinical translation

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

Optimization of a novel and effective antiviral agent targeting Zika NS4B

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

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

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

Baby Toolbox Training and Certification Program

PROJECT SUMMARY Our objective is to improve early childhood outcomes and support the expansion of the NIH Infant and Toddler Toolbox (Baby Toolbox) by providing comprehensive training support to those interested in using it. The Baby Toolbox is a brand new, nationally-normed assessment for infants 1-42 months, commissioned by NICHD and released for public use in 2025. The Baby Toolbox is administered entirely on an iPad and includes 35 measures across six domains using novel technology (e.g., gaze tracking, automatic scoring, computerized adaptive testing). It has the potential to bring harmonization to the developmental fields, but in order for it to become a common currency for developmental research as envisioned, researchers need to know how to administer it and how to train others to administer it. We propose an education program that will include a week-long training workshop, certification activities, and post-workshop support to create expert cohorts of Baby Toolbox test administrators. Individuals who attend the workshops can become certified test trainers, capable of training others at their home institutions to administer the assessment thus creating a self-sufficient training model. Through the proposed educational program, we will provide funding to cover lodging, meals, and incidentals during the workshop, in addition to subsidizing transportation to/from the workshop and provide a one-year subscription to the Baby Toolbox. A portion of slots will also be set aside for those without current grant funding. Our team is highly qualified to complete these tasks because we have led the effort to develop the Baby Toolbox assessment and have already completed multiple training workshops for contract deliverables. This grant would continue the efforts started by the NICHD in funding the Baby Toolbox by helping support its rollout, implementation, and growth. To meet these goals, we have the following aims: Aim 1: Create cohorts of trained Baby Toolbox examiners who can catapult the Baby Toolbox into widespread use by hosting a comprehensive week-long education program (training workshop) yearly for individuals to learn how to administer and train others to administer the Baby Toolbox, Aim 2: Expand the use of the Baby Toolbox by recruiting and financially supporting individuals who will bring the Baby Toolbox into a variety of research and clinical settings. Aim 3: Build a virtual training resource of videos and materials to support ongoing fidelity checks with certified trainers, and future training efforts.

BKCa Channel Contributions to Cerebellar Regulated TSC-Associated Neuropsychiatric Disorders

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

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

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

Cardiorespiratory and autonomic impacts of coolants in e-cigarette aerosols

PROJECT SUMMARY / ABSTRACT Coolants such as menthol, WS-3, and WS-23 are widely used in electronic cigarettes (e-cigs) to reduce irritation and enhance appeal—especially among youth. Despite their prevalence, the cardiopulmonary toxicity of these agents remains poorly characterized. Recent work shows that e-cig aerosols can disrupt autonomic nervous system regulation and cardiac electrophysiology, increasing catecholamine release, enhancing sympathetic regulation of cardiac rhythm, and provoking arrhythmias. Proof is also mounting that nicotine’s sympathomimetic traits mediate these pathogenic effects. Preliminary data from our laboratory show that coolants increase systemic nicotine levels, blunt respiratory reflexes, and potentiate arrhythmias upon exposures to e-cigarette aerosols, suggesting a paradoxical role for coolants in suppressing ventilatory responses while intensifying cardiovascular risk. These findings take on added significance in light of recent case reports of sudden cardiac arrest in young e-cigarette users, including some in otherwise healthy individuals. This project will elucidate how e-cigarette coolants alter exposure to harmful and potentially harmful constituents (HPHCs)—particularly nicotine and aldehydes—concurrent with their effects on cardiovascular and respiratory physiology. Using robust murine models with continuous ECG, blood pressure, and pleural pressure telemetry, we will assess how coolants alter the acute and chronic effects of e-cigarette aerosols on cardiac electrophysiology, autonomic tone, ventilatory function, hemodynamics, and toxicant exposure. We will also evaluate how coolant concentration and device power modulate these effects. In parallel, we will determine whether adolescent mice exhibit heightened susceptibility to these effects compared to adults, with attention to sex differences and the persistence of cardiotoxicity after exposure cessation. This comprehensive, multi-modal approach incorporates novel protocols for arrhythmia inducibility, high-resolution physiologic monitoring, and complementary analyses of biomarkers of exposure and effect. By clarifying how coolants interact with HPHCs—especially nicotine and aldehydes—to drive cardiopulmonary injury across age and sex, this work addresses high-priority research areas identified in RFA-OD-25-001, including the toxicological evaluation of e-cigarette constituents and their cardiopulmonary effects. The results will inform regulatory policy and public health strategies aimed at mitigating cardiovascular risk associated with e-cigarette use, particularly among vulnerable youth.

Circadian regulation of reperfusion efficacy in acute ischemic stroke

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

Clinical Trial Readiness of MEG Biomarkers in Children Across the Autism Spectrum

PROJECT SUMMARY Biological and phenotypic heterogeneity of autism spectrum disorder (ASD) poses a major challenge for clinically focused research and interventions. Brain electrophysiological phenotyping holds promise for parsing this heterogeneity. Using magnetoencephalography (MEG), findings of diminished and delayed auditory evoked responses (e.g. the ~50ms component, M50 and, specifically, its latency: M50L) have reproducibly been shown in ASD, with correlation to behavior. Additionally, abnormal resting state activity and network functional connectivity has been identified as an electrophysiological hallmark. Such passively-acquired signatures may serve as objective biomarkers in subtyping autistic individuals, including stratifying patients for inclusion in clinical trials according to biology, rather than behavior alone. However, despite their abundant promise, these measures are not yet permeating clinical trial design, nor being utilized in clinical practice, in part because of their lack of standardized implementation and analysis. This proposal seeks to remedy this by using rigorous and standardized, scalable and sharable methods with two leading MEG measures to determine their measurement- reliability as well as their sensitivity to inter-individual differences in clinically-relevant aspects of autism features, general cognitive ability and language and communication. Specifically adopting a 12-week repeated scanning design, mimicking the duration of a typical pharmaceutical trial or behavioral intervention, we will acquire each of these two MEG metrics at baseline and 12-week follow-up to assess interval change. Additionally, we will evaluate test-retest variability with an intermediate measurement point 4-weeks after baseline. As such we will characterize both intra-subject variability (measurement precision) and inter-subject variability which will be correlated with dimension axes of autism features, general cognitive ability and language skills, as well as major co-occurring condition confounds. These studies will recruit a broad range of 240 autistic children, paralleling the CDC’s prevalence data on intellectual ability and encompassing the group considered as having “profound autism”. This is enabled by our adoption of MEG-PLAN, a strategy developed over the last decade in our group and demonstrated to enhance inclusive participation in MEG scanning studies, even in non-verbal participants. Data will be compared to a control group of age-matched typically-developing peers. The two MEG measures will also be assessed for their ability to identify clusters of less heterogeneous neurophysiological phenotype as a novel basis for stratification or subtyping of the heterogeneous autism population. In culmination, this study addresses key “clinical readiness” aspects of utilization of MEG biomarkers for ASD including profound autism, for both stratification (inclusion/trial selection) and monitoring of response to intervention, and will, ultimately, pave the way for the adoption of such biomarkers as adjunctive tests in increasingly-routine clinical practice.

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

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

Improving Disease-Modifying Therapy Uptake among Patients with Multiple Sclerosis

Project Summary/Abstract Recent advances in the epidemiology of multiple sclerosis (MS) indicate that its prevalence is similar among White (238 per 100,000) and Black (226 per 100,000) populations. These data challenge historic assumptions about individuals with northern European heritage having higher risk and prevalence of MS. Evidence also suggests that MS incidence may be higher than previously recognized in the United States and increasing over time with more individuals identified and diagnosed year over year. MS continues to impose significant and growing burden on patients, healthcare systems and society. These health differences in the diagnosis, treatment and symptom management of MS in light of the increasing prevalence of MS in the US are an important public health issue that requires broader urgent research and policy attention to reduce the overall disease burden. In this study, we will use real-world data derived from the electronic health records (EHR) from four large academic medical centers (University of Kentucky, University of Virginia, Virginia Commonwealth University, and University of Southern California). Extracted EHR data from these four medical centers will be deidentified, combined, and harmonized. We will use this combined data set to examine (1) whether there are any differences in the timely treatment of disease modifying therapy (DMT) among different MS populations, (2) any disparities in the management of symptoms and comorbidities, (3) how non-medical factors of health such as income, education, and health insurance status (patientlevel), linguistically appropriate care provision (provider-level), and neighborhood factors (system-level) affect these outcomes and influence disparities across populations, and (4) assess whether disparities exist in the risks of cardiovascular disease CVD and mortality in MS subgroups and examine if these disparities can be reduced with improved treatment of MS and vascular comorbidities. In pursuing these objectives, we will identify clinical solutions (e.g., optimal DMT sequences) and non-medical factors such as neighborhood factors such as poverty, educational achievement, crime rates, civic participation, and housing quality, access to care factors, and cultural and linguistic match between providers and patients that substantially contribute to health disparities. For actionable solutions, we will rank-order these factors by their relative importance in addressing disparities, which will guide decision-making at the policy, system, and provider level. Our long-term objective is to develop public health strategies and scalable solutions to reduce overall burden in the management of MS. This project is expected to help policy makers and health system administrators in prioritizing interventions and to have implications for clinical practice in improving care of all patients with MS in neurology clinics, at the healthcare system level, and for national health policy.

Tbx4-Driven Pulmonary Hypertension: Mechanisms and Therapeutic Targets

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

The Pyruvate-Lactate Metabolic Axis in Heart Failure and Recovery

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

Structure-Based Development of Nucleotide-Competing Inhibitors Against HIV-1 and LINE-1 Reverse Transcriptases

PROJECT SUMMARY Reverse transcriptases (RTs) from retroviruses and endogenous retroelements are essential polymerases that catalyze RNA- and DNA-dependent DNA synthesis. Nucleoside inhibitors (NIs) remain central to HIV-1 therapy and are also used against other viral infections and in cancer, but toxicity, limited selectivity, pharmacokinetic (PK) liabilities, and the emergence of drug resistance highlight the need for alternative RT inhibitor mechanisms. In contrast to NIs, nucleotide-competing inhibitors (NCIs) block the polymerase active site without requiring incorporation into nucleic acids. Structural studies by PI Ruiz have defined the NCI mechanism of action for HIV- 1 RT and revealed conserved binding modules shared across multiple polymerase families. These advances now enable rational discovery of improved NCIs. LINE-1 (L1) ORF2 RT is an emerging therapeutic target in cancer, autoimmunity, and aging, yet NIs are the only inhibitors known to act against L1 RT. Notably, the NCI-binding region is structurally similar between HIV-1 RT and L1 RT, suggesting that NCI recognition principles may extend across these two biologically distinct polymerases. This R21 seeks to establish proof-of-concept for NCI development against both enzymes. Aim 1 will discover and structurally optimize NCIs targeting HIV-1 RT by combining binding modules from known NCI chemotypes and determining their biochemical activity and co-crystal structures. Aim 2 will determine whether HIV-1 RT NCI principles translate to L1 RT by solving L1 RT/nucleic acid/NCI structures, evaluating enzymatic inhibition, and applying AI-based structure prediction and generative design to propose L1-specific NCI candidates. Cellular retrotransposition assays will test mechanism of action. Aim 3 will develop a fragment library tailored to protein–nucleic acid interfaces and perform fragment screening of HIV-1 and L1 RT/nucleic acid complexes to identify additional chemotypes that engage the NCI binding region. Successful completion will yield NCI scaffolds and mechanistic insights applicable to HIV-1 RT and L1 RT, define structural principles governing NCI recognition across two evolutionarily related polymerases, and establish new avenues for RT inhibitor development. The PI is highly qualified to lead this work, with extensive expertise in RT structural biology, drug design, and fragment-based discovery.

Enteric virus-induced innate immune responses in oral tolerance

Project Summary The human gut must constantly balance between defending against harmful microbe, including virus infections, and tolerating harmless substances, like food. One important immune process called oral tolerance helps prevent the immune system from overreacting to dietary proteins such as gluten. When this tolerance breaks down, known as loss of oral tolerance (LOT), it can lead to celiac disease, where the body mounts an immune attack against gluten. Viruses that infect the gut, known as enteric viruses, can disturb the intestinal immune homeostasis and contribute to gastrointestinal diseases. Our research has found that one such virus, the Type 1 Lang (T1L) strain of reovirus, capable of infecting human and mice, can induce LOT to gluten. We discovered that T1L triggers a type of inflammatory cell death called necroptosis in intestinal epithelial cells. This cell death sends danger signals to dendritic cells (DCs) presenting dietary antigens, including gluten to T cells. These signals appear to shift DCs from a tolerance-promoting mode to one that drives inflammation and gluten-specific TH1 responses, a hallmark of celiac disease. We believe this process begins when the virus produces a specific form of RNA called Z-RNA, which is sensed by a host protein called ZBP1, triggering necroptosis and inflammation. Our research aims to understand this pathway in detail. Aim 1 will investigate how ZBP1 detects viral Z-RNA and induces necroptosis in intestinal epithelial cells. Aim 2 will examine how this necroptosis leads to LOT and will test whether blocking or engaging the pathway can prevent or induce inflammatory dietary antigen-specific TH1 immune responses. By revealing how a common virus can break oral tolerance and trigger inflammation, this study could lead to new ways to prevent or treat autoimmune and food-related disease such as celiac disease.

Implementing a New Paradigm for Antifungal Drug Development

About 30% of the drugs currently in clinical use function through covalent modification of their target. Yet, until recently, none of these covalent drugs were specifically designed to utilize this irreversible mode of action. It is our hypothesis that the production of a new class of covalent inactivators, designed to selectively modify new drug targets, will lead to novel agents with efficacy against both native and drug-resistant pathogenic fungal species. Because of their novelty these agents will also offer a greater opportunity to bypass the existing mechanisms of drug resistance. Pathogenic fungal infections remain among the leading causes of human mortality, and this threat is rising due to the increasing prevalence of drug- resistance strains and the paucity of effective antifungal drugs against the more virulent fungal species. Our proposed new drug target is an enzyme that plays a critical role in a uniquely microbial pathway that is essential for the survival of fungal organisms. To test our hypothesis and achieve the goals of this project we plan to complete the following specific aims during the initial R21 phase of this project: (1) Optimization of the potency of novel enzyme inactivators. Our goals here are to use our strong preliminary results to address critical barriers that must be overcome to convert potent enzyme inactivators into advanced drug candidates, thereby achieving higher target selectivity and increasing compound reactivity once bound to the target; (2) Enhance the antifungal capability of these enzyme inactivators. Our strategy for this aim is focused on the incorporation of conjugate partners into this new class of covalent inactivators, enabling them to potentially utilize the existing nutrient uptake systems to achieve toxic levels in Candida species; (3) Examine the target selectivity of our new antifungal agents. Results from fungal growth inhibition and fungal killing assays will be used to evaluate and rank the efficacy of our compounds against both wild-type and drug-resistant Candida strains. Specific milestones are presented to evaluate our achievement of these initial aims. Once accomplished we will immediately proceed to the R33 phase of this project, with the aims of: (4) Pharmacological evaluation of lead candidates, though ranking the drug candidates based on their ADME, pharmacokinetic and toxicity properties; and then (5) Evaluate the efficacy of our candidates against pathogenic fungal infections. A systematic infection animal model will be utilized for candidate screening to identify the best agents against disseminated fungal infections, followed by further efficacy screening in an oral infection model. Completion of these aims will produce, refine and evaluate a new class of antifungal agents with a novel mode of action against an unexplored but essential fungal target. The agents with the most promising drug profiles will then be moved into advanced preclinical trials used to select the most effective new antifungal agents.

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.

2-Deoxyglucose Therapy for Organophosphate Intoxication

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

Noninvasive Neuromodulation to Improve Hand Motor Function in Multiple Sclerosis

Project Summary/Abstract Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating, and degenerative disease that affects nearly one million Americans. Although more than 75% of persons with MS (PwMS) experience hand motor impairments that reduce independence and quality of life, current treatments primarily aim to slow disease progression through pharmacological approaches and rehabilitation and often do not improve motor function. Recent evidence shows that reduced corticospinal transmission is strongly associated with motor impairment severity in PwMS, highlighting the need for targeted strategies to strengthen residual corticospinal pathways. Therefore, this project aims to evaluate the therapeutic potential of paired corticospinal-motoneuronal stimulation (PCMS) in improving hand dexterity in PwMS. PCMS, a noninvasive mechanism-driven neuromodulation approach, enhances corticospinal transmission by producing long-term potentiation-like effects at the corticospinal-motoneuronal synapse by precisely pairing transcranial magnetic stimulation (TMS) with peripheral nerve stimulation (PNS). This project first aims to examine the effects of a single PCMS session on corticospinal transmission and hand motor function in PwMS. Using a randomized, crossover design, 25 PwMS will complete two sessions: (1) PCMS and (2) sham-PCMS. Each session will deliver 180 paired TMS-PNS stimuli over 30 minutes. The primary outcome is performance on the 9-Hole Peg Test (9HPT). Secondary outcomes include pinch grip force, maximal voluntary contraction (MVC), MEP amplitude and latency, F-wave parameters, and M- max amplitude. It is hypothesized that PCMS will enhance corticospinal transmission and improve hand motor performance compared to sham stimulation. Second, this project will examine the effects of PCMS combined with hand motor training in PwMS. Forty-eight PwMS will be randomized to receive either PCMS or sham-PCMS combined with motor training over 10 sessions in 3–4 weeks. Outcomes will be assessed at baseline, post- intervention, and one-month follow-up. It is hypothesized that PCMS participants receiving PCMS with motor training to show greater functional gains than those receiving sham-PCMS with motor training and the functional gains will be better maintained in the PCMS with motor training group at follow-up. This project is the first to apply PCMS in PwMS, leveraging a noninvasive neuromodulation strategy to specifically enhance corticospinal output for improving manual dexterity. Findings will establish proof-of-concept for this intervention in PwMS and guide future studies optimizing stimulation protocols and evaluating clinical efficacy on a larger scale. Ultimately, this work may lead to a new therapeutic approach to improve dexterity, independence, and quality of life for people living with MS.

Development of a multi-modal mouse model of cluster headache

PROJECT SUMMARY / ABSTRACT Cluster headache (CH), which affects about 1 in 1,000 people, is a severe and debilitating primary headache disorder characterized by repeated attacks occurring in clusters over weeks or months. CH has clearly defined features: severe pain (worse than childbirth), facial autonomic changes (such as a watery eye), restlessness, and a striking circadian pattern of attacks (at the same time each day like clockwork in approximately 70.5% of patients). CH also has a well-defined pathophysiology of 3 systems: the trigeminovascular pain system, the autonomic nervous system, and the hypothalamic system (in particular the posterior hypothalamus, the first brain area activated during an attack). Despite the well-known features and systems involved in CH, no disease- specific treatments are available: all CH treatments are repurposed medications from other diseases. This lack of CH-specific treatments is due in large part to the lack of a viable animal model that faithfully recapitulates the aforementioned CH features. To develop a specific animal model for CH, we previously studied a trigeminovascular headache model (repeated nitroglycerin injections), and discovered a circadian pattern of pain responses that reflects the clockwork-like pattern of attacks in CH patients. Furthermore, our analysis also identified a recently discovered CH modifier gene Mertk (MER proto-oncogene, tyrosine receptor kinase) to be highly rhythmically expressed in the trigeminal ganglion. Deletion of Mertk (Mertk-KO) altered the normal circadian rhythm of pain sensitivity by increasing pain sensitivity over 24 hours. Finally, activation of the posterior hypothalamus (via c-Fos staining) was observed after NTG administration in wild-type mice. Based on these exciting preliminary findings, we hypothesize that a combination of trigeminovascular (nitroglycerin), genetic (Mertk-KO), and hypothalamic (direct optogenetic activation of the posterior hypothalamus) manipulations will generate the first multi-modal animal model of CH. In Aim 1 (the R61 phase), we will determine the contributions of each aspect of our combined model, alone or in combination (a 4x2 grid of NTG or control, Mertk KO mouse or wild-type control, and optogenetic injection or control). Our milestone for progression to the R33 phase will be significant differences in at least two pain behaviors in our model compared to controls. In Aims 2 and 3 (the R33 phase), we will validate our model through face validity (lacrimation and restlessness), construct validity (CGRP, PACAP, and VIP in the trigeminal ganglion and hypothalamus), and predictive validity (ability of first-line and new treatments to ameliorate the pain behaviors of our model). This project is highly significant and innovative, addressing a profound need for a specific and comprehensive animal model for this devastating yet understudied disease. With the unique combination of complementary expertise in CH (laboratory and clinical), circadian biology, pharmacology, optogenetics and pain, we are ideally suited to generate this combined CH model with the goal of providing insights into CH pathophysiology and developing novel therapeutics.

How the presynapse forms and functions”

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

Decoding ketamine: Neurobiological mechanisms underlying its rapid antidepressant efficacy

Unlike traditional monoamine-based antidepressants that require weeks to exert effects, ketamine alleviates depression within hours, though its clinical use is limited by side effects. While ketamine was initially thought to work primarily through NMDA receptor (NMDAR) inhibition, our research reveals a more complex mechanism. We demonstrate that NMDAR inhibition alone cannot explain ketamine's sustained antidepressant effects, as other NMDAR antagonists like MK-801 lack similar efficacy. Instead, the (2R,6R)-hydroxynorketamine (HNK) metabolite appears critical, exhibiting antidepressant effects without ketamine's side effects. Paradoxically, our findings suggest an inverted U-shaped dose-response relationship where excessive NMDAR inhibition may actually impede antidepressant efficacy, while some level of NMDAR activation is necessary. The antidepressant actions of ketamine and (2R,6R)-HNK require AMPA receptor activation, leading to synaptic potentiation and upregulation of AMPA receptor subunits GluA1 and GluA2. Furthermore, NMDAR subunit GluN2A appears necessary and possibly sufficient for these effects. This research establishes NMDAR-GluN2A activation as a common downstream effector for rapid-acting antidepressants, regardless of their initial targets, offering promising directions for developing next-generation antidepressants with improved efficacy and reduced side effects.

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

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

The Neurobiology of the Addicted Brain

Brain-Wide Compositionality and Learning Dynamics in Biological Agents

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

Prosocial Learning and Motivation across the Lifespan

2024 BACN Early-Career Prize Lecture Many of our decisions affect other people. Our choices can decelerate climate change, stop the spread of infectious diseases, and directly help or harm others. Prosocial behaviours – decisions that help others – could contribute to reducing the impact of these challenges, yet their computational and neural mechanisms remain poorly understood. I will present recent work that examines prosocial motivation, how willing we are to incur costs to help others, prosocial learning, how we learn from the outcomes of our choices when they affect other people, and prosocial preferences, our self-reports of helping others. Throughout the talk, I will outline the possible computational and neural bases of these behaviours, and how they may differ from young adulthood to old age.

Influence of the context of administration in the antidepressant-like effects of the psychedelic 5-MeO-DMT

Psychedelics like psilocybin have shown rapid and long-lasting efficacy on depressive and anxiety symptoms. Other psychedelics with shorter half-lives, such as DMT and 5-MeO-DMT, have also shown promising preliminary outcomes in major depression, making them interesting candidates for clinical practice. Despite several promising clinical studies, the influence of the context on therapeutic responses or adverse effects remains poorly documented. To address this, we conducted preclinical studies evaluating the psychopharmacological profile of 5-MeO-DMT in contexts previously validated in mice as either pleasant (positive setting) or aversive (negative setting). Healthy C57BL/6J male mice received a single intraperitoneal (i.p.) injection of 5-MeO-DMT at doses of 0.5, 5, and 10 mg/kg, with assessments at 2 hours, 24 hours, and one week post-administration. In a corticosterone (CORT) mouse model of depression, 5-MeO-DMT was administered in different settings, and behavioral tests mimicking core symptoms of depression and anxiety were conducted. In CORT-exposed mice, an acute dose of 0.5 mg/kg administered in a neutral setting produced antidepressant-like effects at 24 hours, as observed by reduced immobility time in the Tail Suspension Test (TST). In a positive setting, the drug also reduced latency to first immobility and total immobility time in the TST. However, these beneficial effects were negated in a negative setting, where 5-MeO-DMT failed to produce antidepressant-like effects and instead elicited an anxiogenic response in the Elevated Plus Maze (EPM).Our results indicate a strong influence of setting on the psychopharmacological profile of 5-MeO-DMT. Future experiments will examine cortical markers of pre- and post-synaptic density to correlate neuroplasticity changes with the behavioral effects of 5-MeO-DMT in different settings.

Use of human systems for neuroinflammatory/neurodegenerative diseases

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

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

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

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

Use of brain imaging data to improve prescriptions of psychotropic drugs - Examples of ketamine in depression and antipsychotics in schizophrenia

The use of molecular imaging, particularly PET and SPECT, has significantly transformed the treatment of schizophrenia with antipsychotic drugs since the late 1980s. It has offered insights into the links between drug target engagement, clinical effects, and side effects. A therapeutic window for receptor occupancy is established for antipsychotics, yet there is a divergence of opinions regarding the importance of blood levels, with many downplaying their significance. As a result, the role of therapeutic drug monitoring (TDM) as a personalized therapy tool is often underrated. Since molecular imaging of antipsychotics has focused almost entirely on D2-like dopamine receptors and their potential to control positive symptoms, negative symptoms and cognitive deficits are hardly or not at all investigated. Alternative methods have been introduced, i.e. to investigate the correlation between approximated receptor occupancies from blood levels and cognitive measures. Within the domain of antidepressants, and specifically regarding ketamine's efficacy in depression treatment, there is limited comprehension of the association between plasma concentrations and target engagement. The measurement of AMPA receptors in the human brain has added a new level of comprehension regarding ketamine's antidepressant effects. To ensure precise prescription of psychotropic drugs, it is vital to have a nuanced understanding of how molecular and clinical effects interact. Clinician scientists are assigned with the task of integrating these indispensable pharmacological insights into practice, thereby ensuring a rational and effective approach to the treatment of mental health disorders, signaling a new era of personalized drug therapy mechanisms that promote neuronal plasticity not only under pathological conditions, but also in the healthy aging brain.

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

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

How Intermittent Bioenergetic Challenges Enhance Brain and Body Health

Humans and other animals evolved in habitats fraught with a range of environmental challenges to their bodies and brains. Accordingly, cells and organ systems possess adaptive stress-responsive signaling pathways that enable them to not only withstand environmental challenges, but also to prepare for future challenges and function more efficiently. These phylogenetically conserved processes are the foundation of the hormesis principle in which repeated exposures to low to moderate amounts of an environmental challenge improve cellular and organismal fitness. Here I describe cellular and molecular mechanisms by which cells in the brain and body respond to intermittent fasting and exercise in ways that enhance performance and counteract aging and disease processes. Switching back and forth between adaptive stress response (during fasting and exercise) and growth and plasticity (eating, resting, sleeping) modes enhances the performance and resilience of various organ systems. While pharmacological interventions that engage a particular hormetic mechanism are being developed, it seems unlikely that any will prove superior to fasting and exercise.

Dynamic endocrine modulation of the nervous system

Sex hormones are powerful neuromodulators of learning and memory. In rodents and nonhuman primates estrogen and progesterone influence the central nervous system across a range of spatiotemporal scales. Yet, their influence on the structural and functional architecture of the human brain is largely unknown. Here, I highlight findings from a series of dense-sampling neuroimaging studies from my laboratory designed to probe the dynamic interplay between the nervous and endocrine systems. Individuals underwent brain imaging and venipuncture every 12-24 hours for 30 consecutive days. These procedures were carried out under freely cycling conditions and again under a pharmacological regimen that chronically suppresses sex hormone production. First, resting state fMRI evidence suggests that transient increases in estrogen drive robust increases in functional connectivity across the brain. Time-lagged methods from dynamical systems analysis further reveals that these transient changes in estrogen enhance within-network integration (i.e. global efficiency) in several large-scale brain networks, particularly Default Mode and Dorsal Attention Networks. Next, using high-resolution hippocampal subfield imaging, we found that intrinsic hormone fluctuations and exogenous hormone manipulations can rapidly and dynamically shape medial temporal lobe morphology. Together, these findings suggest that neuroendocrine factors influence the brain over short and protracted timescales.

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

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

How can we treat visceral pain?

Chronic pain is a leading cause of morbidity, common to patients with gastrointestinal diseases such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). Most pain killers are largely ineffective against this type of pain or restricted for use in these patients due to gut related complications and risk of addition. A significant unmet clinical need therefore exists to develop novel non-opioid based visceral analgesics.

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

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

Linking GWAS to pharmacological treatments for psychiatric disorders

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

Investigating activity-dependent processes in cerebral cortex development and disease

The cerebral cortex contains an extraordinary diversity of excitatory projection neuron (PN) and inhibitory interneurons (IN), wired together to form complex circuits. Spatiotemporally coordinated execution of intrinsic molecular programs by PNs and INs and activity-dependent processes, contribute to cortical development and cortical microcircuits formation. Alterations of these delicate processes have often been associated to neurological/neurodevelopmental disorders. However, despite the groundbreaking discovery that spontaneous activity in the embryonic brain can shape regional identities of distinct cortical territories, it is still unclear whether this early activity contributes to define subtype-specific neuronal fate as well as circuit assembly. In this study, we combined in utero genetic perturbations via CRISPR/Cas9 system and pharmacological inhibition of selected ion channels with RNA-sequencing and live imaging technologies to identify the activity-regulated processes controlling the development of different cortical PN classes, their wiring and the acquisition of subtype specific features. Moreover, we generated human induced pluripotent stem cells (iPSCs) form patients affected by a severe, rare and untreatable form of developmental epileptic encephalopathy. By differentiating cortical organoids form patient-derived iPSCs we create human models of early electrical alterations for studying molecular, structural and functional consequences of the genetic mutations during cortical development. Our ultimate goal is to define the activity-conditioned processes that physiologically occur during the development of cortical circuits, to identify novel therapeutical paths to address the pathological consequences of neonatal epilepsies.

Neuroscience of socioeconomic status and poverty: Is it actionable?

SES neuroscience, using imaging and other methods, has revealed generalizations of interest for population neuroscience and the study of individual differences. But beyond its scientific interest, SES is a topic of societal importance. Does neuroscience offer any useful insights for promoting socioeconomic justice and reducing the harms of poverty? In this talk I will use research from my own lab and others’ to argue that SES neuroscience has the potential to contribute to policy in this area, although its application is premature at present. I will also attempt to forecast the ways in which practical solutions to the problems of poverty may emerge from SES neuroscience. Bio: Martha Farah has conducted groundbreaking research on face and object recognition, visual attention, mental imagery, and semantic memory and - in more recent times - has been at the forefront of interdisciplinary research into neuroscience and society. This deals with topics such as using fMRI for lie detection, ethics of cognitive enhancement, and effects of social deprivation on brain development.

Extrinsic control and intrinsic computation in the hippocampal CA1 network

A key issue in understanding circuit operations is the extent to which neuronal spiking reflects local computation or responses to upstream inputs. Several studies have lesioned or silenced inputs to area CA1 of the hippocampus - either area CA3 or the entorhinal cortex and examined the effect on CA1 pyramidal cells. However, the types of the reported physiological impairments vary widely, primarily because simultaneous manipulations of these redundant inputs have never been performed. In this study, I combined optogenetic silencing of unilateral and bilateral mEC, of the local CA1 region, and performed bilateral pharmacogenetic silencing of CA3. I combined this with high spatial resolution extracellular recordings along the CA1-dentate axis. Silencing the medial entorhinal largely abolished extracellular theta and gamma currents in CA1, without affecting firing rates. In contrast, CA3 and local CA1 silencing strongly decreased firing of CA1 neurons without affecting theta currents. Each perturbation reconfigured the CA1 spatial map. Yet, the ability of the CA1 circuit to support place field activity persisted, maintaining the same fraction of spatially tuned place fields. In contrast to these results, unilateral mEC manipulations that were ineffective in impacting place cells during awake behavior were found to alter sharp-wave ripple sequences activated during sleep. Thus, intrinsic excitatory-inhibitory circuits within CA1 can generate neuronal assemblies in the absence of external inputs, although external synaptic inputs are critical to reconfigure (remap) neuronal assemblies in a brain-state dependent manner.

The role of astroglia-neuron interactions in generation and spread of seizures

Astroglia-neuron interactions are involved in multiple processes, regulating development, excitability and connectivity of neural circuits. Accumulating number of evidences highlight a direct connection between aberrant astroglial genetics and physiology in various forms of epilepsies. Using zebrafish seizure models, we showed that neurons and astroglia follow different spatiotemporal dynamics during transitions from pre-ictal to ictal activity. We observed that during pre-ictal period neurons exhibit local synchrony and low level of activity, whereas astroglia exhibit global synchrony and high-level of calcium signals that are anti correlated with neural activity. Instead, generalized seizures are marked by a massive release of astroglial glutamate release as well as a drastic increase of astroglia and neuronal activity and synchrony across the entire brain. Knocking out astroglial glutamate transporters leads to recurrent spontaneous generalized seizures accompanied with massive astroglial glutamate release. We are currently using a combination of genetic and pharmacological approaches to perturb astroglial glutamate signalling and astroglial gap junctions to further investigate their role in generation and spreading of epileptic seizures across the brain.

Ebselen: a lithium-mimetic without lithium side-effects?

Development of new medications for mental health conditions is a pressing need given the high proportion of people not responding to available treatments. We hope that presenting ebselen to a wider audience will inspire further studies on this promising agent with a benign side-effects profile. Laboratory research, animal research and human studies suggest that ebselen shares many features with the mood stabilising drug lithium, creating a promise of a drug that would have a similar clinical effect but without lithium’s troublesome side-effect profile and toxicity. Both drugs have a common biological target, inositol monophosphatase, whose inhibition is thought key to lithium’s therapeutic effect. Both drugs have neuroprotective action and reduce oxidative stress. In animal studies, ebselen affected neurotransmitters involved in the development of mental health symptoms, and in particular, produced effects of serotonin function very similar to lithium. Both ebselen and lithium share behavioural effects: antidepressant-like effects in rodent models of depression and decrease in behavioural impulsivity, a property associated with lithium's anti-suicidal action. Human neuropsychological studies support an antidepressant profile for ebselen based on its positive impact on emotional processing and reward seeking. Our group currently is exploring ebselen’s effects in patients with mood disorders. A completed ‘add-on’ clinical trial in mania showed ebselen’s superiority over placebo after three weeks of treatment. Our ongoing experimental research explores ebselen’s antidepressant profile in patients with treatment resistant depression. If successful, this will lead to a clinical trial of ebselen as an antidepressant augmentation agent, similar to lithium.

The 15th David Smith Lecture in Anatomical Neuropharmacology: Professor Tim Bliss, "Memories of long term potentiation

The David Smith Lectures in Anatomical Neuropharmacology, Part of the 'Pharmacology, Anatomical Neuropharmacology and Drug Discovery Seminars Series', Department of Pharmacology, University of Oxford. The 15th David Smith Award Lecture in Anatomical Neuropharmacology will be delivered by Professor Tim Bliss, Visiting Professor at UCL and the Frontier Institutes of Science and Technology, Xi’an Jiaotong University, China, and is hosted by Professor Nigel Emptage. This award lecture was set up to celebrate the vision of Professor A David Smith, namely, that explanations of the action of drugs on the brain requires the definition of neuronal circuits, the location and interactions of molecules. Tim Bliss gained his PhD at McGill University in Canada. He joined the MRC National Institute for Medical Research in Mill Hill, London in 1967, where he remained throughout his career. His work with Terje Lømo in the late 1960’s established the phenomenon of long-term potentiation (LTP) as the dominant synaptic model of how the mammalian brain stores memories. He was elected as a Fellow of the Royal Society in 1994 and is a founding fellow of the Academy of Medical Sciences. He shared the Bristol Myers Squibb award for Neuroscience with Eric Kandel in 1991, the Ipsen Prize for Neural Plasticity with Richard Morris and Yadin Dudai in 2013. In May 2012 he gave the annual Croonian Lecture at the Royal Society on ‘The Mechanics of Memory’. In 2016 Tim, with Graham Collingridge and Richard Morris shared the Brain Prize, one of the world's most coveted science prizes. Abstract: In 1966 there appeared in Acta Physiologica Scandinavica an abstract of a talk given by Terje Lømo, a PhD student in Per Andersen’s laboratory at the University of Oslo. In it Lømo described the long-lasting potentiation of synaptic responses in the dentate gyrus of the anaesthetised rabbit that followed repeated episodes of 10-20Hz stimulation of the perforant path. Thus, heralded and almost entirely unnoticed, one of the most consequential discoveries of 20th century neuroscience was ushered into the world. Two years later I arrived in Oslo as a visiting post-doc from the National Institute for Medical Research in Mill Hill, London. In this talk I recall the events that led us to embark on a systematic reinvestigation of the phenomenon now known as long-term potentiation (LTP) and will then go on to describe the discoveries and controversies that enlivened the early decades of research into synaptic plasticity in the mammalian brain. I will end with an observer’s view of the current state of research in the field, and what we might expect from it in the future.

Chemistry of the adaptive mind: lessons from dopamine

The human brain faces a variety of computational dilemmas, including the flexibility/stability, the speed/accuracy and the labor/leisure tradeoff. I will argue that striatal dopamine is particularly well suited to dynamically regulate these computational tradeoffs depending on constantly changing task demands. This working hypothesis is grounded in evidence from recent studies on learning, motivation and cognitive control in human volunteers, using chemical PET, psychopharmacology, and/or fMRI. These studies also begin to elucidate the mechanisms underlying the huge variability in catecholaminergic drug effects across different individuals and across different task contexts. For example, I will demonstrate how effects of the most commonly used psychostimulant methylphenidate on learning, Pavlovian and effortful instrumental control depend on fluctuations in current environmental volatility, on individual differences in working memory capacity and on opportunity cost respectively.

Growing a world-class precision medicine industry

Monash Biomedical Imaging is part of the new $71.2 million Australian Precision Medicine Enterprise (APME) facility, which will deliver large-scale development and manufacturing of precision medicines and theranostic radiopharmaceuticals for industry and research. A key feature of the APME project is a high-energy cyclotron with multiple production clean rooms, which will be located on the Monash Biomedical Imaging (MBI) site in Clayton. This strategic co-location will facilitate radiochemistry, PET and SPECT research and clinical use of theranostic (therapeutic and diagnostic) radioisotopes produced on-site. In this webinar, MBI’s Professor Gary Egan and Dr Maggie Aulsebrook will explain how the APME will secure Australia’s supply of critical radiopharmaceuticals, build a globally competitive Australian manufacturing hub, and train scientists and engineers for the Australian workforce. They will cover the APME’s state-of-the-art 30 MeV and 18-24 MeV cyclotrons and radiochemistry facilities, as well as the services that will be accessible to students, scientists, clinical researchers, and pharmaceutical companies in Australia and around the world. The APME is a collaboration between Monash University, Global Medical Solutions Australia, and Telix Pharmaceuticals. Professor Gary Egan is Director of Monash Biomedical Imaging, Director of the ARC Centre of Excellence for Integrative Brain Function and a Distinguished Professor at the Turner Institute for Brain and Mental Health, Monash University. He is also lead investigator of the Victorian Biomedical Imaging Capability, and Deputy Director of the Australian National Imaging Facility. Dr Maggie Aulsebrook obtained her PhD in Chemistry at Monash University and specialises in the development and clinical translation of radiopharmaceuticals. She has led the development of several investigational radiopharmaceuticals for first-in-human application. Maggie leads the Radiochemistry Platform at Monash Biomedical Imaging.

Malignant synaptic plasticity in pediatric high-grade gliomas

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

Can I be bothered? Neural and computational mechanisms underlying the dynamics of effort processing (BACN Early-career Prize Lecture 2021)

From a workout at the gym to helping a colleague with their work, everyday we make decisions about whether we are willing to exert effort to obtain some sort of benefit. Increases in how effortful actions and cognitive processes are perceived to be has been linked to clinically severe impairments to motivation, such as apathy and fatigue, across many neurological and psychiatric conditions. However, the vast majority of neuroscience research has focused on understanding the benefits for acting, the rewards, and not on the effort required. As a result, the computational and neural mechanisms underlying how effort is processed are poorly understood. How do we compute how effortful we perceive a task to be? How does this feed into our motivation and decisions of whether to act? How are such computations implemented in the brain? and how do they change in different environments? I will present a series of studies examining these questions using novel behavioural tasks, computational modelling, fMRI, pharmacological manipulations, and testing in a range of different populations. These studies highlight how the brain represents the costs of exerting effort, and the dynamic processes underlying how our sensitivity to effort changes as a function of our goals, traits, and socio-cognitive processes. This work provides new computational frameworks for understanding and examining impaired motivation across psychiatric and neurological conditions, as well as why all of us, sometimes, can’t be bothered.

Neural Representations of Social Homeostasis

How does our brain rapidly determine if something is good or bad? How do we know our place within a social group? How do we know how to behave appropriately in dynamic environments with ever-changing conditions? The Tye Lab is interested in understanding how neural circuits important for driving positive and negative motivational valence (seeking pleasure or avoiding punishment) are anatomically, genetically and functionally arranged. We study the neural mechanisms that underlie a wide range of behaviors ranging from learned to innate, including social, feeding, reward-seeking and anxiety-related behaviors. We have also become interested in “social homeostasis” -- how our brains establish a preferred set-point for social contact, and how this maintains stability within a social group. How are these circuits interconnected with one another, and how are competing mechanisms orchestrated on a neural population level? We employ optogenetic, electrophysiological, electrochemical, pharmacological and imaging approaches to probe these circuits during behavior.

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.

Extrinsic control and autonomous computation in the hippocampal CA1 circuit

In understanding circuit operations, a key issue is the extent to which neuronal spiking reflects local computation or responses to upstream inputs. Because pyramidal cells in CA1 do not have local recurrent projections, it is currently assumed that firing in CA1 is inherited from its inputs – thus, entorhinal inputs provide communication with the rest of the neocortex and the outside world, whereas CA3 inputs provide internal and past memory representations. Several studies have attempted to prove this hypothesis, by lesioning or silencing either area CA3 or the entorhinal cortex and examining the effect of firing on CA1 pyramidal cells. Despite the intense and careful work in this research area, the magnitudes and types of the reported physiological impairments vary widely across experiments. At least part of the existing variability and conflicts is due to the different behavioral paradigms, designs and evaluation methods used by different investigators. Simultaneous manipulations in the same animal or even separate manipulations of the different inputs to the hippocampal circuits in the same experiment are rare. To address these issues, I used optogenetic silencing of unilateral and bilateral mEC, of the local CA1 region, and performed bilateral pharmacogenetic silencing of the entire CA3 region. I combined this with high spatial resolution recording of local field potentials (LFP) in the CA1-dentate axis and simultaneously collected firing pattern data from thousands of single neurons. Each experimental animal had up to two of these manipulations being performed simultaneously. Silencing the medial entorhinal (mEC) largely abolished extracellular theta and gamma currents in CA1, without affecting firing rates. In contrast, CA3 and local CA1 silencing strongly decreased firing of CA1 neurons without affecting theta currents. Each perturbation reconfigured the CA1 spatial map. Yet, the ability of the CA1 circuit to support place field activity persisted, maintaining the same fraction of spatially tuned place fields, and reliable assembly expression as in the intact mouse. Thus, the CA1 network can maintain autonomous computation to support coordinated place cell assemblies without reliance on its inputs, yet these inputs can effectively reconfigure and assist in maintaining stability of the CA1 map.

MBI Webinar on preclinical research into brain tumours and neurodegenerative disorders

WEBINAR 1 Breaking the barrier: Using focused ultrasound for the development of targeted therapies for brain tumours presented by Dr Ekaterina (Caty) Salimova, Monash Biomedical Imaging Glioblastoma multiforme (GBM) - brain cancer - is aggressive and difficult to treat as systemic therapies are hindered by the blood-brain barrier (BBB). Focused ultrasound (FUS) - a non-invasive technique that can induce targeted temporary disruption of the BBB – is a promising tool to improve GBM treatments. In this webinar, Dr Ekaterina Salimova will discuss the MRI-guided FUS modality at MBI and her research to develop novel targeted therapies for brain tumours. Dr Ekaterina (Caty) Salimova is a Research Fellow in the Preclinical Team at Monash Biomedical Imaging. Her research interests include imaging cardiovascular disease and MRI-guided focused ultrasound for investigating new therapeutic targets in neuro-oncology. - WEBINAR 2 Disposition of the Kv1.3 inhibitory peptide HsTX1[R14A], a novel attenuator of neuroinflammation presented by Sanjeevini Babu Reddiar, Monash Institute of Pharmaceutical Sciences The voltage-gated potassium channel (Kv1.3) in microglia regulates membrane potential and pro-inflammatory functions, and non-selective blockade of Kv1.3 has shown anti-inflammatory and disease improvement in animal models of Alzheimer’s and Parkinson’s diseases. Therefore, specific inhibitors of pro-inflammatory microglial processes with CNS bioavailability are urgently needed, as disease-modifying treatments for neurodegenerative disorders are lacking. In this webinar, PhD candidate Ms Sanju Reddiar will discuss the synthesis and biodistribution of a Kv1.3-inhibitory peptide using a [64Cu]Cu-DOTA labelled conjugate. Sanjeevini Babu Reddiar is a PhD student at the Monash Institute of Pharmaceutical Sciences. She is working on a project identifying the factors governing the brain disposition and blood-brain barrier permeability of a Kv1.3-blocking peptide.

Mechanisms of visual circuit development: aligning topographic maps of space

The neuroscience of lifestyle interventions for mental health: the BrainPark approach

Our everyday behaviours, such as physical activity, sleep, diet, meditation, and social connections, have a potent impact on our mental health and the health of our brain. BrainPark is working to harness this power by developing lifestyle-based interventions for mental health and investigating how they do and don’t change the brain, and for whom they are most effective. In this webinar, Dr Rebecca Segrave and Dr Chao Suo will discuss BrainPark’s approach to developing lifestyle-based interventions to help people get better control of compulsive behaviours, and the multi-modality neuroimaging approaches they take to investigating outcomes. The webinar will explore two current BrainPark trials: 1. Conquering Compulsions - investigating the capacity of physical exercise and meditation to alter reward processing and help people get better control of a wide range of unhelpful habits, from drinking to eating to cleaning. 2. The Brain Exercise Addiction Trial (BEAT) - an NHMRC funded investigation into the capacity of physical exercise to reverse the brain harms caused by long-term heavy cannabis use. Dr Rebecca Segrave is Deputy Director and Head of Interventions Research at BrainPark, the David Winston Turner Senior Research Fellow within the Turner Institute for Brain and Mental Health, and an AHRPA registered Clinical Neuropsychologist. Dr Chao Suo is Head of Technology and Neuroimaging at BrainPark and a Research Fellow within the Turner Institute for Brain and Mental Health.

Dynamic dopaminergic signaling probabilistically controls the timing of self-timed movements

Human movement disorders and pharmacological studies have long suggested molecular dopamine modulates the pace of the internal clock. But how does the endogenous dopaminergic system influence the timing of our movements? We examined the relationship between dopaminergic signaling and the timing of reward-related, self-timed movements in mice. Animals were trained to initiate licking after a self-timed interval following a start cue; reward was delivered if the animal’s first lick fell within a rewarded window (3.3-7 s). The first-lick timing distributions exhibited the scalar property, and we leveraged the considerable variability in these distributions to determine how the activity of the dopaminergic system related to the animals’ timing. Surprisingly, dopaminergic signals ramped-up over seconds between the start-timing cue and the self-timed movement, with variable dynamics that predicted the movement/reward time, even on single trials. Steeply rising signals preceded early initiation, whereas slowly rising signals preceded later initiation. Higher baseline signals also predicted earlier self-timed movement. Optogenetic activation of dopamine neurons during self-timing did not trigger immediate movements, but rather caused systematic early-shifting of the timing distribution, whereas inhibition caused late-shifting, as if dopaminergic manipulation modulated the moment-to-moment probability of unleashing the planned movement. Consistent with this view, the dynamics of the endogenous dopaminergic signals quantitatively predicted the moment-by-moment probability of movement initiation. We conclude that ramping dopaminergic signals, potentially encoding dynamic reward expectation, probabilistically modulate the moment-by-moment decision of when to move. (Based on work from Hamilos et al., eLife, 2021).

‘Autophagy regulates neurotransmission by controlling the axonal endoplasmic reticulum’

Dissecting the neural circuits underlying prefrontal regulation of reward and threat responsivity in a primate

Gaining insight into the overlapping neural circuits that regulate positive and negative emotion is an important step towards understanding the heterogeneity in the aetiology of anxiety and depression and developing new treatment targets. Determining the core contributions of the functionally heterogenous prefrontal cortex to these circuits is especially illuminating given its marked dysregulation in affective disorders. This presentation will review a series of studies in a new world monkey, the common marmoset, employing pathway-specific chemogenetics, neuroimaging, neuropharmacology and behavioural and cardiovascular analysis to dissect out prefrontal involvement in the regulation of both positive and negative emotion. Highlights will include the profound shift of sensitivity away from reward and towards threat induced by localised activations within distinct regions of vmPFC, namely areas 25 and 14 as well as the opposing contributions of this region, compared to orbitofrontal and dorsolateral prefrontal cortex, in the overall responsivity to threat. Ongoing follow-up studies are identifying the distinct downstream pathways that mediate some of these effects as well as their differential sensitivity to rapidly acting anti-depressants.

Sex, drugs, and bad choices: using rodent models to understand decision making

Nearly every aspect of life involves decisions between options that differ in both their expected rewards and the potential costs (such as delay to reward delivery or risk of harm) that accompany those rewards. The ability to choose adaptively when faced with such decisions is critical for well-being and overall quality of life. In neuropsychiatric conditions such as substance use disorders, however, decision making is often compromised, which can prolong and exacerbate their severity and co-morbidities. In this seminar, Dr. Setlow will discuss research in rodent models investigating behavioral and biological mechanisms of cost-benefit decision making. In particular, he will focus on factors (including sex) that contribute to differences in cost-benefit decision making across the population, how variability in decision making is related to substance use, and how substance use can produce long-lasting changes in decision preference.

Monash Biomedical Imaging highlights from 2021 and looking ahead to 2022

Despite the challenges COVID-19 has continued to present, Monash Biomedical Imaging (MBI) has had another outstanding year in terms of publications and scientific output. In this webinar, Professor Gary Egan, Director of MBI, will present an overview of MBI’s achievements during 2021 and outline the biomedical imaging research programs and partnerships in 2022. His presentation will cover: • MBI operational and research achievements during 2021 • Biomedical imaging technology developments and research outcomes during 2021 • Linked laboratories and research teams at MBI • Progress on the development of a cyclotron and precision radiopharmaceutical facility at Clayton • Emerging research opportunities at the Monash Heart Hospital in cardiology and cardiovascular disease. Professor Gary Egan is Director of Monash Biomedical Imaging, Director of the ARC Centre of Excellence for Integrative Brain Function and a Distinguished Professor at the Turner Institute for Brain and Mental Health, Monash University. He is also lead investigator of the Victorian Biomedical Imaging Capability, and Deputy Director of the Australian National Imaging Facility. His substantive body of published work has made a significant impact on the neuroimaging and neuroscience fields. He has sustained success in obtaining significant grants to support his own research and the development of facilities to advance biomedical imaging.

Nonlinear spatial integration in retinal bipolar cells shapes the encoding of artificial and natural stimuli

Vision begins in the eye, and what the “retina tells the brain” is a major interest in visual neuroscience. To deduce what the retina encodes (“tells”), computational models are essential. The most important models in the retina currently aim to understand the responses of the retinal output neurons – the ganglion cells. Typically, these models make simplifying assumptions about the neurons in the retinal network upstream of ganglion cells. One important assumption is linear spatial integration. In this talk, I first define what it means for a neuron to be spatially linear or nonlinear and how we can experimentally measure these phenomena. Next, I introduce the neurons upstream to retinal ganglion cells, with focus on bipolar cells, which are the connecting elements between the photoreceptors (input to the retinal network) and the ganglion cells (output). This pivotal position makes bipolar cells an interesting target to study the assumption of linear spatial integration, yet due to their location buried in the middle of the retina it is challenging to measure their neural activity. Here, I present bipolar cell data where I ask whether the spatial linearity holds under artificial and natural visual stimuli. Through diverse analyses and computational models, I show that bipolar cells are more complex than previously thought and that they can already act as nonlinear processing elements at the level of their somatic membrane potential. Furthermore, through pharmacology and current measurements, I illustrate that the observed spatial nonlinearity arises at the excitatory inputs to bipolar cells. In the final part of my talk, I address the functional relevance of the nonlinearities in bipolar cells through combined recordings of bipolar and ganglion cells and I show that the nonlinearities in bipolar cells provide high spatial sensitivity to downstream ganglion cells. Overall, I demonstrate that simple linear assumptions do not always apply and more complex models are needed to describe what the retina “tells” the brain.

Astrocytes and oxytocin interaction regulates amygdala neuronal network activity and related behaviors”

Oxytocin orchestrates social and emotional behaviors through modulation of neural circuits in brain structures such as the central amygdala (CeA). In this structure, the release of oxytocin modulates inhibitory circuits and subsequently suppresses fear responses and decreases anxiety levels. Using astrocyte-specific gain and loss of function approaches and pharmacology, we demonstrate that oxytocin signaling in the central amygdala relies on a subpopulation of astrocytes that represent a prerequisite for proper function of CeA circuits and adequate behavioral responses, both in rats and mice. Our work identifies astrocytes as crucial cellular intermediaries of oxytocinergic modulation in emotional behaviors related to anxiety or positive reinforcement. To our knowledge, this is the first demonstration of a direct role of astrocytes in oxytocin signaling and challenges the long-held dogma that oxytocin signaling occurs exclusively via direct action on neurons in the central nervous system.

Synapses, Shadows and Stress Contagion

Survival is predicated on the ability of an organism to respond to stress. The reliability of this response is ensured by a synaptic architecture that is relatively inflexible (i.e. hard-wired). Our work has shown that in naive animals, synapses on CRH neurons in the paraventricular nucleus of the hypothalamus are very reluctant to modification. If animals are stressed, however, these synapses become willing to learn. This seminar will focus on mechanisms linking acute stress to metaplastic changes at glutamate synapses, and also show how stress, and these synaptic changes can be transmitted from one individual to another.

Reflex Regulation of Innate Immunity

Reflex circuits in the nervous system integrate changes in the environment with physiology. Compact clusters of brain neuron cell bodies, termed nuclei, are essential for receiving sensory input and for transmitting motor outputs to the body. These nucelii are critical relay stations which process incoming information and convert these signals to outgoing action potentials which regulate immune system functions. Thus, reflex neural circuits maintain parameters of immunological physiology within a narrow range optimal for health. Advances in neuroscience and immunology using optogenetics, pharmacogenetics, and functional mapping offer a new understanding of the importance of neural circuitry underlying immunity, and offer direct paths to new therapies.

Will it keep me awake? Common caffeine intake habits and sleep in real life situations

Daily caffeine consumption and chronic sleep restriction are highly prevalent in society. It is well established that acute caffeine intake under controlled conditions enhances vigilance and promotes wakefulness but can also delay sleep initiation and reduce electroencephalographic (EEG) markers of sleep intensity, particularly in susceptible individuals. To investigate whether these effects are also present during chronic consumption of coffee/caffeine, we recently conducted several complementary studies. We examined whether repeated coffee intake in dose and timing mimicking ‘real world’ habits maintains simple and complex attentional processes during chronic sleep restriction, such as during a busy work week. We found in genetically caffeine-sensitive individuals that regular coffee (300 mg caffeine/day) benefits most attentional tasks for 3-4 days when compared to decaffeinated coffee. Genetic variants were also used in the population-based HypnoLaus cohort, to investigate whether habitual caffeine consumption causally affects time to fall asleep, number of awakenings during sleep, and EEG-derived sleep intensity. The multi-level statistical analyses consistently showed that sleep quality was virtually unaffected when >3 caffeine-containing beverages/day were compared to 0-3 beverages/day. This conclusion was further corroborated by quantifying the sleep EEG in the laboratory in habitual caffeine consumers. Compared to placebo, daily intake of 3 x 150 mg caffeine over 10 days did not strongly impair nocturnal sleep nor subjective sleep quality in good sleepers. Finally, we tested whether an engineered delayed, pulsatile-release caffeine formula can improve the quality of morning awakening in sleep-restricted volunteers. We found that 160 mg caffeine taken at bedtime ameliorated the quality of awakening, increased positive and reduced negative affect scores, and promoted sustained attention immediately upon scheduled wake-up. Such an approach could prevent over-night caffeine withdrawal and provide a proactive strategy to attenuate disabling sleep inertia. Taken together, the studies suggest that common coffee/caffeine intake habits can transiently attenuate detrimental consequences of reduced sleep virtually without disturbing subjective and objective markers of sleep quality. Nevertheless, coffee/caffeine consumption cannot compensate for chronic sleep restriction.

Dancing to a Different Tune: TANGO Gives Hope for Dravet Syndrome

The long-term goal of our research is to understand the mechanisms of SUDEP, defined as Sudden, Unexpected, witnessed or unwitnessed, nontraumatic and non-drowning Death in patients with EPilepsy, excluding cases of documented status epilepticus. The majority of SUDEP patients die during sleep. SUDEP is the most devastating consequence of epilepsy, yet little is understood about its causes and no biomarkers exist to identify at risk patients. While SUDEP accounts for 7.5-20% of all epilepsy deaths, SUDEP risk in the genetic epilepsies varies with affected genes. Patients with ion channel gene variants have the highest SUDEP risk. Indirect evidence variably links SUDEP to seizure-induced apnea, pulmonary edema, dysregulation of cerebral circulation, autonomic dysfunction, and cardiac arrhythmias. Arrhythmias may be primary or secondary to hormonal or metabolic changes, or autonomic discharges. When SUDEP is compared to Sudden Cardiac Death secondary to Long QT Syndrome, especially to LQT3 linked to variants in the voltage-gated sodium channel (VGSC) gene SCN5A, there are parallels in the circumstances of death. To gain insight into SUDEP mechanisms, our approach has focused on channelopathies with high SUDEP incidence. One such disorder is Dravet syndrome (DS), a devastating form of developmental and epileptic encephalopathy (DEE) characterized by multiple pharmacoresistant seizure types, intellectual disability, ataxia, and increased mortality. While all patients with epilepsy are at risk for SUDEP, DS patients may have the highest risk, up to 20%, with a mean age at SUDEP of 4.6 years. Over 80% of DS is caused by de novo heterozygous loss-of-function (LOF) variants in SCN1A, encoding the VGSC Nav1.1  subunit, resulting in haploinsufficiency. A smaller cohort of patients with DS or a more severe DEE have inherited, homozygous LOF variants in SCN1B, encoding the VGSC 1/1B non-pore-forming subunits. A related DEE, Early Infantile EE (EIEE) type 13, is linked to de novo heterozygous gain-of-function variants in SCN8A, encoding the VGSC Nav1.6. VGSCs underlie the rising phase and propagation of action potentials in neurons and cardiac myocytes. SCN1A, SCN8A, and SCN1B are expressed in both the heart and brain of humans and mice. Because of this, we proposed that cardiac arrhythmias contribute to the mechanism of SUDEP in DEE. We have taken a novel approach to the development of therapeutics for DS in collaboration with Stoke Therapeutics. We employed Targeted Augmentation of Nuclear Gene Output (TANGO) technology, which modulates naturally occurring, non-productive splicing events to increase target gene and protein expression and ameliorate disease phenotype in a mouse model. We identified antisense oligonucleotides (ASOs) that specifically increase the expression of productive Scn1a transcript in human and mouse cell lines, as well as in mouse brain. We showed that a single intracerebroventricular dose of a lead ASO at postnatal day 2 or 14 reduced the incidence of electrographic seizures and SUDEP in the F1:129S-Scn1a+/- x C57BL/6J mouse model of DS. Increased expression of productive Scn1a transcript and NaV1.1 protein were confirmed in brains of treated mice. Our results suggest that TANGO may provide a unique, gene-specific approach for the treatment of DS.

Epigenetic regulation of alternative splicing in the context of cocaine reward

Neuronal alternative splicing is a key gene regulatory mechanism in the brain. However, the spliceosome machinery is insufficient to fully specify splicing complexity. In considering the role of the epigenome in activity-dependent alternative splicing, we and others find the histone modification H3K36me3 to be a putative splicing regulator. In this study, we found that mouse cocaine self-administration caused widespread differential alternative splicing, concomitant with the enrichment of H3K36me3 at differentially spliced junctions. Importantly, only targeted epigenetic editing can distinguish between a direct role of H3K36me3 in splicing and an indirect role via regulation of splice factor expression elsewhere on the genome. We targeted Srsf11, which was both alternatively spliced and H3K36me3 enriched in the brain following cocaine self-administration. Epigenetic editing of H3K36me3 at Srsf11 was sufficient to drive its alternative splicing and enhanced cocaine self-administration, establishing the direct causal relevance of H3K36me3 to alternative splicing of Srsf11 and to reward behavior.

Understanding the Assessment of Spatial Neglect and its Treatment Using Prism Adaptation Training

Spatial neglect is a syndrome that is most frequently associated with damage to the right hemisphere, although damage to the left hemisphere can also result in signs of spatial neglect. It is characterised by absent or deficient awareness of the contralesional side of space. The screening and diagnosis of spatial neglect lacks a universal gold standard, but is usually achieved by using various modes of assessment. Spatial neglect is also difficult to treat, although prism adaptation training (PAT) has in the past reportedly showed some promise. This seminar will include highlights from a series of studies designed to identify knowledge gaps, and will suggest ways in which these can be bridged. The first study was conducted to identify and quantify clinicians’ use of assessment tools for spatial neglect, finding that several different tools are in use, but that there is an emerging consensus and appetite for harmonisation. The second study included PAT, and sought to uncover whether PAT can improve engagement in recommended therapy in order to improve the outcomes of stroke survivors with spatial neglect. The final study, a systematic review and meta-analysis, sought to investigate the scientific efficacy (rather than clinical effectiveness) of PAT, identifying several knowledge gaps in the existing literature and a need for a new approach in the study of PAT in the clinical setting.

LONG-ACTING ANTIPSYCHOTICS: OPTION DOWN THE ROCKY ROAD, NICE TO HAVE OR ESSENTIAL CHOICE?

Time and again we are faced with the question at what point in the treatment of schizophrenia a depot formulation should be used. The data on the so-called LAIs (Long-Acting Injectables) has steadily increased in recent years. Today, we have very good evidence for the early use of depot therapies. However, the willingness and consent of the patient for this form of pharmacotherapy remains central to the successful use of LAIs. In his lecture, Prof. Correll will talk about the current evidence for the use of LAIs summarizing the latest studies.

In-Love with Addiction Neuroscience

In this talk series, addiction neuroscientists from across the world share their personal stories/experiences on the beauty of addiction neuroscience and how/why they have decided to invest their scientific life in this field. We hope that this talk series would encourage and support a new generation of young and passionate addiction neuroscientists in different countries to revolutionize the field of addiction medicine.

Developing metal-based radiopharmaceuticals for imaging and therapy

Personalised medicine will be greatly enhanced with the introduction of new radiopharmaceuticals for the diagnosis and treatment of various cancers, as well as cardiovascular disease and brain disorders. The unprecedented interest in developing theranostic radiopharmaceuticals is mainly due to the recent clinical successes of radiometal-based products including: • 177LuDOTA-TATE (trade name Lutathera, FDA approved in 2018), a peptide-based tracer that is used for treating metastatic neuroendocrine tumours • Ga 68 PSMA-11 (FDA approved in 2020), a positron emission tomography agent for imaging prostate-specific membrane antigen positive lesions in men with prostate cancer. In this webinar, Dr Brett Paterson and PhD candidate Mr Cormac Kelderman will present their research on developing the chemistry and radiochemistry to produce new radiometal-based imaging and therapy agents. They will discuss the synthesis of new molecules, the optimisation of the radiochemistry, and results from preclinical evaluations. Dr Brett Paterson is a National Imaging Facility Fellow at Monash Biomedical Imaging and academic group leader in the School of Chemistry, Monash University. His research focuses on the development of radiochemistry and new radiopharmaceuticals. Cormac Kelderman is a PhD candidate under the supervision of Dr Brett Paterson in the School of Chemistry, Monash University. His research focuses on developing new bis(thiosemicarbazone) chelators for technetium-99m SPECT imaging.

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

FENS Forum 2026

Auditory brainstem responses in the Nrxn1 rat and Cntnap2 mouse models for neurodevelopmental disorders: A longitudinal and pharmacological study

Benchmarking of individual-level preprocessing strategies for pharmaco-fUS

Beyond retigabine: design, synthesis, and pharmacological characterization of a potent and chemically-stable neuronal Kv7 channels activator with anticonvulsant activity

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

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

Deep brain stimulation of the medial septum restores blood perfusion following pharmacologic NMDA antagonism in a region-dependent manner

Effect of a combination treatment of Bee venom pharmacopuncture in GB34 and L-dopa in a mouse model of Parkinson’s disease

Emotional Dysregulation and Altered Reward Processing in Self-Harm

HEMPHASIS 2 : A pharmacological signature to selectively determine mGlu7 containing heterodimers

Novel chemogenetic tools for manipulating nicotinic neurotransmission with high spatio-temporal and pharmacological precision in mice

Pharmacological activation of histamine receptor type 2 enhances evoked firing in medium spiny neurons of the nucleus accumbens through downregulation of Kv4.2 channels

Pharmacological enhancement of adult neurogenesis improves pattern separation in young and aged mice

Pharmacological and genetic probing of the role of Nociceptin/orphaninFQ receptors in chronic stress-induced memory deficits

Pharmacological inactivation of the bed nucleus of the stria terminalis increases prosocial behavior in rhesus macaques

Pharmacological inhibition of the aggregation-promoting ATXN1-MED15 protein interaction

Pharmacological modulation of glial phenotype in animal model of Parkinson’s disease

Pharmacological modulation of neuronal activity for the treatment of Rett syndrome

pharmacological stimulation of the serotonin receptor 7 rescues fear generalization in a ptsd mouse model carrying a truncated form of mecp2

Pharmacologically compromising central amygdala astrocytes prevents the beneficial effects of oxytocin on pain-related behaviors

Pharmacology and desensitization properties of α4-containg GABAA receptors

Physiological, neuronal and pharmacological identification of panic and anxiety-like states during freezing

Second-harmonic generation imaging of axon-like neurites in the unfixed, unlabeled retina

Study of the pharmacological inhibition of RPTP β/ζ as a novel strategy to modulate the LPS-induced loss of neuronal progenitors in the dentate gyrus

Temporal and resolved harmonic pitch encoding in auditory cortex

Transfer of a pharmacological neurovascular uncoupling model from mice to rats

In vitro and vivo pharmacological characterization of the clinically viable NOP receptor antagonist BTRX-246040

Aberrant neuronal activity and habituation of the giant fiber escape response circuit in Drosophila NF1 mutants: A pharmacogenetic approach

FENS Forum 2024

Alcohol perturbed locomotor behavior, metabolism, and pharmacokinetics of methamphetamine in rats

FENS Forum 2024

Astrocyte noradrenaline α-1A receptor activation induces changes to inhibitory synaptic transmission in the hippocampus and reduces the frequency of pharmacoresistant spontaneous seizures

FENS Forum 2024

Attempt to pharmacologically induce neurovascular uncoupling in aged, experienced rats

FENS Forum 2024

Characterization of multi-target ligands comprising opioid/non-opioid pharmacophores for the treatment of pain

FENS Forum 2024

Cholinergic modulation of attentional performance on a signal detection task: Pharmacological modulation of nicotinic and muscarinic receptors

FENS Forum 2024

Combined treatment with the glycine transporter-1-inhibitor Org24598, varenicline, and bupropion as a new pharmacological treatment concept for alcohol use disorder

FENS Forum 2024

Cortical miR-16 involvement in the antidepressant effects of pharmacological elevation of anandamide in a rat model for depression

FENS Forum 2024

Design, synthesis and pharmacological evaluation of pyrazole/tacrine derivatives as potential acetylcholinesterase inhibitors

FENS Forum 2024

Development of pharmacologically active nanobodies targeting the mGlu5R-A2AR heteromer in psychosis

FENS Forum 2024

The differential role of the subiculum > hypothalamic paths in diverse contextual fear responses to a live predator and physically harmful events

FENS Forum 2024

Disruption of climbing behavior by harmaline in marker-based 3D motion capture of freely moving mice

FENS Forum 2024

Disruption of treadmill running by harmaline and cannabinoid agonist in marker-based 3D motion capture of mice

FENS Forum 2024