Jackson Laboratory

NIH Research Projects · FY 2025 · 2024-08

Alzheimer’s Disease (AD) is a complex and heterogenous disorder characterized by multiple clinical, neuropathological and molecular phenotypes. Despite considerable effort to date, there is currently a lack of interventions or cure, thus there is a clear need for a better understanding of the heterogenous disease phenotypes and the underlying genetics to identify more effective preclinical strategies. Rapidly expanding genomic, transcriptomic, proteomic, metabolomic and epigenomic data sets, and emerging animal models of AD created by the Model Organism Development and Evaluation for Late-Onset Alzheimer’s Disease (MODEL-AD) consortium, now provide new tools for accelerating our understanding of AD and enable interspecies analysis for mapping findings from mouse model systems of AD to human omics signatures. While multi-omics data sets derived from AD and animal models of AD are available to the scientific community through various data ecosystems including the AD Knowledge Portal, a NIA designated FAIR (Findable, Accessible, Interoperable, and Reusable) data repository, a current barrier is mapping disease relevant molecular signatures between model organisms and humans. Thus, there is an emerging need to provide training in bioinformatics methodologies for systematic interspecies translation of omics-derived signatures of AD. To address this need, we aim to provide a new, and increasingly multidisciplinary, generation of researchers, with awareness of available resources and to provide skills development for integrating multi-scale data from model systems and humans to advance AD research and interventions. We propose a unique, annual, 4-day workshop at The Jackson Laboratory (JAX), that will leverage trainers and expertise from Sage Bionetwork, the host institution for The AD Knowledge Portal and Exceptional Longevity Data Management and Coordinating Center, and the MODEL-AD Center at JAX. The proposed workshop, Computational Techniques and Resources for Effective Translational Research in Alzheimer’s Disease, will focus on enabling utilization of omics-driven computational techniques and analytical principles for cross species functional alignment. Active learning programming sessions will be the core of the workshop, in combination with lectures and interactive forums. Participants will emerge from the workshop equipped with the knowledge and technical skills to conduct rigorous and reproducible computational research for more effective translational strategies in AD. To achieve this, we propose the following aims: 1) foster utilization of existing data resources from human and model organism studies in AD; 2) deliver hands-on training on computational techniques that reinforce rigor and reproducibility principles for translational AD research, and; 3) create an engaging environment for trainees that fosters networking, collaboration, and experiential learning.. .

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT Charcot-Marie-Tooth disease (CMT) is a collection of inherited peripheral neuropathies with a cumulative incidence of ~1:2500 people. There is no approved treatment for any of the 100 genetic subtypes of CMT, presenting a large unmet clinical need. Charcot-Marie-Tooth type 2D is caused by dominant mutations in glycyl tRNA synthetase (GARS), encoding the enzyme that charges glycine onto its cognate tRNAs during translation. Our working model for the disease mechanism is that the mutant enzyme binds its tRNA substrate, but does not release it to the ribosome, effectively sequestering the substrate. This results in ribosome stalling at glycine codons and activation of the integrated stress response. Support for this mechanism comes from genetic studies in Drosophila and mouse models of CMT2D, in which transgenic overexpression of tRNAGlyGCC was able to effectively suppress the neuropathy phenotype. In preliminary studies, we have reproduced this result using AAV9 to deliver tRNAGly genes to three different CMT2D mouse models. Glycine has four codons (GGC, GGG, GGA, and GGU), and therefore four potential anticodons (GCC, CCC, UCC, and ACC respectively, though ACC is likely a nonfunctional tRNA). We made four AAV9 vectors expressing each tRNAGly anticodon driven by a PolIII U6 promoter. We found that GCC was highly effective, almost completely suppressing the neuropathy phenotype even in mice with a severe allele of Gars. Vectors expressing CCC and UCC were intermediate in efficacy, and ACC was ineffective (as anticipated). This profile of efficacy correlates with tRNA abundance and codon usage, and suggests we are replacing the sequestered substrates of GARS with the AAVs. In the R61 phase of this proposal we will optimize the vector payload (Aim 1) and capsid (Aim 2), and in the R33 phase (Aim 3), we will use this optimized vector in rigorous preclinical studies in mouse models of CMT2D. In Aim 1 (R61), we will construct an AAV9 vector that carries all three effective tRNAGly genes (GCC, CCC, UCC) in a single vector. We will compare this against GCC alone, which was very effective. In Aim 2 (R61), we will recreate the U6-GCC vector in a MACPNS capsid in an attempt to create a vector that is effective with systemic delivery, rather than dosing directly into the nervous system. We will compare the MACPNS-GCC vector to AAV9-GCC. In Aim 3 (R33), we will test the optimized vector (GCC or combined tRNAGlys, AAV9 or MACPNS) in two mouse models of CMT2D. We will also allow treated mice to age to show the perdurance of the effect, and we will examine the effects of treating after the onset of neuropathy. The successful completion of these aims will show the in vivo efficacy of an optimized gene therapy treatment for CMT2D in preclinical studies in mouse models. This will position us for further translational research and IND-enabling studies through mechanisms such as the Blueprint Neurotherapeutics Network for Biologicals.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT The prevalence of Alzheimer's disease (AD) in the U.S. is expected to reach 12.7 million by 2050 if successful disease-modifying treatments are not identified. Without a better understanding of the genetic and neuronal mechanisms that drive AD progression, the development of new therapeutics to enhance cognitive longevity will remain limited. Alternatively, therapeutic strategies to promote cognitive resilience in the face of AD pathology are potentially effective methods to lessen the impact of AD. To identify gene candidates of resiliency I quantified neurodegeneration in the brains of the translationally-relevant AD-BXD mouse model of AD via immunohistochemistry (IHC) and used these imaging outcomes to complete genetic mapping. Using this approach, I identified Leucine Rich Repeat And Fibronectin Type III Domain Containing 2 (Lrfn2) as a potentially causal gene modifying AD-related cortical neurodegeneration. I hypothesize that Lrfn2 overexpression will rescue AD-related cortical neurodegeneration, synaptic dysfunction, and cognitive decline in the 5XFAD model of AD. I will test this hypothesis by developing a novel Lrfn2 overexpression transgenic mouse and evaluating the impact of changes in Lrfn2 expression on AD progression in three aims. Aim 1) I will measure the extent of cortical neurodegeneration in 5XFAD and nontransgenic mice with and without Lrfn2 overexpression in both male and female mice at 6 and 14 months of age. Results will indicate whether Lrfn2 is truly a causal modifier of cortical neurodegeneration. Aim 2a) To test the impact of Lrfn2 overexpression on synaptic plasticity I will perform ex vivo whole-cell current-clamp electrophysiology recordings in a separate cohort of mice. I will determine if overexpression of Lrfn2 in excitatory forebrain neurons rescues long-term potentiation deficits in 5XFAD animals. Aim 2b) To investigate the effect of Lrfn2 on synaptic structure, I will image dendritic spines to assess changes in spine morphology and density as a complementary measure of synapse stability and synaptic function associated with cognitive performance. Aim 3) To evaluate the role of Lrfn2 as a potential resilience factor to cognitive decline, I will execute mouse behavioral tasks measuring working, short-term, long-term, and working memory in the same mice used in Aim 1. To our knowledge, this will be the first study to evaluate Lrfn2 expression in the context of aging, AD, and cortical neurodegeneration. With the successful completion of these aims, I will achieve my long-term goal for this project by determining whether Lrfn2 modulation is a novel resilience therapeutic for AD treatment. The proposed work will facilitate the achievement of my training goals to acquire new skills and knowledge related to IHC, imaging, electrophysiology, dendritic spine characterization, behavioral assays, general wet-lab techniques, and professional development. Overall, the guidance of Drs. O'Connell and Kaczorowski; access to the outstanding core resources at The Jackson Laboratory; and the experience gained from this fellowship will be a significant step toward achieving my long-term career goal of becoming an independent academic scientist.

NIH Research Projects · FY 2024 · 2024-06

PROJECT SUMMARY The long-term goal of this proposal is to advance the research of investigators at The Jackson Laboratory (JAX) and in the Maine research community by bringing a new Bruker timsTOF SCP mass spectrometer package for high-resolution, single-cell proteomic analysis to JAX’s Mass Spectrometry and Protein Chemistry Service (MSPC). JAX’s research mission focuses on discovering precise genomic solutions for disease to improve human health in the global community. As part of this mission, there has been an increasing emphasis on the analysis of single cells, as biomedical research has demonstrated that examination of biology at this level is essential for understanding the biological processes underlying development and disease. Accordingly, JAX has technologies for characterization and quantification of gene expression, chromatin accessibility, and individual cell-surface protein molecules present, all at the single-cell level. In addition, recently JAX investigators have had access to increasingly advanced capabilities in the MSPC in mass spectrometry-based proteomics and metabolomics for biological discoveries linking genomics with phenotype. However, despite our numerous mass spectrometry capabilities, there is currently no instrumentation here with the capability to perform unbiased, high-throughput label-free single-cell proteomics analysis. This gap limits our investigators’ ability to understand biology at the single-cell level, including new insights that would be gained by integrating single-cell proteome data with the array of other single-cell data generated here, and by addressing the issue of a generally weak transcriptome-proteome correlation in organisms. The Bruker timsTOF SCP mass spectrometer is the ideal system to address this gap. This instrument offers innovative technologies, including a dramatically improved ion-source concept, an enhanced trapped ion mobility, and mass analyzer that provide ultra-high sensitivity, enabling unbiased single-cell proteomics with high reproducibility. The timsTOF SCP can achieve coverage of about 1,500 proteins per cell, sufficiently identify post-translational modifications in a very small number of morphologically or functionally similar cells, and efficiently analyze low-abundant samples, such as the rare cell populations that many of our investigators study. The timsTOF SCP and its accessories, including the cellenONE for single-cell sorting and proteomic sample processing, will be transformative for research at JAX and other Maine research institutions. Various studies at JAX focused on rare immune-cell populations, stem-cell differentiation, sub-populations of cancer cells, the identification of new therapeutic targets for cancer, and characterizing mechanisms underlying cognitive aging will all benefit from this instrumentation. Progress in these research areas are particularly important given JAX’s broadening emphasis on translating our basic research discoveries into clinical advances that will directly benefit human health. In sum, the Bruker timsTOF SCP platform will substantially enhance JAX’s ability to fulfill its research mission and will also bring this potential to the greater Maine community.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY/ABSTRACT The goal of this project is to provide training and education in human genomics-related topics to the entry-level biomedical workforce, and in particular, to individuals from underrepresented and disadvantaged populations. Entry-level training in human genomics is critical for creating diverse teams of individuals who can be active participants in genomic medicine. In response to this ongoing need, The Jackson Laboratory (JAX), in partnership with Connecticut State Colleges and Universities (CSCU), proposes a curriculum development initiative, Genomics in Action, for community college students that will impart content knowledge and skills in genetics and genomics to better prepare them for a variety of careers, such as laboratory, medical and nursing assistants. Community college students within the CSCU system are a highly diverse group, with approximately 50% of students receiving financial assistance and 50% of students from minoritized populations. To achieve our objective, we propose the following Aims: 1. Develop 15 virtual modules on key topics in human genomics, tailored to community college student needs. These modules will address curricular gaps as identified through needs analysis with our CSCU faculty. Our innovative approach will utilize short video content to introduce topics, demonstrate concepts, as well as provide context for each topic within a career or job in the genomics workforce. Each module will feature a near-peer who is employed in a role for which community college students are training. The modules will thus be designed to connect the genomics content and skills directly to application in the workplace. 2. Disseminate fully digital modules to the broader community. Our team will design modules that can be seamlessly integrated into our CSCU partner institutions learning management systems, and professional development workshops will be provided to facilitate adoption the Genomics in Action modules among the broader CSCU community. We will also expand dissemination through JAX’s online and digital education program that provides free access to our interactive multimedia educational content, making the modules available for use globally. To evaluate the impact of the Genomics in Action modules, pilot adoption will take place during year 1 of the project and surveys will be administered to assess the effectiveness of the module delivery and usability by both faculty and students. Modules will be adapted, refined, and subsequently delivered in courses again in years 2 and 3. We will examine overall uptake and use of the modules by our partner institutions and will track broader dissemination within other programs and institutions within the CSCU. Program impact will also be assessed by tracking learners’ career progress and retention in biomedicine following program participation. By training community college students seeking entry-level positions, Genomics in Action will execute on NIH’s goal of strengthening the future STEM workforce through increasing genomic and health literacy.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY/ABSTRACT Genome-wide association (GWA) and linkage studies have identified >50 genes significantly associated with age related macular degeneration (AMD). However, many questions remain. Namely, are the identified GWA hits disease-causing variants or are they simply closely linked markers? And if the GWA hits are disease-causing variants, how do disruptions in these molecules lead to the observed pathologies? Further, since AMD is multifactorial, to what extent do particular combinations of factors precipitate AMD disease phenotypes or increase disease severity, as has been suggested to be the case in large scale GWA studies, where CFH and ARMS2/HTRA1 (locus on Chr. 10q26) appear to have synergistic effects on disease risk. Together they explain >50% of the genetic variability observed in AMD. Furthermore, since ARMS2 and HTRA1 are in strong linkage disequilibrium, it has been difficult to decipher whether either or both genes contribute to AMD-pathologies. The issues raised above can be addressed, in part, with appropriate animal models. Although mice do not have a macula per se, they faithfully recapitulate many aspects of retinal degenerative diseases and have been used to learn how disruption of certain molecules lead to AMD-like pathologies. In this application, we will seek in vivo confirmation of the cell-type and subcellular localization of ARMS2, and establish whether mice bearing the ARMS2A69S allele independently develop AMD-like sub-phenotypes and explore potential molecular mechanisms underlying the changes. Finally, because AMD is a multifactorial disease, we will examine if AMD- associated risk factors such as such as diet or genetic variants, such as CFH risk alleles, can potentiate AMD- like disease phenotypes. Identifying the pathogenic pathways and mechanisms underlying the disease sub-phenotypes, the goal of this proposal, is critical for developing effective therapies that can target the pre-symptomatic stage to prevent, delay onset or decrease severity of the disease. Animal models serve an important and unique role for furthering our understanding of the genetic underpinnings of disease, and as a resource to examine tissue pathology and to test therapeutics that cannot be readily done in humans.

NIH Research Projects · FY 2025 · 2024-05

PROJECT SUMMARY The laboratory mouse, the best genetically defined experimental vertebrate model organism for humans, continues to be the primary animal research model of human disease and is a key tool for the translational hands-on training required to engage in modern scientific research, veterinary science, and clinical settings. However, while many institutions provide access to online training modules, few if any have coordinated, intensive, hands-on training in the laboratory mouse. Moreover, there are unmet needs to lower the barriers to participation in advantageous training by diverse and disadvantaged learner groups, to provide equitable access and, in alignment with stated NIH priorities, to train and diversify the biomedical workforce. To help address this, The Jackson Laboratory (JAX) proposes to provide a traveling workshop, entitled Foundational Training in the Use of the Laboratory Mouse as a Model of Human Disease, to be held at partner minority-serving institutions (MSI). This workshop will provide learning and skills development focused on fundamental techniques that are employed across a wide variety of animal research and animal models of human disease. The proposed Aims of the workshop are: (1) To deliver foundational, experiential, training in the use of the laboratory mouse that participants can apply to their research and that will facilitate their career development; and (2) To facilitate engagement, active learning, and skills development by diverse and disadvantaged learners by partnering with MSI to bring hands-on workshops and experiential learning to their institutions. Participants completing this workshop will obtain valuable exposure, perspective, and skills in the use of laboratory mice for biomedical research and as models of human disease and they will learn techniques that they can apply to their research and that will facilitate their career development. JAX is uniquely positioned to execute these traveling workshops: the program will be facilitated by certified and experienced lead instructors from JAX and mobile lab equipment has already been acquired in order to be able to hold this workshop at any partner institution that has basic lab space and a vivarium. Indeed, we have successfully piloted this program at a partner MSI, with highly positive participant feedback. We will offer the proposed program at no cost to the host institutions or to the program participants, making the workshop incredibly accessible and inclusive.

NIH Research Projects · FY 2025 · 2024-04

PROJECT SUMMARY Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype that accounts for ~20% of breast cancer diagnoses. Importantly, non-Hispanic-Black (NHB) and Hispanic women are disproportionally affected by TNBC with worse clinical outcomes than non-Hispanic-White women (NHW), even when risks are adjusted for diagnosis and other clinical factors as well as socio-economic factors. This indicates that biological factors also influence the clinical course of TNBC in patients from different racial/ethnic backgrounds. We recently identified robust TNBC prognostic subgroups based on BRCA1/2 status and the levels of an immune-related gene transcriptional signature. Specifically, we demonstrated that BRCA1/2 mutant tumors (BRCAmut) and BRCA1/2 wildtype tumors with high levels of the immune-related gene signature (nonBRCA-ImmuneHigh) associate with the best therapy response rates (as measured by pathological complete response to platinum- based chemotherapy). In contrast, BRCA1 promoter hypermethylation (BRCA1meth), and BRCA1/2 wild type status with low expression of the immune-related gene signature (nonBRCA-ImmuneLow), are linked to poor response rates. Importantly, BRCA1/2 mutations (linked to better prognosis) are less common in both NHB and Hispanic TNBC patients compared to NHW patients, while evidence from the TCGA TNBC dataset shows a higher prevalence of BRCA1meth cancers (linked to poor prognosis) in NHB than in NHW patients, consistent with the disparities in clinical outcomes. In addition, racial/ethnic differences in TNBC tumor immune cell infiltration have recently been reported. Based on these observations, we hypothesize that at least part of the disparities in clinical outcomes between TNBC patients of NHB and Hispanic ancestry vs. those of NHW ancestry is due to different prevalence of TNBC molecular features that associate with better or worse therapeutic outcomes. Here, to test this hypothesis, we will assess BRCA1/2 gene status (BRCA1/2 mutations and BRCA1 promoter methylation, Specific Aim 1) and levels of the immune-related gene signature (a panel of 30 immune genes, Specific Aim 2) in a retrospective cohort of ~300 primary TNBC samples from women of NHW, NHB and Hispanic ancestry. We will then determine the prevalence of the four TNBC prognostic subtypes (BRCAmut, BRCA1meth, nonBRCA-ImmuneHigh, nonBRCA-ImmuneLow) within each race/ethnicity towards ascertaining whether their prevalence accounts for differences in response outcomes between NHW and NHB/Hispanic TNBC patients. Successful completion of this study will generate novel insight into the basic molecular mechanisms underlying cancer health disparities and will provide the groundwork for a future prospective study to expand and validate the prognostic value of this type of patient stratification across more refined race and ethnicity subgroups.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY The Molecular Phenotypes of Null Alleles in Cells (MorPhiC) program is using multiple perturbation strategies to realize the NHGRI's vision of assigning function to every human gene. Strategies include pooled and individual gene knockouts and knockdowns (KDs), generated using CRISPR technologies and auxin-inducible degrons. Following application of such assays, molecular phenotypes of the cells are profiled longitudinally and at individual time points using bulk and single-cell (sc)RNA-seq. Perturbation strategies have intrinsic sources of variability, e.g., KD penetrance, while the single-cell sequencing approaches contribute technical noise, e.g., `drop out.' Quantifying and controlling this variability are crucial to ensure reliable phenotypic assessment and fulfill MorPhiC's goal to accurately catalog gene function. Given the critical role of transcription factors (TFs) in regulating cell state, all four MorPhiC Data Production Centers (DPCs) will perturb TFs and then profile cells using bulk or sc-RNA-seq. A wide range of other `regulatory phenotyping' data, including (bulk or single-cell) ATAC-seq, are being generated within MorPhiC and TF ChIP-seq, HiC, and massively parallel reporter assay (MPRA) data are available in the ENCODE and Impact of Genomic Variation on Function (IGVF) consortia. To robustly define the regulatory impact of TF perturbation, we propose a JAX MorPhiC Data Analysis and Validation Center (DAV) to analyze these multi-modal data. Our team is uniquely positioned to establish this TF-focused DAV: we are co-located with the JAX MorPhiC DPC and have consortium-level collaborations with its PI, while our own work focuses on elucidating transcriptional regulation of genes and on developing robust computational methods through community efforts. In Aim 1, we will quantify and control variability in perturbation-based regulatory phenotyping by using heterogeneous data generated within MorPhiC to isolate their technical noise characteristics and to derive a set of TF-gene target pairs (TF-GTs). We will then computationally simulate large- scale perturbation screens, through which we will perform power analysis to quantify data variability and make recommendations that ameliorate it. In Aim 2, we will evaluate published gene regulatory network (GRN) inference methods. We will also conduct two “crowd-sourced” DREAM Challenges, in which community participants will develop GRN inference methods that we will objectively evaluate with MorPhiC data. Using top- performing methods, as well as a novel approach we are developing based on dynamical systems, we will perform in silico TF perturbation within the GRNs to prioritize TFs for experimental validation in MorPhiC. In Aim 3, we will further improve robustness of inferred TF-GTs by integrating them with TF ChIP-seq, HiC, and MPRA data, knockout mouse phenotyping data (KOMP2), and spatial transcriptomics data from JAX and MorPhiC. We will validate published methods for defining tissue-specific GRNs by overlapping them with relevant MorPhiC model systems and will then use them to predict TF-GTs in systems yet to be profiled by MorPhiC. Our Aims will bolster the field's ability to decipher the regulatory function of the ~ 1600 TF genes within the human genome.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Type 1 diabetes (T1D) is often accompanied by other autoimmune disorders, including autoimmune neuropathies. Findings in both NOD mice and patients have revealed potential overlap between immune responses targeting pancreatic b-cells and nerves. We hypothesized that lymphocyte populations involved in T1D pathogenesis targeting proteins co-expressed in the nervous system may be the earliest responders causing initial damage to peripheral nerves. These first responders provide the necessary trigger to expand immune responses against myelin and other nervous system components traditionally studied in existing mouse models of autoimmune neuritis. A vast majority of islet-infiltrating B-lymphocytes in NOD mice respond to the nervous-system protein peripherin. Antibodies against phosphorylated peripherin have been identified in T1D patients. We recently created a new mouse model (NOD-PerIg) in which B-lymphocytes transgenically express the immunoglobulin molecule from the peripherin-reactive B-cell clone H280 isolated from islets. T1D is accelerated in NOD-PerIg mice. T-cells from NOD-PerIg, but not NOD mice, transfer an autoimmune neuritis similar to chronic demyelinating polyneuropathy (CIDP) to NOD.scid recipients. This new NOD-PerIg à NOD.scid model of T1D-associated autoimmune neuritis provides an experimental system to directly dissect the discreet stages of nerve cell infiltration and damage. We originally hypothesized that insulitis expanded T- cells capable of causing neuritis. However, we have recently determined that T-cells derived from islets or sciatic nerves in primary NOD.scid recipients are only capable of infiltrating the organ from which they were derived. Therefore, experiments in Aim 1 will address the current unknowns regarding the discrete temporal and spatial steps leading to cellular recruitment into peripheral nerves. We have also found that in addition to the presence of IFNg and TNFa producing CD4+ T-cells, there is paradoxically an expansion of T-cells negative for traditional Th1, Th2, and Th17 cytokines within sciatic nerves. An additional open question is whether peripherin remains the antigen towards which T-cells are responding or whether there has been an expansion of responses against other neuronal antigens. Therefore, studies in Aim 2 will dissect the mechanisms by which T-cells destroy peripheral nerves and how an immune response against one shared b- cell/neuronal antigen can spill over to a wider neuronal response. Finally, in large NOD colonies, spontaneous clinical neuritis can be occasionally observed. We have found that at baseline, NOD mice (around the age of T1D onset) already have sciatic nerve infiltrating T-cells. These T-cells are not observed in non-autoimmune prone C57BL/6 mice. This indicates the likelihood that NOD harbors genetic loci contributing to spontaneous nerve infiltration that can lead to spontaneous neuritis. Whether these loci are distinct or overlapping with loci contributing to T1D is unknown. Therefore, studies in Aim 3 will map genes associated with spontaneous nerve infiltration allowing future genetic screening of patients at risk for developing autoimmune neuropathy.

NIH Research Projects · FY 2026 · 2023-09

PROJECT SUMMARY / ABSTRACT Type 2 diabetes (T2D) results when pancreatic islet β-cells fail to secrete sufficient insulin to meet peripheral insulin demand. Mitochondrial bioenergetics is central to the (patho)physiology of β-cell (dys)function, and recent work suggests that β-cell mitochondrial dysfunction precedes the development of T2D in β-cells from donors with impaired glucose tolerance (or pre-diabetes). Mitochondrial defects have been reported in the β-cells of human T2D patients, but the etiology of mitochondrial dysfunction in T2D is unknown. Such mechanistic knowledge is necessary to guide strategies to prevent or treat islet failure and T2D. Importantly, genome-wide association studies (GWAS) link single nucleotide polymorphisms (SNPs) in >500 genetic loci to T2D and islet dysfunction-related metabolic traits. The majority of these SNPs are non-coding and overlap regulatory elements (REs) with broad transcriptional implications for affected cells. In this study, we combine our expertise in the genomics of T2D, (epi)genomic modification, and mitochondrial function in β-cells to bridge the gap from genomic association to mechanistic understanding. We hypothesize that non-coding T2D SNPs cause β-cell dysfunction by altering RE use or activity, thereby changing expression of effector genes that directly impair mitochondrial health. To test this, we propose to use sophisticated (epi)genomic editing tools in human islets and β-cell specific mouse models for physiological relevance and validation in two complementary Aims. In Aim 1, we will test RE– effector gene links in human islets using CRISPR-QTL. In parallel, we will assess T2D risk allele effects on RE chromatin accessibility, activity, transcription factor binding, and β-cell expression of putative mitochondrial T2D effector genes using complementary in vivo (single cell chromatin accessibility, histone acetylation, and expression quantitative trait locus analysis of primary human islets) and in vitro (reporter gene, DNA-binding assay) approaches. Finally, we will determine the consequences of effector gene perturbation on mitochondrial phenotypes, β-cell viability, and insulin content and secretion in human islets and EndoC-βH3 cells. In Aim 2, we harness β-cell-specific knockout mouse models to assign function to two high-priority mitochondrial T2D effector genes in glycemic control, β-cell mass/function, and mitochondrial metabolism. Further, we will address the importance of these mitochondrial T2D effector genes for β-cell compensation to peripheral insulin resistance following diet-induced obesity. Finally, we will use (epi)genomic editing tools in human islets to determine if mitochondrial T2D effector genes impair β-cell function and glycemic control in ex vivo assays as well as after islet transplantation into immunodeficient mice. Completion of this study will generate new variant-to-function connections that assign molecular and cellular functions to T2D risk alleles, identify novel therapeutic targets, and provide important knowledge to guide subsequent strategies to prevent or treat β-cell failure and T2D.

NIH Research Projects · FY 2026 · 2023-09

PROJECT SUMMARY KAND (KIF1A-associated neurological disorder) is caused by mutations in the KIF1A gene - a microtubule- dependent motor protein that is responsible to transport cellular cargos in neurons. The majority of mutations are dominant missense mutations that cluster in the conserved motor domain of the protein and lead to a spectrum of neurological phenotypes beginning in childhood, including muscle weakness, microcephaly, peripheral neuropathy, intellectual disability, autism, optic nerve and cerebellar atrophy. Without treatment, children and adults affected by KAND suffer from the progressive loss of their mobility, vision and even early death due to intractable epilepsy and complications of respiratory illness. Mouse models are a critical component to both understanding disease mechanisms and to serve as a key platform for preclinical testing of novel therapeutics. Unfortunately, mouse models to advance our understanding of KAND biology and therapeutics are severely lacking, although very much in reach. This proposal aims to build on our current knowledge of KAND to design mouse models that will not only provide patient avatars for KAND disease pathophysiology but will also serve to address important questions around the timing of therapeutic rescue, effects of overexpression, what cell types are required for effective treatment and how much genetic correction is required for disease modulation. Importantly, the models will be valuable for pre- clinical testing of therapeutics. The work described in this proposal leverages recently published Natural History Studies by our clinical collaborator, Dr. Wendy Chung, which provides ongoing insight into the clinical features, prevalence and biomarkers associated with this patient community. Our overall goals are to provide the scientific community with well designed, rigorously tested mouse models that recapitulate key aspects of KAND disease manifestations to be used, without restriction, throughout academia and industry for research and therapeutic discovery.

NIH Research Projects · FY 2026 · 2023-09

PROJECT SUMMARY/ABSTRACT Aging is the greatest risk factor for cancer, but it is not known which age-dependent cellular and molecular events drive cancer initiation. Spatial transcriptomic approaches are revolutionizing our understanding of cancer initiation, progression, and drug resistance by revealing expression patterns with tissue morphological context. However, these approaches have not yet been applied to the interdisciplinary biology of aging-driven cancers, despite the likelihood that intratissue and microenvironmental evolution mediate the aging phenotype. Moreover, the majority of current single cell spatial projects are based on 3' short read RNA-sequencing (RNA-seq) and therefore lack the ability to detect full-length spliced isoforms, which are frequently observed in tumors and are known to impact tumor initiation and treatment response. Work from us and others has revealed widespread alterations in alternative RNA splicing in human tumors, including in breast cancer, and that half of all spliced isoforms detected in human breast tumors using long-read sequencing (LR-seq) are missed by RNA-seq and absent from reference transcriptomes. In addition, we have causally linked the upregulation of specific splicing factors with breast tumor initiation both in vitro and in vivo and identified age-dependent changes in spliced isoforms in cancer-associated genes in mammary epithelial cells. Together, these results suggest that alternative splicing is a critical mechanism underlying tumor initiation with age, and that spatial LR-seq approaches are required to resolve these mechanisms. However, standardized approaches and resources to measure, quantify, and visualize expression of full-length isoforms within tissues are lacking. This gap in infrastructure impedes the field's ability to identify cell populations that express age-dependent isoforms, how such isoforms impact cancer initiation for example through changes in receptor-ligand interactions. To address these infrastructure and knowledge gaps, we will first develop approaches to map and analyze full-length RNA isoforms spatially within tissue sections (R21 phase, Aim 1). These tools, which merge LR-seq and spatial transcriptomics, will be applicable across sample types and will therefore be of broad, sustainable utility to the research community. We will then apply these technologies to generate a spatial map of full-length RNA isoforms in healthy breast tissues and tumors during aging (R33 phase, Aims 2 and 3). Finally, we will develop data sharing and visualization tools for spatial isoform expression to enable others to mine our data via a web resource (R33 phase, Aim 4). To achieve these goals, this project will leverage the complementary and interdisciplinary expertise of the Anczukow lab in alternative splicing and breast cancer, and of the Chuang lab in systems biology and spatial transcriptomics analysis. In response to NOT-CA-22-002, Notice of NCI's Participation in PAR-20- 070, this project will deliver a spatial transcriptomic infrastructure for isoform profiling and a critical interdisciplinary data resource for aging and cancer researchers to understand the role of splicing in tissue aging and oncogenesis, thereby advancing approaches for cancer early detection, intervention, and prevention.

NIH Research Projects · FY 2025 · 2023-09

Project Summary: Rafiou Agoro, PhD is a molecular and cellular biologist whose overarching career goal is to identify promising therapeutic targets relevant for the prevention/treatment of chronic kidney disease (CKD). The proposed research in this K99/R00 application aims to identify novel pathways involved in the control of renal oxidative stress with translational applicability on halting CKD progression and improving patient outcomes. Candidate: Dr. Agoro completed a PhD in Immunology at Orléans University (France) followed with a fellowship at NYU before joining Dr. White’s lab at Indiana University School of Medicine (IUSM) as a postdoctoral fellow. Dr. Agoro’s previous work identified iron metabolism and inflammatory mechanisms involved in tuberculosis, asthma, and CKD pathogeneses giving him the strong background knowledge required to conduct the proposed research. In addition, Dr. Agoro outlined a career development roadmap in building skills in bioinformatics during the K99 phase with a vision of leveraging novel technologies to understand the pathogenesis of CKD as an independent investigator. Dr. Agoro proposes four career goals during the K99/training phase: 1) To master the scATACseq analytic pipelines; 2) To generate conditional mouse models; 3) To successfully find a faculty position and 4) To develop leadership and professional skills in communication. Further Dr. Agoro will undergo training activities that include didactic and experiential learning to enable him to gain the necessary skills for genomic data analyses. Mentors/Environment: Dr. Agoro and his primary mentor, Dr. White, PhD, have assembled a strong team formed with a co-mentor, collaborators, advisor, and consultant to assist Dr. Agoro through the proposed training, research activities, and faculty job search. The proposed career development plan will utilize the intellectual and bioinformatics resources at IUSM. In addition, Dr. Agoro will attend national meetings, as well as seminars/courses and workshops locally. Research: CKD is an important public health epidemic affecting approximately 37 million Americans. CKD disease progression is associated with a graded increase in oxidative stress driving highly adverse complications. This proposal will decipher novel pathways involved in renal stress control via the following specific aims: Aim 1 will identify the mechanisms by which Klotho-dependent FGF23 signaling regulates HMOX1. In Aim 2, Dr. Agoro will test the role of Klotho and Hmox1 in CKD pathogenesis with a specific focus on renal oxidative stress, iron metabolism and mitochondria function. Summary: The proposed research will profile the genome-wide chromatin accessibility of renal proximal tubule in Klotho-transgenic vs WT mice and study the effects of Klotho-dependent FGF23 signaling on Nrf2 binding to ARE elements. Dr. Agoro will also determine the role of the FGF23-Klotho-Hmox1 axis on renal oxidative stress during CKD. In sum, this comprehensive plan will provide Dr. Agoro with the training needed to conduct independent research using genomic and transcriptomic approaches to improve CKD patient outcomes.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY KAND (KIF1A-associated neurological disorder) is caused by mutations in the KIF1A gene - a microtubule- dependent motor protein that is responsible to transport cellular cargos in neurons. The majority of mutations are dominant missense mutations that cluster in the conserved motor domain of the protein and lead to a spectrum of neurological phenotypes beginning in childhood, including muscle weakness, microcephaly, peripheral neuropathy, intellectual disability, autism, optic nerve and cerebellar atrophy. Without treatment, children and adults affected by KAND suffer from the progressive loss of their mobility, vision and even early death due to intractable epilepsy and complications of respiratory illness. Mouse models are a critical component to both understanding disease mechanisms and to serve as a key platform for preclinical testing of novel therapeutics. Unfortunately, mouse models to advance our understanding of KAND biology and therapeutics are severely lacking, although very much in reach. This proposal aims to build on our current knowledge of KAND to design mouse models that will not only provide patient avatars for KAND disease pathophysiology but will also serve to address important questions around the timing of therapeutic rescue, effects of overexpression, what cell types are required for effective treatment and how much genetic correction is required for disease modulation. Importantly, the models will be valuable for pre- clinical testing of therapeutics. The work described in this proposal leverages recently published Natural History Studies by our clinical collaborator, Dr. Wendy Chung, which provides ongoing insight into the clinical features, prevalence and biomarkers associated with this patient community. Our overall goals are to provide the scientific community with well designed, rigorously tested mouse models that recapitulate key aspects of KAND disease manifestations to be used, without restriction, throughout academia and industry for research and therapeutic discovery.

NIH Research Projects · FY 2025 · 2023-08

Healthy centenarians carry protective variants that counteract age-related disease risk variants, the former of which are mostly rare. Therefore, markers associated with exceptional longevity (EL) need be discovered through integrative multi-omics data analysis to improve detection power. However, existing integrative analysis method for multi-omics data do not model the relationships among markers in a modality and among studies, muddying the efficient use of pertinent information provided by multi-omics data from heterogeneous studies. We propose a unified AI strategy that models the relationships among markers, modalities, and studies, and learns nonlinear low-dimensional representations of data in a common space via graph neural networks (GNN). We achieve deep integration by enforcing the maximization of similarities between study representations and the phenotype prediction accuracy in a single GNN. The proposal has three specific aims: 1) Develop an explainable unified AI strategy and software for efficient and robust integrative analysis of multi-omics data from highly heterogeneous multiple studies. 2) Apply the methods developed in Aim 1 to Long-Life Family Study (LLFS) and Integrative Longevity Omics (ILO) data provided by the EL consortium to identify EL-associated pathways and biomarkers. 3) Apply the methods developed in Aim 1 to omics data from human and 100 species of diverse lifespan provided by the EL consortium to identify conserved and species-specific EL-associated pathways and markers. The outcome of this work will result in a publicly available integrative omics data analysis software which not only is able to identify robust longevity-associated pathways and biomarkers, but will also be applicable to any complex disease study with similar omics data analysis demands. Our work will contribute significantly to identify therapeutic interventions for improving human health.

NIH Research Projects · FY 2025 · 2023-08

Influenza viruses are rapidly mutating RNA viruses and are the causative agent of about one billion annual respiratory virus infections and 500,000 deaths worldwide. Influenza-related deaths are generally attributable to viral or bacterial pneumonia (from secondary bacterial infections); excessive inflammation resulting in acute respiratory distress syndrome; and severe lung immunopathology, leading to hypoxia and multi-organ failure. Influenza viruses have significant pandemic potential, seasonal epidemics burden the human population, and viral resistance has developed to all available treatment options. Much emphasis is placed on the humoral immune response to influenza, as neutralizing antibodies are the desired vaccine outcome. However, B cell- deficient mice and humans with hyper-IgM syndrome clear influenza virus infections, while T cell-deficient mice do not. Thus, B cell-independent mechanisms protect against influenza virus-related mortality. However, the immune response to influenza virus infection remains poorly understood, and much-needed therapeutics augmenting the antiviral immune response while preventing harmful immunopathology remain to be developed. To address this knowledge gap, we recently generated novel and compelling evidence that Influenza A virus (IAV) infection triggers lung mast cells (MCs) to produce the anti-inflammatory cytokine IL-10 (MC-IL-10). In wild- type (WT) and T- and B-cell deficient (Rag1-KO) mice, IAV/MC-IL-10 induces the expression of the IL-10 receptor (IL-10R) and programmed cell death ligand 1 (PD-L1) on Natural Killer (NK) cells. Notably, in Rag1-KO mice, where NK cells are the sole virus-fighting lymphocytes, PD-L1 blockade, but not PD-1, PD-L2, or CD80 blockade, significantly reduces IAV-related lethality. The IAV/MC-IL10/NK-PD-L1 pathway is also conserved in humans, at least in vitro: IAV infection of human-lung tissue-derived single-primary-cell suspensions or intact human lung tissue slices elicit MC-IL-10 and NK cell-expressed IL-10R and PD-L1. In mice and humans, T cells also upregulate the IL-10R, PD-1, and PD-L1 upon IAV infection. Further, IAV-infected IL-10-KO/Rag-WT mice, whose NK and T cells do not upregulate IL-10R, PD-1, PD-L1, or PD-L2, and IAV-infected WT mice in which PD-L1 is blocked, develop prolonged immune infiltration and immunopathology after IAV clearance. Our findings are novel and surprising. The induction of the PD/PD-L pathway is generally associated with lymphocyte exhaustion (via T cell-expressed PD-1) in cancer or chronic infection rather than the modulation of lymphocyte function in response to an acute viral illness. We hypothesize that influenza virus-induced MC-IL-10 balances helpful antiviral responses with harmful immunopathology through PDL1 signaling in NK cells, and PD-1 and/or PD-L1 signaling in T cells. We propose identifying the mechanisms of IAV/MC/IL-10/PD-L1-mediated NK cell and IAV/MC/IL-10/PD-1 and/or PD-L1-mediated T cell regulation and each pathway's contribution to viral clearance vs. lung tissue damage. Our proposal is highly significant to human health, as it has great potential to identify therapeutic targets for alleviating IAV immunopathology-associated mortality and morbidity.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY This proposal will address the mechanisms underlying neuromuscular degeneration in Charcot-Marie-Tooth disease (CMT). CMT is a genetically and phenotypically heterogeneous neuromuscular disorder with causative mutations found in over 100 genes. While considered a rare disease, CMT is the most common inherited disorder of the peripheral nervous system, affecting ~1 in 3,500 people worldwide. Dominant mutations in 6 different tRNA synthetases (aaRSs) cause forms of CMT (aaRS-CMT), making them the largest family of CMT-associated genes. Each of these genes is involved in protein synthesis suggesting a common mechanism that leads to defects in protein production and ultimately CMT pathologies. Our recently published work uncovered a potential mechanism underlying aaRS-CMT. We found that mutant aaRSs inappropriately sequester tRNAs from the ribosome, which stalls ribosome function and activates an integrated stress response (ISR) via a sensor protein, GCN2. ISR activation causes two major cellular events: 1) shutdown of a major form of protein synthesis and, 2) upregulation of the transcription factor, ATF4, and its target genes. The relative contributions of each of these events is currently unknown. One goal of this project is to determine the role of ATF4 and target genes in the pathophysiology observed in aaRS-CMT. Preliminary results show that ATF4 overexpression is toxic to motor neurons and produces a CMT- like phenotype in mice, evidence that ATF4 could be a viable therapeutic target for aaRS-CMT. In Aim 1 we will manipulate ATF4 expression levels in validated mouse models of aaRS-CMT to determine whether the disease pathology is driven by decreased protein translation or by increased expression of the ATF4 gene. To advance toward therapeutic applications we need to establish that human motor neurons also activate the ISR in response to aaRS-CMT mutations. Therefore, in Aim 2 we will establish and validate human induced pluripotent stem cell (hiPSC)-derived motor neuron cultures which have been genetically engineered to model aaRS-CMT. We will also test therapeutic strategies in these human cell-based models. Interestingly, ATF4 expression is common in many different types of neurodegeneration. Therefore, in Aim 3, we will integrate data from ATF4 mice in Aim 1, and hiPSC-derived motor neurons in Aim 2 to identify common genes and cellular pathways involved in ATF4-mediated neurodegeneration. These hiPSC-based models will be a powerful tool to help identify and develop new targets or pathways for potential therapeutic interventions. This MOSAIC (Maximizing Opportunities for Scientific and Academic Independent Careers) Postdoctoral Career Transition Award to Promote Diversity will be supported by excellent career development resources and a mentoring team of globally recognized experts in CMT (R.W. Burgess) and human stem cells (M.F. Pera) at The Jackson Laboratory for Mammalian Genetics.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY/ABSTRACT The weaponization of Ultrapotent synthetic opioids (UPS) has made finding a novel reversal agent a priority. The current opioid response agent, Naloxone, is not as effective against UPS opioids and does not reverse one of its known effects, wooden chest syndrome. Our long-term goal is to define the biological basis of opioid overdose risk and promote the discovery of safe and effective agents that reverse fentanyl lethality. A key objective is determining the molecular mechanisms underlying individual variability to fentanyl toxicity using genetically diverse mice. In aim 1 we plan to identify genes and variants that modify the influence of Mcoln1 on acute UPS opioid toxicity. Mcoln1 was identified in a GWAS study of overdose risk, and preliminary data support a genetic knockout of Mcoln1 resulting in death more rapidly from morphine or fentanyl. We will create an additional CRISPR knockout of Mcoln1 on a more sensitive genetic background, NOD/ShiLtJ and compare it with the C57BL/6 knockout we have for response to the UPS opioid fentanyl. The LD50 will be determined for these strains using our piezoelectric respiratory depression detection system. We will also study respiratory mechanics and pulmonary and chest wall impedance in response to three doses of fentanyl. In another cohort of mice, they will be tested by plethysmography to acquire respiratory metrics such as tidal volume and minute ventilation. We will also collect arterial blood to measure blood gases of oxygen and carbon dioxide during the plethysmography session to monitor the response to fentanyl at that level. Finally, another cohort of naïve and fentanyl-treated mice will be dissected for brain stems. The pre-bötzinger complex will be identified and analyzed by single-nucleus RNA-Seq, comparing the cellular populations and differential gene expression across genotypes, sexes and treatments. In aim 2 we plan to identify the physiological, neural, and molecular mechanisms of variable fentanyl-induced toxicity and lethality among eight inbred mouse strains. These eight strains, which served as the foundation for the advanced mouse populations of the Collaborative Cross and Diversity Outbred mice, contain approximately 45 million SNPs segregating between them. We have determined that the LD50 for fentanyl varies > 150-fold across both sexes of the eight strains. As in aim 1, in aim 2 we will phenotype cohorts of mice to detect the diverse phenomena associated with UPS opioids, including Opioid- Induced Respiratory Depression (OIRD), Opioid-Induced Persistent Apnea (OIPA), Wooden Chest Syndrome (WCS), closure/collapse of the upper and cardiovascular/hemodynamic disturbances. This phenotyping will be coupled to identifying the cellular populations, through single nucleus RNA-Seq, within the brainstem pre- bötzinger region that varies across naïve and fentanyl-treated strains of both sexes of mice. The differentially expressed genes that define these populations will help us identify targets for therapeutic development associated with the different fentanyl lethality phenotypes.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY/ABSTRACT The retinal pigment epithelium (RPE) plays an important role in the eye transporting nutrients, ions, and water; and serving in absorption of light and protection against photooxidation, recycling of visual cycle components, and providing essential factors for the structural integrity of the retina. Collectively these support metabolic homeostasis and barrier function of the retina. Many RPE disease phenotypes are shared between humans and mouse models carrying mutations in cell- adhesion and extracellular matrix (ECM) molecules. Defining causative pathways and networks that are induced by mutations or pathogenic variants may provide therapeutic targets. Identifying druggable targets that act during the pre-symptomatic stage of the disease is particularly important to enable development of therapies that can prevent, delay onset, or decrease the severity of RPE-associated diseases, irrespective of the initial cause of the disease, before the pathologic changes become irreversible. The goal of this application is to identify shared pre-clinical and end-stage phenotypic and cellular effects of mutations in two genes, Ctnna1 and Lratd2, which are highly expressed in the RPE and lead to similar retinal defects. Mutations in CTNNA1 have been reported in patients with Pattern Dystrophy and Lebers Congenital Amaurosis (LCA), and LRATD2 has been associated with early AMD in a genome wide association study. Our approach is to use clinical, functional and biochemical tests to provide a deep characterization of the models and to analyze associated cellular changes using single-nuclear transcriptomics as well as spatial metabolomic/proteomic measurements. These phenotypic and genomics data will be jointly analyzed using computational methods to identify the shared pathways perturbed in these models. Our use of two models with shared pathologies will let us filter out mutation specific alterations and focus on the shared disease-causing pathways. Using our mouse resources, we also plan to enhance current models. Successful completion of our studies will identify novel pathogenic pathways underlying RPE-related disorders, revealing potential therapeutic targets that could be effective in a broad range of these diseases, regardless of the cause of the disease. These well-characterized models will be made available to the research community for further mechanistic inquiries as well as for testing therapeutic strategies.

NIH Research Projects · FY 2025 · 2023-05

PROJECT SUMMARY OVERALL While the development of high-throughput sequencing technology and its application to clinical diagnostics has yielded the genetic basis for many rare genetic diseases, the development of effective treatments has not kept pace. Although gene replacement and modulation therapies can be powerful, sometimes even lifesaving treatment options, they come with many risks, such as immunogenicity and oncogenicity. Programmable nucleases such as CRISPR/Cas9 have revolutionized our ability to manipulate the genome, and provide the potential to achieve lasting, precise genome modification for therapeutic benefit. The proposed U19 program seeks to address these challenges through the development, validation and translation of gene editing– based therapeutic solutions for rare neurological genetic diseases. We propose to focus on four neurological conditions that each represent a significant unmet clinical need: Spinal Muscular Atrophy, Friedrich's Ataxia, Huntington's Disease, and Rett Syndrome. Members of our team have developed a suite of base and prime genome editing tools that can install precise alterations without creating a DSB or requiring a donor template. We also have developed validated in vivo mouse models for each of these diseases and bring deep expertise in the IND-enabling preclinical evaluation of gene-editing therapeutics. We propose to merge these considerable assets with disease-specific expertise in each of the four neurological conditions, supported by expertise and resources for scaled production of AAV-based delivery vectors for delivery of precision gene- editing therapies to tissues, and for navigating the regulatory path to IND submission. The proposed U19 team has a track record of individual and collaborative success at every step of the preclinical pipeline pathway and is thus well positioned to achieve our milestones, which include an IND package submitted to FDA for at least one therapy and neurological condition. Our Overall Aims are to: 1) Assemble a multi-disciplinary team with unique strengths and expertise to develop and implement innovative genome editing strategies to address important disease of the CNS, including Spinal Muscular Atrophy, Friedreich's Ataxia, Huntington's Disease, and Rett Syndrome; 2) Optimize lead base editor and prime editor candidates for each disease area, utilizing in vitro platforms and validated animal models; 3) Execute definitive preclinical in vivo pharmacology studies on optimized leads to develop reproducible efficacy data, while monitoring biodistribution, PK/PD, tolerability, and toxicology; and 4) Advance one lead candidate to an allowable investigational new drug (IND) application through coordinated communication with the FDA INTERACT program, the research project team, and the project Cores.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY/ABSTRACT The long-term goal of this work is to facilitate the faithful regeneration of damaged human tissues. Regeneration in adult mammals is extremely limited; damaged tissue in most major organs fails to regenerate, and instead undergoes scar-based repair. The lack of adult regenerative capacity is an enormous burden on the healthcare system and society as a whole. Although both human and mouse digit tips can undergo a true regenerative response, this regeneration is positionally restricted to the terminal distal phalanx bone. Importantly, amputations with an axis point below the terminal distal phalanx bone or too close to the nail bed result in regeneration failure. Notably, salamander limbs have an anatomy similar to that of human limbs, but uniquely regenerate after amputation from any position throughout adult life. The biological mechanisms limiting regeneration in adult mammals is poorly understood. Although the immune system is a powerful regulator of wound repair, the exact role of immune-cell networks as a determinant of regenerative success has been grossly understudied. In our regeneration studies, including those proposed here, we use the mouse digit-tip model, examining regeneration following tissue removal at different digit locations. This is a powerful model, as regeneration can be measured non-invasively with high-resolution micro-computed tomography 3D-imaging (bone/soft tissue volume), and analyzed comprehensively using histology and molecular analysis. We identified several lymphoid immune-cell types that inhibit mouse digit-tip regeneration via cytotoxic activity against progenitor cells and showed that T- regulatory cells (Tregs) play a critical role in protecting progenitors from these cells. We also found that in mice lacking lymphoid immunity, novel regeneration is induced, providing new models to identify pro-regenerative cells and molecular pathways that can be exploited therapeutically. Importantly, we also identified several lymphoid-cell types that support regeneration, suggesting the potential to therapeutically enhance human repair through targeted immunomodulation. This project aims to identify and characterize the mechanisms by which lymphoid cells regulate adult regeneration. Specifically, we will: Aim 1: Dissect and characterize lymphoid-cell mechanisms inhibiting regeneration. We will use a range of mouse strains with mutations in cytotoxic function in ex vivo and in vivo analyses. Aim 2: Define mechanisms of pro-regenerative Treg suppression of lymphoid-cell cytotoxicity using Treg-specific deletion of functional genes in vivo. Aim 3: Test the hypothesis that targeted disarming of lymphoid cells could enhance regeneration in vivo. We will test tolerogenic molecules in ex vivo cytotoxicity assays and then evaluate tolerogenic antigen overexpression in vivo both direct transgenic and viral approaches and then via a modified Treg delivery strategy. This project will fuse developmental biology and immunological methods to identify the critical biological pathways and genetic modifiers required for transient immunomodulation strategies directed at inducing latent regenerative potential in adult tissues in mammals. This work will lay the groundwork for translation studies aimed at enhancing tissue repair in human patients.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY Exercise slows the cognitive declines associated with aging and protects against the development and progression of neurodegenerative diseases such as Alzheimer's disease (AD). At the cellular level, exercise enhances synaptic connectivity and reduces markers of neuroinflammation in aging cortical circuits. Exactly how exercise signals in the brain generate these neuroprotective effects remains unknown. Our preliminary experiments have identified a set of neurons in the mouse ventromedial hypothalamus (VMH) expressing Steroidogenic Factor 1 (SF-1) that robustly increase their activity in response to exercise. We have found that the VMH SF-1 neural activity signal is potentiated severalfold following repeated exercise, suggesting that the exercise signals generated by VMH SF-1 neurons are plastic and shaped by experience. Furthermore, we have found that direct stimulation of SF-1 neurons substantially increases subsequent endurance capacity, suggesting VMH Sf-1 neurons are an important neural node controlling the physiological benefits of exercise. However, several important questions remain unknown. First, which features of VMH SF-1 neurons enables plasticity of activity signals following repeated exercise? Second, which specific sets of VMH SF-1 output neurons transmit exercise-relevant signals? Last, is it possible to stimulate VMH SF-1 neurons and generate the neuroprotective effects of exercise on cognition and neural circuitry in the aging brain or in AD-like states? The proposed experiments will leverage advanced neuroanatomical and neurophysiological tools with preclinical genetic models to gain insights into these questions. In Aim 1, we will pair large-volume, high-resolution, and cell-type specific array tomographic neuroanatomical reconstructions with in vivo calcium imaging and neuronal activity perturbations to determine how exercise shapes the synaptic architecture of VMH SF-1 neurons. These experiments will define how changes in the synaptic inputs to these neurons might physically `store' exercise history within VMH circuitry. In Aim 2, we will use advanced viral mapping and in vivo single-cell functional imaging techniques to identify which neurons are activated by exercise and understand how these exercise signals are transmitted to specific circuits downstream of the VMH. These experiments will define the organization and logic by which exercise-related activity in VMH neurons drives functional changes in the brain. In Aim 3, we will take advantage of advanced preclinical genetic mouse models of early- and late-onset AD to determine whether stimulating activity in VMH neurons might recapitulate the neuroprotective effects of exercise observed in cortical circuits. These experiments will increase our understanding of how signals in the VMH could be harnessed for therapeutic manipulation in disease states. By leveraging the synergistic expertise of the team of investigators assembled to address this problem, insights from these experiments will advance our fundamental understanding of how the beneficial effects of exercise are mediated by specific synapses, cell- types, and circuits, and whether these features are potential therapeutic targets for intervention in disease states.

NIH Research Projects · FY 2025 · 2023-01

PROJECT SUMMARY/ABSTRACT The goal of The Jackson Laboratory (JAX) Diversity Action Plan (DAP) post-baccalaureate program in genomics is to provide a transformative, mentored research experience for individuals from underrepresented groups with an emphasis on trainees whose goals are to pursue advanced degrees and careers in genome-related fields. Low participation in STEM (Science, Technology, Engineering, and Mathematics) by individuals from underrepresented groups is a recognized problem nationally (National Academies Press, 2011). Lack of exposure to scientific research resources and mentors, economic factors, and cultural biases are barriers that limit access to quality science education and, in turn, hinder the expansion of our nation's research capacity by limiting contributions from a deep pool of intellectual talent. The program will benefit from JAX's nine decades of success in training scientists at all career stages, including those from underrepresented groups, by its world-leading mouse genetic, genomic, and computational resources, and by its well-funded and productive faculty. The program will build upon programmatic infrastructure for career development and a community of post-bacc peers from an existing post-bacc program at JAX. The genomics DAP will be distinct from the existing post-bacc program as the focus for the proposed program will be specifically on career development for basic research in the areas of genomics, genome technology, and data science. The program will host up to six post-baccalaureate fellows in a year with individuals distributed across the Bar Harbor, Maine and Farmington, Connecticut JAX campuses. The three primary aims for the program will be (1) Provide two-year mentored post-baccalaureate research experiences in genomics, genome technologies, and data science for individuals from underrepresented minority groups; (2) Implement a curriculum that provides knowledge, skills, and training complementary to the mentored laboratory research experience and, (3) Incorporate formal and informal evaluation methods to strengthen the training program. Oversight for the program will be provided by two PIs, one from each campus, and by Program Staff from the JAX Genomic Education team. An Advisory Council comprised of internal and external individuals will evaluate the program's effectiveness and provide guidance on sustaining a research workplace that is inclusive and equitable. The alumni from the post-bacc program will contribute to building a research workforce that is representative of our nation's population. They will bring unique perspectives and individual enterprise to setting and advancing a research agenda in genomics and data science that benefits all people.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Understanding how combinations of genetic risk factors influence risk for late-onset Alzheimer’s disease (LOAD) can lead to targeted strategies for therapeutic intervention. Apolipoprotein E4 (APOE4), a common variant of APOE, is the single largest genetic risk factor for developing LOAD.APOE4 status is linked to increased inflammation and higher β-amyloid burden in LOAD patients. Despite this increased genetic risk profile, APOE4 carriers do not always develop LOAD in the course of their lifetime. Several large-scale genetic studies have identified a common haplotype of the aging factor klotho that modify age of onset and reduce amyloid plaque deposition specifically in APOE4 carriers, suggesting that klotho variants can provide a protective effect against the development of LOAD by counteracting the negative effects of APOE4. In humans, klotho harbors two common missense variants (rs9536314, p.F352V; rs9527025, p.C370S). The combination of these two coding variants define the klotho V/S (KL-V/S) haplotype, which is protective against LOAD in APOE4 carriers, and the klotho F/C (KL-F/C) haplotype, which is not protective against LOAD. The overall objective of this proposal is to determine the physiological processes altered by klotho as an APOE4-specific protective factor in LOAD using a set of recently-created mouse models harboring combinations of relevant human variants in both klotho and APOE. Our central hypothesis is that the protective KL-V/S haplotype will significantly delay age-dependent inflammation and amyloid deposition while the reference KL-F/C haplotype will fail to attenuate these hallmark LOAD pathologies. We will assess multiple LOAD-relevant outcomes to validate and characterize this klotho- APOE genetic interaction with three specific aims: (1) Determine the effects of klotho haplotypes on age-related frailty and klotho isoform levels in blood and CSF in mice; (2) Determine changes in LOAD hallmark pathologies driven by the interaction between klotho and APOE alleles in mice; and (3) Identify molecular signatures shared in human LOAD stratified by klotho haplotype and the novel klotho mouse models. The outcome of this work will result in the characterization of new mouse models of human klotho haplotypes and identify the pathways which are differentially affected by klotho variants in an APOE-dependent manner. This information will provide a biological basis for the epistatic interaction observed in human genetic studies, thereby providing the necessary functional information to guide potential treatments based on KL-V/S protection for APOE4 carriers.