Student External Award for Research Training Details

Ataxia telangiectasia (AT) is a rare neurodegenerative disorder caused by mutations in the ATM gene and is characterized by progressive cerebellar degeneration. Increasing evidence suggests that somatic mutations accumulating in individual neurons may contribute to selective neuronal vulnerability, yet the rate, spectrum, and functional consequences of these mutations in human brain tissue remain poorly understood. This project proposes to apply single cell genomic approaches to quantify somatic mutation rates in cerebellar neurons from individuals with AT compared to neurotypical controls. By leveraging high resolution single cell sequencing and computational analysis, the study aims to identify molecular mechanisms driving mutagenesis, determine how these mutations affect neuronal function, and assess their contribution to neurodegeneration. Results from this work will provide critical insights into how genomic instability impacts neural health and may reveal novel pathways relevant to neurodegenerative disease pathogenesis.

Opioid Use Disorder (OUD) is an ever-increasing public health concern. Despite available treatments, most patients relapse within the first few months. Therefore, there is a substantial need to understand the etiology of OUD and what mechanisms lead to increased opioid use and relapse. Notably, many patients with OUD have reported severe and persistent disruptions to sleep and circadian rhythms. Indeed, long-term opioid use is associated with increased sleep and circadian rhythm disruption, as well as intense cravings and negative affective states leading to vulnerability to relapse. Importantly, the Logan lab has shown that subregions of the striatum, specifically the nucleus accumbens (NAc), display altered molecular rhythms in OUD. Furthermore, preliminary data from the lab suggests that immune signaling in the NAc is altered due to OUD. Microglia, the primary innate immune cells in the central nervous system, have recently been implicated in OUD. Interestingly, microglia exhibit specific molecular rhythms that are dysregulated in OUD, suggesting a potential role for these cells in OUD-related sleep disruption. However, the role of microglial molecular rhythms in the NAc in OUD has yet to been fully elucidated. Thus, this proposal seeks to investigate the molecular rhythms of microglia in the NAc in OUD and how these mechanisms affect opioid-related behaviors. In the aims of this, I will use snRNAseq to characterize circadian rhythms in NAc microglia from human OUD samples and fentanyl-administering mice of both sexes. Additionally, I will disrupt molecular rhythms in microglia using cell type-specific conditional knockout (cKO) of Bmal1 in mice, a master regulator of circadian rhythm, to determine whether inflammatory gene expression and chromatin accessibility is altered. Furthermore, I will examine whether Bmal1 cKO mice display altered drug-taking behavior using fentanyl self-administration. Results from these studies will provide novel insights into microglial molecular rhythms across species and how they contribute to OUD-related behaviors.

Alpha-1 antitrypsin disease (AATD) is caused by mutations in the SERPINA1 gene, which encodes AAT protein. AAT is produced in liver and delivered via serum to lungs, where it inhibits neutrophil elastase. The most common AATD allele—called PI*Z—is a G-to-A mutation that produces a dysfunctional misfolded protein, Z-AAT, that aggregates in hepatocytes, which can cause liver disease; reduced serum AAT causes pulmonary emphysema. Currently, the only approved therapy for AATD emphysema is costly, weekly infusions of purified AAT for life. CRISPR/Cas9-mediated homology directed repair (HDR) can correct PI*Z in the liver and partially restore serum AAT levels in an AATD mouse model. Yet, HDR is limited by the need to deliver a DNA repair template, its inefficiency in non- and slow-dividing cells, and its generation of genotoxic double-strand breaks. By contrast, CRISPR-mediated adenine base editors (ABEs) support precise editing without requiring a DNA donor or double-strand breaks. ABE consists of adenine deaminase (TadA) conjugated to Cas9 nickase. When directed by a guide RNA to a specific sequence, ABE deaminates adenine in a defined editing window. The resulting inosine is read as guanosine, thereby converting A to G. Thus, ABE is a good candidate for PI*Z correction. Preliminary evidence shows that viral delivery of a compact ABE, utilizing an evolved Cas9 nickase derived from Neisseria meningitidis (eNme2-C ABE), to PI*Z transgenic mice leads to efficient editing of PI*Z in hepatocytes to significantly reduce liver disease. Yet, eNme2-C ABE deaminates not only the target adenine but also “bystander” adenines in the designated editing window, leading to mutations of unknown consequence. Moreover, the level of base editing needed to rescue lung disease is undetermined. This project seeks to optimize ABE precision for PI*Z correction and assess the therapeutic potential of ABE in treating emphysema in a mouse model of AATD. Aim 1 will characterize ABE off-target and bystander edits for PI*Z correction. TadA variants with distinct editing windows have been developed, including ABE8e and ABE9e. eNme2-C ABE8e and ABE9e edit both the target adenine and bystander adenines at the PI*Z target locus in PI*Z reporter cells. Off-target editing events by each variant will be detected and validated in PI*Z reporter cells and liver cells by deep sequencing and RNA sequencing. Bystander alleles generated by each variant will be identified, then in vitro approaches will be used to analyze the secretion and activity of each AAT bystander mutant. Aim 2 will characterize ABE-mediated PI*Z correction and lung function in AAT-null PI*Z mice, which exhibit both lung and liver disease. eNme-2 ABE will be delivered by AAV to AAT-null PI*Z mice, and pulmonary mechanics will be measured over 10 weeks. At endpoint, serum, liver, and lung tissue will be collected to measure serum AAT level, PI*Z correction and hepatocyte AAT aggregates, and alveolar morphometry. This proposal will inform the development of base editing strategies to treat AATD and provide the fellow with training in therapeutic genome editing and genetic disease biology.

Pancreatic cancer is highly metastatic, and patients are often not diagnosed until metastases have already formed, with the vast majority (>75%) presenting with oligometastatic disease. Current genetically engineered mouse models of pancreatic cancer are metastatic in 30-40% of animals and harbor only small focal metastases, complicating the study of metastatic drivers in mice. Preliminary experiments identified Calcium/calmodulin- dependent protein kinase II beta, Camk2b, as a gene whose loss enhances tumor metastasis and creates a highly immunosuppressive tumor microenvironment (TME). Genetically engineered mice with tumor cell-specific Camk2b knockout form metastases in 80% of animals and present with >25 metastatic lesions per mouse. Metastatic burden is so profound in this model that the animals succumb to disease twice as fast as control tumor bearing mice. Preliminary results, show that deletion of Camk2b results in tumors that are more metastatic and express higher levels of Lysyl Oxidase (Lox). Aim 1 will focus on exploring tumor cell intrinsic mechanisms through which Camk2b-deletion promotes tumor cell metastasis. The metastatic cascade typically begins by the modulation of tumor cell epithelial identity through programs such as epithelial-to-mesenchymal (EMT). Thus, Subaim 1.1 will investigate the effect of Camk2b loss on tumor cell epithelial cell identity using isogenic cell lines, genetically engineered mouse model, and patient-derived organoid systems. Subaim 1.2 will investigate the functional contribution of LOX on metastatic competency and the ability of LOX-targeting to block metastatic outgrowth. Preliminary experiments indicate that deletion of Camk2b in tumor cells shifts the immune milieu towards a pro- tumor state that is more permissive to tumor metastasis. In this regard, Camk2b-null tumor cells alter their local microenvironment and confer an immune desert phenotype that may facilitate tumor growth and metastasis. To explore mechanisms of this immunosuppressive phenotype, Aim 2 will test the contribution of the immunosuppressive microenvironment in Camk2b-null tumors on metastasis. Subaim 2.1 will selectively deplete macrophages in Camk2b-deletion tumors and evaluate the impact on the metastatic niche and gross tumor cell metastasis. Subaim 2.2 will investigate the contribution of Tbc1d9, a calcium-responsive gene activated in Camk2b deleted tumor cells, to suppression of the NK and T cell response. Collectively, our work will demonstrate that Camk2b is a metastasis suppressing gene whose loss activates pro- metastatic signaling networks. Our functional and mechanistic work will define targetable downstream signaling nodes, within tumor cells and in the microenvironment, to block immunosuppression and metastatic outgrowth.

Project Summary Mutations in genes encoding proteins that are essential for cell processes such as DNA replication can lead to cellular dysfunction and disease. Protein defects can perturb these cell processes by removing or surpassing cell cycle checkpoints, leading to errant growth and proliferation of cells, defining characteristics of cancer. As such, it is essential to investigate Proliferating Cell Nuclear Antigen (PCNA), the central player that coordinates DNA replication, DNA repair, and cell-cycle regulation. PCNA, also known as the sliding clamp, is a homotrimeric ring that slides along DNA to facilitate interactions of over 100 known proteins, many involved in cancer development and other important cellular processes. The sliding clamp is conserved across all life forms, providing insight into the evolution of DNA replication and cell-cycle machinery. Thus, PCNA is an ideal target to investigate mutational effects on protein function and the long-term impacts on the cell. In Aim 1, we will address an interesting paradox related to PCNA. Point mutations in PCNA that result in subtle biochemical effects cause severe disruption of organism fitness, suggesting that PCNA is especially sensitive to mutations. Conversely, PCNA genes across evolution are widely varying in sequence suggesting that PCNA is actually accepting of mutations. To investigate this contradiction, we will perform a mutational scan of all potential point mutations in the yeast PCNA protein. These mutants will then be exposed to DNA-damaging agents to assess the effects of the PCNA mutants on various PCNA functions. I predict that a mutational screen of PCNA will show mutational effects on cell viability and DNA damage response based on residue location in PCNA providing insight into the acceptability of point mutations in PCNA. This data will also provide insights into potential disease mutations that could impact human PCNA. The Kelch lab has previously investigated two disease-associated germline mutations in PCNA. These mutations lead to PCNA-associated DNA repair disorder (PARD), characterized by UV sensitivity, neurodegeneration, premature aging, and, most notably, the development of skin cancer. In Aim 2, we will investigate how patient-associated mutations in PCNA affect biochemical and cellular function. I selected variants based on association with cancer or PARD. I will establish mutant human retinal pigment epithelial (RPE1) cell lines using CRISPR/Cas9 techniques. Once these cell lines are established, I will assess the cellular impacts by using flow cytometry and DNA-damage assays. I will compare these results with tests of the biochemical functions using isothermal titration calorimetry and thermal shift assays. I predict that the mutations will exhibit defects in thermostability, cell regulation, and DNA repair. The impact of this study is two- fold. First, the study will enhance our understanding of how PCNA function and evolution are intertwined. Second, the study will investigate select human PCNA mutants that can inform cancer diagnosis and provide a framework for investigating other proteins.

Amyotrophic lateral sclerosis (ALS) is a devastating degenerative motor neuron disease that is largely untreatable and leads to death within 5 years of diagnosis. ~10% of ALS cases are familial and caused by mutations in various ALS genes. Ultimately, the ideal treatment for genetic diseases such as ALS is somatic gene correction. Recently, advances in CRISPR/Cas systems have shown considerable promise for precise editing of disease loci using base and prime editing systems delivered by AAV. The second-most prevalent cause of familial ALS are mutations in the SOD1 gene. These mutations confer multiple toxic properties onto the protein. This project proposes to develop treatment to achieve somatic gene correction for common missense mutations in SOD1. The aims of this proposal are: (1) To develop AAV-mediated base editing gene correction strategies for the SOD1 A5V mutation in vitro. We will create next-generation base editors with a compact size, increased efficiency, and greater control over bystander editing. (2) To develop AAV-mediated prime editing gene correction strategies for the SOD1 A5V and G94A mutations in vitro. Different prime editor systems will be tested for optimal editing efficiencies and low off-target editing. (3) In in vivo studies, examine and optimize AAV- mediated base and prime editing gene correction strategies for the A5V and G94A mutations in A5V and SOD1G93A mouse models. Mice will receive AAV-mediated base and prime editors through an intracerebroventricular injection. Base and prime editor strategies will first be screened in mutation carrying HEK293T cells and then optimized in patient fibroblasts and mouse models. The effects of gene correction on gain- and loss-of-function molecular and motor phenotypes will next be evaluated. The fundamental hypothesis driving this proposal is that AAV-mediated somatic gene correction strategies, using base editing or prime editing to target the SOD1 mutations A5V and G94A, will decrease toxic GOF pathology and increase WT SOD1 protein levels in vivo, resulting in a balanced treatment for SOD1-ALS and a rescue of motor phenotype. In addition, with mentorship from experts in ALS and gene editing and the wealth of resources available at UMASS Chan, these studies will provide extensive training in gene editing for CNS diseases and project development that will be an essential foundation for a future career as an independent researcher developing gene therapies for a range of genetic CNS diseases.

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is a cytosolic double- stranded DNA (dsDNA) sensing pathway critical for regulating immune homeostasis. A series of gain-of-function (GOF) mutations result in constitutive activation of STING, causing an autoinflammatory disease called STING- Associated Vasculopathy with Onset in Infancy (SAVI). SAVI patients succumb to treatment resistant inflammatory lung disease and respiratory failure. There is little known about the mechanisms by which inflammation occurs. To address the urgent need to develop safe and effective therapies, we have developed a murine model for the most common STING gain-of-function mutation, STINGV154M/WT (VM). These mice recapitulate the lung inflammation exhibited by human SAVI patients. To identify the specific cell types involved in causing lung inflammation, we developed a novel VM conditional knock-in (CKI), allowing specific targeting of the VM mutation to different cell types. We demonstrated that endothelial cell (EC) STING GOF is sufficient in driving bronchus-associated lymphoid tissue (BALT) formation. However, the mechanism of action remains to be elucidated. Moreover, we have previously described SAVI lung disease as independent of type I interferon (IFN) and IRF3, signaling proteins downstream of STING activation. STING activation leads to downstream signaling of other pathways including NF-κB and autophagy. The signaling mechanism causing lung inflammation is also unknown. Additionally, STING GOF in ECs was insufficient to cause the extent of lung inflammation seen in VM mice, suggesting STING GOF in cells other than ECs is required for lung disease. Upon ubiquitous VM expression, we find evidence of fibroblast activation in the lung tissue. Fibroblastic reticular cells (FRCs) are a subset of fibroblasts that define the function and structure of lymphoid organs such as BALT. In addition to ECs, STING is highly expressed in FRCs, yet the role of STING in FRCs and contributions to lung disease is unknown. Thus, we hypothesize that coordinated interactions between ECs and FRCs exacerbate SAVI lung autoinflammation, which is dependent on NF-κB activation. In this proposal, Aim 1 will investigate how STING GOF mutation in ECs initiates immune cell recruitment. Aim 2 will determine the synergistic effects of STING GOF mutation in ECs and FRCs on lung autoinflammation. We propose to utilize in vivo, ex vivo, and in vitro techniques to test our hypothesis. The studies proposed in this application will provide critical insights that will enable us to design the best therapies. Furthermore, these studies will provide an opportunity to study the impact of STING activation on stromal cell types, an area of research that requires further exploration. Our findings will discern the role of ECs and FRCs in VM lung autoinflammation and will broadly provide insight into stromal cell-driven mechanisms of other lung disorders.

Tuberous sclerosis complex (TSC) is caused by Tsc1 or Tsc2 mutations and can affect several organs, including the kidneys, leading to benign tumors or cyst formation. Impaired TSC1/2 protein function promotes hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1), an important cellular nutrient sensor that controls cell growth and proliferation. However, a much deeper mechanistic understanding of mTORC1 regulation and function in kidney cells is required. The mTORC1 kinase complex is activated by the essential amino acid sensors called Rag-GTPases. There are four Rag GTPases isoforms (RagA, RagB, RagC, and RagD), which localize to lysosomes and function in a heterodimeric complex where RagA/RagB binds to RagC/RagD. The Rag-GTPase complex positively regulates mTORC1 by recruiting it to the lysosomal surface in the presence of amino acids, where mTORC1 is subsequently stimulated by another small GTPase called Rheb. The activated mTORC1 complex signals to several substrates that collectively regulate cell metabolism, growth, and proliferation, key factors for developing kidney cysts. Importantly, all current models indicate that RagA/B loss will inhibit mTORC1 signaling by preventing its localization to the lysosome. Unexpectedly, we discovered that deleting RagA and RagB in kidney tubular epithelial cells causes a striking and progressive cystic phenotype resembling TSC. Moreover, RagA/B deletion in the kidney is associated with increased, rather than decreased mTORC1 signaling, as in TSC. This phenotype is not observed when we delete the mTORC1 subunit Raptor in the kidney epithelium. Thus, we hypothesize that RagA/B loss triggers kidney cystogenesis via a non-canonical Rag-GTPase pathway, involving TFEB regulation and possible cellular crosstalk for mTORC1 hyperactivation, which can be a common mechanism of TSC cyst formation. Since aberrant mTORC1 activation is a hallmark of TSC and other kidney cystic diseases, resolving this unexpected mechanism and the Rag-GTPases role in kidney cyst development may have important translational implications for improving upon current mTOR-based therapeutic strategies for the management of TSC and other kidney cystic diseases. In this proposal, I aim to (1) identify the cellular origin and metabolic traits underlying RagA/B deletion-induced kidney cystogenesis, and (2) determine the mechanism linking RagA/B loss and mTORC1 activation, TFEB regulation, and the similarities to the pathophysiology of TSC.

Mitochondria support multifaceted metabolic reactions in mammalian cells1. The vast majority of these metabolic pathways require the electron transport chain (ETC), a series of reactions in which ubiquinone (UQ) carries electrons to oxygen (O2) as the terminal electron acceptor (TEA). Recent studies have refuted this textbook model and have shown fumarate as a TEA2 through two mechanisms. First, under hypoxia, the reduced electron carrier ubiquinol accumulates driving complex II backwards to deliver electrons to fumarate. Second, in normoxic conditions, a novel mammalian metabolite rhodoquinone (RQ) can deliver electrons to fumarate3. Thus, the mammalian ETC is highly flexible and whether other electron acceptors beyond fumarate and O2 can be used has yet to be studied. The thermodynamic favorability of all reduction and oxidation (redox) reactions in the ETC are dictated by the Nernst equation. Electrons favorably transfer from metabolites with low to high reduction potential4. Thus, as RQ has a lower reduction potential than UQ, it can theoretically deliver electrons to other electron acceptors such as sulfites. Moreover, upon hypoxia exposure, UQH2 can build up enough to enable sulfites reduction as well. Oxidized sulfur-based molecules are known to act as TEA in certain sulfate-reducing prokaryotes5. The same molecules are known to interact with the mammalian ETC via enzymes sulfite oxidase (SUOX) and sulfide quinone oxidoreductase (SQOR) with cytochrome c and UQ, respectively. Importantly, SUOX functions in the cysteine catabolic pathway to oxidize sulfite (SO3-2) into sulfate (SO4-2), subsequently transferring electrons to cytochrome c6. Similarly, SQOR oxidizes hydrogen sulfide (H2S) to glutathione persulfide (S2O3-2) by transferring electrons to UQ6. Although these electron donor mechanisms (Fig.1) are well established, the reversibility of these reactions, enabling reduction of sulfate and glutathione persulfide as electron acceptors, have never been examined in mammals. We hypothesize that sulfate via SUOX and glutathione persulfide via SQOR can serve as TEA for UQ-dependent ETC circuits in hypoxic (low oxygen) environments, and RQ-dependent ETC circuits at any oxygen tension (Fig. 2). The proposed research will test the propensity for mammalian cells using the UQ- and RQ-directed ETC circuits to employ oxidized sulfur species as TEA. We will leverage directing UQ vs RQ circuits in vitro to measure reversibility of SUOX and SQOR activities in varying oxygen availabilities (Aim 1) and an established conditional mouse model to examine the tissue specificity of these enzymes and circuits (Aim 2). This work will add to the growing body of literature on novel mechanisms of flexibility in the mammalian ETC and will provide meaningful insights to mitochondrial function. [1]Monzel, Nat Met 2023 [2]Spinelli, Science 2021 [3]Valeros, Cell 2025 [4]Alberts, Mol Bio of the Cell., 2002 [5]Muyzer, Nat Rev Microbiol 2008 [6]Kohl, Br J Pharmacol 2019

Cardiovascular disease is the leading cause of death in the United States. Despite decades of research, it is unclear how the RNase A family nuclease angiogenin stimulates blood vessel formation, and why angiogenin dysfunction is associated with heart failure and poor cardiovascular health. Recent work revealed that angiogenin’s nuclease activity, which is required for its angiogenic function, is stimulated by binding to the ribosome, but it remains unclear whether angiogenin’s ribosome-dependent mechanism is involved in angiogenesis. A bacterial nuclease named ribocin with a strikingly angiogenin-like structure and ribosome- dependent activity holds similar potential for understanding lung health. Nearly all Cystic Fibrosis patients experience Pseudomonas aeruginosa bacterial pneumonia and subsequently suffer from lung tissue inflammation and lasting damage long after the infection has cleared. Ribocin encoded by P. aeruginosa damages human ribosomes specifically at central helix 69 of the 28S rRNA and inhibits translation. We hypothesize that like other ribosome-inactivating proteins, ribocin induces a ribotoxic stress response that causes inflammation and cell death in human lung tissues. To inform future therapeutic studies aimed at treating cardiovascular disease and post-infection pulmonary damage, the mechanisms of translation control by these RNase A-family nucleases must be elucidated in the context of their cellular functions. The goal of this project is to determine the structural basis of translation control by angiogenin during angiogenesis and determine the impact of translation control by ribocin on ribotoxic stress response and cell death. With guidance from the sponsor, an expert in biochemical and structural basis of translation, and collaborators, who are experts in the RNA developmental biology and cryo-EM method development, the trainee will apply cutting edge methods for in-cell cryogenic electron microscopy (cryo-EM) complemented by cell assay and biochemical approaches to visualize structural changes to actively translating ribosomes during angiogenin- stimulated vascularization and ribocin-mediated tissue damage. Aim 1 will determine the contribution of angiogenin’s ribosome-specific activity on tube formation (angiogenesis) in human umbilical vascular endothelial cells (HUVEC). Aim 2 will elucidate the structural mechanism of translation inhibition by ribocin and investigate the impact of selective ribosome damage by ribocin on ribotoxic stress response in human lung cells (IB3). The results of this study will reveal details of mechanisms underlying fundamental cardiovascular function and novel components of pulmonary disfunction, necessary for future work in developing therapeutics for cardiovascular and lung disease.