• Investigating the role of p16Ink4a in Alzheimer's Disease pathogenesis

  Holloway, Kristopher (2024-10-07)

  Advancing age is the most significant risk factor for Alzheimer’s Disease (AD), but how aging contributes to AD development remains unclear. Human induced pluripotent stem cell (iPSC) technology has revolutionized AD modeling, enabling the study of AD cellular pathogenesis in patient-derived cells. However, the epigenetic landscape and aging clock are reset to the embryonic state in iPSCs during reprogramming. Thus, current iPSC-based models lack proper cellular aging and do not fully recapitulate AD hallmark pathologies, including amyloid- beta plaques and neurofibrillary tau tangles, observed in human AD brains. My thesis aims to introduce factors of cellular aging to improve iPSC-based modeling of AD pathogenesis. I focus on CDKN2A (p16Ink4a or p16), an important senescence regulator and aging biomarker. I demonstrated that robust induction of p16 in iPSCs reduces cell proliferation and triggers downstream pathways, including focal adhesion and extracellular matrix organization. The inducible approach enables temporal control of p16 expression during the differentiation of iPSCs to neurons. I found that inducible p16 increases tau phosphorylation in iPSC-derived neurons in a cell-autonomous manner, providing a direct link between p16 and an AD-related cell pathology. This robust system provides a powerful resource to improve AD disease modeling to advance our understanding of the impact of aging in AD and develop innovative AD therapeutic drug discovery.
• 

  STING Gain-of-Function in Endothelial Cells Impairs Wound Healing Responses

  Chuprin, Jane Evelyn (2024-09-19)

  STimulator of Interferon Genes (STING) gain-of-function (GOF) mutations, resulting in constitutive STING activation, have been linked to a rare autoinflammatory disease called STING-Associated Vasculopathy with onset in Infancy (SAVI). SAVI patients present with hallmark skin findings, including chilblains (cold-sensitive lesions on acral surfaces) and progressive ulcerative lesions. We used a murine model of SAVI, STING(V154M/WT)-(VM), to explore the impact of the VM mutant on wound repair using ultraviolet B (UVB) irradiation as a tool for skin injury. Following UVB-induced injury, we found that VM mice developed exacerbated skin inflammation that persisted for 21 days or more. Conversely, WT mice developed mild erythema and erosion, which resolved within 7 days. Despite a strikingly different phenotype, total immune cell infiltration in VM skin was the same as WT within the first 5 days post-UVB irradiation. However, there were differences in the immune composition, including a significant lack of macrophage expansion during healing in VM skin. Further, we discovered that the VM phenotype is independent of T-cell responses and type 1 interferon signaling, challenging prior expectations in the literature. To identify the cellular driver(s) of skin disease, we used busulfan chimera and conditional knock-in mouse models. We determined that STING GOF in endothelial cells was sufficient to induce ulcerative lesions in VM mice. The critical finding in this thesis work is that following UVB-induced skin injury, STING GOF mutation in endothelial cells prevented macrophage expansion and impaired wound healing responses.
• 

  Mapping the ALS Citrullinome: A Proteomic Perspective on Neurodegeneration, Aggregation, and Protein Dysfunction

  Camille, Webb (2024-09-08)

  Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that is characterized by progressive motor neuron loss, muscle wasting, paralysis, and death. Ninety percent of the cases are sporadic (sALS), while the remaining ten percent are familial (fALS). A hallmark of ALS neuropathology is aberrant protein aggregation and inclusion body formation in neurons leading to eventual degeneration of motor neurons in the brain and spinal cord. Previously, we showed that protein citrullination (PC), a post-translational modification (PTM), and peptidyl arginine deiminase 2 (PAD2) expression are altered dynamically in the spinal cord during ALS disease progression, increasing in astrocytes while decreasing in neurons (PMID: 36076282, 38253209). Interestingly, the citrullinated proteins accumulate in myelin protein aggregates, suggesting a role of PC in protein aggregation. Here, we applied proteomic methods to identify citrullinated proteins in an ALS mouse model expressing mutant SOD1G93A. The ALS citrullinome profiles disease progression distinctly from normal aging in mice and highlighted an increase in citrullinated glial proteins and a decrease in citrullinated neuronal proteins, validating PC as a marker of reactive astrogliosis and neurodegeneration. Additional analyses found that soluble citrullinated proteins were enriched in inflammation, membrane traffic and metabolic pathways, whereas for insoluble citrullinated proteins, myelin proteins were enriched. The findings in the soluble fraction were validated in analysis of two human ALS proteomic datasets. Among the highly citrullinated proteins in ALS are heat shock proteins and MBP. PC severely compromises these proteins’ structure and function. These results demonstrate the impact of PC in protein function, and furthermore, suggest that PC could provide candidate biomarkers of early-stage ALS and be targeted with novel ALS therapeutics.
• Characterizing Colibactin Toxicity and the Resulting Cellular Response to DNA Damage in Mammalian and Bacterial Systems

  Lowry, Emily (2024-09-03)

  The bacterial toxin colibactin, produced primarily by the B2 phylogroup of Escherichia coli, crosslinks DNA and can promote colon cancer in human hosts, where it has been extensively studied. A systematic approach to identify the DNA damage response to colibactin-induced toxicity has yet to be applied and colibactin toxicity in bacteria remains underexplored. Using a genome-wide CRISPR screen in colon cancer cells, I found that colibactin activates most DNA repair pathways with key roles for Fanconi anemia/interstrand crosslink repair and fork quality control pathways. I also conducted a genome-wide loss-of-function screen in E. coli that identified a key role for homologous recombination in repairing colibactin-induced damage. I determined that colibactin induces a mutational pattern in E. coli in A/T rich regions, as it does in colon cells, but that the resulting mutational signature differs in E. coli. I then predicted that long- term colibactin exposure will culminate in a genomic bias based on this mutational signature, which may be detected in colibactin-producing bacteria. I tested this prediction by analyzing thousands of E. coli genomes andfound that colibactin-producing strains show skewness in trinucleotide composition. Finally, I used a sensitive DNA damage reporter assay to find that cell-cell contact is not required in bacteria as was previously suggested for both bacteria and mammalian cells, and that this needs to be reevaluated in mammalian cells. Taken together, this work revealed the DNA damage response to colibactin-induced damage in both colon and bacteria cells, a bacteria-specific mutation pattern, and that cell-cell contact is not required in bacteria.
• Investigating the role of the Non-Classical MHCII Molecule HLA-DO in Regulating Exogenous Antigen Presentation in B Cells

  Ramesh, Karthik M. (2024-08-21)

  In B cells, exogenous antigens internalized through the BCR are processed in the endocytic pathway and loaded onto class II MHC protein (MHCII) for presentation to CD4+ T cells. The non-classical MHCII molecule HLA-DM (DM) catalyzes peptide exchange, selecting peptides with high affinity to MHCII, while HLA-DO (DO) binds to and inhibits DM in a pH-dependent manner. DO expression increases the diversity of the MHCII self-peptide repertoire and, through its inhibition of DM, allows for the presentation of low-affinity self-peptides, but the effect on exogenous antigens is unclear. We wanted to understand if DO expression in B cells affects exogenous antigen presentation and determine whether DO promotes peptide loading in particular intracellular compartments. We used an immunopeptidome workflow to identify naturally processed endogenous, exogenous, and virus-derived peptides in DO-sufficient and DO-deficient B cells infected with influenza virus, and we used a T cell assay to track the presentation of a single DR1-bound epitope derived from influenza HA with altered pH of fusion. We found that DO increases the diversity of peptides presented in infected cells favoring presentation of DM-susceptible viral epitopes. Tracking a single epitope, we show that the absence of DO favors exogenous peptide presentation, driving loading to later endocytic compartments. Our study expands on the role of DO in modulating the MHCII peptidome via a DM-dependent peptide-selection mechanism that regulates presentation of exogenous antigens and helps to illuminate its role in humoral responses to infections.
• Racial Disparities and Trends in Anticoagulant Use among Ambulatory Care Patients with Atrial Fibrillation and Atrial Flutter in the United States from 2007-2019

  Kan, Vincent (2024-08-08)

  Introduction Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, significantly increasing the risk of stroke. The introduction of direct oral anticoagulants (DOACs) since 2010 has transformed anticoagulation therapy, offering an alternative to warfarin with improved safety profiles. Despite the increased adoption of DOACs, disparities in their use among different racial and ethnic groups in the United States remain understudied. Methods This study utilized a repeated cross-sectional design, analyzing data from the National Ambulatory Medical Care Survey (NAMCS) from 2007 to 2019. The study population included adults diagnosed with AF or atrial flutter (AFL). We analyzed the temporal trends of DOAC and warfarin use from 2007 to 2019. We examined the prevalence of DOAC versus warfarin use and assessed associations between race/ethnicity, patient characteristics, and DOAC utilization from 2011 to 2019. Multivariable modified Poisson regression models were used to calculate adjusted prevalence ratios (aPR) for the associations. Results From 2011 to 2019, NAMCS recorded 3,224 visits involving AF or AFL, representing a weighted estimate of 103.6 million visits. DOAC use increased significantly, with apixaban becoming the predominant anticoagulant by 2016. Non-Hispanic Black patients were less likely to use DOACs compared to non-Hispanic White patients over time (aPR 0.75; 95% CI, 0.63-0.90). Patients with Medicaid insurance were also less likely to use DOACs (aPR 0.14; 95% CI: 0.04-0.46). Conclusion Despite the shift from warfarin to DOACs for AF and AFL treatment, significant racial and socioeconomic disparities persist. Non-Hispanic Black patients and those with Medicaid insurance are less likely to use DOACs. These findings highlight the need for targeted strategies to ensure equitable access to advanced anticoagulant therapies.
• 

  Identification of WNK1 as a Therapeutic Target to Suppress IgH/MYC Expression in Multiple Myeloma

  Ye, Tianyi (2024-08-08)

  Multiple myeloma (MM) remains an incurable hematological malignancy demanding innovative therapeutic strategies. Targeting MYC, the notorious yet traditionally undruggable oncogene, presents an appealing avenue. This thesis aims to identify and characterize novel regulators of MYC expression as therapeutic targets in MM. Using a genome-scale CRISPR/Cas9 screen, we identify the WNK lysine deficient protein kinase 1 (WNK1) as a regulator of MYC expression in MM cells. Genetic and pharmacological inhibition of WNK1 reduces MYC expression and, further, disrupts the MYC-dependent transcriptional program. Mechanistically, WNK1 inhibition attenuates the activity of the immunoglobulin heavy chain (IgH) enhancer, thus reducing MYC transcription when this locus is translocated near the MYC locus. Furthermore, we show in MM cells possessing distinct translocation profiles that WNK1 inhibition also downregulates other oncogenes frequently translocated near the IgH locus, including CCND1, FGFR3, and NSD2, broadening its potential therapeutic implications. WNK1 inhibition profoundly impacts MM cell behaviors, leading to growth inhibition, cell cycle arrest, senescence, and apoptosis. Importantly, the WNK inhibitor WNK463 inhibits MM growth in primary patient samples as well as xenograft mouse models, and exhibits synergistic effects with various anti-MM compounds. Collectively, this study uncovers WNK1 as a promising therapeutic target in MM, and suggests the utility of IgH translocations as useful biomarkers.
• Computationally Detecting Viral Infection and Characterizing Host-Virus Dynamics in scRNA-seq Datasets

  Cao, Yuming (2024-07-26)

  Viruses pose significant threats to human health, with their impacts varying by type. Advances in single-cell RNA sequencing (scRNA-seq) have enhanced our understanding of viruses and host responses by mapping human and viral transcripts within individual cells. However, ambient RNA contamination complicates the accurate identification of viral infections in scRNA-seq datasets. To address this, we introduced scVirusFinder, a method that uses a zero-inflated negative binomial model followed by a support vector machine classifier to identify virus-infected cells. This approach improves the detection of true viral infections in scRNA-seq datasets of virus infected cells. We applied this method to scRNA-seq data from nasal washes of healthy donors and those with acute influenza during the 2017-18 season. We identified seventeen cell populations, including a novel epithelial cell population with high MHC class II gene expression in infected individuals. Influenza virus infections were found in most cell populations, primarily in epithelial cells and major immune cells such as macrophages and neutrophils. Using viral reads from the scRNA-seq data, we discovered that each donor harbored a unique influenza variant with distinct non-synonymous mutations. Additionally, we observed interferon production and response in infected samples, with type III interferon particularly produced in infected ciliated epithelial cells. This study highlights the challenge of identifying infected cells from scRNA-seq datasets and provides a robust solution applicable to clinical samples, enhancing our understanding of viral infections and paving the way for therapeutic discoveries.
• 

  Ethical Frameworks in Periviable Decision-Making: Patient Perspectives and Provider Patterns

  Delaney, Kathryn (2024-06-25)

  Background: Periviability counseling involves engaging in difficult ethical decisions. The ethical frameworks used by patients and their healthcare providers to discuss this topic have not been previously studied. Objectives: This study assessed the ethical frameworks used by patients and by providers during periviability counseling and subsequent decision making. Study Design: This mixed methods study included patients between gestation ages 21 weeks 0 days through 24 weeks 6 days who required periviability counseling, and the providers performing this counseling. Counseling sessions between providers and patients were recorded, as were semi-structured follow-up interviews with patients. These recordings were transcribed and analyzed using thematic analysis. Demographic surveys were given to providers, and patient demographic, obstetric, and delivery information was abstracted from medical records. Results: A total of 11 patients and 11 providers participated in either a recorded counseling session, a follow-up interview, or both. Qualitative analysis revealed the following themes: 1) decisions regarding periviable intervention are the patient’s to make, 2) desire to avoid pain or suffering of the fetus/newborn, especially as it related to CPR, 3) doing what is best for the family, including partners and other children at home, 4) “doing everything” as a good in itself, and 5) periviable complications disrupt expectations about parenthood or pregnancy which then need to be reimagined. These themes corresponded to four distinct ethical frameworks: principlism, care ethics, virtue ethics, and narrative ethics. All of the patients, and approximately three-quarters of providers used at least one of these ethical frameworks; most used a combination of frameworks. Conclusions: Patients and providers engaging in decision making surrounding periviable intervention use commonly accepted ethical frameworks to discuss and decide how to proceed with these pregnancies.
• Analyzing the Role of MS4A Receptors in Mouse Olfaction

  Jiang, Hao-Ching (2024-06-17)

  How the brain interprets sensory information to generate appropriate behaviors remains a fundamental, unanswered question in neurobiology. In mammals, the olfactory system detects external chemicals through a vast array of odorant receptors expressed by peripheral olfactory sensory neurons (OSNs). These OSNs connect to brain circuits responsible for creating olfactory percepts and triggering odor-driven behaviors. The olfactory system has evolved distinct subsystems to initiate various behaviors, each containing anatomically and molecularly distinct OSNs for different functions. The olfactory epithelium (OE) is the largest and best-studied subsystem, crucial for odor discrimination and learning. Smaller olfactory subsystems detect ethologically relevant odorants, necessary for innate behaviors like foraging, mating, and predator avoidance. Unlike conventional OSNs in the OE, receptors in smaller subsystems are largely uncategorized, leaving their roles in odor-driven behaviors poorly understood. I focused on the function of the MS4A gene family, which is expressed in a specific olfactory subsystem linked to mouse innate behaviors. I demonstrated that multiple genes in this family act as chemoreceptors in vivo. Additionally, I identified one family member, MS4A1, expressed in a subset of previously uncharacterized OSNs in the mouse nose. MS4A1 functions as a non-canonical chemoreceptor, required for evoking innate avoidance responses to certain aversive chemicals. Beyond OSNs, the MS4A gene family is also expressed in various immune cells. Notably, I found that MS4A1/CD20, a tumor marker on B cells, acts as a potential chemoreceptor. This discovery could enhance understanding of MS4A proteins' roles in tissues outside OSNs and provide potential therapeutic targets for human diseases.
• Calcineurin coordinates cell cycle progression with adaptation to environmental stress

  Flynn, Mackenzie J (2024-06-14)

  Sudden exposure to environmental stress threatens the viability of single-celled microbes and cells within complex tissues. In order to survive, cells must sense environmental changes and coordinate a transient cell cycle arrest with the appropriate adaptive response. Cells have several stress-responsive pathways that promote adaptation to distinct stressors, but how these pathways interact with one another is poorly understood. Here, we examined the response to calcium chloride stress, which activates the phosphatase calcineurin and the MAPK Hog1 in Saccharomyces cerevisiae. We discovered that calcineurin extends Hog1 activation, which causes prolonged downregulation of cell cycle-regulated genes and delays progression through multiple cell cycle phases. At the G1/S transition, crosstalk between calcineurin and Hog1 dramatically increases the duration of calcium-induced arrest. I found that Hog1 triggers arrest independent of calcineurin by decreasing G1 cyclin transcription and calcineurin maintains this arrest by extending Hog1-dependent activation of the G1 CDK inhibitor Cip1. These results suggest that stress-response pathway interactions tailor cell cycle arrest with adaptation to environmental stress. The immediate response to stress is well-characterized, but how cells maintain viability in challenging environments after recovering from a stress-induced arrest is unknown. I investigated the response to prolonged growth in calcium stress and found that calcineurin maintains fitness by promoting cell division and suppressing death. I determined that calcineurin helps cells proliferate and survive prolonged calcium exposure by two mechanisms, which differentially require a downstream transcription factor. Together, these findings highlight the importance of stress-response pathways during both acute and chronic environmental stress.
• 

  Location, Location, Location: The Impact of One's Neighborhood on Outcomes Following Proctectomy for Rectal Cancer

  Onyiego, Alexandra (2024-06-13)

  Purpose: The area deprivation index (ADI) is a comprehensive assessment of the social determinants of health. The higher the composite ADI score, the more underserved the area. This study examined the impact of a patient’s ADI score on surgical, pathologic, and survival outcomes following proctectomy for rectal cancer. Methods: Data from a single tertiary care medical center’s targeted National Surgical Quality Improvement Program (NSQIP) were used to identify all patients undergoing proctectomy for rectal cancer between January 2014 – December 2022. A state ADI (ranking 1-10) was assigned to each patient using their residential address. Patients were categorized into Low (1-4), Moderate (5-6), High (7-8), and Very High (9-10) ADI groups. Results: Two-hundred and four patients who underwent proctectomy for rectal cancer were included. There were no significant between group differences in patient’s post-operative outcomes including 30-day morbidity and hospital readmissions. As well as pathologic outcomes, including completeness of mesorectal excision and rate of positive margins. A Cox multivariable adjusted regression model showed those in the Very High group had a higher risk of dying at 5 years than the Low ADI group. Patients in the Very High and High ADI groups were significantly more likely to be discharged somewhere other than home after their surgery than those in the Low ADI group, (13% vs. 0%). The median time from diagnosis to initiating treatment or surgery showed those in the Very High and High ADI groups took longer to obtain treatment. Conclusion: A patient’s ADI has an impact on their overall survival following proctectomy for rectal cancer and influences their time to receiving treatment and their postoperative disposition. Future studies with larger patient cohorts are needed to more clearly define the role of ADI in predicting patient outcomes following proctectomy for rectal cancer.
• 

  Kinetics of Pre-mRNA 3’ End Cleavage

  Torres Ulloa, Leslie (2024-05-31)

  3’ end cleavage and polyadenylation are required steps in pre-mRNA maturation. The rate at which 3’ end cleavage occurs can determine the temporal availability of mRNA for subsequent function throughout the cell and is likely tightly regulated. While there are numerous high-throughput methods for global profiling of RNA maturation rates, the study of pre-mRNA 3’ end cleavage kinetics has remained limited to low-throughput approaches, and the temporal regulation of polyadenylation site choice that determines the composition of the 3’ UTRs of mRNAs remains poorly understood. This research project seeks to address this gap by introducing a novel genome-wide, site-specific methodology for estimating rates of pre-mRNA 3’ end cleavage, using metabolic labeling of nascent RNA, high-throughput sequencing, and mathematical modeling. Using in-silico simulations of nascent RNA-seq data, we show that our approach can accurately and precisely estimate cleavage half-lives for both constitutive and alternative sites. In Drosophila melanogaster S2 cells, we find that cleavage rates are fast but highly variable across sites, with alternative events being slowest. This variability in rates is underpinned by distinctive sequence elements, where an A-rich region upstream of the cleavage site, a U-rich element downstream of the cleavage site, and a higher density of polyadenylation signals, lead to faster cleavage reactions. Assessment of Polymerase II dynamics around cleavage sites reveals that cleavage rates are associated with the localization of RNA Polymerase II at the end of a gene and faster cleavage leads to quicker degradation of downstream read-through RNA. This approach for estimating pre-mRNA 3’ end cleavage kinetics opens new possibilities in the study of co-transcriptional regulation of mRNA expression and transcription termination across cellular states.
• Variation in Depth of Sedation Targeted and Achieved among Mechanically Ventilated Patients and Associated Outcomes

  Rucci, Justin M (2024-05-30)

  Introduction: Sedative agents are commonly administered to patients receiving mechanical ventilation (MV). Practice guidelines recommend provision of light sedation within validated scoring systems (e.g., Richmond Agitation Sedation Scale [RASS]), but recognize some circumstances require deeper sedation. The real-world approaches to depth of sedation, and the impact of hospital sedation practices on patient outcomes, remain uncharacterized. Methods: We used the US based eICU collaborative research database to identify adult patients who received MV > 24 hours, who did not have a diagnosis that may require sedatives for indications other than facilitating MV, and who had recorded RASS goals and scores. We used mixed effects regression models to determine factors associated with initial RASS goals and rates of RASS score-goal concordance. We organized hospitals into quartiles of risk-adjusted RASS score-goal concordance, and used g-computation to evaluate differences in ventilator free days (VFD) at hospital day 28. Results: We identified a study sample of 1,650 adult patients (at 21 hospitals) who met inclusion/exclusion criteria. Hospital-level risk-adjusted initial RASS goals ranged from -1.4 to 0.2, and hospital-level risk-adjusted RASS score-goal concordance ranged from 27% to 64%. Patients admitted to hospitals in the highest quartile of score-goal concordance (quartile 4) were generally targeted for deeper sedation (median RASS goal -1.31) than patients admitted to hospitals in the lowest quartile (quartile 1) (median RASS goal -0.58). Compared to patients admitted to quartile 1 hospitals, patients at quartile 4 hospitals experienced fewer VFDs (adjusted incidence risk difference -2.4, 95% CI -4.26 to -0.36). Conclusion: US hospitals prescribe RASS goals in line with guideline recommendations for light sedation, but there is wide variation in achieving these RASS goals. Hospitals with higher RASS score-goal concordance typically prescribed deeper RASS goals, and patients admitted to these hospitals experienced fewer VFD.
• Synthesis of Chemically Modified Nucleic Acids to Characterize and Inhibit APOBEC3 Enzymes

  Hedger, Adam K (2024-05-28)

  APOBEC3 proteins (A3s) are enzymes that catalyze the deamination of cytidine (C) to uridine (U) in single-stranded DNA (ssDNA) substrates, playing a key role in innate antiviral immunity. However, incomplete A3 restriction of viruses is shown to cause drug resistance, and erroneous deamination of host DNA is strongly implicated in many cancers and therapy resistance. Small molecule drug discovery efforts have so far largely failed in finding potent and selective inhibitors of A3 enzymes. Recent structural advances have informed the design of “substrate mimicking” competitive oligonucleotide inhibitors, with potencies in the low-µM to -nM range. Nevertheless, further medicinal chemistry efforts are needed to improve the cellular stability and potency of these compounds. With that goal, in this thesis I report the first co-crystal structure of an active A3 enzyme bound to an oligonucleotide inhibitor at high-resolution, capturing the transition-state, and further informing inhibitor design. Next, I develop new and efficient synthesis routes to novel cytidine analogs for later incorporation into oligonucleotide inhibitors, including 2′-fluoroarabino- and 2′-difluoro-1,3-diazepinone nucleosides. Finally, I carry out a detailed structure-activity relationship of chemically modified oligonucleotide inhibitors against A3 enzymes. The resulting chemically optimized inhibitors from this work are the most potent reported to date, show greatly enhanced nuclease stability, and display inhibition in a cellular inhibition assay. In summary, my thesis work combines structural biology with nucleic acid chemistry to further A3 inhibitor design on three fronts, ultimately achieving the goal of generating potent and chemically stabilized inhibitors for use in cellular assays and beyond.
• Spatial Transcriptomics Reconstruction of Mouse Olfactory System

  Wang, I-Hao (2024-05-20)

  The olfactory system is crucial for animals in tasks such as foraging, mate selection, and predator avoidance due to its ability to detect and distinguish a vast array of environmental chemicals. Mice detect these chemicals via olfactory receptor (OR) proteins, which are uniquely expressed by olfactory sensory neurons (OSNs); each OSN expresses only one OR type. OSNs with the same OR converge their axons to a specific location in the olfactory bulb (OB), forming a structure known as a glomerulus. This precise organization ensures a consistent, spatially invariant pattern of glomerular activation for each odorant, playing a likely role in the brain's decoding of odor identities. Nevertheless, the exact locations of most glomeruli are unknown, and the mechanisms that create consistent glomerular maps across different animals are not fully understood. In this study, we leveraged spatial transcriptomics and machine learning to map the majority of glomerular positions within the mouse OB. Furthermore, single-cell RNA sequencing revealed distinct transcriptional profiles for each OSN type, characterized not only by their OR gene but also by a unique set of axon guidance genes. These profiles can predict the eventual location of each OSN's glomerulus within the olfactory bulb. We also identified a correlation between the spatial distribution of glomeruli and the characteristics of their corresponding ORs, suggesting a chemotopic arrangement in the mouse olfactory system. Additionally, we probed the complexity of the OB by creating a spatially resolved cell atlas through spatial single-cell transcriptomics, revealing the identity and distribution of neuron subtypes that contribute to odor perception.
• 

  Developing an Adeno-Associated Viral Gene Therapy for Sialidosis Using Small and Large Animal Models

  Gallagher, Jillian (2024-05-17)

  Sialidosis is a rare, fatal lysosomal storage disease caused by mutations in NEU1, encoding neuraminidase 1 (NEU1) resulting in toxic accumulation of sialylated glycoproteins. Type I patients have adolescent onset with myoclonus, ataxia, seizures, cherry-red spot, and vision loss, with death in midlife. Type II patients are more severe with the addition of hepatosplenomegaly, coarse facial features, dysostosis multiplex, developmental delay, and death in childhood. There are no approved therapies for sialidosis. We developed three adeno-associated viral (AAV) gene therapy vector strategies to treat sialidosis encoding: Neu1 alone (AAV9-Neu1), Neu1 modified to enhance secretion (AAV9-Idua-Neu1) or Neu1 in combination with its chaperone protein cathepsin A (AAV9-Ctsa-Bici-Neu1). Neonatal Neu1-KO mice were injected by intracerebroventricular injection. Increased survival and significant improvement in motor ability was noted for all vectors, however seizures were observed in AAV9-Neu1 treated mice at 10 months of age. Reduced sialic acid and PPCA were found in peripheral organs and brains of treated mice. NEU1 was increased in peripheral organs and the brain, however AAV9-Ctsa-Bici-Neu1 treated mice had NEU1 levels similar to WT mice. Pathology of peripheral organs were markedly ameliorated with AAV gene therapy. We generated a sialidosis type II sheep using CRISPR-SpCas9 (A320del), which is in the same location as the amino acid change in patients (A319V). Importantly, the A320del sheep exhibits a neurological phenotype unlike the Neu1-KO mouse. Overall, the most efficacious vector is AAV9-Ctsa-Bici-Neu1, which will be further tested in the sialidosis sheep model. These studies will inform future clinical trials for this devastating disease.
• 

  Progressive CD4 T-Cell Dysfunction During Chronic Tuberculosis is Associated with Bacterial Recrudescence

  Chang, Evelyn (2024-05-13)

  Tuberculosis remains a leading cause of death globally, and approximately 5-10% of those infected develop pulmonary disease, often after initially controlling infection. Why immunity fails is unknown. CD4 T cells are crucial for immunity against Mtb, yet little is known about how Mtb infection might modulate CD4 T cell function, particularly late during infection. We hypothesize that failure of CD4 T cell function permits bacterial recrudescence and the development of active disease. Here, we develop a minimalistic adoptive transfer model to study antigen-specific T cells in the lungs of Mtb-infected mice. Late during infection, a decrease in T cell polyfunctionality and production of IL-2, TNF, and IFNγ is accompanied by the accumulation of PD-1 and TIM-3 expressing T cells. Transcriptional profiling identifies signatures of senescence and exhaustion in these cells. In C57Bl/6 mice, a similar co-inhibitory receptor expression and loss of function is observed on antigen-specific CD4s. Single-cell sequencing reveals that most expanded parenchymal T cells are hypofunctional, even early post-infection. However, no evidence of exhaustion or senescence was found. As TCR affinity and signaling strength affects T cell differentiation, we next examined whether TCR affinity impacts T cell function. Higher affinity CD4 T cells differentiate into polyfunctional Th1 cells while lower affinity clones skew towards a Th17 phenotype. The distinct clusters formed by high and low affinity clones on a UMAP projection show how TCR signaling strength affects T cell differentiation during chronic infection. These data provide insight into mechanisms modulating CD4 T cell dysfunction during chronic infection. Further study into these mechanisms will inform therapeutic options.
• Investigating the function and regulation of SARM1

  Icso, Janneke D (2024-05-07)

  Wallerian degeneration is a process characterized by axonal fragmentation and myelin disintegration, and is the hallmark of neurodegenerative diseases, traumatic axonal injuries, and peripheral neuropathies. SARM1 is a key promoter of Wallerian degeneration. In injured axons, SARM1 cleaves NAD+ to nicotinamide and a mixture of ADPR and cADPR, which ultimately leads to the axonal degradation that characterizes Wallerian degeneration. SARM1 deletion in animal models of degenerative diseases prevents axonal degeneration, indicating that SARM1 is an attractive therapeutic target. Herein, we describe the investigation of the function and regulation of SARM1. In the initial kinetic characterization of TIR-1, the nematode ortholog of SARM1, we show that these enzymes are regulated by a phase transition to a gel/solid-like state that increases the catalytic activity of the enzyme 27-fold. Moreover, we show that the phase transition of SARM1/TIR-1 occurs in vivo in multiple biological contexts: axon degeneration and intestinal immunity. The phase transition also reduces the threshold for NMN-induced SARM1 activation physiologically relevant levels (140-fold). Furthermore, we delineate the catalytic mechanism of SARM1/TIR-1 and demonstrate that these enzymes use an oxocarbenium-like intermediate to catalyze myriad chemical reactions. This intermediate is common to NAD+ hydrolysis and cyclization as well as the base exchange reaction with NADP+ and nicotinic acid. Finally, we investigate mono-ADP-ribosylation as a novel SARM1 function. Given the critical role of SARM1 in axon degeneration, SARM1 inhibition is likely to attenuate the pathophysiology of Wallerian-like diseases. We expect these findings to inform the further development of inhibitors targeting SARM1.
• Identifying Therapeutic Oligonucleotide-Induced Neurotoxicity and Methods for Safe Delivery to the Central Nervous System

  Miller, Rachael (2024-04-29)

  Huntington’s disease (HD) is a hereditary neurodegenerative disorder caused by an autosomal dominant mutation in Exon 1 of the Huntingtin gene (Htt). There are no approved treatments for HD. Oligonucleotide therapeutics (ASOs and siRNAs) offer a new strategy to treat genetically defined CNS diseases. These therapeutics aim to attenuate disease pathogenesis by targeting Htt mRNA to reduce the toxic mutant protein. Recent technological advancements now enable robust distribution and efficacy throughout mouse, sheep, and NHP brains. However, oligonucleotides can cause acute neurotoxicity when injected directly into the CSF. This dissertation aims to optimize oligonucleotide delivery for the treatment of HD by addressing safety issues across species. We used electroencephalography (EEG) and electromyography (EMG) in awake animals to confirm that direct CSF injection of oligonucleotides induces seizures. We hypothesized that this was due to the negatively charged oligonucleotides changing the delicate balance of divalent cations in the CSF. To address this issue, we developed an artificial CSF (aCSF) buffer supplemented with Ca2+ alone, Mg2+ alone, or Ca2+ and Mg2+ in the injected solution to prevent the imbalance. Real-time EEG monitoring in awake mice and lambs confirmed the absence of seizures when oligonucleotides were delivered in the new aCSF buffer. In summary, this dissertation identified a potential cause of oligonucleotide-induced acute neurotoxicity, developed a method to safely deliver oligonucleotides to the CNS with Ca2+/Mg2+-enriched buffers, and demonstrated the viability of this formulation in a large animal model. These findings support a new method for safely delivering oligonucleotides to the CNS to treat neurological diseases.

View more