Structures and Mechanisms of Lysosomal Transporters

Lysosomal membrane transporters are indispensable for maintaining lysosomal homeostasis and proper function. Indeed, mutations in these key proteins can lead to debilitating disorders known as lysosomal storage diseases. Cystinosin and Sialin are two such transporters. Both proteins utilize the low pH environment to transport their main substrates of interest from the lumen to the cytosol where they can be reused by the cell. Understanding how these proteins work at an atomic level can help us understand their overall function, the roles they play in lysosomal signaling pathways, and may enable the future development of therapeutics to treat their associated disorders. Mutations in Cystinosin cause Cystinosis, a neurodegenerative disorder that occurs when Cystinosin's substrate, the dimeric form of cysteine called cystine, builds up in the lysosome. While there currently is a treatment for this disorder, how mutations disrupt this transport and how the protein utilizes the proton gradient remain a mystery. Similarly, Sialin causes a variety of free sialic acid storage diseases that result in developmental delays as well as neurodegeneration. Unlike Cystinosis, there is currently no approved treatment for these disorders. Like Cystinosin, the structure and mechanism of transport remain unknown. The work presented herein reveals the cryo-EM structures of Cystinosin and Sialin at near atomic level resolution. These structures, captured in both cytosol- and lumen-open conformations as well as substrate bound states, reveal not only the mechanisms of conformational changes but also the residues involved in the substrate binding pocket(s). Along with the accompanying functional assays, I demonstrate that the majority of disease-causing mutations in both Cystinosin and Sialin center around their ability to bind their respective substrates. Additionally, I reveal the potential proton sensors of both proteins involved in their substrate symport. In the case of Sialin, I also hypothesize that its proton sensor could potentially double as a membrane potential sensor for its transport of neurotransmitters into synaptic vesicles. This work paves the way for understanding the greater PQ-loop family (Cystinosin) and SLC17 family (Sialin) of transporters. It also builds a foundation upon which future therapeutics can be designed to treat their associated disorders

Pre- and Postsynaptic MEF2C Promote Experience-Dependent, Input-Specific Development of Local Cortical Excitatory Synapses

Complex and specific neocortical circuits mature postnatally through a combination of genetic factors and sensory experience-driven neural activity. Experience- and activity-dependent transcriptional factor activation is a candidate mechanism for the development and refinement of these circuits. Synapses form the basis of neurons. Robust synapse proliferation during development is closely followed by experience-dependent pruning and modification to preserve and strengthen circuits. Neurodevelopmental disorders, including Autism Spectrum Disorder, are characterized by synaptic and circuit properties that give rise to behavioral and cognitive deficits and symptoms. Transcription factor Myocyte Enhancer Factor 2 C (MEF2C) is highly expressed in the cortex during development and into adulthood and has been identified as a regulator of synaptic strength and transmission in a sensory experience-dependent manner. MEF2C has been shown to function in both repressive and activator roles in postsynaptic compartments; however, little is known about presynaptic regulation by MEF2C. Additionally, the mechanisms by which MEF2C regulates synapses in an activity- and input-specific manner are still largely unknown. My work provides evidence that the activity-dependent transcription factor MEF2C is required for experience-dependent development of inputs from Layer (L) 4 to L2/3 neurons in the mouse primary somatosensory barrel cortex (S1). Importantly, MEF2C is required in both presynaptic L4 and postsynaptic L2/3 neurons during the first two postnatal weeks for L4–L2/3 synapse development. MEF2C plays a local L4 input-specific role in postsynaptic L2/3 cells through the mechanism of reduced probability of presynaptic neurotransmitter release for L4 presynaptic MEF2C. Constitutively active MEF2C-VP16 can rescue the lack of whisker sensory input but does not rescue the loss of MEF2C in presynaptic neurons. Together, these results suggest that the activity-dependent transcriptional activation of MEF2C promotes the development of L4–L2/3 synapses. MEF2C is necessary for the activity-dependent expression of genes encoding pre-, post-, and transsynaptic proteins in cortical neurons. I examined the protein tyrosine kinase PYK2, which was found to be elevated in the cortex of MEF2C KO mice but appeared to be insufficient in affecting or regulating synaptic strength, like MEF2C. Altogether, this work provides insights into the mechanisms of MEF2C-mediatied, experience-dependent development of specific cortical circuits

Mechanistic Basis of Skeletal Muscle Wasting Diseases

Skeletal muscle is the largest tissue in the human body by biomass and it is essential for life. Impaired skeletal muscle function can negatively impact one's quality of life but in the case of diseases like cancer cachexia, it can directly contribute to death of patients. Skeletal muscle wasting is a key feature of many monogenic muscle diseases such as Duchenne muscular dystrophy (DMD) and Emery-Dreifuss muscular dystrophy (EDMD) but also more complex conditions such as cancer cachexia, starvation, and various neuromuscular diseases. Throughout this thesis, I focus on two muscle wasting diseases, cancer cachexia and EDMD. While these diseases have been studied for decades, with the disease-causing genes being identified for EDMD, the mechanistic basis has not been elucidated for either disease. Here we propose that downregulation of the nuclear envelope protein, Net39, contributes to EDMD pathogenesis. Adult deletion of Net39 in mice recapitulates many of the key features of EDMD, including nuclear envelope deformations, dysregulated gene expression, altered metabolism, and muscle wasting. Mechanistically, Net39 protects myonuclear envelopes from mechanical stretch and nuclear envelopes deficient of Net39 are structurally compromised, leading to DNA damage. Cancer cachexia on the other hand, is a highly prevalent and systemic wasting condition characterized by skeletal muscle wasting. We profiled the molecular changes at play in mouse and human cachexic muscle at single nuclear resolution, identifying the conserved activation of a denervation gene program. Mechanistically, we discovered that myogenin regulates myostatin during cancer cachexia and the inhibition of myostatin via AAV-Follistatin gene therapy rescues cancer cachexia and prolongs survival in preclinical models of cancer. Overall, our findings here highlight the importance of Net39 to EDMD pathogenesis and the myogenin-myostatin axis to cancer cachexia-induced muscle atrophy

From Stem Cells to Blastoids: Unraveling the Mechanisms of Early Embryonic Development and Gene Silencing

Understanding the mechanisms of early embryonic development is essential for understanding the origins of aging and disease. Advancements in the culture of embryonic stem cells have opened new avenues for modeling critical timepoints of early development in vitro, such as blastocyst formation, implantation, and gastrulation. The work presented here shows the development of stem cell derived blastocyst like structures, termed blastoids, from pluripotent stem cell cultures in both humans and animals. Blastoids have increased our understanding of the signaling, genetic and epigenetic requirements for the formation of the early embryo. Finally, our work helps to uncover the critical epigenetic interplay that happens in the epiblast of the blastocyst before and after implantation, by using naïve and primed embryonic stem cells to respectively model these stages. We show that TASOR (transcription activator suppressor), part of the Human Silencing Hub (HUSH) complex, is a co-transcriptional platform for epigenetic and epitranscriptomic silencing. We show how the H3K9me3 deposition in naïve cells is essential for the proper establishment of long-term silencing via DNA methylation in primed or differentiated cells. Moreover, our work uncovers an innate immune checkpoint that is activated upon the exit of naïve pluripotency against endogenous retroviral LINE-1 elements and repeats. Overall, this dissertation provides significant insights into the genetic, epigenetic, immunological and signaling mechanisms of the early embryo, offering new perspectives on the intricate biological processes that govern the earliest stage of mammalian life

Explaining Racial Discrepancies in Verbal Memory Assessment with Joint Estimation of Life Course Social Inequalities and Measurement Bias

Decades of research in cognitive test performance have demonstrated that racial group differences appear on a wide variety of tests and samples. The development of new tests, statistical adjustments, and incorporation of additional sociocultural information have contributed to varying degrees of success in the resolution of these differences. At the same time, racially minoritized communities are affected by higher rates of dementia, and yet require, on average, greater levels of severity in cognitive impairment to be diagnosed. To improve the detection of dementia at earlier stages where interventions are more likely to be useful and to facilitate better research into treatments where cognitive test performance is the standard outcome of interest, solutions to measurement differences between racial groups need to be identified. This study proposes a novel theoretically informed psychometric approach to separate cognitive test performance into factors related to life course disparities and those related to measurement bias. Utilizing the Consortium to Establish a Registry for Alzheimer's Disease list learning test administered as part of the national Harmonized Cognitive Assessment Protocol (HCAP) study conducted in partnership with the Health and Retirement Study, the pervasive bias model is used to identify sources of measurement differences, quantify the remaining impact of social inequities on performance differences, and evaluate the potential clinical applications of the approach. Utilizing a sample of 2074 cognitively normal adults over the age of 65 who identified as either racially White or Black, the pervasive bias model revealed that race had minimal direct contributions to measurement bias. In contrast, educational attainment was found to cause measurement bias on the list learning test, and since there were significant differences between the racial groups' years of education, this effect explained apparent racial group differences. Importantly, accounting for education's effects on the measurement of memory not only removed evidence for racial group differences but provided positive evidence overall that the two racial groups had equivalent overall memory abilities. While promising as a new approach to understanding test data, additional evaluation, particularly with clinical samples, is needed before the potential of this novel approach is truly understood

The Role of Target-Directed MicroRNA Degradation in Mammalian Development

Pages vi-xvi are misnumbered as pages vii-xvii.MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that play critical roles in development and disease. In animals, miRNAs canonically bind to partially complementary sites in messenger RNA 3′ untranslated regions, resulting in target repression. However, specialized targets, typically exhibiting extensive complementarity to the miRNA, can invert the regulatory logic and trigger degradation of the miRNA. Although this pathway, known as target-directed miRNA degradation (TDMD), has emerged as a potent mechanism of controlling miRNA levels, the biological role and scope of miRNA regulation by TDMD in mammals remains poorly understood. To address these questions, we generated mice with constitutive or conditional deletion of Zswim8, which encodes an essential TDMD factor. Loss of ZSWIM8 resulted in developmental defects in heart and lung, growth restriction, and perinatal lethality. Small RNA sequencing of embryonic tissues revealed widespread miRNA regulation by TDMD and greatly expanded the known catalog of miRNAs regulated by this pathway. These experiments also uncovered novel features of TDMD-regulated miRNAs, including their enrichment in co-transcribed clusters and examples in which TDMD underlies 'arm switching', a phenomenon wherein the dominant strand of a miRNA precursor changes in different tissues or conditions. Importantly, deletion of two TDMD-regulated miRNAs, miR-322 and miR-503, rescued growth of Zswim8 null embryos, directly implicating the TDMD pathway as a regulator of mammalian body size. Together, these data reveal the broad landscape of TDMD in mammals and demonstrate that regulation of miRNA abundance by this pathway is essential for normal mammalian development

AXL-WRNIP1 Mediated Replication Stress Response Promotes Therapy Resistance and Metachronous Metastasis in HER2+ Breast Cancer

Therapy resistance and metastatic progression are primary causes of cancer-related mortality. Disseminated tumor cells possess adaptive traits that enable them to reprogram their metabolism, maintain stemness, and resist cell death, facilitating their persistence to drive recurrence. The survival of disseminated tumor cells also depends on their ability to modulate replication stress in response to therapy while colonizing inhospitable microenvironments. In this study, we discovered that nuclear translocation of AXL, a TAM receptor tyrosine kinase, and its interaction with WRNIP1, a DNA replication stress response factor, promotes the survival of HER2 targeted therapy-resistant breast cancer cells in the brain. Using preclinical models, we demonstrated that knocking down or pharmacologically inhibiting AXL or WRNIP1 increased replication stress and attenuated metastatic latency and relapse. Our findings suggest that targeting the replication stress response, which is a shared adaptive mechanism in therapy-resistant and metastasis-initiating cells, could reduce metachronous metastasis and enhance the response to standard-of-care therapies

From Genetics to Neurodevelopment: Identifying the Role of the Chromatin Regulator KDM5A in Autism Spectrum Disorder

Autism spectrum disorder (ASD) is a constellation of neurodevelopmental disorders with high phenotypic and genetic heterogeneity, complicating the discovery of causative genes. Through a forward genetics approach, we identified KDM5A as a candidate ASD gene, regulating vocalization and nest building in mice. We subsequently analyzed whole exome sequencing data from a clinical cohort and identified pathogenic KDM5A variants in patients with ASD. KDM5A encodes a chromatin regulator that belongs to the KDM5 family of lysine-specific histone H3 demethylases. Epigenetic chromatin regulation is essential for establishing and maintaining cellular identity and differentiation. It is required for normal brain development and proper gene expression as well as wiring of neuronal circuits. Disruptions in chromatin regulators lead to several diseases, including ASD. In fact, it is one of the top pathways disrupted in ASD (e.g., ARID1B, CHD8, KMT5B). To characterize the in vivo function of KDM5A, we developed a Kdm5a knockout mouse model (Kdm5a-/-) and showed that loss of KDM5A leads to severe social communication and interaction deficits, repetitive behaviors, and learning and memory deficits. Kdm5a-/- also showed an abnormal neuronal phenotype in the cortex and hippocampus, as well as disruption of transcriptional networks essential for normal brain functions. Patients with ASD in many cases present with cognitive deficits, which are often mediated by the cortex and the hippocampus. These two brain regions are composed of a variety of different cell types, each unique in its functions and transcriptome. However, the specific cell types that are affected in ASD as well as the cell-type specific transcriptional programs that are disrupted in this disease are unknown. To investigate this, we performed single-nuclei RNA sequencing from wildtype (WT) and Kdm5a-/- hippocampal tissue, and single-nuclei RNA and ATAC multiome sequencing from the cortex. We found that KDM5A is essential in establishing hippocampal and cortical cell identity, where specific subtypes of excitatory, inhibitory, and glial cells are disrupted. Our findings advance our knowledge of the role of epigenetic chromatin regulation in dictating cellular identities in the brain and help inform future efforts to develop therapeutic strategies in this genetic subtype of ASD

Investigating the Role of Standing Blood Pressure Measurement and the Association Between High-Density Lipoprotein and Skeletal Muscle Mitochondrial Function in Pre-Hypertensive Humans

Cardiovascular disease (CVD) is the leading cause of death in the United States. Importantly, two main driving forces behind developing CVD is the presence of hypertension (HTN) and dyslipidemia. Despite this, there are critical knowledge gaps related to 1) obtaining an accurate clinical assessment of blood pressure (BP), and 2) the mechanisms by which dyslipidemia evoke enhanced cardiometabolic disease risk. Specifically, this dissertation aimed to 1) investigate the role of standing office BP measurement in facilitating HTN diagnosis, and 2) characterize the associations between high-density lipoprotein (HDL) and skeletal muscle mitochondrial function. Herein, we are the first to observe that standing BP measurements are highly accurate in detecting HTN when compared to seated BP. These findings demonstrate standing BP as a novel tool to enhance the accuracy of office BP assessment. Likewise, we are the first to demonstrate higher levels of HDL cholesterol and apolipoprotein A-I are independently associated with enhanced skeletal muscle mitochondrial function in humans. These findings are important because disruptions in skeletal muscle metabolism have been associated with exercise intolerance. In conclusion, our studies have direct implications on the screening of HTN and highlight the utility of measuring BP in both the seated and standing positions. Moreover, we provide evidence for future research aimed at establishing the causal relationship between HDL structure and function with exercise endurance in humans. These studies aimed at addressing two critical gaps in knowledge, and upon completion, have further elucidated more optimal methods of assessing office BP and provided an enhanced understanding of the relationship between dyslipidemia and cardiometabolic disorders

Therapeutic Targeting of Protocadherin 7 in Lung Adenocarcinoma

The general metadata -- e.g., title, author, abstract, subject headings, etc. -- is publicly available, but access to the submitted files is restricted to UT Southwestern campus access and/or authorized UT Southwestern users.Pages 38-92 are misnumbered as pages 41-95.Non-small cell lung cancer (NSCLC) is the most common lung cancer subtype and remains the leading cause of cancer associated deaths worldwide. Patients have few therapeutic options, and disease progression is inevitable, underscoring the critical need for the identification of new targets and therapeutic approaches to treat this disease. We identified a critical oncogenic role for PROTOCADHERIN 7 (PCDH7), a cell surface protein and member of the Cadherin superfamily in NSCLC. PCDH7 is frequently overexpressed in lung adenocarcinoma (LUAD) and associates with poor clinical outcome. Depletion of Pcdh7 reduces lung tumor burden and prolongs survival in mouse models of high-grade NSCLC, demonstrating that this cell surface protein is an actionable therapeutic target. Here we report the development and characterization of high affinity anti-PCDH7 monoclonal antibodies (mAbs) that inhibit downstream MAPK pathway activation and suppress tumor growth in multiple mouse models, including KRAS- and EGFR-mutant models. A lead mAb sensitized tumors to the FDA-approved MEK inhibitor Trametinib, the tyrosine kinase inhibitor Osimertinib, and the KRASG12C inhibitor Adagrasib. Moreover, a humanized mAb7 decreased tumor growth in an Fc-dependent manner and enhanced antibody dependent cell-mediated cytotoxicity and granzyme B production. These findings provide an important step towards the clinical development of PCDH7-targeting antibodies for the treatment of NSCLC and other tumor types with high PCDH7 expression