Ph.D. - Cell and Molecular Biology

Permanent URI for this collectionhttps://hdl.handle.net/10125/20029

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FROM SELENIUM TO SYNAPSE: UNDERSTANDING THE IMPACT OF SELENIUM DEFICIENCY ON PARVALBUMIN INTERNEURONS AND PERINEURONAL NETS IN CORTICAL CULTURES
(University of Hawai'i at Manoa, 2024) Sasuclark, Alexandru Radu; Pitts, Matthew; Hoffmann, Peter; Cell and Molecular Biology
Parvalbumin interneurons (PVIs) are a critical subgroup of GABAergic interneurons involved in neural circuitry. They are integral to generating and modulating gamma oscillations, which are necessary for enhancing information processing by reducing circuit noise and amplifying circuit signals. During development, these interneurons are surrounded by unique extracellular matrix structures called perineuronal nets (PNNs). In neurodevelopmental disorders such as Schizophrenia (SCZ), evidence has pointed to damage or loss in both PVIs and PNNs, and a possible culprit is increased oxidative stress, particularly during development. Selenoproteins, a unique clade of proteins identified by the incorporation of the amino acid selenocysteine, are necessary for PVI development. Most selenoproteins are antioxidant enzymes and several, such as GPX4 and SELENOP, are important to PVI development. This relationship between selenoproteins and PVIs has been explored in depth using several knockout (KO) mouse models. However, more research is needed to understand the involvement of nutritional selenium (Se) for maturing PVIs. Additionally, no studies have examined the role of selenium or selenoproteins in the development of PNNs. Here, using in vitro cultures of primary cortical tissue, we show that Se-deficiency driven by reduced bioavailable selenium, decreases the expression of critical antioxidant selenoproteins, drives oxidative stress in PVIs, and reduces the number of mature PNNs. Critically, we find that Se-deficiency influences the formation of synapses around PNN positive PVIs. This results in changes to cortical network activity, which has broad implications for our understanding and emphasizes the importance of micronutrients in developing the central nervous system (CNS).
The Exocyst Complex Coordinates GLUT4 and CD36 Trafficking and Fuel Uptake in Cardiomyocytes
(University of Hawai'i at Manoa, 2024) Ha, Herena Yun Joon; Polgar, Noemi; Cell and Molecular Biology
Increasing the rate of glucose metabolism in the heart muscle protects against cardiomyocyte injury triggered by low oxygen levels, such as during myocardial ischemia. Molecules that promote a shift toward glucose uptake and glycolytic metabolism in heart muscle, thus improving metabolic function and decreasing ischemic injury, represent attractive therapeutic targets.Substrate uptake via glucose transporters and fatty acid translocase on the cell membrane is the first key governing step of substrate metabolism in heart muscle. Levels of the predominant glucose transporter GLUT4 and the fatty acid translocase CD36 on the cardiomyocyte surface increase in response to elevated insulin levels following a meal or via the activation of AMP-activated protein kinase (AMPK) during exercise or energy requirement. It has been established that in adipocytes, the highly conserved, eight-protein exocyst trafficking complex is required for insulin-induced GLUT4 membrane delivery and glucose uptake, and our recent work showed that the exocyst is necessary for insulin- and AMPK-induced GLUT4 and CD36 trafficking in skeletal muscle cells as well. The exocyst is responsible for the targeted delivery of membrane-bound vesicles by docking them to the intracellular surface of the cell membrane prior to their fusion. However, despite the high expression of exocyst subunits in cardiac muscle, the mechanisms by which the exocyst trafficking complex directs glucose transporter delivery to the cardiomyocyte cell surface presented a gap in our knowledge. The overall goal of this study set out to determine how the exocyst regulates GLUT4 and CD36 membrane trafficking in heart muscle cells in response to insulin and AMPK induction and, additionally, how exocyst-mediated trafficking of these transporters contributes to the shift towards glucose utilization during ischemia. Our work has demonstrated that the exocyst is a key component in insulin- and contraction-stimulated glucose and long chain fatty acid (LCFA) uptake in cardiac muscle cells. The exocyst inhibitor endosidin-2 (ES2) impaired induced exocyst assembly and recruitment to GLUT4 and CD36, trafficking of CD36 to the cell surface, and glucose and LCFA uptake in H9c2 cardiomyoblasts. Moreover, in response to both hypoxia and stimulated ischemia, we observed a demand for the exocyst at the mRNA and protein level in these cardiac myoblasts. ES2 inhibition in H9c2 cells hindered hypoxia-induced exocyst assembly and recruitment to GLUT4 as well glucose uptake. From our results we conclude that the exocyst complex has a critical role in stimulated glucose and fatty acid uptake in cardiac myocytes. We conclude that our research on the exocyst complex presents new insights into potential therapeutics for cardiac ischemic injury.
Regulation of Cardiac Metabolism and Inflammation by Pyruvate Kinase Muscle Isoform 2
(University of Hawai'i at Manoa, 2024) Lee, Katie C. Y.; Shohet, Ralph V.; Cell and Molecular Biology
The metabolic pathways in the heart are fine-tuned to supply substantial amounts of energy for contraction. Metabolic dysfunction not only diminishes energy production but can also enhance oxidative stress. Pyruvate kinase (muscle isoform 2, PKM2), a glycolytic enzyme, has emerged as a potentially cardioprotective therapeutic target to reduce oxidative stress. Its physiological role in the healthy heart remains to be explored. Using germline PKM2 knockout mice (PKM2-/-), we characterized the metabolic functions of PKM2 in the heart. Glucose isotopic tracing revealed perturbations in glucose flux that prioritized glucose consumption in lipid synthesis and the pentose phosphate pathway in PKM2-/- cardiomyocytes compared to controls. Removal of PKM2 altered lipid profiles, led to the accumulation of lipid droplets, and impaired insulin-mediated glucose uptake in cardiomyocytes. These alterations impaired mitochondrial respiration, increased mitochondrial superoxide production in cardiomyocytes, and depleted ATP in PKM2-/- hearts. Our study establishes PKM2 as a metabolic regulator of oxidative stress in the healthy heart. We also provide evidence of enhanced cardiac and systemic inflammation in PKM2-/- mice. In addition, PKM2-/- hearts exhibited aggravated fibrosis following myocardial infarction. Our study demonstrates important roles for PKM2 in the unstressed heart in regulating energy production, oxidative stress, and cardiac inflammation. These functions carry over to the stressed heart and reveal PKM2 as a cardioprotective enzyme in limiting cardiac remodeling after infarction.
The Neuroprotective AβCore Rescues Full Length Aβ42 – Linked Alterations in Dendritic Spine Structure , Synaptic Density, and Glutamate Receptor Expression
(University of Hawai'i at Manoa, 2024) Shontell, Ruth Mariko; Nichols, Robert A.; Cell and Molecular Biology
Alzheimer’s disease (AD) is the most common form of dementia and is currently the seventh leading cause of death in the United States. Although first described over a century ago, there are currently no cures. Histopathologically, AD is characterized by the presence of extracellular plaques composed primarily of beta-amyloid (Aβ) peptide in fibrillar form and intracellular neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau organized in paired-helical filaments in regions and subregions of the brain involved in memory processes (episodic and working), attention, spatial orientation and navigation, language (word retrieval and syntax), and higher order reasoning. During AD pathogenesis, the most prominent neurodegenerative change is progressive dysfunction and eventual loss of synapses in these regions, correlated with the accumulation of soluble oligomeric Aβ, but not plaques, and the primary early symptom of compromised short-term memory. Later, there is profound loss of neurons. Across these phases, there is progressive neuroinflammation, which is, in turn, strongly connected with synapse and neuron loss. There is abundant evidence that pathological levels of Aβ can directly compromise synaptic function and then trigger damage leading to synapse death. However, the focus of research to date has primarily been on either decreased innervation due to loss of presynaptic terminals or altered postsynaptic dendritic spine structure, without a complete understanding of the relation between the two sides of the synapses. Here, I incorporated an approach to examine the impact of Aβ on both the presynaptic and postsynaptic elements, focusing, in particular, on the postsynaptic density, using a volumetric assessment via microscopic imaging. As a means to prevent or reverse the impact of Aβ on synapse and neuronal cell death, our laboratory discovered a neuroprotective hexapeptide sequence within the neurotoxic full-length Aβ peptide. We showed that this neuroprotective hexapeptide, named the AβCore, can reverse Aβ-triggered deficits in synapse function, but have only preliminarily examined the prevention of Aβ-induced changes in synapse structure by the AβCore. Moreover, none of the studies on Aβ-linked changes in synaptic structure considered the potential impact of nerve – synapse activity, particularly on-going synaptic dynamics. The studies described here were thus based on the hypothesis that the AβCore protects against full length Aβ42 – linked alteration in synaptic structure. In relation to this working hypothesis, the studies were guided by two aims: Aim 1 - Determining the neuroprotective effects of the AβCore on synaptic structure: Dendritic Spines in relation to presynaptic association and Aim 2 - Determining the impact of the neuroprotective effect of the AβCore on synaptic structure in the context of synaptic plasticity induced by chemical long-term potentiation. As noted, the postsynaptic density (PSD) was a primary focus, as this essential synaptic structure is a critical postsynaptic organizer for excitatory synapse signaling and its size and functional composition change in direct relation to synaptic dynamics, growing with increased synaptic strength and shrinking with decreased synaptic strength. I found that in mouse hippocampal neuron cultures Aβ induced a reduction in postsynaptic dendrite spine volume, consistent with previous findings for a reduction in cross-sectional spine size, and a corresponding reduction in PSD volume and surface area on the spines, without an acute change in PSDs/ spine. All changes were prevented by co-administration of the AβCore. There was a curious trend for the AβCore to increase the PSD counts across the spine population. On the presynaptic side, Aβ had no acute effect on the number or size of presynaptic terminals, while, intriguingly, the AβCore strongly increased the number and volume of the presynaptic terminals, speculated to reflect terminal sprouting. In the context of positive synaptic dynamics, mimicking long-term potentiation via application of select chemical reagents (cLTP) to ex vivo organotypic mouse hippocampal slice cultures, PSD volume was increased with cLTP, consistent with previous observations, and that was compromised by Aβ and prevented by the AβCore. In conclusion, my findings indicate that the AβCore can prevent the Aβ-associated compromise of synaptic structure.
Phosphoglucomutase 5 and hepatocellular carcinoma
(University of Hawai'i at Manoa, 2024) Martinez Rodriguez, Verena; Hoffmann, Peter R.; Cell and Molecular Biology
Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and accounts for the 5th most common cancer and the 3rd leading cause of cancer mortalities worldwide. HCC occurs most commonly in patients with underlying liver diseases such as cirrhosis caused by hepatitis B and C virus infections. Since patients with early-stage HCC do not present overt symptoms, this disease is often detected in very late stages. Curable treatment options are currently very limited and include surgery and liver transplantation, which is why it is of utmost importance to find suitable biomarkers for early detection. Phosphoglucomutase 5 (PGM5) belongs to the α-D-phosphohexomutase (PHM) family comprised of five members in humans and mice. Members of this family play roles in catalyzing essential metabolic reactions with PGM1 representing the most widely expressed and fully characterized in terms of structure and function. In contrast to the other family members, PGM5 is predominantly expressed in muscle tissues and localizes to the adherens junctions of smooth muscle cells (SMCs). Also, in contrast to the other PHM proteins that exhibit enzyme activity, data suggest that PGM5 may play a structural function within cells. This is supported by studies finding interactions between PGM5 and cytoskeletal proteins in a manner that enhances cell-cell adhesion and contractility. For reasons that have not been determined, decreased levels of PGM5 mRNA have been shown to be a common characteristic in various cancers, including HCC. However, little is known regarding the role that PGM5 or its decreased levels play in tumor development and progression. This thesis dissertation presents the results of our investigation of the role that PGM5 may play in HCC progression with the following specific objectives: (i), determine whether PGM5 protein levels change in HCC in both human and mice, (ii) evaluate whether the localization of PGM5 is altered during HCC progression in the liver, and (iii) determine the in vivo effects of PGM5 overexpression and KO on HCC progression using mouse models. The data presented herein show that, in human patient biopsies of HCC and other types of cancer, PGM5 protein levels are decreased. By using a mouse model of HCC, we show that PGM5 protein levels decrease over time as the tumor progresses. In the healthy liver, PGM5 is located in the smooth muscle cells surrounding arteries and veins of healthy blood vessels, most notably at the portal triads of the classic hepatic lobule. When HCC forms and progresses, this organized structure completely changes, causing a decrease in PGM5 protein levels. At late-stage HCC, abnormal masses enriched in PGM5 form. Interestingly, these structures are not characterized by smooth muscle surrounding blood vessels and instead are clusters of bile ducts. Our findings revealed that low PGM5 protein levels decreased with HCC progression and tissue localization was altered from vascular smooth muscle in healthy livers to bile duct enriched clusters found in larger tumor mass. Overexpressing PGM5 in a transgenic mouse model was found to coincide with decreased tumor progression in female mice, but issues were found in this experimental model that limited any conclusive interpretation regarding the direct effects of increasing PGM5 on HCC outcome.
Diet-associated epigenetic modulation of phenotypes relevant to autism spectrum disorders
(University of Hawai'i at Manoa, 2024) Allan, Nina Picher; Maunakea, Alika K.; Cell and Molecular Biology
Autism Spectrum Disorder (ASD) is a complex neurodevelopmental condition influenced by genetic and environmental factors. This study explores the role of dietary factors as an environmental influence in ASD pathogenesis and phenotype. Maternal high-fat diet-induced obesity prior to conception, but not during gestation, is associated with ASD-like behavioral symptoms and transcriptomic alterations in offspring. Additionally, adherence to a modified ketogenic diet in children with ASD leads to improvements in behavior and increases in gut microbiome diversity. Mechanistic investigations suggest a potential link between dietary interventions, gut microbiota, and epigenetic modifications, highlighting the dynamic interplay between diet and ASD phenotype. These findings contribute to our understanding of environmental influences on ASD and offer insights for future therapeutic strategies targeting dietary interventions.
In vitro and in vivo investigation of SARS-CoV-2 associated injury and functional impairments in the testis.
(University of Hawai'i at Manoa, 2023) Giannakopoulos, Stefanos; Verma, Saguna; Cell and Molecular Biology
Traditionally associated with shortness of breath and lung injury, the 2019 severe-acute respiratory syndrome coronavirus 2 pandemic presented several other alarming features in COVID-19 patients, including cytokine storm and injury of other organs such as the kidney and testis. Infected males also experienced symptoms such as orchitis, erectile dysfunction, reduced testosterone levels, and low sperm count. These testicular symptoms are accompanied by leukocyte infiltration, sloughing of the seminiferous tubules, and germ cell death. Cohorts of studies from COVID-19 moderate, severe, and critical patients have revealed additional hormone alterations as well as functional damages to function of the male reproductive tract. These include decreased semen volume, lower sperm count, and decreased testosterone, follicle-stimulating hormone, and luteinizing hormone. Additional sperm qualitative analysis by these studies revealed altered sperm morphology, decreased sperm viability/vitality, and progressive motility as well as increased pro-inflammatory molecules in the semen such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α) and interleukin-1-beta (IL-1β). Further, lower sperm count, and reduced sperm motility was correlated with increased sperm DNA damage and reactive oxygen species. While most of these parameters recover rapidly upon recovery from the disease, long-term follow up of recovered patients suffering from long-COVID revealed lower testosterone levels and reduced sperm parameters up to 12 months after disease onset. Despite these abnormal findings, SARS-CoV-2 RNA or infectious virions are not detected in the semen of COVID-19 individuals. Therefore, it is still unclear what the mechanism of testicular injury is and what are the specific immune mechanisms responsible for contributing to SARS-CoV-2 testicular short-term and long-term pathogenesis. We here demonstrate that neither human multicellular 3D organoid models nor 2D models consisting of different testicular cell types can support productive SARS-CoV-2 infection. This suggests that the mechanism of testicular injury observed in COVID-19 patients is a bystander effect of infection. Therefore, the objective of this study was to utilize novel in vitro and in vivo tools to define the underlying molecular mechanism and potential consequences of SARS-CoV-2 bystander effects in the testes. For Aim 1, we utilized different 2D and 3D testicular models, optimized in our lab, to characterize the effect of the SARS-CoV-2 infection-derived inflammatory mediators and antigens, on the testicular cells. The K18-hACE2 mouse model has been extensively used to study SARS-CoV-2 pathogenesis and partially recapitulates human disease. Therefore, for Aim 2 we used these hACE2 mice to characterize various aspects of testicular pathogenesis associated with SARS-CoV-2 in vivo. These include histopathology studies during the acute stage and short-term after recovery and the correlation between systemic cytokine storm and different markers of testicular injury and function. For Aim 3 we will use high throughput RNA-seq approach to identify the molecular signatures associated with SARS-CoV-2 infection in the K18-hACE2 mouse testis. The outcome of this study provides significant mechanistic insights into the testicular injury and short-term effects of SARS-CoV-2 on testicular function. It also addresses fundamental gaps in our understanding of how this virus affects male reproductive health. Tools and methodologies developed in this study will also provide a platform for investigating the bystander effects of other viruses such as Ebola virus, Dengue, and West Nile as well as guide therapeutic approaches in the future.
THE ROLE OF TCF21 IN CARDIAC FIBROBLAST CELL NUMBER AND EXTRACELLULAR MATRIX REMODELING
(University of Hawai'i at Manoa, 2023) Chen, Jasmine Ka Yan; Tallquist, Michelle D.; Cell and Molecular Biology
Heart disease remains a major health burden in the United States, and complications from injury to the heart, including fibrosis, may lead to heart failure. Cardiac fibroblasts are the cells most intimately implicated in the cardiac injury response. Elucidating the molecular basis behind cardiac fibroblast injury phenotypes can help us to develop more targeted approaches for treating adverse myocardial remodeling. Cardiac fibroblasts reside within the heart and, during homeostatic conditions, will deposit extracellular matrix (ECM) and maintain vascularity. However, when activated following external stimuli, fibroblasts proliferate, remodel ECM, and recruit inflammatory cells to the area. Excessive deposition of ECM proteins can accelerate the progression of uncontrolled fibrosis to heart failure. Our lab has identified transcription factor 21 (Tcf21) as a critical gene required for epicardial cardiac fibroblast differentiation. Based on preliminary data and previously published work, Tcf21 has been shown to regulate proliferation and ECM remodeling. My hypothesis is that cardiac fibroblast specific deletion of Tcf21 will reduce cardiac fibroblast numbers and ECM remodeling, which is exacerbated following injury. In my dissertation work, I have discovered the role of Tcf21 in adult mouse hearts with a transgenic mouse model after cardiac injury.
Activity and Centromere Targeting of a Maize Retrotransposon Integrase
(University of Hawai'i at Manoa, 2023) Shontell, Ryan; Presting, Gernot; Cell and Molecular Biology
Though closely related in their structure and activities, retroviruses and long terminal repeat (LTR) retrotransposons vary significantly in their target site selection. The mechanisms and host co-factors targeted by the integrase protein (IN) that guides this targeting specificity, have been well characterized in many of the retroviruses and several of the LTR retrotransposons of yeast. The mechanisms guiding many of the retrotransposons have yet to be determined, however. In this dissertation I worked to determine whether centromeric histone H3 (cenH3) nucleosomes are the target guiding centromere-specific retrotransposon (CR) IN to the centromeres of their host’s genomes. The determination of the mechanism of CR centromere targeting has been elusive and could provide some answers to long-standing questions about centromere evolution. I leveraged Google Deepmindʻs AlphaFold2 and AlphaFold2-Multimer to predict structures of the protein and to model protein-protein interactions with close attention to the C-terminal domain (CTD) of the CR1, CR2, CR4, and CR5 sub-family members, the latter two having lost centromere specificity in more recent integration events. I found that the non-centromere targeting CR sub-family members contained surface exposed substitutions disrupting a hydrophobic pocket in the SH3 domain and an additional substitution in the CR motif disrupting an additional hydrophobic surface. The alpha-helical CR motif was predicted to act as an arginine anchor, a common feature of chromatin interacting proteins, binding to the acidic patch formed by the H2A-H2B dimer. Through these findings I developed a model that the CR IN interacts directly with the chromatin through the H2A-H2B acidic patch via the CR motif and that the SH3 domain may interact with an additional co-factor guiding centromere-specificity or that the presence of cenH3 on the nucleosome impacts the accessibility of the acidic patch.I next worked on the generation of bioactive CR IN from E. coli. I found that the CR IN is highly insoluble under a large range of conditions in E. coli and requires solubilization of inclusion bodies and refolding of the purified IN to produce bioactive protein. I employed a fractional factorial method by flash dilution to quickly identify refolding additives that allowed the production of soluble IN. With these refolding conditions I produced soluble IN as high as 1.0 mg/mL (17.56 µM) in high salt buffers. Refolded CR IN catalyzed both 3’-end processing and strand transfer as validated by Nanopore sequencing. To our knowledge this is the first reported case of an active plant retrotransposon in vitro. Finally, I studied whether cenH3 nucleosomes are targeted by CR IN. I performed a series of pulldown experiments and found that the CR IN interacted directly with the H2A-H2B dimer as was modeled in the protein-protein interaction models. In vitro nucleosome reconstitution followed by electromobility shift assays and demonstrated that CR IN CTD bound both H3 and cenH3 nucleosomes. Strand transfer assays with naked or nucleosome-bound (H3 and cenH3) target DNAs revealed that CR2 IN is only active on nucleosomal target DNAs while CR5 IN is highly active on naked DNA targets. Moreover, CR5 IN binds to naked DNA in a concentration dependent manner. Taken together, cenH3 nucleosomes alone do not appear to be the specific target of the CR IN although the nucleosome architecture does play a role in the interactions of CR IN with target DNA. It is likely that an additional centromeric co-factor is necessary for specific integration at the centromere.
Methods To Characterize Anti-viral Immune Responses Reveal Potent Adjuvants, Novel Antibody Genes, And Serological Techniques To Address Viral Outbreaks
(University of Hawaii at Manoa, 2022) Haun, Brien Keith; Lehrer, Axel T.; Cell and Molecular Biology
The COVID-19 pandemic has claimed millions of lives, and its causative agent, SARS-CoV-2, has infected hundreds of millions globally. The highly contagious nature of this respiratory virus has spurred massive global efforts to develop vaccines, therapeutics, and tests at record speeds. Recombinant subunit vaccine candidates take longer to produce than mRNA, which has delayed their availability. Additionally, identifying a suitable adjuvant is especially beneficial for this vaccine technology. Passive immunotherapeutics developed for the previous SARS outbreak (caused by SARS-CoV) were initially used while new monoclonal antibodies (mAb) were produced. These SARS-CoV-2 targeted mAb were sourced from convalescent plasma and mice while vaccines were being developed. Diagnostic tests to document viral infection were quickly developed and widely distributed early in the pandemic. Similarly, IgG and IgM testing for serosurveillance commenced almost immediately. However, resource-limited regions of the world were restricted in their access to these assays, resulting in minimal reporting of the dynamics of the pandemic in these regions. To address these key aspects of responding to the COVID-19 pandemic, we conducted a study in BALB/c mice to identify a suitable adjuvant on readily available SARS-CoV-2 spike S1 model antigens in parallel with the development of a prefusion stabilized full-length spike vaccine candidate. We identified CoVaccine HTTM as producing robust Th1 and Th2 responses compared to Alhydrogel or S1 alone. We utilized samples from a Zika virus vaccine study in cynomolgus macaques for antibody discovery. We demonstrated that single-cell RNA sequencing (scRNA-seq) is an effective method for analyzing vaccine responses while collecting antibody V(D)J) genes for recombinant expression and development. We identified several neutralizing antibodies with this technique and revealed light chain V-genes not currently available in public databases. Lastly, we developed and deployed a multiplexed immunoassay (MIA) for serological analysis of exposure to SARS-CoV-2, NL63, Dengue, and Chikungunya viruses in Liberia, West Africa. This assay was also able to quantitate antibody concentrations to SARS-CoV-2. Furthermore, we adapted this assay for a multiplexed inhibition test (MINT) which could determine if serum antibodies could inhibit SARS-CoV-2 spike protein from binding its cognate receptor, human ACE-2, in a multiplexed fashion. This work demonstrates methods to expedite responses to viral outbreaks through synergistic approaches in vaccine, immunotherapeutic, and assay development. Adjuvant testing in tandem with protein engineering allowed for a recombinant protein-based vaccine candidate to rapidly be tested in mice and macaques. Our methods for vaccine analysis stemming from MIA and scRNA-seq enabled single-cell transcriptome and paired heavy chain/ light chain antibody sequence resolution in a single experiment. These efforts may help expedite mAb development in future outbreaks and highlights the potential that the use of cynomolgus macaques may provide for rapid therapeutic antibody discovery and development. Lastly, our MIA and MINT assays developed to support vaccine development could perform effective serological studies in a resource-limited world region. This work, in total, has global health implications for addressing viral outbreaks.
Exocyst Inactivation In Urothelial Cells Leads To Fn14 Upregulation And Cell Death In A Mouse Model Of Prenatal Ureter Obstructions
(University of Hawaii at Manoa, 2022) Villiger, Ross; Fogelgren, Benjamin C.; Cell and Molecular Biology
Congenital obstructive nephropathy (CON) is the leading cause of chronic kidney disease (CKD) and end-stage renal disease in children. This disorder is usually detected as hydronephrosis by prenatal ultrasounds. The most common cause of CON is obstruction at the ureteropelvic junction (UPJ), where the renal pelvis transitions to the upper ureter. EXOC5 is the central stabilizing subunit of the exocyst, an octameric protein complex that regulates exocytosis of secretory vesicles to the plasma membrane. The Fogelgren laboratory previously generated a novel EXOC5-conditional knockout (EXOC5-CKO) mouse using the Cre/loxP technology to inactivate EXOC5 in ureteric bud-derived epithelial cells. The EXOC5-CKO mice had severe bilateral UPJ obstructions, hydronephrosis, and succumbed to neonatal death. In these mice, UPJ obstruction was preceded by failure of urothelial stratification and cell death at E17.5, followed by a fibroproliferative response by the underlying mesenchyme. By transcriptomic microarray, we identified the Fn14 gene as being robustly upregulated in the EXOC5-CKO embryonic ureter at E16.5. Fn14 is a transmembrane tumor necrosis factor (TNF) receptor superfamily member with a single known ligand, TWEAK, which can activate different pathways depending on cell context, including programmed cell death. We demonstrated that ureter obstructions can be rescued in EXOC5-CKO mice by administering a pan-caspase inhibitor zVAD-fmk at E16.5. We also found inhibited urothelial stratification alone did not drive cell death during ureter development. Instead, exocyst-perturbed urothelial cells in vivo and in vitro have disrupted autophagy which activated NF-κB signaling and cell death. To study the role of Fn14 in renal and urological diseases, we generated a global Fn14-knockout (KO) mouse using CRISPR-Cas9 gene editing. Fn14-KO mice had ameliorated renal pathology in a cisplatininduced CKD model. Our findings support the hypothesis that urothelial cell death is a critical event leading to UPJO pathogenesis and that Fn14 signaling contributes to cell death in renal and urological pathologies.
Investigation Of The Protective Role Of The N-terminal Beta Amyloid Fragments Against Beta Amyloid-induced Gliotoxicity In Neuroinflammation
(University of Hawaii at Manoa, 2021) Lantz, Megan Jean; Nichols, Robert A.; Cell and Molecular Biology
Alzheimer’s Disease (AD) is a progressive neurodegenerative disease and the most common form of dementia. It is characterized by the accumulation of amyloid beta (Aβ) and the microtubule-associated protein tau into extracellular senile plaques and intraneuronal neurofibrillary tangles, respectively, in the brain over the course of many years. Additional histopathological hallmarks of AD include synaptic dysfunction and loss, the loss of neurons in select brain regions, brain atrophy and more recently, the induction and chronic presence of neuroinflammation. Symptoms of AD arise decades after Aβ begins to accumulate in the brain and include increasing memory loss, cognitive deficits, personality changes and language dysfunction. AD is the sixth leading cause of death in the United States and poses a tremendous financial burden on the health care system. To date, a cure remains elusive and current treatments are only minimally efficacious, making research into AD imperative. The focus of the majority of AD research has been on the mechanisms involved in neuronal dysfunction and death; however, more recently a paradigm shift has occurred in the field, expanding the research into the role of glial cells in AD as well. Astrocytes and microglia, two types of glial cells, can modulate their phenotypic state depending on the signals present in the brain parenchyma. At physiological concentrations Aβ has positive neuromodulatory functions; however, the accumulation of soluble, oligomeric Aβ peptide to pathological concentrations (high nM to μM) activates astrocytes and microglia from a normal ‘resting’ state in which they provide many diverse and beneficial neuromodulatory functions, to a reactive phenotype that exacerbates neuronal death. In the prodromal stage of AD, when soluble oligomeric Aβ begins to accumulate, astrocytes and microglia activate to a neuroprotective phenotype and begin to phagocytose the peptide to reduce the concentration in the brain and, thus, mitigate the toxic effects of full-length Aβ. However, as the disease progresses, these cells convert to a reactive phenotype and secrete proinflammatory cytokines, reactive oxygen and nitrogen species, and complement proteins that contribute to the persistent neuroinflammation that is characteristic of AD. Our laboratory previously showed that the endogenous N-terminal fragment of Aβ, compassing residues 1-15/16, termed the N-Aβ fragment, retains the neuromodulatory functions of full-length Aβ. Through further structure-function studies we refined the activity of the N-Aβ fragment to a critical hexapeptide core sequence encompassing residues 10-15 (YEVHHQ), termed the N-Aβcore. We also reported that these two N-terminal Aβ fragments (collectively termed N-Aβ fragments), protect against full-length Aβ-induced cellular neurotoxicity and synaptic dysfunction in neurons as well as behavioral dysfunction in whole animals. Here, we aimed to characterize the neuroprotective potential of the N-Aβ fragment and the N-Aβcore against full-length Aβ-induced gliotoxicity via the modulation of the activation state(s) of astrocytes and microglia within a proinflammatory environment. Utilizing primary cortical glia cultures, I show that the N-Aβ fragment and N-Aβcore elicited differential calcium responses compared to full-length Aβ. In addition, concurrent administration of either of these N-Aβ fragments with Aβ mitigated the robust calcium responses in primary astrocytes and microglia with the application of Aβ alone. Moreover, I demonstrated that the N-Aβ fragment or N-Aβcore are able to mitigate the activation of these cells in two model systems, primary cortical glial cultures and organotypic slice cultures as well as reduce the expression of the proinflammatory cytokine TNFα and complement protein C3, two known neurotoxic proteins that contribute to disease progression with continued expression. Furthermore, the N-Aβ fragment and N-Aβcore were shown to attenuate oxidative stress, mitochondrial dysfunction and cellular death induced by pathological levels of full-length Aβ in astrocytes and microglia. Finally, exogenous application of the N-Aβcore reduced the amount of microglia-dependent loss of presynaptic elements in organotypic slice cultures from an APP/PSEN1 mouse model. Taken together, these findings suggest that the N-Aβ fragment and N-Aβcore protect against full-length Aβ-induced toxicity by modulating the activation state of two glial cell types, astrocytes and microglia, broadening our understanding of the cell populations in the brain involved in the potential therapeutic application of these protective peptides to AD.
In-vivo Non-viral Placental Gene Modulation
(University of Hawaii at Manoa, 2020) Elston, Marlee Jo; Urschitz, Johann; Cell and Molecular Biology
The placenta is critical to fetal development, health during pregnancy and the long-term health of the offspring because the placenta contributes to controlling the intra-uterine environment but placental function is still not completely understood. Currently, the lack of minimally invasive gene modulation methods for placental genes limits the types of investigations that can be performed. Safe and efficient gene therapy to the placenta opens a myriad of possibilities. Additionally, the link(s) between placental function, the intrauterine milieu, fetal health and disease development later in life is poorly understood. In this dissertation I demonstrate, the delivery of gene cassettes non-virally by utilizing ultrasound cavitated microbubbles carrying plasmid DNA as well as the ability to modify the placental genetics by piggyBac mediated transgenesis. In previous studies, these methods have been used to modulate gene expression in a variety of different organs but never in the placenta. The transgene cassette is trophoblast specific and spatial targeting is achieved by use of an unfocused ultrasound transducer. This could have applications in the treatment of fetal growth restriction, intra-uterine growth restriction, fetal overgrowth and pre-eclampsia.
Asbestos Induces Mesothelial Cell Transformation Via HMGB1-driven Autophagy
(University of Hawaii at Manoa, 2020) Xue, Jiaming; Yang, Haining; Cell and Molecular Biology
Asbestos causes malignant transformation of primary human mesothelial cells (HM), leading to mesothelioma. The mechanisms of asbestos carcinogenesis remain enigmatic, as asbestos is toxic HM in cell culture. However, some asbestos-exposed HM escape cell death, where they accumulate DNA damage and may eventually become transformed. We have previously demonstrated that upon asbestos exposure, HM and reactive macrophages release the high mobility group box 1 (HMGB1) protein and trigger chronic inflammation. HMGB1 is commonly detected next to asbestos deposits in the staining of tissue biopsy. Also, serum HMGB1 is elevated in the sera of asbestos-exposed individuals and mice. While we seek for other serum biomarkers for early signs of asbestosis, we found higher levels of the autophagy marker ATG5 in sera from asbestos-exposed individuals compared to unexposed controls. This finding prompted us to continue to investigate the underlying mechanisms. We discovered that the release of HMGB1 upon asbestos exposure up-regulated autophagy, allowing an increased number of HM to survive asbestos toxicity. The HMGB1 silencing inhibited autophagy and promoted asbestos-induced HM death, therefore reducing the risk of asbestos-driven HM transformation. We demonstrated that autophagy was activated by the cytoplasmic and extracellular fractions of HMGB1 via the RAGE receptor and the Beclin 1 pathway, while nuclear HMGB1 did not participate in this process. We validated our findings in a novel unique mesothelial cell-specific conditional HMGB1-knockout (HMGB1-cKO) mouse model. Compared to HMGB1 wild-type mice, mesothelial cells from HMGB1-cKO mice showed significantly reduced autophagy and increased cell death. Autophagy inhibitors chloroquine (CQ) and desmethylclomipramine (DCMI) increased cell death and reduced asbestos-led foci formation. In summary, HMGB1 released upon asbestos exposure induces autophagy, which promotes HM survival and malignant transformation.
Investigation Of The Impact Of Hyperglycemia On The Immune System In Type 2 Diabetes
(University of Hawaii at Manoa, 2020) Pang, Alina Psalm Soranaka; Corley, Michael J.; Cell and Molecular Biology
Type 2 diabetes (T2D) is a prominent and chronic cardiometabolic disorder affecting the way the body processes glucose. There is a need for effective therapies as current options target symptoms of T2D in relation to glucose metabolism and hyperglycemia. However, a comprehensive understanding of the effects of hyperglycemia upon the immune system in the pathogenesis of T2D remain unknown. Insight into T2D impact on the immune system will provide a better understanding of related complications and comorbidities that relate to immune dysfunction and a weakened immune system that leads to poorer health outcomes in diabetes and uncover novel interventions to improve disease outcomes. In type 2 diabetes, hyperglycemia, or high blood sugar is a major clinical concern due to long-term complications due to untreated hyperglycemia. Hence, clinical treatments focus on controlling hyperglycemia with an achievable HbA1c target between 7%-8%; however, individuals with T2D vary dramatically in their achievable HbA1c. I hypothesize that hyperglycemia induces epigenetic, transcriptional, and metabolic alterations underlying immune dysfunction and inflammation and may contribute to poor treatment responses and exacerbation of complications. The goal of this dissertation centers on the identification of metabolic, immunological, and epigenetic aberrations in immune cells of individuals with T2D that associated with hyperglycemia to gain insight into biological mechanisms underlying immune dysfunction in T2D. I used real-time live-cell metabolic analyses to examine whether the most prescribed T2D medication to lower glycemia levels, metformin, impacted immune cell metabolism. Results identified that metformin treatment significantly and dose-dependently impacted human monocyte cell metabolism by inhibiting mitochondrial oxidative phosphorylation. In contrast, metformin did not drastically impact T cell metabolism. Next, we used flow cytometry profiling of monocyte, B cells, CD4 T cells, and CD8 T cell populations in 29 individuals, 25 T2D (HbA1c >7) and 4 healthy (HbA1c <7), and examined association with amount of time (years) a participant was diagnosed with T2D in 25 individuals and found that classical monocytes was associated with T2D duration. Epigenetic and transcriptomic assessment of DNA and RNA was isolated from purified cell sorted monocyte cells of T2D (n=25) and non-diabetic individuals (n=4) identified significant relationships between gene expression and DNA methylation states with glycemia. Genes associated with metabolic pathways, cell growth and monocyte immunometabolism were affected. Lastly, using single cell RNA-sequencing to assess the impact of glucose treatment on immune cells at single cell resolution, I identified conserved and differentially expressed immune regulatory, chemokine ligands, and ribosomal genes based on diabetic status. Altogether, this dissertation provides evidence for a dysregulation of molecular and epigenetic mechanisms in peripheral monocytes of individuals with T2D that relates to glycemia control. These novel findings may underlie the diabetes complications and weakened immune features of T2D as sustained hyperglycemia persist across time, possibly contribute to immune-based therapies for treating T2D, and provide insight into uncovering the immune dysfunction basis of T2D pathogenesis.
The Role Of The Fibroblast In Structuring The Cardiac Microenvironment
(University of Hawaii at Manoa, 2020) Smith, Jill T.; Tallquist, Michelle D.; Cell and Molecular Biology
Fibroblasts are important non-muscle cells in the heart. Their contribution to cardiac development, physiology, and pathology is evident. The primary physiological role of the cardiac fibroblast is the production of extracellular matrix (ECM) network that determines cardiac stiffness and structure and also coordinates extracellular signaling. In addition to their critical role in ECM synthesis, fibroblasts produce cytokines and proteases that directly affect ECM organization and turnover, and influence neonatal cardiomyocyte proliferation. Our knowledge of fibroblast biology is based largely on in vitro studies that cannot recapitulate the complex cellular and biophysical milieu of the working heart. In my dissertation work, I have investigated the role of fibroblasts in the perinatal and adult heart using transgenic deletion of these cells. My overall hypothesis is that ECM deposition and organization by cardiac fibroblasts are required for normal heart development and homeostatic maintenance, and that perturbation of these cells leads to accelerated heart dysfunction after injury.
Genetic deletion and inhibition of selenoprotein K reduces melanoma growth and metastasis by altering intracellular calcium
(University of Hawaii at Manoa, 2019) Marciel, Michael P.; Hoffmann, Peter R.; Cell and Molecular Biology
Melanoma is the deadliest form of skin cancer, resulting in over 9,000 deaths annually in the United States alone. Along with limiting ultraviolet exposure, it is vital that new therapies are generated that target melanoma progression. A recent interest in targeting store operated calcium entry (SOCE) as a treatment for different cancers types has recently emerged. SOCE is the main mechanism by which non-excitable cells import calcium (Ca2+) from the extracellular space and this process is critical for normal growth, proliferation, and migration in a variety of cell types. Several studies have demonstrated that knockdown or complete genetic deletion of SELENOK reduced SOCE in immune cells and retarded their ability to differentiate, proliferate, and migrate. However, the effect of inhibiting SELENOK in cancer has not yet been investigated. We hypothesized that deletion of SELENOK in melanoma cells may decrease their ability to proliferate and metastasize by a reducing SOCE. This hypothesis was tested using three approaches (i) SELENOK null mice were crossed with a murine model of melanoma, and melanoma progression in the absence of SELENOK was observed, (ii) SELENOK null melanoma cells were generated using a CRISPR-Cas9 approach in a human melanoma cell line, and tested for their ability to proliferate and migrate in vitro, and, (iii) an inhibitor based on the structure of SELENOK was generated and tested for its ability to inhibit the SELENOK mediated palmitoylation in a cell free environment. The data presented herein show that SELENOK deletion inhibited calcium flux and calcium dependent signaling in vitro which reduced that capacity of melanoma cells to migrate and proliferate. Furthermore, SELENOK deletion decreased primary growth and metastasis of tumor cells in a spontaneous mouse model of melanoma. Last, the inhibitor based on SELENOK was shown to be able to disrupt SELENOK mediated palmitoylation and thus may serve as a basis for future inhibitors. This dissertation will attempt to outline the data representing the first investigation into the role of SELENOK in metastatic melanoma.
Overexpression Of Chitotriosidase-1 Modulates Macrophage Function And Alters Plaque Morphology In Hyperlipidemic Mice
(University of Hawaii at Manoa, 2019) Yap, Jonathan; Boisvert, William A.; Cell and Molecular Biology
Cardiovascular diseases (CVD) are the leading cause of mortality in the world. Responsible for nearly 1/3 of all deaths globally, CVD poses extreme public health challenges and a growing economic burden. Atherosclerosis is fundamental to CVD, and is characterized by the accumulation of plaque within arterial blood vessels. Atherosclerotic plaques are the result of lipid deposition in the subendothelial space, which initiates monocyte infiltration and formation of lipid-laden foam cells derived from macrophages. The inflammatory cascade that follows, promotes plaque formation and development. Disease progression ultimately leads to a number of atherosclerotic pathologies including myocardial infarction and ischemic stroke. Clearly, our greatest efforts are required in exploring novel therapeutic perspectives that ameliorate inflammation and prevent dysfunctional cholesterol metabolism. My work has been directed at investigating the potential role of macrophage chitotriosidase-1 (CHIT1) overexpression in dampening production of pro-inflammatory cytokines and inhibiting macrophage foam cell formation. We generated an atherosclerotic mouse model constitutively overexpressing CHIT1 by crossing hyperlipidemic Ldl-r-/- mice with transgenic, CHIT1 overexpressing mice. We carried out in vitro experiments aimed at elucidating the effect of CHIT1 overexpression on macrophage inflammatory mechanisms, cholesterol metabolism, and invasion. In vitro, macrophage overexpression resulted in diminished mRNA transcription of important pro-inflammatory mediators and increased expression of cytokines involved in immune cell migration and anti-inflammatory processes. We also observed that CHIT1 overexpression in BMDM enhanced macrophage invasiveness. The CHIT1-overexpressing, atherosclerotic mouse model (Ldlr-/--CHIT1-OE) facilitated a long-term in vivo mouse study. Animals in the study were fed a high-fat diet for 12 weeks which resulted in robust atherosclerotic progression. Although CHIT1 overexpression in Ldlr-/- mice did not affect plaque development when compared to control mice, we were able to observe significant differences in plaque morphology related to the deposition of ECM components. This unexpected finding may suggest that CHIT1 overexpression affects plaque stability in atherosclerotic mice. The possibility of CHIT1 mediated athero-protection may in fact lie in its ability to augment ECM composition in atherosclerotic plaques.
Regulation Of Hippocampal NMDA Receptor Signaling, Memory And Synaptic Plasticity By The Jip1 Scaffold Protein
(University of Hawaii at Manoa, 2018-08) Robles, Michael J.; Cell & Molecular Biology (Neuro)
Learning and memory rely upon mechanisms of activity-dependent plasticity such as hippocampal long-term potentiation (LTP) and long-term depression (LTD). At least six types of plasticity exist to function as various types of coincidence detectors. Ultimately, plasticity signaling at the synapse flows, among other pathways, through mitogen-activated protein kinases (MAPKs) signaling networks, interfacing the input (postsynaptic receptors) and the output (phosphorylation of proteins). A wide body of research has contributed to elucidating the role for MAPKs, such as ERK1/2, in memory, but less is known about the function of other MAPKs that are involved with fear memory such as the c-Jun N-terminal kinases (JNKs) that interact with the scaffold protein JNK-interacting proteins (JIPs). JIPs and JNKs have a variety of important functions in the brain that are not yet fully understood. Here, we studied the contribution of JIP1 to JNK-mediated learning and memory. Initial studies of excitotoxicity show that mice harboring mutations in the Jip1 gene that selectively blocks JIP1-mediated JNK activation results in the same phenotype as found for JNK3 isoform-deficient mice. As JNKs are involved in regulating fear memory, this raised the question as to whether such JIP1 mutants have the same changes in memory as that of carrying JNK deletion. We addressed this question by employing behavioral tests for spatial memory, contextual fear conditioning and electrophysiology. As seen with JNK-deficiency or with pharmacological inhibition of JNK in mice, mice with the Jip1 gene that selectively blocks JIP1-mediated JNK activation, displayed similar improvements in spatial memory, fear memory and plasticity. Furthermore, these mutant mice exhibited increased NMDA receptor currents, increased NMDA receptor-mediated gene expression, and a lower threshold for induction of hippocampal long-term potentiation. In addition, our studies with mice carrying mutation in JIP1 kinesin-1 binding domain, which normally facilitates the transport and assembly of JIP1/JNK signaling module to the neuronal processes, showed normal memory suggesting that that JIP1/JNK signaling is sufficient for the regulation of the NMDA receptor function independent of its cellular localization and the function as an adaptor between motor proteins and their
Characterizing The Protection Of An N-Terminal Active Core Peptide Within Β-Amyloid Against Β-Amyloid Neurotoxicity
(University of Hawaii at Manoa, 2018-05) Forest, Kelly; Cell & Molecular Biology (Neuro)
Alzheimer’s disease (AD) is the most common cause of dementia in the aging population. As the disease progresses, AD leads to cognitive deficits affecting memory, changes in personality, and language dysfunction. AD is characterized by the pathological extracellular accumulation of fibril β-amyloid (Aβ) into senile plaques and the intraneuronal accumulation of the microtubule-associated protein tau as neurofibrillary tangles. Initially, insoluble fibrillary Aβ was believed to be central to disease pathogenesis, but more recent evidence implicates soluble oligomeric Aβ as the trigger behind the earliest cognitive deficits in AD. Under normal conditions, Aβ at low, physiological levels (pM) functions as a positive neuromodulator, enhancing synaptic plasticity and function. In addition, mouse models lacking Aβ show deficits in cognitive function and loss of synapses, suggesting, therefore, a vital role of Aβ in the maintenance of synaptic activity. On the other hand, pathological levels of Aβ (high nM-μM) cause irreversible degeneration of neuronal processes and the loss of synaptic function and connections in select areas of the brain. Previously, we reported that an endogenous N-terminal fragment derived from full-length Aβ retains the latter’s positive neuromodulatory activity and, notably, protects against Aβ-induced synaptic and memory deficits. Furthermore, through subsequent mutational analysis, we found a core sequence (YEVHHQ: N-Aβcore) within the N-terminal fragment accounting for its activity. Here, we aimed to characterize the neuroprotective potential of the N-Aβcore against Aβ-induced neuronal and synaptic damage, while elucidating the neuroprotective mechanism(s) of the N-Aβcore. Utilizing a neuronal toxicity model (rodent hybrid neuroblastoma cells transfected with α4β2 nicotinic acetylcholine receptors, nAChRs), the N-Aβcore was shown to retain receptor-linked activity. Subsequent mutational analysis demonstrated that the two histidine residues, and to a lesser degree, the tyrosine residue in the N-Aβcore are essential for this activity. In comparison to the sustained-elevated Ca2+ response elicited by Aβ, the N-Aβcore and N-terminal fragment displayed differential Ca2+ responses, suggesting the activation of an alternative, Aβ-independent pathway. In addition, the N-Aβcore was shown to be neuroprotective against Aβ-induced oxidative stress, ER stress, mitochondrial dysregulation, apoptosis, and synaptic dysfunction. We have also previously reported an increase in activity of various mitogen-activated protein kinases (MAPKs) in response to prolonged, sustained Aβ exposure. We therefore addressed whether the N-Aβcore has an impact on Aβ-linked MAPK activation. In our neuronal toxicity model, the N-Aβcore reduced the Aβ-induced MAPK activity. Interestingly, preliminary evidence indicates that low levels (pM) of the N-Aβcore nearly abolished c-Jun N-terminal kinase (JNK) activity. Furthermore, low levels of the N-Aβcore showed an increase in cAMP response element-binding protein (CREB) activity and expression, which has been shown to be downregulated in AD and AD models. Taken together, these findings suggest that the N-Aβcore is neuroprotective against Aβ-induced neuronal and synaptic toxicity by partially inhibiting Aβ binding to target receptors and subsequently activating an Aβ-independent neuroprotective pathway.

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