Pharmacy Dissertations and Theses

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  • Publication
    Bioenergetic Regulation of Neuronal Vacuolar-type H+- ATPase
    (University of Kansas, 2019-12-31) He, Lucy
    Neuronal vacuolar-type H+-ATPase (V-ATPase) is an ATP-dependent proton pump that functions to acidify intracellular organelles such as lysosomes and synaptic vesicles, creating a proton gradient by which neurotransmitters can enter the vesicle through proton-coupled neurotransmitter transporters, a crucial step in neurotransmission (Moriyama, Maeda, & Futai, 1992). It is composed of two reversible domains, the integral V0 that allows proton translocation and catalytic peripheral V1 that is responsible for ATP hydrolysis. The V-ATPase regulates its activity through a process called reversible disassembly. When the V0 and V1 domains assemble, V-ATPase is activated and allows the influx of protons. When the domains disassemble, V-ATPase is inactivated, and proton transport does not occur (Beltran & Nelson, 1992). V-ATPase assembly was previously demonstrated to be regulated by glucose in yeast (Kane, 1995) and some mammalian cells (Toei, Saum, & Forgac, 2010), but whether and how glucose regulates neuronal V-ATPase is unclear. This study investigates the effect of bioenergetic substrates on neuronal V-ATPase assembly. Neuro2a (N2a) cells were differentiated for 96 hours, glucose-deprived overnight, and then treated with substrates such as glucose, beta-hydroxybutyrate, sodium pyruvate, creatine phosphate, and creatine monohydrate for 20 minutes prior to cell lysate preparation. To study V-ATPase assembly, assembled V-ATPase were captured through co-immunoprecipitation. Equal amounts of cell lysate were incubated with V1 antibody-coupled resin. Immunoblotting was then performed on the eluate to detect the V1 and V0 domains. The density of the bands was quantified and the ratio of V0 and V1 domains was used to determine V0/ V1 assembly. The results demonstrate changes in glucose availability (deprivation/stimulation) did not impact V-ATPase assembly in differentiated N2a cells. Furthermore, the results show other bioenergetic substrates (pyruvate, beta-hydroxybutyrate, and creatine molecules) did not induce changes in neuronal V-ATPase assembly. Our results did not support the well-documented role of glucose regulation of V-ATPase assembly demonstrated in previous studies conducted in yeast and mammalian cells. Several pitfalls of this study may have contributed to the negative results. The glucose concentration for stimulation and incubation time for glucose-deprivation may not have been optimal for differentiated N2a cells; further investigation will be needed to optimize these conditions. The glucose concentration used for stimulation after overnight glucose-deprivation did not exceed the physiological glucose concentration; higher concentrations of glucose should be tested. Previous studies had various incubation times for glucose-deprivation that could be experimentally optimized. Furthermore, the experiments could be replicated in other neuronal cells or in primary neurons to validate the results.
  • Publication
    Receptor for advanced glycation endproducts (RAGE) modulates glyoxalase-1 enzyme activity in mouse models.
    (University of Kansas, 2019-12-31) Gore, Smruti Satishchandra
    Alzheimer’s disease (AD) is the most common cause of dementia in the world. AD is characterized pathologically by the presence of Amyloid-β (Aβ) plaques and tau neurofibrillary tangles (NFTs). Advanced glycation endproducts (AGEs), which are derived from alpha-dicarbonyls such as methylglyoxal (MG), form endogenously with physiological aging. These AGEs have been observed to co-localize with both the plaques and NFTs in AD patients’ brains. Receptor for advanced glycation endproducts (RAGE) has been implicated in the pathogenesis of AD. However, the exact mechanism by which RAGE contributes to AD pathology is only partially known. Glyoxalase-1(GLO1) is an important enzyme involved in the detoxification of precursors of AGEs, which serve as the highly reactive primary ligands for RAGE. GLO1 is found to be downregulated in the brains of advanced stage AD patients whereas RAGE is overexpressed in such brains. Aging is associated with increased generation and deposition of AGEs, resulting from non-enzymatic glycation (or oxidation) of proteins and lipids. Higher AGE formation is associated with a multitude of cellular and synaptic disturbances. Thus, to see the direct effect of age on enzyme activity, we first performed a study of wild type (WT) animals from 3 to 30 months and found that beyond 12 months of age, GLO1 activity significantly decreases. This shows that aging can be a major factor contributing to AD pathology through the downregulation of GLO1 activity. For uniformity, we used 12 months old mice for all further studies. In order to investigate whether RAGE directly modulates GLO1 enzyme activity and protein expression, we used previously generated multiple transgenic (Tg) non-AD mouse models by either genetic deletion of RAGE (RAGE knockout) or the introduction of signal deficient dominant-negative mutant RAGE (DNRAGE). They were, in the past, characterized for RAGE and subsequent RAGE-mediated signal transduction in our lab. GLO1 enzyme activity was measured spectrophotometrically while GLO1 protein expression was determined with western blotting. The Tg mice displayed either a) an increase in GLO1 enzyme activity, and/or b) an increase in GLO1 protein expression when compared with age-matched WT controls. Global RAGE knockout (RO) and neuronal RAGE knockout (nRKO) mice showed significantly higher GLO1 enzyme activity compared to WT controls. RO mice showed a significant increase in the protein expression but nRKO mice did not. Similarly, the mice with DNRAGE targeted to cortical neurons (neuronal DNRAGE) and microglia (DNMSR) exhibited an increase in GLO1 enzyme activity compared with WT mice but showed no significant change in protein expression. This differential effect on protein expression can be due to the difference in post-translational modification such as disulfide bridge formation or presence of GLO1 variations. It could also be the effect of normal or higher GLO1 activity in non-neuronal or microglial cells. Given the clear modulation of GLO1 by RAGE variation, we performed the GLO1 enzyme activity and expression assays in Tg mice modeling amyloid plaque development by expressing a mutant form of human APP leading to overproduction of Aβ (mAPP) to see if similar effects are observed in a mouse model showing AD-like pathology. We also crossed these mAPP mice with RO mice to develop RAGE-deficient mAPP mice (mAPP/RO). The mAPP mice showed a significant reduction in both GLO1 enzyme activity and protein expression. mAPP/RO mice and RO mice showed higher GLO1 activity and protein expression compared to mAPP mice. These findings highlight that RAGE-dependent signaling downregulates GLO1 enzyme activity and the deletion of RAGE protects against this decrease. Also, it supports the hypothesis that loss of RAGE-mediated signaling leads to an increase in GLO1 activity, whereas specific APP mutation (and Aβ overproduction) contributes to the decrease in GLO1 activity. Taken together, these data show that RAGE functions as an active modulator of GLO1 enzyme activity, thereby providing new insights into a mechanism by which the RAGE-dependent signaling cascade contributes to the pathogenesis of AD. Thus, RAGE deletion or blockade of RAGE signaling may be a potential target for developing treatments for preventing the progression of AD and related degenerative disorders through modulation of GLO1 function.
  • Publication
    Respiratory chain deficiency alters cellular proteostasis and triggers Alzheimer’s disease-like tau alterations
    (University of Kansas, 2019-12-31) Weidling, Ian
    Sporadic Alzheimer’s disease (AD) is defined clinically as a progressive brain disorder resulting in memory loss and, eventually, an inability to perform simple tasks. Pathologically, AD brains accumulate insoluble protein aggregates known as neurofibrillary tangles (NFTs) and amyloid plaques. Definitive diagnosis of AD requires the presence of NFTs and amyloid plaques. Furthermore, variants in genes coding for amyloid associate with early onset forms of the disease. For these reasons and more, removing amyloid plaques from sporadic AD patient brains has been the field’s major therapeutic target for decades. Unfortunately, clinical trials focused on treating AD through amyloid reduction continue to fail. The current standard of care for AD typically extends patient lifespan for months rather than years. The field needs new therapeutic targets and rescuing brain energy production represents a reasonable strategy. Reduced glucose utilization occurs early in AD brains and correlates fairly well with disease progression. Widespread mitochondrial dysfunction accompanies decreased glucose consumption. AD mitochondria display changes in number, ultrastructure, and enzyme activity. The evidence for mitochondrial dysfunction in AD is clear, however, the notion that defective mitochondria could initiate pathological cascades remains controversial. Thus, therapies aimed at mitochondrial function have been slow to reach clinical trials. The following studies examine the relationship between mitochondrial defects and AD pathology and provide evidence that mitochondrial dysfunction leads to AD-relevant retrograde responses. Retrograde responses maintain cellular homeostasis by adapting nuclear gene expression and cytosolic signaling pathways to changes in mitochondrial function. AD mitochondrial dysfunction likely initiates numerous retrograde responses, yet few studies examine defective mitochondria’s influence on AD pathology. Here, we provide evidence that reduced mitochondrial respiratory flux leads to AD-like tau alterations, including changes in splicing, conformation and oligomerization. Alzheimer’s disease cybrids recapitulate disease relevant tau alterations. Further experiments suggest mitochondrial function affects cellular proteostasis pathways including the mitochondrial unfolded protein response (mtUPR), integrated stress response (ISR), autophagy/mitophagy, and proteasome function. Although initial studies in C. elegans and rat hepatoma cells established a link between mtDNA depletion and mtUPR activation, we find mammalian cells downregulate the mtUPR upon mtDNA depletion. Instead, mtDNA depleted human cells activate the ISR, a pathway which alters cellular metabolism and halts general protein translation to preserve proteostasis during stress. Finally, we examine how mtDNA depletion affects cytochrome oxidase (COX) and complex I activity. AD tissue displays decreased COX activity, while complex I activity does not change. The reason for specific reductions in COX activity remain unclear. We found COX activity decreases proportionally to declines in mtDNA levels. Whether complex I activity follows the same pattern will give insight into potential mechanisms for reduced COX activity in AD.
  • Publication
    Role of Autophagy in Alcohol-induced Adipose Atrophy and Liver Injury
    (University of Kansas, 2018-12-31) LI, YUAN
    Alcoholic liver disease (ALD) is a worldwide health issue claiming 2 million lives per year. The pathogenesis of ALD is characterized by steatosis, alcoholic hepatitis, fibrosis, cirrhosis, and eventually hepatocellular carcinoma. There is no ideal treatment other than liver transplantation for late stage of ALD, and novel treatment targets especially for early stage of ALD are still needed. In recent years, increasing evidence implicates the role of adipose-liver axis in in the pathogenesis of ALD. Chronic alcohol consumption leads to increased lipolysis, adipose tissue atrophy, and proinflammatory adipokine secretion. However, the mechanisms of how adipose tissue is affected by alcohol and contributes to ALD are largely unknown. In this study, I found that chronic-plus-binge alcohol treatment, a model resulting in liver injury and steatosis, led to smaller adipocytes and decreased adipose tissue mass. Mechanistically, I found that chronic-plus-binge alcohol inhibited mammalian target of rapamycin (mTOR)/Akt signaling pathways and enhanced autophagy degradation in epididymal adipose tissue. Although the adipose-specific autophagy-related gene 5 knockout (A-Atg5 KO) mice with adipose autophagy deficiency did not present adipose atrophy at basal condition, they were resistant to alcohol-induced adipose tissue atrophy. Moreover, A-Atg5 KO mice had increased adipocytes with multilocular lipid droplets in subcutaneous white adipose tissue. Interestingly, A-Atg5 KO mice were more resistant to alcohol-induced liver injury, though they still developed alcohol-induced liver steatosis. Alcohol metabolism and reactive oxygen species generation in liver was not affected in A-Atg5 KO mice. While the chronic-plus-binge alcohol increased liver mRNA levels of several proinflammatory genes in both wild-type and A-Atg5 KO mice, A-Atg5 KO mice had significantly decreased induction of Ccl2. Compared with wild-type mice, A-Atg5 KO mice had similar serum lipids (triglycerides, free fatty acids, free glycerol), but had higher basal levels of adiponectin and fibroblast growth factor 21 (FGF21). Additionally, chronic-plus-binge alcohol did not induce inflammation or cell death in white adipose tissues. I next used cultured 3T3-L1 preadipocytes to further determine the role of autophagy in adipocyte differentiation and how adipogenesis is affected by alcohol. I found that there was increased autophagy degradation along with increased levels of mitochondria proteins during preadipocyte adipogenesis. Using fluorescence microscopy, I found that elongated, sharp mitochondria signal became diffused and enhanced in mature adipocytes. Electron microscopy analysis revealed that the number of elongated mitochondria with clear cristae structure largely disappeared while electron-dense vacuoles containing undegraded cellular components frequently appeared during adipogenesis. Long-term cotreatment using chloroquine, a lysosome inhibitor that blocks autophagy degradation, sufficiently blocked 3T3-L1 adipogenesis. Intriguingly, in chloroquine-treated cells typical mitochondria still decreased while the electron-dense vacuoles containing undegraded cellular components further increased. Moreover, long-term ethanol or acetaldehyde treatment did not inhibit morphological changes or lipid droplet accumulation during adipogenesis in 3T3-L1 cells. Interestingly, short-term ethanol but not acetaldehyde treatment induced autophagic flux, and long-term ethanol but not acetaldehyde treatment increased mitochondria protein levels. The change in mitochondria morphology during adipogenesis was not affected by long-term ethanol or acetaldehyde treatment. These data suggest that autophagy is required for the proper adipogenesis of cultured 3T3-L1 cells, which is associated with dynamic mitochondrial remodeling. In summary, I characterized the effect of chronic-plus-binge alcohol treatment on adipose morphology, mTOR signaling and autophagy in mice. I demonstrated that chronic-plus-binge alcohol inhibited mTOR and increased autophagic flux and adipose tissue atrophy in mice. I further demonstrated that mice with chronic adipose tissue autophagy deficiency were more resistant to alcohol-induced adipose atrophy. These data indicate that autophagy activation contributes to the adipose dysfunction induced by alcohol. In addition, I also demonstrated that A-Atg5 KO mice were more resistant to alcohol-induced liver injury likely due to the increased secretion of adiponectin and FGF21 at the basal levels of A-Atg5 KO mice. These data thus support an important role of adipose-liver axis in the pathogenesis of ALD. These studies further support the notion that targeting adipose tissue autophagy may be helpful in improving alcohol-induced liver injury.
  • Publication
    The Role of Mitochondrial ATP-Binding Cassette Transporter ABCB6 in Metabolism and Energy Balance
    (University of Kansas, 2019-05-31) Tessman, Robert Thomas
    Abstract Obesity and the associated health risks represent a world-wide health and financial crisis. Lack of physical activity combined with excessive caloric intake are the root cause of the problem. Despite the increased advocation for healthy lifestyle choices, the trend has yet to reverse and indeed, seems to be on the rise especially among pre-teens and adolescents, a constituent that had not been previously part of the obesity epidemic. Mitochondria are the “fuel-burners” of the body and like other combustion devices, become inefficient in the context of fuel surplus. Moreover, with chronic over-feeding, the physiological mechanisms that regulate energy balance become permanently dysfunctional leading to the progression of pathologies such as Type II diabetes and cardiovascular disease. Medical and scientific evidence confirms that mitochondria are integral to the responses necessary to adapt to over-nutrition. However, success in mitochondria-based therapies has been extremely limited in the context of metabolic diseases. Our knowledge of the regulation of mitochondrial function, dynamics, signaling, and transport processes in different tissues and organ systems is extremely limited and this knowledge gap is a serious impediment to progress toward targeting mitochondria for treatment of metabolic diseases. In this study, we successfully genetically manipulated the expression of mitochondrial transporter ABCB6. The physiological function of this transporter is unknown but non-functional mutations of this protein have been linked to several heritable human diseases. This study establishes that ABCB6 plays a role in the maintenance of energy homeostasis. Whole-body Abcb6 knockout adult male and female mice have increased body mass with no increases in food consumption. Increased body mass is due to increased adiposity. ABCB6 deficiency results in steatosis, glucose intolerance, insulin resistance, and lower energy expenditure. Exposure to high-fat diet exacerbates these metabolic derangements. Genetically targeting ABCB6 expression specifically in liver results in disruption of whole-body energy metabolism as well with a loss of metabolic flexibility. Loss of hepatic ABCB6 results in fragmented mitochondria while overexpression leads to mitochondrial elongation, dysregulating dynamic mitochondrial functional responses to energy status. In this liver-specific model, hepatic metabolites are significantly altered with either ABCB6 knockdown or overexpression. Metabolites that have a profound impact on energy metabolism such as bile acids, amino acids, and phospholipids were significantly altered in this model. Interestingly, we discovered that ABCB6 expression is responsive to nutrient status and circadian patterns. ABCB6 expression is upregulated in the fasted state and rapidly downregulated in response to feeding. Also, ABCB6 expression is reduced in cases of chronic over-nutrition such as, diet and genetic mouse models of obesity as well as in clinically obese humans. These findings suggest that ABCB6 acts as a nutrient sensor and mediates a homeostatic response through dynamic mitochondrial changes in form and function.
  • Publication
    MECHANISMS OF CYST GROWTH AND FIBROSIS IN CONGENITAL HEPATIC FIBROSIS IN AUTOSOMAL RECESSIVE POLYCYSTIC KIDNEY DISEASE
    (University of Kansas, 2018-08-31) Jiang, Lu
    Autosomal recessive polycystic kidney disease (ARPKD) is a rare genetic disorder that occurs in 1:20,000 live births. Patients present with a broad spectrum of symptoms involving the kidneys, liver, and pancreas. Renal manifestations are characterized by the presence of cysts that are derived from dilated collecting ducts. All patients with ARPKD develop some degree of congenital hepatic fibrosis (CHF) at birth. CHF is characterized by bile duct dilation resulting in eventual development of cysts and pericystic fibrosis in the liver. Accompanying cyst growth and fibrosis, recent reports suggest that inflammation is also present, and likely contributes to disease pathogenesis and/or progression. Therefore, three interrelated processes form a ‘pathogenic triumvirate’ in CHF/ARPKD: cell proliferation (cyst growth), fibrosis, and inflammation. Aside from management of symptoms, and liver and/or kidney transplant, no effective pharmacologic therapies exist for CHF/ARPKD. Therefore, the goal of this research was to explore additional mechanisms of hepatic cyst and pericystic fibrosis progression in CHF/ARPKD to identify new points of therapeutic intervention. We first characterized the gene expression in CHF/ARPKD by performing RNA-seq in whole liver from Sprague Dawley (SD) and PCK rats at postnatal day (PND) 15, 20, 30, and 90. Upstream regulator and pathway analysis of differentially expressed genes was subsequently conducted using Ingenuity Pathways Analysis (IPA). We found the number of differentially expressed genes was increased in PCK rats over time. Among the top genes upregulated in PCK rats, Cd44, the most well-described receptor for the extracellular matrix glycosaminoglycan hyaluronan, and Tead4, a transcription factor required for regulation of genes downstream of the Hippo-kinase- yes-associated protein pathway, were consistently upregulated from PND 15 to PND 90. This led us to investigate the role of hyaluronan and yes associate protein (YAP) in CHD/ARPKD. Upstream regulator analysis predicted activation of several pro-proliferative and pro-inflammatory transcription factors in PCK rats including Ctnnb (catenin beta-1), Mtpn (myotrophin), Tcf7l2 (transcription factor 7 like 2), and several Stat (signal transducer and activator of transcription) family members (z score ≥ 2). Further, PCK rat livers exhibited inhibition of Smad7, an inhibitory SMAD, which is expected to result in a pro-fibrotic effect by preventing the Smad7-induced negative regulation of TGF-β/SMAD signaling. Additionally, a significant suppression of Hnf4a, the master regulator of hepatic differentiation, was also observed in the PCK rat livers. Mast cells (MCs) are immune cells involved in many liver diseases and release mediators such as histamine from preformed granules found in their cytoplasm. In fact, previously published data demonstrate that histamine induces proliferation of cholangiocytes, which are the precursors of the cyst wall epithelial cells (CWEC) in CHF. We observed MC infiltration in the hepatic periportal areas, but not in the kidneys, in polycystic kidney (PCK) rats. Therefore, we hypothesized that MCs contribute to hepatic cyst growth in PCK rats. To test this hypothesis, we treated PCK rats with one of two different mast cell stabilizers, cromolyn sodium (CS) or ketotifen, or saline from PND 15 to PND 30. We confirmed that CS and ketotifen treatment decreased MC degranulation in liver. Interestingly, we observed an increase in liver/body weight ratio after CS and ketotifen treatment paralleled by a significant increase in cyst size. In contrast, we saw a decreased kidney/body weight ratio paralleled by a significant decrease in individual cyst size after CS treatment. We excluded the direct effect of CS on cyst growth by treating isolated cyst wall epithelial cells (CWECs) and a mouse model (no MC observed) of ARPKD with CS. Taken together, these data demonstrate that hepatic and renal cysts are differentially regulated by MC granule contents in PCK rats. RNA-seq analysis suggested that TEA Domain Transcription Factor 4 (Tead4), a transcription factor in Hippo signaling pathway, is upregulated in PCK rats. Hippo signaling pathway is a conserved signaling that plays critical roles in liver size control and cell proliferation. Our preliminary data suggested that YAP, the downstream effector of the Hippo signaling pathway, was increased in proliferating CWECs in PCK rats and in human ARPKD patients. Consistently, there was increased expression of YAP target genes, Ccnd1 (cyclin D1) and Ctgf (connective tissue growth factor), in PCK rat livers. Extensive expression of YAP and its target genes was also detected in human CHF/ARPKD liver samples. Therefore, we hypothesized that YAP plays a role in CWEC proliferation in CHF/ARPKD. We inhibited YAP activity pharmacologically using verteporfin and genetically using short hairpin (sh) RNA in primary liver CWECs. We found that CWEC proliferation was significantly reduced. These data indicate that increased YAP activity, possibly through dysregulation of the Hippo signaling pathway, is associated with hepatic cyst growth in CHF/ARPKD. In RNA-seq analysis, we found that Cd44 was upregulated in PCK rats from PND 15 to PND 90. Hyaluronan (HA), a CD44 ligand, is a ubiquitous, anionic glycosaminoglycan present in the extracellular matrix (ECM). HA is implicated in liver injury, inflammation, and fibrogenesis. Our study showed an increased HA level in livers from PCK rats and polycystic liver disease patients relative to healthy controls. HA accumulation could be due to an increased Has1 (a HA synthase) expression and decreased expression of HA degrading enzymes. Therefore, we hypothesized that HA plays a pathogenic role in progression of CHF/ARPKD. Future studies can be performed to test the role of HA in CHF/ARPKD by genetically deleting Has enzymes or the HA receptor, CD44, in PCK rats or in Pkhd1 mutant mice Taken together, CHF/ARPKD is a hepatobiliary disease that is regulated by a ‘pathogenic triumvirate’ (cell proliferation/cyst growth, inflammation, fibrosis). My studies aimed to discover novel mechanisms driving CHF/ARPKD and investigated how MC, and YAP, regulated the ‘pathogenic triumvirate’ in CHF/ARPKD. Moreover, we identified and upregulation of HA and Cd44 in PCK rats the significance of which can be explored in future studies. Overall, these studies have uncovered several new avenues for additional research into the mechanisms of cell proliferation/cyst growth, inflammation, and fibrosis, in CHF/ARPKD and from which future therapeutic strategies can be developed.
  • Publication
    A Longitudinal View of How Noise-Induced Hearing Loss Impacts Auditory and Non- Auditory CNS Activity and the Relationship to Tinnitus Behavior
    (University of Kansas, 2018-08-31) Freemyer, Andrea
    Tinnitus, defined as the perception of sound when no corresponding external sound is present, affects 50 million people in the United States with 2 million reporting decreased quality of life. Although the etiology of tinnitus is heterogeneous, exposure to a damaging auditory stimulus is the most common cause of the perceptual disorder. In addition to the better known auditory component of tinnitus there is an affective component. Anxiety and depression can occur concomitantly with tinnitus and is often of unknown etiology. Exposure to damaging sound leads to complex changes throughout the central nervous system (CNS) impacting both auditory and non-auditory brain areas. The absence of a complete picture of how tinnitus is manifested and maintained in the CNS continues to hinder the development of effective treatments. The goal of this project is to elucidate the underlying mechanisms that produce neuroplastic changes over time in the central nervous system following sound damage that may or may not be associated with tinnitus. Using an animal model of sound induced tinnitus, this project evaluates both early and long-term changes in behavior, neuronal activity, and early changes to neuroplastic protein marker expression in various auditory and non-auditory brain regions. The findings reported here reveal information about the timeline of peripheral injury (sound damage) to tinnitus onset and changes that take place in six different brain regions encompassing both auditory and non-auditory brain regions. This project has allowed us to enhance our understanding of the development of tinnitus over time in several auditory and non-auditory brain structures at both the molecular and systems level in addition to obtaining corresponding changes in behavior.
  • Publication
    Role of HNF4a in Liver Regeneration, Liver Cancer Pathogenesis and Global Metabolism
    (University of Kansas, 2018-12-31) Huck, Ian
    Liver is the central metabolic organ and performs many functions necessary for survival. Hepatocyte Nuclear Factor 4 alpha (HNF4a) is a nuclear receptor well characterized for its role in embryonic hepatocyte differentiation and maintenance of the adult hepatocyte phenotype. HNF4a regulates genes involved in many basic hepatocyte functions and deletion or decreased expression of HNF4a results in impaired liver function. More recently, HNF4a has been shown to exhibit tumor suppressor activity and deletion of HNF4a results in hepatocyte proliferation. The objectives of these studies were to characterize the role of hepatic HNF4a in hepatocyte proliferation and metabolism during liver regeneration and to harness HNF4a activity as a prognostic tool in liver cancer. The remarkable ability of liver to regenerate is possible due to the innate proliferative potential of quiescent adult hepatocytes. Knowing the role of HNF4a in maintaining hepatocyte function and suppressing hepatocyte proliferation, we hypothesized that HNF4a activity and expression may be adjusted to navigate hepatocytes between proliferative and quiescent states during liver regeneration. Using the partial hepatectomy model of liver regeneration, we identified decreased HNF4a activity in livers during the initiation of hepatocyte proliferation. Overexpression of HNF4a delayed hepatocyte proliferation after partial hepatectomy, and we identified Src-kinase as a potential regulator of HNF4a during the initiation of regeneration. Hepatocyte-specific deletion of HNF4a in mice (HNF4a-KO mice) resulted in 100% mortality after partial hepatectomy. RNA-Seq analysis of livers from these mice revealed significant dedifferentiated, and increased activation of proliferative and carcinogenic pathways. Our results show that HNF4a is required to prevent hepatic failure after liver regeneration, is a critical regulator of termination of regeneration and provide new understanding of HNF4a position in regenerative networks. Downregulation of HNF4a has been well recognized in the progression of liver disease. We hypothesized that HNF4a activity could be used in the diagnosis and prognosis of liver diseases. Under the assumption that HNF4a activity could best be quantified by its target gene transcription, we identified a set of 44 genes by selecting only those genes that were direct HNF4a targets which were differentially regulated between WT and HNF4a-KO livers in normal and in liver tumors. qPCR analysis was used to validate the signature in mouse models of HNF4a deletion and liver cancer. Using this method, we detected lower HNF4a activity in human cirrhotic samples when compared to HCC samples. While no correlation was observed between disease state and the HNF4a staining pattern in diseased human tissues, the signature was able to accurately classify the severity of samples based on HNF4a activity. Further in silico analysis using human datasets confirmed the signatures ability to diagnose the severity of disease according to HNF4a activity. Lastly, when this analysis was performed on a dataset of human cirrhotic samples, we observed increased survival in a subset of cirrhotic samples with high HNF4a activity as determined by the signature. The role of HNF4a in regulating carbohydrate and lipid metabolism has been well studied, but how it effects overall global metabolism is not known. We used indirect calorimetry to examine changes in energy expenditure and substrate utilization between WT and HNF4a-KO mice in fed, fasted, and high fat diet-fed conditions. We observed significant decreases in energy expenditure in HNF4a-KO mice during fed conditions which were exacerbated during high fat diet feeding. The changes were accompanied by significant adipose depletion and hypoglycemia. Our findings bring focus to the central role of liver in metabolism, which is largely regulated by hepatocyte HNF4a. Altogether, these studies indicate that hepatic HNF4a is a critical regulator of liver regeneration and global metabolism. Further, hepatic HNF4a activity can be used as a prognostic tool in chronic liver diseases such as cirrhosis and cancer.
  • Publication
    Characterization of SUMOylation of 5-HT1ARs
    (University of Kansas, 2019-08-31) Sugandha, -
    Serotonin 1A receptors (5-HT1AR) are G-protein coupled receptors involved in the control of mood, cognition and memory. Clinical and animal studies have demonstrated that abnormal levels of 5-HT1AR lead to anxiety-like and depressive-like phenotypes. Previous studies in our lab have shown that 5-HT1AR can undergo SUMOylation, a post-translational modification process analogous to ubiquitylation but involving conjugation of small ubiquitin-like modifiers (SUMOs). SUMOylated 5-HT1AR is located in the cell membrane and in the cytoplasm especially in the trans-Golgi network and endoplasmic reticulum. Differential centrifugation and receptor binding experiments suggest SUMOylation leads to inactivation of the 5-HT1AR, but further studies are needed to confirm this conclusion. Treatment with a 5-HT1AR agonist, increases the SUMOylated receptors which is further increased by treatment with estradiol, treatments that at pharmacological doses or with chronic use lead to desensitization of the 5-HT1A. Protein inhibitor of activated STAT xα (PiasXα) facilitates the SUMOylation of the 5-HT1AR, and treatment with estradiol and a 5-HT1AR agonist increases PiasXα. However, the mechanisms regulating the increase in 5-HT1AR SUMOylation with 5-HT1AR agonist treatment alone are not known. We hypothesize that sentrin proteases (SENPs) catalyze the deSUMOylation of the 5-HT1AR are reduced with agonist treatment; however, currently, there is limited knowledge regarding which of these enzymes are involved in the deSUMOylation of 5-HT1AR. Thus, the goal of this study is to determine which SENPs are involved in the deSUMOylation of 5-HT1AR. We can then determine if those enzymes are altered by treatment with 5-HT1AR agonist. In the present studies, we found that 5-HT1AR expression is maximal at 32 hours post transfection in Neuroblastoma (N2a) cells. Then, we transfected N2a cells to overexpress SENPs to determine which SENPs are involved in the deSUMOylation of the 5-HT1AR. The results suggest that SENP2 catalyzes the deSUMOylation of the 5-HT1AR when 5-HT1AR and SUMO-1 were overexpressed in N2a cells. Further, we determined that there were two isoforms of SENP2 two SENP2 isoforms at 50 kDa and 60 kDa in the rat frontal cortex and examined whether 8-OH-DPAT, a 5-HT1AR agonist, treatment decreased expression of either isoform. Our results suggest that the levels of 60 kDa SENP2 isoform were significantly decreased with 8-OH-DPAT treatment while levels of the 50 kDa did not change. These studies suggest that increase in SUMOylation of 5-HT1ARs due to 8-OH-DPAT treatment may be mediated through a decrease in 60 kDa isoform of SENP2, but further experiments are needed to confirm this conclusion. These studies to understand the mechanism involved in the regulation and specifically SUMOylation of 5-HT1AR will inform the development of new potential targets for the treatment of anxiety and depression.
  • Publication
    The Combined Effects of Arsenite and Ethanol on Brain Endothelial Cells and Microglial Cells
    (University of Kansas, 2019-08-31) Li, Siying
    Arsenic (As) is a natural compound widely distributed in air, water, and soil. Drinking ground water is the major source of As exposure. As exposure causes many health issues, including nausea, vomiting, pain, diarrhea, cancer and neurotoxicity [1]. Alcohol drinks may also contain As because grapes and rice which are used in making wine and beer, take up As from soil, water, and fungicides containing As. Emerged evidence showed that ethanol (EtOH) also impairs neurological functions [2]. However, the combined toxic effects of As and EtOH on the brain is still unclear. Our long-term goal is to understand the effects of As combined with EtOH on the blood brain barrier (BBB). The BBB controls molecule exchange between peripheral and cerebral compartments [3]. Alterations of the BBB are a critical risk factor of pathology and progression of different neurological diseases [4]. Many studies have shown that As as well as EtOH induced BBB abnormalities [5, 6]. Since brain endothelial cells play a crucial role in the BBB, we used Rat Brain Endothelia (RBE4) cells to investigate the combined toxic effects of As and EtOH on the BBB. Overproduction of reactive oxygen species (ROS) results in destruction of cellular structures, lipid, and proteins [7]. Previously, our lab showed that As increased endothelial cell permeability through a ROS-vascular endothelial growth factor pathway in mouse brain vascular endothelial cells (bEnd 3 cells) [8]. Others have shown that EtOH also impairs the barrier function and junctional organization of human brain microvascular endothelial cell monolayer [9]. In neurons, EtOH-induced ROS mainly come from damaged mitochondria [10]. Since mitochondria are major source of ROS generation, we proposed that As-EtOH-combined treatment impairs the BBB through ROS released by damaged mitochondria. Mitochondrial oxidative stress affected microglia-associated neurodegenerative diseases through their role as pro-inflammatory molecules and modulators of pro-inflammatory processes [11]. BBB disruption is mediated by neuroinflammation which is associated with increase in pro-inflammatory cytokines [12]. Research showed that inflammatory mediators control BBB permeability through regulating the structural components [13]. As induced cytotoxicity in brains via regulation of oxidative stress and TNF-a associated inflammatory pathways [14]. Alcohol consumption enhanced oxidative and inflammatory stress, resulting in cognitive deficit [15]. Since microglial cells are the main effectors in the inflammatory process of the central nervous system [16], we used microglial cells (BV2) to investigate the combined effects of As and EtOH on microglia. Our results showed that As and EtOH increased RBE4 cell monolayer permeability. As-EtOH combined treatment increased the permeability more than As or EtOH treatment alone. RBE4 cells and BV2 cells showed an increase in ROS by the combined treatment. Mitochondrial ROS generation was increased by the combined treatment of As and EtOH but reduced by antioxidant Tempol in RBE4 cells and microglia BV2 cells. The combined treatment of As and EtOH decreased mitochondrial bioenergetics (mtBE) in RBE4 cells but increased by antioxidant Tempol. BV2 cells viability decreased as As or EtOH concentration increased and further decreased by the combined treatment. In conclusion, our results suggest that the combined treatment of As and EtOH induced toxicity on both endothelial cells and microglial cells via increased oxidative stress induced by mitochondrial dysfunction.
  • Publication
    The interaction between MsrA and Csn5/Jab1
    (University of Kansas, 2019-08-31) Jiang, Beichen
    Oxidative stress can cause toxic outcomes to cells/organisms’ survival. To counter this toxicity, multiple cellular mechanisms were evolved. Among them is the methionine sulfoxide reductase (Msr) system. Methionine sulfoxide reductase A (MsrA) plays a key role in protecting cells from oxidative stress. In our previous studies we discovered a novel role of MsrA in the regulation of ubiquitin (Ub) and Ub-like modification of proteins. To further understand the role of MsrA in posttranslational modification of proteins, a yeast-2-hybrid (Y2H) screening was performed to screen for potential substrates for MsrA in brain. Accordingly, one of the major identified substrates was a protein denoted as COP9 signalosome subunit 5 (CSN5, also known as Jab1 or COPS5), containing a unique binding site to MsrA. The COP9 signalosome complex (CSN) is an essential regulator of the ubiquitin conjugation pathway by mediating the removal of Nedd8, an Ub-like protein modifier, from proteins (deneddylation process). Importantly, the Csn5/Jab1 contains a domain that provides the catalytic center of the CSN complex. The known substrates for neddylation and deneddylation (by CSN) are the cullin subunits and their homologues (e.g. Cul-1 of the SCF-type E3 ligase complexes). Furthermore, we showed that MsrA interacts with Csn5/Jab1 in mouse brain following immunoprecipitations and pull-down experiments. This interaction was compromised in brain extracts of MsrA knockout (MsrA KO) compared with the parent wild type (WT) brains. A decrease in the levels of neddylated Cul-1 was also observed in liver extract of MsrA KO mouse, while the levels of Csn5/Jab1 in brain were the same in both the WT and MsrA KO strains. These data suggested that MsrA positively regulates Csn5/Jab1 deneddylation activity, presumably via reduction of this protein MetO residue/s. To further study the relationship between neddylation level and MsrA in vivo, we used yeast strains with various expression levels of MsrA. These yeast strains were WT, MsrA KO and MsrA overexpressed (OE) yeast strains. Neddylation levels of yeast under the condition of hydrogen peroxide or human Csn5/Jab1 inhibitor were investigated. The data showed that both oxidative stress and Csn5/Jab1 inhibitor caused inhibition of deneddylation activity of Csn5/Jab1 in the absence of MsrA, while MsrA-containing strains (i.e. WT and OE) showed a strong ability to protect against H2O2/Csn5/Jab1 inhibitor induced neddylation. For in vitro assays, we used artificial Nedd8 conjugates monitoring deneddylation activity in extracts of brains of WT and MsrA KO mice strains as function of incubation time. The acquired data showed that the deneddylation activity was dramatically reduced in the MsrA KO compared with the WT brain extracts. In conclusion, the presented data provide direct and indirect evidence to support our hypothesis that MsrA plays an important role in maintaining the Csn5/Jab1 deneddylation activity. Lastly, we investigated the potential role of MsrA in regulating of neddylation levels in brain cancer. Glioblastoma is one of the most aggressive brain cancers. This cancer type demonstrates a relative low level of neddylation compared with normal brain cells. Accordingly, Csn5/Jab1 could be one of the posttranslational regulated proteins that may contribute to this phenomenon. We have indeed found that the neddylation level was reduced in glioblastoma cells, while the level of Csn5/Jab1 was unchanged compared with normal brain cells. This observation pointed to the possible role of MsrA in enhancing Csn5/Jab1 activity in these cancer cells. This possibility will be further investigated by comparing the MsrA activities in both the normal and glioblastoma cells in the nearest future.
  • Publication
    The effect of irrelevant visual experience on visual memory
    (University of Kansas, 2019-08-31) Indulkar, Shreya Sanjay
    Consolidation of memories for long term storage involves increases in excitatory synaptic strength and connectivity between neurons encoding a novel experience. The increase in neuronal excitability caused by memory consolidation could augment excitability induced by the experience of related stimuli irrelevant to the memory. Therefore, the additional neuronal excitability caused by memory consolidation could perturb neuronal activity homeostasis towards higher neuronal activation levels. Under conditions of neuronal hyperactivity, such as in Alzheimer’s disease, an increase in excitation induced by memory consolidation would further destabilize homeostasis. We hypothesize that memory deficiency, which would result in reduced neuronal excitability, is an adaptation to maintain neuronal activity homeostasis. To test this hypothesis and to identify whether experience-evoked activity contributes to memory impairments, we used a visual recognition memory (VRM) paradigm that involves synaptic plasticity in the primary visual cortex. In this paradigm, mice are repeatedly presented with a visual grating of a specific orientation and the recognition memory is assessed as a decrease in the exploration of the same stimulus over time. We tested the orientation selective behavioral habituation in a mouse model of Alzheimer’s disease (J20 line) and non-transgenic control siblings (wild type). We found that wild type mice display VRM for grating stimulus when tested one day but not at one month after the training period. In contrast, J20 mice did not display VRM even one day after the training period. To examine whether reducing neuronal excitability caused by memory irrelevant visual experience influences the long-term retention of the VRM for grating stimulus, we performed the same task in mice housed in total darkness except during the VRM task. Our preliminary data indicate that dark adaptation rescues the memory deficit in J20 mice whereas disrupts memory in control mice when tested one day after the training. These results suggest that competing experiences promote memory storage in control mice but interferes with it in APP mice.
  • Publication
    Dorsal Cochlear Nucleus Synaptic Reorganization as a Consequence of Noise-Induced Tinnitus: Increases in Somatosensory Influence on Auditory Circuitry
    (University of Kansas, 2016-12-31) Neal, Christopher Andrew
    Tinnitus is the perception of sound with no corresponding external stimulus. It is estimated that at least 17 million Americans experienced approximately 5 minutes of acute tinnitus in the past year, and at least 10 million Americans experience chronic tinnitus. Tinnitus itself is generally recognized as being a symptom of other conditions or diseases, and there are many methods of tinnitus induction. There are no effective cures for tinnitus, and management therapies, although moderately effective, do not address any pathophysiological changes associated with tinnitus symptoms. A primary focus of tinnitus research has been to elucidate the aberrant anatomy and physiology that underlies tinnitus perception, in the hopes of identifying therapeutic targets. Early research efforts focused on the peripheral auditory system (cochlea, auditory nerve) as likely sources of tinnitus, but increasing evidence suggests that tinnitus is generated and maintained by the central auditory system. The dorsal cochlear nucleus (DCN) likely plays a key role in tinnitus induction and maintenance, and many neural correlates (e.g., hyperactivity) of tinnitus have been observed in the DCN. The DCN is a laminar structure that contains a local circuit, which integrates auditory and non-auditory inputs. This first order central auditory nucleus is the first site of multi-sensory integration in the auditory system. A differential distribution of pre-synaptic markers (i.e., VGlut1, VGlut2, and VGat) has been described for the inputs to this circuit, which allows these excitatory and inhibitory components to be specifically studied. The work presented here utilizes a rat model of sound damage, and seeks to quantify peripheral damage and the corresponding central remodeling of both excitatory and inhibitory synapses in the DCN. We are able to successfully assess tinnitus status of our animals utilizing a behavioral assay for tinnitus (i.e., gap detection), granting us the ability to identify differences in peripheral damage or central remodeling that are unique to either noise-induced hearing loss or tinnitus. Peripheral insult was quantified via cochlear hair cell counts, and auditory brainstem response measures. Central insult and reorganization was quantified, in the dorsal cochlear nucleus, using both pre- and post-synaptic markers for excitatory (VGlut1, VGlut2, and PSD95) and inhibitory (VGat, Gephyrin) synapses. All experiments from baseline tinnitus and auditory assessment through final immunohistochemical labeling of the cochleae and DCN were performed on each animal enrolled in the study, instead of each experimental phase in the sequence involving a unique animal population. Using gap detection data, we sorted animals into two main groups: tinnitus negative (-) and tinnitus positive (+), the latter of which also contained a subgroup of severely impaired (++) animals. Regardless of tinnitus status, all animals had significantly elevated hearing thresholds for frequencies 8 kHz. Similarly, all animals exhibited IHC and OHC loss, although OHC loss was more severe in tinnitus (+) and (++) animals. Central changes in synaptic density in the DCN included an increase in the density of inhibitory synapses in tinnitus (+) and (++) animals. Unexpectedly, no changes were observed in any sound exposed animals in the excitatory granule cell/parallel fiber synapses, auditory nerve synapses, or somatosensory mossy fiber synapses. However, we did observe significant increases in excitatory unipolar brush cell synapses in tinnitus (+) and (++) animals, which suggests a possible increased influence of somatosensory input in the local circuitry of the DCN.
  • Publication
    Cyclophilin D as a potential therapeutic target for Alzheimer’s disease
    (University of Kansas, 2019-05-31) Nolte, Erika D
    Alzheimer’s disease (AD) is the sixth leading cause of death in United States, affecting more than five million people every year. Despite substantial research into the topic, including over a hundred thousand research papers on the topic and nearly a billion dollars per year in funding, no drugs have been approved by the FDA to prevent, slow, or cure AD. Upon discovering that patients with Alzheimer’s disease had increased Cyclophilin D (CypD), a key mitochondrial matrix protein responsible for the initiation of mitochondrial apoptosis, we generated models of tau-induced AD with altered CypD expression. CypD overexpression (OE) mice were successfully used to model more severe AD pathology. These mice showed increased levels of hyperphosphorylated tau (HPT). Additionally, these mice showed mitochondrial failure in an age dependent manner. Synaptic and neuronal loss were also identified in an age dependent manner. Finally, cognitive impairments were detected using an open field study and a daily task performance study. CypD knock out (KO) mice were used to model the potential therapeutic effects of lowering CypD in an AD model. These mice showed an amelioration of every AD biomarker tested. CypD KO mice showed lower HPT loads and normalized mitochondrial function. There was no detectable loss of synaptic terminals in the CypD KO line, and there was no loss of cognitive function in either open field or a daily task performance test. A small molecule CypD inhibitor created in our lab was used in a tau-induced model of AD to attempt to create the significant improvements seen with genetic ablation of CypD. This compound was determined to be safe for use in mice after testing in cell cultures revealed that it was non-toxic and effective at preventing Aβ-induced cellular death. In mice, the CypD inhibitor reduced HPT accumulation, restored mitochondrial function, and prevented cognitive declines without inducing any apparent toxicity in the mice.
  • Publication
    Modulating Molecular Chaperones to Treat Demyelinating Neuropathies
    (University of Kansas, 2018-08-31) Zhang, Xinyue
    Peripheral neuropathies can be classified into two categories, demyelinating or axonal neuropathy. Demyelinating neuropathies are characterized by damaged myelin but intact axons. Recent evidence suggests that the leucine zipper transcription factor c-jun is at the center of driving demyelination. c-Jun is required for Schwann cells (SCs) to dedifferentiate after injury, and up-regulation of c-jun has been reported in human neuropathies. It remains to be tested whether c-jun would be a valid target for treating demyelinating neuropathies. Previously, our published work has shown that modulating the expression of heat shock protein 70 (Hsp70) using a novel small molecule drug called KU-32 attenuated the expression of c-jun and the extent of demyelination in SC-dorsal root ganglia (DRG) co-cultures in an Hsp70 dependent manner. To extend these data, this work examined the in vivo effects of modulating molecular chaperones using the next generation novologue KU-596 in two mouse models of demyelinating neuropathies. MPZ-Raf mice are a conditional transgenic mouse line that exhibits a demyelinating neuropathy due to the SC-specific induction of mitogen-activated protein kinase (MAPK) and c-jun induction after tamoxifen (TMX) injections in adult mice. Five days of TMX treatment induced a severe motor deficits starting from day 8 and treating the MPZ-Raf mice with 20 mg/kg of KU-596 every other day reduced c-jun levels in the sciatic nerves. The decrease in c-jun correlated with an improvement in the myelination status of the nerves and motor function. In line with previous findings, the effects of KU-596 were Hsp70-dependent, as MPZ-RAF × Hsp70 knockout (KO) mice did not show improvement following drug treatment. This study provides proof of principal that modulating molecular chaperones would be beneficial in treating demyelinating neuropathies. However, as this model is less relevant to an actual disease, we complemented our study using a model of human X-linked Charcot-Marie-Tooth disease (CMT1X). CMT1X is caused by the mutation of gap junction beta 1 gene (GJB1) that encodes the gap junction protein connexin 32 (Cx32). Recent evidence suggests an elevated c-jun expression is associated with the disease. Since c-jun could promote demyelination, targeting c-jun using KU-596 could provide a potential therapeutic strategy to treat CMT1X. The pathology of Cx32 deficient (Cx32def) mice occurs in two stages where young mice develop a pre-demyelinating axonopathy, which progresses to a more severe demyelinating neuropathy in older mice. We show that in young mice that exhibit a pre-demyelinating axonopathy, one-month of KU-596 treatment decreased c-jun expression and improved motor nerve conduction velocity (MNCV) and compound muscle action potential (CMAP). In older Cx32def mice that developed a demyelinating neuropathy, 3 months of KU-596 treatment decreased c-jun expression and improved grip strength, MNCV and CMAP. Hsp70 is required for drug efficacy as neither young nor old Cx32def × Hsp70 KO mice showed improvement following KU-596 treatment. Collectively, our data indicates that modulating molecular chaperones is beneficial in managing demyelinating neuropathies.
  • Publication
    ApoE2-Mediated Neuroprotective Mechanism Through Up-regulation of Glycolysis
    (University of Kansas, 2018-08-31) Zhang, Xin
    Studies presented here aim at gaining an insight into mechanisms of how human apolipoprotein E (ApoE) isoforms impact glucose metabolism, particularly through regulation of glycolysis, which may ultimately result in pathophysiological alterations in the brain. Consistent with our previous findings, hexokinase, the enzyme that catalyzes the initial and irreversible conversion of glucose to 6-phophoglucose in the first step of glycolysis, is significantly affected by ApoE isoforms in stably human Apo E2, 3, or 4 expressing Neuro-2a cell lines. Results from a time course study indicate that the regulation of hexokinase by ApoE is typically through a chronic pattern. Additionally, glycolytic function was also differentially regulated by three ApoE isoforms with ApoE2 group shows the most robust profile. The data indicate that hApoE2-expressing cells exhibited significantly enhanced glycolytic activity compared to ApoE4-expressing cells, possibly through the upregulation of hexokinase. With the evidence that ApoE isoforms differentially regulate glycolytic function via hexokinase, cell health status was further assessed by both metabolic activity and morphological phenotype. In line with our prediction, the regulation of hexokinase and the differential glycolytic profiles directly correlated to the overall health status of the three ApoE isoforms-expressing cells. Meanwhile, we observed no significant alteration in apoptotic markers and the insulin-regulated glucose transporter, which further supports a neuroprotective role of ApoE2 through up-regulation of glucose metabolism. Furthermore, this thesis employed a differentiated neuronal model to determine the influence of ApoE on the regulation of neuronal glycolysis. In transfected neurons, differential regulation of hexokinase and glycolytic function by hApoE2/3/4 was also observed. hApoE2-transfected neurons exhibited a significantly higher expression and activity of hexokinase as well as lactate production than cells transfected with ApoE3 or ApoE4. Taken together, results from these studies indicate that human ApoE isoforms differentially modulate neuronal glycolysis via regulation of hexokinase, which directly correlates to neuronal metabolic activity and health status. The ApoE2-mediated glycolytic robustness may suggest a mechanistic rationale for its neuroprotective role and consequently provides a novel therapeutic approach against the onset of AD.
  • Publication
    Mechanistic Studies on Arsenic Toxicity in Endothelial Cells
    (University of Kansas, 2018-08-31) Chen, Jiani
    Arsenic is a well-established human toxin. Over 200 million people worldwide are exposed to arsenic, mainly through drinking water. Recently, several epidemiological studies have reported that even low concentrations of arsenic impair neurological functions across a broad age range in human [1]. However, the cellular and molecular mechanisms of arsenic’s effect on the central nervous system (CNS) are not well understood. The blood-brain barrier (BBB) is a complex multicellular structure separating the CNS from the systemic circulation and protects the neural tissue from toxins and pathogens. Alterations in the BBB are an important component of pathology in many neurological disorders [2]. Therefore, arsenic may have a toxic impact on the BBB and thus affect the function of CNS. Since brain endothelial cells (BECs) are the major component of the BBB and play important roles in maintaining the functional integrity of brain tissue under toxic exposure, we focused on arsenic-induced alterations in BECs in our study. Previously, our laboratory has reported that reactive oxygen species (ROS) play an important role in an arsenic-induced increase in brain endothelial cell permeability[3]. However, the underlying mechanism of arsenic-induced ROS generation in endothelial cells and how ROS regulate endothelial cell functions are unclear. Mitochondria are considered as the major source of ROS generation in mammalian cells and arsenic-induced mitochondrial dysfunction has been reported in many studies, which suggest that it may be associated with arsenic-induced oxidative stress in endothelial cells (ECs). In addition, autophagy, which is regulated by oxidative stress, plays an important role to control endothelial permeability and maintain redox balance in ECs and it may also be involved in arsenic toxicity in BECs [4].
  • Publication
    5α-reductase isoenzymes mediate stress-exacerbated Tourette-like responses in animal models
    (University of Kansas, 2018-05-31) Mosher, Laura Jean
    Tourette syndrome is a neurodevelopmental disorder characterized by purposeless, uncontrollable muscle movements known as tics. These tics are extremely sensitive to environmental factors, especially psychosocial stress. Stress has been demonstrated to increase neurosteroids in animal models, but the relationship of these neurosteroids to Tourette syndrome is unknown. The neurosteroid allopregnanolone is a key regulator of the stress cascade but has also been demonstrated to influence dopamine-mediated behaviors in animal models. Clinical results have shown that inhibiting the synthesis of allopregnanolone and other 3α, 5α steroids with the 5α-reductase inhibitor finasteride reduces tics in adult male patients with Tourette syndrome; however, the mechanism of action is largely unidentified. In this dissertation, the mechanism by which stress exacerbates Tourette syndrome symptoms and finasteride attenuates these behaviors was examined. We found that in various animal models of Tourette syndrome, stress exacerbated tic-like behaviors and deficits in prepulse inhibition (PPI), an operational measure of sensorimotor gating aimed at filtering salient information from the environment; this process is also disrupted in Tourette syndrome patients. These stress-induced tic-like behaviors and PPI deficits were ablated by finasteride treatment, which indicated a role for 3α, 5α steroids. We found that one of these steroids, allopregnanolone exacerbated tic-like behaviors and induced PPI deficits in our animal models. In addition, we determined that allopregnanolone is mediating these effects through several possible receptors; specifically we found evidence suggesting that the pregnane xenobiotic receptor and the purinergic P2X4 receptor are involved in these processes. Finally, we demonstrated that the isoenzymes 5α-reductase type 1 and type 2 exert different effects in regulating Tourette syndrome-like symptoms, and specifically that 5α-reductase type 1 may be the more beneficial and safe target for inhibition over 5α-reductase type 2.
  • Publication
    A FRET-Based approach to study the SUMOylation of Serotonin 1A Receptors
    (University of Kansas, 2018-08-31) Kaur, Sukhmanjit
    Serotonin 1A receptors are an inhibitory G-protein coupled receptor that are known to play a key role in the regulation of mood and cognition. Dysregulation of serotonin 1A receptors has been implicated in mood related disorders such as depression and anxiety. Post translational modifications including palmitoylation and phosphorylation are found to regulate the function of serotonin 1A receptors. Previous studies in our lab demonstrated that serotonin 1A receptors are SUMOylated, however the impact of SUMOylation on serotonin 1A receptor function is yet to be elucidated. Acute agonist stimulation of serotonin 1A receptors was found to increase the levels of the SUMOylated receptors in rat cortex. This study employed acceptor photobleach FRET to further investigate the interaction between SUMO-1 and serotonin 1A receptor and identify the sites of SUMOylation on the serotonin 1A receptor. We used cell lines expressing both endogenous (N2A) and transfected serotonin 1A receptors (HEK293 and SHSY5Y) and observed FRET between serotonin 1A receptor and SUMO-1 in all the cell lines. Using acceptor photobleach FRET, we found three lysine residues on the serotonin 1A receptor (232, 235, 324) that are possibly involved in SUMOylation. We also conducted an immunocytochemistry-based approach, to study the effect of agonist stimulation of serotonin 1A receptors on SUMOylation of the receptors in the cell membrane. We observed similar extent of colocalization of serotonin 1A receptor and SUMO-1 antibody in both the 8-OH-DPAT and vehicle treated groups. This was observed due to the limitations of light microscopy to distinguish between objects closer than 100nm as two different entities. Further studies need to be performed using techniques with higher resolution such as electron microscopy to study the effect of agonist stimulation on the SUMOylated serotonin 1A receptors. Our data provides some important insights about the putative sites of SUMOylation on the serotonin 1A receptor. The identification of the primary site of SUMOylation along with the knowledge about the effect of agonist stimulation on the SUMOylation of serotonin 1A receptors would help us decipher the role of SUMOylation in serotonin 1A receptor desensitization. Further understanding the regulation of serotonin 1A receptors by SUMOylation will aid in elucidating the role of serotonin 1A receptors in various mood disorders such as depression.
  • Publication
    Retinoic Acid Plays Critical Roles in the Late Development of the Heart
    (University of Kansas, 2017-12-31) Wang, Suya
    Vitamin A, via its active metabolite retinoic acid (RA), actively participates in many biological processes including cardiogenesis. Yet RA was found to be highly teratogenic as both excess and deficiency of this critical morphogen result in congenital heart defects. Studies presented here aims to gain insight in mechanisms regulating RA metabolism during embryogenesis and enrich our knowledge of the critical roles of RA during late heart development. Previously, our lab reported that a short-chain dehydrogenase/reductase, i.e. DHRS3, is required for preventing excessive accumulation of RA and thus safeguarding mouse embryogenesis during mid-gestation. The current study expanded the investigation and studied the physiological importance of DHRS3 in RA metabolism at multiple embryonic stages. Consistent with the elevated RA synthesis and signaling at E14.5, genetic ablation of Dhrs3 results in an expansion of RA signaling at E10.5 and E12.5 globally; as well as in the fetal hearts at E10.5, 12.5 and 13.5. Dhrs3-null mutants display a spectrum of congenital defects, including defects in the heart, skeleton and cranial nerves, which collectively result in mid-gestational lethality in Dhrs3-/- embryos. Reduction of maternal intake of vitamin A successfully rescued the Dhrs3-/- fetuses and allows them to survive into full-size adults with normal growth rate. These data jointly demonstrated the indispensability of DHRS3 in reducing the accumulation of RA in various developmental stages and in the formation of fetal organs. With the advancement of knowledge in RA metabolism gained in the first part of this dissertation, the critical roles of RA in cardiogenesis was further explored in the current work. During late cardiogenic stages, the major source of RA is the epicardium. Epicardium contributes greatly to the formation of coronary vessels and the myocardium via 1) giving rise to migratory epicardial cells that differentiate into perivascular cells, and 2) secreting cardiogenic factors. This dissertation employed multiple in vivo and in vitro models to determine the influence of RA on epicardial behaviors and the subsequent epicardial-regulated cardiogenic events. In vitro studies demonstrated that inhibition of RA synthesis in epicardial cell disrupted cytoskeletal reorganization and preserved epithelial characteristics, which resulted in a reduction of migration of epicardial cells. On the contrary, addition of RAR agonist to activate RA signaling strongly induced remodeling of cytoskeleton represented by the formation of stress fibers as well as filopodia marked by polymerized F-actin. RA signaling also abolished the membranous distribution of epithelial markers and induces expression of numerous metalloproteases to pave the way for cell migration. Data from in vivo models showed consistent observation in the regulation of epicardial migration by RA: excess RA in Dhrs3-/- embryos enhances the intramyocardial invasion of epicardial cells whereas deficiency of RA largely retained epicardial cells in the intact epicardium on the surface of the myocardium. To further understand the molecular mechanisms underlying the regulation of epicardial EMT by RA, we employed transcriptomic analysis, which in combination with further molecular assays clearly demonstrated that RhoA pathway is activated by RA and proves to be critical for RA-induced morphological changes in epicardial cells. Furthermore, potentially as a consequence of altered epicardial behavior and functions in response to aberrant RA signaling, both deficiency and excess RA led to compromised coronary vessel formation and hypoplastic ventricular myocardium in vivo. Vascular hierarchy was severely impaired and density of intramyocardial vessels was drastically diminished by altered RA signaling. Lack of proper recruitment and differentiation of vascular smooth muscle cells further acerbated the malformations in coronary vessels in response to abnormal RA signaling. These observations provided novel evidence of the teratogenic nature of RA and collectively demonstrated that RA signaling is not only actively involved but also critically important in the late heart development, which may shed light on the discovery of methods in preventing congenital heart diseases as well as the identification of novel targets for treating cardiovascular diseases.