• Diabetic Membrane Repair Deficiency and Repair Promotion By Vitamin E

      Howard, Amber Cyran; Department of Cellular Biology and Anatomy (5/30/2014)
      Myopathy, characterized by muscle necrosis and atrophy, is a diabetic complication. The myopathy of at least one muscular dystrophy is linked to defective membrane repair. We hypothesized that defective membrane repair is also associated with diabetic myopathy. To test this hypothesis, we monitored repair in intact muscle from diabetic type 1 (INS2Akita+/-) and type 2 (db/db) mouse models. Myocytes were laser injured in the presence of a membrane impermeant dye, and cellular dye uptake through the disruption site was monitored. Dye influx of diabetic myocytes was significantly increased, compared to controls, indicating repair deficiency. This defect was mimicked in cultured cell models by high (30 mM) glucose exposure. Inhibiting the high glucose formation of advanced glycation endproducts (AGE) prevented this repair defect, but was induced in the absence of high glucose exposure by enhanced AGE receptor (RAGE) binding. We conclude that high glucose exposure leads to defective membrane repair in skeletal muscle, and that AGE/RAGE interactions underlie this defect. AGE/RAGE binding also induces generation of reactive oxygen species (ROS), which is increased in diabetes. ROS are also produced in skeletal muscle during eccentric contracts, an act that creates muscle membrane disruptions. Using a potent antioxidant, vitamin E (α-tocopherol), we were able to reverse the high glucose exposure repair defect. Interestingly, diets deficient in vitamin E results in a lethal muscular dystrophy. α-Tocopherol partitions into membrane bilayers where it is thought to act as a membrane stabilizer and/or as an antioxidant. We hypothesize that one important biological role of vitamin E is to promote muscle membrane repair. To test this hypothesis, cultured muscle cells were loaded with α-tocopherol and repair assessed with the laser assay. α-Tocopherol loading significantly decreased cellular dye influx, indicating that repair had been promoted. Strikingly, the HeLa cell, a non-muscle cell that normally displays unrestricted dye influx after laser disruption, e.g. not capable of repair via this form of injury, became repair competent after loading with α-tocopherol. Vitamin C, another antioxidant that can be loaded into cells, also significantly decreased dye influx after laser injury. However, horseradish peroxidase, an antioxidant that lacks transport across the plasma membrane was found to be ineffective in promoting repair. Cells injured in the presence of H2O2, displayed significantly more dye influx than controls injured in physiological saline lacking this oxidant. If however cells were loaded with vitamin E the H2O2 did not affect repair. We further tested H2O2 exposure in intact mouse skeletal muscle, and found repair to be significantly impaired. However, comparable to vitamin E loading in the cell model, Trolox (a water soluble analog of vitamin E) pretreatment prevented the H2O2 muscle membrane repair defect. We conclude that vitamin E promotes plasma membrane repair, and that its capacity as an anti-oxidant is crucial in this role.
    • IN VITRO AND IN VIVO STUDIES DEMONSTRATE A ROLE FOR SH3PX1 IN LAMELLIPODIA FORMATION.

      Hicks, Lawrence Joseph; Department of Cellular Biology and Anatomy (5/22/2018)
      Actin remodeling and endocytosis are essential functions for most cells. Defects in these processes present in a variety of diseases. Sorting nexins are known to contribute to endocytic uptake, cytokinesis, the retromer complex, and autophagy. Sorting nexin 9 (Snx9) interacts with major endocytic factors and proteins involved in regulation of actin cytoskeleton dynamics. Nonetheless, Snx9’s exact in vivo roles in these basic cellular processes and disease mechanisms are not known. By examining the roles of Sh3px1, we can better understand the mechanism by which this protein contributes to endocytosis and actin remodeling in vivo. Two additional paralogs, Snx18 and Snx33, complicate studies in mammalian models due to potential redundant mechanisms. Utilizing the single ortholog in Drosophila, sh3px1, this report describes the function of Sh3px1 in membrane organization and actin dynamics. Drosophila S2 cells that are depleted of Sh3px1 fail to form lamellipodia, a process that is also dependent on the actin nucleation factor, Scar. In addition, over-expression of Sh3px1 in S2 cells results in the formation of tubules and also long membrane protrusions, atypical of a classical BAR domain protein. An intact PX-BAR domain is required for these overexpression phenotypes. sh3px1 null flies are viable; however, mutant females have significantly compromised fertility. Female sh3px1 null egg chambers show many morphological defects. The age-dependent degeneration of the null egg chamber is not likely due to compromised endocytosis. Additionally, collective border cell migration is attenuated in the absence of Sh3px1. These cells are known for their reliance on endocytosis and modulation of actin dynamics for migration. We have found that Sh3px1 is essential in efficient lamellipodia production at the start of border cell migration. Our findings also suggest that Scar directly interacts with Sh3px1 and is upregulated in sh3px1 nulls. Mutation of Scar enhances many reproductive defects in sh3px1 nulls. Thus, our work reveals a main in vivo function of Sh3px1 in actin regulation for the production of structures such as lamellipodia.
    • DNA METHYLATION REGULATION IN ACUTE KIDNEY INJURY

      Guo, Chunyuan; Department of Cellular Biology and Anatomy (4/26/2018)
      DNA methylation is a critical epigenetic mechanism, which is heritable during cell division, but does not involve the change of DNA sequence. It plays an essential role in regulating gene transcription in physiological and disease conditions. However, little is known about DNA methylation in renal diseases, especially in acute kidney injury (AKI). In this study, the role of DNA methylation in AKI was determined in both cell culture and mouse models. In cell culture, 5-aza-2’-deoxycytidine (5-aza), a pharmacological DNA methylation inhibitor, was used to inhibit DNA methylation. Interestingly, 5-aza increased both cisplatin- and hypoxia-induced apoptosis. These results suggest pharmacologic blockade of DNA methylation by 5-aza sensitizes renal tubular cells to apoptosis, supporting a cytoprotective role of DNA methylation in AKI. To determine the role of DNA methylation in vivo, we first successfully established conditional knockout mice that were deficient in DNMT1, DNMT3a or both exclusively in proximal tubules. In cisplatin-induced AKI, consistent with the effects of 5-aza in the cell culture, ablation of DNMT1 from proximal tubules exacerbated cisplatin-induced AKI in mice, and primary proximal tubular cells from PT-DNMT1-KO mice were more sensitive to cisplatin-induced apoptosis than wild-type cells. In sharp contrast, PT-DNMT1/3a-DK mice attenuated cisplatin-induced AKI, and primary proximal tubular cells from PT-DNMT1/3a-DK mice were more resistant to cisplatin-induced apoptosis. However, PT-DNMT3a-KO mice and PT-DNMT3a-WT mice showed similar AKI following cisplatin treatment. These results suggest different DNMTs play different roles in cisplatin-induced AKI. In ischemic AKI, none of the conditional knockout models showed differences in response to ischemia-reperfusion injury. Nevertheless, although ablation of both DNMT1 and DNMT3a in proximal tubular cells did not affect ischemia-reperfusion injury, it, indeed, suppressed renal fibroblast activation and ameliorated renal fibrosis. Furthermore, we found that Irf8 was regulated by DNA methylation during cisplatin treatment and knockdown of Irf8 in RPTC cells inhibited cisplatin-induced apoptosis, supporting a pro-death role of Irf8 in renal tubular cells. In ischemic AKI, although Bcl6 is hypermethylated and repressed in mice, overexpression of Bcl6 in RPTC cells had no impact on hypoxia-induced apoptosis. Collectively, these results suggest an important role of DNA methylation in AKI by regulating specific genes expression.
    • THE ROLE OF NEDDYLATION IN EARLY CARDIAC DEVELOPMENT

      Littlejohn, Rodney; Department of Cellular Biology and Anatomy (Augusta University, 2020-07)
      Background. Early cardiac development is a tightly regulated process, involving spatiotemporal coordination of multiple signaling pathways and heterogenous cell populations, both generated de novo and externally sourced. While the roles of transcription, environmental, and epigenetic factors have all been studied extensively in the context of heart development, the roles of post-translational protein modification in regulating this process remain to be elucidated. NEDD8 (neural precursor cell expressed developmentally downregulated 8) is a novel ubiquitin-like protein modifier. Conjugation of NEDD8 to protein targets, a process termed neddylation, has been shown to regulate cell proliferation, cell signaling, and protein homeostasis, and play important roles in multiple physiological and pathological events. We have previously shown that neddylation is developmentally downregulated in the developing heart and is essential for mid-to-late gestational ventricular chamber maturation. However, whether and how neddylation regulates early cardiogenic events remains unknown. Methods and results. Mice with constitutive, cardiac progenitor cell-specific, cardiomyocyte- and vascular smooth muscle cell-specific deletion of NAE1, a regulatory subunit of the NEDD8-specific E1 activating enzyme, were created. Constitutive deletion of NAE1 led to early embryonic lethality before E9.5. Nkx2.5Cre-mediated deletion of NAE1 decreased neddylated proteins in the heart, disrupted normal cardiogenesis and resulted in embryonic lethality by embryonic day (E) 12.5 due to heart failure. Similarly, SM22αCre-driven deletion of NAE1 also caused cardiac failure and embryonic lethality by E13.5. The striking cardiac phenotypes were associated with myocardial hypoplasia, ventricular hypo-trabeculation, and pronounced endocardial and/or epicardial defects in both models. Unbiased transcriptomic analysis revealed dysregulated expression of genes associated with cardiomyocyte differentiation, proliferation, and maturation in NAE1-deficient hearts. Indeed, inhibition of neddylation disturbed cardiomyocyte proliferation, and myofibril assembly in vitro and in vivo. Moreover, defects in cardiomyocyte differentiation and maturation were linked to downregulation of Nkx2.5 and Mef2c, two key transcription factors regulating early cardiogenesis. Conclusion. Collectively, our findings demonstrate that neddylation in cardiac progenitor cells and cardiomyocytes is essential in the regulation of cardiogenesis in transgenic mouse models. Our results uncover a previously unknown role of post-translational modification in the regulation of cardiac development via potential roles in mediating cardiomyocyte proliferation, differentiation, and maturation.
    • DEFINING THE ROLE OF TROPOMYOSIN-1C IN CARGO TRANSPORT IN DROSOPHILA

      Boggupalli, Shankarappa Devi Prasad; Department of Cellular Biology and Anatomy (Augusta University, 2020-05)
      Cell polarity is the asymmetric organization of different organelles in a cell, including the plasma membrane and cytoskeleton. Such organization results from asymmetric sorting of proteins, either post-translationally or pre-translationally by messenger RNA localization. In Drosophila oocytes, posterior localization of oskar mRNA is required for germplasm assembly and establishing antero-posterior polarity. oskar mRNA is transported by Kinesin, however the adaptor that links Kinesin to oskar mRNA was not known. In Aim 1 of this thesis, we demonstrate that a novel isoform of Tropomyosin, namely Tm1C, binds directly to kinesin and functions as the adaptor in linking kinesin to oskar mRNA. Oskar expression is limited to female germline, however Tm1C is also expressed in male flies. This suggests that there might be additional cargoes for Tm1C. We attempted to identify novel cargoes of Tm1C by performing a proteomic assay in Drosophila S2 cells. Apart from Khc, we identified Supernumerary limbs (Slmb) as the main interacting partner. Our further investigation of Slmb suggests that it might not be a cargo. Instead, Slmb which is a component of E3 ubiquitin ligase, might regulate the expression of Tm1C. In Aim 2 of the thesis, we show that Slmb regulates the levels of Tm1C by ubiquitinating it and facilitating its degradation by the Proteasome.
    • THE ROLE OF KYNURENINE, A TRYPTOPHAN METABOLITE THAT INCREASES WITH AGE, IN MUSCLE ATROPHY AND LIPID PEROXIDATION)

      Kaiser, Helen E.; Department of Cellular Biology and Anatomy (Augusta University, 2020-05)
      Loss of mobility and independence are risk factors for falls and mortality, and drastically reduce the quality of life among older adults. The cellular and molecular mechanisms underlying loss of muscle mass and strength with age (sarcopenia) are not well-understood; however, heterochronic parabiosis experiments show that circulating factors are likely to play a role. Kynurenine (KYN) is a circulating tryptophan metabolite that is known to increase with age and is implicated in several age-related pathologies. Here I tested the hypothesis that KYN contributes directly to muscle loss with aging. Results indicate that that KYN treatment of mouse and human myoblasts increased levels of reactive oxygen species (ROS) two-fold, and significantly increased lipid peroxidation enzymes. Small-molecule inhibition of the Aryl hydrocarbon receptor (Ahr), an endogenous KYN receptor, in vitro did not prevent KYN-induced increases in ROS, and homozygous Ahr knockout in vivo did not protect mice from KYN-induced stress, suggesting that KYN can directly increase ROS independent of Ahr activation. In vivo, wild-type mice treated with KYN had reduced skeletal muscle strength, size, and increased oxidative stress and lipid peroxidation. Old wild-type mice treated with 1MT, a small molecule that suppresses KYN production by IDO1, showed an increase in muscle fiber size, peak muscle strength, and oxidative stress. Protein analysis identified mitochondrial lipid peroxidation as a downstream mechanism that is increased upon KYN treatment. Lipid peroxidation enzymes increased with KYN have been shown to produce H2O2 outside of the electron transport chain. Our data suggest that IDO inhibition may represent a novel therapeutic approach for the attenuation of sarcopenia and possibly other age-associated conditions associated with KYN accumulation such as bone loss and neurodegeneration.
    • ROLE OF CLASS III PHOSPHATIDYLINOSITOL 3-KINASE IN THE RENAL PROXIMAL TUBULE

      Liu, Ting; Department of Cellular Biology and Anatomy (Augusta University, 2020-05)
      Yeast only has a single phosphatidylinositol 3-kinase (PI3K) known as vacuolar protein sorting 34 (VPS34); however, mammals have evolved to express three structurally different classes of PI3Ks: class I, II, and III. Although the class III PI3K (Pik3c3) is the only PI3K evolutionarily conserved from yeast to man, its distribution in the mammalian kidney is unknown, and its role in the renal proximal tubule, especially under certain pathophysiological conditions such as nephron loss-induced Compensatory Nephron Hypertrophy (CNH), remains undefined. The goal of Aim 1 was to define the expression pattern and relevant biological function of Pik3c3 in the kidney. We found that the glomerular podocyte expresses the highest level of Pik3c3 in the kidney. Among all renal tubular cells, the specialized distal convoluted tubular epithelial cells called macula densa cells express the highest level of Pik3c3, and the renal proximal tubular cells (RPTC) express the second highest level of Pik3c3. This prompted us to perform additional experiments for Aim 1 that led to the demonstration of an essential function of Pik3c3 in regulating the degradation of epidermal growth factor (EGF) receptor (EGFR) and the termination of EGFR signaling in RPTC following EGF binding with EGFR. The goal of Aim 2 was to determine whether Pik3c3 is essential in mediating uninephrectomy (UNX)-induced compensatory nephron hypertrophy. We generated a global Pik3c3-hypomorphic mouse model and two slightly different proximal tubule-specific Pik3c3 knockout mouse models: Pik3c3Neo-ptKO and Pik3c3ptKO. Interestingly, CNH was markedly inhibited in the global Pik3c3-hypomorphic mouse model and proximal tubule-specific Pik3c3 knockout models. The goal of Aim 3 was to determine the effect and underlying mechanism of complete Pik3c3 deletion in renal proximal tubule cells. We found that complete Pik3c3 deletion in some renal proximal tubule cells resulting in marked cell death that subsequently progressed to tubulointerstitial fibrosis. My project has, for the first time, determined the expression pattern of Pik3c3 in the kidney and provided the first definitive evidence that Pik3c3 controls the degree of CNH and functions upstream of the mTORC1-S6K1-rpS6 pathway in the regulation of CNH. In addition, my project reveals an essential role of Pik3c3 in maintaining the homeostasis and survival of proximal tubule cells.
    • In search of genetic mutations for familial keratoconus

      Khaled, Mariam Lotfy; Department of Cellular Biology and Anatomy (Augusta University, 2019-05)
      Keratoconus (KC) is the most common corneal degenerative disorder and a leading cause of corneal transplantation in developed countries. KC is a multi-factorial disease with involvement of genetic, environmental, and hormonal factors. Although KC has been widely studied, the main cause of the disease and the molecular mechanism remain unknown. We aimed to study the molecular genetics of KC via utilizing next-generation sequencing technology including RNA-Seq, whole exome sequencing, and whole genome sequencing. We used RNA-Seq to study the KC-affected corneal transcriptome. We identified 436 coding RNAs and 584 lncRNAs with differential expression in the KC-affected corneas with a |fold change| ≥ 2 and a false discovery rate ≤ 0.05. Pathway analysis, using WebGestalt, indicated the enrichment of the genes involved in the extracellular matrix, protein binding, glycosaminoglycan binding, and cell migration. Co-expression analysis revealed 296 pairs of genes with significant KC-specific correlations. The RNA-Seq data analysis highlighted the potential roles of several genes (CTGF, SFRP1, AQP5, lnc-WNT4-2:1, and lnc-ALDH3A2-2:1) and pathways (TGF-β, WNT signaling, and PI3K/AKT pathways) in KC pathogenesis. Next, we used whole genome and exome sequencing to figure out the causal mutation(s) in a four-generation KC family with a linkage locus on Chr5q14.3-q21.1. We found a missense mutation in the phosphatase domain of PPIP5K2 (c.1255T>G, p.Ser419Ala). We found another missense mutation in the same domain of PPIP5K2 (c.2528A>G, p.Asn843Ser) in a second KC family. PPIP5K2 is a bifunctional enzyme involved in the inositol phosphate metabolic pathway. In vitro functional assays indicated the impact of the identified mutations on the enzymatic activity of PPIP5K2. PPIP5K2 expresses at a higher level than its homolog PPIP5K1 in both human and mouse corneas. A transgenic mouse model with the loss of phosphatase activity and elevated kinase activity of Ppip5k2 exhibited corneal structural abnormalities emphasizing the important role of PPIP5K2 in the homeostasis of corneal integrity. This study advances our knowledge of KC genetic etiology and helps in identifying a potential therapeutic target for KC.
    • NEUROVASCULAR DEGENERATION FOLLOWING RETINAL ISCHEMIA REPERFUSION INJURY: ROLE OF ARGINASE 2

      Shosha, Esraa; Department of Cellular Biology and Anatomy (2017)
      Ischemic retinopathies such as retinopathy of prematurity, central retinal artery occlusion and diabetic retinopathy are leading causes of visual impairment and blindness. These pathologies share common features of oxidative stress, activation of inflammatory pathways and neurovascular damage. There is no clinically effective treatment for these conditions because the underlying mechanisms are still not fully understood. In the current study, we used a mouse model of retinal ischemia reperfusion (I/R) insult to explore the underlying mechanisms of neurovascular degeneration in ischemic retinopathies. The arginase enzyme utilizes the L-arginine amino acid for the production of L-ornithine and urea. Here, we investigated the role of the mitochondrial arginase isoform, arginase 2 (A2) in retinal I/R induced neurovascular injury. We found that retinal I/R induced neurovascular degeneration, superoxide and nitrotyrosine formation, glial activation, cell death by necroptosis and impairment of inner retinal function in wild type (WT) mice. A2 homozygous deletion (A2-/-) significantly protected against the neurovascular degeneration after retinal I/R. That was attributed to decreased oxidative stress and glial activation. A2 deletion protected against I/R induced retinal function impairment. Using Optical coherence tomography (OCT), we evaluated the retinal structure in live animals and found that A2-/- retinas showed a more preserved structure and less retinal detachment. To investigate the underlying mechanisms of A2 induced vascular damage after I/R, we used an in vitro model of oxygen glucose deprivation/ reperfusion (OGD/R) in bovine retinal endothelial cells (BRECs). Analysis of oxidative metabolism showed impaired mitochondrial function. We also found an increase in dynamin elated protein 1 (Drp1), a mitochondrial fission marker. Mitochondria labeling studies showed fragmented mitochondria after OGD/R. Arginase inhibition reduced mitochondrial fragmentation in OGD/R insult. This dissertation presents A2 as a new therapeutic target in reducing neurovascular damage in ischemic retinopathies.
    • Targeting cyclic GMP signaling for the treatment of gastrointestinal diseases

      Sharman, Sarah Kristen; Department of Biochemistry and Molecular Biology / Cancer Center (2017)
      Continual renewal of the luminal epithelium in the gut is essential for the maintenance of a healthy intestine as it sustains the barrier that protects underlying tissue from infiltration of material passing through the lumen. Dysregulation of homeostatic processes involved in maintenance of the barrier have been implicated in numerous gastrointestinal diseases. The cGMP signaling axis has emerged as an important regulator of homeostasis in the intestinal mucosa, and has been implicated in the suppression of visceral pain, colitis, and colon cancer. While there is considerable interest in exploiting this pathway, until recently the approaches used to increase cGMP have been limited. The present study sought to test the hypothesis that elevation of cGMP in the intestinal epithelium using PDE5 inhibitors will alter epithelial homeostasis and be therapeutic for constipation and preventative for colon cancer. Healthy mice treated with the PDE5 inhibitor sildenafil or the GC-C agonist linaclotide exhibited reduced proliferation and apoptosis, and increased numbers of differentiated secretory cells in the intestinal epithelium. In addition to these homeostatic effects, both drugs normalized intestinal transit and fecal water content in two mouse models of constipation. Furthermore, administration of sildenafil to mice treated with dextran sulfate sodium tightened the disrupted epithelial barrier. Treatment of ApcMin/+ mice with sildenafil or linaclotide significantly reduced the number of polyps per mouse (67% and 50%, respectively). The effect of these cGMP-elevating agents was not on the polyps themselves but was rather on the pre-neoplastic tissue, which was less proliferative and more apoptotic in the presence of the drugs. Taken together, the results of this study demonstrate that increasing cGMP with a pediatric dose of PDE5 inhibitors could be a potential alternative to GC-C agonists for the treatment of gastrointestinal diseases.
    • Oxidation of Dietary Amino Acids Disrupts their Anabolic Effects on Bone Marrow-Derived Mesenchymal Stem Cells

      El Refaey, Mona M.; Department of Cellular Biology and Anatomy (2016-07)
      Age-dependent bone loss has been well documented in both human and animal models. Since it has been proposed that aging is associated with an increase in the generation of damaging reactive oxygen species (ROS), our hypothesis was that the oxidized products of dietary amino acids could play a role in age-induced bone loss by altering osteoprogenitor cell differentiation and function or activating osteoclastic activity. We first examined the effects of the oxidized nutrients on the bone marrow-derived mesenchymal stem cells and our data showed a decrease in the protein and gene expression of osteogenic markers normally stimulated by nutrients. Aromatic amino acids activated signaling pathways involved in protein synthesis in vitro, and thus, in contrast, the oxidized metabolites of these aromatic amino acids had no effect on the activation of these anabolic pathways. We then examined the bone marrow concentration of the oxidized aromatic amino acids in mature (12 months) vs. aged (24 months) C57BL/6 mice and found that kynurenine, the oxidized product of the aromatic amino acid tryptophan, was found in the highest concentration in 12 months mice. Thus, we tested the effects of kynurenine, fed as a dietary supplement, on the bone mass of twelve-month-old C57BL/6 mice compared to a normal protein diet to see if the oxidized amino acid would induce a pattern consistent with age-related bone loss. Twelve-month-old, male C57BL/6 mice were fed one of four diets; 18% protein diet (normal protein diet); 8% protein diet + tryptophan; 8% protein diet + kynurenine (50 μM) and 8% protein diet + kynurenine (100 μM) for 8 wks. Bone densitometry and micro-CT analyses demonstrated bone loss following the kynurenine diet. Histological and histomorphometric studies showed a decreased bone formation and an increased MONA M. EL REFAEY Oxidation of Dietary Amino Acids Disrupts Their Anabolic Effects on Bone Marrow-Derived Mesenchymal Stem Cells (Under the direction of DR. CARLOS M. ISALES) osteoclastic activity in the kynurenine groups; these animals also exhibited an increase in serum pyridinoline, a marker of bone breakdown. Thus, these data demonstrate that feeding an oxidized product of an essential amino acid induces bone loss in a pattern consistent with accelerated aging, and we propose that one of the mechanisms involved in age-induced bone loss may be from alterations of dietary nutrients by the increased generation of ROS associated with aging.
    • Rapamycin, an evolving role in up-regulation of autophagy to improve stroke outcome and increase neuronal survival to stroke type injuries

      Buckley, Kathleen; Department of Cellular Biology and Anatomy (2015-10)
      Rapamycin was shown to reduce infarct size in a non-reperfusion and a slow reperfusion model of murine stroke; it also improved neurological score and survival in the slow-reperfusion model. The rapamycin improvement was 50 percent greater than that observed with chloroquine. In HT22 mouse hippocampal neurons, rapamycin was shown to improve survival to an oxidative/reperfusion injury with H2O2 and a hypoxic/ischemic injury with oxygen and glucose deprivation to a larger degree than chloroquine. Rapamycin treatment increased punctate microtubule light chain associated protein 3, LC3, in the HT22 neurons in an uninjured and oxygen and glucose deprivation injured HT22 neurons compared to untreated neurons. Finally, genetic knockdown of autophagy with shRNA to autophagy protein 5, ATG5, abrogated the rapamycin’s positive effect on survival to injury.
    • Role of microtubules and motor proteins in mRNA localization

      Sanghavi, Paulomi; Department of Cellular Biology and Anatomy (2015-08)
      Establishment of polarity is essential for many cell types to perform their functions. A common mechanism that is used to establish polarity is localization of mRNAs at specific sites. This results in spatial restriction of protein expression. mRNA localization is a widespread phenomenon, occurring in most species. However, the mechanism by which mRNAs are localized is poorly understood. Using Drosophila as the model system, we investigated the localization of one such localized transcript, oskar mRNA. Studying the mechanism by which oskar mRNA is localized is important because many factors involved in localizing this transcript also function in localizing mRNAs in mammalian neurons. oskar mRNA localizes at the posterior pole of the Drosophila oocyte. This results in the posterior restriction of Oskar protein, which is turn functions in establishment of polarity in the oocyte and the future embryo. Localization of oskar mRNA is microtubule-dependent. We, therefore, characterized the polarity of microtubules in the oocyte. Our findings suggest that the posterior region is highly enriched in microtubule plus ends. However, this polarization is not essential for oskar mRNA localization. Secondly, the posterior localization of oskar mRNA was shown to be mediated primarily by the Kinesin-1 motor. Our findings demonstrate the role of an additional motor, Dynein, in this pathway. We found that Dynein associates with oskar mRNA in vivo and depletion of Dynein caused a significant delocalization of oskar mRNA. Next, we examined the role of a Dynein adaptor, Egalitarian (Egl), in the oskar mRNA localization pathway. Egl has been shown to recruit localized mRNAs to the Dynein motor in Drosophila embryos. Our results suggest that Egl associates with oskar mRNA in vivo and is required for the posterior localization of this transcript. Interestingly, one of the mechanisms by which Egl affects the localization of oskar mRNA is by affecting the microtubule polarity in the oocyte. Additionally, depletion of Egl caused precocious translation of oskar mRNA in the oocyte. Thus, our findings revealed a novel function for Egl in organizing oocyte microtubules and in regulating the translation of a localized mRNA.
    • Characterization of the Retinal Phenotype In Methylene Tetrahydrofolate Reductase (Mthfr) Deficient Mice, A Model Of Mild Hyperhomocysteinemia

      Markand, Shanu; Department of Cellular Biology and Anatomy (2015-05)
      Homocysteine (hcy), a sulfur containing amino acid, is an integral part of methionine metabolism. Elevated plasma level of hcy (Hhcy) is identified as a risk factor for cardiovascular disorders and implicated in various retinal diseases such diabetic retinopathy, glaucoma, age related macular degeneration and central retinal vein occlusion. Cystathionine β-synthase (CBS) and methylene tetrahydrofolate reductase (MTHFR) are key enzymes of hcy metabolism. CBS catalyzes the transsulfuration pathway yielding beneficial downstream products such as taurine, H2S and glutathione (GSH). MTHFR is required for methylation of hcy. Mutations in MTHFR are the most common genetic cause for Hhcy. Murine models of CBS and MTHFR are an invaluable tools to understand Hhcy pathophysiology in humans. Our lab has reported the retinal phenotype of CBS mutant mice. Depending upon the loss of one or both alleles, mild to marked retinal neurovascular and functional alterations are observed. The data from CBS mutant mice raise an important question: is the retinal neurovasculopathy observed in absence/deficiency of CBS attributed to excess hcy levels or is it due to decline in availability of taurine, H 2S and GSH? This can be addressed by studying the retinal phenotype of MTHFR mutant mice which have an intact CBS pathway. No information is available is currently available about the retinal expression of MTHFR and current data regarding CBS in the mouse retina is contentious. This thesis work tested the hypothesis that CBS and MTHFR are expressed in the mouse retina at gene and protein levels and that Hhcy would induce retinal functional and neurovascular alterations in MTHFR-deficient mice. For gene and protein expression studies, RNA and protein were isolated from retinas for analysis of Cbs and Mthfr gene expression by RT-PCR and protein expression by Western blotting. Eyes were harvested from C57BL6 mice and used for immunodetection of CBS and MTHFR in the retina. RT-PCR revealed robust Cbs and Mthfr expression in retina. Western blotting detected CBS and MTHFR protein in mouse retina. In immunohistochemical studies of the intact retina, CBS was present most abundantly in the ganglion cell layer of WT retina while MTHFR showed widespread retinal expression. Our immunofluorescence studies revealed presence of CBS and MTHFR in retinal ganglion, Müller and RPE cells. Taken together, we have compelling molecular evidence that CBS and MTHFR are expressed in mouse retina at gene and protein levels. These data indicate the underlying importance of hcy metabolism in the retina. For characterization of the retinal phenotype in MTHFR deficient mice, we employed tools such as ERG, Fundus and FA, OCT, HPLC, morphometric, immunohistochemistry (IHC) and PCR arrays. ERG revealed a significant decrease in positive scotopic threshold response in retinas of Mthfr+/- mice at 24 wks. FA revealed areas of focal vascular leakage in 20% of Mthfr+/- mice at 12-16 wks and 60% by 24 wks suggesting potential vascular damage mediated by Hhcy. SD-OCT revealed a significant decrease in NFL thickness at 24 wks in Mthfr+/- compared to Mthfr+/+ mice. There was a 2-fold elevation in retinal hcy at 24 wks in Mthfr+/- mice by HPLC and IHC. Morphometric analysis revealed ∼20% reduction in cells in the ganglion cell layer of Mthfr+/- mice at 24 wks. IHC indicated significantly-increased GFAP labeling suggestive of Müller cell activation. The similar loss of ganglion cells, focal vascular leakage, 2-fold increase in retinal hcy, gliosis and functional abnormities were reported in Cbs+/- mice. Taken together, these data support our hypothesis that Hhcy induces retinal neurovascular and functional alterations in MTHFR deficient mice. In addition, we explored retinal mitochondrial gene alteration as a possible mechanism of Hhcy mediated retinal alterations. PCR array data analysis revealed upregulation of pro-apoptotic genes and downregulation of genes associated with normal mitochondrial transport function. Future studies will validate these results at protein and functional levels. To conclude, our data support the hypothesis that Hhcy may be causative in certain retinal neurovasculopathies. These data contribute to our understanding of the potential effects of Hhcy on the retina and may prove useful in other disease model systems of Hhcy.
    • Bisphosphonate-Related Osteonecrosis of the Jaw: From Mechanism to Treatment

      Howie, Rebecca; Department of Cellular Biology and Anatomy (2015-04-20)
      With 55 million prescriptions issued each year, bisphosphonates are the second most prescribed class of drug in the United States. They are widely used to treat diseases with excessive osteoclastic resorption, including post-menopausal osteoporosis, Paget’s disease, and tumor metastasis to bone. Unfortunately, with long term intravenous administration of nitrogen-containing bisphosphonates some patients develop bisphosphonate-related osteonecrosis of the jaw (BRONJ). This debilitating disease has limited treatment options once it has manifested and no mechanism for its development has been elucidated. This dissertation explores the novel concept that bisphosphonates cause osteonecrosis of the jaw by impairing osteocyte-induced osteoclastogenesis and, through the physical accumulation of bisphosphonates in bone, impairing the ability of recruited osteoclasts to attach thereby arresting bone healing. Furthermore, it explores the possibility that chelating agents can be used for the removal of bisphosphonate attachment from bone systemically and locally during extractions, potentially leading to a future preventive treatment. It was found that 13 weeks of 80µg/kg intravenous tail vein injections of Zoledronate followed by two mandibular molar extractions caused the clinical presentation of BRONJ as analyzed by the gross, radiographic, and histological methods. Bone dynamic parameters and TRAP staining suggested an impaired ability for the bone to remodel and defective osteoclast attachment in treated groups that persisted eight weeks after the cessation of treatment. Additionally, it was found through the use of a fluorescently tagged bisphosphonate, that the decalcifying agents cadmium, EDTA, and citric acid all had the ability to cause the significant release of bound bisphosphonate from bone. Finally, this dissertation showed that the migration of monocytes treated with low doses of Zoledronate had increased migration, while their migration to conditioned media of osteocytes treated with Zoledronate was impaired. Collectively, these data suggest that invasive trauma by itself consistently precipitated massive bone necrosis in Zoledronate treated animals, possibly through a bisphosphonate driven alteration of monocyte migration and that the use of decalcifying agents could acutely remove bisphosphonate from bone both systemically and locally. This study establishes and effective rodent model for BRONJ and a possible preventive strategy for the side-effects of bisphosphonates during high-risk situations.
    • Role of Autophagy and Apoptosis in the Pathogenesis of Murine Cytomegalovirus Retinitis

      Mo, Juan; Department of Cellular Biology and Anatomy (2014-05)
      This study focused on the roles o f autophagy and apoptosis in the pathogenesis of murine cytomegalovirus (MCMV) retinitis. An overview of MCMV retinitis and the literature concerning autophagy, apoptosis and viral infection are given below, followed by detailed descriptions o f the eye, the retina, MCMV, autophagy and apoptosis.
    • Dynamic Bayesian Network Analysis Reveals Unique and Conserved Elements of Genetic Circuitry Governing Two Different Cell-Specific Regenerative Paradigms in the Retina

      Walker, Steven L.; Department of Cellular Biology and Anatomy (2014-03)
      Regeneration—the capacity to replace lost body parts—has fascinated scientists since the time of Aristotle. Regenerative phenomena became popular with observationalists during the 18th and 19th centuries and a fertile ground for experimentation. In fact, early regenerative biology practitioners such as Abraham Trembley (1710-1784) and his colleagues have been credited with establishing the foundations of modem experimental biology. However, interest faded during the 20th century in part due to the rise of Genetics and later reinforced by the dominance of mammalian model species which have a limited capacity for tissue replacement. At the start of the 21st century, a confluence of “stem cell promise” and new genetic manipulation techniques applicable to a broad range of species has rekindled the field. (Rosenthal, 2003) One aspect, however, remains a consistent and somewhat limiting theme: the emphasis on large-scale injury paradigms (e.g., limb loss). Conversely, the diseases most often cited as potential therapeutic beneficiaries of stem cell/regenerative research advances are typically linked to the loss of specific cell types (e.g., Parkinson’s disease, Type 1 diabetes). We and others have begun to explore cell-specific regenerative paradigms in order to increase understanding of how the loss of individual cell types is detected, how the response to cell loss is regulated, and ultimately how cell-type specific regeneration can be promoted. The goal of this project is to identify genetic networks that regulate the 12 regeneration of individual retinal neuron subtypes. Visual impairment is cited as the second most feared disease following cancer (Office, 2004). While currently considered ‘irreversible’ in mammals, including humans, we posit that retinal cell loss can be ‘cured’ by stimulating dormant regenerative capacities of adult neural stem cells located in the eye (Das et al., 2006); i.e., that reparative therapeutic strategies can be developed that restore visual function to patients by replacing cells lost to degenerative disease or ocular trauma. We developed a system for studying cell-specific loss and replacement in the zebrafish retina, a highly regenerative species (Poss, Wilson, & Keating, 2002), as a means of understanding how the regenerative potential retinal stem cells is regulated. Mammals, including humans, have a limited innate capacity for retinal regeneration (Das et al., 2006). However, recent data suggests that the potential for regeneration is retained in mammals; treatments with discrete molecular factors can enhance retinal cell replacement in mammalian disease models and human retinal stem cells can give rise to new neurons in cell culture. In addition, key cellular and molecular mechanisms governing retinal regeneration appear to be conserved between fish and mammals (e.g., Muller glia acting as injury-induced retinal stem cells) (Reh & Fischer, 2006). Accordingly, we sought to identify genes and genetic networks which regulate the regeneration of individual retinal cell subtypes using temporally resolved differential expression assays combined with cutting-edge statistical methods for establishing connectivity patterns in genetic circuits. By defining pathways which stimulate retinal stem cells to respond to cell losses in a regenerative manner, we aspire to further the development of novel therapies aimed at reversing vision loss in humans.
    • Characterization of the Thioredoxin System in the Diabetic Retina

      Lamoke, Folami; Department of Cellular Biology and Anatomy (2013-07)
      Diabetes is a group of diseases, which are characterized by high blood glucose levels that are a consequence of the inability to produce and/or utilize insulin. Type 1 diabetes (T1D, previously referred to as juvenile diabetes) is typically diagnosed in children and young adults. In this form, the body does not produce insulin primarily due to autoimmune-mediated destruction of pancreatic - cells, leading to insulin deficiency. Type 2 diabetes (T2D, adult onset or noninsulin dependent diabetes) is a chronic condition where the body either resists the effects or does not produce sufficient insulin. This form is more common in African Americans, Latinos, Native Americans, Asian Americans, Pacific Islanders, as well as the aged population.
    • DRP-1 and BIF-1 Regulations in Mitochondrial Dynamics During Apoptosis

      Cho, Sung Gyu; Department of Cellular Biology and Anatomy (2013-06)
      Recent studies have revealed that mitochondrial fragmentation is a critical event in apoptosis. Mitochondria become fragmented and notably, the fragmentation causes the permeabilization o f the mitochondrial outer membrane and consequently contributes to mitochondrial dysfunction and apoptotic cell death. In apoptosis, mitochondrial fragmentation involves the activations of D rpl, a key fission protein, and Bif-1, a protein originally identified to interact with Bax. However, the molecular mechanisms by which Drpl and Bif-1 regulate mitochondrial dynamics during apoptosis remain unclear. In the first study o f my thesis work, I investigated Drpl regulation and its role in apoptosis o f rat proximal tubular cell (RPTC) following ATP depletion. During ATP depletion, Drpl was shown to be dephosphorylated at serine-637. The dephosphorylation could be suppressed by cyclosporine A and FK506, two calcineurin inhibitors, which also prevented mitochondrial fragmentation, Bax accumulation, cytochrome c release and apoptosis in RPTC. The results suggest that Drpl is activated by calcineurin-mediated dephosphorylation at serin-637. Upon activation, Drpl stimulates mitochondrial fragmentation and the permeabilization of outer membrane, resulting in the release o f apoptogenic factors and apoptosis. In the second study, I detected Bif-1 translocation to mitochondria during apoptosis o f RPTC. Notably, apoptotic events including mitochondrial fragmentation, Bax insertion and oligomerization, and cytochrome c release were all suppressed in Bif-1 deficient cells. Mechanistically, we showed that during apoptosis, Bif-1 bound to prohibitin-2 (PHB2), a mitochondrial protein implicated in mitochondrial inner membrane regulation. Furthermore, PHB2 was shown to form hetero-oligomeric complex with prohibitin-l (PHB1) in control cells and the complex broke down upon apoptosis, which was accompanied by the proteolysis of optic atrophy 1 (OPA1), the mitochondrial inner membrane fusion protein. In Bif-1 deficient cells, the breakdown o f PHB complexes and OPA1 proteolysis were both inhibited, supporting a critical role o f Bif-1 in mitochondrial inner membrane fragmentation by regulating PHB2 and OPA1. Our studies have shed new light on the critical molecular mechanisms responsible for the alteration o f mitochondrial dynamics upon cell stress, resulting in mitochondrial fragmentation, injury and apoptosis.
    • The Role of Stromal Cell-Derived Factor-1Β in Osteogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem/Stromal Cells and Bone Formation

      Herberg, Samuel A.; Department of Cellular Biology and Anatomy (2013-03)
      The experiments performed for this dissertation tested the hypotheses that SDF-1β enhances osteogenic differentiation of BMSCs, promotes engraftment and bone formation following whole-body irradiation, and potentiates suboptimal BMP-2 osteoinduction in a model of acute bone injury. We used multipotent primary BMSCs from 18-month-old C57BL/6J mice, genetically modified to overexpress SDF-1β, to ask whether SDF-1β played a role in cell survival and osteogenic differentiation of BMSCs in vitro. Our studies revealed that SDF-1β protected BMSCs from oxidative stress through increasing autophagy and decreasing apoptosis, independent from potential effects on cell proliferation. In support of the hypothesis we also found that SDF-1β enhanced calcium mineral deposition (independent of BMP-2 co-stimulation), upregulated key osteogenic markers, and increased phosphorylation of intracellular Erk1/2 and Smad1/5/8, thereby potentiating BMP-2 signal transduction during osteogenic differentiation, which was attenuated by blocking CXCR4 signaling. We next inquired whether SDF-1β promotes BMSC engraftment and new bone formation. Using direct tibial transplantation in irradiation-preconditioned animals, we found that SDF-1β enhanced new trabecular bone formation upon local BMSC transplantation. The data furthermore suggested that the differential proteolytic clearance of SDF-1 splice variants in the systemic and local environment following myeloablative injury may be an important determinant in the success of stem cell therapy protocols. The suggestion that SDF-1β could regulate BMP-2 osteoinduction through regulating CXCR4 signaling was compelling because several studies have reported a comparable effect using SDF-1α. We examined the direct contribution of SDF-1β to BMP-2 osteoinduction in a critical-size calvaria osteotomy model and found a dose-dependent ability of SDF-1β to potentiate suboptimal BMP-2-induced bone formation to levels comparable to those obtained with the 10-fold higher optimal/benchmark BMP-2 dose, which was blunted by perturbing CXCR4 signaling. These in vitro and in vivo findings expand our understanding of BMP-2 osteoinduction and implicate osteogenesis-enhancing properties of SDF-1β pointing towards its translational potential for cell therapy and regenerative medicine applications. It appears feasible for SDF-1β to improve bone regeneration in a variety of orthopaedic situations and ultimately reduce the burden of musculoskeletal injuries.