Browsing by Subject "Gene Expression Regulation"
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Item Crosstalk Between Transforming Growth Factor Beta-2 and Toll-Like Receptor 4 in the Trabecular Meshwork(ARVO Journals, 2022-03) Hernandez, Humberto; Medina-Ortiz, Wanda E.; Luan, Tomi; Clark, Abbot F.; McDowell, Colleen M.Purpose: The trabecular meshwork (TM) is involved in the outflow of aqueous humor and intraocular pressure (IOP) regulation. Regulation of the extracellular matrix (ECM) by TGFbeta2 signaling pathways in the TM has been extensively studied. Recent evidence has implicated toll-like receptor 4 (TLR4) in the regulation of ECM and fibrogenesis in liver, kidney, lung, and skin. Here, we investigated the role of TGFbeta2-TLR4 signaling crosstalk in the regulation of the ECM in the TM and ocular hypertension. Methods: Cross sections of human donor eyes, primary human TM cells in culture, and dissected mouse TM rings were used to determine Tlr4 expression in the TM. Trabecular meshwork cells in culture were treated with TGFbeta2 (5 ng/mL), TLR4 inhibitor (TAK-242, 15 muM), and a TLR4 ligand (cellular fibronectin isoform [cFN]-EDA). A/J (n = 13), AKR/J (n = 7), BALBc/J (n = 8), C3H/HeJ (n = 20), and C3H/HeOuJ (n = 10) mice were injected intravitreally with adenovirus 5 (Ad5).hTGFbeta2c226s/c228s in one eye, with the uninjected contralateral eye serving as a control. Conscious IOP measurements were taken using a TonoLab rebound tonometer. Results: Toll-like receptor 4 is expressed in the human and mouse TM. Inhibition of TLR4 signaling in the presence of TGFbeta2 decreases fibronectin expression. Activation of TLR4 by cFN-EDA in the presence of TGFbeta2 further increases fibronectin, laminin, and collagen-1 expression, and TLR4 signaling inhibition blocks this effect. Ad5.hTGFbeta2c226s/c228s induces ocular hypertension in wild-type mice but has no effect in Tlr4 mutant (C3H/HeJ) mice. Conclusions: These studies identify TGFbeta2-TLR4 crosstalk as a novel pathway involved in ECM regulation in the TM and ocular hypertension. These data further explain the complex mechanisms involved in the development of glaucomatous TM damage.Item Cystatin C as a potential therapeutic mediator against Parkinson's disease via VEGF-induced angiogenesis and enhanced neuronal autophagy in neurovascular units(Springer Nature, 2017-06-01) Zou, Jing; Chen, Zhaoyu; Wei, Xiaobo; Chen, Zhigang; Fu, Yongmei; Yang, Xiaoyan; Chen, Dan; Wang, Rui; Jenner, Peter; Lu, Jia-Hong; Li, Min; Zhang, Zhuohua; Tang, Beisha; Jin, Kunlin; Wang, QingCystatin C (CYS C, Cst3) is an endogenous cysteine protease inhibitor that plays neuroprotective roles in neurodegenerative diseases. We aimed to explore the association of CYS C with Parkinson's disease (PD) models and investigate its involvement in the role of neurovascular units (NVUs) in PD neuro-pathogenesis. We used A53T alpha-synuclein (SNCA) transgenic mice and 6-hydroxydopamine-lesioned DAergic PC12 cells as experimental PD models to investigate the mechanisms behind this association. The injections of CYS C were administered to the right substantia nigra (SN) of A53T SNCA transgenic mice to measure the effects of CYS C in transgenic A53T SNCA mice. To explore the angiogenesis in vivo and in vitro, we used the chick embryo chorioallantoic membrane (CAM) assay and tube formation (TF) assay. We found that CYS C has a neuroprotective effect in this in vivo PD model. We observed increased VEGF, NURR1 and autophagy markers LC3B and decreased SNCA and apoptosis marker cleaved CASP3 in different brain regions of CYS C-treated A53T SNCA transgenic mice. In vitro, we observed that CYS C-induced VEGF, a secreted protein, attenuated 6-OHDA-lesioned DAergic PC12 cell degeneration by regulating p-PKC-alpha/p-ERK1/2-Nurr1 signaling and inducing autophagy. VEGF-mediated angiogenesis was markedly enhanced in the conditioned media of 6-OHDA-lesioned PC12 cells with CYS C-overexpression, whereas blockage of autophagy in CYS C-overexpressing PC12 cells significantly downregulated VEGF expression and the associated angiogenesis. Our data indicate that CYS C displays dual neuronal-vascular functions, promoting PC12 cell survival and angiogenesis via regulating the level of secreted VEGF in NVUs. Our study provides evidence that may aid in the development of an alternative approach for the treatment of PD through modulation of CYS C-mediated neuronal-vascular pathways.Item Expression of Mutant Myocilin Induces Abnormal Intracellular Accumulation of Selected Extracellular Matrix Proteins in the Trabecular Meshwork(Association for Research in Vision and Ophthalmology, 2016-11-01) Kasetti, Ramesh B.; Phan, Tien N.; Millar, J. Cameron; Zode, Gulab S.PURPOSE: Abnormal accumulation of extracellular matrix (ECM) in the trabecular meshwork (TM) is associated with decreased aqueous humor outflow facility and IOP elevation in POAG. Previously, we have developed a transgenic mouse model of POAG (Tg-MYOCY437H) by expressing human mutant myocilin (MYOC), a known genetic cause of POAG. The purpose of this study is to examine whether expression of mutant myocilin leads to reduced outflow facility and abnormal ECM accumulation in Tg-MYOCY437H mice and in cultured human TM cells. METHODS: Conscious IOP was measured at various ages of Tg-MYOCY437H mice using a rebound tonometer. Outflow facility was measured in 10-month-old Tg-MYOCY437H mice. Selected ECM proteins were examined in human TM-3 cells stably expressing mutant myocilin and primary human TM cells (n = 4) as well as in the TM of Tg-MYOCY437H mice by real-time PCR, Western blotting, and immunostaining. Furthermore, TM cells expressing WT or mutant myocilin were treated with 5 mM sodium 4-phenylbutyrate (PBA), and ECM proteins were examined by Western blot and immunostaining. RESULTS: Starting from 3 months of age, Tg-MYOCY437H mice exhibited significant IOP elevation compared with wild-type (WT) littermates. Outflow facility was significantly reduced in Tg-MYOCY437H mice (0.0195 mul/min/mm Hg in Tg-MYOCY437H vs. 0.0332 mul/min/mm Hg in WT littermates). Increased accumulation of fibronectin, elastin, and collagen type IV and I was observed in the TM of Tg-MYOCY437H mice compared with WT littermates. Furthermore, increased ECM proteins were also associated with induction of endoplasmic reticulum (ER) stress markers, GRP78 and CHOP in the TM of Tg-MYOCY437H mice. Human TM-3 cells stably expressing DsRed-tagged Y437H mutant MYOC exhibited inhibition of myocilin secretion and its intracellular accumulation compared with TM cells expressing WT MYOC. Expression of mutant MYOC in TM-3 cells or human primary TM cells induced ER stress and also increased intracellular protein levels of fibronectin, elastin, laminin, and collagen IV and I. In addition, TM-3 cells expressing mutant myocilin exhibited reduced active forms of matrix metalloproteinase (MMP)-2 and MMP-9 in conditioned medium compared with TM-3 cells expressing WT myocilin. Interestingly, both intracellularly accumulated fibronectin and collagen I colocalized with mutant myocilin and also with ER marker KDEL further suggesting intracellular accumulation of these proteins in the ER of TM cells. Furthermore, reduction of ER stress via PBA decreased selected ECM proteins in primary TM cells. CONCLUSIONS: These studies demonstrate that mutant myocilin induces abnormal ECM accumulation in the ER of TM cells, which may be responsible for reduced outflow facility and IOP elevation in myocilin-associated glaucoma.Item Glucocorticoid Receptor Transactivation Is Required for Glucocorticoid-Induced Ocular Hypertension and Glaucoma(ARVO Journals, 2019-05) Patel, Gaurang C.; Millar, J. Cameron; Clark, Abbot F.Purpose: Glucocorticoid (GC)-induced ocular hypertension (GC-OHT) is a serious side effect of prolonged GC therapy that can lead to glaucoma and permanent vision loss. GCs cause a plethora of changes in the trabecular meshwork (TM), an ocular tissue that regulates intraocular pressure (IOP). GCs act through the glucocorticoid receptor (GR), and the GR regulates transcription both through transactivation and transrepression. Many of the anti-inflammatory properties of GCs are mediated by GR transrepression, while GR transactivation largely accounts for GC metabolic effects and side effects of GC therapy. There is no evidence showing which of the two mechanisms plays a role in GC-OHT. Methods: GRdim transgenic mice (which have active transrepression and impaired transactivation) and wild-type (WT) C57BL/6J mice received weekly periocular dexamethasone acetate (DEX-Ac) injections. IOP, outflow facilities, and biochemical changes to the TM were determined. Results: GRdim mice did not develop GC-OHT after continued DEX treatment, while WT mice had significantly increased IOP and decreased outflow facilities. Both TM tissue in eyes of DEX-treated GRdim mice and cultured TM cells isolated from GRdim mice had reduced or no change in the expression of fibronectin, myocilin, collagen type I, and alpha-smooth muscle actin (alpha-SMA). GRdim mouse TM (MTM) cells also had a significant reduction in DEX-induced cytoskeletal changes, which was clearly seen in WT MTM cells. Conclusions: We provide the first evidence for the role of GR transactivation in regulating GC-mediated gene expression in the TM and in the development of GC-OHT. This discovery suggests a novel therapeutic approach for treating ocular inflammation without causing GC-OHT and glaucoma.Item Mirna Expression in Glaucomatous and TGFbeta2 Treated Lamina Cribrosa Cells(MDPI, 2021-06-08) Lopez, Navita N.; Rangan, Rajiv; Clark, Abbot F.; Tovar-Vidales, TaraGlaucoma is a group of optic neuropathies that leads to irreversible vision loss. The optic nerve head (ONH) is the site of initial optic nerve damage in glaucoma. ONH-derived lamina cribrosa (LC) cells synthesize extracellular matrix (ECM) proteins; however, these cells are adversely affected in glaucoma and cause detrimental changes to the ONH. LC cells respond to mechanical strain by increasing the profibrotic cytokine transforming growth factor-beta 2 (TGFbeta2) and ECM proteins. Moreover, microRNAs (miRNAs or miR) regulate ECM gene expression in different fibrotic diseases, including glaucoma. A delicate homeostatic balance between profibrotic and anti-fibrotic miRNAs may contribute to the remodeling of ONH. This study aimed to determine whether modulation of miRNAs alters the expression of ECM in human LC cells. Primary human normal and glaucoma LC cells were grown to confluency and treated with or without TGFbeta2 for 24 h. Differences in expression of miRNAs were analyzed using miRNA qPCR arrays. miRNA PCR arrays showed that the miR-29 family was significantly decreased in glaucomatous LC cell strains compared to age-matched controls. TGFbeta2 treatment downregulated the expression of multiple miRNAs, including miR-29c-3p, compared to controls in LC cells. LC cells transfected with miR-29c-3p mimics or inhibitors modulated collagen expression.Item Mitochondrial tRNA methylation in Alzheimer's disease and progressive supranuclear palsy(BioMed Central Ltd., 2020-05-19) Silzer, Talisa K.; Pathak, Gita A.; Phillips, Nicole R.BACKGROUND: Methylation of mitochondrial tRNAs (mt-tRNA) at the 9th position ("p9 site") is known to impact translational efficiency and downstream mitochondrial function; however, direct assessment of mt-RNA methylation is challenging. Recent RNA sequence-based methods have been developed to reliably identify post-transcriptional methylation. Though p9 methylation has been studied in healthy human populations and in the context of cancer, it has not yet been analyzed in neurodegenerative disease, where mitochondrial dysfunction is a prominent and early hallmark of disease progression. METHODS: Mitochondrial p9 methylation was inferred from multi-allelic calls in RNA-seq data. Gene-based association studies were performed in FUMA. Correlations between nuclear gene expression and p9 methylation were tested using Spearman's rho. Fisher's Exact test was used in PANTHER and IPA to test for overrepresentation and enrichment of biological processes and pathways in the top nuclear genes correlated with p9 methylation. RESULTS: Variable methylation was observed at 11 p9 sites in post-mortem cerebellar tissue of elderly subjects who were either healthy or diagnosed with Alzheimer's disease (AD), progressive supranuclear palsy (PSP) or pathological aging (PA). Similarities in degree of methylation were observed between AD and PSP. Certain nuclear encoded genes were identified as significantly associated with p9 methylation. Expression of 5300 nuclear encoded genes was significantly correlated with p9 methylation, with AD and PSP subjects exhibiting similar expression profiles. Overrepresentation and enrichment testing using the top transcripts revealed enrichment for a number of molecular processes, terms and pathways including many of which that were mitochondrial-related. CONCLUSION: With mitochondrial dysfunction being an established hallmark of neurodegenerative disease pathophysiology, this work sheds light on the potential molecular underpinnings of this dysfunction. Here we show overlap in cerebellar pathophysiology between common tauopathies such as Alzheimer's disease and progressive supranuclear palsy. Whether p9 hypermethylation is a cause or consequence of pathology remains an area of focus.Item Peripheral Circulating Exosomal miRNAs Potentially Contribute to the Regulation of Molecular Signaling Networks in Aging(MDPI, 2020-03-11) Zhang, Hongxia; Jin, KunlinPeople are living longer than ever. Consequently, they have a greater chance for developing a functional impairment or aging-related disease, such as a neurodegenerative disease, later in life. Thus, it is important to identify and understand mechanisms underlying aging as well as the potential for rejuvenation. Therefore, we used next-generation sequencing to identify differentially expressed microRNAs (miRNAs) in serum exosomes isolated from young (three-month-old) and old (22-month-old) rats and then used bioinformatics to explore candidate genes and aging-related pathways. We identified 2844 mRNAs and 68 miRNAs that were differentially expressed with age. TargetScan revealed that 19 of these miRNAs are predicated to target the 766 mRNAs. Pathways analysis revealed signaling components targeted by these miRNAs: mTOR, AMPK, eNOS, IGF, PTEN, p53, integrins, and growth hormone. In addition, the most frequently predicted target genes regulated by these miRNAs were EIF4EBP1, insulin receptor, PDK1, PTEN, paxillin, and IGF-1 receptor. These signaling pathways and target genes may play critical roles in regulating aging and lifespan, thereby validating our analysis. Understanding the causes of aging and the underlying mechanisms may lead to interventions that could reverse certain aging processes and slow development of aging-related diseases.