1,721,004 research outputs found
Bone Morphogenetic Protein 4 inhibits TGF-beta2 Stimulation of Extracellular Matrix Proteins in Optic Nerve Head Cells: Role of Gremlin in ECM Modulation
Get PDFZode, Gulab Shalikram, Bone Morphogenetic Protein 4 Inhibits TGF-β2 Stimulation of Extracellular Matrix Proteins in Optic Nerve Head Cells: Role of Gremlin in ECM Modulation . Doctor of Philosophy (Cell Biology and Genetics), May 2008; 177pp; 34 figures; bibliography, 192 titles. The glaucomatous neuropathy is caused by irreversible loss of retinal ganglion axons in the optic nerve head (ONH). The extensive remodeling of the extracellular matrix (ECM) in the glaucomatous ONH including increased synthesis and deposition of ECM (increased collagens, basement proteins, and elastin) is associated with loss of axons. Transforming growth factor-beta2 (TGF-β2) is increased in glaucomatous ONH and is thought to be responsible for increased synthesis and deposition of ECM proteins of the ONH. Bone morphogenetic proteins (BMPs) normally maintain the balance of ECM proteins via opposing TGF-β2 stimulated ECM proteins in various cell types. BMP antagonist gremlin inhibits BMPs function, thus may plan an important role in ECM modulation. We previously demonstrated that human ONH expresses BMP-4, BMP receptor and BMP antagonist gremlin. Therefore, we hypothesize that elevated TGF-β2 in the glaucomatous ONH induces gremlin expression that blocks BMP-4 inhibition of TGF-β2 signaling, leading to increased ECM synthesis and deposition. First, we examined whether human ONH tissues and ONH cells express the canonical BMP signaling pathway. This study demonstrated that ONH tissues and ONH cells express BMP-4 and Smad signaling pathway. Treatment of ONH cells with BMP-4 increased phosphorylation of R-Smad/1/58/ phosphorylation and interaction with Co-Smad4 indicating activation of the Smad signaling pathway. Therefore, cells within the human ONH can respond to locally released BMP via activation of Smad signaling. Second, we examined the signaling pathways utilized by TGF-β2 to stimulate ECM in ONH cells. This study demonstrated that TGF-β2 is increased in glaucomatous ONH. Recombinant TGF-β2 increased ECM deposition in ONH cells. TGF-β2 activated phosphorylation of R-smad2/3 but did not alter phosphorylation of ERK1/2, p38, and JNK1/2 in ONH cells. Inhibition of either TGF-β I receptor activity or phosphorylation of R-Smad3 or knockdown of R-Smad2/3 via siRNA reduced TGF-β2 stimulated ECM in ONH cells. Thus, TGF-β2 requires R-Smad2/3 to stimulate ECM proteins in ONH cells. Lastly, we investigated the potential effects of BMP-4 and gremlin on TGF-β2 stimulated ECM in ONH cells. BMP-4 significantly reduced TGF-β2 stimulation of ECM proteins. Addition of gremlin blocked the BMP-4 effect, increasing ECM proteins in ONH cells. Gremlin levels were significantly increased in the human glaucomatous ONH tissues. Interestingly, recombinant gremlin also increased ECM proteins in ONH cells. Gremlin stimulation of ECM proteins required activation of the TGF-β receptor and R-Smad3. TGF-β2 increased gremlin mRNA and protein in ONH cells. Thus, TGF-β2 induced gremlin expression intensifies TGF-β2 effects on ECM metabolism by inhibiting BMP-4 antagonism of TGF-β2 signaling. In conclusion, elevated TGF-β2 and gremlin in the glaucomatous ONH are involved in the pathogenesis of glaucomatous ONH. Elevated TGF-β2 directly increases ECM and also induces gremlin expression, which further aids TGF-β2 to stimulate ECM via inhibiting BMPs antagonism of TGF-β2 signaling, leading to unopposed TGF-β2 stimulated ECM proteins. Interestingly, R-smad3 is required for TGF-β2 or gremlin induced ECM remodeling in ONH cells. Therefore, modulation of R-smad3 provides a novel therapeutic target for preventing ECM remodeling in glaucoma
A Sensitive Assay for Monitoring Wild-Type and Mutant Myocilin Secretion
No full textThe 54th Annual Medical Student Research Forum at UT Southwestern Medical Center (Monday, January 19, 2016, 2-5 p.m., D1.700)Each year the Medical Student Research Program awards students for the best oral presentation and the best poster presentation as judged by faculty across campus. This author received an award as one of the best poster presentations at this forum.Primary open angle glaucoma (POAG)-associated mutations in myocilin (MYOC) cause protein 'non-secretion', rendering secreted MYOC difficult to quantitatively detect using time-consuming conventional techniques. This study focused on developing an assay which could be used to quickly and easily detect mutant and wild-type (WT) MYOC secretion. We fused Gaussia luciferase (eGLuc2) to MYOC variants and expressed the constructs in HEK-293T cells. The secretion, intracellular soluble and insoluble portions of WT and Y437H MYOC eGLuc2 constructs were evaluated by western blotting and compared to FLAG-tagged constructs. Secreted and soluble MYOC eGLuc2 was measured by a GLuc assay. The secretion of nine additional MYOC mutants was assayed in conditioned media from HEK-293T and NTM-5 cells to test the general applicability of the assay. MYOC eGLuc2 behaved similarly to FLAG MYOC with respect to secretion, soluble intracellular levels, and in response to drug treatment. The GLuc assay could sensitively detect Y437H MYOC secretion 30 min after a media change. eGLuc2 fused variants followed predicted trends; non-pathogenic variants (D208E, G244V) were secreted at WT-like levels, whereas predicted disease-causing variants (C245Y, G246R, E300K, Y437H, I477N) demonstrated substantial secretion defects ranging from 0.20 - 4.0% of WT MYOC levels in HEK-293T cells. These variants were slightly more tolerated in NTM-5 cells, resulting in secretion levels ranging from 1.0 - 12% of WT MYOC levels. Secretion defects caused by the C245Y, G246R, and Y437H mutations were partially rescued by permissive growth temperatures. Interestingly, a combination of growth temperature reduction and cycloheximide treatment of transfected cells indicate that the pool of protein that is rescued at lower temperatures arises from intracellular stores, and is not from newly synthesized MYOC under permissive temperature. Fusion of eGLuc2 to MYOC does not significantly change the behavior of MYOC. The use of the eGLuc2-tagged version of MYOC can be utilized to develop a deeper understanding of MYOC folding and secretion. This newly developed MYOC reporter system has the potential to be used in small molecule and/or genetic high-throughput screens to identify modulators of MYOC secretion.Southwestern Medical Foundatio
Endothelin-1 mediated decline in mitochondrial function contributes to neurodegeneration in glaucoma
Get PDFGlaucoma is an optic neuropathy with multifactorial etiologies, commonly associated with elevated intraocular pressure (IOP) and characterized by degeneration of the optic nerve, loss of retinal ganglion cells (RGC), cupping of optic disc and visual field deficits, which could ultimately lead to vision loss. In most cases, glaucoma is a chronic, asymptomatic and gradually progressing neurodegenerative disease, sometimes referred to as the "silent thief of sight," hence, routine eye examinations by an ophthalmologist are critical to determine if there is a likelihood of developing the disease. Elevated IOP is a primary and the only modifiable risk factor in glaucoma. Currently, reducing IOP remains the only proven treatment to delay the progression of RGC death; however, some patients continue to have neurodegenerative effects despite lowering IOP. Therefore, development of novel neuroprotection strategies as an adjunct therapy to IOP-lowering agents will provide a valuable therapeutic strategy in glaucoma. One of the promising targets for neuroprotection is the endothelin system of peptides and their receptors. The endothelin (ET) system comprises of three vasoactive peptides (ET-1, ET-2 and ET-3), which act through two types of G-protein coupled receptors, namely, ETA and ETB receptors. Originally discovered in the cardiovascular system, the diverse expression pattern of endothelin peptides and their receptors implicate their involvement in a variety of physiological processes in the body. A growing body of evidence suggests that endothelins and their receptors are associated with neurodegeneration in glaucoma. Previous studies have demonstrated that ET-1 levels are elevated in aqueous humor (AH) and plasma of glaucoma patients. Our lab previously demonstrated that in an ocular hypertension model in rats, there was an increase in ETB as well as ETA receptor expression primarily in RGCs compared to contralateral eyes. Following IOP elevation, RGC loss was significantly attenuated in the ETB receptor-deficient rats, pointing to a causative role of the ETB receptor in glaucomatous neurodegeneration. However, the precise cellular and molecular mechanisms by which ET-1 promotes neurodegeneration through its actions on the endothelin receptors are not completely understood. Previous studies have shown that ETB receptor stimulation increases the oxidative stress and production of superoxide anions, in sympathetic neurons. Several studies point to the role of mitochondrial dysfunction and oxidative stress as contributors to glaucomatous damage in animal models of glaucoma. To investigate various molecular events contributing to the ET-1 mediated RGC loss in glaucoma, we carried out RNA-seq analysis of the translatome in rat primary RGCs following ET-1 treatment. We identified several key mitochondrial and neurodegenerative gene candidates including Atp5h, Cox17, Foxo1, Moap1 and Map3k11 that were differentially expressed in the translatome by ET-1 treatment in RGCs. Based on our RNA-seq findings, we hypothesized that ET-1 causes an increase in reactive oxygen species (ROS) by acting through the ETB receptor that produces a subsequent decline in mitochondrial function and bioenergetics ultimately predisposing RGCs to cell death. To test this hypothesis, we used an in vitro approach by utilizing rat primary culture of RGCs treated with ET-1 as well as an in vivo approach by intravitreal ET-1 injections in rodents and the Morrison's model of glaucoma in rats. Our data showed that there is a significant decrease in the expression of cytochrome c oxidase 17 copper chaperone (COX17) and ATP synthase, H+ transporting, mitochondrial F0 complex, subunit D (ATP5H), both of which are critical components of the electron transport chain and oxidative phosphorylation pathway. Using a Seahorse mitostress assay, we also found a significant decline of several mitochondrial parameters following ET-1 treatment in primary RGCs, which indicated the possibility of a disruption in the mitochondrial quality control machinery. Hence, we also explored the effect of the ET-1 treatment on the mitophagy pathway, specifically in RGCs. Our findings suggest that there is a decrease in mitophagosome formation in RGCs in the Morrison ocular hypertensive model as well as in GFP-LC3 mice injected with ET-1, indicating an impairment in the mitochondrial quality control mechanism. Our studies reveal several novel candidates that could be targeted for the development of neuroprotective approaches to treat glaucoma
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
The Role of Mitochondrial Respiration in Müller Glia Survival and Function Under Normal and Glaucomatous Conditions In Vivo
No full textSeveral markers of mitochondrial dysfunction have been observed in the retinas of glaucoma patients and experimental animal models. However, these studies have primarily focused on retinal neuron cells even though glial cells too contain significant amounts of mitochondria. Thus, little is known about how glial cell mitochondrial dysfunction contributes to glaucoma pathology. As the principal macroglial cells of the retina, Müller glia (MG) function is essential to maintaining homeostasis in the retina. However, very little is known about how MG generate energy to support their function in vivo. In this study, we address the role of mitochondrial respiration in MG using an inducible Cox10 knockout transgenic mouse model. Cox10 (protoheme IX farnesyltransferase) encodes a component of cytochrome c oxidase (COX), complex IV, of the electron transport chain. Cox10 deficient cells lack functional COX. Disruption of COX function in MG did not affect MG survival nor retinal structure but impaired visual function and upregulated glycolysis pathway protein expression in the retina. These data suggest that MG-specific mitochondrial respiration is essential for whole retinal energy metabolism and visual processing. Hypoxia-inducible factor 1α (HIF-1α) has been shown to be upregulated in the glaucomatous retina and optic nerve, yet its role in glaucoma pathogenesis remains unexplored. HIF-1α is a transcription factor that promotes glycolysis and metabolic adaptation during hypoxia. By blocking HIF-1α degradation through pharmacologic inhibition, we found that prolonged HIF- 1α stabilization led to retinal glycolysis and oxidative phosphorylation (OXPHOS) protein downregulation and AMP-activated protein kinase (AMPK) activation, indicating low energy status. These changes were accompanied by impaired retinal ganglion cell (RGC) function and glial cell activation. Taken together, these results demonstrate the essential role of MG-specific OXPHOS in the retina, as well as pointing to a role for HIF-1α in neurodegeneration in the retina
Targeting Chronic Endoplasmic Reticulum Stress to Ameliorate Glaucomatous Trabecular Meshwork Pathology
Get PDFGlaucoma is a heterogeneous group of diseases characterized by progressive optic neuropathy, ultimately leading to irreversible loss of visual function and potentially, blindness. Primary open angle glaucoma (POAG) is the most common form, contributing to nearly 80% of total glaucoma cases. Chronic elevation in intraocular pressure (IOP) is the predominant and only modifiable risk factor responsible for inducing glaucomatous damage. The glaucomatous damage to trabecular meshwork (TM) is known to increase aqueous humor outflow resistance, elevating IOP in POAG. However, the precise pathological mechanisms of glaucomatous TM damage are poorly understood. Therefore, the current IOP lowering treatments fail to target the mechanisms underlying glaucomatous TM pathophysiology. These treatments may delay the progression of optic nerve degeneration due to temporary lowering of IOP, but the disorder associated with TM dysfunction continues to progress. Restoring the TM's normal homeostatic function will contribute to a true glaucoma cure. There is a clear need to study factors associated with TM pathogenesis, and design treatment modalities to reverse TM damage. Gene therapy technologies can be a viable tool for targeted curative application. An extension to this technology, CRISPR/Cas9 based genome editing permits direct modification of gene variants associated with pathogenicity. Glaucoma is a genetically complex disease and identification of disease-causing genetic alterations can help identify gene correction techniques. Myocilin (MYOC) is the first gene identified that has a genetic anomaly causing glaucoma. The misfolded protein product of mutated MYOC accumulates in the TM causing endoplasmic reticulum (ER) stress and IOP elevation. A recent study (Jain et.al., PNAS, 2017) demonstrated the feasibility of targeting the mutant-MYOC using CRISPR/Cas9 genome editing that reduced ER stress and reversed TM damage in a transgenic mouse model of MYOC-associated POAG. The adenovirus (Ad)-5 vector employed for gene delivery is touted for its selective tropism for the TM. However, the immunogenic effects associated with Ad5 gene delivery make it unsuitable for clinical applications. The biology of lentiviral (LV) vectors and adeno-associated viral (AAV) vectors has been widely explored for their application in delivery of therapeutic cargos. Therefore, part of the current study evaluates the efficiency and selectivity of these vectors to transduce TM and treat MYOC-associated glaucoma using both in vitro and in vivo models. Identification of a common pathological pathway associated with TM dysfunction creates an opportunity to effectively manage and perhaps even cure glaucoma by targeting ongoing disease pathogenesis. Pathological role of chronic ER stress-induced ATF4 and CHOP was previously linked to POAG-associated TM dysfunction in ocular hypertensive mouse models and in human donor eyes. Elevated transforming growth factor (TGF)β2 is also known to be involved in the pathogenesis of POAG causing TM damage and IOP elevation. Therefore, the second part of the current study evaluates the role of chronic ER stress in TGFb2-induced TM dysfunction. This highlights the potential of inhibiting ATF4 and CHOP as a novel therapeutic strategy for curing POAG. We explored this idea by inhibiting ATF4 and CHOP via gene editing (CRISPR/Cas9) and pharmacological means (ISRIB: ER stress inhibitor; PBA: small molecule chaperone) to treat TGFb2-induced glaucomatous TM pathology. Of all the vectors that were investigated, we found LV vectors to be the most efficient in selectively targeting the TM in our mouse model, and in effectively knocking down MYOC via CRISPRguided Cas9 endonuclease activity. MYOC gene editing reduced ER stress in MYOC-associated glaucomatous TM and lowered IOP in the transgenic mouse model. This warrants exploration of the clinical application of our LV-CRISPR/Cas9 constructs by examining their efficacy in the ex vivo human eye perfusion model. The TM cells exposed to TGFb2 developed chronic ER stress, and the inhibition of associated ATF4 and CHOP prevented TGFb2-induced TM dysfunction. LVCRISPR/ Cas9 based gene editing can be employed in the future to target ATF4 and CHOP in POAG. This study also discusses the potential of PBA's non-chaperonin mechanism to prevent the pathological mechanisms associated with TM's actin cytoskeleton and ECM alterations. In conclusion, our studies provide a novel treatment strategy using CRISPR-Cas9 genome editing that ameliorates glaucomatous TM pathology by reducing chronic ER stress
The Effects of Lifelong Glutathione Deficiency on Functional Decline and Redox Signaling
Get PDFPurpose: A recent paradigm shift has implicated redox state as a potential key determinant underlying the aging process. Specifically, a pro-oxidizing shift in the ratio of reduced to oxidized glutathione (key substrate in redox status) is hypothesized to disrupt cellular signaling leading to functional impairments and mortality. Chronic glutathione deficiency is achieved by global knockout of glutamate-cysteine ligase modifier (GCLM), an enzyme subunit at the rate-limiting step in glutathione synthesis. Glutathione levels in GCLM-/- mice are 10-30% of those in GCLM+/+ mice. Our hypothesis stated that diminished glutathione synthesis would be sufficient to produce an accelerated, aging-like pattern effect on function, a shortened lifespan, and negative alterations in redox state. Methods: We characterized GCLM+/+ and GCLM-/- male and female mice with a functional battery (n = 15-23 / sex / age / genotype) measuring motor, cognitive and affective function. We also measured redox state, inflammation, metabolism and autophagy markers in central and peripheral tissues (n = 3-6 / group) at 5, 10, or 20 months of age. Lastly, survivorship and body weights were recorded for all animals (n = 455). Results: Overall, age-related declines in function were observed in all functional tests. In young and adult mice glutathione deficiency did not negatively affect function, rather it decreased anxiety-related behavior, improved coordinated running performance in young females and adult males, and delayed general motor decline in both sexes. In old mice, glutathione deficiency improved balance in males and worsened age-related motordecline in females, yet it had no negative effects on cognition in either sex. Lifespan was also extended in male and female GCLM-/- mice (median and maximum). Lastly, GCLM-/- had reduced liver redox state throughout life but only at 5 months in the young, and had increased inflammatory markers in old mice. Discussion: These data imply that (i) motor and cognitive domains appear to be differentially affected by glutathione deficiency and led to benefits in young/adult GCLM-/- mice, (ii) functional and biochemical outcomes were sexually dimorphic, (iii) glutathione deficiency did not decrease lifespan, but rather extended lifespan, and (iv) redox state was impaired in GCLM-/- mice across the lifespan peripherally, but primarily only at 5 months in central tissues. These data do not support the redox stress hypothesis of aging and require further investigation of the beneficial outcomes associated with chronic glutathione deficiency
- …
