Browsing by Author "Krishnamoorthy, Raghu R."
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Item A Bone and Buccal Sensitivity Study Comparison and Stability Study using the PowerPlex[R] Fusion 6C System(2018-05) McDaniel, Ethan L.; Warren, Joseph E.; Planz, John V.; Krishnamoorthy, Raghu R.; Gaydosh-Combs, LauraA validation study, a bone sensitivity study and a stability study were performed using the PowerPlex[R] Fusion 6C System. These studies were performed on a 7500 Real-Time PCR System, 9700 GeneAmp Thermocycler and 3500xL Genetic Analyzer. Buccal DNA was used to develop a method to analyze the DNA profiles gathered during the bone sensitivity study and stability study. DNA profiles for specific concentrations of DNA in solution were obtained during the bone sensitivity study. The stability study showed profiles exhibiting the effects of metal PCR inhibitors being introduced to the DNA extract solutions. Full profiles were obtained for calcium concentrations less than 7.35 mM, while the instrument was fully inhibited for copper concentrations between 0mM and 7.35 mM. Based on the limited data, the PowerPlex Fusion 6C System cannot tolerate copper when present in DNA solutions; whereas, calcium may be tolerated as an inhibitor up to 7.35mM.Item A feed-forward regulation of endothelin receptors by c-Jun in human non-pigmented ciliary epithelial cells and retinal ganglion cells(PLOS, 2017-09-22) Wang, Junming; Ma, Hai-Ying; Krishnamoorthy, Raghu R.; Yorio, Thomas; He, Shaoqingc-Jun, c-Jun N-terminal kinase(JNK) and endothelin B (ETB) receptor have been shown to contribute to the pathogenesis of glaucoma. Previously, we reported that an increase of c-Jun and CCAAT/enhancer binding protein beta (C/EBPbeta) immunohistostaining is associated with upregulation of the ETB receptor within the ganglion cell layer of rats with elevated intraocular pressure (IOP). In addition, both transcription factors regulate the expression of the ETB receptor in human non-pigmented ciliary epithelial cells (HNPE). The current study addressed the mechanisms by which ET-1 produced upregulation of ET receptors in primary rat retinal ganglion cells (RGCs) and HNPE cells. Treatment of ET-1 and ET-3 increased the immunocytochemical staining of c-Jun and C/EBPbeta in primary rat RGCs and co-localization of both transcription factors was observed. A marked increase in DNA binding activity of AP-1 and C/EBPbeta as well as elevated protein levels of c-Jun and c-Jun-N-terminal kinase (JNK) were detected following ET-1 treatment in HNPE cells. Overexpression of ETA or ETB receptor promoted the upregulation of c-Jun and also elevated its promoter activity. In addition, upregulation of C/EBPbeta augmented DNA binding and mRNA expression of c-Jun, and furthermore, the interaction of c-Jun and C/EBPbeta was confirmed using co-immunoprecipitation. Apoptosis of HNPE cells was identified following ET-1 treatment, and overexpression of the ETA or ETB receptor produced enhanced apoptosis. ET-1 mediated upregulation of c-Jun and C/EBPbeta and their interaction may represent a novel mechanism contributing to the regulation of endothelin receptor expression. Reciprocally, c-Jun was also found to regulate the ET receptors and C/EBPbeta appeared to play a regulatory role in promoting expression of c-Jun. Taken together, the data suggests that ET-1 triggers the upregulation of c-Jun through both ETA and ETB receptors, and conversely c-Jun also upregulates endothelin receptor expression, thereby generating a positive feed-forward loop of endothelin receptor activation and expression. This feed-forward regulation may contribute to RGC death and astrocyte proliferation following ET-1 treatment.Item A Novel Multiplex Assay for an Ancestry-Informative Marker (AIM) Panel of INDELs(2016-05-01) Sturm, Sarah A.; LaRue, Bobby L.; Budowle, Bruce; Krishnamoorthy, Raghu R.The current standard for forensic laboratories in criminal casework is to use Short Tandem Repeat (STR) markers to develop an evidentiary profile. Commercially available STR amplification kits yield amplicons 100 to 500 base pairs (bp) in length. Commonly, forensic DNA samples are highly degraded to approximately 180-200 bps in length, resulting in incomplete STR profiles. Therefore, markers that can be generated with smaller amplicons may be better suited for degraded DNA samples. Additionally, there are cases where no STR match was obtained through a DNA database search and thus no investigative lead is obtained. The bioancestry of a sample donor could aid law enforcement in such cases. A class of markers that could provide investigative value from degraded DNA samples is Ancestry-Informative Marker (AIM) Insertion/Deletions (INDELs). INDELs are polymorphisms that can be amplified from degraded samples due to their smaller amplicon size. AIMs have the ability provide bioancestry information. This project tested the hypothesis that a multiplex PCR-based assay of INDELs can be developed, and subsequently be analyzed by capillary electrophoresis for population identity testing applications. The use of this assay would require no additional tools or machinery than what already is in standard forensic laboratories. To test this hypothesis, a previously developed panel of AIM-INDEL markers was used to develop this multiplex assay.Item An Antiapoptotic Peptide for Neuroprotection in Glaucoma(2017-03-14) Krishnamoorthy, Raghu R.; Sampathkumar, Sruthi; Nagaraj, Ram; Stankowska, Dorota L.Purpose: Axonal degeneration and death of retinal ganglion cells (RGC) are primary contributors to vision loss in glaucoma. The purpose of this study was to determine if intraperitoneal administration of the core peptide derived from small heat shock protein αB-crystallin (ABCP) could inhibit RGC death in animal models of glaucoma. Materials and Methods: Brown Norway rats were retrogradely labeled (to detect RGCs) using Fluoro-gold and IOP was elevated (150 mmHg/days) in one eye using the Morrison’s method, while the contralateral eye served as control. The rats were intraperitoneally injected with 10μg of ABCP (n=3 animals per group) three times per week for five weeks. Surviving RGCs were counted in retinal flat mounts. In another model of ischemia reperfusion (I/R) injury, C57BL/6 mice were subjected to IOP elevation of 120 mmHg for 30 min, followed by rapid reperfusion. Intraperitoneal ABCP injections were given 3h before and immediately after the procedure and then once daily post I/R injury for 14 days. RGC apoptosis was assessed using a TUNEL assay (n=2 animals per group). Results: Intraperitoneal injections of ABCP significantly (p Conclusions: Intraperitoneally administered ABCP peptide was able to significantly attenuate RGC death in two animal models of glaucoma. These findings suggest that ABCP has the potential to be developed as a neuroprotective agent in glaucoma.Item Ancestry Informative Markers Tailored to Hispanic Populations(2020-05) Setser, Casandra H.; Cross, Deanna S.; Planz, John V.; Barber, Robert C.; Phillips, Nicole R.; Krishnamoorthy, Raghu R.Hispanic populations are highly heterogeneous despite being grouped together as a conglomerate population; this makes an accurate panel of ancestry informative markers (AIMs) especially important for human identification. In Chapter 2, the Genomic Origins and Admixture in Latinos (GOAL) dataset containing 494,886 SNPs was used for SNP ascertainment. Utilizing a country attributable variant of Wright's FST, 234 SNPs were selected for biogeographic ancestry (BGA) determination by tailoring each SNP to genetic differentiation of specific populations. Accuracy of BGA prediction was tested using multinomial logistic regression (MLR) and as few as 55 SNPs were robust to 90% for all populations studied. The panel of 234 SNPs was compressed by 65.8% to 80 SNPs by decreasing the influence of Honduras and the Dominican Republic SNPs with high country attributable mean FST values in favor of additional SNPs for Colombia, Cuba, and Puerto Rico; this balanced small panel size with classification accuracy. In Chapter 3, the Setser80 Hispanic AIMs panel was tested against the panels of 128 SNPs developed by the Seldin group and 55 SNPs developed by the Kidd group using STRUCTURE, PCA, a naive Bayesian classifier and MLR. In STRUCTURE, the Setser80 was able to distinguish Honduras, the Dominican Republic, and Colombia at K=4, where the Seldin and Kidd panels were optimized at K=3 and distinguished only Honduras and the Dominican Republic; similar results were obtained by PCA. The GOAL dataset was combined with the Admixed American super-population from the 1000 Genomes Project to test the panel on an expanded dataset of seven populations. Overall, the Setser80 had superior results to the Seldin and Kidd panels with 91.5% accuracy by naive Bayesian classifier and 93.2% by MLR. As an indication of its portability, the Setser80 had accuracies of >98% for Peru and >80% for Mexicans living in Los Angeles, which were not involved in SNP ascertainment. Given its accuracy and lack of overlap, the Setser80 may supplement existing panels for more granular Hispanic BGA determination. In Chapter 4, the application of allele frequencies to forensic genetics, genealogy, and clinical genetics are discussed as well as future directions and ethical considerations.Item CHANGES IN ENDOTHELIN RECEPTOR A EXPRESSION IN A RAT MODEL OF OCULAR HYPERTENSION(2014-03) McGrady, Nolan R.; Minton, Alena Z.; Krishnamoorthy, Raghu R.Glaucoma is the leading cause of blindness in developed countries and is the second leading cause of blindness worldwide. The most common and currently only treatable symptom associated with glaucoma is an increase in intraocular pressure (pressure inside the eye). Rodent models have been routinely used to understand the effects elevated pressure has on the eye, and this study focuses on the changes in expression of a protein molecule (endothelin A receptor) within the retina due to elevated intraocular pressure in a rat model of elevated intraocular pressure. Purpose (a): The endothelin system of peptides and their receptors have been implicated for their neurodegenerative role in glaucoma. The purpose of this study was to determine changes in ETA receptor expression within the retina in the Morrison’s elevated IOP model of glaucoma in rats. Methods (b): IOP was elevated in the left eye of adult male retired breeder Brown Norway rats using the Morrison’s model of glaucoma (by injection of hypertonic saline through episcleral veins) while the contralateral eye served as the control. The rats were maintained for two to four weeks following IOP elevation and sacrificed. Retinal sections were obtained from both control and IOP-elevated eyes, and analyzed for changes in ETA receptor expression using immunohistochemistry. ETA receptor immunostaining was co-localized with β-III-Tubulin, which is selectively expressed in retinal ganglion cells. Results (c): After two weeks, rat eyes with IOP elevation showed an increase in immunostaining for ETA receptors in several retinal layers including the inner and outer plexiform layers with a modest increase in the retinal ganglion cell layer. Following four weeks of IOP elevation, ETA receptor expression was modestly increased in the inner and outer plexiform layers of the retina, compared to that in the corresponding contralateral eyes. Conclusions (d): Elevated intraocular pressure results in a time-dependent change in ETA receptor expression. Increased ETA receptor expression is associated with neurodegenerative changes in glaucoma.Item Characterization and Optimization of Nanoparticles for Polynucleotide delivery(2015-05-01) Conjeevaram Nagarajan, Bhavani Saranya; Lacko, Andras G.; Cistola, David P.; Krishnamoorthy, Raghu R.Nucleic acid therapeutics involves the use of polynucleotides (DNA, RNA) as novel therapeutic agents for the treatment of a wide range of diseases including cancer and several metabolic and genetic disorders. However, the highly unstable nature of RNA molecules necessitates the use of drug carriers to prevent them from nuclease degradation and facilitate targeted delivery in vivo. Hence, this study was conducted to optimize the preparation of nanoparticle carriers in order to improve the stability of the polynucleotides (siRNA and mRNA). Additionally, as heterogeneity and stability of nanoparticle formulations are major issues preventing the clinical approval of therapeutic formulations this study was also focused on improving the homogeneity and the stability of the nanoparticles. In the siRNA study, reconstituted high density lipoprotein (rHDL) nanoparticles were used as the delivery vector. Optimization of siRNA-rHDL formulation was attempted with respect to homogeneity, size of the nanoparticle and entrapment efficiency of siRNA. The results showed that the inclusion of the lyophilization step in the preparation of nanoparticles resulted in a marginal increase in the homogeneity. The size analysis of siRNA rHDL nanoparticles using AFM and TEM imaging revealed the presence of spherical nanoparticles in the range of 10-16nm. Optimization studies with mRNA peptide nanoparticle formulation were conducted using a combination of cationic detergents and peptides at varied concentrations. The particle size analysis via Dynamic Light Scattering (DLS) detector revealed the presence of 268 nm diameter particles as the major component of the mRNA nanoparticle formulation that involved the combination of DOTAP (neutralizer) and Myr-5A (Apo A-I mimetic peptide). Further optimization of this formulation will be required to improve the homogeneity of the nanoparticles.Item Contribution of the Endothelin Receptor A to Neurodegeneration in a Rat Model of Ocular Hypertension(2015-03) McGrady, Nolan R.; Minton, Alena Z.; Krishnamoorthy, Raghu R.Purpose: The endothelin system of peptides and their receptors, primarily the ETB receptor, has been shown to have a neurodegenerative role in glaucoma. The purpose of this study was to examine alterations in ETA receptor expression within the retina following IOP elevation by the Morrison’s model of ocular hypertension in Brown Norway rats. Methods: IOP was elevated in the left eye of adult male retired breeder Brown Norway rats using the Morrison model of ocular hypertension (by injection of hypertonic saline through episcleral veins) while the contralateral eye served as the control. Rats were maintained for either two or four weeks following IOP elevation and sacrificed. Retina sections were obtained from both control and IOP-elevated eyes and analyzed for changes in ETA receptor expression by immunohistochemistry. ETA receptor immunostaining was co-localized with β-III-Tubulin, which is selectively expressed in retinal ganglion cells. In separate experiments a live/dead assay was performed using calcein AM and ethidium homodimer on stable 661W clones overexpressing the ETA receptor to determine the effect an increase in ETA receptors could have on cell viability. Results: After two weeks of IOP elevation rat eyes showed an increase in immunostaining for ETA receptors in multiple retinal layers. The most prominent increase in ETA receptor expression was observed in the outer plexiform layer and a moderate increase was seen in the ganglion cell layer and inner plexiform layer. Following four weeks of IOP elevation an increase in ETA receptor expression was observed primarily in the outer plexiform layer compared to that in the corresponding contralateral eyes. A modest increase in the ganglion cell layer was also observed. Cell culture studies showed that cells overexpressing the ETA receptor had a greater number of dead or dying cells compared to the empty vector expressing cells. Conclusion: Elevated intraocular pressure results in a appreciable change in ETA receptor expression. Overexpression of the ETA receptor results in a decrease in cell viability in cultured 661W cells. Further work is needed to understand the precise role of the ETA receptor in neurodegeneration during ocular hypertension.Item Editorial: Disparate roles of mitochondria in cell survival and cell death: new insight from the CNS(Frontiers Media S.A., 2023-12-04) Krishnamoorthy, Raghu R.Item Effect of Detergent Selection on Quantity of DNA Obtained and on STR Profile Developed from Bone-Derived DNA(2016-05-01) Proctor, Frances N.; Warren, Joseph E.; LaRue, Bobby L.; Krishnamoorthy, Raghu R.Bone is sometimes the only source of DNA in cases of unidentified persons, missing persons, and mass fatality incidents, but bone characteristically provides lower quantities of DNA and lower quality short tandem repeat (STR) profiles than that of other sample types. Bone composition is very different from that of blood and soft tissue, therefore using a method designed for these other sample types is less effective for bone. A bone-specific extraction method is needed in order to improve these results. This study investigated the effects of detergent selection on both the quantity of DNA obtained and on the STR profiles produced from bone-derived DNA, as a part of developing a bone-specific DNA extraction protocol.Item Effects of TGFβ2 and BMP4 Downstream Targets ID1 and ID3 in Trabecular Meshwork: Implications In Lowering IOP(2017-12-01) Mody, Avani A.; Clark, Abbot F.; Millar, J. Cameron; Krishnamoorthy, Raghu R.Purpose: Primary open angle glaucoma (POAG) is one of the most prevalent forms of glaucoma, which is a major cause of irreversible vision loss. The major risk factor associated with POAG is increased intra ocular pressure (IOP). Elevated transforming growth factor β2 (TGFβ2) expression in the trabecular meshwork (TM) increases the deposition of extracellular matrix (ECM) and prevents ECM turnover by increasing expression of plasminogen activator inhibitor I (PAI-1) and ECM cross-linking enzymes. These disruptions in ECM physiology in the TM elevate IOP and decrease aqueous humor outflow facility. Bone morphogenetic proteins (BMPs) regulate TGFβ2 induced profibrotic ECM production. The underlying mechanism for BMP4 inhibition of TGFβ2 induced fibrosis remains undetermined. BMP4 induces inhibitor of DNA binding proteins (ID1, ID3), which are negative transcription regulators that suppress fibrosis by regulating ECM component expression. Our study sought to determine whether ID1and ID3 proteins are downstream targets of BMP4 in TM and thereby attenuate TGFβ2 induction of ECM proteins in TM cells, elevated IOP and decreased outflow facility. Methods: Primary human TM cells were treated with BMP4, and ID1 and ID3 mRNA and protein expression was determined by Q-PCR and western immunoblotting. Intracellular ID1 and ID3 protein localization was studied by immunocytochemistry. GTM3 cells were transfected with ID1 or ID3 expression vectors to determine their potential inhibitory effects on TGFβ2 induced fibronectin and plasminogen activator inhibitor-I (PAI-1) protein expression and promoter activities. IOP and AH outflow facility changes were studied in female BALB/cJ mice. Ad5-CMV-ID1 and Ad5-CMV-ID3 viral vectors along with Ad5-CMV-TGFβ2C226S/C288S were injected intravitreally. Ad5-CMV-TGFβ2C226S/C288S was injected along with Ad5- null vector as positive control, while Ad5-null injected mice were included as a negative control. IOP was measured using a Tonolab impact tonometer and AH outflow facility was measured using a constant flow infusion method. In addition we also performed a luciferase reporter activity array assay to study the effect of ID1, ID3 and TGFβ2 on various transcription factor activity in TM. Results: Basal expression of ID1-3 was detected in primary human TM cells. BMP4 significantly induced early expression of ID1 and ID3 mRNA (p [less than] < 0.05) and protein in primary TM cells, and a BMP receptor inhibitor blocked this induction. Overexpression of ID1 and ID3 significantly inhibited TGFβ2 induced expression of fibronectin and PAI-1 in TM cells (p< 0.01). Transduction of mouse eyes with ID1 or ID3 significantly blocked TGFβ2 induced ocular hypertension (P [less than] 0.0001) and AH outflow facility changes in living mice. Further, ID1 (P [less than] 0.01) and ID3 increase NFκB, Notch and cAMP/PKA activity, even in presence TGFβ2. Conclusions: BMP4 induced ID1 and ID3 expression suppresses TGFβ2 profibrotic activity in human TM cells. In addition ID1 and ID3 suppresses elevated IOP and decreases AH outflow facility induced by TGFβ2. Further, increased activity of NFκB, Notch and cAMP/PKA suggest that ID1 and ID3 may regulate TGFβ2 effects in TM via activation of the MMP pathway, inhibition of PAI-1 and Rho kinase activity, resisting cytoskeleton changes and lead to increased TM cellularity. This strongly supports ID1 and/or ID3 as robust candidates as a basis for developing disease-modifying IOP lowering therapies in POAG.Item ENDOTHELIN B (ETB) RECEPTORS CONTRIBUTE TO NEURODEGENERATION IN A RODENT MODEL OF GLAUCOMA VIA UPREGULATION OF C-JUN AND BAX(2014-03) Minton, Alena Z.; He, Shaoqing; Ma, Hai-Ying; Krishnamoorthy, Raghu R.Glaucoma is a group of eye conditions that, if left untreated, can result in blindness. It is commonly associated with an increased pressure inside the eye, known as intraocular pressure or simply IOP. As the pressure builds up inside the eye, it causes damage to the optic nerve, which in turn results in the death of retinal ganglion cells (RGCs). Studies from our lab and others have shown that endothelin 1 (ET-1), the potent vasoactive peptide, contributes to glaucoma. Currently, our lab is interested in understanding the role of the endothelin receptor B (ETB) in glaucoma. We are using rats that do not have ETB receptor (ETB KO rats) and those that have the receptor (WT rats). To mimic glaucoma, the high salt solution is injected into the special vein in the eye (episcleral vein). This causes the build up of pressure inside the eye within 7 to 10 days. This model of glaucoma is called the Morrison’s ocular hypertension model. Previously, we found that IOP elevation for 4 weeks in WT rats caused an appreciable loss of RGCs, which was significantly attenuated in ETB KO rats. In addition, pathological changes in the optic nerve were greatly reduced in ETB KO rats, as compared to those in WT rats. To find out the molecular mechanisms responsible for the death of RGCs, we elevated the pressure inside one eye of adult WT and ETB KO rats, while the contralateral eye served as control. After 2 weeks of IOP elevation, retinal sections were obtained and stained with specific antibodies to detect the levels of c-Jun (the member of the activator protein-1 (AP-1) family) and Bax (protein involved in cell death). We found that WT rats have higher levels of c-Jun and Bax in the retina (especially in the ganglion cell layer), as compared to ETB KO rats. Interestingly, using the Promo 3 software, we found 15 binding sites for members of the AP-1 family of proteins on the rat 1.95 kb upstream promoter region of Bax. Therefore, the transcription factor c-Jun may be an upstream regulator of Bax. In conclusion, transcription factor AP-1 could be involved in the elevation of the ETB receptor levels in the Morrison's model of glaucoma. Conversely, deletion of the ETB receptor results in the lower expression of c-Jun. Taken together, there may be a reciprocal relationship between the AP-1 and ETB receptors. Purpose (a): Previously, our lab has demonstrated that increased levels of ETB receptors contribute to the death of retinal ganglion cells (RGCs) and degeneration of optic nerve axons in the Morrison's elevated intraocular pressure (IOP) model of glaucoma in rats. Moreover, these pathological changes were greatly attenuated in ETB receptor-deficient transgenic Wistar Kyoto rats. Interestingly, an increase in ETB receptor levels in RGCs, following 2 weeks of IOP elevation in Brown Norway rats, was shown to be associated with increased expression of c-Jun, a member of the activator protein-1 (AP-1) family. The current study was aimed at investigating whether the increased expression of c-Jun observed in wild type rats is reduced in ETBreceptor-deficient Wistar Kyoto rats subjected to the Morrison’s model of glaucoma. The status of another apoptotic protein, Bax, was also assessed in these rats. Methods (b): IOP was elevated in one eye of adult wild type and ETB receptor-deficient transgenic Wistar Kyoto rats using the Morrison’s method (injection of hypertonic saline through episcleral veins), while the contralateral eye served as control. After IOP was elevated, rats were maintained for 2 weeks and sacrificed. Retinal sections were obtained and stained with specific antibodies to detect the expression of c-Jun and Bax by immunohistochemistry. In addition, retinal sections were immunostained using an antibody to βIII-tubulin, which is selectively expressed by RGCs in the retina. Images were taken using Zeiss LSM-510 confocal microscope with Z-scan. Results (c): Immunohistochemical analysis showed that IOP elevation for 2 weeks caused increased expression of c-Jun and Bax mainly in the ganglion cell layer (GCL) of wild type transgenic Wistar Kyoto rats as compared to ETB receptor-deficient transgenic Wistar Kyoto rats. Interestingly, using the Promo 3 software, we found 15 binding sites for members of the AP-1 family of proteins on the rat 1.95 kb upstream promoter region of Bax. Therefore, the transcription factor c-Jun may be an upstream regulator of Bax (pro-apoptotic factor). Conclusions (d): Transcription factor AP-1 could be involved in the elevation of the ETB receptor levels in the Morrison's model of glaucoma. Conversely, deletion of the ETB receptor results in the downregulation of c-Jun. Taken together, there may be a reciprocal feedback loop between the AP-1 and ETB receptors.Item Endothelin receptor-mediated neurodegeneration in glaucoma(2017-08-01) McGrady, Nolan; Krishnamoorthy, Raghu R.; Yorio, Thomas; Clark, Abbot F.Primary open-angle glaucoma (POAG) is a complex set of optic neuropathies which are characterized by the degeneration of the optic nerve, cupping of the optic disk and loss of retinal ganglion cells (RGCs). There are approximately 3 million Americans who currently suffer from this disease although this is most likely an underestimation since many individuals with glaucoma are unaware that they have the disease. POAG is an age-related disease progressing slowly over the course of several decades and is most commonly associated with an elevation in intraocular pressure (IOP). Currently available treatments for glaucoma, both surgical and pharmacological, are solely focused on the regulation of IOP; nevertheless, some individuals continue to show progressive damage despite being on available therapies. In recent years, there has been increased momentum towards the development of neuroprotective strategies for POAG, particularly in preclinical models of glaucoma. Despite these efforts, there is still no neuroprotective treatment currently available for glaucoma patients. A potential target for the development of a neuroprotective approach is the endothelin system of peptides and their receptors. The endothelin (ET) system is composed of three vasoactive peptides (ET-1, ET-2 and ET-3) which are comprised of 21-amino acids. The peptides bind to two G-protein coupled receptors (ETA and ETB receptors) leading to activation of numerous signal transduction pathways. Although originally described for its role in the vasculature, all components of the ET system has been shown to be expressed in multiple tissues and cell types and are responsible for diverse cellular effects. Clinical studies have demonstrated an increase in ET-1 concentrations both in the aqueous humor and plasma of glaucoma patients. A previous study by our lab, using a rodent model of ocular hypertension, showed that endothelin B (ETB) receptor expression is increased when compared to control eyes and contributes to neurodegeneration (Minton et al., 2012). Preliminary data in the current study, using Brown Norway rats, demonstrated that ETA expression is also increased in the IOP elevated eyes, suggesting the possibility that the ETA receptor might also have a degenerative role during ocular hypertension. We hypothesize that the ETA expression increases following IOP elevation and contributes to the neurodegeneration of retinal ganglion cells and their axons. To test this hypothesis we employed a well-characterized in vivo model of glaucoma as well as multiple cellular and molecular approaches to understand the role of the ETA receptor in glaucomatous degeneration. Our data suggest that overexpression of the ETA receptor promotes cell death in cultured RGCs. Since both ETA and ETB receptors appear to contribute to neurodegeneration, we tested the ability of an FDA approved medication, macitentan, for neuroprotection in the Morrison model of glaucoma in rats and found it to promote RGC survival. Our studies raise the possibility of testing macitentan as a neuroprotective treatment for glaucoma patients.Item Endothelin-1 mediated decline in mitochondrial function contributes to neurodegeneration in glaucoma(2020-08) Chaphalkar, Renuka M.; Krishnamoorthy, Raghu R.; Stankowska, Dorota L.; Clark, Abbot F.; Zode, Gulab S.Glaucoma 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.Item Extracellular PACE4 is increased following transient oxygen glucose deprivation in Optic Nerve Astrocytes(2008-05-01) Fuller, John Anthony; Wordinger, Robert J.; Clark, Abbot F.; Krishnamoorthy, Raghu R.Fuller, John Anthony Extracellular PACE4 is increased following transient oxygen glucose deprivation in Optic Nerve Astrocytes. Doctor of Philosophy (Biomedical Sciences), May, 2008, 140 pp., 2 tables, 25 illustrations, bibliography, 218 titles. Primary Open Angle Glaucoma (POAG) is a family of heterogeneous optic neuropathies characterized by progressive retinal ganglion cell (RGC) death that leads to peripheral vision loss and eventually blindness. Various risk factors are associated with glaucoma, however the molecular mechanisms leading to RGC cell death remain unknown. The optic nerve serves as the conduit for the transmission of retinal ganglion action potentials to the brain. The cells that compromise the optic nerve form a scaffold that forms a physical support for the RGC axons. One cell type found throughout the optic nerve and associated with the RGC axon is the optic nerve astrocyte (ONA). Astrocytes are a predominant cell throughout the CNS and are believed to play crucial roles in metabolic, growth factor, and structural support, and respond to protect neurons during injury. The neuronal-glial interface in the optic nerve is poorly understood and believed to plan an important role in POAG pathophysiology, as unmyelenated RGC axons have direct contact with astrocyte processes. IN this study, the subtilisin-like Proprotein Convertases, (SPC) a family of proteases responsible for cleaving a wide variety of protein substrates, were examined in the retina and optic nerve head. PACE4, an SPC found to be secreted and active in the extracellular matrix was found to be highly expressed in the optic nerve, and colocalized to Mϋller cells in the retina and astrocytes in the optic nerve. Exposure of primary optic nerve astrocytes to oxygen-glucose deprivation (OGD) induces an increase in PACE4 mRNA. Furthermore, protein levels of extracellular, processed PACE4 increase following transient ODG, whereas the pro form of the molecule is degraded, and is believed to be chaperoned by the cleaved cysteine rich domain, a product found at high levels in the optic nerve in situ and the ONA in vitro. Due to the extracellular activity of PACE4, we hypothesized that it may regulate the bioactivity of TGF-β2, a growth factor believed to be involved in glaucoma-associated ONH remodeling by inducing the production of extracellular matrix (ECM). When PACE4 is inhibited via siRNA-mediated knockdown, as well as extracellular inactivation, TGF-β2 levels decrease. In addition, fibronectin, a major component of the ECM, is decreased. Furthermore, there is an increase in latent TGF-β2 secreted from the cell. It is therefore possible that PACE4 plays an active role in extracellular growth factor maturation, and may be a central mediator for growth factor bioactivity in the glaucomatous ONA.Item Impact of GLUT1 transporter knockout in optic nerve head astrocytes and retinal ganglion cells(2023-05) Gollamudi, Phani Sree Harsha; Inman, Denise M.; Clark, Abbot F.; Krishnamoorthy, Raghu R.Introduction: Astrocytes and RGC axons are the primary constituents of the optic nerve head, the initial site of neurodegeneration in glaucoma. This study was intended to understand the metabolic relationship between them. We hypothesized that reducing glucose transporter-1 (GLUT1) expression in astrocytes will increase the RGC-associated pathology after ocular hypertension (OHT). Methods: Mice carrying a GLUT1 gene flanked by loxP sites crossed with a mouse carrying the glial fibrillary acidic protein (GFAP) promoter directing a Cre-ERT2 fusion protein (GFAP-GLUT1") were used (n=30) and were divided into 4 groups: GLUT1-knockout+OHT (Tamox OHT), Control OHT, GLUT1-knockout+No OHT (Tamox), and Control+No OHT (Control naïve). Baseline and final intra-ocular pressure (IOP), pattern electroretinogram (PERG), and visual evoked potential (VEP) measurements were taken. OHT was induced via magnetic microbead injection. Retinas, optic nerves, and brains were collected for retinal ganglion cell (RGC) quantification, synapse analysis, biochemical assays, and protein analysis. Results: Statistically significant increases were noted in the IOP data between mice subjected to OHT and the No OHT groups. OHT led to statistically significant decreases in RGC number, regardless of GLUT1 status. Statistically significant decreases in PERG amplitude were noted in all groups subjected to OHT. GLUT1 knockout PERG amplitude was significantly lower than Control at the outset, suggesting a negative impact on retinal physiology from loss of the GLUT1 in astrocytes. Conclusion: Our data indicate glial metabolic homeostasis can impact retinal physiology, but GLUT1 knockout did not appear, on its own, to negatively impact RGC survival. Future studies will give us better understanding of other structural or functional compromise induced by loss of GLUT1 in astrocytes, including potential compensatory mechanisms that enabled astrocytes to meet their metabolic needs.Item Involvement of c-Jun N-terminal kinase 2 (JNK2) in Endothelin-1 (ET-1) Mediated Neurodegeneration of Retinal Ganglion Cells(ARVO Journals, 2021-05-03) Kodati, Bindu; Stankowska, Dorota L.; Krishnamoorthy, Vignesh R.; Krishnamoorthy, Raghu R.Purpose: The goal of this study was to determine whether JNK2 played a causative role in endothelin-mediated loss of RGCs in mice. Methods: JNK2-/- and wild type (C57BL/6) mice were intravitreally injected in one eye with 1 nmole of ET-1, whereas the contralateral eye was injected with the vehicle. At two time points (two hours and 24 hours) after the intravitreal injections, mice were euthanized, and phosphorylated c-Jun was assessed in retinal sections. In a separate set of experiments, JNK2-/- and wild type mice were intravitreally injected with either 1 nmole of ET-1 or its vehicle and euthanized seven days after injection. Retinal flat mounts were stained with antibodies to the RGC marker, Brn3a, and surviving RGCs were quantified. Axonal degeneration was assessed in paraphenylenediamine stained optic nerve sections. Results: Intravitreal ET-1 administration produced a significant increase in immunostaining for phospho c-Jun in wild type mice, which was appreciably lower in the JNK2 -/- mice. A significant (P < 0.05) 26% loss of RGCs was found in wild type mice, seven days after injection with ET-1. JNK2-/- mice showed a significant protection from RGC loss following ET-1 administration, compared to wild type mice injected with ET-1. A significant decrease in axonal counts and an increase in the collapsed axons was found in ET-1 injected wild type mice eyes. Conclusions: JNK2 appears to play a major role in ET-1 mediated loss of RGCs in mice. Neuroprotective effects in JNK2-/- mice following ET-1 administration occur mainly in the soma and not in the axons of RGCs.Item Involvement of Caspase-7 in Photoreceptor and Retinal Ganglion Cell Death(2014-08-01) Choudhury, Shreyasi; Pang, Iok-Hou; Wordinger, Robert J.; Krishnamoorthy, Raghu R.Apoptosis has been implicated in retinal cell death during both retinal differentiation and degeneration. In diseases such as retinitis pigmentosa, glaucoma, age-related macular degeneration, diabetic retinopathy and traumatic optic neuropathy, retinal cell apoptosis plays an important role. Caspases, a family of cysteine proteases, are major players of apoptosis. Thus, one obvious target for modulating apoptosis is the caspase family of proteins. The role of initiator caspases (caspase-1, -2, -8, -9) and effector caspases (caspase-3, -6) in retinal neuronal apoptosis has been studied previously. But the role of a unique effector caspase, caspase-7, has never been studied before. The purpose of this study was to investigate the role of caspase-7 in retinal neuronal cell apoptosis, especially in photoreceptor and retinal ganglion cell (RGC) death. We used the T17M RHO mouse, an animal model for Autosomal Dominant Retinitis Pigmentosa, to study photoreceptor cell apoptosis, and evaluate the role of caspase-7 in a corresponding caspase-7 knockout mouse. Our results show that morphological (evaluated by spectral-domain optical coherence tomography (SD-OCT) and histology) and functional (by electroretinography (ERG)) degenerations in the photo-receptor cells of the T17M RHO mouse are significantly protected by knocking out caspase-7. We further discovered that caspase-7 inhibition reprograms the unfolded-protein response and reduces JNK-induced photoreceptor cell death. To assess the role of caspase-7 in RGC apoptosis, we used the mouse optic nerve crush-induced RGC death as a study model. We found that the insult activates caspase‐7 in RGCs in a time-dependent manner, concomitant with loss of the cells. We also observed the activation of calpain-1, an upstream activator of caspase-7 and the hydrolysis of caspase-7 specific substrates, confirming the involvement of caspase-7. Most importantly, in caspase--‐7 knockout mice, significantly more RGCs survive the optic nerve injury when compared to injured wild type mice as assessed morphologically (immunohistochemistry and SD-OCT) and functionally (ERG) throughout the 28-day post crush study period. Altogether, our findings indicate that caspase-7 appears to play a critical role in photoreceptor and RGC death and inhibition of caspase-7 activity may be a novel therapeutic strategy for retinal degenerative diseases.Item Modulating mitochondrial calcium channels (TRPM2/MCU/NCX) as a therapeutic strategy for neurodegenerative disorders(Frontiers Media S.A., 2023-11-06) Johnson, Gretchen A.; Krishnamoorthy, Raghu R.; Stankowska, Dorota L.Efficient cellular communication is essential for the brain to regulate diverse functions like muscle contractions, memory formation and recall, decision-making, and task execution. This communication is facilitated by rapid signaling through electrical and chemical messengers, including voltage-gated ion channels and neurotransmitters. These messengers elicit broad responses by propagating action potentials and mediating synaptic transmission. Calcium influx and efflux are essential for releasing neurotransmitters and regulating synaptic transmission. Mitochondria, which are involved in oxidative phosphorylation, and the energy generation process, also interact with the endoplasmic reticulum to store and regulate cytoplasmic calcium levels. The number, morphology, and distribution of mitochondria in different cell types vary based on energy demands. Mitochondrial damage can cause excess reactive oxygen species (ROS) generation. Mitophagy is a selective process that targets and degrades damaged mitochondria via autophagosome-lysosome fusion. Defects in mitophagy can lead to a buildup of ROS and cell death. Numerous studies have attempted to characterize the relationship between mitochondrial dysfunction and calcium dysregulation in neurodegenerative diseases such as Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Amyotrophic lateral sclerosis, spinocerebellar ataxia, and aging. Interventional strategies to reduce mitochondrial damage and accumulation could serve as a therapeutic target, but further research is needed to unravel this potential. This review offers an overview of calcium signaling related to mitochondria in various neuronal cells. It critically examines recent findings, exploring the potential roles that mitochondrial dysfunction might play in multiple neurodegenerative diseases and aging. Furthermore, the review identifies existing gaps in knowledge to guide the direction of future research.Item Neuroprotective Effects of Brn3b in PC12 Cells and Retinal Ganglion Cells under Glaucomatous Conditions(2015-08-01) Phatak, Nitasha R.; Krishnamoorthy, Raghu R.; Mao, Weiming; Planz, John V.Glaucoma is a group of chronic progressive optic neuropathies commonly characterized by elevated intraocular pressure (IOP) (a subset of glaucoma patients display neurodegenerative effects at ‘normal’ IOP) leading to axonal degeneration, optic nerve head cupping and apoptosis of retinal ganglion cell death (RGCs), which result in visual field defects and blindness. While there are medications available to lower IOP, there is an unmet need for neuroprotective treatments for glaucoma, since some neurodegenerative effects persist despite lowering IOP. The main focus of this study was on the class 4 POU domain transcription factor, Brn3b, which has been shown to play a key role in the development of RGCs. Two previous studies from other labs showed that a decrease in Brn3b expression occurs in animal model of glaucoma. A recent publication from our laboratory demonstrated neuroprotective effects of adeno-associated virus (AAV) mediated expression of Brn3b in a rat model of ocular hypertension. This research project identified some mechanisms of Brn3b-mediated neuroprotection in cultured PC12 cells (under the condition of hypoxia) and also in vivo in the Morrison’s model of ocular hypertension in rats. In the first part of the study, we demonstrated the effect of overexpression of Brn3b on various markers of synaptic plasticity in PC12 cells under conditions of normoxia as well as hypoxia. Immunoblot as well as immunocytochemical analyses revealed an increase in expression of neurite growth markers, GAP-43 and ac-TUBA, by Brn3b upregulation both under conditions of normoxia as well as hypoxia. . This suggests that transcription factor Brn3b has the ability to upregulate expression genes contributing to synaptic plasticity genes both under ‘normal’ conditions and during a glaucomatous insult (hypoxia). In the concluding part of this study, cell survival factors including, Bcl-2, Bcl-xL and p-AKT were studied as potential targets of Brn3b-mediated neuroprotection. Adeno-associated virus-mediated expression of Brn3b in rat eyes with elevated IOP promoted an upregulation of Bcl-2, Bcl-xL and p-AKT in RGCs, as determined by immunohistochemistry. Taken together, the evidence suggests that Brn3b has the potential to be developed as a therapeutic agent for neuroprotection during ocular neurodegenerative diseases like glaucoma.