Browsing by Subject "Amino Acids, Peptides, and Proteins"
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Item 17 Beta-Estradiol, Integrins, and Synaptic Proteins(2009-05-01) Chandra, Manjari; Simpkins, James W.Item Adaptation of the Genetic Risk Prediction Model BRCAPRO for Primary Care Settings(2017-05-01) Atienza, Philamer M.; Sumihiro Suzuki; Swati Biswas; Karan P. SinghIdentifying women at high risks of carrying the breast cancer susceptibility genes is crucial for providing timely surveillance and necessary health management interventions. BRCAPRO is one of the most widely used statistical models for breast cancer risk prediction in genetic counseling. It provides carrier probabilities of BRCA1/2 mutations and calculates the risks of developing breast and ovarian cancers. This calculation requires extensive personal and family history information, which makes it difficult to use in primary care where a wider population could be reached. Thus, we developed a two-stage approach for the genetic risk prediction of BRCA1/2 mutation. In the first stage, limited information on the counselee and her family history of cancer are used in simplified versions of BRCAPRO. If the risk at this stage is found to be high, the full BRCAPRO model utilizing the complete family history is implemented in the second stage. We aimed to balance the tradeoff between the amount of information used and the accuracy of the predictions. We explored several first stage tools. BRCAPROLYTE uses information on the affected relatives up to the second degree only. BRCAPROLYTE-Plus additionally includes unaffected relatives by imputing their ages. BRCAPROLYTE-Simple eliminates the need to collect information on the numbers and types of unaffected relatives and imputes them and their ages instead. The study cohorts include 1,917 families mostly at high risk from the Cancer Genetics Network, 796 high-risk families from MD Anderson Cancer Center, and 1,344 population-based families from Newton-Wellesley Hospital. To evaluate the models, we used sensitivity, specificity, area under the curve, and observed versus expected number of carriers. We also considered clinical criteria of number of referrals made by each model. We found the proposed two-stage approach (with BRCAPROLYTE, BRCAPROLYTE-Plus, and BRCAPROLYTE-Simple at the first stage) has very limited loss of discrimination and comparable calibration with BRCAPRO. It identifies a similar number of carriers without requiring a full family history evaluation on all probands. Thus, our two-stage approach allows for practical large-scale genetic risk assessment in primary care.Item Effect of Collagenase Type 2 and Proteinase K Digestion on DNA Yield from Bone Samples Purified on the EZ1 Advanced XL(2015-08-01) Barrett, Lisa C.; Arthur J. Eisenberg; Joseph E. Warren; Raghu R. KrishnamoorthyGenetic results from bone samples often yield low quantities of DNA and poor quality of genetic data. Proteinase K is a proteinase that is commonly used in DNA extraction methods, however the target proteins of Proteinase K do not closely align with the makeup of bone. Collagenase Type 2 is a protease that is more specific to the breakdown of collagen, which bone is comprised of. This study looked at the potential effects of Collagenase Type 2 digestion on bone samples compared to the effects of Proteinase K on quantity and quality of genetic typing. This study also incorporates the EZ1 Advanced XL purification platform and the AmpFLSTR Globalfiler Amplification Kit.Item Effect of Phosphorylation on Muscle Physiology and Biophysical Characterization of Mutations Responsible for Familial Hypertrophic Cardiomyopathy(2016-08-01) Duggal, Divya; Borejdo, Julian; Gryczynski, Ignacy; Clark, Abbot F.Familial hypertrophic cardiomyopathy (FHC) is the most common cause of sudden cardiac death in young individuals. Molecular mechanisms underlying this disorder are largely unknown; this study aims at revealing how disruptions in actin-myosin interactions can play a role in the pathogenesis of this disorder. Cross-bridge (XB) kinetics and the degree of order were examined in contracting myofibrils from the ex vivo ventricles of transgenic (Tg) mice expressing FHC regulatory light chain (RLC) mutation K104E and Troponin I mutation, R21C. Because the degree of order and the kinetics are best studied when an individual XB makes a significant contribution to the overall signal, the number of observed XBs in an ex vivo ventricle was minimized to 20. Autofluorescence and photobleaching were minimized by using a relatively long-lived red-emitting dye. In case of K104E, mutated XBs were significantly better ordered during steady-state contraction and during rigor, but the mutation had no effect on the degree of order in relaxed myofibrils. The K104E mutation increased the rate of XB binding to thin filaments and the rate of execution of the power stroke. In case of R21C, differences were investigated in the left (LV) and right ventricle (RV) mutant where it was found that the mutation imposed significant difference in the distribution of angles that actin makes with thin filament axis: during contraction, actin angles from LV were more tightly distributed compared to actin angles from RV. Collectively, the data indicates that the mutation-induced changes in the interaction of myosin with actin during the contraction- relaxation cycle may contribute to altered contractility and the development of FHC. Phenotypic differences of the R21C mutation in the left versus right mouse ventricles, even though both ventricles express the same isoform of the cardiac highlights the importance of functional differences between the two ventricles of cardiac disease.Item Elucidation of the Mechanism of Action of Carisoprodol at GABAA Receptors(2009-05-01) Gonzalez, Lorie A.; Dillon, Glenn H.Carisoprodol is an increasingly abused, centrally-acting muscle relaxant. Its sedative effects, which contribute to its therapeutic and recreational use, are attributed to its metabolite, meprobamate, a controlled substance with barbiturate-like activity at GABAA receptors (GABAARs). GABAARs are ion channel-coupled protein complexes underlying the majority of fast synaptic inhibition in the central nervous system. Recent evidence suggests carisoprodol may act independently of meprobamate. Thus, we used behavioral and pharmacological approaches to investigate carisoprodol’s effects on GABAAR function with the ultimate goal of elucidating its mechanism of action at these receptors. In mice, the time course of locomotor depression was comparable for carisoprodol (intraperitoneal or oral) versus meprobamate (intraperitoneal). GABAergic ligands substituted for carisoprodol in drug discrimination studies using carisoprodol trained rats. As observed in vitro, carisoprodol’s effects were antagonized by bemegride, a barbiturate antagonist, but not by the benzodiazepine site antagonist flumazenil, suggesting carisoprodol produces barbiturate-like effects in vivo. Moreover, whole-cell patch clamp recordings were obtained from HEK293 cells expressing human α1β2 and αxβzγ2 (where x = 1-4 and z = 1-2) GABAARs. Each receptor configuration was directly activated and allosterically modulated by carisoprodol in a barbiturate-like manner. Carisoprodol efficacy, but not potency, was subunit-dependent with α and β isoforms contributing to carisoprodol site(s) of action. Notably, carisoprodol was more efficacious at α1-containing receptors, consistent with its sedative effects and abuse potential. Homomeric glycine α1 and GABA ρ1 receptors were carisoprodol-insensitive. Despite similarities between carisoprodol and barbiturates, their sites of action are likely not equivalent as barbiturate-sensitive ρ1W328M subunits were carisoprodol-insensitive. However, chimeric ρ1/α1 receptors gained sensitivity to modulation, but not direct activation by carisoprodol. Our findings indicate carisoprodol modulates GABAARs in a subunit- and receptor-dependent manner, contributing to its pharmacological profile and possibly its abuse potential. Furthermore, partial restoration of modulation, but not direct gating by carisoprodol suggests this drug may mediate its effects via multiple sites on GABAARs.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 Evaluation of NK Cell – Astrocyte Interactions: Potential Role in HIV-Associated Neurocognitive Disorders and HIV- Associated Dementia(2015-05-01) Bowen, Kelly E.; Mathew, Porunelloor A.; Mathew, Stephen O.; Hodge, Lisa M.NK cells play important roles in immunity against pathogens and cancer. NK cell functions are regulated by inhibitory and activating receptors binding corresponding ligands on the surface of target cells. During pathological conditions, NK cells were shown to be recruited to the CNS and could impact CNS physiology by killing glial cells and by secreting IFN-g. Astrocytes are intimately involved in immunological and inflammatory events occurring in the CNS and reactive astrogliosis is a key feature in HIV-associated neurocognitive disorders (HAND). There is little data on NK cell-astrocyte interactions and ligands expressed on astrocytes that could impact NK cell function. This study aimed to identify NK-associated ligands expressed by human astrocytes that confer this NK-directed cytotoxicity of astrocytes and assay the cytotoxicity differences in presence and absence of HIV 3S peptide. Using a fusion protein consisting of the extracellular domain of NKp44 fused to Fc portion of human IgG, we determined the expression of a novel ligand for NKp44 (NKp44L) on astrocytes. Incubation of astrocytes with 3S peptide downregulated NKp44L expression on astrocytes implicating protection from NK mediated killing. Thus, our study demonstrated that NKp44 has a protective effect on astrocytes from NK cell mediated killing during HIV infection. Astrocytes could also secrete cytokines that affect the expression of NK receptors on NK cells. We evaluated the expression of receptors on NK cells after co-culture with astrocytes. CD38 expression was increased on primary NK cells after incubation with astrocytes. CD38 is expressed on both NK cells and astrocytes and has an important implication in HIV-1 infection. Blocking CD38 signaling in our studies decreased astrocyte lysis, suggesting CD38 signaling has important implications in NK-astrocyte interactions. Future studies providing novel insights into the role of NK cells in the pathogenesis of HAND and other brain disorders might result in the development of NK cell based therapies for brain pathologies.Item Intercellular Nef transfer and HIV-1 infection of astrocytes(2015-05-01) Luo, Xiaoyu; He, Johnny J.; Ghorpade, Anuja; Wordinger, Robert J.Acquired immune deficiency syndrome (AIDS) is a pandemic caused by human immunodeficiency virus type 1 (HIV-1). It is a major health issue in many parts of the world ever since its discovery in 1981. The most devastating effect of HIV-1 infection is the graduate loss of CD4+ T cells, which eventually leads to the dysfunction of the immune system, susceptibility to opportunistic infections and cancer. HIV-1 Nef protein is long known as an essential pathogenic factor for HIV-1/AIDS pathogenesis. A few recent studies including ours have demonstrated that Nef can be transferred to neighboring cells and alters the function of these cells. However, the underlying mechanism of intercellular Nef transfer is in dispute. In the first part of our study, we characterized two potential underlying mechanisms for intercellular Nef transfer: direct cell-cell contact and exosomes using several complementary strategies and a panel of exosomal markers. First, we showed that Nef was transferred from Nef-expressing or HIV-infected CD4+ T lymphocytes to CD4+ T lymphocytes and astrocytes, and that the transfer was mainly associated with tunneling nanotube formation. Then we determined that Nef enhanced virological synapse formation and induced cytoskeleton re-arrangement and cell surface protrusions, suggesting that Nef promotes the establishment of intercellular connection and communication between infected cells and uninfected cells. Thirdly, we examined the possibility of Nef transfer through exosomes. In the exosome uptake assay, Nef transfer was undetectable while exosome marker CD81 transferred rapidly. In contrast, Nef was detected in crude exosomes collected from Nef-transfected 293T. In addition, two different populations of exosomes were successfully separated by OptiPrep gradient fractionation and determined as AChE+/CD81low/TSG101low exosomes and AChE- /CD81high/TSG101high exosomes. We determined that Nef was selectively secreted into the AChE+/CD81low/TSG101low population. Lastly, microscopic imaging showed no significant Nef detection in exosomal vesicle-like structures in and out the cell. Taken together, this study shows that Nef transfer requires direct cell-cell contact such as tunneling nanotubes, not cell-free exosomes. In addition, this study reveals existence of two types of exosomes: AChE+/CD81low/TSG101low exosomes and AChE/CD81high/TSG101high exosomes. In the second part, we characterized HIV-1 infection of astrocytes. Astrocytes are the most abundant cells in the central nervous system (CNS) and play important roles in HIV-1/neuroAIDS. Detection of HIV-1 proviral DNA, RNA and early gene products but not late structural gene products in astrocytes in vivo and in vitro indicates that astrocytes are susceptible to HIV-1 infection albeit in a restricted manner. We, as well as others have shown that cell-free HIV-1 is capable of entering CD4- astrocytes through human mannose receptor-mediated endocytosis. In this study, we took advantage of several newly developed fluorescence protein-based HIV-1 reporter viruses and further characterized HIV-1 interaction with astrocytes. First, we found that HIV-1 was successfully transferred to astrocytes from HIV-infected CD4+ T cells in a cell-cell contact- and gp120-dependent manner. In addition, we demonstrated that compared to endocytosis-mediated cell-free HIV-1 entry and subsequent degradation of endocytosed virions, cell-cell contact between astrocytes and HIV-infected CD4+ T cells led to robust HIV-1 infection of astrocytes but retained the restricted nature of viral gene expression. Furthermore, we showed that HIV-1 latency was established in astrocytes. Lastly, we demonstrated that infectious progeny HIV-1 was readily recovered from latently infected astrocytes in a cell-cell contact-mediated manner. Taken together, our studies point to the importance of the cell-cell contact-mediated HIV-1 interaction with astrocytes and provide direct evidence to support the notion that astrocytes are HIV-1 latent reservoirs in the CNS.Item Intermittent hypoxia induced opioidergic protection of the heart(2015-08-01) Estrada, Juan A.; Robert T. Mallet; Steve W. Mifflin; J. Thomas CunninghamNormobaric intermittent hypoxia conditioning (IHC) induces a robust cardioprotected phenotype in dogs that is remarkably resistant to ischemia and reperfusion induced myocardial infarction and lethal arrhythmias. Previous studies demonstrated that IHC induced cardioprotection requires β1-adrenergic receptor activity. Cardiac opioid systems are stimulated by, and counteract the harmful effects of, excessive stressors such as sympathetic activity. Additional modes of hypoxic conditioning have been shown to induce synthesis of cardiac enkephalins and delta opioid receptors (DOR). The hypothesis that DOR mediates IHC cardioprotection was examined in two studies conducted in intermittent hypoxia conditioned and non-hypoxic sham dogs. For the first study dogs were assigned to groups subjected to non-hypoxic sham conditioning, IHC, IHC plus the aminothiol antioxidant N-acetylcysteine (NAC), and IHC plus the DOR antagonist naltrindole. After IHC or sham conditioning, the dogs were subjected to an left anterior descending coronary artery occlusion/reperfusion protocol and incidence of reperfusion arrhythmias and myocardial infarct size were measured and adjusted for coronary collateral flow. Naltrindole and NAC abolished the anti-infarct and anti-arrhythmia effects of IHC, in a manner independent of collateral blood flow. Intermittent hypoxia conditioning is thus dependent on DOR activity as well as formation of reactive oxygen species (ROS) during cylic hypoxia-reoxygenation. Whether ROS are generated upstream, downstream, or in parallel to DOR activation remains to be determined. DORs are abundant on dog parasympathetic nerves and therefore are ideally positioned to stimulate cardioprotective cholinergic activity. However it is unknown in what direction IHC modulates bimodal DORs, i.e. modulation of synaptic inhibitory or excitatory activity. In the second study dogs were assigned to sham conditioned, IHC, and IHC plus naltrindole groups. IHC resulted in a profound enhancement of vagal bradycardia, in the absence and presence of increasingly vagolytic doses of the DOR agonist MEAP. This result demonstrated that IHC shifts DOR signaling in favor of the vagotonic DOR-1 receptor subtype. However, the fate of the vagolytic DOR-2 receptors was unknown. Immunolabeling of atrial tissue revealed that IHC increased content of the monosialoganglioside GM1 in autonomic nerve fibers associated with parasympathetic fibers, an effect which may shift DOR signaling in favor of the DOR-1 subtype. In addition, IHC increased the number of fibers containing the vesicular acetylcholine transporter within the sinoatrial node. However, DOR positive fibers in both the atria and SAN were decreased after IHC, perhaps reflecting redistribution or intracellular trafficking of DOR1 and/or DOR2 receptors. Immunoblotting revealed decreased content of adrenergic protein tyrosine hydroxylase in the left ventricle following IHC. Collectively, these results indicate IHC is dependent on opioidergic activity to induce cardioprotection by enhancing cholinergic signaling components at the expense of adrenergic proteins, suggesting IHC-induced shifting of autonomic balance in favor of parasympathetic control of the heart.Item Intravenous Pyruvate to Prevent Renal Injury Following Cardiac Arrest and Resuscitation(2014-08-01) Hollrah, Roger A.; Robert T. Mallet; Myoung-Gwi Ryou; Rong MaIntroduction: Cardiac arrest followed by resuscitation and recovery of spontaneous circulation (ROSC) produces systemic ischemia reperfusion (I/R), affecting all internal organs, including the kidney. This type of stress generates both a robust increase in reactive oxygen and nitrogen species (RONS) and an intense inflammatory response, which can result in renal cell death. The glycoprotein erythropoietin (EPO) has been shown to combat renal I/R injury by offering cyto-protection against inflammation and oxidative damage, as well as inhibiting apoptosis. The endogenous intermediary metabolite pyruvate has been observed to stabilize specific genetic machinery responsible for the production of EPO. This study was conducted to test the efficacy of intravenous pyruvate in exploiting these endogenous mechanisms of EPO to protect the kidney from cardiac arrest-induced, I/R injury. Hypothesis: Pyruvate administration during cardiopulmonary resuscitation (CPR), defibrillation, and ROSC will protect the kidneys from I/R injury by suppressing oxidative stress and inflammation via increased EPO production at the renal corticomedullary border. Methods: Yorkshire swine underwent 10 minutes of cardiac arrest, CPR effected by precordial compressions, and defibrillation, and were recovered for either 4 hours (acute) or 3 days (chronic). The animals were randomly assigned to 1 of 4 groups. Two groups underwent the cardiac arrest protocol described above: one group received intravenous infusion of 2M sodium pyruvate at a rate of 0.1 mmol∙kg-1∙min-1 during CPR and the first 60 minutes of recovery; the other group received an equimolar infusion of NaCl. The other two groups were surgically prepared and infused with NaCl or sodium pyruvate, but were not subjected to cardiac arrest, CPR, or defibrillation. For the acute protocol (n=28), animals were sacrificed 4hr after cardiac arrest, while in the chronic protocol (n=18), animals recovered for 3d before sacrifice. To evaluate the impact of cardiac arrest and pyruvate treatment on renal metabolism and antioxidant defense, proteins were extracted from snap-frozen renal corticomedullary border tissue for spectrophotometric activity assays of a panel of 10 metabolic and antioxidant enzymes; myeloperoxidase (MPO), an enzyme marker of pro-inflammatory leukocytes, was analyzed to assess inflammation. Plasma was sampled before cardiac arrest and at the time of biopsy to measure creatinine concentration, an indirect measure of glomerular filtration rate (GFR). Enzyme-linked immunosorbent assay (ELISA) kits were used to measure EPO content and Kidney Injury Molecule-1 (KIM-1) content, a receptor expressed on renal tubular cells that plays an important role in apoptosis. Tissue sections were stained with hematoxylin and eosin (H&E) and examined under light microscopy to count neutrophils and monocytes and to compare structure integrity across the different treatment groups and protocols. Results: In this study global I/R stress imposed on the kidneys by reversible cardiac arrest did not appreciably alter the activity of the 10 panel enzymes. Despite having no histological evidence of neutrophil infiltration (H&E stained slides), an increase in renal MPO activity was evident at 4 h recovery in the NaCl group which was prevented by pyruvate treatment (P [less than] 0.05). There was no evidence of ultrastructural damage to renal cortical and outer medullary structures. There was a noticeable increase in renal EPO content at 4 h ROSC vs. the sham group. An apparent, albeit not statistically significant, increase in KIM-1 content was observed in the two CPR groups vs. the NaCl-infused sham group. Plasma creatinine concentrations did not change appreciably between pre-arrest baseline and 3 d recovery. Interpretation and Conclusion: The I/R stress produced by the present cardiac arrest-resuscitation failed to alter appreciably the activities of the 10 panel enzymes, suggesting the oxidative stress was not sufficient to overwhelm the kidney’s endogenous antioxidant defenses. Plasma creatinine concentrations were also stable, implying the GFR was maintained and the glomerular ultrastructures were unaffected by I/R. The increase in MPO activity at 4 h ROSC implied a transient infiltration of inflammatory leukocytes, although none were visible on histological examination. The increase in KIM-1 content, though not statistically significant, suggests modest renal apoptotic activity after cardiac arrest and reperfusion. The transient increase in renal EPO content in the NaCl-infused post-arrest vs. sham pigs supports the possibility that even a brief period of renal ischemia by cardiac arrest can evoke renal EPO production. Collectively, these results indicate the renal I/R imposed by cardiac arrest and resuscitation does not inflict appreciable damage on the kidneys or its enzyme systems, at least within the first 3 d of post-arrest recovery. Abbreviations: AKI: acute kidney injury; ARF: acute renal failure; CK: creatine kinase; CPR: cardiopulmonary resuscitation; CS: citrate synthase; EPO: erythropoietin; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; G6PDH: glucose 6-phosphate dehydrogenase; GFR: glomerular filtration rate; GP: glutathione peroxidase; GR: glutathione reductase; HIF-1: hypoxia-inducible factor 1; I/R: ischemia-reperfusion; KIM-1: kidney injury molecule 1; LDH: lactate dehydrogenase; MPO: myeloperoxidase; PFK: phosphofructokinase; PHD: prolyl hydroxylase; RONS: reactive oxygen and nitrogen species; ROSC: recovery of spontaneous circulation.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 Neuroprotective Effects of Brn3b in PC12 Cells and Retinal Ganglion Cells under Glaucomatous Conditions(2015-08-01) Phatak, Nitasha R.; Raghu R. Krishnamoorthy; Weiming Mao; John V. PlanzGlaucoma 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.Item Novel androgen receptor splice variant in the substantia nigra(2017-08-01) Contreras, Jo Garza; Cunningham, Rebecca L.; Basha, Riyaz; Salvatore, MichaelTestosterone can increase calcium influx and cell death in dopamine neurons via a putative membrane androgen receptor (mAR). The mAR induced calcium increase may be due to activation of G-proteins involved in calcium mobilization. Previous studies using an N-terminal targeted androgen receptor (AR) antibody yielded low AR expression in dopamine neurons. Studies in our lab show high AR expression using a C-terminal targeted AR antibody. This difference in expression may be due to an AR variant. We hypothesize an AR variant is present in the membrane of dopaminergic neurons and associated with G proteins. To identify the presence of AR in dopaminergic neurons. We performed immunoblot, sucrose gradient, and immunohistochemistry studies. To determine the protein-protein interaction between mAR and G-proteins we performed co-immunoprecipitation studies. Our results show AR45 localizes in the membrane lipid rafts of dopaminergic neurons. Furthermore, AR45 interacts with Gαq and Gαo G-proteins, which can impact calcium signaling.Item Prevention and treatment of diseases: a small molecule discovery and development approach(2016-08-01) Dalwani, Dhwanil A.; John A. SchetzThis work examined the structure-activity relationship, and molecular mechanisms of different structural classes of small molecules at their target receptors. Three different systems were explored and each chapter is devoted to a single system. All three systems utilized similar experimental approaches, and practical application of the same core pharmacological principles. The first system involved the evaluation of the structure-activity space of small molecules acting on the α-like octopamine receptors from the barnacle Balanus improvisus (BiOctR) and the fruit fly Drosophila melanogaster (DmOctR). A number of molecules belonging to the imidazole and imidazole structural class were determined to have high potency for the BiOctR and the DmOctR. This information will be useful in designing new OctR ligands that are highly selective for the OctRs over their mammalian off-targets. Similarly, for the second system, the structure-activity space of different structural classes of sigma-1 receptor (S1R) ligands were evaluated. Four novel EPGN compounds with more than 100-fold selectivity for the S1R over the sigma-2 receptor were identified which were able to stimulate S1R-mediated BDNF secretion. Potential therapeutic applications of these compounds include the treatment of neurodegenerative diseases like Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. The third system involved the identification of receptor off-targets of efavirenz that may be responsible for efavirenz’s neuropsychiatric adverse events (NPAEs). In this study, multiple receptor targets of efavirenz belonging to the serotonin receptor family and the muscarinic receptor family of G protein-coupled receptors (GPCR) were identified, and its mechanism of action at these targets was established. The most prominent finding of this study was that efavirenz functioned as an inverse agonist, antagonist and an allosteric modulator, depending on off-the target receptor. Knowing which off-target receptors efavirenz interacts with may help to understand the molecular mechanisms responsible for efavirenz’s NPAEs. Overall, the insights gained regarding the mechanisms of action of small molecules will aid in the discovery and development of novel compounds, or an improved understanding of known compounds with established or potential therapeutic value.Item Psalmotoxin-1 and nonproton ligand interactions with acid-sensing ion channels(2015-05-01) Smith, Rachel N.; Gonzales, Eric B.; Dillon, Glenn H.; Sumien, NathalieAcid-sensing ion channels (ASICs) are trimeric, sodium-selective channels activated by extracellular protons. Although ASICs are intriguing molecular targets for pharmacological agents, there remains a lack of selective compounds that differentiate ASIC subtypes. The peripherally located ASIC3 activates with the simple removal of calcium. Additionally, nonproton ligands, like 2-guanidine-4-methylquinazoline (GMQ), have been identified to selectively activate ASIC3 via the nonproton ligand sensor domain (NPLSD). A pair of glutamates in rat ASIC3 (E79 and E423) responsible for GMQ activation is present in ASIC1, despite having no direct modulation effect on the channel. We proposed that nonproton ligand activation of ASIC1 may be state dependent, and relies on expansion of the NPLSD in order for GMQ to reach the binding site and exert its effects. We utilized two features of ASICs in order to test our hypothesis with whole cell and outside out patch-clamp electrophysiology. First, we induced a persistent current in chicken ASIC1 (cASIC1) via a natural venom toxin, Psalmotoxin-1 (PcTx1). We determined that GMQ acts as a molecular wedge by prying apart the transmembrane domains of the cASIC1-PcTx1 protein complex and potentiating ASIC-current. This led us to better understand that the NPLSD is intact in cASIC1 and is sensitive to GMQ additions, albeit in a state-dependent manner. We then theorized that direct activation of rASIC3 by GMQ is possible due the channel’s interaction with extracellular calcium, and were interested in introducing feature into the cASIC1 channel. We generated a chimeric ASIC combining the extracellular, transmembrane, and intracellular domains of rASIC3 and cASIC1 in order to individually isolate the calcium and nonproton ligand effects on channel activation. This chimera, termed cASIC1 (rASIC3-TM/ITC), is comprised of the extracellular domain of cASIC1 and the transmembrane/intracellular domains of rASIC3, and can be activated by GMQ in the absence of calcium similarly to wild-type rASIC3. Thus, GMQ activation was introduced in cASIC1 by replacing the transmembrane domains with those of ASIC3 suggesting that the ASIC3 TM domains dictate NPLSD influence on channel activity. Taken together, we identified that channel state influences nonproton ligand interaction with ASICs, and the transmembrane domains are critical for this interaction.Item Statins and PCSK9 Inhibitors: How They Have Shaped Medicine, A Comparative Review(2016-12-01) Escarsega, Phillip R.; Rustin E. Reeves; Patricia A. GwirtzKeeping serum low density lipoprotein cholesterol (LDL-C) levels within recommended ranges has been proven to lower the risk of cardiovascular events from occurring. To lower LDL-C levels, statins have been the drug of choice for the last few decades since they were introduced. However, the discovery of PCSK9 inhibitors and new knowledge about how they are able to lower LDL-C levels may provide physicians a new path to consider when choosing a course of treatment in patients that fit the criteria for PCSK9 inhibition. Furthermore, there are several risks with statins which may turn physicians towards using PCSK9 inhibitors in patients with uncontrolled LDL-C. This practicum report is a review of the latest developments in PCSK9 drugs and how they compare to statins in lowering LDL levels.Item Trace amine associated receptor 1 (TAAR1), a novel astrocyte receptor for METH-mediated neurotoxicity in HIV-1-associated neurocognitive disorders (HAND)(2015-05-01) Cisneros, Irma E.; Ghorpade, Anuja; Wordinger, Robert J.; Forster, Michael J.This dissertation explores the role of astrocyte trace amine associated receptor 1 (TAAR1), a novel G-protein coupled receptor (GPCR), in modulating the effects of methamphetamine (METH) on astrocyte-mediated excitotoxicity, thereby exacerbating HIV-associated neurocognitive disorders (HAND). The rising pandemic of methamphetamine (METH) abuse has multiple effects and interactions with HIV-1 in infected individuals, affecting both the periphery and the central nervous system (CNS). Moreover, there is a high prevalence of HIV-1 infection among METH users. Underlying evidence provides insight into the cellular mechanisms associated with METH and HIV-1 neurodegeneration, including the effects and byproducts of glial cells, specifically astrocytes. While indirect effects of METH and HIV-1 have been proposed in astrocytes the direct mechanisms by which they contribute to neurodegeneration and continue to evolve. Particularly, imbalance in glutamate homeostasis plays a vital role in METH- & HIV-1-mediated neurodegeneration. We propose METH activates a novel GPCR, trace amine associated receptor 1 (TAAR1), thereby regulating astrocyte-mediated glutamate uptake via excitatory amino acid transporter-2 (EAAT-2), exacerbating HIV-1-induced excitotoxicity. Importantly, our data demonstrate astrocyte functions leading to neurotoxic outcomes like excitotoxicity can be directly exacerbated through TAAR1 regulation. Additionally, extrinsic regulation of TAAR1 signaling, including cAMP, calcium, PKA and PKC, not only reduce activation of subsequent signaling factors, but also reduce or eliminate METH- and IL-1β-mediated alterations in astrocytes glutamate clearance abilities. Finally, preliminary studies indicate that astrocyte-TAAR1 may be a novel therapeutic target for the common morbidity of METH abuse in HAND