Year: 2017
Type: article
Abstract: Introduction 61 2.3 Results 63 2.3.1 Increase in GLRX copy number is associated with earlier age of PD onset 63 2.3.2Inflammatory agents induce Grx1 in microglia 65 2.3.3Grx1 levels in microglia govern activation 69 2.3.4Grx1 upregulation in microglia promotes neuronal cell death in co--culture 71 2.3.5 Microglial Grx1 induction in response to LPS is regulated by the AP--1, NFκB / Nurr1 axis 76 2.3.6 Grx1 overexpression in vivo displays a pro--inflammatory phenotype and promotes DA neuronal loss in a model of metabolic syndrome 81 2.4 Discussion 84 2.4.1 Glutaredoxin upregulation --a common factor in pro--inflammatory activation of immune cells 84 2.4.2Mechanism of microglial Grx1 induction and implications for PD 85 2.4.3Interrelationship of Grx1 and TNF--α --role in inflammation 86 2.4.4Exacerbating effect of insulin resistance on dopaminergic viability 88 2.4.5 Genetic evidence for elevated Grx1 in PD onset 91 v 2.5 Materials and Methods 94 2.5.1 Reagents 94 2.5.2Animals and animal tissue 94 2.5.3Immunohistochemistry 95 2.5.4 Isolation of mouse primary neonatal microglia 95 2.5.5 Isolation of rat primary embryonic neurons 96 2.5.6 Cell lines and treatments 96 2.5.7 RNA silencing in BV2 cells 97 2.5.8Adenoviral Grx1 overexpression 97 2.5.9Co--culture 97 2.5.10 Brain tissue homogenization and analysis 98 2.5.11Chromatin immunoprecipitation (ChIP) 99 2.5.12Glutaredoxin activity assay 99 2.5.13ELISA 100 2.5.14 Determination of glutathionylated protein content 100 2.5.15RT--qPCR 100 2.5.16Immunoprecipitation 100 2.5.17Western blotting 101 2.5.18Gene expression analysis 101 2.5.19Copy number variant analysis 102 Chapter 3: Novel Chloroacetamido Compound CWR--J02 is an Anti--Inflammatory Glutaredoxin--1 Inhibitor vi 103 3.1 Abstract 103 Chapter 4: Discussion and Significance 147 4.1 What are the factors regulating Grx1 transcription in microglia?147 4.2 Which S--glutathionylatable proteins mediate the pro--inflammatory effect of Grx1 upregulation in microglia?149 4.3 How can Grx1 induction promote PD? 151 4.4 How does Grx1 synergize with HFHS/T2D to promote PD onset?158 4.5 Grx1 in astrocytes 162 4.6 Predicting therapeutic uses for J02 outside the anti--inflammatory realm 165 4.7 Significance of the work 169Chapter 5: Future Directions 172 5.1 Determining the role of Grx1 induction versus diminution viii in the onset and progression of Parkinson's disease 172 5.2 Designing a Grx1--targeted inhibitor 1 Glutaredoxin--1 As A Therapeutic Target In Neurodegenerative Inflammation Abstract by OLGA GORELENKOVA MILLER Parkinson's disease (PD) is a debilitating movement disorder.Degeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta is the main feature of PD.PD primarily affects the elderly, and it .is incurable.The main treatment for PD is Levodopa (L--DOPA, L--3,4--dihydroxyphenylalanine), a brain-permeable precursor of dopamine, alleviates disease symptoms by replenishing the neurotransmitter lost due to neuronal degeneration.However, L--DOPA does not block disease progression.PD is a social as well as an economic burden.Currently, over 1 million Americans are suffering from PD, and medical care for PD patients costs an estimated $23 billion per year in the US.Therefore, developing better treatments for PD is paramount.Inflammation has been implicated in PD etiology.Increased concentrations of inflammatory cytokines have been detected in the midbrain of PD animal models and PD patients.Anti--inflammatory agents have been found to decrease DA neuronal loss in animal models of PD.Moreover, increased use of non--steroidal anti--inflammatories (NSAIDs) has been correlated with a decrease in the probability of developing PD in humans.Therefore, anti--inflammatory agents represent an attractive avenue for PD treatment.Glutaredoxin--1 (Grx1) is a ubiquitously expressed oxidoreductase enzyme that specifically catalyzes reduction of glutathionyl mixed disulfides.Grx1 has been implicated as a positive regulator of inflammation in a variety of inflammation models.Moreover, Grx1 is upregulated by various inflammatory stimuli in distinct cell types, thereby potentially creating a feed--forward loop of inflammatory propagation.Grx1 silencing inhibits pro--inflammatory cytokine release in cell culture and animal models of inflammatory disease.It is therefore plausible that Grx1 contributes importantly to the inflammatory response observed in PD, and could represent a therapeutic target.Research presented in this thesis focuses on examining Grx1 as an inflammatory mediator and a target for anti--inflammatory therapy.The first part of this thesis research documents the pro--inflammatory role of Grx1 in microglia, resident macrophages of the central nervous system; and discovers a deleterious role for Grx1 upregulation in PD.The second part of this thesis focuses on characterizing a novel Grx1 inhibitor, CWR--J02, and its anti--inflammatory properties.Chapter 1: Background and Introduction Part of the work presented in Chapter 1 was published in Arch Toxicol 89(9): 1439--1467 and Macrophage 2017; 4: e1504 Parkinson's disease (PD) is a debilitating movement disorder.While no single cause of PD has been elucidated, inflammation and redox imbalance have been implicated as important contributors to its etiology.Diabetes mellitus (DM) has been associated with increased risk of PD development.This introductory section describes the role of inflammation in the central nervous system (CNS) as well as in the peripheral nervous system in PD, and considers how inflammatory responses may provide a connection between PD and Diabetes mellitus (DM).Redox signal transduction is a key mechanism of control of cell survival, inflammatory responses, and programmed cell death, and it is regulated to a large part by reversible cysteine modifications.In this section, I describe how cysteine modifications, including S-glutathionylation, of inflammatory proteins may mediate inflammation.Glutaredoxin--1 (Grx1) is the enzyme responsible for regulating protein glutathionylation, and it has been implicated as a positive regulator of inflammation in several contexts, providing the premise for the focus of this thesis on the function of Grx1 in microglial cells.In the concluding portion of this introductory chapter, I review previous evidence for Grx1 in inflammation that forecast its potential role in CNS inflammation and PD and then describe strategies for developing inhibitors
Author: Olga Gorelenkova Miller
Institution: Case Western Reserve University
Language: English
Subfield: Neurology
Field: Neuroscience
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2807448447