RING FINGER PROTEIN 11 (RNF11) MEDIATES NF-κB SIGNALING AND DOPAMINERGIC NEURODEGENERATION Público

Abstract

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by selective loss of dopaminergic neurons in substantia nigra. A number of studies have highlighted chronic inflammation, through activation of NF-κB pathway, as a critical mediator of PD pathogenesis. The A20 complex is a key regulator of NF-κB signaling outside the central nervous system, however the existence as well as the contribution of A20 complex-mediated NF-κB signaling in the brain has not been investigated. Using immunohistochemistry and quantative real-time PCR, we demonstrated that essential components of the A20 complex are present in neurons in human brain. Further, we determined that RING finger protein 11 (RNF11) was the only component of the A20 complex with reduced expression in PD. Given that RNF11 is robustly expressed in neurons and co-localizes with a population of Lewy bodies in PD patients, it became imperative for us to investigate the function of neuronal RNF11 in NF-κB signaling pathway. Luciferase assays and p65 translocation analyses were conducted to assess NF-κB activity under knockdown of RNF11 in neurons, which suggested RNF11 acts as a negative regulator of canonical NF-κB signaling. The association of RNF11, NF-κB signaling, and PD, prompted us to investigate the role of RNF11-mediated NF-κB activation on survival of dopaminergic neurons in the 6-hydroxydopamine rat model of PD. Due to the endogenous neuronal expression of RNF11, we employed targeted modulation of RNF11 expression in dopaminergic neurons using stereotaxic injections of adeno-associated viruses (AAV2) in the substantia nigra. RNF11 overexpression enhanced dopaminergic cell death while RNF11 knockdown substantially protected against 6-hydroxydopamine toxicity. Our results suggested that RNF11 in dopaminergic cells modulates susceptibility to 6-hydroxydopamine toxicity through NF-κB-mediated responses, including induction of antioxidants and anti-apoptotic factors. Together, our in vivo and in vitro studies provide compelling support that RNF11, a component of the A20 complex, can modulate NF-κB activation in dopaminergic neurons with critical influences on PD-associated neurodegeneration. Further investigation into RNF11-mediated NF-κB signaling in context of distinct cell types and a myriad of insults is critical for understanding the contribution of RNF11 to PD pathogenesis.

Table of Contents

CHAPTER 1: INTRODUCTION AND BACKGROUND...1

1.1 History and clinical description of Parkinson's disease...1
1.2 Etiology of Parkinson's disease...8
1.3 Inflammatory signaling in Parkinson's disease...19

NF-κB signaling...22
RNF11...26

1.4 Thesis Outline...28

CHAPTER 2: MATERIALS AND METHODS...36

2.1 Cell culture...36
2.2 Antibodies and reagents...38
2.3 Plasmids and transfections...38
2.4 RNA interference and virus production...39
2.5 Site-directed mutagenesis...40
2.6 TNF-α stimulation and luciferase assays...40
2.7 Immunocytochemistry...41
2.8 Immunoblotting...41
2.9 Co-immunoprecipitation...42
2.10 ELISA...43
2.11 Nuclear fractionation...43
2.12 Cell death assays...44
2.13 Human tissue...44
2.14 Brightfield immunohistochemistry...45
2.15 Fluorescence immunohistochemistry of mammalian tissue...46
2.16 Extraction of RNA from cells or Drosophila melanogaster tissue...46
2.17 Extraction of RNA from tissue...46
2.18 Quantitative real-time PCR...47
2.19 Drosophila stocks and husbandry...47
2.20 Immunohistochemistry of Drosophila tissue...47
2.21 Drosophila immunity paradigm...48
2.22 Drosophila survival assays...48
2.23 Animal studies...49
2.24 Stereotaxic injections...49
2.25 Microdissection and harvest of tissue for qRT-PCR...50
2.26 Rotational behavior analysis...50
2.27 Perfusion and tissue processing for histology...50
2.28 Stereological analysis of nigral dopaminergic neurons...51
2.29 Statistical analysis...51

CHAPTER 3: RNF11 EXPRESSION IS REDUCED IN PARKINSON'S DISEASE...55

3.1 Introduction...56
3.2 Differential expression of A20 and RNF11 in normal human brain...57
3.3 Differential expression of RNF11, Itch, TAX1BP1, and Traf6 in normal human brain...58
3.4 Reduction of RNF11 mRNA expression in Parkinson's disease...60
3.5 Reduction of RNF11 protein expression in Parkinson's disease...61
3.6 Discussion...62

CHAPTER 4: NEURONAL RNF11 IS A NEGATIVE REGULATOR OF NF-ΚB SIGNALING...76

4.1 Introduction...76
4.2 RNF11-mediated regulation of NF-κB signaling...78
4.3 Association of neuronal RNF11 with the A20 ubiquitin editing protein complex...82
4.4 Myristoylation domain is required for the function of RNF11...84
4.5 Altered inflammatory responses with manipulation of RNF11...87
4.6 Discussion...89

CHAPTER 5: CG32850, THE DROSOPHILA MELANOGASTER HOMOLOGUE OF RNF11, IS A NEGATIVE REGULATOR OF NF-ΚB SIGNALING...108

5.1 Introduction...108
5.2 CG32850 is homologous to mammalian RNF11...112
5.3 CG32850 is a negative regulator of NF-κB signaling...113
5.4 Discussion...117

CHAPTER 6: RING FINGER PROTEIN 11 (RNF11) MODULATES SUSCEPTIBILITY TO 6-OHDA-INDUCED NIGRAL DEGENERATION AND BEHAVIORAL DEFICITS THROUGH NF-ΚB SIGNALING IN DOPAMINERGIC CELLS...132

6.1 Introduction...132
6.2 Validation of AAV-mediated targeted knockdown and over-expression of RNF11 in vitro and in vivo...134
6.3 In vivo targeted knockdown of nigral dopaminergic RNF11 imparts neuroprotection against 6-OHDA-induced toxicity and attenuates rotational behavior...136

Validation of AAV2 in vivo...136
Behavioral analysis...137
Histological and mRNA analysis...138

6.4 In vivo targeted knockdown of nigral dopaminergic RNF11 increases NF-κB signaling...140
6.5 In vitro knockdown of RNF11 in dopaminergic cells imparts neuroprotection against 6-OHDA-induced toxicity...142
6.6 Protection against 6-OHDA-induced cell death in PC12 cells is attenuated with inhibition of NF-κB activation...144
6.7 Protection against 6-OHDA-induced cell death in PC12 cells is enhanced with TNF-α pretreatment...145
6.8 Discussion...146

CHAPTER 7: SUMMARY AND FUTURE DIRECTIONS...169

7.1 Summary of results...169
7.2 Expression of the A20 complex in the brain...170
7.3 Neuronal RNF11 as a negative regulator of canonical NF-κB signaling...171
7.4 Activation of neuronal NF-κB signaling is cytoprotective in dopaminergic neurons...173
7.5 Final Thoughts...176

REFERENCES...177

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience Health Sciences, General
Palavra-chave	A20 complex RNF11 Parkinson's disease E3 ligase NF-kB
Committee Chair / Thesis Advisor	Betarbet, Ranjita, Emory University
Committee Members	Tansey, Malu, Emory University Lah, James J, Emory University Greene, James G, Emory University Levey, Allan I, Emory University Seyfried, Nicholas, Emory University

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