Objective/Rationale:
A family of NADPH oxidase (NOX) is the specialized enzyme complex which generates superoxide. We demonstrate that Nox1, a NOX homologue, is upregulated in DA cells under various stress conditions both in vivo and in vitro. Rac1, a key component of the Nox1 system, is also activated. This project will investigate whether inhibition of Nox1/Rac1 reduces oxidative stress and subsequent DA neuronal degeneration in MPTP-induced PD animal model.
Project Description:
Three different target validation strategies will be tested in MPTP-induced mouse PD model. First, a specific NOX inhibitor, apocynin, will be tested. Neuroprotective effects of apocynin have been reported in several CNS disorders. For selective targeting, adeno-associated virus2 (rAAV2)-mediated inhibition of Nox1/Rac1 in the substantia nigra will be tested by stereotaxic delivery of viral particles into the SN. The limitations of this system include, however, variable transduction efficiency and knockdown efficiency, and off-targeting. Thus, lastly, we plan to investigate Nox1 knockout mice treated with MPTP. The following four parameters will be analyzed to evaluate the outcomes: oxidative stress, neuropathology (DA neuronal death), striatal neurochemistry (including dopamine release and levels of biogenic amines), and behavioral changes.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
Although it is generally accepted that reactive oxygen species (ROS) play a cardinal role in degeneration of the nigrostriatal dopaminergic system, the specific therapeutic target molecules have not been identified. The proposed studies will test whether inhibition of Nox1 ameliorate oxidative stress and leads to prevention of dopaminergic neuronal degeneration. If the Nox1 inhibition is successful to halt DA neurodegeneration, the Nox1/Rac1 would serve as a novel therapeutic target for PD.
Anticipated Outcome:
Our recent in vitro and paraquat-mediated in vivo PD model studies demonstrate that apocynin effectively ameliorate DA cell death. Thus, we are anticipating that the proposed studies employing various levels of Nox1 targeting strategies will result in decreased oxidative stress and degeneration in the nigrostriatal DA pathway caused by MPTP administration.
Progress Report
A novel therapeutic target in PD which we want to test in this proposed study is NADPH oxidase 1(Nox1), the specialized enzyme complex generating superoxide. Previously, we found that Nox1 expression is increased in the substantia nigra under various oxidative stress conditions including 6-OHDA, paraquat and MPTP as well as postmortem human PD brain tissue. During the first year of award period, we were able to demonstrate that a NADPH oxidase inhibitor, apocynin, significantly reduces dopaminergic neuronal degeneration induced by paraquat or MPTP. The data strongly support our hypothesis that Nox-derived oxidative stress plays a role in the pathogenesis of PD and could serve as an effective therapeutic target in PD. Our research is now focusing on the specific role of Nox1 in the nigrostriatal pathway using AAV-mediated selective Nox1 knockdown in the SN and Nox1 knockout mice in MPTP-induced PD model.
Final Outcome
During this project period, our research has focused on the validation of Nox1 as a target for Parkinson’s disease therapy. Acute MPTP administration (four- time i.p. injections of 15 mg/kg every two hour) was used to develop the nigrostriatal lesion in pre-clinical models. Administration of apocynin, a putative inhibitor of NADPH oxidases, in the drinking water efficiently ameliorated MPTP-induced dopaminergic neuronal loss. Nox1 knockout model treated with MPTP demonstrated significant reduction in dopaminergic neuronal degeneration compared with wild-type littermates. We were able to efficiently knockdown Nox1 in pre-clinical models, leading us to test other pre-clinical PD models employing 6-OHDA and paraquat. Nox1 knockdown in the substantia nigra substantially diminished dopaminergic neuronal loss. Comprehensive analysis of the results obtained from various PD models and Nox1 inhibitions are currently underway.
Presentations & Publications
Publications
Choi DH, Cristóvão AC, Joh TH, Beal MF, Kim YS. NADPH oxidase 1, a novel molecular source of ROS in dopaminergic neurodegeneration of Parkinson’s disease. (under review by Antioxidants and Redox signaling)
Cristóvão AC, Choi DH, Beal MF, Joh TH, Kim YS. NADPH oxidase 1 relays paraquat-mediated alpha-synuclein regulation in vivo (manuscript in preparation)
Presentation
Society for Neuroscience Annual Meeting 2010
Gordon Research Conference 2010 “Nox Family NADPH Oxidases” : invited as an oral presenter