NADPH Oxidase 1 Mediates α-Synucleinopathy in Parkinson's Disease.

Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, Florida 32827, Health Sciences Research Center, University of Beira Interior, Covilhã 6200-506, Portugal, Experimental Biology and Biomedicine, Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra 3400-516, Portugal, Department of Medical Science, Konkuk University School of Medicine and Center for Neuroscience, SMART Institute of Advanced Biomedical Science, Konkuk University, Seoul 143-701, South Korea, and Department of Physical Medicine and Rehabilitation, School of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience (Impact Factor: 6.34). 10/2012; 32(42):14465-14477. DOI: 10.1523/JNEUROSCI.2246-12.2012
Source: PubMed


Accumulation of misfolded α-synuclein is the pathological hallmark of Parkinson's disease (PD). Nevertheless, little is known about the mechanism contributing to α-synuclein aggregation and its further toxicity to dopaminergic neurons. Since oxidative stress can increase the expression and aggregation levels of α-synuclein, NADPH oxidases (Noxs), which are responsible for reactive oxygen species generation, could be major players in α-synucleinopathy. Previously, we demonstrated that Nox1 is expressed in dopaminergic neurons of the PD animal models as well as postmortem brain tissue of PD patients, and is responsible for oxidative stress and subsequent neuronal degeneration. Here, using paraquat (PQ)-based in vitro and in vivo PD models, we show that Nox1 has a crucial role in modulating the behavior of α-synuclein expression and aggregation in dopaminergic neurons. We observed in differentiated human dopaminergic cells that Nox1 and α-synuclein expressions are increased under PQ exposure. Nox1 knockdown significantly reduced both α-synuclein expression and aggregation, supporting the role of Nox1 in this process. Furthermore, in rats exposed to PQ, the selective knockdown of Nox1 in the substantia nigra, using adeno-associated virus encoding Nox1-specific shRNA, largely attenuated the PQ-mediated increase of α-synuclein and ubiquitin expression levels as well as α-synuclein aggregates (proteinase K resistant) and A11 oligomers. Significant reductions in oxidative stress level and dopaminergic neuronal loss were also observed. Our data reveal a new mechanism by which α-synuclein becomes a neuropathologic protein through Nox1-mediated oxidative stress. This finding may be used to generate new therapeutic interventions that slower the rate of α-synuclein aggregation and the progression of PD pathogenesis.

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    • "Accumulation of α-synuclein has also been suggested to trigger mitochondrial oxidative stress (Devi et al., 2008) and alterations in Ca 2+ permeability (Furukawa et al., 2006; Goldberg et al., 2012). Extracellular α-synuclein also triggers microglia activation and Nox-mediated ROS formation (Zhang et al., 2005), which in turn increase α-synuclein aggregation (Cristovao et al., 2012). It has been proposed that α-synuclein exerts protective effects (Hashimoto et al., 2002), while the A53P mutant α-synuclein sensitizes cells upon oxidative damage (Ko et al., 2000). "
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    ABSTRACT: Oxidative stress is a common hallmark of neuronal cell death associated with neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, as well as brain stroke/ischemia and traumatic brain injury. Increased accumulation of reactive species of both oxygen (ROS) and nitrogen (RNS) has been implicated in mitochondrial dysfunction, energy impairment, alterations in metal homeostasis and accumulation of aggregated proteins observed in neurodegenerative disorders, which lead to the activation/modulation of cell death mechanisms that include apoptotic, necrotic and autophagic pathways. Thus, the design of novel antioxidant strategies to selectively target oxidative stress and redox imbalance might represent important therapeutic approaches against neurological disorders. This work reviews the evidence demonstrating the ability of genetically encoded antioxidant systems to selectively counteract neuronal cell loss in neurodegenerative diseases and ischemic brain damage. Because gene therapy approaches to treat inherited and acquired disorders offer many unique advantages over conventional therapeutic approaches, we discussed basic research/clinical evidence and the potential of virus-mediated gene delivery techniques for antioxidant gene therapy.
    Pharmacology [?] Therapeutics 12/2013; 142(2). DOI:10.1016/j.pharmthera.2013.12.007 · 9.72 Impact Factor
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    • "The present work confirmed that all three animal models of PD trigger the characteristic motor symptoms of the disease, as a consequence of reduced number of TH-positive neurons in the SNpc. Furthermore, on the latter parameter, we found a range of variation from 20 to almost 50% in the reduction of TH-positive cells, which is within the ranges of variance observed in other studies using similar animals models (Kirik et al., 2002; Pioli et al., 2004; Cristovao et al., 2009, 2012; Decressac et al., 2012b). However, we failed to find a significant correlation between the DAergic neuronal degeneration and the severity of the motor deficits; therefore, we cannot make an association between each model and a stage of the disease progression, as have been done in other studies. "
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    ABSTRACT: Parkinson's disease (PD) is classically characterized by motor symptoms; however, non-motor symptoms (NMS) are increasingly recognized as relevant in disease-state, given the associated alterations in mood (depression and anxiety) and cognition. Here, particularly in regards to NMS, we aimed to compare the motor, emotional and cognitive behavior of three animal models of PD that trigger dopaminergic (DAergic) degeneration on both brain hemispheres: (i) the 6-hydroxydopamine (6-OHDA, 8 or 6 μg) lesion model; (ii) the paraquat (PQ) induced model, and (iii) a genetic model based on α-synuclein overexpression (α-syn). 6-OHDA and α-syn vector were injected bilaterally in the substantia nigra pars compacta (SNpc) of adult male Wistar rats; as for PQ delivery, micro-osmotic pumps were implanted in the interscapular region. Motor deficits were observed in all models, with histological analysis of tyrosine hydroxylase positive cells in the SNpc revealing a significant loss of DAergic neurons in all animal models. In addition, the α-syn animal model also presented a reduction in exploratory activity, and the 6-OHDA and PQ animals displayed a significant increase in both depressive- and anxiety-like behavior. Interestingly, cognitive impairment (working memory) was only observed in the 6-OHDA model. Overall, these PD models are suitable for mimicking the motor symptoms associated to PD, with each encompassing other relevant NMS components of the disorder that may prove beneficial for further studies in PD.
    Frontiers in Behavioral Neuroscience 11/2013; 7:175. DOI:10.3389/fnbeh.2013.00175 · 3.27 Impact Factor
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    • "We confirmed the toxicity of TAT-AS whereas the native form had no toxic effect in this kind of assay, as previously reported [5]. The basic toxicity of TAT-AS might be a starting point for experiments aimed at unraveling the relation between AS and other kind of cellular stressors, for instance oxidative stress, proteasome impairment or autophagy, all features potentially linked to PD etiopathogenesis [28-31]. "
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    ABSTRACT: Background Human α-synuclein is a small-sized, natively unfolded protein that in fibrillar form is the primary component of Lewy bodies, the pathological hallmark of Parkinson’s disease. Experimental evidence suggests that α-synuclein aggregation is the key event that triggers neurotoxicity although additional findings have proposed a protective role of α-synuclein against oxidative stress. One way to address the mechanism of this protective action is to evaluate α-synuclein-mediated protection by delivering this protein inside cells using a chimeric protein fused with the Tat-transduction domain of HIV Tat, named TAT-α-synuclein. Results A reliable protocol was designed to efficiently express and purify two different forms of human α-synuclein. The synthetic cDNAs encoding for the native α-synuclein and the fusion protein with the transduction domain of Tat protein from HIV were overexpressed in a BL21(DE3) E. coli strain as His-tagged proteins. The recombinant proteins largely localized (≥ 85%) to the periplasmic space. By using a quick purification protocol, based on recovery of periplasmic space content and metal-chelating chromatography, the recombinant α-synuclein protein forms could be purified in a single step to ≥ 95% purity. Both α-synuclein recombinant proteins form fibrils and the TAT-α-synuclein is also cytotoxic in the micromolar concentration range. Conclusions To further characterize the molecular mechanisms of α-synuclein neurotoxicity both in vitro and in vivo and to evaluate the relevance of extracellular α-synuclein for the pathogenesis and progression of Parkinson’s disease, a suitable method to produce different high-quality forms of this pathological protein is required. Our optimized expression and purification procedure offers an easier and faster means of producing different forms (i.e., both the native and the TAT-fusion form) of soluble recombinant α-synuclein than previously described procedures.
    BMC Biotechnology 04/2013; 13(1):32. DOI:10.1186/1472-6750-13-32 · 2.03 Impact Factor
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