Research ReportAn alpha-synuclein AAV gene silencing vector ameliorates a behavioral deficit in a rat model of Parkinson's disease, but displays toxicity in dopamine neurons
Highlights
► An AAV harboring a hSNCA-specific shRNA silences ectopic hSNCA expression in rat SN. ► hSNCA silencing in rat SN protects against a hSNCA-induced forelimb behavior deficit. ► Chronic expression of shRNA is toxic to DA neurons ► ST-projecting DA neurons outside the AAV-transduced SN region are protected by hSNCA gene silencing. ► hSNCA gene silencing holds promise as a novel PD therapy upon further vector refinement.
Introduction
Alpha-synuclein (SNCA) is a 140 amino acid presynaptic phosphoprotein that is abundant in neurons (Lee and Trojanowski, 2006). Findings from familial and sporadic Parkinson's disease (PD) cases implicate SNCA in PD pathogenesis. Three point mutations in the human (h)SNCA gene, as well as multiplication of the SNCA gene, are included among familial forms of PD. Proteinacious inclusions termed Lewy bodies (Lee and Trojanowski, 2006), of which SNCA is a major component (Spillantini et al., 1997), are present in 90% of PD cases, suggesting that SNCA is also involved in sporadic PD. Further, specific SNCA promoter polymorphisms have been linked to PD (Maraganore et al., 2006).
Experimental SNCA over-expression models that result in loss of dopamine (DA) neurons are valuable for testing the hypothesis that SNCA is a therapeutic target for PD (Chesselet, 2008). Neurotoxin-induced PD models, including exposure to rotenone, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 6-OHDA and paraquat, exhibit increased SNCA expression and aggregation as well as DA neuron loss (Cannon and Greenamyre, 2010). In addition, experimental expression of wild-type and mutant forms of SNCA has been shown to result in DA neuron degeneration and formation of aggregated SNCA cellular inclusions in drosophila (Feany and Bender, 2000), mouse (Chesselet, 2008), rat (Kirik et al., 2002) and monkey (Kirik et al., 2003). In SNCA transgenic (tg) mice, SNCA inclusion formation and DA-related deficits vary depending on the promoter and the form of SNCA used (Fernagut and Chesselet, 2004). Non-tg models of SNCA-induced PD-like symptoms also have been developed using lentiviral (LV, Lo Bianco et al., 2002) and adeno-associated viral (AAV, Kirik et al., 2002, Kirik et al., 2003) vector-mediated delivery of exogenous SNCA, which induces nigrostriatal DA neurodegeneration. In this study, we use AAV to express hSNCA in rat substantia nigra (SN), a model reported previously (Kirik et al., 2002).
Approaches that directly target aberrant SNCA expression may be promising for therapeutic development. Glial cell line-derived neurotrophic factor (GDNF) does not prevent DA neurodegeneration in a LV-based SNCA expression model (Lo Bianco et al., 2004), although GDNF has been shown to ameliorate neurodegeneration in neurotoxin models of PD (Choi-Lundberg et al., 1997, Kearns and Gash, 1995, Mandel et al., 1997, Sauer et al., 1995, Tomac et al., 1995). However, SNCA targeting using a ribozyme in a rat model of PD where 1-methyl-4-phenylpryridinium induced an increase in SNCA expression has been shown to protect against DA neuron loss in rat SN (Hayashita-Kinoh et al., 2006).
RNA interference (RNAi) is a conserved process whereby double-stranded RNA targets mRNA in a sequence-specific manner resulting in degradation or translational inhibition of target mRNA (Fire et al., 1998, Scherr and Eder, 2007). Development of RNAi as a therapeutic approach is expected to provide novel opportunities for treating a wide range of disorders. Exogenous interfering RNAs can be administered as synthetic small interfering (si)RNAs or as short hairpin (sh)RNAs, usually delivered using viral vectors. siRNAs incorporate into the cellular RNAi machinery at the RNA inhibitory silencing complex and have only transient effects. In contrast, shRNAs can be chronically expressed in cells where they undergo processing similar to that of endogenous pre-micro (mi)RNAs (Scherr and Eder, 2007). Several approaches have been used to silence SNCA expression, including use of ribozymes (Hayashita-Kinoh et al., 2006), intracellularly expressed single chain antibodies (Yuan and Sierks, 2009), siRNAs (Lewis et al., 2008, McCormack et al., n.a), miRNAs (Doxakis, 2010, Junn et al., 2009) and viral vector-mediated delivery of an shRNA (Sapru et al., 2006). AAV and LV are ideal viral vectors for delivery of gene silencing molecules to the CNS because they are capable of transducing neurons to elicit long-term transgene expression (Bjorklund et al., 2000).
We previously reported that an shRNA sequence targeting hSNCA effectively silences endogenous hSNCA in SH-SY5Y cells in vitro and ectopically expressed hSNCA in vivo in rat striatum (ST, Sapru et al., 2006). To further explore the potential of SNCA gene silencing for PD, we generated an AAV2 harboring this shRNA. This was used to determine whether SNCA gene silencing can protect SN DA neurons in a rat model of PD where hSNCA is ectopically expressed in SN, leading to DA neuronal degeneration (Kirik et al., 2002).
Section snippets
Silencing of ectopically expressed hSNCA in rat SN and ST
To confirm that ectopic hSNCA expression in rat SN using our AAV vector has a time-dependent toxic effect on nigral DA neurons as reported previously (Kirik et al., 2002), AAV-hSNCA was injected into one SN alone or with a control vector, AAV-humanized green fluorescent protein (hrGFP) or AAV-shRNA-luciferse (Luc)-hrGFP. Expression of hSNCA at 4 and 9 weeks after injection was studied by immunofluorescence using a hSNCA-specific antibody. hSNCA expression was abundant in SN of all
Discussion
Development of techniques to specifically decrease SNCA toxicity holds promise for therapeutic applications in synucleinopathies, including PD. However, it remains a challenge to target aberrant SNCA expression in specific neurons in the brain. Here, we report the first study designed to silence ectopically expressed hSNCA in rat DA neurons. We investigated whether SNCA gene silencing using AAV-mediated delivery of a hSNCA-specific shRNA would protect DA neurons in rat SN from toxic effects of
Generation of AAV shuttle plasmids
The dual expression pAAV gene silencing vectors used, pAAV-H1-shRNA-SNCA-CMV-hrGFP and pAAV-H1-shRNA-Luc-CMV-hrGFP, harbor the human H1 promoter driving a shRNA targeting either nucleotides 288–309 of hSNCA (GenBank Accession No. L08850) or nucleotides 153–173 of firefly GL2 Luc (GenBank Accession No.X65323), respectively, joined by an 11-base pair duplex loop and the cellular reporter hrGFP under control of the cytomegalovirus (CMV) promoter. These were derived from plasmids pBCSK-H1 and
Acknowledgments
This study was supported by the Department of Defense Neurotoxicology Program (NO06079001) and NIH grants (NS31957 and NS054989 to MCB and T32 NS041234 to CEK), the Harry F and Elaine M Chaddick Foundation and the Medical Research Institute Council of Children's Memorial Hospital. Support from the Chicago Biomedical Consortium and the Illinois Excellence in Academic Medicine to the CMRC Viral Vector Core is acknowledged. The technical assistance of Jianping Xie, Xue Song Wang, David George and
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