期刊
BRAIN RESEARCH
卷 1550, 期 -, 页码 47-60出版社
ELSEVIER
DOI: 10.1016/j.brainres.2014.01.010
关键词
Neurodegeneration; RNAi; Substantia nigra; Gene therapy; Tyrosine hydroxylase
资金
- Department of Defense Neurotoxicology Program [NO06079001]
- NIH [NS31957, NS054989, T32 NS041234]
- Harry F. and Elaine M. Chaddick Foundation
- Medical Research Institute Council of Ann and Robert H. Lurie Children's Hospital of Chicago
Background: Alpha-synuclein (SNCA) downregulation shows therapeutic potential for synucleinopathies, including Parkinson's disease (PD). Previously we showed that human (h)SNCA gene silencing using a short hairpin (sh)RNA in rat substantia nigra (SN) protects against a hSNCA-induced forelimb deficit, but not dopamine (DA) neuron loss. Furthermore, the shRNA increases cell death in vitro, but the same target sequence embedded in a microRNA30 transcript (mir30-hSNCA) does not. Objective: Examine hSNCA gene silencing using mir30-hSNCA in vivo. Methods: Rats were stereotaxically injected into one SN with adeno-associated virus serotype 2/8 (AAV)-hSNCA, AAV-hSNCA plus AAV-mir3O-SNCA or AAV-hSNCA plus a control non-silencing mir30-embedded siRNA and DA neuron markers and associated behavior were examined. Results: AAV2/8-mediated SN hSNCA expression induces a forelimb deficit and tyrosine hydroxylase-immunoreactive (TH-IR) neuron loss. hSNCA gene silencing using mir30-hSNCA protects against this forelimb deficit at 2 m and ameliorates TH-IR neuron loss. Striatal (ST) TH-IR fiber density and DA markers, assessed by western blot, are unaffected by AAV-hSNCA alone. Co-expression of either silencing vector reduces ST TH-IR fibers, panTH in SN and Ser40 phosphorylated TH in SN and ST, but does not affect vesicular monoamine transporter-2. However, hSNCA gene silencing promotes partial TH-IR fiber recovery by 2 m. Co-expression of either silencing vector also induces SN inflammation, although some recovery was observed by 2 m in hSNCA-silenced SN. Conclusion: hSNCA gene silencing with AAV-mir30-hSNCA has positive effects on forelimb behavior and SN DA neurons, which are compromised by inflammation and reduced TH expression, suggesting that AAV2/8-mir30-hSNCA-mediated gene silencing, although promising in vitro, is not a candidate for therapeutic translation for PD. (c) 2014 The Authors. Published by Elsevier B.V. All rights reserved.
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