TY - JOUR
T1 - Targeted knock-down of neuronal nitric oxide synthase expression in basal forebrain with RNA interference
AU - Mahairaki, Vasiliki
AU - Xu, Leyan
AU - Farah, Mohamed H.
AU - Hatfield, Glen
AU - Kizana, Eddy
AU - Marbán, Eduardo
AU - Koliatsos, Vassilis E.
N1 - Funding Information:
This work was supported by NIH grants RO1 AG16263 and 3 P50 AG05146.
PY - 2009/5/15
Y1 - 2009/5/15
N2 - Nitric oxide (NO) is a gas messenger with diverse physiological roles in the nervous system, from modulation of synaptic plasticity and neurogenesis to the mediation of neuronal death. NO production in the brain is catalyzed by three isoforms of NO synthase (NOS) including neuronal NOS (nNOS), inducible NOS and endothelial NOS. In this report, we demonstrate a method for in vitro and in vivo silencing of nNOS using RNAi strategies. Because of their efficiency in infecting postmitotic cells like neurons, lentiviral vectors were used as nNOS shRNA carriers. Of the siRNA sequences screened, one corresponding to exon 10 of the rat nNOS specifically and efficiently inhibited nNOS expression at the mRNA and protein level. In vitro experiments using rat cortical neurons showed the general efficacy of shRNA vectors in silencing constitutively expressed nNOS. To demonstrate the anatomical specificity of nNOS silencing in vivo, vectors were used to selectively knock-down the endogenous nNOS expression in cortical GABAergic interneurons of rat piriform cortex. Our findings show that the method reported here can achieve stable and highly effective nNOS suppression in an anatomically defined brain region. The ability of our nNOS silencing vectors to effectively and precisely silence nNOS expression shows their value as research tools for further studies of the role of nNOS in specific brain circuits. Furthermore, our findings raise the possibility for future considerations of lentiviral strategies as therapies for diseases of the nervous system involving NO neurotoxic cascades.
AB - Nitric oxide (NO) is a gas messenger with diverse physiological roles in the nervous system, from modulation of synaptic plasticity and neurogenesis to the mediation of neuronal death. NO production in the brain is catalyzed by three isoforms of NO synthase (NOS) including neuronal NOS (nNOS), inducible NOS and endothelial NOS. In this report, we demonstrate a method for in vitro and in vivo silencing of nNOS using RNAi strategies. Because of their efficiency in infecting postmitotic cells like neurons, lentiviral vectors were used as nNOS shRNA carriers. Of the siRNA sequences screened, one corresponding to exon 10 of the rat nNOS specifically and efficiently inhibited nNOS expression at the mRNA and protein level. In vitro experiments using rat cortical neurons showed the general efficacy of shRNA vectors in silencing constitutively expressed nNOS. To demonstrate the anatomical specificity of nNOS silencing in vivo, vectors were used to selectively knock-down the endogenous nNOS expression in cortical GABAergic interneurons of rat piriform cortex. Our findings show that the method reported here can achieve stable and highly effective nNOS suppression in an anatomically defined brain region. The ability of our nNOS silencing vectors to effectively and precisely silence nNOS expression shows their value as research tools for further studies of the role of nNOS in specific brain circuits. Furthermore, our findings raise the possibility for future considerations of lentiviral strategies as therapies for diseases of the nervous system involving NO neurotoxic cascades.
KW - Lentivirus
KW - Neuronal NOS
KW - Piriform cortex
KW - RNAi
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U2 - 10.1016/j.jneumeth.2009.02.006
DO - 10.1016/j.jneumeth.2009.02.006
M3 - Article
C2 - 19428540
AN - SCOPUS:63049114730
SN - 0165-0270
VL - 179
SP - 292
EP - 299
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
IS - 2
ER -