Suppressing nNOS Enzyme by Small-Interfering RNAs Protects SH-SY5Y Cells and Nigral Dopaminergic Neurons from 6-OHDA Injury.

Abstract

Nitric oxide (NO) has chemical properties that make it uniquely suitable as an intracellular and intercellular messenger. NO is produced by the activity of the enzyme nitric oxide synthases (NOS). There is substantial and mounting evidence that slight abnormalities of NO may underlie a wide range of neurodegenerative disorders. NO participates of the oxidative stress and inflammatory processes that contribute to the progressive dopaminergic loss in Parkinson's disease (PD). The present study aimed to evaluate in vitro and in vivo the effects of neuronal NOS-targeted siRNAs on the injury caused in dopaminergic neurons by the toxin 6-hidroxydopamine (6-OHDA). First, we confirmed (immunohistochemistry and Western blotting) that SH-SY5Y cell lineage expresses the dopaminergic marker tyrosine hydroxylase (TH) and the protein under analysis, neuronal NOS (nNOS). We designed four siRNAs by using the BIOPREDsi algorithm choosing the one providing the highest knockdown of nNOS mRNA in SH-SY5Y cells, as determined by qPCR. siRNA 4400 carried by liposomes was internalized into cells, caused a concentration-dependent knockdown on nNOS, and reduced the toxicity induced by 6-OHDA (p < 0.05). Regarding in vivo action in the dopamine-depleted animals, intra-striatal injection of siRNA 4400 at 4days prior 6-OHDA produced a decrease in the rotational behavior induced by apomorphine. Finally, siRNA 4400 mitigated the loss of TH(+) cells in substantia nigra dorsal and ventral part. In conclusion, the suppression of nNOS enzyme by targeted siRNAs modified the progressive death of dopaminergic cells induced by 6-OHDA and merits further pre-clinical investigations as a neuroprotective approach for PD.