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

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 4 days 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.

The Presence of the Neuronal Nitric Oxide Synthase Isoform in the Intervertebral Disk.

Intervertebral disk degeneration is a progressive and debilitating disease with multifactorial causes. Nitric oxide (NO) might contribute to the cell death pathway. We evaluated the presence of the constitutive form of the neuronal NOS (nNOS) in both health and degenerated intervertebral disk through qPCR and immunohistochemistry. We also analyzed the potential role of nNOS modulation in the tail needle puncture model of intervertebral disk degeneration. Male Wistar rats were submitted to percutaneous disk puncture with a 21-gauge needle of coccygeal vertebras. The selective nNOS pharmacological inhibitor N (ω)-propyl-L-arginine (NPLA) or a nNOS-target siRNA (siRNAnNOShum_4400) was injected immediately after the intervertebral disk puncture with a 30-gauge needle. Signs of disk degeneration were analyzed by in vivo magnetic resonance imaging and histological score. We found that intact intervertebral disks express low levels of nNOS mRNA. Disk injury caused a 4 fold increase in nNOS mRNA content at 5 h post disk lesion. However, NPLA or nNOS-target siRNA slight mitigate the intervertebral disk degenerative progress. Our data show evidence of the nNOS presence in the intervertebral disk and its upregulation during degeneration. Further studies would disclose the nNOS role and its potential therapeutical value in the intervertebral disk degeneration.

Anti-dyskinetic effect of the neuronal nitric oxide synthase inhibitor is linked to decrease of FosB/deltaFosB expression.

Rodents with lesion of dopaminergic pathway when receiving repeated l-3,4-dihydroxiphenylalanine (l-DOPA) treatment develop abnormal involuntary movements called dyskinesia. We demonstrated that nitric oxide synthase (NOS) inhibitors mitigate l-DOPA-induced dyskinesia in rodents. The aim of the present study was to verify if the in vivo preferential neuronal NOS (nNOS) inhibitor 7-nitroindazole (7-NI) affect the expression of the transcription factor FosB/ΔFosB in the lesioned striatum, an indicator of neuronal activity associated with dyskinesia. Male Wistar rats with unilateral microinjection (medial forebrain bundle) of either the neurotoxin 6-hydroxidopamine (6-OHDA; n=4-6/group) or saline (sham; n=6/group) were provided with l-DOPA (30mg/kg plus benserazide 7.5mg/kg/day, oral gavage), once a day during 22 days. 6-OHDA-lesioned animals developed abnormal involuntary movements (AIMs) classified as axial, limb, orofacial and locomotive dyskinesia and presented FosB/ΔFosB increase in the dopamine-depleted striatum. Administration of 7-NI (30mg/kg, i.p.), 30min prior to l-DOPA reduced the severity of AIMs (≈65% for axial, limb and orofacial and 74% for locomotive AIMs scores), without interfering with the rotarod performance. Simultaneously, 7-NI attenuated the expression of FosB/ΔFosB in dopamine-depleted striatum (≈65% in medial and ≈54% in lateral striatum, bregma 0.48mm). FosB/ΔFosB expression in lateral striatum was correlated with l-DOPA-induced dyskinesia. The findings described here corroborate a new approach to the management of l-DOPA-therapy in Parkinson's disease (PD) treatment.

Effects of acute prenatal exposure to ethanol on the postnatal morphology of the prefrontal cortex in wistar rats

Prenatal exposure to ethanol is frequently associated with micronecephaly, hypomyelinization, delayed cell migration, and impaired neuronal and glial maturation in the offspring. The mechanism by which ethanol induces its effects on the development of the nervous system is still not fully understood. In this study, the influence of acute prenatal exposure to ethanol on the prefrontal cortex cells of rats were examined. Three doses of ethanol (3g/kg of body weight) were administered intraperitoneally to pregnant female rats on the 12th day of pregnacy, at 8 hours intervals. Control rats received the same treatment but with a saline solution. Cells in the synthesis phase (S) of the cell cycle were labeled with bromodeoxyuridine. Six controls and 12 ethanol-treated neonates were sacrificed on the 8th day of postnatal life. The distance between nuclear cores in immunohistochemically labeled cells was determined by image analysis. Control rats had a normal neocortex, with six layers in the prefrontal region. Rats treated with ethanol showed ectopia of pyramidal neurons in layers I and II, heterotopia in the basal area of the prefrontal fissure, and a decrease in cellular density in layers I and VI of the cerebral prefrontal cortex. These alterations could help to explain some of the dysfunctions reported in patients with fetal alcohol syndrome.