Signature of Aberrantly Expressed microRNAs in the Striatum of Rotenone-Induced Parkinsonian Rats.

Parkinson's disease (PD) is a highly complex brain disorder regarding clinical presentation, pathogenesis, and therapeutics. The cardinal motor signs, i.e., rigidity, bradykinesia, and unilateral tremors, arise in consequence of a progressive neuron death during the prodromal phase. Although multiple transmission systems are involved in disease neurobiology, patients will cross the line between the prodromal and early stage of diagnosed PD when they had lost half of the dopaminergic nigrostriatal cells. As the neurons continue to die ascending the neuroaxis, patients will face a more disabling disease with motor and nonmotor signs. Shedding light on molecular mechanisms of neuron death is an urgent need for understanding PD pathogenesis and projecting therapeutics. This work examined the expression of microRNAs in the striatum of parkinsonian rats chronically exposed to rotenone (2.5 mg/Kg, i.p., daily for 10 days). Rotenone caused motor deficits, the loss of TH(+) cells in the nigrostriatal pathway, and a marked microgliosis. This parkinsonian rat striatum was examined at 26 days after the last rotenone injection, for quantification of microRNAs, miR-7, miR-34a, miR-26a, miR-132, miR-382, and Let7a, by qPCR. Parkinsonian rats presented a significant increase in miR-26a and miR-34a (1.5 and 2.2 fold, respectively, P < 0.05), while miR-7 (0.5 fold, P < 0.05) and Let7a were downregulated. This work reports for first time microRNAs aberrantly expressed in the striatum of rotenone-induced parkinsonian rats, suggesting that this dysregulation may contribute to PD pathogenesis. Beyond revealing new clues of neurodegeneration, our findings might prime further studies targeting miRNAs for neuroprotection or even for diagnosis proposal.

Delivery of miRNA-Targeted Oligonucleotides in the Rat Striatum by Magnetofection with Neuromag®.

MicroRNAs (miRNAs) regulate gene expression at posttranscriptional level by triggering RNA interference. In such a sense, aberrant expressions of miRNAs play critical roles in the pathogenesis of many disorders, including Parkinson's disease (PD). Controlling the level of specific miRNAs in the brain is thus a promising therapeutic strategy for neuroprotection. A fundamental need for miRNA regulation (either replacing or inhibition) is a carrier capable of delivering oligonucleotides into brain cells. This study aimed to examine a polymeric magnetic particle, Neuromag®, for delivery of synthetic miRNA inhibitors in the rat central nervous system. We injected the miRNA inhibitor complexed with Neuromag® into the lateral ventricles next to the striatum, by stereotaxic surgery. Neuromag efficiently delivered oligonucleotides in the striatum and septum areas, as shown by microscopy imaging of fluorescein isothiocyanate (FITC)-labeled oligos in astrocytes and neurons. Transfected oligos showed efficacy concerning miRNA inhibition. Neuromag®-structured miR-134 antimiR (0.36 nmol) caused a significant 0.35 fold decrease of striatal miR-134, as revealed by real-time quantitative polymerase chain reaction (RT-qPCR). In conclusion, the polymeric magnetic particle Neuromag® efficiently delivered functional miRNA inhibitors in brain regions surrounding lateral ventricles, particularly the striatum. This delivery system holds potential as a promising miRNA-based disease-modifying drug and merits further pre-clinical studies using animal models of PD.

The involvement of Eag1 potassium channels and miR-34a in rotenone-induced death of dopaminergic SH-SY5Y cells.

The loss of dopaminergic neurons and the resultant motor impairment are hallmarks of Parkinson's disease. The SH­SY5Y cell line is a model of dopaminergic neurons, and allows for the study of dopaminergic neuronal injury. Previous studies have revealed changes in Ether à go­go 1 (Eag1) potassium channel expression during p53-induced SH­SY5Y apoptosis, and the regulatory involvement of microRNA­34a (miR­34a) was demonstrated. In the present study, the involvement of Eag1 and miR­34a in rotenone­induced SH­SY5Y cell injury was investigated. Rotenone is a neurotoxin, which is often used to generate models of Parkinson's disease, since it causes the death of nigrostriatal neurons by inducing intracellular aggregation of alpha synuclein and ubiquitin. In the present study, rotenone resulted in a dose­dependent decrease in cell viability, as revealed by 3­(4,5­dimethylthiazol­2­yl)­2,5­diphenyltetrazolium bromide (MTT) and trypan blue cell counting assays. In addition, Eag1 was demonstrated to be constitutively expressed by SH­SY5Y cells, and involved in cell viability. Suppression of Eag1 with astemizole resulted in a dose­dependent decrease in cell viability, as revealed by MTT assay. Astemizole also enhanced the severity of rotenone­induced injury in SH­SY5Y cells. RNA interference against Eag1, using synthetic small interfering RNAs (siRNAs), corroborated this finding, as siRNAs potentiated rotenone­induced injury. Eag1­targeted siRNAs (kv10.1­3 or EAG1hum_287) resulted in a statistically significant 16.4­23.5% increase in vulnerability to rotenone. An increased number of apoptotic nuclei were observed in cells transfected with EAG1hum_287. Notably, this siRNA intensified rotenone­induced apoptosis, as revealed by an increase in caspase 3/7 activity. Conversely, a miR­34a inhibitor was demonstrated to exert neuroprotective effects. The viability of cells exposed to rotenone for 24 or 48 h and treated with miR­34a inhibitor was restored by 8.4­8.8%. In conclusion, Eag1 potassium channels and miR­34a are involved in the response to rotenone-induced injury in SH­SY5Y cells. The neuroprotective effect of mir­34a inhibitors merits further investigations in animal models of Parkinson's disease.

Interferon Gamma potentiates the injury caused by MPP(+) on SH-SY5Y cells, which is attenuated by the nitric oxide synthases inhibition.

This study examined whether the cytokine interferon (IFN) gamma plays a role in the injury of SH-SY5Y cells caused by MPP(+) (1-methyl-4-phenylpyridinium). First of all, IFN-gamma sensitized cells to the neurotoxin MPP(+), as determined by MTT (3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide) assay. MPP(+)-injured cells showed higher reactive oxygen species (ROS) levels, which was reinforced by IFN-gamma. The injury triggered a marked expression of the neuronal NOS (nNOS) enzyme. L-NAME [N(ω)-nitro-L-arginine methyl ester, a non-specific NOS inhibitor] reestablished the cell viability after IFN-gamma challenging, and recovered cells from MPP(+) injury (95.0 vs. 84.7 %; P < 0.05). Seven-NI (7-nitroindazole, a nNOS inhibitor) protected cells against the injury by MPP(+) co-administered with IFN-gamma. Both inhibitors restrained the apoptosis of SH-SY5Y cells caused by MPP(+)/IFN-gamma. Regarding oxidative stress, L-NAME and 7-NI attenuated the increase in ROS levels caused by MPP(+) (45.3 or 48.4 vs. 87.9 %, P < 0.05). Indeed, L-NAME was more effective than 7-NI for reducing oxidative stress caused by MPP(+) under IFN-gamma exposition. The nNOS gene silencing by small-interfering RNAs recovered cells challenged by IFN-gamma (24 h), or MPP(+) (8 h). In conclusion, IFN-gamma sensitizes cells to MPP(+)-induced injury, also causing an increase in ROS levels. Pretreating cells with L-NAME or 7-NI reverts both the oxidative stress and apoptosis triggered by the neurotoxin MPP(+). Taking together, our data reinforce that IFN-gamma and NOS enzymes play a role in oxidative stress and dopaminergic cell death triggered by MPP(+).