Sphingosine kinase 2 and sphingosine-1-phosphate promotes mitochondrial function in dopaminergic neurons of mouse model of Parkinson's disease and in MPP+ -treated MN9D cells in vitro.

Dysregulation of sphingolipid metabolism has been shown to trigger the pathophysiology of many neurodegenerative disorders. The present study focuses on the role of one of the two sphingosine kinases, Sphk2 and its metabolite sphingosine-1-phosphate (S1P) signaling in Parkinson's disease (PD). Our study indicated a marked down regulation of Sphk2 expression in the substantia nigra region of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model and in the cellular PD model. Localization studies indicated that Sphk2 was predominantly present in mitochondria, proposing for its potential role in mitochondrial functions. Since mitochondrial dysfunction has been described to be the major pathological event in PD, the present study focused on the role of Sphk2/S1P signaling in promoting mitochondrial functions in the MPTP-induced mouse model of PD and in 1-methyl-4 phenylpyridinium (MPP(+))-treated MN9D cells. Our study demonstrated that inhibition of Sphk2 decreased the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and its downstream targets nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM) which are the key genes regulating mitochondrial function. In addition, there was also a significant reduction in the total cellular adenosine triphosphate (ATP) and superoxide dismutase 2 (SOD 2) with an associated increase in levels of reactive oxygen species (ROS) in the absence of Sphk2. Interestingly, it was found that treating the cells with exogenous S1P along with MPP(+) exerted a neuroprotective effect by activation of p-CREB, PGC-1α and NRF-1 in the MN9D cells. Moreover, the level of ATP was unaffected in the MPP(+)-treated cells in the presence of S1P. It was also observed that levels of ROS were significantly decreased in the MPP(+)-treated cells in the presence of exogenous S1P. Our study also demonstrated that S1P exerted its protective effect through the S1P1 receptor. Taken together, these results show that Sphk2/S1P has an important role to play in the survival of the dopaminergic neurons, in the pathogenesis of PD.

Downregulation of miR-124 in MPTP-treated mouse model of Parkinson's disease and MPP iodide-treated MN9D cells modulates the expression of the calpain/cdk5 pathway proteins.

Parkinson's disease (PD) is a debilitating neurodegenerative disorder causing severe motor disabilities resulting from the loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) region of the midbrain. MicroRNAs (miRNAs) are small, non-coding RNAs which play a major role in several cellular processes in health and disease by regulating gene expression post-transcriptionally. Aberrant miRNA expression has been detected in post-mortem human PD brain samples, in vitro and in vivo PD models. However, none of the studies have focused on the role of the brain-abundant miR-124 in PD. In this study, we have evaluated the expression changes of miR-124 in the SN of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. MiRNA expression analysis by qPCR revealed a decrease in the expression of brain-enriched miR-124 in the SN of MPTP-treated mice as compared to controls. Further, in vitro study revealed a decrease in the expression of miR-124 in MN9D dopaminergic neurons treated with methyl phenyl pyridinium (MPP) iodide. The expression of calpains 1 and 2 which is modulated by miR-124 was increased in the SNc of MPTP-treated mice as observed at different time points after treatment and in the MN9D dopaminergic neurons treated with MPP iodide leading to increased expression of the p35 cleavage product, p25 and cyclin-dependent kinase 5 (cdk5). Calpain-p25-mediated increase in cdk5 expression leading to dopaminergic neuronal death has been demonstrated in human PD and MPTP-PD models. Increased expression of calpain 1/cdk5 pathway proteins was observed in anti-miR-124-transfected MN9D cells in our studies. Knockdown of miR-124 led to increased production of reactive oxygen species (ROS) and hydrogen peroxide (H2O2) both known to increase oxidative stress. Further, experiments with miR-124 target protector sequences specific to calpain 1 revealed an interaction of miR-124 with calpain 1. Overexpression of miR-124 after MPP iodide treatment on MN9D cells was found to attenuate the expression of the calpain 1/p25/cdk5 proteins while improving cell survival. These results suggest that miR-124 acts to modulate the expression of calpain/cdk5 pathway proteins in the dopaminergic neurons. A better understanding of the mechanisms controlling the expression of miR-124 will aid in targeting miR-124 for better treatment strategies for PD.