Comparative analysis of 1-methyl-4-phenylpyridinium (MPP+) and manganese induced neurotoxic effects on DNA methylation in dopaminergic neurons

Idiopathic Parkinson’s disease (iPD) and manganese-induced atypical Parkinsonism are characterized by movement disorder and nigrostriatal pathology. Although clinical features, brain region involved and responsiveness to L-DOPA differentiate both, the differences at the neuronal level are largely unknown. We investigated the morphological, physiological and molecular differences in dopaminergic neurons exposed to the PD toxin 1-methyl-4-phenylpyridinium ion (MPP+) and manganese (Mn). While Mn was neurotoxic at lower dose, MPP+ toxicity entailed oxidative damage, mitochondria dysfunction and glycolytic shift. Morphological analysis highlighted mitochondrial damage, while morphometric analysis indicated loss of neuronal processes in the MPP+ model and not in the Mn model. Elecrophysiological analysis demonstrated lower number of spikes and firing frequency in MPP+ treated cells, while it was unchanged in the Mn model. High throughput transcriptomic analysis revealed upregulation of 694 and 603 genes and down-regulation of 428 and 255 genes in the MPP+ and Mn models respectively. Many differentially expressed genes were unique to either models and contributed to neuroinflammation, metabolic and mitochondrial function, apoptosis and nuclear function, synaptic plasticity, neurotransmission and cytoskeletal architecture. Analysis of the JAK-STAT pathway with implications for neuritogenesis, neuronal proliferation and nuclear function revealed contrasting profile between Mn and MPP+ models. Genome-wide DNA methylation profile revealed significant differences between both models and substantiated the epigenetic basis of the difference in the JAK-STAT pathway. We conclude that iPD and atypical Parkinsonism represent a divergent neurotoxicological manifestation at the dopaminergic neuronal level with implications for pathobiology and to evolve novel therapeutics DNA methylation analysis of control N27 cells was carried out as described (Kelkar et al. 2009, Rajpathak et al. 2014). DNA methylation analysis of Mn treated N27 cells was carried out as described (Kelkar et al. 2009, Rajpathak et al. 2014). DNA methylation analysis of MPP+ treated N27 cells was carried out as described (Kelkar et al. 2009, Rajpathak et al. 2014). For each group, three independent biological replicates were subjected to methylation array (Agilent technologies Inc). Genomic DNA was isolated from cells of different experimental groups, subjected to proteinase K digestion in the presence of 1% SDS, followed by phenol: chloroform extraction, RNase A treatment and precipitation using NaCl and ethanol. Genomic DNA (2 µg) was sheared into 1.5-2 kb fragments by sonication and the fragments were denatured by heating at 95OC and hybridized on to a Rat GE 4x44K v3 Microarray slides (Agilent Technologies, CA, USA) each with 4x44K arrays, containing 44,000 probes of 22,583 genes. After hybridization at 42OC for 16 h, the slides were washed, blocked and then incubated in anti-5-methylcytosine antibody (1h at 25OC). Slides were then washed and incubated in cy3 labeled anti-mouse IgG (1h at 25OC), washed and scanned at 5 uM resolution at 543 nm. Scanarray software was used for data acquisition, mean or median intensity calculation of each spot and normalization.