Identification of Altered Blood MicroRNAs and Plasma Proteins in a Rat Model of Parkinson's Disease.

Parkinson's disease (PD) is the age-related neurological disorder characterized by the degeneration of dopamine (DA) neurons in the substantia nigra pars compacta (SNpc). PD is based on motor deficits which start to appear when up to 80% of the DA neurons of SNpc have been lost. Effective management of PD requires the development of novel biomarkers. Therefore, the present study aimed to characterize biomarkers of PD using miRNomics, proteomics, and bioinformatics approaches. Rats exposed to rotenone (2.5 mg/kg b.wt) for 2 months were used as an animal model to identify the unbiased set of miRNAs and proteins deregulated in blood samples. OpenArray, a real-time PCR-based array, is used for high-throughput profiling of miRNAs, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to carry out the global protein profiling. Systematic bioinformatics analysis of miRNAs and proteins was also performed, including annotation, functional classification and functional enrichment, network analysis, and miRNA-protein interaction analysis. Expression of 19 miRNAs and 96 proteins was significantly upregulated in the blood, while 22 proteins were significantly downregulated in blood samples of rotenone-exposed rats. In silico pathway analysis of deregulated proteins and miRNAs in rotenone-exposed rats has identified multiple pathways leading to PD. In summary, we have identified a set of miRNAs (miR-144, miR-96, and miR-29a) and proteins (PLP1, TUBB4A, and TUBA1C), which can be used as a potential biomarker of PD, while further validation required large human population studies.

Studies on Regulation of Global Protein Profile and Cellular Bioenergetics of Differentiating SH-SY5Y Cells.

The SH-SY5Y cells differentiated by sequential exposure of retinoic acid (RA) and brain-derived neurotrophic growth factor (BDNF) are a well-employed cellular model for studying the mechanistic aspects of neural development and neurodegeneration. Earlier studies from our lab have identified dramatic upregulation (77 miRNAs) and downregulation (17 miRNAs) of miRNAs in SH-SY5Y cells differentiated with successive exposure of RA + BDNF and demonstrated the essential role of increased levels of P53 proteins in coping with the differentiation-induced changes in protein levels. In continuation to our earlier studies, we have performed unbiased LC-MS/MS global protein profiling of naïve and differentiated SH-SY5Y cells and analyzed the identified proteins in reference to miRNAs identified in our earlier studies to identify the cellular events regulated by both identified miRNAs and proteins. Analysis of LC-MS/MS data has shown a significant increase and decrease in levels of 215 and 163 proteins, respectively, in differentiated SH-SY5Y cells. Integrative analysis of miRNA identified in our previous studies and protein identified in the present study is carried out to discover novel miRNA-protein regulatory modules to elucidate miRNA-protein regulatory relationships of differentiating neurons. In silico network analysis of miRNAs and proteins deregulated upon SH-SY5Y differentiation identified cell cycle, synapse formation, axonogenesis, differentiation, neuron projection, and neurotransmission, as the topmost involved pathways. Further, measuring mitochondrial dynamics and cellular bioenergetics using qPCR and Seahorse XFp Flux Analyzer, respectively, showed that differentiated cells possess increased mitochondrial dynamics and OCR relative to undifferentiated cells. In summary, our studies have identified a novel set of proteins deregulated during neuronal differentiation and establish the role of miRNAs identified in earlier studies in the regulation of proteins identified by LC-MS/MS-based global profiling of differentiating neurons, which will help in future studies related to neural development and neurodegeneration.

A combined microRNA and proteome profiling to investigate the effect of ZnO nanoparticles on neuronal cells.

Zinc oxide nanoparticles (ZnO NPs) are one of the most broadly used engineered nanomaterials. The toxicity potential of ZnO NPs has been explored in several studies; however, its neurotoxicity, especially its molecular mechanism, has not been studied in depth. In this study, we have used a cellular model of neuronal differentiation (nerve growth factor differentiated PC12 cells) to compare the effect of ZnO NPs exposure on neuronal (differentiated or mature neurons) and non-neuronal (undifferentiated) cells. Our studies have shown that the noncytotoxic concentration of ZnO NPs causes neurite shortening and degeneration in differentiated PC12 cells. Brain-specific microRNA (miRNA) array and liquid chromatography with tandem mass spectrometry (LC-MS/MS) are used to carry out profiling of miRNAs and proteins in PC12 cells exposed with ZnO NPs. Exposure of ZnO NPs produced significant deregulation of a higher number of miRNAs (15) and proteins (267) in neuronal cells in comparison to miRNAs (8) and proteins (207) of non-neuronal cells (8). In silico pathway analysis of miRNAs and proteins deregulated in ZnO NPs exposed differentiated PC12 cells have shown pathways leading to neurodegenerative diseases and mitochondrial dysfunctions are primarily targeted pathways. Further, a bioenergetics study carried out using Seahorse XFp metabolic flux analyzer has confirmed the involvement of mitochondrial dysfunctions in ZnO NPs exposed differentiated PC12 cells. In conclusion, differentiated PC12 cells (neuronal) were found more vulnerable than undifferentiated (non-neuronal PC12 cells) toward the exposure of ZnO NPs and deregulation of miRNAs and mitochondrial dysfunctions play a significant role in its toxicity.