Transcriptome Analysis of Rotenone Induced Neurotoxicity in Enriched Rat Primary Ventral Mesencephalic Neurons

Special Issue - Parkinson’s Disease

Austin Neurol & Neurosci. 2017; 2(1): 1019.

Transcriptome Analysis of Rotenone Induced Neurotoxicity in Enriched Rat Primary Ventral Mesencephalic Neurons

Bollimpelli VS, Dholaniya PS and Kondapi AK*

Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, India

*Corresponding author: Kondapi AK, Department of Biotechnology and Bioinformatics, University of Hyderabad, Hyderabad, India

Received: May 05, 2017; Accepted: June 06, 2017; Published: June 13, 2017


Rotenone induced neurotoxicity is being widely investigated in relation to Parkinson’s Disease (PD). However the crucial molecular mechanisms involved in rotenone induced PD still remains elusive. This report details the transcriptome changes on rotenone treatment in enriched rat primary ventral mesencephalic neurons through microarray analysis. Transcriptome analysis had yielded 705 up-regulated genes and 2415 down-regulated genes. This data was further validated by quantitative real time PCR analysis. To further refine this data, association rule mining was used and a gene interaction network among 53 genes was developed. Functional characterization of the top 25 scored genes in the network was done using panther database. Interestingly TNF was the highest scored gene among them and found to be significantly down regulated on rotenone treatment. Along with TNF other inflammation related genes like Il1b, Itpr3 and TNFR2 were also significantly down-regulated on rotenone treatment. These observations suggest that down regulation of neuronal TNF might be a critical cause leading to cellular death via TNFR2, Il1b mediated Pi3Kinase pathway in rotenone induced neurotoxicity. Further investigation in this neuronal TNF related pathways may give novel therapeutic approaches in treatment of PD.

Keywords: Parkinson’s disease; Micro array; Tumor necrosis factor; Neurodegeneration


In-Vitro/In-Vivo treatments with rotenone are known to induce certain features of Parkinson Disease (PD) [1]. Dopaminergic neurodegeneration in substantianigra pars compacta of Ventral Mesencephalic (VM) brain region is the hall mark feature of PD [2]. Rotenone, besides affecting mitochondrial function was also reported to be affecting a variety of cellular processes like cytoskeleton stability, inflammation, oxidative stress and apoptosis [1,3,4]. All these observations were made using directed approaches studying few of the genes involved in those specific pathways. Microarray analysis of whole transcriptome is an alternative approach for identifying key genes and pathways that might not be feasible through single-gene studies. Enriched rat primary VM neurons were well characterized [5] and studied in co-relation with rotenone induced neurotoxicity [6]. In the present study primary VM neurons were analyzed for changes in their genome expression upon rotenone treatment using microarray analysis along with Association Rule Mining (ARM) [7] for discovering the relationship among genes in a large dataset.



Pregnant female wistar rats were procured from the National Institute of Nutrition, Hyderabad, India and maintained at University of Hyderabad animal house facility.

Ethical approval: Animal experiments were carried out according to the norms of Institutional animal ethical committee, University of Hyderabad (Proposal number LS/IAEC/AKK/10/1).

Isolation and culture of VM neurons: VM neuronal culture was done following the previously reported protocol by Bollimpelli VS et al., 2015. VM neurons were seeded at 3X 106 cells in 1ml of Dulbecco’s Minimum Essential Media (DMEM- F12) with 10% fetal bovine serum and 1X pen strep (Gibco, NY, USA) per well in a 6 well plate coated with 0.1mg/ml Poly-D- Lysine (PDL) (Sigma chemical co, MO, USA). Cultures were incubated at 5% CO2 and 37°C. Cultures from 2nd-DIV were supplemented with 2μM of mitotic inhibitor arabinosylcytosine (Sigma chemical co, MO, USA). Rotenone was dissolved in DMSO (Sigma Chemical Co, MO, USA) and added to the culture in a single application at specified concentration for 48 h.


VM neurons at 7th Day In Vitro (DIV) were treated with 15nM rotenone, and the cells were processed at 9thDIV for microarray analysis. DMSO alone treated cells at 7th DIV were harvested at 9th DIV and were considered as control. The samples were processed for microarray analysis at Genotypic Technology Pvt Ltd, Bangalore, India, with whole rat genome microarray kit provided by Agilent. A fold change of 0.6 was used to detect the up-regulation and downregulation with a geomean fold of 0.8.

Real Time quantitative PCR (qRT-PCR)

Total RNA (1μg) extracted from treated and control samples were reverse transcribed by Superscript III Kit (Invitrogen). Primers for the selected genes were given in Supplementary Table 1. PCR reactions were run with SYBR Green Kit (Quiagen) in ABI Prism H7500 fast thermal cycler (Applied Biosystem, CA, USA). 18srRNA was used as internal control. Amplification resulted fluorescence was analyzed and expressed in relative fold change using 2-??CT method [8].


Experiments were performed in triplicates and repeated for three times independently. Data was averaged and presented as mean ± SD. Statistical analysis for micro array data was done by student’s unpaired t test. Spearman rho test were performed to find correlation between micro array data and qRT-PCR data.


Micro array and validation

VM neurons at 7th DIV were treated with 15nM rotenone and incubated for 48hrs. Through micro array analysis transcriptome change in rotenone treated VM neuron was compared with DMSO alone treated VM neurons. Considering the parameters mentioned in methodology, a data with 705 up-regulated genes and 2415downregulated genes was obtained (supplementary Table 2). In order to validate this data, five genes with different folds of expression ranging from high to low were selected randomly and their expression in VM neurons on rotenone treatment was analyzed by qRT-PCR analysis. Comparative qRT-PCR analysis in Figure 1confirmed the expression pattern of microarray for the selected genes. Most of the genes were in a positive correlation with micro array data except Myoc which had shown a higher expression in qRT-PCR than in micro array data.