Neurotoxic Models in Parkinson’s

The dysfunction of mitochondria and oxidative damage play a significant role in the development of Parkinson's disease. Studies on brain samples from deceased Parkinson's patients have shown that mitochondrial damage in the substantia nigra, a part of the prain, is mainly caused by the inhibition of mitochondrial complex I (CI), which is the first enzyme of the electron transport chain. Genetic mutations and post-translational modifications of subunits of this complex could also contribute to the lowered enzyme activity. Exposure to neurotoxins such as pesticides and herbicides could induce acute Parkinson's disease via selective inhibition of CI. However, it is still unknown whether the downstream pathways following exposure to different toxins are comparable and whether oxidative damage of different subunits of CI is common across these models.

To address this gap, in a recently published study researchers compared the downstream signaling pathways triggered after exposure to three toxins in dopaminergic cell lines through a comprehensive proteomic analysis. They also isolated CI from three toxic models and compared post-translational modifications (PTMs) that could potentially characterize inhibition of the complex. Finally, to understand the structural changes induced by selected oxidative PTMs, they used a molecular dynamics simulation (MDS) approach.

Here's how the researchers went about their study: They compared the downstream metabolic pathways elicited in dopaminergic cell lines following exposure to rotenone, paraquat, and MPP+ toxins. A proteomic analysis was carried out to compare the downstream pathways. The researchers also compared the post-translational modifications that could potentially characterize the inhibition of the complex. The structural changes induced by oxidative PTMs were analyzed using molecular dynamic simulations. Furthermore, the study also included procedures for cell culture, measurement of cell viability, ROS, total glutathione estimation, isolation of mitochondria, mitochondrial complex I assay, and total proteomics. The study used Rat dopaminergic 1RB3AN27 (N27) neuronal cell line throughout the research. The chemicals and solvents used were of analytical grade.

For data analysis of raw mass spectrometry data, SEQUEST search engine nodes on Proteome Discoverer 2.2 platform were used to search the data against the Rattus norvegicus database from UniProt, with trypsin as the proteolytic enzyme, and carbamidomethylation of cysteine and TMT labelling at peptide N-terminus and lysine side chain as static modifications. To investigate complex I proteomics, CI was isolated using immunocapture method from N27 mitochondria and analyzed through in-solution tryptic digestion and LC-MS/MS. Data analysis was performed through Proteome Discoverer platform using SEQUEST search algorithm against the Rattus norvegicus protein database from UNIPROT.

Let us now look at the authors' comparative proteomic analysis of the three neurotoxic models, namely Red, Pq, and MPP+, that mimic the pathology of Parkinson's disease via selective inhibition of complex I and mitochondrial dysfunction. Thus, the researchers aimed to determine whether these three neurotoxins induce similar degenerative pathways in dopaminergic neurons and post-translational modifications (PTMs) of complex I.

For this purpose, they used cell viability assays, increased reactive oxygen species (ROS) and decreased total glutathione levels to confirm the neurotoxicity of the three models. They performed proteomic analyses of the untreated control and the three neurotoxic models at LD25 and LD50 and identified 6400 proteins. Functional classification of the differentially expressed proteins revealed that biological processes are involved, mainly:

The authors highlight the potential for cell death pathways, structural and metabolic changes to contribute to neurotoxicity in Parkinson's disease.

Five subunits of an enzyme complex were selected for structural analysis to evaluate the effects of a specific posttranslational modification (PTM), Trp oxidation. Trp433 in one of the subunits, NDUFV1, was found to be oxidized in a neurotoxic model, so the modified sub-complex with this residue replaced by oxyindolylalanine was compared to the unmodified sub-complex using molecular dynamics simulations. The analysis showed significant structural changes in NDUFV1 and its interacting subunit, NDUFS4. It also showed altered hydrogen bonding and long-distance conformational changes affecting the distances between consecutive Fe-S clusters. However, the overall structure of the sub-complex remained relatively unchanged. These findings suggest that Trp oxidation can induce local conformational changes that may potentially affect enzyme activity.