MPP⁺ is the active metabolite of MPTP, a molecule structurally similar to the herbicide Paraquat, known to injure the dopaminergic neurons of the nigrostriatal system in Parkinson’s disease models. Within the cells, MPP⁺ accumulates in mitochondria where it inhibits complex I of the electron transport chain, resulting in ATP depletion and neuronal impairment/death. So far, MPP⁺ is recognized as a valuable tool to mimic dopaminergic degeneration in various cell lines. However, despite a large number of studies, a detailed characterization of mitochondrial respiration in neuronal cells upon MPP⁺ treatment is still missing. By using high-resolution respirometry, we deeply investigated oxygen consumption related to each respiratory state in differentiated neuroblastoma cells exposed to the neurotoxin. Our results indicated the presence of extended mitochondrial damage at the inner membrane level, supported by increased LEAK respiration, and a drastic drop in oxygen flow devoted to ADP phosphorylation in respirometry measurements. Furthermore, prior to complex I inhibition, an enhancement of complex II activity was observed, suggesting the occurrence of some compensatory effect. Overall our findings provide a mechanistic insight on the mitochondrial toxicity mediated by MPP⁺, relevant for the standardization of studies that employ this neurotoxin as a disease model.

Mitochondria from affected tissues of amyotrophic lateral sclerosis (ALS) patients show morphological and biochemical abnormalities. Mitochondrial dysfunction causes oxidative damage and the accumulation of ROS, and represents one of the major triggers of selective death of motor neurons in ALS. We aimed to assess whether oxidative stress in ALS induces post-translational modifications (PTMs) in VDAC1, the main protein of the outer mitochondrial membrane and known to interact with SOD1 mutants related to ALS. In this work, specific PTMs of the VDAC1 protein purified by hydroxyapatite from mitochondria of a NSC34 cell line expressing human SOD1G93A, a suitable ALS motor neuron model, were analyzed by tryptic and chymotryptic proteolysis and UHPLC/High-Resolution ESI-MS/MS. We found selective deamidations of asparagine and glutamine of VDAC1 in ALS-related NSC34-SOD1G93A cells but not in NSC34-SOD1WT or NSC34 cells. In addition, we identified differences in the over-oxidation of methionine and cysteines between VDAC1 purified from ALS model or non-ALS NSC34 cells. The specific range of PTMs identified exclusively in VDAC1 from NSC34-SOD1G93A cells but not from NSC34 control lines, suggests the appearance of important changes to the structure of the VDAC1 channel and therefore to the bioenergetics metabolism of ALS motor neurons. Data are available via ProteomeXchange with identifier .

Voltage-Dependent Anion-selective Channel isoform 1 (VDAC1) is the most abundant isoform of the outer mitochondrial membrane (OMM) porins and the principal gate for ions and metabolites to and from the organelle. VDAC1 is also involved in a number of additional functions, such as the regulation of apoptosis. Although the protein is not directly involved in mitochondrial respiration, its deletion in yeast triggers a complete rewiring of the whole cell metabolism, with the inactivation of the main mitochondrial functions. In this work, we analyzed in detail the impact of VDAC1 knockout on mitochondrial respiration in the near-haploid human cell line HAP1. Results indicate that, despite the presence of other VDAC isoforms in the cell, the inactivation of VDAC1 correlates with a dramatic impairment in oxygen consumption and a re-organization of the relative contributions of the electron transport chain (ETC) enzymes. Precisely, in VDAC1 knockout HAP1 cells, the complex I-linked respiration (N-pathway) is increased by drawing resources from respiratory reserves. Overall, the data reported here strengthen the key role of VDAC1 as a general regulator of mitochondrial metabolism.

α-synuclein (αSyn) is a small neuronal protein whose accumulation correlates with Parkinson’s disease. αSyn A53T mutant impairs mitochondrial functions by affecting substrate import within the organelle, activity of complex I and the maximal respiratory capacity. However, the precise mechanism initiating the bioenergetic dysfunction is not clearly understood yet. By overexpressing αSyn A53T in SH-SY5Y cells, we investigated the specific changes in the mitochondrial respiratory profile using High-Resolution Respirometry. We found that αSyn A53T increases dissipative fluxes across the intermembrane mitochondrial space: this does not compromise the oxygen flows devoted to ATP production while it reduces the bioenergetic excess capacity of mitochondria, providing a possible explanation of the increased cell susceptibility observed in the presence of further stress stimuli.