Quantitative analysis of neuronal mitochondrial movement reveals patterns resulting from neurotoxicity of rotenone and 6-hydroxydopamine.

Alterations in mitochondrial dynamics, including their intracellular trafficking, are common early manifestations of neuronal degeneration. However, current methodologies used to study mitochondrial trafficking events rely on parameters that are primarily altered in later stages of neurodegeneration. Our objective was to establish a reliable applied statistical analysis to detect early alterations in neuronal mitochondrial trafficking. We propose a novel quantitative analysis of mitochondria trajectories based on innovative movement descriptors, including straightness, efficiency, anisotropy, and kurtosis. We evaluated time- and dose-dependent alterations in trajectory descriptors using biological data from differentiated SH-SY5Y cells treated with the mitochondrial toxicants 6-hydroxydopamine and rotenone. MitoTracker Red CMXRos-labelled mitochondria movement was analyzed by total internal reflection fluorescence microscopy followed by computational modelling to describe the process. Based on the aforementioned trajectory descriptors, this innovative analysis of mitochondria trajectories provides insights into mitochondrial movement characteristics and can be a consistent and sensitive method to detect alterations in mitochondrial trafficking occurring in the earliest time points of neurodegeneration.

Alpha-Synuclein Aggregates Associated with Mitochondria in Tunnelling Nanotubes.

The interaction of α-synuclein with mitochondria in both typical and atypical Parkinson's disease is a critical component of degeneration. The mechanism of cell-to-cell propagation of pathological α-synuclein in synucleinopathies is unclear. Intercellular exchange of mitochondria along tunnelling nanotubes has been described in other diseases, such as cancer; however, its role in synucleinopathies is unknown. Pathological α-synuclein species have been demonstrated previously to move from cell to cell via tunnelling nanotubes. This process was further explored using co-culture and monoculture systems to determine if α-synuclein binds to migrating mitochondria within tunnelling nanotubes. Super-resolution analysis via stimulated emission depletion microscopy showed interaction between α-synuclein with the mitochondrial outer membrane and the presence of alpha-synuclein associated with mitochondria in tunnelling nanotubes between 1321N1, differentiated THP-1 and SH-SY5Y cell types. siRNA knockdown of Miro1, a critical protein-bridging mitochondria to the motor adaptor complex, had no effect on mitochondrial density or α-synuclein association with mitochondria in tunnelling nanotubes. The results show that α-synuclein aggregates associate with mitochondria in intercellular tunnelling nanotubes, suggesting that mitochondria-mediated α-synuclein transfer between cells may contribute to cell-to-cell spread of α-synuclein aggregates and disease propagation.

Replication and ribosomal stress induced by targeting pyrimidine synthesis and cellular checkpoints suppress p53-deficient tumors.

p53-mutated tumors often exhibit increased resistance to standard chemotherapy and enhanced metastatic potential. Here we demonstrate that inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme of the de novo pyrimidine synthesis pathway, effectively decreases proliferation of cancer cells via induction of replication and ribosomal stress in a p53- and checkpoint kinase 1 (Chk1)-dependent manner. Mechanistically, a block in replication and ribosomal biogenesis result in p53 activation paralleled by accumulation of replication forks that activate the ataxia telangiectasia and Rad3-related kinase/Chk1 pathway, both of which lead to cell cycle arrest. Since in the absence of functional p53 the cell cycle arrest fully depends on Chk1, combined DHODH/Chk1 inhibition in p53-dysfunctional cancer cells induces aberrant cell cycle re-entry and erroneous mitosis, resulting in massive cell death. Combined DHODH/Chk1 inhibition effectively suppresses p53-mutated tumors and their metastasis, and therefore presents a promising therapeutic strategy for p53-mutated cancers.

Intracellular and Intercellular Mitochondrial Dynamics in Parkinson's Disease.

The appearance of alpha-synuclein-positive inclusion bodies (Lewy bodies) and the loss of catecholaminergic neurons are the primary pathological hallmarks of Parkinson's disease (PD). However, the dysfunction of mitochondria has long been recognized as a key component in the progression of the disease. Dysfunctional mitochondria can in turn lead to dysregulation of calcium homeostasis and, especially in dopaminergic neurons, raised mean intracellular calcium concentration. As calcium binding to alpha-synuclein is one of the important triggers of alpha-synuclein aggregation, mitochondrial dysfunction will promote inclusion body formation and disease progression. Increased reactive oxygen species (ROS) resulting from inefficiencies in the electron transport chain also contribute to the formation of alpha-synuclein aggregates and neuronal loss. Recent studies have also highlighted defects in mitochondrial clearance that lead to the accumulation of depolarized mitochondria. Transaxonal and intracytoplasmic translocation of mitochondria along the microtubule cytoskeleton may also be affected in diseased neurons. Furthermore, nanotube-mediated intercellular transfer of mitochondria has recently been reported between different cell types and may have relevance to the spread of PD pathology between adjacent brain regions. In the current review, the contributions of both intracellular and intercellular mitochondrial dynamics to the etiology of PD will be discussed.

Reactivation of Dihydroorotate Dehydrogenase-Driven Pyrimidine Biosynthesis Restores Tumor Growth of Respiration-Deficient Cancer Cells.

Cancer cells without mitochondrial DNA (mtDNA) do not form tumors unless they reconstitute oxidative phosphorylation (OXPHOS) by mitochondria acquired from host stroma. To understand why functional respiration is crucial for tumorigenesis, we used time-resolved analysis of tumor formation by mtDNA-depleted cells and genetic manipulations of OXPHOS. We show that pyrimidine biosynthesis dependent on respiration-linked dihydroorotate dehydrogenase (DHODH) is required to overcome cell-cycle arrest, while mitochondrial ATP generation is dispensable for tumorigenesis. Latent DHODH in mtDNA-deficient cells is fully activated with restoration of complex III/IV activity and coenzyme Q redox-cycling after mitochondrial transfer, or by introduction of an alternative oxidase. Further, deletion of DHODH interferes with tumor formation in cells with fully functional OXPHOS, while disruption of mitochondrial ATP synthase has little effect. Our results show that DHODH-driven pyrimidine biosynthesis is an essential pathway linking respiration to tumorigenesis, pointing to inhibitors of DHODH as potential anti-cancer agents.

Horizontal transfer of whole mitochondria restores tumorigenic potential in mitochondrial DNA-deficient cancer cells.

Recently, we showed that generation of tumours in syngeneic mice by cells devoid of mitochondrial (mt) DNA (ρ0 cells) is linked to the acquisition of the host mtDNA. However, the mechanism of mtDNA movement between cells remains unresolved. To determine whether the transfer of mtDNA involves whole mitochondria, we injected B16ρ0 mouse melanoma cells into syngeneic C57BL/6Nsu9-DsRed2 mice that express red fluorescent protein in their mitochondria. We document that mtDNA is acquired by transfer of whole mitochondria from the host animal, leading to normalisation of mitochondrial respiration. Additionally, knockdown of key mitochondrial complex I (NDUFV1) and complex II (SDHC) subunits by shRNA in B16ρ0 cells abolished or significantly retarded their ability to form tumours. Collectively, these results show that intact mitochondria with their mtDNA payload are transferred in the developing tumour, and provide functional evidence for an essential role of oxidative phosphorylation in cancer.

Correction: Characterisation of Mesothelioma-Initiating Cells and Their Susceptibility to Anti-Cancer Agents.

[This corrects the article DOI: 10.1371/journal.pone.0119549.].

Horizontal transfer of mitochondria between mammalian cells: beyond co-culture approaches.

Current dogma holds that genes are the property of individual mammalian cells and partition between daughter cells during cell division. However, and rather unexpectedly, recent research has demonstrated horizontal cell-to-cell transfer of mitochondria and mitochondrial DNA in several mammalian cell culture systems. Furthermore, unequivocal evidence that mitochondrial DNA transfer occurs in vivo has now been published. While these studies show horizontal transfer of mitochondrial DNA in pathological settings, it is also possible that intercellular mitochondrial transfer is a fundamental physiological process with a role in development and tissue homeostasis.

Characterisation of mesothelioma-initiating cells and their susceptibility to anti-cancer agents.

Malignant mesothelioma (MM) is an aggressive type of tumour causing high mortality. One reason for this paradigm may be the existence of a subpopulation of tumour-initiating cells (TICs) that endow MM with drug resistance and recurrence. The objective of this study was to identify and characterise a TIC subpopulation in MM cells, using spheroid cultures, mesospheres, as a model of MM TICs. Mesospheres, typified by the stemness markers CD24, ABCG2 and OCT4, initiated tumours in immunodeficient mice more efficiently than adherent cells. CD24 knock-down cells lost the sphere-forming capacity and featured lower tumorigenicity. Upon serial transplantation, mesospheres were gradually more efficiently tumrigenic with increased level of stem cell markers. We also show that mesospheres feature mitochondrial and metabolic properties similar to those of normal and cancer stem cells. Finally, we show that mesothelioma-initiating cells are highly susceptible to mitochondrially targeted vitamin E succinate. This study documents that mesospheres can be used as a plausible model of mesothelioma-initiating cells and that they can be utilised in the search for efficient agents against MM.

Mitochondrial genome acquisition restores respiratory function and tumorigenic potential of cancer cells without mitochondrial DNA.

We report that tumor cells without mitochondrial DNA (mtDNA) show delayed tumor growth, and that tumor formation is associated with acquisition of mtDNA from host cells. This leads to partial recovery of mitochondrial function in cells derived from primary tumors grown from cells without mtDNA and a shorter lag in tumor growth. Cell lines from circulating tumor cells showed further recovery of mitochondrial respiration and an intermediate lag to tumor growth, while cells from lung metastases exhibited full restoration of respiratory function and no lag in tumor growth. Stepwise assembly of mitochondrial respiratory (super)complexes was correlated with acquisition of respiratory function. Our findings indicate horizontal transfer of mtDNA from host cells in the tumor microenvironment to tumor cells with compromised respiratory function to re-establish respiration and tumor-initiating efficacy. These results suggest pathophysiological processes for overcoming mtDNA damage and support the notion of high plasticity of malignant cells.

Mitochondrial targeting of α-tocopheryl succinate enhances its anti-mesothelioma efficacy.

UNLABELLED: Malignant mesothelioma (MM) is a fatal neoplastic disease with no therapeutic option. Therefore, the search for novel therapies is of paramount importance. METHODS: Since mitochondrial targeting of α-tocopheryl succinate (α-TOS) by its tagging with triphenylphosphonium enhances its cytotoxic effects to cancer cells, we tested its effect on MM cells and experimental mesotheliomas. RESULTS: Mitochondrially targeted vitamin E succinate (MitoVES) was more efficient in killing MM cells than α-TOS with IC50 lower by up to two orders of magnitude. Mitochondrial association of MitoVES in MM cells was documented using its fluorescently tagged analogue. MitoVES caused apoptosis in MM cells by mitochondrial destabilization, resulting in the loss of mitochondrial membrane potential, generation of reactive oxygen species, and destabilization of respiratory supercomplexes. The role of the mitochondrial complex II in the activity of MitoVES was confirmed by the finding that MM cells with suppressed succinate quinone reductase were resistant to MitoVES. MitoVES suppressed mesothelioma growth in nude mice with high efficacy. DISCUSSION: MitoVES is more efficient in killing MM cells and suppressing experimental mesotheliomas compared with the non-targeted α-TOS, giving it a potential clinical benefit.