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

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

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

We report that tumor cells devoid of their mitochondrial genome (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 supercomplexes 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 a novel pathophysiological process for overcoming mtDNA damage and support the notion of high plasticity of malignant cells

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

Mesospheres derived from different tumour generations show different mitochondrial features.

<p>Sphere cells representing individual generations were evaluated for respiration using the routine protocol (A) and the protocol for permeabilised cells (B) by means of the Oxygraph 2k high resolution respirometer. The symbols GM_L, GM_P, GMS_P, GMS_E and S(Rot)_E represent routine respiration, respiration via complex I, respiration via complex I+II, uncoupled respiration and uncoupled respiration via CII, respectively. (C) Mitochondrial mass was evaluated using MitoTracker Green as detailed in the Methods section. Superoxide was evaluated using the fluorescent probe MitoSOX (D), glucose uptake using the fluorescent glucose analogue 2-NBDG (E), ATP levels by a luciferase-based assay (F), ΔΨ_m using the fluorescent probe TMRM (G), lactate levels using a commercial kit (H), for SDH (I) and SQR (J) activities using enzymatic assays, and hand-held cells counter for cell size (K). (L) TEM was performed on individual generation sphere cells as detailed in the Methods section. Data in panels A-K are mean values ±S.D., and are derived from three individual experiments. The symbol ‘*’ denotes statistically significant differences with <i>p</i><0.05. Images in panel L are representative of three independent experiments. The white bar in panel K in the upper images represents 500 nm, in the lower images 200 nm.</p

Mesospheres form tumours in serial transplantations.

<p>(A) Ist-Mes-2 sphere cells (generation 1, G1) were subcutaneously grafted into Balb-c/nude mice at 10⁶ cells per animal. When tumours reached about 2,000 mm³, the mice were sacrificed, tumours excised and malignant cells grew <i>in vitro</i> as a cell line. The adherent cells were converted into spheres (G1) and these were grafted into Balb-c/nude mice to form tumours that were used for generation 2 (G2) spheres. This procedure was repeated two more times to derive G3 and G4 spheres. The inset in panel A shows the lag to tumour appearance following cell grafting for individual sphere cell generations. Cells of individual generations were evaluated for stemness markers as shown using qPCR (B) and WB (panel C shows the blots, panel D their densitometric evaluations) and for CS activity (E). (F) Tumours derived from adherent cells and spheres of individual generations were excised, paraffin-embedded and stained for the MM marker mesothelin (upper images show staining with the exclusion of the primary IgG) and with H&E. Data shown in panel A are derived from 5 animals and are mean values ± S.E.M, data in panels B and E are mean values from three independent experiments ± S.D. Images in panel C are representative of two individual experiments and their densitometric evaluations in panel D represent mean values with differences lower than 10%. Images in panel F were obtained using one tumour for each condition (generation). The symbol ‘*’ denotes statistically significant differences with <i>p</i><0.05. Images are representative of three different tumours.</p

Sphere cells derived from MM cell lines show increased levels of ‘stemness’ markers.

<p>(A) Ist-Mes-2, Meso-2 and MM-BI cells were allowed to form spheres, after which these were placed in the complete medium to cause differentiation and re-adhesion of the cells. Relative levels <i>CD24</i>, <i>ABCG2</i> and <i>OCT4</i> mRNAs were assessed in the original adherent cells (set as 1), the spheres and the re-adherent cells. (B) Ist-Mes-2, Meso-2 and MM-BI cells spheres were tested for the level of <i>SOX4</i>, <i>C-MYC</i>, <i>ABCB5</i> and <i>KLF4</i> mRNA and their level expressed relative to that of adherent cells. The data are mean values ± S.D. derived from three independent experiments, the symbol ‘*’ stands significantly different data with <i>p</i><0.05, ‘**’ for those with <i>p</i><0.005.</p

CD24 supports initiation and progression of mesotheliomas.

<p>(A) Mock-transfected and CD24^- Ist-Mes-2 cells (10⁶ per animal) were grafted subcutaneously into NOD/SCID mice and tumour formation followed using USI. (B) CD24^- and mock-transfected Ist-Mes-2 cell-derived tumours were evaluated for CD24, CD47, EpCAM and Oct3/4 by western blotting using anti-actin IgG as a loading control, with panel C documenting densitometric evaluation of the blots. (D) A representative image of a mouse with CD24^- cell-derived tumour and parental cell-derived tumour is shown. (E) Representative USI images of a tumour derived from mock-transfected and CD24^- cells acquired on different days are shown, with the yellow arrows indicating the position of the tumours. Data in panel A are mean values ±S.E.M., and are derived from four animals. The symbol ‘*’ denotes statistically significant differences with <i>p</i><0.05. Images in panel B are representative of two independent experiments with the densitometric evaluation showing average data with differences less than 10%.</p

IC₅₀ values for MitoVES and several other anti-cancer agents for parental and CD24^- adherent and sphere Ist-Mes-2 cells.

<p>^aNumbers in bold indicate significantly different data (<i>p</i><0.05) from adherent CD24^-and parental cells.</p><p>The data are mean values ± S.D. from three independent experiments.</p><p>IC₅₀ values for MitoVES and several other anti-cancer agents for parental and CD24^- adherent and sphere Ist-Mes-2 cells.</p