Cristae dynamics is modulated in bioenergetically compromised mitochondria.

Cristae membranes have been recently shown to undergo intramitochondrial merging and splitting events. Yet, the metabolic and bioenergetic factors regulating them are unclear. Here, we investigated whether and how cristae morphology and dynamics are dependent on oxidative phosphorylation (OXPHOS) complexes, the mitochondrial membrane potential (ΔΨm), and the ADP/ATP nucleotide translocator. Advanced live-cell STED nanoscopy combined with in-depth quantification were employed to analyse cristae morphology and dynamics after treatment of mammalian cells with rotenone, antimycin A, oligomycin A, and CCCP. This led to formation of enlarged mitochondria along with reduced cristae density but did not impair cristae dynamics. CCCP treatment leading to ΔΨm abrogation even enhanced cristae dynamics showing its ΔΨm-independent nature. Inhibition of OXPHOS complexes was accompanied by reduced ATP levels but did not affect cristae dynamics. However, inhibition of ADP/ATP exchange led to aberrant cristae morphology and impaired cristae dynamics in a mitochondrial subset. In sum, we provide quantitative data of cristae membrane remodelling under different conditions supporting an important interplay between OXPHOS, metabolite exchange, and cristae membrane dynamics.

The Antimalarial Drug Artesunate Mediates Selective Cytotoxicity by Upregulating HO-1 in Melanoma Cells.

Despite great efforts to develop new therapeutic strategies to combat melanoma, the prognosis remains rather poor. Artesunate (ART) is an antimalarial drug displaying anti-cancer effects in vitro and in vivo. In this in vitro study, we investigated the selectivity of ART on melanoma cells. Furthermore, we aimed to further elucidate the mechanism of the drug with a focus on the role of iron, the induction of oxidative stress and the implication of the enzyme heme oxygenase 1 (HO-1). ART treatment decreased the cell viability of A375 melanoma cells while it did not affect the viability of normal human dermal fibroblasts, used as a model for normal (healthy) cells. ART's toxicity was shown to be dependent on intracellular iron and the drug induced high levels of oxidative stress as well as upregulation of HO-1. Melanoma cells deficient in HO-1 or treated with a HO-1 inhibitor were less sensitive towards ART. Taken together, our study demonstrates that ART induces oxidative stress resulting in the upregulation of HO-1 in melanoma cells, which subsequently triggers the effect of ART's own toxicity. This new finding that HO-1 is involved in ART-mediated toxicity may open up new perspectives in cancer therapy.

MIC26 and MIC27 are bona fide subunits of the MICOS complex in mitochondria and do not exist as glycosylated apolipoproteins.

Impairments of mitochondrial functions are linked to human ageing and pathologies such as cancer, cardiomyopathy, neurodegeneration and diabetes. Specifically, aberrations in ultrastructure of mitochondrial inner membrane (IM) and factors regulating them are linked to diabetes. The development of diabetes is connected to the 'Mitochondrial Contact Site and Cristae Organising System' (MICOS) complex which is a large membrane protein complex defining the IM architecture. MIC26 and MIC27 are homologous apolipoproteins of the MICOS complex. MIC26 has been reported as a 22 kDa mitochondrial and a 55 kDa glycosylated and secreted protein. The molecular and functional relationship between these MIC26 isoforms has not been investigated. In order to understand their molecular roles, we depleted MIC26 using siRNA and further generated MIC26 and MIC27 knockouts (KOs) in four different human cell lines. In these KOs, we used four anti-MIC26 antibodies and consistently detected the loss of mitochondrial MIC26 (22 kDa) and MIC27 (30 kDa) but not the loss of intracellular or secreted 55 kDa protein. Thus, the protein assigned earlier as 55 kDa MIC26 is nonspecific. We further excluded the presence of a glycosylated, high-molecular weight MIC27 protein. Next, we probed GFP- and myc-tagged variants of MIC26 with antibodies against GFP and myc respectively. Again, only the mitochondrial versions of these tagged proteins were detected but not the corresponding high-molecular weight MIC26, suggesting that MIC26 is indeed not post-translationally modified. Mutagenesis of predicted glycosylation sites in MIC26 also did not affect the detection of the 55 kDa protein band. Mass spectrometry of a band excised from an SDS gel around 55 kDa could not confirm the presence of any peptides derived from MIC26. Taken together, we conclude that both MIC26 and MIC27 are exclusively localized in mitochondria and that the observed phenotypes reported previously are exclusively due to their mitochondrial function.

Involvement of necroptosis in the selective toxicity of the natural compound (±) gossypol on squamous skin cancer cells in vitro.

Cutaneous basal and squamous cell carcinoma reflect the first and second most common type of non-melanoma skin cancer, respectively. Especially cutaneous squamous cell carcinoma has the tendency to metastasize, finally resulting in a rather poor prognosis. Therapeutic options comprise surgery, radiation therapy, and a systemic or targeted chemotherapy. There are some good treatment results, but overall, the response rate of newly developed drugs is still modest. Drug repurposing represents an alternative approach where already available and clinically approved substances are used, which originally intended for other clinical benefits. In this context, we tested the effect of the naturally occurring polyphenolic aldehyde (±) gossypol with concentrations between 1 and 5 µM on the invasive squamous cell carcinoma cell line SCL-1 and normal human epidermal keratinocytes. Gossypol treatment up to 96 h resulted in a selective cytotoxicity of SCL-1 cells (IC50: 1.7 µM, 96 h) compared with normal keratinocytes (IC50: ≥ 5.4 µM, 96 h) which is mediated by mitochondrial dysfunction and finally leading to necroptotic cell death. Taken together, gossypol shows a high potential as an alternative anticancer drug for the treatment of cutaneous squamous cell carcinoma.

Emerging Roles of the MICOS Complex in Cristae Dynamics and Biogenesis.

Mitochondria are double membrane-enclosed organelles performing important cellular and metabolic functions such as ATP generation, heme biogenesis, apoptosis, ROS production and calcium buffering. The mitochondrial inner membrane (IM) is folded into cristae membranes (CMs) of variable shapes using molecular players including the 'mitochondrial contact site and cristae organizing system' (MICOS) complex, the dynamin-like GTPase OPA1, the F1FO ATP synthase and cardiolipin. Aberrant cristae structures are associated with different disorders such as diabetes, neurodegeneration, cancer and hepato-encephalopathy. In this review, we provide an updated view on cristae biogenesis by focusing on novel roles of the MICOS complex in cristae dynamics and shaping of cristae. For over seven decades, cristae were considered as static structures. It was recently shown that cristae constantly undergo rapid dynamic remodeling events. Several studies have re-oriented our perception on the dynamic internal ambience of mitochondrial compartments. In addition, we discuss the recent literature which sheds light on the still poorly understood aspect of cristae biogenesis, focusing on the role of MICOS and its subunits. Overall, we provide an integrated and updated view on the relation between the biogenesis of cristae and the novel aspect of cristae dynamics.

The BH3 mimetic (±) gossypol induces ROS-independent apoptosis and mitochondrial dysfunction in human A375 melanoma cells in vitro.

A major challenge in current cancer therapy is still the treatment of metastatic melanomas of the skin. BH3 mimetics represent a novel group of substances inducing apoptosis. In this study, we investigated the cytotoxic effect of (±) gossypol (GP), a natural compound from cotton seed, on A375 melanoma cells and the underlying biochemical mechanisms. To prevent undesired side effects due to toxicity on normal (healthy) cells, concentrations only toxic for tumor cells have been elaborated. Viability assays were performed to determine the cytotoxicity of GP in A375 melanoma and normal (healthy) cells. For the majority of experiments, a concentration of 2.5 µM GP was used resulting in a ROS-independent but caspase-dependent cell death of A375 melanoma cells. At this level, GP was non-toxic for normal human epidermal melanocytes. GP has a very short half-life, however, it was demonstrated that only the "parent" compound and not decomposition products are responsible for the cytotoxic effect in A375 melanoma cells. GP significantly decreased mitochondrial membrane potential accompanied by a Drp1-dependent loss of mitochondrial integrity (fragmentation) in tumor cells. Taken together, GP induced a ROS-independent intrinsic apoptosis leading to the conclusion that within a specific concentration range, GP may work as effective anticancer drug without harmful side effects.

Individual cristae within the same mitochondrion display different membrane potentials and are functionally independent.

The mitochondrial membrane potential (ΔΨm ) is the main driver of oxidative phosphorylation (OXPHOS). The inner mitochondrial membrane (IMM), consisting of cristae and inner boundary membranes (IBM), is considered to carry a uniform ΔΨm . However, sequestration of OXPHOS components in cristae membranes necessitates a re-examination of the equipotential representation of the IMM. We developed an approach to monitor ΔΨm at the resolution of individual cristae. We found that the IMM was divided into segments with distinct ΔΨm , corresponding to cristae and IBM. ΔΨm was higher at cristae compared to IBM. Treatment with oligomycin increased, whereas FCCP decreased, ΔΨm heterogeneity along the IMM. Impairment of cristae structure through deletion of MICOS-complex components or Opa1 diminished this intramitochondrial heterogeneity of ΔΨm . Lastly, we determined that different cristae within the individual mitochondrion can have disparate membrane potentials and that interventions causing acute depolarization may affect some cristae while sparing others. Altogether, our data support a new model in which cristae within the same mitochondrion behave as independent bioenergetic units, preventing the failure of specific cristae from spreading dysfunction to the rest.

Functional Interplay between Cristae Biogenesis, Mitochondrial Dynamics and Mitochondrial DNA Integrity.

Mitochondria are vital cellular organelles involved in a plethora of cellular processes such as energy conversion, calcium homeostasis, heme biogenesis, regulation of apoptosis and ROS reactive oxygen species (ROS) production. Although they are frequently depicted as static bean-shaped structures, our view has markedly changed over the past few decades as many studies have revealed a remarkable dynamicity of mitochondrial shapes and sizes both at the cellular and intra-mitochondrial levels. Aberrant changes in mitochondrial dynamics and cristae structure are associated with ageing and numerous human diseases (e.g., cancer, diabetes, various neurodegenerative diseases, types of neuro- and myopathies). Another unique feature of mitochondria is that they harbor their own genome, the mitochondrial DNA (mtDNA). MtDNA exists in several hundreds to thousands of copies per cell and is arranged and packaged in the mitochondrial matrix in structures termed mt-nucleoids. Many human diseases are mechanistically linked to mitochondrial dysfunction and alteration of the number and/or the integrity of mtDNA. In particular, several recent studies identified remarkable and partly unexpected links between mitochondrial structure, fusion and fission dynamics, and mtDNA. In this review, we will provide an overview about these recent insights and aim to clarify how mitochondrial dynamics, cristae ultrastructure and mtDNA structure influence each other and determine mitochondrial functions.

Modulation of oxidative phosphorylation and redox homeostasis in mitochondrial NDUFS4 deficiency via mesenchymal stem cells.

BACKGROUND: Disorders of the oxidative phosphorylation (OXPHOS) system represent a large group among the inborn errors of metabolism. The most frequently observed biochemical defect is isolated deficiency of mitochondrial complex I (CI). No effective treatment strategies for CI deficiency are so far available. The purpose of this study was to investigate whether and how mesenchymal stem cells (MSCs) are able to modulate metabolic function in fibroblast cell models of CI deficiency. METHODS: We used human and murine fibroblasts with a defect in the nuclear DNA encoded NDUFS4 subunit of CI. Fibroblasts were co-cultured with MSCs under different stress conditions and intercellular mitochondrial transfer was assessed by flow cytometry and fluorescence microscopy. Reactive oxygen species (ROS) levels were measured using MitoSOX-Red. Protein levels of CI were analysed by blue native polyacrylamide gel electrophoresis (BN-PAGE). RESULTS: Direct cellular interactions and mitochondrial transfer between MSCs and human as well as mouse fibroblast cell lines were demonstrated. Mitochondrial transfer was visible in 13.2% and 6% of fibroblasts (e.g. fibroblasts containing MSC mitochondria) for human and mouse cell lines, respectively. The transfer rate could be further stimulated via treatment of cells with TNF-α. MSCs effectively lowered cellular ROS production in NDUFS4-deficient fibroblast cell lines (either directly via co-culture or indirectly via incubation of cell lines with cell-free MSC supernatant). However, CI protein expression and activity were not rescued by MSC treatment. CONCLUSION: This study demonstrates the interplay between MSCs and fibroblast cell models of isolated CI deficiency including transfer of mitochondria as well as modulation of cellular ROS levels. Further exploration of these cellular interactions might help to develop MSC-based treatment strategies for human CI deficiency.

Loss of the m-AAA protease subunit AFG3L2 causes mitochondrial transport defects and tau hyperphosphorylation.

The m-AAA protease subunit AFG3L2 is involved in degradation and processing of substrates in the inner mitochondrial membrane. Mutations in AFG3L2 are associated with spinocerebellar ataxia SCA28 in humans and impair axonal development and neuronal survival in mice. The loss of AFG3L2 causes fragmentation of the mitochondrial network. However, the pathogenic mechanism of neurodegeneration in the absence of AFG3L2 is still unclear. Here, we show that depletion of AFG3L2 leads to a specific defect of anterograde transport of mitochondria in murine cortical neurons. We observe similar transport deficiencies upon loss of AFG3L2 in OMA1-deficient neurons, indicating that they are not caused by OMA1-mediated degradation of the dynamin-like GTPase OPA1 and inhibition of mitochondrial fusion. Treatment of neurons with antioxidants, such as N-acetylcysteine or vitamin E, or decreasing tau levels in axons restored mitochondrial transport in AFG3L2-depleted neurons. Consistently, tau hyperphosphorylation and activation of ERK kinases are detected in mouse neurons postnatally deleted for Afg3l2. We propose that reactive oxygen species signaling leads to cytoskeletal modifications that impair mitochondrial transport in neurons lacking AFG3L2.