Safari with an Electron Gun: Visualization of Protein and Membrane Interactions in Mitochondria in Natural Environment.

This paper presents new structural data about mitochondria using correlative light and electron microscopy (CLEM) and cryo-electron tomography. These state-of-the-art structural biology methods allow studying biological objects at nanometer scales under natural conditions. Non-invasiveness of these methods makes them comparable to observing animals in their natural environment on a safari. The paper highlights two areas of research that can only be accomplished using these methods. The study visualized location of the Aß42 amyloid aggregates in relation to mitochondria to test a hypothesis of development of mitochondrial dysfunction in Alzheimer's disease. The results showed that the Aß42 aggregates do not interact with mitochondria, although some of them are closely located. Therefore, the study demonstrated that mitochondrial dysfunction is not directly associated with the effects of aggregates on mitochondrial structure. Other processes should be considered as sources of mitochondrial dysfunction. Second unique area presented in this work is high-resolution visualization of the mitochondrial membranes and proteins in them. Analysis of the cryo-ET data reveals toroidal holes in the lamellar structures of cardiac mitochondrial cristae, where ATP synthases are located. The study proposes a new mechanism for sorting and clustering protein complexes in the membrane based on topology. According to this suggestion, position of the OXPHOS system proteins in the membrane is determined by its curvature. High-resolution tomography expands and complements existing ideas about the structural and functional organization of mitochondria. This makes it possible to study the previously inaccessible structural interactions of proteins with each other and with membranes in vivo.

SkQ3 Exhibits the Most Pronounced Antioxidant Effect on Isolated Rat Liver Mitochondria and Yeast Cells.

Oxidative stress is involved in a wide range of age-related diseases. A critical role has been proposed for mitochondrial oxidative stress in initiating or promoting these pathologies and the potential for mitochondria-targeted antioxidants to fight them, making their search and testing a very urgent task. In this study, the mitochondria-targeted antioxidants SkQ1, SkQ3 and MitoQ were examined as they affected isolated rat liver mitochondria and yeast cells, comparing SkQ3 with clinically tested SkQ1 and MitoQ. At low concentrations, all three substances stimulated the oxidation of respiratory substrates in state 4 respiration (no ADP addition); at higher concentrations, they inhibited the ADP-triggered state 3 respiration and the uncoupled state, depolarized the inner mitochondrial membrane, contributed to the opening of the mPTP (mitochondrial permeability transition pore), did not specifically affect ATP synthase, and had a pronounced antioxidant effect. SkQ3 was the most active antioxidant, not possessing, unlike SkQ1 or MitoQ, prooxidant activity with increasing concentrations. In yeast cells, all three substances reduced prooxidant-induced intracellular oxidative stress and cell death and prevented and reversed mitochondrial fragmentation, with SkQ3 being the most efficient. These data allow us to consider SkQ3 as a promising potential therapeutic agent to mitigate pathologies associated with oxidative stress.

Altered Mitochondrial Morphology and Bioenergetics in a New Yeast Model Expressing Aß42.

Alzheimer's disease (AD) is an incurable, age-related neurological disorder, the most common form of dementia. Considering that AD is a multifactorial complex disease, simplified experimental models are required for its analysis. For this purpose, genetically modified Yarrowia lipolytica yeast strains expressing Aß42 (the main biomarker of AD), eGFP-Aß42, Aß40, and eGFP-Aß40 were constructed and examined. In contrast to the cells expressing eGFP and eGFP-Aß40, retaining "normal" mitochondrial reticulum, eGFP-Aß42 cells possessed a disturbed mitochondrial reticulum with fragmented mitochondria; this was partially restored by preincubation with a mitochondria-targeted antioxidant SkQThy. Aß42 expression also elevated ROS production and cell death; low concentrations of SkQThy mitigated these effects. Aß42 expression caused mitochondrial dysfunction as inferred from a loose coupling of respiration and phosphorylation, the decreased level of ATP production, and the enhanced rate of hydrogen peroxide formation. Therefore, we have obtained the same results described for other AD models. Based on an analysis of these and earlier data, we suggest that the mitochondrial fragmentation might be a biomarker of the earliest preclinical stage of AD with an effective therapy based on mitochondria- targeted antioxidants. The simple yeast model constructed can be a useful platform for the rapid screening of such compounds.

Effect of Tau Protein on Mitochondrial Functions.

Alzheimer's disease is the most common age-related progressive neurodegenerative disorder of brain cortex and hippocampus leading to cognitive impairment. Accumulation of extracellular amyloid plaques and intraneuronal neurofibrillary tangles are believed to be the main hallmarks of the disease. Origin of Alzheimer's disease is not totally clear, multiple initiator factors are likely to exist. Intracellular impacts of Alzheimer's disease include mitochondrial dysfunction, oxidative stress, ER-stress, disruption of autophagy, severe metabolic challenges leading to massive neuronal apoptosis. Mitochondria are the key players in all these processes. This formed the basis for the so-called mitochondrial cascade hypothesis. This review provides current data on the molecular mechanisms of the development of Alzheimer's disease associated with mitochondria. Special attention was paid to the interaction between Tau protein and mitochondria, as well as to the promising therapeutic approaches aimed at preventing development of neurodegeneration.

The First Yarrowia lipolytica Yeast Models Expressing Hepatitis B Virus X Protein: Changes in Mitochondrial Morphology and Functions.

Chronic hepatitis B virus infection is the dominant cause of hepatocellular carcinoma, the main cause of cancer death. HBx protein, a multifunctional protein, is essential for pathogenesis development; however, the underlying mechanisms are not fully understood. The complexity of the system itself, and the intricate interplay of many factors make it difficult to advance in understanding the mechanisms underlying these processes. The most obvious solution is to use simpler systems by reducing the number of interacting factors. Yeast cells are particularly suitable for studying the relationships between oxidative stress, mitochondrial dynamics (mitochondrial fusion and fragmentation), and mitochondrial dysfunction involved in HBx-mediated pathogenesis. For the first time, genetically modified yeast, Y. lipolytica, was created, expressing the hepatitis B virus core protein HBx, as well as a variant fused with eGFP at the C-end. It was found that cells expressing HBx experienced stronger oxidative stress than the control cells. Oxidative stress was alleviated by preincubation with the mitochondria-targeted antioxidant SkQThy. Consistent with these data, in contrast to the control cells (pZ-0) containing numerous mitochondrial forming a mitochondrial reticulum, in cells expressing HBx protein, mitochondria were fragmented, and preincubation with SkQThy partially restored the mitochondrial reticulum. Expression of HBx had a significant influence on the bioenergetic function of mitochondria, making them loosely coupled with decreased respiratory rate and reduced ATP formation. In sum, the first highly promising yeast model for studying the impact of HBx on bioenergy, redox-state, and dynamics of mitochondria in the cell and cross-talk between these parameters was offered. This fairly simple model can be used as a platform for rapid screening of potential therapeutic agents, mitigating the harmful effects of HBx.

Mitochondria-targeted 1,4-naphthoquinone (SkQN) is a powerful prooxidant and cytotoxic agent.

An increase in the production of reactive oxygen species (ROS) in mitochondria due to targeted delivery of redox active compounds may be useful in studies of modulation of cell functions by mitochondrial ROS. Recently, the mitochondria-targeted derivative of menadione (MitoK3) was synthesized. However, MitoK3 did not induce mitochondrial ROS production and lipid peroxidation while exerting significant cytotoxic action. Here we synthesized 1,4-naphthoquinone conjugated with alkyltriphenylphosphonium (SkQN) as a prototype of mitochondria-targeted prooxidant, and its redox properties, interactions with isolated mitochondria, yeast cells and various human cell lines were investigated. According to electrochemical measurements, SkQN was more active redox agent and, due to the absence of methyl group in the naphthoquinone ring, more reactive as electrophile than MitoK3. SkQN (but not MitoK3) stimulated hydrogen peroxide production in isolated mitochondria. At low concentrations, SkQN stimulated state 4 respiration in mitochondria, decreased membrane potential, and blocked ATP synthesis, being more efficient uncoupler of oxidative phosphorylation than MitoK3. In yeast cells, SkQN decreased cell viability and induced oxidative stress and mitochondrial fragmentation. SkQN killed various tumor cells much more efficiently than MitoK3. Since many tumors are characterized by increased oxidative stress, the use of new mitochondria-targeted prooxidants may be a promising strategy for anticancer therapy.

Mitochondrial Dysfunctions May Be One of the Major Causative Factors Underlying Detrimental Effects of Benzalkonium Chloride.

Benzalkonium chloride (BAC) is currently the most commonly used antimicrobial preservative in ophthalmic solutions, nasal sprays, and cosmetics. However, a large number of clinical and experimental investigations showed that the topical administration of BAC-containing eye drops could cause a variety of ocular surface changes, from ocular discomfort to potential risk for future glaucoma surgery. BAC-containing albuterol may increase the risk of albuterol-related systemic adverse effects. BAC, commonly present in personal care products, in cosmetic products can induce irritation and dose-dependent changes in the cell morphology. The cationic nature of BAC (it is a quaternary ammonium) suggests that one of the major targets of BAC in the cell may be mitochondria, the only intracellular compartment charged negatively. However, the influence of BAC on mitochondria has not been clearly understood. Here, the effects of BAC on energy parameters of rat liver mitochondria as well as on yeast cells were examined. BAC, being a "weaker" uncoupler, potently inhibited respiration in state 3, diminished the mitochondrial membrane potential, caused opening of the Ca2+/Pi-dependent pore, blocked ATP synthesis, and promoted H2O2 production by mitochondria. BAC triggered oxidative stress and mitochondrial fragmentation in yeast cells. BAC-induced oxidative stress in mitochondria and yeast cells was almost totally prevented by the mitochondria-targeted antioxidant SkQ1; the protective effect of SkQ1 on mitochondrial fragmentation was only partial. Collectively, these data showed that BAC acts adversely on cell bioenergetics (especially on ATP synthesis) and mitochondrial dynamics and that its prooxidant effect can be partially prevented by the mitochondria-targeted antioxidant SkQ1.