Alzheimer's Disease: Significant Benefit from the Yeast-Based Models.

Alzheimer's disease (AD) is an age-related, multifaceted neurological disorder associated with accumulation of aggregated proteins (amyloid Aß and hyperphosphorylated tau), loss of synapses and neurons, and alterations in microglia. AD was recognized by the World Health Organization as a global public health priority. The pursuit of a better understanding of AD forced researchers to pay attention to well-defined single-celled yeasts. Yeasts, despite obvious limitations in application to neuroscience, show high preservation of basic biological processes with all eukaryotic organisms and offer great advantages over other disease models due to the simplicity, high growth rates on low-cost substrates, relatively simple genetic manipulations, the large knowledge base and data collections, and availability of an unprecedented amount of genomic and proteomic toolboxes and high-throughput screening techniques, inaccessible to higher organisms. Research reviewed above clearly indicates that yeast models, together with other, more simple eukaryotic models including animal models, C. elegans and Drosophila, significantly contributed to understanding Aß and tau biology. These models allowed high throughput screening of factors and drugs that interfere with Aß oligomerization, aggregation and toxicity, and tau hyperphosphorylation. In the future, yeast models will remain relevant, with a focus on creating novel high throughput systems to facilitate the identification of the earliest AD biomarkers among different cellular networks in order to achieve the main goal-to develop new promising therapeutic strategies to treat or prevent the disease.

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.

Propagation of Mitochondria-Derived Reactive Oxygen Species within the Dipodascus magnusii Cells.

Mitochondria are considered to be the main source of reactive oxygen species (ROS) in the cell. It was shown that in cardiac myocytes exposed to excessive oxidative stress, ROS-induced ROS release is triggered. However, cardiac myocytes have a network of densely packed organelles that do not move, which is not typical for the majority of eukaryotic cells. The purpose of this study was to trace the spatiotemporal development (propagation) of prooxidant-induced oxidative stress and its interplay with mitochondrial dynamics. We used Dipodascus magnusii yeast cells as a model, as they have advantages over other models, including a uniquely large size, mitochondria that are easy to visualize and freely moving, an ability to vigorously grow on well-defined low-cost substrates, and high responsibility. It was shown that prooxidant-induced oxidative stress was initiated in mitochondria, far preceding the appearance of generalized oxidative stress in the whole cell. For yeasts, these findings were obtained for the first time. Preincubation of yeast cells with SkQ1, a mitochondria-addressed antioxidant, substantially diminished production of mitochondrial ROS, while only slightly alleviating the generalized oxidative stress. This was expected, but had not yet been shown. Importantly, mitochondrial fragmentation was found to be primarily induced by mitochondrial ROS preceding the generalized oxidative stress development.

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.

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.