Introduction: Alpha lipoic acid (ALA) is a sulphur-containing organic compound, derived from octanoic acid, and an important cofactor for mitochondrial respiratory enzymes. It has strong antioxidant properties that improve mitochondrial function. We investigated if ALA improves mitochondrial dysfunction in a cellular model of Alzheimer’s disease (AD). Methods: SH-SY5Y-APP₆₉₅ cells were used as a model for an early stage of AD. Vector-transfected SH-SY5Y-MOCK cells served as controls. Using these cells, we investigated mitochondrial respiration (OXPHOS), mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) production, and citrate synthase activity (CS) in cells treated with ALA. Cells were treated for 24 h with different concentrations of ALA and with or without the complex I inhibitor rotenone. Results: Incubation with ALA showed a significant increase in ATP levels in both SH-SY5Y-APP₆₉₅ and SH-SY5Y-MOCK cells. MMP levels were elevated in SH-SY5Y-MOCK cells, treatment with rotenone showed a reduction in MMP, which could be partly alleviated after incubation with ALA in SH-SY5Y-MOCK cells. ALA treatment showed significant differences in respiration chain complex activities in SH-SY5Y-MOCK cells. Citrate synthase activity was unaffected. ROS levels were significantly lower in both cell lines treated with ALA. Conclusions: ALA increased the activity of the different complexes of the respiratory chain, and consequently enhanced the MMP, leading to increased ATP levels indicating improved mitochondrial function. ALA only marginally protects from additional rotenone-induced mitochondrial stress.

(1) Background: Mitochondria are the cells’ main source of energy. Mitochondrial dysfunction represents a key hallmark of aging and is linked to the development of Alzheimer’s disease (AD). Maintaining mitochondrial function might contribute to healthy aging and the prevention of AD. The Mediterranean diet, including walnuts, seems to prevent age-related neurodegeneration. Walnuts are a rich source of α-linolenic acid (ALA), an essential n3-fatty acid and the precursor for n3-long-chain polyunsaturated fatty acids (n3-PUFA), which might potentially improve mitochondrial function. (2) Methods: We tested whether a lipophilic walnut extract (WE) affects mitochondrial function and other parameters in human SH-SY5Y cells transfected with the neuronal amyloid precursor protein (APP695). Walnut lipids were extracted using a Soxhlet Extraction System and analyzed using GC/MS and HPLC/FD. Adenosine triphosphate (ATP) concentrations were quantified under basal conditions in cell culture, as well as after rotenone-induced stress. Neurite outgrowth was investigated, as well as membrane integrity, cellular reactive oxygen species, cellular peroxidase activity, and citrate synthase activity. Beta-amyloid (Aβ) was quantified using homogenous time-resolved fluorescence. (3) Results: The main constituents of WE are linoleic acid, oleic acid, α-linolenic acid, and γ- and δ-tocopherol. Basal ATP levels following rotenone treatment, as well as citrate synthase activity, were increased after WE treatment. WE significantly increased cellular reactive oxygen species but lowered peroxidase activity. Membrane integrity was not affected. Furthermore, WE treatment reduced Aβ_1–40 and stimulated neurite growth. (4) Conclusions: WE might increase ATP production after induction of mitochondrial biogenesis. Decreased Aβ_1–40 formation and enhanced ATP levels might enhance neurite growth, making WE a potential agent to enhance neuronal function and to prevent the development of AD. In this sense, WE could be a promising agent for the prevention of AD.

Environmentally hazardous substances such as pesticides are gaining increasing interest in agricultural and nutritional research. This study aims to investigate the impact of these compounds on the healthspan and mitochondrial functions in an invertebrate in vivo model and in vitro in SH-SY5Y neuroblastoma cells, and to investigate the potential of polyphenolic metabolites to compensate for potential impacts. Wild-type nematodes (Caenorhabditis elegans, N2) were treated with pesticides such as pyraclostrobin (Pyr), glyphosate (Gly), or fluopyram (Fluo). The lifespans of the nematodes under heat stress conditions (37 °C) were determined, and the chemotaxis was assayed. Energetic metabolites, including adenosine triphosphate (ATP), lactate, and pyruvate, were analyzed in lysates of nematodes and cells. Genetic expression patterns of several genes associated with lifespan determination and mitochondrial parameters were assessed via qRT-PCR. After incubation with environmentally hazardous substances, nematodes were incubated with a pre-fermented polyphenol mixture (Rechtsregulat^®Bio, RR) or protocatechuic acid (PCA) to determine heat stress resistance. Treatment with Pyr, Glyph and Fluo leads to dose-dependently decreased heat stress resistance, which was significantly improved by RR and PCA. The chemotaxes of the nematodes were not affected by pesticides. ATP levels were not significantly altered by the pesticides, except for Pyr, which increased ATP levels after 48 h leads. The gene expression of healthspan and mitochondria-associated genes were diversely affected by the pesticides, while Pyr led to an overall decrease of mRNA levels. Over time, the treatment of nematodes leads to a recovery of the nematodes on the mitochondrial level but not on stress resistance on gene expression. Fermented extracts of fruits and vegetables and phenolic metabolites such as PCA seem to have the potential to recover the vitality of C. elegans after damage caused by pesticides.

The physiological functions of eukaryotic cells rely on energy mainly provided by mitochondria. Mitochondrial dysfunction is linked to metabolic diseases and aging. Oxidative phosphorylation plays a decisive role, as it is crucial for the maintenance of energetic homeostasis. PBMCs have been identified as a minimally invasive sample to measure mitochondrial function and have been shown to reflect disease conditions. However, measurement of mitochondrial bioenergetic function can be limited by several factors in human samples. Limitations are the amount of samples taken, sampling time, which is often spread over several days, and locations. Cryopreservation of the collected samples can ensure consistent collection and measurement of samples. Care should be taken to ensure that the parameters measured are comparable between cryopreserved and freshly prepared cells. Here, we describe methods for isolating and cryopreserving PBMCs from human blood samples to analyze the bioenergetic function of the mitochondria in these cells. PBMC cryopreserved according to the protocol described here show only minor differences in cell number and viability, adenosine triphosphate levels, and measured respiratory chain activity compared with freshly harvested cells. Only 8-24 mL of human blood is needed for the described preparations, making it possible to collect samples during clinical studies multicentrally and determine their bioenergetics on site.