Mutant SPART causes defects in mitochondrial protein import and bioenergetics reversed by Coenzyme Q.

Pathogenic variants in SPART cause Troyer syndrome, characterized by lower extremity spasticity and weakness, short stature and cognitive impairment, and a severe mitochondrial impairment. Herein, we report the identification of a role of Spartin in nuclear-encoded mitochondrial proteins. SPART biallelic missense variants were detected in a 5-year-old boy with short stature, developmental delay and muscle weakness with impaired walking distance. Patient-derived fibroblasts showed an altered mitochondrial network, decreased mitochondrial respiration, increased mitochondrial reactive oxygen species and altered Ca2+ versus control cells. We investigated the mitochondrial import of nuclear-encoded proteins in these fibroblasts and in another cell model carrying a SPART loss-of-function mutation. In both cell models the mitochondrial import was impaired, leading to a significant decrease in different proteins, including two key enzymes involved in CoQ10 (CoQ) synthesis, COQ7 and COQ9, with a severe reduction in CoQ content, versus control cells. CoQ supplementation restored cellular ATP levels to the same extent shown by the re-expression of wild-type SPART, suggesting CoQ treatment as a promising therapeutic approach for patients carrying mutations in SPART.

Sustainable Drug Discovery of Multi-Target-Directed Ligands for Alzheimer's Disease.

The multifactorial nature of Alzheimer's disease (AD) is a reason for the lack of effective drugs as well as a basis for the development of "multi-target-directed ligands" (MTDLs). As cases increase in developing countries, there is a need of new drugs that are not only effective but also accessible. With this motivation, we report the first sustainable MTDLs, derived from cashew nutshell liquid (CNSL), an inexpensive food waste with anti-inflammatory properties. We applied a framework combination of functionalized CNSL components and well-established acetylcholinesterase (AChE)/butyrylcholinesterase (BChE) tacrine templates. MTDLs were selected based on hepatic, neuronal, and microglial cell toxicity. Enzymatic studies disclosed potent and selective AChE/BChE inhibitors (5, 6, and 12), with subnanomolar activities. The X-ray crystal structure of 5 complexed with BChE allowed rationalizing the observed activity (0.0352 nM). Investigation in BV-2 microglial cells revealed antineuroinflammatory and neuroprotective activities for 5 and 6 (already at 0.01 µM), confirming the design rationale.

Root Extracts of Two Cultivars of Paeonia Species: Lipid Composition and Biological Effects on Different Cell Lines: Preliminary Results.

The roots of two cultivars of Paeonia, namely Paeonia officinalis "Rubra Plena" and Paeonia "Pink Hawaiian Coral", have been extracted with chloroform. The composition of the lipid fraction, analyzed by GC-MS technique, revealed the absence of paeonol and the presence of phenol, benzoic acid, fatty acid-and some sterol-derivatives. The chloroformic extracts have been tested on normal and several cancer cell lines but showed antiproliferative activity only on the ovarian carcinoma and the osteosarcoma. The biological activity of extracts was investigated mainly by confocal microscopy, flow cytometry and quantum phase imaging. The results indicated that the root extracts induced a hyperpolarization of mitochondria and an increase in reactive oxygen species levels, without inducing cell death. These effects are associated to an increased doubling time and a retarded confluence.

Coenzyme Q biosynthesis inhibition induces HIF-1α stabilization and metabolic switch toward glycolysis.

Coenzyme Q10 (CoQ, ubiquinone) is a redox-active lipid endogenously synthesized by the cells. The final stage of CoQ biosynthesis is performed at the mitochondrial level by the 'complex Q', where coq2 is responsible for the prenylation of the benzoquinone ring of the molecule. We report that the competitive coq2 inhibitor 4-nitrobenzoate (4-NB) decreased the cellular CoQ content and caused severe impairment of mitochondrial function in the T67 human glioma cell line. In parallel with the reduction in CoQ biosynthesis, the cholesterol level increased, leading to significant perturbation of the plasma membrane physicochemical properties. We show that 4-NB treatment did not significantly affect the cell viability, because of an adaptive metabolic rewiring toward glycolysis. Hypoxia-inducible factor 1α (HIF-1α) stabilization was detected in 4-NB-treated cells, possibly due to the contribution of both reduction in intracellular oxygen tension and ROS overproduction. Exogenous CoQ supplementation partially recovered cholesterol content, HIF-1α degradation, and ROS production, whereas only weakly improved the bioenergetic impairment induced by the CoQ depletion. Our data provide new insights on the effect of CoQ depletion and contribute to shed light on the pathogenic mechanisms of ubiquinone deficiency syndrome.

Castanea sativa Mill. bark extract cardiovascular effects in a rat model of high-fat diet.

Ellagitannins may have a beneficial impact in cardiovascular diseases. The aim of the study was to evaluate the effect of high-fat diet (HFD) and the efficacy of Castanea sativa Mill. bark extract (ENC) on cardiac and vascular parameters. Rats were fed with regular diet, (RD, n = 15), HFD (n = 15), RD + ENC (20 mg/kg/day by gavage, n = 15), and HFD + ENC (same dose, n = 15) and the effects on body weight, biochemical serum parameters, and inflammatory cytokines determined. Cardiac functional parameters and aorta contractility were also assessed on isolated atria and aorta. Results showed that ENC reduced weight gain and serum lipids induced by HFD. In in vitro assays, HFD decreased the contraction force of left atrium, increased right atrium chronotropy, and decreased aorta K+ -induced contraction; ENC induced transient positive inotropic and negative chronotropic effects on isolated atria from RD and HFD rats and a spasmolytic effect on aorta. In ex vivo experiments, ENC reverted inotropic and chronotropic changes induced by HFD and enhanced Nifedipine effect more on aorta than on heart. In conclusion, ENC restores metabolic dysfunction and cardiac cholinergic muscarinic receptor function, and exerts spasmolytic effect on aorta in HFD rats, highlighting its potential as nutraceutical tool in obesity.

Discovery of Sustainable Drugs for Neglected Tropical Diseases: Cashew Nut Shell Liquid (CNSL)-Based Hybrids Target Mitochondrial Function and ATP Production in Trypanosoma brucei.

In the search for effective and sustainable drugs for human African trypanosomiasis (HAT), we developed hybrid compounds by merging the structural features of quinone 4 (2-phenoxynaphthalene-1,4-dione) with those of phenolic constituents from cashew nut shell liquid (CNSL). CNSL is a waste product from cashew nut processing factories, with great potential as a source of drug precursors. The synthesized compounds were tested against Trypanosoma brucei brucei, including three multidrug-resistant strains, T. congolense, and a human cell line. The most potent activity was found against T. b. brucei, the causative agent of HAT. Shorter-chain derivatives 20 (2-(3-(8-hydroxyoctyl)phenoxy)-5-methoxynaphthalene-1,4-dione) and 22 (5-hydroxy-2-(3-(8-hydroxyoctyl)phenoxy)naphthalene-1,4-dione) were more active than 4, displaying rapid micromolar trypanocidal activity, and no human cytotoxicity. Preliminary studies probing their mode of action on trypanosomes showed ATP depletion, followed by mitochondrial membrane depolarization and mitochondrion ultrastructural damage. This was accompanied by reactive oxygen species production. We envisage that such compounds, obtained from a renewable and inexpensive material, might be promising bio-based sustainable hits for anti-trypanosomatid drug discovery.

Liver and intestinal protective effects of Castanea sativa Mill. bark extract in high-fat diet rats.

The effects of Castanea sativa Mill. have been studied in high fat diet (HFD) overweight rats. Natural Extract of Chestnut bark (Castanea sativa Mill.) (ENC®), rich in ellagitannins, has been studied in 120 male Sprague-Dawley rats, divided in four groups. Two groups were controls: regular (RD) and HDF diet. Two groups received ENC® (20 mg/kg/day): RD + ENC® and HFD + ENC®. At baseline and at 7, 14 and 21 days, weight gain, serum lipids, plasma cytokines, liver histology, microsomial enzymes and oxidation, intestinal oxidative stress and contractility were studied. HFD increased body weight, increased pro-inflammatory cytokines, induced hepatocytes microvescicular steatosis, altered microsomial, increased liver and intestinal oxidative stress, deranged intestinal contractility. In HFD-fed rats, ENC® exerted antiadipose and antioxidative activities and normalized intestinal contractility, suggesting a potential approach to overweight management associated diseases.

Role of mitochondrial complex I and protective effect of CoQ10 supplementation in propofol induced cytotoxicity.

Propofol (2,6-diisopropylphenol) is an anaesthetic widely used for human sedation. Due to its intrinsic antioxidant properties, rapid induction of anaesthesia and fast recovery, it is employed in paediatric anaesthesia and in the intensive care of premature infants. Recent studies have pointed out that exposure to anaesthesia in the early stage of life might be responsible of long-lasting cognitive impairment. The apoptotic neurodegeneration induced by general anaesthetics (GA) involves mitochondrial impairment due to the inhibition of the OXPHOS machinery. In the present work, we aim to identify the main mitochondrial respiratory chain target of propofol toxicity and to evaluate the possible protective effect of CoQ10 supplementation. The propofol effect on the mitochondrial functionality was assayed in isolated mitochondria and in two cell lines (HeLa and T67) by measuring oxygen consumption rate. The protective effect of CoQ10 was assessed by measuring cells viability, NADH-oxidase activity and ATP/ADP ratio in cells treated with propofol. Our results show that propofol reduces cellular oxygen consumption rate acting mainly on mitochondrial Complex I. The kinetic analysis of Complex I inhibition indicates that propofol interferes with the Q module acting as a non-competitive inhibitor with higher affinity for the free form of the enzyme. Cells supplemented with CoQ10 are more resistant to propofol toxicity. Propofol exposure induces cellular damages due to mitochondrial impairment. The site of propofol inhibition on Complex I is the Q module. CoQ10 supplementation protects cells against the loss of energy suggesting its possible therapeutic role to minimizing the detrimental effects of general anaesthesia.

Multitarget drug design strategy: quinone-tacrine hybrids designed to block amyloid-ß aggregation and to exert anticholinesterase and antioxidant effects.

We report the identification of multitarget anti-Alzheimer compounds designed by combining a naphthoquinone function and a tacrine fragment. In vitro, 15 compounds displayed excellent acetylcholinesterase (AChE) inhibitory potencies and interesting capabilities to block amyloid-ß (Aß) aggregation. The X-ray analysis of one of those compounds in complex with AChE allowed rationalizing the outstanding activity data (IC50 = 0.72 nM). Two of the compounds showed negligible toxicity in immortalized mouse cortical neurons Neuro2A and primary rat cerebellar granule neurons. However, only one of them was less hepatotoxic than tacrine in HepG2 cells. In T67 cells, both compounds showed antioxidant activity, following NQO1 induction. Furthermore, in Neuro2A, they were able to completely revert the decrease in viability induced by Aß. Importantly, they crossed the blood-brain barrier, as demonstrated in ex vivo experiments with rats. When ex vivo results were combined with in vitro studies, these two compounds emerged to be promising multitarget lead candidates worthy of further pursuit.

Noise-induced hearing loss (NIHL) as a target of oxidative stress-mediated damage: cochlear and cortical responses after an increase in antioxidant defense.

This study addresses the relationship between cochlear oxidative damage and auditory cortical injury in a rat model of repeated noise exposure. To test the effect of increased antioxidant defenses, a water-soluble coenzyme Q10 analog (Qter) was used. We analyzed auditory function, cochlear oxidative stress, morphological alterations in auditory cortices and cochlear structures, and levels of coenzymes Q9 and Q10 (CoQ9 and CoQ10, respectively) as indicators of endogenous antioxidant capability. We report three main results. First, hearing loss and damage in hair cells and spiral ganglion was determined by noise-induced oxidative stress. Second, the acoustic trauma altered dendritic morphology and decreased spine number of II-III and V-VI layer pyramidal neurons of auditory cortices. Third, the systemic administration of the water-soluble CoQ10 analog reduced oxidative-induced cochlear damage, hearing loss, and cortical dendritic injury. Furthermore, cochlear levels of CoQ9 and CoQ10 content increased. These findings indicate that antioxidant treatment restores auditory cortical neuronal morphology and hearing function by reducing the noise-induced redox imbalance in the cochlea and the deafferentation effects upstream the acoustic pathway.

A water soluble CoQ10 formulation improves intracellular distribution and promotes mitochondrial respiration in cultured cells.

BACKGROUND: Mitochondria are both the cellular powerhouse and the major source of reactive oxygen species. Coenzyme Q(10) plays a key role in mitochondrial energy production and is recognized as a powerful antioxidant. For these reasons it can be argued that higher mitochondrial ubiquinone levels may enhance the energy state and protect from oxidative stress. Despite the large number of clinical studies on the effect of CoQ(10) supplementation, there are very few experimental data about the mitochondrial ubiquinone content and the cellular bioenergetic state after supplementation. Controversial clinical and in vitro results are mainly due to the high hydrophobicity of this compound, which reduces its bioavailability. PRINCIPAL FINDINGS: We measured the cellular and mitochondrial ubiquinone content in two cell lines (T67 and H9c2) after supplementation with a hydrophilic CoQ(10) formulation (Qter®) and native CoQ(10). Our results show that the water soluble formulation is more efficient in increasing ubiquinone levels. We have evaluated the bioenergetics effect of ubiquinone treatment, demonstrating that intracellular CoQ(10) content after Qter supplementation positively correlates with an improved mitochondrial functionality (increased oxygen consumption rate, transmembrane potential, ATP synthesis) and resistance to oxidative stress. CONCLUSIONS: The improved cellular energy metabolism related to increased CoQ(10) content represents a strong rationale for the clinical use of coenzyme Q(10) and highlights the biological effects of Qter®, that make it the eligible CoQ(10) formulation for the ubiquinone supplementation.

Alpha-bisabolol: unexpected plant-derived weapon in the struggle against tumour survival?

Despite enormous scientific and economic effort tumour still is one of the most terrible pathologies among human population all over the world. Products derived from the plant kingdom have often offered an opportunity to counteract or alleviate this illness. Here, we summarize the short story of the study of an extraordinary effect of one plant compound towards transformed cells derived from highly malignant tumours. Alpha-bisabolol, a sesquiterpene widely present in plants, selectively kills transformed cells by apoptosis without affecting the viability of normal cells. One of its intracellular targets seems to be situated on mitochondria and is possibly identified as the permeability transition pore, as judged from rapid mitochondrial membrane potential dissipation induced by alpha-bisabolol and the failure to kill cells in the presence of cyclosporine A. Preferential adsorption of alpha-bisabolol into lipid rafts, rich in tumour cells, may explain the selective action of this compounds towards tumour cells. Furthermore, Surface Plasmon Resonance analysis indicates that alpha-bisabolol directly interacts with Bid protein, a member of the Bcl2 family deeply involved in apoptosis, suggesting a possibility that Bid, or similar protein(s), may be involved in a putative intracellular transport system of alpha-bisabolol from plasma membrane to mitochondria. Experiments with animals indicate that alpha-bisabolol is not toxic and is accumulated, through blood flow, in every tissues examined. Further animal studies to test its effect are currently under way.