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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, muscle weakness with impaired walking distance. Patient-derived fibroblasts showed an altered mitochondrial network, decreased mitochondrial respiration and 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.

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Primary CoQ10 deficiency diseases encompass a heterogeneous spectrum of clinical phenotypes. Among these, defect or mutation on COQ2 gene, encoding a para-hydroxybenzoate polyprenyl transferase, have been associated with different diseases. Understanding the functional and metabolic impact of COQ2 mutation and the consequent CoQ10 deficiency is still a matter of debate. To date the aetiology of the neurological phenotypes correlated to CoQ10 deficiency does not present a clear genotype-phenotype association. In addition to the metabolic alterations due to Coenzyme Q depletion, the impairment of mitochondrial function, associated with the reduced CoQ level, could play a significant role in the metabolic flexibility of cancer. This study aimed to characterize the effect of varying degrees of CoQ10 deficiency and investigate the multifaceted aspect of CoQ10 depletion and its impact on cell metabolism. To induced CoQ10 depletion, different cell models were used, employing a chemical and genome editing approach. In T67 and MCF-7 CoQ10 depletion was achieved by a competitive inhibitor of the enzyme, 4-nitrobenzoate (4-NB), whereas in SH-SY5Y the COQ2 gene was edited via CRISPR-Cas9 cutting edge technology.

α-Bisabolol (BSB) is a plant-derived sesquiterpene alcohol able to trigger regulated cell death in transformed cells, while deprived of the general toxicity in several mouse models. Here, we investigated the involvement of lysosomal and mitochondrial compartments in the cytotoxic effects of BSB, with a specific focus on the BH3-only activator protein BID. We found that BSB particularly accumulated in cancer cell lines, displaying a higher amount of lipid rafts as compared to normal blood cells. By means of western blotting and microscopy techniques, we documented rapid BSB-induced BID translocation to lysosomes and mitochondria, both of them becoming dysfunctional. Lysosomal membranes were permeabilized, thus blocking the cytoprotective autophagic flux and provoking cathepsin B leakage into the cytosol. Multiple flow cytometry-based experiments demonstrated the loss of mitochondrial membrane potential due to pore formation across the lipid bilayer. These parallel events converged on neoplastic cell death, an outcome significantly prevented by BID knockdown. Therefore, BSB promoted BID redistribution to the cell death executioner organelles, which in turn activated anti-autophagic and proapoptotic mechanisms. This is an example of how xenohormesis can be exploited to modulate basic cellular programs in cancer.

Abnormal gut motility is a feature of several mitochondrial encephalomyopathies, and mutations in genes such as TYMP and POLG, have been linked to these rare diseases. The human genome encodes three DNA ligases, of which only one, ligase III (LIG3), has a mitochondrial splice variant and is crucial for mitochondrial health. We investigated the effect of reduced LIG3 activity and resulting mitochondrial dysfunction in seven patients from three independent families, who showed the common occurrence of gut dysmotility and neurological manifestations reminiscent of mitochondrial neurogastrointestinal encephalomyopathy. DNA from these patients was subjected to whole exome sequencing. In all patients, compound heterozygous variants in a new disease gene, LIG3, were identified. All variants were predicted to have a damaging effect on the protein. The LIG3 gene encodes the only mitochondrial DNA (mtDNA) ligase and therefore plays a pivotal role in mtDNA repair and replication. In vitro assays in patient-derived cells showed a decrease in LIG3 protein levels and ligase activity. We demonstrated that the LIG3 gene defects affect mtDNA maintenance, leading to mtDNA depletion without the accumulation of multiple deletions as observed in other mitochondrial disorders. This mitochondrial dysfunction is likely to cause the phenotypes observed in these patients. The most prominent and consistent clinical signs were severe gut dysmotility and neurological abnormalities, including leukoencephalopathy, epilepsy, migraine, stroke-like episodes, and neurogenic bladder. A decrease in the number of myenteric neurons, and increased fibrosis and elastin levels were the most prominent changes in the gut. Cytochrome c oxidase (COX) deficient fibres in skeletal muscle were also observed. Disruption of lig3 in zebrafish reproduced the brain alterations and impaired gut transit in vivo. In conclusion, we identified variants in the LIG3 gene that result in a mitochondrial disease characterized by predominant gut dysmotility, encephalopathy, and neuromuscular abnormalities. Bonora et al. identify a new mitochondrial recessive disorder caused by biallelic variants in the LIG3 gene encoding DNA ligase III, which is responsible for mitochondrial DNA repair. Clinical signs include gut dysmotility and neurological features such as leucoencephalopathy, epilepsy and stroke-like episodes.

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.

International audience; Facing the complexity of Alzheimer's disease (AD), it is now currently admitted that a therapeutic pleiotropic intervention is needed to alter its progression. Among the major hallmarks of the disease, the amyloid pathology and the oxidative stress are closely related. We propose in this study to develop original Multi-Target Directed Ligands (MTDL) able to impact at the same time Aβ protein accumulation and toxicity of Reactive Oxygen Species (ROS) in neuronal cells. Such MTDL were obtained by linking on a central piperidine two scaffolds of interest: a typical aminochlorobenzophenone present in numerous 5-HT4R agonists, and diverse antioxidant chemotypes. Interestingly, the most active compound 9g possesses a Ki of 12.7 nM towards 5-HT4R and an antioxidant activity in vitro and in cellulo.

Coenzyme Q biosynthesis is a complex process occurring in both the cytosol and the mitochondrial matrix. The cytosolic pathway is shared with cholesterol biosynthesis through the mevalonate pathway, while the biosynthesis of the benzoquinone ring starts from p-hydroxybenzoic acid (4-HB), derived from tyrosine. A crucial step in the CoQ assembly is the insertion of the isoprenyl chain in the aromatic ring of 4-HB catalysed by 4-para-hydroxybenzoate: polyprenyl transferase (Coq2). We used 4-nitrobenzoic acid (4-NB) as a competitive inhibitor of Coq2 to induce CoQ depletion [1]. We found that CoQ depleted cells showed increased cholesterol levels, increased HIF-1α levels and a reduced intracellular oxygen tension in addition to the well-known effects associated with CoQ depletion (low respiratory capacity, low ATP content and high ROS production). The analysis of the chemical-physical state of the cellular membranes showed an increased membrane rigidity that, in our opinion, is responsible for the reduced oxygen uptake. Moreover, we found increased cytosolic NADH levels that contribute to the stabilization of the hypoxic factor HIF-1α, which is responsible for the rearrangement of cellular metabolic status that cannot be completely restored by CoQ10 supplementation. These findings could be relevant for clinical interest suggesting an explanation for the lack of effectiveness of CoQ10 supplementation therapy observed in a number of patients affected by primary CoQ10 deficiency syndrome. Moreover, our data provide new insights on the effect of CoQ10 depletion in cells and sheds light on the mechanisms that underlie CoQ10 deficiency syndrome pathogenesis.