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Ferroptosis is a cell death mechanism based on extensive cellular membrane peroxidation, implicated in neurodegenerative and other diseases. The essential oil component γ-terpinene, a natural monoterpene with a unique highly oxidizable pro-aromatic 1,4-cyclohexadiene skeleton, inhibits peroxidation of polyunsaturated lipid in model heterogeneous systems (micelles and liposomes). Upon H-atom abstraction, an unstable γ-terpinene-derived peroxyl radical is formed, that aromatizes to p-cymene generating HOO• radicals. As HOO• are small and hydrophilic radicals, they quickly diffuse outside the lipid core, blocking the radical chain propagation of polyunsaturated lipids. This unprecedented antioxidant "slingshot" mechanism explains why γ-terpinene shows a protective activity against ferroptosis, being effective at submicromolar concentrations in human neuroblastoma (SH-SY5Y) cells., (© 2024 Wiley‐VCH GmbH.)

Background & Aims: We recently identified a recessive syndrome due to DNA ligase 3 (LIG3) mutations in patients with chronic intestinal pseudo-obstruction, leukoencephalopathy, and neurogenic bladder. LIG3 mutations affect mitochondrial DNA maintenance, leading to defective energy production. We aimed at identifying altered molecular pathways and developing possible targeted treatments to revert/ameliorate the cellular energy impairment., Methods: Whole transcriptome analysis was performed on patient-derived fibroblasts total RNA and controls. Mitochondrial function, mitophagy, and l-glutamine supplementation effects were analyzed by live cell analysis, immunostaining, and Western blot. Patients were treated with Dipeptiven (Fresenius-Kabi) according to standard protocols. Patients' symptoms were analyzed by the Gastrointestinal Symptom Rating Scale questionnaire., Results: We identified deregulated transcripts in mutant fibroblasts vs controls, including overexpression of genes involved in extracellular matrix development and remodeling and mitochondrial functions. Gut biopsy specimens of LIG3-mutant patients documented collagen and elastic fiber accumulation. Mutant fibroblasts exhibited impaired mitochondrial mitophagy indicative of dysfunctional turnover and altered Ca 2+ homeostasis. Supplementation with l-glutamine (6 mmol/L), previously shown to increase mitochondrial DNA-defective cell survival, improved growth rate and adenosine 5'-triphosphate production in LIG3-mutant fibroblasts. These data led us to provide parenterally a dipeptide containing l-glutamine to 3 siblings carrying biallelic LIG3 mutations. Compared with baseline, gastrointestinal and extra-gastrointestinal symptoms significantly improved after 8 months of treatment., Conclusions: LIG3 deficiency leads to mitochondrial dysfunction. High levels l-glutamine supplementation were beneficial in LIG3-mutant cells and improved symptom severity without noticeable adverse effects. Our results provide a proof of concept to design ad hoc clinical trials with l-glutamine in LIG3-mutant patients., (Copyright © 2024 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The search for new drugs fulfilling One Health and Green Chemistry requirements is an urgent call. Here, for the first time, we envisaged developing SAHA analogues by starting from the cashew nutshell liquid (CNSL) agro-industrial waste and employing a metathesis approach. This sustainable combination (comprising principles #7 and #9) allowed a straightforward synthesis of compounds 13 - 20 . All of them were found to not be toxic on HepG2, IMR-32, and L929 cell lines. Then, their potential against major human and animal vector-borne parasitic diseases (VBPDs) was assessed. Compound 13 emerged as a green hit against the trypomastigote forms of T. b. brucei . In silico studies showed that the T. b. brucei HDAC (TbDAC) catalytic pocket could be occupied with a similar binding mode by both SAHA and 13 , providing a putative explanation for its antiparasitic mechanism of action ( 13 , EC 50 = 0.7 ± 0.2 μM)., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

Lewy body dementia (LBD) represents the second most common neurodegenerative dementia but is a quite underexplored therapeutic area. Nepflamapimod (1) is a brain-penetrant selective inhibitor of the alpha isoform of the mitogen-activated serine/threonine protein kinase (MAPK) p38α, recently repurposed for LBD due to its remarkable antineuroinflammatory properties. Neuroprotective propargylamines are another class of molecules with a therapeutical potential against LBD. Herein, we sought to combine the antineuroinflammatory core of 1 and the neuroprotective propargylamine moiety into a single molecule. Particularly, we inserted a propargylamine moiety in position 4 of the 2,6-dichlorophenyl ring of 1, generating neflamapimod-propargylamine hybrids 3 and 4. These hybrids were evaluated using several cell models, aiming to recapitulate the complexity of LBD pathology through different molecular mechanisms. The N-methyl-N-propargyl derivative 4 showed a nanomolar p38α-MAPK inhibitory activity (IC 50  = 98.7 nM), which is only 2.6-fold lower compared to that of the parent compound 1, while displaying no hepato- and neurotoxicity up to 25 μM concentration. It also retained a similar immunomodulatory profile against the N9 microglial cell line. Gratifyingly, at 5 μM concentration, 4 demonstrated a neuroprotective effect against dexamethasone-induced reactive oxygen species production in neuronal cells that was higher than that of 1., (© 2024 Deutsche Pharmazeutische Gesellschaft.)

Multi-target drug discovery is one of the most active fields in the search for new drugs against Alzheimer's disease (AD). This is because the complexity of AD pathological network might be adequately tackled by multi-target-directed ligands (MTDLs) aimed at modulating simultaneously multiple targets of such a network. In a continuation of our efforts to develop MTDLs for AD, we have been focusing on the molecular hybridization of the acetylcholinesterase inhibitor tacrine with the aim of expanding its anti-AD profile. Herein, we manipulated the structure of a previously developed tacrine-quinone hybrid (1). We designed and synthesized a novel set of MTDLs (2-6) by replacing the naphthoquinone scaffold of 1 with that of 2,5,8-quinolinetrione. The most interesting hybrid 3 inhibited cholinesterase enzymes at nanomolar concentrations. In addition, 3 exerted antioxidant effects in menadione-induced oxidative stress of SH-SY5Y cells. Importantly, 3 also showed low hepatotoxicity and good anti-amyloid aggregation properties. Remarkably, we uncovered the potential of the quinolinetrione scaffold, as a novel anti-amyloid aggregation and antioxidant motif to be used in further anti-AD MTDL drug discovery endeavors., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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 Ca 2+ 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 .

A vaginal microbiota dominated by certain Lactobacillus species may have a protective effect against Chlamydia trachomatis infection. One of the key antimicrobial compounds produced is lactic acid, which is believed to play a central role in host defense. Lactobacillus strains producing the D(-)-lactic acid isomer are known to exert stronger protection. However, the molecular mechanisms underlying this antimicrobial action are not well understood. The aim of this study was to investigate the role of D(-)-lactic acid isomer in the prevention of C. trachomatis infection in an in vitro HeLa cell model. We selected two strains of lactobacilli belonging to different species: a vaginal isolate of Lactobacillus crispatus that releases both D(-) and L(+) isomers and a strain of Lactobacillus reuteri that produces only the L(+) isomer. Initially, we demonstrated that L. crispatus was significantly more effective than L. reuteri in reducing C. trachomatis infectivity. A different pattern of histone acetylation and lactylation was observed when HeLa cells were pretreated for 24 h with supernatants of Lactobacillus crispatus or L. reuteri , resulting in different transcription of genes such as CCND1, CDKN1A, ITAG5 and HER-1. Similarly, distinct transcription patterns were found in HeLa cells treated with 10 mM D(-)- or L(+)-lactic acid isomers. Our findings suggest that D(-) lactic acid significantly affects two non-exclusive mechanisms involved in C. trachomatis infection: regulation of the cell cycle and expression of EGFR and α5β1-integrin.

Background: Metabolic reprogramming is a well-known marker of cancer, and it represents an early event during hepatocellular carcinoma (HCC) development. The recent approval of several molecular targeted agents has revolutionized the management of advanced HCC patients. Nevertheless, the lack of circulating biomarkers still affects patient stratification to tailored treatments. In this context, there is an urgent need for biomarkers to aid treatment choice and for novel and more effective therapeutic combinations to avoid the development of drug-resistant phenotypes. This study aims to prove the involvement of miR-494 in metabolic reprogramming of HCC, to identify novel miRNA-based therapeutic combinations and to evaluate miR-494 potential as a circulating biomarker., Methods: Bioinformatics analysis identified miR-494 metabolic targets. QPCR analysis of glucose 6-phosphatase catalytic subunit (G6pc) was performed in HCC patients and preclinical models. Functional analysis and metabolic assays assessed G6pc targeting and miR-494 involvement in metabolic changes, mitochondrial dysfunction, and ROS production in HCC cells. Live-imaging analysis evaluated the effects of miR-494/G6pc axis in cell growth of HCC cells under stressful conditions. Circulating miR-494 levels were assayed in sorafenib-treated HCC patients and DEN-HCC rats., Results: MiR-494 induced the metabolic shift of HCC cells toward a glycolytic phenotype through G6pc targeting and HIF-1A pathway activation. MiR-494/G6pc axis played an active role in metabolic plasticity of cancer cells, leading to glycogen and lipid droplets accumulation that favored cell survival under harsh environmental conditions. High miR-494 serum levels associated with sorafenib resistance in preclinical models and in a preliminary cohort of HCC patients. An enhanced anticancer effect was observed for treatment combinations between antagomiR-494 and sorafenib or 2-deoxy-glucose in HCC cells., Conclusions: MiR-494/G6pc axis is critical for the metabolic rewiring of cancer cells and associates with poor prognosis. MiR-494 deserves attention as a candidate biomarker of likelihood of response to sorafenib to be tested in future validation studies. MiR-494 represents a promising therapeutic target for combination strategies with sorafenib or metabolic interference molecules for the treatment of HCC patients who are ineligible for immunotherapy., (© 2023. The Author(s).)

CDKL5 (cyclin-dependent kinase-like 5) deficiency disorder (CDD) is a severe neurodevelopmental disease that mostly affects girls, who are heterozygous for mutations in the X-linked CDKL5 gene. Mutations in the CDKL5 gene lead to a lack of CDKL5 protein expression or function and cause numerous clinical features, including early-onset seizures, marked hypotonia, autistic features, gastrointestinal problems, and severe neurodevelopmental impairment. Mouse models of CDD recapitulate several aspects of CDD symptomology, including cognitive impairments, motor deficits, and autistic-like features, and have been useful to dissect the role of CDKL5 in brain development and function. However, our current knowledge of the function of CDKL5 in other organs/tissues besides the brain is still quite limited, reducing the possibility of broad-spectrum interventions. Here, for the first time, we report the presence of cardiac function/structure alterations in heterozygous Cdkl5 +/- female mice. We found a prolonged QT interval (corrected for the heart rate, QTc) and increased heart rate in Cdkl5 +/- mice. These changes correlate with a marked decrease in parasympathetic activity to the heart and in the expression of the Scn5a and Hcn4 voltage-gated channels. Interestingly, Cdkl5 +/- hearts showed increased fibrosis, altered gap junction organization and connexin-43 expression, mitochondrial dysfunction, and increased ROS production. Together, these findings not only contribute to our understanding of the role of CDKL5 in heart structure/function but also document a novel preclinical phenotype for future therapeutic investigation.

Astaxanthin is a red orange xanthophyll carotenoid produced mainly by microalgae but which can also be chemically synthesized. As demonstrated by several studies, this lipophilic molecule is endowed with potent antioxidant properties and is able to modulate biological functions. Unlike synthetic astaxanthin, natural astaxanthin (NAst) is considered safe for human nutrition, and its production is considered eco-friendly. The antioxidant activity of astaxanthin depends on its bioavailability, which, in turn, is related to its hydrophobicity. In this study, we analyzed the water-solubility of NAst and assessed its protective effect against oxidative stress by means of different approaches using a neuroblastoma cell model. Moreover, due to its highly lipophilic nature, astaxanthin is particularly protective against lipid peroxidation; therefore, the role of NAst in counteracting ferroptosis was investigated. This recently discovered process of programmed cell death is indeed characterized by iron-dependent lipid peroxidation and seems to be linked to the onset and development of oxidative-stress-related diseases. The promising results of this study, together with the "green sources" from which astaxanthin could derive, suggest a potential role for NAst in the prevention and co-treatment of chronic degenerative diseases by means of a sustainable approach.

A series of naphthoquinones, namely, 1,4-naphthoquinone, menadione, plumbagin, juglone, naphthazarin, and lawsone, were reacted with N -acetyl- L -cysteine, and except for lawsone, which did not react, the related adducts were obtained. After the tuning of the solvent and reaction conditions, the reaction products were isolated as almost pure from the complex reaction mixture via simple filtration and were fully characterized. Therefore, the aim of this work was to evaluate whether the antitumor activity of new compounds of 1,4-naphthoquinone derivatives leads to an increase in ROS in tumor cell lines of cervical carcinoma (HeLa), neuroblastoma (SH-SY5Y), and osteosarcoma (SaOS2, U2OS) and in normal dermal fibroblast (HDFa). The MTT assay was used to assay cell viability, the DCF-DA fluorescent probe to evaluate ROS induction, and cell-cycle analysis to measure the antiproliferative effect. Compounds 8 , 9 , and 12 showed a certain degree of cytotoxicity towards all the malignant cell lines tested, while compound 11 showed biological activity at higher IC 50 values. Compounds 8 and 11 induced increases in ROS generation after 1 h of exposure, while after 48 h of treatment, only 8 induced an increase in ROS formation in HeLa cells. Cell-cycle analysis showed that compound 8 caused an increase in the number of G0/G1-phase cells in the HeLa experiment, while for the U2OS and SH-SY5Y cell lines, it led to an accumulation of S-phase cells. Therefore, these novel 1,4-naphthoquinone derivatives may be useful as antitumoral agents in the treatment of different cancers.

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., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Coenzyme Q10 (CoQ10) is a lipid-soluble molecule with a dual role: it transfers electrons in the mitochondrial transport chain by promoting the transmembrane potential exploited by the ATPase to synthesize ATP and, in its reduced form, is a membrane antioxidant. Since the high CoQ10 hydrophobicity hinders its bioavailability, several formulations have been developed to facilitate its cellular uptake. In this work, we studied the bioenergetic and antioxidant effects in I407 and H9c2 cells of a CoQ10 phytosome formulation (UBIQSOME ® , UBQ). We investigated the cellular and mitochondrial content of CoQ10 and its redox state after incubation with UBQ. We studied different bioenergetic parameters, such as oxygen consumption, ATP content and mitochondrial potential. Moreover, we evaluated the effects of CoQ10 incubation on oxidative stress, membrane lipid peroxidation and ferroptosis and highlighted the connection between the intracellular concentration of CoQ10 and its antioxidant potency. Finally, we focused on the cellular mechanism that regulates UBQ internalization. We showed that the cell lines used in this work share the same uptake mechanism for UBQ, although the intestinal cell line was less efficient. Given the limitations of an in vitro model, the latter result supports that intestinal absorption is a critical step for the oral administration of Coenzyme Q10 formulations.

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., (© 2020 John Wiley & Sons, Ltd.)

Coenzyme Q 10 (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., (© 2020 Federation of European Biochemical Societies.)

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.

Thanks to the widespread use and safety profile of donepezil (1) in the treatment of Alzheimer's disease (AD), one of the most widely adopted multi-target-directed ligand (MTDL) design strategies is to modify its molecular structure by linking a second fragment carrying an additional AD-relevant biological property. Herein, supported by a proposed combination therapy of 1 and the quinone drug idebenone, we rationally designed novel 1-based MTDLs targeting Aβ and oxidative pathways. By exploiting a bioisosteric replacement of the indanone core of 1 with a 1,4-naphthoquinone, we ended up with a series of highly merged derivatives, in principle devoid of the "physicochemical challenge" typical of large hybrid-based MTDLs. A preliminary investigation of their multi-target profile identified 9, which showed a potent and selective butyrylcholinesterase inhibitory activity, together with antioxidant and antiaggregating properties. In addition, it displayed a promising drug-like profile., (© 2020 Wiley-VCH GmbH.)