High-Content C. elegans Screen Identifies Natural Compounds Impacting Mitochondria-Lipid Homeostasis and Promoting Healthspan

The aging process is concurrently shaped by genetic and extrinsic factors. In this work, we screened a small library of natural compounds, many of marine origin, to identify novel possible anti-aging interventions in Caenorhabditis elegans, a powerful model organism for aging studies. To this aim, we exploited a high-content microscopy platform to search for interventions able to induce phenotypes associated with mild mitochondrial stress, which is known to promote animal’s health- and lifespan. Worms were initially exposed to three different concentrations of the drugs in liquid culture, in search of those affecting animal size and expression of mitochondrial stress response genes. This was followed by a validation step with nine compounds on solid media to refine compounds concentration, which led to the identification of four compounds (namely isobavachalcone, manzamine A, kahalalide F and lutein) consistently affecting development, fertility, size and lipid content of the nematodes. Treatment of Drosophila cells with the four hits confirmed their effects on mitochondria activity and lipid content. Out of these four, two were specifically chosen for analysis of age-related parameters, kahalalide F and lutein, which conferred increased resistance to heat and oxidative stress and extended animals’ healthspan. We also found that, out of different mitochondrial stress response genes, only the C. elegans ortholog of the synaptic regulatory proteins neuroligins, nlg-1, was consistently induced by the two compounds and mediated lutein healthspan effects.

Potential Combination Drug Therapy to Prevent Redox Stress and Mitophagy Dysregulation in Retinal Müller Cells under High Glucose Conditions: Implications for Diabetic Retinopathy

Chronic hyperglycemia-induced thioredoxin-interacting protein (TXNIP) expression, associated oxidative/nitrosative stress (ROS/RNS), and mitochondrial dysfunction play critical roles in the etiology of diabetic retinopathy (DR). However, there is no effective drug treatment to prevent or slow down the progression of DR. The purpose of this study is to examine if a combination drug treatment targeting TXNIP and the mitochondria-lysosome pathway prevents high glucose-induced mitochondrial stress and mitophagic flux in retinal Müller glial cells in culture, relevant to DR. We show that diabetes induces TXNIP expression, redox stress, and Müller glia activation (gliosis) in rat retinas when compared to non-diabetic rat retinas. Furthermore, high glucose (HG, 25 mM versus low glucose, LG 5.5 mM) also induces TXNIP expression and mitochondrial stress in a rat retinal Müller cell line, rMC1, in in vitro cultures. Additionally, we develop a mitochondria-targeted mCherry and EGFP probe tagged with two tandem COX8a mitochondrial target sequences (adenovirus-CMV-2×mt8a-CG) to examine mitophagic flux in rMC1. A triple drug combination treatment was applied using TXNIP-IN1 (which inhibits TXNIP interaction with thioredoxin), Mito-Tempo (mitochondrial anti-oxidant), and ML-SA1 (lysosome targeted activator of transient calcium channel MCOLN1/TRPML1 and of transcription factor TFEB) to study the mitochondrial–lysosomal axis dysregulation. We found that HG induces TXNIP expression, redox stress, and mitophagic flux in rMC1 versus LG. Treatment with the triple drug combination prevents mitophagic flux and restores transcription factor TFEB and PGC1α nuclear localization under HG, which is critical for lysosome biosynthesis and mitogenesis, respectively. Our results demonstrate that 2×mt8a-CG is a suitable probe for monitoring mitophagic flux, both in live and fixed cells in in vitro experiments, which may also be applicable to in vivo animal studies, and that the triple drug combination treatment has the potential for preventing retinal injury and disease progression in diabetes.

Oxidative Stress, Mitochondrial Function and Adaptation to Exercise: New Perspectives in Nutrition

Cells have the ability to adapt to stressful environments as a part of their evolution. Physical exercise induces an increase of a demand for energy that must be met by mitochondria as the main (ATP) provider. However, this process leads to the increase of free radicals and the so-called reactive oxygen species (ROS), which are necessary for the maintenance of cell signaling and homeostasis. In addition, mitochondrial biogenesis is influenced by exercise in continuous crosstalk between the mitochondria and the nuclear genome. Excessive workloads may induce severe mitochondrial stress, resulting in oxidative damage. In this regard, the objective of this work was to provide a general overview of the molecular mechanisms involved in mitochondrial adaptation during exercise and to understand if some nutrients such as antioxidants may be implicated in blunt adaptation and/or an impact on the performance of exercise by different means.

The effects of real and simulated microgravity on cellular mitochondrial function

Astronauts returning from space shuttle missions or the International Space Station have been diagnosed with various health problems such as bone demineralization, muscle atrophy, cardiovascular deconditioning, and vestibular and sensory imbalance including visual acuity, altered metabolic and nutritional status, and immune system dysregulation. These health issues are associated with oxidative stress caused by a microgravity environment. Mitochondria are a source of reactive oxygen species (ROS). However, the molecular mechanisms through which mitochondria produce ROS in a microgravity environment remain unclear. Therefore, this review aimed to explore the mechanism through which microgravity induces oxidative damage in mitochondria by evaluating the expression of genes and proteins, as well as relevant metabolic pathways. In general, microgravity-induced ROS reduce mitochondrial volume by mainly affecting the efficiency of the respiratory chain and metabolic pathways. The impaired respiratory chain is thought to generate ROS through premature electron leakage in the electron transport chain. The imbalance between ROS production and antioxidant defense in mitochondria is the main cause of mitochondrial stress and damage, which leads to mitochondrial dysfunction. Moreover, we discuss the effects of antioxidants against oxidative stress caused by the microgravity environment space microgravity in together with simulated microgravity (i.e., spaceflight or ground-based spaceflight analogs: parabolic flight, centrifugal force, drop towers, etc.). Further studies should be taken to explore the effects of microgravity on mitochondrial stress-related diseases, especially for the development of new therapeutic drugs that can help increase the health of astronauts on long space missions.

Vascular Dysfunction in Preeclampsia

Preeclampsia is a life-threatening pregnancy-associated cardiovascular disorder characterized by hypertension and proteinuria at 20 weeks of gestation. Though its exact underlying cause is not precisely defined and likely heterogenous, a plethora of research indicates that in some women with preeclampsia, both maternal and placental vascular dysfunction plays a role in the pathogenesis and can persist into the postpartum period. Potential abnormalities include impaired placentation, incomplete spiral artery remodeling, and endothelial damage, which are further propagated by immune factors, mitochondrial stress, and an imbalance of pro- and antiangiogenic substances. While the field has progressed, current gaps in knowledge include detailed initial molecular mechanisms and effective treatment options. Newfound evidence indicates that vasopressin is an early mediator and biomarker of the disorder, and promising future therapeutic avenues include mitigating mitochondrial dysfunction, excess oxidative stress, and the resulting inflammatory state. In this review, we provide a detailed overview of vascular defects present during preeclampsia and connect well-established notions to newer discoveries at the molecular, cellular, and whole-organism levels.

Disrupting Mitohormesis As a Novel Therapeutic Strategy for Multiple Myeloma (MM) Including Those with High Risk Disease and Proteosome Inhibitor Resistance

Background: Moderate mitochondrial stress induced by multiple mediators but most notably ROS can lead to activation of persistent mito-protective mechanisms termed "Mitohormesis". As a result of massive protein synthesis, malignant plasma cells (PCs) from MM patients (pts) undergo substantial ER stress but in addition high rates of Ig synthesis contributes to overproduction of ROS. We hypothesized that MM cells exploit mitohormesis to maintain ROS in the hometic zone, thereby increasing mitochondrial fitness to avoid apoptosis. We therefore set out to determine if the processes of mitohormesis are activated in MM and whether unmitigated mitochondrial stress can be exploited as a therapeutic strategy in MM. Results: Protective stress mechanisms of mitohormesis include the activation of the mitochondrial UPR (UPR MT),a mitochondrial-to-nuclear signaling pathway mediated by CHOP and ATF5 that upregulates mitochondrial import proteins, chaperones and proteases to maintain mitochondrial proteastasis. We first demonstrated that UPR MT activation occurs with progression from precursor to overt MM. Using a UPR MTgene signature derived from published gene-sets we observed upregulation of UPR MT genes in single-cell RNA sequencing (scRNA-seq) data generated from PCs derived from Vκ*MYC mice (a transgenic mouse model of MM) spanning the spectrum of the disease. UPR MT gene signature scores in PCs from mice increased with disease progression with the highest levels found in late-MM> int-MM> early MM>wild type mice. Similarly, analysis of publicly available gene expression datasets (GSE6477) that includes normal donors, MGUS and newly diagnosed MM (NDMM) revealed higher expression of UPR MT genes in the majority of NDMM, weak expression in MGUS and absence in normal PCs. To assess the impact of UPR MT expression on pt outcomes we calculated a UPR MT index score derived from the median expression of 12 mtUPR classifier genes across the MMRF CoMMpass dataset of NDMM pts. Stratifying pts by UPR MT expression score we found that pts in the top quartile had a significantly shorter PFS and OS compared to pts with the lowest quartile weighted score. Next, we postulated that perturbation of the mitochondrial import protein, Translocase of the Inner Membrane 23 (TIM23) would exaggerate mitochondrial stress as mitochondrial import efficiency is a key regulator of the UPR MT. First, we demonstrated that TIM23 complex genes are enriched in pts from the CoMMpass dataset with poor risk (1q gain and PR gene signature) and that shorter PFS and OS is associated with a higher weighted score of TIM23 complex genes. We then demonstrated that genetic (shRNA) knockdown or pharmacologic inhibition of TIM23 with MB-10, a small molecule inhibitor of TIM23 induced apoptosis of MM cell lines and primary pt PCs. Further non-transformed cell lines, CD138 - non-MM cells and normal donor hematopoietic progenitor cells were less susceptible to the effects of MB-10. Consistent with activation of the UPR MT, treatment of MM cells resulted in increased cytosolic ATF4, CHOP and a shift of ATF5 to the nuclear fraction. Activation of the CHOP-dependent branch of the UPR MT resulted in in upregulation of mitochondrial-targeted proteins, cpn10 and ClpP. Interestingly, MB-10 also induced XBP1 splicing demonstrating that inhibition of TIM23 complex can simultaneously activate the IRE1/XBP1 branch of integrated stress response (ISR), This led us to hypothesize that targeting TIM23 as an alternative means of activating the ISR could overcome acquired resistance to proteosome inhibitors (PIs). Indeed, PI-resistant and parental isogenic cell lines were equally susceptible to MB-10 as measured by IC50 values of cell growth. Finally, we demonstrated that doxycycline inducible knockdown of TIM23 in a mouse xenograft model induced tumor regression with significantly small tumor volumes at the end of 17 days of doxycycline treatment compared to tumors expressing an inducible control vector. Conclusions: These data demonstrate that mitohormesis and UPR MT activation is associated with MM progression and worse clinical outcomes. Further we show that disrupting mitochondrial protein import results in unmitigated mitochondrial stress that switches the UPR MT from an adaptive cytoprotective to cytotoxic proapoptotic response. Thus, targeting mitochondrial import proteins such as TIM23 may represent novel therapeutic targets for MM. Disclosures Schimmer: Takeda Pharmaceuticals: Consultancy, Research Funding; Medivir AB: Research Funding; Novartis: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Otsuka Pharmaceuticals: Consultancy, Honoraria; UHN: Patents & Royalties. Trudel: Janssen: Honoraria, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Roche: Consultancy; Sanofi: Honoraria; Pfizer: Honoraria, Research Funding; Genentech: Research Funding; BMS/Celgene: Consultancy, Honoraria, Research Funding.