Karin Nowikovsky

Mitochondrial Ca ²⁺ ions are crucial regulators of bioenergetics and cell death pathways. Mitochondrial Ca ²⁺ content and cytosolic Ca ²⁺ homeostasis strictly depend on Ca ²⁺ transporters. In recent decades, the major players responsible for mitochondrial Ca ²⁺ uptake and release have been identified, except the mitochondrial Ca ²⁺ /H ⁺ exchanger (CHE). Originally identified as the mitochondrial K ⁺ /H ⁺ exchanger, LETM1 was also considered as a candidate for the mitochondrial CHE. Defining the mitochondrial interactome of LETM1, we identify TMBIM5/MICS1, the only mitochondrial member of the TMBIM family, and validate the physical interaction of TMBIM5 and LETM1. Cell‐based and cell‐free biochemical assays demonstrate the absence or greatly reduced Na ⁺ ‐independent mitochondrial Ca ²⁺ release in TMBIM5 knockout or pH‐sensing site mutants, respectively, and pH‐dependent Ca ²⁺ transport by recombinant TMBIM5. Taken together, we demonstrate that TMBIM5, but not LETM1, is the long‐sought mitochondrial CHE, involved in setting and regulating the mitochondrial proton gradient. This finding provides the final piece of the puzzle of mitochondrial Ca ²⁺ transporters and opens the door to exploring its importance in health and disease, and to developing drugs modulating Ca ²⁺ exchange.

Mitochondria are fundamental for life and require balanced ion exchange to maintain proper functioning. The mitochondrial cation exchanger LETM1 sparks interest because of its pathophysiological role in seizures in the Wolf Hirschhorn Syndrome (WHS). Despite observation of sleep disorganization in epileptic WHS patients, and growing studies linking mitochondria and epilepsy to circadian rhythms, LETM1 has not been studied from the chronobiological perspective. Here we established a viable letm1 knock-out, using the diurnal vertebrate Danio rerio to study the metabolic and chronobiological consequences of letm1 deficiency. We report diurnal rhythms of Letm1 protein levels in wild-type fish. We show that mitochondrial nucleotide metabolism is deregulated in letm1−/− mutant fish, the rate-limiting enzyme of NAD⁺ production is up-regulated, while NAD⁺ and NADH pools are reduced. These changes were associated with increased expression amplitude of circadian core clock genes in letm1−/− compared with wild-type under light/dark conditions, suggesting decreased NAD(H) levels as a possible mechanism for circadian system perturbation in Letm1 deficiency. Replenishing NAD pool may ameliorate WHS-associated sleep and neurological disorders.

Mitochondrial Ca ²⁺ ions are crucial regulators of bioenergetics, cell death pathways and cytosolic Ca ²⁺ homeostasis. Mitochondrial Ca ²⁺ content strictly depends on Ca ²⁺ transporters. In recent decades, the major players responsible for mitochondrial Ca ²⁺ uptake and release have been identified, except the mitochondrial Ca ²⁺ /H ⁺ exchanger (CHE). Originally identified as the mitochondrial K ⁺ /H ⁺ exchanger, LETM1 was also considered as a candidate for the mitochondrial CHE. Defining the mitochondrial interactome of LETM1, we identified MICS1, the only mitochondrial member of the TMBIM family. Applying cell-based and cell-free biochemical assays, here we demonstrate that MICS1 is responsible for the Na ⁺ - and permeability transition pore-independent mitochondrial Ca ²⁺ release and identify MICS1 as the long-sought mitochondrial CHE. This finding provides the final piece of the puzzle of mitochondrial Ca ²⁺ transporters and opens the door to exploring its importance in health and disease, and to developing drugs modulating Ca ²⁺ exchange.

Cadmium (Cd) accumulates with aging and is elevated in long-lived species. Metallothioneins (MTs), small cysteine-rich proteins involved in metal homeostasis and Cd detoxification, are known to be related to longevity. However, the relationship between Cd accumulation, the role of MTs, and aging is currently unclear. Specifically, we do not know if long-lived species evolved an efficient metal stress response by upregulating their MT levels to reduce the toxic effects of environmental pollutants, such as Cd, that accumulate over their longer life span. It is also unknown if the number of MT genes, their expression, or both protect the organisms from potentially damaging effects during aging. To address these questions, we reanalyzed several cross-species studies and obtained data on MT expression and Cd accumulation in long-lived mouse models. We confirmed a relationship between species maximum life span in captive mammals and their Cd content in liver and kidney. We found that although the number of MT genes does not affect longevity, gene expression and protein amount of specific MT paralogs are strongly related to life span in mammals. MT expression rather than gene number may influence the high Cd levels and longevity of some species. In support of this, we found that overexpression of MT-1 accelerated Cd accumulation in mice and that tissue Cd was higher in long-lived mouse strains with high MT expression. We conclude that long-lived species have evolved a more efficient stress response by upregulating the expression of MT genes in presence of Cd, which contributes to elevated tissue Cd levels.

Due to their high biological activity, thiosemicarbazones have been developed for treatment of diverse diseases, including cancer, resulting in multiple clinical trials especially of the lead compound Triapine. During the last years, a novel subclass of anticancer thiosemicarbazones has attracted substantial interest based on their enhanced cytotoxic activity. Increasing evidence suggests that the double-dimethylated Triapine derivative Me₂NNMe₂ differs from Triapine not only in its efficacy but also in its mode of action. Here we show that Me₂NNMe₂- (but not Triapine)-treated cancer cells exhibit all hallmarks of paraptotic cell death including, besides the appearance of endoplasmic reticulum (ER)-derived vesicles, also mitochondrial swelling and caspase-independent cell death via the MAPK signaling pathway. Subsequently, we uncover that the copper complex of Me₂NNMe₂ (a supposed intracellular metabolite) inhibits the ER-resident protein disulfide isomerase, resulting in a specific form of ER stress based on disruption of the Ca²⁺ and ER thiol redox homeostasis. Our findings indicate that compounds like Me₂NNMe₂ are of interest especially for the treatment of apoptosis-resistant cancer and provide new insights into mechanisms underlying drug-induced paraptosis.