Prohibitin 2 (PHB2) is a highly conserved protein with essential roles in cell homeostasis and survival across different cell types. Previous studies have shown that the deletion of PHB2 results in an arrest in proliferation due to impaired mitochondrial function regulated by the dynamin-like GTPase OPA1. The function of PHB2 in immune cells remains unclear; however, some studies suggest that PHB2 plays a role in the cell membranes of B and T cells. In order to elucidate the role of PHB2 in immune cells, we generated PHB2-deficient T cells. Our findings reveal a pivotal role for PHB2 in the proliferation and differentiation of T cells. PHB2 deficiency inhibits T cell proliferation by inducing a cell cycle arrest at the G1 to S phase, thereby preventing the differentiation into effector T cells. Furthermore, in contrast to previous reports, T cells lacking PHB2 are more resistant to apoptosis. Metabolic analysis reveals that PHB2-deficient T cells fail to boost their energy production through glycolysis and oxidative phosphorylation upon activation, hindering their ability to sustain biosynthetic processes and to proliferate in response to activation.

Mitochondrial Ca2+ signaling regulates metabolism, redox balance, and cell fate. Current models primarily emphasize Ca2+ amplitude and load, yet emerging evidence suggests that the duration of mitochondrial Ca2+ elevation may represent an additional signaling dimension. Recent studies linking altered mitochondrial Ca2+ decay kinetics to neuronal metabolism and memory raise the possibility that mitochondria decode Ca2+ signals temporally rather than relying solely on quantitative measures. Here, we argued that mitochondrial Ca2+ persistence may influence adaptive and pathological responses depending on tissue-specific energetic demand and mitochondrial resilience. We integrated established Ca2+ transport systems with emerging and controversial regulators, including leucine zipper EF hand containing transmembrane protein 1 and transmembrane BAX inhibitor motif containing protein 5, whose precise roles remain unresolved. This framework positions temporal mitochondrial signaling as a potential determinant of metabolic adaptation and disease vulnerability.

BACKGROUND AND PURPOSE:Noise pollution, particularly by aircraft, is a significant risk factor for cardiovascular disease. Aircraft noise activates stress response pathways in the brain, via the amygdala, the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis. EXPERIMENTAL APPROACH:Male C57BL/6J mice were treated with citalopram (a selective serotonin (5-HT)-reuptake inhibitor) or diazepam (a benzodiazepine) 1 day before aircraft noise exposure for 4 days. Aortic vascular function was measured by isometric tension method, microvascular function by video microscopy in pressurized cerebral arterioles, blood pressure by tail cuff, reactive oxygen species formation by dihydrothidium staining of vascular tissue and markers of inflammation and oxidative stress by western blotting. KEY RESULTS:In support of the stress response concept, we report here that noise exposure of mice implicates an increase in activity primarily in the left amygdala, envisaged by [18F]fluorodeoxyglucose positron emission tomography (PET) scan. Both neuro-active drugs, diazepam and citalopram, ameliorated the adverse cardiovascular and neurobiological effects of noise exposure, partially preventing blood pressure increases and endothelial dysfunction in both large (aorta) and small vessels (cerebral arterioles). Diazepam showed slightly greater efficacy. Noise exposure also increased markers of oxidative stress and inflammation in the heart and brain (cortex and hippocampus), and both drugs mostly prevented these pathophysiological changes. CONCLUSION AND IMPLICATIONS:The study provides indirect evidence that modulating the stress response pathway may represent a pharmacological approach to mitigate the negative effects of noise exposure. This may have implications for patients with neuropsychiatric disease suffering from aircraft noise exposure.

Mitochondrial calcium homeostasis involves coordinated uptake via the mitochondrial calcium uniporter (MCU) and efflux through sodium-dependent NCLX (encoded by SLC8B1) and/or TMEM65. We investigated TMBIM5, a proposed bidirectional mitochondrial calcium/proton transporter, by generating zebrafish lacking tmbim5, slc8b1, plus tmbim5/mcu and tmbim5/slc8b1 double knockouts. Tmbim5-deficient fish exhibited growth impairment, muscle atrophy, and increased brain cell death. tmbim5/mcu double knockouts showed no additive effects, arguing against Tmbim5 functioning as an independent calcium uptake pathway. slc8b1 knockouts had no major phenotype but showed attenuated, although not abolished sodium-dependent mitochondrial calcium efflux. tmbim5/slc8b1 double knockouts showed altered mitochondrial calcium handling with reduced uptake and efflux. Remarkably, brain phenotypes were rescued while muscle dysfunction was exacerbated in double mutants, corresponding to restored mitochondrial membrane potential in brain tissue and decreased calcium levels in muscle. These findings suggest that TMBIM5 functions as an auxiliary calcium efflux pathway cooperating with NCLX in a tissue-specific manner.

Ion transport within mitochondria influences their structure, energy production, and cell death regulation. TMBIM5, a conserved calcium/proton exchanger in the inner mitochondrial membrane, contributes to mitochondrial structure, ATP synthesis, and apoptosis regulation. The relationship of TMBIM5 with the mitochondrial calcium uniporter complex formed by MCU, MICU1-3, and EMRE remains undefined. We generated Tmbim5-deficient Drosophila that exhibit disrupted cristae architecture, premature mitochondrial permeability transition pore opening, reduced calcium uptake, and mitochondrial swelling - resulting in impaired mobility and shortened lifespan. Crossing these with flies lacking mitochondrial calcium uniporter complex proteins was generally detrimental, but partial MICU1 depletion ameliorated the Tmbim5-deficiency phenotype. In human cells, MICU1 rescues morphological defects in TMBIM5-knockout mitochondria, while TMBIM5 overexpression exacerbates size reduction in MICU1-knockout mitochondria. Both proteins demonstrated opposing effects on submitochondrial localization and coexisted in the same macromolecular complex. Our findings establish a functional interplay between TMBIM5 and MICU1 in maintaining mitochondrial integrity, with implications for understanding calcium homeostasis mechanisms.

Charcot-Marie-Tooth disease type 4 A ((CMT4A), an autosomal recessive neuropathy, is caused by mutations in ganglioside-induced differentiation-associated protein 1 (GDAP1). GDAP1 resides in the outer mitochondrial membrane facing the cytosol and is involved in mitochondrial dynamics and function. Its perturbation affects mitochondrial shape, contact sites, redox homeostasis and cellular metabolism. In response to GDAP1 knockdown in a human neuronal cell line, we found increased mitochondrial turnover, biogenesis and mitophagy. This was associated with more lysosomal proteins in mitochondrial fractions including BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) and its homolog BNIP3-like (BNIP3L) - proteins involved in the recruitment of autophagy machinery via direct interaction. Flies with neural Gdap1 knockdown also exhibited upregulated levels of the sole BNIP3 ortholog. Neural expression of human BNIP3 reduced the detrimental effects of Gdap1 knockdown on eclosion and climbing ability in adult flies, while simultaneous knockdown of both genes was detrimental. These findings suggest that increased BNIP3-driven mitophagy may act as a protective mechanism, partially counteracting the cellular dysfunction caused by GDAP1 loss of function, and highlight the potential of targeting mitophagy pathways as a therapeutic strategy for CMT4A.

Mitochondria generate ATP by creating a proton gradient across the inner membrane. This gradient also drives calcium uptake by the mitochondrial calcium uniporter (MCU) complex, while NCLX, encoded by SLC8B1, facilitates efflux. Mcu deficiency in various species does not result in obvious phenotypes, suggesting the existence of additional calcium transport systems. We here generated zebrafish lacking Tmbim5, a potential bidirectional mitochondrial calcium transporter, and Slc8b1, the probable NCLX ortholog. tmbim5 knockout fish exhibited impaired growth, muscle atrophy, increased brain cell death, and reduced mitochondrial membrane potential, but no significant changes in steady-state mitochondrial calcium levels. Slc8b1-deficient fish had increased larval mortality but were otherwise normal. Double knockouts of tmbim5/mcu and tmbim5/slc8b1 were viable with normal Mendelian distribution, indicating robust compensatory mechanisms maintain calcium homeostasis. tmbim5 knockout rescued the increased mortality in Slc8b1-deficient fish, and the NCLX inhibitor CGP-37157 lost its effect on behavior in Tmbim5-deficient but not Slc8b1-deficient zebrafish, implying shared, possibly antagonistic pathways. These findings suggest alternative pathways for mitochondrial calcium transport compensate for the loss of these proteins, enabling zebrafish survival. ### Competing Interest Statement The authors have declared no competing interest.

Ferroptosis is a non-apoptotic iron-dependent form of cell death involved in cancer, ischemia-reperfusion injury, inflammation and neurodegeneration. Ferroptosis can be triggered by inhibiting system Xc-, a cystine/glutamate antiporter system, at the plasma membrane (class 1 e.g. erastin) resulting in glutathione depletion or by inhibiting glutathione peroxidase 4 (class 2 e.g. RSL3) resulting in direct lipid peroxidation. In vivo models for ferroptosis are very desirable. We here investigated the effects of erastin and RSL3 on the lifespan, iron metabolism and lipid abundance of the fruitfly Drosophila melanogaster. Both compounds dose-dependently shortened the lifespan of three commonly used fly strains. The reduction in lifespan induced by erastin was rescued by the ferroptosis inhibitor ferrostatin-1. Erastin had no effect on total glutathione levels but increased the Fe2+/Fe3+ ratio and reduced the amount of ferritin in male but not in female w1118 flies. Also RSL3 only altered the lipid composition of male but not female flies. The fly ortholog of GPX4, Gtpx, has a cysteine instead of a selenocysteine in its otherwise very homologous active center and possesses thioredoxin and not glutathione peroxidase activity. We found that its complete knockout in flies is lethal. Supplying food with ferroptosis inhibitors did not rescue homozygous Gtpx knockout flies suggesting that lethality occurs during early development. Together these results characterize male w1118 flies as a suitable in vivo model for ferroptosis.

The N-terminal EF-hand calcium-binding proteins 1–3(NECAB1–3) constitute a family of predominantly neuronal proteins characterized by the presence of at least one EF-hand calcium-binding domain and a functionally less well characterized C-terminal antibiotic biosynthesis monooxygenase domain. All three family members were initially discovered due to their interactions with other proteins. NECAB1 associates with synaptotagmin-1, a critical neuronal protein involved in membrane trafficking and synaptic vesicle exocytosis. NECAB2 interacts with predominantly striatal G-protein-coupled receptors, while NECAB3 partners with amyloid-β A4 precursor protein-binding family A members 2 and 3, key regulators of amyloid-β production. This demonstrates the capacity of the family for interactions with various classes of proteins. NECAB proteins exhibit distinct subcellular localizations: NECAB1 is found in the nucleus and cytosol, NECAB2 resides in endosomes and the plasma membrane, and NECAB3 is present in the endoplasmic reticulum and Golgi apparatus. The antibiotic biosynthesis monooxygenase domain, an evolutionarily ancient component, is akin to atypical heme oxygenases in prokaryotes but is not wellcharacterized in vertebrates. Prokaryotic antibiotic biosynthesis monooxygenase domains typically form dimers, suggesting that calcium-mediated conformational changes in NECAB proteins may induce antibiotic biosynthesis monooxygenase domain dimerization, potentially activating some enzymatic properties. However, the substrate for this enzymatic activity remains uncertain. Alternatively, calcium-mediated conformational changes might influence protein interactions or the subcellular localization of NECAB proteins by controlling the availability of protein–protein interaction domains situated between the EF hands and the antibiotic biosynthesis monooxygenase domain. This review summarizes what is known about genomic organization, tissue expression, intracellular localization, interaction partners, and the physiological and pathophysiological role of the NECAB family.

One mechanism of particular interest to regulate mRNA fate post-transcriptionally is mRNA modification. Especially the extent of m 1 A mRNA methylation is highly discussed due to methodological differences. However, one single m 1 A site in mitochondrial ND5 mRNA was unanimously reported by different groups. ND5 is a subunit of complex I of the respiratory chain. It is considered essential for the coupling of oxidation and proton transport. Here we demonstrate that this m 1 A site might be involved in the pathophysiology of Alzheimer’s disease (AD). One of the pathological hallmarks of this neurodegenerative disease is mitochondrial dysfunction, mainly induced by Amyloid β (Aβ). Aβ mainly disturbs functions of complex I and IV of the respiratory chain. However, the molecular mechanism of complex I dysfunction is still not fully understood. We found enhanced m 1 A methylation of ND5 mRNA in an AD cell model as well as in AD patients. Formation of this m 1 A methylation is catalyzed by increased TRMT10C protein levels, leading to translation repression of ND5. As a consequence, here demonstrated for the first time, TRMT10C induced m 1 A methylation of ND5 mRNA leads to mitochondrial dysfunction. Our findings suggest that this newly identified mechanism might be involved in Aβ-induced mitochondrial dysfunction.

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra of the midbrain. Familial cases of PD are often caused by mutations of PTEN-induced kinase 1 (PINK1) and the ubiquitin ligase Parkin, both pivotal in maintaining mitochondrial quality control. CISD1, a homodimeric mitochondrial iron-sulfur-binding protein, is a major target of Parkin-mediated ubiquitination. We here discovered a heightened propensity of CISD1 to form dimers in Pink1 mutant flies and in dopaminergic neurons from PINK1 mutation patients. The dimer consists of two monomers that are covalently linked by a disulfide bridge. In this conformation CISD1 cannot coordinate the iron-sulfur cofactor. Overexpressing Cisd, the Drosophila ortholog of CISD1, and a mutant Cisd incapable of binding the iron-sulfur cluster in Drosophila reduced climbing ability and lifespan. This was more pronounced with mutant Cisd and aggravated in Pink1 mutant flies. Complete loss of Cisd, in contrast, rescued all detrimental effects of Pink1 mutation on climbing ability, wing posture, dopamine levels, lifespan, and mitochondrial ultrastructure. Our results suggest that Cisd, probably iron-depleted Cisd, operates downstream of Pink1 shedding light on PD pathophysiology and implicating CISD1 as a potential therapeutic target.

Ferroptosis is a pervasive non-apoptotic form of cell death highly relevant in various degenerative diseases and malignancies. The hallmark of ferroptosis is uncontrolled and overwhelming peroxidation of polyunsaturated fatty acids contained in membrane phospholipids, which eventually leads to rupture of the plasma membrane. Ferroptosis is unique in that it is essentially a spontaneous, uncatalyzed chemical process based on perturbed iron and redox homeostasis contributing to the cell death process, but that it is nonetheless modulated by many metabolic nodes that impinge on the cells' susceptibility to ferroptosis. Among the various nodes affecting ferroptosis sensitivity, several have emerged as promising candidates for pharmacological intervention, rendering ferroptosis-related proteins attractive targets for the treatment of numerous currently incurable diseases. Herein, the current members of a Germany-wide research consortium focusing on ferroptosis research, as well as key external experts in ferroptosis who have made seminal contributions to this rapidly growing and exciting field of research, have gathered to provide a comprehensive, state-of-the-art review on ferroptosis. Specific topics include: basic mechanisms, in vivo relevance, specialized methodologies, chemical and pharmacological tools, and the potential contribution of ferroptosis to disease etiopathology and progression. We hope that this article will not only provide established scientists and newcomers to the field with an overview of the multiple facets of ferroptosis, but also encourage additional efforts to characterize further molecular pathways modulating ferroptosis, with the ultimate goal to develop novel pharmacotherapies to tackle the various diseases associated with - or caused by - ferroptosis.

Background Parkinson’s disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra of the midbrain. Familial cases of PD are often caused by mutations of PTEN-induced kinase 1 (PINK1) and the ubiquitin ligase Parkin, both pivotal in maintaining mitochondrial quality control. CISD1, a homodimeric mitochondrial iron-sulfur-binding protein, is a major target of Parkin-mediated ubiquitination. Loss of CISD1 is associated with mitochondrial dysfunction, redox imbalance, and abnormal iron accumulation, all hallmark features of PD. Methods To elucidate a possible involvement of CISD1 in PD pathophysiology, we investigated its role in dopaminergic neurons from PINK1 mutation patients, in fibroblasts lacking CISD1 or expressing a CISD1 mutant lacking its iron/sulfur cluster, and in Pink1 and Parkin mutant flies. Results In both patient-derived dopaminergic neurons and in Pink1 mutant flies, we observed a heightened propensity of CISD1 to form dimers. This corresponded to the iron-depleted state of CISD1. Reintroducing a CISD1 mutant incapable of binding the iron-sulfur cluster into CISD1 knockout cells failed to rescue mitochondrial fragmentation and oxidative distress. When overexpressed in Drosophila, this mutant proved detrimental by disrupting the redox equilibrium. Complete loss of Cisd, the Drosophila orthologue of CISD1, rescued all detrimental effects of Pink1 loss of function on climbing ability, wing posture, dopamine levels, lifespan, and mitochondrial ultrastructure. In Parkin mutant flies, additional loss of Cisd ameliorated climbing and wing posture phenotypes, but did not mitigate the reduction in lifespan. Conclusion Our results suggest that Cisd operates downstream of Pink1 and, partially, Prkn, shedding light on PD pathophysiology and implicating CISD1 as a potential therapeutic target.

Synaptic signaling depends on ATP generated by mitochondria. Dysfunctional mitochondria shift the redox balance towards a more oxidative environment. Due to extensive connectivity, the striatum is especially vulnerable to mitochondrial dysfunction. We found that neuronal calcium-binding protein 2 (NECAB2) plays a role in striatal function and mitochondrial homeostasis. NECAB2 is a predominantly endosomal striatal protein which partially colocalizes with mitochondria. This colocalization is enhanced by mild oxidative stress. Global knockout of Necab2 in the mouse results in increased superoxide levels, increased DNA oxidation and reduced levels of the antioxidant glutathione which correlates with an altered mitochondrial shape and function. Striatal mitochondria from Necab2 knockout mice are more abundant and smaller and characterized by a reduced spare capacity suggestive of intrinsic uncoupling respectively mitochondrial dysfunction. In line with this, we also found an altered stress-induced interaction of endosomes with mitochondria in Necab2 knockout striatal cultures. The predominance of dysfunctional mitochondria and the pro-oxidative redox milieu correlates with a loss of striatal synapses and behavioral changes characteristic of striatal dysfunction like reduced motivation and altered sensory gating. Together this suggests an involvement of NECAB2 in an endosomal pathway of mitochondrial stress response important for striatal function.

Chronic stress has the potential to impair health and may increase the vulnerability for psychiatric disorders. Emerging evidence suggests that specific neurometabolic dysfunctions play a role herein. In mice, chronic social defeat (CSD) stress reduces cerebral glucose uptake despite hyperglycemia. We hypothesized that this metabolic decoupling would be reflected by changes in contact sites between mitochondria and the endoplasmic reticulum, important intracellular nutrient sensors, and signaling hubs. We thus analyzed the proteome of their biochemical counterparts, mitochondria-associated membranes (MAMs) from whole brain tissue obtained from CSD and control mice. This revealed a lack of the glucose-metabolizing enzyme hexokinase 3 (HK3) in MAMs from CSD mice. In controls, HK3 protein abundance in MAMs and also in striatal synaptosomes correlated positively with peripheral blood glucose levels, but this connection was lost in CSD. We conclude that the ability of HK3 to traffic to sites of need, such as MAMs or synapses, is abolished upon CSD and surmise that this contributes to a cellular dysfunction instigated by chronic stress. KEY MESSAGES : Chronic social defeat (CSD) alters brain glucose metabolism CSD depletes hexokinase 3 (HK3) from mitochondria-associated membranes (MAMs) CSD results in loss of positive correlation between blood glucose and HK3 in MAMs and synaptosomes.

Charcot-Marie-Tooth (CMT) disease 4A is an autosomal-recessive polyneuropathy caused by mutations of ganglioside-induced differentiation-associated protein 1 (GDAP1), a putative glutathione transferase, which affects mitochondrial shape and alters cellular Ca 2+ homeostasis. Here, we identify the underlying mechanism. We found that patient-derived motoneurons and GDAP1 knockdown SH-SY5Y cells display two phenotypes: more tubular mitochondria and a metabolism characterized by glutamine dependence and fewer cytosolic lipid droplets. GDAP1 interacts with the actin-depolymerizing protein Cofilin-1 and beta-tubulin in a redox-dependent manner, suggesting a role for actin signaling. Consistently, GDAP1 loss causes less F-actin close to mitochondria, which restricts mitochondrial localization of the fission factor dynamin-related protein 1, instigating tubularity. GDAP1 silencing also disrupts mitochondria-ER contact sites. These changes result in lower mitochondrial Ca 2+ levels and inhibition of the pyruvate dehydrogenase complex, explaining the metabolic changes upon GDAP1 loss of function. Together, our findings reconcile GDAP1-associated phenotypes and implicate disrupted actin signaling in CMT4A pathophysiology.

TMBIM5 deficiency reduces mitochondrial K + /H + exchange. Mutation of the channel pore in mice destabilizes the protein and results in increased embryonic lethality and a skeletal myopathy. Ion fluxes across the inner mitochondrial membrane control mitochondrial volume, energy production, and apoptosis. TMBIM5, a highly conserved protein with homology to putative pH-dependent ion channels, is involved in the maintenance of mitochondrial cristae architecture, ATP production, and apoptosis. Here, we demonstrate that overexpressed TMBIM5 can mediate mitochondrial calcium uptake. Under steady-state conditions, loss of TMBIM5 results in increased potassium and reduced proton levels in the mitochondrial matrix caused by attenuated exchange of these ions. To identify the in vivo consequences of TMBIM5 dysfunction, we generated mice carrying a mutation in the channel pore. These mutant mice display increased embryonic or perinatal lethality and a skeletal myopathy which strongly correlates with tissue-specific disruption of cristae architecture, early opening of the mitochondrial permeability transition pore, reduced calcium uptake capability, and mitochondrial swelling. Our results demonstrate that TMBIM5 is an essential and important part of the mitochondrial ion transport system machinery with particular importance for embryonic development and muscle function.