Novel Purine Derivative ITH15004 Facilitates Exocytosis through a Mitochondrial Calcium-Mediated Mechanism

Upon depolarization of chromaffin cells (CCs), a prompt release of catecholamines occurs. This event is triggered by a subplasmalemmal high-Ca2+ microdomain (HCMD) generated by Ca2+ entry through nearby voltage-activated calcium channels. HCMD is efficiently cleared by local mitochondria that avidly take up Ca2+ through their uniporter (MICU), then released back to the cytosol through mitochondrial Na+/Ca2+ exchanger (MNCX). We found that newly synthesized derivative ITH15004 facilitated the release of catecholamines triggered from high K+-depolarized bovine CCs. Such effect seemed to be due to regulation of mitochondrial Ca2+ circulation because: (i) FCCP-potentiated secretory responses decay was prevented by ITH15004; (ii) combination of FCCP and ITH15004 exerted additive secretion potentiation; (iii) such additive potentiation was dissipated by the MICU blocker ruthenium red (RR) or the MNCX blocker CGP37157 (CGP); (iv) combination of FCCP and ITH15004 produced both additive augmentation of cytosolic Ca2+ concentrations ([Ca2+]c) K+-challenged BCCs, and (v) non-inactivated [Ca2+]c transient when exposed to RR or CGP. On pharmacological grounds, data suggest that ITH15004 facilitates exocytosis by acting on mitochondria-controlled Ca2+ handling during K+ depolarization. These observations clearly show that ITH15004 is a novel pharmacological tool to study the role of mitochondria in the regulation of the bioenergetics and exocytosis in excitable cells.

Regulation of Kidney Mitochondrial Function by Caloric Restriction

Caloric restriction (CR) prevents obesity, promotes healthy aging, and increases resilience against several pathological stimuli in laboratory rodents. At the mitochondrial level, protection promoted by CR in the brain and liver is related to higher calcium uptake rates and capacities, avoiding Ca2+-induced mitochondrial permeability transition. Dietary restriction has also been shown to increase kidney resistance against damaging stimuli such as ischemia/reperfusion, but if these effects are related to similar mitochondrial adaptations had not yet been uncovered. Here, we characterized changes in mitochondrial function in response to six months of CR in rats, measuring bioenergetic parameters, redox balance and calcium homeostasis. CR promoted an increase in mitochondrial oxygen consumption rates under non-phosphorylating and uncoupled conditions. While CR prevents mitochondrial reactive oxygen species production in many tissues, in kidney we found that mitochondrial H2O2 release was enhanced, although levels of carbonylated proteins and methionine sulfoxide were unchanged. Surprisingly, and opposite to the effects observed in brain and liver, mitochondria from CR animals are more prone to Ca2+-induced mitochondrial permeability transition. CR mitochondria also displayed higher calcium uptake rates, which were not accompanied by changes in calcium efflux rates, nor related to altered inner mitochondrial membrane potentials or the amounts of the mitochondrial calcium uniporter (MCU). Instead, increased mitochondrial calcium uptake rates in CR kidneys correlate with a loss of MICU2, an MCU modulator. Interestingly, MICU2 is also modulated by CR in liver, suggesting it has a broader diet-sensitive regulatory role controlling mitochondrial calcium homeostasis. Together, our results highlight the organ-specific bioenergetic, redox, and ionic transport effects of CR. Specifically, we describe the regulation of the expression of MICU2 and its effects on mitochondrial calcium transport as a novel and interesting aspect of the metabolic responses to dietary interventions.

The mitochondrial calcium uniporter regulates mitochondrial mass and dendritic localization in distinct hippocampal circuits

CA2 is an understudied subregion of the hippocampus that is critical for social memory. Previous studies identified multiple components of the mitochondrial calcium uniporter (MCU) complex as selectively enriched in CA2, however the functional significance of this enrichment remains unclear. The MCU complex regulates calcium entry into mitochondria, which in turn regulates mitochondrial transport and localization to active synapses. We found that MCU is strikingly enriched in CA2 distal apical dendrites, precisely where CA2 neurons receive entorhinal cortical input carrying social information. Further, MCU-enriched mitochondria in CA2 distal dendrites are larger compared to mitochondria in CA2 proximal apical dendrites and neighboring CA1 apical dendrites. Genetic knockdown of MCU in CA2 resulted in smaller mitochondria in CA2 distal dendrites, indicating that MCU expression plays a role in regulating mitochondrial mass in CA2. MCU overexpression in neighboring CA1 led to larger mitochondria preferentially in proximal dendrites compared to distal dendrites and GFP controls. Our findings demonstrate that mitochondria are molecularly and structurally diverse across hippocampal cell types and circuits, and that MCU expression cell-autonomously regulates mitochondrial mass, but layer-specific dendritic localization depends on cell type. Our data support the idea that CA2 mitochondria are functionally distinct from CA1 mitochondria, which may confer unique synaptic and circuit properties underlying CA2 function in social memory.

IGF1 Receptor Regulates Upward Firing Rate Homeostasis via the Mitochondrial Calcium Uniporter

Regulation of firing rate homeostasis constitutes a fundamental property of central neural circuits. While intracellular Ca2+ has long been hypothesized to be a feedback control signal, the molecular machinery enabling network-wide homeostatic response remains largely unknown. Here we show that deletion of insulin-like growth factor-1 receptor (IGF1R), a well-known regulator of neurodevelopment and ageing, limits firing rate homeostasis in response to inactivity, without altering the baseline firing rate distribution. Disruption of both synaptic and intrinsic homeostatic plasticity contributed to deficient firing rate homeostatic response. At the cellular level, a fraction of IGF1Rs was localized in mitochondria with the mitochondrial calcium uniporter complex (MCUc). IGF1R deletion suppressed mitochondrial Ca2+ (mitoCa2+) evoked by spike bursts by weakening mitochondria-to-cytosol Ca2+ coupling. This coupling was homeostatically maintained following inactivity in control, but upregulated in IGF1R-deficient neurons. MCUc overexpression in IGF1R-deficient neurons rescued the deficits in spike-to-mitoCa2+ coupling and firing rate homeostasis. Our findings highlight IGF1R as a key regulator of the integrated homeostatic response by tuning mitochondrial temporal filtering. Decline in mitochondrial reliability for burst transfer may drive dysregulation of firing rate homeostasis in brain disorders associated with abnormal IGF1R / MCUc signaling.

Mitochondrial calcium uniporter affects neutrophil bactericidal activity during Staphylococcus aureus infection

Neutrophils simultaneously restrict Staphylococcus aureus dissemination and facilitate bactericidal activity during infection through the formation of neutrophil extracellular traps (NETs). Neutrophils that produce higher levels of mitochondrial superoxide undergo enhanced terminal NET formation (suicidal NETosis) in response to S. aureus ; however, mechanisms regulating mitochondrial homeostasis upstream of neutrophil antibacterial processes are not fully resolved. Here, we demonstrate that mitochondrial calcium uptake 1 (MICU1)-deficient (MICU1 -/- ) neutrophils accumulate higher levels of calcium and iron within the mitochondria in a mitochondrial calcium uniporter (MCU)-dependent manner. Corresponding with increased ion flux through the MCU, mitochondrial superoxide production is elevated, thereby increasing the propensity for MICU1 -/- neutrophils to undergo suicidal NETosis rather than primary degranulation in response to S. aureus . Increased NET formation augments macrophage killing of bacterial pathogens. Similarly, MICU1 -/- neutrophils alone are not more antibacterial towards S. aureus , but rather enhanced suicidal NETosis by MICU1 -/- neutrophils facilitates increased bactericidal activity in the presence of macrophages. Similarly, mice with a deficiency in MICU1 restricted to cells expressing LysM exhibit lower bacterial burdens in the heart with increased survival during systemic S. aureus infection. Coinciding with the decrease in S. aureus burdens, MICU1 -/- neutrophils in the heart produced higher levels of mitochondrial superoxide and undergo enhanced suicidal NETosis. These results demonstrate that ion flux by the MCU affects the antibacterial function of neutrophils during S. aureus infection.

Protective effect of HINT2 on mitochondrial function via repressing MCU complex activation attenuates cardiac microvascular ischemia–reperfusion injury

Current evidence indicates that coronary microcirculation is a key target for protecting against cardiac ischemia–reperfusion (I/R) injury. Mitochondrial calcium uniporter (MCU) complex activation and mitochondrial calcium ([Ca2+]m) overload are underlying mechanisms involved in cardiovascular disease. Histidine triad nucleotide-binding 2 (HINT2) has been reported to modulate [Ca2+]m via the MCU complex, and our previous work demonstrated that HINT2 improved cardiomyocyte survival and preserved heart function in mice with cardiac ischemia. This study aimed to explore the benefits of HINT2 on cardiac microcirculation in I/R injury with a focus on mitochondria, the MCU complex, and [Ca2+]m overload in endothelial cells. The present work demonstrated that HINT2 overexpression significantly reduced the no-reflow area and improved microvascular perfusion in I/R-injured mouse hearts, potentially by promoting endothelial nitric oxide synthase (eNOS) expression and phosphorylation. Microvascular barrier function was compromised by reperfusion injury, but was repaired by HINT2 overexpression via inhibiting VE-Cadherin phosphorylation at Tyr731 and enhancing the VE-Cadherin/β-Catenin interaction. In addition, HINT2 overexpression inhibited the inflammatory response by suppressing vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Mitochondrial fission occurred in cardiac microvascular endothelial cells (CMECs) subjected to oxygen–glucose deprivation/reoxygenation (OGD/R) injury and resulted in mitochondrial dysfunction and mitochondrion-dependent apoptosis, the effects of which were largely relieved by HINT2 overexpression. Additional experiments confirmed that [Ca2+]m overload was an initiating factor for mitochondrial fission and that HINT2 suppressed [Ca2+]m overload via modulation of the MCU complex through directly interacting with MCU in CMECs. Regaining [Ca2+]m overload by spermine, an MCU agonist, abolished all the protective effects of HINT2 on OGD/R-injured CMECs and I/R-injured cardiac microcirculation. In conclusion, the present report demonstrated that HINT2 overexpression inhibited MCU complex-mitochondrial calcium overload-mitochondrial fission and apoptosis pathway, and thereby attenuated cardiac microvascular ischemia–reperfusion injury.