Mitochondrial Calcium Uptake Research Articles

Ru360 Alleviates Postoperative Cognitive Dysfunction in Aged Mice by Inhibiting MCU-Mediated Mitochondrial Dysfunction.

Ru360, a selective inhibitor of mitochondrial calcium uptake, maintains mitochondrial calcium homeostasis. To evaluate whether mitochondrial calcium uniporter (MCU)-mediated mitochondrial function is associated with the pathological process of Postoperative cognitive dysfunction (POCD), elucidate its relationship with neuroinflammation, and observe whether the relevant pathological process can be improved with Ru360. Aged mice underwent experimental open abdominal surgery after anesthesia. Open field tests, Novel object recognition tests and Y Maze Tests were used to conduct behavioral experiments. The reactive oxygen species (ROS) content, the levels of inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), intra-mitochondrial calcium, mitochondrial membrane potential (MMP) and the activity of antioxidant superoxide dismutase (SOD) in the hippocampus of mice were detected using kits. The expression of proteins was detected using Western blot. After treatment with Ru360, MCU-mediated mitochondrial dysfunction was inhibited, neuroinflammation was reduced, and the learning ability of the mice was improved after surgery. Our study demonstrated that mitochondrial function plays a crucial role in the pathology of POCD, and using Ru360 to improve mitochondrial function may be a new and necessary direction for the treatment of POCD.

Reduced mitochondrial calcium uptake in macrophages is a major driver of inflammaging

Mitochondrial dysfunction is linked to age-associated inflammation or inflammaging, but underlying mechanisms are not understood. Analyses of 700 human blood transcriptomes revealed clear signs of age-associated low-grade inflammation. Among changes in mitochondrial components, we found that the expression of mitochondrial calcium uniporter (MCU) and its regulatory subunit MICU1, genes central to mitochondrial Ca2+ (mCa2+) signaling, correlated inversely with age. Indeed, mCa2+ uptake capacity of mouse macrophages decreased significantly with age. We show that in both human and mouse macrophages, reduced mCa2+ uptake amplifies cytosolic Ca2+ oscillations and potentiates downstream nuclear factor kappa B activation, which is central to inflammation. Our findings pinpoint the mitochondrial calcium uniporter complex as a keystone molecular apparatus that links age-related changes in mitochondrial physiology to systemic macrophage-mediated age-associated inflammation. The findings raise the exciting possibility that restoring mCa2+ uptake capacity in tissue-resident macrophages may decrease inflammaging of specific organs and alleviate age-associated conditions such as neurodegenerative and cardiometabolic diseases.

Decreased Expression of Mitochondrial Calcium Uptake Protein 1 Leads to Skeletal Muscle Dysfunction in Septic Mice

To observe the effect of sepsis on skeletal muscle function and to explore the role of skeletal muscle mitochondrial calcium uptake protein 1 (MICU1). A total of 40 specific-pathogen-free (SPF) healthy male C57BL/6J mice were randomly assigned to 4 groups, a sham operation group (Sham group, n=8), a sepsis modeling 6 h group (cecal ligation and puncture [CLP]-6 h group, n=10), a sepsis modeling 12 h group (CLP-12 h group, n=10), and a sepsis modeling 24 h group (CLP-24 h, n=12). The sepsis model was established by CLP. Mice in the Sham group only underwent laparotomic exploration of the cecum. Another 20 SPF mice were selected. The tibialis anterior muscle on one side was empty-transfected with adeno-associated virus (AAV) as controls (AAV-C), and the tibialis anterior muscle on the other side was transfected with AAV to enhance MICU1 expression (AAV-M). The mice were randomly assigned to two groups, a sham operation group (AAV-C-Sham and AAV-M-Sham, n=8) and a sepsis model 24 h group (AAV-C-CLP and AAV-M-CLP, n=12). The grip strength and compound muscle action potential (CMAP) of the tibialis anterior muscle were measured in each group at the corresponding time points. The levels of inflammatory factors, including tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6), in the skeletal muscle were measured by ELISA. The morphological changes of skeletal muscle cells were observed through H&E staining. The expression levels of MICU1 and muscle atrophy-related proteins, including muscle RING-finger containing protein 1 (MuRF1) and muscle atrophy Fbox protein (MAFbx), were determined by Western blot. The expression levels of MICU1 mRNA in skeletal muscle were determined by RT-qPCR. Compared with mice in the Sham group, mice in the CLP group showed decreased body weight ( P<0.05); their grip strength decreased with the prolongation of CLP modeling time ( P<0.05); the amplitude of CMAP decreased, showing prolonged duration and latency ( P<0.05); the expression levels of inflammatory factors, including TNF-α and IL-6, in skeletal muscle increased gradually ( P<0.05); the fiber diameter and cross-sectional area of skeletal muscle decreased gradually with the prolongation of modeling time ( P<0.05); the protein expression levels of MuRF1and MAFbx proteins increased gradually ( P<0.05); the expression levels of MICU1 protein and mRNA decreased gradually ( P<0.05). There was no significant difference in all indices between AAV-M-Sham and AAV-C-Sham groups ( P>0.05). Compared with mice in the AAV-C-CLP group, mice in the AAV-M-CLP group showed increased grip strength ( P<0.05); the amplitude of CMAP increased, showing shortened duration and latency ( P <0.05); the fiber diameter and cross-sectional area of skeletal muscle increased ( P<0.05); the expression levels of MuRF1and MAFbx decreased ( P<0.05). Sepsis leads to skeletal muscle dysfunction, which is related to the decrease in mitochondrial MICU1 expression.

Structure-Activity Relationships of Metal-Based Inhibitors of the Mitochondrial Calcium Uniporter.

The mitochondrial calcium uniporter (MCU) is a transmembrane protein that is responsible for mediating mitochondrial calcium (mCa2+ ) uptake. Given this critical function, the MCU has been implicated as an important target for addressing various human diseases. As such, there has a been growing interest in developing small molecules that can inhibit this protein. To date, metal coordination complexes, particularly multinuclear ruthenium complexes, are the most widely investigated MCU inhibitors due to both their potent inhibitory activities as well as their longstanding use for this application. Recent efforts have expanded the metal-based toolkit for MCU inhibition. This concept paper summarizes the development of new metal-based inhibitors of the MCU and their structure-activity relationships in the context of improving their potential for therapeutic use in managing human diseases related to mCa2+ dysregulation.

UCP2 deficiency impairs podocyte autophagy in diabetic nephropathy

ObjectivePodocytes have been indicated to be a critical factor for the development of diabetic kidney disease. Podocyte loss leads to irreversible glomerular injury and proteinuria in animal models. As terminal differentiated cells, autophagy is crucial for maintaining podocyte homeostasis. Previous studies have shown that Uncoupling proteins 2 (UCP2) regulate fatty acid metabolism, mitochondrial calcium uptake and reactive oxygen species (ROS) production. This study aimed to investigate whether UCP2 promote autophagy in podocyte and further explore the regulation mechanism of UCP2. MethodsFor podocyte-specific UCP2-KO mice, we cross bred UCP2fl/fl mouse strain with the podocin-Cre mice. Diabetic mice were obtained by daily intraperitoneally injections of 40 mg/kg streptozotocin for 3 days. After 6 weeks, mice were scarified, and kidney tissues were analyzed by histological stain, Western blot, Immunofluorescence, and immunohistochemistry. Also, urine samples were collected for protein quantification. For in vitro study, podocytes were primary cultured from UCP2fl/fl mouse or transfected with adeno-associated virus (AAV)-UCP2. ResultsDiabetic kidney showed elevated expression of UCP2 and specific ablation of UCP2 in podocyte aggravates diabetes-induced albuminuria and glomerulopathy. UCP2 protects hyperglycemia-induced podocyte injury by promoting autophagy in vivo and in vitro. Rapamycin treatment significantly ameliorates streptozotocin (STZ)-induced podocyte injury in UCP2−/− mice. ConclusionUCP2 expression in podocyte increased under diabetic condition and appeared to be an initial compensatory response. UCP2 deficiency in podocyte impaired autophagy and exacerbates podocyte injury and proteinuria in diabetic nephropathy.

Mitochondrial calcium uptake 3 mitigates cerebral amyloid angiopathy-related neuronal death and glial inflammation by reducing mitochondrial dysfunction.

Cerebral amyloid angiopathy (CAA) is characterized by the cerebrovascular amyloid-β (Aβ) accumulation, and always accompanied by Alzheimer's disease (AD). Mitochondrial dysfunction-associated cellular events including cell death, inflammation and oxidative stress are implicated in the progression of CAA. Unfortunately, the molecular mechanisms revealing CAA pathogenesis are still obscure, thus requiring further studies. Mitochondrial calcium uptake 3 (MICU3), a regulator of the mitochondrial Ca2+ uniporter (MCU), mediates various biological functions, but its expression and influence on CAA are largely unknown. In the present study, we found that MICU3 expression was gradually declined in cortex and hippocampus of Tg-SwDI transgenic mice. Using stereotaxic operation with AAV9 encoding MICU3, we showed that AAV-MICU3 improved the behavioral performances and cerebral blood flow (CBF) in Tg-SwDI mice, along with markedly reduced Aβ deposition through mediating Aβ metabolism process. Importantly, we found that AAV-MICU3 remarkably improved neuronal death and mitigated glial activation and neuroinflammation in cortex and hippocampus of Tg-SwDI mice. Furthermore, excessive oxidative stress, mitochondrial impairment and dysfunction, decreased ATP and mitochondrial DNA (mtDNA) were detected in Tg-SwDI mice, while being considerably ameliorated upon MICU3 over-expression. More importantly, our in vitro experiments suggested that MICU3-attenuated neuronal death, activation of glial cells and oxidative stress were completely abrogated upon PTEN induced putative kinase 1 (PINK1) knockdown, indicating that PINK1 was required for MICU3 to perform its protective effects against CAA. Mechanistic experiment confirmed an interaction between MICU3 and PINK1. Together, these findings demonstrated that MICU3-PINK1 axis may serve as a key target for CAA treatment mainly through improving mitochondrial dysfunction.

Mitochondrial Ca2+ overload due to altered proteostasis amplifies apoptosis in C2C12 myoblasts under hypoxia: Protective role of nanocurcumin formulation.

Severe hypoxia triggers apoptosis leads to myofibers loss and is attributable to impaired intracellular calcium (iCa2+ ) homeostasis, resulting in reduced muscle activity. Hypoxia increases intracellular Ca2+ by activating the release of Ca2+ from iCa2+ stores, however, the effect of increased [iCa2+ ] on the mitochondria of muscle cells at high-altitude hypoxia is largely unexplored. This study examined mitochondrial Ca2+ overload due to altered expression of mitochondrial calcium uptake 1 (MICU1), that is, a gatekeeper of the mitochondrial Ca2+ uniporter, impaired mitochondrial membrane potential (ΔΨm). p53 stabilization and its translocation to the mitochondria were observed following disrupted mitochondrial membrane integrity in myoblasts under hypoxia. Furthermore, the downstream effects of p53 led to the upregulation of proapoptotic proteins (Bax, Caspase-3, and cytochrome C) in myoblasts under hypoxia. Nanocurcumin-pyrroloquinoline quinone formulation (NCF; Indian patent no. 302877), developed to address hypoxia-induced consequences, was found to be beneficial in maintaining mitochondrial Ca2+ homeostasis and limiting p53 translocation into mitochondria under hypoxia in muscle myoblasts. NCF treatment also modulates heat shock proteins and apoptosis-regulating protein expression in myoblasts. Conclusively, we proposed that mitochondrial Ca2+ overload due to altered MICU1 expression intensifies apoptosis and mitochondrial dysfunctionality. The study also reported that NCF could improve mitochondrial [Ca2+ ] homeostasis and antiapoptotic ability in C2C12 myoblasts under hypoxia.

Downregulation of mitochondrial calcium uptake family 3 attenuates secondary brain injury after intracerebral hemorrhage in rats.

Intracerebral hemorrhage (ICH) is one type of stroke with a high incidence and mortality. Mitochondria provide energy for various life processes and regulate calcium-mediated signaling pathways by taking up calcium ions from cytoplasm. Mitochondrial calcium uptake family 3 (MICU3) is a tissue-specific enhancer of mitochondrial calcium uptake. The effects and mechanisms of MICU3 in ICH are unknown. In this study, we aimed to explore the role of MICU3 in ICH in rats and neuronal models. First, we constructed ICH model both in vivo and in vitro and observed increased expression of MICU3. Then lentivirus was transduced to knock down MICU3. We observed that knockdown of MICU3 significantly reduced mitochondrial uptake of calcium in primary neurons. Moreover, the downregulation of MICU3 attenuated cell apoptosis and decreased the accumulation of reactive oxygen species (ROS). Recovery of neurobehavioral and cognitive function also benefited from downregulation of MICU3. The findings demonstrated that MICU3 played an important role in cell apoptosis, oxidative stress, and maintenance of mitochondrial structure and function, and promoted rehabilitation of neurobehavior. In conclusion, MICU3 is expected to be a molecular marker and a potential therapeutic target for ICH.

Mild Traumatic Brain Injury Induces Mitochondrial Calcium Overload and Triggers the Upregulation of NCLX in the Hippocampus

Traumatic brain injury (TBI) is brain damage due to external forces. Mild TBI (mTBI) is the most common form of TBI, and repeated mTBI is a risk factor for developing neurodegenerative diseases. Several mechanisms of neuronal damage have been described in the cortex and hippocampus, including mitochondrial dysfunction. However, up until now, there have been no studies evaluating mitochondrial calcium dynamics. Here, we evaluated mitochondrial calcium dynamics in an mTBI model in mice using isolated hippocampal mitochondria for biochemical studies. We observed that 24 h after mTBI, there is a decrease in mitochondrial membrane potential and an increase in basal matrix calcium levels. These findings are accompanied by increased mitochondrial calcium efflux and no changes in mitochondrial calcium uptake. We also observed an increase in NCLX protein levels and calcium retention capacity. Our results suggest that under mTBI, the hippocampal cells respond by incrementing NCLX levels to restore mitochondrial function.

Physiological significance of tissue-specific MICU regulation of mitochondrial calcium uptake.

The mitochondrial calcium uniporter is a multi-subunit calcium channel that imports Ca2+ into mitochondria. Its MICU subunits (MICU1, MICU2, and the neuron-specific MICU3) gate the channel by blocking the pore in low Ca2+. Upon local Ca2+ elevation, Ca2+ binds to MICUs to cause MICU unblock, thus opening the pore so Ca2+ can permeate. Previous work using cell lines suggests that the uniporter in mammalian cells is exclusively regulated by a MICU1-MICU2 heterodimer. However, we show here that multiple types of electrically excitable cells, including skeletal muscle and cardiac tissues, can also possess a MICU1-MICU1 homodimer or virtually no MICUs. Kinetic analyses demonstrate that MICU1 has a higher Ca2+ affinity than MICU2, and that without MICUs the uniporter is constitutively open. As a result, uniporters with the MICU1-1 homodimer or no MICUs exhibit higher transport activities, leading to mitochondria accumulating much higher levels of matrix Ca2+. Using a Seahorse assay, we show that cells with MICU1-1 or no MICUs have impaired basal oxidative phosphorylation, likely due to increased ROS and damaged respiratory-complex proteins, including NDUFS3 and COX2. These cells, moreover, are highly susceptible to apoptosis. The disadvantage of employing MICU1-1 or omitting MICUs, however, accompanies strong physiological benefits. We show that in response to intracellular Ca2+ signals, these mitochondria import more Ca2+ and consequently produce more ATP, thus better supplying the energy required for the cellular processes initiated by the Ca2+ signals. In conclusion, this work reveals that tissues can manipulate their mitochondrial calcium uptake properties to suit their unique physiological needs by customizing their MICU regulation of the mitochondrial calcium uniporter.

Modulation of the mitochondrial Ca2+ uniporter complex subunit expression by different shear stress patterns in vascular endothelial cells

Mitochondrial calcium (m Ca2+ ) uptake occurs via the Mitochondrial Ca2+ Uniporter (MCU) complex and plays a critical role in mitochondrial dynamics, mitophagy, and apoptosis. MCU complex activity is in part modulated by the expression of its regulatory subunits. Cardiovascular disease models demonstrated altered gene/protein expression of one or multiple subunits in different cells, including vascular endothelial cells (ECs). MCU complex activity was found necessary for stable flow (s-flow)-induced mitophagy and promotion of an atheroprotective EC phenotype. Disturbed flow (d-flow) is known to lead to an atheroprone phenotype. Despite the role of MCU in flow-regulated EC function, flow-induced alterations in MCU complex subunit expression are currently unknown. We exposed cultured human ECs to atheroprotective (steady shear stress, SS) or atheroprone flow (oscillatory shear stress, OS) and measured mRNA and protein levels of the MCU complex members. SS and OS differentially modulated subunit expression at gene/protein levels. Protein expression changes of the core MCU, m Ca2+ uptake 1 (MICU1) and MCU regulator 1 (MCUR1) subunits in SS- and OS-exposed, compared to static, ECs suggested an enhanced m Ca2+ influx under each flow and a potential contribution to EC dysfunction under OS. In silico analysis of a single-cell RNA-sequencing dataset was employed to extract transcript values of MCU subunits in mouse carotid ECs from regions exposed to s-flow or d-flow. Mcu and Mcur1 genes showed significant differences in expression after prolonged exposure to each flow. The differential expression of MCU complex subunits indicated a tight regulation of the complex activity under physiological and pathological hemodynamic conditions.

Dinuclear nitrido-bridged osmium complexes inhibit the mitochondrial calcium uniporter and protect cortical neurons against lethal oxygen-glucose deprivation.

Dysregulation of mitochondrial calcium uptake mediated by the mitochondrial calcium uniporter (MCU) is implicated in several pathophysiological conditions. Dinuclear ruthenium complexes are effective inhibitors of the MCU and have been leveraged as both tools to study mitochondrial calcium dynamics and potential therapeutic agents. In this study, we report the synthesis and characterization of Os245 ([Os2(μ-N)(NH3)8Cl2]3+) which is the osmium-containing analogue of our previously reported ruthenium-based inhibitor Ru265. This complex and its aqua-capped analogue Os245' ([Os2(μ-N)(NH3)8(OH2)2]5+) are both effective inhibitors of the MCU in permeabilized and intact cells. In comparison to the ruthenium-based inhibitor Ru265 (k obs = 4.92 × 10-3 s-1), the axial ligand exchange kinetics of Os245 are two orders of magnitude slower (k obs = 1.63 × 10-5 s-1) at 37 °C. The MCU-inhibitory properties of Os245 and Os245' are different (Os245 IC50 for MCU inhibition = 103 nM; Os245' IC50 for MCU inhibition = 2.3 nM), indicating that the axial ligands play an important role in their interactions with this channel. We further show that inhibition of the MCU by these complexes protects primary cortical neurons against lethal oxygen-glucose deprivation. When administered in vivo to mice (10 mg kg-1), Os245 and Os245' induce seizure-like behaviors in a manner similar to the ruthenium-based inhibitors. However, the onset of these seizures is delayed, a possible consequence of the slower ligand substitution kinetics for these osmium complexes. These findings support previous studies that demonstrate inhibition of the MCU is a promising therapeutic strategy for the treatment of ischemic stroke, but also highlight the need for improved drug delivery strategies to mitigate the pro-convulsant effects of this class of complexes before they can be implemented as therapeutic agents. Furthermore, the slower ligand substitution kinetics of the osmium analogues may afford new strategies for the development and modification of this class of MCU inhibitors.

Regulatory mechanisms of mitochondrial calcium uptake by the calcium uniporter complex.

Mitochondria play an important role in energy conversion as well as in intracellular calcium (Ca2+) storage. Ca2+ uptake from the cytosol to the mitochondria is mediated by the calcium uniporter, which functions as a Ca2+ ion channel. However, the molecular composition of this uniporter has remained unclear until recently. The Ca2+ ion channel consists of seven subunits. The yeast reconstitution technique revealed that the mitochondrial calcium uniporter (MCU) and essential MCU regulatory element (EMRE) are the core subunits of the complex. Furthermore, detailed structure-function analyses of the core subunits (MCU and EMRE) were performed. In this review, the regulatory mechanism of mitochondrial Ca2+ uptake is discussed.

Genetic Manipulation of CB1 Cannabinoid Receptors Reveals a Role in Maintaining Proper Skeletal Muscle Morphology and Function in Mice.

The endocannabinoid system (ECS) refers to a widespread signaling system and its alteration is implicated in a growing number of human diseases. Cannabinoid receptors (CBRs) are highly expressed in the central nervous system and many peripheral tissues. Evidence suggests that CB1Rs are expressed in human and murine skeletal muscle mainly in the cell membrane, but a subpopulation is present also in the mitochondria. However, very little is known about the latter population. To date, the connection between the function of CB1Rs and the regulation of intracellular Ca2+ signaling has not been investigated yet. Tamoxifen-inducible skeletal muscle-specific conditional CB1 knock-down (skmCB1-KD, hereafter referred to as Cre+/-) mice were used in this study for functional and morphological analysis. After confirming CB1R down-regulation on the mRNA and protein level, we performed in vitro muscle force measurements and found that peak twitch, tetanus, and fatigue were decreased significantly in Cre+/- mice. Resting intracellular calcium concentration, voltage dependence of the calcium transients as well as the activity dependent mitochondrial calcium uptake were essentially unaltered by Cnr1 gene manipulation. Nevertheless, we found striking differences in the ultrastructural architecture of the mitochondrial network of muscle tissue from the Cre+/- mice. Our results suggest a role of CB1Rs in maintaining physiological muscle function and morphology. Targeting ECS could be a potential tool in certain diseases, including muscular dystrophies where increased endocannabinoid levels have already been described.

Manipulation of cardiomyocyte MCU expression reveals distinct consequences of mitochondrial calcium uptake in early versus late adaptation to sustained stress

Rapid mitochondrial calcium (mCa2+) uptake through the mitochondrial calcium uniporter channel (mtCU) matches ATP production to acute increases in cardiac workload and energy demand, but excessive Ca2+ uptake during stress such as ischemia-reperfusion injury initiates permeability transition and cell death.

Carboxylate-Capped Analogues of Ru265 Are MCU Inhibitor Prodrugs.

The mitochondrial calcium uniporter (MCU) is a transmembrane protein that resides on the inner membrane of the mitochondria and mediates calcium uptake into this organelle. Given the critical role of mitochondrial calcium trafficking in cellular function, inhibitors of this channel have arisen as tools for studying the biological relevance of this process and as potential therapeutic agents. In this study, four new analogues of the previously reported Ru-based MCU inhibitor [ClRu(NH3)4(μ-N)Ru(NH3)4Cl]Cl3 (Ru265) are reported. These compounds, which bear axial carboxylate ligands, are of the general formula [(RCO2)Ru(NH3)4(μ-N)Ru(NH3)4(O2CR)]X3, where X = NO3- or CF3SO3- and R = H (1), CH3 (2), CH2CH3 (3), and (CH2)2CH3 (4). These complexes were fully characterized by IR spectroscopy, NMR spectroscopy, and elemental analysis. X-ray crystal structures of 1 and 3 were obtained, revealing the expected presence of both the linear Ru(μ-N)Ru core and axial formate and propionate ligands. The axial carboxylate ligands of complexes 1-4 are displaced by water in buffered aqueous solution to give the aquated compound Ru265'. The kinetics of these processes were measured by 1H NMR spectroscopy, revealing half-lives that span 5.9-9.9 h at 37 °C. Complex 1 with axial formate ligands underwent aquation approximately twice as fast as the other compounds. In vitro cytotoxicity and mitochondrial membrane potential measurements carried out in HeLa and HEK293T cells demonstrated that none of these four complexes negatively affects cell viability or mitochondrial function. The abilities of 1-4 to inhibit mitochondrial calcium uptake in permeabilized HEK293T cells were assessed and compared to that of Ru265. Fresh solutions of 1-4 are approximately 2-fold less potent than Ru265 with IC50 values in the range of 14.7-19.1 nM. Preincubating 1-4 in aqueous buffers for longer time periods to allow for the aquation reactions to proceed increases their potency of mitochondrial uptake inhibition to match that of Ru265. This result indicates that 1-4 are aquation-activated prodrugs of Ru265'. Finally, 1-4 were shown to inhibit mitochondrial calcium uptake in intact, nonpermeabilized cells, revealing their value as tools and potential therapeutic agents for mitochondrial calcium-related disorders.

Understanding the structural basis of mitochondrial calcium uptake by mitochondrial calcium uniporter

Understanding the structural basis of mitochondrial calcium uptake by mitochondrial calcium uniporter

MICU1 and MICU2 potentiation of Ca2+ uptake by the mitochondrial Ca2+ uniporter of Trypanosoma cruzi and its inhibition by Mg2+

The mitochondrial Ca2+ uptake, which is important to regulate bioenergetics, cell death and cytoplasmic Ca2+ signaling, is mediated via the calcium uniporter complex (MCUC). In animal cells the MCUC is regulated by the mitochondrial calcium uptake 1 and 2 dimer (MICU1/MICU2), which has been proposed to act as gatekeeper preventing mitochondrial Ca2+ overload at low cytosolic Ca2+ levels. In contrast to animal cells, knockout of either MICU1 or MICU2 in Trypanosoma cruzi, the etiologic agent of Chagas disease, did not allow Ca2+ uptake at low extramitochondrial Ca2+ concentrations ([Ca2+]ext) and it was though that in the absence of one MICU the other would replace its role. However, previous attempts to knockout both genes were unsuccessful. Here, we designed a strategy to generate TcMICU1/TcMICU2 double knockout cell lines using CRISPR/Cas9 genome editing. Ablation of both genes was confirmed by PCR and Southern blot analyses. The absence of both proteins did not allow Ca2+ uptake at low [Ca2+]ext, significantly decreased the mitochondrial Ca2+ uptake at different [Ca2+]ext, without dissipation of the mitochondrial membrane potential, and increased the [Ca2+]ext set point needed for Ca2+ uptake, as we have seen with TcMICU1-KO and TcMICU2-KO cells. Mg2+ was found to be a negative regulator of MCUC-mediated mitochondrial Ca2+ uptake at different [Ca2+]ext. Occlusion of the MCUC pore by Mg2+ could partially explain the lack of mitochondrial Ca2+ uptake at low [Ca2+]ext in TcMICU1/TcMICU2-KO cells. In addition, TcMICU1/TcMICU2-KO epimastigotes had a lower growth rate, while infective trypomastigotes have a reduced capacity to invade host cells and to replicate within them as amastigotes.

PolyGA targets the ER stress-adaptive response by impairing GRP75 function at the MAM in C9ORF72-ALS/FTD

ER stress signaling is linked to the pathophysiological and clinical disease manifestations in amyotrophic lateral sclerosis (ALS). Here, we have investigated ER stress-induced adaptive mechanisms in C9ORF72-ALS/FTD, focusing on uncovering early endogenous neuroprotective mechanisms and the crosstalk between pathological and adaptive responses in disease onset and progression. We provide evidence for the early onset of ER stress-mediated adaptive response in C9ORF72 patient-derived motoneurons (MNs), reflected by the elevated increase in GRP75 expression. These transiently increased GRP75 levels enhance ER–mitochondrial association, boosting mitochondrial function and sustaining cellular bioenergetics during the initial stage of disease, thereby counteracting early mitochondrial deficits. In C9orf72 rodent neurons, an abrupt reduction in GRP75 expression coincided with the onset of UPR, mitochondrial dysfunction and the emergence of PolyGA aggregates, which co-localize with GRP75. Similarly, the overexpression of PolyGA in WT cortical neurons or C9ORF72 patient-derived MNs led to the sequestration of GRP75 within PolyGA inclusions, resulting in mitochondrial calcium (Ca2+) uptake impairments. Corroborating these findings, we found that PolyGA aggregate-bearing human post-mortem C9ORF72 hippocampal dentate gyrus neurons not only display reduced expression of GRP75 but also exhibit GRP75 sequestration within inclusions. Sustaining high GRP75 expression in spinal C9orf72 rodent MNs specifically prevented ER stress, normalized mitochondrial function, abrogated PolyGA accumulation in spinal MNs, and ameliorated ALS-associated behavioral phenotype. Taken together, our results are in line with the notion that neurons in C9ORF72-ALS/FTD are particularly susceptible to ER–mitochondrial dysfunction and that GRP75 serves as a critical endogenous neuroprotective factor. This neuroprotective pathway, is eventually targeted by PolyGA, leading to GRP75 sequestration, and its subsequent loss of function at the MAM, compromising mitochondrial function and promoting disease onset.

Piezo1 stimulates mitochondrial function via cAMP signaling.

Mechanical signals stimulate mitochondrial function but the molecular mechanisms are not clear. Here, we show that the mechanically sensitive ion channel Piezo1 plays a critical role in mitochondrial adaptation to mechanical stimulation. The activation of Piezo1 induced mitochondrial calcium uptake and oxidative phosphorylation (OXPHOS). In contrast, loss of Piezo1 reduced the mitochondrial oxygen consumption rate (OCR) and adenosine triphosphate (ATP) production in calvarial cells and these changes were associated with increased expression of the phosphodiesterases Pde4a and lower cyclic AMP (cAMP) levels. In addition, Piezo1 increased cAMP production and the activation of a cAMP-responsive transcriptional reporter. Consistent with this, cAMP was sufficient to increase mitochondrial OCR and the inhibition of phosphodiesterases augmented the increase in OCR induced by Piezo1. Moreover, the inhibition of cAMP production or activity of protein kinase A, a kinase activated by cAMP, prevented the increase in OCR induced by Piezo1. These results demonstrate that cAMP signaling contributes to the increase in mitochondrial OXPHOS induced by activation of Piezo1.