Expression of mRNA Encoding Mcu and Other Mitochondrial Calcium Regulatory Genes Depends on Cell Type, Neuronal Subtype, and Ca2+ Signaling.
ABSTRACT: Uptake of Ca2+ into the mitochondrial matrix controls cellular metabolism and survival-death pathways. Several genes are implicated in controlling mitochondrial Ca2+ uptake (mitochondrial calcium regulatory genes, MCRGs), however, less is known about the factors which influence their expression level. Here we have compared MCRG mRNA expression, in neural cells of differing type (cortical neurons vs. astrocytes), differing neuronal subtype (CA3 vs. CA1 hippocampus) and in response to Ca2+ influx, using a combination of qPCR and RNA-seq analysis. Of note, we find that the Mcu-regulating Micu gene family profile differs substantially between neurons and astrocytes, while expression of Mcu itself is markedly different between CA3 and CA1 regions in the adult hippocampus. Moreover, dynamic control of MCRG mRNA expression in response to membrane depolarization-induced Ca2+ influx is also apparent, resulting in repression of Letm1, as well as Mcu. Thus, the mRNA expression profile of MCRGs is not fixed, which may cause differences in the coupling between cytoplasmic and mitochondrial Ca2+, as well as diversity of mitochondrial Ca2+ uptake mechanisms.
Project description:Ca2+ influx into mitochondria is mediated by the mitochondrial calcium uniporter (MCU), whose identity was recently revealed as a 40-kDa protein that along with other proteins forms the mitochondrial Ca2+ uptake machinery. The MCU is a Ca2+-conducting channel spanning the inner mitochondrial membrane. Here, deletion of the MCU completely inhibited Ca2+ uptake in liver, heart, and skeletal muscle mitochondria. However, in brain nonsynaptic and synaptic mitochondria from neuronal somata/glial cells and nerve terminals, respectively, the MCU deletion slowed, but did not completely block, Ca2+ uptake. Under resting conditions, brain MCU-KO mitochondria remained polarized, and in brain MCU-KO mitochondria, the electrophoretic Ca2+ ionophore ETH129 significantly accelerated Ca2+ uptake. The residual Ca2+ uptake in brain MCU-KO mitochondria was insensitive to inhibitors of mitochondrial Na+/Ca2+ exchanger and ryanodine receptor (CGP37157 and dantrolene, respectively), but was blocked by the MCU inhibitor Ru360. Respiration of WT and MCU-KO brain mitochondria was similar except that for mitochondria that oxidized pyruvate and malate, Ca2+ more strongly inhibited respiration in WT than in MCU-KO mitochondria. Of note, the MCU deletion significantly attenuated but did not completely prevent induction of the permeability transition pore (PTP) in brain mitochondria. Expression level of cyclophilin D and ATP content in mitochondria, two factors that modulate PTP induction, were unaffected by MCU-KO, whereas ADP was lower in MCU-KO than in WT brain mitochondria. Our results suggest the presence of an MCU-independent Ca2+ uptake pathway in brain mitochondria that mediates residual Ca2+ influx and induction of PTP in a fraction of the mitochondrial population.
Project description:Calcium (Ca2+) dynamics and oxidative signaling control mitochondrial bioenergetics in the central nervous system, where astrocytes are a major energy source for neurons. Cocaine use exacerbates HIV-associated neurocognitive disorders, but little is known about disruptions in astrocyte metabolism in this context. Our data show that the HIV protein Tat and cocaine induce a metabolic switch from glucose to fatty acid oxidation in astrocytes, thereby limiting lactate transport to neurons. Mechanistic analyses revealed increased Mitochondrial Ca2+ Uniporter (MCU)-mediated Ca2+ uptake in astrocytes exposed to Tat and cocaine due to oxidation of MCU. Since our data suggest that mitochondrial oxidation is dependent in part on MCU-mediated Ca2+ uptake, we targeted MCU to restore glycolysis in astrocytes to normalize extracellular lactate levels. Knocking down MCU in astrocytes prior to Tat and cocaine exposure prevented metabolic switching and protected neurons. These findings identify a novel molecular mechanism underlying neuropathogenesis in HIV and cocaine use.
Project description:Saturated fatty acids contribute to ?-cell dysfunction in the onset of type 2 diabetes mellitus. Cellular responses to lipotoxicity include oxidative stress, endoplasmic reticulum (ER) stress, and blockage of autophagy. Palmitate induces ER Ca2+ depletion followed by notable store-operated Ca2+ entry. Subsequent elevation of cytosolic Ca2+ can activate undesirable signaling pathways culminating in cell death. Mitochondrial Ca2+ uniporter (MCU) is the major route for Ca2+ uptake into the matrix and couples metabolism with insulin secretion. However, it has been unclear whether mitochondrial Ca2+ uptake plays a protective role or contributes to lipotoxicity. Here, we observed palmitate upregulated MCU protein expression in a mouse clonal ?-cell, MIN6, under normal glucose, but not high glucose medium. Palmitate elevated baseline cytosolic Ca2+ concentration ([Ca2+]i) and reduced depolarization-triggered Ca2+ influx likely due to the inactivation of voltage-gated Ca2+ channels (VGCCs). Targeted reduction of MCU expression using RNA interference abolished mitochondrial superoxide production but exacerbated palmitate-induced [Ca2+]i overload. Consequently, MCU knockdown aggravated blockage of autophagic degradation. In contrast, co-treatment with verapamil, a VGCC inhibitor, prevented palmitate-induced basal [Ca2+]i elevation and defective [Ca2+]i transients. Extracellular Ca2+ chelation as well as VGCC inhibitors effectively rescued autophagy defects and cytotoxicity. These observations suggest enhanced mitochondrial Ca2+ uptake via MCU upregulation is a mechanism by which pancreatic ?-cells are able to alleviate cytosolic Ca2+ overload and its detrimental consequences.
Project description:Excitotoxic mechanisms contribute to the degeneration of hippocampal pyramidal neurons after recurrent seizures and brain ischemia. However, susceptibility differs, with CA1 neurons degenerating preferentially after global ischemia and CA3 neurons after limbic seizures. Whereas most studies address contributions of excitotoxic Ca2+ entry, it is apparent that Zn2+ also contributes, reflecting accumulation in neurons either after synaptic release and entry through postsynaptic channels or upon mobilization from intracellular Zn2+-binding proteins such as metallothionein-III (MT-III). Using mouse hippocampal slices to study acute oxygen glucose deprivation (OGD)-triggered neurodegeneration, we found evidence for early contributions of excitotoxic Ca2+ and Zn2+ accumulation in both CA1 and CA3, as indicated by the ability of Zn2+ chelators or Ca2+ entry blockers to delay pyramidal neuronal death in both regions. However, using knock-out animals (of MT-III and vesicular Zn2+ transporter, ZnT3) and channel blockers revealed substantial differences in relevant Zn2+ sources, with critical contributions of presynaptic release and its permeation through Ca2+- (and Zn2+)-permeable AMPA channels in CA3 and Zn2+ mobilization from MT-III predominating in CA1. To assess the consequences of the intracellular Zn2+ accumulation, we used OGD exposures slightly shorter than those causing acute neuronal death; under these conditions, cytosolic Zn2+ rises persisted for 10-30 min after OGD, followed by recovery over ?40-60 min. Furthermore, the recovery appeared to be accompanied by mitochondrial Zn2+ accumulation (via the mitochondrial Ca2+ uniporter MCU) in CA1 but not in CA3 neurons and was markedly diminished in MT-III knock-outs, suggesting that it depended upon Zn2+ mobilization from this protein.The basis for the differential vulnerabilities of CA1 versus CA3 pyramidal neurons is unclear. The present study of events during and after acute oxygen glucose deprivation highlights a possible important difference, with rapid synaptic entry of Ca2+ and Zn2+ contributing more in CA3, but with delayed and long-lasting accumulation of Zn2+ within mitochondria occurring in CA1 but not CA3 pyramidal neurons. These data may be consistent with observations of prominent mitochondrial dysfunction as a critical early event in the delayed degeneration of CA1 neurons after ischemia and support a hypothesis that mitochondrial Zn2+ accumulation in the early reperfusion period may be a critical and targetable upstream event in the injury cascade.
Project description:Mitochondrial calcium uptake proteins 1 and 2 (MICU1 and MICU2) mediate mitochondrial Ca2+ influx via the mitochondrial calcium uniporter (MCU). Its molecular action for Ca2+ uptake is tightly controlled by the MICU1-MICU2 heterodimer, which comprises Ca2+ sensing proteins which act as gatekeepers at low [Ca2+] or facilitators at high [Ca2+]. However, the mechanism underlying the regulation of the Ca2+ gatekeeping threshold for mitochondrial Ca2+ uptake through the MCU by the MICU1-MICU2 heterodimer remains unclear. In this study, we determined the crystal structure of the apo form of the human MICU1-MICU2 heterodimer that functions as the MCU gatekeeper. MICU1 and MICU2 assemble in the face-to-face heterodimer with salt bridges and me-thio-nine knobs stabilizing the heterodimer in an apo state. Structural analysis suggests how the heterodimer sets a higher Ca2+ threshold than the MICU1 homodimer. The structure of the heterodimer in the apo state provides a framework for understanding the gatekeeping role of the MICU1-MICU2 heterodimer.
Project description:MCU is a Ca2+-selective channel that mediates mitochondrial Ca2+ influx. The human channel contains tetrameric pore-forming MCU, regulatory subunits MICU1/2, and EMRE that is required both for channel function and MICU1/2-mediated Ca2+ regulation. A structure of MCU with EMRE revealed a 4:4 stoichiometry, but the stoichiometry in vivo is unknown. Expression of tagged EMRE and MCU at a 1:10 ratio in cells lacking EMRE and MCU restored channel activity but not full channel gatekeeping. Increasing EMRE expression enhanced gatekeeping, raising the cytoplasmic Ca2+ concentration ([Ca2+]c) threshold for channel activation. MCU-EMRE concatemers creating channels with 1EMRE:4MCU restored Ca2+ uptake in cells, whereas cells expressing concatemers that enforced a 4EMRE:4MCU stoichiometry demonstrated enhanced channel gatekeeping. Concatemers enforcing 2EMRE/4MCU recapitulated the activity, gatekeeping, and size of endogenous channels. Thus, MCU does not require four EMRE, with most endogenous channels containing two, but complexes with 1-4 EMRE have activity with full or partial gatekeeping.
Project description:: Intracellular calcium (Ca2+) homeostasis plays a vital role in the preservation of skeletal muscle. In view of the well-maintained skeletal muscle found in Daurian ground squirrels (Spermophilus dauricus) during hibernation, we hypothesized that hibernators possess unique strategies of intracellular Ca2+ homeostasis. Here, cytoplasmic, sarcoplasmic reticulum (SR), and mitochondrial Ca2+ levels, as well as the potential Ca2+ regulatory mechanisms, were investigated in skeletal muscle fibers of Daurian ground squirrels at different stages of hibernation. The results showed that cytoplasmic Ca2+ levels increased in the skeletal muscle fibers during late torpor (LT) and inter-bout arousal (IBA), and partially recovered when the animals re-entered torpor (early torpor, ET). Furthermore, compared with levels in the summer active or pre-hibernation state, the activity and protein expression levels of six major Ca2+ channels/proteins were up-regulated during hibernation, including the store-operated Ca2+ entry (SOCE), ryanodine receptor 1 (RyR1), leucine zipper-EF-hand containing transmembrane protein 1 (LETM1), SR Ca2+ ATPase 1 (SERCA1), mitochondrial calcium uniporter complex (MCU complex), and calmodulin (CALM). Among these, the increased extracellular Ca2+ influx mediated by SOCE, SR Ca2+ release mediated by RyR1, and mitochondrial Ca2+ extrusion mediated by LETM1 may be triggers for the periodic elevation in cytoplasmic Ca2+ levels observed during hibernation. Furthermore, the increased SR Ca2+ uptake through SERCA1, mitochondrial Ca2+ uptake induced by MCU, and elevated free Ca2+ binding capacity mediated by CALM may be vital strategies in hibernating ground squirrels to attenuate cytoplasmic Ca2+ levels and restore Ca2+ homeostasis during hibernation. Compared with that in LT or IBA, the decreased extracellular Ca2+ influx mediated by SOCE and elevated mitochondrial Ca2+ uptake induced by MCU may be important mechanisms for the partial cytoplasmic Ca2+ recovery in ET. Overall, under extreme conditions, hibernating ground squirrels still possess the ability to maintain intracellular Ca2+ homeostasis.
Project description:OBJECTIVE:The main objective of this study is to define the mechanisms by which mitochondria control vascular smooth muscle cell (VSMC) migration and impact neointimal hyperplasia. APPROACH AND RESULTS:The multifunctional CaMKII (Ca2+/calmodulin-dependent kinase II) in the mitochondrial matrix of VSMC drove a feed-forward circuit with the mitochondrial Ca2+ uniporter (MCU) to promote matrix Ca2+ influx. MCU was necessary for the activation of mitochondrial CaMKII (mtCaMKII), whereas mtCaMKII phosphorylated MCU at the regulatory site S92 that promotes Ca2+ entry. mtCaMKII was necessary and sufficient for platelet-derived growth factor-induced mitochondrial Ca2+ uptake. This effect was dependent on MCU. mtCaMKII and MCU inhibition abrogated VSMC migration and mitochondrial translocation to the leading edge. Overexpression of wild-type MCU, but not MCU S92A, mutant in MCU-/- VSMC rescued migration and mitochondrial mobility. Inhibition of microtubule, but not of actin assembly, blocked mitochondrial mobility. The outer mitochondrial membrane GTPase Miro-1 promotes mitochondrial mobility via microtubule transport but arrests it in subcellular domains of high Ca2+ concentrations. In Miro-1-/- VSMC, mitochondrial mobility and VSMC migration were abolished, and overexpression of mtCaMKII or a CaMKII inhibitory peptide in mitochondria (mtCaMKIIN) had no effect. Consistently, inhibition of mtCaMKII increased and prolonged cytosolic Ca2+ transients. mtCaMKII inhibition diminished phosphorylation of focal adhesion kinase and myosin light chain, leading to reduced focal adhesion turnover and cytoskeletal remodeling. In a transgenic model of selective mitochondrial CaMKII inhibition in VSMC, neointimal hyperplasia was significantly reduced after vascular injury. CONCLUSIONS:These findings identify mitochondrial CaMKII as a key regulator of mitochondrial Ca2+ uptake via MCU, thereby controlling mitochondrial translocation and VSMC migration after vascular injury.
Project description:Due to its Ca2+ buffering capacity, the mitochondrion is one of the most important intracellular organelles in regulating Ca2+ dynamic oscillation. Mitochondrial calcium uniporter (MCU) is the primary mediator of Ca2+ influx into mitochondria, manipulating cell energy metabolism, ROS production, and programmed cell death, all of which are critical for carcinogenesis. The understanding of the uniporter complex was significantly boosted by recent groundbreaking discoveries that identified the uniporter pore-forming subunit MCU and its regulatory molecules, including MCU-dominant negative ? subunit (MCUb), essential MCU regulator (EMRE), MCU regulator 1 (MCUR1), mitochondrial calcium uptake (MICU) 1, MICU2, and MICU3. These provide the means and molecular platform to investigate MCU complex (uniplex)-mediated impaired Ca2+ signalling in physiology and pathology. This review aims to summarize the progress of the understanding regulatory mechanisms of uniplex, roles of uniplex-mediated Ca2+ signalling in cancer, and potential pharmacological inhibitors of MCU.
Project description:Rods and cones use intracellular Ca2+ to regulate many functions, including phototransduction and neurotransmission. The Mitochondrial Calcium Uniporter (MCU) complex is thought to be the primary pathway for Ca2+ entry into mitochondria in eukaryotes. We investigate the hypothesis that mitochondrial Ca2+ uptake via MCU influences phototransduction and energy metabolism in photoreceptors using a mcu-/- zebrafish and a rod photoreceptor-specific Mcu-/- mouse. Using genetically encoded Ca2+ sensors to directly examine Ca2+ uptake in zebrafish cone mitochondria, we found that loss of MCU reduces but does not eliminate mitochondrial Ca2+ uptake. Loss of MCU does not lead to photoreceptor degeneration, mildly affects mitochondrial metabolism, and does not alter physiological responses to light, even in the absence of the Na+/Ca2+, K+ exchanger. Our results reveal that MCU is dispensable for vertebrate photoreceptor function, consistent with its low expression and the presence of an alternative pathway for Ca2+ uptake into photoreceptor mitochondria.