Loss of Mitochondrial Ca2+ Uniporter Limits Inotropic Reserve and Provides Trigger and Substrate for Arrhythmias in Barth Syndrome Cardiomyopathy.

BACKGROUND: Barth syndrome (BTHS) is caused by mutations of the gene encoding tafazzin, which catalyzes maturation of mitochondrial cardiolipin and often manifests with systolic dysfunction during early infancy. Beyond the first months of life, BTHS cardiomyopathy typically transitions to a phenotype of diastolic dysfunction with preserved ejection fraction, blunted contractile reserve during exercise, and arrhythmic vulnerability. Previous studies traced BTHS cardiomyopathy to mitochondrial formation of reactive oxygen species (ROS). Because mitochondrial function and ROS formation are regulated by excitation-contraction coupling, integrated analysis of mechano-energetic coupling is required to delineate the pathomechanisms of BTHS cardiomyopathy. METHODS: We analyzed cardiac function and structure in a mouse model with global knockdown of tafazzin (Taz-KD) compared with wild-type littermates. Respiratory chain assembly and function, ROS emission, and Ca2+ uptake were determined in isolated mitochondria. Excitation-contraction coupling was integrated with mitochondrial redox state, ROS, and Ca2+ uptake in isolated, unloaded or preloaded cardiac myocytes, and cardiac hemodynamics analyzed in vivo. RESULTS: Taz-KD mice develop heart failure with preserved ejection fraction (>50%) and age-dependent progression of diastolic dysfunction in the absence of fibrosis. Increased myofilament Ca2+ affinity and slowed cross-bridge cycling caused diastolic dysfunction, in part, compensated by accelerated diastolic Ca2+ decay through preactivated sarcoplasmic reticulum Ca2+-ATPase. Taz deficiency provoked heart-specific loss of mitochondrial Ca2+ uniporter protein that prevented Ca2+-induced activation of the Krebs cycle during ß-adrenergic stimulation, oxidizing pyridine nucleotides and triggering arrhythmias in cardiac myocytes. In vivo, Taz-KD mice displayed prolonged QRS duration as a substrate for arrhythmias, and a lack of inotropic response to ß-adrenergic stimulation. Cellular arrhythmias and QRS prolongation, but not the defective inotropic reserve, were restored by inhibiting Ca2+ export through the mitochondrial Na+/Ca2+ exchanger. All alterations occurred in the absence of excess mitochondrial ROS in vitro or in vivo. CONCLUSIONS: Downregulation of mitochondrial Ca2+ uniporter, increased myofilament Ca2+ affinity, and preactivated sarcoplasmic reticulum Ca2+-ATPase provoke mechano-energetic uncoupling that explains diastolic dysfunction and the lack of inotropic reserve in BTHS cardiomyopathy. Furthermore, defective mitochondrial Ca2+ uptake provides a trigger and a substrate for ventricular arrhythmias. These insights can guide the ongoing search for a cure of this orphaned disease.

Cdc42 localized in the CatSper signaling complex regulates cAMP-dependent pathways in mouse sperm.

Sperm acquire the ability to fertilize in a process called capacitation and undergo hyperactivation, a change in the motility pattern, which depends on Ca2+ transport by CatSper channels. CatSper is essential for fertilization and it is subjected to a complex regulation that is not fully understood. Here, we report that similar to CatSper, Cdc42 distribution in the principal piece is confined to four linear domains and this localization is disrupted in CatSper1-null sperm. Cdc42 inhibition impaired CatSper activity and other Ca2+ -dependent downstream events resulting in a severe compromise of the sperm fertilizing potential. We also demonstrate that Cdc42 is essential for CatSper function by modulating cAMP production by soluble adenylate cyclase (sAC), providing a new regulatory mechanism for the stimulation of CatSper by the cAMP-dependent pathway. These results reveal a broad mechanistic insight into the regulation of Ca2+ in mammalian sperm, a matter of critical importance in male infertility as well as in contraception.

The α2δ-1/NMDA receptor complex is involved in brain injury after intracerebral hemorrhage in mice.

BACKGROUND: Intracerebral hemorrhage (ICH), a common cerebrovascular disease, seriously threatens human health and has severe secondary injuries, while existing treatment methods have many limitations. α2δ-1 is a subunit of voltage-gated Ca2+ channels (VGCCs) and can act on glutamate receptor N-methyl-D-aspartate receptors (NMDARs) to relieve neuropathic pain. METHODS: We first performed ICH modeling on WT mice and Cacna2d1 knockout (KO) mice. The expression levels of GluN1 and α2δ-1 were measured by Western blot and q-PCR, and the interaction between the two proteins was evaluated by co-precipitation. The neuronal apoptosis was detected by the TUNEL assay, and the expression levels of inflammatory factors were assessed by ELISA. The nerve functions of mice were evaluated using behavioral experiments including corner turn test and forelimb use asymmetry. Cerebral hematoma was indicated by brain water content and lesion volume. RESULTS: ICH up-regulated the expression levels of α2δ-1 and GluN1. KO of Cacna2d1 significantly reduced the ICH-induced apoptosis. The treatment of gabapentin on α2δ-1 also significantly reduced the occurrence of apoptosis. KO of Cacna2d1 also reduced the ICH-induced levels of inflammatory factors. Furthermore, neural functions were also significantly improved. CONCLUSION: Cacna2d1 KO alleviates cerebral hematoma in ICH mice, exhibits a significant regulating effect on its secondary injuries such as neuronal apoptosis and inflammation, and restores the nerve functions of ICH mice. Loss of Cacna2d1 can provide useful therapeutic clues for ICH treatment.

Gene editing and synthetically accessible inhibitors reveal role for TPC2 in HCC cell proliferation and tumor growth.

The role of two-pore channel 2 (TPC2), one of the few cation channels localized on endolysosomal membranes, in cancer remains poorly understood. Here, we report that TPC2 knockout reduces proliferation of cancer cells in vitro, affects their energy metabolism, and successfully abrogates tumor growth in vivo. Concurrently, we have developed simplified analogs of the alkaloid tetrandrine as potent TPC2 inhibitors by screening a library of synthesized benzyltetrahydroisoquinoline derivatives. Removal of dispensable substructures of the lead molecule tetrandrine increases antiproliferative properties against cancer cells and impairs proangiogenic signaling of endothelial cells to a greater extent than tetrandrine. Simultaneously, toxic effects on non-cancerous cells are reduced, allowing in vivo administration and revealing a TPC2 inhibitor with antitumor efficacy in mice. Hence, our study unveils TPC2 as valid target for cancer therapy and provides easily accessible tetrandrine analogs as a promising option for effective pharmacological interference.

Cadmium disrupts mitochondrial distribution and activates excessive mitochondrial fission by elevating cytosolic calcium independent of MCU-mediated mitochondrial calcium uptake in its neurotoxicity.

Cadmium (Cd) is a ubiquitous environmental and occupational pollutant that is considered as a high-risk factor for neurodegenerative diseases. However, the mechanism underlying Cd-induced neurotoxicity has not been fully elucidated. Abnormal mitochondrial distribution and excessive mitochondrial fission are increasingly implicated in various neurological pathologies. Herein, by exposing primary cortical neurons to Cd (10 and 100 µM) for various times (0, 6, 12, and 24 h), we observed that the rapid motility of the mitochondria in neurons progressively slowed. Many more mitochondria were transported and distributed to the somas of Cd-treated neurons. Coupled with abnormal mitochondrial distribution, Cd exposure triggered excessive mitochondrial fragmentation, followed by mitochondrial membrane potential loss and neuronal damage. However, BAPTA-AM, a chelator of cytosolic calcium ([Ca2+]c), significantly attenuated Cd-induced abnormal mitochondrial distribution and excessive mitochondrial fission, which protected against Cd-induced mitochondrial damage and neuronal toxicity. In contrast to the increase in [Ca2+]c, Cd exposure had no effect on the level of mitochondrial calcium ([Ca2+]m). Inhibiting [Ca2+]m uptake, either by ruthenium 360 (Ru360) or by knock-out of mitochondrial calcium uniporter (MCU), failed to alleviate Cd-induced mitochondrial damage and neuronal toxicity. Additionally, in MCU knock-out neurons, BAPTA-AM effectively prevented Cd-induced abnormal mitochondrial distribution and excessive mitochondrial fission. Taken together, Cd exposure disrupts mitochondrial distribution and activates excessive mitochondrial fission by elevating [Ca2+]c independent of MCU-mediated mitochondrial calcium uptake, thereby leading to neurotoxicity. Chelating overloaded [Ca2+]c is a promising strategy to prevent the neurotoxicity of Cd.

The calcium channel TRPV6 is a novel regulator of RANKL-induced osteoclastic differentiation and bone absorption activity through the IGF-PI3K-AKT pathway.

OBJECTIVES: Calcium ion signals are important for osteoclast differentiation. Transient receptor potential vanilloid 6 (TRPV6) is a regulator of bone homeostasis. However, it was unclear whether TRPV6 was involved in osteoclast formation. Therefore, the aim of this study was to evaluate the role of TPRV6 in bone metabolism and to clarify its regulatory role in osteoclasts at the cellular level. MATERIALS AND METHODS: Bone structure and histological changes in Trpv6 knockout mice were examined using micro-computed tomography and histological analyses. To investigate the effects of Trpv6 on osteoclast function, we silenced or overexpressed Trpv6 in osteoclasts via lentivirus transfection, respectively. Osteoclast differentiation and bone resorption viability were measured by tartrate-resistant acid phosphatase (TRAP) staining and pit formation assays. The expression of osteoclast marker genes, including cathepsin k, DC-STAMP, Atp6v0d2 and TRAP, was measured by qRT-PCR. Cell immunofluorescence and Western blotting were applied to explore the mechanisms by which the IGF-PI3K-AKT pathway was involved in the regulation of osteoclast formation and bone resorption by Trpv6. RESULTS: We found that knockout of Trpv6 induced osteoporosis and enhanced bone resorption in mice, but did not affect bone formation. Further studies showed that Trpv6, which was distributed on the cell membrane of osteoclasts, acted as a negative regulator for osteoclast differentiation and function. Mechanistically, Trpv6 suppressed osteoclastogenesis by decreasing the ratios of phosphoprotein/total protein in the IGF-PI3K-AKT signalling pathway. Blocking of the IGF-PI3K-AKT pathway significantly alleviated the inhibitory effect of Trpv6 on osteoclasts formation. CONCLUSIONS: Our study confirmed the important role of Trpv6 in bone metabolism and clarified its regulatory role in osteoclasts at the cellular level. Taken together, this study may inspire a new strategy for the treatment of osteoporosis.

Neuronal mitochondrial calcium uniporter deficiency exacerbates axonal injury and suppresses remyelination in mice subjected to experimental autoimmune encephalomyelitis.

High-capacity mitochondrial calcium (Ca2+) uptake by the mitochondrial Ca2+ uniporter (MCU) is strategically positioned to support the survival and remyelination of axons in multiple sclerosis (MS) by undocking mitochondria, buffering Ca2+ and elevating adenosine triphosphate (ATP) synthesis at metabolically stressed sites. Respiratory chain deficits in MS are proposed to metabolically compromise axon survival and remyelination by suppressing MCU activity. In support of this hypothesis, clinical scores, mitochondrial dysfunction, myelin loss, axon damage and inflammation were elevated while remyelination was blocked in neuronal MCU deficient (Thy1-MCU Def) mice relative to Thy1 controls subjected to experimental autoimmune encephalomyelitis (EAE). At the first sign of walking deficits, mitochondria in EAE/Thy1 axons showed signs of activation. By contrast, cytoskeletal damage, fragmented mitochondria and large autophagosomes were seen in EAE/Thy1-MCU Def axons. As EAE severity increased, EAE/Thy1 axons were filled with massively swollen mitochondria with damaged cristae while EAE/Thy1-MCU Def axons were riddled with late autophagosomes. ATP concentrations and mitochondrial gene expression were suppressed while calpain activity, autophagy-related gene mRNA levels and autophagosome marker (LC3) co-localization in Thy1-expressing neurons were elevated in the spinal cords of EAE/Thy1-MCU Def compared to EAE/Thy1 mice. These findings suggest that MCU inhibition contributes to axonal damage that drives MS progression.

TPC1 deficiency or blockade augments systemic anaphylaxis and mast cell activity.

Mast cells and basophils are main drivers of allergic reactions and anaphylaxis, for which prevalence is rapidly increasing. Activation of these cells leads to a tightly controlled release of inflammatory mediators stored in secretory granules. The release of these granules is dependent on intracellular calcium (Ca2+) signals. Ca2+ release from endolysosomal compartments is mediated via intracellular cation channels, such as two-pore channel (TPC) proteins. Here, we uncover a mechanism for how TPC1 regulates Ca2+ homeostasis and exocytosis in mast cells in vivo and ex vivo. Notably, in vivo TPC1 deficiency in mice leads to enhanced passive systemic anaphylaxis, reflected by increased drop in body temperature, most likely due to accelerated histamine-induced vasodilation. Ex vivo, mast cell-mediated histamine release and degranulation was augmented upon TPC1 inhibition, although mast cell numbers and size were diminished. Our results indicate an essential role of TPC1 in endolysosomal Ca2+ uptake and filling of endoplasmic reticulum Ca2+ stores, thereby regulating exocytosis in mast cells. Thus, pharmacological modulation of TPC1 might blaze a trail to develop new drugs against mast cell-related diseases, including allergic hypersensitivity.

Cytosolic, but not matrix, calcium is essential for adjustment of mitochondrial pyruvate supply.

Mitochondrial oxidative phosphorylation (OXPHOS) and cellular workload are tightly balanced by the key cellular regulator, calcium (Ca2+). Current models assume that cytosolic Ca2+ regulates workload and that mitochondrial Ca2+ uptake precedes activation of matrix dehydrogenases, thereby matching OXPHOS substrate supply to ATP demand. Surprisingly, knockout (KO) of the mitochondrial Ca2+ uniporter (MCU) in mice results in only minimal phenotypic changes and does not alter OXPHOS. This implies that adaptive activation of mitochondrial dehydrogenases by intramitochondrial Ca2+ cannot be the exclusive mechanism for OXPHOS control. We hypothesized that cytosolic Ca2+, but not mitochondrial matrix Ca2+, may adapt OXPHOS to workload by adjusting the rate of pyruvate supply from the cytosol to the mitochondria. Here, we studied the role of malate-aspartate shuttle (MAS)-dependent substrate supply in OXPHOS responses to changing Ca2+ concentrations in isolated brain and heart mitochondria, synaptosomes, fibroblasts, and thymocytes from WT and MCU KO mice and the isolated working rat heart. Our results indicate that extramitochondrial Ca2+ controls up to 85% of maximal pyruvate-driven OXPHOS rates, mediated by the activity of the complete MAS, and that intramitochondrial Ca2+ accounts for the remaining 15%. Of note, the complete MAS, as applied here, included besides its classical NADH oxidation reaction the generation of cytosolic pyruvate. Part of this largely neglected mechanism has previously been described as the "mitochondrial gas pedal." Its implementation into OXPHOS control models integrates seemingly contradictory results and warrants a critical reappraisal of metabolic control mechanisms in health and disease.

MCUB Regulates the Molecular Composition of the Mitochondrial Calcium Uniporter Channel to Limit Mitochondrial Calcium Overload During Stress.

BACKGROUND: The mitochondrial calcium uniporter (mtCU) is an ≈700-kD multisubunit channel residing in the inner mitochondrial membrane required for mitochondrial Ca2+ (mCa2+) uptake. Here, we detail the contribution of MCUB, a paralog of the pore-forming subunit MCU, in mtCU regulation and function and for the first time investigate the relevance of MCUB to cardiac physiology. METHODS: We created a stable MCUB knockout cell line (MCUB-/-) using CRISPR-Cas9n technology and generated a cardiac-specific, tamoxifen-inducible MCUB mutant mouse (CAG-CAT-MCUB x MCM; MCUB-Tg) for in vivo assessment of cardiac physiology and response to ischemia/reperfusion injury. Live-cell imaging and high-resolution spectrofluorometery were used to determine intracellular Ca2+ exchange and size-exclusion chromatography; blue native page and immunoprecipitation studies were used to determine the molecular function and impact of MCUB on the high-molecular-weight mtCU complex. RESULTS: Using genetic gain- and loss-of-function approaches, we show that MCUB expression displaces MCU from the functional mtCU complex and thereby decreases the association of mitochondrial calcium uptake 1 and 2 (MICU1/2) to alter channel gating. These molecular changes decrease MICU1/2-dependent cooperative activation of the mtCU, thereby decreasing mCa2+ uptake. Furthermore, we show that MCUB incorporation into the mtCU is a stress-responsive mechanism to limit mCa2+ overload during cardiac injury. Indeed, overexpression of MCUB is sufficient to decrease infarct size after ischemia/reperfusion injury. However, MCUB incorporation into the mtCU does come at a cost; acute decreases in mCa2+ uptake impair mitochondrial energetics and contractile function. CONCLUSIONS: We detail a new regulatory mechanism to modulate mtCU function and mCa2+ uptake. Our results suggest that MCUB-dependent changes in mtCU stoichiometry are a prominent regulatory mechanism to modulate mCa2+ uptake and cellular physiology.

Cardiac-specific deficiency of the mitochondrial calcium uniporter augments fatty acid oxidation and functional reserve.

The mitochondrial calcium uniporter (MCU) relays cytosolic Ca2+ transients to the mitochondria. We examined whether energy metabolism was compromised in hearts from mice with a cardiac-specific deficiency of MCU subjected to an isoproterenol (ISO) challenge. Surprisingly, isolated working hearts from cardiac MCU-deficient mice showed higher cardiac work, both in the presence or absence of ISO. These hearts were not energy-starved, with ISO inducing a similar increase in glucose oxidation rates compared to control hearts, but a greater increase in fatty acid oxidation rates. This correlated with lower levels of the fatty acid oxidation inhibitor malonyl CoA, and to an increased stimulatory acetylation of its degrading enzyme malonyl CoA decarboxylase and of the fatty acid ß-oxidation enzyme ß-hydroxyacyl CoA dehydrogenase. We conclude that impaired mitochondrial Ca2+ uptake does not compromise cardiac energetics due to a compensatory stimulation of fatty acid oxidation that provides a higher energy reserve during acute adrenergic stress.

Cyclophilin D-mediated regulation of the permeability transition pore is altered in mice lacking the mitochondrial calcium uniporter.

Aims: Knockout (KO) of the mitochondrial Ca2+ uniporter (MCU) in mice abrogates mitochondrial Ca2+ uptake and permeability transition pore (PTP) opening. However, hearts from global MCU-KO mice are not protected from ischaemic injury. We aimed to investigate whether adaptive alterations occur in cell death signalling pathways in the hearts of global MCU-KO mice. Methods and results: First, we examined whether cell death may occur via an upregulation in necroptosis in MCU-KO mice. However, our results show that neither RIP1 inhibition nor RIP3 knockout afford protection against ischaemia-reperfusion injury in MCU-KO as in wildtype (WT) hearts, indicating that the lack of protection cannot be explained by upregulation of necroptosis. Instead, we have identified alterations in cyclophilin D (CypD) signalling in MCU-KO hearts. In the presence of a calcium ionophore, MCU-KO mitochondria take up calcium and do undergo PTP opening. Furthermore, PTP opening in MCU-KO mitochondria has a lower calcium retention capacity (CRC), suggesting that the calcium sensitivity of PTP is higher. Phosphoproteomics identified an increase in phosphorylation of CypD-S42 in MCU-KO. We investigated the interaction of CypD with the putative PTP component ATP synthase and identified an approximately 50% increase in this interaction in MCU-KO cardiac mitochondria. Mutation of the novel CypD phosphorylation site S42 to a phosphomimic reduced CRC, increased CypD-ATP synthase interaction by approximately 50%, and increased cell death in comparison to a phospho-resistant mutant. Conclusion: Taken together these data suggest that MCU-KO mitochondria exhibit an increase in phosphorylation of CypD-S42 which decreases PTP calcium sensitivity thus allowing activation of PTP in the absence of an MCU-mediated increase in matrix calcium.

TRPV2 is required for mechanical nociception and the stretch-evoked response of primary sensory neurons.

Mechanotransduction plays important roles in many sensory processes, including touch, pain, hearing, and proprioception. However, the molecular mechanisms of mechanical nociception have remained unclear. Here, we showed that elimination of transient receptor potential vanilloid 2 (TRPV2) in mice resulted in the deficit of mechanical nociception due to the lack of mechanosensitivity in a subclass of adult primary sensory neurons (PSNs). The PSN-specific TRPV2-deficient mice showed behavioural impairment of mechanical nociception in tail-pressure and von Frey hair tests, without defects in axonal growth and neuronal composition. Conversely, the mice displayed normal behaviour to noxious heat and non-noxious tactile stimuli. Furthermore, based on the stretch-evoked Ca2+ response of cultured PSNs, we characterised two types of stretch-activated neurons in normal mice; fast-decay high-threshold and slow-decay low-threshold mechanosensitive. The cultured neurons from TRPV2-deficient mice lacked stretch-evoked Ca2+ responses by fast-decay neurons normally activated by high-threshold mechanical stimulation. These results demonstrated that TRPV2 has a critical role in mechanical nociception in the adult somatosensory system.

Deficiency of PKD2L1 (TRPP3) Exacerbates Pathological Cardiac Hypertrophy by Augmenting NCX1-Mediated Mitochondrial Calcium Overload.

High salt intake is one independent risk factor for cardiac hypertrophy. Polycystic kidney disease 2-like 1 (PKD2L1, also called TRPP3) acts as a sour sensor in taste cells, and its possible role in the cardiovascular system is unknown. Here, we report that knockout of PKD2L1 exacerbated high-salt diet (HSD)-induced cardiac hypertrophy and fibrosis, accompanied by cardiac dysfunction and reduced cardiac mitochondrial oxidative phosphorylation and enzyme activity. Furthermore, knockdown of PKD2L1 led to more serious mitochondrial Ca2+ overload and reduced Ca2+ uptake in cardiomyocytes on high salt loading. Mechanistically, PKD2L1 deficiency increased p300-mediated acetylation of histone 3 lysine 27 on the promoter of sodium/calcium exchange 1 (NCX1) by repressing AMP-activated protein kinase (AMPK) activity, resulting in NCX1 overexpression and mitochondrial Ca2+ overload. These results reveal an inhibitory effect of PKD2L1 on cardiac hypertrophy and provide a mechanistic insight into the link between mitochondrial Ca2+ homeostasis and cardiac hypertrophy.

Deletion of mitochondrial calcium uniporter incompletely inhibits calcium uptake and induction of the permeability transition pore in brain mitochondria.

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.

Greater reductions in fat preferences in CALHM1 than CD36 knockout mice.

Several studies indicate an important role of gustation in intake and preference for dietary fat. The present study compared fat preference deficits produced by deletion of CD36, a putative fatty acid taste receptor, and CALHM1, an ion channel responsible for release of the ATP neurotransmitter used by taste cells. Naïve CD36 knockout (KO) mice displayed reduced preferences for soybean oil emulsions (Intralipid) at low concentrations (0.1-1%) compared with wild-type (WT) mice in 24 h/day two-bottle tests. CALHM1 KO mice displayed even greater Intralipid preference deficits compared with WT and CD36 KO mice. These findings indicate that there may be another taste receptor besides CD36 that contributes to fat detection and preference. After experience with concentrated fat (2.5-5%), CD36 KO and CALHM1 KO mice displayed normal preferences for 0.1-5% fat, although they still consumed less fat than WT mice. The experience-induced rescue of fat preferences in KO mice can be attributed to postoral fat conditioning. Short-term (3-min) two-bottle tests further documented the fat preference deficits in CALHM1 KO mice but also revealed residual preferences for concentrated fat (5-10%), which may be mediated by odor and/or texture cues.

Tamoxifen-induced knockdown of the mitochondrial calcium uniporter in Thy1-expressing neurons protects mice from hypoxic/ischemic brain injury.

The mitochondrial calcium uniporter (MCU) mediates high-capacity mitochondrial calcium uptake that stimulates energy production. However, excessive MCU activity can cause ischemic heart injury. To examine if the MCU is also involved in hypoxic/ischemic (HI) brain injury, we have generated conditional MCU knockout mice by tamoxifen (TMX) administration to adult MCU-floxed (MCUfl/fl) mice expressing a construct encoding Thy1-cre/ERT2-eYFP. Relative to TMX/Thy1-cre/ERT2-eYFP controls, HI-induced sensorimotor deficits, forebrain neuron loss and mitochondrial damage were decreased for conditional MCU knockout mice. MCU knockdown by siRNA-induced silencing in cortical neuron cultures also reduced cell death and mitochondrial respiratory deficits following oxygen-glucose deprivation. Furthermore, MCU silencing did not produce metabolic abnormalities in cortical neurons observed previously for global MCU nulls that increased reliance on glycolysis for energy production. Based on these findings, we propose that brain-penetrant MCU inhibitors have strong potential to be well-tolerated and highly-efficacious neuroprotectants for the acute management of ischemic stroke.

The α2δ-1-NMDA Receptor Complex Is Critically Involved in Neuropathic Pain Development and Gabapentin Therapeutic Actions.

α2δ-1, commonly known as a voltage-activated Ca2+ channel subunit, is a binding site of gabapentinoids used to treat neuropathic pain and epilepsy. However, it is unclear how α2δ-1 contributes to neuropathic pain and gabapentinoid actions. Here, we show that Cacna2d1 overexpression potentiates presynaptic and postsynaptic NMDAR activity of spinal dorsal horn neurons to cause pain hypersensitivity. Conversely, Cacna2d1 knockdown or ablation normalizes synaptic NMDAR activity increased by nerve injury. α2δ-1 forms a heteromeric complex with NMDARs in rodent and human spinal cords. The α2δ-1-NMDAR interaction predominantly occurs through the C terminus of α2δ-1 and promotes surface trafficking and synaptic targeting of NMDARs. Gabapentin or an α2δ-1 C terminus-interfering peptide normalizes NMDAR synaptic targeting and activity increased by nerve injury. Thus, α2δ-1 is an NMDAR-interacting protein that increases NMDAR synaptic delivery in neuropathic pain. Gabapentinoids reduce neuropathic pain by inhibiting forward trafficking of α2δ-1-NMDAR complexes.

TMCO1 is essential for ovarian follicle development by regulating ER Ca2+ store of granulosa cells.

TMCO1 (transmembrane and coiled-coil domains 1) is an endoplasmic reticulum (ER) transmembrane protein that actively prevents Ca2+ stores from overfilling. To characterize its physiological function(s), we generated Tmco1-/- knockout (KO) mice. In addition to the main clinical features of human cerebrofaciothoracic (CFT) dysplasia spectrum, Tmco1-/- females manifest gradual loss of ovarian follicles, impaired ovarian follicle development, and subfertility with a phenotype analogous to the premature ovarian failure (POF) in women. In line with the role of TMCO1 as a Ca2+ load-activated Ca2+ channel, we have detected a supernormal Ca2+ signaling in Tmco1-/- granulosa cells (GCs). Interestingly, although spontaneous Ca2+ oscillation pattern was altered, ER Ca2+ stores of germinal vesicle (GV) stage oocytes and metaphase II (MII) arrested eggs were normal upon Tmco1 ablation. Combined with RNA-sequencing analysis, we also detected increased ER stress-mediated apoptosis and enhanced reactive oxygen species (ROS) level in Tmco1-/- GCs, indicating the dysfunctions of GCs upon TMCO1 deficiency. Taken together, these results reveal that TMCO1 is essential for ovarian follicle development and female fertility by maintaining ER Ca2+ homeostasis of GCs, disruption of which causes ER stress-mediated apoptosis and increased cellular ROS level in GCs and thus leads to impaired ovarian follicle development.

Blockade and knock-out of CALHM1 channels attenuate ischemic brain damage.

Overactivation of purinergic receptors during cerebral ischemia results in a massive release of neurotransmitters, including adenosine triphosphate (ATP), to the extracellular space which leads to cell death. Some hypothetical pathways of ATP release are large ion channels, such as calcium homeostasis modulator 1 (CALHM1), a membrane ion channel that can permeate ATP. Since this transmitter contributes to postischemic brain damage, we hypothesized that CALHM1 activation may be a relevant target to attenuate stroke injury. Here, we analyzed the contribution of CALHM1 to postanoxic depolarization after ischemia in cultured neurons and in cortical slices. We observed that the onset of postanoxic currents in neurons in those preparations was delayed after its blockade with ruthenium red or silencing of Calhm1 gene by short hairpin RNA, as well as in slices from CALHM1 knockout mice. Subsequently, we used transient middle cerebral artery occlusion and found that ruthenium red, a blocker of CALHM1, or the lack of CALHM1, substantially attenuated the motor symptoms and reduced significantly the infarct volume. These results show that CALHM1 channels mediate postanoxic depolarization in neurons and brain damage after ischemia. Therefore, targeting CALHM1 may have a high therapeutic potential for treating brain damage after ischemia.