Essential role of the mitochondrial Na+/Ca2+ exchanger NCLX in mediating PDE2-dependent neuronal survival and learning.

Impaired phosphodiesterase (PDE) function and mitochondrial Ca2+ (i.e., [Ca2+]m) lead to multiple health syndromes by an unknown pathway. Here, we fluorescently monitor robust [Ca2+]m efflux mediated by the mitochondrial Na+/Ca2+ exchanger NCLX in hippocampal neurons sequentially evoked by caffeine and depolarization. Surprisingly, neuronal depolarization-induced Ca2+ transients alone fail to evoke strong [Ca2+]m efflux in wild-type (WT) neurons. However, pre-treatment with the selective PDE2 inhibitor Bay 60-7550 effectively rescues [Ca2+]m efflux similarly to caffeine. Moreover, PDE2 acts by diminishing mitochondrial cAMP, thus promoting NCLX phosphorylation at its PKA site. We find that the protection of neurons against excitotoxic insults, conferred by PDE2 inhibition in WT neurons, is NCLX dependent. Finally, the administration of Bay 60-7550 enhances new object recognition in WT, but not in NCLX knockout (KO), mice. Our results identify a link between PDE and [Ca2+]m signaling that may provide effective therapy for cognitive and ischemic syndromes.

CKII Control of Axonal Plasticity Is Mediated by Mitochondrial Ca2+ via Mitochondrial NCLX.

Mitochondrial Ca2+ efflux by NCLX is a critical rate-limiting step in mitochondria signaling. We previously showed that NCLX is phosphorylated at a putative Casein Kinase 2 (CKII) site, the serine 271 (S271). Here, we asked if NCLX is regulated by CKII and interrogated the physiological implications of this control. We found that CKII inhibitors down-regulated NCLX-dependent Ca2+ transport activity in SH-SY5Y neuronal cells and primary hippocampal neurons. Furthermore, we show that the CKII phosphomimetic mutants on NCLX inhibited (S271A) and constitutively activated (S271D) NCLX transport, respectively, rendering it insensitive to CKII inhibition. These phosphomimetic NCLX mutations also control the allosteric regulation of NCLX by mitochondrial membrane potential (ΔΨm). Since the omnipresent CKII is necessary for modulating the plasticity of the axon initial segment (AIS), we interrogated, in hippocampal neurons, if NCLX is required for this process. Similarly to WT neurons, NCLX-KO neurons can exhibit homeostatic plasticity following M-channel block. However, while WT neurons utilize a CKII-sensitive distal relocation of AIS Na+ and Kv7 channels to decrease their intrinsic excitability, we did not observe such translocation in NCLX-KO neurons. Thus, our results indicate that NCLX is regulated by CKII and is a crucial link between CKII signaling and fast neuronal plasticity.

ASIC1a senses lactate uptake to regulate metabolism in neurons.

Lactate is a major metabolite largely produced by astrocytes that nourishes neurons. ASIC1a, a Na+ and Ca2+-permeable channel with an extracellular proton sensing domain, is thought to be activated by lactate through chelation of divalent cations, including Ca2+, Mg2+ and Zn2+, that block the channel pore. Here, by monitoring lactate-evoked H+ and Ca2+ transport in cultured mouse cortical and hippocampal neurons, we find that stereo-selective neuronal uptake of L-lactate results in rapid intracellular acidification that triggers H+ extrusion to activate plasma membrane ASIC1a channels, leading to propagating Ca2+ waves into the cytosol and mitochondria. We show that lactate activates ASIC1a at its physiological concentrations, far below that needed to chelate divalent cations. The L-isomer of lactate exerts a much greater effect on ASIC1a-mediated activity than the d-isomer and this stereo-selectivity arises from lactate transporters, which prefer the physiologically common L-lactate. The lactate uptake in turn results in intracellular acidification, which is then followed by a robust acid extrusion. The latter response sufficiently lowers the pH in the vicinity of the extracellular domain of ASIC1a to trigger its activation, resulting in cytosolic and mitochondrial Ca2+ signals that accelerate mitochondrial respiration. Furthermore, blocking ASIC1a led to a robust mitochondrial ROS production induced by L-lactate. Together our results indicate that ASIC1a is a metabolic sensor, which by sensing extracellular pH drop triggered by neuronal lactate uptake with subsequent proton extrusion, transmits a Ca2+ response that is propagated to mitochondria to enhance lactate catabolism and suppress ROS production.

Recent studies on NCLX in health and diseases.

The mitochondria is a major hub for cellular Ca 2+ signaling. The identification of MCU, the mitochondrial Ca 2+ influx mediator, and the mitochondrial Ca 2+ extruder NCLX, were major breakthroughs in this field. Their identification provided novel molecular tools and animal models to interrogate their physiological function and mode of regulation. Here we will focus on the mitochondrial Na + / Ca 2+ exchanger NCLX that plays a dual role in mitochondrial Na + and Ca 2+ signaling. We will discuss recent advances in NCLX mods of regulation by kinases and mitochondrial ΔΨ. We will also focus on the heterogeneity of its expression in distinct mitochondrial populations and the pathophysiological implication of its excessive degradation. We will describe the ongoing debate on the stoichiometry of Na + to Ca 2+ transport, mediated by NCLX, and its physiological implication. We will focus on the major effects of mitochondrial Na + signaling by NCLX on mitochondrial metabolism in health; and finally, we will discuss the role NCLX plays in a wide range of health disorders, from heart failure and cancer to Parkinson and Alzheimer disease, making it a prime candidate for therapeutic targeting.

ASIC1a channels regulate mitochondrial ion signaling and energy homeostasis in neurons.

Acid-sensing ion channel 1a (ASIC1a) is well-known to play a major pathophysiological role during brain ischemia linked to acute acidosis of ~pH 6, whereas its function during physiological brain activity, linked to much milder pH changes, is still poorly understood. Here, by performing live cell imaging utilizing Na+ and Ca2+ sensitive and spatially specific fluorescent dyes, we investigated the role of ASIC1a in cytosolic Na+ and Ca2+ signals elicited by a mild extracellular drop from pH 7.4 to 7.0 and how these affect mitochondrial Na+ and Ca2+ signaling or metabolic activity. We show that in mouse primary cortical neurons, this small extracellular pH change triggers cytosolic Na+ and Ca2+ waves that propagate to mitochondria. Inhibiting ASIC1a with Psalmotoxin 1 or ASIC1a gene knockout blocked not only the cytosolic but also the mitochondrial Na+ and Ca2+ signals. Moreover, physiological activation of ASIC1a by this pH shift enhances mitochondrial respiration and evokes mitochondrial Na+ signaling even in digitonin-permeabilized neurons. Altogether our results indicate that ASIC1a is critical in linking physiological extracellular pH stimuli to mitochondrial ion signaling and metabolic activity and thus is an important metabolic sensor.

Mitochondria control store-operated Ca2+ entry through Na+ and redox signals.

Mitochondria exert important control over plasma membrane (PM) Orai1 channels mediating store-operated Ca2+ entry (SOCE). Although the sensing of endoplasmic reticulum (ER) Ca2+ stores by STIM proteins and coupling to Orai1 channels is well understood, how mitochondria communicate with Orai1 channels to regulate SOCE activation remains elusive. Here, we reveal that SOCE is accompanied by a rise in cytosolic Na+ that is critical in activating the mitochondrial Na+/Ca2+ exchanger (NCLX) causing enhanced mitochondrial Na+ uptake and Ca2+ efflux. Omission of extracellular Na+ prevents the cytosolic Na+ rise, inhibits NCLX activity, and impairs SOCE and Orai1 channel current. We show further that SOCE activates a mitochondrial redox transient which is dependent on NCLX and is required for preventing Orai1 inactivation through oxidation of a critical cysteine (Cys195) in the third transmembrane helix of Orai1. We show that mitochondrial targeting of catalase is sufficient to rescue redox transients, SOCE, and Orai1 currents in NCLX-deficient cells. Our findings identify a hitherto unknown NCLX-mediated pathway that coordinates Na+ and Ca2+ signals to effect mitochondrial redox control over SOCE.