King-Ho Cheung

Essential Regulation of Cell Bioenergetics by Constitutive InsP3 Receptor Ca2+ Transfer to Mitochondria

Mechanisms that regulate cellular metabolism are a fundamental requirement of all cells. Most eukaryotic cells rely on aerobic mitochondrial metabolism to generate ATP. Nevertheless, regulation of mitochondrial activity is incompletely understood. Here we identified an unexpected and essential role for constitutive InsP(3)R-mediated Ca(2+) release in maintaining cellular bioenergetics. Macroautophagy provides eukaryotes with an adaptive response to nutrient deprivation that prolongs survival. Constitutive InsP(3)R Ca(2+) signaling is required for macroautophagy suppression in cells in nutrient-replete media. In its absence, cells become metabolically compromised due to diminished mitochondrial Ca(2+) uptake. Mitochondrial uptake of InsP(3)R-released Ca(2+) is fundamentally required to provide optimal bioenergetics by providing sufficient reducing equivalents to support oxidative phosphorylation. Absence of this Ca(2+) transfer results in enhanced phosphorylation of pyruvate dehydrogenase and activation of AMPK, which activates prosurvival macroautophagy. Thus, constitutive InsP(3)R Ca(2+) release to mitochondria is an essential cellular process that is required for efficient mitochondrial respiration and maintenance of normal cell bioenergetics.

Gain-of-Function Enhancement of IP3 Receptor Modal Gating by Familial Alzheimer’s Disease–Linked Presenilin Mutants in Human Cells and Mouse Neurons

By altering IP3 receptor gating, presenilin mutations associated with familial Alzheimer’s disease increase Ca2+ release from the endoplasmic reticulum. Opening the Calcium Floodgates? Alzheimer’s disease (AD), which is the most common cause of dementia, is a neurodegenerative disorder that affects some 5 million Americans. Although most cases of AD are sporadic, an early-onset form of familial AD (FAD) has been linked to mutations in the presenilins (PSs), transmembrane proteins localized to the endoplasmic reticulum (ER). Cheung et al. investigated the effects of wild-type and mutant forms of PS on inositol trisphosphate receptor (IP3R)–mediated Ca2+ release from the ER in various different cellular systems, including human lymphoblasts derived from individuals with FAD and cortical neurons from a mouse model of FAD. They found that FAD-linked PS mutants enhanced Ca2+ release by modulating IP3R channel gating through a gain-of-function mechanism, consistent with the autosomal-dominant inheritance of FAD. FAD-linked PS mutants, but not PS mutants associated with another form of dementia, shifted IP3R channel gating to a mode in which the probability that individual channels were open after stimulation was increased, leading to exaggerated Ca2+ signals. Familial Alzheimer’s disease (FAD) is caused by mutations in amyloid precursor protein or presenilins (PS1 and PS2). Many FAD-linked PS mutations affect intracellular calcium (Ca2+) homeostasis by mechanisms proximal to and independent of amyloid production, although the molecular details are controversial. We found that several FAD-causing PS mutants enhance gating of the inositol trisphosphate receptor (IP3R) Ca2+ release channel by a gain-of-function effect that mirrored the genetics of FAD and was independent of secretase activity. In contrast, wild-type PS or PS mutants that cause frontotemporal dementia had no such effect. FAD-causing PS mutants altered the modes in which the IP3R channel gated. Recordings of endogenous IP3R in lymphoblasts derived from individuals with FAD or cortical neurons of asymptomatic PS1-AD mice revealed that they were more likely than IP3R in cells with wild-type PS to dwell in a high open-probability burst mode, resulting in enhanced Ca2+ signaling. These results indicate that exaggerated Ca2+ signaling through IP3R-PS interaction is a disease-specific and robust proximal mechanism in FAD.

Calcium homeostasis modulator 1 (CALHM1) is the pore-forming subunit of an ion channel that mediates extracellular Ca2+ regulation of neuronal excitability

Extracellular Ca2+ (Ca2+ o) plays important roles in physiology. Changes of Ca2+o concentration ([Ca2+]o) have been observed to modulate neuronal excitability in various physiological and pathophysiological settings, but the mechanisms by which neurons detect [Ca2+]o are not fully understood. Calcium homeostasis modulator 1 (CALHM1) expression was shown to induce cation currents in cells and elevate cytoplasmic Ca2+ concentration ([Ca2+]i) in response to removal of Ca2+o and its subsequent addback. However, it is unknown whether CALHM1 is a pore-forming ion channel or modulates endogenous ion channels. Here we identify CALHM1 as the pore-forming subunit of a plasma membrane Ca2+-permeable ion channel with distinct ion permeability properties and unique coupled allosteric gating regulation by voltage and [Ca2+]o. Furthermore, we show that CALHM1 is expressed in mouse cortical neurons that respond to reducing [Ca2+]o with enhanced conductance and action potential firing and strongly elevated [Ca2+]i upon Ca2+o removal and its addback. In contrast, these responses are strongly muted in neurons from mice with CALHM1 genetically deleted. These results demonstrate that CALHM1 is an evolutionarily conserved ion channel family that detects membrane voltage and extracellular Ca2+ levels and plays a role in cortical neuronal excitability and Ca2+ homeostasis, particularly in response to lowering [Ca2+]o and its restoration to normal levels.

Graded recruitment and inactivation of single InsP3 receptor Ca2+-release channels: implications for quartal Ca2+release

Modulation of cytoplasmic free Ca2+ concentration ([Ca2+]i) by receptor‐mediated generation of inositol 1,4,5‐trisphosphate (InsP3) and activation of its receptor (InsP3R), a Ca2+‐release channel in the endoplasmic reticulum, is a ubiquitous signalling mechanism. A fundamental aspect of InsP3‐mediated signalling is the graded release of Ca2+ in response to incremental levels of stimuli. Ca2+ release has a transient fast phase, whose rate is proportional to [InsP3], followed by a much slower one even in constant [InsP3]. Many schemes have been proposed to account for quantal Ca2+ release, including the presence of heterogeneous channels and Ca2+ stores with various mechanisms of release termination. Here, we demonstrate that mechanisms intrinsic to the single InsP3R channel can account for quantal Ca2+ release. Patch‐clamp electrophysiology of isolated insect Sf9 cell nuclei revealed a consistent and high probability of detecting functional endogenous InsP3R channels, enabling InsP3‐induced channel inactivation to be identified as an inevitable consequence of activation, and allowing the average number of activated channels in the membrane patch (NA) to be accurately quantified. InsP3‐activated channels invariably inactivated, with average duration of channel activity reduced by high [Ca2+]i and suboptimal [InsP3]. Unexpectedly, NA was found to be a graded function of both [Ca2+]i and [InsP3]. A qualitative model involving Ca2+‐induced InsP3R sequestration and inactivation can account for these observations. These results suggest that apparent heterogeneous ligand sensitivity can be generated in a homogeneous population of InsP3R channels, providing a mechanism for graded Ca2+ release that is intrinsic to the InsP3R Ca2+ release channel itself.

Constitutive cAMP response element binding protein (CREB) activation by Alzheimer's disease presenilin-driven inositol trisphosphate receptor (InsP3R) Ca2+ signaling

Mutations in presenilins (PS) account for most early-onset familial Alzheimers disease (FAD). Accumulating evidence suggests that disrupted Ca2+ signaling may play a proximal role in FAD specifically, and Alzheimers disease (AD) more generally, but its links to the pathogenesis of AD are obscure. Here we demonstrate that expression of FAD mutant PS constitutively activates the transcription factor cAMP response element binding protein (CREB) and CREB target gene expression in cultured neuronal cells and AD mouse models. Constitutive CREB activation was associated with and dependent on constitutive activation of Ca2+/CaM kinase kinase β and CaM kinase IV (CaMKIV). Depletion of endoplasmic reticulum Ca2+ stores or plasma membrane phosphatidylinositol-bisphosphate and pharmacologic inhibition or knockdown of the expression of the inositol trisphosphate receptor (InsP3R) Ca2+ release channel each abolished FAD PS-associated constitutive CaMKIV and CREB phosphorylation. CREB and CaMKIV phosphorylation and CREB target gene expression, including nitric oxide synthase and c-fos, were enhanced in brains of M146V-KI and 3xTg-AD mice expressing FAD mutant PS1 knocked into the mouse locus. FAD mutant PS-expressing cells demonstrated enhanced cell death and sensitivity to Aβ toxicity, which were normalized by interfering with the InsP3R–CAMKIV–CREB pathway. Thus, constitutive CREB phosphorylation by exaggerated InsP3R Ca2+ signaling in FAD PS-expressing cells may represent a signaling pathway involved in the pathogenesis of AD.

Mode switching is the major mechanism of ligand regulation of InsP3 receptor calcium release channels.

The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) plays a critical role in generation of complex Ca2+ signals in many cell types. In patch clamp recordings of isolated nuclei from insect Sf9 cells, InsP3R channels were consistently detected with regulation by cytoplasmic InsP3 and free Ca2+ concentrations ([Ca2+]i) very similar to that observed for vertebrate InsP3R. Long channel activity durations of the Sf9-InsP3R have now enabled identification of a novel aspect of InsP3R gating: modal gating. Using a novel algorithm to analyze channel modal gating kinetics, InsP3R gating can be separated into three distinct modes: a low activity mode, a fast kinetic mode, and a burst mode with channel open probability (P o) within each mode of 0.007 ± 0.002, 0.24 ± 0.03, and 0.85 ± 0.02, respectively. Channels reside in each mode for long periods (tens of opening and closing events), and transitions between modes can be discerned with high resolution (within two channel opening and closing events). Remarkably, regulation of channel gating by [Ca2+]i and [InsP3] does not substantially alter channel P o within a mode. Instead, [Ca2+]i and [InsP3] affect overall channel P o primarily by changing the relative probability of the channel being in each mode, especially the high and low P o modes. This novel observation therefore reveals modal switching as the major mechanism of physiological regulation of InsP3R channel activity, with implications for the kinetics of Ca2+ release events in cells.

Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A

Autophagy is a catabolic lysosomal degradation process essential for cellular homeostasis and cell survival. Dysfunctional autophagy has been associated with a wide range of human diseases, e.g., cancer and neurodegenerative diseases. A large number of small molecules that modulate autophagy have been widely used to dissect this process and some of them, e.g., chloroquine (CQ), might be ultimately applied to treat a variety of autophagy-associated human diseases. Here we found that vacuolin-1 potently and reversibly inhibited the fusion between autophagosomes and lysosomes in mammalian cells, thereby inducing the accumulation of autophagosomes. Interestingly, vacuolin-1 was less toxic but at least 10-fold more potent in inhibiting autophagy compared with CQ. Vacuolin-1 treatment also blocked the fusion between endosomes and lysosomes, resulting in a defect in general endosomal-lysosomal degradation. Treatment of cells with vacuolin-1 alkalinized lysosomal pH and decreased lysosomal Ca2+ content. Besides marginally inhibiting vacuolar ATPase activity, vacuolin-1 treatment markedly activated RAB5A GTPase activity. Expression of a dominant negative mutant of RAB5A or RAB5A knockdown significantly inhibited vacuolin-1-induced autophagosome-lysosome fusion blockage, whereas expression of a constitutive active form of RAB5A suppressed autophagosome-lysosome fusion. These data suggest that vacuolin-1 activates RAB5A to block autophagosome-lysosome fusion. Vacuolin-1 and its analogs present a novel class of drug that can potently and reversibly modulate autophagy.

Enhanced ROS generation mediated by Alzheimer's disease presenilin regulation of InsP3R Ca2+ signaling.

Familial Alzheimers disease (FAD) is caused by mutations in amyloid precursor protein and presenilins (PS1, PS2). Many FAD-linked PS mutations affect intracellular calcium (Ca(2+)) homeostasis by proximal mechanisms independent of amyloid production by dramatically enhancing gating of the inositol trisphosphate receptor (InsP(3)R) intracellular Ca(2+) release channel by a gain-of-function effect that mirrors genetics of FAD and is independent of secretase activity. Electrophysiological recordings of InsP(3)R in FAD patient B cells, cortical neurons of asymptomatic PS1-AD mice, and other cells revealed they have higher occupancy in a high open probability burst mode, resulting in enhanced Ca(2+) signaling. Exaggerated Ca(2+) signaling through this mechanism results in enhanced generation of reactive oxygen species, believed to be an important component in AD pathogenesis. Exaggerated Ca(2+) signaling through InsP(3)R-PS interaction is a disease specific and robust proximal mechanism in AD that may contribute to the pathology of AD by enhanced generation of reactive oxygen species.

Calcium signals and calpain-dependent necrosis are essential for release of coxsackievirus B from polarized intestinal epithelial cells

In contrast to nonpolarized cells, coxsackievirus B (CVB)–infected polarized intestinal Caco-2 cells undergo necrotic cell death triggered by inositol 1,4,5-trisphosphate receptor–dependent calcium release. This CVB-induced necrosis depends on Ca2+-activated calpain-2, which is required for disruption of the apical tight junction complex.

Mechanistic study of TRPM2-Ca2+-CAMK2-BECN1 signaling in oxidative stress-induced autophagy inhibition

ABSTRACT Reactive oxygen species (ROS) have been commonly accepted as inducers of autophagy, and autophagy in turn is activated to relieve oxidative stress. Yet, whether and how oxidative stress, generated in various human pathologies, regulates autophagy remains unknown. Here, we mechanistically studied the role of TRPM2 (transient receptor potential cation channel subfamily M member 2)-mediated Ca2+ influx in oxidative stress-mediated autophagy regulation. On the one hand, we demonstrated that oxidative stress triggered TRPM2-dependent Ca2+ influx to inhibit the induction of early autophagy, which renders cells more susceptible to death. On the other hand, oxidative stress induced autophagy (and not cell death) in the absence of the TRPM2-mediated Ca2+ influx. Moreover, in response to oxidative stress, TRPM2-mediated Ca2+ influx activated CAMK2 (calcium/calmodulin dependent protein kinase II) at levels of both phosphorylation and oxidation, and the activated CAMK2 subsequently phosphorylated BECN1/Beclin 1 on Ser295. Ser295 phosphorylation of BECN1 in turn decreased the association between BECN1 and PIK3C3/VPS34, but induced binding between BECN1 and BCL2. Clinically, acetaminophen (APAP) overdose is the most common cause of acute liver failure worldwide. We demonstrated that APAP overdose also activated ROS-TRPM2-CAMK2-BECN1 signaling to suppress autophagy, thereby causing primary hepatocytes to be more vulnerable to death. Inhibiting the TRPM2-Ca2+-CAMK2 cascade significantly mitigated APAP-induced liver injury. In summary, our data clearly demonstrate that oxidative stress activates the TRPM2-Ca2+-CAMK2 cascade to phosphorylate BECN1 resulting in autophagy inhibition.