Dietary zinc supplementation of 3xTg-AD mice increases BDNF levels and prevents cognitive deficits as well as mitochondrial dysfunction

The overall effect of brain zinc (Zn²⁺) in the progression and development of Alzheimer''s disease (AD) is still not completely understood. Although an excess of Zn²⁺ can exacerbate the pathological features of AD, a deficit of Zn²⁺ intake has also been shown to increase the volume of amyloid plaques in AD transgenic mice. In this study, we investigated the effect of dietary Zn²⁺ supplementation (30 p.p.m.) in a transgenic mouse model of AD, the 3xTg-AD, that expresses both β amyloid (Aβ)- and tau-dependent pathology. We found that Zn²⁺ supplementation greatly delays hippocampal-dependent memory deficits and strongly reduces both Aβ and tau pathology in the hippocampus. We also evaluated signs of mitochondrial dysfunction and found that Zn²⁺ supplementation prevents the age-dependent respiratory deficits we observed in untreated 3xTg-AD mice. Finally, we found that Zn²⁺ supplementation greatly increases the levels of brain-derived neurotrophic factor (BDNF) of treated 3xTg-AD mice. In summary, our data support the idea that controlling the brain Zn²⁺ homeostasis may be beneficial in the treatment of AD.     

Demand for Zn2+ in acid-secreting gastric mucosa and its requirement for intracellular Ca2+

Background and Aims

Recent work has suggested that Zn²⁺ plays a critical role in regulating acidity within the secretory compartments of isolated gastric glands. Here, we investigate the content, distribution and demand for Zn²⁺ in gastric mucosa under baseline conditions and its regulation during secretory stimulation.

Methods and Findings

Content and distribution of zinc were evaluated in sections of whole gastric mucosa using X-ray fluorescence microscopy. Significant stores of Zn²⁺ were identified in neural elements of the muscularis, glandular areas enriched in parietal cells, and apical regions of the surface epithelium. In in vivo studies, extraction of the low abundance isotope, ⁷⁰Zn²⁺, from the circulation was demonstrated in samples of mucosal tissue 24 hours or 72 hours after infusion (250 µg/kg). In in vitro studies, uptake of ⁷⁰Zn²⁺ from media was demonstrated in isolated rabbit gastric glands following exposure to concentrations as low as 10 nM. In additional studies, demand of individual gastric parietal cells for Zn²⁺ was monitored using the fluorescent zinc reporter, fluozin-3, by measuring increases in free intracellular concentrations of Zn²⁺ {[Zn²⁺]_i} during exposure to standard extracellular concentrations of Zn²⁺ (10 µM) for standard intervals of time. Under resting conditions, demand for extracellular Zn²⁺ increased with exposure to secretagogues (forskolin, carbachol/histamine) and under conditions associated with increased intracellular Ca²⁺ {[Ca²⁺]_i}. Uptake of Zn²⁺ was abolished following removal of extracellular Ca²⁺ or depletion of intracellular Ca²⁺ stores, suggesting that demand for extracellular Zn²⁺ increases and depends on influx of extracellular Ca²⁺.

Conclusions

This study is the first to characterize the content and distribution of Zn²⁺ in an organ of the gastrointestinal tract. Our findings offer the novel interpretation, that Ca²⁺ integrates basolateral demand for Zn²⁺ with stimulation of secretion of HCl into the lumen of the gastric gland. Similar connections may be detectable in other secretory cells and tissues.     

Amyloid-β and Alzheimer's disease type pathology differentially affects the calcium signalling toolkit in astrocytes from different brain regions

The entorhinal–hippocampal circuit is severely affected in Alzheimer''s disease (AD). Here, we demonstrate that amyloid-β (Aβ) differentially affects primary cultured astrocytes derived from the entorhinal cortex (EC) and from the hippocampus from non-transgenic controls and 3xTg-AD transgenic mice. Exposure to 100 nM of Aβ resulted in increased expression of the metabotropic glutamate receptor type 5 (mGluR5) and its downstream InsP₃ receptor type 1 (InsP₃R1) in hippocampal but not in EC astrocytes. Amplitudes of Ca²⁺ responses to an mGluR5 agonist, DHPG, and to ATP, another metabotropic agonist coupled to InsP₃Rs, were significantly increased in Aβ-treated hippocampal but not in EC astrocytes. Previously we demonstrated that senile plaque formation in 3xTg-AD mice triggers astrogliosis in hippocampal but not in EC astrocytes. The different sensitivities of the Ca²⁺ signalling toolkit of EC versus hippocampal astrocytes to Aβ may account for the lack of astrogliosis in the EC, which in turn can explain the higher vulnerability of this region to AD.     

A minimal model for the mitochondrial rapid mode of Ca²+ uptake mechanism

Mitochondria possess a remarkable ability to rapidly accumulate and sequester Ca²⁺. One of the mechanisms responsible for this ability is believed to be the rapid mode (RaM) of Ca²⁺ uptake. Despite the existence of many models of mitochondrial Ca²⁺ dynamics, very few consider RaM as a potential mechanism that regulates mitochondrial Ca²⁺ dynamics. To fill this gap, a novel mathematical model of the RaM mechanism is developed herein. The model is able to simulate the available experimental data of rapid Ca²⁺ uptake in isolated mitochondria from both chicken heart and rat liver tissues with good fidelity. The mechanism is based on Ca²⁺ binding to an external trigger site(s) and initiating a brief transient of high Ca²⁺ conductivity. It then quickly switches to an inhibited, zero-conductive state until the external Ca²⁺ level is dropped below a critical value (∼100–150 nM). RaM''s Ca²⁺- and time-dependent properties make it a unique Ca²⁺ transporter that may be an important means by which mitochondria take up Ca²⁺ in situ and help enable mitochondria to decode cytosolic Ca²⁺ signals. Integrating the developed RaM model into existing models of mitochondrial Ca²⁺ dynamics will help elucidate the physiological role that this unique mechanism plays in mitochondrial Ca²⁺-homeostasis and bioenergetics.     

Intracellular Ca2+ release through ryanodine receptors contributes to AMPA receptor-mediated mitochondrial dysfunction and ER stress in oligodendrocytes

Overactivation of ionotropic glutamate receptors in oligodendrocytes induces cytosolic Ca²⁺ overload and excitotoxic death, a process that contributes to demyelination and multiple sclerosis. Excitotoxic insults cause well-characterized mitochondrial alterations and endoplasmic reticulum (ER) dysfunction, which is not fully understood. In this study, we analyzed the contribution of ER-Ca²⁺ release through ryanodine receptors (RyRs) and inositol triphosphate receptors (IP₃Rs) to excitotoxicity in oligodendrocytes in vitro. First, we observed that oligodendrocytes express all previously characterized RyRs and IP₃Rs. Blockade of Ca²⁺-induced Ca²⁺ release by TMB-8 following α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated insults attenuated both oligodendrocyte death and cytosolic Ca²⁺ overload. In turn, RyR inhibition by ryanodine reduced as well the Ca²⁺ overload whereas IP₃R inhibition was ineffective. Furthermore, AMPA-triggered mitochondrial membrane depolarization, oxidative stress and activation of caspase-3, which in all instances was diminished by RyR inhibition. In addition, we observed that AMPA induced an ER stress response as revealed by α subunit of the eukaryotic initiation factor 2α phosphorylation, overexpression of GRP chaperones and RyR-dependent cleavage of caspase-12. Finally, attenuating ER stress with salubrinal protected oligodendrocytes from AMPA excitotoxicity. Together, these results show that Ca²⁺ release through RyRs contributes to cytosolic Ca²⁺ overload, mitochondrial dysfunction, ER stress and cell death following AMPA receptor-mediated excitotoxicity in oligodendrocytes.     

Computational Study of Hippocampal-Septal Theta Rhythm Changes Due to Beta-Amyloid-Altered Ionic Channels

Electroencephagraphy (EEG) of many dementia patients has been characterized by an increase in low frequency field potential oscillations. One of the characteristics of early stage Alzheimer’s disease (AD) is an increase in theta band power (4–7 Hz). However, the mechanism(s) underlying the changes in theta oscillations are still unclear. To address this issue, we investigate the theta band power changes associated with β-Amyloid (Aβ) peptide (one of the main markers of AD) using a computational model, and by mediating the toxicity of hippocampal pyramidal neurons. We use an established biophysical hippocampal CA1-medial septum network model to evaluate four ionic channels in pyramidal neurons, which were demonstrated to be affected by Aβ. They are the L-type Ca²⁺ channel, delayed rectifying K⁺ channel, A-type fast-inactivating K⁺ channel and large-conductance Ca²⁺-activated K⁺ channel. Our simulation results demonstrate that only the Aβ inhibited A-type fast-inactivating K⁺ channel can induce an increase in hippocampo-septal theta band power, while the other channels do not affect theta rhythm. We further deduce that this increased theta band power is due to enhanced synchrony of the pyramidal neurons. Our research may elucidate potential biomarkers and therapeutics for AD. Further investigation will be helpful for better understanding of AD-induced theta rhythm abnormalities and associated cognitive deficits.     

Selective modulation of subtype III IP3R by Akt regulates ER Ca2+ release and apoptosis

Ca²⁺ transfer from endoplasmic reticulum (ER) to mitochondria can trigger apoptotic pathways by inducing release of mitochondrial pro-apoptotic factors. Three different types of inositol 1,4,5-trisphosphate receptor (IP₃R) serve to discharge Ca²⁺ from ER, but possess some peculiarities, especially in apoptosis induction. The anti-apoptotic protein Akt can phosphorylate all IP₃R isoforms and protect cells from apoptosis, reducing ER Ca²⁺ release. However, it has not been elucidated which IP₃R subtypes mediate these effects. Here, we show that Akt activation in COS7 cells, which lack of IP₃R I, strongly suppresses IP₃-mediated Ca²⁺ release and apoptosis. Conversely, in SH-SY 5Y cells, which are type III-deficient, Akt is unable to modulate ER Ca²⁺ flux, losing its anti-apoptotic activity. In SH-SY 5Y-expressing subtype III, Akt recovers its protective function on cell death, by reduction of Ca²⁺ release. Moreover, regulating Ca²⁺ flux to mitochondria, Akt maintains the mitochondrial integrity and delays the trigger of apoptosis, in a type III-dependent mechanism. These results demonstrate a specific activity of Akt on IP₃R III, leading to diminished Ca²⁺ transfer to mitochondria and protection from apoptosis, suggesting an additional level of cell death regulation mediated by Akt.     

An inhibitory effect of extracellular Ca2+ on Ca2+-dependent exocytosis

Aim

Neurotransmitter release is elicited by an elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). The action potential triggers Ca²⁺ influx through Ca²⁺ channels which causes local changes of [Ca²⁺]_i for vesicle release. However, any direct role of extracellular Ca²⁺ (besides Ca²⁺ influx) on Ca²⁺-dependent exocytosis remains elusive. Here we set out to investigate this possibility on rat dorsal root ganglion (DRG) neurons and chromaffin cells, widely used models for studying vesicle exocytosis.

Results

Using photolysis of caged Ca²⁺ and caffeine-induced release of stored Ca²⁺, we found that extracellular Ca²⁺ inhibited exocytosis following moderate [Ca²⁺]_i rises (2–3 µM). The IC₅₀ for extracellular Ca²⁺ inhibition of exocytosis (ECIE) was 1.38 mM and a physiological reduction (∼30%) of extracellular Ca²⁺ concentration ([Ca²⁺]_o) significantly increased the evoked exocytosis. At the single vesicle level, quantal size and release frequency were also altered by physiological [Ca²⁺]_o. The calcimimetics Mg²⁺, Cd²⁺, G418, and neomycin all inhibited exocytosis. The extracellular Ca²⁺-sensing receptor (CaSR) was not involved because specific drugs and knockdown of CaSR in DRG neurons did not affect ECIE.

Conclusion/Significance

As an extension of the classic Ca²⁺ hypothesis of synaptic release, physiological levels of extracellular Ca²⁺ play dual roles in evoked exocytosis by providing a source of Ca²⁺ influx, and by directly regulating quantal size and release probability in neuronal cells.     

A Steep Dependence of Inward-Rectifying Potassium Channels on Cytosolic Free Calcium Concentration Increase Evoked by Hyperpolarization in Guard Cells

Inactivation of inward-rectifying K⁺ channels (I_K,in) by a rise in cytosolic free [Ca²⁺] ([Ca²⁺]_i) is a key event leading to solute loss from guard cells and stomatal closure. However, [Ca²⁺]_i action on I_K,in has never been quantified, nor are its origins well understood. We used membrane voltage to manipulate [Ca²⁺]_i (A. Grabov and M.R. Blatt [1998] Proc Natl Acad Sci USA 95: 4778–4783) while recording I_K,in under a voltage clamp and [Ca²⁺]_i by Fura-2 fluorescence ratiophotometry. I_K,in inactivation correlated positively with [Ca²⁺]_i and indicated a K_i of 329 ± 31 nm with cooperative binding of four Ca²⁺ ions per channel. I_K,in was promoted by the Ca²⁺ channel antagonists Gd³⁺ and calcicludine, both of which suppressed the [Ca²⁺]_i rise, but the [Ca²⁺]_i rise was unaffected by the K⁺ channel blocker Cs⁺. We also found that ryanodine, an antagonist of intracellular Ca²⁺ channels that mediate Ca²⁺-induced Ca²⁺ release, blocked the [Ca²⁺]_i rise, and Mn²⁺ quenching of Fura-2 fluorescence showed that membrane hyperpolarization triggered divalent release from intracellular stores. These and additional results point to a high signal gain in [Ca²⁺]_i control of I_K,in and to roles for discrete Ca²⁺ flux pathways in feedback control of the K⁺ channels by membrane voltage.Ca²⁺ underlies many fundamental regulatory processes in plants, including adaptive responses to abiotic environmental stress (Knight et al., 1996; Russell et al., 1996; McAinsh et al., 1997) and programmed cell death evoked by pathogen attack (Low and Merida, 1996; Hammondkosack and Jones, 1997). Coordination of changes in [Ca²⁺]_i and its integration with downstream response elements are central in coupling stimulus input to cellular response in these processes.In stomatal guard cells, the best characterized higher-plant cell model, major downstream targets of [Ca²⁺]_i and their roles in stomatal function have been identified. Increasing [Ca²⁺]_i is known to inactivate I_K,in and to activate Cl⁻ channels, events that bias plasma membrane transport for net efflux of osmotically active solute and a loss of turgor, which drives stomatal closure (Blatt and Grabov, 1997). Furthermore, changes in [Ca²⁺]_i are associated with ABA, CO₂, and the growth hormone auxin (Blatt and Grabov, 1997; McAinsh et al., 1997). These [Ca²⁺]_i signals have been observed to oscillate (McAinsh et al., 1995; Webb et al., 1996), characteristics that may constitute “Ca²⁺ signatures” to encode specific downstream responses (Berridge, 1996). Yet, despite the evidence for [Ca²⁺]_i signaling in guard cells, surprisingly little detail is known about the link between [Ca²⁺]_i changes and ion channel activity at the plasma membrane or about the mechanisms mediating such [Ca²⁺]_i changes. To our knowledge, in no instance have the characteristics of ion channel regulation by Ca²⁺ been quantified directly in any higher-plant cell.We recently described the coupling of membrane voltage to [Ca²⁺]_i, demonstrating that hyperpolarization, whether under a voltage clamp or in the presence of low [K⁺]_o, evoked [Ca²⁺]_i increases in guard cells, and that the voltage threshold for [Ca²⁺]_i rise was profoundly altered by ABA (Grabov and Blatt, 1998). Our observations indicated a link to Ca²⁺ influx across the plasma membrane and raised questions about the efficacy of [Ca²⁺]_i in inactivating I_K,in and about the contributions of intracellular Ca²⁺ release to the [Ca²⁺]_i signal. We have used membrane voltage to experimentally manipulate [Ca²⁺]_i and report that I_K,in is strongly dependent on [Ca²⁺]_i, consistent with a cooperative binding of four Ca²⁺ ions to effect inactivation. Additional experiments indicate that voltage-evoked [Ca²⁺]_i increases depend both on Ca²⁺ influx and on release of Ca²⁺ from intracellular stores. These results underscore the role of [Ca²⁺]_i as a high-gain “switch” in the control of I_K,in, and implicate [Ca²⁺]_i in feedback control linking membrane voltage to the activity of the K⁺ channels.     

Enhancement effects of martentoxin on glioma BK channel and BK channel (α+β1) subtypes

Background

BK channels are usually activated by membrane depolarization and cytoplasmic Ca²⁺. Especially,the activity of BK channel (α+β4) can be modulated by martentoxin, a 37 residues peptide, with Ca²⁺-dependent manner. gBK channel (glioma BK channel) and BK channel (α+β1) possessed higher Ca²⁺ sensitivity than other known BK channel subtypes.

Methodology and Principal Findings

The present study investigated the modulatory characteristics of martentoxin on these two BK channel subtypes by electrophysiological recordings, cell proliferation and Ca²⁺ imaging. In the presence of cytoplasmic Ca²⁺, martentoxin could enhance the activities of both gBK and BK channel (α+β1) subtypes in dose-dependent manner with EC₅₀ of 46.7 nM and 495 nM respectively, while not shift the steady-state activation of these channels. The enhancement ratio of martentoxin on gBK and BK channel (α+β1) was unrelated to the quantitive change of cytoplasmic Ca²⁺ concentrations though the interaction between martentoxin and BK channel (α+β1) was accelerated under higher cytoplasmic Ca²⁺. The selective BK pore blocker iberiotoxin could fully abolish the enhancement of these two BK subtypes induced by martentoxin, suggesting that the auxiliary β subunit might contribute to the docking for martentoxin. However, in the absence of cytoplasmic Ca²⁺, the activity of gBK channel would be surprisingly inhibited by martentoxin while BK channel (α+β1) couldn''t be affected by the toxin.

Conclusions and Significance

Thus, the results shown here provide the novel evidence that martentoxin could increase the two Ca²⁺-hypersensitive BK channel subtypes activities in a new manner and indicate that β subunit of these BK channels plays a vital role in this enhancement by martentoxin.     

Increase in the density of resting microglia precedes neuritic plaque formation and microglial activation in a transgenic model of Alzheimer's disease

The formation of cerebral senile plaques composed of amyloid β peptide (Aβ) is a fundamental feature of Alzheimer''s disease (AD). Glial cells and more specifically microglia become reactive in the presence of Aβ. In a triple transgenic model of AD (3 × Tg-AD), we found a significant increase in activated microglia at 12 (by 111%) and 18 (by 88%) months of age when compared with non-transgenic (non-Tg) controls. This microglial activation correlated with Aβ plaque formation, and the activation in microglia was closely associated with Aβ plaques and smaller Aβ deposits. We also found a significant increase in the area density of resting microglia in 3 × Tg-AD animals both at plaque-free stage (at 9 months by 105%) and after the development of A plaques (at 12 months by 54% and at 18 months by 131%). Our results show for the first time that the increase in the density of resting microglia precedes both plaque formation and activation of microglia by extracellular Aβ accumulation. We suggest that AD pathology triggers a complex microglial reaction: at the initial stages of the disease the number of resting microglia increases, as if in preparation for the ensuing activation in an attempt to fight the extracellular Aβ load that is characteristic of the terminal stages of the disease.     

Cyclic ADP ribose-dependent Ca2+ release by group I metabotropic glutamate receptors in acutely dissociated rat hippocampal neurons

Group I metabotropic glutamate receptors (group I mGluRs; mGluR1 and mGluR5) exert diverse effects on neuronal and synaptic functions, many of which are regulated by intracellular Ca²⁺. In this study, we characterized the cellular mechanisms underlying Ca²⁺ mobilization induced by (RS)-3,5-dihydroxyphenylglycine (DHPG; a specific group I mGluR agonist) in the somata of acutely dissociated rat hippocampal neurons using microfluorometry. We found that DHPG activates mGluR5 to mobilize intracellular Ca²⁺ from ryanodine-sensitive stores via cyclic adenosine diphosphate ribose (cADPR), while the PLC/IP₃ signaling pathway was not involved in Ca²⁺ mobilization. The application of glutamate, which depolarized the membrane potential by 28.5±4.9 mV (n = 4), led to transient Ca²⁺ mobilization by mGluR5 and Ca²⁺ influx through L-type Ca²⁺ channels. We found no evidence that mGluR5-mediated Ca²⁺ release and Ca²⁺ influx through L-type Ca²⁺ channels interact to generate supralinear Ca²⁺ transients. Our study provides novel insights into the mechanisms of intracellular Ca²⁺ mobilization by mGluR5 in the somata of hippocampal neurons.     

KCa2 channels activation prevents [Ca2+]i deregulation and reduces neuronal death following glutamate toxicity and cerebral ischemia

Exacerbated activation of glutamate receptor-coupled calcium channels and subsequent increase in intracellular calcium ([Ca²⁺]_i) are established hallmarks of neuronal cell death in acute and chronic neurological diseases. Here we show that pathological [Ca²⁺]_i deregulation occurring after glutamate receptor stimulation is effectively modulated by small conductance calcium-activated potassium (K_Ca2) channels. We found that neuronal excitotoxicity was associated with a rapid downregulation of K_Ca2.2 channels within 3 h after the onset of glutamate exposure. Activation of K_Ca2 channels preserved K_Ca2 expression and significantly reduced pathological increases in [Ca²⁺]_i providing robust neuroprotection in vitro and in vivo. These data suggest a critical role for K_Ca2 channels in excitotoxic neuronal cell death and propose their activation as potential therapeutic strategy for the treatment of acute and chronic neurodegenerative disorders.     

The effect of OPA1 on mitochondrial Ca²⁺ signaling

The dynamin-related GTPase protein OPA1, localized in the intermembrane space and tethered to the inner membrane of mitochondria, participates in the fusion of these organelles. Its mutation is the most prevalent cause of Autosomal Dominant Optic Atrophy. OPA1 controls the diameter of the junctions between the boundary part of the inner membrane and the membrane of cristae and reduces the diffusibility of cytochrome c through these junctions. We postulated that if significant Ca²⁺ uptake into the matrix occurs from the lumen of the cristae, reduced expression of OPA1 would increase the access of Ca²⁺ to the transporters in the crista membrane and thus would enhance Ca²⁺ uptake. In intact H295R adrenocortical and HeLa cells cytosolic Ca²⁺ signals evoked with K⁺ and histamine, respectively, were transferred into the mitochondria. The rate and amplitude of mitochondrial [Ca²⁺] rise (followed with confocal laser scanning microscopy and FRET measurements with fluorescent wide-field microscopy) were increased after knockdown of OPA1, as compared with cells transfected with control RNA or mitofusin1 siRNA. Ca²⁺ uptake was enhanced despite reduced mitochondrial membrane potential. In permeabilized cells the rate of Ca²⁺ uptake by depolarized mitochondria was also increased in OPA1-silenced cells. The participation of Na⁺/Ca²⁺ and Ca²⁺/H⁺ antiporters in this transport process is indicated by pharmacological data. Altogether, our observations reveal the significance of OPA1 in the control of mitochondrial Ca²⁺ metabolism.     

Disrupted membrane structure and intracellular Ca²⁺ signaling in adult skeletal muscle with acute knockdown of Bin1

Efficient intracellular Ca²⁺ ([Ca²⁺]i) homeostasis in skeletal muscle requires intact triad junctional complexes comprised of t-tubule invaginations of plasma membrane and terminal cisternae of sarcoplasmic reticulum. Bin1 consists of a specialized BAR domain that is associated with t-tubule development in skeletal muscle and involved in tethering the dihydropyridine receptors (DHPR) to the t-tubule. Here, we show that Bin1 is important for Ca²⁺ homeostasis in adult skeletal muscle. Since systemic ablation of Bin1 in mice results in postnatal lethality, in vivo electroporation mediated transfection method was used to deliver RFP-tagged plasmid that produced short –hairpin (sh)RNA targeting Bin1 (shRNA-Bin1) to study the effect of Bin1 knockdown in adult mouse FDB skeletal muscle. Upon confirming the reduction of endogenous Bin1 expression, we showed that shRNA-Bin1 muscle displayed swollen t-tubule structures, indicating that Bin1 is required for the maintenance of intact membrane structure in adult skeletal muscle. Reduced Bin1 expression led to disruption of t-tubule structure that was linked with alterations to intracellular Ca²⁺ release. Voltage-induced Ca²⁺ released in isolated single muscle fibers of shRNA-Bin1 showed that both the mean amplitude of Ca²⁺ current and SR Ca²⁺ transient were reduced when compared to the shRNA-control, indicating compromised coupling between DHPR and ryanodine receptor 1. The mean frequency of osmotic stress induced Ca²⁺ sparks was reduced in shRNA-Bin1, indicating compromised DHPR activation. ShRNA-Bin1 fibers also displayed reduced Ca²⁺ sparks'' amplitude that was attributed to decreased total Ca²⁺ stores in the shRNA-Bin1 fibers. Human mutation of Bin1 is associated with centronuclear myopathy and SH3 domain of Bin1 is important for sarcomeric protein organization in skeletal muscle. Our study showing the importance of Bin1 in the maintenance of intact t-tubule structure and ([Ca²⁺]i) homeostasis in adult skeletal muscle could provide mechanistic insight on the potential role of Bin1 in skeletal muscle contractility and pathology of myopathy.     

Mitochondrial Ca2+ Overload Underlies Aβ Oligomers Neurotoxicity Providing an Unexpected Mechanism of Neuroprotection by NSAIDs

Dysregulation of intracellular Ca²⁺ homeostasis may underlie amyloid β peptide (Aβ) toxicity in Alzheimer''s Disease (AD) but the mechanism is unknown. In search for this mechanism we found that Aβ_1–42 oligomers, the assembly state correlating best with cognitive decline in AD, but not Aβ fibrils, induce a massive entry of Ca²⁺ in neurons and promote mitochondrial Ca²⁺ overload as shown by bioluminescence imaging of targeted aequorin in individual neurons. Aβ oligomers induce also mitochondrial permeability transition, cytochrome c release, apoptosis and cell death. Mitochondrial depolarization prevents mitochondrial Ca²⁺ overload, cytochrome c release and cell death. In addition, we found that a series of non-steroidal anti-inflammatory drugs (NSAIDs) including salicylate, sulindac sulfide, indomethacin, ibuprofen and R-flurbiprofen depolarize mitochondria and inhibit mitochondrial Ca²⁺ overload, cytochrome c release and cell death induced by Aβ oligomers. Our results indicate that i) mitochondrial Ca²⁺ overload underlies the neurotoxicity induced by Aβ oligomers and ii) inhibition of mitochondrial Ca²⁺ overload provides a novel mechanism of neuroprotection by NSAIDs against Aβ oligomers and AD.     

Reduced IRE1α mediates apoptotic cell death by disrupting calcium homeostasis via the InsP3 receptor

The endoplasmic reticulum (ER) is not only a home for folding and posttranslational modifications of secretory proteins but also a reservoir for intracellular Ca²⁺. Perturbation of ER homeostasis contributes to the pathogenesis of various neurodegenerative diseases, such as Alzheimer''s and Parkinson diseases. One key regulator that underlies cell survival and Ca²⁺ homeostasis during ER stress responses is inositol-requiring enzyme 1α (IRE1α). Despite extensive studies on this ER membrane-associated protein, little is known about the molecular mechanisms by which excessive ER stress triggers cell death and Ca²⁺ dysregulation via the IRE1α-dependent signaling pathway. In this study, we show that inactivation of IRE1α by RNA interference increases cytosolic Ca²⁺ concentration in SH-SY5Y cells, leading to cell death. This dysregulation is caused by an accelerated ER-to-cytosolic efflux of Ca²⁺ through the InsP3 receptor (InsP3R). The Ca²⁺ efflux in IRE1α-deficient cells correlates with dissociation of the Ca²⁺-binding InsP3R inhibitor CIB1 and increased complex formation of CIB1 with the pro-apoptotic kinase ASK1, which otherwise remains inactivated in the IRE1α–TRAF2–ASK1 complex. The increased cytosolic concentration of Ca²⁺ induces mitochondrial production of reactive oxygen species (ROS), in particular superoxide, resulting in severe mitochondrial abnormalities, such as fragmentation and depolarization of membrane potential. These Ca²⁺ dysregulation-induced mitochondrial abnormalities and cell death in IRE1α-deficient cells can be blocked by depleting ROS or inhibiting Ca²⁺ influx into the mitochondria. These results demonstrate the importance of IRE1α in Ca²⁺ homeostasis and cell survival during ER stress and reveal a previously unknown Ca²⁺-mediated cell death signaling between the IRE1α–InsP3R pathway in the ER and the redox-dependent apoptotic pathway in the mitochondrion.     

Anthrax lethal toxin suppresses murine cardiomyocyte contractile function and intracellular Ca2+ handling via a NADPH oxidase-dependent mechanism

Objectives

Anthrax infection is associated with devastating cardiovascular sequelae, suggesting unfavorable cardiovascular effects of toxins originated from Bacillus anthracis namely lethal and edema toxins. This study was designed to examine the direct effect of lethal toxins on cardiomyocyte contractile and intracellular Ca²⁺ properties.

Methods

Murine cardiomyocyte contractile function and intracellular Ca²⁺ handling were evaluated including peak shortening (PS), maximal velocity of shortening/ relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR₉₀), intracellular Ca²⁺ rise measured as fura-2 fluorescent intensity (ΔFFI), and intracellular Ca²⁺ decay rate. Stress signaling and Ca²⁺ regulatory proteins were assessed using Western blot analysis.

Results

In vitro exposure to a lethal toxin (0.05 – 50 nM) elicited a concentration-dependent depression on cardiomyocyte contractile and intracellular Ca²⁺ properties (PS, ± dL/dt, ΔFFI), along with prolonged duration of contraction and intracellular Ca²⁺ decay, the effects of which were nullified by the NADPH oxidase inhibitor apocynin. The lethal toxin significantly enhanced superoxide production and cell death, which were reversed by apocynin. In vivo lethal toxin exposure exerted similar time-dependent cardiomyocyte mechanical and intracellular Ca²⁺ responses. Stress signaling cascades including MEK1/2, p38, ERK and JNK were unaffected by in vitro lethal toxins whereas they were significantly altered by in vivo lethal toxins. Ca²⁺ regulatory proteins SERCA2a and phospholamban were also differentially regulated by in vitro and in vivo lethal toxins. Autophagy was drastically triggered although ER stress was minimally affected following lethal toxin exposure.

Conclusions

Our findings indicate that lethal toxins directly compromised murine cardiomyocyte contractile function and intracellular Ca²⁺ through a NADPH oxidase-dependent mechanism.     

Targeting β-tubulin:CCT-β complexes incurs Hsp90- and VCP-related protein degradation and induces ER stress-associated apoptosis by triggering capacitative Ca2+ entry,mitochondrial perturbation and caspase overactivation

We have previously demonstrated that interrupting the protein–protein interaction (PPI) of β-tubulin:chaperonin-containing TCP-1β (CCT-β) induces the selective killing of multidrug-resistant cancer cells due to CCT-β overexpression. However, the molecular mechanism has not yet been identified. In this study, we found that CCT-β interacts with a myriad of intracellular proteins involved in the cellular functions of the endoplasmic reticulum (ER), mitochondria, cytoskeleton, proteasome and apoptosome. Our data show that the targeted cells activate both the heat-shock protein 90 (Hsp90)-associated protein ubiquitination/degradation pathway to eliminate misfolded proteins in the cytoplasm and the valosin-containing protein (VCP)-centered ER-associated protein degradation pathway to reduce the excessive levels of unfolded polypeptides from the ER, thereby mitigating ER stress, at the onset of β-tubulin:CCT-β complex disruption. Once ER stress is expanded, ER stress-associated apoptotic signaling is enforced, as exhibited by cellular vacuolization and intracellular Ca²⁺ release. Furthermore, the elevated intracellular Ca²⁺ levels resulting from capacitative Ca²⁺ entry augments apoptotic signaling by provoking mitochondrial perturbation and caspase overactivation in the targeted cells. These findings not only provide a detailed picture of the apoptotic signaling cascades evoked by targeting the β-tubulin:CCT-β complex but also demonstrate a strategy to combat malignancies with chemoresistance to Hsp90- and VCP-related anticancer agents.