Soluble,Prefibrillar α-Synuclein Oligomers Promote Complex I-dependent,Ca2+-induced Mitochondrial Dysfunction

α-Synuclein (αSyn) aggregation and mitochondrial dysfunction both contribute to the pathogenesis of Parkinson disease (PD). Although recent studies have suggested that mitochondrial association of αSyn may disrupt mitochondrial function, it is unclear what aggregation state of αSyn is most damaging to mitochondria and what conditions promote or inhibit the effect of toxic αSyn species. Because the neuronal populations most vulnerable in PD are characterized by large cytosolic Ca²⁺ oscillations that burden mitochondria, we examined mitochondrial Ca²⁺ stress in an in vitro system comprising isolated mitochondria and purified recombinant human αSyn in various aggregation states. Using fluorimetry to simultaneously measure four mitochondrial parameters, we observed that soluble, prefibrillar αSyn oligomers, but not monomeric or fibrillar αSyn, decreased the retention time of exogenously added Ca²⁺, promoted Ca²⁺-induced mitochondrial swelling and depolarization, and accelerated cytochrome c release. Inhibition of the permeability transition pore rescued these αSyn-induced changes in mitochondrial parameters. Interestingly, the mitotoxic effects of αSyn were specifically dependent upon both electron flow through complex I and mitochondrial uptake of exogenous Ca²⁺. Our results suggest that soluble prefibrillar αSyn oligomers recapitulate several mitochondrial phenotypes previously observed in animal and cell models of PD: complex I dysfunction, altered membrane potential, disrupted Ca²⁺ homeostasis, and enhanced cytochrome c release. These data reveal how the association of oligomeric αSyn with mitochondria can be detrimental to the function of cells with high Ca²⁺-handling requirements.     

Abnormal accumulation of lipid droplets in neurons induces the conversion of alpha-Synuclein to proteolytic resistant forms in a Drosophila model of Parkinson’s disease

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein (αSyn) aggregation and associated with abnormalities in lipid metabolism. The accumulation of lipids in cytoplasmic organelles called lipid droplets (LDs) was observed in cellular models of PD. To investigate the pathophysiological consequences of interactions between αSyn and proteins that regulate the homeostasis of LDs, we used a transgenic Drosophila model of PD, in which human αSyn is specifically expressed in photoreceptor neurons. We first found that overexpression of the LD-coating proteins Perilipin 1 or 2 (dPlin1/2), which limit the access of lipases to LDs, markedly increased triacylglyclerol (TG) loaded LDs in neurons. However, dPlin-induced-LDs in neurons are independent of lipid anabolic (diacylglycerol acyltransferase 1/midway, fatty acid transport protein/dFatp) and catabolic (brummer TG lipase) enzymes, indicating that alternative mechanisms regulate neuronal LD homeostasis. Interestingly, the accumulation of LDs induced by various LD proteins (dPlin1, dPlin2, CG7900 or Klarsicht^LD-BD) was synergistically amplified by the co-expression of αSyn, which localized to LDs in both Drosophila photoreceptor neurons and in human neuroblastoma cells. Finally, the accumulation of LDs increased the resistance of αSyn to proteolytic digestion, a characteristic of αSyn aggregation in human neurons. We propose that αSyn cooperates with LD proteins to inhibit lipolysis and that binding of αSyn to LDs contributes to the pathogenic misfolding and aggregation of αSyn in neurons.     

The Calcium/Calcineurin Pathway Promotes Hemidesmosome Stability through Inhibition of β4 Integrin Phosphorylation

Cell migration depends on cells being able to create and disassemble adhesive contacts. Hemidesmosomes are multiprotein structures that attach epithelia to basal lamina and disassemble during migration and carcinoma invasion. Phosphorylation of the β4 integrin, a hemidesmosome component, induces disassembly. Although kinases involved in β4 phosphorylation have been identified, little is known about phosphatases countering kinase action. Here we report that calcineurin, a serine-threonine protein phosphatase, regulates β4 phosphorylation. Calcineurin inhibitor cyclosporin A (CsA) and calcineurin-siRNA increase β4 phosphorylation, induce hemidesmosome disassembly, and increase migration in HaCat keratinocytes, suggesting that calcineurin negatively regulates β4 phosphorylation. We found no direct dephosphorylation of β4 by calcineurin or association between β4 and calcineurin, suggesting indirect regulation of β4 phosphorylation. We therefore assessed calcineurin influence on MAPK and PKC, known to phosphorylate β4. CsA increased MAPK activity, whereas MAPK inhibitors reduced CsA-induced β4 phosphorylation, suggesting that calcineurin restricts β4 phosphorylation by MAPK. Calcineurin is activated by calcium. Increased [Ca²⁺]_i reduces β4 phosphorylation and stabilizes hemidesmosomes, effects that are reversed by CsA, indicating that calcineurin mediates calcium effects on β4. However, MAPK activation is increased when [Ca²⁺]_i is increased, suggesting that calcineurin activates an additional mechanism that counteracts MAPK-induced β4 phosphorylation. Interestingly, in some squamous cell carcinoma cells, which have reduced hemidesmosomes and increased β4 phosphorylation, an increase in [Ca²⁺]_i using thapsigargin, bradykinin, or acetylcholine can increase hemidesmosomes and reduce β4 phosphorylation in a calcineurin-dependent manner. These findings have implications in calcineurin-inhibitor induced carcinoma, a complication of immunosuppressive therapy.     

Deficiency of Sphingosine-1-phosphate Lyase Impairs Lysosomal Metabolism of the Amyloid Precursor Protein

Progressive accumulation of the amyloid β protein in extracellular plaques is a neuropathological hallmark of Alzheimer disease. Amyloid β is generated during sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. In addition to the proteolytic processing by secretases, APP is also metabolized by lysosomal proteases. Here, we show that accumulation of intracellular sphingosine-1-phosphate (S1P) impairs the metabolism of APP. Cells lacking functional S1P-lyase, which degrades intracellular S1P, strongly accumulate full-length APP and its potentially amyloidogenic C-terminal fragments (CTFs) as compared with cells expressing the functional enzyme. By cell biological and biochemical methods, we demonstrate that intracellular inhibition of S1P-lyase impairs the degradation of APP and CTFs in lysosomal compartments and also decreases the activity of γ-secretase. Interestingly, the strong accumulation of APP and CTFs in S1P-lyase-deficient cells was reversed by selective mobilization of Ca²⁺ from the endoplasmic reticulum or lysosomes. Intracellular accumulation of S1P also impairs maturation of cathepsin D and degradation of Lamp-2, indicating a general impairment of lysosomal activity. Together, these data demonstrate that S1P-lyase plays a critical role in the regulation of lysosomal activity and the metabolism of APP.     

Insulin Secretion and Ca2+ Dynamics in β-Cells Are Regulated by PERK (EIF2AK3) in Concert with Calcineurin

Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) (EIF2AK3) is essential for normal development and function of the insulin-secreting β-cell. Although genetic ablation of PERK in β-cells results in permanent neonatal diabetes in humans and mice, the underlying mechanisms remain unclear. Here, we used a newly developed and highly specific inhibitor of PERK to determine the immediate effects of acute ablation of PERK activity. We found that inhibition of PERK in human and rodent β-cells causes a rapid inhibition of secretagogue-stimulated subcellular Ca²⁺ signaling and insulin secretion. These dysfunctions stem from alterations in store-operated Ca²⁺ entry and sarcoplasmic endoplasmic reticulum Ca²⁺-ATPase activity. We also found that PERK regulates calcineurin, and pharmacological inhibition of calcineurin results in similar defects on stimulus-secretion coupling. Our findings suggest that interplay between calcineurin and PERK regulates β-cell Ca²⁺ signaling and insulin secretion, and that loss of this interaction may have profound implications in insulin secretion defects associated with diabetes.     

Ca2+ Influx through Store-operated Ca2+ Channels Reduces Alzheimer Disease β-Amyloid Peptide Secretion

Alzheimer disease (AD), the leading cause of dementia, is characterized by the accumulation of β-amyloid peptides (Aβ) in senile plaques in the brains of affected patients. Many cellular mechanisms are thought to play important roles in the development and progression of AD. Several lines of evidence point to the dysregulation of Ca²⁺ homeostasis as underlying aspects of AD pathogenesis. Moreover, direct roles in the regulation of Ca²⁺ homeostasis have been demonstrated for proteins encoded by familial AD-linked genes such as PSEN1, PSEN2, and APP, as well as Aβ peptides. Whereas these studies support the hypothesis that disruption of Ca²⁺ homeostasis contributes to AD, it is difficult to disentangle the effects of familial AD-linked genes on Aβ production from their effects on Ca²⁺ homeostasis. Here, we developed a system in which cellular Ca²⁺ homeostasis could be directly manipulated to study the effects on amyloid precursor protein metabolism and Aβ production. We overexpressed stromal interaction molecule 1 (STIM1) and Orai1, the components of the store-operated Ca²⁺ entry pathway, to generate cells with constitutive and store depletion-induced Ca²⁺ entry. We found striking effects of Ca²⁺ entry induced by overexpression of the constitutively active STIM1_D76A mutant on amyloid precursor protein metabolism. Specifically, constitutive activation of Ca²⁺ entry by expression of STIM1_D76A significantly reduced Aβ secretion. Our results suggest that disruptions in Ca²⁺ homeostasis may influence AD pathogenesis directly through the modulation of Aβ production.     

Splice Cassette II of Na+,HCO3? Cotransporter NBCn1 (slc4a7) Interacts with Calcineurin A: IMPLICATIONS FOR TRANSPORTER ACTIVITY AND INTRACELLULAR pH CONTROL DURING RAT ARTERY CONTRACTIONS*

Activation of Na⁺,HCO₃⁻ cotransport in vascular smooth muscle cells (VSMCs) contributes to intracellular pH (pH_i) control during artery contraction, but the signaling pathways involved have been unknown. We investigated whether physical and functional interactions between the Na⁺,HCO₃⁻ cotransporter NBCn1 (slc4a7) and the Ca²⁺/calmodulin-activated serine/threonine phosphatase calcineurin exist and play a role for pH_i control in VSMCs. Using a yeast two-hybrid screen, we found that splice cassette II from the N terminus of NBCn1 interacts with calcineurin Aβ. When cassette II was truncated or mutated to disrupt the putative calcineurin binding motif PTVVIH, the interaction was abolished. Native NBCn1 and calcineurin Aβ co-immunoprecipitated from A7r5 rat VSMCs. A peptide (acetyl-DDIPTVVIH-amide), which mimics the putative calcineurin binding motif, inhibited the co-immunoprecipitation whereas a mutated peptide (acetyl-DDIATAVAA-amide) did not. Na⁺,HCO₃⁻ cotransport activity was investigated in VSMCs of mesenteric arteries after an NH₄⁺ prepulse. During depolarization with 50 mm extracellular K⁺ to raise intracellular [Ca²⁺], Na⁺,HCO₃⁻ cotransport activity was inhibited 20–30% by calcineurin inhibitors (FK506 and cyclosporine A). FK506 did not affect Na⁺,HCO₃⁻ cotransport activity in VSMCs when cytosolic [Ca²⁺] was lowered by buffering, nor did it disrupt binding between NBCn1 and calcineurin Aβ. FK506 augmented the intracellular acidification of VSMCs during norepinephrine-induced artery contractions. No physical or functional interactions between calcineurin Aβ and the Na⁺/H⁺ exchanger NHE1 were observed in VSMCs. In conclusion, we demonstrate a physical interaction between calcineurin Aβ and cassette II of NBCn1. Intracellular Ca²⁺ activates Na⁺,HCO₃⁻ cotransport activity in VSMCs in a calcineurin-dependent manner which is important for protection against intracellular acidification.     

Manganese promotes α-synuclein amyloid aggregation through the induction of protein phase transition

α-Synuclein (α-Syn) is the major protein component of Lewy bodies, a key pathological feature of Parkinson’s disease (PD). The manganese ion Mn²⁺ has been identified as an environmental risk factor of PD. However, it remains unclear how Mn²⁺ regulates α-Syn aggregation. Here, we discovered that Mn²⁺accelerates α-Syn amyloid aggregation through the regulation of protein phase separation. We found that Mn²⁺ not only promotes α-Syn liquid-to-solid phase transition but also directly induces soluble α-Syn monomers to form solid-like condensates. Interestingly, the lipid membrane is integrated into condensates during Mn²⁺-induced α-Syn phase transition; however, the preformed Mn²⁺/α-syn condensates can only recruit lipids to the surface of condensates. In addition, this phase transition can largely facilitate α-Syn amyloid aggregation. Although the Mn²⁺-induced condensates do not fuse, our results demonstrated that they could recruit soluble α-Syn monomers into the existing condensates. Furthermore, we observed that a manganese chelator reverses Mn²⁺-induced α-Syn aggregation during the phase transition stage. However, after maturation, α-Syn aggregation becomes irreversible. These findings demonstrate that Mn²⁺ facilitates α-Syn phase transition to accelerate the formation of α-Syn aggregates and provide new insights for targeting α-Syn phase separation in PD treatment.     

Plasma Membrane Ca2+-ATPase Overexpression Depletes Both Mitochondrial and Endoplasmic Reticulum Ca2+ Stores and Triggers Apoptosis in Insulin-secreting BRIN-BD11 Cells

Ca²⁺ may trigger apoptosis in β-cells. Hence, the control of intracellular Ca²⁺ may represent a potential approach to prevent β-cell apoptosis in diabetes. Our objective was to investigate the effect and mechanism of action of plasma membrane Ca²⁺-ATPase (PMCA) overexpression on Ca²⁺-regulated apoptosis in clonal β-cells. Clonal β-cells (BRIN-BD11) were examined for the effect of PMCA overexpression on cytosolic and mitochondrial [Ca²⁺] using a combination of aequorins with different Ca²⁺ affinities and on the ER and mitochondrial pathways of apoptosis. β-cell stimulation generated microdomains of high [Ca²⁺] in the cytosol and subcellular heterogeneities in [Ca²⁺] among mitochondria. Overexpression of PMCA decreased [Ca²⁺] in the cytosol, the ER, and the mitochondria and activated the IRE1α-XBP1s but inhibited the PRKR-like ER kinase-eIF2α and the ATF6-BiP pathways of the ER-unfolded protein response. Increased Bax/Bcl-2 expression ratio was observed in PMCA overexpressing β-cells. This was followed by Bax translocation to the mitochondria with subsequent cytochrome c release, opening of the permeability transition pore, and apoptosis. In conclusion, clonal β-cell stimulation generates microdomains of high [Ca²⁺] in the cytosol and subcellular heterogeneities in [Ca²⁺] among mitochondria. PMCA overexpression depletes intracellular [Ca²⁺] stores and, despite a decrease in mitochondrial [Ca²⁺], induces apoptosis through the mitochondrial pathway. These data open the way to new strategies to control cellular Ca²⁺ homeostasis that could decrease β-cell apoptosis in diabetes.     

Lysosomes Behave as Ca2+-regulated Exocytic Vesicles in Fibroblasts and Epithelial Cells

Lysosomes are considered to be a terminal degradative compartment of the endocytic pathway, into which transport is mostly unidirectional. However, specialized secretory vesicles regulated by Ca²⁺, such as neutrophil azurophil granules, mast cell–specific granules, and cytotoxic lymphocyte lytic granules, share characteristics with lysosomes that may reflect a common biogenesis. In addition, the involvement of Ca²⁺ transients in the invasion mechanism of the parasite Trypanosoma cruzi, which occurs by fusion of lysosomes with the plasma membrane, suggested that lysosome exocytosis might be a generalized process present in most cell types.

Here we demonstrate that elevation in the intracellular free Ca²⁺ concentration of normal rat kidney (NRK) fibroblasts induces fusion of lysosomes with the plasma membrane. This was verified by measuring the release of the lysosomal enzyme β-hexosaminidase, the appearance on the plasma membrane of the lysosomal glycoprotein lgp120, the release of fluid-phase tracers previously loaded into lysosomes, and the release of the lysosomally processed form of cathepsin D. Exposure to the Ca²⁺ ionophore ionomycin or addition of Ca²⁺containing buffers to streptolysin O–permeabilized cells induced exocytosis of ~10% of the total lysosomes of NRK cells. The process was also detected in other cell types such as epithelial cells and myoblasts. Lysosomal exocytosis was found to require micromolar levels of Ca²⁺ and to be temperature and ATP dependent, similar to Ca²⁺-regulated secretory mechanisms in specialized cells.

These findings highlight a novel role for lysosomes in cellular membrane traffic and suggest that fusion of lysosomes with the plasma membrane may be an ubiquitous form of Ca²⁺-regulated exocytosis.

     

Abnormal Calcium Handling and Exaggerated Cardiac Dysfunction in Mice with Defective Vitamin D Signaling

Aim

Altered vitamin D signaling is associated with cardiac dysfunction, but the pathogenic mechanism is not clearly understood. We examine the mechanism and the role of vitamin D signaling in the development of cardiac dysfunction.

Methods and Results

We analyzed 1α-hydroxylase (1α-OHase) knockout (1α-OHase^−/−) mice, which lack 1α-OH enzymes that convert the inactive form to hormonally active form of vitamin D. 1α-OHase^−/− mice showed modest cardiac hypertrophy at baseline. Induction of pressure overload by transverse aortic constriction (TAC) demonstrated exaggerated cardiac dysfunction in 1α-OHase^−/− mice compared to their WT littermates with a significant increase in fibrosis and expression of inflammatory cytokines. Analysis of calcium (Ca²⁺) transient demonstrated profound Ca²⁺ handling abnormalities in 1α-OHase^−/− mouse cardiomyocytes (CMs), and treatment with paricalcitol (PC), an activated vitamin D₃ analog, significantly attenuated defective Ca²⁺ handling in 1α-OHase^−/− CMs. We further delineated the effect of vitamin D deficiency condition to TAC by first correcting the vitamin D deficiency in 1α-OHase^−/− mice, followed then by either a daily maintenance dose of vitamin D or vehicle (to achieve vitamin D deficiency) at the time of sham or TAC. In mice treated with vitamin D, there was a significant attenuation of TAC-induced cardiac hypertrophy, interstitial fibrosis, inflammatory markers, Ca²⁺ handling abnormalities and cardiac function compared to the vehicle treated animals.

Conclusions

Our results provide insight into the mechanism of cardiac dysfunction, which is associated with severely defective Ca²⁺ handling and defective vitamin D signaling in 1α-OHase^−/− mice.     

Remodeling the Proteostasis Network to Rescue Glucocerebrosidase Variants by Inhibiting ER-Associated Degradation and Enhancing ER Folding

Gaucher’s disease (GD) is characterized by loss of lysosomal glucocerebrosidase (GC) activity. Mutations in the gene encoding GC destabilize the protein’s native folding leading to ER-associated degradation (ERAD) of the misfolded enzyme. Enhancing the cellular folding capacity by remodeling the proteostasis network promotes native folding and lysosomal activity of mutated GC variants. However, proteostasis modulators reported so far, including ERAD inhibitors, trigger cellular stress and lead to induction of apoptosis. We show herein that lacidipine, an L-type Ca²⁺ channel blocker that also inhibits ryanodine receptors on the ER membrane, enhances folding, trafficking and lysosomal activity of the most severely destabilized GC variant achieved via ERAD inhibition in fibroblasts derived from patients with GD. Interestingly, reprogramming the proteostasis network by combining modulation of Ca²⁺ homeostasis and ERAD inhibition remodels the unfolded protein response and dramatically lowers apoptosis induction typically associated with ERAD inhibition.     

Bile Acids Induce Pancreatic Acinar Cell Injury and Pancreatitis by Activating Calcineurin

Biliary pancreatitis is the leading cause of acute pancreatitis in both children and adults. A proposed mechanism is the reflux of bile into the pancreatic duct. Bile acid exposure causes pancreatic acinar cell injury through a sustained rise in cytosolic Ca²⁺. Thus, it would be clinically relevant to know the targets of this aberrant Ca²⁺ signal. We hypothesized that the Ca²⁺-activated phosphatase calcineurin is such a Ca²⁺ target. To examine calcineurin activation, we infected primary acinar cells from mice with an adenovirus expressing the promoter for a downstream calcineurin effector, nuclear factor of activated T-cells (NFAT). The bile acid taurolithocholic acid-3-sulfate (TLCS) was primarily used to examine bile acid responses. TLCS caused calcineurin activation only at concentrations that cause acinar cell injury. The activation of calcineurin by TLCS was abolished by chelating intracellular Ca²⁺. Pretreatment with 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (acetoxymethyl ester) (BAPTA-AM) or the three specific calcineurin inhibitors FK506, cyclosporine A, or calcineurin inhibitory peptide prevented bile acid-induced acinar cell injury as measured by lactate dehydrogenase leakage and propidium iodide uptake. The calcineurin inhibitors reduced the intra-acinar activation of chymotrypsinogen within 30 min of TLCS administration, and they also prevented NF-κB activation. In vivo, mice that received FK506 or were deficient in the calcineurin isoform Aβ (CnAβ) subunit had reduced pancreatitis severity after infusion of TLCS or taurocholic acid into the pancreatic duct. In summary, we demonstrate that acinar cell calcineurin is activated in response to Ca²⁺ generated by bile acid exposure, bile acid-induced pancreatic injury is dependent on calcineurin activation, and calcineurin inhibitors may provide an adjunctive therapy for biliary pancreatitis.     

Ca2+-binding Motif of βγ-Crystallins

βγ-Crystallin-type double clamp (N/D)(N/D)XX(S/T)S motif is an established but sparsely investigated motif for Ca²⁺ binding. A βγ-crystallin domain is formed of two Greek key motifs, accommodating two Ca²⁺-binding sites. βγ-Crystallins make a separate class of Ca²⁺-binding proteins (CaBP), apparently a major group of CaBP in bacteria. Paralleling the diversity in βγ-crystallin domains, these motifs also show great diversity, both in structure and in function. Although the expression of some of them has been associated with stress, virulence, and adhesion, the functional implications of Ca²⁺ binding to βγ-crystallins in mediating biological processes are yet to be elucidated.     

Hsp31 Is a Stress Response Chaperone That Intervenes in the Protein Misfolding Process

The Saccharomyces cerevisiae heat shock protein Hsp31 is a stress-inducible homodimeric protein that is involved in diauxic shift reprogramming and has glyoxalase activity. We show that substoichiometric concentrations of Hsp31 can abrogate aggregation of a broad array of substrates in vitro. Hsp31 also modulates the aggregation of α-synuclein (αSyn), a target of the chaperone activity of human DJ-1, an Hsp31 homolog. We demonstrate that Hsp31 is able to suppress the in vitro fibrillization or aggregation of αSyn, citrate synthase and insulin. Chaperone activity was also observed in vivo because constitutive overexpression of Hsp31 reduced the incidence of αSyn cytoplasmic foci, and yeast cells were rescued from αSyn-generated proteotoxicity upon Hsp31 overexpression. Moreover, we showed that Hsp31 protein levels are increased by H₂O₂, in the diauxic phase of normal growth conditions, and in cells under αSyn-mediated proteotoxic stress. We show that Hsp31 chaperone activity and not the methylglyoxalase activity or the autophagy pathway drives the protective effects. We also demonstrate reduced aggregation of the Sup35 prion domain, PrD-Sup35, as visualized by fluorescent protein fusions. In addition, Hsp31 acts on its substrates prior to the formation of large aggregates because Hsp31 does not mutually localize with prion aggregates, and it prevents the formation of detectable in vitro αSyn fibrils. These studies establish that the protective role of Hsp31 against cellular stress is achieved by chaperone activity that intervenes early in the protein misfolding process and is effective on a wide spectrum of substrate proteins, including αSyn and prion proteins.     

Withdrawal of Essential Amino Acids Increases Autophagy by a Pathway Involving Ca2+/Calmodulin-dependent Kinase Kinase-β (CaMKK-β)

Autophagy is the main lysosomal catabolic process that becomes activated under stress conditions, such as amino acid starvation and cytosolic Ca²⁺ upload. However, the molecular details on how both conditions control autophagy are still not fully understood. Here we link essential amino acid starvation and Ca²⁺ in a signaling pathway to activate autophagy. We show that withdrawal of essential amino acids leads to an increase in cytosolic Ca²⁺, arising from both extracellular medium and intracellular stores, which induces the activation of adenosine monophosphate-activated protein kinase (AMPK) via Ca²⁺/calmodulin-dependent kinase kinase-β (CaMKK-β). Furthermore, we show that autophagy induced by amino acid starvation requires AMPK, as this induction is attenuated in its absence. Subsequently, AMPK activates UNC-51-like kinase (ULK1), a mammalian autophagy-initiating kinase, through phosphorylation at Ser-555 in a process that requires CaMKK-β. Finally, the mammalian target of rapamycin complex C1 (mTORC1), a negative regulator of autophagy downstream of AMPK, is inhibited by amino acid starvation in a Ca²⁺-sensitive manner, and CaMKK-β appears to be important for mTORC1 inactivation, especially in the absence of extracellular Ca²⁺. All these results highlight that amino acid starvation regulates autophagy in part through an increase in cellular Ca²⁺ that activates a CaMKK-β-AMPK pathway and inhibits mTORC1, which results in ULK1 stimulation.     

JNK1/2 regulates ER–mitochondrial Ca2+ cross-talk during IL-1β–mediated cell death in RINm5F and human primary β-cells

Elevated interleukin-1β (IL-1β) induces apoptosis in pancreatic β-cells through endoplasmic reticulum (ER) stress induction and subsequent c-jun-N-terminal kinase 1/2 (JNK1/2) activation. In earlier work we showed that JNK1/2 activation is initiated before ER stress and apoptotic induction in response to IL-1β. However, the detailed regulatory mechanisms are not completely understood. Because the ER is the organelle responsible for Ca²⁺ handling and storage, here we examine the effects of IL-1β on cellular Ca²⁺ movement and mitochondrial dysfunction and evaluate the role of JNK1/2. Our results show that in RINm5F cells and human primary β-cells, IL-1β alters mitochondrial membrane potential, mitochondrial permeability transition pore opening, ATP content, and reactive oxygen species production and these alterations are preceded by ER Ca²⁺ release via IP₃R channels and mitochondrial Ca²⁺ uptake. All these events are prevented by JNK1/2 small interfering RNA (siRNA), indicating the mediating role of JNK1/2 in IL-1β–induced cellular alteration. This is accompanied by IL-1β–induced apoptosis, which is prevented by JNK1/2 siRNA and the IP₃R inhibitor xestospongin C. This suggests a regulatory role of JNK1/2 in modulating the ER-mitochondrial-Ca²⁺ axis by IL-1β in apoptotic cell death.     

Functional Characterization of CaVα2δ Mutations Associated with Sudden Cardiac Death

L-type Ca²⁺ channels play a critical role in cardiac rhythmicity. These ion channels are oligomeric complexes formed by the pore-forming Ca_Vα1 with the auxiliary Ca_Vβ and Ca_Vα2δ subunits. Ca_Vα2δ increases the peak current density and improves the voltage-dependent activation gating of Ca_V1.2 channels without increasing the surface expression of the Ca_Vα1 subunit. The functional impact of genetic variants of CACNA2D1 (the gene encoding for Ca_Vα2δ), associated with shorter repolarization QT intervals (the time interval between the Q and the T waves on the cardiac electrocardiogram), was investigated after recombinant expression of the full complement of L-type Ca_V1.2 subunits in human embryonic kidney 293 cells. By performing side-by-side high resolution flow cytometry assays and whole-cell patch clamp recordings, we revealed that the surface density of the Ca_Vα2δ wild-type protein correlates with the peak current density. Furthermore, the cell surface density of Ca_Vα2δ mutants S755T, Q917H, and S956T was not significantly different from the cell surface density of the Ca_Vα2δ wild-type protein expressed under the same conditions. In contrast, the cell surface expression of Ca_Vα2δ D550Y, Ca_Vα2δ S709N, and the double mutant D550Y/Q917H was reduced, respectively, by ≈30–33% for the single mutants and by 60% for the latter. The cell surface density of D550Y/Q917H was more significantly impaired than protein stability, suggesting that surface trafficking of Ca_Vα2δ was disrupted by the double mutation. Co-expression with D550Y/Q917H significantly decreased Ca_V1.2 currents as compared with results obtained with Ca_Vα2δ wild type. It is concluded that D550Y/Q917H reduced inward Ca²⁺ currents through a defect in the cell surface trafficking of Ca_Vα2δ. Altogether, our results provide novel insight in the molecular mechanism underlying the modulation of Ca_V1.2 currents by Ca_Vα2δ.     

Two-pore Channels (TPC2s) and Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) at Lysosomal-Sarcoplasmic Reticular Junctions Contribute to Acute and Chronic β-Adrenoceptor Signaling in the Heart

Ca²⁺-permeable type 2 two-pore channels (TPC2) are lysosomal proteins required for nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca²⁺ release in many diverse cell types. Here, we investigate the importance of TPC2 proteins for the physiology and pathophysiology of the heart. NAADP-AM failed to enhance Ca²⁺ responses in cardiac myocytes from Tpcn2^−/− mice, unlike myocytes from wild-type (WT) mice. Ca²⁺/calmodulin-dependent protein kinase II inhibitors suppressed actions of NAADP in myocytes. Ca²⁺ transients and contractions accompanying action potentials were increased by isoproterenol in myocytes from WT mice, but these effects of β-adrenoreceptor stimulation were reduced in myocytes from Tpcn2^−/− mice. Increases in amplitude of L-type Ca²⁺ currents evoked by isoproterenol remained unchanged in myocytes from Tpcn2^−/− mice showing no loss of β-adrenoceptors or coupling mechanisms. Whole hearts from Tpcn2^−/− mice also showed reduced inotropic effects of isoproterenol and a reduced tendency for arrhythmias following acute β-adrenoreceptor stimulation. Hearts from Tpcn2^−/− mice chronically exposed to isoproterenol showed less cardiac hypertrophy and increased threshold for arrhythmogenesis compared with WT controls. Electron microscopy showed that lysosomes form close contacts with the sarcoplasmic reticulum (separation ∼25 nm). We propose that Ca²⁺-signaling nanodomains between lysosomes and sarcoplasmic reticulum dependent on NAADP and TPC2 comprise an important element in β-adrenoreceptor signal transduction in cardiac myocytes. In summary, our observations define a role for NAADP and TPC2 at lysosomal/sarcoplasmic reticulum junctions as unexpected but major contributors in the acute actions of β-adrenergic signaling in the heart and also in stress pathways linking chronic stimulation of β-adrenoceptors to hypertrophy and associated arrhythmias.     

The Effect of SERCA1b Silencing on the Differentiation and Calcium Homeostasis of C2C12 Skeletal Muscle Cells

The sarcoplasmic/endoplasmic reticulum Ca²⁺ATPases (SERCAs) are the main Ca²⁺ pumps which decrease the intracellular Ca²⁺ level by reaccumulating Ca²⁺ into the sarcoplasmic reticulum. The neonatal SERCA1b is the major Ca²⁺ pump in myotubes and young muscle fibers. To understand its role during skeletal muscle differentiation its synthesis has been interfered with specific shRNA sequence. Stably transfected clones showing significantly decreased SERCA1b expression (cloneC1) were selected for experiments. The expression of the regulatory proteins of skeletal muscle differentiation was examined either by Western-blot at the protein level for MyoD, STIM1, calsequestrin (CSQ), and calcineurin (CaN) or by RT-PCR for myostatin and MCIP1.4. Quantitative analysis revealed significant alterations in CSQ, STIM1, and CaN expression in cloneC1 as compared to control cells. To examine the functional consequences of the decreased expression of SERCA1b, repeated Ca²⁺-transients were evoked by applications of 120 mM KCl. The significantly higher [Ca²⁺]i measured at the 20^th and 40^th seconds after the beginning of KCl application (112±3 and 110±3 nM vs. 150±7 and 135±5 nM, in control and in cloneC1 cells, respectively) indicated a decreased Ca²⁺-uptake capability which was quantified by extracting the maximal pump rate (454±41 μM/s vs. 144±24 μM/s, in control and in cloneC1 cells). Furthermore, the rate of calcium release from the SR (610±60 vs. 377±64 μM/s) and the amount of calcium released (843±75 μM vs. 576±80 μM) were also significantly suppressed. These changes were also accompanied by a reduced activity of CaN in cells with decreased SERCA1b. In parallel, cloneC1 cells showed inhibited cell proliferation and decreased myotube nuclear numbers. Moreover, while cyclosporineA treatment suppressed the proliferation of parental cultures it had no effect on cloneC1 cells. SERCA1b is thus considered to play an essential role in the regulation of [Ca²⁺]i and its ab ovo gene silencing results in decreased skeletal muscle differentiation.