Luminal Ca2+ depletion during the unfolded protein response in Xenopus oocytes: Cause and consequence

The endoplasmic reticulum (ER) is a Ca2+ storing organelle that plays a critical role in the synthesis, folding and post-translational modifications of many proteins. The ER enters into a condition of stress when the load of newly synthesized proteins exceeds its folding and processing capacity. This activates a signal transduction pathway called the unfolded protein response (UPR) that attempts to restore homeostasis. The precise role of ER Ca2+ in the initiation of the UPR has not been defined. Specifically, it has not been established whether ER Ca2+ dysregulation is a cause or consequence of ER stress. Here, we report that partial depletion of ER Ca2+ stores induces a significant induction of the UPR, and leads to the retention of a normally secreted protein Carboxypeptidase Y. Moreover, inhibition of protein glycosylation by tunicamycin rapidly induced an ER Ca2+ leak into the cytosol. However, blockade of the translocon with emetine inhibited the tunicamycin-induced Ca2+ release. Furthermore, emetine treatment blocked elF2α phosphorylation and reduced expression of the chaperone BiP. These findings suggest that Ca2+ may be both a cause and a consequence of ER protein misfolding. Thus, it appears that ER Ca2+ leak is a significant co-factor for the initiation of the UPR.Fil: Paredes, R. Madelaine. University of Texas; Estados UnidosFil: Bollo, Mariana Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Holstein, Deborah. University of Texas; Estados UnidosFil: Lechleiter, James D.. University of Texas; Estados Unido

Purinergic Receptor Stimulation Reduces Cytotoxic Edema and Brain Infarcts in Mouse Induced by Photothrombosis by Energizing Glial Mitochondria

Treatments to improve the neurological outcome of edema and cerebral ischemic stroke are severely limited. Here, we present the first in vivo single cell images of cortical mouse astrocytes documenting the impact of single vessel photothrombosis on cytotoxic edema and cerebral infarcts. The volume of astrocytes expressing green fluorescent protein (GFP) increased by over 600% within 3 hours of ischemia. The subsequent growth of cerebral infarcts was easily followed as the loss of GFP fluorescence as astrocytes lysed. Cytotoxic edema and the magnitude of ischemic lesions were significantly reduced by treatment with the purinergic ligand 2-methylthioladenosine 5′ diphosphate (2-MeSADP), an agonist with high specificity for the purinergic receptor type 1 isoform (P2Y1R). At 24 hours, cytotoxic edema in astrocytes was still apparent at the penumbra and preceded the cell lysis that defined the infarct. Delayed 2MeSADP treatment, 24 hours after the initial thrombosis, also significantly reduced cytotoxic edema and the continued growth of the brain infarction. Pharmacological and genetic evidence are presented indicating that 2MeSADP protection is mediated by enhanced astrocyte mitochondrial metabolism via increased inositol trisphosphate (IP3)-dependent Ca2+ release. We suggest that mitochondria play a critical role in astrocyte energy metabolism in the penumbra of ischemic lesions, where low ATP levels are widely accepted to be responsible for cytotoxic edema. Enhancement of this energy source could have similar protective benefits for a wide range of brain injuries

Calcineurin Interacts with PERK and Dephosphorylates Calnexin to Relieve ER Stress in Mammals and Frogs

Background: The accumulation of misfolded proteins within the endoplasmic reticulum (ER) triggers a cellular process known as the Unfolded Protein Response (UPR). One of the earliest responses is the attenuation of protein translation. Little is known about the role that Ca 2+ mobilization plays in the early UPR. Work from our group has shown that cytosolic phosphorylation of calnexin (CLNX) controls Ca 2+ uptake into the ER via the sarco-endoplasmic reticulum Ca 2+-ATPase (SERCA) 2b. Methodology/Principal Findings: Here, we demonstrate that calcineurin (CN), a Ca 2+ dependent phosphatase, associates with the (PKR)-like ER kinase (PERK), and promotes PERK auto-phosphorylation. This association, in turn, increases the phosphorylation level of eukaryotic initiation factor-2 a (eIF2-a) and attenuates protein translation. Data supporting these conclusions were obtained from co-immunoprecipitations, pull-down assays, in-vitro kinase assays, siRNA treatments and [ 35 S]-methionine incorporation measurements. The interaction of CN with PERK was facilitated at elevated cytosolic Ca 2+ concentrations and involved the cytosolic domain of PERK. CN levels were rapidly increased by ER stressors, which could be blocked by siRNA treatments for CN-Aa in cultured astrocytes. Downregulation of CN blocked subsequent ER-stress-induced increases in phosphorylated elF2-a. CN knockdown in Xenopus oocytes predisposed them to induction of apoptosis. We also found that CLNX was dephosphorylated by CN when Ca 2+ increased. These data were obtained from [c 32 P]-CLN

Luminal Ca2+ depletion during the unfolded protein response in Xenopus oocytes: Cause and consequence

The endoplasmic reticulum (ER) is a Ca2+ storing organelle that plays a critical role in the synthesis, folding and post-translational modifications of many proteins. The ER enters into a condition of stress when the load of newly synthesized proteins exceeds its folding and processing capacity. This activates a signal transduction pathway called the unfolded protein response (UPR) that attempts to restore homeostasis. The precise role of ER Ca2+ in the initiation of the UPR has not been defined. Specifically, it has not been established whether ER Ca2+ dysregulation is a cause or consequence of ER stress. Here, we report that partial depletion of ER Ca2+ stores induces a significant induction of the UPR, and leads to the retention of a normally secreted protein Carboxypeptidase Y. Moreover, inhibition of protein glycosylation by tunicamycin rapidly induced an ER Ca2+ leak into the cytosol. However, blockade of the translocon with emetine inhibited the tunicamycin-induced Ca2+ release. Furthermore, emetine treatment blocked elF2α phosphorylation and reduced expression of the chaperone BiP. These findings suggest that Ca2+ may be both a cause and a consequence of ER protein misfolding. Thus, it appears that ER Ca2+ leak is a significant co-factor for the initiation of the UPR.Fil: Paredes, R. Madelaine. University of Texas; Estados UnidosFil: Bollo, Mariana Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Holstein, Deborah. University of Texas; Estados UnidosFil: Lechleiter, James D.. University of Texas; Estados Unido

Calcineurin β protects brain after injury by activating the unfolded protein response

The Ca2 +-dependent phosphatase, calcineurin (CN) is thought to play a detrimental role in damaged neurons; however, its role in astrocytes is unclear. In cultured astrocytes, CNβ expression increased after treatment with a sarco/endoplasmic reticulum Ca2 +-ATPase inhibitor, thapsigargin, and with oxygen and glucose deprivation, an in vitro model of ischemia. Similarly, CNβ was induced in astrocytes in vivo in two different mouse models of brain injury - photothrombotic stroke and traumatic brain injury (TBI). Immunoprecipitation and chemical activation dimerization methods pointed to physical interaction of CNβ with the unfolded protein response (UPR) sensor, protein kinase RNA-like endoplasmic reticulum kinase (PERK). In accordance, induction of CNβ resulted in oligomerization and activation of PERK. Strikingly, the presence of a phosphatase inhibitor did not interfere with CNβ-mediated activation of PERK, suggesting a hitherto undiscovered non-enzymatic role for CNβ. Importantly, the cytoprotective function of CNβ was PERK-dependent both in vitro and in vivo. Loss of CNβ in vivo resulted in a significant increase in cerebral damage, and correlated with a decrease in astrocyte size, PERK activity and glial fibrillary acidic protein (GFAP) expression. Taken together, these data reveal a critical role for the CNβ-PERK axis in not only prolonging astrocyte cell survival but also in modulating astrogliosis after brain injury.Fil: Chen, Yanan. University of Texas Health Science Center at San Antonio; Estados UnidosFil: Holstein, Deborah M.. University of Texas Health Science Center at San Antonio; Estados UnidosFil: Aime, Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Bollo, Mariana Ines. University of Texas Health Science Center at San Antonio; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Lechleiter, James D.. University of Texas Health Science Center at San Antonio; Estados Unido

Cyclophilin D over-expression increases mitochondrial complex III activity and accelerates supercomplex formation

Cyclophilin D (CyPD), a mitochondrial matrix protein, has been widely studied for its role in mitochondrial-mediated cell death. Unexpectedly, we previously discovered that overexpression of CyPD in a stable cell line, increased mitochondrial membrane potentials and enhanced cell survival under conditions of oxidative stress. Here, we investigated the underlying mechanisms responsible for these findings. Spectrophotometric measurements in isolated mitochondria revealed that overexpression of CyPD in HEK293 cells increased respiratory chain activity, but only for Complex III (CIII). Acute treatment of mitochondria with the immumosupressant cyclosporine A did not affect CIII activity. Expression levels of the CIII subunits cytochrome b and Rieske-FeS were elevated in HEK293 cells overexpressing CyPD. However, CIII activity was still significantly higher compared to control mitochondria, even when normalized by protein expression. Blue native gel electrophoresis and Western blot assays revealed a molecular interaction of CyPD with CIII and increased levels of supercomplexes in mitochondrial protein extracts. Radiolabeled protein synthesis in mitochondria showed that CIII assembly and formation of supercomplexes containing CIII were significantly faster when CyPD was overexpressed. Taken together, these data indicate that CyPD regulates mitochondrial metabolism, and likely cell survival, by promoting more efficient electrons flow through the respiratory chain via increased supercomplex formation.Fil: Etzler, Julie C.. University of Texas Health Science Center at San Antonio; Estados UnidosFil: Bollo, Mariana Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Holstein, Deborah. University of Texas Health Science Center at San Antonio; Estados UnidosFil: Deng, Janice Jianhong. University of Texas Health Science Center at San Antonio; Estados UnidosFil: Perez, Viviana. State University of Oregon; Estados UnidosFil: Lin, Da-ting. University of Texas Health Science Center at San Antonio; Estados UnidosFil: Richardson, Arlan. Oklahoma State University; Estados UnidosFil: Bai, Yidong. University of Texas Health Science Center at San Antonio; Estados UnidosFil: Lechleiter, James D.. University of Texas Health Science Center at San Antonio; Estados Unido

Ca2+ signalling system initiated by endoplasmic reticulum stress stimulates PERK activation

The accumulation of unfolded proteins within the Endoplasmic Reticulum (ER) activates a signal transduction pathway termed the unfolded protein response (UPR), which attempts to restore ER homoeostasis. If this cannot be done, UPR signalling ultimately induces apoptosis. Ca2+ depletion in the ER is a potent inducer of ER stress. Despite the ubiquity of Ca2+ as an intracellular messenger, the precise mechanism(s) by which Ca2+ release affects the UPR remains unknown. Tethering a genetically encoded Ca2+ indicator (GCamP6) to the ER membrane revealed novel Ca2+ signalling events initiated by Ca2+ microdomains in human astrocytes under ER stress, induced by tunicamycin (Tm), an N-glycosylation inhibitor, as well as in a cell model deficient in all three inositol triphosphate receptor isoforms. Pharmacological and molecular studies indicate that these local events are mediated by translocons and that the Ca2+ microdomains impact (PKR)-like-ER kinase (PERK), an UPR sensor, activation. These findings reveal the existence of a Ca2+ signal mechanism by which stressor-mediated Ca2+ release regulates ER stress.Fil: Feliziani, Constanza. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Fernández, Macarena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Quassollo Infanzon, Gonzalo Emiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Holstein, Deborah. University Of Texas Health Science Center At San Antonio (ut Health San Antonio) ; University Of Texas At San Antonio;Fil: Bairo, Sebastián Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Paton, James C.. University of Adelaide; AustraliaFil: Paton, Adrienne W.. University of Adelaide; AustraliaFil: de Batista, Juan. Instituto Universitario de Ciencias Biomédicas de Córdoba; ArgentinaFil: Lechleiter, James D.. University Of Texas Health Science Center At San Antonio (ut Health San Antonio) ; University Of Texas At San Antonio;Fil: Bollo, Mariana Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentin