Molecular Modulation by Lentivirus-Delivered Specific shRNAs in Endoplasmic Reticulum Stressed Neurons

The accumulation of unfolded proteins within the endoplasmic reticulum (ER), caused by any stress condition, triggers the unfolded protein response (UPR) ...

Universidad Nacional de Córdoba

The endoplasmic reticulum (ER) is a Ca(2+) 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 Ca(2+) in the initiation of the UPR has not been defined. Specifically, it has not been established whether ER Ca(2+) dysregulation is a cause or consequence of ER stress. Here, we report that partial depletion of ER Ca(2+) 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 Ca(2+) leak into the cytosol. However, blockade of the translocon with emetine inhibited the tunicamycin-induced Ca(2+) release. Furthermore, emetine treatment blocked elF2α phosphorylation and reduced expression of the chaperone BiP. These findings suggest that Ca(2+) may be both a cause and a consequence of ER protein misfolding. Thus, it appears that ER Ca(2+) leak is a significant co-factor for the initiation of the UPR.

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

The Ca(2+)-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 Ca(2+)-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. 

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

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.

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

GM2-gangliosidosis, a subgroup of lysosomal storage disorders, is caused by deficiency of hexosaminidase activity, and comprises the closely related Tay-Sachs and Sandhoff diseases. The enzyme deficiency prevents normal metabolization of ganglioside GM2, usually resulting in progressive neurodegenerative disease. The molecular mechanisms whereby GM2 accumulation in neurons triggers neurodegeneration remain unclear. In vitro experiments, using microsomes from Sandhoff mouse model brain, showed that increase of GM2 content negatively modulates sarco/endoplasmic reticulum Ca-ATPase (SERCA) (Pelled et al., 2003). Furthermore, Ca depletion in endoplasmic reticulum (ER) triggers Unfolded Protein Response (UPR), which tends to restore homeostasis in the ER; however, if cellular damage persists, an apoptotic response is initiated. We found that ER GM2 accumulation in cultured neurons induces luminal Ca depletion, which in turn activates PERK (protein kinase RNA [PKR]-like ER kinase), one of three UPR sensors. PERK signaling displayed biphasic activation; i.e., early upregulation of cytoprotective calcineurin (CN) and, under prolonged ER stress, enhanced expression of pro-apoptotic transcription factor C/EBP homologous protein (CHOP). Moreover, GM2 accumulation in neuronal cells induced neurite atrophy and apoptosis. Both processes were effectively modulated by treatment with the selective PERK inhibitor GSK2606414, by CN knockdown, and by CHOP knockdown. Overall, our findings demonstrate the essential role of PERK signaling pathway contributing to neurodegeneration in a model of GM2-gangliosidosis.

Neurite atrophy and apoptosis mediated by PERK signaling after accumulation of GM2-ganglioside.

17β-estradiol (E2) induces axonal growth through extracellular signal-regulated kinase 1 and 2 (ERK1/2)-MAPK cascade in hypothalamic neurons of male rat embryos , but the mechanism that initiates these events is poorly understood. This study reports the intracellular Ca increase that participates in the activation of ERK1/2 and axogenesis induced by E2. Hypothalamic neuron cultures were established from 16-day-old male rat embryos and fed with astroglia-conditioned media for 48 h. E2-induced ERK phosphorylation was completely abolished by a ryanodine receptor (RyR) inhibitor (ryanodine) and partially attenuated by an L-type voltage-gated Ca channel (L-VGCC) blocker (nifedipine), an inositol-1,4,5-trisphosphate receptor (IPR) inhibitor (2-APB), and a phospholipase C (PLC) inhibitor (U-73122). We also conducted Ca imaging recording using primary cultured neurons. The results show that E2 rapidly induces an increase in cytosolic Ca, which often occurs in repetitive Ca oscillations. This response was not observed in the absence of extracellular Ca or with inhibitory ryanodine and was markedly reduced by nifedipine. E2-induced axonal growth was completely inhibited by ryanodine. In summary, the results suggest that Ca mobilization from extracellular space as well as from the endoplasmic reticulum is necessary for E2-induced ERK1/2 activation and axogenesis. Understanding the mechanisms of brain estrogenic actions might contribute to develop novel estrogen-based therapies for neurodegenerative diseases.

Mariana Bollo

Instituto de Investigación Médica M y M Ferreyra,
INIMEC-CONICET,
Instituto de Investigación Médica M y M Ferreyra, INIMEC-CONICET

Universidad Nacional de Córdoba

Molecular Modulation by Lentivirus-Delivered Specific shRNAs in Endoplasmic Reticulum Stressed Neurons

Mariana Bollo

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Universidad Nacional de Córdoba

Molecular Modulation by Lentivirus-Delivered Specific shRNAs in Endoplasmic Reticulum Stressed Neurons

Instituto de Investigación Médica M y M Ferreyra,
INIMEC-CONICET,
Instituto de Investigación Médica M y M Ferreyra, INIMEC-CONICET