Search

Carbon monoxide (CO) is an endogenously produced gasotransmitter, which is associated with cytoprotection and cellular homeostasis in several distinct cell types and tissues. CO mainly targets mitochondria because: (i) mitochondrial heme-proteins are the main potential candidates for CO to bind, (ii) many CO's biological actions are dependent on mitochondrial ROS signaling and (iii) heme is generated in the mitochondrial compartment. Mitochondria are the key cell energy factory, producing ATP through oxidative phosphorylation and regulating cell metabolism. These organelles are also implicated in many cell signaling pathways and the production of reactive oxygen species (ROS). Finally, mitochondria contain several factors activating programmed cell death pathways, which are released from the mitochondrial inter-membrane space upon mitochondrial membrane permeabilization. Therefore, disclosing CO mode of action at mitochondria opens avenues for deeper understanding CO's biological properties. Herein, it is discussed how CO affects the three main aspects of mitochondrial modulation of cell function: metabolism, redox response and cell death. [ABSTRACT FROM AUTHOR]

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) is a crucial component of the cellular machinery responsible for Ca2+ homeostasis. The selective inhibition of SERCA by thapsigargin (TG) leads to perturbations in Ca2+ signaling, which can trigger endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) pathway is activated in response to ER stress and induces an adaptive response to preserve cell survival or committee cells to programmed death, depending on stress duration and/or level. Early stages of ER stress stimulate mitochondrial metabolism to preserve survival but under chronic ER stress conditions, mitochondrial dysfunction is induced, which, in turn, can enhance inflammation through NLRP3 inflammasome activation. This study was aimed at investigating the role of SERCA inhibition on NLRP3 inflammasome activation in human monocytes, which was evaluated in primary monocytes isolated from healthy individuals and in the THP-1 human monocytic cell line. Findings obtained in both THP-1 and primary monocytes demonstrate that SERCA inhibition triggered by TG does not activate the NLRP3 inflammasome in these innate immune cells since IL-1β secretion was not affected. Results from THP-1 monocytes showing that SERCA inhibition increases mitochondrial Ca2+ content and fusion, in the absence of changes in ROS levels and membrane potential, support the view that human monocytes counteract ER stress that arises from inhibition of SERCA through modulation of mitochondrial morphology towards mitochondria fusion, thus preventing NLRP3 inflammasome activation. Overall, this work contributes to a better understanding of the molecular mechanisms that modulate the activity of the NLRP3 inflammasome leading to sterile inflammation, which are still poorly understood. [ABSTRACT FROM AUTHOR]

Prion disorders are progressive neurodegenerative diseases characterized by extensive neuronal loss and by the accumulation of the pathogenic form of prion protein, designated PrPSc. Recently, we have shown that PrP106–126 induces endoplasmic reticulum (ER) stress, leading to mitochondrial cytochrome c release, caspase 3 activation and apoptotic death. In order to further clarify the role of mitochondria in ER stress-mediated apoptotic pathway triggered by the PrP peptide, we investigated the effects of PrP106–126 on the Ntera2 human teratocarcinoma cell line that had been depleted of their mitochondrial DNA, termed NT2 ρ0 cells, characterized by the absence of functional mitochondria, as well as on the parental NT2 ρ+ cells. In this study, we show that PrP106–126 induces ER stress in both cell lines, given that ER Ca2+ content is low, glucose-regulated protein 78 levels are increased and caspase 4 is activated. Furthermore, in parental NT2 ρ+ cells, PrP106–126-activated caspase 9 and 3, induced poly (ADP-ribose) polymerase cleavage and increased the number of apoptotic cells. Dantrolene was shown to protect NT2 ρ+ from PrP106–126-induced cell death, demonstrating the involvement of Ca2+ release through ER ryanodine receptors. However, in PrP106–126-treated NT2 ρ0 cells, apoptosis was not able to proceed. These results demonstrate that functional mitochondria are required for cell death as a result of ER stress triggered by the PrP peptide, and further elucidate the molecular mechanisms involved in the neuronal loss that occurs in prion disorders. [ABSTRACT FROM AUTHOR]

In this study we analysed the effect of Bcl-2 on the cytotoxicity induced by the amyloid-β (Aβ_{25-35}) and prion (PrP_{106-126}) peptides by using GT1-7 puro and GT1-7 bcl-2 (overexpressing the anti-apoptotic protein Bcl-2) neural cells. Exposure to Aβ _{25-35} (1–5 μM) and PrP_{106-126} (25 μM) caused a decrease in cell viability, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. These data were correlated with Aβ_{25-35} and PrP_{106-126}-induced activation of caspase-9, which is linked to the mitochondrial death pathway, and the activation of the effector caspase-3, suggesting cell death by apoptosis. Furthermore, Bcl-2 overexpression protected from loss of cell viability and caspase-9 and -3 activation induced by Aβ_{25-35} and PrP_{106-126}, showing that Bcl-2 is neuroprotective against apoptotic cell death caused by amyloidogenic peptides. [ABSTRACT FROM AUTHOR]

Autophagy is an autodigestive process, promoting cytoprotection by the elimination of dysfunctional organelles, misfolded proteins and toxic aggregates. Carbon monoxide (CO) is an endogenous gasotransmitter that under low concentrations prevents cell death and inflammation. For the first time, the role of autophagy in CO-mediated cytoprotection against oxidative stress was evaluated in the model yeast Saccharomyces cerevisiae. The boron-based CO-releasing molecule, CORM-A1, was used to deliver CO. CORM-A1 partially prevented oxidative stress-induced cell death in yeast. Likewise, CORM-A1 activated autophagy under basal physiological conditions, which were assessed by autophagic flux and the expression of mCherry-Atg8 or GFP-Atg8. Inhibition of autophagy by knocking out key autophagic genes in yeast (ATG8 or ATG11) blocked CORM-A1 cytoprotective effect, indicating the critical role of autophagy in CO-induced cytoprotection. The CO-mediated cytoprotection via autophagy induction observed in yeast was validated in primary cultures of astrocytes, a well-characterized model for CO's cytoprotective functions. As in yeast, CORM-A1 prevented oxidative stress-induced cell death in an autophagy-dependent manner in astrocytes. Overall, our data support the cytoprotective action of CO against oxidative stress. CO promotes cytoprotection in yeast via autophagy, opening new possibilities for the study of molecular mechanisms of CO's biological functions using this powerful eukaryotic model. [ABSTRACT FROM AUTHOR]

Significance: Mitochondria-Associated Membranes (MAMs) are highly dynamic endoplasmic reticulum (ER)-mitochondria contact sites that, due to the transfer of lipids and Ca2+ between these organelles, modulate several physiologic processes, such as ER stress response, mitochondrial bioenergetics and fission/fusion events, autophagy, and inflammation. In addition, these contacts are implicated in the modulation of the cellular redox status since several MAMs-resident proteins are involved in the generation of reactive oxygen species (ROS), which can act as both signaling mediators and deleterious molecules, depending on their intracellular levels. Recent Advances: In the past few years, structural and functional alterations of MAMs have been associated with the pathophysiology of several neurodegenerative diseases that are closely associated with the impairment of several MAMs-associated events, including perturbation of the redox state on the accumulation of high ROS levels. Critical Issues: Inter-organelle contacts must be tightly regulated to preserve cellular functioning by maintaining Ca2+ and protein homeostasis, lipid metabolism, mitochondrial dynamics and energy production, as well as ROS signaling. Simultaneously, these contacts should avoid mitochondrial Ca2+ overload, which might lead to energetic deficits and deleterious ROS accumulation, culminating in oxidative stress-induced activation of apoptotic cell death pathways, which are common features of many neurodegenerative diseases. Future Directions: Given that Sig-1R is an ER resident chaperone that is highly enriched at the MAMs and that controls ER to mitochondria Ca2+ flux, as well as oxidative and ER stress responses, its potential as a therapeutic target for neurodegenerative diseases such as Amyotrophic Lateral Sclerosis, Alzheimer, Parkinson, and Huntington diseases should be further explored. Antioxid. Redox Signal. 37, 758–780. [ABSTRACT FROM AUTHOR]

Doxorubicin (DOX) is one of the most widely used anti-neoplastic agents. However, treatment with DOX is associated with cumulative cardiotoxicity inducing progressive cardiomyocyte death. Sirtuin 3 (Sirt3), a mitochondrial deacetylase, regulates the activity of proteins involved in apoptosis, autophagy and metabolism. Our hypothesis is that pharmacological modulation by berberine (BER) pre-conditioning of Sirt3 protein levels decreases DOX-induced cardiotoxicity. Our results showed that DOX induces cell death in all experimental groups. Increase in Sirt3 content by transfection-mediated overexpression decreased DOX cytotoxicity, mostly by maintaining mitochondrial network integrity and reducing oxidative stress. p53 was upregulated by DOX, and appeared to be a direct target of Sirt3, suggesting that Sirt3-mediated protection against cell death could be related to this protein. BER pre-treatment increased Sirt3 and Sirt1 protein levels in the presence of DOX and inhibited DOX-induced caspase 9 and 3-like activation. Moreover, BER modulated autophagy in DOX-treated H9c2 cardiomyoblasts. Interestingly, mitochondrial biogenesis markers were upregulated in in BER/DOX-treated cells. Sirt3 over-expression contributes to decrease DOX cytotoxicity on H9c2 cardiomyoblasts, while BER can be used as a modulator of Sirtuin function and cell quality control pathways to decrease DOX toxicity. [ABSTRACT FROM AUTHOR]

Abstract: Neurovascular dysfunction arising from endothelial cell damage is an early pathogenic event that contributes to the neurodegenerative process occurring in Alzheimer's disease (AD). Since the mechanisms underlying endothelial dysfunction are not fully elucidated, this study was aimed to explore the hypothesis that brain endothelial cell death is induced upon the sustained activation of the endoplasmic reticulum (ER) stress response by amyloid-beta (Aβ) peptide, which deposits in the cerebral vessels in many AD patients and transgenic mice. Incubation of rat brain endothelial cells (RBE4 cell line) with Aβ1–40 increased the levels of several markers of ER stress-induced unfolded protein response (UPR), in a time-dependent manner, and affected the Ca2+ homeostasis due to the release of Ca2+ from this intracellular store. Finally, Aβ1–40 was shown to activate both mitochondria-dependent and -independent apoptotic cell death pathways. Enhanced release of cytochrome c from mitochondria and activation of the downstream caspase-9 were observed in cells treated with Aβ1–40 concomitantly with caspase-12 activation. Furthermore, Aβ1–40 activated the apoptosis effectors' caspase-3 and promoted the translocation of apoptosis-inducing factor (AIF) to the nucleus demonstrating the involvement of caspase-dependent and -independent mechanisms during Aβ-induced endothelial cell death. In conclusion, our data demonstrate that ER stress plays a significant role in Aβ1–40-induced apoptotic cell death in brain endothelial cells suggesting that ER stress-targeted therapeutic strategies might be useful in AD to counteract vascular defects and ultimately neurodegeneration. [Copyright &y& Elsevier]

Abstract: Previously we reported that amyloid-β (Aβ) leads to endoplasmic reticulum (ER) stress in cultured cortical neurons and that ER-mitochondria Ca2+ transfer is involved in Aβ-induced apoptotic neuronal cell death. In cybrid cells which recreate the defect in mitochondrial cytochrome c oxidase (COX) activity observed in platelets from Alzheimer''s disease (AD) patients, we have shown that mitochondrial dysfunction affects the ER stress response triggered by Aβ. Here, we further investigated the impact of COX inhibition on Aβ-induced ER dysfunction using a neuronal model. Primary cultures of cortical neurons were challenged with toxic concentrations of Aβ upon chemical inhibition of COX with potassium cyanide (KCN). ER Ca2+ homeostasis was evaluated under these conditions, together with the levels of ER stress markers, namely the chaperone GRP78 and XBP-1, a mediator of the ER unfolded protein response (UPR). We demonstrated that COX inhibition potentiates the Aβ-induced depletion of ER Ca2+ content. KCN pre-treatment was also shown to enhance the rise of cytosolic Ca2+ levels triggered by Aβ and thapsigargin, a widely used ER stressor. This effect was reverted in the presence of dantrolene, an inhibitor of ER Ca2+ release through ryanodine receptors. Similarly, the increase in GRP78 and XBP-1 protein levels was shown to be higher in neurons treated with Aβ or thapsigargin in the presence of KCN in comparison with levels determined in neurons treated with the neurotoxins alone. Although the decrease in cell survival, the activation of caspase-9- and -3-mediated apoptotic cell death observed in Aβ- and thapsigargin-treated neurons were also potentiated by KCN, this effect is less pronounced than that observed in Ca2+ signalling and UPR. Furthermore, in neurons treated with Aβ, the potentiating effect of the COX inhibitor in cell survival and death was not prevented by dantrolene. These results show that inhibition of mitochondrial COX activity potentiates Aβ-induced ER dysfunction and, to a less extent, neuronal cell death. Furthermore, data supports that the effect of impaired COX on Aβ-induced cell death occurs independently of Ca2+ release through ER ryanodine receptors. Together, our data demonstrate that mitochondria dysfunction in AD enhances the neuronal susceptibility to toxic insults, namely to Aβ-induced ER stress, and strongly suggest that the close communication between ER and mitochondria can be a valuable future therapeutic target in AD. [Copyright &y& Elsevier]

Prion disorders are progressive neurodegenerative diseases characterized by extensive neuronal loss and by the accumulation of the pathogenic form of prion protein, designated PrPSc. Recently, we have shown that PrP106–126 induces endoplasmic reticulum (ER) stress, leading to mitochondrial cytochrome c release, caspase 3 activation and apoptotic death. In order to further clarify the role of mitochondria in ER stress-mediated apoptotic pathway triggered by the PrP peptide, we investigated the effects of PrP106–126 on the Ntera2 human teratocarcinoma cell line that had been depleted of their mitochondrial DNA, termed NT2 ρ0 cells, characterized by the absence of functional mitochondria, as well as on the parental NT2 ρ+ cells. In this study, we show that PrP106–126 induces ER stress in both cell lines, given that ER Ca2+ content is low, glucose-regulated protein 78 levels are increased and caspase 4 is activated. Furthermore, in parental NT2 ρ+ cells, PrP106–126-activated caspase 9 and 3, induced poly (ADP-ribose) polymerase cleavage and increased the number of apoptotic cells. Dantrolene was shown to protect NT2 ρ+ from PrP106–126-induced cell death, demonstrating the involvement of Ca2+ release through ER ryanodine receptors. However, in PrP106–126-treated NT2 ρ0 cells, apoptosis was not able to proceed. These results demonstrate that functional mitochondria are required for cell death as a result of ER stress triggered by the PrP peptide, and further elucidate the molecular mechanisms involved in the neuronal loss that occurs in prion disorders. [ABSTRACT FROM AUTHOR]