ER-mitochondria interactions: Both strength and weakness within cancer cells

ER-mitochondria contact sites represent hubs for signaling that control mitochondrial biology related to several aspects of cellular survival, metabolism, cell death sensitivity and metastasis, which all contribute to tumorigenesis. Altered ER-mitochondria contacts can deregulate Ca²⁺ homeostasis, phospholipid metabolism, mitochondrial morphology and dynamics. MAM represent both a hot spot in cancer onset and progression and an Achilles' heel of cancer cells that can be exploited for therapeutic perspectives. Over the past years, an increasing number of cancer-related proteins, including oncogenes and tumor suppressors, have been localized in MAM and exert their pro- or antiapoptotic functions through the regulation of Ca²⁺ transfer and signaling between the two organelles. In this review, we highlight the central role of ER-mitochondria contact sites in tumorigenesis and focus on chemotherapeutic drugs or potential targets that act on MAM properties for new therapeutic approaches in cancer.     

Endoplasmic Reticulum Stress and Autophagy in Homocystinuria Patients with Remethylation Defects

Proper function of endoplasmic reticulum (ER) and mitochondria is crucial for cellular homeostasis, and dysfunction at either site as well as perturbation of mitochondria-associated ER membranes (MAMs) have been linked to neurodegenerative and metabolic diseases. Previously, we have observed an increase in ROS and apoptosis levels in patient-derived fibroblasts with remethylation disorders causing homocystinuria. Here we show increased mRNA and protein levels of Herp, Grp78, IP₃R1, pPERK, ATF4, CHOP, asparagine synthase and GADD45 in patient-derived fibroblasts suggesting ER stress and calcium perturbations in homocystinuria. In addition, overexpressed MAM-associated proteins (Grp75, σ-1R and Mfn2) were found in these cells that could result in mitochondrial calcium overload and oxidative stress increase. Our results also show an activation of autophagy process and a substantial degradation of altered mitochondria by mitophagy in patient-derived fibroblasts. Moreover, we have observed that autophagy was partially abolished by antioxidants suggesting that ROS participate in this process that may have a protective role. Our findings argue that alterations in Ca²⁺ homeostasis and autophagy may contribute to the development of this metabolic disorder and suggest a therapeutic potential in homocystinuria for agents that stabilize calcium homeostasis and/or restore the proper function of ER-mitochondria communications.     

The Regulation of the Autophagic Network and Its Implications for Human Disease

Autophagy has attracted a lot of attention in recent years. More and more proteins and signaling pathways have been discovered that somehow feed into the autophagy regulatory pathways. Regulation of autophagy is complex and condition-specific, and in several diseases, autophagic fluxes are changed. Here, we review the most well-established concepts in this field as well as the reported signaling pathways or components which steer the autophagy machinery. Furthermore, we will highlight how autophagic fluxes are changed in various diseases either as cause for or as response to deal with an altered cellular homeostasis and how modulation of autophagy might be used as potential therapy for such diseases.     

Tau localises within mitochondrial sub-compartments and its caspase cleavage affects ER-mitochondria interactions and cellular Ca2+ handling

Intracellular neurofibrillary tangles (NFT) composed by tau and extracellular amyloid beta (Aβ) plaques accumulate in Alzheimer's disease (AD) and contribute to neuronal dysfunction. Mitochondrial dysfunction and neurodegeneration are increasingly considered two faces of the same coin and an early pathological event in AD. Compelling evidence indicates that tau and mitochondria are closely linked and suggests that tau-dependent modulation of mitochondrial functions might be a trigger for the neurodegeneration process; however, whether this occurs either directly or indirectly is not clear. Furthermore, whether tau influences cellular Ca²⁺ handling and ER-mitochondria cross-talk is yet to be explored. Here, by focusing on wt tau, either full-length (2N4R) or the caspase 3-cleaved form truncated at the C-terminus (2N4RΔC₂₀), we examined the above-mentioned aspects. Using new genetically encoded split-GFP-based tools and organelle-targeted aequorin probes, we assessed: i) tau distribution within the mitochondrial sub-compartments; ii) the effect of tau on the short- (8–10?nm) and the long- (40–50?nm) range ER-mitochondria interactions; and iii) the effect of tau on cytosolic, ER and mitochondrial Ca²⁺ homeostasis. Our results indicate that a fraction of tau is found at the outer mitochondrial membrane (OMM) and within the inner mitochondrial space (IMS), suggesting a potential tau-dependent regulation of mitochondrial functions. The ER Ca²⁺ content and the short-range ER-mitochondria interactions were selectively affected by the expression of the caspase 3-cleaved 2N4RΔC₂₀ tau, indicating that Ca²⁺ mis-handling and defects in the ER-mitochondria communications might be an important pathological event in tau-related dysfunction and thereby contributing to neurodegeneration. Finally, our data provide new insights into the molecular mechanisms underlying tauopathies.     

Phosphorylation of Serine 779 in Fibroblast Growth Factor Receptor 1 and 2 by Protein Kinase C? Regulates Ras/Mitogen-activated Protein Kinase Signaling and Neuronal Differentiation

The FGF receptors (FGFRs) control a multitude of cellular processes both during development and in the adult through the initiation of signaling cascades that regulate proliferation, survival, and differentiation. Although FGFR tyrosine phosphorylation and the recruitment of Src homology 2 domain proteins have been widely described, we have previously shown that FGFR is also phosphorylated on Ser⁷⁷⁹ in response to ligand and binds the 14-3-3 family of phosphoserine/threonine-binding adaptor/scaffold proteins. However, whether this receptor phosphoserine mode of signaling is able to regulate specific signaling pathways and biological responses is unclear. Using PC12 pheochromocytoma cells and primary mouse bone marrow stromal cells as models for growth factor-regulated neuronal differentiation, we show that Ser⁷⁷⁹ in the cytoplasmic domains of FGFR1 and FGFR2 is required for the sustained activation of Ras and ERK but not for other FGFR phosphotyrosine pathways. The regulation of Ras and ERK signaling by Ser⁷⁷⁹ was critical not only for neuronal differentiation but also for cell survival under limiting growth factor concentrations. PKCϵ can phosphorylate Ser⁷⁷⁹ in vitro, whereas overexpression of PKCϵ results in constitutive Ser⁷⁷⁹ phosphorylation and enhanced PC12 cell differentiation. Furthermore, siRNA knockdown of PKCϵ reduces both growth factor-induced Ser⁷⁷⁹ phosphorylation and neuronal differentiation. Our findings show that in addition to FGFR tyrosine phosphorylation, the phosphorylation of a conserved serine residue, Ser⁷⁷⁹, can quantitatively control Ras/MAPK signaling to promote specific cellular responses.     

The endogenous caspase-8 inhibitor c-FLIPL regulates ER morphology and crosstalk with mitochondria

Components of the death receptor-mediated pathways like caspase-8 have been identified in complexes at intracellular membranes to spatially restrict the processing of local targets. In this study, we report that the long isoform of the cellular FLICE-inhibitory protein (c-FLIP_L), a well-known inhibitor of the extrinsic cell death initiator caspase-8, localizes at the endoplasmic reticulum (ER) and mitochondria-associated membranes (MAMs). ER morphology was disrupted and ER Ca²⁺-release as well as ER-mitochondria tethering was decreased in c-FLIP^−/− mouse embryonic fibroblasts (MEFs). Mechanistically, c-FLIP ablation resulted in enhanced basal caspase-8 activation and in caspase-mediated processing of the ER-shaping protein reticulon-4 (RTN4) that was corrected by re-introduction of c-FLIP_L and caspase inhibition, resulting in the recovery of a normal ER morphology and ER-mitochondria juxtaposition. Thus, the caspase-8 inhibitor c-FLIP_L emerges as a component of the MAMs signaling platforms, where caspases appear to regulate ER morphology and ER-mitochondria crosstalk by impinging on ER-shaping proteins like the RTN4.Cellular FLICE inhibitory proteins (c-FLIP) inhibit death receptor (DR)-mediated apoptosis, by preventing caspase-8 activation.¹ Among the three identified c-FLIP splicing forms,^{2, 3} c-FLIP_S,R were described as cytosolic, whereas c-FLIP_L was also observed in the nucleus. A pool of membrane-bound c-FLIP_L was also described⁴ suggesting that caspase-8/c-FLIP_L could re-distribute on stimulation, leading to a more subtle regulation of caspase-8 activity depending on substrates localization.⁵ Furthermore, caspase-8 itself and Fas-Associated Death Domain adaptor protein (FADD) were found or were shown to re-loca5lize in local complexes on ER^{6, 7, 8} and mitochondria,^{9, 10} mediating the exchange of signals between the two organelles.^{11, 12, 13} Several molecular platforms containing both membrane-bound proteins and cytosolic apoptosis modulators have been identified at the ER-mitochondria interface (the so-called mitochondria-associated membranes or MAMs),¹⁴ controlling ER-mitochondria anchorage as well as lipid metabolism, Ca²⁺ signaling and apoptosis.¹⁵ MAMs have been recently described as lipid raft-like domains that orient proteins to promote the ER-mitochondria juxtaposition;¹⁶ consequently, alterations in their composition may profoundly affect the physical and functional inter-organelle crosstalk. Furthermore, as mitochondrial and ER membranes are continuously and concertedly remodeled,¹⁷ it is not surprising that membrane-shaping proteins can also exert a function in regulating the ER-mitochondria coupling.^{12, 18} Different families of ER-shaping proteins control the organization of peripheral ER, which consists of sheet-like cisternae and tubules connected by three-way junctions.¹⁹ Among these, Reticulons (RTN) and Deleted in Polyposis locus 1 (DP1) proteins cause the ER membrane to curve and tubulate,^{20, 21} whereas the GTPases Atlastins (ATL) promote the branching of ER tubules;²² finally, ER sheet-enriched proteins such as the 63-kDa cytoskeleton-linking membrane protein (CLIMP63) control the width of ER cisternae, anchoring the organelle to microtubules and maintaining its spatial distribution.^{23, 24} Along with other components of the extrinsic apoptosis, here we described for the first time the enrichment of c-FLIP_L at ER and ER-mitochondria interface. Furthermore, we observed that ER structure and tethering to mitochondria are impaired in cells lacking c-FLIP. Given the importance of membrane-shaping proteins and MAM complexes in regulating organelles structure and ER-mitochondria juxtaposition, we focused on the mechanism underlying this phenotype and we found that c-FLIP_L deficiency induces the caspase-mediated processing of RTN4, thus affecting organelle shape and coupling to mitochondria. We therefore concluded that c-FLIP_L is a novel regulator of ER morphology and ER-mitochondria crosstalk.     

Prion protein—Semisynthetic prion protein (PrP) variants with posttranslational modifications

Deciphering the pathophysiologic events in prion diseases is challenging, and the role of posttranslational modifications (PTMs) such as glypidation and glycosylation remains elusive due to the lack of homogeneous protein preparations. So far, experimental studies have been limited in directly analyzing the earliest events of the conformational change of cellular prion protein (PrP^C) into scrapie prion protein (PrP^Sc) that further propagates PrP^C misfolding and aggregation at the cellular membrane, the initial site of prion infection, and PrP misfolding, by a lack of suitably modified PrP variants. PTMs of PrP, especially attachment of the glycosylphosphatidylinositol (GPI) anchor, have been shown to be crucially involved in the PrP^Sc formation. To this end, semisynthesis offers a unique possibility to understand PrP behavior invitro and invivo as it provides access to defined site‐selectively modified PrP variants. This approach relies on the production and chemoselective linkage of peptide segments, amenable to chemical modifications, with recombinantly produced protein segments. In this article, advances in understanding PrP conversion using semisynthesis as a tool to obtain homogeneous posttranslationally modified PrP will be discussed.     

New directions in ER stress-induced cell death

Endoplasmic reticulum (ER) stress has been implicated in the pathophysiology of many diseases including heart disease, cancer and neurodegenerative diseases such as Alzheimer’s and Huntington’s. Prolonged or excessive ER stress results in the initiation of signaling pathways resulting in cell death. Over the past decade much research investigating the onset and progression of ER stress-induced cell death has been carried out. Owing to this we now have a better understanding of the signaling pathways leading to ER stress-mediated cell death and have begun to appreciate the importance of ER localized stress sensors, IRE1α, ATF6 and PERK in this process. In this article we provide an overview of the current thinking and concepts concerning the various stages of ER stress-induced cell death, focusing on the role of ER localized proteins in sensing and triggering ER stress-induced death signals with particular emphasis on the contribution of calcium signaling and Bcl-2 family members to the execution phase of this process. We also highlight new and emerging directions in ER stress-induced cell death research particularly the role of microRNAs, ER-mitochondria cross talk and the prospect of mitochondria-independent death signals in ER stress-induced cell death.     

微RNAs与内质网应激信号通路的相互调控作用

内质网应激(endoplasmic reticulum stress,ERS)是为恢复稳态和减轻蛋白质负荷的一种细胞防御性反应.过度激活的ERS可诱导细胞分化、增殖、凋亡和自噬等.微RNAs(microRNAs,miRNAs)作为一种内源性的非编码RNA(non-coding RNA,ncRNA),可通过转录后作用调控...     

Caspases and neuronal development

Recent developments have shown that inappropriate activation of apoptotic pathways contributes to many neurodegenerative diseases. The basic mechanisms that underlie apoptosis in neurodegenerative diseases are uncertain, although they likely represent the subversion of normal developmental programs. Several types of neuronal cell death have been reported, including autophagic and caspase-independent cell death. In this review we consider evidence for the participation of apoptotic caspases in neuronal development, and examine the hypothesis that differentiating neurons undergo stage-specific alterations in apoptosis sensitivity that may be due to caspase regulation. In addition, we present data supporting this hypothesis.     

PACS-2 controls endoplasmic reticulum-mitochondria communication and Bid-mediated apoptosis

The endoplasmic reticulum (ER) and mitochondria form contacts that support communication between these two organelles, including synthesis and transfer of lipids, and the exchange of calcium, which regulates ER chaperones, mitochondrial ATP production, and apoptosis. Despite the fundamental roles for ER-mitochondria contacts, little is known about the molecules that regulate them. Here we report the identification of a multifunctional sorting protein, PACS-2, that integrates ER-mitochondria communication, ER homeostasis, and apoptosis. PACS-2 controls the apposition of mitochondria with the ER, as depletion of PACS-2 causes BAP31-dependent mitochondria fragmentation and uncoupling from the ER. PACS-2 also controls formation of ER lipid-synthesizing centers found on mitochondria-associated membranes and ER homeostasis. However, in response to apoptotic inducers, PACS-2 translocates Bid to mitochondria, which initiates a sequence of events including the formation of mitochondrial truncated Bid, the release of cytochrome c, and the activation of caspase-3, thereby causing cell death. Together, our results identify PACS-2 as a novel sorting protein that links the ER-mitochondria axis to ER homeostasis and the control of cell fate, and provide new insights into Bid action.     

Intracellular sodium sensing: SIK1 network,hormone action and high blood pressure

Sodium is the main determinant of body fluid distribution. Sodium accumulation causes water retention and, often, high blood pressure. At the cellular level, the concentration and active transport of sodium is handled by the enzyme Na⁺,K⁺-ATPase, whose appearance enabled evolving primitive cells to cope with osmotic stress and contributed to the complexity of mammalian organisms. Na⁺,K⁺-ATPase is a platform at the hub of many cellular signaling pathways related to sensing intracellular sodium and dealing with its detrimental excess. One of these pathways relies on an intracellular sodium-sensor network with the salt-inducible kinase 1 (SIK1) at its core. When intracellular sodium levels rise, and after the activation of calcium-related signals, this network activates the Na⁺,K⁺-ATPase and expel the excess of sodium from the cytosol. The SIK1 network also mediates sodium-independent signals that modulate the activity of the Na⁺,K⁺-ATPase, like dopamine and angiotensin, which are relevant per se in the development of high blood pressure. Animal models of high blood pressure, with identified mutations in components of multiple pathways, also have alterations in the SIK1 network. The introduction of some of these mutants into normal cells causes changes in SIK1 activity as well. Some cellular processes related to the metabolic syndrome, such as insulin effects on the kidney and other tissues, also appear to involve the SIK1. Therefore, it is likely that this protein, by modulating active sodium transport and numerous hormonal responses, represents a “crossroad” in the development and adaptation to high blood pressure and associated diseases.     

Oxidative stress as a component of chromium-induced cytotoxicity in rat calvarial osteoblasts

It has been documented that medical prosthetic alloys release metal ions into surrounding tissues and cause cytotoxicity, but the mechanisms remain undefined. In that regard the cellular oxidative stress may be a common pathway in cellular responses to metal ions. The objective of this study was to approach the hypothesis that oxidative stress mediates chromium-induced cytotoxicity in rat calvarial osteoblasts. Osteoblasts were exposed to different concentrations of Cr⁶⁺ or Cr³⁺ (5–20 μM) in the presence or absence of the antioxidant N-acetyl-cysteine (NAC; 1–5 mM). Cellular viability, differentiation, and intracellular ultrastructural alterations were evaluated by MTT assay, alkaline phosphatase (ALP) activity assay, and transmission electron microscopy. Cellular oxidative stress was evaluated by intracellular reactive oxygen species (ROS) production. ROS production was monitored by the oxidation-sensitive fluorescent probe 2′7′-dichlorofluorescin diacetate (DCFH-DA). A time- and concentration- dependent increased cytotoxicity, time-dependent increased intracellular ROS production were indicated on exposure to Cr⁶⁺. Pretreatment of osteoblasts with 1–5 mM NAC afforded dose-dependent cytoprotective effects against Cr⁶⁺-induced cytotoxicity in osteoblasts. NAC decreased the level of intracellular ROS induced by Cr⁶⁺, too. While Cr³⁺ and NAC did not have any significant effects on osteoblasts (5–20 μM). These results suggest that oxidative stress is involved in Cr⁶⁺-induced cytotoxicity in osteoblasts, and NAC can provide protection for osteoblasts against Cr⁶⁺-induced oxidative stress. Cr³⁺ (5–20 μM) have no significant cytotoxicity in osteoblasts based on the results of this study.     

Identification of aberrant pathways and network activities from high-throughput data

Many complex diseases such as cancer are associated with changes in biological pathways and molecular networks rather than being caused by single gene alterations. A major challenge in the diagnosis and treatment of such diseases is to identify characteristic aberrancies in the biological pathways and molecular network activities and elucidate their relationship to the disease. This review presents recent progress in using high-throughput biological assays to decipher aberrant pathways and network activities. In particular, this review provides specific examples in which high-throughput data have been applied to identify relationships between diseases and aberrant pathways and network activities. The achievements in this field have been remarkable, but many challenges have yet to be addressed.     

Defective endoplasmic reticulum-mitochondria contacts and bioenergetics in SEPN1-related myopathy

SEPN1-related myopathy (SEPN1-RM) is a muscle disorder due to mutations of the SEPN1 gene, which is characterized by muscle weakness and fatigue leading to scoliosis and life-threatening respiratory failure. Core lesions, focal areas of mitochondria depletion in skeletal muscle fibers, are the most common histopathological lesion. SEPN1-RM underlying mechanisms and the precise role of SEPN1 in muscle remained incompletely understood, hindering the development of biomarkers and therapies for this untreatable disease. To investigate the pathophysiological pathways in SEPN1-RM, we performed metabolic studies, calcium and ATP measurements, super-resolution and electron microscopy on in vivo and in vitro models of SEPN1 deficiency as well as muscle biopsies from SEPN1-RM patients. Mouse models of SEPN1 deficiency showed marked alterations in mitochondrial physiology and energy metabolism, suggesting that SEPN1 controls mitochondrial bioenergetics. Moreover, we found that SEPN1 was enriched at the mitochondria-associated membranes (MAM), and was needed for calcium transients between ER and mitochondria, as well as for the integrity of ER-mitochondria contacts. Consistently, loss of SEPN1 in patients was associated with alterations in body composition which correlated with the severity of muscle weakness, and with impaired ER-mitochondria contacts and low ATP levels. Our results indicate a role of SEPN1 as a novel MAM protein involved in mitochondrial bioenergetics. They also identify a systemic bioenergetic component in SEPN1-RM and establish mitochondria as a novel therapeutic target. This role of SEPN1 contributes to explain the fatigue and core lesions in skeletal muscle as well as the body composition abnormalities identified as part of the SEPN1-RM phenotype. Finally, these results point out to an unrecognized interplay between mitochondrial bioenergetics and ER homeostasis in skeletal muscle. They could therefore pave the way to the identification of biomarkers and therapeutic drugs for SEPN1-RM and for other disorders in which muscle ER-mitochondria cross-talk are impaired.Subject terms: Chaperones, Respiratory tract diseases     

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.     

Isolation,Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca²⁺ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.