Detection of Isolated Mitochondria-Associated ER Membranes Using the Sigma-1 Receptor.
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ABSTRACT:
SUBMITTER: Lewis A
PROVIDER: S-EPMC5547822 | biostudies-other | 2016
REPOSITORIES: biostudies-other
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Project description:In addition to the appearance of senile plaques and neurofibrillary tangles, Alzheimer's disease (AD) is characterized by aberrant lipid metabolism and early mitochondrial dysfunction. We recently showed that there was increased functionality of mitochondria-associated endoplasmic reticulum (ER) membranes (MAM), a subdomain of the ER involved in lipid and cholesterol homeostasis, in presenilin-deficient cells and in fibroblasts from familial and sporadic AD patients. Individuals carrying the ε4 allele of apolipoprotein E (ApoE4) are at increased risk for developing AD compared to those carrying ApoE3. While the reason for this increased risk is unknown, we hypothesized that it might be associated with elevated MAM function. Using an astrocyte-conditioned media (ACM) model, we now show that ER-mitochondrial communication and MAM function-as measured by the synthesis of phospholipids and of cholesteryl esters, respectively-are increased significantly in cells treated with ApoE4-containing ACM as compared to those treated with ApoE3-containing ACM. Notably, this effect was seen with lipoprotein-enriched preparations, but not with lipid-free ApoE protein. These data are consistent with a role of upregulated MAM function in the pathogenesis of AD and may help explain, in part, the contribution of ApoE4 as a risk factor in the disease.
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Project description:Alzheimer disease (AD) is associated with aberrant processing of the amyloid precursor protein (APP) by ?-secretase, via an unknown mechanism. We recently showed that presenilin-1 and -2, the catalytic components of ?-secretase, and ?-secretase activity itself, are highly enriched in a subcompartment of the endoplasmic reticulum (ER) that is physically and biochemically connected to mitochondria, called mitochondria-associated ER membranes (MAMs). We now show that MAM function and ER-mitochondrial communication-as measured by cholesteryl ester and phospholipid synthesis, respectively-are increased significantly in presenilin-mutant cells and in fibroblasts from patients with both the familial and sporadic forms of AD. We also show that MAM is an intracellular detergent-resistant lipid raft (LR)-like domain, consistent with the known presence of presenilins and ?-secretase activity in rafts. These findings may help explain not only the aberrant APP processing but also a number of other biochemical features of AD, including altered lipid metabolism and calcium homeostasis. We propose that upregulated MAM function at the ER-mitochondrial interface, and increased cross-talk between these two organelles, may play a hitherto unrecognized role in the pathogenesis of AD.
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Project description:In eukaryotic cells, mitochondria are closely tethered to the endoplasmic reticulum (ER) at sites called mitochondria-associated ER membranes (MAMs). Ca2+ ion and phospholipid transfer occurs at MAMs to support diverse cellular functions. Unlike those in yeast, the protein complexes involved in phospholipid transfer at MAMs in humans have not been identified. Here, we determine the crystal structure of the tetratricopeptide repeat domain of PTPIP51 (PTPIP51_TPR), a mitochondrial protein that interacts with the ER-anchored VAPB protein at MAMs. The structure of PTPIP51_TPR shows an archetypal TPR fold, and an electron density map corresponding to an unidentified lipid-like molecule probably derived from the protein expression host is found in the structure. We reveal functions of PTPIP51 in phospholipid binding/transfer, particularly of phosphatidic acid, in vitro. Depletion of PTPIP51 in cells reduces the mitochondrial cardiolipin level. Additionally, we confirm that the PTPIP51-VAPB interaction is mediated by the FFAT-like motif of PTPIP51 and the MSP domain of VAPB. Our findings suggest that PTPIP51 is a phospholipid transfer protein with a MAM-tethering function.
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Project description:BackgroundDiabetes is associated with an increased risk of cognitive decline and dementia. These diseases are linked with mitochondrial dysfunction, most likely as a consequence of excessive formation of mitochondria-associated membranes (MAMs). Sirtuin3 (SIRT3), a key mitochondrial NAD+-dependent deacetylase, is critical responsible for mitochondrial functional homeostasis and is highly associated with neuropathology. However, the role of SIRT3 in regulating MAM coupling remains unknown.MethodsStreptozotocin-injected diabetic mice and high glucose-treated SH-SY5Y cells were established as the animal and cellular models, respectively. SIRT3 expression was up-regulated in vivo using an adeno-associated virus in mouse hippocampus and in vitro using a recombinant lentivirus vector. Cognitive function was evaluated using behavioural tests. Hippocampus injury was assessed using Golgi and Nissl staining. Apoptosis was analysed using western blotting and TUNEL assay. Mitochondrial function was detected using flow cytometry and confocal fluorescence microscopy. The mechanisms were investigated using co-immunoprecipitation of VDAC1-GRP75-IP3R complex, fluorescence imaging of ER and mitochondrial co-localisation and transmission electron microscopy of structural analysis of MAMs.ResultsOur results demonstrated that SIRT3 expression was significantly reduced in high glucose-treated SH-SY5Y cells and hippocampal tissues from diabetic mice. Further, up-regulating SIRT3 alleviated hippocampus injuries and cognitive impairment in diabetic mice and mitigated mitochondrial Ca2+ overload-induced mitochondrial dysfunction and apoptosis. Mechanistically, MAM formation was enhanced under high glucose conditions, which was reversed by genetic up-regulation of SIRT3 via reduced interaction of the VDAC1-GRP75-IP3R complex in vitro and in vivo. Furthermore, we investigated the therapeutic effects of pharmacological activation of SIRT3 in diabetic mice via honokiol treatment, which exhibited similar effects to our genetic interventions.ConclusionsIn summary, our findings suggest that SIRT3 ameliorates cognitive impairment in diabetic mice by limiting aberrant MAM formation. Furthermore, targeting the activation of SIRT3 by honokiol provides a promising therapeutic candidate for diabetes-associated cognitive dysfunction. Overall, our study suggests a novel role of SIRT3 in regulating MAM coupling and indicates that SIRT3-targeted therapies are promising for diabetic dementia patients.
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Project description:During autophagy, phagophores grow into double-membrane vesicles called autophagosomes, but the underlying mechanism remains unclear. Here, we show a critical role of Atg2A in phagophore expansion. Atg2A translocates to the phagophore at the mitochondria-associated ER membrane (MAM) through a C-terminal 45-amino acid domain that we have termed the MAM localization domain (MLD). Proteomic analysis identifies the outer mitochondrial membrane protein TOM40 as a MLD-interacting partner. The Atg2A-TOM40 interaction is responsible for MAM localization of Atg2A and requires the TOM receptor protein TOM70. In addition, Atg2A interacts with Atg9A by a region within its N terminus. Inhibition of either Atg2A-TOM40 or Atg2A-Atg9A interactions impairs phagophore expansion and accumulates Atg9A-vesicles in the vicinity of autophagic structures. Collectively, we propose a model that the TOM70-TOM40 complex recruits Atg2A to the MAM for vesicular and/or non-vesicular lipid transport into the expanding phagophore to grow the size of autophagosomes for efficient autophagic flux.
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Project description:Multiple cellular organelles tightly orchestrate intracellular calcium (Ca2+) dynamics to regulate cellular activities and maintain homeostasis. The interplay between the endoplasmic reticulum (ER), a major store of intracellular Ca2+, and mitochondria, an important source of adenosine triphosphate (ATP), has been the subject of much research, as their dysfunction has been linked with metabolic diseases. Interestingly, throughout the cell's cytosolic domain, these two organelles share common microdomains called mitochondria-associated ER membranes (MAMs), where their membranes are in close apposition. The role of MAMs is critical for intracellular Ca2+ dynamics as they provide hubs for direct Ca2+ exchange between the organelles. A recent experimental study reported correlation between obesity and MAM formation in mouse liver cells, and obesity-related cellular changes that are closely associated with the regulation of Ca2+ dynamics. We constructed a mathematical model to study the effects of MAM Ca2+ dynamics on global Ca2+ activities. Through a series of model simulations, we investigated cellular mechanisms underlying the altered Ca2+ dynamics in the cells under obesity. We predict that, as the dosage of stimulus gradually increases, liver cells from obese mice will reach the state of saturated cytosolic Ca2+ concentration at a lower stimulus concentration, compared to cells from healthy mice.
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Project description:Mitochondria-associated ER membranes, or MAMs, define the sites of endoplasmic reticulum/mitochondria juxtaposition that control Ca(2+) flux between these organelles. We found that in a mouse model of the human lysosomal storage disease GM1-gangliosidosis, GM1-ganglioside accumulates in the glycosphingolipid-enriched microdomain (GEM) fractions of MAMs, where it interacts with the phosphorylated form of IP3 receptor-1, influencing the activity of this channel. Ca(2+) depleted from the ER is then taken up by the mitochondria, leading to Ca(2+) overload in this organelle. The latter induces mitochondrial membrane permeabilization (MMP), opening of the permeability transition pore, and activation of the mitochondrial apoptotic pathway. This study identifies the GEMs as the sites of Ca(2+) diffusion between the ER and the mitochondria. We propose a new mechanism of Ca(2+)-mediated apoptotic signaling whereby GM1 accumulation at the GEMs alters Ca(2+) dynamics and acts as a molecular effector of both ER stress-induced and mitochondria-mediated apoptosis of neuronal cells.
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Project description:The prevalence and healthcare burden of obesity and its related metabolic disorders such as type 2 diabetes (T2D) are increasing rapidly. A better understanding of the pathogenesis of these diseases helps to find the therapeutic strategies. Mitochondria and endoplasmic reticulum (ER) are two important organelles involved in the maintenance of intracellular Ca2+ and ROS homeostasis. Their functional defects are thought to participate in the pathogenesis of insulin resistance or T2D. The proper structure and function of the mitochondria-associated ER membranes (MAMs) is required for efficient communication between the ER and mitochondria and defects in MAMs have been shown to play a role in metabolic syndrome and other diseases. However, the detailed mechanism to link MAMs dysfunction and pathogenesis of insulin resistance or T2D remains unclear. In the present study, we demonstrated that the proteins involved in .MAMs structure are upregulated and the formation of MAMs is increased during adipogenic differentiation of 3T3-L1 preadipocytes. Disruption of MAMs by knocking down GRP75, which is responsible for connecting ER and mitochondria, led to the impairment of differentiation and ROS accumulation in 3T3-L1 preadipocytes. Most importantly, the differentiated 3T3-L1 adipocytes with GRP75 knockdown displayed inactivation of insulin signaling pathway upon insulin stimulation. Moreover, GRP75 knockdown impaired thermogenesis and glucose utilization in brown adipocytes, the adipocytes with abundant mitochondria that regulate whole-body energy homeostasis. Taken together, our findings suggest that MAMs formation is essential for promoting mitochondrial function and maintaining a proper redox status to enable the differentiation of preadipocytes and normal functioning such as insulin signaling and thermogenesis in mature adipocytes.
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