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Endoplasmic reticulum-mitochondria lockdown in Wolfram syndrome
No full textWolfram syndrome (WS) is an incurable autosomal recessive disorder originally described as a mitochondriopathy. In a recent work, Liiv and colleagues found that an impaired endoplasmic reticulum (ER)-to-mitochondria calcium shuttling underlies mitochondrial dysfunction in WS models
The endoplasmic reticulum-mitochondria coupling: role of Presenilin-2
Full text linkAlzheimer’s Disease (AD) is the most frequent form of dementia. A small percentage of cases is inherited (Familial AD, FAD) and is due to dominant mutations on three genes, coding for Amyloid Precursor Protein (APP), Presenilin-1 (PS1) and Presenilin-2 (PS2).
Mutations in these proteins cause alterations in the cleavage of APP by a PS1- or PS2- containing enzyme, named У-secretase, thus leading to an increase in the ratio between Aß42 and Aß40, the two main peptides finally derived from APP maturation. This in turn would increase the deposition of the “Amyloid Plaques”, one of the main histopathological feature of AD. To date, the generation of Aß42 peptides, its oligomers and finally amyloid plaques is the core of the most widely accepted pathogenic hypothesis for AD, the “Amyloid Cascade Hypothesis”.
PS1 and PS2 are ubiquitous “9 trans-membrane domains” homologous proteins localized mainly in the membranes of Endoplasmic Reticulum (ER), Golgi apparatus, endosomes and plasma membrane. Despite being the catalytic core of У-secretase, PSs display also some specialized, У-secretase independent activities. On this line, numerous studies reported a role for FAD-linked PS mutations in cellular calcium (Ca2+) alterations.
Ca2+ is a key second messenger in living cells and it regulates a multitude of cell functions; thus, alterations in its signaling cascade can be detrimental for cell fate. Ca2+ mishandling has been proposed as a causative mechanism for different neurodegenerative diseases and in particular for AD. Although supported by several groups for many years, the Ca2+ hypothesis for AD pathogenesis has never been undisputedly accepted, since some data were clearly in contrast, especially those considering PS2 mutations.
In our lab, it was previously shown that several FAD PS2 mutants, but not PS1, reduce ER and Golgi apparatus Ca2+ content, mainly by interfering with SERCA activity.
Over the last decade, evidence has accumulated on the existence of continuous flux of information between the ER and mitochondria, two organelles whose privileged interplay modulate key aspects of cell pathophysiology, ranging from lipid metabolism and Ca2+ homeostasis to cell death.
Several proteins have been suggested to be involved in keeping the ER and mitochondria at a given distance, allowing the correct organization, their mutual interactions and Ca2+ cross-talk. Among them, mitofusin 2 (Mfn2), which is located on both the outer mitochondrial membrane (OMM) and the ER surface, has been shown to take part in homotypic interactions that contribute to the tethering at the level of mitochondria-associated-membranes.(MAMs). Interestingly, also PS1 and PS2 are enriched in MAMs: we have recently demonstrated that PS2, but not PS1, is able to modulate ER-mitochondria tethering and their Ca2+ cross-talk, with PS2-FAD mutants more potent than their wt counterpart in this novel function.
We here investigate the molecular mechanism by which PS2 favours ER-mt tethering, taking into consideration the possibility that PS2 effect depends on the presence of Mfn2.
By crossed genetic complementation and ablation experiments, we found that, in order to modulate ER-mitochondria coupling, PS2 requires the expression of Mfn2 and viceversa. In contrast, their homologues PS1 and Mitofusin 1 (Mfn1) are completely dispensable for these functions. Functional and biochemical evidence indicates that PS2 (wt and FAD) needs to physically interact, via its big cytosolic loop, with Mfn2 at both sides of MAM domains, likely forming, or stabilizing, a triple complex made by itself, ER and mitochondrial Mfn2.
Our results clearly suggest that PS2 and Mfn2 cooperate and need one each other to promote ER-mitochondria apposition. On the contrary, their homologues PS1 and Mfn1 are completely dispensable in this function.
In order to explain the stronger effect of FAD-PS2 compared to wt, we performed protein subcellular fractionation from mouse brains, and we observed that in transgenic (tg) mice, carrying FAD-PS2-N141I mutation, PS2 is strongly enriched in MAMs, compared to controls. Moreover, in tg mice also Mfn2 levels are slightly increased in MAMs, thus possibly explaining the stronger tethering in presence of FAD mutations.
We proposed a model in which, being FAD-PS2 more enriched in MAMs compared to wt, it could here recruit more Mfn2 (by physically interacting with it both in cis and in trans) and form more PS2-Mfn2 complexes critical for determining the apposition between the two organelles.
Finally, the increase in ER-mitochondria coupling was observed not only in FAD-models over-expressing the mutated form of PS2, but also in human fibroblasts from patient carrying the PS2-N141I mutation, thus a condition in which the mutated protein exerts its function independently of any artefact due to its over-expression.
Further investigations will be focused to highlight the mechanism that promotes accumulation into MAMs of FAD-PS2 and whether these stronger FAD-PS2-linked effects on ER-mitochondria coupling are involved in the pathogenesis of A
Calcium, mitochondria and cell metabolism: A functional triangle in bioenergetics
No full textThe versatility of mitochondrial metabolism and its fine adjustments to specific physiological or pathological conditions regulate fundamental cell pathways, ranging from proliferation to apoptosis. In particular, Ca 2+ signalling has emerged as a key player exploited by mitochondria to tune their activity according with cell demand. The functional interaction between mitochondria and endoplasmic reticulum (ER) deeply impacts on the correct mitochondrial Ca 2+ signal, thus modulating cell bioenergetics and functionality. Indeed, Ca 2+ released by the ER is taken up by mitochondria where, both in the intermembrane space and in the matrix, it regulates the activity of transporters, enzymes and proteins involved in organelles' metabolism. In this review, we will briefly summarize Ca 2+ -dependent mechanisms involved in the regulation of mitochondrial activity. Moreover, we will discuss some recent reports, in which alterations in mitochondrial Ca 2+ signalling have been associated with specific pathological conditions, such as neurodegeneration and cancer
The endoplasmic reticulum-mitochondria coupling in heath anddisease: Molecules, functions and significance
No full textMitofusin 2: from functions to disease
Full text linkAbstractMitochondria are highly dynamic organelles whose functions are essential for cell viability. Within the cell, the mitochondrial network is continuously remodeled through the balance between fusion and fission events. Moreover, it dynamically contacts other organelles, particularly the endoplasmic reticulum, with which it enterprises an important functional relationship able to modulate several cellular pathways. Being mitochondria key bioenergetics organelles, they have to be transported to all the specific high-energy demanding sites within the cell and, when damaged, they have to be efficiently removed. Among other proteins, Mitofusin 2 represents a key player in all these mitochondrial activities (fusion, trafficking, turnover, contacts with other organelles), the balance of which results in the appropriate mitochondrial shape, function, and distribution within the cell. Here we review the structural and functional properties of Mitofusin 2, highlighting its crucial role in several cell pathways, as well as in the pathogenesis of neurodegenerative diseases, metabolic disorders, cardiomyopathies, and cancer.</jats:p
Analysis of the Effects of Hexokinase 2 Detachment From Mitochondria-Associated Membranes with the Highly Selective Peptide HK2pep
Full text linkThe crucial role of hexokinase 2 (HK2) in the metabolic rewiring of tumors is now well established, which makes it a suitable target for the design of novel therapies. However, hexokinase activity is central to glucose utilization in all tissues; thus, enzymatic inhibition of HK2 can induce severe adverse effects. In an effort to find a selective anti-neoplastic strategy, we exploited an alternative approach based on HK2 detachment from its location on the outer mitochondrial membrane. We designed a HK2-targeting peptide named HK2pep, corresponding to the N-terminal hydrophobic domain of HK2 and armed with a metalloprotease cleavage sequence and a polycation stretch shielded by a polyanion sequence. In the tumor microenvironment, metalloproteases unleash polycations to allow selective plasma membrane permeation in neoplastic cells. HK2pep delivery induces the detachment of HK2 from mitochondria-associated membranes (MAMs) and mitochondrial Ca2+ overload caused by the opening of inositol-3-phosphate receptors on the endoplasmic reticulum (ER) and Ca2+ entry through the plasma membrane leading to Ca2+-mediated calpain activation and mitochondrial depolarization. As a result, HK2pep rapidly elicits death of diverse tumor cell types and dramatically reduces in vivo tumor mass. HK2pep does not affect hexokinase enzymatic activity, avoiding any noxious effect on non-transformed cells. Here, we make available a detailed protocol for the use of HK2pep and to investigate its biological effects, providing a comprehensive panel of assays to quantitate both HK2 enzymatic activity and changes in mitochondrial functions, Ca2+ flux, and cell viability elicited by HK2pep treatment of tumor cells. Graphical abstract: Flowchart for the analysis of the effects of HK2 detachment from MAMs
Highlighting the Endoplasmic Reticulum-Mitochondria connection: focus on Mitofusin 2
No full textThe endoplasmic reticulum (ER) and the mitochondrial network are two highly interconnected cellular structures. By proteinaceous tethers, specialized membrane domains of the ER are tightly associated with the outer membrane of mitochondria, allowing the assembly of signaling platforms where different cell functions take place or are modulated, such as lipid biosynthesis, Ca2+ homeostasis, inflammation, autophagy and apoptosis. The ER-mitochondria coupling is highly dynamic and contacts between the two organelles can be modified in their number, extension and thickness by different stimuli. Importantly, several pathological conditions, such as cancer, neurodegenerative diseases and metabolic syndromes show alterations in this feature, underlining the key role of ER-mitochondria crosstalk in cell physiology. In this contribution, we will focus on one of the major modulator of ER-mitochondria apposition, Mitofusin 2, discussing the structure of the protein and its debated role on organelles tethering. Moreover, we will critically describe different techniques commonly used to investigate this crucial issue, highlighting their advantages, drawbacks and limits
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