Publications by authors named "Riccardo Filadi"

31 Publications

Loosening ER-Mitochondria Coupling by the Expression of the Presenilin 2 Loop Domain.

Cells 2021 Aug 3;10(8). Epub 2021 Aug 3.

Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy.

Presenilin 2 (PS2), one of the three proteins in which mutations are linked to familial Alzheimer's disease (FAD), exerts different functions within the cell independently of being part of the γ-secretase complex, thus unrelated to toxic amyloid peptide formation. In particular, its enrichment in endoplasmic reticulum (ER) membrane domains close to mitochondria (i.e., mitochondria-associated membranes, MAM) enables PS2 to modulate multiple processes taking place on these signaling hubs, such as Ca handling and lipid synthesis. Importantly, upregulated MAM function appears to be critical in AD pathogenesis. We previously showed that FAD-PS2 mutants reinforce ER-mitochondria tethering, by interfering with the activity of mitofusin 2, favoring their Ca crosstalk. Here, we deepened the molecular mechanism underlying PS2 activity on ER-mitochondria tethering, identifying its protein loop as an essential domain to mediate the reinforced ER-mitochondria connection in FAD-PS2 models. Moreover, we introduced a novel tool, the PS2 loop domain targeted to the outer mitochondrial membrane, Mit-PS2-LOOP, that is able to counteract the activity of FAD-PS2 on organelle tethering, which possibly helps in recovering the FAD-PS2-associated cellular alterations linked to an increased organelle coupling.
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http://dx.doi.org/10.3390/cells10081968DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8394530PMC
August 2021

Analysis of the Effects of Hexokinase 2 Detachment From Mitochondria-Associated Membranes with the Highly Selective Peptide HK2pep.

Bio Protoc 2021 Jul 20;11(14):e4087. Epub 2021 Jul 20.

Department of Biomedical Sciences (DSB), University of Padova, Padova, Italy.

The 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 Ca overload caused by the opening of inositol-3-phosphate receptors on the endoplasmic reticulum (ER) and Ca entry through the plasma membrane leading to Ca-mediated calpain activation and mitochondrial depolarization. As a result, HK2pep rapidly elicits death of diverse tumor cell types and dramatically reduces 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, Ca 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.
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http://dx.doi.org/10.21769/BioProtoc.4087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8329469PMC
July 2021

Defining the molecular mechanisms of the mitochondrial permeability transition through genetic manipulation of F-ATP synthase.

Nat Commun 2021 08 10;12(1):4835. Epub 2021 Aug 10.

Department of Biomedical Sciences, University of Padova, Padova, Italy.

F-ATP synthase is a leading candidate as the mitochondrial permeability transition pore (PTP) but the mechanism(s) leading to channel formation remain undefined. Here, to shed light on the structural requirements for PTP formation, we test cells ablated for g, OSCP and b subunits, and ρ cells lacking subunits a and A6L. Δg cells (that also lack subunit e) do not show PTP channel opening in intact cells or patch-clamped mitoplasts unless atractylate is added. Δb and ΔOSCP cells display currents insensitive to cyclosporin A but inhibited by bongkrekate, suggesting that the adenine nucleotide translocator (ANT) can contribute to channel formation in the absence of an assembled F-ATP synthase. Mitoplasts from ρ mitochondria display PTP currents indistinguishable from their wild-type counterparts. In this work, we show that peripheral stalk subunits are essential to turn the F-ATP synthase into the PTP and that the ANT provides mitochondria with a distinct permeability pathway.
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http://dx.doi.org/10.1038/s41467-021-25161-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8355262PMC
August 2021

Neuronal cell-based high-throughput screen for enhancers of mitochondrial function reveals luteolin as a modulator of mitochondria-endoplasmic reticulum coupling.

BMC Biol 2021 03 24;19(1):57. Epub 2021 Mar 24.

Center for Alzheimer Research, Division of Neurogeriatrics, Department of Neurobiology Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden.

Background: Mitochondrial dysfunction is a common feature of aging, neurodegeneration, and metabolic diseases. Hence, mitotherapeutics may be valuable disease modifiers for a large number of conditions. In this study, we have set up a large-scale screening platform for mitochondrial-based modulators with promising therapeutic potential.

Results: Using differentiated human neuroblastoma cells, we screened 1200 FDA-approved compounds and identified 61 molecules that significantly increased cellular ATP without any cytotoxic effect. Following dose response curve-dependent selection, we identified the flavonoid luteolin as a primary hit. Further validation in neuronal models indicated that luteolin increased mitochondrial respiration in primary neurons, despite not affecting mitochondrial mass, structure, or mitochondria-derived reactive oxygen species. However, we found that luteolin increased contacts between mitochondria and endoplasmic reticulum (ER), contributing to increased mitochondrial calcium (Ca) and Ca-dependent pyruvate dehydrogenase activity. This signaling pathway likely contributed to the observed effect of luteolin on enhanced mitochondrial complexes I and II activities. Importantly, we observed that increased mitochondrial functions were dependent on the activity of ER Ca-releasing channels inositol 1,4,5-trisphosphate receptors (IPRs) both in neurons and in isolated synaptosomes. Additionally, luteolin treatment improved mitochondrial and locomotory activities in primary neurons and Caenorhabditis elegans expressing an expanded polyglutamine tract of the huntingtin protein.

Conclusion: We provide a new screening platform for drug discovery validated in vitro and ex vivo. In addition, we describe a novel mechanism through which luteolin modulates mitochondrial activity in neuronal models with potential therapeutic validity for treatment of a variety of human diseases.
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http://dx.doi.org/10.1186/s12915-021-00979-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7989211PMC
March 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

Excitotoxicity Revisited: Mitochondria on the Verge of a Nervous Breakdown.

Trends Neurosci 2021 05 16;44(5):342-351. Epub 2021 Feb 16.

Department of Cell and Developmental Biology, University College London, London, UK. Electronic address:

Excitotoxicity is likely to occur in pathological scenarios in which mitochondrial function is already compromised, shaping neuronal responses to glutamate. In fact, mitochondria sustain cell bioenergetics, tune intracellular Ca dynamics, and regulate glutamate availability by using it as metabolic substrate. Here, we suggest the need to explore glutamate toxicity in the context of specific disease models in which it may occur, re-evaluating the impact of mitochondrial dysfunction on glutamate excitotoxicity. Our aim is to signpost new approaches, perhaps combining glutamate and pathways to rescue mitochondrial function, as therapeutic targets in neurological disorders.
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http://dx.doi.org/10.1016/j.tins.2021.01.001DOI Listing
May 2021

The yin and yang of mitochondrial Ca signaling in cell physiology and pathology.

Cell Calcium 2021 01 26;93:102321. Epub 2020 Nov 26.

Neuroscience Institute, National Research Council (CNR), 35131 Padua, Italy; Department of Biomedical Sciences, University of Padua, 35131, Padua, Italy. Electronic address:

Mitochondria are autonomous and dynamic cellular organelles orchestrating a diverse range of cellular activities. Numerous cell-signaling pathways target these organelles and Ca is one of the most significant. Mitochondria are able to rapidly and transiently take up Ca, thanks to the mitochondrial Ca uniporter complex, as well as to extrude it through the Na/Ca and H/Ca exchangers. The transient accumulation of Ca in the mitochondrial matrix impacts on mitochondrial functions and cell pathophysiology. Here we summarize the role of mitochondrial Ca signaling in both physiological (yang) and pathological (yin) processes and the methods that can be used to investigate mitochondrial Ca homeostasis. As an example of the pivotal role of mitochondria in pathology, we described the state of the art of mitochondrial Ca alterations in different pathological conditions, with a special focus on Alzheimer's disease.
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http://dx.doi.org/10.1016/j.ceca.2020.102321DOI Listing
January 2021

Presenilin-2 and Calcium Handling: Molecules, Organelles, Cells and Brain Networks.

Cells 2020 09 25;9(10). Epub 2020 Sep 25.

Department of Biomedical Sciences, University of Padua, Via U. Bassi 58/B, 35131 Padua, Italy.

Presenilin-2 (PS2) is one of the three proteins that are dominantly mutated in familial Alzheimer's disease (FAD). It forms the catalytic core of the γ-secretase complex-a function shared with its homolog presenilin-1 (PS1)-the enzyme ultimately responsible of amyloid-β (Aβ) formation. Besides its enzymatic activity, PS2 is a multifunctional protein, being specifically involved, independently of γ-secretase activity, in the modulation of several cellular processes, such as Ca signalling, mitochondrial function, inter-organelle communication, and autophagy. As for the former, evidence has accumulated that supports the involvement of PS2 at different levels, ranging from organelle Ca handling to Ca entry through plasma membrane channels. Thus FAD-linked PS2 mutations impact on multiple aspects of cell and tissue physiology, including bioenergetics and brain network excitability. In this contribution, we summarize the main findings on PS2, primarily as a modulator of Ca homeostasis, with particular emphasis on the role of its mutations in the pathogenesis of FAD. Identification of cell pathways and molecules that are specifically targeted by PS2 mutants, as well as of common targets shared with PS1 mutants, will be fundamental to disentangle the complexity of memory loss and brain degeneration that occurs in Alzheimer's disease (AD).
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http://dx.doi.org/10.3390/cells9102166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7601421PMC
September 2020

Sarcoplasmic Reticulum-Mitochondria Kissing in Cardiomyocytes: Ca, ATP, and Undisclosed Secrets.

Front Cell Dev Biol 2020 26;8:532. Epub 2020 Jun 26.

Department of Biomedical Sciences, University of Padua, Padua, Italy.

In cardiomyocytes, to carry out cell contraction, the distribution, morphology, and dynamic interaction of different cellular organelles are tightly regulated. For instance, the repetitive close apposition between junctional sarcoplasmic reticulum (jSR) and specialized sarcolemma invaginations, called transverse-tubules (TTs), is essential for an efficient excitation-contraction coupling (ECC). Upon an action potential, Ca microdomains, generated in synchrony at the interface between TTs and jSR, underlie the prompt increase in cytosolic Ca concentration, ultimately responsible for cell contraction during systole. This process requires a considerable amount of energy and the active participation of mitochondria, which encompass ∼30% of the cell volume and represent the major source of ATP in the heart. Importantly, in adult cardiomyocytes, mitochondria are distributed in a highly orderly fashion and strategically juxtaposed with SR. By taking advantage of the vicinity to Ca releasing sites, they take up Ca and modulate ATP synthesis according to the specific cardiac workload. Interestingly, with respect to SR, a biased, polarized positioning of mitochondrial Ca uptake/efflux machineries has been reported, hinting the importance of a strictly regulated mitochondrial Ca handling for heart activity. This notion, however, has been questioned by the observation that, in some mouse models, the deficiency of specific molecules, modulating mitochondrial Ca dynamics, triggers non-obvious cardiac phenotypes. This review will briefly summarize the physiological significance of SR-mitochondria apposition in cardiomyocytes, as well as the pathological consequences of an altered organelle communication, focusing on Ca signaling. We will discuss ongoing debates and propose future research directions.
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http://dx.doi.org/10.3389/fcell.2020.00532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7332691PMC
June 2020

Better to keep in touch: investigating inter-organelle cross-talk.

FEBS J 2021 02 30;288(3):740-755. Epub 2020 Jun 30.

Department of Biomedical Sciences, University of Padua, Padua, Italy.

The strategic importance for cellular organelles of being in contact with each other, exchanging messenger molecules, is nowadays well established. Different inter-organelle cross-talk pathways finely regulate multiple physiological cellular mechanisms, and their dysregulation has been found to underlie different pathological conditions. In the last years, a great effort has been made to study such organelle interactions, to understand their functional roles within the cell and the molecules involved in their formation and/or modulation. In this contribution, some examples of organelle cross-talk and their contributions in regulating physiological processes are presented. Moreover, the pro and cons of the available methods for a proper, reliable investigation of membrane contact sites are described.
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http://dx.doi.org/10.1111/febs.15451DOI Listing
February 2021

Hexokinase 2 displacement from mitochondria-associated membranes prompts Ca -dependent death of cancer cells.

EMBO Rep 2020 07 8;21(7):e49117. Epub 2020 May 8.

Department of Biomedical Sciences (DSB), University of Padova, Padova, Italy.

Cancer cells undergo changes in metabolic and survival pathways that increase their malignancy. Isoform 2 of the glycolytic enzyme hexokinase (HK2) enhances both glucose metabolism and resistance to death stimuli in many neoplastic cell types. Here, we observe that HK2 locates at mitochondria-endoplasmic reticulum (ER) contact sites called MAMs (mitochondria-associated membranes). HK2 displacement from MAMs with a selective peptide triggers mitochondrial Ca overload caused by Ca release from ER via inositol-3-phosphate receptors (IP3Rs) and by Ca entry through plasma membrane. This results in Ca -dependent calpain activation, mitochondrial depolarization and cell death. The HK2-targeting peptide causes massive death of chronic lymphocytic leukemia B cells freshly isolated from patients, and an actionable form of the peptide reduces growth of breast and colon cancer cells allografted in mice without noxious effects on healthy tissues. These results identify a signaling pathway primed by HK2 displacement from MAMs that can be activated as anti-neoplastic strategy.
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http://dx.doi.org/10.15252/embr.201949117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7332982PMC
July 2020

Defective Mitochondrial Pyruvate Flux Affects Cell Bioenergetics in Alzheimer's Disease-Related Models.

Cell Rep 2020 02;30(7):2332-2348.e10

Department of Biomedical Sciences, University of Padua, Via U. Bassi 58/B, 35121 Padua, Italy; Neuroscience Institute - Italian National Research Council (CNR), Padua 35121, Italy. Electronic address:

Mitochondria are key organelles for brain health. Mitochondrial alterations have been reported in several neurodegenerative disorders, including Alzheimer's disease (AD), and the comprehension of the underlying mechanisms appears crucial to understand their relationship with the pathology. Using multiple genetic, pharmacological, imaging, and biochemical approaches, we demonstrate that, in different familial AD cell models, mitochondrial ATP synthesis is affected. The defect depends on reduced mitochondrial pyruvate oxidation, due to both lower Ca-mediated stimulation of the Krebs cycle and dampened mitochondrial pyruvate uptake. Importantly, this latter event is linked to glycogen-synthase-kinase-3β (GSK-3β) hyper-activation, leading, in turn, to impaired recruitment of hexokinase 1 (HK1) to mitochondria, destabilization of mitochondrial-pyruvate-carrier (MPC) complexes, and decreased MPC2 protein levels. Remarkably, pharmacological GSK-3β inhibition in AD cells rescues MPC2 expression and improves mitochondrial ATP synthesis and respiration. The defective mitochondrial bioenergetics influences glutamate-induced neuronal excitotoxicity, thus representing a possible target for future therapeutic interventions.
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http://dx.doi.org/10.1016/j.celrep.2020.01.060DOI Listing
February 2020

ER-mitochondria tethering and Ca crosstalk: The IPR team takes the field.

Cell Calcium 2019 Dec 10;84:102101. Epub 2019 Oct 10.

Department of Biomedical Sciences, University of Padua, Via U. Bassi 58/B, 35121, Padua, Italy; Neuroscience Institute - Italian National Research Council (CNR), Padua, 35121, Italy. Electronic address:

Inter-organelle communication represents a booming topic in cell biology research, with endoplasmic reticulum (ER)-mitochondria coupling playing the lion's share. In a recent work, Bartok and colleagues found that inositol trisphosphates receptors (IPRs), in addition to their well-known involvement in ER-mitochondria Ca transfer, are endowed with structural properties at organelles' interface.
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http://dx.doi.org/10.1016/j.ceca.2019.102101DOI Listing
December 2019

Familial Alzheimer's disease presenilin-2 mutants affect Ca homeostasis and brain network excitability.

Aging Clin Exp Res 2021 Jun 12;33(6):1705-1708. Epub 2019 Oct 12.

Neuroscience Institute - Italian National Research Council (CNR), Padua, Italy.

Alzheimer's disease (AD) is the most frequent cause of dementia in the elderly. Few cases are familial (FAD), due to autosomal dominant mutations in presenilin-1 (PS1), presenilin-2 (PS2) or amyloid precursor protein (APP). The three proteins are involved in the generation of amyloid-beta (Aβ) peptides, providing genetic support to the hypothesis of Aβ pathogenicity. However, clinical trials focused on the Aβ pathway failed in their attempt to modify disease progression, suggesting the existence of additional pathogenic mechanisms. Ca dysregulation is a feature of cerebral aging, with an increased frequency and anticipated age of onset in several forms of neurodegeneration, including AD. Interestingly, FAD-linked PS1 and PS2 mutants alter multiple key cellular pathways, including Ca signaling. By generating novel tools for measuring Ca in living cells, and combining different approaches, we showed that FAD-linked PS2 mutants significantly alter cell Ca signaling and brain network activity, as summarized below.
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http://dx.doi.org/10.1007/s40520-019-01341-0DOI Listing
June 2021

Defective autophagy and Alzheimer's disease: is calcium the key?

Neural Regen Res 2019 Dec;14(12):2081-2082

Department of Biomedical Sciences, University of Padua and Neuroscience Institute - Italian National Research Council (CNR), Padua, Italy.

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http://dx.doi.org/10.4103/1673-5374.262584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6788238PMC
December 2019

Calcium, mitochondria and cell metabolism: A functional triangle in bioenergetics.

Biochim Biophys Acta Mol Cell Res 2019 07 26;1866(7):1068-1078. Epub 2018 Oct 26.

Department of Biomedical Sciences, University of Padova, Via U. Bassi 58/B, 35131 Padova, Italy.

The 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 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 signal, thus modulating cell bioenergetics and functionality. Indeed, Ca 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-dependent mechanisms involved in the regulation of mitochondrial activity. Moreover, we will discuss some recent reports, in which alterations in mitochondrial Ca signalling have been associated with specific pathological conditions, such as neurodegeneration and cancer.
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http://dx.doi.org/10.1016/j.bbamcr.2018.10.016DOI Listing
July 2019

PSEN2 (presenilin 2) mutants linked to familial Alzheimer disease impair autophagy by altering Ca homeostasis.

Autophagy 2019 12 27;15(12):2044-2062. Epub 2019 Mar 27.

Department of Biomedical Sciences, University of Padua, Padua, Italy.

PSEN2 (presenilin 2) is one of the 3 proteins that, when mutated, causes early onset familial Alzheimer disease (FAD) cases. In addition to its well-known role within the γ-secretase complex (the enzyme ultimately responsible for Aβ peptides formation), PSEN2 is endowed with some γ-secretase-independent functions in distinct cell signaling pathways, such as the modulation of intracellular Ca homeostasis. Here, by using different FAD-PSEN2 cell models, we demonstrate that mutated PSEN2 impairs autophagy by causing a block in the degradative flux at the level of the autophagosome-lysosome fusion step. The defect does not depend on an altered lysosomal functionality but rather on a decreased recruitment of the small GTPase RAB7 to autophagosomes, a key event for normal autophagy progression. Importantly, FAD-PSEN2 action on autophagy is unrelated to its γ-secretase activity but depends on its previously reported ability to partially deplete ER Ca content, thus reducing cytosolic Ca response upon IP3-linked cell stimulations. Our data sustain the pivotal role for Ca signaling in autophagy and reveal a novel mechanism by which FAD-linked presenilins alter the degradative process, reinforcing the view of a causative role for a dysfunctional quality control pathway in AD neurodegeneration. Aβ: amyloid β; AD: Alzheimer disease; ACTB: actin beta; AMPK: AMP-activated protein kinase; APP: amyloid-beta precursor protein; BafA: bafilomycin A; BAPTA-AM: 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester; CFP: cyan fluorescent protein; EGTA-AM: ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid acetoxymethyl ester; ER: endoplasmic reticulum; EGFP-HDQ74: enhanced GFP-huntingtin exon 1 containing 74 polyglutamine repeats; FAD: familial Alzheimer disease; FCS: fetal calf serum; FRET: fluorescence/Förster resonance energy transfer; GFP: green fluorescent protein; IP3: inositol trisphosphate; KD: knockdown; LAMP1: lysosomal associated membrane protein 1; MAP1LC3-II/LC3-II: lipidated microtubule-associated protein 1 light chain 3; MCU: mitochondrial calcium uniporter; MICU1: mitochondrial calcium uptake 1; MEFs: mouse embryonic fibroblasts; MFN2: mitofusin 2; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; SQSTM1/p62: sequestosome 1; PSEN1: presenilin 1; PSEN2: presenilin 2; RAB7: RAB7A: member RAS oncogene family; RFP: red fluorescent protein; ATP2A/SERCA: ATPase sarcoplasmic/endoplasmic reticulum Ca transporting; siRNA: small interference RNA; V-ATPase: vacuolar-type H-ATPase; WT: wild type.
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http://dx.doi.org/10.1080/15548627.2019.1596489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6844518PMC
December 2019

Mitofusin 2: from functions to disease.

Cell Death Dis 2018 02 28;9(3):330. Epub 2018 Feb 28.

Department of Biomedical Sciences, University of Padova, Padova, Italy.

Mitochondria 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.
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http://dx.doi.org/10.1038/s41419-017-0023-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5832425PMC
February 2018

TOM70 Sustains Cell Bioenergetics by Promoting IP3R3-Mediated ER to Mitochondria Ca Transfer.

Curr Biol 2018 02 27;28(3):369-382.e6. Epub 2018 Jan 27.

Center for Alzheimer Research, Division of Neurogeriatrics, Department of Neurobiology, Care Sciences and Society, Novum 5(th) Floor, Karolinska Institutet, 141 57 Huddinge, Sweden. Electronic address:

The mitochondrial translocase of the outer membrane (TOM) is a protein complex that is essential for the post-translational import of nuclear-encoded mitochondrial proteins. Among its subunits, TOM70 and TOM20 are only transiently associated with the core complex, suggesting their possible additional roles within the outer mitochondrial membrane (OMM). Here, by using different mammalian cell lines, we demonstrate that TOM70, but not TOM20, clusters in distinct OMM foci, frequently overlapping with sites in which the endoplasmic reticulum (ER) contacts mitochondria. Functionally, TOM70 depletion specifically impairs inositol trisphosphates (IP3)-linked ER to mitochondria Ca transfer. This phenomenon is dependent on the capacity of TOM70 to interact with IP3-receptors and favor their functional recruitment close to mitochondria. Importantly, the reduced constitutive Ca transfer to mitochondria, observed in TOM70-depleted cells, dampens mitochondrial respiration, affects cell bioenergetics, induces autophagy, and inhibits proliferation. Our data reveal a hitherto unexpected role for TOM70 in pro-survival ER-mitochondria communication, reinforcing the view that the ER-mitochondria signaling platform is a key regulator of cell fate.
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http://dx.doi.org/10.1016/j.cub.2017.12.047DOI Listing
February 2018

Highlighting the endoplasmic reticulum-mitochondria connection: Focus on Mitofusin 2.

Pharmacol Res 2018 02 5;128:42-51. Epub 2018 Jan 5.

Department of Biomedical Sciences, University of Padua, Via U. Bassi 58/B, 35121 Padua, Italy; Neuroscience Institute - Italian National Research Council (CNR), Padua, 35121, Italy. Electronic address:

The 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, Ca 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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http://dx.doi.org/10.1016/j.phrs.2018.01.003DOI Listing
February 2018

SPLICS: a split green fluorescent protein-based contact site sensor for narrow and wide heterotypic organelle juxtaposition.

Cell Death Differ 2018 06 11;25(6):1131-1145. Epub 2017 Dec 11.

Department of Biomedical Sciences, University of Padova, Padova, Italy.

Contact sites are discrete areas of organelle proximity that coordinate essential physiological processes across membranes, including Ca signaling, lipid biosynthesis, apoptosis, and autophagy. However, tools to easily image inter-organelle proximity over a range of distances in living cells and in vivo are lacking. Here we report a split-GFP-based contact site sensor (SPLICS) engineered to fluoresce when organelles are in proximity. Two SPLICS versions efficiently measured narrow (8-10 nm) and wide (40-50 nm) juxtapositions between endoplasmic reticulum and mitochondria, documenting the existence of at least two types of contact sites in human cells. Narrow and wide ER-mitochondria contact sites responded differently to starvation, ER stress, mitochondrial shape modifications, and changes in the levels of modulators of ER-mitochondria juxtaposition. SPLICS detected contact sites in soma and axons of D. rerio Rohon Beard (RB) sensory neurons in vivo, extending its use to analyses of organelle juxtaposition in the whole animal.
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http://dx.doi.org/10.1038/s41418-017-0033-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5988678PMC
June 2018

The Concerted Action of Mitochondrial Dynamics and Positioning: New Characters in Cancer Onset and Progression.

Front Oncol 2017 22;7:102. Epub 2017 May 22.

Department of Biomedical Sciences, University of Padova, Padova, Italy.

Mitochondria are dynamic organelles whose morphology and activity are extremely variable, depending on the metabolic state of the cell. In particular, their shape and movements within the cell are finely regulated by an increasing number of proteins, which take part in the process of mitochondrial fission/fusion and connect the organelles to the cytoskeleton. As to their activities, mitochondria are considered to be at the crossroad between cell life and death since, on the one hand, they are essential in ATP production and in multiple metabolic pathways but, on the other, they are involved in the intrinsic apoptotic cascade, triggered by different stress conditions. Importantly, the process of mitochondrial Ca uptake, as well as the morphology and the dynamics of these organelles, is known to deeply impact on both pro-survival and pro-death mitochondrial activities. Recently, increasing evidence has accrued on a central role of deregulated mitochondrial functionalities in the onset and progression of different pathologies, ranging from neurodegenerative diseases to cancer. In this contribution, we will present the latest findings connecting alterations in the machineries that control mitochondrial dynamics and localization to specific cancer hallmarks, highlighting the importance of mitochondria for the viability of cancer cells and discussing their role as promising targets for the development of novel anticancer therapies.
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http://dx.doi.org/10.3389/fonc.2017.00102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5439081PMC
May 2017

On the role of Mitofusin 2 in endoplasmic reticulum-mitochondria tethering.

Proc Natl Acad Sci U S A 2017 03 13;114(12):E2266-E2267. Epub 2017 Mar 13.

Department of Biomedical Sciences, University of Padua, 35121 Padua, Italy;

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http://dx.doi.org/10.1073/pnas.1616040114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5373360PMC
March 2017

The endoplasmic reticulum-mitochondria coupling in health and disease: Molecules, functions and significance.

Cell Calcium 2017 03 12;62:1-15. Epub 2017 Jan 12.

Department of Biomedical Sciences, University of Padova, Italy; Neuroscience Institute, National Research Council (CNR), Padova, Italy. Electronic address:

The close apposition between endoplasmic reticulum (ER) and mitochondria represents a key platform, capable to regulate different fundamental cellular pathways. Among these, Ca signaling and lipid homeostasis have been demonstrated over the last years to be deeply modulated by ER-mitochondria cross-talk. Given its importance in cell life/death decisions, increasing evidence suggests that alterations of the ER-mitochondria axis could be responsible for the onset and progression of several diseases, including neurodegeneration, cancer and obesity. However, the molecular identity of the proteins controlling this inter-organelle apposition is still debated. In this review, we summarize the main cellular pathways controlled by ER-mitochondria appositions, focusing on the principal molecules reported to be involved in this interplay and on those diseases for which alterations in organelles communication have been reported.
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http://dx.doi.org/10.1016/j.ceca.2017.01.003DOI Listing
March 2017

Beyond Intracellular Signaling: The Ins and Outs of Second Messengers Microdomains.

Adv Exp Med Biol 2017 ;981:279-322

Department of Biomedical Sciences, University of Padova, Padova, Italy.

A typical characteristic of eukaryotic cells compared to prokaryotes is represented by the spatial heterogeneity of the different structural and functional components: for example, most of the genetic material is surrounded by a highly specific membrane structure (the nuclear membrane), continuous with, yet largely different from, the endoplasmic reticulum (ER); oxidative phosphorylation is carried out by organelles enclosed by a double membrane, the mitochondria; in addition, distinct domains, enriched in specific proteins, are present in the plasma membrane (PM) of most cells. Less obvious, but now generally accepted, is the notion that even the concentration of small molecules such as second messengers (Ca and cAMP in particular) can be highly heterogeneous within cells. In the case of most organelles, the differences in the luminal levels of second messengers depend either on the existence on their membrane of proteins that allow the accumulation/release of the second messenger (e.g., in the case of Ca, pumps, exchangers or channels), or on the synthesis and degradation of the specific molecule within the lumen (the autonomous intramitochondrial cAMP system). It needs stressing that the existence of a surrounding membrane does not necessarily imply the existence of a gradient between the cytosol and the organelle lumen. For example, the nuclear membrane is highly permeable to both Ca and cAMP (nuclear pores are permeable to solutes up to 50 kDa) and differences in [Ca] or [cAMP] between cytoplasm and nucleoplasm are not seen in steady state and only very transiently during cell activation. A similar situation has been observed, as far as Ca is concerned, in peroxisomes.
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http://dx.doi.org/10.1007/978-3-319-55858-5_12DOI Listing
August 2018

Presenilin 2 Modulates Endoplasmic Reticulum-Mitochondria Coupling by Tuning the Antagonistic Effect of Mitofusin 2.

Cell Rep 2016 06 26;15(10):2226-2238. Epub 2016 May 26.

Department of Biomedical Sciences, University of Padua, via U. Bassi 58/B, Padua 35131, Italy. Electronic address:

Communication between organelles plays key roles in cell biology. In particular, physical and functional coupling of the endoplasmic reticulum (ER) and mitochondria is crucial for regulation of various physiological and pathophysiological processes. Here, we demonstrate that Presenilin 2 (PS2), mutations in which underlie familial Alzheimer's disease (FAD), promotes ER-mitochondria coupling only in the presence of mitofusin 2 (Mfn2). PS2 is not necessary for the antagonistic effect of Mfn2 on organelle coupling, although its abundance can tune it. The two proteins physically interact, whereas their homologues Mfn1 and PS1 are dispensable for this interplay. Moreover, PS2 mutants associated with FAD are more effective than the wild-type form in modulating ER-mitochondria tethering because their binding to Mfn2 in mitochondria-associated membranes is favored. We propose a revised model for ER-mitochondria interaction to account for these findings and discuss possible implications for FAD pathogenesis.
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http://dx.doi.org/10.1016/j.celrep.2016.05.013DOI Listing
June 2016

Mitofusin-2 knockdown increases ER-mitochondria contact and decreases amyloid β-peptide production.

J Cell Mol Med 2016 09 20;20(9):1686-95. Epub 2016 May 20.

Center for Alzheimer Research, Division of Neurogeriatrics, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Stockholm, Sweden.

Mitochondria are physically and biochemically in contact with other organelles including the endoplasmic reticulum (ER). Such contacts are formed between mitochondria-associated ER membranes (MAM), specialized subregions of ER, and the outer mitochondrial membrane (OMM). We have previously shown increased expression of MAM-associated proteins and enhanced ER to mitochondria Ca(2+) transfer from ER to mitochondria in Alzheimer's disease (AD) and amyloid β-peptide (Aβ)-related neuronal models. Here, we report that siRNA knockdown of mitofusin-2 (Mfn2), a protein that is involved in the tethering of ER and mitochondria, leads to increased contact between the two organelles. Cells depleted in Mfn2 showed increased Ca(2+) transfer from ER to mitchondria and longer stretches of ER forming contacts with OMM. Interestingly, increased contact resulted in decreased concentrations of intra- and extracellular Aβ40 and Aβ42 . Analysis of γ-secretase protein expression, maturation and activity revealed that the low Aβ concentrations were a result of impaired γ-secretase complex function. Amyloid-β precursor protein (APP), β-site APP-cleaving enzyme 1 and neprilysin expression as well as neprilysin activity were not affected by Mfn2 siRNA treatment. In summary, our data shows that modulation of ER-mitochondria contact affects γ-secretase activity and Aβ generation. Increased ER-mitochondria contact results in lower γ-secretase activity suggesting a new mechanism by which Aβ generation can be controlled.
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http://dx.doi.org/10.1111/jcmm.12863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988279PMC
September 2016

Mitofusin 2 ablation increases endoplasmic reticulum-mitochondria coupling.

Proc Natl Acad Sci U S A 2015 Apr 13;112(17):E2174-81. Epub 2015 Apr 13.

Department of Biomedical Sciences, University of Padua, Padua, 35121, Italy;

The organization and mutual interactions between endoplasmic reticulum (ER) and mitochondria modulate key aspects of cell pathophysiology. Several proteins have been suggested to be involved in keeping ER and mitochondria at a correct distance. Among them, in mammalian cells, mitofusin 2 (Mfn2), located on both the outer mitochondrial membrane and the ER surface, has been proposed to be a physical tether between the two organelles, forming homotypic interactions and heterocomplexes with its homolog Mfn1. Recently, this widely accepted model has been challenged using quantitative EM analysis. Using a multiplicity of morphological, biochemical, functional, and genetic approaches, we demonstrate that Mfn2 ablation increases the structural and functional ER-mitochondria coupling. In particular, we show that in different cell types Mfn2 ablation or silencing increases the close contacts between the two organelles and strengthens the efficacy of inositol trisphosphate (IP3)-induced Ca(2+) transfer from the ER to mitochondria, sensitizing cells to a mitochondrial Ca(2+) overload-dependent death. We also show that the previously reported discrepancy between electron and fluorescence microscopy data on ER-mitochondria proximity in Mfn2-ablated cells is only apparent. By using a different type of morphological analysis of fluorescent images that takes into account (and corrects for) the gross modifications in mitochondrial shape resulting from Mfn2 ablation, we demonstrate that an increased proximity between the organelles is also observed by confocal microscopy when Mfn2 levels are reduced. Based on these results, we propose a new model for ER-mitochondria juxtaposition in which Mfn2 works as a tethering antagonist preventing an excessive, potentially toxic, proximity between the two organelles.
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http://dx.doi.org/10.1073/pnas.1504880112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4418914PMC
April 2015

Generation and functions of second messengers microdomains.

Cell Calcium 2015 Oct 27;58(4):405-14. Epub 2015 Mar 27.

Department of Biomedical Sciences, University of Padova, Italy; CNR Institute of Neuroscience, Padova Section, Padova, Italy; Venetian Institute of Molecular Medicine (VIMM), Padova, Italy. Electronic address:

A compelling example of the mechanisms by which the cells can organize and decipher complex and different functional activities is the convergence of a multitude of stimuli into signalling cascades, involving only few intracellular second messengers. The possibility of restricting these signalling events in distinct microdomains allows a fine and selective tuning of very different tasks. In this review, we will discuss the mechanisms that control the formation and the spatial distribution of Ca(2+) and cAMP microdomains, providing some examples of their functional consequences.
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http://dx.doi.org/10.1016/j.ceca.2015.03.007DOI Listing
October 2015

Modulation of the endoplasmic reticulum-mitochondria interface in Alzheimer's disease and related models.

Proc Natl Acad Sci U S A 2013 May 25;110(19):7916-21. Epub 2013 Apr 25.

Department of Neurobiology, Care Sciences and Society, Karolinska Institutet-Alzheimer's Disease Research Center, Karolinska Institutet, 141 86 Stockholm, Sweden.

It is well-established that subcompartments of endoplasmic reticulum (ER) are in physical contact with the mitochondria. These lipid raft-like regions of ER are referred to as mitochondria-associated ER membranes (MAMs), and they play an important role in, for example, lipid synthesis, calcium homeostasis, and apoptotic signaling. Perturbation of MAM function has previously been suggested in Alzheimer's disease (AD) as shown in fibroblasts from AD patients and a neuroblastoma cell line containing familial presenilin-2 AD mutation. The effect of AD pathogenesis on the ER-mitochondria interplay in the brain has so far remained unknown. Here, we studied ER-mitochondria contacts in human AD brain and related AD mouse and neuronal cell models. We found uniform distribution of MAM in neurons. Phosphofurin acidic cluster sorting protein-2 and σ1 receptor, two MAM-associated proteins, were shown to be essential for neuronal survival, because siRNA knockdown resulted in degeneration. Up-regulated MAM-associated proteins were found in the AD brain and amyloid precursor protein (APP)Swe/Lon mouse model, in which up-regulation was observed before the appearance of plaques. By studying an ER-mitochondria bridging complex, inositol-1,4,5-triphosphate receptor-voltage-dependent anion channel, we revealed that nanomolar concentrations of amyloid β-peptide increased inositol-1,4,5-triphosphate receptor and voltage-dependent anion channel protein expression and elevated the number of ER-mitochondria contact points and mitochondrial calcium concentrations. Our data suggest an important role of ER-mitochondria contacts and cross-talk in AD pathology.
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http://dx.doi.org/10.1073/pnas.1300677110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3651455PMC
May 2013
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