Publications by authors named "Tito Calì"

63 Publications

Calcium Signaling and Mitochondrial Function in Presenilin 2 Knock-Out Mice: Looking for Any Loss-of-Function Phenotype Related to Alzheimer's Disease.

Cells 2021 Jan 21;10(2). Epub 2021 Jan 21.

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

Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder in which learning, memory and cognitive functions decline progressively. Familial forms of AD (FAD) are caused by mutations in amyloid precursor protein (), presenilin 1 () and presenilin 2 () genes. Presenilin 1 (PS1) and its homologue, presenilin 2 (PS2), represent, alternatively, the catalytic core of the γ-secretase complex that, by cleaving APP, produces neurotoxic amyloid beta (Aβ) peptides responsible for one of the histopathological hallmarks in AD brains, the amyloid plaques. Recently, FAD mutations have been associated with a loss-of-function phenotype. To investigate whether this finding can also be extended to FAD mutations, we studied two processes known to be modulated by PS2 and altered by FAD mutations: Ca signaling and mitochondrial function. By exploiting neurons derived from a knock-out (PS2-/-) mouse model, we found that, upon IP-generating stimulation, cytosolic Ca handling is not altered, compared to wild-type cells, while mitochondrial Ca uptake is strongly compromised. Accordingly, PS2-/- neurons show a marked reduction in endoplasmic reticulum-mitochondria apposition and a slight alteration in mitochondrial respiration, whereas mitochondrial membrane potential, and organelle morphology and number appear unchanged. Thus, although some alterations in mitochondrial function appear to be shared between PS2-/- and FAD-PS2-expressing neurons, the mechanisms leading to these defects are quite distinct between the two models. Taken together, our data appear to be difficult to reconcile with the proposal that FAD-PS2 mutants are loss-of-function, whereas the concept that PS2 plays a key role in sustaining mitochondrial function is here confirmed.
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http://dx.doi.org/10.3390/cells10020204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7909802PMC
January 2021

Mitochondria Associated Membranes (MAMs): Architecture and physiopathological role.

Cell Calcium 2021 03 2;94:102343. Epub 2021 Jan 2.

Department of Biomedical Sciences, University of Padova, Padova, Italy. Electronic address:

In the last decades, the communication between the Endoplasmic reticulum (ER) and mitochondria has obtained great attention: mitochondria-associated membranes (MAMs), which represent the contact sites between the two organelles, have indeed emerged as central hub involved in different fundamental cell processes, such as calcium signalling, apoptosis, autophagy and lipid biosynthesis. Consistently, dysregulation of ER-mitochondria crosstalk has been associated with different pathological conditions, ranging from diabetes to cancer and neurodegenerative diseases. In this review, we will try to summarize the current knowledge on MAMs' structure and functions in health and their relevance for human diseases.
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http://dx.doi.org/10.1016/j.ceca.2020.102343DOI Listing
March 2021

An expanded palette of improved SPLICS reporters detects multiple organelle contacts in vitro and in vivo.

Nat Commun 2020 11 27;11(1):6069. Epub 2020 Nov 27.

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

Membrane contact sites between virtually any known organelle have been documented and, in the last decades, their study received momentum due to their importance for fundamental activities of the cell and for the subtle comprehension of many human diseases. The lack of tools to finely image inter-organelle proximity hindered our understanding on how these subcellular communication hubs mediate and regulate cell homeostasis. We develop an improved and expanded palette of split-GFP-based contact site sensors (SPLICS) for the detection of single and multiple organelle contact sites within a scalable distance range. We demonstrate their flexibility under physiological conditions and in living organisms.
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http://dx.doi.org/10.1038/s41467-020-19892-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7699637PMC
November 2020

ER-Mitochondria Contact Sites Reporters: Strengths and Weaknesses of the Available Approaches.

Int J Mol Sci 2020 Oct 31;21(21). Epub 2020 Oct 31.

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

Organelle intercommunication represents a wide area of interest. Over the last few decades, increasing evidence has highlighted the importance of organelle contact sites in many biological processes including Ca signaling, lipid biosynthesis, apoptosis, and autophagy but also their involvement in pathological conditions. ER-mitochondria tethering is one of the most investigated inter-organelle communications and it is differently modulated in response to several cellular conditions including, but not limited to, starvation, Endoplasmic Reticulum (ER) stress, and mitochondrial shape modifications. Despite many studies aiming to understand their functions and how they are perturbed under different conditions, approaches to assess organelle proximity are still limited. Indeed, better visualization and characterization of contact sites remain a fascinating challenge. The aim of this review is to summarize strengths and weaknesses of the available methods to detect and quantify contact sites, with a main focus on ER-mitochondria tethering.
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http://dx.doi.org/10.3390/ijms21218157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7663704PMC
October 2020

Sorcin is an early marker of neurodegeneration, Ca dysregulation and endoplasmic reticulum stress associated to neurodegenerative diseases.

Cell Death Dis 2020 10 15;11(10):861. Epub 2020 Oct 15.

Institute of Molecular Biology and Pathology, Italian National Research Council, IBPM-CNR, Rome, Italy.

Dysregulation of calcium signaling is emerging as a key feature in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD), and targeting this process may be therapeutically beneficial. Under this perspective, it is important to study proteins that regulate calcium homeostasis in the cell. Sorcin is one of the most expressed calcium-binding proteins in the human brain; its overexpression increases endoplasmic reticulum (ER) calcium concentration and decreases ER stress in the heart and in other cellular types. Sorcin has been hypothesized to be involved in neurodegenerative diseases, since it may counteract the increased cytosolic calcium levels associated with neurodegeneration. In the present work, we show that Sorcin expression levels are strongly increased in cellular, animal, and human models of AD, PD, and HD, vs. normal cells. Sorcin partially colocalizes with RyRs in neurons and microglia cells; functional experiments with microsomes containing high amounts of RyR2 and RyR3, respectively, show that Sorcin is able to regulate these ER calcium channels. The molecular basis of the interaction of Sorcin with RyR2 and RyR3 is demonstrated by SPR. Sorcin also interacts with other ER proteins as SERCA2 and Sigma-1 receptor in a calcium-dependent fashion. We also show that Sorcin regulates ER calcium transients: Sorcin increases the velocity of ER calcium uptake (increasing SERCA activity). The data presented here demonstrate that Sorcin may represent both a novel early marker of neurodegenerative diseases and a response to cellular stress dependent on neurodegeneration.
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http://dx.doi.org/10.1038/s41419-020-03063-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566454PMC
October 2020

Play Around with mtDNA.

DNA Cell Biol 2020 Aug 27;39(8):1369. Epub 2020 May 27.

Department of Biology, University of Padova, Padova, Italy.

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http://dx.doi.org/10.1089/dna.2020.29016.tjtDOI Listing
August 2020

PINK1/Parkin Mediated Mitophagy, Ca Signalling, and ER-Mitochondria Contacts in Parkinson's Disease.

Int J Mol Sci 2020 Mar 5;21(5). Epub 2020 Mar 5.

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

Endoplasmic reticulum (ER)-mitochondria contact sites are critical structures for cellular function. They are implicated in a plethora of cellular processes, including Ca signalling and mitophagy, the selective degradation of damaged mitochondria. Phosphatase and tensin homolog (PTEN)-induced kinase (PINK) and Parkin proteins, whose mutations are associated with familial forms of Parkinson's disease, are two of the best characterized mitophagy players. They accumulate at ER-mitochondria contact sites and modulate organelles crosstalk. Alterations in ER-mitochondria tethering are a common hallmark of many neurodegenerative diseases including Parkinson's disease. Here, we summarize the current knowledge on the involvement of PINK1 and Parkin at the ER-mitochondria contact sites and their role in the modulation of Ca signalling and mitophagy.
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http://dx.doi.org/10.3390/ijms21051772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084677PMC
March 2020

Special Issue on Mitochondrial DNA in Health and Disease.

DNA Cell Biol 2019 12 20;38(12):1411-1412. Epub 2019 Nov 20.

University of Padova.

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http://dx.doi.org/10.1089/dna.2019.29011.cfp3DOI Listing
December 2019

A split-GFP tool reveals differences in the sub-mitochondrial distribution of wt and mutant alpha-synuclein.

Cell Death Dis 2019 11 12;10(11):857. Epub 2019 Nov 12.

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

Parkinson's disease (PD), the second most common neurodegenerative disorder, is characterized by dopaminergic neuronal loss that initiates in the substantia nigra pars compacta and by the formation of intracellular inclusions mainly constituted by aberrant α-synuclein (α-syn) deposits known as Lewy bodies. Most cases of PD are sporadic, but about 10% are familial, among them those caused by mutations in SNCA gene have an autosomal dominant transmission. SNCA encodes α-syn, a small 140-amino acids protein that, under physiological conditions, is mainly localized at the presynaptic terminals. It is prevalently cytosolic, but its presence has been reported in the nucleus, in the mitochondria and, more recently, in the mitochondria-associated ER membranes (MAMs). Whether different cellular localizations may reflect specific α-syn activities is presently unclear and its action at mitochondrial level is still a matter of debate. Mounting evidence supports a role for α-syn in several mitochondria-derived activities, among which maintenance of mitochondrial morphology and modulation of complex I and ATP synthase activity. α-syn has been proposed to localize at the outer membrane (OMM), in the intermembrane space (IMS), at the inner membrane (IMM) and in the mitochondrial matrix, but a clear and comparative analysis of the sub-mitochondrial localization of WT and mutant α-syn is missing. Furthermore, the reasons for this spread sub-mitochondrial localization under physiological and pathological circumstances remain elusive. In this context, we decided to selectively monitor the sub-mitochondrial distribution of the WT and PD-related α-syn mutants A53T and A30P by taking advantage from a bimolecular fluorescence complementation (BiFC) approach. We also investigated whether cell stress could trigger α-syn translocation within the different mitochondrial sub-compartments and whether PD-related mutations could impinge on it. Interestingly, the artificial targeting of α-syn WT (but not of the mutants) to the mitochondrial matrix impacts on ATP production, suggesting a potential role within this compartment.
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http://dx.doi.org/10.1038/s41419-019-2092-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851186PMC
November 2019

ER-Mitochondria Calcium Transfer, Organelle Contacts and Neurodegenerative Diseases.

Adv Exp Med Biol 2020 ;1131:719-746

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

It is generally accepted that interorganellar contacts are central to the control of cellular physiology. Virtually, any intracellular organelle can come into proximity with each other and, by establishing physical protein-mediated contacts within a selected fraction of the membrane surface, novel specific functions are acquired. Endoplasmic reticulum (ER) contacts with mitochondria are among the best studied and have a major role in Ca and lipid transfer, signaling, and membrane dynamics.Their functional (and structural) diversity, their dynamic nature as well as the growing number of new players involved in the tethering concurred to make their monitoring difficult especially in living cells. This review focuses on the most established examples of tethers/modulators of the ER-mitochondria interface and on the roles of these contacts in health and disease by specifically dissecting how Ca transfer occurs and how mishandling eventually leads to disease. Additional functions of the ER-mitochondria interface and an overview of the currently available methods to measure/quantify the ER-mitochondria interface will also be discussed.
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http://dx.doi.org/10.1007/978-3-030-12457-1_29DOI Listing
October 2019

Special Issue on Mitochondrial DNA in Health and Disease.

DNA Cell Biol 2019 11 16;38(11):1167-1168. Epub 2019 Oct 16.

University of Padova

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http://dx.doi.org/10.1089/dna.2019.29011.cfpDOI Listing
November 2019

splitGFP Technology Reveals Dose-Dependent ER-Mitochondria Interface Modulation by α-Synuclein A53T and A30P Mutants.

Cells 2019 09 12;8(9). Epub 2019 Sep 12.

Department of Biology, University of Padova, Padova 35131, Italy.

Familial Parkinson's disease (PD) is associated with duplication or mutations of α-synuclein gene, whose product is a presynaptic cytosolic protein also found in mitochondria and in mitochondrial-associated ER membranes. We have originally shown the role of α-syn as a modulator of the ER-mitochondria interface and mitochondrial Ca transients, suggesting that, at mild levels of expression, α-syn sustains cell metabolism. Here, we investigated the possibility that α-syn action on ER-mitochondria tethering could be compromised by the presence of PD-related mutations. The clarification of this aspect could contribute to elucidate key mechanisms underlying PD. The findings reported so far are not consistent, possibly because of the different methods used to evaluate ER-mitochondria connectivity. Here, the effects of the PD-related α-syn mutations A53T and A30P on ER-mitochondria relationship were investigated in respect to Ca handling and mitochondrial function using a newly generated SPLICS sensor and aequorin-based Cameasurements. We provided evidence that A53T and A30P amino acid substitution does not affect the ability of α-syn to enhance ER/mitochondria tethering and mitochondrial Ca transients, but that this action was lost as soon as a high amount of TAT-delivered A53T and A30P α-syn mutants caused the redistribution of α-syn from cytoplasm to foci. Our results suggest a loss of function mechanism and highlight a possible connection between α-syn and ER-mitochondria Ca cross-talk impairment to the pathogenesis of PD.
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http://dx.doi.org/10.3390/cells8091072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6769576PMC
September 2019

Special Issue on Mitochondrial DNA in Health and Disease.

DNA Cell Biol 2019 10 10;38(10):1023-1024. Epub 2019 Sep 10.

University of Padova.

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http://dx.doi.org/10.1089/dna.2019.29010.cfpDOI Listing
October 2019

Impaired Mitochondrial ATP Production Downregulates Wnt Signaling via ER Stress Induction.

Cell Rep 2019 08;28(8):1949-1960.e6

Department of Biology, University of Padova, Padova, Italy. Electronic address:

Wnt signaling affects fundamental development pathways and, if aberrantly activated, promotes the development of cancers. Wnt signaling is modulated by different factors, but whether the mitochondrial energetic state affects Wnt signaling is unknown. Here, we show that sublethal concentrations of different compounds that decrease mitochondrial ATP production specifically downregulate Wnt/β-catenin signaling in vitro in colon cancer cells and in vivo in zebrafish reporter lines. Accordingly, fibroblasts from a GRACILE syndrome patient and a generated zebrafish model lead to reduced Wnt signaling. We identify a mitochondria-Wnt signaling axis whereby a decrease in mitochondrial ATP reduces calcium uptake into the endoplasmic reticulum (ER), leading to endoplasmic reticulum stress and to impaired Wnt signaling. In turn, the recovery of the ATP level or the inhibition of endoplasmic reticulum stress restores Wnt activity. These findings reveal a mechanism that links mitochondrial energetic metabolism to the control of the Wnt pathway that may be beneficial against several pathologies.
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http://dx.doi.org/10.1016/j.celrep.2019.07.050DOI Listing
August 2019

A chloroplast-localized mitochondrial calcium uniporter transduces osmotic stress in Arabidopsis.

Nat Plants 2019 06 10;5(6):581-588. Epub 2019 Jun 10.

Department of Biology, University of Padova, Padova, Italy.

Chloroplasts are integral to sensing biotic and abiotic stress in plants, but their role in transducing Ca-mediated stress signals remains poorly understood. Here we identify cMCU, a member of the mitochondrial calcium uniporter (MCU) family, as an ion channel mediating Ca flux into chloroplasts in vivo. Using a toolkit of aequorin reporters targeted to chloroplast stroma and the cytosol in cMCU wild-type and knockout lines, we provide evidence that stress-stimulus-specific Ca dynamics in the chloroplast stroma correlate with expression of the channel. Fast downstream signalling events triggered by osmotic stress, involving activation of the mitogen-activated protein kinases (MAPK) MAPK3 and MAPK6, and the transcription factors MYB60 and ethylene-response factor 6 (ERF6), are influenced by cMCU activity. Relative to wild-type plants, cMCU knockouts display increased resistance to long-term water deficit and improved recovery on rewatering. Modulation of stromal Ca in specific processing of stress signals identifies cMCU as a component of plant environmental sensing.
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http://dx.doi.org/10.1038/s41477-019-0434-8DOI Listing
June 2019

EMBO Workshop: Membrane Contact Sites in Health and Disease.

Contact (Thousand Oaks) 2019 May 21;2. Epub 2019 Feb 21.

Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Universitè Laval, Quebec, Canada.

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http://dx.doi.org/10.1177/2515256419825931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6544536PMC
May 2019

Calcium, Dopamine and Neuronal Calcium Sensor 1: Their Contribution to Parkinson's Disease.

Front Mol Neurosci 2019 22;12:55. Epub 2019 Mar 22.

Department of Biology, University of Padova, Padua, Italy.

Parkinson's disease (PD) is a debilitating neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra pars compacta. The causes of PD in humans are still unknown, although metabolic characteristics of the neurons affected by the disease have been implicated in their selective susceptibility. Mitochondrial dysfunction and proteostatic stress are recognized to be important in the pathogenesis of both familial and sporadic PD, and they both culminate in bioenergetic deficits. Exposure to calcium overload has recently emerged as a key determinant, and pharmacological treatment that inhibits Ca entry diminishes neuronal damage in chemical models of PD. In this review, we first introduce general concepts on neuronal Ca signaling and then summarize the current knowledge on fundamental properties of substantia nigra pars compacta dopaminergic neurons, on the role of the interplay between Ca and dopamine signaling in neuronal activity and susceptibility to cell death. We also discuss the possible involvement of a "neglected" player, the Neuronal Calcium Sensor-1 (NCS-1), which has been shown to participate to dopaminergic signaling by regulating dopamine dependent receptor desensitization in normal brain but, data supporting a direct role in PD pathogenesis are still missing. However, it is intriguing to speculate that the Ca-dependent modulation of NCS-1 activity could eventually counteract dopaminergic neurons degeneration.
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http://dx.doi.org/10.3389/fnmol.2019.00055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6440390PMC
March 2019

Publisher Correction: Parkin-dependent regulation of the MCU complex component MICU1.

Sci Rep 2019 Mar 12;9(1):4665. Epub 2019 Mar 12.

Department of Biomedical Sciences, University of Padova, via U. Basi 58/b, 35131, Padova, Italy.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-018-37929-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411860PMC
March 2019

The VAPB-PTPIP51 endoplasmic reticulum-mitochondria tethering proteins are present in neuronal synapses and regulate synaptic activity.

Acta Neuropathol Commun 2019 03 6;7(1):35. Epub 2019 Mar 6.

Department of Basic and Clinical Neuroscience, Maurice Wohl Clinical Neuroscience Institute, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, SE5 9RX, UK.

Signaling between the endoplasmic reticulum (ER) and mitochondria regulates a number of key neuronal functions. This signaling involves close physical contacts between the two organelles that are mediated by "tethering proteins" that function to recruit regions of ER to the mitochondrial surface. The ER protein, vesicle-associated membrane protein-associated protein B (VAPB) and the mitochondrial membrane protein, protein tyrosine phosphatase interacting protein-51 (PTPIP51), interact to form one such tether. Recently, damage to ER-mitochondria signaling involving disruption of the VAPB-PTPIP51 tethers has been linked to the pathogenic process in Parkinson's disease, fronto-temporal dementia (FTD) and related amyotrophic lateral sclerosis (ALS). Loss of neuronal synaptic function is a key feature of Parkinson's disease and FTD/ALS but the roles that ER-mitochondria signaling and the VAPB-PTPIP51 tethers play in synaptic function are not known. Here, we demonstrate that the VAPB-PTPIP51 tethers regulate synaptic activity. VAPB and PTPIP51 localise and form contacts at synapses, and stimulating neuronal activity increases ER-mitochondria contacts and the VAPB-PTPIP51 interaction. Moreover, siRNA loss of VAPB or PTPIP51 perturbs synaptic function and dendritic spine morphology. Our results reveal a new role for the VAPB-PTPIP51 tethers in neurons and suggest that damage to ER-mitochondria signaling contributes to synaptic dysfunction in Parkinson's disease and FTD/ALS.
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http://dx.doi.org/10.1186/s40478-019-0688-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6402140PMC
March 2019

Measuring Ca Levels in Subcellular Compartments with Genetically Encoded GFP-Based Indicators.

Methods Mol Biol 2019 ;1925:31-42

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

Ca homeostasis is crucial for the entire life of eukaryotic cells from the beginning to the end. Mishandling in Ca homeostasis is indeed linked with a large number of pathological conditions. Thus, the possibility to specifically monitor cellular calcium fluxes in different subcellular compartments represents a key tool to deeply understand the mechanisms involved in cellular dysfunctions. To cope with this need, several Ca indicators have been developed allowing to accurately measure both basal Ca concentration and agonist-induced Ca signals in a wide spectrum of organelles. Among these, the genetically encoded GFP-based indicators are routinely used to measure Ca transients thanks to their ability to change their spectral properties in response to Ca binding. In this chapter, we will describe a protocol that utilizes the GCaMP6f probe targeted to mitochondria (4mtGCaMP) to measure mitochondrial calcium levels in resting conditions in HeLa cells. This method allows to easily and quickly register alterations of mitochondrial Ca homeostasis in different cell populations and experimental settings, representing a precious tool to unravel the pathological pathways leading to pathogenic conditions.
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http://dx.doi.org/10.1007/978-1-4939-9018-4_3DOI Listing
June 2019

Phosphorylation of nuclear Tau is modulated by distinct cellular pathways.

Sci Rep 2018 12 7;8(1):17702. Epub 2018 Dec 7.

Laboratory for Biomedical Neurosciences, Neurocenter of Southern Switzerland, Ente Cantonale Ospedaliero, Torricella-Taverne, Switzerland.

Post-translational protein modification controls the function of Tau as a scaffold protein linking a variety of molecular partners. This is most studied in the context of microtubules, where Tau regulates their stability as well as the distribution of cellular components to defined compartments. However, Tau is also located in the cell nucleus; and is found to protect DNA. Quantitative assessment of Tau modification in the nucleus when compared to the cytosol may elucidate how subcellular distribution and function of Tau is regulated. We undertook an unbiased approach by combing bimolecular fluorescent complementation and mass spectrometry in order to show that Tau phosphorylation at specific residues is increased in the nucleus of proliferating pluripotent neuronal C17.2 and neuroblastoma SY5Y cells. These findings were validated with the use of nuclear targeted Tau and subcellular fractionation, in particular for the phosphorylation at T, T and S. We also report that the DNA damaging drug Etoposide increases the translocation of Tau to the nucleus whilst reducing its phosphorylation. We propose that overt phosphorylation of Tau, a hallmark of neurodegenerative disorders defined as tauopathies, may negatively regulate the function of nuclear Tau in protecting against DNA damage.
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http://dx.doi.org/10.1038/s41598-018-36374-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286375PMC
December 2018

Parkin-dependent regulation of the MCU complex component MICU1.

Sci Rep 2018 09 21;8(1):14199. Epub 2018 Sep 21.

Department of Biomedical Sciences, University of Padova, via U. Basi 58/b, 35131, Padova, Italy.

The mitochondrial Ca uniporter machinery is a multiprotein complex composed by the Ca selective pore-forming subunit, the mitochondrial uniporter (MCU), and accessory proteins, including MICU1, MICU2 and EMRE. Their concerted action is required to fine-tune the uptake of Ca into the mitochondrial matrix which both sustains cell bioenergetics and regulates the apoptotic response. To adequately fulfil such requirements and avoid impairment in mitochondrial Ca handling, the intracellular turnover of all the MCU components must be tightly regulated. Here we show that the MCU complex regulator MICU1, but not MCU and MICU2, is rapidly and selectively degraded by the Ubiquitin Proteasome System (UPS). Moreover, we show that the multifunctional E3 ubiquitin ligase Parkin (PARK2), whose mutations cause autosomal recessive early-onset Parkinson's disease (PD), is a potential candidate involved in this process since its upregulation strongly decreases the basal level of MICU1. Parkin was found to interact with MICU1 and, interestingly, Parkin Ubl-domain, but not its E3-ubquitin ligase activity, is required for the degradation of MICU1, suggesting that in addition to the well documented role in the control of Parkin basal auto-inhibition, the Ubl-domain might exert important regulatory functions by acting as scaffold for the proteasome-mediated degradation of selected substrates under basal conditions, i.e. to guarantee their turnover. We have found that also MICU2 stability was affected upon Parkin overexpression, probably as a consequence of increased MICU1 degradation. Our findings support a model in which the PD-related E3 ubiquitin ligase Parkin directly participates in the selective regulation of the MCU complex regulator MICU1 and, indirectly, also of the MICU2 gatekeeper, thus indicating that Parkin loss of function could contribute to the impairment of the ability of mitochondria to handle Ca and consequently to the pathogenesis of PD.
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http://dx.doi.org/10.1038/s41598-018-32551-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6155109PMC
September 2018

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

Biochim Biophys Acta Mol Basis Dis 2018 10 11;1864(10):3247-3256. Epub 2018 Jul 11.

Department of Biomedical Sciences, University of Padova, Padova, Italy; Padova Neuroscience Center (PNC), University of Padova, Padova, Italy. Electronic address:

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 Ca 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 (2N4RΔC), 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 Ca 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 Ca content and the short-range ER-mitochondria interactions were selectively affected by the expression of the caspase 3-cleaved 2N4RΔC tau, indicating that Ca 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.
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http://dx.doi.org/10.1016/j.bbadis.2018.07.011DOI Listing
October 2018

The Close Encounter Between Alpha-Synuclein and Mitochondria.

Front Neurosci 2018 7;12:388. Epub 2018 Jun 7.

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

The presynaptic protein alpha-synuclein (α-syn) is unequivocally linked to the development of Parkinson's disease (PD). Not only it is the major component of amyloid fibrils found in Lewy bodies but mutations and duplication/triplication in its gene are responsible for the onset of familial autosomal dominant forms of PD. Nevertheless, the precise mechanisms leading to neuronal degeneration are not fully understood. Several lines of evidence suggest that impaired autophagy clearance and mitochondrial dysfunctions such as bioenergetics and calcium handling defects and alteration in mitochondrial morphology might play a pivotal role in the etiology and progression of PD, and indicate the intriguing possibility that α-syn could be involved in the control of mitochondrial function both in physiological and pathological conditions. In favor of this, it has been shown that a fraction of cellular α-syn can selectively localize to mitochondrial sub-compartments upon specific stimuli, highlighting possible novel routes for α-syn action. A plethora of mitochondrial processes, including cytochrome c release, calcium homeostasis, control of mitochondrial membrane potential and ATP production, is directly influenced by α-syn. Eventually, α-syn localization within mitochondria may also account for its aggregation state, making the α-syn/mitochondria intimate relationship a potential key for the understanding of PD pathogenesis. Here, we will deeply survey the recent literature in the field by focusing our attention on the processes directly controlled by α-syn within mitochondrial sub-compartments and its potential partners providing possible hints for future therapeutic targets.
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http://dx.doi.org/10.3389/fnins.2018.00388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5999749PMC
June 2018

A V1143F mutation in the neuronal-enriched isoform 2 of the PMCA pump is linked with ataxia.

Neurobiol Dis 2018 07 12;115:157-166. Epub 2018 Apr 12.

Venetian Institute of Molecular Medicine, Padova, Italy. Electronic address:

The fine regulation of intracellular calcium is fundamental for all eukaryotic cells. In neurons, Ca oscillations govern the synaptic development, the release of neurotransmitters and the expression of several genes. Alterations of Ca homeostasis were found to play a pivotal role in neurodegenerative progression. The maintenance of proper Ca signaling in neurons demands the continuous activity of Ca pumps and exchangers to guarantee physiological cytosolic concentration of the cation. The plasma membrane CaATPases (PMCA pumps) play a key role in the regulation of Ca handling in selected sub-plasma membrane microdomains. Among the four basic PMCA pump isoforms existing in mammals, isoforms 2 and 3 are particularly enriched in the nervous system. In humans, genetic mutations in the PMCA2 gene in association with cadherin 23 mutations have been linked to hearing loss phenotypes, while those occurring in the PMCA3 gene were associated with X-linked congenital cerebellar ataxias. Here we describe a novel missense mutation (V1143F) in the calmodulin binding domain (CaM-BD) of the PMCA2 protein. The mutant pump was present in a patient showing congenital cerebellar ataxia but no overt signs of deafness, in line with the absence of mutations in the cadherin 23 gene. Biochemical and molecular dynamics studies on the mutated PMCA2 have revealed that the V1143F substitution alters the binding of calmodulin to the CaM-BD leading to impaired Ca ejection.
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http://dx.doi.org/10.1016/j.nbd.2018.04.009DOI Listing
July 2018

Alpha-synuclein aggregates activate calcium pump SERCA leading to calcium dysregulation.

EMBO Rep 2018 05 29;19(5). Epub 2018 Mar 29.

Danish Research Institute of Translational Neuroscience - DANDRITE, Aarhus University, Aarhus, Denmark

Aggregation of α-synuclein is a hallmark of Parkinson's disease and dementia with Lewy bodies. We here investigate the relationship between cytosolic Ca and α-synuclein aggregation. Analyses of cell lines and primary culture models of α-synuclein cytopathology reveal an early phase with reduced cytosolic Ca levels followed by a later Ca increase. Aggregated but not monomeric α-synuclein binds to and activates SERCA , and proximity ligation assays confirm this interaction in cells. The SERCA inhibitor cyclopiazonic acid (CPA) normalises both the initial reduction and the later increase in cytosolic Ca CPA protects the cells against α-synuclein-aggregate stress and improves viability in cell models and in Proximity ligation assays also reveal an increased interaction between α-synuclein aggregates and SERCA in human brains affected by dementia with Lewy bodies. We conclude that α-synuclein aggregates bind SERCA and stimulate its activity. Reducing SERCA activity is neuroprotective, indicating that SERCA and down-stream processes may be therapeutic targets for treating α-synucleinopathies.
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http://dx.doi.org/10.15252/embr.201744617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5934765PMC
May 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

Organelles: The Emerging Signalling Chart of Mitochondrial Dynamics.

Curr Biol 2018 01;28(2):R73-R75

Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy; Department of Cell and Developmental Biology, Consortium for Mitochondrial Research, University College London, London WC1E 6BT, UK; The Francis Crick Institute, London NW1 1AT, UK. Electronic address:

Many molecular and functional details of single events in mitochondrial dynamics have been reported, but little is known about their coordination. A recent study describes how cellular Ca signals, via remodelling the actin cytoskeleton, synchronise the formation of endoplasmic reticulum-mitochondria contacts with inner and outer mitochondrial membrane fission.
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http://dx.doi.org/10.1016/j.cub.2017.11.040DOI Listing
January 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 PMCA pumps in genetically determined neuronal pathologies.

Neurosci Lett 2018 01 17;663:2-11. Epub 2017 Nov 17.

Venetian Institute of Molecular Medicine, Via G. Orus, 2, 35129 Padova, Italy. Electronic address:

Ca signals regulate most aspects of animal cell life. They are of particular importance to the nervous system, in which they regulate specific functions, from neuronal development to synaptic plasticity. The homeostasis of cell Ca must thus be very precisely regulated: in all cells Ca pumps transport it from the cytosol to the extracellular medium (the Plasma Membrane Ca ATPases, hereafter referred to as PMCA pumps) or to the lumen of intracellular organelles (the Sarco/Endoplasmatic Reticulum Ca ATPase and the Secretory Pathway Ca ATPase, hereafter referred to as SERCA and SPCA pumps, respectively). In neurons and other excitable cells a powerful plasma membrane Na/Ca exchanger (NCX) also exports Ca from cells. Quantitatively, the PMCA pumps are of minor importance to the bulk regulation of neuronal Ca. However, they are important in the regulation of Ca in specific sub-plasma membrane microdomains which contain a number of enzymes that are relevant to neuronal function. The PMCA pumps (of which 4 basic isoforms are expressed in animal cells) are P-type ATPases that are characterized by a long C-terminal cytosolic tail which is the site of interaction with most of the regulatory factors of the pump, the most important being calmodulin. In resting neurons, at low intracellular Cathe C-terminal tail of the PMCA interacts with the main body of the protein keeping it in an autoinhibited state. Local Ca increase activates calmodulin that removes the C-terminal tail from the inhibitory sites. Dysregulation of the Ca signals are incompatible with healthy neuronal life. A number of genetic mutations of PMCA pumps are associated with pathological phenotypes, those of the neuron-specific PMCA 2 and PMCA 3 being the best characterized. PMCA 2 mutations are associated with deafness and PMCA 3 mutations are linked to cerebellar ataxias. Biochemical analysis of the mutated pumps overexpressed in model cells have revealed their decreased ability to export Ca. The defect in the bulk cytosolic Ca homeostasis is minor, in keeping with the role of the PMCA pumps in the local control of Ca in specialized plasma membrane microdomains.
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http://dx.doi.org/10.1016/j.neulet.2017.11.005DOI Listing
January 2018