Publications by authors named "Marisa Brini"

83 Publications

Ca handling at the mitochondria-ER contact sites in neurodegeneration.

Cell Calcium 2021 09 5;98:102453. Epub 2021 Aug 5.

Faculty of Biology, Medicine and Health, The University of Manchester, Manchester M13 9PT, UK; Achucarro Centre for Neuroscience, IKERBASQUE, Basque Foundation for Science, 48011 Bilbao, Spain; Department of Stem Cell Biology, State Research Institute Centre for Innovative Medicine, LT-01102 Vilnius, Lithuania. Electronic address:

Mitochondria-endoplasmic reticulum (ER) contact sites (MERCS) are morpho-functional units, formed at the loci of close apposition of the ER-forming endomembrane and outer mitochondrial membrane (OMM). These sites contribute to fundamental cellular processes including lipid biosynthesis, autophagy, apoptosis, ER-stress and calcium (Ca) signalling. At MERCS, Ca ions are transferred from the ER directly to mitochondria through a core protein complex composed of inositol-1,4,5 trisphosphate receptor (InsPR), voltage-gated anion channel 1 (VDAC1), mitochondrial calcium uniporter (MCU) and adaptor protein glucose-regulated protein 75 (Grp75); this complex is regulated by several associated proteins. Deregulation of ER-mitochondria Ca transfer contributes to pathogenesis of neurodegenerative and other diseases. The efficacy of Ca transfer between ER and mitochondria depends on the protein composition of MERCS, which controls ER-mitochondria interaction regulating, for example, the transversal distance between ER membrane and OMM and the extension of the longitudinal interface between ER and mitochondria. These parameters are altered in neurodegeneration. Here we overview the ER and mitochondrial Ca homeostasis, the composition of ER-mitochondrial Ca transfer machinery and alterations of the ER-mitochondria Ca transfer in three major neurodegenerative diseases: motor neurone diseases, Parkinson disease and Alzheimer's disease.
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http://dx.doi.org/10.1016/j.ceca.2021.102453DOI Listing
September 2021

Split Green Fluorescent Protein-Based Contact Site Sensor (SPLICS ) for Heterotypic Organelle Juxtaposition as Applied to ER -Mitochondria Proximities.

Methods Mol Biol 2021 ;2275:363-378

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

In the last decades, membrane contact sites (MCSs) have been the object of intense investigation in different fields of cell physiology and pathology and their importance for the correct functioning of the cell is now widely recognized. MCS between any known intercellular organelles, including endoplasmic reticulum (ER), mitochondria, Golgi, endosomes, peroxisomes, lysosomes, lipid droplets, and the plasma membrane (PM), have been largely documented and in some cases the molecules responsible for the tethering also identified. They represent specific membrane hubs where a tightly coordinated exchange of ions, lipids, nutrients, and factors required to maintain proper cellular homeostasis takes place. Their delicate, dynamic, and sometimes elusive nature prevented and/or delayed the development of tools to easily image interorganelle proximity under physiological conditions and in living organisms. Nowadays, this aspect received great momentum due to the finding that MCSs' dysregulation is involved in several pathological conditions. We have recently developed modular, split-GFP-based contact site sensors (SPLICS) engineered to fluoresce when homo- and heterotypic juxtapositions between ER and mitochondria occur over a range of specific distances. Here we describe in detail, by highlighting strengths and weaknesses, the use and the application of these novel genetically encoded SPLICS sensors and how to properly quantify short- and long-range ER-mitochondria interactions.
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http://dx.doi.org/10.1007/978-1-0716-1262-0_23DOI Listing
August 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

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

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

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

Regulation of ER-mitochondria contacts by Parkin via Mfn2.

Pharmacol Res 2018 12 13;138:43-56. Epub 2018 Sep 13.

Department of Biology, University of Padova, Padova, Italy; Fondazione Ospedale San Camillo, IRCCS, Lido di Venezia, Venezia, Italy. Electronic address:

Parkin, an E3 ubiquitin ligase and a Parkinson's disease (PD) related gene, translocates to impaired mitochondria and drives their elimination via autophagy, a process known as mitophagy. Mitochondrial pro-fusion protein Mitofusins (Mfn1 and Mfn2) were found to be a target for Parkin mediated ubiquitination. Mfns are transmembrane GTPase embedded in the outer membrane of mitochondria, which are required on adjacent mitochondria to mediate fusion. In mammals, Mfn2 also forms complexes that are capable of tethering mitochondria to endoplasmic reticulum (ER), a structural feature essential for mitochondrial energy metabolism, calcium (Ca) transfer between the organelles and Ca dependent cell death. Despite its fundamental physiological role, the molecular mechanisms that control ER-mitochondria cross talk are obscure. Ubiquitination has recently emerged as a powerful tool to modulate protein function, via regulation of protein subcellular localization and protein ability to interact with other proteins. Ubiquitination is also a reversible mechanism, which can be actively controlled by opposing ubiquitination-deubiquitination events. In this work we found that in Parkin deficient cells and parkin mutant human fibroblasts, the tether between ER and mitochondria is decreased. We identified the site of Parkin dependent ubiquitination and showed that the non-ubiquitinatable Mfn2 mutant fails to restore ER-mitochondria physical and functional interaction. Finally, we took advantage of an established in vivo model of PD to demonstrate that manipulation of ER-mitochondria tethering by expressing an ER-mitochondria synthetic linker is sufficient to rescue the locomotor deficit associated to an in vivo Drosophila model of PD.
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http://dx.doi.org/10.1016/j.phrs.2018.09.006DOI Listing
December 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

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

Editorial.

Neurosci Lett 2018 01 13;663. Epub 2017 Oct 13.

Department of Biomedical Sciences, University of Padova, Italy.

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http://dx.doi.org/10.1016/j.neulet.2017.10.017DOI Listing
January 2018

A novel PMCA3 mutation in an ataxic patient with hypomorphic phosphomannomutase 2 (PMM2) heterozygote mutations: Biochemical characterization of the pump defect.

Biochim Biophys Acta Mol Basis Dis 2017 12 12;1863(12):3303-3312. Epub 2017 Aug 12.

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

The neuron-restricted isoform 3 of the plasma membrane Ca ATPase plays a major role in the regulation of Ca homeostasis in the brain, where the precise control of Ca signaling is a necessity. Several function-affecting genetic mutations in the PMCA3 pump associated to X-linked congenital cerebellar ataxias have indeed been described. Interestingly, the presence of co-occurring mutations in additional genes suggest their synergistic action in generating the neurological phenotype as digenic modulators of the role of PMCA3 in the pathologies. Here we report a novel PMCA3 mutation (G733R substitution) in the catalytic P-domain of the pump in a patient affected by non-progressive ataxia, muscular hypotonia, dysmetria and nystagmus. Biochemical studies of the pump have revealed impaired ability to control cellular Ca handling both under basal and under stimulated conditions. A combined analysis by homology modeling and molecular dynamics have revealed a role for the mutated residue in maintaining the correct 3D configuration of the local structure of the pump. Mutation analysis in the patient has revealed two additional function-impairing compound heterozygous missense mutations (R123Q and G214S substitution) in phosphomannomutase 2 (PMM2), a protein that catalyzes the isomerization of mannose 6-phosphate to mannose 1-phosphate. These mutations are known to be associated with Type Ia congenital disorder of glycosylation (PMM2-CDG), the most common group of disorders of N-glycosylation. The findings highlight the association of PMCA3 mutations to cerebellar ataxia and strengthen the possibility that PMCAs act as digenic modulators in Ca-linked pathologies.
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http://dx.doi.org/10.1016/j.bbadis.2017.08.006DOI Listing
December 2017

Lipid-Mediated Modulation of Intracellular Ion Channels and Redox State: Physiopathological Implications.

Antioxid Redox Signal 2018 Apr 21;28(10):949-972. Epub 2017 Sep 21.

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

Ion channels play an important role in the regulation of organelle function within the cell, as proven by increasing evidence pointing to a link between altered function of intracellular ion channels and different pathologies ranging from cancer to neurodegenerative diseases, ischemic damage, and lysosomal storage diseases. A link between these pathologies and redox state as well as lipid homeostasis and ion channel function is in the focus of current research. Ion channels are target of modulation by lipids and lipid messengers, although in most cases the mechanistic details have not been clarified yet. Ion channel function importantly impacts production of reactive oxygen species (ROS), especially in the case of mitochondria and lysosomes. ROS, in turn, may modulate the function of intracellular channels triggering thereby a feedback control under physiological conditions. If produced in excess, ROS can be harmful to lipids and may produce oxidized forms of these membrane constituents that ultimately affect ion channel function by triggering a "circulus vitiosus." The present review summarizes our current knowledge about the contribution of intracellular channels to oxidative stress and gives examples of how these channels are modulated by lipids and how this modulation may affect ROS production in ROS-related diseases. Future studies need to address the importance of the regulation of intracellular ion channels and related oxidative stress by lipids in various physiological and pathological contexts. 28, 949-972.
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http://dx.doi.org/10.1089/ars.2017.7215DOI Listing
April 2018

The ataxia related G1107D mutation of the plasma membrane Ca ATPase isoform 3 affects its interplay with calmodulin and the autoinhibition process.

Biochim Biophys Acta Mol Basis Dis 2017 01 12;1863(1):165-173. Epub 2016 Sep 12.

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

The plasma membrane Ca ATPases (PMCA pumps) have a long, cytosolic C-terminal regulatory region where a calmodulin-binding domain (CaM-BD) is located. Under basal conditions (low Ca), the C-terminal tail of the pump interacts with autoinhibitory sites proximal to the active center of the enzyme. In activating conditions (i.e., high Ca), Ca-bound CaM displaces the C-terminal tail from the autoinhibitory sites, restoring activity. We have recently identified a G1107D replacement within the CaM-BD of isoform 3 of the PMCA pump in a family affected by X-linked congenital cerebellar ataxia. Here, we investigate the effects of the G1107D replacement on the interplay of the mutated CaM-BD with both CaM and the pump core, by combining computational, biochemical and functional approaches. We provide evidence that the affinity of the isolated mutated CaM-BD for CaM is significantly reduced with respect to the wild type (wt) counterpart, and that the ability of CaM to activate the pump in vitro is thus decreased. Multiscale simulations support the conclusions on the detrimental effect of the mutation, indicating reduced stability of the CaM binding. We further show that the G1107D replacement impairs the autoinhibition mechanism of the PMCA3 pump as well, as the introduction of a negative charge perturbs the contacts between the CaM-BD and the pump core. Thus, the mutation affects both the ability of the pump to optimally transport Ca in the activated state, and the autoinhibition mechanism in its resting state.
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http://dx.doi.org/10.1016/j.bbadis.2016.09.007DOI Listing
January 2017

Emerging (and converging) pathways in Parkinson's disease: keeping mitochondrial wellness.

Biochem Biophys Res Commun 2017 02 28;483(4):1020-1030. Epub 2016 Aug 28.

Dept. of Biomedical Sciences, University of Padova, 35131 Padova, Italy. Electronic address:

The selective cell loss in the ventral component of the substantia nigra pars compacta and the presence of alpha-synuclein (α-syn)-rich intraneuronal inclusions called Lewy bodies are the pathological hallmarks of Parkinson's disease (PD), the most common motor system disorder whose aetiology remains largely elusive. Although most cases of PD are idiopathic, there are rare familial forms of the disease that can be traced to single gene mutations that follow Mendelian inheritance pattern. The study of several nuclear encoded proteins whose mutations are linked to the development of autosomal recessive and dominant forms of familial PD enhanced our understanding of biochemical and cellular mechanisms contributing to the disease and suggested that many signs of neurodegeneration result from compromised mitochondrial function. Here we present an overview of the current understanding of PD-related mitochondrial dysfunction including defects in bioenergetics and Ca homeostasis, mitochondrial DNA mutations, altered mitochondrial dynamics and autophagy. We emphasize, in particular, the convergence of many "apparently" different pathways towards a common route involving mitochondria. Understanding whether mitochondrial dysfunction in PD represents the cause or the consequence of the disease is challenging and will help to define the pathogenic processes at the basis of the PD onset and progression.
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http://dx.doi.org/10.1016/j.bbrc.2016.08.153DOI Listing
February 2017

Alpha-synuclein at the intracellular and the extracellular side: functional and dysfunctional implications.

Biol Chem 2017 01;398(1):77-100

Alpha-synuclein (α-syn) is an abundant neuronal protein whose physiological function, even if still not completely understood, has been consistently related to synaptic function and vesicle trafficking. A group of disorders known as synucleinopathies, among which Parkinson's disease (PD), is deeply associated with the misfolding and aggregation of α-syn, which can give rise to proteinaceous inclusion known as Lewy bodies (LB). Proteostasis stress is a relevant aspect in these diseases and, currently, the presence of oligomeric α-syn species rather than insoluble aggregated forms, appeared to be associated with cytotoxicity. Many observations suggest that α-syn is responsible for neurodegeneration by interfering with multiple signaling pathways. α-syn protein can directly form plasma membrane channels or modify with their activity, thus altering membrane permeability to ions, abnormally associate with mitochondria and cause mitochondrial dysfunction (i.e. mitochondrial depolarization, Ca2+ dys-homeostasis, cytochrome c release) and interfere with autophagy regulation. The picture is further complicated by the fact that single point mutations, duplications and triplication in α-syn gene are linked to autosomal dominant forms of PD. In this review we discuss the multi-faced aspect of α-syn biology and address the main hypothesis at the basis of its involvement in neuronal degeneration.
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http://dx.doi.org/10.1515/hsz-2016-0201DOI Listing
January 2017

The plasma membrane calcium pumps: focus on the role in (neuro)pathology.

Biochem Biophys Res Commun 2017 02 29;483(4):1116-1124. Epub 2016 Jul 29.

Dept. of Biomedical Sciences, University of Padova, 35131 Padova, Italy. Electronic address:

The plasma membrane Ca ATPase (PMCA pump) is a member of the superfamily of P-type pumps. It is organized in the plasma membrane with ten transmembrane helices and two main cytosolic loops, one of which contains the catalytic center. It also contains a long C-terminal tail that houses the binding site for calmodulin, the main regulator of the activity of the pump. The pump also contains a number of other regulators, among them acidic phospholipids, kinases, and numerous protein interactors. Separate genes code for 4 basic pump isoforms in mammals, additional isoform complexity being generated by the alternative splicing of primary transcripts. Pumps 1 and 4 are expressed ubiquitously, pumps 2 and 3 are tissue restricted, with preference for the nervous system. In essentially all cells, the pump coexists with much more powerful systems that clear Ca from the cytosol, e.g. the SERCA pump and the Na/Ca exchanger. Its role in the global regulation of cellular Ca homeostasis is thus quantitatively marginal: its main function is the regulation of Ca signaling in selected sub-plasma membrane microdomains where Ca modulated interactors also reside. Malfunctions of the pump linked to genetic mutations are now described with increasing frequency, the disease phenotypes being especially severe in the nervous system where isoforms 2 and 3 predominate. The analysis of the pump defects suggests that the disease phenotypes are likely to be related to the imperfect modulation of Ca signaling in selected sub-plasma membrane microdomains, leading to the defective control of the activity of important Ca dependent interactors.
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http://dx.doi.org/10.1016/j.bbrc.2016.07.117DOI Listing
February 2017
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