Publications by authors named "Domenico Cieri"

12 Publications

  • Page 1 of 1

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-19892-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7699637PMC
November 2020

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41419-019-2092-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851186PMC
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cells8091072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6769576PMC
September 2019

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.nbd.2018.04.009DOI Listing
July 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cub.2017.12.047DOI 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41418-017-0033-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5988678PMC
June 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbadis.2017.08.006DOI Listing
December 2017

Perturbations in cell signaling elicit early cardiac defects in mucopolysaccharidosis type II.

Hum Mol Genet 2017 05;26(9):1643-1655

Department of Molecular Medicine.

Morphogens release and activity can be negatively affected by an impaired glycosaminoglycans (GAGs) turnover and proteoglycans assembly in the extracellular matrix, leading to altered tissue morphogenesis. In this work, we show that loss of Iduronate-2-sulfatase (IDS) activity, affecting GAGs catabolism and responsible for a life-threatening valvulopathy in mucopolysaccharidosis type II (MPSII), triggers early Sonic Hedgehog (Shh) and Wnt/β-catenin signaling defects, leading to aberrant heart development and atrioventricular valve formation in a zebrafish model. In addition, we consistently found impaired Shh signaling activity and cardiac electrophysiological abnormalities in IDS knockout mice at postnatal stages before any evident massive GAGs accumulation. These results suggest that IDS activity substantially affect cardiac morphogenesis through impaired Shh signaling and document an unexplored role of the enzyme in the fine-tuning of cell signaling pathways.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/ddx069DOI Listing
May 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2016.08.153DOI Listing
February 2017

Zebrafish Tg(hb9:MTS-Kaede): a new in vivo tool for studying the axonal movement of mitochondria.

Biochim Biophys Acta 2016 Jun 9;1860(6):1247-55. Epub 2016 Mar 9.

Dipartimento di Biologia, Università degli Studi di Padova, Via Ugo Bassi 58/B, 35121 Padova (PD), Italy.

Objectives: Deregulation of axonal transport in neurons is emerging as the major cause of many neurodegenerative diseases in human, such as Charcot-Marie-Tooth (CMT) neuropathy. However, little is known about how mitochondria move in vivo and whether cell culture systems truly represent what happens in living animals. Here we describe the generation of a new zebrafish transgenic line that specifically allows to study mitochondrial dynamics in motor neurons and its application to analyse mitochondrial movement in zebrafish models expressing CMT2A causing mutations.

Methods: The Tol2 transposon system was used to generate a transgenic zebrafish line expressing the photoconvertible fluorescent protein Kaede in mitochondria of motor neurons. Mitochondrial shape and movement were monitored by time-lapse confocal live imaging and measured by kymograph analysis. The effects of two well-known CMT causing mutations, L76P and R94Q substitutions in MFN2, were then investigated with the same methods.

Results: We generated the transgenic zebrafish Tg(hb9:MTS-Kaede) line with genetically labelled mitochondria in motor neurons. Kaede protein was correctly and stably targeted to mitochondrial matrix while retaining its photoconvertibility, thus qualifying this model for in vivo studies. Expression of the L76P and R94Q mutations reduced mitochondrial movement in axons and altered mitochondrial distribution in distinct ways.

Conclusions And General Significance: These findings confirm previously published data obtained in cell cultures and strengthen the hypothesis of different mechanism of action of the two MFN2 mutations. Considering the number of neurodegenerative diseases associated to mitochondrial dynamics, the Tg(hb9:MTS-Kaede) zebrafish line is a promising model to study in vivo alterations of mitochondrial transport underlying human diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbagen.2016.03.007DOI Listing
June 2016
-->