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 Ca2+ 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 Ca2+ handling and mitochondrial function using a newly generated SPLICS sensor and aequorin-based Ca2+measurements. 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 Ca2+ 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 Ca2+ cross-talk impairment to the pathogenesis of PD.

Plasma membrane Ca 2+ ATPases are single polypeptides of about 1100–1250 amino-acid residues with a molecular mass of 125–140 kDa. They contain ten membrane spanning segments and their Nand C-terminals are both on the cytosolic side. The bulk of their mass is also in the cytoplasm and contains three major intracellular domains: the A (actuator), N (nucleotide-binding), and P (catalytic phosphorylation) domains. Four basic isoforms are encoded by four distinct genes, and their transcripts originated a huge number of alternative splicing variants that in most cases are also translated in the corresponding protein variants. Emerging evidence underlines that PMCA pumps, in addition to maintain resting cytosolic Ca 2+ levels against a steep concentration gradient (i.e., nM versus mM), play a local control in specifi c sub-plasma membrane domains by tethering Ca 2+ -/calmodulindependent enzymes and reducing their activity, i.e., by decreasing Ca 2+ concentration in the microenvironment where they are confi ned. This aspect of pump activity confers to PMCA pump a key role as signal transducer and justifi es the existence of so many PMCA variants that could be specialized in tuning the activity of different partners with different Ca 2+ sensitivity.

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 (Ca2+) transfer between the organelles and Ca2+ 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.

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.

Plasma membrane Ca2+ ATPases are single polypeptides of about 1100-1250 amino-acid residues with a molecular mass of 125-140 kDa. They contain ten membrane spanning segments and their N-and C -terminals are both on the cytosolic side. The bulk of their mass is also in the cytoplasm and contains three major intracellular domains: the A (actuator), N (nucleotide -binding), and P (catalytic phosphorylation) domains. Four basic isoforms are encoded by four distinct genes, and their transcripts originated a huge number of alternative splicing variants that in most cases are also translated in the corresponding protein variants. Emerging evidence underlines that PMCA pumps, in addition to maintain resting cytosolic Ca2+ levels against a steep concentration gradient (i.e., nM versus mM), play a local control in specific sub -plasma membrane domains by tethering Ca2+-/calmodulindependent enzymes and reducing their activity, i.e., by decreasing Ca2+ concentration in the microenvironment where they are confined This aspect of pump activity confers to PMCA pump a key role as signal transducer and justifies the existence of so many PMCA variants that could be specialized in tuning the activity of different partners with different Ca2+ sensitivity.

Ca2+ has a central role in all the cellular functions. Its signal is shaped by the coordinated action of the Ca2+-transporting proteins and the intracellular organelles. Mitochondria have a special role since they are the energy powerhouse of the cells, but also a major hub for cellular Ca2+ signaling crucial for cell life and death. The mitochondrial membrane potential generated by the respiratory chain is used by the ATP synthase for running the endergonic reaction of ADP phosphorylation and by the mitochondrial Ca2+ uniporter to take up Ca2+ into the matrix accordingly its electrochemical gradient. The action of the H+/Ca2+ and the Na+/Ca2+ exchangers prevents the attainment of the electrical equilibrium. Impaired Ca2+ handling can lead to matrix Ca2+ overload and activation of the high conductance mitochondrial permeability transition pore. Mitochondrial Ca2+ overload has deleterious consequences for the cells: increased membrane permeability leads to the release of proapoptotic factors and the activation of the apoptotic pathway. Even the absence of proper Ca2+ transfer from the endoplasmic reticulum to mitochondria could be detrimental since it results in defective metabolism and autophagy. Thus, mitochondrial Ca2+ handling dysfunctions may have important implications in different physiopathological conditions.

The Parkinson's disease-related protein DJ-1 has a role in the protection against oxidative stress and maintenance of mitochondria structure. Whether this action depends on its localization and activity within the mitochondria is not clear. Here we develop an approach to resolve intra-mitochondrial distribution of DJ-1 and monitor its translocation under specific conditions. By a new split-green fluorescent protein (GFP)-based tool, we can observe that a small DJ-1 fraction is located within the mitochondrial matrix and that it consistently increases upon nutrient depletion. We also find that the targeting of DJ-1 to the mitochondrial matrix enhances mitochondrial and cytosolic adenosine triphosphate levels. Intriguingly, DJ-1 pathogenic mutants fail to improve bioenergetics and translocate within the mitochondrial matrix, suggesting that the DJ-1 protective role requires both these actions. By this new split-GFP-based tool, we can resolve mitochondrial compartmentalization of proteins which are not constitutively resident in mitochondria but translocate to them in response to specific stimuli.

Calcium (Ca(2+)) is an universal second messenger that regulates the most important activities of all eukaryotic cells. It is of critical importance to neurons as it participates in the transmission of the depolarizing signal and contributes to synaptic activity. Neurons have thus developed extensive and intricate Ca(2+) signaling pathways to couple the Ca(2+) signal to their biochemical machinery. Ca(2+) influx into neurons occurs through plasma membrane receptors and voltage-dependent ion channels. The release of Ca(2+) from the intracellular stores, such as the endoplasmic reticulum, by intracellular channels also contributes to the elevation of cytosolic Ca(2+). Inside the cell, Ca(2+) is controlled by the buffering action of cytosolic Ca(2+)-binding proteins and by its uptake and release by mitochondria. The uptake of Ca(2+) in the mitochondrial matrix stimulates the citric acid cycle, thus enhancing ATP production and the removal of Ca(2+) from the cytosol by the ATP-driven pumps in the endoplasmic reticulum and the plasma membrane. A Na(+)/Ca(2+) exchanger in the plasma membrane also participates in the control of neuronal Ca(2+). The impaired ability of neurons to maintain an adequate energy level may impact Ca(2+) signaling: this occurs during aging and in neurodegenerative disease processes. The focus of this review is on neuronal Ca(2+) signaling and its involvement in synaptic signaling processes, neuronal energy metabolism, and neurotransmission. The contribution of altered Ca(2+) signaling in the most important neurological disorders will then be considered.

The photoprotein aequorin generates blue light upon binding of Ca2+ ions. Together with its very low Ca2+-buffering capacity and the possibility to add specific targeting sequences, this property has rendered aequorin particularly suitable to monitor Ca2+ concentrations in specific subcellular compartments. Recently, a new generation of genetically encoded Ca2+ probes has been developed by fusing Ca2+-responsive elements with the green fluorescent protein (GFP). Aequorin has also been employed to this aim, resulting in an aequorin-GFP chimera with the Ca2+ sensitivity of aequorin and the fluorescent properties of GFP. This setup has actually solved the major limitation of aequorin, for example, its poor ability to emit light, which rendered it inappropriate for the monitoring of Ca2+ waves at the single-cell level by imaging. In spite of the numerous genetically encoded Ca2+ indicators that are currently available, aequorin-based probes remain the method of election when an accurate quantification of Ca2+ levels is required. Here, we describe currently available aequorin variants and their use for monitoring Ca2+ waves in specific subcellular compartments. Among various applications, this method is relevant for the study of the alterations of Ca2+ homeostasis that accompany oncogenesis, tumor progression, and response to therapy.

A missense mutation in ATP2A1 gene, encoding sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) protein, causes Chianina cattle congenital pseudomyotonia, an exercise-induced impairment of muscle relaxation. Skeletal muscles of affected cattle are characterized by a selective reduction of SERCA1 in sarcoplasmic reticulum membranes. In this study, we provide evidence that the ubiquitin proteasome system is involved in the reduced density of mutated SERCA1. The treatment with MG132, an inhibitor of ubiquitin proteasome system, rescues the expression level and membrane localization of the SERCA1 mutant in a heterologous cellular model. Cells co-transfected with the Ca(2+)-sensitive probe aequorin show that the rescued SERCA1 mutant exhibits the same ability of wild type to maintain Ca(2+) homeostasis within cells. These data have been confirmed by those obtained ex vivo on adult skeletal muscle fibers from a biopsy from a pseudomyotonia-affected subject. Our data show that the mutation generates a protein most likely corrupted in proper folding but not in catalytic activity. Rescue of mutated SERCA1 to sarcoplasmic reticulum membrane can re-establish resting cytosolic Ca(2+) concentration and prevent the appearance of pathological signs of cattle pseudomyotonia.

Calcium (Ca2+) is an almost universal second messenger that regulates important activities of all eukaryotic cells. It is of critical importance to neurons, which have developed extensive and intricate pathways to couple the Ca2+ signal to their biochemical machinery. In particular, Ca2+ participates in the transmission of the depolarizing signal and contributes to synaptic activity. During aging and in neurodegenerative disease processes, the ability of neurons to maintain an adequate energy level can be compromised, thus impacting on Ca2+ homeostasis. In Parkinson's disease (PD), many signs of neurodegeneration result from compromised mitochondrial function attributable to specific effects of toxins on the mitochondrial respiratory chain and/or to genetic mutations. Despite these effects being present in almost all cell types, a distinguishing feature of PD is the extreme selectivity of cell loss, which is restricted to the dopaminergic neurons in the ventral portion of the substantia nigra pars compacta. Many hypotheses have been proposed to explain such selectivity, but only recently it has been convincingly shown that the innate autonomous activity of these neurons, which is sustained by their specific Cav1.3 L-type channel pore-forming subunit, is responsible for the generation of basal metabolic stress that, under physiological conditions, is compensated by mitochondrial buffering. However, when mitochondria function becomes even partially compromised (because of aging, exposure to environmental factors or genetic mutations), the metabolic stress overwhelms the protective mechanisms, and the process of neurodegeneration is engaged. The characteristics of Ca2+ handling in neurons of the substantia nigra pars compacta and the possible involvement of PD-related proteins in the control of Ca2+ homeostasis will be discussed in this review.

Elisa Bianchini, Stefania Testoni, Arcangelo Gentile, Tito Calì , Denis Ottolini , Antonello Villa**, Marisa Brini , Romeo Betto, Francesco Mascarello, Poul Nissen, Dorianna Sandonà, and Roberta Sacchetto From the Departments of Biomedical Sciences and Biology, University of Padova, 35131 Padova, Italy, the Departments of Animal Medicine Production and Health and Comparative Biomedicine and Food Science, University of Padova,35020 Legnaro (Padova), Italy, the Department of Veterinary Medical Sciences, University of Bologna, 40064 Bologna, Italy, the **Consorzio M.I.A., University of Milano Bicocca, 20900 Monza, Italy, the Neuroscience Institute, Consiglio Nazionale delle Ricerche Padova, 35131 Padova, Italy, and the Department of Molecular Biology and Genetics, Centre for Membrane Pumps in Cells and Disease, Aarhus University, 8000 Aarhus, Denmark

DJ-1 was first identified as an oncogene. More recently, mutations in its gene have been found causative for autosomal recessive familial Parkinson disease. Numerous studies support the DJ-1 role in the protection against oxidative stress and maintenance of mitochondria structure; however, the mechanism of its protective function remains largely unknown. We investigated whether mitochondrial Ca2+ homeostasis, a key parameter in cell physiology, could be a target for DJ-1 action. Here, we show that DJ-1 modulates mitochondrial Ca2+ transients induced upon cell stimulation with an 1,4,5-inositol-tris-phosphate agonist by favouring the endoplasmic reticulum (ER)-mitochondria tethering. A reduction of DJ-1 levels results in mitochondria fragmentation and decreased mitochondrial Ca2+ uptake in stimulated cells. To functionally couple these effects with the well-recognized cytoprotective role of DJ-1, we investigated its action in respect to the tumour suppressor p53. p53 overexpression in HeLa cells impairs their ability to accumulate Ca2+ in the mitochondrial matrix, causes alteration of the mitochondrial morphology and reduces ER-mitochondria contact sites. Mitochondrial impairments are independent from Drp1 activation, since the co-expression of the dominant negative mutant of Drp1 failed to abolish them. DJ-1 overexpression prevents these alterations by re-establishing the ER-mitochondria tethering. Similarly, the co-expression of the pro-fusion protein Mitofusin 2 blocks the effects induced by p53 on mitochondria, confirming that the modulation of the ER-mitochondria contact sites is critical to mitochondria integrity. Thus, the impairment of ER-mitochondria communication, as a consequence of DJ-1 loss-of-function, may be detrimental for mitochondria-related processes and be at the basis of mitochondrial dysfunction observed in Parkinson disease.

Mitochondria are key players of many physiological processes and deregulation of mitochondrial and/or mitochondria-related activity is unequivocally associated to numerous ageing-linked neurodegenerative disorders, including Parkinson's disease (PD), Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS). Recently, the endoplasmic reticulum (ER) stress condition is emerging as a common feature relevant to the pathogenesis of this type of diseases. Mitochondria and ER are two compartments physically and functionally tightly interconnected and recent evidence revealed that the impairment in their communication might represent a common hit in different neurodegenerative diseases. ER-mitochondria contact sites are crucial for Ca2+ signaling since, upon the opening of ER Ca2+ release channels, microdomains of high [Ca2+] are generated in their proximity and Ca2+ can be taken up by the low-affinity mitochondrial uniporter. This transfer is essential in stimulated as well as in resting conditions to sustain cell metabolism and bioenergetics. Alterations in the ER-mitochondria juxtaposition are critical not only because they determine mitochondrial dysfunctions, but also because they compromise lipid metabolism, protein synthesis, and folding, thus demonstrating that the interaction between the two compartments is bi-functional. However, the functional consequences of these alterations on Ca2+ signaling and the possible involvement in the development of neurodegenerative conditions are currently largely unexplored. Here we will survey the recent literature in the field and discuss recent insights focusing on some cellular models expressing mutant proteins involved in the pathogenesis of familial forms of PD, AD, and ALS.

The Ca 2+ ATP ases of the plasma membrane ( PMCA pumps) export Ca 2+ from all eukaryotic cells. In mammals they are the products of four separate genes. PMCA types 1 and 4 are distributed ubiquitously; PMCA types 2 and 3 are restricted to some tissues, the most important being the nervous system. Alternative splicing at two sites greatly increases the number of pump isoforms. The two ubiquitous isoforms are no longer considered as only housekeeping pumps as they also perform tissue‐specific functions. The PMCA s are classical P‐type pumps, their reaction cycle repeating that of all other pumps of the family. Their 3D structure has not been solved, but molecular modeling on SERCA pump templates shows the essential structural pattern of the latter. PMCA s are regulated by calmodulin, which interacts with high affinity with their cytosolic C‐terminal tail. A second calmodulin‐binding domain with lower affinity is present in some splicing variants of the pump. The PMCAs are essential to the regulation of cellular Ca 2+ , but the all‐important Ca 2+ signal is ambivalent: defects in its control generate various pathologies, the most thoroughly studied being those of genetic origin. Genetic defects of PMCA function produce disease phenotypes: the best characterized is a form of deafness in mice and in humans linked to PMCA 2 mutations. A cerebellar X‐linked human ataxia has recently been found to be caused by a mutation in the calmodulin‐binding domain of PMCA 3.

Loss-of-function mutations in PINK1 or parkin genes are associated with juvenile-onset autosomal recessive forms of Parkinson disease. Numerous studies have established that PINK1 and parkin participate in a common mitochondrial-quality control pathway, promoting the selective degradation of dysfunctional mitochondria by mitophagy. Upregulation of parkin mRNA and protein levels has been proposed as protective mechanism against mitochondrial and endoplasmic reticulum (ER) stress. To better understand how parkin could exert protective function we considered the possibility that it could modulate the ER–mitochondria inter-organelles cross talk. To verify this hypothesis we investigated the effects of parkin overexpression on ER–mitochondria crosstalk with respect to the regulation of two key cellular parameters: Ca2 + homeostasis and ATP production. Our results indicate that parkin overexpression in model cells physically and functionally enhanced ER–mitochondria coupling, favored Ca2 + transfer from the ER to the mitochondria following cells stimulation with an 1,4,5 inositol trisphosphate (InsP3) generating agonist and increased the agonist-induced ATP production. The overexpression of a parkin mutant lacking the first 79 residues (ΔUbl) failed to enhance the mitochondrial Ca2 + transients, thus highlighting the importance of the N-terminal ubiquitin like domain for the observed phenotype. siRNA-mediated parkin silencing caused mitochondrial fragmentation, impaired mitochondrial Ca2 + handling and reduced the ER–mitochondria tethering. These data support a novel role for parkin in the regulation of mitochondrial homeostasis, Ca2 + signaling and energy metabolism under physiological conditions.

Evolution has exploited the chemical properties of Ca(2+), which facilitate its reversible binding to the sites of irregular geometry offered by biological macromolecules, to select it as a carrier of cellular signals. A number of proteins bind Ca(2+) to specific sites: those intrinsic to membranes play the most important role in the spatial and temporal regulation of the concentration and movements of Ca(2+) inside cells. Those which are soluble, or organized in non-membranous structures, also decode the Ca(2+) message to be then transmitted to the targets of its regulation. Since Ca(2+) controls the most important processes in the life of cells, it must be very carefully controlled within the cytoplasm, where most of the targets of its signaling function reside. Membrane channels (in the plasma membrane and in the organelles) mediate the entrance of Ca(2+) into the cytoplasm, ATPases, exchangers, and the mitochondrial Ca(2+) uptake system remove Ca(2+) from it. The concentration of Ca(2+) in the external spaces, which is controlled essentially by its dynamic exchanges in the bone system, is much higher than inside cells, and can, under conditions of pathology, generate a situation of dangerous internal Ca(2+) overload. When massive and persistent, the Ca(2+) overload culminates in the death of the cell. Subtle conditions of cellular Ca(2+) dyshomeostasis that affect individual systems that control Ca(2+), generate cell disease phenotypes that are particularly severe in tissues in which the signaling function of Ca(2+) has special importance, e.g., the nervous system.

In the last two decades the study of Ca(2+) homeostasis in living cells has been enhanced by the explosive development of genetically encoded Ca(2+)-indicators. The cloning of the Ca(2+)-sensitive photoprotein aequorin and of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has been enormously advantageous. As polypeptides, aequorin and GFP allow their endogenous production in cell systems as diverse as bacteria, yeast, slime molds, plants, and mammalian cells. Moreover, it is possible to specifically localize them within the cell by including defined targeting signals in the amino acid sequence. These two proteins have been extensively engineered to obtain several recombinant probes for different biological parameters, among which Ca(2+) concentration reporters are probably the most relevant. The GFP-based Ca(2+) probes and aequorin are widely employed in the study of intracellular Ca(2+) homeostasis. The new generation of bioluminescent probes that couple the Ca(2+) sensitivity of aequorin to GFP fluorescence emission allows real-time measurements of subcellular Ca(2+) changes in single cell imaging experiments and the video-imaging of Ca(2+) concentrations changes in live transgenic animals that express GFP-aequorin bifunctional probes.