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

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.

Regulation of ER-mitochondria contacts by Parkin via Mfn2.

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 at the intracellular and the extracellular side: functional and dysfunctional implications.

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.

A new split-GFP-based probe reveals DJ-1 translocation into the mitochondrial matrix to sustain ATP synthesis upon nutrient deprivation.

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.

Inhibition of ubiquitin proteasome system rescues the defective sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1) protein causing Chianina cattle pseudomyotonia.

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.

Methods to measure intracellular Ca(2+) fluxes with organelle-targeted aequorin-based probes.

The photoprotein aequorin generates blue light upon binding of Ca(2+) ions. Together with its very low Ca(2+)-buffering capacity and the possibility to add specific targeting sequences, this property has rendered aequorin particularly suitable to monitor Ca(2+) concentrations in specific subcellular compartments. Recently, a new generation of genetically encoded Ca(2+) probes has been developed by fusing Ca(2+)-responsive elements with the green fluorescent protein (GFP). Aequorin has also been employed to this aim, resulting in an aequorin-GFP chimera with the Ca(2+) 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 Ca(2+) waves at the single-cell level by imaging. In spite of the numerous genetically encoded Ca(2+) indicators that are currently available, aequorin-based probes remain the method of election when an accurate quantification of Ca(2+) levels is required. Here, we describe currently available aequorin variants and their use for monitoring Ca(2+) waves in specific subcellular compartments. Among various applications, this method is relevant for the study of the alterations of Ca(2+) homeostasis that accompany oncogenesis, tumor progression, and response to therapy.

Calcium signaling in Parkinson's disease.

Calcium (Ca(2+)) 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 Ca(2+) signal to their biochemical machinery. In particular, Ca(2+) 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 Ca(2+) 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 Ca(2+) handling in neurons of the substantia nigra pars compacta and the possible involvement of PD-related proteins in the control of Ca(2+) homeostasis will be discussed in this review.

Neuronal calcium signaling: function and dysfunction.

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.

Intracellular calcium homeostasis and signaling.

Ca(2+) is a universal carrier of biological information: it controls cell life from its origin at fertilization to its end in the process of programmed cell death. Ca(2+) is a conventional diffusible second messenger released inside cells by the interaction of first messengers with plasma membrane receptors. However, it can also penetrate directly into cells to deliver information without the intermediation of first or second messengers. Even more distinctively, Ca(2+) can act as a first messenger, by interacting with a plasma membrane receptor to set in motion intracellular signaling pathways that involve Ca(2+) itself. Perhaps the most distinctive property of the Ca(2+) signal is its ambivalence: while essential to the correct functioning of cells, Ca(2+) becomes an agent that mediates cell distress, or even (toxic) cell death, if its concentration and movements inside cells are not carefully tuned. Ca(2+) is controlled by reversible complexation to specific proteins, which could be pure Ca(2+) buffers, or which, in addition to buffering Ca(2+), also decode its signal to pass it on to targets. The most important actors in the buffering of cell Ca(2+) are proteins that transport it across the plasma membrane and the membrane of the organelles: some have high Ca(2+) affinity and low transport capacity (e.g., Ca(2+) pumps), others have opposite properties (e.g., the Ca(2+) uptake system of mitochondria). Between the initial event of fertilization, and the terminal event of programmed cell death, the Ca(2+) signal regulates the most important activities of the cell, from the expression of genes, to heart and muscle contraction and other motility processes, to diverse metabolic pathways involved in the generation of cell fuels.

Calcium and endoplasmic reticulum-mitochondria tethering in neurodegeneration.

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 Ca(2+) signaling since, upon the opening of ER Ca(2+) release channels, microdomains of high [Ca(2+)] are generated in their proximity and Ca(2+) 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 Ca(2+) 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 Parkinson disease-related protein DJ-1 counteracts mitochondrial impairment induced by the tumour suppressor protein p53 by enhancing endoplasmic reticulum-mitochondria tethering.

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 Ca(2+) homeostasis, a key parameter in cell physiology, could be a target for DJ-1 action. Here, we show that DJ-1 modulates mitochondrial Ca(2+) 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 Ca(2+) 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 Ca(2+) 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.

The plasma membrane calcium pump in health and disease.

The Ca(2+) ATPases 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 PMCAs 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. PMCAs 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 PMCA2 mutations. A cerebellar X-linked human ataxia has recently been found to be caused by a mutation in the calmodulin-binding domain of PMCA3.

Enhanced parkin levels favor ER-mitochondria crosstalk and guarantee Ca(2+) transfer to sustain cell bioenergetics.

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: Ca(2+) homeostasis and ATP production. Our results indicate that parkin overexpression in model cells physically and functionally enhanced ER-mitochondria coupling, favored Ca(2+) transfer from the ER to the mitochondria following cells stimulation with an 1,4,5 inositol trisphosphate (InsP(3)) 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 Ca(2+) 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 Ca(2+) handling and reduced the ER-mitochondria tethering. These data support a novel role for parkin in the regulation of mitochondrial homeostasis, Ca(2+) signaling and energy metabolism under physiological conditions.

Measurements of Ca²âº concentration with recombinant targeted luminescent probes.

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.

Calcium in health and disease.

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.

Mutation of plasma membrane Ca2+ ATPase isoform 3 in a family with X-linked congenital cerebellar ataxia impairs Ca2+ homeostasis.

Ca(2+) in neurons is vital to processes such as neurotransmission, neurotoxicity, synaptic development, and gene expression. Disruption of Ca(2+) homeostasis occurs in brain aging and in neurodegenerative disorders. Membrane transporters, among them the calmodulin (CaM)-activated plasma membrane Ca(2+) ATPases (PMCAs) that extrude Ca(2+) from the cell, play a key role in neuronal Ca(2+) homeostasis. Using X-exome sequencing we have identified a missense mutation (G1107D) in the CaM-binding domain of isoform 3 of the PMCAs in a family with X-linked congenital cerebellar ataxia. PMCA3 is highly expressed in the cerebellum, particularly in the presynaptic terminals of parallel fibers-Purkinje neurons. To study the effects of the mutation on Ca(2+) extrusion by the pump, model cells (HeLa) were cotransfected with expression plasmids encoding its mutant or wild-type (wt) variants and with the Ca(2+)-sensing probe aequorin. The mutation reduced the ability of the PMCA3 pump to control the cellular homeostasis of Ca(2+). It significantly slowed the return to baseline of the Ca(2+) transient induced by an inositol-trisphosphate (InsP(3))-linked plasma membrane agonist. It also compromised the ability of the pump to oppose the influx of Ca(2+) through the plasma membrane capacitative channels.

Mitochondrial Ca(2+) and neurodegeneration.

Mitochondria are essential for ensuring numerous fundamental physiological processes such as cellular energy, redox balance, modulation of Ca(2+) signaling and important biosynthetic pathways. They also govern the cell fate by participating in the apoptosis pathway. The mitochondrial shape, volume, number and distribution within the cells are strictly controlled. The regulation of these parameters has an impact on mitochondrial function, especially in the central nervous system, where trafficking of mitochondria is critical to their strategic intracellular distribution, presumably according to local energy demands. Thus, the maintenance of a healthy mitochondrial population is essential to avoid the impairment of the processes they regulate: for this purpose, cells have developed mechanisms involving a complex system of quality control to remove damaged mitochondria, or to renew them. Defects of these processes impair mitochondrial function and lead to disordered cell function, i.e., to a disease condition. Given the standard role of mitochondria in all cells, it might be expected that their dysfunction would give rise to similar defects in all tissues. However, damaged mitochondrial function has pleiotropic effects in multicellular organisms, resulting in diverse pathological conditions, ranging from cardiac and brain ischemia, to skeletal muscle myopathies to neurodegenerative diseases. In this review, we will focus on the relationship between mitochondrial (and cellular) derangements and Ca(2+) dysregulation in neurodegenerative diseases, emphasizing the evidence obtained in genetic models. Common patterns, that recognize the derangement of Ca(2+) and energy control as a causative factor, have been identified: advances in the understanding of the molecular regulation of Ca(2+) homeostasis, and on the ways in which it could become perturbed in neurological disorders, may lead to the development of therapeutic strategies that modulate neuronal Ca(2+) signaling.

Ca2+-activated nucleotidase 1, a novel target gene for the transcriptional repressor DREAM (downstream regulatory element antagonist modulator), is involved in protein folding and degradation.

DREAM is a Ca(2+)-dependent transcriptional repressor highly expressed in neuronal cells. A number of genes have already been identified as the target of its regulation. Targeted analysis performed on cerebella from transgenic mice expressing a dominant active DREAM mutant (daDREAM) showed a drastic reduction of the amount of transcript of Ca(2+)-activated nucleotidase 1 (CANT1), an endoplasmic reticulum (ER)-Golgi resident Ca(2+)-dependent nucleoside diphosphatase that has been suggested to have a role in glucosylation reactions related to the quality control of proteins in the ER and the Golgi apparatus. CANT1 down-regulation was also found in neuroblastoma SH-SY5Y cells stably overexpressing wild type (wt) DREAM or daDREAM, thus providing a simple cell model to investigate the protein maturation pathway. Pulse-chase experiments demonstrated that the down-regulation of CANT1 is associated with reduced protein secretion and increased degradation rates. Importantly, overexpression of wtDREAM or daDREAM augmented the expression of the EDEM1 gene, which encodes a key component of the ER-associated degradation pathway, suggesting an alternative pathway to enhanced protein degradation. Restoring CANT1 levels in neuroblastoma clones recovered the phenotype, thus confirming a key role of CANT1, and of the regulation of its gene by DREAM, in the control of protein synthesis and degradation.

Mitochondrial Ca(2+) as a key regulator of mitochondrial activities.

Mitochondria play a central role in cell biology, not only as producers of ATP but also as regulators of the Ca(2+) signal. The translocation by respiratory chain protein complexes of H(+) across the ion-impermeable inner membrane generates a very large H(+) electrochemical gradient that can be employed not only by the H(+) ATPase to run the endoergonic reaction of ADP phosphorylation, but also to accumulate cations into the matrix. Mitochondria can rapidly take up Ca(2+) through an electrogenic pathway, the uniporter, that acts to equilibrate Ca(2+) with its electrochemical gradient, and thus accumulates the cation into the matrix, and they can release it through two exchangers (with H(+) and Na(+), mostly expressed in non-excitable and excitable cells, respectively), that utilize the electrochemical gradient of the monovalent cations to prevent the attainment of electrical equilibrium.The uniporter, due to its low Ca(2+) affinity, demands high local Ca(2+) concentrations to work. In different cell systems these high Ca(2+) concentration microdomains are generated, upon cell stimulation, in proximity of the plasma membrane and the sarco/endoplasmic reticulum Ca(2+) channels.Recent work has revealed the central role of mitochondria in signal transduction pathways: evidence is accumulating that, by taking up Ca(2+), they not only modulate mitochondrial activities but also tune the cytosolic Ca(2+) signals and their related functions. This review analyses recent developments in the area of mitochondrial Ca(2+) signalling and attempts to summarize cell physiology aspects of the mitochondrial Ca(2+) transport machinery.

α-Synuclein controls mitochondrial calcium homeostasis by enhancing endoplasmic reticulum-mitochondria interactions.

α-Synuclein has a central role in Parkinson disease, but its physiological function and the mechanism leading to neuronal degeneration remain unknown. Because recent studies have highlighted a role for α-synuclein in regulating mitochondrial morphology and autophagic clearance, we investigated the effect of α-synuclein in HeLa cells on mitochondrial signaling properties focusing on Ca(2+) homeostasis, which controls essential bioenergetic functions. By using organelle-targeted Ca(2+)-sensitive aequorin probes, we demonstrated that α-synuclein positively affects Ca(2+) transfer from the endoplasmic reticulum to the mitochondria, augmenting the mitochondrial Ca(2+) transients elicited by agonists that induce endoplasmic reticulum Ca(2+) release. This effect is not dependent on the intrinsic Ca(2+) uptake capacity of mitochondria, as measured in permeabilized cells, but correlates with an increase in the number of endoplasmic reticulum-mitochondria interactions. This action specifically requires the presence of the C-terminal α-synuclein domain. Conversely, α-synuclein siRNA silencing markedly reduces mitochondrial Ca(2+) uptake, causing profound alterations in organelle morphology. The enhanced accumulation of α-synuclein into the cells causes the redistribution of α-synuclein to localized foci and, similarly to the silencing of α-synuclein, reduces the ability of mitochondria to accumulate Ca(2+). The absence of efficient Ca(2+) transfer from endoplasmic reticulum to mitochondria results in augmented autophagy that, in the long range, could compromise cellular bioenergetics. Overall, these findings demonstrate a key role for α-synuclein in the regulation of mitochondrial homeostasis in physiological conditions. Elevated α-synuclein expression and/or eventually alteration of the aggregation properties cause the redistribution of the protein within the cell and the loss of modulation on mitochondrial function.