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Coronaviruses Hijack the LC3-I-Positive EDEMosomes, ER-Derived Vesicles Exporting Short-Lived ERAD Regulators, for Replication

Summary Coronaviruses (CoV), including SARS and mouse hepatitis virus (MHV), are enveloped RNA viruses that induce formation of double-membrane vesicles (DMVs) and target their replication and transcription complexes (RTCs) on the DMV-limiting membranes. The DMV biogenesis has been connected with the early secretory pathway. CoV-induced DMVs, however, lack conventional endoplasmic reticulum (ER) or Golgi protein markers, leaving their membrane origins in question. We show that MHV co-opts the host cell machinery for COPII-independent vesicular ER export of a short-living regulator of ER-associated degradation (ERAD), EDEM1, to derive cellular membranes for replication. MHV infection causes accumulation of EDEM1 and OS-9, another short-living ER chaperone, in the DMVs. DMVs are coated with the nonlipidated LC3/Atg8 autophagy marker. Downregulation of LC3, but not inactivation of host cell autophagy, protects cells from CoV infection. Our study identifies the host cellular pathway hijacked for supplying CoV replication membranes and describes an autophagy-independent role for nonlipidated LC3-I.

α-Synuclein Controls Mitochondrial Calcium Homeostasis by Enhancing Endoplasmic Reticulum-Mitochondria Interactions.

Background: α-Synuclein has a central role in Parkinson disease, and it has been proposed to regulate mitochondrial morphology and autophagic clearance. Results: α-Synuclein overexpression augments mitochondrial Ca2+ transients by enhancing endoplasmic reticulum-mitochondria interactions. Conclusion: Physiological levels of α-synuclein are required to sustain mitochondrial function and morphology integrity. Significance: Mitochondrial Ca2+ represents an important site where α-synuclein can modulate cell bioenergetics and survival. 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 Ca2+ homeostasis, which controls essential bioenergetic functions. By using organelle-targeted Ca2+-sensitive aequorin probes, we demonstrated that α-synuclein positively affects Ca2+ transfer from the endoplasmic reticulum to the mitochondria, augmenting the mitochondrial Ca2+ transients elicited by agonists that induce endoplasmic reticulum Ca2+ release. This effect is not dependent on the intrinsic Ca2+ 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 Ca2+ 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 Ca2+. The absence of efficient Ca2+ 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.

Neuronal calcium signaling: function and dysfunction

Calcium (Ca2+) 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 Ca2+ signaling pathways to couple the Ca2+ signal to their biochemical machinery. Ca2+ influx into neurons occurs through plasma membrane receptors and voltage-dependent ion channels. The release of Ca2+ from the intracellular stores, such as the endoplasmic reticulum, by intracellular channels also contributes to the elevation of cytosolic Ca2+. Inside the cell, Ca2+ is controlled by the buffering action of cytosolic Ca2+-binding proteins and by its uptake and release by mitochondria. The uptake of Ca2+ in the mitochondrial matrix stimulates the citric acid cycle, thus enhancing ATP production and the removal of Ca2+ from the cytosol by the ATP-driven pumps in the endoplasmic reticulum and the plasma membrane. A Na+/Ca2+ exchanger in the plasma membrane also participates in the control of neuronal Ca2+. The impaired ability of neurons to maintain an adequate energy level may impact Ca2+ signaling: this occurs during aging and in neurodegenerative disease processes. The focus of this review is on neuronal Ca2+ signaling and its involvement in synaptic signaling processes, neuronal energy metabolism, and neurotransmission. The contribution of altered Ca2+ signaling in the most important neurological disorders will then be considered.

Enhanced parkin levels favor ER-mitochondria crosstalk and guarantee Ca2 + 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.

Segregation and rapid turnover of EDEM1 by an autophagy-like mechanism modulates standard ERAD and folding activities

EDEM1 is a crucial regulator of endoplasmic reticulum (ER)-associated degradation (ERAD) that extracts non-native glycopolypeptides from the calnexin chaperone system. Under normal growth conditions, the intralumenal level of EDEM1 must be low to prevent premature interruption of ongoing folding programs. We report that in unstressed cells, EDEM1 is segregated from the bulk ER into LC3-I-coated vesicles and is rapidly degraded. The rapid turnover of EDEM1 is regulated by a novel mechanism that shows similarities but is clearly distinct from macroautophagy. Cells with defective EDEM1 turnover contain unphysiologically high levels of EDEM1, show enhanced ERAD activity and are characterized by impaired capacity to efficiently complete maturation of model glycopolypeptides. We define as ERAD tuning the mechanisms operating in the mammalian ER at steady state to offer kinetic advantage to folding over disposal of unstructured nascent chains by selective and rapid degradation of ERAD regulators.

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

Ca2+ in neurons is vital to processes such as neurotransmission, neurotoxicity, synaptic development, and gene expression. Disruption of Ca2+ homeostasis occurs in brain aging and in neurodegenerative disorders. Membrane transporters, among them the calmodulin (CaM)-activated plasma membrane Ca2+ ATPases (PMCAs) that extrude Ca2+ from the cell, play a key role in neuronal Ca2+ 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 Ca2+ extrusion by the pump, model cells (HeLa) were cotransfected with expression plasmids encoding its mutant or wild-type (wt) variants and with the Ca2+-sensing probe aequorin. The mutation reduced the ability of the PMCA3 pump to control the cellular homeostasis of Ca2+. It significantly slowed the return to baseline of the Ca2+ transient induced by an inositol-trisphosphate (InsP3)-linked plasma membrane agonist. It also compromised the ability of the pump to oppose the influx of Ca2+ through the plasma membrane capacitative channels.

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 Ca2+ ATPases of the plasma membrane (PMCA pumps) export Ca2+ 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 Ca2+, but the all‐important Ca2+ 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.

Mitochondria, calcium, and endoplasmic reticulum stress in Parkinson's disease

Parkinsons disease (PD) is a progressive neurodegenerative disease characterized by a loss of dopaminergic neurons in the substantia nigra pars compacta (SNPC) and the presence of intracytoplasmatic inclusions known as Lewy bodies, largely composed of alpha-synuclein (α-syn). PD is a multifactorial disease and its etiology remains largely elusive. Although more than 90% of the cases are sporadic, mutations in several nuclear encoded genes have been linked to the development of autosomal recessive and dominant familial parkinsonian syndromes (Bogaerts et al. (2008) Genes Brain Behav 7, 129-151), enhancing our understanding of biochemical and cellular mechanisms contributing to the disease. Many cellular mechanisms are thought to be involved in the dopaminergic neuronal death in PD, including oxidative stress, intracellular Ca(2+) homeostasis impairment, and mitochondrial dysfunctions. Furthermore, endoplasmic reticulum (ER) stress together with abnormal protein degradation by the ubiquitin proteasome system is considered to contribute to the PD pathogenesis. This review covers all the aspects related to the molecular mechanisms underlying the interplay between mitochondria, ER, and proteasome system in PD-associated neurodegeneration.

Mitochondrial Ca2+ and neurodegeneration

Mitochondria are essential for ensuring numerous fundamental physiological processes such as cellular energy, redox balance, modulation of Ca2+ 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 Ca2+ dysregulation in neurodegenerative diseases, emphasizing the evidence obtained in genetic models. Common patterns, that recognize the derangement of Ca2+ and energy control as a causative factor, have been identified: advances in the understanding of the molecular regulation of Ca2+ 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 Ca2+ signaling.