LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF-κB to the nucleus.

Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling; however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here, we show that the NF-κB pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-κB to the nucleus. TNF treatment induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-κB is locally activated and transported to the nucleus by mitochondria, leading to an increase in mitochondria-nucleus contact sites in a HOIP-dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 furthermore contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN. Overall, our study reveals a role for mitochondria in amplifying TNF-mediated NF-κB activation, both serving as a signaling platform, as well as a transport mode for activated NF-κB to the nuclear.

Sorcin promotes migration in cancer and regulates the EGF-dependent EGFR signaling pathways.

The epidermal growth factor receptor (EGFR) is one of the main tumor drivers and is an important therapeutic target for many cancers. Calcium is important in EGFR signaling pathways. Sorcin is one of the most important calcium sensor proteins, overexpressed in many tumors, that promotes cell proliferation, migration, invasion, epithelial-to-mesenchymal transition, malignant progression and resistance to chemotherapeutic drugs. The present work elucidates a functional mechanism that links calcium homeostasis to EGFR signaling in cancer. Sorcin and EGFR expression are significantly correlated and associated with reduced overall survival in cancer patients. Mechanistically, Sorcin directly binds EGFR protein in a calcium-dependent fashion and regulates calcium (dys)homeostasis linked to EGF-dependent EGFR signaling. Moreover, Sorcin controls EGFR proteostasis and signaling and increases its phosphorylation, leading to increased EGF-dependent migration and invasion. Of note, silencing of Sorcin cooperates with EGFR inhibitors in the regulation of migration, highlighting calcium signaling pathway as an exploitable target to enhance the effectiveness of EGFR-targeting therapies.

ATP2B2 de novo variants as a cause of variable neurodevelopmental disorders that feature dystonia, ataxia, intellectual disability, behavioral symptoms, and seizures.

PURPOSE: ATP2B2 encodes the variant-constrained plasma-membrane calcium-transporting ATPase-2, expressed in sensory ear cells and specialized neurons. ATP2B2/Atp2b2 variants were previously linked to isolated hearing loss in patients and neurodevelopmental deficits with ataxia in mice. We aimed to establish the association between ATP2B2 and human neurological disorders. METHODS: Multinational case recruitment, scrutiny of trio-based genomics data, in silico analyses, and functional variant characterization were performed. RESULTS: We assembled 7 individuals harboring rare, predicted deleterious heterozygous ATP2B2 variants. The alleles comprised 5 missense substitutions that affected evolutionarily conserved sites and 2 frameshift variants in the penultimate exon. For 6 variants, a de novo status was confirmed. Unlike described patients with hearing loss, the individuals displayed a spectrum of neurological abnormalities, ranging from ataxia with dystonic features to complex neurodevelopmental manifestations with intellectual disability, autism, and seizures. Two cases with recurrent amino-acid variation showed distinctive overlap with cerebellar atrophy-associated ataxia and epilepsy. In cell-based studies, all variants caused significant alterations in cytosolic calcium handling with both loss- and gain-of-function effects. CONCLUSION: Presentations in our series recapitulate key phenotypic aspects of Atp2b2-mouse models and underline the importance of precise calcium regulation for neurodevelopment and cerebellar function. Our study documents a role for ATP2B2 variants in causing heterogeneous neurodevelopmental and movement-disorder syndromes.

Angiotensin II Promotes SARS-CoV-2 Infection via Upregulation of ACE2 in Human Bronchial Cells.

Blockers of the renin-angiotensin system (RAS) have been reported to increase the angiotensin converting enzyme (ACE)2, the cellular receptor of SARS-CoV-2, and thus the risk and course of COVID-19. Therefore, we investigated if angiotensin (Ang) II and RAS blockers affected ACE2 expression and SARS-CoV-2 infectivity in human epithelial bronchial Calu-3 cells. By infectivity and spike-mediated cell-cell fusion assays, we showed that Ang II acting on the angiotensin type 1 receptor markedly increased ACE2 at mRNA and protein levels, resulting in enhanced SARS-CoV-2 cell entry. These effects were abolished by irbesartan and not affected by the blockade of ACE-1-mediated Ang II formation with ramipril, and of ACE2- mediated Ang II conversion into Ang 1-7 with MLN-4760. Thus, enhanced Ang II production in patients with an activated RAS might expose to a greater spread of COVID-19 infection in lung cells. The protective action of Angiotensin type 1 receptor antagonists (ARBs) documented in these studies provides a mechanistic explanation for the lack of worse outcomes in high-risk COVID-19 patients on RAS blockers.

Regulation of Endoplasmic Reticulum-Mitochondria Tethering and Ca2+ Fluxes by TDP-43 via GSK3ß.

Mitochondria-ER contacts (MERCs), tightly regulated by numerous tethering proteins that act as molecular and functional connections between the two organelles, are essential to maintain a variety of cellular functions. Such contacts are often compromised in the early stages of many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). TDP-43, a nuclear protein mainly involved in RNA metabolism, has been repeatedly associated with ALS pathogenesis and other neurodegenerative diseases. Although TDP-43 neuropathological mechanisms are still unclear, the accumulation of the protein in cytoplasmic inclusions may underlie a protein loss-of-function effect. Accordingly, we investigated the impact of siRNA-mediated TDP-43 silencing on MERCs and the related cellular parameters in HeLa cells using GFP-based probes for MERCs quantification and aequorin-based probes for local Ca2+ measurements, combined with targeted protein and mRNA profiling. Our results demonstrated that TDP-43 down-regulation decreases MERCs density, thereby remarkably reducing mitochondria Ca2+ uptake after ER Ca2+ release. Thorough mRNA and protein analyses did not highlight altered expression of proteins involved in MERCs assembly or Ca2+-mediated ER-mitochondria cross-talk, nor alterations of mitochondrial density and morphology were observed by confocal microscopy. Further mechanistic inspections, however, suggested that the observed cellular alterations are correlated to increased expression/activity of GSK3ß, previously associated with MERCs disruption.

Alpha-synuclein aggregates activate calcium pump SERCA leading to calcium dysregulation.

Aggregation of α-synuclein is a hallmark of Parkinson's disease and dementia with Lewy bodies. We here investigate the relationship between cytosolic Ca2+ and α-synuclein aggregation. Analyses of cell lines and primary culture models of α-synuclein cytopathology reveal an early phase with reduced cytosolic Ca2+ levels followed by a later Ca2+ increase. Aggregated but not monomeric α-synuclein binds to and activates SERCA in vitro, and proximity ligation assays confirm this interaction in cells. The SERCA inhibitor cyclopiazonic acid (CPA) normalises both the initial reduction and the later increase in cytosolic Ca2+ CPA protects the cells against α-synuclein-aggregate stress and improves viability in cell models and in Caenorhabditis elegans in vivo Proximity ligation assays also reveal an increased interaction between α-synuclein aggregates and SERCA in human brains affected by dementia with Lewy bodies. We conclude that α-synuclein aggregates bind SERCA and stimulate its activity. Reducing SERCA activity is neuroprotective, indicating that SERCA and down-stream processes may be therapeutic targets for treating α-synucleinopathies.

ER-Mitochondria Calcium Transfer, Organelle Contacts and Neurodegenerative Diseases.

It is generally accepted that interorganellar contacts are central to the control of cellular physiology. Virtually, any intracellular organelle can come into proximity with each other and, by establishing physical protein-mediated contacts within a selected fraction of the membrane surface, novel specific functions are acquired. Endoplasmic reticulum (ER) contacts with mitochondria are among the best studied and have a major role in Ca2+ and lipid transfer, signaling, and membrane dynamics.Their functional (and structural) diversity, their dynamic nature as well as the growing number of new players involved in the tethering concurred to make their monitoring difficult especially in living cells. This review focuses on the most established examples of tethers/modulators of the ER-mitochondria interface and on the roles of these contacts in health and disease by specifically dissecting how Ca2+ transfer occurs and how mishandling eventually leads to disease. Additional functions of the ER-mitochondria interface and an overview of the currently available methods to measure/quantify the ER-mitochondria interface will also be discussed.

ER-Mitochondria Contact Sites Reporters: Strengths and Weaknesses of the Available Approaches.

Organelle intercommunication represents a wide area of interest. Over the last few decades, increasing evidence has highlighted the importance of organelle contact sites in many biological processes including Ca2+ signaling, lipid biosynthesis, apoptosis, and autophagy but also their involvement in pathological conditions. ER-mitochondria tethering is one of the most investigated inter-organelle communications and it is differently modulated in response to several cellular conditions including, but not limited to, starvation, Endoplasmic Reticulum (ER) stress, and mitochondrial shape modifications. Despite many studies aiming to understand their functions and how they are perturbed under different conditions, approaches to assess organelle proximity are still limited. Indeed, better visualization and characterization of contact sites remain a fascinating challenge. The aim of this review is to summarize strengths and weaknesses of the available methods to detect and quantify contact sites, with a main focus on ER-mitochondria tethering.

PINK1/Parkin Mediated Mitophagy, Ca2+ Signalling, and ER-Mitochondria Contacts in Parkinson's Disease.

Endoplasmic reticulum (ER)-mitochondria contact sites are critical structures for cellular function. They are implicated in a plethora of cellular processes, including Ca2+ signalling and mitophagy, the selective degradation of damaged mitochondria. Phosphatase and tensin homolog (PTEN)-induced kinase (PINK) and Parkin proteins, whose mutations are associated with familial forms of Parkinson's disease, are two of the best characterized mitophagy players. They accumulate at ER-mitochondria contact sites and modulate organelles crosstalk. Alterations in ER-mitochondria tethering are a common hallmark of many neurodegenerative diseases including Parkinson's disease. Here, we summarize the current knowledge on the involvement of PINK1 and Parkin at the ER-mitochondria contact sites and their role in the modulation of Ca2+ signalling and mitophagy.

A V1143F mutation in the neuronal-enriched isoform 2 of the PMCA pump is linked with ataxia.

The fine regulation of intracellular calcium is fundamental for all eukaryotic cells. In neurons, Ca2+ oscillations govern the synaptic development, the release of neurotransmitters and the expression of several genes. Alterations of Ca2+ homeostasis were found to play a pivotal role in neurodegenerative progression. The maintenance of proper Ca2+ signaling in neurons demands the continuous activity of Ca2+ pumps and exchangers to guarantee physiological cytosolic concentration of the cation. The plasma membrane Ca2+ATPases (PMCA pumps) play a key role in the regulation of Ca2+ 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 Ca2+ ejection.

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.

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

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 Ca2+ 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.

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

The plasma membrane Ca2+ ATPase (PMCA pump) is a member of the superfamily of P-type pumps. It is organized in the plasma membrane with ten transmembrane helices and two main cytosolic loops, one of which contains the catalytic center. It also contains a long C-terminal tail that houses the binding site for calmodulin, the main regulator of the activity of the pump. The pump also contains a number of other regulators, among them acidic phospholipids, kinases, and numerous protein interactors. Separate genes code for 4 basic pump isoforms in mammals, additional isoform complexity being generated by the alternative splicing of primary transcripts. Pumps 1 and 4 are expressed ubiquitously, pumps 2 and 3 are tissue restricted, with preference for the nervous system. In essentially all cells, the pump coexists with much more powerful systems that clear Ca2+ from the cytosol, e.g. the SERCA pump and the Na+/Ca2+ exchanger. Its role in the global regulation of cellular Ca2+ homeostasis is thus quantitatively marginal: its main function is the regulation of Ca2+ signaling in selected sub-plasma membrane microdomains where Ca2+ modulated interactors also reside. Malfunctions of the pump linked to genetic mutations are now described with increasing frequency, the disease phenotypes being especially severe in the nervous system where isoforms 2 and 3 predominate. The analysis of the pump defects suggests that the disease phenotypes are likely to be related to the imperfect modulation of Ca2+ signaling in selected sub-plasma membrane microdomains, leading to the defective control of the activity of important Ca2+ dependent interactors.

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

The neuron-restricted isoform 3 of the plasma membrane Ca2+ ATPase plays a major role in the regulation of Ca2+ homeostasis in the brain, where the precise control of Ca2+ 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 Ca2+ 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 Ca2+-linked pathologies.

The ataxia related G1107D mutation of the plasma membrane Ca2+ ATPase isoform 3 affects its interplay with calmodulin and the autoinhibition process.

The plasma membrane Ca2+ ATPases (PMCA pumps) have a long, cytosolic C-terminal regulatory region where a calmodulin-binding domain (CaM-BD) is located. Under basal conditions (low Ca2+), the C-terminal tail of the pump interacts with autoinhibitory sites proximal to the active center of the enzyme. In activating conditions (i.e., high Ca2+), Ca2+-bound CaM displaces the C-terminal tail from the autoinhibitory sites, restoring activity. We have recently identified a G1107D replacement within the CaM-BD of isoform 3 of the PMCA pump in a family affected by X-linked congenital cerebellar ataxia. Here, we investigate the effects of the G1107D replacement on the interplay of the mutated CaM-BD with both CaM and the pump core, by combining computational, biochemical and functional approaches. We provide evidence that the affinity of the isolated mutated CaM-BD for CaM is significantly reduced with respect to the wild type (wt) counterpart, and that the ability of CaM to activate the pump in vitro is thus decreased. Multiscale simulations support the conclusions on the detrimental effect of the mutation, indicating reduced stability of the CaM binding. We further show that the G1107D replacement impairs the autoinhibition mechanism of the PMCA3 pump as well, as the introduction of a negative charge perturbs the contacts between the CaM-BD and the pump core. Thus, the mutation affects both the ability of the pump to optimally transport Ca2+ in the activated state, and the autoinhibition mechanism in its resting state.

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 Novel Mutation in Isoform 3 of the Plasma Membrane Ca2+ Pump Impairs Cellular Ca2+ Homeostasis in a Patient with Cerebellar Ataxia and Laminin Subunit 1α Mutations.

The particular importance of Ca(2+) signaling to neurons demands its precise regulation within their cytoplasm. Isoform 3 of the plasma membrane Ca(2+) ATPase (the PMCA3 pump), which is highly expressed in brain and cerebellum, plays an important role in the regulation of neuronal Ca(2+). A genetic defect of the PMCA3 pump has been described in one family with X-linked congenital cerebellar ataxia. Here we describe a novel mutation in the ATP2B3 gene in a patient with global developmental delay, generalized hypotonia and cerebellar ataxia. The mutation (a R482H replacement) impairs the Ca(2+) ejection function of the pump. It reduces the ability of the pump expressed in model cells to control Ca(2+) transients generated by cell stimulation and impairs its Ca(2+) extrusion function under conditions of low resting cytosolic Ca(2+) as well. In silico analysis of the structural effect of the mutation suggests a reduced stabilization of the portion of the pump surrounding the mutated residue in the Ca(2+)-bound state. The patient also carries two missense mutations in LAMA1, encoding laminin subunit 1α. On the basis of the family pedigree of the patient, the presence of both PMCA3 and laminin subunit 1α mutations appears to be necessary for the development of the disease. Considering the observed defect in cellular Ca(2+) homeostasis and the previous finding that PMCAs act as digenic modulators in Ca(2+)-linked pathologies, the PMCA3 dysfunction along with LAMA1 mutations could act synergistically to cause the neurological phenotype.

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.

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.