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Alpha-synuclein pathology is associated with dopaminergic neuronal loss in the substantia nigra (SN) of Parkinson's patients. Working across human and mouse models, we investigated mechanisms by which the accumulation of soluble α-synuclein oligomers leads to neurodegeneration. Biochemical analysis of the midbrain of α-synuclein overexpressing BAC-transgenic male and female mice revealed age- and region-dependent mitochondrial dysfunction and accumulation of damaged proteins downstream of the RE1 Silencing Transcription Factor (REST). Vulnerable SN dopaminergic neurons displayed low REST levels compared with neighboring protected SN GABAergic neurons, which correlated with the accumulation of α-synuclein oligomers and disrupted mitochondrial morphology. Consistent with a protective role, REST levels were reduced in patient induced pluripotent stem cell-derived dopaminergic neurons carrying theSNCA-Triplicationmutation, which accumulated α-synuclein oligomers and mitochondrial damage, and displayed REST target gene dysregulation. Furthermore, CRISPR-mediated REST KO induced mitochondrial dysfunction and impaired mitophagyin vitro. Conversely, REST overexpression attenuated mitochondrial toxicity and mitochondrial morphology disruption through the transcription factor PGC-1α. Finally, decreased α-synuclein oligomer accumulation and mitochondrial dysfunction in mice correlated with nuclear REST and PGC-1α in protected SN GABAergic neurons compared with vulnerable dopaminergic neurons. Our findings show that increased levels of α-synuclein oligomers cause dopaminergic neuronal-specific dysfunction through mitochondrial toxicity, which can be attenuated by REST in an early model of Parkinsonian pathology. These findings highlight REST as a mediator of dopaminergic vulnerability in PD.SIGNIFICANCE STATEMENTUnderstanding early Parkinsonian pathophysiology through studies of advanced preclinical models is fundamental to the translation of disease-modifying therapies. Here we show disease-relevant levels of α-synuclein expression in mice leads to accumulation of α-synuclein oligomers in the absence of overt aggregation, and mitochondrial dysfunction in dopaminergic neurons lacking the RE1 Silencing Transcription Factor. Our findings identify the mechanism of action of RE1 Silencing Transcription Factor and PGC-1α as mediators of dopaminergic vulnerability in α-synuclein BAC-transgenic mice and induced pluripotent stem cell-derived dopaminergic cultures, highlighting their potential as therapeutic targets.

The causes of Parkinson’s disease (PD) likely involve complex interactions between environmental factors and susceptibility genes with variants at theGBAlocus encoding the glucocerebrosidase (GCase) enzyme being the strongest common genetic risk factor for PD. To understandGBA-related disease mechanisms, we used a novel multipart-enrichment proteomics and post-translational modification workflow to simultaneously identify peptides with phosphorylation, reversible cysteine-modifications or sialylated N-linked glycosylation, alongside unmodified proteins.We identified large numbers of dysregulated proteins and post-translational modifications (PTMs) in heterozygousGBA-N370SPD patient induced pluripotent stem cells (iPSC)-derived dopamine neurons. Alterations in glycosylation status of lysosomal proteins identified disturbances in the autophagy-lysosomal pathway, concurrent with upstream perturbations in mTOR phosphorylation and activity inGBA-N370SiPSC-dopamine neurons. In addition, the strategy revealed several native and modified proteins encoded by PD-associated genes to be dysregulated inGBA-N370Sneurons, enhancing our understanding of the wider role ofGBAmutations on the neuronal proteome. Integrated pathway analysis of all datasets revealed impaired neuritogenesis inGBA-N370SPD iPSC-dopamine neurons and identified tau (MAPT) as a key mediator of this process. Using a functional assay, we confirmed neurite outgrowth deficits inGBA-N370SPD neurons and a central role for tau in this process. Furthermore, pharmacological restoration of GCase activity inGBA-N370SPD patient neurons rescued the neurite outgrowth deficit. Overall, this study demonstrates the potential of PTMomics to elucidate novel neurodegeneration-associated pathways and identify phenotypes and potential drug targets in complex disease models.

Mutations in the PARK2 gene encoding parkin, an E3 ubiquitin ligase, are associated with autosomal recessive early-onset Parkinson’s disease (PD). While parkin has been implicated in the regulation of mitophagy and proteasomal degradation, the precise mechanism leading to neurodegeneration in both sporadic and familial PD upon parkin loss-of-function remains unknown. Cultures of isogenic induced pluripotent stem cell (iPSC) lines with and without PARK2 knockout (KO) enable mechanistic studies of the effect of parkin deficiency in human dopaminergic neurons. We used such cells to investigate the impact of PARK2 KO on the lysosomal compartment and found a clear link between parkin deficiency and lysosomal alterations. PARK2 KO neurons exhibited a perturbed lysosomal morphology with enlarged electron-lucent lysosomes and an increased lysosomal content, which was exacerbated by mitochondrial stress and could be ameliorated by antioxidant treatment. We also found decreased lysosomal enzyme activity and autophagic perturbations, suggesting an impairment of the autophagy-lysosomal pathway in parkin-deficient cells. Interestingly, activity of the GBA-encoded enzyme, β-glucocerebrosidase, was increased, suggesting the existence of a compensatory mechanism. In conclusion, our data provide a unique characterization of the morphology, content, and function of lysosomes in PARK2 KO neurons and reveal an important new connection between mitochondrial dysfunction and lysosomal dysregulation in PD pathogenesis., Innovation Fund Denmark (BrainStem; 4108–00008 A), H. Lundbeck A/S (R167-2013-15778), the Danish Parkinson Foundation (3687), the A.P. Møller Foundation for the Advancement of Medical Science (15–396, 14–427), and the Faculty of Health Sciences at University of Southern Denmark. Work at the AMS group at the CBMSO-Madrid was supported by grants from MINECO (SAF-2017-83241-R), and ISC-III RETICS TerCel (RD16/0011/0032).

Background: Although primarily a neurodegenerative process, there is increasing awareness of peripheral disease mechanisms in Parkinson's disease. To investigate disease processes in accessible patient cells, we studied peripheral blood mononuclear cells in recently diagnosed PD patients and rapid eye movement‐sleep behavior disorder patients who have a greatly increased risk of developing PD. We hypothesized that peripheral blood mononuclear cells may recapitulate cellular pathology found in the PD brain and investigated these cells for mitochondrial dysfunction and oxidative stress. Methods: Peripheral blood mononuclear cells were isolated and studied from PD patients, rapid eye movement‐sleep behavior disorder patients and age‐ and sex‐matched control individuals from the well‐characterized Oxford Discovery cohort. All participants underwent thorough clinical assessment. Results: Initial characterization showed that PD patients had elevated levels of CD14 + monocytes and monocytes expressing C‐C motif chemokine receptor 2. Mitochondrial dysfunction and oxidative stress were increased in PD patient peripheral blood mononuclear cells, with elevated levels of mitochondrial reactive oxygen species specifically in patient monocytes. This was combined with reduced levels of the antioxidant superoxide dismutase in blood cells from PD patients and, importantly, also in rapid eye movement‐sleep behavior disorder patients. This mitochondrial dysfunction was associated with a concomitant increase in glycolysis in both PD and rapid eye movement‐sleep behavior disorder patient blood cells independent of glucose uptake or monocyte activation. Conclusions: This work demonstrates functional bioenergetic deficits in PD and rapid eye movement‐sleep behavior disorder patient blood cells during the early stages of human disease. © 2018 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Neurodegenerative diseases are highly debilitating illnesses and a growing cause of morbidity and mortality worldwide. Mitochondrial dysfunction and impairment of mitochondrial-specific autophagy, namely mitophagy, have emerged as important components of the cellular processes underlying neurodegeneration. Defective mitophagy has been highlighted as the cause of the accumulation of damaged mitochondria, which consequently leads to cellular dysfunction and/or death in neurodegenerative diseases. Here, we highlight the recent advances in the molecular mechanisms of mitochondrial homeostasis and mitophagy in neurodegenerative diseases. In particular, we evaluate how mitophagy is altered in Alzheimer's, Parkinson's, and Huntington's diseases, as well as in amyotrophic lateral sclerosis, and the potential of restoring mitophagy as a therapeutic intervention. We also discuss the interlinked connections between mitophagy and innate immunity (e.g., the involvement of Parkin, interferons and TRIM21) as well as the opportunity these pathways provide to develop combinational therapeutic strategies targeting them and related molecular mechanisms in such neurodegenerative diseases.

Glucose metabolism is essential for the brain: it not only provides the required energy for cellular function and communication but also participates in balancing the levels of oxidative stress in neurons. Defects in glucose metabolism have been described in neurodegenerative disease; however, it remains unclear how this fundamental process contributes to neuronal cell death in these disorders. Here, we investigated the molecular mechanisms driving the selective neurodegeneration in an ataxic mouse model lacking oxidation resistance 1 (Oxr1) and discovered an unexpected function for this protein as a regulator of the glycolytic enzyme, glucose-6-phosphate isomerase (GPI/Gpi1). Initially, we present a dysregulation of metabolites of glucose metabolism at the pre-symptomatic stage in the Oxr1 knockout cerebellum. We then demonstrate that Oxr1 and Gpi1 physically and functionally interact and that the level of Gpi1 oligomerisation is disrupted when Oxr1 is deleted in vivo. Furthermore, we show that Oxr1 modulates the additional and less well-understood roles of Gpi1 as a cytokine and neuroprotective factor. Overall, our data identify a new molecular function for Oxr1, establishing this protein as important player in neuronal survival, regulating both oxidative stress and glucose metabolism in the brain.

Induced pluripotent stem cell (iPSC)-derived dopamine neurons provide an opportunity to model Parkinson's disease (PD), but neuronal cultures are confounded by asynchronous and heterogeneous appearance of disease phenotypes in vitro. Using high-resolution, single-cell transcriptomic analyses of iPSC-derived dopamine neurons carrying the GBA-N370S PD risk variant, we identified a progressive axis of gene expression variation leading to endoplasmic reticulum stress. Pseudotime analysis of genes differentially expressed (DE) along this axis identified the transcriptional repressor histone deacetylase 4 (HDAC4) as an upstream regulator of disease progression. HDAC4 was mislocalized to the nucleus in PD iPSC-derived dopamine neurons and repressed genes early in the disease axis, leading to late deficits in protein homeostasis. Treatment of iPSC-derived dopamine neurons with HDAC4-modulating compounds upregulated genes early in the DE axis and corrected PD-related cellular phenotypes. Our study demonstrates how single-cell transcriptomics can exploit cellular heterogeneity to reveal disease mechanisms and identify therapeutic targets.

A commentary on Parkinson disease-linked GBA mutation effects reversed by molecular chaperones in human cell and fly models by Sanchez-Martinez,A.,Beavan,M.,Gegg,M.E.,Chau,K.-Y.,Whitworth,A.J.,andSchapira,A. H. V.(2016).Sci.Rep.6:31380.doi:10.1038/srep31380

Tissue inflammation results in the production of numerous reactive oxygen, nitrogen and chlorine species, in addition to the products of lipid and sugar oxidation. Some of these products are capable of chemically modifying amino acids. This in turn results in changes to the structure and function of proteins. Increasing evidence demonstrates that such oxidative post-translational modifications result in the generation of neo-epitopes capable of eliciting both innate and adaptive immune responses. In this paper, we focus on how free radicals and related chemical species generated in inflammatory environments modulate the antigenicity of self-proteins, resulting in immune responses which involve the generation of autoantibodies against key autoantigens in autoimmune diseases. As examples, we will focus on Ro-60 and C1q in systemic lupus erythematosus, along with type-II collagen in rheumatoid arthritis. This review also covers some of the emerging literature which demonstrates that neo-epitopes generated by oxidation are conserved, as exemplified by the evolutionarily conserved pathogen-associated molecular patterns (PAMPs). We discuss how these observations relate to the pathogenesis of both human autoimmune diseases and inflammatory disease, such as atherosclerosis. The potential for these neo-epitopes and the immune responses against them to act as biomarkers or therapeutic targets is also discussed., Highlights • Oxidants can generate stable post-translational modifications (PTMs) on proteins. • Oxidative PTMs are recognised in evolutionarily-conserved innate immune responses. • These PTMs can represent neo-epitopes that break tolerance in autoimmune disease. • Antibodies targeting these PTMs in diseases e.g. RA and SLE, can be biomarkers., Graphical abstract

Parkinson's disease (PD) is a neurodegenerative disorder classically characterized by the death of dopamine (DA) neurons in the substantia nigra pars compacta and by intracellular Lewy bodies composed largely of α-synuclein. Approximately 5–10% of PD patients have a familial form of Parkinsonism, including mutations in α-synuclein. To better understand the cell-type specific role of α-synuclein on DA neurotransmission, and the effects of the disease-associated A30P mutation, we generated and studied a novel transgenic model of PD. We expressed the A30P mutant form of human α-synuclein in a spatially-relevant manner from the 111 kb SNCA genomic DNA locus on a bacterial artificial chromosome (BAC) insert on a mouse null (Snca −/−) background. The BAC transgenic mice expressed α-synuclein in tyrosine hydroxylase-positive neurons and expression of either A30P α-synuclein or wildtype α-synuclein restored the sensitivity of DA neurons to MPTP in resistant Snca −/− animals. A30P α-synuclein mice showed no Lewy body-like aggregation, and did not lose catecholamine neurons in substantia nigra or locus coeruleus. However, using cyclic voltammetry at carbon-fiber microelectrodes we identified a deficit in evoked DA release in the caudate putamen, but not in the nucleus accumbens, of SNCA-A30P Snca −/− mice but no changes to release of another catecholamine, norepinephrine (NE), in the NE-rich ventral bed nucleus of stria terminalis. SNCA-A30P Snca −/− mice had no overt behavioral impairments but exhibited a mild increase in wheel-running. In summary, this refined PD mouse model shows that A30P α-synuclein preferentially perturbs the dopaminergic system in the dorsal striatum, reflecting the region-specific change seen in PD., Highlights • SNCA A30P BAC transgenic mice recapitulate endogenous α-synuclein expression pattern • SNCA A30P BAC transgenic mice demonstrate a region specific deficit in evoked DA, but not NE, release. • Expression of A30P or WT α-syn restored the sensitivity of DA neurons to MPTP. • A30P BAC mice had no Lewy body-like aggregation or neuronal loss in SNpc or LC. • Early changes in DA neurotransmission in the absence of aggregation

Autoantibodies have been associated with human pathologies for a long time, particularly with autoimmune diseases (AIDs). Rheumatoid factor (RF) is known since the late 1930s to be associated with rheumatoid arthritis (RA). The discovery of anticitrullinated protein antibodies in the last century has changed this and other posttranslational modifications (PTM) relevant to RA have since been described. Such PTM introduce neoepitopes in proteins that can generate novel autoantibody specificities. The recent recognition of these novel specificities in RA provides a unique opportunity to understand human B-cell developmentin vivo. In this paper, we will review the three of the main classes of PTMs already associated with RA: citrullination, carbamylation, and oxidation. With the advancement of research methodologies it should be expected that other autoantibodies against PTM proteins could be discovered in patients with autoimmune diseases. Many of such autoantibodies may provide significant biomarker potential.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterised by the preferential loss of dopaminergic neurons in the substantia nigra. Mitochondrial dysfunction is increasingly appreciated as a key determinant of dopaminergic neuronal susceptibility in PD and is a feature of both familial and sporadic disease, as well as in toxin-induced Parkinsonism. Recently, the mechanisms by which PD-associated mitochondrial proteins phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-induced putative kinase 1 (PINK1) and parkin function and induce neurodegeneration have been identified. In addition, increasing evidence implicates other PD-associated proteins such as α-synuclein (α-syn) and leucine-rich repeat kinase 2 (LRRK2) in mitochondrial dysfunction in genetic cases of PD with the potential for a large functional overlap with sporadic disease. This review highlights how recent advances in understanding familial PD-associated proteins have identified novel mechanisms and therapeutic strategies for addressing mitochondrial dysfunction in PD.

Extracellular vesicles including exosomes, microvesicles and apoptotic vesicles, are phospholipid bilayer surrounded structures secreted by cells universally, in an evolutionarily conserved fashion. Posttranslational modifications such as oxidation, citrullination, phosphorylation and glycosylation play diverse roles in extracellular vesicle biology. Posttranslational modifications orchestrate the biogenesis of extracellular vesicles. The signals extracellular vesicles transmit between cells also often function via modulating posttranslational modifications of target molecules, given that extracellular vesicles are carriers of several active enzymes catalysing posttranslational modifications. Posttranslational modifications of extracellular vesicles can also contribute to disease pathology by e.g. amplifying inflammation, generating neoepitopes or carrying neoepitopes themselves.

Introduction GTP cyclohydrolase I (GTPCH) catalyses the first and rate-limiting reaction in the synthesis of the enzymatic cofactor, tetrahydrobiopterin (BH4). Loss of function mutations in the GCH1 gene lead to congenital neurological diseases such as DOPA-responsive dystonia and hyperphenylalaninemia. However, little is known about how GTPCH and BH4 affects embryonic development in utero, and in particular whether metabolic replacement or supplementation in pregnancy is sufficient to rescue genetic GTPCH deficiency in the developing embryo. Methods and results Gch1 deficient mice were generated by the insertion of loxP sites flanking exons 2–3 of the Gch1 gene. Gch1fl/fl mice were bred with Sox2cre mice to generate mice with global Gch1 deficiency. Genetic ablation of Gch1 caused embryonic lethality by E13.5. Despite loss of Gch1 mRNA and GTPCH enzymatic activity, whole embryo BH4 levels were maintained until E11.5, indicating sufficient maternal transfer of BH4 to reach this stage of development. After E11.5, Gch1−/− embryos were deficient in BH4, but an unbiased metabolomic screen indicated that the lethality was not due to a gross disturbance in metabolic profile. Embryonic lethality in Gch1−/− embryos was not caused by structural abnormalities, but was associated with significant bradycardia at E11.5. Embryonic lethality was not rescued by maternal supplementation of BH4, but was partially rescued, up to E15.5, by maternal supplementation of BH4 and l-DOPA. Conclusion These findings demonstrate a requirement for Gch1 in embryonic development and have important implications for the understanding of pathogenesis and treatment of genetic BH4 deficiencies, as well as the identification of new potential roles for BH4., Highlights • Generation and developmental analysis of Gch1 knockout mice. • Global deficiency in Gch1 is embryonically lethal between E11.5 and E13.5. • Gch1−/− embryos have no gross structural abnormalities but are bradycardic at E11.5. • Embryo lethality cannot be rescued by maternal supplementation. • There is a requirement for Gch1 and BH4 in embryo development.

There are over 120 types of autoantibodies found in the blood of SLE patients against cellular and extracellular components in both their native and posttranslationally modified forms. In recent years, these autoantibodies have provoked interest as initiators of pathology and as biomarkers of disease activity. Often, the host antigens employed in lab-based and commercially developed immunoassays use non-human antigen or non-modified host antigen as a probe for autoantibodies. Here, we describe methods to posttranslationally modify host antigens, which better represent the antigen recognized by autoantibodies in vivo. This has implications in developing immunoassay with greater sensitivity and specificity.

Parkinson disease (PD) is a multifactorial disease resulting in preferential death of the dopaminergic neurons in the substantia nigra. Studies of PD-linked genes and toxin-induced models of PD have implicated mitochondrial dysfunction, oxidative stress, and the misfolding and aggregation of α-synuclein (α-syn) as key factors in disease initiation and progression. Many of these features of PD may be modeled in cells or animal models using the neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)). Reducing oxidative stress and nitric oxide synthase (NOS) activity has been shown to be protective in cell or animal models of MPP(+) toxicity. We have previously demonstrated that siRNA-mediated knockdown of α-syn lowers the activity of both dopamine transporter and NOS activity and protects dopaminergic neuron-like cells from MPP(+) toxicity. Here, we demonstrate that α-syn knockdown and modulators of oxidative stress/NOS activation protect cells from MPP(+)-induced toxicity via postmitochondrial mechanisms rather than by a rescue of the decrease in mitochondrial oxidative phosphorylation caused by MPP(+) exposure. We demonstrate that MPP(+) significantly decreases the synthesis of the antioxidant and obligate cofactor of NOS and TH tetrahydrobiopterin (BH4) through decreased cellular GTP/ATP levels. Furthermore, we demonstrate that RNAi knockdown of α-syn results in a nearly twofold increase in GTP cyclohydrolase I activity and a concomitant increase in basal BH4 levels. Together, these results demonstrate that both mitochondrial activity and α-syn play roles in modulating cellular BH4 levels.

The pathological end-state of Parkinson disease is well described from postmortem tissue, but there remains a pressing need to define early functional changes to susceptible neurons and circuits. In particular, mechanisms underlying the vulnerability of the dopamine neurons of the substantia nigra pars compacta (SNc) and the importance of protein aggregation in driving the disease process remain to be determined. To better understand the sequence of events occurring in familial and sporadic Parkinson disease, we generated bacterial artificial chromosome transgenic mice (SNCA-OVX) that express wild-type α-synuclein from the complete human SNCA locus at disease-relevant levels and display a transgene expression profile that recapitulates that of endogenous α-synuclein. SNCA-OVX mice display age-dependent loss of nigrostriatal dopamine neurons and motor impairments characteristic of Parkinson disease. This phenotype is preceded by early deficits in dopamine release from terminals in the dorsal, but not ventral, striatum. Such neurotransmission deficits are not seen at either noradrenergic or serotoninergic terminals. Dopamine release deficits are associated with an altered distribution of vesicles in dopaminergic axons in the dorsal striatum. Aged SNCA-OVX mice exhibit reduced firing of SNc dopamine neurons in vivo measured by juxtacellular recording of neurochemically identified neurons. These progressive changes in vulnerable SNc neurons were observed independently of overt protein aggregation, suggesting neurophysiological changes precede, and are not driven by, aggregate formation. This longitudinal phenotyping strategy in SNCA-OVX mice thus provides insights into the region-specific neuronal disturbances preceding and accompanying Parkinson disease.

The breakdown of human immune tolerance to self-proteins occurs by a number of mechanisms, including posttranslational modifications of host molecules by reactive oxygen, nitrogen, or chlorine species. This has led to great interest in detecting serum autoantibodies raised against small quantities of oxidatively modified host proteins in patients with autoimmune inflammatory diseases, such as rheumatoid arthritis. Here, we provide protocols for the preparation and chemical characterization of oxidatively modified protein antigens and procedures for their use in immunoblotting and ELISAs that detect autoantibodies against these antigens in clinical samples. These gel electrophoresis- and plate reader-based immunochemical methods sometimes suffer from low analytical specificity and/or sensitivity when used for serum autoantibody detection. This is often because a single solid-phase protein (antigen) is exposed to a complex mixture of serum proteins that undergo nonspecific binding. Therefore more sensitive/specific techniques are required to detect autoantibodies specifically directed against oxidatively modified proteins. To address this, we describe novel affinity chromatography protocols by which purified autoantibodies are isolated from small volumes (

Reactive species of oxygen, nitrogen and sulfur play cell signalling roles in human health, e.g. recent studies have shown that increased dietary nitrate, which is a source of RNS (reactive nitrogen species), lowers resting blood pressure and the oxygen cost of exercise. In such studies, plasma nitrite and nitrate are readily determined by chemiluminescence. At sites of inflammation, such as the joints of RA (rheumatoid arthritis) patients, the generation of ROS (reactive oxygen species) and RNS overwhelms antioxidant defences and one consequence is oxidative/nitrative damage to proteins. For example, in the inflamed joint, increased RNS-mediated protein damage has been detected in the form of a biomarker, 3-nitrotyrosine, by immunohistochemistry, Western blotting, ELISAs and MS. In addition to NO•, another cell-signalling gas produced in the inflamed joint is H2S (hydrogen sulfide), an RSS (reactive sulfur species). This gas is generated by inflammatory induction of H2S-synthesizing enzymes. Using zinc-trap spectrophotometry, we detected high (micromolar) concentrations of H2S in RA synovial fluid and levels correlated with clinical scores of inflammation and disease activity. What might be the consequences of the inflammatory generation of reactive species? Effects on inflammatory cell-signalling pathways certainly appear to be crucial, but in the current review we highlight the concept that ROS/RNS-mediated protein damage creates neoepitopes, resulting in autoantibody formation against proteins, e.g. type-II collagen and the complement component, C1q. These autoantibodies have been detected in inflammatory autoimmune diseases.

Objective The binding of FasL (CD95L) to its receptor, Fas (CD95), induces apoptosis. Studies have shown that in patients with rheumatoid arthritis (RA), T lymphocytes are resistant to FasL-induced apoptosis in vivo but are susceptible to FasL-induced apoptosis in vitro. Dysfunction in this mechanism may be an important contributor to the pathophysiology of RA. Thus, the present study was undertaken to determine which factors might inhibit FasL–Fas binding in vivo and those that would inhibit apoptosis of T lymphocytes in an in vitro model system. Methods Human Jurkat T cells rendered apoptotic by FasL exposure were analyzed by flow cytometry. Necrosis was determined according to measurement of lactate dehydrogenase release. Quantification of calreticulin in plasma and synovial fluid and of calreticulin–FasL binding was performed by enzyme-linked immunosorbent assay. Measurement of nitrite/nitrate in the plasma and synovial fluid was carried out by chemiluminescence assay. Results Extracellular calreticulin was present at a significantly higher concentration in the plasma (median 10.3 ng/ml, interquartile range [IQR] 14.8 ng/ml) and synovial fluid (median 10.3 ng/ml, IQR 12.0 ng/ml) of RA patients (each P < 0.05) compared with the plasma (median 3.1 ng/ml, IQR 1.3 ng/ml) and synovial fluid (median 2.9 ng/ml, IQR 0.9 ng/ml) of patients with psoriatic arthritis and the plasma of healthy control subjects (median 2.9 ng/ml, IQR 0.9 ng/ml). Calreticulin concentrations in the synovial fluid correlated with the tender and swollen joint counts and the activity scores on the 28-joint Disease Activity Score assessment. Calreticulin also bound directly to FasL. In vitro, calreticulin (2–16 ng/ml) inhibited FasL-induced apoptosis of Jurkat T cells. Conclusion Calreticulin was present at higher concentrations in the plasma and synovial fluid of RA patients. Calreticulin had the capacity to bind directly to FasL and to inhibit FasL-mediated apoptosis of Jurkat T cells, and thus might play a role in inhibiting apoptosis of inflammatory T cells in RA.

Sir, We read with great interest the study titled ‘Parkinson’s disease in GTP cyclohydrolase 1 mutation carriers’ in the September edition of Brain (Mencacci et al. , 2014). The study demonstrates loss-of-function variants in the GCH1 gene are not only a major cause of DOPA-responsive dystonia but are also enriched in relatives with adult-onset parkinsonism. Furthermore, the authors identify, through exome re-sequencing, a number of GCH1 variants that are enriched in patients with Parkinson’s disease compared to control subjects. This elegant study demonstrates the power of exome re-sequencing and highlights the potential role for rare variants in genes such as GCH1 as susceptibility factors in Parkinson’s disease. A genetic role for the GCH1 locus is reinforced by the recent discovery that a SNP at the GCH1 locus is associated with Parkinson’s disease in a large-scale genome-wide association study (GWAS) meta-analysis (Nalls et al. , 2014). The coding variants identified in the exome re-sequencing study increased the risk of Parkinson’s disease by 7.5-fold (2.4–25.3, 95% confidence intervals) and the authors note that this is likely to be an underestimation of the true odds ratio, because of the method of implementing …

Mutations in parkin, encoded by the PARK2 gene, causes early-onset familial Parkinson's disease (PD), but dysfunctional parkin has also been implicated in sporadic PD. By combining human isogenic induced pluripotent stem cells (iPSCs) with and without PARK2 knockout (KO) and a novel large-scale mass spectrometry based proteomics and post-translational modification (PTM)-omics approach, we have mapped changes in protein profiles and PTMs caused by parkin deficiency in neurons. Our study identifies changes to several proteins previously shown to be dysregulated in brains of sporadic PD patients. Pathway analysis and subsequent in vitro assays reveal perturbations in migration and neurite outgrowth in the PARK2 KO neurons. We confirm the neurite defects using long-term engraftment of neurons in the striatum of immunosuppressed hemiparkinsonian adult rats. The GTP-binding protein RhoA was identified as a key upstream regulator, and RhoA activity was significantly increased in PARK2 KO neurons. By inhibiting RhoA signalling the migration and neurite outgrowth phenotypes could be rescued. Our study provides new insight into the pathogenesis of PD and demonstrates the broadly applicable potential of proteomics and PTMomics for elucidating the role of disease-causing mutations.