Mitochondrial Dysfunction and Mitophagy in Parkinson's Disease: From Mechanism to Therapy.

Mitochondrial dysfunction has been associated with neurodegeneration in Parkinson's disease (PD) for over 30 years. Despite this, the role of mitochondrial dysfunction as an initiator, propagator, or bystander remains undetermined. The discovery of the role of the PD familial genes PTEN-induced putative kinase 1 (PINK1) and parkin (PRKN) in mediating mitochondrial degradation (mitophagy) reaffirmed the importance of this process in PD aetiology. Recently, progress has been made in understanding the upstream and downstream regulators of canonical PINK1/parkin-mediated mitophagy, alongside noncanonical PINK1/parkin mitophagy, in response to mitochondrial damage. Progress has also been made in understanding the role of PD-associated genes, such as SNCA, LRRK2, and CHCHD2, in mitochondrial dysfunction and their overlap with sporadic PD (sPD), opening opportunities for therapeutically targeting mitochondria in PD.

Mitochondrial dysfunction and mitophagy defects in LRRK2-R1441C Parkinson's disease models.

Mutations in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene have been identified as one of the most common genetic causes of Parkinson's disease (PD). The LRRK2 PD-associated mutations LRRK2G2019S and LRRK2R1441C, located in the kinase domain and in the ROC-COR domain, respectively, have been demonstrated to impair mitochondrial function. Here, we sought to further our understanding of mitochondrial health and mitophagy by integrating data from LRRK2R1441C rat primary cortical and human induced pluripotent stem cell-derived dopamine (iPSC-DA) neuronal cultures as models of PD. We found that LRRK2R1441C neurons exhibit decreased mitochondrial membrane potential, impaired mitochondrial function and decreased basal mitophagy levels. Mitochondrial morphology was altered in LRRK2R1441C iPSC-DA but not in cortical neuronal cultures or aged striatal tissue, indicating a cell-type-specific phenotype. Additionally, LRRK2R1441C but not LRRK2G2019S neurons demonstrated decreased levels of the mitophagy marker pS65Ub in response to mitochondrial damage, which could disrupt degradation of damaged mitochondria. This impaired mitophagy activation and mitochondrial function were not corrected by the LRRK2 inhibitor MLi-2 in LRRK2R1441C iPSC-DA neuronal cultures. Furthermore, we demonstrate LRRK2 interaction with MIRO1, a protein necessary to stabilize and to anchor mitochondria for transport, occurs at mitochondria, in a genotype-independent manner. Despite this, we found that degradation of MIRO1 was impaired in LRRK2R1441C cultures upon induced mitochondrial damage, suggesting a divergent mechanism from the LRRK2G2019S mutation.

CRISPRi: a way to integrate iPSC-derived neuronal models.

The genetic landscape of neurodegenerative diseases encompasses genes affecting multiple cellular pathways which exert effects in an array of neuronal and glial cell-types. Deconvolution of the roles of genes implicated in disease and the effects of disease-associated variants remains a vital step in the understanding of neurodegeneration and the development of therapeutics. Disease modelling using patient induced pluripotent stem cells (iPSCs) has enabled the generation of key cell-types associated with disease whilst maintaining the genomic variants that predispose to neurodegeneration. The use of CRISPR interference (CRISPRi), alongside other CRISPR-perturbations, allows the modelling of the effects of these disease-associated variants or identifying genes which modify disease phenotypes. This review summarises the current applications of CRISPRi in iPSC-derived neuronal models, such as fluorescence-activated cell sorting (FACS)-based screens, and discusses the future opportunities for disease modelling, identification of disease risk modifiers and target/drug discovery in neurodegeneration.

REST Protects Dopaminergic Neurons from Mitochondrial and α-Synuclein Oligomer Pathology in an Alpha Synuclein Overexpressing BAC-Transgenic Mouse Model.

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 the SNCA-Triplication mutation, which accumulated α-synuclein oligomers and mitochondrial damage, and displayed REST target gene dysregulation. Furthermore, CRISPR-mediated REST KO induced mitochondrial dysfunction and impaired mitophagy in 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 STATEMENT Understanding 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.

Cellular α-synuclein pathology is associated with bioenergetic dysfunction in Parkinson's iPSC-derived dopamine neurons.

Parkinson's disease (PD) is the second most common neurodegenerative disorder and a central role for α-synuclein (αSyn; SNCA) in disease aetiology has been proposed based on genetics and neuropathology. To better understand the pathological mechanisms of αSyn, we generated induced pluripotent stem cells (iPSCs) from healthy individuals and PD patients carrying the A53T SNCA mutation or a triplication of the SNCA locus and differentiated them into dopaminergic neurons (DAns). iPSC-derived DAn from PD patients carrying either mutation showed increased intracellular αSyn accumulation, and DAns from patients carrying the SNCA triplication displayed oligomeric αSyn pathology and elevated αSyn extracellular release. Transcriptomic analysis of purified DAns revealed perturbations in expression of genes linked to mitochondrial function, consistent with observed reduction in mitochondrial respiration, impairment in mitochondrial membrane potential, aberrant mitochondrial morphology and decreased levels of phosphorylated DRP1Ser616. Parkinson's iPSC-derived DAns showed increased endoplasmic reticulum stress and impairments in cholesterol and lipid homeostasis. Together, these data show a correlation between αSyn cellular pathology and deficits in metabolic and cellular bioenergetics in the pathology of PD.

Mitochondrial dysfunction and mitophagy in Parkinson's: from familial to sporadic disease.

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.

Perturbations in RhoA signalling cause altered migration and impaired neuritogenesis in human iPSC-derived neural cells with PARK2 mutation.

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.

Mitochondrial dysfunction and increased glycolysis in prodromal and early Parkinson's blood cells.

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.

Oxidative and other posttranslational modifications in extracellular vesicle biology.

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.

A requirement for Gch1 and tetrahydrobiopterin in embryonic development.

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. Gch1(fl/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.

Deficits in dopaminergic transmission precede neuron loss and dysfunction in a new Parkinson model.

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.

Region-specific deficits in dopamine, but not norepinephrine, signaling in a novel A30P α-synuclein BAC transgenic mouse.

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 111kb 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.

Autoantibodies to posttranslational modifications in rheumatoid arthritis.

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 development in 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.

Multiparameter phenotypic screening for endogenous TFEB and TFE3 translocation identifies novel chemical series modulating lysosome function.

The accumulation of toxic protein aggregates in multiple neurodegenerative diseases is associated with defects in the macroautophagy/autophagy-lysosome pathway. The amelioration of disease phenotypes across multiple models of neurodegeneration can be achieved through modulating the master regulator of lysosome function, TFEB (transcription factor EB). Using a novel multi-parameter high-throughput screen for cytoplasmic:nuclear translocation of endogenous TFEB and the related transcription factor TFE3, we screened the Published Kinase Inhibitor Set 2 (PKIS2) library as proof of principle and to identify kinase regulators of TFEB and TFE3. Given that TFEB and TFE3 are responsive to cellular stress we have established assays for cellular toxicity and lysosomal function, critical to ensuring the identification of hit compounds with only positive effects on lysosome activity. In addition to AKT inhibitors which regulate TFEB localization, we identified a series of quinazoline-derivative compounds that induced TFEB and TFE3 translocation. A novel series of structurally-related analogs was developed, and several compounds induced TFEB and TFE3 translocation at higher potency than previously screened compounds. KINOMEscan and cell-based KiNativ kinase profiling revealed high binding for the PRKD (protein kinase D) family of kinases, suggesting good selectivity for these compounds. We describe and utilize a cellular target-validation platform using CRISPRi knockdown and orthogonal PRKD inhibitors to demonstrate that the activity of these compounds is independent of PRKD inhibition. The more potent analogs induced subsequent upregulation of the CLEAR gene network and cleared pathological HTT protein in a cellular model of proteinopathy, demonstrating their potential to alleviate neurodegeneration-relevant phenotypes. Abbreviations: AD: Alzheimer disease; AK: adenylate kinase; CLEAR: coordinated lysosomal expression and regulation; CQ: chloroquine; HD: Huntington disease; PD: Parkinson disease; PKIS2: Published Kinase Inhibitor Set 2; PRKD: protein kinase D; TFEB: transcription factor EB.

Multi-modal Proteomic Characterization of Lysosomal Function and Proteostasis in Progranulin-Deficient Neurons.

Progranulin (PGRN) is a lysosomal protein implicated in various neurodegenerative diseases. Over 70 mutations discovered in the GRN gene all result in reduced expression of PGRN protein. However, the detailed molecular function of PGRN within lysosomes and the impact of PGRN deficiency on lysosomal biology remain unclear. Here we leveraged multifaceted proteomic techniques to comprehensively characterize how PGRN deficiency changes the molecular and functional landscape of neuronal lysosomes. Using lysosome proximity labeling and immuno-purification of intact lysosomes, we characterized lysosome compositions and interactomes in both human induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (i3Neurons) and mouse brains. Using dynamic stable isotope labeling by amino acids in cell culture (dSILAC) proteomics, we measured global protein half-lives in i3Neurons for the first time and characterized the impact of progranulin deficiency on neuronal proteostasis. Together, this study indicated that PGRN loss impairs the lysosome's degradative capacity with increased levels of v-ATPase subunits on the lysosome membrane, increased catabolic enzymes within the lysosome, elevated lysosomal pH, and pronounced alterations in neuron protein turnover. Collectively, these results suggested PGRN as a critical regulator of lysosomal pH and degradative capacity, which in turn influences global proteostasis in neurons. The multi-modal techniques developed here also provided useful data resources and tools to study the highly dynamic lysosome biology in neurons.

Multi-modal proteomic characterization of lysosomal function and proteostasis in progranulin-deficient neurons.

BACKGROUND: Progranulin (PGRN) is a lysosomal glycoprotein implicated in various neurodegenerative diseases, including frontotemporal dementia and neuronal ceroid lipofuscinosis. Over 70 mutations discovered in the GRN gene all result in reduced expression of the PGRN protein. Genetic and functional studies point toward a regulatory role for PGRN in lysosome functions. However, the detailed molecular function of PGRN within lysosomes and the impact of PGRN deficiency on lysosomes remain unclear. METHODS: We developed multifaceted proteomic techniques to characterize the dynamic lysosomal biology in living human neurons and fixed mouse brain tissues. Using lysosome proximity labeling and immuno-purification of intact lysosomes, we characterized lysosome compositions and interactome in both human induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (i3Neurons) and mouse brains. Using dynamic stable isotope labeling by amino acids in cell culture (dSILAC) proteomics, we measured global protein half-lives in human i3Neurons for the first time. RESULTS: Leveraging the multi-modal proteomics and live-cell imaging techniques, we comprehensively characterized how PGRN deficiency changes the molecular and functional landscape of neuronal lysosomes. We found that PGRN loss impairs the lysosome's degradative capacity with increased levels of v-ATPase subunits on the lysosome membrane, increased hydrolases within the lysosome, altered protein regulations related to lysosomal transport, and elevated lysosomal pH. Consistent with impairments in lysosomal function, GRN-null i3Neurons and frontotemporal dementia patient-derived i3Neurons carrying GRN mutation showed pronounced alterations in protein turnover, such as cathepsins and proteins related to supramolecular polymerization and inherited neurodegenerative diseases. CONCLUSION: This study suggested PGRN as a critical regulator of lysosomal pH and degradative capacity, which influences global proteostasis in neurons. Beyond the study of progranulin deficiency, these newly developed proteomic methods in neurons and brain tissues provided useful tools and data resources for the field to study the highly dynamic neuronal lysosome biology.

Identification of a possible proteomic biomarker in Parkinson's disease: discovery and replication in blood, brain and cerebrospinal fluid.

Biomarkers to aid diagnosis and delineate the progression of Parkinson's disease are vital for targeting treatment in the early phases of the disease. Here, we aim to discover a multi-protein panel representative of Parkinson's and make mechanistic inferences from protein expression profiles within the broader objective of finding novel biomarkers. We used aptamer-based technology (SomaLogic®) to measure proteins in 1599 serum samples, 85 cerebrospinal fluid samples and 37 brain tissue samples collected from two observational longitudinal cohorts (the Oxford Parkinson's Disease Centre and Tracking Parkinson's) and the Parkinson's Disease Brain Bank, respectively. Random forest machine learning was performed to discover new proteins related to disease status and generate multi-protein expression signatures with potential novel biomarkers. Differential regulation analysis and pathway analysis were performed to identify functional and mechanistic disease associations. The most consistent diagnostic classifier signature was tested across modalities [cerebrospinal fluid (area under curve) = 0.74, P = 0.0009; brain area under curve = 0.75, P = 0.006; serum area under curve = 0.66, P = 0.0002]. Focusing on serum samples and using only those with severe disease compared with controls increased the area under curve to 0.72 (P = 1.0 × 10-4). In the validation data set, we showed that the same classifiers were significantly related to disease status (P < 0.001). Differential expression analysis and weighted gene correlation network analysis highlighted key proteins and pathways with known relationships to Parkinson's. Proteins from the complement and coagulation cascades suggest a disease relationship to immune response. The combined analytical approaches in a relatively large number of samples, across tissue types, with replication and validation, provide mechanistic insights into the disease as well as nominate a protein signature classifier that deserves further biomarker evaluation.

Post-translational proteomics platform identifies neurite outgrowth impairments in Parkinson's disease GBA-N370S dopamine neurons.

Variants at the GBA locus, encoding glucocerebrosidase, are the strongest common genetic risk factor for Parkinson's disease (PD). To understand GBA-related disease mechanisms, we use a multi-part-enrichment proteomics and post-translational modification (PTM) workflow, identifying large numbers of dysregulated proteins and PTMs in heterozygous GBA-N370S PD patient induced pluripotent stem cell (iPSC) dopamine neurons. Alterations in glycosylation status show disturbances in the autophagy-lysosomal pathway, which concur with upstream perturbations in mammalian target of rapamycin (mTOR) activation in GBA-PD neurons. Several native and modified proteins encoded by PD-associated genes are dysregulated in GBA-PD neurons. Integrated pathway analysis reveals impaired neuritogenesis in GBA-PD neurons and identify tau as a key pathway mediator. Functional assays confirm neurite outgrowth deficits and identify impaired mitochondrial movement in GBA-PD neurons. Furthermore, pharmacological rescue of glucocerebrosidase activity in GBA-PD neurons improves the neurite outgrowth deficit. Overall, this study demonstrates the potential of PTMomics to elucidate neurodegeneration-associated pathways and potential drug targets in complex disease models.

Measurement and meaning of markers of reactive species of oxygen, nitrogen and sulfur in healthy human subjects and patients with inflammatory joint disease.

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.

Extracellular calreticulin is present in the joints of patients with rheumatoid arthritis and inhibits FasL (CD95L)-mediated apoptosis of T cells.

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.