Zell-zu-Zell-Propagation von Alpha-Synuclein in Bezug auf Morbus Parkinson - Folgen von extrazellulärem Alpha-Synuclein für die Empfängerzelle

Aggregation of the protein alpha synuclein (aSyn) and the resulting neurotoxicity play a crucial role in Parkinson’s disease (PD). Findings of recent studies strongly support the hypothesis that the progression of aSyn from one cell to adjacent cells contribute to the progression of PD. According to this hypothesis, the progression of aSyn includes important steps, like the release of this intracellular protein into the extracellular space, the subsequent uptake in neighboring cells, and the potential to trigger the aggregation of endogenous aSyn in recipient cells. However, in order to interact with intracellular pathways and to enable the following propagation of pathogenic aSyn species, the uptaken aSyn must be able to escape from the protein degradation mechanisms of the recipient cell. Therefore, the uptake and the subsequent processing of extracellular aSyn within the recipient cell are crucial for the propagation of aSyn pathology (aggregation and neurotoxicity). It is essential to understand the mechanisms of the propagation of aSyn, in order to find new therapeutic approaches. This study focused on the uptake and deposition of extracellular aSyn, the following trafficking in recipient cells, as well as the potential effect of exogenous aSyn on recipient cells. For these purposes, H4 neuroglioma cells and rat primary hippocampal neurons were used as recipient cell models. To analyze the aggregation-dependent effects of extracellular aSyn on recipient cells, aSyn in different aggregation states (monomers, oligomers, and fibrils) was applied to cells. All analyzed aSyn species showed a concentration and time dependent uptake pattern in recipient cells. Notably, the uptake of aggregated species (aSyn oligomers and fibrils) occurred more preferentially than monomeric aSyn. This effect was also commonly observed in other cell types. Moreover, aggregated aSyn accumulated in a time dependent manner in recipient cells and was capable of triggering the aggregation of endogenous aSyn. The uptake of extracellular aSyn occurred partly over clathrin dependent endocytosis, and the protein interacted subsequently with endosomes and lysosomes. In addition, part of exogenous aSyn was found in the cytoplasm, suggesting that aSyn partly escape from the endosomal/lysosomal system. In fact, the result from a more in-depth analysis suggested that exogenous aSyn is able to escape from endosomal vesicles through the translocon Sec61. Furthermore, the study provided evidence that the uptaken aSyn interacts with mitochondria, the ubiquitin-proteasome system (UPS), and the autophagy lysosomal pathway (ALP). However, the lysosomal morphology and function were particularly affected after the application of extracellular aSyn. Specifically, lysosomal expansion and accumulation were observed, accompanied by a reduced protein degradation capacity of lysosomes. Moreover, the autophagosomal lysosomal flux was disturbed. Importantly, lysosomal impairments were practically detected in cells exposed to aggregated aSyn species. In contrast to the disturbed lysosomal activity, the endocytosis activity, the proteasomal function and mitochondrial homeostasis of aSyn-exposed cells were not significantly affected. These results suggest that lysosomal impairments induced by extracellular, aggregated aSyn are early pathological events. The devastating lysosomal dysfunction and accumulation of exogenous aSyn can be prevented through a pretreatment of trehalose (a disaccharide molecule to activate autophagy). Taken together, extracellular aggregated aSyn is more preferentially internalized into recipient cells and exhibits a higher potential to impair lysosomes than monomeric aSyn. The decreased protein clearance capacity of recipient cells promotes in turn the deposition of extracellular pathogenic species and the formation of new pathogenic species though aggregation of endogenous aSyn. This study reveals the importance of lysosomes and lysosome-related autophagy in the propagation of aSyn pathology and suggests these pathways as potential therapeutic targets in PD.Die Aggregation des Proteins Alpha-Synuclein (aSyn) und die daraus resultierende Neurotoxizität spielen eine entscheidende Rolle in der Parkinsonerkrankung (parkinson disease – PD). Neuste Studien unterstützen die Hypothese, dass eine Weitergabe des aSyn von einer Zelle zur Nachbarzelle zu der Progression der PD beiträgt. Nach dieser Hypothese beinhaltet die aSyn-Ausbreitung wichtige Schritte, wie die Freisetzung des intrazellulären Proteins in den extrazellulären Raum, dessen Aufnahme in die benachbarte Zelle, sowie das Potential die Anregung der Aggregation von endogenem aSyn in der Empfängerzelle auszulösen. Damit jedoch eine Interaktion mit den intrazellulären Strukturen und eine anschließende Verbreitung des pathogenen aSyn möglich ist, muss das aufgenommene aSyn dazu in der Lage sein, den Proteinabbaumechanismen der Zelle zu entkommen. Die Aufnahme von extrazellulärem aSyn in die Empfängerzellen und die anschließende Prozessierung haben daher für die aSyn-Pathologie (Aggregation und Neurotoxizität) eine äußerst wichtige Bedeutung. Es ist unerlässlich den Mechanismus zu verstehen, welcher der Ausbreitung von aSyn zugrunde liegt, um neue therapeutische Ansätze zu entwickeln. In dieser Arbeit wurde die Aufnahme sowie die Ablagerung von extrazellulärem aSyn in den Empfängerzellen, das anschließende Verhalten dort, sowie die möglichen Auswirkungen auf die Empfängerzelle untersucht. Um das aufgenommene aSyn in den Empfängerzellen zu verfolgen wurden H4-Neuroglioma Zellen und primäre rattenartige Neuronen als Modellsystemzellen verwendet. Um mögliche aggregationsabhängige Effekte von extrazellulärem aSyn auf die Empfängerzelle zu untersuchen, wurden die Zellen mit aSyn in unterschiedlichen Aggregationszuständen (Monomere, Oligomere und Fibrillen) behandelt. Alle untersuchten aSyn-Spezies wurden konzentrations- und zeitabhängig von den Empfängerzellen aufgenommen. Interessanterweise fand die Aufnahme von aggregierten Spezies (aSyn-Oligomere und Fibrillen) bevorzugt statt. Dieser Effekt konnte auch in weiteren Zelltypen beobachtet werden. Hauptsächlich besaß aggregiertes aSyn die Fähigkeit, sich mit der Zeit in den Empfängerzellen abzulagern und darüber hinaus auch die Aggregation von endogenem aSyn anzuregen. Die Aufnahme von extrazellulärem aSyn in die Zelle fand teilweise über clathrinabhängige Endozytose statt und interagierte dann mit den Endosomen und Lysosomen der Zellen. Ein Teil des aSyn lag auch frei im Zytoplasma vor, was bedeutet, dass das aSyn dem endosomalen/lysosomalen Weg entkommt. Weitere Untersuchungen wiesen tatsächlich darauf hin, dass exogenes aSyn die endosomalen Vesikel über das Translokon Sec61 verlassen kann. Des Weiteren interagierte das aSyn auch mit den Mitochondrien der Zellen, sowie mit dem Ubiquitin-Proteasom-System (UPS) und der Autophagiemachinerie. Es zeigten sich nach der Applikation von extrazellulärem aSyn vor allem Veränderungen in der lysosomalen Morphologie und Funktion. Es konnte eine lysosomale Vergrößerung und Akkumulation, welche mit einer reduzierten lysosomalen Abbauaktivität einhergeht, festgestellt werden. Darüber hinaus, wurde auch ein gestörter autophagolysosomaler Fluss beobachtet. Die beobachteten lysosomalen Störungen waren besonders in den Zellen, die den aggregierten aSyn-Spezies ausgesetzt waren, auffällig. Im Gegensatz zu der gestörten lysosomalen Aktivität zeigten sich keine großen Veränderungen in der Endozytose-Aktivität, der proteasomalen Funktion, sowie der mitochondrialen Homöostase. Diese Ergebnisse deuten darauf hin, dass es sich bei den lysosomalem Veränderungen, welche durch extrazelluläres, aggregiertes aSyn hervorrufen werden können, um ein frühes pathologisches Ereignis handelt. Die massiven Störungen der Lysosomen, sowie die Akkumulation von exogenem aSyn konnten durch eine zusätzliche Behandlung mit Trehalose (ein Disaccharid, welches die Autophagie aktiviert) verringert werden. Zusammengefasst wurde extrazelluläres, aggregiertes aSyn bevorzugt von den Zellen internalisiert. Dort inhibierte es die lysosomale Aktivität, wodurch eine Akkumulation und eine weitere Bildung von pathogenem aSyn in den Empfängerzellen begünstigt wurde. Diese Studie unterstreicht deutlich eine wichtige Rolle der Lyososmen und der lysosomenassoziierten Autophagie in der Propagation der aSyn-Pathologie und deutet damit auf Lysosomen als potentielle therapeutische Angriffsziele gegen PD hin

Posttranslational modification and mutation of histidine 50 trigger alpha synuclein aggregation and toxicity

Background Aggregation and aggregation-mediated formation of toxic alpha synuclein (aSyn) species have been linked to the pathogenesis of sporadic and monogenic Parkinson’s disease (PD). A novel H50Q mutation of aSyn, resulting in the substitution of histidine by glutamine, has recently been identified in PD patients. We have previously shown that the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) induces the formation of HNE-aSyn adducts, thereby promoting aSyn oligomerization and increasing its extracellular toxicity to human dopaminergic neurons. Intriguingly, we identified histidine 50 (H50) of aSyn as one of the HNE modification target residues. These converging lines of evidence support the hypothesis that changes in H50 via posttranslational modification (PTM) and mutation trigger the formation of aggregated, toxic aSyn species, which interfere with cellular homeostasis. In the present study, we aim to elucidate 1) the role of H50 in HNE-mediated aSyn aggregation and toxicity, and 2) the impact of H50 mutation on aSyn pathology. Besides the PD-related H50Q, we analyze a PD-unrelated control mutation, in which H50 is replaced by an arginine residue (H50R). Results Analysis of HNE-treated aSyn revealed that H50 is the most susceptible residue of aSyn to HNE modification and is crucial for HNE-mediated aSyn oligomerization. Overexpression of aSyn with substituted H50 in H4 neuroglioma cells reduced HNE-induced cell damage, indicating a pivotal role of H50 in HNE modification-induced aSyn toxicity. Furthermore, we showed in vitro that H50Q/R mutations substantially increase the formation of high density and fibrillar aSyn species, and potentiate the oligomerization propensity of aSyn in the presence of a nitrating agent. Cell-based experiments also revealed that overexpression of H50Q aSyn in H4 cells promotes aSyn oligomerization. Importantly, overexpression of both H50Q/R aSyn mutants in H4 cells significantly increased cell death when compared to wild type aSyn. This increase in cell death was further exacerbated by the application of H2O2. Conclusion A dual approach addressing alterations of H50 showed that either H50 PTM or mutation trigger aSyn aggregation and toxicity, suggesting an important role of aSyn H50 in the pathogenesis of both sporadic and monogenic PD

Contribution of copy number variants to schizophrenia from a genome-wide study of 41,321 subjects

Copy number variants (CNVs) have been strongly implicated in the genetic etiology of schizophrenia (SCZ). However, genome-wide investigation of the contribution of CNV to risk has been hampered by limited sample sizes. We sought to address this obstacle by applying a centralized analysis pipeline to a SCZ cohort of 21,094 cases and 20,227 controls. A global enrichment of CNV burden was observed in cases (OR=1.11, P=5.7×10−15), which persisted after excluding loci implicated in previous studies (OR=1.07, P=1.7 ×10−6). CNV burden was enriched for genes associated with synaptic function (OR = 1.68, P = 2.8 ×10−11) and neurobehavioral phenotypes in mouse (OR = 1.18, P= 7.3 ×10−5). Genome-wide significant evidence was obtained for eight loci, including 1q21.1, 2p16.3 (NRXN1), 3q29, 7q11.2, 15q13.3, distal 16p11.2, proximal 16p11.2 and 22q11.2. Suggestive support was found for eight additional candidate susceptibility and protective loci, which consisted predominantly of CNVs mediated by non-allelic homologous recombination

No Reliable Association between Runs of Homozygosity and Schizophrenia in a Well-Powered Replication Study

It is well known that inbreeding increases the risk of recessive monogenic diseases, but it is less certain whether it contributes to the etiology of complex diseases such as schizophrenia. One way to estimate the effects of inbreeding is to examine the association between disease diagnosis and genome-wide autozygosity estimated using runs of homozygosity (ROH) in genome-wide single nucleotide polymorphism arrays. Using data for schizophrenia from the Psychiatric Genomics Consortium (n = 21,868), Keller et al. (2012) estimated that the odds of developing schizophrenia increased by approximately 17% for every additional percent of the genome that is autozygous (β = 16.1, CI(β) = [6.93, 25.7], Z = 3.44, p = 0.0006). Here we describe replication results from 22 independent schizophrenia case-control datasets from the Psychiatric Genomics Consortium (n = 39,830). Using the same ROH calling thresholds and procedures as Keller et al. (2012), we were unable to replicate the significant association between ROH burden and schizophrenia in the independent PGC phase II data, although the effect was in the predicted direction, and the combined (original + replication) dataset yielded an attenuated but significant relationship between Froh and schizophrenia (β = 4.86,CI(β) = [0.90,8.83],Z = 2.40,p = 0.02). Since Keller et al. (2012), several studies reported inconsistent association of ROH burden with complex traits, particularly in case-control data. These conflicting results might suggest that the effects of autozygosity are confounded by various factors, such as socioeconomic status, education, urbanicity, and religiosity, which may be associated with both real inbreeding and the outcome measures of interest

Gene expression imputation across multiple brain regions provides insights into schizophrenia risk

Transcriptomic imputation approaches combine eQTL reference panels with large-scale genotype data in order to test associations between disease and gene expression. These genic associations could elucidate signals in complex genome-wide association study (GWAS) loci and may disentangle the role of different tissues in disease development. We used the largest eQTL reference panel for the dorso-lateral prefrontal cortex (DLPFC) to create a set of gene expression predictors and demonstrate their utility. We applied DLPFC and 12 GTEx-brain predictors to 40,299 schizophrenia cases and 65,264 matched controls for a large transcriptomic imputation study of schizophrenia. We identified 413 genic associations across 13 brain regions. Stepwise conditioning identified 67 non-MHC genes, of which 14 did not fall within previous GWAS loci. We identified 36 significantly enriched pathways, including hexosaminidase-A deficiency, and multiple porphyric disorder pathways. We investigated developmental expression patterns among the 67 non-MHC genes and identified specific groups of pre- and postnatal expression

Genetic correlation between amyotrophic lateral sclerosis and schizophrenia

A. Palotie on työryhmän Schizophrenia Working Grp Psychiat jäsen.We have previously shown higher-than-expected rates of schizophrenia in relatives of patients with amyotrophic lateral sclerosis (ALS), suggesting an aetiological relationship between the diseases. Here, we investigate the genetic relationship between ALS and schizophrenia using genome-wide association study data from over 100,000 unique individuals. Using linkage disequilibrium score regression, we estimate the genetic correlation between ALS and schizophrenia to be 14.3% (7.05-21.6; P = 1 x 10(-4)) with schizophrenia polygenic risk scores explaining up to 0.12% of the variance in ALS (P = 8.4 x 10(-7)). A modest increase in comorbidity of ALS and schizophrenia is expected given these findings (odds ratio 1.08-1.26) but this would require very large studies to observe epidemiologically. We identify five potential novel ALS-associated loci using conditional false discovery rate analysis. It is likely that shared neurobiological mechanisms between these two disorders will engender novel hypotheses in future preclinical and clinical studies.Peer reviewe

Estimation of Genetic Correlation via Linkage Disequilibrium Score Regression and Genomic Restricted Maximum Likelihood

J. Lönnqvist on työryhmän Psychiat Genomics Consortium jäsen.Genetic correlation is a key population parameter that describes the shared genetic architecture of complex traits and diseases. It can be estimated by current state-of-art methods, i.e., linkage disequilibrium score regression (LDSC) and genomic restricted maximum likelihood (GREML). The massively reduced computing burden of LDSC compared to GREML makes it an attractive tool, although the accuracy (i.e., magnitude of standard errors) of LDSC estimates has not been thoroughly studied. In simulation, we show that the accuracy of GREML is generally higher than that of LDSC. When there is genetic heterogeneity between the actual sample and reference data from which LD scores are estimated, the accuracy of LDSC decreases further. In real data analyses estimating the genetic correlation between schizophrenia (SCZ) and body mass index, we show that GREML estimates based on similar to 150,000 individuals give a higher accuracy than LDSC estimates based on similar to 400,000 individuals (from combinedmeta-data). A GREML genomic partitioning analysis reveals that the genetic correlation between SCZ and height is significantly negative for regulatory regions, which whole genome or LDSC approach has less power to detect. We conclude that LDSC estimates should be carefully interpreted as there can be uncertainty about homogeneity among combined meta-datasets. We suggest that any interesting findings from massive LDSC analysis for a large number of complex traits should be followed up, where possible, with more detailed analyses with GREML methods, even if sample sizes are lesser.Peer reviewe

Schizophrenia-associated somatic copy-number variants from 12,834 cases reveal recurrent NRXN1 and ABCB11 disruptions

While germline copy-number variants (CNVs) contribute to schizophrenia (SCZ) risk, the contribution of somatic CNVs (sCNVs)—present in some but not all cells—remains unknown. We identified sCNVs using blood-derived genotype arrays from 12,834 SCZ cases and 11,648 controls, filtering sCNVs at loci recurrently mutated in clonal blood disorders. Likely early-developmental sCNVs were more common in cases (0.91%) than controls (0.51%, p = 2.68e−4), with recurrent somatic deletions of exons 1–5 of the NRXN1 gene in five SCZ cases. Hi-C maps revealed ectopic, allele-specific loops forming between a potential cryptic promoter and non-coding cis-regulatory elements upon 5′ deletions in NRXN1. We also observed recurrent intragenic deletions of ABCB11, encoding a transporter implicated in anti-psychotic response, in five treatment-resistant SCZ cases and showed that ABCB11 is specifically enriched in neurons forming mesocortical and mesolimbic dopaminergic projections. Our results indicate potential roles of sCNVs in SCZ risk

Interaction Testing and Polygenic Risk Scoring to Estimate the Association of Common Genetic Variants with Treatment Resistance in Schizophrenia

Importance: About 20% to 30% of people with schizophrenia have psychotic symptoms that do not respond adequately to first-line antipsychotic treatment. This clinical presentation, chronic and highly disabling, is known as treatment-resistant schizophrenia (TRS). The causes of treatment resistance and their relationships with causes underlying schizophrenia are largely unknown. Adequately powered genetic studies of TRS are scarce because of the difficulty in collecting data from well-characterized TRS cohorts. Objective: To examine the genetic architecture of TRS through the reassessment of genetic data from schizophrenia studies and its validation in carefully ascertained clinical samples. Design, Setting, and Participants: Two case-control genome-wide association studies (GWASs) of schizophrenia were performed in which the case samples were defined as individuals with TRS (n = 10501) and individuals with non-TRS (n = 20325). The differences in effect sizes for allelic associations were then determined between both studies, the reasoning being such differences reflect treatment resistance instead of schizophrenia. Genotype data were retrieved from the CLOZUK and Psychiatric Genomics Consortium (PGC) schizophrenia studies. The output was validated using polygenic risk score (PRS) profiling of 2 independent schizophrenia cohorts with TRS and non-TRS: a prevalence sample with 817 individuals (Cardiff Cognition in Schizophrenia [CardiffCOGS]) and an incidence sample with 563 individuals (Genetics Workstream of the Schizophrenia Treatment Resistance and Therapeutic Advances [STRATA-G]). Main Outcomes and Measures: GWAS of treatment resistance in schizophrenia. The results of the GWAS were compared with complex polygenic traits through a genetic correlation approach and were used for PRS analysis on the independent validation cohorts using the same TRS definition. Results: The study included a total of 85490 participants (48635 [56.9%] male) in its GWAS stage and 1380 participants (859 [62.2%] male) in its PRS validation stage. Treatment resistance in schizophrenia emerged as a polygenic trait with detectable heritability (1% to 4%), and several traits related to intelligence and cognition were found to be genetically correlated with it (genetic correlation, 0.41-0.69). PRS analysis in the CardiffCOGS prevalence sample showed a positive association between TRS and a history of taking clozapine (r2 = 2.03%; P =.001), which was replicated in the STRATA-G incidence sample (r2 = 1.09%; P =.04). Conclusions and Relevance: In this GWAS, common genetic variants were differentially associated with TRS, and these associations may have been obscured through the amalgamation of large GWAS samples in previous studies of broadly defined schizophrenia. Findings of this study suggest the validity of meta-analytic approaches for studies on patient outcomes, including treatment resistance

Genomic Dissection of Bipolar Disorder and Schizophrenia, Including 28 Subphenotypes

publisher: Elsevier articletitle: Genomic Dissection of Bipolar Disorder and Schizophrenia, Including 28 Subphenotypes journaltitle: Cell articlelink: https://doi.org/10.1016/j.cell.2018.05.046 content_type: article copyright: © 2018 Elsevier Inc