Ancient Origin of the Parkinson Disease Gene LRRK2

Dominant mutations in the LRRK2 gene, a member of the Roco family, cause both familial and sporadic Parkinson disease. LRRK genes had so far been detected only in bilaterian animals. In deuterostomes, including humans, two LRRK genes (LRRK1 and LRRK2) exist, while in protostomes a single LRRK gene has been found. In this study, I combine structural and phylogenetic analyses to show that the cnidarian Nematostella vectensis has four LRRK genes. One of them is a bona fide orthologue of the human LRRK2 gene, demonstrating that this gene has an ancient origin. Two others are, respectively, orthologues of the deuterostome LRRK1 and the protostome LRRK genes. The fourth gene is probably cnidarian-specific. This precise characterization of the early evolution of LRRK genes in animals has important implications, because it indicates that the Drosophila and Caenorhabditis LRRK genes, which are studied to gain an understanding of LRRK2 function, are not true orthologues of the human Parkinson disease gene. Novel functional insights are also gained by comparison of the structures of LRRK2 genes in distantly related species.     

神经炎症在帕金森病中的作用及研究进展

帕金森病(Parkinson disease,PD)是全球第二大神经退行性疾病,病理本质是"黑质多巴胺能神经元选择性、进行性死亡",发病机制不清。研究发现神经炎症在PD病程中发挥重要作用,表现在:PD病人和动物模型中,黑质局部小胶质细胞、星形胶质细胞激活,血脑屏障受损及外周T淋巴细胞浸润。但PD中神经炎症发生的机制尚未阐明。本文将综述神经炎症在PD中的表现及发生机制的研究进展,从神经炎症的角度为PD防治的研究提供可能的思路。     

PET studies of cerebral metabolism in Parkinson Disease

A defect in cerebral energy production due to dysfunction of the mitochondrial electron transport system (ETS) has been postulated to be important in the pathogenesis of Parkinson Disease (PD). However, direct in vivo measurements of cerebral mitochondrial function are scant and inconsistent. We directly investigated cerebral mitochondrial function in vivo with positron emission tomography (PET) in 12 patients with early, never-medicated PD and 12 age-matched normal controls by combined measurements of the cerebral metabolic rate of oxygen (CMRO₂) and the cerebral metabolic rate of glucose (CMRglc). Instead of the decrease in CMRO₂ and CMRO₂/CMRglc molar ratio characteristic of defects in mitochondrial oxidative metabolism, there was a statistically significant 24% general increase in CMRO₂ and no change in CMRO₂/CMRglc. Since PD symptoms were already manifest, reduced oxidative activity of the mitochondrial ETS cannot be a primary mechanism of neuronal death in early PD. This increase in metabolism could reflect the increased energy requirements of an injured brain or an uncoupling of ATP production from oxidation in the terminal stage of oxidative phosphorylation. Which is the case in early PD and whether these metabolic abnormalities are important in the pathogenesis of PD will require further study.     

Replication of association between ELAVL4 and Parkinson disease: the GenePD study

Genetic variants in embryonic lethal, abnormal vision, Drosophila-like 4 (ELAVL4) have been reported to be associated with onset age of Parkinson disease (PD) or risk for PD affection in Caucasian populations. In the current study we genotyped three single nucleotide polymorphisms in ELAVL4 in a Caucasian study sample consisting of 712 PD patients and 312 unrelated controls from the GenePD study. The minor allele of rs967582 was associated with increased risk of PD (odds ratio = 1.46, nominal P value = 0.011) in the GenePD population. The minor allele of rs967582 was also the risk allele for PD affection or earlier onset age in the previously studied populations. This replication of association with rs967582 in a third cohort further implicates ELAVL4 as a PD susceptibility gene.     

Evaluation of Parkinson Disease Risk Variants as Expression-QTLs

The recent Parkinson Disease GWAS Consortium meta-analysis and replication study reports association at several previously confirmed risk loci SNCA, MAPT, GAK/DGKQ, and HLA and identified a novel risk locus at RIT2. To further explore functional consequences of these associations, we investigated modification of gene expression in prefrontal cortex brain samples of pathologically confirmed PD cases (N = 26) and controls (N = 24) by 67 associated SNPs in these 5 loci. Association between the eSNPs and expression was evaluated using a 2-degrees of freedom test of both association and difference in association between cases and controls, adjusted for relevant covariates. SNPs at each of the 5 loci were tested for cis-acting effects on all probes within 250 kb of each locus. Trans-effects of the SNPs on the 39,122 probes passing all QC on the microarray were also examined. From the analysis of cis-acting SNP effects, several SNPs in the MAPT region show significant association to multiple nearby probes, including two strongly correlated probes targeting the gene LOC644246 and the duplicated genes LRRC37A and LRRC37A2, and a third uncorrelated probe targeting the gene DCAKD. Significant cis-associations were also observed between SNPs and two probes targeting genes in the HLA region on chromosome 6. Expanding the association study to examine trans effects revealed an additional 23 SNP-probe associations reaching statistical significance (p<2.8×10⁻⁸) including SNPs from the SNCA, MAPT and RIT2 regions. These findings provide additional context for the interpretation of PD associated SNPs identified in recent GWAS as well as potential insight into the mechanisms underlying the observed SNP associations.     

VPS35 Mutations in Parkinson Disease

The identification of genetic causes for Mendelian disorders has been based on the collection of multi-incident families, linkage analysis, and sequencing of genes in candidate intervals. This study describes the application of next-generation sequencing technologies to a Swiss kindred presenting with autosomal-dominant, late-onset Parkinson disease (PD). The family has tremor-predominant dopa-responsive parkinsonism with a mean onset of 50.6 ± 7.3 years. Exome analysis suggests that an aspartic-acid-to-asparagine mutation within vacuolar protein sorting 35 (VPS35 c.1858G>A; p.Asp620Asn) is the genetic determinant of disease. VPS35 is a central component of the retromer cargo-recognition complex, is critical for endosome-trans-golgi trafficking and membrane-protein recycling, and is evolutionarily highly conserved. VPS35 c.1858G>A was found in all affected members of the Swiss kindred and in three more families and one patient with sporadic PD, but it was not observed in 3,309 controls. Further sequencing of familial affected probands revealed only one other missense variant, VPS35 c.946C>T; (p.Pro316Ser), in a pedigree with one unaffected and two affected carriers, and thus the pathogenicity of this mutation remains uncertain. Retromer-mediated sorting and transport is best characterized for acid hydrolase receptors. However, the complex has many types of cargo and is involved in a diverse array of biologic pathways from developmental Wnt signaling to lysosome biogenesis. Our study implicates disruption of VPS35 and retromer-mediated trans-membrane protein sorting, rescue, and recycling in the neurodegenerative process leading to PD.     

Replication of Nondefective Parvoviruses: Lack of a Virion-Associated DNA Polymerase

We have examined four of the nondefective parvoviruses for an associated DNA polymerase. Virions were purified from neuraminidase-treated infected-cell lysates by isopycnic centrifugation in CsCl or from infected cell material by CaCl(2) precipitation and centrifugation through sucrose into CsCl. Preparations of bovine parvovirus or Kilham rat virus obtained by the former procedure contained DNA polymerase activity but were not free of contaminating cellular proteins. The latter method produced viral preparations free of contaminating cellular proteins, and no DNA polymerase activity was detected in light infectious particles of H-1, LuIII, bovine parvovirus, or Kilham rat virus. Examination of levels of each cellular DNA polymerase in these preparations from each step of both purification procedures revealed that DNA polymerase beta had a greater tendency to copurify with bovine parvovirus and Kilham rat virus than did DNA polymerases alpha or gamma. Disruption of infectious virions obtained by the second purification method with detergents and sonic treatment did not result in the detection of a DNA polymerase activity. The biological activity and purity of each of the four different viruses obtained by the latter procedure were determined by hemagglutination and infectivity assays, polyacrylamide gel electrophoresis, and electron microscopy. In each case, the virions banding at a density of 1.39 to 1.41 g/cm(2) in CsCl were infectious and contained only the virion structural proteins. DNA polymerase activity was not detected in any of these preparations, and we have concluded that a virion-associated DNA polymerase is not required for productive infection with the nondefective parvoviruses.     

Association of Parkinson Disease with Structural and Regulatory Variants in the HLA Region

Historically, association of disease with the major histocompatibility complex (HLA) genes has been tested with HLA alleles that encode antigen-binding affinity. The association with Parkinson disease (PD), however, was discovered with noncoding SNPs in a genome-wide association study (GWAS). We show here that several HLA-region SNPs that have since been associated with PD form two blocks tagged by rs3129882 (p = 9 × 10⁻¹¹) and by rs9268515 and/or rs2395163 (p = 3 × 10⁻¹¹). We investigated whether these SNP-associations were driven by HLA-alleles at adjacent loci. We imputed class I and class II HLA-alleles for 2000 PD cases and 1986 controls from the NeuroGenetics Research Consortium GWAS and sequenced a subset of 194 cases and 204 controls. We were therefore able to assess accuracy of two imputation algorithms against next-generation-sequencing while taking advantage of the larger imputed data sets for disease study. Additionally, we imputed HLA alleles for 843 cases and 856 controls from another GWAS for replication. PD risk was positively associated with the B^∗07:02_C^∗07:02_DRB5^∗01_DRB1^∗15:01_DQA1^∗01:02_DQB1^∗06:02 haplotype and negatively associated with the C^∗03:04, DRB1^∗04:04 and DQA1^∗03:01 alleles. The risk haplotype and DQA1^∗03:01 lost significance when conditioned on the SNPs, but C^∗03:04 (OR = 0.72, p = 8 × 10⁻⁶) and DRB1^∗04:04 (OR = 0.65, p = 4 × 10⁻⁵) remained significant. Similarly, rs3129882 and the closely linked rs9268515 and rs2395163 remained significant irrespective of HLA alleles. rs3129882 and rs2395163 are expression quantitative trait loci (eQTLs) for HLA-DR and HLA-DQ (9 × 10⁻⁵ ≥ P_eQTL ≥ 2 × 10⁻⁷⁹), suggesting that HLA gene expression might influence PD. Our data suggest that PD is associated with both structural and regulatory elements in HLA. Furthermore, our study demonstrates that noncoding SNPs in the HLA region can be associated with disease irrespective of HLA alleles, and that observed associations with HLA alleles can sometimes be secondary to a noncoding variant.     

Decreased Cholesterol Biosynthesis in Fibroblasts from Patients with Parkinson Disease

The underlying cause of cellular degeneration in the substantia nigra of patients with Parkinson disease has not been clearly established. With the objective of investigating whether metabolic abnormalities would be detected in peripheral non-neuronal cells, we began assessing key metabolic parameters in skin fibroblasts of these patients. The present report focuses on the finding of a remarkably reduced cholesterol biosynthetic capability of fibroblasts from patients with Parkinson disease. ¹⁴C-Acetate incorporation into cholesterol of these fibroblasts was 27.8 ± 9.4% that observed in normal fibroblasts, and the reduced cholesterol synthesis was confirmed by measuring the activity of the rate-limiting enzyme HMGCoA reductase which averaged 6.64 ± 2.50 nmol/h/mg protein in the patient′s fibroblasts compared to 14.70 ± 0.69 nmol/h/mg protein in the control fibroblasts. Cholesterol esterifying activity, as cholesteryl oleate formed from ¹⁴C-oleate, of the fibroblasts from Parkinson patients, was reduced by an average 43%. Two hypotheses are put forward to link these findings with the current experimental evidences for both increased lipid per-oxidation and defective mitochondrial respiratory chain complex I activity in a number of cell types from Parkinson patients. Considering that decreased cholesterol biosynthesis has been detected in all the Parkinson cell lines thus far investigated, it is suggested that this may be a hallmark of the disease.     

Interaction of Replication Protein A with Photoreactive DNA Structures

A new photoreactive oligonucleotide derivative was synthesized with a perfluoroarylazido group attached to the 2'-position of the ribose fragment of the 5'-terminal nucleotide. Using this conjugate, photoreactive DNA duplexes were produced which contained single-stranded regions of different length, single-stranded breaks (nicks), and also ds duplex with a photoreactive group inside one of the chains. These structures imitate DNA intermediates generated at different stages of DNA replication and repair. The interaction of replication protein A (RPA) with the resulting DNA structures was studied using photoaffinity modification and gel retardation assay. Independently of the DNA structure, only the large subunit of RPA (p70) was crosslinked to photoreactive DNAs, and the intensity of its labeling increased with decrease in the size of the single-stranded region and was maximal in the case of the nick-containing DNA structure. By gel retardation, the most effective binding of RPA to this structure was shown, whereas the complexing of RPA with DNA containing the unmodified nick and also with the full duplex containing the photoreactive group inside the chain was significantly less effective. The data suggest that RPA should be sensitive to such damages in the double-stranded DNA structure.     

Mcm10 Mediates the Interaction Between DNA Replication and Silencing Machineries

The connection between DNA replication and heterochromatic silencing in yeast has been a topic of investigation for >20 years. While early studies showed that silencing requires passage through S phase and implicated several DNA replication factors in silencing, later works showed that silent chromatin could form without DNA replication. In this study we show that members of the replicative helicase (Mcm3 and Mcm7) play a role in silencing and physically interact with the essential silencing factor, Sir2, even in the absence of DNA replication. Another replication factor, Mcm10, mediates the interaction between these replication and silencing proteins via a short C-terminal domain. Mutations in this region of Mcm10 disrupt the interaction between Sir2 and several of the Mcm2–7 proteins. While such mutations caused silencing defects, they did not cause DNA replication defects or affect the association of Sir2 with chromatin. Our findings suggest that Mcm10 is required for the coupling of the replication and silencing machineries to silence chromatin in a context outside of DNA replication beyond the recruitment and spreading of Sir2 on chromatin.     

脆性组氨酸三联体与复制蛋白A相互作用的鉴定

目的：研究脆性组氨酸三联体（Fhit）对ATR／CHKl通路的影响,确定Fhit与复制蛋白A（RPA）存在相互作用,为进一步研究Fhit特异的信号通路奠定基础。方法：将Fhit全长基因插入含GST基因的原核表达载体中,在大肠杆菌中表达纯化GST-Fhit融合蛋白,并用GST沉降技术研究Fhit与RPA是否在体外存在相互作用;在表达Fhit的人细胞中用免疫共沉淀技术分析Fhit与RPA是否在体内存在相互作用,同时用免疫荧光染色方法研究Fhit与RPA在细胞内是否可以共定位。结果：通过免疫共沉淀、免疫荧光染色及GST沉降技术,确定了Fhit与RPA在体内及体外均可以相互作用。结论：确定了Fhit与RPA之间存在相互作用,为阐明Fhit在维持基因组完整性方面的机理提供了线索。     

Autophagy Benefits the Replication of Newcastle Disease Virus in Chicken Cells and Tissues

Newcastle disease virus (NDV) is an important avian pathogen. We previously reported that NDV triggers autophagy in U251 glioma cells, resulting in enhanced virus replication. In this study, we investigated whether NDV triggers autophagy in chicken cells and tissues to enhance virus replication. We demonstrated that NDV infection induced steady-state autophagy in chicken-derived DF-1 cells and in primary chicken embryo fibroblast (CEF) cells, evident through increased double- or single-membrane vesicles, the accumulation of green fluorescent protein (GFP)-LC3 dots, and the conversion of LC3-I to LC3-II. In addition, we measured autophagic flux by monitoring p62/SQSTM1 degradation, LC3-II turnover, and GFP-LC3 lysosomal delivery and proteolysis, to confirm that NDV infection induced the complete autophagic process. Inhibition of autophagy by pharmacological inhibitors and RNA interference reduced virus replication, indicating an important role for autophagy in NDV infection. Furthermore, we conducted in vivo experiments and observed the conversion of LC3-I to LC3-II in heart, liver, spleen, lung, and kidney of NDV-infected chickens. Regulation of the induction of autophagy with wortmannin, chloroquine, or starvation treatment affects NDV production and pathogenesis in tissues of both lung and intestine; however, treatment with rapamycin, an autophagy inducer of mammalian cells, showed no detectable changes in chicken cells and tissues. Moreover, administration of the autophagy inhibitor wortmannin increased the survival rate of NDV-infected chickens. Our studies provide strong evidence that NDV infection induces autophagy which benefits NDV replication in chicken cells and tissues.     

Evidence for Gender-Specific Transcriptional Profiles of Nigral Dopamine Neurons in Parkinson Disease

Background

Epidemiological data suggest that the male gender is one of the risks factors for the development of Parkinson Disease (PD). Also, differences in the clinical manifestation and the course of PD have been observed between males and females. However, little is known about the molecular aspects underlying gender-specificity in PD. To address this issue, we determined the gene expression profiles of male and female dopamine (DA) neurons in sporadic PD.

Methodology/Principal Findings

We analyzed Affymetrix-based microarrays on laser microdissected DA neurons from postmortem brains of sporadic PD patients and age-matched controls across genders. Pathway enrichment demonstrated that major cellular pathways involved in PD pathogenesis showed different patterns of deregulation between males and females with more prominent downregulation of genes related to oxidative phosporylation, apoptosis, synaptic transmission and transmission of nerve impulse in the male population. In addition, we found upregulation of gene products for metabolic processes and mitochondrial energy consumption in the age-matched male control neurons. On the single cell level, selected data validation using quantitative Real-Time (qRT)-PCR was consistent with microarray raw data and supported some of the observations from data analysis.

Conclusions/Significance

On the molecular level, our results provide evidence that the expression profiles of aged normal and PD midbrain DA neurons are gender-specific. The observed differences in the expression profiles suggest a disease bias of the male gender, which could be in concordance with clinical observations that the male gender represents a risk factor for sporadic PD. Validation of gene expression by qRT-PCR supported the microarray results, but also pointed to several caveats involved in data interpretation.     

Baseline Plasma C-Reactive Protein Concentrations and Motor Prognosis in Parkinson Disease

Background

C-reactive protein (CRP), a blood inflammatory biomarker, is associated with the development of Alzheimer disease. In animal models of Parkinson disease (PD), systemic inflammatory stimuli can promote neuroinflammation and accelerate dopaminergic neurodegeneration. However, the association between long-term systemic inflammations and neurodegeneration has not been assessed in PD patients.

Objective

To investigate the longitudinal effects of baseline CRP concentrations on motor prognosis in PD.

Design, Setting, and Participants

Retrospective analysis of 375 patients (mean age, 69.3 years; mean PD duration, 6.6 years). Plasma concentrations of high-sensitivity CRP were measured in the absence of infections, and the Unified Parkinson’s Disease Rating Scale Part III (UPDRS-III) scores were measured at five follow-up intervals (Days 1–90, 91–270, 271–450, 451–630, and 631–900).

Main Outcome Measure

Change of UPDRS-III scores from baseline to each of the five follow-up periods.

Results

Change in UPDRS-III scores was significantly greater in PD patients with CRP concentrations ≥0.7 mg/L than in those with CRP concentrations <0.7 mg/L, as determined by a generalized estimation equation model (P = 0.021) for the entire follow-up period and by a generalized regression model (P = 0.030) for the last follow-up interval (Days 631–900). The regression coefficients of baseline CRP for the two periods were 1.41 (95% confidence interval [CI] 0.21–2.61) and 2.62 (95% CI 0.25–4.98), respectively, after adjusting for sex, age, baseline UPDRS-III score, dementia, and incremental L-dopa equivalent dose.

Conclusion

Baseline plasma CRP levels were associated with motor deterioration and predicted motor prognosis in patients with PD. These associations were independent of sex, age, PD severity, dementia, and anti-Parkinsonian agents, suggesting that subclinical systemic inflammations could accelerate neurodegeneration in PD.     

Association between GRIN3A Gene Polymorphism in Kawasaki Disease and Coronary Artery Aneurysms in Taiwanese Children

Kawasaki disease (KD) is pediatric systemic vasculitis with the classic complication of coronary artery aneurysm (CAA). It is the leading cause of acquired cardiovascular diseases in children. Some severe cases present with multi-organ involvement or neurological dysfunction. To identify the role of the glutamate receptor, ionotropic, N-methyl-d-aspartate 3A (GRIN3A) in KD, we investigated genetic variations in GRIN3A in a Taiwanese cohort of 262 KD patients (76 with and 186 without CAA complications). We used univariate and multivariate regression analyses to identify the associations between clinical characteristics and GRIN3A genetic variations in KD. According to univariate regression analysis, CAA formation in KD was significantly associated with fever duration (p < 0.0001), first Intravenous immunoglobulin (IVIG) used (days after day one of fever) (p < 0.0001), and the GRIN3A (rs7849782) genetic variant (p < 0.001). KD patients with GG+GC genotype showed a lower rate of developing CAA (GG+GC genotype: odds ratio = 0.26; 95% CI = 0.14–0.46). Significant associations were identified between KD with CAA complication and the GRIN3A (rs7849782) genetic variant by using multivariate regression analysis. Specifically, significant correlations were observed between KD with CAA complications and the presence of GG+GC genotypes for the GRIN3A rs7849782 single-nucleotide polymorphism (full model: odds ratio = 0.25; 95% CI = 0.14–0.46). Our results suggest that a polymorphism of the GRIN3A gene may play a role in KD pathogenesis.