Genome-wide gene-environment study identifies glutamate receptor gene GRIN2A as a Parkinson's disease modifier gene via interaction with coffee

Our aim was to identify genes that influence the inverse association of coffee with the risk of developing Parkinson''s disease (PD). We used genome-wide genotype data and lifetime caffeinated-coffee-consumption data on 1,458 persons with PD and 931 without PD from the NeuroGenetics Research Consortium (NGRC), and we performed a genome-wide association and interaction study (GWAIS), testing each SNP''s main-effect plus its interaction with coffee, adjusting for sex, age, and two principal components. We then stratified subjects as heavy or light coffee-drinkers and performed genome-wide association study (GWAS) in each group. We replicated the most significant SNP. Finally, we imputed the NGRC dataset, increasing genomic coverage to examine the region of interest in detail. The primary analyses (GWAIS, GWAS, Replication) were performed using genotyped data. In GWAIS, the most significant signal came from rs4998386 and the neighboring SNPs in GRIN2A. GRIN2A encodes an NMDA-glutamate-receptor subunit and regulates excitatory neurotransmission in the brain. Achieving P_2df = 10⁻⁶, GRIN2A surpassed all known PD susceptibility genes in significance in the GWAIS. In stratified GWAS, the GRIN2A signal was present in heavy coffee-drinkers (OR = 0.43; P = 6×10⁻⁷) but not in light coffee-drinkers. The a priori Replication hypothesis that “Among heavy coffee-drinkers, rs4998386_T carriers have lower PD risk than rs4998386_CC carriers” was confirmed: OR_Replication = 0.59, P_Replication = 10⁻³; OR_Pooled = 0.51, P_Pooled = 7×10⁻⁸. Compared to light coffee-drinkers with rs4998386_CC genotype, heavy coffee-drinkers with rs4998386_CC genotype had 18% lower risk (P = 3×10⁻³), whereas heavy coffee-drinkers with rs4998386_TC genotype had 59% lower risk (P = 6×10⁻¹³). Imputation revealed a block of SNPs that achieved P_2df<5×10⁻⁸ in GWAIS, and OR = 0.41, P = 3×10⁻⁸ in heavy coffee-drinkers. This study is proof of concept that inclusion of environmental factors can help identify genes that are missed in GWAS. Both adenosine antagonists (caffeine-like) and glutamate antagonists (GRIN2A-related) are being tested in clinical trials for treatment of PD. GRIN2A may be a useful pharmacogenetic marker for subdividing individuals in clinical trials to determine which medications might work best for which patients.     

Evidence for more than one Parkinson's disease-associated variant within the HLA region

Parkinson''s disease (PD) was recently found to be associated with HLA in a genome-wide association study (GWAS). Follow-up GWAS''s replicated the PD-HLA association but their top hits differ. Do the different hits tag the same locus or is there more than one PD-associated variant within HLA? We show that the top GWAS hits are not correlated with each other (0.00≤r²≤0.15). Using our GWAS (2000 cases, 1986 controls) we conducted step-wise conditional analysis on 107 SNPs with P<10⁻³ for PD-association; 103 dropped-out, four remained significant. Each SNP, when conditioned on the other three, yielded P_SNP1 = 5×10⁻⁴, P_SNP2 = 5×10⁻⁴, P_SNP3 = 4×10⁻³ and P_SNP4 = 0.025. The four SNPs were not correlated (0.01≤r²≤0.20). Haplotype analysis (excluding rare SNP2) revealed increasing PD risk with increasing risk alleles from OR = 1.27, P = 5×10⁻³ for one risk allele to OR = 1.65, P = 4×10⁻⁸ for three. Using additional 843 cases and 856 controls we replicated the independent effects of SNP1 (P_{conditioned-on-SNP4} = 0.04) and SNP4 (P_{conditioned-on-SNP1} = 0.04); SNP2 and SNP3 could not be replicated. In pooled GWAS and replication, SNP1 had OR_{conditioned-on-SNP4} = 1.23, P_{conditioned-on-SNP4} = 6×10⁻⁷; SNP4 had OR_{conditioned-on-SNP1} = 1.18, P_{conditioned-on-SNP1} = 3×10⁻³; and the haplotype with both risk alleles had OR = 1.48, P = 2×10⁻¹². Genotypic OR increased with the number of risk alleles an individual possessed up to OR = 1.94, P = 2×10⁻¹¹ for individuals who were homozygous for the risk allele at both SNP1 and SNP4. SNP1 is a variant in HLA-DRA and is associated with HLA-DRA, DRB5 and DQA2 gene expression. SNP4 is correlated (r² = 0.95) with variants that are associated with HLA-DQA2 expression, and with the top HLA SNP from the IPDGC GWAS (r² = 0.60). Our findings suggest more than one PD-HLA association; either different alleles of the same gene, or separate loci.     

Crystal structure of human CD38 extracellular domain

Human CD38 is a multifunctional protein involved in diverse functions. As an enzyme, it is responsible for the synthesis of two Ca2+ messengers, cADPR and NAADP; as an antigen, it is involved in regulating cell adhesion, differentiation, and proliferation. Besides, CD38 is a marker of progression of HIV-1 infection and a negative prognostic marker of B-CLL. We have determined the crystal structure of the soluble extracellular domain of human CD38 to 1.9 A resolution. The enzyme's overall topology is similar to the related proteins CD157 and the Aplysia ADP-ribosyl cyclase, except with large structural changes at the two termini. The extended positively charged N terminus has lateral associations with the other CD38 molecule in the crystallographic asymmetric unit. The analysis of the CD38 substrate binding models revealed two key residues that may be critical in controlling CD38's multifunctionality of NAD hydrolysis, ADP-ribosyl cyclase, and cADPR hydrolysis activities.     

Alkaloids of anuran skin: antimicrobial function?

A variety of alkaloids, most of which occur or are structurally related to alkaloids that occur in skin glands of dendrobatid poison frogs, were assayed for antimicrobial activity against the Gram-positive bacterium Bacillus subtilis, the Gram-negative bacterium Escherichia coli and the fungus Candida albicans. Certain pyrrolidines, piperidines and decahydroquinolines, perhydro-histrionicotoxin, and a synthetic pumiliotoxin were active against B. subtilis. Only 2-n-nonylpiperidine was active against E. coli. One pyrrolidine, two piperidines, two decahydroquinolines, and the synthetic pumiliotoxin were active against the fungus C. albicans. The results suggest that certain of the skin alkaloids of poison frogs, in addition to being noxious to predators, may also benefit the frog through protection against skin infections.     

Radial compression of microtubules and the mechanism of action of taxol and associated proteins

Microtubules (MTs) are hollow cylindrical polymers composed of alphabeta-tubulin heterodimers that align head-to-tail in the MT wall, forming linear protofilaments that interact laterally. We introduce a probe of the interprotofilament interactions within MTs and show that this technique gives insight into the mechanisms by which MT-associated proteins (MAPs) and taxol stabilize MTs. In addition, we present further measurements of the mechanical properties of MT walls, MT-MT interactions, and the entry of polymers into the MT lumen. These results are obtained from a synchrotron small angle x-ray diffraction (SAXRD) study of MTs under osmotic stress. Above a critical osmotic pressure, P(cr), we observe rectangular bundles of MTs whose cross sections have buckled to a noncircular shape; further increases in pressure continue to distort MTs elastically. The P(cr) of approximately 600 Pa provides, for the first time, a measure of the bending modulus of the interprotofilament bond within an MT. The presence of neuronal MAPs greatly increases P(cr), whereas surprisingly, the cancer chemotherapeutic drug taxol, which suppresses MT dynamics and inhibits MT depolymerization, does not affect the interprotofilament interactions. This SAXRD-osmotic stress technique, which has enabled measurements of the mechanical properties of MTs, should find broad application for studying interactions between MTs and of MTs with MAPs and MT-associated drugs.     

Cardioprotective effects of erythropoietin in the reperfused ischemic heart: a potential role for cardiac fibroblasts

Erythropoietin has recently been shown to have effects beyond hematopoiesis such as prevention of neuronal and cardiac apoptosis secondary to ischemia. In this study, we evaluated the in vivo protective potential of erythropoietin in the reperfused rabbit heart following ventricular ischemia. We show that "preconditioning" with erythropoietin activates cell survival pathways in myocardial tissue in vivo and adult rabbit cardiac fibroblasts in vitro. These pathways, activated by erythropoietin in both whole hearts and cardiac fibroblasts, are also activated acutely by ischemia/reperfusion injury. Moreover, in vivo studies indicate that erythropoietin treatment either prior to or during ischemia significantly enhances cardiac function and recovery, including left ventricular contractility, following myocardial ischemia/reperfusion. Our data indicate that a contributing in vivo cellular mechanism of this protection is mitigation of myocardial cell apoptosis. This results in decreased infarct size as evidenced by area at risk studies following in vivo ischemia/reperfusion injury, translating into more viable myocardium and less ventricular dysfunction. Therefore, erythropoietin treatment may offer novel protection against ischemic heart disease and may act, at least in part, by direct action on cardiac fibroblasts and myocytes to alter survival and ventricular remodeling.     

Phosphorylated Rad18 directs DNA polymerase η to sites of stalled replication

The E3 ubiquitin ligase Rad18 guides DNA Polymerase eta (Polη) to sites of replication fork stalling and mono-ubiquitinates proliferating cell nuclear antigen (PCNA) to facilitate binding of Y family trans-lesion synthesis (TLS) DNA polymerases during TLS. However, it is unclear exactly how Rad18 is regulated in response to DNA damage and how Rad18 activity is coordinated with progression through different phases of the cell cycle. Here we identify Rad18 as a novel substrate of the essential protein kinase Cdc7 (also termed Dbf4/Drf1-dependent Cdc7 kinase [DDK]). A serine cluster in the Polη-binding motif of Rad 18 is phosphorylated by DDK. Efficient association of Rad18 with Polη is dependent on DDK and is necessary for redistribution of Polη to sites of replication fork stalling. This is the first demonstration of Rad18 regulation by direct phosphorylation and provides a novel mechanism for integration of S phase progression with postreplication DNA repair to maintain genome stability.     

Effects from shear stress on morphology and growth of early stages of Norway spruce somatic embryos

The shear stress effect on directional expansion of pro embryogenic masses (PEMs) and suspensor cell development of somatic embryos of Norway spruce (Picea abies) at the proliferation stage was studied by a direct and quantitative image analysis system. The experimental system allowed for detailed observations of the effect of hydrodynamic shear stress in rotating and deforming liquid cultures of proliferating Norway spruce somatic embryos. Briefly, somatic embryos at an early development stage comprised only of clusters of meristematic cells without suspensor cells were fixed on an alginate film. The alginate film was affixed on the bottom of a flow cell and the somatic embryos were subjected to laminar flow through the chamber of the flow cell. Magnified images of the cell clusters were collected every 24 h. The image data was processed based on a normalized cross‐correlation method, capable of measuring morphological and size features of individual cell clusters in both temporal and spatial domains. No suspensor cells developed in the cell clusters under shear stress of 140 s^?1 for the duration of the experiments. Cell clusters in the control cultured in stationary liquid conditions developed suspensor cells after 5–9 days in culture. Furthermore, the radial growth of meristematic cell clusters was inhibited by shear rates of 86 and 140 s^?1, corresponding to shear stress of 0.086 and 0.14 N/m², compared to growth under stationary conditions. The shear rate showed a significant negative correlation to growth rate. Control group showed no preference for direction during growth under static conditions. Biotechnol. Bioeng. 2010; 105: 588–599. © 2009 Wiley Periodicals, Inc.     

Epstein-Barr virus BPLF1 deubiquitinates PCNA and attenuates polymerase η recruitment to DNA damage sites

PCNA is monoubiquitinated in response to DNA damage and fork stalling and then initiates recruitment of specialized polymerases in the DNA damage tolerance pathway, translesion synthesis (TLS). Since PCNA is reported to associate with Epstein-Barr virus (EBV) DNA during its replication, we investigated whether the EBV deubiquitinating (DUB) enzyme encoded by BPLF1 targets ubiquitinated PCNA and disrupts TLS. An N-terminal BPLF1 fragment (a BPLF1 construct containing the first 246 amino acids [BPLF1 1-246]) associated with PCNA and attenuated its ubiquitination in response to fork-stalling agents UV and hydroxyurea in cultured cells. Moreover, monoubiquitinated PCNA was deubiquitinated after incubation with purified BPLF1 1-246 in vitro. BPLF1 1-246 dysregulated TLS by reducing recruitment of the specialized repair polymerase polymerase η (Polη) to the detergent-resistant chromatin compartment and virtually abolished localization of Polη to nuclear repair foci, both hallmarks of TLS. Expression of BPLF1 1-246 decreased viability of UV-treated cells and led to cell death, presumably through deubiquitination of PCNA and the inability to repair damaged DNA. Importantly, deubiquitination of PCNA could be detected endogenously in EBV-infected cells in comparison with samples expressing short hairpin RNA (shRNA) against BPLF1. Further, the specificity of the interaction between BPLF1 and PCNA was dependent upon a PCNA-interacting peptide (PIP) domain within the N-terminal region of BPLF1. Both DUB activity and PIP sequence are conserved in the members of the family Herpesviridae. Thus, deubiquitination of PCNA, normally deubiquitinated by cellular USP1, by the viral DUB can disrupt repair of DNA damage by compromising recruitment of TLS polymerase to stalled replication forks. PCNA is the first cellular target identified for BPLF1 and its deubiquitinating activity.     

Effect of methyl jasmonate treatments on alleviation of polyethylene glycol -mediated water stress in banana (Musa acuminata cv. ‘Berangan’, AAA) shoot tip cultures

Although irregular monoterpenes are important and common in the Asteraceae family, little is known about their biosynthesis at the genetic level via the MEP pathway. Chrysanthemyl diphosphate (CPP) is an intermediate in the biosynthesis of pyrethrins which are irregular monoterpenes with excellent insecticidal activity in Tanacetum cinerariaefolium (T. cinerariaefolium). In this study, a chrysanthemyl diphosphate synthase (CDS) gene named CDS_CCI2 (GenBank accession no. HQ235057) was isolated from T. cinerariaefolium. It was homologous to T. cinerariaefolium CPP gene family, and proved to be located in the plastid by the in situ subcellular localization. CDS_CCI2 was found to be expressed in roots, stems, leaves, buds and flowers. Moreover, the expression of CDS_CCI2 can be up-regulated by abscisic acid (ABA), methyl jasmonate and ethrel treatment. Phenotypic and molecular analysis showed that overexpression of CDS_CCI2 in micro-tom tomato resulted in dwarf phenotypes characterized with infertile flowers and seedless fruits. Furthermore, overexpression of CDS_CCI2 altered the production of endogenous secondary metabolites. Our data indicate that CPP affects the synthesis of gibberellic acid (GA) and ABA.