Rare variants in the splicing regulatory elements of EXOC3L4 are associated with brain glucose metabolism in Alzheimer’s disease

Background

Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases that causes problems related to brain function. To some extent it is understood on a molecular level how AD arises, however there are a lack of biomarkers that can be used for early diagnosis. Two popular methods to identify AD-related biomarkers use genetics and neuroimaging. Genes and neuroimaging phenotypes have provided some insights as to the potential for AD biomarkers. While the field of imaging-genomics has identified genetic features associated with structural and functional neuroimaging phenotypes, it remains unclear how variants that affect splicing could be important for understanding the genetic etiology of AD.

Methods

In this study, rare variants (minor allele frequency?<?0.01) in splicing regulatory element (SRE) loci from whole genome sequencing (WGS) in the Alzheimer’s Disease Neuroimaging Initiative (ADNI) cohort, were used to identify genes that are associated with global brain cortical glucose metabolism in AD measured by FDG PET-scans. Gene-based associated analyses of rare variants were performed using the program BioBin and the optimal Sequence Kernel Association Test (SKAT-O).

Results

The gene, EXOC3L4, was identified as significantly associated with global cortical glucose metabolism (FDR (false discovery rate) corrected p?<?0.05) using SRE coding variants only. Three loci that may affect splicing within EXOC3L4 contribute to the association.

Conclusion

Based on sequence homology, EXOC3L4 is likely a part of the exocyst complex. Our results suggest the possibility that variants which affect proper splicing of EXOC3L4 via SREs may impact vesicle transport, giving rise to AD related phenotypes. Overall, by utilizing WGS and functional neuroimaging we have identified a gene significantly associated with an AD related endophenotype, potentially through a mechanism that involves splicing.

     

A 20 nucleotide upstream element is essential for the nopaline synthase (nos) promoter activity

The nopaline synthase (nos) promoter is expressed in a wide range of plant cell types and regulated by various developmental and environmental factors. The nos upstream control region essential for this regulation was studied by means of synthetic oligomers using transient and stable transformation systems. Insertion of a 20 nucleotide sequence containing two hexamer motifs and a spacer region into deletion mutants lacking the upstream control region was essential for promoter activity. Mutation of one or more nucleotides of either hexamer sequence significantly altered the strength of expression of the nos promoter. Point mutations within the spacer region also strongly influenced promoter strength. Insertion of multiple copies of the 20 nucleotide sequence into the nonfunctional deletion mutants proportionally increased the promoter activity. These results suggest that this twenty nucleotide sequence is essential for the nos promoter to function. Substitution of the nos element with the ocs or 35S as-1 which contain similar hexamer motifs restored not only promoter activity but also responses to wounding, auxin, methyl jasmonate, and salicylic acid.     

Characterization of flavonoid 7-O-glucosyltransferase from Arabidopsis thaliana

Most flavonoids found in plants exist as glycosides, and glycosylation status has a wide range of effects on flavonoid solubility, stability, and bioavailability. Glycosylation of flavonoids is mediated by Family 1 glycosyltransferases (UGTs), which use UDP-sugars, such as UDP-glucose, as the glycosyl donor. AtGT-2, a UGT from Arabidopsis thaliana, was cloned and expressed in Escherichia coli as a gluthatione S-transferase fusion protein. Several compounds, including flavonoids, were tested as potential substrates. HPLC analysis of the reaction products indicated that AtGT-2 transfers a glucose molecule into several different kinds of flavonoids, eriodictyol being the most effective substrate, followed by luteolin, kaempferol, and quercetin. Based on comparison of HPLC retention times with authentic flavonoid 7-O-glucosides and nuclear magnetic resonance spectroscopy, the glycosylation position in the reacted flavonoids was determined to be the C-7 hydroxyl group. These results indicate that AtGT-2 encodes a flavonoid 7-O-glucosyltransferase.     

Redox regulation of the tumor suppressor PTEN by glutaredoxin 5 and Ycp4

Human PTEN (phosphatase and tensin homolog deleted on chromosome 10; a phosphatidylinositol 3-phosphatase) expressed in Saccharomyces cerevisiae was oxidized in a time- and H₂O₂-concentration-dependent manner. Oxidized hPTEN was reduced by cellular reductants as in human cells. The reduction rate of oxidized hPTEN was monitored in S. cerevisiae mutants in which the genes involved in redox homeostasis had been disrupted. Reduction of hPTEN was delayed in each of S. cerevisiae grx5Δ and ycp4Δ mutants. Expression of Grx5 and Ycp4 in each of the mutants rescued the reduction rate of oxidized hPTEN. Furthermore, an in vitro assay revealed that the human Grx5/GSH system efficiently catalyzed the reduction of oxidized hPTEN. These results suggest that the reduction of oxidized hPTEN is regulated by Grx5 and Ycp4.     

Effects of Lead Exposure on Nitric Oxide-associated Gene Expression in the Olfactory Bulb of Mice

Lead (Pb) is known to have toxic effects on the brain; however, data regarding its specific toxic effects on the olfactory bulb are lacking. Therefore, we investigated the relationship between acute Pb exposure and alterations in gene expression associated with the nitric oxide signaling pathway in the olfactory bulb of mice. After administration of Pb (intraperitoneal injections of 1 or 10 mg/kg Pb(CH₃CO₂)₂ · 3H₂O once per day for 4 days), body weight, motor activity, and gene expression in the olfactory bulb of mice were examined. High doses of Pb resulted in significant decreases in body weight, but motor coordination was not significantly altered until 11 days after the end of Pb treatment. The expression patterns of dimethylarginine dimethylaminohydrolase 1 (Ddah1), superoxide dismutase 1 (Sod1), and superoxide dismutase (Ccs) were increased, whereas expression of the Stratifin (Sfn) gene was significantly decreased following treatment with 10 mg/kg Pb. The expression patterns of nitric oxide synthases at the mRNA and protein levels, however, were not significantly altered by treatment with 10 mg/kg Pb. These findings indicate that Pb-induced neurotoxicity may be modulated in part by the expression of Ddah1, Sod1, Ccs, and Sfn in the olfactory bulb.     

Therapeutic effect of a TM4SF5-specific peptide vaccine against colon cancer in a mouse model

Molecular-targeted therapy has gained attention because of its high efficacy and weak side effects. Previously, we confirmed that transmembrane 4 superfamily member 5 protein (TM4SF5) can serve as a molecular target to prevent or treat hepatocellular carcinoma (HCC). We recently extended the application of the peptide vaccine, composed of CpG-DNA, liposome complex, and TM4SF5 peptide, to prevent colon cancer in a mouse model. Here, we first implanted mice with mouse colon cancer cells and then checked therapeutic effects of the vaccine against tumor growth. Immunization with the peptide vaccine resulted in robust production of TM4SF5-specific antibodies, alleviated tumor growth, and reduced survival rate of the tumor-bearing mice. We also found that serum levels of VEGF were markedly reduced in the mice immunized with the peptide vaccine. Therefore, we suggest that the TM4SF5-specific peptide vaccine has a therapeutic effect against colon cancer in a mouse model. [BMB Reports 2014; 47(4): 215-220]     

Effects of lipopolysaccharide and CpG-DNA on burn-induced skin injury

Destruction of the skin barrier by thermal injury induces microbial invasion, which can lead to the development of systemic infection and septic shock. Microbial pathogens possess pathogen-associated molecular patterns (PAMPs), which are recognized by conserved receptors. To understand the role of PAMPs in thermal injury-induced mice, LPS or CpG-DNA were topically applied to dorsal skin after thermal injury. We observed an increase in the number of inflammatory cell infiltrates as well as thickening in the dermis upon treatment with LPS or CpG-DNA. We also found that expression of IL-1β, MIP-2, and RANTES induced by thermal injury was enhanced by LPS or CpG-DNA. In addition, the proportions of CD4(+) and CD8(+) T cells in the spleen and lymph nodes were altered by LPS or CpG-DNA. These results provide important information concerning PAMPs-induced inflammation upon thermal injury and provide a basis for studying the role of PAMPs in thermal injury-induced complications.     

Prevention and therapy of hepatocellular carcinoma by vaccination with TM4SF5 epitope-CpG-DNA-liposome complex without carriers

Although peptide vaccines have been actively studied in various animal models, their efficacy in treatment is limited. To improve the efficacy of peptide vaccines, we previously formulated an efficacious peptide vaccine without carriers using the natural phosphodiester bond CpG-DNA and a special liposome complex (Lipoplex(O)). Here, we show that immunization of mice with a complex consisting of peptide and Lipoplex(O) without carriers significantly induces peptide-specific IgG2a production in a CD4(+) cells- and Th1 differentiation-dependent manner. The transmembrane 4 superfamily member 5 protein (TM4SF5) has gained attention as a target for hepatocellular carcinoma (HCC) therapy because it induces uncontrolled growth of human HCC cells via the loss of contact inhibition. Monoclonal antibodies specific to an epitope of human TM4SF5 (hTM4SF5R2-3) can recognize native mouse TM4SF5 and induce functional effects on mouse cancer cells. Pre-immunization with a complex of the hTM4SF5R2-3 epitope and Lipoplex(O) had prophylactic effects against tumor formation by HCC cells implanted in an mouse tumor model. Furthermore, therapeutic effects were revealed regarding the growth of HCC when the vaccine was injected into mice after tumor formation. These results suggest that our improved peptide vaccine technology provides a novel prophylaxis measure as well as therapy for HCC patients with TM4SF5-positive tumors.