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1.
Neuroscience ; 220: 41-6, 2012 Sep 18.
Article in English | MEDLINE | ID: mdl-22732502

ABSTRACT

Alzheimer's Disease (AD) is the most common cause of dementia in elderly people. The presynaptic terminal is an important site of pathological changes in AD, leading to synaptic loss in specific brain regions, such as in the cortex and hippocampus. In this study, we investigated synaptosomal-associated protein, 25-kDa (SNAP25) mRNA levels and promoter DNA methylation in post mortem brain tissues (entorhinal and auditory cortices and hippocampus) from healthy elderly and AD subjects as well as in peripheral blood leukocytes of young, healthy elderly and AD patients. mRNA quantification was performed by quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) using the ΔΔC(T) method and promoter DNA methylation was quantified by mass spectrometry using the Sequenom EpiTYPER platform. We observed a significant decrease in SNAP25 expression in AD across all the three brain regions in relation to the healthy elderly subjects, suggesting impairment in synaptic function. The changes in the auditory cortex reflected those observed in the hippocampus and entorhinal cortex, the primary areas affected in AD. However, no AD-associated differences in SNAP25 promoter DNA methylation were observed suggesting that other mechanisms may be involved in mediating the observed gene expression changes.


Subject(s)
Alzheimer Disease/genetics , Brain/metabolism , DNA Methylation/genetics , Promoter Regions, Genetic/genetics , Synaptosomal-Associated Protein 25/genetics , Aged , Alzheimer Disease/metabolism , Female , Humans , Male , RNA, Messenger/analysis , Real-Time Polymerase Chain Reaction , Synaptosomal-Associated Protein 25/biosynthesis , Synaptosomes/metabolism , Transcriptome
2.
J Biol Chem ; 276(42): 38353-60, 2001 Oct 19.
Article in English | MEDLINE | ID: mdl-11459835

ABSTRACT

Vaccinia virus (VV) triggers a mitogenic signal at an early stage of infection. VV-induced proto-oncogene c-fos mRNA with kinetics paralleling that stimulated by serum. The VV virokine, or vaccinia virus growth factor (VGF), was not crucial for c-fos induction because it was observed upon infection with the virokine-minus mutant VV (VGF(-)). Furthermore, c-fos expression did not require infectious virus particles, as it occurred even with UV-inactivated VV and was equally induced by the different multiplicities of infection, i.e. 1.0, 5.0, and 25.0. c-fos expression was preceded by VV-induced DNA binding activity and was mediated via the cis-acting elements serum response element (SRE), activating protein-1 (AP-1), and cAMP-response element (CRE). VV activated the protein kinases p42MAPK/ERK2 and p44MAPK/ERK1 and the transcription factor ATF1 in a time-dependent manner with kinetics that paralleled those of VV-stimulated DNA-protein complex formation. The mitogenic signal transmission pathways leading to c-fos activation upon VV infection were apparently mediated by the protein kinases MEK, ERK, and PKA. This assumption was based on the findings that: 1) c-fos transcript was down-regulated; 2) the SRE, AP-1, and CRE binding activities were significantly reduced; and 3) the activation of p42MAPK/ERK2, p44MAPK/ERK1, and ATF1 were drastically affected when the viral infections were carried out in the presence of specific protein kinase inhibitor. Moreover, the mutant VV (VGF(-)) was also able to activate ERK1/2. It is noteworthy that virus multiplication was equally affected by the same kinase inhibitors. Taken together, our data provide evidence that the early mitogenic signal triggered upon VV infection relies upon the activation of the protein kinases MEK, ERK, and PKA, which are needed for both signal transduction and virus multiplication.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Mitogen-Activated Protein Kinases/metabolism , Mitogens/metabolism , Vaccinia virus/metabolism , Vaccinia virus/pathogenicity , Animals , Blotting, Northern , Blotting, Western , Cell Line , Cyclic AMP Response Element-Binding Protein/metabolism , DNA/metabolism , DNA, Complementary/metabolism , Down-Regulation , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Kinetics , MAP Kinase Signaling System , Mice , Mice, Inbred BALB C , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Models, Biological , Protein Binding , Proto-Oncogene Proteins c-fos/metabolism , RNA, Messenger/metabolism , Serum Response Element/genetics , Signal Transduction , Time Factors , Transcription, Genetic
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