Association between several immune response-related genes and the effectiveness of biological treatments in patients with moderate-to-severe psoriasis.

Biological therapies are safer and more effective against psoriasis than conventional treatments. Even so, 30-50% of psoriatic patients show an inadequate response, which is associated with individual genetic heterogeneity. Pharmacogenetic studies have identified several single nucleotide polymorphisms (SNPs) as possible predictive and prognostic biomarkers for psoriasis treatment response. The objective of this study was to determine the link between several SNPs and the clinical response to biological therapies in patients with moderate-severe psoriasis. A set of 21 SNPs related to psoriasis and/or other immunological diseases were selected and analysed from salivary samples of patients (n = 88). Treatment effectiveness and patient improvement was assessed clinically through Relative Psoriasis Area and Severity Index (PASI), also called 'PASI response', as well as absolute PASI. Associations between SNPs and PASI factors were assessed at 3 and 12 months for every treatment category of IL-17, IL-23, IL-12&23 and TNF-α inhibitors. Multivariate correlation analysis and Fisher's exact test were used to analyse the relationship between SNPs and therapy outcomes. Several SNPs located in the TLR2, TLR5, TIRAP, HLA-C, IL12B, SLC12A8, TNFAIP3 and PGLYRP4 genes demonstrated association with increased short and long-term therapy-effectiveness rates. Most patients achieved values of PASI response ≥75 or absolute PASI<1, regardless of the biological treatment administered. In conclusion, we demonstrate a relationship between different SNPs and both short- and especially long-term effectiveness of biological treatment in terms of PASI. These polymorphisms may be used as predictive markers of treatment response in patients with moderate-to-severe psoriasis, providing personalized treatment.

Altered Mitochondrial DNA Methylation Pattern in Alzheimer Disease-Related Pathology and in Parkinson Disease.

Mitochondrial dysfunction is linked with the etiopathogenesis of Alzheimer disease and Parkinson disease. Mitochondria are intracellular organelles essential for cell viability and are characterized by the presence of the mitochondrial (mt)DNA. DNA methylation is a well-known epigenetic mechanism that regulates nuclear gene transcription. However, mtDNA methylation is not the subject of the same research attention. The present study shows the presence of mitochondrial 5-methylcytosine in CpG and non-CpG sites in the entorhinal cortex and substantia nigra of control human postmortem brains, using the 454 GS FLX Titanium pyrosequencer. Moreover, increased mitochondrial 5-methylcytosine levels are found in the D-loop region of mtDNA in the entorhinal cortex in brain samples with Alzheimer disease-related pathology (stages I to II and stages III to IV of Braak and Braak; n = 8) with respect to control cases. Interestingly, this region shows a dynamic pattern in the content of mitochondrial 5-methylcytosine in amyloid precursor protein/presenilin 1 mice along with Alzheimer disease pathology progression (3, 6, and 12 months of age). Finally, a loss of mitochondrial 5-methylcytosine levels in the D-loop region is found in the substantia nigra in Parkinson disease (n = 10) with respect to control cases. In summary, the present findings suggest mtDNA epigenetic modulation in human brain is vulnerable to neurodegenerative disease states.

Increased striatal adenosine A2A receptor levels is an early event in Parkinson's disease-related pathology and it is potentially regulated by miR-34b.

Adenosine A2A receptor (A2AR) is a G-protein coupled receptor that stimulates adenylyl cyclase activity. In the brain, A2ARs are found highly enriched in striatal GABAergic medium spiny neurons, related to the control of voluntary movement. Pharmacological modulation of A2ARs is particularly useful in Parkinson's disease (PD) due to their property of antagonizing dopamine D2 receptor activity. Increases in A2AR levels have been described in PD patients showing an important loss of dopaminergic denervation markers, but no data have been reported about A2AR levels in incidental PD brains. In the present report, we show that increased A2ARs protein levels were also detected in the putamen of incidental PD cases (Braak PD stages 1-2) with respect to age-matched controls. By contrast, A2ARs mRNA levels remained unchanged, suggesting that posttranslational mechanisms could be involved in the regulation of A2ARs. It has been described how miR-34b/c downregulation is an early event in PD cases. We found that miR-34b levels are also significantly reduced in the putamen of incidental PD cases and along disease progression. Given that 3'UTR of A2AR contains a predicted target site for miR-34b, the potential role of this miRNA in protein A2AR levels was assessed. In vitro studies revealed that endogenous A2AR protein levels increased when miR-34b function was blocked using a specific anti-miR-34b. Moreover, using a luciferase reporter assay with point mutations in a miR-34b predicted binding site within the 3'UTR region of A2AR mRNA abolished the effect of the miRNA using a miR-34b mimic. In addition, we showed a reduced percentage of DNA methylation in the 5'UTR region of ADORA2A in advanced PD cases. Overall, these findings reveal that increased A2AR protein levels occur in asymptomatic PD patients and provide new insights into the molecular mechanisms underlying A2AR expression levels along the progression of this neurodegenerative disease.

Striatal adenosine A2A receptor expression is controlled by S-adenosyl-L-methionine-mediated methylation.

Adenosine A2A receptor (A2AR) is a G protein-coupled receptor enriched in the striatum for which an increased expression has been demonstrated in certain neurological diseases. Interestingly, previous in vitro studies demonstrated that A2AR expression levels are reduced after treatment with S-adenosyl-L-methionine (SAM), a methyl donor molecule involved in the methylation of important biological structures such as DNA, proteins, and lipids. However, the in vivo effects of SAM treatment on A2AR expression are still obscure. Here, we demonstrated that 2 weeks of SAM treatment produced a significant reduction in the rat striatal A2AR messenger RNA (mRNA) and protein content as well as A2AR-mediated signaling. Furthermore, when the content of 5-methylcytosine levels in the 5'UTR region of ADORA2A was analyzed, this was significantly increased in the striatum of SAM-treated animals; thus, an unambiguous correlation between SAM-mediated methylation and striatal A2AR expression could be established. Overall, we concluded that striatal A2AR functionality can be controlled by SAM treatment, an issue that might be relevant for the management of these neurological conditions that course with increased A2AR expression.

Reduced striatal ecto-nucleotidase activity in schizophrenia patients supports the "adenosine hypothesis".

Schizophrenia (SZ) is a major chronic neuropsychiatric disorder characterized by a hyperdopaminergic state. The hypoadenosinergic hypothesis proposes that reduced extracellular adenosine levels contribute to dopamine D2 receptor hyperactivity. ATP, through the action of ecto-nucleotidases, constitutes a main source of extracellular adenosine. In the present study, we examined the activity of ecto-nucleotidases (NTPDases, ecto-5'-nucleotidase, and alkaline phosphatase) in the postmortem putamen of SZ patients (n = 13) compared with aged-matched controls (n = 10). We firstly demonstrated, by means of artificial postmortem delay experiments, that ecto-nucleotidase activity in human brains was stable up to 24 h, indicating the reliability of this tissue for these enzyme determinations. Remarkably, NTPDase-attributable activity (both ATPase and ADPase) was found to be reduced in SZ patients, while ecto-5'-nucleotidase and alkaline phosphatase activity remained unchanged. In the present study, we also describe the localization of these ecto-enzymes in human putamen control samples, showing differential expression in blood vessels, neurons, and glial cells. In conclusion, reduced striatal NTPDase activity may contribute to the pathophysiology of SZ, and it represents a potential mechanism of adenosine signalling impairment in this illness.

Histone tail acetylation in brain occurs in an unpredictable fashion after death.

Histone acetylation plays a role in the regulation of gene transcription. Yet it is not known whether post-mortem brain tissue is suitable for the analysis of histone acetylation. To examine this question, nucleosomes were isolated from frontal cortex of nine subjects which were obtained at short times after death and immediately frozen at -80°C or maintained at room temperature from 3 h up to 50 h after death and then frozen at -80°C to mimic variable post-mortem delay in tissue processing as currently occurs in normal practice. Chromatin immunoprecipitation assays were performed for two lysine residues, H3K9ac and H3K27ac. Four gene loci were amplified by SyBrGreen PCR: Adenosine A(2A) receptor, UCHL1, α-synuclein and ß-globin. Results showed variability in the histone acetylation level along the post-mortem times and an increase in the acetylation level at an unpredictable time from one case to another and from one gene to another within the first 24 h of post-mortem delay. Similar results were found with three rat brains used to exclude the effects of agonal state and to normalize the start-point as real time zero. Therefore, the present observations show that human post-mortem brain is probably not suitable for comparative studies of histone acetylation.

UV-Induced Somatic Mutations Driving Clonal Evolution in Healthy Skin, Nevus, and Cutaneous Melanoma.

INTRODUCTION: Due to its aggressiveness, cutaneous melanoma (CM) is responsible for most skin cancer-related deaths worldwide. The origin of CM is closely linked to the appearance of UV-induced somatic mutations in melanocytes present in normal skin or in CM precursor lesions (nevi or dysplastic nevi). In recent years, new NGS studies performed on CM tissue have increased the understanding of the genetic somatic changes underlying melanomagenesis and CM tumor progression. METHODS: We reviewed the literature using all important scientific databases. All articles related to genomic mutations in CM as well as normal skin and nevi were included, in particular those related to somatic mutations produced by UV radiation. CONCLUSIONS: CM development and progression are strongly associated with exposure to UV radiation, although each melanoma subtype has different characteristic genetic alterations and evolutionary trajectories. While BRAF and NRAS mutations are common in the early stages of tumor development for most CM subtypes, changes in CDKN2A, TP53 and PTEN, together with TERT promoter mutations, are especially common in advanced stages. Additionally, large genome duplications, loss of heterozygosity, and copy number variations are hallmarks of metastatic disease. Finally, the mutations driving melanoma targeted-therapy drug resistance are also summarized. The complete sequential stages of clonal evolution leading to CM onset from normal skin or nevi are still unknown, so further studies are needed in this field to shed light on the molecular pathways involved in CM malignant transformation and in melanoma acquired drug resistance.

Non-classical Notch signaling by MDA-MB-231 breast cancer cell-derived small extracellular vesicles promotes malignancy in poorly invasive MCF-7 cells.

Aberrant Notch signaling is implicated in breast cancer progression, and recent studies have demonstrated links between the Notch pathway components Notch1 and Notch1 intracellular domain (N1ICD) with poor clinical outcomes. Growing evidence suggests that Notch signaling can be regulated by small extracellular vesicles (SEVs). Here, we used breast cancer cell models to examine whether SEVs are involved in functional Notch signaling. We found that Notch components are packaged into MDA-MB-231- and MCF-7-derived SEVs, although higher levels of N1ICD were detected in SEVs from the more aggressive MDA-MB-231 cell line than from poorly invasive MCF-7 cells. SEV-Notch components were functional, as SEVs cargo from MDA-MB-231 cells induced the expression of Notch target genes in MCF-7 cells and triggered a more invasive and proliferative phenotype concomitant with the acquisition of mesenchymal features. Neutralization of the N1ICD cargo in MDA-MB-231-derived SEVs significantly reduced their potential to enhance the aggressiveness of MCF-7 cells in vitro and in a xenograft model. Overall, our results indicate that a SEV-mediated non-classical pathway of Notch signal transduction in breast cancer models bypasses the need for classical ligand-receptor interactions, which may have important implications in cancer.

DNA methylation and Yin Yang-1 repress adenosine A2A receptor levels in human brain.

Adenosine A(2A) receptors (A(2A) Rs) are G-protein coupled receptors that stimulate adenylyl cyclase activity. The most A(2A) Rs-enriched brain region is the striatum, in which A(2A) Rs are largely restricted to GABAergic neurons of the indirect pathway. We recently described how DNA methylation controls basal A(2A) R expression levels in human cell lines. The present report provides clues about the molecular mechanisms that promote human brain region-specific A(2A) R gene (ADORA2A) basal expression. The transcription factors ZBP-89 and Yin Yang-1 (YY1) have been characterized as regulators of ADORA2A in SH-SY5Y cells by means of specific expression vectors/siRNAs transient transfection and chromatin immunoprecipitation assay. ZBP-89 plays a role as an activator and YY1 as a repressor. No differences were found in ZBP-89 levels with western blot between the putamen and cerebellum of human postmortem brains. However, increased YY1 levels and DNA methylation percentage in the 5' untranslated region of ADORA2A, using SEQUENOM MassArray, were found in the cerebellum with respect to the putamen of human brains, showing an inverse relationship with A(2A) R levels in the two cerebral regions.

DNA methylation regulates adenosine A(2A) receptor cell surface expression levels.

Adenosine A(2A) receptors (A(2A)Rs) appear to play important roles in inflammation and in certain diseases of the nervous system. Pharmacological modulation of A(2A)Rs is particularly useful in Parkinson's disease and has been tested in schizophrenia. However, little is known about the regulation of A(2A)R gene (ADORA2A). A bioinformatic analysis revealed the presence of three CpG islands in the 5' UTR region of human ADORA2A. Next, HeLa, SH-SY5Y and U87-MG cells were treated for 48 h with 5 muM 5-azacytidine (Aza). Increased A(2A)R levels were demonstrated in HeLa and SH-SY5Y cells when compared with non-treated cells. No modifications were seen in U87-MG cells. The increased A(2A)R mRNA and protein levels were accompanied by a loss of DNA methylation pattern in HeLa and SH-SY5Y cells, as measured with the SEQUENOM MassArray platform. The Aza treatment also reduced the affinity of a methyl-CpG-binding protein for ADORA2A by quantitative chromatin immunoprecipitation in HeLa cells. Interestingly, A(2A)R levels were reduced by S-adenosyl-l-methionine treatment in U87-MG and methyl-CpG-binding protein affinity was increased for ADORA2A by quantitative chromatin immunoprecipitation. Therefore, these results show for the first time that DNA methylation plays a role in ADORA2A transcription and, subsequently, in constitutive A(2A)R cell surface levels.

The effect of long-term ultra-endurance exercise and SOD2 genotype on telomere shortening with age.

Telomere shortening, a well-known biomarker of aging, is a complex process influenced by several intrinsic and lifestyle factors. Although habitual exercise may promote telomere length maintenance, extreme endurance exercise has been also associated with increased oxidative stress-presumed to be the major cause of telomere shortening. Therefore, the pace of telomere shortening with age may also depend on antioxidant system efficiency, which is, in part, genetically determined. In this study, we aimed to evaluate the impact of ultra-endurance exercise and oxidative stress susceptibility (determined by the rs4880 polymorphism in the superoxide dismutase 2 (SOD2) gene) on telomere length. Genomic DNA was obtained from 53 sedentary individuals (34 females, 19-67 yr) and 96 ultra-trail runners (31 females, 23-58 yr). Indeed, blood samples before and after finishing a 107-km-trail race were collected from 69 runners to measure c-reactive protein (CRP) levels and, thus, analyze whether acute inflammation response is modulated by the SOD2 rs4880 polymorphism. Our results revealed that telomere length was better preserved in ultra-trail runners compared with controls, especially in elderly runners who have been regularly training for many years. Carrying the SOD2 rs4880*A allele was significantly associated with having shorter telomeres, as well as with having increased CRP levels after the ultra-trail race. In conclusion, habitual ultra-endurance exercise had a beneficial effect on telomere length maintenance, especially at older ages. This study also suggested that the SOD2 rs4880 polymorphism may also have an impact on acute and chronic oxidative-related damage (inflammatory response and telomere length) after an ultra-trail race.NEW & NOTEWORTHY Habitual ultra-endurance exercise seems to promote telomere length maintenance, especially at older ages. In addition, the beneficial effect of ultra-endurance training on biological aging is higher in ultra-trail runners who have been engaged to ultra-endurance training during many years. Finally, and for the first time, this study shows that the SOD2 rs4880 polymorphism has a significant impact on telomere length, as well as on acute inflammatory response to a 107-km trail race.

DNA methylation of Alzheimer disease and tauopathy-related genes in postmortem brain.

DNA methylation occurs predominantly at cytosines that precede guanines in dinucleotide CpG sites; it is one of the most important mechanisms for epigenetic DNA regulation during normal development and for aberrant DNA in cancer. To determine the feasibility of DNA methylation studies in the postmortem human brain, we evaluated brain samples with variable postmortem artificially increased delays up to 48 hours. DNA methylation was analyzed in selected regions of MAPT, APP, and PSEN1 in the frontal cortex and hippocampus of controls (n=26) and those with Alzheimer disease at Stages I to II (n=17); Alzheimer disease at Stages III to IV (n=15); Alzheimer disease at Stages V to VI (n=12); argyrophilic grain disease (n=10); frontotemporal lobar degeneration linked to tau mutations (n=6); frontotemporal lobar degeneration with ubiquitin-immunoreactive inclusions (n=4); frontotemporal lobar degeneration with motor neuron disease (n=3); Pick disease (n=3); Parkinson disease (n=8); dementia with Lewy bodies, pure form (n=5); and dementia with Lewy bodies, common form (n=15). UCHL1 (ubiquitin carboxyl-terminal hydrolase 1 gene) was analyzed in the frontal cortex of controls and those with Parkinson disease and related synucleinopathies. DNA methylation sites were very reproducible in every case. No differences in the percentage of CpG methylation were found between control and disease samples or among the different pathological entities in any region analyzed. Because small changes in methylation of DNA promoters in vulnerable cells might have not been detected in total homogenates, however, these results should be interpreted with caution, particularly as they relate to chronic degenerative diseases in which small modifications may be sufficient to modulate disease progression.

BDNF genetic variants and methylation: effects on cognition in major depressive disorder.

Brain-derived neurotrophic factor (BDNF) gene regulation has been linked to the pathophysiology of major depressive disorder (MDD). MDD patients show cognitive deficits, and altered BDNF regulation has a relevant role in neurocognitive functions. Our goal was to explore the association between BDNF genetic and epigenetic variations with neurocognitive performance in a group of MDD patients and healthy controls considering possible modulating factors. The sample included 134 subjects, 64 MDD patients, and 70 healthy controls. Clinical data, childhood maltreatment, and neurocognitive performance were assessed in all participants. Eleven single nucleotide polymorphisms (SNPs) and two promoter regions in the BDNF gene were selected for genotype and methylation analysis. The role of interactions between BDNF genetic and epigenetic variations with MDD diagnosis, sex, and Childhood Trauma Questionnaire (CTQ) scores was also explored. We observed significant associations between neurocognitive performance and two BDNF SNPs (rs908867 and rs925946), an effect that was significantly mediated by methylation values at specific promoter I sites. We identified significant associations between neurocognitive results and methylation status as well as its interactions with MDD diagnosis, sex, and CTQ scores. Our results support the hypothesis that BDNF gene SNPs and methylation status, as well as their interactions with modulating factors, can influence cognition. Further studies are required to confirm the effect of BDNF variations and cognitive function in larger samples.

Up-regulation of adenosine receptors in the frontal cortex in Alzheimer's disease.

Adenosine receptors are G-protein coupled receptors which modulate neurotransmitter release, mainly glutamate. Adenosine A(1) and A(2A) receptors were studied in post-mortem human cortex in Alzheimer's disease (AD) and age-matched controls. Total adenosine A(1) receptor number, determined by radioligand binding assay, using [(3)H]DPCPX, was significantly increased in AD cases in early and advanced stages without differences with the progression of the disease. A significant increase of A(1)R (37 kDa) levels was also observed by Western blot in early and advanced stages of AD. In addition, increased numbers of adenosine A(2A) receptors were observed in AD samples as determined by a binding assay using [(3)H]ZM 241385 as a radioligand and by Western blot. Increased binding and protein expression levels of adenosine receptors were not associated with increased mRNA levels coding A(1) and A(2A) receptors. Finally, increased A(1) and A(2A) receptor-mediated response was observed. These results show up-regulation of adenosine A(1) and A(2A) receptors in frontal cortex in AD, associated with sensitization of the corresponding transduction pathways.

FKBP5 polymorphisms and hypothalamic-pituitary-adrenal axis negative feedback in major depression and obsessive-compulsive disorder.

Major depressive disorder (MDD) and obsessive-compulsive disorder (OCD) have both been linked to abnormalities in the hypothalamic-pituitary-adrenal (HPA) axis. Polymorphisms in the genes involved in HPA axis activity, such as FKBP5, and their interactions with childhood trauma have been associated with stress-related mental disorders. Our goal was to study the role of FKBP5 genetic variants in HPA axis negative feedback regulation as a possible risk factor for different mental disorders such as MDD and OCD, while controlling for childhood trauma, anxiety and depressive symptoms. The sample included 266 participants divided into three groups: 1) MDD (n = 89 [n = 73 melancholic; n = 3 atypical]), 2) OCD (n = 51; 39% with comorbid MDD [n = 13 melancholic; n = 7 atypical]) and 3) healthy controls (n = 126). Childhood trauma, trait anxiety and depressive symptoms were assessed. HPA negative feedback was analyzed using the dexamethasone suppression test ratio (DSTR) after administration of 0.25 mg of dexamethasone. Twelve SNPs in the FKBP5 gene were selected for genotyping. Multiple linear regressions, after adjusting for the covariates considered, showed a reduced DSTR in individuals with the rs9470079-A variant that was significant after correction for multiple testing. Childhood trauma did not moderate the association between the rs9470079 and DSTR. Our results support the evidence that FKBP5 genetic variation could lead to abnormal HPA axis negative feedback independent of diagnosis. Therefore, this association can be identified as a transdiagnostic feature, offering an interesting opportunity to identify patients with higher stress vulnerability. Further studies focusing on the influence of FKBP5 on measurable biological endophenotypes are needed.

Aquaporin expression in the cerebral cortex is increased at early stages of Alzheimer disease.

Abnormalities in the cerebral microvasculature are common in Alzheimer disease (AD). Expression levels of the water channels aquaporin 1 and aquaporin 4 (AQP1, AQP4) were examined in AD cases by gel electrophoresis and Western blotting, and densitometric values normalized with beta-actin were compared with corresponding values in age-matched controls processed in parallel. In addition, samples of cases with Pick disease (PiD) were examined for comparative purposes. A significant increase in the expression levels of AQP1 was observed in AD stage II (following Braak and Braak classification). Individual variations were seen in advanced stages which resulted in non-significant differences between AD stages V-VI and age-matched controls. No differences in AQP1 levels were observed between familial AD cases (FAD, all of them at advanced stages) and corresponding age-matched controls. Immunohistochemistry showed increased AQP1 in astrocytes at early stages of AD. Double-labelling immunofluorescence and confocal microscopy disclosed AQP1 immunoreactivity at the cell surface of astrocytes which were recognized with anti-glial fibrillary acidic protein antibodies. No differences in the levels of AQP4 were observed in AD, FAD and PiD when compared with corresponding controls. These results indicate abnormal expression of AQP1 in astrocytes in AD, and they add support to the idea that abnormal regulation of mechanisms involved in the control of water fluxes occurs at early stages in AD.

Adenosine A1 receptor protein levels and activity is increased in the cerebral cortex in Creutzfeldt-Jakob disease and in bovine spongiform encephalopathy-infected bovine-PrP mice.

Prion diseases are characterized by neuronal loss, astrocytic gliosis, spongiform change, and abnormal protease-resistant prion protein (PrP) deposition. Creutzfeldt-Jakob disease (CJD) is the most prevalent human prion disease, whereas scrapie and bovine spongiform encephalopathy (BSE) are the most common animal prion diseases. Several candidates have been proposed as mediators of degeneration in prion diseases, one of them glutamate. Recent studies have shown reduced metabotropic glutamate receptor/phospholipase C signaling in the cerebral cortex in CJD, suggesting that this important neuromodulator and neuroprotector pathway is attenuated in CJD. Adenosine is involved in the regulation of different metabolic processes under physiological and pathologic conditions. Adenosine function is mediated by adenosine receptors, which are categorized into 4 types: A1, A2A, A2B, and A3. A1Rs are G-protein-coupled receptors that induce the inhibition of adenylyl cyclase activity. The most dramatic inhibitory actions of adenosine receptors are on the glutamatergic system. For these reasons, we examined the levels of A1Rs in the frontal cortex of 12 patients with CJD and 6 age-matched controls and in BSE-infected bovine-PrP transgenic mice (BoPrP-Tg110 mice) at different postincubation times to address modifications in A1Rs with disease progression. A significant increase in the protein levels of A1Rs was found in the cerebral cortex in CJD and in the murine BSE model at advanced stages of the disease and coincidental with the appearance of PrP expression. In addition, the activity of A1Rs was analyzed by in vitro assays with isolated membranes of the frontal cortex in CJD. Increased activity of the receptor, as revealed by the decreased forskolin-stimulated cAMP production in response to the A1R agonists cyclohexyl adenosine and cyclopentyl adenosine, was observed in CJD cases when compared with controls. Finally, mRNA A1R levels were similar in CJD and control cases, thus suggesting abnormal A1R turnover or dysregulation of raft-associated signaling pathways in CJD. These results show, for the first time, sensitization of A1Rs in prion diseases.

TaqMan PCR assay in the control of RNA normalization in human post-mortem brain tissue.

The brain tissue obtained after death is subjected to several circumstances that can affect RNA integrity. The present study has been directed to reveal possible pitfalls and to control RNA normalization in post-mortem samples in order to recognize the limitations and minimize errors when using TaqMan PCR technology. This has been carried out in samples of the frontal cortex in a series of control and diseased cases covering Parkinson's disease, dementia with Lewy bodies pure form and common form, and Alzheimer's disease. Special attention has been paid to the value of the agonal state, post-mortem delay and pH of the nervous tissue as approximate predictors of the quality of RNA, as well as to the use of the Bioanalyzer to confirm RNA preservation. In addition, since possible disease-modified mRNAs have to be normalized with ideal unaltered RNAs, TaqMan human endogenous control plates have been used to determine the endogenous control most appropriate for the study. beta-glucuronidase (GUS) and beta-actin were good endogenous controls because their expression levels showed a small variation across a representative number of control and pathological cases. RNA stability was also analysed in a paradigm mimicking cumulative delay in tissue processing. GUS mRNA levels were not modified although beta-actin mRNA levels showed degradation at 22 h. Finally, the control of RNA degradation for the normalization of genes of interest was also tested. mRNA expression levels for superoxide dismutase 1 (SOD1) and metalloproteinase domain 22 (ADAM22) were examined at several artificial post-mortem times, and their expression levels compared with those for putative controls beta-actin and GUS. In our paradigm, the expressions of SOD1 and ADAM22 were apparently not modified when normalized with beta-actin. Yet their expression levels were reduced with post-mortem delay when values were normalized with GUS. Taken together, these observations point to practical consequences in TaqMan PCR studies. Short post-mortem delays and acceptable pH of the brain are not sufficient to rule out RNA degradation. The selection of adequate endogenous controls is pivotal in the study. beta-actin and GUS are found to be good endogenous controls in these pathologies, although GUS but not beta-actin expression levels are preserved in samples with long post-mortem delay.

Group I mGluR signaling in BSE-infected bovine-PrP transgenic mice.

Abnormalities of synapses and impaired synaptic transmission appear to be crucial in the pathogenesis of prion diseases. Excitotoxic mechanisms have been postulated as a major cause of neurodegeneration in these conditions. In this line, previous studies have shown abnormal group 1 metabotropic glutamate receptor signaling in Creutzfeldt-Jakob disease (CJD). In the present study, we have examined this pathway by western blotting in the cerebral cortex of bovine-spongiform encephalopathy (BSE)-infected bovine-PrP transgenic mice at different days post-inoculation (dpi). Activation of post-synaptic metabotropic glutamate receptor 1 (mGluR1) promotes phospholipase Cbeta1 (PLCbeta1) activation which may activate, in turn, protein kinase C (PKC), which regulates gene expression. Densitometric analysis of the western blot bands revealed no differences in the protein levels of (mGluR1) through time, but demonstrated decreased levels of PLCbeta1 and protein kinase C delta (nPKCdelta) at 270dpi, at the time when mice showed neurological deficits accompanied by neuropathological changes and PrPres deposition in the brain. The present results show, for the first time impairment of the mGluR1/PLCbeta1/PKCdelta pathway signaling with disease-progression in a murine model of BSE.

Amyloid-beta deposition in the cerebral cortex in Dementia with Lewy bodies is accompanied by a relative increase in AbetaPP mRNA isoforms containing the Kunitz protease inhibitor.

Deposition of amyloid-beta, the fibrillogenic product of the cell surface protein AbetaPP (amyloid-beta protein precursor), occurs in the cerebral cortex of patients with Dementia with Lewy bodies (DLB). Amyloid deposition, basically in the form of senile plaques, occurs not only in the common form (DLBc), which is defined by changes consistent with diffuse Lewy body disease accompanied by Alzheimer's disease (AD), but also in the pure form (DLBp), in which neurofibrillary tangles are absent. The present study analyses the expression of AbetaPP mRNA isoforms with (AbetaPP751 and AbetaPP770) and without (AbetaPP695) the Kunitz-type serine protease inhibitor (KPI) domain, in the cerebral cortex in DLBc (n=4), DLBp (n=4), Parkinson's disease (PD, n=5), AD (n=3 stages I-IIA, and n=4 stage VC of Braak and Braak), amyloid angiopathy (AA, n=2) and progressive supranuclear palsy (PSP, n=4) compared with age-matched controls (n=6). For this purpose, TaqMan RT-PCR assay was used on frozen post-mortem samples of the frontal cortex (area 8) obtained with short post-mortem delays (8.29+/-4.57 h) and strict RNA preservation (A260/280 of 1.78+/-0.15). A 3.66-fold, 6.67-fold, 4.28-fold and 5.24-fold increases, in the (AbetaPP751+AbetaPP770)/AbetaPP695 mRNA ratio were found in DLBc, DLBp, AD stage VC and AA, respectively, when compared with controls. No modifications in the ratio were found in PD, AD stage I-IIA and PSP. These findings suggest that alternative splicing of the AbetaPP mRNA may play a role in betaA4 amyloidogenesis in DLBp, DLBc, AD stage VC and Amyloid angiopathy.