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.

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.

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.

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.

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.

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 adenosine A2A receptor levels define a molecular subgroup in schizophrenia.

Schizophrenia (SZ) is a mental disorder of unknown origin. Some scientific evidence seems to indicate that SZ is not a single disease entity, since there are patient groups with clear symptomatic, course and biomarker differences. SZ is characterized by a hyperdopaminergic state related to high dopamine D2 receptor activity. It has also been proposed that there is a hypoadenosynergic state. Adenosine is a nucleoside widely distributed in the organism with neuromodulative and neuroprotective activity in the central nervous system. In the brain, the most abundant adenosine receptors are A1R and A2AR. In the present report, we characterize the presence of both receptors in human postmortem putamens of patients suffering SZ with real time TaqMan PCR, western blotting and radioligand binding assay. We show that A1R levels remain unchanged with respect to age-matched controls, whereas nearly fifty percent of patients have reduced A2AR, at the transcriptional and translational levels. Moreover, we describe how DNA methylation plays a role in the pathological A2AR levels with the bisulfite-sequencing technique. In fact, an increase in 5-methylcytosine percentage in the 5' UTR region of ADORA2A was found in those SZ patients with reduced A2AR levels. Interestingly, there was a relationship between the A2A/ß-actin ratio and motor disturbances as assessed with some items of the PANSS, AIMS and SAS scales. Therefore, there may be a subgroup of SZ patients with reduced striatal A2AR levels accompanied by an altered motor phenotype.

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.

Increased 5-methylcytosine and decreased 5-hydroxymethylcytosine levels are associated with reduced striatal A2AR levels in Huntington's disease.

Adenosine A2A receptor (A2AR) is a G-protein-coupled receptor highly expressed in basal ganglia. Its expression levels are severely reduced in Huntington's disease (HD), and several pharmacological therapies have shown its implication in this neurodegenerative disorder. The main goal of this study was to gain insight into the molecular mechanisms that regulate A2AR gene (ADORA2A) expression in HD. Based on previous data reported by our group, we measured the methylcytosine (5mC) and hydroxymethylcytosine (5hmC) content in the 5'UTR region of ADORA2A in the putamen of HD patients and in the striatum of R6/1 and R6/2 mice at late stages of the disease. In this genomic region, 5mC and 5hmC remained unchanged in both mice strains, although low striatal A2AR levels were associated with reduced 5mC levels in 30-week-old R6/1 mice and reduced 5hmC levels in 12-week-old R6/2 mice in exon m2. In order to analyze when this mechanism appears during the progression of the disease, a time course for A2AR protein levels was carried out in R6/1 mice striatum (8, 12, and 20 weeks of age). A2AR levels were reduced from 12 weeks of age onwards, and this downregulation was concomitant with reduced 5hmC levels in the 5'UTR region of ADORA2A. Interestingly, increased 5mC levels and reduced 5hmC were found in the 5'UTR region of ADORA2A in the putamen of HD patients with respect to age-matched controls. Therefore, an altered DNA methylation pattern in ADORA2A seems to play a role in the pathologically decreased A2AR expression levels found in HD.

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.

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.

The locus control region of the MHC class II promoter acts as a repressor element, the activity of which is inhibited by CIITA.

The closest region of the promoter of MHC II genes and particularly three conserved boxes (X, Y and S) are fundamental for the transcriptional regulation. A second set of conserved sequences is present approximately 1200-1500 bp upstream in opposite orientation. In transient transfection experiments in IFN-gamma-treated macrophages and in B lymphocytes, we determined the expression of a fragment of 2035 bp of the I-Abeta gene, which contains the upstream boxes. Mutation of the distal boxes increased induction, thereby suggesting a repressive effect on transcription. In vitro, the proximal and distal ends of I-Abeta promoter were ligated in the presence of nuclear extracts from untreated macrophages but not when the extracts were obtained from IFN-gamma-stimulated cells. The mutation of distal or proximal boxes resulted in a decrease in the ligation assay. The addition of recombinant CIITA to untreated nuclear extracts decreased the capacity of the promoter to be ligated. Finally, we observed increased capacity to ligate the promoter in extracts from B cells lacking CIITA, but not from B cells lacking RFXANK. These results allow us to postulate a model where the proteins in the proximal and distal conserved sequences interact. When CIITA is induced, these proteins make an enhanceosome, allowing chromatin to open and initiate transcription.

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.

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.

Brain banks: benefits, limitations and cautions concerning the use of post-mortem brain tissue for molecular studies.

Brain banks are facilities providing an interface between generous donation of nervous tissues and research laboratories devoted to increase our understanding of the diseases of the nervous system, discover new diagnostic targets, and develop new strategies. Considering this crucial role, it is important to learn about the suitabilities, limitations and proper handling of individual brain samples for particular studies. Several factors may interfere with preservation of DNA, RNA, proteins and lipids, and, therefore, special care must be taken first to detect sub-optimally preserved tissues and second to provide adequate material for each specific purpose. Basic aspects related with DNA, RNA and protein preservation include agonal state, post-mortem delay, temperature of storage and procedures of tissue preservation. Examination of DNA and RNA preservation is best done by using bioanalyzer technologies instead of less sensitive methods such as agarose gels. Adequate RNA preservation is mandatory in RNA microarray studies and adequate controls are necessary for proper PCR validation. Like for RNA, the preservation of proteins is not homogeneous since some molecules are more vulnerable than others. This aspect is crucial in the study of proteins including expression levels and possible post-translational modifications. Similarly, the reliability of functional and enzymatic studies in human post-mortem brain largely depends on protein preservation. Much less is known about other aspects, such as the effects of putative deleterious factors on epigenetic events such as methylation of CpGs in gene promoters, nucleosome preservation, histone modifications, and conservation of microRNA species. Most brains are appropriate for morphological approaches but not all brains are useful for certain biochemical and molecular studies.

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.

Target genes of neuron-restrictive silencer factor are abnormally up-regulated in human myotilinopathy.

Myotilinopathy is a subgroup of myofibrillar myopathies caused by mutations in the myotilin gene in which there is aggregation of abnormal cytoskeletal proteins and ubiquitin. We report here on the accumulation of neuron-related proteins such as ubiquitin carboxy-terminal hydrolase L1 (UCHL1), synaptosomal-associated protein 25, synaptophysin, and alpha-internexin in aberrant protein aggregates in myotilinopathy. We have determined that the neuron-restrictive silencer factor (NRSF)/RE1 silencing transcription factor (REST), a transcription factor expressed in non-neuronal tissues repressing the expression of several neuronal genes, is reduced in myotilinopathies. Moreover, NRSF transfection reduces UCHL1, synaptosomal-associated protein 25, synaptophysin, and alpha-internexin mRNA levels in DMS53 cells, whereas short interferring NRSF transfection increases UCHL1 and synaptophysin mRNA levels in U87-MG cells. Chromatin immunoprecipitation assays have shown that NRSF interacts with the UCHL1 promoter in U87-MG and HeLa cells. In silico analysis of the UCHL1 gene promoter sequence using the MatInspector software has predicted three potential neuron-restrictive silencer elements (NRSEs): NRSE1 located in the complementary DNA chain and NRSE2 and NRSE3 in intron 1, in the coding and complementary chains, respectively. Together, these findings show, for the first time, abnormal regulation of NRSF/REST as a mechanism associated with the aberrant expression of selected neuron-related proteins, which in turn accumulate in abnormal protein aggregates, in myotilinopathy.