Aquaporin-4 Expression Switches from White to Gray Matter Regions during Postnatal Development of the Central Nervous System.

Aquaporin-4 (AQP4) is the most abundant water channel in the central nervous system and plays a fundamental role in maintaining water homeostasis there. In adult mice, AQP4 is located mainly in ependymal cells, in the endfeet of perivascular astrocytes, and in the glia limitans. Meanwhile, its expression, location, and function throughout postnatal development remain largely unknown. Here, the expression of AQP4 mRNA was studied by in situ hybridization and RT-qPCR, and the localization and amount of protein was studied by immunofluorescence and western blotting, both in the brain and spinal cord. For this, wild-type mice of the C57BL/6 line, aged 1, 3, 7, 11, 20, and 60 days, and 18 months were used. The results showed a change in both the expression and location of AQP4 in postnatal development compared to those during adult life. In the early stages of postnatal development it appears in highly myelinated areas, such as the corpus callosum or cerebellum, and as the animal grows, it disappears from these areas, passing through the cortical regions of the forebrain and concentrating around the blood vessels. These findings suggest an unprecedented possible role for AQP4 in the early cell differentiation process, during the first days of life in the newborn animal, which will lead to myelination.

Generation of Periventricular Reactive Astrocytes Overexpressing Aquaporin 4 Is Stimulated by Mesenchymal Stem Cell Therapy.

Aquaporin-4 (AQP4) plays a crucial role in brain water circulation and is considered a therapeutic target in hydrocephalus. Congenital hydrocephalus is associated with a reaction of astrocytes in the periventricular white matter both in experimental models and human cases. A previous report showed that bone marrow-derived mesenchymal stem cells (BM-MSCs) transplanted into the lateral ventricles of hyh mice exhibiting severe congenital hydrocephalus are attracted by the periventricular astrocyte reaction, and the cerebral tissue displays recovery. The present investigation aimed to test the effect of BM-MSC treatment on astrocyte reaction formation. BM-MSCs were injected into the lateral ventricles of four-day-old hyh mice, and the periventricular reaction was detected two weeks later. A protein expression analysis of the cerebral tissue differentiated the BM-MSC-treated mice from the controls and revealed effects on neural development. In in vivo and in vitro experiments, BM-MSCs stimulated the generation of periventricular reactive astrocytes overexpressing AQP4 and its regulatory protein kinase D-interacting substrate of 220 kDa (Kidins220). In the cerebral tissue, mRNA overexpression of nerve growth factor (NGF), vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1 (HIF1α), and transforming growth factor beta 1 (TGFß1) could be related to the regulation of the astrocyte reaction and AQP4 expression. In conclusion, BM-MSC treatment in hydrocephalus can stimulate a key developmental process such as the periventricular astrocyte reaction, where AQP4 overexpression could be implicated in tissue recovery.

Predictive Value of Serum Antibodies and Point Mutations of AQP4, AQP1 and MOG in A Cohort of Spanish Patients with Neuromyelitis Optica Spectrum Disorders.

The detection of IgG aquaporin-4 antibodies in the serum of patients with Neuromyelitis optica (NMO) has dramatically improved the diagnosis of this disease and its distinction from multiple sclerosis. Recently, a group of patients have been described who have an NMO spectrum disorder (NMOsd) and who are seronegative for AQP4 antibodies but positive for IgG aquaporin-1 (AQP1) or myelin oligodendrocyte glycoprotein (MOG) antibodies. The purpose of this study was to determine whether AQP1 and MOG could be considered new biomarkers of this disease; and if point mutations in the gDNA of AQP4, AQP1 and MOG genes could be associated with the etiology of NMOsd. We evaluated the diagnostic capability of ELISA and cell-based assays (CBA), and analyzed their reliability, specificity, and sensitivity in detecting antibodies against these three proteins. The results showed that both assays can recognize these antigen proteins under appropriate conditions, but only anti-AQP4 antibodies, and not AQP1 or MOG, appears to be a clear biomarker for NMOsd. CBA is the best method for detecting these antibodies; and serum levels of AQP4 antibodies do not correlate with the progression of this disease. So far, the sequencing analysis has not revealed a genetic basis for the etiology of NMOsd, but a more extensive analysis is required before definitive conclusions can be drawn.

Aquaporin-1 plays important role in proliferation by affecting cell cycle progression.

Aquaporin-1 (AQP1) has been associated with tumor development. Here, we investigated how AQP1 may affect cell proliferation. The proliferative rate of adult carotid body (CB) cells, known to proliferate under chronic hypoxia, was analyzed in wild-type (AQP1(+/+) ) and knock out (AQP1(-/-) ) mice, maintained in normoxia or exposed to hypoxia while BrdU was administered. Fewer numbers of total BrdU(+) and TH-BrdU(+) cells were observed in AQP1(-/-) mice, indicating a role for AQP1 in CB proliferation. Then, by flow cytometry, cell cycle state and proliferation of cells overexpressing AQP1 were compared to those of wild-type cells. In the AQP1-overexpressing cells, we observed higher cell proliferation and percentages of cells in phases S and G2/M and fewer apoptotic cells after nocodazole treatment were detected by annexin V staining. Also in these cells, proteomic assays showed higher expression of cyclin D1 and E1 and microarray analysis revealed changes in many cell proliferation-related molecules, including, Zeb 2, Jun, NF-kß, Cxcl9, Cxcl10, TNF, and the TNF receptor. Overall, our results indicate that the presence of AQP1 modifies the expression of key cell cycle proteins apparently related to increases in cell proliferation. This contributes to explaining the presence of AQP1 in many different tumors.

Comparative Analysis for the Presence of IgG Anti-Aquaporin-1 in Patients with NMO-Spectrum Disorders.

Detection of IgG anti-Aquaporin-4 (AQP4) in serum of patients with Neuromyelitis optica syndrome disorders (NMOSD) has improved diagnosis of these processes and differentiation from Multiple sclerosis (MS). Recent findings also claim that a subgroup of patients with NMOSD, serum negative for IgG-anti-AQP4, present antibodies anti-AQP1 instead. Explore the presence of IgG-anti-AQP1 using a previously developed cell-based assay (CBA) highly sensitive to IgG-anti-AQP4. Serum of 205 patients diagnosed as NMOSD (8), multiple sclerosis (94), optic neuritis (39), idiopathic myelitis (29), other idiopathic demyelinating disorders of the central nervous system (9), other neurological diseases (18) and healthy controls (8), were used in a CBA over fixed HEK cells transfected with hAQP1-EGFP or hM23-AQP4-EGFP, treated with Triton X-100 and untreated. ELISA was also performed. Analysis of serum with our CBA indicated absence of anti-AQP1 antibodies, whereas in cells pretreated with detergent, noisy signal made reliable detection impossible. ELISA showed positive results in few serums. The low number of NMOSD serums included in our study reduces its power to conclude the specificity of AQP1 antibodies as new biomarkers of NMOSD. Our study does not sustain detection of anti-AQP1 in serum of NMOSD patients but further experiments are expected.

An immunoassay that distinguishes real neuromyelitis optica signals from a labeling detected in patients receiving natalizumab.

BACKGROUND: Cell-based assays for neuromyelitis optica (NMO) diagnosis are the most sensitive and specific methods to detect anti-aquaporin 4 (AQP4) antibodies in serum, but some improvements in their quantitative and specificity capacities would be desirable. Thus the aim of the present work was to develop a sensitive quantitative method for detection of anti-AQP4 antibodies that allows clear diagnosis of NMO and distinction of false labeling produced by natalizumab treatment. METHODS: Sera from 167 individuals, patients diagnosed with NMO (16), multiple sclerosis (85), optic neuritis (24), idiopathic myelitis (21), or other neurological disorders (13) and healthy controls (8), were used as the primary antibody in an immunofluorescence assay on HEK cells transfected with the M23 isoform of human AQP4 fused with enhanced green fluorescent protein. Cells used were freshly transfected or stored frozen and then thawed just before adding the serum. RESULTS: Microscopic observation and fluorescence quantification produced similar results in fresh and frozen samples. Serum samples from patients diagnosed with NMO were 100% positive for anti-AQP4 antibodies, while all the other sera were negative. Using serum from patients treated with natalizumab, a small and unspecific fluorescent signal was produced from all HEK cells, regardless of AQP4 expression. CONCLUSIONS: Our cell-based double-label fluorescence immunoassay protocol significantly increases the signal specificity and reduces false diagnosis of NMO patients, especially in those receiving natalizumab treatment. Frozen pretreated cells allow faster detection of anti-AQP4 antibodies.

Impact of aquaporin-4 and CD11c + microglia in the development of ependymal cells in the aqueduct: inferences to hydrocephalus.

AQP4 is expressed in the endfeet membranes of subpial and perivascular astrocytes and in the ependymal cells that line the ventricular system. The sporadic appearance of obstructive congenital hydrocephalus (OCHC) has been observed in the offspring of AQP4-/- mice (KO) due to stenosis of Silvio's aqueduct. Here, we explore whether the lack of AQP4 expression leads to abnormal development of ependymal cells in the aqueduct of mice. We compared periaqueductal samples from wild-type and KO mice. The microarray-based transcriptome analysis reflected a large number of genes with differential expression (809). Gene sets (GS) associated with ependymal development, ciliary function and the immune system were specially modified qPCR confirmed reduced expression in the KO mice genes: (i) coding for transcription factors for ependymal differentiation (Rfx4 and FoxJ1), (ii) involved in the constitution of the central apparatus of the axoneme (Spag16 and Hydin), (iii) associated with ciliary assembly (Cfap43, Cfap69 and Ccdc170), and (iv) involved in intercellular junction complexes of the ependyma (Cdhr4). By contrast, genes such as Spp1, Gpnmb, Itgax, and Cd68, associated with a Cd11c-positive microglial population, were overexpressed in the KO mice. Electron microscopy and Immunofluorescence of vimentin and γ-tubulin revealed a disorganized ependyma in the KO mice, with changes in the intercellular complex union, unevenly orientated cilia, and variations in the planar cell polarity of the apical membrane. These structural alterations translate into reduced cilia beat frequency, which might alter cerebrospinal fluid movement. The presence of CD11c + microglia cells in the periaqueductal zone of mice during the first postnatal week is a novel finding. In AQP4-/- mice, these cells remain present around the aqueduct for an extended period, showing peak expression at P11. We propose that these cells play an important role in the normal development of the ependyma and that their overexpression in KO mice is crucial to reduce ependyma abnormalities that could otherwise contribute to the development of obstructive hydrocephalus.

Full protection from SARS-CoV-2 brain infection and damage in susceptible transgenic mice conferred by MVA-CoV2-S vaccine candidate.

Vaccines against SARS-CoV-2 have been shown to be safe and effective but their protective efficacy against infection in the brain is yet unclear. Here, in the susceptible transgenic K18-hACE2 mouse model of severe coronavirus disease 2019 (COVID-19), we report a spatiotemporal description of SARS-CoV-2 infection and replication through the brain. SARS-CoV-2 brain replication occurs primarily in neurons, leading to neuronal loss, signs of glial activation and vascular damage in mice infected with SARS-CoV-2. One or two doses of a modified vaccinia virus Ankara (MVA) vector expressing the SARS-CoV-2 spike (S) protein (MVA-CoV2-S) conferred full protection against SARS-CoV-2 cerebral infection, preventing virus replication in all areas of the brain and its associated damage. This protection was maintained even after SARS-CoV-2 reinfection. These findings further support the use of MVA-CoV2-S as a promising vaccine candidate against SARS-CoV-2/COVID-19.

Cellular Distribution of Brain Aquaporins and Their Contribution to Cerebrospinal Fluid Homeostasis and Hydrocephalus.

Brain aquaporins facilitate the movement of water between the four water compartments: blood, cerebrospinal fluid, interstitial fluid, and intracellular fluid. This work analyzes the expression of the four most abundant aquaporins (AQPs) (AQP1, AQP4, AQP9, and AQP11) in the brains of mice and discuss their contribution to hydrocephalus. We analyzed available data from single-cell RNA sequencing of the central nervous system of mice to describe the expression of aquaporins and compare their distribution with that based on qPCR, western blot, and immunohistochemistry assays. Expression of AQP1 in the apical cell membrane of choroid plexus epithelial cells and of AQP4 in ependymal cells, glia limitans, and astrocyte processes in the pericapillary end foot is consistent with the involvement of both proteins in cerebrospinal fluid homeostasis. The expression of both aquaporins compensates for experimentally induced hydrocephalus in the animals. Recent data demonstrate that hypoxia in aged animals alters AQP4 expression in the choroidal plexus and cortex, increasing the ventricle size and intraventricular pressure. Cerebral distensibility is reduced in parallel with a reduction in cerebrospinal fluid drainage and cognitive deterioration. We propose that aged mice chronically exposed to hypoxia represent an excellent experimental model for studying the pathophysiological characteristics of idiopathic normal pressure hydrocephalus and roles for AQPs in such disease.

Identification of genetic factors associated with susceptibility to angiotensin-converting enzyme inhibitors-induced cough.

BACKGROUND AND OBJECTIVE: Angiotensin-converting enzyme inhibitors (ACEi) are the first selected drugs for hypertensive patients because of its protective properties against heart and kidney diseases. Persistent cough is a common adverse reaction associated with ACEi, which can bind to the treatment cessation, but its etiology remains an unresolved issue. The most accepted mechanism is that the inhibition of ACEi increases kinins levels, resulting in the activation of proinflammatory mechanisms and nitric oxide generation. However, relatively little is known about the genetic susceptibility to ACEi-induced cough in hypertensive patients. METHODS: We carried out a monogenic association analysis of 39 polymorphisms and haplotypes in genes encoding key proteins related to ACEi activity with the occurrence of ACEi-induced cough. We also carried out a digenic association analysis and investigated the existence of epistatic interactions between the analyzed polymorphisms using a logistic regression procedure. Finally, we investigated the predictive value of the identified associations for ACEi-induced cough. RESULTS: We found that genetic polymorphisms in MME [rs2016848, P=0.002, odds ratio (OR)=1.795], BDKRB2 (rs8012552, P=0.012, OR=1.609), PTGER3 (rs11209716, P=0.002, OR=0.565), and ACE (rs4344) genes are associated with ACEi-related cough. For the latter, the effect is sex specific, having a protective effect in males (P=0.027, OR=0.560) and increasing the risk in females (P=0.031, OR=1.847). In addition, genetic interactions between peptidases involved in kinins levels (CPN1 and XPNPEP1) and proteins related to prostaglandin metabolism (PTGIS and PTGIR) strongly modify the risk of ACEi-induced cough presentation (0.102≤OR≤0.384 for protective combinations and 2.732≤OR≤7.216 for risk combinations). CONCLUSION: These results are consistent with the hypothesis that the mechanism of cough is related to the accumulation of bradykinin, substance P, and prostaglandins.

Genetic association of complement receptor 1 polymorphism rs3818361 in Alzheimer's disease.

Complement receptor 1 gene polymorphism rs3818361 was recently shown to increase the risk of Alzheimer's disease (AD). We performed an independent replication study of this genetic variant in 2,470 individuals from Spain. By applying an allelic model, we observed a trend toward an association between this marker and late-onset AD susceptibility in our case-control study (odds ratio = 1.114, 95% confidence interval: 0.958-1.296, P = .16). Meta-analysis of available studies (n = 31,771 individuals), including previous studies and public genome-wide association study resources (Alzheimer's Disease Neuroimaging Initiative, Translational Genomics Research Institute, and Multi-site Collaborative Study for Genotype-Phenotype Associations in Alzheimer's Disease), strongly supports the effect of rs3818361 (odds ratio = 1.180, 95% confidence interval: 1.113-1.252, P < 2.99E-8) and suggests the existence of between-study heterogeneity (P < .05). We concluded that the complement receptor 1 gene may contribute to AD risk, although its effect size could be smaller than previously estimated.

Evaluation of aquaporins in the cerebrospinal fluid in patients with idiopathic normal pressure hydrocephalus.

Brain aquaporin 1 (AQP1) and AQP4 are involved in cerebrospinal fluid (CSF) homeostasis and might participate in the origin of hydrocephalus. Studies have shown alterations of perivascular AQP4 expression in idiopathic normal pressure hydrocephalus (iNPH) and Alzheimer's disease (AD). Due to the overlapping of clinical signs between iNPH and certain neurological conditions, mainly AD, specific biomarkers might improve the diagnostic accuracy for iNPH. The goal of the present study was to analyze and quantify the presence of AQP1 and AQP4 in the CSF of patients with iNPH and AD to determine whether these proteins can be used as biomarkers of iNPH. We examined AQP1 and AQP4 protein levels in the CSF of 179 participants (88 women) classified into 5 groups: possible iNPH (81 participants), hydrocephalus associated with other neurological disorders (13 participants), AD (41 participants), non-AD dementia (32 participants) and healthy controls (12 participants). We recorded each participant's demographic and clinical variables and indicated, when available in the clinical history, the record of cardiovascular and respiratory complications. An ELISA showed virtually no AQP content in the CSF. Information on the vascular risk factors (available for 61 patients) confirmed some type of vascular risk factor in 86% of the patients with possible iNPH and 58% of the patients with AD. In conclusion, the ELISA analysis showed insufficient sensitivity to detect the presence of AQP1 and AQP4 in CSF, ruling out the possible use of these proteins as biomarkers for diagnosing iNPH.

Genetic structure of the Spanish population.

BACKGROUND: Genetic admixture is a common caveat for genetic association analysis. Therefore, it is important to characterize the genetic structure of the population under study to control for this kind of potential bias. RESULTS: In this study we have sampled over 800 unrelated individuals from the population of Spain, and have genotyped them with a genome-wide coverage. We have carried out linkage disequilibrium, haplotype, population structure and copy-number variation (CNV) analyses, and have compared these estimates of the Spanish population with existing data from similar efforts. CONCLUSIONS: In general, the Spanish population is similar to the Western and Northern Europeans, but has a more diverse haplotypic structure. Moreover, the Spanish population is also largely homogeneous within itself, although patterns of micro-structure may be able to predict locations of origin from distant regions. Finally, we also present the first characterization of a CNV map of the Spanish population. These results and original data are made available to the scientific community.

Genome-wide analysis of genetic loci associated with Alzheimer disease.

CONTEXT: Genome-wide association studies (GWAS) have recently identified CLU, PICALM, and CR1 as novel genes for late-onset Alzheimer disease (AD). OBJECTIVES: To identify and strengthen additional loci associated with AD and confirm these in an independent sample and to examine the contribution of recently identified genes to AD risk prediction in a 3-stage analysis of new and previously published GWAS on more than 35,000 persons (8371 AD cases). DESIGN, SETTING, AND PARTICIPANTS: In stage 1, we identified strong genetic associations (P < 10(-3)) in a sample of 3006 AD cases and 14,642 controls by combining new data from the population-based Cohorts for Heart and Aging Research in Genomic Epidemiology consortium (1367 AD cases [973 incident]) with previously reported results from the Translational Genomics Research Institute and the Mayo AD GWAS. We identified 2708 single-nucleotide polymorphisms (SNPs) with P < 10(-3). In stage 2, we pooled results for these SNPs with the European AD Initiative (2032 cases and 5328 controls) to identify 38 SNPs (10 loci) with P < 10(-5). In stage 3, we combined data for these 10 loci with data from the Genetic and Environmental Risk in AD consortium (3333 cases and 6995 controls) to identify 4 SNPs with P < 1.7x10(-8). These 4 SNPs were replicated in an independent Spanish sample (1140 AD cases and 1209 controls). Genome-wide association analyses were completed in 2007-2008 and the meta-analyses and replication in 2009. MAIN OUTCOME MEASURE: Presence of Alzheimer disease. RESULTS: Two loci were identified to have genome-wide significance for the first time: rs744373 near BIN1 (odds ratio [OR],1.13; 95% confidence interval [CI],1.06-1.21 per copy of the minor allele; P = 1.59x10(-11)) and rs597668 near EXOC3L2/BLOC1S3/MARK4 (OR, 1.18; 95% CI, 1.07-1.29; P = 6.45x10(-9)). Associations of these 2 loci plus the previously identified loci CLU and PICALM with AD were confirmed in the Spanish sample (P < .05). However, although CLU and PICALM were confirmed to be associated with AD in this independent sample, they did not improve the ability of a model that included age, sex, and APOE to predict incident AD (improvement in area under the receiver operating characteristic curve from 0.847 to 0.849 in the Rotterdam Study and 0.702 to 0.705 in the Cardiovascular Health Study). CONCLUSIONS: Two genetic loci for AD were found for the first time to reach genome-wide statistical significance. These findings were replicated in an independent population. Two recently reported associations were also confirmed. These loci did not improve AD risk prediction. While not clinically useful, they may implicate biological pathways useful for future research.

Expression Pattern of Aquaporin 1 and Aquaporin 3 in Melanocytic and Nonmelanocytic Skin Tumors.

OBJECTIVES: Study of aquaporin 1 (AQP1) and aquaporin 3 (AQP3) expression to understand its potential role in the pathophysiology of skin cancer. METHODS: Analysis of AQP1 and AQP3 expression by immunohistochemistry of 72 skin biopsy specimens from melanocytic skin tumors, nonmelanocytic tumors, or healthy samples. RESULTS: AQP1 showed strong labeling in 100% of benign common melanocytic nevi. Small blood vessels, stroma, and melanophages surrounding different types of melanomas tumors also were positive. Tumoral melanocytes in atypical nevi and melanomas were negative for AQP1. AQP3 showed strong labeling in 100% of melanocytic nevi, 100% of atypical melanocytic nevi, and 100% of melanomas. In all basal cell carcinomas and squamous cell carcinomas, staining for AQP3 was positive. CONCLUSIONS: To our knowledge, this work represents the first demonstration of AQP1/AQP3 expression in human melanocytic skin tumors. More studies are needed to understand the underlying molecular mechanisms of expression of both AQPs in melanocytic tumors and their potential as molecular therapeutic targets.

A method for detecting epistasis in genome-wide studies using case-control multi-locus association analysis.

BACKGROUND: The difficulty in elucidating the genetic basis of complex diseases roots in the many factors that can affect the development of a disease. Some of these genetic effects may interact in complex ways, proving undetectable by current single-locus methodology. RESULTS: We have developed an analysis tool called Hypothesis Free Clinical Cloning (HFCC) to search for genome-wide epistasis in a case-control design. HFCC combines a relatively fast computing algorithm for genome-wide epistasis detection, with the flexibility to test a variety of different epistatic models in multi-locus combinations. HFCC has good power to detect multi-locus interactions simulated under a variety of genetic models and noise conditions. Most importantly, HFCC can accomplish exhaustive genome-wide epistasis search with large datasets as demonstrated with a 400,000 SNP set typed on a cohort of Parkinson's disease patients and controls. CONCLUSION: With the current availability of genetic studies with large numbers of individuals and genetic markers, HFCC can have a great impact in the identification of epistatic effects that escape the standard single-locus association analyses.

Interaction between Calpain 5, Peroxisome proliferator-activated receptor-gamma and Peroxisome proliferator-activated receptor-delta genes: a polygenic approach to obesity.

CONTEXT: Obesity is a multifactorial disorder, that is, a disease determined by the combined effect of genes and environment. In this context, polygenic approaches are needed. OBJECTIVE: To investigate the possibility of the existence of a crosstalk between the CALPAIN 10 homologue CALPAIN 5 and nuclear receptors of the peroxisome proliferator-activated receptors family. DESIGN: Cross-sectional, genetic association study and gene-gene interaction analysis. SUBJECTS: The study sample comprise 1953 individuals, 725 obese (defined as body mass index > or = 30) and 1228 non obese subjects. RESULTS: In the monogenic analysis, only the peroxisome proliferator-activated receptor delta (PPARD) gene was associated with obesity (OR = 1.43 [1.04-1.97], p = 0.027). In addition, we have found a significant interaction between CAPN5 and PPARD genes (p = 0.038) that reduces the risk for obesity in a 55%. CONCLUSION: Our results suggest that CAPN5 and PPARD gene products may also interact in vivo.

Methylation alterations are not a major cause of PTTG1 misregulation.

BACKGROUND: On its physiological cellular context, PTTG1 controls sister chromatid segregation during mitosis. Within its crosstalk to the cellular arrest machinery, relies a checkpoint of integrity for which gained the over name of securin. PTTG1 was found to promote malignant transformation in 3T3 fibroblasts, and further found to be overexpressed in different tumor types. More recently, PTTG1 has been also related to different processes such as DNA repair and found to trans-activate different cellular pathways involving c-myc, bax or p53, among others. PTTG1 over-expression has been correlated to a worse prognosis in thyroid, lung, colorectal cancer patients, and it can not be excluded that this effect may also occur in other tumor types. Despite the clinical relevance and the increasing molecular characterization of PTTG1, the reason for its up-regulation remains unclear. METHOD: We analysed PTTG1 differential expression in PC-3, DU-145 and LNCaP tumor cell lines, cultured in the presence of the methyl-transferase inhibitor 5-Aza-2'-deoxycytidine. We also tested whether the CpG island mapping PTTG1 proximal promoter evidenced a differential methylation pattern in differentiated thyroid cancer biopsies concordant to their PTTG1 immunohistochemistry status. Finally, we performed whole-genome LOH studies using Affymetix 50 K microarray technology and FRET analysis to search for allelic imbalances comprising the PTTG1 locus. CONCLUSION: Our data suggest that neither methylation alterations nor LOH are involved in PTTG1 over-expression. These data, together with those previously reported, point towards a post-transcriptional level of misregulation associated to PTTG1 over-expression.

Combined effects of aquaporin-4 and hypoxia produce age-related hydrocephalus.

Aquaporin-4, present in ependymal cells, in glia limiting and abundantly in pericapillary astrocyte foot processes, and aquaporin-1, expressed in choroid plexus epithelial cells, play an important role in cerebrospinal fluid production and may be involved in the pathophysiology of age-dependent hydrocephalus. The finding that brain aquaporins expression is regulated by low oxygen tension led us to investigate how hypoxia and elevated levels of cerebral aquaporins may result in an increase in cerebrospinal fluid production that could be associated with a hydrocephalic condition. Here we have explored, in young and aged mice exposed to hypoxia, whether aquaporin-4 and aquaporin-1 participate in the development of age-related hydrocephalus. Choroid plexus, striatum, cortex and ependymal tissue were analyzed separately both for mRNA and protein levels of aquaporins. Furthermore, parameters such as total ventricular volume, intraventricular pressure, cerebrospinal fluid outflow rate, ventricular compliance and cognitive function were studied in wild type, aquaporin-1 and aquaporin-4 knock-out animals subjected to hypoxia or normoxia. Our data demonstrate that hypoxia is involved in the development of age-related hydrocephalus by a process that depends on aquaporin-4 channels as a main route for cerebrospinal fluid movement. Significant increases in aquaporin-4 expression that occur over the course of animal aging, together with a reduced cerebrospinal fluid outflow rate and ventricular compliance, contribute to produce more severe hydrocephalus related to hypoxic events in aged mice, with a notable impairment in cognitive function. These results indicate that physiological events and/or pathological conditions presenting with cerebral hypoxia/ischemia contribute to the development of chronic adult hydrocephalus.

Calpain-5 gene variants are associated with diastolic blood pressure and cholesterol levels.

BACKGROUND: Genes implicated in common complex disorders such as obesity, type 2 diabetes mellitus (T2DM) or cardiovascular diseases are not disease specific, since clinically related disorders also share genetic components. Cysteine protease Calpain 10 (CAPN10) has been associated with T2DM, hypertension, hypercholesterolemia, increased body mass index (BMI) and polycystic ovary syndrome (PCOS), a reproductive disorder of women in which isunlin resistance seems to play a pathogenic role. The calpain 5 gene (CAPN5) encodes a protein homologue of CAPN10. CAPN5 has been previously associated with PCOS by our group. In this new study, we have analysed the association of four CAPN5 gene variants(rs948976A>G, rs4945140G>A, rs2233546C>T and rs2233549G>A) with several cardiovascular risk factors related to metabolic syndrome in general population. METHODS: Anthropometric measurements, blood pressure, insulin, glucose and lipid profiles were determined in 606 individuals randomly chosen from a cross-sectional population-based epidemiological survey in the province of Segovia in Central Spain (Castille), recruited to investigate the prevalence of anthropometric and physiological parameters related to obesity and other components of the metabolic syndrome. Genotypes at the four polymorphic loci in CAPN5 gene were detected by polymerase chain reaction (PCR). RESULTS: Genotype association analysis was significant for BMI (p < or = 0.041), diastolic blood pressure (p = 0.015) and HDL-cholesterol levels (p = 0.025). Different CAPN5 haplotypes were also associated with diastolic blood pressure (DBP) (0.0005 < or = p < or = 0.006) and total cholesterol levels (0.001 < or = p < or = 0.029). In addition, the AACA haplotype, over-represented in obese individuals, is also more frequent in individuals with metabolic syndrome defined by ATPIII criteria (p = 0.029). CONCLUSION: As its homologue CAPN10, CAPN5 seems to influence traits related to increased risk for cardiovascular diseases. Our results also may suggest CAPN5 as a candidate gene for metabolic syndrome.