Polytopic protease downregulation effect on the brain (rogae-affy-arabi-492460)
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE11148 | GEO | 2008/04/12
SECONDARY ACCESSION(S): PRJNA106947
REPOSITORIES: GEO
Similar Datasets
Project description:Regulation of AD-related genes. Presenilins are intramembrane polytopic proteases. These proteases are critical proteins in pathogenesis of Alzheimer's disease. The function of other polytopic proteases expressed in brains is unknown. Proteins that may potentially interact with PS pathway are of interest to elucidate. In this project we will determine gene expression patterns in the brains of mice with altered expression of polytopic protease. We hypothesize that family of polytopic proteases expressed in brain may potentially interact with presenilin pathway. We will examine 4 RNA samples isolated from brains of mice with significant downregulation of polytopic protease and control animals. Total RNA was extracted, purified according to the manufacturer's protocol and stored at -80C.
2008-06-11 | E-GEOD-11148 | biostudies-arrayexpress
Project description:Regulation of presenilin genes. Presenilins are intramembrane aspartic proteases. These proteases are critical proteins in pathogenesis of Alzheimer's disease. The function of recently identified presenilin-homologous proteases (IMPAS or SPP)s is unknown. Our preliminary data in C.elegans model suggested the role of these proteins in early-development and , perhaps, in pathway related- to cholesterol -regulated signalling (Grigorenko et al, 2004, PNAS). The overall goal is to determine pattern of gene expression alterations in cells with knock-out or knock-down presenilin related proteins (IMPAS). The cultured cells, C.elegans and mouse models are planned to be used in this study. Here , we will determine gene expression patterns in C.elegans strain NL2099, fed with bacteria HT115, producing dsRNA for Ce-imp-2 gene (knock-down of imp-2 gene by RNAi )or GFP gene as a control. We hypothesize that novel family of presenilin-related proteases is important for cholesterol-regulated intracellular signalling and early development (including CNS/neuronal development and function). C.elegans (strain NL2099) on L1 stage were placed on agarose plates with bacteria HT115, producing dsRNA for: 1) Ce-imp-2 gene; 2) GFP as a control gene. After 48 hours at 20C , worms were washed in M9 buffer and total RNA was isolated by TRIzol method. Keywords: dose response
2007-03-23 | GSE7354 | GEO
Project description:Regulation of presenilin genes. Presenilins are intramembrane aspartic proteases. These proteases are critical proteins in pathogenesis of Alzheimer's disease. The function of recently identified presenilin-homologous proteases (IMPAS or SPP)s is unknown. Our preliminary data in C.elegans model suggested the role of these proteins in early-development and , perhaps, in pathway related- to cholesterol -regulated signalling (Grigorenko et al, 2004, PNAS). The overall goal is to determine pattern of gene expression alterations in cells with knock-out or knock-down presenilin related proteins (IMPAS). The cultured cells, C.elegans and mouse models are planned to be used in this study. Initially, we will determine gene expression patterns in human cells embryonic kidney cells (HEK 293 cell line) overexpressing different isoforms of hIMP1 protein. We hypothesize that novel family of presenilin-related proteases is important for cholesterol-regulated intracellular signalling and early development (including CNS/neuronal development and function). We will examine in HEK 293 cells effect of over expression of 1) hIMP1 wt protein, 2) over expression of dominant negative isoform of hIMP1 protein and compare their gene expression patterns to 3) HEK 293 cells treated with mock vector. HEK 293 cells were seeded on 10 cm plates and transfected next day using Lipofectamine Plus reagent. 24 hours after cells were washed twice with ice cold PBS buffer and lysed with TRizol reagent directly on plates. Total RNA was extracted, purified according the manufacturer protocol and stored at -800C. We will provide 2 RNA samples for each hIMP1 isoform used in two different transfection experiment to reduce any spurious expression differences resulting from culture condition or transfection efficiency variations. Keywords: dose response
2006-11-14 | GSE6276 | GEO
Project description:Regulation of presenilin genes. Presenilins are intramembrane aspartic proteases. These proteases are critical proteins in pathogenesis of Alzheimer's disease. The function of recently identified presenilin-homologous proteases (IMPAS or SPP)s is unknown. Our preliminary data in C.elegans model suggested the role of these proteins in early-development and , perhaps, in pathway related- to cholesterol -regulated signalling (Grigorenko et al, 2004, PNAS). The overall goal is to determine pattern of gene expression alterations in cells with knock-out or knock-down presenilin related proteins (IMPAS). The cultured cells, C.elegans and mouse models are planned to be used in this study. Here , we will determine gene expression patterns in C.elegans strain NL2099, fed with bacteria HT115, producing dsRNA for Ce-imp-2 gene (knock-down of imp-2 gene by RNAi )or GFP gene as a control. We hypothesize that novel family of presenilin-related proteases is important for cholesterol-regulated intracellular signalling and early development (including CNS/neuronal development and function). C.elegans (strain NL2099) on L1 stage were placed on agarose plates with bacteria HT115, producing dsRNA for: 1) Ce-imp-2 gene; 2) GFP as a control gene. After 48 hours at 20C , worms were washed in M9 buffer and total RNA was isolated by TRIzol method.
2008-06-15 | E-GEOD-7354 | biostudies-arrayexpress
Project description:Regulation of presenilin genes. Presenilins are intramembrane aspartic proteases. These proteases are critical proteins in pathogenesis of Alzheimer's disease. The function of recently identified presenilin-homologous proteases (IMPAS or SPP)s is unknown. Our preliminary data in C.elegans model suggested the role of these proteins in early-development and , perhaps, in pathway related- to cholesterol -regulated signalling (Grigorenko et al, 2004, PNAS). The overall goal is to determine pattern of gene expression alterations in cells with knock-out or knock-down presenilin related proteins (IMPAS). The cultured cells, C.elegans and mouse models are planned to be used in this study. Initially, we will determine gene expression patterns in human cells embryonic kidney cells (HEK 293 cell line) overexpressing different isoforms of hIMP1 protein. We hypothesize that novel family of presenilin-related proteases is important for cholesterol-regulated intracellular signalling and early development (including CNS/neuronal development and function). We will examine in HEK 293 cells effect of over expression of 1) hIMP1 wt protein, 2) over expression of dominant negative isoform of hIMP1 protein and compare their gene expression patterns to 3) HEK 293 cells treated with mock vector. HEK 293 cells were seeded on 10 cm plates and transfected next day using Lipofectamine Plus reagent. 24 hours after cells were washed twice with ice cold PBS buffer and lysed with TRizol reagent directly on plates. Total RNA was extracted, purified according the manufacturer protocol and stored at -800C. We will provide 2 RNA samples for each hIMP1 isoform used in two different transfection experiment to reduce any spurious expression differences resulting from culture condition or transfection efficiency variations.
2008-06-14 | E-GEOD-6276 | biostudies-arrayexpress
Project description:Schwann cells, expanded in number by exposure to growth factors in vitro, could be useful in nervous system repair. Our previous results suggest that long term exposure to heregulin and forskolin changes the functional properties of the human Schwann cells, including the ability to myelinate axons after transplantation. Here, we propose to determine the molecular changes in the Schwann cells that occur as a result of extended growth with mitogenic factors. We believe that the information obtained in these studies will provide clues about mechanisms underlying the already observed changes in function. This information will aid in the prediction of the safety and efficacy of neural repair approaches that use cultured, expanded Schwann cells. Finally this data may provide clues into the mechanisms underlying normal human Schwann cell function. To use gene array analysis to compare gene expression profiles in early and late passage human Schwann cells exposed to the growth factors heregulin and forskolin. Observed changes in the function of human Schwann cells, including their capacity for growth and differentiation, after prolonged exposure to heregulin and forskolin, are caused by changes in the gene expression profiles in these cells. Nerves from four different donors were obtained within 12-15 h postmortem, with full consent for research from the families of the donors (faxed). IRB approval not required because subjects were officially dead at nerve harvest (IRB confirmation faxed). The nerve tissue obtained consisted of the lower 15 cm of nerve extending from mid calf to ankle. Endoneurial fascicles were dissected from short (1-2 cm) nerve fragments and cultured with heregulin and forskolin for 1 week. The nerve fascicles were then treated overnight with collagenase and dispase and dissociated by gentle trituration. Cells were plated at low density on laminin coated dishes and maintained in culture with mitogens for 3 passages. Schwann cell purities during this period were > 95%. RNA was extracted from the cells at passage 1 ( i.e. after 2 population doublings) and passage 3 (i.e. after 8 population doublings) from the following donors: SN HSC 330, age 51; SN HSC 317, age 29; SN HSC 329, age 20 and SN HSC 351, age 42. Thus eight samples (2 groups, 4 independent samples per group) are to be analyzed. Because both groups in this comparison have been exposed to mitogens, differences in gene expression profiles will be interpreted as indicative of changes caused by prolonged versus short term exposure to mitogens. More RNA was prepared from each group of cells than was needed for gene array analysis to allow confirmation of differentially-expressed gene transcripts by real-time RT-PCR. Since the main purpose of this project is to detect any major changes in the molecular properties of the Schwann cells, we propose that these samples be analyzed using Affymetrix U133 + 2 arrays containing the complete human genome and request Option 1: Start to Finish Profiling of all samples. We have contacted Dr Stanley Nelson at UCLA and discussed the project with him. We request that the samples be analyzed in the UCLA facility. Keywords: time-course
2006-01-13 | GSE4030 | GEO
Project description:In skeletal muscle, the pattern of electrical activity regulates the expression of proteins involved in synaptic transmission, contraction and metabolism. Disruptions in electrical activity, resulting from prolonged bed-rest, cast-immobilization or trauma, inevitably lead to muscle atrophy. The mechanisms that regulate muscle atrophy are poorly understood, but it seems likely that changes in gene expression play a key role in initiating and maintaining a muscle atrophy program. Previously, we found that Runx1, a transcription factor previously termed AML1, was substantially induced in muscle following denervation. More recently, we sought to determine whether this increase in Runx1 expression may be causally related to the morphological changes in skeletal muscle that accompany muscle disuse, notably muscle atrophy. We found that Runx1 is indeed required to sustain muscle and to minimize atrophy following denervation. Experiments described here are designed to identify the genes that are regulated by Runx1 in skeletal muscle with the particular goal of identifying genes that regulate muscle atrophy. We propose to use microarray analysis to identify genes, expressed in skeletal muscle, that are mis-regulated in mice lacking Runx1. We inactivated runx1 selectively in skeletal muscle and found that denervated myofibers in mutant mice atrophy far more (90% atrophy) than in wild-type mice (30% atrophy). We therefore reason that Runx1 activates and/or represses genes that are required to sustain muscle and to minimize atrophy. We generated MCK::cre; runx1f/- and runx1f/- control mice. In normal mice, an increase in runx1 expression is detected by two days after denervation and is maximal by five days after denervation. Muscle atrophy is first evident between one and two weeks after denervation. As we wish to avoid detecting global changes in gene expression that are associated with late stages of muscle atrophy, we plan to denervate muscle for three or five days and to compare gene expression in dissected innervated and denervated muscles from mutant and control mice. We will generate thirty samples for comparison-5 replicates per condition: Samples 1-3 from runx1f/- control mice. (1) innervated tibialis anterior muscles (TA); (2) 3-day-denervated TA; (3) 5-day-denervated TA. Samples 4-6 from MCK::cre; runx1f/- mice. (4) innervated TA; (5) 3-day-denervated TA; (6) 5-day-denervated TA. We obtain sufficient total RNA (10 micrograms) from each dissected muscle to avoid pooling samples. We will analyze adult mice of the same age (~six weeks after birth; most will be littermates) and sex-male. It is difficult to anticipate how many genes will be identified in this screen, as few target genes for Runx1 have been identified in any cell type and none in skeletal muscle. Moreover, although we would prefer to focus our attention on genes that are strongly dependent upon Runx1 expression (e.g. more than 5-fold difference in expression in wild-type and mutant mice), we do not know the extent to which target gene expression will depend upon Runx1. For these reasons, in these experiments, we will analyze expression from five “identical” samples, so that we can be confident that even small (e.g. three-fold) differences in expression can be reliably determined. Importantly, in order to confirm results obtained from the microarray data, we will use other assays (RNase protection) to measure RNA expression of candidate genes in innervated and denervated muscles of wild-type and mutant mice. Keywords: other
2006-03-08 | GSE4411 | GEO
Project description:In our original grant we proposed to use the NR3B-null mouse model to study the role of NR3B subunit in motor neuron function. We have now successfully generated NR3B null mice. Interestingly, NR3B-null mice invariably die at age P4-P8. Our preliminary examination indicates that the motor strength of these mice is severely impaired prior to death. As we continue to explore the cause of death in NR3B null mice, we propose to conduct gene profiling experiments to search for transcription changes in the brain related to ablation of the NR3B gene. We would like to use the facility provided by the NINDS/NIMH Microarray Consortium to identify true outlier genes that show abnormal expression patterns in these mice. Analysis of these outlier genes will help to identify changes in networks and pathways that may cause the death of NR3B-null mice. These studies will further help to elucidate the functional role of NR3B in motor neurons. We will compare samples from the hindbrain and spinal cord of wild type and NR3B null mice to identify true outlier genes that show abnormal expression patterns, which may be implicated in the death of NR3B-null mice We hypothesize that genes with their transcription level changing significantly by ablation of NR3B will be associated with the molecular mechanism underlying the death of motor neurons in NR3B null mice. As NR3B is expressed primarily in the hindbrain and spinal cord, we will first collect and analyze the hindbrain and spinal cord samples from NR3B null mice and wild-type controls in triplicate at two time points (P0 and P5). Total RNA from total 12 samples will be purified. Extracted RNAs will be treated for with DNase I to remove contaminating genomic DNA. The purified RNA will be sent to the NINDS/NIMH Microarray Consortium to generate biotin-labeled probe according to the Affymetrix protocol. These probes will be used to hybridize the GeneChip Mouse Genome 430 2.0 Array. The hybridization, scanning, and initial data analysis of these GeneChips will be conducted by the Consortium staff. We will analyze the collected data further after data collection. We will use computer software, GeneSpring (Silicon Genetics, Redwood City, CA), to analyze data from microarray experiments. We will first identify genes that show significant changes between wild-type and NR3B null mice by applying ANOVA analysis to sample groups of different ages. The genes identified in this manner will then be subjected to hierarchical (Gene tree) and nonhierarchical (K-means) cluster analysis, in order to group them according to similar expression patterns. Another clustering method, the self-organizing map, will be used to determine whether one gene cluster is a variant of another. Finally, the Principal Components Analysis (PCA) will be employed to analyze large sample groups, which may include experimental variability with respect to age, anatomical location and genotype. We will determine from these analysis whether further experiments are needed using different array platforms, such as Agillent Oligo Array. Keywords: time-course
2006-06-09 | GSE5035 | GEO
Project description:The Early Growth Response (Egr) family of transcription factors consists of 4 members (Egr1-4) that are expressed in a wide variety of cell types. A large body of evidence point to a role for Egr transcription factors in growth, survival, and differentiation. A major unanswered question is whether Egr transcription factors serve similar functions in diverse cell types by activating a common set of target genes. Signal transduction cascades in neurons and lymphocytes show striking parallels. Activation of either cell type activates the Ras-MAPK pathway and, in parallel, leads to increases in intracellular calcium stimulating the calcineurin-NFAT pathway. In both cell types, the strength of the activation signal affects the cellular outcomes and very strong stimuli lead to cell death. Notably both these pathways converge on the induction of Egr genes. We believe that downstream targets of Egr transcription factors in lymphocytes may also be activated by Egr factors in activated neurons. There is precedence for common target gene activation in these two cell types: apoptosis in both activated T cells and methamphetamine stimulated neurons occurs via FasL induction by NFAT transcription factors. We propose to use developing T lymphocytes (thymocytes) as a model system for discovery of Egr-dependent target genes for several reasons. First, we have observed a prominent survival defect in thymocytes from mice deficient in both Egr1 and Egr3 (1/3 DKO) and a partial differention block in the immature double negative (DN) stage. In addition, thymocytes are an easily manipulatable cell type, and the DN subpopulation affected in 1/3 DKO mice can be isolated to very high purity. We anticipate that 1/3 DKO thymocytes will provide an excellent experimental system that will provide insight into Egr-dependent transcription in neuronal development, activation, and death. To identify Egr transcription factor target genes in developing thymocytes. Egr1/3 DKO mice show a profound defect in thymocyte survival and differentiation, but the target genes downstream of Egr1 and Egr3 are unknown. Using Egr1/3 double knockout and wild type littermate mice, we will compare gene expression profiles from DN thymocytes to identify genes that are deregulated in Egr1/3 DKO cells. Egr1 and Egr3 expression is necessary for the proper survival and differentiation of developing thymocytes. We hypothesize that we will be able to identify crucial target genes of Egr1 and Egr3 by comparing gene expression profiles from wild type and 1/3 DKO thymocytes. Egr1 and Egr3 are first expressed in the immature double negative (DN) thymocytes. We will isolate DN thymocytes by FACS sorting which allows isolation of cell populations of >99% purity. We will perform microarray analysis using the Mouse 430 2.0 gene array with thymocyte RNA samples from 3 wild type and 3 1/3 DKO mice (6 arrays total). Differentially regulated genes (up-regulated and down-regulated) will be identified from the list of genes with significantly altered expression greater than or equal to 2-fold by paired T test. Interesting genes will be validated by real-time PCR in wild type and 1/3 DKO thymocytes. Recognizing that both direct and indirect target genes may be identified in the list of differentially expressed genes, real-time PCR validated target genes will be further screened using chromatin immunoprecipitation coupled with PCR (ChIP-PCR) to determine whether Egr1 and/or Egr3 are bound to potential regulatory regions of the putative target genes. Keywords: other
2006-08-24 | GSE5587 | GEO
Project description:Primary objectives: This study aims at (further) revealing the pathophysiology of intestinal IR in man, with a specific interest for the role of proteases and protease-activated receptor-2 (PAR-2), cellular and inflammatory changes, barrier function and intestinal permeability, microscopic mucosal changes and gene expression patterns. An important element will be the determination of the effects of protease- and MMP inhibitors. By these means we hope to identify preventive and therapeutic strategies for patients with intestinal IR.
Primary endpoints: The primary endpoint in this study is inflammation (neutrophil influx, complement activation, interleukins, TNF-α, COX 1-2) and protease activity in tissue as well as in blood plasma.
| 2533504 | ecrin-mdr-crc