Biomedical subjects

W J Lukiw

Publications and source records attributed to W J Lukiw.

At least 19 recordsLinked to original sources

PubMed2002

Regulatory region variability in the human presenilin-2 (PSEN2) gene: potential contribution to the gene activity and risk for AD.

We have analyzed the 5'-upstream promoter region of the presenilin 2 gene (PSEN2) for regulatory elements and examined Alzheimer disease (AD) patients and non-demented individuals for polymorphisms in the 5' upstream promoter region of the PSEN2 gene. Direct sequencing analysis detected a common single adenine (A) nucleotide deletion polymorphism in the upstream promoter region of the PSEN2 gene. Examination of cohorts of AD patients and age-matched control individuals revealed no statistically significant differences in the frequency of this polymorphism when compared with the total sample of AD patients and control individuals. However, subgroup and regression analysis suggested that the relatively rare -A/-A genotype increases risk of AD among subjects lacking apolipoprotein E (APOE) epsilon4 and among persons ages 65 years and younger. DNA sequence and DNA-protein binding analysis demonstrated that this mutation negates binding with putative repressor transcription factor (TF), interferon regulatory factor 2 (IRF2), in nuclear extracts prepared from the aged human brain neocortex. However this mutation creates a potential regulatory element, C/EBPbeta, that is responsive to pro-inflammatory (PI) induction. The expression activity assay with luciferase reporter gene into normal human neural progenitor cells in primary culture shows that the mutant PSEN2 regulatory region exhibits a 1.8-fold higher level of basal expression and is sensitive to IL-1beta and Abeta42, but that it is synergistically induced 3.2-fold over the wild-type PSEN2 by [IL-1beta+Abeta42]. These results suggest that under Pl and oxygen stress conditions relatively minor variations in PSEN2 promoter DNA sequence structure can enhance PSEN2 gene expression and that consequently these may play a role in the induction and/or proliferation of a Pl response in AD brain.

5' Untranslated Regions↗

PubMed2001

Metallothionein III is reduced in Alzheimer's disease.

Metallothionein III (MT-III) is a functionally distinct member of the metallothionein family that displays neuroinhibitory activity and is involved in the repair of neuronal damage. Altered expression levels of MT-III have been observed in Alzheimer's disease (AD) which has led to suggestions that it could be a mitigating factor in AD-related neuronal dysfunction. However, conflicting results have been reported on this issue which may be due to methodological differences and/or sampling size. In the current study, we have assessed MT-III expression in a large number of AD cases through the quantification of mRNA as well as by immunohistochemistry and Western blotting using an MT-III specific antibody. The results of this comprehensive study indicate that the mononucleosome DNA encoding MT-III is occluded preventing transcription and that message levels are reduced by approximately 30%. In addition, protein levels were specifically decreased by approximately 55% in temporal cortex. These data support the conclusion that MT-III is significantly downregulated in AD and may contribute to the loss of its protective effects and/or repair functions that lead to an exacerbation of the pathogenic processes.

Alzheimer Disease↗

PubMed2001

Presenilin-2 (PS2) expression up-regulation in a model of retinopathy of prematurity and pathoangiogenesis.

Presenilin-2 (PS2; AD4), a regulator of intercellular signaling during CNS development and cell fate determination, appears to be involved in pathogenic processing of beta-amyloid precursor protein (betaAPP) into potentially neurotoxic beta-amyloid (Abeta) peptides. The PS2 gene promoter contains multiple DNA binding sites for the relatively rare hypoxia-inducible transcription factor HIF-1, suggesting that PS2 expression may be a sensitive indicator of decreased oxygen availability. We have used a cycled hypoxia/hyperoxia (10-50% O2) protocol followed by normoxia (20% O2) as a retinal model of retinopathy of prematurity to induce neovascularization (NV) in rat pups. Retinal cell nuclear extracts from pups undergoing hypoxia exhibited a dramatic increase in HIF-1-DNA binding, followed by a delayed (2-7 day) elevation of PS2 RNA message and protein. PS2 gene activation during hypoxia may direct cellular fate towards pathoangiogenesis and intercellular PS2-mediated signaling dysfunction.

Animals↗

PubMed2001

Gene expression analyzed by microarrays in HSV-1 latent mouse trigeminal ganglion following heat stress.

An understanding of the cellular genes whose expression is altered during HSV reactivation will enable us to better understand host responses and biochemical pathways involved in the process. Furthermore, this knowledge could allow us to develop gene-targeted inhibitors to prevent viral reactivation. Mice latent with HSV-1 strain McKrae and uninfected control mice were subjected to hyperthermic stress (43 degrees C for 10 min) and their trigeminal ganglia (TG) collected 1 h later. Two additional groups included HSV-1 latently infected and uninfected mice not subjected to hyperthermic stress. Poly A+ mRNA was enriched from total mouse TG RNA and reverse transcribed using MMLV RT. Radioactively labeled cDNAs were analyzed by microarray analysis. A stress/toxicology array of 149 mouse genes on a nylon membrane was used. The labeled cDNAs prepared from latently infected, stressed mice demonstrated 3-fold or greater increases in certain mRNA-early response genes (ERGs) compared to cDNAs from uninfected, stressed control mice. The ERG mRNAs that showed increases included two heat shock proteins (HSP60 and HSP40), a basic transcription factor (BTF T62), a DNA repair enzyme, two kinases [MAP kinase and a stress-induced protein kinase (SADK)], an oxidative stress-induced protein, a manganese superoxide dismutase precursor-2 (SOD-2), and cyclooxygenase 2 (COX-2). The gene expression in unstressed, infected TGs was similar to the gene expression in unstressed, uninfected controls. These results suggest that there is a significant difference in the ERG expression profile in latently infected TGs undergoing stress-induced reactivation compared to uninfected TGs.

Animals↗

PubMed2000

Neuroinflammatory signaling upregulation in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive, neurodestructive process of the human neocortex, characterized by the deterioration of memory and higher cognitive function. A progressive and irreversible brain disorder, AD is characterized by three major pathogenic episodes involving (a) an aberrant processing and deposition of beta-amyloid precursor protein (betaAPP) to form neurotoxic beta-amyloid (betaA) peptides and an aggregated insoluble polymer of betaA that forms the senile plaque, (b) the establishment of intraneuronal neuritic tau pathology yielding widespread deposits of agyrophilic neurofibrillary tangles (NFT) and (c) the initiation and proliferation of a brain-specific inflammatory response. These three seemingly disperse attributes of AD etiopathogenesis are linked by the fact that proinflammatory microglia, reactive astrocytes and their associated cytokines and chemokines are associated with the biology of the microtubule associated protein tau, betaA speciation and aggregation. Missense mutations in the presenilin genes PS1 and PS2, implicated in early onset familial AD, cause abnormal betaAPP processing with resultant overproduction of betaA42 and related neurotoxic peptides. Specific betaA fragments such as betaA42 can further potentiate proinflammatory mechanisms. Expression of the inducible oxidoreductase cyclooxygenase-2 and cytosolic phospholipase A2 (cPLA2) are strongly activated during cerebral ischemia and trauma, epilepsy and AD, indicating the induction of proinflammatory gene pathways as a response to brain injury. Neurotoxic metals such as aluminum and zinc, both implicated in AD etiopathogenesis, and arachidonic acid, a major metabolite of brain cPLA2 activity, each polymerize hyperphosphorylated tau to form NFT-like bundles. Further, epidemiological and longitudinal studies have identified a reduced risk for AD in patients (<70 yrs) previously treated with non-steroidal anti-inflammatory drugs for non-CNS afflictions that include arthritis. This review will focus on the interrelationships between the mechanisms of PS1, PS2 and betaAPP gene expression, tau and betaA deposition and the induction, regulation and proliferation in AD of the neuroinflammatory response. Novel therapeutic interventions in AD are discussed.

Aluminum↗

PubMed2000

Cyclooxygenase-2 in synovial tissue and fluid of dysfunctional temporomandibular joints with internal derangement.

PURPOSE: This study investigated cyclooxygenase-2 (COX-2) gene expression in temporomandibular joint (TMJ) synovial tissue and fluid from patients with internal derangement. PATIENTS AND METHODS: Seventeen synovial tissue biopsy specimens and 16 synovial fluid samples were obtained from patients (1 male and 11 female) during arthroscopic TMJ surgery. The samples were frozen at -70 degrees C and, by using Northern and reverse transcription polymerase chain reaction (RT-PCR) analysis, the levels of COX-2 RNA in relation to beta-actin RNA message levels were determined. RESULTS: COX-2 RNA message was detected in 16 of 17 synovial tissue samples (94%) and 12 of 16 synovial fluid samples (75%) by using beta-actin RNA levels in the same sample (either tissue or fluid) as an internal control. Samples were not quantified because of the same sample mass. CONCLUSION: COX-2, an important inflammatory mediator, is present in the TMJ synovial tissue and fluid from patients with internal derangement. Therefore, COX-2 antagonists may be indicated in the treatment of TMJ arthralgia.

Analysis of Variance↗

PubMed2000

Modulation of early response gene expression by prostaglandins in cultured rat retinal pigment epithelium cells.

PURPOSE: To explore the role of prostaglandins (PGs) as modulators of retinal pigment epithelium (RPE) rod outer segment (ROS)-phagocytosis and ROS-phagocytosis-induced gene expression. METHODS: RPE cells in primary cell culture were pre-incubated with PGE( 2), PGD(2), PGF(2)alpha, PGJ(2), 15-deoxy-Delta( 12,14)-PGJ(2) or U-46619 (stable analog of thromboxane A(2)), and fed with a suspension of ROS. Expression of zif-268 and tis-1 mRNA was determined by Northern blotting. DNA-binding activity of TIS-1 protein was assessed by electrophoretic mobility shift assay. Concentration of PGE (2) and PGD (2) in the tissue culture medium was measured by enzyme immuno-assay. Phagocytis-tosis was quantified by counting of double-immunostained bound and ingested ROS. RESULTS: PGE 2, the most potent of PGs, strongly elevated both basal and ROS-phagocytosis-induced levels of tis-1 mRNA, while significantly inhibiting both basal and phagocytosis-induced expression of zif-268 mRNA. PGD(2), PGJ(2) and 15-deoxy-Delta(12,14)-PGJ( 2) elevated ROS-phagocytosis-induced, but not basal, expression of tis-1 mRNA expression. PGF(2alpha) super-induced both phagocytosis-induced and basal tis-1 mRNA expression. U-46619 and carbaprostacyclin had no effect on expression of tis-1 mRNA. PGE(2) was the only PG to affect zif-268 expression. Exogenous PGE(2), PGD( 2) and PGF(2alpha), when added to the medium at 1-microM concentrations, significantly inhibited ingestion of ROS, with PGE(2) being the most potent PG affecting ROS-phagocytosis. CONCLUSIONS: PGs act as selective regulators of phagocytosis-induced transcription factor gene expression in RPE cells, as well as of ROS-phagocytosis itself. This modulation may help to ensure specificity in the differential activation of target genes by ROS-phagocytosis receptor-mediated signal transduction in RPE cells.

Animals↗

PubMed1999

The interleukin-1 type 2 receptor gene displays immediate early gene responsiveness in glucocorticoid-stimulated human epidermal keratinocytes.

Human epidermal keratinocytes (HEKs) in primary culture (P2-P4) were used to study glucocorticoid (GC)-mediated transcription of the genes encoding the constitutively expressed interleukin-1 type 1 receptor (IL-1R1) and the inducible interleukin-1 type 2 receptor (IL-1R2). Utilizing Northern dot blot analysis and a quantitative reverse transcription-polymerase chain reaction protocol for IL-1R1 and IL-1R2, dexamethasone and, in particular, the budesonide epimer R were shown to effectively and rapidly induce transcription from the IL-IR2 gene when compared with IL-1R1 or beta-actin RNA message levels in the same sample. Southern blot analysis of newly generated IL-1R2 reverse transcription-polymerase chain reaction products using end-labeled IL-1R2 intron probes suggested that GC enhancement of IL-1R2 expression was regulated primarily at the level of de novo transcription. GC-induced IL-1R2 gene transcription displayed features characteristic of a classical immediate early gene response, including a signal transduction function, a relatively low basal abundance, a rapid, transient induction, cycloheximide superinduction, actinomycin D suppression, and a rapid decay of IL-1R2 RNA message. Parallel time course kinetic analysis of IL-1R2 RNA message levels with Western immunoblotting revealed tight coupling of de novo IL-IR2 gene transcription with translation of the IL-1R2 RNA message; a newly synthesized ( approximately 46-kDa) IL-1R2 protein was detected in the HEK growth medium as early as 1 h after budesonide epimer R treatment. These data indicate that different GC compounds can variably up-regulate the IL-1R2 response in HEKs through transcription-mediated mechanisms and, for the first time, suggest that a gene encoding a soluble cytokine receptor can respond like an immediate early gene.

Budesonide↗

PubMed1998

Strong nuclear factor-kappaB-DNA binding parallels cyclooxygenase-2 gene transcription in aging and in sporadic Alzheimer's disease superior temporal lobe neocortex.

Cyclooxygenase-2 (COX-2; EC 1.14.99.1) RNA message abundance in 25 control and Consortium to Establish a Registry for Alzheimer's Disease (CERAD)-confirmed sporadic Alzheimer's disease (AD) brains is remarkably heterogeneous when compared with 55 other AD brain RNA message levels that were previously characterized (Lukiw and Bazan: J Neurosci Res 50:937-945, 1997). Examination of nuclear protein extracts (NPXTs) that were derived from control and AD-affected brain neocortical nuclei (n = 20; age range, 60-82 years; postmortem interval, 0.5-6.5 hours) by using gel shift, gel supershift, and cold oligonucleotide competition assay revealed a highly significant relationship between the extent of inflammatory transcription factor, nuclear factor (NF)-kappaB: DNA binding and the abundance of the COX-2 RNA signal (P < 0.0001; analysis of variance). No strong correlation with AP-1-DNA binding was noted (P > 0.045). These data are the first linking inflammation-related transcription factor NF-KB-DNA binding to up-regulation of transcription from a key inflammatory gene, COX-2, in both normally aging brain and in AD-affected neocortex. Systematic deletion of NF-KB-DNA binding sites in human COX-2 promoter constructs attenuates COX-2 transcriptional induction by mediators of inflammation. Strong NF-kappaB-DNA binding has been reported previously to temporally precede COX-2 gene transcription in human epithelial (A549), hamster B-cell (HIT-T15), human endothelial (HUVEC), human lymphoblast (IM9), human fibroblast (IMR90), rat glioma/mouse neuroblastoma (NG108-15), human keratinocyte (NHEK), mouse fibroblast (NIH 3T3), rat neuroblastoma (SH-SY5Y) cell lines and in mouse and rat brain hippocampus, indicating a highly conserved inflammatory signaling pathway that is common to diverse species and cell types. The mouse, rat, and human COX-2 immediate promoters, despite 7.5 x 10(7) years of DNA sequence divergence, each retain multiple recognition sites specific for NF-kappaB-DNA binding. These data suggest that basic gene induction mechanisms, which have been conserved over long periods of evolution, that increase NF-kappaB-DNA binds ing may be fundamental in driving transcription from inflammation-related genes, such as COX-2, that operate in stressed tissues, in normally aging cell lines, and in neurodegenerative disorders that include AD brain.

Aged↗

PubMed1998

Budesonide epimer R or dexamethasone selectively inhibit platelet-activating factor-induced or interleukin 1beta-induced DNA binding activity of cis-acting transcription factors and cyclooxygenase-2 gene expression in human epidermal keratinocytes.

To further understand the molecular mechanism of glucocorticoid action on gene expression, DNA-binding activities of the cis-acting transcription factors activator protein 1 (AP1), AP2, Egr1 (zif268), NF-kappaB, the signal transducers and activators of transcription proteins gamma interferon activation site (GAS), Sis-inducible element, and the TATA binding protein transcription factor II D (TFIID) were examined in human epidermal keratinocytes. The cytokine interleukin 1beta (IL-1beta) and platelet-activating factor (PAF), both potent mediators of inflammation, were used as triggers for gene expression. Budesonide epimer R (BUDeR) and dexamethasone (DEX) were studied as potential antagonists. BUDeR or DEX before IL-1beta- or PAF-mediated gene induction elicited strong inhibition of AP1-, GAS-, and in particular NF-kappaB-DNA binding (P < 0.001, ANOVA). Only small effects were noted on AP2, Egr1 (zif268), and Sis-inducible element-DNA binding (P > 0.05). No significant effect was noted on the basal transcription factor TFIID recognition of TATA-containing core promoter sequences (P > 0.68). To test the hypothesis that changing cis-acting transcription factor binding activity may be involved in inflammatory-response related gene transcription, RNA message abundance for human cyclooxygenase (COX)-1 and -2 (E.C.1.14.99.1) was assessed in parallel by using reverse transcription-PCR. Although the COX-1 gene was found to be expressed at constitutively low levels, the TATA-containing COX-2 gene, which contains AP1-like, GAS, and NF-kappaB DNA-binding sites in its immediate promoter, was found to be strongly induced by IL-1beta or PAF (P < 0.001). BUDeR and DEX both suppressed COX-2 RNA message generation; however, no correlation was associated with TFIID-DNA binding. These results suggest that on stimulation by mediators of inflammation, although the basal transcription machinery remains intact, modulation of cis-activating transcription factor AP1, GAS, and NF-kappaB-DNA binding by the glucocorticoids BUDeR and DEX play important regulatory roles in the extent of specific promoter activation and hence the expression of key genes involved in the inflammatory response.

Anti-Inflammatory Agents↗

PubMed1998

Run-on gene transcription in human neocortical nuclei. Inhibition by nanomolar aluminum and implications for neurodegenerative disease.

The incorporation of [alpha-32P]-uridine triphosphate into DNA transcription products was examined in short post-mortem interval (PMI) human brain neocortical nuclei (n, 22; PMI, 0.5-24 h) using run-on-gene transcription. Reverse Northern dot-blot hybridization of newly synthesized RNA against either total cDNA or Alu repetitive DNA indicated that human brain neocortical nuclei of up to 4-h PMI were efficient in incorporating radiolabel into new transcription products, after which there was a graded decline in de novo RNA biosynthetic capacity. To test the effects of 0-3000 nM concentrations of ambient aluminum on RNA polymerase I (RNAP I) and RNA polymerase II (RNAP II) transcription, dot blots containing 0.5, 1.0, 2.0, and 5.0 micrograms of DNA for (1) the human-specific Alu repetitive element (2) the neurofilament light (NFL) chain, and (3) glial fibrillary acidic protein (GFAP) were Northern hybridized against newly synthesized radiolabeled total RNA. These DNAs represent heterogeneous nuclear RNA (hnRNA), neuronal-, and glial-specific markers, respectively. We report here a dose-dependent repression in the biosynthetic capabilities of brain RNAP II in the range of 50-100 nM aluminum, deficits similar to those previously described using a rabbit neocortical nuclei transcription system and at concentrations that have been reported in Alzheimer's disease (AD) euchromatin. Transcription from RNAP II and the neuron-specific NFL gene in the presence of aluminum was found to be particularly affected. These findings support the hypothesis that brain gene transcription in the presence of trace amounts of ambient aluminum impairs mammalian brain DNA to adequately read out genetic information.

Aged↗

PubMed1997

Cyclooxygenase 2 RNA message abundance, stability, and hypervariability in sporadic Alzheimer neocortex.

Long-term treatment by nonsteroidal anti-inflammatory drugs has been shown to decrease the incidence of Alzheimer's disease (AD). Both platelet-activating factor and interleukin-1beta, potent mediators of the inflammatory and immune response, strongly induce transcription of the cyclooxygenase-2 (COX-2) gene in brain cells. Using Northern and RT-PCR analysis, we have determined in 15 control and 10 sporadic AD human neocortical samples (age range, 60-82 yr; postmortem interval [PMI] range, 0.7-16.0 hr) the levels of COX-2 RNA in relation to the constitutively expressed COX-1 and beta-actin RNA message levels. Our results indicate that in short PMI brain, COX-1 and COX-2 transcripts are relatively low abundance RNA messages, ranging from a mean of 6.8% of the beta-actin signal in controls to 8.5% of the beta-actin signal in AD-affected brain. A large variation in the signal intensity for COX-2 RNA was noted in both control and AD; although there was a trend for higher COX-2 RNA message abundance in AD neocortex to +11.5% of that of controls, it did not reach statistical significance (ANOVA = 0.45). Several human tissues, including heart, skeletal muscle, lung, kidney, and spinal cord, displayed 4.6- and 2.8-kb COX-2 RNA message isoforms; however, the 4.6-kb COX-2 RNA predominated in the hippocampus and association neocortex. COX-2 RNA message was found to be degraded at similar rates in both control and AD tissues, and a strong positive correlation between the PMI and the intensity of the COX-2 RNA signal was noted (ANOVA = 0.006). Linear regression analysis indicated that the 4.6-kb COX-2 RNA is an unstable short-lived RNA species with a half-life of not more than 3.5 hr, a feature characteristic of immediate early gene transcripts. Individual hypervariability in COX-2 RNA message abundance may reflect various degrees of expression of AD-related inflammatory processes.

Aged↗

PubMed1997

Analysis of the 5' sequence, genomic structure, and alternative splicing of the presenilin-1 gene (PSEN1) associated with early onset Alzheimer disease.

Mutations in the human presenilin genes (PSEN1 and PSEN2) are associated with early onset familial Alzheimer disease. The presenilin genes encode integral membrane proteins with similar structures, which suggests that they may have closely related, but as yet unknown functions. Analysis of the 5' upstream sequence and the structure of the PSEN1 gene reveals that the 5' sequence contains multiple putative transcription regulatory elements including clusters of STAT elements involved in transcriptional activation in response to signal transduction. The first four exons contain untranslated sequences, with Exons 1 and 2 representing alternate initial transcription sites. The function of these alternate initial exons is unclear. Exon 4 bears the first ATG sequence. The last 12 bp of Exon 4 is used as an alternative splice donor site. Exon 9 is alternately spliced in leukocytes, but not in most other tissues. Splicing of Exon 9 is predicted to cause significant structural changes to the protein. The majority of transcripts expressed in most tissues are polyadenylated 1127 bp from the TAG stop codon in Exon 13. A small proportion of transcripts contain the same 5'UTR and ORF but are polyadenylated 4435 bp from the stop codon. The longer polyadenylated transcripts contain three additional palindromes and at least one additional stem-loop structure with stabilities greater than -16 kcal/mol.

Alternative Splicing↗

PubMed1996

Selective transcription factor induction in retinal pigment epithelial cells during photoreceptor phagocytosis.

Expression of early response genes during rod outer segment phagocytosis by normal Long Evans and Royal College of Surgeons-rdy+p+ rats and by dystrophic Royal College of Surgeons-p+ rat retinal pigment epithelial cells was studied in primary cell culture. Northern analysis revealed that the abundance of zif-268 (egr-1), c-fos, and tis-1 (NGF1-B) mRNA was rapidly and transiently increased in normal retinal pigment epithelial cells during rod outer segment phagocytosis but not during phagocytosis of latex particles. No increase in gene expression was found in Royal College of Surgeons-p+ dystrophic retinal pigment epithelial cells challenged with rod outer segments. As shown by electrophoretic mobility shift assay, a prominent short term increase in the intensity of the gel-shifted band was detected using nuclear protein extracts derived from rod outer segment-challenged, control retinal pigment epithelial cells and zif-268, AP-1, AP-2, or tis-1 consensus oligonucleotides. No such increase was detected when using nuclear factor kappaB consensus oligonucleotide or when the early response gene prostaglandin H synthase-2 mRNA was measured over the time course studied. The results suggest that in retinal pigment epithelial cells, rod outer segment-specific phagocytosis is accompanied by the selective expression of early response genes coding for transcription factors. The specific pattern of the induction of these transcription factors is predicted to modulate the expression of gene cascades.

Animals↗

PubMed1994

The upstream promoter of the beta-amyloid precursor protein gene (APP) shows differential patterns of methylation in human brain.

The human beta-amyloid protein precursor (beta-APP) gene (symbol APP) shows variable levels of expression in different human tissues, including brain. Because at least a moderate level of beta-APP expression is probably a necessary, although not sufficient, condition for diseases associated with pathologic deposition of beta-APP proteolytic products (such as the A beta peptide), we sought to identify factors in the 5' promoter of the human APP gene that may regulate tissue-specific expression of the APP gene. We report that sequences upstream from -500 bp in the APP promoter display complex, tissue-specific patterns of methylation. Furthermore, different patterns of methylation were observed even in DNA from different regions of brain. These differentially methylated sequences are able to bind nuclear proteins expressed in brain and HeLa cells and are also methylated in neocortex of nonhuman primates. Because these methylation patterns crudely reflect differences in APP expression, they may represent one mechanism for the tissue-specific regulation of APP expression.

5-Methylcytosine↗

PubMed1994

Protein-DNA interactions in the promoter region of the amyloid precursor protein (APP) gene in human neocortex.

We have investigated protein-DNA interactions in the proximal promoter of the human amyloid precursor protein (APP) gene in temporal lobe neocortical nuclei isolated from control and Alzheimer disease (AD) affected brains. We report that the human APP 5' promoter sequence from -203 to +55 bp, which has been previously reported to contain essential regulatory elements for APP gene transcription, lies in a deoxyribonuclease I, micrococcal nuclease- and restriction endonuclease-sensitive, G+C-rich nucleosome-free gap flanked both 5' and 3' by typical nucleosome structures. As analyzed by electrophoretic mobility shift assay, this extended internucleosomal linker DNA is heavily occupied by nuclear protein factors, and interacts differentially with nuclear protein extracts obtained from HeLa and human brain neocortical nuclei. This suggests that the chromatin conformation of the APP gene promoter may vary in different cell types, and may correlate with differences in APP gene expression. Human recombinant transcription factors AP1, SP1 and TFIID (but not AP2 or brain histones H1, H2B and H4) interact with the -203 to +55 bp of the human APP promoter sequence. Only minor differences were observed in the chromatin structure of the immediate APP promoter between non-AD and AD affected neocortical nuclei, suggesting either that post-transcriptional processes, or that regulatory elements lying elsewhere in the APP gene may be important in the aberrant accumulation of the APP gene product.

Aged↗

PubMed1993

Analysis of the c-FOS gene on chromosome 14 and the promoter of the amyloid precursor protein gene in familial Alzheimer's disease.

The c-FOS gene product, a putative transacting transcriptional regulator of the amyloid precursor protein (APP) gene, is a candidate locus for the familial Alzheimer's disease (FAD) mutation on chromosome 14 (FAD14). In light of this functional relationship, we investigated the nucleotide sequence and segregation of c-FOS and the nucleotide sequence of the 5' APP promoter. Single-stranded conformational polymorphisms (SSCPs) in the c-FOS gene revealed that c-FOS closely cosegregates with the FAD14 gene but does not show allelic association with FAD. A conservative third-position T-->C mutation was demonstrated in exon 2 (codon 84) of c-FOS, and a C-->G substitution was detected at -209 bp in the 5' promoter of APP. Neither were unique to FAD and are unlikely to be pathogenic or secondary modifiers of the FAD phenotype. We conclude that the c-FOS open reading frame is probably not the site of the FAD14 locus, but we cannot exclude the existence of modifier loci on chromosome 21.

Adult↗

PubMed1992

BC200 RNA in normal human neocortex, non-Alzheimer dementia (NAD), and senile dementia of the Alzheimer type (AD).

BC200 RNA is a polyadenylated 200 nucleotide primate brain-specific transcript with 80% homology to the left monomer of the human Alu family of repetitive elements. Whether this transcription product contributes anything to normal brain gene function or is a residue of post transcriptional processing of brain heterogeneous nuclear RNA (hnRNA) is uncertain. However, the high abundance, tissue-specific expression and nucleotide sequence characteristics of BC200 RNA suggests that the generation of this small RNA is associated with some brain cell function. Sustained levels of the BC200 RNA transcript may be indicative of a genetically competent and normally functioning cerebral neocortex. In this investigation, we have measured the abundance of the BC200 RNA transcript in total RNA isolated from 18 temporal neocortices (Brodman area 22) of brains with no pathology and those affected with neurodegenerative disease. Neocortices were examined from 3 neurologically normal brains, 5 non-Alzheimer demented [NAD; 3 Huntington's chorea (HC), 1 amyotrophic lateral sclerosis (ALS) and 1 dementia unclassified] and 10 Alzheimer disease (AD) affected brains. Our results indicate a strong BC200 presence in both the normal brains and NAD affected neocortices, but a 70 per cent reduction in BC200 signal strength in AD afflicted brains. These results may be related to the observation that Alzheimer brains exhibit marked deficits in the abundance of neuron-specific DNA transcripts; these deficits are consistent with the idea that AD is characterized by an impairment in the primary generation of brain gene transcription products.

Alzheimer Disease↗