FBN1 isoform expression varies in a tissue and development-specific fashion.

Mutations in FBN1 cause Marfan syndrome, a heritable disorder of connective tissue. FBN1 encodes the extracellular matrix protein, fibrillin. Our objective was to elucidate the extent that variation in RNA splicing contributes to FBN1 isoforms. To identify FBN1 splice variants, we scanned each of its 64 internal exons in a set of pooled human brain cDNA samples. FBN1 splicing is generally efficient as we identified only two variants. Neither variant has previously been reported in the literature and include (i) an isoform which contains a cryptic 105 basepair exon between exons 54 and 55 (54A-FBN1) and (ii) an isoform which contains a cryptic 62 basepair exon between exons 57 and 58 (57A-FBN1). We compared 57A-FBN1 and FBN1 expression in multiple human tissues, including adult skeletal muscle and brain, as well as fetal skeletal muscle, brain, liver, aorta, lung, skin, and heart. 57A-FBN1 represents 8-44% of FBN1 mRNA and varies in a tissue- and development-specific fashion. In adult brain, 57A-FBN1 represented 39±3 (%, mean±SD) of total FBN1 expression. In contrast, 57A-FBN1 represented 19±2 (%, mean±SD) of FBN1 expression in skeletal muscle. In fetal tissue, the 57A-FBN1 proportion was highest in brain (27%) and low elsewhere, e.g., skin, aorta and lung (9-13%). In summary, a significant proportion of FBN1 is expressed as 57A-FBN1 and this proportion varies in a tissue- and development-specific fashion. Since the 57A insertion creates a premature stop codon that mimics Marfan-associated mutations, the protein encoded by 57A-FBN1 is likely to not be functional. These results suggest that altered splicing may modulate disease severity, regulate FBN1 expression, and potentially represent a therapeutic target.

Role of SFRS13A in low-density lipoprotein receptor splicing.

Low-density lipoprotein receptor (LDLR) is a major apolipoprotein E (APOE) receptor and thereby is critical to cholesterol homeostasis and, possibly, Alzheimer disease (AD) development. We previously identified a single nucleotide polymorphism (SNP), rs688:C>T, that modulates LDLR exon 12 splicing and is associated with cholesterol levels in premenopausal women and with Alzheimer disease in men. To gain additional insights into LDLR splicing regulation, we seek to identify splicing factors that modulate LDLR splicing efficiency. By using an in vitro minigene study, we first found that ectopic expression of SFRS3 (SRp20), SFRS13A (SRp38), SFRS13A-2 (SRp38-2), and RBMX (hnRNP G) robustly decreased LDLR splicing efficiency. Although SFRS3 and SFRS13A specifically increased the LDLR transcript lacking exon 11, SFRS13A-2 and RBMX primarily increased the LDLR isoform lacking both exons 11 and 12. When we evaluated the relationship between the expression of these splicing factors and LDLR splicing in human brain and liver specimens, we found that overall SFRS13A expression was significantly associated with LDLR splicing efficiency in vivo. We interpret these results as suggesting that SFRS13A regulates LDLR splicing efficiency and may therefore emerge as a modulator of cholesterol homeostasis.

Expression and regulation of a low-density lipoprotein receptor exon 12 splice variant.

As low-density lipoprotein receptor (LDLR) contributes to cholesterol and amyloid beta homeostasis, insights into LDLR regulation may facilitate our understanding of cardiovascular disease and Alzheimer's disease. Previously, we identified LDLR isoforms that lacked exon 12 or exons 11-12 and that are predicted to encode soluble, dominant negative, LDLR. Moreover, these isoforms were associated with rs688, an exon 12 polymorphism that was associated with LDL-cholesterol and Alzheimer's disease risk. In this study, we present evidence that although the truncated LDLR isoforms are translated in vitro, they represent < 0.1% of CSF proteins. As these LDLR isoforms likely represent a loss of mRNA-encoding functional LDLR, we then focused upon identifying intron-exon boundary and exonic splicing enhancer elements critical to splicing. Exon 12 inclusion is enhanced by altering the 5' splice site in intron 12 towards a consensus splice donor sequence, consistent with its being a weak 5' splice site. Additionally, of the nine evolutionarily conserved putative splicing enhancer regions within exon 12, two regions that flank rs688 were critical to exon 12 inclusion. Overall, these results suggest that LDLR splice variants represent a loss of mRNA encoding functional LDLR and provide insights into the regulatory elements critical for LDLR exon 12 splicing.

Sex-dependent association of a common low-density lipoprotein receptor polymorphism with RNA splicing efficiency in the brain and Alzheimer's disease.

Since apoE allele status is the predominant Alzheimer's disease (AD) genetic risk factor, functional single nucleotide polymorphisms (SNPs) in brain apoE receptors represent excellent candidates for association with AD. Recently, we identified a SNP, rs688, as modulating the splicing efficiency of low-density lipoprotein receptor (LDLR) exon 12 in female human liver and in minigene-transfected HepG2 cells. Moreover, the rs688T minor allele was associated with significantly higher LDL and total cholesterol in women within the Framingham Offspring Study cohort. Since LDLR is a major apoE receptor in the brain, we hypothesized that rs688 modulates LDLR splicing in neural tissues and associates with AD. To evaluate this hypothesis, we first transfected LDLR minigenes into SH-SY5Y neuroblastoma cells and found that the rs688T allele reduces exon 12 inclusion in this neural model. We then evaluated the association of rs688 allele with exon 12 splicing efficiency in vivo by quantifying LDLR splicing in human anterior cingulate tissue obtained at autopsy; the rs688T allele is associated with decreased LDLR exon 12 splicing efficiency in aged males, but not females. Lastly, we evaluated whether rs688 associates with AD by genotyping DNA from 1457 men and 2055 women drawn from three case-control series. The rs688T/T genotype was associated with increased AD odds in males [recessive model, odds ratio (OR) of 1.49, 95% confidence interval (CI) of 1.13-1.97, uncorrected P = 0.005], but not in females. In summary, these studies identify a functional apoE receptor SNP that is associated with AD in a sex-dependent fashion.

Modulation of Aß42 in vivo by γ-secretase modulator in primates and humans.

INTRODUCTION: Ibuprofen is one of the nonsteroidal anti-inflammatory drugs that have been shown to selectively lower pathogenic amyloid beta-peptide (Aß)42 without impairing overall γ-secretase activity in vitro. This γ-secretase modulator (GSM) activity has been hypothesized to contribute to the reduction in risk of developing Alzheimer's disease in chronic users of nonsteroidal anti-inflammatory drugs. However, it is unclear whether ibuprofen, within therapeutic dosing range, demonstrates GSM activity in humans. In this study, we evaluated the effects of ibuprofen and a second-generation GSM, GSM-1, on Aß levels in cerebrospinal fluid and plasma of young nonhuman primates and humans. METHODS: Five to seven conscious cynomolgus monkeys (Macaca fascicularis) were nontreated or treated with 30 mg/kg GSM-1 or 50 or 100 mg/kg ibuprofen and the plasma and cerebrospinal fluid were sampled at -8, 0 (baseline or right before treatment), 2, 4, 6, 8, 12, and 24 h postdosing. In addition, sixteen healthy human subjects were randomly assigned to receive either placebo or 800 mg ibuprofen given by intravenous administration and plasma were collected at 0 (before drug infusion), 0.5, 1, 2, 4, 6, 8, 10, and 24 h after dosing. RESULTS: A single dose of GSM-1 (30 mg/kg) decreased the ratio of Aß42 to Aß40 to 60% in plasma and the ratio of Aß42 to total Aß to 65% in cerebrospinal fluid from baseline to postdosing in monkeys. However, no significant changes were detected following ibuprofen treatment at 100 mg/kg. Consistent with the results from nonhuman primates, ibuprofen did not alter plasma Aß levels in human volunteers after a single 800 mg dose. CONCLUSIONS: GSM-1 exerted potent lowering of the ratio of Aß42 to Aß40 in nonhuman primates but the hypothesized GSM activity of ibuprofen could not be demonstrated in nonhuman primates and humans after acute dosing.

Genetics of clusterin isoform expression and Alzheimer's disease risk.

The minor allele of rs11136000 within CLU is strongly associated with reduced Alzheimer's disease (AD) risk. The mechanism underlying this association is unclear. Here, we report that CLU1 and CLU2 are the two primary CLU isoforms in human brain; CLU1 and CLU2 share exons 2-9 but differ in exon 1 and proximal promoters. The expression of both CLU1 and CLU2 was increased in individuals with significant AD neuropathology. However, only CLU1 was associated with the rs11136000 genotype, with the minor "protective" rs11136000T allele being associated with increased CLU1 expression. Since CLU1 and CLU2 are predicted to encode intracellular and secreted proteins, respectively, we compared their expression; for both CLU1 and CLU2 transfected cells, clusterin is present in the secretory pathway, accumulates in the extracellular media, and is similar in size to clusterin in human brain. Overall, we interpret these results as indicating that the AD-protective minor rs11136000T allele is associated with increased CLU1 expression. Since CLU1 and CLU2 appear to produce similar proteins and are increased in AD, the AD-protection afforded by the rs11136000T allele may reflect increased soluble clusterin throughout life.

Expression of SORL1 and a novel SORL1 splice variant in normal and Alzheimers disease brain.

BACKGROUND: Variations in sortilin-related receptor (SORL1) expression and function have been implicated in Alzheimers Disease (AD). Here, to gain insights into SORL1, we evaluated SORL1 expression and splicing as a function of AD and AD neuropathology, neural gene expression and a candidate single nucleotide polymorphism (SNP). RESULTS: To identify SORL1 splice variants, we scanned each of the 46 internal SORL1 exons in human brain RNA samples and readily found SORL1 isoforms that lack exon 2 or exon 19. Quantification in a case-control series of the more abundant isoform lacking exon 2 (delta-2-SORL1), as well as the "full-length" SORL1 (FL-SORL1) isoform containing exon 2 showed that expression of FL-SORL1 was reduced in AD individuals. Moreover, FL-SORL1 was reduced in cognitively intact individuals with significant AD-like neuropathology. In contrast, the expression of the delta-2-SORL1 isoform was similar in AD and non-AD brains. The expression of FL-SORL1 was significantly associated with synaptophysin expression while delta-2-SORL1 was modestly enriched in white matter. Lastly, FL-SORL1 expression was associated with rs661057, a SORL1 intron one SNP that has been associated with AD risk. A linear regression analysis found that rs661057, synaptophysin expression and AD neuropathology were each associated with FL-SORL1 expression. CONCLUSION: These results confirm that FL-SORL1 expression declines in AD and with AD-associated neuropathology, suggest that FL-SORL1 declines in cognitively-intact individuals with AD-associated neuropathology, identify a novel SORL1 splice variant that is expressed similarly in AD and non-AD individuals, and provide evidence that an AD-associated SNP is associated with SORL1 expression. Overall, these results contribute to our understanding of SORL1 expression in the human brain.

Effect of 5-aminolevulinic acid-mediated photodynamic therapy on MCF-7 and MCF-7/ADR cells.

BACKGROUND AND OBJECTIVES: Photodynamic therapy (PDT) has been proposed as an alternative approach in overcoming multidrug resistance (MDR) phenotype. To verify whether 5-aminolevulinic acid (ALA)-mediated PDT is effective in MDR cells, we studied the protoporphyrin IX (PpIX) content, intracellular localization, and phototoxicity in human breast cancer cells MCF-7 and derived MDR subline, MCF-7/ADR. STUDY DESIGN/MATERIALS AND METHODS: The fluorescence kinetics of ALA-induced PpIX was evaluated by spectrofluorometer. The phototoxicity of MCF-7 and MCF-7/ADR cells was determined by tetrazolium (MTT) assays and clonogenic assay. Furthermore, Annexin V and propidium iodide (PI) binding assays were performed to analyze the characteristics of cell death after ALA-PDT. RESULTS: MCF-7/ADR accumulated a lower level of PpIX as compared to parental MCF-7 cells. Significant phototoxicity was observed in MCF-7 and increased in a fluence-dependent manner with LD(50) around 8 J/cm(2). Compared to its parental counterpart, MCF-7/ADR cells were less sensitive to ALA photodynamic treatment and PDT-induced cytotoxicity did not increase in a dose responsive manner as the concentration of ALA increased or the fluence of light increased. ALA-PDT was less effective for MCF-7/ADR cells than MCF-7 cells even under the condition when these two cell lines contained the similar amounts of PpIX. CONCLUSIONS: These results indicate that, except for the MDR related characteristics, MCF-7/ADR cells might possess intrinsic mechanisms that render them less sensitive to ALA-PDT induced phototoxicity.

Detection of quadruplex DNA structures in human telomeres by a fluorescent carbazole derivative.

Single-stranded telomeric DNA tends to form a four-base-paired planar structure termed G-quadruplex. This structure was easily formed in vitro in the presence of monovalent cations. However, the existence of this structure in native human telomeres is unclear. Here we address this important question through the distinctive properties of 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC) upon binding to various DNA structures. Although the fluorescence of BMVC increases significantly in the presence of DNA, BMVC has high sensitivity and binding preference to quadruplex d(T(2)AG(3))(4) over duplex DNA. In addition, the fluorescent emissions were characterized around 575 nm for quadruplex d(T(2)AG(3))(4) and 545 nm for most of duplex DNA. The 575-nm fluorescence emissions were detected in the mixtures of 2 nM BMVC with the chromosomal DNA that were extracted from human cells, suggesting the presence of quadruplex structure in human nucleus. Further analyzing the BMVC fluorescence at the ends of metaphase chromosomes and other regions of chromosomes, we detected the quadruplex-binding BMVC fluorescence at telomere-proximal regions. Together these results provide the first evidence for the presence of quadruplex structures in human telomeres.