Mikko Hiltunen

Depletion of GGA3 stabilizes BACE and enhances β-secretase activity

Summary β-site APP-cleaving enzyme (BACE) is required for production of the Alzheimers disease (AD)-associated Aβ protein. BACE levels are elevated in AD brain, and increasing evidence reveals BACE as a stress-related protease that is upregulated following cerebral ischemia. However, the molecular mechanism responsible is unknown. We show that increases in BACE and β-secretase activity are due to posttranslational stabilization following caspase activation. We also found that during cerebral ischemia, levels of GGA3, an adaptor protein involved in BACE trafficking, are reduced, while BACE levels are increased. RNAi silencing of GGA3 also elevated levels of BACE and Aβ. Finally, in AD brain samples, GGA3 protein levels were significantly decreased and inversely correlated with increased levels of BACE. In summary, we have elucidated a GGA3-dependent mechanism regulating BACE levels and β-secretase activity. This mechanism may explain increased cerebral levels of BACE and Aβ following cerebral ischemia and existing in AD.

Association of Single Nucleotide Polymorphisms of the Bile Salt Export Pump Gene with Intrahepatic Cholestasis of Pregnancy

BACKGROUND We determined whether genetic variability in the gene encoding the bile salt export pump (BSEP) contributes to individual differences in susceptibility to the development of intrahepatic cholestasis of pregnancy (ICP). METHODS The study involved 57 affected and 115 healthy control pregnant women who were genotyped for two single nucleotide polymorphisms (SNPs) in the BSEP gene. Chi-square analysis was used to assess genotype and allele frequency differences between the cholestatic and control groups. In addition, single locus analysis was expanded to pair of loci haplotype analysis to examine the estimated haplotype frequencies of the two SNPs, of unknown phase, among the cholestatic and control groups. Estimated haplotype frequencies were assessed using the maximum-likelihood method, employing an expectation-maximization (EM) algorithm. RESULTS The genotype and allele frequency distribution of the two intragenic SNPs in the ICP and control groups revealed significant evidence of association with the exon 28 SNP (P=0.04 and P=0.02, respectively). In addition, a borderline allele association was noted with the intron 19 SNP (P=0.08). Although the overall distribution of estimated haplotypes of intron 19 and exon 28 SNPs did not differ between the ICP and control groups, the most common haplotype, A-G, was significantly overrepresented in the ICP group (P=0.02), at an odds ratio of 1.73 (95% CI: 1.08-2.74). CONCLUSIONS The use of two intragenic SNPs in both single locus and haplotype analyses of association suggests that the BSEP gene is a susceptibility gene in intrahepatic cholestasis of pregnancy.Background: We determined whether genetic variability in the gene encoding the bile salt export pump ( BSEP ) contributes to individual differences in susceptibility to the development of intrahepatic cholestasis of pregnancy (ICP). Methods: The study involved 57 affected and 115 healthy control pregnant women who were genotyped for two single nucleotide polymorphisms (SNPs) in the BSEP gene. Chi-square analysis was used to assess genotype and allele frequency differences between the cholestatic and control groups. In addition, single locus analysis was expanded to pair of loci haplotype analysis to examine the estimated haplotype frequencies of the two SNPs, of unknown phase, among the cholestatic and control groups. Estimated haplotype frequencies were assessed using the maximum-likelihood method, employing an expectation-maximization (EM) algorithm. Results: The genotype and allele frequency distribution of the two intragenic SNPs in the ICP and control groups revealed significant evidence of association with the exon 28 SNP ( P r = r 0.04 and P r = r 0.02, respectively). In addition, a borderline allele association was noted with the intron 19 SNP ( P r = r 0.08). Although the overall distribution of estimated haplotypes of intron 19 and exon 28 SNPs did not differ between the ICP and control groups, the most common haplotype, A-G, was significantly overrepresented in the ICP group ( P r = r 0.02), at an odds ratio of 1.73 (95% CI: 1.08-2.74). Conclusions: The use of two intragenic SNPs in both single locus and haplotype analyses of association suggests that the BSEP gene is a susceptibility gene in intrahepatic cholestasis of pregnancy.

Genetic analysis of BDNF and TrkB gene polymorphisms in Alzheimer’s disease

According to previous biochemical and genetic findings, brain–derived neurotrophic factor (BDNF), via activation of its tyrosine kinase receptor B (TrkB), is considered as a plausible candidate for contributing to Alzheimer’s disease (AD). To examine the genetic association of BDNF and TrkB genes with AD, we genotyped multiple single nucleotide polymorphisms (SNPs) within these genes among 375 Finnish AD patients and 460 control subjects. Single locus and multi–loci haplotype association analyses of BDNF and TrkB gene SNPs did not reveal significant differences between unstratified AD and control groups. In the case of BDNF SNPs, different allele and haplotype frequencies were observed when 160 sporadic AD cases were compared with 460 control subjects. However, these differences did not remain statistically significant after multiple corrections. We conclude that BDNF and TrkB genes are not contributing significant risk effect among Finnish AD patients.

Insulin degrading enzyme is genetically associated with Alzheimer's disease in the Finnish population

The gene for insulin-degrading enzyme (IDE), which is located at chromosome 10q24, has been previously proposed as a candidate gene for late-onset Alzheimer’s disease (AD) based on its ability to degrade amyloid β-protein. Genotyping of single nucleotide polymorphisms (SNPs) in the IDE gene in Finnish patients with AD and controls revealed SNPs rs4646953 and rs4646955 to be associated with AD, conferring an approximately two-fold increased risk. Single locus findings were corroborated by the results obtained from haplotype analyses. This suggests that genetic alterations in or near the IDE gene may increase the risk for developing AD.

Butyrylcholinesterase K variant and apolipoprotein E4 genes do not act in synergy in Finnish late-onset Alzheimer's disease patients

Examination of the allelic frequency of the butyrylcholinesterase K (BChE-K) variant gene revealed no increase among Finnish late-onset Alzheimers disease (AD) patients either as a whole or among a subset of AD patients carrying the epsilon4 allele of apolipoprotein E (ApoE4). In contrast, BChE-K allele frequency was significantly reduced in the Finnish AD patient group under 75 years of age carrying the ApoE4 allele when compared to the non-demented controls (chi2, P < 0.05). The proportion of subjects with both BChE-K and ApoE4 alleles was 14% and 41% in AD and control groups, respectively (chi2, P < 0.01; odds-ratio 0.22, 95% CI 0.07-0.71). These results are in contrast to a previous study on English AD patients, in which the genes for BChE-K and ApoE4 were suggested to act in synergy.

Endothelial nitric oxide synthase polymorphism in preeclampsia.

objective: We wished to determine whether genetic variability in the gene encoding endothelial nitric oxide synthase (eNOS) modifies individual susceptibility to the development of preeclampsia. Methods: The study involved 132 preeclamptic and 113 healthy control pregnant women who were genotyped for the Glu298Asp polymorphism in the eNOS gene. X2 analysis was used to assess genotype and allele frequency differences between preeclamptic women and controls. Results: A statistically similar allelic distribution of eNOS Glu298Asp polymorphism was observed in the two groups, with the frequency of the variant G allele being 74.6% in the preeclampsia group and 67.7% in the control group (P = .91; odds rato 1.40, 95% confidence interval 0.95, 2.01). Accordingly, the genotype distribution of the NOS polymorphism in the preeclamptic and control groups was found to be similar (P = .233). Conclusion: These genotype data in subjects from eastem Finland were not suggestive of an important contribution of the Glu298Asp polymorphism in the NOS gene on preeclampsia across populations. However, the observed association between the Gallele and disease risk, of borderline significance, may imply that other polymorphism(s) in the gene may modify disease risk.

APP substitutions V715F and L720P alter PS1 conformation and differentially affect Aβ and AICD generation

The 37–43 amino acid Aβ peptide is the principal component of β‐amyloid deposits in Alzheimers disease (AD) brain, and is derived by serial proteolysis of the amyloid precursor protein (APP) by β‐ and γ‐secretase. γ‐Secretase also cleaves APP at Val50 in the Aβ numbering (ε cleavage), resulting in the release of a fragment called APP intracellular domain (AICD). The aim of this study was to determine whether amino acid substitutions in the APP transmembrane domain differentially affect Aβ and AICD generation. We found that the APPV715F substitution, which has been previously shown to dramatically decrease Aβ40 and Aβ42 while increasing Aβ38 levels, does not affect in vitro generation of AICD. Furthermore, we found that the APPL720P substitution, which has been previously shown to prevent in vitro generation of AICD, completely prevents Aβ generation. Using a fluorescence resonance energy transfer (FRET) method, we next found that both the APPV715F and APPL720P substitutions significantly increase the distance between the N‐ and C‐terminus of presenilin 1 (PS1), which has been proposed to contain the catalytic site of γ‐secretase. In conclusion, both APPV715F and APPL720P change PS1 conformation with differential effects on Aβ and AICD production.

Interaction between presenilin 1 and ubiquilin 1 as detected by fluorescence lifetime imaging microscopy and a high-throughput fluorescent plate reader

Presenilin 1 (PS1) in its active heterodimeric form is the catalytic center of the γ-secretase complex, an enzymatic activity that cleaves amyloid precursor protein (APP) to produce amyloid β (Aβ). Ubiquilin 1 is a recently described PS1 interacting protein, the overexpression of which increases PS1 holoprotein levels and leads to reduced levels of functionally active PS1 heterodimer. In addition, it has been suggested that splice variants of the UBQLN1 gene are associated with an increased risk of developing Alzheimer disease (AD). However, it is still unclear whether PS1 and ubiquilin 1 interact when expressed at endogenous levels under normal physiological conditions. Here, we employ three novel fluorescence resonance energy transfer-based techniques to investigate the interaction between PS1 and ubiquilin 1 in intact cells. We consistently find that the ubiquilin 1 N terminus is in close proximity to several epitopes on PS1. We show that ubiquilin 1 interacts both with PS1 holoprotein and heterodimer and that the interaction between PS1 and ubiquilin 1 takes place near the cell surface. Furthermore, we show that the PS1-ubiquilin 1 interaction can be detected between endogenous proteins in primary neurons in vitro as well as in brain tissue of healthy controls and Alzheimer disease patients, providing evidence of its physiological relevance.

Multidrug resistance 3 gene mutation 1712delT and estrogen receptor α gene polymorphisms in Finnish women with obstetric cholestasis

OBJECTIVE To investigate the contribution of the estrogen receptor alpha (ERalpha) polymorphism in the development of obstetric cholestasis and to determine whether multidrug resistance 3 (MDR3) gene 1712delT mutation detected in French patients is also present in Finnish women with obstetric cholestasis. STUDY DESIGN In a retrospective case-control study, two ERalpha polymorphisms and MDR3 gene mutation 1712delT were investigated in healthy control women (N = 47) and in women with diagnosis of obsteric cholestasis (N = 57). PvuII and XbaI polymorphisms in ERalpha gene were evaluated in genomic DNA by using the polymerase chain reaction (PCR). In addition, the frequencies in the general population in our area are presented for comparison. RESULTS None of the ERalpha genotypes or alleles was significantly over-represented in obstetric cholestasis. When the two ERalpha gene polymorphisms were analyzed in parallel, six genotype combinations were recognized, and the distribution of these genotype combinations did not reveal statistically significant differences between the cases and controls (P = 0.612). No patient or control was heterozygous or homozygous for the mutant allele in the MDR3 gene. CONCLUSION The present data indicate that polymorphism of the ERalpha gene and MDR3 gene 1712delT mutation are unlikely to play any significant role in obstetric cholestasis in affected Finnish women. Further work to identify explanatory factors is of particular interest.

Effects of Ubiquilin 1 on the Unfolded Protein Response

Previous studies have implicated the unfolded protein response (UPR) in the pathogenesis of Alzheimer’s disease (AD). We previously reported that DNA variants in the ubiquilin 1 (UBQLN1) gene increase the risk for AD. Since UBQLN1 has been shown to play a role in the UPR, we assessed the effects of overexpression and downregulation of UBQLN1 splice variants during tunicamycin-induced ER stress. In addition to previously described transcript variants, TV1 and TV2, we identified two novel transcript variants of UBQLN1 in brain: TV3 (lacking exons 2–4) and TV4 (lacking exon 4). Overexpression of TV1–3, but not TV4 significantly decreased the mRNA induction of UPR-inducible genes, C/EBP homologous protein (CHOP), BiP/GRP78, and protein disulfide isomerase (PDI) during the UPR. Stable overexpression of TV1–3, but not TV4, also significantly decreased the induction of CHOP protein and increased cell viability during the UPR. In contrast, downregulation of UBQLN1 did not affect CHOP mRNA induction, but instead increased PDI mRNA levels. These findings suggest that overexpression UBQLN1 transcript variants TV1–3, but not TV4, exert a protective effect during the UPR by attenuating CHOP induction and potentially increasing cell viability.