Pathogenic Aβ production by heterozygous PSEN1 mutations is intrinsic to the mutant protein and not mediated by conformational hindrance of wild-type PSEN1

Presenilin-1 (PSEN1) is the catalytic subunit of the intramembrane protease γ-secretase and undergoes endoproteolysis during its maturation. Heterozygous mutations in the PSEN1 gene cause early-onset familial Alzheimer’s disease (eFAD) and increase the proportion of longer aggregation-prone amyloid-β peptides (Aβ42 and/or Aβ43). Previous studies had suggested that PSEN1 mutants might act in a dominant-negative fashion by functional impediment of wild-type PSEN1, but the exact mechanism by which PSEN1 mutants promote pathogenic Aβ production remains controversial. Using dual recombinase-mediated cassette exchange (dRMCE), here we generated a panel of isogenic embryonic and neural stem cell lines with heterozygous, endogenous expression of PSEN1 mutations. When catalytically inactive PSEN1 was expressed alongside the wild-type protein, we found the mutant accumulated as a full-length protein, indicating that endoproteolytic cleavage occurred strictly as an intramolecular event. Heterozygous expression of eFAD-causing PSEN1 mutants increased the Aβ42/Aβ40 ratio. In contrast, catalytically inactive PSEN1 mutants were still incorporated into the γ-secretase complex but failed to change the Aβ42/Aβ40 ratio. Finally, interaction and enzyme activity assays demonstrated the binding of mutant PSEN1 to other γ-secretase subunits, but no interaction between mutant and wild-type PSEN1 was observed. These results establish that pathogenic Aβ production is an intrinsic property of PSEN1 mutants and strongly argue against a dominant-negative effect in which PSEN1 mutants would compromise the catalytic activity of wild-type PSEN1 through conformational effects.This work was supported in part by the Alzheimer Forschung Initiative (grant number 11811 to S. W.) and the Deutsche Forschungsgemeinschaft (German Research Foundation, grant number 494911640 to S. W.).Peer reviewe

Low-density lipoprotein receptor-related protein interacts with MafB, a regulator of hindbrain development

AbstractThe intracellular domain (ICD) of the low-density lipoprotein receptor-related protein (LRP) functionally interacts with adaptor proteins both as an integral part of the receptor polypeptide and after proteolytic release. Identification of such adaptors has been difficult because the ICD is self-activating in conventional transcription factor-based yeast two-hybrid screens. We adopted an alternative screen for the ICD that depends on the activation of the Ras-signaling pathway and uncovered the transcription factor MafB as novel ICD interacting protein. MafB is a regulator of hindbrain segmentation and interacts with the ICD through a leucine zipper domain. The ICD co-localizes with MafB to the nucleus and negatively regulates its transcriptional activity, suggesting a possible role for LRP in brain development

Development of a monoclonal antibody against recombinant neuroendocrine 7B2 protein

AbstractMouse monoclonal antibody MON-100 was raised against the neuroendocrine protein 7B2 using bacterially produced hybrid proteins. In Western blot analysis, MON-100 reacted with 3 different 7B2 hybrid proteins and not with the respective carrier proteins. Furthermore, MON-100 was reactive with recombinant 7B2 cleaved from a hybrid protein. In an immunohistochemical study, MON-100 exhibited strong reactivity with the intermediate lobe of the Xenopus pituitary gland, a tissue previously shown to contain 7B2 mRNA. Using MON-100, immunoprecipitation analysis of newly synthesized proteins produced by in vitro incubated Xenopus neurointermediate lobes revealed the biosynthesis of a single protein of Mr 24 kDa, the expected size of the 7B2 protein. It appears, therefore, that the anti-7B2 monoclonal antibody MON-100 can be successfully used for Western blot analysis and immunohistochemical analysis as well as for immunoprecipitation experiments

Reduced atherosclerosis development in ApoE^−/−LRP1^n2/n2 mice.

<p>A–B, Spontaneous atherosclerosis development in 26- (A) and 52-week (A–B) old mice. C–E, Different cholesterol levels in lipoprotein fractions (VLDL, LDL and HDL) separated via sequential ultracentrifugation in mice at 52-weeks of age (C), total triglyceride levels (D) and correlation plot between atherogenesis and total cholesterol for individual mice (E). Statistical analysis via determination of the Pearson’s correlation coefficient (Rp) revealed a significant positive correlation between atherosclerosis load in the aorta and circulating cholesterol levels. F, Total plasma cholesterol levels in mice at 12-, 26- or 52-weeks of age. G, Immunoblot analyses of hepatic LDLR and β-actin expression levels in 8- and 52-week old mice. ApoE^−/− (□ or ○) and apoE^−/−LRP1^n2/n2 (▪ or •) mice, n = 7–12 on a chow diet, data are mean±SEM. *<i>P</i><0.005, **<i>P</i><0.0005.</p

VLDL production, postprandial triglyceride response, intestinal lipid absorption and TRL clearance in apoE^−/− (Δ or □) and ApoE^−/−LRP1^n2/n2 (▪) mice.

<p>A–C, VLDL production after a Tyloxapol injection to inhibit lipolysis (A), postprandial triglyceride response after gastric olive oil load (B) and lipid absorption and chylomicron production after a combined gastric olive oil load and Tyloxapol injection (C) (n = 6–10 per genotype). D, Postprandial accumulation of ³H-Triolein in liver, white adipose tissue (WAT) and proximal (Prox.), medial (Med.) and distal (Dist.) intestine 2 h after a gastric load with olive oil mixed with ³H-Triolein (E) (n = 5 per genotype). Data are mean±SEM. *<i>P</i><0.05, **<i>P</i><0.001.</p

Analyses of total cholesterol, triglycerides and lipoprotein profiles in ApoE^−/− (open bars or Δ) and ApoE^−/−LRP1^n2/n2 (filled bars or ▪) mice.

<p>A–C, Plasma lipid levels (A), immunoblot analysis of plasma apolipoproteins (B) and their relative expression levels (C) (n = 8 per genotype). D–G, Lipoprotein profiles in fasted and postprandial state (pooled plasma from six mice per genotype). Plasma lipoprotein distribution of cholesterol (D–E) and triglyceride (F–G) levels in 5 hour fasted apoE^−/− and apoE^−/−LRP1^n2/n2 mice just before (D & F) or 2 hours after receiving a gastric olive oil load (E & G). Data are mean±SEM. *<i>P</i><0.05.</p