Characterization of presenilin complexes from mouse and human brain using Blue Native gel electrophoresis reveals high expression in embryonic brain and minimal change in complex mobility with pathogenic presenilin mutations

Abstract

The presenilin proteins are required for intramembrane cleavage of a subset of type 1 membrane proteins including the Alzheimer's disease amyloid precursor protein. Previous studies indicate presenilin proteins form enzymatically active high molecular mass complexes consisting of heterodimers of N‐ and C‐terminal fragments in association with nicastrin, presenilin enhancer‐2 and anterior pharynx defective‐1 proteins. Using Blue Native gel electrophoresis (BN/PAGE) we have studied endogenous presenilin 1 complex mass, stability and association with nicastrin, presenilin enhancer‐2 and anterior pharynx defective‐1. Solubilization of mouse or human brain membranes with dodecyl‐d‐maltoside produced a 360‐kDa species reactive with antibodies to presenilin 1. Presenilin 1 complex levels were high in embryonic brain. Complex integrity was sensitive to Triton X‐100 and SDS, but stable to reducing agent. Addition of 5 m urea caused complex dissolution and nicastrin to migrate as a subcomplex. Nicastrin and presenilin enhancer‐2 were detected in the presenilin 1 complex following BN/PAGE, electroelution and second‐dimension analysis. Anterior pharynx defective‐1 was detected as an 18‐kDa form and 9‐kDa C‐terminal fragment by standard SDS/PAGE of mouse tissues, and as a predominant 36‐kDa band after presenilin 1 complex second‐dimension analysis. Membranes from brain cortex of Alzheimer's disease patients, or from cases with presenilin 1 missense mutations, indicated no change in presenilin 1 complex mobility. Higher molecular mass presenilin 1‐reactive species were detected in brain containing presenilin 1 exon 9 deletion mutation. This abnormality was confirmed using cells transfected with the same presenilin deletion mutation.

Potent amyloidogenicity and pathogenicity of Aβ43

“…1c: arrowhead). A similar higher 8 molecular weight signal has previously been described in preparations from a patient with a deletion of exon 9 in the PSEN1 gene (PS1-ΔE9) 17 and from SH-SY5Y cells treated with a γ-secretase inhibitor, L-685,458 18 . PS1-ΔE9 and PS1-ΔT440 also cause PS1 endoproteolytic abnormality in a similar manner to the PS1-R278I mutation [19][20][21] .…”

“…3e). It is also noteworthy that the endoproteolysis was partially blocked in the heterozygous PS1-R278I brain, suggesting that the process is at least in part autolytic.We next investigated whether the PS1-R278I mutation affects the assembly of the γ-secretase complex by Blue Native PAGE (BN-PAGE) 17 . A major signal corresponding to a molecular weight of 360 kDa, the normal molecular weight of the native PS1 γ-secretase complex, was detected in both the wild-type and knockin brains in a manner similar to that of the PS2 γ-secretase complex (Fig.…”

Presenilins Promote the Cellular Uptake of Copper and Zinc and Maintain Copper Chaperone of SOD1-dependent Copper/Zinc Superoxide Dismutase Activity

“…PS2 expression was detected using a polyclonal rabbit antibody "00/12," raised to residues 307-336 of the human sequence PS2 loop domain as described previously (22). Polyclonal anti-SOD1 was purchased from Stressgen bioreagents.…”

S100B and S100A6 Differentially Modulate Cell Survival by Interacting with Distinct RAGE (Receptor for Advanced Glycation End Products) Immunoglobulin Domains

“…The membrane fraction and soluble proteins were prepared as described previously (36). SH-SY5Y cells were washed with PBS, scraped from the plates, and centrifuged, and then lysis buffer (250 mM sucrose, 20 mM Hepes, pH 7.4, containing Complete protease inhibitor mixture (Roche Applied Science)) was added to the cell pellet.…”