Amyloid β peptides (Aβs) of Alzheimer’s disease (AD) are formed from the cleavage of amyloid precursor protein (APP) by β- and λ-secretase. λ-Secretase is known to be a complex including members of the presenilin protein family which also have direct effects on IP3-sensitive intracellular Ca2+ stores (Leissring et al. 2000; Smith et al. 2002). Here, we report a novel effect of Aβs on presenilin expression.
SH-SY5Y cells were cultured as previously described and, either plated onto coverslips and loaded with fura-2 AM for the measurement of muscarine-evoked increases in [Ca2+]i or lysed into protein extraction reagent for standard Western immunoblotting (Smith et al. 2001). Blots were probed with antibodies raised against c or n terminal fragments (CTF and NTF) of PS-1 and the NTF of PS-2. All values are means ± S.E.M. and n > 3. Statistical significance (P < 0.01) was tested by one-way ANOVA with a Bonferroni multiple comparison post test.
Treatment with Aβ1-40 (1 mM, 24 h) caused a 1.8 ± 0.3 (NTF)- and 4.0 ± 0.8 (CTF)-fold increase in expression of full length and a 2.2 ± 0.06 (NTF)- and 2.6 ± 0.6 (CTF)-fold increase in cleaved PS-1 relative to control values. The Aβ25-35 fragment mimicked this effect of Aβ1-40 with a 3.3 ± 1.8 (NTF)- and 3.3 ± 0.8 (CTF)- fold increase in full length and a 2.4 ± 0.06 (NTF)- and 1.4 ± 0.1 (CTF)-fold increase in cleaved PS-1. The reverse sequence peptide Aβ40-1 and Aβ1-42 were without effect. This effect of Aβ1-40 could be due to the production of reactive oxygen species (ROS) and so cells were treated with H2O2 (40 or 150 mM, 24 h) which had no effect on PS-1 expression. Co-incubation with both Aβ1-40 (1 mM) and one of the anti-oxidants melatonin (150 mM), ascorbic acid (200 mM) or ebselen (10 mM), for 24 h did not prevent the increase in PS-1 levels. Similar results were obtained for PS-2 expression.
Aβ1-40 and Aβ25-35 (1 mM, 24 h) caused a significant increase in the size of muscarine-sensitive Ca2+ stores in these cells (control, 23.6 ± 5.7; 1-40, 61.7 ± 5.2; 25-35, 65.4 ± 5.6), an effect that was not seen after treatment with the 40-1 or 1-42 peptides. The effect of Aβ1-40 on Ca2+ stores was not prevented by melatonin or ebselen.
The present study shows for the first time that Aβ1-40 positively regulates the expression of the enzyme responsible for the production of Aβ peptides. The mechanism is unknown but does not involve the production of reactive oxygen species. This positive feedback loop may contribute to the deposition of amyloid peptides and so the progressive neurodegeneration seen in AD.
This work was supported by The Wellcome Trust.