Amyloid β-peptide 1-42 modulates the proliferation of mouse neural stem cells: upregulation of fucosyltransferase IX and notch signaling

Abstract

Amyloid β-peptides (Aβs) aggregate to form amyloid plaques, also known as senile plaques, which are a major pathological hallmark of Alzheimer's disease (AD). Aβs are reported to possess proliferation effects on neural stem cells (NSCs); however, this effect remains controversial. Thus, clarification of their physiological function is an important topic. We have systematically evaluated the effects of several putative bioactive Aβs (Aβ1-40, Aβ1-42, and Aβ25-35) on NSC proliferation. Treatment of NSCs with Aβ1-42 significantly increased the number of those cells (149 ± 10 %). This was not observed with Aβ1-40 which did not have any effects on the proliferative property of NSC. Aβ25-35, on the other hand, exhibited inhibitory effects on cellular proliferation. Since cell surface glycoconjugates, such as glycolipids, glycoproteins, and proteoglycans, are known to be important for maintaining cell fate determination, including cellular proliferation, in NSCs and they undergo dramatic changes during differentiation, we examined the effect of Aβs on a number of key glycoconjugate metabolizing enzymes. Significantly, we found for the first time that Aβ1-42 altered the expression of several key glycosyltransferases and glycosidases, including fucosyltransferase IX (FUT9), sialyltransferase III (ST-III), glucosylceramide ceramidase (GLCC), and mitochondrial sialidase (Neu4). FUT9 is a key enzyme for the synthesis of the Lewis X carbohydrate epitope, which is known to be expressed in stem cells. Aβ1-42 also stimulated the Notch1 intracellular domain (NICD) by upregulation of the expression of Musashi-1 and the paired box protein, Pax6. Thus, Aβ1-42 upregulates NSC proliferation by modulating the expression of several glycogenes involved in Notch signaling.

Fig. 1. Effects of soluble Aβs (Aβ1-40 or Aβ1-42) on mouse NSCs in culture

a. Primary structures of Aβ peptides. b. NSCs were cultured for 3 days with or without Aβs. The number of NSCs in the monolayer culture in the presence of soluble Aβs (10 μM) was estimated by the WST-8 assay. The y-axis represents relative absorbance (Abs.), which represents the percentage of absorbance against vehicle treatment. Each bar represents mean ± SD of 8 independent experiments (n = 8). Comparison was made between vehicle versus each treatment. *p<0.05.

Fig. 2. Structures and metabolic pathways of gangliosides

The nomenclature for gangliosides and the components are based on those of Svennerholm (1981) and IUPAC-IUBMB Joint Commission on Biochemical Nomenclature (1997). β-gal, lysosomal acid β-galactosidase; GalNAc-T, N-acetylgalactosaminyltransferase I (GA2/GM2/GD2/GT2-synthase); GalT-I, galactosyltransferase I (lactosylceramide synthase); GalT-II, galactosyltransferase II (GA1/GM1/GD1b/GT1c-synthase); GLCC, glucosylceramidase; GlcT, glucosyl transferase (glucosylceramide synthase); GM2A, GM2 activator protein; HEX, β-N-acetylhexosaminidase; ST-I, sialyltransferase I (GM3-synthase); ST-II, sialyltransferase II (GD3-synthase); ST-III, sialyltransferase III (GT3-synthase); ST-IV; sialyltransferase IV (GM1b/GD1a/GT1b/GQ1c-synthase); ST-V, sialyltransferase V (GD1c/GT1a/GQ1b/GP1c-synthase); ST-VII, sialyltransferase VII (GD1aα/GT1aα/ GQ1bα/GP1cα-synthase).

Fig. 3. Aβ treatment alters glycosyltransferases and glycosidases expression in NSCs

NSCs were cultured for 3 days with 10 μM of Aβ1-40 or Aβ1-42, or without Aβs. RT-PCR analyses were performed using specific primer sets. The enzymes for ganglioside metabolism are shown in Fig.1. Neu1, lysosomal sialidase; Neu3, plasma membrane sialidase; Neu4; mitochondrial sialidase; FUT9, α1–3 fucosyltransferase IX. G3PDH was used as a control. RT-PCR products were resolved on agarose gels and the intensity was quantified with normalization against G3PDH mRNA. The normalized value from control (vehicle) is defined as 1.0. Each bar represents mean ± SD of 3 independent experiments (n = 3). * (p<0.05) indicate the level of significance in two tailed t-tests of differences between vehicle versus each treatment.

Fig. 4. Expressions of neural cell lineage-associated markers

NSCs were cultured for 3 days with 10 μM of Aβ1-40 or Aβ1-42, or without Aβs. a RT-PCR analyses were performed for Nestin, MAP2, GFAP, Pax6 and Musashi-1. RT-PCR products were resolved on agarose gels and intensity was quantified with normalization against G3PDH mRNA. The normalized value from control (vehicle) is defined as 1.0. b Western blot of NSCs treated with Aβs, and immunostained using anti-Notch1 intracellular domain (NICD), and anti-actin antibodies. Values were normalized by setting the NICD/Actin protein ratio for control (vehicle) in each treatment. Each bar represents mean ± SD of 3 independent experiments (n = 3). * (p<0.05) indicate the level of significance in two tailed t-tests of differences between vehicle versus each treatment.