Figure 4.

Provision of extracellular Aβ elicits the phosphorylation of CRMP-2 primarily at the pThr555 site.

(A) Aβ binds to plasma membranes to initiate downstream signaling. Mapping neuronal plasma membrane with AlexaFluor555 cholera toxin B shows co-localization with Fluo488-Aβ_1–40. (B) Aβ_1–42 peptide in culture medium [1 μM following 36 hours at 37°C in DMEM/F12] imaging using tapping mode (TM)-AFM is rich in fibrils of approximately 5–8 nm in height and > 20 nm in length. (C & E) Aβ-induced phosphorylation of CRMP-2 at the T555 site increases with the increase in Aβ_1–40 concentration administered to the cells for 24 hours (n = 3 independent experiments; *P < 0.05, **P < 0.01). Potentiated phosphorylation of both CRMP-2A (75 kDa) and CRMP-2B (62 kDa) isoforms occur after the administration of Aβ1-40, while decreased phosphorylation is observed for both CRMP-2A and CRMP-2B at T555 site after Aβ_1–42 treatment (C & E; n = 3 independent experiments; **P < 0.01). What is evident is the presence of approximately 55 kDa CRMP-2 cleavage product primarily in Aβ_1–42 treated cells (Arrow, C). (C & F) Phosphorylation of CRMP-2B and CRMP-2A at the T514 site after Aβ_1–40 and Aβ_1–42 treatment of cells illustrates no significant differences compared to untreated cells (n = 3 independent experiments). (C & G) The treatment of cells with either Aβ_1–40 or Aβ_1–42 demonstrates no significant difference in CRMP-2B phosphorylation at the S522 site (n = 3 independent experiments). (C & H) Hyperphosphorylated tau levels increase with the increase in Aβ_1–40 concentration (n = 3 independent experiments; *P < 0.05, **P < 0.01). On the other hand, no significant increases can be attributed for tau hyperphosphorylation following Aβ_1–42 at 1.0 μM or at 10.0 μM. (D–G) Treating SH-SY5Y cells with different concentrations of scrambled Aβ_1–40 or Aβ_1–42 peptides show no significant differences in the phosphorylation at either T555, T514 nor S522 sites, when compared to untreated control cells (n = 3 independent experiments). (H) Moreover, SH-SY5Y cells treated with different concentrations of scrambled Aβ_1–40 and Aβ_1–42 peptides show no significant differences in tau phosphorylation compared to untreated control cells (n = 3 independent experiments, *P < 0.05). (I) Neurite (anti-NF200-positive) lengths of differentiated SH-SY5Y human neuroblastoma cells treated with either, Aβ_1–40, Aβ_1–42 or the scrambled peptides of these species (****P < 0.0001, n > 200 neurites counted per treatment in at least n = 3 wells). Statistically significant differences were demonstrated following multiple pair-wise comparisons (one-way analysis of variance with Tukey's post hoc tests performed).