The phosphorylation of Hsp20 enhances its association with amyloid-β to increase protection against neuronal cell death

Abstract

Up-regulation of Hsp20 protein levels in response to amyloid fibril formation is considered a key protective response against the onset of Alzheimer's disease (AD). Indeed, the physical interaction between Hsp20 and Aβ is known to prevent Aβ oligomerisation and protects neuronal cells from Aβ mediated toxicity, however, details of the molecular mechanism and regulatory cell signalling events behind this process have remained elusive. Using both conventional MTT end-point assays and novel real time measurement of cell impedance, we show that Hsp20 protects human neuroblastoma SH-SY5Y cells from the neurotoxic effects of Aβ. In an attempt to provide a mechanism for the neuroprotection afforded by Hsp20, we used peptide array, co-immunoprecipitation analysis and NMR techniques to map the interaction between Hsp20 and Aβ and report a binding mode where Hsp20 binds adjacent to the oligomerisation domain of Aβ, preventing aggregation. The Hsp20/Aβ interaction is enhanced by Hsp20 phosphorylation, which serves to increase association with low molecular weight Aβ species and decrease the effective concentration of Hsp20 required to disrupt the formation of amyloid oligomers. Finally, using a novel fluorescent assay for the real time evaluation of morphology-specific Aβ aggregation, we show that phospho-dependency of this effect is more pronounced for fibrils than for globular Aβ forms and that 25mers corresponding to the Hsp20 N-terminal can be used as Aβ aggregate inhibitors. Our report is the first to provide a molecular model for the Hsp20/Aβ complex and the first to suggest that modulation of the cAMP/cGMP pathways could be a novel route to enhance Hsp20-mediated attenuation of Aβ fibril neurotoxicity.

Keywords: Aβ oligomerisation; Hsp20; Peptide array.

Fig. 1

Mapping the interaction between Hsp20 and Aβ_1–42. Peptide array was used to map the domains responsible for Hsp20/Aβ_1–42 interaction. (A) Diagram of domain structure of Hsp20 highlighting the PKA/PKG site located in the N-terminal domain and the conserved α-crystallin domain located between residues 70 and 144. (B) Peptide array libraries of Hsp20 25mers were probed with either Aβ_1–42 or Aβ_scr. (C) Alanine scanning arrays of peptide 3 (W¹¹–E³⁵) were probed with either Aβ_1–42 (middle panel) or Aβ_scr (upper panel) to determine the Hsp20 amino acids that are essential for Aβ_1–42 binding. The association of Aβ_1–42 with substitution arrays in which serine 16 was replaced by a phospho-serine or phospho-mimetic substitution (serine changed to aspartic acid) was also evaluated (lower panel). * = p < 0.05, ** = p < 0.01 using Student-t-test (n = 4).

Fig. 2

Mapping the interaction between Aβ_1–42 and Hsp20. (A) Diagram of the of Aβ_1–42 peptide with oligomerisation domain highlighted. Peptide array libraries of 25mers that spanned the Aβ_1–42 sequence were probed with either His-Hsp20 or His-RACK1. (B) Alanine scanning arrays of Aβ_1–42 peptide 1 (D¹–G²⁵) were probed with either His-Hsp20 (upper panel) or His-RACK1 (lower panel) to determine the Aβ_1–42 amino acids that are essential for Hsp20 binding. (C) Quantifications of spot density of peptides in B (typical of n = 3).

Fig. 3

Cell viability assays to monitor Aβ_1–42 mediated cytotoxicity. (A) The MTT cell viability assay was used to determine the effect of Aβ_1–42 or Aβ_scr on cell viability of SH-SY5Y cells transfected with various constructs of Hsp20 (see inset for relative levels of expression). ($ = p < 0.001). Significant reduction in viability is denoted by * = p < 0.05 and $ = p < 0.001 using Student-t-test (n = 4). (B) Direct comparison of Aβ_1–42 dose-dependent reduction in SH-SY5Y cell viability measured by MTT or using the xCELLigence real-time monitoring system. Data representative of n = 3.

Fig. 4

Real time monitoring of Aβ_1–42 induced cytotoxicity in SH-SY5Y cells. (A) Impedance growth profiles of SH-SY5Y cells were measured over 48 h following treatment with Aβ_1–42 or Aβ_scr .(B) Impedance growth profiles of SH-SY5Y cells transfected with empty vector or Hsp20 (see inset for relative expression levels) were measured over 48 h following treatment with Aβ_1–42.

Fig. 5

Hsp20 over-expression attenuates Aβ_1–42 induced cytotoxicity in SH-SY5Y cells. (A) A dose response curve of cell viability (as measured by cell index) was constructed over a range of Aβ_1–42 concentrations in SH-SY5Y cells transfected with empty vector or Hsp20. Relative levels of phospho-Hsp20 and total Hsp20 were determined by western blotting (left panels). Impedance growth profiles of SH-SY5Y cells transfected with empty vector, Hsp20Wt, Hsp20S16D, and Hsp20S16A (see inset for relative expression levels) were measured over 48 h following treatment with (B) Aβ_scr or (C) Aβ_1–42. Quantifications of cell index at 48 h compared with the scrambled control in (A) were determined (n = 3) and statistical evaluation undertaken * = p < 0.05 and *** = p < 0.001 using Student-t-test.

Fig. 6

Evaluation of morphology-specific inhibition of Aβ_1–42 aggregation by Hsp20 using a novel fluorescence self-quenching assay. The interaction between Hsp20 variants and Aβ_1–42 labelled at the N-terminus with HiLyte Fluor 555 (Aβ₅₅₅) was monitored using fluorescence self-quenching under globular (A) and fibrillar (C) growing conditions. The interaction between Hsp20 N-terminal 25mers and Aβ_1–42 labelled at the N-terminus with HiLyte Fluor 555 (Aβ₅₅₅) was monitored using fluorescence self-quenching under globular (B) and fibrillar (D) growing conditions. WT = wild type Hsp20, S16D = a phosphomimetic HSP20, RRA = a construct that is defective in binding Aβ_1–42, and P20L = a polymorph (a naturally occurring mutant that is known to reduce the capacity of Hsp20 to be phosphorylated at serine 16).

Fig. 7

Chemical shift analysis of ¹⁵N-Aβ_1–40 co-incubation with Hsp20. A. 2D HSQC experiment showing ¹⁵N-Aβ_1–40 (green); co-incubated with either Hsp20 WT (blue), Hsp20-S16D (purple) or Hsp20-RAA (red) at 4 °C prior to aggregation. B. Chemical shift perturbation plot from same experiment as (A). Data plotted relative to the ¹⁵N-Aβ_1–40 control. C. Hsp20 immunoprecipitations from the NMR samples were probed for Aβ following 4 day incubation under aggregating conditions. WT = wild type Hsp20, S16D = a phosphomimetic HSP20, RRA = a construct that is defective in binding Aβ_1–42.