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.

Selective inhibition of phosphodiesterases 4, 5 and 9 induces HSP20 phosphorylation and attenuates amyloid beta 1–42‐mediated cytotoxicity

“…However, the chaperone's neuroprotective abilities in AD had not previously been linked with its phosphorylation. Recent work from our laboratory has demonstrated that phosphorylation of HSP20 by PKA increases its association with the aggregation domain of Aβ 1–42 , reducing oligomerisation 12 and neuronal toxicity of the peptide 13. We show here that PDE inhibitors, which target specifically PDEs 4, 5 and 9, induce the phosphorylation of HSP20 in a dose‐ and time‐dependent manner.…”

“…Expression of PDE4 13 and PDE5 16 had already been confirmed in our cellular model SH‐SY5Y, although there has been no such confirmation of PDE9. As a result, our first priority was to confirm the expression of PDE9 in this cell line.…”

“…Using the xCELLigence system for label‐free, real‐time monitoring of Aβ‐induced cytotoxicity 13, preliminary growth curves demonstrated that divergence between the Aβ 1–42 and Aβscr growth curves occurred after approximately 6 h of peptide incubation. We concluded that it takes 6 h for significant quantities of soluble Aβ 1–42 oligomers to accumulate within the cells to induce SH‐SY5Y death, hence we added PDE inhibitors 6 h post Aβ 1–42 addition.…”

“…As we have shown that phosphorylation of HSP20 increases its association with Aβ 1–42 and reduces the effective concentration of the chaperone required to inhibit Aβ 1–42 oligomerisation 13, we evaluated the formation of the HSP20‐fluorescein Aβ 1–42 ‐tagged complex in cells using immunofluorescence (Fig. 6A).…”

Location, location, location: PDE4D5 function is directed by its unique N-terminal region

“…Indeed, the HSP20-PDE4D5 disruptor peptide was shown to enhance HSP20-mediated protection against the hypertrophic response induced by chronic isoprenaline treatment in neonatal cardiac myocytes and to attenuate pathological cardiac remodelling in a mouse model of pressure overload [45]. In the future, the strategy of disrupting the PDE4D5-HSP20 complex to promote phosphorylation of HSP20 may also have potential as a therapeutic intervention to combat Alzheimer's disease, where phosphorylation of HSP20 promotes the chaperone's association with beta-amyloid [46] and ischemia/reperfusion injury where phospho-HSP20 prevents autophagy and cell death [47].…”