Probing the mechanism of inhibition of amyloid-β(1–42)–induced neurotoxicity by the chaperonin GroEL

Marielle A Wälti,Joseph Steiner,Fanjie Meng,Hoi Sung Chung,John M Louis,Rodolfo Ghirlando,Vitali Tugarinov,Avindra Nath,G Marius Clore

doi:10.1073/pnas.1817477115

Abstract

The human chaperonin Hsp60 is thought to play a role in the progression of Alzheimer's disease by mitigating against intracellular β-amyloid stress. Here, we show that the bacterial homolog GroEL (51% sequence identity) reduces the neurotoxic effects of amyloid-β(1-42) (Aβ42) on human neural stem cell-derived neuronal cultures. To understand the mechanism of GroEL-mediated abrogation of neurotoxicity, we studied the interaction of Aβ42 with GroEL using a variety of biophysical techniques. Aβ42 binds to GroEL as a monomer with a lifetime of ∼1 ms, as determined from global analysis of multiple relaxation-based NMR experiments. Dynamic light scattering demonstrates that GroEL dissolves small amounts of high-molecular-weight polydisperse aggregates present in fresh soluble Aβ42 preparations. The residue-specific transverse relaxation rate profile for GroEL-bound Aβ42 reveals the presence of three anchor-binding regions (residues 16-21, 31-34, and 40-41) located within the hydrophobic GroEL-consensus binding sequences. Single-molecule FRET analysis of Aβ42 binding to GroEL results in no significant change in the FRET efficiency of a doubly labeled Aβ42 construct, indicating that Aβ42 samples a random coil ensemble when bound to GroEL. Finally, GroEL substantially slows down the disappearance of NMR visible Aβ42 species and the appearance of Aβ42 protofibrils and fibrils as monitored by electron and atomic force microscopies. The latter observations correlate with the effect of GroEL on the time course of Aβ42-induced neurotoxicity. These data provide a physical basis for understanding how Hsp60 may serve to slow down the progression of Alzheimer's disease.

Highlights

The human chaperonin Hsp60 is thought to play a role in the progression of Alzheimer’s disease by mitigating against intracellular β-amyloid stress
We examine the impact of GroEL on Aβ42-induced neuronal cell toxicity and analyze the interaction of Aβ42 with GroEL using a variety of biophysical methods including solution NMR, electron microscopy (EM) and atomic force microscopy (AFM), dynamic light scattering (DLS), and single-molecule fluorescence resonance energy transfer (FRET)
The neurotoxic concentration range of Aβ42 was established by adding varying concentrations of Aβ42 (0– 10 μM) to cell cultures of fluorescently labeled neurons derived from neuronal stem cells and measuring neuronal cell count and neurite length at 72 h postexposure to Aβ42 (SI Appendix, Fig. S1A)

Summary

Neural cell count

In the presence of 3 μM Aβ42, disappearance of neurons is clearly apparent at 48 h, and at 72 h no intact neurons remain (Fig. 2A, Middle row). The latter changes are significantly reduced upon addition of GroEL (Fig. 2A, Bottom row): no morphological changes are observed at 48 h, and only a small amount of neuronal damage is observed at 72 h. Neuron cultures were monitored for nearly 4 wk, and only after consistent neuronal firing activity in all wells were the neurons exposed to Aβ42 in the absence or presence of GroEL.

Results and Discussion

Normalized intensity

Experimental Procedures