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The molecular chaperone Brichos breaks the catalytic cycle that generates toxic Aβ oligomers

Alzheimer’s disease is an increasingly prevalent neurodegenerative disorder whose pathogenesis has been associated with aggregation of the amyloid-β peptide (Aβ42). Recent studies have revealed that once Aβ42 fibrils are generated, their surfaces strongly catalyse the formation of neurotoxic oligomers. Here we show that a molecular chaperone, a Brichos domain, can specifically inhibit this catalytic cycle and limit Aβ42 toxicity. We demonstrate in vitro that Brichos achieves this inhibition by binding to the surfaces of fibrils, thereby redirecting the aggregation reaction to a pathway that involves minimal formation of toxic oligomeric intermediates. We verify that this mechanism occurs in living brain tissue by means of cytotoxicity and electrophysiology experiments. These results reveal that molecular chaperones can help maintain protein homeostasis by selectively suppressing critical microscopic steps within the complex reaction pathways responsible for the toxic effects of protein misfolding and aggregation.

The effect of an inhibitor can in the first instance be semi-empirically captured by altering the microscopic rate constants for primary nucleation (k n ), elongation (k + ) and secondary nucleation (k 2 ) in the integrated rate law in Eq. 1. Perturbing the different microscopic events results in characteristic changes in the shape of the macroscopic reaction profile that can be used to identify the mechanism of action of an inhibitor (Fig. 1a-c). This approach is exact for complete inhibition of a particular process, as shown for secondary nucleation in Fig. 1c (green dashed line). Using this approach (Fig. 1a-c and Supplementary Fig. 2), we were able to identify a Brichos domain which affects specifically the secondary nucleation rate.
In order to further quantify the inhibitory effect of the Brichos domain at chaperone concentrations where inhibition of secondary nucleation is not complete, we then used a firstprinciples approach by introducing into the reaction scheme the reversible binding of the chaperone along the fibril surface. Such binding decreases the surface of the fibrils available to catalyse the oligomer formation, therefore reducing the secondary nucleation rate. The number of bound monomeric units in the fibrils, M bound , was evaluated by considering a Langmuir-type adsorption of the chaperone along the fibril surface: where C c (t) is the concentration of soluble chaperone and k onSurf and k offSurf are the binding and unbinding rate constants, respectively, which have been estimated independently by surface plasmon resonance (Fig. 3b). Since the characteristic time of binding is comparable to the characteristic time of incorporation of a monomeric unit into the fibril by elongation, the binding and unbinding reactions cannot be considered at equilibrium with respect to the microscopic aggregation rates. In order to predict the kinetic profiles at intermediate chaperone concentrations (thin dotted lines, Fig. 1c), Eq. 2 has been solved together with the kinetic equations 26 for the total fibrillar number, P, and mass, M, concentrations, where the secondary nucleation process is proportional to the area of the fibril surface that does not have chaperone bound to it: The set of Eqs. 2-4 has been integrated numerically using the microscopic reaction constants evaluated in our previous kinetic study 19 and the binding and unbinding reaction rates determined by SPR, with the latter allowed to vary in a narrow range to give the best fit to the data (k onSurf = 1 × 10 4 M −1 s −1 and k offSurf = 1 × 10 −4 s −1 ). Remarkably, the integrated global kinetic profiles predict essentially perfectly the inhibitory effect at different concentrations of chaperone, as shown in Fig. 1c.
In addition to affecting the reaction profile, perturbing the different microscopic reaction rates results in dramatically different effects on the generation of low molecular weight oligomeric species. To illustrate this behaviour, we calculated the time evolution of the rate of generation of new fibrils (via on-pathway oligomers) from monomers, with this nucleation rate r n (t) given by ( ) = ( ) + 2 ( ) ( ) 2 . Moreover, since at the end of the reaction no oligomers are detectable in the system 19 , the total number of fibrils present at the end of the process is indicative of the total number of on-pathway oligomers generated during the reaction. This value is calculated by integrating the nucleation rate r n (t) over the reaction.
The kinetic analysis predicts that the inhibitory effect of Brichos reaches a saturation at a critical chaperone concentration corresponding to the complete saturation of the surface of the fibrils. According to this prediction, an increase of the Brichos concentration above this critical threshold value does not have any consequences on the aggregation kinetics. We verified this hypothesis by performing kinetic experiments in the presence of increasing concentrations of Brichos ( Supplementary Fig. 1).

Cell viability and cytotoxicity
Assays were performed on SHSY-5Y human neuroblastoma cells cultured under standard conditions at 37ºC in a humidified incubator with 5% CO2. Cells were seeded at a density of 25,000 per well in a white walled, clear bottomed 96 well plate and cultured for 24 hours in DMEM/10% FBS. The culture media was then replaced with pre-warmed phenol red free DMEM without serum into which the peptide samples or NaH 2 PO 4 buffer were diluted 1:4. Cell viability as measured by the MTS assay is drastically reduced in the presence of 1 µM Aβ42 without or with 10 nM seeds ( Supplementary Fig. 4a), while the addition of 1 µM Brichos leads to essentially full recovery of cell viability. An equivalent trend is observed for the caspase 3/7 assay, although the analysis is less straightforward since the assay suffers from the limitation that the single colorimetric readout represents the global effect of many possible cellular processes. An increase of caspase signal is commonly associated with an increase of apoptosis activity of the cells, although caspase activation has been observed in the absence of apoptosis 63 . Critically, a rapid decrease of cell viability (as observed on addition of Aβ42) results in a decrease in the number of cells able to generate the caspase signal, therefore potentially inducing a decrease of caspase fluorescence rather than an increase. As a consequence, the result of the assay is significantly affected by the time of the readout with respect to the characteristic time of cell death. To account for this behaviour, we used the same assay for two separate clones of the SHSY-5Y cells with widely different sensitivity to perturbations of the growing medium. In Supplementary Fig. 4b we show one example of the caspase activation assay for the more sensitive cells (the same cells used for the MTS assay in Supplementary Fig. 4a) with 1 µM Aβ42 without or with 10 nM seeds. In this case, the caspase activity decreases on addition of Aβ42 relative to untreated cells, likely reflecting that cells die quickly, therefore reducing the number of cells able to generate signal after 24 hours. The effect of Aβ42 on the caspase signal is, however, abolished by 1 µM Brichos. In Supplementary Fig. 4c we show an alternative example of the caspase activation assay for the less sensitive cells with 10 µM Aβ42 without or with 100 nM seeds. In this case the caspase activity is increased by Aβ42 compared to untreated cells, indicating that the cell viability is still high but several cells have initiated the apoptosis processes. Again, the effect of Aβ42 on the caspase signal is abolished in the presence of Brichos. Importantly, despite the two cell lines having different sensitivities to toxic species and exhibiting different relative changes in caspase activity with respect to the control, in both cases the results clearly show that the toxic effects of Aβ42 aggregation are removed by the presence of Brichos. The results of the viability and cytotoxicity assays ( Supplementary Fig. 4) show that the addition of Brichos decreases the toxicity of reactions with or without pre-formed fibrils.
These results are in excellent agreement with the electrophysiology data shown in Fig. 5.