Inhibition of Aβ assembly is an attractive pathway to developing reagents that will block Aβ toxicity and potentially will lead to treatment for AD. Because the assembly process of Aβ is complex and the relationship between assembly size and structure and toxicity are not well understood, it is important to understand the mechanisms by which inhibitors affect Aβ assembly and how the resulting structures correlate with inhibition of toxicity. Such structure–activity analysis may lead eventually to the ability to predict factors necessary for successful inhibition of Aβ toxicity.
Here, we used two complementary methods, PICUP and DLS, for studying the interaction of peptide inhibitors with Aβ42, and compared the data with our previous characterization of inhibition of Aβ42-induced toxicity by these peptides (18
) and the biophysical features of the peptides themselves (19
). The PICUP data showed that Aβ(35–42) and longer CTFs interrupted Aβ42 paranucleus formation, whereas the shorter peptides did not. The order of activity of the CTFs in inhibiting hexamer formation in this assay roughly followed CTF length and did not explain the relatively high potency with which Aβ(30–42), Aβ(31–42), Aβ(39–42), or Aβ(30–40) inhibited Aβ42-induced toxicity.
DLS measurements showed that CTFs interacted with Aβ42 and stabilized two oligomer populations. The data suggested several lines of correlation between inhibition of Aβ42-induced toxicity and the assembly behavior of different CTFs. The two previously-characterized toxicity inhibitors, Aβ(31–42) and Aβ(39–42) showed the strongest reduction of growth rate of P2 particles, dRH2/dt. However, slow growth rate alone did not explain the behavior of other CTFs, such as Aβ(30–42) or Aβ(30–40), which showed strong inhibition of Aβ42-induced toxicity but had little effect on dRH2/dt. A decrease in the size of P1 particles was observed in the presence of Aβ(39–42) or Aβ(30–40), but not other CTFs. Inhibition of formation of putative fibrils measured by the effect of CTFs on the frequency of intensity spikes correlated only partially with inhibition of toxicity and did not provide satisfactory mechanistic explanation for the toxicity results. These analyses suggested that more than one mechanism might be responsible for inhibition of Aβ42-induced toxicity by CTFs.
To gain additional mechanistic insight, we examined potential sets of correlation among the different data sets, including inhibition of paranucleus formation (), abundance of P
2 particles (), inhibition of toxicity ((18
) and Figure S2A
), CTF solubility (19
), CTF conversion to β-sheet-rich fibrils (19
), and CTF particle growth (19
). We calculated linear correlations among these data sets, which, depending on the parameter and the availability of the data, ranged from 4–7 data points. The analysis confirmed a poor correlation between inhibition of paranucleus formation and inhibition of Aβ42-induced neurotoxicity (r2
= 0.01, ). Inhibition of paranucleus formation showed relatively high correlation with CTFs solubility (r2
= 0.72, ), β-sheet formation (r2
= 0.96, Figure S3A
), and particle size increase (r2
= 0.94, Figure S3B
). The error bars of the solubility and particle growth rate of the CTFs alone in and Figure S3
are inherently quite large due to the large variability in amyloid peptide samples (30
). These errors are reflected in the calculated r2
values for the linear correlations. The correlation calculated might raise a concern regarding precipitation of Aβ42 in the presence of the least soluble CTFs. However, neither SDS-PAGE analysis of cross-linked oligomers (Figure S1
) nor the DLS measurements () showed such precipitation or reduced solubility. Thus, our analysis suggests that the same forces that reduce aqueous solubility and promote fibrillogenesis of CTFs in the absence of Aβ42 also facilitate the interaction of the CTFs with Aβ42 leading to inhibition of paranucleus formation.
Figure 4 Correlation analysis. A) Linear regression analysis correlating inhibition of paranucleus formation for Aβ(29–42)–Aβ(35–42) with inhibition of Aβ42-induced toxicity (19) (r2 = 0.01, p = 0.8). B) Linear regression (more ...)
Out of the different parameters we measured in the DLS experiments (dRH2/dt, the abundance of P2 particles, and intensity), we found that inhibition of Aβ42-induced toxicity correlated with a low abundance of P2 particles on day 2 (r2 = 0.90, ) and on day 4–7 (r2 = 0.75, data not shown). Thus, although the particle distribution initially had increased contribution of P2 particles in the presence of all CTFs relative to Aβ42 alone, on subsequent days, the relative contribution of P2 particles was small for strong inhibitors of toxicity and large for weak inhibitors. For reasons that are not entirely clear, Aβ(30–42) was an outlier and therefore was not included in this analysis.
Though the DLS experiments were done under conditions that differ from those of toxicity experiments, the high correlation between low abundance of P2 and Aβ42-induced toxicity at 48 h provides important insights into the mechanism(s) by which CTFs might inhibit the toxicity. This putative mechanism is summarized in . In the absence of CTFs (, top path), Aβ monomers rapidly self-assemble into small oligomers (P1 particles). Association of these oligomers into larger assemblies (P2 particles) is relatively slow, whereas the conversion of P2 assemblies into fibrils or their disassembly back into P1 oligomers is fast. As a result, little accumulation of P2 particles is observed. In the presence of CTFs (, bottom path), Aβ42 and the CTFs co-assemble into heterooligomers. the size of which is generally similar to that of the oligomers formed in the absence of CTFs. The CTFs stabilize both P1 and P2 oligomers and retard the conversion of P2 assemblies into fibrils. However CTFs vary in their effect on the conversion of the small P1 oligomers into the larger P2 oligomers. Effective inhibitors slow down this process and give rise to predominantly P1 oligomers, whereas less effective inhibitors allow for a relatively fast P1→P2 conversion. Thus, the anti-correlation between P2 abundance and inhibition of toxicity suggest that a predominant mechanism by which CTFs inhibit Aβ42 toxicity is by stabilizing P1 heterooligomers.
Figure 5 Schematic representation of a putative mechanism by which CTFs affect Aβ42 assembly. Monomer (M) assembly into P1 particles is a fast process in the absence (top path) or presence (bottom path) of CTFs. CTFs may accelerate the conversion of P (more ...)
Taken together, our data indicate that CTFs affect Aβ42 assembly in different ways, including disruption of paranucleus formation by Aβ(35–42) and longer Aβ42 CTFs, stabilization of P
1 and P
2 particles by all CTFs, alteration of the size and abundance of P
1 and P
2 assemblies, and co-assembly with Aβ42 into heterooligomers. Inhibition of Aβ42 toxicity by CTFs correlates with accumulation of P
1 heterooligomers suggesting attenuation of P
2 conversion. Stabilization of non-toxic Aβ oligomers is a mechanism shared by other inhibitors of Aβ assembly and toxicity, including scyllo
) and (−)-epigallocatechin gallate (32
). Thus, we propose that efforts geared towards designing inhibitors of protein self-assembly should focus on diversion of the process towards formation of non-toxic oligomers (or heterooligomers of Aβ and the inhibitor) that can be degraded by cellular clearance mechanisms rather than attempting to prevent monomer self-association.