Chaperome screening leads to identification of Grp94/Gp96 and FKBP4/52 as modulators of the α-synuclein-elicited immune response.

We have investigated the potential role of molecular chaperones as modulators of the immune response by using α-synuclein (αSyn) as an aggregation-prone model protein. We first performed an in vitro immunoscreening with 21 preselected candidate chaperones and selected 2 from this set as displaying immunological activity with differential profiles, Grp94/Gp96 and FKBP4/52. We then immunized mice with both chaperone/α-synuclein combinations using monomeric or oligomeric α-synuclein (MαSyn or OαSyn, respectively), and we characterized the immune response generated in each case. We found that Grp94 promoted αSyn-specific T-helper (Th)1/Th17 and IgG1 antibody responses (up to a 3-fold increase) with MαSyn and OαSyn, respectively, coupled to a Th2-type general phenotype (generating 2.5-fold higher IgG1/IgG2 levels). In addition, we observed that FKBP4 favored a Th1-skewed phenotype with MαSyn but strongly supported a Th2-type phenotype with OαSyn (with a 3-fold higher IL-10/IFN-γ serum levels). Importantly, results from adoptive transfer of splenocytes from immunized animals in a Parkinson's disease mouse model indicates that these effects are robust, stable in time, and physiologically relevant. Taken together, Grp94 and FKBP4 are able to generate differential immune responses to α-synuclein-based immunizations, depending both on the nature of the chaperone and on the aggregation state of α-synuclein. Our work reveals that several chaperones are potential modulators of the immune response and suggests that different chaperones could be exploited to redirect the amyloid-elicited immunity both for basic studies of the immunological processes associated with neurodegeneration and for immunotherapy of pathologies associated with protein misfolding and aggregation.

A rationally designed six-residue swap generates comparability in the aggregation behavior of α-synuclein and ß-synuclein.

The aggregation process of α-synuclein, a protein closely associated with Parkinson's disease, is highly sensitive to sequence variations. It is therefore of great importance to understand the factors that define the aggregation propensity of specific mutational variants as well as their toxic behavior in the cellular environment. In this context, we investigated the extent to which the aggregation behavior of α-synuclein can be altered to resemble that of ß-synuclein, an aggregation-resistant homologue of α-synuclein not associated with disease, by swapping residues between the two proteins. Because of the vast number of possible swaps, we have applied a rational design procedure to single out a mutational variant, called α2ß, in which two short stretches of the sequence in the NAC region have been replaced in α-synuclein from ß-synuclein. We find not only that the aggregation rate of α2ß is close to that of ß-synuclein, being much lower than that of α-synuclein, but also that α2ß effectively changes the cellular toxicity of α-synuclein to a value similar to that of ß-synuclein upon exposure of SH-SY5Y cells to preformed oligomers. Remarkably, control experiments on the corresponding mutational variant of ß-synuclein, called ß2α, confirmed that the mutations that we have identified also shift the aggregation behavior of this protein toward that of α-synuclein. These results demonstrate that it is becoming possible to control in quantitative detail the sequence code that defines the aggregation behavior and toxicity of α-synuclein.