Chaperoned amyloid proteins for immune manipulation: α-Synuclein/Hsp70 shifts immunity toward a modulatory phenotype.

Adahir Labrador‐Garrido,Erwin J De Genst,Rebecca Klippstein,María M Leal,Laura Tomas‐Gallardo,Cintia Roodveldt,Juan J Toledo‐Aral,David Pozo,Javier Villadiego,Christopher M Dobson,Marta Cejudo‐Guillén

doi:10.1002/iid3.39

Abstract

α-Synuclein (αSyn) is a 140-residue amyloid-forming protein whose aggregation is linked to Parkinson's disease (PD). It has also been found to play a critical role in the immune imbalance that accompanies disease progression, a characteristic that has prompted the search for an effective αSyn-based immunotherapy. In this study, we have simultaneously exploited two important features of certain heat-shock proteins (HSPs): their classical “chaperone” activities and their recently discovered and diverse “immunoactive” properties. In particular, we have explored the immune response elicited by immunization of C57BL/6 mice with an αSyn/Hsp70 protein combination in the absence of added adjuvant. Our results show differential effects for mice immunized with the αSyn/Hsp70 complex, including a restrained αSyn-specific (IgM and IgG) humoral response as well as minimized alterations in the Treg (CD4+CD25+Foxp3+) and Teff (CD4+Foxp3−) cell populations, as opposed to significant changes in mice immunized with αSyn and Hsp70 alone. Furthermore, in vitro-stimulated splenocytes from immunized mice showed the lowest relative response against αSyn challenge for the “αSyn/Hsp70” experimental group as measured by IFN-γ and IL-17 secretion, and higher IL-10 levels when stimulated with LPS. Finally, serum levels of Th1-cytokine IFN-γ and immunomodulatory IL-10 indicated a unique shift toward an immunomodulatory/immunoprotective phenotype in mice immunized with the αSyn/Hsp70 complex. Overall, we propose the use of functional “HSP-chaperoned amyloid/aggregating proteins” generated with appropriate HSP-substrate protein combinations, such as the αSyn/Hsp70 complex, as a novel strategy for immune-based intervention against synucleinopathies and other amyloid or “misfolding” neurodegenerative disorders.

Highlights

Introduction aSynuclein is a highly conserved, soluble protein which is abundant in various regions of the brain, and which is currently believed to play a role in modulating synaptic plasticity, neurotransmitter release, and presynaptic vesicle pool size [1,2,3]
Highly purified aSyn and Hsp70 in buffer (50 mM Tris pH 7.4, 150 mM KCl, 2 mM MgCl2) at a 1:1 molar ratio were incubated at room temperature (RT) in the presence of 4 mM adenosine 50-triphosphate magnesium salt (ATP) for 2 h to induce the opening of the Hsp70 substrate binding pocket, and for a further 2 h with adenosine 50-diphosphate monopotassium salt dehydrate (ADP) added to a final concentration of 2.5 mM to favor the formation of the ‘‘high affinity’’ Hsp70/substrate complex [37]
In order to evaluate the formation of an aSyn/Hsp70 complex, we subjected the incubated mixtures to native PAGE electrophoresis followed by Western blot with antiHsp70 and anti-aSyn specific antibodies (Fig. 1A)

Summary

Introduction

Synuclein (aSyn) is a highly conserved, soluble protein which is abundant in various regions of the brain, and which is currently believed to play a role in modulating synaptic plasticity, neurotransmitter release, and presynaptic vesicle pool size [1,2,3]. The aberrant misfolding and aggregation of this protein and, its conversion into insoluble amyloid-like fibrils, are linked to Parkinson’s disease and other synucleinopathies [4]. These and other ‘‘misfolding/conformational’’ disorders are characterized by conversion of an initially soluble and functional polypeptide. In each particular disorder, unfolding and/or misfolding of specific proteins (for simplicity, referred to as ‘‘aggregating proteins’’) are key initial steps in the aberrant aggregation and amyloid formation process [6]

Methods

Results

Conclusion