Hsp70 Reduces α-Synuclein Aggregation and Toxicity

Abstract

Aggregation and cytotoxicity of misfolded ␣-synuclein is postulated to be crucial in the disease process of neurodegenerative disorders such as Parkinson's disease and DLB (dementia with Lewy bodies). In this study, we detected misfolded and aggregated ␣-synuclein in a Triton X-100 insoluble fraction as well as a high molecular weight product by gel electrophoresis of temporal neocortex from DLB patients but not from controls. We also found similar Triton X-100 insoluble forms of ␣-synuclein in an ␣-synuclein transgenic mouse model and in an in vitro model of ␣-synuclein aggregation. Introducing the molecular chaperone Hsp70 into the in vivo model by breeding ␣-synuclein transgenic mice with Hsp70-overexpressing mice led to a significant reduction in both the high molecular weight and detergent-insoluble ␣-synuclein species. Concomitantly, we found that Hsp70 overexpression in vitro similarly reduced detergent-insoluble ␣-synuclein species and protected cells from ␣-synuclein-induced cellular toxicity. Taken together, these data demonstrate that the molecular chaperone Hsp70 can reduce the amount of misfolded, aggregated ␣-synuclein species in vivo and in vitro and protect it from ␣-synuclein-dependent toxicity.␣-Synuclein is a natively unfolded molecule that can selfaggregate to form oligomers and fibrillar intermediates (1-5) that accumulate to form Lewy bodies (LBs) 1 and Lewy neurites in neurons at risk for degeneration in Parkinson's disease and dementia with Lewy bodies (DLB) (6 -14). For the most part, these ␣-synuclein aggregates are densely compact and can be immunostained for multiple additional components including ubiquitin, synphilin-1, and heat shock proteins (HSPs), which suggests that protein misfolding or degradation is altered in cells that develop LBs. Mouse models of ␣-synuclein aggregation exist that mimic the findings in human brains and show intracellular ␣-synuclein aggregates (15-17). Aggregated ␣-synuclein molecules are less detergent-soluble, and these detergent-insoluble species of ␣-synuclein can be detected in human brain, transgenic mouse models, and in vitro models (18 -21). Although it is known that the conformation of ␣-synuclein in LBs is significantly different from that in the neuropil (22), it is unclear which conformation of ␣-synuclein contributes to inclusion formation. In addition to the formation of intracellular aggregates, ␣-synuclein is also cytotoxic. It has been postulated that ␣-synuclein oligomers found in Parkinson's disease tissue by Western blot analysis represent the toxic species (5).HSPs belong to the family of chaperone proteins and are important in both refolding misfolded proteins and directing proteins toward proteasomal degradation (23-25). HSPs can be protective in several neurodegeneration models (26 -29), and recent data in the fly model suggest that overexpression of the molecular chaperone Hsp70 protects against ␣-synuclein-induced degeneration (26, 30). Hsp70 and its related co-chaperones may be important in ␣-synuclein misfolding. In fact, sever...

Molecular chaperones and neuronal proteostasis

“…In a cell culture model, the overexpression of Hsp70 reduced high molecular weight and detergent insoluble α-synuclein, as well as reducing total α-synuclein levels. This corresponded with a reduction in α-synuclein-induced toxicity [16]. Consistent with these findings, the expression of Hsp70 in an α-synuclein transgenic mouse model was also able to reduce high molecular weight and detergent insoluble species [16].…”

“…This corresponded with a reduction in α-synuclein-induced toxicity [16]. Consistent with these findings, the expression of Hsp70 in an α-synuclein transgenic mouse model was also able to reduce high molecular weight and detergent insoluble species [16]. Further in vivo studies in both Drosophila melanogaster and mice showed that the Hsp70-mediated reduction in α-synuclein aggregation corresponded with an increase in dopaminergic neuron survival, enabling the preservation of striatal dopamine levels [17,18].…”

“…Reduced Aβ aggregation in vitro [22] and in transgenic mice [85]; modest effect on R6/2 Htt mice [31] but increased aggregation on knock-down in HD flies [86]; suppression of α-synuclein toxicity in flies [17] cells and mice [16], but another report found no effect in mice [87]; reduced mutant SOD1 aggregation in cells [88] but had no effect in mice [89]; suppressed TDP-43 toxicity in fly [90]. …”

Firefly luciferase mutants as sensors of proteome stress

“…Hsp70 members are highly multifunctional proteins that have been shown to play a key role in proteome maintenance, such as in de novo protein folding (co-or post-translational), protein translocation across membranes (Lyman and Schekman, 1997;Matlack et al, 1999;Young et al, 2003), refolding of stress damaged proteins (Ben-Zvi et al, 2004;Schroder et al, 1993;Sharma et al, 2010), in preventing protein aggregation (Auluck et al, 2002;Broadley and Hartl, 2009;Klucken et al, 2004;Sakahira et al, 2002;Warrick et al, 1999), disaggregation (Ben-Zvi and Goloubinoff, 2001;Diamant et al, 2000;Liberek et al, 2008;Shorter, 2011) and degradation of irreparable misfolded proteins (Bercovich et al, 1997;Fisher et al, 1997;Urushitani et al, 2004). These essential and diverse cellular functions of Hsp70 are attributed to its physical interaction with various co-chaperones such as Hsp40, NEFs and with proteins such as HIP, HOP and CHIP.…”

An antiaggregation gene therapy strategy for Lewy body disease utilizing β-synuclein lentivirus in a transgenic model

“…1 Among them, three potential candidates have been recently identified, namely b-synuclein, Parkin and heat-shock proteins (Hsp). 1,17 b-Synuclein belongs to a larger family of synaptic proteins that includes a-synuclein and g-synuclein. 18 b-Synuclein is an abundant synaptic protein originally identified as PNP14 in the bovine brain.…”