Hsp110 mitigates α-synuclein pathology in vivo Yumiko V. Taguchia, Erica L. Gorenberga, Maria Nagyb, Drake Thrasherc, Wayne A. Fentonb, Laura Volpicelli-Daleyc, Arthur L. Horwichb,d, and Sreeganga S. Chandraa,e,f,1 aDepartment of Neurology, Yale University, New Haven, CT 06536; bHoward Hughes Medical Institute, Yale University, New Haven, CT 06510; cDepartment of Neurology, University of Alabama, Birmingham, AL 35294; dDepartment of Genetics, Yale University, New Haven, CT 06510; eDepartment of Neuroscience, Yale University, New Haven, CT 06536; and fYale Stem Cell Center, Yale University, New Haven, CT 06510 Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved October 14, 2019 (received for review February 24, 2019) Parkinson’s disease is characterized by the aggregation of the pre- Hsp70, Hsp110, DnaJA, and DnaJB proteins. Subsequently, synaptic protein α-synuclein and its deposition into pathologic Gao et al. (26) described a related Hsc70 complex capable of Lewy bodies. While extensive research has been carried out on breaking down α-synuclein fibrils in vitro. By reconstituting the mediators of α-synuclein aggregation, molecular facilitators of disaggregase with purified proteins, this complex was shown to α-synuclein disaggregation are still generally unknown. We inves- release soluble monomeric α-synuclein from its fibrillized form. tigated the role of molecular chaperones in both preventing and The α-synuclein fibril-specific disaggregase complex is composed α disaggregating -synuclein oligomers and fibrils, with a focus on of Hsc70, the DnaJ protein DnaJB1, and NEF Hsp110 in the the mammalian disaggregase complex. Here, we show that over- molar ratio 1.0:0.5:0.1 (26–29). DnaJB1 binds α-synuclein fibrils, expression of the chaperone Hsp110 is sufficient to reduce α recruiting Hsc70 to the surface of aggregates and promoting ATP -synuclein aggregation in a mammalian cell culture model. Addi- hydrolysis, while Hsp110 accelerates the rate-limiting step of nu- tionally, we demonstrate that Hsp110 effectively mitigates α-synuclein cleotide exchange—the release of ADP and P followed by ATP pathology in vivo through the characterization of transgenic i binding. The release of ADP was shown to cause a power stroke Hsp110 and double-transgenic α-synuclein/Hsp110 mouse models. Unbiased analysis of the synaptic proteome of these mice revealed responsible for fibril disassembly through a combination of fibril that overexpression of Hsp110 can override the protein changes fragmentation and depolymerization (26). driven by the α-synuclein transgene. Furthermore, overexpression Although the Hsc70 disaggregase complex can reverse α of Hsp110 is sufficient to prevent endogenous α-synuclein templat- -synuclein aggregation in vitro, the effects of this complex on α ing and spread following injection of aggregated α-synuclein seeds -synuclein aggregation in vivo have not been assessed. In neurons, into brain, supporting a role for Hsp110 in the prevention and/or Hsc70 is the most abundant molecular chaperone and is consti- disaggregation of α-synuclein pathology. tutively expressed, while DnaJB1 and Hsp110 are present at rela- tively low levels (30). Thus, increasing either DnaJB1 or Hsp110 Lewy body | chaperone | synapse | proteomics | disaggregase are reasonable options to enhance disaggregase activity in the brain (31, 32). Recently, Hsp110 was implicated as the limiting factor in arkinson’s disease (PD) is a progressive neurodegenerative the Hsc70-DnaJB1-Hsp110 disaggregase complex (24, 31). Fur- Pdisease affecting roughly 1% of the population over 65 y old thermore, the Hsp110 knockout mouse exhibits age-dependent (1). PD is characterized by the aberrant aggregation of α-synuclein, protein aggregation in the brain (33). Conversely, in a study fo- a presynaptic protein involved in synaptic vesicle trafficking (2–5). cusing on disaggregase activity in a SOD1-linked amyotrophic Fibrillized α-synuclein is the major component of Lewy bodies, the lateral sclerosis (ALS) mouse model, Hsp110 overexpression sig- aggregative hallmark pathology of PD (6). The discovery of several nificantly improved the survival of ALS mice, increasing mean point mutations (A53T, A53E, A30P, E46K, G51D) (7–12), du- survival by 2 mo (31). Hence, we opted to overexpress Hsp110 plications, and triplication of the α-synuclein gene SNCA (13, 14) resulting in autosomal dominant PD have cemented α-synuclein’s Significance integral role in PD pathophysiology. The SNCA point mutations and multiplications exert their effects through increased protein Recently, the mammalian disaggregase that can disassemble aggregation. Interestingly, several other neurodegenerative dis- α α -synuclein fibrils was reconstituted in vitro. However, it was eases, such as Lewy body dementia, also exhibit -synuclein pa- unclear if this disaggregase can function in vivo. Here we show thology and are classified as synucleinopathies. Strategies to α that overexpressing Hsp110, a limiting component of the dis- prevent and alleviate -synuclein aggregation are therefore crucial aggregase, is beneficial in reducing α-synuclein fibrillar pa- to generating effective therapeutics for the treatment of PD and thology in 2 distinct mouse models of Parkinson’s disease. This other synucleinopathies. reduction in α-synuclein pathology leads to improved survival. Heat shock protein 70 (Hsp70) proteins are the mainstay class Significantly, Hsp110 overexpression is associated with a broad of chaperones that assist in protein folding (15). Constitutive elevation of chaperones and may be an effective therapeutic Heat shock cognate 70 (Hsc70) as well as its inducible form, strategy against synucleinopathies. Hsp70, recognize hydrophobic residues of newly synthesized or misfolded proteins, binding transiently in a cyclic, ATP-dependent Author contributions: Y.V.T., W.A.F., L.V.-D., A.L.H., and S.S.C. designed research; Y.V.T., manner to mediate protein (re)folding (16, 17). Hsp70 activity is E.L.G., M.N., D.T., and L.V.-D. performed research; Y.V.T., E.L.G., L.V.-D., A.L.H., and S.S.C. facilitated by 2 classes of cochaperones: DnaJ proteins, which re- analyzed data; and Y.V.T., E.L.G., W.A.F., L.V.-D., A.L.H., and S.S.C. wrote the paper. cruit appropriate substrates and accelerate Hsp70 ATPase activity, The authors declare no competing interest. and a nucleotide exchange factor (NEF), which promotes ADP This article is a PNAS Direct Submission. and Pi release and ATP binding to the Hsp70 (18–22). Together, Published under the PNAS license. Hsc70 and Hsp70 facilitate proper protein folding and, impor- Data deposition: The data reported in this article have been deposited in the PRIDE tantly, prevent protein misfolding. database, https://www.ebi.ac.uk/pride/archive/ (accession no. PXD016015). Recent studies have identified the mammalian Hsp70 dis- 1To whom correspondence may be addressed. Email: [email protected]. aggregase, a chaperone complex involved in the disaggregation This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of proteins from insoluble aggregates (23, 24), akin to Hsp104 1073/pnas.1903268116/-/DCSupplemental. and ClpB found in nonmetazoans (25). This complex consists of First published November 4, 2019. 24310–24316 | PNAS | November 26, 2019 | vol. 116 | no. 48 www.pnas.org/cgi/doi/10.1073/pnas.1903268116 Downloaded by guest on September 16, 2020 to investigate whether the disaggregase can prevent or reverse α-synuclein pathology in vivo. Previous unbiased proteomic studies of Lewy bodies from pa- tient brains have shown an enrichment of Hsc70, DnaJB1, and Hsp110 (34), suggesting that the disaggregase does recognize misfolded α-synuclein but is insufficient to prevent its aggregation. Here we show that overexpression of Hsp110 both in mammalian culture and in transgenic mice (31) ameliorates α-synuclein ag- gregation in vivo. We also observe strong protective effects of Hsp110 overexpression in α-synuclein spread. Together, these experiments strongly support an ameliorative role for Hsp110 in vivo and indicate that enhancement of Hsp110 levels could be of benefit for PD and other synucleinopathies. Results Hsp110 Overexpression Ameliorates α-Synuclein Aggregation in Mammalian Culture. To establish that the α-synuclein fibril- disaggregase complex functions in a cellular context, we utilized an α-synuclein seeding assay in HEK293T cells that produces visible cytosolic aggregates (35, 36). First, HEK293T cells were transfected with human α-synuclein–GFP with or without Hsp110 (HspA4L/Apg1/Hsph3) constructs, which results in a 51.6- ± 6.1- fold increase in α-synuclein and a 3.7- ± 0.51-fold increase in Hsp110 protein levels (Fig. 1 B and C). Following Hsp110 and α-synuclein–GFP overexpression, aggregated recombinant human untagged α-synuclein seeds (Fig. 1A) were added to the media to induce cytosolic aggregation. Cells were washed and cellular α-synuclein aggregate levels were assessed by Western blotting (35, 36). Consistent with our previous results (36), the addition of NEUROSCIENCE α-synuclein seeds resulted in a significant amount of high molec- ular weight (HMW) α-synuclein by Western blot (Fig. 1 B and D). When Hsp110 was co-overexpressed, these HMW α-synuclein species were significantly reduced by ∼50% (1.0 versus 0.48; P = 0.0003). Next, we immunostained the treated HEK293T cells and assessed the effects of Hsp110 overexpression on GFP-positive aggregates by confocal microscopy. We found that