NEDD4-mediated HSF1 degradation underlies α-synucleinopathy

Human Molecular Genetics, 2016, Vol. 25, No. 2 211–222 doi: 10.1093/hmg/ddv445 Advance Access Publication Date: 26 October 2015 Original Article ORIGINAL ARTICLE NEDD4-mediated HSF1 degradation underlies α-synucleinopathy Eunhee Kim1,2, Bin Wang1,2, Namratha Sastry3, Eliezer Masliah5, Peter T. Nelson4, Huaibin Cai3 and Francesca-Fang Liao1,2, * Department of Pharmacology and 2Department of Anatomy and Neurobiology, University of Tennessee Health Science Center, 874 Union Avenue/Crowe 401, Memphis, TN 38163, USA, 3Transgenics Section, National Institute on Aging, National Institutes of Health, Bethesda, MD 20892, USA, 4Department of Neurology, Sanders-Brown Center on Aging, 800 South Limestone Street, Lexington, KY 40536, USA and 5Department of Neurosciences, University of California San Diego, La Jolla, CA 92093, USA *To whom correspondence should be addressed at: Department of Pharmacology, UTHSC, 874 Union Ave/Crowe Bldg 401, Memphis TN 38163, USA. Tel: +1 9014482752; Fax: +1 901448822; Email: fl[email protected] Abstract Cellular protein homeostasis is achieved by a delicate network of molecular chaperones and various proteolytic processes such as ubiquitin–proteasome system (UPS) to avoid a build-up of misfolded protein aggregates. The latter is a common denominator of neurodegeneration. Neurons are found to be particularly vulnerable to toxic stress from aggregation-prone proteins such as α-synuclein. Induction of heat-shock proteins (HSPs), such as through activated heat shock transcription factor 1 (HSF1) via Hsp90 inhibition, is being investigated as a therapeutic option for proteinopathic diseases. HSF1 is a master stressprotective transcription factor which activates genes encoding protein chaperones (e.g. iHsp70) and anti-apoptotic proteins. However, whether and how HSF1 is dysregulated during neurodegeneration has not been studied. Here, we discover aberrant HSF1 degradation by aggregated α-synuclein (or α-synuclein-induced proteotoxic stress) in transfected neuroblastoma cells. HSF1 dysregulation via α-synuclein was confirmed by in vivo assessment of mouse and in situ studies of human specimens with α-synucleinopathy. We demonstrate that elevated NEDD4 is implicated as the responsible ubiquitin E3 ligase for HSF1 degradation through UPS. Furthermore, pharmacologically induced SIRT1-mediated deacetylation can attenuate aberrant NEDD4-mediated HSF1 degradation. Indeed, we define the acetylation status of the Lys 80 residue located in the DNA-binding domain of HSF1 as a critical factor in modulating HSF1 protein stability in addition to its previously identified role in the transcriptional activity. Together with the finding that preserving HSF1 can alleviate α-synuclein toxicity, this study strongly suggests that aberrant HSF1 degradation is a key neurodegenerative mechanism underlying α-synucleinopathy. Introduction Synucleinopathies are a major class of neurodegenerative diseases, including Parkinson’s disease (PD) and diffuse lewy body (DLB). These pathologic conditions are defined by the presence of pathological α-synuclein aggregates known as Lewy bodies. Disruption of protein quality control, interconnected cellular strategies of the ubiquitin–proteasome system (UPS) and molecular chaperones, has been postulated to be involved in the pathogenesis of neurodegeneration underlying proteinopathies (1). Molecular chaperones composed of a set of heat shock proteins (HSPs) are considered a first line of defense against misfolded and aberrantly accumulated proteins like α-synuclein aggregates Received: July 17, 2015. Revised: September 14, 2015. Accepted: October 19, 2015 © The Author 2015. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 211 1 212 | Human Molecular Genetics, 2016, Vol. 25, No. 2 Results Overexpressed α-syn protein promotes ubiquitination and degradation of HSF1 protein via UPS To determine whether α-synuclein (α-syn, herein after) aggregation altered HSF1 expression levels, we transiently transfected SH-SY5Y neuroblastoma and HEK293 cells with GFP-tagged wild-type (WT) α-syn (GFP-α-syn WT) or GFP-tagged A53T mutant α-syn (GFP-α-syn A53T). In both cell lines, A53T α-syn overexpression 48 h post transfection caused more dramatic reduction (>70% loss) in HSF1 protein expression than WT α-syn (Figs 1A, C and 2A), without decreasing the mRNA levels of hsf1 gene (Fig. 1B). Similar results were obtained when HSF1 protein levels were determined at 24 or 72 h after transfection (Supplementary Material, Fig. S1). A53T mutant form of α-syn is known to aggregate more rapidly than WT form. We could detect both Triton X-soluble and detergent-insoluble α-syn aggregates in α-syn transfected SH-SY5Y cells (Fig. 1D). Insoluble WT α-syn expression was increased by transfection of double concentration of WT α-syn (WT 2 μg) (Fig. 1D). This higher WT α-syn aggregation resulted in ∼50% loss of HSF1 protein (Fig. 1A and C). In contrast to A53T α-syn which resulted in HSF1 loss in both nuclear and cytoplasmic compartments, WT α-syn only caused reduced HSF1 in the nucleus (Fig. 1C). HSF1 loss appears to be a common phenomenon upon proteotoxic stress since other aggregation-prone proteins such as mutant huntingtin (Htt), but not transactive response DNA-binding protein (TDP-43), also led to reduced HSF1 protein levels (Supplementary Material, Fig. S2). We next assessed HSF1 protein stability by treating transfected cells with cyclohexamide (CHX). While HSF1 protein was stable in SH-SY5Y cells transfected with empty vector, it was greatly reduced in cells overexpressing A53T over time (Fig. 1E), which was largely rescued by proteasome inhibitor MG132 but not by chloroquine (CQ, a lysosomal inhibitor). Of note, we noticed upshifted HSF1 bands in both control and A53Ttrasnfected cells when blocking proteasomal degradation with MG132 (Fig. 1E; denoted by *). This slower mobility-shifted band may be due to hyperphosphorylated HSF1 which correlated with hyperactivated HSF1 following heat shock and proteasomal stress (15). Polyubiquitination is a pre-requisite step for proteasome system to degrade target proteins. We found that HSF1 proteins to be degraded by proteasome were indeed highly polyubiquitinated, as revealed by MG132 treatment (Fig. 1F). HSF1 ubiquitination was further proven to be involved in HSF1 degradation by in vivo ubiquitination assay. As HA-Ub was increasingly acquired in A53T-transfected cells, HSF1 protein became more polyubiquitinated, correlating with decreased HSF1 protein levels (Fig. 1F). On the contrary, GFP-α-syn A53T expression was increased by exogenous ubiquitin overexpression, consistent with a positive role of HSF1 in α-syn clearance (Fig. 6). α-Syn-induced HSF1 degradation i (...truncated)