© 2020. Published by The Company of Biologists Ltd | Journal of Cell Science (2020) 133, jcs243709. doi:10.1242/jcs.243709 RESEARCH ARTICLE Neurodegenerative disease-associated protein aggregates are poor inducers of the heat shock response in neuronal cells Rebecca San Gil1,2,3, Dezerae Cox4, Luke McAlary1,2, Tracey Berg1,2, Adam K. Walker3, Justin J. Yerbury1,2, Lezanne Ooi1,2 and Heath Ecroyd1,2,* ABSTRACT monomers can nucleate the formation of toxic protein oligomers Protein aggregates that result in inclusion formation are a pathological that are the precursors of inclusions (Kopito, 2000). Inclusions can hallmark common to many neurodegenerative diseases, including be diverse with regards to their size, localisation, and composition. amyotrophic lateral sclerosis, Parkinson’s disease and Huntington’s Specific inclusion subtypes have been identified, including those disease. Under conditions of cellular stress, activation of the heat known as IPOD (Insoluble Protein Deposit), JUNQ (Juxtanuclear shock response (HSR) results in an increase in the levels of Quality Control Compartment) and RISCI (RNA Interactor Specific molecular chaperones and is a first line of cellular defence against Compartments/Inclusions) (Farrawell et al., 2015; Kaganovich inclusion formation. It remains to be established whether et al., 2008; Kayatekin et al., 2014; Polling et al., 2014; Matsumoto neurodegenerative disease-associated proteins and inclusions are et al., 2006; Whiten et al., 2016). The roles of different inclusion themselves capable of inducing an HSR in neuronal cells. To address types may include interacting with proteostasis pathways and acting this, we generated a neuroblastoma cell line that expresses a as terminal storage sites for destabilised proteins (Kayatekin et al., fluorescent reporter protein under conditions of heat shock 2014; Polling et al., 2014; Matsumoto et al., 2006; Farrawell et al., transcription factor 1 (HSF1)-mediated HSR induction. We show 2015). There is increasing evidence to suggest that the misfolding of that the HSR is not induced by exogenous treatment with aggregated monomers and subsequent formation of oligomeric intermediates forms of recombinant α-synuclein or the G93A mutant of superoxide are the processes responsible for generating toxic species that trigger dismutase-1 (SOD1G93A) nor intracellular expression of SOD1G93A or cellular stress and neurotoxicity: inclusion formation might actually a pathogenic form of polyglutamine-expanded huntingtin (Htt72Q). represent a neuroprotective process (Winner et al., 2011; Leitman These results suggest that pathogenic proteins evade detection or et al., 2013; Zhu et al., 2018; Bolognesi et al., 2010). impair induction of the HSR in neuronal cells. A failure of protein The process of protein aggregation has been strongly correlated aggregation to induce an HSR might contribute to the development of with the death of neurons in neurodegenerative diseases inclusion pathology in neurodegenerative diseases. (Brettschneider et al., 2013, 2014; Braak et al., 2006, 2003). The progression of inclusion pathology, from a focal site of onset to This article has an associated First Person interview with the first other regions of the central nervous system (CNS), may be driven by author of the paper. the movement of aggregated proteins in the extracellular space (Vaquer-Alicea and Diamond, 2019). Therefore, aggregation is KEY WORDS: HSF1, Heat shock response, Inclusions, seeded in neighbouring cells (including neurons) via prion-like Neurodegenerative disorders, Protein aggregation propagation (Victoria and Zurzolo, 2017; Jucker and Walker, 2013; Zeineddine and Yerbury, 2015; Hanspal et al., 2017; McAlary et al., INTRODUCTION 2019; Peng et al., 2020). This spread of disease pathology The formation of intracellular protein inclusions is a characteristic throughout the CNS suggests a widespread inability of cells to hallmark of neurodegenerative diseases, such as amyotrophic lateral prevent the initial events leading to protein aggregation and the sclerosis (ALS), Parkinson’s disease and Huntington’s disease propagative seeding events that follow. (Chiti and Dobson, 2017). In these diseases, intrinsically disordered Cells have several mechanisms of defense against disturbances in or partially unfolded proteins self-associate through hydrophobic proteostasis (Yerbury et al., 2016). These mechanisms usually interactions between regions that are usually buried in the native function to ensure the correct folding, function and turnover of conformation (Hipp et al., 2019). Interactions between misfolded proteins in the cell, and prevent the negative effects of proteostasis imbalance on cell viability. The heat shock response (HSR) is one such mechanism of proteostasis and acts as a first line of defense 1Illawarra Health and Medical Research Institute, Wollongong, NSW 2522, Australia. 2Molecular Horizons and School of Chemistry and Molecular Bioscience, against protein destabilisation, misfolding and aggregation (San Gil University of Wollongong, Wollongong, NSW, Australia. 3Neurodegeneration et al., 2017b). Under conditions of cellular stress, an increasing Pathobiology Laboratory, Queensland Brain Institute, University of Queensland, St intracellular abundance of destabilised proteins can lead to the Lucia, QLD 4072, Australia. 4Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, activation of heat shock transcription factor 1 (HSF1). HSF1 Parkville, VIC 3052, Australia. translocates into the nucleus and binds heat shock elements (HSEs; a pentameric sequence nGAAn, where ‘n’ represents any *Author for correspondence ([email protected]) nucleotide) in the promoter elements of HSF1-target genes R.S.G., 0000-0001-5725-5512; D.C., 0000-0002-5345-8360; L.M., 0000-0002- (Pelham, 1982; Sorger et al., 1987; Amin et al., 1988; Xiao and 1764-3809; A.K.W., 0000-0001-7954-5801; J.J.Y., 0000-0003-2528-7039; L.O., Lis, 1988). Induction of the HSR results in rapid and dramatic 0000-0001-9241-8268; H.E., 0000-0001-7574-0044 upregulation of a subset of the heat shock protein (Hsp) family of Handling Editor: Giampietro Schiavo molecular chaperones. Molecular chaperones are capable of Received 12 May 2020; Accepted 30 June 2020 stabilising and re-folding misfolded proteins, as well as trafficking Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs243709. doi:10.1242/jcs.243709 damaged proteins to the proteasomal or autophagy-lysosomal activated HSR. The relatively poor induction of the HSR by pathways for degradation (Leak, 2014). There is a wealth of pathogenic proteins compared to non-pathogenic Fluc could evidence in cell-based and in vivo models demonstrating that the explain, at least in part, the ability of disease-related inclusions to overexpression of individual Hsps significantly ameliorates evade proteostasis pathways. neurodegenerative disease-associated protein aggregation (San Gil et al., 2017b; Webster et al., 2019; Kakkar et al., 2014). For RESULTS example, overexpression of HSPB1 (Hsp27) or HSPB5 (αB- Generation and validation of an HSR reporter cell line crystallin, also known as CRYAB) results in a 30–40% decrease in To investigate the activation of the HSR, stable Neuro-2a cell lines Neuro-2a cells with α-synuclein inclusions (Cox and Ecroyd, 2017). were generated in which expression of a fluorescent protein (EGFP) Moreover, overexpression of HSF1 itself can prevent protein was used to report on HSR induction. These cells also constitutively aggregation, presumably via its capacity to increase the express mCherry to account for changes in cell number or expression of molecular chaperones. For example, in a mutant transcription rates over time with different treatment paradigms. superoxide dismutase-1 (SOD1) mouse model of ALS, To validate that this stable cell line reports on HSR induction, the overexpression of HSF1 leads to a 34% decrease in the level of cells were first treated with known inducers of the HSR, namely H46R/H48Q insoluble SOD1 in spinal cord tissue compared to controls CdCl2, heat shock, and celastrol (a compound identified in a drug (Lin et al., 2013). The overexpression of constitutively active HSF1 screen as a potent inducer of the HSR) (Westerheide et al., 2004; in the R6/2 mouse model of Huntington’s disease leads to a 21% Heemskerk et al., 2002). The fluorescent reporter, EGFP, has a half- decrease in the proportion of nuclei with inclusions and extended life of ∼27 h (Corish and Tyler-Smith, 1999), therefore these survival (mean survival 122 d in R6/2-HSF1Tg compared to 107 d experiments were performed over a timeframe that would facilitate in R6/2 mice) (Fujimoto et al., 2005). Whilst the activation of HSF1 the accumulation of EGFP after HSR induction in order to assess the or increased expression of Hsps can ameliorate protein aggregation magnitude of the HSR. Concentration-response experiments were and extend survival in models of neurodegenerative disease, it conducted to determine the concentration of CdCl2 (0–33 µM) and remains to be definitively established whether aggregation-prone celastrol (0–1 µM) that induces a maximal HSR (Fig. S2). Based on proteins or the inclusions they form are themselves capable of these results, subsequent experiments used 10 µM CdCl2 and inducing an endogenous HSF1-mediated HSR. 0.75 µM celastrol over a 24 h timeframe, which were the lowest At the whole tissue level, there is evidence to suggest that