**The Hsp70 Chaperone System in Parkinson's Disease**

Adahir Labrador-Garrido1, Carlos W. Bertoncini2 and Cintia Roodveldt1 *1CABIMER-Andalusian Center for Molecular Biology & Regenerative Medicine CSIC-University of Seville-UPO-Junta de Andalucía, Seville, 2Laboratory of Molecular Biophysics, Institute for Research in Biomedicine (IRB), Barcelona, Spain* 

#### **1. Introduction**

220 Etiology and Pathophysiology of Parkinson's Disease

Yu, T.; Scully, S.; Yu, Y.; Fox, GM.; Jing, S. & Zhou, R. (1998). Expression of GDNF

Yurek, DM. & Fletcher-Turner, A. (2000). Lesion-induced increase of BDNF is greater in the

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striatum of young versus old rat brain. Exp Neurol, Vol.161, No.1, pp.392-396, ISSN

Several neurodegenerative diseases are associated with a build up of misfolded or abnormal proteins and the formation of distinct aggregates, resulting in a putative pathological protein load on the nervous system (Chiti & Dobson, 2006). This aberrant accumulation of amyloid or amyloid-like aggregates occurs in Parkinson's (PD), Alzheimer's (AD), and Huntington's (HD) diseases, amyotrophic lateral sclerosis, and frontotemporal dementia, among others. A broad array of cellular defence mechanisms operate to counteract this effect, including antioxidant proteins, the stress-inducible response and, in particular, molecular chaperones (Morimoto, 2008; Voisine et al., 2010). Molecular chaperones are responsible for maintaining normal protein homeostasis within the cell by assisting protein folding, inhibiting protein aggregation, and modulating protein degradation pathways (Hartl & Hayer-Hartl, 2009). Currently, there is substantial evidence supporting the involvement of these protein aggregational processes and a role of molecular chaperones, and especially of Hsp70, in PD pathogenesis (Bandopadhyay & de Belleroche, 2010; Broadley & Hartl, 2009; Witt, 2009). Firstly, extensive colocalization of Hsp70 with αsynuclein (αSyn), the major component of Lewy bodies (LBs) (Spillantini et al., 1998), within the intraneuronal inclusions in PD brains has been demonstrated (Auluck et al., 2002; McLean et al., 2002). Secondly, patients with PD show highly perturbed expression of different members of the Hsp70 family in the *substantia nigra pars compacta* (SN) of the brain, which is precisely the target of neurodegeneration (Grunblatt et al., 2001; Hauser et al., 2005). Finally, there is a considerable amount of data derived from studies performed *in vitro*, in cell culture and with animal models of PD (Arawaka et al., 2010; Witt, 2009), that support the protective effects of Hsp70 against αSyn aggregation and toxicity, considered to be central in the aetiology of the disease.

The discovery within the last few years of three different missense mutations (A30P, E46K and A53T) in the αSyn gene as causative of early onset PD unambiguously linked this protein to disease onset and progression (Kruger et al., 1998; Polymeropoulos et al., 1997; Zarranz et al., 2004). Additionally, a locus triplication causing an increased dosage of the wild-type (Wt) αSyn gene has been found to potentiate neurodegeneration (Singleton et al.,

The Hsp70 Chaperone System in Parkinson's Disease 223

addition to acting as enhancer of the basal ATPase rate of Hsp70, the Hsp40 family of cochaperones has a key role in the canonical model of the Hsp70 machinery mechanism, given the ability of some of them to also recognize and bind to unfolded client proteins and 'deliver' them to Hsp70 (Kampinga & Craig, 2010). Other important co-chaperones are Hop, which binds to the C-termini of Hsp70 and Hsp90 and assists substrate transfer between the two chaperones (Scheufler et al., 2000), and Hip (ST13) which has been shown to specifically bind to and stabilize, the ADP-bound state of Hsp70 (Hohfeld et al., 1995; Prapapanich et al., 1996), and has been suggested to increase the half-life of Hsp70-substrate complexes (Hohfeld et al., 1995). Finally, CHIP (Ballinger et al., 1999) acts as an E3-ubiquitin ligase that ubiquitinates HSPA8/Hsc70/Hsp73 (i.e. the constitutive cytosolic Hsp70) substrates,

PD is currently thought to involve different pathogenic mechanisms that eventually lead to neurodegeneration, as discussed elsewhere in this book series.. There is substantial evidence supporting a prominent role in PD-related cell death of αSyn toxic oligomers. One hypothesis postulates that certain αSyn species can affect the homeostasis of cell membranes (Gupta et al., 2008; Lashuel et al., 2002b; Volles & Lansbury, 2002), and produce ER and oxidative stress, UPS and mitochondrial dysfunction (Gupta et al., 2008; Jellinger, 2010), as well as neuroinflammation (Jellinger, 2010; Roodveldt et al., 2008) processes, all of which have been linked, to a lower or larger extent, to the amyloid-like aggregation of αSyn. As will be described below, the Hsp70 system has been found to be a key player in counteracting most of these processes, not only by physically interacting with αSyn, but also

Fig. 1. Links between Hsp70 and the multiple αSyn-mediated processes in PD pathogenesis. CMA: chaperone-mediated autophagy; UPS: ubiquitin-proteasome system; LB: Lewy bodies; LN: Lewy neurites. Discontinuous lines depict the possible 'sequestration' of certain

promoting their degradation by the proteasome (Hohfeld et al., 2001).

**2. Links between Hsp70 and the pathogenesis of PD** 

by promoting aggregation clearance (Figure 1).

chaperones by αSyn aggregating species.

2003). Finally, as mentioned above, αSyn is the major component of intracellular proteinrich aggregates found in the brain of post-mortem patients of PD, the LBs and Lewy neurites (LNs). The appealing hypothesis for LBs formation is that αSyn monomers combine to form oligomers (or protofibrils), which coalesce into fibrils and then co-aggregate with other proteins into (intracellular) inclusions (Conway et al., 1998; Wood et al., 1999). While the monomers and oligomers of αSyn are soluble, the fibrils and LBs are insoluble in the neuronal cytoplasm. Some controversy arises, however, from the roles of the various physical forms or species of αSyn in PD pathogenesis. LBs have been proposed to be both neurotoxic (El-Agnaf et al., 1998), and protective (Mouradian, 2002; Rochet et al., 2000). Other hypotheses state that the pre-fibrillar intermediates, composed of αSyn oligomers, are the main toxic species towards dopaminergic neurons (Conway et al., 2000; Volles & Lansbury, 2003). Lansbury and co-workers have shown that αSyn oligomers can form annular, elliptical, or circular amyloid pores in cell membranes (Lashuel et al., 2002a; Volles & Lansbury, 2003), and cell culture studies have demonstrated that αSyn oligomers reduce cell viability, disrupt lysosomes and induce Golgi fragmentation (Gosavi et al., 2002), as well as toxicity in animal models (Karpinar et al., 2009). In line with these findings, the more neurotoxic A30P and A53T mutants of αSyn share an increased tendency to form soluble oligomeric intermediates, whereas the E46K and A53T mutants fibrillate faster than the wild-type protein (Conway et al., 2000; Choi et al., 2004).

The heat-shock-protein 70 (Hsp70) family of chaperones (Mayer & Bukau, 2005; Young, 2010) is well conserved from bacteria to higher eukaryotes (where it is found within different organelles), having critical roles in a range of cellular processes such as promoting the folding of newly synthesized proteins and assisting the rescue of misfolded aggregated proteins. Hsp70 is highly relevant in the context of protein conformational diseases given that stress-induced cytosolic Hsp70 can prevent protein aggregation and enables the cell to avoid the accumulation of potentially toxic aggregates (Hartl, 1996).

The structures of several Hsp70 homologues are similar and consist of an actin-like ATPase domain (nucleotide-binding domain, NBD) and a and a C-terminal substrate-binding domain (SBD), which are connected by a short linker region (Mayer & Bukau, 2005). The substrate binding pocket recognizes and binds to unstructured or partially folded stretches within polypeptides (Bukau & Horwich, 1998), with the current view that Hsp70s could prevent misfolding by binding to certain patterns in the polypeptide chain of the substrate that are highly enriched in hydrophobic residues (Maeda et al., 2007; Rudiger et al., 1997). Even though most of our current understanding of the Hsp70 molecular mechanism has largely derived from studies performed with the bacterial orthologue (DnaK), the outlines of the mechanism appear conserved (Hartl & Hayer-Hartl, 2002; Young, 2010). The ATPase cycle of Hsp70 involves alternation between an ATP-bound state which has low affinity and fast exchange rates for peptide substrates ('open' state), and an ADP-bound state with high affinity and low exchange rates for substrates ('closed' state) (Mayer & Bukau, 2005). This alternation is achieved by a bidirectional structural communication between the NBD and the SBD domains, driven by a complex allosteric mechanism (Mayer & Bukau, 2005; Young, 2010).

The ATPase cycle is typically modulated by several co-chaperones, most notably the 'Jdomain' protein Hsp40/DNAJB1 (or DnaJ, the bacterial orthologue), resulting in increase of the ATPase activity (Bukau & Horwich, 1998; Mayer & Bukau, 2005; Minami et al., 1996), and the BAG family of proteins which function as nucleotide-exchange factors (NEFs) and promote the ADP release from the the Hsp70 NBD (Takayama et al., 1999; Young, 2010). In

2003). Finally, as mentioned above, αSyn is the major component of intracellular proteinrich aggregates found in the brain of post-mortem patients of PD, the LBs and Lewy neurites (LNs). The appealing hypothesis for LBs formation is that αSyn monomers combine to form oligomers (or protofibrils), which coalesce into fibrils and then co-aggregate with other proteins into (intracellular) inclusions (Conway et al., 1998; Wood et al., 1999). While the monomers and oligomers of αSyn are soluble, the fibrils and LBs are insoluble in the neuronal cytoplasm. Some controversy arises, however, from the roles of the various physical forms or species of αSyn in PD pathogenesis. LBs have been proposed to be both neurotoxic (El-Agnaf et al., 1998), and protective (Mouradian, 2002; Rochet et al., 2000). Other hypotheses state that the pre-fibrillar intermediates, composed of αSyn oligomers, are the main toxic species towards dopaminergic neurons (Conway et al., 2000; Volles & Lansbury, 2003). Lansbury and co-workers have shown that αSyn oligomers can form annular, elliptical, or circular amyloid pores in cell membranes (Lashuel et al., 2002a; Volles & Lansbury, 2003), and cell culture studies have demonstrated that αSyn oligomers reduce cell viability, disrupt lysosomes and induce Golgi fragmentation (Gosavi et al., 2002), as well as toxicity in animal models (Karpinar et al., 2009). In line with these findings, the more neurotoxic A30P and A53T mutants of αSyn share an increased tendency to form soluble oligomeric intermediates, whereas the E46K and A53T mutants fibrillate faster than the

The heat-shock-protein 70 (Hsp70) family of chaperones (Mayer & Bukau, 2005; Young, 2010) is well conserved from bacteria to higher eukaryotes (where it is found within different organelles), having critical roles in a range of cellular processes such as promoting the folding of newly synthesized proteins and assisting the rescue of misfolded aggregated proteins. Hsp70 is highly relevant in the context of protein conformational diseases given that stress-induced cytosolic Hsp70 can prevent protein aggregation and enables the cell to

The structures of several Hsp70 homologues are similar and consist of an actin-like ATPase domain (nucleotide-binding domain, NBD) and a and a C-terminal substrate-binding domain (SBD), which are connected by a short linker region (Mayer & Bukau, 2005). The substrate binding pocket recognizes and binds to unstructured or partially folded stretches within polypeptides (Bukau & Horwich, 1998), with the current view that Hsp70s could prevent misfolding by binding to certain patterns in the polypeptide chain of the substrate that are highly enriched in hydrophobic residues (Maeda et al., 2007; Rudiger et al., 1997). Even though most of our current understanding of the Hsp70 molecular mechanism has largely derived from studies performed with the bacterial orthologue (DnaK), the outlines of the mechanism appear conserved (Hartl & Hayer-Hartl, 2002; Young, 2010). The ATPase cycle of Hsp70 involves alternation between an ATP-bound state which has low affinity and fast exchange rates for peptide substrates ('open' state), and an ADP-bound state with high affinity and low exchange rates for substrates ('closed' state) (Mayer & Bukau, 2005). This alternation is achieved by a bidirectional structural communication between the NBD and the SBD domains,

wild-type protein (Conway et al., 2000; Choi et al., 2004).

avoid the accumulation of potentially toxic aggregates (Hartl, 1996).

driven by a complex allosteric mechanism (Mayer & Bukau, 2005; Young, 2010).

The ATPase cycle is typically modulated by several co-chaperones, most notably the 'Jdomain' protein Hsp40/DNAJB1 (or DnaJ, the bacterial orthologue), resulting in increase of the ATPase activity (Bukau & Horwich, 1998; Mayer & Bukau, 2005; Minami et al., 1996), and the BAG family of proteins which function as nucleotide-exchange factors (NEFs) and promote the ADP release from the the Hsp70 NBD (Takayama et al., 1999; Young, 2010). In addition to acting as enhancer of the basal ATPase rate of Hsp70, the Hsp40 family of cochaperones has a key role in the canonical model of the Hsp70 machinery mechanism, given the ability of some of them to also recognize and bind to unfolded client proteins and 'deliver' them to Hsp70 (Kampinga & Craig, 2010). Other important co-chaperones are Hop, which binds to the C-termini of Hsp70 and Hsp90 and assists substrate transfer between the two chaperones (Scheufler et al., 2000), and Hip (ST13) which has been shown to specifically bind to and stabilize, the ADP-bound state of Hsp70 (Hohfeld et al., 1995; Prapapanich et al., 1996), and has been suggested to increase the half-life of Hsp70-substrate complexes (Hohfeld et al., 1995). Finally, CHIP (Ballinger et al., 1999) acts as an E3-ubiquitin ligase that ubiquitinates HSPA8/Hsc70/Hsp73 (i.e. the constitutive cytosolic Hsp70) substrates, promoting their degradation by the proteasome (Hohfeld et al., 2001).
