**4. The Hsp70 system as a therapeutic tool for PD**

As described in this chapter, in the last few years it has become evident that HSPs play an important role in the initiation and progression of PD and other neurodegenerative diseases. This cumulative evidence has prompted the development of therapeutic tools based on the Hsp70 machinery. Different strategies have been tested to manipulate Hsp70 as a therapeutic approximation for PD and related neurodegenerative diseases (reviewed in Kalia 2010). Three general approaches have been explored, namely, to increase the intracellular activity of Hsp70, to overexpress Hsp70 and/or other co-chaperones, and to deliver chaperones or regulatory factors using cell-penetrating peptides (CPPs).

#### **4.1 Increasing the intracellular activity of Hsp70**

Theoretically, it should be feasible to control the activity of the cellular chaperone machinery by using different types of drugs. The mechanism of most of such chemical compounds is based on activating HSF-1, a key transcriptional regulator of the heat shock response (HSR) that activates the gene expression of inducible HSPs. One way of activating HSF-1 is by inhibiting Hsp90 activity; as a result, HSF-1 becomes active and increases the expression of inducible chaperones like HSPA1A/Hsp72 and others. Geldanamycin (GA) and its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) are antibiotics with high affinity for the ATPase domain of Hsp90, which blocks its interaction with HSF-1 and subsequently allows its activation (Waza et al., 2006; Zou et al., 1998). GA prevents αSyninduced dopaminergic cell loss in cell culture (McLean et al., 2004) and in animal models of PD (Auluck et al., 2005; Shen et al., 2005), while the less toxic 17-AAG has also proven to be neuroprotective in PD cellular models (Danzer et al., 2011; Riedel et al., 2010) and in two animal models of PD-related neurodegenerative diseases (Fujikake et al., 2008; Waza et al., 2005). Even though 17-AAG is currently under phase-II clinical trials as an anti-tumour drug (Pacey et al., 2010; Richardson et al., 2010; Solit et al., 2008), its use in patients with neurodegenerative diseases could be hampered by its toxicity and unavailability for oral administration (Pacey et al., 2010). Another family of inhibitors of Hsp90 activity, is SNX-2112 and its analogues, which are orally available and present improved blood brain barrier (BBB) permeability. In particular, SNX-0723 was shown to prevent αSyn oligomer formation and αSyn-induced toxicity in cell culture (Putcha et al., 2010), and preclinical studies for cancer therapy are proving their safety (Zhai et al., 2011), although similar studies in neurodegenerative animal models are still needed.

Other drugs are able to activate HSF-1 without inhibiting Hsp90 activity, which could represent a less toxic approximation for neurodegenerative pathologies. Arimoclomol, for example, has already been tested in phase I- and IIa-clinical trials for treating ALS, and shown to be safe and tolerable (Cudkowicz et al., 2008; Phukan, 2010). Another example is HSF-1A (Neef et al., 2010), shown to upregulate Hsp70 expression and to reduce poly-Qcitotoxicity in cell and fly models of poly-Q neurodegenerative disorders (Neef et al., 2010). Celastrol, yet a similar drug, and some structural relatives, appear as promising drugs due to their rapid kinetics and low EC50 (Westerheide et al., 2004), although further studies are needed.

Besides these approaches, HSF-1 co-inducers could represent more tolerable drugs for therapy. These are molecules that partially activate HSF-1, reducing its activation threshold and often working in conjunction with secondary stress signals to fully induce HSR. Non steroidal anti-

The Hsp70 Chaperone System in Parkinson's Disease 235

protect cells against heat stress *in vitro* (Hou & Zou, 2009). Although further investigation is needed, CPPs tagging could prove a powerful tool in therapy against neurodegenerative

There is strong experimental support to propose the Hsp70 chaperone system as a key player in pathogenesis and progression of PD. This central role seems to be especially linked to αSyn, although alternative connections between the Hsp70 machinery and different pathogenic mechanisms underlying PD might exist. Up to this point, there is substantial evidence supporting an active role of Hsp70 in well established PD-related processes. First, in the inhibition or modulation of αSyn aggregation pathway that results in the formation of LBs and the suppression of αSyn-mediated toxicity to cells that leads to neurodegeneration. Second, a prominent role in the control of the activity of the UPS machinery, the general protein degradation and disposal system in the cell. And third, a central role in CMA, which handles the lysosomal degradation of selected cytosolic proteins, including αSyn. In addition, emerging mechanisms for Hsp70 in relation to PD include its participation in the CSPα-Hsc70-SGT complex in the neuron, and the increasing attention paid to the UPR and

The accumulated studies thus far suggest that Hsp70 chaperone is a highly versatile protein whose anti-aggregation activity seems to involve different interactions and the formation of transient and highly dynamic complexes with various αSyn species, presumably early oligomers and probably monomers, along the aggregation pathway. This activity can be certainly modulated by the presence of nucleotides and by certain co-chaperones, in particular, Hip. Clearly, further *in vitro* and in cell studies with Hsp70 and co-chaperones, to better understand the full molecular mechanism of mammalian Hsp70 in managing αSyn

As a biomarker of PD, Hsp70 seems not to represent a good candidate itself, probably due to its central role in maintaining cellular proteostasis which is perturbed in several amyloidoses and related diseases. However, the Hsp70 co-chaperone Hip might represent a potentially useful biomarker for early diagnosis of PD, although more studies are needed in this direction. Given that PD is a complex disease, a 'complex biomarker' (i.e. composed of various markers) appears to be the only reliable option. Finally, the Hsp70 machinery can be indirectly enhanced by HSF-1 pharmacological activation, which represents one of the most promising therapeutic approaches for treating this complex and highly debilitating disease.

We acknowledge financial support provided by the Spanish Ministry of Health according to the 'Plan Nacional de I+D+I 2008-2011', Instituto Nacional Carlos III (ISCIII, project CP10/00527 to C.R.), and cofunding by FEDER funds., and the PAIDI Program from the Andalusian Government (CTS-677). A.L.G. holds a FPU Fellowship from the Spanish Ministry of Science (MICINN). C.W.B. acknowledges funding from the EU FP7 (MC-IEF 236721). The authors are grateful to Christopher M. Dobson (University of Cambridge, UK) and John Christodoulou (University College London, UK) for helpful and highly

diseases by allowing the efficient transduction of cytoprotective proteins and factors.

mitochondrial dysfunction processes in PD, both of which rely on Hsp70.

**5. Conclusions** 

aggregation, are needed.

**6. Acknowledgements** 

stimulating discussion of results.

inflammatory drugs (NSAIDs) are well known co-inducers of HSR. For example, Sodium salicylate and Indomethacin induce HSF-1 DNA binding and reduce the temperature required for triggering the HSR (Jurivich et al., 1995; Lee et al., 1995). The association of NSAIDs use with a lower risk of common neurodegenerative diseases such as AD and PD has been analysed in several studies (Etminan et al., 2008; Gagne & Power, 2010). According to one hypothesis, this negative correlation could be due to continuous up-regulation of HSR and consequently continued cytoprotection against neurodegenereration (Westerheide et al., 2004).

#### **4.2 Overexpression of Hsp70 and/or related co-chaperones**

In principle, it should be possible to design a gene therapy approach for the treatment of PD and other conformational neurodegenerative diseases based on HSPs, considering the substantial number of reports having characterized the molecular pathways by which Hsp70 acts in the context of disease. Intriguingly, even though overexpression of Hsp70 has been shown to be protective in animal models of PD (Dong et al., 2005; Jung et al., 2008), a recent study in mouse indicates otherwise (Shimshek et al.). Up to date, one phase-I study in PD patients using recombinant Adeno-Associated Virus (AAV) to deliver aromatic aminoacid decarboxylase enzyme into the putamen, supports the proof-of-principle for the use of gene therapy in PD (Christine et al., 2009). With this precedent, gene therapy could potentially be employed to overexpress other chaperones and co-chaperones that may improve Hsp70 function and its neuroprotective properties. Indeed, recombinant AAV has been already used to transduce Hsp104 (Vashist et al., 2010), a non-mammalian chaperone, in a rat model of PD and proven to be neuroprotective by disaggregating protein inclusions and synergizing with endogenous Hsp70 (Lo Bianco et al., 2008).

Given that co-chaperone BAG-5 is known as a negative regulator of Hsp70, downregulation of its expression has been tested in a mouse model of PD. In this study, direct expression in the SN by recombinant AAV delivery of BAG-5(DARA), a BAG-5 mutant which inhibits wild-type BAG-5 activity, resulted in increased dopaminergic neuron survival (Kalia et al., 2004). Another possible approach to increase Hsp70 function by using gene therapy techniques could be gene silencing by RNA interference to knockdown Hsp70 downregulators. Although these techniques have not been extensively tested for neurodegenerative therapy, they remain a potentially useful tool (Manfredsson et al., 2006).

#### **4.3 Chaperones or regulatory factors delivery using CPPs**

Cell penetrating peptides (CPPs) are the most recent approximation that shows promise towards increasing Hsp70 activity within cells. These are peptide motifs that allow cell transduction of macromolecules including functional full-length proteins. The basic domain of the trans-activator of transcription (TAT) from HIV-1 is the best known among CPPs and it has been shown that fusion with TAT allows proteins to penetrate cell membranes of several cell types and even to cross the BBB (Fawell et al., 1994; Schwarze et al., 1999). In the last few years, TAT-Hsp70 transduction has been reported to be neuroprotective against different kinds of stress in cell models (Lai et al., 2005; Nagel et al., 2008) as well as in a MPTP mouse model of PD (Nagel et al., 2008). In addition, transduction of TAT-Hsp40 has also been shown to be cytoprotective against oxidative stress in cells (Kim et al., 2008). Finally, transduction of HSF-1(+)-TAT, an HSF-1 mutant fused to TAT, capable of activating HSR by itself by itself, was demonstrated to induce to induce Hsp70 expression and to protect cells against heat stress *in vitro* (Hou & Zou, 2009). Although further investigation is needed, CPPs tagging could prove a powerful tool in therapy against neurodegenerative diseases by allowing the efficient transduction of cytoprotective proteins and factors.
