Heat shock proteins in neurodegenerative diseases: Pathogenic roles and therapeutic implications

Abstract

Neurodegenerative diseases including amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, and polyglutamine (polyQ) diseases are thought to be caused by protein misfolding. Heat shock proteins (HSPs), which function mainly as molecular chaperones, play an important role in the folding and quality control of proteins. The histopathological hallmark of neurodegenerative diseases is accumulation and/or inclusions of the disease-causing proteins in residual neurons in targeted regions of the nervous system. The inclusions combine with many components of molecular chaperone pathways and ubiquitin-proteasome, raising the possibility that misfolding and altered degradation of mutant proteins may be involved in the pathogenesis of neurodegenerative diseases. Overexpression of HSPs has been reported to reduce the number and size of inclusions and accumulation of disease-causing proteins, and ameliorate the phenotypes in neuronal cell and mouse models. Hsp90 inhibitors also exert therapeutic effects through selective proteasome degradation of its client proteins. Elucidation of its pathophysiology using animal models has led to the development of disease-modifying drugs, i.e., Hsp90 inhibitor and HSP inducer, which inhibit the pathogenic process of neuronal degeneration. These findings may provide the basis for development of an HSP-related therapy for neurodegenerative diseases.

• neurodegenerative diseases
• heat shock protein
• geranylgeranylacetone
• Hsp90 inhibitor
• ubiquitin proteasome system

Introduction

In chronic neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), Alzheimer's disease (AD), and polyglutamine (polyQ) diseases, abnormal accumulation and inclusions of disease-causing protein are a commonly observed characteristic. Under pathological conditions, the level of such misfolded proteins may exceed the protective machinery of the cell to either maintain them in a soluble form or degrade them, resulting in their accumulation and inclusion. ALS is an adult-onset motor neuron disease and most forms of ALS are sporadic, but ∼10% of patients have an inherited familial form of the disease of dominant causative mutations in the gene encoding copper/zinc superoxide dismutase 1 (SOD1) or 43 kDa TAR DNA-binding protein (TDP-43). Ubiquitinated protein aggregates found in many patients with sporadic or familial ALS are mainly composed of SOD1 or TDP-43 proteins [1]. The α-synuclein gene is mutated in rare familial forms of PD, and α-synuclein is a major component of the pathological protein aggregates characteristic of the disease, known as Lewy bodies and Lewy neuritis [2]. PolyQ diseases are caused by the expansion of a trinucleotide CAG repeat encoding glutamine in the causative genes, and to date, nine disorders have been identified as polyQ diseases [3]. Many molecular biological studies have been undertaken to elucidate the pathogenesis of these diseases and to develop treatment methods. Among these treatments, pharmacological induction of molecular chaperones may ameliorate these disorders. Here we highlight the research findings from which the concept of the chaperone-dependent pathophysiology of neurodegenerative diseases has emerged, and discuss disease-modifying therapeutic approaches targeting molecular chaperones.

Involvement of the molecular chaperone pathway and the ubiquitin-proteasome system in the histopathology of neurodegenerative disorders

In patients with neurodegenerative diseases, neurons are selectively depleted in the nervous system. A pathological hallmark of most neurodegenerative diseases is the presence of nuclear and cytoplasmic inclusions. For example, nuclear inclusions (NIs) containing the mutant androgen receptor (AR) are detected in residual motor neurons in the brainstem and spinal cord [4–6] as well as in the skin, testis and some other visceral organs in patients with spinal and bulbar muscular atrophy (SBMA), one of the polyQ diseases [7], [8]. These NIs are detectable by antibodies that recognise many components of the molecular chaperone pathway and the ubiquitin-proteasome system (UPS), implying that these proteins are involved in neurodegeneration in these diseases [9–11]. Heat shock proteins (HSPs) are classified into the following families according to molecular size: Hsp100, Hsp90, Hsp70, Hsp60, Hsp40, and small HSPs [12]. These molecular chaperones are either constitutively expressed or inducibly synthesised after cellular stress. HSPs play important roles in suppressing protein misfolding by synergistically assisting misfolded proteins in the refolding process, as well as maintaining correct folding, assembly, and intracellular transport of newly synthesised proteins (Figure 1). However, several studies suggest that misfolded proteins interfere with the protective cellular responses against cytotoxic stress [13]. Transfected cells expressing truncated polyQ-expanded AR showed delayed induction of Hsp70 after heat shock compared with that in transfected cells expressing the full-length AR [14]. Progressive reductions in the expression of Hsp70, Hsp40 and α B-crystallin in the brain lesion of a mouse Huntington's Disease (HD) model were reported [15–17], and such reductions were also observed in SBMA model mice [18]. Reduced expression of heat shock cognate protein (Hsc)-70 was found in PD patients [19], reduced expression of Hsp27 and Hsp70 in transformed lymphoblastoid cells from patients with spinocerebellar ataxia type 7 (SCA7) [20], and reduced expression of Hsp27 in ALS-SOD1 mice [21]. In keeping with the importance of Hsp70 in defining neuronal susceptibility to degeneration, an inverse correlation between the relative amount of Hsp70 in neuronal subtypes and susceptibility to mutant Huntington-induced cell death was reported in a mouse model of HD [22]. Taken together, impairment of the capability of HSP induction is implicated in the pathogenesis of neuron degeneration in neurodegenerative diseases (Table I).

Figure 1. Diagram showing the roles of chaperones in folding of proteins and degradation of misfolded proteins. It has been suggested that a pathogenic process leads to formation of misfolded conformations of the target protein in neurodegenerative diseases. Correct protein folding is an essential biological process, and to achieve this, many proteins interact with molecular chaperones in the cellular environment. The Hsp70 class of molecular chaperones recognises the unfolded substrate early in the folding process, and maintains the polypeptide in the soluble conformation. To facilitate protein folding, Hsp70 interacts with its co-chaperones that regulate its chaperone activity. A major class of Hsp70 co-chaperone proteins is the Hsp40 family. Both Hsp70 and the Hsp40 family of chaperones have been shown to interact with the E3 ubiquitin ligases, particularly the quality-control E3 ligases, and are implicated in the biology of neurodegenerative disorders involving protein misfolding. Quality-control E3 ligases like CHIP could specifically target misfolded mutant disease proteins for proteasomal degradation and thereby could potentially slow disease progression.

Table I.  Changes in chaperone expression in neurodegenerative diseases.

Disease	Chaperones	References
SBMA	Hsp70↓	Katsuno et al. [18]
HD	Hsp70↓, Hsp40↓	Hay et al. [15]
	α B-crystalin↓	Zabel et al. [16]
SCA7	Hsp70↓, Hsp27↓	Tsai et al. [20]
ALS	Hsp25↓	Maatkamp et al. [21]
PD	Hsc70↓	Grunblatt et al. [19]

SBMA, spinal and bulbar muscular atrophy; HD, Huntington's disease; SCA7, spinocerebellar ataxia type 7; ALS, amyotrophic lateral sclerosis; PD, Parkinson's disease.

Induction of heat shock proteins

Accumulation of mutant proteins has been considered to be central to the pathogenesis of neurodegenerative diseases including polyQ diseases. It has been postulated that expanded polyQ confers monomeric protein conformational change from random coil to β-sheet, leading to formation of polyQ oligomer [23]. The misfolded monomer and oligomer that are resistant to denaturing detergents possess toxic property by means of interacting with normal cellular proteins [24]. Direct inhibition of polyQ oligomerisation by Congo red has been demonstrated to exert therapeutic effects in a mouse model of HD [25]. Oligomerisation of causative protein has thus been implicated in the pathogenic process of neurodegeneration, whereas formation of inclusion body, or matured amyloid fibrils, appears to possess cytoprotective property [26], [27]. Based on this hypothesis, HSPs have been drawing a great deal of attention, because they inhibit oligomer assembly and thereby mitigate its toxicity [28]. These HSPs have been proposed to prevent the initial conformation conversion of mutant polyQ-containing protein from a random coil to a ß-sheet, leading to attenuation of toxic oligomer formation [13]. For example, HSPs, particularly Hsp70, were shown to suppress aggregate formation and cellular toxicity in polyQ disease [18], [45–47], ALS [34] and PD models [35], [36]. Hsp104 also reduced α-synuclein aggregation and dopaminergic degeneration in a rat model of PD [37]. Moreover, Hsp70 overexpression enhanced degradation of polyQ-expanded proteins via its interaction with the UPS [32], [38]. CHIP (carboxyl terminus of Hsc70-interacting protein) might be one such coupling factor between the Hsp70 chaperone system and the machinery responsible for degrading mutant proteins [39], [40]. Thus, enhancement of chaperone expression under preserved UPS function is a reasonable clinical approach for the treatment of pathogenic mutant or modified protein-mediated neurodegenerative diseases (Figure 1).

Favourable effects obtained by genetic modulation of HSPs suggest that pharmacological induction of molecular chaperones might be a promising approach for the treatment of neurodegenerative diseases. Hsp90 inhibitors and celastrol function as HSP inducers, resulting in the dissociation of HSF-1 from the Hsp90 complex and subsequent HSF-1 trimerisation, thereby leading to HSP activation [41], and exerting potentially neuroprotective effects in models of polyQ diseases [15], [33], [45–47], tauopathies [45–47], ALS [48], [49] and PD [50–52]. Geranylgeranylacetone (GGA), an acyclic isoprenoid compound with a retinoid skeleton, has been shown to strongly induce HSP expression in various tissues [53]. This compound has been used as an oral anti-ulcer drug. Oral administration of GGA up-regulated the levels of Hsp70, Hsp90, and Hsp105 via activation of heat shock factor-1 in the central nervous system and inhibited nuclear accumulation of the pathogenic AR protein, resulting in amelioration of polyQ-dependent neuromuscular phenotypes of the SBMA mouse model [18]. Thus, enhancement of cellular defences using Hsp90 inhibitors and GGA is a reasonable clinical approach for the treatment of neurodegenerative diseases (Table II).

Table II.  Compounds that induce chaperone expression.

Compound	Molecular weight	Therapeutic effects	Change in chaperones	Clinical use
Cell			Animal
GGA	330.55	SBMA [18]	SBMA [18]	Hsp105↑↑	Anti-ulcer drug
				Hsp90↑
				Hsp70↑↑↑
17-AAG	585.69	SBMA [41], [61]	SBMA [41], [61]	Hsp90→	Anti-cancer drug
17-DMAG	652	HD [42]	PD [50]	Hsp70↑↑↑
		ALS [48]		Hsp40↑↑↑
Celastrol	450.61	HD [33]	HD [36]	Hsp70↑↑
			PD [36]
			ALS [49]
Arimoclomol	313.78		ALS [34]	Hsp90↑↑	ALS
				Hsp70↑↑↑
				Hsp27↑↑

GGA, Geranylgeranylacetone; 17-AAG, 17-allylamino-17-demethoxygeldanamycin; 17-DMAG, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin; SBMA, spinal and bulbar muscular atrophy; HD, Huntington's disease; PD, Parkinson's disease; ALS: amyotrophic lateral sclerosis.

Hsp90-dependent pathogenesis

Heat shock protein 90, a cytosolic molecular chaperone, is involved in the maturation and activation of a number of proteins, known as client proteins. Hsp90 functions in multi-component complexes of chaperone proteins including Hsp70, Hop, Cdc37, and p23 [54]. Two main Hsp90 complexes are thought to exist: one complex is a stabilising form with Cdc37 and p23 and this complex stabilises Hsp90 client proteins, while the other complex is a proteasome-targeting form with Hsp70 and Hop and it directs Hsp90 client proteins to proteasome degradation [55–58]. Hsp90 inhibitors inhibit the ATP-dependent progression of the Hsp90 complex toward the stabilising form and shift it to the proteasome-targeting form, resulting in proteasomal degradation of the Hsp90 client proteins, particularly their mutant versions [56]. Another possibility is that Hsp90 client proteins may also be recognised initially by the Hsp40/Hsp70 complex with CHIP, and transferred to the Hsp90 complex by Hop [59]. There remain two fates for the client proteins: refolding or ubiquitin-dependent proteasomal degradation. For example, dephosphorylation and refolding of p-tau is initially facilitated by an Hsp90/P23/Pin1 complex, however, when refolding is not accomplished, p-tau is transferred to the Hsp70/CHIP complex, and polyubiquitination mediates their degradation [59]. Steroid receptors and numerous oncoproteins belonging to the Hsp90 client protein family are selectively degraded in the UPS by Hsp90 inhibitors, and 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-dimethylamino-17-demethoxy-geldanamycin (17-DMAG) are now under clinical trials as novel molecular-targeted agents for a wide range of malignancies [60]. We previously examined the effectiveness of 17-AAG and 17-DMAG in a mouse model of SBMA [41], [61]. These compounds significantly reduced the amount of the insoluble high-molecular-weight complex of mutant AR as well as the amount of soluble monomer of mutant AR in the spinal cord and skeletal muscle, and significantly reduced the amount of diffuse nuclear-accumulated AR. Alternatively, Hsp90 inhibitors may inhibit aggregation of mutant AR by inducing Hsp70 and Hsp40 expression, as described above. Of interest is the finding that Hsp90 inhibitors preferentially targeted mutant AR rather than wild-type AR to proteasomal degradation. A high association between p23 and AR containing expanded-polyQ tract renders the mutant AR more sensitive to Hsp90 inhibitors than wild-type AR [41].

On the other hand, hsp90 inhibitors were reported to degrade the Hsp90 client proteins via macroautophagy (referred to here as autophagy) or chaperone-mediated autophagy (CMA). Macroautophagy is a process in which cells form double-membrane vesicles, called autophagosomes, around a portion of cytoplasm. These autophagosomes ultimately fuse with lysosomes, resulting in degradation of their substrates. CMA is selective for specific cytosolic proteins that contain a KFERQ-like motif [62]. This motif is recognised by Hsc70, which transfers their substrates to the lysosomal membrane where they are translocated into the lysosomal lumen and degraded via binding to the receptor lysosome-associated membrane protein 2A (LAMP2A) [63]. The IkB kinase (IKK), an essential activator of NF-κB, was selectively degraded by autophagy following geldanamycin (GA) treatment [64]. Inactivation of ubiquitination or proteasome failed to block IKK degradation, however, inhibition of autophagy by an autophagy inhibitor or knockout of Atg5, a key component of the autophagy pathway, significantly rescued IKK from GA-induced degradation [64]. GA increased total cellular proteolysis through an induction of CMA, which was not 3-MA sensitive [65]. Another Hsp90 inhibitor, apratoxin A (oz-apraA), a cyclodepsipeptide isolated from a marine cyanobacterium, also promoted the degradation of epidermal growth factor receptor (EGFR), which has a KFERQ-like motif in the kinase domain, through CMA [66]. Mutation of this motif prevented EGFR degradation by CMA and promoted its proteasomal degradation in oz-apraA–treated cells. Oz-apraA modulates Hsp90 chaperone complexes and enhances the interaction of Hsp90 client proteins and Hsc70, which can then be recognised by LAMP-2A for degradation via CMA [66]. On the contrary, not an autophagic inhibitor but a proteasome inhibitor was able to prevent the degradation of p35 and mutant tau over a treatment with the Hsp90 inhibitors [67]. These observations suggest that a subset of Hsp90 client proteins may be degraded via autophagy following Hsp90 inhibition.

In the case of neurodegenerative diseases, phosphorylated tau would be one of the target proteins of Hsp90 inhibitors, since Hsp90 inhibitors significantly reduce the total amount of phosphorylated tau [45], [46], [59] due to a dose- and time-dependent degradation of p35 by Hsp90 inhibitors [67], and also reduce aggregated form of tau [45], [67]. In AD, tau hyperphosphorylation is suggested to be a pathogenic process caused by aberrant activation of several kinases, in particular cyclin-dependent protein kinase 5 (Cdk5), leading to phosphorylation of tau on pathogenic sites. Reduction of p35 levels by Hsp90 inhibition affected the activity of the Cdk5/p35 complex [67]. Leucine-rich repeat kinase 2 (LRRK2) would be another target protein of Hsp90 inhibitors [68]. Although the majority of PD cases are sporadic, some are inherited, including those causing LRRK2 mutations. LRRK2 forms a complex with Hsp90 and its co-chaperone Cdc37, and inhibition of Hsp90 disrupts the association of Hsp90 with LRRK2 and leads to proteasomal degradation of LRRK2. Hsp90 inhibitors preferentially reduced the amount of the mutant LRRK2 by integrating into the Hsp90 chaperone complex compared with the wild-type LRRK2, and rescued the axon growth retardation caused by overexpression of the LRRK2 G2019S mutation in neurons [68]. On the other hand, the steady expression level of α-synuclein, a common constituent of Lewy bodies (LBs) and Lewy neurites (LNs) in PD, was not affected by Hsp90 inhibition [50]. We regard this general versatility as very important for the development of Hsp90 inhibitors as a treatment for neurological disorders. The strategy behind Hsp90 inhibitors differs from previous strategies employed against neurodegenerative diseases, which unavoidably allowed abnormal protein to remain and placed much value mainly on inhibition of protein aggregation. We consider that the ability to facilitate degradation of disease-causing or disease-related proteins by modulation of Hsp90 function would be of value when applied to neurodegenerative diseases. There is no doubt that reduction of the amount of the main culprit protein would have a curative effect against various neurodegenerative diseases. In fact, one therapeutic approach that directly reduces the level of abnormal protein by RNA interference, has already proven beneficial in various mouse models of polyQ diseases and amyotrophic lateral sclerosis [69–71].

Hsp90 inhibitor-induced degradation requires well-preserved proteasome function; however, the question of whether the UPS is impaired in patients with neurodegenerative diseases has been raised with respect to this UPS-dependent therapy [72]. The UPS is responsible for the turnover of most soluble proteins and plays an essential role in degrading short-lived regulatory proteins and damaged or misfolded proteins [73]. It is generally accepted that the UPS is involved in the pathology of neurodegenerative diseases, since many components of the UPS and molecular chaperones are known to co-localise with neuronal inclusions [29], [74]. The function of the UPS has been evaluated in various assays that provided divergent results, ranging from impaired [75–78] to preserved [79], [80] UPS function. If the function of the UPS was impaired, Hsp90 inhibitors would not be able to exert their pharmacological effect on neurodegenerative diseases. It is possible that mutant polyQ-expanded proteins may directly affect proteasome function within the specific subcellular compartment of synaptic UPS activities [81], although it has been demonstrated that neuronal dysfunction can develop without significant impairment of the UPS in a mouse model of SCA7 [82]. Consistent with this are data showing that proteasome impairment did not contribute to the pathogenesis of HD in a mouse model [83]. Taking these data together, we consider that treatment with Hsp90 inhibitors, which would enhance a self-clearing system of target disease-causing proteins via the UPS, is a reasonable therapeutic strategy against neurodegenerative diseases.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.