Expanding role of molecular chaperones in regulating α-synuclein misfolding; implications in Parkinson’s disease

Abstract

Protein misfolding under stressful environmental conditions cause several cellular problems owing to the disturbed cellular protein homeostasis, which may further lead to neurological disorders like Parkinson’s disease (PD), Alzheimer’s disease (AD), Amyloid lateral sclerosis and Huntington disease (HD). The presence of cellular defense mechanisms like molecular chaperones and proteasomal degradation systems prevent protein misfolding and aggregation. Molecular chaperones plays primary role in preventing protein misfolding by mediating proper native folding, unfolding and refolding of the polypeptides along with vast number of cellular functions. In past few years, the understanding of molecular chaperone mechanisms has been expanded enormously although implementation to prevent protein aggregation diseases is still deficient. We in this review evaluated major classes of molecular chaperones and their mechanisms relevant for preventing protein aggregation, specific case of α-synuclein aggregation. We also evaluate the molecular chaperone function as a novel therapeutic approach and the chaperone inhibitors or activators as small molecular drug targets.

Introduction

Protein quality control of the cell is regulated by various components to maintain homeostasis and cell viability, cumulatively forming a protein homeostasis network coordinated by several proteins [1, 2]. The protein homeostasis network spans over all the subcellular compartments and finely communicates with each other to ensure the correct folding and degradation of nearly 10,000 different proteins to maintain cell viability and organism health [3]. Protein homeostasis regulates the cellular protein activities and ensures that every protein in the cell is functional during its lifetime or, once stress-damaged, is directed for elimination to prevent toxicity of misfolded polypeptides. The efficacy of cellular protein homeostasis is known to decline naturally during aging [4] or due to external stress conditions resulting in deregulation of de novo protein biogenesis, protein quality control systems for protein repair and the degradation pathways resulting in altered protein activities leading to “loss of function” diseases, e.g., cancer or “gain-of-toxic-function” diseases, e.g., Parkinson’s disease (PD), Alzheimer’s disease (AD), Amyloid lateral sclerosis and Huntington disease (HD) [5].

Parkinson’s disease and α-synuclein homeostasis

Despite of the elegant pathways in place to prevent protein misfolding and aggregation, problems in protein folding presents a major health burden in aging population [2]. PD is the second most common neurodegenerative disorder after AD. The disease affects up to 10 % of the human population over 65 years and is caused by specific loss of dopaminergic neurons within the substantia nigra pars compacta (SNpc) region of brain [6]. PD is one of the protein aggregation diseases caused by α-synuclein misfolding and is characterized by the formation of insoluble α-synuclein aggregates that coalesce into nuclear and cytoplasmic inclusions [7]. The pathological hallmark of the disease is the presence of intracellular protein inclusions termed as Lewy bodies, comprising largely of α-synuclein aggregates [8, 9] along with other proteins such as synphilin-1 [10], ubiquitin [11], torsin A [12] and Hsp70 and Hsp90 chaperones [13, 14]. The presence of large amounts of ubiquitinated α-synuclein and molecular chaperones in Lewy bodies indicate a cellular attempt to sequester toxic misfolded or aggregated α-synuclein.

Aggregation of α-synuclein; perturbed protein homeostasis

Initially, the mitochondrial impairment and oxidative stress [15] were considered to underlie neuronal death in PD, however, the continued research on genetic mutations in single genes, such as α-synuclein [16–18], parkin [19], DJ-1 [20], PINK1 [21], ATP13A2 [22, 23] and leucine-rich repeat kinase-2 (LRRK2) [24, 25] indicate that protein misfolding plays a pivotal role in causing neurodegenerative PD. Earlier, Lewy bodies were considered to be pathogenic, yet the research in following years showed presynaptic α-synuclein aggregates as toxic species [26] and later oligomeric α-synuclein were found to be toxic conformers in the α-synuclein aggregation pathway [27].

α-Synuclein is localized in the presynaptic termini of neurons and physiologically regulates the release of neurotransmitters, synaptic functions and the plasticity of neurons [28]. In neuronal cells, α-synuclein is found in two structurally different isoforms; a free disordered cytosolic form prevalent under physiological conditions and a membrane bound α-helix-rich form with a high propensity to aggregate under the conditions of cellular stress [29–31]. These α-synuclein aggregates act as seeds for further aggregation of free cytosolic α-synuclein, however, the mechanism of initiation and transition from unfolded α-synuclein to β-sheet rich fibrils is still not clear [32, 33]. α-Synuclein from Lewy bodies of PD brains revealed occurrence of a wide array of posttranslational modifications, including nitration of tyrosines, oxidation of methionines, covalent modification of histidines and lysines, phosphorylation, ubiquitination, or SUMOylation and their impact on toxicity and aggregation of α-synuclein is being explored [34–37]. Oxidative stress being a hallmark feature of PD, has been reported to initiate phosphorylation of α-synuclein [36, 37], which affects the neurotransmitter recycling, mitochondrial function and dynamics (fission/fusion), α-synuclein degradation and the transfer of modified α-synuclein to neighboring cells [38, 39]. The oxidative environment of dopaminergic neurons may also be a key factor in initiating oxidative modifications of α-synuclein and its toxic gain-of-functions, thus making nigrostriatal pathway highly vulnerable in PD [34].

α-Synuclein aggregation pathway is composed of several misfolding events a protein can undergo resulting in various degrees of conformers formed. In cytosol, unfolded monomers of α-synuclein interact and form oligomers of various orders and morphologies, which owing to the hydrophobic nature assemble into fibrils and accumulation of fibrils, generate intracellular inclusions called as Lewy bodies [33]. However, the exact mechanism of α-synuclein aggregation causing neurodegeneration is still unclear, but its effects include alteration in calcium homeostasis, mitochondrial fragmentation leading to dysfunction, oxidative stress, inhibition of core cellular processes of protein refolding, transport and degradation (Fig. 1a) [40, 41], which could ultimately cause neurodegeneration. Strict cellular protein quality control mechanisms act to coordinate the rates of protein synthesis and degradation, and prevent the formation of intracellular aggregates, thus maintaining cellular protein homeostasis [2, 42]. However, prolonged exposure to various stresses and failure of these protein quality control mechanisms results in the accumulation of toxic α-synuclein aggregates in PD [7].

Fig. 1.

a Pathologically toxic species in the α-synuclein aggregation pathway. α-Synuclein polypeptide transforms from physiological monomeric helical state to misfolded monomers, leading to the formation of small and large oligomers and finally stack as fibrils, which accumulate in cell in the form of Lewy bodies. The least compact oligomeric species are reported to be the most toxic by affecting a number of cellular processes to disrupt cellular functions [27, 33]. Fibrils are not as toxic as oligomers but there quantitative presence in the cell interferes with the normal cellular functions and ultimately leads to neuronal loss. b Role of chaperones in maintaining α-synuclein protein homeostasis. Molecular chaperones act on misfolded α-synuclein monomers which are cytotoxic in two ways: first, they unfold them back to a natively foldable conformation as assisted by Hsp70 and co-chaperones. Second, they may prevent α-synuclein misfolding and aggregation by mere “holding”, as in the case of the small heat shock proteins [70, 130, 162]. Further, already formed stable small oligomeric α-synuclein aggregates, which are toxic can become disaggregated and unfolded by the Hsp104 + Hsp70 + Hsp40 chaperone machinery or in the human cytosol by the Hsp110 + Hsp70 + Hsp40 chaperone machinery and disaggregated into monomers that may reach their harmless native state [91, 96, 109, 163]

Molecular chaperones are essential components in maintaining protein homeostasis by acting as molecular nanomachines which modulate the kinetic partitioning of polypeptides between the pathways that lead to native, inactive misfolded and aggregated protein conformations [43].

Aggregation of α-synuclein; role of molecular chaperones

For proteins to function optimally, they must fold and be stably maintained in their native conformations. The information contained within the primary amino acid sequence can dictate the three-dimensional structure of the protein [44], which together with the environment of the cell ensures that proteins are assembled, processed and transported accordingly. The pathway by which a protein achieves its folded low energy conformation is complex and involves ensemble of intermediates and conformations [45]. Protein misfolding occurs because of the inappropriate self-associations of hydrophobic residues leading to oligomerization and aggregation, which are stable and low in energy [46]. Molecular chaperones are highly conserved and ubiquitously expressed in all subcellular compartments, cells, and tissues. They are necessary for the stability of the proteome under normal and stressful conditions [47, 48]. The expression of many molecular chaperones is regulated by different forms of environmental and physiological stresses such as heat shock that can interfere with the folding stability of proteins. Thus, molecular chaperones are often referred as heat shock proteins in the literature, although many but not all are induced by heat stress [49].

In unstressed cells, the molecular chaperones play a central role in physiological protein homeostasis. They may regulate structural transitions between “native” and so called “alternative” conformations of the proteins [50, 51]. In stressed cells, the molecular chaperones become a primary line of cellular defenses against stress-induced protein misfolding and aggregation events [52] that otherwise become increasingly toxic by compromising with the stability of other proteins and the integrity of membranes [53].

The molecular chaperones fall into five families of highly conserved proteins: the Hsp100s (ClpB), the Hsp90s (HtpG), the Hsp70/Hsp110 (DnaK), Hsp60/CCTs (GroEL), and the α-crystallin-containing domain generally called the “small Hsps” (IbpA/B) (Escherichia coli orthologues shown in parentheses). Each family comprised multiple members that share sequence homology, have common functional domains, expressed in different subcellular compartments and at different levels in the tissues [48, 54].

All molecular chaperones can bind the misfolding intermediates, and as such, prevent directly or indirectly protein misfolding and aggregation events [55–57]. Yet they may achieve this by different complementary mechanisms that optimize their concerted action within the larger network of molecular chaperones and proteases controlling the cellular protein homeostasis [49, 58, 59]. Apparently, all chaperone families share the ability to screen for proteins with hydrophobic residues that are abnormally exposed to the aqueous phase, and are thus prone to self-association and thereby forming stable inactive aggregates [46, 51, 60]. With the exception of the sHsps, all major classes of molecular chaperones are ATPases, suggesting that their function can implicate an ATP-driven increase of the free energy in their bound misfolded or alternatively folded polypeptide substrates [61]. Recent advances in the mechanisms of molecular chaperones revealed an ATP-independent refolding activity of Hsp60 class of chaperones [62].

In PD, first evidence of the involvement of molecular chaperones was provided by pathological studies that identified Hsp90, Hsp70, Hsp60, Hsp40 and Hsp27 as the components of Lewy bodies [63]. In some reports, rotenone, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and lactacystin induced PD models in cultured neuronal cells and mice, a heat shock like transient response is observed which leads to overexpression of Hsp70 and Hsp90 chaperones [64]. Also, heat shock induced expression of chaperones demonstrated that the induction of Hsp70 can prevent α-synuclein-induced cell death in yeast, Drosophila and mouse models of PD [65–67].

The possible mechanism of action for molecular chaperones in PD is to protect neurons against misfolded α-synuclein-induced toxicity as the depletion of molecular chaperones exacerbates protein toxicity and neurodegeneration [64]. Many reports confirmed the association of molecular chaperones with α-synuclein aggregates in vitro and in vivo [68], and showed their binding to profibrillar species of α-synuclein [69]. In solution, under controlled conditions, the α-synuclein oligomers with high β-sheet content strongly inhibit both the bacterial and human Hsp70/Hsp40 unfolding/refolding activities, whereas no inhibition was observed by unstructured α-synuclein monomers even if provided in large molar excess [70].

The network of chaperones in cell ‘The Chaperome’ constitutes nearly 10 % of the total protein mass of the human cancer cell, half of which are Hsp70s and 90s [71]. The chaperone network functions in accord under cellular conditions, first the Hsp70 and co-chaperones prevent protein misfolding, sHsps also prevent accumulation of aggregation prone proteins and assist in their unfolding and refolding, whereas on larger oligomers Hsp100 class of chaperone act as an ATP-fuelled disaggregating enzyme converting already formed stable aggregated substrates into natively refoldable products, Hsp90 can help in refolding or targeting the irreparable misfolded species to elimination by the ubiquitin proteasomal degradation system (Fig. 1b). A direct correlation between chaperone levels and detergent soluble α-synuclein aggregates is also reported, which makes proteotoxic aggregates bioavailable for repair with coordinated Hsp response [72]. As the activity of chaperones and proteasomal degradation of cell declines, the proteotoxic species such as α-synuclein aggregates starts accumulating in the cells [73].

The highly conserved molecular chaperone machinery and degradation pathways maintain a fine balance in protein homeostasis in PD. The exact molecular mechanism of protein misfolding and aggregation is not well understood but the significant involvement of chaperones in not doubted. Molecular chaperones and co-chaperones are now well documented for their precise mechanism of action and their role in cellular protein homeostasis. Here, we revisit the role of molecular chaperone systems in reference to the recent mechanisms elucidated for their possible involvement in the prevention of α-synuclein aggregation in PD.

Hsp70 system

The heat shock protein 70 (Hsp70) is a conserved family of proteins from bacteria to eukaryotes having a critical role in various cellular processes beyond defenses against stress damage, such as de novo protein folding, protein translocation, vesicular trafficking and cellular signaling [74]. Hsp70 collaborates with a J domain co-chaperone, Hsp40 (DnaJ, CpbA or DjlA in E. coli) that targets the chaperone onto its misfolded protein substrates and, together with a nucleotide exchange factor (NEF, GrpE in E. coli) controls the coupling between ATP consumption and the unfolding work of the Hsp70 chaperone [74–76]. As such, Hsp70 along with Hsp40 chaperone are powerful modulators of proteome integrity and cell viability [77]. In protein aggregation disorders such as PD, the Hsp70 system plays an important role in regulating α-synuclein aggregation at the first place. The co-localization of Hsp70 with α-synuclein aggregates in PD brain is evident [67]. Hsp70 can prevent dopaminergic neuronal loss in D. melanogaster model of PD [67], where overexpression of Hsp70 is able to reduce α-synuclein aggregation in in vitro and in vivo conditions [68, 78–80].

Mechanistically, Hsp70 reduces α-synuclein toxicity by modulating α-synuclein oligomers [81] and helps in the clearance by aggresome formation [80]. Hsp70 can interact with α-synuclein fibrils [82], however, it cannot disaggregate them alone [69]. The interactions between Hsp70 and α-synuclein are transient as reported by FRET studies [83] and Hsp70-α-synuclein complex from rat brains failed to co-immunoprecipitate [84]. It has been shown that in vitro assembly of α-synuclein was efficiently inhibited by sub-stoichiometric concentrations of Hsp70 because of the interactions between the Hsp70 substrate binding domain and the core hydrophobic region of α-synuclein [84].

Interestingly, Hsp70 alone was able to reduce Aβ(1-42) aggregate induced cytotoxicity specifically by reducing apoptosis and ROS in human neuroblastoma cells which were preincubated with Hsp70 and treated with Aβ-aggregates [85]. The in vitro efficiency of Hsp70 at sub-stoichiometric concentrations was comparable with earlier reports of Hsp70 (DnaK) mediated refolding of misfolded luciferase [74], emphasizing the low energy requirements of molecular chaperones and their efficiency as polypeptide unfolding enzymes [86].

The upregulation of Hsp70 in PD models is exploited as a therapeutic target for long now. Heat shock factor 1 (Hsf1) is the main transcription factor that regulates the inducible expression of Hsp70. Under physiological conditions, Hsf1 is bound to Hsp90, whereas under stress conditions Hsp90 binds to high affinity misfolded substrates leaving Hsf1 active, which subsequently induces the expression of Hsps including Hsp70. In PD, the overexpression of Hsp70 is also reported in a number of studies to reduce α-synuclein aggregation and toxicity. Transgenic overexpression of Hsp70 in PD model of D. melanogaster completely maintains the number of normal dopaminergic neurons in aged flies [67]. In Hsp70-overexpressing transgenic mice, a significant reduction in detergent soluble and high molecular weight α-synuclein aggregates was observed [66]. Brain permeable chemical inducers such as Geranylgeranylacetone (GGA), Hsp90 inhibitors geldanamycin and its derivatives are also reported to induce Hsp70 through activation of Hsf1 and reduce α-synuclein aggregation [79, 87]. Recently, a known neuroprotective agent in ischemia, carbenoxolone (CBX), is shown to induce Hsf1 in α-synuclein overexpressing neuroglioma cells and reduce α-synuclein aggregate toxicity [88]. A possible therapeutic target to reduce α-synuclein toxicity can be explored among the growing number of chemical inducers of Hsp70.

Despite of the several reports on Hsp70-α-synuclein interactions, pull down of the complex failed because of the poor mechanistic understanding of Hsp70 system. In cell, Hsp70 system functions as a network with the members of the HSPA (Hsp70) and DnaJ (Hsp40) chaperone families, still in neurodegenerative diseases, the role of co-chaperones is understated. Hsp40 proteins DnaJB6 and DnaJB8 are predominantly expressed in the neurons and are known to suppress polyglutamine aggregation and toxicity [89, 90]. Hsp40 binds to the misfolded polypeptide and is further acted upon by Hsp70 for ATP fueled forceful unfolding and spontaneous refolding thereafter [74]. A mechanistic understanding of how α-synuclein oligomers inhibit Hsp70 system reported in Hinault et al. [70] explains that the toxic α-synuclein oligomers halt Hsp70/40 unfolding machinery, and toxic oligomers preferably interact with J domain co-chaperones. Human Hsp70 along with DnaJB1 also interacts with Hsp110 (Apg2) and forms a disaggregase machinery which could efficiently depolymerize stable α-synuclein aggregates and convert them into less toxic species [91]. Hsp110 is structurally and functionally related to Hsp70 and share a conserved nucleotide-binding domain (NBD) [92]. It is classified as a nucleotide exchange factor of Hsp70 along with the other NEFs including HspA1, 2 3 and BAG1. The remarkable feature of Hsp110 is its disaggregation activity, which is lacking in other NEFs. The Hsp110 can disassemble the amyloids and protein aggregates to nontoxic monomers at physiological rates in an ATP-dependent manner. In C. elegans, depletion of Hsp110 family proteins by RNAi [93] or Hsp105 knockout mouse cells shows severe defects in protein aggregation clearance indicating the role of Hsp110 beyond a mere NEF [94]. Hsp110 (Hsp70-Z) in Plasmodium falciparum exhibit an independent chaperone activity as well as NEF activity for Hsp70 and both activities are necessary for its survival [95]. Hsp110 can unfold smaller misfolded species with Hsp40 and disaggregate larger aggregates with Hsp70, suggesting a specialized unfolding/refolding chaperone activity [96, 97].

Remodeling of detergent insoluble aggregates [91] is an extraordinary feature of Hsp110-Hsp70-Hsp40, which needs to be further verified in the animal models of PD. This recently discovered disaggregase activity of Hsp110-Hsp70-Hsp40 could possibly have vast implications in modulating α-synuclein homeostasis in PD and therapeutic applications.

Hsp100 system

Hsp100 class of chaperones belong to the AAA+ superfamily of ATPases, and is defined by the presence of a basic core of ~200 to 250 amino acids that comprise α-helical domain and a Walker-type nucleotide-binding domain. They have the ability to remodel the protein substrates in an ATP-dependent manner [55]. The Hsp100 chaperones are categorized into two classes; class I proteins with two AAA+ modules, including Hsp104 yeast protein, bacterial ClpB and their distant relatives ClpA, ClpC, whereas class II chaperones include ClpX and HslU [98]. Their role in protein aggregation diseases is crucial as they can rapidly disassemble various amyloids and prefibrillar oligomers and reactivate proteins from aggregates [99]. Hsp100 exert an ATP-driven threading activity and translocate protein substrates through their central channel and form proteolytic complexes with peptidases (e.g., E. coli ClpA or ClpX with the ClpP peptidase) to feed substrates into the associated proteolytic chamber for degradation. ClpB and Hsp104 specifically do not associate with peptidases and use their threading power to disentangle the polypeptide chains trapped within the protein aggregates [100].

Hsp104 was initially identified as a factor behind thermotolerance in yeast [101]. The first chaperone like activity of Hsp104 was discovered to solubilize luciferase aggregates, although no other chaperone possesses such disaggregating activity. Hsp104 has a remarkable ability to synergize with mammalian Hsp70 and Hsp40 [102] and to interact with various misfolded polypeptide species, prions and amyloids. Hsp104 can efficiently disaggregate prions in yeast model and enhance its survival up to 10,000 folds [103, 104]. In AD, the Aβ42 oligomerization and aggregation is reported to be significantly inhibited at sub-stoichiometric concentrations of Hsp104. [105]. There has been a conflict whether Hsp104 can interact with α-synuclein or not, but toxic A30P pre-amyloid oligomers were reported to be disassembled by Hsp104 alone in vitro as observed by anti-oligomeric immuno-reactivity and EM analysis [99]. Being a yeast protein, the role of Hsp104 has always been criticized for its function as a mammalian disaggregase, although Hsp104 along with Hsp70 system showed enhanced disaggregation in mammalian cell cultures without exerting toxicity [99, 106, 107]. In rat model of PD, simultaneous overexpression of Hsp 104 and A30P α-synuclein in the brain significantly reduced tyrosine hydroxylase (TH) immune reactivity and phosphorylated inclusions of α-synuclein at P129 indicating the protective role of Hsp104 in vivo [99].

The structural variation by missense mutation in Hsp104 middle domain is known to enhance its optimal ATPase activity and the intrinsic disaggregase activity [108]. Wild-type Hsp104 has no effect on eliminating cytoplasmic α-synuclein inclusions and translocations, whereas its variants with mutations in the middle domain (Hsp104^A503V, Hsp104^A503S and Hsp104^A503V−DPLF) can eliminate cytoplasmic inclusions and restore plasma membrane localization of α-synuclein. Also, these potentiated variants possess enhanced unfoldase activity and do not require Hsp70-40 for disaggregation activity [109].

Crude whole animal extracts from C. elegans are able to slowly disaggregate and degrade Aβ42 fibers [110]; however, the factor(s) involved remain unidentified, which might be Hsp110 class of chaperones. A relatively distinct homolog p97 of Hsp104 was observed to collaborate with Hsp70 and 40 but optimum disaggregase activity is not yet established [103, 111].

The yeast Hsp104 and ClpB are reported to partially antagonize protein misfolding and neurodegeneration in metazoans [98, 108, 112]. In mammals, Hsp104 does not exist; instead, the disaggregase activity of Hsp110 in synergism with Hsp70 may be an evolutionary strategy under stress conditions.

Small heat shock proteins

The small heat shock proteins (sHsps) form oligomers of small (15–45 kDa) polypeptide subunits with a conserved α-crystallin domain composed of a stable antiparallel β-sheet domain flanked by a highly variable N-terminal domain and a short flexible C-terminal extension [71, 113]. sHsps are known to bind or hold misfolded polypeptides and maintain them in their non-aggregated yet inactive state and also help in the dissociation and unfolding/refolding by other chaperones such as Hsp70, Hsp60 and Hsp104 [114, 115]. As such, sHsps are considered passive anti-aggregation chaperones serving as the first line of cellular defenses against stress- and mutation-induced protein aggregation [43]. In humans, ten different sHSPs (HspB1–HspB10) have been identified, among which the best characterized are Hsp27, αA and αB-crystallin (HspB4 and B5) and Hsp20 (HspB6). In mouse brain, five sHsps are mainly expressed and are shown to have neuroprotective effects, among which the expression of Hsp27 and αB crystallin is highly induced in response to neurological stress [116]. αB crystallin (HspB5) is a major constituent of the eye lens, where it functions as a structural protein and a chaperone specialized in the prevention of light scattering by aggregating polypeptides [117, 118]. sHsps are upregulated in protein aggregation diseases and are known to interact with the partially folded proteins [119]. Mass spectrometry reveals that in transgenic C. elegans, three Hsp16 family members in addition to other molecular chaperones co-immunoprecipitate with human Aβ [120] and sHsps are also found in protein aggregates of AD, PD affected patients [121]. αB Crystallin and Hsp27 have been known to protect against α-synuclein-induced toxicity and aggregation [122, 123]. αB-crystallin interacts with α-synuclein and leads to the formation of large nonfibrillar aggregates, thus redirecting α-synuclein from a fibril-forming pathway to an amorphous aggregation pathway, and reducing the amount of physiologically stable fibrils and increasing the amounts of easily degradable amorphous aggregates [122]. Hsp27 is also the most strongly induced protein across several brain regions in AD and PD patients, and along with αB crystallin it chaperones the Aβ(1–40) and (1–42) aggregation [124, 125]. The exact mechanism and the role of sHsps in amyloid fibril diseases is unclear, however, αB-crystallin binds to wild-type Aβ(1-42) fibrils with micromolar affinity along the entire length and ends of the fibrils. Binding of αB-crystallin to the fibril seeds strongly inhibit their elongation indicating that this could be effective in inhibiting fibril proliferation [126–128]. Hsp27 reduces the size of fibrils and interacts with multiple species formed in the α-synuclein aggregation pathway and retards α-synuclein aggregation in the initial stages most likely by binding to the partially folded monomers as observed by reduced Th-T binding. [129, 130]. In in vivo studies, the expression of murine Hsp25 was upregulated in A53T overexpressing mice conforming their role in preventing toxic protein aggregation [131].

Overexpression of sHsps in mammalian, yeast and bacterial cells enhances stress tolerance, whereas a knockout mutant or lack of sHsp activity neither produces a strong mutant phenotype nor is necessary for thermotolerance in E. coli or S. cerevisiae [132] sHsps cooperate with other chaperone machineries present in the cell. In prokaryotes, sHsps IBPA/B assist in reactivation of the aggregated protein substrates with ClpB (Hsp104), DnaK (Hsp70) and DnaJ (Hsp40) systems [112, 115, 133]. In the presence of IbpA/IbpB, the disaggregation of insoluble protein aggregates is accelerated by ClpB/KJE chaperone system. Under in vivo conditions, sHsps and ClpB are essential for cell viability and protein resolubilization at elevated temperatures where DnaK levels are limiting [133, 134].

Upon heat shock, S. cerevisiae Hsp26 becomes insoluble and its solubility depends on Hsp104 expression. Hsp104–Hsp26 together solubilizes the protein aggregates and is effective on yeast polyglutamine aggregates [135, 136]. The sHsps probably renders Hsp104/Hsp70/Hsp40 system a more accountability, and thus defend the cells against protein misfolding problems [135]. In mammalian models where Hsp104 is absent, exogenous overexpression of sHsps with Hsp104 showed a positive collaboration in disaggregating protein aggregates. Hsp27 and Hsp104 expression in lentiviral mouse model of HD restored the normal positive nuclei cell density like wild-type [137]. The newly discovered Hsp110 disaggregase activity of mammalian systems is still to be explored for cooperation with sHsps, although Hsp110 is related to Hsp70 and possibly enhance the chaperone activity along with sHsps by remodeling substrates. This activity may be remarkable in maintaining α-synuclein proteins homeostasis in PD.

Posttranslational modifications of sHsps is another important aspect, as reversible phosphorylation of some sHsps occurs under conditions of cellular stress and is thought to have an important functional role in dynamically altering the oligomeric distribution and chaperoning ability of sHsps like αB crystallin in response to the stress [138]. Structural flexibility of N-terminal domain (NTD) is regulated by phosphorylation, which destabilizes the inter-subunit interactions and remodels αB crystallin to active oligomers predominantly consisting of 12-mers and 6-mers [139]. Phosphorylation also affects the cellular distribution of some sHsps. For example, following stress, phosphorylation of αB-crystallin and Hsp27 causes them to be translocated into the nucleus, presumably to protect the nuclear proteins important for the cell survival [140]. Several observations indicated that phosphorylation pathways are important in the pathogenesis of PD where α-synuclein is phosphorylated at several sites; phosphorylation at serine 129 constitutes major form of the protein in Lewy bodies [35]. A familial PD mutation in α-synuclein Ala53Thr, increases the amount of S-129 phosphorylation and reportedly enhances its aggregation [141]. Therefore, it would be interesting to check whether phosphorylation of sHsps and α-synuclein have an effect on the disaggregation activities of chaperones.

Molecular chaperones need to be explored for potential therapeutics

Since α-synuclein association with PD has been discovered in 1997, its various aggregated forms are now regarded as the major pathogenic species in PD. Researchers have developed many prevention strategies against α-synuclein toxicity [9] and several clinical trials focusing on α-synuclein homeostasis have been initiated [41]. Molecular chaperone-based approaches to target aggregation and clearance of the aggregates are logical targets for drug development. Induction of chaperones by various chemicals has been validated to reduce protein aggregation in PD and AD. Geldanamycin-mediated upregulation of Hsp70 was shown to inhibit α-synuclein aggregation and reduce neurodegeneration [67]. A new class of aggregation inhibitor molecules and proteins [142] opened new horizons in this research area. The insulin-degrading enzyme is known to maintain glucose concentration in blood, in addition, it degrades amyloidogenic peptides such as Αβ and Islet amyloid polypeptide (IAPP) associated with AD and type 2 diabetes, respectively. Recently, an additional chaperone like activity of insulin degrading enzyme (IDE) was proposed, where it forms stable and irreversible complex with Αβ and α-synuclein and inhibit their aggregation [142, 143]. CsgC a bacterial protein involved in bacterial film formation also inhibit α-synuclein fibrillization and renders α-synuclein as chaperone amenable substrate [144]. Chemical compounds studied in compound library screens reported efficient neuroprotectors, such as epigallocatechin-3-gallate (EGCG) and 3-(1,3-benzodioxol-5-yl)-5-(3-bromophenyl)-1H-pyrazol (anle138b), CLR01 and pyrolyloligopeptidase inhibitor KYP2047 [145]. Further leads from cancer and other disease should be taken where the molecular chaperone-based therapeutics have already reached advanced phases of clinical trials [146, 147].

GBA encodes glucocerebrosidase (GCase), a 497-residue lysosomal hydrolase that catalyzes the metabolism of glycolipid glucosylceramide to ceramide and glucose [148]. GBA1 gene mutation is known for causing Gaucher’s disease, a lysosomal storage disorder; however, in recent reports mutation in GBA gene has been linked with a higher risk of PD [149–153]. Mutated or misfolded GCase gets trapped in the endoplasmic reticulum and its translocation to the lysosomes fails resulting in the misfolding of other proteins, abnormal chaperone recognition, early accumulation of α-synuclein and premature self-degradation. GCase and α-synuclein interact in a bi-directional feedback loop manner where deficient activity of GCase enhances α-synuclein oligomerization, and α-synuclein oligomers in turn decrease the activity of GCase [153–155]. Preventing the misfolding of GCase with the help of molecular chaperones could be an alternative approach to inhibit α-synuclein aggregation. Hsp27 has been shown to interact with misfolded GCase and directs it for 26S mediated proteasomal degradation [156]. Pharmacological chaperones, such as glucosylceramide mimicking GCase inhibitors [157], isofagomine and 2,5-anhydro-2,5-imino-d-glucitol related compounds [158] have been reported to assist in the proper folding and translocation of GCase to lysosomes and subsequently reduces the aggregation of α-synuclein. Ambroxol, a small molecule acts as a chaperone to upregulate the activity of GCase and to ensure its proper translocation from endoplasmic reticulum leading to reduced levels of α-synuclein in the cells [159, 160].

Molecular chaperones perform their physiological functions in stressed and unstressed and are shown to collaborate with other classes of molecular chaperones resulting in enhanced chaperoning abilities. Recent reports explored chaperone functions beyond holding, to refolding polypeptide by unfolding and cooperation of chaperones to disentangle aggregates [62, 74, 91, 96, 100, 109, 161]. Hsp70 in addition to refolding a misfolded polypeptide, acts as a disaggregase with Hsp110. Hsp110 itself acts as a NEF with remarkable chaperone activity and depends on Hsp70 and Hsp40 for substrate selection. Likewise, sHsps alone prevent misfolding and aggregation but enhances the disaggregation potential of Hsp104 by remodeling toxic protein aggregates. Hsp104 cooperates with Hsp70 and Hsp40 to form powerful disaggregase machinery. In PD, where α-synuclein aggregation poses severe cellular stress, these chaperone machineries must be functional behind the scene and need to be explored further for collaborative chaperone networks acting under disease conditions in vivo and to implement the mechanistic knowledge of chaperones in maintaining the homeostasis of α-synuclein. Drugs that would be able to specifically induce the chaperone network similar to their natural expression profile in young stressed tissues would be expected to combat the formation of toxic α-synuclein aggregates, thereby retarding the onset of degenerative synucleinopathies. Such interventions may hold promise in the prevention of further α-synuclein aggregation and may play an augmenting role in strategies aimed at the prevention of dopaminergic neuronal loss.