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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Prog Neurobiol. Author manuscript; available in PMC Oct 1, 2011.
Published in final edited form as:
PMCID: PMC2939168
NIHMSID: NIHMS210993

Heat Shock Proteins: Cellular and molecular mechanisms in the CNS

Abstract

Emerging evidence describe heat shock proteins (HSPs) as critical regulators in normal neural physiological function as well as in cell stress responses. The functions of HSPs represent an enormous and diverse range of cellular activities, far beyond the originally identified role in protein folding and chaperoning. Now understood to be involved in processes such as synaptic transmission, autophagy, ER stress response, protein kinase and cell death signaling as well as protein chaperone and folding, manipulation of HSPs have robust effects on the fate of cells in neurological injury and disease states. The ongoing exploration of multiple HSP superfamilies has underscored the pluripotent nature of HSPs in the cellular context, and demanded the recent restructuring of the nomenclature referring to these families to reflect a re-organization based on structure and function. In keeping with this re-organization, we have first discussed the HSP superfamilies in terms of protein structure, regulation and expression and distribution in the brain. We then explore major cellular functions of HSPs that are relevant to neural physiological states, and from there discuss known and proposed HSP impact on major neurological disease states. This review article presents a three-part discussion on the array of HSPs families relevant to neuronal tissue, their cellular functions, and the exploration of therapeutic targets of these proteins in the context of neurological diseases.

Keywords: heat shock protein, synaptic transmission, protein degradation, cell death, cerebral ischemia, Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, prion, Charcot-Marie-Tooth

1. Introduction

Critical functions and neuroprotective properties of heat shock proteins (HSPs) in the brain have been explored for several decades, yet studies of the precise mechanistic control and function of HSPs are continually yielding new surprises. Moreover, the HSP superfamily of proteins includes a multitude of subgroups and closely related members. The emerging literature related to HSP functions in protein management and cell death, in particular within the neuronal context, creates a focal point for reviewing these newfound roles in light of systemic functions.

Within the context of this review, we will 1) strive to apply current nomenclature to a review of previously published literature, 2) highlight important recent advances in the regulation of HSP function, 3) illustrate relevant physiological roles of HSPs in the context of the brain, and 4) describe the alteration of HSP function by neuropathological conditions, and thus explore the therapeutic potential of HSPs in the context of neuropathology.

1.1. Nomenclature

The overall understanding of heat shock proteins in terms of gene organization, phylogenic branching of protein families and regulation has expanded dramatically in the past several years. While multiple review articles underscore the relevance of HSPs in neurological stress conditions, the rapid expansion of knowledge based on specific chaperone subtypes creates a need for continued review of new data and incorporation of alterations in terminology.

The original discovery that heat-inducible (and constitutive) chaperone proteins function in the folding of proteins to correct native structures was an exciting leap in the understanding of protein-protein interactions. However, the molecular tools available at the time precluded a detailed analysis of full gene sequences, specific function and/or regulation, or the development of antibodies able to differentiate between highly homologous members. In the ensuing 40 years of research devoted to HSPs, more than 80 chaperones grouped into several distinctive functional families have been discovered. Many of these family members were not previously dissociated from each other; thus, the literature dating within even recent years can be somewhat nebulous in terms of the specific family members.

In the present review, we will attempt to bring up to date advances in the regulation and function of HSPs in the neuronal context, and we will try to evaluate the roles of specific family members in light of experimental findings (Table 1). In order to minimize confusion, we will adhere to the recently proposed guidelines for nomenclature of heat shock proteins put forth by (Kampinga et al., 2009). When the specific family member cannot be determined based on the experimental evidence, the general family name will be used followed by the referenced name, in order to minimize possible misassignment of function.

2. HSP family catagorization: Untangling the family tree

HSPs were originally discovered as stress-inducible proteins. However, it is now understood that most of the stress-inducible HSPs have highly homologous constitutively expressed (also called “cognate”) relatives that perform critical cellular housekeeping functions. Due to the sequence similarity and overlap in function, the term HSP now refers to both constitutive and induced family members.

Originally thought to be largely controlled at the transcriptional level, substantial mechanistic data have confirmed that HSPs have multiple levels of regulation. The stress-induced transcription of HSPs lies primarily under the control of the heat shock factor-1 (HSF1), a transcription factor that, when bound to DNA in a multichaperone complex, is activated and upregulates target heat shock genes. HSF1 itself is under the negative regulation of HSPC/HSP90, which allows suppression of stress-responsive gene transcription in control settings at the same time as setting the stage for rapid induction of gene expression within minutes of cellular stress. Many HSPs also contain alternative promoter recognition sites, allowing for transcriptional activation via multiple signaling pathways and cellular environments.

In addition to transcriptional control, it is becoming increasingly clear that many members of the HSP superfamily are regulated by posttranslational modifications, including nucleotide binding, carbonylation, phosphorylation, oligomerization and protein-protein interactions. The HSPA and HSPC families are functionally regulated by the interaction with several diverse classes of co-chaperones, including the DNAJ/HSP40 family, the HSPH/HSP110 nucleotide exchange factors, and smaller co-chaperones such as the Bag family, the HSP-organizing protein (Hop) and HSP-interacting protein (Hip). Several of these interacting proteins contain tetratricopeptide repeats (TPR) that recognize EEVD C-terminal sequence motifs in HSP70 and HSP90 (Brinker et al., 2002; Carrello et al., 2004; Michels et al., 1999). These interactions and posttranslational modifications make HSPs sensitive and immediate responders to the cellular environment, thus allowing for intricate control and communication, ultimately balancing cellular responses for cell survival and, in some cases, death.

Within this section, we will briefly introduce the major HSP families, focusing on the neuronally relevant members (Table 1). The HSPA and HSPC families, which perform the classical chaperoning function and are most widely described in the literature, will be discussed first, followed by the major co-chaperone HSP families, DNAJ and HSPH. The more recently described small HSP family, HSPB, and the mitochondrial associated HSPs, HSPD and its co-chaperone HSPE, will then be addressed. Finally, the mitochondrial HSP families, HSPD, HSPE and the yeast HSP100 will be addressed.

2.1. HSPA family (HSP70s)

The HSPA/HSP70 family of chaperones is by far the most widely studied group of heat shock proteins, and is currently known to be comprised of at least 13 highly related proteins that are either constitutively expressed or induced upon cell stress. These proteins are found in multiple subcellular compartments, playing critical roles in the mitochondria, endoplasmic reticulum, cytosol, lysosomes and extracellular compartments. Their activity is associated with a variety of cell functions and is regulated by the interaction and control of various co-chaperone families in an intricate manner. The HSPA family has received extensive attention and in-depth review; therefore, following a brief introduction, the focus of the discussion will be on more recently discovered functionality in the context of neurological disease.

2.1.1. Expression

In addition to rapid induction following stress, a number of HSPA family members are constitutively expressed and perform critical cellular housekeeping functions. Either the loss or overstimulation of these functions contributes heavily to activation of multiple possible cell death pathways. The expression patterns of HSPA proteins extend to nearly all subcellular compartments, as well as secretion into the extracellular milieu and surrounding cells. HSPA1A/HSP72, HSPA2/HSP70-2, HSPA6/HSP70B’ and HSPA8/Hsc70 are primarily cytosolic, while HSPA9/Grp75/mortalin is associated with the mitochondria, and HSPA5/Grp78/BiP is resident in the endoplasmic reticulum (ER). However, many of these members can shuttle between compartments. For example, in addition to its cytosolic localization, HSPA1 is also expressed at the luminal side of the lysosomal membrane, particularly under stress, both to stabilize the lysosomal membrane and to facilitate import of proteins being degraded via chaperone-mediated autophagy (CMA). HSPA5/Grp78, though classically defined as the primary ER chaperone, may also have a cytosolic function as an alternative splice variant found in ER-stressed leukocytes (Ni et al., 2009). This isoform promoted cellular survival, though the mechanism and potential relevance in brain is unknown. Similarly, HSPA9/Grp75 serves as a major mitochondrial chaperone and plays an essential role in translocation of nuclear encoded mitochondria-targeted proteins through double mitochondrial membranes into matrix. HSPA9/Grp75 coordinates with HSPD/HSP60 and HSPE/HSP10 to maintain correct protein folding in mitochondria (see below), illustrating the interaction between HSP families. However, HSPA9/Grp75 is not exclusively expressed in mitochondria, but is also docked at extramitochondrial sites, such as the ER (Ran et al., 2000), cytosol/cytoplasmic vesicles and plasma membrane (Ran et al., 2000; Wadhwa et al., 2002). The activity and function of these chaperones are largely determined through binding partners, which in turn depend on subcellular localization in the cell.

2.1.2. Structure and function

The chaperone function of HSPA family members relies heavily on ATP hydrolysis and nucleotide exchange, managed via the coordination of several accessory co-chaperone families, including the HSPH (HSP110) and DNAJ (HSP40) families, in a cellular and environmental context-specific manner. Accordingly, HSPA family members contain several fairly well-conserved domains: the ATPase domain in the N-terminal, a substrate binding domain (also referred to as the “chaperone function”) and a C-terminal region that regulates the release of the substrate upon nucleotide exchange (Figure 1). In addition to the function of these domains in chaperoning, HSPA members have been implicated in the binding and alteration of various other proteins, including cell death signaling pathways, as discussed below.

Figure 1
Major protein domains defining HSP families.

Under normal conditions, many constitutive members of the HSPA– including HSPA8/HSC70, HSPA5/Grp78 and HSPA9/Grp75/mortalin – form the critical compartmental-specific protein-folding machines. They function in concert with specific binding partners, particularly the DNAJ family of chaperones, and with specific nucleotide exchange factors (NEFs) in normal cellular environments. However, in addition to protein folding, HSPA family members recognize and bind exposed hydrophobic residues of misfolded or denatured proteins. These proteins are often held by HSPAs for ubiquination and subsequent targeting to the proteasome for degradation. Alternatively, HSPA family members are responsible for the recognition of proteins containing a KFERQ-like pentapeptide. These proteins are then passed into lysosomes by HSPA proteins via CMA.

2.2. HSPC (HSP90)

Similar to the HSPA family, HSPC/HSP90 family members contain both constitutive and inducible members, all of which are highly homologous in sequence, structural domains and regulation. HSPC members are also found in various subcellular locations, including the cytosol, ER and mitochondria. The overlap in HSPA and HSPC families in location and expression has translated to evidence of interplay between these two families. The mechanisms and implications of these interactions are still under investigation, and will likely reveal a highly interconnected model of HSP coordination across cellular functions.

2.2.1. Expression

HSP90 is one of the most highly abundant constitutively expressed proteins, comprising 1–2% of cellular proteins (Sreedhar et al., 2004). HSP90 is widespread and constitutively expressed in rat brain, and present in almost all neurons (Gass et al., 1994; Itoh et al., 1993; Izumoto and Herbert, 1993). By in situ hybridization, HSP90 mRNA was found abundant in limbic system-related structures, such as the hippocampus, amygdala and mamillary body. The highest abundance of mRNA was detected in the Purkinje cell layer of the cerebellum (Izumoto and Herbert, 1993). At the cellular level, HSP90 is predominantly found in cell bodies, but to a lesser extent also in dendrites and nuclei (Gass et al., 1994). In mammalian cells, several HSP90 members have been identified to date, including the two major cytoplasmic homologues, HSPC1/HSP90α (inducible) and HSPC3/HSP90β (constitutive), the glucose-regulated protein 94 (HSPC4/GPR94) in the endoplasmic reticulum, and tumor necrosis factor receptor-associated protein 1 (HSPC5/TRAP1) in mitochondrial matrix (Sreedhar et al., 2004). Until recently, the literature has not distinguished much between HSPC1/HSP90α and HSPC3/HSP90β in terms of function, with the exception that HSPC1/HSP90α appears to be inducible, while HSPC3/HSP90β is constitutively expressed. HSPC has also been localized to the cell surface in the developing nervous system (Cid et al., 2004; Cid et al., 2009; Sidera et al., 2004).

2.2.2. Structure and function

The major function of HSPC/HSP90 centers on the regulation of substrate activity, typically via multichaperone complex tethering and sequestration in active or inactive states (Pratt and Toft, 2003). The functional HSPC/HSP90 molecule is a homodimer. Each monomer consists of three domains, the N-terminal domain containing the ATP binding site, and a long linker middle (M) domain followed by a C-terminal domain (Figure 1) (Pearl and Prodromou, 2006). While the M-domain possesses limited capacity for protein-protein interactions, the C-terminal domain is essential for HSP90 dimerization and is the primary domain for substrate binding. In eukaryotes, the C-terminal domain also provides an extreme segment containing the MEEVD motif implicated in binding to tetratricopeptide repeat (TPR) domain-containing co-chaperones, including the proteins Hop, protein phosphatase 5 (PP5) and the large peptidylprolyl isomerases (PPIases) (Pearl and Prodromou, 2006; Wandinger et al., 2008). The interactions with specific co-chaperones diversify and target HSP90 cellular functions.

Similar to HSPA family members, HSP90 operates in a complex cycle driven by ATP binding and hydrolysis and by ATP/ADP exchange. The N-terminal domain possesses a deep binding pocket for ATP. This ATP-binding can be competitively inhibited by the antibiotic geldanamycin (GA). Upon ATP binding, the N-terminal domain undergoes a conformational change leading to a twisted and compacted dimer in which N- and M-domains associate. The association of N- and M-domains fulfills the ATPase activity, which is essential for the function of HSP90. Additionally, the C-terminus of HSP90 has a second ATP-binding site, which can be inhibited by cisplatin, novobiocin, EGCG and taxol (Donnelly and Blagg, 2008).

Serine/threonine phosphorylation of HSP90 has been observed in vivo (Garnier et al., 2001; Lees-Miller and Anderson, 1989a, b; Szyszka et al., 1989), indicating that, like other HSP families, HSP90 can be controlled via posttranslational covalent modifications. The phosphorylation of HSP90 complexes was observed in neural tissue, suggesting a possible target for neuronal studies. However, the exact mechanisms leading to the phosphorylation – and subsequent dephosphorylation – are not well studied in brain. Several potential kinases, including casein kinase-2 (CK2) (Lees-Miller and Anderson, 1989b), double-stranded DNA-activated protein kinase (DNA-PK) (Lees-Miller and Anderson, 1989a) and autophosphorylation by HSP90 itself (Langer et al., 2002), have been suggested to mediate HSP90 phosphorylation, but the exact regulatory mechanisms – particularly under neurological dysfunction – are, as yet, unknown.

Accordingly, dephosphorylation may then be an alternate target of modulating HSP90 function. The protein phosphatase 5 (PP5) is present in the functional heterocomplexes containing HSP90 (Chen et al., 1996b; Silverstein et al., 1997), and the related yeast phosphatase PPT1 specifically dephosphorylates HSP90. Without PPT1, yeast HSP90 is hyperphosphorylated and unable to correctly fold substrate proteins (Wandinger et al., 2006). Interestingly, the C-terminal domain of HSP90, which binds to the TPR domain in PP5, can in turn activate PP5 activity. This presents a possible self-regulatory mechanism for HSP90 activity. The functional significance of HSP90 phosphorylation appears to be in the substrate binding and release, where the phosphorylated state is associated with substrate release. Phosphorylation of HSP90 can be inhibited with the HSP90 inhibitor geldanamycin (GA). GA treatment led to the inhibition of substrate release from HSP90 (Zhao et al., 2001).

In addition to phosphorylation, HSPC can be acetylated, resulting in a decreased affinity for substrate binding (Scroggins et al., 2007). Under normal circumstances, the histone deacetylase HDAC6 maintains HSPC in a deacetylated form. While acetylation represents another level of control over HSPC, the relation to neuronal models has not been explored.

2.2.3. Crosstalk with HSPA family

As will be discussed below, the HSPC and HSPA families coordinate many activities via “coordinating” proteins, effectively balancing chaperone activities across cellular compartments. In addition, the perturbation of HSPC almost invariably leads to the upregulation of HSPA expression and activity. For example, one of the major tools in HSPC functional research is the use of HSPC/HSP90 inhibition, most notably by the small molecule inhibitor geldanamycin (GA). GA targets HSPC1 fairly specifically by binding to and inhibiting the ATP binding pocket. However, the inhibition of HSP90 invokes a cell stress response and induces HSPA transcription and activity (Lu et al., 2002). Thus, when using inhibition of HSPC to determine function, it is often difficult to determine if the effect is from a direct effect on HSPC endogenous activity, or an indirect effect due to an increase in HSPA activity.

2.3. DNAJ (HSP40)

In contrast to other HSP families, the DNAJ/HSP40 family members function as partner chaperones in the modulation and control of HSPA/HSP70 and HSPC/HSP90 families. Part of these activities is the recruitment of HSPA members to protein complexes, harnessing ATPase activity necessary for conformational changes on substrate proteins. This section will discuss the structure and subclassification of DNAJ proteins and the expression pattern, and will briefly touch on binding partners.

The DNAJ/HSP40 family contains a large number of members, thereby comprising the largest and most diverse heat shock family. To date, 37 protein members have been identified that harbor the typical J-domain unique to the DNAJ family. An additional seven members contain a partially conserved J domain (Qiu et al., 2006). Moreover, many of these proteins reside in specific cellular locales and in specific cell types. For a list of their names and intracellular locations, please refer to Qiu et al., 2006 (Qiu et al., 2006). In our review, we will focus on members of this family that are present in or directly impact neurons.

2.3.1. Structure and subclassification

Essentially, the J-domain is comprised of 70 amino acids folded into three coiled α-helices, which enables the interaction between DNAJ members and HSPA and HSPC families (Li et al., 2009). Mechanistically, this interaction assists the co-chaperone DNAJ in harnessing and regulating the ATPase activity on HSPA or HSPC molecules. Conversion of ATP to ADP on HSPA/HSPC results in the stabilization of binding to its substrate, in turn providing sufficient time for the proper folding of its substrates (Li et al., 2009; Qiu et al., 2006). DNAJ family members are subgrouped as DNAJA, DNAJB and DNAJC (corresponding to types I, II and III, respectively) based on the location of specific domains within the molecule (Figure 1) (Mitra et al., 2009; Qiu et al., 2006; Zhao et al., 2008). Both DNAJA and DNAJB subclasses contain a glycine/phenylalanine (G/F)-rich domain, which is thought to function as a linker region between the J domain and the rest of the molecule and appears to modulate the interaction with HSPA (Cheetham and Caplan, 1998). In the DNAJA subclass, the J domain is located at the N-terminal and is followed by the G/F domain. The DNAJA subclass is the only DNAJ group that contains cysteine repeats forming zinc binding sites located after the G/F domain (Cheetham and Caplan, 1998). On the other hand, the DNAJB subclass has its G/F domain located at the N-terminal followed by the J-domain. Finally, the DNAJC subclass contains the J-domain located within the molecule but lacks the G/F domain. In general, DNAJ molecules also contain specific polypeptide peptide domains (PPD), enabling them to associate with specific substrates in lieu of a more generic binding motif.

Consistent with distinct structural elements, each subclass of DNAJ proteins has different functional properties. DNAJA proteins can bind to and chaperone non-native polypeptides independent of the participation of ATP. It is the only subclass of DNAJ proteins that can function as an independent chaperone without the involvement of HSPA or HSPC. Similarly, DNAJB proteins associate with non-native polypeptides independent of ATP. However, this DNAJ subclass can only complete its chaperoning function in conjunction with HSPA. Finally, DNAJC chaperones function by a mechanism similar to that of DNAJB, but due to their unique PPD, they have the capacity to bind and stabilize specific substrates before shuttling them to HSPA to complete the proper folding of these substrates (Qiu et al., 2006; Zhao et al., 2008).

2.3.2. Function and binding partners

To date, eight members of the mammalian DNAJ family have been found expressed in neurons. These members consist of representatives from each subclass, including DNAJC6/auxilin, DNAJC26/GAK, DNAJC5/CSPα, DNAJA2/Rdj2, DNAJC13/RME-8, DNAJB2/Hsj1 DNAJB6/Mrj and DNAJB1/Hdj-1/HSP40. Our understanding of these proteins is cursory at best, but ongoing efforts to characterize each of them will further elucidate the mechanistic action of heat shock protein machinery in neurological disease.

Subclass C members are the most variable and numerous of DNAJ proteins. Due to the combination of highly specialized PPDs and expression patterns, DNAJC molecules are postulated to dictate the specific compartmentalization and/or function of HSPA members (Kampinga et al., 2009). The neuronally expressed members of the DNAJC subclass include DNAJC6/auxilin, DNAJC26/GAK, DNAJC5/CSPα and DNAJC13/Rme-8. DNAJC6/auxilin is a 100-Kda cytosolic protein whose expression is specific to neurons. As a member of the subclass C, it contains a PPD that enables it to associate with clathrin, adaptor protein 2 (AP2) and dynamin. Upstream of the PPD is a PTEN-like domain that binds to phosphatidylinositol (4,5)-biphosphate (PIP2) whereas downstream of the PPD resides the J domain (Zhao et al., 2008). DNAJC26/GAK is a 150-kDa ubiquitously expressed subclass C protein present in various cell types not limited to neurons. It is localized both in the cytosol and in the nucleus. Apart from the J domain, it encompasses a PTEN-like domain similar to DNAJC6/auxilin (Lee et al., 2006a) that associates with clathrin. DNAJC5/CSPα is a 35-kDa subclass C protein that is involved in synaptic vesicle function (Zhao et al., 2008). In addition, it contains a distinctive string of cysteines (from which its name is derived) that are palmitoylated, enabling DNAJC5/CSPα to anchor onto the synaptic vesicle membrane (Chamberlain and Burgoyne, 1998; Greaves and Chamberlain, 2006; Gundersen et al., 1994). DNAJC13/Rme-8 is a 220-Kda subclass C protein that is ubiquitously expressed in various cell types including neuronal cells. Its role appears to be relevant to endosomal trafficking.

Compared to the neuronal subclass C DNAJ members that are best associated with synaptic and vesicular functions, subclass B DNAJ members expressed in the brain have described roles in proteasome and in prevention of accumulation of protein aggregates. DNAJB1/HSP40 is a 40-kDa subclass B cytosolic protein that is widely expressed in all cell types, including neural cells, and its expression is responsive to stress and heat shock. In neurons, the heat shock response for DNAJB1/HSP40 involves not just a change in expression level but also translocation from cytosol to nucleus (Manzerra and Brown, 1996), and, in some instances, it involves the association of DNAJB1/HSP40 with the lipid raft (Chen et al., 2005). Another neuronally expressed subclass B member, DNAJB2/Hsj1, pairs with HSPA in the shuttling and sorting of proteins destined for degradation by the proteasome (Westhoff et al., 2005; Zhao et al., 2008). The last member, DNAJB6/Mrj is a 26-kDa subclass B protein that resides mainly in the cytosol. It is expressed in various cell types and is abundant in the brain (Chuang et al., 2002; Hunter et al., 1999). While its function is less described, DNAJB6/Mrj has demonstrated protective capabilities against protein aggregate diseases.

The subclass A DNAJ proteins include only one neuronally expressed member found thus far – DNAJA2/Rdj2. To date, we have a very limited understanding of this subclass A DNAJ protein. Localization studies have indicated that DNAJA2/Rdj2 is a neuroprotein expressed in all cell types of the whole rodent brain (Rosales-Hernandez et al., 2009). Moreover, DNAJA2/Rdj2 has been reported to be part of the multimeric chaperone complex with HSPA, HSPH, HSPC, HSP-interacting protein (Hip) and HSP organizing protein (Hop) (Craig et al., 2006). In addition, DNAJA2/Rdj2 has been shown to associate with Gα and Gβ, which suggests that it is involved in G-protein signaling (Rosales-Hernandez et al., 2009). Ongoing efforts are underway to identify the specific G-protein signaling pathways and other functions of DNAJA2/Rdj2.

In addition to neuronally expressed DNAJ members, DNAJ proteins expressed in non-neuronal tissue can indirectly affect the morphology and function of neuronal tissue. This is most apparent in the context of the neuromuscular junction (NMJ). The DNAJ family member DNAJA3/Tid1 has recently been identified as a muscle-specific kinase binding partner ((Linnoila et al., 2008), see below for discussion). DNAJA3/Tid1 is expressed in both the mitochondria and cytosol of non-neuronal cells (Lu et al., 2006). In the postsynaptic muscle cell, DNAJA3/Tid1 localizes to the site of acetylcholine receptor clusters and interacts with several kinases (Linnoila et al., 2008). DNAJA3/Tid1 binds to HSPA family members (Lu et al., 2006), and perturbations in DNAJA3/Tid1 expression disorganized AChR clustering, which elicited morphological changes in motor nerve terminals (Linnoila et al., 2008). Thus, DNAJA3/Tid1 function may indirectly affect neuronal activity via NMJ formation and maintenance.

2.4. HSPH (HSP110/105), nucleotide exchange factors

The HSPH family represents the first of the HSPs identified, but little has been understood about its members’ function or structural biochemistry. Until recently, HSPH family members were assumed to be analogues of the HSPA family due to the high degree of homology that exists between the two families. However, HSPH family members now appear to function most prominently in the role of nucleotide exchange factors for HSPA, acting as co-regulators of chaperone function in balance with other co-chaperones, such as DNAJ proteins.

The expression of HSPH1 is remarkably high in brain. Originally thought to be predominantly cytoplasmic, the beta splice variant of HSPH1/HSP105 localizes to the nucleus (Saito et al., 2007) and is associated with the induction of HSPA/HSP70 via the signal transducer and activator of transcription (STAT)-3 pathway (Saito et al., 2009; Yamagishi et al., 2009). While the N-terminal ATP-binding domain and substrate-binding domain appear to evolve from the HSPA family, the HSPH molecule differs in that it contains an acidic region and divergent C-terminal regions, and the ATP substrate-binding domain is extended (Figure 1). HSPH family members have no identified client substrates for folding, but have been found to associate with HSPA/HSP70 and possibly HSPC/HSP90 families (Dragovic et al., 2006; Liu et al., 1999; Wang et al., 2000; Yamagishi et al., 2004). This interaction with HSPH both accelerated the DNAJ-mediated ATP hydrolysis of HSPA/HSP70 when added in equimolar amounts, and suppressed substrate refolding when added in excess (Dragovic et al., 2006). Subsequent studies have determined that HSPH family members function as a nucleotide exchange factor (NEF) for the HSPA family (D’Silva et al., 2004; Dragovic et al., 2006; Raviol et al., 2006), whereby the nucleotide-binding domains of the HSPA and HSPH proteins interact when HSPA is bound to ADP (Andreasson et al., 2008a). This interaction decreases the affinity of nucleotide binding to HSPA via a conformation change and subsequent release of ADP. Once ATP binds to HSPA, the interaction with HSPH is lost (Andreasson et al., 2008b), allowing DNAJ co-chaperones access to the HSPA ATP-bound molecule, promoting hydrolysis of ATP to ADP, and cycling back to an HSPH-binding competent state.

2.5. HSPB family (small HSPs)

The small HSP family of proteins has only recently been described. Originally overlooked due to high variation in sequence homology, the regulation and function of HSPB family members is unique and presents an interesting expansion in the understanding of HSP mechanisms. While the endogenous role of HSPB members in central nervous system neurons is likely limited, the possibilities for HSPBs as therapeutic reagents appear promising, and will be discussed below.

2.5.1. Expression

The HSPB family includes 11 members in the human and mouse genomes. Of these, the expression of HSPB1 (HSP27, HSP25), HSPB5 (alpha B crystallin), HSPB6 (HSP20) and HSPB8 (HSP22) were confirmed in brain tissue (Quraishe et al., 2008), and HSPB2 expression has been observed in smooth muscle in vessel walls of the human brain (Wilhelmus et al., 2006c). Notably, HSPB1 and HSPB5 have garnered the most attention in terms of stress responsiveness under neurodegenerative conditions, although recent evidence suggests that HSPB6 and HSPB8 may also contribute to neuroprotection. The transcriptional regulation of HSPB members includes the standard HSE promoter elements that recruit HIF1 or HSF2, but some members, such as HSPB1, also appear to contain other promoter elements, including SP1-binding sequences (Gaestel et al., 1993).

HSPB1 and HSPB8 are expressed in motor and sensory neurons in the brainstem, cranial nerve nuclei and cerebellum, with only limited expression present in other postmitotic neuronal phenotypes ((Chen and Brown, 2007; Pareyson and Marchesi, 2009; Plumier et al., 1997). HSPB8 is expressed in astrocytes in both grey and white matter, as well as in cerebrovascular cells forming vessel walls (Wilhelmus et al., 2006b). Both HSPB1 and HSPB5 are expressed in glial cells under control conditions (Wilhelmus et al., 2006c); their expression is also highly inducible under neurological stress ((Iwaki et al., 1992; Kato et al., 1994)). During normal aging and degenerative conditions, HSPB1, HSPB5 and HSPB8 are expressed in reactive astrocytes proximal to senile plaques ((Wilhelmus et al., 2006b; Wilhelmus et al., 2006c), see below). Despite the lack of robust endogenous expression in neurons, multiple studies now suggest that exogenous overexpression may confer neuroprotection in pathological settings, as will be discussed below (An et al., 2008; Stetler et al., 2008; van der Weerd et al., 2009). Thus, understanding the mechanistic role of HSPB may aid in therapeutic interventions.

2.5.2. Structure and function

The HSPB family is structurally defined by the presence of a conserved crystallin domain in the C terminus (Figure 1), which often forms a β sandwich structure (HSPB8 is an exception to the β folding). In contrast, the N-terminal region is highly variable among family members, but typically is alpha helical in nature, possesses a conserved hydrophobic motif (WDPF) and contains phosphorylation sites critical to regulation of function. The C-terminus ends in a short flexible extension. Although poorly conserved in sequence, this extension is typically polar in nature and is thought to stabilize HSPB under thermal stress (Lindner et al., 2000; Smulders et al., 1995). While the crystallin domain is most associated with protein-protein interactions, the N-terminal in some contexts is sufficient for protein interaction (Stetler et al., 2008).

In contrast to other chaperone superfamilies, HSPB family members do not appear to require ATP. HSPB family members oligomerize via association of the C-terminal regions under normal conditions in both hetero- and homodimeric high molecular weight structures. It is the higher oligomeric state that is most associated with the quasi-chaperoning capacity that holds proteins in a foldable state. Upon phosphorylation at serine residues in the N-terminal region, however, the high molecular weight structures disassemble to lower oligomeric structures and are associated with the interaction and suppression of intracellular signaling to promote cell survival under stress (Stetler et al., 2009).

The upstream signaling pathways leading to HSPB phosphorylation and subsequent dephosphorylation are poorly understood. Canonical kinase signaling pathways, e.g. ERK1/2 and p38, have been implicated in different cell types, but appear to be highly cell specific. Phosphatases, including PP2A, PP1 and PP2B, have been demonstrated in vitro to dephosphorylate HSPB1 (Cairns et al., 1994), but the precise mechanism of regulation in the neural context has yet to be elucidated.

2.6. HSPD1/HSP60 and HSPE1/HSP10

A separate class of chaperones and co-chaperones exists for mitochondrial protein folding. While the mitochondrially targeted HSPA9/mortalin assists in the import of nuclear-encoded proteins to the mitochondrial space, the bulk of matrix protein refolding lies in the hands of the HSPD1/HSP60/GroEL and its co-chaperone, HSPE1/HSP10/GroES. As such, HSPD1 and HSPE1 are vital for the health and maintenance of mitochondrial function, as well as for proper mitochondrial biogenesis. In addition to the constitutive mitochondrial chaperone function, HSPD1/HSP60 and HSPE1 are stress-inducible (Truettner et al., 2009), suggesting that these proteins may also participate in non-housekeeping functions.

2.6.1. Expression

HSPD1/HSP60 and HSPE1/HSP10 genes are arranged in a head-to-toe configuration separated by a bidirectional promoter (Hansen et al., 2003; Ryan et al., 1997). This arrangement allows for stoichiometric balance to maintain the expression of HSPD1/HSP60 as approximately twofold higher than the expression of HSPE1. The promoter is under the control of an HSE, CHOP binding site, STAT3 binding site and two SP1-binding sites (Kim and Lee, 2007), allowing the expression of HSPD1 and HSPE1 to be controlled by both quiescent and stressed cellular signaling.

In brain, HSPD1/HSP60 is endogenously expressed in astrocytes, neurons, microglia, oligodendrocytes and ependymal cells. Neural expression increases over the course of development, consistent with the changes of mitochondrial content in the brain (D’Souza and Brown, 1998). Subcellular expression of HSPD1/HSP60 presents in a typically punctate staining pattern indicative of mitochondrial localization. Although the majority of HSPD1/HSP60 protein resides in the mitochondria, 15% to 20% of cellular HSPD1/HSP60 is located in extramitochondrial sites, such as in the cytosol and the cell surface of non-neuronal cells, as well as in the extracellular space and in plasma circulation (Stefano, 2009 #129; Soltys, 1997 #130; Soltys, 1996 #1314; Gupta, 2008 #1309; Pfister, 2005 #131}. The extramitochondrial form retains the N-terminal 26-amino-acid stretch normally cleaved upon import to the mitochondria.

2.6.2. Structure and function

Most of the structural understanding of HSPD1/HSP60 is based on the E. coli form of HSPD1, GroEL. Each subunit of GroEL contains three domains: equatorial, intermediate and apical (Figure 1). First, the equatorial domain contains a binding site for ATP and a heptameric ring structure. Second, the intermediate domain is also heptameric in structure and determines the transitional states of GroEL: peptide-accepting state, which facilitates the capture of non-native peptides into the cavity; and peptide-folding state, which combines with the lid-like HSPE1/GroES to refold the substrate. Third, the apical domain binds to the substrate and HSPE1/GroES (Ranford et al., 2000; Sigler et al., 1998; Weissman et al., 1995). Similar to the HSPA and HSPC families, the conformational change required for chaperone activity for HSPD1/GroEL is ATP dependent. HSPE1/GroES caps the ATP-activated HSPD1/GroEL on the top of the apical domain, which contributes to the formation of a hydrophobic cavity for substrate folding/refolding.

2.7. Non-mammalian heat shock family HSP100/mtHSP78/HSP104

A separate class of HSPs with no known mammalian homologue has recently emerged as a potential therapeutic agent in neurological disorders. Characterized primarily in yeast, the HSP100/mtHSP78/HSP104 proteins are homologues of the bacterial ClpB gene related by the presence of an ATP binding site (200–250 aa) and several conserved motifs (for review, see (Doyle and Wickner, 2009)) (Figure 1). The members of this class of HSPs are of the family of ATPases associated with various cellular activities (AAA+). They are localized to the mitochondria and appear to function in protein quality control by recognizing and solubilizing aggregated proteins and restoring them to their native conformation.

Interestingly, the exogenous presence of HSP100/mtHSP78/HSP104 in mammalian cells appears to be capable of disaggregating insoluble protein aggregates (Lo Bianco et al., 2008), a process thought to be impossible until recently. This function could both play an important role in neurological disease attributable to aggregated protein and aid in understanding the pathological consequence of insoluble aggregates. The pathological effects of the latter have long been a point of contention in the field, and determination of the role of protein aggregates in various pathological settings would help to determine therapeutic pursuits and disease etiology.

2.8. Non-HSP co-chaperones

A number of non-HSP molecules associate with and are critical in the function of HSPs. These include the BAG protein family, the HSP-interacting protein (Hip), and the HSP70-HSP90 organizing protein (Hop). Although these proteins are not considered HSPs due to their lack of conserved structural elements present in the defined HSP families, they are important co-chaperone systems nonetheless. They will be briefly introduced here, and discussed in their functional contexts further below.

Hip and Hop are HSPA- and HSPC-interacting proteins that help to coordinate functions between these two HSP superfamilies. Hip typically binds to the N-terminus of HSPA or HSPC proteins, whereas Hop competes with other cofactors such as DNAJ proteins for the C-terminal domain. The BAG proteins (BAG1, BAG2 and BAG3) interact and bind with high affinity to the HSPA N-terminal ATPase domain in a manner consistent with a NEF (Alberti et al., 2003). This process, similar to HSPH, promotes the release of the substrate from HSPA. Once ATP binds to HSPA again, the NEF dissociates, allowing the DNAJ co-chaperone to function and HSPA to bind to a new protein. The consequences of Bag proteins binding HSPs are context dependent, but are thought to compete with other cofactors such as Hip for HSPA binding and substrate protein targeting to degradation pathways. Furthermore, Bag3 may also participate in the coordination of HSPs in the stimulation of macroautophagy (Carra et al., 2008b; Gamerdinger et al., 2009).

3. HSP functions in the cellular context

HSPs were first described as heat shock induced genes, and were originally thought to be a generalized cellular defense response to combat denaturizing of folded proteins. The presence of constitutive HSPs was likewise thought to primarily serve in nascent protein folding. However, HSPs now appear capable of providing a plethora of cellular functions in normal cells, as well as interacting within the stressed cell in a more directed and targeted manner to promote cell survival. Due to the abundance of literature describing the canonical protein folding activity, we will focus on the more recently described literature, and the emerging functions relevant to the neuropathological environments discussed in the final section of this review.

3.1. Degrade, digest or deaggregate: Dealing with protein substrates

Hand in hand with the recognition of nascent unfolded proteins, heat shock proteins have long been implicated in the recognition of unfolded proteins as a result of cell stress. The unfolded protein response (UPR) initiated by HSPs resident in the endoplasmic reticulum (ER), proteasomal degradation of cytosolic proteins mediated by HSP transport, chaperone-mediated autophagy (CMA) as well as potential roles in macroautophagy and mitochondrial protein degradation, highlight the multifunctional and pan-cellular roles of HSPs in overall protein degradation (Figure 2). It is tempting to speculate that HSPs may function in the communication between specific modes of protein degradation – and by extension, cell death phenotypes – but this has yet to be directly tested and will likely involve multiple binding partners that crosstalk in changing cellular environments.

Figure 2
ER unfolded protein response and protein degradation pathways mediated by HSPs

3.1.1. Unfolded protein response

Upon cell stress, multiple points in the translational pathway can be interrupted, leading to the accumulation of unfolded or misfolded nascent proteins in the lumen of the ER and triggering the UPR. The UPR includes the repression of translation, upregulation of ER chaperones, and shuttling of misfolded proteins out of the ER to the proteasome (Mori, 2000).

HSPA5/BiP/Grp78, a major ER-resident HSP, plays a critical role in the UPR. Under normal protein translation, HSPA5 recognizes hydrophobic residues of nascent proteins in order to direct proper folding and formation of disulfide bonds. A significant proportion of HSPA5 protein is also tethered to transmembrane receptors, such as the activating transcriptional factor-6 (ATF6), PKR-like ER kinase (PERK) and inositol-requiring 1 (Ire1), in the ER of unstressed cells (Figure 2A) (Bertolotti et al., 2000; Shen et al., 2002; Shen et al., 2005b). The accumulation of unfolded proteins exposes more hydrophobic surfaces, increasing the requirement for HSPA5/BiP. The available molecules of HSPA5/BiP normally tethered to the luminal transmembrane receptors dissociate in order to bind to unfolded proteins. The disassociation of HSPA5, in turn, activates the transmembrane receptors PERK and Ire1: (Bertolotti et al., 2000), and releases ATF6 (Shen et al., 2002; Shen et al., 2005b). Activation of PERK leads to phosphorylation of EIF2a-alpha and suppression of translation. Additionally, the release ATF6 by HSPA5/BiP results in ATF6 cleavage and translocation first to the Golgi and subsequently to the nucleus, where ATF6 acts as a transcription factor to upregulate essential ER genes, including BiP. The upregulation of HSPA5/BiP acts as a potential negative feedback, wherein overexpression of HSP5A/BiP suppresses the continued UPR signaling (Bertolotti et al., 2000). The activation of the heat shock response and upregulation of critical HSPs has been demonstrated to relieve ER stress (Liu and Chang, 2008). Consistent with crosstalk between major HSP families in cellular function, non-neuronal studies have indicated that HSPC/HSP90 may counterbalance the UPR on the cytosolic side of the ER membrane. HSPC/HSP90 binds to the cytoplasmic side of PERK and Ire1 in order to stabilize the proteins and suppress the continuation of the UPR signal (Marcu et al., 2002). Furthermore, crosstalk with the cytosolic members of the HSPA and DNAJ families occurs following retrotranslocation of misfolded proteins out of the ER for targeting to the proteasome for degradation (see below). In addition to the link between the UPR and proteasome pathways, the UPR may also crosstalk with autophagic pathways. For example, inhibition of UPR in ALS models increased autophagy, and delayed disease progression. Similarly, in non-neuronal cells, activation of UPR via knockdown of HSPA5/Bip blocked autophagic vacuole formation following ER stress (Li et al., 2008). These findings indicate that these pathways crosstalk and affect cellular fate, potentially using HSPs as sensing or cross-communication molecules. As HSPs participate in both the UPR and autophagy (see below), they may be well positioned to serve as mediators between cell responses.

3.1.2. Proteasome

The proteasome complex is one of the major degradation structures for many cellular proteins under various cellular conditions. The complex itself is comprised of 20S core particle composed of four stacked heptameric structures comprising both structural and catalytic elements. The association of the 19S regulatory particle stimulates proteolytic activity by the 20S core by allowing entry of the protein substrate into the catalytic chamber. Degradation of proteins is accomplished by nucleophilic attack, leaving short peptides that can be either further degraded or reused in new protein synthesis.

While HSPs do not directly function in the proteolysis of substrates by the proteasomes, HSPA and/or HSPC and related co-chaperones are required for recognition of misfolded or mis-localized proteins that are subsequently degraded by the proteasome (Esser et al., 2004; Park et al., 2007). The exact mechanism linking the chaperone and proteasomal systems and the subsequent consequences are still under investigation and are hampered by the compensatory effects of molecular manipulation of HSPs, but several lines point to the coordination between the chaperone recognition of misfolded proteins and degradation via the proteasome. In purified proteasome systems, depletion of HSPA or HSPC severely decreased the proteasomal degradation of substrate proteins, illustrating a requirement for these two chaperones in targeting their substrates to the proteasome. In cellular models, HSPA and its DNAJ co-chaperones were also found to be required for proteasomal degradation of client proteins (Esser et al., 2004; Park et al., 2007). For example, yeast strains deficient in HSPA could not effectively degrade substrate proteins (Park et al., 2007) and the co-chaperone DNAJB2/Hsj1 when paired with HSPA is involved in shuttling and sorting proteins destined for degradation by the proteasome (Westhoff et al., 2005; Zhao et al., 2008).

Co-factor binding to HSPA or HSPC has been demonstrated to initiate ubiquitination and recruit the proteasome (Figure 2B). CHIP is one of the major co-chaperones implicated in linking the HSPA and HSPC families of chaperones to the proteasome system. CHIP interacts with HSPA and HSPC via its amino-terminal tetratricopeptide repeat (TPR) and effectively inhibits the ATPase function necessary for chaperone function (Ballinger et al., 1999; Connell et al., 2001; Esser et al., 2004). In addition, the binding of CHIP maintains HSPC in an intermediary state that is chaperone-compromised by prevention of interaction with TPR-containing co-factors, such as HOP. The absence of these co-factors can lead to altered steroid-receptor binding and increased degradation of the client protein. Given that CHIP contains a U-box – a structure that is reminiscent of ubiquitin ligases – the binding of CHIP to a substrate-bound chaperone appears to lead to ubiquitination of the substrate and subsequent targeting to the proteasome for degradation. Proteasomal targeting may also necessitate binding by other co-factors. For example, Bag1 allows HSPA to bind to the proteasome (Luders et al., 2000)

Currently, the balance between chaperone-assisted folding versus degradation appears to involve the competition between specific co-factors to the client-bound HSPA or HSPC molecule that further steers the client protein toward the proper pathway. In this model, the client protein is first recognized and bound by HSPA or HSPC. Then, in a poorly understood mechanism, co-factors compete for binding sites on the chaperone to promote degradation or folding. Folding is promoted by association of Hop on the C-terminus and/or Hip on the N-terminus, whereas degradation is promoted by CHIP competition with Hop on the C-terminus and Bag-1 competition with Hip on the N-terminal ATPase domain. An additional level of degradation control has been found at the ternary level by further association with either HSP70-binding protein 1 (HSPBP1) or Bag-2 with HSPA on its N-terminal ATPase domain, which, in the presence of CHIP bound to the C-terminus, inhibits the ligase activity of CHIP and blocks client protein degradation (Alberti et al., 2004; Arndt et al., 2005).

The inhibition of proteasome degradation may elicit compensation by alternative protein degradation mechanisms, such as lysosomal autophagy. For example, Parkin deficiency increased the resistance of midbrain neurons and glia to mild proteasome inhibition, and led to an increase in the expression of autophagic markers (Casarejos et al., 2009). Consistent with this, inhibition of HSPC/HSP90 in non-neuronal cells activated CMA (Finn et al., 2005), and shifted HSP90 client degradation to an autophagic process (Qing et al., 2007). Together with the above-mentioned crosstalk involving UPR pathways and autophagy, these data suggest crosstalk between degradation pathways correlated with HSP activity.

3.1.3. Autophagy

Autophagy is the controlled and intended degradation of proteins and/or subcellular organelles by lysosomal proteases. While several distinct autophagic pathways exist under different molecular and environmental contexts, it appears that significant levels of crosstalk and shared components connect the individual autophagic mechanisms and allow compensation in the face of failure of individual pathways.

Of these pathways, chaperone-mediated autophagy (CMA) has been generally understood to be the constitutive autophagic process present under normal conditions in mammals (Majeski and Dice, 2004), although it is also inducible under cellular stress (Kiffin et al., 2004). CMA is a selective process where proteins are removed from the cytosol and delivered to the lysosome for degradation. Mediated by a chaperone/co-chaperone complex containing HSPA8/Hsc70 and several other chaperones, including HSP90 (Finn et al., 2005), CMA involves the recognition by HSPA8/Hsc70 of proteins containing a KFERQ-like pentapeptide (Figure 2C). Once bound, HSPA8/Hsc70 shuttles the protein to the outer lysosomal membrane, binds to the lysosome-associated membrane protein 2 (LAMP2A) which is complexed with HSPC/HSP90 (Bandyopadhyay et al., 2008), and the substrate protein to be degraded is unfolded and transported into the lysosome with the help of the lysosomally expressed HSPA8/HSC70 inside the lumen (Agarraberes et al., 1997).

The involvement of CMA in neuronal stress has only recently been investigated, and will be discussed in a more disease-specific manner below. Impairment of CMA likely increases susceptibility to any cellular stressor (Massey et al., 2006). Consistent with this concept, recent studies have implicated inhibition of CMA in synucleiopathies (Martinez-Vicente et al., 2008; Xilouri et al., 2009; Yang et al., 2009). CMA appears to be a major degradation mechanism of wild-type alpha-synuclein, both endogenous and exogenously overexpressed, in neuronal cultures (Vogiatzi et al., 2008). Overexpression of mutant alpha-synuclein inhibited CMA and led to cellular toxicity in both PC12 and SH-SY5Y neuronal cell lines (Xilouri et al., 2009). Likewise, dopamine-modified alpha-synuclein inhibited CMA (Martinez-Vicente et al., 2008), leading to the possibility that impaired CMA may be involved in the pathogenesis of PD. Indeed, impairment of CMA leads to the accumulation of myocyte enhancer factor (MEF2D), a transcription factor necessary for neuronal survival (Yang and Mao, 2009); accumulation of MEF2D has been observed in mouse models and human PD brains.

In addition to CMA, macroautophagic processes may also utilize HSPs to degrade larger cellular constituents. The activation of macroautophagy is often associated with a cell stress response, as it represents a way to degrade and recycle large portions of the cytoplasm and/or organelles, but may also be a mechanism for clearance under normal conditions in some settings. The molecular mechanisms of macroautophagic processes are just beginning to emerge, and have recently been found to include the HSPB family via the interaction with the non-HSP co-factor Bag-3 (Figure 2D). Briefly, the current evidence suggests that either HSPB8 or HSPB6 binds to substrate proteins and subsequently complexes with Bag-3 (Fuchs et al., 2010). The bound Bag-3 then is proposed to recruit and stimulate local autophagic machinery (Carra et al., 2008b). However, the details of how HSPB proteins specifically recognize its substrates are still unknown, as is the recruitment of Bag-3.

Crosstalk between the above-mentioned protein degradation pathways has been proposed on several fronts. Regarding polyubiquinated proteins targeted for degradation, the interplay between the proteasomal system and macroautophagy appears to involve at least in part a switch from Bag-1-associated proteasomal dominant activity to Bag-3 macroautophagy recruitment (Gamerdinger et al., 2009). Inhibition of CMA via LAMP2A downregulation has also been demonstrated to stimulate macroautophagy (Massey et al., 2006). Interestingly, the perceived crosstalk between autophagic processes may be bidirectional, as blocking macroautophagy by the genetic deletion of a critical component of the autophagosome increased CMA activity (Kaushik et al., 2008).

3.1.4. Protein aggregation and disaggregation

The aggregation of misfolded proteins is a hallmark of many neurological disease states (see below), including neurodegenerative diseases and prion infection where large masses of protein aggregates form, and acute oxidative insults such as stroke, where damaged proteins and mRNA from the translational complex form intracellular stress granules (Jamison et al., 2008; Liu et al., 2005a). The accumulation of unfolded proteins can lead to the unfolded protein response (UPR, see above) and activation of ER-associated degradation (ERAD) (Friedlander et al., 2000; Travers et al., 2000), resulting in the degradation of ER proteins using the proteasome system (Hiller et al., 1996). In this context, the proteasome system may become overloaded, and protein aggregation and/or cell death is thought to arise.

The consequential effect of intracellular aggregates is still debatable. On one hand, aggregation of proteins may represent a means by which the cell can safely store or sequester misfolded proteins to avoid toxic gain-of-function mutations. This is supported by observations that aggregates can exist in cells that continue to survive (see below). However, large accumulations of aggregates or inclusions may also inhibit normal cellular functions, including trafficking or intracellular communication, due to macromolecular crowding. Despite the debate on the consequences of protein aggregation, HSPs – both in the chaperone and stress-inducible contexts – have been associated with aggregation. We will present disease-specific associations in sections below, but will take the opportunity here to present ideas on the possible function of HSPs in the context of aggregation.

The HSPB family is perhaps the best described in terms of preventing aggregation of substrate proteins, but several other HSP families may also have effects on aggregate formation or cellular toxicity in aggregate disease. HSPB proteins were found to prevent aggregation of actin, polyglutamine, beta-amyloid and alpha-synuclein (Carra et al., 2005; Hayes et al., 2009; Lee et al., 2006b; Outeiro et al., 2006; Pivovarova et al., 2007). In cultures, overexpression of DNAJ prevented aggregation of polyglutamine repeats. On the other hand, overexpression of HSPA members does not appear to robustly suppress aggregates compared to other HSPs, despite the observation that HSPA proteins could suppress toxicity in aggregate disease models (Jana et al., 2000). Thus, the cellular effects of HSPs on aggregate disease may be molecule-dependent, in that one subset of HSPs may block aggregate formation whereas another subset may block toxicity.

Interestingly, the connection between aggregate formation and HSPs may also lie in the induction of HSP protein expression. Formation of stress granules – aggregates comprised of protein and RNA – has been associated with heat shock (Jamison et al., 2008). Stress granules often contain HuR, a protein that recognizes AU-rich elements (AREs) in mRNA. Unbound AREs cause rapid mRNA turnover, but when bound to HuR, the mRNA is stabilized to promote protein synthesis. HSPA contains ARE sequence (Laroia et al., 1999), and the induction of HSPA protein expression following stress correlates with the recruitment of HuR to stress granules (Jamison et al., 2008). This suggests a stress aggregate mechanism to specifically recruit and induce an HSP response.

In terms of disaggregation of already-formed aggregates, recent studies are borrowing a unique HSP member from yeast mitochondrial chaperone systems. A yeast mitochondrial chaperone protein, HSP100/mtHSP78/HSP104, not only contributes to the degradation of foreign and misfolded proteins in matrix, but is also involved in the disaggregation of denatured proteins under stress, previously thought not possible in cellular conditions. HSP100/mtHSP78/HSP104 cannot refold misfolded proteins by itself; instead, its chaperone activity focuses more on the disaggregation of proteins, and partly relies on the reactivation of mitochondrial HSP70 (Ssc1p) machinery for completion of native folding ((Krzewska et al., 2001). There is no currently known homologue of HSP100/mtHSP78/HSP104 in the mitochondria of mammals. However, recent studies overexpressing the yeast genes HSP104 in mammalian neurons have suggested that this heat shock protein family may be able to reduce aggregated proteins in neurological disorders associated with the accumulation of protein aggregates (Lo Bianco et al., 2008; Vashist et al., 2010).

3.2. Chaperoning vesicles: Synaptic transmission and endosomal function

Members across several classes of HSPs have been implicated in the sustenance of synaptic function beyond general roles in cell survival and maintenance. The function of HSPs at the synapse is underscored by the observation that HSPs may be involved in several neurodegenerative diseases involving axonal degeneration as a primary feature (described below). Accordingly, both the HSPA and DNAJ families are highly involved in synaptic transmission, clathrin uncoating and endosomal trafficking.

Intracellular vesicular storage of neurotransmitters is integral in neuronal synaptic function. The presynaptic terminal contains vesicular structures that are preloaded with neurotransmitters in preparation for synaptic transmission, and specific transporters control the contents of these vesicles. Supporting a role for HSPA members, HSPA8/Hsc70 and HSPA1/HSP70 interact with vesicular transporter in neurons (Figure 3). The interaction of HSPA with the GABA transporter is thought to provide linkage between GABA synthesis and packaging via recruitment of synthesis enzymes to the transporter (Hsu et al., 2000; Jin et al., 2003). Interestingly, HSPA1/HSP70 and HSPA8/Hsc70 co-localize with VMAT2 in DA neurons within processes and are present in the same fractions as synaptic vesicles (Requena et al., 2009). Using a recombinant in vitro system, HSPAs can bind to and inhibit the activity of the vesicular monoamine transporter-2 (VMAT2) (Requena et al., 2009). This interaction requires both substrate binding domain and C-terminal domain. The functional implications of HSPs on vesicular storage are still unclear, but may be useful in elucidating the mechanisms of vesicle loading and stress response or altered neuronal environments such as in addiction or degeneration.

Figure 3
Synaptic functions of HSP family members

During synaptic transmission of vesicular contents, such as neurotransmitters, HSP proteins play critical roles. DNAJC5/CSPα participates in the assembly of the SNARE complex, which mediates vesicular fusion to the synaptic membrane, by binding with syntaxin, HSPA, and G-proteins (Figure 3) (Evans et al., 2001). In this process, DNAJC5/CSPα anchors onto the synaptic vesicle membrane (Chamberlain and Burgoyne, 1998; Greaves and Chamberlain, 2006; Gundersen et al., 1994) and recruits several co-factors. When bound to a complex including HSPA8/Hsc70 and Hip, hydrolysis of ATP occurs on HSPA8 and assists in the coordination of synaptic transmission, although the exact mechanism is still controversial. However, the functional significance of DNAJC5/CSPα in synaptic transmission has been established using mouse models. Deletion of DNAJC5/CSPα causes a degenerative and fatal sensorimotor phenotype with impaired synaptic transmission that does not appear to be caused by initial synaptic formation (Fernandez-Chacon et al., 2004). Thus, although DNAJC5/CSPα is critical for regulated synaptic transmission, it is likely required for maintaining synaptic function rather than the initial formation of the synapse itself. The impairment of synaptic transmission in DNAJC5/CSPα-deficient mice was partially reversed by alpha-synuclein overexpression (Chandra et al., 2005), suggesting that DNAJC5/CSPα and alpha-synuclein coordinate under normal conditions in the synapse. It is thus this interaction that may be involved in the dysregulation of neurotransmission present in synucleopathies.

After the release of vesicular contents into the synaptic cleft, the fused vesicular membrane is reformed and recycled via clathrin-mediated endocytosis. During this process, the plasma membrane invaginates and buds via binding of AP2 and clathrin and recruitment of dynamin. HSPA and DNAJ members coordinate subsequent clathrin uncoating and disassembly of membrane structures within the synapse to complete the reformation of functional vesicles (Chappell et al., 1986; Ungewickell et al., 1995; Ungewickell and Hinrichsen, 2007). The recycled clathrin-coated vesicle is recognized and bound by HSPJC6/auxilin and recruits HSPA8/Hsc70 for the removal of clathrin monomer (Holstein et al., 1996; Umeda et al., 2000), in turn facilitating the disassembly of the clathrin cage that forms the scaffold of synaptic vesicles during endocytosis (Eisenberg and Greene, 2007; Ungewickell and Hinrichsen, 2007). Similar to DNAJC6/auxilin, the brain-specific DNAJC26/GAK functions with HSPA/HSP70 and assists in the disassembly of the clathrin scaffold of vesicles from the Golgi apparatus in brain (Greener et al., 2000; Korolchuk and Banting, 2002).

In addition to a role in removing clathrin-coated vesicles from the plasma membrane and Golgi, the J-domain protein DNAJC13/Rme-8 is localized to endosomes (Figure 3). Several studies have indicated that DNAJC13/Rme-8 is required for endocytosis and endosomal trafficking (Chuang et al., 2002; Girard et al., 2005; Silady et al., 2008; Zhang et al., 2001), although the interacting target for DNAJC13/Rme-8 is as yet uncertain. Demonstrating its function with endosomes, alteration of the endogenous level of Rme-8 can change the level of epidermal growth factor receptor via changes in its trafficking through early endosomes (Girard et al., 2005). However, the mechanism by which DNAJC13/Rme-8 alters endosomal trafficking is not completely understood.

Besides contributing to the neuronal side of synaptic connections, HSPs on the postsynaptic muscle terminal may contribute to motor nerve reorganization. DNAJA3/Tid1 has recently been identified as a J-domain protein critical to acetylcholine receptor (AChR) clustering in the postsynaptic muscle fiber (Figure 3) (Linnoila et al., 2008). When knocked down, absence of DNAJA3/Tid1 resulted in the dispersion of motor neuron terminals, suggesting that postsynaptic heat shock proteins may contribute to formation of functional NMJ. A splice variant of DNAJA3/Tid1 was cloned from a fetal brain cDNA library, and hippocampal cultures expressed endogenous DNAJA3/Tid1 (Liu et al., 2005b), but the possible effects of Tid1 in the brain are unknown.

While there currently is no functional evidence for HSPB members in the synapse, there is an emerging possibility that HSPB proteins may be present in synaptic regions and that exogenous expression of HSPBs in neurons may cause morphological alterations. This is consistent with the concept that HSPB1 interacts with actin and contributes to the cytoskeleton structure (Pivovarova et al., 2007). Overexpression of HSPB1/HSP27 in cultured cortical neurons increased neurite growth (King et al., 2009). Following hyperthermia, HSPB1/HSP27 and HSP32 localize to glial radial fibers in rat brain, proximal to synapse-rich regions (Bechtold and Brown, 2000). Furthermore, mutations in HSPB1 and HSPB8 have been associated with the neuromuscular degenerative disease Charcot-Marie-Tooth syndrome (described below). Although none of these findings can establish that HSPB family members are involved in synaptic function, the localization and cytoskeletal interactions of HSPB proteins make it tempting to investigate this possibility.

3.3. Maintaining the mitochondrial microcosm

In contrast to other stress-inducible HSP family members, the mitochondrial heat shock proteins, HSPA9/mortalin, HSPD1/HSP60 and HSPE1/HSP10, are constitutively expressed and function as molecular chaperones that facilitate the mitochondrial protein import and folding systems to maintain the functional activities of healthy mitochondria.

3.3.1. Import and quality control

Although mitochondria have their own protein translation system, the majority of mitochondrial proteins are encoded by nuclear DNA, synthesized in cytosol and then imported into mitochondria. This process involves two channels, the translocase of outer membrane (TOM) and translocase of inner membrane (TIM), and the unfolding and subsequent refolding of mitochondrial proteins. In this process, cytosolic HSPA members recognize mitochondrial-targeted proteins and transport them to the outer membrane complex, TOM (Figure 4). The protein is unfolded as it passes through the complex, and transferred to and shuttled through the inner membrane complex, TIM. HSPA9/mortalin interacts with the matrix side of Tim-44 and Tim-23 to facilitate the import of nuclear encoded mitochondria proteins. In addition to facilitation of protein import into the matrix, HSPA9/mortalin also initiates proper folding of imported proteins, releasing the protein substrate to be further folded by HSPD1/HSP60, a critical component of the matrix protein folding system that assists in the maintenance of mitochondrial proteome integrity (D’Silva et al., 2004; Manning-Krieg et al., 1991; Schneider et al., 1994).

Figure 4
Mitochondrial chaperone-mediated protein import and folding

HSPA9/mortalin is considered to be the core factor of mitochondrial transport machinery while HSPD1/HSP60 is the major player in protein folding in the matrix (Figure 4). The cooperation between the HSPA and HSPD families within the mitochondria is important in the prevention of accumulated misfolded proteins and in the maintenance of mitochondrial function. Oxidative stress results in the deficiency of ATP that is necessary for mitochondrial chaperone (HSPA9/mortalin and HSPD1/HSP60) activities. Similar to the UPR in the ER, accumulation of misfolded proteins in the mitochondria may elicit a similar mitochondrial unfolded protein response (mtUPR) (Tatsuta, 2009). For example, oxidative stress or abnormalities of HSPA9/mortalin or HSPD1/HSP60 will induce the accumulation of misfolded proteins and lead to the transactivation of genes with a heat shock element (HSE). Though the targeting of HSPD1/HSP60 to unfolded proteins alleviates stress-induced damage, the increased affinity of HSPD1/HSP60 with target substrates exacerbates the accumulation of misfolded proteins due to depletion of available HSPD1/HSP60 to deal with incoming translocated proteins. While “mtUPR” remains a descriptive title, it would be interesting to determine potential mechanistic signaling arms and a possible role in mitochondrial cell stress and death signaling.

The functional protein import and folding systems comprised of chaperones are critical to the health and maintenance of mitochondria. Without proper import of nuclear-encoded genes, mitochondrial processes and cellular energy production are impossible. Thus, the mitochondrial HSPA/HSP70 machinery, the HSPD/HSPE machinery, and – at least in yeast – HSP100/HSP104 should play important roles in mitochondria biogenesis, protein refolding, protein degradation, and in the protection of proteins against stress (Tatsuta, 2009). Aberrations in the chaperone function lead to a mitochondrial-UPR (mtUPR), triggering pathways as yet unknown to signal cell stress. Even though the yeast HSP100 and HSPA system cannot prevent the denaturation of matrix proteins under stress, their cooperation efficiently preserves mitochondrial DNA synthesis and mediates the reactivation of stress-denatured proteins (Germaniuk et al., 2002; Krzewska et al., 2001). Further studies are critical in understanding the role of chaperones in mitochondrial dysfunction.

3.3.2. Biogenesis

Currently, the biochemical presence of HSPD1/HSP60 associated with survival populations and aging is viewed as a marker of mitochondrial biogenesis, and HSPA9/mortalin is likely to be essential in biogenesis in light of its critical role in nuclear-encoded protein import. HSPD1/HSP60 expression increased in correlation with an increased number of mitochondria following hypoxia/ischemia in neonatal rats (Yin et al., 2008). Knockdown of the C. elegans homologue of HSPA9/mortalin produced a phenotype with characteristics of progeria, or premature aging (Kimura et al., 2007). The exact molecular underpinnings in initiating and orchestrating mitochondrial biogenesis are far from understood, and at this point, the role of HSPs in mitochondria may be either epiphenomenal or simply a function of the critical nature of the protein to mitochondrial viability. While mitochondrial biogenesis and cellular survival are likely to be correlated, it may be interesting to see if HSPs play a functional role in triggering and mediating mitochondrial biogenesis and the consequential effect on cellular survival, rather than simply being assigned a role as an epiphenomenon.

3.4. Nuclear functions

The nuclear localization of several heat shock proteins – most notably HSPC/HSP90 – has uncovered roles as transcriptional elements as well as other functions for HSPs in the nucleus.

3.4.1. Disinhibition of heat shock factor-1 and the heat shock response

The heat shock response (HSR) leads to transcriptional upregulation of molecular chaperones and related elements, such as the ubiquitin–proteasome system. Although originally discovered as a response to thermal stress, HSR is triggered by a variety of stress conditions that interfere with folding and result in accumulation of misfolded or aggregated proteins (Liu and Chang, 2008). HSR is mediated by the heat shock factor-1 (Hsf1) transcription factor. The mechanism of control of Hsf1 is a chaperone-mediated model whereby a multichaperone complex interacts with Hsf1 and prevents its transcription activity. This complex is under the negative control of HSPC/HSP90, as treatment with HSP90 inhibitors was reported to enhance Hsf1 binding to HSE, and to induce HSPs, including HSPA/HSP70, DNAJ/HSP40 and HSPB/HSP25, in neurons (Lu et al., 2002; Shen et al., 2005a; Taylor et al., 2007). The proposed mechanism of HSPC/HSP90 inhibition of Hsf1 is twofold. First, HSPC/HSP90 is thought to tether Hsf1 in the cytosol, preventing its function as a transcription factor (Zou et al., 1998). Second, HSPC/HSP90 itself can translocate into the nucleus, where it can associate with the multichaperone complex and inhibit activation of DNA-bound Hsf1 (Guo et al., 2001; Taylor et al., 2007). These data indicate that HSP90 plays a gene-regulation role at the nuclear level by affecting DNA binding and transcriptional activation of client proteins.

Furthermore, HSP90 has been shown to interact with telomeres and is required for their function (Dezwaan and Freeman, 2008; Zhao and Houry, 2005). Telomeres are specialized nucleoprotein complexes that serve DNA end protection and linear DNA length maintenance. It is suggested that HSP90 is involved in the formation and maintenance of a certain state of the chromatin. HSP90 has also been found to play an essential role in chromosome segregation during mitosis (Zhao and Houry, 2005).

3.4.2. Transcriptional co-activation

In addition to nuclear localization of HSPC/HSP90, other HSPs, including HSPB, have been found localized in the nucleus. The functional significance of HSPB1/HSP27 in neurons is typically focused on its cytoplasmic localization. Although the expression of HSPB1/HSP27 is low in neurons, its forced overexpression is neuroprotective against a variety of insults, including cerebral ischemia (Stetler et al., 2008), beta-amyloid (King et al., 2009), and polyglutamine expansions (Kadin and Said, 1990). Interestingly, nuclear accumulation of HSPB family members has been observed in several cell types: (Bryantsev et al., 2007); and (den Engelsman et al., 2005), and was recently described in models of SCA17 (a polyglutamine repeat neurological disorder) following forced overexpression (Friedman et al., 2009). In the polyglutamine repeat model, HSPB1/HSP27 physically interacted with the transcription factor Sp1 and was associated with activation of gene transcription (Friedman et al., 2009). The functional consequence of the nuclear localization of HSPB is still unsettled, although it appears to be independent of the classical chaperone function as the phosphorylated form of HSPBs are nuclear-targeted (Bryantsev et al., 2007; den Engelsman et al., 2005)). The non-chaperone (i.e., phosphorylated form) and possible nuclear function of HSPB remains an interesting study.

Similar to HSPB, the beta splice variant of HSPH1/HSP105 translocates to the nucleus following stress (Saito et al., 2009). In non-neuronal cells, this translocation is associated with the induction of HSPA/HSP70 expression via the phosphorylation and translocation of signal transducer and activator of transcription (STAT)-3 (Saito et al., 2009; Yamagishi et al., 2009). Taken with the above data, a novel role for HSPs in transcriptional co-activation appears plausible.

3.5. Bringing it all together: HSPs as modulators of protein signaling

Intracellular signaling involves complex cascades that are regulated not only by post-translational modifications but also by tethering and scaffolding molecules that can sequester or gather signaling molecules. As discussed above, HSPs are often observed as scaffolding elements in the cell, and thus the presence of HSPs in signaling protein complexes is not surprising. However, the functional role and contribution in terms of pathology is still under investigation.

3.5.1. Protein scaffolding

One family of major adaptor molecules in intracellular protein signaling is the 14-3-3 group of proteins. 14-3-3 proteins bind a diverse array of membrane receptors, kinases, phosphatases and cell death signaling molecules. Recently, HSP family members have been found to interact with 14-3-3 proteins under both normal and pathological conditions. As discussed above, DNAJC5/CSPα plays a critical but as yet unidentified role in the coordination of neurotransmitter release and synaptic function. 14-3-3 proteins have also been postulated to be involved in synaptic transmission and exocytosis (Broadie et al., 1997). Brain 14-3-3 isoforms were recently identified as binding partners of a phosphorylated form of DNAJC5/CSPα (Prescott et al., 2008), suggesting that this interaction may have implications in synaptic transmission regulation.

Both HSPD1/HSP60 and 14-3-3zeta colocalize with mitochondria (Reading et al., 1989; Wang et al., 2009), and HSPD1/HSP60 binds to 14-3-3zeta in neuronal cells (Satoh et al., 2005). The impact of this association is unknown, but the localization of 14-3-3zeta in the mitochondria has been suggested to activate tyrosine hydroxylase and stimulate dopamine synthesis within the mitochondrial compartment (Wang et al., 2009).

In non-neuronal systems, functional interactions between 14-3-3 and HSPs have also been observed. 14-3-3gamma can associate with HSPD1/HSP60 or phosphorylated HSPB6, but the implications are unknown. 14-3-3gamma is predominantly cytoplasmic (Satoh et al., 2005), suggesting that its interaction with HSPD1/HSP60 is independent of HSPD1’s role in the mitochondria. Thus, HSP association with the 14-3-3 family may have different functional implications based on intracellular locality.

14-3-3zeta may be induced by HSF under thermal stress and cooperate with HSP40 and HSP70. In this context, it appears to potentially solubilize aggregated proteins in the presence of HSP40 and HSP70, analogous to the yeast HSP100 family (Yano et al., 2006). The implications of aggregate solubilization in neuronal disease could be exciting if these findings are extended into neuronal contexts.

3.5.2. Protein kinases

In addition to adaptor molecule interactions, HSPs have well-described direct interaction with kinases. A number of protein kinases depend on interaction with HSP90 for stability and function. In neuronal cells, HSPC/HSP90 has been found to interact with various protein kinases, including Akt (Cen et al., 2006), wide-type and mutant LRRK (Wang et al., 2008a), GRK3 and GRK2 (Salim and Eikenburg, 2007), MLK3 and Src (Wen et al., 2008a). Enhancement of the association between HSP90 and the protein kinases MLK3 and Src was observed following global cerebral ischemia (Wen et al., 2008a). The consequences of the interaction between HSP90 and target kinases are likely context-specific. For example, HSP90 association led to an increase in Akt activation (Cen et al., 2006) but a decrease in Src activity (Wen et al., 2008a) by altering the phosphorylation level. HSP90 was also demonstrated to interact with the cofilin phosphatase chronophin (CIN) in primary neurons. During anoxic stress, attenuated interaction between CIN and HSP90 enhanced cofilin dephosphorylation and consequent cofilin/actin rod assembly (Huang et al., 2008).

HSPA and HSPB members also interact with and affect the mitogen-activated protein kinase (MAPK) pathway (Figure 5). ASK1, an upstream signaling MAPK kinase kinase of MAPK kinase4/7 and the c-jun N-terminal kinase (JNK), is capable of interacting with and being regulated by HSPB1 in neurons (Stetler et al., 2008). Using cortical neuronal cultures and a purified recombinant system, activated (i.e., phosphorylated) ASK1 physically interacted with HSPB1, leading to an inhibition of ASK1 activity and activation of downstream kinases. In a recombinant system and non-neuronal cells, HSPA/HSP72 also binds and inhibits both ASK1 (Park et al., 2002) and JNK (Park et al., 2001). While ASK1 signaling pathways are often described as “pro-death,” the PI3K/Akt kinase cascade is thought to be a “pro-survival” arm of kinase signaling. In some model systems, HSPB1 has also been found to interact with and increase Akt activation (Konishi et al., 1997; Mearow et al., 2002; Nakagomi et al., 2003; Rane et al., 2003; Zhang and Shen, 2007), possibly via a scaffolding function allowing the association of the upstream activator MK2 with Akt (Wu et al., 2007). Interestingly, in the context of cerebral ischemia, exogenous expression of HSPB1 was neuroprotective independent of Akt activity, suggesting that HSPB1 may function via different pathways in different contexts in the brain. Thus, HSPB1 may have a pluripotent nature that can either suppress deleterious kinase signaling or activate survival signaling.

Figure 5
HSP interaction with cell death signaling

In addition to direct physical association between HSPs and kinases, perturbation of HSPs may lead to altered kinase expression levels. Inhibiting HSP90 activity by GA in neural contexts significantly decreased the protein level of p-Akt (Cen et al., 2006), wide-type and mutant LRRK (Wang et al., 2008a), GRK3 (Salim and Eikenburg, 2007), c-Raf-1 (Xiao et al., 1999), and MLK3 (Wen et al., 2008a). In some cases, this down-regulation effect of GA was reported to be due to an increased proteasome-mediated degradation of these client proteins (Salim and Eikenburg, 2007; Wang et al., 2008a).

The multiple interactions between HSPs and protein kinases further underscore the pleiotropic nature of HSPs in cellular function.

3.6. Running interference: Chaperones tackle cell death machinery

In the past few years, more conclusive evidence has suggested HSPs function in neuroprotection independent of their traditional roles in protein folding. Although the exact mechanism of action appears to be highly cell type- and context-dependent, it appears now that multiple HSP families can specifically target cell death machinery and signaling to exert neuroprotective functions under stress.

Cell death signaling involves the activation or suppression of multiple upstream pathways, and then converging on so-called ‘apoptotic’ pathways that ultimately trigger cell death. Many of these pathways include the release of mitochondrial proteins into the cytosol, including cytochrome c and apoptosis-inducing factor (AIF). The release of these proteins is associated with the convergence of pro-apoptotic members of the bcl-2 family, such as Bax or caspase-8 cleaved Bid, on the mitochondria that are controlled by various upstream signaling pathways. HSPA/Hsp70 with its co-chaperones DNAJB1/Hdj-1 can directly associate with Bax (Gotoh et al., 2004), or indirectly suppresses Bax or Bid translocation via inhibition of pro-death kinases, such as ASK1 and JNK (Gabai et al., 2002; Park et al., 2002; Park et al., 2001; Stankiewicz et al., 2005) (Figure 5). Similarly, HSPB/Hsp27 may also inhibit upstream of Bid or Bax translocation via indirect mechanisms, such as cytoskeletal stabilization (Paul et al., 2002) or direct inhibition of ASK1 (Stetler et al., 2008) or association with prodeath molecules such as the death domain associated protein, DAXX (Charette et al., 2000). HSPC/Hsp90 may also function upstream of effector molecules by the inhibition of caspase-2 activation (Bouchier-Hayes et al., 2009). Recently, HSPC In addition to predominant localization in mitochondrial, HSPD1/HSP60 may form a macromolecular complex with Bax and Bak in the cytosol (Gupta and Knowlton, 2002). During hypoxia and ATP depletion in myocytes, cytosolic HSPD1/Hsp60 moves from cytosol to plasma membrane, coincident with the redistribution of Bax from cytosol to mitochondria (Lin et al., 2007), suggesting that the interaction between HSPD1/Hsp60 and Bax in the cytosol is associated with protection.

Instigation of mitochondrial cell death-signaling results in the release of cytochrome c into the cytosol, allowing it to complex with Apaf-1 and recruit the zymogen form of caspase-9. Upon recruitment, caspase-9 is altered to an enzymatically active form, which can in turn cleave and activate caspase-3, the quintessential executioner enzyme in classical apoptotic settings. Direct interaction between HSPs and apoptotic machinery has also been described (Figure 5). HSPB1/Hsp27 can interact with the procaspase-3, inhibiting its activation (Concannon et al., 2001; Pandey et al., 2000a)), and may directly interfere with the formation of the apoptosome complex via interaction with cytochrome c (Concannon et al., 2001; Garrido et al., 1999; Samali et al., 2001). HSPA/Hsp70 and HSPC/Hsp90, on the other hand, complex with Apaf-1 to inhibit apoptosome formation (Pandey et al., 2000b; Saleh et al., 2000), but HSPA may also directly associate with and inhibit activation of procaspase-3 (Komarova et al., 2004).

As an alternative mechanism to caspase activation, release of AIF into the cytosol leads to a caspase-independent execution pathway of cell death. The overexpression of HSPA appears to inhibit AIF-dependent cell death in several ways. HSPA inhibited upstream of mitochondrial AIF-leakage via suppression of Bax activation (Ruchalski et al., 2006). HSPA also binds directly to AIF, sequestering leaked AIF in cytosol in order to block its translocation to nucleus (Gurbuxani et al., 2003; Ruchalski et al., 2006).

These actions of HSPs on cell death signaling may be highly model-specific. There exists a significant amount of conflicting evidence on the HSP domains required for interaction with cell death signaling molecules, reflecting cell- or model-specific nuances, or experimental method (Chow et al., 2009). Many of these findings have not been replicated across a broad range of cell types or cell-death challenges; therefore the general applicability of these observations and extension to neuronal cell death is lagging. In the following section, we will describe neuronal disease-specific contexts and involvement of HSPs.

4. HSP functions in the context of neurological diseases

4.1. Acute injury states: Cerebral ischemia and epilepsy

4.1.1. Cerebral ischemia – HSP expression

The loss of blood flow to and subsequent reperfusion of neural tissue elicits a complex, multicellular pathophysiological state. Cerebral ischemic insults incur an array of cellular damage, ranging from acute excitotoxic stress to delayed programmed cell death. In addition, the induction of reactive oxygen species (ROS) as well as intracellular calcium overload activates numerous intracellular stress signaling pathways, leading to rapid and compensatory response mechanisms. Thus, the pathophysiology and subsequent role of particular proteins is complex, and likely involves the overlapping and crosstalk of multiple signaling pathways. Given the role of HSPs in a vast array of cellular functions, this wide group of proteins is well poised to transmit and coordinate stress signals following cerebral ischemia and to function in neuroprotective capacities.

Evidence for the induction of heat shock proteins following cerebral ischemia has been extensively documented and described. Expression and induction of HSPs, particularly HSPA and its associated co-chaperones, have been largely correlated with penumbral regions or in regions that ultimately survive the injury. Both mRNA and protein expression of HSPA/HSP70 were observed in resistant hippocampal regions following global ischemia (Tanaka et al., 2002) and in penumbral regions following focal ischemia (Kinouchi et al., 1993; Wagstaff et al., 1996) prior to morphological cell damage (Li et al., 1992). The co-chaperone DNAJ/HSP40 expression (mRNA, protein) overlapped with HSPA8/Hsc70 following global ischemia (Tanaka et al., 2002). ER-associated HSP members are also induced following global and focal ischemia in brain, including HSPA5/GRP78 and HSPC4/GRP94 (Nakka et al., 2010; Truettner et al., 2009). In contrast to the rapid transcription of cytosolic HSPA family members, HSPA5/GRP78 and HSPC/GRP94 display a delayed induction at the mRNA level following global ischemia (Truettner et al., 2009). Consistent with the expression of HSPs in penumbral or resistant regions in injurious ischemic models, HSP induction has also been observed in sublethal ischemia models such as preconditioning. Induction of HSPA/HSP72 was observed in cortical neurons by focal ischemic preconditioning stimulus alone in a timeframe consistent with the tolerant window (Chen et al., 1996a; Kato et al., 1994; Schlegel, 1976).

Expression of HSPD/HSP60 and HSPE/HSP10 is increased after brain damage, such as in the brain stem after subarachnoid hemorrhage, forebrain or focal cerebral ischemia, and neonatal hypoxia-ischemia (Hwang et al., 2007; Izaki et al., 2001; Okubo et al., 2000; Satoh et al., 2003; Yin et al., 2008). Furthermore, increased HSP60 concentration was found in the cerebrospinal fluid of pediatric TBI patients, and the peak concentration correlated with the severity of injury (Lai et al., 2006). Compared with the early induction of cytosolic HSPA/HSP72, the induction of HSPD/HSP60 is delayed and sustained in injured regions (Izaki et al., 2001; Okubo et al., 2000; Truettner et al., 2009) The continued presence of induced HSPD/HSP60 in injured cells suggests that mitochondrial stress overlaps cellular stress in ischemic models (Izaki et al., 2001; Okubo et al., 2000), or may indicate alternative functions of HSPD/HSP60 in stressed cells.

In addition to neuronal HSP induction, reactive astrocytes exhibit strong induction of HSPB following sublethal ischemia (Kato et al., 1994; Schlegel, 1976; Wagstaff et al., 1996) as well as delayed induction of HSPD/HSP60 following global ischemia. Similar results were observed in other acute neuronal injury models, such as traumatic brain injury (Chen et al., 1998) and epilepsy (Akbar et al., 2001), although the latter in particular demonstrated induction in regions sensitive to infarct as well as in resistant regions (Yang et al., 2008).

Much of the induction of the heat shock response appears to involve upregulation at both the transcriptional and translational levels (Wagstaff et al., 1996). The HSPC/HSP90 inhibitor GA leads to the release of HSF1, which is then able to bind to HSE elements present in inducible HSP promoter regions. Pretreatment with GA led to increased expression of both HSPA in neurons and HSPB in glia and vascular cells following MCAO, as well as decreased infarct (Lu et al., 2002). Taken together with the correlation of expression in regions that survive injury, these observations serve as a starting point for investigating the notion that HSP induction may contribute to cellular survival following acute neuronal injury, in particular ischemic injury.

4.1.2. Seizure – HSP expression

Seizure activity – typically defined as excessive and synchronous excitatory neurotransmission of cortical neurons – has also been associated with HSP induction. In particular, the classical seizure model using kainic acid induced both HSPA/HSP72 and the ER-associated HSPA5/Grp78 mRNA. Endogenous HSP induction appears to correlate as a stress response rather than as a protective mechanism, as expression of HSPs occurs in both surviving cells and cells destined to die. Furthermore, knockdown of HSPA/HSP70 did not appear to have an effect in vitro against kainic acid (KA) toxicity (Yang et al., 2008). However, exogenous upregulation of HSPs may elicit neuroprotective mechanisms against degeneration subsequent to seizure activity (discussed below).

Interestingly, little is known about any possible contribution of HSP induction to febrile seizures. Febrile seizures are seemingly benign seizures induced by a sudden hyperthermic state in the immature brain in the context of an immune reaction. Unlike epilepsy, short (<10 minutes) generalized febrile seizure does not appear to associate with later development of epilepsy, brain damage or developmental delays, although longer and focal febrile seizure may have a correlation with development of temporal lobe epilepsy in the adult. Hyperthermia is the classical model of the heat shock response, and induces both seizures and HSP expression in brain. However, the induction of febrile seizure may not be synonymous with seizure modeled by elevated body temperature (hyperthermia). Rather, febrile seizure typically is associated with an immunological challenge concurrent with high fever. This presents an interesting possibility for heat shock response: the presence of an immunological challenge may activate stress proteins (or other molecules) that function to buffer the hyperthermic seizure state in the immature brain. Given the recent developments linking HSPs to regulation of inflammatory signals, febrile seizures may present an interesting model in which to explore HSP actions.

4.1.3. Therapeutic advantages of HSPs against acute neurological injury

Exogenous overexpression of HSPs confers neuroprotection against many acute injury states. Increased HSP levels have been attained via transgene overexpression, viral transduction and protein transduction systems or via preconditioning stimuli. While many of these studies provide only histological endpoints, there are some mechanistic clues to the neuroprotection afforded by HSP overexpression. We will first discuss evidence for the therapeutic benefit of HSP overexpression in models of acute neuronal injury, and then posit potential mechanistic implications.

Overexpression of HSPA via gene transfection, induction by heat shock, preconditioning or pharmacology, virally encoded infection, protein transduction and transgene expression have consistently proven to enhance cellular protection against a variety of acute neuronal insults. Transgenic overexpression of HSP70 protected against focal and global cerebral ischemia and KA-induced seizure brain injury in adult mice (Tsuchiya et al., 2003a) and against hypoxia/ischemia brain injury in the neonate (Matsumori et al., 2005; Matsumori et al., 2006). In addition to cellular protection, protein transduction of HSP70 linked to the TAT domain increased functional outcomes and increased the number of newly generated NPC in striatum following focal ischemia (Doeppner et al., 2009). Using HSPA mutant vectors, the ATPase domain does not appear critical for cellular protection against focal ischemia (Sun et al., 2006). Interestingly, although viral overexpression of HSPA consistently increased cell survival, it did not always decrease infarct size, in part dependent on the assay used to measure infarct or the HSPA family member (Badin et al., 2006; Badin et al., 2009; Sun et al., 2006; Yenari et al., 1998). In addition, the mitochondrial-targeted HSPA, HSPA5/mortalin, may also protect neural cells from ischemic injury (Voloboueva et al., 2008; Xu et al., 2009).

HSPB and HSPD families are also neuroprotective when exogenously overexpressed in multiple models of acute neuronal injury. Transgenic overexpression or protein transduction of HSPB1/HSP27 protects against focal and global cerebral ischemia (An et al., 2008; Badin et al., 2009; Stetler et al., 2008; van der Weerd et al., 2009) and KA-induced seizures (Akbar et al., 2003; Kalwy et al., 2003). Furthermore, pre- and postischemic viral delivery of HSPB1/HSP27 was effective at reducing lesion volume following focal ischemia (Badin et al., 2006; Badin et al., 2009). Post-ischemic delivery of HSPB1/HSP27 also improved sensorimotor outcomes (Badin et al., 2009).

Overexpression of HSPD/HSP60 via adenovirus protected CA1 pyramidal neurons from ischemic damage in transient global ischemia (Hwang et al., 2007), and administration of the HSPD/HSP60 homolog GroEL reduced infarct volume and improved neurological outcome after middle cerebral artery occlusion (Xu et al., 2006). This effect was observed even with a folding-deficient mutant, suggesting that protection is not necessarily associated with co-chaperone binding. These findings argue against a deleterious role for HSPD/HSP60, which has been posited based on the prolonged expression of HSPD/HSP60 in injured regions.

4.1.4. Targets for HSP neuroprotective mechanisms following acute injury

As described previously, HSPs are involved in a multitude of critical cellular functions that promote survival under normal and stressed conditions. In the context of acute neurological injury, many of these processes are disturbed, allowing speculation that HSP neuroprotection may occur through corresponding cellular pathways. The literature thus far has only limited mechanistic studies detailing potential pathways for HSP neuroprotection in acute brain injury. Thus, we will briefly describe the possible cellular events in which HSPs could be promoting cell survival.

4.1.4.1. Protein degradation and aggregation

Cerebral ischemia has long been noted to be associated with proteasomal dysfunction and protein aggregation (Asai et al., 2002; Ge et al., 2007). The dysfunction of the proteasome and aggregation of proteins can be a consequence of ER stress, where the accumulation of unfolded proteins may lead to UPR and degradation via activation of ER-associated degradation (ERAD) (Friedlander et al., 2000; Travers et al., 2000). The activation of the heat shock response and upregulation of critical HSPs has been demonstrated to relieve ER stress in non-neuronal cells (Liu and Chang, 2008), and several studies point to the role of HSPs related to ER stress in models of cerebral ischemia.

As mentioned in the previous section, HSPs have integral roles in the targeting of proteins to the proteasome, activation of the UPR, and the prevention and recognition of protein aggregates. Indeed, protein aggregates and markers of ER stress and UPR induction, including increased HSPA5/Grp78 and ATF-6 expression and processing of the x-box binding protein (XBP1) transcription factor, are observed following cerebral ischemia (Morimoto et al., 2007; Nakka et al., 2010; Roberts et al., 2007; Truettner et al., 2009; Urban et al., 2009) and traumatic brain injury (Paschen et al., 2004; Truettner et al., 2007). Increased expression of ER proteins such as HSPA5/Grp78 is correlated with neuroprotective paradigms, such as ischemic preconditioning or neuroprotective drugs (Lehotsky et al., 2009; Urban et al., 2009). However, whether these ER stress markers are a truly functional induction of the UPR has not yet been determined. Dysfunction of the UPR could lead to the incapacity to deal with damaged protein and contribute to cellular toxicity. Thus, amelioration of the UPR has the possibility of assisting damaged cells in overcoming ER stress and avoiding cell death.

Autophagic markers have also been observed following neonatal hypoxia/ischemia and in adult models of cerebral ischemia (Carloni et al., 2008; Wen et al., 2008b) and seizure (Wang et al., 2008b). Interestingly, in the neonatal model, the inhibition of autophagy led to a switch in cell death phenotype from apoptotic to necrotic (Balduini et al., 2009; Carloni et al., 2008), but overall tissue loss was unaffected. Tissue protection was obtained, however, by pharmacological stimulation of autophagy using rapamycin (Carloni et al., 2008), suggesting that the stimulation of autophagic processes may serve neuroprotective purposes in neonatal hypoxia/ischemia (Balduini et al., 2009). To date, the role of HSPs in autophagy following acute models has not been defined, nor has the process of CMA been explored in cerebral ischemic models. Given the inclusion of ER stress and autophagic markers as well as the neurotherapeutic observations of HSP overexpression, the role of CMA and the potential involvement of HSPs in autophagic stimulation should be addressed.

4.1.4.2. Cell death signaling

Given the volumes of data underscoring the role of cell death signaling following acute neurological injury, the neuroprotective mechanisms of HSPs against cerebral ischemia and/or seizure are likely to involve the direct or indirect suppression of signaling molecules leading to cell death. Consistent with this, in various models of ischemia, transgenic overexpression of HSPA/HSP70 increased binding with Apaf-1 (Matsumori et al., 2006) and reduced cytochrome c release (Matsumori et al., 2006; Tsuchiya et al., 2003b). HSPA/HSP70 overexpression also led to increased binding with AIF and suppression of AIF translocation to the nucleus in several ischemia models (Matsumori et al., 2005; Sun et al., 2006). HSPB/HSP27 has also been observed to block cell death signaling in acute neurological injury models. In particular, HSPB/HSP27 physically interacts with the upstream kinase ASK-1 after ASK-1 has been activated (Stetler et al., 2008). This interaction then suppresses ASK-1 activity, blocking the activation of pro-death JNK kinases and mitochondrial cell death signaling. While this mechanism is upstream of mitochondrial cell death signaling, it is possible that HSPB1/HSP27 may also function via direct interaction with cell death molecules if overexpressed at later stages in pathology. In contrast to HSPA and HSPB actions, HSPC/HSP90 bound to and promoted the activation of the mixed lineage kinase-3 (MLK3) and Src, which led to activation of ASK1/JNK signaling pathways (Wen et al., 2008a). Pharmacological inhibition of HSPC with GA led to decreased cell death signaling and increased Akt activation, although this could also be due to consequential upregulation of HSPA following GA administration.

The targeting of HSPs in acute neurological injury thus presents a possible multilayered approach to neurotherapeutics. Detailed mechanistic explorations into the protective capacities of HSPs against acute injury could feasibly lay the groundwork for development of clinically relevant therapeutic molecules.

4.2. Aggregate-associated neurodegenerative diseases

The aggregation of misfolded proteins is a hallmark of many neurodegenerative diseases, including Parkinson’s disease (PD), Alzheimer’s disease (AD), Huntington’s disease (HD), and transmissible spongiform encephalopathies (TSE, or prion disease). These diseases are marked with a long-term and specific degeneration of neuronal populations, and the etiology is poorly understood.

Along with the degeneration of neurons that occurs in brain regions specific to the neurological condition, the morphological features associated with many of these diseases often include the presence of insoluble protein aggregates. These aggregates are made up largely of misfolded and proteolytically resistant molecules, which often contain structural elements that promote the recruitment and incorporation of additional molecules, resulting in a positive feedback loop and increased aggregation. The physiological relevance of aggregates is still a matter of debate, where one theory postulates that aggregation of proteins serves as a quasi-protective function (i.e., sequestration of proteolytically resistant abnormal proteins), and the other postulates that the formation of cellular inclusions is toxic in nature. Furthermore, aggregates are by no means homogenous – in addition to involving different proteins and morphological localization, they are heterogeneous in terms of solubility even within an aggregate. Regardless of the pathological relevance, the aggregation and sequestration of misfolded proteins remain in the domain of HSPs; thus, the potential role of HSPs in aggregate-associated neurodegenerative diseases has been investigated through the years.

Several classes of HSPs have been found to colocalize with aggregate formations; whether or not these associations are causative, responsive or epiphenomenal is of debate. We will discuss the evidence of HSPs and aggregation and/or survival, bearing in mind that there is still much to explore.

4.2.1. Parkinson’s disease and synucleinopathies

Parkinson’s disease (PD) is pathologically identified postmortem as a selective loss of dopaminergic neurons in the brain, particularly those localized in the substantia nigra (SN) pars compacta, but is also associated with distal aberrations in the olfactory bulb and enteric plexus (Hawkes et al., 2009). Aggregate formation is observed in classic PD, but may be absent in cases that present with clinical symptoms identical to classic PD but upon postmortem analysis differ in morphology (parkinsonism). These aggregates (called Lewy bodies) are composed primarily of α-synuclein, thought to be modified post-translationally. The aberration of α-synuclein structure is not specific to PD, but rather leads to various synucleinopathies (Marti et al., 2003). Likewise, the majority of PD cases are idiopathic, although a small percentage have been linked to genetic or environmental sources. Despite the unknown etiology, several observations related to HSPs appear relevant in PD and/or synucleinopathology.

Several lines of evidence point to an associated loss of HSPA members in PD progression. For example, decreased expression of HSPA8/Hsc70 and other critical members of the ubiquitin-proteasome system have been associated with idiopathic PD (Mandel et al., 2005). HSPA9/mortalin expression levels are decreased in PD postmortem brain, in both frontal cortex and substantia nigra (Jin et al., 2006; Shi et al., 2008), where degeneration is most often observed. Furthermore, HSPA9/mortalin was covalently modified by dopamine following exposure of isolated rat brain mitochondria to the dopamine quinone (Van Laar et al., 2009), and protein levels were selectively decreased in cells exposed to dopamine toxicity or isolated mitochondria exposed to the dopamine quinone (Van Laar et al., 2008). Dopamine oxidation and quinone formation has been associated with PD models, and dopamine modification of HSPA9/mortalin may underscore mitochondrial dysfunction consistent with PD-related pathology.

Conversely, HSP90 expression levels are positively correlated with increased amounts of insoluble α-synuclein in PD brains and mouse models (Uryu et al., 2006). HSP90 co-localizes with α-synuclein filaments of Lewy bodies in PD (Uryu et al., 2006), and reduced phosphorylation of HSP90 has been observed in the SN of PD brains and in response to α-synuclein accumulation (Kulathingal et al., 2009). The associations of reduced HSP90 phosphorylation, α-synuclein aggregation, and HSP90 interaction with aggregates are consistent with the need for HSP90 to be phosphorylated in order to release its substrate, and indicate that HSPC/HSP90 may be unable to dissociate from aggregated proteins.

Inhibition of HSPC/HSP90 and consequential upregulation of HSPA/HSP70 using pharmacological HSPC inhibitors such as GA has consistently proved to decrease aggregate formation and inhibit cytotoxicity in models of synucleinopathies. In MES cells, GA completely blocked the recycling of extracellular α-synuclein, which has been suggested to play a role in amplifying neurotoxicity, and inhibited cytotoxicity (Liu et al., 2009). Consistent with the side effect of GA increasing HSPA expression, transgenic overexpression of HSPA/HSP70 inhibited formation of α-synuclein aggregates in vivo and decreased cytotoxicity due to α-synuclein overexpression in vitro (Klucken et al., 2004). HSPA/HSP70 directly interacts with α-synuclein prior to aggregation (Dedmon et al., 2005; Huang et al., 2006; Luk et al., 2008), suggesting that HSPA may abrogate aggregate formation by recognizing the misfolded α-synuclein protein. Brain permeable small molecule inhibitors of HSPC/HSP90, when used in culture systems, prevented α-synuclein oligomer formation and rescued α-synuclein-induced toxicity (Putcha et al., 2009). Furthermore, pretreatment with GA prevented α-synuclein aggregation in cultured cells, but GA treatment against pre-existing inclusions did not result in a reduction in the number of cells containing inclusions (McLean et al., 2004). This study indicates that HSPC/HSP90 (or inadequate levels of HSPA/HSP70) facilitates the formation of inclusions, but is not required for inclusion maintenance.

However, the relevance of aggregate formation and number to cell toxicity has been a long-standing debate. Using a Drosophila model of PD, Auluck et al. demonstrated that GA uncouples neuronal toxicity from Lewy body and Lewy neurite formation. In their study, GA protected dopaminergic neurons from the effects of α-synuclein expression, despite the continued presence of (and even increase in) inclusion pathology (Auluck et al., 2005). The possible effects of crosstalk with HSPA/HSP70 were tentatively excluded as the concentration sufficient to protect neurons against α-synuclein toxicity in flies was below the level required for HSP70 induction (Auluck et al., 2005). Further mechanistic studies demonstrated that GA sensitized the stress response within normal physiological parameters to enhance chaperone activation, and the subsequent neuroprotection required HSF (Hay et al., 2004).

Overall, although the role of HSPC/HSP90 remains unclear, the findings seem to indicate that inhibition of HSPC/HSP90 largely reduces aggregate formation and/or confers neuroprotection in models of PD. In addition to cell models using α-synuclein toxicity, mutations in LRRK2 have been linked to both familial and apparently sporadic forms of PD, and have been identified in aggregate formations in PD. LRRK2 forms a complex with HSPC/HSP90 via its kinase domain, and the disruption of the HSPC/HSP90-LRRK2 complex with GA treatment dramatically increased the proteasome-mediated degradation of both endogenous and mutant LRRK2 in neurons (Wang et al., 2008a). Thus, HSPC/HSP90 may stabilize LRKK2 protein expression, leading to its inclusion in aggregate formations.

Support for a link between decreased aggregates and neuroprotection was observed by overexpression the yeast HSP, HSP100/HSP104. As mentioned above, this non-mammalian chaperone possesses the capacity to disassemble pre-existing aggregates. Lentiviral overexpression of HSP100/HSP104 suppressed dopaminergic neurotoxicity and reduced α-synculein inclusions in rats co-injected with mutant α-synculein (Lo Bianco et al., 2008). Furthermore, in vitro data indicate that HSP100/HSP104 recognizes and remodels mutant α-synculein pre-amyloid oligomers, suggesting that this HSP may be functional at the hypothetical critical early process of amyloid generation. While the possibility remains that HSP100/HSP104 may function in neuroprotective capacities other than disaggregating inclusions, the use of this molecule in neurodegenerative diseases is emerging, and will likely yield interesting mechanistic details.

Other links between HSPs and PD have been identified, but are somewhat more nebulous. Overexpression of HSPB proteins led to protection and decreased aggregation in a culture system of α-synuclein toxicity (Outeiro et al., 2006). HSPD1 interacts with parkin, a protein that, when mutated, leads to early-onset PD (Davison et al., 2009), and may provide an interesting clue in the mitochondrial dysfunction associated with PD.

Impairment in CMA appears to be related to α-synuclein pathology (Xilouri et al., 2009; Xilouri et al., 2008), but the role of HSP-directed therapies has not yet been investigated. Consistent with this concept, recent studies have implicated inhibition of CMA in synucleiopathies (Martinez-Vicente et al., 2008; Xilouri et al., 2009; Yang et al., 2009). CMA appears to be a major degradation mechanism of wild-type α-synuclein, both endogenous and exogenously overexpressed, in neuronal cultures (Vogiatzi et al., 2008). Overexpression of mutant α-synuclein inhibited CMA and led to cellular toxicity in both PC12 and SH-SY5Y neuronal cell lines (Xilouri et al., 2009). Likewise, dopamine-modified α-synuclein inhibited CMA (Martinez-Vicente et al., 2008), leading to the possibility that impaired CMA may be involved in the pathogenesis of PD. Indeed, impairment of CMA leads to the accumulation of inactive myocyte enhancer factor (MEF2D), a transcription factor necessary for neuronal survival (Yang and Mao, 2009); accumulation of inactive MEF2D has been observed in mouse models and human PD brains (Yang et al., 2009). In order to avoid accumulation of inactive MEF2D, interactions with HSPA/Hsc70 and CMA degradation occur. Given that overexpression of HSPA was neuroprotective against α-synuclein toxicity, it would be interesting to explore whether HSPA overexpression restores CMA.

Detailed mechanistic studies, such as the involvement of autophagic processes, ER stress, cell death signaling or inflammation, and potential interplay with HSP family members, still have yet to be widely explored in PD models.

4.2.2. Huntington’s disease and polyglutamine repeat neuropathologies

The expansion of a trinucleotide repeat sequence within several genes beyond a clinically normal threshold leads to insoluble aggregate formations and associated neuropathies, including Huntington’s disease (HD) and spinocerebellar ataxias (SCA). The cause of the pathology is still under intense study, and the relevance of the aggregates to cell death has been a point of contention. The most commonly studied expansion is the CAG repeat, which leads to a translation of an extended polyglutamine (polyQ) tract and subsequent alteration of protein structure, function and interactions. PolyQ repeat neuropathies are delineated from other aggregate-associated diseases such as PD or AD due to the genetic basis of the aggregate-forming repeat. HSPs of varying families have been associated both with cytoprotection and with aggregates in polyQ diseases.

In cell culture models of HD, treatments with GA (Sittler et al., 2001) and its derivatives 17-DMAG and 17-AAG (Herbst and Wanker, 2007) were demonstrated to lead to enhanced expression of various HSPs, including HSPA/HSP70, DNAJ/HSP40, and HSPC/HSP90, and to inhibit mutant huntingtin protein aggregation. Oral supplementation with GA suppressed photoreceptor neuronal loss in a Drosophila HD model (Agrawal et al., 2005). Demonstrating that pharmacological manipulation of HSP expression may be more potent than selective transgene overexpression, Hay et al. found that constant exposure to HSPC/HSP90 inhibitors successfully induced the expression of both DNAJ proteins and HSPA/HSP70, and increased the level of soluble exon 1 huntingtin in a period of three weeks compared to the insoluble aggregates found in the absence of HSPC inhibitors (Hay et al., 2004). In contrast, HSP70 transgenic overexpression had no effect on the change in the aggregate solubility in organotypic slice culture, and did not improve the phenotype in a mouse model of HD (Hay et al., 2004). This suggests that the actions of GA are not exclusive to the induction of HSPA/HSP70.

Consistent with the induction of DNAJ proteins and coordination with HSPA family members, overexpression of DNAJ family members has been associated with protection against polyglutamine repeats. DNAJB6/Mrj paired with HSPPA/HSP70 can associate with keratin-intermediate filaments (Izawa et al., 2000; Watson et al., 2007) as well as huntingtin (Chuang et al., 2002). Over-expression of DNAJB6/Mrj attenuated cell death induced by huntingtin aggregation (Chuang et al., 2002) as well as polyglutamine toxicity (Fayazi et al., 2006), demonstrating its capacity to prevent protein aggregation. Furthermore, DNAJB1/HSP40 suppressed the pathogenicity of polyQ (Bonini, 2002). While these studies indicate that DNAJ members may attenuate polyQ pathology, it is currently unknown if the mechanism is via interaction with HSPA or HSPC family members, or if DNAJ proteins are functioning independently.

Polyglutamine-mediated cell death may also be inhibited by overexpression of HSPB1/HSP27 in a manner distinct from HSPA and DNAJ. As described above, HSPA and DNAJ overexpression attenuated both cell toxicity and polyQ aggregation. HSPB1/HSP27, however, attenuated cell death without an effect on polyQ aggregates in vitro. The effects of HSPB1/HSP27 in this case are consistent with its role in suppression of cell death (described above), but likely do not involve direct interaction with cytochrome c (Wyttenbach et al., 2002). Interestingly, the unphosphorylated form of HSPB1/HSP27 appeared to be required for the neuroprotective effects against polyQ toxicity, which is associated more with chaperone function as opposed to interaction with cell death signaling. Lentiviral overexpression of HSPB1/HSP27 protected neurons against co-overexpression of polyQ-expanded huntingtin fragment (Perrin et al., 2007). However, the neuroprotective effects of HSPB1/HSP27 were not observed in a cross between HSPB1/HSP27 transgenic mice and the R6/2 HD mouse model (Zourlidou et al., 2007), suggesting model variation that should be accounted for in further experiments.

Another HSPB member, HSPB8/HSP22, prevented the accumulation of a polyQ-expanded huntingtin fragment when overexpressed in non-neuronal cultures (Carra et al., 2005). HSPB8/HSP22 was found to interact with the non-HSP co-factor Bag-3 in the same model systems, where Bag-3 induced degradation of the polyQ-expanded huntingtin fragment (Carra et al., 2008a). HSPB8/HSP22 did not seem to be required for the increased polyQ degradation, but could nonetheless act as a chaperone in the handling of polyQ proteins.

The particular genes containing trinucleotide repeats often encode very large, multifunctional critical protein products. As such, cellular and animal models often utilize the overexpression of solely the repeat sequence due to the technical difficulties of packaging and overexpressing large sequences. Thus, while the models help identify possible avenues of research, they are often limited in extrapolation to the disease state.

4.2.3. Alzheimer’s disease and tauopathies

Alzheimer’s disease is perhaps one of the least understood prevalent neurodegenerative diseases in terms of molecular mechanisms of pathology. Stemming largely from the limited familial AD genetic associations and the observations on postmortem tissue, research has primarily focused on mutations in the amyloid precursor protein (APP) and presenilin-1 (PS1). One of the most notable hallmarks of AD is the formation of extracellular amyloid plaques, formed by the secretion of the 42-amino acid peptide (A-beta) derived from aberrant APP processing. The familial mutations in APP and PS1, when overexpressed as transgenes in mice, lead to A-beta secretion, formation of amyloid plaques and neurotoxicity consistent with AD. In addition to amyloid plaques, neurofibrillary tangles comprised largely of the tau protein are also a hallmark of AD. Originally thought to be an epiphenomenal event in response to general neurotoxicity, tau tangle formation has been highly correlated with neuronal loss and cognitive decline and is now understood to be capable of inducing toxicity, rather than being a side event (Ramsden et al., 2005). Interestingly, HSPs have been associated with multiple layers of AD-associated pathology, and thus may represent several intervention points (Koren et al., 2009), although the current mechanistic understanding of the role of HSPs in AD is still in the early stages.

As the native APP is folded in the ER, it associates with the ER-resident HSPA, HSPA5/Grp78 (Yang et al., 1998). Given that exposure of cells to A-beta elicits an ER stress response, and that mice deficient in caspase-12 (the downstream effector of ER stress) were resistant to A-beta toxicity (Nakagawa et al., 2000), the ER and ER-stress sensitive HSP response cascades may be involved in the propagation of A-beta toxicity. Overexpression of HSPA5/Grp78 decreased the aberrant processing of APP and subsequent formation of A-beta (Hoshino et al., 2007), suggesting that HSPA5/Grp78 may interact with or suppress the proteolysis of APP. However, the targeting of APP proteolysis may be insufficient to affect AD progression, as pharmacological inhibition of the initial proteolytic step in A-beta production was ineffective in clinical trials (Koren et al., 2009).

Following APP processing and production of the 1–42 A-beta peptide, the formation of A-beta fibrils occurs and is secreted into the extracellular space, eventually forming larger and more stable amyloid plaques. Cytosolic expression of HSPB members inhibited A-beta fibril formation (Kudva et al., 1997; Lee et al., 2005; Wilhelmus et al., 2006c), and overexpression of HSPB inhibited A-beta cellular toxicity in both cultured cortical neurons (King et al., 2009) and cerebrovascular cells (Wilhelmus et al., 2006a). Likewise, HSPA and HSPC proteins inhibited A-beta aggregation in a recombinant system, and overexpression of HSPA prevented A-beta toxicity in cultured neurons (Magrane et al., 2004). These data suggest that HSPs may have the capacity to function downstream of the initial pathological event (APP cleavage) to suppress formation of aggregates and neurotoxicity.

In terms of the tauopathy aspect of AD, many HSPs recognize and promote the degradation of abnormal tau. HSPB1/HSP27 bound to and promoted the degradation of abnormally phosphorylated tau in a cortical neuronal cell line (Shimura et al., 2004). In AD postmortem brain, HSPB1/HSP27 associated with neurofibrillary tangles (Nemes et al., 2004), suggesting that the recognition of abnormal tau by HSPB1 exists in human brain. HSPA8/Hsc70 co-immunoprecipitated with tau in vitro and increased its incorporation of tau into microtubules, thereby inhibiting aggregation (Dou et al., 2003). Recently, chemical manipulation of HSPA/HSP70 ATPase activity was also found to regulate tau stability (Jinwal et al., 2009). Inhibition of the HSPA ATPase activity induced degradation of tau, whereas stimulators of the ATPase activity maintained tau expression levels.

HSPC/HSP90 has the capacity to complex with mutant tau and promote tau aggregation. A recent paper reported that binding of HSP90 to tau facilitated a conformational change that resulted in its phosphorylation by glycogen synthase kinase 3 and subsequent aggregation into filamentous structures (Tortosa et al., 2009). Treatment with an HSPC/HSP90 inhibitor induced the proteasome-mediated degradation of p35 and mutant tau. Inhibition of HSPC/HSP90 in cellular and mouse models of tauopathies led to a reduction of the pathogenic activity of these proteins and elimination of aggregated tau (Luo et al., 2007). Dickey et al. reported that inhibition of HSPC/HSP90 promoted selective proteasome-dependent degradation of hyperphosphorylated tau species in a cell culture model (Dickey et al., 2006) and in a mouse model of tauopathy (Dickey et al., 2007). They further identified a constitutive chaperone complex involved in the refolding and dephosphorylation of aberrant tau protein and HSPC/HSP90 inhibitor-mediated tau degradation. It was suggested that phosphorylated tau is processed via the constitutive HSP90 refolding system rather than being dependent on de novo transcription stimulated by HSF1 (Dickey et al., 2007). Thus, abnormal tau protein may be preferentially repaired through restorative chaperone activity rather than by protein degradation pathways (Dickey et al., 2007). Consistent with this concept, inhibition of PI3K activity by LY294002 reduced Hsf1 activity but did not affect tau degradation (Dickey et al., 2008).

4.3. Prion disease (transmissible spongiform encephalopathies)

The emergence of prion disease – a transmissible spongiform encephalopathy leading to severe neurodegeneration and death – has led to surprising biological discoveries and fascinating possibilities in the function of proteins. Unfortunately, the base of knowledge concerning both the mode of transmission and the normal function of the implicated protein is limited. In humans, pathological hallmarks include spongiform alterations, neuronal degeneration, astrogliosis and, importantly, amyloid plaque formation. It is the latter of these that raised the possibility that HSPs could affect either the disease formation itself, or serve as possible therapeutic agents. While we can discuss these possibilities, much more research is necessary to fully understand the pathogenesis of prion disease as well as HSP involvement.

Transmissible prion disease occurs with the post-translational alteration of the endogenous cellular glycoprotein PrPC to a protease-resistant conformation (PrPSc) that induces further conversion of PrPC to the toxic form, providing a positive feedback loop in the formation of PrPSc. It is this positive feedback that may underlie transmission and disease progression. PrPSc recruits multiple PrPC molecules to form aggregates and is found in amyloid plaques in brain. In addition to refolding, recent work also suggests that neurotoxicity of PrP occurs following mislocalization to the cytosol. PrPC is normally localized to the ER prior to insertion into the outer leaflet of the plasma membrane, where it is thought to function in synaptic communication and cell survival. Because of the nature of the disease – i.e., protein refolding and compartmentalization – HSPs may be involved in both the propagation of prion disease and, possibly, its treatment.

The HSPA superfamily, responsible for refolding damaged proteins, and its functional co-chaperones have gained some attention in prion disease. In purified systems, the DNAJ member DNJA2/Rdj2 associates with PrPC independent of ATP, whereas DNAC5/Cspα can also bind PrPC but only in the presence of ATP (Beck et al., 2006). In addition, DNAJB1/HSP40 as well as overexpression of HSPA directly bound and consequently suppressed PrP toxicity (Fernandez-Funez et al., 2009; Rambold et al., 2006).

Another level of involvement of HSPs in prion disease may also involve the induction of stress response. Prion disease has been associated with induction of ER stress-related proteins and some elements of the UPR (Hetz et al., 2003; Steele et al., 2007), both of which typically result in the transcriptional upregulation of HSPs via activation of the heat shock response. Accordingly, HSF1 knockout mice inoculated with prions had a shortened lifespan compared to control mice inoculated with prions (Steele et al., 2008). Surprisingly, however, the reduction in lifespan was not correlated with exacerbated pathology or behavioral deficits, suggesting that lifespan ameliorated by heat shock induction is disconnected from the disease onset (Steele et al., 2008). Furthermore, several important markers of the UPR were not detectable in vivo (Samali et al., 2010; Unterberger et al., 2006), which may indicate that the upregulation of HSPs may be independent of ER stress signaling in brain. Clearly, more research is necessary to determine the origin and/or effects of the heat shock response on prion propagation and disease toxicity.

PrPC has been primarily localized to the plasma membrane where it remains attached via its glycosylphosphatidylinositol (GPI) anchor. Several studies indicate that PrPC may interact with the proteins 14-3-3 and HSP60 both in recombinant systems and in neurons (Edenhofer et al., 1996; Satoh et al., 2005) or under artificial systems where PrPC was highly overexpressed. Under these settings, 14-3-3 interacts with both HSP60 and PrPC in a phosphorylation-independent manner. (Satoh et al., 2005) Alluding to a potential function, the bacterial HSP60, GroEL, has the capacity of promoting aggregation of the toxic PrPSc molecule. However, as discussed below, the pathological implication of protein aggregation is a debatable issue. While this complex between HSP60 and PrP could play interesting regulatory roles in prion disease, further studies are needed to fully understand the implications of these associations.

4.4. Charcot- Marie-Tooth (CMT)

CMT is the most commonly inherited neuromuscular disorder, affecting approximately 1 in 2500 persons. Although patients diagnosed with CMT share commonalities in their clinical phenotype, the underlying genetic pathology is quite heterogeneous. Identification of the genetic mutations linked with CMT phenotypes has led to a more detailed classification system and molecular diagnoses (Pareyson and Marchesi, 2009). To date, more than 25 genes have been linked to CMT, two of which – HSPB8 and, more recently, HSPB1 – are HSPB family members.

The pathology of CMT lies in the degeneration of axonal projections, although it is possible that the primary pathology lies either in the neuronal axon itself or in the myelinating axonal support. Both HSPB1 and HSPB8 are expressed in motor neurons as well as glia (Armstrong et al., 2001; Plumier et al., 1997; Wilhelmus et al., 2006b; Wilhelmus et al., 2006c) and play significant roles in prevention of aggregate formation and cell survival against toxic stimuli (Carra et al., 2005; Concannon et al., 2001; Lee et al., 2006b; Outeiro et al., 2006; Pandey et al., 2000a; Paul et al., 2002; Pivovarova et al., 2007; Stetler et al., 2008). Several mutations in both of these HSPB members, in particular HSPB1, have been observed that are positively associated with CMT and distal hereditary motor neuropathy (dHMN). Mutations led to increased interactions between HSPB family members (Fontaine et al., 2006) and to subtle substrate-dependent changes in chaperone function (Kasakov et al., 2007; Kim et al., 2006). Additionally, one of the point mutations in HSPB1 (P182L) associated with CMT occurs in the C-terminal IXI/V motif, which is required for stabilization of sHSP dimers. The P182L mutation found in CMT patients was related to neurofilament disruption and decreased viability of neuronal cultures (Ackerley et al., 2006; Evgrafov et al., 2004). HSPB family members are highly involved in cell survival and inhibition of apoptotic signaling, and thus it is equally possible that mutation in these proteins could render cells more susceptible to injury through inability to suppress apoptotic signaling. Perhaps critical in the case of neuromuscular degeneration, small HSPs, including HSPB1, have been localized in synapses and glia (Bechtold and Brown, 2000), suggesting that the aberrant role of HSPBs in the neuromuscular synaptic context could contribute to CMT disease. More detailed molecular studies of CMT pathology in terms of HSPB dysfunction may further the understanding of small heat shock proteins in neuromuscular function.

5.0. Concluding remarks

HSPs have been intensely studied over the past half-century, and are now understood to function in a wide array of cellular activities. However, the mechanisms regarding these roles are still yielding new surprises and twists. As we have described in this review, protein folding, degradation targeting, sequestration and scaffolding represent major identified roles for HSPs in the cellular context, now understood to impact transcriptional control, cell death and survival, synaptic transmission, organelle viability and intracellular – and possibly inflammatory – signaling. However, how these function impact neurodegenerative disease is as yet unclear. The expression of HSPs has been correlated with cell survival in many neuronal injury contexts, and mutation or deletion of HSP function is correlated with several neurodegenerative conditions. In addition, exogenous overexpression or targeted deletion of HSPs has a profound impact on cellular survival in the neuronal context. Currently, two major arms of research are critically needed in furthering the pursuit of HSPs as translational targets in neuronal contexts – the exploration of dysregulation of cellular processes and the mechanisms of HSP-mediated neuroprotection. These two aims are fairly synergistic, and complement each other well considering the tantalizing groundwork that has already been laid, outlined in the present review. Putting the pieces of these two puzzles together simultaneously may lead then to clarity in translational approaches. While HSPs have been demonstrated to confer neuroprotection in a variety of settings, the development of small molecules targeting mechanistic molecular interactions remains to be explored, and is hopefully just around the corner.

Acknowledgments

We thank Armando Signore for the artwork, Carol Culver and Sulaiman Hassan for editorial assistance, and Pat Strickler for secretarial support. This work was supported by funds from the American Heart Association (09POST22006065 to R.A.S.), the National Institutes of Health (NS36736, NS43802 and NS45048 to J.C.), VA Merit Review Grant (to J.C.) and the Chinese National Science Foundation (30870794, 30670642 to Y.G.).

Footnotes

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