Therapeutic potential of Hsp27 in neurological diseases

Background

Heat shock proteins (Hsps) are widely reported in normal cellular dynamics under stress and non-stress conditions, and parallelly, the studies regarding its role in disease condition are also progressing steadily. The function of Hsps in neurodegenerative disorders is puzzling and not fully understood. This review aims to focus on the role of Hsp27 in normal and diseased conditions and emphasize its therapeutic potential.

Hsp27

Hsp27, in particular, has shown to be involved in cell viability and actin cytoskeleton remodeling and also shown to improve many disease conditions. Phosphorylated Hsp27 modulates the p53 pathway by downregulating cellular senescence and also lowers reactive oxygen species to protect TNFα-mediated apoptosis. Hsp27 is also known to interfere with mitochondria-dependent and mitochondria-independent cell apoptotic stimulation.

Conclusion

This article will highlight the various functions of Hsp27 especially as an anti-apoptotic factor and stress response factor and its therapeutic potential in preventing neuronal apoptosis in neurological diseases. This review also includes a comparison of the therapeutic potential of Hsp27 with regard to other small Hsps.

Heat shock proteins (Hsps) were discovered by Ferruccio Ritossa in 1960 in Drosophila melanogaster. Hsps are a group of proteins expressed by cells in response to any environmental stress. For example, during stressful conditions such as exposure to toxins or hypoxia, Hsps are upregulated to generate proteins that will be identified as antigens to stabilize the condition. Hsps also function as molecular chaperones that are involved in folding and unfolding of different proteins to prevent the aggregation of unwanted protein and facilitate proper refold of damaged proteins. Hsps constitute of many different proteins separated based on their molecular masses. The main Hsps comprise of Hsp100, Hsp90, Hsp70, Hsp60, Hsp40, and small heat shock proteins (sHsps). Small heat shock proteins (HspB1–HspB10) have a molecular weight of monomeric forms between 12 and 43 kDa and are characterized by the presence of α-crystallin domain that is flanked by less conserved N-terminal domain and C-terminal extension. Among the sHSPs, Hsp27 (also known as HspB1) has a significant responsibility in increasing the cell viability and acts in multiple roles as an anti-apoptotic protein and antioxidant as well as being involved in actin cytoskeleton remodeling [1, 2].

By targeting Hsp27, apoptosis of α-synuclein may be prevented and regulated which makes it useful in treating neurodegenerative disorders like Parkinson’s disease. With the use of its stress response, it is possible to eliminate the aggregation of amyloid plaques and thus prevent the progression of Alzheimer’s disease. Due to its numerous roles within the cell, Hsp27 is a promising therapeutic candidate for neurological diseases. This review discusses the possible therapeutic strategies of Hsp27 against few neurological diseases and also includes a critical comparison of other Hsps having therapeutic activity.

Cells activate various signaling pathways when exposed to environmental stress conditions. This stress can cause damage to the cellular mechanism by causing mitochondrial dysfunction, protein misfolding, and finally neuronal cell death. During stress conditions, Hsp27 stabilizes the cytoskeleton with actin capping to prevent cellular injury. It scavenges the reactive oxygen species (ROS) by raising the levels of intracellular glutathione and is associated with independent and dependent apoptotic pathways in mitochondria. In unstressed cells, these Hsps are actively involved in folding, assembly, intracellular localization, secretion, regulation, and degradation of proteins [3].

The functions of Hsp27 are influenced by post-translational modifications via phosphorylation with differential effects in cellular functions. The expression of Hsp27 gene during stress is caused by the binding of HSF1 (heat shock transcription factor-1) to HSE (heat shock element) which incorporates the promoters of Hsp27 [4]. During mitosis, HSF2 (heat shock transcription factor-2) binds to HSE to induce normal stress-inducible expression of genes [5]. The main role of HSF1 is to induce the expression of Hsps. This can be seen in studies where HSF1 protein levels are elevated in cancer, whereas in neurodegenerative disorders, the levels are depleted. The elevated expression of HSF1 has shown to enhance the pro-survival function in neurodegenerative disorder studies [4].

The expression levels of Hsp27 vary among different cells as well as species based on their sites of phosphorylation. In humans, Hsp27 gets phosphorylated at specific serine residues such as Ser15 (Serine 15), Ser78 (Serine 78), and Ser82 (Serine 82) which acts as a common substrate for MK2 (Mapkap kinase 2), MK3 (Mapkap kinase 3), and MK5 (Mapkap kinase 5), respectively [6, 7]. Hsp27 occurs in two pathways—p38MAPK (p38 mitogen-activated protein kinases) and PKA (p21-activated protein kinases) pathways. MK2, a major Hsp27 kinase, phosphorylates Hsp27 by an activated p38MAPK pathway in response to cellular stress. Similarly MK5, another Hsp27 kinase, also phosphorylates Hsp27 by the PKA pathway [7, 8].

Upon phosphorylation, Hsp27 has a role in actin filament regulation where it promotes polymerization contributing to microfilament network maintenance by preventing filament degeneration and also blocking the early response of actin to growth factors [9]. In the unphosphorylated form [10], Hsp27 has an alternative role in actin capping thereby inhibiting assembly of wild-type proteins [11]. During platelet activation, Hsp27 undergoes conformational changes by phosphorylation, promotes interaction between Hsp27 and actin or between Hsp27 and other actin-associated proteins, and facilitates translocation of proteins to the cytoskeleton [12].

Hsp27 phosphorylation also modulates the p53 pathway by inhibiting the accumulation of p21 which downregulates cellular senescence. p21 is an inhibitor of cyclin-dependent kinases that are important for the progression of the cell cycle. On the inhibition of Hsp27 phosphorylation, an increased accrual of p21 may occur, due to decreased p53, leading to apoptosis [1]. During stress conditions, as an antiapoptotic agent, Hsp27 expression inhibits mitochondrial injury and apoptosis of cells [13]. When Hsp27 expression is suppressed, it will lead to an increase in cellular susceptibility to apoptosis contributing to organ dysfunction [14]. This makes Hsp27 hold a pivotal post that can control cell death and cell survival. Hsp27 interferes with the apoptotic signaling pathway by interacting with both mitochondria-dependent and mitochondria-independent pathways (Fig. 1). It affects different key components of the apoptotic pathway in various ways as follows: (a) It enhances PI3-K which activates Akt-Bax interaction, inhibiting Bax activation and translocation to mitochondria preventing the release of cytochrome c from mitochondria [14]. (b) It inhibits the Ask1-JNK pathway leading to cytochrome c leakage [15]. (c) It prevents apoptosome formation [16]. (d) It interacts with Daxx in the nucleus preventing its translocation to cytosol which is essential for interaction with the Fas receptor involving apoptosis [17]. (e) Hsp27 seems to be involved in inhibition of the Smac from mitochondria, and this may lead to inhibition of caspases [14]. (f) It also downregulates tBID released from caspase 8 [16].

Schematic representation showing the regulatory and inhibitory actions of Hsp27. Through induction of stress, the expression of hsp27 gene occurs when HSF-1 trimer binds to HSE. Hsp27 expression level gets varied when phosphorylated at specific serine residues such as Ser15, Ser78, and Ser82. Phosphorylated Hsp27 interferes with apoptotic signaling pathway by affecting its key components like cytochrome c, apoptosome, Daxx, Smac, tBID, caspase 9, and ASK1-JNK through interaction with both mitochondria-dependent and mitochondria-independent pathways of apoptosis

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Besides its activity in apoptosis inhibition, Hsp27 also acts as an antioxidant by lowering the levels of ROS and iron by raising intracellular glutathione levels [18]. ROS plays a role in intracellular signaling and regulation and acts as redox messengers at cellular concentrations. Under normal conditions, ROS is produced in small amounts, during the formation of ATP, in the electron transport chain (ETC) by oxidative phosphorylation in mitochondria [19]. Many non-cellular processes like inflammatory reactions and ionizing radiations, and cellular processes like mitochondrial oxidative respiratory reactions, NADPH oxidases, and nitric oxide synthases (NOSs) contribute greatly to oxidative stress. These processes generate excess ROS resulting in imbalanced redox state and mitochondrial dysfunction. Hsp27 promotes a balanced redox state by reducing the ROS levels to avoid mitochondrial dysfunction, cell damage, and cell death [19].

A study was performed to demonstrate Hsp27 neuroprotective effects by mutating serine residues to either alanine (Hsp27-A) or aspartate (Hsp27-D). In both in vivo and in vitro conditions, overexpression of Hsp27 wild-type and Hsp-D by phosphorylation provided neuroprotection by inhibiting the Ask-1 signaling pathway. In unphosphorylated form, it was insufficient to suppress the Ask-1 pathway or to provide protection [20]. In a study involving glial cell inclusion bodies, the response of Hsp27 to induced stress was studied. The bodies were transfected with OLN-3 cells carrying plasmids encoding Hsp27 expressed in three ways—wild-type, pseudophosphorylated form, and nonphosphorylatable form. The study revealed that Hsp27 regulated by phosphorylation protected the cytoskeleton and provided resistance from apoptotic stimuli upon stress conditions [21].

In another study, human neuroblastoma cell lines IMR-32 were treated with Cu²⁺ resulting in increased stress to the cells. This indirectly stimulated an increased level of endogenous Hsp27 and the overexpression of Hsp27, in turn, protecting the cells from Cu²⁺-induced cell death. Hsp27 hindered the upstream mechanism by inhibiting ROS production, decreasing the cascade events of Cu²⁺-induced inflammation and oxidative stress, and playing a protective role in maintaining Cu²⁺ homeostasis [22]. Studies have also shown that activated microglia-derived TNFα causes inflammation and degeneration processes [23]. When treated with Hsp27, which is an antioxidant, the cells were protected from TNFα-mediated apoptosis by lowering the levels of reactive oxygen intermediate (ROI) formation by modulating glutathione content.

Alzheimer’s disease (AD) is neuropathologically characterized by memory impairment, and it is the most common type of dementia which alters cognitive abilities. The characteristics observed in the brains of people with AD are amyloid plaques and neurofibrillary tangles [24]. The amyloid plaques are primarily composed of Aβ42 (42 residues β-amyloid peptide) which is hydrophobic in nature. Increased amounts of insoluble β-amyloid peptides cause an imbalance between Aβ production and clearance in the central nervous system [25]. The most important function of tau protein is providing stability and promoting assembly of microtubules. Neurofibrillary tangles in AD indicate neuronal dysfunction induced by hyperphosphorylation of tau which aggregates into insoluble paired helical filaments (PHFs) [26]. The altered structure of tau in AD is due to abnormal post-translational modifications like hyperphosphorylation, acetylation, glycosylation, and truncation [27]. Tau hyperphosphorylation can also lead to several other events like Aβ-mediated toxicity, inflammation, an increase in oxidative stress, and covalent modifications of tau [28]. Increase in oxidative stress results in overproduction of ROS which in turn leads to a cascade of events ultimately ending in apoptosis [29].

Other than Hsp27, sHsps like Hsp20, Hsp22, and α-crystallins also have similar functions as anti-apoptotic; chaperone activity and preventing cell death pathway through the effect of the sHsps varies widely. Table 1 gives a detailed account of the phosphorylation sites and the different modes of action against the aggregation of amyloid proteins. Though being small in size, Hsp27 has a wide range of therapeutic potential. Limited studies were focused on its therapeutic abilities on AD, but there have been reports that have shown a restoration in the amyloid plaque and tangle formation [31,32,33,34].

Research has shown that the inhibition of tau hyperphosphorylation restores neuronal dysfunction and modifies disease progression in AD which could be a therapeutic target for treatment. A study [35] in human neuroblastoma cell line SH-SY5Y was performed by inducing hyperphosphorylation of tau with okadaic acid. For effective delivery, Hsp27 protein was fused with HIV Tat protein (Tat-Hsp27) and introduced to the hyperphosphorylated tau aggregates where it resulted in a reduction of hyperphosphorylated tau levels conferring protection against apoptotic cell death. It was also demonstrated that without ubiquitination, degradation of hyperphosphorylated tau was carried out by Hsp27 [31]. Abisambra et al. confirmed that tau fibril formation can be prevented in in vitro condition by the addition of recombinant Hsp27. Similarly, he also verified that tau protein levels were reduced in in vivo conditions by overexpressing Hsp27 inhibiting the formation of tau fibrils [36]. A study by Chang et al. showed that synthetic indole derivatives upregulated Hsp27 expression which in turn reduced tau misfolding [32].

Several researches are also carried out to study the inhibition of Aβ aggregation and to attenuate Aβ toxicity with Hsp27 as the target protein. Hsp27 binds to Aβ inhibiting its aggregation into mature fibrils [30]. A study was also performed to determine and compare the mechanism of interaction among three sHsps namely Hsp20 from Babesia bovis, Hsp17.7 from carrot, and Hsp27 from humans and their capability in lowering toxicity of Aβ aggregation. It was seen that Hsp27 interacts only at a later stage after forming Hsp27-Aβ mixture thus inhibiting the formation of fibrils [37]. In a mouse model of AD, overexpression of Hsp27 improved the learning abilities, increased the excitability of synaptic neurons, and decreased the Aβ aggregates [33]. Additionally, it was also observed that the increase in Hsp27 expression after exercises prevented the aggregation of plaques and greatly improved brain function of elderly women [34].

The above studies exhibited that Hsp27 provides an almost untouched avenue for therapeutic intervention by exerting a beneficial effect in reducing oxidative stress as an antagonistic effect on apoptosis and its inhibitory actions through interaction with the amyloid formation and also in tau pathologies by providing neuronal protection.

The deposition of α-synuclein (α-syn) into fibrillar protein aggregates is the characteristics of many neurodegenerative diseases collectively called α-synucleinopathies including Parkinson, dementia with Lewy bodies, and multiple system atrophy. α-Synuclein is a neuronal protein found in presynaptic terminals which modulates the synaptic activities like neurotransmitter release and vesicular trafficking [38]. The α-synuclein aggregation was sensitive to inhibition of autophagy and the proteasome which lead to an increase in proportions of α-synuclein inclusion cells. Parkinson’s disease (PD) is a neurodegenerative disorder which is characterized by selective degeneration of dopaminergic neurons with Lewy bodies composed of α-synuclein and sHsps [39].

Studies have revealed that sHsp upregulation in disease progression of PD prevents the degeneration of neurons [40]. Overexpression of α-B crystallin and Hsp27 [41] by using bicistronic expression plasmids prevented the intracellular aggregation of α-syn. It was also suggested that the effectiveness of Hsp27 was dependent on the kinetics of α-syn aggregation. Hsp27 was found to be less effective at a faster rate of aggregation [38]. A study showed that the expression of Hsp27/70 in SH-SY5Y cells induced by FLZ (a synthetic novel derivative of squamosamide from a Chinese herb) provided neuroprotective effects against MPP+-induced cytotoxicity where MPP+ is a neurotoxin used in mimicking PD model [42].

A recent study revealed that Hsp27 binds to the α-syn fibrils thus decreasing their hydrophobicity and cellular toxicity. It was also shown that Hsp27 was capable of inhibiting the elongation of α-syn [43]. Additionally, Hsp27 also prevents the aggregation of monomeric α-syn fibrils along with α-crystallins [38]. All these properties highlight the therapeutic potential of Hsp27 in preventing aggregation of α-synuclein and the progression of α-synucleinopathies.

Amyotrophic lateral sclerosis

Hsp27 may also have a therapeutic role in amyotrophic lateral sclerosis (ALS). ALS is a rare neurodegenerative disorder characterized by progressive muscle weakness and atrophy due to the death of motor neurons in the spinal cord, cortex, and brainstem. In a study conducted, Hsp27 levels were increased and delivered to ND7 cells. When these cells were subjected to serum removal to induce apoptosis, overexpressed Hsp27 protected G93A or G93R SOD1 mutants from apoptotic cell death [44].

Neuronal injury

Neuronal injury after ischemia initiates a series of signaling cascades contributing to delayed neuronal death. In Hsp27 transgenic mice, overexpression of Hsp27 protected the cells against subsequent neuronal injury by inhibiting ASK1-dependent MKK4/JNK activation. This reflects that Hsp27 has a therapeutic potential during a stroke [45].

Ataxia telangiectasia

Ataxia telangiectasia is a neuromotor dysfunction neurodegenerative disorder of childhood caused by the disruption of gene ATM. In this disease, neurons lose their ability to divide and function. A study revealed that differential expression of Hsp27 in the frontal cortex can protect cortical neurons from degeneration, whereas in the cerebellum, proliferating glial cells were found to synthesize Hsp27 [46]. This again opens up the possibilities of using Hsp27 in treatment purposes.

Charcot-Marie-Tooth disease

Charcot-Marie-Tooth (CMT) neuropathies constitute a group of monogenic diseases that primarily affect the peripheral nervous system [47]. Mutations in Hsp27 can lead to adverse pathological neuromuscular disease as in distal hereditary motor neuropathy (dHMN) and also have been reported to cause CMT. These may be either due to toxic gain of function as a result of misfolding and aggregation or due to loss of function leading to decrease in the ability of cells to tolerate stress. Four mutant transgenic mouse models of dHMN were developed, and treatment with a selective HDAC6 inhibitor showed a reversal of the clinical phenotype of both S135F and P182L transgenic mice [48].

The therapeutic possibilities of Hsp27 are still not fully understood in most neurological diseases, but their role in these diseases cannot be ignored. Though not a lot of focus is placed on these small molecules, it is possible that these tiny molecules can shift the typical paradigm and open a new pathway to developing therapies for neurological diseases. Hsp27 has shown, in multiple diseases, that there is a chance to improve the condition of the cells affected and not just temporarily relieve the modifications. From the abovementioned studies, it is clear that Hsp27 may be a promising novel therapeutic target in treating neurological diseases.

All data and materials are available in the manuscript and with the corresponding author.

AD:

Alzheimer’s disease

ALS:

Amyotrophic lateral sclerosis

CMT:

Charcot-Marie-Tooth

dHMN:

Distal hereditary motor neuropathy

ETC:

Electron transport chain

HSE:

Heat shock element

HSF1:

Heat shock transcription factor-1

HSF2:

Heat shock transcription factor-2

Hsp:

Heat shock protein

NOS:

Nitric oxide synthase

PD:

Parkinson’s disease

PHF:

Paired helical filament

ROI:

Reactive oxygen intermediate

ROS:

Reactive oxygen species

sHsp:

Small heat shock protein

α-syn:

α-Synuclein

The authors would like to thank Bharathiar University, India, for providing the necessary infrastructure facilities required for completing the article.

Funding

Not applicable. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sector.

Affiliations

1. Human Molecular Cytogenetics and Stem Cell Laboratory, Department of Human Genetics and Molecular Biology, Bharathiar University, Coimbatore, Tamil Nadu, 641046, India

   • Anila Venugopal
   •  & Balachandar Vellingiri

2. School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, 625021, India

   • Kasthuri Sundaramoorthy

Contributions

AV and BV contributed to the concept development. AV and KS contributed to the manuscript writing and figure generation. BV contributed to the final approval. All authors have read and approved the manuscript.

Corresponding author

Correspondence to Balachandar Vellingiri.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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