Small heat shock proteins in inherited peripheral neuropathies

Abstract

Small heat shock proteins (small HSPs) are molecular chaperones that protect cells against stress by assisting in the correct folding of denatured proteins and thus prevent aggregation of misfolded proteins. Small HSPs also modulate apoptotic pathways by interacting with components of programmed cell death. Furthermore, some small HSPs interact with the cytoskeleton to assist in spatial organization and dynamics of its structural elements. The role of small HSPs has been studied in many disorders, including neurodegenerative disease. Recently, mutations in HSPB1 (HSP27) and HSPB8 (HSP22), two members of the small HSP superfamily, have been associated with inherited peripheral neuropathies. In this review, we will summarize the current knowledge of small HSPs, in particular HSPB1 and HSPB8, and discuss their role in health and disease.

• Neurodegeneration
• peripheral neuropathy
• small heat shock proteins

Introduction

Heat shock proteins (HSP) act as molecular chaperones and protect intracellular components during stress conditions. Even in unstressed cells, HSPs are constitutively expressed to properly fold and assemble polypeptides. The name ‘heat shock protein’ in fact refers to the rapid induction of these proteins in response to elevated temperatures, although other stress stimuli (e.g. oxidative stress, heavy metals and ischemia injury) are also capable of inducing expression of HSPs. HSPs are classified according to their molecular weight and currently five major classes are recognized: HSP100, HSP90, HSP70, HSP60 and the small heat shock proteins (small HSPs), which have a monomer size between 12 and 43 kDa. In this review we will focus on the small HSPs. In vitro these small HSPs have a chaperone‐like activity, i.e. the capacity to interact with partially denatured proteins and to prevent protein misfolding and aggregation. They are also involved in diverse cellular activities such as modulation of actin and intermediate filament dynamics 1, 2, cellular growth and differentiation 3, 4 and apoptosis 5.

Genes encoding small HSPs are found in all organisms, from bacteria to vertebrates. In addition, all members of the small HSP superfamily share a conserved C‐terminal α‐crystallin domain, containing 80–100 amino acid residues, a more variable N‐terminal sequence and a short variable C‐terminal tail. The combination of a conserved domain and their universal presence, indicate a crucial, evolutionary role in cellular homeostasis. In human, the ten genes encoding small HSPs are named HSPB1 to HSPB10 according to the HUGO Gene Nomenclature Committee 6, 7. Recently, five novel small HSPs were identified in other vertebrates, HSPB11–15, but these do not have a mammalian orthologue 8. In retrospect, the first members of the small HSP superfamily, αA‐crystallin/HSPB4 and αB‐crystallin/HSPB5, were identified more than a century ago as ‘α‐crystallins’ 9. Only in 1982 the relationship of these proteins with the small HSP superfamily was recognized 10. HSPB4 is almost exclusively expressed in the lens and is not heat‐inducible, while HSPB5 is distributed ubiquitously and is heat‐inducible 11. HSPB1 (HSP27), a heat‐inducible small HSP that was identified 20 years ago, is ubiquitously expressed in human tissues and interacts with HSPB8, another small HSP 12. HSPB8, also known as HSP22, is a heat‐inducible small HSP, expressed in various tissues including muscle, motor and sensory neurons 13–16. The remaining small HSPs will not be discussed in detail since so far they have not been implicated in human disorders.

During the last decade, the involvement of small HSPs has been studied in many disorders, including neurodegenerative diseases. The first studies focused on the potential protective effect of upregulation of HSP expression. Increased levels of wild‐type small HSPs, especially HSPB5 and HSPB1, were indeed found in individuals with neurodegenerative diseases, such as Alzheimer 17, Parkinson 18, Alexander disease 19 and amyotrophic lateral sclerosis (ALS) 20. These neurodegenerative disorders are all marked by the deposition of improperly folded proteins, inclusion bodies or plaques in the nervous system.

On the other hand, mutations in four small HSPs have been associated with autosomal dominant and recessive hereditary disorders. Mutations in the crystallin genes, HSPB4 and HSPB5, are associated with congenital cataract and desmin‐related myopathy 21, 22. Mutations in the interacting small HSPs, HSPB1 and HSPB8, have been shown to cause distal hereditary motor neuropathy (distal HMN type II) and Charcot‐Marie‐Tooth disease type 2 (CMT2) (Table I) 23–25. It is not clear what the underlying pathological mechanism is in these disorders: misfolding and aggregation of the mutant HSP, a defective cytoprotective activity leading to misfolding and aggregation of other proteins, a dysfunction in cell death pathways, or a combination of these effects.

Table I. Mutations in HSPB1 and HSPB8 and the associated phenotypes.

Small HSP	Nucleotide change	AA change	Domain	Phenotype	Reference
HSPB1	c.379C>T	R127W	α‐crystallin	distal HMN	24
	c.404C>T	S135F	α‐crystallin	distal HMN, CMT2F	24
	c.406C>T	R136W	α‐crystallin	CMT2	24
	c.452C>T	T151I	α‐crystallin	distal HMN	24
	c.545C>T	P182L	IXI/V	distal HMN	24
HSPB8	c.421A>G	K141E	α‐crystallin	distal HMN II	23
	c.423G>C	K141N	α‐crystallin	distal HMN II	23
	c.423G>T	K141N	α‐crystallin	CMT2L	25

AA change = amino acid change; CMT2F and CMT2L = Charcot‐Marie‐Tooth type 2 neuropathies; distal HMN II = distal hereditary motor neuropathy type II.

Because mutations in HSPB1 and HSPB8 were only recently identified as a cause of inherited peripheral neuropathies, we will concentrate on these two small HSPs and discuss how mutations in these genes affect motor and sensory neurons.

Key messages

• Small heat shock proteins (small HSPs) have cytoprotective roles including chaperone‐like activity, anti‐apoptotic activity and preservation of cytoskeletal elements.

• The neuroprotective role of small HSPs has been studied in many disorders, including neurodegenerative disease.

• Recently, mutations in HSPB1 (HSP27) and HSPB8 (HSP22), two members of the small HSP superfamily, have been associated with inherited peripheral neuropathies.

HSPB1 and HSPB8 mutations in inherited peripheral neuropathies

Distal HMN, also known as hereditary distal spinal muscular atrophy (distal SMA) is a pure motor disorder of the peripheral nervous system (PNS) 26. Distal HMN is clinically characterized by progressive weakness and atrophy of the muscles in the lower limbs. Later on weakness is also present in the distal upper limbs. In a related inherited peripheral neuropathy, CMT2, sensory neurons are also involved. On clinical examination, distal HMN patients are hard to distinguish from CMT2 patients, because in CMT2 patients sensory symptoms are often absent. Therefore electrophysiological examinations are essential to confirm the diagnosis of either distal HMN, where only motor neurons are involved, or CMT2, where motor and sensory neurons are involved 27. Both disorders are clinically and genetically heterogeneous 28, 29. Recently, missense mutations in HSPB1 and HSPB8 have been associated with distal HMN and CMT2 (Table I) 23–25. Initially two distinct missense mutations (K141N and K141E), involving the same amino acid in the α‐crystallin domain of HSPB8 were found in four distal HMN type II families. Subsequently, the heterozygous amino acid change (K141N) in HSPB8 was also reported in a large Chinese CMT2 family (CMT type 2L) which was previously linked to the same chromosomal region as distal HMN type II 25, 30. These observations raise two important points. First, the same HSPB8 missense mutation (K141N) is associated with two distinct phenotypes, i.e. distal HMN and CMT2. Secondly, all reported pathogenic mutations so far are missense mutations that target the same amino acid. The conserved K141 residue in HSPB8 corresponds to R116 and R120 of the HSPB4 and HSPB5 proteins respectively (Figure 1). Interestingly, these arginine residues are mutated in patients with congenital cataract and desmin‐related myopathy 21, 22. Biochemical analysis of the R116C and R120G missense mutations in HSPB4 and HSPB5 shows significant changes in the secondary and tertiary structure of these small HSPs, a defective chaperone‐like activity and a higher aggregate size 31. The R116C mutation in HSPB4 also reduces the protective ability in lens epithelial cell apoptosis 32. Members of the small HSP superfamily interact with each other and changes in these interactions may be a culprit mechanism in pathological conditions. The interaction between mutant HSPB4 (R116C) and wild‐type HSPB1 and HSPB5 is increased, whereas the interaction with HSPB4 wild‐type proteins shows little change 33. Co‐immunoprecipitation studies demonstrated also that mutant HSPB8 (K141N/E) has an increased interaction with HSPB1 and thus might interfere with the activity of HSPB1 23. These studies underline the importance of the preservation of a positively charged amino acid at that position in these small HSPs. In vitro studies have also shown that mutant HSPB8 promotes the formation of cytoplasmatic and perinuclear aggregates, which correlated with a significant decrease in neuronal cell viability 23. Aggregation of desmin filaments is also induced by mutant HSPB5 (R120G) 34. Accumulation of these misfolded proteins can have deleterious effects on neuronal cell function by disrupting the axonal transport and can ultimately lead to cell death (Figure 2).

Figure 1 Structure‐based sequence alignments of HSPB1 and HSPB8 orthologues and homologues HSPB4 and HSPB5. The N‐terminal WD/EPF and SRLDQF/AFG domain, the α‐crystallin domain and the C‐terminal IXI/V domain are indicated. The peripheral neuropathy‐associated mutations are boxed in grey. The cytochrome C binding motifs are indicated by a dotted line.

Figure 2 Suggested pleiotropic actions of mutant HSPB1 and HSPB8. Mutations in HSPB1 and HSPB8 can interfere with the chaperone‐like activity, causing insoluble cellular aggregates and subsequent induction of neuronal cell death. HSPB1 and HSPB8 regulate different apoptotic pathways. Mutant HSPB1 can no longer bind cytochrome C, pro‐caspase 3 and Daxx and is thus unable to prevent apoptotic cell death. Mutations in HSPB8 may result in enhanced apoptosis by inhibiting the pro‐survival activity of casein kinase‐2 or by negatively influencing the anti‐apoptotic activity of its interacting partner, HSPB1. Furthermore, mutant HSPB1 is able to disrupt cytoskeletal functions and neurofilament assembly, resulting in neuronal cell death or altered axonal transport. Ultimately, these pathological mechanisms will cause peripheral neuropathy in humans.

Not entirely unexpected, missense mutations in HSPB1, the molecular partner of HSPB8, were also associated with distal HMN and CMT type 2F 24. Four mutations in HSPB1 target the highly conserved α‐crystallin domain (R127W, S135F, R136W and T151I) and one is positioned in the C‐terminal part (P182L) (Table I; Figure 1). Experimental studies with HSPB5 showed that mutations in the core α‐crystallin domain decrease the chaperone function 35. The HSPB1 mutations found in distal HMN and CMT2F patients may therefore interfere with the chaperone activity. The C‐terminus of several small HSPs contains a conserved IXI/V motif (Figure 1). This motif is relevant for inter‐subunit interactions and oligomerization, either with regions in the α‐crystallin domain or with IXI/V motifs on other monomers 36. Possibly, the C‐terminal missense mutation (P182L) in HSPB1 interferes with subunit‐interactions of small HSPs, which are important to maintain the oligomeric state of the protein. Furthermore, this IXI/V motif is critical in the chaperone‐like activity of several bacterial small HSPs. Deletion of this motif or mutations in this motif, result in a complete loss of chaperone activity and the truncated protein was unable to form oligomers 37. In addition, a site‐directed mutation of P160 in HSPB5, which corresponds to P182 in HSPB1, also influences the chaperone function 35. These effects may contribute to the reduced cell viability of neuronal cells seen in transient transfection studies with mutated HSPB1 24.

Cellular and cytoprotective properties of HSPB1 and HSPB8

Under normal conditions HSPB1, as most small HSPs, forms large oligomers consisting of about 24 subunits. These oligomeric complexes are formed by dimeric building blocks composed of small HSP monomers. The well conserved N‐terminal SRLFDQFFG motif determines the oligomeric size of small HSPs and contributes to their structural stability (Figure 1) 38. A unique cysteine residue in Hspb1 (C141) regulates the formation of dimers 39. Hspb1 with a mutation at this cysteine residue showed decreased ability to multimerize, and was less efficient in inhibiting staurosporine‐induced apoptosis 39. Interestingly, the cysteine residue, corresponding to C137 in HSPB1, is located next to the amino acids S135 and R136, which are mutated in individuals with a motor and sensory neuropathy 24. Mutations located near this functionally important cysteine residue, possibly interfere with oligomerization. The oligomeric structure of HSPB8 is not clear. An in vitro study showed that Hspb8 exists as a monomer in contrast to other small HSPs 40, however in other studies, HSPB8 predominantly formed dimers 15, 41 and high molecular mass complexes in the heart 42. The oligomerization of mammalian small HSPs is regulated by phosphorylation 1. Mitogen activating proteins (MAP), MAPKAP kinase 2 and 3, which are activated by p38 MAP kinase, are the main kinases involved in the phosphorylation of small HSPs. Stress‐induced phosphorylation and oligomerization may thus play an important role in the biological activities of small HSPs 43, 44.

We will now summarize the three main cytoprotective functions of small HSPs: 1) the chaperone‐like activity, 2) the capacity to interfere with cellular death pathways and 3) the role in stabilizing the cytoskeleton (Figure 2).

HSPB1 and HSPB8 act as molecular chaperones

Small HSPs contribute to protection of the cell against stress by their chaperone‐like activity, i.e. their capacity to bind non‐native proteins. In vitro studies have demonstrated that mammalian small HSPs recognize misfolded proteins and prevent intracellular protein aggregation by keeping them in a soluble phase 45. The in vitro molecular chaperone activity of HSPB1 and HSPB8 has been extensively documented 34, 40, 41, 44, 46. They prevented aggresome formation in cells expressing mutant HSPB5 (R120G) by forming complexes with misfolded HSPB5 34. Interestingly, in contrast to HSPB1, HSPB8 did not remain associated with the mutant HSPB5 complexes. This suggests that HSPB8 is involved in protein folding, and therefore can be considered as a true chaperoning molecule 34.

Several studies demonstrated an important, but not sufficient role of the α‐crystallin domain in the chaperone‐activity of small HSPs 35, since additional parts of the protein are required, such as the N‐terminal proline‐phenylalanine‐rich region containing a WD/EPF (single letter amino acid code) motif in HSPB1 and the C‐terminal region (Figure 1) 47, 48. The chaperone‐like activity of small HSPs, as documented in in vitro studies, may of course be highly relevant for their role in human disorders. However this activity still needs to be confirmed by in vivo studies 45.

HSPB1 and HSPB8 act as modulators of apoptosis

The protective effect of small HSPs is also related to their interference with apoptotic pathways. HSPB1 prevents the formation of apoptosomes and prevents cell death triggered by various stimuli. HSPB1 maintains the redox status and mitochondrial stability in the cell and prevents apoptosis due to reactive oxygen species 44, 49. It also inhibits apoptosis by direct binding to molecular components of cell death pathways. HSPB1 binds to both cytochrome C, when it is released from the mitochondria to the cytosol, and to pro‐caspase‐3, thus preventing the activation of the caspase cascade (Figure 2) 50, 51. The cysteine residue at position 137, as well as the N‐terminal region, seem to be essential for binding to cytochrome C (Figure 1) 50. HSPB1 also affects the caspase‐independent apoptotic pathway, by its interference in the translocation of Daxx to the membrane and its interaction with Fas 52. Daxx is a nuclear protein that translocates to the membrane during Fas‐mediated caspase‐independent apoptosis.

Another function of HSPB1 related to cell death, is its role in proteasome‐mediated degradation of selected proteins. HSPB1 is able to interact with specific components of the proteasome and to bind ubiquitin, thus facilitating protein degradation 53. The interaction between HSPB1 and ubiquitin explains the co‐localization with ubiquitinated proteins in cytoplasmatic inclusions that characterize some neurodegenerative diseases.

In contrast to the anti‐apoptotic activity of most small HSPs, a pro‐apoptotic activity was assigned to HSPB8 when forced to express in tumor cell lines 54. HSPB8 triggered apoptosis is caspase‐ and p38 MAP kinase‐dependent and cell‐type specific since not all cell‐types overexpressing HSPB8 showed an enhanced apoptosis 54. This is well documented in the heart were HSPB8 plays a dual role; at low doses it induces myocardial hypertrophy through the activation of the Akt or protein kinase B (Akt/PKB) pathway, while at high doses it has a pro‐apoptotic activity via protein kinase‐dependent mechanisms 55. HSPB8 also interacts with casein kinase‐2 (CK‐2), which has anti‐apoptotic effects. It is speculated that HSPB8 promotes apoptosis by inhibition of the CK‐2 activity in cardiac myocytes 55. Since HSPB8 interacts with HSPB1 and other small HSPs, mutant HSPB8 may induce apoptosis by disturbing the anti‐apoptotic effect of other small HSPs 23, 42. Although the apoptotic activity of HSPB8 in motor neurons is unknown, both HSPB8 and HSPB1 associate with the Akt/PKB kinase pathway, a major signaling pathway in non‐neuronal and neuronal cells for growth and survival 56, 57. This pathway is also activated in spinal motor neurons after nerve injury, wherein HSPB1 plays a role in promoting cell survival 56.

HSPB1 and HSPB8 act as protectors of the cytoskeleton

A cytoprotective function of HSPB1 is also to regulate cytoskeletal dynamics. The cytoskeleton maintains the shape of the cell by modulating the spatial arrangement and dynamics of its structural elements, consisting of microtubules, intermediate filaments and actin microfilaments. During stress, the integrity of this cytoskeletal network is disrupted by disorganization and aggregation of the filament system. Abnormalities in cytoskeletal organization and aggregates are frequent histopathological hallmarks in neurodegenerative disorders. HSPB1 and microfilaments interact to prevent disruption of the cytoskeleton during exposure to stress stimuli 2. Although the molecular mechanism by which HSPB1 stabilizes the actin network is not fully understood, an inhibition of actin polymerization was demonstrated 58. The depolymerization of actin is regulated by the structural organization of HSPB1 and its p38 MAP kinase‐mediated phosphorylation 59. Unphosphorylated HSPB1 monomers effectively block polymerization, whereas phosphorylated monomers and unphosphorylated oligomers are not able to prevent actin polymerization 58. The interaction between HSPB1 and actin, and the related depolymerization effect were assigned to amino acid sequences at the N‐terminus of HSPB1 60.

Besides the interaction with actin microfilaments, HSPB1 also associates with intermediate filaments. In vitro studies show that this interaction controls filament‐filament interactions, which are important for cell survival by preventing intermediate filament aggregation 61. The fact that mutant HSPB1 (S135F) results in disruption of the cellular neurofilament (NF‐L) network, provides evidence for an essential role of HSPB1 in regulating this dynamic network 24. Furthermore, the R120G mutation in HSPB5, is also associated with impaired neurofilament assembly, resulting in pathological aggregates of desmin filaments 34. These findings underscore the functional relationship between small HSPs and the cytoskeleton, and link several neuronal diseases with intermediate filaments. Interestingly, mutations in the neurofilament light chain gene (NEFL) cause another variant of CMT2 (CMT2E) 62 and mutations in the peripherin gene (PRPH) are associated with ALS 63. At present, HSPB8 has not been shown to interact directly with cytoskeletal elements, however since small HSPs operate in association with each other, it is possible that HSPB8 does interact with microfilaments and intermediate filaments via its molecular partner HSPB1. The missense mutations in HSPB1 and HSPB8 in distal HMN and CMT2 could therefore interfere either directly or indirectly with cytoskeletal functions and affect axonal transport in motor and sensory neurons (Figure 2). Of note is that mutations in dynamin 2 (DNM2) and dynactin (DCTN1), proteins that are involved in axonal transport, result in peripheral neuropathy 64, 65.

Neuroprotective role of HSPB1

HSPB1 is expressed in the PNS and might be important for axonal outgrowth, since it is induced in Schwann cells and regenerated axons 66, 67. Furthermore, expression of HSPB1 is upregulated after damage to adult peripheral sensory and motor neurons 66, 68, 69. In addition, exogenous delivery of HSPB1 is able to rescue injured sensory, sympathetic and motor neurons in vitro and in vivo69, 70. This neuroprotective activity of HSPB1 is crucial, because in vivo antisense RNA studies showed that HSPB1 is necessary to inhibit neuronal cell death 69. As for other cellular functions of HSPB1, the cell survival‐promoting activity seems to be regulated by phosphorylation 69. It has been shown that p38 MAP kinase, which regulates the phosphorylation of HSPB1, plays a fundamental role in the PNS by regulating cell shape and gene expression associated with Schwann cell myelination 71.

Increased neuronal cell survival by HSPB1 is not limited to the PNS because several studies indicate its upregulation and protective action also in the central nervous system. In hippocampal and retinal ganglion neurons, Hspb1 is also upregulated after injury 72, 73. Exogenous HSPB1 protects against neuronal loss in an in vivo model of epilepsy, in a cellular model of Huntington disease, and in an in vitro mammalian cell model of α‐synuclein‐neurotoxicity 49, 74, 75. Furthermore, the neuroprotective effect of increased HSPB1 levels was demonstrated in transgenic HSPB1 mice, which had reduced kainate‐induced seizures and hippocampal cell death 76.

It is assumed that loss of constitutive HSPB1 correlates with and promotes neurodegeneration. In the ‘paralyse mouse mutant’, a model of early onset SMA, the expression level of Hspb1 is decreased compared with control mice 77. Furthermore, in a cellular model for spinocerebellar ataxia type 3, expressing mutant ataxin‐3, HSPB1 was down‐regulated, suggesting that loss of HSPB1 has a pathologic effect 78. HSPB1 expression is also differentially and temporally regulated in mouse models of ALS. Recently, decreased Hspb1 expression was demonstrated several weeks prior to onset of motor neuron death in Cu/Zn superoxide dismutase‐1 (SOD1) mutant mice 79. Other studies demonstrated that expression of Hspb1 was gradually upregulated in spinal cords of early and late symptomatic SOD1 mutant mice relative to non‐symptomatic mutant mice 20, 80.

Until now, the exact mechanism of protection against neuronal cell death is unclear, but the cytoprotective roles of HSPB1 including chaperone‐like activity, anti‐apoptotic activity and preservation of cytoskeletal stability, may all contribute to neuroprotection. The survival‐promoting effect may be associated with the anti‐apoptotic actions of HSPB1. More recently, a direct anti‐apoptotic activity of HSPB1 was shown in a cellular model of Parkinson disease. In this model overexpression of HSPB1 delays the release of cytochrome C, reduces caspase activity and prevents apoptosis 81.

Until now there is no direct evidence that HSPB8 is also neuroprotective. Since HSPB8 acts as a cytoprotective component in non‐neuronal cell lines 34, it is plausible that it has the ability to protect neurons from cell death. The specific mechanisms underlying this neuroprotection are still to be defined. It will be important to characterize the mechanisms by which HSPB1 and HSPB8 contribute to neuronal survival, because this may lead to the identification of new strategies to prevent neuronal death.

Indeed, compounds inducing HSPs may be useful as a potential treatment for human neurodegenerative diseases. Recently a co‐inducer of HSP expression, arimoclomol, significantly delayed disease progression in a mouse model of ALS, even when administered after the disease onset 82. Furthermore, geldanamycin, a naturally occurring anticancer drug, induces HSP expression in different neuronal cell types and suppresses huntingtin protein aggregation in a cellular model of Huntington disease by modulating the expression of HSP40, HSP70 and HSP90 83. Also immunophilin‐ligands, which are used in transplantation to suppress allograft such as FK506, rapidly induce the expression of HSP70 and HSPB1. The administration of FK506 dramatically reduces signs of neuropathy and markedly protects against axonal loss during acrylamide induced neuropathy 84. Major drawbacks of these components are the considerable side effects, which limit their application in humans 85.

Concluding remarks

The identification of mutations in small HSPs associated with human motor and sensory neuropathies, indicates their pivotal role in the PNS. The diversity of interactions and cytoprotective functions of small HSPs make identifying the underlying pathologic mechanism of these neuromuscular disorders difficult. The mutations could interfere with the chaperone‐like activity or oligomerization property of these small HSPs and may be responsible for the aggregates found in cultured cells expressing the mutant proteins (Figure 2) 23, 34. Another possibility to explain the induction of neuronal cell death by mutant small HSPs, is their capacity to modulate apoptotic pathways, either directly or indirectly (Figure 2). Therefore, the HSPB1 mutations observed in patients with inherited neuropathies could interfere with binding of cytochrome C 24, 50. In this respect, mutations in these small HSPs could also influence the pro‐survival activity of HSPB1 or the pro‐apoptotic activity of HSPB8. Furthermore, the disruption of neurofilament assembly in cells expressing mutant HSPB1 is an indication that this small HSP has a role in the maintenance of cytoskeletal dynamics and axonal transport, which could also be relevant for the pathologic mechanism in these inherited peripheral neuropathies. The length of sensory and motor axons make these cell‐types highly susceptible to defects in axonal transport. The identification of the complete molecular and functional components of small HSPs in motor and sensory neurons will allow understanding why these neurons are specifically affected by mutations in HSPB1 and HSPB8, and will subsequently be useful to develop therapeutic approaches in the future.

If mutations in HSPB1 and HSPB8 result in a loss‐of‐function phenotype, components that upregulate HSP‐expression will be useful for hereditary peripheral neuropathies. However, if mutant small HSPs act as toxic components that induce neuronal cell death, an upregulation of these mutant proteins could make the pathology even worse. The observed mutant phenotype could also be a complex combination of loss‐of‐function and dominant‐negative effects. Therefore, the ideal therapeutic strategy would be to selectively inhibit the expression of the mutant protein and maintain the expression of wild‐type proteins. Hypothetically, since small HSPs are structurally closely related, development of safe therapeutic approaches with the aim to upregulate HSPB1 in patients with mutant HSPB8 and HSPB8 in patients with mutant HSPB1 may improve the neuronal loss observed in distal HMN and CMT2.

Acknowledgements

ID is supported by a PhD fellowship of the Institute for Science and Technology (IWT), and JI is supported by a postdoctoral fellowship of the Fund for Scientific Research (FWO‐Flanders), Belgium.