Vicky De Winter

Small Heat-Shock Protein HSPB1 Mutants Stabilize Microtubules in Charcot-Marie-Tooth Neuropathy

Mutations in the small heat shock protein HSPB1 (HSP27) are causative for Charcot-Marie-Tooth (CMT) neuropathy. We previously showed that a subset of these mutations displays higher chaperone activity and enhanced affinity to client proteins. We hypothesized that this excessive binding property might cause the HSPB1 mutant proteins to disturb the function of proteins essential for the maintenance or survival of peripheral neurons. In the present work, we explored this hypothesis further and compared the protein complexes formed by wild-type and mutant HSPB1. Tubulin came out as the most striking differential interacting protein, with hyperactive mutants binding more strongly to both tubulin and microtubules. This anomalous binding leads to a stabilization of the microtubule network in a microtubule-associated protein-like manner as reflected by resistance to cold depolymerization, faster network recovery after nocodazole treatment, and decreased rescue and catastrophe rates of individual microtubules. In a transgenic mouse model for mutant HSPB1 that recapitulates all features of CMT, we could confirm the enhanced interaction of mutant HSPB1 with tubulin. Increased stability of the microtubule network was also clear in neurons isolated from these mice. Since neuronal cells are particularly vulnerable to disturbances in microtubule dynamics, this mechanism might explain the neuron-specific CMT phenotype caused by HSPB1 mutations.

Increased Monomerization of Mutant HSPB1 Leads to Protein Hyperactivity in Charcot-Marie-Tooth Neuropathy

Small heat shock proteins are molecular chaperones capable of maintaining denatured proteins in a folding-competent state. We have previously shown that missense mutations in the small heat shock protein HSPB1 (HSP27) cause distal hereditary motor neuropathy and axonal Charcot-Marie-Tooth disease. Here we investigated the biochemical consequences of HSPB1 mutations that are known to cause peripheral neuropathy. In contrast to other chaperonopathies, our results revealed that particular HSPB1 mutations presented higher chaperone activity compared with wild type. Hyperactivation of HSPB1 was accompanied by a change from its wild-type dimeric state to a monomer without dissociation of the 24-meric state. Purification of protein complexes from wild-type and HSPB1 mutants showed that the hyperactive isoforms also presented enhanced binding to client proteins. Furthermore, we show that the wild-type HSPB1 protein undergoes monomerization during heat-shock activation, strongly suggesting that the monomer is the active form of the HSPB1 protein.

Mutant HSPB8 causes motor neuron-specific neurite degeneration.

Missense mutations (K141N and K141E) in the α-crystallin domain of the small heat shock protein HSPB8 (HSP22) cause distal hereditary motor neuropathy (distal HMN) or Charcot-Marie-Tooth neuropathy type 2L (CMT2L). The mechanism through which mutant HSPB8 leads to a specific motor neuron disease phenotype is currently unknown. To address this question, we compared the effect of mutant HSPB8 in primary neuronal and glial cell cultures. In motor neurons, expression of both HSPB8 K141N and K141E mutations clearly resulted in neurite degeneration, as manifested by a reduction in number of neurites per cell, as well as in a reduction in average length of the neurites. Furthermore, expression of the K141E (and to a lesser extent, K141N) mutation also induced spheroids in the neurites. We did not detect any signs of apoptosis in motor neurons, showing that mutant HSPB8 resulted in neurite degeneration without inducing neuronal death. While overt in motor neurons, these phenotypes were only very mildly present in sensory neurons and completely absent in cortical neurons. Also glial cells did not show an altered phenotype upon expression of mutant HSPB8. These findings show that despite the ubiquitous presence of HSPB8, only motor neurons appear to be affected by the K141N and K141E mutations which explain the predominant motor neuron phenotype in distal HMN and CMT2L.

Charcot–Marie–Tooth causing HSPB1 mutations increase Cdk5-mediated phosphorylation of neurofilaments

Mutations in the small heat shock protein HSPB1 (HSP27) are a cause of axonal Charcot–Marie–Tooth neuropathy (CMT2F) and distal hereditary motor neuropathy. To better understand the effect of mutations in HSPB1 on the neuronal cytoskeleton, we stably transduced neuronal cells with wild-type and mutant HSPB1 and investigated axonal transport of neurofilaments (NFs). We observed that mutant HSPB1 affected the binding of NFs to the anterograde motor protein kinesin, reducing anterograde transport of NFs. These deficits were associated with an increased phosphorylation of NFs and cyclin-dependent kinase Cdk5. As Cdk5 mediates NF phosphorylation, inhibition of Cdk5/p35 restored NF phosphorylation level, as well as NF binding to kinesin in mutant HSPB1 neuronal cells. Altogether, we demonstrate that HSPB1 mutations induce hyperphosphorylation of NFs through Cdk5 and reduce anterograde transport of NFs.

Nlrp6 promotes recovery after peripheral nerve injury independently of inflammasomes

BackgroundNOD-like receptors (Nlrs) are key regulators of immune responses during infection and autoimmunity. A subset of Nlrs assembles inflammasomes, molecular platforms that are activated in response to endogenous danger and microbial ligands and that control release of interleukin (IL)-1β and IL-18. However, their role in response to injury in the nervous system is less understood.MethodsIn this study, we investigated the expression profile of major inflammasome components in the peripheral nervous system (PNS) and explored the physiological role of different Nlrs upon acute nerve injury in mice.ResultsWhile in basal conditions, predominantly members of NOD-like receptor B (Nlrb) subfamily (NLR family, apoptosis inhibitory proteins (NAIPs)) and Nlrc subfamily (ICE-protease activating factor (IPAF)/NOD) are detected in the sciatic nerve, injury causes a shift towards expression of the Nlrp family. Sterile nerve injury also leads to an increase in expression of the Nlrb subfamily, while bacteria trigger expression of the Nlrc subfamily. Interestingly, loss of Nlrp6 led to strongly impaired nerve function upon nerve crush. Loss of the inflammasome adaptor apoptosis-associated speck-like protein containing a CARD (ASC) and effector caspase-1 and caspase-11 did not affect sciatic nerve function, suggesting that Nlrp6 contributed to recovery after peripheral nerve injury independently of inflammasomes. In line with this, we did not detect release of mature IL-1β upon acute nerve injury despite potent induction of pro-IL-1β and inflammasome components Nlrp3 and Nlrp1. However, Nlrp6 deficiency was associated with increased pro-inflammatory extracellular regulated MAP kinase (ERK) signaling, suggesting that hyperinflammation in the absence of Nlrp6 exacerbated peripheral nerve injury.ConclusionsTogether, our observations suggest that Nlrp6 contributes to recovery from peripheral nerve injury by dampening inflammatory responses independently of IL-1β and inflammasomes.

HSPB1 Facilitates the Formation of Non-Centrosomal Microtubules

The remodeling capacity of microtubules (MT) is essential for their proper function. In mammals, MTs are predominantly formed at the centrosome, but can also originate from non-centrosomal sites, a process that is still poorly understood. We here show that the small heat shock protein HSPB1 plays a role in the control of non-centrosomal MT formation. The HSPB1 expression level regulates the balance between centrosomal and non-centrosomal MTs. The HSPB1 protein can be detected specifically at sites of de novo forming non-centrosomal MTs, while it is absent from the centrosomes. In addition, we show that HSPB1 binds preferentially to the lattice of newly formed MTs in vitro, suggesting that its function occurs by stabilizing MT seeds. Our findings open new avenues for the understanding of the role of HSPB1 in the development, maintenance and protection of cells with specialized non-centrosomal MT arrays.

Molecular chaperones in the pathogenesis of amyotrophic lateral sclerosis : the role of HSPB1

Genetic discoveries in amyotrophic lateral sclerosis (ALS) have a significant impact on deciphering molecular mechanisms of motor neuron degeneration but, despite recent advances, the etiology of most sporadic cases remains elusive. Several cellular mechanisms contribute to the motor neuron degeneration in ALS, including RNA metabolism, cellular interactions between neurons and nonneuronal cells, and seeding of misfolded protein with prion‐like propagation. In this scenario, the importance of protein turnover and degradation in motor neuron homeostasis gained increased recognition. In this study, we evaluated the role of the candidate gene HSPB1, a molecular chaperone involved in several proteome‐maintenance functions. In a cohort of 247 unrelated Italian ALS patients, we identified two variants (c.570G>C, p.Gln190His and c.610dupG, p.Ala204Glyfs*6). Functional characterization of the p.Ala204Glyfs*6 demonstrated that the mutant protein alters HSPB1 dynamic equilibrium, sequestering the wild‐type protein in a stable dimer and resulting in a loss of chaperone‐like activity. Our results underline the relevance of identifying rare but pathogenic variations in sporadic neurodegenerative diseases, suggesting a possible correlation between specific pathomechanisms linked to HSPB1 mutations and the associated neurological phenotype. Our study provides additional lines of evidence to support the involvement of HSPB1 in the pathogenesis of sporadic ALS.

A knock-in/knock-out mouse model of HSPB8-associated distal hereditary motor neuropathy and myopathy reveals toxic gain-of-function of mutant Hspb8

Mutations in the small heat shock protein B8 gene (HSPB8/HSP22) have been associated with distal hereditary motor neuropathy, Charcot–Marie–Tooth disease, and recently distal myopathy. It is so far not clear how mutant HSPB8 induces the neuronal and muscular phenotypes and if a common pathogenesis lies behind these diseases. Growing evidence points towards a role of HSPB8 in chaperone-associated autophagy, which has been shown to be a determinant for the clearance of poly-glutamine aggregates in neurodegenerative diseases but also for the maintenance of skeletal muscle myofibrils. To test this hypothesis and better dissect the pathomechanism of mutant HSPB8, we generated a new transgenic mouse model leading to the expression of the mutant protein (knock-in lines) or the loss-of-function (functional knock-out lines) of the endogenous protein Hspb8. While the homozygous knock-in mice developed motor deficits associated with degeneration of peripheral nerves and severe muscle atrophy corroborating patient data, homozygous knock-out mice had locomotor performances equivalent to those of wild-type animals. The distal skeletal muscles of the post-symptomatic homozygous knock-in displayed Z-disk disorganisation, granulofilamentous material accumulation along with Hspb8, αB-crystallin (HSPB5/CRYAB), and desmin aggregates. The presence of the aggregates correlated with reduced markers of effective autophagy. The sciatic nerve of the homozygous knock-in mice was characterized by low autophagy potential in pre-symptomatic and Hspb8 aggregates in post-symptomatic animals. On the other hand, the sciatic nerve of the homozygous knock-out mice presented a normal morphology and their distal muscle displayed accumulation of abnormal mitochondria but intact myofiber and Z-line organisation. Our data, therefore, suggest that toxic gain-of-function of mutant Hspb8 aggregates is a major contributor to the peripheral neuropathy and the myopathy. In addition, mutant Hspb8 induces impairments in autophagy that may aggravate the phenotype.

Mutant HSPB1 causes loss of translational repression by binding to PCBP1, an RNA binding protein with a possible role in neurodegenerative disease

The small heat shock protein HSPB1 (Hsp27) is an ubiquitously expressed molecular chaperone able to regulate various cellular functions like actin dynamics, oxidative stress regulation and anti-apoptosis. So far disease causing mutations in HSPB1 have been associated with neurodegenerative diseases such as distal hereditary motor neuropathy, Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis. Most mutations in HSPB1 target its highly conserved α-crystallin domain, while other mutations affect the C- or N-terminal regions or its promotor. Mutations inside the α-crystallin domain have been shown to enhance the chaperone activity of HSPB1 and increase the binding to client proteins. However, the HSPB1-P182L mutation, located outside and downstream of the α-crystallin domain, behaves differently. This specific HSPB1 mutation results in a severe neuropathy phenotype affecting exclusively the motor neurons of the peripheral nervous system. We identified that the HSPB1-P182L mutant protein has a specifically increased interaction with the RNA binding protein poly(C)binding protein 1 (PCBP1) and results in a reduction of its translational repressive activity. RNA immunoprecipitation followed by RNA sequencing on mouse brain lead to the identification of PCBP1 mRNA targets. These targets contain larger 3′- and 5′-UTRs than average and are enriched in an RNA motif consisting of the CTCCTCCTCCTCC consensus sequence. Interestingly, next to the clear presence of neuronal transcripts among the identified PCBP1 targets we identified known genes associated with hereditary peripheral neuropathies and hereditary spastic paraplegias. We therefore conclude that HSPB1 can mediate translational repression through interaction with an RNA binding protein further supporting its role in neurodegenerative disease.

Sensory neuropathy-causing mutations in ATL3 affect ER–mitochondria contact sites and impair axonal mitochondrial distribution

Abstract Axonopathies are neurodegenerative disorders caused by axonal degeneration, affecting predominantly the longest neurons. Several of these axonopathies are caused by genetic defects in proteins involved in the shaping and dynamics of the endoplasmic reticulum (ER); however, it is unclear how these defects impinge on neuronal survival. Given its central and widespread position within a cell, the ER is a pivotal player in inter-organelle communication. Here, we demonstrate that defects in the ER fusion protein ATL3, which were identified in patients suffering from hereditary sensory and autonomic neuropathy, result in an increased number of ER–mitochondria contact sites both in HeLa cells and in patient-derived fibroblasts. This increased contact is reflected in higher phospholipid metabolism, upregulated autophagy and augmented Ca2+ crosstalk between both organelles. Moreover, the mitochondria in these cells display lowered motility, and the number of axonal mitochondria in neurons expressing disease-causing mutations in ATL3 is strongly decreased. These results underscore the functional interdependence of subcellular organelles in health and disease and show that disorders caused by ER-shaping defects are more complex than previously assumed.