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

Nlrp6 promotes recovery after peripheral nerve injury independently of inflammasomes

Background: NOD-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)-1 beta and IL-18. However, their role in response to injury in the nervous system is less understood. Methods: In 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. Results: While 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-1 beta upon acute nerve injury despite potent induction of pro-IL-1 beta 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. Conclusions: Together, our observations suggest that Nlrp6 contributes to recovery from peripheral nerve injury by dampening inflammatory responses independently of IL-1 beta and inflammasomes

Microtubule dynamics in the peripheral nervous system: A matter of balance

The special architecture of neurons in the peripheral nervous system, with axons extending for long distances, represents a major challenge for the intracellular transport system. Two recent studies show that mutations in the small heat shock protein HSPB1, which cause an axonal type of Charcot-Marie-Tooth (CMT) neuropathy, affect microtubule dynamics and impede axonal transport. Intriguingly, while at presymptomatic age the neurons in the mutant HSPB1 mouse show a hyperstable microtubule network, at postsymptomatic age, the microtubule network completely lost its stability as reflected by a marked decrease in tubulin acetylation levels. We here propose a model explaining the role of microtubule stabilization and tubulin acetylation in the pathogenesis of HSPB1 mutations

A systematic comparison of all mutations in hereditary sensory neuropathy type I (HSAN I) reveals that the G387A mutation is not disease associated

Hereditary sensory neuropathy type 1 (HSAN I) is an autosomal dominant inherited neurodegenerative disorder of the peripheral nervous system associated with mutations in the SPTLC1 subunit of the serine palmitoyltransferase (SPT). Four missense mutations (C133W, C133Y, V144D and G387A) in SPTLC1 were reported to cause HSAN I. SPT catalyses the condensation of Serine and Palmitoyl-CoA, which is the first and rate-limiting step in the de novo synthesis of ceramides. Earlier studies showed that C133W and C133Y mutants have a reduced activity, whereas the impact of the V144D and G387A mutations on the human enzyme was not tested yet. In this paper, we show that none of the HSAN I mutations interferes with SPT complex formation. We demonstrate that also V144D has a reduced SPT activity, however to a lower extent than C133W and C133Y. In contrast, the G387A mutation showed no influence on SPT activity. Furthermore, the growth phenotype of LY-B cells—a SPTLC1 deficient CHO cell line—could be reversed by expressing either the wild-type SPTLC1 or the G387A mutant, but not the C133W mutant. This indicates that the G387A mutation is most likely not directly associated with HSAN I. These findings were genetically confirmed by the identification of a nuclear HSAN family which showed segregation of the G387A variant as a non-synonymous SN

HSPB1 facilitates ERK-mediated phosphorylation and degradation of BIM to attenuate endoplasmic reticulum stress-induced apoptosis

BIM, a pro-apoptotic BH3-only protein, is a key regulator of the intrinsic (or mitochondrial) apoptosis pathway. Here, we show that BIM induction by endoplasmic reticulum (ER) stress is suppressed in rat PC12 cells overexpressing heat shock protein B1 (HSPB1 or HSP27) and that this is due to enhanced proteasomal degradation of BIM. HSPB1 and BIM form a complex that immunoprecipitates with p-ERK1/2. We found that HSPB1-mediated proteasomal degradation of BIM is dependent on MEK-ERK signaling. Other studies have shown that several missense mutations in HSPB1 cause the peripheral neuropathy, Charcot-Marie-Tooth (CMT) disease, which is associated with nerve degeneration. Here we show that cells overexpressing CMT-related HSPB1 mutants exhibited increased susceptibility to ER stress-induced cell death and high levels of BIM. These findings identify a novel function for HSPB1 as a negative regulator of BIM protein stability leading to protection against ER stress-induced apoptosis, a function that is absent in CMT-associated HSPB1 mutants

Sensory-neuropathy-causing mutations in ATL3 cause aberrant ER membrane tethering

The endoplasmic reticulum (ER) is a complex network of sheets and tubules that is continuously remodeled. The relevance of this membrane dynamics is underscored by the fact that mutations in atlastins (ATLs), the ER fusion proteins in mammals, cause neurodegeneration. How defects in this process disrupt neuronal homeostasis is unclear. Using electron microscopy (EM) volume reconstruction of transfected cells, neurons, and patient fibroblasts, we show that hereditary sensory and autonomic neuropathy (HSAN)-causing ATL3 mutants promote aberrant ER tethering hallmarked by bundles of laterally attached ER tubules. In vitro, these mutants cause excessive liposome tethering, recapitulating the results in cells. Moreover, ATL3 variants retain their dimerization-dependent GTPase activity but are unable to promote membrane fusion, suggesting a defect in an intermediate step of the ATL3 functional cycle. Our data show that the effects of ATL3 mutations on ER network organization go beyond a loss of fusion and shed light on neuropathies caused by atlastin defects

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

Molecular Chaperones in the Pathogenesis of Amyotrophic Lateral Sclerosis: The Role of HSPB1

open15siGenetic 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.openCapponi, Simona; Geuens, Thomas; Geroldi, Alessandro; Origone, Paola; Verdiani, Simonetta; Cichero, Elena; Adriaenssens, Elias; De Winter, Vicky; Bandettini di Poggio, Monica; Barberis, Marco; Chiò, Adriano; Fossa, Paola; Mandich, Paola; Bellone, Emilia; Timmerman, VincentCapponi, Simona; Geuens, Thomas; Geroldi, Alessandro; Origone, Paola; Verdiani, Simonetta; Cichero, Elena; Adriaenssens, Elias; De Winter, Vicky; BANDETTINI DI POGGIO, MONICA LAURA; Barberis, Marco; Chiò, Adriano; Fossa, Paola; Mandich, Paola; Bellone, Emilia; Timmerman, Vincen

Profiling peripheral nerve macrophages reveals two macrophage subsets with distinct localization, transcriptome and response to injury

The authors identify two subsets of peripheral nerve macrophages residing in the endoneurium and the epineurium and displaying a distinct transcriptome and response to injury. These cells lack the main microglia identity and have a distinct origin. While CNS microglia have been extensively studied, relatively little is known about macrophages populating the peripheral nervous system. Here we performed ontogenic, transcriptomic and spatial characterization of sciatic nerve macrophages (snMacs). Using multiple fate-mapping systems, we show that snMacs do not derive from the early embryonic precursors colonizing the CNS, but originate primarily from late embryonic precursors and become replaced by bone-marrow-derived macrophages over time. Using single-cell transcriptomics, we identified a tissue-specific core signature of snMacs and two spatially separated snMacs: Relm alpha(+)Mgl1(+) snMacs in the epineurium and Relm alpha(-)Mgl1(-) snMacs in the endoneurium. Globally, snMacs lack most of the core signature genes of microglia, with only the endoneurial subset expressing a restricted number of these genes. In response to nerve injury, the two resident snMac populations respond differently. Moreover, and unlike in the CNS, monocyte-derived macrophages that develop during injury can engraft efficiently in the pool of resident peripheral nervous system macrophages