Titel Deelnemers "Korte inhoud" "Histone Deacetylase 6 (HDAC6) as therapeutic target in axonal Charcot-Marie-Tooth disease" "Veronick Benoy" "Charcot-Marie-Tooth disease (CMT) is the most common inherited disorder of the peripheral nervous system, affecting 1 in 2500 people. Progressive degeneration of the motor nerves leads to the development of motor problems such as muscle wasting and weakness, steppage gate and deformities of hand and feet. Also the sensory nerves are affected leading to reduced sensation of touch, pain and temperature. When only motor axons are affected, the disease is referred to as distal Hereditary Motor Neuropathy (distal HMN). Currently, no curative treatment exists for CMT or distal HMN patients. Previously, it was shown that pharmacologic inhibition of Histone Deacetylase 6 (HDAC6) is beneficial in a mouse model for the axonal form of CMT (CMT2), expressing mutations in small Heat Shock Protein B1 (HSPB1). HDAC6 is a major α-tubulin deacetylating enzyme and plays a role in the regulation of axonal transport. Defects in mitochondrial axonal transport are often associated with neurodegenerative disorders and peripheral neuropathies in particular. Specifically, we wanted to study if HDAC6 could serve as a potential target for the development of a therapeutic strategy. This research question was tackled in two different ways. CMT and distal HMN can be caused by mutations in more than 70 genes. Therefore, in a first step, we wanted to investigate whether pharmacological inhibition of HDAC6 has beneficial effects when motor and sensory axonal degeneration arises as a consequence of other genetic alterations in HSPB1 and in Glycyl-tRNA Synthetase (GARS). In a second step, we focused on the translation of selective HDAC6 inhibition into a clinical application. To investigate our first approach, we selected different mouse models for distal HMN or CMT2 in which we could study the effect of selective HDAC6 inhibition on the motor and sensory behavioral defects. First, we focused on other mutations in the gene encoding HSPB1 as a cause of distal HMN. Therefore, we used the previously developed and characterized mouse model for distal HMN, caused by the neuronal overexpression of human HSPB1P182L. Secondly, we extended our research to another animal model representative for CMT2. This mouse model was developed by inducing a mutation in the gene encoding GARS and became an interesting tool to study the pathogenesis of CMT2 as the motor and sensory deficits are caused by an endogenous mutation in Gars and thus no overexpression is needed to develop a phenotype. In both animal models for distal HMN and CMT2 we were able to demonstrate that the therapeutic potential of HDAC6 inhibition extends beyond mutant HSPB1-induced CMT2. We could show that inhibition of HDAC6 restores the motor and sensory problems on the behavioral level and also on the electrophysiological level. Moreover, these mouse models allowed us to study the presence of specific pathological deficits, such as alterations in the acetylation of α-tubulin and defects in the mitochondrial axonal transport, as possible hallmarks of peripheral nerve degeneration. Decreased acetylation of α-tubulin was present only in peripheral nerve tissue and in both the mutant HSPB1-induced distal HMN and in the mutant Gars-induced CMT2 mouse models. Also the disturbances in the axonal transport of mitochondria were observed in DRG neurons cultured from both disease models. Interestingly, these defects could be restored by selective inhibition of HDAC6. This indicates that decreased α-tubulin acetylation and mitochondrial transport defects are part of pathology common to peripheral nerve degeneration. Additionally, this can form the basis for the development of new therapeutic strategies for CMT. Lastly, to focus on the translation of HDAC6 inhibition into a clinical therapeutic strategy, we developed a compound screening based on the pathological findings of the mutant HSPB1-induced CMT2 mouse model. This screening method was used to develop and characterize HDAC6 inhibitors with improved pharmacokinetic properties suited for testing in clinical trials. In total, 70 chemical structures were tested in our screening method. The testing results of 35 compounds are summarized in this work. All the compounds were similar in their functional group, a hydroxamic acid, but different in their capping group which is responsible for HDAC6 surface recognition. The compound screening resulted in a selection of 3 molecules as potent and selective HDAC6 inhibitors that were able to restore the motor and sensory deficits of the mutant HSPB1-induced CMT2 mouse model. Interestingly, one of the inhibitors is already being tested in a phase IIb clinical trial for multiple myeloma. Thus, the positive results obtained in this screening will advance this compound faster into clinical trials for CMT patients. In summary, we were able to demonstrate that selective inhibition of HDAC6 is beneficial in several animal models of distal HMN and CMT2, further supporting the therapeutic potential of pharmacological inhibition of HDAC6 in specific subtypes of distal HMN and CMT patients. These data encourage further investigation of the effects of HDAC6 inhibition in other subforms of distal HMN and CMT, eventually aiming at the development of a curative treatment for distal HMN and CMT. Moreover, decreased acetylation of α-tubulin and defects in mitochondrial axonal transport are part of pathology common to mutant HSPB1-induced distal HMN/CMT2 and mutant GARS-induced CMT2. This indicates a more general mechanism for peripheral neuropathies involving decreased acetylation of α-tubulin and mitochondrial axonal transport defects. Finally, through our compound screening we were able to identify ACY-1215 with a good drug-like profile that acts as a potent and selective HDAC6 inhibitor for the treatment of CMT and distal HMN." "Increased monomerization of mutant HSPB1 leads to protein hyperactivity in Charcot-Marie-tooth neuropathy" "Leonardo Almeida-Souza, Sofie Goethals, Vicky de Winter, Ines Dierick, Rodrigo Gallardo, Joost Van Durme, Joy Irobi, Jan Gettemans, Frederic Rousseau, Joost Schymkowitz, Vincent Timmerman, Sophie Janssens" "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 wildtype HSPB1 protein undergoes monomerization during heat-shock activation, strongly suggesting that the monomer is the active form of the HSPB1 protein." "An adapted GeneSwitch toolkit for comparable cellular and animal models" "Laura Morant, Maria-Luise Petrovic-Erfurth, Albena Jordanova" "Investigating the impact of disease-causing mutations, their affected pathways, and/or potential therapeutic strategies using disease modeling often requires the generation of different in vivo and in cellulo models. To date, several approaches have been established to induce transgene expression in a controlled manner in different model systems. Several rounds of subcloning are, however, required, depending on the model organism used, thus bringing labor-intensive experiments into the technical approach and analysis comparison. The GeneSwitch (TM) technology is an adapted version of the classical UAS-GAL4 inducible system, allowing the spatial and temporal modulation of transgene expression. It consists of three components: a plasmid encoding for the chimeric regulatory pSwitch protein, Mifepristone as an inducer, and an inducible plasmid. While the pSwitch-containing first plasmid can be used both in vivo and in cellulo, the inducible second plasmid can only be used in cellulo. This requires a specific subcloning strategy of the inducible plasmid tailored to the model organism used. To avoid this step and unify gene expression in the transgenic models generated, we replaced the backbone vector with standard pUAS-attB plasmid for both plasmids containing either the chimeric GeneSwitch (TM) cDNA sequence or the transgene cDNA sequence. We optimized this adapted system to regulate transgene expression in several mammalian cell lines. Moreover, we took advantage of this new system to generate unified cellular and fruit fly models for YARS1-induced Charco-Marie-Tooth neuropathy (CMT). These new models displayed the expected CMT-like phenotypes. In the N2a neuroblastoma cells expressing YARS1 transgenes, we observed the typical ""teardrop"" distribution of the synthetase that was perturbed when expressing the YARS1CMT mutation. In flies, the ubiquitous expression of YARS1CMT induced dose-dependent developmental lethality and pan-neuronal expression caused locomotor deficit, while expression of the wild-type allele was harmless. Our proof-of-concept disease modeling studies support the efficacy of the adapted transgenesis system as a powerful tool allowing the design of studies with optimal data comparability." "Modeling CMT1A and investigating HDAC inhibitors as a therapeutic strategy" "Robert Ciarán Prior" "Charcot-Marie-Tooth disease (CMT) is the most common inherited neurological disorder of the peripheral nervous system (PNS). Patients suffer from mild-to-severe muscle loss at the most distal regions of their body in a 'stock-glove' distribution. Duplications in the gene coding for peripheral myelin protein 22 (PMP22) causes the most common form of CMT, CMT type 1A (CMT1A). PMP22 is an essential protein for the initiation and maintenance of Schwann cell myelination in the PNS. Myelin is a multilamellar lipid dense structure Schwann cells produce to enables fast and efficient 'signalling' of PNS sensory and motor axons so that we may take in sensory information and respond to our environment. In CMT1A, the duplication PMP22 is known to cause lipid metabolism defects that ultimately impar CMT1A Schwann cells in their ability to myelinate PNS axons, however, how PMP22 duplications caused this is unknown. In the first part of this thesis, we investigated whether inhibiting known negative regulator of myelination, histone deacetylase inhibitor 3 (HDAC3), was a valuable treatment strategy for CMT1A using the C3 CMT1A mouse model. We demonstrated that early treatment of CMT1A mice with the selective HDAC3 inhibitor (HDAC3i) increased myelination and myelin g-ratios, and improved electromyography recordings. However, a high dose of the HDAC3i caused a decline in rotarod performance and a decline in overall grip strength. Additionally, macrophage presence in peripheral nerves was increased in HDAC3i treated CMT1A mice. We conclude that HDAC3 does not only play a role in regulating myelination but is also important in neuroimmune modulation. Overall, our results indicate that correct dosing of HDAC3 inhibitors is of crucial importance if translated to a clinical setting for demyelinating forms of CMT, or other neurological disorders. In the second part of the thesis, we aimed to investigate the molecular mechanisms that lead to the a lipid metabolic defect in CMT1A. Bulk RNA-sequencing data of sciatic nerves from the C3 and the C22 CMT1A mouse models, which overexpress 5 and 8 copies of human PMP22 respectively, demonstrated that cholesterol metabolism is the most dysregulated pathway throughout their development. PMP22 overexpression had a dose-dependent effect on the suppression of cholesterol metabolism. Lipidomic analysis of sciatic nerves of 5 weeks old C3 mice revealed dysregulated relative expression of lipids regulating lipid storage/droplet homeostasis, plasma membrane identity, and very-long-chain polyunsaturated fatty acids (VLC-PUFAs). To model this lipid metabolism phenotype in patient and patient-corrected (isogenic) derived cells, we developed an induced pluripotent stem cell (iPSC) to Schwann cell lineage protocol. We differentiated iPSCs to Schwann cell precursors (iPSC-SCPs) and showed via bulk RNA-sequencing that CMT1A patient-derived iPSC-SCPs have dysregulated expression of plasma membrane-associated genes in comparison to their isogenic control iPSC-SCPs. Lipidomic analysis of these cells similarly revealed alteration in the relative expression of lipids regulating plasma membrane identity, VLC-PUFAs, and lipid storage/droplet homeostasis. Using electron microscopy, we identified lipid accumulations in late endosome-lysosomes in the CMT1A iPSC-SCPs. Moreover, Di-4-ANEPPDHQ flow cytometry analysis highlighted increased CMT1A iPSC-SCP membranes are more disordered compared to their isogenic iPSC-SCP. We further investigated these alterations via high-content confocal immunofluorescence imaging to analyze cholesterol distribution as well as LD and lysosome dynamics. We discovered that CMT1A iPSC-SCPs have reduced membrane cholesterol and, upon oleic acid stimulation, generate a larger size and quantity of LDs. This LD/storage phenotype was confirmed using Western blot analysis which showed alterations in DGAT1, perilipin-2, seipin, LAMP1, and NPC1. As a proof-of-concept, we showed that forskolin or progesterone antagonist treatment can modulate and improve the deficits in lipolysis/autophagy in CMT1A iPSC-SCPs. Moreover, we show that lipolysis/autophagy modulation in CMT1A iPSC-SCPs attenuates lipid recycling homeostasis and restores their membranes' cholesterol content. In conclusion, in this thesis, we highlight the therapeutic potential of HDAC3i for the treatment of CMT1A and shed light on some of the molecular mechanisms underlying lipid metabolic phenotype in CMT1A. Further research is needed to better understand how lipids are processed by CMT1A Schwann cells, as this may lead to more effective therapies being developed for this currently untreatable disease." "De novo PMP2 mutations in families with type 1 CharcotMarieTooth disease" "William W. Motley, Paulius Palaima, Sabrina W. Yum, Els De Vriendt, Albena Jordanova, et al." "We performed whole exome sequencing on a patient with CharcotMarieTooth disease type 1 and identified a de novo mutation in PMP2, the gene that encodes the myelin P2 protein. This mutation (p.Ile52Thr) was passed from the proband to his one affected son, and segregates with clinical and electrophysiological evidence of demyelinating neuropathy. We then screened a cohort of 136 European probands with uncharacterized genetic cause of CharcotMarieTooth disease and identified another family with CharcotMarieTooth disease type 1 that has a mutation affecting an adjacent amino acid (p.Thr51Pro), which segregates with disease. Our genetic and clinical findings in these kindred demonstrate that dominant PMP2 mutations cause CharcotMarieTooth disease type 1." "Mutations in HspB1 and hereditary neuropathies" "Sergei Strelkov" "Charcot-Marie-Tooth (CMT) disease is major hereditary neuropathy. CMT has been linked to mutations in a range of proteins, including the small heat shock protein HspB1. Here we review the properties of several HspB1 mutants associated with CMT. In vitro, mutations in the N-terminal domain lead to a formation of larger HspB1 oligomers when compared with the wild-type (WT) protein. These mutants are resistant to phosphorylation-induced dissociation and reveal lower chaperone-like activity than the WT on a range of model substrates. Mutations in the α-crystallin domain lead to the formation of yet larger HspB1 oligomers tending to dissociate at low protein concentration and having variable chaperone-like activity. Mutations in the conservative IPV motif within the C-terminal domain induce the formation of very large oligomers with low chaperone-like activity. Most mutants interact with a partner small heat shock protein, HspB6, in a manner different from that of the WT protein. The link between the altered physico-chemical properties and the pathological CMT phenotype is a subject of discussion. Certain HspB1 mutations appear to have an effect on cytoskeletal elements such as intermediate filaments and/or microtubules, and by this means damage the axonal transport. In addition, mutations of HspB1 can affect the metabolism in astroglia and indirectly modulate the viability of motor neurons. While the mechanisms of pathological mutations in HspB1 are likely to vary greatly across different mutations, further in vitro and in vivo studies are required for a better understanding of the CMT disease at molecular level." "Characterization of human small heat shock protein HSPB1 alpha-crystallin domain localized mutants associated with hereditary motor neuron diseases" "Stephen Weeks, Michelle Heirbaut, Sergei Strelkov" "Congenital mutations in human small heat shock protein HSPB1 (HSP27) have been linked to Charcot-Marie-Tooth disease, a commonly occurring peripheral neuropathy. Understanding the molecular mechanism of such mutations is indispensable towards developing future therapies for this currently incurable disorder. Here we describe the physico-chemical properties of the autosomal dominant HSPB1 mutants R127W, S135F and R136W. Despite having a nominal effect on thermal stability, the three mutations induce dramatic changes to quaternary structure. At high concentrations or under crowding conditions, the mutants form assemblies that are approximately two times larger than those formed by the wild-type protein. At low concentrations, the mutants have a higher propensity to dissociate into small oligomers, while the dissociation of R127W and R135F mutants is enhanced by MAPKAP kinase-2 mediated phosphorylation. Specific differences are observed in the ability to form hetero-oligomers with the homologue HSPB6 (HSP20). For wild-type HSPB1 this only occurs at or above physiological temperature, whereas the R127W and S135F mutants form hetero-oligomers with HSPB6 at 4 °C, and the R136W mutant fails to form hetero-oligomers. Combined, the results suggest that the disease-related mutations of HSPB1 modify its self-assembly and interaction with partner proteins thus affecting normal functioning of HSPB1 in the cell." "The chaperone-assisted selective autophagy complex dynamics and dysfunctions" "Barbara Tedesco, Leen Vendredy, Vincent Timmerman, Angelo Poletti" "Each protein must be synthesized with the correct amino acid sequence, folded into its native structure, and transported to a relevant subcellular location and protein complex. If any of these steps fail, the cell has the capacity to break down aberrant proteins to maintain protein homeostasis (also called proteostasis). All cells possess a set of well-characterized protein quality control systems to minimize protein misfolding and the damage it might cause. Autophagy, a conserved pathway for the degradation of long-lived proteins, aggregates, and damaged organelles, was initially characterized as a bulk degradation pathway. However, it is now clear that autophagy also contributes to intracellular homeostasis by selectively degrading cargo material. One of the pathways involved in the selective removal of damaged and misfolded proteins is chaperone-assisted selective autophagy (CASA). The CASA complex is composed of three main proteins (HSPA, HSPB8 and BAG3), essential to maintain protein homeostasis in muscle and neuronal cells. A failure in the CASA complex, caused by mutations in the respective coding genes, can lead to (cardio)myopathies and neurodegenerative diseases. Here, we summarize our current understanding of the CASA complex and its dynamics. We also briefly discuss how CASA complex proteins are involved in disease and may represent an interesting therapeutic target." "CMT-associated mutations in glycyl- and tyrosyl-tRNA synthetases exhibit similar pattern of toxicity and share common genetic modifiers in Drosophila" "William W. Motley, Ricardo Leitão-Gonçalves, Bob Asselbergh, Kristel Sleegers, Tinne Ooms, Vincent Timmerman, Albena Jordanova, et al." "Aminoacyl-tRNA synthetases are ubiquitously expressed proteins that charge tRNAs with their cognate amino acids. By ensuring the fidelity of protein synthesis, these enzymes are essential for the viability of every cell. Yet, mutations in six tRNA synthetases specifically affect the peripheral nerves and cause Charcot-Marie-Tooth (CMT) disease. The CMT-causing mutations in tyrosyl- and glycyl-tRNA synthetases (YARS and GARS, respectively) alter the activity of the proteins in a range of ways (some mutations do not impact charging function, while others abrogate it), making a loss of function in tRNA charging unlikely to be the cause of disease pathology. It is currently unknown which cellular mechanisms are triggered by the mutant enzymes and how this leads to neurodegeneration. Here, by expressing two pathogenic mutations (G240R, P234KY) in Drosophila, we generated a model for GARS-associated neuropathy. We observed compromised viability, and behavioral, electrophysiological and morphological impairment in flies expressing the cytoplasmic isoform of mutant GARS. Their features recapitulated several hallmarks of CMT pathophysiology and were similar to the phenotypes identified in our previously described Drosophila model of YARS-associated neuropathy. Furthermore, CG8316 and CG15599 - genes identified in a retinal degeneration screen to modify mutant YAKS, also modified the mutant GARS phenotypes. Our study presents genetic evidence for common mutant-specific interactions between two CMT-associated aminoacyl-tRNA synthetases, lending support for a shared mechanism responsible for the synthetase-induced peripheral neuropathies. (C) 2014 Elsevier Inc. All rights reserved." "Tyrosyl-tRNA synthetase has a noncanonical function in actin bundling" "Patrick Callaerts, Patrik Verstreken" "Dominant mutations in tyrosyl-tRNA synthetase (YARS1) and six other tRNA ligases cause Charcot-Marie-Tooth peripheral neuropathy (CMT). Loss of aminoacylation is not required for their pathogenicity, suggesting a gain-of-function disease mechanism. By an unbiased genetic screen in Drosophila, we link YARS1 dysfunction to actin cytoskeleton organization. Biochemical studies uncover yet unknown actin-bundling property of YARS1 to be enhanced by a CMT mutation, leading to actin disorganization in the Drosophila nervous system, human SH-SY5Y neuroblastoma cells, and patient-derived fibroblasts. Genetic modulation of F-actin organization improves hallmark electrophysiological and morphological features in neurons of flies expressing CMT-causing YARS1 mutations. Similar beneficial effects are observed in flies expressing a neuropathy-causing glycyl-tRNA synthetase. Hence, in this work, we show that YARS1 is an evolutionary-conserved F-actin organizer which links the actin cytoskeleton to tRNA-synthetase-induced neurodegeneration."