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Inherited peripheral neuropathies (IPNs) are a group of diseases associated with mutations in various genes with fundamental roles in the development and function of peripheral nerves. Over the past 10 years, significant advances in identifying molecular disease mechanisms underlying axonal and myelin degeneration, acquired from cellular biology studies and transgenic fly and rodent models, have facilitated the development of promising treatment strategies. However, no clinical treatment has emerged to date. This lack of treatment highlights the urgent need for more biologically and clinically relevant models recapitulating IPNs. For both neurodevelopmental and neurodegenerative diseases, patient-specific induced pluripotent stem cells (iPSCs) are a particularly powerful platform for disease modeling and preclinical studies. In this review, we provide an update on different in vitro human cellular IPN models, including traditional two-dimensional monoculture iPSC derivatives, and recent advances in more complex human iPSC-based systems using microfluidic chips, organoids, and assembloids.
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Células Madre Pluripotentes Inducidas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Animales , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedades del Sistema Nervioso Periférico/patología , Enfermedades del Sistema Nervioso Periférico/terapia , Organoides/metabolismo , Modelos BiológicosRESUMEN
Torin1, a selective kinase inhibitor targeting the mammalian target of rapamycin (mTOR), remains widely used in autophagy research due to its potent autophagy-inducing abilities, regardless of its unspecific properties. Recognizing the impact of mTOR inhibition on metabolism, our objective was to develop a reliable and thorough untargeted metabolomics workflow to study torin1-induced metabolic changes in mouse embryonic fibroblast (MEF) cells. Crucially, our quality assurance and quality control (QA/QC) protocols were designed to increase confidence in the reported findings by reducing the likelihood of false positives, including a validation experiment replicating all experimental steps from sample preparation to data analysis. This study investigated the metabolic fingerprint of torin1 exposure by using liquid chromatography-high resolution mass spectrometry (LC-HRMS)-based untargeted metabolomics platforms. Our workflow identified 67 altered metabolites after torin1 exposure, combining univariate and multivariate statistics and the implementation of a validation experiment. In particular, intracellular ceramides, diglycerides, phosphatidylcholines, phosphatidylethanolamines, glutathione, and 5'-methylthioadenosine were downregulated. Lyso-phosphatidylcholines, lyso-phosphatidylethanolamines, glycerophosphocholine, triglycerides, inosine, and hypoxanthine were upregulated. Further biochemical pathway analyses provided deeper insights into the reported changes. Ultimately, our study provides a valuable workflow that can be implemented for future investigations into the effects of other compounds, including more specific autophagy modulators.
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International consortia collaborating on the genetics of rare diseases have significantly boosted our understanding of inherited neurological disorders. Historical clinical classification boundaries were drawn between disorders with seemingly different etiologies, such as inherited peripheral neuropathies (IPNs), spastic paraplegias, and cerebellar ataxias. These clinically defined borders are being challenged by the identification of mutations in genes displaying wide phenotypic spectra and by shared pathomechanistic themes, which are valuable indications for therapy development. We highlight common cellular alterations that underlie this genetic landscape, including alteration of cytoskeleton, axonal transport, mitochondrial function, and DNA repair response. Finally, we discuss venues for future research using the long axonopathies of the PNS as a model to explore other neurogenetic disorders.
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Ataxia Cerebelosa , Enfermedades del Sistema Nervioso Periférico , Paraplejía Espástica Hereditaria , Humanos , Ataxia Cerebelosa/genética , Paraplejía Espástica Hereditaria/genética , Enfermedades del Sistema Nervioso Periférico/genética , Mutación/genética , ParaplejíaRESUMEN
Myelin protein zero (MPZ/P0) is a major structural protein of peripheral nerve myelin. Disease-associated variants in the MPZ gene cause a wide phenotypic spectrum of inherited peripheral neuropathies. Previous nerve biopsy studies showed evidence for subtype-specific morphological features. Here, we aimed at enhancing the understanding of these subtype-specific features and pathophysiological aspects of MPZ neuropathies. We examined archival material from two Central European centers and systematically determined genetic, clinical, and neuropathological features of 21 patients with MPZ mutations compared to 16 controls. Cases were grouped based on nerve conduction data into congenital hypomyelinating neuropathy (CHN; n = 2), demyelinating Charcot-Marie-Tooth (CMT type 1; n = 11), intermediate (CMTi; n = 3), and axonal CMT (type 2; n = 5). Six cases had combined muscle and nerve biopsies and one underwent autopsy. We detected four MPZ gene variants not previously described in patients with neuropathy. Light and electron microscopy of nerve biopsies confirmed fewer myelinated fibers, more onion bulbs and reduced regeneration in demyelinating CMT1 compared to CMT2/CMTi. In addition, we observed significantly more denervated Schwann cells, more collagen pockets, fewer unmyelinated axons per Schwann cell unit and a higher density of Schwann cell nuclei in CMT1 compared to CMT2/CMTi. CHN was characterized by basal lamina onion bulb formation, a further increase in Schwann cell density and hypomyelination. Most late onset axonal neuropathy patients showed microangiopathy. In the autopsy case, we observed prominent neuromatous hyperinnervation of the spinal meninges. In four of the six muscle biopsies, we found marked structural mitochondrial abnormalities. These results show that MPZ alterations not only affect myelinated nerve fibers, leading to either primarily demyelinating or axonal changes, but also affect non-myelinated nerve fibers. The autopsy case offers insight into spinal nerve root pathology in MPZ neuropathy. Finally, our data suggest a peculiar association of MPZ mutations with mitochondrial alterations in muscle.
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Enfermedad de Charcot-Marie-Tooth , Proteína P0 de la Mielina , Humanos , Proteína P0 de la Mielina/genética , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/patología , Mutación/genética , Proteínas/genética , BiopsiaRESUMEN
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.
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Actinas , Tirosina-ARNt Ligasa , Animales , Humanos , Actinas/metabolismo , Enfermedad de Charcot-Marie-Tooth/genética , Drosophila/genética , Glicina-ARNt Ligasa/genética , Mutación , ARN de Transferencia , Tirosina-ARNt Ligasa/genética , Tirosina-ARNt Ligasa/metabolismo , Línea Celular TumoralRESUMEN
Chaperone-assisted selective autophagy (CASA) is a highly selective pathway for the disposal of misfolding and aggregating proteins. In muscle, CASA assures muscle integrity by favoring the turnover of structural components damaged by mechanical strain. In neurons, CASA promotes the removal of aggregating substrates. A crucial player of CASA is HSPB8 (heat shock protein family B (small) member 8), which acts in a complex with HSPA, their cochaperone BAG3, and the E3 ubiquitin ligase STUB1. Recently, four novel HSPB8 frameshift (fs) gene mutations have been linked to neuromyopathies, and encode carboxy-terminally mutated HSPB8, sharing a common C-terminal extension. Here, we analyzed the biochemical and functional alterations associated with the HSPB8_fs mutant proteins. We demonstrated that HSPB8_fs mutants are highly insoluble and tend to form proteinaceous aggregates in the cytoplasm. Notably, all HSPB8 frameshift mutants retain their ability to interact with CASA members but sequester them into the HSPB8-positive aggregates together with two autophagy receptors SQSTM1/p62 and TAX1BP1. This copartitioning process negatively affects the CASA capability to remove its clients and causes a general failure in proteostasis response. Further analyses revealed that the aggregation of the HSPB8_fs mutants occurs independently of the other CASA members or from the autophagy receptors interaction, but it is an intrinsic feature of the mutated amino acid sequence. HSPB8_fs mutants aggregation alters the differentiation capacity of muscle cells and impairs sarcomere organization. Collectively, these results shed light on a potential pathogenic mechanism shared by the HSPB8_fs mutants described in neuromuscular diseases.Abbreviations : ACD: α-crystallin domain; ACTN: actinin alpha; BAG3: BAG cochaperone 3; C: carboxy; CASA: chaperone-assisted selective autophagy; CE: carboxy-terminal extension; CLEM: correlative light and electron microscopy; CMT2L: Charcot-Marie-Tooth type 2L; CTR: carboxy-terminal region; dHMNII: distal hereditary motor neuropathy type II; EV: empty vector; FRA: filter retardation assay; fs: frameshift; HSPA/HSP70: heat shock protein family A (Hsp70); HSPB1/Hsp27: heat shock protein family B (small) member 1; HSPB8/Hsp22: heat shock protein family B (small) member 8; HTT: huntingtin; KO: knockout; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MD: molecular dynamics; MTOC: microtubule organizing center; MYH: myosin heavy chain; MYOG: myogenin; NBR1: NBR1 autophagy cargo receptor; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; NSC34: Neuroblastoma X Spinal Cord 34; OPTN: optineurin; polyQ: polyglutamine; SQSTM1/p62: sequestosome 1; STUB1/CHIP: STIP1 homology and U-box containing protein 1; TARDBP/TDP-43: TAR DNA binding protein; TAX1BP1: Tax1 binding protein 1; TUBA: tubulin alpha; WT: wild-type.
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Enfermedad de Charcot-Marie-Tooth , Enfermedades Neuromusculares , Humanos , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismo , Autofagia/genética , Proteínas de Choque Térmico/metabolismo , Enfermedad de Charcot-Marie-Tooth/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismoRESUMEN
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.Abbreviation ALP: autophagy lysosomal pathway; ALS: amyotrophic lateral sclerosis; AMOTL1: angiomotin like 1; ARP2/3: actin related protein 2/3; BAG: BAG cochaperone; BAG3: BAG cochaperone 3; CASA: chaperone-assisted selective autophagy; CMA: chaperone-mediated autophagy; DNAJ/HSP40: DnaJ heat shock protein family (Hsp40); DRiPs: defective ribosomal products; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; EIF2AK1/HRI: eukaryotic translation initiation factor 2 alpha kinase 1; GABARAP: GABA type A receptor-associated protein; HDAC6: histone deacetylase 6; HSP: heat shock protein; HSPA/HSP70: heat shock protein family A (Hsp70); HSP90: heat shock protein 90; HSPB8: heat shock protein family B (small) member 8; IPV: isoleucine-proline-valine; ISR: integrated stress response; KEAP1: kelch like ECH associated protein 1; LAMP2A: lysosomal associated membrane protein 2A; LATS1: large tumor suppressor kinase 1; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOC: microtubule organizing center; MTOR: mechanistic target of rapamycin kinase; NFKB/NF-κB: nuclear factor kappa B; NFE2L2: NFE2 like bZIP transcription factor 2; PLCG/PLCγ: phospholipase C gamma; polyQ: polyglutamine; PQC: protein quality control; PxxP: proline-rich; RAN translation: repeat-associated non-AUG translation; SG: stress granule; SOD1: superoxide dismutase 1; SQSTM1/p62: sequestosome 1; STUB1/CHIP: STIP1 homology and U-box containing protein 1; STK: serine/threonine kinase; SYNPO: synaptopodin; TBP: TATA-box binding protein; TARDBP/TDP-43: TAR DNA binding protein; TFEB: transcription factor EB; TPR: tetratricopeptide repeats; TSC1: TSC complex subunit 1; UBA: ubiquitin associated; UPS: ubiquitin-proteasome system; WW: tryptophan-tryptophan; WWTR1: WW domain containing transcription regulator 1; YAP1: Yes1 associated transcriptional regulator.
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Autofagia , Factor 2 Relacionado con NF-E2 , Autofagia/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ubiquitinas/metabolismoRESUMEN
Mitochondria are complex organelles with different compartments, each harbouring their own protein quality control factors. While chaperones of the mitochondrial matrix are well characterized, it is poorly understood which chaperones protect the mitochondrial intermembrane space. Here we show that cytosolic small heat shock proteins are imported under basal conditions into the mitochondrial intermembrane space, where they operate as molecular chaperones. Protein misfolding in the mitochondrial intermembrane space leads to increased recruitment of small heat shock proteins. Depletion of small heat shock proteins leads to mitochondrial swelling and reduced respiration, while aggregation of aggregation-prone substrates is countered in their presence. Charcot-Marie-Tooth disease-causing mutations disturb the mitochondrial function of HSPB1, potentially linking previously observed mitochondrial dysfunction in Charcot-Marie-Tooth type 2F to its role in the mitochondrial intermembrane space. Our results reveal that small heat shock proteins form a chaperone system that operates in the mitochondrial intermembrane space.
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Proteínas de Choque Térmico Pequeñas , Proteínas de Choque Térmico Pequeñas/genética , Proteínas de Choque Térmico Pequeñas/metabolismo , Chaperonas Moleculares/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismoRESUMEN
Charcot-Marie-Tooth disease is the most common inherited disorder of the PNS. CMT1A accounts for 40-50% of all cases and is caused by a duplication of the PMP22 gene on chromosome 17, leading to dysmyelination in the PNS. Patient-derived models to study such myelination defects are lacking as the in vitro generation of human myelinating Schwann cells has proved to be particularly challenging. Here, we present an induced pluripotent stem cell-derived organoid culture, containing various cell types of the PNS, including myelinating human Schwann cells, which mimics the human PNS. Single-cell analysis confirmed the PNS-like cellular composition and provides insight into the developmental trajectory. We used this organoid model to study disease signatures of CMT1A, revealing early ultrastructural myelin alterations, including increased myelin periodic line distance and hypermyelination of small axons. Furthermore, we observed the presence of onion-bulb-like formations in a later developmental stage. These hallmarks were not present in the CMT1A-corrected isogenic line or in a CMT2A iPSC line, supporting the notion that these alterations are specific to CMT1A. Downregulation of PMP22 expression using short-hairpin RNAs or a combinatorial drug consisting of baclofen, naltrexone hydrochloride and D-sorbitol was able to ameliorate the myelin defects in CMT1A-organoids. In summary, this self-organizing organoid model can capture biologically meaningful features of the disease and capture the physiological complexity, forms an excellent model for studying demyelinating diseases and supports the therapeutic approach of reducing PMP22 expression.
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Enfermedad de Charcot-Marie-Tooth , Células Madre Pluripotentes Inducidas , Humanos , Vaina de Mielina/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Regulación hacia Abajo , Proteínas de la Mielina/genética , Proteínas de la Mielina/metabolismo , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/metabolismo , Organoides/metabolismo , Células de SchwannRESUMEN
Genetic pain loss includes congenital insensitivity to pain (CIP), hereditary sensory neuropathies and, if autonomic nerves are involved, hereditary sensory and autonomic neuropathy (HSAN). This heterogeneous group of disorders highlights the essential role of nociception in protecting against tissue damage. Patients with genetic pain loss have recurrent injuries, burns and poorly healing wounds as disease hallmarks. CIP and HSAN are caused by pathogenic genetic variants in >20 genes that lead to developmental defects, neurodegeneration or altered neuronal excitability of peripheral damage-sensing neurons. These genetic variants lead to hyperactivity of sodium channels, disturbed haem metabolism, altered clathrin-mediated transport and impaired gene regulatory mechanisms affecting epigenetic marks, long non-coding RNAs and repetitive elements. Therapies for pain loss disorders are mainly symptomatic but the first targeted therapies are being tested. Conversely, chronic pain remains one of the greatest unresolved medical challenges, and the genes and mechanisms associated with pain loss offer new targets for analgesics. Given the progress that has been made, the coming years are promising both in terms of targeted treatments for pain loss disorders and the development of innovative pain medicines based on knowledge of these genetic diseases.
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Canalopatías , Neuropatías Hereditarias Sensoriales y Autónomas , Insensibilidad Congénita al Dolor , Neuropatías Hereditarias Sensoriales y Autónomas/complicaciones , Neuropatías Hereditarias Sensoriales y Autónomas/diagnóstico , Neuropatías Hereditarias Sensoriales y Autónomas/genética , Humanos , Dolor/genética , Insensibilidad Congénita al Dolor/genéticaRESUMEN
BACKGROUND: The contribution of native or modified oligodendroglia-derived extracellular vesicles (OL-EVs) in controlling chronic inflammation is poorly understood. In activated microglia, OL-EVs contribute to the removal of cytotoxic proteins following a proteotoxic stress. Intracellular small heat shock protein B8 (HSPB8) sustain this function by facilitating autophagy and protecting cells against oxidative stress mediated cell death. Therefore, secretion of HSPB8 in OL-EVs could be beneficial for neurons during chronic inflammation. However, how secreted HSPB8 contribute to cellular proteostasis remains to be elucidated. METHODS: We produced oligodendroglia-derived EVs, either native (OL-EVs) or HSPB8 modified (OL-HSPB8-EVs), to investigate their effects in controlling chronic inflammation and cellular homeostasis. We analyzed the impact of both EV subsets on either a resting or activated microglial cell line and on primary mixed neural cell culture cells. Cells were activated by stimulating with either tumor necrosis factor-alpha and interleukin 1-beta or with phorbol-12-myristate-13-acetate. RESULTS: We show that OL-EVs and modified OL-HSPB8-EVs are internalized by C20 microglia and by primary mixed neural cells. The cellular uptake of OL-HSPB8-EVs increases the endogenous HSPB8 mRNA expression. Consistently, our results revealed that both EV subsets maintained cellular homeostasis during chronic inflammation with an increase in the formation of autophagic vesicles. Both EV subsets conveyed LC3B-II and BAG3 autophagy markers with an enhanced effect observed for OL-HSPB8-EVs. Moreover, stimulation with either native or modified OL-HSPB8-EVs showed a significant reduction in ubiquitinated protein, reactive oxygen species and mitochondrial depolarization, with OL-HSPB8-EVs exhibiting a more protective effect. Both EV subsets did not induce cell death in the C20 microglia cell line or the primary mixed neural cultures. CONCLUSION: We demonstrate that the functions of oligodendroglia secreted EVs enriched with HSPB8 have a supportive role, comparable to the native OL-EVs. Further development of engineered oligodendroglia derived EVs could be a novel therapeutic strategy in countering chronic inflammation. Video Abstract.
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Vesículas Extracelulares , Proteínas de Choque Térmico , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia , Vesículas Extracelulares/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Inflamación/metabolismo , Chaperonas Moleculares/metabolismo , Oligodendroglía/metabolismo , Oligodendroglía/patología , Estrés OxidativoRESUMEN
BACKGROUND: Pathogenic variants in SPTAN1 have been linked to a remarkably broad phenotypical spectrum. Clinical presentations include epileptic syndromes, intellectual disability, and hereditary motor neuropathy. OBJECTIVES: We investigated the role of SPTAN1 variants in rare neurological disorders such as ataxia and spastic paraplegia. METHODS: We screened 10,000 NGS datasets across two international consortia and one local database, indicative of the level of international collaboration currently required to identify genes causative for rare disease. We performed in silico modeling of the identified SPTAN1 variants. RESULTS: We describe 22 patients from 14 families with five novel SPTAN1 variants. Of six patients with cerebellar ataxia, four carry a de novo SPTAN1 variant and two show a sporadic inheritance. In this group, one variant (p.Lys2083del) is recurrent in four patients. Two patients have novel de novo missense mutations (p.Arg1098Cys, p.Arg1624Cys) associated with cerebellar ataxia, in one patient accompanied by intellectual disability and epilepsy. We furthermore report a recurrent missense mutation (p.Arg19Trp) in 15 patients with spastic paraplegia from seven families with a dominant inheritance pattern in four and a de novo origin in one case. One further patient carrying a de novo missense mutation (p.Gln2205Pro) has a complex spastic ataxic phenotype. Through protein modeling we show that mutated amino acids are located at crucial interlinking positions, interconnecting the three-helix bundle of a spectrin repeat. CONCLUSIONS: We show that SPTAN1 is a relevant candidate gene for ataxia and spastic paraplegia. We suggest that for the mutations identified in this study, disruption of the interlinking of spectrin helices could be a key feature of the pathomechanism. © 2022 International Parkinson and Movement Disorder Society.
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Proteínas Portadoras , Ataxia Cerebelosa , Discapacidad Intelectual , Proteínas de Microfilamentos , Paraplejía Espástica Hereditaria , Proteínas Portadoras/genética , Ataxia Cerebelosa/genética , Humanos , Discapacidad Intelectual/genética , Proteínas de Microfilamentos/genética , Mutación/genética , Paraplejía/genética , Linaje , Fenotipo , Paraplejía Espástica Hereditaria/genética , Espectrina/genéticaRESUMEN
Molecular markers scalable for clinical use are critical for the development of effective treatments and the design of clinical trials. Here, we identify proteins in sera of patients and mouse models with Charcot-Marie-Tooth disease (CMT) with characteristics that make them suitable as biomarkers in clinical practice and therapeutic trials. We collected serum from mouse models of CMT1A (C61 het), CMT2D (GarsC201R, GarsP278KY), CMT1X (Gjb1-null), CMT2L (Hspb8K141N) and from CMT patients with genotypes including CMT1A (PMP22d), CMT2D (GARS), CMT2N (AARS) and other rare genetic forms of CMT. The severity of neuropathy in the patients was assessed by the CMT Neuropathy Examination Score (CMTES). We performed multitargeted proteomics on both sample sets to identify proteins elevated across multiple mouse models and CMT patients. Selected proteins and additional potential biomarkers, such as growth differentiation factor 15 (GDF15) and cell free mitochondrial DNA, were validated by ELISA and quantitative PCR, respectively. We propose that neural cell adhesion molecule 1 (NCAM1) is a candidate biomarker for CMT, as it was elevated in Gjb1-null, Hspb8K141N, GarsC201R and GarsP278KY mice as well as in patients with both demyelinating (CMT1A) and axonal (CMT2D, CMT2N) forms of CMT. We show that NCAM1 may reflect disease severity, demonstrated by a progressive increase in mouse models with time and a significant positive correlation with CMTES neuropathy severity in patients. The increase in NCAM1 may reflect muscle regeneration triggered by denervation, which could potentially track disease progression or the effect of treatments. We found that member proteins of the complement system were elevated in Gjb1-null and Hspb8K141N mouse models as well as in patients with both demyelinating and axonal CMT, indicating possible complement activation at the impaired nerve terminals. However, complement proteins did not correlate with the severity of neuropathy measured on the CMTES scale. Although the complement system does not seem to be a prognostic biomarker, we do show complement elevation to be a common disease feature of CMT, which may be of interest as a therapeutic target. We also identify serum GDF15 as a highly sensitive diagnostic biomarker, which was elevated in all CMT genotypes as well as in Hspb8K141N, Gjb1-null, GarsC201R and GarsP278KY mouse models. Although we cannot fully explain its origin, it may reflect increased stress response or metabolic disturbances in CMT. Further large and longitudinal patient studies should be performed to establish the value of these proteins as diagnostic and prognostic molecular biomarkers for CMT.
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Antígeno CD56 , Enfermedad de Charcot-Marie-Tooth , Factor 15 de Diferenciación de Crecimiento , Animales , Ratones , Biomarcadores , Antígeno CD56/genética , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/diagnóstico , Factor 15 de Diferenciación de Crecimiento/genética , Proteínas , HumanosRESUMEN
Axonal Charcot-Marie-Tooth neuropathies (CMT type 2) are caused by inherited mutations in various genes functioning in different pathways. The types of genes and multiplicity of mutations reflect the clinical and genetic heterogeneity in CMT2 disease, which complicates its diagnosis and has inhibited the development of therapies. Here, we used CMT2 patient-derived pluripotent stem cells (iPSCs) to identify common hallmarks of axonal degeneration shared by different CMT2 subtypes. We compared the cellular phenotypes of neurons differentiated from CMT2 patient iPSCs with those from healthy controls and a CRISPR/Cas9-corrected isogenic line. Our results demonstrated neurite network alterations along with extracellular electrophysiological abnormalities in the differentiated motor neurons. Progressive deficits in mitochondrial and lysosomal trafficking, as well as in mitochondrial morphology, were observed in all CMT2 patient lines. Differentiation of the same CMT2 iPSC lines into peripheral sensory neurons only gave rise to cellular phenotypes in subtypes with sensory involvement, supporting the notion that some gene mutations predominantly affect motor neurons. We revealed a common mitochondrial dysfunction in CMT2-derived motor neurons, supported by alterations in the expression pattern and oxidative phosphorylation, which could be recapitulated in the sciatic nerve tissue of a symptomatic mouse model. Inhibition of a dual leucine zipper kinase could partially ameliorate the mitochondrial disease phenotypes in CMT2 subtypes. Altogether, our data reveal shared cellular phenotypes across different CMT2 subtypes and suggests that targeting such common pathomechanisms could allow the development of a uniform treatment for CMT2.
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Enfermedad de Charcot-Marie-Tooth/metabolismo , Mitocondrias/metabolismo , Neuronas Motoras/metabolismo , Enfermedad de Charcot-Marie-Tooth/patología , Genotipo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Mitocondrias/patología , Neuronas Motoras/patología , Mutación , LinajeRESUMEN
HSP27 is a human molecular chaperone that forms large, dynamic oligomers and functions in many aspects of cellular homeostasis. Mutations in HSP27 cause Charcot-Marie-Tooth (CMT) disease, the most common inherited disorder of the peripheral nervous system. A particularly severe form of CMT disease is triggered by the P182L mutation in the highly conserved IxI/V motif of the disordered C-terminal region, which interacts weakly with the structured core domain of HSP27. Here, we observed that the P182L mutation disrupts the chaperone activity and significantly increases the size of HSP27 oligomers formed in vivo, including in motor neurons differentiated from CMT patient-derived stem cells. Using NMR spectroscopy, we determined that the P182L mutation decreases the affinity of the HSP27 IxI/V motif for its own core domain, leaving this binding site more accessible for other IxI/V-containing proteins. We identified multiple IxI/V-bearing proteins that bind with higher affinity to the P182L variant due to the increased availability of the IxI/V-binding site. Our results provide a mechanistic basis for the impact of the P182L mutation on HSP27 and suggest that the IxI/V motif plays an important, regulatory role in modulating protein-protein interactions.
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Enfermedad de Charcot-Marie-Tooth/genética , Proteínas de Choque Térmico/química , Chaperonas Moleculares/química , Adulto , Sitios de Unión , Células Cultivadas , Células HeLa , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Simulación de Dinámica Molecular , Neuronas Motoras/citología , Neuronas Motoras/metabolismo , Mutación Missense , Unión Proteica , Multimerización de ProteínaRESUMEN
Three missense mutations targeting the same proline 209 (Pro209) codon in the co-chaperone Bcl2-associated athanogene 3 (BAG3) have been reported to cause distal myopathy, dilated cardiomyopathy or Charcot-Marie-Tooth type 2 neuropathy. Yet, it is unclear whether distinct molecular mechanisms underlie the variable clinical spectrum of the rare patients carrying these three heterozygous Pro209 mutations in BAG3. Here, we studied all three variants and compared them to the BAG3_Glu455Lys mutant, which causes dilated cardiomyopathy. We found that all BAG3_Pro209 mutants have acquired a toxic gain-of-function, which causes these variants to accumulate in the form of insoluble HDAC6- and vimentin-positive aggresomes. The aggresomes formed by mutant BAG3 led to a relocation of other chaperones such as HSPB8 and Hsp70, which, together with BAG3, promote the so-called chaperone-assisted selective autophagy (CASA). As a consequence of their increased aggregation-proneness, mutant BAG3 trapped ubiquitinylated client proteins at the aggresome, preventing their efficient clearance. Combined, these data show that all BAG3_Pro209 mutants, irrespective of their different clinical phenotypes, are characterized by a gain-of-function that contributes to the gradual loss of protein homeostasis.
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Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Reguladoras de la Apoptosis/genética , Cardiomiopatía Dilatada/genética , Enfermedad de Charcot-Marie-Tooth/genética , Miopatías Distales/genética , Chaperonas Moleculares/metabolismo , Mutación Missense , Agregado de Proteínas , Agregación Patológica de Proteínas/genética , Autofagia , Codón , Células HEK293 , Humanos , Prolina , Agregación Patológica de Proteínas/metabolismo , Transporte de Proteínas , UbiquitinaciónRESUMEN
Small heat shock proteins are ubiquitously expressed chaperones, yet mutations in some of them cause tissue-specific diseases. Here, we will discuss how small heat shock proteins give rise to neurodegenerative disorders themselves while we will also highlight how these proteins can fulfil protective functions in neurodegenerative disorders caused by protein aggregation. The first half of this paper will be focused on how mutations in HSPB1, HSPB3, and HSPB8 are linked to inherited peripheral neuropathies like Charcot-Marie-Tooth (CMT) disease and distal hereditary motor neuropathy (dHMN). The second part of the paper will discuss how small heat shock proteins are linked to neurodegenerative disorders like Alzheimer's, Parkinson's, and Huntington's disease.
Asunto(s)
Enfermedad de Alzheimer , Enfermedad de Charcot-Marie-Tooth , Proteínas de Choque Térmico Pequeñas/genética , Enfermedad de Huntington , Enfermedad de Parkinson , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/metabolismo , Proteínas de Choque Térmico/genética , Humanos , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Chaperonas Moleculares/genética , Mutación , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismoRESUMEN
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: Relmα+Mgl1+ snMacs in the epineurium and Relmα-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.
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Macrófagos/citología , Macrófagos/fisiología , Nervio Ciático/inmunología , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Compresión Nerviosa , TranscriptomaRESUMEN
OBJECTIVE: Intraplaque neovascularization is an important feature of unstable human atherosclerotic plaques. However, its impact on plaque formation and stability is poorly studied. Because proliferating endothelial cells generate up to 85% of their ATP from glycolysis, we investigated whether pharmacological inhibition of glycolytic flux by the small-molecule 3PO (3-[3-pyridinyl]-1-[4-pyridinyl]-2-propen-1-one) could have beneficial effects on plaque formation and composition. Approach and Results: ApoE-/- (apolipoprotein E deficient) mice treated with 3PO (50 µg/g, ip; 4×/wk, 4 weeks) showed a metabolic switch toward ketone body formation. Treatment of ApoE-/-Fbn1C1039G+/- mice with 3PO (50 µg/g, ip) either after 4 (preventive, twice/wk, 10 weeks) or 16 weeks of Western diet (curative, 4×/wk, 4 weeks) inhibited intraplaque neovascularization by 50% and 38%, respectively. Plaque formation was significantly reduced in all 3PO-treated animals. This effect was independent of intraplaque neovascularization. In vitro experiments showed that 3PO favors an anti-inflammatory M2 macrophage subtype and suppresses an M1 proinflammatory phenotype. Moreover, 3PO induced autophagy, which in turn impaired NF-κB (nuclear factor-kappa B) signaling and inhibited TNF-α (tumor necrosis factor-alpha)-mediated VCAM-1 (vascular cell adhesion molecule-1) and ICAM-1 (intercellular adhesion molecule-1) upregulation. Consistently, a preventive 3PO regimen reduced endothelial VCAM-1 expression in vivo. Furthermore, 3PO improved cardiac function in ApoE-/-Fbn1C1039G+/- mice after 10 weeks of treatment. CONCLUSIONS: Partial inhibition of glycolysis restrained intraplaque angiogenesis without affecting plaque composition. However, less plaques were formed, which was accompanied by downregulation of endothelial adhesion molecules-an event that depends on autophagy induction. Inhibition of coronary plaque formation by 3PO resulted in an overall improved cardiac function.
Asunto(s)
Arterias/efectos de los fármacos , Aterosclerosis/tratamiento farmacológico , Glucólisis/efectos de los fármacos , Neovascularización Patológica , Placa Aterosclerótica , Piridinas/farmacología , Animales , Arterias/metabolismo , Arterias/patología , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Autofagia/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Femenino , Fibrilina-1/genética , Fibrilina-1/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Ratones Noqueados para ApoE , FN-kappa B/metabolismo , Fenotipo , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Microglia, the immunocompetent cells of the central nervous system (CNS), play an important role in maintaining cellular homeostasis in the CNS. These cells secrete immunomodulatory factors including nanovesicles and participate in the removal of cellular debris by phagocytosis or autophagy. Accumulating evidence indicates that specifically the cellular exchange of small extracellular vesicles (EVs), participates in physiology and disease through intercellular communication. However, the contribution of microglial-derived extracellular vesicles (M-EVs) to the maintenance of microglia homeostasis and how M-EVs could influence the phenotype and gene function of other microglia subtypes is unclear. In addition, knowledge of canonical signalling pathways of inflammation and immunity gene expression patterns in human microglia exposed to M-EVs is limited. Here, we analysed the effects of M-EVs produced in vitro by either tumour necrosis factor alpha (TNFα) activated or non-activated microglia BV2 cells. We showed that M-EVs are internalized by both mouse and human C20 microglia cells and that the uptake of M-EVs in microglia induced autophagic vesicles at various stages of degradation including autophagosomes and autolysosomes. Consistently, stimulation of microglia with M-EVs increased the protein expression of the autophagy marker, microtubule-associated proteins 1A/1B light chain 3B isoform II (LC3B-II), and promoted autophagic flux in live cells. To elucidate the biological activities occurring at the transcriptional level in C20 microglia stimulated with M-EVs, the gene expression profiles, potential upstream regulators, and enrichment pathways were characterized using targeted RNA sequencing. Inflammation and immunity transcriptome gene panel sequencing of both activated and normal microglia stimulated with M-EVs showed involvement of several canonical pathways and reduced expression of key genes involved in neuroinflammation, inflammasome and apoptosis signalling pathways compared to control cells. In this study, we provide the perspective that a beneficial activity of in vitro cell culture produced EVs could be the modulation of autophagy during cellular stress. Therefore, we use a monoculture system to study microglia-microglia crosstalk which is important in the prevention and propagation of inflammation in the brain. We demonstrate that in vitro produced microglial EVs are able to influence multiple biological pathways and promote activation of autophagy in order to maintain microglia survival and homeostasis.