RESUMO
Patients with the neurological disorder HSAN-I suffer frequent infections, attributed to a lack of pain sensation and failure to seek care for minor injuries. Whether protective CD8+ T cells are affected in HSAN-I patients remains unknown. Here, we report that HSAN-I-associated mutations in serine palmitoyltransferase subunit SPTLC2 dampened human T cell responses. Antigen stimulation and inflammation induced SPTLC2 expression, and murine T-cell-specific ablation of Sptlc2 impaired antiviral-T-cell expansion and effector function. Sptlc2 deficiency reduced sphingolipid biosynthetic flux and led to prolonged activation of the mechanistic target of rapamycin complex 1 (mTORC1), endoplasmic reticulum (ER) stress, and CD8+ T cell death. Protective CD8+ T cell responses in HSAN-I patient PBMCs and Sptlc2-deficient mice were restored by supplementing with sphingolipids and pharmacologically inhibiting ER stress-induced cell death. Therefore, SPTLC2 underpins protective immunity by translating extracellular stimuli into intracellular anabolic signals and antagonizes ER stress to promote T cell metabolic fitness.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Neuropatias Hereditárias Sensoriais e Autônomas/genética , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Serina C-Palmitoiltransferase/genética , Animais , Proliferação de Células , Células Cultivadas , Citocinas/biossíntese , Estresse do Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/imunologia , Feminino , Humanos , Coriomeningite Linfocítica/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Transdução de Sinais/imunologia , Esfingolipídeos/biossínteseRESUMO
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.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Proteínas de Choque Térmico/química , Chaperonas Moleculares/química , Adulto , Sítios de Ligação , Células Cultivadas , Células HeLa , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Simulação de Dinâmica Molecular , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Mutação de Sentido Incorreto , Ligação Proteica , Multimerização ProteicaRESUMO
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.
Assuntos
Doença de Charcot-Marie-Tooth , Células-Tronco Pluripotentes Induzidas , Humanos , Bainha de Mielina/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Regulação para Baixo , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Organoides/metabolismo , Células de SchwannRESUMO
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.
Assuntos
Antígeno CD56 , Doença de Charcot-Marie-Tooth , Fator 15 de Diferenciação de Crescimento , Animais , Camundongos , Biomarcadores , Antígeno CD56/genética , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/diagnóstico , Fator 15 de Diferenciação de Crescimento/genética , Proteínas , HumanosRESUMO
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.
Assuntos
Proteínas de Transporte , Ataxia Cerebelar , Deficiência Intelectual , Proteínas dos Microfilamentos , Paraplegia Espástica Hereditária , Proteínas de Transporte/genética , Ataxia Cerebelar/genética , Humanos , Deficiência Intelectual/genética , Proteínas dos Microfilamentos/genética , Mutação/genética , Paraplegia/genética , Linhagem , Fenótipo , Paraplegia Espástica Hereditária/genética , Espectrina/genéticaRESUMO
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.
Assuntos
Vesículas Extracelulares , Proteínas de Choque Térmico , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia , Vesículas Extracelulares/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Inflamação/metabolismo , Chaperonas Moleculares/metabolismo , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Estresse OxidativoRESUMO
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.
Assuntos
Doença de Charcot-Marie-Tooth/metabolismo , Mitocôndrias/metabolismo , Neurônios Motores/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Genótipo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Mitocôndrias/patologia , Neurônios Motores/patologia , Mutação , LinhagemRESUMO
Mutations in the MFN2 gene encoding Mitofusin 2 lead to the development of Charcot-Marie-Tooth type 2A (CMT2A), a dominant axonal form of peripheral neuropathy. Mitofusin 2 is localized at both the outer membrane of mitochondria and the endoplasmic reticulum and is particularly enriched at specialized contact regions known as mitochondria-associated membranes (MAM). We observed that expression of MFN2R94Q induces distal axonal degeneration in the absence of overt neuronal death. The presence of mutant protein leads to reduction in endoplasmic reticulum and mitochondria contacts in CMT2A patient-derived fibroblasts, in primary neurons and in vivo, in motoneurons of a mouse model of CMT2A. These changes are concomitant with endoplasmic reticulum stress, calcium handling defects, and changes in the geometry and axonal transport of mitochondria. Importantly, pharmacological treatments reinforcing endoplasmic reticulum-mitochondria cross-talk, or reducing endoplasmic reticulum stress, restore the mitochondria morphology and prevent axonal degeneration. These results highlight defects in MAM as a cellular mechanism contributing to CMT2A pathology mediated by mutated MFN2.
Assuntos
Doença de Charcot-Marie-Tooth/metabolismo , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Animais , Axônios/metabolismo , Transporte Biológico , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/fisiopatologia , Modelos Animais de Doenças , Retículo Endoplasmático/ultraestrutura , Feminino , Marcha , Locomoção/genética , Masculino , Camundongos , Camundongos Transgênicos , Mitocôndrias/ultraestrutura , Neurônios Motores/metabolismo , Denervação Muscular , Fibras Musculares de Contração Lenta , Transdução de SinaisRESUMO
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.
Assuntos
Axônios/metabolismo , Retículo Endoplasmático/genética , GTP Fosfo-Hidrolases/genética , Neuropatias Hereditárias Sensoriais e Autônomas/genética , Autofagia/genética , Axônios/patologia , Cálcio/metabolismo , Sinalização do Cálcio/genética , Retículo Endoplasmático/metabolismo , Fibroblastos/metabolismo , Células HeLa , Neuropatias Hereditárias Sensoriais e Autônomas/metabolismo , Neuropatias Hereditárias Sensoriais e Autônomas/patologia , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação , Neurônios/metabolismo , Neurônios/patologiaRESUMO
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.
Assuntos
Artérias/efeitos dos fármacos , Aterosclerose/tratamento farmacológico , Glicólise/efeitos dos fármacos , Neovascularização Patológica , Placa Aterosclerótica , Piridinas/farmacologia , Animais , Artérias/metabolismo , Artérias/patologia , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Autofagia/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Fibrilina-1/genética , Fibrilina-1/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos Knockout para ApoE , NF-kappa B/metabolismo , Fenótipo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Much has been achieved in terms of understanding the complex clinical and genetic heterogeneity of Charcot-Marie-Tooth neuropathy (CMT). Since the identification of mutations in the first CMT associated gene, PMP22, the technological advancement in molecular genetics and gene technology has allowed scientists to generate diverse animal models expressing monogenetic mutations that closely resemble the CMT phenotype. Additionally, one can now culture patient-derived neurons in a dish using cellular reprogramming and differentiation techniques. Nevertheless, despite the fact that finding a disease-causing mutation offers a precise diagnosis, there is no cure for CMT at present. This review will shed light on the exciting advancement in CMT disease modelling, the breakthroughs, pitfalls, current challenges for scientists and key considerations to move the field forward towards successful therapies.
Assuntos
Doença de Charcot-Marie-Tooth/fisiopatologia , Modelos Animais de Doenças , Células-Tronco Pluripotentes Induzidas/metabolismo , Animais , Doença de Charcot-Marie-Tooth/genética , Humanos , Camundongos , Modelos Biológicos , Mutação , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismoRESUMO
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.
Assuntos
Miopatias Distais/metabolismo , Mutação com Ganho de Função , Proteínas de Choque Térmico HSP20/genética , Proteínas de Choque Térmico HSP20/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miopatias Congênitas Estruturais/metabolismo , Doenças do Sistema Nervoso Periférico/metabolismo , Animais , Atrofia/metabolismo , Atrofia/patologia , Autofagia/fisiologia , Modelos Animais de Doenças , Miopatias Distais/patologia , Feminino , Proteínas de Choque Térmico , Camundongos Transgênicos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Chaperonas Moleculares , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Miopatias Congênitas Estruturais/patologia , Nervo Isquiático/metabolismo , Nervo Isquiático/patologiaRESUMO
BACKGROUND: Charcot-Marie-Tooth type 2 (CMT2) neuropathy is characterised by a vast clinical and genetic heterogeneity complicating its diagnosis and therapeutic intervention. Identification of molecular signatures that are common to multiple CMT2 subtypes can aid in developing therapeutic strategies and measuring disease outcomes. METHODS: A proteomics-based approach was performed on lymphoblasts from CMT2 patients genetically diagnosed with different gene mutations to identify differentially regulated proteins. The candidate proteins were validated through real-time quantitative PCR and western blotting on lymphoblast samples of patients and controls, motor neurons differentiated from patient-derived induced pluripotent stem cells (iPSCs) and sciatic nerves of CMT2 mouse models. RESULTS: Proteomic profiling of patient lymphoblasts resulted in the identification of profilin 2 (PFN2) and guanidinoacetate methyltransferase (GAMT) as commonly downregulated proteins in different genotypes compared with healthy controls. This decrease was also observed at the transcriptional level on screening 43 CMT2 patients and 22 controls, respectively. A progressive decrease in PFN2 expression with age was observed in patients, while in healthy controls its expression increased with age. Reduced PFN2 expression was also observed in motor neurons differentiated from CMT2 patient-derived iPSCs and sciatic nerves of CMT2 mice when compared with controls. However, no change in GAMT levels was observed in motor neurons and CMT2 mouse-derived sciatic nerves. CONCLUSIONS: We unveil PFN2 and GAMT as molecular determinants of CMT2 with possible indications of the role of PFN2 in the pathogenesis and disease progression. This is the first study describing biomarkers that can boost the development of therapeutic strategies targeting a wider spectrum of CMT2 patients.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Genótipo , Guanidinoacetato N-Metiltransferase/genética , Mutação , Profilinas/genética , Adulto , Idoso , Axônios/patologia , Doença de Charcot-Marie-Tooth/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Proteômica , Adulto JovemRESUMO
Small heat shock proteins are molecular chaperones that exert diverse cellular functions. To date, mutations in the coding regions of HSPB1 (Hsp27) and HSPB8 (Hsp22) were reported to cause distal hereditary motor neuropathy and Charcot-Marie-Tooth disease. Recently, the clinical spectrum of HSPB1 and HSPB8 mutations was expanded to also include myopathies. Here we provide an update on the molecular genetics and biology of small heat shock protein mutations in neuromuscular diseases.
Assuntos
Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico/genética , Mutação/genética , Doenças Neuromusculares/genética , Proteínas Serina-Treonina Quinases/genética , Idoso , Idoso de 80 Anos ou mais , Humanos , Masculino , Modelos Moleculares , Chaperonas MolecularesRESUMO
In this study, we describe the phenotypic spectrum of distal hereditary motor neuropathy caused by mutations in the small heat shock proteins HSPB1 and HSPB8 and investigate the functional consequences of newly discovered variants. Among 510 unrelated patients with distal motor neuropathy, we identified mutations in HSPB1 (28 index patients/510; 5.5%) and HSPB8 (four index patients/510; 0.8%) genes. Patients have slowly progressive distal (100%) and proximal (13%) weakness in lower limbs (100%), mild lower limbs sensory involvement (31%), foot deformities (73%), progressive distal upper limb weakness (29%), mildly raised serum creatine kinase levels (100%), and central nervous system involvement (9%). We identified 12 HSPB1 and four HSPB8 mutations, including five and three not previously reported. Transmission was either dominant (78%), recessive (3%), or de novo (19%). Three missense mutations in HSPB1 (Pro7Ser, Gly53Asp, and Gln128Arg) cause hyperphosphorylation of neurofilaments, whereas the C-terminal mutant Ser187Leu triggers protein aggregation. Two frameshift mutations (Leu58fs and Ala61fs) create a premature stop codon leading to proteasomal degradation. Two mutations in HSPB8 (Lys141Met/Asn) exhibited increased binding to Bag3. We demonstrate that HSPB1 and HSPB8 mutations are a major cause of inherited motor axonal neuropathy. Mutations lead to diverse functional outcomes further demonstrating the pleotropic character of small heat shock proteins.
Assuntos
Estudos de Associação Genética , Proteínas de Choque Térmico Pequenas/genética , Mutação , Adolescente , Adulto , Alelos , Substituição de Aminoácidos , Biomarcadores , Linhagem Celular , Criança , Análise Mutacional de DNA , Feminino , Frequência do Gene , Genótipo , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico/genética , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Chaperonas Moleculares , Doença dos Neurônios Motores/diagnóstico , Doença dos Neurônios Motores/genética , Família Multigênica , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Adulto JovemRESUMO
OBJECTIVE: To identify the unknown genetic cause in a large pedigree previously classified with a distinct form of axonal Charcot-Marie-Tooth disease type 2G (CMT2G) and to explore its transcriptional consequences. METHODS: Clinical reevaluation of the pedigree was performed, followed by linkage analysis with the redefined disease statuses, and whole genome and exome sequencing. The impact of the mutation was investigated by immunoblotting and transcriptome sequencing. RESULTS: Thirteen affected individuals over 3 generations displayed mild and quiescent lower-limb axonal sensorimotor neuropathy. Magnetic resonance imaging (MRI) of lower-limb musculature systematically showed fatty atrophy in clinical and subclinical mutation carriers. We redefined the disease-linked region to chr9q31.3-q34.2 and subsequently identified a novel missense variant in the E3 ubiquitin-protein ligase LRSAM1 (p.Cys694Tyr). Unlike previous reports, we demonstrated in patients' lymphoblasts that the mutation does not influence overall protein levels of LRSAM1, nor of its ubiquitylation target TSG101. The mutation is associated with several transcriptional changes, including a significant upregulation of another E3 ubiquitin-protein ligase, NEDD4L, and of TNFRSF21, a key regulator of axonal degeneration. INTERPRETATION: Our findings demonstrate that the isolated genetic entity CMT2G is caused by a missense mutation in LRSAM1 and should be reclassified as CMT2P. MRI of lower-limb musculature can be used to detect minimal signs of the disease. Transcriptome analysis of patients' cells highlights novel molecular players associated with LRSAM1 dysfunction, and reveals pathways and therapeutic targets shared with amyotrophic lateral sclerosis and Alzheimer disease. Ann Neurol 2016;80:823-833.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Ubiquitina-Proteína Ligases Nedd4 , Condução Nervosa/genética , Condução Nervosa/fisiologia , Linhagem , Regulação para CimaRESUMO
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.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Mutação , Idoso , Esclerose Lateral Amiotrófica/metabolismo , Feminino , Predisposição Genética para Doença , Proteínas de Choque Térmico HSP27/química , Proteínas de Choque Térmico , Humanos , Itália , Pessoa de Meia-Idade , Chaperonas Moleculares , Multimerização ProteicaRESUMO
Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a large family of RNA-binding proteins (RBPs) that contribute to multiple aspects of nucleic acid metabolism including alternative splicing, mRNA stabilization, and transcriptional and translational regulation. Many hnRNPs share general features, but differ in domain composition and functional properties. This review will discuss the current knowledge about the different hnRNP family members, focusing on their structural and functional divergence. Additionally, we will highlight their involvement in neurodegenerative diseases and cancer, and the potential to develop RNA-based therapies.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas/genética , Neoplasias/genética , Doenças Neurodegenerativas/genética , Processamento Alternativo/genética , Humanos , Neoplasias/patologia , Doenças Neurodegenerativas/patologia , Biossíntese de Proteínas , Estabilidade de RNA/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA , Transcrição GênicaRESUMO
The most common form of spinal muscular atrophy (SMA) is a recessive disorder caused by deleterious SMN1 mutations in 5q13, whereas the genetic etiologies of non-5q SMA are very heterogeneous and largely remain to be elucidated. In a Bulgarian family affected by autosomal-dominant proximal SMA, we performed genome-wide linkage analysis and whole-exome sequencing and found a heterozygous de novo c.320C>T (p.Ser107Leu) mutation in bicaudal D homolog 2 (Drosophila) (BICD2). Further analysis of BICD2 in a cohort of 119 individuals with non-5q SMA identified a second de novo BICD2 mutation, c.2321A>G (p.Glu774Gly), in a simplex case. Detailed clinical and electrophysiological investigations revealed that both families are affected by a very similar disease course, characterized by early childhood onset, predominant involvement of lower extremities, and very slow disease progression. The amino acid substitutions are located in two interaction domains of BICD2, an adaptor protein linking the dynein molecular motor with its cargo. Our immunoprecipitation and localization experiments in HeLa and SH-SY5Y cells and affected individuals' lymphoblasts demonstrated that p.Ser107Leu causes increased dynein binding and thus leads to accumulation of BICD2 at the microtubule-organizing complex and Golgi fragmentation. In addition, the altered protein had a reduced colocalization with RAB6A, a regulator of vesicle trafficking between the Golgi and the endoplasmic reticulum. The interaction between p.Glu744Gly altered BICD2 and RAB6A was impaired, which also led to their reduced colocalization. Our study identifies BICD2 mutations as a cause of non-5q linked SMA and highlights the importance of dynein-mediated motility in motor neuron function in humans.
Assuntos
Proteínas de Transporte/genética , Genes Dominantes , Atrofia Muscular Espinal/genética , Mutação de Sentido Incorreto , Adulto , Sequência de Bases , Proteínas de Transporte/metabolismo , Criança , Pré-Escolar , Feminino , Estudos de Associação Genética , Células HeLa , Humanos , Masculino , Proteínas Associadas aos Microtúbulos , Pessoa de Meia-Idade , Atrofia Muscular Espinal/metabolismo , Linhagem , Transporte Proteico , Análise de Sequência de DNA , Adulto Jovem , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
There is a growing appreciation that membrane-bound organelles in eukaryotic cells communicate directly with one another through direct membrane contact sites. Mitochondria-associated membranes are specialized subdomains of the endoplasmic reticulum that function as membrane contact sites between the endoplasmic reticulum and mitochondria. These sites have emerged as major players in lipid metabolism and calcium signaling. More recently also autophagy and mitochondrial dynamics have been found to be regulated at ER-mitochondria contact sites. Neurons critically depend on mitochondria-associated membranes as a means to exchange metabolites and signaling molecules between these organelles. This is underscored by the fact that genes affecting mitochondrial and endoplasmic reticulum homeostasis are clearly overrepresented in several hereditary neurodegenerative disorders. Conversely, the processes affected by the contact sites between the endoplasmic reticulum and mitochondria are widely implicated in neurodegeneration. This review will focus on the most recent data addressing the structural composition and function of the mitochondria-associated membranes. In addition, the 3D morphology of the contact sites as observed using volume electron microscopy is discussed. Finally, it will highlight the role of several key proteins associated with these contact sites that are involved not only in dementias, amyotrophic lateral sclerosis and Parkinson's disease, but also in axonopathies such as hereditary spastic paraplegia and Charcot-Marie-Tooth disease.