RESUMO
Haplo-insufficiency of the gene encoding the myelin protein PMP22 leads to focal myelin overgrowth in the peripheral nervous system and hereditary neuropathy with liability to pressure palsies (HNPP). Conversely, duplication of PMP22 causes Charcot-Marie-Tooth disease type 1A (CMT1A), characterized by hypomyelination of medium to large caliber axons. The molecular mechanisms of abnormal myelin growth regulation by PMP22 have remained obscure. Here, we show in rodent models of HNPP and CMT1A that the PI3K/Akt/mTOR-pathway inhibiting phosphatase PTEN is correlated in abundance with PMP22 in peripheral nerves, without evidence for direct protein interactions. Indeed, treating DRG neuron/Schwann cell co-cultures from HNPP mice with PI3K/Akt/mTOR pathway inhibitors reduced focal hypermyelination. When we treated HNPP mice in vivo with the mTOR inhibitor Rapamycin, motor functions were improved, compound muscle amplitudes were increased and pathological tomacula in sciatic nerves were reduced. In contrast, we found Schwann cell dedifferentiation in CMT1A uncoupled from PI3K/Akt/mTOR, leaving partial PTEN ablation insufficient for disease amelioration. For HNPP, the development of PI3K/Akt/mTOR pathway inhibitors may be considered as the first treatment option for pressure palsies.
Assuntos
Artrogripose , Doença de Charcot-Marie-Tooth , Neuropatia Hereditária Motora e Sensorial , Fosfatidilinositol 3-Quinases , Camundongos , Animais , Proteínas Proto-Oncogênicas c-akt , Roedores/metabolismo , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/patologia , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Serina-Treonina Quinases TORRESUMO
We have previously shown that targeting endoneurial macrophages with the orally applied CSF-1 receptor specific kinase (c-FMS) inhibitor PLX5622 from the age of 3 months onwards led to a substantial alleviation of the neuropathy in mouse models of Charcot-Marie-Tooth (CMT) 1X and 1B disease, which are genetically-mediated nerve disorders not treatable in humans. The same approach failed in a model of CMT1A (PMP22-overexpressing mice, line C61), representing the most frequent form of CMT. This was unexpected since previous studies identified macrophages contributing to disease severity in the same CMT1A model. Here we re-approached the possibility of alleviating the neuropathy in a model of CMT1A by targeting macrophages at earlier time points. As a proof-of-principle experiment, we genetically inactivated colony-stimulating factor-1 (CSF-1) in CMT1A mice, which resulted in lower endoneurial macrophage numbers and alleviated the neuropathy. Based on these observations, we pharmacologically ablated macrophages in newborn CMT1A mice by feeding their lactating mothers with chow containing PLX5622, followed by treatment of the respective progenies after weaning until the age of 6 months. We found that peripheral neuropathy was substantially alleviated after early postnatal treatment, leading to preserved motor function in CMT1A mice. Moreover, macrophage depletion affected the altered Schwann cell differentiation phenotype. These findings underscore the targetable role of macrophage-mediated inflammation in peripheral nerves of inherited neuropathies, but also emphasize the need for an early treatment start confined to a narrow therapeutic time window in CMT1A models and potentially in respective patients.
Assuntos
Doença de Charcot-Marie-Tooth , Lactação , Animais , Diferenciação Celular , Doença de Charcot-Marie-Tooth/genética , Feminino , Humanos , Macrófagos/metabolismo , Camundongos , Nervos Periféricos/metabolismoRESUMO
The neuregulin 1 (NRG1) ErbB4 module is at the core of an "at risk" signaling pathway in schizophrenia. Several human studies suggest hyperstimulation of NRG1-ErbB4 signaling as a plausible pathomechanism; however, little is known about the significance of stage-, brain area-, or neural cell type-specific NRG1-ErbB4 hyperactivity for disease-relevant brain endophenotypes. To address these spatiotemporal aspects, we generated transgenic mice for Cre recombinase-mediated overexpression of cystein-rich domain (CRD) NRG1, the most prominent NRG1 isoform in the brain. A comparison of "brain-wide" vs cell type-specific CRD-NRG1 overexpressing mice revealed that pathogenic CRD-NRG1 signals for ventricular enlargement and neuroinflammation originate outside glutamatergic neurons and suggests a subcortical function of CRD-NRG1 in the control of body weight. Embryonic onset of CRD-NRG1 in glutamatergic cortical networks resulted in reduced inhibitory neurotransmission and locomotor hyperactivity. Our findings identify ventricular enlargement and locomotor hyperactivity, 2 main endophenotypes of schizophrenia, as specific consequences of spatiotemporally distinct expression profiles of hyperactivated CRD-NRG1 signaling.
Assuntos
Encéfalo , Endofenótipos , Ácido Glutâmico/metabolismo , Rede Nervosa , Neuregulina-1/metabolismo , Agitação Psicomotora , Receptor ErbB-4/metabolismo , Esquizofrenia , Animais , Comportamento Animal/fisiologia , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Embrião de Mamíferos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Rede Nervosa/metabolismo , Rede Nervosa/fisiopatologia , Agitação Psicomotora/metabolismo , Agitação Psicomotora/fisiopatologia , Esquizofrenia/metabolismo , Esquizofrenia/fisiopatologia , Transdução de Sinais/fisiologiaRESUMO
Charcot-Marie-Tooth disease 1 A (CMT1A) results from a duplication of the PMP22 gene in Schwann cells and a deficit of myelination in peripheral nerves. Patients with CMT1A have reduced nerve conduction velocity, muscle wasting, hand and foot deformations and foot drop walking. Here, we evaluate the safety and efficacy of recombinant adeno-associated viral vector serotype 9 (AAV2/9) expressing GFP and shRNAs targeting Pmp22 mRNA in animal models of Charcot-Marie-Tooth disease 1 A. Intra-nerve delivery of AAV2/9 in the sciatic nerve allowed widespread transgene expression in resident myelinating Schwann cells in mice, rats and non-human primates. A bilateral treatment restore expression levels of PMP22 comparable to wild-type conditions, resulting in increased myelination and prevention of motor and sensory impairments over a twelve-months period in a rat model of CMT1A. We observed limited off-target transduction and immune response using the intra-nerve delivery route. A combination of previously characterized human skin biomarkers is able to discriminate between treated and untreated animals, indicating their potential use as part of outcome measures.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/terapia , Proteínas da Mielina/antagonistas & inibidores , Proteínas da Mielina/genética , Animais , Doença de Charcot-Marie-Tooth/patologia , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Inativação Gênica , Terapia Genética/métodos , Vetores Genéticos , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/genética , Ratos , Ratos Mutantes , Células de Schwann/metabolismo , Células de Schwann/patologia , Nervo Isquiático/metabolismo , Nervo Isquiático/patologiaRESUMO
Peripheral nerves contain axons and their enwrapping glia cells named Schwann cells (SCs) that are either myelinating (mySCs) or nonmyelinating (nmSCs). Our understanding of other cells in the peripheral nervous system (PNS) remains limited. Here, we provide an unbiased single cell transcriptomic characterization of the nondiseased rodent PNS. We identified and independently confirmed markers of previously underappreciated nmSCs and nerve-associated fibroblasts. We also found and characterized two distinct populations of nerve-resident homeostatic myeloid cells that transcriptionally differed from central nervous system microglia. In a model of chronic autoimmune neuritis, homeostatic myeloid cells were outnumbered by infiltrating lymphocytes which modulated the local cell-cell interactome and induced a specific transcriptional response in glia cells. This response was partially shared between the peripheral and central nervous system glia, indicating common immunological features across different parts of the nervous system. Our study thus identifies subtypes and cell-type markers of PNS cells and a partially conserved autoimmunity module induced in glia cells.
Assuntos
Neurônios/fisiologia , Nervos Periféricos/citologia , Animais , Doenças Autoimunes/metabolismo , Biomarcadores , Comunicação Celular , Linhagem da Célula , Regulação da Expressão Gênica/fisiologia , Homeostase , Humanos , Leucócitos/fisiologia , Macrófagos/fisiologia , Camundongos , RatosRESUMO
In contrast to acute peripheral nerve injury, the molecular response of Schwann cells in chronic neuropathies remains poorly understood. Onion bulb structures are a pathological hallmark of demyelinating neuropathies, but the nature of these formations is unknown. Here, we show that Schwann cells induce the expression of Neuregulin-1 type I (NRG1-I), a paracrine growth factor, in various chronic demyelinating diseases. Genetic disruption of Schwann cell-derived NRG1 signalling in a mouse model of Charcot-Marie-Tooth Disease 1A (CMT1A), suppresses hypermyelination and the formation of onion bulbs. Transgenic overexpression of NRG1-I in Schwann cells on a wildtype background is sufficient to mediate an interaction between Schwann cells via an ErbB2 receptor-MEK/ERK signaling axis, which causes onion bulb formations and results in a peripheral neuropathy reminiscent of CMT1A. We suggest that diseased Schwann cells mount a regeneration program that is beneficial in acute nerve injury, but that overstimulation of Schwann cells in chronic neuropathies is detrimental.
Assuntos
Doenças Desmielinizantes/genética , Neuregulina-1/genética , Comunicação Parácrina , Células de Schwann/metabolismo , Nervo Sural/metabolismo , Animais , Animais Geneticamente Modificados , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Diabetes Mellitus Tipo 1/complicações , Neuropatias Diabéticas/etiologia , Neuropatias Diabéticas/genética , Neuropatias Diabéticas/metabolismo , Neuropatias Diabéticas/patologia , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Atividade Motora , Proteínas da Mielina/genética , Neuregulina-1/metabolismo , Neurite Autoimune Experimental/genética , Neurite Autoimune Experimental/metabolismo , Neurite Autoimune Experimental/patologia , Neuroglia/metabolismo , Ratos , Receptor ErbB-2/metabolismo , Células de Schwann/ultraestrutura , Nervo Isquiático/lesões , Transdução de Sinais , Nervo Sural/ultraestrutura , Nervo TibialRESUMO
BACKGROUND: Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited neuropathy, a debilitating disease without known cure. Among patients with CMT1A, disease manifestation, progression and severity are strikingly variable, which poses major challenges for the development of new therapies. Hence, there is a strong need for sensitive outcome measures such as disease and progression biomarkers, which would add powerful tools to monitor therapeutic effects in CMT1A. METHODS: We established a pan-European and American consortium comprising nine clinical centres including 311 patients with CMT1A in total. From all patients, the CMT neuropathy score and secondary outcome measures were obtained and a skin biopsy collected. In order to assess and validate disease severity and progression biomarkers, we performed qPCR on a set of 16 animal model-derived potential biomarkers in skin biopsy mRNA extracts. RESULTS: In 266 patients with CMT1A, a cluster of eight cutaneous transcripts differentiates disease severity with a sensitivity and specificity of 90% and 76.1%, respectively. In an additional cohort of 45 patients with CMT1A, from whom a second skin biopsy was taken after 2-3 years, the cutaneous mRNA expression of GSTT2, CTSA, PPARG, CDA, ENPP1 and NRG1-Iis changing over time and correlates with disease progression. CONCLUSIONS: In summary, we provide evidence that cutaneous transcripts in patients with CMT1A serve as disease severity and progression biomarkers and, if implemented into clinical trials, they could markedly accelerate the development of a therapy for CMT1A.
Assuntos
Doença de Charcot-Marie-Tooth/terapia , Progressão da Doença , Marcadores Genéticos/genética , Pele/patologia , Resultado do Tratamento , Adulto , Idoso , Biópsia , Catepsina A/genética , Doença de Charcot-Marie-Tooth/sangue , Doença de Charcot-Marie-Tooth/genética , Feminino , Glutationa Transferase/genética , Glicoproteínas/genética , Humanos , Masculino , Pessoa de Meia-Idade , Neuregulina-1/genética , Proteínas Nucleares , PPAR gama/genética , Diester Fosfórico Hidrolases/genética , Prognóstico , Pirofosfatases/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Gênica/genéticaRESUMO
Duplication of the gene encoding the peripheral myelin protein of 22 kDa (PMP22) underlies the most common inherited neuropathy, Charcot-Marie-Tooth 1A (CMT1A), a disease without a known cure. Although demyelination represents a characteristic feature, the clinical phenotype of CMT1A is determined by the degree of axonal loss, and patients suffer from progressive muscle weakness and impaired sensation. CMT1A disease manifests within the first two decades of life, and walking disabilities, foot deformities and electrophysiological abnormalities are already present in childhood. Here, we show in Pmp22-transgenic rodent models of CMT1A that Schwann cells acquire a persistent differentiation defect during early postnatal development, caused by imbalanced activity of the PI3K-Akt and the Mek-Erk signaling pathways. We demonstrate that enhanced PI3K-Akt signaling by axonally overexpressed neuregulin-1 (NRG1) type I drives diseased Schwann cells toward differentiation and preserves peripheral nerve axons. Notably, in a preclinical experimental therapy using a CMT1A rat model, when treatment is restricted to early postnatal development, soluble NRG1 effectively overcomes impaired peripheral nerve development and restores axon survival into adulthood. Our findings suggest a model in which Schwann cell differentiation within a limited time window is crucial for the long-term maintenance of axonal support.
Assuntos
Doença de Charcot-Marie-Tooth/fisiopatologia , Modelos Animais de Doenças , Neuregulina-1/fisiologia , Animais , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos TransgênicosRESUMO
Pelizaeus-Merzbacher disease (PMD) is a severe hypomyelinating disease, characterized by ataxia, intellectual disability, epilepsy, and premature death. In the majority of cases, PMD is caused by duplication of PLP1 that is expressed in myelinating oligodendrocytes. Despite detailed knowledge of PLP1, there is presently no curative therapy for PMD. We used a Plp1 transgenic PMD mouse model to test the therapeutic effect of Lonaprisan, an antagonist of the nuclear progesterone receptor, in lowering Plp1 mRNA overexpression. We applied placebo-controlled Lonaprisan therapy to PMD mice for 10 weeks and performed the grid slip analysis to assess the clinical phenotype. Additionally, mRNA expression and protein accumulation as well as histological analysis of the central nervous system were performed. Although Plp1 mRNA levels are increased 1.8-fold in PMD mice compared to wild-type controls, daily Lonaprisan treatment reduced overexpression at the RNA level to about 1.5-fold, which was sufficient to significantly improve the poor motor phenotype. Electron microscopy confirmed a 25% increase in the number of myelinated axons in the corticospinal tract when compared to untreated PMD mice. Microarray analysis revealed the upregulation of proapoptotic genes in PMD mice that could be partially rescued by Lonaprisan treatment, which also reduced microgliosis, astrogliosis, and lymphocyte infiltration.
Assuntos
Estrenos/uso terapêutico , Antagonistas de Hormônios/uso terapêutico , Doença de Pelizaeus-Merzbacher/tratamento farmacológico , Progesterona/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Estrenos/farmacocinética , Estrenos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Antagonistas de Hormônios/farmacocinética , Antagonistas de Hormônios/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Proteolipídica de Mielina/genética , Fenótipo , RNA Mensageiro/genéticaRESUMO
Glycogen synthase kinase 3ß (GSK3ß) inhibitors, especially the mood stabilizer lithium chloride, are also used as neuroprotective or anti-inflammatory agents. We studied the influence of LiCl on the remyelination of peripheral nerves. We showed that the treatment of adult mice with LiCl after facial nerve crush injury stimulated the expression of myelin genes, restored the myelin structure, and accelerated the recovery of whisker movements. LiCl treatment also promoted remyelination of the sciatic nerve after crush. We also demonstrated that peripheral myelin gene MPZ and PMP22 promoter activities, transcripts, and protein levels are stimulated by GSK3ß inhibitors (LiCl and SB216763) in Schwann cells as well as in sciatic and facial nerves. LiCl exerts its action in Schwann cells by increasing the amount of ß-catenin and provoking its nuclear localization. We showed by ChIP experiments that LiCl treatment drives ß-catenin to bind to T-cell factor/lymphoid-enhancer factor response elements identified in myelin genes. Taken together, our findings open perspectives in the treatment of nerve demyelination by administering GSK3ß inhibitors such as lithium.
Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Cloreto de Lítio/farmacologia , Bainha de Mielina/química , Nervos Periféricos/metabolismo , Animais , Núcleo Celular/metabolismo , Glicogênio Sintase Quinase 3 beta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína P0 da Mielina/metabolismo , Nervos Periféricos/efeitos dos fármacos , Placebos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células de Schwann/metabolismo , Nervo Isquiático/lesões , Transdução de SinaisRESUMO
Charcot-Marie-Tooth disease is the most common inherited neuropathy and a duplication of the peripheral myelin protein 22 gene causes the most frequent subform Charcot-Marie-Tooth 1A. Patients develop a slowly progressive dysmyelinating and demyelinating peripheral neuropathy and distally pronounced muscle atrophy. The amount of axonal loss determines disease severity. Although patients share an identical monogenetic defect, the disease progression is strikingly variable and the impending disease course can not be predicted in individual patients. Despite promising experimental data, recent therapy trials have failed. Established clinical outcome measures are thought to be too insensitive to detect amelioration within trials. Surrogate biomarkers of disease severity in Charcot-Marie-Tooth 1A are thus urgently needed. Peripheral myelin protein 22 transgenic rats harbouring additional copies of the peripheral myelin protein 22 gene ('Charcot-Marie-Tooth rats'), which were kept on an outbred background mimic disease hallmarks and phenocopy the variable disease severity of patients with Charcot-Marie-Tooth 1A. Hence, we used the Charcot-Marie-Tooth rat to dissect prospective and surrogate markers of disease severity derived from sciatic nerve and skin tissue messenger RNA extracts. Gene set enrichment analysis of sciatic nerve transcriptomes revealed that dysregulation of lipid metabolism associated genes such as peroxisome proliferator-activated receptor gamma constitutes a modifier of present and future disease severity. Importantly, we directly validated disease severity markers from the Charcot-Marie-Tooth rats in 46 patients with Charcot-Marie-Tooth 1A. Our data suggest that the combination of age and cutaneous messenger RNA levels of glutathione S-transferase theta 2 and cathepsin A composes a strong indicator of disease severity in patients with Charcot-Marie-Tooth 1A, as quantified by the Charcot-Marie-Tooth Neuropathy Score. This translational approach, utilizing a transgenic animal model, demonstrates that transcriptional analysis of skin biopsy is suitable to identify biomarkers of Charcot-Marie-Tooth 1A.