RESUMO
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
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
Isovaleryl coenzymeâ A (IV-CoA) is an important precursor for iso-fatty acids and lipids. It acts in the development of myxobacteria, which can produce this compound from acetyl-CoA through alternative IV-CoA biosynthesis (aib). A central reaction of aib is catalyzed by AibA/AibB, which acts as a cofactor-free decarboxylase despite belonging to the family of CoA-transferases. We developed an efficient expression system for AibA/AibB that allowed the determination of high-resolution crystal structures in complex with different ligands. Through mutational studies, we show that an active-site cysteine previously proposed to be involved in decarboxylation is not required for activity. Instead, AibA/AibB seems to induce an intramolecular decarboxylation by binding its substrate in a hydrophobic cavity and forcing it into a bent conformation. Our study opens opportunities for synthetic biology studies, since AibA/AibB may be suitable for the production of isobutene, a precursor of biofuels and chemicals.
Assuntos
Carboxiliases/metabolismo , Myxococcus xanthus/metabolismo , Ácidos Pentanoicos/metabolismo , Acil Coenzima A/metabolismo , Domínio Catalítico , Coenzima A-Transferases/metabolismo , Descarboxilação , Hemiterpenos , Interações Hidrofóbicas e Hidrofílicas , LigantesRESUMO
Oligosialic and polysialic acid (oligoSia and polySia) of the glycocalyx of neural and immune cells are linear chains, in which the sialic acid monomers are α2.8-glycosidically linked. Sialic acid-binding immunoglobulin-like lectin-11 (SIGLEC-11) is a primate-lineage specific receptor of human tissue macrophages and microglia that binds to α2.8-linked oligoSia. Here, we show that soluble low molecular weight polySia with an average degree of polymerization 20 (avDP20) interacts with SIGLEC-11 and acts anti-inflammatory on human THP1 macrophages involving the SIGLEC-11 receptor. Soluble polySia avDP20 inhibited the lipopolysaccharide (LPS)-induced gene transcription and protein expression of tumor necrosis factor-α (Tumor Necrosis Factor Superfamily Member 2, TNFSF2). In addition, polySia avDP20 neutralized the LPS-triggered increase in macrophage phagocytosis, but did not affect basal phagocytosis or endocytosis. Moreover, polySia avDP20 prevented the oxidative burst of human macrophages triggered by neural debris or fibrillary amyloid-ß1-42. In a human macrophage-neuron co-culture system, polySia avDP20 also reduced loss of neurites triggered by fibrillary amyloid-ß1-42. Thus, treatment with polySia avDP20 might be a new anti-inflammatory therapeutic strategy that also prevents the oxidative burst of macrophages.