RESUMO
Botulinum neurotoxins (BoNTs) have been widely used clinically as a muscle relaxant. These toxins target motor neurons and cleave proteins essential for neurotransmitter release like Synaptosomal-associated protein of 25 kDa (SNAP-25). In vitro assays for BoNT testing using rodent cells or immortalized cell lines showed limitations in accuracy and physiological relevance. Here, we report a cell-based assay for detecting SNAP-25-cleaving BoNTs by combining human induced Pluripotent Stem Cells (hiPSC)-derived motor neurons and a luminescent detection system based on split NanoLuc luciferase. This assay is convenient, rapid, free-of-specialized antibodies, with a detection sensitivity of femtomolar concentrations of toxin, and can be used to study the different steps of BoNT intoxication.
Assuntos
Toxinas Botulínicas Tipo A , Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Toxinas Botulínicas Tipo A/toxicidade , Toxinas Botulínicas Tipo A/metabolismo , Neurônios Motores/metabolismo , Transporte BiológicoRESUMO
The olfactory bulb (OB) is a highly plastic structure that can change organizational networks depending on environmental inputs in adult mammals. Particularly, in rodents, adult neurogenesis underlies plastic changes in the OB circuitry by continuously adding new interneurons to the network. We addressed the question of whether microglia, the immune cells of the brain, were involved in pruning OB neurons. Using lentiviral labeling of neurons in neonatal or adult mice and confocal analysis, we showed that microglia engulfed parts of neonatal-born and adult-born neurons in the healthy OB. We demonstrated that OB deafferentation by Dichlobenil administration induced sensory deprivation. It also increased phagocytosis of adult-born, but not neonatal-born neurons, by activated microglia. Conversely, intranasal lipopolysaccharide administration induced activation of microglia but changed neither adult neurogenesis nor olfaction. Our data reveal that steady-state microglia eliminate adult-born neurons and their synapses in both healthy and sensory deprived OBs, thereby adapting neuronal connections to the sensory experience.
Assuntos
Microglia/citologia , Neurônios/citologia , Bulbo Olfatório/citologia , Fagocitose/fisiologia , Privação Sensorial/fisiologia , Animais , Masculino , Camundongos Endogâmicos C57BL , Neurogênese/fisiologia , Plasticidade Neuronal/fisiologia , Olfato/fisiologia , Sinapses/metabolismoRESUMO
Fast nerve conduction relies on successive myelin segments that electrically isolate axons. Segment geometry-diameter and length-is critical for the optimization of nerve conduction and the molecular mechanisms allowing this optimized geometry are partially known. We show here that peripheral myelin elongation is dynamically regulated by stimulation of YAP (Yes-associated protein) transcription cofactor activity during axonal elongation and limited by inhibition of YAP activity via the Hippo pathway. YAP promotes myelin and non-myelin genes transcription while the polarity protein Crb3, localized at the tips of the myelin sheath, activates the Hippo pathway to temper YAP activity, therefore allowing for optimal myelin growth. Dystrophic Dy(2j/2j) mice mimicking human peripheral neuropathy with reduced internodal lengths have decreased nuclear YAP which, when corrected, leads to longer internodes. These data show a novel mechanism controlling myelin growth and nerve conduction, and provide a molecular ground for disease with short myelin segments.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Axônios/metabolismo , Proteínas de Membrana/metabolismo , Bainha de Mielina/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Animais , Axônios/ultraestrutura , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Células HEK293 , Via de Sinalização Hippo , Humanos , Glicoproteínas de Membrana , Camundongos , Fenótipo , Fosforilação , Ratos , Células de Schwann/metabolismo , Transcrição Gênica , Proteínas de Sinalização YAPRESUMO
The thickness of the myelin sheath that insulates axons is fitted for optimal nerve conduction velocity. Here, we show that, in Schwann cells, mammalian disks large homolog 1 (Dlg1) interacts with PTEN (phosphatase and tensin homolog deleted on chromosome 10) to inhibit axonal stimulation of myelination. This mechanism limits myelin sheath thickness and prevents overmyelination in mouse sciatic nerves. Removing this brake results also in myelin outfoldings and demyelination, characteristics of some peripheral neuropathies. Indeed, the Dlg1 brake is no longer functional in a mouse model of Charcot-Marie-Tooth disease. Therefore, negative regulation of myelination appears to be essential for optimization of nerve conduction velocity and myelin maintenance.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Membrana/metabolismo , Bainha de Mielina/fisiologia , Proteínas do Tecido Nervoso/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Células de Schwann/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Axônios/fisiologia , Técnicas de Cocultura , Proteína 1 Homóloga a Discs-Large , Gânglios Espinais/citologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Bainha de Mielina/ultraestrutura , Proteínas do Tecido Nervoso/genética , Condução Nervosa , Neuregulina-1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Ratos , Proteínas Associadas SAP90-PSD95 , Nervo Isquiático/fisiologiaRESUMO
Diameter, organization, and length of the myelin sheath are important determinants of the nerve conduction velocity, but the basic molecular mechanisms that control these parameters are only partially understood. Cell polarization is an essential feature of differentiated cells, and relies on a set of evolutionarily conserved cell polarity proteins. We investigated the molecular nature of myelin sheath polarization in connection with the functional role of the cell polarity protein pals1 (Protein Associated with Lin Seven 1) during peripheral nerve myelin sheath extension. We found that, in regard to epithelial polarity, the Schwann cell outer abaxonal domain represents a basolateral-like domain, while the inner adaxonal domain and Schmidt-Lanterman incisures form an apical-like domain. Silencing of pals1 in myelinating Schwann cells in vivo resulted in a severe reduction of myelin sheath thickness and length. Except for some infoldings, the structure of compact myelin was not fundamentally affected, but cells produced less myelin turns. In addition, pals1 is required for the normal polarized localization of the vesicular markers sec8 and syntaxin4, and for the distribution of E-cadherin and myelin proteins PMP22 and MAG at the plasma membrane. Our data show that the polarity protein pals1 plays an essential role in the radial and longitudinal extension of the myelin sheath, likely involving a functional role in membrane protein trafficking. We conclude that regulation of epithelial-like polarization is a critical determinant of myelin sheath structure and function.