RESUMEN

Adult mammalian central nervous system axons have intrinsically poor regenerative capacity, so axonal injury has permanent consequences. One approach to enhancing regeneration is to increase the axonal supply of growth molecules and organelles. We achieved this by expressing the adaptor molecule Protrudin which is normally found at low levels in non-regenerative neurons. Elevated Protrudin expression enabled robust central nervous system regeneration both in vitro in primary cortical neurons and in vivo in the injured adult optic nerve. Protrudin overexpression facilitated the accumulation of endoplasmic reticulum, integrins and Rab11 endosomes in the distal axon, whilst removing Protrudin's endoplasmic reticulum localization, kinesin-binding or phosphoinositide-binding properties abrogated the regenerative effects. These results demonstrate that Protrudin promotes regeneration by functioning as a scaffold to link axonal organelles, motors and membranes, establishing important roles for these cellular components in mediating regeneration in the adult central nervous system.

Asunto(s)

Axones/fisiología , Sistema Nervioso Central/fisiología , Retículo Endoplásmico/metabolismo , Regeneración Nerviosa , Proteínas de Transporte Vesicular/metabolismo , Animales , Axones/metabolismo , Células Cultivadas , Retículo Endoplásmico/genética , Endosomas/metabolismo , Femenino , Humanos , Integrinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Mutación , Regeneración Nerviosa/efectos de los fármacos , Neuronas/metabolismo , Neuronas/fisiología , Fármacos Neuroprotectores/administración & dosificación , Traumatismos del Nervio Óptico/tratamiento farmacológico , Traumatismos del Nervio Óptico/metabolismo , Traumatismos del Nervio Óptico/patología , Fosforilación , Dominios Proteicos , Ratas , Ratas Sprague-Dawley , Retina/efectos de los fármacos , Retina/fisiología , Proteínas de Transporte Vesicular/administración & dosificación , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética

RESUMEN

Most endosomal sorting complex required for transport (ESCRT)-III proteins are not fully functional when expressed as fusion of fluorescent or epitope tags, frequently making the use of specific antibodies the only available method for their detection. Heterologous expression of ESCRT-III proteins in bacteria often results in the formation of insoluble aggregates or inclusion bodies that interfere with their purification. However, inclusion bodies are usually pure protein aggregates with high antigenicity. In addition, since proteins within inclusion bodies are presented in a range of folding states, immunization with inclusion bodies can potentially result in antibodies with specificity for different folding states of the protein under study. We describe here a protocol to isolate bacterial inclusion bodies of plant ESCRT-III proteins for production of polyclonal antibodies. We also describe a nitrocellulose-based immunoaffinity purification method that allows the immobilization of ESCRT-III proteins and the subsequent isolation of specific antibodies from a crude serum.

Asunto(s)

Anticuerpos/aislamiento & purificación , Proteínas de Arabidopsis/aislamiento & purificación , Complejos de Clasificación Endosomal Requeridos para el Transporte/aislamiento & purificación , Cuerpos de Inclusión/metabolismo , Proteínas de Transporte Vesicular/aislamiento & purificación , Animales , Anticuerpos/inmunología , Proteínas de Arabidopsis/administración & dosificación , Proteínas de Arabidopsis/inmunología , Proteínas de Arabidopsis/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/administración & dosificación , Complejos de Clasificación Endosomal Requeridos para el Transporte/inmunología , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Escherichia coli/genética , Vectores Genéticos/genética , Inmunización/métodos , Plásmidos/genética , Pliegue de Proteína , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Purificación por Afinidad en Tándem/métodos , Transformación Bacteriana , Proteínas de Transporte Vesicular/administración & dosificación , Proteínas de Transporte Vesicular/inmunología , Proteínas de Transporte Vesicular/metabolismo

RESUMEN

Niemann-Pick type C (NPC) disease is a fatal inherited neurodegenerative disorder caused by loss-of-function mutations in the NPC1 or NPC2 gene. There is no effective way to treat NPC disease. In this study, we used adeno-associated virus (AAV) serotype 9 (AAV9) to deliver a functional NPC1 gene systemically into NPC1-/- mice at postnatal day 4. One single AAV9-NPC1 injection resulted in robust NPC1 expression in various tissues, including brain, heart, and lung. Strikingly, AAV9-mediated NPC1 delivery significantly promoted Purkinje cell survival, restored locomotor activity and coordination, and increased the lifespan of NPC1-/- mice. Our work suggests that AAV-based gene therapy is a promising means to treat NPC disease.

Asunto(s)

Técnicas de Transferencia de Gen , Terapia Genética , Enfermedad de Niemann-Pick Tipo C/genética , Enfermedad de Niemann-Pick Tipo C/terapia , Proteínas/genética , Animales , Encéfalo/metabolismo , Supervivencia Celular/genética , Dependovirus/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular , Locomoción/genética , Pulmón/metabolismo , Ratones , Miocardio/metabolismo , Proteína Niemann-Pick C1 , Enfermedad de Niemann-Pick Tipo C/patología , Proteínas/administración & dosificación , Células de Purkinje/metabolismo , Células de Purkinje/patología , Proteínas de Transporte Vesicular/administración & dosificación , Proteínas de Transporte Vesicular/genética

RESUMEN

Heparin is a carbohydrate anticoagulant used clinically to prevent thrombosis, however impurities can limit its efficacy. Here we report the biosynthesis of heparin-like heparan sulfate via the recombinant expression of human serglycin in human cells. The expressed serglycin was also decorated with chondroitin/dermatan sulfate chains and the relative abundance of these glycosaminoglycan chains changed under different concentrations of glucose in the culture medium. The recombinantly expressed serglycin produced with 25mM glucose present in the culture medium was found to possess anticoagulant activity one-seventh of that of porcine unfractionated heparin, demonstrating that bioengineered human heparin-like heparan sulfate may be a safe next-generation pharmaceutical heparin.

Asunto(s)

Coagulación Sanguínea/efectos de los fármacos , Ingeniería Genética/métodos , Heparina/análogos & derivados , Proteoglicanos/administración & dosificación , Proteoglicanos/biosíntesis , Proteínas de Transporte Vesicular/administración & dosificación , Proteínas de Transporte Vesicular/biosíntesis , Anticoagulantes/administración & dosificación , Anticoagulantes/metabolismo , Células HEK293 , Heparina/administración & dosificación , Heparina/biosíntesis , Heparina/genética , Humanos , Ingeniería Metabólica , Proteoglicanos/genética , Proteínas de Transporte Vesicular/genética

RESUMEN

LYVE-1 (lymphatic vessel endothelial hyaluronan receptor-1) is a homolog of the hyaluronan receptor CD44, and one of the most widely used markers of lymphatic endothelial cells in normal and tumor tissues. However, the physiologic role of LYVE-1 in the lymphatic system still remains unclear. It is well established that fibroblast growth factor 2 (FGF2) induces lymphangiogenesis. Based on the known interaction between FGF2 and CD44 and based on the structural similarity of CD44 and LYVE-1, we investigated whether FGF2 might interact with LYVE-1. We found that FGF2 is able to bind LYVE-1 using AlphaScreen, or after surface-immobilization or in solution. FGF2 binds to LYVE-1 with a higher affinity than any other known LYVE-1­binding molecules, such as hyaluronan or PDGF-BB. Glycosylation of LYVE-1 is important for FGF2 binding. Furthermore, FGF2 interacts with LYVE-1 when overexpressed in CHO cells. Soluble LYVE-1 and knockdown of LYVE-1 in lymphatic endothelial cells impaired FGF2 signaling and functions. In addition, FGF2 but not VEGF-C-induced in vivo lymphangiogenesis, was also inhibited. Conversely, FGF2 also modulates LYVE-1 expression in cells and ex vivo. Thus, our data demonstrate a functional relationship to the interaction between FGF2 and LYVE-1.

Asunto(s)

Células Endoteliales/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animales , Biomarcadores/metabolismo , Células CHO , Línea Celular , Cricetinae , Cricetulus , Células Endoteliales/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/administración & dosificación , Factor 2 de Crecimiento de Fibroblastos/genética , Células Endoteliales de la Vena Umbilical Humana , Humanos , Receptores de Hialuranos/metabolismo , Linfangiogénesis/efectos de los fármacos , Linfangiogénesis/fisiología , Ratones , Ratones Endogámicos BALB C , Mapas de Interacción de Proteínas , ARN Interferente Pequeño/genética , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Transporte Vesicular/administración & dosificación , Proteínas de Transporte Vesicular/antagonistas & inhibidores , Proteínas de Transporte Vesicular/genética