Papel de cpt1c en el desarrollo y en el transporte de endosomas tardíos

Resumen

Introduction: Carnitine palmitoyltransferase 1C (CPTIC) is an endoplasmic reticulum protein that has no catalytic activity but maintains the ability to bind to malonyl-CoA, an intermediary in the synthesis of fatty acids, whose levels highly fluctuate depending on the energetic status of the cell. Consequently, CPTIC has been proposed to be a sensor of malonyl-CoA in neurons. A CPTIC mutation in the human gene has been described to cause pure hereditary spastic paraplegia (HSP), a group of neurological disorders characterized by slowly progressive weakness and spasticity of the muscles of the legs, caused by axonopathy of corticospinal motor neurons. Genetic analysis has identified more than 50 different loci involved in HSP, with the mutations in the Protrudin gene being responsible for a large number of cases. Protrudin has been recently involved in late endosome (LE) transport along the axon and in neurite growth. We aimed to demonstrate: 1) the interaction between CPTIC and Protrudin, 2) CPTIC regulation of LE transport depending on malonyl-CoA levels, and 3) CPTIC involvement in axon growth. Material and methods: CPTIC-Protrudin interaction was monitored by fluorescence resonance energy transfer (FRET) analysis. Neurite outgrowth measurements were performed in primary culture mouse cortical neurons from WT or CPTIC KO embryos. Analysis of the LE velocity and distance was determined in living Hela cells. Results: We demonstrate that CPT1C is necessary for the proper growth of axons and dendritic arborization in cortical neurons. FYCO1-positive LEs show a strong movement towards the cell periphery only in CPT1C overexpressing cells and this effect is dependent on malonyl-CoA levels. When CPTIC and Protrudin are overexpressed and malonyl-CoA levels are decreased, LES are localized in perinuclear area. Moreover, FRET studies demonstrate the interaction between CPTIC and Protrudin. Conclusions: 1. CPTIC is necessary for the correct axonal and dendritic growth in primary cultures of cortical neurons. 2. CPTIC acts as a malonyl-CoA sensor. High levels of malonyl-CoA favor axonal growth, while low levels or the CPTIC mutation at the malonyl-CoA binding site reduce axonal growth. 3. CPTIC favors the initial polarization of the axon. 4. CPT1C interacts with protrudin. 5. The interaction of CPTIC with protrudin or with atlastin-1 does not depend on malonyl-CoA levels. 6. CPTIC does not contact directly with late endosomes. 7. CPTIC overexpression in HeLa cells promotes the peripheral localization of late endosomes and this effect requires the binding of CPTIC to malonyl-CoA. 8. CPTIC overexpression in HeLa cells favors anterograde transport of LEs. This action is also dependent on the binding of CPT1C to malonyl-CoA.