Novel regulation of AMPA receptor function by interacting protein CPT1C

AMPA receptors (AMPARs) are responsible for the 90% of synaptic transmission and are involved in plasticity, developmental and neurological processes. Their function depends on the proteins interacting with the AMPAR complex, which determine their specific gating and trafficking properties and hence their specific roles. In addition to well-known interacting proteins of AMPARs, recent studies have described the CPT1C as an associated partner in the outer core of the AMPAR complex in the hippocampus, cortex and cerebellum. CPT1C is a neuron specific homologue of the carnitine acyltransferase family of enzymes, which are involved in fatty acid oxidation at the mitochondria. Contrary to the rest of the CPT1 family, CPT1C localizes at the endoplasmic reticulum and apparently is not related with the functions that other CPT1s carry out. To elucidate the physiological role of CPT1C isoform different studies have been performed, showing that CPT1C is involved in energy homeostasis, control of body weight and motor function as well as behavioral learning mechanisms. Of note, CPT1C KO animals display impairments in spatial learning along with immature dendritic spines. Despite all these studies with CPT1C, including its interaction with AMPARs, it is unknown the CPT1C physiological relevance and role, particularly in the regulation of the AMPAR function and its implication in neurological diseases. In this thesis we have described a novel role of CPT1C in AMPA receptor function regulation. More specifically, the results show that glutamate-evoked currents of the recombinant GluA1 receptors are increased when CPT1C is present and this effect is specific of the CPT1 isoform CPT1C, since CPT1A does not share the same pattern. The ER location of CPT1C seems to be crucial to modulate AMPAR surface expression since mislocalization of CPT1C avoids its AMPAR modulation. Co-localization studies confirmed that GluA1-CPT1C interaction happens at ER level but not at the cell surface. On the other hand, no changes in current density have been found in cells expressing GluA2 subunit along with CPT1C indicating AMPA subunit specificity. Additionally, electrophysiological experiments have determined that GluA1 channel properties are no altered, thus indicating that the increased current is probably due to a rise in AMPAR number at the cell surface. Indeed, this hypothesis is corroborated by the findings from the immunofluorescence experiments, where surface expression of GluA1 is increased in the presence of CPT1C in heterologous systems and cortical neurons. In agreement with a putative role of CPT1C in determining the AMPAR content at synapse level, we have demonstrated that synaptic transmission is altered in CPT1C KO neurons. In parallel, we have studied the molecular mechanisms of CPT1C effect on AMPARs. We have shown that the palmitoylable cysteine 585 of the GluA1 subunit is crucial for the CPT1C enhancement of AMPA receptors trafficking by using immunofluorescence and electrophysiological techniques. However, the palmitoylation state of this residue does not determine AMPAR-CPT1C interaction. We have found evidences for a supposed depalmitoylation activity by CPT1C studying the role of the C-terminus of CPT1C, which contains the residue His469 with catalytic activity. Specifically we have found that CPT1C H469A mutant form does not alter GluA1 induced whole-cell currents as the wild type CPT1C does indicating that the C-terminal catalytic domain plays a crucial role in GluA1 modulation. This is supported by the fact that Palmostatin B – a newly described palmitoyl thioesterase inhibitor – decreases the CPT1C effect on GluA1 induced currents, most likely by inhibiting its PTE activity. In summary, this thesis unravels a novel regulation of AMPA receptor function by the interacting protein CPT1C, which modulates AMPA receptor trafficking and this effect depends on the catalytic domain of CPT1C C-terminal acting on the cysteine 585 of GluA1 AMPAR subunit.Los receptores de glutamato tipo AMPA son fundamentales en la transmisión excitatoria rápida que se da en el sistema nervioso central. A parte de su papel crucial en la comunicación neuronal, los receptores AMPA son responsables de ciertos tipos de plasticidad sináptica, siendo importantes en el desarrollo del sistema nervioso central además de estar involucrados en multitud de procesos patológicos y enfermedades neurodegenerativas. La función de los receptores AMPA depende principalmente de dos factores: la composición de las subunidades que lo conforman (el receptor propiamente dicho) y la presencia de proteínas que interactúan con el receptor y actúan como subunidades auxiliares. Estos dos factores establecen las características intrínsecas del canal así como las interacciones de los receptores AMPA con otras proteínas intracelulares que determinarán sus propiedades de tráfico (ensamblaje, exocitosis, endocitosis y anclaje sináptico) y en último término sus funciones en la neurona. Entre la gran cantidad de proteínas que interaccionan con los receptores AMPA, recientes estudios proteómicos han demostrado que la proteína CPT1C forma parte del conjunto macromolecular de los receptores AMPA en el tejido neuronal. El objetivo principal de esta tesis se ha focalizado en el estudio esta proteína (CPT1C) en el contexto de la función de los receptores AMPA debido a su interacción. Para ello se han utilizado técnicas electrofisiológicas, de inmunofluorescencia y de biología molecular y celular tanto en sistemas de expresión heterólogos como en cultivos de células neuronales. Los experimentos llevados al cabo durante la tesis han confirmado la interacción entre las dos proteínas y han atribuido un papel modulador de CPT1C en el trafico de los receptores AMPA. El efecto regulador de la CPT1C es dependiente de la composición del receptor, afectando de manera diferencial subtipos distintos de receptores AMPA. La proteína CPT1C aumenta el tráfico de los receptores AMPA a la superficie celular sin alterar sus propiedades biofísicas y sin interaccionar a nivel de membrana plasmática ambas proteínas. Además en esta tesis se han descrito los mecanismos implicados en la modulación de dichos receptores por parte de CPT1C, desentrañando un residuo cisteína concreto determinante para la modulación de los receptores AMPA por la CPT1C. Finalmente, los experimentos llevados a cabo en esta tesis nos indican que los efectos observados se deben a una posible depalmitoilación del receptor, lo cual lleva a un aumento de su capacidad de acumulación en la membrana. En resumen, los resultados obtenidos durante esta tesis demuestran que el número de receptores AMPA en las sinapsis está aumentado en presencia de CPT1C y este efecto es debido a la actividad catalítica de CPT1C