Study of the physiological function of carnitine palmitoyltransferase 1C enzyme

Carnitine palmitoyl transferase 1 (CPT1) enzymes catalyze the conversion of long-chain acyl-CoA to acyl-carnitines, thus facilitating the entry of long-chain fatty acids to the mitochondria, where they undergo β-oxidation. There are three isoforms: the liver isoform CPT1A (Esser, V. 1993), the muscle isoform CPT1B (Yamazaki, N. 1995) and the brain-specific isoform CPT1C (Price, N. 2002). CPT1A and CPT1B are localized in the outer mitochondrial membrane and are rate-limiting enzymes in fatty-acid β-oxidation. The CPT1C isoform, was first described in 2002, is expressed exclusively in the central nervous system, with a homogeneous distribution in all areas such as hippocampus, cortex, hypothalamus, cerebellum and others. CPT1C enzyme highly differs from the two other isozymes. Its C-terminal region is longer than that of the other CPTs (Price, N. 2002). It is located in the endoplasmic reticulum (ER) of cells, rather than in mitochondria, and so it does not facilitate fatty acid oxidation (Sierra, A.Y. 2008). Analysis of amino sequence of CPT1C reveals that all important residues for CPT1 activity are conserved in CPT1C enzyme, despite this, no catalytic activity was found (Price, N. 2002; Wolfgang, M.J. 2006), but it binds the CPT1 physiological inhibitor malonyl-CoA with the same affinity as CPT1A (Wolfgang, M.J. 2006). Finally, CPT1C is only present in mammals and appears to stem from a relatively recent CPT1A gene duplication (Price, N. 2002). The other isozymes are expressed in such organisms as fish, reptiles, amphibians or insects. This suggests a specific role for CPT1C in more evolved brains. At the physiological level, CPT1C contributes to the control of food intake and energy homeostasis (Wolfgang, M.J. 2006; Gao, X.F. 2009). Two independent groups developed a CPT1C-KO mouse, and both lines showed decreased food intake respect to wild-type animals (WT). However, when fed a high-fat diet, they were more susceptible to obesity and diabetes, presenting lower rates of peripheral fatty acid oxidation. All these effects were attributed to the hypothalamic function of CPT1C, since ectopic over-expression of CPT1C in hypothalamus protected mice from adverse weight gain caused by high-fat diet (Dai, Y. 2007). Moreover, the involvement of CPT1C in energy homeostasis has also been confirmed in transgenic animals over-expressing CPT1C specifically in brain (Reamy, A.A. 2011). At the molecular level, in collaboration with the group of Dr. Gary Lopaschuk, we showed that CPT1C is involved in the anorectic action of leptin, by modulating ceramide synthesis in the arcuate nucleus (ARC) of the hypothalamus (Gao, S. 2011). Interestingly, recent findings in tumor cells showed a new, unexpected role of CPT1C in the metabolic transformations reported in tumor cell growth (Zaugg, K. 2011). The authors demonstrated that CPT1C is frequently expressed in human lung tumors and protects cancerous cells from death induced by glucose deprivation or hypoxia. The results suggest that CPT1C might provide unidentified fatty-acid derived products that would be beneficial for cell survival under metabolic stress. However, despite these recent findings about CPT1C, little is known about its catalytic activity or its physiological function in other brain areas. We demonstrate that CPT1C has low CPT1 activity although it has similar affinity for its substrates: carnitine and palmitoyl-CoA than CPT1A isoform. The present study also shows that CPT1-KO mice have reduced long-chain acyl-carnitine levels in the hippocampus, hypothalamus or cerebellum. We examined whether CPT1C is expressed in the peripheral nervous system: in the ventral horn of the spinal cord (motor neurons) and in the sensitive ganglions, in addition to the brain. We found that CPT1C is expressed in both regions, albeit at lower levels than in the brain. We also examined CPT1C expression along mouse development, and we found that CPT1C protein expression is present in early stage of embryos at day 15, is increased postnatally and reaches its expression peak in adulthood. Moreover, CPT1C is expressed in pyramidal neurons of hippocampus and is located in ER throughout the neuron, even inside dendritic spines. We used molecular, cellular and behavioral approaches to determine CPT1C function. First, we analyzed the implication of CPT1C in ceramide metabolism. CPT1C over-expression in primary hippocampal cultured neurons increased ceramide levels, an effect that was blocked by treatment with myriocin, an inhibitor of the de novo synthesis of ceramide. Correspondingly, CPT1C knock-out (KO) mice showed reduced ceramide levels in hippocampus, cerebellum, striatum and motor cortex, mainly during fasting. At the cellular level, CPT1C deficiency altered dendritic spine morphology by increasing immature filopodia and reducing mature mushroom and stubby spines. Total protrusion density and spine head area in mature spines were unaffected. Treatment of cultured neurons with exogenous ceramide reverted the KO phenotype, as did ectopic over-expression of CPT1C, indicating that CPT1C regulation of spine maturation is mediated by ceramide. To study the repercussions of the KO phenotype on cognition and motor function, we performed the hippocampus-dependent Morris Water Maze (MWM) test and some motor tests on mice. Results show that CPT1C-KO mice are hypoactive and exhibit clear deficits in motor function, especially in coordination skills and strength. Moreover, CPT1C deficiency strongly impairs spatial learning without affecting memory or cognitive flexibility. So, all these results demonstrate that CPT1C regulates the de novo synthesis of ceramide in ER of hippocampal neurons and this is a relevant mechanism for the correct maturation of dendritic spines and for proper spatial learning.ESTUDIO DE LA FUNCIÓN FISIOLÓGICA DE LA ENZIMA CPT1C La isoforma carnitina palmitoil transferasa 1C (CPT1C) se expresa únicamente en cerebro y ha sido implicada en la regulación hipotalámica de la ingesta de alimentos y la homeostasis energética. No obstante, su función molecular y su papel en otras áreas del cerebro son desconocidas. Hemos demostrado que CPT1C se expresa en las neuronas piramidales del hipocampo y se localiza en el retículo endoplásmico a lo largo de la neurona, incluso dentro de las espinas dendríticas. Hemos utilizado métodos moleculares, celulares y conductuales para determinar la función de CPT1C. En primer lugar, analizamos la implicación de CPT1C en el metabolismo de la ceramida. La sobre-expresión de CPT1C en neuronas de hipocampo aumentó los niveles de ceramidas, un efecto que fue bloqueado por el tratamiento con miriocina, un inhibidor de la síntesis de novo de la ceramida. En consecuencia, los ratones CPT1C knock-out (CPT1C-KO) demostraron una reducción de los niveles de ceramidas en el hipocampo, cerebelo, estriado y corteza motora principalmente durante el ayuno. A nivel celular, la deficiencia en CPT1C afecta a la morfología de las espinas dendríticas mediante el aumento de filopodios inmaduros y reduciendo el número de espinas maduras. La densidad de protrusiones totales o el área de la cabeza de la espinas dendrítica no se vieron afectadas. El tratamiento de las neuronas en cultivo con ceramida exógena, como la sobre-expresión ectópica de CPT1C, revirtieron el fenotipo de las espinas CPT1C-KO, lo que indica que CPT1C regula la maduración de las espinas dendríticas a través de las ceramidas. Para estudiar las repercusiones del fenotipo CPT1C-KO en la cognición y en la habilidad motora se realizaron diferentes test conductuales. Los resultados del test cognitivo demostraron que la deficiencia de CPT1C perjudica al aprendizaje espacial. Por otra parte, la realización de test motores demostraron que los ratones CPT1C son hipoactivos y tienen disminuida tanto la coordinación motora como la fuerza muscular. Todos estos resultados demuestran que CPT1C regula la síntesis de novo de ceramidas en el retículo endoplásmico de las neuronas y éste es un mecanismo necesario para la correcta maduración de las espinas dendríticas y para el adecuado procedimiento del aprendizaje espacial y la función motora