Ceramide levels regulated by carnitine palmitoyltransferase 1C control dendritic spine maturation and cognition

Abstract

The brain-specific isoform carnitine palmitoyltransferase 1C (CPT1C) has been implicated in the hypothalamic regulation of food intake and energy homeostasis. Nevertheless, its molecular function is not completely understood, and its role in other brain areas is unknown. We demonstrate that CPT1C is expressed in pyramidal neurons of the hippocampus and is located in the endoplasmic reticulum 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 overexpression in primary hippocampal cultured neurons increased ceramide levels, whereas in CPT1C-deficient neurons, ceramide levels were diminished. Correspondingly, CPT1C knock-out (KO) mice showed reduced ceramide levels in the hippocampus. 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 overexpression of CPT1C, indicating that CPT1C regulation of spine maturation is mediated by ceramide. To study the repercussions of the KO phenotype on cognition, we performed the hippocampus-dependent Morris water maze test on mice. Results show that CPT1C deficiency strongly impairs spatial learning. All of these results demonstrate that CPT1C regulates the levels of ceramide in the endoplasmic reticulum of hippocampal neurons, and this is a relevant mechanism for the correct maturation of dendritic spines and for proper spatial learning.

FIGURE 1.

CPT1C location in hippocampal neurons. A, CPT1C is present in neurons of the hippocampus, mainly pyramidal cells. Brain sections were double-immunodetected with anti-CPT1C antibody (green) and anti-glial fibrillary acidic protein antibody (red). B, hippocampal cultured neurons were double-transfected with pCPT1C-EGFP and pDS-Red at 11 DIV and visualized at 15 DIV. Images show that CPT1C is present in neuronal body, dendritic shaft, and spines (marked with arrows). pDs-Red transfection was performed to display the outline of the neuron. C, hippocampal cultured neurons were transfected with pDS-ER-Red to stain the ER. At 15 DIV, cells were immunodetected with anti-CPT1C antibodies (green). The merge image (yellow) demonstrates that CPT1C is localized to the ER membrane. D, Western blot analysis of CPT1C and CPT1A proteins in isolated microsomes and mitochondria from hippocampus of WT, heterozygous (HT), and KO mice.

FIGURE 2.

Regulation of ceramide levels by CPT1C. A, levels of ceramides in hippocampal neurons transduced with AAV1-GFP (as a control) or AAV1-CPT1C at 7 DIV. Cells were collected at 14 DIV. B, levels of ceramides in hippocampal neurons from WT and CPT1C KO mice. Cells were collected at 14 DIV. C, time course of incorporation of serine-d₇ into ceramide C18:0-d₃. Hippocampal cultured neurons from WT animals were treated with 4 mm serine-d₇ at DIV14. Ceramides C18:0 and C18:0-d₃ were analyzed at different times, and the percentage of incorporation is shown. D, effect of CPT1C overexpression and CPT1C deficiency on serine-d₇ incorporation into ceramide C18:0-d₃. Hippocampal cultured cells were transduced with AAV1-GFP (as a control) or AAV1-CPT1C at 7 DIV. Cells were treated with serine-d₇ at DIV 14 and collected after 2.5 h of treatment. The percentage of variation in ceramide C18:0-d₃ levels compared with the control cells is shown. Error bars, S.E.; n = 6; *, p < 0.05.

FIGURE 3.

Ceramide levels in hippocampus from ad libitum and fasted CPT1C KO and WT mice. Fasted mice were deprived of food for 15 h. Different ceramide species were measured: ceramide C16:0, ceramide C18:0, ceramide C18:1, ceramide C20:0, and ceramide C24:1. Error bars, S.E. n = 6; *, p < 0.05; **, p < 0.005; ***, p < 0.001, ANOVA test.

FIGURE 4.

Dendritic spine density and morphology from CPT1C KO and WT hippocampal neurons. Hippocampal neurons were transfected (13 DIV) with pEGFP to visualize the outline of the cell. Protrusions were analyzed 2 days after transfection. Protrusion density (A) and protrusion length (B and C) were measured. Mature spines (A) and filopodia (B) are indicated. Spine morphology (D–F) was assayed by analysis of types of protrusions: filopodia (without head), mushroom (with head and neck), and stubby (with only head). G, percentage of mature spines (mushroom and stubby) relative to the total number of protrusions was also measured. H, spine head area was measured in mushroom and stubby spines. I, a representative image of dendritic spines from WT and CPT1C KO neurons. For the quantification of protrusion density, spine length, and morphology, ∼100 dendrites from independent transfections were selected randomly. Student's t tests were used to assess statistical significance of the differences. Error bars, S.E.; ***, p < 0.001.

FIGURE 5.

Rescue of CPT1C KO phenotype on spine morphology by CPT1C expression or ceramide treatment. A, hippocampal neurons treated with 1.5 μm C6-ceramide at 7 DIV and transfected with pEGFP (BD Biosciences) at 12 DIV, fixed, and analyzed for the morphology of dendritic protrusions at 15 DIV. B, hippocampal neurons were transfected with pIRES-CPT1C at 7 DIV and analyzed for spine morphology at 15 DIV. pIRES-CPT1C vector expresses both CPT1C and GFP proteins, which permits us to visualize in green the cells overexpressing CPT1C. C, hippocampal neurons at DIV9 were treated with 10 μm myriocin until 15 DIV. Cells were transfected with pEGFP at 12 DIV and analyzed for the morphology of dendritic protrusions at 15 DIV. D, a representative image showing dendritic spines from WT mice, KO mice, KO mice treated with C6-ceramide, KO mice transfected with pIRES-CPT1C, and WT mice treated with myriocin. For the quantification of spine morphology, ∼100 dendrites from independent transfections were selected randomly. Student's t tests and ANOVA post hoc were used to assess statistical significance of the differences. Error bars, S.E.; *, p < 0.05; ***, p < 0.001.

FIGURE 6.

Spatial learning and memory measured by MWM test. A, MWM performance of CPT1C KO and WT mice during the learning sessions as latency (s) to find the platform along the acquisition phase (A), removal (Rem), and cued sessions (Cue). PT, pretraining. B, visual pathway traced by all animals. The white round platform is located in the northeast (NE) quadrant. C, mean swimming speed along acquisition sessions. D, percentage of time spent in the target quadrant (NE) during the removal session; discontinuous lines represent the chance level in this session. E, percentage of permanence in quadrants during the reversal (Rev) session. Data are represented as mean ± S.E. (error bars); *, p < 0.05; **, p < 0.05; ***, p < 0.001, ANOVA test.