Brain cholesterol turnover required for geranylgeraniol production and learning in mice

Abstract

The mevalonate pathway produces cholesterol and nonsterol isoprenoids, such as geranylgeraniol. In the brain, a fraction of cholesterol is metabolized in neurons by the enzyme cholesterol 24-hydroxylase, and this depletion activates the mevalonate pathway. Brains from mice lacking 24-hydroxylase excrete cholesterol more slowly, and the tissue compensates by suppressing the mevalonate pathway. Here we report that this suppression causes a defect in learning. 24-Hydroxylase knockout mice exhibit severe deficiencies in spatial, associative, and motor learning, and in hippocampal long-term potentiation (LTP). Acute treatment of wild-type hippocampal slices with an inhibitor of the mevalonate pathway (a statin) also impairs LTP. The effects of statin treatment and genetic elimination of 24-hydroxylase on LTP are reversed by a 20-min treatment with geranylgeraniol but not by cholesterol. We conclude that cholesterol turnover in brain activates the mevalonate pathway and that a constant production of geranylgeraniol in a small subset of neurons is required for LTP and learning.

Fig. 1.

Schematic of the mevalonate pathway. Arrows, flow of intermediates; brakes, pharmacologic and genetic inhibition of the indicated steps. PP, diphosphate.

Fig. 2.

Assessment of behavioral learning. (A) Spatial learning in Morris water maze tests. Mean time to find a submerged platform (latency) is shown as a function of trial day. Data were collected in four different experiments involving 40 WT (+/+) and 30 KO (−/−) mice. Error bars in this experiment and all other experiments represent SEM values. (B) After removal of the platform on the indicated day, the number of times animals of the two genotypes swam across the former location of the platform was determined. (C) Associative learning in cued conditioning fear tests. Percent freezing was measured in response to an auditory cue 1 h after the training period (Upper left) and 24 h after training period (Lower left), or in response to the context in which the training was performed after 1 h (Upper right) or 24 h (Lower right). Data were collected in two different experiments involving 30 +/+ mice and 30 −/− mice. (D) Motor learning in rotating rod tests. The mean times mice of the indicated genotypes stayed on the rotating rod are plotted as a function of trial day. Data were collected from 15 +/+ and 15 −/− mice.

Fig. 3.

Histology and protein expression in the hippocampus. (A Left) Hematoxylin and eosin staining of hippocampal sections from WT (+/+) and KO (−/−) mice. CA, cornu ammonis; DG, dentate gyrus. (Scale bar, 50 μm.) (A Center) Histochemical staining for cell nuclei (DAPI, blue), and double immunofluorescent staining for microtubule-associated protein 2 (MAP2) (antibody AP20, green) and synaptogyrin (antibody P925, red) in the CA1 region of the hippocampus. (Scale bar, 20 μm.) (A Right) Electron microscopic analyses of synaptic contacts on pyramidal cell dendrites in CA1 stratum radiatum. Arrows indicate synaptic clefts. (Scale bar, 400 nm.) (B) Protein expression assessed in hippocampal extracts by immunoblotting. The levels of three glutamate receptor subunits (NR1, GluR1, and GluR2/3), a small GTP binding protein (Rab3A), two synaptic vesicle proteins (synaptotagmin and synaptogyrin), glutamic acid decarboxylase (GAD67), and calnexin were determined.

Fig. 4.

Synaptic transmission in WT and 24-hydroxylase KO mice. (A) The indicated hippocampal membrane fractions from WT (+/+) and KO (−/−) mice were probed for 24-hydroxylase, the GluR1 glutamate receptor subunit, and synaptophysin. (B) Spontaneous miniature synaptic currents in single CA1 pyramidal neurons in the presence of 1 μM tetrodotoxin. Representative traces from recordings made from 11 +/+ (○) and 14 −/− (•) neurons. (C) Input-output curves. Field potentials were determined in WT (○, n = 14) and KO slices (•, n = 13) over a stimulus intensity range of 5 to 40 μA. (D) Short term plasticity assessed by paired-pulse facilitation. Data from two different experiments with 43 +/+ (○) and 28 −/− (•) slices.

Fig. 5.

Impaired hippocampal LTP and normal LTD in 24-hydroxylase KO mice. (A) LTP in hippocampal slices from WT (○, n = 19) and KO (•, n = 13) mice. The arrow marks the point of high frequency stimulation (θ burst). Insets in this experiment and all subsequent experiments show representative recorded potentials immediately before (dashed lines) and 55 min after (solid lines) tetanization. (B) LTD in hippocampal slices from WT (○, n = 13) and KO (•, n = 10) mice.

Fig. 6.

Restoration of LTP by mevalonate and isoprenoids. (A) Hippocampal slices from WT mice were incubated beginning at time 0 with no additions (○, n = 11) or 12.5 μM compactin (statin; •, n = 17), and LTP was induced after 20 min. (B) Reversal of compactin-mediated LTP inhibition by mevalonate (Mev) but not cholesterol (Chol). Hippocampal slices from WT mice were incubated with 12.5 μM compactin, 0.2 mM mevalonate, and 5 μg/ml cholesterol (○, n = 13), or 12.5 μM compactin and 5 μg/ml cholesterol (•, n = 10). (C) Mevalonate restores LTP in 24-hydroxylase KO mice. Hippocampal slices were incubated in the absence of additions (•, n = 10) or in the presence of 0.2 mM mevalonate (○, n = 12). (D) Geranylgeraniol (GG) restores LTP to WT levels. Hippocampal slices from WT mice (○, n = 18) were incubated with no additions. Slices from KO mice (•, n = 12) were incubated with 0.2 mM geranylgeraniol.