Presynaptic defects underlying impaired learning and memory function in lipoprotein lipase-deficient mice

Abstract

Lipoprotein lipase (LPL) is predominantly expressed in adipose and muscle where it plays a crucial role in the metabolism of triglyceride-rich plasma lipoproteins. LPL is also expressed in the brain with highest levels found in the pyramidal cells of the hippocampus, suggesting a possible role for LPL in the regulation of cognitive function. However, very little is currently known about the specific role of LPL in the brain. We have generated a mouse model of LPL deficiency which was rescued from neonatal lethality by somatic gene transfer. These mice show no exogenous and endogenous LPL expression in the brain. To study the role of LPL in learning and memory, the performance of LPL-deficient mice was tested in two cognitive tests. In a water maze test, LPL-deficient mice exhibited increased latency to escape platform and increased mistake frequency. Decreased latency to platform in the step-down inhibitory avoidance test was observed, consistent with impaired learning and memory in these mice. Transmission electron microscopy revealed a significant decrease in the number of presynaptic vesicles in the hippocampus of LPL-deficient mice. The levels of the presynaptic marker synaptophysin were also reduced in the hippocampus, whereas postsynaptic marker postsynaptic density protein 95 levels remained unchanged in LPL-deficient mice. Theses findings indicate that LPL plays an important role in learning and memory function possibly by influencing presynaptic function.

Figure 1.

Impaired hippocampal learning and memory in LPL-deficient mice. A, Latency to find the platform in water maze escape task. B, Frequency of entries into the no-exit arms. C, Correlation between latency and error number. D, Retention times in step-down passive avoidance task (left) and training trial. Data were expressed as mean ± SEM, n = 6 for each group. Statistical significance was determined using the Student's t test. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2.

Ultrastrucural changes of synapses from WT and LPL-deficient neonatal mice detected by transmission electron microscopy. Synaptic vesicles within the presynaptic part are highlighted by arrows; the edges of the active zone/postsynaptic density complexes are marked by arrowheads. A–C are representative photos showing the total and docked synaptic vesicles in the hippocampal terminals of WT mice, whereas D–F are those of LPL-deficient ones. The table below shows the morphological analysis of synapses in CA3 regions of hippocampi from WT and LPL-deficient mice. L, Active zone length (μm); DV, number of docked vesicles per active zone; DV/μm, number of docked vesicles per micrometer of active zone length; TV, total vesicle number per terminal; TV/μm², total vesicle number per micrometer square of synapse area. Numbers are mean ± SEM; 30 synapses from each mouse were analyzed in WT group (n = 4); 50 synapses from each mouse were analyzed in LPL deficiency group (n = 4). ***p < 0.001.

Figure 3.

Immunofluorescence of presynaptic vesicles and neurofilaments in hippocampi of LPL-deficient and WT mice. Paraffin-embedded sections were immunostained and quantitated for presynaptic vesicle marker synaptophysin and cytoskeleton marker neurofilaments M. A, From neonatal mice (n = 5 for WT and n = 3 for LPL deficiency); B, from adult mice (n = 6 for WT and n = 3 for LPL deficiency). Scale bar, 50 μm. Western blotting detection of synaptophysin, PSD-95, and neurofilament M (150 kDa) levels in the hippocampus from WT and LPL-deficient mice. C, D, The hippocampal protein homogenates of neonatal (C) and adult (D) mice were subjected to SDS-PAGE, electrotransferred to PVDF membranes, and detected with antibodies against neurofilaments, PSD-95, and synaptophysin. Quantitation of blots was done by image analysis against GAPDH.

Figure 4.

Vitamin E contents in plasma and the brain. Vitamin E was extracted and measured by commercial kit. A, Plasma vitamin E contents in adult mice (n = 6 for both WT and LPL deficiency). B, Vitamin E levels in hippocampus of adult mice (n = 4 for each group). C, Vitamin E levels in the brain in fetal mice (n = 6 for each group). *p < 0.05, ***p < 0.001.