Molecular markers of brain cholesterol homeostasis are unchanged despite a smaller brain mass in a mouse model of cholesteryl ester storage disease

Abstract

Lysosomal acid lipase (LAL), encoded by the gene LIPA, facilitates the intracellular processing of lipids by hydrolyzing cholesteryl esters and triacylglycerols present in newly internalized lipoproteins. Loss-of-function mutations in LIPA result in cholesteryl ester storage disease (CESD) or Wolman disease when mutations cause complete loss of LAL activity. Although the phenotype of a mouse CESD model has been extensively characterized, there has not been a focus on the brain at different stages of disease progression. In the current studies, whole-brain mass and the concentrations of cholesterol in both the esterified (EC) and unesterified (UC) fractions were measured in Lal^-/- and matching Lal^+/+ mice (FVB-N strain) at ages ranging from 14 up to 280 days after birth. Compared to Lal^+/+ controls at 50, 68-76, 140-142, and 230-280 days of age, Lal^-/- mice had brain weights that averaged approximately 6%, 7%, 18%, and 20% less, respectively. Brain EC levels were higher in the Lal^-/- mice at every age, being elevated 27-fold at 230-280 days. Brain UC concentrations did not show a genotypic difference at any age. The elevated brain EC levels in the Lal^-/- mice did not reflect EC in residual blood. An mRNA expression analysis for an array of genes involved in the synthesis, catabolism, storage, and transport of cholesterol in the brains of 141-day old mice did not detect any genotypic differences although the relative mRNA levels for several markers of inflammation were moderately elevated in the Lal^-/- mice. The possible sites of EC accretion in the central nervous system are discussed.

Keywords: brain mass; cholesterol-esterifying enzymes; esterified cholesterol sequestration; lysosomal acid lipase; residual blood volume; unesterified cholesterol.

Fig. 1.

Body (a) and whole-brain (b) weights, and concentrations in brain of esterified (c) and unesterified (d) cholesterol in male and female Lal^+/+ and Lal^−/− mice at 68 to 72 days of age. The measurements (for c, d) were made in extracts of the entire brain using a combination of column and gas chromatography as described in Materials and Methods. Values represent the mean ± SEM of measurements for 5 Lal^+/+ and 6 Lal^−/− males, and 4 Lal^+/+ and 6 Lal^−/− females, all of which were of the FVB/N strain. *Significantly different from the value for Lal^+/+ controls of the same gender (p < 0.05).

Fig. 2.

Concentration of esterified (a) and unesterified (b) cholesterol in the brain, and whole-brain cholesterol contents (c) in Lal^+/+ and Lal^−/− mice from pre-weaning to late-stage disease. All mice were of the FVB/N strain. The brain tissue was derived from the same sets of mice as described in Table 1. Values represent the mean ± SEM of measurements for the number of mice specified. *Significantly different from the value for Lal^+/+ controls at that age (p< 0.05).

Fig. 3.

Concentration of total cholesterol in whole blood (a) and in plasma (b) of Lal^+/+ and Lal^−/− mice in different studies. In one experiment the concentrations of EC and UC in whole blood were measured in mice aged from 70 to 76 days and summed to obtain the total cholesterol (TC) level. In another study plasma TC data were obtained directly without separation of the EC and UC fractions from mice at four different ages ranging from 49 to 111 days. In both studies all mice were of the FVB/N strain. Values for whole blood represent the mean ± SEM of data from 4 Lal^+/+ (3 males /1 female) and 4 Lal^−/− (3 males /1 female) mice, while those for the plasma TC levels at four age points were from males only. The numbers of mice at each age were respectively: 49 d: 12 Lal^+/+ and 15 Lal^−/−, 71 d: 5 Lal^+/+ and 6 Lal^−/−, 91 d: 5 Lal^+/+ and 6 Lal^−/−, 111 d: 4 Lal^+/+ and 3 Lal^−/−.*Significantly different from the value for the matching Lal^+/+ controls (p < 0.05).

Fig. 4.

Rate of cholesterol synthesis in the brains of Lal^+/+ and Lal^−/− mice at two different ages. These rates were measured in vivo using [³H]water as described in Materials and Methods. They reflect the rate of incorporation of water into new sterols throughout the entire brain over the course of 1 h. Values represent the mean ± SEM of measurements in 3 Lal^+/+ (1 male /2 females) and 5 Lal^−/− (3 males /2 females) mice at 23 days, and in 6 Lal^+/+ (all females) and 6 Lal^−/− (all females) mice at 50 days. All mice were of the FVB/N strain. At neither age was the value for the Lal^−/− mice significantly different from that for the matching Lal^+/+ controls (p >0.05).

Fig. 5.

Relative mRNA levels for multiple genes involved in the regulation of cholesterol homeostasis (a), or that serve as markers of inflammation (b) in the brains of 141-day old Lal^+/+ and Lal^−/− mice. The details of these analyses are given in Materials and Methods. For each genotype brains were obtained from 2 male and 2 female mice, all of the FVB/N strain. The expression level for each gene in the Lal^+/+ mice was arbitrarily set at 1.0 (dashed line) so bars reflect fold-change observed in the Lal^−/− mice. Values represent the mean ± SEM. *Significantly different from the value for the Lal^+/+ controls (p<0.05). The full name of each gene in the order as it appears in the figure is: Lipa, gene that encodes LAL; Npc2, Niemann-Pick type C2; Npc1, Niemann-Pick type C1; Srebp2, sterol regulatory element-binding protein 2; Hmgcs, 3-hydroxy-3-methylglutaryl coenzyme A synthase; Hmgcr, 3-hydroxy-3-methylglutaryl-coenzyme A reductase; Cyp46a1, cholesterol 24-hydroxylase; Apoe, apolipoprotein E; Ldlr, low-density lipoprotein receptor; Soat1, sterol O-acyltransferase 1 (previously called Acat1); Lcat, lecithin:cholesterol acyltransferase; Abca1, ATP-binding cassette transporter A1; Abca2, ATP-binding cassette transporter A2; Abcg1, ATP-binding cassette transporter G1; Ccl3, chemokine (C-C motif) ligand 3 (also known as Mip1α); TNFα, tumor necrosis factor alpha; ltgax, integrin subunit alpha X (also known as CD11c); Cd14 (Cluster of differentiation 14 antigen).