Apolipoprotein A-I in mouse cerebrospinal fluid derives from the liver and intestine via plasma high-density lipoproteins assembled by ABCA1 and LCAT

Abstract

Apolipoprotein (apo) A-I, the major structural protein of high-density lipoprotein (HDL), is present in human and mouse cerebrospinal fluid (CSF) despite its lack of expression in brain cells. To identify the origin of apoA-I in CSF, we generated intestine-specific and liver-specific Apoa1 knockout mice (Apoa1^ΔInt and Apoa1^Δliv mice, respectively). Lipoprotein profiles of Apoa1^ΔInt and Apoa1^ΔLiv mice resembled those of control littermates, whereas knockout of Apoa1 in both intestine and liver (Apoa1^ΔIntΔLiv ) resulted in a 60-percent decrease in HDL-cholesterol levels, thus strongly mimicking the Apoa1^-/- mice. Immunoassays revealed that mouse apoA-I was not present in the CSF of the Apoa1^ΔIntΔLiv mice. Furthermore, apoA-I levels in CSF were highly correlated with plasma spherical HDL levels, which were regulated by ABCA1 and LCAT. Collectively, these results suggest that apoA-I protein in CSF originates in liver and small intestine and is taken up from the plasma.

Keywords: apoA-I; cerebrospinal fluid; intestine; liver; mice.

Figure 1.

Genetic Design A: Insertion of loxP in mouse Apoa1 allele at Intron 1 and Intron 3 using CRISPR/Cas9 technique. The sequence indicated at genome alleles are complementally sequence for sgRNA and PAM sequences. Donor oligonucleotides including loxP sequence were described, respectively. The open arrow indicated the cleavage sites by cas9. B: The genotyping primers positions of Apoa1^fl/fl mice and cre transgenic mice. Solid blue boxes indicated the loxP insertion sites. The primer sets for each allele’s genotyping were indicated in arrows.

Figure 2.

Typical Lipoprotein profiles of mice plasma. The apoA-I floxed (Apoa1^fl/fl) mice without cre expression(A), with villin-cre expression, Apoa1^ΔInt (B), with albumin-cre expression, Apoa1^ΔLiv (C), with both villin-cre and albumin-cre expression, Apoa1^ΔIntΔLiv (D), with CAG-cre expression, Apoa1^/- (E), Abca1-null mice(F), and Lcat-null mice(G) plasma were analyzed by the gel-permeation HPLC equipped with an online enzymatic colorimetric lipid assay system (Skylight Biotech Inc, Japan). Solid burgundy line, total cholesterol. The dotted blue line, triacylglycerol. Solid triangles, open arrows, and solid arrows point to the typical retention time of human VLDL, human LDL, and human HDL, respectively.

Figure 3.

The adrenal gland of apoA-I conditional knockout mice. Adrenal glands store cholesterol in the cortex to supply cholesterol at the acute steroid generations. Apoa1^fl/fl mice with Vil-cre Alb-cre expressed mice and with CAG-cre expressed mice showed reddish adrenal gland as hypoalphalipoproteinemia mice model, Abca1-null, and Lcat-null mice.

Figure 4.

The cryo-injury score of the mouse brain. P values were obtained by a two-tailed t-test. Asterisks indicate the statistical difference compared to the Apoa1^fl/fl mice (*: p<0.05, **: p<0.01).

Figure 5.

Negative staining transmission electron microscopy (TEM) image of HDL in cerebrospinal fluid (CSF) and in plasma. The contrast was obtained by a 2% uranyl acetate solution. The typical HDL particles in mouse CSF (A), in human plasma (B), and in rat CSF (C, D). The scale bar indicates 100 nm. Short bars in the image D was used for calibrations of each particle diameter lengths which indicated by side.

Figure 6.

ApoA-I and apoE level in mouse plasma and mouse CSF. A: apoA-I level in mouse plasma. B: apoE level in mouse plasma. C: apoA-I level in mouse CSF. D: apoE level in mouse CSF. Asterisks indicate the statistical difference compared to the Apoa1^fl/fl mice (**: p<0.01, ***: p<0.001, ****:p<0.0001). Daggers indicate the statistical difference compared to the C57BL/6 N mice (††: p<0.01, ††††: p<0.0001).

Figure 7.

Scattered plots of plasma and CSF apolipoproteins. Mean values of apoA-I and apoE in plasma and in CSF (Figure 5A and 5C, Figure 5B and 5D) were replotted. Open symbol; apoA-I floxed mouse group. Solid symbol; mouse group of C57BL/6N background.

Figure 8.

Illustrated summary sketch of apoA-I fates in biofluid and the mouse genotypes.