Overexpression of ABCA1 reduces amyloid deposition in the PDAPP mouse model of Alzheimer disease (original) (raw)
Creation and characterization of PrP-mAbca1 Tg mice. Thirteen founders tested positive for the PrP-mAbca1 transgene by PCR. One founder died before breeding, 1 founder was sterile, 3 founders failed to exhibit germline transmission of the transgene, and 1 founder did not show overexpression of ABCA1 in the brain. The 7 remaining founders (designated B, D, E, G, I, J, and L) all produced pups that were positive for the PrP-mAbca1 transgene and demonstrated ABCA1 overexpression in the brain. The progeny of 5 founders bred well and were used for experiments (lines D, E, L, J, and G).
The 5 lines of PrP-mAbca1 Tg mice that we examined had different levels of ABCA1 overexpression in the cortex, ranging from approximately 2-fold in line D to approximately 14-fold in line G (Figure 1A). Analysis of tissue from the occipital cortex, frontal cortex, hippocampus, cerebellum, striatum, septum, and thalamus showed that PrP-mAbca1 Tg mice overexpressed ABCA1 throughout the brain (Figure 1B).
Characterization of 3-month-old PrP-mAbca1 mice. (A) Levels of ABCA1 in the cortex of 5 different lines of PrP-mAbca1 mice (lines D, E, L, J, and G) were visualized by Western blotting. RIPA lysates were made from cortex, and equal amounts of total protein (10 μg) were electrophoresed. Western blotting of ABCA1 was performed using the HJ1 antibody. Samples from Tg (+) and non-Tg mice (–) were compared. Liver lysates from Abca1+/+ and Abca1–/– mice were used as positive and negative controls, respectively. The fold difference in ABCA1 overexpression was estimated by diluting the Tg sample until the level of ABCA1 equaled that of the non-Tg sample. (B) ABCA1 expression in multiple brain regions was assessed by Western blotting in PrP-mAbca1 line E mice. (C) ABCA1 expression in major body tissues was examined in mice expressing no ABCA1 (bottom panel), the PrP-mAbca1 transgene with no endogenous ABCA1 (the top panel), and endogenous ABCA1 (middle panel). Liver lysates from Abca1+/+ and Abca1–/– mice were used as positive and negative controls, respectively.
Representative tissues from the major organ systems were collected to determine the extent of ABCA1 overexpression throughout the body. Using Western blot analyses, we detected high endogenous mouse ABCA1 expression in lung, liver, spleen, bladder, testis, and brain (Figure 1C, middle panel). In comparison, high expression of the PrP-mAbca1 transgene was found in the brain, as well as in kidney, testis, and muscle (Figure 1C, upper panel). Since the PrP-mAbca1 Tg mice used in these experiments express endogenous ABCA1 in addition to the PrP-mAbca1 transgene, they have normal to increased ABCA1 activity in organs outside the brain.
The 5 PrP-mAbca1 Tg lines we studied produced equal numbers of male and female pups with no overt differences in growth or health. However, male mice from all but the lowest overexpressing line (line D) were sterile. Gross examination of the testes from PrP-mAbca1 line E mice revealed severe atrophy in Tg mice, with an approximately 50% reduction in testes mass compared with non-Tg mice (Figure 2, A and B). To further characterize this phenotype, we performed histological examination of the testes of Tg and non-Tg mice. Compared with non-Tg mice (Figure 2C, top left panel), the seminiferous tubules of the testes from the Tg mice showed atrophy with marked degenerative changes highlighted by a large increase in multinucleated giant cells and cells with condensed nuclei (Figure 2C, top right panel). These degenerative changes were present in almost every section of the seminiferous tubules in the Tg testes compared with their rare presence in the non-Tg testes. TUNEL staining showed approximately 3-fold more apoptotic cells in testes from Tg mice (Figure 2C, bottom right panel), as compared with non-Tg mice (Figure 2C, bottom left panel, and Figure 2D). Further, testes from Tg mice exhibited maturation arrest due to absence of mature products of spermatogenesis such as elongated spermatids and spermatozoa (Figure 2C, top right panel). These findings are consistent with the marked reduction in germ cells detected by immunohistochemistry with an anti-GCNA1 antibody (Figure 2E). The numbers of Sertoli cells, detected by GATA4 immunohistochemistry, were similar in Tg and wild-type testes (data not shown). A previous study has shown that Abca1–/– mice have abnormalities in sperm production (28), and our data suggest that an excess of ABCA1 is also detrimental to male fertility. However, previous reports on other ABCA1 Tg mice have not reported any abnormalities in testicle size or male fertility (25, 27, 29). It is interesting that the spermatogenesis defects reported here are highly similar to those observed in mice with aberrant glial cell line–derived neurotrophic factor–Ret receptor tyrosine kinase (GDNF/Ret) signaling (30, 31). Thus, it is possible that ABCA1 activity directly or indirectly influences the GDNF/Ret signaling system in the testes.
Spermatogenesis defects in 3-month-old PrP-mAbca1 line E Tg mice. (A) Testes from PrP-mAbca1 line E mice (n = 5) and non-Tg mice (n = 6) were dissected and weighed. (B) A representative testis from a Tg mouse and a non-Tg mouse. Scale bar: 5 mm. (C) H&E-stained sections show normal testicular histology in non-Tg mice (top left panel) and marked degeneration of seminiferous tubules of Tg mice (top right panel), with several multinucleated giant cells (arrowheads) present in almost every tubule compared with their rare occurrence in non-Tg mice. No elongated spermatids were present in testes from Tg mice. TUNEL staining shows occasional apoptotic cells in testes from non-Tg mice (bottom left panel) and frequent apoptotic cells in testes from Tg mice (bottom right panel). Scale bar: 50 μm for the top panels and 100 μm for the bottom panels. (D) Quantification of TUNEL staining in the testes of Tg (n = 4) and non-Tg littermate control mice (n = 4). (E) Sections of testes from Tg (n = 3) and non-Tg littermate control mice (n = 4) underwent immunohistochemical staining with an anti-GCNA1 antibody that stains germ cells. The number of GCNA1-positive cells per tubule was tabulated. Statistical analyses of differences between Tg and non-Tg mice were performed using 2-tailed Student’s t test. **P < 0.01; ****P < 0.0001.
Amyloid deposition in the brains of PDAPP/Abca1 Tg mice. Mice from PrP-mAbca1 lines D, E, and J, which overexpressed mABCA1 by approximately 2-, 6-, and 12-fold, respectively, were bred to the PDAPP mouse model of AD. The resulting PDAPP/Abca1 Tg mice and their PDAPP/Abca1 non-Tg littermates were sacrificed at either 3 or 12 months. Brain sections from 12-month-old PDAPP/Abca1 line E mice were stained for Aβ. The PDAPP mice with wild-type ABCA1 levels (PDAPP/Abca1 non-Tg mice) had frequent punctate Aβ deposits in the cortex and hippocampus (Figure 3A, top left panel), including the molecular layer of the hippocampus, but almost no Aβ deposits in the hilus of the dentate gyrus of the hippocampus (Figure 3A, middle left panel). In contrast, the PDAPP mice with ABCA1 overexpression (PDAPP/Abca1 Tg mice) had rare Aβ deposition in the cortex (Figure 3A, top middle panel), occasional Aβ deposition in the molecular layer, and frequent Aβ deposition in the hilus of the dentate gyrus (Figure 3A, middle panel). This is interesting because PDAPP mice rarely deposit Aβ in the hilus of the dentate gyrus unless they also lack Apoe (Figure 3A, top right and middle right panels) (32–34).
Aβ staining and thioflavine S fluorescence in the brains of 12-month-old PDAPP/Abca1 line E mice. (A) Sections of brain from PDAPP/Abca1 non-TG mice, PDAPP/Abca1 line E mice, and PDAPP/Apoe–/– mice were stained for Aβ using the 3D6 antibody. True amyloid was visualized by thioflavine S fluorescence. The arrows shown in the middle panels indicate the upper and lower limits of the hilus of the dentate gyrus. Scale bar: 125 μm for the top and bottom panels and 250 μm for the middle panels. (B) Brain sections from PDAPP/Abca1 line E mice were immunostained with an antibody against the microglial marker CD45. Scale bar: 625 μm for all panels.
Brain sections from the 12-month-old mice were also stained with thioflavine S to detect fibrillar Aβ. As detected via fluorescence microscopy, frequent amyloid plaques were seen in the cortex and hippocampus of the PDAPP/Abca1 non-Tg mice (Figure 3A, bottom left panel). However, almost no thioflavine S–positive amyloid plaques were seen in the PDAPP/Abca1 Tg mice (Figure 3A, bottom middle panel) or in PDAPP/Apoe–/– mice (Figure 3A, bottom right panel), even in sections with extensive Aβ deposits. This demonstrates that Aβ deposits in the PDAPP/Abca1 Tg mice are almost all diffuse, with very little true amyloid. In the PDAPP/Abca1 Tg mice, the preferential deposition of Aβ in the hilus of the dentate gyrus of the hippocampus, along with the lack of thioflavine S–positive Aβ deposits, is virtually identical to the pattern of Aβ deposition in PDAPP/Apoe–/– mice. This suggests that increasing ABCA1-mediated lipidation of apoE has an effect on Aβ deposition very similar to that seen in APP Tg mice lacking apoE.
Consistent with previous work (35), the thioflavine S–positive plaques in the brains of PDAPP/Abca1 non-Tg mice were associated with clusters of reactive microglia (Figure 3B, upper left and lower left panels). The PDAPP/Abca1 Tg mice, which had very few thioflavine S–positive deposits, had few to no observable clusters of reactive microglia in their brains (Figure 3B, upper right and lower right panels). These findings demonstrate that the paucity of fibrillar Aβ in the brains of PDAPP/Abca1 Tg mice is associated with decreased microglial activation.
Quantitative stereological analyses of Aβ deposition demonstrated markedly and significantly lower Aβ load in the cortex of 12-month-old PDAPP/Abca1 Tg mice as compared with PDAPP/Abca1 non-Tg littermate control mice (Figure 4A). The Aβ load in the entire hippocampus did not vary significantly according to the presence of the mABCA1 transgene, but the distribution of Aβ within the hippocampus was altered by the transgene. PDAPP/Abca1 Tg mice had more Aβ deposition in the hilus of the dentate gyrus and less in the molecular layer (Figure 4A). Quantification of thioflavine S–positive amyloid plaques confirmed the near absence of true amyloid plaques in PDAPP/Abca1 Tg mice, in contrast to the robust presence of amyloid plaques in PDAPP/non-Tg mice (Figure 4B). Furthermore, thioflavine S–positive amyloid plaques were quantified in PDAPP/Abca1 lines D, E, and J, which overexpress ABCA1 by 2-, 6-, and 12-fold in the brain, respectively. Two-fold overexpression of ABCA1 in the brain decreased deposition of thioflavine S–positive amyloid by approximately 50%. Overexpression of ABCA1 by 6-fold or more decreased deposition of thioflavine S–positive amyloid to almost 0. These data demonstrate that overexpression of ABCA1 protects PDAPP mice from developing thioflavine S–positive amyloid plaques.
Aβ load and thioflavine S–positive plaque load in 12-month-old PDAPP/Abca1 mice. (A and B) Using stereological methods, Aβ staining and thioflavine S fluorescence were quantified in the cortex, hippocampus, and 2 subregions of the hippocampus (molecular layer and hilus of the dentate gyrus) of PDAPP/Abca1 line E mice. (C and D) Thioflavine S fluorescence was quantified in the cortex and hippocampus of PDAPP/Abca1 line D, line E, and line J mice. n = 10 for all groups. Statistical analyses of differences between Tg and non-Tg mice were performed using the Mann-Whitney U test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
The amount of Aβ in the brains of the PDAPP/Abca1 mice was quantified by ELISA at 3 months of age, when Aβ had not yet deposited, and after 12 months, when extensive Aβ deposition had occurred. At 3 months of age, both the PDAPP/Abca1 line D and line E Tg mice, which overexpress ABCA1 by 2- and 6-fold, respectively, had significantly (15%–50%) higher Aβ levels than non-Tg littermate control mice (Figure 5, A and B, and Table 1). These higher levels do not represent deposited Aβ, as the mice did not have histological evidence of Aβ deposition at this age. To determine whether the Aβ levels in the young PDAPP/Abca1 Tg mice were higher due to increased Aβ generation from APP, levels of APP and the C-terminal fragments (CTFs) created by enzymatic cleavage of APP were assessed by Western blotting. There were no differences between PDAPP/Abca1 line E Tg and PDAPP/non-Tg mice (Figure 5E), suggesting that Aβ production was not likely to account for the differences in Aβ levels at 3 months. Previously, our group found that PDAPP/Apoe–/– mice also have higher brain Aβ levels at 3 months of age, probably because apoE is required for normal transport and clearance of soluble Aβ (36).
Aβ as detected by ELISA in the hippocampus of PDAPP/Abca1 mice. (A–D) Aβ was serially extracted from the hippocampus using carbonate and guanidine buffers and measured by ELISA. The Aβ in the carbonate and guanidine extracts was summed, and total Aβ40 and Aβ42 ares represented. TP, total protein in the tissue lysate. (A and B) Aβ in the hippocampus of 3-month-old mice from PDAPP/Abca1 lines D and E, respectively. n = 11 in all groups. (C and D) Aβ in the hippocampus of 12-month-old mice from PDAPP/Abca1 lines D and E, respectively. n = 10–14 in all groups. Statistical analyses of differences between Tg and non-Tg mice were performed using 2-tailed Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (E) Levels of APP and APP C-terminal fragments in hippocampus from PDAPP/Abca1 line E mice were visualized by Western blotting. n = 7 for both groups. β-Tubulin was used as a loading control. Quantification of the APP and CTF bands using image analysis software revealed no significant differences between PDAPP/Abca1 Tg and PDAPP/Abca1 non-Tg mice.
Aβ levels in brain extracts as measured by ELISA
By 12 months of age, large amounts of Aβ had accumulated in the brains of all mice with the PDAPP transgene (more than 100-fold compared with 3 months of age). However, mice that also overexpressed ABCA1 developed significantly less Aβ deposition than non-Tg littermates. Aβ levels in PDAPP/Abca1 line D Tg mice were approximately 2.5-fold lower compared with those in PDAPP/non-Tg littermate control mice (Figure 5C and Table 1), and the PDAPP/Abca1 line E Tg mice had approximately 3-fold less Aβ in the brain compared with PDAPP/non-Tg littermate control mice (Figure 5D and Table 1). PDAPP/Abca1 line J Tg mice had a decrease in Aβ levels at 12 months of age similar to that seen in PDAPP/Abca1 line E Tg mice (data not shown). As shown in previous studies, PDAPP/Apoe–/– mice also develop approximately 2- to 3-fold less Aβ deposition than PDAPP/Apoe+/+ mice (32–34). Thus, the PDAPP/Abca1 Tg mice have a phenotype similar to that seen in PDAPP/Apoe–/– mice.
ApoE levels and solubility in PrP-mAbca1 Tg mice. Previously, we and others found that Abca1–/– mice have reduced levels of apoE in the CNS (18–20). This may reflect increased brain catabolism of poorly lipidated apoE produced in the absence of ABCA1, similar to the rapid turnover of poorly lipidated peripheral apoA-I in ABCA1-deficient states. We hypothesized that excess ABCA1 function in the PrP-mAbca1 Tg mice would result in increased apoE lipidation, which would decrease apoE catabolism. Theoretical support for this hypothesis was given by the elevated levels of HDL-associated apolipoproteins in the plasma of previously generated ABCA1 Tg models (25, 27). Although we expected that the PrP-mAbca1 Tg mice would have increased levels of apoE in the CNS compared with non-Tg littermate controls, this was not the case. While 2-fold overexpression of ABCA1 in PrP-mAbca1 line D Tg mice did not result in altered apoE levels (Figure 6A), overexpression of ABCA1 by 6-fold or greater resulted in reductions in hippocampal apoE of approximately 40% compared with non-Tg littermate control mice (Figure 6A). The size of the decrease did not vary between lines that overexpressed ABCA1 6-fold to 14-fold, indicating that the effect is saturable. Also, the extent that ABCA1 overexpression results in lower levels of apoE varies according to brain region. In the cortex, apoE was decreased by only approximately 20% in Tg mice from PrP-mAbca1 lines L, J, and G and was not decreased significantly in lines D or E (Figure 6B). In the CSF, apoE levels were decreased by approximately 40%–70% in all lines overexpressing ABCA1 (Figure 6C). PDAPP/Abca1 Tg mice with 6-fold or greater levels of overexpression also had lower levels of apoE in the brain at 3 months of age, and the amount of this change was not different from that in non-PDAPP mice (data not shown). To determine whether the decreased apoE levels in the PrP-mAbca1 Tg mice were due to decreased transcription of apoE, we performed real-time quantitative RT-PCR to determine the relative levels of apoE mRNA in PrP-mAbca1 line E and line J mice. We found no difference between Tg and non-Tg apoE mRNA levels in either the hippocampus or cortex of either line (Figure 6D and data not shown). In addition to decreasing apoE levels, overexpression of ABCA1 affected the biochemical properties of apoE. In all groups of PrP-mAbca1 Tg mice that overexpressed ABCA1 by 6-fold or greater, approximately twice as much apoE in the hippocampus and cortex was insoluble in carbonate buffer and required 5 M guanidine for extraction compared with non-Tg littermate control groups (data not shown). The relative insolubility of brain-associated apoE from PrP-mAbca1 mice in carbonate buffer suggests a difference in apoE metabolism induced by ABCA1 overexpression.
Levels of apoE in the hippocampus, cortex, and CSF of 3-month-old PrP-mAbca1 Tg mice. (A and B) apoE was serially extracted from brain tissue using carbonate and guanidine buffers and measured by ELISA. The apoE in the carbonate and guanidine extracts was summed, and total apoE is represented in A and B. (C) CSF was diluted, and apoE levels were measured by ELISA. (D) RNA was extracted from the hippocampus and cortex of PrP-mAbca1 line E mice. Real-time quantitative RT-PCR was performed for mouse apoE mRNA and total rRNA. Mouse apoE mRNA was normalized to total rRNA. (A–D) For all groups, the n = 7–9. Tg and non-Tg samples from the same line were processed at the same time. Statistical analyses of differences between Tg and non-Tg mice were performed using 2-tailed Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Plasma levels of cholesterol and apoE were measured in 3-month-old mice of PrP-Abca1 lines D, E, L, J, and G. No differences in plasma apoE levels between Tg and non-Tg mice were observed except in line J, the second-highest-expressing line, which exhibited elevated plasma apoE levels of 97 ± 6 μg/ml in Tg mice compared with 78 ± 6 μg/ml in non-Tg mice (mean ± SEM; P = 0.04). Plasma cholesterol levels were also not different between Tg and non-Tg mice except for the 2 highest-expressing lines (J and G), which had higher plasma cholesterol than non-Tg mice. In PrP-Abca1 line J mice, levels of plasma cholesterol were 647 ± 47 μg/ml in Tg mice and 348 ± 50 μg/ml in non-Tg mice (P = 0.0007). In PrP-Abca1 line G mice, levels of plasma cholesterol were 586 ± 42 μg/ml in Tg mice and 418 ± 48 μg/ml in non-Tg mice (P = 0.02). These findings suggest that integration of multiple copies of the PrP-mAbca1 transgene can affect peripheral cholesterol metabolism through mechanisms that remain to be identified. In addition to plasma cholesterol, plasma HDL was assessed in Tg and non-Tg mice by fractionating plasma from 3-month-old PrP-mAbca1 line E mice using size-exclusion gel chromatography. HDL-associated cholesterol and phospholipids were not significantly different in Tg and non-Tg mice. Finally, plasma apoA-I levels were assessed by Western blotting in mAbca1 line E Tg mice versus non-Tg littermates. There was no significant difference between the groups (data not shown).
apoE-containing lipoprotein particles from PrP-mAbca1 Tg mice. We previously found that mice lacking ABCA1 had abnormally small apoE-containing lipoprotein particles in their CSF and that primary astrocyte cultures derived from Abca1–/– mice contained small, lipid-poor apoE-containing lipoprotein particles (18). We hypothesized that overexpression of ABCA1 in the brain would result in greater lipidation of apoE-containing lipoprotein particles in the CSF and that primary astrocytes derived from PrP-mAbca1 Tg mice would secrete larger, more lipid-rich apoE-containing lipoprotein particles. Nondenaturing gradient gel electrophoresis of CSF from PrP-mAbca1 mice, followed by Western blotting for apoE, was performed. apoE-containing lipoprotein particles from the Tg mice tended to be larger in size than particles from non-Tg mice (Figure 7A). This suggests that apoE particles in the CNS are more lipidated in Tg mice. To further characterize the effects of ABCA1 overexpression on nascent apoE particles, primary cultures of astrocytes were prepared from PrP-mAbca1 Tg mice and non-Tg littermate control mice and allowed to secrete apoE-containing lipoprotein particles into serum-free media over 72 hours. Astrocyte-conditioned media (ACM) from PrP-mAbca1 Tg mice and non-Tg littermate controls was then subjected to nondenaturing gradient gel electrophoresis followed by Western blotting for apoE. The apoE-containing lipoprotein particles from the Tg mice had a greater proportion of lipoprotein particles larger than 11 nm than that seen in media derived from non-Tg mice (Figure 7B).
Analysis of apoE-containing lipoprotein particles derived from PrP-mAbca1 line E mice. (A) CSF (2 μl) from 3-month-old PrP-mAbca1 line E mice was run on a nondenaturing gradient gel, and Western blot analysis for mouse apoE was performed. (B) Media conditioned by primary astrocytes derived from PrP-mAbca1 line E mice was run on a nondenaturing gradient gel, and Western blot analysis for mouse apoE was performed. Samples containing equal amounts of apoE were loaded. (C and D) Primary astrocytes derived from PrP-mAbca1 line E mice secreted apoE-containing lipoprotein particles into the media over 72 hours. Lipoprotein particles in the ACM were separated by size using gel filtration chromatography. The levels of apoE and cholesterol were then measured in the fractions and normalized to total protein in the cell pellet. n = 3 for non-Tg and n = 5 for Tg. (E) The amount of cholesterol and apoE in lipoprotein fractions 20–50 was totaled, and lipoprotein cholesterol was divided by lipoprotein apoE. Two-tailed Student’s t test was performed to evaluate the statistical significance of differences between particles from Tg and non-Tg mice. ****P < 0.0001.
To determine whether the astrocyte-derived apoE-containing lipoprotein particles from PrP-mAbca1 mice contained more lipid, ACM was subjected to size-exclusion gel chromatography to separate the different classes of lipoproteins. Compared with astrocytes derived from non-Tg mice, astrocytes derived from Tg mice secreted less apoE and associated cholesterol in HDL-like lipoproteins (Figure 7, C and D). However, within the lipoprotein fractions, there was a significantly greater ratio of cholesterol to apoE (Figure 7E). This demonstrates that ABCA1 overexpression resulted in increased lipidation of nascent apoE particles.
Effects of brain overexpression of ABCA1 on other proteins. We used Western blotting of ABCA1 Tg line E brain lysates to assess whether overexpression of mAbca1 influenced the levels of other proteins that are potentially involved in apoE and Aβ metabolism. No differences were found in the levels of low-density lipoprotein receptor (LDLR), LDLR-related protein 1 (LRP1), and apoA-I when comparing ABCA1 line E Tg mice to non-Tg littermates (Figure 8, A and B). The level of ABCG1 in brain lysates was also compared by Western blot in samples from multiple mAbca1 transgenic lines, and there was no consistent difference found between mAbca1 Tg and non-Tg mice (data not shown). However, levels of apoJ (also called clusterin) were significantly lower in PrP-mAbca1 line E Tg brain lysates versus non-Tg control brain lysates (Figure 8, A and B). Because this difference in apoJ levels was not observed in ABCA1 line D mice (data not shown), a 2-fold overexpression of ABCA1 in the brain is insufficient to significantly alter either apoJ or apoE levels (Figure 6A). However, lines with greater than 2-fold ABCA1 overexpression exhibited changes in both apoE and apoJ levels. Notably, because 2-fold overexpression of ABCA1 was sufficient to reduce Aβ levels in PDAPP/Abca1 line D mice (Figure 5, A and C) without significant alterations in either apoE or apoJ levels, the beneficial effects of excess ABCA1 function in Aβ reduction appear to be largely independent of the absolute levels of apoE and apoJ.
Comparison of lipoprotein receptors and lipoproteins in PrP-mAbca1 line E Tg versus non-Tg mice. (A) Samples of cortex from 3-month-old mice were lysed in RIPA buffer. Equal amounts of total protein from each mouse were loaded into each well of the gel and then assessed by Western blotting for LDLR, LRP, apoJ, apoA-I, and tubulin. Three representative lanes are shown for Tg and non-Tg mice. (B) Quantitative analysis of Western blots was performed using Kodak Image Station software, with n = 3–6 per group. ****P < 0.0001.