Figure 1.
LDLR deficiency results in increased Thioflavine-S plaque load in the presence or in the absence of ApoE.
A–B. Representative pictures of Thioflavine-S staining in cortices (A) and hippocampi (B) of the analyzed groups (n = 5−7, 6–7 sections per animal, 240 mm apart). The absence of LDLR results in increased Thioflavine-S positive staining in the 5XFAD mice in the presence (A and B upper photos) or the absence of ApoE (A and B lower photos). Scale bar 0.5 mm. C. Magnification of the subiculum of the 5XFAD/ApoE-/- and the 5XFAD/ApoE-/-LDLR-/- mice, showing the difference in abundance of Thioflavine-S positive plaques. Scale bar 0.1 mm. D. Quantitation of Thioflavine-S positive staining in the hippocampi (left) and cortices (right) of female mice showing the increase in the amyloid plaques in the 5XFAD/LDL-/- and 5XFAD/ApoE-/-LDLR-/- mice. One-way ANOVA showed a significant difference among groups (P<0.0001) followed by Student's t-test. * P<0.05. P-values among groups are analysed in Table 1.
Table 1.
Results of Student's t-test for Thioflavine-S positive amyloid deposits of the analyzed groups.
Figure 2.
Lack of LDLR increases brain ApoE levels in the 5XFAD/LDLR-/- mice and has no effect on the APP processing.
A. Western blot for ApoE of protein extracts of 5XFAD and 5XFAD/LDLR-/- mouse brains. Tubulin was used as a loading control. 30 µg from the guanidine fraction of brain homogenates was loaded in each lane. In the absence of LDLR the levels of brain ApoE are increased. B. Densitometry of western blots shows a significant increase in the levels of ApoE in the guanidine fraction of total brain extracts. Statistical analysis was performed with the Student's t-test (** P = 0.0041). C. 10 µg of protein from the lysis fraction of total brain homogenates were loaded in each lane and immunoblotted for full length APP and CTFs. GAPDH was used as a loading control. No difference was observed in the full length APP or the CTFs when normalised to the control indicating that LDLR deficiency has no effect on the steady-state levels of full-length APP or on α- and β-secretase activity (5-7 animals per genotype were analysed and the experiments were repeated 3 times).
Figure 3.
LDLR deletion increases Aβ deposition in the mouse hippocampus.
A. Immunohistochemistry for total Aβ (6E10) in the brains of female mice. Representative pictures are shown for each genotype. The Aβ deposition is increased in the subiculum of the 5XFAD/LDLR-/- mice compared to the 5XFAD mice. The same effect is observed in the 5XFAD/ApoE-/-LDLR-/- mice where Aβ deposition is more intense in the subiculum. Aβ deposition in the 5XFAD/ApoE-/-LDLR-/- cortices appears more ‘dense’ and “compact” compared to the 5XFAD/ApoE-/- mice where Aβ deposition is more “diffuse”. Scale bar 0.5 mm (n = 5−7, 6–7 sections per animal, 240 mm apart). B. Quantitation of Aβ immunoreactivity in the hippocampi (left) of the analysed groups shows a significant increase in the Aβ deposition in the 5XFAD/LDLR-/- and the 5XFAD/ApoE-/-LDLR-/- mice. In the cortices (right) there is no significant difference between 5XFAD and 5XFAD/LDLR-/- as well as between 5XFAD/ApoE-/- and 5XFAD/ApoE-/-/LDLR-/-.*P<0.05. P-values among all groups are analysed in Table 2 and Table 3 for hippocampi and cortices respectively. C. Quantitation of Aβ42 and Aβ40 levels by ELISA in the 5XFAD and the 5XFAD/LDLR-/- mouse brain extracts showed an increase both in the guanidine and lysis fraction in the 5XFAD/LDLR-/- mice. A similar increase was also observed in the guanidine fraction of the 5XFAD/ApoE-/-LDLR-/- mice compared to the 5XFAD/ApoE-/- but not in the lysis fraction (n = 5−7) *P<0.05, **P<0.001.
Table 2.
Results of Student's t-test for Aβ immunostaining in the hippocampi of the analyzed groups.
Table 3.
Results of Student's t-test for Aβ immunostaining in the cortices of the analyzed groups.
Figure 4.
Astrocytosis is decreased in the brains of 5XFAD/LDLR-/- and 5XFAD/ApoE-/- LDLR-/- mice.
Immunohistochemistry for GFAP (red) and Thioflavine-S (green) in the hippocampi (A) and the cortices (B) of mouse brains. Representative pictures are shown for each genotype. In both areas examined the absence of LDLR results in reduction of GFAP immunoreactivity. Astrocytosis in both the cortex and the hippocampus is more intense in the 5XFAD and the 5XFAD/ApoE-/- compared to the 5XFAD/LDLR-/- and the 5XFAD/ApoE-/-LDLR-/- sections respectively. Scale bar 250 µm. The pictures were taken under the same conditions of intensity and the experiment was repeated 3 times. (n = 5−7, 3−4 independent sections per animal were analyzed). C. Quantitation of GFAP positive staining in the hippocampi and cortices showing a decrease in the 5XFAD/LDL-/- and 5XFAD/ApoE-/-LDLR-/- mice compared to the control mice. P-values in the hippocampi (left graph) ** P = 0.0040 for 5XFAD vs. 5XFAD/LDLR-/- and P = 0.0031 for 5XFAD/ApoE-/- vs. 5XFAD/ApoE-/-LDLR-/-. In the cortices (right graph) * P = 0.0399 for 5XFAD vs. 5XFAD/LDLR-/- and P = 0.0205 for 5XFAD/ApoE-/- vs. 5XFAD/ApoE-/-LDLR-/-.
Figure 5.
Microgliosis is decreased in the brains of 5XFAD/LDLR-/- and 5XFAD/ApoE-/-LDLR-/- mice.
A. Immunohistochemistry for Iba1 (red) and Thioflavine-S (green) in the hippocampus and cortex of mouse brains. Representative pictures are shown for each genotype. LDLR deficiency results in the reduction of microgliosis both in the hippocampi (A) and the cortices (B) in 5XFAD/LDLR-/- and 5XFAD/ ApoE-/- LDLR-/- mice. C. Iba1 positive round cell formations surrounding Thioflavine-S positive amyloid deposits in the thalamus of 5XFAD/LDLR-/- mice. Scale bar 75 µm. The pictures were taken under the same conditions of intensity and the experiment was repeated 3 times. (n = 5-7, 3-4 independent sections per animal were analyzed). D. Quantitation of Iba1 positive staining in the hippocampi and cortices showing a decrease in the 5XFAD/LDL-/- and 5XFAD/ApoE-/-LDLR-/- mice compared to the control mice. P-values in the hippocampi (left graph) ** P = 0.0043 for 5XFAD vs. 5XFAD/LDLR-/- and ***P<0.0001 for 5XFAD/ApoE-/- vs. 5XFAD/ApoE-/-LDLR-/-. In the cortices (right graph) *** P<0.0001 for 5XFAD vs. 5XFAD/LDLR-/- and *P = 0.0487 for 5XFAD/ApoE-/- vs. 5XFAD/ApoE-/-LDLR-/-.