Loss of Endothelial APOE4 Dysregulates Neural Function In Vivo

Abstract

Background: We recently found that loss of endothelial cell APOE3 disrupts neurovascular and synaptic function. However, whether endothelial APOE4 is detrimental or protective for neural function under physiological conditions is unknown. Therefore, the goal of this study was to determine the role of endothelial cell APOE4 in regulating brain function in vivo.

Methods and results: We developed APOE4^fl/fl/Cdh5(PAC)-CreERT2^+/- and APOE4^fl/fl/Cdh5(PAC)-CreERT2^-/- (control) mice. Knockdown of endothelial cell APOE4 was induced at ≈4 to 5 weeks of age. Experiments were conducted at 9 months of age to evaluate neurovascular and neuronal function via biochemistry, immunohistochemistry, behavior tests, and electrophysiology. Endothelial cell APOE4 knockdown resulted in higher neurovascular permeability, lower claudin-5 vessel coverage, impaired trace fear memory extinction, and disruption of cortical excitatory-inhibitory balance of synaptic activity.

Conclusions: Our data support the novel concept that endothelial cell APOE4 is protective for brain function when other cell types express APOE4.

Keywords: ApoE4; behavior; brain endothelial cells.

Figure 1. Endothelial cell APOE4 knockdown results in higher neurovascular permeability.

Neurovascular permeability was measured in APOE4 ^fl/fl/Cdh5(PAC)‐CreERT2^−/− (APOE4 ^Cre−/−) and APOE4 ^fl/fl/Cdh5(PAC)‐CreERT2^+/− (APOE4 ^Cre+/−) mice. A, ApoE protein levels in ex vivo‐isolated brain endothelial cells evaluated by western blot. ApoE protein levels are ≈69% lower in APOE4 ^Cre+/− mice compared with APOE4 ^Cre−/− mice [F _{(1, 18)}=31.729; P<0.001], n≈5. B and C, Cortical plasma protein levels measured by ELISA. B, There is an effect of both Cre Genotype [F _{(1, 38)}=6.424; P=0.016] and Sex [F _{(1, 38)}=22.573; P<0.001], n≈10, on IgG levels in cortical homogenates. Cortical IgG levels are ≈17% higher in APOE4 ^Cre+/− compared with APOE4 ^Cre−/− mice (middle) and ≈34% higher in female mice compared with males (right). Representative images demonstrate higher levels of IgG in APOE4 ^Cre+/− (middle) and female (right) mice. Scale bar=10 μm. C, Cortical fibrinogen levels trend ≈20% higher in APOE4 ^Cre+/− mice compared with APOE4 ^Cre−/− mice [F _{(1, 41)}=3.067; P=0.088], n≈10. Representative images highlight higher levels of fibrinogen in APOE4 ^Cre+/− mice. Scale bar=10 μm. D, Quantitative immunohistochemistry of claudin‐5 levels in the cortical neurovasculature. Claudin‐5 levels are ≈10% lower with endothelial APOE4 knockdown when normalized to CD31+ vessel area [F _{(1, 22)}=5.264; P=0.033], n≈6. Representative images reveal less claudin‐5 in the cortical vasculature and a greater number of areas lacking claudin‐5 entirely (white arrowheads) in APOE4 ^Cre+/− mice compared with APOE4 ^Cre−/− mice. Scale bar=20 μm. All data are expressed as a box plot depicting the minimum score, the lower quartile (25%), the median (50%, horizontal line), the upper quartile (75%), maximum values, and mean (+). Data were analyzed using GLM. *P<0.05 for APOE4 ^Cre+/− vs APOE4 ^Cre−/− mice or female vs male mice. ApoE indicates apolipoprotein E (protein); APOE4, apolipoprotein E4 (gene); APOE4 ^Cre−/− and Cre−, APOE4 ^fl/fl/Cdh5(PAC)‐CreERT2^−/−; APOE4 ^Cre+/− and Cre+APOE4 ^fl/fl/Cdh5(PAC)‐CreERT2^+/−; F, female; GLM, general linear models; IgG, immunoglobulin G; and M, male.

Figure 2. Loss of endothelial cell APOE4 disrupts fear extinction and neural function.

Fear memory extinction and neural function were measured in APOE4 ^fl/fl/Cdh5(PAC)‐CreERT2^−/− (APOE4 ^Cre−/−) and APOE4 ^fl/fl/Cdh5(PAC)‐CreERT2^+/− (APOE4 ^Cre+/−) mice. A through D, Acquisition and extinction of fear conditioned freezing behavior measured in trace fear conditioning. A, There are no differences in the acquisition of fear memory with endothelial APOE4 knockdown; “H” designates the habituation phase. B, During the extinction phase, there is both a Trial×Cre Genotype [F _(3.564)=3.723; P=0.009] and a Trial×Sex interaction [F _(3.564)=3.016; P=0.025], n≈9. The Trial×Cre Genotype interaction (middle) is driven by the fact that APOE4 ^Cre−/− mice begin to extinguish fear memory by trials 11–15 whereas APOE4 ^Cre+/− mice do not begin to extinguish until trials 21 to 25. In addition, freezing responses are higher in APOE4 ^Cre+/− compared with APOE4 ^Cre−/− mice in trials 16 to 30. The Trial×Sex interaction (right) is due to female mice freezing to a greater extent than male mice in trials 1 to 20. C, Plots of fear memory extinction separated by Cre Genotype and Sex to visualize differences in freezing behavior between the first and last trials. Qualitatively, the extent of overall extinction appears less in APOE4 ^Cre+/− mice when comparing the final trials (26, 27, 28, 29, 30) to the first trials (1, 2, 3, 4, 5). D, Overall extinction was quantified by an EI(EI=(1−[% Freezing_{Trials 26–30}/% Freezing_{Trials 1–5}])×100). Extinction of fear memory is lower in APOE4 ^Cre+/− compared with APOE4 ^Cre−/− mice [F _{(1, 33)}=17.578; P<0.001], n ≈ 9. E through H, Ex vivo recordings of spontaneous PSC from excitatory (−60 mV) and inhibitory (+15 mV) synapses onto layer V pyramidal neurons from the prefrontal cortex (n=17 neurons per Cre genotype; 5 mice per Cre genotype). E, There is an interaction of Holding Potential×Cre Genotype on the frequency of PSCs driven by both a lower frequency of excitatory synaptic events and a higher frequency of inhibitory synaptic events in APOE4 ^Cre+/− mice compared with APOE4 ^Cre−/− mice [F _(1,67)=37.358; P<0.001]. F, Thus, the E‐I ratio (PSC_‐60mV/PSC_+15mV) was imbalanced (< 1) in APOE4 ^Cre+/− mice [F _{(1, 33)}=276.937; P<0.001]. G, Simple linear regression of inhibitory vs excitatory synaptic events. Visually the slope calculated from APOE4 ^Cre+/− (slope=1.877) mice is supralinear compared with the curve derived from APOE4 ^Cre−/− mice (slope=0.9373). H, Representative traces of spontaneous postsynaptic events recorded at both −60 mV and +15 mV holding potentials (scale bar=25pA/1s). A and B, Data expressed as mean±SEM analyzed using GLM with repeated measures. Asterisks indicating statistical significance (*// P<0.05) from Bonferroni post hoc analysis are color coded: *black for APOE4 ^Cre+/− vs APOE4 ^Cre−/− mice or male vs female mice, dark red for trials 1 to 5 vs other trials within APOE4 ^Cre−/− or male sex, and light red for trials 1–5 vs other trials within APOE4 ^Cre+/− or female sex. D through G, Data are expressed as a box plot depicting the minimum score, the lower quartile (25%), the median (50%, horizontal line), the upper quartile (75%), maximum values, and mean (+). Data were analyzed using GLM. Asterisks indicating statistical significance (*// P<0.05) from Bonferroni post hoc analysis are color coded: *black for APOE4 ^Cre+/− vs APOE4 ^Cre−/− mice (within a holding potential for D through G), dark red is within APOE4 ^Cre−/−, and <light red is within APOE4 ^Cre+/−. ApoE indicates apolipoprotein E (protein); APOE4; apolipoprotein E4 (gene); APOE4 ^Cre−/− and Cre‐ indicate APOE4^fl/fl/Cdh5(PAC)‐CreERT2^−/−; APOE4 ^Cre+/− and Cre+, APOE4 ^fl/fl/Cdh5(PAC)‐CreERT2^+/−; E‐I, excitatory‐inhibitory; EI, extinction index; F, female; GLM, general linear models; H, habituation; M, male; and PSC, postsynaptic current.