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Literature Interpretation | APOE4 does not depend on β‑amyloid deposition; instead, it accelerates late‑stage vascular alterations and neurodegeneration in aged AD mouse models via cyclophilin A.

Introduction:

Apolipoprotein E4 (APOE4) is a major genetic risk factor for Alzheimer’s disease (AD), and it contributes to vascular dysfunction, amyloid‑β pathology, neurodegeneration, and dementia. However, the underlying pathogenic mechanisms remain unclear.

The team led by Berisłav V. Zloković at the University of Southern California, through crossbreeding APOE knock-in mice with 5xFAD mice and examining vascular dysfunction, Aβ pathology, neuronal impairment, and behavioral deficits in APOE3- and APOE4-knockout mice, found that APOE4 mice exhibit accelerated disruption of the blood–brain barrier (BBB), loss of cerebral blood flow, neuronal loss, and behavioral deficits independent of β‑amyloid. This BBB disruption is associated with the activation of a CypA–MMP‑9–mediated BBB degradation pathway in pericytes.

Therefore, APOE4 can accelerate cerebrovascular pathology, thereby driving the progression of Alzheimer’s disease; this process is independent of β‑amyloid deposition, and its inhibition can slow disease progression. These findings have important implications for understanding the pathogenesis and developing therapies for both vascular and neurodegenerative aspects of Alzheimer’s disease.

 

Blood–brain barrier disruption in APOE4 and APOE4;5xFAD mice

First, the researchers examined blood–brain barrier (BBB) integrity in the cortex and hippocampus of 18- to 24-month-old APOE3 and APOE4 knock-in control mice, as well as APOE3;5xFAD and APOE4;5xFAD mice. Using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), they found that, compared with the corresponding APOE3 strains, APOE4 mice exhibited accelerated BBB breakdown in both cortical and hippocampal regions—both in the absence and presence of Aβ pathology (Fig. 1a–c). Specifically, the BBB permeability parameter Ktrans was 43% and 52% higher in APOE4 mice than in APOE3 mice, and 29% and 34% higher than in mice heterozygous for 5xFAD, respectively (Fig. 1b,c). Furthermore, compared with their APOE3 and APOE4 littermate controls, APOE3;5xFAD and APOE4;5xFAD mice showed a substantial increase in cortical and hippocampal BBB Ktrans values—35% and 36%, and 22% and 20%, respectively (Fig. 1c)—consistent with previously reported BBB disruption in 5xFAD mice.

Figure 1: Age-related disruption of the blood–brain barrier in APOE4 and APOE4;5xFAD mice

 

Compared with APOE3 mice, APOE4 mice exhibited increased levels of perivascular capillary deposition of blood-derived fibrinogen in the cortex and hippocampus, confirming these findings (Fig. 1d,e). In the cortex and hippocampus of APOE4 mice, fibrinogen deposition was elevated by 111%, 122%, 135%, and 137% relative to APOE3 mice, respectively (Fig. 1e).

Compared with littermate controls, Aβ pathology was evident in both genotypes (Fig. 1d,e), consistent with previously reported blood–brain barrier disruption in 5xFAD mice.

Analysis of tight junction proteins in the blood–brain barrier revealed that, compared with APOE4 mice, APOE3 mice exhibited reduced expression of occludin and ZO‑1, with significant deficits in the length of these proteins (Fig. 1l, m). The researchers found that, in APOE3 and APOE4 littermates, cortical and hippocampal ZO‑1 levels were decreased by 34% and 57%, respectively, while occludin levels were reduced by 45% and 42%, respectively; these mice were crossed with 5×FAD mice (Fig. 1k, m). These findings are consistent with previous reports of BBB tight junction protein loss observed in APOE4 and 5×FAD mice.

 

Reduced cerebral blood flow in APOE4 and APOE4;5xFAD mice

The researchers examined changes in cortical and hippocampal cerebral blood flow (CBF) in 18- to 24-month-old APOE3- and APOE4-knock-in littermate controls, as well as in APOE3;5xFAD and APOE4;5xFAD mice. Compared with APOE3 mice, APOE4 mice exhibited moderate reductions in CBF in both the cortex and hippocampus, under conditions with and without amyloid‑β pathology (Fig. 2a,b). Specifically, local cortical and hippocampal CBF in APOE4 mice was reduced by 16%, 13%, 24%, and 24% relative to APOE3 mice, respectively (Fig. 2a,b). Furthermore, the researchers found that, compared with APOE3 and APOE4 mice, 5xFAD mice showed additional reductions in cortical and hippocampal CBF of 23%, 23%, 31%, and 33% (Fig. 2b). Consistent with the breakdown of the blood–brain barrier, sex did not influence regional cerebral blood flow alterations in aged mice (Extended Data Fig. 2).

Figure 2: Reduced cerebral blood flow in aged APOE4 and APOE4;5xFAD mice.

Supplementary Figure 2: Additional characterization of reduced cerebral blood flow in aged APOE4 and APOE4;5xFAD male and female mice.

 

Compared with APOE3 mice, APOE4 mice exhibited a moderate reduction in capillary density in both the cortex and hippocampus (Fig. 2c,d). Specifically, the cortical and hippocampal capillary lengths were reduced by 17% and 11%, respectively, in APOE4 mice relative to APOE3 mice, and by 10% and 9%, respectively, compared with 5xFAD mice. Furthermore, when compared with APOE3‑expressing 5xFAD mice, APOE4 mice showed reductions in capillary length of 17% in the cortex and 11% in the hippocampus (Fig. 2d). The decrease in cerebral blood flow (CBF) was positively correlated with the reduction in capillary length (Fig. 2e,f). This observed correlation is consistent with previously reported findings linking pericyte dysfunction and the association between reduced CBF and microvascular density in APOE4 mice.

 

Peroxiredoxin A–matrix metalloproteinase-9 pathway

Immunocytochemical analysis revealed that, compared with the corresponding APOE3‑expressing mice, APOE4 mice exhibited higher levels of CypA‑positive immunofluorescence in CD13‑positive pericytes within the cortex and hippocampus, both in the presence and absence of Aβ pathology (Fig. 3a,b). The increased CypA‑positive signal in cortical and hippocampal CD13‑positive pericytes of APOE4 mice was negatively correlated with reduced expression of the tight junction proteins ZO‑1 (Fig. 3c,d) and occludin (Fig. 3e,f), a relationship that was even more pronounced in mice expressing the 5xFAD transgene.

Compared with their respective APOE3‑expressing counterparts, the activation of the CypAMMP9 pathway in pericytes of both the old APOE4 and APOE4;5xFAD mice suggests a potential mechanism underlying APOE4‑mediated blood–brain barrier disruption and loss of capillary density.

Figure 3: APOE4 and APOE4–expressing pericytes exhibit elevated levels of cyclophilin A and matrix metalloproteinase‑9; these changes are associated with the loss of tight junction proteins in 5xFAD mice.

 

 

Aβ Pathology and Vascular Dysfunction

Researchers found no substantial differences in the levels of the major Aβ42 peptide in the cortex and hippocampus of 18- to 24-month-old APOE4;5xFAD and APOE3;5xFAD mice (Figure 4a). Tissue analysis confirmed that these mice exhibited comparable Aβ burden in both the cortical and hippocampal regions (Figures 4b and 4c).

According to the report, female APOE3;5xFAD and APOE4;5xFAD mice exhibited significantly elevated Aβ42 levels compared with their male counterparts (Figure 4d). Consistent with previous findings in the 5xFAD cell line, Aβ40 levels were markedly lower than Aβ42 levels in both APOE4;5xFAD and APOE3;5xFAD mice (Extended Data Figure 3a). Similar to Aβ42, we also observed that, when crossed with 5xFAD mice, female APOE4 and APOE3 mice displayed significantly higher Aβ40 levels than males (Extended Data Figure 3b).

Supplementary Figure 3: Additional characterization of Aβ pathology in male and female APOE3;5xFAD and APOE4;5xFAD mice, as well as Aβ‑independent vascular alterations.

 

Compared with the cross between APOE3 mice and 5xFAD mice, the increased Ktrans values of blood–brain barrier permeability and the moderate reduction in cerebral blood flow in APOE4 mice were not associated with elevated cortical or hippocampal Aβ42 levels (Fig. 4e–h) or Aβ40 levels (Extended Data Fig. 3e,f), suggesting that, relative to APOE3;5xFAD mice, Aβ pathology may not drive vascular dysfunction in older APOE4;5xFAD mice.

Figure 4: Age‑related APOE3;5xFAD and APOE4;5xFAD mice | Aβ pathology and Aβ‑independent vascular changes.

 

APOE4 mediates neurodegeneration

Compared with APOE3 mice, APOE4 mice exhibit accelerated neuronal and axonal loss (Fig. 5a–f). When compared with APOE3 control mice and mice crossed with 5xFAD mice, APOE4 mice show reductions in cortical neuron counts of 18%, 34%, 11%, and 22%, respectively, and decreases in neuronal density of 20%, 27%, 27%, and 43%, respectively (Fig. 5b,c), as well as in the hippocampus (Fig. 5e,f).

Figure 5: Age-related neurodegenerative changes in APOE4 and APOE4; 5xFAD mice are associated with blood–brain barrier disruption but not with Aβ pathology.

 

APOE4-Related Behavioral Deficits

Compared with APOE3, APOE4 mice exhibited behavioral deficits and hippocampus‑related memory impairments in daily activity tests (Figure 6).

Figure 6: In aged APOE4 and APOE4;5xFAD mice, behavioral deficits are associated with vascular dysfunction but not with Aβ pathology.

 

After administering Debio-025 to mice for 30 consecutive days, researchers observed significant improvements in cerebral vascular pathology, neuronal damage, and associated behavioral deficits in E4‑FAD mice, while β‑amyloid levels remained persistently elevated. These findings indicate that inhibition of the CypA–MMP‑9 signaling pathway can ameliorate ApoE4‑induced vascular abnormalities, thereby delaying the onset of Alzheimer’s disease.

Figure 7: Debio-025 inhibits the pericyte CypA–MMP9 pathway, thereby enhancing blood–brain barrier integrity in APOE4;5xFAD mice.

 

Discussion

The findings indicate that the APOE4 gene can accelerate cerebrovascular pathology and neurodegeneration in the brains of mice with advanced Alzheimer’s disease by activating the CypA–MMP‑9 signaling pathway in pericytes, a process that is independent of β‑amyloid deposition. Both ApoE4 and cerebrovascular pathology are highly prevalent in individuals with Alzheimer’s disease; thus, this discovery offers new avenues for the development of therapeutic strategies for the condition.

 

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Editor-in-chief  |  Aaron, Bian Zhouzi

Editor  |  Lemon

 

 

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