Apolipoprotein E4 (APOE4), the main Alzheimer’s disease susceptibility gene, is associated with a host of problems: increased amyloid deposition, breakdown of the blood-brain barrier (BBB), cerebral blood flow (CBF) dysfunction, and cognitive deficits. And yet, despite the clearly important role that APOE4 plays in Alzheimer’s disease, how exactly the gene may be contributing to disease has remained unclear. A recent study published in Nature Aging by Berislav Zlokovic, M.D., Ph.D., from the University of Southern California (USC) now sheds some light on the relationship between APOE4 and AD progression.

To better understand the potential contribution of APOE4 to the emergence of late-stage AD pathology, Zlokovic and colleagues crossed (mated) heterozygous 5xFAD (+/-) mice with either homozygous human APOE3(+/+) or APOE4(+/+) mice. 5xFAD mice express a total of five familial AD-linked mutations whereas the APOE3(+/+) and APOE4(+/+) mice contain a knocked-in version of the humanized APOE3 or APOE4 gene, respectively.

In total, the team generated four mouse lines: E3, E3FAD, E4, E4FAD (see table below for full genotypes). Previous research has shown that 5xFAD mice exhibit early breakdown of the blood brain barrier (BBB), reductions in cerebral blood flow (CBF) and suffer from a loss of synapses and neurons in later stages of life. In order to better understand to what degree (if at all) APOE4 specifically contributes to some of these same late-stage outcomes, it was important to directly compare E4 mice with or without 5xFAD and to compare E4 mice (E4 and E4FAD) to E3 mice (E3 and E3FAD).

Notably, E3FAD and E4FAD mice have comparable pathologies at early ages. However, the picture starts to measurably change as the mice grow older. At 18 to 24-month-old APOE4 knock-in mice (E4 and E4FAD) exhibited an accelerated breakdown of the blood-brain barrier (BBB); a loss of pericyte capillary coverage; and reductions in cerebral blood flow (CBF) compared to the APOE3 mouse lines (E3 and E3FAD). Importantly, all of these outcomes were observed regardless of Aβ pathology status in the mice, which suggested to the research team that another key mechanism was at play.

After analyzing the tissue, the researchers identified the cyclophilin A (CypA)-MMP-9 pathway as a key reason for the observed BBB breakdown. When pericytes (cells that are important for BBB maintenance) activate the CypA-MMP-9 pathway, the MMP-9 enzyme breaks down important tight junction proteins in the BBB. This breakdown, in turn, leads to leakage of blood-derived toxic proteins, causing stress and dysfunction. Notably, the CypA-MMP-9 pathway is not only activated in the pericytes of transgenic APOE4 mouse lines but also in AD patients who carry APOE4 (confirmed via both postmortem tissue analysis of human and CSF analysis).

Curious about the relationship, if any, between the observed vascular dysfunction in older E4 mice and Aβ pathology, the team analyzed the relationship between the elevated levels of Aβ42 and Aβ40 present in the FAD mice. They found no correlation between Aβ levels and BBB breakdown or CBF reductions, suggesting that these late-stage vascular dysfunctions are not being driven by changes in Aβ. Instead, the team found a strong correlation between BBB dysfunction in the E4 mice and neuronal (and behavioral) deficits. Given that the Aβ42 levels did not correlate well at all, this suggests that the presence of APOE4 is a larger driving factor of BBB breakdown or vascular dysfunction in older animals than Aβ pathology alone.

Zlokovic and colleagues are optimistic that their findings may offer new strategies for treating late-stage Alzheimer’s disease. Promisingly, when they treated their APOE4 mice with a CypA inhibitor, effectively suppressing the CypA-MMP9 pathway in pericytes, they observed improved BBB integrity and reduced behavioral deficits, regardless of Aβ pathology status

Collectively, the results of this study suggest that treatments and interventions designed to directly repair the BBB and improve vascular dysfunction could potentially slow down or even halt cognitive decline in advanced AD stages. For APOE4 carriers who are at an advanced stage of Alzheimer’s disease, targeting the CypA pathway to improve BBB integrity offers another potential therapeutic strategy that doesn’t hinge on targeting Aβ alone.