Background
The Apolipoprotein-E ε4 (APOE4) allele is the strongest genetic risk factor for late-onset Alzheimer’s disease (LOAD) and may contribute to neurodegeneration through a multi-hit hypothesis, in which vascular dysfunction, glial activation, and impaired lipid metabolism play central roles. Alterations in neurovascular unit (NVU) have emerged as an early APOE4-related phenotype, independent of amyloid and tau pathology. Astrocytes, as the primary source of APOE in the brain and key regulators of NVU homeostasis, may play a central role in these processes. This study investigates APOE4-associated NVU water exchange dynamics and astrocyte-vascular interactions using integrated in vivo MRI, ex vivo histology, and transcriptomic profiling.

Methods
Non-contrast multimodal MRI, including multi-echo time arterial spin labeling (multi-TE ASL), T1-weighted imaging, and diffusion-weighted MRI, were applied in 6-9-month-old APOE3-KI and APOE4-KI mice. Multi-TE ASL was used to estimate regional NVU water exchange dynamics, while diffusion MRI assessed tissue microstructural alterations. Immunohistochemistry evaluated perivascular matrix metalloproteinase-9 (MMP9) activity, vascular-associated markers, astrocytic AQP4 expression, and glial reactivity. Single-nucleus RNA sequencing (snRNAseq) characterized cell-type-specific transcriptional profiles, and inferred cell-cell communication analysis between astrocytes, pericytes, and other NVU components. Integrated analyses compared MRI-derived measures with molecular and cellular findings.

Results
APOE4-KI mice showed regionally specific alterations in NVU water exchange dynamics, particularly in the hippocampus, accompanied by trends toward altered microstructural complexity. Immunohistochemistry demonstrated increased perivascular MMP9 expression and evidence of extracellular matrix remodeling without prominent structural disruption of blood-brain barrier (BBB) markers in APOE4 mice. Astrocytes showed increased AQP4 expression, heightened proinflammatory gene signatures, and morphological reactivity. Molecular findings aligned with MRI, supporting the sensitivity of non-contrast MRI to early NVU alterations. Exploratory snRNAseq suggested an APOE4-enriched astrocyte subpopulation associated with immune activation and matrix-related pathways and suggested potential glial-vascular interactions that require validation in larger samples.

Conclusions
This integrated imaging and molecular analysis suggests that non-contrast multimodal MRI detects early APOE4-related changes in NVU exchange dynamics and glial-vascular interactions. By providing converging multiscale neuroimaging and cellular observations, this work provides a foundation for developing non-invasive biomarkers to monitor neurovascular vulnerability and guide early intervention strategies in individuals at risk for LOAD.