Background: Clearance at the blood-brain barrier (BBB) plays an important role in removal of Alzheimer’s amyloid-β (Aβ) toxin from brain both in humans and animal models. Apolipoprotein E (apoE), the major genetic risk factor for AD, disrupts Aβ clearance at the BBB. The cellular and molecular mechanisms, however, still remain unclear, particularly whether the BBB-associated brain capillary pericytes can contribute to removal of aggregated Aβ from brain capillaries, and whether removal of Aβ aggregates by pericytes requires apoE, and if so, is Aβ clearance on pericytes apoE isoform-specific. Methods: We performed immunostaining for Aβ and pericyte biomarkers on brain capillaries (< 6 μm in diameter) on tissue sections derived from AD patients and age-matched controls, and APP^Swe/0 mice and littermate controls. Human Cy3-Aβ42 uptake by pericytes was studied on freshly isolated brain slices from control mice, pericyte LRP1-deficient mice (Lrp^lox/lox;Cspg4-Cre) and littermate controls. Clearance of aggregated Aβ42 by mouse pericytes was studied on multi-spot glass slides under different experimental conditions including pharmacologic and/or genetic inhibition of the low density lipoprotein receptor related protein 1 (LRP1), an apoE receptor, and/or silencing mouse endogenous Apoe in the presence and absence of human astrocyte-derived lipidated apoE3 or apoE4. Student’s t-test and one-way ANOVA followed by Bonferroni's post-hoc test were used for statistical analysis. Results: First, we found that 35% and 60% of brain capillary pericytes accumulate Aβ in AD patients and 8.5-month-old APP^Sw/0 mice, respectively, compared to negligible uptake in controls. Cy3-Aβ42 species were abundantly taken up by pericytes on cultured mouse brain slices via LRP1, as shown by both pharmacologic and genetic inhibition of LRP1 in pericytes. Mouse pericytes vigorously cleared aggregated Cy3-Aβ42 from multi-spot glass slides via LRP1, which was inhibited by pharmacologic and/or genetic knockdown of mouse endogenous apoE. Human astrocyte-derived lipidated apoE3, but not apoE4, normalized Aβ42 clearance by mouse pericytes with silenced mouse apoE. Conclusions: Our data suggest that BBB-associated pericytes clear Aβ aggregates via an LRP1/apoE isoform-specific mechanism. These data support the role of LRP1/apoE interactions on pericytes as a potential therapeutic target for controlling Aβ clearance in AD.

Amyloid beta (Aβ) homeostasis in the brain is governed by its production and clearance mechanisms. An imbalance in this homeostasis results in pathological accumulations of cerebral Aβ, a characteristic of Alzheimer’s disease (AD). While Aβ may be cleared by several physiological mechanisms, a major route of Aβ clearance is the vascular-mediated removal of Aβ from the brain across the blood-brain barrier (BBB). Here, we discuss the role of the predominant Aβ clearance protein – low-density lipoprotein receptor-related protein 1 (LRP1) – in the efflux of Aβ from the brain. We also outline the multiple factors that influence the function of LRP1-mediated Aβ clearance, such as its expression, shedding, structural modification and transcriptional regulation by other genes. Finally, we summarize approaches aimed at restoring LRP1-mediated Aβ clearance from the brain.

The low-density lipoprotein receptor–related protein 1 (LRP1) is an endocytic and cell signaling transmembrane protein. Endothelial LRP1 clears proteinaceous toxins at the blood–brain barrier (BBB), regulates angiogenesis, and is increasingly reduced in Alzheimer’s disease associated with BBB breakdown and neurodegeneration. Whether loss of endothelial LRP1 plays a direct causative role in BBB breakdown and neurodegenerative changes remains elusive. Here, we show that LRP1 inactivation from the mouse endothelium results in progressive BBB breakdown, followed by neuron loss and cognitive deficits, which is reversible by endothelial-specific LRP1 gene therapy. LRP1 endothelial knockout led to a self-autonomous activation of the cyclophilin A–matrix metalloproteinase-9 pathway in the endothelium, causing loss of tight junctions underlying structural BBB impairment. Cyclophilin A inhibition in mice with endothelial-specific LRP1 knockout restored BBB integrity and reversed and prevented neuronal loss and behavioral deficits. Thus, endothelial LRP1 protects against neurodegeneration by inhibiting cyclophilin A, which has implications for the pathophysiology and treatment of neurodegeneration linked to vascular dysfunction.

Past clinical trials of docosahexaenoic Acid (DHA) supplements for the prevention of Alzheimer's disease (AD) dementia have used lower doses and have been largely negative. We hypothesized that larger doses of DHA are needed for adequate brain bioavailability and that APOE4 is associated with reduced delivery of DHA and eicosapentaenoic acid (EPA) to the brain before the onset of cognitive impairment. 33 individuals were provided with a vitamin B complex (1 mg vitamin B12, 100 mg of vitamin B6 and 800 mcg of folic acid per day) and randomized to 2,152 mg of DHA per day or placebo over 6 months. 26 individuals completed both lumbar punctures and MRIs, and 29 completed cognitive assessments at baseline and 6 months. The primary outcome was the change in CSF DHA. Secondary outcomes included changes in CSF EPA levels, MRI hippocampal volume and entorhinal thickness; exploratory outcomes were measures of cognition. A 28% increase in CSF DHA and 43% increase in CSF EPA were observed in the DHA treatment arm compared to placebo (mean difference for DHA (95% CI): 0.08 µg/mL (0.05, 0.10), p<0.0001; mean difference for EPA: 0.008 µg/mL (0.004, 0.011), p<0.0001). The increase in CSF EPA in non-APOE4 carriers after supplementation was three times greater than APOE4 carriers. The change in brain volumes and cognitive scores did not differ between groups. Dementia prevention trials using omega-3 supplementation doses equal or lower to 1 g per day may have reduced brain effects, particularly in APOE4 carriers. Trial Registration: NCT02541929. HNY was supported by R01AG055770, R01AG054434, R01AG067063 from the National Institute of Aging and NIRG-15-361854 from the Alzheimer's Association, and MGH by the L. K. Whittier Foundation. This work was also supported by P50AG05142 (HCC) from the National Institutes of Health. Funders had no role in study design, data collection, data analysis, interpretation, or writing of the report.

In humans, apolipoprotein E (apoE) has 3 isoforms: apoE2, apoE3, and apoE4. APOE4 is a major genetic risk factor for Alzheimer disease (AD).¹ Apolipoprotein E4 has direct effects on the cerebrovascular system, resulting in microvascular lesions and blood-brain barrier (BBB) damage, as recently reviewed.² Neurovascular dysfunction is also present in cognitively normal APOE4 carriers and individuals with APOE4-associated disorders including AD.^1-3 Moreover, postmortem brain tissue analysis has indicated that BBB breakdown in patients with AD is more pronounced in APOE4 carriers compared with APOE3 or APOE2.^4-6 Our recent studies in transgenic mice have demonstrated that apoE4 leads to BBB breakdown by activating the proinflammatory cyclophilin A (CypA)–matrix metalloproteinase 9 (MMP-9) pathway in brain pericytes, which in turn results in degradation of the BBB tight junctions and basement membrane proteins.⁷ It has also been shown that apoE4-mediated BBB breakdown leads to secondary neuronal injury and cognitive decline in transgenic mice.⁷ Apolipoprotein E2 and apoE3 maintained normal BBB integrity in transgenic mice by suppressing the CypA–MMP-9 pathway.⁷ Here, we studied the cerebrospinal fluid (CSF)/plasma albumin quotient (Q_Alb), an established marker of BBB breakdown,⁸ and CypA and active MMP-9 levels in the CSF of cognitively normal individuals with different APOE genotypes to determine whether apoE4-dependent changes in BBB permeability and CypA–MMP-9 pathway as shown in APOE4, but not APOE3 and APOE2 transgenic mice, also occur in humans.