Several pieces of evidence suggest that blood-brain barrier (BBB) dysfunction is implicated in the pathophysiology of Alzheimer's disease (AD), exemplified by the frequent occurrence of cerebral amyloid angiopathy (CAA) and the defective clearance of Aβ peptides. However, the specific role of brain microvascular cells in these anomalies remains elusive. In this study, we validated by Western, ELISA and immunofluorescence analyses a procedure to generate microvasculatureenriched fractions from frozen samples of human cerebral cortex. We then investigated Aβ and proteins involved in its clearance or production in microvessel extracts generated from the parietal cortex of 60 volunteers in the Religious Orders Study. Volunteers were categorized as AD (n = 38) or controls (n = 22) based on the ABC scoring method presented in the revised guidelines for neuropathological diagnosis of AD. Higher ELISA-determined concentrations of vascular Aβ40 and Aβ42 were found in persons with a neuropathological diagnosis of AD, in apoE4 carriers and in participants with advanced parenchymal CAA, compared to respective age-matched controls. Vascular levels of two proteins involved in Aβ clearance, ABCB1 and neprilysin, were lower in persons with AD and positively correlated with cognitive function, while being inversely correlated to vascular Aβ40. In contrast, BACE1, a protein necessary for Aβ production, was increased in individuals with AD and in apoE4 carriers, negatively correlated to cognitive function and positively correlated to Aβ40 in microvessel extracts. The present report indicates that concentrating microvessels from frozen human brain samples facilitates the quantitative

The transferrin receptor (TfR) is highly expressed by brain capillary endothelial cells (BCECs) forming the blood–brain barrier (BBB) and is therefore considered as a potential target for brain drug delivery. Monoclonal antibodies binding to the TfR, such as clone Ri7, have been shown to internalize into BCECs in vivo. However, since Alzheimer’s disease (AD) is accompanied by a BBB dysfunction, it raises concerns about whether TfR-mediated transport becomes inefficient during the progression of the disease. Measurements of TfR levels using Western blot analysis in whole homogenates from human post-mortem parietal cortex and hippocampus did not reveal any significant difference between individuals with or without a neuropathological diagnosis of AD (respectively, n = 19 and 22 for the parietal cortex and n = 12 and 14 for hippocampus). Similarly, TfR concentrations in isolated human brain microvessels from parietal cortex were similar between controls and AD cases. TfR levels in isolated murine brain microvessels were not significantly different between groups of 12- and 18-month-old NonTg and 3xTg-AD mice, the latter modeling Aβ and τ neuropathologies. In situ brain perfusion assays were then conducted to measure the brain uptake and internalization of fluorolabeled Ri7 in BCECs upon binding. Consistently, TfR-mediated uptake in BCECs was similar between 3xTg-AD mice and nontransgenic controls (∼0.3 μL·g– 1·s–1) at 12, 18, and 22 months of age. Fluorescence microscopy analysis following intravenous administration of fluorolabeled Ri7 highlighted that the signal from the antibody was widely distributed throughout the cerebral vasculature but not in neurons or astrocytes. Overall, our data suggest that both TfR protein levels and TfR-dependent internalization mechanisms are preserved in the presence of Aβ and τ neuropathologies, supporting the potential of TfR as a vector target for drug delivery into BCECs in AD.

AimsBrain mural cells (BMC), smooth muscle cells and pericytes, interact closely with endothelial cells and modulate numerous cerebrovascular functions. A loss of BMC function is suspected to play a role in the pathophysiology of Alzheimer’s Disease (AD).MethodsBMC markers, namely smooth muscle alpha actin (α‐SMA) for smooth muscle cells, as well as platelet‐derived growth factor receptor β (PDGFRβ) and aminopeptidase N (ANPEP or CD13) for pericytes, were assessed by Western immunoblotting in microvessel extracts from the parietal cortex of 60 participants of the Religious Orders study, with ages at death ranging from 75 to 98 years old.ResultsParticipants clinically diagnosed with AD had lower vascular levels of α‐SMA, PDGFRβ and CD13. These reductions were correlated with lower cognitive scores for global cognition, episodic and semantic memory, perceptual speed and visuospatial ability. In addition, α‐SMA, PDGFRβ and CD13 were negatively correlated with vascular Aβ40 concentrations. Vascular levels of BMC markers were also inversely correlated with insoluble cleaved phosphorylated transactive response DNA binding protein 43 (TDP‐43) (25 kDa) and positively correlated with soluble cleaved phosphorylated TDP‐43 (35 kDa) in cortical homogenates, suggesting strong association between BMC loss and cleaved phosphorylated TDP‐43 aggregation.ConclusionsThe results of this study highlight a loss of BMC in AD. The associations between α‐SMA, PDGFRβ and CD13 vascular levels with cognitive scores, TDP‐43 aggregation and cerebrovascular accumulation of Aβ in the parietal cortex suggest that BMC loss contributes to both AD symptoms and pathology, further strengthening the link between cerebrovascular defects and dementia.

No effective preventive treatment is available for age-related cognitive decline and Alzheimer's disease (AD). Epidemiological studies indicate that a diet rich in fruit is associated with cognitive improvement. It was thus proposed that high polyphenol concentrations found in berries can prevent cognitive impairment associated with aging and AD. Therefore, the Neurophenols project aimed at investigating the effects of a polyphenolic extract from blueberries and grapes (PEBG) in the triple-transgenic (3xTg-AD) mouse model of AD, which develops AD neuropathological markers, including amyloid-β plaques and neurofibrillary tangles, leading to memory deficits. In this study, 12-month-old 3xTg-AD and NonTg mice were fed a diet supplemented with standardized PEBG (500 or 2500 mg/kg) for 4 months (n = 15-20/group). A cognitive evaluation with the novel object recognition test was performed at 15 months of age and mice were sacrificed at 16 months of age. We observed that PEBG supplementation with doses of 500 or 2500 mg/kg prevented the decrease in novel object recognition observed in both 15-month-old 3xTg-AD mice and NonTg mice fed a control diet. Although PEBG treatment did not reduce Aβ and tau pathologies, it prevented the decrease in mature BDNF observed in 16-month-old 3xTg-AD mice. Finally, plasma concentrations of phenolic metabolites, such as dihydroxyphenyl valerolactone, a microbial metabolite of epicatechin, positively correlated with memory performances in supplemented mice. The improvement in object recognition observed in 3xTg-AD mice after PEBG administration supports the consumption of polyphenols-rich extracts to prevent memory impairment associated with age-related disease, without significant effects on classical AD neuropathology.

IntroductionHigh levels of plasmatic branched-chain amino acids (BCAA), commonly used as dietary supplements, are linked to metabolic risk factors for Alzheimer's disease (AD). BCAA directly influence amino acid transport to the brain and, therefore, neurotransmitter levels. We thus investigated the impact of BCAA on AD neuropathology in a mouse model.Methods3xTg-AD mice were fed either a control diet or a high-fat diet from 6 to 18 months of age. For the last 2 months, dietary BCAA content was adjusted to high (+50%), normal (+0%), or low (−50%).ResultsMice fed a BCAA-supplemented high-fat diet displayed higher tau neuropathology and only four out of 13 survived. Mice on the low-BCAA diet showed higher threonine and tryptophan cortical levels while performing better on the novel object recognition task.DiscussionThese preclinical data underscore a potential risk of combining high-fat and high BCAA consumption, and possible benefits from BCAA restriction in AD.

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Although the causal role of Amyloid-β (Aβ) in Alzheimer’s disease (AD) is unclear, it is still reasonable to expect that lowering concentrations of Aβ in the brain may decrease the risk of developing the neurocognitive symptoms of the disease. Brain capillary endothelial cells forming the blood-brain barrier (BBB) express transporters regulating the efflux of Aβ out of the cerebral tissue. Age-related BBB dysfunctions, that have been identified in AD patients, might impair Aβ clearance from the brain. Thus, targeting BBB outward transport systems has been suggested as a way to stimulate the clearance of Aβ from the brain. Recent data indicate that the increase in soluble brain Aβ and behavioral impairments in 3×Tg-AD mice generated by months of intake of a high-fat diet can be acutely reversed by the administration of a single dose of insulin. A concomitant increase in plasma Aβ suggests that clearance from the brain through the BBB is a likely mechanism for this rapid effect of insulin. Here, we review how BBB insulin response pathways could be stimulated to decrease brain Aβ concentrations and improve cognitive performance, at least on the short term.

Central response to insulin is suspected to be defective in Alzheimer’s disease. As most insulin is secreted in the bloodstream by the pancreas, its capacity to regulate brain functions must, at least partly, be mediated through the cerebral vasculature. However, how insulin interacts with the blood–brain barrier and whether alterations of this interaction could contribute to Alzheimer’s disease pathophysiology both remain poorly defined. Here, we show that human and murine cerebral insulin receptors (INSRs), particularly the long isoform INSRα-B, are concentrated in microvessels rather than in the parenchyma. Vascular concentrations of INSRα-B were lower in the parietal cortex of subjects diagnosed with Alzheimer’s disease, positively correlating with cognitive scores, leading to a shift towards a higher INSRα-A/B ratio, consistent with cerebrovascular insulin resistance in the Alzheimer’s disease brain. Vascular INSRα was inversely correlated with amyloid-β plaques and β-site APP cleaving enzyme 1, but positively correlated with insulin-degrading enzyme, neprilysin and P-glycoprotein. Using brain cerebral intracarotid perfusion, we found that the transport rate of insulin across the blood–brain barrier remained very low (<0.03 µl/g·s) and was not inhibited by an insulin receptor antagonist. However, intracarotid perfusion of insulin induced the phosphorylation of INSRβ that was restricted to microvessels. Such an activation of vascular insulin receptor was blunted in 3xTg-AD mice, suggesting that Alzheimer’s disease neuropathology induces insulin resistance at the level of the blood–brain barrier. Overall, the present data in post-mortem Alzheimer’s disease brains and an animal model of Alzheimer’s disease indicate that defects in the insulin receptor localized at the blood–brain barrier strongly contribute to brain insulin resistance in Alzheimer’s disease, in association with β-amyloid pathology.