APOE4 leads to blood-brain barrier dysfunction predicting cognitive decline.

Vascular contributions to dementia and Alzheimer's disease are increasingly recognized1-6. Recent studies have suggested that breakdown of the blood-brain barrier (BBB) is an early biomarker of human cognitive dysfunction7, including the early clinical stages of Alzheimer's disease5,8-10. The E4 variant of apolipoprotein E (APOE4), the main susceptibility gene for Alzheimer's disease11-14, leads to accelerated breakdown of the BBB and degeneration of brain capillary pericytes15-19, which maintain BBB integrity20-22. It is unclear, however, whether the cerebrovascular effects of APOE4 contribute to cognitive impairment. Here we show that individuals bearing APOE4 (with the ε3/ε4 or ε4/ε4 alleles) are distinguished from those without APOE4 (ε3/ε3) by breakdown of the BBB in the hippocampus and medial temporal lobe. This finding is apparent in cognitively unimpaired APOE4 carriers and more severe in those with cognitive impairment, but is not related to amyloid-ß or tau pathology measured in cerebrospinal fluid or by positron emission tomography23. High baseline levels of the BBB pericyte injury biomarker soluble PDGFRß7,8 in the cerebrospinal fluid predicted future cognitive decline in APOE4 carriers but not in non-carriers, even after controlling for amyloid-ß and tau status, and were correlated with increased activity of the BBB-degrading cyclophilin A-matrix metalloproteinase-9 pathway19 in cerebrospinal fluid. Our findings suggest that breakdown of the BBB contributes to APOE4-associated cognitive decline independently of Alzheimer's disease pathology, and might be a therapeutic target in APOE4 carriers.

Evidence that blood-CSF barrier transport, but not inflammatory biomarkers, change in migraine, while CSF sVCAM1 associates with migraine frequency and CSF fibrinogen.

OBJECTIVE: Our objective is to explore whether blood-cerebrospinal fluid (CSF) barrier biomarkers differ in episodic migraine (EM) or chronic migraine (CM) from controls. BACKGROUND: Reports of blood-brain barrier and blood-cerebrospinal fluid barrier (BCSFB) disruption in migraine vary. Our hypothesis is that investigation of biomarkers associated with blood, CSF, brain, cell adhesion, and inflammation will help elucidate migraine pathophysiology. METHODS: We recruited 14 control volunteers without headache disorders and 42 individuals with EM or CM as classified using the International Classification of Headache Disorders, 3rd edition, criteria in a cross-sectional study located at our Pasadena and Stanford headache research centers in California. Blood and lumbar CSF samples were collected once from those diagnosed with CM or those with EM during two states: during a typical migraine, before rescue therapy, with at least 6/10 level of pain (ictal); and when migraine free for at least 48 h (interictal). The average number of headaches per month over the previous year was estimated by those with EM; this enabled comparison of biomarker changes between controls and three headache frequency groups: <2 per month, 2-14 per month, and CM. Blood and CSF biomarkers were determined using antibody-based methods. RESULTS: Antimigraine medication was only taken by the EM and CM groups. Compared to controls, the migraine group had significantly higher mean CSF-blood quotients of albumin (Qalb : mean ± standard deviation (SD): 5.6 ± 2.3 vs. 4.1 ± 1.9) and fibrinogen (Qfib mean ± SD: 1615 ± 99.0 vs. 86.1 ± 55.0). Mean CSF but not plasma soluble vascular cell adhesion molecule-1 (sVCAM-1) levels were significantly higher in those with more frequent migraine: (4.5 ng/mL ± 1.1 in those with <2 headache days a month; 5.5 ± 1.9 with 2-14 days a month; and 7.1 ± 2.9 in CM), while the Qfib ratio was inversely related to headache frequency. We did not find any difference in individuals with EM or CM from controls for CSF cell count, total protein, matrix metalloproteinase-9, soluble platelet-derived growth factor receptor ß, tumor necrosis factor-alpha, interferon-gamma, interleukin (IL)-6, IL-8, IL-10, or C-reactive protein. CONCLUSIONS: The higher Qalb and Qfib ratios may indicate that the transport of these blood-derived proteins is disturbed at the BCSFB in persons with migraine. These changes most likely occur at the choroid plexus epithelium, as there are no signs of typical endothelial barrier disruption. The most striking finding in this hypothesis-generating study of migraine pathophysiology is that sVCAM-1 levels in CSF may be a biomarker of higher frequency of migraine and CM. An effect from migraine medications cannot be excluded, but there is no known mechanism to suggest they have a role in altering the CSF biomarkers.

A novel sensitive assay for detection of a biomarker of pericyte injury in cerebrospinal fluid.

INTRODUCTION: Blood-brain barrier (BBB) breakdown and loss of brain capillary pericytes contributes to cognitive impairment. Pericytes express platelet-derived growth factor receptor-ß (PDGFRß) that regulates brain angiogenesis and blood vessel stability. Elevated soluble PDGFRß (sPDGFRß) levels in cerebrospinal fluid (CSF) indicate pericyte injury and BBB breakdown, which is an early biomarker of human cognitive dysfunction. METHODS: A combination of reagents and conditions were tested, optimized, and validated on the Meso Scale Discovery electrochemiluminescence platform to develop a new sPDGFRß immunoassay that was used to measure sPDGFRß in human CSF from 147 individuals. RESULTS: We developed standard operating procedures for a highly sensitive and reproducible sPDGFRß immunoassay with a dynamic range from 100 to 26,000 pg/mL, and confirmed elevated CSF sPDGFRß levels in individuals with cognitive dysfunction. DISCUSSION: This assay could be applied at different laboratories to study brain pericytes and microvascular damage in relation to cognition in disorders associated with neurovascular and cognitive dysfunction.

Vascular dysfunction-The disregarded partner of Alzheimer's disease.

Increasing evidence recognizes Alzheimer's disease (AD) as a multifactorial and heterogeneous disease with multiple contributors to its pathophysiology, including vascular dysfunction. The recently updated AD Research Framework put forth by the National Institute on Aging-Alzheimer's Association describes a biomarker-based pathologic definition of AD focused on amyloid, tau, and neuronal injury. In response to this article, here we first discussed evidence that vascular dysfunction is an important early event in AD pathophysiology. Next, we examined various imaging sequences that could be easily implemented to evaluate different types of vascular dysfunction associated with, and/or contributing to, AD pathophysiology, including changes in blood-brain barrier integrity and cerebral blood flow. Vascular imaging biomarkers of small vessel disease of the brain, which is responsible for >50% of dementia worldwide, including AD, are already established, well characterized, and easy to recognize. We suggest that these vascular biomarkers should be incorporated into the AD Research Framework to gain a better understanding of AD pathophysiology and aid in treatment efforts.

Apolipoprotein E controls cerebrovascular integrity via cyclophilin A.

Human apolipoprotein E has three isoforms: APOE2, APOE3 and APOE4. APOE4 is a major genetic risk factor for Alzheimer's disease and is associated with Down's syndrome dementia and poor neurological outcome after traumatic brain injury and haemorrhage. Neurovascular dysfunction is present in normal APOE4 carriers and individuals with APOE4-associated disorders. In mice, lack of Apoe leads to blood-brain barrier (BBB) breakdown, whereas APOE4 increases BBB susceptibility to injury. How APOE genotype affects brain microcirculation remains elusive. Using different APOE transgenic mice, including mice with ablation and/or inhibition of cyclophilin A (CypA), here we show that expression of APOE4 and lack of murine Apoe, but not APOE2 and APOE3, leads to BBB breakdown by activating a proinflammatory CypA-nuclear factor-κB-matrix-metalloproteinase-9 pathway in pericytes. This, in turn, leads to neuronal uptake of multiple blood-derived neurotoxic proteins, and microvascular and cerebral blood flow reductions. We show that the vascular defects in Apoe-deficient and APOE4-expressing mice precede neuronal dysfunction and can initiate neurodegenerative changes. Astrocyte-secreted APOE3, but not APOE4, suppressed the CypA-nuclear factor-κB-matrix-metalloproteinase-9 pathway in pericytes through a lipoprotein receptor. Our data suggest that CypA is a key target for treating APOE4-mediated neurovascular injury and the resulting neuronal dysfunction and degeneration.

Neurovascular dysfunction and neurodegeneration in dementia and Alzheimer's disease.

Vascular insults can initiate a cascade of molecular events leading to neurodegeneration, cognitive impairment, and dementia. Here, we review the cellular and molecular mechanisms in cerebral blood vessels and the pathophysiological events leading to cerebral blood flow dysregulation and disruption of the neurovascular unit and the blood-brain barrier, which all may contribute to the onset and progression of dementia and Alzheimer's disease (AD). Particularly, we examine the link between neurovascular dysfunction and neurodegeneration including the effects of AD genetic risk factors on cerebrovascular functions and clearance of Alzheimer's amyloid-ß peptide toxin, and the impact of vascular risk factors, environment, and lifestyle on cerebral blood vessels, which in turn may affect synaptic, neuronal, and cognitive functions. Finally, we examine potential experimental treatments for dementia and AD based on the neurovascular model, and discuss some critical questions to be addressed by future studies. This article is part of a Special Issue entitled: Vascular Contributions to Cognitive Impairment and Dementia edited by M. Paul Murphy, Roderick A. Corriveau and Donna M. Wilcock.

Blood-spinal cord barrier disruption contributes to early motor-neuron degeneration in ALS-model mice.

Humans with ALS and transgenic rodents expressing ALS-associated superoxide dismutase (SOD1) mutations develop spontaneous blood-spinal cord barrier (BSCB) breakdown, causing microvascular spinal-cord lesions. The role of BSCB breakdown in ALS disease pathogenesis in humans and mice remains, however, unclear, although chronic blood-brain barrier opening has been shown to facilitate accumulation of toxic blood-derived products in the central nervous system, resulting in secondary neurodegenerative changes. By repairing the BSCB and/or removing the BSCB-derived injurious stimuli, we now identify that accumulation of blood-derived neurotoxic hemoglobin and iron in the spinal cord leads to early motor-neuron degeneration in SOD1(G93A) mice at least in part through iron-dependent oxidant stress. Using spontaneous or warfarin-accelerated microvascular lesions, motor-neuron dysfunction and injury were found to be proportional to the degree of BSCB disruption at early disease stages in SOD1(G93A) mice. Early treatment with an activated protein C analog restored BSCB integrity that developed from spontaneous or warfarin-accelerated microvascular lesions in SOD1(G93A) mice and eliminated neurotoxic hemoglobin and iron deposits. Restoration of BSCB integrity delayed onset of motor-neuron impairment and degeneration. Early chelation of blood-derived iron and antioxidant treatment mitigated early motor-neuronal injury. Our data suggest that BSCB breakdown contributes to early motor-neuron degeneration in ALS mice and that restoring BSCB integrity during an early disease phase retards the disease process.

Low levels of copper disrupt brain amyloid-ß homeostasis by altering its production and clearance.

Whereas amyloid-ß (Aß) accumulates in the brain of normal animals dosed with low levels of copper (Cu), the mechanism is not completely known. Cu could contribute to Aß accumulation by altering its clearance and/or its production. Because Cu homeostasis is altered in transgenic mice overexpressing Aß precursor protein (APP), the objective of this study was to elucidate the mechanism of Cu-induced Aß accumulation in brains of normal mice and then to explore Cu's effects in a mouse model of Alzheimer's disease. In aging mice, accumulation of Cu in brain capillaries was associated with its reduction in low-density lipoprotein receptor-related protein 1 (LRP1), an Aß transporter, and higher brain Aß levels. These effects were reproduced by chronic dosing with low levels of Cu via drinking water without changes in Aß synthesis or degradation. In human brain endothelial cells, Cu, at its normal labile levels, caused LRP1-specific down-regulation by inducing its nitrotyrosination and subsequent proteosomal-dependent degradation due in part to Cu/cellular prion protein/LRP1 interaction. In APP(sw/0) mice, Cu not only down-regulated LRP1 in brain capillaries but also increased Aß production and neuroinflammation because Cu accumulated in brain capillaries and, unlike in control mice, in the parenchyma. Thus, we have demonstrated that Cu's effect on brain Aß homeostasis depends on whether it is accumulated in the capillaries or in the parenchyma. These findings should provide unique insights into preventative and/or therapeutic approaches to control neurotoxic Aß levels in the aging brain.

A lipoprotein receptor cluster IV mutant preferentially binds amyloid-ß and regulates its clearance from the mouse brain.

Soluble low density lipoprotein receptor-related protein-1 (sLRP1) binds ~70% of amyloid ß-peptide (Aß) in human plasma. In Alzheimer disease (AD) and individuals with mild cognitive impairment converting to AD, plasma sLRP1 levels are reduced and sLRP1 is oxidized, which results in diminished Aß peripheral binding and higher levels of free Aß in plasma. Experimental studies have shown that free circulating Aß re-enters the brain and that sLRP1 and/or its recombinant wild type cluster IV (WT-LRPIV) prevent Aß from entering the brain. Treatment of Alzheimer APPsw(+/0) mice with WT-LRPIV has been shown to reduce brain Aß pathology. In addition to Aß, LRPIV binds multiple ligands. To enhance LRPIV binding for Aß relative to other LRP1 ligands, we generated a library of LRPIV-derived fragments and full-length LRPIV variants with glycine replacing aspartic acid residues 3394, 3556, and 3674 in the calcium binding sites. Compared with WT-LRPIV, a lead LRPIV-D3674G mutant had 1.6- and 2.7-fold higher binding affinity for Aß40 and Aß42 in vitro, respectively, and a lower binding affinity for other LRP1 ligands (e.g. apolipoprotein E2, E3, and E4 (1.3-1.8-fold), tissue plasminogen activator (2.7-fold), matrix metalloproteinase-9 (4.1-fold), and Factor Xa (3.8-fold)). LRPIV-D3674G cleared mouse endogenous brain Aß40 and Aß42 25-27% better than WT-LRPIV. A 3-month subcutaneous treatment of APPsw(+/0) mice with LRPIV-D3674G (40 µg/kg/day) reduced Aß40 and Αß42 levels in the hippocampus, cortex, and cerebrospinal fluid by 60-80% and improved cerebral blood flow responses and hippocampal function at 9 months of age. Thus, LRPIV-D3674G is an efficient new Aß clearance therapy.

Anti-malaria drug artesunate prevents development of amyloid-ß pathology in mice by upregulating PICALM at the blood-brain barrier.

BACKGROUND: PICALM is one of the most significant susceptibility factors for Alzheimer's disease (AD). In humans and mice, PICALM is highly expressed in brain endothelium. PICALM endothelial levels are reduced in AD brains. PICALM controls several steps in Aß transcytosis across the blood-brain barrier (BBB). Its loss from brain endothelium in mice diminishes Aß clearance at the BBB, which worsens Aß pathology, but is reversible by endothelial PICALM re-expression. Thus, increasing PICALM at the BBB holds potential to slow down development of Aß pathology. METHODS: To identify a drug that could increase PICALM expression, we screened a library of 2007 FDA-approved drugs in HEK293t cells expressing luciferase driven by a human PICALM promoter, followed by a secondary mRNA screen in human Eahy926 endothelial cell line. In vivo studies with the lead hit were carried out in Picalm-deficient (Picalm+/-) mice, Picalm+/-; 5XFAD mice and Picalmlox/lox; Cdh5-Cre; 5XFAD mice with endothelial-specific Picalm knockout. We studied PICALM expression at the BBB, Aß pathology and clearance from brain to blood, cerebral blood flow (CBF) responses, BBB integrity and behavior. RESULTS: Our screen identified anti-malaria drug artesunate as the lead hit. Artesunate elevated PICALM mRNA and protein levels in Eahy926 endothelial cells and in vivo in brain capillaries of Picalm+/- mice by 2-3-fold. Artesunate treatment (32 mg/kg/day for 2 months) of 3-month old Picalm+/-; 5XFAD mice compared to vehicle increased brain capillary PICALM levels by 2-fold, and reduced Aß42 and Aß40 levels and Aß and thioflavin S-load in the cortex and hippocampus, and vascular Aß load by 34-51%. Artesunate also increased circulating Aß42 and Aß40 levels by 2-fold confirming accelerated Aß clearance from brain to blood. Consistent with reduced Aß pathology, treatment of Picalm+/-; 5XFAD mice with artesunate improved CBF responses, BBB integrity and behavior on novel object location and recognition, burrowing and nesting. Endothelial-specific knockout of PICALM abolished all beneficial effects of artesunate in 5XFAD mice indicating that endothelial PICALM is required for its therapeutic effects. CONCLUSIONS: Artesunate increases PICALM levels and Aß clearance at the BBB which prevents development of Aß pathology and functional deficits in mice and holds potential for translation to human AD.

A "multi-omics" analysis of blood-brain barrier and synaptic dysfunction in APOE4 mice.

Apolipoprotein E4 (APOE4), the main susceptibility gene for Alzheimer's disease, leads to blood-brain barrier (BBB) breakdown in humans and mice. Remarkably, BBB dysfunction predicts cognitive decline and precedes synaptic deficits in APOE4 human carriers. How APOE4 affects BBB and synaptic function at a molecular level, however, remains elusive. Using single-nucleus RNA-sequencing and phosphoproteome and proteome analysis, we show that APOE4 compared with APOE3 leads to an early disruption of the BBB transcriptome in 2-3-mo-old APOE4 knock-in mice, followed by dysregulation in protein signaling networks controlling cell junctions, cytoskeleton, clathrin-mediated transport, and translation in brain endothelium, as well as transcription and RNA splicing suggestive of DNA damage in pericytes. Changes in BBB signaling mechanisms paralleled an early, progressive BBB breakdown and loss of pericytes, which preceded postsynaptic interactome disruption and behavioral deficits that developed 2-5 mo later. Thus, dysregulated signaling mechanisms in endothelium and pericytes in APOE4 mice reflect a molecular signature of a progressive BBB failure preceding changes in synaptic function and behavior.

Protection of ischemic white matter and oligodendrocytes in mice by 3K3A-activated protein C.

Subcortical white matter (WM) stroke accounts for 25% of all strokes and is the second leading cause of dementia. Despite such clinical importance, we still do not have an effective treatment for ischemic WM stroke, and the mechanisms of WM postischemic neuroprotection remain elusive. 3K3A-activated protein C (APC) is a signaling-selective analogue of endogenous blood protease APC that is currently in development as a neuroprotectant for ischemic stroke patients. Here, we show that 3K3A-APC protects WM tracts and oligodendrocytes from ischemic injury in the corpus callosum in middle-aged mice by activating protease-activated receptor 1 (PAR1) and PAR3. We show that PAR1 and PAR3 were also required for 3K3A-APC's suppression of post-WM stroke microglia and astrocyte responses and overall improvement in neuropathologic and functional outcomes. Our data provide new insights into the neuroprotective APC pathway in the WM and illustrate 3K3A-APC's potential for treating WM stroke in humans, possibly including multiple WM strokes that result in vascular dementia.

Endothelial LRP1 protects against neurodegeneration by blocking cyclophilin A.

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.

APOE4 accelerates advanced-stage vascular and neurodegenerative disorder in old Alzheimer's mice via cyclophilin A independently of amyloid-ß.

Apolipoprotein E4 (APOE4), the main susceptibility gene for Alzheimer's disease (AD), leads to vascular dysfunction, amyloid-ß pathology, neurodegeneration and dementia. How these different pathologies contribute to advanced-stage AD remains unclear. Using aged APOE knock-in mice crossed with 5xFAD mice, we show that, compared to APOE3, APOE4 accelerates blood-brain barrier (BBB) breakdown, loss of cerebral blood flow, neuronal loss and behavioral deficits independently of amyloid-ß. BBB breakdown was associated with activation of the cyclophilin A-matrix metalloproteinase-9 BBB-degrading pathway in pericytes. Suppression of this pathway improved BBB integrity and prevented further neuronal loss and behavioral deficits in APOE4;5FAD mice while having no effect on amyloid-ß pathology. Thus, APOE4 accelerates advanced-stage BBB breakdown and neurodegeneration in Alzheimer's mice via the cyclophilin A pathway in pericytes independently of amyloid-ß, which has implication for the pathogenesis and treatment of vascular and neurodegenerative disorder in AD.

Low-density lipoprotein receptor-related protein-1: a serial clearance homeostatic mechanism controlling Alzheimer's amyloid ß-peptide elimination from the brain.

Low-density lipoprotein receptor-related protein-1 (LRP1), a member of the low-density lipoprotein receptor family, has major roles in the cellular transport of cholesterol, endocytosis of 40 structurally diverse ligands, transcytosis of ligands across the blood-brain barrier, and transmembrane and nuclear signaling. Recent evidence indicates that LRP1 regulates brain and systemic clearance of Alzheimer's disease (AD) amyloid ß-peptide (Aß). According to the two-hit vascular hypothesis for AD, vascular damage precedes cerebrovascular and brain Aß accumulation (hit 1) which then further amplifies neurovascular dysfunction (hit 2) preceding neurodegeneration. In this study, we discuss the roles of LRP1 during the hit 1 and hit 2 stage of AD pathogenesis and describe a three-level serial LRP1-dependent homeostatic control of Aß clearance including (i) cell-surface LRP1 at the blood-brain barrier and cerebrovascular cells mediating brain-to-blood Aß clearance (ii) circulating LRP1 providing a key endogenous peripheral 'sink' activity for plasma Aß which prevents free Aß access to the brain, and (iii) LRP1 in the liver mediating systemic Aß clearance. Pitfalls in experimental Aß brain clearance measurements with the concurrent use of peptides/proteins such as receptor-associated protein and aprotinin are also discussed. We suggest that LRP1 has a critical role in AD pathogenesis and is an important therapeutic target in AD.

Retinal nerve fiber layer thickness predicts CSF amyloid/tau before cognitive decline.

BACKGROUND: Alzheimer's disease (AD) pathology precedes symptoms and its detection can identify at-risk individuals who may benefit from early treatment. Since the retinal nerve fiber layer (RNFL) is depleted in established AD, we tested whether its thickness can predict whether cognitively healthy (CH) individuals have a normal or pathological cerebrospinal fluid (CSF) Aß42 (A) and tau (T) ratio. METHODS: As part of an ongoing longitudinal study, we enrolled CH individuals, excluding those with cognitive impairment and significant ocular pathology. We classified the CH group into two sub-groups, normal (CH-NAT, n = 16) or pathological (CH-PAT, n = 27), using a logistic regression model from the CSF AT ratio that identified >85% of patients with a clinically probable AD diagnosis. Spectral-domain optical coherence tomography (OCT) was acquired for RNFL, ganglion cell-inner plexiform layer (GC-IPL), and macular thickness. Group differences were tested using mixed model repeated measures and a classification model derived using multiple logistic regression. RESULTS: Mean age (± standard deviation) in the CH-PAT group (n = 27; 75.2 ± 8.4 years) was similar (p = 0.50) to the CH-NAT group (n = 16; 74.1 ± 7.9 years). Mean RNFL (standard error) was thinner in the CH-PAT group by 9.8 (2.7) µm; p < 0.001. RNFL thickness classified CH-NAT vs. CH-PAT with 87% sensitivity and 56.3% specificity. CONCLUSIONS: Our retinal data predict which individuals have CSF biomarkers of AD pathology before cognitive deficits are detectable with 87% sensitivity. Such results from easy-to-acquire, objective and non-invasive measurements of the RNFL merit further study of OCT technology to monitor or screen for early AD pathology.

Correction: Retinal nerve fiber layer thickness predicts CSF amyloid/tau before cognitive decline.

[This corrects the article DOI: 10.1371/journal.pone.0232785.].