Transport across the blood–brain barrier

The endothelial cells that form the BBB are connected by tight and adherens junctions, and it is the tight junctions that confer the low paracellular permeability of the BBB¹. Small lipophilic molecules, oxygen and carbon dioxide diffuse freely across the endothelial cells, and hence the BBB, but normal brain endothelium lacks fenestrae and has limited vesicular transport.

The high number of mitochondria in endothelial cells reflects a high energy demand for active ATP-dependent transport, conferred by transporters such as the sodium pump ((Na⁺+K⁺) ATPase) and the ATP-binding cassette (ABC) efflux transporters. Sodium influx and potassium efflux across the abluminal side of the BBB is controlled by (Na⁺+K⁺) ATPase (FIG. 2). Changes in sodium and potassium levels in the ISF influence the generation of action potentials in neurons and thus directly affect neuronal and synaptic functions^1,12.

Brain endothelial cells express transporters that facilitate the transport of nutrients down their concentration gradients, as described in detail elsewhere (FIG. 2). Glucose transporter 1 (GLUT1; also known as solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1)) — the BBB-specific glucose transporter — is of special importance because glucose is a key energy source for the brain. Monocarboxylate transporter 1 (MCT1), which transports lactate, and the L1 and y+ amino acid trans- porters are expressed at the luminal and abluminal membranes. Sodium-dependent excitatory amino acid transporter 1 (EAAT1), EAAT2 and EAAT3 are expressed at the abluminal side of the BBB¹⁵ and enable removal of glutamate, an excitatory neurotransmitter, from the brain (FIG. 2). Glutamate clearance at the BBB is essential for protecting neurons from overstimulation of glutaminergic receptors, which is neurotoxic¹⁶. ABC transporters limit the penetration of many drugs into the brain¹⁷. For example, multidrug resistance protein 1 (ABCB1; also known as ATP-binding cassette subfamily B member 1) controls the rapid removal of ingested toxic lipophilic metabolites¹⁷ (FIG. 2). Some ABC transporters also mediate the efflux of nutrients from the endothelium into the ISF. For example, solute carrier organic anion transporter family member 1C1 (OATP1C1) transports thyroid hormones into the brain. MCT8 mediates influx of thyroid hormones from blood into the endothelium¹⁸ (FIG. 2).

The transport of circulating peptides across the BBB into the brain is restricted or slow compared with the transport of nutrients¹⁹. Carrier-mediated transport of neuroactive peptides controls their low levels in the ISF^20–24 (FIG. 2). Some proteins, including transferrin, insulin, insulin-like growth factor 1 (IGF1), leptin^25–27 and activated protein C (APC)28, cross the BBB by receptor-mediated transcytosis (FIG. 2).

Blood–brain barrier breakdown

Disruption to tight and adherens junctions, an increase in bulk-flow fluid transcytosis, and/or enzymatic degradation of the capillary basement membrane cause physical breakdown of the BBB. The levels of many tight junction proteins, their adaptor molecules and adherens junction proteins decrease in Alzheimer’s disease and other diseases that cause dementia^1,9, ALS³¹, multiple sclerosis³² and various animal models of neurological disease^8,33. These decreases might be partly explained by the fact that vascular-associated matrix metalloproteinase (MMP) activity rises in many neurodegenerative disorders and after ischaemic CNS injury^34,35; tight junction proteins and basement membrane extracellular matrix proteins are substrates for these enzymes³⁴. Lowered expression of messenger RNAs that encode several key tight junction proteins, however, has also been reported in some neurodegenerative disorders, such as ALS³¹.

Endothelial cell–pericyte interactions are crucial for the formation^36,37 and maintenance of the BBB^33,38. Pericyte deficiency can lead to a reduction in expression of certain tight junction proteins, including occludin, claudin 5 and ZO1³³, and to an increase in bulk- flow transcytosis across the BBB, causing BBB breakdown³⁸. Both processes can lead to extravasation of multiple small and large circulating macromolecules (up to 500 kDa) into the brain parenchyma^33,38. Moreover, in mice, an age-dependent progressive loss of pericytes can lead to BBB disruption and microvasular degeneration and, subsequently, neuronal dysfunction, cognitive decline and neurodegenerative changes³³. In their lysosomes, pericytes concentrate and degrade multiple circulating exogenous³⁹ and endogenous proteins amplify BBB breakdown in cases of pericyte deficiency.

BBB breakdown typically leads to an accumulation of various molecules in the brain. The build up of serum proteins such as immunoglobulins and albumin can cause brain oedema and suppression of capillary blood flow whereas high concentrations of thrombin lead to neurotoxicity and memory impairment⁴⁰, and accelerate vascular damage and BBB disruption⁴¹. The accumulation of plasmin (derived from circulating plasminogen) can catalyze the degradation of neuronal laminin and, hence, promote neuronal injury⁴², and high fibrin levels accelerate neurovascular damage⁶. Finally, an increase in the number of RBCs causes deposition of haemoglobin-derived neurotoxic products including iron, which generates neurotoxic reactive oxygen species (ROS)^8,43 (FIG. 3a). In addition to protein-mediated vasogenic oedema, local tissue ischaemia–hypoxia depletes ATP stores, causing (Na⁺+K⁺) ATPase pumps and Na⁺-dependent ion channels to stop working and, consequently, the endothelium and astrocytes to swell (known as cytotoxic oedema)⁴⁴. Upregulation of aquaporin 4 water channels in response to ischaemia facilitates the development of cytotoxic oedema in astrocytes⁴⁵.

Hypoperfusion and hypoxia

CBF is regulated by local neuronal activity and metabolism, known as neurovascular coupling⁴⁶. The pial and intracerebral arteries control the local increase in CBF that occurs during brain activation, which is termed ‘functional hyperaemia’. Neurovascular coupling requires intact pial circulation, and for VSMCs and pericytes to respond normally to vasoactive stimuli^33,46,47. In addition to VSMC-mediated constriction and vasodilation of cerebral arteries, recent studies have shown that pericytes modulate brain capillary diameter through constriction of the vessel wall⁴⁷, which obstructs capillary flow during ischaemia⁴⁸. Astrocytes regulate the contractility of intracerebral arteries^49,50.

Progressive CBF reductions have increasingly serious consequences for neurons (FIG. 3b). Briefly, mild hypoperfusion — termed oligaemia — affects protein synthesis, which is required for the synaptic plasticity mediating learning and memory⁴⁶. Moderate to severe CBF reductions and hypoxia affect ATP synthesis, diminishing (Na⁺+K⁺) ATPase activity and the ability of neurons to generate action potentials⁹. In addition, such reductions can lower or increase pH, and alter electrolyte balances and water gradients, leading to the development of oedema and white matter lesions, and the accumulation of glutamate and proteinaceous toxins (for example, amyloid-β and hyperphopshorylated tau) in the brain. A reduction of greater than 80% in CBF results in neuronal death².

The effect of CBF reductions has been extensively studied at the molecular and cellular levels in relation to Alzheimer’s disease. Reduced CBF and/or CBF dysregulation occurs in elderly individuals at high risk of Alzheimer’s disease before cognitive decline, brain atrophy and amyloid-β accumulation^{10,46,51–54}. In animal models, hypoperfusion can induce or amplify Alzheimer’s disease-like neuronal dysfunction and/or neuropathological changes. For example, bilateral carotid occlusion in rats causes memory impairment, neuronal dysfunction, synaptic changes and amyloid-β oligomerization⁵⁵, leading to accumulation of neurotoxic amyloid-β oligomers⁵⁶. In a mouse model of Alzheimer’s disease, oligaemia increases neuronal amyloid-β levels and neuronal tau phosphophorylation at an epitope that is associated with Alzheimer’s disease-type paired helical filaments⁵⁷. In rodents, ischaemia leads to the accumulation of hyperphosphorylated tau in neurons and the formation of filaments that resemble those present in human neurodegenerative tauopathies and Alzheimer’s disease⁵⁸. Mice expressing amyloid-β precursor protein (APP) and transforming growth factor β1 (TGFβ1) develop deficient neurovascular coupling, cholinergic denervation, enhanced cerebral and cerebrovascular amyloid-β deposition, and age-dependent cognitive decline⁵⁹.

Recent studies have shown that ischaemia–hypoxia influences amyloidogenic APP processing through mechanisms that increase the activity of two key enzymes that are necessary for amyloid-β production; that is, β-secretase and γ-secretase^60–63. Hypoxia-inducible factor 1α (HIF1α) mediates transcriptional increase in β-secretase expression⁶¹. Hypoxia also promotes phosphorylation of tau through the mitogen-activated protein kinase (MAPK; also known as extracellular signal-regulated kinase (ERK)) pathway⁶⁴, downregulates neprilysin — an amyloid-β-degrading enzyme65 — and leads to alterations in the expression of vascular-specific genes, including a reduction in the expression of the homeobox protein MOX2 gene mesenchyme homeobox 2 (MEOX2) in brain endothelial cells⁵ and an increase in the expression of the myocardin gene (MYOCD) in VSMCs⁶⁶. In patients with Alzheimer’s disease and in models of this disorder, these changes cause vessel regression, hypoperfusion and amyloid-β accumulation resulting from the loss of the key amyloid-β clearance lipoprotein receptor (see below). In addition, hypoxia facilitates alternative splicing of Eaat2 mRNA in Alzheimer’s disease transgenic mice before amyloid-β deposition⁶⁷ and suppresses glutamate reuptake by astrocytes independently of amyloid formation⁶⁸, resulting in glutamate-mediated neuronal injury that is independent of amyloid-β.

In response to hypoxia, mitochondria release ROS that mediate oxidative damage to the vascular endothelium and to the selective population of neurons that has high metabolic activity. Such damage has been suggested to occur before neuronal degeneration and amyloid-β deposition in Alzheimer’s disease^69,70. Although the exact triggers of hypoxia-mediated neurodegeneration and the role of HIF1α in neurodegeneration versus preconditioning-mediated neuroprotection remain topics of debate, mitochondria-generated ROS seem to have a primary role in the regulation of the HIF1α-mediated transcriptional switch that can activate an array of responses, ranging from mechanisms that increase cell survival and adaptation to mechanisms inducing cell cycle arrest and death⁷¹. Whether inhibition of hypoxia- mediated pathogenic pathways will delay onset and/or control progression in neurodegenerative conditions such as Alzheimer’s disease remains to be determined.

When comparing the contributions of BBB break-down and hypoperfusion to neuronal injury, it is interesting to consider Meox2^+/− mice. Such animals have normal pericyte coverage and an intact BBB but a substantial perfusion deficit⁵ that is comparable to that found in pericyte-deficient mice that develop BBB breakdown³³. Notably, however, Meox2^+/− mice show less pronounced neurodegenerative changes than pericyte- deficient mice, indicating that chronic hypoperfusion– hypoxia alone can cause neuronal injury, but not to the same extent as hypoperfusion–hypoxia combined with BBB breakdown.

Age-associated neurovascular changes

Normal ageing diminishes brain circulatory functions, including a detectable decay of CBF in the limbic and association cortices that has been suggested to underlie age-related cognitive changes⁷⁷. Alterations in the cerebral microvasculature, but not changes in neural activity, have been shown to lead to age-dependent reductions in functional hyperaemia in the visual system in cats⁷⁸ and in the sensorimotor cortex in pericyte-deficient mice³³. Importantly, a recent longitudinal CBF study in neurologically normal individuals revealed that people bearing the apolipoprotein E (APOE) ε4 allele — the major genetic risk factor for late-onset Alzheimer’s disease^79–81 — showed greater regional CBF decline in brain regions that are particularly vulnerable to pathological changes in Alzheimer’s disease than did people without this allele⁸².

A meta-analysis of BBB permeability in 1,953 individuals showed that neurologically healthy humans had an age-dependent increase in vascular permeability⁸³. Moreover, patients with vascular or Alzheimer’s dis- ease-type dementia and leucoaraiosis — a small-vessel disease of the cerebral white matter — had an even greater age-dependent increase in vascular permeability⁸³. Interestingly, an increase in BBB permeability in brain areas with normal white matter in patients with leukoaraiosis has been suggested to play a causal part in disease and the development of lacunar strokes⁸⁴. Age-related changes in the permeability of the blood– CSF barrier and the choroid plexus have been reported in sheep⁸⁵.

Vascular pathology

Patients with Alzheimer’s disease or other dementia-causing diseases frequently show focal changes in brain microcirculation. These changes include the appearance of string vessels (collapsed and acellular membrane tubes), a reduction in capillary density, a rise in endothelial pinocytosis, a decrease in mitochondrial content, accumulation of collagen and perlecans in the basement membrane, loss of tight junctions and/or adhe-rens junctions^{3–6,9,46,86}, and BBB breakdown with leakage of blood-borne molecules^4,6,7,9. The time course of these vascular alterations and how they relate to dementia and Alzheimer’s disease pathology remain unclear, as no protocol that allows the development of the diverse brain vascular pathology to be scored, and hence to be tracked with ageing, has so far been developed and widely validated⁸⁷. Interestingly, a recent study involving 500 individuals who died between the ages of 69 and 103 years showed that small-vessel disease, infarcts and the presence of more than one vascular pathological change were associated with Alzheimer’s disease-type pathological lesions and dementia in people aged 75 years of age⁸⁷. These associations were, however, less pronounced in individuals aged 95 years of age, mainly because of a marked ageing-related reduction in Alzheimer’s disease neuropathology relative to a moderate but insignificant ageing-related reduction in vascular pathology⁸⁷.

Accumulation of amyloid-β and amyloid deposition in pial and intracerebral arteries results in CAA, which is present in over 80% of Alzheimer’s disease cases⁸⁸. Inpatients who have Alzheimer’s disease with established CAA in small arteries and arterioles, the VSMC layer frequently shows atrophy, which causes a rupture of the vessel wall and intracerebral bleeding in about 30% of these patients^89,90. These intracerebral bleedings contribute to, and aggravate, dementia. Patients with hereditary cerebral β-amyloidosis and CAA of the Dutch, Iowa, Arctic, Flemish, Italian or Piedmont L34V type have accelerated VSMC degeneration resulting in haemorrhagic strokes and dementia⁹¹. Duplication of the gene encoding APP causes early-onset Alzheimer’s disease dementia with CAA and intracerebral haemorrhage⁹².

Early studies of serum immunoglobulin leakage reported that patients with ALS had BSCB break- down and BBB breakdown in the motor cortex⁹³. Microhaemorrhages and BSCB breakdown have been shown in the spinal cord of transgenic mice expressing mutant variants of human superoxide dismutase 1 (SOD1), which in mice cause an ALS-like disease^8,94,95. In mice with ALS-like disease and in patients with ALS, BSCB breakdown has been shown to occur before motor neuron degeneration or brain atrophy^8,11,95.

BBB breakdown in the substantia nigra and the striatum has been detected in murine models of Parkinson’s disease that are induced by administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)^96–98. However, the temporal relationship between BBB breakdown and neurodegeneration in Parkinson’s disease is currently unknown. Notably, the prevalence of CAA and vascular lesions increases in Parkinson’s dis- ease^99,100. Vascular lesions in the striatum and lacunar infarcts can cause vascular parkinsonism syndrome¹⁰¹. A recent study reported BBB breakdown in a rat model of Huntington’s disease that is induced with the toxin 3-nitropropionic acid¹⁰².

Several studies have established disruption of BBB with a loss of tight junction proteins during neuroinflammatory conditions such as multiple sclerosis and its murine model, experimental allergic encephalitis. Such disruption facilitates leukocyte infiltration, leading to oliogodendrocyte death, axonal damage, demyelination and lesion development³².

Functional changes in the vasculature

In individuals with Alzheimer’s disease, GLUT1 expression at the BBB decreases¹⁰³, suggesting a shortage in necessary metabolic substrates. Studies using 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) have identified reductions in glucose uptake in asymptomatic individuals with a high risk of dementia^104,105. Several studies have suggested that reduced glucose uptake across the BBB, as seen by FDG PET, precedes brain atrophy^104–108.

Amyloid-β constricts cerebral arteries¹⁰⁹. In a mouse model of Alzheimer’s disease, impairment of endothelium-dependent regulation of neocortical microcirculation^110,111 occurs before amyloid-β accumulation. Recent studies have shown that CD36, a scavenger receptor that binds amyloid-β, is essential for the vascular oxidative stress and diminished functional hyperaemia that occurs in response to amyloid-β exposure¹¹². Neuroimaging studies in patients with Alzheimer’s disease have shown that neurovascular uncoupling occurs before neurode-generative changes^10,51–53. Moreover, cognitively normal APOE ε4 carriers at risk of Alzheimer’s disease show impaired CBF responses to brain activation in the absence of neurodegenerative changes or amyloid-β accumulation⁵⁴. Recently, patients with Alzheimer’s disease as well as mouse models of this disease with high cerebrovascular levels of serum response factor (SRF) and MYOCD, the two transcription factors that control VSMC differentiation, have been shown to develop a hypercontractile arterial phenotype resulting in brain hypoperfusion, diminished functional hyperaemia and CAA^66,113. More work is needed to establish the exact role of SRF and MYOCD in the vascular dysfunction that results in the Alzheimer’s disease phenotype and CAA.

PET studies with ¹¹C-verapamil, an ABCB1 substrate, have indicated that the function of ABCB1, which removes multiple drugs and toxins from the brain, decreases with ageing¹¹⁴ and is particularly compromised in the midbrain of patients with Parkinson’s disease, progressive supranuclear palsy or multiple system atrophy¹¹⁵. More work is needed to establish the exact roles of ABC BBB transporters in neurodegeneration and whether their failure precedes the loss of dopaminergic neurons that occurs in Parkinson’s disease.

In mice with ALS-like disease and in patients with ALS, hypoperfusion and/or dysregulated CBF have been shown to occur before motor neuron degeneration or brain atrophy^8,116. Reduced regional CBF in basal ganglia and reduced blood volume have been reported in pre-symptomatic gene-tested individuals at risk for Huntington’s disease¹¹⁷. Patients with Huntington’s disease display a reduction in vasomotor activity in the cerebral anterior artery during motor activation¹¹⁸.

Vascular and neuronal common growth factors

Blood vessels and neurons share common growth factors and molecular pathways that regulate their development and maintenance^119,120. Angioneurins are growth factors that exert both vasculotrophic and neurotrophic activities¹²¹. The best studied angioneurin is vascular endothelial growth factor (VEGF). VEGF regulates vessel formation, axonal growth and neuronal survival¹²⁰. Ephrins, semaphorins, slits and netrins are axon guidance factors that also regulate the development of the vascular system¹²¹. During embryonic development of the neural tube, blood vessels and choroid plexus secrete IGF2 into the CSF, which regulates the proliferation of neuronal progenitor cells¹²². Genetic and pharmacological manipulations of angioneurin activity yielded various vascular and cerebral phenotypes¹²¹. Given the dual nature of angioneurin action, these studies have not been able to address whether neuronal dysfunction results from a primary insult to neurons and/or whether it is secondary to vascular dysfunction.

Increased levels of VEGF, a hypoxia-inducible angiogenic factor, were found in the walls of intraparenchymal vessels, perivascular deposits, astrocytes and intrathecal space of patients with Alzheimer’s disease, and were consistent with the chronic cerebral hypoperfusion and hypoxia that were observed in these individuals⁷³. In addition to VEGF, brain microvessels in Alzheimer’s disease release several molecules that can influence angiogenesis, including IL-1β, IL-6, IL-8, TNF, TGFβ, MCP1, thrombin, angiopoietin 2, αVβ3 and αVβ5 integrins, and HIF1α⁷³. However, evidence for increased vascularity in Alzheimer’s disease is lacking. On the contrary, several studies have reported that focal vascular regression and diminished microvascular density occur in Alzheimer’s disease^4,5,73 and in Alzheimer’s disease transgenic mice¹²³. The reason for this discrepancy is not clear. The anti-angiogenic activity of amyloid-β, which accumulates in the brains of individuals with Alzheimer’s disease and Alzheimer’s disease models, may contribute to hypo-vascularity¹²³. Conversely, genome-wide transcriptional profiling of brain endothelial cells from patients with Alzheimer’s disease revealed that extremely low expression of vascular-restricted MEOX2 mediates aberrant angiogenic responses to VEGF and hypoxia, leading to capillary death⁵. This finding raises the interesting question of whether capillary degeneration in Alzheimer’s disease results from unsuccessful vascular repair and/or remodelling. Moreover, mice that lack one Meox2 allele have been shown to develop a primary cerebral endothelial hypoplasia with chronic brain hypoperfusion⁵, resulting in secondary neurodegenerative changes³³.

Alzheimer’s disease

Amyloid-β clearance from the brain by the BBB is the best studied example of clearance of a proteinaceous toxin from the CNS. Multiple pathways regulate brain amyloid-β levels, including its production and clearance (FIG. 4). Recent studies^127–129 have confirmed earlier findings in multiple rodent and non-human primate models demonstrating that peripheral amyloid-β is an important precursor of brain amyloid-β^130–136. Moreover, peripheral amyloid-β sequestering agents such as soluble LRP1¹³⁷, anti-amyloid-β antibodies^138–140, gelsolin and the ganglioside GM1¹⁴¹, or systemic expression of neprilysin have been shown to reduce the amyloid burden in Alzheimer’s disease mice by eliminating contributions of the peripheral amyloid-β pool to the total brain pool of this peptide.

The receptor for advanced glycation end products (RAGE) mediates amyloid-β transport in brain and the propagation of its toxicity. RAGE expression in brain endothelium provides a mechanism for influx of amyloid-β^144,145 and amyloid-β-laden monocytes¹⁴⁶ across the BBB, as shown in Alzheimer’s disease models (FIG. 4). The amyloid-β-rich environment in Alzheimer’s disease and models of this disorder increases RAGE expression at the BBB and in neurons^147,148, amplifying amyloid-β-mediated pathogenic responses. Blockade of amyloid-β–RAGE signaling in Alzheimer’s disease is a promising strategy to control self-propagation of amyloid-β-mediated injury.

Several studies in animal models of Alzheimer’s disease and, more recently, in patients with this disorder¹⁴⁹ have shown that diminished amyloid-β clearance occurs in brain tissue in this disease. LRP1 plays an important part in the three-step serial clearance of this peptide from brain and the rest of the body¹⁵⁰ (FIG. 4). In step one, LRP1 in brain endothelium binds brain-derived amyloid-β at the abluminal side of the BBB, initiating its clearance to blood, as shown in many animal models1^51–156 and BBB models in vitro^151,157,158. The vasculotropic mutants of amyloid-β that have low binding affinity for LRP1 are poorly cleared from the brain or CSF^151,159,160. APOE4, but not APOE3 or APOE2, blocks LRP1-mediated amyloid-β clearance from the brain and, hence, promotes its retention¹⁶¹, whereas clusterin (also known as apolipoprotein J (APOJ)) mediates amyloid-β clearance across the BBB via LRP2¹⁵³. APOE and clusterin influence amyloid-β aggregation^162,163. Reduced LRP1 levels in brain microvessels, perhaps in addition to altered levels of ABCB1, are associated with amyloid-β cerebrovascular and brain accumulation during ageing in rodents, non-human primates, humans, Alzheimer’s disease mice and patients with Alzheimer’s disease^{66,151,152,164–166}. Moreover, recent work has shown that brain LRP1 is oxidized in Alzheimer’s disease¹⁶⁷, and may contribute to amyloid-β retention in brain because the oxidized form cannot bind and/or trans- port amyloid-β¹³⁷. LRP1 also mediates the removal of amyloid-β from the choroid plexus¹⁶⁸.

In step two, circulating soluble LRP1 binds more than 70% of plasma amyloid-β in neurologically normal humans¹³⁷. In patients with Alzheimer’s disease or mild cognitive impairment (MCI), and in Alzheimer’s disease mice, amyloid-β binding to soluble LRP1 is compromised due to oxidative changes^137,169, resulting in elevated plasma levels of free amyloid-β isoforms comprising 40 or 42 amino acids (amyloid-β_1–40 and amyloid-β_1–42). These peptides can then re-enter the brain, as has been shown in a mouse model of Alzheimer’s disease¹³⁷. Rapid systemic removal of amyloid-β by the liver is also mediated by LRP1 and comprises step three of the clearance process¹⁷⁰.

In brain, amyloid-β is enzymatically degraded by neprilysin¹⁷¹, insulin-degrading enzyme¹⁷², tissue plasminogen activator ¹⁷³ and MMPs^173,174 in various cell types, including endothelial cells, pericytes, astrocytes, neurons and microglia. Cellular clearance of this peptide by astrocytes and VSMCs is mediated by LRP1 and/or another lipoprotein receptor^66,175. Clearance of amyloid-β aggregates by microglia has an important role in amyloid-β-directed immunotherapy¹⁷⁶ and reduction of the amyloid load in brain¹⁷⁷. Passive ISF–CSF bulk flow and subsequent clearance through the CSF might contribute to 10–15% of total amyloid-β removal^152,153,178. In the injured human brain, increasing soluble amyloid-β concentrations in the ISF correlated with improvements in neurological status, suggesting that neuronal activity might regulate extracellular amyloid-β levels¹⁷⁹.

The role of BBB dysfunction in amyloid-β accumulation, as discussed above, underlies the contribution of vascular dysfunction to Alzheimer’s disease (see FIG. 5 for a model of vascular damage in Alzheimer’s disease). The amyloid hypothesis for the pathogenesis of Alzheimer’s disease maintains that this peptide initiates a cascade of events leading to neuronal injury and loss and, eventually, dementia^180,181. Here, I present an alternative hypothesis — the two-hit vascular hypothesis of Alzheimer’s disease — that incorporates the vascular contribution to this disease, as discussed in this Review (BOX 1). This hypothesis states that primary damage to brain microcirculation (hit one) initiates a non-amyloidogenic pathway of vascular-mediated neuronal dysfunction and injury, which is mediated by BBB dysfunction and is associated with leakage and secretion of multiple neurotoxic molecules and/or diminished brain capillary flow that causes multiple focal ischaemic or hypoxic micro-injuries. BBB dysfunction also leads to impairment of amyloid-β clearance, and oligaemia leads to increased amyloid-β generation. Both processes contribute to accumulation of amyloid-β species in the brain (hit two), where these peptides exert vasculotoxic and neurotoxic effects. According to the two-hit vascular hypothesis of Alzheimer’s disease, tau pathology develops secondary to vascular and/or amyloid-β injury.

The author wishes to thank the US National Institutes of Health (grants R37 AG023084, R37 NS34467 and HL63290), the ALS Association (grant 1859) and the Zilkha family for research support. The author also wishes to thank A. Sagare for preparing the illustrations and for valuable help with the reference list.