William A Banks 12Naoko Nonaka JSusan A Farr and John E Morley

1Geriatric Research, Education, and Clinical Center, Veteran Affairs Medical Center—St. Louis and Department of Internal Medicine,

Division of Geriatrics, Saint Louis University School of Medicine, St. Louis, Missouri. 2Department of Oral Anatomy, Showa University School of Dentistry, Tokyo, Japan, and Department of 1st Anatomy, Showa University

School of Medicine, Tokyo, Japan

The dynamic functioning of the central nervous system (CNS) is increasingly viewed to include cell types in addition to neurons, such as astrocytes and microglia. The endothelial cells that make up the microvasculature must be included in this new view. The microvasculature of the CNS is specially modified to play roles that it seldom undertakes in other tissue beds. Specifically, it forms the blood-brain barrier (BBB), which acts to prevent the unrestricted entry of circulating substances into the brain. But the BBB also is endowed with numerous saturable transport systems. All of the vitamins, minerals, glucose, amino acids, free fatty acids, and other nutrients needed by the CNS are delivered to it by the BBB. The BBB also plays a homeostatic role for the CNS, exporting toxins, regulating electrolyte levels, and controlling pH. Finally, the BBB has a role in communication between the CNS and peripheral tissues. Both blood-to-brain influx and brain-to-blood efflux systems participate in this communication. By regulating the exchange between the CNS and blood of information molecules such as cytokines and peptides, the BBB acts as a key regulatory point in an endocrine-like communication between the CNS and the peripheral tissues. Failure of the microcirculation, especially of some aspect of BBB function, has figured largely in several theories of the etiology of Alzheimer's disease (AD). It should be noted that the theories are not mutually exclusive. Indeed, if some driving force, such as amyloid beta protein (ABP) is invoked, it is easy to imagine how the various theories can be interlinked.

Perturbances in Microvascular Circulation and Endothelial Cell Function

The microvasculature is clearly perturbed in AD. With normal aging, there is an increased thinning of the endothelial wall, a decreased number of endothelial cells forming the capillary bed, a decreased number of pericytes, and an increased susceptibility to events that can disrupt the BBB. In AD, many of these findings are accentuated. In particular, the microvasculature is extremely tortuous. It has been suggested that the tortuosity is so severe that perturbed hemodynamics occur, with a shift from normal laminar flow to one of chaotic flow [1]. Such chaotic flow could perturb the exchange of solutes across the BBB, leading to deficiencies in brain of critical substances, which, in turn, would lead to cognitive impairments.

Brain endothelial cells from AD patients show a number of abnormalities. For example, they have decreased protein kinase C activity, decreased capacity for transporting glucose, an increased density of ß-1 adrenergic receptors, and increased nitric oxide synthase activity. They secrete more proinflammatory cytokines in culture, and the perturbance in protein kinase C activity is associated with the secretion of an unidentified neurotoxic protein.

ABP, the leading candidate as an etiologic agent in AD [2], has a wide variety of effects that could alter the functions of brain endothelial cells. ABP acts as an ionophore, inducing channels capable of transporting calcium and potassium. ABP can generate free radicals and affect nitric oxide synthase activity. ABP decreases glucose uptake by brain endothelial cells, inhibits their proliferation, and induces apoptosis and secretion of proinflammatory cytokines, including endothelin-1. ABP-induced reductions in cerebral blood flow are likely mediated by release of endothelin-1.

Altered Permeability of the Blood-Brain Barrier

Disruption of the Blood-Brain Barrier

One of the most investigated areas of AD and the BBB is whether the BBB is disrupted to blood-borne proteins. One of the main findings supporting a disruption of the BBB is that the cerebrospinal fluid (CSF)/serum ratio for albumin is increased in AD. Albumin is virtually excluded from the CNS by the BBB, with only small amounts entering by way of the extracellular pathways. An increase in the CSF/serum ratio for albumin is a hallmark for BBB disruption. However, not all studies have found an elevation in the CSF/serum ratio for albumin. Furthermore, other causes for an increase in the CSF/serum ratio for albumin, such as a decrease in the rate at which the CSF is replaced, have not been ruled out. Overall, the majority of studies have concluded that the BBB remains intact in AD. This is largely supported to the degree it has been investigated in animal models of AD.

Alterations in the Saturable Transport Properties of the Blood-Brain Barrier

Studies in Humans

Other CSF/serum ratios are also abnormal in AD. For example, ratios for both vitamin B12 and insulin are decreased in AD. Both of these substances are transported by saturable systems across the BBB. Therefore, disturbances in their levels suggests that selective impairments in the transport properties of the BBB can be perturbed in AD. This is clearly supported by work in animal models of AD.

Studies in Animal Models of Alzheimer's Disease

Animal models have been key in supporting a causal link between ABP and AD. Studies using animal models of AD also support the idea that transporters are altered in this disease. Whereas transgenics have been the primary model in investigating ABP as a cause of AD, a natural mutation has also been used in examining questions related to BBB. The SAMP8 is a natural mutation that develops age-related learning and memory deficits. At 4 months of age, SAMP8 mice have normal cognition, but develop severe deficits between 8 and 12 months of age. These deficits are reversed by antibodies or antisense molecules directed at ABP or its precursor. Several transporters for key peptides or regulatory proteins have been examined in the SAMP8 mouse. The transport of insulin and that of interleukin-1 show no changes with aging in the SAMP8 mouse. Tumor necrosis factor is transported more rapidly into the occipital cortex, midbrain, and striatum of aged SAMP8 mice. Pituitary adenylate cyclase activating polypeptide (PACAP) is transported into brain by peptide transport system-6, or PTS-6. Enough PACAP is transported into brain by PTS-6 to prevent apoptosis of CA-1 hippocampal neurons even when given intravenously 24 hours after four-vessel stroke. PTS-6 is present throughout the brain excepting the pons medulla of CD-1 mice. With aging, SAMP8 mice lose PTS-6 activity in the thalamus, midbrain, and olfactory bulbs.

in contrast to the selective changes in some saturable transport systems, even very old SAMP8 mice do not show disruption of the BBB. The BBB remains largely intact to both albumin and sucrose.

Transport of Amyloid Beta Protein

Blood-to-Brain Transport

Traditionally, peptides arising in the periphery have been thought to only affect peripheral receptors. But with the appreciation that biologically relevant amounts of peptides are able to cross the BBB, it has become clear that peptides arising on one side of the BBB can interact with receptors on the other side. The possibility that ABP can cross the BBB was considered early on. An immediate question that arises is which ABP should one study. ABP is cleaved irregularly from its precursor so that a series of peptides of between 39 and 43 amino acids is formed. The 1-40 and 142 forms are the most abundant and of greatest interest. These two forms of ABP differ substantially in their biological and chemical properties. ABP1-40 is the more abundant form in human plasma and cerebrospinal fluid. However, ABP1-42 is considered the more toxic form. It more readily forms fibrils than does ABP1-40, which is likely related to its neurotoxicity. Fibrillation makes it more difficult to work with, and so most studies have used ABP1-40. However, a single amino acid substitution can substantially alter the properties of ABP, so it cannot be assumed that work with one form of ABP can be directly applied to another form.

Another potential problem in examining the ability of a peptide to cross the BBB is the number of related interactions. For example, if a peptide is degraded rapidly in blood or by the BBB, sequestered by the vasculature, binds avidly with circulating substances, or has multiple molecular forms because of fibrillation, it may appear to have different interactions with the BBB when studied by different techniques. The various ABPs undergo a great number of these interactions (Figure 1). By comparing and contrasting results from different methods, a great deal of information had been gathered about the interactions of the ABPs with the BBB.

Radioactively labeled ABP 1-40 given by intravenous injection can be recovered from both brain and cerebrospinal fluid [3], and radioactively labeled ABP1-42 has been recovered from brain after brain perfusion [4]. This is in contrast to ABP 1-28, originally the only form of ABP commercially available for study, which is so rapidly degraded in blood or at the BBB as to likely preclude its

Figure 1 Postulated and proven pathways: interactions between amyloid beta protein (ABP) and the blood-brain barrier (BBB). 1, Peripheral and CNS sources of ABP are in dynamic equilibrium because of bidirectional transport of ABP across the BBB. 2, ABP binds to circulating substances in blood; 3, these binding proteins may themselves either be transported across the BBB, thus forming an alternate route for the transport of ABP across the BBB, or may be less permeable to the BBB than unbound ABP, thus retarding ABP transport across the BBB. 4, ABP can be sequestered by or accumulate on cells forming the BBB. 5, Some forms of ABP are especially rapidly degraded by enzymes in blood, at the BBB, and in the CNS. 6, ABP in the CSF may enter the circulation by way of bulk flow, the process of reabsorption of CSF back into the blood.

Figure 1 Postulated and proven pathways: interactions between amyloid beta protein (ABP) and the blood-brain barrier (BBB). 1, Peripheral and CNS sources of ABP are in dynamic equilibrium because of bidirectional transport of ABP across the BBB. 2, ABP binds to circulating substances in blood; 3, these binding proteins may themselves either be transported across the BBB, thus forming an alternate route for the transport of ABP across the BBB, or may be less permeable to the BBB than unbound ABP, thus retarding ABP transport across the BBB. 4, ABP can be sequestered by or accumulate on cells forming the BBB. 5, Some forms of ABP are especially rapidly degraded by enzymes in blood, at the BBB, and in the CNS. 6, ABP in the CSF may enter the circulation by way of bulk flow, the process of reabsorption of CSF back into the blood.

entry into brain. Uptake of radioactively labeled human ABP1-40 is not saturable in the mouse when given by intravenous injection but is in the guinea pig and mouse when given by brain perfusion. This discrepancy could be caused by much of the unlabeled ABP being sequestered by blood-borne binding proteins. In vitro studies with monolayers of brain endothelial cells and in vivo studies have shown that the receptor for advanced glycation end products, or RAGE, plays a major role in the transport of ABP into brain. There is also evidence that the scavenger receptor type A can bind and transport ABP.

Bloodborne ABP 1-40 taken up by the BBB is largely sequestered by the capillaries. Only about 20 percent completely crosses the BBB to reach the brain parenchymal and brain interstitial fluid space, with about 80 percent being retained by the capillaries. ABP 1-28 is similarly sequestered, but not the synthetic reverse peptide ABP40-1.

ABP1-40 binds to Apo J, which itself is transported by a saturable system across the BBB by glycoprotein 330/mega-lin. However, the circulating levels of Apo J far exceed the Km of its saturable transporter so that the transporter is completely saturated under physiological conditions. Therefore, the net effect of Apo J binding is to retard the entry of ABP.

Brain-to-Blood Transport

Normal Physiology

Several studies with ABP1-40 have shown that it is transported rapidly out of the brain by a saturable efflux system.

The efflux system has been suggested to be LDL receptor-related protein-1, and others have suggested it to be P-glycoprotein. Studies in monkeys and mice have suggested that efflux is impaired with aging and in AD. This has led to the vasculogenic hypothesis stating that ABP accumulates in brain because of an impairment in efflux transport [5].

Animal Models of AD

DeMattos et al. used transgenic mice overexpressing the precursor to ABP to dramatically demonstrate brain-to-blood efflux of ABP [6]. Rapid clearance of ABP from blood prevents serum levels from being very useful as an indicator of brain levels of ABP. DeMattos et al. peripherally administered an ABP-binding antibody to mice. After injection, serum ABP1-40 levels increased dramatically and correlated with levels of ABP and of amyloid load in the hippocampus. It is thought that because of clearance from blood was slowed, ABP was able to accumulate in blood. This accumulation would be related to the amount of ABP entering the blood from the brain, which, in turn, would be dependent on the amount of ABP in brain. This modeling of ABP efflux and clearance from blood suggests that administration of antibody to ABP could be used to determine brain levels of ABP in a diagnostic setting. If blood-to-brain influx of ABP is a significant contributor to brain levels of ABP, this model would also support the use of antibodies as a therapeutic intervention.

The preceding studies with ABP1-40 raise the question of whether ABP1-42 is also transported across the BBB and whether its transport is impaired in animal models of AD. We recently addressed both these questions [3] in the CD-1 and in the SAMP8 mouse strains. Human ABP 1-40, the form almost exclusively studied to date, and mouse ABP1-42 were each transported out of the brain by a saturable process in young CD-1 mice, young SAMP8 mice, and aged SAMP8 mice. The young SAMP8 mouse had impaired transport of the ABP 1-42 and ABP 1-40 and the aged SAMP8 mouse had impaired transport of ABP1-42 (Figure 2). Other forms of ABP (mouse ABP1-40 and human ABP1-42) showed statistically significant differences only with aging in the SAMP8, with total loss of efflux. These results support the hypothesis that impaired efflux is associated with AD. Since decreased efflux occurred prior to the development of cognitive impairments in the SAMP8, decreased efflux could play its postulated role in leading to ABP accumulation. Finally, the results show that the BBB does not interact in the same way with all forms of ABP


The vasculature of the central nervous system (CNS) is specially modified to form the blood-brain barrier (BBB). The BBB not only prevents the unrestricted leakage of serum proteins into the CNS, it also transports into the brain vitamins, minerals, and nutrients while transporting out of the brain toxins and other substances. The BBB also aids communication between the CNS and peripheral tissues



SAM-Young SAM-Aged r



Figure 2 Decreased brain-to-blood efflux in SAMP8 mice of ABP. The SAMP8 mouse, a natural mutation used as an animal model of AD, has an impaired ability of the BBB to transport out the various forms ABP, especially the more toxic ABP1-42. Values for (—) slopes are shown, which are inversely related to half-time clearance from brain; a larger value means more rapid clearance.

through its transport of peptides and regulatory proteins. The majority of papers find the barrier function of the BBB to remain intact with Alzheimer's disease (AD). However, other aspects are altered and have been postulated to play a causal role in AD. Hemodynamic alterations in blood flow caused by vascular tortuosity could interfere with the transport of substances across the BBB. The individual brain endothelial cells that make up the BBB have alterations in their biochemical functions. Amyloid beta protein (ABP), considered widely to play a causal role in AD, induces many changes, including cell death, in brain endothelial cells. Animal models of Alzheimer's disease have shown alterations in the transporter properties of ABP. Perhaps most relevant of these are a loss of the ability to transport out the most toxic form: ABP1-42. This last finding supports the vascu-logenic theory, which states that loss of the ability of the BBB to rid the brain of ABP leads to its accumulation and, ultimately, to AD.



Alzheimer's disease: A degenerative disease of the brain characterized by the insidious onset of dementia.

Amyloid beta protein: A peptide of 39 to 43 amino acids thought to play a central role in the cause of Alzheimer's disease.

Blood—brain barrier: The modified vasculature of the CNS that prevents the unrestricted entry of blood-borne proteins into the CNS and also transports into and out of the CNS vitamins, minerals, glucose, amino acids, free fatty acids, peptides, and regulatory proteins.

CD-1: The official strain name for the standard albino outbred white lab mouse used since about 1926 and sold by Charles River.

Central nervous system: Consisting of the brain, spinal cord, and cranial nerves.

SAMP8: A mouse strain with a natural mutation that leads to an age-related decline in learning and memory. Used as an animal model of Alzheimer's disease.


1. de la Torre, J. C., and Mussivand, T. (1993). Can disturbed brain microcirculation cause Alzheimer's disease? Neurol. Res. 15, 146-153.

2. Rosenberg, R. N. (2000). The molecular and genetic basis of AD: The end of the beginning. The 2000 Wartenberg lecture. Neurology 54, 2045-2054. In-depth review of etiologies for Alzheimer's disease with emphasis on amyloid beta protein.

3. Banks, W. A., Robinson, S. M., Verma, S., and Morley, J. E. (2004) Efflux of human and mouse amyloid b proteins 1-40 and 1-42 from brain: Impairment in a mouse model of Alzheimer's disease. Neuroscience, in press. An in-depth study of the brain-to-blood transport kinetics of the various forms of amyloid beta proteins in an animal model of Alzheimer's disease. Neurosci. 121, 487-492.

4. Deane, R., Yan, S. D., Submamaryan, R. K., LaRue, B., Jovanovic, S., Hogg, E., Welch, D., Manness, L., Lin, C., Yu, J., Zhu, H., Ghiso, J., Frangione, B., Stern, A., Schmidt, A. M., Armstrong, D. L., Arnold, B., Liliensiek, B., Nawroth, P., Hofman, F., Kindy, M., Stern, D., and Zlokovic, B. (2003). RAGE mediates amyloid-b peptide transport across the blood-brain barrier and accumulation in brain. Nat. Med. 7, 907-913. A classic study of the role of blood-to-brain transport in affecting brain levels of amyloid beta protein with emphasis on mediation of the receptor for advanced glycation end products.

5. Zlokovic, B. V., Yamada, S., Holtzman, D., Ghiso, J., and Frangione, B. (2000). Clearance of amyloid b-peptide from brain: Transport or metabolism? Nat. Med. 6, 718-719.

6. DeMattos, R. B., Bales, K. R., Cummins, D. J., Paul, S. M., and Holtzman, D. M. (2002). Brain to plasma amyloid-b efflux: A measure of brain amyloid burden in a mouse model of Alzheimer's disease. Science 295, 2264.

This review has considered how the microvasculature is altered in AD. It has particularly addressed theories in which those alterations play a pathologic or even causal role in AD. Observations in humans has been greatly aided by work with both transgenic and natural mutation animal models of AD. Overall, work shows alterations in the morphology, cellular biology, and transport characteristics of the BBB. ABP is toxic to brain endothelial cells and could induce many of these changes. The loss of the ability of the BBB to transport ABP out of the CNS could be an immediate cause of ABP accumulation in the brain. Thus, the brain microvas-culature likely plays a central role in the pathogenesis of AD.

Further Reading

Banks, W. A., Kastin, A. J., Maness, L. M., Banks, M. F., and Shayo, M. (1997). In Interactions of ß-Amyloids with the Blood—Brain Barrier (J. C. de la Torre and V. Hachinski, eds.), Vol. 826, pp. 190-199. New York: New York Academy of Sciences.

Capsule Biography

Dr. William A. Banks is a staff physician and a principal investigator at the Veterans Affairs Medical Center—Saint Louis. He is also a professor in the Division of Geriatrics in the Department of Internal Medicine and in the Department of Physiological and Pharmacological Sciences. He is editorin-chief of Current Pharmaceutical Design and has more than 250 publications in the area of blood-brain barrier.

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