Keeping brain amyloid-β (Aβ) levels in check means controlling both the means of production and the method of disposal. On the disposal side, a few years ago researchers recognized for the first time that Aβ is actively cleared from the brain via the LDL receptor-related protein-1 (LRP1) (Shibata et al., 2000). Now, David Holtzman and colleagues from Washington University in St. Louis have identified a second pump. In a paper that appeared online in the Journal of Clinical Investigation on October 20, the scientists, working with collaborators at Eli Lilly and at the University of Rochester Medical Center, New York, report that P-glycoprotein (Pgp), best known as the membrane transporter that confers multidrug resistance on tumor cells, also contributes to Aβ efflux from brain. Their work shows that blocking Pgp function enhances amyloid deposition in a mouse model of AD. Since genetic polymorphisms and many common drugs modulate Pgp activity, both positively and negatively, understanding the role of this protein could lead to the identification of new risk factors, or protective mechanisms, for AD in people.

The Pgp transporter is an important component of the capillary endothelial cell blood-brain barrier. There, it acts like a sump pump, that is, as fast as substrate drugs and peptides pour in, the pump escorts them back out. Hints that Pgp could also be an Aβ exporter included in-vitro work (Lam et al., 2001), as well as histological studies showing that Pgp expression inversely mirrored the distribution of Aβ in human brain (Vogelgesang et al., 2002). These intriguing results led Holtzman and colleagues to directly measure the contribution of Pgp to Aβ efflux in vivo.

Using different experimental approaches, first author John Cirrito and colleagues built the case for Pgp’s involvement in Aβ clearance. First, they measured efflux in wild-type versus Pgp knockout mice by following the fate of radioactive Aβ after injection into the brain. When they looked at what remained 30 minutes after injection, they found that about twice as much Aβ40 or Aβ42 cleared the blood-brain barrier in the wild-type mice as in Pgp knockout mice. In another experiment, they crossed APPsw AD transgenic mice with the Pgp knockouts. At one year of age, the Pgp-null offspring had larger plaque areas, more thioflavin S reactivity, and double the levels of Aβ42 in the hippocampus compared to AD mice with normal Pgp.

Though suggestive, these experiments were complicated by the observation that Pgp-null mice also had much lower levels of the other Aβ transporter, LRP1, on capillary endothelial cells. To sort out the contribution of Pgp alone to Aβ efflux, the investigators turned to a pharmacological inhibitor to acutely reduce Pgp activity. After administering the inhibitor XR9576 intravenously, they followed Aβ levels in brain interstitial fluid for 10 hours by microdialysis. During this period, they saw Aβ levels climb, with no change in LRP1 protein levels. Taking these results together with previous studies showing the role of LRP1 in Aβ clearance, the authors speculate that the two efflux proteins may actually act synergistically, with LRP1 functioning on the basolateral surface of brain endothelial cells, and Pgp on the luminal surface.

The implication of Pgp in Aβ clearance and AD pathology brings up some pressing questions. First, Pgp polymorphisms that affect drug handling are well known. If the changes also affect Aβ clearance, they could represent genetic risk or protection for AD. Because of this, the authors stress the need for a detailed genetic analysis of Pgp polymorphisms and AD risk.

Second, many common drugs alter Pgp function, and conceivably, their use could carry an increased or decreased risk of AD. Among that group is the antibiotic rifampin, which upregulates Pgp. Rifampin was shown in a clinical trial to lessen cognitive decline in people with mild to moderate AD after one year of treatment (Loeb et al., 2004). These new results raise the possibility that the drug could be beneficial because it increases clearance of Aβ. On the flip side, many drugs inhibit Pgp (indeed, some cancer treatments have been designed to do exactly that). Whether the use of such agents will be associated with an increased risk of AD needs to be investigated.—Pat McCaffrey

Comments

Make a Comment

To make a comment you must login or register.

Comments on this content

  1. This paper is a typically well-reasoned and very logical study from the group of David Holtzman. The authors show how P-glycoprotein (Pgp), which is involved in efflux transport of cytotoxic agents from tumor cells, is also involved in the transport of Aβ from brain into blood.

    The accumulation of insoluble material as plaques in gray matter of the brain in Alzheimer disease has been known for 100 years and this material was identified as amyloid-β 20 years ago. With the characterization of genetic defects in the amyloid precursor protein (APP) gene in relatively small numbers of cases of familial AD, the amyloid hypothesis was born, suggesting that overproduction of Aβ was a major factor in the pathogenesis of AD. However, there is little firm evidence that overproduction of Aβ occurs in the large number of cases of sporadic AD or in cognitively normal elderly individuals who also accumulate Aβ in the brain. Attention has turned, therefore, during the last few years towards failure of elimination of Aβ from the brain as a major factor in the pathogenesis of AD.

    Aβ is produced by neurons and other cells in the brain, and a number of different pathways for the elimination of Aβ from the brain have been identified. They include (a) active transport of Aβ from brain directly into the blood (Cirrito et al., 2005; Shibata et al., 2000); (b) proteolytic degradation of Aβ by enzymes in brain parenchyma (Iwata et al., 2002); and (c) removal of Aβ by bulk flow drainage with interstitial fluid (ISF) along capillary and artery walls (Weller et al., 1998).

    There are several exciting aspects of the present paper showing the involvement of P-glycoprotein (Pgp) in transport of Aβ out of the brain into the blood. The authors suggest that some polymorphisms in Pgp may restrict the elimination of Aβ, whereas others may enhance it. Perhaps of more immediate application to therapy of Alzheimer disease is the suggestion that some drugs, such as rifampin, are Pgp substrates and inducers. Because Pgp is located on the luminal surface of the endothelial cell, it is accessible to pharmacological influences as the drug is not required to cross the blood-brain barrier.

    So what of the future? Perhaps it is timely to look at the balance of the different known pathways for the elimination of Aβ from the human brain. Knowing how, why and to what extent each mechanism for elimination of Aβ fails in the elderly and in AD will guide the development of therapies to prevent AD and possibly to ameliorate established disease. There is some indication from studies in mice that active transport of Aβ from brain directly into the blood fails with age (Shibata et al., 2000), as does proteolytic degradation of Aβ by enzymes in brain parenchyma (Iwata et al., 2002), but less is known in this area in elderly humans. Removal of Aβ by bulk flow drainage with interstitial fluid along capillary and artery walls appears to fail with age in humans and results in deposition of Aβ in the walls of capillaries and arteries as cerebral amyloid angiopathy (CAA). Aging and stiffening of blood vessels with cerebrovascular disease may be a factor in the failure of elimination of Aβ by the perivascular route and the development of CAA (Weller et al., 2002; Weller and Nicoll, 2003; Schley et al., 2005). At the moment, the age changes that occur in human cerebral arteries are not built into mouse models used to study the elimination of Aβ.

    In devising suitable therapies for AD, can the absorption into the blood be enhanced in the elderly, as suggested by Cirrito et al.? Or can the elimination of Aβ along perivascular pathways be enhanced by suitable chaperone molecules? Removal of insoluble Aβ from brain parenchyma in AD does seem to occur following immunization against Aβ, both in mouse (Wilcock et al., 2004) and humans (Nicoll et al., 2003). At the same time, the severity of CAA increases following treatment (Wilcock et al., 2004), possibly because the soluble Aβ derived from amyloid plaques overloads perivascular drainage pathways.

    Many questions remain unanswered. For example: What is the relationship between the extracellular accumulation of Aβ in the brain in Alzheimer disease and the intracellular accumulation of tau protein and ubiquitin as neurofibrillary tangles (NFT)? Does the deposition of Aβ induce the formation of NFTs, as is suggested by some transgenic mouse models, or are they independent aging processes? Another question revolves around what exactly interferes with neuronal function in Alzheimer disease. Is it the insoluble plaques of Aβ or the NFTs, or is it the high levels of soluble Aβ in the interstitial fluid of the brain?

    One final point to consider is whether Aβ is actually the main toxic influence on neurons or whether its main role in AD is to block the extracellular spaces in brain parenchyma and the drainage pathways in vessel walls so that there is failure of elimination of a wide variety of metabolites in the interstitial fluid. Such failure of elimination of solutes and ions may significantly alter the composition of the extracellular fluid and the extracellular environment of neurons in the brain, and this may be a factor that induces neuronal dysfunction.

    Whatever the answers to the questions posed above, it does seem that a coordinated effort is required to understand how the various mechanisms of elimination of Aβ from the brain are balanced, why they appear to collectively fail in AD, and how elimination of Aβ can be most suitably enhanced for the prevention and treatment of this disease.

    References:

    . P-glycoprotein deficiency at the blood-brain barrier increases amyloid-beta deposition in an Alzheimer disease mouse model. J Clin Invest. 2005 Nov;115(11):3285-90. PubMed.

    . Region-specific reduction of A beta-degrading endopeptidase, neprilysin, in mouse hippocampus upon aging. J Neurosci Res. 2002 Nov 1;70(3):493-500. PubMed.

    . Neuropathology of human Alzheimer disease after immunization with amyloid-beta peptide: a case report. Nat Med. 2003 Apr;9(4):448-52. PubMed.

    . Mechanisms to explain the reverse perivascular transport of solutes out of the brain. J Theor Biol. 2006 Feb 21;238(4):962-74. PubMed.

    . Clearance of Alzheimer's amyloid-ss(1-40) peptide from brain by LDL receptor-related protein-1 at the blood-brain barrier. J Clin Invest. 2000 Dec;106(12):1489-99. PubMed.

    . Cerebral amyloid angiopathy: amyloid beta accumulates in putative interstitial fluid drainage pathways in Alzheimer's disease. Am J Pathol. 1998 Sep;153(3):725-33. PubMed.

    . Cerebral amyloid angiopathy: pathogenesis and effects on the ageing and Alzheimer brain. Neurol Res. 2003 Sep;25(6):611-6. PubMed.

    . Cerebrovascular disease is a major factor in the failure of elimination of Abeta from the aging human brain: implications for therapy of Alzheimer's disease. Ann N Y Acad Sci. 2002 Nov;977:162-8. PubMed.

    . Passive immunotherapy against Abeta in aged APP-transgenic mice reverses cognitive deficits and depletes parenchymal amyloid deposits in spite of increased vascular amyloid and microhemorrhage. J Neuroinflammation. 2004 Dec 8;1(1):24. PubMed.

  2. A recurring theme in the pathogenesis of diverse degenerative disorders is the accumulation of certain proteins in cells and tissues. Although many details remain to be ironed out, the pathogenic importance of cerebral Aβ accumulation in Alzheimer disease is now beyond reasonable dispute. Simply increasing the concentration of aggregation-prone proteins such as Aβ raises the odds that they will multimerize and deposit, as is demonstrated by various disease states and transgenic models. Thus, the more we know about how proteopathic proteins are made, transported, and dismantled, the better the chances that we can manipulate these processes for the benefit of patients.

    Aβ production and degradation have received the lion's share of attention in AD, and many of the cellular and molecular players in these processes have been identified. Less consideration has been given to the transport of the peptide, despite burgeoning evidence that it can be actively conveyed across cell membranes. Low-density lipoprotein receptor-related protein (LRP1) is one such Aβ-efflux transporter, and now Cirrito, Holtzman, and colleagues furnish compelling evidence that P-glycoprotein (Pgp) is another. Their experiments demonstrate that pharmacologic or genetic diminution of Pgp function decreases transport and increases the amount of Aβ in vivo, and that crossing Pgp-null mice with APP-transgenic mice results in an augmentation of age-associated cerebral Aβ deposition.

    Pgp is a promiscuous transporter that regulates the levels of xenobiotics and other substances in tissues that have a barrier and/or excretory role, such as intestine, kidney, liver, and (in the brain) the blood-brain barrier. Aβ was found to be a substrate for Pgp by Lam and colleagues, and Aβ deposition subsequently was noted to be increased in the brains of elderly humans who had reduced endothelial Pgp immunoreactivity (Vogelgesang et al., 2002; Vogelgesang et al., 2004). Because it can be induced or inhibited by various agents, Pgp presents an interesting and accessible target for regulating Aβ levels in brain. Ideally, of course, enhancement of Pgp activity should be selective for brain endothelial cells, and even if this tall order can be filled, the (theoretically) possible side effects of upregulating such a vital molecular ferry are an issue. On the other hand, the apparent benignity of existing upregulators suggests that Pgp could be a reasonably safe target, at least under most circumstances. I hope that the Cirrito study will stimulate further work in this arena. Finally, it is worth noting that, in addition to genetic and pharmacologic influences on Pgp function, the amount of the transporter in brain vessels appears to diminish with age (Vogelgesang et al., 2004), suggesting one potential means by which senescence increases the risk of idiopathic AD.

    References:

    . Deposition of Alzheimer's beta-amyloid is inversely correlated with P-glycoprotein expression in the brains of elderly non-demented humans. Pharmacogenetics. 2002 Oct;12(7):535-41. PubMed.

    . The role of P-glycoprotein in cerebral amyloid angiopathy; implications for the early pathogenesis of Alzheimer's disease. Curr Alzheimer Res. 2004 May;1(2):121-5. PubMed.

  3. P-glycoprotein (Pgp) is a multifaceted protein that functions as an efflux pump of a variety of endogenous and exogenous compounds in different systemic organs. It is also well recognized in endothelial cells of the brain capillaries, thus playing an important role in the integrity of the blood-brain barrier (BBB). Since the accumulation of insoluble beta amyloid (Aβ) in the brains of patients with Alzheimer disease is thought to be, at least in part, due to insufficient clearance at the BBB, many efforts have been made to find the mechanisms by which Aβ is transported out of the brain. In addition to Pgp, other potential transport proteins such as LRP have been investigated by several research groups.

    On the basis of the results of Lam et al. (2001), we examined the relationship between Pgp expression and the amount of Aβ deposition in the brains of 243 non-demented elderly people, and found an inverse correlation between vascular Pgp and the quantity of Aβ-positive plaques, suggesting that Pgp might indeed play an important role in the pathogenesis of AD (Vogelgesang et al., 2002). In a subsequent study of the correlation between Pgp expression and cerebral amyloid angiopathy (CAA), we found an age-dependent decrease of Pgp expression in cases without CAA and an upregulation of endothelial Pgp in cases with CAA. Pgp and vascular Aβ were never colocalized, and the upregulation of Pgp was detected only in vessels that were not involved in the deposition of Aβ (Vogelgesang et al., 2004).

    In a series of elegant experiments, Cirrito et al. now have provided the strongest evidence to date that Pgp acts as a transporter for Aβ in the living brain, and that the activity of this transporter can minimize the accumulation of the peptide into amyloid lesions in the brains of transgenic mice. Since the expression of Pgp can be modulated by a range of pharmacological substances, these findings support efforts to develop therapeutic strategies directed toward the regulation of Aβ-transport to prevent Aβ accumulation, and thus, the development of AD. Because Pgp expels some therapeutic agents from target tissues, such as tumors or seizure-prone regions in medically intractable epilepsy, most efforts have been made to find substances that inhibit Pgp. In contrast, the goal of the treatment of AD must be the development of Pgp inducers. Such treatment will need to take into account each patient's global medication requirements, since Pgp inducers can reduce the efficacy of some other agents by promoting their removal.

    A second point that makes the Pgp story intriguing is that Pgp expression is believed to be influenced by polymorphisms in the MDR1 gene that encodes Pgp. It has been shown that the MDR1 gene is highly polymorphic. An alteration of transport function could be demonstrated for a C3435T single nucleotide polymorphism in the intestine, where the T allele was found to be associated with decreased Pgp levels, whereas the C allele yielded increased Pgp expression. In our cohort of non-demented elderly cases, we investigated several polymorphisms of the MDR1 gene, but we have not yet found a significant association between Pgp expression and the C3435T polymorphism (Vogelgesang et al., 2002).

    As nicely demonstrated by Cirrito and colleagues, it is now evident that Pgp plays an important role in the clearance of Aβ from brain, and hence may be involved in the development of AD as well as CAA. We hope that larger future efforts will be made to determine the role of Pgp in the pathogenesis of AD, to find possible risk factors for AD, and to develop new therapeutic strategies in the treatment of AD.

    References:

    . beta-Amyloid efflux mediated by p-glycoprotein. J Neurochem. 2001 Feb;76(4):1121-8. PubMed.

    . Deposition of Alzheimer's beta-amyloid is inversely correlated with P-glycoprotein expression in the brains of elderly non-demented humans. Pharmacogenetics. 2002 Oct;12(7):535-41. PubMed.

    . The role of P-glycoprotein in cerebral amyloid angiopathy; implications for the early pathogenesis of Alzheimer's disease. Curr Alzheimer Res. 2004 May;1(2):121-5. PubMed.

  4. This study by the Holtzman lab is most interesting. I had proposed that inhibition of P-glycoprotein together with Gleevec may be a useful therapy in AD. The study I'd referenced in my poster found that levels of Gleevec in the brain were higher in mdr1a/b (/) knockout mice. Perhaps if P-glycoprotein is already reduced in AD, then Gleevec may be expected to cross the BBB in greater concentration and may be, in fact, a therapeutic option on its own.

    References: Gleevec for Alzheimer's?

References

Paper Citations

  1. . Clearance of Alzheimer's amyloid-ss(1-40) peptide from brain by LDL receptor-related protein-1 at the blood-brain barrier. J Clin Invest. 2000 Dec;106(12):1489-99. PubMed.
  2. . beta-Amyloid efflux mediated by p-glycoprotein. J Neurochem. 2001 Feb;76(4):1121-8. PubMed.
  3. . Deposition of Alzheimer's beta-amyloid is inversely correlated with P-glycoprotein expression in the brains of elderly non-demented humans. Pharmacogenetics. 2002 Oct;12(7):535-41. PubMed.
  4. . A randomized, controlled trial of doxycycline and rifampin for patients with Alzheimer's disease. J Am Geriatr Soc. 2004 Mar;52(3):381-7. PubMed.

Further Reading

No Available Further Reading

Primary Papers

  1. . P-glycoprotein deficiency at the blood-brain barrier increases amyloid-beta deposition in an Alzheimer disease mouse model. J Clin Invest. 2005 Nov;115(11):3285-90. PubMed.