How Does Excess Aβ Leave the Brain, How Does It Get In, And Can We Trap It Outside?
Berislav Zlokovic led this live discussion on 23 June 2003. Readers are invited to submit additional comments by using our Comments form at the bottom of the page.
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Lary Walker — Posted 26 August 2006
By Berislav Zlokovic
In yesterday's online version of Nature Medicine, our lab and colleagues published a report about blocking Ab brain import from the periphery across the blood-brain barrier via the endothelial receptor RAGE. The text below first summarizes the main news in this paper, and then lays out the transport-clearance hypothesis of Ab and related Ab-lowering strategies in more breadth and detail.
Synposis of Deane et al. (15a)
We found that RAGE in brain endothelium mediates transport of circulating unbound (free) Ab across the blood-brain barrier (BBB). At pathophysiological levels, this transport results in neurovascular stress and reductions in cerebral blood flow (CBF). Both a soluble form of RAGE (sRAGE) and RAGE-specific IgG block Ab transport at the BBB and the resulting reductions in CBF in wild-type and Tg2576 mice. Treatment of PD-hAbPP mice with sRAGE reduces amyloid load and Ab levels in the brain. Data suggest that peripheral non-immune scavenging agents such as sRAGE efficiently shift Ab exchanges across the BBB, favoring egress of peptide from brain. Flux calculations indicate that at pathophysiological Ab plasma levels in Tg2576 mice or PD-hAbPP mice treated with an antibody or sRAGE, the Ab plasma pool can still rapidly replenish Ab brain levels at remarkably high rates close to 0.15 micromoles/kg brain interstitial fluid per day. Thus, neutralizing the peripheral pool of Ab and blocking its transport across the BBB should lower the risk of generalized neuroinflammation and of compromise of the blood flow during Ab-lowering therapeutic interventions associated with increases in circulating free Ab.
Ab Transport-Clearance Hypothesis
Increases in Ab production can explain a small percentage of early-onset cases of familial AD in those people who carry inherited mutations in the AbPP gene that flank the Ab coding region (i.e., Swedish mutation) or the presenilin 1 or 2 genes. (27) Increases in production have not, however, been found in sporadic AD, or in familial AD/CAA where people inherited mutations inside the Ab coding region (e.g., Dutch, Iowa); an exception here is the Flemish mutation. Consequently, we could regard b-amyloidosis in sporadic and even some familial AD as a "storage" disease caused by inefficient clearance of a peptide that is normally produced in the CNS. (28,36,37) In general, any mismatch between Ab production, transport of circulating Ab into the CNS and clearance-whether resulting from increased production, increased transport of blood-borne peptide or inadequate clearance-may result in Ab accumulation in the CNS, and the two plausible hypotheses for its clearance from the brain are metabolism (41,42,89) and transport out. (36,37) Here we will discuss Ab transport, and why our current understanding of this process lends support for the therapeutic strategy of the "peripheral sink."
Transport of Ab in the CNS: Part Drifting, Part Shipping
Nonspecific bulk flow of brain interstitial fluid seems to be responsible for about 10-15 percent of Ab clearance from normal brain. (29) The blood-brain-barrier (BBB) normally prohibits free exchange of polar solutes, such as Ab, between brain and blood or blood and brain, mostly because of the presence of tight junctions between brain endothelial cells that form a continuous monolayer. Therefore, carrier-mediated or receptor-mediated transport system(s) for Ab must exist at the BBB to remove Ab from the CNS and inject it into circulation shortly after its physiological production (37), or to shuttle circulating Ab into the CNS. In 1993 we suggested that carrier and/or receptor-mediated transport across the BBB regulates brain Ab (34), and since then numerous reports from different groups have verified this hypothesis. (1-5,8,9,11,16-23,25,26,29,34-37)
1. Brain Export
Recent studies in PDAbPP mice have demonstrated that a single intravenous injection of the m266 monoclonal anti-Ab antibody promotes a rapid outflow of Ab from the CNS into plasma, increasing plasma Ab from baseline levels of 200 pg/ml to 5-10 ng/ml within 24 hours. (3) Given the similarity in plasma and CSF levels of Ab between humans and PDAbPP mice (6), DeMattos et al. suggested that Ab efflux measurements from brain to plasma after challenge with an anti-Ab antibody may be useful for quantifying brain amyloid burden in patients at risk for Alzheimer's, or diagnosed with the disease (see ARF related news story). As plaques develop in primate models of b-amyloidosis and in transgenic mice, soluble Ab from brain and plasma settles onto amyloid deposits in the CNS and around blood vessels and, consequently, the transport equilibrium for Ab between the CNS and plasma shifts (1,3-5,18). Using a squirrel monkey model of cerebral amyloid angiopathy (CAA), our lab recently confirmed that Ab is rapidly eliminated from brain into plasma across the BBB, and we noticed an age-dependent decline in this Ab clearance via the BBB that correlates with increases in amyloid deposition and Ab cerebrovascular immunoreactivity. (1,18)
How Does Ab Get Out? Ask LRP-1
LRP-1 is a large, multifunctional scavenger and signaling receptor belonging to the LDL receptor family. (10) LRP-1 was first discovered as a key endocytic receptor for the transport and metabolism of cholesterol and ApoE-containing lipoproteins. Its 515 kDa heavy chain contains four ligand-binding domains (clusters I-IV) that bind numerous structurally unrelated ligands, including ApoE, a-macroglobulin, tissue plasminogen activator, plasminogen activator inhibitor-1, AbPP, factor VIII, and lactoferrin. The 85 kDa light chain of LRP-1 contains a transmembrane domain and a cytoplasmic tail. The latter can be phosphorylated on serine, which has been linked to enhanced endocytosis, or on tyrosine; see review. (10)
We recently showed that LRP-1 functions as a clearance receptor for Ab at the BBB. (29) The LRP-1-mediated crossing (or transcytosis) of Ab begins at the brain side of the endothelium and is, therefore, directly responsible for eliminating Ab from the brain's interstitial fluid into blood (Fig. 1). It is not understood what the exact molecular mechanisms are that regulate interactions of Ab?with LRP-1 at the BBB, but we do know that the LRP-1 ligands ApoE and a-macroglobulin can influence Ab clearance.
The expression of brain endothelial LRP-1 appears to go down during normal aging in rodents, nonhuman primates, and in AD patients associated with positive staining of vessels for Ab40 and Ab42. (29) There is a genetic association between LRP-1 and the development of AD, but the biochemical mechanisms by which LRP-1 could affect the onset of the disease remain unknown. (10) Our most recent in vitro surface plasmon resonance studies indicate that Ab40 and Ab42, as well as Dutch and Dutch/Iowa mutants of Ab40, are direct ligands for sLRP-1 clusters II and IV; moreover, all Ab peptides bind directly to the abluminal site of the BBB via LRP-1 with high affinity. (38)
2. Brain Import
The autosomal dominant mutations that cause early-onset AD all increase Ab42 in plasma and brain. A late-onset AD locus on chromosome 10 acts to increase plasma Ab. The few studies that have analyzed plasma Ab levels in AD patients vs. age-matched controls suggest either no change or increased levels in AD, and/or an increased risk for AD in cognitively normal elderly individuals with high levels of plasma Ab as reviewed. (6)
Tg2576 mice overexpressing AbPP develop high plasma levels of Ab40 and 42 (4 nM and 0.5 nM, respectively), between three and nine months of age. (14) PDAbPP mice have significantly lower baseline values of plasma Ab (~200 pg/ml), but these levels shoot up 40-fold after a single intravenous injection of anti-Ab antibody (3) or sRAGE. (33). Ab circulating at such pathophysiological concentrations can be rapidly transported back into the CNS. (11,15,20,34) Therefore, trapping Ab in plasma is critical to reducing Ab levels in the CNS and to shift the plasma-CNS equilibrium of Ab, at least in these models.
Agents that bind Ab in plasma but do not themselves penetrate the BBB may promote the outflow of a rapidly mobilized, soluble pool of Ab, acting as a peripheral "sink." If such agents do enter the CNS, they may bind soluble brain Ab. This would promote resolubilization of previously aggregated Ab as the brain equilibrium between soluble and aggregated Ab shifts towards the soluble side, and this in turn should result in Ab elimination from the CNS provided the clearance systems are intact.
How Does Ab Get In? Ask RAGE
RAGE is a receptor in the immunoglobulin superfamily. In addition to Ab, it binds a broad repertoire of ligands, including products of nonenzymatic glycoxidation (AGE), the S100/calgranulin family of proinflammatory cytokine-like mediators, and the high-mobility group 1 DNA-binding protein amphoterin. (31) RAGE biology is largely dictated by its ligands in that mature animals show little RAGE expression in most tissues until deposition of ligands triggers expression. When pathogenic Ab species accumulate in AD (32) or transgenic models of b-amyloidosis, RAGE expression increases in affected cerebral vessels, neurons, or microglia. In contrast to the ligand-mediated receptor downregulation observed with LDL receptors in a lipoprotein-rich environment (10), or LRP-1 in an Ab-rich environment (29), RAGE is upregulated by its ligands. This mechanism could exacerbate cellular dysfunction.
RAGE binds soluble Ab in the nanomolar range, and then mediates pathophysiologic cellular responses. (31) RAGE is upregulated in the AD brain vasculature (32) and it regulates binding and transport of Ab in a human model of the BBB. (16) In light of these findings, we have recently shown that, in vivo, Ab binding to RAGE at the brain endothelium may increase transport of circulating Ab into the CNS. We also think RAGE is involved in diminishing cerebral blood flow, accompanying amyloid angiopathy, and proinflammatory events as observed in AD brain. In support of this hypothesis, we have found that RAGE mediates transport of physiological and pathophysiological concentrations of plasma Ab across the BBB into the brain, and that the latter leads to expression of proinflammatory cytokines in neurovascular cells and elaboration of endothelin-1, causing decreased cerebral blood flow. (15,15a)
Finally, there is also LRP-2 at the BBB. It may import plasma Ab complexed with apolipoprotein J (ApoJ). (35) Yet LRP-2 is normally saturated by high levels of plasma ApoJ , which precludes significant influx of Ab into the CNS. This leaves RAGE as the major Ab influx receptor at the BBB.
Where does all this leave us? Peripheral Ab-binding agents may well promote clearance of brain-derived Ab, thereby reducing Ab levels and amyloid load in the CNS of different AbPP-overexpressing mice. In the current online Nature Medicine, we have shown that sRAGE completely prevents transport of circulating Ab into the CNS, and since it does not penetrate the CNS in appreciable amounts it may act as a sink, favoring egress of Ab from brain. (15,15a) Moreover treatment with sRAGE improves the blood flow in Tg2576 mice, reduces neuroinflammation caused by pathophysiological levels of plasma Ab, and reduces amyloid load and Ab40/42 levels in PD-hAbPP mice. (15a,33) Serum amyloid P (SAP) component can be removed from human amyloid deposits in peripheral tissues by drugs that are competitive inhibitors of SAP and may enable its rapid clearance (24) (see ARF related news story). It has been also suggested that insulin-like growth factor I may induce clearance of brain Ab, probably by enhancing the import into the CNS of Ab-carrier proteins such as albumin and transthyretin. (2) This therapeutic approach is alive and well, and we will discuss which gaps we must still fill in our basic understanding of Ab transport, which models are best suited to test the therapeutic approach, and whether current experimental compounds are promising.
Thumbnail Summary of Transport-Clearance Hypothesis
Out of the Brain: LRP-1 mediates rapid Ab transcytosis across the BBB. (29) In parallel, the interstitial fluid bulk flow into the CSF slowly removes soluble Ab.
Into the Brain: RAGE mediates influx of free, circulating Ab across the BBB into the CNS. (11,15,16)
Ab-sequestering agents such as sRAGE (15,33), anti-Ab IgG (3,30), gelsolin and/or GM1 (22), or sLRP-1 clusters II and IV (38) can mop up Ab in plasma, reducing its influx across the BBB. Eliminating contributions of the circulating pool of Ab to its central pool may promote Ab's flow from brain into blood; this could be particularly important in cases of sporadic AD where the efflux transport systems are defective, such as down-regulated LRP-1. (29)
We suggest these questions for discussion:
- What should the next experiments be to advance this potential therapeutic approach?
- Do receptor-mediated transport mechanisms respond in the same way to peripheral sequestration as passive flow would, i.e., will CNS levels go down if Ab is removed from the peripheral pool?
- Is serum Ab truly out of the picture once it is bound? Could transport systems "sense" that bound serum Ab is increasing, and reduce Ab efflux in response? If so, peripheral sequestration could lead to an unintended increase of CNS Ab. Have animal models ruled this out?
- Do peripheral Ab-antibody complexes get degraded faster or slower than Ab alone? Could they pile up and cause problems?
- The m266 work is a promising approach. How far along is it?
- Are there ways of stimulating peripheral degradation of Ab in the liver and the kidney?
- Does the importance of Ab clearance and peripheral degradation make AD a systemic disease?
- Is it desirable to solubilize aggregated Ab? Might soluble intermediates be toxic?
1. Bading JR, Yamada S, Mackic JB, Kirkman L, Miller C, Calero M, Ghiso J, Frangione B, Zlokovic BV. Brain clearance of Alzheimer's amyloid-ß40 in the squirrel monkey: a SPECT study in a primate model of cerebral amyloid angiopathy. J Drug Targeting. 2002 10;4:359-368. Abstract
2. Carro E, Trejo JL, Gomez-Isla T, LeRoith D, Torres-Aleman I. Serum insulin-like growth factor I regulates brain amyloid-beta levels. Nature Med. 2002;8:1390-1397. Abstract
3. DeMattos RB, Bales KR, Cummins DJ, Paul SM, Holtzman DM. Brain to plasma amyloid-beta: a measure of brain amyloid burden in a mouse model of Alzheimer's disease. Science. 2002;295:2264-2267. Abstract
4. DeMattos RB, Bales KR, Cummins DJ, Dodart JC, Paul SM, Holtzman DM. Peripheral anti-Ab antibody alters CNS and plasma Aß clearance and decreases brain Ab burden in a mouse model of
lzheimer's disease. Proc Natl Acad Sci USA. 2001;98:8850-8855. Abstract
5. DeMattos RB, Bales KR, Parsadanian M, O'Dell MA, Foss EM, Paul SM, Holtzman DM. Plaque-associated disruption of CSF and plasma amyloid-beta equilibrium in a mouse model of Alzheimer's disease. J Neurochem. 2002;81:229-236. Abstract
6. DeMattos RB, Bales KR, Paul SM, Holtzman DM. Potential role of endogenous and exogenous Ab binding molecules in Ab clearance and metabolism. Ab Metabolism in Alzheimer's Disease. (Ed. T. Saido). Landes Bioscience. 2003;127-139.
7. Ertekin-Taner N, Graff-Radford N, Younkin LH, et al. Linkage of plasma Ab42 to a quantitative locus on chromosome 10 in late-onset Alzheimer's disease pedigrees. Science. 2000;290:2303-2304. Abstract
8. Ghersi-Egea JF, Gorevic PD, Ghiso J, Frangione B, Patlak CS, Fenstermacher JD. Fate of cerebrospinal fluid-borne amyloid b-peptide: rapid clearance into blood and appreciable accumulation by cerebral arteries. J Neurochem. 1996;67:880-83. Abstract
9. Ghilardi JR, Catton M, Stimson ER, Rogers S, Walker LC, Maggio JE, Mantyh PW. Intra arterial infusion of [125I]Ab1 40 labels amyloid deposits in the aged primate brain in vivo. Neuroreport. 1996;7:2607-2611. Abstract
10. Herz J, Strickland DK. LRP: a multifunctional scavenger and signaling receptor. J Clin Invest. 2001;108:779-784. Abstract
11. Hogg E, Kumar S, Holtzman J, et al. A model of brain arterial infusion in mice for the measurements of cerebrovascular functions: applications to amyloid b1-40 peptide transport in apolipoprotein E knockout mice. Soc Neurosci Abst. 2000;26:342.
12. Iwata N, Tsubuki S, Takaki Y, Watanabe K, Sekiguchi M, Hosoki E, Kawashima-Morishima M, Lee HJ, Hama E, Sekine-Aizawa Y, Saido TC. Y. Identification of the major Ab1-42-degrading catabolic pathway in brain parenchyma: suppression leads to biochemical and pathological deposition. Nature Med. 2000;6:143-150. Abstract
13. Iwata N, Tsubuki S, Takaki Y, Shirotani K, Lu B, Gerard NP, Gerard C, Hama E, Lee HJ, Saido TC. Metabolic regulation of brain Ab by neprilysin. Science. 2001;292:1550-1552. Abstract
14. Kawarabayashi T, Younkin LH, Saido TC, Shoji M, Ashe KH, Younkin SG. Age-dependent changes in brain, CSF, and plasma amyloid b protein in the Tg2576 transgenic mouse model of Alzheimer's Disease. J Neuroscience. 2001;21:372-381. Abstract
15. Kumar R, Miao W, Ghiso J, et al. RAGE at the blood-brain barrier mediates neurovascular dysfunction caused by amyloid-b1-40 peptide. Soc Neuorsci Abst. 2000;26:741.
15A. Deane R, Yan SD, Kumar RS, LaRue B, Jovanovic S, Hogg E, Welch D, Maness L, Lin C, Yu J, Zhu H, Ghiso J, Frangione B, Stern A, Schmidt AM, Armstrong D, Arnold B, Liliensiek B, Nawroth P, Hofman F, Kindy M, Stern D, Zlokovic BV. RAGE mediates amyloid-b peptide transport across the blood-brain barrier and accumulation in brain. Nature Medicine 2003, 9(7) in press. Advanced Online Publication June 15, 2003.
16. Mackic JB, Stins M, McComb JG, Calero M, Ghiso J, Kim KS, Yan SD, Stern D, Schmidt AM, Frangione B, Zlokovic BV. Human blood-brain barrier receptors for Alzheimer's amyloid-b. J Clin Invest. 1998;102:734-743. Abstract
17. Mackic JB, Weiss MH, Miao W, Kirkman E, Ghiso J, Calero M, Bading J, Frangione B, Zlokovic BV. Cerebrovascular accumulation and increased blood-brain barrier permeability to circulating Alzheimer's amyloid b-peptide in aged squirrel monkey with cerebral amyloid angiopathy. J. Neurochem. 1998;70:210-215. Abstract
18. Mackic JB, Bading J, Ghiso J, Walker L, Wisniewski T, Frangione B, Zlokovic BV. Differential cerebrovascular sequestration and enhanced blood-brain barrier permeability to circulating Alzheimer's amyloid-beta peptide in aged Rhesus vs. aged Squirrel monkey. Gen Pharmacology Vasc Biol. 2002;18:303-313.
19. Maness LM, Banks WA, Podlisny MB, Selkoe DJ, Kastin AJ. Passage of human amyloid-b protein 1-40 across the murine blood-brain barrier. Life Sci. 1994;55:1643-1650. Abstract
20. Martel CL, Mackic JB, McComb JG, Ghiso J, Zlokovic BV. Blood-brain barrier uptake of the 40 and 42 amino acid sequences of circulating Alzheimer's amyloid-b in guinea pigs. Neurosci Lett. 1996;206:157-160. Abstract
21. Martel CL, Mackic JB, Matsubara E, Governale S, Miguel C, Miao W, McComb JG, Frangione B, Ghiso J, Zlokovic BV. Isoform-specific effects of apolipoproteins E2, E3, E4 on cerebral capillary sequestration and blood-brain barrier transport of circulating Alzheimer's amyloid b. J Neurochem. 1997;69:1995-2004. Abstract
22. Matsuoka Y, Saito M, LaFrancois J, Saito M, Gaynor K, Olm V, Wang L, Casey E, Lu Y, Shiratori C, Lemere C, Duff K. Novel therapeutic approach for the treatment of Alzheimer's disease by peripheral administration of agents with an affinity to beta-amyloid. J Neurosci. 2003;23:29-33. Abstract (See also ARF related news story)
23. Monro OR, Mackic JB, Yamada S, Segal MB, Ghiso J, Maurer C, Calero M, Frangione B, Zlokovic BV. Substitution at codon 22 reduces clearance of Alzheimer's amyloid-b peptide from the cerebrospinal fluid and prevents its transport from the central nervous system into blood. Neurobiol Aging. 2002;23:405-412. Abstract
24. Pepys MB, Herbert J, Hutchinson GA, et al. Targeted pharmacological deletion of serum amyloid P component for treatment of human amyloidosis. Nature. 2002;417:254-259. Abstract
25. Poduslo JF, Curran GL, Haggard JJ, Biere AL, Selkoe DJ. Permeability and residual plasma volume of human, Dutch variant, and rat amyloid b-protein 1-40 at the blood-brain barrier. Neurobiol Dis. 1997;4:27-34. Abstract
26. Poduslo JF, Curran GL. Amyloid-b peptide as a vaccine for Alzheimer's disease involves receptor-mediated transport at the blood-brain barrier. Neuroreport. 2001;12:3197-3200. Abstract
27. Selkoe DJ. The cell biology of beta-amyloid precursor protein and presenilin in Alzheimer's disease. Trends Cell Biol. 1998;8:447-453. Abstract
28. Selkoe DJ. Clearing the brain's amyloid cobwebs. Neuron. 2001;32:177-180. Abstract
29. Shibata M, Yamada S, Kumar SR, Calero M, Bading J, Frangione B, Holtzman DM, Miller CA, Strickland DK, Ghiso J, Zlokovic BV. Clearance of Alzheimer's amyloid-b1- 40 peptide from brain by LDL receptor-related protein-1 at the blood-brain barrier. J Clin Invest. 2000;106:1489-1499. Abstract
30. Sigurdsson EM, Scholtzova H, Mehta PD, et al. Immunization with a nontoxic/nonfibrillar amyloid-beta homologous peptide reduces Alzheimer's disease-associated pathology in transgenic mice. Am J Pathol. 2001;159:439-447. Abstract
31. Stern D, Yan SD, Yan SF, et al. Receptor for advanced glycation endproducts: a multiligand receptor magnifying cell stress in diverse pathologic settings. Adv Drug Del Rev. 2002;54:1615-1625. Abstract
32. Yan SD, Chen X, Fu J, Chen M, Zhu H, Roher A, Slattery T, Zhao L, Nagashima M, Morser J, Migheli A, Nawroth P, Stern D, Schmidt AM. RAGE and amyloid-beta peptide neurotoxicity in Alzheimer's disease. Nature. 1996;382:685-691. Abstract
33. Yu J, Zhu H, Pettigrew LC, et al. Infusion of soluble RAGE inhibits Ab amyloid deposition in AbPP transgenic mice. Soc Neurosci Abstr. 2001;27:856.
34. Zlokovic BV, Ghiso J, Mackic JB, McComb JG, Weiss MH, Frangione B. Blood-brain barrier transport of circulating Alzheimer's amyloid-beta. Biochem Biophys Res Commun. 1993;197:1034-40. Abstract
35. Zlokovic BV, Martel CL, Matsubara E, McComb JG, Zheng G, McCluskey RT, Frangione B, Ghiso J. Glycoprotein 330/megalin: probable role in receptor-mediated transport of apolipoprotein J alone and in a complex with Alzheimer's disease amyloid-b ?at the blood-brain and blood-cerebrospinal fluid barriers. Proc Natl Acad Sci USA. 1996;93:4229-4234. Abstract
36. Zlokovic BV, Yamada S, Holtzman D, Ghiso J, Frangione B. Clearance of amyloid-beta peptide from brain: transport or metabolism? Nature Med. 2000;6:718-719. Abstract
37. Zlokovic BV, Frangione B. Transport-clearance hypothesis for Alzheimer's disease and potential therapeutic implications. Ab Metabolism in Alzheimer's Disease. (Ed. T. Saido). Landes Bioscience. 2003;114-122.
38. Zlokovic BV, Wu Z, Barclay DR, Lenting PJ, Yan S, Deane R, Pinkert CA. LRP-1 binds free Ab and promotes its clearance across the blood-brain barrier in wild type and Tie-2 LRP-1 Tg mice. Soc. Neurosci. 2003 (in press).
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