Human apoE isoforms differentially regulate brain amyloid-β peptide clearance

Science Translational Medicine

Volumn 3, Issue 89, 2011, Pages

  Castellano, Joseph M ^a   Kim, Jungsu ^a   Stewart, Floy R ^a   Jiang, Hong ^a   DeMattos, Ronald B ^b   Patterson, Bruce W ^a   Fagan, Anne M ^a   Morris, John C ^a   Mawuenyega, Kwasi G ^a   Cruchaga, Carlos ^a   Goate, Alison M ^a   Bales, Kelly R ^c   Paul, Steven M ^d   Bateman, Randall J ^a   Holtzman, David M ^a  

^a WASHINGTON UNIVERSITY SCHOOL OF MEDICINE   (United States)

^b ELI LILLY AND COMPANY   (United States)

^c PFIZER INC   (United States)

^d WEILL CORNELL MEDICAL COLLEGE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

AMYLOID BETA PROTEIN; AMYLOID BETA PROTEIN[1-40]; AMYLOID BETA PROTEIN[1-42]; AMYLOID PRECURSOR PROTEIN; APOLIPOPROTEIN E; APOLIPOPROTEIN E2; APOLIPOPROTEIN E3; APOLIPOPROTEIN E4; BIOLOGICAL MARKER; MOLECULAR MARKER; STABLE ISOTOPE; ISOPROTEIN;

ADULT; AGED; ALLELE; ALZHEIMER DISEASE; AMYLOID PLAQUE; AMYLOID PRECURSOR PROTEIN GENE; AMYLOIDOSIS; ANIMAL EXPERIMENT; ANIMAL MODEL; ANIMAL TISSUE; APOLIPOPROTEIN E GENE; APOLIPOPROTEIN E2 GENE; APOLIPOPROTEIN E3 GENE; APOLIPOPROTEIN E4 GENE; ARTICLE; BIOACCUMULATION; CEREBROSPINAL FLUID LEVEL; CONTROLLED STUDY; DISEASE MARKER; FEMALE; GENE; GENE OVEREXPRESSION; GENETIC RISK; GENOTYPE; HUMAN; IN VIVO STUDY; ISOTOPE LABELING; MALE; MICRODIALYSIS; MOUSE; NEUROIMAGING; NONHUMAN; NORMAL HUMAN; PRIORITY JOURNAL; PROTEIN SYNTHESIS REGULATION; RISK FACTOR; ANIMAL; BRAIN; C57BL MOUSE; CEREBROSPINAL FLUID; GENETICS; METABOLISM; MIDDLE AGED; PATHOLOGY; TRANSGENIC MOUSE;

ADULT; AGED; ALLELES; ALZHEIMER DISEASE; AMYLOID BETA-PEPTIDES; ANIMALS; APOLIPOPROTEIN E4; BIOLOGICAL MARKERS; BRAIN; FEMALE; GENOTYPE; HUMANS; MALE; MICE; MICE, INBRED C57BL; MICE, TRANSGENIC; MICRODIALYSIS; MIDDLE AGED; PROTEIN ISOFORMS;

EID: 79959772357     PISSN: 19466234     EISSN: 19466242     Source Type: Journal    
DOI: 10.1126/scitranslmed.3002156     Document Type: Article

References (66)

1. J. Hardy, D. J. Selkoe, The amyloid hypothesis of Alzheimer's disease: Progress and problems on the road to therapeutics. Science 297, 353-356 (2002).
2. E. H. Corder, A. M. Saunders, W. J. Strittmatter, D. E. Schmechel, P. C. Gaskell, G. W. Small, A. D. Roses, J. L. Haines, M. A. Pericak-Vance, Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families. Science 261, 921-923 (1993).
3. A. M. Saunders, W. J. Strittmatter, D. Schmechel, P. H. George-Hyslop, M. A. Pericak-Vance, S. H. Joo, B. L. Rosi, J. F. Gusella, D. R. Crapper-MacLachlan, M. J. Alberts, C. Hulette, B. Crain, D. Goldgaber, A. D. Roses, Association of apolipoprotein E allele e4 with late-onset familial and sporadic Alzheimer's disease. Neurology 43, 1467-1472 (1993).
4. E. H. Corder, A. M. Saunders, N. J. Risch, W. J. Strittmatter, D. E. Schmechel, P. C. Gaskell Jr., J. B. Rimmler, P. A. Locke, P. M. Conneally, K. E. Schmader, G. W. Small, A. D. Roses, J. L. Haines, M. A. Pericak-Vance, Protective effect of apolipoprotein E type 2 allele for late onset Alzheimer disease. Nat. Genet. 7, 180-184 (1994).
5. P. B. Verghese, J. M. Castellano, D. M. Holtzman, Apolipoprotein E in Alzheimer's disease and other neurological disorders. Lancet Neurol. 10, 241-252 (2011).
6. E.M. Reiman, K. Chen, X. Liu, D. Bandy, M. Yu, W. Lee, N. Ayutyanont, J. Keppler, S. A. Reeder, J. B. Langbaum, G. E. Alexander, W. E. Klunk, C. A. Mathis, J. C. Price, H. J. Aizenstein, S. T. DeKosky, R. J. Caselli, Fibrillar amyloid-b burden in cognitively normal people at 3 levels of genetic risk for Alzheimer's disease. Proc. Natl. Acad. Sci. U.S.A. 106, 6820-6825 (2009).
7. J. C. Morris, C. M. Roe, C. Xiong, A. M. Fagan, A. M. Goate, D. M. Holtzman, M. A. Mintun, APOE predicts amyloid-β but not tau Alzheimer pathology in cognitively normal aging. Ann. Neurol. 67, 122-131 (2010).
8. D. E. Schmechel, A. M. Saunders, W. J. Strittmatter, B. J. Crain, C. M. Hulette, S. H. Joo, M. A. Pericak-Vance, D. Goldgaber, A. D. Roses, Increased amyloid β-peptide deposition in cerebral cortex as a consequence of apolipoprotein E genotype in late-onset Alzheimer disease. Proc. Natl. Acad. Sci. U.S.A. 90, 9649-9653 (1993).
9. P. Tiraboschi, L. A. Hansen, E. Masliah, M. Alford, L. J. Thal, J. Corey-Bloom, Impact of APOE genotype on neuropathologic and neurochemical markers of Alzheimer disease. Neurology 62, 1977-1983 (2004).
10. D. M. Holtzman, K. R. Bales, T. Tenkova, A. M. Fagan, M. Parsadanian, L. J. Sartorius, B. Mackey, J. Olney, D. McKeel, D. Wozniak, S. M. Paul, Apolipoprotein E isoform-dependent amyloid deposition and neuritic degeneration in a mouse model of Alzheimer's disease. Proc. Natl. Acad. Sci. U.S.A. 97, 2892-2897 (2000).
11. A. M. Fagan, M. Watson, M. Parsadanian, K. R. Bales, S. M. Paul, D. M. Holtzman, Human and murine ApoE markedly alters Aβ metabolism before and after plaque formation in a mouse model of Alzheimer's disease. Neurobiol. Dis. 9, 305-318 (2002).
12. J. C. Dodart, R. A. Marr, M. Koistinaho, B. M. Gregersen, S. Malkani, I. M. Verma, S. M. Paul, Gene delivery of human apolipoprotein E alters brain Aβ burden in a mouse model of Alzheimer's disease. Proc. Natl. Acad. Sci. U.S.A. 102, 1211-1216 (2005).
13. J. D. Fryer, K. Simmons, M. Parsadanian, K. R. Bales, S. M. Paul, P. M. Sullivan, D. M. Holtzman, Human apolipoprotein E4 alters the amyloid-β 40:42 ratio and promotes the formation of cerebral amyloid angiopathy in an amyloid precursor protein transgenic model. J. Neurosci. 25, 2803-2810 (2005).
14. K. R. Bales, F. Liu, S. Wu, S. Lin, D. Koger, C. DeLong, J. C. Hansen, P. M. Sullivan, S. M. Paul, Human APOE isoform-dependent effects on brain β-amyloid levels in PDAPP transgenic mice. J. Neurosci. 29, 6771-6779 (2009).
15. C. C. Rowe, S. Ng, U. Ackermann, S. J. Gong, K. Pike, G. Savage, T. F. Cowie, K. L. Dickinson, P. Maruff, D. Darby, C. Smith, M. Woodward, J. Merory, H. Tochon-Danguy, G. O'Keefe, W. E. Klunk, C. A. Mathis, J. C. Price, C. L. Masters, V. L. Villemagne, Imaging β-amyloid burden in aging and dementia. Neurology 68, 1718-1725 (2007).
16. C. R. Jack Jr., V. J. Lowe, M. L. Senjem, S. D. Weigand, B. J. Kemp, M. M. Shiung, D. S. Knopman, B. F. Boeve, W. E. Klunk, C. A. Mathis, R. C. Petersen, 11C PiB and structural MRI provide complementary information in imaging of Alzheimer's disease and amnestic mild cognitive impairment. Brain 131, 665-680 (2008).
17. L. M. Shaw, H. Vanderstichele, M. Knapik-Czajka, C. M. Clark, P. S. Aisen, R. C. Petersen, K. Blennow, H. Soares, A. Simon, P. Lewczuk, R. Dean, E. Siemers, W. Potter, V. M. Lee, J. Q. Trojanowski; Alzheimer's Disease Neuroimaging Initiative, Cerebrospinal fluid biomarker signature in Alzheimer's disease neuroimaging initiative subjects. Ann. Neurol. 65, 403-413 (2009).
18. 42 in humans. Ann. Neurol. 59, 512-519 (2006).
19. 42 ratio as a prediction of cognitive decline in nondemented older adults. Arch. Neurol. 64, 343-349 (2007).
20. 1-42 and increased tau levels in cerebrospinal fluid of patients with Alzheimer disease. JAMA 289, 2094-2103 (2003).
21. C. M. Clark, S. Xie, J. Chittams, D. Ewbank, E. Peskind, D. Galasko, J. C. Morris, D. W. McKeel Jr., M. Farlow, S. L. Weitlauf, J. Quinn, J. Kaye, D. Knopman, H. Arai, R. S. Doody, C. DeCarli, S. Leight, V. M. Lee, J. Q. Trojanowski, Cerebrospinal fluid tau and β-amyloid: How well do these biomarkers reflect autopsy-confirmed dementia diagnoses? Arch. Neurol. 60, 1696-1702 (2003).
22. 42 correlates with brain atrophy in cognitively normal elderly. Ann. Neurol. 65, 176-183 (2009).
23. W. E. Klunk, H. Engler, A. Nordberg, Y. Wang, G. Blomqvist, D. P. Holt, M. Bergström, I. Savitcheva, G. F. Huang, S. Estrada, B. Ausén, M. L. Debnath, J. Barletta, J. C. Price, J. Sandell, B. J. Lopresti, A. Wall, P. Koivisto, G. Antoni, C. A. Mathis, B. Långström, Imaging brain amyloid in Alzheimer's disease with Pittsburgh Compound-B. Ann. Neurol. 55, 306-319 (2004).
24. M. D. Ikonomovic, W. E. Klunk, E. E. Abrahamson, C. A. Mathis, J. C. Price, N. D. Tsopelas, B. J. Lopresti, S. Ziolko, W. Bi, W. R. Paljug, M. L. Debnath, C. E. Hope, B. A. Isanski, R. L. Hamilton, S. T. DeKosky, Post-mortem correlates of in vivo PiB-PET amyloid imaging in a typical case of Alzheimer's disease. Brain 131, 1630-1645 (2008).
25. V. Leinonen, I. Alafuzoff, S. Aalto, T. Suotunen, S. Savolainen, K. Någren, T. Tapiola, T. Pirttilä, J. Rinne, J. E. Jää skeläinen, H. Soininen, J. O. Rinne, Assessment of β-amyloid in a frontal cortical brain biopsy specimen and by positron emission tomography with carbon 11-labeled Pittsburgh Compound B. Arch. Neurol. 65, 1304-1309 (2008).
26. T. Tapiola, I. Alafuzoff, S. K. Herukka, L. Parkkinen, P. Hartikainen, H. Soininen, T. Pirttilä, Cerebrospinal fluid β-amyloid 42 and tau proteins as biomarkers of Alzheimer-type pathologic changes in the brain. Arch. Neurol. 66, 382-389 (2009).
27. 11C]PIB in a nondemented population: Potential antecedent marker of Alzheimer disease. Neurology 67, 446-452 (2006).
28. K. R. Bales, T. Verina, R. C. Dodel, Y. Du, L. Altstiel, M. Bender, P. Hyslop, E. M. Johnstone, S. P. Little, D. J. Cummins, P. Piccardo, B. Ghetti, S. M. Paul, Lack of apolipoprotein E dramatically reduces amyloid β-peptide deposition. Nat. Genet. 17, 263-264 (1997).
29. J. R. Cirrito, P. C. May, M. A. O'Dell, J. W. Taylor, M. Parsadanian, J. W. Cramer, J. E. Audia, J. S. Nissen, K. R. Bales, S. M. Paul, R. B. DeMattos, D. M. Holtzman, In vivo assessment of brain interstitial fluid with microdialysis reveals plaque-associated changes in amyloid-β metabolism and half-life. J. Neurosci. 23, 8844-8853 (2003).
30. P. Yan, A. W. Bero, J. R. Cirrito, Q. Xiao, X. Hu, Y. Wang, E. Gonzales, D. M. Holtzman, J. M. Lee, Characterizing the appearance and growth of amyloid plaques in APP/PS1 mice. J. Neurosci. 29, 10706-10714 (2009).
31. J. Kim, J. M. Castellano, H. Jiang, J. M. Basak, M. Parsadanian, V. Pham, S. M. Mason, S. M. Paul, D. M. Holtzman, Overexpression of low-density lipoprotein receptor in the brain markedly inhibits amyloid deposition and increases extracellular Ab clearance. Neuron 64, 632-644 (2009).
32. R. B. DeMattos, J. R. Cirrito, M. Parsadanian, P. C. May, M. A. O'Dell, J. W. Taylor, J. A. Harmony, B. J. Aronow, K. R. Bales, S. M. Paul, D. M. Holtzman, ApoE and clusterin cooperatively suppress Ab levels and deposition: Evidence that ApoE regulates extracellular Aβ metabolism in vivo. Neuron 41, 193-202 (2004).
33. S. Menacherry, W. Hubert, J. B. Justice Jr., In vivo calibration of microdialysis probes for exogenous compounds. Anal. Chem. 64, 577-583 (1992).
34. E. Ronne-Engström, K. G. Cesarini, P. Enblad, G. Hesselager, N. Marklund, P. Nilsson, K. Salci, L. Persson, L. Hillered, Intracerebral microdialysis in neurointensive care: The use of urea as an endogenous reference compound. J. Neurosurg. 94, 397-402 (2001).
35. L. Hillered, P. M. Vespa, D. A. Hovda, Translational neurochemical research in acute human brain injury: The current status and potential future for cerebral microdialysis. J. Neurotrauma 22, 3-41 (2005).
36. D. L. Brody, S. Magnoni, K. E. Schwetye, M. L. Spinner, T. J. Esparza, N. Stocchetti, G. J. Zipfel, D. M. Holtzman, Amyloid-β dynamics correlate with neurological status in the injured human brain. Science 321, 1221-1224 (2008).
37. K. E. Schwetye, J. R. Cirrito, T. J. Esparza, C. L. Mac Donald, D. M. Holtzman, D. L. Brody, Traumatic brain injury reduces soluble extracellular amyloid-β in mice: A methodologically novel combined microdialysis- controlled cortical impact study. Neurobiol. Dis. 40, 555-564 (2010).
38. J. E. Kang, M. M. Lim, R. J. Bateman, J. J. Lee, L. P. Smyth, J. R. Cirrito, N. Fujiki, S. Nishino, D. M. Holtzman, Amyloid-β dynamics are regulated by orexin and the sleep-wake cycle. Science 326, 1005-1007 (2009).
39. J. Kim, J. M. Basak, D. M. Holtzman, The role of apolipoprotein E in Alzheimer's disease. Neuron 63, 287-303 (2009).
40. R. Craig-Schapiro, M. Kuhn, C. Xiong, E. H. Pickering, J. Liu, T. P. Misko, R. J. Perrin, K. R. Bales, H. Soares, A. M. Fagan, D. M. Holtzman, Multiplexed immunoassay panel identifies novel CSF biomarkers for Alzheimer's disease diagnosis and prognosis. PLoS One 6, e18850 (2011).
41. S. E. Wahrle, H. Jiang, M. Parsadanian, J. Kim, A. Li, A. Knoten, S. Jain, V. Hirsch-Reinshagen, C. L. Wellington, K. R. Bales, S. M. Paul, D. M. Holtzman, Overexpression of ABCA1 reduces amyloid deposition in the PDAPP mouse model of Alzheimer disease. J. Clin. Invest. 118, 671-682 (2008).
42. A. L. Biere, B. Ostaszewski, H. Zhao, S. Gillespie, S. G. Younkin, D. J. Selkoe, Co-expression of β-amyloid precursor protein (βAPP) and apolipoprotein E in cell culture: Analysis of βAPP processing. Neurobiol. Dis. 2, 177-187 (1995).
43. R. J. Bateman, L. Y. Munsell, J. C. Morris, R. Swarm, K. E. Yarasheski, D. M. Holtzman, Human amyloid-β synthesis and clearance rates as measured in cerebrospinal fluid in vivo. Nat. Med. 12, 856-861 (2006).
44. D. M. Holtzman, Alzheimer's disease: Moving towards a vaccine. Nature 454, 418-420 (2008).
45. R. J. Perrin, A. M. Fagan, D. M. Holtzman, Multimodal techniques for diagnosis and prognosis of Alzheimer's disease. Nature 461, 916-922 (2009).
46. A. D. Roses, Apolipoprotein E alleles as risk factors in Alzheimer's disease. Annu. Rev. Med. 47, 387-400 (1996).
47. 1-antichymotrypsin and apolipoprotein E promote assembly of Alzheimer β-protein into filaments. Nature 372, 92-94 (1994).
48. T. Wisniewski, E. M. Castano, A. Golabek, T. Vogel, B. Frangione, Acceleration of Alzheimer's fibril formation by apolipoprotein E in vitro. Am. J. Pathol. 145, 1030-1035 (1994).
49. U. Beffert, J. Poirier, ApoE associated with lipid has a reduced capacity to inhibit β-amyloid fibril formation. Neuroreport 9, 3321-3323 (1998).
50. S. J. Wood, W. Chan, R. Wetzel, Seeding of Aβ fibril formation is inhibited by all three isotypes of apolipoprotein E. Biochemistry 35, 12623-12628 (1996).
51. M. J. LaDu, M. T. Falduto, A. M. Manelli, C. A. Reardon, G. S. Getz, D. E. Frail, Isoform-specific binding of apolipoprotein E to β-amyloid. J. Biol. Chem. 269, 23403-23406 (1994).
52. D. S. Yang, J. D. Smith, Z. Zhou, S. E. Gandy, R. N. Martins, Characterization of the binding of amyloid-β peptide to cell culture-derived native apolipoprotein E2, E3, and E4 isoforms and to isoforms from human plasma. J. Neurochem. 68, 721-725 (1997).
53. S. Aleshkov, C. R. Abraham, V. I. Zannis, Interaction of nascent ApoE2, ApoE3, and ApoE4 isoforms expressed in mammalian cells with amyloid peptide β (1-40). Relevance to Alzheimer's disease. Biochemistry 36, 10571-10580 (1997).
54. T. Tokuda, M. Calero, E. Matsubara, R. Vidal, A. Kumar, B. Permanne, B. Zlokovic, J. D. Smith, M. J. Ladu, A. Rostagno, B. Frangione, J. Ghiso, Lipidation of apolipoprotein E influences its isoform-specific interaction with Alzheimer's amyloid β peptides. Biochem. J. 348, 359-365 (2000).
55. R. D. Bell, A. P. Sagare, A. E. Friedman, G. S. Bedi, D. M. Holtzman, R. Deane, B. V. Zlokovic, Transport pathways for clearance of human Alzheimer's amyloid β-peptide and apolipoproteins E and J in the mouse central nervous system. J. Cereb. Blood Flow Metab. 27, 909-918 (2007).
56. R. Deane, A. Sagare, K. Hamm, M. Parisi, S. Lane, M. B. Finn, D. M. Holtzman, B. V. Zlokovic, apoE isoform-specific disruption of amyloid β peptide clearance from mouse brain. J. Clin. Invest. 118, 4002-4013 (2008).
57. M. J. Sadowski, J. Pankiewicz, H. Scholtzova, P. D. Mehta, F. Prelli, D. Quartermain, T. Wisniewski, Blocking the apolipoprotein E/amyloid-β interaction as a potential therapeutic approach for Alzheimer's disease. Proc. Natl. Acad. Sci. U.S.A. 103, 18787-18792 (2006).
58. U. Beffert, N. Aumont, D. Dea, S. Lussier-Cacan, J. Davignon, J. Poirier, β-Amyloid peptides increase the binding and internalization of apolipoprotein E to hippocampal neurons. J. Neurochem. 70, 1458-1466 (1998).
59. D. S. Yang, D. H. Small, U. Seydel, J. D. Smith, J. Hallmayer, S. E. Gandy, R. N. Martins, Apolipoprotein E promotes the binding and uptake of β-amyloid into Chinese hamster ovary cells in an isoform-specific manner. Neuroscience 90, 1217-1226 (1999).
60. Q. Jiang, C. Y. Lee, S. Mandrekar, B. Wilkinson, P. Cramer, N. Zelcer, K. Mann, B. Lamb, T. M. Willson, J. L. Collins, J. C. Richardson, J. D. Smith, T. A. Comery, D. Riddell, D. M. Holtzman, P. Tontonoz, G. E. Landreth, ApoE promotes the proteolytic degradation of Aβ. Neuron 58, 681-693 (2008).
61. K. M. Korwek, J. H. Trotter, M. J. Ladu, P. M. Sullivan, E. J. Weeber, ApoE isoform-dependent changes in hippocampal synaptic function. Mol. Neurodegener. 4, 21 (2009).
62. J. D. Fryer, R. B. Demattos, L. M. McCormick, M. A. O'Dell, M. L. Spinner, K. R. Bales, S. M. Paul, P. M. Sullivan, M. Parsadanian, G. Bu, D. M. Holtzman, The low density lipoprotein receptor regulates the level of central nervous system human and murine apolipoprotein E but does not modify amyloid plaque pathology in PDAPP mice. J. Biol. Chem. 280, 25754-25759 (2005).
63. D. R. Riddell, H. Zhou, K. Atchison, H. K. Warwick, P. J. Atkinson, J. Jefferson, L. Xu, S. Aschmies, Y. Kirksey, Y. Hu, E. Wagner, A. Parratt, J. Xu, Z. Li, M. M. Zaleska, J. S. Jacobsen, M. N. Pangalos, P. H. Reinhart, Impact of apolipoprotein E (ApoE) polymorphism on brain ApoE levels. J. Neurosci. 28, 11445-11453 (2008).
64. D. M. Hatters, J. C. Voss, M. S. Budamagunta, Y. N. Newhouse, K. H. Weisgraber, Insight on the molecular envelope of lipid-bound apolipoprotein E from electron paramagnetic resonance spectroscopy. J. Mol. Biol. 386, 261-271 (2009).
65. K. G. Mawuenyega, W. Sigurdson, V. Ovod, L. Munsell, T. Kasten, J. C. Morris, K. E. Yarasheski, R. J. Bateman, Decreased clearance of CNS β-amyloid in Alzheimer's disease. Science 330, 1774 (2010).
66. 42. LY411,575 was a gift from P. C. May (Eli Lilly and Co.). Funding: This work was supported by NIH grants AG13956 (D.M.H.), NS034467 (D.M.H.), AG034004 (J.M.C.), and P30NS069329 (J.K.); the American Health Assistance Foundation (J.K. and D.M.H.); DK56341 (B.W.P., Nutrition Obesity Research Center); NIH grants AG05681 (J.C.M.), AG03991 (J.C.M.), AG026276 (J.C.M.), P30NS069329-01 (C.C.), and K23-AG03094601 (R.J.B.); NIH Neuroscience Blueprint Center Core Grant P30-NS057105; and Eli Lilly and Pfizer to Washington University (D.M.H.). Author contributions: J.M.C. wrote and edited the manuscript with critical evaluation/interpretation from co-authors. J.M.C., F.R.S., H.J., and D.M.H. performed/designed research in Figs. 1 to 6 and the Supplementary Material. A.M.F. and J.C.M. provided human subject data for Fig. 1 and Table 1. C.C., A.M.G., and K.R.B. provided human data for fig. S3A. J.K., J.M.C., K.G.M., and R.J.B. designed/performed experiments for Fig. 6. J.M.C., J.K., and D.M.H. analyzed the data. R.B.D. and S.M.P. contributed valuable reagents. Competing interests: D.M.H. and R.J.B. are scientific advisors to C2N Diagnostics, which uses the SILK methodology in human studies and are co-inventors on U.S. patent 7,892,845 "Methods for measuring the metabolism of neurally derived biomolecules in vivo." Washington University, with D.M.H. and R.J.B. as co-inventors, has also submitted the U.S. nonprovisional patent application "Methods for measuring the metabolism of CNS derived biomolecules in vivo," serial #12/267,974. D.M.H. is also on the scientific advisory boards of Satori, En Vivo. D.M.H., J.C.M., and A.M.G. consult for Pfizer and Bristol Myers Squibb. D.M.H. also consults for Innogenetics. J.C.M. also consults for AstraZeneca, Eisai, Janssen, Genentech, Eli Lilly, Merck, Novartis, Otsuka, and Schering Plough. A.M.G. also consults for AstraZeneca and Genentech. All other authors declare no competing interests.