Neuroprotective natural antibodies to assemblies of amyloidogenic peptides decrease with normal aging and advancing Alzheimer’s disease M. Britschgia, C. E. Olina, H. T. Johnsa, Y. Takeda-Uchimuraa, M. C. LeMieuxb, K. Rufibachc, J. Rajadasa, H. Zhanga, B. Tomookad, W. H. Robinsond,e, C. M. Clarkf, A. M. Fagang, D. R. Galaskoh, D. M. Holtzmang, M. Juteli, J. A. Kayej, C. A. Lemerek, J. Leszekl,G.Lim,n, E. R. Peskindm,n, J. F. Quinnj, J. A. Yesavagea,e, J. A. Ghisoo, and T. Wyss-Coraya,e,1 Departments of aNeurology and Neurological Sciences and dMedicine, Stanford University School of Medicine, Stanford, CA 94305; bDepartment of Chemical Engineering, Stanford University, Stanford, CA 94305-5025; cInstitute of Social and Preventive Medicine, Biostatistics Unit, University of Zurich, CH-8001 Zurich, Switzerland; eGeriatric Research Education and Clinical Center, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304; fDepartment of Neurology, Alzheimer’s Disease Center, and Institute on Aging, University of Pennsylvania, Philadelphia, PA 19104; gDepartment of Neurology, Washington University, St. Louis, MO 63110; hDepartment of Neurosciences, University of California at San Diego, La Jolla, CA 92093; iDepartment of Internal Medicine and Allergology, Wroclaw Medical University, 50-417 Wroclaw, Poland; jLayton Aging and Alzheimer’s Disease Center, Oregon Health Sciences University, Portland, OR 97201; kBrigham and Women’s Hospital and Center for Neurologic Diseases, Harvard Medical School, Boston, MA 02115; lDepartment of Psychiatry, Wroclaw Medical University, 51-622 Wroclaw, Poland; mAlzheimer’s Disease Research Center, University of Washington, Seattle, WA 98108; nVeterans Affairs Puget Sound Health Care System, Seattle, WA 98108; and oDepartments of Pathology and Psychiatry, New York University Langone Medical Center, New York, NY 10016 Edited by L. L. Iversen, University of Oxford, Oxford, United Kingdom, and approved May 28, 2009 (received for review May 8, 2009) A number of distinct ␤-amyloid (A␤) variants or multimers have been antibodies correlated with attenuated cognitive decline (16). It has implicated in Alzheimer’s disease (AD), and antibodies recognizing also been suggested that antibodies recognizing different domains such peptides are in clinical trials. Humans have natural A␤-specific (12, 13, 17) or conformations (18, 19) of A␤ may have different antibodies, but their diversity, abundance, and function in the general efficacy in humans. population remain largely unknown. Here, we demonstrate with Interestingly, antibodies against A␤ occur naturally in blood and peptide microarrays the presence of natural antibodies against cerebrospinal fluid (CSF) in free form or in complex with A␤, both NEUROSCIENCE known toxic A␤ and amyloidogenic non-A␤ species in plasma sam- in AD patients and healthy individuals (20–26). The titers of these ples and cerebrospinal fluid of AD patients and healthy controls aged antibodies are low, and their origin and physiological or patholog- 21–89 years. Antibody reactivity was most prominent against oligo- ical role is unknown. Using crude methods A␤ antibody titers were meric assemblies of A␤ and pyroglutamate or oxidized residues, and found to be increased (22), decreased (23, 27, 28), or unchanged IgGs specific for oligomeric preparations of A␤1-42 in particular (21) in AD compared with healthy controls, but a large-scale declined with age and advancing AD. Most individuals showed analysis of A␤ antibody subspecificities or changes in antibody unexpected antibody reactivities against peptides unique to autoso- repertoire with age or in response to A␤ vaccination has not been mal dominant forms of dementia (mutant A␤, ABri, ADan) and IgGs described. Based on the presence of A␤ antibodies in normal isolated from plasma of AD patients or healthy controls protected plasma and the promise of therapeutic A␤ vaccines polyclonal i.v. primary neurons from A␤ toxicity. Aged vervets showed similar immune globulins (IVIg) are being tested in clinical trials for the patterns of plasma IgG antibodies against amyloid peptides, and after treatment of AD (25, 29). immunization with A␤ the monkeys developed high titers not only against A␤ peptides but also against ABri and ADan peptides. Our Results findings support the concept of conformation-specific, cross-reactive Plasma A␤ Antibodies Predominantly Recognize High Molecular Mass antibodies that may protect against amyloidogenic toxic peptides. If Assemblies in Oligomeric Preparations of A␤1-42. To characterize the a therapeutic benefit of A␤ antibodies can be confirmed in AD specificity of human plasma A␤ antibodies we isolated IgGs, and patients, stimulating the production of such neuroprotective anti- analyzed their reactivity to assemblies of A␤1-42 peptides on bodies or passively administering them to the elderly population may Western blots. Some IgG samples reacted distinctively with a provide a preventive measure toward AD. nonfibrillar A␤ assembly between 55 and 78 kDa, reminiscent of the previously described 56-kDa A␤*56 (30) but the main reactivity in lzheimer’s disease (AD) is the most common cause of demen- all samples was against entities Ͼ210 kDa (Fig. 1A). The lack of Atia, affecting an estimated 5.3 million individuals in the United binding to fibrillar A␤ preparations (Fig. 1A) indicates that most of States alone. Deposits of ␤-amyloid peptide (A␤) in extracellular the IgG antibodies are specific to smaller, potentially oligomeric plaques characterize the AD brain, but soluble oligomeric A␤ conformations. The A␤1-5 specific antibody 3D6 detected various species appear to be more neurotoxic than plaques and interfere assemblies in the oligomeric and fibrillar A␤ preparations (Fig. 1B). with synaptic function (reviewed in ref. 1). Notably, most A␤ Atomic force microscopy confirmed the presence of predominantly peptides isolated from AD brains are posttranslationally modified fibrillar structures in fibrillar preparations (Fig. 1C) or nonfibrillar and truncated (2–6), and some are proposed to be oxidized (7, 8) A␤ species in oligomeric preparations of A␤1-42 (Fig. 1D). To- or cross-linked at Tyr-10 (9). Although the pathogenic conse- quences of these modifications need to be resolved, most of them can stabilize A␤ assemblies, interfere with proteolytic degradation, Author contributions: M.B. and T.W.-C. designed research; M.B., C.E.O., H.T.J., Y.T.-U., ␤ M.C.L., J.R., H.Z., B.T., and J.A.G. performed research; J.R., W.H.R., C.M.C., A.M.F., D.R.G., and increase A toxicity in vitro (7, 8, 10). D.M.H., M.J., J.A.K., C.A.L., J.L., G.L., E.R.P., J.F.Q., J.A.Y., and J.A.G. contributed new One line of defense against toxic A␤ species could be neutralizing reagents/analytic tools; M.B., C.E.O., K.R., and J.A.G. analyzed data; and M.B. and T.W.-C. antibodies. Stimulating the production of A␤ antibodies by active wrote the paper. immunization with synthetic A␤ (11) or administering monoclonal The authors declare no conflict of interest. A␤ antibodies (12, 13) reduced amyloid pathology and inflamma- This article is a PNAS Direct Submission. tion and improved cognitive function in mouse models of AD (14). 1To whom correspondence should be addressed. E-mail: [email protected]. In patients with mild to moderate AD active immunization appears This article contains supporting information online at www.pnas.org/cgi/content/full/ to reduce plaque load (15), and in some patients production of A␤ 0904866106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904866106 PNAS ͉ July 21, 2009 ͉ vol. 106 ͉ no. 29 ͉ 12145–12150 Aβ1-40_1 500 IgG: mean of Aβ1-42 oligo ABBiotinylated human IgG 3D6 monoclonal antibody F C DLMH Probing Reprobing β Donor 1 Donor 2 Donor 3 Donor 4 A 1-40_2 400 NDC NDC AD AD 1st 2nd Donor 1 IgG fofo fo fo fo o o Aβ1-40_3 300 Donor 3 IgG β kDa A 1-42_1 Donor 4 IgG β 200 210 A 1-42_2 500 nm 1 µm 105 β 200 nm 200 nm A 1-42_4 reactivity (DFU) 100 78 cut here Relative antibody 55 45 >55 kDa Aβ1-42_5 34 Non-demented controls (NDC) AD patients 0 cut here β A 1-42_6 Mean of all non-aggregated Mean + standard deviation of all 0 0 0 0 17 >17 kDa β β β β 1:15 1:45 1:75 10 A 1-40 and A 1-42 in NDC non-aggregated A 1-40 and A 1-42 in NDC 1:1050 1:135 Aβ1-40 dimer Antibody titer 7 Aβ1-40 polymer Aβ1-40 (D7N) Tottori G I 500 IgM: mean of Aβ1-42 oligo 4 Aβ1-42 polymer Aβ1-40 (D23N) Iowa 1 β 400 A 1-42 oligo 3 Aβ1-40 (D23N) Iowa 2 Donor 1 IgM Aβ1-42 oligo 4 Aβ1-40 (A21G) Flemish 300 Donor 3 IgM Aβ1-42 oligo 5 Aβ1-40 (E22Q) Dutch 1 Donor 4 IgM Aβ1-40_1 200 Aβ1-42 CAPS 2 Aβ1-40 (E22Q) Dutch 2 E β A 1-40_2 reactivity (DFU) 100 Aβ1-42 CAPS 3 Aβ1-40 (E22Q) Dutch 3 Relative antibody Aβ1-40_3 Aβ1-42 CAPS 4 Aβ1-42 (E22Q) Dutch 0 Aβ1-42_1 0 Aβ1-42 CAPS 5 β A 1-42 (E22G) Arctic 1:15 1:450 1:750 Aβ1-42_2 1:1050 1:1350 Aβ1-42 CAPS 6 Aβ1-42 (E22K) Italian Antibody titer Aβ1-42_3 Aβ1-42 fibril 3 Aβ1-40 rodent J 104 *** Aβ1-40 oligo 1 Aβ1-42 fibril 4 Aβ1-42 rodent *** *** *** *** β β 103 A 1-40 oligo 2 Aβ10-20 A 1-55 Aβ1-42 oligo 1 β 102 A 11-42 ABri1(pE)-34 Aβ1-42 oligo 2 β 1 A 22-35 ABri1(pE)-34 (Ox) 10 Aβ1-42 CAPS 1 Aβ33-42 0 ABri3-34 reactivity (DFU) 10 Aβ1-40 fibril Relative antibody Aβ3(pE)-40 ABri22-34 –1 Aβ1-42 fibril 1 10 Aβ11(pE)-40 ABri24-34 1-42 Aβ1-42 fibril 2 1-40 11-42 β β Aβ3(pE)-42 ADan1(pE)-25 β Mean of A 3(pE)-40 3(pE)-42 Mean of 1-42 fibril 11(pE)-40 11(pE)-42 1-42 oligo β Aβ3(pE)-40 β β A 11(pE)-42 ADan1(pE)-34 β β β β and A A A A A A Aβ11(pE)-40 Aβ1-42 (Ox) ADan22-34 A β A Mean of β A 29-35(MetSox) ADan1(pE)-25 SS cyclized A 1-40 (D7N) Tottori