Neuroprotective Sirtuin ratio reversed by ApoE4 Veena Theendakaraa,1, Alexander Patenta,1, Clare A. Peters Libeua, Brittany Philpota, Sonia Floresa, Olivier Descampsa, Karen S. Poksaya, Qiang Zhanga, Gabriellee Cailinga, Matthew Hartb, Varghese Johna, Rammohan V. Raoa,2,3, and Dale E. Bredesena,c,2,3 aThe Buck Institute for Research on Aging, Novato, CA 94945; bDepartment of Molecular Medicine, University of Texas Health Science Center, San Antonio, TX 78245; and cDepartment of Neurology, University of California, San Francisco, CA 94143 Edited by Solomon H. Snyder, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved September 26, 2013 (received for review July 26, 2013) The canonical pathogenesis of Alzheimer’s disease links the ex- In contrast, cleavage at the α site produces the trophic peptide pression of apolipoprotein E e4 allele (ApoE) to amyloid precursor sAPPα and the inhibitor of APP γ-site cleavage, alpha-COOH- protein (APP) processing and Aβ peptide accumulation by a set of terminal fragment of APP (αCTF) (1, 6). The decision between mechanisms that is incompletely defined. The development of these two proteolytic pathways is governed at least in part by a simple system that focuses not on a single variable but on mul- ligand binding: interaction with the axon guidance and trophic tiple factors and pathways would be valuable both for dissecting factor netrin-1 increases α-site cleavage, whereas interaction with the underlying mechanisms and for identifying candidate thera- the antitrophin Aβ inhibits α-site cleavage and increases net pro- peutics. Here we show that, although both ApoE3 and ApoE4 duction of the four neurite-retractive peptides (1, 6). According associate with APP with nanomolar affinities, only ApoE4 signifi- to a recent study, the level of cerebrospinal fluid (CSF) sAPPα is cantly (i) reduces the ratio of soluble amyloid precursor protein significantly lower in AD patients possessing one or two ApoE4 alpha (sAPPα)toAβ;(ii) reduces Sirtuin T1 (SirT1) expression, alleles than in those not possessing the ApoE4 allele (7). There- resulting in markedly differing ratios of neuroprotective SirT1 to fore, it was of interest to determine whether ApoE isoforms im- neurotoxic SirT2; (iii) triggers Tau phosphorylation and APP phos- pact this trophic–antitrophic peptide balance differentially, and, if phorylation; and (iv) induces programmed cell death. We describe so, by what mechanism. a subset of drug candidates that interferes with the APP–ApoE Our data reveal differential effects of ApoE4 vs. ApoE3 on interaction and returns the parameters noted above to normal. APP interaction, signaling, and processing and are compatible NEUROSCIENCE Our data support the hypothesis that neuronal connectivity, as with the notion that the Alzheimer’s phenotype represents an reflected in the ratios of critical mediators such as sAPPα:Aβ, imbalance between the trophic and antitrophic signaling of APP, SirT1:SirT2, APP:phosphorylated (p)-APP, and Tau:p-Tau, is pro- reflected by the ratio of the four APP-derived neurite-retractive grammatically altered by ApoE4 and offer a simple system peptides to the two APP-derived trophic peptides. Our current for the identification of program mediators and therapeutic results show that ApoE4 interacts with high affinity with APP, candidates. shifting the processing balance in the antitrophic direction, de- creasing sAPPα secretion, and reducing sAPPα/Aβ and sAPPα/ lzheimer’s disease (AD) poses a health problem of pan- sAPPβ ratios in comparison with ApoE3. In addition, the pres- Ademic proportions. It is estimated that by the year 2030 ence of ApoE4 results in increased APP-Thr668 phosphorylation there will be greater than 60 million people worldwide with AD, (p-APP) and Tau phosphorylation (p-Tau). Some of these and $375 billion will be spent annually in the United States to ApoE4-mediated events were blocked by proteasomal inhibitors, care for affected individuals. The tedious and expensive process and in a small pilot study, the proteasomal inhibitor disulfiram of drug discovery in AD is complicated by the fact that the causes and a CDK inhibitor were shown to be effective in reversing and underlying mechanisms of the disease are still incompletely some of the ApoE4-mediated effects. Furthermore, we identified defined, and presymptomatic diagnosis is not yet in routine clinical use (1). Significance The apolipoprotein E e4 allele (ApoE; chromosomal locus 19q13) is the single most important genetic risk factor associated This manuscript links ApoE4-mediated signaling with Sirtuin with AD. This allele confers increased risk for sporadic and fa- function. Specifically, we show that ApoE4, but not ApoE3, milial AD (2). Individuals with two copies of the ApoE e4 allele reduces neuroprotective SirT1 levels. Our data support the hy- have an approximately eightfold increased risk of AD and have fi pothesis that neuronal connectivity, as reflected in the ratios of a signi cantly lower age of onset compared with AD patients not α β carrying this allele (2). Recent data indicate that the greater risk critical mediators such as sAPP :A , SirT1:SirT2, APP:p-APP, and of AD associated with the ApoE4 isoform might relate to ApoE’s Tau:p-Tau, is programmatically altered by ApoE4. Thus ApoE4, susceptibility to proteolysis and neurotoxicity or through its role SirT1/2, p-Tau, and p-APP, all may be part of a signaling network in inhibiting Aβ clearance and/or stimulating Aβ deposition, that is affected in AD, providing a model for therapeutic candi- leading to plaque formation (3, 4). Contrary to the previously date screening in AD drug discovery. These findings offer a reported work, one recent study provides evidence that ApoE and unique insight into the mechanism by which ApoE4 confers risk soluble Aβ have very minimal direct interaction, thus ApoE may for the development of Alzheimer’s disease. influence soluble Aβ metabolism through its interactions with other receptors or transporters (5). Thus, despite knowing for Author contributions: R.V.R. and D.E.B. designed research; V.T., A.P., C.A.P.L., B.P., S.F., over a decade that the ApoE e4 allele is somehow contributory to O.D., K.S.P., Q.Z., G.C., M.H., and R.V.R. performed research; V.J., R.V.R., and D.E.B. ana- lyzed data; and R.V.R. and D.E.B. wrote the paper. the disease process, the precise molecular mechanisms underlying fl ApoE and APP interactions, direct or indirect, resulting in The authors declare no con ict of interest. ApoE4-mediated toxicity, remain unclear. This article is a PNAS Direct Submission. The amyloid precursor protein, APP, has been shown to 1V.T. and A.P. contributed equally to this work. function as a molecular switch: cleavage at the β, γ, and caspase 2R.V.R. and D.E.B. contributed equally to this work. — 3 sites results in the production of four pro-AD-peptides soluble To whom correspondence may be addressed. E-mail: [email protected] or amyloid precursor protein beta (sAPPβ) (from which N-APP is [email protected]. derived), Aβ, Jcasp, and C31—that mediate neurite retraction, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. synaptic reorganization, and ultimately programmed cell death. 1073/pnas.1314145110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1314145110 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 initial therapeutic candidates—F03 and F05—that normalize some ApoE4 Binds to APP at Endocytic pH with a Significantly Higher of the ApoE4 effects. Affinity than ApoE3. We also confirmed the interaction of ApoE In addition to these effects on APP processing and signaling, and APP by surface plasmon resonance (SPR) to measure the ApoE4 expression was associated with a marked reduction in the binding of recombinant ApoE isoforms with recombinant pro- ratio of SirT1 to SirT2, both in cultured neural cells and in the tein fragments of the ectodomain of APP, as well as the full brains of patients with AD. Because SirT1 has been implicated in ectodomain of APP695 (eAPP19–624) at pHs more characteristic neuroprotection and SirT2 in neurodegeneration (8), this effect of intracellular compartments. Analysis of the recombinant of ApoE4 may be important from both mechanistic and thera- ApoE with a calibrated Superdex S-200 size-exclusion chroma- peutic development standpoints. tography column gave the expected molecular weight of the Our data support the view that ApoE4 modulates the con- ApoE tetramer for both ApoE3 and ApoE4, indicating that nectivity balance, as reflected in the ratios of sAPPα:sAPPβ, the amounts of lipid retained were small in comparison with the sAPPα:Aβ, SirT1:SirT2, APP:p-APP, and Tau:p-Tau. The net- mass of the protein. The E2 domain of APP and the Aβ-cognate work of proteins that mediates this balance thus represents a set region (trx-eAPP290–624) gave an effective KD of 80 nM for A B of candidate targets for the prevention and treatment of ApoE4- ApoE4 and 300 nM for ApoE3 (Fig. 2 and , Table S1, and associated processes such as mild cognitive impairment and AD. Fig. S3). Although both ApoE and APP self-associate (10), the amount of ApoE–APP complex formed at equilibrium approxi- Results mated the isotherm expected for a single-binding equilibrium C D ApoE Isoforms Associate with APP. Although the lipidation status (Fig. 2 and ), consistent with previous observations regarding of ApoE affects its structure (9), it is not yet clear whether the the association of ApoE-containing VLDL particles at pH risk associated with ApoE4 is related solely to lipidated ApoE4, greater than 7 (11). At pH 6.5, we found a consistent trend that ApoE4 displayed tighter binding than ApoE3 independent of to poorly lipidated ApoE4, to unlipidated ApoE4, or to a com- whether the ApoE or APP was attached to the flow cell surface.