medRxiv preprint doi: https://doi.org/10.1101/2020.11.20.20235051; this version posted November 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license . Protein phosphatase 2A, complement component 4, and APOE genotype linked to Alzheimer’s disease using a systems biology approach Gyungah R. Jun1,2,8,*, Yang You5, Congcong Zhu1, Gaoyuan Meng10, Jaeyoon Chung1, Rebecca Panitch1, Junming Hu1, Weiming Xia5,10, The Alzheimer’s Disease Genetics Consortium, David A. Bennett11, Tatiana M. Foroud12, Li-San Wang13, Jonathan L. Haines14, Richard Mayeux15, Margaret A. Pericak-Vance16, Gerard D. Schellenberg13, Rhoda Au3,6,9, Kathryn L. Lunetta8, Tsuneya Ikezu3,5,7, Thor D. Stein4,10, Lindsay A. Farrer1,2,3,8,9,* Departments of 1Medicine (Biomedical Genetics), 2Ophthalmology, 3Neurology, 4Pathology & Laboratory Medicine, 5Pharmacology & Experimental Therapeutics,6Anatomy & Neurobiology, and7Center for Systems Neuroscience, Boston University School of Medicine, 72 East Concord Street, Boston, MA 02118, USA. Departments of 8Biostatistics and 9Epidemiology, Boston University School of Public Health, 715 Albany Street, Boston, MA 02118, USA. 10Department of Veterans Affairs Medical Center, Bedford, MA 01730, USA. 11Rush Alzheimer’s Disease Center, Rush University Medical Center, 1750 W. Harrison Street, Suite 1000, Chicago, IL 60612, USA. 12Department of Medical and Molecular Genetics, Indiana University, 410 W 10th Street, Indianapolis, IN 46202, USA. 13Penn Neurodegeneration Genomics Center, Department of Pathology and Laboratory Medicine, University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104, USA. 1 NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. medRxiv preprint doi: https://doi.org/10.1101/2020.11.20.20235051; this version posted November 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license . 14Department of Population & Quantitative Health Sciences, Case Western Reserve University, 10900 Euclid Ave, Cleveland, OH 44106, USA. 15Taub Institute on Alzheimer's Disease and the Aging Brain, Gertrude H. Sergievsky Center Department of Neurology, Columbia University, 710 West 168th Street, New York, NY 10032, USA. 16John P. Hussman Institute for Human Genomics, Department of Human Genetics, and Dr. John T. Macdonald Foundation, University of Miami, 1501 NW 10th Ave, Miami, FL 33136, USA. * To whom correspondence should be addressed Gyungah R. Jun, PhD, Department of Medicine (Biomedical Genetics), Boston University School of Medicine, 72 East Concord Street, Boston, MA 02118, USA 02118, email: [email protected]; Lindsay A. Farrer, PhD, Department of Medicine (Biomedical Genetics), Boston University School of Medicine, 72 East Concord Street, Boston, MA 02118, USA 02118, email: [email protected]. 2 medRxiv preprint doi: https://doi.org/10.1101/2020.11.20.20235051; this version posted November 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license . ABSTRACT Background: Recent reports suggest that the rare apolipoprotein E (APOE) Christchurch mutation and ɛ2 allele protect against Alzheimer’s disease (AD) pathology by reducing the burden of tau pathology. However, the mechanism(s) underlying the ɛ2 protective effect linking to tau is largely unknown. Methods: The role of the ɛ2 allele in Alzheimer’s disease (AD) was investigated a genome-wide association study (GWAS) for AD among 2,120 ɛ2 carriers from the Alzheimer Disease Genetics Consortium (ADGC), and then prioritized by gene network analysis, differential gene expression analysis at tissue- and cell-levels as well as methylation profiling of CpG sites, in prefrontal cortex tissue from 761 brains of the Religious Orders Study and Memory and Aging Project (ROSMAP) and the Framingham Heart Study (FHS), Boston University Alzheimer’s Disease Center (BUADC). The levels of two catalytic subunit proteins from protein phosphatase 2A (PPP2CA and PPP2CB) were validated in prefrontal cortex area of 193 of the FHS/BUADC brains. The findings from human autopsied brains were further validated by a co-culture experiment of human isogenic APOE induced pluripotent stem cell (iPSC) derived neurons and astrocytes. Results: Of the significantly associated loci with AD among APOE ɛ2 carriers (P<10-6), PPP2CB (P=1.1x10-7) was the key node in the APOE ɛ2-related gene network and contained the most significant CpG site (P=7.3x10-4) located 2,814 base pair upstream of the top-ranked GWAS variant. Among APOE ɛ3/ɛ4 subjects, the level of Aβ42 was negatively correlated with protein levels of PPP2CA (P=9.9x10-3) and PPP2CB (P=2.4x10-3), and PPP2CA level was correlated with the level of pTau231 level (P=5.3x10-3). Significant correlations were also observed for PPP2CB with complement 4B (C4B) protein levels (P=3.3x10-7) and PPP2CA with cross reactive protein -4 (CRP) levels (P=6.4x10 ). C1q level was not associated with Aβ42, pTau231, PPP2CB, or C4B 3 medRxiv preprint doi: https://doi.org/10.1101/2020.11.20.20235051; this version posted November 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license . levels. We confirmed the significant correlation of PPP2CB expression with pTau231/tTau ratio (P=0.01) and C4A/B (P=2.0x10-4) expression observed in brain tissue in a co-culture experiment of iPSC derived neurons and astrocytes. Conclusion: We demonstrated for the first time a molecular link between a tau phosphatase and the classical complement pathway, especially C4, and AD-related tau pathology. Key words: Alzheimer disease, APOE genotype, PPP2CB, complement component 4B, tau pathology 4 medRxiv preprint doi: https://doi.org/10.1101/2020.11.20.20235051; this version posted November 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license . BACKGROUND Alzheimer disease (AD) is a progressive neurodegenerative disorder and accounts for 60-80% of all causes of dementia. None of the prescribed medications for symptomatic treatment of AD retard or stop neuronal degeneration [1]. AD currently affects about 5.8 million Americans age 65 and older and will increase to 13.8 million by 2050 if current trends continue [1]. AD is the sixth leading cause of death in the United States and its mortality rate increased 146% between 2000 and 2018 [1]. More than 16 million caregivers provided about 18.6 billion hours of care (valued around $244 billion) to people with AD or other dementias in 2019, and total health care payments in 2020 for people age 65 and older with dementia are estimated to be $305 billion [1]. Apolipoprotein E (APOE) genotype is the strongest risk factor for the common form of AD that occurs after age 65 years [1]. Combinations of amino acid residues at 112 (rs429358) and 158 (rs7412) determine three common APOE alleles (ɛ2, ɛ3 and ɛ4), where ɛ4 increases but ɛ2 decreases AD risk [2, 3]. Lifetime risk of AD among female ɛ4 homozygotes is approximately 60% and 10 times higher than for ɛ2 carriers matched for sex and age [2, 3]. Neuropathological hallmarks of AD are neurofibrillary tangles consisting of decomposed microtubules and phosphorylated tau (p-tau) and neuritic plaques containing toxic beta-amyloid peptides (Aβ) [1]. Frequencies of APOE ɛ2/2, ɛ3/3 and ɛ4/4 homozygotes among non-Hispanic whites are about 0.5%, 38.2%, and 12.6%, respectively [3, 4]. Odds ratios of AD among clinically and neuropathologically confirmed ɛ4 and ɛ2 homozygotes in this group are approximately 31 and 0.13, respectively, compared to persons with the common ɛ3/ɛ3 genotype [5]. Common variants in the region of MAPT (the gene encoding tau protein) have been associated with AD among individuals who lack ɛ4 [6]. The protective effect of ɛ2 on tangle burden is independent from that on plaque burden and specific to AD pathology [5]. An exaggerated modifying effect of APOE 5 medRxiv preprint doi: https://doi.org/10.1101/2020.11.20.20235051; this version posted November 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license . alleles on clinical and pathological manifestations of AD was demonstrated by the recent discovery of the rare APOE Christchurch (APOEch) mutation on a ɛ3 chromosome background in a carrier of the deleterious presenilin 1 (PSEN1) E280A mutation that causes early onset AD typically between ages 30 and 60 among members of a large kindred with autosomal dominant AD. Notably, the E280A mutation carrier who was homozygous for the APOEch variant had hyperlipoproteinemia Type III that is typically associated with ɛ2 homozygosity, presented with delayed cognitive impairment in her seventies, and showed profound plaque burden but limited neurofibrillary tangle involvement by positron emission tomography (PET) imaging [7]. In fact, accumulation of tau protein (the primary constituent of tangles) measured by PET is strongly associated with memory decline and most prominent in the medial temporal lobe [8].