Phospholipase D3 contributes to Alzheimer’s disease risk via disruption of Aβ clearance and microglia response to amyloid plaques

Alzheimer’s disease (AD) is characterized by the accumulation of amyloid-β (Aβ) plaques and neurofibrillary tangles in the brain. AD is also the result of complex genetic architecture that can be leveraged to understand pathways central to disease processes. We have previously identified coding variants in the phospholipase D3 ( PLD3 ) gene that double the late-onset AD risk. However, the mechanism by which PLD3 impacts AD risk is unknown. One AD risk variant, PLD3 p.A442A, disrupts a splicing enhancer-binding site and reduces PLD3 splicing in human brains. Using differentiated induced pluripotent stem cells from a PLD3 p.A442A carrier and CRISPR-reverted, isogenic control, we show that PLD3 p.A442A cortical neurons exhibit a PLD3 splicing defect and a significant increase in Aβ42 and Aβ40, both of which are corrected upon reversion of the risk allele in isogenic control neurons. Thus, PLD3 p.A442A is sufficient to alter PLD3 splicing and Aβ metabolism. While the normal function of PLD3 is poorly understood, PLD3 is highly expressed in neurons and brain regions most susceptible to amyloid pathology. PLD3 expression is significantly lower in AD brains than controls, suggesting that PLD3 may play a role in sporadic AD. Thus, we sought to determine whether PLD3 contributes to Aβ accumulation in AD. In a mouse model of amyloid accumulation, loss of Pld3 increases interstitial fluid (ISF) Aβ and reduces Aβ turnover. AAV-mediated overexpression of PLD3 in the hippocampus decreased ISF Aβ levels and accelerated Aβ turnover. To determine whether PLD3-mediated reduction of ISF Aβ impacts amyloid accumulation, we measured amyloid plaque abundance and size after significant Aβ deposition. We found that in the absence of Pld3 , amyloid plaques were less compact and more diffuse. Additionally, we observed reduced recruitment of microglia to amyloid plaques in the absence of Pld3 . PLD3 may impact amyloid accumulation and AD risk through disrupted microglia function as PLD3 is enriched in disease associated microglia in human brains. Together, our findings demonstrate that PLD3 regulates Aβ clearance through cell-autonomous and non-cell-autonomous pathways in a manner that likely contributes to AD risk. ### Competing Interest Statement D.M.H. co-founded and is on the scientific advisory board of C2N Diagnostics. D.M.H. is on the scientific advisory board of Denali and Cajal Neuroscience and consults for Genentech and Alector. CC has received research support from: Biogen, EISAI, Alector and Parabon. The funders of the study had no role in the collection, analysis, or interpretation of data; in the writing of the report; or in the decision to submit the paper for publication. CC is a member of the advisory board of Vivid genetics, Halia Therapeutics and ADx Healthcare. AMG is on the scientific advisory boards of Genentech and Muna Therapeutics. M. K. has filed a patent application related to CRISPRi and CRISPRa screening (PCT/US15/40449) and serves on the Scientific Advisory Board of Engine Biosciences, Casma Therapeutics, and Cajal Neuroscience, and is an advisor to Modulo Bio and Recursion Therapeutics. The remaining authors have no disclosures. ### Funding Statement This work was funded by BrightFocus Foundation (CMK), the Alzheimer's Association (CMK), NIH U01 AG052411 (AG), R01 AG062359 (CMK), P50 AG005681 (JM, CMK), R56AG067764 (CMK, OH, CC), U01 AG072464 (ST, CMK, OH, MK), UL1 TR002345, NS090934 (DMH), AG047644 (DMH), NS094692 (JML), AG062359 (MK). This work was supported by access to equipment made possible by the Hope Center for Neurological Disorders and the Departments of Neurology and Psychiatry at Washington University School of Medicine. We thank the Washington University in St Louis Mouse Genetics Core for assistance in generating the Pld3 KO mice. The gRNAs were generated by the Genome Engineering and iPSC Center (GEiC) at the Washington University in St. Louis. We thank the Hope Center Viral Vector Core for generating the AAV8 particles and the Hope Center Animal Surgery Core for performing the AAV8 injections. Confocal was generated on a Zeiss LSM 880 Airyscan Confocal Microscope, which was purchased with support from the Office of Research Infrastructure Programs (ORIP), a part of the NIH Office of the Director under grant OD021629. The recruitment and clinical characterization of research participants at Washington University were supported by NIH P50 AG05681, P01 AG03991, and P01 AG026276. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Washington University School of Medicine Institutional Review Board and Ethics Committee approved the informed consent (IRB 201104178, 201306108). The consent allows for the use of tissue by all parties, commercial and academic, for research but not for human therapy. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors. Data can be publicly accessed at .