p53 prevents neurodegeneration by regulating synaptic genes Paola Merloa, Bess Frosta, Shouyong Pengb,c, Yawei J. Yangd,e,f, Peter J. Parkc,g, and Mel Feanya,1 Departments of aPathology and cMedicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115; bDepartment of Medical Oncology, Dana–Farber Cancer Institute, Boston, MA 02115; dDivision of Genetics and Manton Center for Orphan Disease Research, Children’s Hospital Boston, Boston, MA 02115; eBroad Institute of MIT and Harvard, fHoward Hughes Medical Institute, Cambridge, MA 02142; and gCenter for Biomedical Informatics, Harvard Medical School, Boston, MA 02115 Edited by Nancy M. Bonini, University of Pennsylvania, Philadelphia, PA, and approved October 30, 2014 (received for review October 8, 2014) DNA damage has been implicated in neurodegenerative disorders, Here we show that p53 is neuroprotective in an in vivo model including Alzheimer’s disease and other tauopathies, but the con- of tauopathy. Through chromatin immunoprecipitation (ChIP)- sequences of genotoxic stress to postmitotic neurons are poorly chip analyses we determine that p53 controls the transcription of understood. Here we demonstrate that p53, a key mediator of the a group of genes involved in synaptic function. Genetic manipu- DNA damage response, plays a neuroprotective role in a Drosophila lation of these genes modifies tau neurotoxicity. We find that the model of tauopathy. Further, through a whole-genome ChIP-chip transcriptional control by p53 of these synaptic genes is conserved analysis, we identify genes controlled by p53 in postmitotic neu- in murine neurons and human brain. Our results thus implicate rons. We genetically validate a specific pathway, synaptic function, synaptic function as a critical p53 target in neuroprotection. in p53-mediated neuroprotection. We then demonstrate that the control of synaptic genes by p53 is conserved in mammals. Collec- Results tively, our results implicate synaptic function as a central target in p53 Protects from Neurodegeneration During Aging in Vivo. To in- p53-dependent protection from neurodegeneration. vestigate the role of p53 in tauopathy pathogenesis, we first expressed tau in flies lacking p53 (Fig. 1A). Although p53-null p53 transcriptional function | ChIP-chip | neurodegeneration | tauopathy | flies showed no evidence of neurodegeneration in the absence of synaptic genes transgenic human tau (Fig. 1 B–D), cell death, as measured by the number of TUNEL-positive neurons, significantly increased 11-1B-1 eurodegenerative diseases are a group of inexorably pro- in the brains of tau-expressing, p53 (p53 )-null animals Ngressive disorders characterized by ongoing dysfunction and compared with tau-transgenic animals with normal levels of en- death of postmitotic neurons and are among the principal causes of dogenous p53 (Fig. 1B and Fig. S1A, Left). Neurodegeneration in morbidity and mortality associated with aging (1–3). Tauopathies, our model, as in mouse tauopathy models and in Alzheimer’s including Alzheimer’s disease, are defined neuropathologically by disease, is associated with abnormal reactivation of the cell cycle aggregation and deposition of the microtubule-associated protein in postmitotic neurons (Fig. S1B) (5, 6, 14, 15). Removing p53 tau accompanying neuronal loss (1–3). DNA damage has been from tau-transgenic flies significantly increased cell-cycle reac- NEUROSCIENCE documented in neurodegenerative disorders, including tauopathies, tivation, as measured by proliferating cell nuclear antigen but the effects of DNA damage on the function and viability of (PCNA) reexpression in neurons (Fig. 1C and Fig. S1A, Right). nondividing neurons are poorly understood (4–6). Enhancement of human tau-driven neuronal death and cell-cycle reactivation was also observed when a second p53 null allele In this study, we used a Drosophila model of tauopathy based 5A-1-4 on expression of human tau carrying the R406W mutation, which (p53 ; Fig. S1 C and D) was used to remove p53 function. To is found in patients with the familial tauopathy frontotemporal dementia with Parkinsonism linked to chromosome 17 (7). Our Significance model recapitulates several key features of human tauopathies, including accumulation of abnormally phosphorylated and con- The transcription factor p53 plays a critical role in the cellular formationally altered tau, age-dependent neurodegeneration, response to DNA damage and has thus been studied in- and early death (7). We have previously shown that expression of tensively in oncogenesis research. However, the role that p53 human tau in Drosophila neurons induces the DNA damage plays in the response of postmitotic neurons to cellular stress response, including p53 up-regulation, and that reducing the has received less attention. Here we describe an unexpected function of the DNA damage checkpoint significantly increases neuroprotective role for p53 in an in vivo model of tau-medi- neurodegeneration (5). These data suggest an unexpected neu- ated neurodegeneration relevant to Alzheimer’s disease and roprotective role for p53 in postmitotic neurons. We were related disorders. Further, we identify synaptic function as therefore motivated to examine the transcriptional function of a novel target pathway for p53 in aging neurons, consistent p53 in the context of progressive neurodegeneration. with the growing evidence for synaptic pathology as an early p53, together with p63 and p73, comprises a family of tran- event in neurodegenerative disease. Our study defines mech- scription factors, which regulate fundamental processes including anistically a new, conserved role for p53 in protecting post- proliferation, differentiation, senescence, and cell death (8, 9). The mitotic neurons from degeneration during aging and disease. p53 family has been extensively studied in tumorigenesis, but the Author contributions: P.M. and M.F. designed research; P.M., B.F., Y.J.Y., and M.F. per- role of family members in postmitotic neurons is poorly defined, formed research; P.M. contributed new reagents/analytic tools; P.M., S.P., P.J.P., and M.F. with an emerging body of literature suggesting involvement of the analyzed data; and P.M. and M.F. wrote the paper. p73 subfamily in aspects of neuronal development and neuro- The authors declare no conflict of interest. – degeneration (10 12). Using Drosophila to study p53-dependent This article is a PNAS Direct Submission. transcription in neurodegeneration has two significant advantages. Data deposition: The data reported in this paper have been deposited in the Gene Ex- First, transcription can be studied in aging neurons in vivo. Second, pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE40418). analysis of overall p53 family function in a simplified genome, 1To whom correspondence should be addressed. Email: [email protected]. “ ” which contains a single p53 gene, may reveal ancestral activities This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of the family (9, 13). 1073/pnas.1419083111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1419083111 PNAS | December 16, 2014 | vol. 111 | no. 50 | 18055–18060 Downloaded by guest on September 30, 2021 AB with p53 if they had binding peaks in the promoter region, de- fined as 2 kb upstream and 1 kb downstream of the annotated transcriptional start site. Examination of the ChIP-chip data 60 *** ctr p53 nulltau tau + p53 null 50 revealed that p53 binds the promoter region of ∼1,500 genes in ∼ a-tau 40 tau-transgenic flies, of which 550 showed significant enrich- 30 ment in tau-expressing neurons compared with controls [false a-actin 20 discovery rate (FDR) < 0.01; Fig. 2B]. To provide a compre- 10 hensive picture of p53 transcriptional function in postmitotic p53 wt # TUNEL positive cells TUNEL # 0 neurons, we included the entire list of 1,500 genes bound in tau- p53 mut ctr p53 null tau tau + transgenic animals in our subsequent analyses (Dataset S1). p53 null An important physiological role for the p53 targets we iden- CD tified here is consistent with our previous recovery of th, par-1, 120 *** 35 *** Tao, cdi, chb, aop, dally, and Fmr1 in an unbiased forward genetic 100 30 screen for modifiers of tau neurotoxicity (19). Each of these 80 25 genes was bound by p53 in the present ChIP-chip experiment. 60 20 Similarly, a number of the genes that we have previously shown 15 *** 40 10 to control tau neurotoxicity in pathway-focused studies are also # PCNA foci # PCNA 20 (cm / 30 sec) walking speed 5 p53 targets: hep, Act5C, Drp1, sqh, WASp, zip, PHGPx, CycD,and 0 0 eIF-4E (5, 6, 20–22). In addition, the list of genes bound by p53 ctr p53 null tau tau + ctr p53 null tau tau + in our ChIP-chip experiment includes cell-cycle regulators (p53, p53 null p53 null Gadd45, CycD, Cdt1, and Msh2). Recovery of these cell-cycle genes is consistent with genetic evidence supporting a neuro- Fig. 1. p53 is neuroprotective in Drosophila tauopathy. (A, Upper) Tau protein levels do not change in p53-null flies. (Lower) Genomic PCR analysis protective, rather than proapoptotic, role for the DNA damage confirms genotypes at the p53 locus for the animals used. (B) Apoptosis, checkpoint in postmitotic neurons (5, 6). Of note, vertebrate indicated by TUNEL-positive neurons, increases in the brains of tau-trans- homologs of p53, Gadd45, CycD, Cdt1, and Msh2 are known p53 genic, p53-null animals. (C) Cell-cycle reactivation, indicated by PCNA-posi- targets, supporting conservation of p53 binding and function tive foci, increases in the brains of tau-transgenic, p53-null animals. In B and from flies to mammals (18, 23, 24). C, flies are 10-d-old (n ≥ 6 per genotype). (D) Removing p53 in tau-transgenic We next determined whether particular cellular pathways were animals worsens locomotion, measured as walking speed. Flies are 2-d-old enriched for p53 binding. We used the list of p53 target genes (n = 18 per genotype). In the graph, values represent mean ± SEM. Asterisks indicate significant differences compared with control by ANOVA with Student–Newman–Keuls post hoc test for multiple comparisons.