Extended Data Figure 7. Induction of REST by Wnt signaling.

a. Cell non-autonomous induction of REST by aged brain extracts is partially inhibited by the Wnt antagonist Dickkopf (DKK). Extracts were derived from young adult (Y), aged (O) or AD PFC of the indicated ages and then incubated with SH-SY5Y cells in the absence (−) or presence (+) of DKK (250 ng/ml). b. Conditioned medium transferred from SH-SY5Y cells, treated with either wortmannin (2 μM), tunicamycin (2 μM), H₂O₂ (100 μM) or LiCl (10 mM), induce REST when added to naïve cells, which is inhibited by DKK. c. REST levels are increased by treatment of SH-SY5Y cells with Wnt 3a or 7a (250 ng/ml, 16 hrs; upper Western blot), and by LiCl (5 or 10 mM, 6 hrs) or Chiron 99021 (20 nM, 6 hrs) (lower Western blot).

d. REST ChIP-seq shows that the Wnt activator LiCl (5 mM) broadly increases REST binding to target genes. Shown are REST targets P<10⁻⁵ with REST-RE1 site binding within 10 kb of the transcription start site. They include genes related to AD pathology, such as the gamma secretase presenilin-2 (PSEN2), and pro-apoptotic genes (PUMA, BAX, DAXX, TRADD and BCL2L11). e, f. SH-SY5Y cells incubated with LiCl (10 mM, 24 hrs) or Chiron 99021 (100 nM, 24 hrs) exhibit increased nuclear REST. Nuclei are stained dark blue with DAPI, which become light blue when there is overlap with the green REST staining (e). Quantitative analysis of % REST-positive nuclei (f). g. Increased nuclear β-catenin in the PFC in normal aging, and reduced levels in AD. Shown is FACS analysis of neuronal nuclei isolated from PFC. Values are the mean±S.E.M. ^*P<0.05, ^**P<0.01 by Student’s unpaired t-test. Young, n=13; Aged, n=18; AD, n=10. h. Co-localization of REST and β-catenin in the nucleus of aging neurons in the PFC. Aged and AD cases were double-labeled for REST (green) and β-catenin (red). Scale bars, 20μm.