Neprilysin gene expression requires binding of the amyloid precursor protein intracellular domain to its promoter: implications for Alzheimer disease

Abstract

Amyloid beta-peptide (Abeta) accumulation leads to neurodegeneration and Alzheimer disease; however, amyloid metabolism is a dynamic process and enzymic mechanisms exist for Abeta removal. Considerable controversy surrounds whether the intracellular domain of the amyloid precursor protein (AICD) regulates expression of the Abeta-degrading metalloprotease, neprilysin (NEP). By comparing two neuroblastoma cell lines differing substantially in NEP expression, we show by chromatin immunoprecipitation (ChIP) that AICD is bound directly to the NEP promoter in high NEP-expresser (NB7) cells but not in low-expresser (SH-SY5Y) cells. The methylation status of the NEP promoter does not regulate expression in these cells, whereas the histone deacetylase inhibitors trichostatin A and valproate partly restore NEP expression and activity in SH-SY5Y cells. ChIP analysis also reveals AICD binding to the NEP promoter in rat primary neurons but not in HUVEC cells. Chromatin remodelling of crucial Alzheimer disease-related genes by valproate could provide a new therapeutic strategy.

Figure 1

Comparative analysis of NEP, APP and Fe65 expression in SH-SY5Y and NB7 cells. NEP expression is substantially higher in NB7 cells compared with SH-SY5Y cells at the level of (A) mRNA by conventional PCR, (B) protein immunoblotting (20 μg cell lysate) and (C) enzyme activity (mean of three experiments, each assayed in triplicate for enzyme activity). AzaC does not affect NEP mRNA expression in either cell line (A). (D) Immunoblotting of cell extracts (50 μg protein) with antibodies against human APP and Fe65. (E) Effect of APP gene silencing by APP siRNA on NEP mRNA expression in NB7 and SH-SY5Y cells, assessed by real-time PCR (siRNA treatment, see Methods), compared with effects of GAPDH or a scrambled siRNA (mean of three experiments). APP, amyloid precursor protein; azaC, 5-aza-2′-deoxycytidine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA; NEP, neprilysin; siRNA, small-interfering RNA.

Figure 2

Chromatin immunoprecipitation analysis of the NEP promoters in SH-SY5Y and NB7 cells. (A,B) ChIP and conventional DNA analysis shows that the NEP promoter 2 in NB7, but not in SH-SY5Y cells, has enriched lysine acetylation of histone H4 in positions K8 and K16, and is marked by AICD, whereas the SH-SY5Y NEP promoter 2 is marked by HDAC1. ChIP with antibody to H3 was used as a positive control in (B) and IgG as a negative control. (C) ChIP analysis of the NEP promoters 1 and 2 in NB7 and SH-SY5Y cells with antibodies to AICD and HDAC1. (D,E) ChIP followed by real-time PCR analysis with (D) anti-AICD and (E) anti-HDAC1 of the NEP promoters 1 and 2 in NB7 and SH-SY5Y cells (mean of five experiments). (F) Relative luciferase luminescence from NB7 or SH-SY5Y cells transfected with either NEP promoters 1- or 2-luciferase constructs (mean of three experiments). (G) Immunocytochemical detection of AICD. Localization of AICD was observed in the nuclei of NB7 cells (upper panel, a), whereas only predominantly cytoplasmic and weak detection of AICD was observed in SH-SY5Y cells (lower panel, d). Cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAP1; b,e); captured images were digitally merged and are shown in (c,f). AICD, amyloid precursor protein intracellular domain; ChIP, chromatin immunoprecipitation; HDAC1, histone deacetylase 1; NEP, neprilysin.

Figure 3

Chromatin immunoprecipitation analysis of AICD and HDAC1 binding to the NEP promoters in primary cells, and effects of the γ-secretase inhibitor L685,458 in NB7 and SH-SY5Y cells. (A) NB7 and SH-SY5Y cells were treated with (+) or without (−) L685,458 (10 μM, 48 h), and NEP expression was compared using conventional PCR. (B) Gel densitometry relative to β-actin for NB7 cells from (A) is presented graphically (mean of three experiments; ^*P<0.05). (C) Real-time PCR of RNA extracts from NB7 and SH-SY5Y cells incubated with or without L685,458 (10 μM, 48 h; mean of five experiments). (D) ChIP with anti-AICD followed by conventional DNA analysis of NB7 cells treated with or without L685,458 (ChIP with IgG used as a negative control). (E) ChIP with anti-AICD or anti-HDAC1 followed by real-time PCR DNA analysis with primers to NEP promoter 2 in NB7 and SH-SY5Y cells treated with or without L685,458 (mean of three experiments). (F,G) AICD and HDAC1 ChIP followed by real-time PCR analysis with primers to the NEP promoters 1 and 2, and a coding region of the NEP gene, in (F) rat primary cortical neurons and (G) HUVEC cells (mean of three experiments). AICD, amyloid precursor protein intracellular domain; ChIP, chromatin immunoprecipitation; HDAC1, histone deacetylase 1; NEP, neprilysin.

Figure 4

Effect of the HDAC inhibitors VA and TSA on NEP expression and enzyme activity. (A) SH-SY5Y and NB7 cells were incubated without (control) or with VA (10 μM, 48 h), and NEP mRNA expression was assessed using conventional PCR. (B) Effects of TSA (100 nM) and VA (10 μM) treatment for 48 h on NEP mRNA levels in SH-SY5Y and NB7 cells, as assessed using real-time PCR (mean of five experiments). (C,D) Effects of TSA and VA on NEP activity in (C) SH-SY5Y and (D) NB7 cells (mean of three experiments, each assayed in triplicate for enzyme activity). (E) ChIP analysis with anti-AICD and anti-HDAC1, followed by real-time PCR of pulled-down DNA with primers to the NEP promoter 2 in NB7 and SH-SY5Y cells treated with TSA (100 nM, 48 h) compared with control (mean of three experiments; ^*P<0.05). AICD, amyloid precursor protein intracellular domain; HDAC, histone deacetylase; mRNA, messenger RNA; NEP, neprilysin; TSA, trichostatin A; VA, sodium valproate.