The Alzheimer's disease risk factors apolipoprotein E and TREM2 are linked in a receptor signaling pathway.

BACKGROUND: Triggering receptor expressed on myeloid cells 2 (TREM2) and apolipoprotein E (APOE) are genetically linked to Alzheimer's disease. Here, we investigated whether human ApoE mediates signal transduction through human and murine TREM2 and sought to identify a TREM2-binding domain in human ApoE. METHODS: To investigate cell signaling through TREM2, a cell line was used which expressed an NFAT-inducible ß-galactosidase reporter and human or murine TREM2, fused to CD8 transmembrane and CD3ζ intracellular signaling domains. ELISA-based binding assays were used to determine binding affinities of human ApoE isoforms to human TREM2 and to identify a TREM2-binding domain in ApoE. RESULTS: ApoE was found to be an agonist to human TREM2 with EC50 in the low nM range, and to murine TREM2 with reduced potency. In the reporter cells, TREM2 expression was lower than in nontransgenic mouse brain. Human ApoE isoforms ε2, ε3, and ε4 bound to human TREM2 with K d in the low nM range. The binding was displaced by an ApoE-mimetic peptide (amino acids 130-149). CONCLUSIONS: An ApoE-mediated dose-dependent signal transduction through TREM2 in reporter cells was demonstrated, and a TREM2-binding region in ApoE was identified. The relevance of an ApoE-TREM2 receptor signaling pathway to Alzheimer's disease is discussed.

Analyzing microglial-associated Aß in Alzheimer's disease transgenic mice with a novel mid-domain Aß-antibody.

The mechanisms of amyloid-ß (Aß)-degradation and clearance in Alzheimer's disease (AD) pathogenesis have been relatively little studied. Short Aß-fragments form by enzymatic cleavage and alternate amyloid-beta precursor protein (APP)-processing. Here we characterized a novel polyclonal Aß-antibody raised against an Aß mid-domain and used it to investigate microglial Aß-uptake in situ by microscopy at the light- and ultrastructural levels. The rabbit Aß-mid-domain antibody (ab338), raised against the mid-domain amino acids 21-34 (Aß21-34), was characterized with biochemical and histological techniques. To identify the epitope in Aß recognized by ab338, solid phase and solution binding data were compared with peptide folding scores as calculated with the Tango software. The ab338 antibody displayed high average affinity (KD: 6.2 × 10-10 M) and showed preference for C-terminal truncated Aß-peptides ending at amino acid 34 and Aß-mid domain peptides with high scores of ß-turn structure. In transgenic APP-mouse brain, ab338 labelled amyloid plaques and detected Aß-fragments in microglia at the ultra- and light microscopic levels. This reinforces a role of microglia/macrophages in Aß-clearance in vivo. The ab338 antibody might be a valuable tool to study Aß-clearance by microglial uptake and Aß-mid-domain peptides generated by enzymatic degradation and alternate production.

Cerebrospinal fluid soluble TREM2 in aging and Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) neuropathology is associated with neuroinflammation, but there are few useful biomarkers. Mutant variants of triggering receptor expressed on myeloid cells 2 (TREM2) have recently been linked to late-onset AD and other neurodegenerative disorders. TREM2, a microglial receptor, is involved in innate immunity. A cleaved fragment, soluble TREM2 (sTREM2), is present in the cerebrospinal fluid (CSF). METHODS: We developed and used a novel enzyme-linked immunosorbent assay to investigate the potential value of CSF sTREM2 as an AD biomarker in two independent cohorts: an AD/mild cognitive impairment (MCI)/control cohort (n = 100) and an AD/control cohort (n = 50). RESULTS: We found no significant difference in sTREM2 levels between groups of controls and patients with AD or MCI. However, among all controls there was a positive correlation between sTREM2 and age (Spearman rho = 0.50; p < 0.001; n = 75). In the AD/MCI/control cohort, CSF sTREM2 correlated positively with total Tau (T-tau) (Spearman rho 0.57; p < 0.001; n = 50), phosphorylated Tau (P-tau) (Spearman rho 0.63; p < 0.001; n = 50) and amyloid-ß1-42 (Aß42) (Spearman rho 0.35; p = 0.01; n = 50) in control subjects. Among controls with a CSF Aß42 above a cut-off value (700 pg/ml) in this cohort, the positive correlation between sTREM2 and Aß42 was stronger (Spearman rho = 0.44; p = 0.002; n = 46). CONCLUSIONS: sTREM2 in CSF correlates with aging in controls, and with the neurodegenerative markers CSF T-tau/P-tau among controls who are negative for AD CSF core biomarkers Aß42, T-tau or P-tau.

Nucleosome binding by the bromodomain and PHD finger of the transcriptional cofactor p300.

The PHD finger and the bromodomain are small protein domains that occur in many proteins associated with phenomena related to chromatin. The bromodomain has been shown to bind acetylated lysine residues on histone tails. Lysine acetylation is one of several histone modifications that have been proposed to form the basis for a mechanism for recording epigenetically stable marks in chromatin, known as the histone code. The bromodomain is therefore thought to read a part of the histone code. Since PHD fingers often occur in proteins next to bromodomains, we have tested the hypothesis that the PHD finger can also interact with nucleosomes. Using two different in vitro assays, we found that the bromodomain/PHD finger region of the transcriptional cofactor p300 can bind to nucleosomes that have a high degree of histone acetylation. In a nucleosome retention assay, both domains were required for binding. Replacement of the p300 PHD finger with other PHD fingers resulted in loss of nucleosome binding. In an electrophoretic mobility shift assay, each domain alone showed, however, nucleosome-binding activity. The binding of the isolated PHD finger to nucleosomes was independent of the histone acetylation levels. Our data are consistent with a model where the two domains cooperate in nucleosome binding. In this model, both the bromodomain and the PHD finger contact the nucleosome while simultaneously interacting with each other.