(a) NEP2 and (b) NEP mRNA expression in NI, MCI, and AD male and female groups in the MTG and MFG (n=12 for NI males, n=6 for MCI males, n=5 for AD males, n=17 for NI females, n=22 for MCI females, n=18 for AD females). (c) NEP2 (solid bars) and NEP (open bars) regional mRNA expression levels for the MTG, MFG, caudate, and cerebellum. All NEP2 and NEP regional comparisons were significantly different (p<0.05) (n=80 for MTG, n=76 for MFG, n=33 for caudate, n=33 for cerebellum). Values are means ± SEM. For cross-diagnostic group comparisons, *p<0.05, **p<0.01. For cross-sex classification comparisons within each diagnostic group, #p<0.05, ##p<0.01.

a) NEP2 detected in blood cells by endpoint PCR. NEP2 detected in human peripheral blood mononuclear cells (PBMC) of three NI individuals using qRT-PCR NEP2-specific primers. NEP2-expressing 293T (293) cell lysate was used as a positive control and mock reverse transcription (−) was used as a negative control. b) Immunoprecipitation/Western blot hybridization (IP-Western) of NEP2-β and NEP2-γ. Western blot hybridization of NEP2-β, NEP2-γ, and GFP transfected 293T cell lysates (rows 1–3) and NEP2-β, NEP2-γ, and GFP transfected 293T cell lysates (rows 4–6) immunoprecipitated with NEP2-specific antibody (AF2340). NEP2-β and NEP2-γ were both detected in the cell lysate (rows 1–2) and were both present at higher levels following immunoprecipitation (rows 4–5).
c) Recombinant human NEP2 (rhNEP2) immunoprecipitation activity assay. rhNEP2 shows no remaining activity following AF2340 immunoprecipitation validating the specificity of the AF2340 antibody to NEP2. Samples were run in triplicate (n=3). **p<0.01. d) Recombinant human NEP (rhNEP) immunoprecipitation activity assay. rhNEP remains fully active following AF2340 and SN5c immunoprecipitation indicating that there is no cross reactivity of rhNEP to NEP2-specific and GFP-specific antibodies. Samples were run in triplicate (n=3).

NEP2 activity in the MTG (a) or MFG (b) of NI, MCI, and AD male and female groups as measured by immunoprecipitation/fluorogenic substrate enzymatic activity assay (n=4 for NI males, n=3 for MCI males, n=4 for AD males, n=11 for NI females, n=15 for MCI females, n=10 for AD females). (c) Sex-independent comparisons of NEP2 activity in the MTG and MFG of NI, MCI, and AD groups (n=15 for NI, n=18 for MCI, n=14 for AD). Values are means ± SEM. For cross-diagnostic group comparisons, *p<0.05, **p<0.01. For cross-sex classification comparisons within each diagnostic group, #p<0.05, ##p<0.01. (d) Linear regression model analysis correlating NEP2 activity in the MFG and MTG with cognitive function domains (EST = estimated slope).

Linear regression model graphs correlating NEP2 activity with cognitive function domains. NEP2 activity in the MFG shows a positive correlation with global cognition (p=0.029) (a), episodic memory (p=0.018) (b), perceptual speed (p=0.021) (c), and visuospatial ability (p=0.011) (d). NEP2 activity in the MTG shows a positive correlation with episodic memory (p=0.006) (e).