Fig 2: Plasma AChE levels in healthy controls (wild-type) after BuChE immunodepletion and in BuChE-silent individuals.(A) Control plasma was immunoprecipitated with anti-BuChE antibody and cholinesterase activity levels determined beforeb and aftera immunoprecipitation (n = 6; 46±4 yrs). AChE activity level in plasma from BuChE-silent individuals is also shown (n = 3; 30±5 yrs). The anti-BuChE antibody does not immunoprecipitate AChE in BuChE-silent plasma (not shown). Values are means ± SEM. (B) Immunoprecipitation of control plasma with antibody, followed by immunoblotting with the anti-AChE antibody, N-19. The presence (+) or absence (-) of the anti-BuChE antibody linked to the resin is indicated in the top margin. Prior to electrophoretic analysis, proteins abundant in plasma were depleted by immunoaffinity-based protein subtraction chromatography with IgY microbeads (Seppro™). The anti-BuChE antibody does not immunoprecipitate AChE. Extracts incubated with protein A-Sepharose, in the absence of the antibody, were analyzed in parallel as negative controls. (C) Representative profiles of AChE and (D) BuChE molecular forms (G4 = tetramers; G1+G2 = monomers and dimers) in control plasma samples before (•) and after (?) BuChE-immunoprecipitation, and in BuChE-silent plasma (?). (E) Representative immunoblot of individual AChE G4 and G1+G2 peak-fractions separated by sucrose gradient centrifugation from control plasma and detected with the N-19 antibody (a similar volume for both the G4 and G1+G2 peaks was loaded in each lane). (F) Comparison of the AChE-banding pattern detected with the N-19 antibody, for fractions bound and unbound to the anti-AChE antibody HR2 and to the Fas2-Sepharose affinity matrix.

Fig 3: Correlation of ChAT expression ratios with CFP reporter expression levels for hM4Di_CFP-mirE treated regions.a Confocal multichannel image for colocalization of ChAT, AChE and hM4Di_CFP reporter protein. White arrows indicate the same cells in the four panels. b ChAT categorized cells (strong, medium, weak), categorized for co-staining with reporter expression—RU untreated, RN reporter not detected (treated), RW weak reporter (treated), RM medium reporter (treated), and RS strong reporter (treated). c AChE categorized cells (strong, medium, weak) were further categorized co-staining with reporter expression—labels as above. Distributions were compared to distribution of ChAT categories in surrounding untreated regions (RU). X2 Chi Square number. Asterisk (*) indicates significant with p < 0.001 (Chi square test). See Supplementary Table 2 for Raw Data.

Fig 4: Rare cell types detected by single-cell RNA sequencing of the adventitia. A, Feature plot of lymphatic endothelium markers Pecam1 and Lyve1 in wt (wild type) and ApoE (apolipoprotein E)-/- adventitial mesenchyme cells. B, En face staining of wt and ApoE-/- aortic adventitia of PECAM1 and LYVE1. C, Feature plot of neuronal markers Rbfox3 and Ache in wt and ApoE-/- adventitial mesenchyme cells. D, En face staining of wt and ApoE-/- aortic adventitia for RBFOX3 and ACHE. ACHE indicates acetylcholinesterase; ApoE, apolipoprotein E; DAPI, 4',6-diamidino-2-phenylindole; LYVE1, lymphatic vessel endothelial hyaluronan receptor 1; PECAM1, platelet and endothelial cell adhesion molecule 1; RBFOX3, RNA-binding fox-1 homolog 3; and t-SNE, t-distributed stochastic neighbor embedding.

Fig 5: Altered AChE immunoreactivity in plasma of non-demented controls (ND) and Alzheimer's disease (AD) patients.(A) Representative blot of plasma-AChE from AD and ND using the antibody N-19, and (B) densitometric quantification of the AChE-immunoreactive bands, expressed in arbitrary units (a u.), from 15 ND (▪; n = 15) and 14 AD (□; n = 14) subjects. Proteins abundant in plasma were depleted by immunoaffinity-based protein subtraction chromatography with IgY microbeads (Seppro™) and equivalent amounts of protein were loaded in each lane. Columns represent means ± SEM *p = 0.02 significantly different from NDs as assessed by Student's t test. (C) Representative blot of plasma AChE detected with the anti-AChE antibody to the C-terminal, ab31276.