Figure 1.

Expression of sTLR4 and sTLR5 reduces Aβ plaques in TgCRND8 mice. (A) Table depicting the details of sequences used for generating rAAV2 constructs used in the study. MW, molecular weight. (B) Linear diagram of human full-length TLR5 molecule with four major domains: secretion signal peptide, leucine repeat–rich (LRR) domain, LRR C terminus (LRR CT), transmembrane domain (TMD), and Toll/IL-1 receptor (TIR) domain. The numbers depict the amino acid corresponding to each of the domains. (C) sTLR protein was detected with anti-FLAG antibody in HEK cell lysates. (D) Immunofluorescence analysis from primary neuroglial cultures transduced with rAAV2/1-sTLR shows sTLR expression (Alexa Fluor 488 nm) in MAP2-immunopositive (Alexa Fluor 594 nm) neurons (arrows). Immunofluorescence was used to confirm expression of these rAAV constructs in 6-mo-old CRND8 mouse forebrains. (E) sTLR expression (FLAG antibody, Alexa Fluor 594 nm) could be detected in β-tubulin III–immunopositive neurons (Alexa Fluor 488 nm; arrows). Arrowheads mark putative blood vessel staining. (D and E) DAPI marks nuclei. Scale bars, 50 µm (D); 25 µm (E). (F–M) Neonatal CRND8 mice were injected with AAV2/1-sTLR or AAV2/1-EGFP (control) in the cerebral ventricles and analyzed after 5 mo for biochemical Aβ levels (F and G) and Aβ plaques (H–K). (F and G) Biochemical analyses showed that sTLR4 and sTLR5 tandem affinity purification reduces formic acid– and SDS-extractable insoluble Aβ42 levels. Data (normalized to percentage of control mice in each cohort) represent mean ± SEM; n = 5–7/group. (H–K) Both anti-Aβ mAb 33.1.1 (H and I) and thioflavin S (ThioS) staining (J and K) depict reduced Aβ deposition in sTLR4- and sTLR5-expressing mice compared with controls. (L and M) Representative GFAP (L) and Iba-1 (M) immunoreactivity in AAV-expressing mice. Scale bars, 125 µm (H and J); 100 µm (L and M). n = 5–6/group. Data represent mean ± SEM. ***P < 0.001, **P < 0.01, *P < 0.05; one-way ANOVA.