Fig 1.
Stomach tissue from 15 month V30M animal stained with Thioflavin-S, α-hTTR antibody and Congo red in consecutive sections (Ai-Avi). The area of Thioflavin-S and α-hTTR antibody co-localization which was included in measurements as TTR plaques is indicated in (Avi). Stomach tissue from 5 month V30M animal stained with stained with Thioflavin-S and α-hTTR antibody indicating solely the presence of pre-fibrillar hTTR (Bi-Biii) (A&B Scale bar = 75mm). C Quantification of the amyloid plaques found in the stomach measured through Thioflavin-S and α-hTTR antibody co-localization for both the V30M and V30M C1q KO mice (n = 15/age group/line). Data presented as mean ± 1SD.
Fig 2.
hTTR levels in serum and stomach.
hTTR immunoblotting exhibiting a single band at 14KDa (A). hTTR levels measured via immunoblotting in serum of V30M and V30M C1q KO animals of all three age groups indicating higher hTTR in the V30M mice and a gradual decrease in the amount of hTTR present as the animals age (B). hTTR levels measured via immunoblotting in stomach tissue of the V30M and V30M C1q KO animals exhibit a severe decrease in the second age group (C). B&C n = 15/age group/line. Data presented as mean ± 1SD.
Fig 3.
Expression of complement markers.
The expression of Properdin (A) and C5b-9 (B) measured via immunoblotting in both V30M and V30M C1q KO animals stomach tissue in all three age groups exhibiting an age related increase of both markers, as well as a higher expression in the V30M C1q KO animals (n = 15/age group/line, data presented as mean ± 1SD).
Fig 4.
Expression of apoptotic markers.
The expression of Fas protein measured by immunoblotting (A) in stomach tissue of both V30M and V30M C1q KO animals in all three age groups indicating an age related steady increase, as well as a higher expression in the V30M C1q KO mice. The same was observed by immunofluorescence (B). Similarly, the expression of Caspase-3 was also measured by immunoblotting (C) again indicating an age related increase with a markedly increased expression in the V30M C1q KO mice. Similar observations were made through immunofluorescence (D). A&C n = 15/age group/line, data presented as mean ± 1SD. B&D Scale bar = 150mm.
Fig 5.
The expression of BiP protein measured by immunoblotting (A) in stomach tissue of both V30M and V30M C1q KO animals in all three age groups depicting an increased expression in V30M C1q KO mice of all ages. The same was observed by immunofluorescence (B). Furthermore, the presence of the 4-HNE was quantified via immunoblotting (C) in stomach tissues of both V30M and V30M C1q KO animals in all three age groups, again indicating the higher expression of 4-HNE in V30M C1q KO mice. The same was observed by immunofluorescence (D). A&C n = 15/age group/line, data presented as mean ± 1SD. B&D Scale bar = 150mm.
Fig 6.
Immunofluorescence indicating the absence of the pan-macrophage marker CD68 on hTTR positive plaques from the V30M C1q KO mice contrary to V30M mice where macrophages co-localize with C1q (A) n = 15/age group/line, data presented as mean ± 1SD. By Western blot expression of the marker CD68 was severely decreased in stomach tissue of the V30M C1q KO mice when compared to the V30M mice (B), Scale bar = 30mm.
Fig 7.
The expression of the CD88 receptor (A) and its ligand C5a (B) were measured by immunoblotting in stomach tissues of both V30M and V30M C1q KO animals in all three age groups. Both markers exhibit an astounding reduction in the V30M C1q KO mice. A&B n = 15/age group/line, data presented as mean ± 1SD.
Fig 8.
Model of observed C1q ablation effects.
Classical and alternative pathway activation lead to the hydrolysis of C3 initiating the formation of the membrane attack complex (MAC) and production of the chemoattractant C5a. C1q and C5a recruit and activate phagocytic cells such as macrophages through receptors located on their surfaces (A). In the absence of C1q, properdin expression increases along with the entire alternative pathway and the terminal MAC complex. Concurrently, the presence of CD68 positive phagocytes was decreased along with the expression of C5a anaphylatoxin and its receptor CD88 (B). As a result, amyloid deposition increases following C1q ablation (b) versus the original transgenic model (a).