Table 1.
Primary antibodies used in this study.
Fig 1.
FO supplementation ameliorates neuritic dystrophy through suppression of abnormal tau hyperphosphorilation in the parietal cortex of 5xFAD mice.
(A and D) Axonal dystrophies (arrows) surrounding amyloid plaques (P) in 4-month-old 5xFAD mice; (B and E) Brains of FO-supplemented 5xFAD mice showing significant suppression of dystrophic axons around plaques; (C and F) wild type mice showing absence of dystrophic neurites (G-I) Print screen of ImageJ SMI31 analysis (Max Entropy threshold). (J) Percentage of SMI31-positive spheroids larger than 50μm2 in untreated and FO-treated 5xFAD mice. (K) Percentage of SMI31 coverage of parietal cortex in untreated and FO-treated 5xFAD mice (low surface area limit set to 50μm2). (L) Representative immunoblot of p-Tau (Ser416) in untreated and FO-treated 5xFAD mice. (M) Changes in p-Tau (Ser416) protein in the cortex of 5xFAD and 5xFAD FO mice were revealed by Western blot analysis. The data represent mean ± S.E.M. value; *p < 0.05. Scale bar (D-F) - 5μm.
Fig 2.
FO supplementation significantly reduces Aβ42 in the parietal cortex of 5xFAD mice.
(A) The Aβ42 coverage of the parietal cortex in untreated and in (B) FO treated 5xFAD mice. (C-E) Representative images of Aβ42 clusters in parietal cortex of untreated and treated (F-H) 5xFAD mice. (I) Quantification of Aβ42 coverage of parietal cortex of untreated vs. treated 5xFAD mice from 8 animals for each group. Data represents mean ± s.e.m. (J) Quantification of average Aβ42 cluster surface in the parietal cortex of untreated vs. treated 5xFAD mice. Scale bar 10μm.
Fig 3.
Decreased Aβ42 halo overlaps with the decreased incidence of DNs in FO treated mice.
(A-D) Double staining of untreated 5xFAD mice brain sections with the 4g8 and SMI31 antibodies revealed that the areas with higher incidence of DNs display the greater total Aβ halo around the plaques (AmyloGlo+). (E-H) In the FO-treated 5xFAD animals the decreased surface of the Aβ halo overlaps with the decreased incidence of swollen, dystrophic neurites.
Fig 4.
FO supplementation does not affect the phagocytic properties of microglia/macrophages and levels of proinflammatory cytokines.
((A,B) Representative confocal images showing absence of phagocytosis of Aβ42 by microglia/macrophages as assessed by measuring colocalization of Iba-1 and Aβ42 immunostaining. (C)The obtained PCC values for non-supplemented and (D) FO-supplemented animals from n>90 plaques from 8 animals for each group. (E) The RealTime PCR analysis of TNFα an IL-1β. (F) TNFa protein levels as revealed by ELISA Scale bar (A,B) - 10μm.
Fig 5.
FO supplementation affects the total number of microglia/macrophages, average number of plaque associated microglia/macrophages and plaque envelopment.
(A) Representative confocal images of Iba-1 immunolabeled microglia/macrophage in parietal cortex in untreated and (B) FO treated 5xFAD mouse. (C) Quantification of number of Iba-1 positive microglia/macrophages in parietal cortex of untreated vs. treated 5xFAD mice in from 8 animals for each group. Data represents mean ± s.e.m.(D) Representative confocal images of Iba-1 immunolabeled microglia/macrophages around AmyloGlo-labeled amyloid plaques in untreated and (E) FOtreated 5xFAD mice, larger magnification. (F) Quantification of number of plaque-associated microglia/macrophage in untreated vs. treated 5xFAD mice in n>90 plaques from 8 animals for each group. Data represents mean ± s.e.m. (G) Representative confocal images of Iba-1 immunolabeled microglial/macrophage processes (green) contacting AmyloGlo-labeled amyloid plaques in untreated and (H) FO-treated 5xFAD mice. (I) Microglia/macrophage coverage was quantified as the percentage of plaque perimeter contacted by microglial/macrophage processes in n>90 plaques from 8 animals for each group. Data represents mean ± s.e.m. (J,K) Representative confocal images of the degree of microglia coverage (Iba-1, green) versus extent of neuritic dystrophy (SMI31, red) around the plaque. (L,M) Representative confocal images of Iba-1 immunolabeled microglia/macrophages (green) and Aβ42 (red) mutual exclusion. Scale bar (A, B) - 50μm. Scale bar (D,E,G,H)– 10 μm. Scale bar (J,K,L-M)– 5μm.