Fig 1: Effects of circulating amylin modulation on AD-like pathology in vivo and in human amylin-treated neonatal neurons(A–D) Heat maps comparing the brain tissue levels of pT231-tau, tau, Aβ40, and Aβ42 in the same mice as in Figures 2, 3, 4, 5, and 6 (i.e., hAON vs. hAi−OFF mice and hAOFF vs. hAi−ON mice).(E) Aβ immunofluorescence signal for Aβ (green) of cultured primary neurons incubated with human amylin (hAmylin; 1 μM for 4 h) or under control conditions (control). MAP2 (blue) was used to identify the neurons. n = 86 neurons from 3 primary neonatal rat neuron cultures.(F) Total Aβ level secreted in the culture media by primary neurons incubated with human amylin (hAmylin; 1 μM for 2 h) or under control conditions (control). Aβ was enriched by immunoprecipitation and measured by Western blot.(G) pTau and total Tau lysates from primary neurons incubated with human amylin (hAmylin; 1 μM for 2 h) or under control conditions (control). pTau and Tau were enriched by immunoprecipitation and measured by western blot. Individual data points and mean ± s.e.m for three neuronal cultures.(H) Effect of amylin on pS214-tau in cultured murine astrocytes. Cells were incubated for 30 min under control conditions or in the presence of human amylin (15 μM) with and without the amylin receptor antagonist AC187 (10 μM) or PKA inhibitor H-89 (10 μM). pS214-tau was measured by ELISA. Individual data points and mean ± s.e.m for three cell cultures.(I) Representative images of IHC analyses of cortical slices from hAON mice stained for amylin (top) and for total Aβ (bottom) (n = 5 slices/mouse from n = 3 mice). Statistical analyses were performed using one-way ANOVA followed by Dunnett’s multiple-comparisons test (H) and two-tail t test (E–G). See also Figures S4–S6.
Fig 2: Cerebral glycolysis impairment and AD-like pathology in rats with genetically elevated pancreatic human amylin secretion(A) Schematic of the experimental approach for assessing cerebral glycolytic flux, Aβ40, Aβ42, pTau, and total tau levels in rats expressing WT rat amylin vs. pancreatic human amylin (HIP rats) vs. amylin knockout (AKO) rats. All rats were maintained on chow diet through the endpoint (16 months of age).(B) Endpoint blood glucose concentrations in HIP, WT, and AKO rats.(C) Brain tissue amylin levels in HIP and WT rats measured at the endpoint.(D–F) Comparative analyses of brain tissue G6P levels (D), glycolytic amino acids (Ser), glycine (Gly), and alanine (Ala) (E) and cerebral glycolytic flux (F) in the same rats as in (B).(G–J) Brain tissue levels of Aβ40, Aβ42, pTau, and total Tau in the same rats as in (B).(K and L) Representative images of immunohistochemistry analysis of pTau in HIP brain tissue (K) and confocal microscopy analysis of brain sections from the same rats stained with a combination of anti-amylin and anti-pTau antibodies (L). Three sections/brain. The diagram in (A) was created using BioRender. Data are mean ± s.e.m from 7 to 10 male mice/group. Statistical analyses were performed using One-way ANOVA followed by Dunnett’s multiple-comparisons test (B and D–J) and two-tail t test (C).
from Cell Signaling Technology for PathScan ® Phospho-Tau (Ser214) Sandwich ELISA Kit