Fig 2: APP/PS1/TRPC6 mice showed improvement in spatial learning ability at 11 months.(a) Representative images of Golgi staining of hippocampal CA1 neurons in the four groups of mice at 11 month. Scale bar, 5 µm. (b) Quantification of spine number per 10 µm of dendrite (n=40 neurons from 3–5 mice). (c) Cumulative probability analysis of the spine density shown in b. (d) Escape latency of the four groups of mice at 11 months in the training session of Morris water maze (n=15–19). (e) Platform crossing number of the mice at 11 month in the probe session of Morris water maze (n=14–19). Data were presented as means±s.e.m. One way ANOVA with Newman–Keuls post hoc test was performed for spine quantification and repeated measures and multivariate analysis in general linear model with LSD post hoc test was performed for behavioural study. *P<0.05, **P<0.01, ***P<0.001 versus WT or APP/PS1.

Fig 3: TAT-TM2 reduced Aß levels in vitro and in vivo.(a) ELISA examination of Aß levels in the medium of HEK293APP cells transfected with TRPC6 or its mutants within second transmembrane domain (TM2) for 2 days (n=3–6). (b) Lysates of HEK293APP stable cells transfected with C6-mut1 for 2 days precipitated with APP antibody and immunoblotted with indicated antibodies. (c) The Aß levels in the medium of HEK293APP cells treated with 2 µM indicated peptides for 12 h (n=3–6). (d) Lysates of HEK293APP stable cells incubated with 5 µM TAT-biotin or TAT-TM2-bitoin for 6 h precipitated with avidin beads and immunoblotted with indicated antibodies. (e) ELISA examination in a blinded manner of Aß levels in the brain lysates of 5 month male APP/PS1 mice intra-peritoneally injected with 200 µl 2.5 mM TAT or TAT-TM2 for 3 h (n=8 mice). (f) The Aß levels generated in the in vitro purified C99 cleavage assay treated with 2 µM of indicated peptides for 2 h (n=4). (g) Aß levels in the medium of COS7C99 stable cells treated with 5 µM TAT-TM2 for 12 h (n=3–4). (h) Immunoblots of AICD in HEK293C99 cells treated with 5 µM TAT-TM2 for 12 h or NICD in HEK293APP cells transfected with Notch?E-myc for 1 day and then treated with 5 µM TAT-TM2 for 12 h. (i) Quantification of the ratio of AICD/C99 or NICD/Notch?E (n=3–4). CTRL, transfection with YFP. Data were presented as means±s.e.m. of indicated numbers of independent experiments. Two-tailed Student's t-test was performed for two groups, and one way ANOVA with Newman–Keuls post hoc test was performed for more than two groups. *P<0.05, ***P<0.001 versus CTRL or TAT.

Fig 4: TRPC6 interacted with APP (C99).(a) Immunocytochemical analysis of HEK293 cells transfected with APP-Myc and TRPC6-HA for 3 days. EEA1, Calnexin and GM130 are markers for early endosome, ER and Golgi, respectively. Arrow heads indicated the co-localized signals. Scale bar, 20 µm. (b) Immunoblots of indicated proteins after fractionation of mouse brain lysates. Bip and Cathepsin D are markers for ER and lysosome, respectively. Arrow indicated alignment of two gels with 11 lanes running in parallel. (c) Wild-type mouse brain lysates precipitated with the antibody against APP, PS1 or Notch, and immunoblotted with indicated antibodies. (d) Lysates of HEK293APP stable cells transfected with TRPC5 or 6 for 2 days precipitated with APP antibody, and immunoblotted with indicated antibodies. (e) Lysates of HEK293TRPC6 stable cells transfected with C99-Myc or Notch?E-Myc for 1 day, treated with 10 µM L685,458 for 12 h and then precipitated with Myc or TRPC6 antibody, and immunoblotted with indicated antibodies. (f) Lysates of HEK293C99-Myc stable cells transfected with PS1-HA together with YFP or TRPC6 for 2 days and precipitated with Myc or HA antibody, and immunoblotted with indicated antibodies. Lower, quantification of the reciprocal precipitated protein levels of C99 and PS1 (n=4). CTRL, transfection with YFP. Data were presented as means±s.e.m. of indicated numbers of independent experiments. Two-tailed Student's t-test was performed. *P<0.05, **P<0.01 versus CTRL.

Fig 5: The Aß levels were decreased in APP/PS1/TRPC6 mice.(a) Representative images of amyloid plaque immunostained with Aß antibody (6E10) on brain sections from 6 months female APP/PS1 and APP/PS1/TRPC6 mice. Scale bar, 500 µm. (b) Quantification of the plaque load in indicated mouse brain sections shown in a (n=8). ELISA of total (c) TBST soluble (d) or insoluble (e) Aß levels in the forebrain lysates of 6 month female indicated mice (n=8). Representative images (f) and quantification (g) of thioflavin S staining of amyloid plaque in the hippocampal region of 6 and 11 months APP/PS1 and APP/PS1/TRPC6 mice with B6C3 background (n=4–5 mice). Scale bar, 200 µm. Data were presented as means±s.e.m. Two-tailed Student's t-test was performed. *P<0.05, **P<0.01 versus APP/PS1.