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TRPC6 specifically interacts with APP to inhibit its cleavage by γ-secretase and reduce Aβ production.

Wang J, Lu R, Yang J, Li H, He Z, Jing N, Wang X, Wang Y - Nat Commun (2015)

Bottom Line: A fusion peptide derived from TRPC6 also reduces Aβ levels without effect on Notch cleavage.Crossing APP/PS1 mice with TRPC6 transgenic mice leads to a marked reduction in both plaque load and Aβ levels, and improvement in structural and behavioural impairment.Thus, TRPC6 specifically modulates γ-secretase cleavage of APP and preventing APP (C99) interaction with PS1 via TRPC6 could be a novel strategy to reduce Aβ formation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Neural Signal Transduction, Institute of Neuroscience, State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, SIBS, CAS, 320 Yue-Yang Road, Shanghai 200031, China.

ABSTRACT
Generation of β-amyloid (Aβ) peptide in Alzheimer's disease involves cleavage of amyloid precursor protein (APP) by γ-secretase, a protease known to cleave several substrates, including Notch. Finding specific modulators for γ-secretase could be a potential avenue to treat the disease. Here, we report that transient receptor potential canonical (TRPC) 6 specifically interacts with APP leading to inhibition of its cleavage by γ-secretase and reduction in Aβ production. TRPC6 interacts with APP (C99), but not with Notch, and prevents C99 interaction with presenilin 1 (PS1). A fusion peptide derived from TRPC6 also reduces Aβ levels without effect on Notch cleavage. Crossing APP/PS1 mice with TRPC6 transgenic mice leads to a marked reduction in both plaque load and Aβ levels, and improvement in structural and behavioural impairment. Thus, TRPC6 specifically modulates γ-secretase cleavage of APP and preventing APP (C99) interaction with PS1 via TRPC6 could be a novel strategy to reduce Aβ formation.

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TRPC6 inhibited γ-secretase cleavage of APP (C99), but not that of other substrates.(a) Aβ levels in the medium of COS7C99 stable cells transfected with YFP or TRPC6 for 2 days (n=3–4). (b) Immunoblot analysis of AICD in HEK293C99 cells transfected with YFP or TRPC6 for 2 days. (c) Quantification of the ratio of AICD/C99 (n=3). (d) Immunoblots of presenilin 1 (PS1), PS2, nicastrin (Nct) and presenilin enhance 2 (Pen2) in HEK293APP cells transfected with TRPC6 for 2 days. (e) γ-Secretase activity in the in vitro fluorogenic substrate assay of HEK293APP cells transfected with TRPC6 for 2 days (n=3–5). (f) Immunoblot analysis of Notch intracellular domain (NICD) in HEK293APP cells transfected with NotchΔE-myc for 1 day and then further transfected with YFP or TRPC6 for 2 days. (g) Quantification of the ratio of NICD/NotchΔE (n=4). (h) Immunoblots of CTF1 and CTF2 of E-/N-Cadherin in HEK293APP cells transfected with YFP or TRPC6 for 2 days. (i) Quantification of CTF2/CTF1 of E-/N-Cadherin (n=3–4). CTRL, transfection with YFP or pcDNA3.1. L658,458: γ-secretase inhibitor. 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.
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f2: TRPC6 inhibited γ-secretase cleavage of APP (C99), but not that of other substrates.(a) Aβ levels in the medium of COS7C99 stable cells transfected with YFP or TRPC6 for 2 days (n=3–4). (b) Immunoblot analysis of AICD in HEK293C99 cells transfected with YFP or TRPC6 for 2 days. (c) Quantification of the ratio of AICD/C99 (n=3). (d) Immunoblots of presenilin 1 (PS1), PS2, nicastrin (Nct) and presenilin enhance 2 (Pen2) in HEK293APP cells transfected with TRPC6 for 2 days. (e) γ-Secretase activity in the in vitro fluorogenic substrate assay of HEK293APP cells transfected with TRPC6 for 2 days (n=3–5). (f) Immunoblot analysis of Notch intracellular domain (NICD) in HEK293APP cells transfected with NotchΔE-myc for 1 day and then further transfected with YFP or TRPC6 for 2 days. (g) Quantification of the ratio of NICD/NotchΔE (n=4). (h) Immunoblots of CTF1 and CTF2 of E-/N-Cadherin in HEK293APP cells transfected with YFP or TRPC6 for 2 days. (i) Quantification of CTF2/CTF1 of E-/N-Cadherin (n=3–4). CTRL, transfection with YFP or pcDNA3.1. L658,458: γ-secretase inhibitor. 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.

Mentions: We then examined whether γ-secretase cleavage of APP was affected by TRPC6. Cleavage of C99 by γ-secretase releases Aβ peptides and APP intracellular domain (AICD)28. TRPC6 greatly reduced both Aβ40 and Aβ42 levels in the culture medium of COS7 cells stably expressing C99 (Fig. 2a). TRPC6 also downregulated AICD generation in HEK293C99 stable cells (Supplementary Figs 2g, 11 and 15; Fig. 2b,c). In addition, TRPC6 did not affect CTF (C-terminal fragments of APP) levels either in HEK293APP cells (Supplementary Fig. 2h,I; Supplementary Fig. 15) or in APP/PS1 mice (Supplementary Fig. 2j,k; Supplementary Fig. 15). Further, expression of PS1, PS2, nicastrin or presenilin enhancer 2, proteins known as the γ-secretase components, was not changed by TRPC6 (Fig. 2d; Supplementary Figs 2l and 11). Consistently, the γ-secretase activity determined by in vitro assay using the fluorogenic substrate was unchanged by TRPC6 (Fig. 2e). We also examined whether TRPC6 affected γ-secretase cleavage of Notch by detecting Notch intracellular domain level generated from NotchΔE, an immediate substrate of γ-secretase29. As shown in Fig. 2f,g and Supplementary Fig. 11, TRPC6 did not alter the Notch intracellular domain level, suggesting that TRPC6 did not inhibit γ-secretase cleavage of Notch. We further examined the CTF1 and CTF2 levels of E-/N-Cadherin, known as the endogenous substrates of γ-secretase. As shown in Fig. 2h,i and Supplementary Fig. 11, TRPC6 did not alter the CTF2/CTF1 ratio of E-/N-Cadherin, suggesting that TRPC6 did not inhibit γ-secretase cleavage of E-/N-Cadherin. Together, these results suggest that TRPC6 specifically reduces γ-secretase cleavage of C99, but does not act as a general γ-secretase inhibitor.


TRPC6 specifically interacts with APP to inhibit its cleavage by γ-secretase and reduce Aβ production.

Wang J, Lu R, Yang J, Li H, He Z, Jing N, Wang X, Wang Y - Nat Commun (2015)

TRPC6 inhibited γ-secretase cleavage of APP (C99), but not that of other substrates.(a) Aβ levels in the medium of COS7C99 stable cells transfected with YFP or TRPC6 for 2 days (n=3–4). (b) Immunoblot analysis of AICD in HEK293C99 cells transfected with YFP or TRPC6 for 2 days. (c) Quantification of the ratio of AICD/C99 (n=3). (d) Immunoblots of presenilin 1 (PS1), PS2, nicastrin (Nct) and presenilin enhance 2 (Pen2) in HEK293APP cells transfected with TRPC6 for 2 days. (e) γ-Secretase activity in the in vitro fluorogenic substrate assay of HEK293APP cells transfected with TRPC6 for 2 days (n=3–5). (f) Immunoblot analysis of Notch intracellular domain (NICD) in HEK293APP cells transfected with NotchΔE-myc for 1 day and then further transfected with YFP or TRPC6 for 2 days. (g) Quantification of the ratio of NICD/NotchΔE (n=4). (h) Immunoblots of CTF1 and CTF2 of E-/N-Cadherin in HEK293APP cells transfected with YFP or TRPC6 for 2 days. (i) Quantification of CTF2/CTF1 of E-/N-Cadherin (n=3–4). CTRL, transfection with YFP or pcDNA3.1. L658,458: γ-secretase inhibitor. 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.
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f2: TRPC6 inhibited γ-secretase cleavage of APP (C99), but not that of other substrates.(a) Aβ levels in the medium of COS7C99 stable cells transfected with YFP or TRPC6 for 2 days (n=3–4). (b) Immunoblot analysis of AICD in HEK293C99 cells transfected with YFP or TRPC6 for 2 days. (c) Quantification of the ratio of AICD/C99 (n=3). (d) Immunoblots of presenilin 1 (PS1), PS2, nicastrin (Nct) and presenilin enhance 2 (Pen2) in HEK293APP cells transfected with TRPC6 for 2 days. (e) γ-Secretase activity in the in vitro fluorogenic substrate assay of HEK293APP cells transfected with TRPC6 for 2 days (n=3–5). (f) Immunoblot analysis of Notch intracellular domain (NICD) in HEK293APP cells transfected with NotchΔE-myc for 1 day and then further transfected with YFP or TRPC6 for 2 days. (g) Quantification of the ratio of NICD/NotchΔE (n=4). (h) Immunoblots of CTF1 and CTF2 of E-/N-Cadherin in HEK293APP cells transfected with YFP or TRPC6 for 2 days. (i) Quantification of CTF2/CTF1 of E-/N-Cadherin (n=3–4). CTRL, transfection with YFP or pcDNA3.1. L658,458: γ-secretase inhibitor. 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.
Mentions: We then examined whether γ-secretase cleavage of APP was affected by TRPC6. Cleavage of C99 by γ-secretase releases Aβ peptides and APP intracellular domain (AICD)28. TRPC6 greatly reduced both Aβ40 and Aβ42 levels in the culture medium of COS7 cells stably expressing C99 (Fig. 2a). TRPC6 also downregulated AICD generation in HEK293C99 stable cells (Supplementary Figs 2g, 11 and 15; Fig. 2b,c). In addition, TRPC6 did not affect CTF (C-terminal fragments of APP) levels either in HEK293APP cells (Supplementary Fig. 2h,I; Supplementary Fig. 15) or in APP/PS1 mice (Supplementary Fig. 2j,k; Supplementary Fig. 15). Further, expression of PS1, PS2, nicastrin or presenilin enhancer 2, proteins known as the γ-secretase components, was not changed by TRPC6 (Fig. 2d; Supplementary Figs 2l and 11). Consistently, the γ-secretase activity determined by in vitro assay using the fluorogenic substrate was unchanged by TRPC6 (Fig. 2e). We also examined whether TRPC6 affected γ-secretase cleavage of Notch by detecting Notch intracellular domain level generated from NotchΔE, an immediate substrate of γ-secretase29. As shown in Fig. 2f,g and Supplementary Fig. 11, TRPC6 did not alter the Notch intracellular domain level, suggesting that TRPC6 did not inhibit γ-secretase cleavage of Notch. We further examined the CTF1 and CTF2 levels of E-/N-Cadherin, known as the endogenous substrates of γ-secretase. As shown in Fig. 2h,i and Supplementary Fig. 11, TRPC6 did not alter the CTF2/CTF1 ratio of E-/N-Cadherin, suggesting that TRPC6 did not inhibit γ-secretase cleavage of E-/N-Cadherin. Together, these results suggest that TRPC6 specifically reduces γ-secretase cleavage of C99, but does not act as a general γ-secretase inhibitor.

Bottom Line: A fusion peptide derived from TRPC6 also reduces Aβ levels without effect on Notch cleavage.Crossing APP/PS1 mice with TRPC6 transgenic mice leads to a marked reduction in both plaque load and Aβ levels, and improvement in structural and behavioural impairment.Thus, TRPC6 specifically modulates γ-secretase cleavage of APP and preventing APP (C99) interaction with PS1 via TRPC6 could be a novel strategy to reduce Aβ formation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Neural Signal Transduction, Institute of Neuroscience, State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, SIBS, CAS, 320 Yue-Yang Road, Shanghai 200031, China.

ABSTRACT
Generation of β-amyloid (Aβ) peptide in Alzheimer's disease involves cleavage of amyloid precursor protein (APP) by γ-secretase, a protease known to cleave several substrates, including Notch. Finding specific modulators for γ-secretase could be a potential avenue to treat the disease. Here, we report that transient receptor potential canonical (TRPC) 6 specifically interacts with APP leading to inhibition of its cleavage by γ-secretase and reduction in Aβ production. TRPC6 interacts with APP (C99), but not with Notch, and prevents C99 interaction with presenilin 1 (PS1). A fusion peptide derived from TRPC6 also reduces Aβ levels without effect on Notch cleavage. Crossing APP/PS1 mice with TRPC6 transgenic mice leads to a marked reduction in both plaque load and Aβ levels, and improvement in structural and behavioural impairment. Thus, TRPC6 specifically modulates γ-secretase cleavage of APP and preventing APP (C99) interaction with PS1 via TRPC6 could be a novel strategy to reduce Aβ formation.

Show MeSH
Related in: MedlinePlus