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Induction of autophagy by a novel small molecule improves aβ pathology and ameliorates cognitive deficits.

Chu C, Zhang X, Ma W, Li L, Wang W, Shang L, Fu P - PLoS ONE (2013)

Bottom Line: GTM-1 modulates autophagy in an Akt-independent and mTOR-independent manner.In addition, we demonstrated that GTM-1 enhances autophagy clearance and reverses the downregulation of autophagy flux by thapsigargin and asparagine.Furthermore, administration of GTM-1 attenuated Aβ pathology and ameliorated cognitive deficits in AD mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, The First People's Hospital of Yangzhou, Jiang Su, PR China.

ABSTRACT
Growing evidence has demonstrated a neuroprotective role of autophagy in Alzheimer's disease (AD). Thus, autophagy has been regarded as a potential therapeutic target, attracting increasing interest in pharmaceutical autophagy modulation by small molecules. We designed a two-cycle screening strategy on the basis of imaging high-throughout screening (HTS) and cellular toxicity assay, and have identified a novel autophagy inducer known as GTM-1. We further showed that GTM-1 exhibits dual activities, such as autophagy induction and antagonism against Aβ-oligomer toxicity. GTM-1 modulates autophagy in an Akt-independent and mTOR-independent manner. In addition, we demonstrated that GTM-1 enhances autophagy clearance and reverses the downregulation of autophagy flux by thapsigargin and asparagine. Furthermore, administration of GTM-1 attenuated Aβ pathology and ameliorated cognitive deficits in AD mice.

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Related in: MedlinePlus

GTM-1 modulates autophagy in neurons using a different mechanism from rapamycin.A–B, SH-SY5Y cells were treated with the indicated compounds for 12 hrs. Next, the cell lysates were analyzed using western blotting with specific antibodies. DMSO (0.1%), GTM-1 (20 µM), Rap: Rapamycin (0.2 µM), A-AM (0.1 µM). β-tubulin was used a control. C–D, SH-SY5Y (4C) or MC-65 (4D) cells were incubated with GTM-1 (20 µM) for the indicated times. Cells treated with vehicle (0.1% DMSO) for 24 hrs were used as a negative control (NC). Cell viability was assessed using MTT, and all of the data were normalized to the NC. E, MC65 cells were grown in the presence (Tet+) or absence (Tet−) of tetracycline and under Tet− with GTM-1 (20 µM) for 8 hrs. The indicated compounds were then added within 2 hrs. Soluble Aβ oligomers were assessed using ELISA in MC65 cells. Bafilomycin (100 nM), NH4Cl (10 mM). F, MC65 cells were grown in the presence (Tet+) or absence (Tet−) of tetracycline and under Tet− with GTM-1 (at indicated concentrations) for 5 hrs, and the indicated compounds were added within 5 hrs. The soluble Aβ oligomers were assessed using ELISA. Rap: Rapamycin (at the indicated concentrations), thapsigargin (3 µM). Stars indicate significant differences between the model group and control or treatment groups for specific time points. *P<0.05, **P<0.01.
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pone-0065367-g004: GTM-1 modulates autophagy in neurons using a different mechanism from rapamycin.A–B, SH-SY5Y cells were treated with the indicated compounds for 12 hrs. Next, the cell lysates were analyzed using western blotting with specific antibodies. DMSO (0.1%), GTM-1 (20 µM), Rap: Rapamycin (0.2 µM), A-AM (0.1 µM). β-tubulin was used a control. C–D, SH-SY5Y (4C) or MC-65 (4D) cells were incubated with GTM-1 (20 µM) for the indicated times. Cells treated with vehicle (0.1% DMSO) for 24 hrs were used as a negative control (NC). Cell viability was assessed using MTT, and all of the data were normalized to the NC. E, MC65 cells were grown in the presence (Tet+) or absence (Tet−) of tetracycline and under Tet− with GTM-1 (20 µM) for 8 hrs. The indicated compounds were then added within 2 hrs. Soluble Aβ oligomers were assessed using ELISA in MC65 cells. Bafilomycin (100 nM), NH4Cl (10 mM). F, MC65 cells were grown in the presence (Tet+) or absence (Tet−) of tetracycline and under Tet− with GTM-1 (at indicated concentrations) for 5 hrs, and the indicated compounds were added within 5 hrs. The soluble Aβ oligomers were assessed using ELISA. Rap: Rapamycin (at the indicated concentrations), thapsigargin (3 µM). Stars indicate significant differences between the model group and control or treatment groups for specific time points. *P<0.05, **P<0.01.

Mentions: Previous reports have shown that mTOR is hyperactive in select neurons in AD brains, although the link between mTOR hyperactivity and AD progression is still unclear. Rapamycin and several other autophagy inducers, such as curcumin [32], have been found to induce autophagy by inhibiting the Akt/mTOR/p70S6K pathway. To test whether GTM-1 exhibits a similar ability to inhibit Akt, the phosphorylation levels of Akt (phosphoryl-threonine 308) were assayed using the known Akt inhibitor A-AM (Sigma) as a positive control [33]. As shown in Figure 4A, in contrast to A-AM, GTM-1 treatment had no effects on the phosphorylation levels of Akt. Further assays also demonstrated that GTM-1 induced autophagy, but did not stimulate phosphorylation of mTOR or p70S6K (Fig. 4B). These results demonstrated that GTM-1 upregulated autophagy in neurons in an Akt- and mTOR independent manner.


Induction of autophagy by a novel small molecule improves aβ pathology and ameliorates cognitive deficits.

Chu C, Zhang X, Ma W, Li L, Wang W, Shang L, Fu P - PLoS ONE (2013)

GTM-1 modulates autophagy in neurons using a different mechanism from rapamycin.A–B, SH-SY5Y cells were treated with the indicated compounds for 12 hrs. Next, the cell lysates were analyzed using western blotting with specific antibodies. DMSO (0.1%), GTM-1 (20 µM), Rap: Rapamycin (0.2 µM), A-AM (0.1 µM). β-tubulin was used a control. C–D, SH-SY5Y (4C) or MC-65 (4D) cells were incubated with GTM-1 (20 µM) for the indicated times. Cells treated with vehicle (0.1% DMSO) for 24 hrs were used as a negative control (NC). Cell viability was assessed using MTT, and all of the data were normalized to the NC. E, MC65 cells were grown in the presence (Tet+) or absence (Tet−) of tetracycline and under Tet− with GTM-1 (20 µM) for 8 hrs. The indicated compounds were then added within 2 hrs. Soluble Aβ oligomers were assessed using ELISA in MC65 cells. Bafilomycin (100 nM), NH4Cl (10 mM). F, MC65 cells were grown in the presence (Tet+) or absence (Tet−) of tetracycline and under Tet− with GTM-1 (at indicated concentrations) for 5 hrs, and the indicated compounds were added within 5 hrs. The soluble Aβ oligomers were assessed using ELISA. Rap: Rapamycin (at the indicated concentrations), thapsigargin (3 µM). Stars indicate significant differences between the model group and control or treatment groups for specific time points. *P<0.05, **P<0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672196&req=5

pone-0065367-g004: GTM-1 modulates autophagy in neurons using a different mechanism from rapamycin.A–B, SH-SY5Y cells were treated with the indicated compounds for 12 hrs. Next, the cell lysates were analyzed using western blotting with specific antibodies. DMSO (0.1%), GTM-1 (20 µM), Rap: Rapamycin (0.2 µM), A-AM (0.1 µM). β-tubulin was used a control. C–D, SH-SY5Y (4C) or MC-65 (4D) cells were incubated with GTM-1 (20 µM) for the indicated times. Cells treated with vehicle (0.1% DMSO) for 24 hrs were used as a negative control (NC). Cell viability was assessed using MTT, and all of the data were normalized to the NC. E, MC65 cells were grown in the presence (Tet+) or absence (Tet−) of tetracycline and under Tet− with GTM-1 (20 µM) for 8 hrs. The indicated compounds were then added within 2 hrs. Soluble Aβ oligomers were assessed using ELISA in MC65 cells. Bafilomycin (100 nM), NH4Cl (10 mM). F, MC65 cells were grown in the presence (Tet+) or absence (Tet−) of tetracycline and under Tet− with GTM-1 (at indicated concentrations) for 5 hrs, and the indicated compounds were added within 5 hrs. The soluble Aβ oligomers were assessed using ELISA. Rap: Rapamycin (at the indicated concentrations), thapsigargin (3 µM). Stars indicate significant differences between the model group and control or treatment groups for specific time points. *P<0.05, **P<0.01.
Mentions: Previous reports have shown that mTOR is hyperactive in select neurons in AD brains, although the link between mTOR hyperactivity and AD progression is still unclear. Rapamycin and several other autophagy inducers, such as curcumin [32], have been found to induce autophagy by inhibiting the Akt/mTOR/p70S6K pathway. To test whether GTM-1 exhibits a similar ability to inhibit Akt, the phosphorylation levels of Akt (phosphoryl-threonine 308) were assayed using the known Akt inhibitor A-AM (Sigma) as a positive control [33]. As shown in Figure 4A, in contrast to A-AM, GTM-1 treatment had no effects on the phosphorylation levels of Akt. Further assays also demonstrated that GTM-1 induced autophagy, but did not stimulate phosphorylation of mTOR or p70S6K (Fig. 4B). These results demonstrated that GTM-1 upregulated autophagy in neurons in an Akt- and mTOR independent manner.

Bottom Line: GTM-1 modulates autophagy in an Akt-independent and mTOR-independent manner.In addition, we demonstrated that GTM-1 enhances autophagy clearance and reverses the downregulation of autophagy flux by thapsigargin and asparagine.Furthermore, administration of GTM-1 attenuated Aβ pathology and ameliorated cognitive deficits in AD mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, The First People's Hospital of Yangzhou, Jiang Su, PR China.

ABSTRACT
Growing evidence has demonstrated a neuroprotective role of autophagy in Alzheimer's disease (AD). Thus, autophagy has been regarded as a potential therapeutic target, attracting increasing interest in pharmaceutical autophagy modulation by small molecules. We designed a two-cycle screening strategy on the basis of imaging high-throughout screening (HTS) and cellular toxicity assay, and have identified a novel autophagy inducer known as GTM-1. We further showed that GTM-1 exhibits dual activities, such as autophagy induction and antagonism against Aβ-oligomer toxicity. GTM-1 modulates autophagy in an Akt-independent and mTOR-independent manner. In addition, we demonstrated that GTM-1 enhances autophagy clearance and reverses the downregulation of autophagy flux by thapsigargin and asparagine. Furthermore, administration of GTM-1 attenuated Aβ pathology and ameliorated cognitive deficits in AD mice.

Show MeSH
Related in: MedlinePlus