Limits...
Using Human iPSC-Derived Neurons to Model TAU Aggregation.

Verheyen A, Diels A, Dijkmans J, Oyelami T, Meneghello G, Mertens L, Versweyveld S, Borgers M, Buist A, Peeters P, Cik M - PLoS ONE (2015)

Bottom Line: TAU aggregation and phosphorylation was quantified using AlphaLISA technology.To validate our model, activity of two autophagy inducers was tested.Both rapamycin and trehalose significantly reduced TAU aggregation levels suggesting that iPSC-derived neurons allow for the generation of a biologically relevant human Tauopathy model, highly suitable to screen for compounds that modulate TAU aggregation.

View Article: PubMed Central - PubMed

Affiliation: Janssen Research & Development, a division of Janssen Pharmaceutica N.V, Beerse, Belgium.

ABSTRACT
Alzheimer's disease and frontotemporal dementia are amongst the most common forms of dementia characterized by the formation and deposition of abnormal TAU in the brain. In order to develop a translational human TAU aggregation model suitable for screening, we transduced TAU harboring the pro-aggregating P301L mutation into control hiPSC-derived neural progenitor cells followed by differentiation into cortical neurons. TAU aggregation and phosphorylation was quantified using AlphaLISA technology. Although no spontaneous aggregation was observed upon expressing TAU-P301L in neurons, seeding with preformed aggregates consisting of the TAU-microtubule binding repeat domain triggered robust TAU aggregation and hyperphosphorylation already after 2 weeks, without affecting general cell health. To validate our model, activity of two autophagy inducers was tested. Both rapamycin and trehalose significantly reduced TAU aggregation levels suggesting that iPSC-derived neurons allow for the generation of a biologically relevant human Tauopathy model, highly suitable to screen for compounds that modulate TAU aggregation.

Show MeSH

Related in: MedlinePlus

Model validation: autophagy inducers reduce TAU hyperphosphorylation and aggregation in TAU-P301L neurons.(A) CellTiter-Glo® data showing that rapamycin is not toxic (P = NS). (B, C) Rapamycin dose-dependently reduces hTAU10 (P<0,001; B) and AT8 aggregated TAU measured with AlphaLISA (P = 0,025 at 10 nM and P<0.001 at 1 μM; C) and compared to DMSO. (D, E) Also general TAU phosphorylation is reduced (AT8/hTAU10; P<0,001; D) to a similar extent as the reduction in total TAU (HT7/hTAU10; P<0,001; E). (F) CellTiter-Glo® results show that trehalose is highly toxic at 250 mM (P<0,001). (G, H) AlphaLISA results reveal that trehalose significantly reduces hTAU10 (P<0,001) and AT8 TAU aggregation levels versus control (P = 0,006 at 31,5 mM and P = 0,014 at 125 mM). (I, J) Only at 125 mM of trehalose, both phosphorylated TAU (P<0,001, I) and total TAU levels (P<0,001, J) are decreased. ***P<0,001; **P<0,01; *P<0,05; #P<0,001 due to toxicity; 1-way ANOVA with Dunnett’s post hoc; n≥3 independent experiments
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4697850&req=5

pone.0146127.g005: Model validation: autophagy inducers reduce TAU hyperphosphorylation and aggregation in TAU-P301L neurons.(A) CellTiter-Glo® data showing that rapamycin is not toxic (P = NS). (B, C) Rapamycin dose-dependently reduces hTAU10 (P<0,001; B) and AT8 aggregated TAU measured with AlphaLISA (P = 0,025 at 10 nM and P<0.001 at 1 μM; C) and compared to DMSO. (D, E) Also general TAU phosphorylation is reduced (AT8/hTAU10; P<0,001; D) to a similar extent as the reduction in total TAU (HT7/hTAU10; P<0,001; E). (F) CellTiter-Glo® results show that trehalose is highly toxic at 250 mM (P<0,001). (G, H) AlphaLISA results reveal that trehalose significantly reduces hTAU10 (P<0,001) and AT8 TAU aggregation levels versus control (P = 0,006 at 31,5 mM and P = 0,014 at 125 mM). (I, J) Only at 125 mM of trehalose, both phosphorylated TAU (P<0,001, I) and total TAU levels (P<0,001, J) are decreased. ***P<0,001; **P<0,01; *P<0,05; #P<0,001 due to toxicity; 1-way ANOVA with Dunnett’s post hoc; n≥3 independent experiments

Mentions: To validate our assay for screening purposes, we selected two autophagy-inducing compounds rapamycin and trehalose described to reduce TAU aggregation and phosphorylation in vitro and in vivo [22–25] and tested different concentrations in the human iPSC-derived neuronal TAU aggregation assay. Rapamycin at the concentrations tested appears to be not toxic for the cells (Fig 5A) and induces a concentration-dependent reduction in TAU aggregation (Fig 5B and 5C). At 1 μM we observe an approximately 32% decrease in hTAU10/hTAU10 (Fig 5B) signals while phosphorylated aggregates (AT8/AT8) are reduced by 16% (Fig 5C). Furthermore, phosphorylation at the AT8 epitope is inhibited by almost 50% (Fig 5D). Notably, total TAU is also reduced (Fig 5E). Trehalose shows significant toxicity at 250 mM and a trend at 125 mM (P = NS) (Fig 5F). The lowest tested concentration significantly reduces both hTAU10 and AT8 TAU aggregation Fig 5G and 5H). Only at 125 mM of trehalose, a reduction of TAU phosphorylation and total TAU (Fig 5I and 5J) levels are observed.


Using Human iPSC-Derived Neurons to Model TAU Aggregation.

Verheyen A, Diels A, Dijkmans J, Oyelami T, Meneghello G, Mertens L, Versweyveld S, Borgers M, Buist A, Peeters P, Cik M - PLoS ONE (2015)

Model validation: autophagy inducers reduce TAU hyperphosphorylation and aggregation in TAU-P301L neurons.(A) CellTiter-Glo® data showing that rapamycin is not toxic (P = NS). (B, C) Rapamycin dose-dependently reduces hTAU10 (P<0,001; B) and AT8 aggregated TAU measured with AlphaLISA (P = 0,025 at 10 nM and P<0.001 at 1 μM; C) and compared to DMSO. (D, E) Also general TAU phosphorylation is reduced (AT8/hTAU10; P<0,001; D) to a similar extent as the reduction in total TAU (HT7/hTAU10; P<0,001; E). (F) CellTiter-Glo® results show that trehalose is highly toxic at 250 mM (P<0,001). (G, H) AlphaLISA results reveal that trehalose significantly reduces hTAU10 (P<0,001) and AT8 TAU aggregation levels versus control (P = 0,006 at 31,5 mM and P = 0,014 at 125 mM). (I, J) Only at 125 mM of trehalose, both phosphorylated TAU (P<0,001, I) and total TAU levels (P<0,001, J) are decreased. ***P<0,001; **P<0,01; *P<0,05; #P<0,001 due to toxicity; 1-way ANOVA with Dunnett’s post hoc; n≥3 independent experiments
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4697850&req=5

pone.0146127.g005: Model validation: autophagy inducers reduce TAU hyperphosphorylation and aggregation in TAU-P301L neurons.(A) CellTiter-Glo® data showing that rapamycin is not toxic (P = NS). (B, C) Rapamycin dose-dependently reduces hTAU10 (P<0,001; B) and AT8 aggregated TAU measured with AlphaLISA (P = 0,025 at 10 nM and P<0.001 at 1 μM; C) and compared to DMSO. (D, E) Also general TAU phosphorylation is reduced (AT8/hTAU10; P<0,001; D) to a similar extent as the reduction in total TAU (HT7/hTAU10; P<0,001; E). (F) CellTiter-Glo® results show that trehalose is highly toxic at 250 mM (P<0,001). (G, H) AlphaLISA results reveal that trehalose significantly reduces hTAU10 (P<0,001) and AT8 TAU aggregation levels versus control (P = 0,006 at 31,5 mM and P = 0,014 at 125 mM). (I, J) Only at 125 mM of trehalose, both phosphorylated TAU (P<0,001, I) and total TAU levels (P<0,001, J) are decreased. ***P<0,001; **P<0,01; *P<0,05; #P<0,001 due to toxicity; 1-way ANOVA with Dunnett’s post hoc; n≥3 independent experiments
Mentions: To validate our assay for screening purposes, we selected two autophagy-inducing compounds rapamycin and trehalose described to reduce TAU aggregation and phosphorylation in vitro and in vivo [22–25] and tested different concentrations in the human iPSC-derived neuronal TAU aggregation assay. Rapamycin at the concentrations tested appears to be not toxic for the cells (Fig 5A) and induces a concentration-dependent reduction in TAU aggregation (Fig 5B and 5C). At 1 μM we observe an approximately 32% decrease in hTAU10/hTAU10 (Fig 5B) signals while phosphorylated aggregates (AT8/AT8) are reduced by 16% (Fig 5C). Furthermore, phosphorylation at the AT8 epitope is inhibited by almost 50% (Fig 5D). Notably, total TAU is also reduced (Fig 5E). Trehalose shows significant toxicity at 250 mM and a trend at 125 mM (P = NS) (Fig 5F). The lowest tested concentration significantly reduces both hTAU10 and AT8 TAU aggregation Fig 5G and 5H). Only at 125 mM of trehalose, a reduction of TAU phosphorylation and total TAU (Fig 5I and 5J) levels are observed.

Bottom Line: TAU aggregation and phosphorylation was quantified using AlphaLISA technology.To validate our model, activity of two autophagy inducers was tested.Both rapamycin and trehalose significantly reduced TAU aggregation levels suggesting that iPSC-derived neurons allow for the generation of a biologically relevant human Tauopathy model, highly suitable to screen for compounds that modulate TAU aggregation.

View Article: PubMed Central - PubMed

Affiliation: Janssen Research & Development, a division of Janssen Pharmaceutica N.V, Beerse, Belgium.

ABSTRACT
Alzheimer's disease and frontotemporal dementia are amongst the most common forms of dementia characterized by the formation and deposition of abnormal TAU in the brain. In order to develop a translational human TAU aggregation model suitable for screening, we transduced TAU harboring the pro-aggregating P301L mutation into control hiPSC-derived neural progenitor cells followed by differentiation into cortical neurons. TAU aggregation and phosphorylation was quantified using AlphaLISA technology. Although no spontaneous aggregation was observed upon expressing TAU-P301L in neurons, seeding with preformed aggregates consisting of the TAU-microtubule binding repeat domain triggered robust TAU aggregation and hyperphosphorylation already after 2 weeks, without affecting general cell health. To validate our model, activity of two autophagy inducers was tested. Both rapamycin and trehalose significantly reduced TAU aggregation levels suggesting that iPSC-derived neurons allow for the generation of a biologically relevant human Tauopathy model, highly suitable to screen for compounds that modulate TAU aggregation.

Show MeSH
Related in: MedlinePlus