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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.

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Related in: MedlinePlus

Differentiation of hiPSC into cortical neurons and efficient transduction with AAV-TAU-P301L (A) Immunostaining for OCT4 and NANOG shows that iPSC0028 is pluripotent. Scale bar represents 50μm. (B-C) Immunostaining for Nestin and PAX6 revealing NPC stage at DIV25. Scale bar = 50μm for both. (D-F) Immunostaining on DIV70 visualizes the neuronal marker TUBB3 and cortical markers TBR1 and CTIP2 (D) as well as the dendritic marker MAP2 (E-F) together with either vGLUT2 (E) or vGAT (F). Scale bar = 25μm. (G-I) Representative traces of intrinsic neuronal properties of DIV70 neurons showing evoked responses in current clamp (G) as well as sodium and potassium currents (H) in voltage clamp (n = 13 cells). (I) Example of spontaneous EPSCs recorded at a holding of -65mV in the presence of 50μM PTX in voltage clamp mode. (J) Quantitative RTPCR data showing that transduced neurons express both 3R and 4R TAU mRNA, represented by an increased 4R/3R TAU ratio compared to non-transduced control cells (P = 0.04; n≥3 from different experiments). Values were normalized to PGK1 before analyses. * P<0,05 (K) Western Blot with a 4R TAU specific antibody depicts 2N4R TAU bands, only in transduced (2N4R-P301L) cells. TAU ladder and marker for band sizes are represented by (T) and (M) respectively. (L) Immunostaining with 4R TAU specific antibody confirms the presence of the 2N4R P301L TAU on the cellular level, only in transduced neurons, while total TAU (red) is present also in control neurons. Scale bar = 25 μm. DAPI stains the nuclei
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pone.0146127.g001: Differentiation of hiPSC into cortical neurons and efficient transduction with AAV-TAU-P301L (A) Immunostaining for OCT4 and NANOG shows that iPSC0028 is pluripotent. Scale bar represents 50μm. (B-C) Immunostaining for Nestin and PAX6 revealing NPC stage at DIV25. Scale bar = 50μm for both. (D-F) Immunostaining on DIV70 visualizes the neuronal marker TUBB3 and cortical markers TBR1 and CTIP2 (D) as well as the dendritic marker MAP2 (E-F) together with either vGLUT2 (E) or vGAT (F). Scale bar = 25μm. (G-I) Representative traces of intrinsic neuronal properties of DIV70 neurons showing evoked responses in current clamp (G) as well as sodium and potassium currents (H) in voltage clamp (n = 13 cells). (I) Example of spontaneous EPSCs recorded at a holding of -65mV in the presence of 50μM PTX in voltage clamp mode. (J) Quantitative RTPCR data showing that transduced neurons express both 3R and 4R TAU mRNA, represented by an increased 4R/3R TAU ratio compared to non-transduced control cells (P = 0.04; n≥3 from different experiments). Values were normalized to PGK1 before analyses. * P<0,05 (K) Western Blot with a 4R TAU specific antibody depicts 2N4R TAU bands, only in transduced (2N4R-P301L) cells. TAU ladder and marker for band sizes are represented by (T) and (M) respectively. (L) Immunostaining with 4R TAU specific antibody confirms the presence of the 2N4R P301L TAU on the cellular level, only in transduced neurons, while total TAU (red) is present also in control neurons. Scale bar = 25 μm. DAPI stains the nuclei

Mentions: In this study, we used induced pluripotent stem cells (iPSC) derived from healthy donors. Immunostaining for the pluripotency markers OCT4 and NANOG reveals nuclear expression of both transcription factors (Fig 1A). Further differentiation into cortical neural precursor cells (NPC’s) and immunostaining for PAX6 and Nestin around DIV25 (Fig 1B and 1C) confirms the NPC stage. At this point, NPC’s were either frozen or further differentiated into cortical neurons [16]. Neural identity of the cells around DIV70 is confirmed by immunostaining for the neuronal markers TUBB3 and MAP2 (Fig 1D–1F). Furthermore, TBR1 and CTIP2 staining reveals a cortical identity of the neurons while vGAT and vGLUT2 suggest the presence of both GABAergic and glutamatergic subtypes (Fig 1E and 1F). Western Blot with specific antibodies against 3R and 4R TAU shows that around DIV90 only the embryonic 0N3R TAU isoform is present (S1A Fig). To assess the functionality of the neurons, NPC’s were co-cultured with human astrocytes. Using current clamp, single or multiple action potentials were evoked in 85% of the neurons (n = 13) and with voltage clamp, around 85% of patched neurons had measurable sodium at -20mV (-1.283 ± 0.075nA) and all cells showed potassium currents at 40mV (1.361 ± 0.062nA). Moreover, spontaneous excitatory activity was observed at -65mV confirming functionality and network activity of the neurons [16] (Fig 1G–1I).


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)

Differentiation of hiPSC into cortical neurons and efficient transduction with AAV-TAU-P301L (A) Immunostaining for OCT4 and NANOG shows that iPSC0028 is pluripotent. Scale bar represents 50μm. (B-C) Immunostaining for Nestin and PAX6 revealing NPC stage at DIV25. Scale bar = 50μm for both. (D-F) Immunostaining on DIV70 visualizes the neuronal marker TUBB3 and cortical markers TBR1 and CTIP2 (D) as well as the dendritic marker MAP2 (E-F) together with either vGLUT2 (E) or vGAT (F). Scale bar = 25μm. (G-I) Representative traces of intrinsic neuronal properties of DIV70 neurons showing evoked responses in current clamp (G) as well as sodium and potassium currents (H) in voltage clamp (n = 13 cells). (I) Example of spontaneous EPSCs recorded at a holding of -65mV in the presence of 50μM PTX in voltage clamp mode. (J) Quantitative RTPCR data showing that transduced neurons express both 3R and 4R TAU mRNA, represented by an increased 4R/3R TAU ratio compared to non-transduced control cells (P = 0.04; n≥3 from different experiments). Values were normalized to PGK1 before analyses. * P<0,05 (K) Western Blot with a 4R TAU specific antibody depicts 2N4R TAU bands, only in transduced (2N4R-P301L) cells. TAU ladder and marker for band sizes are represented by (T) and (M) respectively. (L) Immunostaining with 4R TAU specific antibody confirms the presence of the 2N4R P301L TAU on the cellular level, only in transduced neurons, while total TAU (red) is present also in control neurons. Scale bar = 25 μm. DAPI stains the nuclei
© Copyright Policy
Related In: Results  -  Collection

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

pone.0146127.g001: Differentiation of hiPSC into cortical neurons and efficient transduction with AAV-TAU-P301L (A) Immunostaining for OCT4 and NANOG shows that iPSC0028 is pluripotent. Scale bar represents 50μm. (B-C) Immunostaining for Nestin and PAX6 revealing NPC stage at DIV25. Scale bar = 50μm for both. (D-F) Immunostaining on DIV70 visualizes the neuronal marker TUBB3 and cortical markers TBR1 and CTIP2 (D) as well as the dendritic marker MAP2 (E-F) together with either vGLUT2 (E) or vGAT (F). Scale bar = 25μm. (G-I) Representative traces of intrinsic neuronal properties of DIV70 neurons showing evoked responses in current clamp (G) as well as sodium and potassium currents (H) in voltage clamp (n = 13 cells). (I) Example of spontaneous EPSCs recorded at a holding of -65mV in the presence of 50μM PTX in voltage clamp mode. (J) Quantitative RTPCR data showing that transduced neurons express both 3R and 4R TAU mRNA, represented by an increased 4R/3R TAU ratio compared to non-transduced control cells (P = 0.04; n≥3 from different experiments). Values were normalized to PGK1 before analyses. * P<0,05 (K) Western Blot with a 4R TAU specific antibody depicts 2N4R TAU bands, only in transduced (2N4R-P301L) cells. TAU ladder and marker for band sizes are represented by (T) and (M) respectively. (L) Immunostaining with 4R TAU specific antibody confirms the presence of the 2N4R P301L TAU on the cellular level, only in transduced neurons, while total TAU (red) is present also in control neurons. Scale bar = 25 μm. DAPI stains the nuclei
Mentions: In this study, we used induced pluripotent stem cells (iPSC) derived from healthy donors. Immunostaining for the pluripotency markers OCT4 and NANOG reveals nuclear expression of both transcription factors (Fig 1A). Further differentiation into cortical neural precursor cells (NPC’s) and immunostaining for PAX6 and Nestin around DIV25 (Fig 1B and 1C) confirms the NPC stage. At this point, NPC’s were either frozen or further differentiated into cortical neurons [16]. Neural identity of the cells around DIV70 is confirmed by immunostaining for the neuronal markers TUBB3 and MAP2 (Fig 1D–1F). Furthermore, TBR1 and CTIP2 staining reveals a cortical identity of the neurons while vGAT and vGLUT2 suggest the presence of both GABAergic and glutamatergic subtypes (Fig 1E and 1F). Western Blot with specific antibodies against 3R and 4R TAU shows that around DIV90 only the embryonic 0N3R TAU isoform is present (S1A Fig). To assess the functionality of the neurons, NPC’s were co-cultured with human astrocytes. Using current clamp, single or multiple action potentials were evoked in 85% of the neurons (n = 13) and with voltage clamp, around 85% of patched neurons had measurable sodium at -20mV (-1.283 ± 0.075nA) and all cells showed potassium currents at 40mV (1.361 ± 0.062nA). Moreover, spontaneous excitatory activity was observed at -65mV confirming functionality and network activity of the neurons [16] (Fig 1G–1I).

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