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Inhibition of p25/Cdk5 Attenuates Tauopathy in Mouse and iPSC Models of Frontotemporal Dementia

Seo, Jinsoo, Kritskiy, Oleg, Watson, L. Ashley, Barker, Scarlett J., Dey, Dilip, Raja, Waseem K., Lin, Yuan-Ta, Ko, Tak, Cho, Sukhee, Penney, Jay, Silva, M. Catarina, Sheridan, Steven D., Lucente, Diane, Gusella, James F., Dickerson, Bradford C., Haggarty, Stephen J., Tsai, Li-Huei·2017·The Journal of Neuroscience
DOI 10.1523/jneurosci.0621-17.2017
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Abstract

Increased p25, a proteolytic fragment of the regulatory subunit p35, is known to induce aberrant activity of cyclin-dependent kinase 5 (Cdk5), which is associated with neurodegenerative disorders, including Alzheimer's disease. Previously, we showed that replacing endogenous p35 with the noncleavable mutant p35 (Δp35) attenuated amyloidosis and improved cognitive function in a familial Alzheimer's disease mouse model. Here, to address the role of p25/Cdk5 in tauopathy, we generated double-transgenic mice by crossing mice overexpressing mutant human tau (P301S) with Δ p35KI mice. We observed significant reduction of phosphorylated tau and its seeding activity in the brain of double transgenic mice compared with the P301S mice. Furthermore, synaptic loss and impaired LTP at hippocampal CA3 region of P301S mice were attenuated by blocking p25 generation. To further validate the role of p25/Cdk5 in tauopathy, we used frontotemporal dementia patient-derived induced pluripotent stem cells (iPSCs) carrying the Tau P301L mutation and generated P301L:Δp35KI isogenic iPSC lines using CRISPR/Cas9 genome editing. We created cerebral organoids from the isogenic iPSCs and found that blockade of p25 generation reduced levels of phosphorylated tau and increased expression of synaptophysin. Together, these data demonstrate a crucial role for p25/Cdk5 in mediating tau-associated pathology and suggest that inhibition of this kinase can remedy neurodegenerative processes in the presence of pathogenic tau mutation. SIGNIFICANCE STATEMENT Accumulation of p25 results in aberrant Cdk5 activation and induction of numerous pathological phenotypes, such as neuroinflammation, synaptic loss, Aβ accumulation, and tau hyperphosphorylation. However, it was not clear whether p25/Cdk5 activity is necessary for the progression of these pathological changes. We recently developed the Δ p35KI transgenic mouse that is deficient in p25 generation and Cdk5 hyperactivation. In this study, we used this mouse model to elucidate the role of p25/Cdk5 in FTD mutant tau-mediated pathology. We also used a frontotemporal dementia patient-derived induced pluripotent stem cell carrying the Tau P301L mutation and generated isogenic lines in which p35 is replaced with noncleavable mutant Δp35. Our data suggest that p25/Cdk5 plays an important role in tauopathy in both mouse and human model systems.

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Figure 1 Inhibition of p25 generation abolishes Cdk5 hyperactivation in P301S mice brain. A , Levels of p25 in WT, P301S , and P301S; 𝚫 p35KI hippocampus. 𝚫 p35 expression in P301S; 𝚫 p35KI was confirmed by immunoblotting with an anti-HA antibody. *Nonspecificbackgroundband. B , Bar graph represents relative immunoreactivity of p25/p35 comparedwithWT(Student's t test). C , Immunoprecipitation-linked Cdk5 kinase assays were performed on WT, P301S , and P301S; 𝚫 p35KI hippocampal lysates ( n = 3-5pergroup; p = 0.0009 by ANOVA). * p < 0.05 (Student's t test or Tukey's post hoc analysis). ** p < 0.01 (Student's t test or Tukey's post hoc analysis). *** p < 0.001 (Student's t test or Tukey's post hoc analysis). Error bars indicate ± SEM.
Figure 1 Inhibition of p25 generation abolishes Cdk5 hyperactivation in P301S mice brain. A , Levels of p25 in WT, P301S , and P301S; 𝚫 p35KI hippocampus. 𝚫 p35 expression in P301S; 𝚫 p35KI was confirmed by immunoblotting with an anti-HA antibody. *Nonspecificbackgroundband. B , Bar graph represents relative immunoreactivity of p25/p35 comparedwithWT(Student's t test). C , Immunoprecipitation-linked Cdk5 kinase assays were performed on WT, P301S , and P301S; 𝚫 p35KI hippocampal lysates ( n = 3-5pergroup; p = 0.0009 by ANOVA). * p < 0.05 (Student's t test or Tukey's post hoc analysis). ** p < 0.01 (Student's t test or Tukey's post hoc analysis). *** p < 0.001 (Student's t test or Tukey's post hoc analysis). Error bars indicate ± SEM.CC-BY-NC-SA · From the PDF
Figure2 Inhibition of p25/Cdk5 attenuates hyperphosphorylation of tau and its seeding activity in P301S micebrain. A , Immunohistochemistry with an anti-pTau T181 antibody in hippocampal CA1 of WT, P301S , and P301S; 𝚫 p35KI mice. Scale bar, 20 𝛍 m. B , Relative levels of pTau T181, pTau S202, and total tau in P301S and P301S; 𝚫 p35KI hippocampus normalized to P301S ( n = 4-8, per group; Student's t test). C , Cortical lysates from 2-month-old WT, P301S , and P301S; 𝚫 p35KI mice were added to biosensor HEK293T cells, and their tau seeding activity was quantified by measuring FRET signals ( n = 4 per group; p < 0.0001 by ANOVA). * p < 0.05 (Student's t test or Tukey's post hoc analysis). ** p < 0.01 (Student's t test or Tukey's post hoc analysis). *** p < 0.001 (Student's t test or Tukey's post hoc analysis). Error bars indicate ± SEM.
Figure2 Inhibition of p25/Cdk5 attenuates hyperphosphorylation of tau and its seeding activity in P301S micebrain. A , Immunohistochemistry with an anti-pTau T181 antibody in hippocampal CA1 of WT, P301S , and P301S; 𝚫 p35KI mice. Scale bar, 20 𝛍 m. B , Relative levels of pTau T181, pTau S202, and total tau in P301S and P301S; 𝚫 p35KI hippocampus normalized to P301S ( n = 4-8, per group; Student's t test). C , Cortical lysates from 2-month-old WT, P301S , and P301S; 𝚫 p35KI mice were added to biosensor HEK293T cells, and their tau seeding activity was quantified by measuring FRET signals ( n = 4 per group; p < 0.0001 by ANOVA). * p < 0.05 (Student's t test or Tukey's post hoc analysis). ** p < 0.01 (Student's t test or Tukey's post hoc analysis). *** p < 0.001 (Student's t test or Tukey's post hoc analysis). Error bars indicate ± SEM.CC-BY-NC-SA · From the PDF
Figure 3 Blockade of p25 generation restores synaptic integrity and function in hippocampal CA3 region of P301S mice. A , Immunohistochemistry with an anti-synaptophysin antibody in hippocampal CA3 of WT, P301S , and P301S; 𝚫 p35KI mice. Right, Bar graphs represent the relative immunoreactivity of synaptophysin in WT, P301S , and P301S; 𝚫 p35KI normalized to WT ( n = 3-5 per group; p = 0.0005 by ANOVA). Scale bar, 20 𝛍 m. B , Left, Input- output curves from the fEPSP amplitude against the fiber volley amplitude at a range of stimulus intensities. Right, Paired-pulse facilitation (fEPSP2/fEPSP1) was measured at various interstimulus intervals. C , LTP was induced by 3 × high-frequency stimulation at mossy fiber-CA3 synapses. Sample traces represent fEPSPs at 1 min before (gray) and 1 h after (block) high-frequency stimulation. Calibration: 0.2 mV, 10 ms. Right, The magnitude of LTP was calculated by comparing the average slopes of fEPSPs during the last 5 min of recordings with those recorded before stimulation ( n = 4-6pergroup; p = 0.0013 by ANOVA). * p < 0.05 (Student's t test or Tukey's post hoc analysis). ** p < 0.01 (Student's t test or Tukey's post hoc analysis). Error bars indicate ± SEM.
Figure 3 Blockade of p25 generation restores synaptic integrity and function in hippocampal CA3 region of P301S mice. A , Immunohistochemistry with an anti-synaptophysin antibody in hippocampal CA3 of WT, P301S , and P301S; 𝚫 p35KI mice. Right, Bar graphs represent the relative immunoreactivity of synaptophysin in WT, P301S , and P301S; 𝚫 p35KI normalized to WT ( n = 3-5 per group; p = 0.0005 by ANOVA). Scale bar, 20 𝛍 m. B , Left, Input- output curves from the fEPSP amplitude against the fiber volley amplitude at a range of stimulus intensities. Right, Paired-pulse facilitation (fEPSP2/fEPSP1) was measured at various interstimulus intervals. C , LTP was induced by 3 × high-frequency stimulation at mossy fiber-CA3 synapses. Sample traces represent fEPSPs at 1 min before (gray) and 1 h after (block) high-frequency stimulation. Calibration: 0.2 mV, 10 ms. Right, The magnitude of LTP was calculated by comparing the average slopes of fEPSPs during the last 5 min of recordings with those recorded before stimulation ( n = 4-6pergroup; p = 0.0013 by ANOVA). * p < 0.05 (Student's t test or Tukey's post hoc analysis). ** p < 0.01 (Student's t test or Tukey's post hoc analysis). Error bars indicate ± SEM.CC-BY-NC-SA · From the PDF
Figure 4 pTau levels in organoids derived from an FTD patient iPSCs are reduced by inhibition of p25 expression. A , Relative levels of p35 and p25 in organoids derived from nonmutant control (MGH 2069) or Tau P301L (MGH 2046) iPSCs ( n = 3 biological replicates; 3 or 4 organoids were pooled for each blot). B , Schematics of 𝚫 p35KI isogenic line generation from an FTD patient iPSCs carrying the Tau P301L mutation. C , Sanger sequencing confirms the insertion of a desired repair template into an FTD patient iPSCs. D , Relative levels of p35, p25, synaptophysin, NeuN, pTau T181, pTau S202, and total tau in organoids derived from P301L or P301L; 𝚫 p35KI iPSCs. Right, Bar graph represents the quantification of relative immunoreactivity for each protein in organoids normalized to P301L organoids ( n = 4biological replicates; 3 or 4 organoids were pooled for each blot). E , Immunohistochemistry with an anti-pTau T181 antibody in organoids derived from P301L or P301L; 𝚫 p35KIiPSCs.Scalebar,5 𝛍 m.Right,BargraphrepresentstherelativeimmunoreactivityofpTauT181ineachgroup( n = 6pergroup).* p < 0.05(Student's t test). ** p < 0.01(Student's t test). *** p < 0.001 (Student's t test). Error bars indicate ± SEM.
Figure 4 pTau levels in organoids derived from an FTD patient iPSCs are reduced by inhibition of p25 expression. A , Relative levels of p35 and p25 in organoids derived from nonmutant control (MGH 2069) or Tau P301L (MGH 2046) iPSCs ( n = 3 biological replicates; 3 or 4 organoids were pooled for each blot). B , Schematics of 𝚫 p35KI isogenic line generation from an FTD patient iPSCs carrying the Tau P301L mutation. C , Sanger sequencing confirms the insertion of a desired repair template into an FTD patient iPSCs. D , Relative levels of p35, p25, synaptophysin, NeuN, pTau T181, pTau S202, and total tau in organoids derived from P301L or P301L; 𝚫 p35KI iPSCs. Right, Bar graph represents the quantification of relative immunoreactivity for each protein in organoids normalized to P301L organoids ( n = 4biological replicates; 3 or 4 organoids were pooled for each blot). E , Immunohistochemistry with an anti-pTau T181 antibody in organoids derived from P301L or P301L; 𝚫 p35KIiPSCs.Scalebar,5 𝛍 m.Right,BargraphrepresentstherelativeimmunoreactivityofpTauT181ineachgroup( n = 6pergroup).* p < 0.05(Student's t test). ** p < 0.01(Student's t test). *** p < 0.001 (Student's t test). Error bars indicate ± SEM.CC-BY-NC-SA · From the PDF

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Metadata

Authors
Seo, J., Kritskiy, O., Watson, L. A., Barker, S. J., Dey, D., Raja, W. K., Lin, Y.-T., Ko, T., Cho, S., Penney, J., Silva, M. C., Sheridan, S. D., Lucente, D., Gusella, J. F., Dickerson, B. C., Haggarty, S. J., & Tsai, L.-H.
ORCID
Venue
The Journal of Neuroscience
Publisher
Society for Neuroscience
Published
2017-09-14
Volume
37
Issue
41
Pages
9917-9924
ISSN
  • 0270-6474
  • 1529-2401
DOI
10.1523/jneurosci.0621-17.2017
Cited by
156
References
10
Language
en
Licence
creativecommons.org/licenses/by-nc-sa/4.0/ (vor, 209 d)

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