Journal: Frontiers in cellular neuroscience

Article Title: APPsα rescues CDK5 and GSK3β dysregulation and restores normal spine density in Tau transgenic mice.

doi: 10.3389/fncel.2023.1106176

Figure Lengend Snippet: FIGURE 2 Assessment of kinase activity in THY-Tau22 mice using a radioactive kinase assay. (A) Schematic overview of assay format. GSK3β or CDK5-activator complexes are immunoprecipitated from hippocampal tissue homogenates using protein G/A coupled beads. Immunoprecipitated kinases were incubated with [γ −32P]-ATP and recombinant Tau, leading to radioactive labeling. For further analysis samples were separated by SDS-PAGE, transferred to a PVDF membrane and visualized using Western blot and phosphorimaging. (B,C) Western blot (WB) and phosphorimaging (PI) analysis after immunoprecipitation of (B) CDK5 or (C) GSK3β from littermate controls and THY-Tau22 mice at 12 months of age. 650 µg of total hippocampal lysate was used as input for each IP when employing the α-CDK5 antibody (B). The remaining lysate (amounting to about 300–600 µg) was used for GSK3β-IP (C). As total protein yield varied between the hippocampal samples the second IP was performed with more variable input, as reflected in the more variable GSK3β WB signal. To account for input variability signal intensity of 32P-recTau (PI) was normalized to total amount of immunoprecipitated CDK5 (α-CDK5 WB signal intensity) (B) or to total amount of immunoprecipitated GSK3β (α-GSK3β WB signal intensity) (C), respectively. Kinase activity was plotted as arbitrary units (A.U.). Note the absence of GSK3β after immunoprecipitation with an CDK5-specific antibody (B) and the absence of CDK5 after immunoprecipitation with an GSK3β-specific antibody (C). The asterisk ∗indicates an unspecific band detected after α-GSK3β staining. (D) CDK5 activity increased in THY-Tau22 mice without reaching significance (Littermate vs. THY-Tau22, p = 0.29, ns). (E) GSK3β activity was significantly increased in THY-Tau22 mice compared to littermate controls (Littermate vs. THY-Tau22, ∗p = 0.04). Data are depicted as mean ± SEM; N, number of animals; age, 12 months; data were analyzed using a two-tailed Student’s t-test.

Article Snippet: For this assay the following primary antibodies were used: α-CDK5 (mouse monoclonal, 1:1000, #sc-6247, Santa Cruz Biotechnology), α-GSK3β (mouse monoclonal, 1:1500, #9832, Cell Signaling Technology), HT7 (mouse monoclonal, 1:1000, #MN1000, Thermo Fisher Scientific).

Techniques: Activity Assay, Kinase Assay, Immunoprecipitation, Incubation, Recombinant, Labeling, SDS Page, Membrane, Western Blot, Staining, Two Tailed Test

Journal: Frontiers in cellular neuroscience

Article Title: APPsα rescues CDK5 and GSK3β dysregulation and restores normal spine density in Tau transgenic mice.

doi: 10.3389/fncel.2023.1106176

Figure Lengend Snippet: FIGURE 6 APPsα rescues CDK5 hyperactivation in THY-Tau22 mice. (A) Schematic overview of CDK5 regulation under physiological (left) and pathological conditions (right). Under physiological conditions, CDK5 is activated by binding to the myristoylated p35 activator, anchoring the CDK5-p35 complex to the plasma membrane. Pathological conditions lead to increased cytosolic calcium, activating the protease calpain. Active calpain cleaves p35 into p10 and p25 that remains bound to CDK5. The hyperactive CDK5-p25 complex now dissociates from the plasma membrane, shows a prolonged half-life compared to CDK5-p35 and may phosphorylate additional substrates. (B) Western blot analysis of hippocampal lysates of AAV-Venus or AAV-APPsα injected THY-Tau22, or littermate control mice. CDK5 and the activator proteins p35, p25 were detected using specific monoclonal antibodies. Note that a longer exposure was necessary to visualize p25 that is visible as a very faint band below p35 in the WB above. Vinculin is depicted as a qualitative loading control. Note that for quantification of immunoreactive bands a normalization was performed against total protein level per lane (stain-free method, Bio-Rad). (C–E) Quantitative analysis of the Western blot depicted in (B) revealed (C) a similar expression level of CDK5 between all three groups. (D) The abundance of p35 was significantly increased in THY-Tau22-Venus mice (LM-Venus vs. THY-Tau22-Venus, ∗∗p = 0.004). (E) Note that THY-Tau22-Venus mice show increased expression of the hyperactivating regulatory p25 subunit that is rescued upon APPsα expression (LM-Venus vs. THY-Tau22-Venus, ∗p = 0.01; THY-Tau22-Venus vs. THY-Tau22-APPsα, ∗p = 0.01). (F) Radioactive kinase assay involving Western blot (WB) and phosphorimaging (PI) analysis after immunoprecipitation of CDK5 from AAV-Venus or AAV-APPsα injected littermates and THY-Tau22 mice. Radioactively labeled Tau was visualized using PI. Recombinant Tau (HT7), GSK3β and CDK5 were visualized by immunodetection using specific monoclonal antibodies. Note the absence of GSK3β after immunoprecipitation of CDK5. (G) Quantitative analysis revealed a slight increase in CDK5 activity (PI signal normalized to total immunoprecipitated CDK5, WB signal) in THY-Tau22-Venus mice compared to LM-Venus mice (LM-Venus vs. THY-Tau22-Venus, p = 0.13, ns). AAV-APPsα significantly reduced CDK5 activity to a level not different from controls (THY-Tau22-Venus vs. THY-Tau22-APPsα, ∗∗p = 0.009; THY-Tau22-APPsα vs. LM-Venus, p = 0.414, ns). (H) Quantitative analysis (radioactive CDK5 kinase assay) of pooled data from non-injected and AAV-Venus injected THY-Tau22 and WT littermate mice indicates increased CDK5 activity in THY-Tau22 mice (Littermate vs. THY-Tau22, ∗p = 0.02). Data are depicted as mean ± SEM; N, number of animals; age, 12 months; data were analyzed using one-way ANOVA with Tukey post hoc test.

Article Snippet: For this assay the following primary antibodies were used: α-CDK5 (mouse monoclonal, 1:1000, #sc-6247, Santa Cruz Biotechnology), α-GSK3β (mouse monoclonal, 1:1500, #9832, Cell Signaling Technology), HT7 (mouse monoclonal, 1:1000, #MN1000, Thermo Fisher Scientific).

Techniques: Binding Assay, Clinical Proteomics, Membrane, Western Blot, Injection, Control, Bioprocessing, Staining, Expressing, Kinase Assay, Immunoprecipitation, Labeling, Recombinant, Immunodetection, Activity Assay

Journal: PLoS Biology

Article Title: Cdk5 Regulates Accurate Maturation of Newborn Granule Cells in the Adult Hippocampus

doi: 10.1371/journal.pbio.0060272

Figure Lengend Snippet: Control cells (A) and DNcdk5 -overexpressing cells (B) colabeled with the granule cell markers Prox-1 (red) and Calbindin (blue), demonstrating that newborn neurons were dentate granule cells. Small panels in (A and B) show the single channels for Prox-1 (red) and calbindin (blue). Arrows point towards the GFP-labeled, newborn cells. HL, hilus. Scale bar in (B) represents 50 μm.

Article Snippet: Primary antibodies used were rat α-BrdU (Harlan Seralab), mouse α-MAP2ab (Sigma), rabbit α-GFP (Molecular Probes), chicken α-GFP (Aves), rabbit α-Prox-1 (Chemicon), mouse α-Calbindin (Swant), rabbit α-synapsin (Calbiochem), mouse α-NeuN (Chemicon), rabbit α-cdk5 (Santa Cruz), rabbit α-p35 (Santa Cruz), and goat α-DCX (Santa Cruz).

Techniques: Labeling

Journal: PLoS Biology

Article Title: Cdk5 Regulates Accurate Maturation of Newborn Granule Cells in the Adult Hippocampus

doi: 10.1371/journal.pbio.0060272

Figure Lengend Snippet: (A–E) Stable transduced cell lines expressing a control virus (A), cdk5 (B), cdk5 plus coelectroporation with p35 (red in [C]), and DNcdk5 (D) showed no statistically significant differences in BrdU (red in [A, B, and D], blue in [C]) uptake after a 1-h pulse (E). Percentages of BrdU-labeled cells (E) were 54.5 ± 5.3% (Con), 65.7 ± 4.9% (cdk5), 63.2 ± 4.7% (cdk5/p35), and 51.0 ± 2.2% (DNcdk5, p > 0.2). Left and middle panels in (A–D) show single channels for GFP (green), BrdU (red in [A, B, and D]; blue in [C]), and p35/CAG-RFP coelectroporation (red in [C]). Merged images in (A, B, and D) also include DAPI (blue). Note that the total number of GFP-labeled cells expressing p35 decreased due to electroporation-associated toxicity, whereas the relative proportion of BrdU-labeled cells was not altered with p35 electroporation. (F) Stable transduced cell lines expressing a control virus, cdk5 , or DNcdk5 were treated for 4 d with retinoic acid and forskolin to induce neuronal differentiation. There were no significant differences upon neuronal induction between the groups ( p > 0.08). Percentages of MAP2ab-expressing cells were 5.8 ± 0.8% (Con), 4.5 ± 0.5% (cdk5), 6.3 ± 1.0% (cdk5/p35), and 4.6 ± 0.8% (DNcdk5). Scale bar in (D) represents 50 μm. Error bars represent the standard error of the mean (s.e.m.).

Article Snippet: Primary antibodies used were rat α-BrdU (Harlan Seralab), mouse α-MAP2ab (Sigma), rabbit α-GFP (Molecular Probes), chicken α-GFP (Aves), rabbit α-Prox-1 (Chemicon), mouse α-Calbindin (Swant), rabbit α-synapsin (Calbiochem), mouse α-NeuN (Chemicon), rabbit α-cdk5 (Santa Cruz), rabbit α-p35 (Santa Cruz), and goat α-DCX (Santa Cruz).

Techniques: Expressing, Control, Virus, Labeling, Electroporation

Journal: Frontiers in Cellular Neuroscience

Article Title: APPsα rescues CDK5 and GSK3β dysregulation and restores normal spine density in Tau transgenic mice

doi: 10.3389/fncel.2023.1106176

Figure Lengend Snippet: Assessment of kinase activity in THY-Tau22 mice using a radioactive kinase assay. (A) Schematic overview of assay format. GSK3β or CDK5-activator complexes are immunoprecipitated from hippocampal tissue homogenates using protein G/A coupled beads. Immunoprecipitated kinases were incubated with [γ− 32 P]-ATP and recombinant Tau, leading to radioactive labeling. For further analysis samples were separated by SDS-PAGE, transferred to a PVDF membrane and visualized using Western blot and phosphorimaging. (B,C) Western blot (WB) and phosphorimaging (PI) analysis after immunoprecipitation of (B) CDK5 or (C) GSK3β from littermate controls and THY-Tau22 mice at 12 months of age. 650 μg of total hippocampal lysate was used as input for each IP when employing the α-CDK5 antibody (B) . The remaining lysate (amounting to about 300–600 μg) was used for GSK3β-IP (C) . As total protein yield varied between the hippocampal samples the second IP was performed with more variable input, as reflected in the more variable GSK3β WB signal. To account for input variability signal intensity of 32 P-recTau (PI) was normalized to total amount of immunoprecipitated CDK5 (α-CDK5 WB signal intensity) (B) or to total amount of immunoprecipitated GSK3β (α-GSK3β WB signal intensity) (C) , respectively. Kinase activity was plotted as arbitrary units (A.U.). Note the absence of GSK3β after immunoprecipitation with an CDK5-specific antibody (B) and the absence of CDK5 after immunoprecipitation with an GSK3β-specific antibody (C) . The asterisk * indicates an unspecific band detected after α-GSK3β staining. (D) CDK5 activity increased in THY-Tau22 mice without reaching significance (Littermate vs. THY-Tau22, p = 0.29, ns). (E) GSK3β activity was significantly increased in THY-Tau22 mice compared to littermate controls (Littermate vs. THY-Tau22, * p = 0.04). Data are depicted as mean ± SEM; N, number of animals; age, 12 months; data were analyzed using a two-tailed Student’s t -test.

Article Snippet: For this assay the following primary antibodies were used: α-CDK5 (mouse monoclonal, 1:1000, #sc-6247, Santa Cruz Biotechnology), α-GSK3β (mouse monoclonal, 1:1500, #9832, Cell Signaling Technology), HT7 (mouse monoclonal, 1:1000, #MN1000, Thermo Fisher Scientific).

Techniques: Activity Assay, Kinase Assay, Immunoprecipitation, Incubation, Recombinant, Labeling, SDS Page, Membrane, Western Blot, Staining, Two Tailed Test

Journal: Frontiers in Cellular Neuroscience

Article Title: APPsα rescues CDK5 and GSK3β dysregulation and restores normal spine density in Tau transgenic mice

doi: 10.3389/fncel.2023.1106176

Figure Lengend Snippet: APPsα restores normal GSK3β activity and modulates the Akt/GSK3β pathway in THY-Tau22 mice. (A) Schematic overview of the regulation of GSK3β activity. Activated Akt (pAkt Ser473 ) negatively regulates the activity of GSK3β through phosphorylation of Ser 9 , which leads to GSK3β inactivation. (B) Western blot analysis of hippocampi from AAV-Venus or AAV-APPsα injected littermates (LM) or THY-Tau22 mice. Specific antibodies were used to detect total GSK3β and inactive pGSK3β Ser9 . Vinculin is depicted as a qualitative loading control. Note that for quantification of immunoreactive bands a normalization was performed against total protein level per lane (stain-free method, Bio-Rad). (C) No differences were detected for total GSK3β between groups. (D) THY-Tau22-Venus mice revealed a strong trend toward reduced GSK3β activity, as shown by signal intensities of inactive pGSK3β Ser9 normalized to that of total GSK3β (LM-Venus vs. THY-Tau22-Venus, p = 0.060). AAV-APPsα expression restored GSK3β activity to littermate control level (THY-Tau22-Venus vs. THY-Tau22-APPsα, p = 0.051). (E) Radioactive kinase assay involving Western blot (WB) and phosphorimaging (PI) analysis after immunoprecipitation of GSK3β from AAV-Venus or AAV-APPsα injected littermates and THY-Tau22 mice. Radioactively labeled Tau was visualized using PI. Recombinant Tau (HT7), GSK3β and CDK5 were visualized by immunodetection using specific monoclonal antibodies. Note the absence of CDK5 after immunoprecipitation of GSK3β. (F) Quantitative analysis revealed significantly increased GSK3β activity (PI signal normalized to total immunoprecipitated GSK3β, WB signal) in THY-Tau22-Venus mice compared to LM-Venus mice (LM-Venus vs. THY-Tau22-Venus, ** p = 0.007). AAV-APPsα restored normal GSK3β activity (THY-Tau22-Venus vs. THY-Tau22-APPsα, ** p = 0.007). (G) Western blot analysis of total Akt and active Akt (pAkt Ser473 ) in THY-Tau22 mice after AAV-Venus or AAV-APPsα injection. Vinculin is depicted as a qualitative loading control. Note that for quantification of immunoreactive bands a normalization was performed against total protein level per lane (stain-free method, Bio-Rad). (H,I) Quantitative analysis of the Western blot depicted in (G) . THY-Tau22 mice showed a reduction in (H) the total expression of Akt (LM-Venus vs. THY-Tau22-Venus, ** p = 0.003) and (I) for the activating Ser 473 phosphorylation of Akt (LM-Venus vs. THY-Tau22-Venus, *** p = 0.0005). AAV-APPsα rescued both total Akt and pAkt 473 (THY-Tau22-Venus vs. THY-Tau22-APPsα, *** p = 0.0002 and *** p = 0.0009), respectively. Data are depicted as mean ± SEM; N, number of animals; age of analysis, 12 months, data were analyzed using one-way ANOVA with Tukey post hoc test.

Article Snippet: For this assay the following primary antibodies were used: α-CDK5 (mouse monoclonal, 1:1000, #sc-6247, Santa Cruz Biotechnology), α-GSK3β (mouse monoclonal, 1:1500, #9832, Cell Signaling Technology), HT7 (mouse monoclonal, 1:1000, #MN1000, Thermo Fisher Scientific).

Techniques: Activity Assay, Phospho-proteomics, Western Blot, Injection, Control, Staining, Expressing, Kinase Assay, Immunoprecipitation, Labeling, Recombinant, Immunodetection, Bioprocessing

Journal: Frontiers in Cellular Neuroscience

Article Title: APPsα rescues CDK5 and GSK3β dysregulation and restores normal spine density in Tau transgenic mice

doi: 10.3389/fncel.2023.1106176

Figure Lengend Snippet: APPsα rescues CDK5 hyperactivation in THY-Tau22 mice. (A) Schematic overview of CDK5 regulation under physiological (left) and pathological conditions (right). Under physiological conditions, CDK5 is activated by binding to the myristoylated p35 activator, anchoring the CDK5-p35 complex to the plasma membrane. Pathological conditions lead to increased cytosolic calcium, activating the protease calpain. Active calpain cleaves p35 into p10 and p25 that remains bound to CDK5. The hyperactive CDK5-p25 complex now dissociates from the plasma membrane, shows a prolonged half-life compared to CDK5-p35 and may phosphorylate additional substrates. (B) Western blot analysis of hippocampal lysates of AAV-Venus or AAV-APPsα injected THY-Tau22, or littermate control mice. CDK5 and the activator proteins p35, p25 were detected using specific monoclonal antibodies. Note that a longer exposure was necessary to visualize p25 that is visible as a very faint band below p35 in the WB above. Vinculin is depicted as a qualitative loading control. Note that for quantification of immunoreactive bands a normalization was performed against total protein level per lane (stain-free method, Bio-Rad). (C–E) Quantitative analysis of the Western blot depicted in (B) revealed (C) a similar expression level of CDK5 between all three groups. (D) The abundance of p35 was significantly increased in THY-Tau22-Venus mice (LM-Venus vs. THY-Tau22-Venus, ** p = 0.004). (E) Note that THY-Tau22-Venus mice show increased expression of the hyperactivating regulatory p25 subunit that is rescued upon APPsα expression (LM-Venus vs. THY-Tau22-Venus, * p = 0.01; THY-Tau22-Venus vs. THY-Tau22-APPsα, * p = 0.01). (F) Radioactive kinase assay involving Western blot (WB) and phosphorimaging (PI) analysis after immunoprecipitation of CDK5 from AAV-Venus or AAV-APPsα injected littermates and THY-Tau22 mice. Radioactively labeled Tau was visualized using PI. Recombinant Tau (HT7), GSK3β and CDK5 were visualized by immunodetection using specific monoclonal antibodies. Note the absence of GSK3β after immunoprecipitation of CDK5. (G) Quantitative analysis revealed a slight increase in CDK5 activity (PI signal normalized to total immunoprecipitated CDK5, WB signal) in THY-Tau22-Venus mice compared to LM-Venus mice (LM-Venus vs. THY-Tau22-Venus, p = 0.13, ns). AAV-APPsα significantly reduced CDK5 activity to a level not different from controls (THY-Tau22-Venus vs. THY-Tau22-APPsα, ** p = 0.009; THY-Tau22-APPsα vs. LM-Venus, p = 0.414, ns). (H) Quantitative analysis (radioactive CDK5 kinase assay) of pooled data from non-injected and AAV-Venus injected THY-Tau22 and WT littermate mice indicates increased CDK5 activity in THY-Tau22 mice (Littermate vs. THY-Tau22, * p = 0.02). Data are depicted as mean ± SEM; N, number of animals; age, 12 months; data were analyzed using one-way ANOVA with Tukey post hoc test.

Article Snippet: For this assay the following primary antibodies were used: α-CDK5 (mouse monoclonal, 1:1000, #sc-6247, Santa Cruz Biotechnology), α-GSK3β (mouse monoclonal, 1:1500, #9832, Cell Signaling Technology), HT7 (mouse monoclonal, 1:1000, #MN1000, Thermo Fisher Scientific).

Techniques: Binding Assay, Clinical Proteomics, Membrane, Western Blot, Injection, Control, Bioprocessing, Staining, Expressing, Kinase Assay, Immunoprecipitation, Labeling, Recombinant, Immunodetection, Activity Assay