Journal: Investigative ophthalmology & visual science

Article Title: Vps35 Deficiency Impairs Cdk5/p35 Degradation and Promotes the Hyperphosphorylation of Tau Protein in Retinal Ganglion Cells.

doi: 10.1167/iovs.61.1.1

Figure Lengend Snippet: FIGURE 1. The expression of Vps35, p35, CDK5, and tau s396 in retina and primary cultured RGCs. (A, B) Vps35 and p35 were colo- calized in the GCL and the signals of Vps35 decreased while the signals of p35 increased 1 week after intravitreal injection of 50 nmol glutamate. (C, D) Vps35 and p35, and Vps35 and tau s396 colocalized in the GCL. (E, F) The expression of p35 and Cdk5 in primary cultured RGCs. (G, H) The colocalization of Vps35 and p35, Vps35 and tau s396, p35 and Cdk5, and Vps35 and Cdk5 in primary cultured RGCs.

Article Snippet: Abcam Rabbit anti-tau antibody ab32057 1 to 300 Rabbit anti-tau phospho s396 ab109390 1 to 200 Rabbit anti-tau phospho s404 ab131338 1 to 200 Goat anti-Vps35 ab10099 1 to 200 Rabbit anti-Vps35 ab157220 1 to 250 Rabbit anti-LAMP1 ab24170 1 to 200 Goat anti-mouse IgG H&L (Cy3) ab97035 1 to 300 Donkey anti-goat IgG H&L (Cy3) ab6949 1 to 500 Goat anti-rabbit IgG H&L (Alexa Fluor 488) ab150077 1 to 300 Donkey anti-rabbit IgG H&L (Alexa Fluor 488) ab150073 1 to 300 Rabbit anti-beta Ш tubulin ab18207 1 to 500 Mouse anti-CD90/Thy1 ab225 1 to 300 Goat anti-GFP ab6662 1 to 300 Cell Signaling Technology Rabbit anti-p35/25 (C64B10) mAb #2680 1 to 200 Rabbit anti-UBE1a/b #4891 1 to 25 Rabbit anti-EEA1 (C45B10) mAb #3288 1 to 100 Santa Cruz Biotechnology Mouse anti-p35 (4G11) sc-293184 1 to 200 Mouse anti-Cdk5 (J-3) sc-6247 1 to 200 The aim of the present study was to investigate the mechanism involved in the hyperphosphorylation of tau protein in the retina caused by Vps35 deficiency.

Techniques: Expressing, Cell Culture, Injection

Journal: Investigative ophthalmology & visual science

Article Title: Vps35 Deficiency Impairs Cdk5/p35 Degradation and Promotes the Hyperphosphorylation of Tau Protein in Retinal Ganglion Cells.

doi: 10.1167/iovs.61.1.1

Figure Lengend Snippet: FIGURE 2. The relative mRNA and protein expression of Vps35 decreased, while the expression of p35, p-tau s396 increased with glutamate excitotoxity both in vivo (especially 7D and 14D after intravitreal injection of glutamate 50 nmol) (A–C) and in vitro (D, E). **P < 0.01; *P < 0.05.

Article Snippet: Abcam Rabbit anti-tau antibody ab32057 1 to 300 Rabbit anti-tau phospho s396 ab109390 1 to 200 Rabbit anti-tau phospho s404 ab131338 1 to 200 Goat anti-Vps35 ab10099 1 to 200 Rabbit anti-Vps35 ab157220 1 to 250 Rabbit anti-LAMP1 ab24170 1 to 200 Goat anti-mouse IgG H&L (Cy3) ab97035 1 to 300 Donkey anti-goat IgG H&L (Cy3) ab6949 1 to 500 Goat anti-rabbit IgG H&L (Alexa Fluor 488) ab150077 1 to 300 Donkey anti-rabbit IgG H&L (Alexa Fluor 488) ab150073 1 to 300 Rabbit anti-beta Ш tubulin ab18207 1 to 500 Mouse anti-CD90/Thy1 ab225 1 to 300 Goat anti-GFP ab6662 1 to 300 Cell Signaling Technology Rabbit anti-p35/25 (C64B10) mAb #2680 1 to 200 Rabbit anti-UBE1a/b #4891 1 to 25 Rabbit anti-EEA1 (C45B10) mAb #3288 1 to 100 Santa Cruz Biotechnology Mouse anti-p35 (4G11) sc-293184 1 to 200 Mouse anti-Cdk5 (J-3) sc-6247 1 to 200 The aim of the present study was to investigate the mechanism involved in the hyperphosphorylation of tau protein in the retina caused by Vps35 deficiency.

Techniques: Expressing, In Vivo, Injection, In Vitro

Journal: Investigative ophthalmology & visual science

Article Title: Vps35 Deficiency Impairs Cdk5/p35 Degradation and Promotes the Hyperphosphorylation of Tau Protein in Retinal Ganglion Cells.

doi: 10.1167/iovs.61.1.1

Figure Lengend Snippet: FIGURE 3. Vps35 alters the activity of Cdk5/p35 and the phosphorylation of tau protein. (A, B, E) Downregulation of Vps35 in primary cultured RGC could statistically enhance the levels of Cdk5/p35 and tau s396. (C, D, F) Overexpression of Vps35 in primary cultured RGC could considerably decrease the levels of Cdk5/p35 and tau s396. (G, H) Roscovitine significantly inhibited Cdk5 (P < 0.05), resulting in a decrease in the p35 and p-tau s396 levels in primary cultured RGCs. **P < 0.01; *P < 0.05. DMSO, dimethylsulfoxide.

Article Snippet: Abcam Rabbit anti-tau antibody ab32057 1 to 300 Rabbit anti-tau phospho s396 ab109390 1 to 200 Rabbit anti-tau phospho s404 ab131338 1 to 200 Goat anti-Vps35 ab10099 1 to 200 Rabbit anti-Vps35 ab157220 1 to 250 Rabbit anti-LAMP1 ab24170 1 to 200 Goat anti-mouse IgG H&L (Cy3) ab97035 1 to 300 Donkey anti-goat IgG H&L (Cy3) ab6949 1 to 500 Goat anti-rabbit IgG H&L (Alexa Fluor 488) ab150077 1 to 300 Donkey anti-rabbit IgG H&L (Alexa Fluor 488) ab150073 1 to 300 Rabbit anti-beta Ш tubulin ab18207 1 to 500 Mouse anti-CD90/Thy1 ab225 1 to 300 Goat anti-GFP ab6662 1 to 300 Cell Signaling Technology Rabbit anti-p35/25 (C64B10) mAb #2680 1 to 200 Rabbit anti-UBE1a/b #4891 1 to 25 Rabbit anti-EEA1 (C45B10) mAb #3288 1 to 100 Santa Cruz Biotechnology Mouse anti-p35 (4G11) sc-293184 1 to 200 Mouse anti-Cdk5 (J-3) sc-6247 1 to 200 The aim of the present study was to investigate the mechanism involved in the hyperphosphorylation of tau protein in the retina caused by Vps35 deficiency.

Techniques: Activity Assay, Phospho-proteomics, Cell Culture, Over Expression

Journal: Investigative ophthalmology & visual science

Article Title: Vps35 Deficiency Impairs Cdk5/p35 Degradation and Promotes the Hyperphosphorylation of Tau Protein in Retinal Ganglion Cells.

doi: 10.1167/iovs.61.1.1

Figure Lengend Snippet: FIGURE 5. Vp35 interacts with p35 in vivo and in vitro. (A, B) Vps35 and p35 were colocalized in the cytoplasm of RGCs in vivo and in vitro by immunofluorescence. (C) The colocalization of Cdk5 and p35 in primary cultured RGCs. (D) The interaction between Vps35 and p35 by IP was obvious 1 week after the intravitreal injection of 50 nmol glutamate. Cdk5 interacted with p35 both in control group and one week after the intravitreal injection of 50 nmol of glutamate.

Article Snippet: Abcam Rabbit anti-tau antibody ab32057 1 to 300 Rabbit anti-tau phospho s396 ab109390 1 to 200 Rabbit anti-tau phospho s404 ab131338 1 to 200 Goat anti-Vps35 ab10099 1 to 200 Rabbit anti-Vps35 ab157220 1 to 250 Rabbit anti-LAMP1 ab24170 1 to 200 Goat anti-mouse IgG H&L (Cy3) ab97035 1 to 300 Donkey anti-goat IgG H&L (Cy3) ab6949 1 to 500 Goat anti-rabbit IgG H&L (Alexa Fluor 488) ab150077 1 to 300 Donkey anti-rabbit IgG H&L (Alexa Fluor 488) ab150073 1 to 300 Rabbit anti-beta Ш tubulin ab18207 1 to 500 Mouse anti-CD90/Thy1 ab225 1 to 300 Goat anti-GFP ab6662 1 to 300 Cell Signaling Technology Rabbit anti-p35/25 (C64B10) mAb #2680 1 to 200 Rabbit anti-UBE1a/b #4891 1 to 25 Rabbit anti-EEA1 (C45B10) mAb #3288 1 to 100 Santa Cruz Biotechnology Mouse anti-p35 (4G11) sc-293184 1 to 200 Mouse anti-Cdk5 (J-3) sc-6247 1 to 200 The aim of the present study was to investigate the mechanism involved in the hyperphosphorylation of tau protein in the retina caused by Vps35 deficiency.

Techniques: In Vivo, In Vitro, Immunofluorescence, Cell Culture, Injection, Control

Journal: Investigative ophthalmology & visual science

Article Title: Vps35 Deficiency Impairs Cdk5/p35 Degradation and Promotes the Hyperphosphorylation of Tau Protein in Retinal Ganglion Cells.

doi: 10.1167/iovs.61.1.1

Figure Lengend Snippet: FIGURE 6. Deficiency of Vps35 leads to a decrease in LAMP1 of retina. (A, B) The expression of LAMP1 and EEA1 in the retina significantly decreased, while p35 increased, 1 week after the intravitreal injection of 50 nmol of glutamate. (C, D) The inhibition of Vps35 resulted in a decrease in LAMP1 in primary RGCs. **P < 0.01; *P < 0.05.

Article Snippet: Abcam Rabbit anti-tau antibody ab32057 1 to 300 Rabbit anti-tau phospho s396 ab109390 1 to 200 Rabbit anti-tau phospho s404 ab131338 1 to 200 Goat anti-Vps35 ab10099 1 to 200 Rabbit anti-Vps35 ab157220 1 to 250 Rabbit anti-LAMP1 ab24170 1 to 200 Goat anti-mouse IgG H&L (Cy3) ab97035 1 to 300 Donkey anti-goat IgG H&L (Cy3) ab6949 1 to 500 Goat anti-rabbit IgG H&L (Alexa Fluor 488) ab150077 1 to 300 Donkey anti-rabbit IgG H&L (Alexa Fluor 488) ab150073 1 to 300 Rabbit anti-beta Ш tubulin ab18207 1 to 500 Mouse anti-CD90/Thy1 ab225 1 to 300 Goat anti-GFP ab6662 1 to 300 Cell Signaling Technology Rabbit anti-p35/25 (C64B10) mAb #2680 1 to 200 Rabbit anti-UBE1a/b #4891 1 to 25 Rabbit anti-EEA1 (C45B10) mAb #3288 1 to 100 Santa Cruz Biotechnology Mouse anti-p35 (4G11) sc-293184 1 to 200 Mouse anti-Cdk5 (J-3) sc-6247 1 to 200 The aim of the present study was to investigate the mechanism involved in the hyperphosphorylation of tau protein in the retina caused by Vps35 deficiency.

Techniques: Expressing, Injection, Inhibition

Journal: Investigative ophthalmology & visual science

Article Title: Vps35 Deficiency Impairs Cdk5/p35 Degradation and Promotes the Hyperphosphorylation of Tau Protein in Retinal Ganglion Cells.

doi: 10.1167/iovs.61.1.1

Figure Lengend Snippet: FIGURE 7. There was a direct interaction between Vps35 and LAMP1 and between LAMP1 and p35. (A–C) The colocalization of Vps35 and LAMP1, p35 and LAMP1, and p35 and EEA1 in the GCL of retina. (D–F) The colocalization of Vps35 and LAMP1, p35 and LAMP1, and p35 and EEA1 in primary cultured RGC. (G) The coimmunoprecipitation showed a direct interaction of Vps35 and LAMP1, and LAMP1 and p35, with protein extracted from rat retinae.

Article Snippet: Abcam Rabbit anti-tau antibody ab32057 1 to 300 Rabbit anti-tau phospho s396 ab109390 1 to 200 Rabbit anti-tau phospho s404 ab131338 1 to 200 Goat anti-Vps35 ab10099 1 to 200 Rabbit anti-Vps35 ab157220 1 to 250 Rabbit anti-LAMP1 ab24170 1 to 200 Goat anti-mouse IgG H&L (Cy3) ab97035 1 to 300 Donkey anti-goat IgG H&L (Cy3) ab6949 1 to 500 Goat anti-rabbit IgG H&L (Alexa Fluor 488) ab150077 1 to 300 Donkey anti-rabbit IgG H&L (Alexa Fluor 488) ab150073 1 to 300 Rabbit anti-beta Ш tubulin ab18207 1 to 500 Mouse anti-CD90/Thy1 ab225 1 to 300 Goat anti-GFP ab6662 1 to 300 Cell Signaling Technology Rabbit anti-p35/25 (C64B10) mAb #2680 1 to 200 Rabbit anti-UBE1a/b #4891 1 to 25 Rabbit anti-EEA1 (C45B10) mAb #3288 1 to 100 Santa Cruz Biotechnology Mouse anti-p35 (4G11) sc-293184 1 to 200 Mouse anti-Cdk5 (J-3) sc-6247 1 to 200 The aim of the present study was to investigate the mechanism involved in the hyperphosphorylation of tau protein in the retina caused by Vps35 deficiency.

Techniques: Cell Culture

Journal: Cell Death and Differentiation

Article Title: IRF4 is a novel mediator for neuronal survival in ischaemic stroke

doi: 10.1038/cdd.2014.9

Figure Lengend Snippet: SRF-deficiency abrogates IRF4-mediated neuroprotection in vivo . ( a – c ) SRF ablation reversed cerebroprotection upon IRF4 overexpression. ( a ) TTC-stained brains from IRF4-TG, SRF-KO and IRF4nTG/SRF-KO mice 24 h after I/R. Scale bar: 10 mm. ( b and c ) Quantification of infarct volumes ( b ) and neurological scores ( c ) 24 and 72 h after I/R. Four to six independent experiments were performed. ( d ) IRF4-TG, SRF-KO and IRF4nTG/SRF-KO mice were subjected to 24-h I/R. The Fluoro-jade B- (left panel) and TUNEL-positive (right panel) neurons in brains were quantified. Three to four independent experiments were performed. ( e ) Immunoblotting and quantification of the BDNF, Bcl2 and cleaved caspase-3 levels in the brains of IRF4-TG, SRF-KO or IRF4nTG/SRF-KO mice subjected to a sham operation or 6-h I/R. GAPDH served as a loading control. Three independent experiments were performed. For b – e , * P <0.0001 compared with IRF4-TG mice. NS: not significant, post hoc Tukey's test with Bonferroni correction. The values are the means±S.E

Article Snippet: Finally, an Odyssey infrared imaging system (LI-COR Biosciences) was employed to detect the blot signals; signals were quantified using an Odyssey software (LI-COR).The primary antibodies used were as follows: rabbit anti-cleaved caspase-3 (Asp 175; #9661), anti-fosB (#2251), anti-Bcl2 (#2870), anti-Bax (#2772), anti-PSD95 (#2507), anti-EGR1 (#4153) and anti-Cdk5 (#2680; all from Cell Signaling Technology, Beverly, MA, USA; 1 : 1000); mouse anti-GAPDH (MB001; both from Bioworld Technology, Minneapolis, MN, USA); rabbit anti-BDNF (ab72439; from Abcam); and rabbit anti-IRF4 (sc28696), anti-SRF (H300; sc13029), anti-JunB (sc46), anti-Gelsolin (H-70, sc48769), anti-Egr-2 (H-220, sc20690), anti-Nur77 (M-210, sc5569) and goat anti-CTGF (L-20, sc14939; all from Santa Cruz Biotechnology).

Techniques: In Vivo, Over Expression, Staining, TUNEL Assay, Western Blot

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: The following antibodies were used: AT8 (mouse monoclonal, 1:500, #MN1020, Thermo Fisher Scientific), AT180 (mouse monoclonal, 1:1000, #MN1040, Thermo Fisher Scientific), HT7 (mouse monoclonal, 1:1000, #MN1000, Thermo Fisher Scientific), MC1 (mouse monoclonal, 1:500, kindly provided by Peter Davies), α-CDK5 (mouse monoclonal, 1:1000, #sc-6247, Santa Cruz Biotechnology), α-p35/p25 (rabbit monoclonal, 1:1000, #2680, Cell Signaling Technology), α-Vinculin (mouse monoclonal, 1:1000, #sc-73614, Santa Cruz Biotechnology), α-GAPDH (rabbit polyclonal, 1:2000, #ABS16, Merck Millipore), α-beta-Tubulin (mouse monoclonal, 1:10000, #MAB3408, Merck Millipore), α-GSK3β (mouse monoclonal, 1:1500, #9832, Cell Signaling Technology), α-pGSK3β Ser9 (rabbit monoclonal, 1:1000, #9322, Cell Signaling Technology), α-GFAP (rabbit polyclonal, 1:1000, #173002, Synaptic Systems), α-HA-tag (mouse monoclonal, 1:1000, #2367, Cell Signaling Technology), α-PSD95 (mouse monoclonal, 1:1000, #MAB1598, Merck Millipore), α-β-catenin (mouse monoclonal, 1:500, #sc-7963, Santa Cruz Biotechnology), α-pβ-catenin Ser33/37/Thr41 (rabbit polyclonal, 1:1000, #9561, Cell Signaling Technology), M3.2 (mouse monoclonal, 1:1000, a kind gift from Paul Mathews), α-Akt (rabbit monoclonal, 1:1500, #4691, Cell Signaling Technology), α-pAkt Ser473 (rabbit monoclonal, 1:1000, #4060, Cell Signaling Technology).

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: The following antibodies were used: AT8 (mouse monoclonal, 1:500, #MN1020, Thermo Fisher Scientific), AT180 (mouse monoclonal, 1:1000, #MN1040, Thermo Fisher Scientific), HT7 (mouse monoclonal, 1:1000, #MN1000, Thermo Fisher Scientific), MC1 (mouse monoclonal, 1:500, kindly provided by Peter Davies), α-CDK5 (mouse monoclonal, 1:1000, #sc-6247, Santa Cruz Biotechnology), α-p35/p25 (rabbit monoclonal, 1:1000, #2680, Cell Signaling Technology), α-Vinculin (mouse monoclonal, 1:1000, #sc-73614, Santa Cruz Biotechnology), α-GAPDH (rabbit polyclonal, 1:2000, #ABS16, Merck Millipore), α-beta-Tubulin (mouse monoclonal, 1:10000, #MAB3408, Merck Millipore), α-GSK3β (mouse monoclonal, 1:1500, #9832, Cell Signaling Technology), α-pGSK3β Ser9 (rabbit monoclonal, 1:1000, #9322, Cell Signaling Technology), α-GFAP (rabbit polyclonal, 1:1000, #173002, Synaptic Systems), α-HA-tag (mouse monoclonal, 1:1000, #2367, Cell Signaling Technology), α-PSD95 (mouse monoclonal, 1:1000, #MAB1598, Merck Millipore), α-β-catenin (mouse monoclonal, 1:500, #sc-7963, Santa Cruz Biotechnology), α-pβ-catenin Ser33/37/Thr41 (rabbit polyclonal, 1:1000, #9561, Cell Signaling Technology), M3.2 (mouse monoclonal, 1:1000, a kind gift from Paul Mathews), α-Akt (rabbit monoclonal, 1:1500, #4691, Cell Signaling Technology), α-pAkt Ser473 (rabbit monoclonal, 1:1000, #4060, Cell Signaling Technology).

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: The following antibodies were used: AT8 (mouse monoclonal, 1:500, #MN1020, Thermo Fisher Scientific), AT180 (mouse monoclonal, 1:1000, #MN1040, Thermo Fisher Scientific), HT7 (mouse monoclonal, 1:1000, #MN1000, Thermo Fisher Scientific), MC1 (mouse monoclonal, 1:500, kindly provided by Peter Davies), α-CDK5 (mouse monoclonal, 1:1000, #sc-6247, Santa Cruz Biotechnology), α-p35/p25 (rabbit monoclonal, 1:1000, #2680, Cell Signaling Technology), α-Vinculin (mouse monoclonal, 1:1000, #sc-73614, Santa Cruz Biotechnology), α-GAPDH (rabbit polyclonal, 1:2000, #ABS16, Merck Millipore), α-beta-Tubulin (mouse monoclonal, 1:10000, #MAB3408, Merck Millipore), α-GSK3β (mouse monoclonal, 1:1500, #9832, Cell Signaling Technology), α-pGSK3β Ser9 (rabbit monoclonal, 1:1000, #9322, Cell Signaling Technology), α-GFAP (rabbit polyclonal, 1:1000, #173002, Synaptic Systems), α-HA-tag (mouse monoclonal, 1:1000, #2367, Cell Signaling Technology), α-PSD95 (mouse monoclonal, 1:1000, #MAB1598, Merck Millipore), α-β-catenin (mouse monoclonal, 1:500, #sc-7963, Santa Cruz Biotechnology), α-pβ-catenin Ser33/37/Thr41 (rabbit polyclonal, 1:1000, #9561, Cell Signaling Technology), M3.2 (mouse monoclonal, 1:1000, a kind gift from Paul Mathews), α-Akt (rabbit monoclonal, 1:1500, #4691, Cell Signaling Technology), α-pAkt Ser473 (rabbit monoclonal, 1:1000, #4060, Cell Signaling Technology).

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