Specific inhibition of p25/Cdk5 activity by the Cdk5 inhibitory peptide reduces neurodegeneration in vivo

Abstract

The aberrant hyperactivation of Cyclin-dependent kinase 5 (Cdk5), by the production of its truncated activator p25, results in the formation of hyperphosphorylated tau, neuroinflammation, amyloid deposition, and neuronal death in vitro and in vivo. Mechanistically, this occurs as a result of a neurotoxic insult that invokes the intracellular elevation of calcium to activate calpain, which cleaves the Cdk5 activator p35 into p25. It has been shown previously that the p25 transgenic mouse as a model to investigate the mechanistic implications of p25 production in the brain, which recapitulates deregulated Cdk5-mediated neuropathological changes, such as hyperphosphorylated tau and neuronal death. To date, strategies to inhibit Cdk5 activity have not been successful in targeting selectively aberrant activity without affecting normal Cdk5 activity. Here we show that the selective inhibition of p25/Cdk5 hyperactivation in vivo, through overexpression of the Cdk5 inhibitory peptide (CIP), rescues against the neurodegenerative pathologies caused by p25/Cdk5 hyperactivation without affecting normal neurodevelopment afforded by normal p35/Cdk5 activity. Tau and amyloid pathologies as well as neuroinflammation are significantly reduced in the CIP-p25 tetra transgenic mice, whereas brain atrophy and subsequent cognitive decline are reversed in these mice. The findings reported here represent an important breakthrough in elucidating approaches to selectively inhibit the p25/Cdk5 hyperactivation as a potential therapeutic target to reduce neurodegeneration.

Figure 1.

Characterization of CIPTg mice. A, The CIPTg construct contains a 3′-FLAG-tagged CIP transgene incorporated in to the ROSA26 locus whose 5′ regulatory elements were separated from the coding region with a floxed stop sequence. A frt-flanked neomycin resistance (Neo) cassette was inserted next to the transgene before the 3′ homology arm. Representative confocal images of frontal cortex (layer 2/3) (B) and hippocampus (CA3 region) (C) from 6-week-old and 18-week-old WT and CIPTg mice. The sections were immunostained with anti-FLAG antibody (red), and nuclei were counterstained with DAPI (blue). D, Immunoblot analyses of brain lysates from WT and CIPTg mice using anti-Cdk5 (C8) and anti-FLAG antibodies. Equal amounts of protein loading were confirmed by reprobing the membrane with anti-tubulin antibody. E, Quantification of immunoblot analyses in D by densitometric scanning (n = 3) (***p < 0.001 and ^NSp > 0.05). F, Kinase assays using active kinase (Cdk5) from WT and CIPTg mice brains to phosphorylate a high-molecular-weight neurofilament (NF-H) peptide (^NSp > 0.05). G, WT and CIPTg mice brain lysates were immunoprecipitated (IP) using anti-Cdk5 (J3) antibody. Samples were separated by SDS-PAGE and immunoprobed with anti-p35, anti-p39, and anti-Cdk5 (C8) antibodies. H, Quantification of immunoblot analyses in G by densitometric scanning (^NSp > 0.05). MRI scanning images of coronal slices (I) and Nissl staining images of frontal cortex (J) from WT and CIPTg mice brains. Scale bars, 20 μm. Data are representative of n = 4 mice. Error bars indicate ±SEM.

Figure 2.

Characterization of TetraTg mice. A, Immunofluorescence images of frontal cortex (layer 2/3) from 12 week induced p25Tg mice, TetraTg, and their respective age-matched CIPTg control mice. The sections were immunostained with anti-GFP antibody (green) and anti-FLAG antibody (red). Nuclei were counterstained with DAPI (blue). B, Immunoblot analyses of brain lysates from 12 week induced p25Tg, TetraTg, and the CIPTg mice using anti-GFP, anti-FLAG, and anti-Cdk5 (C8) antibodies. C, Representative in vitro kinase assays using active kinase (Cdk5) immunoprecipitated from the p25Tg, TetraTg, and CIPTg mice (**p < 0.001, ***p < 0.0001, ^NSp > 0.05). D, Western blot analyses of brain lysates from 12 week induced p25Tg, TetraTg, and the CIPTg mice using anti-phospho-GSK-3β (Ser9) antibody. E, Quantification of immunoblots in D by densitometric scanning (*p < 0.05, **p < 0.001, ^NSp > 0.05). Scale bars, 20 μm. Error bars indicate ±SEM.

Figure 3.

Reduced neuroinflammation in TetraTg mice. A, Representative confocal images of frontal cortex from 12 week induced p25Tg, TetraTg, and CIPTg mice. The brain sections were labeled with anti-GFAP antibody (green), anti-Cd11b antibody (red), and nuclei were stained with DAPI (blue). B, Western blot analyses of brain lysates from p25Tg, TetraTg, and the CIPTg mice using anti-GFAP and anti-Cd11b antibodies. C, Quantification of immunoblots in B by densitometric scanning (***p < 0.001). D, Immunoblot analyses were performed on lysates from the samples same as in B using anti-cPLA2 antibody. E, Quantification of immunoblot analyses in D (*p < 0.05, ^NSp > 0.05). F, cPLA2 activity assays were performed with lysates from p25Tg, TetraTg, and CIPTg mice (*p < 0.05, ^NSp > 0.05, ***p < 0.001). G, Results from the mass spectrometric analyses for the p25Tg, TetraTg, and CIPTg mice brain samples. Results were normalized against the internal standards of LPC (*p < 0.05, **p < 0.01). Scale bars, 20 μm. Data are representative of n = 4 mice. Error bars indicate ±SEM.

Figure 4.

Inhibition of p25-mediated phospho-tau and β-amyloid accumulation in TetraTg mice. A, Immunofluorescence images of frontal cortex from p25Tg, TetraTg, and their respective age-matched CIPTg mice using AT8 (PHF–tau) and AT180 (PHF–tau) (red) antibodies. DAPI-stained nuclei appear blue. B, Hyperphosphorylated tau protein levels were analyzed by Western blot analyses using AT8 and AT180 antibodies. C, Quantification of immunoblots in B by densitometric scanning (***p < 0.001). D, Brain sections from p25Tg, TetraTg, and CIPTg mice were immunostained with Aβ_1–42 and 6E10 (Aβ_1–16) (green). Nuclei were stained with DAPI (blue). E, Thioflavin-S staining (top) and Bielschowsky silver staining (bottom) images of the brain sections from p25Tg, TetraTg, and CIPTg mice. Scale bars, 20 μm. Data are representative of n = 4 mice. Error bars indicate ±SEM.

Figure 5.

CIP expression rescues p25-induced brain atrophy in TetraTg mice. A, Representative confocal images of frontal cortex from 12 week induced p25Tg, TetraTg, and CIPTg mice. Top shows the TUNEL staining images (red), and the bottom shows the immunofluorescence staining with cleaved caspase-3 antibody (green). Nuclei were counterstained with DAPI (blue). B, Percentage cell death in bottom of A was calculated by counting the TUNEL-positive cells normalized with DAPI from 10 independent fields (***p < 0.001). C, Western blot analyses of brain lysates from 12 week induced p25Tg, TetraTg, and CIPTg mice using anti-cleaved caspase-3 antibody. D, Quantification of immunoblots in C by densitometric scanning (***p < 0.001). E, Whole-brain photos showing the levels of forebrain atrophy in CIPTg, p25Tg, and TetraTg mice. F, Quantification of brain weight from p25Tg, TetraTg, and CIPTg mice (***p < 0.0001 and ^NSp > 0.05). G, Nissl staining images of frontal cortex as well as CA3 region of the hippocampus from the p25Tg, TetraTg, and CIPTg control mice. H, I, Representative magnetic resonance images of sagittal, transverse, and axial planes of the CIPTg, p25Tg, and TetraTg mice. White rectangular boxes, arrowheads, and arrows indicate the level of cortical atrophy between the three groups of mice. Scale bars, 20 μm. Data are representative of n = 4 mice. Error bars indicate ±SEM.

Figure 6.

Recovery of p25-induced neurocognitive deficits in TetraTg mice. A, Graphical representation of reference memory errors (8-arm radial maze performance) made by mice groups. Data points show mean reference memory errors in blocks of two trials [p < 0.0001 between the groups (repeated-measures ANOVA), *p < 0.05 compared with CIPTg, ^#p < 0.05 compared with TetraTg, and ^±p < 0.05 compared with WT mice (Student's t test)]. B, Graph shows the working memory errors made by mice groups. Columns represent mean working memory errors from 10 sessions (*p < 0.01, one-way ANOVA, followed by Tukey's test). n = 6 mice for each group. Error bars indicate ±SEM.