CIP specifically inhibited p25/Cdk5 but not p35/Cdk5 activity in coinfected primary cortical neurons. Infections of primary cortical neurons were carried out and Cdk5 was immunoprecipitated using C-8 and J-3 antibodies from equal amounts of lysates. Immunoprecipitates were then subjected to in vitro histone H1 kinase assays and Western blot for immunodetection. All bar graphs represent mean optical density measurements of phospho-histone (³²P-histone H1) of both Cdk5 activities from C-8 and J-3 IPs measured from autoradiographs (first and fourth panels in (A–C)). Data are expressed as mean±s.e.m. of four separate experiments. Coomassie-stained gels show equal amounts of histone present in all assays (histone H1; second and fifth panels in (A–C)). (A) NI (lane 1), EV (lane 2), p25+wild-type Cdk5 (p25/WT; lane 3), p25+dominant-negative Cdk5 (p25/DN; lane 4), p25+WT+CIP (p25/Cdk5/CIP; lane 5), p25 only (lane 6) and p25+CIP (p25/CIP; lane 7). Cdk5 was immunodetected using polyclonal J-3 antibody (third panel) in the C-8 IP. In the J-3 IP, p25 and p35 were immunodetected using polyclonal C-19 antibody (sixth panel); Cdk5 was detected using polyclonal C-8 antibody (seventh panel). (B) The CIP effect on infected p35/Cdk5 activity was investigated using in vitro kinase assays after infections of 7-DIC cortical neurons. EV, lane 1; p35/Cdk5, lane 2; p35/Cdk5/CIP, lane 3; p35/Cdk5+roscovitine, lane 4. Cdk5 IP and Western blot immunodetection were performed using the same antibodies as in (A). (C) CIP effect on endogenous Cdk5 activity was investigated using in vitro kinase assays in neurons. NI (lane 1), EV infected (lane 2), NI+roscovitine (lane 3) and CIP infected (lane 4). Cdk5 IP and Western blot immunodetection were performed using the same antibodies as in (A).

CIP inhibited Aβ₁₋₄₂-induced endogenous p25/Cdk5 hyperactivity and tau hyperphosphorylation in cortical neurons. (A) 7-DIC cortical neurons were infected with EV or CIP and treated with 10 μM Aβ₁₋₄₂ for 6 h. Cdk5 was immunoprecipitated from equal amounts of lysates with polyclonal C-8 and J-3 antibodies, and subjected to histone H1 kinase assays. The bar graph represents average mean optical density±s.e.m. of phospho-histone levels in four separate experiments. Coomassie panels show equal amounts of histone present in the assay. To determine Cdk5, p25 and p35 levels in the Cdk5 immunoprecipitates, Cdk5 was immunodetected using polyclonal J-3 antibody in the C-8 IP. In the J-3 IP, p25 and p35 were immunodetected using polyclonal C-19 antibody; Cdk5 was detected using rabbit polyclonal C-8. (B) Western blots were performed to detect the presence of p35, p25 and Cdk5 using lysates from (A). The bar graph shows mean density±s.e.m. measurements of each of the three proteins of four independent experiments. (C) Lysates from (A) were resolved by SDS–PAGE. Lane 1: EV; lane 2: EV+Aβ₁₋₄₂; lane 3: CIP+Aβ₁₋₄₂. The top three panels show phospho-tau using different phospho-tau antibodies (anti-pS199/202, anti-pS404 and AT8), the fourth panel shows total tau (Tau5) and the bottom panel shows tubulin. The bottom two panels show equal amounts of total tau (Tau5) and tubulin in the lysates. The bar graph shows mean optical densities of phospho-tau ps199/202, ps404 and AT-8 of four separate experiments.

CIP reduces neuronal apoptosis induced by Aβ₁₋₄₂ treatment in cortical neurons as measured by TUNEL assay and ICC assay of cleaved caspase-3. p35 Promoter-CIP-hrIRES-GFP(CIP-GFP) and p35 Promoter-empty vector hrIRES-GFP (GFP) were transfected into 3-DIC cortical neurons and after 24 h neurons were treated with 10 μM Aβ₁₋₄₂ for 6 h. Neurons were fixed and stained in separate experiments for TUNEL and caspase-3 (cleaved) to show apoptotic neurons. In the first row (A–D), neurons were stained for TUNEL. (A) CIP-GFP; (B) TUNEL-TMR; (C) nuclear staining with DAPI; (D) overlay of CIP and TUNEL. In the second row (E–H), neurons are stained for cleaved caspase-3 expression. (E) CIP-GFP; (F) cleaved caspase-3; (G) nuclear staining with DAPI; (H) overlay of CIP and cleaved caspase-3. Cell counts were performed as follows: 10 independent fields were analyzed with a total of 500 neurons where TUNEL and cleaved caspase-3 could be counted. DAPI staining gave the total number of neurons and CIP was identified by GFP. The bar graph shows the quantity of neuronal apoptosis expressed as mean±s.e.m. from four separate transfections.

Cdk5, p35, p25 and CIP expression in coinfected primary cortical neurons. E18 rat embryonic cortical neurons were infected with the following expression constructs: empty LV-GFP vector (EV), p25 and wild-type Cdk5 (Cdk5WT) with or without CIP, p35 and wild-type Cdk5 with or without CIP, p25 and dominant-negative Cdk5 (Cdk5DN) and p35 and Cdk5DN. (A) After 3 days of infection, neuronal lysates were resolved on 4–20% SDS–PAGE gradient gels and Western blotting was employed to detect endogenous and transfected Cdk5 (C-8; top panel), p35 and p25 (C-19; bottom panel). Lane 1: EV only; lanes 2 and 3: p25+Cdk5WT with and without CIP respectively; lanes 4 and 5: p35+Cdk5WT with and without CIP respectively; lane 6: p25+Cdk5DN; lane 7: p35+Cdk5DN. (B) Confocal images illustrate the cortical neurons single infections with Cdk5, p35, p25 and CIP (panels a–d, respectively), double infection of Cdk5 and p25 (panels e–h), double infection of Cdk5 and p35 (panels j–m), triple infections of Cdk5, p25 and CIP (panels n–r) and Cdk5, p35 and CIP (panels s–w). The scale bar represents 20 μm.

CIP specifically inhibits tau hyperphosphorylation induced by p25/Cdk5 but not tau phosphorylation induced by p35/Cdk5. (A) Cortical neurons were infected with the following expression constructs: EV (lane 2), p25+Cdk5 (lane 3), p25+Cdk5+CIP (lane 4), p25+Cdk5+roscovitine treatment (lane 5), p25 only (lane 6) and p25+CIP (lane 7). The top three panels show phospho-tau with three different phospho-tau antibodies (anti-pS199/202, anti-pS404 and AT8), while the bottom panel shows total tau (Tau5). (B) Cortical neurons were infected with the following expression constructs: EV (lane 1), p35+Cdk5 (lane 2), p35+Cdk5+CIP (lane 3) and p35+Cdk5 treated with roscovitine (lane 4). The top three panels show Western blots for phospho-tau using the same phospho-tau antibodies mentioned above and the bottom panel shows total tau (Tau5). The bar graphs show the mean optical density of phospho-tau (ps199/202, ps404 and AT-8). The results are expressed as mean±s.e.m. of four independent experiments.

CIP reduced neuronal apoptosis induced by p25/Cdk5 overexpression as well as Aβ₁₋₄₂ treatment of cortical neurons. (A) Levels of cleaved caspase-3, a well-established marker of apoptosis, were investigated after E18 rat cortical neurons were infected with the following constructs: lane 1, EV; lane 2, p25+wild-type Cdk5 (p25/WT); lane 3, p25+wild-type Cdk5+CIP (p25/WT/CIP); lane 4, p25+dominant-negative Cdk5 (p25/DN); lane 5, p35+wild-type Cdk5 (p35/WT); lane 6, p35+wild-type Cdk5+CIP (p35/WT/CIP); lane 7, NI control. The bar graph shows measurements of cleaved caspase-3 expressed as mean density±s.e.m. of four separate experiments. (B) Cortical neurons were infected with EV (lane 2), EV and treated with Aβ₁₋₄₂ (lane 3), infected with CIP and treated with Aβ₁₋₄₂ (lane 4), NI, treated with roscovitine and then 1 h later with Aβ₁₋₄₂ (lane 5) and NI control (lane 1). All Aβ₁₋₄₂ treatments were carried out for 6 h. Cell lysates were resolved by SDS–PAGE and cleaved caspase-3 (top panel) and tubulin (bottom panel) were immunodetected. The bar graph shows mean density measurements of cleaved caspase-3 expressed as ±s.e.m. of four separate experiments. (C) To confirm the Western blot data, we performed immunocytochemical assays of the infections as in (A, B) using cleaved caspase-3 as a marker. The bar graphs show cell counts of cleaved caspase-3-positive neurons with the coinfections and Aβ₁₋₄₂ treatment with or without CIP in cortical neurons. (D) As another measure of apoptosis/survival afforded by CIP, we used phospho-Akt, a well-established marker of survival. Lysates from (A, B) were resolved by SDS–PAGE and immunodetected using anti-phospho-Akt antibody. The bar graph shows mean optical density±s.e.m. of four separate experiments of phospho-Akt.

CIP did not affect endogenous neuronal Cdc2 activity nor the activity of other cyclin-dependent kinases, but inhibited Cdk5 hyperactivity in a dose-dependent manner. (A) 7-DIC cortical neurons were treated with 10 μM Aβ₁₋₄₂ or PBS (UT) for 6 h and subjected to in vitro kinase assays using 5 μg GST-CIP to investigate its inhibitory properties. Cdk5 was immunoprecipitated using C-8 antibody and Cdc2 was immunoprecipitated using the PSTAIRE antibody. In lanes 7 and 8, we infected CIP prior to treatment with Aβ₁₋₄₂ for 6 h, immunoprecipitated Cdc2 and performed histone H1 kinase assays. The top panel shows the autoradiograph, while the bottom panel shows Coomassie-stained histone H1 bands. (B) 7-DIC cortical neurons were infected with the CIP expression construct using the lentiviral system (lane 3 only) and neurons were treated with 10 μM Aβ₁₋₄₂ or PBS (UT) for 6 h. Endogenous Cdk2, Cdk4 and Cdk6 were immunoprecipitated from equal amounts of lysates and immunoprecipitates were then subjected to in vitro histone H1 kinase assays. The top panel shows the autoradiograph while the bottom panel shows Coomassie-stained histone H1 bands. (C) CIP affects p25/Cdk5 activity in a dose-dependent manner. Cdk5 was immunoprecipitated using C-8 antibody from equal amounts of lysates of cortical neurons treated or untreated with 10 μM Aβ₁₋₄₂ for 6 h. Immunoprecipitates were then subjected to in vitro histone H1 kinase assays with the addition of increasing amounts of GST-CIP in a reaction volume of 60 μl. The top panel is the autoradiograph and the bottom panel shows the corresponding Coomassie-stained histone H1 bands. GST-CIP titration experiments were reproducible in four separate assays.