p10, the N-terminal domain of p35, protects against CDK5/p25-induced neurotoxicity

Abstract

Cyclin-dependent kinase 5(CDK5) in complex with its activator, p35 (protein of 35 kDa), is essential for early neurodevelopment in mammals. However, endogenous cleavage of p35 to p25 is associated with neuron death and neurodegenerative disease. Here we show that a peptide (p10') encoding the N-terminal domain of p35 protects against CDK5/p25-induced toxicity in neurons. p10' also prevented the death of neurons treated with the neurotoxin, 1-methyl-4-phenylpyridinium (MPP(+)), which induces conversion of endogenous p35 to p25, and Parkinson disease (PD)-like symptoms in animals. MPP(+) induces CDK5/p25-dependent phosphorylation of peroxiredoxin 2 (Prx2), resulting in inhibition of its peroxireductase activity and accumulation of reactive oxygen species (ROS). We found that p10' expression inhibited both Prx2 phosphorylation and ROS accumulation in neurons. In addition, p10' inhibited the p25-induced appearance of antigen of the Ki67 antibody (Ki67) and phosphohistone H2AX (γH2AX), classic markers of cell cycle activity and DNA double-strand breakage, respectively, associated with neuron death. Our results suggest that p10 (protein of 10 kDa) is a unique prosurvival domain in p35, essential for normal CDK5/p35 function in neurons. Loss of the p10 domain results in CDK5/p25 toxicity and neurodegeneration in vivo.

Fig. 1.

p10′ protects against CDK5/p25 toxicity. (A) COS7 cells were cotransfected with gfp-CDK5 + myc-p35 (Top) or myc-p25 (Middle); COS7 cells stably expressing p10′ (COS7^p10′) were cotransfected with gfp-CDK5 + myc-p25 (Bottom). Arrows indicate cotransfectants. Mag = 400× . (B) Condensed vs. normal nuclei in A were quantified by counting >300 cells per experimental condition. (C) COS7 or COS7^p10′ cells were transfected with the indicated genes and Western analysis was conducted on whole cell lysates using the indicated antibodies. (D) SY5Y^p10′ cells were coinfected with LV-CDK5 + LV-p25 at decreasing multiplicity of infection (MOI) of both, and cell viability was assessed by using the CellTiter-Glo Assay (Promega) for cellular ATP. (E) Rat primary cortical neurons were infected with LV- p10′ or LV-GFP, then subsequently coinfected with LV-CDK5 + LV-p25. Cell viability was measured 48 h after coinfection by MTT reduction. More than 90% of cells were neurons. All error bars represent SD about the mean from three separate experiments.

Fig. 2.

p10′ protects against MPP⁺ toxicity. (A) Rat cortical neurons were treated with 50 μM MPP⁺ for the indicated times, and p25/p35 was visualized by Western blot using anti-p25 (C-19). loading was normalized by anti-MAP2 staining (B) LV-p10′ infected or uninfected neurons were treated with MPP⁺ (50 μM), and the presence of live neurons (dead neurons are washed away) was measured after 24 h by neuronal-specific tubulin staining on Western blot using anti–Tuj-1. Anti-GFAP (glial fibrillary acidic protein) shows equal loading (glial cells are not sensitive to MPP⁺); anti-p10 (N-20) shows correlation between cell survival and p10′ expression. (C) In the presence of MPP⁺ (50 μM) cell viability was alternatively analyzed by MTT reduction.

Fig. 3.

MG132 protects against MPP⁺ toxicity. (A) Rat cortical neurons were infected for 72 h with LV-p10′ at which time p10′ expression levels were constant. Neurons were then treated with 10 μM MG132 for 0, 3, or 6 h. p10′ levels were measured by Western blot using anti-p10 (N-20). (B) Uninfected neurons were stained with anti-p10 (N-20) or anti-p25 (C-19) (Insets) in the presence of MPP⁺, MG132, or both, and Cy3 (red) second antibody staining was observed by confocal microscopy. Nuclei were stained with DAPI (blue). (C) Neuron death was determined by Guava ViaCount assay for dying cells after a 1-h pretreatment with 10 μM MG132, followed by a 24-h treatment with 50 μM MPP⁺.

Fig. 4.

p10′ prevents phosphorylation of Prx2 and generation of reactive oxygen species (ROS) in response to CDK5/p25 or MPP⁺. (A) SH-SY5Y cells were infected with CDK5, p25, or p35 and/or p10′ for 48 h. Western analysis was performed on cell lysates using a Prx2–pThr⁸⁹-specific (first row), or Prx2-specific (second row) antibody. (B) ROS production was monitored by DCF staining in cells expressing GFP, p10′, CDK5/p25 + GFP, or CDK5/p25 + p10′. (C) DCF fluorescence at 480 nm was quantified by FACS. A total of 5,000 cells per condition were flow sorted using a Guava EasyCyte instrument. (D) Rat cortical neurons were treated with MPP⁺ (50 μM), and Prx2 phosphorylation at varying times was monitored by Western blot. (E) Neurons were infected with p10′ for 48 h, then treated with 50 μM MPP⁺ for 24 h. Cell lysates were analyzed by Western blot for pPrx2. In the presence of MPP⁺ (lanes 1 and 3), p10′ reduced the levels of pPrx2 by 3.2-fold (determined by densitometry). (F) ROS production was monitored by DCF staining, as described in B.

Fig. 5.

p10′ prevents both Ki67 accumulation and H2AX phosphorylation. (A) Ki67. Neurons were isolated and cultured as described in Materials and Methods. Neurons were identified by anti-MAP2 staining. Ki67-expressing cells were identified by anti-Ki67 staining (Fig. S5). Percent of Ki67⁺ neurons in uninfected control, p25, or p10′ + p25-infected cells was determined by counting >500 neurons. Values are mean ±SD, n = 3. (B) H2AX. Western blot analysis of whole neurons was performed with the indicated antibodies.