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Peptide TFP5/TP5 derived from Cdk5 activator P35 provides neuroprotection in the MPTP model of Parkinson's disease.

Binukumar BK, Shukla V, Amin ND, Grant P, Bhaskar M, Skuntz S, Steiner J, Pant HC - Mol. Biol. Cell (2015)

Bottom Line: To date, strategies to specifically inhibit Cdk5 hyperactivity have not been successful without affecting normal Cdk5 activity.Previously we reported that TFP5 peptide has neuroprotective effects in animal models of Alzheimer's disease.The neuroprotective effect of TFP5/TP5 peptide is also associated with marked reduction in neuroinflammation and apoptosis.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.

No MeSH data available.


Related in: MedlinePlus

TFP5 inhibits hyperactive Cdk5/p25 in MPP+-induced mesencephalic primary cultures. A dose-dependent study. (A) Ventral mesencephalic neuronal-enriched cultures were pretreated with TFP5 (500 nM) or scrambled peptide for 12 h and then coincubated with different concentrations of MPP+ and TFP5 for 24 h. Cdk5 was immunoprecipitated from equal amounts of lysates using C-8 antibody. Immunoprecipitates were then subjected to in vitro kinase pad assays with histone H1 as substrate. Activity, as counts/minute, was quantified from three separate experiments and summarized in the bar graphs (***p < 0.001; **p < 0.01; *p < 0.05). (B) Ventral mesencephalic neuronal-enriched cultures were treated as in A, after which SDS–PAGE and Western blots were prepared with the Cdk5 and p35 antibodies. Note p25 expression in all MPP+ lanes. The results are expressed as mean ± SEM of three independent experiments (***p < 0.001). (C) Mesencephalic tissues from E14 mouse embryos were cultured and grown on polylysine-coated cover slips. Representative images. The neuronal cultures were pretreated with TFP5 (500 nM) or scrambled peptide for 12 h and then coincubated in the presence of 10 μM concentration of MPP+ and TFP5 for 24 h. The cells were fixed and stained. (D) Numbers of TH-immunoreactive (IR) neurons. Scale bar, 100 μm (C).
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Figure 1: TFP5 inhibits hyperactive Cdk5/p25 in MPP+-induced mesencephalic primary cultures. A dose-dependent study. (A) Ventral mesencephalic neuronal-enriched cultures were pretreated with TFP5 (500 nM) or scrambled peptide for 12 h and then coincubated with different concentrations of MPP+ and TFP5 for 24 h. Cdk5 was immunoprecipitated from equal amounts of lysates using C-8 antibody. Immunoprecipitates were then subjected to in vitro kinase pad assays with histone H1 as substrate. Activity, as counts/minute, was quantified from three separate experiments and summarized in the bar graphs (***p < 0.001; **p < 0.01; *p < 0.05). (B) Ventral mesencephalic neuronal-enriched cultures were treated as in A, after which SDS–PAGE and Western blots were prepared with the Cdk5 and p35 antibodies. Note p25 expression in all MPP+ lanes. The results are expressed as mean ± SEM of three independent experiments (***p < 0.001). (C) Mesencephalic tissues from E14 mouse embryos were cultured and grown on polylysine-coated cover slips. Representative images. The neuronal cultures were pretreated with TFP5 (500 nM) or scrambled peptide for 12 h and then coincubated in the presence of 10 μM concentration of MPP+ and TFP5 for 24 h. The cells were fixed and stained. (D) Numbers of TH-immunoreactive (IR) neurons. Scale bar, 100 μm (C).

Mentions: We first determined whether 24 h MPP+ incubation induces Cdk5 hyperactivation in mesencephalic primary cultures in the presence or absence of TFP5 pretreatment and coincubation. Seven days in culture (DIC) mesencephalic neuron–enriched primary cultures pretreated with TFP5 were coincubated with different micromolar concentrations of MPP+ and TFP5, followed by Cdk5 immunoprecipitation and kinase assay. From 0.01 to 10.0 μM, a concentration-dependent increase in the activity was observed in the presence of MPP+ treatment (Figure 1A); the decline at 100 μM is probably due to cell toxicity. Pretreatment and coincubation with 0.5 μM TFP5, however, was sufficient to significantly reduce the elevated activity (30%) at each concentration (Figure 1A). In Western blots of the same lysates as in Figure 1A, we see a decline of p35 expression from 0.01 to 10 μM with modest expression of p25 (Figure 1B). The kinase activities, however, are reduced at all concentrations (20–30%) by TFP5 treatment. Because 10 μM MPP+ induced maximum activity, we selected this as the measure of hyperactivity in vitro for further study. It is noteworthy that scrambled peptide did not show any inhibitory ­effect (Figure 1A).


Peptide TFP5/TP5 derived from Cdk5 activator P35 provides neuroprotection in the MPTP model of Parkinson's disease.

Binukumar BK, Shukla V, Amin ND, Grant P, Bhaskar M, Skuntz S, Steiner J, Pant HC - Mol. Biol. Cell (2015)

TFP5 inhibits hyperactive Cdk5/p25 in MPP+-induced mesencephalic primary cultures. A dose-dependent study. (A) Ventral mesencephalic neuronal-enriched cultures were pretreated with TFP5 (500 nM) or scrambled peptide for 12 h and then coincubated with different concentrations of MPP+ and TFP5 for 24 h. Cdk5 was immunoprecipitated from equal amounts of lysates using C-8 antibody. Immunoprecipitates were then subjected to in vitro kinase pad assays with histone H1 as substrate. Activity, as counts/minute, was quantified from three separate experiments and summarized in the bar graphs (***p < 0.001; **p < 0.01; *p < 0.05). (B) Ventral mesencephalic neuronal-enriched cultures were treated as in A, after which SDS–PAGE and Western blots were prepared with the Cdk5 and p35 antibodies. Note p25 expression in all MPP+ lanes. The results are expressed as mean ± SEM of three independent experiments (***p < 0.001). (C) Mesencephalic tissues from E14 mouse embryos were cultured and grown on polylysine-coated cover slips. Representative images. The neuronal cultures were pretreated with TFP5 (500 nM) or scrambled peptide for 12 h and then coincubated in the presence of 10 μM concentration of MPP+ and TFP5 for 24 h. The cells were fixed and stained. (D) Numbers of TH-immunoreactive (IR) neurons. Scale bar, 100 μm (C).
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Related In: Results  -  Collection

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Figure 1: TFP5 inhibits hyperactive Cdk5/p25 in MPP+-induced mesencephalic primary cultures. A dose-dependent study. (A) Ventral mesencephalic neuronal-enriched cultures were pretreated with TFP5 (500 nM) or scrambled peptide for 12 h and then coincubated with different concentrations of MPP+ and TFP5 for 24 h. Cdk5 was immunoprecipitated from equal amounts of lysates using C-8 antibody. Immunoprecipitates were then subjected to in vitro kinase pad assays with histone H1 as substrate. Activity, as counts/minute, was quantified from three separate experiments and summarized in the bar graphs (***p < 0.001; **p < 0.01; *p < 0.05). (B) Ventral mesencephalic neuronal-enriched cultures were treated as in A, after which SDS–PAGE and Western blots were prepared with the Cdk5 and p35 antibodies. Note p25 expression in all MPP+ lanes. The results are expressed as mean ± SEM of three independent experiments (***p < 0.001). (C) Mesencephalic tissues from E14 mouse embryos were cultured and grown on polylysine-coated cover slips. Representative images. The neuronal cultures were pretreated with TFP5 (500 nM) or scrambled peptide for 12 h and then coincubated in the presence of 10 μM concentration of MPP+ and TFP5 for 24 h. The cells were fixed and stained. (D) Numbers of TH-immunoreactive (IR) neurons. Scale bar, 100 μm (C).
Mentions: We first determined whether 24 h MPP+ incubation induces Cdk5 hyperactivation in mesencephalic primary cultures in the presence or absence of TFP5 pretreatment and coincubation. Seven days in culture (DIC) mesencephalic neuron–enriched primary cultures pretreated with TFP5 were coincubated with different micromolar concentrations of MPP+ and TFP5, followed by Cdk5 immunoprecipitation and kinase assay. From 0.01 to 10.0 μM, a concentration-dependent increase in the activity was observed in the presence of MPP+ treatment (Figure 1A); the decline at 100 μM is probably due to cell toxicity. Pretreatment and coincubation with 0.5 μM TFP5, however, was sufficient to significantly reduce the elevated activity (30%) at each concentration (Figure 1A). In Western blots of the same lysates as in Figure 1A, we see a decline of p35 expression from 0.01 to 10 μM with modest expression of p25 (Figure 1B). The kinase activities, however, are reduced at all concentrations (20–30%) by TFP5 treatment. Because 10 μM MPP+ induced maximum activity, we selected this as the measure of hyperactivity in vitro for further study. It is noteworthy that scrambled peptide did not show any inhibitory ­effect (Figure 1A).

Bottom Line: To date, strategies to specifically inhibit Cdk5 hyperactivity have not been successful without affecting normal Cdk5 activity.Previously we reported that TFP5 peptide has neuroprotective effects in animal models of Alzheimer's disease.The neuroprotective effect of TFP5/TP5 peptide is also associated with marked reduction in neuroinflammation and apoptosis.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.

No MeSH data available.


Related in: MedlinePlus