Cyclin-dependent kinases as a therapeutic target for stroke

Abstract

Cyclin-dependent kinases (CDKs) are commonly known to regulate cell proliferation. However, previous reports suggest that in cultured postmitotic neurons, activation of CDKs is a signal for death rather than cell division. We determined whether CDK activation occurs in mature adult neurons during focal stroke in vivo and whether this signal was required for neuronal death after reperfusion injury. Cdk4/cyclin D1 levels and phosphorylation of its substrate retinoblastoma protein (pRb) increase after stroke. Deregulated levels of E2F1, a transcription factor regulated by pRb, are also observed. Administration of a CDK inhibitor blocks pRb phosphorylation and the increase in E2F1 levels and dramatically reduces neuronal death by 80%. These results indicate that CDKs are an important therapeutic target for the treatment of reperfusion injury after ischemia.

Figure 1

(A) Cyclin D1 (CD1), Cdk4, Ser-795-phosphorylated pRb (phosRb), and E2F1 levels increase during reperfusion. Representative sections obtained from rats 24 h after reperfusion at the level of the striatum were analyzed for the indicated cell cycle markers by immunohistochemistry. The area of the ischemic core is shown. (The black bar represents 50 μm.) MAP2 staining is included as a control marker that does not increase during reperfusion. (B) Diagram indicating regions of quantitation after stroke (Left), and representative section with MAP2 staining (Right) indicating infarct. (C–F) Quantification of cyclin D1- (C), Cdk4- (D), phospho-pRb- (E), and E2F1- (F) positive cells at the indicated regions at various time points was performed as described in Materials and Methods. “Contralateral” indicates the area of uninfarcted cortex on the contralateral side. Each data point is the mean ± SEM (n = 5). * indicates significance (P < 0.05; paired t test with Bonferroni correction) as compared with contralateral control values.

Figure 2

Cyclin D1 immunoreactivity is localized to neurons in a confocal image of the core infarct region 24 h after reperfusion. (A and B) Sections were double labeled with cyclin D1 (A) and the neuronal marker NeuN (B). (C) The merged image. (The white bar represents 50 μm.)

Figure 3

TUNEL labeling and phospho-pRb reactivity colocalize in neurons after reperfusion injury in a confocal image of the core infarct region 24 h after reperfusion. Sections were triple labeled with TUNEL, phospho-pRb (phoRb), and 4′,6-diamidino-2-phenylindole (DAPI). The merged image of TUNEL and phospho-Rb is indicated. (The white bar represents 50 μm.)

Figure 4

Blood flow and select physiological parameters do not change with flavopiridol treatment. (A) Table of physiological parameters of animals treated with flavopiridol or vehicle. Blood pressure is given in mmHg and body temperature in °C. (B) Flavopiridol does not affect cerebral blood flow, as measured by hydrogen clearance. Each data point represents a mean ± SEM (n = 3).

Figure 5

Flavopiridol prevents neuronal death after 24 h of reperfusion. (A and B) Representative sections of core infarct region stained for TUNEL from vehicle-treated (A) or flavopiridol-treated (500 μM) (B) animals 24 h after reperfusion. (The black bar represents 100 μm.) (C) Quantitation of TUNEL-positive cells in the ipsilateral cortical hemisphere of animals treated with vehicle or flavopiridol. Each data point represents a mean ± SEM (n = 4) and is expressed relative to the number of TUNEL-positive cells present in vehicle-treated ischemic animals at 24 h. * indicates significance (P < 0.01 as determined by independent t test). (D) H&E analyses of sections from the core infarct area of vehicle-treated or flavopiridol-treated (500 μM) animals 24 h after reperfusion. (The black bar represents 50 μm.)

Figure 6

Flavopiridol (500 μM) treatment inhibits pRb phosphorylation and the rise in E2F1 levels. (A) Representative sections of cortex ipsilateral to occlusion from animals treated with vehicle or flavopiridol. Sections were obtained 24 h after reperfusion. (The black bar indicates 50 μm.) (B and C) Phospho-pRb-positive (B) and E2F1-positive (C) cells were quantified as described in the legend of Fig. 1. Each point represents a mean ± SEM (n = 4). * indicates significance (P < 0.05 as determined by repeated-measures ANOVA, followed by post hoc Tukey's highly significant difference) as compared with vehicle-treated controls.

Figure 7

Time course of TUNEL-positive neurons after reperfusion. Each data point represents a mean ± SEM (n = 5) and is expressed relative to the number of TUNEL-positive cells present in the ipsilateral hemisphere at 24 h.

Figure 8

Flavopiridol (500 μM) treatment reduces cortical infarct 6 days after reperfusion. (A) Photograph of exterior of the cortex and striatal sections from representative animals treated with vehicle or flavopiridol as indicated. The brackets indicate the length of infarct along the cortical surface. (B) Quantification of infarct as described in Materials and Methods. Each point represents a mean ± SEM (n = 4). * indicates significance (P < 0.01 as determined by independent t test).