Ischemic stroke injury is mediated by aberrant Cdk5

Abstract

Ischemic stroke is one of the leading causes of morbidity and mortality. Treatment options are limited and only a minority of patients receive acute interventions. Understanding the mechanisms that mediate neuronal injury and death may identify targets for neuroprotective treatments. Here we show that the aberrant activity of the protein kinase Cdk5 is a principal cause of neuronal death in rodents during stroke. Ischemia induced either by embolic middle cerebral artery occlusion (MCAO) in vivo or by oxygen and glucose deprivation in brain slices caused calpain-dependent conversion of the Cdk5-activating cofactor p35 to p25. Inhibition of aberrant Cdk5 during ischemia protected dopamine neurotransmission, maintained field potentials, and blocked excitotoxicity. Furthermore, pharmacological inhibition or conditional knock-out (CKO) of Cdk5 prevented neuronal death in response to ischemia. Moreover, Cdk5 CKO dramatically reduced infarctions following MCAO. Thus, targeting aberrant Cdk5 activity may serve as an effective treatment for stroke.

Keywords: Cdk5; biomarker; calpain; ischemia; neuroprotection; stroke.

Figure 1.

Stroke induces dysregulation of Cdk5. A, p35/p25 aa sequence. B, Staining of stroked brain hemispheres 6 h after reperfusion for FJB and 24 h after reperfusion for GFAP. Scale bars: left, 2 mm; center, right, 100 μm. C, Quantitative immunoblots of p25/p35 in lysates from striatum and prefrontal cortex of control and stroked brain hemispheres at 6 and 48 h after unilateral MCAO. D, E, Quantitative immunoblots of cleaved fodrin (D) and phospho-Thr75 DARPP-32 (E) from lysates of striatum and prefrontal cortex of control and stroked brain hemispheres at 6 h after unilateral MCAO. Aged (9–12 months) rats were used for studies in B–E. Data represent means ± SEM; n = 6–8; *p < 0.05, **p < 0.01, ***p < 0.005, versus control; Student's t test.

Figure 2.

Ischemic Cdk5 dysregulation in acute brain slices is dependent upon calpain. A, Time-dependent p25 generation in striatal slices in response to OGD. Quantitative immunoblotting of lysates from slices subjected to OGD for the indicated period is shown. Controls (Con) were incubated in oxygenated buffer for 60 min. B, GFP and p25 detection in costained GFP-expression vector-transfected primary cultured striatal neurons before and after OGD. Scale bars, 25 μm. C, Attenuation of OGD-induced p25 generation by Ca²⁺ removal. D, Inhibition of OGD-dependent p25 generation by calpain inhibitors calpeptin or calpain inhibitor 3. E, F, Quantitative immunoblots of cleaved fodrin (E) and phospho-Thr75 DARPP-32 (F) in lysates from acutely prepared mouse striatal slices left untreated (Control) or exposed to OGD (20 min). Data represent means ± SEM; n = 4–6; *p < 0.05, **p < 0.01, ***p < 0.005; Student's t test versus control.

Figure 3.

Inhibition of Cdk5 prevents neuronal cell death from ischemia in acute brain slices. A, Dose–response inhibition of striatal Cdk5 by Indo A (1 h), as assessed by blotting phospho-Thr75 DARPP-32 (PT75 D32). B, Viability staining (TTC) of coronal slices after 30 or 60 min of OGD in the absence or presence of calpeptin or Indo A. C, Quantitation of the effects of calpeptin, Indo A, or indolinone B (Indo B) on viability. Data represent means ± SEM; n = 4–6; *p < 0.05 versus control; #p < 0.05 versus same period of OGD treatment alone; Student's t test.

Figure 4.

Ischemia-induced deficits in dopamine neurotransmission are Cdk5 dependent. A, B, Effect of 20 min OGD and 10 min reperfusion on (A) phospho-Thr34 DARPP-32 and (B) phospho-Ser845 GluR1 in striatal slices that were untreated or incubated with the D1 dopamine receptor agonist SKF81297 (SKF). C, Effects of 0, 3, or 10 min OGD and 10 min reperfusion on stimulation of phospho-Ser845 GluR1 by SKF81297 in the absence or presence of Indo A (1 h, 10 μm). Data represent means ± SEM; n = 6; *p < 0.05 versus control; Student's t test.

Figure 5.

Inhibition of Cdk5 prevents ischemia-induced neurophysiological deficits. A, Effect of 3 min OGD on membrane potential and EPSP amplitude (upward deflections) in control (top) and Indo A-treated striatal slices (bottom). Note the lack of increase in EPSP amplitude 50 min post-OGD in the Indo A-treated slice relative to pre-OGD conditions. B, Mean OGD-induced depolarization amplitude (white bars) and mean recovery time (gray bars) of MSNs after 3 min OGD in untreated and Indo A-treated slices (n = 9, p > 0.05). C, Single tracings show the EPSP amplitude 5 min before, during, and 50 min after OGD in control and Indo A-treated slices. D, Time course of EPSP amplitudes revealing i-LTP after 3 min OGD (Control). The i-LTP is abolished by Indo A (n = 5, p < 0.05). E, Tracings show FPs 5 min before, during, and 50 min after OGD in control and in Indo A-treated slices. F, The plot shows the effect of 10 min of OGD on the time course of FP amplitudes recorded in control and Indo A-treated slices. Data represent means ± SEM.

Figure 6.

Cdk5 CKO is neuroprotective against ischemia and stroke. A, TTC staining of striatal slices from Cdk5+ (WT) or Cdk5− (CKO) mice subjected to the indicated period of OGD is shown with quantitation. Data represent means ± SEM; n = 6; *p < 0.05, **p < 0.01 versus 0 OGD control, and compared with same treatment for Cdk5+; n = 6. B, TTC-stained coronal brain sections from Cdk5+ or Cdk5− mice after MCAO (2 h), reperfusion, and 24 h survival. Top, Ventral view of brains from Cdk5+ and Cdk5− littermates showing a representative embolism of the MCA (arrows) verifying placement. Quantitation of infarct size is shown (right). Data represent means ± SEM; n = 9; ***p < 0.001, *p < 0.05; ANOVA with Bonferroni's post hoc, for each group.