Distinct changes in glycogen synthase kinase-3 (GSK-3) signalling can regulate neuronal morphogenesis including the determination and maintenance of axonal identity, and are required for neurotrophin-mediated axon elongation. In addition, we have previously shown a dependency on GSK-3 activation in the semaphorin 3A (Sema3A)-mediated growth-cone-collapse response of sensory neurons. Regulation of GSK-3 activity involves the intermediate signalling lipid phosphatidylinositol 3,4,5-trisphosphate, which can be modulated by phosphatidylinositol 3-kinase (PI3K) and the tumour suppressor PTEN. We report here the involvement of PTEN in the Sema3A-mediated growth cone collapse. Sema3A suppresses PI3K signalling concomitant with the activation of GSK-3, which depends on the phosphatase activity of PTEN. PTEN is highly enriched in the axonal compartment and the central domain of sensory growth cones during axonal extension, where it colocalises with microtubules. Following exposure to Sema3A, PTEN accumulates rapidly at the growth cone membrane suggesting a mechanism by which PTEN couples Sema3A signalling to growth cone collapse. These findings demonstrate a dependency on PTEN to regulate GSK-3 signalling in response to Sema3A and highlight the importance of subcellular distributions of PTEN to control growth cone behaviour.

The expression and function of the 8 distinct catalytic isoforms of PI 3-kinase (PI3K) in the nervous system are unknown. Whereas most PI3Ks have a broad tissue distribution, the tyrosine kinase-linked p110δ isoform has previously been shown to be enriched in leukocytes. Here we report that p110δ is also highly expressed in the nervous system. Inactivation of p110δ in mice did not affect gross neuronal development but led to an increased vulnerability of dorsal root ganglia neurons to exhibit growth cone collapse and decreases in axonal extension. Loss of p110δ activity also dampened axonal regeneration following peripheral nerve injury in adult mice and impaired functional recovery of locomotion. p110δ inactivation resulted in reduced neuronal signaling through the Akt protein kinase, and increased activity of the small GTPase RhoA. Pharmacological inhibition of ROCK, a downstream effector of RhoA, restored axonal extension defects in neurons with inactive p110δ, suggesting a key role of RhoA in p110δ signaling in neurons. Our data identify p110δ as an important signaling component for efficient axonal elongation in the developing and regenerating nervous system.

Abstract-The inactivation of glycogen synthase kinase-3␤ (GSK-3␤) is proposed as the event integrating protective pathways initiated by preconditioning and other interventions. The inactivation of GSK-3 is thought to decrease the probability of opening of the mitochondrial permeability transition pore. The aim of this study was to verify the role of GSK-3 using a targeted mouse line lacking the critical N-terminal serine within GSK-3␤ (Ser9) and the highly homologous GSK-3␣ (Ser21), which when phosphorylated results in kinase inactivation. Postconditioning with 10 cycles of 5 seconds of reperfusion/5 seconds of ischemia and preconditioning with 6 cycles of 4 minutes of ischemia/6 minutes of reperfusion, similarly reduced infarction of the isolated perfused mouse heart in response to 30 minutes of global ischemia and 120 minutes of reperfusion. Preconditioning caused noticeable inactivating phosphorylation of GSK-3. However, both preconditioning and postconditioning still protected hearts of homozygous GSK-3 double knockin mice. Moreover, direct pharmacological inhibition of GSK-3 catalytic activity with structurally diverse inhibitors before or after ischemia failed to recapitulate conditioning protection. Nonetheless, cyclosporin A, a direct mitochondrial permeability transition pore inhibitor, reduced infarction in hearts from both wild-type and homozygous GSK-3 double knockin mice. Furthermore, in adult cardiac myocytes from GSK-3 double knockin mice, insulin exposure was still as effective as cyclosporin A in delaying mitochondrial permeability transition pore opening. Our results, which include a novel genetic approach, suggest that the inhibition of GSK-3 is unlikely to be the key determinant of cardioprotective signaling in either preconditioning or postconditioning in the mouse. (Circ Res. 2008;103:307-314.)Key Words: postconditioniong Ⅲ preconditioning Ⅲ GSK-3 Ⅲ mPTP I schemic preconditioning is a powerful and established modulator of intrinsic myocardial resistance to lethal ischemia. 1 Its clinical application in patients with acute myocardial infarction is limited as a result of the inability to predict the moment of coronary artery occlusion. More recently, Zhao et al described the concept of postconditioning as a novel strategy for protecting the heart against reperfusion-injury. 2 They demonstrated that a similar regimen of brief periods of ischemia after a lethal index ischemic insult was as protective as preconditioning. This phenomenon has subsequently been confirmed in a variety of animal models, both in vivo and in isolated heart preparations, 3 as well as in preliminary clinical interventional studies. 4,5 The endogenous cellular and molecular mechanisms involved in cardioprotection against ischemia have been extensively investigated in the field of preconditioning. 6 Because pre-and postconditioning are temporally remote, the signaling pathways might be expected to differ considerably. 2,7 However, recent evidence suggests that the early reperfusion phase after lethal ischemia is crucial i...