Differential Modulation of Akt/Glycogen Synthase Kinase-3β Pathway Regulates Apoptotic and Cytoprotective Signaling Responses

Abstract

We have previously reported that specific dopamine agonists mediate protection against apoptosis induced by oxidative stress by activating the D 2 receptor-coupled phosphoinositide 3-kinase (PI-3K)/Akt pathway. In the present study we examined the downstream effectors of PI-3K/Akt signaling and their role in cell death after oxidative stress and protection provided by ropinirole, a D 2 receptor agonist in PC12 cells and primary cultures of dopamine neurons. Ropinirole treatment was associated with rapid translocation and phosphorylation of the PI-3K substrate Akt and phosphorylation of Akt substrates. One of these Akt downstream substrates was identified as the proapoptotic factor glycogen synthase kinase-3␤ (GSK-3␤). Ropinirole-induced protection was associated with phosphorylation of GSK-3␤ (inactivation). In contrast, inhibition of PI-3K blocked the phosphorylation of Akt and GSK-3␤ (activation) and prevented the protection mediated by ropinirole. Suppression of Akt with specific short hairpin RNA in normal PC12 cells caused cell death, which was associated with reduced phosphorylation of GSK-3␤ and reduced levels of ␤-catenin, a transcriptional activator that is regulated by GSK-3␤. Knock-out of GSK-3␤ expression with a short hairpin RNA alone was itself sufficient to cause cell death. We further demonstrated that oxidative stress induced by hydrogen peroxide (H 2 O 2 ) dephosphorylates Akt and GSK-3␤, increases GSK-3␤ activity, and promotes an interaction with ␤-catenin and its degradation. Inhibition of GSK-3␤ activity by inhibitor VIII protects cells from H 2 O 2 similar to ropinirole. These results indicate that GSK-3␤ downstream of Akt plays a critical role in cell death and survival in these models.The characteristic pathology of Parkinson disease (PD) 2 is degeneration of dopaminergic neurons coupled with Lewy body inclusions in the substantia nigra pars compacta (1). The mechanism underlying dopaminergic cell death in PD has not been elucidated. A variety of cellular and molecular changes indicative of mitochondrial dysfunction, oxidative stress, proteasomal dysfunction, and apoptosis have been identified in the parkinsonian brain (for review, see Refs. 2 and 3). Specifically, a large body of evidence suggests that oxidative stress or reactive oxygen species-mediated apoptosis may contribute to the progressive and selective neuronal degeneration observed in PD (4). Brains of PD patients have increased iron, which promotes free radical formation, decreased levels of reduced glutathione, which is the major anti-oxidant in the brain, and evidence of oxidative damage to DNA, lipids, and proteins (5). Furthermore, in the substantia nigra pars compacta of PD patients there are increased levels of cyclooxygenase, which contribute to formation of the oxidant species dopamine-quinone (5), and reduced mitochondrial complex I activity, which promotes free radical formation (6 -8).Current therapies for PD are primarily based on a dopamine replacement strategy. Although they provide effective anti-parkinsonian ef...

ERK5/KLF4 signaling as a common mediator of the neuroprotective effects of both nerve growth factor and hydrogen peroxide preconditioning

“…The rat pheochromocytoma neuron-like PC12 cells have been widely used as a model to study H 2 O 2 -induced oxidative stress (Li et al 2008;Nair and Olanow 2008;Aliaghaei et al 2014), therefore, we started our investigation of PC neuroprotection with this model. To examine the effects of different concentrations of H 2 O 2 on cell viability, PC12 cells were exposed to various concentrations of H 2 O 2 (50 to 1000 μM) for 24 h, and cell viability was determined by CalceinAM assay.…”

Inhibition of glycogen synthase kinase-3β enhances cognitive recovery after stroke: the role of TAK1

“…Earlier studies that utilized GSK-3b inhibitor VIII were performed in in vitro cell culture systems (Nair and Olanow 2008) but in vivo rat models have also demonstrated the efficacy of this agent when administered through the external jugular vein (Koh et al 2007(Koh et al , 2008. Although a few studies have shown a benefit of nonselective GSK inhibitors in stroke, these were limited by lack of pharmacological specificity of the inhibitor used, and the mechanism was not clearly identified.…”

Signaling Pathways Leading to Phosphorylation of Akt and GSK-3β by Activation of Cloned Human and Rat Cerebral D2and D3Receptors

“…Although the PI3-K/Akt pathway is known to be activated by native D 2 and D 3 receptors in primary cultures of striatal neurons (Brami-Cherrier et al, 2002), by D 2L and D 3 receptors stably expressed in PC12 cells (Nair and Sealfon, 2003;Nair and Olanow, 2008;Chen et al, 2009), and by GFPtagged D 3 receptors expressed in EM4 cells (Zapata et al, 2007), the intracellular cascades leading to recruitment of Akt remain to be characterized. Furthermore, although activation of D 2 receptors expressed in PC12 and CHO cells (Welsh et al, 1998;Nair and Olanow, 2008) enhanced phosphorylation of GSK-3␤ and p70S6 kinase, there is otherwise little information about D 2 receptor-recruited pathways that interact with Akt signaling, such as Wnt and ␤-catenin. Information is even sparser for D 3 receptors, because there is only fragmentary evidence that stimulation leads to the phosphorylation of Akt and GSK-3␤ (Zapata et al, 2007;Chen et al, 2008).…”