Study of the pathways involved in apoptosis induced by PI3K inhibition in cerebellar granule neurons

Abstract

In the present study we focused in the PI3K/Akt pathway which plays a key role in neuronal survival. Here we show that inhibition of PI3K/Akt by means of LY294002 induces apoptosis via a caspase-dependent and calpain-independent pathway in cerebellar granule neurons (CGNs). This finding was confirmed using zVAD-fmk, a widely caspase inhibitor that prevents apoptosis. For this purpose, we compared two models of apoptosis in CGNs, namely inhibition of PI3K/Akt, and serum potassium deprivation (S/K deprivation). In contrast to the S/K deprivation model, caspase-3 was not activated when PI3K is inhibited. Likewise, CDK5 activation was not involved in this apoptotic process, because calpain activation is responsible for the formation of CDK5/p25 neurotoxic form. However, S/K deprivation activated calpain, as it is shown by α-spectrin breakdown, and favoured the formation of CDK5/p25. Moreover, although PI3K/Akt inhibition enhanced pRbser780 phosphorylation, no increase in the expression of cell-cycle proteins, namely: cyclin D, cyclin E, CDK2 or CDK4, was detected. Furthermore, BrdU incorporation assay did not shown any increase in DNA synthesis. Likewise, PI3K/Akt inhibition increased GSK3β activity and c-Jun phosphorylation, which implicates these two pathways in this apoptotic route. Although previous reports suggest that apoptosis induced in CGNs by LY294002 and S/K deprivation causes PI3K inhibition and increases GSK3β activity and c-Jun phosphorylation activation, our results demonstrate substantial differences between them and point to a key role of GSK3β in the apoptosis induced in CGNs in the two models tested.

Introduction

Understanding the regulation of neuronal cell death and survival is crucial for the development of drugs for neurodegenerative disorders. In this context, it is accepted that apoptosis is the main mechanism involved in neuronal loss in diseases such as Alzheimer’s and Parkinson’s (Galluzzi et al., 2009a, Galluzzi et al., 2009b, Camins et al., 2010). Thus, inhibition of the intrinsic apoptotic pathway is the object of intense research into strategies to prevent neuronal loss in neurodegenerative diseases (Ribe et al., 2008, Hisatomi et al., 2009, Galluzzi et al., 2009a, Galluzzi et al., 2009b). Key intracellular components are involved in this pathway, including mitochondria, which against apoptotic stimuli respond to a fall in membrane potential by releasing cytochrome c thus activating the caspase cascade (Galluzzi et al., 2009a, Galluzzi et al., 2009b). The release of cytochrome c is an important point in this apoptotic pathway (also called intrinsic pathway) as it constitutes the point of no return in the apoptotic cascade through the recruitment of Apaf-1 and procaspase-9 to form the apoptosome (Ribe et al., 2008). Three caspases are activated in this apoptotic route, namely caspase-9, -6 and -3, the last two being caspase effectors involved in intracellular substrate degradation leading to apoptotic cell death. While considerable effort has been devoted to the synthesis of caspase inhibitors for neuroprotection, such compounds act too late in the pathway to be effective (Camins et al., 2010, Sureda et al., 2011).

Another family of cysteine proteases involved in apoptosis and necrosis are calpains (Camins et al., 2006, Camins et al., 2009, Samantaray et al., 2008). These enzymes are activated in a calcium-dependent manner. Cyclin-dependent kinase 5 (CDK5) is a well characterized substrate of calpains. Given that calpains are activated in Alzheimer’s disease and Parkinson’s disease, CDK5 has been implicated in several neurodegenerative disorders Camins et al., 2006, Camins et al., 2009, Camins et al., 2010. Moreover, CDK5 is involved in the regulation of apoptosis through tau phosphorylation in Alzheimer’s disease and the regulation of transcription factors, such as p53 and myocyte-inducing factor 2 (Camins et al., 2006, Ikiz and Przedborski, 2008, Kim et al., 2008, Vosler et al., 2008).

In addition, the phosphatidylinositol-3 kinase (PI3K)/Akt pathway is a critical transducer for several major survival signals in central nervous system neurons (Miller et al., 1997, Shimoke et al., 1998, Watson et al., 1998, Datta et al., 1999, Bondy and Cheng, 2004, Vasudevan and Garraway, 2010, Miyamoto et al., 2009, Tajes et al., 2009). In these cells, it has been widely demonstrated that this pathway mediates the pro-survival effects of several agents such as neurotrophins, insulin, insulin-like growth factor-I (IGF-1) and vascular endothelial growth factor (Miller et al., 1997, Shimoke et al., 1998, Bondy and Cheng, 2004). In this context, the PI3K pathway is crucial in neuroprotection in cerebellar granular neurons (CGNs) (Mora et al., 1999, Subramaniam et al., 2003, Cao et al., 2007, Tajes et al., 2009, Yeste-Velasco et al., 2009a, Yeste-Velasco et al., 2009b). For instance, lithium and IGF-1 show anti-apoptotic effects against serum potassium deprivation (S/K deprivation) by the activation of PI3K (Miller et al., 1997). Moreover, PI3K/Akt signalling is involved in the pro-survival effects of N-methyl-d-aspartate receptor stimulation in CGNs (Bondy and Cheng, 2004). Thus PI3K/Akt activation is a promising strategy to achieve neuronal protection or the prevention of cell death.

In contrast, inhibition of this prosurvival pathway, using LY294002, a reversible and highly specific inhibitor of PI3K, induces apoptosis. The mechanisms involved in the apoptotic process after inhibition of PI3K are not well understood. Previous studies using LY294002 identified two pathways in neuron apoptosis: GSK3β activation and c-Jun N-terminal kinase (JNK) phosphorylation (Shimoke et al., 1998, Watson et al., 1998, Tajes et al., 2009).

Likewise, glycogen synthase kinase 3β (GSK3β) is an important regulator of neuronal apoptosis. Pharmacological inhibition of this kinase, for example with lithium, offers protection against S/K deprivation in CGNs, HIV proteins and β-amyloid peptides (Mora et al., 1999, Subramaniam et al., 2003, Cao et al., 2007, Yeste-Velasco et al., 2007, Mendes et al., 2009, Tajes et al., 2009, Yeste-Velasco et al., 2009a, Yeste-Velasco et al., 2009b, Hughes et al., 2010). The activity of GSK3β is modulated mainly by survival signalling such as Akt, and its inhibition is achieved by phosphorylation at the Ser9 residue (Mora et al., 1999, O’Brien and Klein, 2009, Hughes et al., 2010).

In addition to CDK5, cyclin-dependent kinases associated with cyclins involved in cell cycle regulation, such as CDK4/cyclin D, CDK2/cyclyn E, are also involved in neuronal apoptosis (Perry et al., 1998, Zhu et al., 1999, Shimoke et al., 1999, Harris et al., 2002, Hongisto et al., 2003, Kruman et al., 2004, Verdaguer et al., 2005, Bauer and Patterson, 2005, Majd et al., 2008, Thakur et al., 2008, Lopes et al., 2009, Bonda et al., 2010). Thus, aberrant neuronal cell cycle re-entry has also been demonstrated in the brains of patients with Alzheimer’s, Parkinson’s and other neurodegenerative disorders (Bauer and Patterson, 2005, Neve and McPhie, 2006, Thakur et al., 2008, Lopes et al., 2009, Bonda et al., 2010).

Here we compare the pathways involved in the apoptosis induced by PI3K/Akt inhibition and S/K deprivation. For this purpose, we used LY294002, which competes with ATP for binding to PI3K as PI3K/Akt inhibitor. Moreover, we demonstrate that although LY294002 and S/K deprivation induced GSK3β activation, there are significant differences between these apoptotic routes.