Study of the pathways involved in apoptosis induced by PI3K inhibition in cerebellar granule neurons
Highlights
► This is the first report where is described in detail the route by which PI3K inhibition causes apoptosis in CGNs. ► The JNK pathway and cell cycle re-entry do not play a key role in the apoptosis induced by PI3K inhibition. ► We demonstrate that PI3K inhibition causes apoptosis in CGNs is caspase-6 dependent and calpain independent.
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.
Section snippets
Preparation of cell cultures
Primary cultures of cerebellar granule neurons (CGNs) from Sprague–Dawley rats and wild-type mice, and JNK3 Knockout, were prepared from postnatal day 7 rat and mice pups as described elsewhere. Cells were dissociated in the presence of trypsin and DNase I (Sigma–Aldrich) and placed in poly-l-lysine (100 μg/ml)-coated dishes at a density of 5 × 105 cells/ml in basal Eagle’s medium supplemented with 10% heat-inactivated foetal bovine serum, 0.1 mg/ml gentamicin, 2 mM l-glutamine and 25 mM KCl. Cytosine-
Inhibition of PI3K by LY294002 and S/K deprivation-induced CGNs apoptosis via different pathways
Inhibition of the PI3K/Akt pathway in CGNs by LY294002 induces apoptosis (Miller et al., 1997) (Fig. 1A). However, the mechanism by which these cells die in this apoptotic model is unclear. Here we studied the implication of cysteine proteases calpain and caspases in apoptosis. For this purpose CGNs were pretreated with calpain inhibitor III (1–50 μM), or zVAD-fmk, a caspase inhibitor (10–100μM). Our results show that inhibition of caspases prevents the apoptosis induced by LY294002 in these
Discussion
Here we demonstrated that in response to PI3K inhibition in CGNs: (i) calpain activation is not involved in this apoptotic process; however, caspase activation, mainly caspase-6 plays a prominent role in CGN cell death; (ii) although the retinoblastoma protein is phosphorylated, the canonical cell cycle pathway is not activated; and (iii) c-Jun activity is increased after PI3K inhibition but it is not the main pathway involved in this model.
It has been suggested that neuronal death pathways
Acknowledgments
This study was supported by Grants from the Spanish Ministry of Education and Science SAF-2009-13093, BFU2010-19119 and BFU2010-22149 the Fondo de Investigación Sanitaria and the Instituto de Salud Carlos III (PI080400 and PS09/01789) (FEDER FOUNDS). We thank the Catalan Government (Generalitat de Catalunya) for supporting the research groups (2009/SGR00853). 610RT0405 from Programa Iberoamericano de Ciencia y Tecnología para el Desarrollo (CYTED). We thank the University of Barcelona Language
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