BDNF regulates BIM expression levels in 3-nitropropionic acid-treated cortical neurons

Abstract

3-Nitropropionic acid (3-NP) is an irreversible inhibitor of succinate dehydrogenase that has been used to explore the primary mechanisms of cell death associated with mitochondrial dysfunction and neurodegeneration in Huntington's disease. In this study we investigated the ability of brain-derived neurotrophic factor (BDNF) to suppress mitochondrial-dependent cell death induced by 3-NP in primary cortical neurons. This neurotrophin prevented 3-NP-induced release of cytochrome c and Smac/Diablo, caspase-3-like activity and nuclear condensation/fragmentation. Furthermore, it greatly increased phosphorylation of Akt and MAPK, suggesting the involvement of these signalling pathways in BDNF neuroprotection. Interestingly, BDNF decreased the levels of the pro-apoptotic protein Bim in mitochondrial and total cell lysates through the activation of the MEK1/2 pathway. This effect was due to an increase in the degradation rates of Bim. Our data support an important role for BDNF, in protecting cortical neurons against apoptotic cell death caused by inhibition of mitochondrial complex II.

Introduction

Mitochondrial dysfunction has been linked to the pathogenic mechanisms of several neurodegenerative diseases (Schapira, 2006, Keating, 2008). Neurons are particularly sensitive to alterations in normal mitochondrial function because of their high levels of activity and subsequent need for energy. 3-Nitropropionic acid (3-NP), an inhibitor of succinate dehydrogenase (Coles et al., 1979, Huang et al., 2006), has been widely used to analyse the mechanisms by which metabolic impairment leads to the degeneration of neurons. In particular, 3-NP has been used in the context of Huntington's disease (HD), a hereditary neurodegenerative disease affecting the striatum and the cerebral cortex, since its administration in rodents and non-human primates causes abnormal movement, cognitive deficits and neuronal degeneration similar to that seen in HD patients (Beal et al., 1993, Brouillet et al., 1995, Blum et al., 2002). In addition, post-mortem brain extracts of HD patients showed reduced activity and expression levels of succinate dehydrogenase (Gu et al., 1996, Browne et al., 1997, Benchoua et al., 2006). Although not entirely elucidated, the mechanisms of 3-NP-induced neurotoxicity involve depletion of ATP, mitochondrial membrane depolarization, dysregulation of intracellular calcium homeostasis, calpain activation, and release of pro-apoptotic proteins from mitochondria with the consequent activation of caspases and apoptotic pathways (Lee et al., 2002, Bizat et al., 2003, Almeida et al., 2004, Almeida et al., 2006). Although these changes may underlie massive striatal degeneration following 3-NP in vivo administration, a reduction in the activities of complexes I and II was also recently described in the cerebral cortex (Pandey et al., 2008), implicating neuronal dysfunction in this brain area. Indeed, early cortical dysfunction, linked to striatal excitotoxicity and decreased trophic support, appears to have a fundamental role in the onset and progression of HD.

The mitochondrial death pathway is regulated by a fine balance between pro-apoptotic and pro-survival Bcl-2 family members (Cory and Adams, 2002). Pro-apoptotic proteins such as Bax and Bak can disrupt the outer mitochondrial membrane and promote the release of apoptogenic factors such as cytochrome c or Smac/Diablo, an event that ultimately activates the caspase cascade. Apoptosis-inducing factor (AIF) can also be released from the mitochondria, leading to caspase-independent cell death (Susin et al., 1999). In viable cells and under normal conditions, pro-apoptotic proteins are normally repressed by binding to pro-survival proteins such as Bcl-2 and Bcl-xL. In response to stress, BH3-only proteins, such as Bim, Bid, and Bad, bind to Bcl-2 or Bcl-xL, that then release Bax or Bak. These pro-apoptotic proteins then change their conformation and oligomerize in the mitochondrial membrane to promote cell death (Puthalakath and Strasser, 2002).

Trophic support to neurons largely influences neuronal survival and function. Members of the neurotrophin family, namely brain-derived neurotrophic factor (BDNF), have been suggested as therapeutic candidates to treat neurodegenerative disorders because they promote neuronal survival in different lesion models (e.g. Connor and Dragunow, 1998). BDNF is particularly relevant in HD since its transcription (Zuccato et al., 2001) and axonal transport (Gauthier et al., 2004) are decreased by the presence of mutant huntingtin, affecting the survival of both striatal and cortical neurons. BDNF was previously shown to prevent the death of different populations of striatal projection neurons in a quinolinic acid model of HD (Perez-Navarro et al., 2000, Kells et al., 2004). BDNF was also reported to protect striatal neurons from 3-NP toxicity (Ryu et al., 2004). However, the mechanism used by BDNF leading to neuroprotection is not clear. The effects of BDNF are mainly mediated by TrkB receptor-induced activation of key signalling pathways, including PLC-γ, Ras/MEK/MAPK and PI3K/Akt pathways (Huang and Reichardt, 2001). These pathways have been shown to regulate apoptotic cell death by increasing the transcription of neuroprotective proteins such as Bcl-2 (Pugazhenthi et al., 2000) and/or by posttranslational modifications of proteins such as Bad and Bim (del Peso et al., 1997, Scheid et al., 1999, Luciano et al., 2003, Qi et al., 2006).

Taking into account the importance of cortical dysfunction in HD and the potential benefit of BDNF against mitochondrial-driven neuronal degeneration, in this study we analysed the mechanisms by which BDNF protects primary cortical neurons against mild neurotoxicity induced by 3-NP. Our data show that BDNF protects cortical neurons from 3-NP toxicity through the activation of PI3K and MEK1/2 intracellular signalling pathways and the regulation of Bim turnover.