Bcl2-A1 interacts with pro-caspase-3: Implications for amyotrophic lateral sclerosis
Graphical abstract
Research Highlights
► ALS-linked mutant SOD1 induces Bcl2-A1 expression via the redox-sensitive AP1 transcription factor in motor neuronal cells. ► Bcl2-A1 can interact with pro-caspase-3, via its C-terminal helix α9. ► Bcl2-A1/pro-caspase-3 interaction can prevents the completion of caspase-3 processing in vitro. ► Bcl2-A1 lacking helix α9 has impaired anti-apoptotic activity in motor neuronal cells expressing ALS-linked mutant SOD1.
Introduction
Amyotrophic Lateral sclerosis (ALS) is the most common motor neuron disease and it is invariably fatal since no effective therapy is available (Carri et al., 2006). Most ALS cases are sporadic, but 5–10% of cases are familial and among these 20% of cases show mutations within the SOD1 gene (OMIM #105400). Notably SOD1 is also responsible of 1.5% of sALS, suggesting a possible role of this protein in both forms of the disease (Pasinelli and Brown, 2006, Rothstein, 2009). Recently mutations in other genes, including TDP-43 and FUS/TLS, have been associated to ALS, but they are present at lower percentages (Lagier-Tourenne et al., 2010, Mackenzie et al., 2010). Current hypotheses for the biology underlying both sporadic and familiar ALS forms outline a model in which non-competing mechanisms are likely to converge in various unfortunate patterns to mediate selective motor neuron degeneration. The proposed mechanisms include oxidative stress, mitochondrial impairment, protein aggregation, glutamate cytotoxicity, transcription dysfunction, alterations in RNA processing, inflammation and apoptotic cell death. Moreover the assessment of the different cell types implicated in the development of ALS and their interactions with motor neurons has led to the identification of several additional pathogenic mechanisms (Boillee et al., 2006, Rothstein, 2009). These significant progresses in understanding the cellular mechanisms of motor neuron degeneration in ALS have not been matched with an effective disease-modifying pharmacotherapy, mostly because the molecular basis of selective vulnerability of motor neurons upon mutant SOD1 expression is still debated. A crucial step in filling this gap is the identification of genes whose expression is altered by mutant SOD1 in motor neurons. We previously demonstrated that motor neuronal death prompted by the expression of mutant SOD1 induces specific up-regulation of the Bcl2 family member Bcl2-A1 gene (Crosio et al., 2006). Bcl2-A1 (also known as Bcl2-related protein A1, BFL1; A1; Bfl-1/A1) is the only member of Bcl2 family to be up-regulated selectively in spinal motor neurons of mice transgenic for G93A–SOD1 already at the asymptomatic stage. Bcl2-A1 is protective against death of neuronal cells induced by expression of G93A–SOD1, but is detrimental upon stimulation of those cells with TNFα (Crosio et al., 2006). Although Bcl2-A1 is the unique example of apoptotic protein specifically over-expressed in motor neurons of mouse model of ALS, the molecular mechanisms of Bcl2-A1 pro/anti-apoptotic action is still unclear. The pro- and anti-apoptotic behavior of Bcl2-A1 seems to be mediated by two independent pathways (Kucharczak et al., 2005). Both in man and mouse Bcl2-A1 has a helical structure typical of other pro-survival Bcl-2 proteins, with a C-terminal tail-anchor domain corresponding to helix α9, needed for its association with mitochondria and for the prosurvival activity (Brien et al., 2009, Fan et al., 2010, Ko et al., 2007, Ko et al., 2011, Kucharczak et al., 2005, Smits et al., 2008). In B lymphocytes stimulated with TNFα, the Bcl2-A1 anti-apoptotic mechanism seems to be mediated by cytoplasmic protein partners, while the pro-apoptotic function seems to be regulated by post-translational modifications, including proteolytic cleavage and phosphorylation, that can alter Bcl2-A1 localization or its ability to interact with other factors (Kucharczak et al., 2005).
In this context, we have investigated the molecular pathways leading to Bcl2-A1 transcriptional activation upon mutant SOD1 expression and the molecular mechanisms underlying the anti-apoptotic action of Bcl2-A1 in ALS cellular models.
Section snippets
Animals and tissue dissection
We used SOD1G93A transgenic mice (strain B6.Cg-Tg(SOD1–G93A)-1Gur from The Jackson Laboratory, 99.99% C57BL/6 genetic background, 50% survive at 157.1 ± 9.3 days, http://jaxmice.jax.org/strain/004435.html). Non-transgenic littermates were used as control. All animal procedures have been performed according to the European Guidelines for the use of animals in research (86/609/CEE) and the requirements of Italian laws (D.L. 116/92).
At early symptomatic stage of the disease (120 days), mice were
Mutant SOD1 induces Bcl2-A1 expression via AP1
We have previously shown that Bcl2-A1 is specifically induced in motor neurons of SOD1G93A mice (Crosio et al., 2006). Since mice have multiple Bcl2-A1 genes (the isoforms -a, -b and -d consist of two exons and encode a full length protein, while isoform -c lacks putative exon 2 and it is most likely a pseudogene), in a preliminary experiment we carried out the classification and relative quantification of Bcl2-A1a, -A1b and -A1d mRNA subtypes in spinal cord and lymphocytes of mice expressing
Discussion
This study demonstrates that mutant SOD1 induces the expression of Bcl2-A1, which plays an important role in the modulation of caspase-3 activity, in a cell-specific manner via AP1 transcription factor. Bcl2-A1 is a member of the Bcl2 family, the key regulators of the mitochondria-dependent apoptotic pathway and of outer mitochondrial membrane integrity (Chipuk et al., 2010). Different lines of evidence link Bcl2-A1 overexpression to ALS, indicating that injured motor neurons may counteract
Acknowledgments
This work was supported by “Fondazione Banco di Sardegna” and PRIN 2008 (Grant no 20083R593R_002) to C.C. and by Ministero della Salute to M.T.C.
We wish to thank Manuela Galioto for invaluable technical support and Cristiana Valle, Alberto Ferri and Mauro Cozzolino for constant support and critical reading of the manuscript.
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C.I. and M.E.M. contributed equally to this work.