Novel survivin mutant protects differentiated SK-N-SH human neuroblastoma cells from activated T-cell neurotoxicity Sara Baratchi, Rupinder K. Kanwar, Jagat R. Kanwar ⁎ Laboratory of Immunology and Molecular Biomedical Research, Centre for Biotechnology and Interdisciplinary Biosciences (BioDeakin), Institute for Technology Research and Innovation (ITRI), Deakin University, Waurn Ponds, Victoria 3217, Australia abstract article info Article history: Received 19 July 2010 Received in revised form 27 October 2010 Accepted 29 October 2010 Keywords: SurR9-C84A Neuroprotection Multiple sclerosis Neuro-degeneration Currently, there are no known treatments for protection of axonal loss associated with neuroinflammatory diseases such as multiple sclerosis (MS). Survivin is a member of the inhibitors of the apoptosis (IAP) family of proteins that its neuroprotective effects have not been studied. We demonstrate here that SurR9-C84A, a survivin mutant, exhibits a neuroprotective role against the cytotoxic effects of activated T-cell infiltrates, such as granzyme B (GrB). The activated T-cell supernatants induce toxicity on differentiated SK-N-SH cells, which is associated with the loss of Ca 2+ homeostasis, the increased population of dead cells, mitochondrial membrane depolarisation, and the accelerated expression of cyclinD1, caspase3 and Fas, as observed for most apoptotic cells. Alternatively, the pre-treatment with SurR9-C84A reduces the population of dead cells by balancing the cytosolic Ca 2+ homeostasis, decreasing the level of mitochondrial depolarisation, and also reducing the expression of cyclinD1 and caspase3. Our findings suggest that SurR9-C84A has a neuroprotective effect against the cytotoxins existing in activated T-cell supernatants including GrB. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved. 1. Introduction Multiple sclerosis (MS) is a debilitating long term disability that is associated with severe axonal damage during its early stages, as well as inflammatory responses (Dhib-Jalbut et al., 2006; Kanwar et al., 2009). Due to clinical variability and unpredictable nature of MS, prescribing treatments for specific individuals is complicated, and so far no therapy has been found with the capability of halting disease progression (Cook, 2003; Kanwar, 2005; Vartanian et al., 2004). To successfully stop the progression or reverse MS, a treatment must simultaneously target a combination of inflammatory and degenerative factors (Kanwar, 2005; Kanwar et al., 2000, 2004a). The approved treatments for MS include the application of conventional agents such as interferon-β, glatiramer acetate and mitoxantrone, as well as the treatments still under trial such as monoclonal antibodies (i.e., Atalizumab, Alemtuzumab and Rituximab) and oral drugs, which all target inflammatory components of MS pathogenesis (Gold and Voskuhl, 2009). Until now, no direct neuroprotective treatment has been available, especially because administering direct neuroprotective therapies needs to penetrate the blood brain barrier (Baratchi et al., 2009; Kanwar et al., 2009). Targeting members of the apoptosis family of proteins and evaluating their neuroprotective effects have been the subject of much attention (Charriaut-Marlangue, 2004). The apoptosis pathway is negatively regulated by Bcl-2 and the IAP family of proteins. The neuroprotective role of IAPs in degenerating neurons has been studied by different groups (Hefti, 1997; Simons et al., 1999; Xu et al., 1999). Among the different members of IAPs, NIAP and XIAP are shown to have essential roles in the intracellular signalling of glial cell line-derived neurotrophic factor (GDNF) (Perrelet et al., 2002). In addition, adenoviral delivery of XIAP into axotomised retinal ganglion cells can significantly decrease neuronal death (Kato et al., 2000; Perrelet et al., 2004). Survivin is another member of the IAP family, which has been shown to have a role in early brain development (Jiang et al., 2005), and serves a bifunctional role during mitosis and inhibition of apoptosis (Altieri, 2008; Baratchi et al., 2010a; Kanwar et al., 2010). Survivin consists of two defined domains, including the amino- terminal BIR domain that is composed of an extensive dimerisation interface (Verdecia et al., 2000). This interface is associated with XIAP and allows the formation of survivin–XIAP complex, which increases the stability of XIAP against ubiquitination/proteasomal destruction and the synergistic inhibition of apoptosis (Dohi et al., 2004). However, using wild-type IAPs for the purpose of human clinical trial raises concerns due to their role in cancer formation (Kanwar et al., 2004b, 2010). Nevertheless, developing IAP mutants capable of increasing the cell proliferation will provide a new therapeutic window for neuronal repair and proliferation therapy. Different forms of survivin mutants (such as C84A, Δ106 and T34A) have been used for the purpose of targeting survivin over- expression in cancer cells (Altieri, 2003b; Cheung et al., 2006; Kanwar et al., 2001; Khan et al., 2009; Li et al., 1998; Pisarev et al., 2003). Journal of Neuroimmunology 233 (2011) 18–28 ⁎ Corresponding author. Tel.: +61 3 52271148; fax: +61 3 52273402. E-mail address: jagat.kanwar@deakin.edu.au (J.R. Kanwar). 0165-5728/$ – see front matter. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2010.10.036 Contents lists available at ScienceDirect Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim