The Journal of Neuroscience, March 15, 2002, 22(6):2035–2043 The Neuronal Apoptosis Inhibitory Protein Is a Direct Inhibitor of Caspases 3 and 7 Johannes K. X. Maier,1,2 Zahia Lahoua,1 Nathalie H. Gendron,1 Raouf Fetni,1 Anne Johnston,1 Jamshid Davoodi,1 Dita Rasper,4 Sophie Roy,4 Ruth S. Slack,3 Donald W. Nicholson,4 and Alex E. MacKenzie1,2,5 1Solange Gauthier Karsh Laboratory, Children’s Hospital of Eastern Ontario Research Institute, Ottawa, Ontario, Canada K1H 8L1, 2Department of Biochemistry, Microbiology, and Immunology, and 3Neuroscience Research Institute, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5, 4Merck Frosst Canada Inc., Kirkland, Quebec, Canada H9H 3L1, and 5Aegera Therapeutics Inc., Ottawa, Ontario, Canada K1H 8M5 The neuronal apoptosis inhibitory protein (NAIP) was identified cells transiently transfected with a NAIP N-terminal construct and as a candidate gene for the inherited neurodegenerative disor- treated to undergo a caspase-3-dependent cell death. To examine der spinal muscular atrophy. NAIP is the founding member of a whether NAIP inhibits caspases directly, recombinant N-terminal human protein family that is characterized by highly conserved NAIP protein containing BIR domains was overexpressed, puri- N-terminal motifs called baculovirus inhibitor of apoptosis repeats fied, and tested for caspase inhibition potential. Our results dem- (BIR). Five members of the human family of inhibitor of apoptosis onstrate that inhibition of caspases is selective and restricted to ϳ proteins including NAIP have been shown to be antiapoptotic in the effector group of caspases, with Ki values as low as 14 nM for various systems. To date, a mechanism for the antiapoptotic effect caspase-3 and ϳ45 nM for caspase-7. Additional investigations of NAIP has not been elucidated. To investigate NAIP function, we with NAIP fragments containing either one or two NAIP BIRs found cytoprotection of NAIP-expressing primary cortical neurons revealed that the second BIR and to a lesser extent the third BIR treated to undergo caspase-3-dependent apoptosis. The addi- alone are sufficient to mediate full caspase inhibition. tional treatment of these neurons with the pancaspase inhibitor boc-aspartyl(OMe)-fluoromethylketone did not result in increased Key words: BIR domains; NAIP; caspase inhibition; neuronal survival. Similar cytoprotective effects were obtained using HeLa apoptosis; inhibitor of apoptosis protein family; cytoprotection With the discovery of biochemical and histological hallmarks of baculoviral inhibitor of apoptosis proteins (IAPs) Cp-IAP and apoptosis, it has become increasingly evident that the intrinsic Op-IAP. NAIP is the founding member of the human IAP cellular suicide program is not only required for normal CNS protein family (Roy et al., 1995), which typically express one to development (Oppenheim, 1991) but also, if executed inappro- three motifs in the N-terminal region termed baculovirus inhibi- priately, contributes to the pathology of neurodegenerative dis- tor of apoptosis repeat (BIR) domains that are defined by a orders such as Alzheimer’s disease (Cotman, 1998), Parkinson’s CX2CX16HX6–8C consensus sequence (Uren et al., 1998). disease (Hartmann et al., 2000), or the childhood spinal muscular Recent studies examining neuronal cell death indicate a key atrophy (SMA; Morrison, 1996). The defining characteristic of role for caspase-3 in the apoptotic process. For example, it has SMA is a progressive loss of motor neurons leading to wasting of been shown that caspase-3 knock-out mice show severe alter- the voluntary muscles. SMA patients are clinically heterogeneous ations of brain structures in regions where neuronal apoptosis is and are classified into three types (types I–III) on the basis of age predominantly believed to occur. The changes include an overall of onset, severity, and clinical progression (Morrison, 1996). The brain mass increase, disorganized cell deployment, and dupli- ϳ combined frequency for all three types of SMA is 1:10,000, cated brain structures (Kuida et al., 1996). In addition, upregula- making it one of the most common inherited neurodegenerative tion and activation of caspase-3 or a caspase-3-like protease has diseases (Emery, 1991). Analysis of the complex SMA locus that been shown to be a key mediator of neuronal death in neurons maps to chromosome 5q13 has led to the discovery of two can- after excitotoxic and hypoxic injuries, as well as in the hippocam- didate genes for SMA, the SMA-causing gene survival motor pal CA1 sector after transient global ischemia (Yakovlev et al., neuron (SMN) and the potential modifier neuronal apoptosis 1997; Chen et al., 1998; Namura et al., 1998; Ni et al., 1998). inhibitory protein (NAIP; Lefebvre et al., 1995; Roy et al., 1995). An antiapoptotic effect of NAIP and other members of the The NAIP gene coding region spans 4212 nucleotides encoding a human IAP family has been shown in cell culture systems (Liston 1403-amino acid 156 kDa protein with strong homology to the et al., 1996; Deveraux and Reed, 1999). Apoptotic hippocampal neurons can be rescued by stereotactic microinjection of NAIP- Received May 18, 2001; revised Oct. 30, 2001; accepted Nov. 6, 2001. expressing adenovirus in the CA1 region, suggesting that the This work was supported by grants from the Canadian Institutes of Health Research, the Muscular Dystrophy Association of Canada, and Aegera Therapeutics antiapoptotic activity of NAIP shown in vitro extends to the in Inc. A.E.M. is a Burroughs-Wellcome clinical translation awardee. vivo situation (Xu et al., 1997). Recent evidence suggests that in Correspondence should be addressed to Dr. Alex E. MacKenzie, Children’s addition to supraphysiologic levels of NAIP conferring neuronal Hospital of Eastern Ontario Research Institute, 401 Smyth Road, Ottawa, Ontario, Canada K1H 8L1. E-mail: [email protected]. protection, the loss of endogenous NAIP results in enhanced Copyright © 2002 Society for Neuroscience 0270-6474/02/222035-09$15.00/0 neuronal vulnerability (Holcik et al., 2000), indicating that NAIP 2036 J. Neurosci., March 15, 2002, 22(6):2035–2043 Maier et al. • Inhibition of Effector Caspases by NAIP BIR Domains plays an important role in regulation of neuronal apoptosis. at room temperature in continuous readings for 30 min [five readings per minute; Cytofluor II; PerkinElmer Life Sciences, Emeryville, CA; ␭ However, a mechanism for antiapoptotic function of NAIP has ␭ excitation (ex), 380 nm; emission (em), 460 nm]. IC50 values were not yet been identified. Although other IAP family members have calculated using the curve-fitting program PROFIT 5.5 for MacIntosh been shown to inhibit caspases directly (Deveraux et al., 1997; (QuantumSoft Inc., Zurich, Switzerland); Ki values were calculated ac- Roy et al., 1997; Tamm et al., 1998), a similar interaction could cording to the formula developed by Cheng and Prusoff (1973). This not be demonstrated for NAIP (Roy et al., 1997), suggesting an formula is valid when the binding is competitive and reversible; we know alternate mechanism in mediating cytoprotection. The present that this is the case for capase-3 and XIAP given the recently published structural analyses of IAP and caspases (Chai et al., 2001; Huang et al., study demonstrates the direct inhibition of effector caspases by 2001; Riedl et al., 2001). Because substrate concentration and Km were ␮ NAIP BIR domains and thus provides a mechanistic explanation kept constant in the assay (both at 10 M), the Ki should approximate half for the cytoprotective effect of NAIP and its function as an of the obtained IC50 value. Ki values were also calculated from the important regulator protein for neuronal apoptosis. steady-state velocities of the enzyme inhibitor complexes using the equa- tions developed by Morrison and Walsh (1988) for the analysis of slow MATERIALS AND METHODS and tight binding inhibitors. This was done in the event that the binding had a marked time dependence and was not wholly competitive and Generation and cloning of NAIP BIR constructs. NAIP constructs encom- reversible, in which case the Cheng and Prusoff (1973) equation would passing one to three BIR domains (see Fig. 2A) were generated by PCR not obtain. The fact that equivalent results were obtained by both using the following primer pairs: B123xt, 5Ј forward primer GGATCC Ј statistical methods supports the accuracy of the inhibitory constants. ATG GCC ACC CAG CAG AAA GCC and 3 reverse primer CTCGAG Culture and survival of primary cortical neurons. Mouse cortical neurons CCA GAT GCC CAC AGA AAA GCT AT; B1, 5Ј forward primer Ј (CD1) were cultured from embryonic day 16 (E16) and grown in defined GGATCC GCA GTT CAG TTG GCA AGG and 3 reverse primer CTC- serum-free medium as described previously (Keramaris et al., 2000). For GAG CTC AGC CTG CTC TTC AGA TT; B2, 5Ј forward primer Ј optimal infection of neurons with minimal cytotoxicity, recombinant GGATCC AGC AGG CTG AGA GAG GT and 3 reverse primer CTC- adenoviral vectors (carrying lacZ, NAIP,orXIAP), at a multiplicity of GAG GTA ATT TCC TCT GAG GAT TTC; B3, 5Ј forward primer Ј infection (MOI) of 10 or 20 pfu/cell, were added to cell suspensions GGATCC TCC TCA GAG GAA ATT ACC and 3 reverse primer CTC- immediately before plating. Neurons were cultured up to day 3 and GAG ATA GGA CCA ACT GCA TTG AA; B12, 5Ј forward primer ␮ Ј subsequently treated either with camptothecin (10 M) alone or with GGATCC GCA GTT CAG TTG GCA AGG and 3 reverse primer CTC- camptothecin and boc-aspartyl(OMe)-fluoromethylketone (BAF; 100 GAG GTA ATT TCC TCT GAG GAT TTC; B23, 5Ј forward primer ␮ Ј M). Cell survival was assessed by using the colorimetric (3-(4,5- GGATCC AGC AGG CTG AGA GAG GT and 3 reverse primer CTC- dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) survival GAG ATA GGA CCA ACT GCA TTG AA; B2A, 5Ј forward primer Ј assay (Cell Titer Kit; Promega, Madison, WI), which measures the GGATCC GCA TCC TTC AGG AAC TGG and 3 reverse primer CTC- conversion by mitochondrial enzymes of the tetrazolium salt to a blue GAG TAT GTC AAC AAA TCC C; B2B, 5Ј forward primer GGATCC Ј formizan salt 24 hr after treatment in an adaptation of the method of ATG AGG TAC CAA GAA GAGG and 3 reverse primer CTCGAG GGG Keramaris et al.