NAIP interacts with hippocalcin and protects neurons against calcium-induced cell death through caspase-3-dependent and –independent pathways

Citation

Eric A. Mercer, Laura Korhonen, Ylva Skoglösa, Per-Anders Olsson, Jyrki P. Kukkonen and Dan Lindholm. Department of Neuroscience, Neurobiology, Box 587, Biomedical Centre, Uppsala University and Department of Physiology, Division of Cellular Physiology, Uppsala University, S-751 23 Uppsala, Sweden. The EMBO Journal, Vol. 19, No. 14 pp. 3597-3607 (2000).

Summary

This series of experiments was designed to investigate the mechanism by which NAIP, neuronal apoptosis inhibitory protein, protects against calcium-induced cell death. Inhibitor-of-apoptosis proteins (IAPs) share one or more “baculovirus inhibitory repeat” (BIR) domains which, when over-expressed, display anti-apoptotic attributes. Although the BIR domains of other known IAPs are known to inhibit specific caspases in the apoptosis cascade, the mechanism by which NAIP prevents apoptosis was yet unknown, as it lacks the C-terminal RING motif. The absence of this motif prompts the consideration that NAIP’s BIR domains may be used for an alternate function – they may interact elsewhere besides the caspases of the apoptotic cascade, or they may require additional proteins to activate their protective service.

Initially, the BIR domains of NAIP were shown to significantly arrest cell death in vitro by stably transfecting motor neuron cells with the construct coding for the BIR domains (NAIP-BIR1-3) and exposing these cells and controls to different concentrations of the lethal ionomycin. Although over-expression of the BIR domains of NAIP did indeed hinder apoptosis, “the effect was not complete,” yielding less cell viability than those cells transformed with Bcl-2, a domain which has previously been revealed to inhibit cell death.

Subsequently, a yeast two-hybrid system was used to determine which, if any, molecules may potential interact with NAIP to stimulate a response. The BIR domains of NAIP were screened with the human fetal library, which yielded that hippocalcin, “a member of [a] family of neuron-specific proteins” that can bind to calcium, binds the most strongly to NAIP. In addition, a Western blot was used to determine that this interaction is enhanced by the presence of calcium, an ion essential to both neuronal survival and termination. Also, hippocalcin was proven to be expressed in spinal cord motor neurons, in proximity to NAIP, by in situ hybridization.

Furthermore, the study proves a synergic relationship between hippocalcin and NAIP by transfecting a motor neuron-like cell line (NSC-34) with NAIP-BIR1-3 and hippocalcin in tandem so that the two proteins would be co-translated. The results when exposing the cell line to ionomycin revealed that “over-expression of this hybrid vector construct was significantly more effective in counteracting cell death induced by ionomycin than NAIP-BIR1-3 alone, as determined by MTT assay.” The combination of NAIP-BIR1-3 and hippocalcin also afforded more cell viability than did over-expression of hippocalcin alone, as well. The data of the MTT assay was quantified by measuring the concentration of lactate dehydrogenase (LDH) released from the cells, which correlates to cell viability. As seen in Figure F, the synchronized efforts of NAIP and hippocalcin accrued 40% less LDH than did the control wild-type cells.

It was then found that hippocalcin binds to NAIP via the BIR3 domain by comparing the binding efficiency of each of the BIR domains of NAIP with hippocalcin in a yeast two-hybrid assay. The BIR3 region alone was found to be sufficient for hippocalcin binding by fusing BIR3 constructs with GST immobilized on glutathione beads and incubating these beads in lysate of motor neurons. The results were determined via Western blot to be that BIR3 is crucial for hippocalcin binding, as seen in Figures A and B1. Finally, the authors tested if the NAIP-hippocalcin interaction protected against apoptosis through multiple apoptotic pathways, namely caspase-3-dependent and –independent pathways. Cells containing NAIP-BIR alone and NAIP-BIR + hippocalcin constructs, as well as controls, were exposed to two different toxins, one of which killed cells along caspase-3-dependent pathways, while the other stimulated apoptosis through caspase-3-independent pathways. As shown in Figure B2, NAIP augmented with hippocalcin showed the greatest caspase 3/7 activity reduction, meaning that the synergic interaction helps prevent apoptosis along those pathways with greater effect than NAIP alone.

Significance

This study provides insight into the function of NAIP that may prove useful in producing solutions to neurodegenerative disorders, such as spinal muscular atrophy. These diseases are marked by deterioration of motor neurons in the spinal cord, similar to apoptotic degeneration. Because motor neurons are so vulnerable to calcium, understanding the mechanism by which NAIP protects neurons against calcium-induced cell death is crucial to further development of neurodegenerative disorder therapy.

Critique

The authors are very thorough in the design of their experiments, even providing a list of materials and methods at the close of the article that goes into great detail describing each assay. Their step-wise structure made the paper easy to follow and cohesively connected each experiment, guiding the reader through the rather dense background information. An initial complaint arose early; when the authors were testing the synergic interaction of NAIP and hippocalcin, they did not use actual motor neurons, instead opting for a cell line that “exhibits properties of spinal cord motor neurons.” If I were to repeat this experiment, I would make sure to do more in vivo testing using proper spinal motor neurons in hopes of obtaining more accurate, if not more reputable results. In addition, only one control (wild-type cells) was commonly used. I prefer to see both a positive and a negative control in experiments because I feel that the more controls used, the less likely one is to skew data one way or another to fit the anticipated results.

Furthermore, the long-term effects of calcium were not tested in this study, and it was often alluded that calcium may differ in the control cells and those transfected with NAIP/hippocalcin. The authors use indecisive words like “may” and “per se” to describe the basal calcium concentrations and its effect on NAIP. Without the in vivo testing, the authors can only speculate if NAIP and hippocalcin interact when intracellular calcium is increased. It would be worth researching the long-term effects of calcium both intracellularly and externally on the efficiency of NAIP.