We report here that activation of the caspase-3 apoptotic cascade in spinal cord injury is regulated, in part, by calcineurin-mediated BAD dephosphorylation. BAD, a proapoptotic member of the bcl-2 gene family, is rapidly dephosphorylated after injury, dissociates from 14-3-3 in the cytosol, and translocates to the mitochondria of neurons where it binds to Bcl-x(L). Pretreatment of animals with FK506, a potent inhibitor of calcineurin activity, or MK801, an NMDA glutamate receptor antagonist, blocked BAD dephosphorylation and abolished activation of the caspase-3 apoptotic cascade. These findings extend previous in vitro observations and are the first to implicate the involvement of glutamate-mediated calcineurin activation and BAD dephosphorylation as upstream, premitochondrial signaling events leading to caspase-3 activation in traumatic spinal cord injury.

Abstract:Immunocytochemical and immunoblotting techniques were used to investigate calpain I activation and the stability of the calpain-sensitive cytoskeletal proteins microtubule-associated protein 2 (MAP2) and spectrin at 1, 4, and 24 h after contusion injury to the spinal cord. Spinal cord injury resulted in the activation of calpain I at all time points examined, with the highest level of activation occurring at 1 h. At the same early time point, there was a loss of dendritic MAP2 staining in spinal cord sections, accompanied by pronounced perikaryal accumulation. The loss in MAP2 staining in the injured spinal cord progressed over the 24-h survival period to affect regions 3 mm distant to the site of injury. The presence of calpain I-specific spectrin degradation was apparent in neuronal cell bodies and fibers as early as 1 h after injury, with the most intense staining occurring within and juxtaposed to the injury site. Spectrin breakdown products in neuronal cell bodies declined rapidly at 4 h and were nearly undetectable at 24 h after injury. Immunoblot studies confirmed the immunocytochemical results by demonstrating a significant increase in calpain I activation, a significant decrease in MAP2 levels, and a significant increase in spectrin breakdown. Finally, treatment of animals with riluzole, an inhibitor of glutamate release, before surgery reduced significantly the loss of MAP2 levels observed at 24 h after injury. These results demonstrate that Ca 2~-dependentprotease activation and degradation of critical cytoskeletal proteins are early events after spinal cord injury and that treatments that minimize the actions of glutamate may limit their breakdown. Key Words: Microtubule-associated protein 2-Spectrin -Cytoskeleton -Glutamate-Calcium -Calpain l-RiluzoIe-CNS injury.

The nickel(II) complex of the encapsulating ligand 1 -methyl-8-amino-3,13-dithia-6,10,16,19-tetraazabicyclo[6.6.6]icosane, [Ni(AMN4S2sar)]2+, is reported. Crystals of the complex are monoclinic, space group P2\/c, with Z = 4,a = 9.167(6) Á, b = 19.885(4) Á, c = 13.068(8) Á, ß = 95.37(3)°, and R = 0.055. The low-temperature (~10 K) single-crystal absorption spectrum and ligand field analysis of the complex are reported. The spin-forbidden 3A2g -*• 'Eg.'Aig transitions were observed at low temperature, and from the spectroscopic analysis, a significant differential nephelauxetic effect is shown to occur resulting in much lower values for the Racah B and C parameters for these spin-forbidden transitions in relation to the spin-allowed transitions. introduction Encapsulated transition metal complexes have been studied extensively. In the majority of cases the ligands have been hexaamines of the sar type (sar = 3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane),M5 although metal complexes of ligands with donors other than Ng have been reported.16-21 As part of a study directed at the synthesis and properties of potentially octahedral encapsulating ligands with unsymmetrical nitrogen and sulfur donor sets, we have prepared the encapsulating ligands