sq-23377 and Brain-Injuries

Neural-cadherin (N-cadherin), a member of the classical cadherin family of transmembrane glycoproteins, mediates cellular recognition and cell-cell adhesion through calcium-dependent homophilic interactions and plays important roles in the development and maintenance of the nervous system. Metalloproteinase is known to cleave N-cadherin, which is further cleaved by gamma-secretase. The intracellular domain of N-cadherin interacts with beta-catenin, and beta-catenin stability is critical for cell-cell adhesion and cell survival. In the present study, we showed that N-cadherin is cleaved specifically by calpain, resulting in the generation of a novel 110 kDa fragment. The cleavage occurred in ischemic brain lesions and in vitro neural cells in the presence of NMDA and ionomycin, and was restored by calpain inhibitors but not matrix metalloproteinase or gamma-secretase inhibitors. Calpain directly cleaved N-cadherin in in vitro calpain assays, and calpain inhibitors prevented its cleavage in a dose-dependent manner. Using N-cadherin deletion mutants, we found that calpain cleavage sites exist in at least four regions of the cytoplasmic domain. Treatment with NMDA induced neuronal death, and it suppressed the expression of surface N-cadherin and the N-cadherin/beta-catenin interaction, effects that were prevented by calpain inhibitor. Furthermore, calpain-mediated N-cadherin cleavage significantly affected cell-cell adhesion, AKT signaling, the N-cadherin/beta-catenin interaction and the Wnt target gene expressions through the accumulation of nuclear beta-catenin.

Topics: Animals; Animals, Newborn; beta Catenin; Biotinylation; Brain Injuries; Cadherins; Calcium; Calpain; Cells, Cultured; Disease Models, Animal; Dizocilpine Maleate; Embryo, Mammalian; Endocytosis; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Gene Expression Regulation; Green Fluorescent Proteins; In Vitro Techniques; Ionomycin; Ionophores; Mice; Models, Biological; Mutation; N-Methylaspartate; Neurons; Protein Structure, Tertiary; Proto-Oncogene Proteins c-akt; Signal Transduction; Subcellular Fractions; Transfection; Wnt Proteins

Experimental traumatic brain injury (TBI) damages cerebral vascular endothelium and reduces cerebral blood flow (CBF). The nitric oxide synthase (NOS) substrate, L-arginine, prevents CBF reductions after TBI, but the mechanism is not known. This study examined the possibility that post-traumatic hypoperfusion is due to reductions in the substrate sensitivity of NOS which are overcome by L-arginine. Isoflurane-anesthetized rats were prepared for TBI (midline fluid-percussion, 2.2 atm), sham-TBI, or no surgery (control), and were decapitated 30 min after injury or sham injury. The brains were removed and homogenized or minced for measurements of crude soluble or cell-dependent stimulated NOS activity, respectively. Baseline arterial oxygen, carbon dioxide, pH, or hemoglobin levels did not differ among control, sham, or TBI groups. Total cortical soluble NOS activity in TBI-treated rats was not significantly different from either untreated or sham groups when 0.45 microM or 1.5 microM L-arginine was added. Also, there were no differences in cell-dependent NOS activity among the three groups stimulated by 300 microM N-methyl-D-aspartate, 50 mM K+, or 10 microM ionomycin. These data suggest that TBI reduces CBF by a mechanism other than altering the substrate specificity or activation of nNOS.

Topics: Analysis of Variance; Animals; Arginine; Blood Pressure; Brain Injuries; Cerebral Cortex; Cerebrovascular Circulation; Disease Models, Animal; Excitatory Amino Acid Agonists; Ionomycin; Ionophores; Male; N-Methylaspartate; Nitric Oxide Synthase; Potassium Chloride; Rats; Rats, Sprague-Dawley