SUPPRESSION OF FRIZZLED-2-MEDIATED WNT/CA2+ SIGNALING SIGNIFICANTLY ATTENUATES INTRACELLULAR CALCIUM ACCUMULATION IN VITRO AND IN A RAT MODEL OF TRAUMATIC BRAIN INJURY

Traumatic brain injury (TBI) can dramatically increase levels of intracellular calcium (Ca2+). The association between Wnt5a/Frizzled-2 (wingless-type mouse mammary tumor virus integration site family member 5a/Fzd2) signaling and Ca2+ cellular homeostasis in lower vertebrates has been well documented. However, little is known about Wnt5a/Fzd2 signaling in mammalian nerve cells, or whether Ca2+ accumulation after TBI is mediated through this pathway. We hypothesized that an activated Wnt5a/Fzd2 pathway following TBI may play a role in Ca2+ overloading. To elucidate the influence of Fzd2 and the Wnt5a signal transduction pathway on an increase in intracellular Ca2+, we assessed the expression of Wnt5a/Fzd2 in rat hippocampal cells both in vitro and in vivo. We found that transfection of the rat Fzd2 gene in rat neonatal hippocampal astrocytes significantly increased gene expressions of both Wnt5a and Frd2 by fourfold when compared to non-transfected cells (P < 0.01 in both cases). Expressions of the proteins Wnt5a and Fzd2 were significantly increased approximately two- and threefold, respectively, when compared to non-transfected control cells (P < 0.01 in both cases). Moreover, intracellular Ca2+, as manifested by the fluorescent intensity of the intracellular Ca2+ indicator Fluo-3/AM, was significantly increased by 1.75-fold (P < 0.01). The blocking of Fzd2 signaling using Stealth RNAi markedly inhibited the elevated gene and protein expression of Wnt5a in the transfected cells by two- and fourfold, respectively (P < 0.01), and suppressed intracellular Ca2+ by 1.5-fold (P < 0.01). Furthermore, in vivo, we demonstrated that TBI-induced dramatic upregulation of gene and protein expression of Wnt5a/Fzd2 by two- and five-fold (P < 0.01) in injured hippocampi, and intracellular Ca2+ increased in isolated injured hippocampal cells. Whereas, the in vivo blocking of Fzd2 signaling by hippocampal delivery of Stealth RNA1 and Invivofectamine significantly suppressed the increased gene and protein expression of Wnt5a and Fzd2 induced by TBI by 1- to 3.5-fold (P < 0.01) and also inhibited Ca(2+)accumulation by 1.5-fold (P < 0.01). These findings demonstrated that the Wnt5a/Fzd2 signaling pathway contributed to increasing intracellular Ca2+ in nerve cells under physiological and pathological conditions. Furthermore, our findings provide evidence that specifically expressed components of this signal pathway, such as Wnt5a and Fzd2, are potential therapeutic targets following brain trauma. (C) 2012 IBRO. Published by Elsevier Ltd. All rights reserved.