Brandy L. Wilkinson, Ph.D. - US grants

Neuronal survival in developing animals is often dependent on afferent activity. In the posthatch chick, elimination of eighth nerve activity leads to the death of 20-40% of the neurons in the cochlear nucleus, nucleus magnocellularis (NM). The factors that determine whether an individual NM neuron will live or die are largely unknown. Previous studies indicate that both cell death and cell survival mechanisms compete to determine an individual cell's fate. This proposal seeks to examine the underlying cellular and molecular cascades that are activated following deafferentation. Several studies have implicated bcl-2 family members in the regulation of common cell death pathways. This gene family includes both cell "protectors" (bcl-2 and bcl-xL) and "executioners" (bax and bak). The effect of deafferentation on the activation and expression of pro-survival bcl-2 gene and gene product will be examined using both in situ hybridization and immunocytochemistry. Additional studies will be performed to assess the effect of deafferentation on other members of the bcl-2 family. For example, pro-apoptotic bax will be investigated for differential expression from the pro-survival bcl-2 protein. Another line of research will focus on molecules thought to participate in apoptosis downstream of bcl-2 genes. Mitochondrial integrity is pivotal to the maintenance of healthy cells; compromise of mitochondrial bioenergetics can result in the release of cytochrome-c. Cytosolic cytochrome-c activates what is commonly referred to as the apoptosome. This complex of molecules including cytochrome-c, Apaf-1, and caspase-9 will initiate proteolytic caspase cascades that ultimately lead to cell death. Afferent regulation of all the molecules involved in the apoptosome will be investigated through a combination of immuncytochemistry and Western blotting techniques. It is the hope that this research will elucidate some of the underlying mechanisms involved with deafferentation-induced cell death.

DESCRIPTION (provided by applicant): The goal of this proposal is to elucidate mechanisms mediating the microglia pro-inflammatory response to fibrillar beta amyloid (fAa). The interaction of microglia with Abeta plaques leads to the induction of proinflammatory signaling cascades and the release of neurotoxic secretory products. Previous studies have shown fAbeta-stimulated ROS production from the activation of microglial NADPH oxidase. The NADPH oxidase consists of the membrane associated flavoprotein cytochrome b558 and the cytosolic, p47phox, p67phox, p40phox, and Rac1. Rac must be GTP-bound to function as part of the oxidase. The cytosolic components, p47phox and p67phox, under go phosphorylation and translocation to the membrane to initiate ROS generation. Little is known about the Abeta-stimulated intracellular signaling pathways responsible for this activation. We hypothesize that fAbeta engagement of a newly discovered multireceptor cell surface complex on microglia leads to the production of ROS and subsequent neuronal damage. This proposal seeks to identify tyrosine kinase-based signaling cascades that activate intracellular signaling elements that function to stimulate assembly and activation of the NADPH oxidase following exposure to fAbeta.

DESCRIPTION (provided by applicant): The goal of this proposal is to elucidate mechanisms mediating the microglia pro-inflammatory response to fibrillar beta amyloid (fAa). The interaction of microglia with Abeta plaques leads to the induction of proinflammatory signaling cascades and the release of neurotoxic secretory products. Previous studies have shown fAbeta-stimulated ROS production from the activation of microglial NADPH oxidase. The NADPH oxidase consists of the membrane associated flavoprotein cytochrome b558 and the cytosolic, p47phox, p67phox, p40phox, and Rac1. Rac must be GTP-bound to function as part of the oxidase. The cytosolic components, p47phox and p67phox, under go phosphorylation and translocation to the membrane to initiate ROS generation. Little is known about the Abeta-stimulated intracellular signaling pathways responsible for this activation. We hypothesize that fAbeta engagement of a newly discovered multireceptor cell surface complex on microglia leads to the production of ROS and subsequent neuronal damage. This proposal seeks to identify tyrosine kinase-based signaling cascades that activate intracellular signaling elements that function to stimulate assembly and activation of the NADPH oxidase following exposure to fAbeta.