Lipid raft signaling in cofilin-actin rod formation induced by amyloid-β and TNFα
Date
2012
Authors
Mi, Jianjie, author
Bamburg, James R., advisor
Chen, Chaoping, committee member
Partin, Kathryn M., committee member
Journal Title
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Volume Title
Abstract
Rod-like inclusions (rods), composed of actin saturated with cofilin, are induced in neurons by energetic and oxidative stresses, excitotoxic levels of glutamate, and amyloid beta treatment. Cofilin is an F-actin assembly regulatory protein critical to various actin-dependent processes, such as cytokinesis, cell migration, and neurite formation. Overexpression or hyperactivation (excessive dephosphorylation) of cofilin coupled with its oxidation can lead to formation of rods. Rods represent a likely mechanism to explain the synaptic loss associated with early stages of Alzheimer's disease (AD) and thus represent a novel target for therapeutic intervention. In live neurons, the study of cofilin-actin rod formation induced by specific mediators of stress has been limited because overexpression of fluorescent protein-tagged wild type (WT) cofilin results in formation of considerable numbers of spontaneous rods. A fluorescent cofilin mutant that could incorporate into induced rods but form no spontaneous rods even when overexpressed would offer a useful alternative for live-cell imaging. The R21Q mutant cofilin-RFP has been reported to not induce rods when overexpressed but incorporates into rods containing endogenous cofilin, thus serving as a rod marker in live cells. Here we show that expression of WT cofilin driven by promoters that result in a high or moderate steady-state level of exogenous protein produces a significant number of spontaneous rods, three to four fold over controls. However, R21Q cofilin-RFP expressed behind these same promoters will only incorporate into rods formed from endogenous protein, but not enhance spontaneous rod, even when accompanied by the photo stress induced by microscopic observation. Using the R21Q cofilin- RFP to measure rod formation, we then showed that the proinflammatory cytokine (TNFα) induced about a 3 fold increase in rod formation over untreated controls quantified either as the percent of neurons with rods (percent rod index) or as the number of rods per field (number rod index). Amyloid beta dimer/trimer (Aβd/t) induced about a 2.5 fold increase over controls in the percent of neurons with rods, and close to a 2 fold increase in the number of rods per field. To determine the fidelity of the R21Q cofilin-RFP in labeling all of the rods, we induced rods in control infected or R21Q cofilin-RFP expressing neurons with ATP depletion for 30 min, or with either Aβd/t (250 pM) or TNFα (50 ng/ml; 2.9 nM) for 24 h. Neurons were fixed and immunostained with a primary antibody for cofilin and an Alexa 647 nm-labeled secondary antibody. The percent of rods in RFP expressing cells that co-labeled with mRFP and Alexa 647 were then quantified. Although 100% of rods induced by ATP depletion co-labeled, surprisingly only 48% of the rods induced by TNFα co-labeled, similar to Aβd/t treatment. The reasons for this are not clear but taken together, our results demonstrate that R21Q cofilin-RFP can be used for a live cell marker for following induced rod formation but not as a quantitative measure of the total rod response. Induction of cofilin-actin rods by amyloid beta and TNFα is mediated by the cellular prion protein, a component of lipid raft domains which can signal to activate NADPH oxidase. Lipid rafts are cholesterol/sphingolipid enriched detergent resistant membrane domains in which many membrane receptors associate. Rafts can be visualized with an Alexa labeled cholera toxin B subunit which binds to GM1 ganglioside. Here we used neurons expressing R21Q cofilin-RFP to determine if rod formation is associated with coalesced lipid raft domains and if the coalesced lipid rafts form before or after rods are visible. In the three rods we visualized forming during the period in which lipid rafts were labeled we saw no lipid raft coalescence at sites of the newly formed rods. If we looked at the total R21Q cofilin-RFP labeled rods, about 45% of them co-localize with enlarged lipid raft domains. Thus results suggest that rods may bring about the reorganization of the membrane raft domains, although more data are required to make a definitive conclusion.
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Subject
lipid raft
cellular prion protein
cofilin-actin rod
Alzheimer's disease