Neuronal cytoskeletal alterations evoked by a platelet-activating factor (PAF) analogue

Platelet-activating factor (PAF), a phospholipid signaling molecule found in brain, modulates several neural functions and is implicated in the human developmental brain disorder Miller-Dieker Lissencephaly (MDL). Exposure to PAF, and a non-hydrolyzable analogue, methyl carbamyl PAF (mc-PAF), produces the following rapid, reversible effects upon cultured hippocampal neurites: growth cone collapse, neurite retraction, and neurite varicosity formation. In this study, the cytoskeletal alterations that mediate these shape changes were investigated by comparing the effects of mc-PAF with other cytoskeletal-altering drugs, through the fluorescent labeling of cytoskeletal proteins and mitochondria, and by electron microscopy. Results indicate that rearrangements of microtubules (MTs), F-actin, and mitochondria underlie the neurite shape changes produced by mc-PAF. Evidence for MT alteration was obtained by comparing the effects of mc-PAF with nocodozole and taxol. Exposure to nocodazole, a MT-depolymerizing agent, produced growth cone collapse and neurite varicosity formation similar to mc-PAF, whereas pre-incubation of neurites in taxol, a MT-stabilizing drug, was effective in blocking mc-PAF-induced neurite effects. Immunofluorescent labeling and EM revealed MT splaying and unbundling within neurite varicosities following mc-PAF treatment. Immunofluorescent labeling also revealed that F-actin shifted from concentration in the growth cone to a diffuse distribution along the neurite shaft following mc-PAF exposure. Fluorescent labeling and EM also revealed retrograde movement and morphological alterations of mitochondria following mc-PAF exposure, resulting in mitochondrial aggregates within neurite varicosities. These cytoskeletal rearrangements may provide insights into the mechanisms by which PAF influences neuronal activity, and could have important implications for the impairment of neuronal motility observed in MDL.