ALS IGG EFFECTS ON MOTORNEURON UNTRASTRUCTURE

点击翻译按钮获取中文摘要

Amyotrophic lateral sclerosis is a devastating neuromuscular disease which produces progressive weakness leading eventually to respiratory failure and death. Our long term objective is to define the role of immunological factors in the etiology and pathogenesis of ALS. Although the etiology is still unknown, recent evidence suggests that immune mechanisms may play a role in triggering motoneuron injury and cell death. Results of in vitro experiments suggest that ALS IgG interact with neuronal voltage gated calcium channels resulting in excess calcium influx and calcium mediated neuronal injury. However, ALS in man is a slowly progressive pathologic condition whereas ALS IgG in vitro has a fast killing potency. Our present investigations are aimed at characterizing the evolution of ultrastructural changes and alterations of calcium levels of motoneurons exposed to ALS IgG in vivo by passive transfer to mice. Such investigations should help to define neuronal injury at its earliest stage, and may fill the gap; between the slowly evolving changes in ALS and the result of model experiments. Accordingly, the specific aims are: (1) To determine the time/dose dependence of effects of ALS IgG on ultrastructure and intracellular calcium level of motoneurons of mice injected with ALS IgG (2) To determine the specificity of ALS IgG by substituting ALS IgG with disease control IgG, by preadsorption of ALS IgG with potential membrane targets (purified calcium channels) and by antagonizing their effect with Ca-channel blockers. (3) To provide a detailed electrophysiological and ultrastructural study of transmitter release and vesicle recycling after in vivo IgG injection into mice. For these studies, mice will be injected intraperitoneally with different dosages of ALS IgG followed by perfusion fixation for ultrastructural analysis at different time points. An oxalate-ion containing glutaraldehyde fixative will be applied to provide good ultrastructural preservation and simultaneous conservation of in situ calcium distribution. Morphological and intracellular calcium distributional changes will be assayed by quantitative electron microscopy at motor nerve terminals, intramuscular axons and cell bodies of motoneurons with different susceptibility to ALS and the changes will be related to appropriate controls. In parallel experiments ALS IgG induced changes in neuromuscular transmission will be characterized by measuring changes in miniature end- plate potential frequency and by tracking synaptic vesicle recycling with immunohistochemical methods utilizing antibodies against vesicle proteins.