这项工作旨在阐明不同ataxin-3(ATX3)聚集体的形态和理化性质与其细胞毒性之间的关系。我们调查了一种非病理性ATX3形式(ATX3Q24),病理扩展形式(ATX3Q55),和在残基291处截短的缺乏polyQ扩展的ATX3变体(ATX3/291Δ)。溶解度,对寡聚聚集体的形态和疏水暴露进行了表征。然后,我们监测了细胞内Ca(2)水平的变化以及与培养的大鼠小脑颗粒细胞的聚集体相互作用引起的异常Ca(2)信号传导。ATX3Q55,ATX3/291Δ和,在较小程度上,ATX3Q24寡聚体表现出相似的形态和物理化学特征,并在质量上诱导了相当的时间依赖性细胞内Ca(2)反应。然而,只有扩增的ATX3(形成成熟原纤维束的唯一变体)的前纤维聚集体在后期触发了特征性的Ca(2+)反应,这与相对于其他两种变体而言更大的疏水性暴露相关.与早期寡聚体的细胞相互作用涉及谷氨酸能受体,电压门控通道和富含单唾液酸四己糖神经节苷脂(GM1)的膜结构域,而细胞与更多老化的ATX3Q55前纤维状聚集体的相互作用通过仅涉及富含GM1区域的机制导致膜分解。暴露于ATX3Q55和ATX3/291Δ聚集体导致细胞凋亡,而ATX3Q24基本上是无害的。我们的发现提供了对ATX3聚集机制的洞察,暴露小脑细胞的聚集细胞毒性和钙水平改变。
This work aims at elucidating the relation between morphological and physicochemical properties of different ataxin-3 (ATX3) aggregates and their cytotoxicity. We investigated a non-pathological ATX3 form (ATX3Q24), a pathological expanded form (ATX3Q55), and an ATX3 variant truncated at residue 291 lacking the polyQ expansion (ATX3/291Δ). Solubility, morphology and hydrophobic exposure of oligomeric aggregates were characterized. Then we monitored the changes in the intracellular Ca(2+) levels and the abnormal Ca(2+) signaling resulting from aggregate interaction with cultured rat cerebellar granule cells. ATX3Q55, ATX3/291Δ and, to a lesser extent, ATX3Q24 oligomers displayed similar morphological and physicochemical features and induced qualitatively comparable time-dependent intracellular Ca(2+) responses. However, only the pre-fibrillar aggregates of expanded ATX3 (the only variant which forms bundles of mature fibrils) triggered a characteristic Ca(2+) response at a later stage that correlated with a larger hydrophobic exposure relative to the two other variants. Cell interaction with early oligomers involved glutamatergic receptors, voltage-gated channels and monosialotetrahexosylganglioside (GM1)-rich membrane domains, whereas cell interaction with more aged ATX3Q55 pre-fibrillar aggregates resulted in membrane disassembly by a mechanism involving only GM1-rich areas. Exposure to ATX3Q55 and ATX3/291Δ aggregates resulted in cell apoptosis, while ATX3Q24 was substantially innocuous. Our findings provide insight into the mechanisms of ATX3 aggregation, aggregate cytotoxicity and calcium level modifications in exposed cerebellar cells.