Rideout Hardy J, Lang-Rollin Isabelle, Stefanis Leonidas
Department of Neurology, Columbia University, New York, USA.
Int J Biochem Cell Biol. 2004 Dec;36(12):2551-62. doi: 10.1016/j.biocel.2004.05.008.
Ubiquitinated inclusions are a common feature of many neurodegenerative conditions. We have proposed that, at least in part, such inclusions may be formed due to dysfunction of the proteasome. We have modeled such proteasomal dysfunction by applying pharmacological inhibitors to cultured embryonic rat cortical neurons. This treatment leads to neuronal death and formation of ubiquitin/alpha-synuclein-positive cytoplasmic inclusions. At late time points following proteasomal inhibition such inclusions are no longer discerned. Instead, many neurons accumulate small ubiquitinated aggregates, which may represent remnants of the inclusions. In this work we have examined a potential mechanism for inclusion dissolution. Electron microscopy images showed activation of macroautophagy at late time points after proteasomal inhibition. Labeling with LysoTracker Red, a dye that accumulates in acidic compartments, or immunostaining for the lysosomal enzyme Cathepsin D, showed an increase in globular staining. Cathepsin D co-localized partially with small ubiquitinated aggregates, but not inclusions. Application of an inhibitor of macroautophagy or of the vacuolar ATPase led to an increase in the number of inclusions and a decrease in small aggregates, whereas an activator of autophagy had the opposite effects. There was no significant change in apoptotic death following these manipulations. We conclude that, following proteasomal inhibition of cultured cortical neurons, there is activation of macroautophagy and of the lysosomal pathway. This activation results in dissolution of ubiquitinated inclusions into small aggregates, without directly impacting neuronal cell death. These data further support the idea that in this model inclusions and neuronal cell death are independent processes.
泛素化包涵体是许多神经退行性疾病的常见特征。我们提出，至少部分此类包涵体可能是由于蛋白酶体功能障碍形成的。我们通过将药理抑制剂应用于培养的胚胎大鼠皮质神经元来模拟这种蛋白酶体功能障碍。这种处理导致神经元死亡并形成泛素/α-突触核蛋白阳性的细胞质包涵体。在蛋白酶体抑制后的晚期时间点，此类包涵体不再可见。相反，许多神经元积累了小的泛素化聚集体，这可能代表包涵体的残余物。在这项工作中，我们研究了包涵体溶解的潜在机制。电子显微镜图像显示在蛋白酶体抑制后的晚期时间点自噬被激活。用LysoTracker Red（一种积聚在酸性区室的染料）标记或对溶酶体酶组织蛋白酶D进行免疫染色，显示球状染色增加。组织蛋白酶D与小的泛素化聚集体部分共定位，但不与包涵体共定位。应用自噬抑制剂或液泡ATP酶抑制剂导致包涵体数量增加和小聚集体数量减少，而自噬激活剂则产生相反的效果。这些操作后凋亡性死亡没有显著变化。我们得出结论，在对培养的皮质神经元进行蛋白酶体抑制后，自噬和溶酶体途径被激活。这种激活导致泛素化包涵体溶解为小聚集体，而不会直接影响神经元细胞死亡。这些数据进一步支持了在该模型中包涵体和神经元细胞死亡是独立过程的观点。
