PRNP/prion protein regulates the secretion of exosomes modulating CAV1/caveolin-1-suppressed autophagy

Prion protein modulates many cellular functions including the secretion of trophic factors by astrocytes. Some of these factors are found in exosomes, which are formed within multivesicular bodies (MVBs) and secreted into the extracellular space to modulate cell-cell communication. The mechanisms underlying exosome biogenesis were not completely deciphered. Here, we demonstrate that primary cultures of astrocytes and fibroblasts from prnp-null mice secreted lower levels of exosomes than wild-type cells. Furthermore, prnp-null astrocytes exhibited reduced MVB formation and increased autophagosome formation. The reconstitution of PRNP expression at the cell membrane restored exosome secretion in PRNP-deficient astrocytes, whereas macroautophagy/autophagy inhibition via BECN1 depletion reestablished exosome release in these cells. Moreover, the PRNP octapeptide repeat domain was necessary to promote exosome secretion and to impair the formation of the CAV1-dependent ATG12–ATG5 cytoplasmic complex that drives autophagosome formation. Accordingly, higher levels of CAV1 were found in lipid raft domains instead of in the cytoplasm in prnp-null cells. Collectively, these findings demonstrate that PRNP supports CAV1-suppressed autophagy to protect MVBs from sequestration into phagophores, thus facilitating exosome secretion.

朊蛋白（Prion protein）可调控诸多细胞功能，其中包括星形胶质细胞（astrocyte）分泌营养因子。部分此类营养因子存在于外泌体（exosome）中——外泌体形成于多囊泡体（multivesicular bodies，MVBs），并被分泌至细胞外间隙以调控细胞间通讯。此前，外泌体生物发生的具体机制尚未完全阐明。本研究证实，来自prnp基因敲除（prnp-null）小鼠的星形胶质细胞与成纤维细胞原代培养物，其外泌体分泌水平显著低于野生型细胞。进一步研究发现，prnp基因敲除的星形胶质细胞中，多囊泡体形成减少而自噬体形成增多。在细胞膜上重构PRNP的表达，可恢复PRNP缺陷型星形胶质细胞的外泌体分泌能力；而通过BECN1耗竭抑制巨自噬/自噬（macroautophagy/autophagy），则可重建这些细胞的外泌体释放功能。此外，PRNP的八肽重复结构域是促进外泌体分泌、以及阻碍依赖于CAV1的ATG12–ATG5胞质复合物形成所必需的——该复合物可驱动自噬体的形成。相应地，在prnp基因敲除细胞中，更高水平的CAV1分布于脂筏结构域而非细胞质内。综上，本研究结果表明，PRNP可通过维持CAV1介导的自噬抑制作用，使多囊泡体免于被吞噬泡包裹，从而促进外泌体的分泌。