Yap Chan Choo, Digilio Laura, McMahon Lloyd, Winckler Bettina
Department of Cell Biology, University of Virginia, 1340 Jefferson Park Avenue, Pinn Hall 3226, Charlottesville, VA 22908, USA.
bioRxiv. 2023 Mar 9:2023.03.08.531742. doi: 10.1101/2023.03.08.531742.
In neurons, degradation of dendritic cargos requires RAB7 and dynein-mediated retrograde transport to somatic lysosomes. In order to test if the dynein adaptor RILP (RAB-interacting lysosomal protein) mediated the recruitment of dynein to late endosomes for retrograde transport in dendrites, we obtained several knockdown reagents which had been previously validated in non-neuronal cells. We found that striking endosomal phenotypes elicited by one shRILP plasmid were not reproduced by another one. Furthermore, we discovered a profound depletion of Golgi/TGN markers for both shRILP plasmids. This Golgi disruption was only observed in neurons and could not be rescued by re-expression of RILP. This Golgi phenotype was also not found in neurons treated with siRILP or gRILP/Cas9. Lastly, we tested if a different RAB protein that interacts with RILP, namely the Golgi-associated RAB34, might be responsible for the loss of Golgi markers. Expression of a dominant-negative RAB34 did indeed cause changes in Golgi staining in a small subset of neurons but manifested as fragmentation rather than loss of markers. Unlike in non-neuronal cells, interference with RAB34 did not cause dispersal of lysosomes in neurons. Based on multiple lines of experimentation, we conclude that the neuronal Golgi phenotype observed with shRILP is likely off-target in this cell type specifically. Any observed disruptions of endosomal trafficking caused by shRILP in neurons might thus be downstream of Golgi disruption. Different approaches will be needed to test if RILP is required for late endosomal transport in dendrites. Cell type-specific off-target phenotypes therefore likely occur in neurons, making it prudent to re-validate reagents that were previously validated in other cell types.
在神经元中，树突货物的降解需要RAB7和动力蛋白介导的逆行运输至胞体溶酶体。为了测试动力蛋白衔接蛋白RILP（RAB相互作用溶酶体蛋白）是否介导动力蛋白募集至晚期内体以进行树突中的逆行运输，我们获得了几种先前已在非神经元细胞中验证过的敲低试剂。我们发现，一种shRILP质粒引发的显著内体表型未被另一种质粒重现。此外，我们发现两种shRILP质粒均导致高尔基体/TGN标志物大量减少。这种高尔基体破坏仅在神经元中观察到，且无法通过重新表达RILP来挽救。在用siRILP或gRILP/Cas9处理的神经元中也未发现这种高尔基体表型。最后，我们测试了与RILP相互作用的另一种RAB蛋白，即高尔基体相关的RAB34，是否可能导致高尔基体标志物的丢失。显性负性RAB34的表达确实在一小部分神经元中引起了高尔基体染色的变化，但表现为碎片化而非标志物丢失。与非神经元细胞不同，干扰RAB34不会导致神经元中的溶酶体分散。基于多方面的实验，我们得出结论，用shRILP观察到的神经元高尔基体表型可能在这种细胞类型中具有特异性脱靶效应。因此，shRILP在神经元中观察到的任何内体运输破坏可能是高尔基体破坏的下游效应。需要采用不同方法来测试RILP是否是树突中晚期内体运输所必需的。因此，细胞类型特异性脱靶表型可能在神经元中出现，这使得重新验证先前在其他细胞类型中验证过的试剂变得谨慎。
