Title: “Disruption of Golgi markers by two RILP-directed shRNAs in neurons: a new role for RILP or a neuron-specific off-target phenotype?” Authors:

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. Introduction Short hairpin (sh) plasmids are powerful tools to study loss-of-function phenotypes by downregulating the expression of proteins of interest and are thus widely used (1). Compared to generating knockout animals or stable knockout lines, the advantages of using sh-plasmids include the ability to rapidly test many candidates, the capacity of relatively acute downregulation, and the ability to achieve partial, timed downregulation for examining the roles of essential genes. In addition, it has been shown that complete knockout phenotypes are sometimes less severe than knockdown approaches because only knockouts but not knockdowns activate cellular compensation pathways, thus dampening loss of function phenotypes (2). The disadvantages of sh-mediated knockdown approaches include incomplete downregulation and the need to carefully distinguish on-target from potential off-target phenotypes (3). This necessary validation is time-consuming and requires tools not always readily available. We previously showed that downregulation of RAB7 in cultured rat hippocampal neurons impairs maturation of early endosomes (EEs) to late endosomes (LEs), transport of LEs to the soma, and leads to degradation delays of short-lived cargos (e.g. the neuronal membrane receptor NSG2) in dendrites (4). Similar phenotypes were observed when dynein was inhibited (5). Since RILP has a known role as a dynein adaptor for RAB7 (6,7), we wanted to ask if RILP is necessary for degradative flux in dendrites. We already showed that overexpression of RILP causes increased retrograde transport of LEs in dendrites and clustering of LEs and lysosomes in the soma (5), suggesting the hypothesis that RILP serves as the endogenous dynein adaptor for late endosomes. CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 9, 2023. ; https://doi.org/10.1101/2023.03. 08.531742 doi: bioRxiv preprint