# OCRL1 Deficiency Affects the Intracellular Traffic of ApoER2 and Impairs Reelin-Induced Responses
## 3 Results
### OCRL KO Cells Show Significant Alterations in ApoER2 Protein Levels and Trafficking
Data were normalized to wild-type ratios and plotted with GraphPad. Significant differences were calculated with a Mann-Whitney test from three different experiments. *** \(p\) < 0.001. (**F**) Wild-type and _OCRL_ KO i3 neurons were differentiated for 14 days, fixed, and stained for ApoER2 (green), ANK G (cyan), and MAP2 (magenta). Scale bar = 20 \(\upmu\)m (**G-I**) ApoER2 intensities were measured in (**G**) Soma, (**H**) Dendrites (MAP2-positive structures) and (**I**) Axons (MAP2-negative structures). Data from 25 neurons from two independent experiments were analyzed and plotted. Statistical significance \(t\)-test with [PERSON]'s correction. ns: \(p\) > 0.05, * \(p\) < 0.05, **** \(p\) < 0.0001. Original images can be found in Supplementary Materials. So far, our results support the idea that the loss of OCRL1 perturbs the intracellular traffic of receptors, including ApoER2, which accumulates in EEA1- and Retrompositive endosomes. Considering what has been described in other OCRL1 deficient cells, we measured the levels of recycled ApoER2 using a chase protocol [41]. We detected a 70% reduction in recycled ApoER2 in KO vs. wild-type cells (Figure 5A,B). A less efficient recycling explains both the increased endosomal localization of the receptor and its reduced cell surface expression. It was previously demonstrated that endosomal-associated protein SNX17 binds to ApoER2, promoting its recycling towards the cell surface [41]. Total levels of SNX17 did not change by western blot in wild-type vs. _OCRL_ KO H4 cells (Figure 5C,D), which supports that the recycling impairment is primarily due to alterations caused by the lack of OCRL1. Further, we also analyzed the expression of SNX17, wild-type and OCRL1 deficient i3 neurons, finding that the total levels of SNX17 remain the same (Figure 5E,F). However, in _OCRL_ KO i3 neurons, we found a decrease in the intensity of SNX17 in dendrites (Figure 5G,H), suggesting a specific alteration in the dendritic localization of the protein. Since the deficiency of OCRL1 induced a defect in recycling and in the protein levels of ApoER2 (Figure 3), we determined if part of the receptor could be diverted to the degradation pathway. For internalized ApoER2, 3% was present in LAMP1-positive structures in wild-type H4 cells. In the _OCRL_ KO cells, 5% of the internalized ApoER2 colocalized with LAMP1 after 30 min of internalization (Figure 6A,B), and this difference was significant. It has been shown that in the absence of Reelin as a ligand, most of the receptor recycles and is poorly directed to the degradation pathway, explaining the low presence of ApoER2 in late endosomes/lysosomes [41]. To evaluate the behavior of ApoER2 in a more physiologic model that also expresses endogenous Reelin [67], we determined the receptor half-life in i3 neurons (differentiated for 21 days) using a cycloheximide treatment. ApoER2 protein levels decreased significantly faster in _OCRL_ KO than in wild-type i3 neurons, with half-lives of 5 h and 16 h, respectively (Figure 6C,D). Our results suggest that the loss of OCRL1 disrupts ApoER2 intracellular trafficking, affecting its availability at the cell surface and decreasing its half-life, explaining the significant reduction of the receptor in the absence of the phosphatase. Figure 4: OCRL1 is necessary for ApoER2 intracellular traffic. H4 cells expressing HA-ApoER2 were incubated with anti-HA (green) at 4 \({}^{\circ}\)C for 45 min, followed by internalization at 37 \({}^{\circ}\)C for indicated times. Cells were acid-washed, fixed, and stained for indicated proteins (magenta): (**A**) EEA1; (**C**) TfR; (**E**) VPS26. Confocal images were deconvolved and analyzed with JACoP from Fiji. Scale bar = 10 \(\upmu\)m. (**B,D,F**) Manders coefficients were analyzed for HA and each protein (corresponding images on the left). ANOVA with Sidak’s multiple comparisons test was calculated from three different experiments. ns: \(p\) > 0.05, * \(p\) < 0.05, *** \(p\) < 0.0001. Figure 5: The loss of OCRL1 decreases ApoER2 recycling.