# 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
In _OCRL_ KO i3 neurons, ApoE2 intensity in dendrites (MAP2-positive areas) and the soma were remarkably decreased, but not in the axon (MAP2-negative regions) (Figure 3F-I). As expected for ApoE2 in neurons, nuclear staining should correspond to the receptor intracellular domain [35]. Thus, the loss of OCRL1 reduces ApoE2 total and cell surface levels and its somatodendritic localization. Given the alterations of ApoE2 in the absence of OCRL1, we determined if the phosphatase deficiency affects the receptor's endocytic traffic. Consequently, we measured the colocalization of internalized ApoE2 by following the transfected HA-ApoER2. In wild-type H4 cells, after 15 min of internalization, ApoE2 colocalized less than 20% with EEA1; later (30 min), the percentage increased to 30%. In contrast, in _OCRL_ KO cells, the receptor was significantly increased in EEA1-positive structures, compared to the control cells, with 35% and 41% after 15 and 30 min of internalization, respectively (Figure 4A,B). To better characterize the changes in the intracellular traffic of the receptor upon OCRL1 loss, we colocalized the internalized ApoE2 with TfR, another receptor affected in LS [21], that at steady state is present in early/recycling endosomes. In wild-type H4 cells, 45% of internalized ApoE2 was present in TfR-positive structures after 15 min, whereas at 30 min post internalization, the colocalization increased to 60% (Figure 4C,D). Meanwhile, in _OCRL_ KO cells, 60% of ApoE2 colocalized with TfR at 15 min after internalization, and the proportion was maintained at 30 min post-internalization (Figure 4C,D). Moreover, as expected for a recycling receptor [21], the presence of TfR in EEA1-positive endosomes was significantly increased in _OCRL_ KO cells (Figure S3A-D). The Retromer is a physiologically relevant recycling complex responsible for sorting and trafficking different cargoes from the early endosome to the trans-Golgi network and the cell surface [82; 83]. We determined the colocalization of internalized ApoE2 with the vacuolar protein sorting-associated protein (VPS26), a specific Retromer complex subunit [84; 85]. In wild-type H4 cells, we found that 20% of ApoE2 was present in VPS26-positive structures after 15 min of internalization, whereas the percentage of colocalization decreased to 14% 30 min after internalization (Figure 4E,F). Strikingly, we detected a rise in colocalization of ApoE2 and VPS26 after 30 min of endocytosis (Figure 4E,F) together with increased detection of EEA1 and VPS26 (Figure S3E-H), indicating a loss of identity of the endosomal compartments in the absence of OCRL1 in H4 cells, as was described in kidney cells [22]. Figure 3: Decreased ApoE2 protein levels in _OCRL_ KO cells. Wild-type and _OCRL_ KO H4 and i3 neurons were grown for indicated days; then cell lysates were prepared. (**A**) Proteins were separated by SDS-PAGE, followed by western blot, and visualized by the UVITEC photo-documentation system to detect ApoE2 and \(\alpha\)-tubulin. (**B**) Signal intensities from ApoE2 and \(\alpha\)-tubulin in H4 cells (**A**, left) and  
(**C**) i3 neurons differentiated for 21 days (**A**, right) were measured by Fiji as the area below the curve for each band. The ratio of ApoEER2 bands relative to \(\alpha\)-tubulin was normalized to wild-type, plotted, and analyzed with GraphPad. Significant differences were calculated with a \(t\)-test for three independent experiments * \(p\) < 0.05, ** \(p\) < 0.01. (**D**) H4 cells were transfected with plasmids for HA-ApoER2 and RAP expression. 24 h later, the surface receptor (green) was stained, incubating the cells with Alexa fluor 488-coupled anti-HA for 45 min at 4 \({}^{\circ}\)C, followed by fixation, permeabilization, and staining of total ApoER2 (magenta) with chicken anti-HA and corresponding secondary antibody and DAPI (blue). Confocal images were deconvolved and analyzed with Fiji. Scale bar = 20 \(\upmu\)m. (**E**) Ratios of surface vs. total signals were calculated from at least 20 cells per experiment.