# 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  
The endosomal traffic of several receptors is affected in LS [21; 22; 23]. In human neurons, there is no information about alterations of relevant receptors and their associated signaling pathways that could underlie the neurological phenotype of LS patients. Thus, we evaluated protein levels and localization of ApoER2 as a crucial receptor of Reelin involved in neuronal development and function [81]. Figure 1: H4 _OCRL_ KO cells recapitulate LS cellular phenotype. H4 cells were transfected with a plasmid containing Cas9 and specific sgRNA to target OCRL, selected, and propagated to perform further experiments. (**A**) Whole-cell lysate protein samples from wild-type or _OCRL_ KO cells were separated by SDS-PAGE followed by western blot to detect OCRL1 and tubulin. (**B**,**D**) 48 h after seeding, the cells were fixed and stained for (**B**) EEA1 or (**D**) LAMP1. Scale bar = 10 μm. (**C**) EEA1 signal from (**B**) was analyzed with Fiji to measure the relative size of EEA1-positive endosomes. Data from 3 experiments was analyzed with GraphPad. Error bars indicate SEM. Mann–Whitney test, ***** \(p\) < 0.0001. (**E**) LAMP1 signal from (**D**) was used to perform shell analysis with Fiji. At least 50 cells were analyzed from three experiments. Error bars indicate SEM. Significant differences were estimated using ANOVA, and multiple comparisons were calculated using [PERSON]’s test. ***** \(p\) < 0.0001. Original images can be found in Supplementary Materials. Figure 2: Characterization of _OCRL_ KO iPSC and i3 neurons. iPSCs were transfected with a plasmid containing Cas9 and sgRNA directed to _OCRL_, selected, and then differentiated for 2I days to obtain i3 neurons. **(A)** Protein extracts from wild-type and _OCRL_ KO cells were subjected to SDS-PAGE and  
western blot to detect OCRL1 and tubulin or \(\beta\)-actin. (**B**) Wild-type and _OCRL_ KO iPSC at 70% confluency were detached and seeded. Following an incubation of 30 min, the cells were fixed and stained with phalloidin and DAPI. Images were captured using a wild-field microscope. Arrowheads indicate actin accumulations. Scale bar = 20 \(\upmu\)m. (**C**) The total area of each cell, determined by phalloidin labeling, was quantified. At least 50 cells from three independent experiments were analyzed. Statistical significance was calculated using a \(t\)-test (****\(p<0.0001\)). (**D**) Wild-type and _OCRL_ KO iPSC were differentiated for three days, transfected to express GFP, and fixed 48 h later. Images were captured using a wild-field microscope. Scale bar = 100 \(\upmu\)m. (**E**) Neurite length was measured from the GFP signal using Neuron J. Data from seven cells per experimental condition were analyzed, and statistical significance was calculated using a \(t\)-test (* \(p<0.05\)). (**F**) Wild-type and _OCRL_ KO i3 neurons were differentiated for 14 days, fixed, and stained for EEA1 (green), MAP2 (magenta) and DAPI (blue). Scale bar = 20 \(\upmu\)m. (**G**) EEA1-positive particles were measured in dendrites (MAP2-positive structures, grey box). Data from 50 ROIs from three independent experiments were analyzed and plotted. Statistical significance was calculated using a \(t\)-test. ****\(p<0.0001\). (**H**) EEA1 signal intensity was measured in dendrites (MAP2-positive structures). Data from 50 ROIs from three independent experiments were analyzed and plotted. Statistical significance was calculated using a \(t\)-test. ns: \(p>0.05\). Original images can be found in Supplementary Materials. First, we determined if ApoE2 protein levels were altered in _OCRL_ KO H4 cells and i3 neurons. We found a marked decrease in ApoE2 in both OCRL1 deficient cellular models (Figure 3A-C), while ApoE2 mRNA expression in i3 neurons was not modified (Figure S2A). Moreover, ApoE2 surface levels, evaluated by antibody binding in non-permeabilized cells (Figure 3D,E) or cell surface biotinylation (Figure S2B,C), were reduced in OCRL1 deficient cells.