# OCRL1 Deficiency Affects the Intracellular Traffic of ApoER2 and Impairs Reelin-Induced Responses
## 3 Results
### Neuronal Responses to Realin Are Affected in OCRL KO Cells  
We previously reported that decreased levels of cell surface ApoER2 led to a reduction of the Reelin/ApoER2 signaling pathway [41; 75]. Hence, our next aim was to determine if Reelin responses are affected by the lack of OCRL1. Two branches of the signaling pathway that involve ApoER2 were measured in i3 neurons differentiated for 21 days. For both AKT and ERK activation, we detected significantly reduced responses in the absence of OCRL1 (Figure 7). Furthermore, we evaluated whether the deficiency in OCRL1 affects the deployment of Golgi to the primary dendrite, a developmental relevant ApoER2/Reelin response that depends on the activation of PI3-K and Cdc42 [48]. We found a significant induction of deployment of Golgi in the wild-type neurons (Figure 8). In contrast, Golgi appeared more fragmented in the soma of _OCRL_ KO i3 neurons, and Reelin did not induce the entrance of the Golgi membrane into the main MAP2-positive process. Taken together, our results show that OCRL1 is required for ApoER2 functions as a Reelin receptor, suggesting that this signaling pathway could be less active in neurons from LS individuals.  
Figure 6: The loss of OCRL1 increases ApoER2 degradation. Wild-type and OCRL1-deficient H4 cells were transfected with a plasmid to express HA-ApoER2. (**A**) After anti-HA (green) internalization, cells were fixed and stained for LAMP1 (magenta). Confocal images were deconvolved and analyzed with Fiji. Scale bar = 10 \(\upmu\)m. (**B**) Manders coefficients were analyzed for HA and LAMP1. ANOVA with Sidak’s multiple comparisons test was calculated from two different experiments. ns: \(p>0.05\), * \(p<0.05\). (**C**) To measure the half-life of ApoER2, wild-type, and OCRL1 deficient i3 neurons were differentiated for 21 days and then treated with 25 \(\upmu\)M cycloheximide for specified times in a complete cortical medium. Protein samples were subjected to SDS-PAGE and western blot to determine the ApoER2 levels. (**D**) The relative expression of ApoER2, calculated as the ratio of ApoER2 and matching tubulin signals, was normalized to time 0, plotted, and analyzed with GraphPadPad. ANOVA with [PERSON]’s multiple comparisons test was calculated for data from three independent experiments. * \(p<0.05\). Original images can be found in Supplementary Materials.  
Figure 7: AKT and ERK activation induced by Reelin are decreased in \(OCRL\) KO i3 neurons. Wild-type and OCRL1 deficient i3 neurons were cultured in differentiation media for 21 days, depleted for 1 h, and incubated with 10 nM Reelin or Mock for the indicated times. (**A**,**C**) i3 neurons were lysed with phosphatase inhibitors, and proteins were separated by SDS-PAGE, followed by a western blot. Phosphorylated and total levels of AKT (**A**) and ERK (**C**) were determined (**B**,**D**). Signal intensity was measured with Fiji as the area below the curve. Phosphorylated values were normalized to their respective total levels and plotted. Three-way ANOVA with [PERSON]’s multiple comparisons test was calculated for three independent experiments * \(p\) < 0.05, **_p_ < 0.01, ***_p_ < 0.001. (**E**,**F**) For each time point, the mock value was subtracted from the Reelin value in (**B**,**D**) to create the curves presented in (**E**,**F**). Two-way ANOVA with [PERSON]’s multiple comparisons test was calculated from 3 independent experiments * \(p\) < 0.05, **_p_ < 0.01. Original images can be found in Supplementary Materials.