Fig 2: OCRL knockdown. Left panel shows a western blot of OCRL along with a control protein, GAPDH, after NPCs were exposed to an OCRL siRNA and a scrambled control. Two independent KD experiments were carried out. The right three panels are qPCR results for OCRL, MEIS2 and EFEMP1 carried out on two independent RNA samples, both analyzed in duplicate, as described in the methods section an in the Fig. 3 legend

Fig 3: Rab35 stimulates retrograde trafficking of BACE1 via OCRL.A Schematic representation of BACE1 internalization assay, in which cell-surface BACE1 was labeled with FLAG antibody, cells were incubated for 0, 10, 30, or 60 min to allow for BACE1 internalization, and finally, cells were immunostained with syntaxin-6 antibodies to label the TGN. B, C Representative high-resolution images (Zeiss Airyscan) and quantification of BACE1 internalization in N2a cells expressing FLAG-BACE1 and either HA Control, HA-Rab35 wild-type (WT), or HA-Rab35 dominant-negative (DN). Internalized BACE1 (red) and syntaxin-6 (blue) are shown at 0 and 60 min time points post-labeling, with cells outlined in gray, the TGN outlined in blue, and white arrowheads pointing to areas of colocalization in insets (*PDN-0 min = 0.0392, **PDN-10 min = 0.0011, **PWT-30 min = 0.0025, ****PWT-60 min < 0.0001; 2-way ANOVA and Sidak post hoc analysis, n = 45–155 cells per condition/timepoint, 2–5 experiments. Time × Rab35 interaction F6,1253 = 8.002, P < 0.0001, overall Rab35 effect F2,1253 = 10.43, P < 0.0001). D, E Representative high-resolution images (Zeiss Airyscan) and quantification of BACE1 internalization in N2a cells expressing FLAG-BACE1 and HA or HA-Rab35 together with control siRNA (siCtrl) or siRNA to knockdown OCRL (siOCRL). Internalized BACE1 (red) and syntaxin-6 (blue) are shown at 0 and 60 min time points post-labeling (*PHA+siCtrl vs. Rab35+siCtrl = 0.0151, 2-way ANOVA and Tukey’s multiple comparisons test, n = 50–61 cells per condition/time point, two experiments. Time × Condition interaction F6,635 = 3.861, P = 0.009). Scale bars: 5 µm; 1 µm for zoomed insets. All numeric data represent mean ± SEM.

Fig 4: Working model summarizing the interplay between Rab35 and stress/GC on APP trafficking and processing.AD etiopathological factors such as advanced age, Aß accumulation, or exposure to chronic stress and/or high levels of glucocorticoids (GC), lead to a significant reduction of Rab35 levels [1]. This reduction inhibits APP and BACE1 recycling with the plasma membrane [2], increasing APP-BACE1 association in the endosomal network and stimulating amyloidogenic APP processing and Aß production [3]. Alternatively, high levels of Rab35 promote APP internalization [4] and recycling to the plasma membrane via the effector ACAP2 [5], and in parallel stimulate BACE1 retrograde trafficking to the trans-Golgi network via the effector OCRL [6]. These trafficking events reduce APP-BACE1 interaction within the endosomal network, thereby decreasing production of APP CTFs and Aß.