(A) Schematic illustration of the in utero electroporation experiments. Control and miRNA-Vps35 (miR-Vps35) constructs were in utero electroporated into E15.5 brains, and neurons and their dendrites in the hippocampal CA1 region were examined at P10, P14, or P25 postnatal stages. All images were counter-stained with Topro-3 (blue). (B) Shortened apical dendrites at P10 by miR-Vps35-1 and -3 electroporation compared to control dendrites. A blank area (yellow arrows) between the distal end of dendrites and pia surface (dotted line) was observed in Vps35-suppressed CA1 regions. Scale bar: 100 µm. (C) Quantification of apical dendritic lengths at different postnatal stages. *P<0.01, n = 3–4 brains for each group. (D) Impaired spine morphology in basal and apical dendrites at P25 by miR-Vps35-1 electroporation compared to the control. Scale bar: 10 µm. (E,F) Quantification of spine density (E) and spine head size (F) in basal and apical dendrites. *P<0.01, n = 300 spines from 3 different brains for each group.

(A) In utero electroporation of control, miR-Vps35-1 or -3 was performed at E15.5 and brains were examined at P10, P14, and P25. All images were counter-stained with Topro-3 (blue). Axonal morphology in HCC region was examined. (B) Representative images for increased axonal spheroids in HCC region at P10 by miR-Vps35-1 and -3. Dotted blue lines indicate the midline of the brain. Scale bar: 50 µm. (C) Quantification of axonal swelling size. *P<0.01, n = 300 spheroids from 6 different brains for each group. (D) More severe spheroid formation on the contralateral side of HCC than that on the ipsilateral side. The spatial distribution pattern of axonal spheroids by miR-Vps35-1 electroporation was quantified and illustrated, and each dot represents a spheroid with size > 10 µm². Data were from 6 brains at P10.

(A,B) In utero electroporation with miR-Vps35-1 was performed at E15.5 and brain slices at P14 were immunostained with different antibodies (APP, SORLA, EEA1 and LAMP1). Projected images from z-stack confocal scanning showing immunosignal (red) in HCC (A) and cell body (B) regions. Note that APP and LAMP1, but not SORLA and EEA1, appeared to be enriched in spheroids (A). Scale bar: 2.5 µm (A); 5.0 µm (B). (C) Co-electroporation of miR-Vps35-1 with BACE1-mCherry was performed at E15.5 and brain slices at P10 were scanned under confocal microscope. Note that BACE1-mCherry fusion proteins (red) were enriched in spheroids. Scale bar: 50 µm. (D) The percentages of spheroids enriched in indicated proteins were quantified. APP: 93.8%, n = 16; SORLA: 0%, n = 18; EEA1: 0%, n = 10; LAMP1: 95.2%, n = 21; BACE1-mCherry: 100%, n = 50.

Co-transfection of BACE1-mCherry with control or miR-Vps35-1 was performed in primary rat hippocampal neurons (E18) at DIV 5. Confocal imaging analysis of transfected neurons at DIV 9 was carried out and representative images were shown (A,D). Scale bar: 5 µm. The middle and lower panels showed the amplified images of the boxed areas. (B) Quantification analysis of the density of BACE1-mCherry fluorescence crossing the lines indicated in lower panels of A. (C) Quantification analysis of the percentage of BACE1-mCherry puncta in soma (black column) vs dendrite (orange column) regions. (E) Quantification analysis of the size of GFP-aggregates from D. The sizes of the top 100 GFP aggregates were showed as the grouped column scatter. The line in the scatter indicated the median. *, P<0.05.

Co-electroporation of BACE1-mCherry with control miRNA or miR-Vps35-1 was performed at E15.5 embryos in utero and brain slices were examined at P10. (A) Representative images showing BACE1-mCherry distribution in the proximal apical dendrites of the CA1 neurons. Inserts of enlarged images showing basal shift of BACE-mCherry signal in VPS35 deficient neurons. Scale bar: 5 µm. (B) Sample images at higher magnification showing BACE1-mCherry aggregation and basally shifted redistribution. Scale bar: 2.5 µm. (C) Quantification analysis of BACE1-mCherry distribution in miR-VPS35-1 neurons and control neurons. Basal shift index (BSI; see Materials and Methods) was introduced to judge the degree of BACE-mCherry redistribution from apical to basal side of the neuron. Bars showing average BSI (Control: 46.8; miR-VPS35-1: 87.5; n = 54 from 3 brains for each group). (D) Quantification analysis of BACE1-mCherry aggregation in miR-VPS35-1 neurons and control neurons. The size of BACE1-mCherry puncta was measured (see Materials and Methods) (n = 300 from 3 brains for each group). The bars showing average size of BACE1-mCherry puncta (Control: 0.57 µm²; miR-VPS35-1: 1.29 µm²). (E) Representative images of BACE1-mCherry distribution in control and Vps35 deficient CA1 axons in HCC region. Scale bar: 50 µm.

(A) Representative images showing distribution patterns of BACE1-labeled vesicles in control and miR-Vps35-1 expressing hippocampal neurons. Neurons were co-transfected with BACE1-mCherry with control and miR-Vps35-1 at DIV5 and followed by time-lapse imaging analysis 48 hours after transfection. Scale bar: 2 µm. (B) Representative kymographs showing the mobility of BACE1 positive vesicles/endosomes during 15-min recordings in control and miR-Vps35 expressing neurons. Vertical lines represent stationary BACE1-vesicles; oblique lines or curves to the right represent anterograde movements and lines to the left indicate retrograde transport. (C–E) Relative mobility (anterograde, retrograde, and stationary) of BACE1-vesicles in control and miR-Vps35-1 expressing neurons. Data were quantified from the total number of 17 BACE1-vesicles in neurons from >3 experiments, as indicated in parentheses. Error bars: S.D. *P<0.01.