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Research Article
VPS35 regulates developing mouse hippocampal neuronal morphogenesis by promoting retrograde trafficking of BACE1
Chun-Lei Wang, Fu-Lei Tang, Yun Peng, Cheng-Yong Shen, Lin Mei, Wen-Cheng Xiong
Biology Open 2012 1: 1248-1257; doi: 10.1242/bio.20122451
Chun-Lei Wang
1Institute of Molecular Medicine and Genetics, and Department of Neurology, Medical College of Georgia, Georgia Health Sciences University, Augusta, GA 30912, USA
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Fu-Lei Tang
1Institute of Molecular Medicine and Genetics, and Department of Neurology, Medical College of Georgia, Georgia Health Sciences University, Augusta, GA 30912, USA
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Yun Peng
1Institute of Molecular Medicine and Genetics, and Department of Neurology, Medical College of Georgia, Georgia Health Sciences University, Augusta, GA 30912, USA
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Cheng-Yong Shen
1Institute of Molecular Medicine and Genetics, and Department of Neurology, Medical College of Georgia, Georgia Health Sciences University, Augusta, GA 30912, USA
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Lin Mei
1Institute of Molecular Medicine and Genetics, and Department of Neurology, Medical College of Georgia, Georgia Health Sciences University, Augusta, GA 30912, USA
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Wen-Cheng Xiong
1Institute of Molecular Medicine and Genetics, and Department of Neurology, Medical College of Georgia, Georgia Health Sciences University, Augusta, GA 30912, USA
2Charlie Norwood VA Medical Center, Augusta, GA 30912, USA
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  • For correspondence: wxiong@georgiahealth.edu
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    Fig. 1. Vps35 expression in developing mouse hippocampus.

    (A) Detection of enzymatic LacZ activity in developing Vps35+/m hippocampus. At the neonatal brain (e.g., P10–P15), LacZ activity detected in CA1–3 hippocampus was at its peak level. DG and CA1–3 in hippocampus are indicated. Scale bar: 200 µm. (B) Western blot analysis of VPS35 protein levels in lysates from Vps35+/+ and +/m mouse hippocampus during development. Again, a highest level of VPS35 protein was detected in P15 hippocampus. Note that ∼50% reduction of VPS35 protein was found in lysates from Vps35+/m mice, demonstrating the antibody specificity.

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    Fig. 2. Defective apical dendritic arborization and impaired spine morphology in Vps35 deficient mouse CA1 neurons.

    (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.

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    Fig. 3. Axonal spheroids in Vps35 deficient mouse CA1 neurons.

    (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.

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    Fig. 4. Protein components in axonal spheroids induced by Vps35 deficiency.

    (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.

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    Fig. 5. Altered BACE1 distribution in primary hippocampal neurons expressing miR-Vps35-1.

    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.

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    Fig. 6. Altered BACE1 distribution in Vps35 deficient mouse CA1 neurons.

    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.

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    Fig. 7. Defective BACE1-mCherry retrograde trafficking in primary hippocampal neurons expressing miR-Vps35-1.

    (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.

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    Fig. 8. Rescue of Vps35 deficiency induced axonal spheroids and apical dendritic arborization defect by suppressing BACE1 expression.

    Co-electroporation of miR-VPS35-1 with control construct or miR-BACE1 was performed at E15.5 in utero and brains were examined at P10. (A) Effects of miR-BACE1 on miR-VPS35 induced axon- and dendrite-defects. Upper panels, similar distribution pattern and cell density of electroporated cells between miR-VPS35-1+control and miR-VPS35-1+miR-BACE1 shown in low magnification. Middle panels, apical dendrites in the distal region were much longer in miR-VPS35-1+miR-BACE1 than in miR-VPS35-1+control. Lower panels, spheroids in miR-VPS35-1+miR-BACE1 expressing axons in HCC and CC regions were greatly reduced compared to that in miR-VPS35-1+control. Scale bar: 50 µm. (B) Quantification of apical dendrite growth by measuring normalized length of apical dendrites. miR-VPS35-1+miR-BACE1 expression dendrites were significantly longer than control miR-VPS35-1 dendrites (*P<0.01). (C) Quantification of axon spheroid formation by measuring the size of swellings in commissural axons (n = 300 from 3 brains for each group). Bars showing average size of selected swellings (miR-VPS35-1+control: 9.16 µm2; miR-VPS35-1+miR-BACE1: 5.36 µm2). The percentage of spheroids (>10 µm2) is 29% for miR-VPS35-1+control and 5% for miR-VPS35-1+miR-BACE1. (D) Schematic illustration of a working model for VPS35 containing retromer in promoting retrograde transport of BACE1.

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Research Article
VPS35 regulates developing mouse hippocampal neuronal morphogenesis by promoting retrograde trafficking of BACE1
Chun-Lei Wang, Fu-Lei Tang, Yun Peng, Cheng-Yong Shen, Lin Mei, Wen-Cheng Xiong
Biology Open 2012 1: 1248-1257; doi: 10.1242/bio.20122451
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Research Article
VPS35 regulates developing mouse hippocampal neuronal morphogenesis by promoting retrograde trafficking of BACE1
Chun-Lei Wang, Fu-Lei Tang, Yun Peng, Cheng-Yong Shen, Lin Mei, Wen-Cheng Xiong
Biology Open 2012 1: 1248-1257; doi: 10.1242/bio.20122451

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