The online version of this article (doi:10.1186/1750-1326-9-1) contains supplementary material, which is available to authorized users.
Celia G Fernandez, Sean Riordan contributed equally to this work.
The authors declare that they have no competing interests.
VBP, CGF, and GT designed experiments, performed live cell imaging, confocal microscopy, and analyzed the data. VBP, CGF, KSV, and JR generated expression plasmids and immunostaining. SR and RV generated and characterized BACE1-YFP transgenic mice. VBP, SR, and JW performed multiphoton imaging. VBP and GT wrote the manuscript. GT conceived of the study, coordinated data analysis, and prepared the final manuscript. All authors read and approved the final manuscript.
BACE1 is one of the two enzymes that cleave amyloid precursor protein to generate Alzheimer's disease (AD) beta amyloid peptides. It is widely believed that BACE1 initiates APP processing in endosomes, and in the brain this cleavage is known to occur during axonal transport of APP. In addition, BACE1 accumulates in dystrophic neurites surrounding brain senile plaques in individuals with AD, suggesting that abnormal accumulation of BACE1 at presynaptic terminals contributes to pathogenesis in AD. However, only limited information is available on BACE1 axonal transport and targeting.
By visualizing BACE1-YFP dynamics using live imaging, we demonstrate that BACE1 undergoes bi-directional transport in dynamic tubulo-vesicular carriers along axons in cultured hippocampal neurons and in acute hippocampal slices of transgenic mice. In addition, a subset of BACE1 is present in larger stationary structures, which are active presynaptic sites. In cultured neurons, BACE1-YFP is preferentially targeted to axons over time, consistent with predominant in vivo localization of BACE1 in presynaptic terminals. Confocal analysis and dual-color live imaging revealed a localization and dynamic transport of BACE1 along dendrites and axons in Rab11-positive recycling endosomes. Impairment of Rab11 function leads to a diminution of total and endocytosed BACE1 in axons, concomitant with an increase in the soma. Together, these results suggest that BACE1 is sorted to axons in endosomes in a Rab11-dependent manner.
Our results reveal novel information on dynamic BACE1 transport in neurons, and demonstrate that Rab11-GTPase function is critical for axonal sorting of BACE1. Thus, we suggest that BACE1 transcytosis in endosomes contributes to presynaptic BACE1 localization.
Additional file 1: Time-lapse Analysis of Axonal Transport of BACE1 in Hippocampal Mossy Fibers. Acute hippocampal slices of BACE1-YFP bigenic mice were maintained at 30°C by perfusion with ACSF, and imaged on Olympus FV1000 MPE multiphoton confocal microscope under 920 nm excitation. Time-lapse images of the hippocampal mossy fibers were acquired at a rate of 0.8 frames/sec (4 µsec/pixel sampling speed) for 4 min, and displayed at 15 frames/sec. BACE1-YFP containing carriers moving away from the hilus (anterograde) or toward the hilus (retrograde) are shown. Representative still images are shown in Figure 2C. (MOV 430 KB)
Additional file 2: Dynamic Axonal Transport of BACE1 in Cultured Hippocampal Neurons. Hippocampal neurons transfected with BACE1-YFP (DIV12) were monitored under a temperature-controlled environment at 37°C on a Nikon TE 2000 microscope. Images were acquired at a rate of 1 frame/sec for 5 min, with an exposure time of 300 ms, and displayed at 10 frames/sec. Arrows indicate BACE1 anterograde (blue) and retrograde transport (red). Representative still image and image montages are shown in Figure 4A and 4C. (MOV 1 MB)
Additional file 3: Presynaptic Localization of BACE1. Hippocampal neurons (DIV14) cotransfected with BACE1-YFP and Synaptophysin-Cerulean were observed by live-cell imaging. Sequential time-lapse images were acquired at a rate of 1 frame/sec for 3 min on the same axon, with an exposure time of 300 ms, and displayed at 10 frames/sec. Arrowheads point to stationary structures that contain BACE1 and synaptophysin. The anterograde direction of movement is toward the right. Representative still images are shown in Figure 4D. (MOV 890 KB)
Additional file 4: Dynamic Colocalization BACE1with Rab11b in Dendrites and Axons. Hippocampal neurons (DIV14) cotransfected with BACE1-YFP and mCherry-Rab11b were observed by live-cell imaging. Dual-color time-lapse images of a dendritic branch and an axon were acquired using the Dual View imaging system at a rate of 1 frame/sec for 3 min, with an exposure time of 300 ms, and displayed at 7 frames/sec. In the dendrite (top), bidirectional movement of a carrier containing BACE1 and Rab11b, emerging from a larger stationary structure, which also contains both the proteins, is indicated by a pink arrowhead; a rapidly moving vesicle containing both BACE1 and Rab11 is indicated by a blue arrowhead. In the axon (bottom), blue and pink arrowheads indicate rapidly moving carriers that contain both the proteins. The anterograde direction of movement is toward the right. Scale bar 20 µm. Representative kymographs are shown in Figure 5C. (MOV 1 MB)
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- Axonal BACE1 dynamics and targeting in hippocampal neurons: a role for Rab11 GTPase
Celia G Fernandez
Kulandaivelu S Vetrivel
Vytautas P Bindokas
- BioMed Central
Neu in den Fachgebieten Neurologie und Psychiatrie
Meistgelesene Bücher in der Neurologie & Psychiatrie