Abstract
Proteolytic processing of amyloid precursor protein (APP) generates amyloid-β peptide and has been implicated in the pathogenesis of Alzheimer's disease1. However, the normal function of APP, whether this function is related to the proteolytic processing of APP, and where this processing takes place in neurons in vivo remain unknown. We have previously shown that the axonal transport of APP in neurons is mediated by the direct binding of APP to the kinesin light chain subunit of kinesin-I, a microtubule motor protein2. Here we identify an axonal membrane compartment that contains APP, β-secretase and presenilin-1. The fast anterograde axonal transport of this compartment is mediated by APP and kinesin-I. Proteolytic processing of APP can occur in the compartment in vitro and in vivo in axons. This proteolysis generates amyloid-β and a carboxy-terminal fragment of APP, and liberates kinesin-I from the membrane. These results suggest that APP functions as a kinesin-I membrane receptor, mediating the axonal transport of β-secretase and presenilin-1, and that processing of APP to amyloid-β by secretases can occur in an axonal membrane compartment transported by kinesin-I.
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Acknowledgements
We thank G. Stokin for the Kif5b antibody, S. Brady for the 63-90 Klc antibody, S. Sisodia for providing the Ps1, Aplp1 and Aplp2 antibodies, and Merck for the APP-null mice. We thank D. Cleveland for discussions, and S. Emr for use of the deconvolution microscope. This work is supported by a grant from the National Institutes for Health. L.S.B.G. is an investigator of the Howard Hughes Medical Institute.
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Figure 1
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a, APP, BACE and PS1 accumulate proximal to a sciatic ligation. The sciatic nerves of wild-type mice were ligated unilaterally at the midpoint for 6hrs, sections were processed for immunohistochemistry as described in methods, and stained with antibodies to APP, BACE and PS1. Bar is 100 microns. b, APP, BACE, and PS1 co-localize proximal to a sciatic ligation. Wild-type mouse sciatic nerves were ligated unilaterally at the midpoint for 6hrs, sections were processed for immunohistochemistry as described in methods, co-stained with antibodies to BACE and APP or BACE and PS1. Arrows show co-localization whereas arrowheads point to nerve fibers where this is little co-localization. Bar is 2 microns.
Figure 2
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Immunofluoresence localization of kinesin-I, APP, BACE, and PS1 in mouse sciatic nerves. Cross-sections of mouse sciatic nerves were double-stained with antibodies to KLC1 and axonal marker SMI-31, KLC1 and Schwann marker S100, APP and S100, BACE and S100, or PS1 and S100, and then detected with FITC (green) or Texas-red (red) secondary antibodies; merged images in each panel are shown on the right. Arrows indicate axons, and arrowheads indicate Schwann cells. Comparable staining (not shown) of APP null mice with the APP antibody revealed a much reduced intensity of staining, which is presumably due to cross-reactivity with APLP1 and APLP2. Bar is 10 micron.
Figure 3
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APP, BACE, PS1 and kinesin-I are present in a specific subset of vesicles in mouse sciatic nerves. a, Fractionation profile of vesicles. Mouse sciatic nerves were fractionated as described in methods. 50ug each of the various fractions were analyzed by SDS-PAGE and Western blots with the indicated antibodies. b, Immunoisolation of vesicles from P3. Vesicles were immunoisolated with either no antibody (No Ab) or antibodies to KLC (63-90), KIF5B, APP (C-terminal pAb), or synaptotagmin (SYT). Equal amounts of bound (vesicle IP) and unbound vesicles were analyzed by SDS-PAGE and Western blots with the indicated antibodies. c, Immunoisolation of vesicle proteins in the presence of detergent. Vesicles were prepared and immunoisolated in the presence of 1% NP-40 detergent. The unbound supernatant (supe) and detergent vesicle IP’s were analyzed by SDS-PAGE and Western blots using the indicated antibodies.
Figure 4
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Ab is present mouse sciatic nerves. a, Ab40 and Ab42 immunoreactivity is present in mouse sciatic nerve extracts. Mouse sciatic nerve extracts were prepared from wild-type (wt) and mutant (-/-) APP mice, and analyzed on Tris-tricine gels and Western blots using the three Ab antibodies, Ab40 or Ab42 or 4G8. b, Ab40 and Ab42 antibodies recognize synthetic peptides and can co-immunoprecipitate Ab40 and Ab42 from sciatic nerve extracts. Wild-type mouse sciatic nerves were immunoprecipitated with the antibodies Ab40 or Ab42 or 4G8, and the immunoprecipitates (IP) and supernatant (supe) remaining were analyzed by Tris-tricine gels and Western blots using the indicated antibodies; Ab40 and Ab42 synthetic peptides (Calbiochem) were loaded as controls. c, Detection of Ab fragments in mouse sciatic nerves by SELDI mass spectroscopy. Spectra from SELDI analyses of immunoprecipitates of Ab from sciatic nerves of wild-type (wt) or APP null mutant mice using either Ab40 or Ab42 or 4G8 antibodies (see Table I for sequence assignments).
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Kamal, A., Almenar-Queralt, A., LeBlanc, J. et al. Kinesin-mediated axonal transport of a membrane compartment containing β-secretase and presenilin-1 requires APP. Nature 414, 643–648 (2001). https://doi.org/10.1038/414643a
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DOI: https://doi.org/10.1038/414643a
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