The amyloid precursor protein of Alzheimer’s disease clusters at the organelle/microtubule interface on organelles that bind microtubules in an ATP dependent manner

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS One 11 (2016): e0147808, doi:10.1371/journal.pone.0147808.The amyloid precursor protein (APP) is a causal agent in the pathogenesis of Alzheimer’s disease and is a transmembrane protein that associates with membrane-limited organelles. APP has been shown to co-purify through immunoprecipitation with a kinesin light chain suggesting that APP may act as a trailer hitch linking kinesin to its intercellular cargo, however this hypothesis has been challenged. Previously, we identified an mRNA transcript that encodes a squid homolog of human APP770. The human and squid isoforms share 60% sequence identity and 76% sequence similarity within the cytoplasmic domain and share 15 of the final 19 amino acids at the C-terminus establishing this highly conserved domain as a functionally import segment of the APP molecule. Here, we study the distribution of squid APP in extruded axoplasm as well as in a well-characterized reconstituted organelle/microtubule preparation from the squid giant axon in which organelles bind microtubules and move towards the microtubule plus-ends. We find that APP associates with microtubules by confocal microscopy and co-purifies with KI-washed axoplasmic organelles by sucrose density gradient fractionation. By electron microscopy, APP clusters at a single focal point on the surfaces of organelles and localizes to the organelle/microtubule interface. In addition, the association of APP-organelles with microtubules is an ATP dependent process suggesting that the APP-organelles contain a microtubule-based motor protein. Although a direct kinesin/APP association remains controversial, the distribution of APP at the organelle/microtubule interface strongly suggests that APP-organelles have an orientation and that APP like the Alzheimer’s protein tau has a microtubule-based function.Research reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103430

Frequency histogram of APP-gold labeling on free organelles and on organelles attached to microtubules in axoplasmic spreads.

<p>The number of gold particles on organelles attached to microtubules and organelles free from microtubules in the cytoplasm were counted on axoplasmic touch preparations. The number of gold particles on each organelle was plotted against the percentage of total organelles for both attached and unattached categories.</p

APP distribution in reconstituted organelle/microtubule complexes.

<p>Isolated KI-organelles were added to paclitaxel stabilized microtubules at a 1:1 ratio volume:volume and incubated at room temperature for 30 min to form reconstituted organelle/MT complexes. Formvar carbon coated copper EM grids were placed on 30 μl drops of complexes to adhere complexes to the grid surface. Complexes were labeled for APP using an Anti-APP primary antibody and a 12 nm colloidal gold conjugated secondary antibody. Images were taken at 50,000X with a Jeol 200CX transmission electron microscope and an AMT digital camera. Representative photos are presented in the montage. Gold particles appear in large foci on each organelle forming a cluster along the organelle surface, while the adjacent side of the organelle remains free of gold particles.</p

Distribution of APP label in axoplasmic spreads (particles/μm²).

<p>Distribution of APP label in axoplasmic spreads (particles/μm²).</p

Immuno-localization of APP to the surfaces of isolated axoplasmic organelles.

<p>An APP antibody raised against the C-terminal of human APP was used to determine whether APP is directly bound to isolated axoplasmic organelles. Using a colloidal gold secondary antibody, gold particles were found to cluster on the surfaces of axoplasmic organelles through transmission electron microscopy.</p

Immuno-gold labeling of axoplasmic touch preparations.

<p>Total axoplasm was extruded from a freshly dissected squid giant axon onto parafilm and formvar carbon-coated copper EM grids touch to the axoplasm to obtain a thin layer of axoplasmic components on the formvar surface. The sample was labeled with immuno-gold for the presence of APP using a C-terminal APP antibody.</p