Walsh Rylie B.
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ArticleThe amyloid precursor protein of Alzheimer’s disease clusters at the organelle/microtubule interface on organelles that bind microtubules in an ATP dependent manner(Public Library of Science, 2016-01-27) Stevenson, James W. ; Conaty, Eliza A. ; Walsh, Rylie B. ; Poidomani, Paul J. ; Samoriski, Colin M. ; Scollins, Brianne J. ; DeGiorgis, Joseph A.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.
ArticleAcute increase of α-synuclein inhibits synaptic vesicle recycling evoked during intense stimulation(American Society for Cell Biology, 2014-10-01) Busch, David J. ; Oliphint, Paul A. ; Walsh, Rylie B. ; Banks, Susan M. L. ; Woods, Wendy S. ; George, Julia M. ; Morgan, Jennifer R.Parkinson's disease is associated with multiplication of the α-synuclein gene and abnormal accumulation of the protein. In animal models, α-synuclein overexpression broadly impairs synaptic vesicle trafficking. However, the exact steps of the vesicle trafficking pathway affected by excess α-synuclein and the underlying molecular mechanisms remain unknown. Therefore we acutely increased synuclein levels at a vertebrate synapse and performed a detailed ultrastructural analysis of the effects on presynaptic membranes. At stimulated synapses (20 Hz), excess synuclein caused a loss of synaptic vesicles and an expansion of the plasma membrane, indicating an impairment of vesicle recycling. The N-terminal domain (NTD) of synuclein, which folds into an α-helix, was sufficient to reproduce these effects. In contrast, α-synuclein mutants with a disrupted N-terminal α-helix (T6K and A30P) had little effect under identical conditions. Further supporting this model, another α-synuclein mutant (A53T) with a properly folded NTD phenocopied the synaptic vesicle recycling defects observed with wild type. Interestingly, the vesicle recycling defects were not observed when the stimulation frequency was reduced (5 Hz). Thus excess α-synuclein impairs synaptic vesicle recycling evoked during intense stimulation via a mechanism that requires a properly folded N-terminal α-helix.