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P1–368: The identification neuronal genes and proteins responsive to insulin and WNT signalling
Author(s) -
Killick Richard,
Hooper Claudie,
Cocks Graham,
Lovestone Simon
Publication year - 2006
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2006.05.746
Subject(s) - wnt signaling pathway , transcriptome , biology , gsk 3 , proteome , microbiology and biotechnology , kinase , insulin receptor , insulin , xenopus , dna microarray , gene , gene expression , signal transduction , biochemistry , endocrinology , insulin resistance
of these vesicles is severely impaired in APP deficient mice (Kamal et al., 2001). Intriguingly, recent data reported a defect in synapse morphology accompanied by reduction of synaptic vesicle density in mice deficient for APP and its homologue APLP2 (Yang et al., 2005. Neurosci. lett. 384:6671). Together these findings raise the possibility that APP-dependent axonally-transported vesicles could play an important role in regulating the establishment and maintenance of proper synaptic structures. Objective: To test further the nature of APP axonally-transported vesicles and to explore their functional role we are characterizing their protein content and morphology by mass spectrometry and electron microscopy. Methods and Results: To this end, we developed a biochemical approach to purify enriched populations of APP vesicles using subcellular fractionation from mouse brain cortices. We used sucrose density gradients to separate distinct populations of APP vesicles based on their density properties followed by affinity purification using specific APP antibodies attached to magnetic beads. Additionally, we performed Western blot analysis to monitor our fractionation steps and to test for the presence or absence of the APP secretase presenilin in each vesicle population. Parallel silver staining of the gels resulted in a specific protein band pattern when compared to control samples. Finally, we performed electron microscopy and mass spectrometry analysis to confirm the existence of morphologically distinct APP vesicles and to determine their specific protein content. Conclusion: We believe that the isolation and the biochemical and morphological characterization of distinct populations of axonally-transported APP-containing vesicles will shed new light on the normal function of APP in neuronal cells and, ultimately, improve our understanding of the development of Alzheimer’s disease.