title: Synaptotagmins interact with APP and promote Aβ generation.
abstract: BACKGROUND
  Accumulation of the β-amyloid peptide (Aβ) is a major pathological hallmark of Alzheimer's disease (AD). Recent studies have shown that synaptic Aβ toxicity may directly impair synaptic function. However, proteins regulating Aβ generation at the synapse have not been characterized. Here, we sought to identify synaptic proteins that interact with the extracellular domain of APP and regulate Aβ generation.
  
  
  RESULTS
  Affinity purification-coupled mass spectrometry identified members of the Synaptotagmin (Syt) family as novel interacting proteins with the APP ectodomain in mouse brains. Syt-1, -2 and -9 interacted with APP in cells and in mouse brains in vivo. Using a GST pull-down approach, we have further demonstrated that the Syt interaction site lies in the 108 amino acids linker region between the E1 and KPI domains of APP. Stable overexpression of Syt-1 or Syt-9 with APP in CHO and rat pheochromocytoma cells (PC12) significantly increased APP-CTF and sAPP levels, with a 2 to 3 fold increase in secreted Aβ levels in PC12 cells. Moreover, using a stable knockdown approach to reduce the expression of endogenous Syt-1 in PC12 cells, we have observed a ~ 50% reduction in secreted Aβ generation. APP processing also decreased in these cells, shown by lower CTF levels. Lentiviral-mediated knock down of endogenous Syt-1 in mouse primary neurons also led to a significant reduction in both Aβ40 and Aβ42 generation. As secreted sAPPβ levels were significantly reduced in PC12 cells lacking Syt-1 expression, our results suggest that Syt-1 regulates Aβ generation by modulating BACE1-mediated cleavage of APP.
  
  
  CONCLUSIONS
  Altogether, our data identify the synaptic vesicle proteins Syt-1 and 9 as novel APP-interacting proteins that promote Aβ generation and thus may play an important role in the pathogenesis of AD.
authors.affiliation: Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, 02129, USA. gautam.vivek@mgh.harvard.edu.
authors.forename: Vivek
authors.initials: V
authors.lastname: Gautam
authors.affiliation: Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, 02129, USA. cdavanzo@mgh.harvard.edu.
authors.forename: Carla
authors.initials: C
authors.lastname: D'Avanzo
authors.affiliation: MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, 02129, USA. oberezovska@mgh.harvard.edu.
authors.forename: Oksana
authors.initials: O
authors.lastname: Berezovska
authors.affiliation: Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, 02129, USA. tanzi@helix.mgh.harvard.edu.
authors.forename: Rudolph E
authors.initials: RE
authors.lastname: Tanzi
authors.affiliation: Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, 02129, USA. dora_kovacs@hms.harvard.edu.
authors.forename: Dora M
authors.initials: DM
authors.lastname: Kovacs
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000502:physiology; D016229:Amyloid beta-Peptides / Q000096:biosynthesis / Q000378:metabolism; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000502:physiology; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D051379:Mice; D009474:Neurons / Q000378:metabolism; D016716:PC12 Cells; D010446:Peptide Fragments / Q000096:biosynthesis / Q000378:metabolism; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013045:Species Specificity; D050863:Synaptotagmin I / Q000172:deficiency / Q000235:genetics / Q000502:physiology; D050861:Synaptotagmin II / Q000502:physiology; D050857:Synaptotagmins / Q000502:physiology*
chemical_list: D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D010446:Peptide Fragments; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D050863:Synaptotagmin I; D050861:Synaptotagmin II; C494311:Syt1 protein, mouse; C494312:Syt1 protein, rat; C494263:Syt2 protein, mouse; C494309:Syt9 protein, mouse; C483193:Syt9 protein, rat; C078592:amyloid beta-protein (1-40); C075222:amyloid beta-protein (1-42); D050857:Synaptotagmins; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; C509281:Bace1 protein, rat