title: Synaptic vesicle dynamic changes in a model of fragile X.
abstract: BACKGROUND
  Fragile X syndrome (FXS) is a single-gene disorder that is the most common heritable cause of intellectual disability and the most frequent monogenic cause of autism spectrum disorders (ASD). FXS is caused by an expansion of trinucleotide repeats in the promoter region of the fragile X mental retardation gene (Fmr1). This leads to a lack of fragile X mental retardation protein (FMRP), which regulates translation of a wide range of messenger RNAs (mRNAs). The extent of expression level alterations of synaptic proteins affected by FMRP loss and their consequences on synaptic dynamics in FXS has not been fully investigated.
  
  
  METHODS
  Here, we used an Fmr1 knockout (KO) mouse model to investigate the molecular mechanisms underlying FXS by monitoring protein expression changes using shotgun label-free liquid-chromatography mass spectrometry (LC-MS(E)) in brain tissue and synaptosome fractions. FXS-associated candidate proteins were validated using selected reaction monitoring (SRM) in synaptosome fractions for targeted protein quantification. Furthermore, functional alterations in synaptic release and dynamics were evaluated using live-cell imaging, and interpretation of synaptic dynamics differences was investigated using electron microscopy.
  
  
  RESULTS
  Key findings relate to altered levels of proteins involved in GABA-signalling, especially in the cerebellum. Further exploration using microscopy studies found reduced synaptic vesicle unloading of hippocampal neurons and increased vesicle unloading in cerebellar neurons, which suggests a general decrease of synaptic transmission.
  
  
  CONCLUSIONS
  Our findings suggest that FMRP is a regulator of synaptic vesicle dynamics, which supports the role of FMRP in presynaptic functions. Taken together, these studies provide novel insights into the molecular changes associated with FXS.
authors.affiliation: Cambridge Centre for Neuropsychiatric Research, Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, UK.
authors.forename: Jantine A C
authors.initials: JAC
authors.lastname: Broek
authors.affiliation: Department of Neurosciences, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Zhanmin
authors.initials: Z
authors.lastname: Lin
authors.affiliation: Erasmus Optical Imaging Center, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: H Martijn
authors.initials: HM
authors.lastname: de Gruiter
authors.affiliation: Cambridge Centre for Neuropsychiatric Research, Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, UK.
authors.forename: Heleen
authors.initials: H
authors.lastname: van 't Spijker
authors.affiliation: Department of Neurosciences, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Elize D
authors.initials: ED
authors.lastname: Haasdijk
authors.affiliation: Cambridge Centre for Neuropsychiatric Research, Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, UK.
authors.forename: David
authors.initials: D
authors.lastname: Cox
authors.affiliation: Cambridge Centre for Neuropsychiatric Research, Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, UK.
authors.forename: Sureyya
authors.initials: S
authors.lastname: Ozcan
authors.affiliation: Erasmus Optical Imaging Center, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Gert W A
authors.initials: GWA
authors.lastname: van Cappellen
authors.affiliation: Erasmus Optical Imaging Center, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Adriaan B
authors.initials: AB
authors.lastname: Houtsmuller
authors.affiliation: Department of Clinical Genetics, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Rob
authors.initials: R
authors.lastname: Willemsen
authors.affiliation: Department of Neurosciences, Erasmus MC, Rotterdam, The Netherlands.|Netherlands Institute for Neurosciences, Royal Academy for Arts and Sciences, Amsterdam, The Netherlands.
authors.forename: Chris I
authors.initials: CI
authors.lastname: de Zeeuw
authors.affiliation: Cambridge Centre for Neuropsychiatric Research, Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, UK.|Department of Neurosciences, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Sabine
authors.initials: S
authors.lastname: Bahn
mesh_terms: D000818:Animals; D020296:Animals, Congenic; D002478:Cells, Cultured; D002531:Cerebellum / Q000473:pathology / Q000503:physiopathology; D005456:Fluorescent Dyes; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics / Q000502:physiology*; D005600:Fragile X Syndrome / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology*; D006624:Hippocampus / Q000473:pathology / Q000503:physiopathology; D000069416:Intravital Microscopy; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008818:Mice, Neurologic Mutants; D008854:Microscopy, Electron; D023421:Models, Animal; D009419:Nerve Tissue Proteins / Q000032:analysis; D017729:Presynaptic Terminals / Q000378:metabolism; D020543:Proteome; D011689:Purkinje Cells / Q000502:physiology / Q000648:ultrastructure; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D015398:Signal Transduction; D009435:Synaptic Transmission; D013572:Synaptic Vesicles / Q000378:metabolism*; D013574:Synaptosomes / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D051860:Fragile X Messenger Ribonucleoprotein 1; D009419:Nerve Tissue Proteins; D020543:Proteome; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; C073804:FM1 43; C497684:Fmr1 protein, mouse
keywords: Electron microscopy; Fragile X syndrome (FXS); Mass spectrometry (MS); Quantitative live-cell imaging; Synaptic transmission