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Molecular Autism

, 7:17

First Online: 01 March 2016Received: 19 January 2016Accepted: 20 February 2016


BackgroundFragile 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.

MethodsHere, 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 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.

ResultsKey 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.

ConclusionsOur 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.

KeywordsFragile X syndrome FXS Synaptic transmission Mass spectrometry MS Quantitative live-cell imaging Electron microscopy AbbreviationsAT1A1sodium potassium transporting ATPase alpha 1

AT1A3sodium potassium transporting ATPase alpha 3

AT1B1sodium potassium transporting ATPase beta 1

AT1B2sodium potassium transporting ATPase beta 2

AT2B2plasma membrane calcium-transporting ATPase 2

AZactive zone

BASP1brain acid soluble protein 1


CYFPcytoplasmic FMRP-interacting protein

DCE1glutamate decarboxylase 1

DCE2glutamate decarboxylase 2

EAA1excitatory amino acid transporter 1

FM1-43n-3-triethylammoniumpropyl-4-4-dibutylamino styryl pyridinium dibromide

Fmr1fragile X mental retardation 1 gene

FMRPfragile X mental retardation protein

FXR2fragile x mental retardation syndrome-related 2

FXSfragile X syndrome

GABAgamma-aminobutyric acid

GRIAglutamate receptor

H1Thistone H1t


KCD12BTB POZ domain- containing protein KCTD12


LC-MSliquid-chromatography mass spectrometry

mGluRmetabotropic glutamate receptor


PCpeptide count

Q-TOFquadrupole time-of-flight

RIrelative intensity

ROIsregions of interest

SRM-MSselected reaction monitoring mass spectrometry

SSDHsuccinate semialdehyde dehydrogenase

STPshort-term plasticity

SV2Asynaptic vesicle glycoprotein 2A

SV2Bsynaptic vesicle glycoprotein 2B

VGLU1vesicular glutamate transporter 1

WTwild type

Electronic supplementary materialThe online version of this article doi:10.1186-s13229-016-0080-1 contains supplementary material, which is available to authorized users.

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Autor: Jantine A. C. Broek - Zhanmin Lin - H. Martijn de Gruiter - Heleen van ‘t Spijker - Elize D. Haasdijk - David Cox - Sur


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