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|Title: ||The differential character of protein expression in the developing mouse brain and beyond: a proteomic approach and validation|
|Authors: ||Van de Plas, Babs|
Van Hove, Inge
Van den Bergh, Gert
Arckens, Lut #
|Issue Date: ||May-2006 |
|Conference: ||BSCDB Spring Meeting: Molecular & Cellular Basis of Neuroconnectivity location:Leuven, Belgium date:May 4-6, 2006|
|Abstract: ||Sensory systems in the mammalian brain have a remarkable capacity to adapt to input changes by modifying neuronal connectivity within a ‘critical period’ during development.
This goal-directed strengthening, remodelling and elimination of synapses ultimately leads to the neuronal circuitry specific for the adult brain. In adulthood, this connectional plasticity appears greatly reduced. Detailed molecular mechanisms underlying these age-dependent differences in brain plasticity are not known yet. We are studying the (dis)similarities among molecular mechanisms of brain plasticity in young and adult mice by means of fluorescent two-dimensional differential gel electrophoresis (2D-DIGE) and mass spectrometry (MS).
As developmental plasticity model, paired samples were analysed by 2D-DIGE. Protein samples from 2 whole forebrain extracts of 10-day-old pups (P10) and adult mice were
compared after separate labelling with Cy3 or Cy5 and consecutive pairing. Only spots switching colour in forward and reverse labelling were considered to contain differentially expressed proteins. The spots visually identified as differential were statistically analysed using Phoretics 2D spot analysis software. These proteins were identified by means of trypsin digestion and both MALDI (Reflex IV, Bruker), MALDI-TOF/TOF (Ultraflex II, Bruker) and nano-LC-MS/MS (Ultimate nano LC, LCPackings; on-line with a Q-TOF, Micromass) analyses. Both PMF and MS/MS sequence data were searched against protein sequence databases using the Mascot search engine (Matrixscience).
Approximately 140 significantly differential spots were identified. Apart from metabolic enzymes, the list contained proteins involved in the outgrowth of axons and in growth cone collapse. Moreover they are involved in neuronal differentiation during the development of the nervous system. Proteins selected for validation via Western blotting and immunocytochemistry were dynamin-1, 5 times more abundant in adult brain, and involved in endocytosis and maintenance of the mature neuronal structure, fascin with a 5 times higher expression in pup and involved in the dendritic development of neurons,
and NSE which was 6 times more abundant in adult compared to pup.
By means of Western blotting we compared the expression levels of the 3 proteins of interest between P1, P5, P10, P30, adult (A) and old (O) mice (7 months). Fascin
showed the highest expression level at P1, which decreased during development to reach the lowest level in old mice, whereas the NSE expression showed an upregulation
during development. Dyn-1 was upregulated at P30, A and O, and showed lower but comparable levels of expression in P1, P5, and P10.
The distribution pattern of fascin was studied with immunocytochemistry on frontal sections of the entire mouse brain (P10 & A). At P10, ICC resulted in neuropil staining.
In adult brain, the fascin antibody stained neurons with an immunopositive perikaryon and proximal part of the apical dendrite, but no immunostaining was seen in the nucleus.
The laminar staining pattern differed between adjacent areas, which allows us to distinguish several cortical regions.
The differential proteins found by comparing the protein expression profiles of P10 and adult mouse brain were successfully validated with 2 different techniques. The proteomic approach is therefore an excellent tool to study the molecular mechanisms of brain plasticity and development.
|Publication status: ||published|
|KU Leuven publication type: ||AMa|
|Appears in Collections:||Animal Physiology and Neurobiology Section - miscellaneous|
Research Group Neuroplasticity and Neuroproteomics (-)
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