Alternative experimental design with an applied normalization scheme can improve statistical power in 2D-DIGE experiments.

2D-DIGE experiments are a high-throughput technique for measuring protein abundances based on gel separation. Traditionally three samples are multiplexed per gel: two biological test samples and a third internal standard sample consisting of a pool of all test samples. We demonstrate that the use of an internal standard helps to account for technical variation caused by spatial intensity biases that exist in the gels and propose a novel data-preprocessing technique, a spatial intensity bias removal (SIBR), which can approximate these biases using only the data of biological replicates loaded on the gel. Using this technique, we show that by replacing the internal standard with additional biological replicates, a significant increase in statistical power can be achieved compared to traditional 2D-DIGE designs. This boost in statistical power can be used to reduce the false positive rate for identifying differential protein abundances without compromising sensitivity, or to improve sensitivity without compromising false positive rate. A software implementation of SIBR can be downloaded at http://ibiza.biw.kuleuven.be/SIBR .

Distinct and simultaneously active plasticity mechanisms in mouse hippocampus during different phases of Morris water maze training.

Although the Morris water maze (MWM) is the most frequently used protocol to examine hippocampus-dependent learning in mice, not much is known about the spatio-temporal dynamics of underlying plasticity processes. Here, we studied molecular and cellular hippocampal plasticity mechanisms during early and late phases of spatial learning in the MWM. Quantitative in situ hybridization for the immediate early genes zif268 and Homer1a (H1a) revealed phase-dependent differences in their expression between areas CA1 and CA3. During the initial learning phase, CA1 expression levels of the molecular plasticity marker H1a, but not of the activity reporter gene zif268, were related to task proficiency; whereas no learning-specific changes could be detected in CA3. Simultaneously, the ratio of surface-expressed NMDAR subunits NR2A and NR2B was downregulated as measured by acute slice biotinylation assay, while the total number of surface NMDARs was unaltered. When intrinsic 'somatic' and synaptic plasticity in the CA1-region of hippocampal slices were examined, we found that early learning promotes intrinsic neuronal plasticity as manifested by a reduction of spike frequency adaptation and postburst afterhyperpolarization. At the synaptic level, however, maintenance of long-term potentiation (LTP) in all learning groups was impaired which is most likely due to 'intrinsic' learning-induced LTP which occluded any further electrically induced LTP. Late learning, in contrast, was characterized by re-normalized H1a, NR2A and NR2B expression and neuronal firing, yet a further strengthening of learning-induced LTP. Together, our data support a precisely timed cascade of complex molecular and subcellular transformations occurring from early to late MWM learning.

Protein expression dynamics during postnatal mouse brain development.

We explored differential protein expression profiles in the mouse forebrain at different stages of postnatal development, including 10-day (P10), 30-day (P30), and adult (Ad) mice, by large-scale screening of proteome maps using two-dimensional difference gel electrophoresis. Mass spectrometry analysis resulted in the identification of 251 differentially expressed proteins. Most molecular changes were observed between P10 compared to both P30 and Ad. Computational ingenuity pathway analysis (IPA) confirmed these proteins as crucial molecules in the biological function of nervous system development. Moreover, IPA revealed Semaphorin signaling in neurons and the protein ubiquitination pathway as essential canonical pathways in the mouse forebrain during postnatal development. For these main biological pathways, the transcriptional regulation of the age-dependent expression of selected proteins was validated by means of in situ hybridization. In conclusion, we suggest that proteolysis and neurite outgrowth guidance are key biological processes, particularly during early brain maturation.

Proteomic analysis of global protein expression changes in the endothelin-1 rat model for cerebral ischemia: rescue effect of mild hypothermia.

Mild hypothermia is a promising neuroprotective therapy in stroke management. However, little is known about its effects on the global protein expression patterns in brain regions affected by ischemic stroke. We investigated protein expression changes associated with the neuroprotective effects of hypothermia via a functional proteomics approach through the analysis of the core (striatum) and the penumbra (cortex) after an ischemic insult in rats induced by endothelin-1 (Et-1). Functional outcome, infarct volume and related global protein expression changes were assessed 24h after the insult using two-dimensional difference gel electrophoresis. Mild hypothermia, induced 20 min after endothelin-1 infusion, improved the neurological outcome, reflected by a 36% reduction in infarct volume and a significantly better neurological deficit score. Hypothermia was typically associated with opposite protein expression changes inthe cortex to those induced by stroke under normothermic conditions, but not in the striatum. The main cellular processes rescued by hypothermia and potentially involved in the protection of the cortex are cellular assembly and organization, followed by cell signaling, thereby confirming that hypothermia is neuroprotective through multiple molecular and cellular pathways.

Identification of new regional marker proteins to map mouse brain by 2-D difference gel electrophoresis screening.

To screen for new region-specific protein markers we compared the proteome maps of the primary visual and somatosensory areas V1 and S1 in mouse brain using 2-D difference gel electrophoresis (2-D DIGE). Twenty-three protein spots showed a statistically significant difference in expression level between V1 and S1, with 52% appearing more abundantly in V1. Twenty-six proteins were mass spectrometrically identified in 22 spots. To assess the validity of this list of potential areal markers generated by 2-D DIGE, the effective area-specific distribution profile of creatine kinase brain subtype (CKB), a protein with a clearly higher expression level in S1, was monitored with in situ hybridization. The mRNA expression profile of CKB displayed a clear area-specific distribution, which allowed demarcation of S1 and its topographical borders with neighboring neocortical areas. This proteomic study demonstrates the innovative application of 2-D DIGE and MS to select new regional markers for neuroscience research.

Age-dependent expression of collapsin response mediator proteins (CRMPs) in cat visual cortex.

The functional properties and anatomical organization of the mammalian visual cortex are immature at birth and develop gradually during the first postnatal weeks. There is a 'critical period' where the cortex is plastic and susceptible to changes in visual input. Knowledge of proteins with a high expression during this period has great importance for the understanding of activity-driven maturation of the brain. The collapsin response mediator protein family consists of five cytosolic phosphoproteins (CRMP1-5) that are involved in neuronal differentiation during the development of the nervous system. They have been implicated in axon guidance and growth cone collapse through their action in the signalling pathway of collapsin/semaphorin. We examined the distribution of the CRMPs throughout the visual cortex of kitten and adult cat by in situ hybridization. While CRMP3 could not be detected in cat forebrain, the other CRMPs showed a higher expression in the immature brain compared to the adult state. Western blotting allowed the quantification of the observed age-dependent differences in the expression of CRMP2, 4 and 5. Moreover, for CRMP2 and 5 we observed a number of development-dependent post-translational modifications. We thus conclude that CRMPs might be important during the normal postnatal development of the visual cortex possibly for the fine-tuning of the specific connections in the brain.