Gert Van den Bergh

Proteomics in Drosophila melanogaster: first 2D database of larval hemolymph proteins

A proteomic approach was used for the identification of larval hemolymph proteins of Drosophila melanogaster. We report the initial establishment of a two-dimensional gel electrophoresis reference map for hemolymph proteins of third instar larvae of D. melanogaster. We used immobilized pH gradients of pH 4-7 (linear) and a 12-14% linear gradient polyacrylamide gel. The protein spots were silver-stained and analyzed by nanoLC-Q-Tof MS/MS (on-line nanoscale liquid chromatography quadrupole time of flight tandem mass spectrometry) or by Matrix assisted laser desorption time of flight MS (MALDI-TOF MS). Querying the SWISSPROT database with the mass spectrometric data yielded the identity of the proteins in the spots. The presented proteome map lists those protein spots identified to date. This map will be updated continuously and will serve as a reference database for investigators, studying changes at the protein level in different physiological conditions.

Proteomics Analysis of Cytokine-induced Dysfunction and Death in Insulin-producing INS-1E Cells New Insights into the Pathways Involved

Cytokines released by islet-infiltrating immune cells play a crucial role in β-cell dysfunction and apoptotic cell death in the pathogenesis of type 1 diabetes and after islet transplantation. RNA studies revealed complex pathways of genes being activated or suppressed during this β-cell attack. The aim of the present study was to analyze protein changes in insulin-producing INS-1E cells exposed to inflammatory cytokines in vitro using two-dimensional DIGE. Within two different pH ranges we observed 2214 ± 164 (pH 4–7) and 1641 ± 73 (pH 6–9) spots. Analysis at three different time points (1, 4, and 24 h of cytokine exposure) revealed that the major changes were taking place only after 24 h. At this time point 158 proteins were altered in expression (4.1%, n = 4, p ≤ 0.01) by a combination of interleukin-1β and interferon-γ, whereas only 42 and 23 proteins were altered by either of the cytokines alone, giving rise to 199 distinct differentially expressed spots. Identification of 141 of these by MALDI-TOF/TOF revealed proteins playing a role in insulin secretion, cytoskeleton organization, and protein and RNA metabolism as well as proteins associated with endoplasmic reticulum and oxidative stress/defense. We investigated the interactions of these proteins and discovered a significant interaction network (p < 1.27e−05) containing 42 of the identified proteins. This network analysis suggests that proteins of different pathways act coordinately in a β-cell dysfunction/apoptotic β-cell death interactome. In addition the data suggest a central role for chaperones and proteins playing a role in RNA metabolism. As many of these identified proteins are regulated at the protein level or undergo post-translational modifications, a proteomics approach, as performed in this study, is required to provide adequate insight into the mechanisms leading to β-cell dysfunction and apoptosis. The present findings may open new avenues for the understanding and prevention of β-cell loss in type 1 diabetes.

Fluorescent two-dimensional difference gel electrophoresis and mass spectrometry identify age-related protein expression differences for the primary visual cortex of kitten and adult cat

The recent introduction of fluorescent two‐dimensional difference gel electrophoresis, combined with mass spectrometry, has greatly simplified the analysis and identification of differentially expressed proteins by eliminating intergel variability. In this report, we describe the successful application of this functional proteomics approach to compare protein expression levels in visual cortical area 17 of adult cats and 30‐day‐old kittens, in order to identify proteins expressed in an age‐related fashion. We identified 16 proteins that were more abundantly expressed in kitten striate cortex and 12 proteins with a pronounced expression in adult cat area 17. Among those isolated from kitten area 17 were proteins related to axon growth and growth cone guidance and to the formation of cytoskeletal filaments. Glial fibrillary acidic protein, as identified in adult cat area 17, has been implicated previously in the termination of the critical period for cortical plasticity in kittens. In situ hybridization experiments for two of the identified proteins, glial fibrillary acidic protein and collapsin response mediator protein 5, confirmed and extended their differential expression to the mRNA level. Our findings show that two‐dimensional difference gel electrophoresis combined with mass spectrometry is a powerful approach that permits the identification of small protein expression differences correlated to different physiological conditions.

Recent advances in 2D electrophoresis: an array of possibilities

2D electrophoresis is currently the most widespread technique used for performing functional proteomics (i.e., the large-scale analysis of alterations in protein expression levels). Nevertheless, several limitations inherent to this technology have restricted the full potential of this protein differential display methodology for years. This has even led to the abandonment of 2D electrophoresis by several groups that switched to performing gel-free functional proteomics analyses based on liquid chromatography and mass spectrometry. Meanwhile, important recent advances in 2D electrophoresis, such as the introduction of fluorescent 2D difference gel electrophoresis and numerous protein prefractionation techniques, have thoroughly modernized 2D electrophoresis, making it again one of the preferred methods for the analysis of protein expression differences in many laboratories.

Receptive-field properties of V1 and V2 neurons in mice and macaque monkeys.

We report the results of extracellular single‐unit recording experiments where we quantitatively analyzed the receptive‐field (RF) properties of neurons in V1 and an adjacent extrastriate visual area (V2L) of anesthetized mice with emphasis on the RF center‐surround organization. We compared the results with the RF center‐surround organization of V1 and V2 neurons in macaque monkeys. If species differences in spatial scale are taken into consideration, mouse V1 and V2L neurons had remarkably fine stimulus selectivity, and the majority of response properties in V2L were not different from those in V1. The RF center‐surround organization of mouse V1 neurons was qualitatively similar to that for macaque monkeys (i.e., the RF center is surrounded by extended suppressive regions). However, unlike in monkey V2, a significant proportion of cortical neurons, largely complex cells in V2L, did not exhibit quantifiable RF surround suppression. Simple cells had smaller RF centers than complex cells, and the prevalence and strength of surround suppression were greater in simple cells than in complex cells. These findings, particularly on the RF center‐surround organization of visual cortical neurons, give new insights into the principles governing cortical circuits in the mouse visual cortex and should provide further impetus for the use of mice in studies on the genetic and molecular basis of RF development and synaptic plasticity. J. Comp. Neurol. 518:2051–2070, 2010.

Differential expression of brain proteins in glycogen synthase kinase-3 transgenic mice: a proteomics point of view.

One of the landmarks of Alzheimer’s disease are neurofibrillary tangles (NFT) in the brain. NFT mainly consist of a hyperphosphorylated form of the protein tau, which is responsible for stabilisation of the neuronal cytoskeleton by microtubule binding and is unable to function properly in its hyperphosphorylated form. Glycogen synthase kinase‐3β (GSK3β) is able to phosphorylate tau in a cellular context which could play a role in the formation of these NFT. In order to learn more about the effect of GSK‐3β in the brain, two‐dimensional electrophoresis patterns of cerebrum extracts of GSK3β[S9A] transgenic mice and wild type mice were compared quantitatively. Fifty‐one spots were identified as being different in integrated intensity by at least a factor 1.5. The spots were subsequently identified by mass spectrometry. Identification of several proteins linked to signal transduction pathways in which GSK3β plays a role, indicates that our population of identified proteins includes some down stream proteins of GSK3β. This study may contribute to filling the gaps between GSK3β, its substrates and finally the phosphorylation of tau.

Differential display implicates cyclophilin A in adult cortical plasticity

Removal of retinal input from a restricted region of adult cat visual cortex leads to a substantial reorganization of the retinotopy within the sensory‐deprived cortical zone. Little is known about the molecular mechanisms underlying this reorganization. We used differential mRNA display (DDRT‐PCR) to compare gene expression patterns between normal control and reorganizing visual cortex (area 17–18), 3 days after induction of central retinal lesions. Systematic screening revealed a decrease in the mRNA encoding cyclophilin A in lesion‐affected cortex. In situ hybridization and competitive PCR confirmed the decreased cyclophilin A mRNA levels in reorganizing cortex and extended this finding to longer postlesion survival times as well. Western blotting and immunocytochemistry extended these data to the protein level. In situ hybridization and immunocytochemistry further demonstrated that cyclophilin A mRNA and protein are present in neurons. To exclude the possibility that differences in neuronal activity per se can induce alterations in cyclophilin A mRNA and protein expression, we analyzed cyclophilin A expression in the dorsal lateral geniculate nucleus (dLGN) of retinally lesioned cats and in area 17 and the dLGN of isolated hemisphere cats. In these control experiments cyclophilin A mRNA and protein were distributed as in normal control subjects indicating that the decreased cyclophilin A levels, as observed in sensory‐deprived area 17 of retinal lesion cats, are not merely a reflection of changes in neuronal activity. Instead our findings identify cyclophilin A, classically considered a housekeeping gene, as a gene with a brain plasticity‐related expression in the central nervous system.

Behavioural alterations relevant to developmental brain disorders in mice with neonatally induced ventral hippocampal lesions

Neonatal lesioning of the ventral hippocampus (vHc) in rats has served as a useful heuristic animal model to elucidate neurodevelopmental mechanisms of schizophrenia (SCZ). In the current study we have established that this procedure can be applied to model SCZ symptomatology in mice. Neonatal mice (postnatal day 6) were anaesthetised by hypothermia and electrolytic lesions of the vHc were induced. We observed locomotor hyperactivity at prepubertal and adult age and hypersensitivity to amphetamine. Furthermore, working memory deficits were observed in Y-maze (spontaneous alternation) and T-maze (exploration of a novel arm) test protocols. Decreased anxious behaviour in the elevated plus maze and increased sociability were also observed. These changes were dependent on lesion size. No differences were observed in prepulse inhibition of the startle reflex, latent inhibition, spatial memory (Morris water maze), problem solving capacities (syringe puzzle) and ability to discriminate between different unfamiliar mice. The presented findings might further help to identify neurobiological mechanisms of neurodevelopmental disorders.

Age- and experience-dependent expression of Dynamin I and Synaptotagmin I in cat visual system

Dynamin I (Dyn I) and Synaptotagmin I (Syt I) are essential for endocytosis‐exocytosis processes, thus for neurotransmission. Despite their related function at presynaptic terminals, Dyn I and Syt I displayed opposite expression patterns during visual cortex maturation in the cat. Dyn I was more abundantly expressed in adults, while Syt I exhibited higher levels in kittens of postnatal day 30 (P30). In area 17 this developmental difference was most obvious in layers II/III. Layer VI displayed a strong hybridization signal for both molecules, independent of age. In addition, Syt I levels were higher in posterior compared to anterior area 17 in adult subjects. Moreover, in higher‐order visual areas Syt I was unevenly distributed over the cortical layers, thereby setting clear areal boundaries in mature cortex. In contrast, Dyn I was rather homogeneously distributed over extrastriate areas at both ages. Both molecules thus demonstrated a widespread but different distribution and an opposite temporal expression pattern during visual system development. Notably, monocular deprivation during the critical period of ocular dominance plasticity significantly decreased Syt I expression levels in area 17 ipsilateral to the deprived eye, while no effect was observed on Dyn I expression. We therefore conclude that visual experience induces changes in Syt I expression that may reflect changes in constitutive exocytosis involved in postnatal structural refinements of the visual cortex. On the other hand, the spatial and temporal expression patterns of Dyn I correlate with the establishment and maintenance of the mature neuronal structure rather than neurite remodeling. J. Comp. Neurol. 504:254–264, 2007.

Recovery from Retinal Lesions: Molecular Plasticity Mechanisms in Visual Cortex Far beyond the Deprived Zone

In cats with central retinal lesions, deprivation of the lesion projection zone (LPZ) in primary visual cortex (area 17) induces remapping of the cortical topography. Recovery of visually driven cortical activity in the LPZ involves distinct changes in protein expression. Recent observations, about molecular activity changes throughout area 17, challenge the view that its remote nondeprived parts would not be involved in this recovery process. We here investigated the dynamics of the protein expression pattern of remote nondeprived area 17 triggered by central retinal lesions to explore to what extent far peripheral area 17 would contribute to the topographic map reorganization inside the visual cortex. Using functional proteomics, we identified 40 proteins specifically differentially expressed between far peripheral area 17 of control and experimental animals 14 days to 8 months postlesion. Our results demonstrate that far peripheral area 17 is implicated in the functional adaptation to the visual deprivation, involving a meshwork of interacting proteins, operating in diverse pathways. In particular, endocytosis/exocytosis processes appeared to be essential via their intimate correlation with long-term potentiation and neurite outgrowth mechanisms.