Early dysregulation of hippocampal proteins in transgenic rats with Alzheimer's disease-linked mutations in amyloid precursor protein and presenilin 1.

The response of the hippocampal proteome to expression of mutant proteins present in familial forms of Alzheimer's disease (AD) was studied using transgenic rats. These animals carry both the amyloid precursor protein Swedish and 717 mutation (APP(SW+717)) as well as the presenilin 1 Finnish mutation (PS1(FINN)). This transgenic rat model displays intracellular amyloid beta (Abeta) in neurons of the neocortex and the hippocampus (CA2 and CA3). The hippocampus was selected as it is one of the first brain regions affected in AD and is involved in the processing of short-term memory and spatial memory. Applying a proteomic approach, we demonstrate that the expression of APP(SW+717) and PS1(FINN) transgenes causes changes in expression of hippocampal proteins, some of which have been previously linked to learning and memory formation. The protein alterations documented here occur in the absence of plaque formation and prior to the onset of cognitive deficits later observed in these transgenic rats. This indicates that molecular changes take place in the hippocampal neurons in response to expression of mutant proteins APP(SW+717) and PS1(FINN), which precede the occurrence of overt extracellular accumulation of extracellular amyloid. The implications of these findings on our understanding of the early stages of AD are discussed.

Proteomic approaches in brain research and neuropharmacology.

Numerous applications of genomic technologies have enabled the assembly of unprecedented inventories of genes, expressed in cells under specific physiological and pathophysiological conditions. Complementing the valuable information generated through functional genomics with the integrative knowledge of protein expression and function should enable the development of more efficient diagnostic tools and therapeutic agents. Proteomic analyses are particularly suitable to elucidate posttranslational modifications, expression levels and protein-protein interactions of thousands of proteins at a time. In this review, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) investigations of brain tissues in neurodegenerative diseases such as Alzheimer's disease, Down syndrome and schizophrenia, and the construction of 2D-PAGE proteome maps of the brain are discussed. The role of the Human Proteome Organization (HUPO) as an international coordinating organization for proteomic efforts, as well as challenges for proteomic technologies and data analysis are also addressed. It is expected that the use of proteomic strategies will have significant impact in neuropharmacology over the coming decade.

Time-dependent changes in the expression of the MEF2 transcription factor family during topographic map reorganization in mammalian visual cortex.

Removal of retinal input from a restricted region of adult mammalian visual cortex leads to a substantial reorganization of the retinotopy within the lesion projection zone (LPZ) of primary visual cortex (area 17). Little is known about the molecular mechanisms underlying such cortical plasticity. We investigated whether small but homonymous central retinal lesions induced differences in gene expression patterns between central area 17, the LPZ, vs. peripheral area 17 of the adult cat. Systematic differential mRNA display screening revealed higher levels for the mRNA encoding the transcription factor MEF2A in the LPZ. Semi-quantitative PCR confirmed this dependency of mef2A mRNA expression on visual eccentricity in area 17 of animals with retinal lesions in contrast to normal animals. Western blotting experiments extended these data to the protein level and to two other members of the MEF2 transcription factor family, i.e. MEF2C and MEF2D. Quantitative analysis of the Western blotting experiments further revealed a post-lesion survival time-dependent change in expression for all three MEF2 family members. The lesion effect was maximal at 3 days and 1 month post-lesion, but only minor at 2 weeks post-lesion. Interestingly, complete removal of retinal input from area 17 by surgery did not significantly alter the expression of the MEF2 transcription factors, excluding a definite correlation between neuronal activity and MEF2A expression levels. MEF2A immunocytochemistry confirmed both qualitatively and quantitatively the Western blotting observations in all animal models. Together, our findings identified a brain plasticity-related expression pattern for the MEF2 transcription factor family in adult mammalian neocortex.

Ovoinhibitor in the chicken bursa of Fabricius: identification, isolation, and localization.

A monoclonal antibody (Mab) developed against a partially purified bursal protein extract was found to bind specifically to a single cell type in the cortico-medullary border region of the chicken bursa of Fabricius. These cells were microscopically similar to the bursal secretory dendritic-like cells. A product with an apparent molecular weight of approximately 56 kDa on SDS-polyacrylamide gel electrophoresis was immunopurified from bursal extracts by utilizing this Mab. This product was subjected to peptide digestion and protein sequencing. The two resulting sequences perfectly matched the known sequence of chicken ovoinhibitor. Gene-specific polymerase chain reaction (PCR) primers were designed for the ovoinhibitor, RNA was purified from chicken bursae, and reverse transcription/PCR was performed. Two amplicons with the expected size for ovoinhibitor mRNA were obtained. These data suggest that the gene for ovoinhibitor is expressed in the bursa of Fabricius, and that the bursal secretory dendritic-like cells may be a previously unreported source of ovoinhibitor.

Heavy metal exposure affects the humoral immune response in a free-living small songbird, the great tit (Parus major).

Although many studies have investigated possible effects of heavy metal contamination on components of the immune system in captive birds, studies on the effects of chronic exposure to heavy metals on the immune system of free-living birds are rare. Therefore we studied the effect of heavy metal exposure on the humoral immune responsiveness in free-living great tit (Parus major) populations from four study sites along a pollution gradient near a metallurgic smelter. Although there were no differences in body condition or hematocrit values among great tits from the four study sites, the heavy metal exposure appeared to affect an individual's humoral immune responsiveness, as measured by antibody titers to sheep red blood cells. Great tits from the study site farthest away from the smelter complex had a significantly higher immune responsiveness than birds from the two areas closest to the metallurgic smelter. Further work is now necessary to establish a causal association between heavy metal contamination and immunosuppression.

Identification of 26RFa, a hypothalamic neuropeptide of the RFamide peptide family with orexigenic activity.

A neuropeptide was isolated from a frog brain extract by HPLC purification and characterized by mass spectrometry. This 26-aa neuropeptide, which belongs to the RFamide peptide family, was designated 26RFa, and its primary structure was established as VGTALGSLAEELNGYNRKKGGFSFRF-NH2. Research in databases revealed the presence of sequences homologous to frog 26RFa in the human genome and in rat ESTs. On the basis of this sequence information, the cDNAs encoding the human and rat 26RFa precursors were cloned. The two preproteins show a similar organization, with the 26RFa sequence located in the C-terminal region of the precursor. Human preprotein (prepro)-26RFa encodes an additional putative RFamide peptide that is not found in the rat precursor. The primary structures of human, rat, and frog 26RFa exhibit approximately 80% identity, and the C-terminal octapeptide has been fully conserved from amphibians to mammals. In situ hybridization histochemistry revealed that, in the rat brain, the 26RFa gene is exclusively expressed in the ventromedial hypothalamic nucleus and in the lateral hypothalamic area. 26RFa induced a dose-dependent stimulation in cAMP production by rat pituitary cells in vitro and markedly increased food intake in mice. The conservation of the primary structure of 26RFa during vertebrate evolution, the discrete localization of the mRNA encoding its precursor in hypothalamic nuclei involved in the control of feeding behavior, and the observation that 26RFa possesses orexigenic properties indicate that this neuropeptide may play important biological functions.

Distribution and morphological characterization of phosphate-activated glutaminase-immunoreactive neurons in cat visual cortex.

Phosphate-activated glutaminase (PAG) is the major enzyme involved in the synthesis of the excitatory neurotransmitter glutamate in cortical neurons of the mammalian cerebral cortex. In this study, the distribution and morphology of glutamatergic neurons in cat visual cortex was monitored through immunocytochemistry for PAG. We first determined the specificity of the anti-rat brain PAG polyclonal antibody for cat brain PAG. We then examined the laminar expression profile and the phenotype of PAG-immunopositive neurons in area 17 and 18 of cat visual cortex. Neuronal cell bodies with moderate to intense PAG immunoreactivity were distributed throughout cortical layers II-VI and near the border with the white matter of both visual areas. The largest and most intensely labeled cells were mainly restricted to cortical layers III and V. Careful examination of the typology of PAG-immunoreactive cells based on the size and shape of the cell body together with the dendritic pattern indicated that the vast majority of these cells were pyramidal neurons. However, PAG immunoreactivity was also observed in a paucity of non-pyramidal neurons in cortical layers IV and VI of both visual areas. To further characterize the PAG-immunopositive neuronal population we performed double-stainings between PAG and three calcium-binding proteins, parvalbumin, calbindin and calretinin, to determine whether GABAergic non-pyramidal cells can express PAG, and neurofilament protein, a marker for a subset of pyramidal neurons in mammalian neocortex. We here present PAG as a neurochemical marker to map excitatory cortical neurons that use the amino acid glutamate as their neurotransmitter in cat visual cortex.

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.

Extracellular GABA concentrations in area 17 of cat visual cortex during topographic map reorganization following binocular central retinal lesioning.

Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system of mammals, plays an important role in cortical reorganization following sensory deprivation, by regulating the level of cortical inhibition and gating changes in receptive field size and synaptic efficacy. In cats it has been shown that 2 weeks after the induction of binocular retinal lesions, GABAergic inhibition, as determined by immunocytochemistry, is decreased in the deafferented region of area 17, whereas 3 months post-lesion, normal GABAergic control is restored within the cortical scotoma. In this study we used in vivo microdialysis to investigate the extracellular GABA concentrations 1-2 months post-lesion, in the sensory-deprived and remote, non-deprived region of area 17. Data were collected at those sample times and sites for which the extracellular glutamate concentrations had been determined in a previous investigation to elucidate the role of this excitatory neurotransmitter in cortical reorganization. As for glutamate, we observed significantly increased extracellular GABA concentrations in non-deprived area 17, whereas in deafferented area 17, extracellular GABA concentrations were comparable to those observed in normal, control subjects. These data suggest that 1-2 months post-lesion the deafferented cortex behaves like normal visual cortex, in contrast to remote, non-deprived cortex. Notwithstanding the increase in extracellular GABA concentration of 134%, the parallel increase in glutamate concentration of 269% could give rise to a net increase in excitability in remote area 17. We therefore suggest that LTP-like mechanisms, and thereby cortical reorganization, might still be facilitated, while possible excessive hyperexcitability is balanced by the moderately increased GABAergic control.

Reversed-phase high-performance liquid chromatography prefractionation prior to two-dimensional difference gel electrophoresis and mass spectrometry identifies new differentially expressed proteins between striate cortex of kitten and adult cat.

Two-dimensional difference gel electrophoresis (2-D DIGE), in combination with mass spectrometry, is a highly effective method for the rapid and reproducible detection of differentially expressed proteins. This approach, however, has the unfortunate drawback that it preferentially displays rather abundantly expressed proteins. Nevertheless, comparison of the protein expression levels of the striate cortex of adult cats and 30-day-old kittens, resulted in the identification of several proteins related to postnatal brain development and possibly age-dependent plasticity as well (Van den Bergh et al., J. Neurochem. 2003, in press). The goal of the present study was the selective enrichment and identification of less abundant proteins within the same paradigm. Hereto, we performed a reversed-phase chromatography prefractionation of our tissue lysate to separate the proteins in four fractions based on their hydrophobicity prior to 2-D DIGE analysis. This approach not only confirmed the differential expression levels of a number of proteins from the previous study, but also identified three additional proteins preferentially expressed in kitten visual cortex and five additional proteins with higher expression levels in adult cat visual cortex. These spots were not visible on the total tissue lysate protein maps, indicating that the high-performance liquid chromatography (HPLC) prefractionation enabled us to visualize additional, less abundant proteins.

Molecular cloning and differential expression of the cat immediate early gene c-fos.

Recently, the effect of binocular central retinal lesions on the expression of immediate early genes in the visual system of adult cats was demonstrated using in situ hybridization and immunocytochemistry. The present study was undertaken to quantify cat c-fos mRNA expression differences in the cat primary visual cortex after sensory deafferentation. Prior to quantification, DNA fragments obtained using reverse transcription-polymerase chain reaction (RT-PCR) in combination with rapid amplification of complementary DNA ends (RACE) were cloned and sequenced. This provided us with the necessary sequence(1) information to prepare cat-specific c-fos primers for the development of a new quantitative RT-PCR assay. We optimized a reverse transcription-competitive polymerase chain reaction (RT-cPCR) method with a heterologous DNA fragment (competitor) as external standard to quantify relative amounts of cat c-fos mRNA expression levels. Internal standardization was accomplished by quantifying, in a parallel RT-cPCR, a well-characterized housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This cat-specific RT-cPCR assay allowed us to measure c-fos mRNA expression levels in central and peripheral regions of primary visual cortex in normal and retinal lesion cats.

Glutamate levels and transport in cat (Felis catus) area 17 during cortical reorganization following binocular retinal lesions.

Glutamate is known to play a crucial role in the topographic reorganization of visual cortex after the induction of binocular central retinal lesions. In this study we investigated the possible involvement of the glial high-affinity Na+/K+-dependent glutamate transporters in cortical plasticity using western blotting and intracortical microdialysis. Basal extracellular glutamate levels and the re-uptake activity for glutamate have been determined by comparing the extracellular glutamate concentration before and during the blockage of glutamate removal from the synaptic cleft with the potent transporter inhibitor l-trans-pyrrolidine-3,4-dicarboxylic acid. In cats with central retinal lesions we observed increased basal extracellular glutamate concentrations together with a decreased re-uptake activity in non-deprived, peripheral area 17, compared with the sensory-deprived, central cortex of the same animal as well as the topographically matching regions of area 17 in normal subjects. Western blotting experiments revealed a parallel decrease in the expression level of the glial glutamate transporter proteins GLT-1 and GLAST in non-deprived cortex compared with sensory-deprived cortex of lesion cats and the corresponding regions of area 17 of normal subjects. This study shows that partial sensory deprivation of the visual cortex affects the removal of glutamate from the synaptic cleft and implicates a role for glial-neuronal interactions in adult brain plasticity.

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.

Identification of cGnRH-II in the median eminence of Japanese quail (Coturnix coturnix japonica).

In a previous paper, we described the presence of cGnRH-II in the quail (Coturnix coturnix japonica) and chicken (Gallus gallus) median eminence using highly specific antibodies directed against a polypeptide corresponding to the C-terminal portion of cGnRH-II (van Gils et al., 1993). This finding remained very controversial, since no other study, with any other antibody, had ever reported the presence of cGnRH-II immunoreactive fibers in the median eminence of birds. In this study, the cGnRH-II immunoreactive substances in quail median eminence were isolated by RP-HPLC and identified by RIA. To eliminate the possibility that the cGnRH-II-like immunoreactivity in the median eminence was due to a cross-reaction of our anti-cGnRH-II antiserum with an unknown peptide, the cGnRH-II immunoreactive substances, present in a quail median eminence extract, were isolated by immunoaffinity chromatography using immunoaffinity-purified antibodies. In the eluate of the immunoaffinity column only one peptide could be detected by mass spectrometry. This peptide had a mass of 1235.56 Da, which is the same as synthetic cGnRH-II. In addition, MS/MS fragmentation generated an amino acid sequence corresponding to the sequence of cGnRH-II. The present study therefore identified indisputably cGnRH-II in the median eminence of the quail.

Retinal lesions affect extracellular glutamate levels in sensory-deprived and remote non-deprived regions of cat area 17 as revealed by in vivo microdialysis.

This study aimed at gaining insight into the role of the excitatory neurotransmitter glutamate in topographic map reorganization in the sensory systems of adult mammals after restricted deafferentations. Hereto, in vivo microdialysis was used to sample extracellular glutamate from sensory-deprived and non-deprived visual cortex of adult awake cats 18 to 53 days after the induction of restricted binocular retinal lesions, and in topographically corresponding cortical regions of control animals. A microbore HPLC-ED method was applied for the analysis of the microdialysates. In normal subjects, the visual cortex subserving central and peripheral vision showed similar extracellular fluid glutamate concentrations. In contrast, in animals with homonymous central retinal lesions, the extracellular glutamate concentration was significantly lower in central, sensory-deprived cortex compared to peripheral, non-deprived cortex. Compared to control regions in normal subjects, glutamate decreased in the extracellular fluid of deprived cortex but increased significantly in remote non-deprived visual cortex. These results not only suggest an activity-dependent regulation of the glutamate levels in visual cortex but also imply a role for perilesional cortical regions in topographic map reorganization following sensory deafferentation.

Distribution of the AMPA2 glutamate receptor subunit in adult cat visual cortex.

In this study, we revealed the distribution of the AMPA2 glutamate receptor subunit (AMPA2) in the visual cortical areas 17 and 18 of the adult cat by means of different techniques. In situ hybridization, with a cat-specific radioactively labeled oligonucleotide probe, showed that AMPA2 mRNA was expressed mainly in cortical layers II/III and V/VI with a lower expression in layer IV and practically no signal in layer I. Immunocytochemistry, using a polyclonal AMPA2 subunit-specific antibody, showed immunoreactivity almost exclusively in the somata and dendrites of pyramidal neurons in cortical layers II/III and V/VI. Only a very faint signal was detected in layer IV. Neurons with little or no AMPA2 have AMPA receptors that are highly permeable to calcium. By determining the location of AMPA2, this study therefore provides a clear examination of the distribution of Ca(2+)-impermeable AMPA receptors over the supra- and infragranular layers of cat visual cortex. The functional implication of the absence of AMPA2 in cortical layer IV and thus the presence of Ca(2+)-permeable AMPA receptors in this layer, is still speculative and has yet to be elucidated.

Differential expression of c-fos in subtypes of GABAergic cells following sensory stimulation in the cat primary visual cortex.

Recent immunocytochemical stainings on cat visual cortex, visually stimulated for 1 h, showed a strong induction of Fos expression in cortical neurons. We initiated immunocytochemical double staining experiments with different cytochemical markers to investigate the neurochemical and morphological character of these activated neurons showing Fos induction after sensory stimulation. Double staining with Fos and glutamic acid decarboxylase (GAD) demonstrated the presence of Fos in the nuclei of GABAergic neurons of the primary visual cortex. To further subdivide this Fos/GABAergic cell population we investigated whether Fos colocalized with parvalbumin, calbindin or calretinin. Colocalization of Fos with these calcium-binding proteins delineated distinct neuronal subclasses of Fos-immunoreactive neurons in supra- and infragranular layers of cat area 17. Quantitative analysis of the proportion of immunoreactive local circuit neurons revealed that 35% of the GABAergic neurons showed Fos induction in supragranular layers, whereas in infragranular layers a mere 10% of the GABAergic cells revealed Fos expression. Fos coexisted in about 24% of the calbindin-immunopositive cells within supra- and infragranular layers, but only a minority of the parvalbumin and the calretinin neuronal subgroups were immunopositive for Fos in the corresponding layers of area 17. These findings suggest that visual stimulation induces Fos expression in distinct subsets of inhibitory neurons in cat primary visual cortex.

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-3beta (GSK3beta) 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-3beta in the brain, two-dimensional electrophoresis patterns of cerebrum extracts of GSK3beta[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 GSK3beta plays a role, indicates that our population of identified proteins includes some down stream proteins of GSK3beta. This study may contribute to filling the gaps between GSK3beta, its substrates and finally the phosphorylation of tau.