A daily single dose of a novel modafinil analogue CE-123 improves memory acquisition and memory retrieval.

Dopamine reuptake inhibitors have been shown to improve cognitive parameters in various tasks and animal models. We recently reported a series of modafinil analogues, of which the most promising, 5-((benzhydrylsulfinyl)methyl) thiazole (CE-123), was selected for further development. The present study aims to characterize pharmacological properties of CE-123 and to investigate the potential to enhance memory performance in a rat model. In vitro transporter assays were performed in cells expressing human transporters. CE-123 blocked uptake of [3H] dopamine (IC50 = 4.606 µM) while effects on serotonin (SERT) and the norepinephrine transporter (NET) were negligible. Blood-brain barrier and pharmacokinetic studies showed that the compound reached the brain and lower elimination than R-modafinil. The Pro-cognitive effect was evaluated in a spatial hole-board task in male Sprague-Dawley rats and CE-123 enhances memory acquisition and memory retrieval, represented by significantly increased reference memory indices and shortened latency. Since DAT blockers can be considered as indirect dopamine receptor agonists, western blotting was used to quantify protein levels of dopamine receptors D1R, D2R and D5R and DAT in the synaptosomal fraction of hippocampal subregions CA1, CA3 and dentate gyrus (DG). CE-123 administration in rats increased total DAT levels and D1R protein levels were significantly increased in CA1 and CA3 in treated/trained groups. The increase of D5R was observed in DG only. Dopamine receptors, particularly D1R, seem to play a role in mediating CE-123-induced memory enhancement. Dopamine reuptake inhibition by CE-123 may represent a novel and improved stimulant therapeutic for impairments of cognitive functions.

Reduced Levels of the Synaptic Functional Regulator FMRP in Dentate Gyrus of the Aging Sprague-Dawley Rat.

Fragile X mental retardation protein (FMRP) encoded by Fragile X mental retardation 1 (FMR1) gene is a RNA-binding regulator of mRNA translation, transport and stability with multiple targets responsible for proper synaptic function. Epigenetic silencing of FMR1 gene expression leads to the development of Fragile X syndrome (FXS) that is characterized by intellectual disability and other behavioral problems including autism. In the rat FXS model, the lack of FMRP caused a deficit in hippocampal-dependent memory. However, the hippocampal changes of FMRP in aging rats are not fully elucidated. The current study addresses the changes in FMRP levels in dentate gyrus (DG) from young (17 weeks) and aging (22 months) Sprague - Dawley rats. The aging animal group showed significant decline in spatial reference memory. Protein samples from five rats per each group were analyzed by quantitative proteomic analysis resulting in 153 significantly changed proteins. FMRP showed significant reduction in aging animals which was confirmed by immunoblotting and immunofluorescence microscopy. Furthermore, bioinformatic analysis of the differential protein dataset revealed several functionally related protein groups with individual interactions with FMRP. These include high representation of the RNA translation and processing machinery connected to FMRP and other RNA-binding regulators including CAPRIN1, the members of Pumilio (PUM) and CUG-BP, Elav-like (CELF) family, and YTH N(6)-methyladenosine RNA-binding proteins (YTHDF). The results of the current study point to the important role of FMRP and regulation of RNA processing in the rat DG and memory decline during the aging process.

Comprehensive identification of age-related lipidome changes in rat amygdala during normal aging.

Brain lipids are integral components of brain structure and function. However, only recent advancements of chromatographic techniques together with mass spectrometry allow comprehensive identification of lipid species in complex brain tissue. Lipid composition varies between the individual areas and the majority of previous reports was focusing on individual lipids rather than a lipidome. Herein, a mass spectrometry-based approach was used to evaluate age-related changes in the lipidome of the rat amygdala obtained from young (3 months) and old (20 months) males of the Sprague-Dawley rat strain. A total number of 70 lipid species with significantly changed levels between the two animal groups were identified spanning four main lipid classes, i.e. glycerolipids, glycerophospholipids, sphingolipids and sterol lipids. These included phospholipids with pleiotropic brain function, such as derivatives of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine. The analysis also revealed significant level changes of phosphatidic acid, diacylglycerol, sphingomyelin and ceramide that directly represent lipid signaling and affect amygdala neuronal activity. The amygdala is a crucial brain region for cognitive functions and former studies on rats and humans showed that this region changes its activity during normal aging. As the information on amygdala lipidome is very limited the results obtained in the present study represent a significant novelty and may contribute to further studies on the role of lipid molecules in age-associated changes of amygdala function.

R-Modafinil exerts weak effects on spatial memory acquisition and dentate gyrus synaptic plasticity.

Modafinil is a wake promoting drug approved for clinical use and also has cognitive enhancing properties. Its enantiomer R-Modafinil (R-MO) is not well studied in regard to cognitive enhancing properties. Hence we studied its effect in a spatial memory paradigm and its possible effects on dentate gyrus long-term potentiation (DG-LTP). Clinically relevant doses of R-MO, vehicle dimethyl sulfoxide (DMSO) or saline were administered for three days during the hole-board test and in in vivo DG-LTP. Synaptic levels of dopamine receptors D1R, D2R, dopamine transporter (DAT), and its phosphorylated form (ph-DAT) in DG tissue 4 h after LTP induction were quantified by western blot analysis. Monoamine reuptake and release assays were performed by using transfected HEK-293 cells. Possible neurotoxic side effects on general behaviour were also studied. R-MO at both doses significantly enhanced spatial reference memory during the last training session and during memory retrieval compared to DMSO vehicle but not when compared to saline treated rats. Similarly, R-MO rescues DG-LTP from impairing effects of DMSO. DMSO reduced memory performance and LTP magnitude when compared to saline treated groups. The synaptic DR1 levels in R-MO groups were significantly decreased compared to DMSO group but were comparable with saline treated animals. We found no effect of R-MO in neurotoxicity tests. Thus, our results support the notion that LTP-like synaptic plasticity processes could be one of the factors contributing to the cognitive enhancing effects of spatial memory traces. D1R may play an important regulatory role in these processes.

Hippocampal levels of GluR1 and GluR2 complexes are modulated by training in the Multiple T-maze in C57BL/6J mice.

A series of studies has shown the importance of AMPA-type glutamate receptors (AMPARs) for memory formation. The aim of the current study was to show whether GluR1 and GluR2 complexes rather than subunits in mouse hippocampus were involved in training in the multiple T-Maze (MTM). C57BL/6J mice were trained in the MTM and compared to yoked controls. 6 h following the completion of the fourth day training, mice were euthanized, hippocampi were taken and proteins extracted, run on blue native gels with subsequent immunoblotting with antibodies against mouse GluR1 and GluR2. On blue-native western blotting, GluR1 protein was represented by a single band at the apparent molecular weight of about 480 kDa probably indicating a tetrameric assembly. GluR2 protein was represented by a single band between apparent molecular weights of 480 and 720 kDa indicating a homo- or heteropolymer probably with other AMPAR or regulatory subunits. In mice trained in the MTM, protein levels for GluR1 were significantly increased while GluR2 levels were significantly decreased. On two-dimensional (2D) gel electrophoresis, the presence of GluR1 and GluR2 were identified by mass spectrometry, and 2D immunoblotting revealed several expression forms of these receptor subunits. Findings unequivocally show that GluR1 and GluR2 complexes are linked to training in the MTM in C57BL/6J mice. These results may not only form the basis for studying receptor complexes rather than receptor subunits in memory formation or mechanisms of potential cognitive enhancers but represent a tool for investigations into pharmacological studies including the use of glutamate receptor agonists and antagonists.

Hippocampal lipids linked to spatial memory in the C57BL/6J mouse.

Although the role of individual brain lipids for learning and memory has been reported, no systematic approach associating brain lipids with spatial memory has been carried out. It was therefore the aim of the study to determine brain lipids in hippocampus of mice forming and yoked controls that did not form spatial memory using the probe trial as the endpoint. 10 animals were trained in the Morris water maze (MWM) and 10 mice were serving as yoked controls i.e. no platform was used during the whole experiment. Hippocampal tissue lipids were extracted and data were acquired with Fourier transformation ion cyclotron resonance mass spectrometry (LTQ-FT) coupled to HPLC. Glycerophosphatidylethanolamines (18:0/22:6, 18:0/20:4 and 18:1/18:1), plasmalogens (16:0-10/22:6 and 18:0-10/22:6) and ceramides (18:0) showed higher levels in the trained group, while glycerolysophosphatidylcholines (16:0, 18:1, 18:0, 20:4), sphingomyelins (16:0, 24:1), ether linked glycerophosphatidylcholines (16:0-10/18:0), glycerophosphatidylcholines (16:0/18:1, 16:0/18:0, 18:0/18:1, 38:7, 18:1/20:1, 20:4/20:4, 22:1/18:1, 22:0/18:1, 20:4/22:6, 22:6/22:6), glucosylceramide (24:1) and plasmalogen (18:0-10/20:1) revealed lower levels in the trained group. Decreased levels of certain species of lysophosphatidylcholine, sphingomyelin, plasmenylphosphatidylcholine, phosphatidylcholine, glycosylceramide and plasmalogen at the probe trial for spatial memory may indicate catabolism in terms of consumption during this process. Increased hippocampal levels of long chain highly unsaturated phosphatidylethanolamines, plasmalogens and ceramides may reflect increased synthesis or decreased degradation at the endpoint of memory testing, probably representing interactions in the brain lipid pathways. The study shows pathways involved in spatial memory, may propose the use of individual brain lipids as probable cognitive enhancers and forms the basis for further studies on the role of brain lipids per se.

Strain-dependent expression of metabolic proteins in the mouse hippocampus.

Individual mouse strains differ significantly in terms of behavior and cognitive function. Strain-specific variation of metabolic protein levels in the hippocampus among various commonly used mouse strains, however, has not been investigated yet. A proteomic approach based on two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry [high capacity ion trap (HCT)] has been chosen to address this question by determining strain-dependent levels of metabolic proteins in hippocampal tissue of four inbred and one outbred mouse strain. Statistical analysis of protein spots on 2-DE gels of the individual strains (n=10) revealed significant strain-dependent differences in densities of 39 spots. Subsequent HCT analysis led to the identification of 22 different metabolic proteins presenting with differential protein levels among the five mouse strains investigated. Among those are proteins concerned with the metabolism of amino acid, nucleic acid, carbohydrate and energy. Moreover, proteins known to play a pivotal role in the processes of learning and memory, such as calcium/calmodulin-dependent protein kinase type II alpha chain, were found to present with significant inter-strain variability, which is also in agreement with our previous reports. Strain-specific protein levels of metabolic proteins in the mouse hippocampus may provide some insight into the molecular underpinnings and genetic determination of strain-dependent neuronal function. Furthermore, data presented herein emphasize the significance of the genetic background for the analysis of metabolic pathways in the hippocampus in wild-type mice as well as in gene-targeting experiments.

Inhibition of oligodendrocyte precursor cell differentiation by myelin-associated proteins.

OBJECT: Promoting repair of central nervous system (CNS) white matter represents an important approach to easing the course of a number of tragic neurological diseases. For this purpose, strategies are currently being evaluated for transplanting cells capable of generating new oligodendrocytes into areas of demyelination and/or enhancing the potential of endogenous stem/precursor cells to give rise to new oligodendrocytes. Emerging evidence, however, indicates that increasing the presence of cells capable of forming new myelin sheaths is not sufficient to promote repair because of unknown inhibitors that accumulate in lesions as a consequence of myelin degeneration and impair the generation of new oligodendrocytes. The aim of the present study was to characterize the nature of the inhibitory molecules present in myelin. METHODS: Differentiation of primary rat oligodendrocyte precursor cells (OPCs) in the presence of CNS and peripheral nervous system myelin was assessed by immunocytochemical methods. The authors further characterized the nature of the inhibitors by submitting myelin membrane preparations to biochemical precipitation and digestion. Finally, OPCs were grown on purified Nogo-A, oligodendrocyte myelin glycoprotein, and myelin-associated glycoprotein, the most prominent inhibitors of axon regeneration. RESULTS: Myelin membrane preparations induced a differentiation block in OPCs that was associated with down-regulation of expression of the transcription factor Nkx2.2. The inhibitory activity in myelin was restricted to the CNS and was predominantly associated with white matter. Furthermore, the results demonstrate that myelin proteins that are distinct from the most prominent inhibitors of axon outgrowth are specific inhibitors of OPC differentiation. CONCLUSIONS: The inhibitory effect of unknown myelin-associated proteins should be considered in future treatment strategies aimed at enhancing CNS repair.

Mass spectrometrical characterization of NDRG2 protein (N-myc-downstream regulated gene 2) and description of two novel phosphorylation sites.

Antidepressant-related protein (NDRG2) is a member of the N-myc downstream-regulated gene family and a role for differentiation and signaling has been proposed. Performing protein profiling we observed NDRG2 and decided to characterize this important biomolecule. Estrous cycle phases were determined in Sprague-Dawley rats and the hippocampus was taken. Proteins were extracted, run on two-dimensional gel electrophoresis with subsequent multi-enzyme digestion followed by MALDI-TOF-TOF and nano-LC-ESI-MS/MS analysis of spots. Spots identified as NDRG2 were quantified by specific software. Five spots were identified as NDRG2 and two novel phosphorylation sites (T330 and T334) were detected. Gender and estrous cycle-dependent NDRG2 levels were observed. Results are of importance for further qualitative and quantitative studies at the protein level as well as for the design of antibodies for immunochemical applications and for the interpretation of previous studies on NDRG2 that did not take into account different expression forms and posttranslational modifications.

Involvement of endothelial NO in the dilator effect of VIP on rat isolated pulmonary artery.

The endothelium and its interaction with smooth muscle play a central role in the local control of the pulmonary vasculature, and endothelial dysfunction is thought to contribute to pulmonary hypertension and chronic obstructive pulmonary disease. Vasoactive intestinal peptide (VIP), a 28-amino acid neuropeptide, relaxes the rat pulmonary artery, but there is controversy as to whether or not this action of VIP depends on the endothelium. The aim of this study, therefore, was to investigate the role of the endothelium and nitric oxide (NO), the major endothelium-derived relaxing factor, in the dilator action of VIP on the rat isolated pulmonary artery. Pulmonary artery preparations pre-contracted by the alpha(1)-adrenoceptor agonist L-phenylephrine were relaxed by VIP (0.003-1 microM) and acetylcholine (0.003-10 microM) in a concentration-dependent manner. Mechanical removal of the endothelium reduced the maximal response to VIP by about 50% and practically abolished the response to acetylcholine. Inhibition of NO synthesis by N(omega)-nitro-L-arginine methyl ester (0.5 mM) had a similar effect, abolishing the vasorelaxation caused by acetylcholine and attenuating the vasorelaxation caused by VIP by about 50%. From these data it is concluded that the relaxant action of VIP on the rat isolated pulmonary artery depends in part on the presence of the endothelium and that this part is mediated by endothelial NO.

An integrated map of the murine hippocampal proteome based upon five mouse strains.

With the advent of proteomics technologies it is possible to simultaneously demonstrate the expression of hundreds of proteins. The information offered by proteomics provides context-based understanding of cellular protein networks and has been proven to be a valuable approach in neuroscience studies. The mouse hippocampus has been a major target of analysis in the search for molecular correlates to neuronal information storage. Although human and rat hippocampal samples have been successfully subjected to proteomic profiling, no elaborate analysis providing the fundamental experimental basis for protein-expression studies in the mouse hippocampus has been carried out as yet. This led us to construct a master map generated from the individual hippocampal proteomes of five different mouse strains. A proteomic approach, based upon 2-DE coupled to MS (MALDI-TOF/TOF) has been chosen in an attempt to establish a comprehensive reference database of proteins expressed in the mouse hippocampus. 469 individual proteins, represented by 1156 spots displaying various functional states of the respective gene products were identified. Proteomic profiling of the hippocampus, a brain region with a pivotal role for neuronal information processing and storage may provide insight into the characteristics of proteins serving this highly sophisticated function.

Components of the protein quality control system are expressed in a strain-dependent manner in the mouse hippocampus.

Inbred mouse strains are used in forward-genetic experiments, designed to uncover genes contributing to their highly distinct neurophenotypes and multiple reports of variations in mutant phenotypes due to genetic background differences in reverse-genetic approaches have been published. Information on strain-specific protein expression-phenotypes however, is limited and a comprehensive screen of an effect of strain on brain protein levels has not yet been carried out. Herein a proteomic approach, based upon two-dimensional gel electrophoresis (2-DE) coupled to mass spectrometry (MALDI-TOF/TOF) was used to show significant genetic variation in hippocampal protein levels between five mouse strains. Considering recent evidence for the importance of the intracellular protein quality control system for synaptic plasticity-related mechanism we decided to focus on the analysis of molecular chaperones and components of the ubiquitin-proteasome system. Sixty-six spots, depicting 36 proteins have been unambiguously identified by mass spectrometry. Quantification revealed strain-dependent levels of 18 spots, representing 12 individual gene products. We thus present proteome analysis of hippocampal tissues of several mouse strains as suitable tool to address fundamental questions about genetic control of protein levels and to demonstrate molecular networks of protein metabolism and chaperoning. The findings are useful for designing future studies on these cascades and interpretation of results show that data on brain protein levels cannot be simply extrapolated among different mouse strains.

Strain-dependent regulation of plasticity-related proteins in the mouse hippocampus.

Inbred mouse strains have different genetic backgrounds that can result in impairment of synaptic plasticity and memory. Strain-dependent performance in behavioral and cognitive tasks is well-documented. Hippocampal long-term potentiation (LTP), an activity-dependent enhancement of synaptic transmission that may underlie some forms of learning and memory has been shown to differ significantly between inbred mouse strains. However, an effect of strain on the expression of proteins, critically involved in synaptic plasticity, learning and memory has not been described yet. We have been addressing this question by determining expressional levels of a panel of proteins involved in neuronal information processing in hippocampus of five mouse strains by immunoblotting. Four inbred strains (FVB/N, C57Bl/6J, 129S2/Sv and Balb/c), commonly used for generating genetically modified mice and for conventional experiments in pharmacology and toxicology and one outbred strain (OF1) have been selected. A significant effect of strain was detected for total and phosphorylated calcium-calmodulin dependent kinase IIalpha (CaMKII, pCaMKII), phosphorylated mitogen-activated protein kinase (pMAPK), total and phosphorylated calcium-responsive element binding 1 (creb, pcreb), early-growth response protein 1 (egr 1), brain derived neurotrophic factor (BDNF), drebrin and postsynaptic density-95 (PSD-95). These results may indicate genetic determination of synaptic plasticity-related mechanisms relevant for the molecular events mediating hippocampal information processing and storage. Data presented herein highlight the importance of careful selection of the mouse strain for studies of synaptic plasticity.

Behavioral testing upregulates pCaMKII, BDNF, PSD-95 and egr-1 in hippocampus of FVB/N mice.

Several protein cascades are proposed to be involved in the formation of synaptic plasticity and have been linked to neuronal information processing and storage. Although modified expression of specific proteins following behavioral testing has been shown, no systematic approach for their concomitant determination has been reported. We therefore determined hippocampal expression of signaling proteins, transcription factors and synaptosomal-associated proteins representing key elements of neuronal plasticity in mice following behavioral training. Male FVB/N mice, 12 weeks of age, were used for behavioral testing. After completion of tests mice were sacrificed and hippocampi were dissected. Levels of total and autophosphorylated (T286) alphacalcium-calmodulin dependent kinase II (CaMKII, pCaMKII), total and phosphorylated mitogen-activated protein kinase (MAPK, pMAPK), total and phosphorylated calcium-responsive element binding (creb, pcreb), early-growth response protein 1 (egr-1), brain derived neurotrophic factor (BDNF), tyrosine kinase receptor B (trk B), drebrin and postsynaptic density-95 (PSD-95) were quantified in hippocampi of behavior trained animals (n=7) and naïve caged controls (n=7). Expression of pCaMKII, BDNF, PSD-95 and egr-1 was significantly increased in the behavior-trained group. Expression of total CaMKII, total and pMAPK, total and pcreb, trk B and drebrin was comparable between groups. Detection of significantly increased pCaMKII, BDNF, PSD-95 and egr-1 induced by behavioral training at the protein level per se is intriguing and supports the proposed importance of these molecules for neuronal information storage.

Proteome analysis in hippocampus of mice overexpressing human Cu/Zn-superoxide dismutase 1.

Cu/Zn-superoxide dismutase 1 (SOD1), encoded on chromosome 21, is a key enzyme in metabolism of oxygen free radicals and oxidative stress. Transgenic mice overexpressing human SOD1 (Tg-hSOD1) are useful model for Down syndrome (trisomy 21) and familial amyotrophic lateral sclerosis (ALS). It was shown recently that Tg-hSOD1 mice develop a characteristic set of neurodegenerative changes in hippocampus and we therefore decided to study differential protein expression patterns, constructing a mouse hippocampal proteome map using two-dimensional electrophoresis (2-DE) with in-gel digestion of spots followed by matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) identification and quantitatively compared protein profiles between non-transgenic mice, hemizygous and homozygous Tg-hSOD1 mice. In total 1056 spots were analysed, resulting in the identification of 445 polypeptides that were the products of 157 different genes. Among these a series of proteins involved in scaffolding, metabolism, signaling and other functions were deranged. Our findings suggest that overexpressed SOD1 directly or by generating reactive oxygen species may lead to aberrant protein expressional patterns that in turn may lead to or reflect neurodegeneration observed in this animal model.

Cerebellar protein expression in three different mouse strains and their relevance for motor performance.

The present study uses a proteomic approach to link motor function to cerebellar protein expression in 129X1/SvJ, C57BL/6J and nNOS WT mice. Poor performance on the Rota rod, the standard test for motor coordination, was detected in 129X1/SvJ mice. No gross impairments of neurological, cognitive and behavioural functions were observed. Identification and quantification of 48 proteins revealed reduced expression of calbindin, septin 5 and syntaxin binding protein 1 in 129X1/SvJ. In nNos WT glucose-6-phosphate 1 dehydrogenase X was decreased whereas dihydropyrimidinase-related protein-4 was increased. In C57BL/6J stress-70 protein, alpha enolase, NAD-dependent deacetylase sirtuin 2, septin 2, dihydropyrimidinase-related protein-2 and brain derived neurotrophic factor showed elevated levels. Neurological examination, Rota rod test, Morris Water Maze, Multiple-T-Maze, Open field and Elevated plus-maze were employed to study motor, cognitive and behavioural function. Mice were sacrificed and cerebellar tissue was homogenized. Proteins were extracted and separated on two-dimensional gel electrophoresis with subsequent in-gel digestion followed by mass spectrometrical analysis of peptides (MALDI-TOF/TOF-TOF). Quantification of spots was carried out by specific software. A strong association of impaired motor function with altered cerebellar protein expression of calbindin, septin 5 and syntaxin binding protein 1in 129X1/SvJ was observed and is in agreement with previous observations of motor deficiencies in a calbindin knock-out mouse. These results have to be taken into account when using 129X1/SvJ for biochemical, toxicological or gene targeting experiments as well as when studying the above-mentioned proteins or corresponding pathways and cascades in this mouse strain.

Impaired cognitive performance in neuronal nitric oxide synthase knockout mice is associated with hippocampal protein derangements.

Nitric oxide is implicated in modulation of memory and pharmacological as well as genetic inhibition of neuronal nitric oxide synthase (nNOS) leads to impaired cognitive function. We therefore decided to study learning and memory functions and cognitive flexibility in the Morris water maze (MWM) in 1-month-old male mice lacking nNOS (nNOS KO). Hippocampal protein profiling was carried out to possibly link protein derangement to impaired cognitive function. Two-dimensional gel electrophoresis with in-gel digestion of spots and subsequent MALDI-TOF identification of proteins and quantification of proteins using specific software was applied. In the memory as well as in the relearning task of the MWM, most of the nNOS KO failed to find the submerged platform within a given time. Proteomic evaluation of hippocampus, the main anatomical structure computing cognitive functions, revealed aberrant expression of a synaptosomal associated protein of the exocytotic machinery (NSF), glycolytic enzymes, chaperones 78 kDa glucose-regulated protein, T-complex protein 1; the signaling structure guanine nucleotide-binding protein G(I)/G(S)/G(T) and heterogeneous nuclear ribonucleoprotein H of the splicing machinery. We conclude that nNOS knockout mice show impaired spatial performance in the MWM, a finding that may be either linked to direct effects of nNOS/NO and/or to specific hippocampal protein derangements.

Life-long effects of perinatal asphyxia on stress-induced proteins and dynamin 1 in rat brain.

In previous work, we have shown that perinatal asphyxia (PA) in the rat leads to life-long neurotransmitter deficits and impairment of cognitive functions and behavior. This observation made us examine protein expression in hippocampus of rats with PA at the end of the life span. We applied a well-documented and characterized animal model of PA. Pups, normoxic and asphyxiated for 20 min, were brought up until the age of 24 months and then were sacrificed. Hippocampal tissue was dissected from the brains, and proteins were run on two-dimensional gel electrophoresis with in-gel digestion and subsequent identification of proteins by MALDI-TOF followed by quantification of protein spots by specific software. In hippocampus of rats with PA, the stress proteins protein disulfide isomerase A3 precursor and stress-induced phosphoprotein-1 were significantly increased, whereas the microtubule-associated protein dynamin-1 was significantly reduced. Increased stress protein levels may represent long-term effects of PA or, alternatively, could reflect conditioning of the stress protein machinery known to occur as a neuroprotective principle following hypoxic-ischemic conditions. Decreased dynamin-1 levels may be considered as a long-term effect on the exocytotic system possibly reflecting or leading to impaired neuronal transport and vesicle-trafficking in PA of the rat of advanced age.

Long-term influence of perinatal asphyxia on the social behavior in aging rats.

BACKGROUND: Various groups have been addressing the question of whether perinatal asphyxia (PA) affects the behavior of young animals, but no information is available on long-term effects of PA on the behavior in aged rats, although it has been postulated that PA may lead to neurological and psychiatric deficits in adult life. OBJECTIVE: We, therefore, decided to study the effects of PA on social and anxiety-related behaviors in 2-year-old rats, using a noninvasive animal model resembling the clinical situation. METHODS: For the behavioral studies, the open-field test, the elevated plus-maze test, and a social interaction test in pairs were performed. Magnetic resonance imaging of the brain was selected to rule out neuropathological changes due to the aging process per se, as well as asphyxia-induced pathologies in the brain areas known to play an important role in the modulation of behavior. RESULTS: The social interaction test revealed a statistically significant increase in the number of social grooming episodes and the time spent running alone, whereas the numbers of social sniffing and fighting episodes and the time spent running together were decreased in the asphyxiated group. The elevated plus- maze test revealed a higher presence of entries into the closed arm. Furthermore, sniffing and self-grooming episodes were significantly increased in the asphyxiated group. CONCLUSIONS: We found a significantly decreased social aggressiveness and an increased social contact behavior as well as increased anxiety levels in the asphyxiated animals. The present findings may provide important information on the long-term behavioral sequelae of PA in the aged individual.

Altered expression of hypothetical proteins in hippocampus of transgenic mice overexpressing human Cu/Zn-superoxide dismutase 1.

BACKGROUND: Cu/Zn-superoxide dismutase 1 (SOD1), encoded on chromosome 21, is a key enzyme in the metabolism of reactive oxygen species (ROS) and pathogenetically relevant for several disease states including Down syndrome (DS; trisomy 21). Systematically studying protein expression in human brain and animal models of DS we decided to carry out "protein hunting" for hypothetical proteins, i.e. proteins that have been predicted based upon nucleic sequences only, in a transgenic mouse model overexpressing human SOD1. RESULTS: We applied a proteomics approach using two-dimensional electrophoresis (2-DE) with in-gel digestion of spots followed by mass spectrometric (matrix-assisted laser desorption/ionization-time of flight) identification and quantification of hypothetical proteins using specific software. Hippocampi of wild type, hemizygous and homozygous SOD1 transgenic mice (SOD1-TGs) were analysed.We identified fourteen hypothetical proteins in mouse hippocampus. Of these, expression levels of 2610008O03Rik protein (Q9D0K2) and 4632432E04Rik protein (Q9D358) were significantly decreased (P < 0.05 and 0.001) and hypothetical protein (Q99KP6) was significantly increased (P < 0.05) in hippocampus of SOD1-TGs as compared with non-transgenic mice. CONCLUSIONS: The biological meaning of aberrant expression of these proteins may be impairment of metabolism, signaling and transcription machinery in SOD1-TGs brain that in turn may help to explain deterioration of these systems in DS brain.