Alcoholism: protein expression profiles in a human hippocampal model

It is well known that chronic, excessive consumption of alcohol can cause brain damage/structural changes in the regions important for neurocognitive function. Some of the damages are permanent, while others are reversible. Molecular mechanisms underlying alcohol-induced and/or -related brain damage are largely unknown, although it is generally believed that three factors (ethanol, nutritious and hepatic factors) play important roles. Recently, we have been employing a high-throughput proteomics technology to investigate several alcohol-sensitive brain regions from uncomplicated and hepatic cirrhosis-complicated alcoholics to understand the mechanisms of alcohol effects on the CNS at the level of protein expression. The changes of protein expression profiles in the hippocampus of alcoholic subjects were firstly demonstrated using 2D gel electrophoresis-based proteomics. Protein expression profiles identified in the hippocampus of alcoholic subjects were significantly different from those previously identified by our group in other brain regions of the same alcoholic cases, possibly indicating that these different brain regions react differently to chronic alcohol ingestion at the level of protein expression. Identified changes of protein expression associated with astrocyte and oxidative stress may indicate the possibility that increased levels of CNS ammonia and reactive oxygen species induced by alcoholic mild hepatic damage/dysfunction could cause selective damage in astrocytes of the hippocampus. Although our data did not demonstrate any evidence of direct alcohol effects to induce the alteration of protein expression in association with brain damage, high-throughput neuroproteomics approaches have proved to have the potential to dissect the mechanisms of complex brain disorders. Proteomics studies on human hippocampus, an important region for neurocognitive function and psychiatric illnesses (e.g., Alzheimer's disease, alcoholism and schizophrenia) are still sparse, and further investigation is warranted to understand the underlying mechanisms.     

Differential protein expression in the corpus callosum (body) of human alcoholic brain

Neuroimage analysis in alcoholic corpus callosum (CC) suggests that microstructural abnormalities are higher in the genu followed by the body and the splenium. Molecular mechanisms underlying these dysmorphologys are still unclear. Protein expression was performed using the CC body samples [(nine controls, seven uncomplicated, and six complicated (with liver cirrhosis) alcoholics] through proteomics approach. Thirty-nine protein spots in uncomplicated and 60 in complicated alcoholics were differentially altered compared with the control ( p  < 0.05). Comparison between alcoholic groups revealed that 40% more protein showed altered expression in complicated compared with uncomplicated. This result suggests that alcohol-related liver dysfunction has synergetic effects on brain protein expression. Subregional expression profiles indicate that the highest numbers of region-specific proteins were in the genus followed by the CC body and the splenium. Interestingly, abnormal thiamine cascade was strongly suggested in the genu, and to a lesser extent in the CC body, but no such cascade was observed in the splenium. Therefore, alcohol-induced microstructural damage detected by image analysis in the CC, possibly involves multiple biochemical mechanisms.     

Alterations of cerebral cortex and hippocampal proteasome subunit expression and function in a traumatic brain injury rat model

Following cellular stress or tissue injury, the proteasome plays a critical role in protein degradation and signal transduction. The present study examined the β-subunit expression of constitutive proteasomes (β1, β2, and β5), immunoproteasomes (β1i, β2i, and β5i) and the 11S proteasome activator, PA28α, in the rat CNS after traumatic brain injury (TBI). Concomitant measures assessed changes in proteasome activities. Quantitative real time PCR results indicated that β1 and β2 mRNA levels were not changed, while β5 mRNA levels were significantly decreased in injured CNS following TBI. However, β1i, β2i, β5i, and PA28α mRNA levels were significantly increased in the injured CNS. Western blotting studies found that β1, β2, β5, β2i, and β5i subunit protein levels remained unchanged in the injured CNS, but β1i and PA28α protein levels were significantly elevated in ipsilateral cerebral cortex and hippocampus. Proteasome activity assays found that peptidyl glutamyl peptide hydrolase-like and chymotrypsin-like activity were significantly reduced in the CNS after TBI, and that trypsin-like proteasome activity was increased in the injured cerebral cortex. Our results demonstrated that both proteasome composition and function in the CNS were affected by trauma. Treatments that preserve proteasome function following CNS injury may be beneficial as an approach to cerebral neuroprotection.     

Altered brain protein expression profiles are associated with molecular neurological dysfunction in the PKU mouse model

Phenylketonuria (PKU), if not detected and treated in newborns, causes severe neurological dysfunction and cognitive and behavioral deficiencies. Despite the biochemical characterization of PKU, the molecular mechanisms underlying PKU‐associated brain dysfunction remain poorly understood. The aim of this study was to gain insights into the pathogenesis of this neurological damage by analyzing protein expression profiles in brain tissue of Black and Tan BRachyury‐PahEnu2 mice (a mouse model of PKU). We compared the cerebral protein expression of homozygous PKU mice with that of their heterozygous counterparts using two‐dimensional difference gel electrophoresis analysis, and identified 21 differentially expressed proteins, four of which were over‐expressed and 17 under‐expressed. An in silico bioinformatic approach indicated that protein under‐expression was related to neuronal differentiation and dendritic growth, and to such neurological disorders as progressive motor neuropathy and movement disorders. Moreover, functional annotation analyses showed that some identified proteins were involved in oxidative metabolism. To further investigate the proteins involved in the neurological damage, we validated two of the proteins that were most strikingly under‐expressed, namely, Syn2 and Dpysl2, which are involved in synaptic function and neurotransmission. We found that Glu2/3 and NR1 receptor subunits were over‐expressed in PKU mouse brain. Our results indicate that differential expression of these proteins may be associated with the processes underlying PKU brain dysfunction, namely, decreased synaptic plasticity and impaired neurotransmission.

     

Identification of conserved modes of expression profiles during hippocampal development and neuronal differentiation in vitro

Gene expression profiles can be regarded as sums of simpler modes, analogous to the modes of a vibrating violin string. Decomposition of temporal gene expression profiles into modes by singular value decomposition (SVD) was reported before, but the question as to what degree the SVD modes can be interpreted in terms of biology remains open. We report and compare the results of SVD of published datasets from hippocampal development, neuronal differentiation in vitro, and a control time-series hippocampal dataset. We demonstrate that the first SVD mode reflects the magnitude of expression, interpretable on the Affymetrix platform. In the datasets from gene profiling of hippocampal development and neuronal differentiation, the second mode reflects a monotonous change in expression, either up- or down-regulation, in the time course of experiment. We demonstrate that the top two SVD modes are conserved between datasets and therefore, likely reflect properties of the underlying system (gene expression in hippocampus) rather than of a particular experiment or dataset. Our results also indicate that the magnitude of expression, and the direction of change in expression during hippocampal development, are uncorrelated, suggesting that they are regulated by largely independent mechanisms.     

Analysis of protein expression in cell microarrays: a tool for antibody-based proteomics.

Tissue microarray (TMA) technology provides a possibility to explore protein expression patterns in a multitude of normal and disease tissues in a high-throughput setting. Although TMAs have been used for analysis of tissue samples, robust methods for studying in vitro cultured cell lines and cell aspirates in a TMA format have been lacking. We have adopted a technique to homogeneously distribute cells in an agarose gel matrix, creating an artificial tissue. This enables simultaneous profiling of protein expression in suspension- and adherent-grown cell samples assembled in a microarray. In addition, the present study provides an optimized strategy for the basic laboratory steps to efficiently produce TMAs. Presented modifications resulted in an improved quality of specimens and a higher section yield compared with standard TMA production protocols. Sections from the generated cell TMAs were tested for immunohistochemical staining properties using 20 well-characterized antibodies. Comparison of immunoreactivity in cultured dispersed cells and corresponding cells in tissue samples showed congruent results for all tested antibodies. We conclude that a modified TMA technique, including cell samples, provides a valuable tool for high-throughput analysis of protein expression, and that this technique can be used for global approaches to explore the human proteome.     

Seasonal variation in hippocampal volume in a food-storing bird,the black-capped chickadee

Black-capped chickadees (Parus atricapillus) in upstate New York show a peak in food-hoarding intensity in October. We caught chickadees at six different times of the year and measured the volume of several brain structures. We found that the hippocampal formation, which is involved in spatial memory for cached food items, has a larger volume, relative to the rest of the brain, in October than at any other time of the year. We conclude that there is an association between the intensity of food hoarding and the volume of the hippocampal formation and suggest that the enhanced anatomy might be caused by the increased use of spatial memory. © 1995 John Wiley & Sons, Inc.     

IgLON cell adhesion molecules regulate synaptogenesis in hippocampal neurons

IgLON cell adhesion molecules (CAMs) belonging to the immunoglobulin superfamily comprise of LAMP, neurotrimin (Ntm), OBCAM, and Kilon. In the present study, we performed the single and double transfection of IgLON gene constructs into hippocampal neurons in vitro and evaluated synaptic number. The quantitative analysis showed that the single over‐expression of LAMP or OBCAM increased synaptic number, while the over‐expression of Kilon reduced synaptic number and Ntm had no effects. The double over‐expression of Kilon‐Ntm, Kilon‐OBCAM, LAMP‐Ntm, and Ntm‐OBCAM decreased synaptic number and that of Kilon‐LAMP and LAMP‐OBCAM had no effect. These results suggest that IgLON CAMs participate in regulating synapse formation in hippocampal neurons. Copyright © 2009 John Wiley & Sons, Ltd.     

ARG3.1/ARC expression in hippocampal dentate gyrus astrocytes: ultrastructural evidence and co-localization with glial fibrillary acidic protein

Synaptic efficacy following long-term potentiation (LTP) and memory consolidation is associated with changes in the expression of immediate early genes (IEGs). These changes are often accompanied by increased expression of glial fibrillary acidic protein (GFAP). While the protein products of the majority of IEGs are mainly restricted to the cell body, Arg3.1/Arc product is rapidly delivered to dendrites, where it accumulates close to synaptic sites. Arg3.1/Arc protein was originally considered neurone specific; however, we have recently found Arg3.1/Arc immunoreactivity (Arg3.1/Arc-IR) within glial cells and demonstrated its increased expression after LTP in the hippocampal dentate gyrus (DG). Here, we have further investigated this novel finding, using electron microscopic immunocytochemistry to determine the localization and sub-cellular distribution of Arg3.1/Arc protein in GFAP positive glia (GFAP-IR) in the DG. Arg3.1/Arc labelling was seen prominently in GFAP-IR glial cell bodies and in large- and medium-sized glial filamentous processes. GFAP-labelled medium-small peri-synaptic glial profiles also displayed Arg3.1/Arc-IR; however, the very thin and distal glial filaments only displayed Arc-IR. Arc-IR was distributed throughout the cytoplasm, often associated with GFAP filaments, and along the plasma membrane of glial processes. Peri-synaptic glial Arg3.1/Arc-IR processes were apposed to pre- and/or post-synaptic profiles at asymmetric axospinous synapses. These data, taken with our earlier study which provided evidence for an increase in astrocytic Arg3.1/Arc-IR after the induction of LTP, suggest a role for glial Arg3.1/Arc in structural and synaptic plasticity which may be critical for the maintenance of cognitive functions.     

Differential protein expression profiles in the hippocampus of human alcoholics

Mild to severe cognitive impairments are frequently observed symptoms in chronic alcoholics. Decline of cognitive function significantly affects patients' recovery process and prognosis. The hippocampal region is sensitive to the effects of alcohol and it has been suggested that alcohol-induced hippocampal damage and/or changes in neuronal circuitry play an important role in generating these symptoms. Although various hypotheses have been proposed, molecular mechanisms underlying these alterations in the hippocampus are largely unknown. In the present study, we employed a 2DE-based proteomics approach to compare the protein expression profiles of the hippocampus in human alcoholic and healthy control brains. In the alcoholic hippocampus, 20 protein spots were found to be differentially regulated, 2 increased and 18 decreased. Seventeen proteins were identified using mass spectroscopy and were subcategorized into three energy metabolic proteins, six protein metabolic proteins, four signalling proteins, two oxidative stress-related proteins, one vesicle trafficking protein and one cytoskeletal protein. Some of these proteins have been previously implicated in alcohol-induced brain pathology. Based upon the results, several hypotheses were generated to explain the mechanisms underlying possible functional and/or structural alterations induced by chronic alcohol use in this brain region.     

Cross-talk of vitamin D and glucocorticoids in hippocampal cells

There is growing evidence for a role of vitamin D3 signalling in the brain. In this study, we investigated the influence of vitamin D3, in combination with glucocorticoids, on differentiation of the hippocampal progenitor line HIB5, as well as survival of rat primary hippocampal cells. In HIB5, pre-treatment with dexamethasone (Dex) alone inhibited neurite outgrowth and abolished activation of the mitogen-activated protein kinase (MAPK) pathway during platelet-derived growth factor (PDGF)-induced differentiation, consistent with previous findings. Interestingly, pre-treating HIB5 with vitamin D3 significantly reduced these effects of Dex and, in addition, lowered the transactivational function of the glucocorticoid receptor (GR) in transient reporter gene assays. A further impact of vitamin D3 on glucocorticoid effects was observed in a rat primary hippocampal culture known to be particularly sensitive to prolonged GR activation. In this model, Dex induced considerable cell death after 72 h of exposure in vitro. However, 24 h of pre-treatment with low doses of vitamin D3 substantially reduced the degree of Dex-induced apoptosis in primary hippocampal cells. Taken together, our experiments demonstrate a cross-talk between vitamin D3 and glucocorticoids in two hippocampal models, a feature that may have important implications in disorders with dysregulated glucocorticoid signalling, including major depression.     

Proteomic analysis of rat hippocampal plasma membrane: characterization of potential neuronal-specific plasma membrane proteins

The hippocampus is a distinct brain structure that is crucial in memory storage and retrieval. To identify comprehensively proteins of hippocampal plasma membrane (PM) and detect the neuronal-specific PM proteins, we performed a proteomic analysis of rat hippocampus PM using the following three technical strategies. First, proteins of the PM were purified by differential and density-gradient centrifugation from hippocampal tissue and separated by one-dimensional electophoresis, digested with trypsin and analyzed by electrospray ionization (ESI) quadrupole time-of-flight (Q-TOF) tandem mass spectrometry (MS/MS). Second, the tryptic peptide mixture from PMs purified from hippocampal tissue using the centrifugation method was analyzed by liquid chromatography ion-trap ESI-MS/MS. Finally, the PM proteins from primary hippocampal neurons purified by a biotin-directed affinity technique were separated by one-dimensional electrophoresis, digested with trypsin and analyzed by ESI-Q-TOF-MS/MS. A total of 345, 452 and 336 non-redundant proteins were identified by each technical procedure respectively. There was a total of 867 non-redundant protein entries, of which 64.9% are integral membrane or membrane-associated proteins. One hundred and eighty-one proteins were detected only in the primary neurons and could be regarded as neuronal PM marker candidates. We also found some hypothetical proteins with no functional annotations that were first found in the hippocampal PM. This work will pave the way for further elucidation of the mechanisms of hippocampal function.     

Brain asymmetry: both sides of the story

Biological systems demonstrate asymmetry, while lateralization has been observed from humans to lower animals structurally, functionally and behaviorally. This may be derived from evolutionary, genetic, developmental, epigenetic and pathologic factors. However, brain structure and function is complex, and macroscopic or microscopic asymmetries are hard to discern from random fluctuations. In this article, we discuss brain laterality and lateralization, beginning with a brief review of the literature on brain structural and functional asymmetries. We conclude with methods to detect and quantify asymmetry, focusing on neuroproteomics, for retrieval of protein-expression patterns, as a method of diagnosis and treatment monitoring. We suggest inter-hemispheric differential proteomics as a valid method to assess the experimental and biological variations in the healthy brain, and neurologic and neuropsychiatric disorders.     

Investigation of protein expression in magnetic field-treated human glioma cells

We previously reported phenotypic changes in human breast cancer cells following low-level magnetic field (MF) exposure. Here proteomic methods were used to investigate the biochemical effect of MF exposure in SF767 human glioma cells. Protein alterations were studied after exposure to 1.2 microTesla (microT) MF [12 milliGauss (mG), 60 Hertz (Hz)] +/- epidermal growth factor (EGF). SF767 cells were exposed for 3 h to sham conditions (<0.2 microT ambient field strength) or 1.2 microT MF (+/-EGF; 10 ng/ml). Solubilized protein fractions (sham; 1.2 microT; sham + EGF; 1.2 microT + EGF) were loaded for electrophoresis by 2D-PAGE and stained using a colloidal Coomassie blue technique to resolve and characterize the proteins. Protein patterns were compared across groups via Student's t-test using PDQUEST software. Cell profiles revealed significant alterations in the spot density of a subset of treated cells. Automated spot excision and processing was performed prior to peptide mass fingerprinting proteins of interest. Fifty-seven proteins from the detectable pool were identified and/or found to differ significantly across treatment groups. The mean abundance of 10 identified proteins was altered following 1.2 microT exposure. In the presence of EGF six proteins were altered after low magnetic field treatment by increasing (4) or decreasing (2) in abundance. The results suggest that the analysis of differentially expressed proteins in SF767 cells may be useful as biomarkers for biological changes caused by exposure to magnetic fields.     

Alterations in the gut microbiota contribute to cognitive impairment induced by the ketogenic diet and hypoxia

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ESPD: a pattern detection model underlying gene expression profiles

MOTIVATION: DNA arrays permit rapid, large-scale screening for patterns of gene expression and simultaneously yield the expression levels of thousands of genes for samples. The number of samples is usually limited, and such datasets are very sparse in high-dimensional gene space. Furthermore, most of the genes collected may not necessarily be of interest and uncertainty about which genes are relevant makes it difficult to construct an informative gene space. Unsupervised empirical sample pattern discovery and informative genes identification of such sparse high-dimensional datasets present interesting but challenging problems. RESULTS: A new model called empirical sample pattern detection (ESPD) is proposed to delineate pattern quality with informative genes. By integrating statistical metrics, data mining and machine learning techniques, this model dynamically measures and manipulates the relationship between samples and genes while conducting an iterative detection of informative space and the empirical pattern. The performance of the proposed method with various array datasets is illustrated.