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.     

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.     

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.     

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.     

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.     

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.     

Characteristics of hippocampal glycine release in cell-damaging conditions in the adult and developing mouse

The release of preloaded [³H]glycine from hippocampal slices from 7-day-old and 3-month-old (adult) mice was studied in different cell-damaging conditions, including hypoxia, hypoglycemia, ischemia, oxidative stress and the presence of free radicals and metabolic poisons, using a superfusion system. Glycine release was greatly enhanced in all the above conditions in both age groups, with the exception of hypoxia in developing mice. This coincides with the increased susceptibility to seizures and excitotoxicity during postnatal development. The ischemia-induced release of glycine was Ca²⁺-independent at both ages. The release was potentiated by exogenously applied glycine but not in Na⁺-free conditions, indicating the involvement of Na⁺-dependent transporters operating outwards. The Cl^– channel blockers 4-acetamido-4-isothiocyanostilbene-2,2-disulphonate and diisothiocyanostilbene-2,2-disulphonate generally reduced the ischemia-induced release, suggesting that this occurs through anion channels in both developing and adult mice. Furthermore, in the adult hippocampus riluzole and amiloride inhibited the release, indicating that Na⁺ channels also contribute to the ischemia-evoked release. Since glycine is an essential factor in glutamate-induced Ca²⁺ channel opening at the N-methyl-D-aspartate receptor, the elevated levels of glycine, together with the increased release of excitatory amino acids, must obviously collaborate in the development of ischemic neuronal damage.     

Altered hippocampal morphology in unmedicated patients with major depressive illness

Despite converging evidence that major depressive illness is associated with both memory impairment and hippocampal pathology, findings vary widely across studies and it is not known whether these changes are regionally specific. In the present study we acquired brain MRIs (magnetic resonance images) from 31 unmedicated patients with MDD (major depressive disorder; mean age 39.2±11.9 years; 77% female) and 31 demographically comparable controls. Three-dimensional parametric mesh models were created to examine localized alterations of hippocampal morphology. Although global volumes did not differ between groups, statistical mapping results revealed that in MDD patients, more severe depressive symptoms were associated with greater left hippocampal atrophy, particularly in CA1 (cornu ammonis 1) subfields and the subiculum. However, previous treatment with atypical antipsychotics was associated with a trend towards larger left hippocampal volume. Our findings suggest effects of illness severity on hippocampal size, as well as a possible effect of past history of atypical antipsychotic treatment, which may reflect prolonged neuroprotective effects. This possibility awaits confirmation in longitudinal studies.     

热性癫痫发作大鼠海马齿状回外侧支的可塑性和再可塑性

热性癫痫发作是儿童常见病,能损害认知功能,而突触可塑性和再可塑性(metaplasticity)是维系大脑认知功能的重要神经基础.本文通过脑片灌流和细胞外场电位记录术研究了热性癫痫发作大鼠海马齿状回外侧支的突触可塑性和再可塑性.制作对照组和热性癫痫发作组大鼠的脑切片后,记录电极置于齿状回外侧支的外分子层获取兴奋性突触后...     

Maternal behavior regulates long-term hippocampal expression of BAX and apoptosis in the offspring

Naturally occurring variations in maternal care influence hippocampal development in the rat. In the present study we found that variations in maternal licking/grooming (LG) during the first week of life are associated with altered hippocampal expression of BAX (group-1 tumor necrosis factor family mediated cell death effector) in 90-day-old male offspring. BAX-like immunoreactivity on western blots is significantly increased in the adult offspring of low-level LG mothers. There is no effect of maternal care on levels of either B-cell lymphoma-2 (BCL-2) (group-II mitochondria mediated cell death suppressor) or BAD (group-III endoplasmic reticulum mediated cell death effector). The most striking biochemical event in apoptosis is DNA fragmentation. Terminal deoxynucleotidyl transerferase (Tdt)-mediated dUTP-biotin nick-end labeling (TUNEL) and 4',6'-diamidino-2-phenylindole hydrochloride (DAPI) staining showed that the number of TUNEL-positive cells in both the dentate gyrus and CA1 region of the hippocampus is significantly increased in the adult offspring of low-level LG mothers. In conclusion, we propose that hippocampal neurons in the offspring of low-level LG mothers may be more vulnerable to loss through apoptosis.     

Hypothyroidism-evoked shifts in hippocampal adrenergic receptors: Implications to ischemia-induced hippocampal damage

Hypothyroidism was induced in a group of male Fischer 344 rats by administration of 0.05% propylthiouracil (PTU) in the drinking water for 12 weeks. Control rats were not treated. Plasma levels of thyroid hormones indicated that PTU treatment had produced severe thyroid hormone deficiency. In PTU-treated rats compared to control rats, levels of total T₃ and total T₄ were reduced 54.5% and 53.7%; while levels of free T₃ and free T₄ were reduced 87.1% and 96.5%. Functional hypothyroidism was demonstrated by: (i) a 49.1% decrease in hepatic plasma membrane ₁-adrenergic receptor binding, and (ii) a 11.2- fold increase in hepatic -glutamyltranspeptidase activity; relative to the expression of these parameters in control rats. Membranes were isolated from hippocampi of control, PTUinduced hypothyroid and thyroxine-replaced rats and specific adrenergic receptor binding determined by radioligand binding techniques. Hypothyrodism resulted in a shift in the balance of ₁ and ₂ adrenergic receptor binding by evoking: an increase in ₁- adrenergic receptor binding to 1.57-fold of control levels; and, a decrease in ₂-adrenergic receptor binding to 64% of control levels. Thyroid hormone replacement carried out in PTU-treated hypothyroid rats at 30 g/kg s.c. per day for the last 3 days of the 12 week PTU-treatment protocol, which reversed physical and functional hypothyroidism, reversed the observed changes in hippocampal adrenergic receptor binding, indicating them to be thyroid hormone, and not PTU, -dependent. This receptor shift evoked by hypothyroidism may, in part, explain the protective effect of hypothyroidism on ischemia-induced hippocampal damage by favoring inhibitory input and limiting excitotoxic input by catecholamines.