Proteomic Analysis of Hippocampus and Cortex in Streptozotocin-Induced Diabetic Model Mice Showing Dementia.

AIM: Diabetes with its associated hyperglycemia induces various type of peripheral damage and also impairs the central nervous system (CNS). This study is aimed at clarifying the precise mechanism of diabetes-induced dementia as an impairment of CNS. METHODS: The proteomic analysis of the hippocampus and cortex in streptozotocin- (STZ-) treated mouse diabetic model showing dementia was performed using two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (n = 3/group). RESULTS: Significant changes in the expression of 32 proteins and 7 phosphoproteins were observed in the hippocampus and cortex. These identified proteins and phosphoproteins could be functionally classified as cytoskeletal protein, oxidoreductase, protein deubiquitination, energy metabolism, GTPase activation, heme binding, hydrolase, iron storage, neurotransmitter release, protease inhibitor, transcription, glycolysis, antiapoptosis, calcium ion binding, heme metabolic process, protein degradation, vesicular transport, and unknown in the hippocampus or cortex. Additionally, Western blotting validated the changes in translationally controlled tumor protein, ATP-specific succinyl-CoA synthetase beta subunit, and gamma-enolase isoform 1. CONCLUSIONS: These findings showed that STZ-induced diabetes changed the expression of proteins and phosphoproteins in the hippocampus and cortex. We propose that alterations in expression levels of these proteins play an important role in diabetes-induced dementia.

The protective role of protein L-isoaspartyl (D-aspartate) O-methyltransferase for maintenance of mitochondrial morphology in A549 cell.

PURPOSE: The increasing amounts of evidence with abnormal aging process have been involved in the pathogenesis of chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Mice with deficient protein L-isoaspartate (D-aspartate) O-methyl transferase 1 (PCMT1) expression reveal acceleration of aging and result in the increased proportion of D-aspartate (D-Asp) residues and dysfunction in proteins. Furthermore, mitochondrial morphology and functions are associated with COPD and IPF pathogenesis. The purpose of the current study was to investigate the role of PCMT1 on mitochondrial morphology using A549 cells. MATERIALS AND METHODS: We investigated PCMT1, prohibitin1 (PHB1), mitochondrial membrane proteins expression, mitochondrial morphology, and the proportion of D-Asp residues in PHB1 in A549 cells with (PCMT1-KD) and without the context of decreased PCMT1 expression (PCMT1-Cont) using electron microscopy, fluorescence staining, Western blot analysis, and the ATP content per cells. To investigate the effects of the PCMT1-KD cells, we developed double-transfected cell lines containing either the cytosolic or the endoplasmic isoform of PCMT1. RESULTS: We found a significantly higher proportion of D-Asp residues in PHB1 in PCMT1-KD cells than that in PCMT1-Cont cells. The PCMT1-KD cells without cigarette smoke extract exposure were characterized by a significantly increased proportion of the D-Asp residues in PHB1, damaged mitochondrial ultrastructure, and a tendency toward the fission direction of the mitochondrial dynamics followed by a significant decrease in the cellular ATP content. CONCLUSIONS: The increased proportion of the D-Asp residues may contribute to COPD pathogenesis, via irreversible protein conformational changes, followed by mitochondrial dysfunction.

The influence of chronic nicotine treatment on proteins expressed in the mouse hippocampus and cortex.

Chronic treatment with nicotine, the primary psychoactive substance in tobacco smoke, affects central nervous system functions, such as synaptic plasticity. Here, to clarify the effects of chronic nicotine treatment on the higher brain functions, proteomic analysis of the hippocampus and cortex of mice treated for 6 months with nicotine was performed using two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry. There was significant change in the expression of 16 proteins and one phosphoprotein in the hippocampus (increased tubulin ß-5, atp5b, MDH1, cytochrome b-c1 complex subunit 1, Hsc70, dynamin, profilin-2, 4-aminobutyrate aminotransferase, mitochondrial isoform 1 precursor, calpain small subunit 1, and vacuolar adenosine triphosphatase subunit B and decreased γ-actin, α-tubulin isotype M-α-2, putative ß-actin, tubulin ß-2A, NDUFA10, and G6PD) and 24 proteins and two phosphoproteins in the cortex (increased spectrin α chain, non-erythrocytic 1 isoform 1, tubulin ß-5, γ-actin, creatine kinase B-type, LDH-B, secernin-1, UCH-L1, 14-3-3 γ, type II peroxiredoxin 1, PEBP-1, and unnamed protein product and decreased tubulin α-1C, α-internexin, γ-enolase, PDHE1-B, DPYL2, vacuolar adenosine triphosphatase subunit A, vacuolar adenosine triphosphatase subunit B, TCTP, NADH dehydrogenase Fe-S protein 1, protein disulfide-isomerase A3, hnRNP H2, γ-actin, atp5b, and unnamed protein product). Additionally, Western blotting validated the changes in dynamin, Hsc70, MDH1, NDUFA10, α-internexin, tubulin ß-5 chain, and secernin-1. Thus, these findings indicate that chronic nicotine treatment changes the expression of proteins and phosphoproteins in the hippocampus and cortex. We propose that effect of smoking on higher brain functions could be mediated by alterations in expression levels of these proteins.

Proteomic analysis of mouse choroid plexus cell line ECPC-4 treated with lipid A.

OBJECTIVES: Choroid plexus (CP) epithelial cells have multiple functions in the cerebral ventricles, including cerebrospinal fluid (CSF) production and forming part of the blood-CSF barrier. They are also responsible for producing inflammatory mediators involved in meningitis. The present study aimed to elucidate the functions of the CP epithelial cells during CNS inflammation. MATERIALS AND METHODS: We analyzed the proteome and phosphoproteome in lipid A-treated ECPC-4 mouse CP cells by gel electrophoresis and mass spectrometry. RESULTS: Levels of 10 proteins and seven phosphoproteins were significantly altered by lipid A in time-dependent manners, including V-type proton ATPase subunit B (ATP6V), protein 40 kD, elongation factor-1δ, coatomer subunit ε (COPE), vimentin (isoform CRA a), purine nucleoside phosphorylase, eukaryotic initiation factor-4F splicing variant, put. ß-actin, peroxiredoxin-6 isoform 1, and immunoglobulin heavy chain variable region. These proteins could be classified as having cytoskeleton/intermediate filament, protein-folding, signal-transduction, cell-growth, metabolism, and redox-regulation functions. The identified phosphoproteins were HSP 84, γ-actin, HSP 70 cognate, vimentin, tubulin ß-4B chain, protein disulfide-isomerase A6 precursor, and heterogenous nuclear ribonucleoprotein, which could be classified as having cytoskeleton/intermediate filament, protein-folding, and metabolism functions. CONCLUSIONS: These results indicate that lipid A can change the levels of proteins and phosphoproteins in ECPC-4 cells, suggesting that the identified proteins and phosphoproteins may play important roles in inflammation of the CP.

Changes in the expression of collapsin response mediator protein-2 during synaptic plasticity in the mouse hippocampus.

We have previously reported that nicotine application to the adult mouse causing long-term potentiation-like facilitation in vivo in the hippocampus can serve as a model of synaptic plasticity. The present study clarifies the involvement of collapsin response mediator protein-2 (CRMP2) in synaptic plasticity. CRMP2 was detected in hippocampal neurons of adult mice. The levels of CRMP2 mRNA and protein were increased 2-24 hr and 4-24 hr, respectively, after application of nicotine (3 mg/kg, i.p.), finally returning to the basal level by 48 hr. Furthermore, the ratio of phosphorylated CRMP2 (pCRMP2) at Thr514 residue, an inactive form, to total CRMP2 levels was not changed during synaptic plasticity expressed by nicotine, indicating an enhanced level of non-pCRMP2. This increase of CRMP2 was inhibited by blockade of nicotinic acetylcholine receptors (nAChRs) and required activation of both α4ß2 and α7 nAChRs. Although the level of ubiquitinated CRMP2 was increased 8 hr after nicotine treatment, the ratio of ubiquitinated CRMP2 to total CRMP2 protein was similar for nicotine-treated and nontreated mice. This study demonstrates that the expression of CRMP2 increases in hippocampal neurons during synaptic plasticity and that the increment is due mainly to mRNA expression. We propose that CRMP2, particularly non-pCRMP2, could contribute to long-lasting synaptic plasticity.

The influence of chronic ibuprofen treatment on proteins expressed in the mouse hippocampus.

Ibuprofen is a nonsteroidal anti-inflammatory drug (NSAID), treatment with which has been shown to delay the onset, slows the cognitive decline, and decreases the incidence of Alzheimer׳s disease (AD) in epidemiological and clinical studies. However, a comprehensive understanding of its mechanism of action remains unclear. To elucidate the prophylactic effect of ibuprofen on the onset of the learning and memory disturbances of AD, we performed proteomic analysis of the hippocampus of chronic ibuprofen-treated mice using two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry. Twenty-eight proteins and seven phosphoproteins were identified to be significantly changed in the hippocampus of chronic ibuprofen-treated mice: translationally controlled tumor protein, thioredoxin-dependent peroxide reductase, and peroxiredoxin 6 were increased, and glial fibrillary acidic protein, dihydropyrimidinase-related protein 2, EF-hand domain-containing protein D2, and 14-3-3ζ were decreased. These identified proteins and phosphoproteins could be classified as cytoskeletal, neuronal development, chaperone, metabolic, apoptosis, neurotransmitter release, ATP synthase, deubiquitination, proteasome, NOS inhibitor, adapter, vesicle transport, signal transduction, antioxidant enzyme, proton transport, synaptogenesis, and serine/threonine phosphatase types. Western blot analysis showed the changes in dihydropyrimidinase-related protein 2, heat shock protein 8, ubiquitin carboxyl-terminal hydrolase PGP9.5, and γ-enolase levels in the hippocampus of chronic ibuprofen-treated mice. These findings showed that the chronic treatment with ibuprofen changed the levels of some proteins and phosphoproteins in the hippocampus. We propose that these identified proteins and phosphoproteins play an important role in decreasing the incidence of AD, especially impaired learning and memory functions.

Proteomic analysis of time-dependent changes in proteins expressed in mouse hippocampus during synaptic plasticity induced by GABAA receptor blockade.

Protein synthesis is required for long-lasting synaptic plasticity. We examined the time-dependent changes in protein expression that occurred in the hippocampus during synaptic plasticity using two-dimensional gel electrophoresis followed by mass spectrometry. The levels of 15 proteins were significantly changed in mouse hippocampus 8h after bicuculline application (1.0mg/kg, i.p.). Expression of 14 proteins (i.e., dihydropyrimidinase-related protein 2, α-tubulin isotype M-α-2, tubulin ß-1 chain, tubulin ß-2A chain, protein disulfide-isomerase ERp61 precursor, chaperonin-containing T complex polypeptide 1 ß subunit, T complex polypeptide 1 [partial], creatine kinase B-type, cytosolic malate dehydrogenase [partial], vacuolar adenosine triphosphatase subunit A, and uncharacterized protein LOC433182) was increased and expression of one protein (i.e., actin γ, cytoplasmic 1) was decreased. Western blotting also validated the changes in dihydropyrimidinase-related protein 2, creatine kinase B-type, and vacuolar adenosine triphosphatase subunit A levels in mouse hippocampus 8h after bicuculline application. The identified proteins were effectors of cellular functions including neuronal differentiation, cytoskeletal dynamics, folding of proteins, stress response, energy metabolism, synapse formation, and unknown function. Taken together, these findings indicate that the identified proteins play an important role in synaptic plasticity in the hippocampus.

Proteomic analysis of the hippocampus in Alzheimer's disease model mice by using two-dimensional fluorescence difference in gel electrophoresis.

We previously identified the E693Δ mutation in amyloid precursor protein (APP) in patients with Alzheimer's disease (AD) and then generated APP-transgenic mice expressing this mutation. As these mice possessed abundant Aß oligomers from 8 months of age but no amyloid plaques even at 24 months of age, they are a good model to study pathological effects of amyloid ß (Aß) oligomers. The two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) technology, using a mixed-sample internal standard, is now recognized as an accurate method to determine and quantify proteins. In this study, we examined the proteins for which levels were altered in the hippocampus of 12-month-old APP(E693Δ)-transgenic mice using 2D-DIGE and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Fourteen proteins were significantly changed in the hippocampus of APP(E693Δ)-transgenic mice. Actin cytoplasmic 1 (ß-actin), heat shock cognate 71kDa, γ-enolase, ATP synthase subunit ß, tubulin ß-2A chain, clathrin light chain B (clathrin) and dynamin-1 were increased. Heat shock-related 70kDa protein 2, neurofilament light polypeptide (NFL), stress-induced-phosphoprotein 2, 60kDa heat shock protein (HSP60), α-internexin, protein kinase C and casein kinase substrate in neurons protein 1 (Pacsin 1), α-enolase and ß-actin were decreased. Western blotting also validated the changed levels of HSP60, NFL, clathrin and Pacsin 1 in APP(E693Δ)-transgenic mice. The identified proteins could be classified as cytoskeleton, chaperons, neurotransmission, energy supply and signal transduction. Thus, proteomics by 2D-DIGE and LC-MS/MS has provided knowledge of the levels of proteins in the early stages of AD brain.

The expression changes of EphA3 receptor during synaptic plasticity in mouse hippocampus through activation of nicotinic acetylcholine receptor.

We have reported that systemic application of nicotinic agonists results in expression of a long-term potentiation-like facilitation, a model of synaptic plasticity, in the mouse hippocampus in vivo. Eph receptors and their ephrin ligands, are thought to participate in synaptic plasticity. The present study was conducted to clarify the involvement of EphA3 receptor in synaptic plasticity by investigating the time-dependent change of the expression levels of EphA3 receptor during long-term potentiation-like facilitation in the mouse hippocampus. EphA3 receptor mRNA and protein expression was found in adult mouse hippocampus. EphA3 receptor was localized in neuronal cells but not astrocytes or microglia of hippocampus. After intraperitoneal application of nicotine (3 mg/kg), the protein expression of EphA3 receptor in hippocampus increased during 2-24-h period, significantly increasing during 2-12-h period, and finally returned to the basal level in 72 h, although the mRNA expression of EphA3 receptor was not changed for 24 h. This enhanced expression of EphA3 receptor protein at 4 h was inhibited by pretreatment of mecamylamine (0.5 mg/kg, intraperitoneally), a nonselective nicotinic acetylcholine receptor antagonist. Our findings demonstrated that EphA3 receptor localized only in neuronal cells of the hippocampus was enhanced without transcriptional regulation during synaptic plasticity through activation of the nicotinic acetylcholine receptor. These results suggest that the enhancement of EphA3 receptor after synaptic plasticity may contribute to long-lasting synaptic plasticity through positive, feedforward mechanisms.

Proteomic analysis of the brain tissues from a transgenic mouse model of amyloid ß oligomers.

Amyloid ß (Aß) oligomers are presumed to be one of the causes of Alzheimer's disease (AD). Previously, we identified the E693Δ mutation in amyloid precursor protein (APP) in patients with AD who displayed almost no signals of amyloid plaques in amyloid imaging. We generated APP-transgenic mice expressing the E693Δ mutation and found that they possessed abundant Aß oligomers from 8months of age but no amyloid plaques even at 24months of age, indicating that these mice are a good model to study pathological effects of Aß oligomers. To elucidate whether Aß oligomers affect proteome levels in the brain, we examined the proteins and phosphoproteins for which levels were altered in 12-month-old APP(E693Δ)-transgenic mice compared with age-matched non-transgenic littermates. By two-dimensional gel electrophoresis (2DE) followed by staining with SYPRO Ruby and Pro-Q Diamond and subsequent mass spectrometry techniques, we identified 17 proteins and 3 phosphoproteins to be significantly changed in the hippocampus and cerebral cortex of APP(E693Δ)-transgenic mice. Coactosin like-protein, SH3 domain-bind glutamic acid-rich-like protein 3 and astrocytic phosphoprotein PEA-15 isoform 2 were decreased to levels less than 0.6 times those of non-transgenic littermates, whereas dynamin, profilin-2, vacuolar adenosine triphosphatase and creatine kinase B were increased to levels more than 1.5 times those of non-transgenic littermates. Furthermore, 2DE Western Blotting validated the changed levels of dynamin, dihydropyrimidinase-related protein 2 (Dpysl2), and coactosin in APP(E693Δ)-transgenic mice. Glyoxalase and isocitrate dehydrogenase were increased to levels more than 1.5 times those of non-transgenic littermates. The identified proteins could be classified into several groups that are involved in regulation of different cellular functions, such as cytoskeletal and their interacting proteins, energy metabolism, synaptic component, and vesicle transport and recycling. These findings indicate that Aß oligomers altered the levels of some proteins and phosphoproteins in the hippocampus and cerebral cortex, which could illuminate novel therapeutic avenues for the treatment of AD.

Identification of phosphorylated serine-15 and -82 residues of HSPB1 in 5-fluorouracil-resistant colorectal cancer cells by proteomics.

To identify the proteins involved in 5-fluorouracil (5-FU) resistance, a comparison of the total and phosphorylated proteins between the human colorectal cancer (CRC) cell line DLD-1 and its 5-FU-resistant subclone DLD-1/5-FU was performed. Using 2-DE and MALDI-TOF/TOF-based proteomics, 17 up-regulated and 19 down-regulated protein spots were identified in the 5-FU-resistant DLD-1/5-FU cells compared with the parent cell lines. In DLD-1/5-FU cells, 7 anti-apoptotic proteins (HSPB1, proteasome subunit α-5, transitional endoplasmic reticulum ATPase, 14-3-3 ß, 14-3-3 γ, 14-3-3 σ, and phosphoglycerate kinase 1) were up-regulated and 4 proapoptotic proteins (cofilin-1, pyruvate kinase M2, glyceraldehyde-3-phosphate dehydrogenase, and nucleophosmin) were down-regulated. The results show that the acquired drug resistance of DLD-1/5-FU cells is caused by the prevention of drug-induced apoptosis, in particular through the enhanced constitutive expression of HSPB1 and its phosphorylated form. Short interfering RNA knockdown of endogenous HSPB1 in DLD-1/5-FU cells restored the sensitivity to 5-FU. Furthermore, MALDI-TOF/TOF and 2-DE Western blot analysis identified the phosphorylated residues of HSPB1 as Ser-15 and Ser-82 in the main (diphosphorylated) form and Ser-15, Ser-78, and Ser-82 in the minor (triphosphorylated) form. The current findings indicate that phosphorylated HSPB1 may play an important role in 5-FU resistance.

Caenorhabditis elegans p97 controls germline-specific sex determination by controlling the TRA-1 level in a CUL-2-dependent manner.

p97 (CDC-48 in Caenorhabditis elegans) is a ubiquitin-selective AAA (ATPases associated with diverse cellular activities) chaperone and its key function is to disassemble protein complexes. p97 functions in diverse cellular processes including endoplasmic reticulum (ER)-associated degradation, membrane fusion, and meiotic and mitotic progression. However, its cellular functions in development have not yet been clarified. Here, we present data that p97 is involved in the switch from spermatogenesis to oogenesis in the germline of the C. elegans hermaphrodite. We found that the cdc-48.1 deletion mutant produced less sperm than the wild type and thus showed a decreased brood size. The cdc-48.1 mutation suppressed the sperm-overproducing phenotypes of fbf-1 and fem-3(gf) mutants. In addition, the p97/CDC-48-UFD-1-NPL-4 complex interacted with the E3 ubiquitin ligase CUL-2 complex via NPL-4 binding to Elongin C. Furthermore, TRA-1A, which is the terminal effector of the sex determination pathway and is regulated by CUL-2-mediated proteolysis, accumulated in the cdc-48.1 mutant. Proteasome activity was also required for the brood size determination and sperm-oocyte switch. Our results demonstrate that the C. elegans p97/CDC-48-UFD-1-NPL-4 complex controls the sperm-oocyte switch by regulating CUL-2-mediated TRA-1A proteasome degradation.

Germ cell specific protein VASA is over-expressed in epithelial ovarian cancer and disrupts DNA damage-induced G2 checkpoint.

OBJECTIVE: Cancer cells have characteristics, such as high telomerase activity and high levels of migration activity and proliferation, which are very similar to those of germ cell lineages. In this study, we examined the expression of VASA, a germ cell lineage specific marker and evaluated its clinical significance in epithelial ovarian cancer (EOC). METHODS: We investigated VASA expression in 75 EOC tissues by immunohistochemistry, correlating results with clinicopathological factors. To clarify the effects of VASA on cellular phenotypes, we compared the protein expression profiles between SKOV-3 cells stably expressing VASA (SKOV-3-VASA) and vector-control cell lines by coupling 2D fingerprinting and identification of proteins by mass spectrometry. RESULTS: VASA expression in tumor cells was found in 21 of 75 cases and was positively correlated with high age and serous histology. Significant down-regulation of 14-3-3sigma was observed in SKOV-3-VASA versus control cells. Over-expression of VASA abrogates the G2 checkpoint, induced by DNA damage, by down-regulating the expression of 14-3-3sigma. CONCLUSIONS: These results suggest that VASA may either play a direct role in the progression of EOC or serve as a valuable marker of tumorigenesis.

Involvement of HMG-12 and CAR-1 in the cdc-48.1 expression of Caenorhabditis elegans.

Caenorhabditis elegans possesses two p97/VCP/Cdc48p homologues, named CDC-48.1 (C06A1.1) and CDC-48.2 (C41C4.8), and their expression patterns and levels are differently regulated. To clarify the regulatory mechanisms of differential expression of two p97 proteins of C. elegans, we performed detailed deletion analysis of their promoter regions. We found that the promoter of cdc-48.1 contains two regions necessary for embryonic and for post-embryonic expression, while the promoter of cdc-48.2 contains the single region necessary for embryonic expression. In particular, two elements (Element A and Element B) and three conserved boxes (Box a, Box b and Box c) were essential for cdc-48.1 expression in embryos and at post-embryonic stages, respectively. By using South-Western blotting and MALDI-TOF MS analysis, we identified HMG-12 and CAR-1 as proteins that bind to Element A and Element B, respectively, from the embryonic nuclear extract. Importantly, we found the decreased expression of p97 in embryos prepared from hmg-12(RNAi) or car-1(RNAi) worms. These results indicate that both HMG-12 and CAR-1 play important roles in embryonic expression of cdc-48.1.

Lipopolysaccharide activates the kallikrein-kinin system in mouse choroid plexus cell line ECPC4.

Regulation of the kallikrein-kinin system in cerebral inflammation is still unclear. Here, we used reverse-transcription polymerase chain reaction (RT-PCR) techniques to show that lipopolysaccharide (LPS) activates the kallikrein-kinin system by enhancing liberation of bradykinin (BK), and alters mRNA levels of kallikrein-kinin system components, including high molecular weight (H-) and low molecular weight (L-) kininogens, in ECPC4 cells, a cell line of mouse choroid plexus epithelium. LPS treatment increased liberation of immunoreactive bradykinin in the supernatant of ECPC4 cells, and addition of LPS (500 ng/ml) to cultures resulted in elevation of H- and L-kininogen mRNA levels in ECPC4 cells within 24-48 h. Furthermore, LPS treatment elevated bradykinin type 2 and type 1 receptor mRNA levels within 4h, but did not change tissue kallikrein or plasma kallikrein mRNA levels. On the other hand, expression of pro-inflammatory mediators interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and cyclooxygenase-2 mRNA increased within 4-8h after addition of LPS to ECPC4 cells. The addition of IL-1beta and TNF-alpha to investigate the major mediator for kininogen expression in ECPC4 cells remarkably induced expression of H- and L-kininogen mRNAs in ECPC4 cells. These results suggest that LPS activates the kallikrein-kinin system in the choroid plexus via autocrine induction of IL-1beta and TNF-alpha.