Upregulation of Glycolytic Enzymes,Mitochondrial Dysfunction and Increased Cytotoxicity in Glial Cells Treated with Alzheimer’s Disease Plasma

Alzheimer’s disease (AD) is a neurodegenerative disorder associated with increased oxidative stress and neuroinflammation. Markers of increased protein, lipid and nucleic acid oxidation and reduced activities of antioxidant enzymes have been reported in AD plasma. Amyloid plaques in the AD brain elicit a range of reactive inflammatory responses including complement activation and acute phase reactions, which may also be reflected in plasma. Previous studies have shown that human AD plasma may be cytotoxic to cultured cells. We investigated the effect of pooled plasma (n = 20 each) from healthy controls, individuals with amnestic mild cognitive impairment (aMCI) and Alzheimer’s disease (AD) on cultured microglial cells. AD plasma and was found to significantly decrease cell viability and increase glycolytic flux in microglia compared to plasma from healthy controls. This effect was prevented by the heat inactivation of complement. Proteomic methods and isobaric tags (iTRAQ) found the expression level of complement and other acute phase proteins to be altered in MCI and AD plasma and an upregulation of key enzymes involved in the glycolysis pathway in cells exposed to AD plasma. Altered expression levels of acute phase reactants in AD plasma may alter the energy metabolism of glia.     

A liver X receptor and retinoid X receptor heterodimer mediates apolipoprotein E expression, secretion and cholesterol homeostasis in astrocytes

Apolipoprotein E (apoE) is an important protein involved in lipoprotein clearance and cholesterol redistribution. ApoE is abundantly expressed in astrocytes in the brain and is closely linked to the pathogenesis of Alzheimer's disease (AD). We report here that small molecule ligands that activate either liver X receptors (LXR) or retinoid X receptor (RXR) lead to a dramatic increase in apoE mRNA and protein expression as well as secretion of apoE in a human astrocytoma cell line (CCF-STTG1 cells). Examination of primary mouse astrocytes also revealed significant induction of apoE mRNA, and protein expression and secretion following incubation with LXR/RXR agonists. Moreover, treatment of mice with a specific synthetic LXR agonist T0901317 resulted in up-regulation of apoE mRNA and protein in both hippocampus and cerebral cortex, indicating that apoE expression in brain can be up-regulated by LXR agonists in vivo. Along with a dramatic induction of ABCA1 cholesterol transporter expression, these ligands effectively mediate cholesterol efflux in both CCF-STTG1 cells and mouse astrocytes in the presence or absence of apolipoprotein AI (apoAI). Our studies provide strong evidence that small molecule LXR/RXR agonists can effectively mediate apoE synthesis and secretion as well as cholesterol homeostasis in astrocytes. LXR/RXR agonists may have significant impact on the pathogenesis of multiple neurological diseases, including AD.     

Investigation of plasma biomarkers in HIV-1/HCV mono- and coinfected individuals by multiplex iTRAQ quantitative proteomics

The analysis of plasma samples from HIV-1/HCV mono- and coinfected individuals by quantitative proteomics is an efficient strategy to investigate changes in protein abundances and to characterize the proteins that are the effectors of cellular functions involved in viral pathogenesis. In this study, the infected and healthy plasma samples (in triplicate) were treated with ProteoMiner beads to equalize protein concentrations and subjected to 4-plex iTRAQ labeling and liquid chromatography/mass spectrometry (LC-MS/MS) analysis. A total of 70 proteins were identified with high confidence in the triplicate analysis of plasma proteins and 65% of the proteins were found to be common among the three replicates. Apolipoproteins and complement proteins are the two major classes of proteins that exhibited differential regulation. The results of quantitative analysis revealed that APOA2, APOC2, APOE, C3, HRG proteins were upregulated in the plasma of all the three HIV-1 mono-, HCV mono-, and coinfected patient samples compared to healthy control samples. Ingenuity pathway analysis (IPA) of the upregulated proteins revealed that they are implicated in the hepatic lipid metabolism, inflammation, and acute-phase response signaling pathways. Thus, we identified several differentially regulated proteins in HIV-1/HCV mono and coinfected plasma samples that may be potential biomarkers for liver disease.     

Plasma protein profiling of Mild Cognitive Impairment and Alzheimer’s disease using iTRAQ quantitative proteomics

Background

With the promise of disease modifying treatments, there is a need for more specific diagnosis and prognosis of Alzheimer’s disease (AD) and mild cognitive impairment (MCI). Plasma biomarkers are likely to be utilised to increase diagnostic accuracy and specificity of AD and cognitive decline.

Methods

Isobaric tags (iTRAQ) and proteomic methods were used to identify potential plasma biomarkers of MCI and AD. Relative protein expression level changes were quantified in plasma of 411 cognitively normal subjects, 19 AD patients and 261 MCI patients. Plasma was pooled into 4 groups including normal control, AD, amnestic single and multiple domain MCI (aMCI), and nonamnestic single and multiple domain MCI (nMCI). Western-blotting was used to validate iTRAQ data. Integrated function and protein interactions were explored using WEB based bioinformatics tools (DAVID v6.7 and STRING v9.0).

Results

In at least two iTRAQ replicate experiments, 30 proteins were significantly dysregulated in MCI and AD plasma, relative to controls. These proteins included ApoA1, ApoB100, complement C3, C4b-binding protein, afamin, vitamin D-binding protein precursor, isoform 1 of Gelsolin actin regulator, Ig mμ chain C region (IGHM), histidine-rich glycoprotein and fibrinogen β and γ chains. Western-blotting confirmed that afamin was decreased and IGHM was increased in MCI and AD groups. Bioinformatics results indicated that these dysregulated proteins represented a diversity of biological processes, including acute inflammatory response, cholesterol transport and blood coagulation.

Conclusion

These findings demonstrate that expression level changes in multiple proteins are observed in MCI and AD plasma. Some of these, such as afamin and IGHM, may be candidate biomarkers for AD and the predementia condition of MCI.     

The Proteome of Pancreatic Cancer‐Derived Exosomes Reveals Signatures Rich in Key Signaling Pathways

Exosomes are membrane‐bound vesicles that traffic small molecular cargos. These cargos participate in cell–cell communication and contribute to the pathogenesis of many disease including cancer. How these mechanisms contribute to communication within the pancreatic adenocarcinoma (PDAC) microenvironment and how they contribute to PDAC biology are poorly understood. Performed in this study are comprehensive, quantitative comparisons of the proteomes of three PDAC cell lines to those of the exosomes they produce. Approximately 35% of whole cell proteins sort into exosomes. Analysis of composition of microbiomes (ANCOM) determined a cluster of 98 enriched pancreatic cancer exosome core proteins (ePC‐ECPs). Further, these proteins are predicted by ingenuity pathway analysis (IPA) as actively involved in signaling pathways regulating cell death and survival, cellular movement, and cell‐to‐cell signaling and interaction in particular (top three p‐value significant pathways). Significant enrichment of canonical pathways of acute phase response signaling (inflammatory response signaling pathways) and FXR and RXR activation in biosynthetic pathways are also predicted; 97 ePC‐ECPs are associated with cancer and among them, 34 are specifically associated with PDAC. In conclusion, exosomes from PDAC are enriched with cancer‐associated signaling proteins. Further assessment of these proteins as PDAC biomarkers or therapeutic targets is warranted.     

Proteomic identification of rainbow trout blood plasma proteins and their relationship to seminal plasma proteins

The characterisation of fish blood proteomes is important for comparative studies of seminal and blood proteins as well as for the analysis of fish immune mechanisms and pathways. In this study, LC‐MS/MS and 2D‐DIGE were applied to compare rainbow trout seminal (SP) and blood plasma (BP) proteomes. The 54 differentially abundant proteins identified in SP are involved in a variety of signalling pathways, including protein ubiquitination, liver X receptor/retinoid X receptor (LXR/RXR) and farnesoid X receptor activation, cell cycle and acute phase signalling. These findings may indicate the prevalence of acute phase signalling pathways in trout SP, and its essential role in protecting spermatozoa and reproductive tissues. Our study provides the first in‐depth analysis of the trout BP proteome, with a total of 119 proteins identified. The major proteins of rainbow trout BP were recognised as acute phase proteins. Analysis of BP proteins indicated that acute phase response signalling, the complement system, liver X receptor/retinoid X receptor and farnesoid X receptor activation and the coagulation system are the top canonical pathways. This study enhances knowledge of the blood origin of trout SP proteins and understanding of fish reproductive biology. Our results provide new insight into blood proteins specifically important for fish physiology and innate immunity. The mass spectrometry data are available via ProteomeXchange with the identifier PXD005988 and https://doi.org/10.6019/PXD005988 .     

Quantitative Proteomic Analysis Reveals That Anti-Cancer Effects of Selenium-Binding Protein 1 In Vivo Are Associated with Metabolic Pathways

Previous studies have shown the tumor-suppressive role of selenium-binding protein 1 (SBP1), but the underlying mechanisms are unclear. In this study, we found that induction of SBP1 showed significant inhibition of colorectal cancer cell growth and metastasis in mice. We further employed isobaric tags for relative and absolute quantitation (iTRAQ) to identify proteins that were involved in SBP1-mediated anti-cancer effects in tumor tissues. We identified 132 differentially expressed proteins, among them, 53 proteins were upregulated and 79 proteins were downregulated. Importantly, many of the differentially altered proteins were associated with lipid/glucose metabolism, which were also linked to Glycolysis, MAPK, Wnt, NF-kB, NOTCH and epithelial-mesenchymal transition (EMT) signaling pathways. These results have revealed a novel mechanism that SBP1-mediated cancer inhibition is through altering lipid/glucose metabolic signaling pathways.     

Plasma biomarkers of brain atrophy in Alzheimer's disease

Peripheral biomarkers of Alzheimer''s disease (AD) reflecting early neuropathological change are critical to the development of treatments for this condition. The most widely used indicator of AD pathology in life at present is neuroimaging evidence of brain atrophy. We therefore performed a proteomic analysis of plasma to derive biomarkers associated with brain atrophy in AD. Using gel based proteomics we previously identified seven plasma proteins that were significantly associated with hippocampal volume in a combined cohort of subjects with AD (N = 27) and MCI (N = 17). In the current report, we validated this finding in a large independent cohort of AD (N = 79), MCI (N = 88) and control (N = 95) subjects using alternative complementary methods—quantitative immunoassays for protein concentrations and estimation of pathology by whole brain volume. We confirmed that plasma concentrations of five proteins, together with age and sex, explained more than 35% of variance in whole brain volume in AD patients. These proteins are complement components C3 and C3a, complement factor-I, γ-fibrinogen and alpha-1-microglobulin. Our findings suggest that these plasma proteins are strong predictors of in vivo AD pathology. Moreover, these proteins are involved in complement activation and coagulation, providing further evidence for an intrinsic role of these pathways in AD pathogenesis.     

Genome-wide profiling of liver X receptor, retinoid X receptor, and peroxisome proliferator-activated receptor α in mouse liver reveals extensive sharing of binding sites

The liver X receptors (LXRs) are nuclear receptors that form permissive heterodimers with retinoid X receptor (RXR) and are important regulators of lipid metabolism in the liver. We have recently shown that RXR agonist-induced hypertriglyceridemia and hepatic steatosis in mice are dependent on LXRs and correlate with an LXR-dependent hepatic induction of lipogenic genes. To further investigate the roles of RXR and LXR in the regulation of hepatic gene expression, we have mapped the ligand-regulated genome-wide binding of these factors in mouse liver. We find that the RXR agonist bexarotene primarily increases the genomic binding of RXR, whereas the LXR agonist T0901317 greatly increases both LXR and RXR binding. Functional annotation of putative direct LXR target genes revealed a significant association with classical LXR-regulated pathways as well as peroxisome proliferator-activated receptor (PPAR) signaling pathways, and subsequent chromatin immunoprecipitation-sequencing (ChIP-seq) mapping of PPARα binding demonstrated binding of PPARα to 71 to 88% of the identified LXR-RXR binding sites. The combination of sequence analysis of shared binding regions and sequential ChIP on selected sites indicate that LXR-RXR and PPARα-RXR bind to degenerate response elements in a mutually exclusive manner. Together, our findings suggest extensive and unexpected cross talk between hepatic LXR and PPARα at the level of binding to shared genomic sites.     

Proteomic Profiles in Acute Respiratory Distress Syndrome Differentiates Survivors from Non-Survivors

Acute Respiratory Distress Syndrome (ARDS) continues to have a high mortality. Currently, there are no biomarkers that provide reliable prognostic information to guide clinical management or stratify risk among clinical trial participants. The objective of this study was to probe the bronchoalveolar lavage fluid (BALF) proteome to identify proteins that differentiate survivors from non-survivors of ARDS. Patients were divided into early-phase (1 to 7 days) and late-phase (8 to 35 days) groups based on time after initiation of mechanical ventilation for ARDS (Day 1). Isobaric tags for absolute and relative quantitation (iTRAQ) with LC MS/MS was performed on pooled BALF enriched for medium and low abundance proteins from early-phase survivors (n = 7), early-phase non-survivors (n = 8), and late-phase survivors (n = 7). Of the 724 proteins identified at a global false discovery rate of 1%, quantitative information was available for 499. In early-phase ARDS, proteins more abundant in survivors mapped to ontologies indicating a coordinated compensatory response to injury and stress. These included coagulation and fibrinolysis; immune system activation; and cation and iron homeostasis. Proteins more abundant in early-phase non-survivors participate in carbohydrate catabolism and collagen synthesis, with no activation of compensatory responses. The compensatory immune activation and ion homeostatic response seen in early-phase survivors transitioned to cell migration and actin filament based processes in late-phase survivors, revealing dynamic changes in the BALF proteome as the lung heals. Early phase proteins differentiating survivors from non-survivors are candidate biomarkers for predicting survival in ARDS.     

Human Alzheimer’s disease synaptic <Emphasis Type="Italic">O</Emphasis>-GlcNAc site mapping and iTRAQ expression proteomics with ion trap mass spectrometry

Neuronal synaptic functional deficits are linked to impaired learning and memory in Alzheimer’s disease (AD). We recently demonstrated that O-GlcNAc, a novel cytosolic and nuclear carbohydrate post-translational modification, is enriched at neuronal synapses and positively regulates synaptic plasticity linked to learning and memory in mice. Reduced levels of O-GlcNAc have been observed in AD, suggesting a possible link to deficits in synaptic plasticity. Using lectin enrichment and mass spectrometry, we mapped several human cortical synaptic O-GlcNAc modification sites. Overlap in patterns of O-GlcNAcation between mouse and human appears to be high, as previously mapped mouse synaptic O-GlcNAc sites in Bassoon, Piccolo, and tubulin polymerization promoting protein p25 were identified in human. Novel O-GlcNAc modification sites were identified on Mek2 and RPN13/ADRM1. Mek2 is a signaling component of the Erk 1/2 pathway involved in synaptic plasticity. RPN13 is a component of the proteasomal degradation pathway. The potential interplay of phosphorylation with mapped O-GlcNAc sites, and possible implication of those sites in synaptic plasticity in normal versus AD states is discussed. iTRAQ is a powerful differential isotopic quantitative approach in proteomics. Pulsed Q dissociation (PQD) is a recently introduced fragmentation strategy that enables detection of low mass iTRAQ reporter ions in ion trap mass spectrometry. We optimized LTQ ion trap settings for PQD-based iTRAQ quantitation and demonstrated its utility in O-GlcNAc site mapping. Using iTRAQ, abnormal synaptic expression levels of several proteins previously implicated in AD pathology were observed in addition to novel changes in synaptic specific protein expression including Synapsin II.     

Adenosine Augmentation Evoked by an ENT1 Inhibitor Improves Memory Impairment and Neuronal Plasticity in the APP/PS1 Mouse Model of Alzheimer’s Disease

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by cognitive impairment and synaptic dysfunction. Adenosine is an important homeostatic modulator that controls the bioenergetic network in the brain through regulating receptor-evoked signaling pathways, bioenergetic machineries, and epigenetic-mediated gene regulation. Equilibrative nucleoside transporter 1 (ENT1) is a major adenosine transporter that recycles adenosine from the extracellular space. In the present study, we report that a small adenosine analogue (designated J4) that inhibited ENT1 prevented the decline in spatial memory in an AD mouse model (APP/PS1). Electrophysiological and biochemical analyses further demonstrated that chronic treatment with J4 normalized the impaired basal synaptic transmission and long-term potentiation (LTP) at Schaffer collateral synapses as well as the aberrant expression of synaptic proteins (e.g., NR2A and NR2B), abnormal neuronal plasticity-related signaling pathways (e.g., PKA and GSK3β), and detrimental elevation in astrocytic A_2AR expression in the hippocampus and cortex of APP/PS1 mice. In conclusion, our findings suggest that modulation of adenosine homeostasis by J4 is beneficial in a mouse model of AD. Our study provides a potential therapeutic strategy to delay the progression of AD.     

Quantitative Proteomics and Targeted Fatty Acids Analysis Reveal the Damage of Triptolide in Liver and Kidney

Triptolide (TP), the major active component in Tripterygium wilfordii Hook. f., is widely used for the treatment of rheumatoid arthritis and autoimmune diseases. However, organ toxicity, especially hepatotoxicity and nephrotoxicity, limits its clinical application. To fully understand the mechanism underlying TP toxicity, iTRAQ‐based 2D‐LC‐MS/MS proteomics is used to detect differentially expressed proteins in the livers and kidneys of mice administered the LD50 dose of TP. Functional annotation revealed that multiple pathways are involved in TP toxicity, including acute‐phase response signaling, the antigen presentation pathway, FXR/RXR activation, LPS/IL‐1‐mediated inhibition of RXR function, and EIF2 signaling. Members of the cytochrome P450 protein family that are involved in fatty acid (FA) metabolism, such as CYP2E1, show significant differences in expression among groups. Additionally, the proteomics data suggested that FAs are involved in TP‐induced toxicity. FA analysis is conducted using HPLC‐MRM to characterize the differences among various groups exposed to TP for different times. It has been found that 20 FAs in the liver show significant differences in abundance among groups, whereas in the kidneys, six FAs show significant differences in abundance. By integrating the proteomic and targeted FA analyses, it has been found that differently expressed proteins and FAs both participate in pathways including cellular lipolytic activity, peroxisomal fatty acid beta‐oxidation, and so on. Our data contribute to understanding the mechanisms underlying TP‐induced organ toxicity. The results may help to improve the clinical efficacy and safety of TP in the future.     

iTRAQ is a useful method to screen for membrane-bound proteins differentially expressed in human natural killer cell types

We are interested in the biological as well as the molecular processes involved in natural killer (NK) cell development and function. Determining the proteomic complement could be a useful tool in predicting cellular function and fate. For the first time shown here, we have utilized iTRAQ, a new method that allows identification and quantification of proteins between multiple samples, to determine the expression of membrane-bound proteins in two previously characterized human NK cell populations. One population was derived from umbilical cord blood (UCB) stem cells (CD34+38-Lin-) and the other from expanded CD3-depleted adult peripheral blood. iTRAQ was employed for multiplex peptide labeling of proteins from fractionated membranes followed by two-dimensional high-performance liquid chromatography (2D-HPLC), and tandem mass spectrometry was used to identify protein signatures. We were able to identify and quantify differences in expression levels of 400-800 proteins in a typical experiment. Ontology analysis showed the majority of the proteins to be involved in cell signaling, nucleic acid binding, or mitochondrial function. Nearly all proteins were associated with the plasma membrane, membrane-bound organelle (lysosome or mitochondria), or nucleus. We found several novel proteins highly expressed in UCB stem cell derived NK cells compared to adult NK cells including CD9, alpha-2 macroglobulin, brain abundant signaling protein (BASP1), and allograft inflammatory factor-1 (AIF-1). In addition, we were able to confirm several of our iTRAQ results by RT-PCR, Western blot, and fluorescence-activated cell-sorting (FACS) analysis. This is the first demonstration and verification using iTRAQ to screen for membrane-bound protein differences in human NK cells and represents a powerful new tool in the field of proteomics.     

Effects of DISC1 on Alzheimer’s disease cell models assessed by iTRAQ proteomics analysis

Alzheimer’s disease (AD) is a form of neurodegenerative disease in the elderly with no cure at present. In a previous study, we found that the scaffold protein, disrupted in Schizophrenia 1 (DISC1) is down-regulated in the AD brains, and ectopic expression of DISC1 can delay the progression of AD by protecting synaptic plasticity and down-regulating BACE1. However, the underlying mechanisms remain not to be elucidated. In the present study, we compared the proteomes of normal and DISC1^high AD cells expressing the amyloid precursor protein (APP) using isobaric tag for relative and absolute quantitation (iTRAQ) and mass spectrometry (MS). The differentially expressed proteins (DEPs) were identified, and the protein–protein interaction (PPI) network was constructed to identify the interacting partners of DISC1. Based on the interaction scores, NDE1, GRM3, PTGER3 and KATNA1 were identified as functionally or physically related to DISC1, and may therefore regulate AD development. The DEPs were functionally annotated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases with the DAVID software, and the Non-supervised Orthologous Groups (eggNOG) database was used to determine their evolutionary relationships. The DEPs were significantly enriched in microtubules and mitochondria-related pathways. Gene set enrichment analysis (GSEA) was performed to identify genes and pathways that are activated when DISC1 is overexpressed. Our findings provide novel insights into the regulatory mechanisms underlying DISC1 function in AD.     

YAP1 regulates PPARG and RXR alpha expression to affect the proliferation and differentiation of ovine preadipocyte

Adipose tissue development is regulated by a serial of developmental signaling pathways. The Hippo pathway is a novel signaling cascade closely associated with adipogenesis. While most of Hippo pathway components had been verified that have a vital role in preadipocytes proliferation and differentiation, little is known about the function of Yes-associated protein 1 (YAP1) in mammalian adipose tissue development. Therefore, we investigated the role of YAP1 in ovine adipose tissue development by in vitro and in vivo experiments. We observed that the adipocyte size in subcutaneous adipose tissue increased with development. YAP1 expression increased during adipose tissue development, while decreased during the differentiation of ovine preadipocytes in vitro. YAP1 knockdown notably promoted lipid accumulation and suppressed ovine preadipocyte proliferation. In addition, we observed that YAP1 deficiency significantly upregulated peroxisome proliferator-activated receptor gamma (PPARG) and retinoid X receptor alpha (RXR alpha) expression. By contrast, overexpression of YAP1 led to the suppression of preadipocyte differentiation, lipid droplets formation, and PPARG expression. In brief, our findings demonstrated that YAP1 regulates the proliferation and differentiation of ovine preadipocyte via altering PPARG and RXR alpha expression.     

Retinoic Acid Isomers Facilitate Apolipoprotein E Production and Lipidation in Astrocytes through the Retinoid X Receptor/Retinoic Acid Receptor Pathway

Apolipoprotein E (apoE) is the major cholesterol transport protein in the brain. Among the three human APOE alleles (APOE2, APOE3, and APOE4), APOE4 is the strongest genetic risk factor for late-onset Alzheimer disease (AD). The accumulation of amyloid-β (Aβ) is a central event in AD pathogenesis. Increasing evidence demonstrates that apoE isoforms differentially regulate AD-related pathways through both Aβ-dependent and -independent mechanisms; therefore, modulating apoE secretion, lipidation, and function might be an attractive approach for AD therapy. We performed a drug screen for compounds that modulate apoE production in immortalized astrocytes derived from apoE3-targeted replacement mice. Here, we report that retinoic acid (RA) isomers, including all-trans-RA, 9-cis-RA, and 13-cis-RA, significantly increase apoE secretion to ∼4-fold of control through retinoid X receptor (RXR) and RA receptor. These effects on modulating apoE are comparable with the effects recently reported for the RXR agonist bexarotene. Furthermore, all of these compounds increased the expression of the cholesterol transporter ABCA1 and ABCG1 levels and decreased cellular uptake of Aβ in an apoE-dependent manner. Both bexarotene and 9-cis-RA promote the lipidation status of apoE, in which 9-cis-RA promotes a stronger effect and exhibits less cytotoxicity compared with bexarotene. Importantly, we showed that oral administration of bexarotene and 9-cis-RA significantly increases apoE, ABCA1, and ABCG1 levels in mouse brains. Taken together, our results demonstrate that RXR/RA receptor agonists, including several RA isomers, are effective modulators of apoE secretion and lipidation and may be explored as potential drugs for AD therapy.