Sigmund J. Haidacher

Cognitive Enhancement with Rosiglitazone Links the Hippocampal PPARγ and ERK MAPK Signaling Pathways

We previously reported that the peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone (RSG) improved hippocampus-dependent cognition in the Alzheimers disease (AD) mouse model, Tg2576. RSG had no effect on wild-type littermate cognitive performance. Since extracellular signal-regulated protein kinase mitogen-activated protein kinase (ERK MAPK) is required for many forms of learning and memory that are affected in AD, and since both PPARγ and ERK MAPK are key mediators of insulin signaling, the current study tested the hypothesis that RSG-mediated cognitive improvement induces a hippocampal PPARγ pattern of gene and protein expression that converges with the ERK MAPK signaling axis in Tg2576 AD mice. In the hippocampal PPARγ transcriptome, we found significant overlap between peroxisome proliferator response element-containing PPARγ target genes and ERK-regulated, cAMP response element-containing target genes. Within the Tg2576 dentate gyrus proteome, RSG induced proteins with structural, energy, biosynthesis and plasticity functions. Several of these proteins are known to be important for cognitive function and are also regulated by ERK MAPK. In addition, we found the RSG-mediated augmentation of PPARγ and ERK2 activity during Tg2576 cognitive enhancement was reversed when hippocampal PPARγ was pharmacologically antagonized, revealing a coordinate relationship between PPARγ transcriptional competency and phosphorylated ERK that is reciprocally affected in response to chronic activation, compared with acute inhibition, of PPARγ. We conclude that the hippocampal transcriptome and proteome induced by cognitive enhancement with RSG harnesses a dysregulated ERK MAPK signal transduction pathway to overcome AD-like cognitive deficits in Tg2576 mice. Thus, PPARγ represents a signaling system that is not crucial for normal cognition yet can intercede to restore neural networks compromised by AD.

Diabetes-Induced Activation of Canonical and Noncanonical Nuclear Factor-κB Pathways in Renal Cortex

Evidence of diabetes-induced nuclear factor-κB (NF-κB) activation has been provided with DNA binding assays or nuclear localization with immunohistochemistry, but few studies have explored mechanisms involved. We examined effects of diabetes on proteins comprising NF-κB canonical and noncanonical activation pathways in the renal cortex of diabetic mice. Plasma concentrations of NF-κB–regulated cytokines were increased after 1 month of hyperglycemia, but most returned to control levels or lower by 3 months, when the same cytokines were increased significantly in renal cortex. Cytosolic content of NF-κB canonical pathway proteins did not differ between experimental groups after 3 months of diabetes, while NF-κB noncanonical pathway proteins were affected, including increased phosphorylation of inhibitor of κB kinase-α and several fold increases in NF-κB–inducing kinase and RelB, which were predominantly located in tubular epithelial cells. Nuclear content of all NF-κB pathway proteins was decreased by diabetes, with the largest change in RelB and p50 (approximately twofold decrease). Despite this decrease, measurable increases in protein binding to DNA in diabetic versus control nuclear extracts were observed with electrophoretic mobility shift assay. These results provide evidence for chronic NF-κB activation in the renal cortex of db/db mice and suggest a novel, diabetes-linked mechanism involving both canonical and noncanonical NF-κB pathway proteins.

Altered Retinoic Acid Metabolism in Diabetic Mouse Kidney Identified by 18O Isotopic Labeling and 2D Mass Spectrometry

Background Numerous metabolic pathways have been implicated in diabetes-induced renal injury, yet few studies have utilized unbiased systems biology approaches for mapping the interconnectivity of diabetes-dysregulated proteins that are involved. We utilized a global, quantitative, differential proteomic approach to identify a novel retinoic acid hub in renal cortical protein networks dysregulated by type 2 diabetes. Methodology/Principal Findings Total proteins were extracted from renal cortex of control and db/db mice at 20 weeks of age (after 12 weeks of hyperglycemia in the diabetic mice). Following trypsinization, 18O- and 16O-labeled control and diabetic peptides, respectively, were pooled and separated by two dimensional liquid chromatography (strong cation exchange creating 60 fractions further separated by nano-HPLC), followed by peptide identification and quantification using mass spectrometry. Proteomic analysis identified 53 proteins with fold change ≥1.5 and p≤0.05 after Benjamini-Hochberg adjustment (out of 1,806 proteins identified), including alcohol dehydrogenase (ADH) and retinaldehyde dehydrogenase (RALDH1/ALDH1A1). Ingenuity Pathway Analysis identified altered retinoic acid as a key signaling hub that was altered in the diabetic renal cortical proteome. Western blotting and real-time PCR confirmed diabetes-induced upregulation of RALDH1, which was localized by immunofluorescence predominantly to the proximal tubule in the diabetic renal cortex, while PCR confirmed the downregulation of ADH identified with mass spectrometry. Despite increased renal cortical tissue levels of retinol and RALDH1 in db/db versus control mice, all-trans-retinoic acid was significantly decreased in association with a significant decrease in PPARβ/δ mRNA. Conclusions/Significance Our results indicate that retinoic acid metabolism is significantly dysregulated in diabetic kidneys, and suggest that a shift in all-trans-retinoic acid metabolism is a novel feature in type 2 diabetic renal disease. Our observations provide novel insights into potential links between altered lipid metabolism and other gene networks controlled by retinoic acid in the diabetic kidney, and demonstrate the utility of using systems biology to gain new insights into diabetic nephropathy.

Cognitive Enhancing Treatment with a PPARγ Agonist Normalizes Dentate Granule Cell Presynaptic Function in Tg2576 APP Mice

Hippocampal network hyperexcitability is considered an early indicator of Alzheimers disease (AD) memory impairment. Some AD mouse models exhibit similar network phenotypes. In this study we focused on dentate gyrus (DG) granule cell spontaneous and evoked properties in 9-month-old Tg2576 mice that model AD amyloidosis and cognitive deficits. Using whole-cell patch-clamp recordings, we found that Tg2576 DG granule cells exhibited spontaneous EPSCs that were higher in frequency but not amplitude compared with wild-type mice, suggesting hyperactivity of DG granule cells via a presynaptic mechanism. Further support of a presynaptic mechanism was revealed by increased I–O relationships and probability of release in Tg2576 DG granule cells. Since we and others have shown that activation of the peroxisome proliferator-activated receptor gamma (PPARγ) axis improves hippocampal cognition in mouse models for AD as well as benefitting memory performance in some humans with early AD, we investigated how PPARγ agonism affected synaptic activity in Tg2576 DG. We found that PPARγ agonism normalized the I–O relationship of evoked EPSCs, frequency of spontaneous EPSCs, and probability of release that, in turn, correlated with selective expression of DG proteins essential for presynaptic SNARE function that are altered in patients with AD. These findings provide evidence that DG principal cells may contribute to early AD hippocampal network hyperexcitability via a presynaptic mechanism, and that hippocampal cognitive enhancement via PPARγ activation occurs through regulation of presynaptic vesicular proteins critical for proper glutamatergic neurotransmitter release, synaptic transmission, and short-term plasticity.

Biomarker Discovery for Early Detection of Hepatocellular Carcinoma in Hepatitis C–infected Patients

Chronic hepatic disease damages the liver, and the resulting wound-healing process leads to liver fibrosis and the subsequent development of cirrhosis. The leading cause of hepatic fibrosis and cirrhosis is infection with hepatitis C virus (HCV), and of the patients with HCV-induced cirrhosis, 2% to 5% develop hepatocellular carcinoma (HCC), with a survival rate of 7%. HCC is one of the leading causes of cancer-related death worldwide, and the poor survival rate is largely due to late-stage diagnosis, which makes successful intervention difficult, if not impossible. The lack of sensitive and specific diagnostic tools and the urgent need for early-stage diagnosis prompted us to discover new candidate biomarkers for HCV and HCC. We used aptamer-based fractionation technology to reduce serum complexity, differentially labeled samples (six HCV and six HCC) with fluorescent dyes, and resolved proteins in pairwise two-dimensional difference gel electrophoresis. DeCyder software was used to identify differentially expressed proteins and spots picked, and MALDI-MS/MS was used to determine that ApoA1 was down-regulated by 22% (p < 0.004) in HCC relative to HCV. Differential expression quantified via two-dimensional difference gel electrophoresis was confirmed by means of 18O/16O stable isotope differential labeling with LC-MS/MS zoom scans. Technically independent confirmation was demonstrated by triple quadrupole LC-MS/MS selected reaction monitoring (SRM) assays with three peptides specific to human ApoA1 (DLATVYVDVLK, WQEEMELYR, and VSFLSALEEYTK) using 18O/16O-labeled samples and further verified with AQUA peptides as internal standards for quantification. In 50 patient samples (24 HCV and 26 HCC), all three SRM assays yielded highly similar differential expression of ApoA1 in HCC and HCV patients. These results validated the SRM assays, which were independently confirmed by Western blotting. Thus, ApoA1 is a candidate member of an SRM biomarker panel for early diagnosis, prognosis, and monitoring of HCC. Future multiplexing of SRM assays for other candidate biomarkers is envisioned to develop a biomarker panel for subsequent verification and validation studies.

Steroid receptor coactivator-2 expression in brain and physical associations with steroid receptors

Estradiol and progesterone bind to their respective receptors in the hypothalamus and hippocampus to influence a variety of behavioral and physiological functions, including reproduction and cognition. Work from our lab and others has shown that the nuclear receptor coactivators, steroid receptor coactivator-1 (SRC-1) and SRC-2, are essential for efficient estrogen receptor (ER) and progestin receptor (PR) transcriptional activity in brain and for hormone-dependent behaviors. While the expression of SRC-1 in brain has been studied extensively, little is known about the expression of SRC-2 in brain. In the present studies, we found that SRC-2 was highly expressed throughout the hippocampus, amygdala and hypothalamus, including the medial preoptic area (MPOA), ventral medial nucleus (VMN), arcuate nucleus (ARC), bed nucleus of the stria terminalis, supraoptic nucleus and suprachiasmatic nucleus. In order for coactivators to function with steroid receptors, they must be expressed in the same cells. Indeed, SRC-2 and ER(alpha) were coexpressed in many cells in the MPOA, VMN and ARC, all brain regions known to be involved in female reproductive behavior and physiology. While in vitro studies indicate that SRC-2 physically associates with ER and PR, very little is known about receptor-coactivator interactions in brain. Therefore, we used pull-down assays to test the hypotheses that SRC-2 from hypothalamic and hippocampal tissue physically associate with ER and PR subtypes in a ligand-dependent manner. SRC-2 from both brain regions interacted with ER(alpha) bound to agonist, but not in the absence of ligand or in the presence of the selective ER modulator, tamoxifen. Analysis by mass spectrometry confirmed these ligand-dependent interactions between ER(alpha) and SRC-2 from brain. In dramatic contrast, SRC-2 from brain showed little to no interaction with ERbeta. Interestingly, SRC-2 from both brain regions interacted with PR-B, but not PR-A, in a ligand-dependent manner. Taken together, these findings reveal that SRC-2 is expressed in brain regions known to mediate a variety of steroid-dependent functions. Furthermore, SRC-2 is expressed in many ER(alpha) containing cells in the hypothalamus. Finally, SRC-2 from brain interacts with ER and PR in a subtype-specific manner, which may contribute to the functional differences of these steroid receptor subtypes in brain.

Comprehensive analysis of the mouse renal cortex using two-dimensional HPLC – tandem mass spectrometry

BackgroundProteomic methodologies increasingly have been applied to the kidney to map the renal cortical proteome and to identify global changes in renal proteins induced by diseases such as diabetes. While progress has been made in establishing a renal cortical proteome using 1-D or 2-DE and mass spectrometry, the number of proteins definitively identified by mass spectrometry has remained surprisingly small. Low coverage of the renal cortical proteome as well as our interest in diabetes-induced changes in proteins found in the renal cortex prompted us to perform an in-depth proteomic analysis of mouse renal cortical tissue.ResultsWe report a large scale analysis of mouse renal cortical proteome using SCX prefractionation strategy combined with HPLC – tandem mass spectrometry. High-confidence identification of ~2,000 proteins, including cytoplasmic, nuclear, plasma membrane, extracellular and unknown/unclassified proteins, was obtained by separating tryptic peptides of renal cortical proteins into 60 fractions by SCX prior to LC-MS/MS. The identified proteins represented the renal cortical proteome with no discernible bias due to protein physicochemical properties, subcellular distribution, biological processes, or molecular function. The highest ranked molecular functions were characteristic of tubular epithelium, and included binding, catalytic activity, transporter activity, structural molecule activity, and carrier activity. Comparison of this renal cortical proteome with published human urinary proteomes demonstrated enrichment of renal extracellular, plasma membrane, and lysosomal proteins in the urine, with a lack of intracellular proteins. Comparison of the most abundant proteins based on normalized spectral abundance factor (NSAF) in this dataset versus a published glomerular proteome indicated enrichment of mitochondrial proteins in the former and cytoskeletal proteins in the latter.ConclusionA whole tissue extract of the mouse kidney cortex was analyzed by an unbiased proteomic approach, yielding a dataset of ~2,000 unique proteins identified with strict criteria to ensure a high level of confidence in protein identification. As a result of extracting all proteins from the renal cortex, we identified an exceptionally wide range of renal proteins in terms of pI, MW, hydrophobicity, abundance, and subcellular location. Many of these proteins, such as low-abundance proteins, membrane proteins and proteins with extreme values in pI or MW are traditionally under-represented in 2-DE-based proteomic analysis.

Detection of Structural and Metabolic Changes in Traumatically Injured Hippocampus by Quantitative Differential Proteomics

Traumatic brain injury (TBI) is a complex and common problem resulting in the loss of cognitive function. In order to build a comprehensive knowledge base of the proteins that underlie these cognitive deficits, we employed unbiased quantitative mass spectrometry, proteomics, and bioinformatics to identify and quantify dysregulated proteins in the CA3 subregion of the hippocampus in the fluid percussion model of TBI in rats. Using stable isotope 18O-water differential labeling and multidimensional tandem liquid chromatography (LC)-MS/MS with high stringency statistical analyses and filtering, we identified and quantified 1002 common proteins, with 124 increased and 76 decreased. The ingenuity pathway analysis (IPA) bioinformatics tool identified that TBI had profound effects on downregulating global energy metabolism, including glycolysis, the Krebs cycle, and oxidative phosphorylation, as well as cellular structure and function. Widespread upregulation of actin-related cytoskeletal dynamics was also found. IPA indicated a common integrative signaling node, calcineurin B1 (CANB1, CaNBα, or PPP3R1), which was downregulated by TBI. Western blotting confirmed that the calcineurin regulatory subunit, CANB1, and its catalytic binding partner PP2BA, were decreased without changes in other calcineurin subunits. CANB1 plays a critical role in downregulated networks of calcium signaling and homeostasis through calmodulin and calmodulin-dependent kinase II to highly interconnected structural networks dominated by tubulins. This large-scale knowledge base lays the foundation for the identification of novel therapeutic targets for cognitive rescue in TBI.

Using Power Spectrum Analysis to Evaluate 18O-Water Labeling Data Acquired from Low Resolution Mass Spectrometers

We describe a method for ratio estimations in (18)O-water labeling experiments acquired from low resolution isotopically resolved data. The method is implemented in a software package specifically designed for use in experiments making use of zoom-scan mode data acquisition. Zoom-scan mode data allow commonly used ion trap mass spectrometers to attain isotopic resolution, which makes them amenable to use in labeling schemes such as (18)O-water labeling, but algorithms and software developed for high resolution instruments may not be appropriate for the lower resolution data acquired in zoom-scan mode. The use of power spectrum analysis is proposed as a general approach that may be uniquely suited to these data types. The software implementation uses a power spectrum to remove high-frequency noise and band-filter contributions from coeluting species of differing charge states. From the elemental composition of a peptide sequence, we generate theoretical isotope envelopes of heavy-light peptide pairs in five different ratios; these theoretical envelopes are correlated with the filtered experimental zoom scans. To automate peptide quantification in high-throughput experiments, we have implemented our approach in a computer program, MassXplorer. We demonstrate the application of MassXplorer to two model mixtures of known proteins and to a complex mixture of mouse kidney cortical extract. Comparison with another algorithm for ratio estimations demonstrates the increased precision and automation of MassXplorer.

PPARgamma agonists rescue increased phosphorylation of FGF14 at S226 in the Tg2576 mouse model of Alzheimer's disease

Background Cognitive impairment in humans with Alzheimers disease (AD) and in animal models of A&bgr;‐pathology can be ameliorated by treatments with the nuclear receptor peroxisome proliferator‐activated receptor‐gamma (PPAR&ggr;) agonists, such as rosiglitazone (RSG). Previously, we demonstrated that in the Tg2576 animal model of AD, RSG treatment rescued cognitive deficits and reduced aberrant activity of granule neurons in the dentate gyrus (DG), an area critical for memory formation. Methods We used a combination of mass spectrometry, confocal imaging, electrophysiology and split‐luciferase assay and in vitro phosphorylation and Ingenuity Pathway Analysis. Results Using an unbiased, quantitative nano‐LC‐MS/MS screening, we searched for potential molecular targets of the RSG‐dependent rescue of DG granule neurons. We found that S226 phosphorylation of fibroblast growth factor 14 (FGF14), an accessory protein of the voltage‐gated Na+ (Nav) channels required for neuronal firing, was reduced in Tg2576 mice upon treatment with RSG. Using confocal microscopy, we confirmed that the Tg2576 condition decreased PanNav channels at the AIS of the DG, and that RSG treatment of Tg2576 mice reversed the reduction in PanNav channels. Analysis from previously published data sets identified correlative changes in action potential kinetics in RSG‐treated T2576 compared to untreated and wildtype controls. In vitro phosphorylation and mass spectrometry confirmed that the multifunctional kinase GSK–3&bgr;, a downstream target of insulin signaling highly implicated in AD, phosphorylated FGF14 at S226. Assembly of the FGF14:Nav1.6 channel complex and functional regulation of Nav1.6‐mediated currents by FGF14 was impaired by a phosphosilent S226A mutation. Bioinformatics pathway analysis of mass spectrometry and biochemistry data revealed a highly interconnected network encompassing PPAR&ggr;, FGF14, SCN8A (Nav 1.6), and the kinases GSK–3 &bgr;, casein kinase 2&bgr;, and ERK1/2. Conclusions These results identify FGF14 as a potential PPAR&ggr;‐sensitive target controlling A&bgr;‐induced dysfunctions of neuronal activity in the DG underlying memory loss in early AD. HighlightsPhosphorylation of FGF14 at S226 in Tg2576 animals is reduced by rosiglitazone (RSG).Tg2576 condition decreases PanNav channels at AIS, which is reversed by RSG.GSK‐3&bgr; phosphorylates FGF14 at S226.Assembly of FGF14:Nav1.6 channel complex is reduced by a S226A mutationFunctional regulation of Nav1.6‐mediated currents was impaired by a S226A mutation.