BackgroundThe National NeuroAIDS Tissue Consortium (NNTC) performed a brain gene expression array to elucidate pathophysiologies of Human Immunodeficiency Virus type 1 (HIV-1)-associated neurocognitive disorders.MethodsTwenty-four human subjects in four groups were examined A) Uninfected controls; B) HIV-1 infected subjects with no substantial neurocognitive impairment (NCI); C) Infected with substantial NCI without HIV encephalitis (HIVE); D) Infected with substantial NCI and HIVE. RNA from neocortex, white matter, and neostriatum was processed with the Affymetrix® array platform.ResultsWith HIVE the HIV-1 RNA load in brain tissue was three log10 units higher than other groups and over 1,900 gene probes were regulated. Interferon response genes (IFRGs), antigen presentation, complement components and CD163 antigen were strongly upregulated. In frontal neocortex downregulated neuronal pathways strongly dominated in HIVE, including GABA receptors, glutamate signaling, synaptic potentiation, axon guidance, clathrin-mediated endocytosis and 14-3-3 protein. Expression was completely different in neuropsychologically impaired subjects without HIVE. They had low brain HIV-1 loads, weak brain immune responses, lacked neuronally expressed changes in neocortex and exhibited upregulation of endothelial cell type transcripts. HIV-1-infected subjects with normal neuropsychological test results had upregulation of neuronal transcripts involved in synaptic transmission of neostriatal circuits.InterpretationTwo patterns of brain gene expression suggest that more than one pathophysiological process occurs in HIV-1-associated neurocognitive impairment. Expression in HIVE suggests that lowering brain HIV-1 replication might improve NCI, whereas NCI without HIVE may not respond in kind; array results suggest that modulation of transvascular signaling is a potentially promising approach. Striking brain regional differences highlighted the likely importance of circuit level disturbances in HIV/AIDS. In subjects without impairment regulation of genes that drive neostriatal synaptic plasticity reflects adaptation. The array provides an infusion of public resources including brain samples, clinicopathological data and correlative gene expression data for further exploration (http://www.nntc.org/gene-array-project).

We previously reported that the peroxisome-proliferator-activated receptor gamma (PPARγ) agonist rosiglitazone (RSG) improved hippocampus-dependent cognition in the Alzheimer's disease (AD) mouse model, Tg2576. RSG had no effect on wildtype littermate cognitive performance. Since 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 PPRE-containing PPARγ target genes and ERK-regulated, CRE-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 pERK that is reciprocally affected in response to chronic activation, compared to 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.

Evidence of diabetes-induced nuclear factor-B (NF-B)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-B canonical and noncanonical activation pathways in the renal cortex of diabetic mice. Plasma concentrations of NF-B-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-B canonical pathway proteins did not differ between experimental groups after 3 months of diabetes, while NF-B noncanonical pathway proteins were affected, including increased phosphorylation of inhibitor of B kinase-␣ and several fold increases in NF-B-inducing kinase and RelB, which were predominantly located in tubular epithelial cells. Nuclear content of all NF-B 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-B activation in the renal cortex of db/db mice and suggest a novel, diabetes-linked mechanism involving both canonical and noncanonical NF-B pathway proteins.

BackgroundNumerous 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 FindingsTotal 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/SignificanceOur 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.

Hippocampal network hyperexcitability is considered an early indicator of Alzheimer's 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 (PPAR␥) axis improves hippocampal cognition in mouse models for AD as well as benefitting memory performance in some humans with early AD, we investigated how PPAR␥ agonism affected synaptic activity in Tg2576 DG. We found that PPAR␥ 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 PPAR␥ activation occurs through regulation of presynaptic vesicular proteins critical for proper glutamatergic neurotransmitter release, synaptic transmission, and short-term plasticity.

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 allows commonly used ion trap mass spectrometers to attain isotopic resolution, which make 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 which may be uniquely suited to these data types. The software implementation uses power spectrum to remove high-frequency noise, and band-filter contributions from co-eluting 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.

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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 18 O-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, CaNBa, 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.