Adult Human Glia,Pericytes and Meningeal Fibroblasts Respond Similarly to IFNy but Not to TGFβ1 or M-CSF

The chemokine Interferon gamma-induced protein 10 (IP-10) and human leukocyte antigen (HLA) are widely used indicators of glial activation and neuroinflammation and are up-regulated in many brain disorders. These inflammatory mediators have been widely studied in rodent models of brain disorders, but less work has been undertaken using human brain cells. In this study we investigate the regulation of HLA and IP-10, as well as other cytokines and chemokines, in microglia, astrocytes, pericytes, and meningeal fibroblasts derived from biopsy and autopsy adult human brain, using immunocytochemistry and a Cytometric Bead Array. Interferonγ (IFNγ) increased microglial HLA expression, but contrary to data in rodents, the anti-inflammatory cytokine transforming growth factor β1 (TGFβ₁) did not inhibit this increase in HLA, nor did TGFβ₁ affect basal microglial HLA expression or IFNγ-induced astrocytic HLA expression. In contrast, IFNγ-induced and basal microglial HLA expression, but not IFNγ-induced astrocytic HLA expression, were strongly inhibited by macrophage colony stimulating factor (M-CSF). IFNγ also strongly induced HLA expression in pericytes and meningeal fibroblasts, which do not basally express HLA, and this induction was completely blocked by TGFβ₁, but not affected by M-CSF. In contrast, TGFβ₁ did not block the IFNγ-induced increase in IP-10 in pericytes and meningeal fibroblasts. These results show that IFNγ, TGFβ₁ and M-CSF have species- and cell type-specific effects on human brain cells that may have implications for their roles in adult human brain inflammation.     

Population-specific expression analysis (PSEA) reveals molecular changes in diseased brain

Human diseases are often accompanied by histological changes that confound interpretation of molecular analyses and identification of disease-related effects. We developed population-specific expression analysis (PSEA), a computational method of analyzing gene expression in samples of varying composition that can improve analyses of quantitative molecular data in many biological contexts. PSEA of brains from individuals with Huntington's disease revealed myelin-related abnormalities that were undetected using standard differential expression analysis.     

Fragments of HdhQ150 Mutant Huntingtin Form a Soluble Oligomer Pool That Declines with Aggregate Deposition upon Aging

Cleavage of the full-length mutant huntingtin (mhtt) protein into smaller, soluble aggregation-prone mhtt fragments appears to be a key process in the neuropathophysiology of Huntington’s Disease (HD). Recent quantification studies using TR-FRET-based immunoassays showed decreasing levels of soluble mhtt correlating with an increased load of aggregated mhtt in the aging HdhQ150 mouse brain. To better characterize the nature of these changes at the level of native mhtt species, we developed a detection method that combines size exclusion chromatography (SEC) and time-resolved fluorescence resonance energy transfer (TR-FRET) that allowed us to resolve and define the formation, aggregation and temporal dynamics of native soluble mhtt species and insoluble aggregates in the brain of the HdhQ150 knock-in mouse. We found that mhtt fragments and not full-length mhtt form oligomers in the brains of one month-old mice long before disease phenotypes and mhtt aggregate histopathology occur. As the HdhQ150 mice age, brain levels of soluble full-length mhtt protein remain similar. In contrast, the soluble oligomeric pool of mhtt fragments slightly increases during the first two months before it declines between 3 and 8 months of age. This decline inversely correlates with the formation of insoluble mhtt aggregates. We also found that the pool-size of soluble mhtt oligomers is similar in age-matched heterozygous and homozygous HdhQ150 mouse brains whereas insoluble aggregate formation is greatly accelerated in the homozygous mutant brain. The capacity of the soluble mhtt oligomer pool therefore seems exhausted already in the heterozygous state and likely kept constant by changes in flux and, as a consequence, increased rate of insoluble aggregate formation. We demonstrate that our novel findings in mice translate to human HD brain but not HD patient fibroblasts.     

Mapping the cofilin binding site on yeast G-actin by chemical cross-linking

Cofilin is a major cytoskeletal protein that binds to both monomeric actin (G-actin) and polymeric actin (F-actin) and is involved in microfilament dynamics. Although an atomic structure of the G-actin-cofilin complex does not exist, models of the complex have been built using molecular dynamics simulations, structural homology considerations, and synchrotron radiolytic footprinting data. The hydrophobic cleft between actin subdomains 1 and 3 and, alternatively, the cleft between actin subdomains 1 and 2 have been proposed as possible high-affinity cofilin binding sites. In this study, the proposed binding of cofilin to the subdomain 1/subdomain 3 region on G-actin has been probed using site-directed mutagenesis, fluorescence labeling, and chemical cross-linking, with yeast actin mutants containing single reactive cysteines in the actin hydrophobic cleft and with cofilin mutants carrying reactive cysteines in the regions predicted to bind to G-actin. Mass spectrometry analysis of the cross-linked complex revealed that cysteine 345 in subdomain 1 of mutant G-actin was cross-linked to native cysteine 62 on cofilin. A cofilin mutant that carried a cysteine substitution in the α3-helix (residue 95) formed a cross-link with residue 144 in actin subdomain 3. Distance constraints imposed by these cross-links provide experimental evidence for cofilin binding between actin subdomains 1 and 3 and fit a corresponding docking-based structure of the complex. The cross-linking of the N-terminal region of recombinant yeast cofilin to actin residues 346 and 374 with dithio-bis-maleimidoethane (12.4 Å) and via disulfide bond formation was also documented. This set of cross-linking data confirms the important role of the N-terminal segment of cofilin in interactions with G-actin.     

A new endogenous form of PYY isolated from canine ileum: Gly-extended PYY(1-36)

We purified and identified the peptide YY (PYY) forms present and determined their levels from a portion of the canine ileum directly adjacent to the cecum by a new extraction method designed to prevent and evaluate degradation of endogenous peptides. We used three reverse phase chromatography steps with radioimmunoassay of fractions for PYY-like-immunoreactivity (PYY-LI). The purified fractions underwent intact protein/peptide mass spectrometry identification and sequencing (i.e. "top-down" MS analysis). This analysis confirmed the identity of a new form of PYY, PYY(1-36)-Gly, which co-elutes with PYY(1-36)-NH(2) through all three of separation steps used. The PYY(1-36)-Gly form represents approximately 20% of the total PYY found in this region of the canine intestine. In addition, we also found that the PYY(3-36)-NH(2) form represents 6% of the total PYY in the canine ileo-cecal junction. The physiological implication of the Gly-extended form of PYY(1-36) warrants further investigation.     

Nascent RNA antagonizes the interaction of a set of regulatory proteins with chromatin

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N-Benzylindole-2-carboxylic acids: potent functional antagonists of the CCR2b chemokine receptor

Screening of the corporate database led to the discovery of a novel series of N-benzylindole-2-carboxylic acid CCR2b chemokine receptor antagonists. These compounds demonstrate high affinity and functional inhibition of the CCR2b receptor. A discussion of the structure-activity relationships is presented, together with evidence for a highly selective receptor binding profile.     

Characterization of [(3)H]Quisqualate binding to recombinant rat metabotropic glutamate 1a and 5a receptors and to rat and human brain sections

We have investigated the binding properties of [(3)H]quisqualate to rat metabotropic glutamate (mGlu) 1a and 5a receptors and to rat and human brain sections. Saturation isotherms gave K:(D) values of 27 +/- 4 and 81 +/- 22 nM: for mGlu1a and mGlu5a receptors, respectively. Several compounds inhibited the binding to mGlu1a and mGlu5a receptors concentration-dependently. (S:)-4-Carboxyphenylglycine, (S:)-4-carboxy-3-hydroxyphenylglycine, and (R,S)-1-aminoindan-1,5-dicarboxylic acid, which completely inhibited [(3)H]quisqualate binding to the mGlu5a receptor, were inactive in a functional assay using this receptor. The distribution and abundance of binding sites in rat and human brain sections were studied by quantitative receptor radioautography and image analysis. Using 10 nM: [(3)H]quisqualate, a high density of binding was detected in various brain regions with the following rank order of increasing levels: medulla, thalamus, olfactory bulb, cerebral cortex, spinal cord dorsal horn, olfactory tubercle, dentate gyrus molecular layer, CA1-3 oriens layer of hippocampus, striatum, and cerebellar molecular layer. The ionotropic component of this binding could be inhibited by 30 microM: kainate, revealing the distribution of mGlu1+5 receptors. The latter were almost completely inhibited by the group I agonist (S:)-3,5-dihydroxyphenylglycine. The binding profile correlated well with the cellular sites of synthesis and regional expression of the respective group I receptor proteins revealed by in situ hybridization histochemistry and immunohistochemistry, respectively.