Luiz M. Camargo

Chondroitin 6-sulphate synthesis is up-regulated in injured CNS, induced by injury-related cytokines and enhanced in axon-growth inhibitory glia.

Chondroitin sulphate proteoglycans (CSPGs) are up‐regulated in the CNS after injury and inhibit axon regeneration mainly through their glycosaminoglycan (CS‐GAG) chains. We have analysed the mRNA levels of the CS‐GAG synthesizing enzymes and measured the CS‐GAG disaccharide composition by chromatography and immunocytochemistry. Chondroitin 6‐sulfotransferase 1 (C6ST1) is up‐regulated in most glial types around cortical injuries, and its sulphated product CS‐C is also selectively up‐regulated. Treatment with TGFα and TGFβ, which are released after brain injury, promotes the expression of C6ST1 and the synthesis of 6‐sulphated CS‐GAGs in primary astrocytes. Oligodendrocytes, oligodendrocyte precursors and meningeal cells are all inhibitory to axon regeneration, and all express high levels of CS‐GAG, including high levels of 6‐sulphated GAG. In axon growth‐inhibitory Neu7 astrocytes C6ST1 and 6‐sulphated GAGs are expressed at high levels, whereas in permissive A7 astrocytes they are not detectable. These results suggest that the up‐regulation of CSPG after CNS injury is associated with a specific sulphation pattern on CS‐GAGs, mediating the inhibitory properties of proteoglycans on axonal regeneration.

Fenton chemistry and oxidative stress mediate the toxicity of the β-amyloid peptide in a Drosophila model of Alzheimer’s disease

The mechanism by which aggregates of the β‐amyloid peptide (Aβ) mediate their toxicity is uncertain. We show here that the expression of the 42‐amino‐acid isoform of Aβ (Aβ1–42) changes the expression of genes involved in oxidative stress in a Drosophila model of Alzheimer’s disease. A subsequent genetic screen confirmed the importance of oxidative stress and a molecular dissection of the steps in the cellular metabolism of reactive oxygen species revealed that the iron‐binding protein ferritin and the H2O2 scavenger catalase are the most potent suppressors of the toxicity of wild‐type and Arctic (E22G) Aβ1–42. Likewise, treatment with the iron‐binding compound clioquinol increased the lifespan of flies expressing Arctic Aβ1–42. The effect of iron appears to be mediated by oxidative stress as ferritin heavy chain co‐expression reduced carbonyl levels in Aβ1–42 flies by 65% and restored the survival and locomotion function to normal. This was achieved despite the presence of elevated levels of the Aβ1–42. Taken together, our data show that oxidative stress, probably mediated by the hydroxyl radical and generated by the Fenton reaction, is essential for Aβ1–42 toxicity in vivo and provide strong support for Alzheimer’s disease therapies based on metal chelation.

Progranulin Is a Chemoattractant for Microglia and Stimulates Their Endocytic Activity

Mutations resulting in progranulin haploinsufficiency cause disease in patients with a subset of frontotemporal lobar degeneration; however, the biological functions of progranulin in the brain remain unknown. To address this subject, the present study initially assessed changes in gene expression and cytokine secretion in rat primary cortical neurons treated with progranulin. Molecular pathways enriched in the progranulin gene set included cell adhesion and cell motility pathways and pathways involved in growth and development. Secretion of cytokines and several chemokines linked to chemoattraction but not inflammation were also increased from progranulin-treated primary neurons. Therefore, whether progranulin is involved in recruitment of immune cells in the brain was investigated. Localized lentiviral expression of progranulin in C57BL/6 mice resulted in an increase of Iba1-positive microglia around the injection site. Moreover, progranulin alone was sufficient to promote migration of primary mouse microglia in vitro. Primary microglia and C4B8 cells demonstrated more endocytosis of amyloid β1-42 when treated with progranulin. These data demonstrate that progranulin acts as a chemoattractant in the brain to recruit or activate microglia and can increase endocytosis of extracellular peptides such as amyloid β.

Heparan sulphate proteoglycans in glia and in the normal and injured CNS: expression of sulphotransferases and changes in sulphation

Heparan sulphate proteoglycans (HSPGs) have multiple functions relevant to the control of the CNS injury response, particularly in modulating the effects of growth factors and localizing molecules that affect axon growth. We examined the pattern of expression and glycanation of HSPGs in the normal and damaged CNS, and in astrocytes and oligodendrocyte precursors because of their participation in the injury reaction. The composition of HS glycosaminoglycan (GAG) chains was analysed by biochemical analysis and by the binding of antibodies that recognize sulphated epitopes. We also measured levels of HS sulphotransferases and syndecans. Compared with oligodendrocytes, oligodendrocyte precursors have more 2‐O‐sulphation in their HS GAG. This is accompanied by higher expression of the enzyme responsible for 2‐O‐sulphation, HS 2‐O‐sulphotransferase (HS2ST) and a fall in syndecan‐1. Astrocytes treated with tumour growth factor (TGF)α or TGFβ to mimic the injury response showed upregulation of syndecan‐1 and HS2ST correlating with an increase in 2‐O‐sulphate residues in their HS GAGs. This also correlated with increased staining with AO4B08 anti‐GAG antibody that recognizes high sulphation, and reduced staining with RB4EA12 recognizing low sulphation. After injury to the adult rat brain there was an overall increase in the quantity of HSPG around the injury site, mRNA for HS2ST was increased, and the changes in staining with sulphation‐specific antibodies were consistent with an increase in 2‐O‐sulphated HS. Syndecan‐1 was upregulated in astrocytes. The major injury‐related change, seen in injured brain and cultured glia, was an increase in 2‐O‐sulphated HS and increased syndecan‐1, suggesting novel approaches to modulating scar formation.

Pathway-Based Analysis of Genome-Wide siRNA Screens Reveals the Regulatory Landscape of App Processing

The progressive aggregation of Amyloid-β (Aβ) in the brain is a major trait of Alzheimers Disease (AD). Aβ is produced as a result of proteolytic processing of the β-amyloid precursor protein (APP). Processing of APP is mediated by multiple enzymes, resulting in the production of distinct peptide products: the non-amyloidogenic peptide sAPPα and the amyloidogenic peptides sAPPβ, Aβ40, and Aβ42. Using a pathway-based approach, we analyzed a large-scale siRNA screen that measured the production of different APP proteolytic products. Our analysis identified many of the biological processes/pathways that are known to regulate APP processing and have been implicated in AD pathogenesis, as well as revealing novel regulatory mechanisms. Furthermore, we also demonstrate that some of these processes differentially regulate APP processing, with some mechanisms favouring production of certain peptide species over others. For example, synaptic transmission having a bias towards regulating Aβ40 production over Aβ42 as well as processes involved in insulin and pancreatic biology having a bias for sAPPβ production over sAPPα. In addition, some of the pathways identified as regulators of APP processing contain genes (CLU, BIN1, CR1, PICALM, TREM2, SORL1, MEF2C, DSG2, EPH1A) recently implicated with AD through genome wide association studies (GWAS) and associated meta-analysis. In addition, we provide supporting evidence and a deeper mechanistic understanding of the role of diabetes in AD. The identification of these processes/pathways, their differential impact on APP processing, and their relationships to each other, provide a comprehensive systems biology view of the “regulatory landscape” of APP.

A Genome-Wide siRNA Screen to Identify Modulators of Insulin Sensitivity and Gluconeogenesis

Background Hepatic insulin resistance impairs insulin’s ability to suppress hepatic glucose production (HGP) and contributes to the development of type 2 diabetes (T2D). Although the interests to discover novel genes that modulate insulin sensitivity and HGP are high, it remains challenging to have a human cell based system to identify novel genes. Methodology/Principal Findings To identify genes that modulate hepatic insulin signaling and HGP, we generated a human cell line stably expressing beta-lactamase under the control of the human glucose-6-phosphatase (G6PC) promoter (AH-G6PC cells). Both beta-lactamase activity and endogenous G6PC mRNA were increased in AH-G6PC cells by a combination of dexamethasone and pCPT-cAMP, and reduced by insulin. A 4-gene High-Throughput-Genomics assay was developed to concomitantly measure G6PC and pyruvate-dehydrogenase-kinase-4 (PDK4) mRNA levels. Using this assay, we screened an siRNA library containing pooled siRNA targeting 6650 druggable genes and identified 614 hits that lowered G6PC expression without increasing PDK4 mRNA levels. Pathway analysis indicated that siRNA-mediated knockdown (KD) of genes known to positively or negatively affect insulin signaling increased or decreased G6PC mRNA expression, respectively, thus validating our screening platform. A subset of 270 primary screen hits was selected and 149 hits were confirmed by target gene KD by pooled siRNA and 7 single siRNA for each gene to reduce G6PC expression in 4-gene HTG assay. Subsequently, pooled siRNA KD of 113 genes decreased PEPCK and/or PGC1alpha mRNA expression thereby demonstrating their role in regulating key gluconeogenic genes in addition to G6PC. Last, KD of 61 of the above 113 genes potentiated insulin-stimulated Akt phosphorylation, suggesting that they suppress gluconeogenic gene by enhancing insulin signaling. Conclusions/Significance These results support the proposition that the proteins encoded by the genes identified in our cell-based druggable genome siRNA screen hold the potential to serve as novel pharmacological targets for the treatment of T2D.

Comparative transcriptomics of choroid plexus in Alzheimer’s disease, frontotemporal dementia and Huntington’s disease: implications for CSF homeostasis

BackgroundIn Alzheimer’s disease, there are striking changes in CSF composition that relate to altered choroid plexus (CP) function. Studying CP tissue gene expression at the blood–cerebrospinal fluid barrier could provide further insight into the epithelial and stromal responses to neurodegenerative disease states.MethodsTranscriptome-wide Affymetrix microarrays were used to determine disease-related changes in gene expression in human CP. RNA from post-mortem samples of the entire lateral ventricular choroid plexus was extracted from 6 healthy controls (Ctrl), 7 patients with advanced (Braak and Braak stage III–VI) Alzheimer’s disease (AD), 4 with frontotemporal dementia (FTD) and 3 with Huntington’s disease (HuD). Statistics and agglomerative clustering were accomplished with MathWorks, MatLab; and gene set annotations by comparing input sets to GeneGo (http://www.genego.com) and Ingenuity (http://www.ingenuity.com) pathway sets. Bonferroni-corrected hypergeometric p-values of < 0.1 were considered a significant overlap between sets.ResultsPronounced differences in gene expression occurred in CP of advanced AD patients vs. Ctrls. Metabolic and immune-related pathways including acute phase response, cytokine, cell adhesion, interferons, and JAK-STAT as well as mTOR were significantly enriched among the genes upregulated. Methionine degradation, claudin-5 and protein translation genes were downregulated. Many gene expression changes in AD patients were observed in FTD and HuD (e.g., claudin-5, tight junction downregulation), but there were significant differences between the disease groups. In AD and HuD (but not FTD), several neuroimmune-modulating interferons were significantly enriched (e.g., in AD: IFI-TM1, IFN-AR1, IFN-AR2, and IFN-GR2). AD-associated expression changes, but not those in HuD and FTD, were enriched for upregulation of VEGF signaling and immune response proteins, e.g., interleukins. HuD and FTD patients distinctively displayed upregulated cadherin-mediated adhesion.ConclusionsOur transcript data for human CP tissue provides genomic and mechanistic insight for differential expression in AD vs. FTD vs. HuD for stromal as well as epithelial components. These choroidal transcriptome characterizations elucidate immune activation, tissue functional resiliency, and CSF metabolic homeostasis. The BCSFB undergoes harmful, but also important functional and adaptive changes in neurodegenerative diseases; accordingly, the enriched JAK-STAT and mTOR pathways, respectively, likely help the CP in adaptive transcription and epithelial repair and/or replacement when harmed by neurodegeneration pathophysiology. We anticipate that these precise CP translational data will facilitate pharmacologic/transgenic therapies to alleviate dementia.

An accessible pharmacodynamic transcriptional biomarker for notch target engagement

γ‐Secretase mediates amyloid production in Alzheimers disease (AD) and oncogenic activity of Notch. γ‐Secretase inhibitors (GSIs) are thus of interest for AD and oncology. A peripheral biomarker of Notch activity would aid determination of the therapeutic window and dosing regimen for GSIs, given toxicities associated with chronic Notch inhibition. This study examined the effects of GSI MK‐0752 on blood and hair follicle transcriptomes in healthy volunteers. The effects of a structurally diverse GSI on rhesus blood and hair follicles were also compared. Significant dose‐related effects of MK‐0752 on transcription were observed in hair follicles, but not blood. The GSI biomarker identified in follicles exhibited 100% accuracy in a clinical test cohort, and was regulated in rhesus by a structurally diverse GSI. This study identified a translatable, accessible pharmacodynamic biomarker of GSI target engagement and provides proof of concept of hair follicle RNA as a translatable biomarker source.

Alterations in choroid plexus gene expression in Alzheimer’s disease provide inferences for CSF composition and dynamics

Alterations in choroid plexus gene expression in Alzheimer’s disease provide inferences for CSF composition and dynamics Miles C Miller, Edward G Stopa, Elena V Nikonova, Keith Q Tanis, Alexei A Podtelezhnikov, Eva M Finney, David J Stone, Luiz M Camargo, Lisan Parker, Ajay Verma, Andrew Baird, John E Donahue, Ana Maria Gonzalez, Brian Eliceiri, Gerald D Silverberg, Petra M Klinge, Conrad E Johanson