Richard E. Higgs

Apolipoprotein E promotes astrocyte colocalization and degradation of deposited amyloid-β peptides

We have previously shown that apolipoprotein E (Apoe) promotes the formation of amyloid in brain and that astrocyte-specific expression of APOE markedly affects the deposition of amyloid-β peptides (Aβ) in a mouse model of Alzheimer disease. Given the capacity of astrocytes to degrade Aβ, we investigated the potential role of Apoe in this astrocyte-mediated degradation. In contrast to cultured adult wild-type mouse astrocytes, adult Apoe−/− astrocytes do not degrade Aβ present in Aβ plaque–bearing brain sections in vitro. Coincubation with antibodies to either Apoe or Aβ, or with RAP, an antagonist of the low-density lipoprotein receptor family, effectively blocks Aβ degradation by astrocytes. Phase-contrast and confocal microscopy show that Apoe−/− astrocytes do not respond to or internalize Aβ deposits to the same extent as do wild-type astrocytes. Thus, Apoe seems to be important in the degradation and clearance of deposited Aβ species by astrocytes, a process that may be impaired in Alzheimer disease.

Experimental Designs for Selecting Molecules from Large Chemical Databases

Recent developments in high-throughput screening and combinatorial chemistry have generated interest in experimental design methods to select subsets of molecules from large chemical databases. In this manuscript three methods for selecting molecules from large databases are described:  edge designs, spread designs, and coverage designs. Two algorithms with linear time complexity that approximate spread and coverage designs are described. These algorithms can be threaded for multiprocessor systems, are compatible with any definition of molecular distance, and may be applied to very large chemical databases. For example, ten thousand molecules were selected using the maximum dissimilarity approximation to a spread design from a sixty-dimensional simulated molecular database of one million molecules in approximately 6 h on a UNIX workstation.

Rapid Method To Estimate the Presence of Secondary Metabolites in Microbial Extracts

ABSTRACT Screening microbial secondary metabolites is an established method to identify novel biologically active molecules. Preparation of biological screening samples from microbial fermentation extracts requires growth conditions that promote synthesis of secondary metabolites and extraction procedures that capture the secondary metabolites produced. High-performance liquid chromatography (HPLC) analysis of fermentation extracts can be used to estimate the number of secondary metabolites produced by microorganisms under various growth conditions but is slow. In this study we report on a rapid (approximately 1 min per assay) surrogate measure of secondary metabolite production based on a metabolite productivity index computed from the electrospray mass spectra of samples injected directly into a spectrometer. This surrogate measure of productivity was shown to correlate with an HPLC measure of productivity with a coefficient of 0.78 for a test set of extracts from 43 actinomycetes. This rapid measure of secondary metabolite productivity may be used to identify improved cultivation and extraction conditions by analyzing and ranking large sets of extracts. The same methods may also be used to survey large collections of extracts to identify subsets of highly productive organisms for biological screening or additional study.

Identification of druggable cancer driver genes amplified across TCGA datasets.

The Cancer Genome Atlas (TCGA) projects have advanced our understanding of the driver mutations, genetic backgrounds, and key pathways activated across cancer types. Analysis of TCGA datasets have mostly focused on somatic mutations and translocations, with less emphasis placed on gene amplifications. Here we describe a bioinformatics screening strategy to identify putative cancer driver genes amplified across TCGA datasets. We carried out GISTIC2 analysis of TCGA datasets spanning 14 cancer subtypes and identified 461 genes that were amplified in two or more datasets. The list was narrowed to 73 cancer-associated genes with potential “druggable” properties. The majority of the genes were localized to 14 amplicons spread across the genome. To identify potential cancer driver genes, we analyzed gene copy number and mRNA expression data from individual patient samples and identified 40 putative cancer driver genes linked to diverse oncogenic processes. Oncogenic activity was further validated by siRNA/shRNA knockdown and by referencing the Project Achilles datasets. The amplified genes represented a number of gene families, including epigenetic regulators, cell cycle-associated genes, DNA damage response/repair genes, metabolic regulators, and genes linked to the Wnt, Notch, Hedgehog, JAK/STAT, NF-KB and MAPK signaling pathways. Among the 40 putative driver genes were known driver genes, such as EGFR, ERBB2 and PIK3CA. Wild-type KRAS was amplified in several cancer types, and KRAS-amplified cancer cell lines were most sensitive to KRAS shRNA, suggesting that KRAS amplification was an independent oncogenic event. A number of MAP kinase adapters were co-amplified with their receptor tyrosine kinases, such as the FGFR adapter FRS2 and the EGFR family adapter GRB7. The ubiquitin-like ligase DCUN1D1 and the histone methyltransferase NSD3 were also identified as novel putative cancer driver genes. We discuss the patient tailoring implications for existing cancer drug targets and we further discuss potential novel opportunities for drug discovery efforts.

Changes in Osteoblast, Chondrocyte, and Adipocyte Lineages Mediate the Bone Anabolic Actions of PTH and Small Molecule GSK‐3 Inhibitor

Parathyroid hormone (PTH) and glycogen synthase kinase‐3 (GSK‐3) inhibitor 603281‐31‐8, administered once daily increased bone formation in vivo. We investigated the molecular mechanisms of the anabolic responses of PTH and 603281‐31‐8 in rat osteopenia model. Female 6‐month‐old rats were ovariectomized (Ovx) and permitted to lose bone for 1 month, followed by treatment with PTH (1–38) at 10 µg/kg/day s.c. or 603281‐31‐8 at 3 mg/kg/day p.o. for 60 days. Twenty‐four hours after the last treatment, RNA from distal femur metaphysis was subjected to gene expression analysis. Differentially expressed genes (P < 0.05) were subjected to pathway analysis to delineate relevant bio‐processes involved in skeletal biology. Genes involved in morphogenesis, cell growth/differentiation, and apoptosis were significantly altered by Ovx and the treatments. Analysis of morphogenesis genes showed an overrepresentation of genes involved in osteogenesis, chondrogenesis, and adipogenesis. A striking finding was that Ovx decreased several markers of osteogenesis/chondrogenesis and increased markers of adipogenesis/lipid metabolism. Treatment with either PTH or the GSK‐3 inhibitor reversed these effects, albeit at different levels. Histological analysis confirmed that osteopenia in Ovx animals was associated with three‐fold increase in marrow adiposity. PTH and GSK‐3 inhibitor restored bone volume, and reversed or normalized marrow adiposity. Ex vivo studies showed that PTH and GSK‐3 inhibitor increased the ratio of colony forming marrow stromal progenitors (CFU‐fs) that were alkaline phosphatase positive (putative osteoblasts). Our results suggest that the bone anabolic actions of PTH and GSK‐3 inhibitor in vivo involve concerted effects on mesenchymal lineages; osteoblasts, chondrocytes, and adipocytes. J. Cell. Biochem. 102: 1504–1518, 2007.

M1 and M2 Muscarinic Receptor Subtypes Regulate Antidepressant-Like Effects of the Rapidly Acting Antidepressant Scopolamine

Scopolamine produces rapid and significant symptom improvement in patients with depression, and most notably in patients who do not respond to current antidepressant treatments. Scopolamine is a nonselective muscarinic acetylcholine receptor antagonist, and it is not known which one or more of the five receptor subtypes in the muscarinic family are mediating these therapeutic effects. We used the mouse forced-swim test, an antidepressant detecting assay, in wild-type and transgenic mice in which each muscarinic receptor subtype had been genetically deleted to define the relevant receptor subtypes. Only the M1 and M2 knockout (KO) mice had a blunted response to scopolamine in the forced-swim assay. In contrast, the effects of the tricyclic antidepressant imipramine were not significantly altered by gene deletion of any of the five muscarinic receptors. The muscarinic antagonists biperiden, pirenzepine, and VU0255035 (N-[3-oxo-3-[4-(4-pyridinyl)-1-piper azinyl]propyl]-2,1,3-benzothiadiazole-4-sulfonamide) with selectivity for M1 over M2 receptors also demonstrated activity in the forced-swim test, which was attenuated in M1 but not M2 receptor KO mice. An antagonist with selectivity of M2 over M1 receptors (SCH226206 [(2-amino-3-methyl-phenyl)-[4-[4-[[4-(3 chlorophenyl)sulfonylphenyl]methyl]-1-piperidyl]-1-piperidyl]methanone]) was also active in the forced-swim assay, and the effects were deleted in M2−/− mice. Brain exposure and locomotor activity in the KO mice demonstrated that these behavioral effects of scopolamine are pharmacodynamic in nature. These data establish muscarinic M1 and M2 receptors as sufficient to generate behavioral effects consistent with an antidepressant phenotype and therefore as potential targets in the antidepressant effects of scopolamine.

Pyroglutamyl apelin-13 identified as the major apelin isoform in human plasma.

Apelin is emerging as an important hormone regulator of cardiovascular homoeostasis and an important biomarker for heart failure. Apelin concentrations have historically been measured by immunoassays; however, reported apelin concentrations measured in healthy volunteers show a large disparity from a few picograms per milliliter (pg/ml) to several nanograms per milliliter (ng/ml). Apelin exists in several isoforms ranging in size from 12 to 36 residues, and immunoassays generally cannot distinguish the specific forms present. In this study, an optimized method for enriching apelin peptides with cation-exchange beads followed with mass spectrometry analysis is presented. Apelin peptides are labile in plasma at physiological conditions; however, by lowering the plasma pH to 4.5, the recovery of apelin peptides can be increased significantly. Through optimizing the cation-exchange extraction process, we improved the lower limit of detection for most of the apelin peptides monitored to a few pg/ml. Using the improved method, we detected pyroglutamyl apelin-13 [(pyr)apelin-13] as the major apelin isoform present in plasma from several healthy volunteers at concentrations ranging from 7.7 to 23.3pg/ml.

A method for quantitatively differentiating crude natural extracts using high-performance liquid chromatography-electrospray mass spectrometry.

This paper describes a method for quantitatively differentiating crude natural extracts using high-performance liquid chromatography-electrospray mass spectrometry (HPLC-ESI-MS). The method involves performing an HPLC-MS analysis using standard reversed-phase C18 gradient separation on the crude extract. The HPLC system used in this study was a dual-column system designed to optimize throughput. Using image analysis techniques, the data are reduced to a list containing the m/z value and retention time of each ion. The ion lists are then compared in a pairwise fashion to compute a sample similarity index between two samples. The similarity index is based on the number of ions common to both and is scaled from 0 to 1. Extract controls were analyzed throughout a run of 88 unknown fungal extracts. The controls provided information about column and spectrometer stability and overall sensitivity. Pairwise comparison of all control samples indicates that the similarity index is high (0.8) for replicate samples. Comparison between the unknown extract samples produces a distribution of similarities ranging from replicates (0.8) to very dissimilar (0.1). This information can be used to judge the chemical diversity of natural extract samples, which is one approach to determining the quality of libraries being used for drug discovery via high-throughput screening.

Quantitative Measurement of Intact Alpha-Synuclein Proteoforms from Post-Mortem Control and Parkinson's Disease Brain Tissue by Intact Protein Mass Spectrometry

A robust top down proteomics method is presented for profiling alpha-synuclein species from autopsied human frontal cortex brain tissue from Parkinsons cases and controls. The method was used to test the hypothesis that pathology associated brain tissue will have a different profile of post-translationally modified alpha-synuclein than the control samples. Validation of the sample processing steps, mass spectrometry based measurements, and data processing steps were performed. The intact protein quantitation method features extraction and integration of m/z data from each charge state of a detected alpha-synuclein species and fitting of the data to a simple linear model which accounts for concentration and charge state variability. The quantitation method was validated with serial dilutions of intact protein standards. Using the method on the human brain samples, several previously unreported modifications in alpha-synuclein were identified. Low levels of phosphorylated alpha synuclein were detected in brain tissue fractions enriched for Lewy body pathology and were marginally significant between PD cases and controls (p = 0.03).

A proteomic analysis of adult rat bone reveals the presence of cartilage/chondrocyte markers

The non‐mineral component of bone matrix consists of 90% collagenous, 10% non‐collagenous proteins. These proteins regulate mineralization, growth, cell signaling and differentiation, and provide bone with its tensile strength. Expression of bone matrix proteins have historically been studied individually or in small numbers owing to limitations in analytical technologies. Current mass‐spectrometric and separations technologies allow a global view of protein expression patterns in complex samples. To our knowledge, no proteome profile of bone matrix has yet been reported. Therefore, we have used mass spectrometry as a tool to generate a profile of proteins present in the extracellular matrix of adult rat bone. Overall, 108 and 25 proteins were identified with high confidence in the metaphysis and diaphysis, respectively, using a bottom up proteomic technique. Twenty‐one of these proteins were present in both the metaphysis and diaphysis including the bone specific proteins, osteocalcin, type I collagen, osteopontin, osteoregulin, and bone sialoprotein. Interestingly, type II collagen, a protein thought to be exclusively expressed in cartilage, was identified in both the metaphysis and diaphysis. This observation was validated by Western blot. Additionally, the presence of aggrecan, another protein expressed in cartilage was identified in the bone matrix extracts by Western blot. The proteome profile generated using this technology represents an initial survey of the acid soluble proteins of bone matrix which provides a reference for the analysis of deviations from the normal composition due to perturbations or disease states. J. Cell. Biochem. 101: 466–476, 2007.