Andrew K. Powell

Targeting Cellular Prion Protein Reverses Early Cognitive Deficits and Neurophysiological Dysfunction in Prion-Infected Mice

Currently, no treatment can prevent the cognitive and motor decline associated with widespread neurodegeneration in prion disease. However, we previously showed that targeting endogenous neuronal prion protein (PrP(C)) (the precursor of its disease-associated isoform, PrP(Sc)) in mice with early prion infection reversed spongiform change and prevented clinical symptoms and neuronal loss. We now show that cognitive and behavioral deficits and impaired neurophysiological function accompany early hippocampal spongiform pathology. Remarkably, these behavioral and synaptic impairments recover when neuronal PrP(C) is depleted, in parallel with reversal of spongiosis. Thus, early functional impairments precede neuronal loss in prion disease and can be rescued. Further, they occur before extensive PrP(Sc) deposits accumulate and recover rapidly after PrP(C) depletion, supporting the concept that they are caused by a transient neurotoxic species, distinct from aggregated PrP(Sc). These data suggest that early intervention in human prion disease may lead to recovery of cognitive and behavioral symptoms.

Stabilization of Sialic Acids in N‐linked Oligosaccharides and Gangliosides for Analysis by Positive Ion Matrix‐assisted Laser Desorption/Ionization Mass Spectrometry

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of oligosaccharides and gangliosides normally causes loss of sialic acid, particularly when alpha-cyano-4-hydroxycinnamic acid is used as the matrix. In addition, the potential signal is split because both positive and, to a greater extent, negative ions are formed while signals are frequently complicated as the result of partial alkali-salt formation. In order to stabilize the sialic acid moieties under MALDI conditions and to divert all of the signal into the positive-ion mode, a method involving their conversion into methyl esters has been developed. The method is relatively rapid and produces strong positive-ion signals from N-linked oligosaccharides containing sialic acid and from gangliosides. The latter compounds are stable, even in the presence of alpha-cyano-4-hydroxycinnamic acid. They give abundant molecular (MNa+) ions, but with sufficient residual in-source fragmentation to allow the sequence of the sugar chain to be determined. The sialic acid residue is stable after methylation, irrespective of its linkage to the parent molecule.

A Molecular Mechanism for the Heparan Sulfate Dependence of Slit-Robo Signaling

Slit is a large secreted protein that provides important guidance cues in the developing nervous system and in other organs. Signaling by Slit requires two receptors, Robo transmembrane proteins and heparan sulfate (HS) proteoglycans. How HS controls Slit-Robo signaling is unclear. Here we show that the second leucine-rich repeat domain (D2) of Slit, which mediates binding to Robo receptors, also contains a functionally important binding site for heparin, a highly sulfated variant of HS. Heparin markedly enhances the affinity of the Slit-Robo interaction in a solid-phase binding assay. Analytical gel filtration chromatography demonstrates that Slit D2 associates with a soluble Robo fragment and a heparin-derived oligosaccharide to form a ternary complex. Retinal growth cone collapse triggered by Slit D2 requires cell surface HS or exogenously added heparin. Mutation of conserved basic residues in the C-terminal cap region of Slit D2 reduces heparin binding and abolishes biological activity. We conclude that heparin/HS is an integral component of the minimal Slit-Robo signaling complex and serves to stabilize the relatively weak Slit-Robo interaction.

Fibroblast growth factor receptors 1 and 2 interact differently with heparin/heparan sulfate: implications for dynamic assembly of a ternary signaling complex

Heparan sulfate (HS) regulates the kinetics of fibroblast growth factor 2 (FGF2)-stimulated intracellular signaling and differentially activates cell proliferation of cells expressing different FGF receptors (FGFRs). Evidence suggests that HS interacts with both FGFs and FGFRs to form active ternary signaling complexes. Here we compare the interactions of two FGFRs with HS. We show that the ectodomains of FGFR1 IIIc and FGFR2 IIIc exhibit specific interactions with different characteristics for both heparin and porcine mucosal HS. These glycans are both known to activate FGF signaling via these receptors. FGFR2 interacts with a higher apparent affinity than FGFR1 despite both involving 6-O-, 2-O-, andN-sulfates. FGFR1 and FGFR2 bind heparin with mean association rate constants of 1.9 × 105 and 2.1 × 106 m −1s−1, respectively, and dissociation rate constants of 1.2 × 10−2 and 2.7 × 10−2 s−1, respectively. These produced calculated affinities of 63 and 13 nm, respectively. Hence, FGFR1 and FGFR2 bind to heparin chains with markedly different kinetics and affinities. We propose a mechanistic model where the kinetic parameters of the HS/FGFR interaction are a key element regulating the formation of ternary complexes and the resulting FGF signaling outcomes.

A versatile gold surface approach for fabrication and interrogation of glycoarrays

Glycoarrays on gold: A designer gold surface incorporating a self-assembled monolayer with weak protein absorption properties has been optimised for rapid display and interrogation of both native and derivatised glycans in array formats. This rapid, facile approach has diverse applications in glycomics, through exploitation of fluorescence, SPR and MALDI-ToF MS detection methods.

Inhibition of RhoA pathway rescues the endocytosis defects in Oligophrenin1 mouse model of mental retardation

The patho-physiological hypothesis of mental retardation caused by the deficiency of the RhoGAP Oligophrenin1 (OPHN1), relies on the well-known functions of Rho GTPases on neuronal morphology, i.e. dendritic spine structure. Here, we describe a new function of this Bin/Amphiphysin/Rvs domain containing protein in the control of clathrin-mediated endocytosis (CME). Through interactions with Src homology 3 domain containing proteins involved in CME, OPHN1 is concentrated to endocytic sites where it down-regulates the RhoA/ROCK signaling pathway and represses the inhibitory function of ROCK on endocytosis. Indeed disruption of Ophn1 in mice reduces the endocytosis of synaptic vesicles and the post-synaptic α-amino-3-hydroxy-5-methylisoazol-4-propionate (AMPA) receptor internalization, resulting in almost a complete loss of long-term depression in the hippocampus. Finally, pharmacological inhibition of this pathway by ROCK inhibitors fully rescued not only the CME deficit in OPHN1 null cells but also synaptic plasticity in the hippocampus from Ophn1 null model. Altogether, we uncovered a new patho-physiological mechanism for intellectual disabilities associated to mutations in RhoGTPases linked genes and also opened new directions for therapeutic approaches of congenital mental retardation.

Heparan sulfate and heparin interactions with proteins

Heparan sulfate (HS) polysaccharides are ubiquitous components of the cell surface and extracellular matrix of all multicellular animals, whereas heparin is present within mast cells and can be viewed as a more sulfated, tissue-specific, HS variant. HS and heparin regulate biological processes through interactions with a large repertoire of proteins. Owing to these interactions and diverse effects observed during in vitro, ex vivo and in vivo experiments, manifold biological/pharmacological activities have been attributed to them. The properties that have been thought to bestow protein binding and biological activity upon HS and heparin vary from high levels of sequence specificity to a dependence on charge. In contrast to these opposing opinions, we will argue that the evidence supports both a level of redundancy and a degree of selectivity in the structure–activity relationship. The relationship between this apparent redundancy, the multi-dentate nature of heparin and HS polysaccharide chains, their involvement in protein networks and the multiple binding sites on proteins, each possessing different properties, will also be considered. Finally, the role of cations in modulating HS/heparin activity will be reviewed and some of the implications for structure–activity relationships and regulation will be discussed.

Generating heparan sulfate saccharide libraries for glycomics applications

Natural and semi-synthetic heparan sulfate (HS) saccharide libraries are a valuable resource for investigating HS structure–function relationships, enabling high-throughput glycomics studies. Owing to the difficulty of chemical or in vitro enzymatic synthesis of HS saccharides, the structural diversity displayed in saccharides from tissue or cell sources cannot be readily accessed. In contrast, saccharide libraries can be generated by partial digestion of tissue-derived HS polysaccharide chains and chromatographic fractionation of the resulting saccharide mixtures. Fractionation is initially on the basis of hydrodynamic volume, using size exclusion chromatography. Further fractionation, on the basis of charge using strong anion exchange, can subsequently be applied. Desalting and sample concentration follows each fractionation step. Chromatographic fractions are generated that contain purified, or partially purified, saccharides. Here we describe a comprehensive protocol for generation of structurally diverse natural saccharide libraries from HS variants that is fast (∼3 weeks) and reproducible.

The conformation and structure of GAGs: recent progress and perspectives

The glycosaminoglycan (GAG) family of linear sulphated polysaccharides are involved in most regulatory processes in the extracellular matrix of higher organisms. The relationship between GAG substitution pattern and activity, however, remains unclear and experimental evidence suggests that subtle conformational factors play an important role. The difficulty of modelling these complex charged molecules shifts the burden of investigation towards experimental techniques. Recent advances in complementary physical-chemical, particularly spectroscopy-based approaches are reviewed, together with methods for analysing the resulting complex data. The prospects for combining some of these approaches and fitting them into the wider context of interactions, are also discussed.

Microwave enhanced reaction of carbohydrates with amino-derivatised labels and glass surfaces

The reaction between carbohydrates and amino-derivatised labels has been improved through microwave heating. We show it proceeds principally via solvent mediated heating rather than either direct microwave heating of the reagents or microwave influenced changes in the rates of mutarotation in the sugars beyond those obtained by conventional thermal heating. The method is applied to the attachment of sugars to an aminosilane-derivatised glass surface suitable for the construction of carbohydrate microarrays.