Pharmacological Inhibition of polysialyltransferase ST8SiaII Modulates Tumour Cell Migration

YesPolysialic acid (polySia), an α-2,8-glycosidically linked polymer of sialic acid, is a developmentally regulated posttranslational modification predominantly found on NCAM (neuronal cell adhesion molecule). Whilst high levels are expressed during development, peripheral adult organs do not express polySia-NCAM. However, tumours of neural crest-origin re-express polySia-NCAM: its occurrence correlates with aggressive and invasive disease and poor clinical prognosis in different cancer types, notably including small cell lung cancer (SCLC), pancreatic cancer and neuroblastoma. In neuronal development, polySia-NCAM biosynthesis is catalysed by two polysialyltransferases, ST8SiaII and ST8SiaIV, but it is ST8SiaII that is the prominent enzyme in tumours. The aim of this study was to determine the effect of ST8SiaII inhibition by a small molecule on tumour cell migration, utilising cytidine monophosphate (CMP) as a tool compound. Using immunoblotting we showed that CMP reduced ST8iaII-mediated polysialylation of NCAM. Utilizing a novel HPLC-based assay to quantify polysialylation of a fluorescent acceptor (DMB-DP3), we demonstrated that CMP is a competitive inhibitor of ST8SiaII (Ki = 10 μM). Importantly, we have shown that CMP causes a concentration-dependent reduction in tumour cell-surface polySia expression, with an absence of toxicity. When ST8SiaII-expressing tumour cells (SH-SY5Y and C6-STX) were evaluated in 2D cell migration assays, ST8SiaII inhibition led to significant reductions in migration, while CMP had no effect on cells not expressing ST8SiaII (DLD-1 and C6-WT). The study demonstrates for the first time that a polysialyltransferase inhibitor can modulate migration in ST8SiaII-expressing tumour cells. We conclude that ST8SiaII can be considered a druggable target with the potential for interfering with a critical mechanism in tumour cell dissemination in metastatic cancers.Yorkshire Cancer Research; EPSRC; Association for International Cancer Research; Jordanian Government PhD scholarshi

Polüsialüülitud närviraku adhesioonimolekuli (PSA-NCAMi) roll silma võrkkesta ganglionirakkude elulemusele täiskasvanueas

Väitekirja elektrooniline versioon ei sisalda publikatsiooneNärviraku adhesioonimolekul (NCAM) on glükoproteiin, mis kuulub immunoglobuliinide superperekonda. Kolm peamist NCAMi isovormi on jaotatud molekulmassi alusel (NCAM-180, NCAM-140, NCAM-120) ning need erinevad üksteisest rakumembraani läbiva domeeni poolest. Närviraku adhesioonimolekulide peamine roll on närvirakkude vaheliste kontaktide loomine ja nende stabiliseerimine. Polüsiaalhappe (PSA) seondumine NCAMi viienda immunoglobuliini domeeni külge toimub polüsialüültransferaas II (ST8SiaII) ja IV (ST8SiaIV) vahendusel, ning selle tulemusena väheneb rakkudevaheline adhesioon. Lisaks on näidatud, et PSA-NCAM ei mõjuta mitte üksnes rakkudevahelisi adhesiivseid omandusi, vaid sellel on oluline roll ka protsessides, mille kaudu reguleeritakse rakkude elulemust, proliferatsiooni, diferentseerumist, migratsiooni, aksonite väljakasvu ning sünaptilist plastilisust. PSA-NCAMi leidub valdavalt kesknärvisüsteemi neuronitel ning gliiarakkudel ja olles närvirakkudevahelise struktuurse dünaamilisuse vahendaja, omab ta olulist rolli aju plastilisuse regulatsioonis nii arenevas ajukoes kui ka täiskasvanueas. Silma võrkkestas esineb PSA-NCAMi täiskasvanueas kõige rohkem Mülleri rakkudel ja astrotsüütidel ganglionirakkude (RGC) juures. Silma võrkkest koosneb mitmetest erineva funktsiooniga rakukihtidest, millest ühe kihi moodustavad ganglionirakud. Selles kihis paiknevad RGCde kehad, mille aksonid moodustavad nägemisnärvi. On näidatud, et silma võrkkesta PSA-NCAM võib mõjutada RGC elulemust, kuid milline on PSA-NCAMi täpne roll täiskasvanueas silma võrkkesta funktsiooni tagamisel, pole teada. Töö eesmärgid: 1. Tuvastada, kas NCAM, ST8SiaII, ST8SiaIV puudulikel hiirtel ja endosialidaas-N-i (Endo-N) indutseeritud PSA defitsiit metsiktüüpi hiirtel esinevad muutused PSA-NCAMi ekspressionis ja silma võrkkesta RGCde arvus. 2. Selgitada, kas ja kuidas mõjutab NCAM-/-, ST8SiaII-/-, ST8SiaIV-/- või Endo-N-iga tekitatud PSA kadu silma võrkkesta RGCde elulemust neurotoksiini kaiinhappe intravitreaalse manustamise järgselt. 3. Selgitada, millise mehhanismi vahendusel väheneb PSA-NCAMi tase ning tekib RGCde degeneratsioon kaiinhappe intravitreaalse manustamise järgselt. 4. Teha kindlaks PSA-NCAMi ja maatriksi metalloproteinaas-9 (MMP-9) roll RGCde elulemusele hiire streptosototsiiniga indutseeritud diabeedi mudelis. Töö tulemused: 1. Antud töö tulemused näitasid, et vastsündinud ST8SiaII-/- ja ST8SiaIV-/- hiirtel on PSA-NCAMi ekspressioon oluliselt vähenenud, kuid täiskasvanud hiirtel ekspressioonis muutusi ei täheldatud. NCAM-/- hiirtel PSA-NCAMi ei leitud. Tuvastati, et NCAM-/- hiirtel on RGCde arv oluliselt suurenenud, vastupidiselt ST8SiaII-/- hiirtele, kellel RGCde arv on vähenenud, kuid ST8SiaIV-/- hiirtel RGCde arvus muutusi ei esinenud. Samuti selgus, et PSA eemaldamine NCAMilt Endo-N-iga ei mõjutanud RGCde elulemust. 2. Ilmnes, et ST8SiaII-, ST8SiaIV- või NCAM-defitsiitsetel loomadel ei mõjutanud kaiinhappe poolt põhjustatud toksilisus RGCde elulemust, mille põhjuseks võib olla olemasolevate sialüültransferaaside aktivatsioon. Samas, Endo-N-iga tekitatud PSA kadu suurendas kaiinhappe toksilist toimet RGCdele, mis viitab, et polüsialüülitud NCAMi vorm on vajalik RGCde elulemuse tagamiseks. 3. Selgus, et MMP-9 osaleb kaiinhappest indutseeritud PSA-NCAMi taseme vähenemises, põhjustades PSA eemaldamist NCAMilt ning selle mehhanismi kaudu mõjutatakseb RGCde elulemust. 4. Tuvastati, et streptosototsiiniga indutseeritud diabeedi mudelis on RGCde arv vähenenud ja see on seotud PSA-NCAMi taseme vähenemisega ning MMP-9 aktivatsiooni suurenemisega RGCde piirkonnas.Retinal ganglion cells (RGCs) undergo degeneration in many human diseases, such as glaucoma, diabetic retinopathy, optic nerve atrophy, some inherited diseases (dominant optic atrophy, Leber hereditary optic neuropathy). There is no effective treatment for progressive vision loss due to RGC degeneration. Polysialylated neural cell adhesion molecule (PSA-NCAM) is expressed abundantly in the retina in close proximity to the RGCs. PSA is attached to NCAM by two specific polysialyltransferases, II and IV (ST8SiaII, ST8SiaIV). It is thought that PSA-NCAM affects not only adhesive properties between cells, but also plays a role in the intracellular signaling, which influences cell survival, proliferation, differentiation, migration, axon outgrowth and pathfinding, and synaptic plasticity. The functions of PSA-NCAM in the retina in the adult are not clearly understood. One of the putative roles of PSA-NCAM in the retina is in the maintenance of RGC survival. The aims of this study were to investigate in detail (a) the survival of RGCs in mice deficient for NCAM and ST8SiaII or ST8SiaIV; (b) the protective roles of NCAM/PSA-NCAM during excitotoxic retinal damage induced by kainic acid (KA); (c) the protective roles of PSA-NCAM in the mouse model of diabetic retinopathy (DR). Our study demonstrates that the viability and survival of RGCs is dependent on the presence of PSA-NCAM expressed by astroglial and Müller cells. Excitotoxic retinal damage or diabetic retinopathy induces a decrease in the levels of PSA-NCAM and thereby promotes RGC degeneration. The reduction of PSA-NCAM levels in these models is mediated by matrix metalloproteinase-9 (MMP-9), which induces shedding of the extracellular domain of PSA-NCAM. Our study demonstrates that PSA-NCAM and MMP-9 might be new pharmacological targets to combat retinal degeneration.  https://www.ester.ee/record=b529321

Role of polysialic acid and NCAM in interneuron development

[no abstract

The role of the amygdala in emotional memories:a multidisciplinary approach

This thesis investigates the role of the amygdala for the establishment of fear memories with a multidisciplinary approach, including behavioural, psychopharmacological, genetic, molecular, and electrophysiological techniques in rats or mice, under healthy or pathological conditions. This research program aims to shed light on the acquisition and storage of emotional memories in the amygdala and closely interconnected brain areas. In one line of experiments, the molecular mechanisms leading to the establishment of fear memory traces in the amygdala were investigated. For this purpose, the functional role of the polysialylated neural cell adhesion molecule PSA-NCAM, expressed in the synaptic junction, was assessed in the amygdala – and also prefrontal cortex and hippocampus – with psychopharmacological and genetic approaches and tasks that strongly rely on these brain areas. Two lines of studies were followed: 1) amygdala-targeted cleavage and enhancement of PSA-NCAM in rats and 2) general cleavage of PSA-NCAM throughout the brain using genetically modified mice. Taken together, both approaches show that amygdaloid PSA-NCAM plays no role in the acquisition and storage of fear memories, but is rather involved in their extinction. Furthermore, the results confirm the importance of PSA-NCAM in hippocampus mediated learning and for the first time show that prefrontal cortex mediated learning depends on PSA-NCAM. These results suggest that PSA-NCAM is selectively involved in some, but not all, synaptic plasticity processes in the brain. In another line of experiments, the valproic acid (VPA) animal model of autism was used to investigate a possible contribution of the amygdala towards the autistic pathology. VPA was injected once at a specific time point during gestation, the time of neural tube closure. The offspring of such treated rats were first characterized in a broad set of behavioural tasks. It was found that VPA-treated offspring exhibited very specific behavioural anomalies closely resembling autistic symptomotology, such as impaired social interaction, exploration and recognition, enhanced repetitive behaviours, impaired sensorimotor gating and increased anxiety, while other behavioural parameters were left unharmed. Once the validity of the model was established, amygdala functionality was assessed. The results demonstrated that VPA-treated offspring exhibited highly enhanced conditioned fear memories, which generalized to other stimuli and were resistant to extinction. Electrophysiological in vitro recordings in the amygdala revealed hyper-reactivity towards stimulation and enhanced activity-induced synaptic plasticity. These results imply that enhanced activity and plasticity in the amygdala may underlie the exaggerated fear memories. Furthermore it is suggested in this thesis that a hyper-reactive amygdala may underlie some of the most basic symptoms observed in autism: reduced social interactions and resistance to rehabilitation

Generation and Characterization of Recombinantly Polysialylated Antibody

With high affinity and specificity, antibodies are now proven biotherapuetics for a wide range of diseases, such as cancer and immunological conditions. However, antibody Fc-domain mediated cross reactivity with associated side effects has hindered the development of antibody therapy in a number of applications. The development of engineered recombinant antibody fragments to address the problems seen with whole monoclonal antibodies (mAbs). Their smaller size enables rapid antigen localisation, tissue penetration and higher specificity ratios. Whereas, poor pharmacokinetics due to fast blood clearance from the circulation is an important obstacle to these small molecules, and modification of antibody fragments is required for improved tissue exposure and uptake. In order to improve protein pharmacokinetics whilst maintaining good tissue/tumour to blood ratios and low cross-reactivity, conjugation of antibody fragments with a natural polymer polysialic acid (PSA) composed by N-acetylneuraminic acid (Neu5Ac or NANA), has been investigated. Compared to polyethylene glycol (PEG) conjugation, which is the predominant approach, PSA appears to be a better alternative because it is biodegradable, non-toxic and highly hydrophilic. In this project, an anti-carcinoembryonic antigen scFv (MFE-23), and an anti-HER2 scFv (C6.5) were used as model antibody fragments to conjugate with PSA. To overcome the limitations of chemical conjugations, this project was focused on the investigation of recombinant polysialylation of single chain antibodies. ScFv genes were firstly incorporated with a naturally polysialylatable (Ig5 and FN1) domains from human neuronal cell adhesion molecule. After transfections into polysialyltransferases-expressing HEK-293 cells, the biosynthesized sialic acid was enzymatically added to the N-glycosylated fusion protein Ig5 domain for elongating the attached PSA chain. Expressed by monoclonal cell secretion, polysialylated scFvs were successfully generated and purified. Based on the negative charge of PSA, an ion exchange method was developed for separating different polysialylated scFv isoforms. The degree of polymerization (DP) was detected by mass spectrometry, which showed an average of recombinant DP of up to 20 NANA residues attached to a complex Nglycan core. Affinity binding tests by ELISA confirmed the antibody KD was significantly retained at approximately 5nM after polysialylation, however BIAcore kinetic analysis demonstrated influencing change to the KD due to the strong negative charge of PSA. In vitro experiments using both FACS and confocal microscopy suggested that recombinant polysialylation has no affect of targeting antigens expressed on the live cell surface. Molecular hydrodynamic radius increases of polysialylated scFvs were detected by size exclusion chromatography, which led to corresponding pharmacokinetic improvement from in vivo mice studies with enhanced serum half-lives

Developmental regulation of GABAergic interneuron branching and synaptic development in the prefrontal cortex by soluble neural cell adhesion molecule

Neural cell adhesion molecule, NCAM, is an important regulator of neuronal process outgrowth and synaptic plasticity. Transgenic mice that overexpress the soluble NCAM extracellular domain (NCAM-EC) have reduced GABAergic inhibitory and excitatory synapses, and altered behavioral phenotypes. Here, we examined the role of dysregulated NCAM shedding, modeled by overexpression of NCAM-EC, on development of GABAergic basket interneurons in the prefrontal cortex. NCAM-EC overexpression disrupted arborization of basket cells during the major period of axon/dendrite growth, resulting in decreased numbers of GAD65- and synaptophysin-positive perisomatic synapses. NCAM-EC transgenic protein interfered with interneuron branching during early postnatal stages when endogenous polysialylated (PSA) NCAM was converted to non-PSA isoforms. In cortical neuron cultures, soluble NCAM-EC acted as a dominant inhibitor of NCAM-dependent neurite branching and outgrowth. These findings suggested that excess soluble NCAM-EC reduces perisomatic innervation of cortical neurons by perturbing axonal/dendritic branching during cortical development

Abnormal brain development in mice with region- and cell-type-specific inactivation of ST8SIA2

[no abstract

NCAM180 Regulates Ric8A Membrane Localization and Potentiates β-Adrenergic Response

Cooperation between receptors allows integrated intracellular signaling leading to appropriate physiological responses. The Neural Cell Adhesion Molecule (NCAM) has three main isoforms of 120, 140 and 180 kDa, with adhesive and signaling properties, but their respective functions remains to be fully identified. Here we show that the human NCAM180 intracellular domain is a novel interactor of the human guanosine exchange factor (GEF) Ric8A using the yeast two hybrid system and immunoprecipitation. Furthermore, NCAM, Ric8A and Gαs form a tripartite complex. Colocalization experiments by confocal microscopy revealed that human NCAM180 specifically induces the recruitment of Ric8A to the membrane. In addition, using an in vitro recombinant system, and in vivo by comparing NCAM knock-out mouse brain to NCAM heterozygous and wild type brains, we show that NCAM expression dose dependently regulates Ric8A redistribution in detergent resistent membrane microdomains (DRM). Previous studies have demonstrated essential roles for Ric8 in Gα protein activity at G protein coupled receptors (GPCR), during neurotransmitter release and for asymmetric cell division. We observed that inhibition of Ric8A by siRNA or its overexpression, decreases or increases respectively, cAMP production following β-adrenergic receptor stimulation. Furthermore, in human HEK293T recombinant cells, NCAM180 potentiates the Gαs coupled β-adrenergic receptor response, in a Ric8A dependent manner, whereas NCAM120 or NCAM140 do not. Finally, in mouse hippocampal neurons expressing endogenously NCAM, NCAM is required for the agonist isoproterenol to induce cAMP production, and this requirement depends on Ric8A. These data illustrate a functional crosstalk between a GPCR and an IgCAM in the nervous system