A membrane network of receptors and enzymes for adenine nucleotides and nucleosides

AbstractMost cells express more than one receptor plus degrading enzymes for adenine nucleotides or nucleosides, and cellular responses to purines are rarely compatible with the actions of single receptors. Therefore, these receptors are viewed as components of a combinatorial receptor web rather than self-dependent entities, but it remained unclear to what extent they can associate with each other to form signalling units. P2Y1, P2Y2, P2Y12, P2Y13, P2X2, A1, A2A receptors and NTPDase1 and -2 were expressed as fluorescent fusion proteins which were targeted to membranes and signalled like the unlabelled counterparts. When tested by FRET microscopy, all the G protein-coupled receptors proved able to form heterooligomers with each other, and P2Y1, P2Y12, P2Y13, A1, A2A, and P2X2 receptors also formed homooligomers. P2Y receptors did not associate with P2X, but G protein-coupled receptors formed heterooligomers with NTPDase1, but not NTPDase2. The specificity of prototypic interactions (P2Y1/P2Y1, A2A/P2Y1, A2A/P2Y12) was corroborated by FRET competition or co-immunoprecipitation. These results demonstrate that G protein-coupled purine receptors associate with each other and with NTPDase1 in a highly promiscuous manner. Thus, purinergic signalling is not only determined by the expression of receptors and enzymes but also by their direct interaction within a previously unrecognized multifarious membrane network

P2 receptor-mediated modulation of neurotransmitter release—an update

Presynaptic nerve terminals are equipped with a number of presynaptic auto- and heteroreceptors, including ionotropic P2X and metabotropic P2Y receptors. P2 receptors serve as modulation sites of transmitter release by ATP and other nucleotides released by neuronal activity and pathological signals. A wide variety of P2X and P2Y receptors expressed at pre- and postsynaptic sites as well as in glial cells are involved directly or indirectly in the modulation of neurotransmitter release. Nucleotides are released from synaptic and nonsynaptic sites throughout the nervous system and might reach concentrations high enough to activate these receptors. By providing a fine-tuning mechanism these receptors also offer attractive sites for pharmacotherapy in nervous system diseases. Here we review the rapidly emerging data on the modulation of transmitter release by facilitatory and inhibitory P2 receptors and the receptor subtypes involved in these interactions

Cognitive decline in Huntington's disease expansion gene carriers

BACKGROUND: In Huntington's Disease (HD) cognitive decline can occur before unequivocal motor signs become apparent. As cognitive decline often starts early in the course of the disease and has a progressive nature over time, cognition can be regarded as a key target for symptomatic treatment. The specific progressive profile of cognitive decline over time is unknown. OBJECTIVE: The aim of this study is to quantify the progression of cognitive decline across all HD stages, from pre-motormanifest to advanced HD, and to investigate if CAG length mediates cognitive decline. METHODS: In the European REGISTRY study 2669 HD expansion gene carriers underwent annual cognitive assessment. General linear mixed models were used to model the cognitive decline for each cognitive task across all disease stages. Additionally, a model was developed to evaluate the cognitive decline based on CAG length and age rather than disease stage. RESULTS: There was significant cognitive decline on all administered tasks throughout pre-motormanifest (close to estimated disease onset) participants and the subsequent motormanifest participants from stage 1 to stage 4. Performance on the Stroop Word and Stroop Color tests additionally declined significantly across the two pre-motormanifest groups: far and close to estimated disease onset. The evaluation of cognition performance in relation to CAG length and age revealed a more rapid cognitive decline in participants with longer CAG length than participants with shorter CAG length over time. CONCLUSION: Cognitive performance already shows decline in pre-motormanifest HD gene expansion carriers and gradually worsens to late stage HD. HD gene expansion carriers with certain CAG length have their own cognitive profile, i.e., longer CAG length is associated with more rapid decline

Reduced Cancer Incidence in Huntington's Disease: Analysis in the Registry Study

Background: People with Huntington’s disease (HD) have been observed to have lower rates of cancers. Objective: To investigate the relationship between age of onset of HD, CAG repeat length, and cancer diagnosis. Methods: Data were obtained from the European Huntington’s disease network REGISTRY study for 6540 subjects. Population cancer incidence was ascertained from the GLOBOCAN database to obtain standardised incidence ratios of cancers in the REGISTRY subjects. Results: 173/6528 HD REGISTRY subjects had had a cancer diagnosis. The age-standardised incidence rate of all cancers in the REGISTRY HD population was 0.26 (CI 0.22–0.30). Individual cancers showed a lower age-standardised incidence rate compared with the control population with prostate and colorectal cancers showing the lowest rates. There was no effect of CAG length on the likelihood of cancer, but a cancer diagnosis within the last year was associated with a greatly increased rate of HD onset (Hazard Ratio 18.94, p < 0.001). Conclusions: Cancer is less common than expected in the HD population, confirming previous reports. However, this does not appear to be related to CAG length in HTT. A recent diagnosis of cancer increases the risk of HD onset at any age, likely due to increased investigation following a cancer diagnosis

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation

Feldforschung im nördlichen Wipptal (Silltal) : Trachtenbestandteile von der späten Bronzezeit bis ans Ende der römischen Kaiserzeit

Christoph HusslUniversität Innsbruck, Masterarbeit, 2019(VLID)338464

Release of nucleotides from neuroendocrine cells and oligomerization of receptors and enzymes for nucleosides and nucleotides

Viele Zellen setzen Nucleotide in den Extrazellularraum frei, welche dort mannigfaltige Funktionen via ionotroper P2X und metabotroper P1 und P2Y Rezeptoren ausüben. NTPDasen hydrolysieren ATP zu Nukleosiden und sind oft mit Purinrezeptoren kolokalisiert. Zuerst widmeten wir uns der Frage, ob die Freisetzung von ATP aus neuroendokrinen PC12 Zellen ausschließlich von Vesikelexozytose abhängt, oder ob auch nicht vesikuläre Komponenten beteiligt sind. Wir kreierten PC12 Zellen, die stabil die leichte Kette von Botulinum Toxin C1 exprimierten. Dieses bewirkt durch die Spaltung von Syntaxin 1A eine verringerte Vesikelexozytose. Die Expression wurde durch eine reduzierte Syntaxin 1A Expression in Zellmembranen von PC12 Zellen bestätigt. Die transfizierten Zellen zeigten eine deutlich reduzierte spontane und durch Depolarisation ausgelöste Freisetzung von [3H]Noradrenalin. Wir untersuchten die autokrine/parakrine Aktivierung von P2Y12 Rezeptoren als Biosensor für die Nukleotidfreisetzung aus PC12 Zellen. ADP reduzierte die Synthese von cAMP vergleichbar in transfizierten und untransfizierten Zellen. Die Steigerung der cAMP Synthese durch den Abbau von extrazellulären Nukleotiden mittels Apyrase sowie durch P2Y12-Rezeptor Antagonisten war ebenso vergleichbar. Die Hemmung der cAMP Synthese durch depolarisationinduzierte Freisetzung endogener Nukleotide jedoch war deutlich reduziert in Zellen mit eingeschränkter Vesikelexozytose. Demnach hängt in PC12 Zellen die depolarisationsinduzierte Nukleotidfreisetzung im wesentlichen von Vesikelexozytose ab, während die spontane Nukleotidfreisetzung auch nicht exozytotische Komponenten enthält. Weiters beschäftigten wir uns mit der Frage, ob Rezeptoren für Nukleoside und Nukleotide mit NTPDasen, sowie mit anderen Rezeptor Subtypen interagieren. Wir konstruierten Plasmide fluoreszierender Fusionsproteine von A1, A2A, P2Y1, P2Y2, P2Y12, P2Y13 und P2X2 Rezeptoren sowie von NTPDase1 und -2. In HEK293 Zellen wurden die Fusionsproteine an die Zellmembran transportiert und zeigten vergleichbare Funktionalität wie die nicht fluoreszierenden Konstrukte. FRET Microskopie ergab, dass A1, A2A, P2Y1, P2Y12, P2Y13 und P2X2-Rezeptoren Homooligomere bilden. Weiters interagierten alle getesteten G-Protein-gekoppelten Rezeptoren miteinander sowie mit NTPDase1, nicht jedoch mit ionotropen P2X-Rezeptoren und NTPDase2. Purinerge Signalübertragung wird demnach durch die Freisetzung von Nukleosiden und Nukleotiden, durch deren enzymatische Hydrolyse sowie durch die direkte Interaktion ihrer Rezeptoren und abbauenden Enzyme in Zellmembranen reguliert.Extracellular nucleosides and nucleotides released from various types of cells exert a plethora of functions via ionotropic P2X and metabotropic P1 and P2Y receptors. NTPDases are hydrolysing nucleoside triphosphates towards nucleosides in the extracellular space. First we addressed the question, if the release of ATP from neuroendocrine PC12 cells exclusively depends on vesicle exocytosis or if it also includes non vesicular factors. Therefore we created PC12 cells stably expressing botulinum toxin C1 light chain, which lead to a decreased vesicle exocytosis via cleavage of syntaxin 1A. Reduced expression was detected by immunoblotting of PC12 membranes. Transfected cells showed significantly reduced spontaneous and depolarization-evoked release of [3H]noradrenaline compared to untransfected cells. We examined the autocrine/paracrine activation of P2Y12 receptors as a biosensor for nucleotide release from PC12 cells. ADP reduced cAMP synthesis to the same extent in transfected and non-transfected cells. Additionally, the enhancement of cAMP synthesis via degradation of extracellular nucleotides by apyrase and via P2Y12 receptor antagonists was not different in wild-type and transfected cells. In contrast, the inhibition of cAMP synthesis by depolarization-evoked release of endogenous nucleotides was significantly reduced in cells with impaired vesicle exocytosis. This indicates that in neuroendocrine cells, depolarization-evoked nucleotide release depends essentially on vesicle exocytosis, whereas spontaneous nucleotide release can arise non-exocytotically. The second part of the thesis copes with the question, if P1 and P2 receptors are able to physically interact with NTPDases as well as with other receptor subtypes. We constructed plasmids coding for A1, A2A, P2Y1, P2Y2, P2Y12, P2Y13, P2X2 receptors as well as for NTPDase1 and -2 fused to fluorescent proteins. The constructs were expressed in HEK293 cells, where they were targeted to membranes and showed comparable functionality as their unlabeled counterparts. FRET microscopy revealed that A1, A2A, P2X2 P2Y1, P2Y12 and P2Y13 receptors form homooligomers and all tested GPCRs show heterooligomerization with each other and with NTPDase1, but not with ionotropic P2X receptors and NTPDase2. Co-immunoprecipitation and FRET competition verified the specificity of the interactions. The results indicate that G protein-coupled purine receptors are able to interact variably with each other and with NTPDase1. Purinergic signaling is therefore regulated by the release of nucleotides, their enzymatic hydrolysis and the direct interaction of their receptors and enzymes expressed within the same membrane compartments.eingereicht von Mag. pharm. Simon HusslZusammenfassung in deutscher SpracheAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität, Dissertation, 2016OeB