Aufklärung der dreidimensionalen Struktur der EGF-like Domäne von ADAM17 mittels mehrdimensionaler NMR-Spektroskopie

ADAM17 gehört zu den membrangebundenen zinkabhängigen Metalloproteasen, auch ADAMs (a disintegrin and metalloprotease) genannt. Die Mitglieder der ADAM-Proteasefamilie zeichnen sich durch eine ähnliche Domänenstruktur aus, die aus einer Pro-, Protease-, Disintegrin-, cysteinreichen-, EGF-like-, Transmembran- und einer cytoplasmatischen Domäne aufgebaut ist. Während bei den meisten Mitgliedern der ADAM-Proteasefamilie alle diese Domänen vorhanden sind, fehlt bei ADAM17 und ADAM10 die cysteinreiche- oder die EGF-like Domäne. Welche der Domänen nicht vorhanden ist, wurde bis heute experimentell nicht geklärt. Um die Domäne von ADAM17 der Familie EGF-like Domänen zuordnen zu können, wurde im Rahmen dieser Arbeit diese Domäne rekombinant exprimiert und gereinigt. Mittels mehrdimensionaler NMR-Spektroskopie konnte die Tertiärstruktur der Domäne bestimmt werden. Diese Struktur wurde durch Vergleich der Sekundärstrukturelemente der Superfamilie der EGF/ Laminin-Proteine zugeordnet. In der Ausbildung der Disulfidbrücken zeigt die EGF-like Domäne von ADAM17 und ADAM10 Unterschiede zu allen anderen EGF-like Domänen. Dadurch bilden diese Domänen innerhalb der Superfamilie der EGF/ Laminin-Proteine eine eigene Familie aus

Timing is not Everything: Neuromodulation Opens the STDP Gate

Spike timing dependent plasticity (STDP) is a temporally specific extension of Hebbian associative plasticity that has tied together the timing of presynaptic inputs relative to the postsynaptic single spike. However, it is difficult to translate this mechanism to in vivo conditions where there is an abundance of presynaptic activity constantly impinging upon the dendritic tree as well as ongoing postsynaptic spiking activity that backpropagates along the dendrite. Theoretical studies have proposed that, in addition to this pre- and postsynaptic activity, a “third factor” would enable the association of specific inputs to specific outputs. Experimentally, the picture that is beginning to emerge, is that in addition to the precise timing of pre- and postsynaptic spikes, this third factor involves neuromodulators that have a distinctive influence on STDP rules. Specifically, neuromodulatory systems can influence STDP rules by acting via dopaminergic, noradrenergic, muscarinic, and nicotinic receptors. Neuromodulator actions can enable STDP induction or – by increasing or decreasing the threshold – can change the conditions for plasticity induction. Because some of the neuromodulators are also involved in reward, a link between STDP and reward-mediated learning is emerging. However, many outstanding questions concerning the relationship between neuromodulatory systems and STDP rules remain, that once solved, will help make the crucial link from timing-based synaptic plasticity rules to behaviorally based learning

Timing is not Everything: Neuromodulation Opens the STDP Gate

Spike timing dependent plasticity (STDP) is a temporally specific extension of Hebbian associative plasticity that has tied together the timing of presynaptic inputs relative to the postsynaptic single spike. However, it is difficult to translate this mechanism to in vivo conditions where there is an abundance of presynaptic activity constantly impinging upon the dendritic tree as well as ongoing postsynaptic spiking activity that backpropagates along the dendrite. Theoretical studies have proposed that, in addition to this pre- and postsynaptic activity, a “third factor” would enable the association of specific inputs to specific outputs. Experimentally, the picture that is beginning to emerge, is that in addition to the precise timing of pre- and postsynaptic spikes, this third factor involves neuromodulators that have a distinctive influence on STDP rules. Specifically, neuromodulatory systems can influence STDP rules by acting via dopaminergic, noradrenergic, muscarinic, and nicotinic receptors. Neuromodulator actions can enable STDP induction or – by increasing or decreasing the threshold – can change the conditions for plasticity induction. Because some of the neuromodulators are also involved in reward, a link between STDP and reward-mediated learning is emerging. However, many outstanding questions concerning the relationship between neuromodulatory systems and STDP rules remain, that once solved, will help make the crucial link from timing-based synaptic plasticity rules to behaviorally based learning

Changing the responses of cortical neurons from sub- to suprathreshold using single spikes in vivo

Action Potential (APs) patterns of sensory cortex neurons encode a variety of stimulus features, but how can a neuron change the feature to which it responds? Here, we show that in vivo a spike-timing-dependent plasticity (STDP) protocol-consisting of pairing a postsynaptic AP with visually driven presynaptic inputs-modifies a neurons' AP-response in a bidirectional way that depends on the relative AP-timing during pairing. Whereas postsynaptic APs repeatedly following presynaptic activation can convert subthreshold into suprathreshold responses, APs repeatedly preceding presynaptic activation reduce AP responses to visual stimulation. These changes were paralleled by restructuring of the neurons response to surround stimulus locations and membrane-potential time-course. Computational simulations could reproduce the observed subthreshold voltage changes only when presynaptic temporal jitter was included. Together this shows that STDP rules can modify output patterns of sensory neurons and the timing of single-APs plays a crucial role in sensory coding and plasticity

Dominance of the lurcher mutation in heteromeric kainate and AMPA receptor channels

Homomeric glutamate receptor (GluR) channels become spontaneously active when the last alanine residue within the invariant SYTANLAAF-motif in the third membrane segment is substituted by threonine. The same mutation in the orphan GluRdelta2 channel is responsible for neurodegeneration in Lurcher (Lc) mice. Since most native GluRs are composed of different subunits, we investigated the effect of an Lc-mutated subunit in heteromeric kainate and AMPA receptors expressed in HEK293 cells. Kainate receptor KA2 subunits, either wild type or carrying the Lc mutation (KA2Lc), are retained inside the cell but are surface-expressed when assembled with GluR6 sununits. Importantly, KA2Lc dominates the gating of KA2Lc/GluR6WT channels, as revealed by spontaneous activation and by slowed desensitization and deactivation kinetics of ligand-activated whole-cell currents. Moreover, the AMPA receptor subunit GluR-BLc(Q) which forms spontaneously active homomeric channels with rectifying current-voltage relationships, dominates the gating of heteromeric GluR-BLc(Q)/GluR-A(R) channels. The spontaneous currents of these heteromeric AMPAR channels show linear current-voltage relationships, and the ligand-activated whole-cell currents display slower deactivation and desensitization kinetics than the respective wild-type channels. For heteromeric Lc-mutated kainate and AMPA receptors, the effects on kinetics were reduced relative to the homomeric Lc-mutated forms. Thus, an Lc-mutated subunit can potentially influence heteromeric channel function in vivo, and the severity of the phenotype will critically depend on the levels of homomeric GluRLc and heteromeric GluRLc/GluRWT channels

Ca2+ and Na+ dependence of 3-hydroxyglutarate-induced excitotoxicity in primary neuronal cultures from chick embryo telencephalons

Glutaryl-CoA dehydrogenase deficiency (also known as glutaric aciduria type I) is an autosomal, recessively inherited neurometabolic disorder with a distinct neuropathology characterized by acute encephalopathy during a vulnerable period of brain development. Neuronal damage in this disease was demonstrated to involve N-methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity of the endogenously accumulating metabolite 3-hydroxyglutarate (3-OH-GA). However, it remained unclear whether NMDA receptors are directly or indirectly activated and whether 3-OH-GA disturbs the intracellular Ca(2+) homeostasis. Here we report that 3-OH-GA activated recombinant NMDA receptors (e.g. NR1/NR2A) but not recombinant alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (e.g. GluR-A/GluR-B) in HEK293 cells. Fluorescence microscopy using fura-2 as Ca(2+) indicator revealed that 3-OH-GA increased intracellular Ca2+ concentrations in the presence of extracellular Ca2+ in cultured chick neurons. Similar to glutamate-induced cell damage, 3-OH-GA neurotoxicity was modulated by extracellular Na+. The large cation N-methyl-D-glucamine, which does not permeate NMDA receptor channels, enhanced 3-OH-GA-induced Ca2+ increase and cell damage. In contrast, 3-OH-GA-induced neurotoxicity was reduced after replacement of Na+ by Li+, which permeates NMDA channels but does not affect the Na+ /Ca2+ exchanger in the plasma membrane. Spectrophotometric analysis of respiratory chain complexes I-V in submitochondrial particles from bovine heart revealed only a weak inhibition of 3-OH-GA on complex V at the highest concentration tested (10 mM). In conclusion, the present study revealed that NMDA receptor activation and subsequent disturbance of Ca2+ homeostasis contribute to 3-OH-GA-induced cell damag