GABAB receptor, localization and regulation

GABAB receptors are G-protein coupled receptors for gamma-amino butyric acid, the main inhibitory neurotransmitter in the brain. Functional GABAB receptors are obligate heterodimers composed of GABAB1 and GABAB2 subunits. The GABAB1 subunit exists in two isoforms, GABAB1a and GABAB1b, that can be differentiated by a pair of sushi domains exclusively located on the ectodomain of GABAB1a. As a consequence, two distinct receptor subtypes, GABAB(1a,2) and GABAB(1b,2), are present in the brain. Depending on their subcellular localization, GABAB receptors exert distinct regulatory effects on synaptic transmission. Presynaptically, GABAB receptors inhibit Ca2+ influx by closing voltage-gated Ca2+-channels therefore regulating neurotransmitter release. Postsynaptically, GABAB receptors activate inwardly rectifying Kir3-type K+-channels leading to hyperpolarisation of the postsynaptic membrane. Recently, it has become clear that GABAB(1a,2) and GABAB(1b,2) receptors convey individual functions, which are, at least in part, related to their distinct subcellular distribution. The aim of this thesis was to gain further insight into the function of GABAB receptors by characterizing their localization at the ultrastructural level in respect to effector channels and subtype composition. Moreover, it was of interest to study the dynamic regulation of GABAB receptors in response to synaptic activity. In the first part of this thesis, the localization of GABAB receptors and Kir3-type effector channels was investigated in the CA1 region of the hippocampus. It could be demonstrated that postsynaptic GABAB receptors colocalize with the Kir3.2 subunit of K+-channels in dendritic spines, but not in dendritic shafts of CA1 pyramidal cells (chapter 6.1.; Kulik et al., 2006). The differential distribution of GABAB1 subunit isoforms at the mossy fiber-CA3 pyramidal neuron synapse was investigated in the second part of this work. Due to the lack of isoform specific antibodies, mice selectively expressing GABAB1a or GABAB1b were used. It could be shown that mainly the GABAB1a subunit isoform contributes to the composition of presynaptic GABAB receptors whereas GABAB1b is the predominant GABAB1 subunit isoform on the postsynaptic side. Electrophysiological recordings were used to assess the contribution of the two different GABAB1 subunit isoforms to functional pre- and postsynaptic receptors in response to pharmacological as well as physiological GABAB receptor activation. The findings illustrate that the spatial segregation of GABAB1 subunit isoforms at mossy fiber terminals is sufficient to produce a strictly subtype–specific response (chapter 6.2.; Guetg et al., 2009). In the third part of this work, a new mouse model containing a GABAB1-eGFP transgene, allowing the visualization of GABAB receptors, was generated. Crossing the GABAB1-eGFP transgene into the GABAB1 deficient background allowed the study of GABAB receptors tagged with a fluorescent protein under expression of endogenous promoter elements. Therefore these mice provide a useful tool to visualize the spatio-temporal distribution of GABAB receptors in vivo and in vitro (chapter 6.3.; Casanova et al., 2009). The dynamic regulation of surface GABAB receptors induced by glutamate was investigated in primary hippocampal neurons and the results are presented in the last part of this thesis. Activation of NMDA receptors resulted in a decrease of surface GABAB receptor levels. This decrease involved Ca2+-dependent activation of CaMKII. A CaMKII phosphorylation site within the cytoplasmic domain of the GABAB1 subunit was identified. Evidence that phosphorylation of this site is essential for the observed effect of NMDA receptor activation on GABAB surface receptors is presented in this thesis. In conclusion, it could be demonstrated that GABAB receptors are dynamically regulated and interact with other receptors and kinases. The results obtained, implicate that activity-dependent regulation of GABAB receptors is potentially involved in the modulation of synaptic strength (chapter 6.4.)

Wake Measurements of a Dechirper Jaw with Non-Zero Tilt Angle

The RadiaBeam/SLAC dechirper at the Linac Coherent Light Source (LCLS) is being used as a fast kicker, by inducing transverse wakefields, to e.g. facilitate Fresh-slice, two-color laser operation. The dechirper jaws are independently adjustable at both ends, and it is difficult to avoid leaving residual (longitudinal) tilt in them during set-up. In this report we develop a model independent method of removing unknown tilt in a jaw. In addition, for a short uniform bunch passing by a single dechirper plate, we derive an explicit analytical formula for the transverse wake kick as function of average plate offset and tilt angle. We perform wake kick measurements for the different dechirper jaws of the RadiaBeam/SLAC dechirper, and find that the agreement between measurement and theory is excellent.Comment: 17 pages, 10 figure

Measurements of wake-induced electron beam deflection in a dechirper at the Linac Coherent Light Source

The RadiaBeam/SLAC dechirper, a structure consisting of pairs of flat, metallic, corrugated plates, %a corrugated structure in flat geometry, has been installed just upstream of the undulators in the Linac Coherent Light Source (LCLS). As a dechirper, with the beam passing between the plates on axis, longitudinal wakefields are induced that can remove unwanted energy chirp in the beam. However, with the beam passing off axis, strong transverse wakes are also induced. This mode of operation has already been used for the production of intense, multi-color photon beams using the Fresh-Slice technique, and is being used to develop a diagnostic for attosecond bunch length measurements. Here we measure, as function of offset, the strength of the transverse wakefields that are excited between the two plates, and also for the case of the beam passing near to a single plate. We compare with analytical formulas from the literature, and find good agreement. This report presents the first systematic measurements of the transverse wake strength in a dechirper, one that has been excited by a bunch with the short pulse duration and high energy found in an X-ray free electron laser.Comment: 8 pages, double column, 6 figure

Use of a corrugated beam pipe as a passive deflector for bunch length measurements

We report the experimental demonstration of bunch length measurements using a corrugated metallic beam pipe as a passive deflector. The corrugated beam pipe has been adopted for reducing longitudinal chirping after the bunch compressors in several XFEL facilities worldwide. In the meantime, there have been attempts to measure the electron bunch's longitudinal current profile using the dipole wakefields generated in the corrugated pipe. Nevertheless, the bunch shape reconstructed from the nonlinearly deflected beam suffers from significant distortion, particularly near the head of the bunch. In this paper, we introduce an iterative process to improve the resolution of the bunch shape reconstruction. The ASTRA and ELEGANT simulations have been performed for pencil beam and cigar beam cases, in order to verify the effectiveness of the reconstruction process. To overcome the undesirable effects of transverse beam spreads, a measurement scheme involving both the corrugated beam pipe and the spectrometer magnet has been employed, both of which do not require a dedicated (and likely very expensive) rf system. A proof-of-principle experiment was carried out at Pohang Accelerator Laboratory (PAL) Injector Test Facility (ITF), and its results are discussed together with a comparison with the rf deflector measurement

Differential Regulation of GABABReceptor Trafficking by Different Modes ofN-methyl-d-aspartate (NMDA) Receptor Signaling

Inhibitory GABAB receptors (GABABRs) can down-regulate most excitatory synapses in the CNS by reducing postsynaptic excitability. Functional GABABRs are heterodimers of GABAB1 and GABAB2 subunits and here we show that the trafficking and surface expression of GABABRs is differentially regulated by synaptic or pathophysiological activation of NMDA receptors (NMDARs). Activation of synaptic NMDARs using a chemLTP protocol increases GABABR recycling and surface expression. In contrast, excitotoxic global activation of synaptic and extrasynaptic NMDARs by bath application of NMDA causes the loss of surface GABABRs. Intriguingly, exposing neurons to extreme metabolic stress using oxygen/glucose deprivation (OGD) increases GABAB1 but decreases GABAB2 surface expression. The increase in surface GABAB1 involves enhanced recycling and is blocked by the NMDAR antagonist AP5. The decrease in surface GABAB2 is also blocked by AP5 and by inhibiting degradation pathways. These results indicate that NMDAR activity is critical in GABABR trafficking and function and that the individual subunits can be separately controlled to regulate neuronal responsiveness and survival

Epigenetic Silencing of Nucleolar rRNA Genes in Alzheimer's Disease

Background: Ribosomal deficits are documented in mild cognitive impairment (MCI), which often represents an early stage Alzheimer’s disease (AD), as well as in advanced AD. The nucleolar rRNA genes (rDNA), transcription of which is critical for ribosomal biogenesis, are regulated by epigenetic silencing including promoter CpG methylation. Methodology/Principal Findings: To assess whether CpG methylation of the rDNA promoter was dysregulated across the AD spectrum, we analyzed brain samples from 10 MCI-, 23 AD-, and, 24 age-matched control individuals using bisulfite mapping. The rDNA promoter became hypermethylated in cerebro-cortical samples from MCI and AD groups. In parietal cortex, the rDNA promoter was hypermethylated more in MCI than in advanced AD. The cytosine methylation of total genomic DNA was similar in AD, MCI, and control samples. Consistent with a notion that hypermethylation-mediated silencing of the nucleolar chromatin stabilizes rDNA loci, preventing their senescence-associated loss, genomic rDNA content was elevated in cerebrocortical samples from MCI and AD groups. Conclusions/Significance: In conclusion, rDNA hypermethylation could be a new epigenetic marker of AD. Moreover, silencing of nucleolar chromatin may occur during early stages of AD pathology and play a role in AD-related ribosoma

Binding hotspots of BAZ2B bromodomain: Histone interaction revealed by solution NMR driven docking.

Bromodomains are epigenetic reader domains, which have come under increasing scrutiny both from academic and pharmaceutical research groups. Effective targeting of the BAZ2B bromodomain by small molecule inhibitors has been recently reported, but no structural information is yet available on the interaction with its natural binding partner, acetylated histone H3K14ac. We have assigned the BAZ2B bromodomain and studied its interaction with H3K14ac acetylated peptides by NMR spectroscopy using both chemical shift perturbation (CSP) data and clean chemical exchange (CLEANEX-PM) NMR experiments. The latter was used to characterize water molecules known to play an important role in mediating interactions. Besides the anticipated Kac binding site, we consistently found the bromodomain BC loop as hotspots for the interaction. This information was used to create a data-driven model for the complex using HADDOCK. Our findings provide both structure and dynamics characterization that will be useful in the quest for potent and selective inhibitors to probe the function of the BAZ2B bromodomain.This is the final published version of the article. It has been published by the American Chemical Society in Biochemistry. The article can be accessed on their website here: http://pubs.acs.org/doi/abs/10.1021/bi500909d. It is freely available under a CC BY licence