Spinal cord synaptic plasticity by GlyRß release from receptor fields and syndapin I-dependent uptake.

Glycine receptor-mediated inhibitory neurotransmission is key for spinal cord function. Recent observations suggested that by largely elusive mechanisms also glycinergic synapses display synaptic plasticity. We imaged receptor fields at ultra-high resolution at freeze-fractured membranes, tracked surface and internalized glycine receptors (GlyR) and studied differential regulations of GlyRß interactions with the scaffold protein gephyrin and the F-BAR domain protein syndapin I and thereby reveal key principles of this process. S403 phosphorylation of GlyRß, known to be triggered by synaptic signaling, caused a decoupling from gephyrin scaffolds but simultaneously promoted association of syndapin I with GlyRß. In line, kainate-treatments used to trigger rearrangements of glycine receptors in murine syndapin I KO spinal cords (mixed sex) showed even more severe receptor field fragmentation than already observed in untreated syndapin I KO spinal cords. Syndapin I KO furthermore resulted in more dispersed receptors and increased receptor mobility also pointing out an important contribution of syndapin I in the organization of GlyRß fields. Strikingly, syndapin I KO also led to a complete disruption of kainate-induced GlyRß internalization. Accompanying quantitative ultra-high resolution studies in dissociated spinal cord neurons strongly suggested that the observed defects in GlyR internalization observed in syndapin I KO spinal cords are directly caused by syndapin I deficiency within murine spinal cord neurons. Together our results unveiled important mechanisms organizing and altering glycine receptor fields during both steady-state and particularly upon kainate-induced synaptic rearrangement - principles organizing and fine-tuning synaptic efficacy and plasticity of glycinergic synapses in the spinal cord.SIGNIFICANCE STATEMENTInitial observations suggested that also glycinergic synapses - key for spinal cord and brain stem functions - may display some form of synaptic plasticity. Imaging receptor fields at ultra-high resolution at freeze-fractured membranes, tracking surface and internalized glycine receptors (GlyR) and studying regulations of GlyRß interactions we here reveal key principles of these kainate-inducible adaptations. A switch from gephyrin-mediated receptor scaffolding to syndapin I-mediated GlyRß scaffolding and internalization allows for modulating synaptic receptor availability. In line, kainate-induced GlyRß internalization was completely disrupted and GlyRß receptor fields were distorted upon syndapin I KO. These results unveiled important mechanisms during both steady-state and kainate-induced alterations of synaptic GlyR fields - principles underlying synaptic efficacy and plasticity of synapses in the spinal cord.

Syndapin I Loss-of-Function in Mice Leads to Schizophrenia-Like Symptoms.

Schizophrenia is associated with cognitive and behavioral dysfunctions thought to reflect imbalances in neurotransmission systems. Recent screenings suggested that lack of (functional) syndapin I (PACSIN1) may be linked to schizophrenia. We therefore studied syndapin I KO mice to address the suggested causal relationship to schizophrenia and to analyze associated molecular, cellular, and neurophysiological defects. Syndapin I knockout (KO) mice developed schizophrenia-related behaviors, such as hyperactivity, reduced anxiety, reduced response to social novelty, and an exaggerated novel object response and exhibited defects in dendritic arborization in the cortex. Neuromorphogenic deficits were also observed for a schizophrenia-associated syndapin I mutant in cultured neurons and coincided with a lack of syndapin I-mediated membrane recruitment of cytoskeletal effectors. Syndapin I KO furthermore caused glutamatergic hypofunctions. Syndapin I regulated both AMPAR and NMDAR availabilities at synapses during basal synaptic activity and during synaptic plasticity-particularly striking were a complete lack of long-term potentiation and defects in long-term depression in syndapin I KO mice. These synaptic plasticity defects coincided with alterations of postsynaptic actin dynamics, synaptic GluA1 clustering, and GluA1 mobility. Both GluA1 and GluA2 were not appropriately internalized. Summarized, syndapin I KO led to schizophrenia-like behavior, and our analyses uncovered associated molecular and cellular mechanisms.

Comparison of Multiscale Imaging Methods for Brain Research.

A major challenge in neuroscience is how to study structural alterations in the brain. Even small changes in synaptic composition could have severe outcomes for body functions. Many neuropathological diseases are attributable to disorganization of particular synaptic proteins. Yet, to detect and comprehensively describe and evaluate such often rather subtle deviations from the normal physiological status in a detailed and quantitative manner is very challenging. Here, we have compared side-by-side several commercially available light microscopes for their suitability in visualizing synaptic components in larger parts of the brain at low resolution, at extended resolution as well as at super-resolution. Microscopic technologies included stereo, widefield, deconvolution, confocal, and super-resolution set-ups. We also analyzed the impact of adaptive optics, a motorized objective correction collar and CUDA graphics card technology on imaging quality and acquisition speed. Our observations evaluate a basic set of techniques, which allow for multi-color brain imaging from centimeter to nanometer scales. The comparative multi-modal strategy we established can be used as a guide for researchers to select the most appropriate light microscopy method in addressing specific questions in brain research, and we also give insights into recent developments such as optical aberration corrections.

An AKAP-Lbc-RhoA interaction inhibitor promotes the translocation of aquaporin-2 to the plasma membrane of renal collecting duct principal cells.

Stimulation of renal collecting duct principal cells with antidiuretic hormone (arginine-vasopressin, AVP) results in inhibition of the small GTPase RhoA and the enrichment of the water channel aquaporin-2 (AQP2) in the plasma membrane. The membrane insertion facilitates water reabsorption from primary urine and fine-tuning of body water homeostasis. Rho guanine nucleotide exchange factors (GEFs) interact with RhoA, catalyze the exchange of GDP for GTP and thereby activate the GTPase. However, GEFs involved in the control of AQP2 in renal principal cells are unknown. The A-kinase anchoring protein, AKAP-Lbc, possesses GEF activity, specifically activates RhoA, and is expressed in primary renal inner medullary collecting duct principal (IMCD) cells. Through screening of 18,431 small molecules and synthesis of a focused library around one of the hits, we identified an inhibitor of the interaction of AKAP-Lbc and RhoA. This molecule, Scaff10-8, bound to RhoA, inhibited the AKAP-Lbc-mediated RhoA activation but did not interfere with RhoA activation through other GEFs or activities of other members of the Rho family of small GTPases, Rac1 and Cdc42. Scaff10-8 promoted the redistribution of AQP2 from intracellular vesicles to the periphery of IMCD cells. Thus, our data demonstrate an involvement of AKAP-Lbc-mediated RhoA activation in the control of AQP2 trafficking.

Deciphering caveolar functions by syndapin III KO-mediated impairment of caveolar invagination.

Several human diseases are associated with a lack of caveolae. Yet, the functions of caveolae and the molecular mechanisms critical for shaping them still are debated. We show that muscle cells of syndapin III KO mice show severe reductions of caveolae reminiscent of human caveolinopathies. Yet, different from other mouse models, the levels of the plasma membrane-associated caveolar coat proteins caveolin3 and cavin1 were both not reduced upon syndapin III KO. This allowed for dissecting bona fide caveolar functions from those supported by mere caveolin presence and also demonstrated that neither caveolin3 nor caveolin3 and cavin1 are sufficient to form caveolae. The membrane-shaping protein syndapin III is crucial for caveolar invagination and KO rendered the cells sensitive to membrane tensions. Consistent with this physiological role of caveolae in counterpoising membrane tensions, syndapin III KO skeletal muscles showed pathological parameters upon physical exercise that are also found in CAVEOLIN3 mutation-associated muscle diseases.

A-kinase anchoring proteins as potential drug targets.

A-kinase anchoring proteins (AKAPs) crucially contribute to the spatial and temporal control of cellular signalling. They directly interact with a variety of protein binding partners and cellular constituents, thereby directing pools of signalling components to defined locales. In particular, AKAPs mediate compartmentalization of cAMP signalling. Alterations in AKAP expression and their interactions are associated with or cause diseases including chronic heart failure, various cancers and disorders of the immune system such as HIV. A number of cellular dysfunctions result from mutations of specific AKAPs. The link between malfunctions of single AKAP complexes and a disease makes AKAPs and their interactions interesting targets for the development of novel drugs. LINKED ARTICLES This article is part of a themed section on Novel cAMP Signalling Paradigms. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.166.issue-2.

Current inhibition of human EAG1 potassium channels by the Ca2+ binding protein S100B.

Voltage-dependent human ether à go-go (hEAG1) potassium channels are implicated in neuronal signaling as well as in cancer cell proliferation. Unique sensitivity of the channel to intracellular Ca(2+) is mediated by calmodulin (CaM) binding to the intracellular N- and C-termini of the channel. Here we show that application of the acidic calcium-binding protein S100B to inside-out patches of Xenopus oocytes causes Ca(2+)-dependent inhibition of expressed hEAG1 channels. Protein pull-down assays and fluorescence correlation spectroscopy (FCS) revealed that S100B binds to hEAG1 and shares the same binding sites with CaM. Thus, S100B is a potential alternative calcium sensor for hEAG1 potassium channels.