Loss of Extrasynaptic Inhibitory Glycine Receptors in the Hippocampus of an AD Mouse Model Is Restored by Treatment with Artesunate.

Alzheimer's disease (AD) is characterized by synaptic failure and neuronal loss. Recently, we demonstrated that artemisinins restored the levels of key proteins of inhibitory GABAergic synapses in the hippocampus of APP/PS1 mice, a model of cerebral amyloidosis. In the present study, we analyzed the protein levels and subcellular localization of α2 and α3 subunits of GlyRs, indicated as the most abundant receptor subtypes in the mature hippocampus, in early and late stages of AD pathogenesis, and upon treatment with two different doses of artesunate (ARS). Immunofluorescence microscopy and Western blot analysis demonstrated that the protein levels of both α2 and α3 GlyRs are considerably reduced in the CA1 and the dentate gyrus of 12-month-old APP/PS1 mice when compared to WT mice. Notably, treatment with low-dose ARS affected GlyR expression in a subunit-specific way; the protein levels of α3 GlyR subunits were rescued to about WT levels, whereas that of α2 GlyRs were not affected significantly. Moreover, double labeling with a presynaptic marker indicated that the changes in GlyR α3 expression levels primarily involve extracellular GlyRs. Correspondingly, low concentrations of artesunate (≤1 µM) also increased the extrasynaptic GlyR cluster density in hAPPswe-transfected primary hippocampal neurons, whereas the number of GlyR clusters overlapping presynaptic VIAAT immunoreactivities remained unchanged. Thus, here we provide evidence that the protein levels and subcellular localization of α2 and α3 subunits of GlyRs show regional and temporal alterations in the hippocampus of APP/PS1 mice that can be modulated by the application of artesunate.

Strengths and Weaknesses of Non-enhanced and Contrast-enhanced Cadaver Computed Tomography Scans in the Teaching of Gross Anatomy in an Integrated Curriculum.

Cadaver-specific postmortem computed tomography (PMCT) has become an integral part in anatomy teaching at several universities. Recently, the feasibility of contrast-enhanced (CE)-PMCT has been demonstrated. The purpose of this study was to identify particular strengths and weaknesses of both non-enhanced and contrast-enhanced PMCT compared to conventional cadaver dissection. First, the students' perception of the learning effectiveness of the three different modalities have been assessed using a 34-item survey (five-point Likert scale) covering all anatomy course modules. Results were compared using the nonparametric Friedman Test. Second, the most frequent artifacts in cadaver CT scans, were systematically analyzed in 122 PMCT and 31 CE-PMCT data sets to quantify method-related limitations and characteristics. Perfusion quality was assessed in 57 vascular segments (38 arterial and 19 venous). The survey was answered by n = 257/320 (80.3%) students. Increased learning benefits of PMCT/ CE-PMCT compared to cadaver dissection were found in osteology (2/3 categories, P < 0.001), head and neck (2/5 categories, P < 0.01), and brain anatomy (3/3 categories, P < 0.01). Contrast-enhanced-PMCT was perceived particularly useful in learning vascular anatomy (10/10 categories, P < 0.01). Cadaver dissection received significantly higher scores compared to PMCT and CE-PMCT in all categories of the abdomen and thorax (7/7 categories, P < 0.001), as well as the majority of muscular anatomy (5/6 categories, P < 0.001). Frequent postmortem artifacts (total n = 28, native-phase n = 21, contrast injection-related n = 7) were identified and assessed. The results of this work contribute to the understanding of the value of integrating cadaver-specific PMCT in anatomy teaching.

Binding of gephyrin to microtubules is regulated by its phosphorylation at Ser270.

Gephyrin is a multifunctional scaffolding protein anchoring glycine- and subtypes of GABA type A- receptors at inhibitory postsynaptic membrane specializations by binding to the microtubule (MT) and/or the actin cytoskeleton. However, the conditions under which gephyrin can bind to MTs and its regulation are currently unknown. Here, we demonstrate that during the purification of MTs from rat brain by sedimentation of polymerized tubulin using high-speed centrifugation a fraction of gephyrin was bound to MTs, whereas gephyrin phosphorylated at the CDK5-dependent site Ser270 was detached from MTs and remained in the soluble protein fraction. Moreover, after collybistin fostered phosphorylation at Ser270 the binding of a recombinant gephyrin to MTs was strongly reduced in co-sedimentation assays. Correspondingly, upon substitution of wild-type gephyrin with recombinant gephyrin carrying alanine mutations at putative CDK5 phosphorylation sites the binding of gephyrin to MTs was increased. Furthermore, the analysis of cultured HEK293T and U2OS cells by immunofluorescence-microscopy disclosed a dispersed and punctuated endogenous gephyrin immunoreactivity co-localizing with MTs which was evidently not phosphorylated at Ser270. Thus, our study provides additional evidence for the binding of gephyrin to MTs in brain tissue and in in vitro cell systems. More importantly, our findings indicate that gephyrin-MT binding is restricted to a specific gephyrin fraction and depicts phosphorylation of gephyrin as a regulatory mechanism of this process by showing that soluble gephyrin detached from MTs can be detected specifically with the mAb7a antibody, which recognizes the Ser270 phosphorylated- version of gephyrin.

Gephyrin: a key regulatory protein of inhibitory synapses and beyond.

Scaffolding proteins underlying postsynaptic membrane specializations are important structural and functional components of both excitatory and inhibitory synapses. At inhibitory synapses, gephyrin was identified as anchoring protein. Gephyrin self-assembles into a complex flat submembranous lattice that slows the lateral mobility of glycine and GABAA receptors, thus allowing for their clustering at postsynaptic sites. The structure and stability of the gephyrin lattice is dynamically regulated by posttranslational modifications and interactions with binding partners. As gephyrin is the core scaffolding protein for virtually all inhibitory synapses, any changes in the structure or stability of its lattice can profoundly change the packing density of inhibitory receptors and, therefore, alter inhibitory drive. Intriguingly, gephyrin plays a completely independent role in non-neuronal cells, where it facilitates two steps in the biosynthesis of the molybdenum cofactor. In this review, we provide an overview of the role of gephyrin at inhibitory synapses and beyond. We discuss its dynamic regulation, the nanoscale architecture of its synaptic lattice, and the implications of gephyrin dysfunction for neuropathologic conditions, such as Alzheimer's disease and epilepsy.

Hands-on or no hands-on training in ultrasound imaging: A randomized trial to evaluate learning outcomes and speed of recall of topographic anatomy.

Medical students have difficulties in interpreting two-dimensional (2D) topographic anatomy on sectional images. Hands-on and no hands-on training in ultrasound imaging facilitate learning topographic anatomy. Hands-on training is linked with active search for patterns of anatomical structures and might train pattern recognition for image interpretation better although the added value on learning outcomes is unclear. This study explores first year medical students' knowledge in topographic anatomy of the upper abdomen after attending hands-on or no hands-on training in ultrasound in a randomized trial. While students in the hands-on ultrasound group (N = 21) generated and interpreted standardized planes of ultrasound imaging, students in the no hands-on seminar group (N = 22) interpreted provided ultrasound images by correlation to three-dimensional (3D) anatomical prosections. Afterwards knowledge in topographic anatomy was measured repetitively by text and ultrasound image-based multiple choice (MC) examinations. As surrogate for pattern recognition, students rated whether answers were known after reflection or instantly. While intrinsic motivation was higher in the ultrasound group, no differences in the MC-examination score were found between ultrasound and seminar group instantly (66.5 ±10.9% vs. 64.5% ±11.0%, P = 0.551) or six weeks (62.9% ±12.3% vs. 61.5% ±11.0%, P = 0.718) after training. In both groups scores in text-based questions declined (P < 0.001) while scores in image-based questions remained stable (P = 0.895) with time. After six weeks more image-based questions were instantly known in the hands-on ultrasound compared to seminar-group (28% ±17.3% vs. 16% ±13.5%, P = 0.047). Hands-on ultrasound-training is linked with faster interpreting of ultrasound images without loss in accuracy. The added value of hands-on training might be facilitation of pattern recognition.

Contrast-enhanced cadaver-specific computed tomography in gross anatomy teaching.

OBJECTIVES: To establish contrast-enhanced (CE) cadaver-specific post-mortem computed tomography (PMCT) in first-year gross anatomy teaching and quantitatively evaluate its learning benefit. METHODS: 132 first-year medical students were included in this IRB-approved study and randomly assigned to an intervention group (n=59) provided with continuous access to CE and non-enhanced (NE) cadaver-specific PMCT-scans during the first-semester gross anatomy course, and a control group (n=73) that had only NE cadaver-specific PMCT data available. Four multiple-choice tests were carried out (15 questions each) subsequent to completion of the corresponding anatomy module: Head and neck anatomy, extremities, thorax, and abdomen. Median test results were compared in each module between the groups using the Wilcoxon rank-sum test. Additionally, participants of the intervention group answered a 15-item feedback-questionnaire. RESULTS: The intervention group achieved significantly higher test scores in head and neck anatomy (median=12.0, IQR=10.0-13.0) versus the control group (median=10.5, IQR=9.0-12.0) (p<0.01). There were no significant differences in the comparison of other modules. CEPMCT was highly appreciated by undergraduate medical students. CONCLUSIONS: The incorporation of contrast-enhanced cadaver-specific PMCT-scans in gross anatomy teaching was proven to be feasible in the framework of the medical curriculum and significantly improved the students' learning performance in head and neck anatomy. KEY POINTS: • Cadaver-specific contrast-enhanced post-mortem CT (CEPMCT) is feasible in the medical curriculum. • CEPMCT yields significantly improved learning performance in head and neck anatomy (p<0.01). • CEPMCT is highly appreciated by medical students and used in tutor- or self-guided modes.

Forebrain-specific loss of synaptic GABAA receptors results in altered neuronal excitability and synaptic plasticity in mice.

Mutations that result in the defective trafficking of γ2 subunit containing GABAA receptors (γ2-GABAARs) are known to reduce synaptic inhibition. Whether perturbed clustering of non-mutated GABAARs similarly reduces synaptic inhibition in vivo is less clear. In this study we provide evidence that the loss of postsynaptic γ2-GABAARs upon postnatal ablation of gephyrin, the major scaffolding protein of inhibitory postsynapses, from mature principal neurons within the forebrain results in reduced induction of long-term potentiation (LTP) and impaired network excitability within the hippocampal dentate gyrus. The preferential reduction in not only synaptic γ2-GABAAR cluster number at dendritic sites but also the decrease in γ2-GABAAR density within individual clusters at dendritic inhibitory synapses suggests that distal synapses are more sensitive to the loss of gephyrin expression than proximal synapses. The fact that these mice display behavioural features of anxiety and epilepsy emphasises the importance of postsynaptic γ2-GABAAR clustering for synaptic inhibition.

Aging-dependent reduction in glyoxalase 1 delays wound healing.

Methylglyoxal (MG), the major dicarbonyl substrate of the enzyme glyoxalase 1 (GLO1), is a reactive metabolite formed via glycolytic flux. Decreased GLO1 activity in situ has been shown to result in an accumulation of MG and increased formation of advanced glycation endproducts, both of which can accumulate during physiological aging and at an accelerated rate in diabetes and other chronic degenerative diseases. To determine the physiological consequences which result from elevated MG levels and the role of MG and GLO1 in aging, wound healing in young (≤12 weeks) and old (≥52 weeks) wild-type mice was studied. Old mice were found to have a significantly slower rate of wound healing compared to young mice (74.9 ± 2.2 vs. 55.4 ± 1.5% wound closure at day 6; 26% decrease; p < 0.0001). This was associated with decreases in GLO1 transcription, expression and activity. The importance of GLO1 was confirmed in mice by inhibition of GLO1. Direct application of MG to the wounds of young mice, decreased wound healing by 24% compared to untreated mice, whereas application of BSA modified minimally by MG had no effect. Treatment of either young or old mice with aminoguanidine, a scavenger of free MG, significantly increased wound closure by 16% (66.8 ± 1.6 vs. 77.2 ± 3.1%; p < 0.05) and 64% (40.4 ± 7.9 vs. 66.4 ± 5.2%; p < 0.05), respectively, by day 6. As a result of the aminoguanidine treatment, the overall rate of wound healing in the old mice was restored to the level observed in the young mice. These findings were confirmed in vitro, as MG reduced migration and proliferation of fibroblasts derived from young and old, wild-type mice. The data demonstrate that the balance between MG and age-dependent GLO1 downregulation contributes to delayed wound healing in old mice.

Integration of interactive three-dimensional image post-processing software into undergraduate radiology education effectively improves diagnostic skills and visual-spatial ability.

PURPOSE: Integrating interactive three-dimensional post-processing software into undergraduate radiology teaching might be a promising approach to synergistically improve both visual-spatial ability and radiological skills, thereby reducing students' deficiencies in image interpretation. The purpose of this study was to test our hypothesis that a hands-on radiology course for medical students using interactive three-dimensional image post-processing software improves radiological knowledge, diagnostic skills and visual-spatial ability. MATERIALS AND METHODS: A hands-on radiology course was developed using interactive three-dimensional image post-processing software. The course consisted of seven seminars held on a weekly basis. The 25 participating fourth- and fifth-year medical students learnt to systematically analyse cross-sectional imaging data and correlated the two-dimensional images with three-dimensional reconstructions. They were instructed by experienced radiologists and collegiate tutors. The improvement in radiological knowledge, diagnostic skills and visual-spatial ability was assessed immediately before and after the course by multiple-choice tests comprising 64 questions each. Wilcoxon signed rank test for paired samples was applied. RESULTS: The total number of correctly answered questions improved from 36.9±4.8 to 49.5±5.4 (p<0.001) which corresponded to a mean improvement of 12.6 (95% confidence interval 9.9-15.3) or 19.8%. Radiological knowledge improved by 36.0% (p<0.001), diagnostic skills for cross-sectional imaging by 38.7% (p<0.001), diagnostic skills for other imaging modalities - which were not included in the course - by 14.0% (p=0.001), and visual-spatial ability by 11.3% (p<0.001). CONCLUSION: The integration of interactive three-dimensional image post-processing software into undergraduate radiology education effectively improves radiological reasoning, diagnostic skills and visual-spatial ability, and thereby even diagnostic skills for imaging modalities not included in the course.

Phosphorylation of gephyrin in hippocampal neurons by cyclin-dependent kinase CDK5 at Ser-270 is dependent on collybistin.

Gephyrin is a scaffold protein essential for the postsynaptic clustering of inhibitory glycine and different subtypes of GABA(A) receptors. The cellular and molecular mechanisms involved in gephyrin-mediated receptor clustering are still not well understood. Here we provide evidence that the gephyrin-binding protein collybistin is involved in regulating the phosphorylation of gephyrin. We demonstrate that the widely used monoclonal antibody mAb7a is a phospho-specific antibody that allows the cellular and biochemical analysis of gephyrin phosphorylation at Ser-270. In addition, another neighbored epitope determinant was identified at position Thr-276. Analysis of the double mutant gephyrin(T276A,S277A) revealed significant reduction in gephyrin cluster formation and altered oligomerization behavior of gephyrin. Moreover, pharmacological inhibition of cyclin-dependent kinases in hippocampal neurons reduced postsynaptic gephyrin mAb7a immunoreactivities. In vitro phosphorylation assays and phosphopeptide competition experiments revealed a phosphorylation at Ser-270 depending on enzyme activities of cyclin-dependent kinases CDK1, -2, or -5. These data indicate that collybistin and cyclin-dependent kinases are involved in regulating the phosphorylation of gephyrin at postsynaptic membrane specializations.

Expression and subcellular distribution of gephyrin in non-neuronal tissues and cells.

Gephyrin is a scaffolding protein required for the accumulation of inhibitory neurotransmitter receptors at neuronal postsynaptic membranes. In non-neuronal tissues, gephyrin is indispensible for the biosynthesis of molybdenum cofactor, the prosthetic group of oxidoreductases including sulfite oxidase and xanthine oxidase. However, the molecular and cellular basis of gephyrin's non-neuronal function is poorly understood; in particular, the roles of its splice variants remain enigmatic. Here, we used cDNA screening as well as Northern and immunoblot analyses to show that mammalian liver contains only a limited number of gephyrin splice variants, with the C3-containing variant being the predominant isoform. Using new and established anti-gephyrin antibodies in immunofluorescence and subcellular fractionation studies, we report that gephyrin localizes to the cytoplasm of both tissue hepatocytes and cultured immortalized cells. These findings were corroborated by RNA interference studies in which the cytosolic distribution was found to be abolished. Finally, by blue-native PAGE we show that cytoplasmic gephyrin is part of a ~600 kDa protein complex of yet unknown composition. Our data suggest that the expression pattern of non-neuronal gephyrin is simpler than indicated by previous evidence. In addition, gephyrin's presence in a cytosolic 600 kDa protein complex suggests that its metabolic and/or other non-neuronal functions are exerted in the cytoplasm and are not confined to a particular subcellular compartment.

A three-day anatomy revision course taught by senior peers effectively prepares junior students for their national anatomy exam.

OBJECTIVES: this study examines whether peer-teaching, in the setting of a three-day revision course in anatomy, is effective in preparing medical students for their national anatomy exam. METHODS: the anatomy course was designed for candidates taking the first part of the German national medical exam. Increase of knowledge during the course was assessed by tests before and after the course (group A). To test equivalence, two control groups participated in the pre-test (group B) or in the course and in the post-test (group C). Participants anonymously rated 14 feedback items on a five-point Likert scale ranging from 1 (full agreement) to 5 (full disagreement). RESULTS: group A students' performance improved significantly during the course with a mean increase of 7.15 points (11.9% improvement; p<0.001). Equivalence testing showed that performance of group A students in the pre-/post-tests was equal to those of group B pre-tests and group C post-tests, respectively. Agreement on the 14 feedback items was highly significant (p<0.001 for all items), with a global median of 1. CONCLUSIONS: this study shows that a three-day anatomy revision course is effective and highly appreciated by medical students in their preparation for the national exam. Moreover, peer-teaching is reliable at this stage of the medical curriculum.

Molecular architecture of glycinergic synapses.

Synapses can be considered chemical machines, which are optimized for fast and repeated exocytosis of neurotransmitters from presynaptic nerve terminals and the reliable electrical or chemical transduction of neurotransmitter binding to the appropriate receptors in the postsynaptic membrane. Therefore, synapses share a common repertoire of proteins like, e.g., the release machinery and certain cell adhesion molecules. This basic repertoire must be extended in order to generate specificity of neurotransmission and allow plastic changes, which are considered the basis of developmental and/or learning processes. Here, we focus on these complementary molecules located in the presynaptic terminal and postsynaptic membrane specializations of glycinergic synapses. Moreover, as specificity of neurotransmission in this system is established by the specific binding of the neurotransmitter to its receptor, we review the molecular properties of glycine receptor subunits and their assembly into functional glycine receptors with different functional characteristics. The past years have revealed that the molecular machinery underlying inhibitory and especially glycinergic postsynaptic membrane specializations is more complex and dynamic than previously anticipated from morphological studies. The emerging features include structural components as well as signaling modules, which could confer the plasticity required for the proper function of distinct motor and sensory functions.

Mover is a novel vertebrate-specific presynaptic protein with differential distribution at subsets of CNS synapses.

Presynaptic nerve terminals contain scaffolding proteins that orchestrate neurotransmitter release at active zones. Here we describe mover, a yet unknown non-transmembrane protein that is targeted to presynaptic terminals when overexpressed in cultured neurons. Confocal immunomicroscopy revealed that mover colocalizes with presynaptic markers in the calyx of Held. In the hippocampus, mover localizes to mossy fibre terminals, but is absent from inhibitory nerve terminals. By contrast, mover localizes to inhibitory terminals throughout the cerebellar cortex. Our results suggest that mover may act in concert with generally expressed scaffolding proteins in distinct sets of presynaptic terminals.

Assembly of active zone precursor vesicles: obligatory trafficking of presynaptic cytomatrix proteins Bassoon and Piccolo via a trans-Golgi compartment.

Neurotransmitter release from presynaptic nerve terminals is restricted to specialized areas of the plasma membrane, so-called active zones. Active zones are characterized by a network of cytoplasmic scaffolding proteins involved in active zone generation and synaptic transmission. To analyze the modes of biogenesis of this cytomatrix, we asked how Bassoon and Piccolo, two prototypic active zone cytomatrix molecules, are delivered to nascent synapses. Although these proteins may be transported via vesicles, little is known about the importance of a vesicular pathway and about molecular determinants of cytomatrix molecule trafficking. We found that Bassoon and Piccolo co-localize with markers of the trans-Golgi network in cultured neurons. Impairing vesicle exit from the Golgi complex, either using brefeldin A, recombinant proteins, or a low temperature block, prevented transport of Bassoon out of the soma. Deleting a newly identified Golgi-binding region of Bassoon impaired subcellular targeting of recombinant Bassoon. Overexpressing this region to specifically block Golgi binding of the endogenous protein reduced the concentration of Bassoon at synapses. These results suggest that, during the period of bulk synaptogenesis, a primordial cytomatrix assembles in a trans-Golgi compartment. They further indicate that transport via Golgi-derived vesicles is essential for delivery of cytomatrix proteins to the synapse. Paradigmatically this establishes Golgi transit as an obligatory step for subcellular trafficking of distinct cytoplasmic scaffolding proteins.

Complex formation between the postsynaptic scaffolding protein gephyrin, profilin, and Mena: a possible link to the microfilament system.

Gephyrin is an essential component of the postsynaptic cortical protein network of inhibitory synapses. Gephyrin-based scaffolds participate in the assembly as well as the dynamics of receptor clusters by connecting the cytoplasmic domains of glycine and GABA(A) receptor polypeptides to two cytoskeletal systems, microtubules and microfilaments. Although there is evidence for a physical linkage between gephyrin and microtubules, the interaction between gephyrin and microfilaments is not well understood so far. Here, we show that neuronal gephyrin interacts directly with key regulators of microfilament dynamics, profilin I and neuronal profilin IIa, and with microfilament adaptors of the mammalian enabled (Mena)/vasodilator stimulated phosphoprotein (VASP) family, including neuronal Mena. Profilin and Mena/VASP coprecipitate with gephyrin from tissue and cells, and complex formation requires the E-domain of gephyrin, not the proline-rich central domain. Consequently, gephyrin is not a ligand for the proline-binding motif of profilins, as suspected previously. Instead, it competes with G-actin and phospholipids for the same binding site on profilin. Gephyrin, profilin, and Mena/VASP colocalize at synapses of rat spinal cord and cultivated neurons and in gephyrin clusters expressed in transfected cells. Thus, Mena/VASP and profilin can contribute to the postulated linkage between receptors, gephyrin scaffolds, and the microfilament system and may regulate the microfilament-dependent receptor packing density and dynamics at inhibitory synapses.

Defective integrin switch and matrix composition at alpha 7-deficient myotendinous junctions precede the onset of muscular dystrophy in mice.

Force transmission at the myotendinous junction requires a strong link between the muscle cytoskeleton and the extracellular matrix. At the adult junction, two splice variants of the laminin-binding integrins, alpha7Abeta1D and alpha7Bbeta1D, are highly enriched. The alpha7 subunits are critical for the integrity of the junctional sarcolemma because integrin alpha7-deficient mice develop muscular dystrophy, primarily affecting this site of the muscle. Here, we report that beta1D integrin coimmunoprecipitates and colocalizes with the alpha5 subunit at alpha7-deficient junctions, but does not associate with alpha3, alpha6 or alphav integrins. By immunogold labelling we show that the basement membranes of integrin alpha7-deficient muscles recruit abnormally high levels of fibronectin, the ligand of alpha5beta1D. Finally, we demonstrate that alpha5beta1D is down-regulated at the normal postnatal junction and is displaced by alpha7beta1D. These results suggest that the alpha7 subunit is implicated in the down-regulation of alpha5beta1D and in the removal of fibronectin from the maturing myotendinous junction, thus providing an alpha7beta1D-based link to laminin. We propose that the persistence of alpha5beta1D in alpha7-deficient mice is not compatible with normal muscle function and leads to muscle wasting.