Tobias M. Böckers

Importance of Shank3 Protein in Regulating Metabotropic Glutamate Receptor 5 (mGluR5) Expression and Signaling at Synapses

Shank3/PROSAP2 gene mutations are associated with cognitive impairment ranging from mental retardation to autism. Shank3 is a large scaffold postsynaptic density protein implicated in dendritic spines and synapse formation; however, its specific functions have not been clearly demonstrated. We have used RNAi to knockdown Shank3 expression in neuronal cultures and showed that this treatment specifically reduced the synaptic expression of the metabotropic glutamate receptor 5 (mGluR5), but did not affect the expression of other major synaptic proteins. The functional consequence of Shank3 RNAi knockdown was impaired signaling via mGluR5, as shown by reduction in ERK1/2 and CREB phosphorylation induced by stimulation with (S)-3,5-dihydroxyphenylglycine (DHPG) as the agonist of mGluR5 receptors, impaired mGluR5-dependent synaptic plasticity (DHPG-induced long-term depression), and impaired mGluR5-dependent modulation of neural network activity. We also found morphological abnormalities in the structure of synapses (spine number, width, and length) and impaired glutamatergic synaptic transmission, as shown by reduction in the frequency of miniature excitatory postsynaptic currents (mEPSC). Notably, pharmacological augmentation of mGluR5 activity using 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide as the positive allosteric modulator of these receptors restored mGluR5-dependent signaling (DHPG-induced phosphorylation of ERK1/2) and normalized the frequency of mEPSCs in Shank3-knocked down neurons. These data demonstrate that a deficit in mGluR5-mediated intracellular signaling in Shank3 knockdown neurons can be compensated by 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide; this raises the possibility that pharmacological augmentation of mGluR5 activity represents a possible new therapeutic approach for patients with Shank3 mutations.

ProSAP/Shank postsynaptic density proteins interact with insulin receptor tyrosine kinase substrate IRSp53

The ProSAP/Shank family of multidomain proteins of the postsynaptic density (PSD) can either directly or indirectly interact with NMDA‐type and metabotropic glutamate receptors and the actin‐based cytoskeleton. In a yeast two hybrid screen utilizing a proline‐rich domain that is highly conserved among the ProSAP/Shank family members, we isolated several cDNA clones coding for the insulin receptor substrate IRSp53. The specificity of this interaction was confirmed in transfected COS cells. Co‐immunoprecipitation of IRSp53 and ProSAP2 solubilized from rat brain membranes indicates that the interaction occurs in vivo. The C‐terminal SH3 domain of IRSp53 is responsible for the interaction with a novel proline‐rich consensus sequence of ProSAP/Shank that was characterized by mutational analysis. IRSp53 is a substrate for the insulin receptor in the brain and acts downstream of small GTPases of the Rho family. Binding of Cdc42Hs to IRSp53 induces actin filament assembly, reorganization and filopodia outgrowth in neuronal cell lines. Our data suggest that IRSp53 can be recruited to the PSD via its ProSAP/Shank interaction and may contribute to the morphological reorganization of spines and synapses after insulin receptor and/or Cdc42Hs activation.

A dynein mutation attenuates motor neuron degeneration in SOD1G93A mice

Cu/Zn SOD1(G93A) transgenic mice develop phenotypical hallmarks of ALS and serve therefore as an established model to study the molecular mechanisms underlying this disease. Recent reports demonstrate that mutations in the motor protein dynein in Legs at odd angles (Loa) and Cramping (Cra1) mice lead to similar but milder phenotypes. Surprisingly, double transgenic mice (Loa/SOD1(G93A)) have been recently shown to attenuate rather than to accelerate the phenotypical expression of motor neuron degeneration. These results raise the question whether other functional relevant mutations in dynein cause a similar effect. To address this question, we have cross-bred SOD1(G93A) with Cra1/+ mice. These double transgenic mice show an attenuated decline of both motor activity and body weight and an increase of survival time compared to SOD1(G93A) mice. Thus, this study confirms that mechanisms associated with dynein such as retrograde axonal transport may play an important role in SOD1(G93A-) toxicity on motor neurons.

Synaptic contacts between identified neurons visualized in the confocal laserscanning microscope. Neuroanatomical tracing combined with immunofluorescence detection of post-synaptic density proteins and target neuron-markers

The axons of neurons in the CNS with their delicate ramification patterns and terminal boutons can be visualized with conventional neuroanatomical techniques with a high degree of accuracy. Whether identified terminal boutons form synaptic contacts with target neurons identified by a second and different marker needs resolution beyond that offered by conventional light microscopy. The morphological elements associated with synaptic connectivity consist of specialized pre- and post-synaptic junctional complexes known as the pre- and post-synaptic densities. Electron microscopy of these junctional complexes consumes much time and resources. In an attempt to increase the speed with which we can analyze networks of neurons we developed a high-resolution triple-fluorescence approach including neuroanatomical tracing, immunofluorescence, confocal laserscanning and 3D-computer reconstruction to pinpoint at the light microscopic level the three elements involved in synaptic connectivity: afferent fibers and their terminal boutons, close apposition with neurons identified by the presence of a fluorescent marker, and sandwiched in between a post-synaptic density marker. We used morphological criteria for the detection of axon terminals (swellings on fibers). Antibodies against ProSAP2/Shank3, a post-synaptic density-associated scaffolding protein, were used to pinpoint the location of the synaptic junctions. The results show the existence of sandwich-like configurations: pre-synaptic fiber, ProSAP2/Shank3, post-synaptic neuron. Thus we feel that we can minimize (and perhaps completely eliminate) the need for electron microscopy and hence dramatically increase the overall efficiency of neuroanatomical tracing and network analysis.

Thiel-fixation preserves the non-linear load-deformation characteristic of spinal motion segments, but increases their flexibility.

Human cadaveric specimens are recommended as the best option for in-vitro tests. However, fresh human spine specimens are often difficult to obtain. Further problems are the potential risk of infection and they can only be used over a limited test period. Therefore, the use of embalmed specimens is often discussed. The most common method is formalin fixation. However, this type of embalming can result in failure, because the biomechanical properties of the tissue is partially influenced. In recent years the development of the new method, the fixation according to Thiel, could provide an alternative to fresh or formalin-fixed specimens. The aim of the present study is to compare the biomechanical properties between fresh and Thiel-fixed spine specimens, and to compare the data to previous data of a test with formalin fixation. For the study, six L1-L2 spinal segments from 16-week-old calves were biomechanically tested. The parameters, range of motion and neutral zone, were determined in flexion/extension, right/left lateral bending and left/right axial rotation. The results showed that the specimens kept their non-linear load-deformation-characteristic after Thiel fixation. The range of motion of Thiel-fixed specimens increased relative to the unembalmed state by approximately 22% in flexion-extension, 23% in lateral bending (p<0.05) and 45% in axial rotation (p<0.05). In conclusion, the results still suggest a preference for fresh cadaveric spine specimens for quantitative biomechanical in-vitro testing, because they provide the best physiological conditions. However, for preliminary tests, which may only be used for orientation, embalmed specimens using the Thiel fixation method might serve as an alternative. Compared to formalin-fixated specimens which become approximately 5 times stiffer and completely lose their non-linear load-deformation-characteristic, as found in a previous study; the Thiel fixation maintains the non-linear load-deformation-characteristic but increases the range of motion.

Does learning in clinical context in anatomical sciences improve examination results, learning motivation, or learning orientation?

The preclinical compulsory elective course “Ready for the Operating Room (OR)!?” [in German]: “Fit für den OP (FOP)”] was implemented for students in their second year, who were simultaneously enrolled in the gross anatomy course. The objective of the study was to determine whether the direct practical application of anatomical knowledge within the surgical context of the course led to any improvement in learning motivation, learning orientation, and ultimately examination results in the gross anatomy course, as compared with a control group. Within the scope of five teaching sessions, the students learned surgical hand disinfection, suturing techniques, and the identification of commonly used surgical instruments. In addition, the students attended five surgical demonstrations performed by surgical colleagues on cadavers. Successful learning of these basic skills was then assessed based on an Objectively Structured Practical Examination. Learning motivation and learning orientation in both subgroups was determined using the SELLMO‐ST motivation test and the Approaches and Study Skills Inventory test. While a significant increase in work avoidance was identified in the control group, this was not the case for FOP participants. Similarly, an increase in the “deep approach” to learning, as well as a decrease in the “surface approach,” was able to be documented among the FOP participants following completion of the course. The results suggest that students enrolled in the gross anatomy course, who were simultaneously provided with the opportunity to learn in clinical context, were more likely to be successful at maintaining learning motivation and learning orientation required for the learning process, than students who attended the gross anatomy course alone. Anat Sci Educ. 7: 3–11.

SPAR2, a novel SPAR-related protein with GAP activity for Rap1 and Rap2

Spine‐associated RapGAP 2 (SPAR2) is a novel GTPase activating protein (GAP) for the small GTPase Rap that shows significant sequence homology to SPAR, a synaptic RapGAP that was reported to regulate spine morphology in hippocampal neurons. SPAR2, like SPAR, interacts with the recently described synaptic scaffolding protein ProSAP‐interacting protein (ProSAPiP), which in turn binds to the PDZ domain of ProSAP/Shank post‐synaptic density proteins. In subcellular fractionation experiments, SPAR2 is enriched in synaptosomes and post‐synaptic density fractions indicating that it is a synaptic protein. Furthermore, we could show using in vitro GAP assays that SPAR2 has GAP activity for Rap1 and Rap2. Expression in COS‐7 cells, however, revealed different actin‐binding properties of SPAR2 and SPAR. Additionally, over‐expression of SPAR2 in cultured hippocampal neurons did not affect spine morphology as it was reported for SPAR. In situ hybridization studies also revealed a differential tissue distribution of SPAR and SPAR2 with SPAR2 transcripts being mainly expressed in cerebellar and hippocampal granule cells. Moreover, in the cerebellum SPAR2 is developmentally regulated with a peak of expression around the period of synapse formation. Our results imply that SPAR2 is a new RapGAP with specific functions in cerebellar and hippocampal granule cells.

Neuroprotective Function of Cellular Prion Protein in a Mouse Model of Amyotrophic Lateral Sclerosis

Transgenic mice expressing human mutated superoxide dismutase 1 (SOD1) linked to familial forms of amyotrophic lateral sclerosis are frequently used as a disease model. We used the SOD1G93A mouse in a cross-breeding strategy to study the function of physiological prion protein (Prp). SOD1G93APrp-/- mice exhibited a significantly reduced life span, and an earlier onset and accelerated progression of disease, as compared with SOD1G93APrp+/+ mice. Additionally, during disease progression, SOD1G93APrp-/- mice showed impaired rotarod performance, lower body weight, and reduced muscle strength. Histologically, SOD1G93APrp-/- mice showed reduced numbers of spinal cord motor neurons and extended areas occupied by large vacuoles early in the course of the disease. Analysis of spinal cord homogenates revealed no differences in SOD1 activity. Using an unbiased proteomic approach, a marked reduction of glial fibrillary acidic protein and enhanced levels of collapsing response mediator protein 2 and creatine kinase were detected in SOD1G93APrp-/- versus SOD1G93A mice. In the course of disease, Bcl-2 decreases, nuclear factor-kappaB increases, and Akt is activated, but these changes were largely unaffected by Prp expression. Exclusively in double-transgenic mice, we detected a significant increase in extracellular signal-regulated kinase 2 activation at clinical onset. We propose that Prp has a beneficial role in the SOD1G93A amyotrophic lateral sclerosis mouse model by influencing neuronal and/or glial factors involved in antioxidative defense, rather than anti-apoptotic signaling.

Morphometric analysis of the relationships between intervertebral disc and vertebral body heights: an anatomical and radiographic study of the human thoracic spine

The main aim of this study was to provide anatomical data on the heights of the human intervertebral discs for all levels of the thoracic spine by direct and radiographic measurements. Additionally, the heights of the neighboring vertebral bodies were measured, and the prediction of the disc heights based only on the size of the vertebral bodies was investigated. The anterior (ADH), middle (MDH) and posterior heights (PDH) of the discs were measured directly and on radiographs of 72 spine segments from 30 donors (age 57.43 ± 11.27 years). The radiographic measurement error and the reliability of the measurements were calculated. Linear and non‐linear regression analyses were employed for investigation of statistical correlations between the heights of the thoracic disc and vertebrae. Radiographic measurements displayed lower repeatability and were shorter than the anatomical ones (approximately 9% for ADH and 37% for PDH). The thickness of the discs varied from 4.5 to 7.2 mm, with the MDH approximately 22.7% greater. The disc heights showed good correlations with the vertebral body heights (R2, 0.659–0.835, P‐values < 0.005; anova), allowing the generation of 10 prediction equations. New data on thoracic disc morphometry were provided in this study. The generated set of regression equations could be used to predict thoracic disc heights from radiographic measurement of the vertebral body height posterior. For the creation of parameterized models of the human thoracic discs, the use of the prediction equations could eliminate the need for direct measurement on intervertebral discs. Moreover, the error produced by radiographic measurements could be reduced at least for the PDH.

Reduction of mental distress in the dissection course by introducing the body donor experience through anatomical demonstrations of organ systems

The practice of dissection teaches students not only the foundations of anatomical knowledge but also encourages the development of professional competencies. Yet, the dissection of cadavers in the gross anatomy course can be a stress factor for medical students. There are a minor proportion of students who demonstrate strong emotional reactions in anticipation of being confronted with a cadaver. Therefore, in 2008, the authors implemented a voluntary course entitled, “Anatomical demonstrations of organ systems” (AD‐OS) in advance of the dissection course to ease this psychological burden. The question of whether attendees of AD‐OS showed less mental distress at the start of the dissection course compared with those that had not or only infrequently visited AD‐OS was addressed. AD‐OS attendees assessed their expected mental distress using a five‐point Likert scale before starting the dissection course and a second time at the end of their first day, after they had been confronted with a cadaver. AD‐OS was evaluated as excellent and the majority of students participated actively during teaching sessions. Overall, female students showed higher levels of mental distress. AD‐OS attendees assessed themselves as being less burdened by mental distress than members of the control group. Longitudinal analysis revealed that students who visited AD‐OS showed a marked decrease of their mental distress level, comparing prospective and retrospective ratings. This was significantly (P < 0.001; Z = −6.061) different from nonattendees or those who visited AD‐OS only infrequently. AD‐OS satisfied its intended teaching goals and proved that a step‐by‐step introduction of dissection through anatomical demonstrations helped to reduce the mental distress of students. Future studies are planned to measure mental distress with objective instruments. Anat Sci Educ