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What determines the shape of neuronal contact sites?

Tobias M. Boeckers, an anatomist at the University of Ulm, works on an important aspect of basic neurobiological research - the transmission of signals between nerve cells. Two years ago, his findings suddenly began to be seen in a different light and became important for clinical research.

Boeckers, director of the Department of Anatomy and Cell Biology at the University of Ulm does research into the glutamatergic, excitatory contact sites on the postsynaptic nerve cells, the signalling pathways and the molecules involved in these processes. His major interest is the mechanisms of dynamic development and the alteration of the contact sites (synaptic plasticity) and the signalling pathways involved. Boeckers hypothesises that these morphological alterations might be induced by cytoskeleton elements.

Building blocks for the understanding of higher brain performance

Prof. Dr. Tobias M. Boeckers (Photo: University of Ulm)
The members of the protein family of small GTPases play a major role in this process. These molecular switches are important for the dynamic alteration of skeleton proteins such as actin. Boeckers is looking into what happens when these small GTPases are switched on and off and hopes to increase the understanding of the morphological alterations of synaptic contacts, which themselves are of great interest in the understanding of higher brain processes such as learning and memory. Common belief holds that chemical synapses transmit information from one neurone to another.

The molecular biology of the protein family of small GTPases has been known for several years. These enzymes are activated by neighbouring molecules containing a so-called GEF protein domain. They are inactivated by a group of molecules with a GAP protein domain. These molecules are part of a macromolecular signalling complex at the postsynaptic membrane of synapses that also involves ProSAP/Shank molecules which in turn form a tight network (through which the electrons cannot pass) with several hundred other proteins underneath the postsynaptic membrane.

Molecular clarification of postsynaptic density

Electronmicrograph of a synapse. The arrow shows the synaptic cleft between the neurons.
Ultrastructural composition of an excitatory synapse. The picture shows two neurones in the hippocampus of a rat. (Photo: Boeckers, University of Ulm)
Experts call this macromolecular complex, which looks like a thick membrane under the electron microscope, postsynaptic density (PSD). This neuronal segment, which was described for the first time in the 1960s, has been investigated in detail by many researchers, Boeckers included, since the 1990s.

The PSD molecules form a tight network that functions as interface between accumulated membrane receptors, cell adhesion molecules, components of signalling cascades and cytoskeleton-based elements. In addition, this area, which consists of dendritic spines, postsynaptic density kinases, phosphatases, several signal transduction proteins and regulatory proteins, also houses small GTPases.

The four figures of this schema show processes of snaptic acitvation.
Schematic showing the changes in synaptic contacts following strong synaptic activation. Vesicles are located at the presynaptic membrane; underneath the postsynaptic membrane there is a platform of proSAP/Shank molecules, which organise the PSD (A). Further postsynaptic proteins are attached to the postsynaptic platform (B) and the structure transforms into a mature, fungus-shaped synaptic connection (C). This structure has the potential to divide (D) and can thus lead to the duplication of synapses. (Photo: Boeckers, University of Ulm)

The king of switches

In the last few years, Boeckers’ research group has managed to identify some of these PSD molecules. In an SFB project, the Ulm researchers dealt with a family of new molecules that affect these small GTPases and which can induce morphological changes in neuronal contact sites during neuronal development or following activation. Boeckers and his team of researchers are mainly interested in the molecules SerSAP2 and SerSAP3 of this gene family and have since cloned the proteins and investigated their chemical and biological properties. Using knock-out mice and vector-based methods, Boeckers is working on finding out more about the tasks of individual GTPases at the synapses.

More active and bigger

Boeckers assumes that the cytoskeleton is adapted to the cell’s requirements by way of active or inactive GTPases: An active synapse that is frequently used becomes bigger, of necessity; other less frequently used synapses are, of necessity, degraded. This is based on the idea that at any point of the learning process an active synapse, which receives a lot of information from the receptors, is locally induced to expand as a reflection of its importance.

Many researchers are trying to find answers using the fruitfly Drosophila as a model organism where they switch small GTPases on and off. Boeckers and his team use a different cellular model system. They explant neurones that are not yet fully differentiated from the hippocampus of rats or mice shortly before birth and put them into a Petri dish. Prenatal cells are well suited for this purpose because many synapses are created in this early stage.

Using GFP to monitor the changes in synaptic contacts

Immunostaining of in-vitro neurones with antibodies directed against synaptic proteins. The dot-shaped labelling along the dendrites is visible. An antibody (green) directed against a protein of the neurotransmitter vesicles reveals the presynapse. Postsynaptic density (lilac) is made visible through antibodies against proteins of the ProSAP/Shank family. The magnified picture shows that green and lilac stains are opposite each other (arrow). (Photo: Boeckers)
The small GTPases can be modulated (switched on and off) by way of transfection. Using green fluorescent protein (GFP), Boeckers’ team is investigating how the synaptic contacts change under specific conditions. The Ulm researchers are mainly interested in the different types of small GTPases in the synaptic contacts. Since it is still unknown which synapse is responsible for which structural change, Boeckers will initially focus on the categorisation of GTPases. He hopes to find out which group of small GTPases affects the morphology of the neurones, in other words he is trying to create a molecule-structure relationship.

Initial hypotheses with rough models

If one of these five small GTPase subgroups is permanently activated through minor mutations, then the dendritic spines and the postsynaptic density increase. In this early stage of research, Boeckers is initially interested in creating rough models that can be used to develop hypotheses for later obtaining information on the fine tuning of the morphological change at the synapses.

Overall goal: molecular investigation of synapses and PSD

Boeckers’ small GTPase research goes far beyond this SFB project. His overall goal is the molecular clarification of synapses and postsynaptic densities. However, the role and function of the more than 500 different molecules is not yet known in detail. Boeckers hopes to characterise some of them using cell culture and mouse models. He is more than aware that this is time-consuming work.

The clinical perspective

Two years ago, Boeckers saw how quickly classical basic research can take on a clinical perspective. In 2006, Boeckers’ French colleague, Thomas Bourgeron, recounted an unexpected discovery. A mutation of one of the molecules that Boeckers had examined can lead to forms of autism that can be inherited. Bourgeron discovered the mutation in a copy of the SHANK 3 gene (which is also called ProSAP2) on chromosome 22q13 and immediately realised that there was a direct correlation with autistic diseases.

Hope for the treatment of autism for the first time

Boeckers considered this finding exciting for two reasons. First, this was evidence that autism, a complex and barely understood disease, could be the result of a mutation of a synaptic molecule. Second, this finding was extremely exciting for Boeckers because it was directly linked to his own work. For the very first time, there was a slight hope of being able to treat this complex disease.

Theory: Glutamatergic synapses are rearranged

Boeckers hypothesises that the balance of generation and degradation, the strengthening or weakening of these synapses, is plastic. This hypothesis is based on the idea that many of the glutamatergic synapses are rearranged upon certain cues and are not created once and for all. Boeckers assumes that such rearrangements will then be balanced out at one level or another. This might be one of the reasons for behavioural problems or altered behaviour in those suffering from autism.

Humans need two functional SHANK3 copies

Since only one of the two SHANK3 copies had this mutation, Boeckers assumed that humans require two functional copies in order to be able to maintain the balance of protein concentration in any given nerve cell. If one copy is defective, then the balance is affected in some cell populations.

The SHANK molecules belong to the scaffold proteins and are located in the excitatory synapses, directly underneath the membrane where they hold fast molecules such as adhesion molecules and receptors. The members of the SHANK protein family are regarded as the main organisers of postsynaptic density.

Further focus on synaptic diseases

Boeckers’ research group is part of a European project on synaptic diseases which also involves groups from Montpellier (France) and Italy. It now seems clear that Boeckers’ basic neurobiological research has an unarguable applicable side.


Literature:

Boeckers, T.M, The postsynaptic density, in: Cell and Tissue Research, Vol. 326, 2. Nov. 2006, p. 409-422 (DOI: 10.1007/s00441-006-0274-5)

Durand, Ch, Betancour, C. T. Boeckers et al.: Mutations in the gene encoding the synaptic scaffolding protein SHANK3 are associated with autism spectrum disorders, in: Nature genetics, vol. 39, No. 1, Jan. 2007, p. 25-27.

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/what-determines-the-shape-of-neuronal-contact-sites