Major research topics
- Investigating the involvement of protein kinase D (PKD) in the regulation of neuronal intracellular transport and protein localisation
- Myosin V motor protein in neurons: potential role during neuronal differentiation and intracellular transport processes
- The role of NMDA receptors during initial neurite extension and migration of developing neurons
- How the subunit composition of NMDA receptors influences pain perception – investigations in transgenic mouse lines possessing altered NMDA receptor subunit composition
Investigating the involvement of protein kinase D (PKD) in the regulation of neuronal intracellular transport and protein localisation
Protein kinase D (PKD) isoforms are alternative diacyl-glycerol (DAG) receptors which recently have been grouped into a novel family of serine/threonine kinases. In mammalian cells, so far three PKD isoforms (PKD1, PKD2, PKD3) have been described which can regulate various cellular processes such as cell migration and tumour metastasis, cell survival and apoptosis, growth factor signalling or secretory transport. PKD has been shown to regulate secretory vesicle fission from the Golgi complex and furthermore, is known to selectively direct vesicles towards the basolateral membrane surface. Despite the fact that PKD isoforms are expressed in the central nervous system already from early embryonic ages, not much is known so far about PKD-directed cellular processes in neurons.
Neurons are extremely polarized cells, having strikingly different dendritic and axonal compartments which differ fundamentally in protein and lipid composition as well as in their cellular role. The maintenance of the enormous membrane surface needs strictly regulated secretory machinery and highly selective intracellular transport. Based on the known role of PKD in directed transport processes in non-neuronal cells, we have recently started to investigate PKD-regulated processes in hippocampal neurons.
In a close collaboration with A. Hausser´s group at the Institute of Cell Biology and Immunology, University of Stuttgart, we have introduced fluorescently tagged PKD mutants into mouse embryonal hippocampal neurons by Lipofectamine transfection and investigated Golgi structure and dendritic arborization by several microscopic techniques. We also use transgenic mouse lines expressing a dominant-negative kinase inactive form of PKD1 (PKD1kd) in a neuron-specific manner upon tetracycline-induction. Our recent results indicate that the expression of PKD1kd leads to the disruption of the normally filamentous Golgi structure in neurons and leads to the shrinkage of the dendritic tree.
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Myosin V motor protein in neurons: potential role during neuronal differentiation and intracellular transport processes
The role of myosinVa (myoVa) in neuronal cells is investigated with a close collaboration with L. Nyitray at the Department of Biochemistry, Eotvos Lorand University. MyoVa is expressed abundantly in the mammalian central nervous system. This motor protein subtype can be involved in the transport and recycling of synaptic vesicles in the axon terminals and has been detected in the growth cones, in the postsynaptic densities or in the dendrites and is also present along the axon. The involvement of myoVa-connected transport processes during early neuronal differentiation and early neurite extension, preceding the formation of synaptic connections has not yet been investigated.
DLC is a highly conserved protein which was originally described as a subunit of dynein light chain. Since then it has been discovered that DLC can be also found as a subunit of myosinV thereby it is a feasible idea that DLC can fulfil a regulatory and/or cargo-binding role in both types of motor proteins.
The expression pattern and the importance of DLC and myoVa during the time-course of retinoic acid induced differentiation of NE-4C neuroepithelial stem cell line are investigated either by detecting changes in their endogenous mRNA levels or by introducing dominant negative or constitutive active constructs into the cells. The transport of myoVa is followed by live cell imaging and by FRAP experiments in transfected mouse embryonal hippocampal neurons.
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The role of NMDa receptors during initial neurite extension and migration of developing neurons
NMDA (N-methyl-D-aspartate) receptors belong to the ionotropic glutamate receptors. Upon stimulation by its endogenous ligand, glutamate, NMDA receptor can regulate a broad plethora of cellular events starting from neurite extension, neuronal migration, development, survival or excitotoxic cell death. One explanation for the diversified effects of NMDA receptors is that these receptors are highly permeable to Ca2+ and their activation can lead to localised changes in the intracellular Ca2+ level. Besides functioning as ion channels, NMDA receptors can also interact with many intracellular signalling pathways via their C-terminal intracellular part. The subunit composition of NMDA receptors determines characteristically the properties of the receptor complex. Besides the compulsory NR1 subunits, NMDA receptors contain at least one type of NR2 subunits (NR2A, NR2B, NR2C or NR2D). Additionally, NR3 subunits can also modulate the receptor's activity. During CNS development and maturation, NMDA receptor subunit composition changes characteristically in a spatio-temporal manner.
One of the best described switches between the NMDA receptor subunits takes place during the development of the cerebellum. During the intensive migration of cerebellar granule cells, NR2B is present while its expression is gradually replaced by NR2C once the cells reach their final position and start to form their synaptic connections. In order to understand the importance of the subunit change during neuronal development and migration, we compared two transgenic mouse lines with altered NMDA receptor subunit composition. The NR2C k.o. (knock out) mice lack the NR2C subunit completely, while the NR2C/2B mice express the NR2B instead of the NR2C subunit (so-called knock-in mice). In the latter case, NR2B subunit is ectopically expressed in every cell where normally the NR2C subunit is present.
According to our histological and electronmicroscopy studies, long-term NR2B expression leads to abnormal cerebellar development: granule cell number and the dendritic arborization of Purkinje cells is reduced together with an overall decrease in the thickness of the molecular layer (MOL). Increased expression of NR2B subunits in granule cells during early postnatal development increases their migratory activity both in vivo and in vitro. Videomicroscopic observations in developing granule cell cultures showed that neurite extension rate is not altered by the genetically manipulated subunit switch while soma translocation speed is specifically increased by increased NR2B subunit levels. According to pharmacological studies with NR2B-specific antagonists we propose that NR2B receptors are specifically needed for the migration of young postmitotic neurons to their final position.
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How the subunit composition of NMDA receptors influences pain perception – investigations in transgenic mouse lines possessing altered NMDA receptor subunit composition
NMDA receptors are known to play important role in chronic pain transmission implying the possibility that modulating NMDA receptor activity can serve as a potential pain therapy. NMDA receptor antagonists, however, can not be used for antinociception according to their severe side effects - in recent years, however, it has been concluded that subunit-specific modulators provide better clinical results. So far most of the results have focused on the importance of NR2B subunit and several NR2B-subunit specific antagonists are already under thorough investigations regarding their selective anti-nociceptive action.
Previous work carried out in transgenic mice indicated that forebrain-specific overexpression of the NR2B subunit led to elevated pain responses in chronic pain tests. Accordingly, we also tested chronic pain reactions of NR2C/2B knock-in mice which express the NR2B subunit in exchange for the NR2C subunit and found elevated pain responses. However, the kinetics of elevated pain responses in the formalin test differed between mice with the forebrain-specific NR2B overexpression or with the exchange between the NR2C - NR2B subunits: in NR2C/2B mice, elevated pain reactions were observed earlier, indicating a different mode of action between the two transgenic mouse lines. Currently we carry out behavioural as well as biochemical and histochemical analyses to decide whether the increase in pain perception is due to the elevated NR2B levels and/or to the lack of NR2C subunit.
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