Current Research

Ontogeny and Regulation of Glycinergic Synapses in Spinal Cord and Hippocampus

Components of the translational machinery are associated with juvenile glycine receptors and are redistributed to the cytoskeleton upon aging and synaptic activity. Whereas adult-type glycine receptors (GlyR) are hetero-oligomeric ligand-gated ion channels composed of α and ß subunits, juvenile/early postnatal GlyR are homo-oligomeric channels composed of α2 subunits. The physiological role of juvenile/early postnatal GlyR expression for the functional differentiation of glycinergic synapses in the developing rat brain is not well understood. It is believed that the activation of the neonatal GlyR by taurine is depolarizing and accompanied by an increase in intracellular Ca²⁺ concentrations. Moreover the failure of neonatal GlyR activation as a consequence of taurine deprivation can severely influence the migration of cortical neurons and thus the development of the cortical architecture. In the developing rodent brain glycinergic and GABAergic neurotransmission was shown to be depolarizing leading to Ca²⁺ -influx through L-type channels or NMDA receptors. We could show that activation of juvenile/early postnatal GlyR is crucial for the formation of postsynaptic gephyrin scaffolds, which subsequently trap adult-type glycine receptors via binding of the GlyR ß subunit cytoplasmic pole. Moreover, the formation of gephyrin scaffolds in cultured embryonic rat spinal cord neurons requires Ca²⁺ -influx through L-type channels. Recently, we could demonstrate, that the GlyR α2 subunit is associated with translation eukaryotic elongation factor 1α (eEF1A) in pulldown experiments with rat brain extracts. Moreover, additional proteins involved in translation like ribosomal S6 protein and p70 ribosomal S6 protein kinase as well as ERK1/2 and calcineurin were identified in the same pulldown approaches. Moreover, GlyR activation in young spinal cord neurons resulted in an increased phosphorylation of ribosomal S6 protein. Immunocytochemistry showed that eEF1A and ribosomal S6 protein are localized in the soma, dendrites. Their immunoreactivities were partially overlapping with that of the GlyR at synapses of cultured hippocampal and spinal cord neurons.. Surprisingly, eEF1A immunoreactivity was redistributed to the cytoskeleton in about 45% of neurons after 5 weeks in culture. Notably, the degree of redistribution could be increased at earlier stages of in vitro differentiation by inhibition of either the ERK1/2 pathway or GlyRs and simultaneous N-methyl-D-aspartate receptor activation. Our findings suggest a functional coupling of eEF1A with both inhibitory and excitatory receptors, possibly involving the ERK-signaling pathway. As the monomeric GTPase eEF1A is not only involved in protein synthesis but also thought to participate in other cellular functions such as actin bundling, cell cycle regulation, and apoptosis, we investigate its cellular functions in neurons with respect to dendrite morpholgy and synapse formation and maintainance.

Fig 1: Localization of eEF1 A in 28 days cultured hippocampal neurons

Identification of novel cytomatrix proteins

In analyzing the candidates identified in the screen we focused on a cDNA encoding an as yet uncharacterized protein, which we termed mover. Analysis of the deduced primary structure revealed, that mover is a vertebrate-specific non-transmembrane protein. Biochemical data suggest that it can associate with synaptic vesicles. Uponoverexpression in cultured neurons it is targeted to presynaptic terminals. Confocal immunomicroscopy revealed a differential localization of mover at distinct subsets of CNS synapses. Whereas mover immunoreactivity colocalizes with presynaptic markers in the calyx of Held and localizes to mossy fibre terminals in the hippocampus, it is absent from inhibitory nerve terminals in hippocampus but present at 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. Future work aims at the identification of mover interaction partners and at investigating the role of mover at distict subsets of synapses. Moreover, we want to elucidate by RNAi experiments , which step of synaptic vesicle transport, fusion or recycling can be influenced by mover.

Proteomics studies on brain peroxisomes

Peroxisomes are single membrane enclosed organelles that catalyze a broad variety of catabolic as well as anabolic reactions. Peroxisomal genetic diseases, like X-linked adrenoleukodystophy (X-ALD) or the Zellweger syndrome are characterized by severe malformations of the CNS, indicating an important role of these organelles in brain development. Whereas hepatic peroxisomes were functionally characterized in detail in the past, there is still lack of information on the protein pattern of peroxisomes in neuronal tissue. Since peroxisomes of high purity are a prerequisite of accurate protein identification, protocols for peroxisome isolation from brain tissue are currently under development using a combination of centrifugation techniques and free flow electrophoresis. Quantitative mass spectrometry will be used to unravel detailed changes in the protein pattern of ALDp-knock out and wild type mice, to obtain further insights in the process disturbed in the most prevalent peroxisomal disorder X-ALD.