Gudrun Ahnert-Hilger

Differentiation of pluripotent embryonic stem cells into the neuronal lineage in vitro gives rise to mature inhibitory and excitatory neurons.

Embryonic stem (ES) cells represent a suitable model to analyze cell differentiation processes in vitro. Here, we report that pluripotent ES cells of the line BLC 6 differentiate in vitro into neuronal cells possessing the complex electrophysiological and immunocytochemical properties of postmitotic nerve cells. In the course of differentiation BLC 6-derived neurons differentially express voltage-dependent (K+, Na+, Ca2+) and receptor-operated (GABAA, glycine, AMPA, NMDA receptors) ionic channels. They generate fast Na(+)-driven action potentials and are functionally coupled by inhibitory (GABAergic) and excitatory (glutamatergic) synapses as revealed by measurements of postsynaptic currents. Moreover, BLC 6-derived neurons express neuron-specific cytoskeletal, cell adhesion and synaptic vesicle proteins and exhibit a Ca(2+)-dependent GABA secretion. Thus, the ES cell model enables the investigation of cell lineage determination and signaling mechanisms in the developing nervous system from a pluripotential stem cell to a differentiated postmitotic neuron. The in vitro differentiation of neurons from ES cells may be an excellent approach to study by targeted gene disruption a variety of neuronal functions.

Regulation of Monoamine Oxidase A by Circadian-Clock Components Implies Clock Influence on Mood

The circadian clock has been implicated in addiction and several forms of depression [1, 2], indicating interactions between the circadian and the reward systems in the brain [3-5]. Rewards such as food, sex, and drugs influence this system in part by modulating dopamine neurotransmission in the mesolimbic dopamine reward circuit, including the ventral tegmental area (VTA) and the ventral striatum (NAc). Hence, changes in dopamine levels in these brain areas are proposed to influence mood in humans and mice [6-10]. To establish a molecular link between the circadian-clock mechanism and dopamine metabolism, we analyzed the murine promoters of genes encoding key enzymes important in dopamine metabolism. We find that transcription of the monoamine oxidase A (Maoa) promoter is regulated by the clock components BMAL1, NPAS2, and PER2. A mutation in the clock gene Per2 in mice leads to reduced expression and activity of MAOA in the mesolimbic dopaminergic system. Furthermore, we observe increased levels of dopamine and altered neuronal activity in the striatum, and these results probably lead to behavioral alterations observed in Per2 mutant mice in despair-based tests. These findings suggest a role of circadian-clock components in dopamine metabolism highlighting a role of the clock in regulating mood-related behaviors.

N-Methyl-d-Aspartate Receptor Antibodies in Herpes Simplex Encephalitis

To determine the presence and kinetics of antibodies against synaptic proteins in patients with herpes simplex virus encephalitis (HSE).

The synaptophysin/synaptobrevin interaction critically depends on the cholesterol content

Synaptophysin interacts with synaptobrevin in membranes of adult small synaptic vesicles. The synaptophysin/synaptobrevin complex promotes synaptobrevin to built up functional SNARE complexes thereby modulating synaptic efficiency. Synaptophysin in addition is a cholesterol‐binding protein. Depleting the membranous cholesterol content by filipin or β‐methylcyclodextrin (β‐MCD) decreased the solubility of synaptophysin in Triton X‐100 with less effects on synaptobrevin. In small synaptic vesicles from rat brain the synaptophysin/synaptobrevin complex was diminished upon β‐MCD treatment as revealed by chemical cross‐linking. Mice with a genetic mutation in the Niemann–Pick C1 gene developing a defect in cholesterol sorting showed significantly reduced amounts of the synaptophysin/synaptobrevin complex compared to their homo‐ or heterozygous littermates. Finally when using primary cultures of mouse hippocampus the synaptophysin/synaptobrevin complex was down‐regulated after depleting the endogenous cholesterol content by the HMG‐CoA‐reductase inhibitor lovastatin. Alternatively, treatment with cholesterol up‐regulated the synaptophysin/synaptobrevin interaction in these cultures. These data indicate that the synaptophysin/synaptobrevin interaction critically depends on a high cholesterol content in the membrane of synaptic vesicles. Variations in the availability of cholesterol may promote or impair synaptic efficiency by interfering with this complex.

The tetanus toxin light chain inhibits exocytosis

The intracellular action on exocytosis of various forms of tetanus toxin was studied using adrenal medullary chromaffin cells, the membrane barrier of which has been removed by permeabilization with streptolysin O. Such cells still release catecholamines on stimulation with calcium. The two‐chain form of tetanus toxin (67 nmol/l) strongly inhibited exocytosis, but only if dithiothreitol was present as a reducing agent. Purified light chain completely prevented [3H]noradrenaline release with a half‐maximal effect at about 5 nmol/1. Heavy chain (up to 11 nmol/l) and unprocessed single‐chain toxin (up to 133 nmol/l) were without effect. It is concluded that the original single‐chain form of tetanus toxin has to be processed by proteolysis and reduction to yield a light chain which inhibits transmitter release.

Differential effects of Rho GTPases on axonal and dendritic development in hippocampal neurones.

Formation of neurites and their differentiation into axons and dendrites requires precisely controlled changes in the cytoskeleton. While small GTPases of the Rho family appear to be involved in this regulation, it is still unclear how Rho function affects axonal and dendritic growth during development. Using hippocampal neurones at defined states of differentiation, we have dissected the function of RhoA in axonal and dendritic growth. Expression of a dominant negative RhoA variant inhibited axonal growth, whereas dendritic growth was promoted. The opposite phenotype was observed when a constitutively active RhoA variant was expressed. Inactivation of Rho by C3‐catalysed ADP‐ribosylation using C3 isoforms (Clostridium limosum, C3lim or Staphylococcus aureus, C3stau2), diminished axonal branching. By contrast, extracellularly applied nanomolar concentrations of C3 from C. botulinum (C3bot) or enzymatically dead C3bot significantly increased axon growth and axon branching. Taken together, axonal development requires activation of RhoA, whereas dendritic development benefits from its inactivation. However, extracellular application of enzymatically active or dead C3bot exclusively promotes axonal growth and branching suggesting a novel neurotrophic function of C3 that is independent from its enzymatic activity.

Synaptic and Vesicular Coexistence of VGLUT and VGAT in Selected Excitatory and Inhibitory Synapses

The segregation between vesicular glutamate and GABA storage and release forms the molecular foundation between excitatory and inhibitory neurons and guarantees the precise function of neuronal networks. Using immunoisolation of synaptic vesicles, we now show that VGLUT2 and VGAT, and also VGLUT1 and VGLUT2, coexist in a sizeable pool of vesicles. VGAT immunoisolates transport glutamate in addition to GABA. Furthermore, VGLUT activity enhances uptake of GABA and monoamines. Postembedding immunogold double labeling revealed that VGLUT1, VGLUT2, and VGAT coexist in mossy fiber terminals of the hippocampal CA3 area. Similarly, cerebellar mossy fiber terminals harbor VGLUT1, VGLUT2, and VGAT, while parallel and climbing fiber terminals exclusively contain VGLUT1 or VGLUT2, respectively. VGLUT2 was also observed in cerebellar GABAergic basket cells terminals. We conclude that the synaptic coexistence of vesicular glutamate and GABA transporters allows for corelease of both glutamate and GABA from selected nerve terminals, which may prevent systemic overexcitability by downregulating synaptic activity. Furthermore, our data suggest that VGLUT enhances transmitter storage in nonglutamatergic neurons. Thus, synaptic and vesicular coexistence of VGLUT and VGAT is more widespread than previously anticipated, putatively influencing fine-tuning and control of synaptic plasticity.

Effects of brain-derived neurotrophic factor (BDNF) on glial cells and serotonergic neurones during development.

Serotonergic neurones are among the first to develop in the central nervous system. Their survival and maturation is promoted by a variety of factors, including serotonin itself, brain‐derived neurotrophic factor (BDNF) and S100β, an astrocyte‐specific Ca2+ binding protein. Here, we used BDNF‐deficient mice and cell cultures of embryonic raphe neurones to determine whether or not BDNF effects on developing serotonergic raphe neurones are influenced by its action on glial cells. In BDNF–/– mice, the number of serotonin‐immunoreactive neuronal somata, the amount of the serotonin transporter, the serotonin content in the striatum and the hippocampus, and the content of 5‐hydroxyindoleacetic acid in all brain regions analysed were increased. By contrast, reduced immunoreactivity was found for myelin basic protein (MBP) in all brain areas including the raphe and its target region, the hippocampus. Exogenously applied BDNF increased the number of MBP‐immunopositive cells in the respective culture systems. The raphe area displayed selectively reduced immunoreactivity for S100β. Accordingly, S100β was increased in primary cultures of pure astrocytes by exogenous BDNF. In glia‐free neuronal cultures prepared from the embryonic mouse raphe, addition of BDNF supported the survival of serotonergic neurones and increased the number of axon collaterals and primary dendrites. The latter effect was inhibited by the simultaneous addition of S100β. These results suggest that the presence of BDNF is not a requirement for the survival and maturation of serotonergic neurones in vivo. BDNF is, however, required for the local expression of S100β and production of MBP. Therefore BDNF might indirectly influence the development of the serotonergic system by stimulating the expression of S100β in astrocytes and the production MBP in oligodendrocytes.

Quantitative comparison of glutamatergic and GABAergic synaptic vesicles unveils selectivity for few proteins including MAL2, a novel synaptic vesicle protein.

Synaptic vesicles (SVs) store neurotransmitters and release them by exocytosis. The vesicular neurotransmitter transporters discriminate which transmitter will be sequestered and stored by the vesicles. However, it is unclear whether the neurotransmitter phenotype of SVs is solely defined by the transporters or whether it is associated with additional proteins. Here we have compared the protein composition of SVs enriched in vesicular glutamate (VGLUT-1) and GABA transporters (VGAT), respectively, using quantitative proteomics. Of >450 quantified proteins, ∼50 were differentially distributed between the populations, with only few of them being specific for SVs. Of these, the most striking differences were observed for the zinc transporter ZnT3 and the vesicle proteins SV2B and SV31 that are associated preferentially with VGLUT-1 vesicles, and for SV2C that is associated mainly with VGAT vesicles. Several additional proteins displayed a preference for VGLUT-1 vesicles including, surprisingly, synaptophysin, synaptotagmins, and syntaxin 1a. Moreover, MAL2, a membrane protein of unknown function distantly related to synaptophysins and SCAMPs, cofractionated with VGLUT-1 vesicles. Both subcellular fractionation and immunolocalization at the light and electron microscopic level revealed that MAL2 is a bona-fide membrane constituent of SVs that is preferentially associated with VGLUT-1-containing nerve terminals. We conclude that SVs specific for different neurotransmitters share the majority of their protein constituents, with only few vesicle proteins showing preferences that, however, are nonexclusive, thus confirming that the vesicular transporters are the only components essential for defining the neurotransmitter phenotype of a SV.

Molecular aspects of tetanus and botulinum neurotoxin poisoning.

Clostridial neurotoxins, tetanus and the botulinum toxins A-G, are high molecular weight proteins consisting of a heavy chain which is responsible for the internalisation and a light chain possessing a zinc-dependent proteolytic activity. They exclusively proteolyse either the vesicle membrane protein, synaptobrevin or two integral plasma membrane proteins, SNAP 25 and syntaxin. Together with cytosolic proteins these proteins form the SNARE complex involved in vesicle exocytosis, and their cleavage blocks the latter process. Clostridial neurotoxins have now become powerful tools to investigate the final events occurring during secretion in neuronal, endocrine, and non-neuronal cells. They are applied to dissect the specific interactions of the SNARE protein complex with cytosolic fusogens and other modulators of exocytosis. Whereas exocytosis is not essential for the survival of cells, the organism as a whole will fall victim to a few nanograms since interneuronal and neuromuscular transmission is vital to muscular control, especially in respiration. Although all clostridial neurotoxins by their light chains attack proteins of the SNARE complex, tetanus toxin and the various botulinum toxins differ dramatically in their clinical symptoms. The biological information for this difference resides on the respective heavy chains which select different transport routes carrying the light chain from the place of entrance to the final compartment of action. So far the different transport vesicles used either by the various botulinum neurotoxins or by tetanus toxin are not yet defined. Nevertheless at least one of the botulinum toxins serves as a beneficial drug in the treatment of severe neuromuscular spasms.