Markus Höltje

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).

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.

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.

Role of Rho GTPase in astrocyte morphology and migratory response during in vitro wound healing

Small Rho GTPases are key regulators of the cytoskeleton in a great variety of cells. Rho function mediates morphological changes as well as locomotor activity. Using astrocyte cultures established from neonatal mice we investigated the role of Rho in process formation during astrocyte stellation. Using a scratch‐wound model, we examined the impact of Rho on a variety of morphological and functional variables such as stellation and migratory activity during wound healing. C3 proteins are widely used to study cellular Rho functions. In addition, C3 derived from Clostridium botulinum (C3bot) is considered selectively to promote neuronal regeneration. Because the latter requires a balanced activity of neurones and glial cells, the effects of C3 protein on glial cells such as astrocytes have to be considered carefully. Low nanomolar concentrations of C3 proteins significantly promoted process outgrowth and increased process branching. Besides enzymatic inactivation of Rho by ADP‐ribosylation, changes in protein levels of the various Rho GTPases may also contribute to the observed effects. Furthermore, incubation of scratch‐wounded astrocyte cultures with C3bot accelerated wound healing. By inhibiting the Rho downstream effector ROCK with the selective inhibitor Y27632 we were able to demonstrate that the accelerated wound closure resulted from both enhanced polarized process formation and increased migratory activity of astrocytes into the lesion site. These results suggest that Rho negatively regulates astrocytic process growth and migratory responses after injury and that its inactivation by C3bot in nanomolar concentrations promotes astrocyte migration.

C3 peptide enhances recovery from spinal cord injury by improved regenerative growth of descending fiber tracts

Functional recovery and regeneration of corticospinal tract (CST) fibers following spinal cord injury by compression or dorsal hemisection in mice was monitored after application of the enzyme-deficient Clostridium botulinum C3-protein-derived 29-amino-acid fragment C3bot154-182. This peptide significantly improved locomotor restoration in both injury models as assessed by the open-field Basso Mouse Scale for locomotion test and Rotarod treadmill experiments. These data were supported by tracing studies showing an enhanced regenerative growth of CST fibers in treated animals as visualized by anterograde tracing. Additionally, C3bot154-182 stimulated regenerative growth of raphespinal fibers and improved serotonergic input to lumbar α-motoneurons. These in vivo data were confirmed by in vitro data, showing an enhanced axon outgrowth of α-motoneurons and hippocampal neurons cultivated on normal or growth-inhibitory substrates after application of C3bot154-182. The observed effects were probably caused by a non-enzymatic downregulation of active RhoA by the C3 peptide as indicated by pull-down experiments. By contrast, C3bot154-182 did not induce neurite outgrowth in primary cultures of dorsal root ganglion cells. In conclusion, C3bot154-182 represents a novel, promising tool to foster axonal protection and/or repair, as well as functional recovery after traumatic CNS injury.

The first lumenal domain of vesicular monoamine transporters mediates G-protein-dependent regulation of transmitter uptake

The activity of vesicular monoamine transporters (VMATs) is down-regulated by the G-protein α-subunits of Go2 and Gq, but the signaling pathways are not known. We show here that no such regulation is observed when VMAT1 or VMAT2 are expressed in Chinese hamster ovary (CHO) cells. However, when the intracellular compartments of VMAT-expressing CHO cells are preloaded with different monoamines, transport becomes susceptible to G-protein-dependent regulation, with differences between the two transporter isoforms. Epinephrine induces G-protein-mediated inhibition of transmitter uptake in CHOVMAT1 cells but prevents inhibition induced by dopamine in CHOVMAT2 cells. Epinephrine also antagonizes G-protein-mediated inhibition of monoamine uptake by VMAT2 expressing platelets or synaptic vesicles. In CHOVMAT2 cells G-protein-mediated inhibition of monoamine uptake can be induced by 5-hydroxytryptamine (serotonin) 1B receptor agonists, whereas α1 receptor agonists modulate uptake into CHOVMAT1 cells. Accordingly, 5-hydroxytryptamine 1B receptor antagonists prevent G-proteinmediated inhibition of uptake in partially filled platelets and synaptic vesicles expressing VMAT2. CHO cells expressing VMAT mutants with a shortened first vesicular loop transport monoamines. However, no or a reduced G-protein regulation of uptake can be initiated. In conclusion, vesicular content is involved in the activation of vesicle associated G-proteins via a structure sensing the luminal monoamine content. The first luminal loop of VMATs may represent a G-protein-coupled receptor that adapts vesicular filling.

High prevalence of NMDA receptor IgA/IgM antibodies in different dementia types

To retrospectively determine the frequency of N‐Methyl‐D‐Aspartate (NMDA) receptor (NMDAR) autoantibodies in patients with different forms of dementia.

A 29-amino acid fragment of Clostridium botulinum C3 protein enhances neuronal outgrowth, connectivity, and reinnervation

Small GTPases of the Rho family play versatile roles in the formation and development of axons and dendrites, effects often studied by the Rho‐inactivating C3 transferase (C3bot) from Clostridium botulinum. Recently, we reported that transferasedeficient C3bot also exerted axonotrophic activity. Using overlapping peptides from the C3bot sequence, we identified a small peptide of 29 amino acids (covering residues 154‐182) from the C‐terminal region of C3bot that promotes both axonal and dendritic growth, as well as branching of hippocampal neurons, at sub‐micromolar concentrations. Several C3bot constructs, including the short peptide, enhanced the number of axonal segments from mid‐ to higher‐order segments. C3bot154‐182 also increased the number of synaptophysin‐expressing terminals, up‐regulated various synaptic proteins, and functionally increased the glutamate uptake. Staining against the vesicular glutamate and GABA transporters further revealed that the effect was attributable to a higher number of glutamatergic and GABAergic inputs on proximal dendrites of enhanced green fluorescent protein (EGFP)‐transfected neurons. Using organotypical slice cultures, we also detected trophic effects of C3bot154‐182 on length and density of outgrowing fibers from the entorhinal cortex that were comparable to the effects elicited by full‐length C3bot. In addition, an enhanced reinnervation was observed in a hippocampal‐entorhinal lesion model. In summary, the neurotrophic effect of C3bot is executed by a C‐terminal peptide fragment covering aa 154‐182 of C3; it triggers dendritic and axonal growth and branching as well as increased synaptic connectivity. In contrast to full‐length C3, this C3 peptide selectively acts on neurons but not on glial cells. Holtje, M., Djalali, S., Hofmann, F., Munster‐Wandowski, A., Hendrix, S., Boato, F., Dreger, S. C., Große, G., Henneberger, C., Grantyn, R., Just, I., Ahnert‐Hilger, G. A 29‐amino acid fragment of Clostridium botulinum C3 protein enhances neuronal outgrowth, connectivity, and reinnervation. FASEB J. 23, 1115–1126 (2009)

G alpha(o2) regulates vesicular glutamate transporter activity by changing its chloride dependence

Classical neurotransmitters, including monoamines, acetylcholine, glutamate, GABA, and glycine, are loaded into synaptic vesicles by means of specific transporters. Vesicular monoamine transporters are under negative regulation by α subunits of trimeric G-proteins, including Gαo2 and Gαq. Furthermore, glutamate uptake, mediated by vesicular glutamate transporters (VGLUTs), is decreased by the nonhydrolysable GTP-analog guanylylimidodiphosphate. Using mutant mice lacking various Gα subunits, including Gαo1, Gαo2, Gαq, and Gα11, and a Gαo2-specific monoclonal antibody, we now show that VGLUTs are exclusively regulated by Gαo2. G-protein activation does not affect the electrochemical proton gradient serving as driving force for neurotransmitter uptake; rather, Gαo2 exerts its action by specifically affecting the chloride dependence of VGLUTs. All VGLUTs show maximal activity at ∼5 mm chloride. Activated Gαo2 shifts this maximum to lower chloride concentrations. In contrast, glutamate uptake by vesicles isolated from Gαo2-/- mice have completely lost chloride activation. Thus, Gαo2 acts on a putative regulatory chloride binding domain that appears to modulate transport activity of vesicular glutamate transporters.

Anti-DPPX encephalitis Pathogenic effects of antibodies on gut and brain neurons

Objective: To characterize pathogenic effects of antibodies to dipeptidyl-peptidase-like protein 6 (DPPX), a subunit of Kv4.2 potassium channels, on gut and brain neurons. Methods: We identified a new patient with anti-DPPX encephalitis and analyzed the effects of the patients serum and purified immunoglobulin G (IgG), and of serum of a previous patient with anti-DPPX encephalitis, on the activity of enteric neurons by voltage-sensitive dye imaging in guinea pig myenteric and human submucous plexus preparations. We studied the subcellular localization of DPPX by immunocytochemistry in cultured murine hippocampal neurons using sera of 4 patients with anti-DPPX encephalitis. We investigated the influence of anti-DPPX-containing serum and purified IgG on neuronal surface expression of DPPX and Kv4.2 by immunoblots of purified murine hippocampal neuron membranes. Results: The new patient with anti-DPPX encephalitis presented with a 2-month episode of diarrhea, which was followed by tremor, disorientation, and mild memory impairment. Anti-DPPX-IgG-containing sera and purified IgG increased the excitability and action potential frequency of guinea pig and human enteric nervous system neurons. Patient sera revealed a somatodendritic and perisynaptic neuronal surface staining that colocalized with the signal of commercial anti-DPPX and Kv4.2 antibodies. Incubation of hippocampal neurons with patient serum and purified IgG resulted in a decreased expression of DPPX and Kv4.2 in neuronal membranes. Conclusions: Hyperexcitability of enteric nervous system neurons and downregulation of DPPX and Kv4.2 from hippocampal neuron membranes mirror the clinical phenotype of patients with anti-DPPX encephalitis and support a pathogenic role of anti-DPPX antibodies in anti-DPPX encephalitis.