Jost Leemhuis

Cellular uptake of Clostridium difficile toxin B. Translocation of the N-terminal catalytic domain into the cytosol of eukaryotic cells.

Clostridium difficile toxin B (269 kDa) is one of the causative agents of antibiotic-associated diarrhea and pseudomembranous colitis. Toxin B acts in the cytosol of eukaryotic target cells where it inactivates Rho GTPases by monoglucosylation. The catalytic domain of toxin B is located at the N terminus (amino acid residues 1–546). The C-terminal and the middle region of the toxin seem to be involved in receptor binding and translocation. Here we studied whether the full-length toxin or only a part of the holotoxin is translocated into the cytosol. Vero cells were treated with recombinant glutathione S-transferase-toxin B, and thereafter, toxin B fragments were isolated by affinity precipitation of the glutathione S-transferase-tagged protein from the cytosolic fraction of intoxicated cells. The toxin fragment (∼65 kDa) was recognized by an antibody against the N terminus of toxin B and was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis as the catalytic domain of toxin B. The toxin fragment located in the cytosol possessed glucosyltransferase activity that could modify RhoA in vitro, but it was not able to intoxicate intact cells. After treatment of Vero cells with a radiolabeled fragment of toxin B (amino acid residues 547–2366), radioactivity was identified in the membrane fraction of Vero cells but not in the cytosolic fraction of Vero cells. Furthermore, analysis of cells by fluorescence microscopy revealed that the C terminus of toxin B was located in endosomes, whereas the N terminus was detected in the cytosol. Protease inhibitors, which were added to the cell medium, delayed intoxication of cells by toxin B and pH-dependent translocation of the toxin from the cell surface across the cell membrane. The data indicate that toxin B is proteolytically processed during its cellular uptake process.

Rho GTPases and phosphoinositide 3-kinase organize formation of branched dendrites

Neurons receive information from other neurons via their dendritic tree. Dendrites and their branches result from alternating outgrowth and retraction. The Rho GTPases Rac and Cdc42 (cell division cycle 42) facilitate the outgrowth of branches, whereas Rho attenuates it. The mechanism of neurite retraction is unknown. Because the adenylyl cyclase activator forskolin causes numerous branched extensions in NG108-15 cells, we have investigated the underlying mechanism in this cell line. In additional studies, we used cultured hippocampal neurons in which forskolin induces branched dendrites. In both cell types, forskolin enhanced the activity of Cdc42, but not that of Rac, although both GTPases were necessary for the formation of branched extensions. Time lapse microscopy showed that forskolin did not increase the rate of addition of new extensions or branches, but it reduced the rate of the retraction so that more branched extensions persisted. Inhibition of phosphoinositide 3-kinase activity by wortmannin or LY294002 also reduced the rate of retraction and thus facilitated dendritic arborization. Forskolin diminished the activity of phosphoinositide 3-kinases. Inhibitors of phosphoinositide 3-kinases not only reduced the retraction but also the addition of new dendrites and branches. This reduction was no longer present when Rho kinase was simultaneously inactivated, suggesting an interaction of phosphoinositide 3-kinases and Rho kinase. The present results show a central role of phosphoinositide 3-kinases in dendrite formation. In neuronal cells, increased levels of cAMP can support dendritic arborization by modulating the activity of the lipid kinase.

Vasoactive Intestinal Peptide and PACAP38 Control N-Methyl-D-aspartic Acid-induced Dendrite Motility by Modifying the Activities of Rho GTPases and Phosphatidylinositol 3-Kinases

Dendrite morphogenesis is highly dynamic and characterized by the addition and elongation of processes and also by their selective maintenance, retraction, and elimination. Glutamate can influence these events via N-methyl-d-aspartic acid (NMDA) receptors. The neuropeptides vasoactive intestinal peptide and pituitary adenylyl cyclase-activating polypeptide-38 (PACAP38) affect neurogenesis and differentiation in the developing nervous system. We report here that the peptides and NMDA acted synergistically on dendrite and branch formation. In stage III hippocampal neurons, NMDA increased not only the addition but also the elimination of new dendrites and branches by activating Rac and Cdc42 and phosphatidylinositol 3-kinases, respectively. When applied alone, the neuropeptides did not influence dendrite or branch formation. However, they reduced the elimination of newly formed dendrites and branches caused by NMDA by preventing the NMDA-induced activation of phosphatidylinositol 3-kinases. This led to the formation of persistent dendrites and branches. Additional timelapse studies on the dynamics of dendrite elongation showed alternating periods of elongation and retraction. Phosphatidylinositol 3-kinases increased the velocities of dendrite elongation and retraction, whereas the neuropeptides prolonged the periods of elongation. By modifying NMDA-induced activation of Rho GTPases and phosphatidylinositol 3-kinases, vasoactive intestinal peptide and PACAP38 could play an important role in the control of dendrite growth and branching during development and in response to neuronal activity.

Gabapentin-Lactam Induces Dendritic Filopodia and Motility in Cultured Hippocampal Neurons

Gabapentin is currently used as a therapeutic agent against epilepsy as well as neuropathic pain. In contrast to gabapentin, its derivative gabapentin-lactam has a pronounced neuroprotective activity. We have studied in cultured hippocampal neurons whether gabapentin-lactam has also neurotrophic effects. Gabapentin-lactam enhanced the formation of dendritic filopodia, which are necessary for synapse formation. It also induced a network of F-actin-containing neurites. In studies with time lapse microscopy, gabapentin-lactam increased the addition but also the elimination of new branches. Affinity precipitation assays showed that gabapentin-lactam increased the GTP binding of the small GTPases Rac and Cdc42, which facilitate branch addition. Gabapentin-lactam also activated RhoA and phosphatidylinositol 3-kinases. In neurons transfected with dominant-negative RhoA or treated with the RhoA-inactivating C3 toxin, gabapentin-lactam increased the number of dendrites and branches. In the presence of Y-27632, which inhibits Rho kinase, newly added branches induced by gabapentin-lactam were no longer eliminated so that gabapentin-lactam increased the number of branches. Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide] also prevented the gabapentin-lactam induced activation of phosphatidylinositol 3-kinases. The phosphatidylinositol 3-kinase inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride] reduced the elimination of newly added branches caused by gabapentin-lactam and thus facilitated branch formation. In contrast to gabapentin-lactam, gabapentin had no effect on dendritic filopodia or motility. The effects exerted by gabapentin-lactam on dendritic arborization may be of potential therapeutic interest.

The small GTPase Rac is involved in clustering of hippocampal neurons and fasciculation of their neurites

In hippocampal neurons cultured from brains of newborn rats, the glutamate receptor agonist N-methyl-d-aspartate induced the clustering of neuronal perikarya and the fasciculation of neurites. In addition, N-methyl-d-aspartate activated the small GTPase Rac1. Other stimuli of Rac activity, such as the Rho kinase inhibitors Y-27632, H-1152, and H89, as well as the cytotoxic necrotizing factor-1 from Escherichia coli, also caused neuronal clustering and neurite bundling. In neurons transiently transfected with dominant negative Rac1N17 neither N-methyl-d-aspartate nor Y-27632 induced clustering and fasciculation. In addition, the PI3-kinase inhibitors wortmannin and LY-294002 prevented these effects, as did a dominant negative form of p110PI3-Kγ. Time-lapse microscopy showed that lethal toxin from Clostridium sordellii, which inhibits Rac, and wortmannin blocked the neuronal migration induced by Y-27632. In contrast, only lethal toxin reversed the clustering and fasciculation induced by pre-treatment with Y-27632. This effect of the toxin may be due to inactivation of Ras, since FTI-277, which prevents the farnesylation of Ras and thereby inactivates the GTPase, also dissolved the preformed clusters. We suggest that active Rac and a PI3-kinase synergistically induce neuronal migration, whereas a Ras isoform is responsible for the lasting attachment of neurons necessary for clustering and neurite fasciculation.