Cytotoxic necrotizing factor-2 of Escherichia coli alters the morphology of cultured hippocampal neurons.

Certain pathogenic strains of E. coli produce the cytotoxic necrotizing factors-1 or -2. Cytotoxic necrotizing factor-1 irreversibly activates the small GTPases of the Rho family Rho, Rac and Cdc42. Cytotoxic necrotizing factor-2 may have similar effects. Since the Rho proteins play an important role in the organization of the actin cytoskeleton and neuronal differentiation, we have investigated whether cytotoxic necrotizing factor-2 affects the morphology of cultured hippocampal neurons. The toxin indeed caused dendrite retraction and axon shortening. Within 4 h of application, cytotoxic necrotizing factor-2 induced a transient formation of short finger-like extensions. To study the role of the Rho proteins in the morphological changes caused by cytotoxic necrotizing factor-2, we transfected neurons with recombinant Rho proteins. Dominant-negative forms of Rac or Rho but not of Cdc42 prevented the formation of short extensions induced by cytotoxic necrotizing factor-2, indicating synergistic effects of Rac and Rho. In contrast, the retraction of dendrites induced by cytotoxic necrotizing factor-2 was only prevented by dominant-negative Rho. Analysis with pull-down assays showed that cytotoxic necrotizing factor-2 strongly activated Rac and Rho, whereas an effect on Cdc42 was not observed. Cytotoxic necrotizing factor-2 also diminished the total amount of Rac and Rho. The degradation of Rac was so pronounced that the increase in Rac activity was only transient. In organotypic cultures of the hippocampus, cytotoxic necrotizing factor-2 reduced the number of neurites per neuron, suggesting that neurons in the tissue context were also vulnerable. We conclude that cytotoxic necrotizing factor-2 has pronounced effects on neuronal morphology, which are due to activation of the GTPases Rho and Rac.

Regulation of somatodendritic GABAA receptor channels in rat hippocampal neurons: evidence for a role of the small GTPase Rac1.

The role of the cytoskeleton in the activity of GABA(A) receptors was investigated in cultured hippocampal neurons. Receptor currents were measured with the whole-cell patch-clamp technique during repetitive stimulation with 1 microm muscimol. After destruction of the microtubular system with nocodazol, muscimol-induced currents showed a rundown by 78%. A similar rundown was observed when actin fibers were destroyed with latrunculin B or C2 toxin of Clostridium botulinum. Because the small GTPases of the Rho family RhoA, Rac1, and Cdc42 are known to control the organization of actin fibers, we investigated their possible involvement. Inactivation of the GTPases with clostridial toxins, as well as intracellular application of recombinant Rho GTPases, indicated that active Rac1 was necessary for full GABA(A) receptor activity. Immunocytochemical labeling of the receptors showed that the disappearance of receptor clusters in the somatic membrane as induced by muscimol stimulation was enhanced by Rac1 inactivation. It is suggested that Rac1 participates in the regulation of GABA(A) receptor clustering and/or recycling.

Evidence for cell specific regulation by PACAP38 of the proenkephalin gene expression in neocortical cells.

During the first postnatal week, glial cell production for the neocortex continues in the neocortical subventricular zone. During this time, the proenkephalin gene (PEnk) is expressed in numerous cells of the subventricular zone and of the adjacent neocortex. When neocortical astroglial cells are brought into dissociation culture, they also produce PEnk mRNA. We have investigated the effect of pituitary adenylate cyclase activating peptide-38 (PACAP38) on PEnk gene expression in dissociation cultures as well as in slice cultures, which contained the subventricular zone and the adjacent neocortex. PACAP38 enhanced the levels of PEnk mRNA in both culture systems. In dissociated astroglial cells, inhibition of protein kinase A, of p44,42 mitogen-activated protein kinase as well as of the EGF-receptor tyrosine kinase by H89, PD98059 and AG1478, respectively, reduced the PACAP38-induced expression in a synergistic manner. In the neocortical part of the slice cultures, the effect of PACAP38 on PEnk gene expression was inhibited only by H89 and PD98059. Here, protein kinase A and p44,42 MAP kinases shared a mechanism which increased the gene expression. Surprisingly, the expression of the PEnk gene in the glial progenitors of the subventricular zone as induced by PACAP38 was not affected by any of the three protein kinase inhibitors, but was blocked by the unspecific kinase inhibitor H7. It is concluded that PACAP38 induced the PEnk gene expression in both culture systems in a cell-type specific manner.

Neosynthesis and activation of Rho by Escherichia coli cytotoxic necrotizing factor (CNF1) reverse cytopathic effects of ADP-ribosylated Rho.

Clostridium botulinum exoenzyme C3 inactivates the small GTPase Rho by ADP-ribosylation. We used a C3 fusion toxin (C2IN-C3) with high cell accessibility to study the kinetics of Rho inactivation by ADP-ribosylation. In primary cultures of rat astroglial cells and Chinese hamster ovary cells, C2IN-C3 induced the complete ADP-ribosylation of RhoA and concomitantly the disassembly of stress fibers within 3 h. Removal of C2IN-C3 from the medium caused the recovery of stress fibers and normal cell morphology within 4 h. The regeneration was preceded by the appearance of non-ADP-ribosylated RhoA. Recovery of cell morphology was blocked by the proteasome inhibitor lactacystin and by the translation inhibitors cycloheximide and puromycin, indicating that intracellular degradation of the C3 fusion toxin and the neosynthesis of Rho were required for reversal of cell morphology. Escherichia coli cytotoxic necrotizing factor CNF1, which activates Rho by deamidation of Gln(63), caused reconstitution of stress fibers and cell morphology in C2IN-C3-treated cells within 30-60 min. The effect of CNF1 was independent of RhoA neosynthesis and occurred in the presence of completely ADP-ribosylated RhoA. The data show three novel findings; 1) the cytopathic effects of ADP-ribosylation of Rho are rapidly reversed by neosynthesis of Rho, 2) CNF1-induced deamidation activates ADP-ribosylated Rho, and 3) inhibition of Rho activation but not inhibition of Rho-effector interaction is a major mechanism underlying inhibition of cellular functions of Rho by ADP-ribosylation.

Neocortical projections regulate the neostriatal proenkephalin gene expression.

There is controversial evidence that neocortical projections to the neostriatum may regulate the neostriatal expression of the proenkephalin (PEnk) gene. Therefore, we have studied PEnk gene expression in organotypic neocortico-neostriatal co-cultures as well as cultures of isolated neostriata. PEnk mRNA was determined with in situ hybridization. Removal of the neocortex caused a time-dependent reduction in the number of neostriatal cells which showed expression of the PEnk gene. A maximal decrease was seen after 3 days. Within 2 days after blockade of glutamate receptors of the NMDA type significantly fewer neostriatal cells expressed the PEnk gene, indicating that NMDA receptors mediated the expression of the gene. In isolated neostriatal slices in which the expression of the PEnk gene had been down-regulated, NMDA increased the number of cells which expressed the gene in a time-dependent manner. Maximal expression was observed after 3 days. This induction was reduced by nimodipine, which blocks L-type Ca2(+)-channels. The slow increase in PEnk gene expression caused by NMDA resembled a recruiting process. It seems to be specific for the neostriatum and may be due to the latter's neuronal organization.

Differential expression of the small GTP-binding proteins RhoA, RhoB, Cdc42u and Cdc42b in developing rat neocortex.

Studies with cultured cells indicate that small GTPases of the Rho family may be involved in cell proliferation, differentiation, as well as migration. Therefore, we have studied the expression of four members of this protein family, i.e., RhoA, RhoB, the ubiquitous Cdc42u, and brain specific Cdc42b, during the embryonic and early postnatal development of rat neocortex. A clear isoform specificity of expression was found during the prenatal development. Thus, RhoA and Cdc42u were present in the proliferation zone while RhoB and Cdc42b were expressed only in the cortical plate where neural cells settle and differentiate. After birth, this isoform specificity quickly disappeared so that already at postnatal day 8 the adult pattern of expression was present. Our findings of a differential expression of the small GTP-binding proteins RhoA, RhoB, Cdc42u and Cdc42b in developing brain neocortex suggest isoform specific functions during neurogenesis and differentiation.

Glutamatergic control of the expression of the proenkephalin gene in rat frontoparietal cortical slice cultures.

The expression of the proenkephalin (PEnk) gene in rat neocortex develops during the first two postnatal weeks in an outside first-inside last mode that is opposite to the gradient of neurogenesis. To test whether the distribution of PEnk gene expression depends on the formation of the local circuitry, we examined the role of glutamate neurons in the expression of the gene in slice cultures of rat frontoparietal cortex. In situ and Northern blot hybridization were used for analysis. In slices explanted at postnatal day 6, the neuronal expression of the PEnk gene developed as in vivo. The expression responded to glutamate receptor agonists and antagonists in a time-dependent manner. After 2 days in vitro the expression of the gene was only enhanced by N-methyl-D-aspartate receptors, whereas after 7 days in vitro AMPA receptors also regulated the expression. We concluded that glutamate neurons are involved in the development and maintenance of the PEnk gene expression in the neocortex.

Glial expression of the proenkephalin gene in slice cultures of the subventricular zone.

The proenkephalin (PEnk) gene is expressed in rats in the neocortical subventricular zone (nSVZ) of the lateral ventricle during the first postnatal week, when precursors of astro- and oligoglial cells of the rat neocortex proliferate in this area. To study the expression of the gene in the glial precursors, slices containing the nSVZ were prepared from the brains of newborn and 7-day-old rats. After 1-5 d of cultivation, numerous cells that expressed PEnk mRNA were found in the nSVZ with in situ hybridization. Some of these cells coexpressed the glial fibrillary acidic protein (GFAP), indicating that they were of astroglial origin. Activation of protein kinase A with 8Br.cAMP strongly enhanced the number of cells that expressed the PEnk gene in slices prepared from the brains of newborn or 7-d-old rats. Also pituitary adenylate cyclase activating polypeptide (PACAP) proved to be effective. After stimulation with 8Br.cAMP or PACAP-38, PEnk mRNA-containing cells were found in the subventricular zone as well as in the adjacent area through which glial cells migrate on their way to the neocortex. It has therefore been concluded that protein kinase A may regulate the expression of the PEnk gene expression in glial precursors in the nSVZ.

The N-terminal part of the enzyme component (C2I) of the binary Clostridium botulinum C2 toxin interacts with the binding component C2II and functions as a carrier system for a Rho ADP-ribosylating C3-like fusion toxin.

The binary actin-ADP-ribosylating Clostridium botulinum C2 toxin consists of the enzyme component C2I and the binding component C2II, which are separate proteins. The active component C2I enters cells through C2II by receptor-mediated endocytosis and membrane translocation. The N-terminal part of C2I (C2IN), which consists of 225 amino acid residues but lacks ADP-ribosyltransferase activity, was identified as the C2II contact site. A fusion protein (C2IN-C3) of C2IN and the full-length C3-like ADP-ribosyltransferase from Clostridium limosum was constructed. The fusion protein C2IN-C3 ADP-ribosylated Rho but not actin in CHO cell lysates. Together with C2II, C2IN-C3 induced complete rounding up of CHO and HeLa cells after incubation for 3 h. No cell rounding was observed without C2II or with the original C3-like transferase from C. limosum. The data indicate that the N-terminal 225 amino acid residues of C2I are sufficient to cause the cellular uptake of C. limosum transferase via the binding component of C2II, thereby increasing the cytotoxicity of the C3-like exoenzyme several hundred-fold.

Role of Rho protein in lovastatin-induced breakdown of actin cytoskeleton.

The Rho GTPases are involved in actin cytoskeleton organization and signal transduction. They need polyisoprenylation for membrane association and activation. Lovastatin, a hydroxymethylglutaryl coenzyme A inhibitor, prevents isoprene synthesis and thereby lipid modification of the Rho protein carboxy terminus. Because lovastatin causes rounding up of cultured cells, we investigated whether the compound acts on the actin cytoskeleton through Rho proteins. Lovastatin treatment decreased F-actin content in a time- and concentration-dependent manner. G-actin content remained unchanged. In lovastatin-treated NIH 3T3 cells, the amount of Rho protein which was ADP-ribosylated by Clostridium botulinum exoenzyme C3 decreased in membranes and increased in the cytosol fraction. Cycloheximide prevented lovastatin-induced rounding up of cells. However, after microinjection or direct application of exoenzyme C3, cells treated with cycloheximide and lovastatin rounded up again. On the contrary, lovastatin-treated, round Swiss 3T3 cells reverted to a flat morphology when microinjected with dominant active RhoA (Val14RhoA). Escherichia coli cytotoxic necrotizing factor (CNF1) which activates Rho proteins caused flattening of round, lovastatin-treated NIH 3T3 cells. These results suggest that lovastatin affects the actin cytoskeleton through inactivation of Rho proteins.

Neurons are generated in confluent astroglial cultures of rat neonatal neocortex.

Cells of the telencephalon are generated in specific proliferative zones from which neuronal and glial precursors migrate to their destinations. Recent evidence indicates that some precursors do not turn into differentiated cells but keep their ability to proliferate. Here, we report that neurons can originate in primary cultures of astroglial cells prepared from neocortex of newborn rats. The first neuronal cells appeared shortly before confluence, when a glial monolayer was being formed. After confluence, these still undifferentiated cells increased in number. Later, they became immunohistochemically positive for the neuron-specific marker microtubule-associated protein 2a,b. They also contained neurofilament-L protein as well as the specific messenger RNA coding for neurofilament-H. The observation that they took up bromo-deoxyuridine indicated that they synthesized DNA, i.e. they proliferated. When Dulbecco's modified essential medium was substituted with fetal calf serum, the appearance of neurons depended on the seeding density of the dispersed cells. This was no longer the case, when the cultures were maintained in Dulbecco's modified essential medium/F12 medium to which transferrin, insulin and selenium chloride had been added. It is concluded that neuronal precursors can survive in primary astroglial cultures. After confluence of the astroglial cells the precursors proliferate if appropriate conditions are present. Our observation provides a new model for the investigation of cultured neurons and neuronal-glial interactions.

Expression of the proenkephalin gene in cultured astroglial cells: analysis of cell cycle dependence.

Glial progenitors strongly express the proenkephalin (PEnk) gene during their proliferation in the subventricular zone of the neocortex. Also in primary culture, astroglial cells from rat neocortex produce PEnk mRNA. Since the basal expression sharply declines after the cultured cells reach confluence, it seems to be related to cell proliferation. In contrast, activation of protein kinases A and C strongly enhances the levels of PEnk mRNA in confluent cultures. Therefore, it was investigated in cultured neocortical astroglial cells, whether the basal and stimulated expression of the PEnk gene occurred during different phases of the cell cycle. Activation of protein kinases A and C with 8Br.cAMP and tetradecanoylphorbolacetate, respectively, enhanced the PEnk gene expression only during the G1 phase. Unstimulated astroglial cells contained PEnk mRNA during the G1 as well as the S phase of the cell cycle. Since confluent astroglial cells are arrested in the G1 phase, they should have a basal expression of the gene which is comparable to that in preconfluent cells. Thus, the reduced basal expression after confluence indicates that other mechanisms may play a role, such as humoral factors or contact inhibition.

Development of proenkephalin gene expression in rat neocortex: a non-radioactive in situ hybridization study.

Products of the proenkephalin gene are not only neurotransmitters but may also influence brain development. The ontogeny of the expression of the proenkephalin gene in neocortex was studied in embryonic and postnatal rats with in situ hybridization. At embryonic day 14, the proliferating cells in the ventricular zone strongly expressed the gene. Thereafter, the expression decreased and was hardly detectable up to embryonic day 21. At the day of birth and during the subsequent week, proliferating cells in the subventricular zone were labelled. The expression of the proenkephalin gene in proliferating neuronal and glial progenitors indicates that gene products may affect proliferation and/or commitment. In the neocortex, cells which strongly expressed the gene were first seen at postnatal day 7 in the outer part of the neocortex. Seven days later, a second band of positive cells had appeared in the inner part of the cortex, i.e. the adult pattern of distribution had been established. Thus, in rat neocortex the expression of the proenkephalin gene developed in an outside-first, inside-last mode.

Gene expression of the small GTP-binding proteins RhoA, RhoB, Rac1, and Cdc42 in adult rat brain.

GTPases of the Rho subfamily, i.e. Rho, Rac and Cdc42, are molecular switches in various signaling pathways. Best characterized are their functions in the regulation of the actin cytoskeleton. In neuronal cell lines they are involved in the mechanisms leading to synapse formation and plasticity. It is still unknown whether they have respective functions in the mammalian CNS. In this case, they should be present in the adult brain, especially in areas known for their synaptic remodeling. We have studied the expression of the Rho GTPases in adult rat brain with in situ hybridization and Western blot analysis. High amounts of RhoA, RhoB, Rac1 and Cdc42 mRNAs were detected in neurons of the hippocampus, i.e. in pyramidal cells of the CA1-CA4 regions as well as in granule cells of the dentate gyrus and in hilar cells. Also in cerebellum, Purkinje and granular cells expressed the four mRNAs. Strong gene expression was also found in brainstem, thalamus and neocortex. Using Western blot analysis, RhoA and Cdc42 proteins were detected in hippocampus, cerebellum, thalamus and neocortex. It is concluded that GTPases of the Rho family play a role in the regulation of cellular functions in the adult brain.

Regulation of the expression of the proenkephalin gene in cultured meningeal fibroblasts: opposite effects of alpha 1- and beta 2-adrenoceptors.

Meningeal fibroblasts express the proenkephalin gene during embryonal development but terminate the expression shortly before birth. When brought into primary culture at postnatal day 1, the fibroblasts again express the gene. Activation of protein kinase A reduces this expression and thus may contribute to its prenatal termination. Since the noradrenergic innervation of the meninges begins around the time of birth, it was investigated in the present study, how adrenergic agonists affected the levels of proenkephalin mRNA in cultured fibroblasts. The beta 2-adrenoceptor agonists salbutamol and procaterol increased the levels of endogenous cAMP and diminished the concentration of proenkephalin mRNA indicating that the cultured fibroblasts possessed this beta-subtype. In contrast, noradrenaline increased the level of proenkephalin mRNA in a concentration-dependent manner. This effect was independent of endogenous cAMP and was mediated by alpha 1-adrenoceptors. The data indicate that the noradrenergic innervation of the meninges at the time of birth is not responsible for the termination of the proenkephalin gene expression.

Noradrenaline increases the expression of the proenkephalin gene in cultured astroglial cells by acting on beta 1- and alpha 1-adrenoceptors.

In primary culture, type I astroglial cells from neocortex of newborn rats express the proenkephalin gene. The glial cells are not homogeneous but differ in their morphology; i.e., polygonal and process-bearing cells are found. Transcription of the proenkephalin gene is increased via protein kinase A upon stimulation with cyclic AMP (cAMP) analogues. In the present study, how noradrenaline affected the expression of the proenkephalin gene in both cell types was investigated. Noradrenaline enhanced the levels of proenkephalin mRNA in a concentration-dependent manner. Experiments with subtype-selective antagonists suggested that beta 1-adrenoceptors were involved. In situ hybridization showed that proenkephalin mRNA was induced only in polygonal cells. Nor-adrenaline also increased the levels of cAMP. However, concentrations of noradrenaline that produced a maximal increase in cAMP caused only submaximal elevations of proenkephalin mRNA. This discrepancy was explained by the finding that noradrenaline increased the expression of the proenkephalin gene also via alpha 1-adrenoceptors. It is concluded that beta 1- and alpha 1-adrenoceptors can act in a synergistic manner on the expression of the proenkephalin gene in astroglial cells.

Expression of the proenkephalin A gene in organotypic cultures of neocortex from newborn rats.

In rats, the proenkephalin A gene is expressed in proliferating cells of the neuroepithelial zone which later give rise to neocortical neurones and glial cells. Therefore, organotypic cultures of neocortex of newborn rats were used in the present study to examine whether neurones as well as glial cells expressed the gene. The slices were prepared at birth and kept in culture for 7-13 days. Proenkephalin mRNA was visualised by in situ hybridisation, while immunocytochemical staining for MAP-2 and GFAP was used to identify neurones and astroglial cells, respectively. In the analysed slices, only neurones contained proenkephalin mRNA. Activation of protein kinase C with tetradecanoylphorbol acetate (1 mumol/l) caused a strong increase in the number of neurones expressing proenkephalin mRNA. Our results indicate that a large number of neurones is able to express the proenkephalin gene under these conditions. However, only a few of them have a basal expression which is strong enough to be detected with in situ hybridisation.

Evidence for the involvement of 5-lipoxygenase products in the regulation of the expression of the proenkephalin gene in cultured astroglial cells.

Cultured astroglial cells secrete eicosanoids which are produced by the cyclooxygenase and lipoxygenases. These cells also transcribe the proenkephalin gene. In the present study, it was investigated whether agents which inhibit the metabolism of arachidonic acid affect the basal and stimulated expression of the gene. Tetradecanoyl phorbol acetate (TPA; 1-1000 nmol/l) increases the concentration of proenkephalin mRNA in these cells by activating protein kinase C. The enhancement in proenkephalin mRNA caused by TPA (10 nmol/l) was not affected by the cyclooxygenase inhibitor indomethacin (5 mumol/l). However, nordihydroguaiaretic acid, which blocks cyclooxygenase and lipoxygenases, potentiated the effect of TPA on proenkephalin mRNA, when used at concentrations of 0.5-50 mumol/l. Two selective inhibitors of 5-lipoxygenase, i.e. MK886 (5 mumol/l) and BAY X1005 (1 mumol/l), also enhanced the effect of TPA (10 nmol/l) without affecting the basal expression of the gene. When added to the incubation medium, leukotriene E4 (10-1000 nmol/l) diminished in a dose-dependent manner the basal and TPA-induced expression of the proenkephalin gene. It is concluded that in astroglial cells derived from cortex of new-born rats products of 5-lipoxygenase can diminish the action of protein kinase C on the proenkephalin gene.

Rat meningeal fibroblasts in primary culture express the proenkephalin gene.

When brought into primary culture, rat meningeal fibroblasts contained proenkephalin-mRNA detected with Northern blot hybridization. In contrast, splenic fibroblasts did not express the gene under the same culture conditions. In situ hybridization showed that the meningeal fibroblasts did not uniformly express the gene: groups of positive cells were surrounded by cells with low or no proenkephalin-mRNA. Some fibroblasts which contained the mRNA species took up bromo-deoxyuridine indicating that the expression of the gene also occurred in proliferating cells, but was not restricted to this group. In chromaffin and astroglial cells, activation of protein kinase A or C with 8 Br.cAMP or O-tetradecanoyl 13-phorbolacetate, respectively, increases the expression of the gene. In meningeal fibroblasts, however, both agents reduced the levels of proenkephalin-mRNA. In the case of 8Br.cAMP, this effect was blocked by the protein synthesis inhibitor cycloheximide indicating that a newly-synthesized protein was involved. Cultured meningeal fibroblasts appear to be useful for studies on the cell specificity of the expression of this peptide gene as well on its regulation.

Interaction of protein kinases A and C in their effects on the proenkephalin gene in astroglial cells.

In several cell types, the expression of the proenkephalin (PEnk) gene is enhanced after activation of protein kinase A. In the present study, astroglial cells cultured from rat cortex were used to investigate whether protein kinases A and C can act in a synergistic manner on the endogenous proenkephalin gene. The activator of protein kinase C tetradecanoylphorbolacetate (0.001-1 microM) increased the level of proenkephalin-mRNA in a concentration dependent manner. When used together with the phosphodiesterase inhibitor Rolipram (1 microM), the effect of tetradecanoylphorbolacetate (0.01 microM) was potentiated. 8-Bromoadenosine 3',5'-cyclic monophosphate (0.01-1 mM) also enhanced the expression of the proenkephalin gene. When used together with tetradecanoylphorbolacetate (0.01 and 0.1 microM), respectively, both agents had additive effects. Inhibition of protein synthesis with cycloheximide (35 microM) significantly changed the effects of both agents. While the effect of 8Br.cAMP (1 mM) on PEnk-mRNA was enhanced, that of tetradecanoylphorbolacetate (0.1 microM) was abolished. The results provide evidence for a synergistic effect of protein kinase A and C on the expression of the proenkephalin gene in astroglial cells. However, the protein kinases seem to act via different transcription factors on the expression of the proenkephalin gene.