Claudia Olenik

ENHANCED RATE OF EXPRESSION AND BIOSYNTHESIS OF NEUROPEPTIDE Y AFTER KAINIC ACID-INDUCED SEIZURES

Abstract: Recent studies have shown marked increases in brain content of neuropeptide Y (NPY) after seizures induced by intraperitoneal injection of kainic acid and after pentylenetetrazole kindling in the rat. We have now investigated possible changes in the rate of biosynthesis of NPY after kainic acid treatment, by using pulse‐labeling of the peptide and by determining prepro‐NPY mRNA concentrations. For pulse labeling experiments, [3H]tyrosine was injected into the frontal cortex, and the incorporation of the amino acid into NPY was determined after purifying the peptide by gel filtration chromatography, antibody affinity chromatography, and reversed‐phase HPLC. At 2 and 30 days after kainic acid treatment, the rate of tyrosine incorporation was enhanced by ∼380% in the cortex. In addition, concentrations of prepro‐NPY mRNA were determined in four different brain areas by hybridization of Northern blots with a complementary 32P‐labeled RNA probe 2, 10, 30, and 60 days after kainic acid treatment. Marked increases were observed in the frontal cortex (by up to 350% of controls), in the dorsal hippocampus (by 750%), and in the amygdala/pyriform cortex (by 280%) at all intervals investigated. In the striatum only a small, transient increase was observed. The data demonstrate increased expression of prepro‐NPY mRNA and an enhanced rate of in vivo synthesis of NPY as a result of seizures induced by the neurotoxin kainic acid.

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.

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 colicytotoxic necrotizing factor CNF1, which activates Rho by deamidation of Gln63, 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.

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.

Effects of unilateral cortex lesions on gene expression of rat cortical cholecystokinin neurons

In rat neocortex, the gene encoding preprocholecystokinin is expressed in interneurons which also synthetize gamma-aminobutyric acid. An injury to the meninges and the underlying cortex increased the concentration of mRNA coding for preprocholecystokinin in all ipsilateral cortical areas. Simultaneous treatment of the rats with the anti-inflammatory agent diclofenac did not affect the injury-induced change in gene expression indicating that inflammatory processes were not involved. The injury also enhanced the expression of the immediate early gene c-fos in the ipsilateral cortex in a time-dependent manner. There was an increase in c-fos mRNA 1 h after the operation, which was no longer observed 3 h later. Twenty-four hours after the operation, cells containing c-fos mRNA were found in cortical layers II, III, V and VI. The neurons which showed an increased expression of preprocholecystokinin were also in these layers. The N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 prevented the injury-induced increases in both preprocholecystokinin and c-fos gene expression, indicating that stimulation of this glutamate receptor subtype may initiate the changes in expression of both genes. It is hypothetized that the immediate early gene c-fos is activated first and this then leads to the increase in preprocholecystokinin mRNA.

Secretogranin II Is Synthesized and Secreted in Astrocyte Cultures

Abstract: Astrocyte cultures from rat brain were analyzed for their ability to synthesize and secrete secretogranin II (chromogranin C). Northern blot analysis of polyA‐selected RNA established the presence of secretogranin II mRNA in these cells. By radioimmunoassay, 11.6 fmol/106 astrocytes of secretogranin II was found in these cells. About twice the amount was released into the medium within 3 days. Secretogranin II within the astrocytes was practically unprocessed, as shown by HPLC. These results establish for the first time that astrocytes in vitro synthesize and sec rete a protein of the acidic chromogranin family.

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.

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.

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 Dulbeccos 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 Dulbeccos 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 cholecystokinin gene in organotypic slice cultures of immature rat somatosensory cortex.

The preprocholecystokinin gene is expressed in a subpopulation of cortical interneurons containing gamma-aminobutyric acid. Slices of neonatal rat cortex were cultivated for 12 +/- 2 days and examined for the presence and distribution of these neurons by in situ hybridization and immunocytochemistry. Like in situ, two layers of preprocholecystokinin-mRNA-expressing cells were present. Immunopositive fibers formed a dense network and established symmetric contacts on dendritic shafts and spines. It is concluded that cholecystokinin-expressing interneurons survive in cultured slices of rat cerebral cortex. These organotypic cultures may be useful to study the cellular interactions which regulate neuronal cholecystokinin expression.