Geraldine MacGibbon

Is c-Jun involved in nerve cell death following status epilepticus and hypoxic-ischaemic brain injury?

Neurons undergoing delayed neuronal death produced by hypoxia-ischaemia (HI) or status epilepticus (SE) showed a massive expression of c-Jun in their nuclei 24 h after the insult. With SE there was also a weaker induction of c-Fos and Jun B in dying neurons. SE induced in the presence of the NMDA antagonist MK-801 produced no delayed c-Jun expression in the hippocampus and nerve cell death did not occur in this region, although there was a delayed c-jun expression in the amygdala/piriform region, and cell death occurred in this area. Activation of central muscarinic receptors with pilocarpine, or block of D2 dopamine receptors with haloperidol, treatments which do not cause neuronal damage, strongly induced Fos and Jun B in hippocampal and striatal neurons, but only induced c-Jun very weakly. Thus, c-Jun may participate in the genetic cascade of events that produce programmed cell death in neurons.

Clozapine and haloperidol produce a differential pattern of immediate early gene expression in rat caudate-putamen, nucleus accumbens, lateral septum and islands of Calleja

Acute administration of the typical neuroleptic haloperidol (HAL, 2 mg/kg) induced the immediate-early gene proteins (IEGPs) c-Fos, Fos-related antigens (FRAs), FosB, JunB, JunD and Krox24 in the striatum and nucleus accumbens of the rat brain. In contrast, acute administration of the atypical antipsychotic drug clozapine (CLOZ, 30 mg/kg) induced only FRAs, JunB and Krox24 IEGPs in the striatum, and c-Fos, FRAs, and Krox24 IEGPs in the nucleus accumbens. c-Jun was not induced by acute administration of HAL or CLOZ in the rat brain. Differential induction of IEGs by HAL and CLOZ was also observed in the lateral septal nucleus and the islands of Calleja complex of the rat brain. These differences in IEG induction by HAL and CLOZ may be related to the different clinical profiles of the two drugs. Specifically, CLOZ induces FRAs in the islands of Calleja and lateral septum and this action may be involved in its therapeutic effects on the negative symptoms of schizophrenia, whereas HAL produces a coordinate induction of Fos and JunB in striatal neurons and this dimer combination may be involved in producing the extrapyramidal side-effects of typical neuroleptics.

Bax expression in mammalian neurons undergoing apoptosis, and in Alzheimer's disease hippocampus.

Recent studies indicate that the proto-oncogene Bax, and other related proteins (eg Bcl-2) may play a major role in determining whether cells will undergo apoptosis under conditions which promote cell death. Increased expression of Bax has been found to promote apoptosis, while over-expression of Bcl-2 can inhibit apoptosis. To investigate the role of Bax in nerve cell death in the rat brain we examined the level of Bax expression in cells undergoing apoptosis, using a hypoxic-ischemic stroke model. We found that Bax was expressed at high levels in the nuclei of neurons in the hippocampus, cortex, cerebellum, and striatum on the control side, and that Bax levels increased in hippocampal neurons undergoing apoptosis on the stroke side, and then declined (correlating with cell loss). In the Alzheimers disease hippocampi we found a concentrated localisation of Bax in senile plaques, which correlated with the localisation of beta-amyloid protein in adjacent sections from the same brains. beta-Amyloid positive plaques are thought to contribute to the Alzheimers disease process, possibly via an apoptotic mechanism, and this may occur via an increase in Bax in these areas. Bax was also strongly stained in tau-positive tangles in Alzheimers disease hippocampi, suggesting Bax may play a role in tangle formation. In addition, we observed a loss of Bax expression in the dentate granule cells of Alzheimers disease hippocampi compared with moderate Bax expression in control hippocampi, and this loss may be related to the survival of these neurons in Alzheimers disease. Finally, we observed substantially different staining patterns of Bax using three different commercially available antisera to Bax, indicating the need for caution when interpreting results in this area.

c-Jun promotes neurite outgrowth and survival in PC12 cells.

We investigated the function of c-Jun in PC12 cells by transfecting them with a plasmid containing a c-Jun cDNA transcription cassette. Transfected cells expressed high levels of c-Jun mRNA and protein and demonstrated an increase in both AP-1 DNA binding and gene activation. The c-Jun over-expressing cells showed marked neurite outgrowth but no evidence of spontaneous cell death. In fact, c-Jun over-expressing cells were more resistant to okadaic acid-induced apoptosis. The process outgrowth was not indicative of a full neuronal differentiation response as the transfected PC12 cells did not display action potentials when examined with whole-cell patch-clamping. The phosphorylation of c-Jun on serine 73 appears to be important for this neurite sprouting effect as mutagenesis at this site reduced sprouting whereas a serine 63 mutant tended to increase sprouting. Thus, in PC12 cells c-Jun expression does not induce apoptosis, but rather functions as a neurite outgrowth and neuronal survival signal.

The toxicity of 6-hydroxydopamine on PC12 and P19 cells.

Considerable evidence implicates the involvement of mitochondrial dysfunction in neurodegenerative diseases. 6OHDA is a mitochondrial complex I inhibitor which is frequently used to model Parkinsons disease-like cell loss. We investigated the cell death pathways triggered by 6OHDA in PC12 and P19 cells with a view to shedding light on the molecular basis of Parkinsons disease. We found that 6OHDA triggered mostly necrosis and less than 5% apoptosis in PC12 cells, whereas 6OHDA-induced death in P19 cells was apoptotic. While desipramine, a dopamine uptake blocker, attenuated 6OHDA-induced apoptosis in PC12 cells, this compound had no effect on the large scale necrotic death. Furthermore, desipramine failed to reduce apoptosis in 6OHDA-treated P19 cells, suggesting that the mechanism of 6OHDA toxicity does not require uptake via the dopamine transporter. As cell death triggered by 6OHDA was not blocked by free radical scavengers or NMDA receptor antagonists, a non-specific extracellular mechanism may be involved.

Increased expression and activation of c-Jun contributes to human amylin-induced apoptosis in pancreatic islet β-cells

The role of c-Jun in apoptosis evoked by human amylin was investigated using human and rat insulinoma beta-cell lines. Two transient increases in the levels of c-jun mRNA were detected at 30 minutes and eight hours after treatment with human amylin. The level of c-Jun protein was also up-regulated in a time-dependent manner, reaching maximal levels after eight hours of exposure. However, no c-Jun induction was detected in cells treated with vehicle only or with rat amylin, indicating that the amyloidogenic feature of the human peptide may be important for c-Jun induction. We found that c-Jun was activated by phosphorylation specifically at Ser63 at four hours, but not at Ser73, after treatment with human amylin, preceding increased c-Jun protein. Furthermore, expression of an antisense c-jun (AS-c-jun), which suppressed protein levels of both c-Jun and phosphorylated-c-Jun, caused a marked reduction in apoptotic cell death, whereas the corresponding sense c-jun (S-c-jun) had no effect on changes of either c-Jun production or apoptosis. This indicated that increased expression and activation of c-Jun is required for human amylin-induced apoptosis. Immunocytochemical studies showed a significant increase in nuclear staining for c-Jun, phosphorylated-c-Jun (Ser63) and phosphorylated-JNK, suggesting that c-Jun may be activated through activation of JNK. In addition, electrophoretic mobility-shift assays showed that the increase in expression and phosphorylation of c-Jun was associated with increased AP-1 DNA binding activity. Supershift assays demonstrated that c-Jun, c-Fos and ATF-2 are part of the AP-1 complex, indicating that activated c-Jun is dimerized with c-Fos or ATF-2 for control of its target gene expression. Finally, we showed that human amylin triggers AP-1-mediated transcriptional activation. Our results suggest strongly that human amylin induces apoptosis through stimulation of expression and activation of c-Jun, and that co-expression and dimerization of c-Jun and c-fos or ATF-2 may be important for activation of the downstream apoptotic process.

Do c-Jun, c-Fos, and amyloid precursor protein play a role in neuronal death or survival?

A unilateral hypoxic‐ischemic (HI) episode in immature rat brain was used to investigate the role of the immediate early genes c‐fos and c‐jun in delayed neuronal death and survival. This HI paradigm results in an apoptotic cell death in selectively vulnerable areas, in particular the hippocampal CA1 pyramidal cell layer. In susceptible regions undergoing delayed neuronal death there was a prolonged induction of both c‐Jun and c‐Fos (mRNA and protein). This expression occurred in parallel with a pronounced increase in AP‐1 DNA binding activity but was not associated with either increased levels of Jun NH2‐terminal kinase or phosphorylation of c‐Jun (ser‐63). In addition to changes in immediate early gene expression, the CA1 neurons showed a delayed increase in the expression of amyloid precursor protein (APP751) mRNA, suggesting that APP, which contains an AP‐1 site, might be a down‐stream gene regulated by the Jun transcription factor in neurons dying by apoptosis. The surviving dentate granule cells also showed an increase in Fos, Jun, and APP751 although this expression occurred earlier than in the CA1 neurons and declined rapidly. These results are discussed with respect to the role of these proteins in neuronal death and survival. J. Neurosci. Res. 53:330–342, 1998.

Differential expression of inducible transcription factors in basal ganglia neurons

The dopamine receptor antagonist, haloperidol, produced a time-dependent differential induction of inducible transcription factors (ITFs) in rat striatal neurons: Fos, Fos B, Jun B, Jun D, Krox 20, and Krox 24, but not c-Jun, were induced in the caudate putamen and nucleus accumbens with varying time courses. The induction of Fos by haloperidol was stronger in anterior versus posterior regions of the striatum. In contrast, induction of Fos by the muscarinic agonist pilocarpine was stronger in the posterior regions of the striatum suggesting that muscarinic receptors do not play a role in the induction of ITFs in striatal neurons by haloperidol. Although c-Jun was not induced in caudate neurons by haloperidol it was strongly induced in these neurons following prolonged seizure activity. The differential pattern of Jun protein expression suggests that haloperidol induces a specific transcriptional program in basal ganglia neurons. These effects of haloperidol may be involved in producing its extrapyramidal side effects.

CCAAT-enhancer binding proteinα is expressed in activated microglial cells after brain injury

Abstract Microglial cells play important roles in brain injury and repair and are implicated in diseases such as Alzheimers disease, Creutzfeldt–Jacob disease, multiple sclerosis, the Aids Dementia Complex and stroke. Despite their importance in neuropathology, the underlying molecular basis for the activation of microglia after brain injury is not understood. We show, using RT-PCR, in situ hybridisation, immunocytochemistry, and electrophoretic mobility shift assay, that the CCAAT-enhancer binding proteinα (C/EBPα), a sequence specific DNA-binding protein, is induced in microglial cells, but not astrocytes or neurons, after hypoxic–ischemic brain injury. These results suggest that C/EBPα might regulate gene expression and consequentially have a role in the activation and/or proliferation of microglia following brain injury.

Etoposide-induced PC12 cell death: apoptotic morphology without oligonucleosomal DNA fragmentation or dependency upon de novo protein synthesis.

Etoposide, a topoisomerase II inhibitor used in cancer therapy, has been shown to induce apoptosis in vitro in a variety of cell types. In the present study, we have characterized the effects of etoposide on undifferentiated rat pheochromocytoma PC12 cells. Etoposide killed PC12 cells in a time- and concentration-dependent manner. 20-24 h incubation with 10 micrograms/ml etoposide induced 25-50% cell death. Hoechst 33258 staining revealed apoptotic morphology in dying cells. No evidence was found of either oligonucleosomal DNA fragmentation, as shown by agarose gel electrophoresis, or endonuclease involvement, as shown by the inability of aurintricarboxylic acid to prevent cell death. Cycloheximide and actinomycin-D were unable to prevent etoposide cytotoxicity indicating that the process is not dependent upon de novo protein or mRNA synthesis. NGF (5 ng/ml) prevented etoposide-induced PC12 cell death. These results offer an example of how the morphological features of apoptosis are not necessarily associated with oligonucleosomal DNA fragmentation or with de novo macromolecule synthesis.