Activated c-Jun is present in neurofibrillary tangles in Alzheimer's disease brains.

Alzheimer's disease (AD) pathology is characterized by the presence of insoluble beta-amyoid deposits and neurofibrillary tangles containing hyperphosphorylated tau. Increased expression of the immediate early gene product c-Jun has also been reported in post-mortem AD brains, and the presence of upstream regulators of c-Jun has been described in tangle formations. Here, we report the presence of c-Jun specifically phosphorylated on ser-63, but not ser-73, in tangle-bearing neurons and in 'late-stage' extracellular tangles in AD brains. Western blot analysis confirmed the presence of c-Jun phosphorylated on ser-63 but not on ser-73 in AD brain tissue. The expression of differentially phosphorylated c-Jun in the AD brain may reflect the contradictory roles of these phosphorylation sites in neurons. Furthermore, the inappropriate sequestration of phosphorylated c-Jun in tangles in AD brains may contribute to AD pathology and neurodegeneration.

Immediate-early gene response to methamphetamine, haloperidol, and quinolinic acid is not impaired in Huntington's disease transgenic mice.

Striatal neurons in symptomatic Huntington's disease (HD) transgenic mice are resistant to a variety of toxic insults, including quinolinic acid (QA), kainic acid and 3-nitropropionic acid. The basis for this resistance is currently unknown. To investigate the possibility that the immediate-early gene (IEG) response is defective in symptomatic HD mice leading to a lack of response to these compounds, we examined the expression of c-Fos and Krox 24 after administration of the indirect dopamine agonist methamphetamine, the dopamine D(2) receptor antagonist haloperidol and the neurotoxin QA in 5- and 10-week-old R6/2 transgenic HD and wild-type mice. Unlike wild-type and pre-symptomatic R6/2 transgenic HD mice, 10-week-old symptomatic HD mice were resistant to methamphetamine-induced gliosis and QA lesion. There was, however, no difference in the number or distribution of c-Fos-immunoreactive nuclei 2 hr after single injections of methamphetamine or haloperidol among 5- and 10-week-old wild-type mice and 5- and 10-week-old R6/2 HD mice. Similarly, despite their resistance to QA-induced lesioning and lower basal levels of krox-24 mRNA, the symptomatic R6/2 mice had equivalent increases in the amount of c-fos and krox-24 mRNA compared to wild-type and pre-symptomatic R6/2 HD mice as determined by in situ hybridization and densitometry 2 hr after QA administration. These data demonstrate that the c-Fos and Krox 24 IEG response to dopamine agonists, dopamine antagonists and neurotoxic insult is functional in symptomatic R6/2 HD mice. Resistance to toxic insult in R6/2 mice may be conferred by interactions of mutant huntingtin with proteins or transcriptional processes further along the toxic cascade.

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.

PU.1 expression in microglia.

The transcription factor PU.1 has a pivotal role in both the generation and function of macrophages. To determine whether PU.1 is also involved in microglial regulation, we investigated its expression following hypoxic-ischemia (HI) brain injury and in the BV-2 microglial cell line. We found that microglia constitutively expressed high levels of PU.1 protein in both their 'resting' and 'activated' states.

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 Parkinson's 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 Parkinson's 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.

Inducible transcription factor expression in a cell culture model of apoptosis.

We have developed a model of nerve cell death based on the toxicity of okadaic acid, a compound that triggers apoptosis in PC12 cells via a protein synthesis-dependent mechanism. The cell death process is accompanied by induction of JunB, c-Jun, JunD and Fos proteins. Phosphorylation-specific antibodies were used to demonstrate that c-Jun is phosphorylated at serine 63 and serine 73. Electrophoretic gel mobility shift and pAP1-Luc luciferase assays showed that expression of ITFs is associated with increases in AP-1 binding and in AP-1 transcriptional activity. In addition, dose response and time course studies provided strong correlative evidence that Fos and Jun proteins are involved in the apoptotic death cascades. Thus, this model provides a useful system to investigate the role of inducible transcription factor proteins in apoptosis.

CCAAT-enhancer binding protein alpha is expressed in activated microglial cells after brain injury.

Microglial cells play important roles in brain injury and repair and are implicated in diseases such as Alzheimer's 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 protein alpha (C/EBP alpha), 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/EBP alpha might regulate gene expression and consequentially have a role in the activation and/or proliferation of microglia following brain injury.

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.

Expression of Fos, Jun, and Krox family proteins in Alzheimer's disease.

Apoptosis is an active process of cell death characterized by distinct morphological features and is often the end result of a genetic program of events, i.e., programmed cell death (PCD). There is growing evidence supporting a role for apoptosis and/or PCD in Alzheimer's disease (AD), based on DNA fragmentation studies and recent findings of increased levels of inducible transcription factors (ITFs) such as c-Jun in AD brains. We have characterized the expression of a large range of ITFs (c-Fos, Fos B, Fos-related antigens, c-Jun, Jun B, Jun D, Krox20, and Krox24) using multiple antisera in AD postmortem hippocampi and compared this with human control hippocampi as well as Huntington's disease hippocampi and human epilepsy biopsy tissue. We found little evidence of nuclear expression of any ITF except c-Jun in the human postmortem tissue, compared with nuclear staining in biopsy tissue. We found some evidence for increased levels of c-Jun and Krox24 protein and krox24 mRNA in the CA1 region of AD hippocampi, suggesting that PCD may be involved in the pathogenesis of AD. In general, staining characteristics of ITFs varied with different antisera directed against the same protein, indicating the need for caution when interpreting results.

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.

Apoptosis, neurotrophic factors and neurodegeneration.

Apoptosis is an active process of cell death characterized by distinct morphological features, and is often the end result of a genetic programme of events, i.e. programmed cell death (PCD). There is growing evidence supporting a role for apoptosis in some neurodegenerative diseases. This conclusion is based on DNA fragmentation studies and findings of increased levels of pro-apoptotic genes in human brain and in in vivo and in vitro model systems. Additionally, there is some evidence for a loss of neurotrophin support in neurodegenerative diseases. In Alzheimer's disease, in particular, there is strong evidence from human brain studies, transgenic models and in vitro models to suggest that the mode of nerve cell death is apoptotic. In this review we describe the evidence implicating apoptosis in neurodegenerative diseases with a particular emphasis on Alzheimer's disease.

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 Alzheimer's 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 Alzheimer's 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 Alzheimer's 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 Alzheimer's disease hippocampi compared with moderate Bax expression in control hippocampi, and this loss may be related to the survival of these neurons in Alzheimer's 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.

Acute application of human amylin, unlike beta-amyloid peptides, kills undifferentiated PC12 cells by apoptosis.

Recent studies suggest that Alzheimer's disease and non-insulin-dependent (type 2) diabetes mellitus may share a common cell death mechanism, related to the toxicity of beta-amyloid (Abeta) and amylin, respectively. Both Abeta and amylin cause apoptosis in different cell culture systems, which may be related to the amyloidogenic properties of these peptides. We have further characterized the actions of a variety of Abeta peptides (Abeta25-35, Abeta1-40, Abeta1-42), human amylin and rat amylin (which does not form fibrils) on undifferentiated PC12 cells. Although all peptides except rat amylin compromised mitochondrial function as assessed by MTT reduction, only human amylin decreased cell viability at a concentration of 10 microM, as measured by lactate dehydrogenase release or trypan blue exclusion assay. The cell death caused by human amylin was determined to be predominantly of an apoptotic nature, with a possibility of a portion of necrotic cell death, which was not accompanied by increased expression of c-Jun or c-Fos inducible transcription factors.

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.

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.

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.