Kirsten Mielke

JNK and p38 stresskinases--degenerative effectors of signal-transduction-cascades in the nervous system.

The c-Jun N-terminal kinases (JNKs, also called stress activated protein kinases. SAPKs) and p38 kinases constitute together with extracellular signal-regulated kinases (ERKs) the family of MAP kinases. Whereas the functions of JNKs under physiological conditions are largely unknown, there is raising evidence that JNKs are potent effectors of apoptosis or degeneration of neurons in vitro and in the brain. The activation of the inducible transcription factor c-Jun by N-terminal phosphorylation is a central event in JNK-mediated degenerative processes that depend on de novo protein synthesis. At the post-translational level, cytoplasmic degenerative actions of JNKs might comprise inhibition of Bcl-2 and steroid hormone-receptor signaling or hyperphosphorylation of tau; and at transcriptional level, JNKs might trigger the induction of the apoptotic effectors p53 and Fas-Ligand by phosphorylation of c-Jun. The role of p38 is the nervous system is poorly understood, but its activation is also considered as part of the neuronal stress response. This review informs about the genetic processing, the regulation of activity and the biochemical actions of JNK and p38 isoforms in general. In the second part, we summarize the findings on expression and activation of JNKs and p38 under neurodegenerative condition. A particular focus is also put on the putative function of JNK under physiological conditions and for neuroprotection.

c-Jun N-terminal kinases (JNKs) mediate pro-inflammatory actions of microglia.

The activation and function of c‐Jun N‐terminal kinases (JNKs) were investigated in primary microglia cultures from neonatal rat brain, which express all three JNK isoforms. Lipopolysaccharide (LPS), tumor necrosis factor‐α (TNF‐α), and thrombin preparations induced a rapid and lasting activation of JNKs in the cytoplasm. In the nucleus, the activation patterns were rather complex. In untreated microglia, the small pool of nuclear JNKs was strongly activated, while the high‐affinity JNK substrate c‐Jun was only weakly phosphorylated. Stimulation with LPS increased the total amount of nuclear JNKs and the phosphorylation of the transcription factor c‐Jun. Levels of activated JNKs in the nucleus, however, rapidly decreased. Analysis of the nuclear JNK isoforms revealed that the amount of JNK1 declined, while JNK2 increased, and the weakly expressed JNK3 did not vary. This observation suggests that JNK2 is mainly responsible for the activation of c‐Jun in this context. Upstream of JNKs, LPS induced a lasting activation of the constitutively present JNK kinase MKK4. The function of JNKs in LPS‐triggered cellular reactions was investigated using SP600125 (0.5–5 μM), a direct inhibitor of JNKs. Inhibition of JNKs reduced the LPS‐induced metabolic activity and induction of the AP‐1 target genes cyclooxygenase‐2 (Cox‐2), TNF‐α, monocyte chemoattractant protein‐1 (MCP‐1), and interleukin‐6 (IL‐6) in response to LPS, while ERK1/2 and p38α had a more pronounced effect on LPS‐induced cellular enlargement than JNKs. In summary, JNKs are essential mediators of relevant pro‐inflammatory functions in microglia with different contributions of the JNK isoforms.

Activity and expression of JNK1, P38 and ERK kinases. c-Jun N-terminal phosphorylation, and c-jun promoter binding in the adult rat brain following kainate-induced seizures

The activity and/or expression of the mitogen-activated protein kinases c-Jun N-terminal kinase 1, p38 and extracellular signal-regulated kinases 1/2, as well as their substrates, the transcription factors c-Jun and activating transcription factor-2, were examined following systemic application of kainate in the cortex and hippocampus of the adult rat brain. The protein expression levels of all three mitogen-activated protein kinases remained constant during the observation period. Unexpectedly, c-Jun N-terminal kinase 1 was the only mitogen-activated protein kinase activated in this model of excitotoxicity, its activity raised from between 1 and 3 h moderate basal to maximal levels between 6 and 12 h. In contradistinction, activity of extracellular signal-regulated kinases 1/2 fell from their substantial basal levels and did not recover; activity of p38 was characterized by a high basal level that almost entirely disappeared and did not return to basal levels even 10 days after kainate application. c-Jun protein was rapidly expressed, with a maximum after 3 h and a slow decline after 12 h. Supershift assays revealed that, during the early induction phase of the c-jun gene, the proximal activator protein-1 (jun1) site of the c-jun promoter was mainly occupied by the constitutively expressed activating transcription factor-2, whereas the late induction correlated with the predominant binding of c-Jun and, to a lesser extent, activating transcription factor-2 to the distal activator protein-1 (jun2) site. The time-course of the N-terminal phosphorylation of c-Jun as determined by immunocytochemistry paralleled the activity of c-Jun N-terminal kinase 1 and showed a compartment-specific regulation between 3 and 12 h. A second set of supershift experiments demonstrated that c-Jun, but not activating transcription factor 2, bound to activator protein-1 sites in the promoter of substance P and collagenase genes, but not of the cyclo-oxygenase-2 gene. Our results demonstrate that activation of c-Jun N-terminal kinase 1, phosphorylation of c-Jun and selective occupation of the c-jun promoter by activating transcription factor-2 or c-Jun are part of the neuronal response following excitotoxicity that is considered as the mechanism for neuronal apoptosis in vivo. Some of these findings differ substantially from in vitro experiments and underline the necessity to analyse the neuronal stress pathways in the adult brain.

Angiotensin AT2 receptor stimulates ERK1 and ERK2 in quiescent but inhibits ERK in NGF-stimulated PC12W cells

To investigate the influence of AT(2) receptor stimulation on the ERK pathway and elucidate potential mechanisms of angiotensin II (ANG II)-mediated neuronal differentiation, we analysed tyrosine phosphorylation and activity of ERK after ANG II treatment of both quiescent and NGF-treated PC12W cells. Tyrosine phosphorylation of ERK1 and ERK2 corresponded with the activity of ERK. While ANG II induced an initial activation of ERK in quiescent cells, the NGF-mediated plateau of ERK-stimulation was lowered by costimulation with ANG II. All effects of ANG II were sensitive to AT(2) - but not AT(1) receptor blockade. Ang II-mediated neurite outgrowth in PC12W cells was inhibited by co-treatment with the MEK inhibitor PD 098059. These findings demonstrate that the AT(2) receptor modulates ERK activity depending on the overall cellular input. The distinct regulation of ERK by ANG II and NGF further indicates basic differences in AT(2) receptor- and NGF-induced neuronal differentiation.

Selective expression of JNK isoforms and stress-specific JNK activity in different neural cell lines.

The function of c-Jun N-terminal kinases (JNKs) in the nervous system is poorly understood and the majority of the data has been gained in neuronal and non-neuronal cell lines. Thus, it is not clear to which extent the expression pattern and the degree of activation of the three JNK isoforms in different cell lines are representative for their activation in the adult brain. In the present study, the expression of JNK isoforms and the activity of JNK1 were determined following UV irradiation and exposure to H(2)O(2) and TNFalpha in three neural cell lines, rat PC12, murine Neuro2A and human SHSY5Y. These cell lines differ in their expression of JNK isoforms: PC12 cells express JNK1 and JNK2, whereas Neuro2A and SHSY5Y cells displays the expression of JNK1, JNK2 and JNK3. JNK3 was not inducible following stress and differentiation in PC12 cells. The stimulation paradigms evoked different degree of cell death: UV irradiation resulted in death of around 50% in all three cell lines; exposure to 200 microM H(2)O(2) for 6 h resulted in the death of 43% Neuro2A cells and 31% PC12 cells, SHSY5Y cells are less sensitive to H(2)O(2) since only 5 mM H(2)O(2) killed 59% of SHSY5Y cells after 6 h. Exposure to 50 ng/ml TNFalpha did not induce cell death in SHSY5Y, Neuro2A and naive PC12 cells. Although differentiated PC12 cells exhibit a similar activation of JNK1 compared to naive PC12 cells after exposure to TNFalpha, 42% of differentiated PC12 cells died after 24 h. H(2)O(2) that evoked only a moderate JNK1 activity in Neuro2A and PC12 cells induced only a moderate cell death. In contrast, SHSY5Y cells exhibit a much stronger JNK1 activation accompanied with a higher degree in cell death after exposure to H(2)O(2). JNK1 activity induced by UV irradiation, however, could not be correlated with the extend of cell death. These data clearly demonstrate that expression and activation of JNK depends on the neuronal cell type and the applied stress paradigms, and that JNK activity is not simply linked to cell death.

The c-Jun N-terminal kinases in cerebral microglia: immunological functions in the brain

The c-Jun N-terminal kinases (JNKs) exert a pleiotrophy of physiological and pathological actions. This is also true for the immune system. Disruption of the JNK locus results in substantial functional deficits of peripheral T-cells. In contrast to circulating immune cells and the role of p38, the presence and function of JNKs in the immune cells of the brain remain to be defined. Here, we report on the expression and activation of JNKs in cultivated microglia from neonatal rats and from mice with targeted disruption of the JNK locus and the N-terminal mutation of c-Jun (c-JunAA), respectively. JNK1, 2 and 3 mRNA and proteins were all expressed in microglia. Following stimulation with LPS (100 ng/mL), a classical activator of microglia, JNKs were rapidly activated and this activation returns to basal levels within 4 hr. Following LPS and other stimuli such as thrombin (10-50 unit/mL), the activation of JNKs went along with the N-terminal phosphorylation of c-Jun which persisted for at least 8 hr. Indirect inhibition of JNK by CEP-11004 (0.5-2 microM), an inhibitor of mixed-lineage kinases (MLK), reduced the LPS-induced phosphorylation of both, JNK and c-Jun, by around 50%, and attentuated the LPS-induced the alterations in microglial morphology. Finally, JNKs are involved in the control of cytokine release since both, incubation with CEP-11004 and disruption of the JNK1 locus enhanced the release of TNFalpha, IL-6 and IL-12. Our findings provide insight in so far unknown functions of JNKs in cerebral immune cells. These observations are also important for the wide spread efforts to develop JNK-inhibitors as neuroprotective drugs which, however, might trigger pro-inflammatory processes.

Caspase-3 activation and DNA fragmentation in primary hippocampal neurons following glutamate excitotoxicity

Excitotoxic glutamate CNS stimulation can result in neuronal cell death. Contributing mechanisms and markers of cell death are the activation of caspase-3 and DNA fragmentation. It remains to be resolved to which extent both cellular reactions overlap and/or indicate different processes of neurodegeneration. In this study, mixed neuronal cultures from newborn mice pubs (0-24 h) were stimulated with glutamate, and the co-localization of active caspase-3 and DNA fragmentation was investigated by immunocytochemistry and the TUNEL nick-end labelling. In untreated cultures, 8% scattered neurons (marked by MAP-2) displayed activated caspase-3 at different morphological stages of degeneration. TUNEL staining was detected in 5% of cell nuclei including GFAP-positive astrocytes. However, co-localization of active caspase-3 with TUNEL was less than 2%. After glutamate stimulation (125 microM), the majority of neurons was dying between 12 and 24 h. The absolute number of active caspase-3 neurons increased only moderately but in relation of surviving neurons after 24 h from 8 to 36% (125 microM), to 53% (250 microM) or to 32% (500 microM). TUNEL staining also increased after 24 h following glutamate treatment to 37% but the co-localization with active caspase-3 remained at the basal low level of 2%. In our system, glutamate-mediated excitotoxicity effects the DNA fragmentation and caspase-3 activation. Co-localization of both parameters, however, is very poor. Active caspase-3 in the absence of TUNEL indicates a dynamic degenerative process, whereas TUNEL marks the end stage of severe irreversible cell damage regardless to the origin of the cell.

Expression of c-jun, junB, c-fos, fra-1 and fra-2 mRNA in the rat brain following seizure activity and axotomy

The present study has investigated the congruence of mRNA induction and protein expression of inducible transcription factors (ITFs). The patterns of c-jun, junB, c-fos, fra-1 and fra-2 mRNAs were studied by radioactive and non-radioactive in situ hybridization in the adult rat brain following kainate-induced seizure activity and axotomy. In the same animals, the expression of c-Jun, JunB and c-Fos proteins was compared with the respective mRNA signals. Using radioactive labeled probes all investigated mRNAs showed an onset within 1 h after systemic kainate application and the maximal levels were generally reached after 3 h. Each mRNA displayed a specific temporo-spatial expression pattern. Whereas fra-1 and fra-2 were restricted to the hippocampus, c-jun, junB and c-fos were additionally induced in the cortex, amygdala and thalamus. The areas with maximal labeling were the dentate gyrus and the hippocampal CA1 and CA3 subfields. The expression patterns between c-jun, junB and c-fos mRNA were virtually congruent with the respective protein. Labeling of the junB and fra-2 probes with digoxigenin yielded similar results. Twenty-four hours, 3 and 10 days following transection of the medial forebrain bundle and the mamillo-thalamic tract, high levels of c-jun mRNA (either digoxigenin or radioactive labeled probes) and protein were seen in the axotomized neurons of the substantia nigra pars compacta and mamillary body whereas the other mRNAs studied and the JunB or c-Fos proteins could not be detected. These findings demonstrate that mRNAs encoding for ITFs are translated into the respective proteins following excitotoxic seizures and axotomy, and that the antisera used for immunocytochemistry yield specific expression patterns of homologous proteins.

Fatal Shift of Signal Transduction Is an Integral Part of Neuronal Differentiation: JNKs Realize TNFα-Mediated Apoptosis in Neuronlike, but Not Naive, PC12 Cells

Tumor necrosis factor-alpha (TNFalpha, 10-100 ng/ml) provokes a dramatic cell death in differentiated PC12 cells (dPC12), but it does not affect the viability and the proliferation of naive PC12 cells (nPC12). We have analyzed the molecular alterations of the TNFalpha-signal cascade underlying this developmental switch toward propagation of apoptosis. The transcriptional inhibitor actinomycin D rendered nPC12 responsive for TNFalpha-induced death, but was hardly effective in dPC12, suggesting that TNFalpha evokes its harmful action in dPC12 predominantly by posttranslational modification of existing molecules. This suggestion was supported by the finding that differentiation of PC12 per se went along with the increased expression of the proapoptotic TNFalpha-receptor I (p55) and its adapter protein Traf-2, whereas expression and phosphorylation of the antiapoptotic Akt (PKB) declined. We could demonstrate that the c-Jun N-terminal kinases (JNKs) mediate this enhanced capacity of apoptotic signaling in dPC12. TNFalpha induced in dPC12, but not nPC12, a biphasic activation of JNKs with a rapid transient JNK1 activation and a second persistent activation of JNK1 and JNK2 paralleled by phosphorylation of c-Jun; in contrast, TNFalpha did not activate p38 kinase. Block of JNKs by CEP-11004, a MLK antagonist and subsequently indirect inhibitor of JNK activation, or L-JNK11, a direct peptidergic inhibitor of JNK activity, almost completely rescued dPC12. Summarizing, the NGF-triggered formation of neurites during differentiation of PC12 includes the reinforced propensity for apoptosis, with JNK2 as the effector in JNK3-negative PC12. These findings offer novel insights into the increased risk of neuronal death, which is linked to the potential to regenerate.

Repetitive electroconvulsive seizures induce activity of c-Jun N-terminal kinase and compartment-specific desensitization of c-Jun phosphorylation in the rat brain

Electroconvulsive seizures (ECS) are used for therapy of pharmacoresistent depression and are supposed to induce long-lasting neuronal alterations in morphology and gene expression. In this study, we have investigated the phosphorylation of the transcription factor protein c-Jun at its serine 73 residue by immunohistochemistry and the activity of the c-Jun N-terminal kinase 1 (JNK1) by immunocomplex assay following repetitive ECS in adult rats. In untreated controls, nuclear c-Jun immunoreactivity, but not N-terminal phosphorylation, was present in a variety of neuronal populations including the hippocampus, the temporobasal cortex and the amygdalar complex. Daily ECS for 1, 5 or 10 days (1x, 5x or 10x ECS) did not alter the expression of c-Jun but caused a substantial N-terminal phosphorylation of c-Jun (phospho-c-Jun). Nuclear phospho-c-Jun immunoreactivity was maximal within 15 min following ECS, and became absent after 30 min. The highest levels of phospho-c-Jun labeling were found after 1x ECS in the amygdalar complex, the dorsomedial hypothalamus and the piriform cortex. The inducibility of c-Jun N-terminal phosphorylation was preserved in the medial amygdala and piriform cortex, but significantly declined in the basal amygdala and medial hypothalamus with progressive ECS stimulation. One single ECS 3 or 5 days following 10x ECS yielded a pattern of phospho-c-Jun as seen following 10x ECS; thus, a lag of 5 days was not sufficient to provoke the initial level of N-terminal phosphorylation of c-Jun. In the rostral hippocampus, c-Jun was not phosphorylated at any investigated time inspite of its high constitutive expression. In some contrast with this compartment-specific phosphorylation of c-Jun, immunocomplex assays revealed that the JNK1 activity was strongly enhanced in both amygdala and hippocampus. Our findings demonstrate that rapid JNK activation and phosphorylation of c-Jun as stand-by transcription factor characterize the beginning of neuroplastic changes, e.g., following ECS, a classic treatment of mental disorders. The N-terminal phosphorylation is compartment specific and can habituate following repetitive stimulation suggesting that the differential activation of the JNK/c-Jun axis is part of the neuronal strategy to integrate transynaptic excitation.