Vicki Waetzig

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.

Specific pathophysiological functions of JNK isoforms in the brain

We have investigated the effect of JNK1 ko, JNK2 ko, JNK3 ko, JNK2+3 ko and c‐JunAA mutation on neuronal survival in adult transgenic mice following ischemia, 6‐hydroxydopamine induced neurotoxicity, axon transection and kainic acid induced excitotoxicity. Deletion of JNK isoforms indicated the compartment‐specific expression of JNK isoforms with 46‐kDa JNK1 as the main phosphorylated JNK isoform. Permanent occlusion of the MCA significantly enlarged the infarct area in JNK1 ko, which showed an increased expression of JNK3 in the penumbra. Survival of dopaminergic neurons in the substantia nigra compacta (SNC) following intrastriatal injection of 6‐hydroxydopamine was transiently improved in JNK3 ko and c‐JunAA mice after 7 days, but not 60 days. Following transection of the medial forebrain bundle, however, JNK3 ko conferred persisting neuroprotection of axotomised SNC neurons. None of the JNK ko and c‐JunAA mutation affected the survival of facial motoneurons following peripheral axotomy when investigated after 90 days. Finally, we determined the impact of JNK ko on the survival of animals and the degeneration of hippocampal neurons following kainic acid. JNK3 ko mice were substantially resistant against and survived kainic acid‐induced seizures. JNK3 ko and JNK1 ko showed a nonsignificant tendency for decreased or increased death of hippocampal neurons, respectively. Surprisingly, the deletion of a single JNK isoform did not attenuate the immunocytochemical signal of phosphorylated c‐Jun irrespective on the experimental set‐up. This comprehensive study provides novel insights into the context‐dependent physiological and pathological functions of JNK isoforms.

AP-1 proteins in the adult brain: facts and fiction about effectors of neuroprotection and neurodegeneration

Jun and Fos proteins are induced and activated following most physiological and pathophysiological stimuli in the brain. Only few data allow conclusions about distinct functions of AP-1 proteins in neurodegeneration and neuroregeneration, and these functions mainly refer to c-Jun and its activation by JNKs. Apoptotic functions of activated c-Jun affect hippocampal, nigral and primary cultured neurons following excitotoxic stimulation and destruction of the neuron-target-axis including withdrawal of trophic molecules. The inhibition of JNKs might exert neuroprotection by subsequent omission of c-Jun activation. Besides endogenous neuronal functions, the c-Jun/AP-1 proteins can damage the nervous system by upregulation of harmful programs in non-neuronal cells (e.g. microglia) with release of neurodegenerative molecules. In contrast, the differentiation with neurite extension and maturation of neural cells in vitro indicate physiological and potentially neuroprotective functions of c-Jun and JNKs including sensoring for alterations in the cytoskeleton. This review summarizes the multiple molecular interfunctions which are involved in the shift from the physiological role to degenerative effects of the Jun/JNK-axis such as cell type-specific expression and intracellular localization of scaffold proteins and upstream activators, antagonistic phosphatases, interaction with other kinase systems, or the activation of transcription factors competing for binding to JNK proteins and AP-1 DNA elements.

JNK2 translocates to the mitochondria and mediates cytochrome c release in PC12 cells in response to 6-hydroxydopamine

6-Hydroxydopamine (6-OHDA) causes death of dopaminergic neurons by mitochondrial dysfunction with JNKs as central mediators. Here we provide novel insights into specific actions of JNK isoforms in 6-OHDA-induced death of PC12 cells. Twenty five μm 6-OHDA enhanced total JNK activity in the cytoplasm, nucleus, and at the mitochondria. Inhibition of JNKs by 2 μm SP600125 or transfection with dominant-negative JNK2 (dnJNK2) rescued more than 60% of the otherwise dying PC12 cells after 24 h, whereas transfection with dnJNK1 had no protective effects. In contrast to constitutively present JNK1, JNK2 amounts increased in the nucleus and at the mitochondria after 6-OHDA stimulation. JNK inhibition by SP600125 or transfection of dnJNK2 reduced the pool of active JNKs in the nucleus, the release of cytochrome c, as well as the cleavage of caspase-3 and its substrate poly(ADP-ribose) polymerase-1. Transfection with dnJNK1, however, had no effects on the translocation of JNKs to the mitochondria or the release of cytochrome c. Our data provide novel functional insights into the pathological role of individual JNK isoforms, the signalosome at the mitochondria, and the mode of JNK-induced release of cytochrome c.

The bottleneck of JNK signaling: molecular and functional characteristics of MKK4 and MKK7.

The functions of mitogen-activated protein kinases (MKKs) 4 and 7 are typically associated with the c-Jun N-terminal kinase (JNK) signaling pathway. Both MKKs synergistically phosphorylate different JNK isoforms and are therefore involved in numerous physiological (e.g. differentiation and proliferation) and pathological (e.g. apoptosis and tumorigenesis) processes. MKK4 and MKK7 share similar molecular characteristics as well as several upstream activators and scaffold proteins. However, their functions are non-redundant and determined by different stimuli, biochemical interactions and differential tissue distribution. The central question is how two MKKs regulate or affect the multiple actions of their JNK substrates. Similar to JNKs, MKK4 and MKK7 can simultaneously exert divergent functions in different cellular compartments and signalosomes. It is also important to realize that the MKK effects are splice variant-specific. The present review not only summarizes the various modes of MKK4 and MKK7 activation and activity, but also their functions. We also extensively describe their impact on JNK signaling, their molecular interactions resulting in the formation of context-specific signalosomes and the functional consequences of JNK deficiency.

The concerted signaling of ERK1/2 and JNKs is essential for PC12 cell neuritogenesis and converges at the level of target proteins

Mitogen-activated protein kinase (MAPK) pathways are central signaling elements, which translate and integrate stimuli from cell surface receptors into cytoplasmic and transcriptional responses. Here, we systematically compare the role of MAPKs in the nerve growth factor-induced long-term differentiation of PC12 cells and show the persistent nuclear and dose-dependent cytoplasmic activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the increasing nuclear and cytoplasmic activation of c-Jun N-terminal kinases (JNKs). Inhibition of ERK1/2 and JNKs significantly reduced neurite outgrowth. Both synergistically controlled the expression of c-Jun, the induction and/or phosphorylation of neurofilament, and the phosphorylation of Elk-1. JNKs alone were responsible for the phosphorylation of c-Jun and activating transcription factor 2 as well as for the expression of MAPK phosphatase 1. In contrast, p38alpha was only transiently activated and marginally involved in these processes. Thus, JNKs and ERK1/2 accomplish differentiation by signaling in parallel cascades that converge only at the target level.

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.

MEKK1 controls neurite regrowth after experimental injury by balancing ERK1/2 and JNK2 signaling.

After injury, peripheral neuronal cells initiate complex signaling cascades to promote survival and regeneration. In the present study, we have identified the mitogen-activated protein kinase (MAPK) isoforms which are necessary for nerve growth factor (NGF)-induced neurite regrowth after injury of differentiated PC12 cells. Extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the usually pro-apoptotic c-Jun N-terminal kinase 2 (JNK2) are crucial for neurite regrowth, while p38 plays no role in this context. Surprisingly, the MEK1 inhibitors PD 98059 and U 0126 blocked both ERK1/2 and JNK phosphorylation, indicating a novel form of balancing MAPK cascade cross-talk. Results from RNAi experiments excluded direct ERK/JNK interactions. We identified the upstream kinase MEKK1 as an activator of both the ERK1/2 and JNK2 pathways, whereby the ERK1/2 kinase MEK1 and the JNK kinase MKK7 bind to MEKK1 in a competing fashion. Our findings suggest an important role of JNK2 and MAPK pathway cross-talk in neurite regeneration.

Neurodegenerative and physiological actions of c-Jun N-terminal kinases in the mammalian brain.

The research in the field of AP-1 transcription factor expression, such as Jun or Fos proteins, in the brain was a milestone in neurosciences. The last years have provided growing insights into the upstream signal transduction which controls the expression and activation of these transcriptional regulators. In particular, the c-Jun N-terminal kinases (JNKs) were considered to confer degeneration by activation of c-Jun. Recent findings, however, demonstrate an essential physiological role of JNKs in the nervous system. Here we review the specific control and dual functions of JNK isoforms which are relevant for the development of the intact brain on the one hand, and which can confer dramatic neurodegenerative effects and microglial activation on the other hand.

c-Jun N-terminal kinases (JNKs) and the cytoskeleton: functions beyond neurodegeneration

The c‐Jun N‐terminal kinases (JNKs) are important mediators of neurodegeneration and their actions include the activation of genetic programs by phosphorylation of the nuclear transcription factor c‐Jun/AP‐1, the release of cytochrome c or the pro‐inflammatory actions of microglia. Recent data, however, provide evidence for physiological functions of JNKs in particular JNK1, and this involves a role of JNKs in the development of the brain and the (functional and/or structural) integrity of the cytoskeleton.