Neuroprotective Effects of the Absence of JNK1 or JNK3 Isoforms on Kainic Acid-Induced Temporal Lobe Epilepsy-Like Symptoms.

The activation of c-Jun-N-terminal kinases (JNK) pathway has been largely associated with the pathogenesis and the neuronal death that occur in neurodegenerative diseases. Altogether, this justifies why JNKs have become a focus of screens for new therapeutic strategies. The aim of the present study was to identify the role of the different JNK isoforms (JNK1, JNK2, and JNK3) in apoptosis and inflammation after induction of brain damage. To address this aim, we induced excitotoxicity in wild-type and JNK knockout mice (jnk1 -/- , jnk2 -/- , and jnk3 -/- ) via an intraperitoneal injection of kainic acid, an agonist of glutamic-kainate-receptors, that induce status epilepticus.Each group of animals was divided into two treatments: a single intraperitoneal dose of saline solution, used as a control, and a single intraperitoneal dose (30 mg/kg) of kainic acid. Our results reported a significant decrease in neuronal degeneration in the hippocampus of jnk1 -/- and jnk3 -/- mice after kainic acid treatment, together with reduced or unaltered expression of several apoptotic genes compared to WT treated mice. In addition, both jnk1 -/- and jnk3 -/- mice exhibited a reduction in glial reactivity, as shown by the lower expression of inflammatory genes and a reduction of JNK phosphorylation. In addition, in jnk3 -/- mice, the c-Jun phosphorylation was also diminished.Collectively, these findings provide compelling evidence that the absence of JNK1 or JNK3 isoforms confers neuroprotection against neuronal damage induced by KA and evidence, for the first time, the implication of JNK1 in excitotoxicity. Accordingly, JNK1 and/or JNK3 are promising targets for the prevention of cell death and inflammation during epileptogenesis.

Excitatory transmission onto AgRP neurons is regulated by cJun NH2-terminal kinase 3 in response to metabolic stress.

The cJun NH2-terminal kinase (JNK) signaling pathway is implicated in the response to metabolic stress. Indeed, it is established that the ubiquitously expressed JNK1 and JNK2 isoforms regulate energy expenditure and insulin resistance. However, the role of the neuron-specific isoform JNK3 is unclear. Here we demonstrate that JNK3 deficiency causes hyperphagia selectively in high fat diet (HFD)-fed mice. JNK3 deficiency in neurons that express the leptin receptor LEPRb was sufficient to cause HFD-dependent hyperphagia. Studies of sub-groups of leptin-responsive neurons demonstrated that JNK3 deficiency in AgRP neurons, but not POMC neurons, was sufficient to cause the hyperphagic response. These effects of JNK3 deficiency were associated with enhanced excitatory signaling by AgRP neurons in HFD-fed mice. JNK3 therefore provides a mechanism that contributes to homeostatic regulation of energy balance in response to metabolic stress.

JNK inhibition of VMAT2 contributes to rotenone-induced oxidative stress and dopamine neuron death.

Treatment with rotenone, both in vitro and in vivo, is widely used to model dopamine neuron death in Parkinson's disease upon exposure to environmental neurotoxicants and pesticides. Mechanisms underlying rotenone neurotoxicity are still being defined. Our recent studies suggest that rotenone-induced dopamine neuron death involves microtubule destabilization, which leads to accumulation of cytosolic dopamine and consequently reactive oxygen species (ROS). Furthermore, the c-Jun N-terminal protein kinase (JNK) is required for rotenone-induced dopamine neuron death. Here we report that the neural specific JNK3 isoform of the JNKs, but not JNK1 or JNK2, is responsible for this neuron death in primary cultured dopamine neurons. Treatment with taxol, a microtubule stabilizing agent, attenuates rotenone-induced phosphorylation and presumably activation of JNK. This suggests that JNK is activated by microtubule destabilization upon rotenone exposure. Moreover, rotenone inhibits VMAT2 activity but not VMAT2 protein levels. Significantly, treatment with SP600125, a pharmacological inhibitor of JNKs, attenuates rotenone inhibition of VMAT2. Furthermore, decreased VMAT2 activity following in vitro incubation of recombinant JNK3 protein with purified mesencephalic synaptic vesicles suggests that JNK3 can inhibit VMAT2 activity. Together with our previous findings, these results suggest that rotenone induces dopamine neuron death through a series of sequential events including microtubule destabilization, JNK3 activation, VMAT2 inhibition, accumulation of cytosolic dopamine, and generation of ROS. Our data identify JNK3 as a novel regulator of VMAT2 activity.

JNK3 perpetuates metabolic stress induced by Aß peptides.

Although Aß peptides are causative agents in Alzheimer's disease (AD), the underlying mechanisms are still elusive. We report that Aß42 induces a translational block by activating AMPK, thereby inhibiting the mTOR pathway. This translational block leads to widespread ER stress, which activates JNK3. JNK3 in turn phosphorylates APP at T668, thereby facilitating its endocytosis and subsequent processing. In support, pharmacologically blocking translation results in a significant increase in Aß42 in a JNK3-dependent manner. Thus, JNK3 activation, which is increased in human AD cases and a familial AD (FAD) mouse model, is integral to perpetuating Aß42 production. Concomitantly, deletion of JNK3 from FAD mice results in a dramatic reduction in Aß42 levels and overall plaque loads and increased neuronal number and improved cognition. This reveals AD as a metabolic disease that is under tight control by JNK3.

c-Jun N-terminal kinase induces axonal degeneration and limits motor recovery after spinal cord injury in mice.

c-Jun N-terminal kinase (JNK) mediates neuronal death in response to stress and injury in the CNS and peripheral nervous system. Here, we show that JNK also regulates retrograde axonal degeneration (axonal dieback) after spinal cord injury (SCI) in mice. Activated phospho-JNK was highly expressed in damaged corticospinal tract (CST) axons after thoracic SCI by hemisection. Local administration of SP600125, a JNK inhibitor, prevented accumulation of amyloid-ß precursor protein and retraction of the severed CST axons as well as preserved the axonal arbors rostral to the injury site. The treatment with SP600125 also improved functional recovery of the hindlimbs, assessed by Basso mouse scale open-field scores and the grid-walking test. In Jnk1(-/-) and Jnk3(-/-) mice, we observed prevention of axonal degeneration and enhancement of motor recovery after SCI. These results indicate that both JNK1 and JNK3 induce axonal degeneration and limit motor recovery after SCI. Thus, a JNK inhibitor may be a suitable therapeutic agent for SCI.

Activity of all JNK isoforms contributes to neurite growth in spiral ganglion neurons.

Jun N-terminal kinase (JNK) is a multifunctional protein kinase crucial for neuronal apoptosis as well as neurite growth. We have previously shown that JNK activity is correlated with spiral ganglion neuron (SGN) apoptosis following hair cell loss in rats (Alam et al., 2007) implying that JNK inhibition may have therapeutic potential to protect SGNs in deaf individuals. Here we investigated the role of JNK in neurite outgrowth from cultured neonatal rat and mouse SGNs. We show that JNK is required for initial growth of neurites and for continued extension of already established neurites. The effect of JNK inhibition on neurite growth is rapid and is also rapidly reversible after washout of the inhibitor. Using phosphoJNK immunoreactivity as an indicator, we show that JNK is activated in growth cones within 30 min after transfer to medium lacking neurotrophic stimuli (5 K medium) but activation in the nucleus and soma requires hours. By transfecting epitope-tagged JNK1, JNK2, or JNK3 isoforms into SGNs, we found that all are present in the nucleus and cytoplasm and that there is no preferential redistribution to the nucleus after transfer to 5 K medium. Cotransfection of dominant-negative (dn) JNK1 and JNK2 into SGNs reduced neurite growth, although transfection of dnJNK1 or dnJNK2 alone had no significant effect. SGNs cultured from JNK3(-/-) mice showed reduced neurite growth that was further reduced by transfection of dnJNK1 and dnJNK2. This indicates that all three JNK isoforms promote SGN neurite growth although there may be functional redundancy between JNK1 and JNK2.

Gene expression profile in JNK3 null mice: a novel specific activation of the PI3K/AKT pathway.

JNK3 is mainly expressed in the CNS and it plays a crucial role in neuronal death in several neurodegenerative diseases. By contrast, the isoforms JNK1 and JNK2 seem to be involved in brain development. The lack of Jnk3 confers neuroprotection, although mechanisms responsible are unknown. The present study analyzes the gene expression profile in hippocampus from mice lacking Jnk3 in comparison to wild-type mice. The microarray analysis showed that 22 genes are differentially expressed (z-score>2 in two independent arrays) in Jnk3 null mice. Among these, we focused on pi3kcb, as it is directly related to the prosurvival phosphoinositide-3-kinase (PI3K)/AKT pathway. Results from Jnk3 null mice showed an increase in pik3cb transcript and protein, together with an increase in PI3K activity and phosphorylation of AKT. By contrast, these changes were not observed in Jnk1 null mice, which do not present neuroresistance to certain neurodegenerative insults. Therefore, our results indicate that the activation of PI3K/AKT pathway in hippocampus because of the increase in pik3cb transcription and that this mechanism is specifically related to the lack of Jnk3.

Hippocampal c-Jun-N-terminal kinases serve as negative regulators of associative learning.

In the adult mouse, signaling through c-Jun N-terminal kinases (JNKs) links exposure to acute stress to various physiological responses. Inflammatory cytokines, brain injury and ischemic insult, or exposure to psychological acute stressors induce activation of hippocampal JNKs. Here we report that exposure to acute stress caused activation of JNKs in the hippocampal CA1 and CA3 subfields, and impaired contextual fear conditioning. Conversely, intrahippocampal injection of JNKs inhibitors sp600125 (30 µm) or D-JNKI1 (8 µm) reduced activity of hippocampal JNKs and rescued stress-induced deficits in contextual fear. In addition, intrahippocampal administration of anisomycin (100 µg/µl), a potent JNKs activator, mimicked memory-impairing effects of stress on contextual fear. This anisomycin-induced amnesia was abolished after cotreatment with JNKs selective inhibitor sp600125 without affecting anisomycin's ability to effectively inhibit protein synthesis as measured by c-Fos immunoreactivity. We also demonstrated milder and transient activation of the JNKs pathway in the CA1 subfield of the hippocampus during contextual fear conditioning and an enhancement of contextual fear after pharmacological inhibition of JNKs under baseline conditions. Finally, using combined biochemical and transgenic approaches with mutant mice lacking different members of the JNK family (Jnk1, Jnk2, and Jnk3), we provided evidence that JNK2 and JNK3 are critically involved in stress-induced deficit of contextual fear, while JNK1 mainly regulates baseline learning in this behavioral task. Together, these results support the possibility that hippocampal JNKs serve as a critical molecular regulator in the formation of contextual fear.

Differences in activation of ERK1/2 and p38 kinase in Jnk3 null mice following KA treatment.

The MAPK family is formed by extracellular signal-regulated kinases p38 kinase and stress-activated protein kinases (SAPK/JNK). There are three genes that encode for three JNK proteins. JNK3 is mainly expressed in the central nervous system and has been related to various processes in that tissue. Specifically, JNK3 plays a crucial role in neuronal death in several neurodegenerative diseases. The activation of this kinase has been described in epilepsy, Alzheimer's disease, Parkinson's disease and Huntington's disease. Different studies have shown that the lack of the Jnk3 gene confers neuroprotection. However, the specific mechanism involved in such neuroprotection has not yet been elucidated. Therefore, in the present study, we analyzed the neuroprotection in mice lacking Jnk3 against neuronal death induced by kainic acid. Moreover, we analyzed the activation of different MAPKs. The results revealed that neuronal death was attenuated and different activation/inactivation of p38 and extracellular signal-regulated kinases 1/2 was reported with respect to control. Therefore, the data indicate that the lack of the JNK3 protein modulates other MAPKs and these changes could also have a pivotal role in neuroprotection.

JNK3 mediates paraquat- and rotenone-induced dopaminergic neuron death.

Mechanistic studies underlying dopaminergic neuron death may identify new drug targets for the treatment of Parkinson disease. Epidemiological studies have linked pesticide exposure to increased risk for sporadic Parkinson disease. Here, we investigated the role of c-Jun-N-terminal kinase 3 (JNK3), a neural-specific JNK isoform, in dopaminergic neuron death induced by the pesticides rotenone and paraquat. The role of JNK3 was evaluated using RNA silencing and gene deletion to block JNK3 signaling. Using an antibody that recognizes all isoforms of activated JNKs, we found that paraquat and rotenone stimulate JNK phosphorylation in primary cultured dopaminergic neurons. In cultured neurons transfected with Jnk3-specific siRNA and in neurons from Jnk3 mice, JNK phosphorylation was nearly abolished, suggesting that JNK3 is the main JNK isoform activated in dopaminergic neurons by these pesticides. Paraquat- and rotenone-induced death of dopaminergic neurons was also significantly reduced by Jnk3 siRNA or Jnk3 gene deletion, and deletion of the Jnk3 gene completely attenuated paraquat-induced dopaminergic neuron death and motor deficits in vivo. Our data identify JNK3 as a common and critical mediator of dopaminergic neuron death induced by paraquat and rotenone, suggesting that it is a potential drug target for Parkinson disease treatment.

JNK2 and JNK3 combined are essential for apoptosis in dopamine neurons of the substantia nigra, but are not required for axon degeneration.

Activation of c-jun N-terminal kinase (JNK) by the mitogen-activated protein kinase cascade has been shown to play an important role in the death of dopamine neurons of the substantia nigra, one of the principal neuronal populations affected in Parkinson's disease. However, it has remained unknown whether the JNK2 and JNK3 isoforms, either singly or in combination, are essential for apoptotic death, and, if so, the mechanisms involved. In addition, it has been unclear whether they play a role in axonal degeneration of these neurons in disease models. To address these issues we have examined the effect of single and double jnk2 and jnk3 null mutations on apoptosis in a highly destructive neurotoxin model, that induced by intrastriatal 6-hydroxydopamine. We find that homozygous jnk2/3 double null mutations result in a complete abrogation of apoptosis and a prolonged survival of the entire population of dopamine neurons. In spite of this complete protection at the cell soma level, there was no protection of axons. These studies provide a striking demonstration of the distinctiveness of the mechanisms that mediate cell soma and axon degeneration, and they illustrate the need to identify and target pathways of axon degeneration in the development of neuroprotective therapeutics.

Opposite regulation of oligodendrocyte apoptosis by JNK3 and Pin1 after spinal cord injury.

Although oligodendrocytes undergo apoptosis after spinal cord injury, molecular mechanisms responsible for their death have been unknown. We report that oligodendrocyte apoptosis is regulated oppositely by c-Jun N-terminal kinase 3 (JNK3) and protein interacting with the mitotic kinase, never in mitosis A I (Pin1), the actions of which converge on myeloid cell leukemia sequence-1 (Mcl-1). Activated after injury, JNK3 induces cytochrome c release by facilitating the degradation of Mcl-1, the stability of which is maintained in part by Pin1. Pin1 binds Mcl-1 at its constitutively phosphorylated site, Thr163Pro, and stabilizes it by inhibiting ubiquitination. After injury JNK3 phosphorylates Mcl-1 at Ser121Pro, facilitating the dissociation of Pin1 from Mcl-1. JNK3 thus induces Mcl-1 degradation by counteracting the protective binding of Pin1. These results are confirmed by the opposing phenotypes observed between JNK3-/- and Pin1-/- mice: oligodendrocyte apoptosis and cytochrome c release are reduced in JNK3-/- but elevated in Pin1-/- mice. This report thus unveils a mechanism by which cytochrome c release is under the opposite control of JNK3 and Pin1, regulators for which the activities are intricately coupled.

c-Jun N-terminal kinase 3 deficiency protects neurons from axotomy-induced death in vivo through mechanisms independent of c-Jun phosphorylation.

Both the transcription factor c-Jun and the c-Jun N-terminal kinases (JNKs) have been associated with neuronal loss in several death paradigms. JNK are key regulators of c-Jun and a common accepted model has been that JNKs mediate neuronal death through modulation of c-Jun activation. In the present study, we examined whether JNK2 and -3 (JNK members most associated with neuronal loss) deficiency can rescue neuronal loss caused by facial and sciatic nerve axotomy in the neonate in vivo. JNK2, JNK3, and JNK2/3 double-deficient neurons displayed significantly less death in the facial nerves of the CNS when compared with controls. JNK2 and JNK2/3 double-deficient animals also showed reduced c-Jun phosphorylation and induction following axotomy, consistent with the model that JNK acts to regulate death by activating c-Jun. Of significance, however, protection of facial nerves in JNK3-deficient animals was not accompanied by reduction in c-Jun activation. These results suggest that JNKs can mediate death independently of c-Jun. Importantly, the lack of correlation between JNK3 deficiency and c-Jun induction was not universal. In a sciatic axotomy model of neuronal injury in the neonate, death of DRG neurons was also reduced by JNK3 deficiency. However, in this case, c-Jun activation was also eliminated.