JNK inhibition of VMAT2 contributes to rotenone-induced oxidative stress and dopamine neuron death.
ABSTRACT: 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.
Project description: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.
Project description:JNK is a protein kinase, which induces transactivation of c-jun. The three isoforms of JNK, JNK1, JNK2, and JNK3, are encoded by three distinct genes. JNK1 and JNK2 are expressed ubiquitously throughout the body. By contrast, the expression of JNK3 is limited and observed mainly in the brain, heart, and testes. Concerning the biological properties of JNKs, the contribution of upstream regulators and scaffold proteins plays an important role in the activation of JNKs. Since JNK signaling has been described as a form of stress-response signaling, the contribution of JNK3 to pathophysiological events, such as stress response or cell death including apoptosis, has been well studied. However, JNK3 also regulates the physiological functions of neurons and non-neuronal cells, such as development, regeneration, and differentiation/reprogramming. In this review, we shed light on the physiological functions of JNK3. In addition, we summarize recent advances in the knowledge regarding interactions between JNK3 and cellular reprogramming.
Project description:Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of dopamine-containing neurons, but the molecular pathways underlying its pathogenesis remain uncertain. Here, we show that by eliminating c-Jun N-terminal kinases (JNKs) we can prevent neurodegeneration and improve motor function in an animal model of PD. First, we found that c-Jun is activated in dopaminergic neurons from PD patients and in the 1-methyl-4-phenyl-1,2,4,6-tetrahydropyridine (MPTP) mouse model of PD. Examination of various JNK-deficient mice shows that both JNK2 and JNK3, but not JNK1, are required for MPTP-induced c-Jun activation and dopaminergic cell demise. Furthermore, we have identified cyclooxygenase (COX) 2 as a molecular target of JNK activation and demonstrated that COX-2 is indispensable for MPTP-induced dopaminergic cell death. Our data revealed that JNK2- and JNK3-induced COX-2 may be a principle pathway responsible for neurodegeneration in PD.
Project description:Parkinson's disease is characterized by selective and progressive loss of midbrain DAergic neurons (MDN) in the substantia nigra and degeneration of its nigrostriatal projections. Whereas the cellular pathophysiology has been closely linked to an activation of c-Jun N-terminal kinases (JNKs) and c-Jun, the involvement of JNKs in regenerative processes of the nigrostriatal pathway is controversially discussed. In our study, we utilized a mechanical scratch lesion paradigm of midbrain DAergic neurons in vitro and studied regenerative neuritic outgrowth. After a siRNA-mediated knockdown of each of the three JNK isoforms, we found that JNKs differentially regulate neurite regeneration. Knockdown of JNK3 resulted in the most prominent neurite outgrowth impairment. This effect was attenuated again by plasmid overexpression of JNK3. We also evaluated cell survival of the affected neurons at the scratch border. JNK3 was found to be also relevant for survival of MDN which were lesioned by the scratch. Our data suggest that JNK isoforms are involved in differential regulation of cell death and regeneration in MDN depending on their neurite integrity. JNK3 appears to be required for regeneration and survival in the case of an environment permissive for regeneration. Future therapeutic approaches for the DAergic system may thus require isoform specific targeting of these kinases.
Project description:Mutation of the Survival Motor Neuron 1 (SMN1) gene causes spinal muscular atrophy (SMA), an autosomal recessive neurodegenerative disorder that occurs in early childhood. Degeneration of spinal motor neurons caused by SMN deficiency results in progressive muscle atrophy and death in SMA. The molecular mechanism underlying neurodegeneration in SMA is unknown. No treatment is available to prevent neurodegeneration and reduce the burden of illness in SMA. We report that the c-Jun NH2-terminal kinase (JNK) signaling pathway mediates neurodegeneration in SMA. The neuron-specific isoform JNK3 is required for neuron degeneration caused by SMN deficiency. JNK3 deficiency reduces degeneration of cultured neurons caused by low levels of SMN. Genetic inhibition of JNK pathway in vivo by Jnk3 knockout results in amelioration of SMA phenotype. JNK3 deficiency prevents the loss of spinal cord motor neurons, reduces muscle degeneration, improves muscle fiber thickness and muscle growth, improves motor function and overall growth and increases lifespan of mice with SMA that shows a systemic rescue of phenotype by a SMN-independent mechanism. JNK3 represents a potential (non-SMN) therapeutic target for the treatment of SMA.
Project description:The cJun N-terminal kinases (JNKs) are encoded by three genes generating ten protein kinase polypeptides and are activated in settings of cell stress, mitogenesis, differentiation and morphogenesis. The specific role of the JNK family members in these diverse cell programmes is largely undefined. In this study, we tested the hypothesis that individual JNK isoforms would exhibit distinct patterns of regulation within cells. The cDNAs encoding five haemagglutinin (HA)-tagged JNK isoforms (p46JNK1alpha, p54JNK2alpha, p54JNK2beta, p46JNK3 and p54JNK3) were expressed in cultured rat PC12 phaeochromocytoma cells and human small-cell lung cancer (SCLC) cells by retrovirus-mediated gene transfer. In addition, HA-tagged forms of the dual-specificity mitogen-activated protein kinase kinases (MKKs), MKK4 and MKK7, which are specific activators of the JNK enzymes, were similarly expressed. Reverse transcription and PCR revealed that JNK3 is endogenously expressed in SCLC cells, but not in either chromaffin or neuronally differentiated PC12 cells. MKK4 and MKK7 were endogenously expressed in both PC12 cells and SHP77 cells. Immunoprecipitation and analysis of the JNKs expressed in SCLC cells revealed strong stimulation of all five JNK isoforms by UV radiation. Hypertonic stress, elicited by mannitol, also significantly stimulated these same JNKs, although the JNK3 isoforms were most strongly activated. In PC12 cell transfectants, however, selective and equal activation of p54JNK2alpha and p54JNK3 by UV and osmotic stress was observed, with little or no activation of JNK1alpha or JNK2beta. In contrast with the broad activation of the JNK enzymes by UV in SCLC cells, only HA-MKK4 was stimulated by UV exposure in these cells, whereas osmotic stress stimulated both HA-MKK4 and HA-MKK7. These findings indicate selective activation of JNK and MKK isoforms in a manner that is dependent upon the specific cell stress and the cell type.
Project description:Previous studies have reported that the regulatory function of the different c-Jun N-terminal kinases isoforms (JNK1, JNK2, and JNK3) play an essential role in neurological disorders, such as epilepsy and metabolic-cognitive alterations. Accordingly, JNKs have emerged as suitable therapeutic strategies. In fact, it has been demonstrated that some unspecific JNK inhibitors exert antidiabetic and neuroprotective effects, albeit they usually show high toxicity or lack therapeutic value. In this sense, natural specific JNK inhibitors, such as Licochalcone A, are promising candidates. Nonetheless, research on the understanding of the role of each of the JNKs remains mandatory in order to progress on the identification of new selective JNK isoform inhibitors. In the present review, a summary on the current gathered data on the role of JNKs in pathology is presented, as well as a discussion on their potential role in pathologies like epilepsy and metabolic-cognitive injury. Moreover, data on the effects of synthetic small molecule inhibitors that modulate JNK-dependent pathways in the brain and peripheral tissues is reviewed.
Project description:During the development of the sympathetic nervous system, the p75 neurotrophin receptor (p75NTR) has a dual function: promoting survival together with TrkA in response to NGF, but inducing cell death upon binding pro or mature brain-derived neurotrophic factor (BDNF). Apoptotic signaling through p75NTR requires activation of the stress kinase, JNK. However, the receptor also undergoes regulated proteolysis, first by a metalloprotease, and then by gamma-secretase, in response to pro-apoptotic ligands and this is necessary for receptor mediated neuronal death (Kenchappa, R. S., Zampieri, N., Chao, M. V., Barker, P. A., Teng, H. K., Hempstead, B. L., and Carter, B. D. (2006) Neuron 50, 219-232). Hence, the relationship between JNK activation and receptor proteolysis remains to be defined. Here, we report that JNK3 activation is necessary for p75NTR cleavage; however, following release of the intracellular domain, there is a secondary activation of JNK3 that is cleavage dependent. Receptor proteolysis and apoptosis were prevented in sympathetic neurons from jnk3(-/-) mice, while activation of JNK by ectopic expression of MEKK1 induced p75NTR cleavage and cell death. Proteolysis of the receptor was not detected until 6 h after BDNF treatment, suggesting that JNK3 promotes cleavage through a transcriptional mechanism. In support of this hypothesis, BDNF up-regulated tumor necrosis factor-alpha-converting enzyme (TACE)/ADAM17 mRNA and protein in wild-type, but not jnk3(-/-) sympathetic neurons. Down-regulation of TACE by RNA interference blocked BDNF-induced p75NTR cleavage and apoptosis, indicating that this metalloprotease is responsible for the initial processing of the receptor. Together, these results demonstrate that p75NTR-mediated activation of JNK3 is required for up-regulation of TACE, which promotes receptor proteolysis, leading to prolonged activation of JNK3 and subsequent apoptosis in sympathetic neurons.
Project description:The major components of the mitogen-activated protein kinase (MAPK) cascades are MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). Recent rapid progress in identifying members of MAPK cascades suggests that a number of such signaling pathways exist in cells. To date, however, how the specificity and efficiency of the MAPK cascades is maintained is poorly understood. Here, we have identified a novel mouse protein, termed Jun N-terminal protein kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), by a yeast two-hybrid screen, using JNK3 MAPK as the bait. Of the mammalian MAPKs tested (JNK1, JNK2, JNK3, ERK2, and p38alpha), JSAP1 preferentially coprecipitated with the JNKs in cotransfected COS-7 cells. JNK3 showed a higher binding affinity for JSAP1, compared with JNK1 and JNK2. In similar cotransfection studies, JSAP1 also interacted with SEK1 MAPKK and MEKK1 MAPKKK, which are involved in the JNK cascades. The regions of JSAP1 that bound JNK, SEK1, and MEKK1 were distinct from one another. JNK and MEKK1 also bound JSAP1 in vitro, suggesting that these interactions are direct. In contrast, only the activated form of SEK1 associated with JSAP1 in cotransfected COS-7 cells. The unstimulated SEK1 bound to MEKK1; thus, SEK1 might indirectly associate with JSAP1 through MEKK1. Although JSAP1 coprecipitated with MEK1 MAPKK and Raf-1 MAPKKK, and not MKK6 or MKK7 MAPKK, in cotransfected COS-7 cells, MEK1 and Raf-1 do not interfere with the binding of SEK1 and MEKK1 to JSAP1, respectively. Overexpression of full-length JSAP1 in COS-7 cells led to a considerable enhancement of JNK3 activation, and modest enhancement of JNK1 and JNK2 activation, by the MEKK1-SEK1 pathway. Deletion of the JNK- or MEKK1-binding regions resulted in a significant reduction in the enhancement of the JNK3 activation in COS-7 cells. These results suggest that JSAP1 functions as a scaffold protein in the JNK3 cascade. We also discuss a scaffolding role for JSAP1 in the JNK1 and JNK2 cascades.
Project description:The c-Jun N-terminal kinases (JNKs) are members of a larger group of serine/threonine (Ser/Thr) protein kinases from the mitogen-activated protein kinase family. JNKs were originally identified as stress-activated protein kinases in the livers of cycloheximide-challenged rats. Their subsequent purification, cloning, and naming as JNKs have emphasized their ability to phosphorylate and activate the transcription factor c-Jun. Studies of c-Jun and related transcription factor substrates have provided clues about both the preferred substrate phosphorylation sequences and additional docking domains recognized by JNK. There are now more than 50 proteins shown to be substrates for JNK. These include a range of nuclear substrates, including transcription factors and nuclear hormone receptors, heterogeneous nuclear ribonucleoprotein K, and the Pol I-specific transcription factor TIF-IA, which regulates ribosome synthesis. Many nonnuclear substrates have also been characterized, and these are involved in protein degradation (e.g., the E3 ligase Itch), signal transduction (e.g., adaptor and scaffold proteins and protein kinases), apoptotic cell death (e.g., mitochondrial Bcl2 family members), and cell movement (e.g., paxillin, DCX, microtubule-associated proteins, the stathmin family member SCG10, and the intermediate filament protein keratin 8). The range of JNK actions in the cell is therefore likely to be complex. Further characterization of the substrates of JNK should provide clearer explanations of the intracellular actions of the JNKs and may allow new avenues for targeting the JNK pathways with therapeutic agents downstream of JNK itself.