Emulsified silicone oil is taken up by and induces pro-inflammatory response in primary retinal microglia.

PURPOSE: Silicone oil is used as endotamponade in combination with vitrectomy. Thinning of retinal layers and loss of retinal cells under silicone oil use have been found. Here, we investigate the influence of silicone oil on primary microglia cells. METHODS: Primary microglia cells were prepared from the porcine retina. Microglia identity was assessed with Iba1 staining. Silicone oil was emulsified by sonification. Cell morphology and silicone oil uptake were evaluated by light microscopy after Coomassie blue staining. Cytokine secretion was evaluated with ELISA. Toxicity of silicone oil on microglia and toxic effect of silicone oil-treated microglia on neuronal cell line PC12 were evaluated by MTT or WST assay, respectively. RESULTS: Microglia took up silicone oil droplets after 72 h of incubation. Silicone oil induced no toxicity but increased the metabolism in microglial cells. In addition, the secretion of IL-6 and IL-8, but not of IL-1ß or TNF-α, was induced. Silicone oil-treated microglia did not exert any neurotoxic effect on differentiated PC12 cells but induced an increase in metabolism. CONCLUSION: Emulsified silicone oil changes the activity level of microglia and induces the secretion of IL-6 and IL-8. Neurotoxicity is not induced. Further experiments are required to investigate the long-term effect of silicone oil on microglia and their consequent effect on neuronal cells.

Retinoic acid-induced survival effects in SH-SY5Y neuroblastoma cells.

Neuroblastoma is a malignant childhood cancer arising from the embryonic sympathoadrenal lineage of the neural crest. Retinoic acid (RA) is included in the multimodal therapy of patients with high-risk neuroblastoma to eliminate minimal residual disease. However, the formation of RA-resistant cells substantially lowers 5-year overall survival rates. To examine mechanisms that lead to treatment failure, we chose human SH-SY5Y cells, which are known to tolerate incubation with RA by activating the survival kinases Akt and extracellular signal-regulated kinase 1/2. Characterization of downstream pathways showed that both kinases increased the phosphorylation of the ubiquitin ligase mouse double minute homolog 2 (Mdm2) and thereby enhanced p53 degradation. When p53 signaling was sustained by blocking complex formation with Mdm2 or enhancing c-Jun N-terminal kinase (JNK) activation, cell viability was significantly reduced. In addition, Akt-mediated phosphorylation of the cell-cycle regulator p21 stimulated complex formation with caspase-3, which also contributed to cell protection. Thus, treatment with RA augmented survival signaling and attenuated basal apoptotic pathways in SH-SY5Y cells, which increased cell viability.

Neurodegenerative effects of azithromycin in differentiated PC12 cells.

Azithromycin is a widely used macrolide antibiotic with sustained and high tissue penetration and intracellular accumulation. While short-term exposure to low-dose azithromycin is usually well tolerated, prolonged treatment can lead to unwanted neurological effects like paresthesia and hearing loss. However, the mechanism causing neurodegeneration is still unknown. Here, we show that even low therapeutically relevant azithromycin concentrations like 1µg/ml decreased cell viability by 15% and induced neurite loss of 47% after 96h in differentiated PC12 cells, which are a well-established model system for neuronal cells. When higher concentrations were used, the drug-induced effects occurred earlier and were more pronounced. Thereby, azithromycin altered tropomyosin-related kinase A (TrkA) signaling and attenuated protein kinase B (Akt) activity, which subsequently induced autophagy. Simultaneously, the antibiotic impaired lysosomal functions by blocking the autophagic flux, and this concurrence reduced cell viability. In good agreement with reversible effects observed in patients, PC12 cells could completely recover if azithromycin was removed after 24h. In addition, the detrimental effects of azithromycin were limited to differentiated cells, as confirmed in the human neuronal model cell line SH-SY5Y. Thus, azithromycin alters cell surface receptor signaling and autophagy in neuronal cells, but does not automatically induce irreversible damage when used in low concentrations and for a short time.

Crosstalk control and limits of physiological c-Jun N-terminal kinase activity for cell viability and neurite stability in differentiated PC12 cells.

The c-Jun N-terminal kinases (JNKs) are important mediators of cell viability and structural integrity in postmitotic neurons, which is required for maintaining synaptic connections and neural plasticity. In the present study, we chose differentiated PC12 cells as a well-characterised neuronal model system to selectively examine the regulation of basal JNK activity by extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt. We detected a complex interaction between the kinases to prevent cell death and neurite loss. Especially the appropriate level of JNK activation determined cellular survival. Basal activity of ERK1/2 attenuated the potentiation of JNK phosphorylation and thereby the induction of apoptosis. Importantly, when JNK activity was too low, cell viability and the number of neurite-bearing cells also decreased, even though the activation of ERK1/2 was enhanced. In this case, the JNK-mediated survival signals via activating transcription factor-3 (ATF3) were inhibited. Furthermore, the phosphorylation of ERK1/2 induced by the JNK inhibitor SP600125 inhibited the basal activity of Akt, which normally supported cell viability. Thus, controlling JNK activity is crucial to promote survival and neurite stability of differentiated neuronal cells.

miRNA-187-3p-Mediated Regulation of the KCNK10/TREK-2 Potassium Channel in a Rat Epilepsy Model.

Regulatory RNAs play a key role in the regulation of protein expression patterns in neurological diseases. Here we studied the regulation of miRNAs in a chronic rat model of temporal lobe epilepsy. The analysis was focused on a putative link with pharmacoresponsiveness as well as the functional implications of the regulation of a selected miRNA. The findings did not reveal a difference in hippocampal miRNA expression between phenobarbital responders and nonresponders. However, when comparing rats following status epilepticus with control rats we identified 13 differentially expressed miRNAs with miRNA-187-3p being most strongly regulated. mRNAs encoding KCNK10/TREK-2 as well as DYRK2 were confirmed as targets of miRNA-187-3p. Expression of the potassium channel protein KCNK10/TREK-2 negatively correlated with hippocampal miRNA-187-3p expression and proved to be upregulated in the chronic phase of the epilepsy model. In conclusion, our data do not suggest a relevant impact of miRNA expression patterns on pharmacoresponsiveness. However, we confirmed regulation of miRNA-187-3p and demonstrated that it impacts the expression of the two-pore domain potassium channel protein KCNK10/TREK-2. Considering evidence from brain ischemia models, KCNK10/TREK-2 upregulation might serve a protective function with a beneficial impact on astrocytic potassium and glutamate homeostasis.

Map2k4δ - identification and functional characterization of a novel Map2k4 splice variant.

Mitogen-activated protein kinase kinase 4 (Map2k4) is a dual specificity serin/threonine protein kinase that is unique among all MAP2Ks in activating two different subfamilies of mitogen-activated protein kinases, the c-Jun N-terminal kinases (JNKs) and p38 kinases. Map2k4 is essential during embryogenesis and involved in a variety of physiological and pathological processes. However, studies on its role in cancer development revealed partially conflicting data. In the present study, we report the identification of a novel splice variant of Map2k4, Map2k4δ, with an additional exon in front of the substrate binding D-domain. Map2k4δ is expressed together with Map2k4 in various tissues from rat, mouse and human. In PC12 cells, both splice variants control cell cycle progression and basal apoptosis by using different signaling pathways. If expression and activation of Map2k4 and Map2k4δ are at a certain, cell type-specific equilibrium, an appropriate cell growth is ensured. Overexpression of one kinase disrupts the intricate balance and either results in a highly proliferative or pro-apoptotic phenotype, partially reflecting the discrepancies in the literature on Map2k4 and its role in tumor development. Our findings contribute to the understanding of previous studies and point out that Map2k4 has not always a definite function, but rather triggers a cellular reaction in concert with other modulators.

The JNK inhibitor XG-102 protects against TNBS-induced colitis.

The c-Jun N-terminal kinase (JNK)-inhibiting peptide D-JNKI-1, syn. XG-102 was tested for its therapeutic potential in acute inflammatory bowel disease (IBD) in mice. Rectal instillation of the chemical irritant trinitrobenzene sulfonic acid (TNBS) provoked a dramatic acute inflammation in the colon of 7-9 weeks old mice. Coincident subcutaneous application of 100 µg/kg XG-102 significantly reduced the loss of body weight, rectal bleeding and diarrhoea. After 72 h, the end of the study, the colon was removed and immuno-histochemically analysed. XG-102 significantly reduced (i) pathological changes such as ulceration or crypt deformation, (ii) immune cell pathology such as infiltration and presence of CD3- and CD68-positive cells, (iii) the production of tumor necrosis factor (TNF)-α in colon tissue cultures from TNBS-treated mice, (iv) expression of Bim, Bax, FasL, p53, and activation of caspase 3, (v) complexation of JNK2 and Bim, and (vi) expression and activation of the JNK substrate and transcription factor c-Jun. A single application of subcutaneous XG-102 was at least as effective or even better depending on the outcome parameter as the daily oral application of sulfasalazine used for treatment of IBD.The successful and substantial reduction of the severe, TNBS-evoked intestinal damages and clinical symptoms render the JNK-inhibiting peptide XG-102 a powerful therapeutic principle of IBD.

Macin family of antimicrobial proteins combines antimicrobial and nerve repair activities.

The tertiary structures of theromacin and neuromacin confirmed the macin protein family as a self-contained family of antimicrobial proteins within the superfamily of scorpion toxin-like proteins. The macins, which also comprise hydramacin-1, are antimicrobially active against Gram-positive and Gram-negative bacteria. Despite high sequence identity, the three proteins showed distinct differences with respect to their biological activity. Neuromacin exhibited a significantly stronger capacity to permeabilize the cytoplasmic membrane of Bacillus megaterium than theromacin and hydramacin-1. Accordingly, it is the only macin that displays pore-forming activity and that was potently active against Staphylococcus aureus. Moreover, neuromacin and hydramacin-1 led to an aggregation of bacterial cells that was not observed with theromacin. Analysis of the molecular surface properties of macins allowed confirmation of the barnacle model as the mechanistic model for the aggregation effect. Besides being antimicrobially active, neuromacin and theromacin, in contrast to hydramacin-1, were able to enhance the repair of leech nerves ex vivo. Notably, all three macins enhanced the viability of murine neuroblastoma cells, extending their functional characteristics. As neuromacin appears to be both a functional and structural chimera of hydramacin-1 and theromacin, the putative structural correlate responsible for the nerve repair capacity in leech was located to a cluster of six amino acid residues using the sequence similarity of surface-exposed regions.

MKK7γ1 reverses nerve growth factor signals: proliferation and cell death instead of neuritogenesis and protection.

c-Jun N-terminal kinases (JNKs) are the exclusive downstream substrates of mitogen-activated protein kinase kinase 7 (MKK7). Recently, we have shown that a single MKK7 splice variant, MKK7γ1, substantially changes the functions of JNKs in naïve PC12 cells. Here we provide evidence that MKK7γ1 blocks NGF-mediated differentiation and sustains proliferation by interfering with the NGF-triggered differentiation programme at several levels: (i) down-regulation of the NGF receptors TrkA and p75; (ii) attenuation of the differentiation-promoting pathways ERK1/2 and AKT; (iii) increase of JNK1 and JNK2, especially the JNK2 54kDa splice variants; (iv) repression of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1), which normally supports NGF-mediated cell cycle arrest; (v) strong induction of the cell cycle promoter CyclinD1, and (vi) profound changes of p53 functions. Moreover, MKK7γ1 substantially changes the responsiveness to stress. Whereas NGF differentiation protects PC12 cells against taxol-induced apoptosis, MKK7γ1 triggers an escape from cell cycle arrest and renders transfected cells sensitive to taxol-induced death. This stress response completely differs from naïve PC12 cells, where MKK7γ1 protects against taxol-induced cell death. These novel aspects on the regulation of JNK signalling emphasise the importance of MKK7γ1 in its ability to reverse basic cellular programmes by simply using JNKs as effectors. Furthermore, our results highlight the necessity for the cells to balance the expression of JNK activators to ensure precise intracellular processes.

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.

Specific regulation of JNK signalling by the novel rat MKK7gamma1 isoform.

The c-Jun N-terminal kinases (JNKs) mediate a diversity of physiological and pathophysiological effects. Apart from isoform-specific JNK activation, upstream kinases are supposed to be the relevant regulators, which are involved in the context- and signalosome-depending functions. In the present study we report the cloning and characterization of the novel rat MKK7gamma1, a splice variant of MKK7 with an additional exon in the N-terminal region, in the neuronal pheochromocytoma cell line PC12. Transfected MKK7gamma1 increased basal JNK activity, in particular phosphorylation of JNK2. Consequently, JNK signalling was changed in mRNA-, protein- and activation-levels of JNK targets, such as transcription factors (c-Jun, p53, c-Myc), cell cycle regulators (p21, CyclinD1) and apoptotic proteins (Fas, Bim, Bcl-2, Bcl-xl). These alterations promote the sensitivity of MKK7gamma1-transfected cells towards cell death and repress cell proliferation under normal cell growth conditions. Complexes of JIP-1, MKK7 and JNK2 were the major JNK signalosomes under basal conditions. After stimulation with taxol (5muM) and tunicamycin (1.4mug/ml), MKK7gamma1- but not MKK7beta1-transfection, reduced cell death and even increased cell proliferation. Cellular stress also led to an increased phosphorylation of JNK1 and the almost complete abrogation of complexes of JIP-1, MKK7 and JNK2 in MKK7gamma1-transfected PC12 cells. Summarizing, MKK7gamma1 affects the function and activity of individual JNK isoforms and the formation of their signalosomes. This study demonstrates for the first time that one splice-variant of MKK7 tightly controls JNK signalling and effectively adapts JNK functions to the cellular context.

Deferoxamine mesylate is toxic for retinal pigment epithelium cells in vitro, and its toxicity is mediated by p38.

Deferoxamine mesylate is clinically used as a chelating agent but might induce retinopathy. To evaluate its effect on the retinal pigment epithelium (RPE), porcine RPE cells were stimulated with deferoxamine. Cell death was assessed with trypan blue exclusion assay. To investigate the pathway of cell death, the mitogen-activated protein kinases (MAPKs) Erk, JNK, and p38 were inhibited with U0126, SP600125, and SB203580, respectively. Their activity was determined by Western blot. Deferoxamine induces significant cell death in RPE cells, accompanied by phosphorylation of p38 and Erk. Inhibition of p38 attenuates cell death. In conclusion, deferoxamine is directly toxic on RPE cells, its toxicity depending on p38.

FK506 protects against various immune responses and secondary degeneration following cerebral ischemia.

The immunosuppressant FK506 (1 mg/kg, i.p.) reduces the infarct size following 90 min occlusion of the middle cerebral artery (MCAo) in adult rat brain. Here we have investigated the effect of FK506 on cerebral immune cells that are considered to contribute to neurodegeneration. FK506 substantially attenuated the response of resident and peripheral immune cells following transient ischemia. Between 24 hr and 5 days after MCAo, FK506 reduced the T-cell infiltration in the infarct area as well as the presence of activated and/or phagocytic OX-18, OX-42, GSA-IB4, Iba1, and ED1 positive microglia/macrophages. FK506 also lowered the protein levels of TNFalpha and IL-2 in ischemic brain areas. Repetitive application of FK506 over 20 days attenuated the activation of microglia in the substantia nigra (SN), an area of secondary degeneration. Importantly, FK506 conferred also lasting protection of the neurons of SN; these neurons degenerate by withdrawal of neurotrophic factors from the striatum that undergoes necrotic death as part of the ischemic core. To understand the molecular basis of FK506 effects in cerebral immune cells, we determined in primary postnatal day 0/1 (P0/P1) microglia (i) the expression of the FK506 binding proteins FKBP12, FKBP52, and FKPB65 and (ii) that FK506 (1-100 ng/mL) lowered the number of resting or lipopolysaccharide stimulated microglia as well as we induced the lipopolysaccharide release of TNFalpha in a dose-dependent manner. In summary, FK506 confers rescue of brain tissue following cerebral ischemia not only by neuronal protection, but also by suppression of microglial activation and peripheral immune responses.

Concurrent protective and destructive signaling of JNK2 in neuroblastoma cells.

Investigation of the c-Jun N-terminal kinases (JNKs) has mainly focused on their response to stress and their pro-apoptotic effects. In this regard, JNKs are crucial mediators of chemotherapy-induced killing of tumor cells. Importantly, however, JNKs also have physiological functions in cancer involving cell cycle regulation or oncogenesis. Hypothetically, the composition of JNK signalosomes determines the signaling outcome which,in turn, implies a multitude of different, sometimes opposing and interfering functions. In the present study,the well-characterized human neuroblastoma cell line SH-SY5Y served as a model system to separate physiological and pro-apoptotic JNK actions in the response to the cytoskeleton-interfering substances colchicine, cytochalasin D and taxol. Basically, JNKs mediated both cell death and proliferation. Using the chemical JNK inhibitor SP600125 as well as compartment-specific JNK-inhibiting constructs and dominant negative isoform mutants, we show that the nuclear subgroup of JNK2 is the dominant effector in colchicine and taxol-induced apoptosis, while cell cycle promotion is mediated by both cytoplasmic and nuclear JNK2.In contrast, cytochalasin D-triggered apoptosis is independent of JNK signaling. Interestingly, the data of the present study demonstrate for the first time that both cell protective (cell cycle progression) and destructive mechanisms (apoptosis) are simultaneously controlled by a single JNK isoform in the same cell system even under the influence of one stimulus. This has implications for the therapeutic application of JNK inhibitors and cytoskeleton-interfering substances in oncologic disorders.

c-Jun N-terminal kinases mediate Fas-induced neurite regeneration in PC12 cells.

In response to injury, peripheral neuronal cells initiate complex signalling cascades to promote survival and regeneration. In the present study, we used a model of experimental injury in the rat pheochromocytoma cell line PC12 to investigate receptor signals that lead to neurite outgrowth. Nerve growth factor (NGF) dose-dependently induced sprouting and the expression of the NGF receptors Trk tyrosine kinase receptor (TrkA) and p75 neurotrophin receptor (p75(NTR)) as well as Fas and Fas ligand. Neurite regeneration was decreased by chemical inhibition of TrkA, but not p75(NTR), and by the Fas inhibitor protein Fas-Fc. The mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinases (JNKs) were activated in response to NGF and both significantly contributed to neurite re-growth. Interestingly, otherwise apoptotic Fas ligation supported neuronal recovery exclusively via JNKs and promoted sprouting parallel to NGF. These findings suggest a novel signal integration from the NGF and Fas pathways in the JNK axis of MAPK signalling, where JNKs function as "physiological" mediators of normally apoptotic signals.

The bright side of JNKs-Multitalented mediators in neuronal sprouting, brain development and nerve fiber regeneration.

The c-Jun N-terminal kinases (JNKs) are important regulators of physiological and pathological processes in the central and peripheral nervous system. In general, JNKs are considered as mediators of neuronal degeneration in response to stress and injury. However, recent data have provided substantial evidence that JNKs are also essential for physiological and regenerative signalling in neurons. This review summarizes the importance of JNKs for neurite formation and outgrowth, brain development, dendritic architecture and regeneration of nerve fibers after injury. We discuss putative mechanisms which control the bipartite actions of individual JNK isoforms for neuronal death and repair after nerve fiber injury with a particular focus on the role of the transcription factor c-Jun.

Truncation of the CNS-expressed JNK3 in a patient with a severe developmental epileptic encephalopathy.

We have investigated the breakpoints in a male child with pharmacoresistant epileptic encephalopathy and a de novo balanced translocation t(Y;4)(q11.2;q21). By fluorescence in situ hybridisation, we have identified genomic clones from both chromosome 4 and chromosome Y that span the breakpoints. Precise mapping of the chromosome 4 breakpoint indicated that the c-Jun N-terminal kinase 3 (JNK3) gene is disrupted in the patient. This gene is predominantly expressed in the central nervous system, and it plays an established role in both neuronal differentiation and apoptosis. Expression studies in the patient lymphoblastoid cell line show that the truncated JNK3 protein is expressed, i.e. the disrupted transcript is not immediately subject to nonsense-mediated mRNA decay, as is often the case for truncated mRNAs or those harbouring premature termination codons. Over-expression studies with the mutant protein in various cell lines, including neural cells, indicate that both its solubility and cellular localisation differ from that of the wild-type JNK3. It is plausible, therefore, that the presence of the truncated JNK3 disrupts normal JNK3 signal transduction in neuronal cells. JNK3 is one of the downstream effectors of the GTPase-regulated MAP kinase cascade, several members of which have been implicated in cognitive function. In addition, two known JNK3-interacting proteins, beta-arrestin 2 and JIP3, play established roles in neurite outgrowth and neurological development. These interactions are likely affected by a truncated JNK3 protein, and thereby provide an explanation for the link between alterations in MAP kinase signal transduction and brain disorders.

Context-specific inhibition of JNKs: overcoming the dilemma of protection and damage.

The c-Jun N-terminal kinases (JNKs), which are essential regulators of physiological and pathological processes, are involved in several diseases including diabetes, atherosclerosis, stroke, and Parkinson's and Alzheimer's diseases. Inhibition of JNKs suppresses pathological features of these diseases but the many physiological functions of these enzymes argue against the use of sustained, systemic, nonspecific inhibition in the treatment of these diseases. For example, deletion of the gene that encodes JNK1 prevents insulin resistance but disrupts neuronal cytoarchitecture and initiates the pathology of Alzheimer's disease. Thus, it is not sufficient to inhibit selectively either JNKs or individual isoforms of JNK. Instead, the aim is to inhibit the damaging actions of JNK. This can be achieved using peptides that selectively block molecular domains of individual JNK signaling complexes (exclusively) that form under pathological conditions. To date, peptide inhibitors of JNK have been successful in protecting against ischemia-induced brain damage and insulin resistance following obesity. In this review, we discuss novel pharmacological strategies to inhibit JNK and the limitations of these strategies.

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.