Neurotransmitter signaling in the pathophysiology of microglia

Erratun publicado en Frontiers in Cellular Neuroscience 7 : (2013) // Article ID 107

TWEAK/Fn14 Signaling Is Required for Liver Regeneration after Partial Hepatectomy in Mice

Background & Aims: Pro-inflammatory cytokines are important for liver regeneration after partial hepatectomy (PH). Expression of Fibroblast growth factor-inducible 14 (Fn14), the receptor for TNF-like weak inducer of apoptosis (TWEAK), is induced rapidly after PH and remains elevated throughout the period of peak hepatocyte replication. The role of Fn14 in post-PH liver regeneration is uncertain because Fn14 is expressed by liver progenitors and TWEAK-Fn14 interactions stimulate progenitor growth, but replication of mature hepatocytes is thought to drive liver regeneration after PH. Methods: To clarify the role of TWEAK-Fn14 after PH, we compared post-PH regenerative responses in wild type (WT) mice, Fn14 knockout (KO) mice, TWEAK KO mice, and WT mice treated with anti-TWEAK antibodies. Results: In WT mice, rare Fn14(+) cells localized with other progenitor markers in peri-portal areas before PH. PH rapidly increased proliferation of Fn14(+) cells; hepatocytic cells that expressed Fn14 and other progenitor markers, such as Lgr5, progressively accumulated from 12-8 h post-PH and then declined to baseline by 96 h. When TWEAK/Fn14 signaling was disrupted, progenitor accumulation, induction of pro-regenerative cytokines, hepatocyte and cholangiocyte proliferation, and over-all survival were inhibited, while post-PH liver damage and bilirubin levels were increased. TWEAK stimulated proliferation and increased Lgr5 expression in cultured liver progenitors, but had no effect on either parameter in cultured primary hepatocytes. Conclusions: TWEAK-FN14 signaling is necessary for the healthy adult liver to regenerate normally after acute partial hepatectomy.

RCeveierwamide and ceramide 1-phosphate in health and disease

Sphingolipids are essential components of cell membranes, and many of them regulate vital cell functions. In particular, ceramide plays crucial roles in cell signaling processes. Two major actions of ceramides are the promotion of cell cycle arrest and the induction of apoptosis. Phosphorylation of ceramide produces ceramide 1-phosphate (C1P), which has opposite effects to ceramide. C1P is mitogenic and has prosurvival properties. In addition, C1P is an important mediator of inflammatory responses, an action that takes place through stimulation of cytosolic phospholipase A2, and the subsequent release of arachidonic acid and prostaglandin formation. All of the former actions are thought to be mediated by intracellularly generated C1P. However, the recent observation that C1P stimulates macrophage chemotaxis implicates specific plasma membrane receptors that are coupled to Gi proteins. Hence, it can be concluded that C1P has dual actions in cells, as it can act as an intracellular second messenger to promote cell survival, or as an extracellular receptor agonist to stimulate cell migration.

Intracellular Ca2+ release through ryanodine receptors contributes to AMPA receptor-mediated mitochondrial dysfunction and ER stress in oligodendrocytes

Overactivation of ionotropic glutamate receptors in oligodendrocytes induces cytosolic Ca2+ overload and excitotoxic death, a process that contributes to demyelination and multiple sclerosis. Excitotoxic insults cause well-characterized mitochondrial alterations and endoplasmic reticulum (ER) dysfunction, which is not fully understood. In this study, we analyzed the contribution of ER-Ca2+ release through ryanodine receptors (RyRs) and inositol triphosphate receptors (IP(3)Rs) to excitotoxicity in oligodendrocytes in vitro. First, we observed that oligodendrocytes express all previously characterized RyRs and IP(3)Rs. Blockade of Ca2+-induced Ca2+ release by TMB-8 following alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated insults attenuated both oligodendrocyte death and cytosolic Ca2+ overload. In turn, RyR inhibition by ryanodine reduced as well the Ca2+ overload whereas IP3R inhibition was ineffective. Furthermore, AMPA-triggered mitochondrial membrane depolarization, oxidative stress and activation of caspase-3, which in all instances was diminished by RyR inhibition. In addition, we observed that AMPA induced an ER stress response as revealed by alpha subunit of the eukaryotic initiation factor 2 alpha phosphorylation, overexpression of GRP chaperones and RyR-dependent cleavage of caspase-12. Finally, attenuating ER stress with salubrinal protected oligodendrocytes from AMPA excitotoxicity. Together, these results show that Ca2+ release through RyRs contributes to cytosolic Ca2+ overload, mitochondrial dysfunction, ER stress and cell death following AMPA receptor-mediated excitotoxicity in oligodendrocytes. Cell Death and Disease (2010) 1, e54; doi:10.1038/cddis.2010.31; published online 15 July 2010

Functional analysis of cancer-associated EGFR mutants using a cellular assay with YFP-tagged EGFR intracellular domain

Background: The presence of EGFR kinase domain mutations in a subset of NSCLC patients correlates with the response to treatment with the EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Although most EGFR mutations detected are short deletions in exon 19 or the L858R point mutation in exon 21, more than 75 different EGFR kinase domain residues have been reported to be altered in NSCLC patients. The phenotypical consequences of different EGFR mutations may vary dramatically, but the majority of uncommon EGFR mutations have never been functionally evaluated. Results: We demonstrate that the relative kinase activity and erlotinib sensitivity of different EGFR mutants can be readily evaluated using transfection of an YFP-tagged fragment of the EGFR intracellular domain (YFP-EGFR-ICD), followed by immunofluorescence microscopy analysis. Using this assay, we show that the exon 20 insertions Ins770SVD and Ins774HV confer increased kinase activity, but no erlotinib sensitivity. We also show that, in contrast to the common L858R mutation, the uncommon exon 21 point mutations P848L and A859T appear to behave like functionally silent polymorphisms. Conclusion: The ability to rapidly obtain functional information on EGFR variants of unknown relevance using the YFP-EGFR-ICD assay might prove important in the future for the management of NSCLC patients bearing uncommon EGFR mutations. In addition, our assay may be used to determine the response of resistant EGFR mutants to novel second-generation TKIs.

CGP37157, an inhibitor of the mitochondrial Na+/Ca2+ exchanger, protects neurons from excitotoxicity by blocking voltage-gated Ca2+ channels

Inhibition of the mitochondrial Na+/Ca2+ exchanger (NCLX) by CGP37157 is protective in models of neuronal injury that involve disruption of intracellular Ca2+ homeostasis. However, the Ca2+ signaling pathways and stores underlying neuroprotection by that inhibitor are not well defined. In the present study, we analyzed how intracellular Ca2+ levels are modulated by CGP37157 (10 mu M) during NMDA insults in primary cultures of rat cortical neurons. We initially assessed the presence of NCLX in mitochondria of cultured neurons by immunolabeling, and subsequently, we analyzed the effects of CGP37157 on neuronal Ca2+ homeostasis using cameleon-based mitochondrial Ca2+ and cytosolic Ca2+ ([Ca2+](i)) live imaging. We observed that NCLX-driven mitochondrial Ca2+ exchange occurs in cortical neurons under basal conditions as CGP37157 induced a decrease in [Ca-2](i) concomitant with a Ca2+ accumulation inside the mitochondria. In turn, CGP37157 also inhibited mitochondrial Ca2+ efflux after the stimulation of acetylcholine receptors. In contrast, CGP37157 strongly prevented depolarization-induced [Ca2+](i) increase by blocking voltage-gated Ca2+ channels (VGCCs), whereas it did not induce depletion of ER Ca2+ stores. Moreover, mitochondrial Ca2+ overload was reduced as a consequence of diminished Ca2+ entry through VGCCs. The decrease in cytosolic and mitochondrial Ca2+ overload by CGP37157 resulted in a reduction of excitotoxic mitochondrial damage, characterized here by a reduction in mitochondrial membrane depolarization, oxidative stress and calpain activation. In summary, our results provide evidence that during excitotoxicity CGP37157 modulates cytosolic and mitochondrial Ca2+ dynamics that leads to attenuation of NMDA-induced mitochondrial dysfunction and neuronal cell death by blocking VGCCs.

TWEAK/Fn14 Signaling Is Required for Liver Regeneration after Partial Hepatectomy in Mice

Background & Aims: Pro-inflammatory cytokines are important for liver regeneration after partial hepatectomy (PH). Expression of Fibroblast growth factor-inducible 14 (Fn14), the receptor for TNF-like weak inducer of apoptosis (TWEAK), is induced rapidly after PH and remains elevated throughout the period of peak hepatocyte replication. The role of Fn14 in post-PH liver regeneration is uncertain because Fn14 is expressed by liver progenitors and TWEAK-Fn14 interactions stimulate progenitor growth, but replication of mature hepatocytes is thought to drive liver regeneration after PH. Methods: To clarify the role of TWEAK-Fn14 after PH, we compared post-PH regenerative responses in wild type (WT) mice, Fn14 knockout (KO) mice, TWEAK KO mice, and WT mice treated with anti-TWEAK antibodies. Results: In WT mice, rare Fn14(+) cells localized with other progenitor markers in peri-portal areas before PH. PH rapidly increased proliferation of Fn14(+) cells; hepatocytic cells that expressed Fn14 and other progenitor markers, such as Lgr5, progressively accumulated from 12-8 h post-PH and then declined to baseline by 96 h. When TWEAK/Fn14 signaling was disrupted, progenitor accumulation, induction of pro-regenerative cytokines, hepatocyte and cholangiocyte proliferation, and over-all survival were inhibited, while post-PH liver damage and bilirubin levels were increased. TWEAK stimulated proliferation and increased Lgr5 expression in cultured liver progenitors, but had no effect on either parameter in cultured primary hepatocytes. Conclusions: TWEAK-FN14 signaling is necessary for the healthy adult liver to regenerate normally after acute partial hepatectomy.