Real-time imaging analysis and functional characterisation of transcription factor NF-kappaB activation in a model of excitotoxic injury in rat neurons.

Nuclear factor kappaB (NF-кB) is a transcription factor that regulates neuronal development, plasticity and survival. Increased NF-кB activation has been linked to excitotoxic injury that contributes to cerebral ischemia, seizure-induced injury and neurodegeneration. Interestingly, elevated NF-кB activity has been reported to exert both beneficial and detrimental effects on neuronal survival. Differences in pathway-specific functions of NF-кB, as well as differences in the contribution of individual NF-кB subunits, have been suggested to explain this discrepancy. In order to identify and characterise differential pathways of NF-KB activation, we used real-time imaging of NF-кB nuclear translocation. NF-кB subunit p65 was expressed with an eGFP tag (p65-eGFP) in cultured, rat hippocampal neurons. The co-expressed NF-кB inhibitor lкBa-Cerulean retained p65-eGFP in the cytoplasm of sham and non-treated neurons. Excitotoxic injury was induced by N-methyl-D-aspartate (NMDA, 100 µM NMDA and 10 µM glycine for 15-30 min). NMDA receptor stimulation was terminated by application of MK-801 (1 0 µM) and MgCI2 (1.2 mM). NMDA induced depolarisation of the mitochondrial membrane potential was visualised with tetramethyl rhodamine methyl ester (TMRM). Resultant cell death was determined by nuclear staining with propidium iodide (PI). In the canonical pathway of NF-кB activation, p65 translocates to the nucleus after signal-induced phosphorylation and subsequent degradation of IKBα. Interestingly, in our model, NMDA-induced nuclear translocation of p65- eGFP was preceded by nuclear translocation of IKBα -Cerulean, rather than a reduction of IKBα -Cerulean fluorescence intensity. Furthermore, coexpression of an IкBα -mutant inert to phosphorylation reduced nuclear translocation of p65-eGFP only partially. Additionally, knockdown of the NF-кB subunits p65 and c-Rel in cortical neurons attenuated NMDA-induced excitotoxicity. On the contrary, p65 knockdown reduced survival under physiological conditions. Our experimental results suggest that the detrimental effects of NF-кB in our model of excitotoxic injury are mediated by a non-canonical activation pathway. Previous work in our laboratory has revealed an intriguing clustering of activated IKKα/β and downstream elements of the NF-кB signalling cascade (S32,36-phosphorylated IкBα (p - IкBα)n d S536-phosphorylated p65) in the axon initial segment (AIS). Extraction of live neurons with a buffer containing Triton X-100 (1 %) and ca2+ (3 mM) resulted in the destruction of the microtubule network, which only remained intact in the AIS. Subsequent analyses revealed that immunoreactivity for p65 and p-IкBα was retained in this compartment after extraction. We designed a photo-activatable GFP-p65 fusion protein (p65-paGFP) to investigate the movement rates of p65 in different subcellular neuronal regions. Comparison with paGFP revealed significantly reduced mobility of p65-paGFP in the AIS and dendrites, but not the distal axon. Our findings support the idea that AISlocalised structures are involved in the regulation of neuronal NF-кB activity

Fibroblast growth factor homologous factor 1 interacts with NEMO to regulate NF-κB signaling in neurons.

Neuronal survival and plasticity critically depend on constitutive activity of the transcription factor nuclear factor-κB (NF-κB). We here describe a role for a small intracellular fibroblast growth factor homologue, the fibroblast growth factor homologous factor 1 (FHF1/FGF12), in the regulation of NF-κB activity in mature neurons. FHFs have previously been described to control neuronal excitability, and mutations in FHF isoforms give rise to a form of progressive spinocerebellar ataxia. Using a protein-array approach, we identified FHF1b as a novel interactor of the canonical NF-κB modulator IKKγ/NEMO. Co-immunoprecipitation, pull-down and GAL4-reporter experiments, as well as proximity ligation assays, confirmed the interaction of FHF1 and NEMO and demonstrated that a major site of interaction occurred within the axon initial segment. Fhf1 gene silencing strongly activated neuronal NF-κB activity and increased neurite lengths, branching patterns and spine counts in mature cortical neurons. The effects of FHF1 on neuronal NF-κB activity and morphology required the presence of NEMO. Our results imply that FHF1 negatively regulates the constitutive NF-κB activity in neurons

NRP/Optineurin Cooperates with TAX1BP1 to Potentiate the Activation of NF-κB by Human T-Lymphotropic Virus Type 1 Tax Protein

Nuclear factor (NF)-κB is a major survival pathway engaged by the Human T-Lymphotropic Virus type 1 (HTLV-1) Tax protein. Tax1 activation of NF-κB occurs predominantly in the cytoplasm, where Tax1 binds NF-κB Essential Modulator (NEMO/IKKγ) and triggers the activation of IκB kinases. Several independent studies have shown that Tax1-mediated NF-κB activation is dependent on Tax1 ubiquitination. Here, we identify by co-immunoprecipitation assays NEMO-Related Protein (NRP/Optineurin) as a binding partner for Tax1 in HTLV-1 infected and Tax1/NRP co-expressing cells. Immunofluorescence studies reveal that Tax1, NRP and NEMO colocalize in Golgi-associated structures. The interaction between Tax1 and NRP requires the ubiquitin-binding activity of NRP and the ubiquitination sites of Tax1. In addition, we observe that NRP increases the ubiquitination of Tax1 along with Tax1-dependent NF-κB signaling. Surprisingly, we find that in addition to Tax1, NRP interacts cooperatively with the Tax1 binding protein TAX1BP1, and that NRP and TAX1BP1 cooperate to modulate Tax1 ubiquitination and NF-κB activation. Our data strongly suggest for the first time that NRP is a critical adaptor that regulates the assembly of TAX1BP1 and post-translationally modified forms of Tax1, leading to sustained NF-κB activation

eIF4A2 drives repression of translation at initiation by Ccr4-Not through purine-rich motifs in the 5'UTR

Background: Regulation of the mRNA life cycle is central to gene expression control and determination of cell fate. miRNAs represent a critical mRNA regulatory mechanism, but despite decades of research, their mode of action is still not fully understood. Results: Here, we show that eIF4A2 is a major effector of the repressive miRNA pathway functioning via the Ccr4-Not complex. We demonstrate that while DDX6 interacts with Ccr4-Not, its effects in the mechanism are not as pronounced. Through its interaction with the Ccr4-Not complex, eIF4A2 represses mRNAs at translation initiation. We show evidence that native eIF4A2 has similar RNA selectivity to chemically inhibited eIF4A1. eIF4A2 exerts its repressive effect by binding purine-rich motifs which are enriched in the 5′UTR of target mRNAs directly upstream of the AUG start codon. Conclusions: Our data support a model whereby purine motifs towards the 3′ end of the 5′UTR are associated with increased ribosome occupancy and possible uORF activation upon eIF4A2 binding

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field