Royal College of Surgeons in Ireland
Real-time imaging analysis and functional characterisation of tra.pdf (15.71 MB)

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

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posted on 2019-11-22, 17:49 authored by Robert F. Schwamborn

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.


Science Foundation Ireland


First Supervisor

Professor Jochen HM Prehn

Second Supervisor

Dr. Hans-Georg Konig


A theses submitted to the Royal College of Surgeons in Ireland In Fulfilment of the requirements for the Degree of Doctor of Philosophy March 2011.

Published Citation

Schwamborn RF. Real-time imaging analysis and functional characterisation of transcription factor NF-kappaB activation in a model of excitotoxic injury in rat neurons [PhD Thesis]. Dublin: Royal College of Surgeons in Ireland; 2011.

Degree Name

  • Doctor of Philosophy (PhD)

Date of award


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