Meffert MK, Chang JM, Wiltgen BJ, Fanselow MS, Baltimore D.
NF-kappa B functions in synaptic signaling and behavior.
Nat Neurosci. 2003 Oct;6(10):1072-8.
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Meffert et al. have provided a fascinating report, but the data are not compelling. The most intriguing finding, that RelA-GFP diffuses in dendrites in a distal-->proximal direction, is represented by photomicrographs (and quantifications thereof) that show only the distal border of the photobleached area. More importantly, techniques applied to whole-culture lysates or extracts were performed with cultures that almost certainly contained large numbers of glia. We have documented in three publications that cultures of nearly pure cortical neurons do NOT show an induction of NF-κB DNA-binding activity in response to glutamate;(1-3) all the glutamate-evoked NF-κB activity detected by gel shifts can be attributed to glial contamination of cultures. The recalcitrant tendencies of neuronal NF-κB has since been confirmed by others (e.g., ref. 4). In addition to the gel shifts, luciferase reporter gene assays obviously would be confounded by glial contamination, as well.
Reply to comment by Steve Barger:
Activation of NF-κB by basal synaptic activity (as well as glutamate, depolarization, and bicuculline) was observed in high-density neuronal cultures maintained with defined media in the absence of serum.(1) Under these conditions, glial contribution to the culture is minimal; staining for markers of glia and neurons (GFAP and neurofilament, respectively) shows the cultures to be roughly 90-95 percent hippocampal neurons. In addition, NF-κB activation was observed in physiologically active preparations of synaptosomes, the isolated synaptic subcompartment of neurons (Fig.1 c-f). Under our stimulation conditions, activation of glial NF-κB by glutamate was not observed when glia were cultured separately (EMSA Fig.1b) or when they were co-cultured with neurons and examined by microscopy (GFPp65 translocation, Sup.Fig.1). Multiple differences exist between our experimental systems and those of Barger(2), including, but not limited to, duration and magnitude of stimulation, neuronal cell type, age of culture, and method of extract preparation. The activation of neuronal NF-κB by glutamate or glutamate analogs has also been observed by other researchers in a variety of systems (see, for example, refs 3-6).
Regarding our photobleaching experiments: Examining movement of the bleached border most proximal to the cell body initially sounds attractive. However, the difficulty with this approach is that fluorescence feeds into this bleached edge from unbleached dendrites branching off in the vicinity, making the edge indistinct and precluding quantification of border movement. By instead choosing to examine FRAP, we have been able to quantify GFPp65 movement (Fig.2a-d) and have used separate bleaching experiments to monitor nuclear accumulation of the GFPp65 from distal processes (Fig.2e,f).
1. Meffert MK, Chang JM, Wiltgen BJ, Fanselow MS, Baltimore D. NF-kappaB functions in synaptic signaling and behavior. Nat Neurosci. 2003 Oct;6(10):1072-8. Epub 2003 Aug 31. Abstract
2. Mao X, Moerman AM, Barger SW. Neuronal kappa B-binding factors consist of Sp1-related proteins. Functional implications for autoregulation of N-methyl-D-aspartate receptor-1 expression. J Biol Chem. 2002 Nov 22;277(47):44911-9. Epub 2002 Sep 18. Abstract
3. Kaltschmidt C, Kaltschmidt B, Baeuerle PA. Stimulation of ionotropic glutamate receptors activates transcription factor NF-kappa B in primary neurons. Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9618-22. Abstract
4. Cruise L, Ho LK, Veitch K, Fuller G, Morris BJ. Kainate receptors activate NF-kappaB via MAP kinase in striatal neurones. Neuroreport. 2000 Feb 7;11(2):395-8. Abstract
5. Lipsky RH, Xu K, Zhu D, Kelly C, Terhakopian A, Novelli A, Marini AM. Nuclear factor kappaB is a critical determinant in N-methyl-D-aspartate receptor-mediated neuroprotection. J Neurochem. 2001 Jul;78(2):254-64. Abstract
6. Pizzi M, Goffi F, Boroni F, Benarese M, Perkins SE, Liou HC, Spano P. Opposing roles for NF-kappa B/Rel factors p65 and c-Rel in the modulation of neuron survival elicited by glutamate and interleukin-1beta. J Biol Chem. 2002 Jun 7;277(23):20717-23. Epub 2002 Mar 23. Abstract
I am not certain if this is the proper venue for this discussion or how Dr. Baltimore feels about continuing this dialog. But, I appreciate this opportunity to tell our story—a tale for which it has been difficult to find a receptive audience. I would like to emphasize that my goal here is not to be confrontational but to uncover an explanation for the discrepancies; therefore, I simply want to explain our findings and how they contrast with the literature. I would also like to clarify that I take issue only with the whole-cell EMSAs of NF-κB in glutamate-treated neurons; activation of NF-κB in synaptosomes is not something we have analyzed. Still, synaptic activation of NF-κB would have to be considered irrelevant to nuclear transcription if one cannot demonstrate that its DNA-binding activity reaches the nucleus.
Let me simply reemphasize the fact that we do not see glutamate activation of NF-κB DNA binding in nuclear extracts made from nearly pure cultures of cerebral (i.e., neocortical or hippocampal) neurons. Furthermore, in our hands, glutamate does not activate a NF-κB-responsive reporter gene transfected into cerebral neurons. Failing to scour all our papers is forgivable for someone as busy as the president of a major university must be. Nevertheless, I feel compelled to respond that we did indeed perform experiments with glutamate applications (i.e., duration and magnitude) nearly identical to those used by Meffert et al. (One obvious difference was our omission of the cocktail of tetrodotoxin and glutamate receptor antagonists.) These data were reported in papers other than the one Dr. Baltimore cited. As regards the neuronal cell type, we have extensively tested hippocampal and neocortical neurons from rat and mouse. As for the age of the neurons, it is true that we established our cultures from fetuses (E18) rather than the neonates used by Meffert et al. We then allowed the neurons to mature in culture for approximately a week and a half before using them in experiments. By using fetuses, we can more efficiently limit astrocyte numbers by killing their progenitors while the numbers are still low (as the largest wave of astrogliogenesis occurs after E18). This approach also makes it possible to include the mitotic inhibitor only briefly, so that its potential inhibition of NF-κB (1) can be removed several days before the experiments. At the time of birth, astrocyte numbers are nearly as high as those of neurons, and both cell types survive quite well in the serum-free medium used by Meffert et al. Indeed, serum requirements for astrocyte proliferation in culture are generally overemphasized; clearly, astrocytes proliferate just fine in the absence of serum in vivo! Dr. Baltimore mentions GFAP staining to determine the glial contamination, a measure that would overlook non-astrocytic glia. Greg Brewer, a pioneer in culturing postnatal cerebrocortical cells, finds that adult rat brains maintained under conditions that appear similar to those used by Meffert et al. produce cultures that are five percent GFAP-positive yet still only 80 percent neurons (2). While I would be surprised if half of the glia died during the culture period, as Dr. Baltimore’s numbers suggest, I would not be surprised if all the NF-κB detected by EMSA came from the remaining glia, which he admits may have been as high as 10 percent. By comparison, our methods produce cultures that are less than one percent glia; omitting mitotic poisons results in nearly 30 percent astrocytes, even in serum-free medium.
With respect to the extraction protocol, it should be noted that our extraction and assay conditions are perfectly capable of detecting NF-κB in other cell types or in mixed neuron/glia co-cultures. It is impossible to determine what extraction procedure was used by Meffert et al.; at the time of this writing, the online supplemental material to which the reader is referred contains no information about extraction procedures or EMSA conditions.
Dr. Baltimore has mentioned the evidence for glutamate-evoked activation of neuronal NF-κB that was published by Kaltschmidt and others. In fact, the approaches used in those reports are dramatically different from those used by either Meffert et al. or ourselves. Namely, the other investigators either relied on immunocytochemically detected translocation of NF-κB proteins to the neuronal nucleus (Kaltschmidt, Cruise, Pizzi), a phenomenon that is not tantamount to DNA binding, or they used cerebellar granule cells (Lipsky, Pizzi), which do not show cerebral responses to excitatory amino acids.
So, it would seem that the controversy persists. In my opinion, it is difficult to ascribe biological significance to an effect that would be so finicky as to depend on tetrodotoxin or small differences in age or extraction procedures. But, of course, there is still a caveat to our data, as well. By removing the glia, we may have changed the intrinsic phenotype of the neurons so that they no longer respond to glutamate in the same way. However, we can at least be confident that such an effect of the glia cannot be attributed to diffusible paracrine factors, as we have tested co-cultures in which the glia are separated from neuronal contact only by a permeable membrane (3).
Regarding the movement of RelA-GFP into photobleached dendrites, Dr. Baltimore replies that “fluorescence feeds into this [proximal] bleached edge from unbleached dendrites branching off in the vicinity.” I appreciate this correction of the intial Alzforum News report stating that the authors “found that the green chimera diffused into the bleached section only from the distal, outer end of the dendrite.”