Neuronal Profilin Isoforms Are Addressed by Different Signalling Pathways

Profilins are prominent regulators of actin dynamics. While most mammalian cells express only one profilin, two isoforms, PFN1 and PFN2a are present in the CNS. To challenge the hypothesis that the expression of two profilin isoforms is linked to the complex shape of neurons and to the activity-dependent structural plasticity, we analysed how PFN1 and PFN2a respond to changes of neuronal activity. Simultaneous labelling of rodent embryonic neurons with isoform-specific monoclonal antibodies revealed both isoforms in the same synapse. Immunoelectron microscopy on brain sections demonstrated both profilins in synapses of the mature rodent cortex, hippocampus and cerebellum. Both isoforms were significantly more abundant in postsynaptic than in presynaptic structures. Immunofluorescence showed PFN2a associated with gephyrin clusters of the postsynaptic active zone in inhibitory synapses of embryonic neurons. When cultures were stimulated in order to change their activity level, active synapses that were identified by the uptake of synaptotagmin antibodies, displayed significantly higher amounts of both isoforms than non-stimulated controls. Specific inhibition of NMDA receptors by the antagonist APV in cultured rat hippocampal neurons resulted in a decrease of PFN2a but left PFN1 unaffected. Stimulation by the brain derived neurotrophic factor (BDNF), on the other hand, led to a significant increase in both synaptic PFN1 and PFN2a. Analogous results were obtained for neuronal nuclei: both isoforms were localized in the same nucleus, and their levels rose significantly in response to KCl stimulation, whereas BDNF caused here a higher increase in PFN1 than in PFN2a. Our results strongly support the notion of an isoform specific role for profilins as regulators of actin dynamics in different signalling pathways, in excitatory as well as in inhibitory synapses. Furthermore, they suggest a functional role for both profilins in neuronal nuclei

P65 controls NF-κB activity by regulating cellular localization of IκBβ

The nuclear factor κB (NF-κB) controls diverse cellular processes and is frequently misregulated in chronic immune diseases or cancer. The activity of NF-κB is regulated by inhibitory κB (IκB) proteins which control nuclear-cytoplasmic shuttling and DNA binding of NF-κB. Here, we describe a novel role of p65 as a critical regulator of the cellular localization and functions of NF-kB and its inhibitor IκBβ. In genetically modified p65-/- cells, the localization of ectopic p65 is not solely regulated by IκBα, but is largely dependent on the nuclear localization signal (NLS) and the nuclear export signal (NES) of p65. Furthermore, unlike IκBα, IκBβ does not contribute to nuclear export of p65. In fact, the cellular localization and degradation of IκBβ is controlled by the p65 specific NLS and NES. Our data also reveal that, in addition to stimulus-induced redistribution of NF-κB, changes in the constitutive localization of p65 and IκBβ specifically modulate activation of inflammatory genes. This is a consequence of differences in the DNA binding activity and signal responsiveness between the nuclear and cytoplasmic NF-κB/IκBβ complexes. Together, our findings indicate that the p65 subunit controls transcriptional competence of NF-κB by regulating the NF-κB/IκBβ pathway