Specification of the NF-kappaB transcriptional response by p65 phosphorylation and TNF-induced nuclear translocation of IKK epsilon.

Here we investigated the regulation of NF-κB activity by post-translational modifications upon reconstitution of NF-κB p65-deficient cells with the wild-type protein or phosphorylation-defect mutants. Analysis of NF-κB target gene expression showed that p65 phosphorylations alone or in combination function to direct transcription in a highly target gene-specific fashion, a finding discussed here as the NF-κB barcode hypothesis. High-resolution microscopy and surface rendering revealed serine 536 phosphorylated p65 predominantly in the cytosol, while serine 468 phosphorylated p65 mainly localized in nuclear speckles. TNF stimulation resulted in the translocation of the cytosolic p65 kinase IKKε to the nucleus and also to promyelocytic leukemia (PML) nuclear bodies. This inducible IKKε translocation was dependent on p65 phosphorylation and was prevented by the oncogenic PML-RARα fusion protein. Chromatin immunoprecipitation experiments revealed the inducible association of IKKε to the control regions of several NF-κB target genes. In the nucleus, the kinase contributes to the expression of a subset of NF-κB-regulated genes, thus revealing a novel role of IKKε for the control of nuclear NF-κB activity.

AnkyrinG is required to maintain axo-dendritic polarity in vivo.

Neurons are highly polarized cells that extend a single axon and several dendrites. Studies with cultured neurons indicate that the proximal portion of the axon, denoted as the axon initial segment (AIS), maintains neuronal polarity in vitro. The membrane-adaptor protein ankyrinG (ankG) is an essential component of the AIS. To determine the relevance of ankG for neuronal polarity in vivo, we studied mice with a cerebellum-specific ankG deficiency. Strikingly, ankG-depleted axons develop protrusions closely resembling dendritic spines. Such axonal spines are enriched with postsynaptic proteins, including ProSAP1/Shank2 and ionotropic and metabotropic glutamate receptors. In addition, immunofluorescence indicated that axonal spines are contacted by presynaptic glutamatergic boutons. For further analysis, double mutants were obtained by crossbreeding ankG(-/-) mice with L7/Purkinje cell-specific promoter 2 (PCP2) mice expressing enhanced green fluorescent protein (EGFP) in Purkinje cells (PCs). This approach allowed precise confocal microscopic mapping of EGFP-positive spiny axons and their subsequent identification at the electron microscopic level. Ultrastructurally, axonal spines contained a typical postsynaptic density and established asymmetric excitatory synapses with presynaptic boutons containing synaptic vesicles. In the shaft of spiny axons, typical ultrastructural features of the AIS, including the membrane-associated dense undercoating and cytoplasmic bundles of microtubules, were absent. Finally, using time-lapse imaging of organotypic cerebellar slice cultures, we demonstrate that nonspiny PC axons of EGFP-positive/ankG(-/-) mice acquire a spiny phenotype within a time range of only 3 days. Collectively, these findings demonstrate that axons of ankG-deficient mice acquire hallmark features of dendrites. AnkG thus is important for maintaining appropriate axo-dendritic polarity in vivo.

Inhibitor kappaB Kinase beta deficiency in primary nociceptive neurons increases TRP channel sensitivity.

Inhibitor kappaB kinase (IKK) regulates the activity of the transcription factor nuclear factor-kappa B that normally protects neurons against excitotoxicity. Constitutively active IKK is enriched at axon initial segments and nodes of Ranvier (NR). We used mice with a Cre-loxP-mediated specific deletion of IKKbeta in sensory neurons of the dorsal root ganglion (SNS-IKKbeta(-/-)) to evaluate whether IKK plays a role in sensory neuron excitability and nociception. We observed increased sensitivity to mechanical, cold, noxious heat and chemical stimulation in SNS-IKKbeta(-/-) mice, with normal proprioceptive and motor functions as revealed by gait analysis. This was associated with increased calcium influx and increased inward currents in small- and medium-sized primary sensory neurons of SNS-IKKbeta(-/-) mice during stimulation with capsaicin or Formalin, specific activators of transient receptor potentials TRPV1 and TRPA1 calcium channels, respectively. In vitro stimulation of saphenous nerve preparations of SNS-IKKbeta(-/-) mice showed increased neuronal excitability of A- and C-fibers but unchanged A- and C-fiber conduction velocities, normal voltage-gated sodium channel currents, and normal accumulation of ankyrin G and the sodium channels Nav1.6 at NR. The results suggest that IKKbeta functions as a negative modulator of sensory neuron excitability, mediated at least in part by modulation of TRP channel sensitivity.

Ankyrin-B structurally defines terminal microdomains of peripheral somatosensory axons.

Axons are subdivided into functionally organized microdomains, which are required for generation and propagation of action potentials (APs). In the central nervous system (CNS), APs are generated near the soma in the axon initial segment (AIS) and propagated by nodes of Ranvier (noR). The crucial role of the membrane adapter proteins ankyrin-B and ankyrin-G as organizers of AIS and noR is now well established. By comparison, little is known on the localization and function of these proteins in sensory axon terminals of the peripheral nervous systems (PNS). Here, we tested the hypothesis that somatosensory PNS terminals are organized by distinct members of the ankyrin protein family. We discovered a specific distribution of ankyrin-B in somatosensory axon terminals of skin and muscle. Specifically, ankyrin-B was localized along the membrane of axons innervating Meissner corpuscles, Pacinian corpuscles and hair follicle receptors. Likewise, proprioceptive terminals of muscle spindles exhibited prominent ankyrin-B expression. Furthermore, ankyrin-B expression extended into nociceptive and thermoceptive intraepidermal nerve fibers. Interestingly, all studied somatosensory terminals were largely devoid of ankyrin-G, indicating that this scaffolding protein does not contribute to organization of mechanoelectric transduction zones in peripheral somatosensory neurons. Instead, we propose that ankyrin-B serves as a major membrane organizer in mechanoreceptive and nociceptive terminals of the PNS.