Single-particle tracking uncovers dynamics of glutamate-induced retrograde transport of NF-κB p65 in living neurons

Retrograde transport of NF-κB from the synapse to the nucleus in neurons is mediated by the dynein/dynactin motor complex and can be triggered by synaptic activation. The calibre of axons is highly variable ranging down to 100 nm, aggravating the investigation of transport processes in neurites of living neurons using conventional light microscopy. In this study we quantified for the first time the transport of the NF-κB subunit p65 using high-density single-particle tracking in combination with photoactivatable fluorescent proteins in living mouse hippocampal neurons. We detected an increase of the mean diffusion coefficient (Dmean) in neurites from 0.12 ± 0.05 µm2/s to 0.61 ± 0.03 µm2/s after stimulation with glutamate. We further observed that the relative amount of retrogradely transported p65 molecules is increased after stimulation. Glutamate treatment resulted in an increase of the mean retrograde velocity from 10.9 ± 1.9 to 15 ± 4.9 µm/s, whereas a velocity increase from 9 ± 1.3 to 14 ± 3 µm/s was observed for anterogradely transported p65. This study demonstrates for the first time that glutamate stimulation leads to an increased mobility of single NF-κB p65 molecules in neurites of living hippocampal neurons

Establishing a Large-Scale Field Experiment to Assess the Effects of Artificial Light at Night on Species and Food Webs

Artificial light at night (ALAN) is one of the most obvious hallmarks of human presence in an ecosystem. The rapidly increasing use of artificial light has fundamentally transformed nightscapes throughout most of the globe, although little is known about how ALAN impacts the biodiversity and food webs of illuminated ecosystems. We developed a large-scale experimental infrastructure to study the effects of ALAN on a light-naïve, natural riparian (i.e., terrestrial-aquatic) ecosystem. Twelve street lights (20 m apart) arranged in three rows parallel to an agricultural drainage ditch were installed on each of two sites located in a grassland ecosystem in northern Germany. A range of biotic, abiotic, and photometric data are collected regularly to study the short- and long-term effects of ALAN on behavior, species interactions, physiology, and species composition of communities. Here we describe the infrastructure setup and data collection methods, and characterize the study area including photometric measurements. None of the measured parameters differed significantly between sites in the period before illumination. Results of one short-term experiment, carried out with one site illuminated and the other acting as a control, demonstrate the attraction of ALAN by the immense and immediate increase of insect catches at the lit street lights. The experimental setup provides a unique platform for carrying out interdisciplinary research on sustainable lighting

Clinical and biological behaviour of vestibular schwannomas: signalling cascades involved in vestibular schwannoma resemble molecular and cellular mechanisms of injury-induced Schwann cell dedifferentiation.

Klenke C, Widera D, Sepehrnia A, et al. Clinical and biological behaviour of vestibular schwannomas: signalling cascades involved in vestibular schwannoma resemble molecular and cellular mechanisms of injury-induced Schwann cell dedifferentiation. Head & Neck Oncology. 2013;5(2):20

NOD-Like Receptor Signaling in Cholesteatoma

Background. Cholesteatoma is a destructive process of the middle ear resulting in erosion of the surrounding bony structures with consequent hearing loss, vestibular dysfunction, facial paralysis, or intracranial complications. The etiopathogenesis of cholesteatoma is controversial but is associated with recurrent ear infections. The role of intracellular innate immune receptors, the NOD-like receptors, and their associated signaling networks was investigated in cholesteatoma, since mutations in NOD-like receptor-related genes have been implicated in other chronic inflammatory disorders. Results. The expression of NOD2 mRNA and protein was significantly induced in cholesteatoma compared to the external auditory canal skin, mainly located in the epithelial layer of cholesteatoma. Microarray analysis showed significant upregulation for NOD2, not for NOD1, TLR2, or TLR4 in cholesteatoma. Moreover, regulation of genes in an interaction network of the NOD-adaptor molecule RIPK2 was detected. In addition to NOD2, NLRC4, and PYCARD, the downstream molecules IRAK1 and antiapoptotic regulator CFLAR showed significant upregulation, whereas SMAD3, a proapoptotic inducer, was significantly downregulated. Finally, altered regulation of inflammatory target genes of NOD signaling was detected. Conclusions. These results indicate that the interaction of innate immune signaling mediated by NLRs and their downstream target molecules is involved in the etiopathogenesis and growth of cholesteatoma

Hsc70 is a novel interactor of NF-kappaB p65 in living hippocampal neurons

Klenke C, Widera D, Engelen T, et al. Hsc70 is a novel interactor of NF-kappaB p65 in living hippocampal neurons. PLoS ONE. 2013;8(6): e65280.Signaling via NF-κB in neurons depends on complex formation with interactors such as dynein/dynactin motor complex and can be triggered by synaptic activation. However, so far a detailed interaction map for the neuronal NF-κB is missing. In this study we used mass spectrometry to identify novel interactors of NF-κB p65 within the brain. Hsc70 was identified as a novel neuronal interactor of NF-κB p65. In HEK293 cells, a direct physical interaction was shown by co-immunoprecipitation and verified via in situ proximity ligation in healthy rat neurons. Pharmacological blockade of Hsc70 by deoxyspergualin (DSG) strongly decreased nuclear translocation of NF-κB p65 and transcriptional activity shown by reporter gene assays in neurons after stimulation with glutamate. In addition, knock down of Hsc70 via siRNA significantly reduced neuronal NF-κB activity. Taken together these data provide evidence for Hsc70 as a novel neuronal interactor of NF-κB p65

1,8-Cineol Reduces Mucus-Production in a Novel Human <i>Ex Vivo</i> Model of Late Rhinosinusitis

<div><p>Inflammatory diseases of the respiratory system such as rhinosinusitis, chronic obstructive pulmonary disease, or bronchial asthma are strongly associated with overproduction and hypersecretion of mucus lining the epithelial airway surface. 1,8-cineol, the active ingredient of the pharmaceutical drug Soledum, is commonly applied for treating such inflammatory airway diseases. However, its potential effects on mucus overproduction still remain unclear.In the present study, we successfully established <i>ex vivo</i> cultures of human nasal turbinate slices to investigate the effects of 1,8-cineol on mucus hypersecretion in experimentally induced rhinosinusitis. The presence of acetyl-α-tubulin-positive cilia confirmed the integrity of the <i>ex vivo</i> cultured epithelium. Mucin-filled goblet cells were also detectable in nasal slice cultures, as revealed by Alcian Blue and Periodic acid-Schiff stainings. Treatment of nasal slice cultures with lipopolysaccharides mimicking bacterial infection as observed during late rhinosinusitis led to a significantly increased number of mucin-filled goblet cells. Notably, the number of mucin-filled goblet cells was found to be significantly decreased after co-treatment with 1,8-cineol. On a molecular level, real time PCR-analysis further showed 1,8-cineol to significantly reduce the expression levels of the mucin genes MUC2 and MUC19 in close association with significantly attenuated NF-κB-activity. In conclusion, we demonstrate for the first time a 1,8-cineol-dependent reduction of mucin-filled goblet cells and MUC2-gene expression associated with an attenuated NF-κB-activity in human nasal slice cultures. Our findings suggest that these effects partially account for the clinical benefits of 1,8-cineol-based therapy during rhinosinusitis. Therefore, topical application of 1,8-cineol may offer a novel therapeutic approach to reduce bacteria-induced mucus hypersecretion.</p></div

Cultured human nasal slices show unimpaired epithelium containing mucus-filled goblet cells.

<p><b>A,B</b>: Overview images of the established nasal slice culture system showing sliced nasal tissue cultured in culture plate inserts within a 12-well-plate. <b>C,D</b>: Immunohistochemical staining revealed the presence of acetyl-α-tubulin-positive cilia in nasal slice cultures. <b>E</b>: Hematoxylin and eosin-staining displayed the integrity of the <i>ex vivo</i> cultured epithelium containing ciliated epithelial cells (arrowheads), goblet cells (arrows) and a basal membrane (BM). Scale Bar: 20 μm. <b>F, G</b>: Mucin-filled goblet cells (arrows) were detected in cultivated nasal slices by Alcian Blue-staining and Periodic acid-Schiff stain. Scale Bar: 20 μm.</p

Schematic view on nasal epithelium containing mucus-filled goblet cells during LPS-induced rhinosinusitis and after co-treatment with 1,8-cineol.

<p>Co-treatment with LPS and 1,8-cineol leads to reduced production of mucin and decreased expression levels of mucin genes closely associated with attenuated NF-κB-activity.</p

Increased number of mucus-filled cells in LPS-treated nasal slice cultures is significantly reduced by co-treatment with 1,8-cineol.

<p><b>A</b>: Representative Alcian Blue-staining of an untreated nasal slice culture revealed no increased amount of mucus-filled goblet cells (arrows). <b>B</b>: Representative Alcian Blue-staining of LPS-treated nasal slices showed highly increased numbers of mucus-filled goblet cells (Arrows). <b>C</b>: Representative Alcian Blue-staining of cultured nasal slices co-treated with LPS and 1,8-cineol displayed a highly decreased number of mucus-filled goblet cells (Arrows). Scale Bar: 20 μm. <b>D</b>: Quantification of total areas of Alcian Blue-stained slice cultures from four independent donors revealed a significantly increased number of mucin-filled goblet cells in LPS-treated nasal slice cultures, which was significantly decreased after co-treatment with 1,8-cineol. *p < 0.5, **p < 0.01 were considered significant (t-test); ns: not significant (t-test).</p

Primer Sequences.

<p>Primer Sequences.</p