Rat Anti-Mouse Thy1 (CD90) Monoclonal antibody, clone T24/31 Functional Grade

A key component allowing a neuron to function properly within its dynamic environment is the axon initial segment (AIS), the site of action potential generation. In visual cortex, AIS of pyramidal neurons undergo periods of activity-dependent structural plasticity during development. However, it remains unknown how AIS morphology is organized during development for downstream cells in the visual pathway (retinal ganglion cells; RGCs) and whether AIS retain the ability to dynamically adjust to changes in network state. Here, we investigated the maturation of AIS in RGCs during mouse retinal development, and tested putative activity-dependent mechanisms by applying visual deprivation with a focus on the AIS-specific cisternal organelle (CO), a presumed Ca2+ -store. Whole-mount retinae from wildtype and Thy1-GFP transgenic mice were processed for multi-channel immunofluorescence using antibodies against AIS scaffolding proteins ankyrin-G, beta IV-spectrin and the CO marker synaptopodin (synpo). Confocal microscopy in combination with morphometrical analysis of AIS length and position as well as synpo cluster size was performed. Data indicated that a subset of RGC AIS contains synpo clusters and that these show significant dynamic regulation in size during development as well as after visual deprivation. Using super resolution microscopy, we addressed the subcellular localization of synpo in RGC axons. Similar to cortical neurons, RGCs show a periodic distribution of AIS scaffolding proteins. A previously reported scaffold-deficient nanodomain correlating with synpo localization is not evident in all RGC AIS. In summary, our work demonstrates a dynamic regulation of both the AIS and synpo in RGCs during retinal development and after visual deprivation, providing first evidence that the AIS and CO in RGCs can undergo structural plasticity in response to changes in network activity.

Mesangial proliferative glomerulonephritis (MsPGN) is an inflammatory disease, but both the nature of disease progression and its regulation remain unclear. In the present study, we monitored the course of anti-Thy1 nephritis from days 1 to 5 and established gene expression profiles at each time point using microarrays to explore the development of inflammation. According to the gene expression profiles, macrophage infiltration (triggered by CCL2 activation) was evident on day 1 and enhanced inflammation over the next few days. We screened for genes with expression levels similar to CCL2 and found that the upregulation of the circadian gene albumin D-site-binding protein (DBP) was involved in CCL2 activation in mesangial cells. More importantly, CCL2 expression showed oscillatory changes similar to DBP, and DBP induced peak CCL2 expression at 16: 00 a clock on day 1 in the anti-Thy1 nephritis model. We knocked down DBP through transfection with a small interfering RNA (siRNA) and used RNA sequencing to identify the DBP-regulated TNF-alpha-CCL2 pathway. We performed chromatin immunoprecipitation sequencing (ChIP-Seq) and the dual luciferase assay to show that DBP bound to the TRIM55 promoter, regulating gene expression and in turn controlling the TNF-alpha-CCL2 pathway. In conclusion, DBP-regulated circadian CCL2 expression by the TRIM55-TNF pathway in injured mesangial cells at an early stage, which promoted macrophage recruitment and in turn triggered infiltration and inflammation in a model of anti-Thy1 nephritis.