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Even though the molecular mechanisms of gene expression in neurons are quite broadly described in the literature, little is known about the relationship between these processes and the architecture of the neuronal cell nucleus. For example, it is firmly established that waves of gene expression occurring after neuronal stimulation comprise plenty of genes playing important roles in cognitive and epileptic phenomena. However, it has never been examined whether these bursts of transcriptional activity involve any regulation at the level of higher-order nuclear structure. Accordingly, we have performed studies on the structure of neuronal nucleus in epileptic animals. Based on the literature we chose the genes for BDNF and TRKB that play key roles in synaptic plasticity in the brain. We have investigated clustering of these genes in the nucleus by means of fluorescent in situ hybridization. We found that in hippocampal neurons the bdnf-trkb distance decreases 2 hours after seizure induction compared to control. Moreover, using bioinformatical approach we selected 2 linear clusters of the genes that are upregulated after seizures. One of them was selected on the chromosome 1 and the other one on chromosome 20. Those linear gene clusters appeared to associate 4 weeks after seizure compared to control. Our results suggest that upon epileptogenesis there is a reorganization of neuronal nucleus involving clustering of the genes in three-dimensional space. The phenomenon could occur also in other forms of synaptic plasticity, and should be a topic to follow up in the future.
CD44 is a multifunctional cell surface glycoprotein which regulates cell-cell and cell-matrix interactions in a variety of tissues. CD44 was implicated in the development of peripheral nerves, functioning as a coreceptor for ErbB class of growth factor receptors. However, it is not known whether CD44-ErbB interaction may occur at the adult peripheral synapses. Here we studied, using Fluorescence Lifetime Imaging Microscopy, the proximity between CD44 and ErbB3 at the rat neuromuscular junction (NMJ). This was performed in muscle sections co-immunostained for CD44 and ErbB3, using secondary antibodies coupled to Alexa488 and Alexa647 respectively. Neuromuscular junctions were visualized using Alexa555-bound α-BT. The FRET between Alexa488 (donor) and Alexa647 (acceptor) was judged by measuring an accompanying changes in the donor fl uorescence lifetime. We found that the mean fl uorescence lifetime of the donor fl uorophore labeling CD44 protein was considerably shorter over the NMJ than in nonsynaptic sites. Then we compared normal rat muscle to the muscle affected by denervation in the transgenic model of amyotrophic lateral sclerosis (ALS). Importantly, ALS-like neurodegeneration resulted in signifi cant increase in molecular proximity of CD44 and ErbB3 at the NMJ. The specifi c complex formation between the two proteins was confi rmed using immunoprecipitation analysis. Our study provides novel data on the molecular architecture of the neuromuscular synapse in both health and disease.
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