Regulation of actin polymerization is crucial for neuronal morphogenesis, in particular in neurite elongation and branching, in synaptogenesis and in synaptic plasticity. The heterogeneous nuclear ribonucleoprotein (hnRNP) K protein is an RNA-binding protein characterized by three K homology domains that mediate RNA-binding. hnRNP K is involved in a host of processes that comprise gene expression, such as chromatin remodeling, transcription, pre-mRNA splicing, mRNA export and translation. Moreover in heterologous cells hnRNP K regulates negatively N-WASP, a protein that interacts with Arp2/3 complex . The main aim of this project was the characterization of hnRNP K function in neurons, we analyzed its role on neuron morphology, subsequently on synaptic function. We found that hippocampal neurons overexpressing hnRNP K siRNA show normal spine morphology, but reduced spine density than control neurons, moreover the knockdown of hnRNP K increases the spine turnover. Furthermore we observed a significant decrease in PSD95 and GluA2 in silenced neurons while no effect was detected on VGAT and VGLUT expression; moreover the hnRNP K knockdown impairs the postsynaptic compartment in excitatory synapse, while the presynaptic compartment was not affected. Since the involvement of GluA2 subunit, we performed electrophysiological recordings in order to investigate functional changes in AMPARs physiology. Preliminary results show a dramatic decrease in excitatory, AMPARs mediated, miniature postsynaptic currents (mEPSCs) frequency in hnRNP K-silenced neurons. The importance of hnRNP K in synaptic activity was confirmed by its increase after chemical LTP induction; moreover the role of hnRNP K in LTP was also demonstrated by the impairment of this phenomena in silenced neurons. Now we are investigating the molecular mechanism underling these alterations studying the interaction with N-WASP. We demonstrated the directly interaction between hnRNP K and N-WASP in neurons by coimmunoprecipitation. The alterations observed in hnRNP K silenced neurons were described from a morphological and a functional perspective, evidencing a strong impairment of excitatory synaptic structures. Besides, our data suggest a strong link between this protein and actin dynamics, suggesting that these effects could eventually be consequent to the actin cytoskeleton rearrangements caused by hnRNP K loss.
HNRNP K: A NEW PROTEIN IN NEURON DEVELOPMENT AND FUNCTION / A.c. Folci ; coordinatore: E. Ginelli ; correlatore: D. Fornasari ; tutor M. Passafaro. - Milano : Università degli studi di Milano. Universita' degli Studi di Milano, 2012 Mar 02. ((24. ciclo, Anno Accademico 2011.
|Titolo:||HNRNP K: A NEW PROTEIN IN NEURON DEVELOPMENT AND FUNCTION|
|Supervisori e coordinatori interni:||GINELLI, ENRICO|
|Data di pubblicazione:||2-mar-2012|
|Parole Chiave:||RNA binding protein ; hnRNP K ; excitatory synapse ; long-term potentiation|
|Settore Scientifico Disciplinare:||Settore BIO/14 - Farmacologia|
|Citazione:||HNRNP K: A NEW PROTEIN IN NEURON DEVELOPMENT AND FUNCTION / A.c. Folci ; coordinatore: E. Ginelli ; correlatore: D. Fornasari ; tutor M. Passafaro. - Milano : Università degli studi di Milano. Universita' degli Studi di Milano, 2012 Mar 02. ((24. ciclo, Anno Accademico 2011.|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.13130/folci-alessandra-chiara_phd2012-03-02|
|Appare nelle tipologie:||Tesi di dottorato|