Institutional creator:

Instytut Medycyny Doświadczalnej i Klinicznej im. M. Mossakowskiego PAN

Contributor:

Gruszczyńska-Biegała, Joanna (Promotor)

Publisher:

Instytut Medycyny Doswiadczalnej i Klinicznej im. Miroslawa Mossakowskiego PAN

Place of publishing:

Warszawa

Degree name:

doktor

Level of degree:

2

Degree discipline :

nauki medyczne

Abstract:

According to the excitotoxicity concept, excessive glutamate release and impairment of the mechanisms responsible for its removal result in the accumulation of this neurotransmitter in synapses, leading to excessive activation of ionotropic glutamatergic receptors (mainly NMDAR) and an uncontrolled influx of Ca2+ into the cell. This in turn disrupts Ca2+ signalling, changes protein phosphorylation and gene expression, which leads to the degradation of neurons and contributes to the development of neurodegenerative diseases. Consequently, it is currently postulated that stimulation of the NMDAR endocytosis may reduce Ca2+ influx into the cell and thus have a neuroprotective effect on neurons. An example of proteins that could potentially regulate NMDAR endocytosis are STIM proteins, which act as Ca2+ sensors in the endoplasmic reticulum. Recent research has shown that STIM proteins directly bind with NMDARs and can reduce NMDAR-mediated Ca2+ influx. Therefore, this dissertation aimed to verify a research hypothesis regarding the role of STIM proteins in NMDAR endocytosis. The experiments were conducted on primary rat cortical neurons in vitro. The research was focused on the most abundant NMDARs in this area of the rat brain, i.e., receptors composed of GluN1 and GluN2A (GluN2A-NMDAR) and GluN1 and GluN2B (GluN2B-NMDAR). The experimental data collected during the study show that short-term, NMDAR overactivation (i) decreases the immunoreactivity of GluN1, GluN2A and GluN2B in the plasma membrane and synaptosomes, (ii) increases the colocalization of GluN1, GluN2A, and GluN2B with the marker of early endosomes (EEA1) and (iii) reduces the amplitude of NMDAR-mediated currents. This means that short-term, NMDAR overactivation leads to endocytosis of GluN2A-NMDAR and GluN2B-NMDAR. Moreover, it was shown that, unlike STIM1, STIM2 colocalizes and coimmunoprecipitates with GluN2A and GluN2B, which indicates direct interactions of STIM2 with NMDAR. More importantly, STIM2-GluN2B interactions increased after NMDAR endocytosis. Since STIM proteins reduce NMDAR-mediated Ca2+ influx and the interactions between STIM2 and NMDAR increase after receptor endocytosis, the second part of the study examined the impact of Stim1 and Stim2 silencing on this process. Firstly, it was shown that the reduction of STIM protein level does not affect the immunoreactivity of GluN1, GluN2A, and GluN2B in cell lysates. Then it was revealed that, as in wild-type neurons, short-term, NMDAR overactivation in neurons transduced with a control virus (scrRNA) seems to reduce the immunoreactivity of GluN1, GluN2A, and GluN2B in the plasma membrane and synaptosomes and causes a decrease in the amplitude of NMDAR-mediated current. Stim1 silencing, similar to scrRNA transduction, did not affect the immunoreactivity of GluN1, GluN2A, and GluN2B in the plasma membrane and synaptosomes or the amplitude of the NMDAR-mediated current. However, Stim2 silencing and short-term, NMDAR overactivation (i) did not decrease GluN1, GluN2A, and GluN2B immunoreactivity in the plasma membrane and synaptosomes and (ii) did not decrease the amplitude of the NMDAR-mediated current. These results may indicate that STIM2, but not STIM1, can regulate GluN2A-NMDAR and GluN2B-NMDAR endocytosis. To sum up, the results presented in this study show a new, previously unidentified role of the STIM2 protein, which is the regulation of NMDAR endocytosis after short-term, overactivation of this receptor. Since excessive NMDAR activation leads to neurotoxicity and neurodegeneration, the stimulation of NMDAR endocytosis may have a neuroprotective effect. Thus, further research in this area may contribute to the development of neuroprotective therapies for patients suffering from neurodegenerative diseases in the future.

Detailed Resource Type:

PhD Dissertations

Resource Identifier:

oai:rcin.org.pl:240803

Source:

IMDiK PAN, sygn. ZS 435

Language:

pol

Language of abstract:

eng

Rights:

Creative Commons Attribution BY 4.0 license

Terms of use:

Copyright-protected material. [CC BY 4.0] May be used within the scope specified in Creative Commons Attribution BY 4.0 license, full text available at: ; -

Digitizing institution:

Mossakowski Medical Research Institute PAS

Original in:

Library of the Mossakowski Medical Research Institute PAS

Access:

Open