Effect of miRNA-mediated knockdown of Myo16 gene on pre- and postsynaptic inhibitory synapses in hippocampal neurons

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http://urn.fi/URN:NBN:fi:hulib-202001291180
Title: Effect of miRNA-mediated knockdown of Myo16 gene on pre- and postsynaptic inhibitory synapses in hippocampal neurons
Author: Anastasiadou, Maria
Contributor: University of Helsinki, Faculty of Biological and Environmental Sciences
Publisher: Helsingin yliopisto
Date: 2019
Language: eng
URI: http://urn.fi/URN:NBN:fi:hulib-202001291180
http://hdl.handle.net/10138/310627
Thesis level: master's thesis
Degree program: Neurotieteiden maisteriohjelma
Master's Programme in Neuroscience
Magisterprogrammet i neurovetenskap
Specialisation: Neurotiede
Neuroscience
Neurovetenskap
Abstract: Tiivistelmä – Referat – Abstract Genetic variations within the MYO16 gene indicate a common predisposition to severe psychiatric, neurocognitive and neurodevelopmental disorders (NDD), as well as bipolar disorders (BD) and schizophrenia spectrum disorders (SSD). Myosin XVI’s ability to regulate actin and its involvement in cytoskeleton remodeling highlights the protein’s significance in neuronal circuitry development and signaling. Mutations in actin regulator protein-encoding genes like MYO16 have been found to shift cytoskeletal dynamics, as well as cause irregular dendritic spine and excitation/inhibition (E/I) synapse phenotypes. Interestingly, altered actin dynamics and E/I synapse dysregulation are two commonly detected molecular deficits associated with neuropathologies, namely autism spectrum disorders (ASD), SSD, and intellectual disability (ID). Therefore, synaptic E/I profiles are good candidates for investigating the neuropathologies they accompany, and also for revealing potential functional abnormalities. Hence, we determined that quantifying the levels of inhibitory synaptic proteins VGAT and gephyrin is the most suitable approach to investigate inhibitory synapse profiles and their relation to pathologies. Specifically, we investigated how microRNA (miRNA)-mediated myosin XVI protein knockdown (KD) affects pre- and postsynaptic inhibitory synapse density in rat primary hippocampal neurons. We achieved this by analyzing the density of VGAT and gephyrin puncta, signifying pre- and postsynaptic inhibitory synapses, respectively, and also by measuring their diameter to determine differences in inhibitory synapse size. Moreover, we quantified and assessed inhibitory synapse density and size differences between groups by comparing Myo16 KD-plasmid expressing hippocampal neurons to scrambled control cells. Common for both Myo16 KD plasmids was the active suppression of myosin XVI by 33%. However, Myo16 KD plasmids did not affect inhibitory synapse density and size to the same degree. Specifically, there was a significant reduction of inhibitory synapse density in the Myo16 KD3-plasmid expressing neurons, yet, no changes were observed in Myo16 KD5-plasmid expressing neurons. Finally, pre- and postsynaptic inhibitory synapse size differences were not significant between groups for either Myo16 KD plasmid when compared to scrambled control. Aberrant actin cytoskeleton remodeling, as well as altered E/I synapse ratios may lead to hyper/hypo-transmissive neuronal states or cause E/I imbalance, suggesting a complex relationship between actin regulator genes and inhibitory synapses. Our understanding behind their interplay is fairly limited, thus, gaining insight into the mechanisms associated with altered E/I balance remains the primary aim.
Subject: inhibitory synapses
excitation inhibition imbalance
WRC
WAVE complex
autism spectrum disorders
schizophrenia spectrum disorders


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