Dysregulation of miRNAs Levels in Glycogen Synthase Kinase-3β Overexpressing Mice and the Role of miR-221-5p in Synaptic Function

Highlights

• GSK-3β overactivity leads to dysregulation of miRNA network with a downregulation of miR-221-5p (miR-221*).

• Downregulation of miR-221* increases excitatory synaptic transmission in hippocampal neurons.

• We show a new mechanism by which GSK-3β and miRNAs might regulate synaptic function and therefore also synaptic plasticity.

Abstract

Glycogen synthase kinase-3β (GSK-3β) is a highly expressed kinase in the brain, where it has an important role in synaptic plasticity. Aberrant activity of GSK-3β leads to synaptic dysfunction which results in the development of several neuropsychiatric and neurological diseases. Notably, overexpression of constitutively active form of GSK-3β (GSK-3β[S9A]) in mice recapitulates the cognitive and structural defects characteristic for neurological and psychiatric disorders. However, the mechanisms by which GSK-3β regulates synaptic functions are not clearly known. Here, we investigate the effects of GSK-3β overactivity on neuronal miRNA expression in the mouse hippocampus. We found that GSK-3β overactivity downregulates miRNA network with a potent effect on miR-221-5p (miR-221*). Next, characterization of miR-221* function in primary hippocampal cell culture transfected by miR-221* inhibitor, showed no structural changes in dendritic spine shape and density. Using electrophysiological methods, we found that downregulation of miR-221* increases excitatory synaptic transmission in hippocampal neurons, probably via postsynaptic mechanisms. Thus, our data reveal potential mechanism by which GSK-3β and miRNAs might regulate synaptic function and therefore also synaptic plasticity.

Introduction

MicroRNAs (miRNAs) belong to small non-coding RNAs that play an essential role in modulating genes expression by blocking the translation of the target mRNAs and/or promoting their degradation. miRNAs are generated as a result of a multistep process by Polymerase II/III and then processed into ∼70-nt-long precursor miRNA (pre-miRNA) by Multiprocessor complex containing Drosha ribonuclease. Mature ∼20-nt-long miRNA duplex is formed via removal the terminal loop by Dicer enzyme. Single-stranded miRNA is recruited to the target messenger RNA (mRNA) at its 3′UTR (untranslated region) based on partial complementarity of sequences (Bartel, 2004). miRNAs are particularly abundant in the brain and show distinct expression profiles within brain regions and neuronal sub-populations (Sempere et al., 2004). Moreover, neuronal compartments such as axons, dendrites and the soma display localized expression of specific miRNAs that regulate local protein synthesis (Lugli et al., 2008, Natera-Naranjo et al., 2010, Sasaki et al., 2014, Xu et al., 2013). miRNAs are important in developing and mature brains (Cho et al., 2019, Saugstad, 2013). Brain-enriched miRNAs regulate fundamental processes such as cell fate and type determination; neuronal migration and cortical layering; neuronal polarization including axon development, dendritogenesis, synapse formation and dendritic spine maturation, and finally, neurotransmission (Kiltschewskij and Cairns, 2019). Consequently, miRNAs also play a role in learning and memory (Konopka et al., 2010).

GSK-3β was first discovered for its role in the regulation of glycogen metabolism (Embi et al., 1980, Rayasam et al., 2009), while further research has shown that this kinase is highly enriched in the brain (Pandey et al., 2009). A role of GSK-3β in brain function has been demonstrated for maturation, development, differentiation and functioning at multiple levels, ranging from a cell, to the brain structure level and the mature brain (Salcedo-Tello et al., 2011). Aberrant activity of GSK-3β leads to cognition and memory dysfunctions (Jaworski, 2020, Jaworski et al., 2019). Disturbed GSK-3β activity has been observed in several neurodegenerative and neuropsychiatric disorders, such as Alzheimer’s disease, Parkinson’s disease, schizophrenia and other affective disorder (Dewachter et al., 2009, Inkster et al., 2009, Levchenko et al., 2018). Furthermore, expression of distinct miRNAs are altered in GSK-3β-related diseases (Alural et al., 2017, Banach et al., 2017, Beveridge et al., 2008, Ding et al., 2016), thus suggesting a relationship between miRNAs and GSK-3β.

Emerging evidence indicates a potential role of GSK-3β in regulation of miRNA biogenesis (Fletcher et al., 2017, Guo et al., 2013, Ogórek et al., 2018, Tang et al., 2011). Importantly, nuclear localization of Drosha microprocessor complex, a critical component of miRNA biogenesis, depend on its phosphorylation in Ser300 and Ser 302 positions by GSK-3β (Tang et al., 2011). Thus, inhibition of GSK-3β activity leads to downregulation of Drosha activity reducing expression of several miRNAs, whereas, an increase in GSK-3β activity promotes miRNAs biogenesis (Fletcher et al., 2017, Ogórek et al., 2018).

Although previous studies suggested that GSK-3β regulates miRNA expression in non-neuronal cells, it is unknown whether the same phenomenon occurs in neuronal cells. Furthermore, it is unclear if the miRNAs regulated by GSK-3β regulate synaptic functions. Therefore, we investigated if GSK-3β overactivity may influence neuronal miRNA expression profile in hippocampus of GSK-3β[S9A] mice and studied the effects of miRNA modification on synaptic structure and function. First, we performed the analysis of miRNA expression profile using Next Generation Sequencing (NGS). Next, we confirmed downregulation of miR-221-5p (miR-221*) in hippocampus of GSK-3β[S9A] mice by qPCR. In the subsequent step, we found that the inhibition of miR-221* in hippocampal cell cultures had no detectable effects on spine morphology and density. Finally, we used electrophysiological experiments and showed that inhibition of miR-221* in hippocampal cell cultures influences the amplitude of miniature excitatory postsynaptic currents (mEPSCs). Thus, our study reveals that miR-221* plays a role in synaptic function by the regulation of excitatory synaptic transmission.