The role of miR-379-410 cluster-mediated PRR7 inhibition in homeostatic synaptic depression
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2022
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Doctoral Thesis
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Abstract
Homeostatic plasticity is a neuroprotective process that allows neurons and neural circuits to maintain stable activity in the face of destabilizing stimuli. This self-regulation is carried out through modulation of excitatory synapses at the neuronal level and excitation-inhibition balance at the circuit level. Many neuropsychiatric and neurodegenerative diseases exhibit abnormal excitation and inhibition, therefore suggesting an aberrancy in homeostatic plasticity and highlighting the importance of studying the molecular mechanisms governing this ability. In my thesis, I focused on Homeostatic Synaptic Depression (HSD), a type of homeostatic plasticity. HSD is characterized by dendritic spine elimination and decreased excitatory transmission in response to chronically elevated network activity in a cell-autonomous manner. Recent findings (Fiore et al., 2014; Cohen et al., 2011) suggested that members of the miRNA cluster miR379-410 are important regulators of HSD through inhibiting translation of synaptic proteins locally. Therefore, I sought to explore the role of cluster miRNAs in HSD in more detail. Here, I studied the synaptic protein Proline-rich protein 7 (Prr7), a candidate predicted to be a highly specific target of the miRNA cluster, with the aim of elucidating a novel molecular pathway underlying HSD.
Through qPCR, western blot, and immunostaining analyses, I found that Prr7 was downregulated at both RNA and protein levels in dendrites of hippocampal neurons treated with picrotoxin (PTX) for 48h, a stimulus that induces HSD. Furthermore, Prr7 knockdown led to a reduction in dendritic spine numbers to levels comparable to that induced by 48h PTX, as well as a downregulation of the GluA1 subunit of AMPA-type glutamate receptors. These findings suggested that Prr7 loss in dendrites is necessary and sufficient for spine elimination during HSD. Next, through luciferase assays and immunostaining, I found that Prr7 reduction by PTX was robustly prevented through transfection of a cocktail of inhibitors against cluster members miR-329-3p and miR-495-3p. The PTX-induced spine elimination was also prevented with this miRNA inhibitor cocktail. Moreover, through a dual sensor assay I found that both miRNAs are activated during HSD. These results indicated that miR-329-3p and miR-495-3p are induced and required for Prr7 downregulation and spine elimination by PTX. Finally, I found that Prr7 knockdown reduces expression of the synaptic scaffolding protein SPAR, an effect that was prevented via CDK5 inhibition, thereby tying Prr7 protein reduction together with a previously established HSD mechanism.
In summary, these results indicate a novel pathway wherein cluster members miR-329-3p and miR-495-3p target Prr7 locally, which leads to SPAR destabilization and spine elimination in HSD. Future experiments may be directed to explore the pathophysiological significance of the miR-329/495/Prr7 regulation in neuropsychiatric and neurodegenerative disorders, as well as seizure development and sleep disturbances often comorbid with these conditions.
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ETH Zurich
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homeostasis; synaptic plasticity; microRNA (miRNA); proline-rich protein 7; transmembrane adapter protein 3; AMPA receptor GluR1; spine-associated Rap GTPase-activating protein; Neuron; hippocampus
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09498 - Schratt, Gerhard / Schratt, Gerhard