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PPRP-1, a regulatory subunit for Protein Phosphatase 1(PP1) regulating the synaptic transmission.

par Stratigi, Aikaterini
Président du jury Brion, Jean Pierre
Promoteur Laurent, Patrick
Co-Promoteur Pandolfo, Massimo
Publication Non publié, 2021-08-26

Thèse de doctorat

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Résumé :

Mutations in genes regulating neurotransmission in the brain are implicated in neurological disorders and neurodegeneration. Presynaptically, neurotransmission is regulated by phosphorylation and dephosphorylation of key factors of synaptic function. Comparatively to kinases, little is known on phosphatases regulation. Protein Phosphatase 1 (PP1) signalling is a key player of synaptic plasticity: it controls synaptogenesis, Synaptic Vesicles(SV) endocytic machinery, actin cytoskeleton, receptor recycling, Long-Term-Potentiation(LTP) and Long-Term-Depression(LTD). As PP1 have poor affinity for their substrate by themselves, each of these multiple activities of PP1 likely involves specific regulatory subunits required to specify PP1 substrates. PhospHatase and ACTin Regulator (PHACTR) is a family of four regulatory subunits for PP1, forming a holoenzyme with PP1. PHACTR1 mutations are associated in humans with West syndrome, PHACTR4 with Hirshprung disease and PHACTR2 with Parkinson disease, but until now the molecular role of those proteins for neuronal regulation remain poorly understood. PHACTR4 was proposed to control local G-actin concentration when actin treadmilling is high, e.g. during cell migration.In this thesis I used C. elegans as a genetic model which bears a unique PHACTR ortholog (Protein Phosphatase Regulatory Protein-1/pprp-1). During development, PPRP-1 contributes to neuronal migration and to dendritic arborizations, but doesn’t play a major role in synaptogenesis. In adult, PPRP-1 act at the Neuromuscular Junction (NMJ) to regulate neurotransmission. We demonstrate that mutations reducing G-actin binding to PPRP-1 generate a constitutively active PPRP-1-PP1 holoenzyme. Using a pharmacological assay, we show that loss of PPRP-1, increases acetylcholine release, whereas constitutively active PPRP-1 decreases acetycholine release. PPRP-1 loss and constitutive activation had opposite effects in the amount and the localization of synapsin-1(SNN-1 in C. elegans) and intersectin (ITSN-1 in C. elegans) markers in the NMJ. Those two proteins are proposed to have a molecular link controlling neurotransmission through their phosphoregulation. Their dephosphorylation/phosphorylation cycles affect their formation of complexes with other proteins, changing their functions in an inactive or active synapse. Using electrophysiological and imaging techniques, we show that PPRP-1 influenced synaptic endocytosis during period of high activity. These results, together with genetic interactions, suggest that PPRP-1 might regulate the endocytic mode which is used at synapse to recycle the SVs. We propose that dephosphorylation of one or more still unknown substrates by PPRP-1-PP1 regulates the synaptic recycling pathways.