Modifications in AP2 and clathrin-mediated endocytosis as a mechanism of synaptic plasticity
by Göksemin Sengül
Date of Examination:2022-06-22
Date of issue:2022-07-01
Advisor:Prof. Dr. Peter Schu
Referee:Prof. Dr. Silvio O. Rizzoli
Referee:Prof. Dr. Martin Oppermann
Referee:Prof. Dr. Michael Thumm
Referee:Prof. Dr. Susanne Lutz
Referee:Dr. Alexander Stein
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EnglishAP2 CME fulfills essential functions in embryonic development and cell survival. The AP2 complex and clathrin form AP2 CCV together with sixty other coat proteins. Due to this complex protein network, the regulation of CCV life cycle still remains elusive. The tissue-specific AP1/σ1B complex mediates intracellular protein sorting and coordinates endolysosomal protein transport together with the ubiquitous AP1/σ1A complex. The AP1/σ1B complex knockout mice are viable and fertile, but they have severe learning-memory and motor coordination deficiencies. Hippocampal AP1/σ1B ko synapses showed impaired synaptic vesicle recycling and enhanced endolysosomal protein transport. These alterations in AP1/σ1B ko synapses stimulates protein endocytosis by canonical (canCCV) and by stable, longer-lived AP2 CCV as an indirect, secondary phenotype. In addition, the longer-lasting stable AP2 CCV of these ko synapses are even more stabilized than the corresponding wt stable AP2 CCV and thus, they are named as stCCV. The comparisons of the coat protein composition between wt and ko synaptic AP2 CCV classes revealed three molecular mechanisms, which enhance AP2 stCCV stability, thereby extending their lifetime. These regulatory mechanisms influence all three layers of a CCV: the outer clathrin-basket, the middle AP2 layer and the inner layer, the vesicle membrane phospholipid composition. Firstly, the ko stCCV contain only half the amount of the clathrin basket disassembly protein Hsc70 compared to wt stable CCV. Secondly, the AP2 membrane and cargo binding are hyperactivated via the phosphorylation of its μ2 adaptin by the AAK1 kinase, whose amount and activity are increased in stCCV compared to wt stable CCV. Thirdly, stCCV contain much less of the PI-4,5-P2 phosphatase Synaptojanin1 than wt stable CCV, whose activity is required for AP2 membrane dissociation. Firstly, we analyzed which protein(s) of the Synaptojanin1 CCV interactome determines its level in AP2 CCV. Our results show that the reduction of ITSN1 closely resembles the reduction of Synaptojanin1 levels in ko stCCV. Therefore, ITSN1 regulates the recruitment of Synaptojanin1 into stCCV. Additionally, the excess of Sgip1/AP2 complex over ITSN1 can efficiently compete with Synaptojanin1 for ITSN1 binding lowering Synaptojanin1 level even further. The ITSN1 AP2 CCV levels are controlled by Eps15 only, whereas its close homolog Eps15L1 is not involved. This was not expected because Eps15L1 is indispensable for neurodevelopment and can substitute the absence of Eps15, but not vice versa. Apparently, Eps15 has specific functions in selected protein transport routes. Pacsin1 is, like ITSN1, a member of the Synaptojanin1 CCV interactome. Its level in a stCCV is also reduced, but its reduction is counter-balanced by its higher phosphorylation and thus its higher activation level. This compensatory activation highlights the complexity of the mechanisms regulating the AP2 CME pathways. The mechanism controlling the CCV-associated Hsc70 levels and activity was analyzed next. The levels of the Hsc70 CCV co-chaperons, auxilin1 and GAK/auxilin2, its NEF Hsp110 and its co-worker Hsp90 are not changed in stCCV, in ways which would enable a CCV class specific regulation of Hsc70 levels and its uncoating activity. Hsc70 clathrin cage disassembly activity is regulated by differences in its phosphorylation patterns. All AP2 CCV from wt and ko synapses have two main Hsc70 pools, a hyper- and a hypo-phosphorylated pool, each of them contains Hsc70 proteins with different phosphorylation patterns. Only the Hsc70 homodimerization and activation of hypo-Pi Hsc70 proteins associated with the stCCV is inhibited by their binding of CaM/Ca2+ and most likely also by their binding of CaM. The regulation of Hsc70 and Pacsin1 activities via phosphorylation and the stimulation of AAK1 kinase activity led us to analyze the CCV class specific kinome. The amounts of DYRK1A, CaMK-IIδ, STK38L and SPAK(STK39)/CAB39 kinases are specifically reduced in ko stCCV, suggesting their involvement in the destabilization of AP2 CCV. Of all 12 investigated kinases, only AAK1 levels and its activity are specifically increased in ko stCCV, while being reduced in ko canCCV. Thus, AAK1 appears to be one of the master kinases regulating the AP2 CCV life cycle. Lastly, among the investigated neuronal cell adhesion proteins, only CHL1 and Neurocan are identified as novel, specific stCCV cargo proteins emphesizing particular functions of the AP2 stCCV pathway in synaptic plasticity. Overall, our findings will contribute to better understandings of diverse functions and mechanisms of AP2 CME pathways. Eventually, these data will provide more insights for developing treatments against neurological disorders caused by altered protein transport in endocytic routes.
Keywords: AAK1; AP1/σ1B ko mice; AP2; Calmodulin; clathrin-mediated endocytosis; CCV life cycle; Hsc70; ITSN1; kinases; NCAMs; stabilized CCV; synaptic platicity; Synaptojanin1; X-linked mental retardation disorder