Project Code:
2022-SIDB-COUSIN
Background:
Synaptic transmission is dependent on the efficient and reliable coupling of neurotransmitter release to a spectrum of neuronal activity patterns. It is sustained at the presynapse by a series of discrete endocytosis modes from which new SVs are generated. During periods of high activity, the dominant endocytosis mode is activity-dependent bulk endocytosis (ADBE). This places ADBE at the heart of synaptic plasticity, since many events that modify both synaptic strength and stability are encoded by such activity patterns. We have identified a selective deficit in ADBE in a series of preclinical models of monogenic autism spectrum disorders (ASDs), suggesting a convergence in dysfunction around this endocytosis mode.
Rationale & hypothesis:
ArfGAP With GTPase Domain, Ankyrin Repeat And PH Domain 2 (AGAP2, as also known as PIKE or Centaurin-g1) is a candidate gene for ASD (SFARI gene score 2). It is modular protein with established postsynaptic roles, and is also linked to fragile X syndrome, sharing a series of interactions and signalling cascades with fragile mental retardation protein.
We have shown that AGAP2 is essential for ADBE at the presynapse. Furthermore, the ADBE protein kinase, glycogen synthase kinase 3, controls AGAP2 interactions with the essential ADBE adaptor molecule PI4KIIa. Finally, AGAP2 controls AP-1-dependent protein trafficking via the endolysosomal system, which we recently discovered is intimately integrated with ADBE. Therefore, we hypothesise that AGAP2 performs a key role in ADBE and that ADBE dysfunction is a key convergence point in ASDs.
Aims:
Training outcomes:
To achieve these aims the student will be trained in a series of optical (using genetically encoded reporters such as synaptopHluorins) and morphological assays (electron microscopy) to monitor presynaptic function (synaptic vesicle exocytosis, endocytosis, ADBE, protein cargo turnover) in primary neuronal hippocampal cultures of Agap2 knockout neurons. The majority of the work will be focussed on molecular replacement experiments, via the transient transfection of either structure / function mutants or AGAP2 variants identified in human disease. The student will also be trained in slice electrophysiology and basic molecular biology techniques.
