The serine/threonine kinase AKT switches between functional modes of the vesicle release machinery

Abstract

Neurotransmitter release is a fundamental process in the nervous system by which neurons are enabled to communicate with postsynaptic cells in a highly controlled manner. This communication is mediated by three types of neurotransmitter release: synchronous, asynchronous and spontaneous vesicle releases, of which each has its own functional importance. A misbalance of these forms of transmitter release is therefore likely associated with improper information processing and may hence lead to network malfunction or even psychiatric conditions. While the fundamental principles of synaptic neurotransmission have been well clarified, questions remain whether for example the different release modes are based on distinct identities of vesicular release machineries or whether they reflect different functional states of the same vesicle release machinery. In this thesis we approached these and related questions at the larval (neuromuscular junction) NMJ of Drosophila melanogaster. We found that the membrane-associated serine/threonine kinase AKT, a nodal part of the phosphatidylinositol-3-kinase (PI3K) signaling pathway, is required to maintain the release machineries of presynaptic vesicles in a tightly clamped and calcium-sensitive status. In this status vesicle release can be evoked synchronously or asynchronously by action potentials and associated calcium influxes. Lost or reduced AKT activity switches the release machinery into a calcium-insensitive and fusogenic status resulting in spontaneous vesicle release that is independent of action potentials or the presence of calcium. AKT-mediated switching of the functional status of vesicle release machineries is a rapid process that acts acutely on readily releasable vesicles and hence can dynamically influence synaptic communication. Mechanistically, we show that the calcium sensor Synaptotagmin 1 that is also part of the release machineriesʼ fusion clamp mediates the AKT effects and hence may be a direct target of AKT phosphorylation. We further show that the clustered and functionally intact voltage gated calcium channel Cacophony is required for the AKT interaction with the release machinery, suggesting that only docked and primed vesicles are accessible to and regulated by AKT. These results demonstrate that AKT is an essential direct regulator of the mode by which synaptic vesicles can be released. In chapter II, with an attempt to identify potential synaptic regulators of AKT we assessed whether the recently identified ATP-dependent on-off switch of AKT might play a role in the regulation of its synaptic function. We found that treatment with oligomycin resulted in enhanced spontaneous vesicle release that was similar to that elicited by AKT blockade. Short trains of nerve stimulation during oligomycin incubation triggered an instantaneous increase in the rate of spontaneous vesicle release whereas a similar stimulation without oligomycin was without effect. These data indicate that nerve stimulation strongly enhances the ATP consumption in nerve terminals resulting in ATP-depletion and perhaps in AKT-inactivation. To test this hypothesis we made use of an alternative ATP-depletion strategy that allowed us to make postsynaptic recordings during periods of intense nerve stimulation. We found that stimulation-induced ATP-depletion triggered a strong enhancement of spontaneous vesicle release that was indeed AKT- and PI3K-dependent, which was mediated by Synaptotagmin 1 and depended on the functional presence of the Cacophony. These results confirmed the functional existence of a Cacophony/AKT/release machinery complex and they suggest that AKT is regulated by ATP. They further suggest that AKT may serve at vesicle release sites as a local energy sensor that depending on the availability of ATP switches individual vesicle release machinery either into a tightly clamped mode for evoked release or at low ATP levels into a loosely clamped mode generating spontaneous release. This novel and evolutionarily conserved synaptic role of AKT could shed new light onto the pathogenesis of Schizophrenia or Autism Spectrum Disorders in which AKT activities seem to be reduced. It also needs to be considered in recent approaches to treat several forms of cancer with AKT-inhibitors. In parallel to the above work, I was involved in a collaborative project that aimed at establishing a three-dimensional computational model of the glutamatergic synapses of larval NMJs. Chapter III summarizes the experimental data that formed the basis of the computational model and showed that high frequency nerve stimulation leads to a disproportional decay of evoked EJP amplitudes due to limited vesicle supply. The model is described in the discussion.