The studies in this revised proposal focus on understanding the regulation and physiological role of a diacylglycerol kinase (DGK), DGK-theta. The completion of the proposed studies will fill several important gaps in our current knowledge about the regulation and physiological role of this DGK. This is particularly significant for DGK-q as it is predominately localized to neurons and its regulation and physiological role are unknown. The overarching hypothesis is that DGK- theta is regulated by a polybasic region (PBR) - containing neuronal protein and this regulation mediates the modulation of glutamate release from neurons in the central nervous system. We further hypothesize that the regulation of glutamate release involves modulation of the synaptic vesicle cycle. This hypothesis is based on the following observations: (a) DGK- theta is regulated, (b) this regulation involves an accessory protein that contains a PBR, (c) the enzyme alters lipids known to be involved in synaptic vesicle release, (d) the enzyme has been implicated in modulating acetylcholine release, (e) suppression of DGK- theta attenuates induced release of neuronal glutamate, and (f) DGK- theta suppression results in an apparent slowing of synaptic vesicle cycling. The studies outlined in this proposal will test our hypothesis and examine the molecular mechanisms involved in the regulation of DGK- theta activity, and the modulation of glutamate release and synaptic vesicle cycling. This will be approached in three specific aims.
In specific aim I, we wil examine the regulation of DGK- theta by (a) quantifying the kinetic parameters of DGK-, theta in the presence and absence of activators, as well as (b) the effect of specific phospholipids on these parameters. We will then (c) delve further into the effect of phospholipids and protein activators on enzyme activity by determining whether activators affect the enzyme's intrinsic activity, membrane association, or both. We will also identify neuronal proteins that interact with and activate DGK- theta in specific aim II, we will determine DGK theta -'s role in modulating glutamate release from cortical and hippocampal neurons using in vitro and in situ studies with primary cultured neurons and brain slices from wild-type and DGK- theta knockout mice. We will also examine the effect of DGK- theta ablation on synaptic vesicle cycling.
In specific aim III, we will examine the mechanism by which DGK theta - modulates glutamate by evaluating the effect of DGK- theta ablation on the levels of lipids involved in synaptic vesicle cycling and calcium homeostasis. These studies will forge new mechanistic and physiological links between the regulation and function of neuronal DGK- theta.
The studies in this proposal will examine the regulation of a lipid metabolizing enzyme localized in neurons called diacylglycerol kinase theta. Our data indicates that the activity of this enzyme is regulated by a selective set of proteins. Further, ths enzyme regulates neurotransmitter (glutamate) release from neurons in the central nervous system (CNS) by modulating synaptic vesicle cycling. The studies outlined in this proposal will lead to a molecular understanding of the regulation and role of this enzyme, which will likely lead to the identification of new therapeutic targets for neuronal diseases that include dysregulation of neurotransmitter release in the CNS.