Involvement of phosphatidylinositol 4,5-bisphosphate in neurotransmission

Abstract

Phosphatidylinositol 4,5-bisphosphate (PI4,5P 2 ) is an important regulator in membrane trafficking and signal transductions. In this research, the role of PI4,5P 2 in neurotransmitter release from both dense-core granules (DCVs) and synaptic vesicles (SVs) was studied by using SLO-perforated synaptosomes. Antibody F ab fragments against PI4,5P 2 significantly inhibit both [ 3 H]-NA and [ 14 C]-glu release. Neomycin, a PI4,5P 2 blocking reagent, also inhibits release, supporting the antibody results. Blockage of phosphatidic acid production by 1-butanol inhibits both [ 3 H]-NA and [ 14 C]-glu release. These findings suggest that not only PI4,5P 2 , but also the synthesis of PI4,5P 2 , is required for both DCG and SV exocytosis. ADP-ribosylation factor6 has been shown to regulate chromaffin granule exocytosis through PLD activation. In this study, the role of ARF6 in neurotransmitter release from both DCG and SV is investigated. ARF6 in rat brain cortex is mainly membrane associated. Inhibition of ARF6 by a myristoylated N-terminal ARF6 peptide or by ARF6 antibody significantly decreased both [ 3 H]-Noradrenaline and [ 14 C]-glutamate release. Addition of PA and PIP2 partially restored release. These findings suggest that ARF6 has important regulatory role for both DCG and SV exocytosis, possibly through activating PLD and PIP-5K to regulate PIP 2 synthesis. The involvement of PI-3 kinase (PI3K) and Casein kinase 2 (CK2) in neurotransmission was also examined. LY294002, a PI3K and Ck2 inhibitor, and DRB, a specific CK2 inhibitor, do not affect [ 3 H]-Noradrenaline and [ 14 C]-glutamate release, suggesting that both kinases are not directly involved in DCG and SV exocytosis. LY294002 also increases spontaneous release from intact nerve endings, suggesting it acts on the plasma membrane, possibly through a K + channel. Beta-Amyloid peptide (A?) is a major component of Alzheimer's disease (AD) senile plaques. The effects of A? 42 on neurotransmitter release was studied. A? 42 increased both [ 3 H]-NA and [ 14 C]-glu release by activation of N-type Ca 2+ channels, without affecting reactive oxygen species formation, membrane perforation, and internal Ca 2+ store release in nerve endings. This supports the idea that elevations of A? could chronically increase excitatory neurotransmitter release to produce the neurotoxic effects that eventually lead to pathological lesions that characterize Alzheimer's Disease.