Abstract
The neurotrophic factor neuregulin 1 (NRG1) regulates neuronal development, glial differentiation, and excitatory synapse maturation. NRG1 is synthesized as a membrane-anchored precursor and is then liberated by proteolytic processing or exocytosis. Mature NRG1 then binds to its receptors expressed by neighboring neurons or glial cells. However, the molecular mechanisms that govern this process in the nervous system are not defined in detail. Here we prepared neuron-enriched and glia-enriched cultures from embryonic rat neocortex to investigate the role of neurotransmitters that regulate the liberation/release of NRG1 from the membrane of neurons or glial cells. Using a two-site enzyme immunoassay to detect soluble NRG1, we show that, of various neurotransmitters, glutamate was the most potent inducer of NRG1 release in neuron-enriched cultures. NRG1 release in glia-enriched cultures was relatively limited. Furthermore, among glutamate receptor agonists, N-Methyl-D-Aspartate (NMDA) and kainate (KA), but not AMPA or tACPD, mimicked the effects of glutamate. Similar findings were acquired from analysis of the hippocampus of rats with KA-induced seizures. To evaluate the contribution of members of a disintegrin and metalloproteinase (ADAM) families to NRG1 release, we transfected primary cultures of neurons with cDNA vectors encoding NRG1 types I, II, or III precursors, each tagged with the alkaline phosphatase reporter. Analysis of alkaline phosphatase activity revealed that the NRG1 type II precursor was subjected to tumor necrosis factor-α-converting enzyme (TACE) / a Disintegrin And Metalloproteinase 17 (ADAM17) -dependent ectodomain shedding in a protein kinase C-dependent manner. These results suggest that glutamatergic neurotransmission positively regulates the ectodomain shedding of NRG1 type II precursors and liberates the active NRG1 domain in an activity-dependent manner.
Highlights
The inability of dopamine to induce neuregulin 1 (NRG1) release contrasts with its ability to induce epidermal growth factor (EGF) release from cultured striatal neurons, both factors belong to the same gene family and share the same protein structure [17]
Our significant findings are as follows: (1) Glutamate is a potent inducer of NRG1 release from cultured neocortical and hippocampal neurons; (2) NRG1 release from cultured glial cells is limited; (3) the activation of the NMDA receptor, kainite receptors, or both is involved in NRG1 release; (4) NRG1 release is inhibited by an MPP inhibitor but not by an exocytosis inhibitor; (5) among the membrane-anchored NRG1 precursors, we were able to confirm that the type II precursor is subjected to glutamate-induced ectodomain shedding; (6) and the shedding of the NRG1 type II precursor requires activation of the NMDA receptor followed by PKC activation and a Disintegrin And Metalloproteinase 17 (ADAM17)/ADAM9 enzyme activity
The present study indicates that the excitatory neurotransmitter glutamate is a potent regulator of NRG1 release and shedding from developing neocortical and hippocampal neurons
Summary
The neurotrophic factor neuregulin 1 (NRG1) is a member of the epidermal growth factor (EGF) family, which is widely distributed along with its receptors (ErbB3, ErbB4) in the central. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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