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Tonic ATP-mediated growth suppression in peripheral nerve glia requires arrestin-PP2 and is evaded in NF1.

ABSTRACT: Normal Schwann cells (SCs) are quiescent in adult nerves, when ATP is released from the nerve in an activity dependent manner. We find that suppressing nerve activity in adult nerves causes SC to enter the cell cycle. In vitro, ATP activates the SC G-protein coupled receptor (GPCR) P2Y2. Downstream of P2Y2, β-arrestin-mediated signaling results in PP2-mediated de-phosphorylation of AKT, and PP2 activity is required for SC growth suppression. NF1 deficient SC show reduced growth suppression by ATP, and are resistant to the effects of β-arrestin-mediated signaling, including PP2-mediated de-phosphorylation of AKT. In patients with the disorder Neurofibromatosis type 1, NF1 mutant SCs proliferate and form SC tumors called neurofibromas. Elevating ATP levels in vivo reduced neurofibroma cell proliferation. Thus, the low proliferation characteristic of differentiated adult peripheral nerve may require ongoing, nerve activity-dependent, ATP. Additionally, we identify a mechanism through which NF1 SCs may evade growth suppression in nerve tumors.

SUBMITTER: Coover RA

PROVIDER: S-EPMC6251093 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Tonic ATP-mediated growth suppression in peripheral nerve glia requires arrestin-PP2 and is evaded in NF1.

Coover Robert A RA Healy Tabitha E TE Guo Li L Chaney Katherine E KE Hennigan Robert F RF Thomson Craig S CS Aschbacher-Smith Lindsey E LE Jankowski Michael P MP Ratner Nancy N

Acta neuropathologica communications 20181123 1

Normal Schwann cells (SCs) are quiescent in adult nerves, when ATP is released from the nerve in an activity dependent manner. We find that suppressing nerve activity in adult nerves causes SC to enter the cell cycle. In vitro, ATP activates the SC G-protein coupled receptor (GPCR) P2Y2. Downstream of P2Y2, β-arrestin-mediated signaling results in PP2-mediated de-phosphorylation of AKT, and PP2 activity is required for SC growth suppression. NF1 deficient SC show reduced growth suppression by AT ...[more]

PMID: 30470263

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Project description:PurposeThe G-protein coupled receptor (GPCR) Dopamine Receptor D4 (DRD4) plays an essential role in cAMP regulation and gap junctional coupling in the photoreceptors, where DRD4 expression is under circadian control. Previous in vitro transfection studies of human DRD4 desensitization have reported that DRD4 is not internalized upon dopamine stimulation when beta-arrestin is co-transfected with DRD4. We hypothesized that the visual arrestins, ARR1 and ARR4, play a modulatory role in DRD4 desensitization in the photoreceptors.MethodsTo test this hypothesis, immunohistochemistry analysis of mouse retinas was used to determine the cellular localization of beta-arrestins and DRD4 in photoreceptors. In vitro studies were performed in HEK293T cells transiently transfected with human DRD4 and arrestins. First, co-immunoprecipitation experiments were executed to test protein-protein interactions and to investigate the effect of dopamine stimulation. Second, immunohistochemistry analysis was implemented to study DRD4 internalization and translocation of ARR4.ResultsImmunohistochemistry studies of mouse retinas confirmed the expression of beta-arrestin 2, ARR1 and ARR4, as well as DRD4 in mouse cone photoreceptor inner segments. Co-immunoprecipitation experiments revealed a dopamine-dependent protein-protein interaction between human DRD4 and ARR4. In vitro internalization experiments showed that no detectable internalization of DRD4 was observed with any single arrestin co-transfected. However, a dopamine-dependent internalization of DRD4 was observed with three out of six sets of two arrestins co-transfected with DRD4. Each of these pairs of arrestins contained one visual arrestin and one beta-arrestin, and no internalization was observed with either two visual arrestins or two beta-arrestins. Additional time-course experiments revealed that in vitro, ARR4 translocates to co-localize with DRD4 at the plasma membrane in response to 30min of dopamine stimulation.ConclusionsThe results have functional implications and we hypothesize that the desensitization and internalization of DRD4 in photoreceptors are synergistically mediated by both visual and beta-arrestins. These results are additionally unique because they demonstrate for the first time that at least one G-protein coupled receptor, DRD4, requires two arrestins for desensitization and internalization, and opens up the possibility that other G-protein coupled receptors may require more than one arrestin for desensitization and/or internalization.

Project description:Communication between neuronal and glial cells is important for many brain functions. Astrocytes can modulate synaptic strength via Ca(2+)-stimulated release of various gliotransmitters, including glutamate and ATP. A physiological role of ATP release from astrocytes was suggested by its contribution to glial Ca(2+)-waves and purinergic modulation of neuronal activity and sleep homeostasis. The mechanisms underlying release of gliotransmitters remain uncertain, and exocytosis is the most intriguing and debated pathway. We investigated release of ATP from acutely dissociated cortical astrocytes using "sniff-cell" approach and demonstrated that release is vesicular in nature and can be triggered by elevation of intracellular Ca(2+) via metabotropic and ionotropic receptors or direct UV-uncaging. The exocytosis of ATP from neocortical astrocytes occurred in the millisecond time scale contrasting with much slower nonvesicular release of gliotransmitters via Best1 and TREK-1 channels, reported recently in hippocampus. Furthermore, we discovered that elevation of cytosolic Ca(2+) in cortical astrocytes triggered the release of ATP that directly activated quantal purinergic currents in the pyramidal neurons. The glia-driven burst of purinergic currents in neurons was followed by significant attenuation of both synaptic and tonic inhibition. The Ca(2+)-entry through the neuronal P2X purinoreceptors led to phosphorylation-dependent down-regulation of GABAA receptors. The negative purinergic modulation of postsynaptic GABA receptors was accompanied by small presynaptic enhancement of GABA release. Glia-driven purinergic modulation of inhibitory transmission was not observed in neurons when astrocytes expressed dn-SNARE to impair exocytosis. The astrocyte-driven purinergic currents and glia-driven modulation of GABA receptors were significantly reduced in the P2X4 KO mice. Our data provide a key evidence to support the physiological importance of exocytosis of ATP from astrocytes in the neocortex.

Dataset Information

Tonic ATP-mediated growth suppression in peripheral nerve glia requires arrestin-PP2 and is evaded in NF1.

Publications

Tonic ATP-mediated growth suppression in peripheral nerve glia requires arrestin-PP2 and is evaded in NF1.

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