Anatomical localization of protease-activated receptor-1 and protease-mediated neuroglilal crosstalk on peri-synaptic astrocytic endfeet

Authors

  • Efrat Shavit,

    1. Department of Physiology and Pharmacology, Tel Aviv University, Israel
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  • Daniel M. Michaelson,

    1. Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Israel
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  • Joab Chapman

    1. Department of Physiology and Pharmacology, Tel Aviv University, Israel
    2. Department of Neurology, Sagol Neuroscince Center, Chaim Sheba Medical Center, Israel
    3. Department of Neurology, Sackler Faculty of Medicine, Tel Aviv University, Israel
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Address correspondence and reprint requests to Prof. Joab Chapman, Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv 69978, Israel. E-mail: jchapman@post.tau.ac.il

Abstract

J. Neurochem. (2011) 119, 460–473.

Abstract

We studied the localization, activation and function of protease-activated receptor 1 (PAR-1) at the CNS synapse utilizing rat brain synaptosomes and slices. Confocal immunofluoresence and transmission electron microscopy in brain slices with pre-embedding diaminobenzidine (DAB) immunostaining found PAR-1 predominantly localized to the peri-synaptic astrocytic endfeet. Structural confocal immunofluorescence microscopy studies of isolated synaptosomes revealed spherical structures stained with anti-PAR-1 antibody which co-stained mainly for glial-filament acidic protein compared with the neuronal markers synaptophysin and PSD-95. Immunoblot studies of synaptosomes demonstrated an appropriate major band corresponding to PAR-1 and activation of the receptor by a specific agonist peptide (SFLLRN) significantly modulated phosphorylated extracellular signal-regulated kinase. A significant membrane potential depolarization was produced by thrombin (1 U/mL) and the PAR-1 agonist (100 μM) and depolarization by high K+ elevated extracellular thrombin-like activity in the synaptosomes preparation. The results indicate PAR-1 localized to the peri-synaptic astrocytic endfeet is most likely activated by synaptic proteases and induces cellular signaling and modulation of synaptic electrophysiology. A protease mediated neuron-glia pathway may be important in both physiological and pathological regulation of the synapse.

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