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

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.

Role of Protease-Activated Receptor-1 in Brain Injury After Experimental Global Cerebral Ischemia

“…Thrombin activity was determined according to the method described by Chapman and coworkers 17 with some modification. Briefly, animals were reanesthetized, perfused with saline and the brain removed.…”

Coagulation in Inflammatory Diseases of the Central Nervous System

“…There is much evidence pointing to the importance of blocking this pathway in inflammatory brain diseases: PAR-1 is localized on glia at the node of Ranvier in peripheral nerve and its activation causes conduction block which is a common feature of inflammatory neuropathies. 68 At the synapse PAR-1 is also localized mainly on glia 69 and its activation causes hyperexcitation, long-term potentiation leading to epileptic seizures Fig. 1 The potential activation and inhibition of the thrombin receptor PAR-1 by factors involved in coagulation and inflammation in the central nervous system.…”

Ketorolac Reduces Spinal Astrocytic Activation and PAR1 Expression Associated with Attenuation of Pain after Facet Joint Injury

“…15,16 Expression of PAR1 has been confirmed on neurons, astrocytes, and microglia, all of which are cells known to contribute to pain. [17][18][19][20][21] Yet the role of PAR1 in the Departments of 1 Bioengineering and 2 Neurosurgery, University of Pennsylvania, Philadelphia, Pennsylvania. initiation and maintenance of pain is still not clear.…”

“…24 Although many studies implicate PAR1 activation and its role in certain types of peripheral neuropathic and inflammatory pain states, no study has determined if PAR1 has a role in joint-mediated pain and whether it can be modulated in the presence or absence of pain. PAR1 is expressed by astrocytes and activated microglia, 18,19,21 both of which have been shown to contribute to the initiation and maintenance of pain. [25][26][27] Ketorolac, a non-steroidal anti-inflammatory drug (NSAID) with strong analgesic activity, has been used clinically to treat postoperative, inflammatory, and neuropathic pain.…”