Is tissue-type plasminogen activator a neuromodulator?

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Abstract

In the last few years, it has been evidenced that serine proteases play key roles in the mammalian brain, both in physiological and pathological conditions. It has been well established that among these serine proteases, the tissue-type plasminogen activator (t-PA) is critically involved in development, plasticity, and pathology of the nervous system. However, its mechanism of action remains to be further investigated. By using pharmacological and immunological approaches, we have evidenced in the present work that t-PA should be considered as a neuromodulator. Indeed, we have observed that: (i) neuronal depolarization induces a release of t-PA; (ii) this release of t-PA is sensitive to exocytosis inhibition and calcium chelation; (iii) released t-PA modulates NMDA receptor signaling and (iv) astrocytes are able to recapture extracellular t-PA through a low-density lipoprotein (LDL) receptor-related protein (LRP)-dependent mechanism.

Introduction

Serine proteases are a family of proteins mainly involved in the regulation of fibrinolysis (Carmeliet and Collen, 1996) and in the degradation of the extracellular matrix. In addition to their existence in the blood and in many peripheric tissues, several of these proteases have been detected in the central nervous system Davies et al., 1998, Sappino et al., 1993. Among them, the tissue-type plasminogen activator (t-PA) has been proposed as a candidate contributing to the cellular cascade that converts electrical signals into the formation of new synaptic connections (Baranes et al., 1998). Indeed, t-PA is secreted by neurons during neurite outgrowth Monard, 1988, Seeds et al., 1990, working as an important regulatory molecule in neuronal development. The most common idea is that t-PA facilitates axon elongation by degrading the extracellular matrix. More recently, the use of t-PA transgenic mice has been decisive to enlighten the involvement of t-PA in processes associated to activity-dependent synaptic plasticity such as learning and memory Frey et al., 1996, Huang et al., 1996, Madani et al., 1999 and to the control of excitotoxin-induced neuronal death (Tsirka et al., 1995). For example, by using cultured cortical neurons from either wild type or t-PA deficient mice, it was evidenced that t-PA interacts with and cleaves the NR1 subunit of the NMDA receptor, leading to an exacerbation of NMDA-induced calcium influx (Nicole et al., 2001). These results open the possibility of a direct interaction of glutamatergic transmission with t-PA.

In the blood, the t-PA is derived from vascular endothelial cells (Emeis et al., 1997). Its physiological role is to convert plasminogen into plasmin, a protease that controls proteolysis of a large number of targets such as fibrin or metalloproteases. In the brain, glial and neuronal cells are capable to synthetize and release plasminogen activators Krystosek and Seeds, 1984, Siao et al., 2003. t-PA released from microglia has been suggested to have a critical role in triggering neurodegeneration Flavin et al., 2000, Rogove and Tsirka, 1997. On the other hand, in PC12 cells, Gualandris et al. (1996) have shown that membrane depolarization induces a calcium-dependent secretion of t-PA. In this cell type, it was clearly evidenced that t-PA is targeted to the regulated secretory pathway and co-released with cathecolamine storage vesicles (Parmer et al., 1997). In addition, we have shown that NMDA treatment also leads to a release of t-PA in the extracellular media of cultured cortical neurons (Nicole et al., 2001). Thus, these data suggest that microglial activation, or neuronal activity, could lead to an accumulation of t-PA in the extracellular space which in turn could alter neurotransmission.

In the present study, we demonstrated that t-PA can be released from cortical neurons after depolarization through an exocytotic mechanism, and potentiates NMDA-induced calcium influx. In addition, we evidenced that astrocytes could promote the clearance of extracellular t-PA through a low-density lipoprotein (LDL) receptor-related protein (LRP)-dependent mechanism.

Section snippets

Cortical neurons exhibit t-PA

To address the question of whether cortical neurons were a source of t-PA in the brain parenchyma, we have investigated both mRNA and protein for t-PA. Immunostaining against MAP-2 (neuronal marker), GFAP (astrocytic marker) and lectine (microglial marker) was performed to estimate the percentage of astrocytes and microglia in our cultures of cortical neurons. Immunostaining for GFAP and lectine was also performed in cultured astrocytes and microglia as control (data not shown). As shown in

Discussion

In the nervous system, the main pathway of communication between neurons is synaptic transmission. It is now generally accepted that glutamate is the major fast excitatory neurotransmitter in the brain. In fact, 70% of the fast excitatory central nervous system synapses use glutamate as a transmitter (Watkins and Evans, 1981). Glutamate is capable to activate two major classes of receptors: ionotropic (NMDA, AMPA and Kainate) and metabotropic receptors. Activation of such receptors leads to a

Materials

Polymerase chain reaction and reverse transcriptase system kits were purchased from Promega (Charbonnières, France). RNeasy RNA extraction kit was from Qiagen (Courtaboeuf, France). Dulbecco's Eagle's minimal essential medium (DMEM), poly-d-lysine, laminin, anti-GFAP and anti-MAP-2 antibodies, iso-lectine, cytosine-β-d-arabinofuranoside (Ara-C), and N-methyl-d-aspartate (NMDA) were obtained from Sigma Co. (L'Isle D'Abeau, France). Horse serum (HS) and fetal bovine serum (FBS) were from Life

Acknowledgements

This work was supported by grants from the Medical Research Foundation (M. Fernández-Monreal), the French Ministry of Research and Technology (K. Benchenane), the Regional Council of Lower Normandy (J.P. López-Atalaya, M. Cacquevel), the University of Caen and FEDER.

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