ABSTRACT
Tissue-type plasminogen activator (tPA) is a pleiotropic serine protease of the central nervous system (CNS) with reported neurotrophic and neurotoxic functions. Produced and released under its single chain form (sc), the sc-tPA can be cleaved by plasmin or kallikrein in a two chain form, tc-tPA. Although both sc-tPA and tc-tPA display a similar fibrinolytic activity, we postulated here that these two conformations of tPA (sc-tPA and tc-tPA) could differentially control the effects of tPA on neuronal survival. Using primary cultures of mouse cortical neurons, our present study reveals that sc-tPA is the only one capable to promote N-methyl-D-aspartate receptor (NMDAR)-induced calcium influx and subsequent excitotoxicity. In contrast, both sc-tPA and tc-tPA are capable to activate epidermal growth factor receptors (EGFRs), a mechanism mediating the antiapoptotic effects of tPA. Interestingly, we revealed a tPA dependent crosstalk between EGFR and NMDAR in which a tPA-dependent activation of EGFRs leads to downregulation of NMDAR signaling and to subsequent neurotrophic effects.
Subject(s)
ErbB Receptors/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Tissue Plasminogen Activator/physiology , Apoptosis , Calcium Signaling , Cell Survival , Female , Humans , Protein Conformation , Receptor Cross-Talk , Tissue Plasminogen Activator/chemistryABSTRACT
BACKGROUND: Thrombolysis with tissue-type plasminogen activator (t-PA) is the only treatment approved for acute ischemic stroke. Although t-PA is an efficient clot lysis enzyme, it also causes damage to the neurovascular unit, including hemorrhagic transformations and neurotoxicity. OBJECTIVES: On the basis of the mechanism of action of t-PA on neurotoxicity, we aimed at studying the molecular requirements to generate safer thrombolytics. METHODS: We produced original t-PA-related mutants, including a non-cleavable single-chain form with restored zymogenicity (sc*-t-PA) and a t-PA modified in the kringle 2 lysine-binding site (K2*-t-PA). Both sc*-t-PA and K2*-t-PA showed fibrinolytic activities similar to that of wild-type t-PA on both euglobulin-containing and plasma-containing clots. In contrast to wild-type t-PA, the two mutants did not promote N-methyl-d-aspartate receptor-mediated neurotoxicity. CONCLUSIONS: We designed t-PA mutants with molecular properties that, in contrast to t-PA, do not induce neurotoxicity.
Subject(s)
Bioengineering , Fibrinolysis/drug effects , Fibrinolytic Agents/pharmacology , Neurotoxicity Syndromes/prevention & control , Thrombolytic Therapy , Tissue Plasminogen Activator/pharmacology , Amino Acid Sequence , Animals , Bioengineering/instrumentation , Bioengineering/methods , Bioreactors , Cell Death/drug effects , Drug Design , Fibrinolytic Agents/metabolism , Fibrinolytic Agents/toxicity , HEK293 Cells , Humans , Kringles , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Neurons/drug effects , Neurons/metabolism , Neurons/pathology , Neurotoxicity Syndromes/etiology , Neurotoxicity Syndromes/metabolism , Neurotoxicity Syndromes/pathology , Point Mutation , Rats , Receptors, N-Methyl-D-Aspartate/drug effects , Receptors, N-Methyl-D-Aspartate/metabolism , Recombinant Proteins/pharmacology , Structure-Activity Relationship , Thrombolytic Therapy/adverse effects , Tissue Plasminogen Activator/biosynthesis , Tissue Plasminogen Activator/genetics , Tissue Plasminogen Activator/toxicity , TransfectionABSTRACT
Unlike other serine proteases that are zymogens, the single-chain form of tissue plasminogen activator (sc-tPA) exhibits an intrinsic activity similar to that of its cleaved two-chain form (tc-tPA), especially in the presence of fibrin. In the central nervous system tPA controls brain functions and dysfunctions through its proteolytic activity. We demonstrated here, both in vitro and in vivo, that the intrinsic activity of sc-tPA selectively modulates N-methyl-D-aspartate receptor (NMDAR) signaling as compared with tc-tPA. Thus, sc-tPA enhances NMDAR-mediated calcium influx, Erk(½) activation and neurotoxicity in cultured cortical neurons, excitotoxicity in the striatum and NMDAR-dependent long-term potentiation in the hippocampal CA-1 network. As the first demonstration of a differential function for sc-tPA and tc-tPA, this finding opens a new area of investigations on tPA functions in the absence of its allosteric regulator, fibrin.
Subject(s)
Neurons/drug effects , Receptors, N-Methyl-D-Aspartate/metabolism , Signal Transduction/drug effects , Tissue Plasminogen Activator/pharmacology , Animals , Calcium/metabolism , Cells, Cultured , Humans , Mice , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , N-Methylaspartate/toxicity , Neurons/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Tissue Plasminogen Activator/genetics , Tissue Plasminogen Activator/metabolismABSTRACT
The synthesis and pharmacological evaluation of methoxyindazoles as new inhibitors of neuronal nitric oxide synthase are presented. The 7-methoxyindazole, although less potent than 7-NI, is the most active compound of the series in an in vitro enzymatic assay of neuronal nitric oxide synthase activity. This result shows that the nitro-substitution is not indispensable to the biological activity of the indazole ring. 7-Methoxyindazole possesses in vivo NOS inhibitory as well and related antinociceptive properties.
