Nature Medicine
9, 372 - 373 (2003)
doi:10.1038/nm0403-372
There is a Corrigendum (July 2003) associated with this Letters to Editor.
Reply to "Tissue plasminogen activator and NMDA receptor cleavage"Denis Vivien, Monica Fernandez Monréal, Olivier Nicole
& Alain BuissonUniversité de Caen, UMR 6551 Cyceron, France
Correspondence should be addressed to Alain Buisson a.buisson@neuro.unicaen.frNicole et al. reply
We showed that TPA, a serine protease known for its fibrinolytic activity, enhances NMDA-mediated calcium signaling and subsequent excitotoxic neuronal injury. This potentiation of excitotoxicity is concomitant with the cleavage of the NR1 subunit of NMDA receptor by TPA5.
Although Matys and Strickland are not questioning the deleterious influence of TPA on excitotoxic neuronal cell death, they raise an important concern about a possible misinterpretation of our results: does the TPA-induced cleavage of the NMDA receptor NR1 subunit involve the plasminogen-plasmin axis? We believe that we have strong arguments against this possibility.
First, whereas our primary cortical neuronal cultures were maintained in a serum-supplemented solution that may have contained plasminogen, the excitotoxic injury and cleavage experiments were all conducted in serum-free solutions. A casein gel zymography assay did not detect the presence of active plasmin in the culture media or in crude extracts of neuronal cultures, thereby excluding a possible contamination of our samples (data not shown).
Second, we used a bacterial system to produce a recombinant protein corresponding to the N-terminal end of NR1. Treatment of the purified recombinant protein with recombinant TPA (20  g/ml) led to the appearance of a lower molecular weight product identified by mass-spectrometric analysis as a cleaved form of the NR1 N terminus11. These results prove irrefutably that TPA can cleave the N-terminal moiety of the NR1 subunit of the NMDA receptor without the involvement of plasmin.
Our results show that it is very unlikely that plasminogen has a role in the TPA-mediated cleavage of the NR1 subunit. Plasminogen, the preferential target of TPA in blood, seems to have important structural similarities with NR1, as indicated by Matys and Strickland. This raises the possibility that this protein may be a putative substrate for TPA proteolysis.
In the blood, the physiological role of TPA is to activate the zymogen plasminogen into the broad-spectrum protease plasmin; in the brain, the participation of plasminogen in TPA-dependent effects remains under intense debate. Recent publications strengthen the idea that in the brain, TPA has normal and pathological roles independent of plasminogen activation12,
13. We cannot, however, rule out that generation of plasmin may be necessary in some cases14. Further investigation of the presence of plasminogen in the brain is necessary to re-evaluate the mechanism of action of serine proteases in the brain.
See "Tissue plasminogen activator and NMDA receptor cleavage" by Matys and Strickland.
REFERENCES
- Gingrich, M.B. & Traynelis, S.F. Serine proteases and brain damage - is there a link? Trends. Neurosci. 23, 399–407 (2000). | Article | PubMed | ISI | ChemPort |
- Chen, Z.L. et al. Neuronal death and blood-brain barrier breakdown after excitotoxic injury are independent processes. J. Neurosci. 19, 9813–9820 (1999). | PubMed | ISI | ChemPort |
- Pawlak, R. & Strickland, S. Tissue plasminogen activator and seizures: a clot-buster's secret life. J. Clin. Invest. 109, 1529–1531 (2002). | Article | PubMed | ISI | ChemPort |
- Centonze, D. et al. Tissue plasminogen activator is required for corticostriatal long-term potentiation. Eur. J. Neurosci. 16, 713–721 (2002). | Article | PubMed | ISI |
- Nicole, O. et al. The proteolytic activity of tissue-plasminogen activator enhances NMDA receptor-mediated signaling. Nat. Med. 7, 59–64 (2001). | Article | PubMed | ISI | ChemPort |
- Ponting, C.P., Marshall, J.M. & Cederholm-Williams, S.A. Plasminogen: a structural review. Blood. Coagul. Fibrinolysis 3, 605–614 (1992). | PubMed | ISI | ChemPort |
- Traynelis, S.F. & Lipton, S.A. Is tissue plasminogen activator a threat to neurons? Nat. Med. 7, 17–18 (2001). | Article | PubMed | ISI | ChemPort |
- Gingrich, M.B., Junge, C.E., Lyuboslavsky, P. & Traynelis, S.F. Potentiation of NMDA receptor function by the serine protease thrombin. J. Neurosci. 20, 4582–4595 (2000). | PubMed | ISI | ChemPort |
- Inoue, K., Koizumi, S., Nakajima, K., Hamanoue, M. & Kohsaka, S. Modulatory effect of plasminogen on NMDA-induced increase in intracellular free calcium concentration in rat cultured hippocampal neurons. Neurosci. Lett. 179, 87–90 (1994). | Article | PubMed | ISI | ChemPort |
- Mizutani, A., Tanaka, T., Saito, H. & Matsuki, N. Postsynaptic blockade of inhibitory postsynaptic currents by plasmin in CA1 pyramidal cells of rat hippocampus. Brain. Res. 761, 93–96 (1997). | Article | PubMed | ISI | ChemPort |
- Fernandez-Monreal, M. et al. Modulation of the NMDA receptor function by tissue-type plasminogen activator. Soc. Neurosci. Abstr. 747, 11 (2002).
- Nagai, N., De Mol, M., Lijnen, H.R., Carmeliet, P. & Collen, D. Role of plasminogen system components in focal cerebral ischemic infarction: a gene targeting and gene transfer study in mice. Circulation 99, 2440–2444 (1999). | PubMed | ISI | ChemPort |
- Rogove, A.D., Siao, C., Keyt, B., Strickland, S. & Tsirka, S.E. Activation of microglia reveals a non-proteolytic cytokine function for tissue plasminogen activator in the central nervous system. J. Cell. Sci. 112, 4007–4016 (1999). | PubMed | ISI | ChemPort |
- Tsirka, S.E., Rogove, A.D., Bugge, T.H., Degen, J.L. & Strickland, S. An extracellular proteolytic cascade promotes neuronal degeneration in the mouse hippocampus. J. Neurosci. 17, 543–552 (1997). | PubMed | ISI | ChemPort |
|
