An extracellular proteolytic cascade promotes neuronal degeneration in the mouse hippocampus

Abstract

Mice lacking the serine protease tissue plasminogen activator (tPA) are resistant to excitotoxin-mediated hippocampal neuronal degeneration. We have used genetic and cellular analyses to study the role of tPA in neuronal cell death. Mice deficient for the zymogen plasminogen, a known substrate for tPA, are also resistant to excitotoxins, implicating an extracellular proteolytic cascade in degeneration. The two known components of this cascade, tPA and plasminogen, are both synthesized in the mouse hippocampus. tPA mRNA and protein are present in neurons and microglia, whereas plasminogen mRNA and protein are found exclusively in neurons. tPA-deficient mice exhibit attenuated microglial activation as a reaction to neuronal injury. In contrast, the microglial response of plasminogen-deficient mice was comparable to that of wild-type mice, suggesting a tPA-mediated, plasminogen-independent pathway for activation of microglia. Infusion of inhibitors of the extracellular tPA/plasmin proteolytic cascade into the hippocampus protects neurons against excitotoxic injury, suggesting a novel strategy for intervening in neuronal degeneration.

Fig. 1.

Plasminogen-deficient mice are resistant to kainate-induced neuronal degeneration. Cresyl violet-stained coronal sections through the hippocampus reveal the neuronal degeneration generated by kainate. a, Hippocampus from heterozygous plg^+/− mouse (wt) 5 d after the injection, showing substantial degeneration on the injected side (ipsilateral), whereas the uninjected (contralateral) side remains unaffected (number of mice injected = 15). CA1, CA2, andCA3 denote the hippocampal subfields; DG, dentate gyrus. b, Hippocampus fromplg^−/− mouse 5 d after the injection, showing minimal degeneration on the injected side (n = 4). Arrows show the site of injection. c, High-magnification photomicrograph of part of the ipsilateral CA1 subfield of hippocampus from heterozygous plg^+/− mouse (wt) 12 hr after the injection (n = 3). The pyknotic and refractile neuronal cells in the wild-type mouse have disappeared completely by 24 hr, showing that they are not glial cells. d, High-magnification photomicrograph of part of the ipsilateral CA1 subfield of hippocampus from heterozygous plg^−/− mouse (wt) 12 hr after the injection (n = 3). e, TUNEL labeling, indicating DNA fragmentation in dying cells (arrows), is observed in the CA1 pyramidal subfield in wt mice (n = 2) and is absent in plg^−/− mice (f) 2 d after kainate injection (n = 2).

Fig. 2.

tPA mRNA is present in neurons and microglia, whereas plasminogen mRNA is found only in neurons. Coronal sections through the hippocampus displaying the site of synthesis of tPA (c, e, g) and plasminogen (d, f, h). Low magnification of coronal sections (a–d) and higher magnification of similar sections (e, f). Other mice were unilaterally injected with kainate to cause neuronal degeneration on one side, and subjected to mRNA ISH 5 d later (g, h). Cresyl violet staining of kainate-injected consecutive sections confirmed the complete elimination of the neurons in CA1–CA3 (data not shown). Ina, the probe was control plasmid (pBluescript KS II, Stratagene, La Jolla, CA), and in b the probe was digoxygenin-labeled sense tPA cDNA. The arrows inf indicate dendritic localization of plasminogen mRNA.

Fig. 3.

tPA and plasminogen proteins are synthesized locally in the mouse hippocampus. Immunodetection of tPA and plasminogen proteins in coronal sections through the hippocampus 12 hr after kainate injection of a wild-type mouse unilaterally into the hippocampus. High-magnification photomicrographs of the CA1 field reacting with (a) IgG, (b) normal sheep serum, (c) anti-tPA antibody at the contralateral side, (d) anti-plasminogen antibody at the contralateral side, (e) anti-tPA antibody at the ipsilateral side, and (f) anti-plasminogen antibody at the ipsilateral side. Scale bar, 50 μm.

Fig. 4.

Normal activation of microglia in kainate-injected plasminogen-deficient mice. Left panels, Low-magnification F4/80 immunostaining of coronal sections through the hippocampus 5 d after injection of a wild-type mouse with PBS (a, b) or with kainate (c, d), and of a plg^−/− mouse (e, f) with kainate. Extensive activation is observed on the ipsilateral side (shown) and around the injection site. Microglia on the contralateral side are also activated (not shown), but to a much lower level. Right panels, High-magnification photomicrographs of representative activated microglia in the CA1 field of stratum radiatum on the ipsilateral side of a wild-type (d) and a plg^−/− mouse (f).

Fig. 5.

α₂-antiplasmin can prevent kainate-induced neuronal degeneration. Cresyl violet-stained coronal sections through the hippocampus of wild-type mice. The mice were infused with buffer (aCSF) or α₂-antiplasmin for 2 d, kainate was injected, and then the infusion continued for 5 d more, and the mice were analyzed. Top, Section from a wild-type mouse infused with aCSF showing extensive kainate-induced degeneration. Bottom, Section from a wild-type mouse infused with α₂-antiplasmin, showing resistance to kainate-induced degeneration. Arrowheads point to the site of injection; arrows indicate the site of infusion.