The tissue plasminogen activator (tPA)/plasmin extracellular proteolytic system regulates seizure-induced hippocampal mossy fiber outgrowth through a proteoglycan substrate

Abstract

Short seizure episodes are associated with remodeling of neuronal connections. One region where such reorganization occurs is the hippocampus, and in particular, the mossy fiber pathway. Using genetic and pharmacological approaches, we show here a critical role in vivo for tissue plasminogen activator (tPA), an extracellular protease that converts plasminogen to plasmin, to induce mossy fiber sprouting. We identify DSD-1-PG/phosphacan, an extracellular matrix component associated with neurite reorganization, as a physiological target of plasmin. Mice lacking tPA displayed decreased mossy fiber outgrowth and an aberrant band at the border of the supragranular region of the dentate gyrus that coincides with the deposition of unprocessed DSD-1-PG/phosphacan and excessive Timm-positive, mossy fiber termini. Plasminogen-deficient mice also exhibit the laminar band and DSD- 1-PG/phosphacan deposition, but mossy fiber outgrowth through the supragranular region is normal. These results demonstrate that tPA functions acutely, both through and independently of plasmin, to mediate mossy fiber reorganization.

Figure 1

Neo-Timm staining (Holm and Geneser 1991) visualizing mossy fiber sprouting in coronal sections of the hippocampal formation of wild-type and tPA^−/− mice 20 d after unilateral injection of KA in the amygdala. Note the strong silver deposits at the border of the molecular layer in tPA^−/− mice (arrows). GCL, Supragranular cell layer; mol, molecular cell layer.

Figure 2

A, High magnification of Timm-stained horizontal sections of the injected side of DG of wild-type, tPA^−/−, and plg^−/− mice near the tip of the injected DG. The arrows point to the newly formed mossy fiber sprouts, demonstrating that they accumulate and extend along the border of the granule cell and molecular layers. B, PSA-NCAM immunohistochemistry on horizontal sections of the injected side of DG of wild-type, tPA^−/−, and plg^−/− mice near the tip of the injected DG. The arrows point to the extending neurites through the granule cell layer (GCL), the arrowheads point to the accumulated PSA-NCAM expression on the border between GCL and molecular layer (mol).

Figure 3

In vitro cleavage of neurocan and DSD-1-PG/phosphacan by plasmin, but not tPA. Radiolabeled with ¹²⁵I, purified neurocan (∼9.75 ng) and DSD-1-PG/phosphacan (∼6.5 ng) were incubated with recombinant mouse tPA or human plasmin in the absence or presence of PAI-1 or α₂-antiplasmin. The inhibitors were preincubated with the recombinant enzymes for 5 min at 37°C. The samples were analyzed by SDS-PAGE and autoradiography. Lane 1, tPA 1 ng; lane 2, tPA 10 ng; lane 3, tPA 100 ng; lane 4, tPA 100 ng and PAI1 100 ng; lane 5, proteoglycan incubated with buffer alone; lane 6, proteoglycan by itself; lane 7, plasmin 1 ng; lane 8, plasmin 10 ng; lane 9, plasmin 100 ng; lane 10, plasmin 10 ng and α ₂-antiplasmin 50 ng; lane 11, plasmin 10 ng and α₂-antiplasmin 100 ng. The arrows point to bands that disappear after incubation with plasmin.

Figure 4

Proteolytic processing of DSD-1-PG/phosphacan in wild-type mice after KA injection. Partially purified protein extracts from KA-injected (inj) or PBS-injected (uninj) wild-type (wt), tPA^−/− and plg^−/− hippocampi, as described in Materials and Methods, were analyzed by SDS-PAGE and immunoblotting, using an anti-DSD-1-PG/phosphacan antibody. Note the processing that has occurred in the wild-type injected lane, and the absence of such processing in the corresponding tPA^−/− and plg^−/− lanes. The arrows point to the 400-kD DSD-1-PG/phosphacan protein band (upper arrow), and the DSD-1-PG/phosphacan protein core of 180 kD (lower arrow), which appears more susceptible to plasmin cleavage. Top, Coomassie-stained gel showing comparable loading among the different lanes; bottom, Western blot of the Coomassie-stained gel (top). This experiment has been repeated five times.

Figure 5

DSD-1-PG/phosphacan repels mossy fiber outgrowth in culture. Hippocampal neurons were plated onto nitrocellulose-coated dishes containing: A, laminin in the center (bottom of A), and laminin in a surrounding annulus (top of A); B, laminin and DSD-1-PG/phosphacan in the center, and laminin alone in a surrounding annulus; C, laminin and plasmin-proteolyzed DSD-1/phosphacan in the center, and laminin alone in a surrounding annulus. The cultures were photographed after 5 d of growth. Phase-contrast image of hippocampal neuronal cells attached to the laminin region that are extending neurites (blue arrows denote neurites crossing through the layers, whereas red arrows indicate neurites that are being repelled). D, Phase-contrast image of neuronal cells attached to the laminin region and extending neurites through the laminin, but are being repelled and changing direction at the border of the DSD-1-PG/phosphacan/laminin region (red arrows). Immunohistochemistry with anti-DSD-1-PG/phosphacan antibody was used to confirm that the localization of DSD-1-PG/phosphacan coincided with the boundary at which neurite outgrowth terminated (data not shown). E, Immunostaining of mossy fiber-like axons with dynorphin A. F, Overlap of D and E, showing the avoidance of DSD-1-PG/phosphacan by the mossy fiber axons. Blue bar indicates the laminin region, and the red bar the DSD-1-PG/phosphacan/laminin region.

Figure 6

The tPA^−/− mossy fiber sprouting phenotype can be reversed to that of wild-type mice by infusion of recombinant tPA. Sections of KA-injected wild-type (wt) and tPA^−/− mice are used as controls. When recombinant tPA (tPA^−/−/tPA) is infused into the hippocampus and the intraamygdala KA injection follows, the Timm-positive mossy fiber sprouting pattern is comparable to that of wild-type mice. However, infusion of catalytically inactive S478A tPA (tPA^−/−/S478A tPA) rescues only the neurite pathfinding through the supragranular region (arrowheads), but the aberrant laminar band (arrows) persists.

Figure 7

Inhibition of tPA activity in wild-type mice generates only part of the aberrant sprouting phenotype observed in tPA^−/− mice. A, Section of a KA-injected tPA^−/− mouse as control. B, tPA Stop was infused into wild-type mice, which were then injected with KA into their amygdala. Strong, dense, and tight compacted Timm-positive sprouts are evident through the granular layer, as well as a laminar band at the border of granular and molecular layer of the injected side. The arrows point to the excessive sprouts present through the supragranular layer. The arrowheads point to the Timm-positive sprouts along the border of the molecular layer.