Extracellular proteolysis of reelin by tissue plasminogen activator following synaptic potentiation

Abstract

The secreted glycoprotein reelin plays an indispensable role in neuronal migration during development and in regulating adult synaptic functions. The upstream mechanisms responsible for initiating and regulating the duration and magnitude of reelin signaling are largely unknown. Here we report that reelin is cleaved between EGF-like repeats 6-7 (R6-7) by tissue plasminogen activator (tPA) under cell-free conditions. No changes were detected in the level of reelin and its fragments in the brains of tPA knockouts, implying that other unknown proteases are responsible for generating reelin fragments found constitutively in the adult brain. Induction of NMDAR-independent long-term potentiation with the potassium channel blocker tetraethylammonium chloride (TEA-Cl) led to a specific up-regulation of reelin processing at R6-7 in wild-type mice. In contrast, no changes in reelin expression and processing were observed in tPA knockouts following TEA-Cl treatment. These results demonstrate that synaptic potentiation results in tPA-dependent reelin processing and suggest that extracellular proteolysis of reelin may regulate reelin signaling in the adult brain.

Keywords: Hippocampus; Long-term potentiation; Reelin; Tetraethylammonium chloride; Tissue plasminogen activator (tPA).

Fig. 1

Modulation of Reelin processing by tPA. (A) Reelin is cleaved between epidermal growth factor (EGF) repeats 2–3 (R2–3) and 6–7 (R6–7), resulting in 5 potential fragments (370, 270, 190, 180, and 80 kDa). The anti-reelin G10 primary antibody detects full-length reelin (450 kDa), and the 370- and 180-kDa fragments. Ab14 was used to detect the 80-kDa fragment. (B) Recombinant reelin (50 nM) was incubated with 0–400 nM tPA at 37 °C for 15 min. tPA concentration-dependently increased processing of recombinant reelin between R6 and 7. Both the 370 and 80-kDa reelin fragments were increased, while the level of full-length reelin was decreased by tPA treatment. The 180-kDa fragment was not altered. (C) Inclusion of the broad-spectrum Halt protease inhibitor cocktail (1X P.I.C.) with tPA (200 nM and 400 nM) inhibited reelin processing. Experiments were performed in triplicate.

Fig. 2

Effects of tPA and plasminogen on reelin processing under cell-free conditions. Recombinant reelin (50 nM) was incubated with tPA (50 nM), plasminogen (Plg, 18 μg/μl), plasminogen (18 μg/μl)/tpa (50 nM), and activated plasmin (0.5 U/ml) at 37 °C for 15 and 45 min under cell-free conditions. (A) At both the 15- and 45-min time points, tPA promoted the generation of the 370 fragment. Plasminogen alone had no effect. The Plg/tPA combination and active plasmin converted reelin to 180-kDa and sub-180-kDa fragments. (B) The above-mentioned reactions were co-incubated with serine protease inhibitors or CR-50 for 45 min at 37 °C. Plasminogen activator inhibitor (PAI-1, 1 ng/μl) blocked the cleavage of reelin by tPA and the plg/tPA combination, whereas it had no effect on already active plasmin. DIFP (100 μM) inhibited the processing of reelin by all protease combinations. Aprotinin (40 μM) blocked the effects of tPA-activated plasmin and purified plasmin, but did not prevent tPA-mediated reelin processing. The dimerization-inhibiting CR-50 antibody (0.02 μg/μl) had no effect on reelin processing by tPA, plg/tPA, or active plasmin. Experiments were performed in triplicate.

Fig. 3

tPA does not modulate reelin processing in acute hippocampal slices. Wild-type hippocampal slices (n=4 per treatment) were treated with 50 or 100 nM tPA for 60 min. No significant differences were found in full-length reelin or fragment levels.

Fig. 4

Loss of tPA in vivo does not affect basal reelin processing. The level of full-length reelin and the 370- and 180-kDa fragments were measured in the brains of 4-month-old wild-type (n=6) and tPA knockout (n=6) mice. No significant differences were found in the levels of full-length reelin or fragments in the cortex (A), cerebellum (B), or hippocampus (C). The values are shown as a relative density and error bars represent the mean±the standard error of the mean.

Fig. 5

cLTP induces reelin processing in wild-type mice but tPA is required for reelin processing. Acute hippocampal slices from wild-type mice (Panels A, B) or tPA KO mice (Panels C, D) were treated with TEA-Cl (25 mM; at least 2 slices from 3 different mice) for 10 min and recovered in aCSF for 5, 15, and 45 min (wild-type mice) or 15 min (tPA KO mice). Proteins from dissected hippocampal area CA1 were evaluated for changes in reelin processing and ERK1/2 activation. (A) The level of the 370-kDa fragment was significantly increased at 15 min following TEA-Cl treatment. (B) The level of p-ERK1 (44 kDa) was increased at 5-min post-treatment compared to the non-treated group and 45-min recovery group. The levels of p-ERK2 (42 kDa) were significantly increased at 5, 15, and 45 min following TEA-Cl treatment when compared to non-treatment. The 5-min group was also significantly higher than the 15 and 45-min recovery groups. (C) There were no significant changes in full-length reelin or either of the fragments in tPA KO mice 15 min following TEACl treatment. (D) The levels of p-ERK1 (44 kDa) and p-ERK2 (42 kDa) were significantly increased 15 min following TEA-Cl treatment in the tPA KO mice. The values are shown as a relative density and error bars represent the mean±the standard error of the mean. (*) denotes p<0.05 as indicated by the post-hoc analysis (wild-type mice) or as indicated by the independent samples two-tailed t-test (tPA KO mice).