The Janus face of endogenous neuronal tPA: promoting self-protection and worsening the death of neighboring neurons

Abstract

Recombinant tissue-type plasminogen activator (r-tPA/Actilyse) stands as the prevailing pharmacological solution for treating ischemic stroke patients, of whom because their endogenous circulating tPA alone is not sufficient to rescue reperfusion and to promote favorable outcome. Beyond the tPA contributed by circulating endothelial cells and hepatocytes, neurons also express tPA, sparking debates regarding its impact on neuronal fate ranging from pro-survival to neurotoxic properties. In order to investigate the role of neuronal tPA during brain injuries, we developed models leading to its conditional deletion in neurons, employing AAV9-pPlat-GFP and AAV9-pPlat-Cre-GFP along with tPA floxed mice. These models were subjected to N-methyl-D-aspartate (NMDA)-induced excitotoxicity or thromboembolic ischemic stroke in mice. Initially, we established that our AAV9 constructs selectively transduce neurons, bypassing other brain cell types. Subsequently, we demonstrated that tPA-expressing neurons exhibit greater resistance against NMDA-induced excitotoxicity compared to tPA negative neurons. The targeted removal of tPA in neurons heightened the susceptibility of these neurons to cell death and prevented a paracrine neurotoxic effect on tPA non-expressing neurons. Under ischemic conditions, the self-neuroprotective influence of tPA encompassed both excitatory (GFP⁺/Tbr1⁺) and inhibitory (GFP⁺/GABA⁺) neurons. Our data indicate that endogenous neuronal tPA is a protective or deleterious factor against neuronal death in an excitotoxic/ischemic context, depending on whether it acts as an autocrine or a paracrine mediator.

Fig. 1. tPA is expressed by cortical neurons in vivo.

A Schematic experimental protocol showing the unilateral injection of 2 × 0.25 µL of 1.25 × 10¹³ vp/mL of AAV9-pPlat-GFP in tPA flox^−/− mice (WT, -0.3 mm AP, ±3.3 mm ML, –0.8 & 0.4 mm DV). B Representative images of immunohistochemical analyses showing that AAV9-pPlat-GFP does not transduce oligodendrocytes (magenta), microglia (red), astrocytes (gray), and blood vessels (cyan). Scale bar = 100 µm, ×20. C Representative images and corresponding quantifications of immunohistochemical analyses showing that AAV9-Plat -GFP transduces around one third of neurons (yellow; NeuN and Blue; Neurotrace). Scale bar = 100 µm, objective = ×40. D Quantification of the proportion of neurons GABA-expressing (a marker of inhibitory neurons) or tbr1 (a marker of excitatory neurons) among the population of GFP (tPA) positive neurons, in the transduced area. A white dashed arrow indicates the colocalization between the TBR1 marker and GFP, whereas an isolated white triangle denotes the colocalization between the GABA marker and GFP. Scale bar=100 µm, ×20.

Fig. 2. tPA cortical neurons are more resistant to excitotoxicity than non tPA neurons.

A Schematic experimental protocol showing the AAV9-pPlat-GFP injection in both somatosensory cortices of swiss mice, followed by the unilateral injection of NMDA into the right somatosensory cortex. Brain were collected at 30 min, 1 and 2 h after the NMDA injection to immunohistochemical analyses. B, C Representative images and quantification of the number of tPA non-expressing neurons (red) and tPA-expressing neurons (green) in the lesion area compared to the contralateral area, at 30 min, 1 and 2 h post NMDA injection (N = 4 mice for 30 min and 1 h groups, N = 5 mice for 2 h group; Two-way ANOVA, multiple comparison, Bonferroni post-hoc; p < 0.05 = *, p < 0.001 = ***, p < 0.0001 = ****). Scale bar = 100 µm, ×20. D. Quantification of the percentage of tPA non-expressing (red) and tPA-expressing neurons (green) mortality at 30 min, 1 and 2 h post NMDA injection (N = 4 mice for 30 min and 1 h groups, N = 5 mice for 2 h group; two-way ANOVA, Multiple Comparison, Bonferroni post-hoc; p < 0.01 = **, p < 0.0001 = ****).

Fig. 3. tPA from cortical neurons promotes neuronal survival at the acute phase of excitotoxicity.

A Schematic experimental protocol showing first, the AAV9-Plat-Cre-GFP injection in tPA ^−/− (WT) or flox^+/+ (tPA-cKO^Neu) mice and second, the NMDA injection followed by IHC. B, C Representative images and quantification of the number of tPA non-expressing neurons (red) and tPA-expressing neurons (green) in the lesion area compared to the contralateral area, at 1 and 2 h post NMDA injection (N = 5 (+1 h) and N = 6 (+2 h) mice per time for WT and N = 4 (+1 h) and 4 (+2 h) for tPA-cKO^Neu; Two-way ANOVA, Multiple comparison, Bonferroni post hoc; p < 0.0001 = ****). The percentage of mortality was calculated for both genotypes at 1 and 2 h post excitotoxicity induction (N = 5 (+1 h) and N = 6 (+2 h) mice per time for WT and N = 4 (+1 h) and 4 (+2 h) for tPA-cKO^Neu. Mann–Whitney test; p < 0.01 = ** for NeuN⁺/GFP⁻ cells and Unpaired t-test, p < 0.0001 = **** for NeuN⁺/GFP⁺ cells. Scale bar = 100 µm, ×20. D Representative images and quantification of the tPA non-expressing (red) and tPA-expressing (green) cortical neuronal circularity in the lesion area and the contralateral area of WT or tPA-cKO^Neu mice, 1 and 2 h post excitotoxicity induction (N = 5 (+1 h) and N = 6 (+2 h) mice per time for WT and N = 4 (+1 h) and 4 (+2 h) for tPA-cKO^Neu; Two-way ANOVA, Multiple comparison, Bonferroni post hoc; p < 0.01 = **, p < 0.001 = ***, p < 0.0001 = ****). Scale bar = 100 µm, ×40.

Fig. 4. Neuronal tPA acts as a neuroprotective mediator at the acute phase of ischemic stroke.

A Schematic experimental protocol showing the AAV9-Plat-Cre-GFP injection, the induction of the thromboembolic model of stroke, the MRI T2 sequences and the IHC performed 6 h after stroke. B Illustration Representative images and quantification of the total lesion volume and the lesion volume in the AAV area injection for WT and tPA-cKO^Neu mice (N = 13 for WT and N = 12 for tPA-cKO^Neu; Unpaired t-test; p < 0.01= **, p < 0.001 = ***). C Representative images and quantification of tPA non-expressing neurons (red) and tPA-expressing neurons (green) in the lesion area compared to the contralateral area, 6-h post ischemia (N = 5 WT and N = 4 tPA-cKO^Neu; Two-way ANOVA, Multiple comparison, Bonferroni post hoc; p < 0.01 = **, p < 0.0001 = ****. The percentage of mortality was calculated for both genotypes at 6-h post ischemia (N = 5 for WT and N = 6 for tPA-cKO^Neu mice. Mann–Whitney test for NeuN⁺/GFP^- cells and Unpaired t-test, p < 0.0001 = **** for NeuN⁺/GFP⁺ cells. Scale bar = 100 µm, ×20.

Fig. 5. Excitatory and inhibitory tPA-expressing neurons have a similar sensitivity to excitotoxicity.

A, B Representative images and quantification (C) of the percentage of tPA-expressing neurons (green), inhibitory tPA-expressing neurons (GABA⁺, red) and tPA-expressing neurons (red) and excitatory tPA-expressing neurons (tbr1⁺, yellow) mortality 6-h post ischemia for WT and tPA-cKO^Neu mice (N = 5 for WT and 4 for tPA-cKO^Neu mice; unpaired t-test; p < 0.05 = *). Scale bar = 100 µm, ×20.