Urokinase-type plasminogen activator (uPA) protects the tripartite synapse in the ischemic brain via ezrin-mediated formation of peripheral astrocytic processes

Abstract

Cerebral ischemia has a harmful effect on the synapse associated with neurological impairment. The “tripartite synapse” is assembled by the pre- and postsynaptic terminals, embraced by astrocytic elongations known as peripheral astrocytic processes (PAPs). Ischemic stroke induces the detachment of PAPs from the synapse, leading to synaptic dysfunction and neuronal death. Ezrin is a membrane-associated protein, required for the formation of PAPs, that links the cell surface to the actin cytoskeleton. Urokinase-type plasminogen activator (uPA) is a serine proteinase that upon binding to its receptor (uPAR) promotes neurite growth during development. In the adult brain, neurons release uPA and astrocytes recruit uPAR to the plasma membrane during the recovery phase from an ischemic stroke, and uPA/uPAR binding promotes functional improvement following an ischemic injury. We found that uPA induces the synthesis of ezrin in astrocytes, with the subsequent formation of PAPs that enter in direct contact with the synapse. Furthermore, either the release of neuronal uPA or intravenous treatment with recombinant uPA (ruPA) induces the formation of PAPs in the ischemic brain, and the interaction of these PAPs with the pre- and postsynaptic terminals protects the integrity of the “tripartite synapse” from the harmful effects of the ischemic injury.

Keywords

Peripheral astrocytic processes ezrin urokinase-type plasminogen activator plasmin neurorepair

Introduction

A substantial body of experimental evidence indicates that astrocytes regulate synaptic function.¹ Indeed, astrocytes can induce synaptic plasticity,² modulate synaptic transmission,³ and promote structural plasticity,⁴ and the formation and stabilization of new synapses.^5–8

The interaction between astrocytes and the synapse occurs via highly motile structures known as peripheral astrocytic processes (PAPs) that ensheath the pre- and postsynaptic terminals in an activity-dependent manner.^9,10 PAPs constitute approximately 80% of the astrocytic membrane,¹¹ and although they are devoid of glial fibrillary acidic protein (GFAP)-positive filament rich in glutamine synthetase (GS)¹² and harbor the molecular machinery required for local messenger RNA (mRNA) translation.¹³

Cerebral ischemia has a deleterious effect on the synapse¹⁴ that leads to the impairment in neurological function observed in ischemic stroke patients. Interestingly, although it has long been recognized that astrocytes play a crucial role in the response of the synapse to the ischemic injury,¹⁵ and that the onset of cerebral ischemia induces the detachment of PAPs from the synapse,¹⁶ the role of PAPs in the ischemic brain is still unclear.

Ezrin belongs to a group of evolutionary conserved proteins that regulate the reorganization of the actin cytoskeleton¹⁷ and the formation of microvilli, filopodia, and lamellipodia in the cell.¹⁸ The N-terminus of ezrin binds to membrane proteins, while its C-terminal domain binds to F-actin in the cytoplasm.¹⁹ In the cytosol ezrin exists in an inactive conformation, with its F-actin- and membrane-binding sites concealed by its C-terminus domain. However, following its recruitment to areas of the plasma membrane with high concentrations of phosphoinositides, ezrin is activated by phosphorylation at a conserved Thr567 residue.¹⁹ Importantly, ezrin is abundantly found in astrocytic filopodia,²⁰ and its activation is required for the formation of PAPs.¹¹

Urokinase-type plasminogen activator (uPA) is a serine proteinase that upon binding to its receptor (uPAR) catalyzes the conversion of plasminogen into plasmin on the cell surface.²¹ However, besides regulating the plasminogen activating system, uPA binding to uPAR also promotes tissue remodeling²² via activation of cell signaling pathways by plasminogen-dependent- and -independent mechanisms.²¹ Significantly, uPA anchors uPAR to the actin cytoskeleton in human corneal fibroblasts²³ and uPA-uPAR binding promotes the formation of F-actin bundles in dendritic spines,²⁴ and astrocytic activation and synaptic recovery in the ischemic brain.²⁵

In this study, we show that uPAR is abundantly found in PAPs, and that treatment with uPA induces the synthesis of ezrin in astrocytes by a plasminogen-independent mechanism. We found that this is followed by the recruitment of ezrin to the astrocytic plasma membrane and its subsequent activation by Rho-associated protein kinase (ROCK)-mediated phosphorylation at Thr567. In line with these observations, our data indicate that uPA induces ezrin- and ROCK-mediated formation of PAPs, and that these astrocytic elongations enter in direct contact with the synapse. The translational relevance of these data is underscored by the finding that either endogenous uPA/uPAR binding or treatment with recombinant uPA (ruPA) promotes the formation of PAPs in the ischemic brain. Furthermore, our results indicate that the interaction of these PAPs with the pre- and postsynaptic terminals preserves the integrity of those synapses that have suffered an acute ischemic injury. In summary, these data reveal that uPA induces the formation of “tripartite synapses” in the central nervous system, and that the uPA/uPAR system is a target for the design of therapeutic strategies to protect the synapse in the ischemic brain.

Materials and methods

Animals and reagents

Animal strains were 8- to 12-week-old male wild-type (Wt) mice on a C57BL/6J background and a mouse developed by Dr. Thomas H Bugge (Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health; Bethesda, MD) on a C57BL/6J background (Plau^GFDhu/GFDhu) in which a 4-amino acid substitution into the growth factor domain of uPA abrogates its binding to uPAR while preserving other functions of the protease and its receptor.²⁶ Experiments were approved by and conducted following rules and regulations of the Institutional Animal Care & Use Committee of Emory University and reported according to the guidelines from ARRIVE (Animal Research: Reporting In Vivo Experiments). Recombinant murine uPA (ruPA) and uPA’s amino terminal fragment (ATF) were purchased from Molecular Innovations (Novi, MI). Other reagents were ezrin small interfering RNA (siRNA) and scramble siRNA (Dharmacon; Lafayette, CO); the ROCK inhibitor Y-27632 (Millipore; Burlington, MA); the nuclear marker Hoechst 33342, secondary Alexa antibodies anti-rabbit 488, anti-goat 594, anti-mouse 488, and anti-rabbit 350 (Invitrogen; Waltham, MA); cycloheximide (Sigma-Aldrich; St. Louis, MO); the GS inhibitor L-methionine sulfoximine (Acros Organics, New Jersey); and antibodies against GFAP (DAKO; Santa Clara, CA); uPAR and normal goat IgG (R&D Systems; Minneapolis, MN); ezrin, GS, synaptophysin, F4/80, glutamate transporter 1 (GLT-1), radixin, and moesin (Abcam; Cambridge, MA); actin (Sigma-Aldrich; St. Louis, MO); PSD-95 and normal rabbit IgG control (Cell Signaling; MA); normal mouse IgG (Santa Cruz, Dallas, TX); and ezrin, radixin, and moesin phosphorylated at Thr567/Thr564/Thr558, respectively (pERM, Cell Signaling; Denvers, MA). TaqMAn primers for murine ezrin (Mm00447761_m1) and GAPDH mRNA (Mm99999915_g1) were purchased from Thermofisher (Waltham, MA).

Astrocytic and neuronal cultures

Wt astrocytes and Wt cerebral cortical neurons were cultured, as described elsewhere, from 1-day-old and E16-18 mice, respectively.²⁷ Briefly, the cerebral cortex was dissected, transferred into Hanks’ balanced salt solution containing 100 units/ml penicillin, 100 µg/ml streptomycin, and 10 mM HEPES, and incubated in trypsin containing 0.02% DNase at 37℃ for 15 min. Tissue was triturated, and the supernatant was resuspended in GS21-supplemented neurobasal medium containing 2 mM l-glutamine and plated onto 0.1 mg/ml poly-l-lysine-coated wells. For astrocytes, the triturated tissue was resuspended in 10% fetal bovine serum Dulbecco’s Modified Eagle’s Medium and filtered through a 70 -µm pore membrane. Then cells were plated onto poly-l-lysine-coated T75 flasks. Fourteen days later, astrocytes were plated and used for experiments upon confluency 7 to 14 days thereafter. To test the purity of glial cultures, astrocytes plated on coverslips were stained with antibodies against GFAP and F4/80 and counterstained with Hoechst. We found that from a total of 1038 Hoechst-positive cells examined, 96.41 ± 1.44% were astrocytes, 2.5 ± 1.8% were microglia, and 0.6 ± 1.0% were other cell types.

Animal model of cerebral ischemia

Transient occlusion of the middle cerebral artery (tMCAo) was induced in Wt and Plau^GFDhu/GFDhu mice with a 6-0 silk silicone-coated suture advanced from the external carotid artery into the internal carotid artery and then until the origin of the middle cerebral artery (MCA) as described.²⁵ The suture was withdrawn after 5 or 30 min of cerebral ischemia. Cerebral perfusion (CP) in the distribution of the MCA was monitored throughout the surgical procedure and after reperfusion with a laser Doppler (Perimed), and only animals with a > 80% decrease in CP after occlusion and complete recovery after suture withdrawn were included in this study. The rectal and masseter muscle temperatures were controlled at 37℃ with a homoeothermic blanket. Heart rate and systolic, diastolic, and mean arterial blood pressures were controlled throughout the surgical procedure with an IITC 229 System (IITC-Life Science; Woodland Hills, CA).

Immunocytochemistry

Wt astrocytes were left untreated or incubated during 30 min with 5 nM of uPA or a comparable volume of vehicle (control). At the end of the experiment, cells were fixed with 4% paraformaldehyde, washed 3 times in tris buffered saline (TBS) without triton for samples that were not permeabilized and incubated during 30 min in a blocking solution containing 1 ml of 0.2 mM glycine, 20 μl/ml casein and 5 μl/ml of donkey serum. Then samples were kept overnight in a solution containing antibodies against either GFAP (1:1000) and uPAR (1:100), or GFAP, uPAR and GS (1:100), or GFAP and GS, or ezrin (1:400) and GFAP, or pERM (1:100) and GFAP, or ezrin (1:400) and ICAM-5 (1:200), or normal rabbit IgG control (1:100), or normal goat IgG control (1:100). Secondary antibodies were anti-rabbit Alexa 488 (1:500), anti-goat Alexa 594 (1:500), and anti-mouse Alexa 488 (1:500). To study the uPAR/GFAP and GFAP/uPAR immunostaining ratios, heat maps were created with the HeatMap histogram plugin of ImageJ from pictures taken with a 40 × lens. To obtain the uPAR/GFAP and GFAP/uPAR ratios, we used the Olympus CellSens Dimension 1.17 software to divide the red channel (uPAR immunostaning) by the green channel (GFAP immunoreactivity; uPAR/GFAP ratio) and the green channel by the red channel (GFAP/uPAR ratio). To quantify the intensity profile of uPAR and GFAP immunostaning, a 60 µm line was drawn over 250 pixels from the cell border until the perinuclear region of Wt astrocytes. Then the intensities of uPAR and GFAP immunostaining were quantified in each pixel with the profile tool of ImageJ. To quantify the mean GFAP−/GS+ area in Wt astrocytes treated with uPA and immunostained with anti-GFAP and -GS antibodies as described above, the areas immunoreactive to GFAP and GS antibodies were traced and measured separately with the OLYMPUS CellSens Dimension 1.17 software. Then, the GFAP+ area was subtracted from the GS +area, and the value was divided by the GS+ area. To quantify the number of ezrin-positive processes, pictures were taken with a 60 × lens using an Olympus BX51 microscope from astrocytes immunostained as described above with antibodies against ezrin and GFAP following 30 min of treatment with uPA or vehicle (control). Processes were manually quantified using the ImageJ software. To study the interaction between PAPs and the synapse, Wt astrocytes (10,000) were added to plates containing 10,000 DIV (days in vitro) 12–14 Wt neurons and treated three days later with 5 nM of uPA or a comparable volume of vehicle (control). Thirty minutes later, cells were fixed and immunostained with antibodies against ezrin and ICAM-5 as described above. Images were taken with the 60× lens of a Fluoview FV10i confocal laser-scanning microscope (Olympus), with the pinhole configured at 1X UA. For image analysis, images were electronically enhanced 282 times, traces were drawn over the dendrites surrounding an astrocyte, and the number of PAPs-dendritic contacts denoted by ezrin/ICAM-5-positive puncta/20 µm was manually quantified.

Immunohistochemistry

To quantify the number of PAPs in contact with the synapse and intact synaptic contacts, Wt mice underwent tMCAo as described above. Thirty minutes later, the suture was withdrawn and animals were intravenously treated with 0.1 mg/kg of ruPA or a comparable volume of saline solution (SS). A subgroup of mice was intraperitoneally treated immediately after the onset of transient middle cerebral artery occlusion and following suture withdrawal with 10 mg/kg of the GS inhibitor L-methionine sulfoximine. One hour later, brains were harvested, cut onto 30 µm slices, incubated during 2 h in 0.5% triton/TBS, treated with pepsin for 2 min at 37 C, blocked with 3% bovine serum albumin/TBS, and incubated overnight with antibodies against either ezrin (1:100) and synaptophysin (1:100), or synatophysin and PSD-95 (1:100), or anti-GLT-1 (1:100) and synaptophysin (1:100), followed by the addition of secondary anti-mouse Alexa Fluor 488 (1:500) and anti-rabbit Alexa 594 (1:500) antibodies for 1 h. Images were obtained with the 60× lens of a Fluoview FV10i confocal laser-scanning microscope (Olympus), with a pinhole configured at 1X UA, at bregma 0.14 mm, −0.46 mm, and −1.46 mm.²⁸ The number of ezrin/synaptophysin-, synaptophysin/PSD-95 -, and GLT-1/PSD-95-positive puncta was quantified in images enlarged 4.7 times with the plugin puncta analyzer of ImageJ.

Quantification of astrocytic stellation

Changes in the shape of Wt astrocytes were continuously monitored during 60 min following treatment with 5 nM of uPA or vehicle (control), with a live IX83 Olympus microscope connected to an incubator (Tokai Hit, Japan) with a 21% FiO₂. Images were recorded with a DP80 camera (Olympus, Waltham, Massachusetts) and analyzed with the drawing tool of the software OLYMPUS CellSens Dimension 1.17. In a subgroup of experiments, Wt astrocytes were stained with anti-GS antibodies as described above following 30 min of incubation with 5 nM of uPA, alone or in combination with 10 µM of the ROCK inhibitor Y-27632, or with uPA’s ATF (devoid of proteolytic activity), or with a comparable volume of vehicle (control). A subgroup of astrocytes was treated three days before the experiment with 1 µM of ezrin-siRNA or scramble (Sc)-siRNA and then incubated with uPA or PBS as described above. Ezrin downregulation was confirmed 72 h after siRNA treatment with immunocytochemistry with anti-ezrin antibodies. In all experimental groups, the GS-immunoreactive area was traced with the drawing tool and the area, perimeter, and shape factor were obtained with the software OLYMPUS CellSens Dimension 1.17.

Preparation of synaptoneurosomes

Wt astrocytes (300,000) were added to wells containing 750,000 DIV 14 Wt neurons and treated during 30 min with 5 nM of uPA or a comparable volume of vehicle (control). The left frontoparietal cortex of Wt and Plau^GFDhu/GFDhu mice was dissected following 5 min of tMCAo or sham operation. In both cases, synapse-enriched fractions containing the sealed presynaptic terminal and the attached postsynaptic membrane (synaptoneurosomes) were prepared according to a modification of published protocols.^29–32 Briefly, cells and brains were homogenized and centrifuged at 2000g during 5 min. Pellets were discarded and the supernatants centrifuged in an SS-20 fixed angle rotor at 32,000 g for 10 min. Pellets were resuspended and layered on the top of a 5, 9, and 10% discontinuous Ficoll (Fisher, Fair Lawn, NJ) gradient and centrifuged at 25,000 g for 25 min at 4℃ in a TLS 55 rotor using a Beckman Optima TLX tabletop ultracentrifuge. Synaptoneurosomes were collected from the 5/9% and 9/10% interfaces and then centrifuged at 35,000 r/min for 10 min. The pellet was resuspended in radioimmunoprecipitation assay buffer and used for Western blot analysis.

Western blot analysis

Wt astrocytes were treated during 30 min with 5 nM of uPA or ATF, or with uPA in combination with either 5 µg/ml of cycloheximide, or 10 µM of Y-27632, or with Y-27632 alone. Synaptoneurosomes were prepared from Wt neurons-Wt astrocytes cocultures, or from the left frontoparietal cortex of Wt and Plau^GFDhu/GFDhu mice after tMCAo or sham operation as described above. Protein concentration was quantified using the BCA assay, and 30 µg (brains) or 15 µg (cells) was loaded per sample, separated in a 4% to 20% precast linear gradient polyacrylamide gel (Bio-Rad, Hercules, CA), transferred to a polyvinylidene fluoride membrane by semi-dry transfer system, blocked with 5% nonfat dry milk in Tris-buffered saline pH 8.0 with 0.1% Tween 20 buffer, and immunoblotted with antibodies against actin and either GS (1:1000), or ezrin (1:2000), or pERM (1:1000), or moesin (1:1000), or radixin (1:1000). Membranes were developed in a Li-COR Odyssey Imaging System (Lincoln, NE). Densitometry analysis was performed in each band using the Image Studio (Li-COR).

Statistical analysis

Statistical analysis was performed with the t test and one- or two-way analysis of variance (ANOVA) with Dunnett’s, Kruskal–Wallis, or Tukey’s corrections, as appropriate. p values of < 0.05 were considered as significant.

Results

uPAR is found in PAPs

Our previous work shows that astrocytes express uPAR on their plasma membrane.²⁵ To further characterize these observations, nonpermeabilized Wt cerebral cortical astrocytes were immunostained with antibodies against uPAR followed by permeabilization and staining with anti-GFAP antibodies. Strikingly, these studies revealed that uPAR is found in the membrane of astrocytic elongations that are not immunoreactive to GFAP antibodies (Figure 1(A) and (B)). Because PAPs are highly motile structures immunoreactive to GS but not to GFAP,¹² we performed similar observations in astrocytes triple-stained with antibodies against GFAP, GS, and uPAR. Our results indicate that uPAR is abundantly expressed in PAPs (Figure 1(C)).

Figure 1.

uPA is found in peripheral astrocytic processes. (A) Representative micrographs taken with a 40× lens of a Wt astrocyte stained with antibodies against GFAP (green; panels a, c, and d) and uPAR (red; panels b, c, and d). Panel c is a merged image, and panel d corresponds to an electronic magnification of the area demarcated by the dashed square in c. Arrows denote an example of an uPAR+/GFAP− astrocytic elongation. (B) Heat maps of the Wt astrocyte depicted in (A). Panels a and b denote the uPAR/GFAP and GFAP/uPAR immunostaining intensity ratio, respectively. Panel c indicates the mean intensity of GFAP (green line) and uPAR (red line) immunostaining in each pixel over the dashed line denoted in panel b. n = 15 cells examined per group; p = 0.0001 when the average of the intensity of GFAP staining over the 250 pixels examined per cell is compared to that of uPAR immunoreactivity; t test. Panel d corresponds to a representative micrograph of an astrocyte stained with normal rabbit IgG and normal goat Ig G (isotype controls for anti-GFAP and -uPAR antibodies), at the same concentrations used for the anti-GFAP and -uPAR antibodies. (C) Representative micrographs taken with a 60× lens of an astrocytic elongation immunostained with antibodies against GFAP (white in a and blue in c and d), GS (white in b and green in c and d), and uPAR (red in c and d). Arrows denote the presence of uPAR in a GS+/GFAP− astrocytic elongation. GFAP: glial fibrillary acidic protein; uPAR: upon binding to its receptor; GS: glutamine synthetase.

uPA induces the formation of PAPs

Because uPA is the ligand for uPAR, then we measured the area immunoreactive to GS but not GFAP (denotes PAPs) in Wt astrocytes incubated 30 min with 5 nM of uPA or a comparable volume of vehicle (control). Our data indicate that the mean percentage of the total astrocytic area immunoreactive to GS but not to GFAP increases from 24.9 ± 16% in control cells to 38.8 ± 24.3% in uPA-treated astrocytes (Figure 2(A) and (B); n = 55 astrocytes; p = 0.02; t test). Furthermore, our Western blot analysis shows that the expression of GS increases in uPA-treated astrocytes (Figure 2(C) and (D)) and that this effect is independent of plasmin generation as it is also observed upon treatment with uPA’s ATF (devoid of proteolytic activity; Figure 2(E) and (F)).

Figure 2.

uPA induces the formation of glutamine synthetase-rich peripheral astrocytic processes. (A) Representative micrographs taken with a 40× lens (panels a and c) and electronic magnification of the dashed squares drawn in each corresponding micrograph (panels b and d), of GFAP (red) and glutamine synthetase (GS; green) expression in a Wt astrocyte treated during 30 min with 5 nM of uPA or a comparable volume of vehicle (control: c). Arrowheads in b and d depict GFAP−/GS+ astrocytic elongations. (B) Mean percentage of the total astrocytic area immunoreactive to GS but not to GFAP in 55 Wt cells treated 30 min with uPA or vehicle (control: c). Lines denote SD. (C to F) Representative Western blot analysis of GS expression (C and E) and quantification of the mean intensity of the band (D and F) in extracts from Wt astrocytes incubated 30 min with 5 nM of either uPA (C and D) or its amino terminal fragment (ATF; E and F) or with a comparable volume of vehicle (control). n = 6 observations per experimental group in (C) and (D) and 4 in (E) and (F); lines in (D) and (F) denote SD. GFAP: glial fibrillary acidic protein; uPA: urokinase-type plasminogen activator; GS: glutamine synthetase.

uPA induces the synthesis of ezrin in astrocytes

Ezrin is a member of the ERM (ezrin, radixin and moesin) cystoskeleton-associated family of proteins¹⁹ that is required for the formation of PAPs.¹¹ Thus, to determine whether uPA has an effect on its expression, Wt astrocytes incubated 30 min with 5 nM of uPA or vehicle (control) were immunostained with antibodies against ezrin and GFAP. Our data indicate that uPA increases the expression of ezrin in astrocytes (Figure 3(A) and (B)) and, in line with these observations, that the mean number of ezrin+/GFAP− extensions per cell increases from 80.75 ± 15.5 in control astrocytes to 164.1 ± 24.68 in uPA-treated cells (Figure 3(C); n = 16 cells examined per experimental group; p = 0.01, t test). To further characterize these results, we studied the expression of ezrin, radixin and moesin in extracts from Wt astrocytes incubated 30 min with 5 nM of uPA. We found that uPA induces the expression of ezrin (Figure 3(D) and (E)) but not radixin (Figure 3(F) and (G)) or moesin (Figure 3(I) and (J)); and that the effect of uPA on the expression of radixin is plasminogen-independent (Figure 3(J) and (K)). Furthermore, although our quantitative RT-PCR studies indicate that uPA does not increase the abundance of ezrin mRNA in astrocytes (Figure 3(L)), we found that the effect of uPA on the expression of ezrin is abrogated by cycloheximide (Figure 3(M) and (N)). Together, these data reveal that uPA induces the translation of ezrin mRNA in astrocytes.

Figure 3.

Effect of uPA on the expression of astrocytic ezrin. (A) Panels a and d are representative micrographs taken with a 40 × lens of ezrin (red), GFAP (green), and Hoechst (blue) in a Wt astrocyte treated during 30 min with 5 nM of uPA or a comparable volume of vehicle (control). Panels b and e are an electronic magnification of the dashed rectangles denoted in panels a and b. Arrowheads in panel e denote ezrin +/GFAP− astrocytic elongations in uPA-treated cells. Panels c and f are heat maps of ezrin expression in the micrographs shown in panels a and d. Panels g and h correspond to a representative micrograph of an immunocytochemical staining (g) and corresponding heat map (h) of astrocytes stained with normal mouse IgG and normal rabbit IgG (isotype controls for anti-GFAP and -ezrin antibodies), at the same concentrations used for the anti-GFAP and -uPAR antibodies. (B) Mean intensity of ezrin immunostaining quantified in heat maps of 15 astrocytes incubated during 30 min with uPA or a comparable volume of vehicle (control). Lines denote SD. (C) Mean number of ezrin+/GFAP− processes per cell in Wt astrocytes treated with vehicle (control; n = 16) or 5 nM of uPA (n = 15). Lines denote SD. (D to I) Representative Western blot analyses (D, F, and H) and quantification of the mean intensity of the band (E, G, and I) of ezrin (D and E), radixin (F and G), and moesin (H and I) expression in extracts from Wt astrocytes treated with 5 nM of uPA or a comparable volume of vehicle (control: c). n = 5 observations in (D) and (E) and 4 in (F) to (I). Lines in (E), (G), and (I) denote SD. (J and K) Representative Western blot analyses (J) and quantification of the mean intensity of the band (K) of ezrin expression in extracts from Wt astrocytes treated with 5 nM uPA’s amino terminal fragment (ATF). (C): control-treated samples. n = 5 observations per experimental group. Lines in (K) denote SD. (L) Mean ezrin mRNA expression in Wt astrocytes treated 0 to 30 min with 5 nM of uPA. n = 4 observations per experimental group. (M and N) Representative Western blot analysis (M) and mean intensity of the band (N) of ezrin expression in Wt astrocytes incubated with 5 nM of uPA, alone or in the presence of 10 mg/ml of cycloheximide (CHX). n = 5 observations per experimental group. Lines in (N) denote SD. GFAP: glial fibrillary acidic protein; uPA: urokinase-type plasminogen activator; GS: glutamine synthetase; mRNA: messenger RNA.

uPA induces ROCK-mediated activation of ezrin in astrocytes

Because for its activation ezrin needs to be phosphorylated at Thr567 (pEzrin),¹¹ then we used immunocytochemistry and Western blot analysis with an antibody that recognizes ezrin, radixin, and moesin phosphorylated at Thr567/Thr564/Thr558 (pERM), respectively, to study the expression of pEzrin in Wt astrocytes incubated 30 min with 5 nM of uPA. Our studies indicate that uPA induces the recruitment of ezrin to the astrocytic plasma membrane (Figure 4(A)) and its subsequent activation by phosphorylation (Figure 4(B) and (C)). Because uPA activates the RhoA/ROCK pathway,²¹ and since this pathway induces the phosphorylation of ezrin,³³ we then decided to study the phosphorylation of ezrin in Wt cerebral cortical treated with uPA, alone or in the presence of the ROCK inhibitor Y-27632. We found that ROCK activation mediates the effect of uPA on ezrin phosphorylation (Figure 4(D) and (E)).

Figure 4.

uPA induces ROCK-mediated ezrin phosphorylation. (A) Representative micrographs taken with a 40 × lens of ERM phosphorylated at Thr567/Thr564/Thr558, respectively (pERM; white in panels a and d and green in panels b, c, e, and f), and GFAP (red in b, c, e, and f) in a Wt astrocyte treated with 5 nM of uPA or vehicle (control: c). Panels c and f are electronic magnifications of the area demarcated by the dashed squares in b and e. Arrows in f denote phosphorylated ezrin in GFAP-negative astrocytic processes. (B to E) Representative Western blot analysis (B and D) and quantification of the mean intensity of the band (C and E) of pERM expression in Wt astrocytes incubated during 30 min with 5 nM of uPA, alone (B and C) or in the presence of 10 μM of the ROCK inhibitor Y-27632 (D and E). n = 4 observations per experimental group. Lines denote SD. GFAP: glial fibrillary acidic protein; uPA: urokinase-type plasminogen activator; pERM: phosphorylated ezrin, radixin, and moesin.

uPA induces ezrin-mediated formation of PAPs

To determine whether ezrin mediates the effect of uPA on the formation of PAPs, we used live confocal microscopy to study changes in the perimeter/surface area and mean shape factor (indicative of PAPs formation) in Wt astrocytes incubated 0 to 30 min with 5 nM of uPA or its ATF. Our data indicate that uPA induces the formation of PAPs in Wt cells by a mechanism that does not require plasmin generation, as denoted by an increase in the perimeter/surface area and decrease in the mean shape factor in uPA- and ATF-treated astrocytes (Figure 5(A) to (C)). To study whether the synthesis of ezrin and its subsequent activation by ROCK-mediated phosphorylation mediate this effect, we performed similar observations in Wt astrocytes treated with uPA in the presence of either ezrin siRNA (Figure 5(D) to (F)), or the ROCK inhibitor Y-27362. Our data indicate that synthesis of ezrin (Figure 5(E) and (F)) and its subsequent activation by ROCK (Figure 5(G) and (H)) mediate the effect of uPA on the formation of PAPs.

Figure 5.

Ezrin mediates the effect of uPA on the formation of peripheral astrocytic processes. (A) Representative live confocal micrographs taken with a 40× lens from Wt astrocytes treated with 5 nM of uPA or a comparable volume of vehicle (control). The red lines outline the margins of each astrocyte. (B and C) Mean perimeter/area ratio (B) and shape factor (C) in Wt astrocytes treated 30 min with 5 nM of uPA (n = 80) or its amino terminal fragment (ATF; n = 72), or vehicle (control: c; n = 91). (D) Representative micrographs taken with a 20× lens of ezrin immunostaining (white) in Wt astrocytes treated with ezrin-siRNA or Sc-siRNA. (E to H) Mean perimeter/area ratio (E and G) and shape factor (F and H) in Wt astrocytes treated with 5 nM of uPA, alone or in the presence of either Sc-siRNA or ezrin siRNA (E and F), or 10 μM of the ROCK inhibitor Y-27632 (G and H). n = 75 per experimental group in (E) and (F) and 89 in (G) and (H). uPA: urokinase-type plasminogen activator; ATF: amino terminal fragment; siRNA: small interfering RNA.

Synaptic effect of uPA-induced formation of PAPs

Because PAP’s are highly dynamic structures that embrace the pre- and postsynaptic terminals,³⁴ we then studied whether those PAPs formed in uPA-treated astrocytes enter in direct contact with the synapse. Wt astrocytes were transferred to coverslips containing DIV 14 Wt cerebral cortical neurons and treated three days later with 5 nM of uPA or vehicle (control). Thirty minutes later samples were divided in two groups. The first was used for confocal microscopy studies to quantify the number of crossings of ezrin-positive PAPs with ICAM-5-positive synapses (ICAM-5 detects only the postsynaptic terminal of excitatory neurons³⁵). We found that the mean number of ezrin/ICAM-5-positive crossings/20 µm increases from 4.6 ± 2.5 in control cells to 8.9 ± 2.3 in uPA-treated astrocytes (Figure 6(A) to (C); n = 30 cells examined per experimental group; p < 0.0001, t test). The second sample was used to study the expression of GS in synaptoneurosomes prepared as described in the Methods section. Importantly, because synaptoneurosomes are assembled by the sealed axonal bouton and the attached postsynaptic terminal, and since GS is found in astrocytes but not in neurons,¹² then the detection of GS in these preparations is indicative of PAPs in direct contact with the synapse. In agreement with our immunocytochemical observations, our Western blot analyses revealed an increase in GS immunoreactivity in synaptoneurosomes prepared from uPA-treated cells (Figure 6(D) and (e)).

Figure 6.

Effect of uPA-induced formation of peripheral astrocytic processes on the ischemic synapse. (A and B) Panels a and c correspond to representative confocal micrographs taken with a 40× lens of ezrin (red) and ICAM-5 (green) immunoreactivity in Wt astrocytes added to wells containing DIV 14 Wt neurons and treated three days later during 30 min with 5 nM of uPA or a comparable volume of vehicle (control). Panels b and d correspond to an electronic magnification of the white squares in a and c. Arrows in d denote ezrin-positive peripheral astrocytic processes crossing with ICAM-5-positive postsynaptic terminals. (C) Mean number of ezrin/ICAM-5 crossings in Wt astrocytes/Wt neurons cocultures exposed to the experimental conditions described in (A) and (B). n = 30 observations per experimental group. Lines denote SD. (D to G) Representative Western blot analysis (D and F) and quantification of the mean intensity of the band (E and G) of GS expression in synaptoneurosomes prepared from either wells in which Wt astrocytes were added to Wt neurons and treated during 30 min with 5 nM of uPA or a comparable volume of vehicle (control: c; n = 4 observations per group; D and E), or the left frontoparietal cortex of Wt and Plau^GDDhu/GFDhu mice 5 min after either sham operation or transient occlusion of the middle cerebral artery (tMCAo; F and G). n = 5 animals per experimental group. ICAM-5: intercellular adhesion molecule 5; tMCAo: transient occlusion of the middle cerebral artery; Wt: wild type; uPA: urokinase-type plasminogen activator; GS: glutamine synthetase.

Because degradation of PAPs has been shown to have a deleterious effect in the ischemic brain,¹⁶ then to study the in vivo relevance of these findings we performed similar observations in synaptoneurosomes prepared from the left frontoparietal cortex of Wt and Plau^GFDhu/GFDhu mice [in which endogenous uPA does not bind to uPAR], 5 min after either tMCAo or sham operation. We chose 5 min because our previous studies indicate that this length of cerebral ischemia has an impact on synaptic structure without causing cell death.²⁵ We found that cerebral ischemia induces the formation of GS-positive PAPs that enter in direct contact with the ischemic synapse, and that this effect requires binding of endogenous uPA to uPAR (Figure 6(F) and (G)).

Effect of treatment with ruPA on the formation of PAPs in the ischemic brain

To investigate whether treatment with ruPA induces the formation of “tripartite synapses” in the ischemic brain, we used confocal microscopy to quantify the number of ezrin/synaptophysin-positive puncta (indicative of PAPs—synapse contacts) in the left frontoparietal cortex of Wt mice after 30 min of cerebral ischemia or sham operation followed by 1 h of treatment with ruPA or a comparable volume of SS (n = 3 per experimental group). Our data indicate that the number of PAPs/synapse contacts/2100 µm² of tissue increases from 7.2 ± 1.04 in nonischemic brains (n = 47 observations) to 19.13 ± 1.4 and 89.66 ± 33.09 in SS (n = 32 observations)- and ruPA (n = 35 observations)-treated animals, respectively (Figure 7(A) and (B); p = 0.94 when SS-treated tMCAo brains are compared to nonischemic brains and p = 0.01 when ruPA-treated tMCAo brains are compared to SS-tMCAo brains; two-way ANOVA with Tukey correction). Because the uptake and degradation by GS-rich PAPs of glutamate released in the synaptic cleft during the excitotoxic injury may have a protective effect on the synapse,^36,37 we quantified with confocal microscopy the number of intact synaptic contacts (denoted by synaptophysin/PSD-95-positive puncta) in the left frontoparietal cortex of Wt mice subjected to 30 min of tMCAo or sham operation and intravenous treatment with SS or ruPA, alone or in the presence of the GS inhibitor L-methionine sulfoximine, or with the inhibitor alone (n = 3 animals per experimental group). We found that the number of intact synaptic contacts/2100 µm² of tissue decreases from 434.9 ± 91.05 in the nonischemic brain (n = 29 observations), to 13.48 ± 2.85 and 145 ± 35.77 following cerebral ischemia and treatment with either SS (n = 29 observations) or ruPA (n = 30 observations; Figure 7(C); p < 0.0001 when comparing nonischemic brains to SS-treated tMCAOo animals, and p = 0.0008 when SS-treated tMCAo brains are compared to ruPA-treated tMCAo animals). Remarkably, the effect of treatment with ruPA on the number of intact synaptic contacts was abrogated by GS inhibition (27.74 ± 10.56; p = 0.03 when ruPA-treated brains are compared to animals treated with ruPA in the presence of a GS inhibitor; n = 29 observations; two-way ANOVA with Kruskal–Wallis correction). Importantly, the number of intact synaptic contacts did not decrease when animals kept under nonischemic conditions were treated with the inhibitor alone (n = 29 observations; p > 0.9 two-way ANOVA with Kruskal–Wallis correction). Because GS is an intracellular enzyme, thus glutamate transporters need to be expressed in PAPs to facilitate the uptake of glutamate released in the synaptic cleft into the PAP to be metabolized by GS. Then, we decided to use confocal microscopy to quantify the number of GLT-1/synaptophysin-positive crossings (indicative of GLT-1-containing PAPs in close proximity to the synapse) in nonpermeabilized brain sections of Wt mice subjected to the experimental conditions described above. Our data indicate that the number of GLT-1/synaptophysin crossings increases in the ischemic brain, and that this effect is significantly enhanced following ruPA treatment (Figure 7(D) and (E); n = 40 observations in 4 different animals; p = 0.008 when untreated tMCAo animals are compared to ruPA-treated tMCAo mice; two-way ANOVA with Tukey correction).

Figure 7.

Effect of treatment with recombinant uPA on the formation of peripheral astrocytic processes in the ischemic brain. (A and B) Representative confocal micrographs taken with a 60× lens and electronically magnified 4.7 times of ezrin (white in a, c, e, and g; and red in b, d, f, and h) and synaptophysin (green) expression (A), and mean number of ezrin/synaptophysin (SYP)-positive puncta (B) per 2100 µm² of tissue in the left frontoparietal cortex of Wt mice (n = 3 per group) after 30 min of tMCAo (panels c, d, g, and h) or sham operation (panels a, b, e, and f) followed by intravenous treatment with ruPA (e and f) or saline solution (SS; c and d). (C) Mean number of intact synaptic contacts denoted by their immunoreactivity to synaptophysin and PSD-95 in the left frontoparietal cortex of Wt mice (n = 3 per group) following 30 min of tMCAo and treatment with SS or ruPA, alone or in the presence of the GS inhibitor (GS-i) L-methionine sulfoximine, or with the inhibitor alone. (D and E) Representative confocal micrographs taken with a 60× lens and electronically magnified 4.7 times of GLT-1 (red) and synaptophysin (green) expression (D), and mean number of GLT-1/synaptophysin crossings (E) per 2100 µm² of tissue in the left frontoparietal cortex of Wt mice (n = 40 observations per group for four different animals) subjected to the experimental conditions described above. GFAP: glial fibrillary acidic protein; tMCAo: transient occlusion of the middle cerebral artery; GS: glutamine synthetase; ruPA: recombinant urokinase-type plasminogen activator.

Discussion

Despite the fact that it has been widely recognized that cerebral ischemia has a profound effect on synaptic structure and function that is directly linked to the development of neurological impairment following an acute ischemic stroke,¹⁴ to this date, there is no therapeutic strategy to promote synaptic recovery in the ischemic brain. Our earlier studies indicate that neurons release uPA and astrocytes recruit uPAR to their plasma membrane during the recovery phase from an ischemic stroke.^24,25 Here, we show that uPA released by the injured synapse acts as a signal that promotes the formation of PAPs in neighboring astrocytes, and that the interaction of these astrocytic elongations with the pre- and postsynaptic terminals has a protective effect on the synapse that has suffered an acute ischemic injury.

The concept of “tripartite synapse” was proposed to describe the existence of a bidirectional communication between neurons and astrocytes³⁸ and to conceptualize the fact that astrocytes regulate synaptic function. Indeed, an increase in astrocytic Ca²⁺ leads to the release of neurotransmitters,³⁹ and it has been shown that astrocytes promote structural plasticity,⁴ and the formation and stabilization of new synapses.^5–8 In contrast, the role of astrocytes in the ischemic brain is less well understood. However, a growing body of experimental evidence indicates that astrocytes play a central role in the development of the ischemic injury,¹⁵ and that their activation is crucial for both the formation of new synapses⁴⁰ and the repair of those that have survived an acute ischemic stroke.²⁵ Our data indicate that the binding of neuronal uPA to astrocytic uPAR plays a central in the formation of “tripartite synapses” in the ischemic brain, and that the interaction between astrocytes and the pre- and postsynaptic terminals promoted by either the release of endogenous uPA or the treatment with ruPA has a protective effect on the integrity of those synapses that have suffered an acute ischemic injury.

Astrocytes interact with the synapse via highly motile elongations known as PAPs.²⁰ Significantly, although the interaction of PAPs with the synapse is crucial for the processing of neuronal information,³⁴ induction of synaptic plasticity,⁴¹ synapse formation and maturation,⁴² and maintenance of synaptic stability,⁴ their role in the ischemic brain is poorly understood. Nevertheless, it has been shown that cerebral ischemia induces the detachment of PAPs from the synapse of neurons that subsequently show signs of necrotic cell death,¹⁶ and that astrocytes have a protective effect in the ischemic brain.⁴³ Our studies indicate that uPA/uPAR binding induces the formation of PAPs in the ischemic brain and that these elongations protect the synapse, most likely by GS-mediated metabolism of glutamate that has been released in excitotoxic conditions in the synaptic cleft after the onset of the ischemic injury. Indeed, GS is found only in astrocytes,¹² and its main role is to catalyze the conversion of glutamate to glutamine.³⁶ However, to become available for degradation by GS, glutamate should enter the astrocytes via specific transporters, most likely GLT-1 (EAAT2). Our data indicating that treatment with ruPA increases the number of GLT-1/synaptophysin crossings in the ischemic brain support our hypothesis that ruPA-induced formation of PAPs contributes to the uptake of glutamate from the synaptic cleft and its subsequent degradation by GS. Importantly, our results do not indicate that uPA has a direct effect on the expression of GLT-1 but only in the formation of GLT-1-expressing PAPs that enter in close proximity with the synapse. Therefore, based on the data presented here, we propose a model whereby uPA protects the synapse in the ischemic brain by inducing the formation of GS-rich PAPs that upon entering in contact with the synapse scavenge from the synaptic cleft the glutamate that has been released at high concentrations during the excitotoxic injury. This hypothesis is supported by our observation that the protective effect of uPA on the synapse is abrogated by GS inhibition.

Ezrin is a member of the ERM (ezrin, radixin, and moesin) family of proteins which is involved in several cellular processes including cell migration, reorganization of the actin cytoskeleton, and formation of membrane protrusions.¹⁷ A substantial amount of evidence indicates that ezrin promotes tumor metastasis, which has been associated with changes in junctional remodeling and stability.¹⁹ In contrast with these observations, the role of ezrin in the brain is less well understood. However, it is known that it is required for the formation of PAPs,¹¹ and that its expression is associated with the growth of astrocytic tumors.⁴⁴ In this study, we show that uPA induces the translation of ezrin mRNA in astrocytes, which agrees with reports indicating the capability of these cells to translate transcripts in their PAPs.¹³ Most importantly, these data describe a new mechanism for the regulation of ezrin in the central nervous system. Significantly, we found that uPA does not have an effect on the expression of other ERM proteins in astrocytes, namely radixin and moesin.

The activation of ezrin begins with its recruitment to membrane regions rich in phosphatidyl-inositol 4,5-biphosphate, which renders its Thr567 residue accessible to phosphorylation by several kinases.⁴⁵ Our data indicate that uPA not only induces the translation of ezrin mRNA but also the recruitment of ezrin protein to the astrocytic plasma membrane and that this is followed by its phosphorylation via the RhoA-ROCK protein kinase pathway. These data agree with previous reports indicating that Rho-kinase phosphorylates ERM proteins,⁴⁶ and that ROCK mediates several biological functions of uPA.⁴⁷

The data presented here suggest a model whereby uPA either released from neurons or intravenously administered (ruPA) induces the synthesis and activation of ezrin in astrocytes, with the subsequent ezrin-mediated formation of PAPs that upon contacting the synapse protect its structural stability from the harmful effects on the ischemic injury. In summary, our results indicate that by regulating the expression of astrocytic ezrin and promoting ezrin-mediated formation of “tripartite synapses” uPA protects the synapse in the ischemic brain. This is a novel finding that not only unveils an unknown role for uPA but also has significant translational relevance for the treatment of acute ischemic stroke patients.

Footnotes

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported in part by National Institutes of Health Grants NS-091201 (to MY) and NS-079331 (to MY), and VA MERIT Award IO1BX003441 (to MY).

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Authors’ contributions

Ariel Diaz: designed experiments and performed research; Paola Merino: performed research; Luis Guillermo Manrique: performed research; Lihong Cheng: performed research; Manuel Yepes: designed research and wrote the paper.

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