Background
Astrocytes are the main type of glial cell in the central nervous system (CNS). Their functions range from nursing neurons to forming the blood-brain barrier. Under pro-inflammatory conditions induced by injury or neurodegenerative disorders, such as Alzheimer’s disease and amyotrophic lateral sclerosis (ALS), astrocytes become reactive and take on a more stellate shape, while increasing in number and size. At the same time, the expression of several proteins is enhanced, including that of glial fibrillary acidic protein (GFAP) and inducible nitric oxide synthase (iNOS) [
1‐
5]. This reactive process is referred to as astrogliosis [
6‐
9]. Astrogliosis has been recognized as an inhibitory cue for axon regeneration. Nevertheless, it may have initial benefits for the injured area, since astrocytes restrict any damage to a small area, repair the blood brain barrier, and eliminate toxic factors from the surrounding area [
8‐
10]. During astrogliosis, several molecules are released into the extracellular space, including ATP, adenosine, and a number of cytokines [
8,
9]. One of the most important cytokines released to the injured area is tumor necrosis factor (TNF) [
9,
11], which acts as an alert signal that induces reactivity of the neighboring astrocytes [
12,
13].
Reactive astrocytes are a heterogeneous population of cells, and recent in vivo studies indicate that some astrocytes proliferate, while for another subset of labeled astrocytes hypertrophy and polarization, but not overt migration towards the site of injury are detected [
14‐
16]. However, several previously reported studies indicate that reactive astrocytes migrate to the damaged zone and form the glial scar, repairing the wounded area [
6,
10,
17]. The protein osteopontin is among the various stimuli that induce cell migration; this requires interaction with α
Vβ
3 Integrin [
18,
19]. Importantly, reactive astrocytes have been shown to express increased levels of α
Vβ
3 Integrin, the P2X7 receptor (P2X7R), and the hemichannels Connexin-43 and Pannexin-1 [
4,
18,
20]. All these molecules are reportedly involved in astrocyte migration [
21,
22].
The neuronal surface glycoprotein Thy-1 binds to astrocytes and thereby promotes adhesion and migration, processes that our group has studied in depth. Thy-1-induced adhesion and migration require interaction with the α
Vβ
3 Integrin and Syndecan-4 receptors. These interactions trigger the recruitment of focal adhesion (FA) proteins and activate a signaling module, which in turn leads to the activation of the small GTPase RhoA and its effector ROCK and results in changes in the astrocyte actin cytoskeleton [
23‐
28]. The signaling cascade also includes the opening of the hemichannels Connexin-43 and Pannexin-1 and the release of ATP to the extracellular milieu, where it activates P2X7R, allowing calcium entry [
21,
22]. After a more prolonged stimulation, Thy-1 induces the activation of FAK, PI3-kinase, and the GTPase Rac1, thus leading to astrocyte migration [
26].
We have previously studied the Thy-1-induced signaling cascade using the DITNC1 astrocyte cell line. Considering that, in primary astrocytes, many of the molecules forming part of the Thy-1-induced signaling pathway are only present in low amounts [
29,
30], we hypothesized that primary astrocytes would be refractory to Thy-1 stimulus. Additionally, many signaling proteins, including the Thy-1 receptors, are upregulated under pro-inflammatory conditions [
3,
4,
18,
20,
30]. We therefore decided to directly study the effect of a pro-inflammatory environment on the response of primary astrocytes to Thy-1, and on the Thy-1-induced signaling cascade in these cells. This was achieved in two ways. Firstly, primary astrocytes were treated with the pro-inflammatory signaling factor TNF prior to Thy-1 application, comparing the results to those obtained from controls that had not been exposed to TNF. Secondly, astrocytes were used derived from an ALS model, a neurodegenerative disorder marked by neuroinflammation and the presence of reactive astrocytes [
31].
ALS is a late-onset disease, which generally appears after 50 years of age. It is characterized by motor neuron dysfunction and degeneration and leads to death from respiratory failure [
32‐
34]. Astrocytes from ALS patient samples are activated [
35] and show increased levels of Connexin-43 and GFAP [
3,
36]. In murine ALS models, astrocytes are activated within a time frame before the onset of disease [
31,
37]. Additionally, these reactive astrocytes are now recognized to assist ALS motor neuronal death by secreting inflammatory factors [
33,
36,
38‐
42]. To study the effect of Thy-1 signaling on astrocytes developing under the ALS neuroinflammatory conditions, the transgenic mouse carrying the human G93A mutated super oxide dismutase 1 (hSOD1
G93A) was employed, the most widely used ALS model [
1,
3,
33,
36,
39,
41‐
43].
As discussed above, Thy-1-mediated astrocyte adhesion and migration relies on the α
Vβ
3 Integrin receptor. Because β
3 Integrin is upregulated in the presence of TNF in various cell types [
18,
44,
45], it could be hypothesized that the effect of TNF on astrocyte reactivity is related to β
3 Integrin expression. To further explore the role of this receptor molecule, Thy-1 signaling in primary astrocytes was therefore investigated, in which β
3 Integrin expression had been either silenced or upregulated, both in the presence and absence of TNF.
Here, we show that primary astrocytes only responded to Thy-1 in a pro-inflammatory environment (+TNF), where proteins participating in the Thy-1 response were upregulated. In addition, neonatal primary astrocytes derived from a mouse model of ALS (hSOD1G93A) were found to express higher levels of reactivity markers and β3 Integrin, Syndecan-4, Connexin-43, and Pannexin-1 than those derived from wild-type non-transgenic littermates. The hSOD1G93A-derived astrocytes responded to Thy-1 without the need of cytokine addition. Importantly, in investigating the role of β3 Integrin in Thy-1-induced FA and astrocyte migration, over-expression of β3 Integrin was found to be sufficient to render primary astrocytes reactive and consequently responsive to Thy-1 stimulation in the absence of TNF. Conversely, silencing of β3 Integrin precluded the effect of Thy-1 on TNF-treated astrocytes. These results favor a working model in which inflammation enhances β3 Integrin expression, rendering astrocytes responsive to Thy-1 and thereby promoting their adhesion and migration, as required during the repair of the injured brain. Controlling β3 Integrin levels modulated astrocyte reactivity.
Methods
Animals
The care and use of rodents is detailed in protocols approved by the bioethical Committees of the Universidad de Chile and Universidad Andres Bello. Wistar neonatal rats (P1-P2) were obtained from the Universidad de Chile animal facility. Transgenic mice hemizygous for human SOD1
G93A (hSOD1
G93A) or non-transgenic mice (used as a control) were obtained from the Jackson Laboratory (Bar Harbor, USA). Transgenes were detected by PCR [
39].
Primary cultures
Astrocytes were obtained from 1- to 2-day-old wild-type rats or non-transgenic and hSOD1
G93A mice. Briefly, wild-type rats were sacrificed by decapitation. Brains were extracted and placed in ice-cold PBS under a microscope. Cortices were separated from brain and meninges and kept in fresh ice-cold PBS. To isolate the cells, cortices were treated with trypsin and mechanically disrupted. The resulting cell suspensions were passed through a 70-μm cell strainer, washed with DMEMF-12 (Gibco, Life Technologies, Grand Island, NY) supplemented with 10% FBS, and centrifuged at 1000 rpm. Following this, cells were counted and seeded at a density of 1.5 million cells per T-25 cell flask. The cell medium was changed every 48 h. Once confluent, neurons and other cell types were released to the supernatant by shaking in an orbital shaker at 180 rpm overnight. To induce a pro-inflammatory environment, rat primary astrocytes were stimulated with 10 ng/ml of TNF for 48 h. IL-1β, IL-6, or IFN-γ (R&D systems) were also used (kindly donated by Dr. Rommy von Bernhardi, P. Universidad Católica de Chile). Murine astrocyte cultures were prepared from the ventral spinal cord of P1-P2 hSOD1
G93A transgenic mice, or from non-transgenic littermates as previously described [
39,
41,
42]. Cultures were maintained in DMEM (Gibco, Life Technologies, Grand Island, NY) containing 10% FBS and 1% penicillin-streptomycin at 37 °C, 5% CO
2. Cultures reached confluence after 2–3 weeks and contained > 95% GFAP
+ astrocytes. Residual microglia were removed from both mouse and rat astrocytes by shaking cultures in an orbital shaker overnight (200 rpm).
Thy-1-Fc and Trail-R2-Fc preparation
Thy-1-Fc (wild-type and mutants) and Trail-R2-Fc fusion proteins were obtained as previously described [
21,
26]. Prior to their use, Thy-1-Fc and Trail-R2-Fc were incubated with Protein A in a 10:1 ratio wile rotating gently on a shaker for 1 h at 4 °C. Trail-R2-Fc is a fusion protein of the receptor for the soluble apoptosis-inducing ligand Trail-R2, which is used as a control for possible non-specific effects caused by the Fc portion of the Thy-1 fusion protein [
21]. Prior to each experiment involving Thy-1-Fc or Trail-R2-Fc treatment, astrocytes were serum-starved for at least 30 min in DMEM without serum.
Focal adhesion assay
Primary astrocytes were seeded on 12-mm coverslips and left to adhere for 24 h. Cells were then pre-treated with 10 ng/ml of TNF for 48 h to induce reactivity. Afterwards, astrocytes were rinsed with DMEM and left in serum-free medium for 30 min. This medium was then removed, and cells were incubated for 15 min with 4 μg of either Thy-1-Fc or Trail-R2-Fc coupled to Protein A in serum-free medium. FBS (3% for 5 min) was used as a positive control. Astrocytes were then washed and fixed for 10 min with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 for 10 min, and blocked in 2% bovine serum albumin (BSA)-PBS for 1 h. Cells were then incubated with anti-vinculin antibody (1:200, Sigma-Aldrich Co., Saint Louis, MO) for 1 h at 37 °C to stain for focal adhesions. Rhodamine-conjugated Phalloidin (Sigma-Aldrich Co., Saint Louis, MO) and DAPI (Sigma-Aldrich Co.) (0.025 μg/ml) were used to stain F-actin and nuclei, respectively. Quantification of focal adhesion area and number was performed as described previously [
21,
26].
Transfection of primary astrocytes
Astrocytes were transfected with pEGFP-β
3 Integrin (full length β
3 Integrin subunit kindly donated by Dr. C. Rüegg, University of Friboug, Switzerland [
46]) or a pool of three siRNAs against β
3 Integrin (Ambion) using the Amaxa Nucleofector system following the manufacturer’s instructions for the VPI-1006 transfection kit and the program T-020 (Lonza, Cologne, Germany).
Wound-healing assay
Primary astrocytes were seeded in 24-well plates (100,000 cells per well). After 48 h of incubation in complete medium with or without TNF (10 ng/ml), two parallel wounds were introduced with a micropipette tip. Detached cells were washed away with serum-free medium, and after 30 min of starvation, cells were stimulated with 4 μg of Thy-1-Fc, Thy-1(RLE)-Fc, or Trail-R2-Fc (negative control) in serum-free medium for 24 h. All proteins were coupled to Protein A as indicated above. Alternatively, primary astrocytes were transfected with pEGFP-β
3 Integrin or siRNA against β
3 Integrin, prior to wounding cell monolayers and stimulating as above without TNF pre-treatment. Wound healing was evaluated based on images of the cell-free area at 0 and 24 h, as previously described [
21,
26].
Western blot
Protein extracts were prepared in a lysis buffer (150 mM NaCl, 0.1% SDS, 0.25% sodium deoxycholate, 1% Triton-X100, in 50 mM Tris-HCl pH 7.4) supplemented with protease and phosphatase inhibitor cocktail (Biotool, Houston, TX). Extracts were electrophoretically separated on 10% SDS-PAGE gels and transferred to nitrocellulose (Millipore, Billerica, USA). The nitrocellulose was blocked with 5% w/v nonfat, dry milk in PBS containing 0.1% Tween-20 and subsequently incubated with the following primary antibodies: anti-β3 Integrin (1:2000, Millipore, Billerica, USA), anti-Syndecan-4 (1:2000, Abbexa, Cambridge, UK), anti-iNOS (Abbexa), anti-Connexin-43 (1:500; Santa Cruz Biotechnologies Dallas, TX), anti-Pannexin-1 (1:500; Santa Cruz Biotechnologies), anti-P2X7R (1:1000, Santa Cruz Biotechnologies), anti-GFAP (1:2000, Sigma-Aldrich Co.), and anti-misfolded human SOD1 (C4F6) (MediMabs, Quebec, Canada). The membrane was then washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000; Abbexa, Cambridge, UK) or donkey anti-goat IgG (1:5000; Abbexa) for 1 h at room temperature. Bands were visualized with a chemiluminescence kit (Pierce, Thermo Scientific, Rockford, IL), according to the manufacturer’s instructions. We performed Western blot quantification by measuring band intensity in Photoshop 7 (Adobe) and normalizing to the corresponding loading control. To determine fold increase, all values were normalized to the average value obtained for control bands after any treatment.
Indirect immunofluorescence assay
Astrocytes were seeded on 12-mm coverslips and left to adhere for 24 h. Cells were then washed and fixed as indicated for the focal adhesion assay. Next, cells were stained with anti-Connexin-43 (1:200), anti-Pannexin-1 (1:200), anti-Syndecan-4 (1:200) or anti-P2X7R (1:200) antibodies followed by secondary antibodies (1:400) conjugated to IF488 or IF594 (Abbexa), and DAPI (0.025 μg/ml). Samples were analyzed using a confocal microscope C2+ (Nikon, Japan), with a 60×/1.40 objective and using the NIS-Elements software.
Twelve thousand primary astrocytes were seeded in 48-well plates. After 24 h, cells were incubated in serum-free media containing 100 μM of the exonuclease inhibitor ARL-67156 (Santa Cruz Biotechnologies) for 30 min at 37 °C. Following this, cells were stimulated with Thy-1-Fc:Protein A for 15 min. Where indicated, cells were incubated with a combination of Heptanol (Sigma-Aldrich Co.) (500 μM) and Probenecid (Sigma-Aldrich Co.) (1 mM). Next, culture medium was recovered and centrifuged for 5 min at 1000×g. The supernatants were incubated in the dark with 50 μl CellTiter-Glo® reaction mix (Promega, Madison, WI). Luminescence intensity was determined in a Synergy2 multi-mode reader (Biotek Instruments, Inc., Vermont), and the values were interpolated using a calibration curve obtained from different ATP concentrations (0.01, 0.1, 1, and 10 μM).
Measurement of intracellular calcium kinetics
Primary astrocytes were seeded on 25-mm coverslips (400,000 cells) and left to adhere for 24 h. The cells were then incubated with 5 μM Fluo-4-AM (Life Technologies, Grand Island, NY) in Ringer solution at 37 °C for 30 min, after which they were washed and left in the same solution. Following this, astrocytes were treated with the hemichannel blockers Heptanol (500 μM) and Probenecid (1 mM) for 30 min. Alternatively, they were treated with the P2X7R blocker BBG (American Bioanalytical, Natick, MA) (5 μM for 1 h). Images were acquired at 2-s intervals with an XM10 camera (Olympus Corp.). Thy-1-Fc or Trail-R2-Fc, both coupled to Protein A, were added 120 s after initiating acquisition. Fluorescence intensity was quantified in 100 cells per condition using ImageJ. The results were expressed as (F-F0)/F0, where F is the change in fluorescence and F0 is the basal fluorescence. To measure calcium in astrocytes transfected with pEGFP-β3 Integrin, cells were loaded with the red probe Cal-590 AM (AAT Bioquest) at 4 μM following the manufacturers’ protocol.
Statistical analyses
Results are shown as the means ± standard error of the mean (s.e.m.) for n = 3 or more experiments. The results were analyzed using Kruskall-Wallis test and Dunn’s post-test. Statistical significance was set at p < 0.05.
Discussion
Inflammatory cytokines are widely considered causative in triggering astrocyte reactivity [
52]. Here, we show that wild-type primary astrocytes respond to Thy-1 by adhering and migrating only under inflammatory conditions (+TNF). We also show that in primary astrocytes, Thy-1 induces similar signaling pathways to those reported for DITNC1 astrocytes, namely, hemichannel opening, ATP release, activation of P2X7R, Ca
2+ entry, early cell adhesion, and finally, cell polarization and migration [
21,
26,
53,
54]. In addition, hSOD1
G93A-derived astrocytes respond to Thy-1 without any requirement for additional cytokines. Of note, ectopically expressed β
3 Integrin increases the levels of other important players of the Thy-1-induced signaling pathways, including Syndecan-4 and the Connexin-43/Pannexin-1 hemichannels, as well as markers of reactive astrocytes, such as iNOS and GFAP. Furthermore, over-expression of β
3 Integrin is sufficient to render astrocytes responsive to Thy-1. Therefore, we propose that β
3 Integrin plays a preponderant role in determining Thy-1-induced effects in astrocytes and possibly also in controlling astrocyte reactivity.
After injury to the CNS, microglia generate a pro-inflammatory environment by liberating TNF [
11]. This has been shown to enhance the expression of integrins [
55] and cell adhesion molecules, such as ICAM-1, VCAM-1, and E-selectin [
52,
56,
57]. In our studies, TNF induced astrocyte reactivity, increasing the expression levels of reported markers of astrocytosis as well as the Thy-1 receptors α
Vβ
3 Integrin and Syndecan-4 [
25,
28,
58]. Thy-1 receptor upregulation enables cells to respond to the ligand and thereby initiate astrocyte adhesion and migration. These findings suggest that a pro-inflammatory environment is required for astrocytes to become responsive to neuronal signals such as Thy-1. The low levels of β
3 Integrin observed in primary astrocytes may explain the unresponsiveness of these cells to Thy-1. In this context, it is of interest that TNF also induces α
Vβ
3 Integrin expression, along with a α
Vβ
3 Integrin-dependent cell migration, in endothelial cells [
44]; this indicates that increased α
Vβ
3 Integrin expression is relevant to migration under pro-inflammatory conditions in other cellular models.
In the present study, the application of TNF to primary astrocytes leads to an increased expression of α
Vβ
3 Integrin, Syndecan-4, P2X7R, Connexin-43, and Pannexin-1. All of these molecules have previously been shown to be required for Thy-1-induced cell migration [
21]. Others have reported that Connexin-43 hemichannnels are activated by treating astrocytes with a mixture of TNF and IL-1β. This leads to neuronal death, an effect not observed in astrocytes lacking Connexin-43 [
59]. Moreover, prenatal exposure to LPS, a well-known pro-inflammatory agent, induces activation of astrocytes with increased levels of Connexin-43 and Pannexin-1 [
4]. Additionally, treating astrocytes with TNF for only 3 h induces expression of Connexin-43 hemichannels [
60]. These results suggest that primary astrocytes exposed to a pro-inflammatory environment upregulate surface receptors involved in the Thy-1-Integrin-induced signaling pathways.
ATP release from hemichannels activated P2X7R and thus increased intracellular calcium, only after TNF treatment. ATP release and P2X7R activation has been previously shown to be involved in Thy-1-induced astrocyte responses, first inducing cell adhesion [
22] and subsequently migration [
21]. Of note, increased levels of ATP have been associated with brain damage in humans. Patients with radiation-induced brain injury show high levels of extracellular ATP, IL-6, and TNF in cephalorachidian fluid [
61]. In addition, exposure of primary astroglial cultures to radiation increases extracellular levels of ATP, IL-6, and TNF, unless P2X7R is inhibited or silenced [
61]. This suggests an important role for ATP and P2X7R in the activation of glial cultures and progression of brain injuries. Recently, increased expression of P2X7R and Connexin-43 has been linked to astrogliosis in autoimmune encephalomyelitis, where the increase of P2X7R coincided with the appearance of astrocyte reactivity markers, such as GFAP and S100β. The addition of P2X7R blockers led to a reduction in astrogliosis markers [
20], suggesting that the increase in P2X7R is involved in astrocyte reactivity. In line with this, published evidence indicates that P2X7R over-expression leads to microglia activation [
62,
63]. In the present study, P2X7R protein levels increased after TNF treatment, while the inhibition of P2X7R and the apyrase-catalyzed reduction of extracellular ATP both decreased Thy-1-induced astrocyte migration. However, it remains to be determined whether
P2X7R is a direct or an indirect target of TNF.
The increase in [Ca
2+]
i induced by Thy-1 stimulus is necessary for astrocyte migration. Ca
2+ influx and Ca
2+ release from intracellular stores via stimulation of IP3R participate in Thy-1-induced astrocyte migration, since P2X7R silencing as well as the addition of IP3R inhibitors prevent the migration of astrocytes with a reactive phenotype [
21]. In addition, the calcium-chelator BAPTA-AM blocks Thy-1-induced hemichannel opening and ATP release, two key steps in the process of Thy-1-induced DITNC1 astrocyte migration [
21]. In agreement with our observations, Fang and coworkers published that Orexin, a neuropeptide that evokes responses similar to those triggered by Thy-1 in astrocytes, induces migration in rat astrocytes in a Ca
2+-dependent manner, which can be blocked by either the addition of BAPTA-AM or an IP3R blocker, such as 2-APB [
64], supporting the notion that increased [Ca
2+]
i is necessary for astrocyte migration.
In primary astrocytes, TNF treatment is required for the Thy-1 response; however, TNF by itself neither induces migration nor increases Ca
2+ levels (Figs.
1c, d and
2d), but rather promotes changes in protein expression to generate a migratory phenotype responsive to Thy-1. Of note, α
Vβ
3 Integrin, Syndecan-4, and P2X7 receptors trigger parallel and different signaling pathways [
21]; thus, although elevated [Ca
2+]
i is required for Thy-1-stimulated astrocyte migration, it is not expected to be sufficient alone to induce the migratory phenotype, which results from more complex and interconnected signaling cascades.
Thy-1 elicits responses by engaging its receptors α
Vβ
3 Integrin and Syndecan-4 [
21,
22,
26]. Interestingly, β
3 Integrin over-expression was sufficient to enable primary astrocytes to respond to Thy-1, thereby showing an effect similar to that of TNF treatment. Others have reported an increased cell migration, invasion and tumorigenesis of cancer cells over-expressing β
3 Integrin following stimulation with osteopontin [
65], an extracellular matrix protein, which is upregulated and secreted after TNF treatment, and which induces directional astrocyte migration in an α
Vβ
3 Integrin-dependent manner [
18,
19]. This is in line with our findings, since it indicates that increased levels of β
3 Integrin protein make cells more responsive to external cues. However, over-expression of β
3 Integrin alone does not induce tumorigenesis in the absence of osteopontin [
65]. This is paralleled by our own observation that in the absence of Thy-1, migration was not increased in β
3 Integrin over-expressing astrocytes, confirming that elevated levels of β
3 Integrin render astrocytes sensitive to the neuronal Thy-1, rather than increasing cell autonomous migration. The intriguing possibility that increased α
Vβ
3 Integrin expression might guide reactive astrocytes to regions rich in “damage” signals, allowing them to interact with neuronal Thy-1 in the damaged zone, awaits further studies.
Furthermore, β
3 Integrin appears to control the expression of other proteins involved in the Thy-1 response. It has been reported that α
Vβ
3 Integrin acts upstream of NF-κB in several cell types [
66‐
68] and that tyrosine 759 of β
3 Integrin is required for NF-κB activation [
68], suggesting that this pathway, or a similar one, might be activated upon over-expression of β
3 Integrin. Like α
Vβ
3 Integrin, the expression of the Thy-1 co-receptor, Syndecan-4, is also increased in an NF-κB-dependent manner when EAhy926 cells are treated with TNF [
69]. It is possible that NF-κB activation might increase expression of the surface proteins reported in the present study in response to TNF. In hepatocytes from acute-on-chronic liver failure, increased levels of NF-κB are associated with increased expression of Connexin-43, but not Connexin-26 or Connexin-32 [
70]. In isolated aorta sections, Connexin-43 levels are increased downstream of NF-κB and ERK1/2 activation [
71]. Additionally, IL-8 regulates α
Vβ
3 Integrin expression in a PI3K/Akt/NF-κB-dependent manner in MDA-MB-231 cancer cells [
45]. Similarly, when glioblastoma multiforme cells are treated with the phorbol ester PMA, NF-κB induces the expression of α
Vβ
3 Integrin along with fibronectin and vitronectin [
72]. This suggests a possible role for NF-κB in upregulating the expression of components in the Thy-1 activated pathway. Consequently, β
3 Integrin could be placed upstream or downstream of NF-κB. Considering that β
3 Integrin over-expression is sufficient to increase the expression of reactive astrocyte markers and to render astrocytes sensitive to Thy-1 stimulation in the absence of TNF, and since β
3 Integrin silencing precludes the effect of Thy-1 in TNF-treated cells, we propose that β
3 Integrin expression can control astrocyte reactivity. Whether it does so by activating NF-κB remains to be assessed.
To place our findings into a patho-physiological context, astrocytes were analyzed from the hSOD1
G93A ALS mouse model. In this model, astrocytes are reported to be reactive before the onset motor symptoms, and the degree of reactivity correlates with the neurodegenerative process [
50]. Elevated expression levels of GFAP, Vimentin, and CD44 in tissue sections from post-mortem spinal cord in ALS patients also indicate astrocyte reactivity [
36]. Here, Thy-1 is shown to induce adhesion and migration of hSOD1
G93A-derived astrocytes without the need of prior TNF treatment. As might be expected, Connexin-43 levels substantially increased in these cells (Fig.
4c; see also [
3]). For P2X7R, modest levels of expression are associated with increased ALS severity, as well as increased astrocyte and microglia reactivity [
1]. Consistent with this, we observed low levels of P2X7R in hSOD1
G93A astrocytes, suggesting that these levels are sufficient for Thy-1-induced migration. Therefore, astrocytes derived from hSOD1
G93A mice are reactive and responsive to Thy-1. Notably, unlike P2X7R, Pannexin-1, Syndecan-4, and α
Vβ
3 Integrin expression levels were increased. Such alterations have not been associated with ALS to date, and further studies are required to clarify the specific contribution of each of these proteins.