Impacts of PI3K/protein kinase B pathway activation in reactive astrocytes: from detrimental effects to protective functions.

ABSTRACT: Astrocytes are the most abundant type of glial cell In the central nervous system. Upon Injury and inflammation, astrocytes become reactive and undergo morphological and functional changes. Depending on their phenotypic classification as A1 or A2, reactive astrocytes contribute to both neurotoxic and neuroprotective responses, respectively. However, this binary classification does not fully capture the diversity of astrocyte responses observed across different diseases and injuries. Transcriptomic analysis has revealed that reactive astrocytes have a complex landscape of gene expression profiles, which emphasizes the heterogeneous nature of their reactivity. Astrocytes actively participate in regulating central nervous system inflammation by interacting with microglia and other cell types, releasing cytokines, and influencing the immune response. The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway is a central player in astrocyte reactivity and impacts various aspects of astrocyte behavior, as evidenced by in silico, in vitro, and in vivo results. In astrocytes, inflammatory cues trigger a cascade of molecular events, where nuclear factor-κΒ serves as a central mediator of the pro-inflammatory responses. Here, we review the heterogeneity of reactive astrocytes and the molecular mechanisms underlying their activation. We highlight the involvement of various signaling pathways that regulate astrocyte reactivity, including the PI3K/AKT/ mammalian target of rapamycin (mTOR), αvß3 integrin/PI3K/AKT/connexin 43, and Notch/ PI3K/AKT pathways. While targeting the inactivation of the PI3K/AKT cellular signaling pathway to control reactive astrocytes and prevent central nervous system damage, evidence suggests that activating this pathway could also yield beneficial outcomes. This dual function of the PI3K/AKT pathway underscores its complexity in astrocyte reactivity and brain function modulation. The review emphasizes the importance of employing astrocyte-exclusive models to understand their functions accurately and these models are essential for clarifying astrocyte behavior. The findings should then be validated using in vivo models to ensure real-life relevance. The review also highlights the significance of PI3K/AKT pathway modulation in preventing central nervous system damage, although further studies are required to fully comprehend its role due to varying factors such as different cell types, astrocyte responses to inflammation, and disease contexts. Specific strategies are clearly necessary to address these variables effectively.

Thy-1 (CD90)-regulated cell adhesion and migration of mesenchymal cells: insights into adhesomes, mechanical forces, and signaling pathways.

Cell adhesion and migration depend on the assembly and disassembly of adhesive structures known as focal adhesions. Cells adhere to the extracellular matrix (ECM) and form these structures via receptors, such as integrins and syndecans, which initiate signal transduction pathways that bridge the ECM to the cytoskeleton, thus governing adhesion and migration processes. Integrins bind to the ECM and soluble or cell surface ligands to form integrin adhesion complexes (IAC), whose composition depends on the cellular context and cell type. Proteomic analyses of these IACs led to the curation of the term adhesome, which is a complex molecular network containing hundreds of proteins involved in signaling, adhesion, and cell movement. One of the hallmarks of these IACs is to sense mechanical cues that arise due to ECM rigidity, as well as the tension exerted by cell-cell interactions, and transduce this force by modifying the actin cytoskeleton to regulate cell migration. Among the integrin/syndecan cell surface ligands, we have described Thy-1 (CD90), a GPI-anchored protein that possesses binding domains for each of these receptors and, upon engaging them, stimulates cell adhesion and migration. In this review, we examine what is currently known about adhesomes, revise how mechanical forces have changed our view on the regulation of cell migration, and, in this context, discuss how we have contributed to the understanding of signaling mechanisms that control cell adhesion and migration.

Protein kinase B (AKT) upregulation and Thy-1-αvß3 integrin-induced phosphorylation of Connexin43 by activated AKT in astrogliosis.

BACKGROUND: In response to brain injury or inflammation, astrocytes undergo hypertrophy, proliferate, and migrate to the damaged zone. These changes, collectively known as "astrogliosis", initially protect the brain; however, astrogliosis can also cause neuronal dysfunction. Additionally, these astrocytes undergo intracellular changes involving alterations in the expression and localization of many proteins, including αvß3 integrin. Our previous reports indicate that Thy-1, a neuronal glycoprotein, binds to this integrin inducing Connexin43 (Cx43) hemichannel (HC) opening, ATP release, and astrocyte migration. Despite such insight, important links and molecular events leading to astrogliosis remain to be defined. METHODS: Using bioinformatics approaches, we analyzed different Gene Expression Omnibus datasets to identify changes occurring in reactive astrocytes as compared to astrocytes from the normal mouse brain. In silico analysis was validated by both qRT-PCR and immunoblotting using reactive astrocyte cultures from the normal rat brain treated with TNF and from the brain of a hSOD1G93A transgenic mouse model. We evaluated the phosphorylation of Cx43 serine residue 373 (S373) by AKT and ATP release as a functional assay for HC opening. In vivo experiments were also performed with an AKT inhibitor (AKTi). RESULTS: The bioinformatics analysis revealed that genes of the PI3K/AKT signaling pathway were among the most significantly altered in reactive astrocytes. mRNA and protein levels of PI3K, AKT, as well as Cx43, were elevated in reactive astrocytes from normal rats and from hSOD1G93A transgenic mice, as compared to controls. In vitro, reactive astrocytes stimulated with Thy-1 responded by activating AKT, which phosphorylated S373Cx43. Increased pS373Cx43 augmented the release of ATP to the extracellular medium and AKTi inhibited these Thy-1-induced responses. Furthermore, in an in vivo model of inflammation (brain damage), AKTi decreased the levels of astrocyte reactivity markers and S373Cx43 phosphorylation. CONCLUSIONS: Here, we identify changes in the PI3K/AKT molecular signaling network and show how they participate in astrogliosis by regulating the HC protein Cx43. Moreover, because HC opening and ATP release are important in astrocyte reactivity, the phosphorylation of Cx43 by AKT and the associated increase in ATP release identify a potential therapeutic window of opportunity to limit the adverse effects of astrogliosis.

Antidepressant-relevant behavioral and synaptic molecular effects of long-term fasudil treatment in chronically stressed male rats.

Several lines of evidence suggest that antidepressant drugs may act by modulating neuroplasticity pathways in key brain areas like the hippocampus. We have reported that chronic treatment with fasudil, a Rho-associated protein kinase inhibitor, prevents both chronic stress-induced depressive-like behavior and morphological changes in CA1 area. Here, we examined the ability of fasudil to (i) prevent stress-altered behaviors, (ii) influence the levels/phosphorylation of glutamatergic receptors and (iii) modulate signaling pathways relevant to antidepressant actions. 89 adult male Sprague-Dawley rats received intraperitoneal fasudil injections (10 mg/kg/day) or saline vehicle for 18 days. Some of these animals were daily restraint-stressed from day 5-18 (2.5 h/day). 24 hr after treatments, rats were either evaluated for behavioral tests (active avoidance, anxiety-like behavior and object location) or euthanized for western blot analyses of hippocampal whole extract and synaptoneurosome-enriched fractions. We report that fasudil prevents stress-induced impairments in active avoidance, anxiety-like behavior and novel location preference, with no effect in unstressed rats. Chronic stress reduced phosphorylations of ERK-2 and CREB, and decreased levels of GluA1 and GluN2A in whole hippocampus, without any effect of fasudil. However, fasudil decreased synaptic GluA1 Ser831 phosphorylation in stressed animals. Additionally, fasudil prevented stress-decreased phosphorylation of GSK-3ß at Ser9, in parallel with an activation of the mTORC1/4E-BP1 axis, both in hippocampal synaptoneurosomes, suggesting the activation of the AKT pathway. Our study provides evidence that chronic fasudil treatment prevents chronic stress-altered behaviors, which correlated with molecular modifications of antidepressant-relevant signaling pathways in hippocampal synaptoneurosomes.

Thy-1 (CD90)-Induced Metastatic Cancer Cell Migration and Invasion Are ß3 Integrin-Dependent and Involve a Ca2+/P2X7 Receptor Signaling Axis.

Cancer cell adhesion to the vascular endothelium is an important step in tumor metastasis. Thy-1 (CD90), a cell adhesion molecule expressed in activated endothelial cells, has been implicated in melanoma metastasis by binding to integrins present in cancer cells. However, the signaling pathway(s) triggered by this Thy-1-Integrin interaction in cancer cells remains to be defined. Our previously reported data indicate that Ca2+-dependent hemichannel opening, as well as the P2X7 receptor, are key players in Thy-1-αVß3 Integrin-induced migration of reactive astrocytes. Thus, we investigated whether this signaling pathway is activated in MDA-MB-231 breast cancer cells and in B16F10 melanoma cells when stimulated with Thy-1. In both cancer cell types, Thy-1 induced a rapid increase in intracellular Ca2+, ATP release, as well as cell migration and invasion. Connexin and Pannexin inhibitors decreased cell migration, implicating a requirement for hemichannel opening in Thy-1-induced cell migration. In addition, cell migration and invasion were precluded when the P2X7 receptor was pharmacologically blocked. Moreover, the ability of breast cancer and melanoma cells to transmigrate through an activated endothelial monolayer was significantly decreased when the ß3 Integrin was silenced in these cancer cells. Importantly, melanoma cells with silenced ß3 Integrin were unable to metastasize to the lung in a preclinical mouse model. Thus, our results suggest that the Ca2+/hemichannel/ATP/P2X7 receptor-signaling axis triggered by the Thy-1-αVß3 Integrin interaction is important for cancer cell migration, invasion and transvasation. These findings open up the possibility of therapeutically targeting the Thy-1-Integrin signaling pathway to prevent metastasis.

Region-Specific Reduction of BDNF Protein and Transcripts in the Hippocampus of Juvenile Rats Prenatally Treated With Sodium Valproate.

Autism is a neurodevelopmental disorder characterized by a deep deficit in language and social interaction, accompanied by restricted, stereotyped and repetitive behaviors. The use of genetic autism animal models has revealed that the alteration of the mechanisms controlling the formation and maturation of neural circuits are points of convergence for the physiopathological pathways in several types of autism. Brain Derived Neurotrophic Factor (BDNF), a key multifunctional regulator of brain development, has been related to autism in several ways. However, its precise role is still elusive, in part, due to its extremely complex posttranscriptional regulation. In order to contribute to this topic, we treated prenatal rats with Valproate, a well-validated model of autism, to analyze BDNF levels in the hippocampus of juvenile rats. Valproate-treated rats exhibited an autism-like behavioral profile, characterized by a deficit in social interaction, anxiety-like behavior and repetitive behavior. In situ hybridization (ISH) experiments revealed that Valproate reduced BDNF mRNA, especially long-3'UTR-containing transcripts, in specific areas of the dentate gyrus (DG) and CA3 regions. At the same time, Valproate reduced BDNF immunoreactivity in the suprapyramidal and lucidum layers of CA3, but improved hippocampus-dependent spatial learning. The molecular changes reported here may help to explain the cognitive and behavioral signs of autism and reinforce BDNF as a potential molecular target for this neurodevelopmental disorder.

Connexins in Astrocyte Migration.

Astrocytes have long been considered the supportive cells of the central nervous system, but during the last decades, they have gained much more attention because of their active participation in the modulation of neuronal function. For example, after brain damage, astrocytes become reactive and undergo characteristic morphological and molecular changes, such as hypertrophy and increase in the expression of glial fibrillary acidic protein (GFAP), in a process known as astrogliosis. After severe damage, astrocytes migrate to the lesion site and proliferate, which leads to the formation of a glial scar. At this scar-forming stage, astrocytes secrete many factors, such as extracellular matrix proteins, cytokines, growth factors and chondroitin sulfate proteoglycans, stop migrating, and the process is irreversible. Although reactive gliosis is a normal physiological response that can protect brain cells from further damage, it also has detrimental effects on neuronal survival, by creating a hostile and non-permissive environment for axonal repair. The transformation of astrocytes from reactive to scar-forming astrocytes highlights migration as a relevant regulator of glial scar formation, and further emphasizes the importance of efficient communication between astrocytes in order to orchestrate cell migration. The coordination between astrocytes occurs mainly through Connexin (Cx) channels, in the form of direct cell-cell contact (gap junctions, GJs) or contact between the extracellular matrix and the astrocytes (hemichannels, HCs). Reactive astrocytes increase the expression levels of several proteins involved in astrocyte migration, such as αvß3 Integrin, Syndecan-4 proteoglycan, the purinergic receptor P2X7, Pannexin1, and Cx43 HCs. Evidence has indicated that Cx43 HCs play a role in regulating astrocyte migration through the release of small molecules to the extracellular space, which then activate receptors in the same or adjacent cells to continue the signaling cascades required for astrocyte migration. In this review, we describe the communication of astrocytes through Cxs, the role of Cxs in inflammation and astrocyte migration, and discuss the molecular mechanisms that regulate Cx43 HCs, which may provide a therapeutic window of opportunity to control astrogliosis and the progression of neurodegenerative diseases.

IL-17A levels increase in the infarcted region of the left ventricle in a rat model of myocardial infarction.

Th17 cells, a recently described subtype of CD4+ effector lymphocytes, have been linked to cell-mediated autoimmune and inflammatory diseases as well as to cardiovascular diseases. However, the participation of IL-17A in myocardial ischemic injury has not been clearly defined. We therefore conducted the present study to evaluate IL-17A and Th17-related cytokine levels in a rat model of myocardial infarction (MI). MI was induced in male Sprague Dawley rats by coronary artery ligation. Controls were sham-operated (Sh) or non-operated (C). Blood and samples from the left ventricle (LV) were collected at weeks 1 and 4 post-MI. At week 1, MI animals exhibited increased IL-6, IL-23 and TGF-ß mRNA levels with no apparent change in IL-17 mRNA or protein levels in whole LV. Only TGF-ß mRNA remained elevated at week 4 post-MI. However, further analysis revealed that IL-17A mRNA and protein levels as well as IL-6 and IL-23 mRNA were indeed increased in the infarcted region, though not in the remote non infarcted region of the LV, except for IL-23 mRNA. The increased expression of IL-17A and Th17-related cytokines in the infarcted region of LV, suggests that this proinflammatory pathway might play a role in early stages of post MI cardiac remodelling.

IL-17A levels increase in the infarcted region of the left ventricle in a rat model of myocardial infarction

Th17 cells, a recently described subtype of CD4+ effector lymphocytes, have been linked to cell-mediated autoimmune and inflammatory diseases as well as to cardiovascular diseases. However, the participation of IL-17A in myocardial ischemic injury has not been clearly defined. We therefore conducted the present study to evaluate IL-17A and Th17-related cytokine levels in a rat model of myocardial infarction (MI). MI was induced in male Sprague Dawley rats by coronary artery ligation. Controls were sham-operated (Sh) or non-operated (C). Blood and samples from the left ventricle (LV) were collected at weeks 1 and 4 post-MI. At week 1, MI animals exhibited increased IL-6, IL-23 and TGF-β mRNA levels with no apparent change in IL-17 mRNA or protein levels in whole LV. Only TGF-β mRNA remained elevated at week 4 post-MI. However, further analysis revealed that IL-17A mRNA and protein levels as well as IL-6 and IL-23 mRNA were indeed increased in the infarcted region, though not in the remote non infarcted region of the LV, except for IL-23 mRNA. The increased expression of IL-17A and Th17-related cytokines in the infarcted region of LV, suggests that this proinflammatory pathway might play a role in early stages of post MI cardiac remodelling.

Regulation of autoreactive B cell responses to endogenous TLR ligands.

Immune complexes containing DNA and RNA are responsible for disease manifestations found in patients with systemic lupus erythematosus (SLE). B cells contribute to SLE pathology through BCR recognition of endogenous DNA- and RNA- associated autoantigens and delivery of these self-constituents to endosomal TLR9 and TLR7, respectively. B cell activation by these pathways leads to production of class-switched DNA- and RNA-reactive autoantibodies, contributing to an inflammatory amplification loop characteristic of disease. Intriguingly, self-DNA and RNA are typically non-stimulatory for TLR9/7 due to the absence of stimulatory sequences or the presence of molecular modifications. Recent evidence from our laboratory and others suggests that B cell activation by BCR/TLR pathways is tightly regulated by surface-expressed receptors on B cells, and the outcome of activation depends on the balance of stimulatory and inhibitory signals. Either IFNalpha engagement of the type I IFN receptor or loss of IgG ligation of the inhibitory FcgammaRIIB receptor promotes B cell activation by weakly stimulatory DNA and RNA TLR ligands. In this context, autoreactive B cells can respond robustly to common autoantigens. These findings have important implications for the role of B cells in vivo in the pathology of SLE.

Neuronal Thy-1 induces astrocyte adhesion by engaging syndecan-4 in a cooperative interaction with alphavbeta3 integrin that activates PKCalpha and RhoA.

Clustering of alphavbeta3 integrin after interaction with the RGD-like integrin-binding sequence present in neuronal Thy-1 triggers formation of focal adhesions and stress fibers in astrocytes via RhoA activation. A putative heparin-binding domain is present in Thy-1, raising the possibility that this membrane protein stimulates astrocyte adhesion via engagement of an integrin and the proteoglycan syndecan-4. Indeed, heparin, heparitinase treatment and mutation of the Thy-1 heparin-binding site each inhibited Thy-1-induced RhoA activation, as well as formation of focal adhesions and stress fibers in DI TNC(1) astrocytes. These responses required both syndecan-4 binding and signaling, as evidenced by silencing syndecan-4 expression and by overexpressing a syndecan-4 mutant lacking the intracellular domain, respectively. Furthermore, lack of RhoA activation and astrocyte responses in the presence of a PKC inhibitor or a dominant-negative form of PKCalpha implicated PKCalpha and RhoA activation in these events. Therefore, combined interaction of the astrocyte alphavbeta3-integrin-syndecan-4 receptor pair with Thy-1, promotes adhesion to the underlying matrix via PKCalpha- and RhoA-dependent pathways. Importantly, signaling events triggered by such receptor cooperation are shown here to be the consequence of cell-cell rather than cell-matrix interactions. These observations are likely to be of widespread biological relevance because Thy-1-integrin binding is reportedly relevant to melanoma invasion, monocyte transmigration through endothelial cells and host defense mechanisms.

Toll-like receptor 9-dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE.

Increased concentrations of DNA-containing immune complexes in the serum are associated with systemic autoimmune diseases such as lupus. Stimulation of Toll-like receptor 9 (TLR9) by DNA is important in the activation of plasmacytoid dendritic cells and B cells. Here we show that HMGB1, a nuclear DNA-binding protein released from necrotic cells, was an essential component of DNA-containing immune complexes that stimulated cytokine production through a TLR9-MyD88 pathway involving the multivalent receptor RAGE. Moreover, binding of HMGB1 to class A CpG oligodeoxynucleotides considerably augmented cytokine production by means of TLR9 and RAGE. Our data demonstrate a mechanism by which HMGB1 and RAGE activate plasmacytoid dendritic cells and B cells in response to DNA and contribute to autoimmune pathogenesis.

Aggregation of integrins and RhoA activation are required for Thy-1-induced morphological changes in astrocytes.

Thy-1, a cell adhesion molecule abundantly expressed in mammalian neurons, binds to a beta(3)-containing integrin on astrocytes and thereby stimulates the assembly of focal adhesions and stress fibers. Such events lead to morphological changes in astrocytes that resemble those occurring upon injury in the brain. Extracellular matrix proteins, typical integrin ligands, bind to integrins and promote receptor clustering as well as signal transduction events that involve small G proteins and cytoskeletal changes. Here we investigated the possibility that the cell surface protein Thy-1, when interacting with a beta(3)-containing integrin on astrocytes, could trigger signaling events similar to those generated by extracellular matrix proteins. DI-TNC(1) astrocytes were stimulated with Thy-1-Fc immobilized on beads, and increased RhoA activity was confirmed using an affinity precipitation assay. The effect of various inhibitors on the cellular response was also studied. The presence of Y-27632, an inhibitor of Rho kinase (p160ROCK), a key downstream effector of RhoA, significantly reduced focal adhesion and stress fiber formation induced by Thy-1. Similar effects were obtained when astrocytes were treated with C3 transferase, an inhibitor of RhoA. Alternatively, astrocytes were transfected with an expression vector encoding fusion proteins of enhanced green fluorescent protein with either the Rho-binding domain of Rhotekin, which blocks RhoA function, or the dominant-negative N19RhoA mutant. In both cases, Thy-1-induced focal adhesion formation was inhibited. Furthermore, we observed that RhoA activity after stimulation with soluble Thy-1-Fc molecule was augmented upon further cross-linking using protein A-Sepharose beads. The same was shown by cross-linking beta(3)-containing integrin with anti-beta(3) antibodies. Together, these results indicate that Thy-1-mediated astrocyte stimulation depended on beta(3) integrin clustering and the resulting increase in RhoA activity.

Signaling triggered by Thy-1 interaction with beta 3 integrin on astrocytes is an essential step towards unraveling neuronal Thy-1 function.

Thy-1 is an abundant neuronal glycoprotein in mammals. Despite such prevalence, Thy-1 function remains largely obscure in the absence of a defined ligand. Recently described evidence that Thy-1 interacts with beta 3 integrin on astrocytes will be discussed. Thy-1 binding to beta 3 integrin triggers tyrosine phosphorylation of focal adhesion proteins in astrocytes, thereby promoting focal adhesion formation, cell attachment and spreading. Thy-1 has been reported to modulate neurite outgrowth by triggering a cellular response in neurons. However, our data indicate that Thy-1 can also initiate signaling events that promote adhesion of adjacent astrocytes to the underlying surface. Preliminary results suggest that morphological changes observed in the actin cytoskeleton of astrocytes as a consequence of Thy-1 binding is mediated by small GTPases from the Rho family. Our findings argue that Thy-1 functions in a bimodal fashion, as a receptor on neuronal cells and as a ligand for beta 3 integrin receptor on astrocytes. Since Thy-1 is implicated in the inhibition of neurite outgrowth, signaling events in astrocytes are likely to play an important role in this process.

Signaling triggered by Thy-1 interaction with beta 3 integrin on astrocytes is an essential step towards unraveling neuronal Thy-1 function

Thy-1 is an abundant neuronal glycoprotein in mammals. Despite such prevalence, Thy-1 function remains largely obscure in the absence of a defined ligand. Recently described evidence that Thy-1 interacts with beta 3 integrin on astrocytes will be discussed. Thy-1 binding to beta 3 integrin triggers tyrosine phosphorylation of focal adhesion proteins in astrocytes, thereby promoting focal adhesion formation, cell attachment and spreading. Thy-1 has been reported to modulate neurite outgrowth by triggering a cellular response in neurons. However, our data indicate that Thy-1 can also initiate signaling events that promote adhesion of adjacent astrocytes to the underlying surface. Preliminary results suggest that morphological changes observed in the actin cytoskeleton of astrocytes as a consequence of Thy-1 binding is mediated by small GTPases from the Rho family. Our findings argue that Thy-1 functions in a bimodal fashion, as a receptor on neuronal cells and as a ligand for beta 3 integrin receptor on astrocytes. Since Thy-1 is implicated in the inhibition of neurite outgrowth, signaling events in astrocytes are likely to play an important role in this process