Pedro Tranque

The inhibition of Glycogen Synthase Kinase 3 β (GSK3β) by Ser⁹ phosphorylation affects many physiological processes, including the immune response. However, the consequences of GSK3β inhibition by alternative Ser³⁸⁹ phosphorylation remain poorly characterized. Here we have examined neuroinflammation in GSK3β Ser³⁸⁹ knock-in (KI) mice, in which the phosphorylation of Ser³⁸⁹ GSK3β is impaired. The number of activated microglia/infiltrated macrophages, astrocytes, and infiltrated neutrophils was significantly higher in these animals compared to C57BL/6J wild-type (WT) counterparts, which suggests that the failure to inactivate GSK3β by Ser³⁸⁹ phosphorylation results in sustained low-grade neuroinflammation. Moreover, glial cell activation and brain infiltration of immune cells in response to lipopolysaccharide (LPS) failed in GSK3β Ser³⁸⁹ KI mice. Such effects were brain-specific, as peripheral immunity was not similarly affected. Additionally, phosphorylation of the IkB kinase complex (IKK) in response to LPS failed in GSK3β Ser³⁸⁹ KI mice, while STAT3 phosphorylation was fully conserved, suggesting that the NF-κB signaling pathway is specifically affected by this GSK3β regulatory pathway. Overall, our findings indicate that GSK3β inactivation by Ser³⁸⁹ phosphorylation controls the brain inflammatory response, raising the need to evaluate its role in the progression of neuroinflammatory pathologies.

The study of factors that regulate the survival, proliferation, and differentiation of neural precursor cells (NPCs) is essential to understand neural development as well as brain regeneration. The Nuclear Factor of Activated T Cells (NFAT) is a family of transcription factors that can affect these processes besides playing key roles during development, such as stimulating axonal growth in neurons, maturation of immune system cells, heart valve formation, and differentiation of skeletal muscle and bone. Interestingly, NFAT signaling can also promote cell differentiation in adults, participating in tissue regeneration. The goal of the present study is to evaluate the expression of NFAT isoforms in NPCs, and to investigate its possible role in NPC survival, proliferation, migration, and differentiation. Our findings indicate that NFAT proteins are active not only in neurogenic brain regions such as hippocampus and subventricular zone (SVZ), but also in cultured NPCs. The inhibition of NFAT activation with the peptide VIVIT reduced neurosphere size and cell density in NPC cultures by decreasing proliferation and increasing cell death. VIVIT also decreased NPC migration and differentiation of astrocytes and neurons from NPCs. In addition, we identified NFATc3 as a predominant NFAT isoform in NPC cultures, finding that a constitutively‐active form of NFATc3 expressed by adenoviral infection reduces NPC proliferation, stimulates migration, and is a potent inducer of NPC differentiation into astrocytes and neurons. In summary, our work uncovers active roles for NFAT signaling in NPC survival, proliferation and differentiation, and highlights its therapeutic potential for tissue regeneration. GLIA 2015;63:987–1004

Increase in intracellular calcium ([Ca²⁺]_i) is a key mediator of astrocyte signaling, important for activation of the calcineurin (CN)/nuclear factor of activated T cells (NFAT) pathway, a central mediator of inflammatory events. We analyzed the expression of matrix metalloproteinase 3 (Mmp3) in response to increases in [Ca²⁺]_iand the role of the CN/NFAT pathway in this regulation. AstrocyteMmp3expression was induced by overexpression of a constitutively active form of NFATc3, whereas other MMPs and tissue inhibitor of metalloproteinases (TIMP) were unaffected.Mmp3mRNA and protein expression was also induced by calcium ionophore (Io) and 2′(3′)‐O‐(4‐benzoylbenzoyl) adenosine 5′‐triphosphate (Bz‐ATP) andMmp3upregulation was prevented by the CN inhibitor cyclosporin A (CsA). Ca²⁺‐dependent astrocyteMmp3expression was also inhibited by actinomycin D, and aMmp3promoter luciferase reporter was efficiently activated by increased [Ca²⁺]_i, indicating regulation at the transcriptional level. Furthermore, Ca²⁺/CN/NFAT dependentMmp3expression was confirmed in pure astrocyte cultures derived from neural stem cells (Ast‐NSC), demonstrating that the inducedMmp3expression occurs in astrocytes, and not microglial cells. In anin vivostab‐wound model of brain injury, MMP3 expression was detected in NFATc3‐positive scar‐forming astrocytes. Because [Ca²⁺]_iincrease is an early event in most brain injuries, these data support an important role for Ca²⁺/CN/NFAT‐induced astrocyte MMP3 expression in the early neuroinflammatory response. Understanding the molecular pathways involved in this regulation could provide novel therapeutic targets and approaches to promoting recovery of the injured brain.

Selenoprotein S (SelS) is an endoplasmic reticulum (ER)‐resident protein involved in the unfolded protein response. Besides reducing ER‐stress, SelS attenuates inflammation by decreasing pro‐inflammatory cytokines. We have recently shown that SelS is responsive to ischemia in cultured astrocytes. To check the possible association of SelS with astrocyte activation, here we investigate the expression of SelS in two models of brain injury: kainic acid (KA) induced excitotoxicity and cortical mechanical lesion. The regulation of SelS and its functional consequences for neuroinflammation, ER‐stress, and cell survival were further analyzed using cultured astrocytes from mouse and human. According to our immunofluorescence analysis, SelS expression is prominent in neurons and hardly detectable in astrocytes from control mice. However, brain injury intensely upregulates SelS, specifically in reactive astrocytes. SelS induction by KA was evident at 12 h and faded out after reaching maximum levels at 3–4 days. Analysis of mRNA and protein expression in cultured astrocytes showed SelS upregulation by inflammatory stimuli as well as ER‐stress inducers. In turn, siRNA‐mediated SelS silencing combined with adenoviral overexpression assays demonstrated that SelS reduces ER‐stress markers CHOP and spliced XBP‐1, as well as inflammatory cytokines IL‐1β and IL‐6 in stimulated astrocytes. SelS overexpression increased astrocyte resistance to ER‐stress and inflammatory stimuli. Conversely, SelS suppression compromised astrocyte viability. In summary, our results reveal the upregulation of SelS expression in reactive astrocytes, as well as a new protective role for SelS against inflammation and ER‐stress that can be relevant to astrocyte function in the context of inflammatory neuropathologies.

Astrocytes react to brain injury triggering neuroinflammatory processes that determine the degree of neuronal damage. However, the signaling events associated to astrocyte activation remain largely undefined. The nuclear factor of activated T‐cells (NFAT) is a transcription factor family implicated in activation of immune cells. We previously characterized the expression of NFAT isoforms in cultured astrocytes, and NFAT activation in response to mechanical lesion. Here we analyze NFATc3 in two mouse models of inflammatory brain damage: hippocampal excitotoxicity induced by intracerebral kainic acid (KA) injection and cortical mechanical lesion. Immunofluorescence results demonstrated that NFATc3 is specifically induced in a subset of reactive astrocytes, and not in microglia or neurons. In KA‐treated brains, NFATc3 expression is transient and NFATc3‐positive astrocytes concentrate around damaged neurons in areas CA3 and CA1. Complementary Western blot and RT‐PCR analysis revealed an NFAT‐dependent induction of RCAN1‐4 and COX‐2 in hippocampus as soon as 6 h after KA exposure, indicating that NFAT activation precedes NFATc3 over‐expression. Moreover, activation of NFAT by ATP increased NFATc3 mRNA levels in astrocyte cultures, suggesting that NFATc3 expression is controlled through an auto‐regulatory loop. Meanwhile, stab wound enhanced NFATc3 expression specifically in a subclass of reactive astrocytes confined within the proximal layer of the glial scar, and GFAP immunoreactivity was attenuated in NFATc3‐expressing astrocytes. In conclusion, our work establishes NFATc3 as a marker of activation for a specific population of astrocytes in response to brain damage, which may have consequences for neuronal survival.

Astrocytes are glial cells in the central nervous system (CNS) that play key roles in brain physiology, controlling processes, such as neurogenesis, brain energy metabolism and synaptic transmission. Recently, immune functions have also been demonstrated in astrocytes, influencing neuronal survival in the course of neuroinflammatory pathologies. In this regard, PKCepsilon (PKCε) is a protein kinase with an outstanding role in inflammation. Our previous findings indicating that PKCε regulates voltage-dependent calcium channels as well as morphological stellation imply that this kinase controls multiple signalling pathways within astrocytes, including those implicated in activation of immune functions. The present study applies proteomics to investigate new protein targets of PKCε in astrocytes. Primary astrocyte cultures infected with an adenovirus that expresses constitutively active PKCε were compared with infection controls. Two-dimensional gel electrophoresis clearly detected 549 spots in cultured astrocytes, and analysis of differential protein expression revealed 18 spots regulated by PKCε. Protein identification by mass spectrometry (nano-LC–ESI-MS/MS) showed that PKCε targets molecules with heterogeneous functions, including chaperones, cytoskeletal components and proteins implicated in metabolism and signalling. These results support the notion that PKCε is involved in astrocyte activation; also suggesting that multiple astrocyte-dependent processes are regulated by PKCε, including those associated to neuroinflammation.

Astrocytes are essential cells for maintaining brain integrity. We have recently shown that the transcription factor C/EBP homologous protein (CHOP), associated with endoplasmic reticulum (ER) stress, plays a key role in the astrocyte death induced by ischemia. Meanwhile, mediators of apoptosis downstream of CHOP in the ER stress‐dependent pathway remain to be elucidated. Our aim in this work was to determine whether caspase‐11, able to activate apoptotic and proinflammatory pathways, is implicated in ER stress‐dependent astrocyte death in ischemic conditions. According to our results, caspase‐11 is up‐regulated in primary astrocyte cultures following either oxygen and glucose deprivation (OGD) or treatment with the ER‐stress inducers thapsigargin and tunicamycin. Moreover, these same stimuli increased caspase‐11 mRNA levels and luciferase activity driven by a caspase‐11 promoter, indicating that caspase‐11 is regulated at the transcriptional level. Our data also illustrate the involvement of ER stress‐associated CHOP in caspase‐11 regulation, insofar as CHOP overexpression by means of an adenoviral vector caused a significant raise in caspase‐11. In turn, caspase‐11 suppression with siRNA rescued astrocytes from OGD‐ and ER stress‐induced death, supporting the idea that caspase‐11 is responsible for the deleterious effects of ischemia on astrocytes. Finally, inhibition of caspase‐1 and caspase‐3 significantly reduced astrocyte death, which indicates that these proteases act as death effectors of caspase‐11. In conclusion, our work contributes to clarifying the pathways leading to astrocyte death in response to ischemia by defining caspase‐11 as a key mediator of the ER stress response acting downstream of CHOP.

Contrarily to neurons, astrocytes can survive short periods of ischemia. We have searched for genes implicated in astrocyte resistance to ischemia using oxygen and glucose deprivation (OGD) as a stroke model. A RNA differential display approach uncovered the OGD induction of selenoprotein-S-encoding gene SEPS1. This endoplasmic reticulum (ER) resident protein is known to promote cell survival regulating the ER stress as well as inflammation. We found that suppression of SEPS1 by small interfering RNA severely increases astrocyte injure caused by OGD, suggesting that selenoprotein S protects astrocytes against ischemia. Our data also support that modulation of ER stress is implicated in this effect.