Malgorzata Zawadzka

MAPK Signal Transduction Underlying Brain Inflammation and Gliosis as Therapeutic Target

A majority, if not all, acute and progressive neurodegenerative diseases are accompanied by local microglia‐mediated inflammation, astrogliosis, infiltration of immune cells, and activation of the adaptive immunity. These processes progress by the expression of cytokines, adhesion molecules, proteases, and other inflammation mediators. In response to brain injury or infection, intracellular signaling pathways are activated in microglia, which turn on inflammatory and antigen‐presenting cell functions. Different extrinsic signals shape microglial activation toward neuroprotective or neurotoxic phenotype under pathological conditions. This review discusses recent advances regarding molecular mechanisms of inflammatory signal transduction in neurological disorders and in in vitro models of inflammation/gliosis. Mitogen‐activated protein kinases (MAPKs) are a family of serine/threonine protein kinases responsible for most cellular responses to cytokines and external stress signals and crucial for regulation of the production of inflammation mediators. Increased activity of MAPKs in activated microglia and astrocytes, and their regulatory role in the synthesis of inflammatory cytokines mediators, make them potential targets for novel therapeutics. MAPK inhibitors emerge as attractive anti‐inflammatory drugs, because they are capable of reducing both the synthesis of inflammation mediators at multiple levels and are effective in blocking inflammatory cytokine signaling. Small molecule inhibitors targeting of p38 MAPK and JNK pathways have been developed and offer a great potential as potent modulators of brain inflammation and gliosis in neurological disorders, where cytokine overproduction contributes to disease progression. Many of the pharmacological MAPK inhibitors can be administered orally and initial results show therapeutic benefits in preclinical animal models. Anat Rec, 292:1902–1913, 2009.

Molecular mechanisms of neuroprotective action of immunosuppressants - facts and hypotheses

Cyclosporin A (CsA) and FK506 (Tacrolimus) are short polypeptides which block the activation of lymphocytes and other immune system cells. Immunosuppressants exert neuroprotective and neurotrophic action in traumatic brain injury, sciatic nerve injury, focal and global ischemia in animals. Their neuroprotective actions are not understood and many hypotheses have been formed to explain such effects. We discuss a role of drug target ‐ calcineurin in neuroprotective action of immunosuppressants. Protein dephosphorylation by calcineurin plays an important role in neuronal signal transduction due to its ability to regulate the activity of ion channels, glutamate release, and synaptic plasticity. In vitro FK506 protects cortex neurons from NMDA‐induced death, augments NOS phosphorylation inhibiting its activity and NO synthesis. However, in vivo experiments demonstrated that FK506 in neuroprotective doses did not block excitotoxic cell death nor did it alter NO production during ischemia/reperfusion. Tissue damage in ischemia is the result of a complex pathophysiological cascade, which comprises a variety of distinct pathological events. Resident non‐neuronal brain cells respond rapidly to neuronal cell death and may have both deleterious and useful role in neuronal damage. There is increasing evidence that reactive gliosis and post‐ischemic inflammation involving microglia contribute to ischemic damage. We have demonstrated that FK506 modulates hypertrophic/proliferative responses and proinflammatory cytokine expression in astrocytes and microglia in vitro and in focal transient brain ischemia. Our findings suggest that astrocytes and microglia are direct targets of FK506 and modulation of glial response and inflammation is a possible mechanism of FK506‐mediated neuroprotection in ischemia.

A novel mechanism of FK506‐mediated neuroprotection: Downregulation of cytokine expression in glial cells

Immunosuppressant FK506 is neuroprotective in experimental models of cerebral ischemia, but the molecular mechanisms underlying this neuroprotection remain unknown. We have demonstrated that FK506 inhibits the signaling pathways that regulate hypertrophic/proliferative responses in cultured astrocytes. Ischemia/reperfusion injury is associated with the proliferation and hypertrophy of astrocytes and with inflammatory responses. In the present work, we sought to determine whether FK506 neuroprotection after middle cerebral artery occlusion (MCAo) in rat is mediated via suppression of glia activation and changes in cytokine expression. Neurological deficits, infarct size, and astrocyte/microglial response were quantified in rats subjected to 90 min of MCAo. Changes in the mRNA expression of interleukin‐1β (IL‐1β), IL‐6, and tumor necrosis factor‐α (TNF‐α) in ipsilateral and contralateral cortices were determined by reverse transcription‐polymerase chain reaction (RT‐PCR). FK506 administered at 1 mg/kg, 60 min after MCAo, produced a significant improvement in neurological function and reduction of infarct volume. In FK506‐treated rats, a significant reduction of IL‐1β, IL‐6, and TNF‐α expression was observed 12 h after reperfusion. FK506 neuroprotection was associated with a significant downregulation of IL‐1β expression in astrocytes and microglia in the injured side. FK506 selectively decreased the levels of TNF‐α, and IL‐1β mRNAs in astrocytes in vitro, with no effect on transforming growth factor‐β 1 (TGF‐β1) and IL‐6 expression. Moreover, FK506 inhibits lipopolysaccharide (LPS)‐induced activation and cytokine expression in microglia in vitro. Our findings suggest that astrocytes and microglia are targets for FK506, and that modulation of glial response and inflammation may be a mechanism of FK506‐mediated neuroprotection in ischemia.

Distinct roles of CSF family cytokines in macrophage infiltration and activation in glioma progression and injury response

Gliomas attract brain‐resident (microglia) and peripheral macrophages and reprogram these cells into immunosuppressive, pro‐invasive cells. M‐CSF (macrophage colony‐stimulating factor, encoded by the CSF1 gene) has been implicated in the control of recruitment and polarization of macrophages in several cancers. We found that murine GL261 glioma cells overexpress GM‐CSF (granulocyte–macrophage colony‐stimulating factor encoded by the CSF2 gene) but not M‐CSF when compared to normal astrocytes. Knockdown of GM‐CSF in GL261 glioma cells strongly reduced microglia‐dependent invasion in organotypical brain slices and growth of intracranial gliomas and extended animal survival. The number of infiltrating microglia/macrophages (Iba1+ cells) and intratumoural angiogenesis were reduced in murine gliomas depleted of GM‐CSF. M1/M2 gene profiling in sorted microglia/macrophages suggests impairment of their pro‐invasive activation in GM‐CSF‐depleted gliomas. Deficiency of M‐CSF (op/op mice) did not affect glioma growth in vivo and the accumulation of Iba1+ cells, but impaired accumulation of Iba1+ cells in response to demyelination. These results suggest that distinct cytokines of the CSF family contribute to macrophage infiltration of tumours and in response to injury. The expression of CSF2 (but not CSF1) was highly up‐regulated in glioblastoma patients and we found an inverse correlation between CSF2 expression and patient survival. Therefore we propose that GM‐CSF triggers and drives the alternative activation of tumour‐infiltrating microglia/macrophages in which these cells support tumour growth and angiogenesis and shape the immune microenvironment of gliomas. Copyright

Immunosuppressant FK506 affects multiple signaling pathways and modulates gene expression in astrocytes.

Brain injury is often associated with proliferation and hypertrophic response of glial cells (reactive gliosis). We have previously reported immunosuppressant effects on survival of glioma cells and adult reactive astrocytes. In the present study, we demonstrate growth-inhibitory effect of FK506 on cortical astrocytes from newborn rats. FK506 inhibits Erk and PI-3K/Akt signaling, two crucial pro-survival pathways. The levels of phosphorylated Akt and p42/44 Erk decline in few hours after FK506 addition. Furthermore, in FK506-treated astrocyte cultures the levels of mRNA encoding PDGF, bFGF, and CNTF decreased. Downregulation of growth factor expression by FK506 may play a role in the inhibition of mitogenic/hypertrophic responses. FasL mRNA level was elevated and interaction of FasL with Fas receptor expressed in astrocytes may trigger cell death. Interestingly, expression of BDNF increased in a dose-dependent manner in FK506-treated astrocytes. Upregulation of BDNF mRNA and protein level in astrocytes exposed to FK506 may underlie neuroprotective action of FK506.

Neuroprotectant FK506 inhibits glutamate-induced apoptosis of astrocytes in vitro and in vivo

Neuron–astrocyte interactions are critical for signalling, energy metabolism, extracellular ion and glutamate homeostasis, volume regulation and neuroprotection in the CNS. Glutamate uptake by astrocytes may prevent excitotoxic glutamate elevation and determine neuronal survival. However, an excess of glutamate can cause the death of astrocytes. FK506, an inhibitor of calcineurin, and an immunosuppressive drug, is neuroprotective in animal models of neurologic diseases, including focal and global ischaemia. In the present work, we demonstrate that a single injection of FK506 60 min after a transient middle cerebral artery occlusion (MCAo) significantly decreases the number of terminal deoxynucleotidyl transferase nick‐end labelling (TUNEL)‐positive cells in the ischaemic cortex and striatum. Using 3‐D confocal microscopy we found that, 24 h after MCAo, many TUNEL‐positive cells in the ischaemic striatum and cortex are astrocytes. Furthermore, we demonstrate that exposure of cultured cortical astrocytes to 50–100 mM Glu for 24 h induces apoptotic alterations in nuclear morphology, DNA fragmentation, dissipation of mitochondrial transmembrane potential (ΔΨ) and caspase activation. FK506 (1 μM) efficiently inhibits Glu‐induced apoptosis of cultured astrocytes, DNA fragmentation and changes in mitochondrial ΔΨ. Our findings suggest that modulation of glutamate‐induced astrocyte death early after reperfusion may be a novel mechanism of FK506‐mediated neuroprotection in ischaemia.

Influence of chemical and ischemic preconditioning on cytokine expression after focal brain ischemia

Inflammation, upregulation of cytokines, proapoptotic molecules, and apoptosis are accepted widely as crucial players in stroke‐induced brain damage. Induction of brain tolerance against ischemia by pretreatment with nonlethal stressors (preconditioning) has been found to influence expression of different molecules, in addition to reduction of infarct size. It remains unclear, however, whether and how preconditioning changes expression of cytokines after subsequent brain ischemia. We sought to analyze cortical expression of interleukin (IL)‐1β, IL‐6, tumor necrosis factor (TNF)‐α, transforming growth factor (TGF)‐β, Fas, and Fas ligand (FasL) mRNA after a transient, focal brain ischemia in rats subjected to preconditioning. The mRNA levels were determined using a semiquantitative RT‐PCR in the ischemic and contralateral cortex, separately. Transient ischemia was induced by 90‐min middle cerebral artery occlusion (MCAo) and neurologic deficits as well as infarct size were quantified. Preconditioning was carried out by a short‐term MCAo or an injection of 3‐nitropropionic acid 3 days before MCAo. In both preconditioning paradigms, similar effects on investigated mRNA levels were observed. IL‐1β and IL‐6 levels were decreased in tolerant rats compared to those in nontolerant ones. Changes in TNF‐α, TGF‐β, and Fas levels were comparable independently of tolerance state. FasL mRNA was at similar level in rats subjected to chemical preconditioning but lower after ischemic preconditioning. Our findings demonstrate that both preconditioning methods exert a very similar effect on the expression of investigated cytokines. Interestingly, we observed a selective effect of preconditioning on IL‐1β and IL‐6 expression that suggests different functional properties as well as different regulation of analyzed molecules during an induction of the brain tolerance against ischemia.

Prolonged activation of ERK triggers glutamate‐induced apoptosis of astrocytes: neuroprotective effect of FK506

J. Neurochem. (2010) 113, 904–918.

Early steps of microglial activation are directly affected by neuroprotectant FK506 in both in vitro inflammation and in rat model of stroke

Neuroprotective and/or neuroregenerative activity of FK506, its derivatives, and to a lesser extent cyclosporin A (CsA) in animal models of neurodegenerative diseases of different etiology have been reported. Here, we verified a hypothesis that the most likely mechanism of their neuroprotective action is inhibition of the early steps of inflammatory activation of microglia by interference with mitogen-activated protein kinase (MAPK) signaling. The effect of immunosuppressants on lipopolysaccharide (LPS)-induced changes in morphology, proliferation, and motility of rat primary microglial cultures was evaluated. FK506 and CsA directly inhibited LPS-induced microglia activation and inflammatory responses. While both drugs efficiently reduced the expression of iNOS and the release of nitric oxide, only FK506 strongly inhibited the expression of Cox-2 and secretion of the mature form of IL-1β. FK506 strongly reduced LPS-induced activation of MAPK, and its downstream signaling crucial for inflammatory responses. Comparative analysis of global gene expression in rat ischemic brains and in LPS-stimulated microglial cultures revealed many genes and signaling pathways regulated in the same way in both systems. FK506 treatment blocked a majority of genes induced by an ischemic insult in the cortex, in particular inflammatory/innate immunity and apoptosis-related genes. Microglia-mediated inflammation is considered as one of the most important components of brain injury after trauma or stroke; thus, effective and multifaceted blockade of microglial activation by FK506 has clinical relevance and potential therapeutic implications.

Neurotrophic factor expression after focal brain ischemia preceded by different preconditioning strategies.

Background: Both prolonged brain ischemia and preconditioning (PC) induce expression of neurotrophic factors. However, the influence of PC on their expression after a long-term ischemia remains vague. Previously, we have found various effects of PC on mRNA levels of different cytokines after focal brain ischemia. Thus, we investigated mRNA expression of nerve growth factor, brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor after 90-min middle cerebral artery occlusion (MCAo) preceded by ischemic or chemical PC. Methods: MCAo was induced in rats using the suture method. PC had been carried out 3 days earlier. There were 4 experimental groups: MCAo alone; ischemic PC and MCAo; chemical PC and MCAo, and sham-operated rats. Expression of mRNAs in the ipsi- and contralateral cortex was studied by semiquantitative RT-PCR at 12 and 24 h after MCAo. Results: Despite clearly neuroprotective effects of both PC strategies, mRNA levels of neurotrophic factors were similar in tolerant and nontolerant rats. Only BDNF mRNA expression, 12 h after reperfusion, was lower when ischemic PC was applied prior to long-term ischemia. Conclusions: These results suggest that PC generally does not change the expression of neurotrophic factor expression after a long-term focal brain ischemia compared to the nontolerant state.