Yun-Bi Lu

Viscoelastic properties of individual glial cells and neurons in the CNS

One hundred fifty years ago glial cells were discovered as a second, non-neuronal, cell type in the central nervous system. To ascribe a function to these new, enigmatic cells, it was suggested that they either glue the neurons together (the Greek word “γλια” means “glue”) or provide a robust scaffold for them (“support cells”). Although both speculations are still widely accepted, they would actually require quite different mechanical cell properties, and neither one has ever been confirmed experimentally. We investigated the biomechanics of CNS tissue and acutely isolated individual neurons and glial cells from mammalian brain (hippocampus) and retina. Scanning force microscopy, bulk rheology, and optically induced deformation were used to determine their viscoelastic characteristics. We found that (i) in all CNS cells the elastic behavior dominates over the viscous behavior, (ii) in distinct cell compartments, such as soma and cell processes, the mechanical properties differ, most likely because of the unequal local distribution of cell organelles, (iii) in comparison to most other eukaryotic cells, both neurons and glial cells are very soft (“rubber elastic”), and (iv) intriguingly, glial cells are even softer than their neighboring neurons. Our results indicate that glial cells can neither serve as structural support cells (as they are too soft) nor as glue (because restoring forces are dominant) for neurons. Nevertheless, from a structural perspective they might act as soft, compliant embedding for neurons, protecting them in case of mechanical trauma, and also as a soft substrate required for neurite growth and facilitating neuronal plasticity.

Reactive glial cells: increased stiffness correlates with increased intermediate filament expression

Increased stiffness of reactive glial cells may impede neurite growth and contribute to the poor regenerative capabilities of the mammalian central nervous system. We induced reactive gliosis in rodent retina by ischemia‐reperfusion and assessed intermediate filament (IF) expression and the viscoelastic properties of dissociated single glial cells in wild‐type mice, mice lacking glial fibrillary acidic protein and vimentin (GFAP−/−Vim−/−) in which glial cells are consequently devoid of IFs, and normal Long‐Evans rats. In response to ischemia‐reperfusion, glial cells stiffened significantly in wild‐type mice and rats but were unchanged in GFAP−/− Vim−/− mice. Cell stiffness (elastic modulus) correlated with the density of IFs. These results support the hypothesis that rigid glial scars impair nerve regeneration and that IFs are important determinants of cellular viscoelasticity in reactive glia. Thus, therapeutic suppression of IF up‐regulation in reactive glial cells may facilitate neuroregeneration.—Lu, Y.‐B., Iandiev, I., Hollborn, M., Korber, N., Ulbricht, E., Hirrlinger, P. G., Pannicke, T., Wei, E.‐Q., Bringmann, A., Wol‐burg, H., Wilhelmsson, U., Pekny, M., Wiedemann, P., Reichenbach, A., Kas, J. A. Reactive glial cells: increased stiffness correlates with increased intermediate filament expression. FASEB J. 25, 624–631 (2011). www.fasebj.org

Growth cones as soft and weak force generators

Many biochemical processes in the growth cone finally target its biomechanical properties, such as stiffness and force generation, and thus permit and control growth cone movement. Despite the immense progress in our understanding of biochemical processes regulating neuronal growth, growth cone biomechanics remains poorly understood. Here, we combine different experimental approaches to measure the structural and mechanical properties of a growth cone and to simultaneously determine its actin dynamics and traction force generation. Using fundamental physical relations, we exploited these measurements to determine the internal forces generated by the actin cytoskeleton in the lamellipodium. We found that, at timescales longer than the viscoelastic relaxation time of τ = 8.5 ± 0.5 sec, growth cones show liquid-like characteristics, whereas at shorter time scales they behaved elastically with a surprisingly low elastic modulus of E = 106 ± 21 Pa. Considering the growth cone’s mechanical properties and retrograde actin flow, we determined the internal stress to be on the order of 30 pN per μm2. Traction force measurements confirmed these values. Hence, our results indicate that growth cones are particularly soft and weak structures that may be very sensitive to the mechanical properties of their environment.

Activation of CysLT receptors induces astrocyte proliferation and death after oxygen-glucose deprivation.

We recently found that 5‐lipoxygenase (5‐LOX) is activated to produce cysteinyl leukotrienes (CysLTs), and CysLTs may cause neuronal injury and astrocytosis through activation of CysLT1 and CysLT2 receptors in the brain after focal cerebral ischemia. However, the property of astrocyte responses to in vitro ischemic injury is not clear; whether 5‐LOX, CysLTs, and their receptors are also involved in the responses of ischemic astrocytes remains unknown. In the present study, we performed oxygen‐glucose deprivation (OGD) followed by recovery to induce ischemic‐like injury in the cultured rat astrocytes. We found that 1‐h OGD did not injure astrocytes (sub‐lethal OGD) but induced astrocyte proliferation 48 and 72 h after recovery; whereas 4‐h OGD moderately injured the cells (moderate OGD) and led to death 24–72 h after recovery. Inhibition of phospholipase A2 and 5‐LOX attenuated both the proliferation and death. Sub‐lethal and moderate OGD enhanced the production of CysLTs that was inhibited by 5‐LOX inhibitors. Sub‐lethal OGD increased the expressions of CysLT1 receptor mRNA and protein, while moderate OGD induced the expression of CysLT2 receptor mRNA. Exogenously applied leukotriene D4 (LTD4) induced astrocyte proliferation at 1–10 nM and astrocyte death at 100–1,000 nM. The CysLT1 receptor antagonist montelukast attenuated astrocyte proliferation, the CysLT2 receptor antagonist BAY cysLT2 reversed astrocyte death, and the dual CysLT receptor antagonist BAY u9773 exhibited both effects. In addition, LTD4 (100 nM) increased the expression of CysLT2 receptor mRNA. Thus, in vitro ischemia activates astrocyte 5‐LOX to produce CysLTs, and CysLTs result in CysLT1 receptor‐mediated proliferation and CysLT2 receptor‐mediated death.

Dihydroartemisinin exerts cytotoxic effects and inhibits hypoxia inducible factor-1α activation in C6 glioma cells

Artemisinin and its analogue dihydroartemisinin exert cytotoxic effects in some kinds of cancer cell lines. Here we determined whether dihydroartemisinin inhibits the growth and induces apoptosis of rat C6 glioma cells. We found dihydroartemisinin (5–25 μM) inhibited the growth and induced apoptosis of C6 cells in a concentration‐ and time‐dependent manner; however, it was much less toxic to rat primary astrocytes. Dihydroartemisinin (5–25 μM) also increased the generation of reactive oxygen species in C6 cells. These effects of dihydroartemisinin were enhanced by ferrous ions (12.5–100 μM) and reduced by the iron chelator deferoxamine (25–200 μM). Immunoblotting analysis revealed that dihydroartemisinin (5–25 μM) significantly reduced hypoxia‐ and deferoxamine‐induced expression of hypoxia inducible factor‐1α and its target gene protein, vascular endothelial growth factor, in C6 cells. The results showed that dihydroartemisinin exerts a selective cytotoxic effect on C6 cells by increasing the reactive oxygen species and inhibiting hypoxia inducible factor‐1α activation.

Dihydroartemisinin Potentiates the Cytotoxic Effect of Temozolomide in Rat C6 Glioma Cells

Gliomas are the most common primary brain tumor in adults, but the efficacy of chemotherapy is limited. Artemisinin and its analogs, such as dihydroartemisinin (DHA), can kill cancer cells via generating free radicals. In the present study, we determined whether DHA at low concentrations potentiates the cytotoxic effect of temozolomide in rat glioma C6 cells. We found that the IC50 values of DHA and temozolomide for cell viability were 23.4 and 560 µmol/l, respectively. The cytotoxic effect of temozolomide was enhanced by 177% at a nontoxic DHA concentration (1 µmol/l), and by 321% at a low-toxic DHA concentration (5 µmol/l). DHA substantially increased temozolomide-induced apoptosis and necrosis. The generation of intracellular reactive oxygen species (ROS) was increased by temozolomide combined with DHA at noneffective concentrations of both agents. Edaravone (20 µmol/l), a ROS scavenger, reversed the effects of temozolomide/DHA on both ROS generation and cell viability reduction. These results indicate that DHA at low concentrations potentiates the cytotoxic effects of temozolomide in C6 cells partly via generating ROS, suggesting a beneficial combination for the chemotherapy of gliomas.

The new P2Y-like receptor G protein-coupled receptor 17 mediates acute neuronal injury and late microgliosis after focal cerebral ischemia in rats.

G protein-coupled receptor 17 (GPR17), the new P2Y-like receptor, is phylogenetically related to the P2Y and cysteinyl leukotriene receptors, and responds to both uracil nucleotides and cysteinyl leukotrienes. GPR17 has been proposed to be a damage sensor in ischemic stroke; however, its role in brain inflammation needs further detailed investigation. Here, we extended previous studies on the spatiotemporal profiles of GPR17 expression and localization, and their implications for brain injury after focal cerebral ischemia. We found that in the ischemic core, GPR17 mRNA and protein levels were upregulated at both 12-24 h and 7-14 days, but in the boundary zone the levels increased 7-14 days after reperfusion. The spatiotemporal pattern of GPR17 expression well matched the acute and late (subacute/chronic) responses in the ischemic brain. According to previous findings, in the acute phase, after ischemia (24 h), upregulated GPR17 was localized in injured neurons in the ischemic core and in a few microglia in the ischemic core and boundary zone. In the late phase (14 days), it was localized in microglia, especially in activated (ED1-positive) microglia in the ischemic core, but weakly in most microglia in the boundary zone. No GPR17 was detectable in astrocytes. GPR17 knockdown by a small interfering RNA attenuated the neurological dysfunction, infarction, and neuron loss at 24 h, and brain atrophy, neuron loss, and microglial activation at 14 days after reperfusion. Thus, GPR17 might mediate acute neuronal injury and late microgliosis after focal cerebral ischemia.

CysLT2 receptor-mediated AQP4 up-regulation is involved in ischemic-like injury through activation of ERK and p38 MAPK in rat astrocytes

AIMS We previously reported that cysteinyl leukotriene receptor 2 (CysLT(2)) mediates ischemic astrocyte injury, and leukotriene D(4)-activated CysLT(2) receptor up-regulates the water channel aquaporin 4 (AQP4). Here we investigated the mechanism underlying CysLT(2) receptor-mediated ischemic astrocyte injury induced by 4-h oxygen-glucose deprivation and 24-h recovery (OGD/R). MAIN METHODS Primary cultures of rat astrocytes were treated by OGD/R to construct the cell injury model. AQP4 expression was inhibited by small interfering RNA (siRNA). The expressions of AQP4 and CysLTs receptors, and the MAPK signaling pathway were determined. KEY FINDINGS OGD/R induced astrocyte injury, and increased expression of the CysLT(2) (but not CysLT(1)) receptor and AQP4. OGD/R-induced cell injury and AQP4 up-regulation were inhibited by a CysLT(2) receptor antagonist (Bay cysLT2) and a non-selective CysLT receptor antagonist (Bay u9773), but not by a CysLT(1) receptor antagonist (montelukast). Knockdown of AQP4 by siRNA attenuated OGD/R injury. Furthermore, OGD/R increased phosphorylation of ERK1/2 and p38, whose inhibitors relieved the cell injury and AQP4 up-regulation. SIGNIFICANCE The CysLT(2) receptor mediates AQP4 up-regulation in astrocytes, and up-regulated AQP4 leads to OGD/R-induced injury, which results from activation of the ERK1/2 and p38 MAPK pathways.

Cysteinyl leukotriene receptor 2 is spatiotemporally involved in neuron injury, astrocytosis and microgliosis after focal cerebral ischemia in rats.

Cysteinyl leukotrienes (CysLTs), potent inflammatory mediators, are released from ischemic brain, and may regulate ischemic injury through activating CysLT1 and CysLT2 receptors. The CysLT1 receptor is closely associated with ischemic injury and post-ischemic repair; however, the CysLT2 receptor-mediated responses remain unknown. Here, we investigated the spatiotemporal profiles and implications of CysLT2 receptor expression and localization in rat brain after focal cerebral ischemia. CysLT2 receptors were normally localized in astrocytes in the cortex and around the ventricles. After focal cerebral ischemia, CysLT2 receptor expression was up-regulated in concert with neuronal and glial responses. In the acute phase (6-24 h), up-regulated CysLT2 receptors were restricted to injured neurons in the ischemic core; while in the late phase (3-28 days), the up-regulation was restricted to hypertrophic microglia (ischemic core) and mainly localized in hypertrophic astrocytes (boundary zone). Thus, the spatiotemporal profiles of CysLT2 receptor expression suggest that it plays regulatory roles in acute neuron injury, and astrocytosis and microgliosis in the late phase.

HAMI 3379, a CysLT2 Receptor Antagonist, Attenuates Ischemia-Like Neuronal Injury by Inhibiting Microglial Activation

The cysteinyl leukotrienes (CysLTs) are inflammatory mediators closely associated with neuronal injury after brain ischemia through the activation of their receptors, CysLT1R and CysLT2R. Here we investigated the involvement of both receptors in oxygen-glucose deprivation/recovery (OGD/R)-induced ischemic neuronal injury and the effect of the novel CysLT2R antagonist HAMI 3379 [3-({[(1S,3S)-3- carboxycyclohexyl]amino}carbonyl)-4-(3-{4-[4-(cyclo-hexyloxy)butoxy]phenyl}propoxy)benzoic acid] in comparison with the CysLT1R antagonist montelukast. In primary neurons, neither the nonselective agonist leukotriene D4 (LTD4) nor the CysLT2R agonist N-methyl-leukotriene C4 (NMLTC4) induced neuronal injury, and HAMI 3379 did not affect OGD/R-induced neuronal injury. However, in addition to OGD/R, LTD4 and NMLTC4 induced cell injury and neuronal loss in mixed cultures of cortical cells, and neuronal loss and necrosis in neuron-microglial cocultures. Moreover, they induced phagocytosis and cytokine release (interleukin-1β and tumor necrosis factor-α) from primary microglia, and conditioned medium from the treated microglia induced neuronal necrosis. HAMI 3379 inhibited all of these responses, and its effects were the same as those of CysLT2R interference by CysLT2R short hairpin RNA, indicating CysLT2R dependence. In comparison, montelukast moderately inhibited OGD/R-induced primary neuronal injury and most OGD/R- and LTD4-induced (but not NMLTC4-induced) responses in mixed cultures, cocultures, and microglia. The effects of montelukast were both dependent and independent of CysLT1Rs because interference by CysLT1R small interfering RNA had limited effects on neuronal injury in neuron-microglial cocultures and on cytokine release from microglia. Our findings indicated that HAMI 3379 effectively blocked CysLT2R-mediated microglial activation, thereby indirectly attenuating ischemic neuronal injury. Therefore, CysLT2R antagonists may represent a new type of therapeutic agent in the treatment of ischemic stroke.