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 ''␥␣'' 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.biomechanics ͉ elasticity ͉ viscosity ͉ retina ͉ hippocampus

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 ¼ 8.5 AE 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 ¼ 106 AE 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 μm 2 . 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.motility | neuron | elasticity | mechanics | scanning force microscopy N euronal growth is a fundamental event during ontogenetic development and nerve regeneration. The mechanical forces required for neuronal pathfinding and translocation are generated in the growth cone, a motile extension at the neurite tip (1). As in other motile structures, growth cone movement involves actin polymerization and myosin motor mediated contraction of the actin network in the lamellipodium, a flat structure internally made up by a cross-linked actin network (2). The movement of the leading edge is determined by the difference between outward pushing actin polymerization and the continuous centripetal flow of actin, called the retrograde flow (3) (Fig. S1). Local measurements of the retrograde flow (3, 4) and traction forces of filopodia (5, 6) have been reported on various substrate stiffness. These observations and the detailed analysis of neuron branching on substrates of varying stiffness (7) highlight the importance of the mechanical environment for neuronal growth. Furthermore, the constant neurite tension that is maintained by molecular motors in the neurite cytoskeleton has been extensively studied (8-10). However, neither the internal forces, which are the forces generated and acting within the growth cone, nor the growth cone's lamellipodial traction forces have yet been quantified. To understand the mechanics of neuronal growth, knowledge of the relation between the growth cone's viscoelastic material properties, the internally generated forces and the traction forces transmitted to the substrate is of fundamental importance.A recent study used speckle microscopy and a simple spring model to convert data from retro...

Recent results indicate that, in addition to chemical cues, mechanical stimuli may also impact neuronal growth. For instance, unlike most other cell types, neurons prefer soft substrates. However, the mechanisms responsible for the neuronal affinity for soft substrates have not yet been identified. In this study, we show that, in vitro, neurons continuously probe their mechanical environment. Growth cones visibly deform substrates with a compliance commensurate with their own. To understand the sensing of stiff substrates by growth cones, we investigated their precise temporal response to well-defined mechanical stress. When the applied stress exceeded a threshold of 274 +/- 41 pN/microm(2), neurons retracted and re-extended their processes, thereby enabling exploration of alternative directions. A calcium influx through stretch-activated ion channels and the detachment of adhesion sites were prerequisites for this retraction. Our data illustrate how growing neurons may detect and avoid stiff substrates--as a mechanism involved in axonal branch pruning--and provide what we believe is novel support of the idea that mechanics may act as guidance cue for neuronal growth.

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.

Ubiquitin (Ub) is an important signaling protein. Recent studies have shown that Ub can be enzymatically phosphorylated at S65, and that the resulting pUb exhibits two conformational states-a relaxed state and a retracted state. However, crystallization efforts have yielded only the structure for the relaxed state, which was found similar to that of unmodified Ub. Here we present the solution structures of pUb in both states obtained through refinement against state-specific NMR restraints. We show that the retracted state differs from the relaxed state by the retraction of the last β-strand and by the extension of the second α-helix. Further, we show that at 7.2, the pK a value for the phosphoryl group in the relaxed state is higher by 1.4 units than that in the retracted state. Consequently, pUb exists in equilibrium between protonated and deprotonated forms and between retracted and relaxed states, with protonated/relaxed species enriched at slightly acidic pH and deprotonated/retracted species enriched at slightly basic pH. The heterogeneity of pUb explains the inability of phosphomimetic mutants to fully mimic pUb. The pHsensitive conformational switch is likely preserved for polyubiquitin, as single-molecule FRET data indicate that pH change leads to quaternary rearrangement of a phosphorylated K63-linked diubiquitin. Because cellular pH varies among compartments and changes upon pathophysiological insults, our finding suggests that pH and Ub phosphorylation confer additional target specificities and enable an additional layer of modulation for Ub signals., a 76-residue signaling protein, is found ubiquitously in cells. Two or more Ub molecules can be covalently linked to form a diubiquitin (diUb) and then a polyubiquitin (polyUb), as an isopeptide bond is formed between the carboxylate group of one Ub (called the distal Ub) and the amine group of another Ub (called the proximal Ub). Owing to the characteristic quaternary structures of polyUb and specific interactions between polyUb and its target proteins (1, 2), a polyUb with a specific linkage can be involved in a distinctive set of cellular functions (3).The heterogeneity of Ub also arises from other types of covalent modifications. Proteomics studies have indicated that Ub is phosphorylated at multiple sites (4, 5). However, PINK1 is the only Ub kinase known to date, which specifically phosphorylates Ub at S65 (6, 7). Under normal conditions, only a fraction of Ub is S65-phosphorylated. However, upon oxidative stress, neurodegeneration, or aging, the level of S65 phosphorylation increases significantly (4, 8). S65-phosphorylated Ub (pUb) in turn can activate PARKIN, a ubiquitin ligase, and induce mitophagy (9-12). However, no other pUb-specific targets have been clearly identified, and how phosphorylation affects Ub signaling in general remains unclear.Previously, Komander and coworkers showed that pUb gives two distinct sets of NMR peaks, which correspond to the two conformational states of pUb exchanging at a slow timescale (13). Based on NMR long-r...

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 CysLT(1) and CysLT(2) 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 A(2) 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 CysLT(1) receptor mRNA and protein, while moderate OGD induced the expression of CysLT(2) receptor mRNA. Exogenously applied leukotriene D(4) (LTD(4)) induced astrocyte proliferation at 1-10 nM and astrocyte death at 100-1,000 nM. The CysLT(1) receptor antagonist montelukast attenuated astrocyte proliferation, the CysLT(2) receptor antagonist BAY cysLT2 reversed astrocyte death, and the dual CysLT receptor antagonist BAY u9773 exhibited both effects. In addition, LTD(4) (100 nM) increased the expression of CysLT(2) receptor mRNA. Thus, in vitro ischemia activates astrocyte 5-LOX to produce CysLTs, and CysLTs result in CysLT(1) receptor-mediated proliferation and CysLT(2) receptor-mediated death.

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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 microM) 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 microM) also increased the generation of reactive oxygen species in C6 cells. These effects of dihydroartemisinin were enhanced by ferrous ions (12.5-100 microM) and reduced by the iron chelator deferoxamine (25-200 microM). Immunoblotting analysis revealed that dihydroartemisinin (5-25 microM) significantly reduced hypoxia- and deferoxamine-induced expression of hypoxia inducible factor-1alpha 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-1alpha activation.