A Novel Localization of METTL7A in Bergmann Glial Cells in Human Cerebellum.

Methyltransferase-like protein 7A (METTL7A) is a member of the METTL family of methyltransferases.Little information is available regarding the cellular expression of METTL7A in the brain. METTL7A is commonly located in the endoplasmic reticulum and to a lesser extent, in the lipid droplets of some cells. Several studies have reported altered protein and RNA levels in different brain areas in schizophrenia. One of these studies found reduced protein levels of METTL7A in the cerebellar cortex in schizophrenia and stress murine models. Since there is limited information in the literature about METTL7A, we characterized its cellular and subcellular localizations in the human cerebellum using immunohistochemical analysis with laser confocal microscopy. Our study reveals a novel METTL7A localization in GFAP-positive cells, with higher expression in the end-feet of the Bergmann glia, which participate in the cerebrospinal fluid-brain parenchyma barrier. Further 3D reconstruction image analysis showed that METTL7A was expressed in the contacts between the Bergmann glia and Purkinje neurons. METTL7A was also detected in lipid droplets in some cells in the white matter. The localization of METTL7A in the human cerebellar glia limitans could suggest a putative role in maintaining the cerebellar parenchyma homeostasis and in the regulation of internal cerebellar circuits by modulating the synaptic activity of Purkinje neurons.

Optical Control of Cardiac Function with a Photoswitchable Muscarinic Agonist.

Light-triggered reversible modulation of physiological functions offers the promise of enabling on-demand spatiotemporally controlled therapeutic interventions. Optogenetics has been successfully implemented in the heart, but significant barriers to its use in the clinic remain, such as the need for genetic transfection. Herein, we present a method to modulate cardiac function with light through a photoswitchable compound and without genetic manipulation. The molecule, named PAI, was designed by introduction of a photoswitch into the molecular structure of an M2 mAChR agonist. In vitro assays revealed that PAI enables light-dependent activation of M2 mAChRs. To validate the method, we show that PAI photoisomers display different cardiac effects in a mammalian animal model, and demonstrate reversible, real-time photocontrol of cardiac function in translucent wildtype tadpoles. PAI can also effectively activate M2 receptors using two-photon excitation with near-infrared light, which overcomes the scattering and low penetration of short-wavelength illumination, and offers new opportunities for intravital imaging and control of cardiac function.

GSEA of mouse and human mitochondriomes reveals fatty acid oxidation in astrocytes.

The prevalent view in neuroenergetics is that glucose is the main brain fuel, with neurons being mostly oxidative and astrocytes glycolytic. Evidence supporting that astrocyte mitochondria are functional has been overlooked. Here we sought to determine what is unique about astrocyte mitochondria by performing unbiased statistical comparisons of the mitochondriome in astrocytes and neurons. Using MitoCarta, a compendium of mitochondrial proteins, together with transcriptomes of mouse neurons and astrocytes, we generated cell-specific databases of nuclear genes encoding for mitochondrion proteins, ranked according to relative expression. Standard and in-house Gene Set Enrichment Analyses (GSEA) of five mouse transcriptomes revealed that genes encoding for enzymes involved in fatty acid oxidation (FAO) and amino acid catabolism are consistently more expressed in astrocytes than in neurons. FAO and oxidative-metabolism-related genes are also up-regulated in human cortical astrocytes versus the whole cortex, and in adult astrocytes versus fetal astrocytes. We thus present the first evidence of FAO in human astrocytes. Further, as shown in vitro, FAO coexists with glycolysis in astrocytes and is inhibited by glutamate. Altogether, these analyses provide arguments against the glucose-centered view of energy metabolism in astrocytes and reveal mitochondria as specialized organelles in these cells.

Cell survival during complete nutrient deprivation depends on lipid droplet-fueled ß-oxidation of fatty acids.

Cells exposed to stress of different origins synthesize triacylglycerols and generate lipid droplets (LD), but the physiological relevance of this response is uncertain. Using complete nutrient deprivation of cells in culture as a simple model of stress, we have addressed whether LD biogenesis has a protective role in cells committed to die. Complete nutrient deprivation induced the biogenesis of LD in human LN18 glioblastoma and HeLa cells and also in CHO and rat primary astrocytes. In all cell types, death was associated with LD depletion and was accelerated by blocking LD biogenesis after pharmacological inhibition of Group IVA phospholipase A2 (cPLA2α) or down-regulation of ceramide kinase. Nutrient deprivation also induced ß-oxidation of fatty acids that was sensitive to cPLA2α inhibition, and cell survival in these conditions became strictly dependent on fatty acid catabolism. These results show that, during nutrient deprivation, cell viability is sustained by ß-oxidation of fatty acids that requires biogenesis and mobilization of LD.

Brain specific kinase-1 BRSK1/SAD-B associates with lipid rafts: modulation of kinase activity by lipid environment.

Brain specific kinases 1 and 2 (BRSK1/2, also named SAD kinases) are serine-threonine kinases specifically expressed in the brain, and activated by LKB1-mediated phosphorylation of a threonine residue at their T-loop (Thr189/174 in human BRSK1/2). BRSKs are crucial for establishing neuronal polarity, and BRSK1 has also been shown to regulate neurotransmitter release presynaptically. How BRSK1 exerts this latter function is unknown, since its substrates at the synaptic terminal and the mechanisms modulating its activity remain to be described. Key regulators of neurotransmitter release, such as SNARE complex proteins, are located at membrane rafts. Therefore we initially undertook this work to check whether BRSK1 also locates at these membrane microdomains. Here we show that brain BRSK1, but not BRSK2, is palmitoylated, and provide biochemical and pharmacological evidences demonstrating that a pool of BRSK1, but not BRSK2 or LKB1, localizes at membrane lipid rafts. We also show that raft-associated BRSK1 has higher activity than BRSK1 from non-raft environment, based on a higher T-loop phosphorylation at Thr-189. Further, recombinant BRSK1 activity increased 3-fold when assayed with small multilamellar vesicles (SMV) generated with lipids extracted from synaptosomal raft fractions. A similar BRSK1-activating effect was obtained with synthetic SMV made with phosphatidylcholine, cholesterol and sphingomyelin, mixed in the same molar ratio at which these three major lipids are present in rafts. Importantly, SMV also enhanced the activity of a constitutively active BRSK1 (T189E), underpinning that interaction with lipid rafts represents a new mechanism of BRSK1 activity modulation, additional to T-loop phosphorylation.

Rational Design of Photochromic Analogues of Tricyclic Drugs.

Tricyclic chemical structures are the core of many important drugs targeting all neurotransmitter pathways. These medicines enable effective therapies to treat from peptic ulcer disease to psychiatric disorders. However, when administered systemically, they cause serious adverse effects that limit their use. To obtain localized and on-demand pharmacological action using light, we have designed photoisomerizable ligands based on azobenzene that mimic the tricyclic chemical structure and display reversibly controlled activity. Pseudo-analogues of the tricyclic antagonist pirenzepine demonstrate that this is an effective strategy in muscarinic acetylcholine receptors, showing stronger inhibition upon illumination both in vitro and in cardiac atria ex vivo. Despite the applied chemical modifications to make pirenzepine derivatives sensitive to light stimuli, the most potent candidate of the set, cryptozepine-2, maintained a moderate but promising M1 vs M2 subtype selectivity. These photoswitchable "crypto-azologs" of tricyclic drugs might open a general way to spatiotemporally target their therapeutic action while reducing their systemic toxicity and adverse effects.

Control of Brain State Transitions with a Photoswitchable Muscarinic Agonist.

The ability to control neural activity is essential for research not only in basic neuroscience, as spatiotemporal control of activity is a fundamental experimental tool, but also in clinical neurology for therapeutic brain interventions. Transcranial-magnetic, ultrasound, and alternating/direct current (AC/DC) stimulation are some available means of spatiotemporal controlled neuromodulation. There is also light-mediated control, such as optogenetics, which has revolutionized neuroscience research, yet its clinical translation is hampered by the need for gene manipulation. As a drug-based light-mediated control, the effect of a photoswitchable muscarinic agonist (Phthalimide-Azo-Iper (PAI)) on a brain network is evaluated in this study. First, the conditions to manipulate M2 muscarinic receptors with light in the experimental setup are determined. Next, physiological synchronous emergent cortical activity consisting of slow oscillations-as in slow wave sleep-is transformed into a higher frequency pattern in the cerebral cortex, both in vitro and in vivo, as a consequence of PAI activation with light. These results open the way to study cholinergic neuromodulation and to control spatiotemporal patterns of activity in different brain states, their transitions, and their links to cognition and behavior. The approach can be applied to different organisms and does not require genetic manipulation, which would make it translational to humans.

JNK and ceramide kinase govern the biogenesis of lipid droplets through activation of group IVA phospholipase A2.

The biogenesis of lipid droplets (LD) induced by serum depends on group IVA phospholipase A(2) (cPLA(2)alpha). This work dissects the pathway leading to cPLA(2)alpha activation and LD biogenesis. Both processes were Ca(2+)-independent, as they took place after pharmacological blockade of Ca(2+) transients elicited by serum or chelation with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester). The single mutation D43N in cPLA(2)alpha, which abrogates its Ca(2+) binding capacity and translocation to membranes, did not affect enzyme activation and formation of LD. In contrast, the mutation S505A did not affect membrane relocation of the enzyme in response to Ca(2+) but prevented its phosphorylation, activation, and the appearance of LD. Expression of specific activators of different mitogen-activated protein kinases showed that phosphorylation of cPLA(2)alpha at Ser-505 is due to JNK. This was confirmed by pharmacological inhibition and expression of a dominant-negative form of the upstream activator MEKK1. LD biogenesis was accompanied by increased synthesis of ceramide 1-phosphate. Overexpression of its synthesizing enzyme ceramide kinase increased phosphorylation of cPLA(2)alpha at Ser-505 and formation of LD, and its down-regulation blocked the phosphorylation of cPLA(2)alpha and LD biogenesis. These results demonstrate that LD biogenesis induced by serum is regulated by JNK and ceramide kinase.

Diacylglycerol is required for the formation of COPI vesicles in the Golgi-to-ER transport pathway.

Diacylglycerol is necessary for trans-Golgi network (TGN) to cell surface transport, but its functional relevance in the early secretory pathway is unclear. Although depletion of diacylglycerol did not affect ER-to-Golgi transport, it led to a redistribution of the KDEL receptor to the Golgi, indicating that Golgi-to-ER transport was perturbed. Electron microscopy revealed an accumulation of COPI-coated membrane profiles close to the Golgi cisternae. Electron tomography showed that the majority of these membrane profiles originate from coated buds, indicating a block in membrane fission. Under these conditions the Golgi-associated pool of ARFGAP1 was reduced, but there was no effect on the binding of coatomer or the membrane fission protein CtBP3/BARS to the Golgi. The addition of 1,2-dioctanoyl-sn-glycerol or the diacylglycerol analogue phorbol 12,13-dibutyrate reversed the effects of endogenous diacylglycerol depletion. Our findings implicate diacylglycerol in the retrograde transport of proteins from Golgi to the ER and suggest that it plays a critical role at a late stage of COPI vesicle formation.

Fluorination of Photoswitchable Muscarinic Agonists Tunes Receptor Pharmacology and Photochromic Properties.

Red-shifted azobenzene scaffolds have emerged as useful molecular photoswitches to expand potential applications of photopharmacological tool compounds. As one of them, tetra- ortho-fluoro azobenzene is well compatible for the design of visible-light-responsive systems, providing stable and bidirectional photoconversions and tissue-compatible characteristics. Using the unsubstituted azobenzene core and its tetra- ortho-fluorinated analogue, we have developed a set of uni- and bivalent photoswitchable toolbox derivatives of the highly potent muscarinic acetylcholine receptor agonist iperoxo. We investigated the impact of the substitution pattern on receptor activity and evaluated the different binding modes. Compounds 9b and 15b show excellent photochemical properties and biological activity as fluorination of the azobenzene core alters not only the photochromic behavior but also the pharmacological profile at the muscarinic M1 receptor. These findings demonstrate that incorporation of fluorinated azobenzenes not just may alter photophysical properties but can exhibit a considerably different biological profile that has to be carefully investigated.

Analyzing ligand depletion in a saturation equilibrium binding experiment.

I present a proposal for a laboratory practice to generate and analyze data from a saturation equilibrium binding experiment addressed to advanced undergraduate students. [(3) H]Quinuclidinyl benzilate is a nonselective muscarinic ligand with very high affinity and very low nonspecific binding to brain membranes, which contain a high density of muscarinic receptors. These features allow the instructor to devote especial emphasis to evaluate ligand depletion, and therefore, stress the subtle but fundamental difference between total (added) ligand and free ligand concentration at equilibrium.

Mutation of the 3-Phosphoinositide-Dependent Protein Kinase 1 (PDK1) Substrate-Docking Site in the Developing Brain Causes Microcephaly with Abnormal Brain Morphogenesis Independently of Akt, Leading to Impaired Cognition and Disruptive Behaviors.

The phosphoinositide (PI) 3-kinase/Akt signaling pathway plays essential roles during neuronal development. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) coordinates the PI 3-kinase signals by activating 23 kinases of the AGC family, including Akt. Phosphorylation of a conserved docking site in the substrate is a requisite for PDK1 to recognize, phosphorylate, and activate most of these kinases, with the exception of Akt. We exploited this differential mechanism of regulation by generating neuron-specific conditional knock-in mice expressing a mutant form of PDK1, L155E, in which the substrate-docking site binding motif, termed the PIF pocket, was disrupted. As a consequence, activation of all the PDK1 substrates tested except Akt was abolished. The mice exhibited microcephaly, altered cortical layering, and reduced circuitry, leading to cognitive deficits and exacerbated disruptive behavior combined with diminished motivation. The abnormal patterning of the adult brain arises from the reduced ability of the embryonic neurons to polarize and extend their axons, highlighting the essential roles that the PDK1 signaling beyond Akt plays in mediating the neuronal responses that regulate brain development.

NAADP mediates ATP-induced Ca2+ signals in astrocytes.

Intracellular Ca(2+) signals provide astrocytes with a specific form of excitability that enables them to regulate synaptic transmission. In this study, we demonstrate that NAADP-AM, a membrane-permeant analogue of the new second messenger nicotinic acid-adenine dinucleotide phosphate (NAADP), mobilizes Ca(2+) in astrocytes and that the response is blocked by Ned-19, an antagonist of NAADP signalling. We also show that NAADP receptors are expressed in lysosome-related acidic vesicles. Pharmacological disruption of either NAADP or lysosomal signalling reduced Ca(2+) responses induced by ATP and endothelin-1, but not by bradykinin. Furthermore, ATP increased endogenous NAADP levels. Overall, our data provide evidence for NAADP being an intracellular messenger for agonist-mediated calcium signalling in astrocytes.

Lipid droplet biogenesis induced by stress involves triacylglycerol synthesis that depends on group VIA phospholipase A2.

This work investigates the metabolic origin of triacylglycerol (TAG) formed during lipid droplet (LD) biogenesis induced by stress. Cytotoxic inhibitors of fatty acid synthase induced TAG synthesis and LD biogenesis in CHO-K1 cells, in the absence of external sources of fatty acids. TAG synthesis was required for LD biogenesis and was sensitive to inhibition and down-regulation of the expression of group VIA phospholipase A(2) (iPLA(2)-VIA). Induction of stress with acidic pH, C(2)-ceramide, tunicamycin, or deprivation of glucose also stimulated TAG synthesis and LD formation in a manner dependent on iPLA(2)-VIA. Overexpression of the enzyme enhanced TAG synthesis from endogenous fatty acids and LD occurrence. During stress, LD biogenesis but not TAG synthesis required phosphorylation and activation of group IVA PLA(2) (cPLA(2)alpha). The results demonstrate that iPLA(2)-VIA provides fatty acids for TAG synthesis while cPLA(2)alpha allows LD biogenesis. LD biogenesis during stress may be a survival strategy, recycling structural phospholipids into energy-generating substrates.

Cytokine secretion requires phosphatidylcholine synthesis.

Choline cytidylyltransferase (CCT) is the rate-limiting enzyme in the phosphatidylcholine biosynthetic pathway. Here, we demonstrate that CCT alpha-mediated phosphatidylcholine synthesis is required to maintain normal Golgi structure and function as well as cytokine secretion from the Golgi complex. CCT alpha is localized to the trans-Golgi region and its expression is increased in lipopolysaccharide (LPS)-stimulated wild-type macrophages. Although LPS triggers transient reorganization of Golgi morphology in wild-type macrophages, similar structural alterations persist in CCT alpha-deficient cells. Pro-tumor necrosis factor alpha and interleukin-6 remain lodged in the secretory compartment of CCT alpha-deficient macrophages after LPS stimulation. However, the lysosomal-mediated secretion pathways for interleukin-1 beta secretion and constitutive apolipoprotein E secretion are unaltered. Exogenous lysophosphatidylcholine restores LPS-stimulated secretion from CCT alpha-deficient cells, and elevated diacylglycerol levels alone do not impede secretion of pro-tumor necrosis factor alpha or interleukin-6. These results identify CCT alpha as a key component in membrane biogenesis during LPS-stimulated cytokine secretion from the Golgi complex.

Group IVA phospholipase A2 is necessary for the biogenesis of lipid droplets.

Lipid droplets (LD) are organelles present in all cell types, consisting of a hydrophobic core of triacylglycerols and cholesteryl esters, surrounded by a monolayer of phospholipids and cholesterol. This work shows that LD biogenesis induced by serum, by long-chain fatty acids, or the combination of both in CHO-K1 cells was prevented by phospholipase A(2) inhibitors with a pharmacological profile consistent with the implication of group IVA cytosolic phospholipase A(2) (cPLA(2)alpha). Knocking down cPLA(2)alpha expression with short interfering RNA was similar to pharmacological inhibition in terms of enzyme activity and LD biogenesis. A Chinese hamster ovary cell clone stably expressing an enhanced green fluorescent protein-cPLA(2)alpha fusion protein (EGFP-cPLA(2)) displayed higher LD occurrence under basal conditions and upon LD induction. Induction of LD took place with concurrent phosphorylation of cPLA(2)alpha at Ser(505). Transfection of a S505A mutant cPLA(2)alpha showed that phosphorylation at Ser(505) is key for enzyme activity and LD formation. cPLA(2)alpha contribution to LD biogenesis was not because of the generation of arachidonic acid, nor was it related to neutral lipid synthesis. cPLA(2)alpha inhibition in cells induced to form LD resulted in the appearance of tubulo-vesicular profiles of the smooth endoplasmic reticulum, compatible with a role of cPLA(2)alpha in the formation of nascent LD from the endoplasmic reticulum.

Lysophosphatidic acid rescues RhoA activation and phosphoinositides levels in astrocytes exposed to ethanol.

Long-term ethanol treatment substantially impairs glycosylation and membrane trafficking in primary cultures of rat astrocytes. Our previous studies indicated that these effects were attributable to a primary alteration in the dynamics and organization of the actin cytoskeleton, although the molecular mechanism(s) remains to be elucidated. As small Rho GTPases and phosphoinositides are involved in the actin cytoskeleton organization, we now explore the effects of chronic ethanol treatment on these pathways. We show that chronic ethanol treatment of rat astrocytes specifically reduced endogenous levels of active RhoA as a result of the increase of in the RhoGAP activity. Furthermore, ethanol-treated astrocytes showed reduced phosphoinositides levels. When lysophosphatidic acid was added to ethanol-treated astrocytes, it rapidly reverted actin cytoskeleton reorganization and raised active RhoA levels and phosphoinositides content to those observed in untreated astrocytes. Overall, our results indicate that the harmful effects of chronic exposure to ethanol on a variety of actin dynamics-associated cellular events are primarily because of alterations of activated RhoA and phosphoinositides pools.

Prevalence of necrosis in C2-ceramide-induced cytotoxicity in NB16 neuroblastoma cells.

The mechanism of cell death triggered by C2-ceramide was investigated using the NB16 neuroblastoma cell line. Treatment of NB16 cells with 20 microM C2-ceramide for 20 h resulted in approximately 75% loss of cell viability, but only 25% of cells were scored as apoptotic based on terminal deoxynucleotidyl transferase nick-end labeling. Ultrastructural analysis revealed early development of necrotic cytoplasmic vacuolization. After 20 h of treatment with C2-ceramide, the majority of cells possessed necrotic morphology with pronounced cytoplasmic vacuolization and without any nuclear changes, although a quarter of the cell population also exhibited clear perinuclear chromatin condensation characteristic of apoptosis. Flow cytometric analysis of cells labeled with both annexin V and propidium iodide showed the rapid accumulation of C2-ceramide-treated cells in the necrotic/late apoptotic fraction. In contrast, cells treated with tumor necrosis factor alpha plus cycloheximide (TNFalpha + CHX) first appeared in the early apoptotic fraction and then accumulated in the necrotic/late apoptotic fraction. Both C2-ceramide and TNFalpha + CHX increased caspase 8- and 3-like activities in cytosolic extracts; however, treatment of cells with the broad-spectrum caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone protected NB16 cells from TNFalpha + CHX-induced cell death but did not prevent C2-ceramide cytotoxicity. Although C2-ceramide triggered apoptosis in a fraction of the cells, cell death in the population was primarily caused by necrosis. Thus, C2-ceramide does not faithfully mimic the effects of apoptotic ligands such as TNFalpha, which are thought to be mediated by an accumulation of endogenous ceramide. The inhibition of phosphatidylcholine synthesis is a target for C2-ceramide-mediated cytotoxicity, and this work suggests that other agents that kill cells by inhibiting this pathway may also use a mixture of mechanisms, including necrosis as well as apoptosis.

Inhibition of CTP:phosphocholine cytidylyltransferase by C(2)-ceramide and its relationship to apoptosis.

Apoptosis induced by antitumor phospholipid analogs takes place after the inhibition of the CTP:phosphocholine cytidylyltransferase (CCT; EC 2.7.7.15) catalyzed step of phosphatidylcholine (PtdCho) biosynthesis. Exposure of cells to synthetic short-chain ceramide analogs also triggers apoptosis concomitant with decreased PtdCho biosynthesis, and the present study was undertaken to ascertain whether C(2)-ceramide inhibition of PtdCho synthesis is direct or secondary to other ceramide-mediated cellular responses. The exposure of COS-7 cells to either C(2)-ceramide, ET-18-OCH(3), or farnesol resulted in time- and dose-dependent apoptotic cell death. Cells treated with C(2)-ceramide or ET-18-OCH(3) selectively and immediately accumulated phosphocholine, whereas CDP-choline increased with farnesol treatment. In vitro assays of CCT activity demonstrated that C(2)-ceramide directly inhibited CCT. Comparison of different N-linked sphingosine derivatives suggests an inverse relationship between the length of the N-linked carbon chain and the derivatives ability to trigger apoptosis and inhibit CCT. Taken together, our results suggest CCT as a primary target for C(2)-ceramide inhibition that accounts for its cytotoxic effects.

Opposed effects of lithium on the MEK-ERK pathway in neural cells: inhibition in astrocytes and stimulation in neurons by GSK3 independent mechanisms.

Lithium is widely used in the treatment of bipolar disorder, but despite its proven therapeutic efficacy, the molecular mechanisms of action are not fully understood. The present study was undertaken to explore lithium effects of the MEK/ERK cascade of protein kinases in astrocytes and neurons. In asynchronously proliferating rat cortical astrocytes, lithium decreased time- and dose-dependently the phosphorylation of MEK and ERK, with 1 mM concentrations achieving 60 and 50% inhibition of ERK and MEK, respectively, after a 7-day exposure. Lithium also inhibited [3H]thymidine incorporation into DNA and induced a G2/M cell cycle arrest. In serum-deprived, quiescent astrocytes, pre-exposure to lithium resulted in the inhibition of cell cycle re-entry as stimulated by the mitogen endothelin-1: under this experimental setting, lithium did not affect the rapid, peak phosphorylation of MEK taking place after 3-5 min, but was effective in inhibiting the long-term, sustained phosphorylation of MEK. Lithium inhibition of the astrocyte MEK/ERK pathway was independent of inositol depletion. Further, compound SB216763 inhibited Tau phosphorylation at Ser396 and stabilized cytosolic beta-catenin, consistent with the inhibition of glycogen synthase kinase-3 beta (GSK-3 beta), but failed to reproduce lithium effects on MEK and ERK phosphorylation and cell cycle arrest. In cerebellar granule neurons, millimolar concentrations of lithium enhanced MEK and ERK phosphorylation in a concentration-dependent manner, again through an inositol and GSK-3 beta independent mechanism. These opposing effects in astrocytes and neurons make lithium treatment a promising strategy to favour neural repair and reduce reactive gliosis after traumatic injury.