Chew L. Lau

Effects of lipopolysaccharide on glial phenotype and activity of glutamate transporters: Evidence for delayed up-regulation and redistribution of GLT-1.

Excitatory amino acid transporters (EAATs) are responsible for homeostasis of extracellular L-glutamate, and the glial transporters are functionally dominant. EAAT expression or function is altered in acute and chronic neurological conditions, but little is known about the regulation of EAATs in reactive astroglia found in such neuropathologies. These studies examined the effects of the bacterial endotoxin lipopolysaccharide (LPS) on glial EAATs in vitro. The effects of LPS (1 microg/ml, 24-72 h) on EAAT activity and expression were examined in primary cultures of mouse astrocytes. [(3)H]D-aspartate uptake increased to 129% of control by 72 h treatment with LPS. Saturation analysis revealed that apparent K(m) was unchanged whilst V(max) was significantly increased to 172% of control by 72 h LPS treatment. Biotinylation and Western blotting indicated that cell-surface expression of GLT-1 was significantly elevated (146% control) by LPS treatment whereas GLAST expression was unchanged. Confocal analyses revealed that LPS treatment resulted in cytoskeletal changes and stellation of astrocytes, with rearrangement of F-actin (as shown by phalloidin labelling). Immunocytochemistry revealed clustering of GLAST, and increased expression and redistribution of GLT-1 to the cell-surface following treatment with LPS. Similar experiments were conducted in microglia, where LPS (50 ng/ml) was found to up-regulate expression of GLT-1 at 24 and 72 h in concert with cytoskeletal changes accompanying activation. These findings suggest an association of cytoskeletal changes in glia with EAAT activity, with the predominant adaptation involving up-regulation and redistribution of GLT-1.

Transcriptomic profiling of astrocytes treated with the Rho kinase inhibitor Fasudil reveals cytoskeletal and pro-survival responses†

Inhibitors of Rho kinase (ROCK) have potential for management of neurological disorders by inhibition of glial scarring. Since astrocytes play key roles in brain physiology and pathology, we determined changes in the astrocytic transcriptome produced by the ROCK inhibitor Fasudil to obtain mechanistic insights into its beneficial action during brain injury. Cultured murine astrocytes were treated with Fasudil (100 µM) and morphological analyses revealed rapid stellation by 1 h and time‐dependent (2–24 h) dissipation of F‐actin‐labelled stress fibres. Microarray analyses were performed on RNA and the time‐course of global gene profiling (2, 6, 12 and 24 h) provided a comprehensive description of transcriptomic changes. Hierarchical clustering of differentially expressed genes and analysis for over‐represented gene ontology groups using the DAVID database focused attention on Fasudil‐induced changes to major biological processes regulating cellular shape and motility (actin cytoskeleton, axon guidance, transforming growth factor‐β (TGFβ) signalling and tight junctions). Bioinformatic analyses of transcriptomic changes revealed how these biological processes contributed to changes in astrocytic motility and cytoskeletal reorganisation. Here genes associated with extracellular matrix were also involved, but unexpected was a subset of alterations (EAAT2, BDNF, anti‐oxidant species, metabolic and signalling genes) indicative of adoption by astrocytes of a pro‐survival phenotype. Expression profiles of key changes with Fasudil and another ROCK inhibitor Y27632 were validated by real‐time PCR. Although effects of ROCK inhibition have been considered to be primarily cytoskeletal via reduction of glial scarring, we demonstrate additional advantageous actions likely to contribute to their ameliorative actions in brain injury. J. Cell. Physiol. 227: 1199–1211, 2012.

3D Electrospun scaffolds promote a cytotrophic phenotype of cultured primary astrocytes.

Astrocytes are a target for regenerative neurobiology because in brain injury their phenotype arbitrates brain integrity, neuronal death and subsequent repair and reconstruction. We explored the ability of 3D scaffolds to direct astrocytes into phenotypes with the potential to support neuronal survival. Poly‐ε‐caprolactone scaffolds were electrospun with random and aligned fibre orientations on which murine astrocytes were sub‐cultured and analysed at 4 and 12 DIV. Astrocytes survived, proliferated and migrated into scaffolds adopting 3D morphologies, mimicking in vivo stellated phenotypes. Cells on random poly‐ε‐caprolactone scaffolds grew as circular colonies extending processes deep within sub‐micron fibres, whereas astrocytes on aligned scaffolds exhibited rectangular colonies with processes following not only the direction of fibre alignment but also penetrating the scaffold. Cell viability was maintained over 12 DIV, and cytochemistry for F‐/G‐actin showed fewer stress fibres on bioscaffolds relative to 2D astrocytes. Reduced cytoskeletal stress was confirmed by the decreased expression of glial fibrillary acidic protein. PCR demonstrated up‐regulation of genes (excitatory amino acid transporter 2, brain‐derived neurotrophic factor and anti‐oxidant) reflecting healthy biologies of mature astrocytes in our extended culture protocol. This study illustrates the therapeutic potential of bioengineering strategies using 3D electrospun scaffolds which direct astrocytes into phenotypes supporting brain repair.

Regulation of glutamate transporters in astrocytes: evidence for a relationship between transporter expression and astrocytic phenotype.

The astrocytic glutamate transporters, EAAT1 and EAAT2, remove released L-glutamate from the synaptic milieu thereby maintaining normal excitatory transmission. EAAT dysfunction during the excitotoxicity and oxidative stress of neurological insults may involve homoeostatic mechanisms associated with astrocytic function. We investigated aspects of EAAT function and expression in concert with astrocytic phenotype in primary cultures of cortical astrocytes and mixed cells of the spinal cord. In spinal cord mixed cultures, hydrogen peroxide (300 µM) reduced both EAAT activity and cellular viability to half of their basal values at 24 h post-treatment, but at 2 h EAAT activity was unaltered, while cellular viability was significantly decreased, suggestive of a mechanism for the maintenance of EAAT activity. Cytochemistry for MAP2, GFAP and propidium iodide revealed that neurons and astrocytes were damaged in a time-dependent manner. A change in astrocyte morphology was observed, with astrocyte cell bodies becoming larger and processes becoming more stellate and often shorter in length. EAAT1 immunoreactivity was reduced at 24 h post-treatment and a re-distribution of the protein was noted after 2 h treatment. In pure astrocytes, lipopolysaccharide (1 µg/ml, 3 d) increased [3H]D-aspartate uptake by 90%, as well as EAAT1 immunoreactivity and astrocyte stellation, as shown by immunofluorescent labelling for GFAP. In both culture systems, prominent changes were noted in EAAT function and localization in conjunction with altered astrocytic phenotype. Our findings are indicative of a relationship between astrocytic phenotype and the level of EAAT activity that may be a vital component of astrocytic homeostatic responses in brain injury.

Links between l-glutamate transporters, Na+/K+-ATPase and cytoskeleton in astrocytes: Evidence following inhibition with rottlerin

Astrocytes are plastic cells that play key roles in brain physiology and pathology, including via their glutamate transporters, excitatory amino acid transporter (EAAT)1 and EAAT2, maintaining low extracellular glutamate concentrations and protecting against excitotoxic neuronal injury. Alterations in cell surface expression of EAATs and astrocytic cytoskeleton are important for regulating transporter activity. This study employed the actions of rottlerin, to interrogate the regulation of EAAT activity, expression and localization, and interfaces with Na(+)/K(+)-ATPase and astrocytic morphology. EAAT activity and expression were determined in primary cultures of mouse astrocytes in the presence of and after rottlerin removal, with or without trafficking inhibitors, using uptake ([(3)H]d-aspartate, (86)Rb(+)) and molecular analyses. Astrocytic morphology and EAAT localization were investigated using Western blotting and immunocytochemistry, in concert with image analysis of glial fibrillary acidic protein, F-actin and EAAT1/2. Rottlerin induced a time-dependent inhibition of glutamate transport (Vmax). Rapid changes in cytoskeletal arrangement were observed and immunoblotting revealed increases in EAAT2 total and cell surface expression, despite reduced EAAT activity. Rottlerin-induced inhibition was reversible and its rate was increased by monensin co-treatment. Rottlerin inhibited, while monensin stimulated Na(+)/K(+)-ATPase. Removal of rottlerin rapidly elevated Na(+)/K(+)-ATPase activity beyond control levels, while co-treatment with monensin failed to stimulate the Na(+)/K(+)-ATPase. These data reveal inhibition of EAAT activity by rottlerin is not associated with loss of EAATs at the cell surface, but rather linked to cytoskeletal rearrangement, and inhibition of the Na(+)/K(+)-ATPase. Rapid recovery of Na(+)/K(+)-ATPase activity, and subsequent restoration of glutamate uptake indicates that astrocytic morphology and EAAT activity are co-regulated by a tightly coupled, homeostatic relationship between l-glutamate uptake, the electrochemical gradient and the activity of the Na(+)/K(+)-ATPase.

Transcriptomic analysis and 3D bioengineering of astrocytes indicate ROCK inhibition produces cytotrophic astrogliosis

Astrocytes provide trophic, structural and metabolic support to neurons, and are considered genuine targets in regenerative neurobiology, as their phenotype arbitrates brain integrity during injury. Inhibitors of Rho kinase (ROCK) cause stellation of cultured 2D astrocytes, increased L-glutamate transport, augmented G-actin, and elevated expression of BDNF and anti-oxidant genes. Here we further explored the signposts of a cytotrophic, “healthy” phenotype by data-mining of our astrocytic transcriptome in the presence of Fasudil. Gene expression profiles of motor and autophagic cellular cascades and inflammatory/angiogenic responses were all inhibited, favoring adoption of an anti-migratory phenotype. Like ROCK inhibition, tissue engineered bioscaffolds can influence the extracellular matrix. We built upon our evidence that astrocytes maintained on 3D poly-ε-caprolactone (PCL) electrospun scaffolds adopt a cytotrophic phenotype similar to that produced by Fasudil. Using these procedures, employing mature 3D cultured astrocytes, Fasudil (100 μM) or Y27632 (30 μM) added for the last 72 h of culture altered arborization, which featured numerous additional minor processes as shown by GFAP and AHNAK immunolabelling. Both ROCK inhibitors decreased F-actin, but increased G-actin labeling, indicative of disassembly of actin stress fibers. ROCK inhibitors provide additional beneficial effects for bioengineered 3D astrocytes, including enlargement of the overall arbor. Potentially, the combined strategy of bio-compatible scaffolds with ROCK inhibition offers unique advantages for the management of glial scarring. Overall these data emphasize that manipulation of the astrocyte phenotype to achieve a “healthy biology” offers new hope for the management of inflammation in neuropathologies.

Silent information regulator 1 modulator resveratrol increases brain lactate production and inhibits mitochondrial metabolism, whereas SRT1720 increases oxidative metabolism.

Silent information regulators (SIRTs) have been shown to deacetylate a range of metabolic enzymes, including those in glycolysis and the Krebs cycle, and thus alter their activity. SIRTs require NAD+ for their activity, linking cellular energy status to enzyme activity. To examine the impact of SIRT1 modulation on oxidative metabolism, this study tests the effect of ligands that are either SIRT‐activating compounds (resveratrol and SRT1720) or SIRT inhibitors (EX527) on the metabolism of 13C‐enriched substrates by guinea pig brain cortical tissue slices with 13C and 1H nuclear magnetic resonance spectroscopy. Resveratrol increased lactate labeling but decreased incorporation of 13C into Krebs cycle intermediates, consistent with effects on AMPK and inhibition of the F0/F1‐ATPase. By testing with resveratrol that was directly applied to astrocytes with a Seahorse analyzer, increased glycolytic shift and increased mitochondrial proton leak resulting from interactions of resveratrol with the mitochondrial electron transport chain were revealed. SRT1720, by contrast, stimulated incorporation of 13C into Krebs cycle intermediates and reduced incorporation into lactate, although the inhibitor EX527 paradoxically also increased Krebs cycle 13C incorporation. In summary, the various SIRT1 modulators show distinct acute effects on oxidative metabolism. The strong effects of resveratrol on the mitochondrial respiratory chain and on glycolysis suggest that caution should be used in attempts to increase bioavailability of this compound in the CNS.

GABAergic striatal neurons exhibit caspase-independent, mitochondrially mediated programmed cell death.

GABAergic striatal neurons are compromised in basal ganglia pathologies and we analysed how insult nature determined their patterns of injury and recruitment of the intrinsic mitochondrial pathway during programmed cell death (PCD). Stressors affecting targets implicated in striatal neurodegeneration [3‐morpholinylsydnoneimine (SIN‐1), 3‐nitropropionic acid (3‐NP), NMDA, 3,5‐dihydroxyphenylglycine (DHPG), and staurosporine (STS)] were compared in cultured GABAergic neurons from murine striatum by analyzing the progression of injury and its correlation with mitochondrial involvement, the redistribution of intermembrane space (IMS) proteins, and patterns of protease activation. Stressors produced PCD exhibiting slow‐onset kinetics with time‐dependent annexin‐V labeling and eventual DNA fragmentation. IMS proteins including cytochrome c were differentially distributed, although stressors except STS produced early redistribution of apoptosis‐inducing factor and Omi, suggestive of early recruitment of both caspase‐dependent and caspase‐independent signaling. In general, Bax mobilization to mitochondria appeared to promote IMS protein redistribution. Caspase 3 activation was prominent after STS, whereas NMDA and SIN‐1 produced mainly calpain activation, and 3‐NP and DHPG elicited a mixed profile of protease activation. PCD and redistribution of IMS proteins in striatal GABAergic neurons were canonical and insult‐dependent, reflecting differential interplay between the caspase cascade and alternate cell death pathways.

Rilmenidine promotes MTOR-independent autophagy in the mutant SOD1 mouse model of amyotrophic lateral sclerosis without slowing disease progression

ABSTRACT Macroautophagy/autophagy is the main intracellular catabolic pathway in neurons that eliminates misfolded proteins, aggregates and damaged organelles associated with ageing and neurodegeneration. Autophagy is regulated by both MTOR-dependent and -independent pathways. There is increasing evidence that autophagy is compromised in neurodegenerative disorders, which may contribute to cytoplasmic sequestration of aggregation-prone and toxic proteins in neurons. Genetic or pharmacological modulation of autophagy to promote clearance of misfolded proteins may be a promising therapeutic avenue for these disorders. Here, we demonstrate robust autophagy induction in motor neuronal cells expressing SOD1 or TARDBP/TDP-43 mutants linked to amyotrophic lateral sclerosis (ALS). Treatment of these cells with rilmenidine, an anti-hypertensive agent and imidazoline-1 receptor agonist that induces autophagy, promoted autophagic clearance of mutant SOD1 and efficient mitophagy. Rilmenidine administration to mutant SOD1G93A mice upregulated autophagy and mitophagy in spinal cord, leading to reduced soluble mutant SOD1 levels. Importantly, rilmenidine increased autophagosome abundance in motor neurons of SOD1G93A mice, suggesting a direct action on target cells. Despite robust induction of autophagy in vivo, rilmenidine worsened motor neuron degeneration and symptom progression in SOD1G93A mice. These effects were associated with increased accumulation and aggregation of insoluble and misfolded SOD1 species outside the autophagy pathway, and severe mitochondrial depletion in motor neurons of rilmenidine-treated mice. These findings suggest that rilmenidine treatment may drive disease progression and neurodegeneration in this mouse model due to excessive mitophagy, implying that alternative strategies to beneficially stimulate autophagy are warranted in ALS.

MDMA-induced neurotoxicity of serotonin neurons involves autophagy and rilmenidine is protective against its pathobiology

Abstract Toxicity of 3,4‐methylenedioxymethamphetamine (MDMA) towards biogenic amine neurons is well documented and in primate brain predominantly affects serotonin (5‐HT) neurons. MDMA induces damage of 5‐HT axons and nerve fibres and intracytoplasmic inclusions. Whilst its pathobiology involves mitochondrially‐mediated oxidative stress, we hypothesised MDMA possessed the capacity to activate autophagy, a proteostatic mechanism for degradation of cellular debris. We established a culture of ventral pons from embryonic murine brain enriched in 5‐HT neurons to explore mechanisms of MDMA neurotoxicity and recruitment of autophagy, and evaluated possible neuroprotective actions of the clinically approved agent rilmenidine. MDMA (100 &mgr;M–1 mM) reduced cell viability, like rapamycin (RM) and hydrogen peroxide (H2O2), in a concentration‐ and time‐dependent manner. Immunocytochemistry revealed dieback of 5‐HT arbour: MDMA‐induced injury was slower than for RM and H2O2, neuritic blebbing occurred at 48 and 72 h and Hoechst labelling revealed nuclear fragmentation with 100 &mgr;M MDMA. MDMA effected concentration‐dependent inhibition of [3H]5‐HT uptake with 500 &mgr;M MDMA totally blocking transport. Western immunoblotting for microtubule associated protein light chain 3 (LC3) revealed autophagosome formation after treatment with MDMA. Confocal analyses and immunocytochemistry for 5‐HT, Hoechst and LC3 confirmed MDMA induced autophagy with abundant LC3‐positive puncta within 5‐HT neurons. Rilmenidine (1 &mgr;M) protected against MDMA‐induced injury and image analysis showed full preservation of 5‐HT arbours. MDMA had no effect on GABA neurons, indicating specificity of action at 5‐HT neurons. MDMA‐induced neurotoxicity involves autophagy induction in 5‐HT neurons, and rilmenidine via beneficial actions against toxic intracellular events represents a potential treatment for its pathobiology in sustained usage. Graphical abstract Figure. No caption available. HighlightsMDMA reduced cellular viability producing dieback of 5‐HT neuronal arbours and blockade of 5‐HT uptake.MDMA‐induced injury of 5‐HT neurones involved nuclear fragmentation with recruitment of autophagy.The autophagy activator rilmenidine fully and selectively protected 5‐HT neurones against MDMA‐induced injury.Beneficial actions of rilmenidine represent a potential treatment for 5‐HT dysfunction in MDMA pathobiology in humans.