Milestone review: GABA, from chemistry, conformations, ionotropic receptors, modulators, epilepsy, flavonoids, and stress to neuro-nutraceuticals.

Arising out of a PhD project more than 50 years ago to synthesise analogues of the neurotransmitter GABA, a series of new chemical entities were found to have selective actions on ionotropic GABA receptors. Several of these neurochemicals are now commercially available. A new subtype of these receptors was discovered that could be a target for the treatment of myopia, the facilitation of learning and memory, and the improvement of post-stroke motor recovery. The development of these new chemical entities over many years demonstrates the importance of neurochemicals with which to investigate selective aspects of GABA receptors and illustrates the significance of collaboration between chemists and biologists in neurochemistry. Vital were the improvements in synthetic organic chemistry and the use of functional human receptors expressed in oocytes. Current interest in ionotropic GABA receptors includes the clinical development of subtype-specific agents and the role of gain-of-function receptor variants in epilepsy. Dietary flavonoids were found to cross the blood-brain barrier to influence brain function. Natural and synthetic flavonoids had a range of effects on GABA receptors, ranging from positive, silent, and negative allosteric modulators, to even second-order modulation of first-order modulators. Flavonoids have been called "a new family of benzodiazepines." Like benzodiazepines, flavonoids reduce stress. Stress produces changes in GABA receptors in the brain that may be because of changes in endogenous modulators, such as neurosteroids and corticosteroids. GABA also occurs naturally in the diet leading to studies of the effects of oral GABA on brain function. This finding has resulted in studies of GABA and related neurochemicals as neuro-nutraceuticals. GABA systems in the gut microbiome are essential to such studies. The actions of oral GABA and of GABA-enriched beverages and foodstuffs are now an area of considerable scientific and commercial interest. GABA is a deceptively simple chemical that can take up many shapes, which may underlie its complex functions. The need for new chemical entities with selective actions for further studies highlights the need for continuing collaboration between chemists and biologists.

LRRK2 impairs PINK1/Parkin-dependent mitophagy via its kinase activity: pathologic insights into Parkinson's disease.

Mutations of LRRK2, encoding leucine-rich repeat kinase 2 (LRRK2), are the leading cause of autosomal dominant Parkinson's disease (PD). The most frequent of these mutations, G2019S substitution, increases kinase activity, but it remains unclear how it causes PD. Recent studies suggest that LRRK2 modulates mitochondrial homeostasis. Mitochondrial dysfunction plays a key role in the pathogenesis of autosomal recessive PD forms linked to PARK2 and PINK1, encoding the cytosolic E3 ubiquitin-protein ligase Parkin and the mitochondrial kinase PINK1, which jointly regulate mitophagy. We explored the role of LRRK2 and its kinase activity in PINK1/Parkin-dependent mitophagy. LRRK2 increased mitochondrial aggregation and attenuated mitochondrial clearance in cells coexpressing Parkin and exposed to the protonophore carbonylcyanide m-chlorophenylhydrazone. Förster resonance energy transfer imaging microscopy showed that LRRK2 impaired the interactions between Parkin and Drp1 and their mitochondrial targets early in mitophagy. The inhibition of LRRK2 kinase activity by a 'kinase-dead' LRRK2 mutation or with a pharmacological inhibitor (LRRK2-IN-1) restored these interactions. The monitoring of mitophagy in human primary fibroblasts with the novel dual-fluorescence mtRosella reporter and a new hypothermic shock paradigm revealed similar defects in PD patients with the G2019S LRRK2 substitution or PARK2 mutations relative to healthy subjects. This defect was restored by LRRK2-IN-1 treatment in LRRK2 patients only. Our results suggest that PD forms due to LRRK2 and PARK2 mutations involve pathogenic mechanisms converging on PINK1/Parkin-dependent mitophagy.

Autophagy in neurodegeneration: New insights underpinning therapy for neurological diseases.

In autophagy long-lived proteins, protein aggregates or damaged organelles are engulfed by vesicles called autophagosomes prior to lysosomal degradation. Autophagy dysfunction is a hallmark of several neurodegenerative diseases in which misfolded proteins or dysfunctional mitochondria accumulate. Excessive autophagy can also exacerbate brain injury under certain conditions. In this review, we provide specific examples to illustrate the critical role played by autophagy in pathological conditions affecting the brain and discuss potential therapeutic implications. We show how a singular type of autophagy-dependent cell death termed autosis has attracted attention as a promising target for improving outcomes in perinatal asphyxia and hypoxic-ischaemic injury to the immature brain. We provide evidence that autophagy inhibition may be protective against radiotherapy-induced damage to the young brain. We describe a specialized form of macroautophagy of therapeutic relevance for motoneuron and neuromuscular diseases, known as chaperone-assisted selective autophagy, in which heat shock protein B8 is used to deliver aberrant proteins to autophagosomes. We summarize studies pinpointing mitophagy mediated by the serine/threonine kinase PINK1 and the ubiquitin-protein ligase Parkin as a mechanism potentially relevant to Parkinson's disease, despite debate over the physiological conditions in which it is activated in organisms. Finally, with the example of the autophagy-inducing agent rilmenidine and its discrepant effects in cell culture and mouse models of motor neuron disorders, we illustrate the importance of considering aspects such a disease stage and aggressiveness, type of insult and load of damaged or toxic cellular components, when choosing the appropriate drug, timepoint and duration of treatment.

The origins and early history of neurochemistry and its societies.

At the 2017 joint meeting of the International Society for Neurochemistry (ISN) and the European Society for Neurochemistry, 150 years of neurochemistry - the 50th anniversary of ISN, 40 years of European Society for Neurochemistry, and 60 years of the Journal of Neurochemistry (JNC) - was celebrated with a historical symposium that explored the foundations of neurochemical societies, key international figures in the discipline of neurochemistry, and the pre-eminent role of the JNC. The foundations of neurochemistry were laid in Europe, notably France and Germany, in the late 18th and early 19th centuries. Neurochemists in the United Kingdom made globally relevant contributions before and after the Second World War, and Swedish contributions were especially prominent in the 1950s and 1960s. As neurochemistry is a truly international branch of neuroscience, the important contributions of neurochemists in the Americas and the Asia-Pacific were also recognized, as were the seminal roles of the American, Asia-Pacific, and Japanese Societies of Neurochemistry. Although ISN was only formed in 1967, earlier international meetings in Europe and the Americas reflected the growing recognition of the importance of chemistry and biochemistry for understanding and responding to the pathophysiology of clinical conditions and diseases of the central and peripheral nervous systems. JNC was first published in 1956, but the ISN only assumed complete ownership of the journal under tempestuous circumstances in 1970. The ISN-JNC interface and the sterling work of the JNC Editors has meant that the income generated by the journal has allowed the ISN Council to implement diverse programs for supporting neurochemistry internationally, including sustaining regional neurochemical societies, and supporting neurochemists in the developing world and schools of neurochemistry.

Mutant TDP-43 Expression Triggers TDP-43 Pathology and Cell Autonomous Effects on Primary Astrocytes: Implications for Non-cell Autonomous Pathology in ALS.

Motor neuron degeneration in amyotrophic lateral sclerosis (ALS) caused by mutations in superoxide dismutase 1 (SOD1) is partly non-cell autonomous, involving cellular dysfunction of astrocytes. Whether non-cell autonomous effects occur in other forms of ALS, such as TAR DNA binding protein 43 (TDP-43)-related disease, remains unclear. Here, we characterised the impact of mutant TDP-43 expression on primary astrocytes derived from transgenic TDP-43A315T mice. Mutant TDP-43 astrocytes revealed evidence for TDP-43 pathology, shown by cytoplasmic TDP-43 inclusions and accumulation in insoluble cell fractions which was exacerbated by proteasomal inhibition. L-glutamate uptake, measured using an [3H]D-aspartate assay, was impaired in mutant TDP-43 astrocytes, while ATP accumulation was abnormal, suggesting mutant TDP-43 induced astrocytic dysfunction. Astrocyte activation coupled with spinal and cortical motor neuron loss in transgenic TDP-43A315T mice could imply non-cell autonomous effects of astrocytes in vivo. These data demonstrate mutant TDP-43-mediated cell autonomous effects on astrocytes that may contribute to motor neuron pathology in ALS.

Inhibition of bioenergetics provides novel insights into recruitment of PINK1-dependent neuronal mitophagy.

Contributions of damaged mitochondria to neuropathologies have stimulated interest in mitophagy. We investigated triggers of neuronal mitophagy by disruption of mitochondrial energy metabolism in primary neurons. Mitophagy was examined in cultured murine cerebellar granule cells after inhibition of mitochondrial respiratory chain by drugs rotenone, 3-nitropropionic acid, antimycin A, and potassium cyanide, targeting complexes I, II, III, and IV, respectively. Inhibitor concentrations producing slow cellular demise were determined from analyses of cellular viability, morphology of neuritic damage, plasma membrane permeability, and oxidative phosphorylation. Live cell imaging of dissipation of mitochondrial membrane potential (ΔΨm ) by drugs targeting mitochondrial complexes was referenced to complete depolarization by carbonyl cyanide m-chlorophenyl hydrazone. While inhibition of complexes I, III and IV effected rapid dissipation of ΔΨm , inhibition of complex II using 3-nitropropionic acid led to minimal depolarization of mitochondria. Nonetheless, all respiratory chain inhibitors triggered mitophagy as indicated by increased aggregation of mitochondrially localized PINK1. Mitophagy was further analyzed using a dual fluorescent protein biosensor reporting mitochondrial relocation to acidic lysosomal environment. Significant acidification of mitochondria was observed in neurons treated with rotenone or 3-nitropropionic acid, revealing mitophagy at distal processes. Neurons treated with antimycin A or cyanide failed to show mitochondrial acidification. Minor dissipation of ΔΨm by 3-nitropropionic acid coupled with vigorous triggering of mitophagy suggested depolarization of mitochondria is not a necessary condition to trigger mitophagy. Moreover, weak elicitation of mitophagy by antimycin A, subsequent to loss of ΔΨm , suggested that mitochondrial depolarization is not a sufficient condition for triggering robust neuronal mitophagy. Our findings provide new insight into complexities of mitophagic clearance of neuronal mitochondria.

Inflammation in Traumatic Brain Injury: Roles for Toxic A1 Astrocytes and Microglial-Astrocytic Crosstalk.

Traumatic brain injury triggers neuroinflammation that may contribute to progressive neurodegeneration. We investigated patterns of recruitment of astrocytes and microglia to inflammation after brain trauma by firstly characterising expression profiles over time of marker genes following TBI, and secondly by monitoring glial morphologies reflecting inflammatory responses in a rat model of traumatic brain injury (i.e. the lateral fluid percussion injury). Gene expression profiles revealed early elevation of expression of astrocytic marker glial fibrillary acidic protein relative to microglial marker allograft inflammatory factor 1 (also known as ionized calcium-binding adapter molecule 1). Adult rat brains collected at day 7 after injury were processed for immunohistochemistry with allograft inflammatory factor 1, glial fibrillary acidic protein and complement C3 (marker of bad/disruptive astrocytic A1 phenotype). Astrocytes positive for glial fibrillary acidic protein and complement C3 were significant increased in the injured cortex and displayed more complex patterns of arbourisation with significantly increased bifurcations. Our observations suggested that traumatic brain injury changed the phenotype of microglia from a ramified appearance with long, thin, highly branched processes to a swollen amoeboid shape in the injured cortex. These findings suggest differential glial activation with astrocytes likely undergoing strategic changes in morphology and function. Whilst a detailed analysis is needed of temporal patterns of glial activation, ours is the first evidence of a role for the bad/disruptive astrocytic A1 phenotype in an open head model of traumatic brain injury.

SOD1 Mutations Causing Familial Amyotrophic Lateral Sclerosis Induce Toxicity in Astrocytes: Evidence for Bystander Effects in a Continuum of Astrogliosis.

Astrocytes contribute to the death of motor neurons via non-cell autonomous mechanisms of injury in amyotrophic lateral sclerosis (ALS). Since mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) underlie the neuropathology of some forms of familial ALS, we explored how expression of mutant SOD1 protein A4V SOD1-EGFP affected the biology of secondary murine astrocytes. A4V SOD1-EGFP expressing astrocytes (72 h after transfection) displayed decreased mitochondrial activity (~45%) and L-glutamate transport (~25%), relative to cells expressing wild-type SOD1-EGFP. A4V SOD1-EGFP altered F-actin and Hoechst staining, indicative of cytoskeletal and nuclear changes, and altered GM130 labelling suggesting fragmentation of Golgi apparatus. SOD1 inclusion formation shifted from discrete to "punctate" over 72 h with A4V SOD1-EGFP more rapidly producing inclusions than G85R SOD1-EGFP, and forming more punctate aggregates. A4V, not wild-type SOD1-EGFP, exerted a substantial, time-dependent effect on GFAP expression, and ~60% of astrocytes became stellate and hypertrophic at 72 h. Spreading toxicity was inferred since at 72 h ~80% of bystander cells exhibited hypertrophy and stellation. This evidence favours mutant SOD1-containing astrocytes releasing destructive species that alter the biology of adjacent astrocytes. This panoply of mutant SOD1-induced destructive events favours recruitment of astrocytes to non-cell autonomous injury in ALS.

Synaptic signalling and its interface with neuropathologies: snapshots from the past, present and future.

This 'Past to Future' Review as part of the 60th anniversary year of the Journal of Neurochemistry focuses on synaptic transmission and associated signalling, and seeks to identify seminal progress in neurochemistry over the last 10 years which has advanced our understanding of neuronal communication in brain. The approach adopted analyses neurotransmitters on a case by case basis (i.e. amino acids, monoamines, acetylcholine, neuropeptides, ATP/purines and gasotransmitters) to highlight novel findings that have changed the way we view each type of transmitter, to explore commonalities and interactions, and to note how new insights have changed the way we view the biology of degenerative, psychiatric and behavioural conditions. Across all transmitter systems there was remarkable growth in the identification of targets likely to provide therapeutic benefit and which undoubtedly was driven by the elucidation of circuit function and new vistas of synaptic signalling. There has been an increasing trend to relate signalling to disease, notably for Alzheimer's and Parkinson's disease and related conditions, and which has occurred for each transmitter family. Forebrain circuitry and tonic excitatory control have been the centre of great attention yielding novel findings that will impact upon cognitive, emotional and addictive behaviours. Other impressive insights focus on gasotransmitters integrating activity as volume transmitters. Exciting developments in how serotonin, cholinergic, l-glutamate, galanin and adenosine receptors and their associated signalling can be beneficially targeted should underpin the development of new therapies. Clearly integrated, multifaceted neurochemistry has changed the way we view synaptic signalling and its relevance to pathobiology. Highlighted are important advances in synaptic signalling over the last decade in the Journal of Neurochemistry. Across all transmitter systems elucidation of circuit function, and notably molecular insights, have underpinned remarkable growth in the identification of targets likely to provide therapeutic benefit in neuropathologies. Another commonality was wide interest in forebrain circuitry and its tonic excitatory control. Increasingly observations relate to signalling in disease and behavioural conditions. This article is part of the 60th Anniversary special issue.

Walking the tightrope: proteostasis and neurodegenerative disease.

A characteristic of many neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS), is the aggregation of specific proteins into protein inclusions and/or plaques in degenerating brains. While much of the aggregated protein consists of disease specific proteins, such as amyloid-ß, α-synuclein, or superoxide dismutase1 (SOD1), many other proteins are known to aggregate in these disorders. Although the role of protein aggregates in the pathogenesis of neurodegenerative diseases remains unknown, the ubiquitous association of misfolded and aggregated proteins indicates that significant dysfunction in protein homeostasis (proteostasis) occurs in these diseases. Proteostasis is the concept that the integrity of the proteome is in fine balance and requires proteins in a specific conformation, concentration, and location to be functional. In this review, we discuss the role of specific mechanisms, both inside and outside cells, which maintain proteostasis, including molecular chaperones, protein degradation pathways, and the active formation of inclusions, in neurodegenerative diseases associated with protein aggregation. A characteristic of many neurodegenerative diseases is the aggregation of specific proteins, which alone provides strong evidence that protein homeostasis is disrupted in these disease states. Proteostasis is the maintenance of the proteome in the correct conformation, concentration, and location by functional pathways such as molecular chaperones and protein degradation machinery. Here, we discuss the potential roles of quality control pathways, both inside and outside cells, in the loss of proteostasis during aging and disease.

Comparative Microarray Analysis Identifies Commonalities in Neuronal Injury: Evidence for Oxidative Stress, Dysfunction of Calcium Signalling, and Inhibition of Autophagy-Lysosomal Pathway.

Mitochondrial dysfunction, ubiquitin-proteasomal system impairment and excitotoxicity occur during the injury and death of neurons in neurodegenerative conditions. The aim of this work was to elucidate the cellular mechanisms that are universally altered by these conditions. Through overlapping expression profiles of rotenone-, lactacystin- and N-methyl-D-aspartate-treated cortical neurons, we have identified three affected biological processes that are commonly affected; oxidative stress, dysfunction of calcium signalling and inhibition of the autophagic-lysosomal pathway. These data provides many opportunities for therapeutic intervention in neurodegenerative conditions, where mitochondrial dysfunction, proteasomal inhibition and excitotoxicity are evident.

Modulating Astrocyte Transition after Stroke to Promote Brain Rescue and Functional Recovery: Emerging Targets Include Rho Kinase.

Stroke is a common and serious condition, with few therapies. Whilst previous focus has been directed towards biochemical events within neurons, none have successfully prevented the progression of injury that occurs in the acute phase. New targeted treatments that promote recovery after stroke might be a better strategy and are desperately needed for the majority of stroke survivors. Cells comprising the neurovascular unit, including blood vessels and astrocytes, present an alternative target for supporting brain rescue and recovery in the late phase of stroke, since alteration in the unit also occurs in regions outside of the lesion. One of the major changes in the unit involves extensive morphological transition of astrocytes resulting in altered energy metabolism, decreased glutamate reuptake and recycling, and retraction of astrocyte end feed from both blood vessels and neurons. Whilst globally inhibiting transitional change in astrocytes after stroke is reported to result in further damage and functional loss, we discuss the available evidence to suggest that the transitional activation of astrocytes after stroke can be modulated for improved outcomes. In particular, we review the role of Rho-kinase (ROCK) in reactive gliosis and show that inhibiting ROCK after stroke results in reduced scar formation and improved functional recovery.

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 (13)C-enriched substrates by guinea pig brain cortical tissue slices with (13)C and (1)H nuclear magnetic resonance spectroscopy. Resveratrol increased lactate labeling but decreased incorporation of (13)C 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 (13)C into Krebs cycle intermediates and reduced incorporation into lactate, although the inhibitor EX527 paradoxically also increased Krebs cycle (13)C 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.

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. Astrocytes exist in phenotypes with pro-survival and destructive components, and their biology can be modulated by changing phenotype. Our findings demonstrate murine astrocytes adopt a healthy phenotype when cultured in 3D. Astrocytes proliferate and extend into poly-ε-caprolactone scaffolds displaying 3D stellated morphologies with reduced GFAP expression and actin stress fibres, plus a cytotrophic gene profile. Bioengineered 3D scaffolds have potential to direct inflammation to aid regenerative neurobiology.

The Concise Guide to PHARMACOLOGY 2023/24: Transporters.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and over 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org/), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16182. Transporters are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Multifaceted role of nitric oxide in an in vitro mouse neuronal injury model: transcriptomic profiling defines the temporal recruitment of death signalling cascades.

Nitric oxide is implicated in the pathogenesis of various neuropathologies characterized by oxidative stress. Although nitric oxide has been reported to be involved in the exacerbation of oxidative stress observed in several neuropathologies, existent data fail to provide a holistic description of how nitrergic pathobiology elicits neuronal injury. Here we provide a comprehensive description of mechanisms contributing to nitric oxide induced neuronal injury by global transcriptomic profiling. Microarray analyses were undertaken on RNA from murine primary cortical neurons treated with the nitric oxide generator DETA-NONOate (NOC-18, 0.5 mM) for 8-24 hrs. Biological pathway analysis focused upon 3672 gene probes which demonstrated at least a ±1.5-fold expression in a minimum of one out of three time-points and passed statistical analysis (one-way anova, P < 0.05). Numerous enriched processes potentially determining nitric oxide mediated neuronal injury were identified from the transcriptomic profile: cell death, developmental growth and survival, cell cycle, calcium ion homeostasis, endoplasmic reticulum stress, oxidative stress, mitochondrial homeostasis, ubiquitin-mediated proteolysis, and GSH and nitric oxide metabolism. Our detailed time-course study of nitric oxide induced neuronal injury allowed us to provide the first time a holistic description of the temporal sequence of cellular events contributing to nitrergic injury. These data form a foundation for the development of screening platforms and define targets for intervention in nitric oxide neuropathologies where nitric oxide mediated injury is causative.

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-ß (TGFß) 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.