Exploring germline recombination in Nestin-Cre transgenic mice using floxed androgen receptor.

The Cre-loxP strategy for tissue selective gene deletion has become a widely employed tool in neuroscience research. The validity of these models is largely underpinned by the temporal and spatial selectivity of recombinase expression under the promoter of the Cre driver line. Ectopic Cre-recombinase expression gives rise to off-target effects which can confound results and is especially detrimental if this occurs in germline cells. The Nestin-Cre transgenic mouse is broadly used for selective gene deletion in neurons of the central and peripheral nervous systems. Here we have crossed this mouse with a floxed androgen receptor (AR) transgenic to generate double transgenic neuronal ARKO mice (ARflox ::NesCre) to study germline deletion in male and female transgenic breeders. In male ARflox ::NesCre breeders, a null AR allele was passed on to 86% of progeny regardless of the inheritance of the NesCre transgene. In female ARflox/wt ::NesCre breeders, a null AR allele was passed on to 100% of progeny where ARflox was expected to be transmitted. This surprisingly high incidence of germline recombination in the Nestin-Cre driver line warrants caution in devising suitable breeding strategies, consideration of accurate genotyping approaches and highlights the need for thorough characterization of tissue-specific gene deletion in this model.

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.

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.

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.

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.

Mapping Motor Neuron Vulnerability in the Neuraxis of Male SOD1G93A Mice Reveals Widespread Loss of Androgen Receptor Occurring Early in Spinal Motor Neurons.

Sex steroid hormones have been implicated as disease modifiers in the neurodegenerative disorder amyotrophic lateral sclerosis (ALS). Androgens, signalling via the androgen receptor (AR), predominate in males, and have widespread actions in the periphery and the central nervous system (CNS). AR translocates to the cell nucleus when activated upon binding androgens, whereby it regulates transcription of target genes via the classical genomic signalling pathway. We previously reported that AR protein is decreased in the lumbar spinal cord tissue of symptomatic male SOD1G93A mice. Here, we further explored the changes in AR within motor neurons (MN) of the CNS, assessing their nuclear AR content and propensity to degenerate by endstage disease in male SOD1G93A mice. We observed that almost all motor neuron populations had undergone significant loss in nuclear AR in SOD1G93A mice. Interestingly, loss of nuclear AR was evident in lumbar spinal MNs as early as the pre-symptomatic age of 60 days. Several MN populations with high AR content were identified which did not degenerate in SOD1G93A mice. These included the brainstem ambiguus and vagus nuclei, and the sexually dimorphic spinal MNs: cremaster, dorsolateral nucleus (DLN) and spinal nucleus of bulbocavernosus (SNB). In conclusion, we demonstrate that AR loss directly associates with MN vulnerability and disease progression in the SOD1G93A mouse model of ALS.

Transportable and non-transportable inhibitors of L-glutamate uptake produce astrocytic stellation and increase EAAT2 cell surface expression.

Astrocytic excitatory amino acid transporters (EAATs) regulate excitatory transmission and limit excitotoxicity. Evidence for a functional interface between EAATs and glial fibrillary acidic protein (GFAP) relevant to astrocytic morphology led to investigations of actions of transportable (D-Aspartate (D-Asp) and (2S,3S,4R)-2-(carboxycyclopropyl)glycine (L-CCG-III)) and non-transportable (DL-threo-beta-benzyloxyaspartate (DL-TBOA)) inhibitors of Glu uptake in murine astrocytes. D-Asp (1 mM), L-CCG-III (0.5 mM) and DL-TBOA (0.5 mM) produced time-dependent (24-72 h) reductions in (3)[H]D-Asp uptake (approximately 30-70%) with little or no gliotoxicity. All drugs induced a profound change in phenotype from cobblestone to stellate morphology and image analysis revealed increases in the intensity of GFAP immunolabelling for L-CCG-III and DL-TBOA. Cytochemistry indicated localized changes in F-actin distribution. Cell surface expression of EAAT2, but not EAAT1, was elevated at 72 h. Blockade of Glu uptake by both types of EAAT inhibitor exerts longer-term effects on astrocytic morphology and a compensatory homeostatic rise in EAAT2 abundance.

Dysregulation of Steroid Hormone Receptors in Motor Neurons and Glia Associates with Disease Progression in ALS Mice.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease targeting motor neurons which shows sexual dimorphism in its incidence, age of onset, and progression rate. All steroid hormones, including androgens, estrogens, and progestogens, have been implicated in modulating ALS. Increasing evidence suggests that steroid hormones provide neuroprotective and neurotrophic support to motor neurons, either directly or via surrounding glial cell interactions, by activating their respective nuclear hormone receptors and initiating transcriptional regulatory responses. The SOD1G93A transgenic mouse also shows sex-specific differences in age of onset and progression, and remains the most widely used model in ALS research. To provide a more comprehensive understanding of the influences of steroid hormone signaling in ALS, we systemically characterized sex hormone receptor expression at transcript and protein levels, cellular localization, and the impact of disease course in lumbar spinal cords of male and female SOD1G93A mice. We found that spinal motor neurons highly express nuclear androgen receptor (AR), estrogen receptor (ER)α, ERß, and progesterone receptor with variations in glial cell expression. AR showed the most robust sex-specific difference in expression and was downregulated in male SOD1G93A mouse spinal cord, in association with depletion in 5α-reductase type 2 isoform, which primarily metabolizes testosterone to 5α-dihydrotestosterone. ERα was highly enriched in reactive astrocytes of SOD1G93A mice and ERß was strongly upregulated. The 5α-reductase type 1 isoform was upregulated with disease progression and may influence local spinal cord hormone levels. In conclusion, steroid hormone receptor expression is dynamic and cell-type specific in SOD1G93A mice which may provide targets to modulate progression in ALS.

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.

Androgen receptor antagonism accelerates disease onset in the SOD1G93A mouse model of amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease typically more common in males, implicating androgens in progression of both patients and mouse models. Androgen effects are mediated by androgen receptor which is highly expressed in spinal motor neurons and skeletal muscles. To clarify the role of androgen receptors in ALS, we therefore examined the effect of androgen receptor antagonism in the SOD1G93A mouse model. EXPERIMENTAL APPROACH: The androgen receptor antagonist, flutamide, was administered to presymptomatic SOD1G93A mice as a slow-release subcutaneous implant (5 mg·day-1 ). Testosterone, flutamide, and metabolite levels were measured in blood and spinal cord tissue by LC-MS-MS. Effects on disease onset and progression were assessed using motor function tests, survival, muscle, and neuropathological analyses. KEY RESULTS: Flutamide was metabolised to 2-hydroxyflutamide achieving steady-state plasma levels across the study duration and reached the spinal cord at pharmacologically active concentrations. Flutamide treatment accelerated disease onset and locomotor dysfunction in male SOD1G93A mice, but not female mice, without affecting survival. Analysis of hindlimb muscles revealed exacerbation of myofibre atrophy in male SOD1G93A mice treated with flutamide, although motor neuron pathology was not affected. CONCLUSION AND IMPLICATIONS: The androgen receptor antagonist accelerated disease onset in male SOD1G93A mice, leading to exacerbated muscle pathology, consistent with a role of androgens in modulating disease severity, sexual dimorphism, and peripheral pathology in ALS. These results also demonstrate a key contribution of skeletal muscle pathology to disease onset, but not outcome, in this mouse model of ALS.

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

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.

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 µM-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 µM MDMA. MDMA effected concentration-dependent inhibition of [3H]5-HT uptake with 500 µM 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 µM) 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.

Galactose-functionalised PCL nanofibre scaffolds to attenuate inflammatory action of astrocytes in vitro and in vivo.

Astrocytes represent an attractive therapeutic target for the treatment of traumatic brain injury in the glial scar, which inhibits functional repair and recovery if persistent. Many biomaterial systems have been investigated for neural tissue engineering applications, including electrospun nanofibres, which are a favourable biomaterial as they can mimic the fibrous architecture of the extracellular matrix, and are conveniently modified to present biologically relevant cues to aid in regeneration. Here, we synthesised a novel galactose-presenting polymer, poly(l-lysine)-lactobionic acid (PLL-LBA), for use in layer-by-layer (LbL) functionalisation of poly(ε-caprolactone) (PCL) nanofibres, to covalently attach galactose moieties to the nanofibre scaffold surface. We have assessed the use of this novel biomaterial system in vitro and in vivo, and have shown, for the first time, the ability of galactose to maintain an attenuated inflammatory profile of astrocytes in culture, and to increase the survival of neurons after traumatic injury, as compared to control PCL nanofibres. This study highlights the importance of galactose in controlling the astrocytic response, and provides a promising biomaterial system to deliver the essential morphological and biological cues to achieve functional repair after traumatic brain injury.

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.

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 microM) 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 microg/ml, 3 d) increased [3H]D-aspartate uptake by 90%, as well 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.

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.