The Emerging Role of the Gut-Brain-Microbiota Axis in Neurodevelopmental Disorders.

Autism spectrum disorder (ASD; autism) is a prevalent neurodevelopmental disorder associated with changes in gut-brain axis communication. Gastrointestinal (GI) symptoms are experienced by a large proportion of individuals diagnosed with autism. Several mutations associated with autism modify cellular communication via neuronal synapses. It has been suggested that modifications to the enteric nervous system, an intrinsic nervous system of the GI tract, could contribute to GI dysfunction. Changes in gut motility, permeability, and the mucosal barrier as well as shifts in the large population of microbes inhabiting the GI tract could contribute to GI symptoms. Preclinical research has demonstrated that mice expressing the well-studied R451C missense mutation in Nlgn3 gene, which encodes cell adhesion protein neuroligin-3 at neuronal synapses, exhibit GI dysfunction. Specifically, NL3R451C mice show altered colonic motility and faster small intestinal transit. As well as dysmotility, macrophages located within the gut-associated lymphoid tissue of the NL3R451C mouse caecum show altered morphology, suggesting that neuro-inflammation pathways are modified in this model. Interestingly, NL3R451C mice maintained in a shared environment demonstrate fecal microbial dysbiosis indicating a role for the nervous system in regulating gut microbial populations. To better understand host-microbe interactions, further clarification and comparison of clinical and animal model profiles of dysbiosis should be obtained, which in turn will provide better insights into the efforts taken to design personalized microbial therapies. In addition to changes in neurophysiological measures, the mucosal component of the GI barrier may contribute to GI dysfunction more broadly in individuals diagnosed with a wide range of neurological disorders. As the study of GI dysfunction advances to encompass multiple components of the gut-brain-microbiota axis, findings will help understand future directions such as microbiome engineering and optimisation of the mucosal barrier for health.

Metal chaperones: a novel therapeutic strategy for brain injury?

OBJECTIVE: This study sought to assess the potential efficacy of a novel class of metal chaperone on the outcomes in an animal model of a controlled cortical impact. This work was predicated on previous observations that this class of compound has exhibited neuroprotective potential in other models of aging and neurodegeneration. RESEARCH DESIGN: The study employed a controlled cortical impact traumatic brain injury in three month old mice with subsequent behavioral and cellular assessments to determine therapeutic efficacy. METHODS: Cognitive (Y-maze) and motor assessments (Rotarod and Open Field) were employed to determine behavioral end points. Histological-based methods were utilized to assess neuronal integrity, astrocytosis, and lesion volume. OUTCOMES: We demonstrate here that acute post-injury treatment with PBT2 (Prana Biotechnology) is sufficient to maintain neuronal integrity (evidenced by decreased lesion area and increased numbers of neurons; decreased astrocytosis was also present) and to normalize performance in cognitive testing (Y-maze). These effects occurred within days and were maintained for the entire duration of the study (26 days post-injury). These data support the further interrogation of the utility of metal chaperones for the treatment and/or prevention of the neuroanatomical, biochemical, and behavioral deficits that occur following brain injuries of different etiologies.

The σ 1 receptor agonist 4-PPBP elicits ERK1/2 phosphorylation in primary neurons: a possible mechanism of neuroprotective action.

Although sigma 1 (σ(1)) receptors and mitogen-activated protein kinases (MAPKs) are known modulators of neuroprotection, a role for MAPK signaling pathways in σ receptor-mediated neuroprotection has not been investigated in detail.The present study aims to investigate the possible link between σ(1) receptors and MAPKs in neuroprotection. Primary mixed cortical and hippocampal neurons were treated with σ(1) receptor agonists PRE-084 or 4-PPBP in a time- and concentration-dependent manner; and in another set of experiments, cells were pre-incubated with σ(1) receptor antagonist BD1047 or MEK inhibitor PD98059 in a concentration-dependent manner prior to PRE-084 or 4-PPBP treatment. Levels of phosphorylated and total ERK1/2, JNK and p38-MAPK were determined with western blotting and ERK1/2 phosphorylation was confirmed with immunofluorescence. To investigate neuroprotection by σ(1) receptors, cells were pre-treated with PRE-084 or 4-PPBP and glucose-starved for various times: in the presence or absence of pre-incubated BD1047 or PD98059. Cell viability was then measured with MTT assay. Both PRE-084 and 4-PPBP caused phosphorylation of ERK1/2, but not p38-MAPK and JNK. ERK1/2 phosphorylation was inhibited by BD1047 and PD98059 in a concentration-dependent manner. Immunofluorescence confirmed the phosphorylation of ERK1/2 by PRE-084 and 4-PPBP and its inhibition by BD1047 and PD98059. Pre-treating glucose-deprived neurons with 4-PPBP, but not PRE-084; caused neuroprotection which was inhibited by BD1047 and PD98059. 4-PPBP, but not PRE-084; causes ERK1/2 phosphorylation-mediated neuroprotection. This presents a novel mechanism by σ(1) receptors in modulating neuroprotection.

Reduction of cerebral infarct volume by apocynin requires pretreatment and is absent in Nox2-deficient mice.

BACKGROUND AND PURPOSE: Reactive oxygen species (ROS) derived from Nox2-containing reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity is reportedly detrimental in cerebrovascular disease. However, ROS generation by other Nox isoforms may have a physiological role. No Nox2-selective inhibitors have yet been identified, and thus it is unclear whether isoform non-selective Nox inhibitors would necessarily improve outcome after stroke. We assessed the effect of apocynin on cerebrovascular ROS production and also on outcome following cerebral ischaemia when administered either before ischaemia or after cerebral reperfusion. The involvement of Nox2-containing NADPH oxidase in the effects of apocynin was assessed using Nox2(-/-) mice. EXPERIMENTAL APPROACH: Transient cerebral ischaemia was induced by 0.5 h middle cerebral artery occlusion followed by 23.5 h reperfusion. Mice received apocynin (2.5 mg.kg(-1), i.p.) either 0.5 h before ischaemia or 1 h after reperfusion. In situ superoxide production after cerebral ischaemia-reperfusion was measured in brain sections of wild-type mice at 24 h using dihydroethidium fluorescence. KEY RESULTS: Treatment with apocynin 0.5 h before ischaemia reduced total infarct volume, neurological impairment and mortality in wild-type but not Nox2(-/-) mice. Conversely, treatment with apocynin 1 h after initiation of reperfusion had no protective effect. Cerebral ischaemia and reperfusion increased superoxide production in the brain at 24 h, and pretreatment but not posttreatment with apocynin reduced superoxide levels. CONCLUSIONS AND IMPLICATIONS: Apocynin improves outcome following stroke when administered before ischaemia in wild-type but not Nox2(-/-) mice.

Glutathione peroxidase-1 contributes to the protection of glutamine synthetase in astrocytes during oxidative stress.

Glutamine synthetase (GS) is an astrocytic enzyme that is essential for the glutamate-glutamine cycle between neurons and astrocytes. To measure the effects of oxidative stress on the activity of GS in astrocytes, astrocyte-rich primary cultures from the brains of wild-type and glutathione peroxidase-1 deficient mice (GPx1(-/-)) were exposed to a chronic hydrogen peroxide-generating system consisting of xanthine oxidase, hypoxanthine and superoxide dismutase. The specific activity of GS was strongly diminished by chronic exposure to hydrogen peroxide in astrocytes cultured from both mouse lines. After 60 min of oxidative stress in the presence of 5 mU/mL, 10 mU/mL and 20 mU/mL of xanthine oxidase, the specific GS activity of wild-type astrocytes was reduced to 47%, 22% and 13% of the initial activity, respectively. For all activities of xanthine oxidase applied, astrocytes from GPx1(-/-) mice experienced a significantly greater rate of GS inactivation compared to their wild-type counterparts. These results confirm that GS is sensitive to inactivation by chronic peroxide stress in viable astrocytes and show that glutathione peroxidase-1 helps to protect GS from inactivation by oxidative stress.

Lack of glutathione peroxidase-1 exacerbates Abeta-mediated neurotoxicity in cortical neurons.

The aetiologies of Alzheimer's disease (AD) are complex and multifactorial. Current therapies are largely ineffective, as the pathophysiological pathways are poorly understood. Observations in AD autopsies, as well as in vivo and in vitro observations in transgenic mice, have implicated oxidative stress as pathogenic in AD. This study used the Glutathione Peroxidase-1 knockout mouse (Gpx1--/--) model to investigate the role of antioxidant disparity in neuropathologies. Cultured neurons from control and Gpx1--/-- embryos were treated with AD-related peptides and the degree of cell loss compared. Results show that antioxidant disparity makes Gpx1--/-- cells more susceptible to Abeta toxicity. Surrogate replacement of Gpx1 with the reactive oxygen species scavenger N-acetyl cysteine and the Gpx1 mimetic ebselen, reverses the Gpx1--/-- increased susceptibility to Abeta toxicity. Such results support a role for oxidative stress in AD-related neuronal loss. This study is the first to report such findings using the Gpx1--/-- model, and supports a role for oxidative stress as one of the contributing factors, in development of AD-like pathologies.

Overexpression of the chromosome 21 transcription factor Ets2 induces neuronal apoptosis.

Down syndrome (trisomy 21) neurons display an increased rate of apoptosis in vitro. The genes on chromosome 21 that mediate this increased cell death remain to be elucidated. Here we show that the chromosome 21 transcription factor Ets2, a gene that is overexpressed in Down syndrome, is expressed in neurons, and that moderate overexpression of Ets2 leads to increased apoptosis of primary neuronal cultures from Ets2 tg mice that involves activation of caspase-3. Our data therefore suggest that overexpression of ETS2 may contribute to the increased rate of apoptosis of neurons in Down syndrome.

Increased infarct size and exacerbated apoptosis in the glutathione peroxidase-1 (Gpx-1) knockout mouse brain in response to ischemia/reperfusion injury.

Glutathione peroxidase is an antioxidant enzyme that is involved in the control of cellular oxidative state. Recently, unregulated oxidative state has been implicated as detrimental to neural cell viability and involved in both acute and chronic neurodegeneration. In this study we have addressed the importance of a functional glutathione peroxidase in a mouse ischemia/reperfusion model. Two hours of focal cerebral ischemia followed by 24 h of reperfusion was induced via the intraluminal suture method. Infarct volume was increased three-fold in the glutathione peroxidase-1 (Gpx-1) -/- mouse compared with the wild-type mouse; this was mirrored by an increase in the level of apoptosis found at 24 h in the Gpx-1 -/- mouse compared with the wild-type mouse. Neuronal deficit scores correlated to the histologic data. We also found that activated caspase-3 expression is present at an earlier time point in the Gpx-1 -/- mice when compared with the wild-type mice, which suggests an enhanced susceptibility to apoptosis in the Gpx-1 -/- mouse. This is the first known report of such a dramatic increase, both temporally and in level of apoptosis in a mouse stroke model. Our results suggest that Gpx-1 plays an important regulatory role in the protection of neural cells in response to the extreme oxidative stress that is released during ischemia/reperfusion injury.

The association of metalloendopeptidase EC 3.4.24.15 at the extracellular surface of the AtT-20 cell plasma membrane.

Endopeptidase EC 3.4.24.15 (EP24.15) is a soluble, neuropeptide-degrading metalloenzyme, widely expressed in the brain, pituitary and gonads. For the physiological metabolism of neuropeptides, the enzyme should be located extracellularly, either associated with the plasma membrane or in the extracellular milieu. Western immunoblot analyses of crude cytosolic and post-nuclear membrane fractions prepared by differential centrifugation revealed a slightly smaller molecular mass ( approximately 2 kDa) for EP24.15 in the post-nuclear membrane fraction. This smaller EP24.15 species was also present in an enriched fraction of plasma membrane prepared by Percoll gradient centrifugation. To ascertain whether EP24.15 is associated with the extracellular surface of plasma membrane, two sets of experiments were carried out. First, Western immunoblot analysis of AtT-20 cells treated with the membrane-impermeable, thiol-cleavable cross-linker, 3, 3'-dithio-bis(sulpho-succinimidyl-propionate) (DTSSP), indicated an extracellular membrane association. After cross-linking and thiol-reduction, a distinct band corresponding to EP24.15 was significantly diminished under non-reducing conditions. Second, immunocytochemical studies performed at 4 degrees C on non-permeabilized AtT-20 cells (i.e., non-fixed to prevent antibody internalization), indicated that EP24.15 was expressed on the surface of the AtT-20 cells. We furthermore determined that EP24.15 enzymatic activity is present on the extracellular surface of the cell discernable from the secreted enzyme. These results suggest that the EP24.15 is associated with the extracellular surface of the AtT-20 cell plasma membrane and is enzymatically active. Taken together, the results are consistent with a putative role in the degradation of neuropeptides acting at the external cell surface.

Oxidative stress and neural dysfunction in Down syndrome.

Total or partial trisomy of chromosome 21 occurs with relatively high frequency and is responsible for the occurrence of Down syndrome. Phenotypically, individuals with Down syndrome display characteristic morphological features and a variety of clinical disorders. One of the challenges for researchers in this field has been to ascertain and understand the relationship between the Down syndrome phenotype with the gene dosage effect resulting from trisomy of chromosome 21. Much attention therefore, has been given towards investigating the consequences of overexpressing chromosome 21-linked genes. In particular, an extensive analysis of SOD1 and APP have provided important insights as to how perturbations in the expression of their respective genes may contribute to the Down syndrome phenotype. In this review we will highlight studies which support a key role for SOD1 and APP in the pathogenesis of neural abnormalities observed in individuals with Down syndrome. Central to this relationship is how the redox state of the cell is affected and its consequences to neural function and integrity.

Purification, characterisation and distribution of ovine neuronal nitric oxide synthase.

Nitric oxide (NO) is an ubiquitous intercellular messenger molecule synthesised from the amino acid L-arginine by the enzyme nitric oxide synthase (NOS). A number of NOS iso-enzymes have been identified, varying in molecular size, tissue distribution and possible biological role. To further understand the role of NO in the regulation of neuroendocrine function in the sheep, we have purified and characterised ovine neuronal NOS (nNOS) using anion exchange, affinity and size-exclusion chromatography. SDS-PAGE reveals that ovine nNOS has an apparent denatured molecular weight of 150 kDa which correlates well with the other purified nNOS forms such as rat, bovine and porcine. The native molecular weight predicted by size-exclusion chromatography was 200 kD which is in close agreement with that found for porcine and rat nNOS. Internal amino acid sequences generated from tryptic digests of the purified ovine nNOS are highly homologous to rat nNOS. There was no significant difference in the cofactor dependence and kinetic characteristics of ovine nNOS when compared to rat and bovine nNOS, (K(m) for L-arginine 2.8, 2.0 and 2.3 microM respectively). A polyclonal anti-peptide antibody directed toward the C-terminal end of the rat nNOS sequence showed full cross-reactivity with the purified ovine nNOS. Immunohistochemical and Western analysis using this antiserum demonstrate the expression of nNOS in the cortex, cerebellum, hypothalamus and pituitary of the sheep. The lack of staining in the neural and anterior lobes of the pituitary seems to suggest that NOS plays a varied role in the control of endocrine systems between species.

The involvement of nitric oxide in the secretion of beta-endorphin from the pituitary intermediate lobe of the rat.

Nitric oxide (NO) generated by the enzyme nitric oxide synthase (NOS) has been implicated in the regulation of a variety of endocrine functions. A number of biochemical and anatomical studies have demonstrated the presence of neuronal NOS (nNOS) in the neuroendocrine axis and have shown significant effects of NO on the release of hypothalamic and pituitary hormones. Using a C-terminal directed peptide antibody that is specific for nNOS we have found a predominance of nNOS in the neural lobe of the pituitary and in a single layer of epithelial cells, possibly a remnant of Rathke's pouch that form a border between the intermediate lobe and the anterior lobe. Furthermore, we have examined the effect of sodium nitroprusside (SNP), a donor of NO on the secretion of beta-endorphin (beta-EP) from the isolated neuro-intermediate lobe (NIL) of the rat and cultured rat melanotrophs. It was shown that in explant cultures of intact neuro-intermediate lobes, SNP (100 microM) was able to cause an inhibition of beta-EP secretion. In the presence of sulpiride (10 microM), a dopamine D2-receptor antagonist, there was a partial reversal of the SNP effect. On the other hand SNP did not affect beta-EP secretion in primary cultures of melanotrophs that no longer possessed any innervation. Taken together these data suggest that NO has an indirect inhibitory effect on the secretion of beta-EP by the intermediate lobe via the release of dopamine.

Characterisation of neurons with nitric oxide synthase immunoreactivity that project to prevertebral ganglia.

Retrograde dye tracing was combined with immunohistochemistry to determine the distributions of nitric oxide synthase (NOS) immunoreactive nerve cells that project to prevertebral ganglia from the gastrointestinal tract and spinal cord of the guinea pig. An antiserum was raised against the neuronal form of NOS by selecting an amino-acid sequence specific to this form as immunogen. The antiserum recognised a single band at 150 kDa on Western blots of rat brain extract. Enteric nerve cells that were labelled by Fast Blue injected into the coeliac ganglion were not NOS immunoreactive in the small intestine, whereas 40-70% were reactive in the large intestine. Retrograde dye injected into the inferior mesenteric ganglion labels cells in the colon and rectum; 60-70% were immunoreactive for NOS. The NOS-immunoreactive nerve fibres arising in the intestine appear to end selectively around somatostatin-immunoreactive nerve cells in the coeliac and inferior mesenteric ganglia. Preganglionic nerve cell bodies in the intermediolateral column and dorsal commissural nucleus from T12 to L2 were labelled from the inferior mesenteric ganglion. Nearly 70% of neurons at each level were NOS immunoreactive. Thus, two sources of NOS terminals in prevertebral ganglia have been identified, intestinofugal neurons of the large, but not the small intestine, and sympathetic preganglionic neurons.