Juliet M. Taylor

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.

Neuroinflammation and oxidative stress: Co-conspirators in the pathology of Parkinson’s disease

Parkinsons disease (PD) is a complex disease, with genetics and environment contributing to the disease onset. Recent studies of causative PD genes have confirmed the involvement of cellular mechanisms engaged in mitochondrial and UPS dysfunction, oxidative stress and apoptosis in the progressive degeneration of the dopaminergic neurons in PD. In addition, clinical, epidemiological and experimental evidence has implicated neuroinflammation in the disease progression. This review will discuss neuroinflammation in PD, with particular focus on the genetic and toxin-based models of the disease. These studies have confirmed elevated oxidative stress and the pro-inflammatory response occurs early in the disease and these processes contribute to and/or exacerbate the nigro-striatal degeneration. In addition, the experimental models discussed here have also provided strong evidence that these pathways are an important link between the familial and sporadic causes of PD. The potential application of anti-inflammatory interventions in limiting the dopaminergic neuronal cell death in these models is discussed with evidence suggesting that the further investigation of their use as part of multi-targeted clinical trials is warranted.

The contribution of neuroinflammation to amyloid toxicity in Alzheimer's disease

Alzheimers disease (AD) is a progressive neurodegenerative disease and the most common cause of dementia. Deposition of amyloid‐β (Aβ) remains a hallmark feature of the disease, yet the precise mechanism(s) by which this peptide induces neurotoxicity remain unknown. Neuroinflammation has long been implicated in AD pathology, yet its contribution to disease progression is still not understood. Recent evidence suggests that various Aβ complexes interact with microglial and astrocytic expressed pattern recognition receptors that initiate innate immunity. This process involves secretion of pro‐inflammatory cytokines, chemokines and generation of reactive oxygen species that, in excess, drive a dysregulated immune response that contributes to neurodegeneration. The mechanisms by which a neuroinflammatory response can influence Aβ production, aggregation and eventual clearance are now becoming key areas where future therapeutic intervention may slow progression of AD. This review will focus on evidence supporting the combined neuroinflammatory‐amyloid hypothesis for pathogenesis of AD, describing the key cell types, pathways and mediators involved.

The contribution of astrocytes and microglia to traumatic brain injury

Traumatic brain injury (TBI) represents a major cause of death and disability in developed countries. Brain injuries are highly heterogeneous and can also trigger other neurological complications, including epilepsy, depression and dementia. The initial injury often leads to the development of secondary sequelae; cellular hyperexcitability, vasogenic and cytotoxic oedema, hypoxia‐ischaemia, oxidative stress and inflammation, all of which influence expansion of the primary lesion. It is widely known that inflammatory events in the brain following TBI contribute to the widespread cell death and chronic tissue degeneration. Neuroinflammation is a multifaceted response involving a number of cell types, both within the CNS and in the peripheral circulation. Astrocytes and microglia, cells of the CNS, are considered key players in initiating an inflammatory response after injury. These cells are capable of secreting various cytokines, chemokines and growth factors, and following injury to the CNS, undergo changes in morphology. Ultimately, these changes can influence the local microenvironment and thus determine the extent of damage and subsequent repair. This review will focus on the roles of microglia and astrocytes following TBI, highlighting some of the key processes, pathways and mediators involved in this response. Additionally, both the beneficial and the detrimental aspects of these cellular responses will be examined using evidence from animal models and human post‐mortem TBI studies.

Potential Contribution of NF-κB in Neuronal Cell Death in the Glutathione Peroxidase-1 Knockout Mouse in Response to Ischemia-Reperfusion Injury

Background and Purpose— We have previously identified an increased susceptibility of Gpx1−/− mice to increased infarct size after middle cerebral artery occlusion (MCAO). This study was designed to elucidate the mechanisms involved in elevated neuronal cell death arising from an altered endogenous oxidant state. Methods— Gpx1−/− mice were exposed to transient MCAO and reperfusion by intraluminal suture blockade. Protein expression of the p65 subunit of transcription factor nuclear factor-&kgr;B (NF-&kgr;B) was examined by immunohistochemistry and Western Analysis. NF-&kgr;B DNA-protein activity was assessed by electrophoretic mobility shift assays (EMSA). Wild-type and Gpx1−/− mice were exposed to MCAO with or without the NF-&kgr;B inhibitor, pyrrolidinedithiocarbamate (PDTC). Results— Upregulation of the p65 subunit of NF-&kgr;B and subsequent p65 phosphorylation at serine 536 was detected in the Gpx1−/− brains after stroke. EMSAs revealed that increased ischemia-enhanced DNA binding of NF-&kgr;B was observed in Gpx1−/− mice compared with wild-type. Supershift assays indicated that the p50 and p65 subunits participated in the bound NF-&kgr;B complex. The NF-&kgr;B inhibitor PDTC, a potential antioxidant, was able to afford partial neuroprotection in the Gpx1−/− mice. Conclusions— NF-&kgr;B is upregulated in the Gpx1−/− mouse, and this upregulation contributes to the increased cell death seen in the Gpx1−/− after MCAO. The activation of NF-&kgr;B may increase the expression of downstream target genes that are involved in the progression of neural injury after MCAO.

Glutathione Peroxidase-1 Contributes to the Neuroprotection Seen in the Superoxide Dismutase-1 Transgenic Mouse in Response to Ischemia/Reperfusion Injury:

The authors hypothesized that glutathione peroxidase-1 (Gpx-1) contributes to the neuroprotection seen in the superoxide dismutase-1 transgenic (Sod-1 tg) mouse. To investigate this hypothesis, they crossed the Gpx-1 -/- mouse with the Sod-1 tg and subjected the cross to a mouse model of ischemia/reperfusion. Two hours of focal cerebral ischemia followed by 24 hours of reperfusion was induced via intraluminal suture. The Sod-1 tg/Gpx-1 -/- cross exhibited no neuroprotection when infarct volume was measured; indeed, infarct volume increased in the Sod-1 tg/Gpx-1 -/- cross compared with the wild-type mouse. Our results suggest that Gpx-1 plays an important regulatory role in the protection of neural cells in response to ischemia/reperfusion injury.

Impact of oxidative stress on neuronal survival

1. Reactive oxygen species and oxidative state are slowly gaining acceptance in having a physiological relevance rather than just being the culprits in pathophysiological processes. The control of the redox environment of the cell provides for additional regulation in relation to signal transduction pathways. Conversely, aberrant regulation of oxidative state manifesting as oxidative stress can predispose a cell to adverse outcome.

Diminished Akt phosphorylation in neurons lacking glutathione peroxidase-1 (Gpx1) leads to increased susceptibility to oxidative stress-induced cell death.

We have previously identified an increased susceptibility of glutathione peroxidase‐1 (Gpx1)–/– mice to neuronal apoptosis following mid‐cerebral artery (MCA) occlusion. This study was designed to elucidate the mechanisms involved in elevated neuronal cell death arising from an altered endogenous oxidant state. This was addressed in both an in vitro and in vivo model of oxidative stress in the form of exogenous H2O2 and cerebral ischaemia, respectively. Increased levels of cell death were detected in primary neurons lacking Gpx1 following the addition of exogenous H2O2. This increased apoptosis correlated with a down‐regulation in the activation of the phospho‐inositide 3‐kinase [PI(3)K]–Akt survival pathway. The importance of this pathway in protecting against H2O2‐induced cell death was highlighted by the increased susceptibility of wildtype neurons to apoptosis when treated with the PI(3)K inhibitor, LY294002. The Gpx1–/– mice also demonstrated elevated neuronal cell death following MCA occlusion. Although Akt phosphorylation was detected in the Gpx1–/– brains, activation was not seen in later reperfusion events, as demonstrated in wildtype brains. Previous studies have highlighted the importance of Akt phosphorylation in protecting against neuronal cell death following cerebral ischaemia–reperfusion. Our results suggest that the increased susceptibility of Gpx1–/– neurons to H2O2‐induced apoptosis and neuronal cell death in vivo following cerebral ischaemia–reperfusion injury can be attributed in part to diminished activation of Akt. Perturbations in key anti‐apoptotic mechanisms as a result of an altered redox state may have implications in the study of oxidative stress‐mediated neuropathologies.

Parkin Co-Regulated Gene (PACRG) is regulated by the ubiquitin-proteasomal system and is present in the pathological features of Parkinsonian diseases.

Mutations in parkin are a common cause of early-onset autosomal recessive Parkinsons disease. Parkin Co-Regulated Gene (PACRG) is a novel gene that was discovered because of its close genetic proximity to parkin and the two genes were subsequently demonstrated to be regulated by a bi-directional promoter. However the role of PACRG has not been well characterized and the distribution of the protein in both normal and diseased brain is not known. Here, we report that like parkin, PACRG is regulated by the ubiquitin-proteasomal system. Immunohistochemistry identified PACRG in astrocytes throughout the brain and in pigmented noradrenergic neurons of the locus coeruleus. PACRG was also detected in Lewy bodies and glial cytoplasmic inclusions in patients with Parkinsons disease and Multiple System Atrophy, respectively. Together, these results demonstrate that PACRG is regulated by the ubiquitin-proteasomal system and may play a role in the pathogenesis of Parkinsons disease.

Causes of progressive cerebellar ataxia: prospective evaluation of 1500 patients

Background Cerebellar ataxias are the result of diverse disease processes that can be genetic or acquired. Establishing a diagnosis requires a methodical approach with expert clinical evaluation and investigations. We describe the causes of ataxia in 1500 patients with cerebellar ataxia. Methods All patients were referred to the Sheffield Ataxia Centre, UK, and underwent extensive investigations, including, where appropriate genetic testing using next-generation sequencing (NGS). Patients were followed up on a 6-monthly basis for reassessment and further investigations if indicated. Results A total of 1500 patients were assessed over 20 years. Twenty per cent had a family history, the remaining having sporadic ataxia. The commonest cause of sporadic ataxia was gluten ataxia (25%). A genetic cause was identified in 156 (13%) of sporadic cases with other causes being alcohol excess (12%) and cerebellar variant of multiple system atrophy (11%). Using NGS, positive results were obtained in 32% of 146 patients tested. The commonest ataxia identified was EA2. A genetic diagnosis was achieved in 57% of all familial ataxias. The commonest genetic ataxias were Friedreichs ataxia (22%), SCA6 (14%), EA2 (13%), SPG7 (10%) and mitochondrial disease (10%). The diagnostic yield following attendance at the Sheffield Ataxia Centre was 63%. Conclusions Immune-mediated ataxias are common. Advances in genetic testing have significantly improved the diagnostic yield of patients suspected of having a genetic ataxia. Making a diagnosis of the cause of ataxia is essential due to potential therapeutic interventions for immune and some genetic ataxias.