Karen L. Philpott

Inhibition of p38 mitogen-activated protein kinase provides neuroprotection in cerebral focal ischemia

Mitogen‐activated protein kinases (MAPKs) are involved in many cellular processes. The stress‐activated MAPK, p38, has been linked to inflammatory cytokine production and cell death following cellular stress. Here, we demonstrate focal ischemic stroke‐induced p38 enzyme activation (i.e., phosphorylation) in the brain. The second generation p38 MAPK inhibitor SB 239063 was identified to exhibit increased kinase selectivity and improved cellular and in vivo activity profiles, and thus was selected for evaluation in two rat models of permanent focal ischemic stroke. SB 239063 was administered orally pre‐ and post‐stroke and intravenously post‐stroke. Plasma concentration levels were achieved in excess of those that effectively inhibit p38 activity. In both moderate and severe stroke, SB 239063 reduced infarct size by 28–41%, and neurological deficits by 25–35%. In addition, neuroprotective plasma concentrations of SB 239063 that reduced p38 activity following stroke also reduced the stroke‐induced expression of IL‐1β and TNFα (i.e., cytokines known to contribute to stroke‐induced brain injury). SB 239063 also provided direct protection of cultured brain tissue to in vitro ischemia. This robust SB 239063‐induced neuroprotection emphasizes a significant opportunity for targeting MAPK pathways in ischemic stroke injury, and also suggests that p38 inhibition be evaluated for protective effects in other experimental models of nervous system injury and neurodegeneration.

The use of quantitative RT-PCR to measure mRNA expression in a rat model of focal ischemia--caspase-3 as a case study.

Quantitative reverse transcription and polymerisation chain reaction (RT-PCR) using Taqman¿trade mark omitted¿ fluorogenic probes has been used to measure changes in gene expression in the cerebral cortex of rats in the permanent middle cerebral artery occlusion (pMCAO) model of focal ischemia. The mRNA levels of three housekeeping genes have been analysed in this model to determine which gene showed least change following experimental insult. In the lesioned cortex, beta-actin mRNA increased at 24 h, while the levels of cyclophilin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) did not change. We have also used this methodology to examine modulations in the level of caspase-3 mRNA during focal ischemia in the rat. Caspase-3 mRNA showed a 41% increase at 6 h post-MCAO, which was specific to the lesioned cortex. This change became more pronounced with time, showing an increase of 220% at 24 h. This methodology enables changes in mRNA expression to be analysed more sensitively and quantitatively than other available techniques and highlights the need for careful choice of control or housekeeping genes used for RNA comparisons.

Stroke Genomics: Approaches to Identify, Validate, and Understand Ischemic Stroke Gene Expression

Sequencing of the human genome is nearing completion and biologists, molecular biologists, and bioinformatics specialists have teamed up to develop global genomic technologies to help decipher the complex nature of pathophysiologic gene function. This review will focus on differential gene expression in ischemic stroke. It will discuss inheritance in the broader stroke population, how experimental models of spontaneous stroke might be applied to humans to identify chromosomal loci of increased risk and ischemic sensitivity, and also how the gene expression induced by stroke is related to the poststroke processes of brain injury, repair, and recovery. In addition, we discuss and summarise the literature of experimental stroke genomics and compare several approaches of differential gene expression analyzes. These include a comparison of representational difference analysis we have provided using an experimental stroke model that is representative of stroke evolution observed most often in man, and a summary of available data on stroke differential gene expression. Issues regarding validation of potential genes as stroke targets, the verification of message translation to protein products, the relevance of the expression of neuroprotective and neurodestructive genes and their specific timings, and the emerging problems of handling novel genes that may be discovered during differential gene expression analyses will also be addressed.

Dynamic association of tau with neuronal membranes is regulated by phosphorylation

Tau is an abundant cytosolic protein which regulates cytoskeletal stability by associating with microtubules in a phosphorylation-dependent manner. We have found a significant proportion of tau is located in the membrane fraction of rat cortical neurons and is dephosphorylated, at least at Tau-1 (Ser199/Ser202), AT8 (Ser199/Ser202/Thr205) and PHF-1 (Ser396/Ser404) epitopes. Inhibition of tau kinases casein kinase 1 (CK1) or glycogen synthase kinase-3 decreased tau phosphorylation and significantly increased amounts of tau in the membrane fraction. Mutation of serine/threonine residues to glutamate to mimic phosphorylation in the N-terminal, but not C-terminal, region of tau prevented its membrane localization in transfected cells, demonstrating that the phosphorylation state of tau directly impacts its localization. Inhibiting CK1 in neurons lacking the tyrosine kinase fyn also induced tau dephosphorylation but did not affect its membrane association. Furthermore, inhibition of CK1 increased binding of neuronal tau to the fyn-SH3 domain. We conclude that trafficking of tau between the cytosol and the neuronal membrane is dynamically regulated by tau phosphorylation through a mechanism dependent on fyn expression.

Caspase mRNA expression in a rat model of focal cerebral ischemia

Proteins of the caspase family are involved in the signalling pathway that ultimately leads to programmed cell death (apoptosis), which has been reported to occur in some experimental models of stroke. In a previous paper we used quantitative reverse transcription and polymerase chain reaction (RT-PCR) to characterise changes in the mRNA expression of one member of this family, caspase-3, in a rat model of permanent focal ischemia. Here we have used this technique to study the expression of a further three caspases which are involved in different aspects of caspase signalling. Caspase-8, involved in Fas-mediated apoptosis, was upregulated in the cortex of ischemic rats. Caspase-11, which leads to the synthesis of the functional form of the cytokine interleukin-1 beta, also showed increased expression, but with a different temporal profile from caspase-8. In contrast, caspase-9, which forms part of the pathway signalling through the mitochondria, showed a decrease in expression. The expression of a further four caspases (1, 2, 6 and 7) has also been characterised in a simpler experiment. These caspases all showed distinctive patterns of expression following the induction of ischemia. These data lead us to conclude that caspase expression as a whole is under very strict transcriptional control in this model. Certain elements of caspase signalling, such as the Fas-induced pathway and the events upstream of IL-1 beta processing, are upregulated, while others are not. This may be due to some form of genetic program activated in response to ischemia in the brain and may highlight which biological pathways are modulated.

FTY720 ameliorates MOG-induced experimental autoimmune encephalomyelitis by suppressing both cellular and humoral immune responses

FTY720, an oral sphingosine 1‐phosphate (S1P) receptor modulator, has shown efficacy in phase II trials in patients with relapsing‐remitting multiple sclerosis (MS). Although this molecule is thought to immunosuppress by inhibiting lymphocyte egress from the lymph nodes, the full spectrum of FTY720s actions has not yet been uncovered. In this study, we investigated the effects of FTY720 treatment on disease severity and histopathology of MOG‐induced experimental autoimmune encephalomyelitis (EAE) in the dark agouti (DA) rat, a model that closely mimics several features of MS. The effects of FTY720 on T‐cell subsets, anti‐MOG antibody production, and mRNA expression of a number of cytokines and other genes were also examined. Commencement of treatment before disease onset prevented the appearance of clinical disease. Therapeutic treatment after established disease reduced clinical scores and substantially attenuated inflammation, demyelination, and axon loss. EAE suppression was associated with a reduction in all measured T‐cell subsets in blood and spleen and a significant decrease in serum IgG2a levels. However, in the lymph nodes, all T‐cell subsets except for naïve T cells and recent thymic emigrants remained unaffected. In addition, FTY720 treatment led to a significant inhibition in interferon‐γ, inducible nitric oxide synthase, and glial cell line‐derived neurotrophic factor mRNA expression in the MOG‐EAE spinal cord. In conclusion, our findings indicate that FTY720‐mediated S1P receptor modulation ameliorates chronic relapsing MOG‐EAE by suppressing both cellular and humoral immune responses.

Determination of changes in mRNA expression in a rat model of neuropathic pain by Taqman quantitative RT-PCR.

The aim of this study was to develop a rapid and accurate high throughput method of screening multiple genes across a single sample set to detect changes in gene expression in the dorsal root ganglion (DRG) following partial sciatic nerve ligation in the rat. Using Taqman quantitative RT-PCR, we show that expression of a number of genes, including galanin, vasointestinal peptide and neuropeptide Y are rapidly increased 24 h post-operation in the DRGs on the ligated side only. Other genes tested, including vanilloid receptor-1, substance P, galanin receptor-2 and housekeeping genes did not alter. Analysis of the expression of ASIC4 showed a small difference in expression at 7 days post ligation. By applying a statistical method for analysis of multiple variables, partial least squares, we show that the expression change of ASIC4 was significantly altered on the ligated side even though the change was small. This method will allow us to rapidly identify changes in expression of candidate genes that may be involved in adaptive responses in the DRG due to nerve injury.

An alternatively spliced form of glycogen synthase kinase-3β is targeted to growing neurites and growth cones

The serine/threonine kinase glycogen synthase kinase-3beta (GSK-3beta) is expressed in two, alternatively spliced, isoforms: a short form (GSK-3beta1) and a long form containing a 13 amino acid insert in the catalytic domain (GSK-3beta2). We examined the expression of these isoforms in the rat using specific antibodies and found that GSK-3beta2, in contrast to GSK-3beta1, is only expressed in the nervous system. The highest levels of GSK-3beta2 are found in the developing nervous system but expression persists into adulthood. In the adult central nervous system the highest expression of GSK-3beta2 occurs in regions with a high proportion of white matter, suggesting that GSK-3beta2 is expressed in axons. Consistent with this finding, sub-cellular fractionation of neonatal rat brain showed that GSK-3beta2 is present in fractions enriched in neurites and growth cones. Furthermore, we found that when we separated neuronal cell bodies from neurites by culturing embryonic cortical neurons in neurite outgrowth inserts, GSK-3beta2 was present in both compartments. Finally, a rabbit polyclonal antibody raised to the 13 amino acid insert of GSK-3beta2 (anti-8A) that specifically recognises GSK-3beta2, labels the cell body, including the nucleus, neurites and growth cones of embryonic neurons in culture. To compare functionally the two isoforms, we performed in vitro kinase assays. These showed that GSK-3beta1 is more efficient at phosphorylating the microtubule-associated protein MAP1B than GSK-3beta2, consistent with previous findings with the microtubule-associated protein tau. However, when co-expressed with MAP1B in COS-7 cells, both GSK-3beta isoforms equally efficiently phosphorylated MAP1B and had a similar influence on the regulation of microtubule dynamics by MAP1B in these cells. We conclude that the alternatively spliced isoform of GSK-3beta, GSK-3beta2, is neuron-specific and has overlapping activities with GSK-3beta1.

MAP kinase pathways in neuronal cell death.

The signaling pathways which contribute to neuronal death during development, aging and disease have been extensively studied. While initial efforts focused on developmental death, increasing evidence suggests that mitogen-activated protein kinase pathways play a role in human pathology. In particular, the c-Jun N-terminal kinases (JNKs), mitogen-activated protein kinases activated by extracellular stimuli including stress, are a major focus. Knock-out mouse studies have demonstrated that removing particular JNK genes can reduce the severity in various disease scenarios, including those which are used to model Parkinsons disease and cerebral ischemia. In addition, activation of JNKs can be seen in human disease tissue. In this review we bring together the evidence for JNK being an important regulator of neuronal loss and outline the advancement of small molecule inhibitors for future therapeutic intervention.

Upregulation of death pathway molecules in rat cerebellar granule neurons undergoing apoptosis.

Cerebellar granule neurons can be maintained in culture in a medium containing high serum and depolarising levels of KCl. When serum is removed and the KCl levels lowered from 25 to 5 mM, the cells undergo apoptosis. Apoptosis can be prevented by inhibitors of transcription or translation, suggesting a need for macromolecular synthesis in the apoptotic process. Using quantitative reverse transcription-polymerase chain reaction the levels of mRNA for a range of genes postulated to be important in apoptosis have been examined. Elevated levels of caspase 3, c-Jun, and Fas ligand were found, in addition to a corresponding increase in c-Jun protein and activation of caspase-3. These results suggest that cerebellar granule neurons upregulate components of both death receptor-mediated and the mitochondrial-mediated death pathways.