Characterisation of amyloid-induced inflammatory responses in the rat retina.

Amyloid-induced inflammation is thought to play a critical and early role in the pathophysiology of Alzheimer's disease. As such, robust models with relevant and accessible compartments that provide a means of assessing anti-inflammatory agents are essential for the development of therapeutic agents. In the present work, we have characterised the induction of inflammation in the rat retina following intravitreal administration of amyloid-beta protein (Aß). Histology and mRNA endpoints in the retina demonstrate Aß1-42-, but not Aß42-1-, induced inflammatory responses characterised by increases in markers for microglia and astrocytes (ionised calcium-binding adaptor molecule 1 (iba-1), GFAP and nestin) and increases in mRNA for inflammatory cytokines and chemokines such as IL1-ß, MIP1α and TNFα. Likewise, analysis of vitreal cytokines also revealed increases in inflammatory cytokines and chemokines, including IL1-ß, MIP1α and MCP1, induced by Aß1-42 but not Aß42-1. This profile of pro-inflammatory gene and protein expression is consistent with that observed in the Alzheimer's disease brain and suggest that this preclinical model may provide a useful relevant tool in the development of anti-inflammatory approaches directed towards Alzheimer's disease therapy.

Principles of early drug discovery.

Developing a new drug from original idea to the launch of a finished product is a complex process which can take 12-15 years and cost in excess of $1 billion. The idea for a target can come from a variety of sources including academic and clinical research and from the commercial sector. It may take many years to build up a body of supporting evidence before selecting a target for a costly drug discovery programme. Once a target has been chosen, the pharmaceutical industry and more recently some academic centres have streamlined a number of early processes to identify molecules which possess suitable characteristics to make acceptable drugs. This review will look at key preclinical stages of the drug discovery process, from initial target identification and validation, through assay development, high throughput screening, hit identification, lead optimization and finally the selection of a candidate molecule for clinical development.

Differential effects of amyloid-ß peptide aggregation status on in vivo retinal neurotoxicity.

The present study examined the relationship between amyloid beta (Aß)-peptide aggregation state and neurotoxicity in vivo using the rat retinal-vitreal model. Following single unilateral intravitreal injection of either soluble Aß1-42 or Aß1-42 preaggregated for different periods, retinal pathology was evaluated at 24 hours, 48 hours, and 1-month postinjection. Injection of either soluble Aß (sAß) or preaggregated Aß induced a rapid reduction in immunoreactivity (IR) for synaptophysin, suggesting that direct contact with neurons is not necessary to disrupt synapses. Acute neuronal ionic and metabolic dysfunction was demonstrated by widespread loss of IR to the calcium buffering protein parvalbumin (PV) and protein gene product 9.5, a component of the ubiquitin-proteosome system. Injection of sAß appeared to have a more rapid impact on PV than the preaggregated treatments, producing a marked reduction in PV cell diameters at 48 hours, an effect that was only observed for preaggregated Aß after 1-month survival. Extending the preaggregation period from 4 to 8 days to obtain highly fibrillar Aß species significantly increased the loss of choline acteyltransferase IR, but had no effect on PV-IR. These findings prompt the conclusion that Aß assembly state has a significant impact on in vivo neurotoxicity by triggering distinct molecular changes within the cell.

Selective increases in cytokine expression in the rat brain in response to striatal injection of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and interleukin-1.

A number of cytokines contribute to acute experimental neurodegeneration. The cytokine response can have detrimental or beneficial effects depending on the temporal profile and balance between pro- and anti-inflammatory molecules. Our recent data suggest that the pro-inflammatory cytokine interleukin-1beta (IL-1beta) acts at specific sites (e.g., the striatum) in the rat brain to cause distant cortical injury, when co-administered with the potent excitotoxin alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (S-AMPA). The objective of the present study was to investigate changes in the expression of several cytokines simultaneously in the rat striatum and cortex after intrastriatal administration of vehicle, S-AMPA or human recombinant (hr) IL-1beta alone or S-AMPA co-injected with hrIL-1beta using reverse transcription-polymerase chain reaction (RT-PCR; Taqman fluorogenic probes) and enzyme-linked immunosorbent assay (ELISA). Injection of S-AMPA alone increased IL-6 mRNA expression in the ipsilateral striatum after 8 h, whilst striatal injection of IL-1beta alone increased local IL-1beta and IL-1ra mRNAs. The levels of mRNA encoding IL-1alpha, IL-1beta, IL-1ra, IL-6, IL-10 and TNFalpha were markedly elevated in the ipsilateral cortex 8 h after co-injection of S-AMPA and hrIL-1beta. Cortical mRNA levels for IL-4, IL-18, TGFbeta and IFNgamma were not significantly different between treatment groups after 2 h or 8 h. A similar pattern of change in the levels of IL-1alpha and IL-6 protein was observed 8 h after treatment. These data demonstrate selective increases in the expression of cytokines in areas of remote cell death in response to administration of hrIL-1beta and S-AMPA. Such cytokines may be involved in the ensuing damage, and further clarification of their actions could aid future therapeutic strategies for several acute neurodegenerative disorders.

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.

AXOR12, a novel human G protein-coupled receptor, activated by the peptide KiSS-1.

A novel human G protein-coupled receptor named AXOR12, exhibiting 81% homology to the rat orphan receptor GPR54, was cloned from a human brain cDNA library. Heterologous expression of AXOR12 in mammalian cells permitted the identification of three surrogate agonist peptides, all with a common C-terminal amidated motif. High potency agonism, indicative of a cognate ligand, was evident from peptides derived from the gene KiSS-1, the expression of which prevents metastasis in melanoma cells. Quantitative reverse transcriptase-polymerase chain reaction was used to study the expression of AXOR12 and KiSS-1 in a variety of tissues. The highest levels of expression of AXOR12 mRNA were observed in brain, pituitary gland, and placenta. The highest levels of KiSS-1 gene expression were observed in placenta and brain. A polyclonal antibody raised to the C terminus of AXOR12 was generated and used to show localization of the receptor to neurons in the cerebellum, cerebral cortex, and brainstem. The biological significance of these expression patterns and the nature of the putative cognate ligand for AXOR12 are discussed.

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.

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.

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.

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-1beta and TNFalpha (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.

Speeding up and ( relatively ) slowing down an internal clock in humans.

Two experiments used click-trains to manipulate the subjective duration of stimuli they preceded, in attempts to demonstrate relative slowing down of the pacemaker of a hypothesized internal clock. Experiment 1 used a pair comparison procedure, where two tones presented on each trial in fact had the same duration. In the conditions of particular interest, the first tone was preceded by clicks (thus putatively timed with a faster clock), the other presented without (thus timed normally). The reverse condition (no-clicks/clicks) was also used. Judgements of the relative duration of the stimuli were shifted in both directions (i.e. first tone longer than second and vice versa) by the manipulation, consistent with relative speeding up and slowing down of the pacemaker. Experiment 2 used the popular bisection method, with 200- and 800-ms tones used as the Short and Long standards for the task. After standard presentations, subjects were required to classify a range of comparison stimuli (from 200 to 800 ms in 100-ms steps) in terms of their similarity to one or the other of the standards. In one condition the comparison stimuli were preceded by clicks (thus timed 'fast') and the standards were presented without clicks (thus timed 'normally'); in another condition the clicks preceded the standards but not the comparisons. The psychophysical function obtained from the bisection procedure shifted in opposite directions with the different manipulations, consistent with both relative 'speeding up' and 'slowing down' of the pacemaker of the internal clock.

Blocking cytochrome c activity within intact neurons inhibits apoptosis.

Cytochrome c has been shown to play a role in cell-free models of apoptosis. During NGF withdrawal-induced apoptosis of intact rat superior cervical ganglion (SCG) neurons, we observe the redistribution of cytochrome c from the mitochondria to the cytoplasm. This redistribution is not inhibited by the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (ZVADfmk) but is blocked by either of the neuronal survival agents 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate (CPT-cAMP) or cycloheximide. Moreover, microinjection of SCG neurons with antibody to cytochrome c blocks NGF withdrawal-induced apoptosis. However, microinjection of SCG neurons with cytochrome c does not alter the rate of apoptosis in either the presence or absence of NGF. These data suggest that cytochrome c is an intrinsic but not limiting component of the neuronal apoptotic pathway.

Activated phosphatidylinositol 3-kinase and Akt kinase promote survival of superior cervical neurons.

The signaling pathways that mediate the ability of NGF to support survival of dependent neurons are not yet completely clear. However previous work has shown that the c-Jun pathway is activated after NGF withdrawal, and blocking this pathway blocks neuronal cell death. In this paper we show that over-expression in sympathetic neurons of phosphatidylinositol (PI) 3-kinase or its downstream effector Akt kinase blocks cell death after NGF withdrawal, in spite of the fact that the c-Jun pathway is activated. Yet, neither the PI 3-kinase inhibitor LY294002 nor a dominant negative PI 3-kinase cause sympathetic neurons to die if they are maintained in NGF. Thus, although NGF may regulate multiple pathways involved in neuronal survival, stimulation of the PI 3-kinase pathway is sufficient to allow cells to survive in the absence of this factor.

Involvement of caspases in sympathetic neuron apoptosis.

In order to study the involvement of caspases in neuronal cell death, we have examined the effects of the viral caspase inhibitor p35 and peptide caspase inhibitors on sympathetic neurons isolated from the superior cervical ganglion (SCG). In these neurons, apoptosis can be induced by the withdrawal of nerve growth factor (NGF) and also by the addition of the kinase inhibitor staurosporine. p35 has been shown to be a broad spectrum inhibitor of the caspase family and promotes the survival of SCG neurons withdrawn from NGF. We show that p35 is also protective when apoptosis is induced by staurosporine. In addition, p35 inhibits a number of the morphological features associated with apoptosis, such as nuclear condensation, TUNEL labelling, and externalisation of phosphatidylserine. The tri-peptide caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (O-methyl)-fluoromethylketone (zVAD-fmk) was effective at inhibiting NGF withdrawal-induced and staurosporine-induced apoptosis of SCG neurons. Two other peptide inhibitors, acetyl-Tyr-Val-Ala-Asp-aldehyde (Ac-YVAD-CHO) and acetyl-Asp-Glu-Ala-Asp-aldehyde (Ac-DEVD-CHO), also inhibited apoptosis induced by both means when microinjected into SCG neurons but peptides derived from the caspase cleavage site in p35 were not protective. We present data to suggest that apoptosis induced by separate death stimuli can result either in the activation of distinct caspases or in differences in the time of activation of the family members.

Increased surface phosphatidylserine is an early marker of neuronal apoptosis.

Neuronal apoptosis is the subject of intense investigation and is beginning to be understood in some molecular detail. In the present study, we show that PC12 cells, like certain other cell types, redistribute phosphatidylserine (PS) from the inner leaflet to the outer leaflet of the plasma membrane early in the process of apoptosis. The externalised PS can be readily visualised by incubating intact cells with a fluorescent derivative of the protein annexin V. When apoptosis is blocked with an inhibitor of interleukin-1beta-converting-enzyme-like proteases, the increased annexin binding is also blocked. Fluorescent annexin V binding provides a rapid and convenient way to identify apoptotic neurones.

Morphological and biochemical changes in neurons: apoptosis versus mitosis.

Apoptosis and mitosis are often thought to share certain morphological similarities and therefore to be regulated by similar sets of enzymes. In this study, the Golgi apparatus and nuclear lamina were examined in PC12 cells and rat superior cervical ganglion neurons undergoing apoptosis in response to withdrawal of nerve growth factor or addition of staurosporine. We found that the Golgi apparatus disperses during apoptosis, without obvious degradation, in a manner similar to that occurring in mitosis. In contrast, the nuclear lamina did not become completely solubilized during apoptosis, as occurs in mitosis, but remained as a distinct structure around the nucleus, although some degradation of nuclear lamins was seen. To assess the integrity of the nuclear envelope, fluorescent probes were introduced into the cytoplasm of live and dying cells. High molecular weight tracers were still excluded from the nuclei of apoptotic cells, demonstrating the continued existence of a functional nuclear barrier. These data suggest, therefore, that cell death is unlikely to occur simply as a result of inappropriate activation of cell cycle enzymes.

Expression of major histocompatibility complex class I antigens at low levels in the thymus induces T cell tolerance via a non-deletional mechanism.

Transgenic CBA (H-2k haplotype) mice expressing the H-2 Kb major histocompatibility complex (MHC) class I gene under control of transcriptional promoter elements from a milk protein gene display high-level H-2 Kb transcription in lactating mammary glands and low-level transcription in skin and thymus of male and virgin female transgenic mice. However, H-2 Kb antigen could be detected only in lactating mammary gland epithelial cells by immunohistological methods. All transgenic mice are tolerant of H-2 Kb since they fail to reject skin grafts from mice expressing H-2 Kb molecules. Furthermore, anti-H-2 Kb cytotoxic responses could not be generated using responder T cells from transgenic mice but T cells from the same mice proliferated, in the presence of interleukin-2, in response to stimulator cells expressing H-2 Kb. Tolerance to H-2 Kb is induced in the thymus since CBA mice grafted with thymus tissue from transgenic mice fail to reject H-2 Kb disparate skin grafts. However, experiments with double-transgenic mice also expressing a T cell receptor with anti-H-2 Kb specificity reveal that tolerance induction is not brought about by elimination of thymocytes bearing H-2 Kb-reactive receptors. Instead, a non-deletional mechanism which results in down-modulation of both CD8 and T cell receptor expression in peripheral T cells correlates with the induction of tolerance in these mice. These data reveal that extremely low levels of self-antigen expression in the thymus are sufficient to induce tolerance via non-deletional mechanisms.