A. Jacqueline Hunter

Novel phenotypes identified by plasma biochemical screening in the mouse

We used ENU mutagenesis in the mouse for the rapid generation of novel mutant phenotypes for both gene function studies and use as new animal models of human disease (Nolan et al. 2000b). One focus of the program was the development of a blood biochemistry screen. At 8–12 weeks of age, approximately 300 ml of blood was collected from F1 offspring of ENU mutagenized male mice. This yielded approximately 125 ml of plasma, used to perform a profile of 17 standard biochemical tests on an Olympus analyzer. Cohorts of F1 mice were also aged and then retested to detect late onset phenotypes. In total, 1,961 F1s were screened. Outliers were identified by running means and standard deviations. Of 70 mice showing consistent abnormalities in plasma biochemistry, 29 were entered into inheritance testing. Of these, 9 phenotypes were confirmed as inherited, 10 found not to be inherited, and 10 are still being tested. Inherited mutant phenotypes include abnormal lipid profiles (low total and HDL cholesterol, high triglycerides); abnormalities in bone and liver metabolism (low ALP, high ALP, high ALT, and AST); abnormal plasma electrolyte levels (high sodium and chloride); as well as phenotypes of interest for the study of diabetes (high glucose). The gene loci bearing the mutations are currently being mapped and further characterized. Our results have validated our biochemical screen, which is applicable to other mutagenesis projects, and we have produced a new set of mutants with defined metabolic phenotypes.

Cellular uptake and spread of the cell‐permeable peptide penetratin in adult rat brain

Investigation of normal and pathological diseases of the central nervous system (CNS) has been hampered by the inability to effectively manipulate protein function in vivo. In order to address this important topic, we have evaluated the ability of penetratin, a novel cell‐permeable peptide consisting of a 16‐amino acid sequence derived from a Drosophila homeodomain protein, to act as a carrier system to introduce a cargo into brain cells. Fluorescently tagged penetratin was injected directly into rat brain, either into the striatum or the lateral ventricles, and rats were perfusion‐fixed 24 h later in order to assess the brain response to the peptide. Immunohistochemistry following intrastriatal injection showed that injection of 10 μg penetratin caused neurotoxic cell death and triggered recruitment of inflammatory cells in a dose‐dependent fashion. Doses of 1 μg or less resulted in reduced toxicity and recruitment of inflammatory cells, but interestingly, there was some spread of the penetratin. Injections of an inactive peptide sequence, derived from the same homeodomain, caused little toxicity but could still, however, trigger an inflammatory response. Intraventricular injections showed extensive inflammatory cell recruitment but minimal spread of either peptide. These results suggest that a dose of 1 μg of penetratin peptide is suitable for directing agents to small, discrete areas of the brain and as such is an interesting new system for analysing CNS function.

Proteomic identification and early validation of complement 1 inhibitor and pigment epithelium‐derived factor: Two novel biomarkers of Alzheimer's disease in human plasma

Emerging disease modifying therapeutic strategies for Alzheimers disease (AD) have generated a critical need for biomarkers of early stage disease. Here, we describe the identification and assessment of a number of candidate biomarkers in patients with mild to moderate probable AD. Plasma from 47 probable Alzheimers patients and 47 matched controls were analysed by proteomics to define a significant number of proteins whose expression appeared to be associated with AD. These were compared to a similar proteomic comparison of a mouse transgenic model of amyloidosis, which showed encouraging overlap with the human data. From these studies a prioritised list of 31 proteins were then analysed by immunoassay and/or functional assay in the same human cohort to verify the changes observed. Eight proteins continued to show significance by either immunoassay or functional assay in the human plasma and these were tested in a further set of 100 probable AD patients and 100 controls from the original cohort. From our data it appeared that two proteins, serpin F1 (pigment epithelium‐derived factor) and complement C1 inhibitor are down‐regulated in plasma from AD patients.

Identification of Neuroprotective Properties of Anti-MAG Antibody: A Novel Approach for the Treatment of Stroke?

The inhibitory activity of myelin-associated glycoprotein (MAG) on neurons is thought to contribute to the lack of regenerative capacity of the CNS after injury. The interaction of MAG and its neuronal receptors mediates bidirectional signaling between neurons and oligodendrocytes. The novel finding that an anti-MAG monoclonal antibody not only possesses the ability to neutralise the inhibitory effect of MAG on neurons but also directly protects oligodendrocytes from glutamate-mediated oxidative stress-induced cell death is reported here. Furthermore, administration of anti-MAG antibody (centrally and systemically) starting 1 hour after middle cerebral artery occlusion in the rat significantly reduced lesion volume at 7 days. This neuroprotection was associated with a robust improvement in motor function compared with animals receiving control IgG1. Together, these data highlight the potential for the use of anti-MAG antibodies as therapeutic agents for the treatment of stroke.

A behavioural characterisation of the FVB/N mouse strain

The use of transgenic models in scientific research has made an enormous contribution to our understanding of the causes and symptoms of many diseases, including neurodegenerative conditions such as Alzheimers Disease (AD) and Parkinsons Disease (PD). In the creation of transgenic models of neurodegenerative disease, effects of the background strain of the animal on the resulting genotype must be taken into consideration. This is particularly true for behavioural studies in which the background strain of the mouse may mask the phenotype of the genetic manipulation. Here, the behaviour of two mouse strains used in transgenic models, FVB/N and C57BL6/J, were compared. Studies of circadian wheel activity, cognition and aggression revealed considerable phenotypic differences between strains. These data also indicate that the FVB/N strain is not appropriate as a background strain in the behavioural assessment of transgenic mouse models.

Treatments for stroke

Stroke is the third leading cause of death and the leading cause of disability in developed countries, yet remains a poorly treated condition. Treatments for stroke can be aimed at acutely improving blood flow or protecting brain tissue against ischaemia, enhancing stroke recovery or reducing the risk of stroke recurrence. This paper reviews each of these approaches, particularly focusing on mechanisms for which there are agents in clinical trials. There are a number of appealing neuroprotective agents in Phase II and III clinical trials. However, the majority of acute treatments are likely to suffer from a narrow therapeutic time window and hence limited patient access. Combinations of acute approaches are likely to offer the greatest benefit, but present challenges in development. Promotion of recovery following stroke offers enormous potential for successful therapeutic intervention. Excitingly, new developments in preclinical research have identified possible ways in which this may be achieved.

Characterisation of SB-221420-A — a neuronal Ca2+ and Na+ channel antagonist in experimental models of stroke

For progression to clinical trials in stroke, putative neuroprotective compounds should show robust efficacy post-ischaemia in several experimental models of stroke. This paper describes the characterisation of (+)(1S, 2R)-cis-1-[4-(1-methyl-1-phenylethyl)phenoxy]-2-methylamino indane hydrochloride (SB-221420-A), a Ca(2+) and Na(+) channel antagonist. SB-221420-A inhibited (IC(50)=2.2 microM) N-type voltage-operated Ca(2+) channel currents in cultured superior cervical ganglion neurons, which were pretreated with 10 microM nimodipine to block L-type voltage-operated Ca(2+) channel currents. In dorsal root ganglion neurons pretreated with 1 microM omega-conotoxin GVIA to block N-type voltage-operated Ca(2+) channel currents, SB-221420-A inhibited the residual Ca(2+) current with an IC(50) of 7 microM. SB-221420-A also inhibited Na(+) currents in dorsal root ganglion neurons with an IC(50) of 8 microM. In rats, the pharmacokinetic profile of SB-221420-A shows that it has a half-life of 6.4 h, a high volume of distribution, is highly brain penetrating, and has no persistent metabolites. Following bilateral carotid artery occlusion in gerbils, SB-221420-A significantly reduced the level of ischaemia-induced hyperlocomotor activity and the extent of hippocampal CA1 cell loss compared to the ischaemic vehicle-treated group. SB-221420-A was also effective in focal models of ischaemia. In the mouse permanent middle cerebral artery occlusion model, SB-221420-A (10 mg/kg) administered intravenously, post-ischaemia significantly (P<0.05) reduced lesion volume compared to the ischaemic vehicle-treated group. In the normotensive rat permanent middle cerebral artery occlusion model, SB-221420-A (10 mg/kg) administered intravenously over 1 h, beginning 30 min postmiddle cerebral artery occlusion, significantly (P<0.05) reduced lesion volume from 291+/-16 to 153+/-30 mm(3), compared to ischaemic vehicle-treated controls when measured 24 h postmiddle cerebral artery occlusion. Efficacy was maintained when the same total dose of SB-221420-A was infused over a 6-h period, beginning 30 min postmiddle cerebral artery occlusion. SB-221420-A also significantly (P<0.05) reduced lesion volume following transient middle cerebral artery occlusion in normotensive rats and permanent middle cerebral artery occlusion in spontaneously hypertensive rats (SHR). Investigation of the side effect profile using the Irwin screen in mice revealed that, at neuroprotective doses, there were no overt behavioural or cardiovascular changes. These data demonstrate that robust neuroprotection can be seen post-ischaemia with SB-221420-A in both global and focal ischaemia with no adverse effects at neuroprotective doses, and indicate the potential utility of a mixed cation blocker to improve outcome in cerebral ischaemia.