Non-associative learning in larval zebrafish.

Habituation, where a response is reduced when exposed to a continuous stimulus is one of the simplest forms of non-associative learning and has been shown in a number of organisms from sea slugs to rodents. However, very little has been reported in the zebrafish, a model that is gaining popularity for high-throughput compound screens. Furthermore, since most of the studies involving learning and memory in zebrafish have been conducted in adults, we sought to determine if zebrafish larvae could display non-associative learning and whether it could be modulated by compounds identified in previous rodent studies. We demonstrated that zebrafish larvae (7 days post fertilization) exhibit iterative reduction in a startle response to a series of acoustic stimuli. Furthermore, this reduction satisfied criteria for habituation: spontaneous recovery, more rapid reductions in startle to shorter intertrial intervals and dishabituation. We then investigated the pathways mediating this behavior using established compounds in learning and memory. Administration of rolipram (PDE4 inhibitor), donepezil (acetylcholinesterase inhibitor), and memantine (N-methyl-D-aspartic acid (NMDA) receptor antagonist) all increased the acoustic startle response and decreased habituation in the larvae, similar to previous rodent studies. Further studies demonstrated that NMDA blocked the memantine response and the effect of donepezil was blocked by mecamylamine but not atropine suggesting that the donepezil response was mediated by nicotinic rather than muscarinic receptors. Zebrafish larvae possess numerous advantages for medium to high-throughput screening; the model described herein therefore offers the potential to screen for additional compounds for further study on cognition function.

Circadian programs of transcriptional activation, signaling, and protein turnover revealed by microarray analysis of mammalian cells.

Many aspects of physiology and behavior are temporally organized into daily 24 hr rhythms, driven by an endogenous circadian clock. Studies in eukaryotes have identified a network of interacting genes forming interlocked autoregulatory feedback loops which underlie overt circadian organization in single cells. While in mammals the master oscillator resides in the suprachiasmatic nuclei of the hypothalamus, semiautonomous circadian oscillators also exist in peripheral tissues and in immortalized fibroblasts, where rhythmicity is induced following a serum shock. We used this model system in combination with high-density cDNA microarrays to examine the magnitude and quality of clock control of gene expression in mammalian cells. Supported by application of novel bioinformatics tools, we find approximately 2% of genes, including expected canonical clock genes, to show consistent rhythmic circadian expression across five independent experiments. Rhythmicity in most of these genes is novel, and they fall into diverse functional groups, highlighted by a predominance of transcription factors, ubiquitin-associated factors, proteasome components, and Ras/MAPK signaling pathway components. When grouped according to phase, 68% of the genes were found to peak during estimated subjective day, 32% during estimated subjective night, with a tendency to peak at a phase corresponding to anticipation of dawn or dusk.

A comparative assessment of gamma-secretase activity in transgenic and non-transgenic rodent brain.

Amyloid-beta (Abeta) deposits are one of the hallmarks of the neuropathological degeneration observed in Alzheimer's disease (AD) and Abeta concentrations have been reported to vary in different brain regions of AD patients. Abeta is produced by the sequential cleavage of amyloid precursor protein (APP) by beta-secretase and gamma-secretase, respectively. Previous studies have shown that over-expression of the gamma-secretase complex leads to increased gamma-secretase proteolytic activity increasing Abeta production. However, it is not known whether brain regions with highest Abeta concentration also express relatively higher levels of gamma-secretase activity. Accordingly, the relationship between Abeta levels and gamma-secretase activity across brain regions was investigated and correlated in the brains of transgenic and non-transgenic rodents commonly used in AD research. The data demonstrated that Abeta levels do vary in different brain regions in both transgenic and non-transgenic mice but are not correlated with regional gamma-secretase activity. Furthermore, this study demonstrated that while mutations in the APP and PS1 sequences affect the absolute Abeta levels this is not reflected in an increase in gamma-secretase proteolytic activity. The data in the current paper indicate that this assay is able to measure the level of gamma-secretase activity in rodent species. Using this methodology will aid our understanding of physiological gamma-secretase function.

The Epigenetics of Normal Pregnancy.

Epigenetic modifications to chromatin are essential for the specification and maintenance of cell fate, enabling the same genome to programme a variety of cellular outcomes. Epigenetic modulation of gene expression is also a critical mechanism by which cells stabilize their responses to environmental stimuli, including both nutritional cues and hormonal signalling. Unsurprisingly, epigenetics is proving to be vitally important in fetal development, and this review addresses our current understanding of the roles of epigenetic regulation in the prenatal phase. It is striking that while there has been a major interest in the intersection of fetal health with epigenetics, there has been relatively little discussion in the literature on epigenetic changes in the pregnant woman, and we attempt to redress this balance, drawing on the fragmented but intriguing experimental literature in this field.

Epigenetic opportunities and challenges in cancer.

Epigenetic covalent modifications of DNA and chromatin proteins strongly affect gene expression and cellular activity, and epigenetic misregulation occurs in several diseases, especially cancer. First-generation drugs targeting the relatively promiscuous DNA methylation and histone acetylation modifiers have had successes in the treatment of haematological cancers. Second-generation drug programmes are in the discovery phase, targeting epigenetic enzymes with more tightly defined modes of action. This review highlights some of the challenges in identifying the most appropriate new targets and the issues that need to be addressed to facilitate the successful entry of second-generation epigenetic drugs into the clinic.

Epigenetic therapies for non-oncology indications.

Chronic and degenerative disorders are a major, and growing, human health burden, and current treatments are in many cases inadequate or very expensive. Epigenetic therapies are attractive options for treating such disorders because they manipulate the processes that maintain cells in an abnormal transcriptional state. The challenges lie in identifying the most appropriate diseases and the enzymes that should be targeted. This review describes the different approaches that can be used to address this problem, focusing particularly on CNS disorders (especially mental retardation, neurodegenerative disease, psychiatric disorders and drug addiction), diabetes and diabetic complications, and autoimmunity and inflammatory diseases.

Novel orally bioavailable gamma-secretase inhibitors with excellent in vivo activity.

The development of potent gamma-secretase inhibitors having substituted heterocycles attached to a benzobicyclo[4.2.1]nonane core is described. This work led to the identification of [6S,9R,11R]-2',3',4',5,5',6,7,8,9,10-decahydro-2-(5-(4-fluorophenyl)-1-methylpyrazol-3-yl)-5'-(2,2,2-trifluoroethyl)spiro[6,9-methanobenzocyclooctene-11,3'-[1,2,5]thiadiazole] 1',1'-dioxide (16), which has excellent in vitro potency (0.06 nM) and which reduced amyloid-beta in APP-YAC mice with an ED(50) of 1 mg/kg (po). 16 had a good pharmacokinetic profile in three preclinical species.

The novel gamma secretase inhibitor N-[cis-4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK-560) reduces amyloid plaque deposition without evidence of notch-related pathology in the Tg2576 mouse.

There is a substantial body of evidence indicating that beta-amyloid peptides (Abeta) are critical factors in the onset and development of Alzheimer's disease (AD). One strategy for combating AD is to reduce or eliminate the production of Abeta through inhibition of the gamma-secretase enzyme, which cleaves Abeta from the amyloid precursor protein (APP). We demonstrate here that chronic treatment for 3 months with 3 mg/kg of the potent, orally bioavailable and brain-penetrant gamma-secretase inhibitor N-[cis-4-[(4-chlorophenyl)-sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK-560) attenuates the appearance of amyloid plaques in the Tg2576 mouse. These reductions in plaques were also accompanied by a decrease in the level of reactive gliosis. The morphometric and histological measures agreed with biochemical analysis of Abeta(40) and Abeta(42) in the cortex. Interestingly, the volume of the plaques across treatment groups did not change, indicating that reducing Abeta levels does not significantly alter deposit growth once initiated. Furthermore, we demonstrate that these beneficial effects can be achieved without causing histopathological changes in the ileum, spleen, or thymus as a consequence of blockade of the processing of alternative substrates, such as the Notch family of receptors. This indicates that in vivo a therapeutic window between these substrates seems possible--a key concern in the development of this approach to AD. An understanding of the mechanisms whereby MRK-560 shows differentiation between the APP and Notch proteolytic pathway of gamma-secretase should provide the basis for the next generation of gamma-secretase inhibitors.

In vivo characterization of Abeta(40) changes in brain and cerebrospinal fluid using the novel gamma-secretase inhibitor N-[cis-4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK-560) in the rat.

Plaques in the parenchyma of the brain containing Abeta peptides are one of the hallmarks of Alzheimer's disease. These Abeta peptides are produced by the final proteolytic cleavage of the amyloid precursor protein by the intramembraneous aspartyl protease gamma-secretase. Thus, one approach to lowering levels of Abeta has been via the inhibition of the gamma-secretase enzyme. Here, we report a novel, bioavailable gamma-secretase inhibitor, N-[cis-4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK-560) that displayed oral pharmacokinetics suitable for once-a-day dosing. It was able to markedly reduce Abeta in the brain and cerebrospinal fluid (CSF) in the rat, with ED(50) values of 6 and 10 mg/kg, respectively. Time-course experiments using MRK-560 demonstrated these reductions in Abeta could be maintained for 24 h, and comparable temporal reductions in rat brain and CSF Abeta(40) further suggested that these two pools of Abeta are related. This relationship between the brain and CSF Abeta was maintained when MRK-560 was dosed once a day for 2 weeks, and accordingly, when all the data for the dose-response curve and time courses were correlated, a strong association was observed between the brain and CSF Abeta levels. These results demonstrate that MRK-560 is an orally bioavailable gamma-secretase inhibitor with the ability to markedly reduce Abeta peptide in the brain and CSF of the rat and confirm the utility of the rat for assessing the effects of gamma-secretase inhibitors on central nervous system Abeta(40) levels in vivo.

4-substituted cyclohexyl sulfones as potent, orally active gamma-secretase inhibitors.

The protease gamma-secretase plays a pivotal role in the synthesis of pathogenic amyloid-beta in Alzheimer's disease (AD). Here, we report a further extension to a series of cyclohexyl sulfone-based gamma-secretase inhibitors which has allowed the preparation of highly potent compounds which also demonstrate robust Abeta(40) lowering in vivo (e.g., compound 32, MED 1mg/kg p.o. in APP-YAC mice).

Quantitative measurement of changes in amyloid-beta(40) in the rat brain and cerebrospinal fluid following treatment with the gamma-secretase inhibitor LY-411575 [N2-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N1-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide].

The efficacy of gamma-secretase inhibitors in vivo has, to date, been generally assessed in transgenic mouse models expressing increased levels of amyloid-beta (Abeta) peptide thereby allowing the detection of changes in Abeta production. However, it is not clear whether the in vivo potency of gamma-secretase inhibitors is independent of the level of amyloid precursor protein expression. In other words, does a gamma-secretase inhibitor have the same effect in nontransgenic physiological animals versus transgenic overexpressing animals? In the present study, an immunoassay has been developed which can detect Abeta(40) in the rat brain, where concentrations are much lower than those seen in transgenic mice such as Tg2576 (c. 0.7 and 25 nM, respectively) and in cerebrospinal fluid (CSF, c. 0.3 nM). Using this immunoassay, the effects of the gamma-secretase inhibitor LY-411575 [N(2)-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N(1)-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide] were assessed and robust dose-dependent reductions in rat brain and CSF Abeta(40) levels were observed with ID(50) values of 1.3 mg/kg for both brain and CSF. These values were comparable with those calculated for LY-411575 in transgenic mice. Time course experiments using LY-411575 demonstrated comparable temporal reductions in rat brain and CSF Abeta(40), further suggesting these two pools of Abeta are related. Accordingly, when all the data for the dose-response curve and time course were correlated, a strong association was observed between the brain and CSF Abeta(40) levels. These data demonstrate the utility of the rat as a novel approach for assessing the effects of gamma-secretase inhibitors on central nervous system Abeta(40) levels in vivo.

A novel series of potent gamma-secretase inhibitors based on a benzobicyclo[4.2.1]nonane core.

A new series of gamma-secretase inhibitors was developed from an in-house screening hit based on a benzobicyclo[4.2.1]nonane core. Lead optimisation studies led to the development of a series of potent inhibitors and in vivo efficacy was demonstrated.