David B. Sattelle

Nicotinic Acetylcholine Receptor Signalling: Roles in Alzheimer's Disease and Amyloid Neuroprotection

Alzheimers disease (AD), the major contributor to dementia in the elderly, involves accumulation in the brain of extracellular plaques containing the β-amyloid protein (Aβ) and intracellular neurofibrillary tangles of hyperphosphorylated tau protein. AD is also characterized by a loss of neurons, particularly those expressing nicotinic acetylcholine receptors (nAChRs), thereby leading to a reduction in nAChR numbers. The Aβ1–42 protein, which is toxic to neurons, is critical to the onset and progression of AD. The discovery of new drug therapies for AD is likely to be accelerated by an improved understanding of the mechanisms whereby Aβ causes neuronal death. We examine the evidence for a role in Aβ1–42 toxicity of nAChRs; paradoxically, nAChRs can also protect neurons when activated by nicotinic ligands. Aβ peptides and nicotine differentially activate several intracellular signaling pathways, including the phosphatidylinositol 3-kinase/v-akt murine thymoma viral oncogene homolog pathway, the extracellular signal-regulated kinase/mitogen-activated protein kinase, and JAK-2/STAT-3 pathways. These pathways control cell death or survival and the secretion of Aβ peptides. We propose that understanding the differential activation of these pathways by nicotine and/or Aβ1–42 may offer the prospect of new routes to therapy for AD.

Molecular biology of insect neuronal GABA receptors

Ionotropic gamma-aminobutyric acid (GABA) receptors are distributed throughout the nervous systems of many insect species. As with their vertebrate counterparts, GABAA receptors and GABAC receptors, the binding of GABA to ionotropic insect receptors elicits a rapid, transient opening of anion-selective ion channels which is generally inhibitory. Although insect and vertebrate GABA receptors share a number of structural and functional similarities, their pharmacology differs in several aspects. Recent studies of cloned Drosophila melanogaster GABA receptors have clarified the contribution of particular subunits to these differences. Insect ionotropic GABA receptors are also the target of numerous insecticides and an insecticide-resistant form of a Drosophila GABA-receptor subunit has enhanced our understanding of the structure-function relationship of one aspect of pharmacology common to both insect and vertebrate GABA receptors, namely antagonism by the plant-derived toxin picrotoxinin.

Genomic insights into the Ixodes scapularis tick vector of Lyme disease

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1u2009Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing ∼57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick–host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host ‘questing, prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent.

Actions of the insecticide fipronil, on dieldrin-sensitive and- resistant GABA receptors of Drosophila melanogaster.

1 Blocking actions of the novel insecticide, fipronil, were examined on GABA responses recorded from Xenopus oocytes expressing either wild type (dieldrin‐sensitive) or mutant (dieldrin‐resistant) forms of the Drosphila melanogaster GABA‐gated chloride channel homo‐oligomer, RDL (the product of the resistance to dieldrin locus: Rdl). 2 In the case of the wild type receptor, fipronil blocked GABA‐induced currents inducing both a shift to the right in the GABA dose‐response curve and depressing the maximum amplitude of responses to GABA. The potency of fipronil was dependent on the GABA concentration but was unaffected by membrane potential. 3 Mutant RDL GABA‐receptors, which have a naturally occurring amino acid substitution (A302→SS) in the putative ion‐channel lining region, conferring resistance to dieldrin and picrotoxinin, were markedly less sensitive to fipronil than the wild‐type receptors. 4 Fipronil antagonism is qualitatively similar to that produced by the structurally distinct compound, picrotoxinin. As the mutation A302→S reduces the potency of both fipronil and picrotoxinin, homo‐oligomeric RDL receptors should facilitate detailed studies of the molecular basis of convulsant/ insecticide antagonist actions on GABA receptors.

Genome of the house fly, Musca domestica L., a global vector of diseases with adaptations to a septic environment

BackgroundAdult house flies, Musca domestica L., are mechanical vectors of more than 100 devastating diseases that have severe consequences for human and animal health. House fly larvae play a vital role as decomposers of animal wastes, and thus live in intimate association with many animal pathogens.ResultsWe have sequenced and analyzed the genome of the house fly using DNA from female flies. The sequenced genome is 691 Mb. Compared with Drosophila melanogaster, the genome contains a rich resource of shared and novel protein coding genes, a significantly higher amount of repetitive elements, and substantial increases in copy number and diversity of both the recognition and effector components of the immune system, consistent with life in a pathogen-rich environment. There are 146 P450 genes, plus 11 pseudogenes, in M. domestica, representing a significant increase relative to D. melanogaster and suggesting the presence of enhanced detoxification in house flies. Relative to D. melanogaster, M. domestica has also evolved an expanded repertoire of chemoreceptors and odorant binding proteins, many associated with gustation.ConclusionsThis represents the first genome sequence of an insect that lives in intimate association with abundant animal pathogens. The house fly genome provides a rich resource for enabling work on innovative methods of insect control, for understanding the mechanisms of insecticide resistance, genetic adaptation to high pathogen loads, and for exploring the basic biology of this important pest. The genome of this species will also serve as a close out-group to Drosophila in comparative genomic studies.

Allosteric modulation of an expressed homo‐oligomeric GABA‐gated chloride channel of Drosophila melanogaster

1 Functional GABA‐gated chloride channels are formed when cRNA encoding the Drosophila melanogaster GABA receptor subunit RDL is injected into the cytoplasm of Xenopus oocytes. Two‐electrode voltage‐clamp was used to investigate allosteric modulation of GABA‐induced currents recorded from the expressed, bicuculline‐insensitive, RDL homo‐oligomers. 2 Flunitrazepam (0.1 μm to 100 μm) had no effect on the amplitude of responses to 10 μm GABA (approximately EC10), whereas 4′chlorodiazepam (100 μm) enhanced the amplitude of submaximal responses to GABA. 3‐Hydroxymethyl‐β‐carboline (1 μm) and ethyl‐β‐carboline‐3‐carboxylate (both 1 and 100 μm) had no effect on currents induced by 30 μm (approximately EC50) GABA. However 100 μm 3‐hydroxymethyl‐β‐carboline reduced potentiation by 4′chlorodiazepam. 3 The sodium salts of pentobarbitone (10 μm to 1 mM) and phenobarbitone (50 μm to 1 mM) dose‐dependently enhanced submaximal GABA responses. Neither barbiturate activated currents in the absence of GABA. 4 At 10 μm, the steroids 5α‐pregnan‐3α‐ol‐20‐one and alphaxalone (5α‐pregnan‐3α‐ol‐11,20‐dione), potentiated submaximal GABA responses. The stereoselectivity of steroid action seen on vertebrate GABAA receptors was observed on RDL homo‐oligomers as 5α‐pregnan‐3α‐ol‐20‐one (10 μm) was without effect. None of the three steroids tested activated currents in the absence of GABA. 5 The novel anticonvulsant, loreclezole (100 μm), potentiated the response to 10 μm GABA, but not that of saturating concentrations of GABA. δ‐Hexachlorocyclohexane (0.1 μm to 30 μm) was a potent enhancer of submaximal responses to GABA of RDL. 6 The potencies of barbiturates and steroids on RDL homo‐oligomers resemble those observed for several in situ insect GABA receptors, whereas those of benzodiazepine binding‐site ligands are considerably reduced. The differences in the benzodiazepine pharmacology of RDL homo‐oligomers and native GABA receptors, may reflect roles of other subunits in native insect receptors.

Cotinine reduces amyloid-β aggregation and improves memory in Alzheimer's disease mice

Alzheimers disease (AD) affects millions of people world-wide and new effective and safe therapies are needed. Cotinine, the main metabolite of nicotine, has a long half-life and does not have cardiovascular or addictive side effects in humans. We studied the effect of cotinine on amyloid-β (Aβ) aggregation as well as addressed its impact on working and reference memories. Cotinine reduced Aβ deposition, improved working and reference memories, and inhibited Aβ oligomerization in the brains of transgenic (Tg) 6799 AD mice. In vitro studies confirmed the inhibitory effect of cotinine on Aβ1-42 aggregation. Cotinine stimulated Akt signaling, including the inhibition of glycogen synthase kinase 3β (GSK3β), which promotes neuronal survival and the synaptic plasticity processes underlying learning and memory in the hippocampus and cortex of wild type and Tg6799 AD mice. Simulation of the cotinine-Aβ1-42 complex using molecular dynamics showed that cotinine may interact with key histidine residues of Aβ1-42, altering its structure and inhibiting its aggregation. The good safety profile in humans and its beneficial effects suggest that cotinine may be an excellent therapeutic candidate for the treatment of AD.

Automated, high-throughput, motility analysis in Caenorhabditis elegans and parasitic nematodes: Applications in the search for new anthelmintics

The scale of the damage worldwide to human health, animal health and agricultural crops resulting from parasitic nematodes, together with the paucity of treatments and the threat of developing resistance to the limited set of widely-deployed chemical tools, underlines the urgent need to develop novel drugs and chemicals to control nematode parasites. Robust chemical screens which can be automated are a key part of that discovery process. Hitherto, the successful automation of nematode behaviours has been a bottleneck in the chemical discovery process. As the measurement of nematode motility can provide a direct scalar readout of the activity of the neuromuscular system and an indirect measure of the health of the animal, this omission is acute. Motility offers a useful assay for high-throughput, phenotypic drug/chemical screening and several recent developments have helped realise, at least in part, the potential of nematode-based drug screening. Here we review the challenges encountered in automating nematode motility and some important developments in the application of machine vision, statistical imaging and tracking approaches which enable the automated characterisation of nematode movement. Such developments facilitate automated screening for new drugs and chemicals aimed at controlling human and animal nematode parasites (anthelmintics) and plant nematode parasites (nematicides).

Tick Genome Assembled: New Opportunities for Research on Tick-Host-Pathogen Interactions

As tick-borne diseases are on the rise, an international effort resulted in the sequence and assembly of the first genome of a tick vector. This result promotes research on comparative, functional and evolutionary genomics and the study of tick-host-pathogen interactions to improve human, animal and ecosystem health on a global scale.

Actions of picrotoxinin analogues on an expressed, homo-oligomeric GABA receptor of Drosophila melanogaster.

The actions of picrotoxinin and four of its analogues were tested on a Drosophila melanogaster homo-oligomeric GABA (gamma-aminobutyric acid) receptor formed when RDL (resistance to dieldrin) subunits were expressed in Xenopus oocytes. In agreement with previously reported studies on native insect GABA receptors and native expressed vertebrate GABA receptors, acetylation of the bridgehead hydroxyl group (picrotoxinin acetate) greatly reduced the activity of the molecule, but surprisingly, substitution with flourine at the same position also reduced the activity. Conversion of the terminal isopropenyl group to an acetyl (alpha-picrotoxinone) or hydration of the double bond (picrotin) also reduced activity, in agreement with findings for native insect and mammalian receptors. The present results suggest that interactions of convulsants with homo-oligomeric and multimeric GABA receptors are qualitatively similar. Thus, the RDL homo-oligomer exhibits a pharmacological profile for picrotoxinin analogues resembling that of native GABA receptors.