Investigating the molecular basis of resistance and pyrethroid selectivity at acarine sodium channels

Many acarine (tick and mite) species are ectoparasites of humans, livestock and domestic pets, where they spread disease and impact adversely on health. They are normally controlled through the application of acaricides; however, the prolonged use of individual compounds has resulted in many species developing resistance to specific pesticides. This thesis describes investigations into the molecular properties that determine the resistance to and selectivity of pyrethroids, an important class of pesticides that act on the voltage-gated sodium channels (VGSCs) of arthropod neurons. Comparison of insect and acarine VGSC sequences, coupled with molecular modelling studies, have identified a residue at amino-acid position 933 (M. domestica numbering) found within a putative pyrethroid binding pocket that may contribute to a greater selectivity of pyrethroids with comparatively larger halogenated groups for acarine VGSCs compared to those of insects. This is due to the presence of a smaller glycine residue at position 933 in acarine channels, compared to a cysteine residue in insect channels, which may enhance the binding of such pyrethroids (O'Reilly et al., 2014). This model is supported by the findings of Jonsson et al 2010, that R. microplus cattle ticks carrying the amino acid substitution G933V, are resistant to the pyrethroid flumethrin, which has a comparatively larger halogenated group, but not the pyrethroid cypermethrin, which has a comparatively smaller halogenated group. Work in this thesis describes progress made in the investigation of such specificity; involving sequencing studies, two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes involving insect and acarine VGSCs, and whole arthropod bioassays. While this work cannot conclusively disprove the model proposed by O’Reilly et al 2014, it suggests that the mechanisms of selectivity for pyrethroids in arthropods may involve the interplay of several factors, rather than being solely based upon structural variations in their VGSCs

Investigating the molecular basis of resistance and pyrethroid selectivity at acarine sodium channels

Many acarine (tick and mite) species are ectoparasites of humans, livestock and domestic pets, where they spread disease and impact adversely on health. They are normally controlled through the application of acaricides; however, the prolonged use of individual compounds has resulted in many species developing resistance to specific pesticides. This thesis describes investigations into the molecular properties that determine the resistance to and selectivity of pyrethroids, an important class of pesticides that act on the voltage-gated sodium channels (VGSCs) of arthropod neurons. Comparison of insect and acarine VGSC sequences, coupled with molecular modelling studies, have identified a residue at amino-acid position 933 (M. domestica numbering) found within a putative pyrethroid binding pocket that may contribute to a greater selectivity of pyrethroids with comparatively larger halogenated groups for acarine VGSCs compared to those of insects. This is due to the presence of a smaller glycine residue at position 933 in acarine channels, compared to a cysteine residue in insect channels, which may enhance the binding of such pyrethroids (O'Reilly et al., 2014). This model is supported by the findings of Jonsson et al 2010, that R. microplus cattle ticks carrying the amino acid substitution G933V, are resistant to the pyrethroid flumethrin, which has a comparatively larger halogenated group, but not the pyrethroid cypermethrin, which has a comparatively smaller halogenated group. Work in this thesis describes progress made in the investigation of such specificity; involving sequencing studies, two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes involving insect and acarine VGSCs, and whole arthropod bioassays. While this work cannot conclusively disprove the model proposed by O’Reilly et al 2014, it suggests that the mechanisms of selectivity for pyrethroids in arthropods may involve the interplay of several factors, rather than being solely based upon structural variations in their VGSCs

Damaging coding variants within kainate receptor channel genes are enriched in individuals with schizophrenia, autism and intellectual disabilities

Schizophrenia (Scz), autism spectrum disorder (ASD) and intellectual disability are common complex neurodevelopmental disorders. Kainate receptors (KARs) are ionotropic glutamate ion channels involved in synaptic plasticity which are modulated by auxiliary NETO proteins. Using UK10K exome sequencing data, we interrogated the coding regions of KAR and NETO genes in individuals with Scz, ASD or intellectual disability and population controls; performed follow-up genetic replication studies; and, conducted in silico and in vitro functional studies. We found an excess of Loss-of-Function and missense variants in individuals with Scz compared with control individuals (p = 1.8 x 10-10), and identified a significant burden of functional variants for Scz (p < 1.6 x 10-11) and ASD (p = 6.9 x 10-18). Single allele associations for 6 damaging missense variants were significantly replicated (p < 5.0 x 10-15) and confirmed GRIK3 S310A as a protective genetic factor. Functional studies demonstrated that three missense variants located within GluK2 and GluK4, GluK2 (K525E) and GluK4 (Y555N, L825W), affect agonist sensitivity and current decay rates. These findings establish that genetic variation in KAR receptor ion channels confers risk for schizophrenia, autism and intellectual disability and provide new genetic and pharmacogenetic biomarkers for neurodevelopmental diseas

Physiological changes throughout an insect ear due to age and noise - a longitudinal study

Hearing loss is not unique to humans and is experienced by all animals in the face of wild and eclectic differences in ear morphology. Here we exploited the high throughput and accessible tympanal ear of the desert locust, Schistocerca gregaria to rigorously quantify changes in the auditory system due to noise exposure and age. In this exploratory study we analysed tympanal displacements, morphology of the auditory Müller’s organ and measured activity of the auditory nerve, the transduction current and electrophysiological properties of individual auditory receptors. This work shows that hearing loss manifests as a complex disorder due to differential effects of age and noise of several processes and cell types within the ear. The ‘middle-aged deafness’ pattern of hearing loss found in locusts mirrors that found for humans exposed to noise early in their life suggesting a fundamental interaction of the use of an auditory system (noise) and its aging

Symbiotic microbes from marine invertebrates: Driving a new era of natural product drug discovery.

Invertebrates account for more than 89% of all extant organisms in the marine environment, represented by over 174,600 species (recorded to date). Such diversity is mirrored in (or more likely increased by) the microbial symbionts associated with this group and in the marine natural products (or MNPs) that they produce. Since the early 1950s over 20,000 MNPs have been discovered, including compounds produced by symbiotic bacteria, and the chemical diversity of compounds produced from marine sources has led to them being referred to as "blue gold" in the search for new drugs. For example, 80% of novel antibiotics stemming from the marine environment have come from Actinomycetes, many of which can be found associated with marine sponges, and compounds with anti-tumorigenic and anti-diabetic potential have also been isolated from marine symbionts. In fact, it has been estimated that marine sources formed the basis of over 50% of FDA-approved drugs between 1981 and 2002. In this review, we explore the diversity of marine microbial symbionts by examining their use as the producers of novel pharmaceutical actives, together with a discussion of the opportunities and constraints offered by “blue gold” drug discovery.N

Multitarget drug design strategy in Alzheimer’s disease: focus on cholinergic transmission and amyloid-β aggregation

Background: Alzheimer pathogenesis has been associated with a network of processes working simultaneously and synergistically. Over time, much interest has been focused on cholinergic transmission and its mutual interconnections with other active players of the disease. Besides the cholinesterase mainstay, the multifaceted interplay between nicotinic receptors and amyloid is actually considered to have a central role in neuroprotection. Thus, the multitarget drug-design strategy has emerged as a chance to face the disease network. Results: By exploiting the multitarget approach, the present study provides new molecules able to target the cholinergic pathway, by joining direct nicotinic receptor stimulation to acetylcholinesterase inhibition, and to inhibit Aβ aggregation. Conclusions: These new compounds emerged as a suitable starting point for a further optimization process

Metabolic decline in an insect ear: correlative or causative for age-related auditory decline?

One leading hypothesis for why we lose our hearing as we age is a decrease in ear metabolism. However, direct measurements of metabolism across a lifespan in any auditory system are lacking. Even if metabolism does decrease with age, a question remains: is a metabolic decrease a cause of age-related auditory decline or simply correlative? We use an insect, the desert locust Schistocerca gregaria, as a physiologically versatile model to understand how cellular metabolism correlates with age and impacts on age-related auditory decline. We found that auditory organ metabolism decreases with age as measured fluorometrically. Next, we measured the individual auditory organ’s metabolic rate and its sound-evoked nerve activity and found no correlation. We found no age-related change in auditory nerve activity, using hook electrode recordings, and in the electrophysiological properties of auditory neurons, using patch-clamp electrophysiology, but transduction channel activity decreased. To further test for a causative role of the metabolic rate in auditory decline, we manipulated metabolism of the auditory organ through diet and cold-rearing but found no difference in sound-evoked nerve activity. We found that although metabolism correlates with age-related auditory decline, it is not causative. Finally, we performed RNA-Seq on the auditory organs of young and old locusts, and whilst we found enrichment for Gene Ontology terms associated with metabolism, we also found enrichment for a number of additional aging GO terms. We hypothesize that age-related hearing loss is dominated by accumulative damage in multiple cell types and multiple processes which outweighs its metabolic decline