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Calcium (Ca2+) and Calmodulin (CaM)-dependent serine/threonine kinase II (CaMKII) plays a central role in synaptic plasticity and memory due to its ability to phosphorylate itself and regulate its own kinase activity. Autophosphorylation at threonine 287 (T287) switches CaMKII to a Ca2+ independent and constitutively active state replicated by overexpression of a phosphomimetic CaMKII-T287D transgene or blocked by expression of a T287A transgene. A second pair of sites, T306 T307 in the CaM binding region once autophosphorylated, prevents CaM binding and inactivates the kinase during synaptic plasticity and memory, and can be blocked by a TT306/7AA transgene. Recently the synaptic scaffolding molecule called CASK (Ca2+/CaM-associated serine kinase) has been shown to control both sets of CaMKII autophosphorylation events during neuronal growth, Ca2+ signalling and memory in Drosophila. Deletion of either full length CASK or just its CaMK-like and L27 domains removed middle-term memory (MTM) and long-term memory (LTM), with CASK function in the α’/ß’ mushroom body neurons being required for memory. In a similar manner directly changing the levels of CaMKII autophosphorylation (T287D, T287A or TT306/7AA) in the α’/ß’ neurons also removed MTM and LTM. In the CASK null mutant expression of either the Drosophila or human CASK transgene in the α’/ß’ neurons was found to completely rescue memory, confirming that CASK signalling in α’/β’ neurons is necessary and sufficient for Drosophila memory formation and that the neuronal function of CASK is conserved between Drosophila and human. Expression of human CASK in Drosophila also rescued the effect of CASK deletion on the activity state of CaMKII, suggesting that human CASK may also regulate CaMKII autophosphorylation. Mutations in human CASK have recently been shown to result in intellectual disability and neurological defects suggesting a role in plasticity and learning possibly via regulation of CaMKII autophosphorylation.

Calcium (Ca2+)/calmodulin (CaM)-dependent kinase II (CaMKII) activity plays a fundamental role in learning and memory. A key feature of CaMKII in memory formation is its ability to be regulated by autophosphorylation, which switches its activity on and off during synaptic plasticity. The synaptic scaffolding protein CASK (calcium (Ca2+)/calmodulin (CaM) associated serine kinase) is also important for learning and memory, as mutations in CASK result in intellectual disability and neurological defects in humans. We show that in Drosophila larvae, CASK interacts with CaMKII to control neuronal growth and calcium signalling. Furthermore, deletion of the CaMK-like and L27 domains of CASK (CASK β null) or expression of overactive CaMKII (T287D) produced similar effects on synaptic growth and Ca2+ signalling. CASK overexpression rescues the effects of CaMKII overactivity, consistent with the notion that CASK and CaMKII act in a common pathway that controls these neuronal processes. The reduction in Ca2+ signalling observed in the CASK β null mutant caused a decrease in vesicle trafficking at synapses. In addition, the decrease in Ca2+ signalling in CASK mutants was associated with an increase in Ether-à-go-go (EAG) potassium (K+) channel localisation to synapses. Reducing EAG restored the decrease in Ca2+ signalling observed in CASK mutants to the level of wildtype, suggesting that CASK regulates Ca2+ signalling via EAG. CASK knockdown reduced both appetitive associative learning and odour evoked Ca2+ responses in Drosophila mushroom bodies, which are the learning centres of Drosophila. Expression of human CASK in Drosophila rescued the effect of CASK deletion on the activity state of CaMKII, suggesting that human CASK may also regulate CaMKII autophosphorylation.

CaMKII is a central molecule in mechanisms of synaptic plasticity and memory. A vital feature of CaMKII in plasticity is its ability to switch to a calcium (Ca2+) independent constitutively active state after autophosphorylation at threonine 287 (T287). A second pair of sites, T306 T307 in the calmodulin (CaM) binding region once autophosphorylated, prevent subsequent CaM binding and inactivates the kinase during synaptic plasticity and memory. Recently a synaptic molecule called CASK has been shown to control both sets of CaMKII autophosphorylation events and hence is well poised to be a key regulator of memory. We show deletion of full length CASK or just its CaMK-like and L27 domains disrupts middle-term memory (MTM) and long-term memory (LTM), with CASK function in the α’/ß’ subset of mushroom body neurons being required for memory. Likewise directly changing the levels of CaMKII autophosphorylation in these neurons removed MTM and LTM. The requirement of CASK and CaMKII autophosphorylation was not developmental as their manipulation just in the adult α’/ß’ neurons was sufficient to remove memory. Overexpression of CASK or CaMKII in the α’/ß’ neurons also occluded MTM and LTM. Overexpression of either Drosophila or human CASK in the α’/ß’ neurons of the CASK mutant completely rescued memory, confirming that CASK signalling in α’/β’ neurons is necessary and sufficient for Drosophila memory formation and that the neuronal function of CASK is conserved between Drosophila and human. At the cellular level CaMKII overexpression in the α’/ß’ neurons increased activity dependent Ca2+ responses while reduction of CaMKII decreased it. Likewise reducing CASK or directly expressing a phosphomimetic CaMKII T287D transgene in the α’/ß’ similarly decreased Ca2+ signalling. Our results are consistent with CASK regulating CaMKII autophosphorylation in a pathway required for memory formation that involves activity dependent changes in Ca2+ signalling in the α’/ß’ neurons.

Ion channels are the determinants of excitability; therefore, manipulation of their levels and properties provides an opportunity for the investigator to modulate neuronal and circuit function. There are a number of ways to suppress electrical activity in Drosophila neurons, for instance, over-expression of potassium channels (i.e. Shaker Kv1, Shaw Kv3, Kir2.1 and DORK) that are open at resting membrane potential. This will result in increased potassium efflux and membrane hyperpolarisation setting resting membrane potential below the threshold required to fire action potentials. Alternatively over-expression of other channels, pumps or co-transporters that result in a hyperpolarised membrane potential will also prevent firing. Lastly, neurons can be inactivated by, disrupting or reducing the level of functional voltage-gated sodium (Nav1 paralytic) or calcium (Cav2 cacophony) channels that mediate the depolarisation phase of action potentials. Similarly, strategies involving the opposite channel manipulation should allow net depolarisation and hyperexcitation in a given neuron. These changes in ion channel expression can be brought about by the versatile transgenic (i.e. Gal4/UAS based) systems available in Drosophila allowing fine temporal and spatial control of (channel) transgene expression. These systems are making it possible to electrically inactivate (or hyperexcite) any neuron or neural circuit in the fly brain, and much like an exquisite lesion experiment, potentially elucidate whatever interesting behaviour or phenotype each network mediates. These techniques are now being used in Drosophila to reprogram electrical activity of well-defined circuits and bring about robust and easily quantifiable changes in behaviour, allowing different models and hypotheses to be rapidly tested.

Alzheimer’s disease (AD) is the most common neurodegenerative disease which is becoming increasingly prevalent due to ageing populations resulting in huge social, economic, and health costs to the community. Despite the pathological processing of genes such as Amyloid Precursor Protein (APP) into Amyloid-β and Microtubule Associated Protein Tau (MAPT) gene, into hyperphosphorylated Tau tangles being known for decades, there remains no treatments to halt disease progression. One population with increased risk of AD are people with Down syndrome (DS), who have a 90% lifetime incidence of AD, due to trisomy of human chromosome 21 (HSA21) resulting in three copies of APP and other AD-associated genes, such as DYRK1A (Dual specificity tyrosine-phosphorylation-regulated kinase 1A) overexpression. This suggests that blocking DYRK1A might have therapeutic potential. However, it is still not clear to what extent DYRK1A overexpression by itself leads to AD-like phenotypes and how these compare to Tau and Amyloid-β mediated pathology. Likewise, it is still not known how effective a DYRK1A antagonist may be at preventing or improving any Tau, Amyloid-β and DYRK1a mediated phenotype. To address these outstanding questions, we characterised Drosophila models with targeted overexpression of human Tau, human Amyloid-β or the fly orthologue of DYRK1A, called minibrain (mnb). We found targeted overexpression of these AD-associated genes caused degeneration of photoreceptor neurons, shortened lifespan, as well as causing loss of locomotor performance, sleep, and memory. Treatment with the experimental DYRK1A inhibitor PST-001 decreased pathological phosphorylation of human Tau [at serine (S) 262]. PST-001 reduced degeneration caused by human Tau, Amyloid-β or mnb lengthening lifespan as well as improving locomotion, sleep and memory loss caused by expression of these AD and DS genes. This demonstrated PST-001 effectiveness as a potential new therapeutic targeting AD and DS pathology.

Neonicotinoids are the most widely used insecticides in the world and are implicated in the widespread population declines of insects including pollinators. Neonicotinoids target nicotinic acetylcholine receptors which are expressed throughout the insect central nervous system, causing a wide range of sub-lethal effects on non-target insects. Here, we review the potential of the fruit fly Drosophila melanogaster to model the sub-lethal effects of neonicotinoids on pollinators, by utilizing its well-established assays that allow rapid identification and mechanistic characterization of these effects. We compare studies on the effects of neonicotinoids on lethality, reproduction, locomotion, immunity, learning, circadian rhythms and sleep in D. melanogaster and a range of pollinators. We also highlight how the genetic tools available in D. melanogaster, such as GAL4/UAS targeted transgene expression system combined with RNAi lines to any gene in the genome including the different nicotinic acetylcholine receptor subunit genes, are set to elucidate the mechanisms that underlie the sub-lethal effects of these common pesticides. We argue that studying pollinators and D. melanogaster in tandem allows rapid elucidation of mechanisms of action, which translate well from D. melanogaster to pollinators. We focus on the recent identification of novel and important sublethal effects of neonicotinoids on circadian rhythms and sleep. The comparison of effects between D. melanogaster and pollinators and the use of genetic tools to identify mechanisms make a powerful partnership for the future discovery and testing of more specific insecticides.

The cellular underpinnings of memory deficits in Alzheimer’s disease (AD) are poorly understood. We utilized the tractable neural circuits sub-serving memory in Drosophila to investigate the role of impaired Ca2+ handling in memory deficits caused by expression of human 0N4R isoform of tau which is associated with AD. Expression of tau in mushroom body neuropils, or a subset of mushroom body output neurons, led to impaired memory. By using the Ca2+ reporter GCaMP6f, we observed changes in Ca2+ signaling when tau was expressed in these neurons, an effect that could be blocked by the L-type Ca2+ channel antagonist nimodipine or reversed by RNAi knock-down of the L-type channel gene. The L-type Ca2+ channel itself is required for memory formation, however, RNAi knock-down of the L-type Ca2+ channel in neurons overexpressing human tau resulted in flies whose memory is restored to levels equivalent to wild-type. Expression data suggest that Drosophila L-type Ca2+ channel mRNA levels are increased upon tau expression in neurons, thus contributing to the effects observed on memory and intracellular Ca2+ homeostasis. Together, our Ca2+ imaging and memory experiments suggest that expression of the 0N4R isoform of human tau increases the number of L-type Ca2+ channels in the membrane resulting in changes in neuronal excitability that can be ameliorated by RNAi knockdown or pharmacological blockade of L-type Ca2+ channels. This highlights a role for L-type Ca2+ channels in tauopathy and their potential as a therapeutic target for AD.

Alzheimer’s disease (AD) is the most common form of dementia and is characterized by intracellular neurofibrillary tangles of hyperphosphorylated Tau, including the 0N4R isoform and accumulation of extracellular amyloid beta (Aβ) plaques. However, less than 5% of AD cases are familial, with many additional risk factors contributing to AD including aging, lifestyle, the environment and epigenetics. Recent epigenome-wide association studies (EWAS) of AD have identified a number of loci that are differentially methylated in the AD cortex. Indeed, hypermethylation and reduced expression of the Ankyrin 1 (ANK1) gene in AD has been reported in the cortex in numerous different post-mortem brain cohorts. Little is known about the normal function of ANK1 in the healthy brain, nor the role it may play in AD. We have generated Drosophila models to allow us to functionally characterize Drosophila Ank2, the ortholog of human ANK1 and to determine its interaction with human Tau and Aβ. We show expression of human Tau 0N4R or the oligomerizing Aβ 42 amino acid peptide caused shortened lifespan, degeneration, disrupted movement, memory loss, and decreased excitability of memory neurons with co-expression tending to make the pathology worse. We find that Drosophila with reduced neuronal Ank2 expression have shortened lifespan, reduced locomotion, reduced memory and reduced neuronal excitability similar to flies overexpressing either human Tau 0N4R or Aβ42. Therefore, we show that the mis-expression of Ank2 can drive disease relevant processes and phenocopy some features of AD. Therefore, we propose targeting human ANK1 may have therapeutic potential. This represents the first study to characterize an AD-relevant gene nominated from EWAS.