Keep It Simple: Using README Files to Advance Standardization in Chronobiology.

Standardization plays a crucial role in ensuring the reliability, reproducibility, and interoperability of research data in the biomedical sciences. Metadata standards are one foundation for the FAIR (Findable, Accessible, Interoperable, and Reusable) principles of data management. They facilitate data discovery, understanding, and reuse. However, the adoption of metadata standards in biological research lags in practice. Barriers such as complexity, lack of incentives, technical challenges, resource constraints, and resistance to change hinder widespread adoption. In the field of chronobiology, standardization is essential but faces particular challenges due to the longitudinal nature of experimental data, diverse model organisms, and varied measurement techniques. To address these challenges, we propose an approach that emphasizes simplicity and practicality: the development of README templates tailored for particular data types and species. Through this opinion article, our intention is to initiate a dialogue and commence a community-driven standardization process by engaging potential contributors and collaborators.

Increased interaction between endoplasmic reticulum and mitochondria following sleep deprivation.

BACKGROUND: Prolonged cellular activity may overload cell function, leading to high rates of protein synthesis and accumulation of misfolded or unassembled proteins, which cause endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR) to re-establish normal protein homeostasis. Previous molecular work has demonstrated that sleep deprivation (SD) leads to ER stress in neurons, with a number of ER-specific proteins being upregulated to maintain optimal cellular proteostasis. It is still not clear which cellular processes activated by sleep deprivation lead to ER- stress, but increased cellular metabolism, higher request for protein synthesis, and over production of oxygen radicals have been proposed as potential contributing factors. Here, we investigate the transcriptional and ultrastructural ER and mitochondrial modifications induced by sleep loss. RESULTS: We used gene expression analysis in mouse forebrains to show that SD was associated with significant transcriptional modifications of genes involved in ER stress but also in ER-mitochondria interaction, calcium homeostasis, and mitochondrial respiratory activity. Using electron microscopy, we also showed that SD was associated with a general increase in the density of ER cisternae in pyramidal neurons of the motor cortex. Moreover, ER cisternae established new contact sites with mitochondria, the so-called mitochondria associated membranes (MAMs), important hubs for molecule shuttling, such as calcium and lipids, and for the modulation of ATP production and redox state. Finally, we demonstrated that Drosophila male mutant flies (elav > linker), in which the number of MAMs had been genetically increased, showed a reduction in the amount and consolidation of sleep without alterations in the homeostatic sleep response to SD. CONCLUSIONS: We provide evidence that sleep loss induces ER stress characterized by increased crosstalk between ER and mitochondria. MAMs formation associated with SD could represent a key phenomenon for the modulation of multiple cellular processes that ensure appropriate responses to increased cell metabolism. In addition, MAMs establishment may play a role in the regulation of sleep under baseline conditions.

DYRK1A antagonists rescue degeneration and behavioural deficits of in vivo models based on amyloid-ß, Tau and DYRK1A neurotoxicity.

Alzheimer's disease (AD) involves pathological processing of amyloid precursor protein (APP) into amyloid-ß and microtubule associated protein Tau (MAPT) into hyperphosphorylated Tau tangles leading to neurodegeneration. Only 5% of AD cases are familial making it difficult to predict who will develop the disease thereby hindering our ability to treat the causes of the disease. A large population who almost certainly will, are those with Down syndrome (DS), who have a 90% lifetime incidence of AD. DS is caused by trisomy of chromosome 21 resulting in three copies of APP and other AD-associated genes, like dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) overexpression. This implies that DYRK1a inhibitors may have therapeutic potential for DS and AD, however It is not clear how overexpression of each of these genes contributes to the pathology of each disease as well as how effective a DYRK1A inhibitor would be at suppressing any of these. To address this knowledge gap, we used Drosophila models with human Tau, human amyloid-ß or fly DYRK1A (minibrain (mnb)) neuronal overexpression resulting in photoreceptor neuron degeneration, premature death, decreased locomotion, sleep and memory loss. DYRK1A small molecule Type 1 kinase inhibitors (DYR219 and DYR533) were effective at suppressing these disease relevant phenotypes confirming their therapeutic potential.

Hereditary spastic paraparesis presenting as cerebral palsy due to ADD3 variant with mechanistic insight provided by a Drosophila γ-adducin model.

Cerebral palsy (CP) causes neurological disability in early childhood. Hypoxic-ischaemic injury plays a major role in its aetiology, nevertheless, genetic and epigenetic factors may contribute to the clinical presentation. Mutations in ADD3 (encoding γ-adducin) gene have been described in a monogenic form of spastic quadriplegic cerebral palsy (OMIM 601568). We studied a 16-year-old male with spastic diplegia. Several investigations including neurometabolic testing, brain and spine magnetic resonance imaging (MRI) and CGH-Array were normal. Further, clinical genetics assessment and whole exome sequencing (WES) gave the diagnosis. We generated an animal model using Drosophila to study the effects of γ-adducin loss and gain of function. WES revealed a biallelic variant in the ADD3 gene, NM_016824.5(ADD3): c.1100G > A, p.(Gly367Asp). Mutations in this gene have been described as an ultra-rare autosomal recessive, which is a known form of inherited cerebral palsy. Molecular modelling suggests that this mutation leads to a loss of structural integrity of γ-adducin and is therefore expected to result in a decreased level of functional protein. Pan-neuronal over-expression or knock-down of the Drosophila ortholog of ADD3 called hts caused a reduction of life span and impaired locomotion thereby phenocopying aspects of the human disease. Our animal experiments present a starting point to understand the biological processes underpinning the clinical phenotype and pathogenic mechanisms, to gain insights into potential future methods for treating or preventing ADD3 related spastic quadriplegic cerebral palsy.

DYRK1a Inhibitor Mediated Rescue of Drosophila Models of Alzheimer's Disease-Down Syndrome Phenotypes.

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-ß 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-ß mediated pathology. Likewise, it is still not known how effective a DYRK1A antagonist may be at preventing or improving any Tau, Amyloid-ß and DYRK1a mediated phenotype. To address these outstanding questions, we characterised Drosophila models with targeted overexpression of human Tau, human Amyloid-ß 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-ß 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.

Effects of Eph/ephrin signalling and human Alzheimer's disease-associated EphA1 on Drosophila behaviour and neurophysiology.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease placing a great burden on people living with it, carers and society. Yet, the underlying patho-mechanisms remain unknown and treatments limited. To better understand the molecular changes associated with AD, genome-wide association studies (GWAS) have identified hundreds of candidate genes linked to the disease, like the receptor tyrosine kinase EphA1. However, demonstration of whether and how these genes cause pathology is largely lacking. Here, utilising fly genetics, we generated the first Drosophila model of human wild-type and P460L mutant EphA1 and tested the effects of Eph/ephrin signalling on AD-relevant behaviour and neurophysiology. We show that EphA1 mis-expression did not cause neurodegeneration, shorten lifespan or affect memory but flies mis-expressing the wild-type or mutant receptor were hyper-aroused, had reduced sleep, a stronger circadian rhythm and increased clock neuron activity and excitability. Over-expression of endogenous fly Eph and RNAi-mediated knock-down of Eph and its ligand ephrin affected sleep architecture and neurophysiology. Eph over-expression led to stronger circadian morning anticipation while ephrin knock-down impaired memory. A dominant negative form of the GTPase Rho1, a potential intracellular effector of Eph, led to hyper-aroused flies, memory impairment, less anticipatory behaviour and neurophysiological changes. Our results demonstrate a role of Eph/ephrin signalling in a range of behaviours affected in AD. This presents a starting point for studies into the underlying mechanisms of AD including interactions with other AD-associated genes, like Rho1, Ankyrin, Tau and APP with the potential to identify new targets for treatment.

The opposing chloride cotransporters KCC and NKCC control locomotor activity in constant light and during long days.

Cation chloride cotransporters (CCCs) regulate intracellular chloride ion concentration ([Cl-]i) within neurons, which can reverse the direction of the neuronal response to the neurotransmitter GABA.1 Na+ K+ Cl- (NKCC) and K+ Cl- (KCC) cotransporters transport Cl- into or out of the cell, respectively. When NKCC activity dominates, the resulting high [Cl-]i can lead to an excitatory and depolarizing response of the neuron upon GABAA receptor opening, while KCC dominance has the opposite effect.1 This inhibitory-to-excitatory GABA switch has been linked to seasonal adaption of circadian clock function to changing day length,2-4 and its dysregulation is associated with neurodevelopmental disorders such as epilepsy.5-8 In Drosophila melanogaster, constant light normally disrupts circadian clock function and leads to arrhythmic behavior.9 Here, we demonstrate a function for CCCs in regulating Drosophila locomotor activity and GABA responses in circadian clock neurons because alteration of CCC expression in circadian clock neurons elicits rhythmic behavior in constant light. We observed the same effects after downregulation of the Wnk and Fray kinases, which modulate CCC activity in a [Cl-]i-dependent manner. Patch-clamp recordings from the large LNv clock neurons show that downregulation of KCC results in a more positive GABA reversal potential, while KCC overexpression has the opposite effect. Finally, KCC and NKCC downregulation reduces or increases morning behavioral activity during long photoperiods, respectively. In summary, our results support a model in which the regulation of [Cl-]i by a KCC/NKCC/Wnk/Fray feedback loop determines the response of clock neurons to GABA, which is important for adjusting behavioral activity to constant light and long-day conditions.

Using radio frequency identification and locomotor activity monitoring to assess sleep, locomotor, and foraging rhythmicity in bumblebees.

Bumblebees are a key pollinator. Understanding the factors that influence the timing of sleep and foraging trips is important for efficient foraging and pollination. Here, we illustrate how individual locomotor activity monitoring and colony-wide radio frequency identification tracking can be combined to analyze the effects of agrochemicals like neonicotinoids on locomotor and foraging rhythmicity and sleep quantity/quality in bumblebees. We also highlight aspects of the design that can be adapted for other invertebrates or agrochemicals, allowing broader application of these techniques. For complete details on the use and execution of this protocol, please refer to Tasman et al. (2020).

The Power of Drosophila melanogaster for Modeling Neonicotinoid Effects on Pollinators and Identifying Novel Mechanisms.

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 Drosophila ortholog of the schizophrenia-associated CACNA1A and CACNA1B voltage-gated calcium channels regulate memory, sleep and circadian rhythms.

Schizophrenia exhibits up to 80% heritability. A number of genome wide association studies (GWAS) have repeatedly shown common variants in voltage-gated calcium (Cav) channel genes CACNA1C, CACNA1I and CACNA1G have a major contribution to the risk of the disease. More recently, studies using whole exome sequencing have also found that CACNA1B (Cav2.2 N-type) deletions and rare disruptive variants in CACNA1A (Cav2.1 P/Q-type) are associated with schizophrenia. The negative symptoms of schizophrenia include behavioural defects such as impaired memory, sleep and circadian rhythms. It is not known how variants in schizophrenia-associated genes contribute to cognitive and behavioural symptoms, thus hampering the development of treatment for schizophrenia symptoms. In order to address this knowledge gap, we studied behavioural phenotypes in a number of loss of function mutants for the Drosophila ortholog of the Cav2 gene family called cacophony (cac). cac mutants showed several behavioural features including decreased night-time sleep and hyperactivity similar to those reported in human patients. The change in timing of sleep-wake cycles suggested disrupted circadian rhythms, with the loss of night-time sleep being caused by loss of cac just in the circadian clock neurons. These animals also showed a reduction in rhythmic circadian behaviour a phenotype that also could be mapped to the central clock. Furthermore, reduction of cac just in the clock resulted in a lengthening of the 24 h period. In order to understand how loss of Cav2 function may lead to cognitive deficits and underlying cellular pathophysiology we targeted loss of function of cac to the memory centre of the fly, called the mushroom bodies (MB). This manipulation was sufficient to cause reduction in both short- and intermediate-term associative memory. Memory impairment was accompanied by a decrease in Ca2+ transients in response to a depolarizing stimulus, imaged in the MB presynaptic terminals. This work shows loss of cac Cav2 channel function alone causes a number of cognitive and behavioural deficits and underlying reduced neuronal Ca2+ transients, establishing Drosophila as a high-throughput in vivo genetic model to study the Cav channel pathophysiology related to schizophrenia.

Assessing olfactory, memory, social and circadian phenotypes associated with schizophrenia in a genetic model based on Rim.

Schizophrenia shows high heritability and several of the genes associated with this disorder are involved in calcium (Ca2+) signalling and synaptic function. One of these is the Rab-3 interacting molecule-1 (RIM1), which has recently been associated with schizophrenia by Genome Wide Association Studies (GWAS). However, its contribution to the pathophysiology of this disorder remains unexplored. In this work, we use Drosophila mutants of the orthologue of RIM1, Rim, to model some aspects of the classical and non-classical symptoms of schizophrenia. Rim mutants showed several behavioural features relevant to schizophrenia including social distancing and altered olfactory processing. These defects were accompanied by reduced evoked Ca2+ influx and structural changes in the presynaptic terminals sent by the primary olfactory neurons to higher processing centres. In contrast, expression of Rim-RNAi in the mushroom bodies (MBs), the main memory centre in flies, spared learning and memory suggesting a differential role of Rim in different synapses. Circadian deficits have been reported in schizophrenia. We observed circadian locomotor activity deficits in Rim mutants, revealing a role of Rim in the pacemaker ventral lateral clock neurons (LNvs). These changes were accompanied by impaired day/night remodelling of dorsal terminal synapses from a subpopulation of LNvs and impaired day/night release of the circadian neuropeptide pigment dispersing factor (PDF) from these terminals. Lastly, treatment with the commonly used antipsychotic haloperidol rescued Rim locomotor deficits to wildtype. This work characterises the role of Rim in synaptic functions underlying behaviours disrupted in schizophrenia.

Neonicotinoids disrupt memory, circadian behaviour and sleep.

Globally, neonicotinoids are the most used insecticides, despite their well-documented sub-lethal effects on beneficial insects. Neonicotinoids are nicotinic acetylcholine receptor agonists. Memory, circadian rhythmicity and sleep are essential for efficient foraging and pollination and require nicotinic acetylcholine receptor signalling. The effect of field-relevant concentrations of the European Union-banned neonicotinoids: imidacloprid, clothianidin, thiamethoxam and thiacloprid were tested on Drosophila memory, circadian rhythms and sleep. Field-relevant concentrations of imidacloprid, clothianidin and thiamethoxam disrupted learning, behavioural rhythmicity and sleep whilst thiacloprid exposure only affected sleep. Exposure to imidacloprid and clothianidin prevented the day/night remodelling and accumulation of pigment dispersing factor (PDF) neuropeptide in the dorsal terminals of clock neurons. Knockdown of the neonicotinoid susceptible Dα1 and Dß2 nicotinic acetylcholine receptor subunits in the mushroom bodies or clock neurons recapitulated the neonicotinoid like deficits in memory or sleep/circadian behaviour respectively. Disruption of learning, circadian rhythmicity and sleep are likely to have far-reaching detrimental effects on beneficial insects in the field.

Impaired Pre-Motor Circuit Activity and Movement in a Drosophila Model of KCNMA1-Linked Dyskinesia.

BACKGROUND: Paroxysmal dyskinesias (PxDs) are characterized by involuntary movements and altered pre-motor circuit activity. Causative mutations provide a means to understand the molecular basis of PxDs. Yet in many cases, animal models harboring corresponding mutations are lacking. Here we utilize the fruit fly, Drosophila, to study a PxD linked to a gain-of-function (GOF) mutation in the KCNMA1/hSlo1 BK potassium channel. OBJECTIVES: We aimed to recreate the equivalent BK (big potassium) channel mutation in Drosophila. We sought to determine how this mutation altered action potentials (APs) and synaptic release in vivo; to test whether this mutation disrupted pre-motor circuit function and locomotion; and to define neural circuits involved in locomotor disruption. METHODS: We generated a knock-in Drosophila model using homologous recombination. We used electrophysiological recordings and calcium-imaging to assess AP shape, neurotransmission, and the activity of the larval pre-motor central pattern generator (CPG). We used video-tracking and automated systems to measure movement, and developed a genetic method to limit BK channel expression to defined circuits. RESULTS: Neuronal APs exhibited reduced width and an enhanced afterhyperpolarization in the PxD model. We identified calcium-dependent reductions in neurotransmitter release, dysfunction of the CPG, and corresponding alterations in movement, in model larvae. Finally, we observed aberrant locomotion and dyskinesia-like movements in adult model flies, and partially mapped the impact of GOF BK channels on movement to cholinergic neurons. CONCLUSION: Our model supports a link between BK channel GOF and hyperkinetic movements, and provides a platform to dissect the mechanistic basis of PxDs. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Thermoresponsive motor behavior is mediated by ring neuron circuits in the central complex of Drosophila.

Insects are ectothermal animals that are constrained in their survival and reproduction by external temperature fluctuations which require either active avoidance of or movement towards a given heat source. In Drosophila, different thermoreceptors and neurons have been identified that mediate temperature sensation to maintain the animal's thermal preference. However, less is known how thermosensory information is integrated to gate thermoresponsive motor behavior. Here we use transsynaptic tracing together with calcium imaging, electrophysiology and thermogenetic manipulations in freely moving Drosophila exposed to elevated temperature and identify different functions of ellipsoid body ring neurons, R1-R4, in thermoresponsive motor behavior. Our results show that warming of the external surroundings elicits calcium influx specifically in R2-R4 but not in R1, which evokes threshold-dependent neural activity in the outer layer ring neurons. In contrast to R2, R3 and R4d neurons, thermogenetic inactivation of R4m and R1 neurons expressing the temperature-sensitive mutant allele of dynamin, shibireTS, results in impaired thermoresponsive motor behavior at elevated 31 °C. trans-Tango mediated transsynaptic tracing together with physiological and behavioral analyses indicate that integrated sensory information of warming is registered by neural activity of R4m as input layer of the ellipsoid body ring neuropil and relayed on to R1 output neurons that gate an adaptive motor response. Together these findings imply that segregated activities of central complex ring neurons mediate sensory-motor transformation of external temperature changes and gate thermoresponsive motor behavior in Drosophila.

The Neonicotinoid Insecticide Imidacloprid Disrupts Bumblebee Foraging Rhythms and Sleep.

Neonicotinoids have been implicated in the large declines observed in insects such as bumblebees, an important group of pollinators. Neonicotinoids are agonists of nicotinic acetylcholine receptors that are found throughout the insect central nervous system and are the main mediators of synaptic neurotransmission. These receptors are important for the function of the insect central clock and circadian rhythms. The clock allows pollinators to coincide their activity with the availability of floral resources and favorable flight temperatures, as well as impact learning, navigation, and communication. Here we show that exposure to the field-relevant concentration of 10 µg/L imidacloprid caused a reduction in bumblebee foraging activity, locomotion, and foraging rhythmicity. Foragers showed an increase in daytime sleep and an increase in the proportion of activity occurring at night. This could reduce foraging and pollination opportunities, reducing the ability of the colony to grow and reproduce, endangering bee populations and crop yields.

The behavioral and neurochemical characterization of a Drosophila dysbindin mutant supports the contribution of serotonin to schizophrenia negative symptoms.

Mutations in the dystrobrevin binding protein 1 (DTNBP1) gene that encodes for the dysbindin-1 protein, are associated with a higher risk for schizophrenia. Interestingly, individuals carrying high-risk alleles in this gene have been associated with an increased incidence of negative symptoms for the disease, which include anhedonia, avolition and social withdrawal. Here we evaluated behavioral and neurochemical changes in a hypomorphic Drosophila mutant for the orthologue of human Dysbindin-1, dysb1. Mutant dysb1 flies exhibit altered social space parameters, suggesting asocial behavior, accompanied by reduced olfactory performance. Moreover, dysb1 mutant flies show poor performance in basal and startle-induced locomotor activity. We also report a reduction in serotonin brain levels and changes in the expression of the Drosophila serotonin transporter (dSERT) in dysb1 flies. Our data show that the serotonin-releasing amphetamine derivative 4-methylthioamphetamine (4-MTA) modulates social spacing and locomotion in control flies, suggesting that serotonergic circuits modulate these behaviors. 4-MTA was unable to modify the behavioral deficiencies in mutant flies, which is consistent with the idea that the efficiency of pharmacological agents acting at dSERT depends on functional serotonergic circuits. Thus, our data show that the dysb1 mutant exhibits behavioral deficits that mirror some aspects of the endophenotypes associated with the negative symptoms of schizophrenia. We argue that at least part of the behavioral aspects associated with these symptoms could be explained by a serotonergic deficit. The dysb1 mutant presents an opportunity to study the molecular underpinnings of schizophrenia negative symptoms and reveals new potential targets for treatment of the disease.

Shaw and Shal voltage-gated potassium channels mediate circadian changes in Drosophila clock neuron excitability.

As in mammals, Drosophila circadian clock neurons display rhythms of activity with higher action potential firing rates and more positive resting membrane potentials during the day. This rhythmic excitability has been widely observed but, critically, its regulation remains unresolved. We have characterized and modelled the changes underlying these electrical activity rhythms in the lateral ventral clock neurons (LNvs). We show that currents mediated by the voltage-gated potassium channels Shaw (Kv3) and Shal (Kv4) oscillate in a circadian manner. Disruption of these channels, by expression of dominant negative (DN) subunits, leads to changes in circadian locomotor activity and shortens lifespan. LNv whole-cell recordings then show that changes in Shaw and Shal currents drive changes in action potential firing rate and that these rhythms are abolished when the circadian molecular clock is stopped. A whole-cell biophysical model using Hodgkin-Huxley equations can recapitulate these changes in electrical activity. Based on this model and by using dynamic clamp to manipulate clock neurons directly, we can rescue the pharmacological block of Shaw and Shal, restore the firing rhythm, and thus demonstrate the critical importance of Shaw and Shal. Together, these findings point to a key role for Shaw and Shal in controlling circadian firing of clock neurons and show that changes in clock neuron currents can account for this. Moreover, with dynamic clamp we can switch the LNvs between morning-like and evening-like states of electrical activity. We conclude that changes in Shaw and Shal underlie the daily oscillation in LNv firing rate.

Restoration of Olfactory Memory in Drosophila Overexpressing Human Alzheimer's Disease Associated Tau by Manipulation of L-Type Ca2+ Channels.

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-associated tau alters Drosophila circadian activity, sleep and clock neuron electrophysiology.

Alzheimer's disease (AD) is the most common cause of dementia, which is associated with an enormous personal, social and economic burden worldwide. However, there are few current treatments with none of them targeting the underlying causes of the disease. Sleep and circadian rhythm defects are not only distressing symptoms of AD and other tauopathies and are a leading cause of care home admission but are also thought to accelerate AD pathology. Despite this, little is understood about the underlying causes of these behavioural changes. Expression of the 0N4R isoform of tau has been associated with AD pathology and we show that expressing it in the Drosophila clock network gives rise to circadian and sleep phenotypes which closely match the behavioural changes seen in human AD patients. Tauopathic flies exhibited greater locomotor activity throughout the day and night and displayed a loss of sleep, particularly at night. Under constant darkness, the locomotor behaviour of tau-expressing flies was less rhythmic than controls indicating a defect in their intrinsic circadian rhythm. Current clamp recordings from wake-promoting, pigment dispersing factor (PDF)-positive large lateral ventral clock neurons (l-LNvs) revealed elevated spontaneous firing throughout the day and night which likely underlies the observed hyperactive circadian phenotype. Interestingly, expression of tau in only the PDF-positive pacemaker neurons, which are thought to be the most important for behaviour under constant conditions, was not sufficient or even necessary to affect circadian rhythmicity. This work establishes Drosophila as a model to investigate interactions between human pathological versions of tau and the machinery that controls neuronal excitability, allowing the identification of underlying mechanisms of disease that may reveal new therapeutic targets.

Alzheimer's Disease Associated Genes Ankyrin and Tau Cause Shortened Lifespan and Memory Loss in Drosophila.

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 (Aß) 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 Aß. We show expression of human Tau 0N4R or the oligomerizing Aß 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 Aß42. 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.