high-throughput-small-molecule-drug-screening-for-age-related-sleep

High-Throughput Small Molecule Drug Screening For Age-Related Sleep Disorders Using <em>Drosophila melanogaster</em>

Sleep, an essential component of health and overall well-being, often presents challenges for older individuals who frequently experience sleep disorders ...

Southeast University

The Drosophila visual transduction is the fastest known G protein-coupled signaling cascade and has been served as a model for understanding the molecular mechanisms of other G protein-coupled signaling cascades. Numbers of components in visual transduction machinery have been identified. Based on the functional characterization of these genes, a model for Drosophila phototransduction has been outlined, including rhodopsin activation, phosphoinoside signaling, and the opening of TRP and TRPL channels. Recently, the characterization of mutants, showing slow termination, revealed the physiological significance and the mechanism of rapid termination of light response.

Phototransduction in Drosophila.

Neurexins are cell adhesion molecules involved in synapse formation and synaptic regulation. Mutations in the neurexin genes are linked to a number of neurodevelopmental disorders such as autism. Here, we show that the Drosophila homolog of α-Neurexin is critical for fly visual function. Lack of Neurexin leads to significantly impaired visual function due to reduced rhodopsin levels. We show that the decreased chromophore levels cause deficits in rhodopsin maturation and that Neurexin is required for retinoid transport. Using yeast two-hybrid screening, we identify that Neurexin interacts with apolipoprotein I (ApoL I), a product generated by cleavage of retinoid- and fatty acid-binding glycoprotein (RFABG) that functions in retinoid transport. Finally, we demonstrate that Neurexin is essential for the apolipoproteins level. Our results reveal a role for Neurexin in mediating retinoid transport and subsequent rhodopsin maturation and suggest that Neurexin regulates lipoprotein function.

Neurexin regulates visual function via mediating retinoid transport to promote rhodopsin maturation.

Although Neurexins, which are cell adhesion molecules localized predominantly to the presynaptic terminals, are known to regulate synapse formation and synaptic transmission, their roles in the regulation of synaptic vesicle release during repetitive nerve stimulation are unknown. Here, we show that nrx mutant synapses exhibit rapid short term synaptic depression upon tetanic nerve stimulation. Moreover, we demonstrate that the intracellular region of NRX is essential for synaptic vesicle release upon tetanic nerve stimulation. Using a yeast two-hybrid screen, we find that the intracellular region of NRX interacts with N-ethylmaleimide-sensitive factor (NSF), an enzyme that mediates soluble NSF attachment protein receptor (SNARE) complex disassembly and plays an important role in synaptic vesicle release. We further map the binding sites of each molecule and demonstrate that the NRX/NSF interaction is critical for both the distribution of NSF at the presynaptic terminals and SNARE complex disassembly. Our results reveal a previously unknown role of NRX in the regulation of short term synaptic depression upon tetanic nerve stimulation and provide new mechanistic insights into the role of NRX in synaptic vesicle release.

The Neurexin/N-Ethylmaleimide-sensitive Factor (NSF) Interaction Regulates Short Term Synaptic Depression.

Columnar restriction of neurites is critical for forming nonoverlapping receptive fields and preserving spatial sensory information from the periphery in both vertebrate and invertebrate nervous systems, but the underlying molecular mechanisms remain largely unknown. Here, we demonstrate that Drosophila homolog of α-neurexin (DNrx) plays an essential role in columnar restriction during L4 axon branching. Depletion of DNrx from L4 neurons resulted in misprojection of L4 axonal branches into neighboring columns due to impaired ephrin clustering. The proper ephrin clustering requires its interaction with the intracellular region of DNrx. Furthermore, we find that Drosophila neuroligin 4 (DNlg4) in Tm2 neurons binds to DNrx and initiates DNrx clustering in L4 neurons, which subsequently induces ephrin clustering. Our study demonstrates that DNrx promotes ephrin clustering and reveals that ephrin/Eph signaling from adjacent L4 neurons restricts axonal branches of L4 neurons in columns.

Neurexin Restricts Axonal Branching in Columns by Promoting Ephrin Clustering.

In the past decade, numerous genes associated with autism spectrum disorders (ASDs) have been identified. These genes encode key regulators of synaptogenesis, synaptic function, and synaptic plasticity. Drosophila is a prominent model system for ASD studies to define novel genes linked to ASDs and decipher their molecular roles in synaptogenesis, synaptic function, synaptic plasticity, and neural circuit assembly and consolidation. Here, we review Drosophila studies on ASD genes that regulate synaptogenesis, synaptic function, and synaptic plasticity through modulating chromatin remodeling, transcription, protein synthesis and degradation, cytoskeleton dynamics, and synaptic scaffolding.

Drosophila Studies on Autism Spectrum Disorders.

The functions of autism-associated Neuroligins (Nlgs) are modulated by their post-translational modifications, such as proteolytic cleavage. A previous study has shown that there are different endogenous forms of DNlg3 in Drosophila, indicating it may undergo proteolytic processing. However, the molecular mechanism underlying DNlg3 proteolytic processing is unknown. Here, we report a novel proteolytic mechanism that is essential for DNlg3 maturation and function in the nervous system.

Proteolytic maturation of Drosophila Neuroligin 3 by tumor necrosis factor α-converting enzyme in the nervous system.

Light-induced endocytosis of rhodopsin in the retina is critical for preventing photoreceptor hyperactivity and for the survival of photoreceptor cells. In , this process is mediated by arrestin1 (Arr1). Because Arr1 lacks a clathrin-binding domain required for receptor internalization and the C-terminal sequence that interacts with the β-subunit of the clathrin adaptor protein AP2, the mechanism of how Arr1 mediates endocytosis of the major rhodopsin Rh1 is unclear. Here, using several approaches, including Arr binding and pulldown assays, immunofluorescence techniques, and EM imaging, we found that  metallophosphoesterase (dMPPE) is involved in light-induced rhodopsin endocytosis. We observed that the photoreceptor cells of a  mutant exhibit impaired light-induced rhodopsin endocytosis and that this impairment is independent of dMPPE phosphoesterase activity. Furthermore, dMPPE directly interacted with Arr1 and promoted the association of Arr1 with AP2. Of note, genetic  deletion largely prevented retinal degeneration in  (encoding phospholipase C) mutants, which were reported previously to contribute to retinal degeneration, by suppressing Rh1 endocytosis. Our findings demonstrate that Arr1 interacts with AP2 and that dMPPE functions as a critical regulator in Rh1 endocytosis and retinal degeneration.

Metallophosphoesterase regulates light-induced rhodopsin endocytosis by promoting an association between arrestin and the adaptor protein AP2.

Heterotrimeric G proteins mediate a variety of signaling processes by coupling G protein-coupled receptors to intracellular effector molecules. In  the α gene encodes several Gαq splice variants, with the Gαq1 isoform protein playing a major role in fly phototransduction. However, α null mutant flies still exhibit a residual light response, indicating that other Gαq splice variants or additional Gq α subunits are involved in phototransduction. Here, we isolated a mutant fly with no detectable light responses, decreased rhodopsin (Rh) levels, and rapid retinal degeneration. Using electrophysiological and genetic studies, biochemical assays, immunoblotting, real-time RT-PCR, and EM analysis, we found that mutations in the α gene disrupt light responses and demonstrate that the Gαq3 isoform protein is responsible for the residual light response in α null mutants. Moreover, we report that Gαq3 mediates rhodopsin synthesis. Depletion of all Gαq splice variants led to rapid light-dependent retinal degeneration, due to the formation stable Rh1-arrestin 2 (Arr2) complexes. Our findings clarify essential roles of several different Gαq splice variants in phototransduction and retinal integrity in  and reveal that Gαq3 functions in rhodopsin synthesis.

Gαq splice variants mediate phototransduction, rhodopsin synthesis, and retinal integrity in .

Sleep is an essential and evolutionarily conserved behavior that is modulated by many environmental factors. Ambient temperature shifting usually occurs during climatic or seasonal change or travel from high-latitude area to low-latitude area that affects animal physiology. Increasing ambient temperature modulates sleep in both humans and Drosophila. Although several thermosensory molecules and neurons have been identified, the neural mechanisms that integrate temperature sensation into the sleep neural circuit remain poorly understood. Here, we reveal that prolonged increasing of ambient temperature induces a reversible sleep reduction and impaired sleep consolidation in Drosophila via activating the internal thermosensory anterior cells (ACs). ACs form synaptic contacts with a subset of posterior dorsal neuron 1 (DN1p) neurons and release acetylcholine to promote wakefulness. Furthermore, we identify that this subset of DN1ps promotes wakefulness by releasing CNMamide (CNMa) neuropeptides to inhibit the Dh44-positive pars intercerebralis (PI) neurons through CNMa receptors. Our study demonstrates that the AC-DN1p-PI neural circuit is responsible for integrating thermosensory inputs into the sleep neural circuit. Moreover, we identify the CNMa signaling pathway as a newly recognized wakefulness-promoting DN1 pathway.

A subset of DN1p neurons integrates thermosensory inputs to promote wakefulness via CNMa signaling.

 phototransduction is a well-established model for characterizing biological processes, such as calcium signaling. Here, we present a protocol for electroretinogram (ERG) recording, an  physiological assay extensively used in  phototransduction research to measure light-evoked field potential responses of photoreceptors and neurons in the lamina neuropil. We describe fly preparation and electrode placement, followed by pretest setup, ERG recording, and data analysis. This protocol enables assessment of photoreceptor performance and visual synaptic transmission between photoreceptors and downstream lamina neurons. For complete details on the use and execution of this protocol, please refer to Han et al. (2006), Hu et al. (2012), and Wu et al. (2021).

Protocol for electroretinogram recording of the  compound eye.

In Drosophila, mutations in genes that prevent normal Ca influx after light stimulation usually cause light-dependent retinal degeneration or neurodegeneration, detectable by defects in eye morphology. Here, we present a protocol based on electron microscopy (EM) to observe the morphological structure of photoreceptor cells in Drosophila. We detail how to fix, dehydrate, embed, and polymerize compound eye samples, followed by sectioning, post-staining, and image acquisition, to assess the eye morphology at the ultrastructural level. For complete details on the use and execution of this protocol, please refer to Gu et al. (2020).

Protocol for electron microscopy of Drosophila photoreceptor cells.

Sleep is a fundamental, evolutionarily conserved, plastic behavior that is regulated by circadian and homeostatic mechanisms as well as genetic factors and environmental factors, such as light, humidity, and temperature. Among environmental cues, temperature plays an important role in the regulation of sleep. This review presents an overview of thermoreception in animals and the neural circuits that link this process to sleep. Understanding the influence of temperature on sleep can provide insight into basic physiologic processes that are required for survival and guide strategies to manage sleep disorders.

Article	Year
Phototransduction in Drosophila. Science China. Life sciences\| PubMed ID: 22314488	2012
Neurexin regulates visual function via mediating retinoid transport to promote rhodopsin maturation. Neuron\| PubMed ID: 23352167	2013
The Neurexin/N-Ethylmaleimide-sensitive Factor (NSF) Interaction Regulates Short Term Synaptic Depression. The Journal of biological chemistry\| PubMed ID: 25953899	2015
Neurexin Restricts Axonal Branching in Columns by Promoting Ephrin Clustering. Developmental cell\| PubMed ID: 28366281	2017
Drosophila Studies on Autism Spectrum Disorders. Neuroscience bulletin\| PubMed ID: 28795356	2017
Proteolytic maturation of Drosophila Neuroligin 3 by tumor necrosis factor α-converting enzyme in the nervous system. Biochimica et biophysica acta. General subjects\| PubMed ID: 29107812	2018
Metallophosphoesterase regulates light-induced rhodopsin endocytosis by promoting an association between arrestin and the adaptor protein AP2. The Journal of biological chemistry\| PubMed ID: 31324721	2019
Gαq splice variants mediate phototransduction, rhodopsin synthesis, and retinal integrity in . The Journal of biological chemistry\| PubMed ID: 32198182	2020
A subset of DN1p neurons integrates thermosensory inputs to promote wakefulness via CNMa signaling. Current biology : CB\| PubMed ID: 33740429	2021
Protocol for electroretinogram recording of the compound eye. STAR protocols\| PubMed ID: 35463470	2022
Protocol for electron microscopy of Drosophila photoreceptor cells. STAR protocols\| PubMed ID: 35776650	2022
How Temperature Influences Sleep. International journal of molecular sciences\| PubMed ID: 36293048	2022

Yao Tian

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