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.

Correction to: An integrative phylogenomic approach to elucidate the evolutionary history and divergence times of Neuropterida (Insecta: Holometabola).

An amendment to this paper has been published and can be accessed via the original article.

An integrative phylogenomic approach to elucidate the evolutionary history and divergence times of Neuropterida (Insecta: Holometabola).

BACKGROUND: The latest advancements in DNA sequencing technologies have facilitated the resolution of the phylogeny of insects, yet parts of the tree of Holometabola remain unresolved. The phylogeny of Neuropterida has been extensively studied, but no strong consensus exists concerning the phylogenetic relationships within the order Neuroptera. Here, we assembled a novel transcriptomic dataset to address previously unresolved issues in the phylogeny of Neuropterida and to infer divergence times within the group. We tested the robustness of our phylogenetic estimates by comparing summary coalescent and concatenation-based phylogenetic approaches and by employing different quartet-based measures of phylogenomic incongruence, combined with data permutations. RESULTS: Our results suggest that the order Raphidioptera is sister to Neuroptera + Megaloptera. Coniopterygidae is inferred as sister to all remaining neuropteran families suggesting that larval cryptonephry could be a ground plan feature of Neuroptera. A clade that includes Nevrorthidae, Osmylidae, and Sisyridae (i.e. Osmyloidea) is inferred as sister to all other Neuroptera except Coniopterygidae, and Dilaridae is placed as sister to all remaining neuropteran families. Ithonidae is inferred as the sister group of monophyletic Myrmeleontiformia. The phylogenetic affinities of Chrysopidae and Hemerobiidae were dependent on the data type analyzed, and quartet-based analyses showed only weak support for the placement of Hemerobiidae as sister to Ithonidae + Myrmeleontiformia. Our molecular dating analyses suggest that most families of Neuropterida started to diversify in the Jurassic and our ancestral character state reconstructions suggest a primarily terrestrial environment of the larvae of Neuropterida and Neuroptera. CONCLUSION: Our extensive phylogenomic analyses consolidate several key aspects in the backbone phylogeny of Neuropterida, such as the basal placement of Coniopterygidae within Neuroptera and the monophyly of Osmyloidea. Furthermore, they provide new insights into the timing of diversification of Neuropterida. Despite the vast amount of analyzed molecular data, we found that certain nodes in the tree of Neuroptera are not robustly resolved. Therefore, we emphasize the importance of integrating the results of morphological analyses with those of sequence-based phylogenomics. We also suggest that comparative analyses of genomic meta-characters should be incorporated into future phylogenomic studies of Neuropterida.

PDXK mutations cause polyneuropathy responsive to pyridoxal 5'-phosphate supplementation.

OBJECTIVE: To identify disease-causing variants in autosomal recessive axonal polyneuropathy with optic atrophy and provide targeted replacement therapy. METHODS: We performed genome-wide sequencing, homozygosity mapping, and segregation analysis for novel disease-causing gene discovery. We used circular dichroism to show secondary structure changes and isothermal titration calorimetry to investigate the impact of variants on adenosine triphosphate (ATP) binding. Pathogenicity was further supported by enzymatic assays and mass spectroscopy on recombinant protein, patient-derived fibroblasts, plasma, and erythrocytes. Response to supplementation was measured with clinical validated rating scales, electrophysiology, and biochemical quantification. RESULTS: We identified biallelic mutations in PDXK in 5 individuals from 2 unrelated families with primary axonal polyneuropathy and optic atrophy. The natural history of this disorder suggests that untreated, affected individuals become wheelchair-bound and blind. We identified conformational rearrangement in the mutant enzyme around the ATP-binding pocket. Low PDXK ATP binding resulted in decreased erythrocyte PDXK activity and low pyridoxal 5'-phosphate (PLP) concentrations. We rescued the clinical and biochemical profile with PLP supplementation in 1 family, improvement in power, pain, and fatigue contributing to patients regaining their ability to walk independently during the first year of PLP normalization. INTERPRETATION: We show that mutations in PDXK cause autosomal recessive axonal peripheral polyneuropathy leading to disease via reduced PDXK enzymatic activity and low PLP. We show that the biochemical profile can be rescued with PLP supplementation associated with clinical improvement. As B6 is a cofactor in diverse essential biological pathways, our findings may have direct implications for neuropathies of unknown etiology characterized by reduced PLP levels. ANN NEUROL 2019;86:225-240.

Clinical, pathological and functional characterization of riboflavin-responsive neuropathy.

Brown-Vialetto-Van Laere syndrome represents a phenotypic spectrum of motor, sensory, and cranial nerve neuropathy, often with ataxia, optic atrophy and respiratory problems leading to ventilator-dependence. Loss-of-function mutations in two riboflavin transporter genes, SLC52A2 and SLC52A3, have recently been linked to Brown-Vialetto-Van Laere syndrome. However, the genetic frequency, neuropathology and downstream consequences of riboflavin transporter mutations are unclear. By screening a large cohort of 132 patients with early-onset severe sensory, motor and cranial nerve neuropathy we confirmed the strong genetic link between riboflavin transporter mutations and Brown-Vialetto-Van Laere syndrome, identifying 22 pathogenic mutations in SLC52A2 and SLC52A3, 14 of which were novel. Brain and spinal cord neuropathological examination of two cases with SLC52A3 mutations showed classical symmetrical brainstem lesions resembling pathology seen in mitochondrial disease, including severe neuronal loss in the lower cranial nerve nuclei, anterior horns and corresponding nerves, atrophy of the spinothalamic and spinocerebellar tracts and posterior column-medial lemniscus pathways. Mitochondrial dysfunction has previously been implicated in an array of neurodegenerative disorders. Since riboflavin metabolites are critical components of the mitochondrial electron transport chain, we hypothesized that reduced riboflavin transport would result in impaired mitochondrial activity, and confirmed this using in vitro and in vivo models. Electron transport chain complex I and complex II activity were decreased in SLC52A2 patient fibroblasts, while global knockdown of the single Drosophila melanogaster riboflavin transporter homologue revealed reduced levels of riboflavin, downstream metabolites, and electron transport chain complex I activity. This in turn led to abnormal mitochondrial membrane potential, respiratory chain activity and morphology. Riboflavin transporter knockdown in Drosophila also resulted in severely impaired locomotor activity and reduced lifespan, mirroring patient pathology, and these phenotypes could be partially rescued using a novel esterified derivative of riboflavin. Our findings expand the genetic, clinical and neuropathological features of Brown-Vialetto-Van Laere syndrome, implicate mitochondrial dysfunction as a downstream consequence of riboflavin transporter gene defects, and validate riboflavin esters as a potential therapeutic strategy.

Regulation of synaptic development and function by the Drosophila PDZ protein Dyschronic.

Synaptic scaffold proteins control the localization of ion channels and receptors, and facilitate molecular associations between signaling components that modulate synaptic transmission and plasticity. Here, we define novel roles for a recently described scaffold protein, Dsychronic (DYSC), at the Drosophila larval neuromuscular junction. DYSC is the Drosophila homolog of whirlin/DFNB31, a PDZ domain protein linked to Usher syndrome, the most common form of human deaf-blindness. We show that DYSC is expressed presynaptically and is often localized adjacent to the active zone, the site of neurotransmitter release. Loss of DYSC results in marked alterations in synaptic morphology and cytoskeletal organization. Moreover, active zones are frequently enlarged and misshapen in dysc mutants. Electrophysiological analyses further demonstrate that dysc mutants exhibit substantial increases in both evoked and spontaneous synaptic transmission. We have previously shown that DYSC binds to and regulates the expression of the Slowpoke (SLO) BK potassium channel. Consistent with this, slo mutant larvae exhibit similar alterations in synapse morphology, active zone size and neurotransmission, and simultaneous loss of dysc and slo does not enhance these phenotypes, suggesting that dysc and slo act in a common genetic pathway to modulate synaptic development and output. Our data expand our understanding of the neuronal functions of DYSC and uncover non-canonical roles for the SLO potassium channel at Drosophila synapses.

dyschronic, a Drosophila homolog of a deaf-blindness gene, regulates circadian output and Slowpoke channels.

Many aspects of behavior and physiology are under circadian control. In Drosophila, the molecular clock that regulates rhythmic patterns of behavior has been extensively characterized. In contrast, genetic loci involved in linking the clock to alterations in motor activity have remained elusive. In a forward-genetic screen, we uncovered a new component of the circadian output pathway, which we have termed dyschronic (dysc). dysc mutants exhibit arrhythmic locomotor behavior, yet their eclosion rhythms are normal and clock protein cycling remains intact. Intriguingly, dysc is the closest Drosophila homolog of whirlin, a gene linked to type II Usher syndrome, the leading cause of deaf-blindness in humans. Whirlin and other Usher proteins are expressed in the mammalian central nervous system, yet their function in the CNS has not been investigated. We show that DYSC is expressed in major neuronal tracts and regulates expression of the calcium-activated potassium channel SLOWPOKE (SLO), an ion channel also required in the circadian output pathway. SLO and DYSC are co-localized in the brain and control each other's expression post-transcriptionally. Co-immunoprecipitation experiments demonstrate they form a complex, suggesting they regulate each other through protein-protein interaction. Furthermore, electrophysiological recordings of neurons in the adult brain show that SLO-dependent currents are greatly reduced in dysc mutants. Our work identifies a Drosophila homolog of a deaf-blindness gene as a new component of the circadian output pathway and an important regulator of ion channel expression, and suggests novel roles for Usher proteins in the mammalian nervous system.

A review of the current state of knowledge of fossil Mantispidae (Insecta: Neuroptera).

There are 32 individual specimens of Mantispidae (Insecta: Neuroptera) currently recorded from the fossil record, the oldest of which dates back to the Lower Jurassic. These include 19 described species (in 16 genera), 1 specimen described to genus level and 9 unnamed specimens The specimens have been assigned to the extant subfamilies Drepanicinae (4), Mantispinae (10), Symphrasinae (1), and the extinct subfamily Mesomantispinae (16), with one incertae sedis within Mantispidae. There are currently no known fossil representatives of the subfamily Calomantispinae. Mesithoninae has been removed from Mantispidae and placed back within Berothidae. The species Mesithone carnaria and M. monstruosa, however, are true mantispids and have been removed from Mesithone and placed within a new genus Karataumantispa gen. nov. in the subfamily Mesomantispinae. The current state of knowledge of the fossil record of Mantispidae is reviewed and a key to the genera of Mesomantispinae is provided.

Visualizing adenosine-to-inosine RNA editing in the Drosophila nervous system.

Informational recoding by adenosine-to-inosine RNA editing diversifies neuronal proteomes by chemically modifying structured mRNAs. However, techniques for analyzing editing activity on substrates in defined neurons in vivo are lacking. Guided by comparative genomics, here we reverse-engineered a fluorescent reporter sensitive to Drosophila melanogaster adenosine deaminase that acts on RNA (dADAR) activity and alterations in dADAR autoregulation. Using this artificial dADAR substrate, we visualized variable patterns of RNA-editing activity in the Drosophila nervous system between individuals. Our results demonstrate the feasibility of structurally mimicking ADAR substrates as a method to regulate protein expression and, potentially, therapeutically repair mutant mRNAs. Our data suggest variable RNA editing as a credible molecular mechanism for mediating individual-to-individual variation in neuronal physiology and behavior.

Modulation of a critical period for motor development in Drosophila by BK potassium channels.

Critical periods are windows of heightened plasticity occurring during neurodevelopment. Alterations in neural activity during these periods can cause long-lasting changes in the structure, connectivity, and intrinsic excitability of neurons, which may contribute to the pathology of neurodevelopmental disorders. However, endogenous regulators of critical periods remain poorly defined. Here, we study this issue using a fruit fly (Drosophila) model of an early-onset movement disorder caused by BK potassium channel gain of function (BK GOF). Deploying a genetic method to place robust expression of GOF BK channels under spatiotemporal control, we show that adult-stage neuronal expression of GOF BK channels minimally disrupts fly movement. In contrast, limiting neuronal expression of GOF BK channels to a short window during late neurodevelopment profoundly impairs locomotion and limb kinematics in resulting adult flies. During this critical period, BK GOF perturbs synaptic localization of the active zone protein Bruchpilot and reduces excitatory neurotransmission. Conversely, enhancing neural activity specifically during development rescues motor defects in BK GOF flies. Collectively, our results reveal a critical developmental period for limb control in Drosophila that is influenced by BK channels and suggest that BK GOF causes movement disorders by disrupting activity-dependent aspects of synaptic development.

Fossil Neuropterida (Insecta: Neuroptera and Raphidioptera) from the middle Eocene Kishenehn Formation, Montana, USA.

The neuropterid (Neuroptera and Raphidioptera) fauna of the middle Eocene Coal Creek Member (Kishenehn Formation), U.S.A. is documented. Three families of Neuroptera (Ascalaphidae, Chrysopidae, Hemerobiidae) and two families of Raphidioptera (Raphidiidae, Inocelliidae) are recorded. Five new species and three new genera are described: Pseudoameropterus ambiguus gen. et sp. nov. (Ascalaphidae), Minimochrysa latialata gen. et sp. nov., Palaeochrysa greenwalti sp. nov., P. minor sp. nov. (Chrysopidae: Nothochrysinae), Macrostigmoraphia diluta gen. et sp. nov. (Raphidiidae). Two indeterminate species are also recorded: Megalomus-group gen. et sp. indet. (Hemerobiidae) and Neuroptera fam. gen. et sp. indet. The only previously described neuropterid from the deposit is the raphidiopteran Paraksenocellia australis Makarkin et al. 2019 (Inocelliidae). The neuropterid assemblage suggests a subtropical semi-arid climate of the Coal Creek Member.

Engineered alterations in RNA editing modulate complex behavior in Drosophila: regulatory diversity of adenosine deaminase acting on RNA (ADAR) targets.

Select proteins involved in electrical and chemical neurotransmission are re-coded at the RNA level via the deamination of particular adenosines to inosine by adenosine deaminases acting on RNA (ADARs). It has been hypothesized that this process, termed RNA editing, acts to "fine-tune" neurophysiological properties in animals and potentially downstream behavioral outputs. However, the extreme phenotypes resulting from deletions of adar loci have precluded investigations into the relationship between ADAR levels, target transcripts, and complex behaviors. Here, we engineer Drosophila hypomorphic for ADAR expression using homologous recombination. A substantial reduction in ADAR activity (>80%) leads to altered circadian motor patterns and abnormal male courtship, although surprisingly, general locomotor coordination is spared. The altered phenotypic landscape in our adar hypomorph is paralleled by an unexpected dichotomous response of ADAR target transcripts, i.e. certain adenosines are minimally affected by dramatic ADAR reduction, whereas editing of others is severely curtailed. Furthermore, we use a novel reporter to map RNA editing activity across the nervous system, and we demonstrate that knockdown of editing in fruitless-expressing neurons is sufficient to modify the male courtship song. Our data demonstrate that network-wide temporal and spatial regulation of ADAR activity can tune the complex system of RNA-editing sites and modulate multiple ethologically relevant behavioral modalities.

Sleep: Astrocytes Take Their Toll on Tired Flies.

Calcium signalling in astrocytes modulates sleep, yet how astrocytes communicate with neural circuits that control sleep is unclear. A new study now uncovers a calcium-dependent relay between astrocytes and neurons that promotes sleep homeostasis in fruit flies.

Adenosine-to-inosine genetic recoding is required in the adult stage nervous system for coordinated behavior in Drosophila.

Adenosine deaminases acting on RNA (ADARs) catalyze the deamination of adenosine to inosine in double-stranded RNA templates, a process known as RNA editing. In Drosophila, multiple ADAR isoforms are generated from a single locus (dAdar) via post-transcriptional modifications. Collectively, these isoforms act to edit a wide range of transcripts involved in neuronal signaling, as well as the precursors of endogenous small interfering RNAs. The phenotypic consequences of a loss of dADAR activity have been well characterized and consist of profound behavioral defects manifested at the adult stage, including extreme uncoordination, seizures, and temperature-sensitive paralysis. However, the spatio-temporal requirements of adenosine to inosine editing for correct behavior are unclear. Using transgenic RNA interference, we show that network-wide editing in the nervous system is required for normal adult locomotion. Regulated restoration of editing activity demonstrates that the neuronal requirement of dADAR activity has a significant adult stage component. Furthermore we show that in relation to behavior there are no observable genetic interactions between dAdar and several loci encoding RNA interference components, suggesting that editing of neuronal transcripts is the key mode of ADAR activity for normal behavior in Drosophila.

Antagonistic Regulation of Circadian Output and Synaptic Development by JETLAG and the DYSCHRONIC-SLOWPOKE Complex.

Circadian output genes act downstream of the clock to promote rhythmic changes in behavior and physiology, yet their molecular and cellular functions are not well understood. Here we characterize an interaction between regulators of circadian entrainment, output, and synaptic development in Drosophila that influences clock-driven anticipatory increases in morning and evening activity. We previously showed the JETLAG (JET) E3 ubiquitin ligase resets the clock upon light exposure, whereas the PDZ protein DYSCHRONIC (DYSC) regulates circadian locomotor output and synaptic development. Surprisingly, we find that JET and DYSC antagonistically regulate synaptic development at the larval neuromuscular junction, and reduced JET activity rescues arrhythmicity of dysc mutants. Consistent with our prior finding that DYSC regulates SLOWPOKE (SLO) potassium channel expression, jet mutations also rescue circadian and synaptic phenotypes in slo mutants. Collectively, our data suggest that JET, DYSC, and SLO promote circadian output in part by regulating synaptic morphology.

RNA editing in regulating gene expression in the brain.

Adenosine to inosine RNA editing, catalyzed by Adenosine Deaminases Acting on RNA (ADARs), represents an evolutionary conserved post-transcriptional mechanism which harnesses RNA structures to produce proteins that are not literally encoded in the genome. The species-specific alteration of functionally important residues in a multitude of neuronal ion channels and pre-synaptic proteins through RNA editing has been shown to have profound importance for normal nervous system function in a wide range of invertebrate and vertebrate model organisms. ADARs have also been shown to regulate neuronal gene expression through a remarkable variety of disparate processes, including modulation of the RNAi pathway, the creation of alternative splice sites, and the abolition of stop codons. In addition, ADARs have recently been revealed to have a novel role in the primate lineage: the widespread editing of Alu elements, which comprise approximately 10% of the human genome. Thus, as well as enabling the cell-specific regulation of RNAi and selfish genetic elements, the unshackling of the proteome from the constraints of the genome through RNA editing may have been fundamental to the evolution of complex behavior.

Mechanisms of Neurological Dysfunction in GOSR2 Progressive Myoclonus Epilepsy, a Golgi SNAREopathy.

Successive fusion events between transport vesicles and their target membranes mediate trafficking of secreted, membrane- and organelle-localised proteins. During the initial steps of this process, termed the secretory pathway, COPII vesicles bud from the endoplasmic reticulum (ER) and fuse with the cis-Golgi membrane, thus depositing their cargo. This fusion step is driven by a quartet of SNARE proteins that includes the cis-Golgi t-SNARE Membrin, encoded by the GOSR2 gene. Mis-sense mutations in GOSR2 result in Progressive Myoclonus Epilepsy (PME), a severe neurological disorder characterised by ataxia, myoclonus and seizures in the absence of significant cognitive impairment. However, given the ubiquitous and essential function of ER-to-Golgi transport, why GOSR2 mutations cause neurological dysfunction and not lethality or a broader range of developmental defects has remained an enigma. Here we highlight new work that has shed light on this issue and incorporate insights into canonical and non-canonical secretory trafficking pathways in neurons to speculate as to the cellular and molecular mechanisms underlying GOSR2 PME. This article is part of a Special Issue entitled: SNARE proteins: a long journey of science in brain physiology and pathology: from molecular.

Small Animal Video Tracking for Activity and Path Analysis Using a Novel Open-Source Multi-Platform Application (AnimApp).

Experimental biological model system outcomes such as altered animal movement capability or behaviour are difficult to quantify manually. Existing automatic movement tracking devices can be expensive and imposing upon the typical environment of the animal model. We have developed a novel multiplatform, free-to-use open-source application based on OpenCV, called AnimApp. Our results show that AnimApp can reliably and reproducibly track movement of small animals such as rodents or insects, and quantify parameters of action including distance and speed in order to detect activity changes arising from handling, environment enrichment, or temperature alteration. This system offers an accurate and reproducible experimental approach with potential for simple, fast and flexible analysis of movement and behaviour in a wide range of model systems.

A substitute name for a genus of fossil mantispid (Insecta: Neuroptera: Mesomantispinae) from the Jurassic of Kazakhstan.

In a recent paper (Jepson et al., 2018) a new genus of Mesomantispinae from Karatau, Kazakhstan was described. The name given to the genus, Longicollum, is unavailable, because it is preoccupied by a senior homonym, Longicollum Yamaguti, 1935 (Acanthocephala: Pomphorhynchidae). In accordance with Art. 60.1. of the ICZN, we herein propose a replacement name for the genus: Longipronotum nom. nov.

New Mesomantispinae (Insecta: Neuroptera: Mantispidae) from the Jurassic of Karatau, Kazakhstan.

Two new genera and species, and one indeterminate genera and species of fossil Mantispidae, Mesomantispinae are described from the Upper Jurassic of Karatau, Kazakhstan: Longicollum benmaddoxi gen. et sp. nov., Ovalofemora abbottae gen. et sp. nov., and Mesomantispinae sp. et gen. indet. Karataumantispa monstruosa is removed from the genus Karataumantispa and placed in the new genus Ovalofemora gen. nov. due to its different foreleg morphology (stout coxae, trochanter, and oval shaped femur), in addition to differences in wing venation. These taxa are all placed within the subfamily Mesomantispinae. An updated key to the genera of Mesomantispinae is given.