Belly roll, a GPI-anchored Ly6 protein, regulates Drosophila melanogaster escape behaviors by modulating the excitability of nociceptive peptidergic interneurons.

Appropriate modulation of escape behaviors in response to potentially damaging stimuli is essential for survival. Although nociceptive circuitry has been studied, it is poorly understood how genetic contexts affect relevant escape responses. Using an unbiased genome-wide association analysis, we identified an Ly6/α-neurotoxin family protein, Belly roll (Bero), which negatively regulates Drosophila nociceptive escape behavior. We show that Bero is expressed in abdominal leucokinin-producing neurons (ABLK neurons) and bero knockdown in ABLK neurons resulted in enhanced escape behavior. Furthermore, we demonstrated that ABLK neurons responded to activation of nociceptors and initiated the behavior. Notably, bero knockdown reduced persistent neuronal activity and increased evoked nociceptive responses in ABLK neurons. Our findings reveal that Bero modulates an escape response by regulating distinct neuronal activities in ABLK neurons.

Computational model predicts the neural mechanisms of prepulse inhibition in Drosophila larvae.

Prepulse inhibition (PPI) is a behavioural phenomenon in which a preceding weaker stimulus suppresses the startle response to a subsequent stimulus. The effect of PPI has been found to be reduced in psychiatric patients and is a promising neurophysiological indicator of psychiatric disorders. Because the neural circuit of the startle response has been identified at the cellular level, investigating the mechanism underlying PPI in Drosophila melanogaster larvae through experiment-based mathematical modelling can provide valuable insights. We recently identified PPI in Drosophila larvae and found that PPI was reduced in larvae mutated with the Centaurin gamma 1A (CenG1A) gene, which may be associated with autism. In this study, we used numerical simulations to investigate the neural mechanisms underlying PPI in Drosophila larvae. We adjusted the parameters of a previously developed Drosophila larvae computational model and demonstrated that the model could reproduce several behaviours, including PPI. An analysis of the temporal changes in neuronal activity when PPI occurs using our neural circuit model suggested that the activity of specific neurons triggered by prepulses has a considerable effect on PPI. Furthermore, we validated our speculations on PPI reduction in CenG1A mutants with simulations.

Knockdown of Golgi Stress-Responsive Caspase-2 Ameliorates HLD17-Associated AIMP2 Mutant-Mediated Inhibition of Oligodendroglial Cell Morphological Differentiation.

Hypomyelinating leukodystrophy 17 is an autosomal recessive disease affecting myelin-forming oligodendroglial cells in the central nervous system. The gene responsible for HLD17 encodes aminoacyl-tRNA synthase complex-interacting multifunctional protein 2, whose product proteins form a scaffold that supports aminoacyl-tRNA synthetases throughout the cell body. Here we show that the HLD17-associated nonsense mutation (Tyr35-to-Ter [Y35X]) of AIMP2 localizes AIMP2 proteins as aggregates into the Golgi bodies in mouse oligodendroglial FBD-102b cells. Wild type AIMP2 proteins, in contrast, are distributed throughout the cell body. Expression of the Y35X mutant proteins, but not the wild type proteins, in cells upregulates Golgi stress signaling involving caspase-2 activation. Cells expressing the wild type proteins exhibit differentiated phenotypes with web-like structures bearing many processes following the induction of differentiation, whereas cells expressing the Y35X mutant proteins fail to differentiate. Furthermore, CASP2 knockdown but not control knockdown reverses the phenotypes of cells expressing the mutant proteins. These results suggest that HLD17-associated AIMP2 mutant proteins are localized in the Golgi bodies where their proteins stimulate Golgi stress-responsive CASP2 to inhibit differentiation; this effect is ameliorated by knockdown of CASP2. These findings may reveal some of the molecular and cellular pathological mechanisms underlying HLD17 and possible approaches to ameliorating the disease's effects.

Phospholipase D and phosphatidylinositol-4-phosphate 5-kinase 1 are involved in the regulation of oligodendrocyte morphological differentiation.

Oligodendroglial cells (oligodendrocytes) differentiate to form the myelin that wraps neuronal axons in the central nervous system (CNS). This myelin sheath supports the propagation of saltatory conduction and protects axons from physical stresses. When oligodendrocytes do not normally differentiate to myelinate axons, their key functions as oligodendrocytes in the CNS are severely impaired. The molecular mechanics that control differentiation still remain to be clarified. Arf6 belongs to the small GTPase family and is known to be a positive regulator of oligodendrocyte differentiation. Here, we show that the phospholipase D (PLD) and phosphatidylinositol-4-phosphate 5-kinase 1 (PIP5K1) molecules, the major effectors of Arf6, are involved in the regulation of oligodendrocyte differentiation. Knockdown of PLD1 or PIP5K type 1γ (PIP5K1C) by their respective specific siRNAs in mouse oligodendroglial FBD-102b cells inhibited morphological differentiation into structures bearing myelin-like processes; this finding is consistent with the concurrent changes in expression of differentiation and myelin marker proteins. Treatment with VU0155069 or UNC3230, specific inhibitors of PLD and PIP5K1, respectively, blunted morphological differentiation and decreased expression of myelin and differentiation marker proteins. Similar results have been obtained in studies using primary oligodendrocytes. These results suggest that the major Arf6 effector molecules PLD and PIP5K1 are among the molecules involved in the regulation of morphological differentiation in oligodendrocytes prior to myelination.

Spectral response properties of higher visual neurons in Drosophila melanogaster.

The fruit fly Drosophila melanogaster can process chromatic information for true color vision and spectral preference. Spectral information is initially detected by a few distinct photoreceptor channels with different spectral sensitivities and is processed through the visual circuit. The neuroanatomical bases of the circuit are emerging. However, only little information is available in chromatic response properties of higher visual neurons from this important model organism. We used in vivo whole-cell patch-clamp recordings in response to monochromatic light stimuli ranging from 300 to 650 nm with 25-nm steps. We characterized the chromatic response of 33 higher visual neurons, including their general response type and their wavelength tuning. Color-opponent-type responses that had been typically observed in primates and bees were not identified. Instead, the majority of neurons showed excitatory responses to broadband wavelengths. The UV (300-375 nm) and middle wavelength (425-575 nm) ranges could be separated at the population level owing to neurons that preferentially responded to a specific wavelength range. Our results provide a first mapping of chromatic information processing in higher visual neurons of D. melanogaster that is a suitable model for exploring how color-opponent neural mechanisms are implemented in the visual circuits.

Dopamine modulates the optomotor response to unreliable visual stimuli in Drosophila melanogaster.

State-dependent modulation of sensory systems has been studied in many organisms and is possibly mediated through neuromodulators such as monoamine neurotransmitters. Among these, dopamine is involved in many aspects of animal behaviour, including movement control, attention, motivation and cognition. However, the precise neural mechanism underlying dopaminergic modulation of behaviour induced by sensory stimuli remains poorly understood. Here, we used Drosophila melanogaster to show that dopamine can modulate the optomotor response to moving visual stimuli including noise. The optomotor response is the head-turning response to moving objects, which is observed in most sight-reliant animals including mammals and insects. First, the effects of the dopamine system on the optomotor response were investigated in mutant flies deficient in dopamine receptors D1R1 or D1R2, which are involved in the modulation of sleep-arousal in flies. We examined the optomotor response in D1R1 knockout (D1R1 KO) and D1R2 knockout (D1R2 KO) flies and found that it was not affected in D1R1 KO flies; however, it was significantly reduced in D1R2 KO flies compared with the wild type. Using cell-type-specific expression of an RNA interference construct of D1R2, we identified the fan-shaped body, a part of the central complex, responsible for dopamine-mediated modulation of the optomotor response. In particular, pontine cells in the fan-shaped body seemed important in the modulation of the optomotor response, and their neural activity was required for the optomotor response. These results suggest a novel role of the central complex in the modulation of a behaviour based on the processing of sensory stimulations.

Data on the effects of Charcot-Marie-Tooth disease type 2N-associated AARS missense mutation (Arg329-to-His) on the cell biological properties.

Charcot-Marie-Tooth (CMT) diseases are genetic neuropathies in the peripheral nervous system (PNS). Type 1 CMT diseases are neuropathies in Schwann cells, PNS myelinating glial cells, whereas type 2 CMT diseases are axonal neuropathies. In addition, there are other types of categories in CMT diseases. CMT diseases are associated with approximately 100 responsible genes. Taiwanese mutation (Asn71-to-Tyr) of alanyl-tRNA synthetase (AARS) in type 2N CMT disease has been reported to have several pathological effects on properties of AARS proteins themselves [1]. Also, some mutations in other responsible genes affect cell biological properties of their gene products [2,3]. Herein we provide the data regarding the effects of another type 2N CMT disease-associated AARS mutation (Arg329-to-His) in French family on the cellular properties.

Prepulse inhibition in Drosophila melanogaster larvae.

The neural mechanisms of psychiatric diseases like autism spectrum disorder and schizophrenia have been intensively studied, and a number of candidate genes have been identified. However, the relationship between genes and neural system functioning remains unclear. Model organisms may serve as a powerful tool for addressing this question due to the availability of established genetic tools. Here, we report prepulse inhibition (PPI) in Drosophila larvae for the first time. PPI is a neurological phenomenon found in humans and other organisms and is used in the diagnosis of schizophrenia and other psychiatric disorders. A weaker prestimulus (prepulse) inhibits the reaction to a subsequent strong, startling stimulus (pulse). Using the larval startle response to the buzz of a predator (wasp), we examined PPI in wild-type flies and two mutants: an fmr1 mutant, which is implicated in Fragile X syndrome, and a centaurin gamma 1A (CenG1A) mutant, which is associated with GTPase, PH, ArfGAP, and ANK domains and implicated in autism. Both mutants showed decreased PPI, whereas, interestingly, double mutants showed substantial PPI. The PPI phenomenon described here can provide a useful tool for the study of neural mechanisms of synaptic modification and psychiatric diseases.

Pull down assay for GTP-bound form of Sar1a reveals its activation during morphological differentiation.

The intracellular molecular transport system is a basic and general cellular mechanism that is regulated by an array of signaling molecules. Sar1 small GTPases are molecules that play a key role in controlling vehicle transport between the endoplasmic reticulum (ER) and Golgi bodies. Like other small GTPases, the activities of Sar1a depend on their guanine-nucleotide-binding states, which are regulated by guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Despite the well-known function of mammalian Sar1 in the intracellular transport system, little is known about when and how Sar1 is activated during cell morphological changes. Here we show that the C-terminal, but not the N-terminal, regions of Sec23A and Sec23B, the effector proteins of Sar1a, specifically bind to the active, GTP-bound form of Sar1a. An affinity precipitation (pull-down) assay using a recombinant C-terminal region of Sec23B reveals that Sar1a is activated following differentiation in neuronal cell lines. In neuronal N1E-115 cells, GTP-bound Sar1a is increased when cells elongate neuronal processes. Similar results are observed in morphological differentiation in oligodendroglial FBD-102b cells. Additionally, prolactin regulatory element binding (PREB), the GEF for Sar1 (Sar1 activator), increases the binding ability to the nucleotide-free form of Sar1a when morphological differentiation occurs. Nucleotide-free small GTPases preferentially interact with the cognate, active GEFs. These results provide evidence that using previously unreported pull down assays reveals that Sar1 and PREB are upregulated following the induction of morphological differentiation, suggesting the potential role of signaling through Sar1a during morphological differentiation.

Data on the effect of knockout of neruregulin-1 type III on Remak bundle structure.

Schwann cells in the peripheral nervous system wrap around large diameter axons to form the myelin sheath, that contains one axon. Schwann cells also wrap around small diameter axons to form the Remak bundle, that contains many axons. Neuregulin-1 (NRG1) type III binds Schwann cell plasma membrane ErbB2/3 receptor to regulate morphological changes of Schwann cells. Herein we provide the data on the effect of NRG1 type III knockout (Miyamoto et al., 2017) [1] on the Remak bundle structure. Since complete knockout mice of NRG1type III are embryonically lethal, we have usedNRG1type III (+/-) mice's sciatic nerves in these experiments.

Treacher Collins syndrome 3 (TCS3)-associated POLR1C mutants are localized in the lysosome and inhibits chondrogenic differentiation.

Treacher Collins syndrome (TCS) is a craniofacial developmental disorder whose key feature is a combination of symptoms. For example, a patient could have bilateral downward slanting of the palpebral fissures, colobomas of the lower eyelids, hypoplasia of the facial bones, cleft palate, malformation of the external ears, and atresia of the external auditory canals. TCS3 is caused by mutations of the polr1c gene, which encodes RNA polymerase I and III subunit C (POLR1C). There have been two known missense mutations (Arg279-to-Gln [R279Q] and Arg279-to-Trp [R279W]) at the Arg-279 position. However, it remains to be clarified whether or how both or each individual mutation affects the cellular properties of POLR1C. Here we show that TCS3-associated missense mutations cause aberrant intracellular localization of POLR1C, inhibiting chondrogenic differentiation. The wild type POLR1C is normally localized in the nuclei. The R279Q or R279W mutant is primarily found to be localized in the lysosome. Expression of the R279Q or R279W mutant in mouse chondrogenic ATDC5 cells decreases phosphorylation of 4E-BP1 and ribosomal S6 proteins, which belong to the mammalian target of rapamycin (mTOR) signaling involved in critical roles in the lysosome. Furthermore, expression of the R279Q or R279W mutant inhibits chondrogenic differentiation in ATDC5 cells. Taken together, TCS3-associated mutation leads to the localization of POLR1C into the lysosome and inhibits chondrogenic differentiation, possibly explaining a portion of the pathological molecular basis underlying Treacher Collins syndrome.

Dystonia-4 (DYT4)-associated TUBB4A mutants exhibit disorganized microtubule networks and inhibit neuronal process growth.

Dystonia-1 (DYT1) is an autosomal dominant early-onset torsion form of dystonia, a neurological disease affecting movement. DYT1 is the prototypic hereditary dystonia and is caused by the mutation of the tor1a gene. The gene product has chaperone functions important for the control of protein folding and stability. Dystonia-4 (DYT4) is another autosomal dominant dystonia that is characterized by onset in the second to third decade of progressive laryngeal dysphonia. DYT4 is associated with the mutation of the tubb4a gene, although it remains to be understood how disease-associated mutation affects biochemical as well as cell biological properties of the gene product as the microtubule component (a tubulin beta subunit). Herein we demonstrate that DYT4-associated TUBB4A missense mutants (Arg2-to-Gly or Ala271-to-Thr) form disorganized tubulin networks in cells. Transfected mutants are indeed expressed in cytoplasmic regions, as observed in wild-type transfectants. However, mutant proteins do not exhibit typical radial tubulin networks. Rather, they have diminished ability to interact with tubulin alpha subunits. Processes do not form in sufficient amounts in cells of the N1E-115 neuronal cell line expressing each of these mutants as compared to parental cells. Together, DYT4-associated TUBB4A mutants themselves form aberrant tubulin networks and inhibit neuronal process growth, possibly explaining progress through the pathological states at cellular levels.

Data on the effect of knockout of cytohesin-1 in myelination-related protein kinase signaling.

Cytohesin-1 is the guanine-nucleotide exchange factor of Arf6, a small GTPase of Arf family, and participates in cellular morphological changes. Knockout mice of cytohesin-1 exhibit decreased myelination of neuronal axons in the peripheral nervous system (PNS) "Phosphorylation of cytohesin-1 by Fyn is required for initiation of myelination and the extent of myelination during development (Yamauchi et al., 2012) [1]". Herein we provide the data regarding decreased phosphorylation levels of protein kinases involved in two major myelination-related kinase cascades in cytohesin-1 knockout mice.

Data on the effect of in vivo knockdown using artificial ErbB3 miRNA on Remak bundle structure.

Mature Schwann cells, the peripheral nervous system (PNS) glial cells, have two major roles for neuronal axons (Bunge, 1993) [1]. For large diameter axons, Schwann cells form myelin sheaths with multiple layers. For small diameter axons, they form Remak bundle composed only of single layer of the Schwann cell plasma membrane. In the PNS, ErbB3 forms a dimer with ErbB2 on the Schwann cell plasma membrane. ErbB3 plays a key role in myelination by myelinating Schwann cells, that is to say, its role in myelin thickness. Herein we provide the data regarding the effect of in vivo knockdown of ErbB3 on the thickness between an axon and a neighboring axon in Remak bundle, which is formed by non-myelinating Schwann cells. Since ErbB3 knockout mice are embryonically lethal, Schwann cell lineage-specific transgenic mice transcribing ErbB3 shRNA with an artificial miRNA backbone were generated and used in these experiments (Torii et al., 2014) [2].

Vps35 in cooperation with LRRK2 regulates synaptic vesicle endocytosis through the endosomal pathway in Drosophila.

Mutations of the retromer component Vps35 and endosomal kinase LRRK2 are linked to autosomal dominant forms of familial Parkinson's disease (PD). However, the physiological and pathological roles of Vps35 and LRRK2 in neuronal functions are poorly understood. Here, we demonstrated that the loss of Drosophila Vps35 (dVps35) affects synaptic vesicle recycling, dopaminergic synaptic release and sleep behavior associated with dopaminergic activity, which is rescued by the expression of wild-type dVps35 but not the PD-associated mutant dVps35 D647N. Drosophila LRRK2 dLRRK together with Rab5 and Rab11 is also implicated in synaptic vesicle recycling, and the manipulation of these activities improves the Vps35 synaptic phenotypes. These findings indicate that defects of synaptic vesicle recycling in which two late-onset PD genes, Vps35 and LRRK2, are involved could be key aspects of PD etiology.

Data on the effect of hypomyelinating leukodystrophy 6 (HLD6)-associated mutations on the TUBB4A properties.

Hypomyelinating leukodystrophy (HLD) is genetic demyelinating or dysmyelinating disease and is associated with at least 13 responsible genes. The mutations seem likely cause the functional deficiency of their gene products. HLD4- and HLD5-associated HSPD1 and FAM126A mutations affect biochemical properties of the gene products (Miyamoto et al. (2015,2014) [[1], [2]]). Herein we provide the data regarding the effects of HLD6-associated tubulin beta 4A (TUBB4A) mutations on the properties.

Data supporting the role of Fyn in embryonic sciatic nerve fasciculation.

Fyn is the cytoplasmic tyrosine kinase that has critical roles in many aspects of biological functions. In the central [1] and peripheral nervous systems [2], [3], Fyn plays the key role in initiating myelination by myelin-forming glial cells (Schwann cells and oligodendrocytes). Herein we provide the data regarding the role of Fyn in fasciculation and branching of embryonic peripheral nerves.

Individual differences in sensory responses influence decision making by Drosophila melanogaster larvae on exposure to contradictory cues.

Animals make decisions on behavioral choice by evaluating internal and external signals. Individuals often make decisions in different ways, but the underlying neural mechanisms are not well understood. Here, we describe a system for observing the behavior of individual Drosophila melanogaster larvae simultaneously presented with contradictory signals, in this case attractive (yeast paste) and aversive (NaCl) signals. Olfaction was used to detect the yeast paste, whereas the ENaC/Pickpocket channel was important for NaCl detection. We found that wild-type (Canton-S) larvae fall into two decision making groups: one group decided to approach the yeast paste by overcoming the aversive signal, whereas the other group decided to forgo the yeast paste because of the aversive signal. Our findings indicate that different endogenous sensitivities to NaCl contribute to make differences between two groups and that diverse decision making steps occur in individual animals.

Organization of projection neurons and local neurons of the primary auditory center in the fruit fly Drosophila melanogaster.

Acoustic communication between insects serves as an excellent model system for analyzing the neuronal mechanisms underlying auditory information processing. The detailed organization of auditory neural circuits in the brain has not yet been described. To understand the central auditory pathways, we used the brain of the fruit fly Drosophila melanogaster as a model and performed a large-scale analysis of the interneurons associated with the primary auditory center. By screening expression driver strains and performing single-cell labeling of these strains, we identified 44 types of interneurons innervating the primary auditory center. Five types were local interneurons whereas the other 39 types were projection interneurons connecting the primary auditory center with other brain regions. The projection neurons comprised three frequency-selective pathways and two frequency-embracive pathways. Mapping of their connection targets revealed that five neuropils in the brain-the wedge (WED), anterior ventrolateral protocerebrum, posterior ventrolateral protocerebrum (PVLP), saddle (SAD), and gnathal ganglia (GNG)-were intensively connected with the primary auditory center. In addition, several other neuropils, including visual and olfactory centers in the brain, were directly connected to the primary auditory center. The distribution patterns of the spines and boutons of the identified neurons suggest that auditory information is sent mainly from the primary auditory center to the PVLP, WED, SAD, GNG, and thoracico-abdominal ganglia. Based on these findings, we established the first comprehensive map of secondary auditory interneurons, which indicates the downstream information flow to parallel ascending pathways, multimodal pathways, and descending pathways.