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Friday, October 30th, 2020 - Disease Models

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Blosser, J. A., Podolsky, E. and Lee, D. (2020). L-DOPA-Induced Dyskinesia in a Genetic Drosophila Model of Parkinson's Disease. Exp Neurobiol 29(4): 273-284. PubMed ID: 32921640
Motor symptoms in Parkinson's disease (PD) are directly related to the reduction of a neurotransmitter dopamine. Therefore, its precursor L-DOPA became the gold standard for PD treatment. However, chronic use of L-DOPA causes uncontrollable, involuntary movements, called L-DOPA-induced dyskinesia (LID) in the majority of PD patients. LID is complicated and very difficult to manage. Current rodent and non-human primate models have been developed to study LID mainly using neurotoxins. Therefore, it is necessary to develop a LID animal model with defects in genetic factors causing PD in order to study the relation between LID and PD genes such as α-synuclein. This study first showed that a low concentration of L-DOPA (100 μM) rescues locomotion defects (i.e., speed, angular velocity, pause time) in Drosophila larvae expressing human mutant α-synuclein (A53T). This A53T larval model of PD was used to further examine dyskinetic behaviors. High concentrations of L-DOPA (5 or 10 mM) causes hyperactivity such as body bending behavior (BBB) in A53T larva, which resembles axial dyskinesia in rodents. Using ImageJ plugins and other third party software, dyskinetic BBB has been accurately and efficiently quantified. Further, a dopamine agonist pramipexole (PRX) partially rescues BBB caused by high L-DOPA. This Drosophila genetic LID model will provide an important experimental platform to examine molecular and cellular mechanisms underlying LID, to study the role of PD causing genes in the development of LID, and to identify potential targets to slow/reverse LID pathology.
Russi, M., Martin, E., D'Autreaux, B., Tixier, L., Tricoire, H. and Monnier, V. (2020). A Drosophila model of Friedreich Ataxia with CRISPR/Cas9 insertion of GAA repeats in the frataxin gene reveals in vivo protection by N-acetyl cysteine. Hum Mol Genet. PubMed ID: 32744307
Friedreich Ataxia (FA) is caused by GAA repeat expansions in the first intron of FXN, the gene encoding frataxin, which results in decreased gene expression. Thanks to the high degree of frataxin conservation, the Drosophila melanogaster fruitfly appears as an adequate animal model to study this disease and to evaluate therapeutic interventions. Here, this study generated a Drosophila model of FA with CRISPR/Cas9 insertion of approximately 200 GAA in the intron of the fly frataxin gene fh. These flies exhibit a developmental delay and lethality associated with decreased frataxin expression. It was possible to by-pass preadult lethality using genetic tools to overexpress frataxin only during the developmental period. These frataxin-deficient adults are short-lived and present strong locomotor defects. RNA-Seq analysis identified deregulation of genes involved in amino-acid metabolism and transcriptomic signatures of oxidative stress. In particular, a progressive increase was observed of Tspo expression, fully rescued by adult frataxin expression. Thus, Tspo expression constitutes a molecular marker of the disease progression in the fly model and might be of interest in other animal models or in patients. Finally, in a candidate drug screening, it was observed that N-acetyl cysteine improved the survival, locomotor function, resistance to oxidative stress and aconitase activity of frataxin-deficient flies. Therefore, this model provides the opportunity to elucidate in vivo the protective mechanisms of this molecule of therapeutic potential. This study also highlights the strength of the CRISPR/Cas9 technology to introduce human mutations in endogenous orthologous genes, leading to Drosophila models of human diseases with improved physiological relevance.
Li, S., Wu, Z., Li, Y., Tantray, I., De Stefani, D., Mattarei, A., Krishnan, G., Gao, F. B., Vogel, H. and Lu, B. (2020). Altered MICOS Morphology and Mitochondrial Ion Homeostasis Contribute to Poly(GR) Toxicity Associated with C9-ALS/FTD. Cell Rep 32(5): 107989. PubMed ID: 32755582
Amyotrophic lateral sclerosis (ALS) manifests pathological changes in motor neurons and various other cell types. Compared to motor neurons, the contribution of the other cell types to the ALS phenotypes is understudied. G4C2 repeat expansion in C9ORF72 is the most common genetic cause of ALS along with frontotemporal dementia (C9-ALS/FTD), with increasing evidence supporting repeat-encoded poly(GR) in disease pathogenesis. This study shows in Drosophila muscle that poly(GR) enters mitochondria and interacts with components of the Mitochondrial Contact Site and Cristae Organizing System (MICOS), altering MICOS dynamics and intra-subunit interactions. This impairs mitochondrial inner membrane structure, ion homeostasis, mitochondrial metabolism, and muscle integrity. Similar mitochondrial defects are observed in patient fibroblasts. Genetic manipulation of MICOS components or pharmacological restoration of ion homeostasis with nigericin effectively rescue the mitochondrial pathology and disease phenotypes in both systems. These results implicate MICOS-regulated ion homeostasis in C9-ALS pathogenesis and suggest potential new therapeutic strategies.
M, I. O. d. S., Lopes, C. S. and Liz, M. A. (2020). Transthyretin interacts with actin regulators in a Drosophila model of familial amyloid polyneuropathy. Sci Rep 10(1): 13596. PubMed ID: 32788615
Familial amyloid polyneuropathy (FAP) is a neurodegenerative disorder whose major hallmark is the deposition of mutated transthyretin (TTR) in the form of amyloid fibrils in the peripheral nervous system (PNS). The exposure of PNS axons to extracellular TTR deposits leads to an axonopathy that culminates in neuronal death. However, the molecular mechanisms underlying TTR-induced neurodegeneration are still unclear, despite the extensive studies in vertebrate models. This work used a Drosophila FAP model, based on the expression of the amyloidogenic TTR (V30M) in the fly retina, to uncover genetic interactions with cytoskeleton regulators. TTR interacts with actin regulators and induces cytoskeleton alterations, leading to axonal defects. Moreover, this study pinpoints an interaction between TTRV30M and members of Rho GTPase signaling pathways, the major actin regulators. Based on these findings it is proposed that actin cytoskeleton alterations may mediate the axonopathy observed in FAP patients, and highlight a molecular pathway, mediated by Rho GTPases, underlying TTR-induced neurodegeneration. This work should prompt novel studies and approaches towards FAP therapy.
Qiao, J. D. and Mao, Y. L. (2020). Knockout of PINK1 altered the neural connectivity of Drosophila dopamine PPM3 neurons at input and output sites. Invert Neurosci 20(3): 11. PubMed ID: 32766952
Impairment of the dopamine system is the main cause of Parkinson disease (PD). PTEN-induced kinase 1 (PINK1) is possibly involved in pathogenesis of PD. However, its role in dopaminergic neurons has not been fully established yet. In the present investigation, the PINK1 knockout Drosophila model to explore the role of PINK1 in dopaminergic neurons. Electrophysiological and behavioral tests indicated that PINK1 elimination enhances the neural transmission from the presynaptic part of dopaminergic neurons in the protocerebral posterior medial region 3 (PPM3) to PPM3 neurons (which are homologous to those in the substantia nigra in humans). Firing properties of the action potential in PPM3 neurons were also altered in the PINK1 knockout genotypes. Abnormal motor ability was also observed in these PINK1 knockout animals. These results indicate that knockout of PINK1 could alter both the input and output properties of PPM3 neurons.
Rahul, Naz, F., Jyoti, S. and Siddique, Y. H. (2020). Effect of kaempferol on the transgenic Drosophila model of Parkinson's disease. Sci Rep 10(1): 13793. PubMed ID: 32796885
The present study was aimed to study the effect of kaempferol, on the transgenic Drosophila model of Parkinson's disease. Kaempferol was added in the diet at final concentration of 10, 20, 30 and 40 µM and the effect was studied on various cognitive and oxidative stress markers. The results of the study showed that kaempferol, delayed the loss of climbing ability as well as the activity of PD flies in a dose dependent manner compared to unexposed PD flies. A dose-dependent reduction in oxidative stress markers was also observed. Histopathological examination of fly brains using anti-tyrosine hydroxylase immunostaining has revealed a significant dose-dependent increase in the expression of tyrosine hydroxylase in PD flies exposed to kaempferol. Molecular docking results revealed that kaempferol binds to human alpha synuclein at specific sites that might results in the inhibition of alpha synuclein aggregation and prevents the formation of Lewy bodies.

Thursday, October 29th - Adult Brain Development and Function

Lee, W. P., Chiang, M. H., Chang, L. Y., Lee, J. Y., Tsai, Y. L., Chiu, T. H., Chiang, H. C., Fu, T. F., Wu, T. and Wu, C. L. (2020). Mushroom body subsets encode CREB2-dependent water-reward long-term memory in Drosophila. PLoS Genet 16(8): e1008963. PubMed ID: 32780743
Long-term memory (LTM) formation depends on the conversed cAMP response element-binding protein (CREB)-dependent gene transcription followed by de novo protein synthesis. Thirsty fruit flies can be trained to associate an odor with water reward to form water-reward LTM (wLTM), which can last for over 24 hours without a significant decline. The role of de novo protein synthesis and CREB-regulated gene expression changes in neural circuits that contribute to wLTM remains unclear. This study shows that acute inhibition of protein synthesis in the mushroom body (MB) αβ or γ neurons during memory formation using a cold-sensitive ribosome-inactivating toxin disrupts wLTM. Furthermore, adult stage-specific expression of dCREB2b in αβ or γ neurons also disrupts wLTM. The MB αβ and γ neurons can be further classified into five different neuronal subsets including αβ core, αβ surface, αβ posterior, γ main, and γ dorsal. This stuyd observed that the neurotransmission from αβ surface and γ dorsal neuron subsets is required for wLTM retrieval, whereas the αβ core, αβ posterior, and γ main are dispensable. Adult stage-specific expression of dCREB2b in αβ surface and γ dorsal neurons inhibits wLTM formation. In vivo calcium imaging revealed that αβ surface and γ dorsal neurons form wLTM traces with different dynamic properties, and these memory traces are abolished by dCREB2b expression. These results suggest that a small population of neurons within the MB circuits support long-term storage of water-reward memory in Drosophila.
Sampson, M. M., Myers Gschweng, K. M., Hardcastle, B. J., Bonanno, S. L., Sizemore, T. R., Arnold, R. C., Gao, F., Dacks, A. M., Frye, M. A. and Krantz, D. E. (2020). Serotonergic modulation of visual neurons in Drosophila melanogaster. PLoS Genet 16(8): e1009003. PubMed ID: 32866139
Sensory systems rely on neuromodulators, such as serotonin, to provide flexibility for information processing as stimuli vary, such as light intensity throughout the day. Serotonergic neurons broadly innervate the optic ganglia of Drosophila. This study mapped of patterns of serotonin receptors in the visual system, focusing on a subset of cells with processes in the first optic ganglion, the lamina. Serotonin receptor expression was found in several types of columnar cells in the lamina including 5-HT2B in lamina monopolar cell L2, required for spatiotemporal luminance contrast, and both 5-HT1A and 5-HT1B in T1 cells, whose function is unknown. Subcellular mapping with GFP-tagged 5-HT2B and 5-HT1A constructs indicated that these receptors localize to layer M2 of the medulla, proximal to serotonergic boutons, suggesting that the medulla neuropil is the primary site of serotonergic regulation for these neurons. Exogenous serotonin increased basal intracellular calcium in L2 terminals in layer M2 and modestly decreased the duration of visually induced calcium transients in L2 neurons following repeated dark flashes, but otherwise did not alter the calcium transients. Flies without functional 5-HT2B failed to show an increase in basal calcium in response to serotonin. 5-HT2B mutants also failed to show a change in amplitude in their response to repeated light flashes but other calcium transient parameters were relatively unaffected. While serotonin receptor expression in L1 neurons was not detected, they, like L2, underwent serotonin-induced changes in basal calcium, presumably via interactions with other cells. These data demonstrate that serotonin modulates the physiology of interneurons involved in early visual processing in Drosophila.
Nandakumar, S., Grushko, O. and Buttitta, L. A. (2020). Polyploidy in the adult Drosophila brain. Elife 9. PubMed ID: 32840209
Long-lived cells such as terminally differentiated postmitotic neurons and glia must cope with the accumulation of damage over the course of an animal's lifespan. How long-lived cells deal with ageing-related damage is poorly understood. This study shows that polyploid cells accumulate in the adult fly brain and that polyploidy protects against DNA damage-induced cell death. Multiple types of neurons and glia that are diploid at eclosion, become polyploid in the adult Drosophila brain. The optic lobes exhibit the highest levels of polyploidy, associated with an elevated DNA damage response in this brain region. Inducing oxidative stress or exogenous DNA damage leads to an earlier onset of polyploidy, and polyploid cells in the adult brain are more resistant to DNA damage-induced cell death than diploid cells. These results suggest polyploidy may serve a protective role for neurons and glia in adult Drosophila melanogaster brains.
Liu, C., Trush, O., Han, X., Wang, M., Takayama, R., Yasugi, T., Hayashi, T. and Sato, M. (2020). Dscam1 establishes the columnar units through lineage-dependent repulsion between sister neurons in the fly brain. Nat Commun 11(1): 4067. PubMed ID: 32792493
The brain is organized morphologically and functionally into a columnar structure. According to the radial unit hypothesis, neurons from the same lineage form a radial unit that contributes to column formation. However, the molecular mechanisms that link neuronal lineage and column formation remain elusive. This study shows that neurons from the same lineage project to different columns under control of Down syndrome cell adhesion molecule (Dscam) in the fly brain, in the development of the medulla in the optic lobe. Dscam1 is temporally expressed in newly born neuroblasts and is inherited by their daughter neurons. The transient transcription of Dscam1 in neuroblasts enables the expression of the same Dscam1 splice isoform within cells of the same lineage, causing lineage-dependent repulsion. In the absence of Dscam1 function, neurons from the same lineage project to the same column. When the splice diversity of Dscam1 is reduced, column formation is significantly compromised. Thus, Dscam1 controls column formation through lineage-dependent repulsion.
Sheng, L., Shields, E. J., Gospocic, J., Glastad, K. M., Ratchasanmuang, P., Berger, S. L., Raj, A., Little, S. and Bonasio, R. (2020). Social reprogramming in ants induces longevity-associated glia remodeling. Sci Adv 6(34): eaba9869. PubMed ID: 32875108
In social insects, workers and queens arise from the same genome but display profound differences in behavior and longevity. In Harpegnathos saltator ants, adult workers can transition to a queen-like state called gamergate, which results in reprogramming of social behavior and life-span extension. Using single-cell RNA sequencing, the distribution of neuronal and glial populations was compared before and after the social transition. This study found that the conversion of workers into gamergates resulted in the expansion of neuroprotective ensheathing glia. Brain injury assays revealed that activation of the damage response gene Mmp1 was weaker in old workers, where the relative frequency of ensheathing glia also declined. On the other hand, long-lived gamergates retained a larger fraction of ensheathing glia and the ability to mount a strong Mmp1 response to brain injury into old age. Molecular and cellular changes were observed suggestive of age-associated decline in ensheathing glia in Drosophila.
Turner-Evans, D. B., Jensen, K. T., Ali, S., Paterson, T., Sheridan, A., Ray, R. P., Wolff, T., Lauritzen, J. S., Rubin, G. M., Bock, D. D. and Jayaraman, V. (2020). The Neuroanatomical Ultrastructure and Function of a Biological Ring Attractor. Neuron. PubMed ID: 32916090
Neural representations of head direction (HD) have been discovered in many species. Theoretical work has proposed that the dynamics associated with these representations are generated, maintained, and updated by recurrent network structures called ring attractors. This theorized structure-function relationship was evaluated by performing electron-microscopy-based circuit reconstruction and RNA profiling of identified cell types in the HD system of Drosophila melanogaster. Motifs were identified that have been hypothesized to maintain the HD representation in darkness, update it when the animal turns, and tether it to visual cues. Functional studies provided support for the proposed roles of individual excitatory or inhibitory circuit elements in shaping activity. This study also discovered recurrent connections between neuronal arbors with mixed pre- and postsynaptic specializations. These results confirm that the Drosophila HD network contains the core components of a ring attractor while also revealing unpredicted structural features that might enhance the network's computational power.

Wednesday, October 28th - Chromatin

Das, P. and Bhadra, M. P. (2020). Histone deacetylase (Rpd3) regulates Drosophila early brain development via regulation of Tailless. Open Biol 10(9): 200029. PubMed ID: 32873153
Tailless is a committed transcriptional repressor and principal regulator of the brain and eye development in Drosophila. Rpd3, the histone deacetylase, is an established repressor that interacts with co-repressors like Sin3a, Prospero, Brakeless and Atrophin. This study aims at deciphering the role of Rpd3 in embryonic segmentation and larval brain development in Drosophila. It delineates the mechanism of Tailless regulation by Rpd3, along with its interacting partners. There was a significant reduction in Tailless in Rpd3 heteroallelic mutant embryos, substantiating that Rpd3 is indispensable for the normal Tailless expression. The expression of the primary readout, Tailless was correlative to the expression of the neural cell adhesion molecule homologue, Fascilin2 (Fas2). Rpd3 also aids in the proper development of the mushroom body. Both Tailless and Fas2 expression are reported to be antagonistic to the epidermal growth factor receptor (EGFR) expression. The decrease in Tailless and Fas2 expression highlights that EGFR is upregulated in the larval mutants, hindering brain development. This study outlines the axis comprising Rpd3, dEGFR, Tailless and Fas2, which interact to fine-tune the early segmentation and larval brain development. Therefore, Rpd3 along with Tailless has immense significance in early embryogenesis and development of the larval brain.
Samborskaia, M. D., Galitsyna, A., Pletenev, I., Trofimova, A., Mironov, A. A., Gelfand, M. S. and Khrameeva, E. E. (2020). Cumulative contact frequency of a chromatin region is an intrinsic property linked to its function. PeerJ 8: e9566. PubMed ID: 32864204
One of the drawbacks of Hi-C (high-throughput chromosomes conformation capture) analysis and interpretation is the presence of systematic biases, such as different accessibility to enzymes, amplification, and mappability of DNA regions, which all result in different visibility of the regions. Iterative correction (IC) is one of the most popular techniques developed for the elimination of these systematic biases. IC is based on the assumption that all chromatin regions have an equal number of observed contacts in Hi-C. In other words, the IC procedure is equalizing the experimental visibility approximated by the cumulative contact frequency (CCF) for all genomic regions. However, the differences in experimental visibility might be explained by biological factors such as chromatin openness, which is characteristic of distinct chromatin states. This study shows that CCF is positively correlated with active transcription. It is associated with compartment organization, since compartment A demonstrates higher CCF and gene expression levels than compartment B. Notably, this observation holds for a wide range of species, including human, mouse, and Drosophila. Moreover, the CCF state for syntenic blocks were tracked between human and mouse; it is concluded that active state assessed by CCF is an intrinsic property of the DNA region, which is independent of local genomic and epigenomic context. These findings establish a missing link between Hi-C normalization procedures removing CCF from the data and poorly investigated and possibly relevant biological factors contributing to CCF.
Sarthy, J. F., Meers, M. P., Janssens, D. H., Henikoff, J. G., Feldman, H., Paddison, P. J., Lockwood, C. M., Vitanza, N. A., Olson, J. M., Ahmad, K. and Henikoff, S. (2020). Histone deposition pathways determine the chromatin landscapes of H3.1 and H3.3 K27M oncohistones. Elife 9. PubMed ID: 32902381
Lysine 27-to-methionine (K27M) mutations in the H3.1 or H3.3 histone genes are characteristic of pediatric diffuse midline gliomas (DMGs). These oncohistone mutations dominantly inhibit histone H3K27 trimethylation and silencing, but it is unknown how oncohistone type affects gliomagenesis. This study shows that the genomic distributions of H3.1 and H3.3 oncohistones in human patient-derived DMG cells are consistent with the DNAreplication-coupled deposition of histone H3.1 and the predominant replication-independent deposition of histone H3.3. Although H3K27 trimethylation is reduced for both oncohistone types, H3.3K27M-bearing cells retain some domains, and only H3.1K27M-bearing cells lack H3K27 trimethylation. Neither oncohistone interferes with PRC2 binding. Using Drosophila as a model, this study demonstrated that inhibition of H3K27 trimethylation occurs only when H3K27M oncohistones are deposited into chromatin and only when expressed in cycling cells. It is proposed that oncohistones inhibit the H3K27 methyltransferase as chromatin patterns are being duplicated in proliferating cells, predisposing them to tumorigenesis.
Fresan, U., Rodriguez-Sanchez, M. A., Reina, O., Corces, V. G. and Espinas, M. L. (2020). Haspin kinase modulates nuclear architecture and Polycomb-dependent gene silencing. PLoS Genet 16(8): e1008962. PubMed ID: 32750047
Haspin, a highly conserved kinase in eukaryotes, has been shown to be responsible for phosphorylation of histone H3 at threonine 3 (H3T3ph) during mitosis, in mammals and yeast. This study reports that haspin is the kinase that phosphorylates H3T3 in Drosophila melanogaster and it is involved in sister chromatid cohesion during mitosis. The data reveal that haspin also phosphorylates H3T3 in interphase. H3T3ph localizes in broad silenced domains at heterochromatin and lamin-enriched euchromatic regions. Loss of haspin compromises insulator activity in enhancer-blocking assays and triggers a decrease in nuclear size that is accompanied by changes in nuclear envelope morphology. Haspin is a suppressor of position-effect variegation involved in heterochromatin organization. These results also demonstrate that haspin is necessary for pairing-sensitive silencing and it is required for robust Polycomb-dependent homeotic gene silencing. Haspin associates with the cohesin complex in interphase, mediates Pds5 binding to chromatin and cooperates with Pds5-cohesin to modify Polycomb-dependent homeotic transformations. Therefore, this study uncovers an unanticipated role for haspin kinase in genome organization of interphase cells and demonstrates that haspin is required for homeotic gene regulation.
DeLuca, S. Z., Ghildiyal, M., Pang, L. Y. and Spradling, A. C. (2020). Differentiating Drosophila female germ cells initiate Polycomb silencing by regulating PRC2-interacting proteins. Elife 9. PubMed ID: 32773039
Polycomb silencing represses gene expression and provides a molecular memory of chromatin state that is essential for animal development. This study shows that Drosophila female germline stem cells (GSCs) provide a powerful system for studying Polycomb silencing. GSCs have a non-canonical distribution of PRC2 activity and lack silenced chromatin like embryonic progenitors. As GSC daughters differentiate into nurse cells and oocytes, nurse cells, like embryonic somatic cells, silence genes in traditional Polycomb domains and in generally inactive chromatin. Developmentally controlled expression of two Polycomb repressive complex 2 (PRC2)-interacting proteins, Pcl and Scm, initiate silencing during differentiation. In GSCs, abundant Pcl inhibits PRC2-dependent silencing globally, while in nurse cells Pcl declines and newly induced Scm concentrates PRC2 activity on traditional Polycomb domains. These results suggest that PRC2-dependent silencing is developmentally regulated by accessory proteins that either increase the concentration of PRC2 at target sites or inhibit the rate that PRC2 samples chromatin.
Evans, M. K., Matsui, Y., Xu, B., Willis, C., Loome, J., Milburn, L., Fan, Y., Pagala, V. and Peng, J. C. (2020). Ybx1 fine-tunes PRC2 activities to control embryonic brain development. Nat Commun 11(1): 4060. PubMed ID: 32792512
Chromatin modifiers affect spatiotemporal gene expression programs that underlie organismal development. The Polycomb repressive complex 2 (PRC2) is a crucial chromatin modifier in executing neurodevelopmental programs. This study finds that PRC2 interacts with the nucleic acid-binding protein Ybx1 (see Drosophila Ybx1). In the mouse embryo in vivo, Ybx1 is required for forebrain specification and restricting mid-hindbrain growth. In neural progenitor cells (NPCs), Ybx1 controls self-renewal and neuronal differentiation. Mechanistically, Ybx1 highly overlaps PRC2 binding genome-wide, controls PRC2 distribution, and inhibits H3K27me3 levels. These functions are consistent with Ybx1-mediated promotion of genes involved in forebrain specification, cell proliferation, or neuronal differentiation. In Ybx1-knockout NPCs, H3K27me3 reduction by PRC2 enzymatic inhibitor or genetic depletion partially rescues gene expression and NPC functions. These findings suggest that Ybx1 fine-tunes PRC2 activities to regulate spatiotemporal gene expression in embryonic neural development and uncover a crucial epigenetic mechanism balancing forebrain-hindbrain lineages and self-renewal-differentiation choices in NPCs.

Monday, October 27th - Disease Models

Shimizu, J., Kasai, T., Yoshida, H., Huynh, A. M., Nakao-Azuma, Y., Shinomoto, M., Tokuda, T., Mizuno, T. and Yamaguchi, M. (2020). Novel Drosophila model for parkinsonism by targeting phosphoglycerate kinase. Neurochem Int 139: 104816. PubMed ID: 32758590
Patients with Parkinson's disease (PD) show a common progressive neurodegenerative movement disorder characterized by rigidity, tremors, postural instability, and bradykinesia due to the loss of dopaminergic neurons in the substantia nigra, and is often accompanied by several non-motor symptoms, called parkinsonism. Several lines of recent evidence support the hypothesis that mutations in the gene encoding phosphoglycerate kinase (PGK) play an important role in the PD mechanism. PGK is a key enzyme in the glycolytic pathway that catalyzes the reaction from 1,3-diphosphoglycerate to 3-phosphoglycerate. This study established a parkinsonism model targeting Drosophila Pgk. Dopaminergic (DA) neuron-specific Pgk knockdown lead to locomotive defects in both young and aged adult flies and was accompanied by progressive DA neuron loss with aging. Pgk knockdown in DA neurons decreased dopamine levels in the central nervous system (CNS) of both young and aged adult flies. These phenotypes are similar to the defects observed in human PD patients, suggesting that the Pgk knockdown flies established herein are a promising model for parkinsonism. Furthermore, pan-neuron-specific Pgk knockdown induced low ATP levels and the accumulation of reactive oxygen species (ROS) in the CNS of third instar larvae. Collectively, these results indicate that a failure in the energy production system of Pgk knockdown flies causes locomotive defects accompanied by neuronal dysfunction and degeneration in DA neurons.
Wang, X. and Davis, R. L. (2020). Early Mitochondrial Fragmentation and Dysfunction in a Drosophila Model for Alzheimer's Disease. Mol Neurobiol. PubMed ID: 32909149
Many different cellular systems and molecular processes become compromised in Alzheimer's disease (AD) including proteostasis, autophagy, inflammatory responses, synapse and neuronal circuitry, and mitochondrial function. This study focused on mitochondrial dysfunction owing to the toxic neuronal environment produced by expression of Aβ42, and its relationship to other pathologies found in AD including increased neuronal apoptosis, plaque deposition, and memory impairment. Using super-resolution microscopy, mitochondrial status was assayed in the three distinct neuronal compartments (somatic, dendritic, axonal) of mushroom body neurons of Drosophila expressing Aβ42. The mushroom body neurons comprise a major center for olfactory memory formation in insects. Calcium imaging was employed to measure mitochondrial function, immunohistochemical and staining techniques to measure apoptosis and plaque formation, and olfactory classical conditioning to measure learning. Mitochondria become fragmented at a very early age along with decreased function measured by mitochondrial calcium entry. Increased apoptosis and plaque deposition also occur early, yet interestingly, a learning impairment was found only after a much longer period of time-10 days, which is a large fraction of the fly's lifespan. This is similar to the pronounced delay between cellular pathologies and the emergence of a memory dysfunction in humans. These studies are consistent with the model that mitochondrial dysfunction and/or other cellular pathologies emerge at an early age and lead to much later learning impairments. The results obtained further develop this Drosophila model as a useful in vivo system for probing the mechanisms by which Aβ42 produces mitochondrial and other cellular toxicities that produce memory dysfunction.
Xue, J., Wang, H. L. and Xiao, G. (2020). Transferrin1 modulates rotenone-induced Parkinson's disease through affecting iron homeostasis in Drosophila melanogaster. Biochem Biophys Res Commun 531(3): 305-311. PubMed ID: 32800558
Mitochondrial dysfunction and oxidative stress are pathophysiologic mechanisms implicated in Parkinson's disease (PD). In recent years, environmental toxins are employed to increase oxidative stress mediated neuropathology and sporadic PD. Disruption of iron homeostasis has been implicated in PD patients for many years, but the functional role of iron in sporadic PD pathogenesis is still not well clarified in vivo. To address this question, the effect of iron on a Drosophila rotenone model of sporadic PD was investigated. Iron homeostasis is maintained by many transporters. Inhibition of transferrin1 (Tsf1) expression in the central nervous system (CNS) results in reduced iron levels in brains and significantly ameliorates the neurodegenerative phenotypes of rotenone exposure Drosophila; moreover, the rotenone induced reactive oxygen species (ROS) levels in the brain, the damaged complex I activity and the decreased ATP generation were dramatically rescued by Tsf1 knockdown. Further study indicated that all the rescue effects of Tsf1 knockdown on sporadic PD could be inhibited by malvolio (Mvl) overexpression, an iron transporter responsible for iron uptake. These results imply that Tsf1 knockdown in the CNS could attenuate rotenone toxicity by decreasing the ROS levels in brains through reducing iron levels, and manipulation of iron transporters in brains may provide a novel therapeutic strategy for sporadic PD.
Agudelo, A., St Amand, V., Grissom, L., Lafond, D., Achilli, T., Sahin, A., Reenan, R. and Stilwell, G. (2020). Age-dependent degeneration of an identified adult leg motor neuron in a Drosophila SOD1 model of ALS. Biol Open. PubMed ID: 32994185
Mutations in superoxide dismutase 1 (SOD1) cause familial Amyotrophic lateral sclerosis (ALS) in humans. ALS is a neurodegenerative disease characterized by progressive motor neuron loss leading to paralysis and inevitable death in affected individuals. Using a gene replacement strategy to introduce disease mutations into the orthologous Drosophila sod1 (dsod1) gene, This study characterize changes at the neuromuscular junction using longer lived dsod1 mutant adults. Homozygous dsod1 (H71Y/H71Y) or dsod1 (null/null) flies display progressive walking defects with paralysis of the 3rd metathoracic leg. In dissected legs, age-dependent changes were assessed in a single identified motor neuron (MN-I2) innervating the tibia levitator muscle. At adult eclosion, MN-I2 of dsod1 (H71Y/H71Y) or dsod1 (null/null) flies is patterned similar to wild type flies indicating no readily apparent developmental defects. Over the course of 10 days post-eclosion, MN-I2 shows an overall reduction in arborization with bouton swelling and loss of the post-synaptic marker Discs-large (Dlg) in mutant dsod1 adults. In addition, increases in polyubiquitinated proteins correlate with the timing and extent of MN-I2 changes. Because similar phenotypes are observed between flies homozygous for either dsod1 (H71Y) or dsod1 (null) alleles, it is concluded these NMJ changes are mainly associated with sod loss of function. Together these studies characterize age-related morphological and molecular changes associated with axonal retraction in a Drosophila model of ALS that recapitulate an important aspect of the human disease.
Wan, Z., Xu, J., Huang, Y., Zhai, Y., Ma, Z., Zhou, B. and Cao, Z. (2020). Elevating bioavailable iron levels in mitochondria suppresses the defective phenotypes caused by PINK1 loss-of-function in Drosophila melanogaster. Biochem Biophys Res Commun 532(2): 285-291. PubMed ID: 32873392
Parkinson's disease (PD) is the second most common progressive neurodegenerative disease, which is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Iron deposit was found in the SNpc of PD patients and animal models, however, the mechanisms involved in disturbed iron metabolism remain unknown. Identifying the relationship between iron metabolism and PD is important for finding new therapeutic strategies. This study found that transgenic overexpression (OE) of Drosophila mitoferrin (dmfrn) or knockdown of Fer3HCH significantly mitigated the reduced mitochondrial aconitase activity, abnormal wing posture, flight deficits and mitochondrial morphology defects associated with PINK1 loss-of-function (LOF). Further work demonstrated that dmfrn OE or Fer3HCH knockdown significantly rescued the impaired mitochondrial respiration in PINK1 LOF flies, indicating that dmfrn or Fer3HCH may rescue PINK1 LOF phenotypes through elevating mitochondrial bioavailable iron levels to promote mitochondrial respiration.
Yamauchi, T., Oi, A., Kosakamoto, H., Akuzawa-Tokita, Y., Murakami, T., Mori, H., Miura, M. and Obata, F. (2020). Gut Bacterial Species Distinctively Impact Host Purine Metabolites during Aging in Drosophila. iScience 23(9): 101477. PubMed ID: 32916085
Gut microbiota impacts the host metabolome and affects its health span. How bacterial species in the gut influence age-dependent metabolic alteration has not been elucidated. This study shows in Drosophila melanogaster that allantoin, an end product of purine metabolism, is increased during aging in a microbiota-dependent manner. Allantoin levels are low in young flies but are commonly elevated upon lifespan-shortening dietary manipulations such as high-purine, high-sugar, or high-yeast feeding. Removing Acetobacter persici in the Drosophila microbiome attenuated age-dependent allantoin increase. Mono-association with A. persici, but not with Lactobacillus plantarum, increased allantoin in aged flies. A. persici increased allantoin via activation of innate immune signaling IMD pathway in the renal tubules. On the other hand, analysis of bacteria-conditioned diets revealed that L. plantarum can decrease allantoin by reducing purines in the diet. These data together demonstrate species-specific regulations of host purine levels by the gut microbiome.

Monday, October 26th - Signaling

Strutt, H. and Strutt, D. (2020). DAnkrd49 and Bdbt act via Casein kinase Iepsilon to regulate planar polarity in Drosophila. PLoS Genet 16(8): e1008820. PubMed ID: 32750048
The core planar polarity proteins are essential mediators of tissue morphogenesis, controlling both the polarised production of cellular structures and polarised tissue movements. During development the core proteins promote planar polarisation by becoming asymmetrically localised to opposite cell edges within epithelial tissues, forming intercellular protein complexes that coordinate polarity between adjacent cells. This study describes a novel protein complex that regulates the asymmetric localisation of the core proteins in the Drosophila pupal wing. DAnkrd49 (an ankyrin repeat protein) and Bride of Doubletime (Bdbt, a non-canonical FK506 binding protein family member) physically interact, and regulate each other's levels in vivo. Loss of either protein results in a reduction in core protein asymmetry and disruption of the placement of trichomes at the distal edge of pupal wing cells. Post-translational modifications are thought to be important for the regulation of core protein behaviour and their sorting to opposite cell edges. Consistent with this, it was found that loss of DAnkrd49 or Bdbt leads to reduced phosphorylation of the core protein Dishevelled and to decreased Dishevelled levels both at cell junctions and in the cytoplasm. Bdbt has previously been shown to regulate activity of the kinase Discs Overgrown (Dco, also known as Doubletime or Casein Kinase Iε), and Dco itself has been implicated in regulating planar polarity by phosphorylating Dsh as well as the core protein Strabismus. This study demonstrates that DAnkrd49 and Bdbt act as dominant suppressors of Dco activity. These findings support a model whereby Bdbt and DAnkrd49 act together to modulate the activity of Dco during planar polarity establishment.
Korona, D., Nightingale, D., Fabre, B., Nelson, M., Fischer, B., Johnson, G., Lees, J., Hubbard, S., Lilley, K. and Russell, S. (2020). Characterisation of protein isoforms encoded by the Drosophila Glycogen Synthase Kinase 3 gene shaggy. PLoS One 15(8): e0236679. PubMed ID: 32760087
The Drosophila shaggy gene (sgg, GSK-3) encodes multiple protein isoforms with serine/threonine kinase activity and is a key player in diverse developmental signalling pathways. Currently it is unclear whether different Sgg proteoforms are similarly involved in signalling or if different proteoforms have distinct functions. This study used CRISPR/Cas9 genome engineering to tag eight different Sgg proteoform classes and determined their localization during embryonic development. Proteomic analysis was performed of the two major proteoform classes and mutant lines for both of these were generated for transcriptomic and phenotypic analysis. Distinct tissue-specific localization patterns were uncovered for all of the tagged proteoforms examined, most of which have not previously been characterised directly at the protein level, including one proteoform initiating with a non-standard codon. Collectively, this suggests complex developmentally regulated splicing of the sgg primary transcript. Further, affinity purification followed by mass spectrometric analyses indicate a different repertoire of interacting proteins for the two major proteoforms examined, one with ubiquitous expression (Sgg-PB) and one with nervous system specific expression (Sgg-PA). Specific mutation of these proteoforms shows that Sgg-PB performs the well characterised maternal and zygotic segmentations functions of the sgg locus, while Sgg-PA mutants show adult lifespan and locomotor defects consistent with its nervous system localisation. These findings provide new insights into the role of GSK-3 proteoforms and intriguing links with the GSK-3α and GSK-3β proteins encoded by independent vertebrate genes. This analysis suggests that different proteoforms generated by alternative splicing are likely to perform distinct functions.
Portela, M., Mitchell, T. and Casas-Tinto, S. (2020). Cell-to-cell communication mediates glioblastoma progression in Drosophila. Biol Open 9(9). PubMed ID: 32878880
Glioblastoma (GB) is the most aggressive and lethal tumour of the central nervous system (CNS). GB cells grow rapidly and display a network of projections, ultra-long tumour microtubes (TMs), that mediate cell to cell communication. GB-TMs infiltrate throughout the brain, enwrap neurons and facilitate the depletion of the signalling molecule wingless (Wg)/WNT from the neighbouring healthy neurons. GB cells establish a positive feedback loop including Wg signalling upregulation that activates cJun N-terminal kinase (JNK) pathway and matrix metalloproteases (MMPs) production, which in turn promote further TMs infiltration, GB progression and neurodegeneration. Thus, cellular and molecular signals other than primary mutations emerge as central players of GB. Using a Drosophila model of GB, this study describes the temporal organisation of the main cellular events that occur in GB, including cell-to-cell interactions, neurodegeneration and TM expansion. The progressive activation of JNK pathway signalling is described in GB mediated by the receptor Grindelwald (Grnd) and activated by the ligand Eiger (Egr)/TNFα produced by surrounding healthy brain tissue. It is proposed that cellular interactions of GB with the healthy brain tissue precede TM expansion and it is concluded that non-autonomous signals facilitate GB progression. These results contribute to deciphering the complexity and versatility of these incurable tumours.
Sun, L., Zhang, J., Chen, W., Chen, Y., Zhang, X., Yang, M., Xiao, M., Ma, F., Yao, Y., Ye, M., Zhang, Z., Chen, K., Chen, F., Ren, Y., Ni, S., Zhang, X., Yan, Z., Sun, Z. R., Zhou, H. M., Yang, H., Xie, S., Haque, M. E., Huang, K. and Yang, Y. (2020). Attenuation of epigenetic regulator SMARCA4 and ERK-ETS signaling suppresses aging-related dopaminergic degeneration. Aging Cell 19(9): e13210. PubMed ID: 32749068
How complex interactions of genetic, environmental factors and aging jointly contribute to dopaminergic degeneration in Parkinson's disease (PD) is largely unclear. This study applied frequent gene co-expression analysis on human patient substantia nigra-specific microarray datasets to identify potential novel disease-related genes. In vivo Drosophila studies validated two of 32 candidate genes, a chromatin-remodeling factor SMARCA4 and a biliverdin reductase BLVRA. Inhibition of SMARCA4 was able to prevent aging-dependent dopaminergic degeneration not only caused by overexpression of BLVRA but also in four most common Drosophila PD models. Furthermore, down-regulation of SMARCA4 specifically in the dopaminergic neurons prevented shortening of life span caused by α-synuclein and LRRK2. Mechanistically, aberrant SMARCA4 and BLVRA converged on elevated ERK-ETS activity, attenuation of which by either genetic or pharmacological manipulation effectively suppressed dopaminergic degeneration in Drosophila in vivo. Down-regulation of SMARCA4 or drug inhibition of MEK/ERK also mitigated mitochondrial defects in PINK1 (a PD-associated gene)-deficient human cells. These findings underscore the important role of epigenetic regulators and implicate a common signaling axis for therapeutic intervention in normal aging and a broad range of age-related disorders including PD.
Li, H., Wang, W., Zhang, W. and Wu, G. (2020). Structural insight into the recognition between Sufu and fused in the Hedgehog signal transduction pathway. J Struct Biol 212(2): 107614. PubMed ID: 32911070
Hedgehog signaling plays a crucial role in embryogenesis and adult tissue homeostasis, and mutations of its key components such as Suppressor of fused (Sufu) are closely associated with human diseases. The Ser/Thr kinase Fused (Fu) promotes Hedgehog signaling by phosphorylating the Cubitus interruptus (Ci)/Glioma-associated oncogene homologue (Gli) family of transcription factors. Sufu associates with both Fu and Ci/Gli, but the recognition mechanism between Sufu and Fu remains obscure. The structure of the N-terminal domain (NTD) of Drosophila Sufu (dSufu) in complex with the Sufu-binding site (SBS) of Fu reveals that both main-chain β sheet formation and side-chain hydrophobic interactions contribute to the recognition between Sufu and Fu, and point mutations of highly conserved interface residues eliminated their association. Structural comparison suggests that Fu and Ci/Gli bind on opposite sides of dSufu-NTD, allowing the formation of a Fu-dSufu-Ci ternary complex which facilitates the phosphorylation of Ci/Gli by Fu. Hence, these results provide insights into the Sufu-Fu recognition mechanism.
Molnar, C., Louzao, A. and Gonzalez, C. (2020). Context-Dependent Tumorigenic Effect of Testis-Specific Mitochondrial Protein Tiny Tim 2 in Drosophila Somatic Epithelia. Cells 9(8). PubMed ID: 32781577
A study was undertaken towards understanding the effect of ectopic expression of testis proteins in the soma in Drosophila. In the larval neuroepithelium, ectopic expression of the germline-specific component of the inner mitochondrial translocation complex tiny tim 2 (ttm2) brings about cell autonomous hyperplasia and extension of G2 phase. In the wing discs, cells expressing ectopic ttm2 upregulate Jun N-terminal kinase (JNK) signaling, present extended G2, become invasive, and elicit non-cell autonomous G2 extension and overgrowth of the wild-type neighboring tissue. Ectopic tomboy20, a germline-specific member of the outer mitochondrial translocation complex is also tumorigenic in wing discs. These results demonstrate the tumorigenic potential of unscheduled expression of these two testis proteins in the soma. They also show that a unique tumorigenic event may trigger different tumor growth pathways depending on the tissular context.

Friday, October 23rd - Behavior

Cellini, B. and Mongeau, J. M. (2020). Active vision shapes and coordinates flight motor responses in flies. Proc Natl Acad Sci U S A 117(37): 23085-23095. PubMed ID: 32873637
Animals use active sensing to respond to sensory inputs and guide future motor decisions. In flight, flies generate a pattern of head and body movements to stabilize gaze. How the brain relays visual information to control head and body movements and how active head movements influence downstream motor control remains elusive. Using a control theoretic framework, the optomotor gaze stabilization reflex was studied in tethered flight and quantified how head movements stabilize visual motion and shape wing steering efforts in fruit flies (Drosophila). By shaping visual inputs, head movements increased the gain of wing steering responses and coordination between stimulus and wings, pointing to a tight coupling between head and wing movements. Head movements followed the visual stimulus in as little as 10 ms-a delay similar to the human vestibulo-ocular reflex-whereas wing steering responses lagged by more than 40 ms. This timing difference suggests a temporal order in the flow of visual information such that the head filters visual information eliciting downstream wing steering responses. Head fixation significantly decreased the mechanical power generated by the flight motor by reducing wingbeat frequency and overall thrust. By simulating an elementary motion detector array, this study showd that head movements shift the effective visual input dynamic range onto the sensitivity optimum of the motion vision pathway. Taken together, these results reveal a transformative influence of active vision on flight motor responses in flies. This work provides a framework for understanding how to coordinate moving sensors on a moving body.
Yanagawa, A., Huang, W., Yamamoto, A., Wada-Katsumata, A., Schal, C. and Mackay, T. F. C. (2020). Genetic Basis of Natural Variation in Spontaneous Grooming in Drosophila melanogaster. G3 (Bethesda). PubMed ID: 32727922
Spontaneous grooming behavior is a component of insect fitness. This study quantified spontaneous grooming behavior in 201 sequenced lines of the Drosophila melanogaster Genetic Reference Panel and observed significant genetic variation in spontaneous grooming, with broad-sense heritabilities of 0.25 and 0.24 in females and males, respectively. Although grooming behavior is highly correlated between males and females, significant sex by genotype interactions were observed, indicating that the genetic basis of spontaneous grooming is partially distinct in the two sexes. Genome-wide association analyses of grooming behavior was performed, and 107 molecular polymorphisms associated with spontaneous grooming behavior were mapped, of which 73 were in or near 70 genes and 34 were over 1 kilobase from the nearest gene. The candidate genes were associated with a wide variety of gene ontology terms, and several of the candidate genes were significantly enriched in a genetic interaction network. Functional assessments were performed of 29 candidate genes using RNA interference, and 11 were found to affecte spontaneous grooming behavior. The genes associated with natural variation in Drosophila grooming are involved with glutamate metabolism (Gdh) and transport (Eaat); interact genetically with (CCKLR-17D1) or are in the same gene family as (PGRP-LA) genes previously implicated in grooming behavior; are involved in the development of the nervous system and other tissues; or regulate the Notch and Epidermal growth factor receptor signaling pathways. Several DGRP lines exhibited extreme grooming behavior. Excessive grooming behavior can serve as a model for repetitive behaviors diagnostic of several human neuropsychiatric diseases.
Mezzera, C., Brotas, M., Gaspar, M., Pavlou, H. J., Goodwin, S. F. and Vasconcelos, M. L. (2020). Ovipositor Extrusion Promotes the Transition from Courtship to Copulation and Signals Female Acceptance in Drosophila melanogaster. Curr Biol. PubMed ID: 32795437
Communication between male and female fruit flies during courtship is essential for successful mating, but, as with many other species, it is the female who decides whether to mate. This study shows a novel role for ovipositor extrusion in promoting male copulation attempts in virgin and mated females and signaling acceptance in virgins. It was first shown that ovipositor extrusion is only displayed by sexually mature females, exclusively during courtship and in response to the male song. A pair of descending neurons was identified that controls ovipositor extrusion in mated females. Genetic silencing of the descending neurons shows that ovipositor extrusion stimulates the male to attempt copulation. A detailed behavioral analysis revealed that during courtship, the male repeatedly licks the female genitalia, independently of ovipositor extrusion, and that licking an extruded ovipositor prompts a copulation attempt. However, if the ovipositor is not subsequently retracted, copulation is prevented, as it happens with mated females. This study has revealed a dual function of the ovipositor: while its extrusion is necessary for initiating copulation by the male, its retraction signals female acceptance. This stduy thus uncover the significance of the communication between male and female that initiates the transition from courtship to copulation.
Pegoraro, M., Flavell, L. M. M., Menegazzi, P., Colombi, P., Dao, P., Helfrich-Forster, C. and Tauber, E. (2020). The genetic basis of diurnal preference in Drosophila melanogaster. BMC Genomics 21(1): 596. PubMed ID: 32862827
Most animals restrict their activity to a specific part of the day, being diurnal, nocturnal or crepuscular. The genetic basis underlying diurnal preference is largely unknown. Under laboratory conditions, Drosophila melanogaster is crepuscular, showing a bi-modal activity profile. However, a survey of strains derived from wild populations indicated that high variability among individuals exists, including flies that are nocturnal. Using a highly diverse population, an artificial selection experiment was performed, selecting flies with extreme diurnal or nocturnal preference. After 10 generations, highly diurnal and nocturnal strains were obtained. Whole-genome expression analysis was used to identify differentially expressed genes in diurnal, nocturnal and crepuscular (control) flies. Other than one circadian clock gene (pdp1), most differentially expressed genes were associated with either clock output (pdf, to) or input (Rh3, Rh2, msn). This finding was congruent with behavioural experiments indicating that both light masking and the circadian pacemaker are involved in driving nocturnality. This study demonstrates that genetic variation segregating in wild populations contributes to substantial variation in diurnal preference. Candidate genes associated with diurnality/nocturnality, while data emerging from expression analysis and behavioural experiments suggest that both clock and clock-independent pathways are involved in shaping diurnal preference. The diurnal and nocturnal selection strains provide a unique opportunity to understand the genetic architecture of diurnal preference.
Yost, R. T., Robinson, J. W., Baxter, C. M., Scott, A. M., Brown, L. P., Aletta, M. S., Hakimjavadi, R., Lone, A., Cumming, R. C., Dukas, R., Mozer, B. and Simon, A. F. (2020). Abnormal Social Interactions in a Drosophila Mutant of an Autism Candidate Gene: Neuroligin 3. Int J Mol Sci 21(13). PubMed ID: 32610435
Social interactions are typically impaired in neuropsychiatric disorders such as autism, for which the genetic underpinnings are very complex. Social interactions can be modeled by analysis of behaviors, including social spacing, sociability, and aggression, in simpler organisms such as Drosophila melanogaster. This study examined the effects of mutants of the autism-related gene neuroligin 3 (nlg3) on fly social and non-social behaviors. Startled-induced negative geotaxis is affected by a loss of function nlg3 mutation. Social space and aggression are also altered in a sex- and social-experience-specific manner in nlg3 mutant flies. In light of the conserved roles that neuroligins play in social behavior, these results offer insight into the regulation of social behavior in other organisms, including humans.
Liu, C., Zhang, B., Zhang, L., Yang, T., Zhang, Z., Gao, Z. and Zhang, W. (2020). A neural circuit encoding mating states tunes defensive behavior in Drosophila. Nat Commun 11(1): 3962. PubMed ID: 32770059
Social context can dampen or amplify the perception of touch, and touch in turn conveys nuanced social information. However, the neural mechanism behind social regulation of mechanosensation is largely elusive. This study reports that fruit flies exhibit a strong defensive response to mechanical stimuli to their wings. In contrast, virgin female flies being courted by a male show a compromised defensive response to the stimuli, but following mating the response is enhanced. This state-dependent switch is mediated by a functional reconfiguration of a neural circuit labelled with the Tmc-L gene in the ventral nerve cord. The circuit receives excitatory inputs from peripheral mechanoreceptors and coordinates the defensive response. While male cues suppress it via a doublesex (dsx) neuronal pathway, mating sensitizes it by stimulating a group of uterine neurons and consequently activating a leucokinin-dependent pathway (see Lkr). Such a modulation is crucial for the balance between defense against body contacts and sexual receptivity.

Thursday, October 22nd - Synapse and Vesicles

Platenkamp, A., Detmar, E., Sepulveda, L., Ritz, A., Rogers, S. L. and Applewhite, D. A. (2020). The Drosophila melanogaster Rab GAP RN-tre cross-talks with the Rho1 signaling pathway to regulate nonmuscle myosin II localization and function. Mol Biol Cell 31(21): 2379-2397. PubMed ID: 32816624
To identify novel regulators of nonmuscle myosin II (NMII) an image-based RNA interference screen was performed using stable Drosophila melanogaster S2 cells expressing the enhanced green fluorescent protein (EGFP)-tagged regulatory light chain (RLC) of NMII and mCherry-Actin. The Rab-specific GTPase-activating protein (GAP) RN-tre was identified as necessary for the assembly of NMII RLC into contractile actin networks. Depletion of RN-tre led to a punctate NMII phenotype, similar to what is observed following depletion of proteins in the Rho1 pathway. Depletion of RN-tre also led to a decrease in active Rho1 and a decrease in phosphomyosin-positive cells by immunostaining, while expression of constitutively active Rho or Rho-kinase (Rok) rescues the punctate phenotype. Functionally, RN-tre depletion led to an increase in actin retrograde flow rate and cellular contractility in S2 and S2R+ cells, respectively. Regulation of NMII by RN-tre is only partially dependent on its GAP activity as overexpression of constitutively active Rabs inactivated by RN-tre failed to alter NMII RLC localization, while a GAP-dead version of RN-tre partially restored phosphomyosin staining. Collectively, these results suggest that RN-tre plays an important regulatory role in NMII RLC distribution, phosphorylation, and function, likely through Rho1 signaling and putatively serving as a link between the secretion machinery and actomyosin contractility.
Rushton, E., Kopke, D. L. and Broadie, K. (2020). Extracellular heparan sulfate proteoglycans and glycan-binding lectins orchestrate trans-synaptic signaling. J Cell Sci 133(15). PubMed ID: 32788209
The exceedingly narrow synaptic cleft (<20nm) and adjacent perisynaptic extracellular space contain an astonishing array of secreted and membrane-anchored glycoproteins. A number of these extracellular molecules regulate intercellular trans-synaptic signaling by binding to ligands, acting as co-receptors or modulating ligand-receptor interactions. Recent work has greatly expanded understanding of extracellular proteoglycan and glycan-binding lectin families as key regulators of intercellular signaling at the synapse. These secreted proteins act to regulate the compartmentalization of glycoprotein ligands and receptors, crosslink dynamic extracellular and cell surface lattices, modulate both exocytosis and endocytosis vesicle cycling, and control postsynaptic receptor trafficking. This study focused closely on the Drosophila glutamatergic neuromuscular junction (NMJ) as a model synapse for understanding extracellular roles of the many heparan sulfate proteoglycan (HSPG; see Dally) and lectin proteins that help determine synaptic architecture and neurotransmission strength. This study particularly concentrated on the roles of extracellular HSPGs and lectins in controlling trans-synaptic signaling, especially that mediated by the Wnt and BMP pathways. These signaling mechanisms are causally linked to a wide spectrum of neurological disease states that impair coordinated movement and cognitive functions.
Patel, P. H., Wilkinson, E. C., Starke, E. L., McGimsey, M. R., Blankenship, J. T. and Barbee, S. A. (2020). Vps54 regulates Drosophila neuromuscular junction development and interacts genetically with Rab7 to control composition of the postsynaptic density. Biol Open 9(8). PubMed ID: 32747448
Vps54 is a subunit of the Golgi-associated retrograde protein (GARP) complex, which is involved in tethering endosome-derived vesicles to the trans-Golgi network (TGN). In the wobbler mouse, a model for human motor neuron (MN) disease, reduction in the levels of Vps54 causes neurodegeneration. However, it is unclear how disruption of the GARP complex leads to MN dysfunction. To better understand the role of Vps54 in MNs, this study has disrupted expression of the Vps54 ortholog in Drosophila and examined the impact on the larval neuromuscular junction (NMJ). Surprisingly, it was shown that both null mutants and MN-specific knockdown of Vps54 leads to NMJ overgrowth. Reduction of Vps54 partially disrupts localization of the t-SNARE, Syntaxin-16, to the TGN but has no visible impact on endosomal pools. MN-specific knockdown of Vps54 in MNs combined with overexpression of the small GTPases Rab5, Rab7, or Rab11 suppresses the Vps54 NMJ phenotype. Conversely, knockdown of Vps54 combined with overexpression of dominant negative Rab7 causes NMJ and behavioral abnormalities including a decrease in postsynaptic Dlg and GluRIIB levels without any effect on GluRIIA. Taken together, these data suggest that Vps54 controls larval MN axon development and postsynaptic density composition through a mechanism that requires Rab7.
Lu, Y., West, R. J. H., Pons, M., Sweeney, S. T. and Gao, F. B. (2020). Ik2/TBK1 and Hook/Dynein, an adaptor complex for early endosome transport, are genetic modifiers of FTD-associated mutant CHMP2B toxicity in Drosophila. Sci Rep 10(1): 14221. PubMed ID: 32848189
Mutations in CHMP2B, encoding a protein in the endosomal sorting complexes required for transport (ESCRT) machinery, causes frontotemporal dementia linked to chromosome 3 (FTD3). FTD, the second most common form of pre-senile dementia, can also be caused by genetic mutations in other genes, including TANK-binding kinase 1 (TBK1). How FTD-causing disease genes interact is largely unknown. This study found that partial loss function of Ik2, the fly homologue of TBK1 also known as I-kappaB kinase ε (IKKε), enhanced the toxicity of mutant CHMP2B in the fly eye and that Ik2 overexpression suppressed the effect of mutant CHMP2B in neurons. Partial loss of function of Spn-F, a downstream phosphorylation target of Ik2, greatly enhanced the mutant CHMP2B phenotype. An interactome analysis to understand cellular processes regulated by Spn-F identified a network of interacting proteins including Spn-F, Ik2, dynein light chain, and Hook, an adaptor protein in early endosome transport. Partial loss of function of dynein light chain or Hook also enhanced mutant CHMP2B toxicity. These findings identify several evolutionarily conserved genes, including ik2/TBK1, cut up (encoding dynein light chain) and hook, as genetic modifiers of FTD3-associated mutant CHMP2B toxicity and implicate early endosome transport as a potential contributing pathway in FTD.
Metwally, E., Zhao, G., Wang, Q. and Zhang, Y. Q. (2020). Ttm50 facilitates calpain activation by anchoring it to calcium stores and increasing its sensitivity to calcium. Cell Res. PubMed ID: 32848200
Calcium-dependent proteolytic calpains are implicated in a variety of physiological processes, as well as pathologies associated with calcium overload. However, the mechanism by which calpain is activated remains elusive since intracellular calcium levels under physiological conditions do not reach the high concentration range required to trigger calpain activation. From a candidate screening using the abundance of the calpain target glutamate receptor GluRIIA at the Drosophila neuromuscular junction as a readout, this study uncovered that calpain activity was inhibited upon knockdown of Ttm50, a subunit of the Tim23 complex known to be involved in the import of proteins across the mitochondrial inner membrane. Unexpectedly, Ttm50 and calpain are co-localized at calcium stores Golgi and endoplasmic reticulum (ER), and Ttm50 interacts with calpain via its C-terminal domain. This interaction is required for calpain localization at Golgi/ER, and increases calcium sensitivity of calpain by roughly an order of magnitude. These findings reveal the regulation of calpain activation by Ttm50, and shed new light on calpain-associated pathologies.
Oliva, M. K., Perez-Moreno, J. J., O'Shaughnessy, J., Wardill, T. J. and O'Kane, C. J. (2020). Endoplasmic Reticulum Lumenal Indicators in Drosophila Reveal Effects of HSP-Related Mutations on Endoplasmic Reticulum Calcium Dynamics. Front Neurosci 14: 816. PubMed ID: 32903680
Genes for endoplasmic reticulum (ER)-shaping proteins are among the most commonly mutated in hereditary spastic paraplegia (HSP). Mutation of these genes in model organisms can lead to disruption of the ER network. To investigate how the physiological roles of the ER might be affected by such disruption, tools were developed to interrogate its Ca(2+) signaling function. GAL4-driven Ca(2+) sensors were developed targeted to the ER lumen to record ER Ca(2+) fluxes in identified Drosophila neurons. Using GAL4 lines specific for Type Ib or Type Is larval motor neurons, this study compared the responses of different lumenal indicators to electrical stimulation, in axons and presynaptic terminals. The most effective sensor, ER-GCaMP6-210, had a Ca(2+) affinity close to the expected ER lumenal concentration. Repetitive nerve stimulation generally showed a transient increase of lumenal Ca(2+) in both the axon and presynaptic terminals. Mutants lacking neuronal reticulon and REEP proteins, homologs of human HSP proteins, showed a larger ER lumenal evoked response compared to wild type; mechanisms are proposed by which this phenotype could lead to neuronal dysfunction or degeneration. These lines are useful additions to a Drosophila Ca(2+) imaging toolkit, to explore the physiological roles of ER, and its pathophysiological roles in HSP and in axon degeneration more broadly.

Wednesday October 21 - Embryonic Neural Development-

Dow, E., Jacobo, A., Hossain, S., Siletti, K. and Hudspeth, A. J. (2018). Connectomics of the zebrafish's lateral-line neuromast reveals wiring and miswiring in a simple microcircuit. Elife 7. PubMed ID: 29893686
The lateral-line neuromast of the zebrafish displays a restricted, consistent pattern of innervation that facilitates the comparison of microcircuits across individuals, developmental stages, and genotypes. This study used serial blockface scanning electron microscopy to determine from multiple specimens the neuromast connectome, a comprehensive set of connections between hair cells and afferent and efferent nerve fibers. This analysis delineated a complex but consistent wiring pattern with three striking characteristics: each nerve terminal is highly specific in receiving innervation from hair cells of a single directional sensitivity; the innervation is redundant; and the terminals manifest a hierarchy of dominance. Mutation of the canonical planar-cell-polarity gene vangl2 (Van Gogh), which decouples the asymmetric phenotypes of sibling hair-cell pairs, results in randomly positioned, randomly oriented sibling cells that nonetheless retain specific wiring. Because larvae that overexpress Notch exhibit uniformly oriented, uniformly innervating hair-cell siblings, wiring specificity is mediated by the Notch signaling pathway.
Murate, M., Tomishige, N. and Kobayashi, T. (2020). Wrapping axons in mammals and Drosophila: Different lipids, same principle. Biochimie. PubMed ID: 32800899
Plasma membranes of axon-wrapping glial cells develop specific cylindrical bilayer membranes that surround thin individual axons or axon bundles. Axons are wrapped with single layered glial cells in lower organisms whereas in the mammalian nervous system, axons are surrounded with a characteristic complex multilamellar myelin structure. The high content of lipids in myelin suggests that lipids play crucial roles in the structure and function of myelin. The most striking feature of myelin lipids is the high content of galactosylceramide (GalCer). Serological and genetic studies indicate that GalCer plays a key role in the formation and function of the myelin sheath in mammals. In contrast to mammals, Drosophila lacks GalCer. Instead of GalCer, ceramide phosphoethanolamine (CPE) has an important role to ensheath axons with glial cells in Drosophila. GalCer and CPE share similar physical properties: both lipids have a high phase transition temperature and high packing, are immiscible with cholesterol and form helical liposomes. These properties are caused by both the strong headgroup interactions and the tight packing resulting from the small size of the headgroup and the hydrogen bonds between lipid molecules. These results suggest that mammals and Drosophila wrap axons using different lipids but the same conserved principle.
Dominici, C., Rappeneau, Q., Zelina, P., Fouquet, S. and Chedotal, A. (2018). Non-cell autonomous control of precerebellar neuron migration by Slit and Robo proteins. Development 145(2). PubMed ID: 29343636
During development, precerebellar neurons migrate tangentially from the dorsal hindbrain to the floor plate. Their axons cross it but their cell bodies stop their ventral migration upon reaching the midline. It has previously been shown that Slit chemorepellents and their receptors, Robo1 and Robo2 (see Drosophila Robo), might control the migration of precerebellar neurons in a repulsive manner. This study used a conditional knockout strategy in mice to test this hypothesis. The targeted inactivation of the expression of Robo1 and Robo2 receptors in precerebellar neurons does not perturb their migration, and they still stop at the midline. The selective ablation of the expression of all three Slit proteins in floor-plate cells has no effect on pontine neurons and only induces the migration of a small subset of inferior olivary neurons across the floor plate. Likewise, this study shows that the expression of Slit proteins in the facial nucleus is dispensable for pontine neuron migration. Together, these results show that Robo1 and Robo2 receptors act non-cell autonomously in migrating precerebellar neurons and that floor-plate signals, other than Slit proteins, must exist to prevent midline crossing.
Del Toro, D., Carrasquero-Ordaz, M. A., Chu, A., Ruff, T., Shahin, M., Jackson, V. A., Chavent, M., Berbeira-Santana, M., Seyit-Bremer, G., Brignani, S., Kaufmann, R., Lowe, E., Klein, R. and Seiradake, E. (2020). Structural Basis of Teneurin-Latrophilin Interaction in Repulsive Guidance of Migrating Neurons. Cell 180(2): 323-339 e319. PubMed ID: 31928845
Teneurins are ancient metazoan cell adhesion receptors that control brain development and neuronal wiring in higher animals. The extracellular C terminus binds the adhesion GPCR Latrophilin, forming a trans-cellular complex with synaptogenic functions. However, Teneurins (see Drosophila Ten-m), Latrophilins (see Drosophila Cirl), and FLRT proteins are also expressed during murine cortical cell migration at earlier developmental stages. This study presents crystal structures of Teneurin-Latrophilin complexes that reveal how the lectin and olfactomedin domains of Latrophilin bind across a spiraling beta-barrel domain of Teneurin, the YD shell. Structure-based protein engineering was coupled to biophysical analysis, cell migration assays, and in utero electroporation experiments to probe the importance of the interaction in cortical neuron migration. Binding of Latrophilins to Teneurins and FLRTs directs the migration of neurons using a contact repulsion-dependent mechanism. The effect is observed with cell bodies and small neurites rather than their processes. The results exemplify how a structure-encoded synaptogenic protein complex is also used for repulsive cell guidance.
Barak, R., Yom-Tov, G., Guez-Haddad, J., Gasri-Plotnitsky, L., Maimon, R., Cohen-Berkman, M., McCarthy, A. A., Perlson, E., Henis-Korenblit, S., Isupov, M. N. and Opatowsky, Y. (2019). Structural Principles in Robo Activation and Auto-inhibition. Cell 177(2): 272-285. PubMed ID: 30853216
Proper brain function requires high-precision neuronal expansion and wiring, processes controlled by the transmembrane Roundabout (Robo) receptor family and their Slit ligands (see Drosophila Robo and Slit). Despite their great importance, the molecular mechanism by which Robos' switch from "off" to "on" states remains unclear. This study reports a 3.6 Å crystal structure of the intact human Robo2 ectodomain (domains D1-8). Robo cis dimerization via D4 is conserved through hRobo1, 2, and 3 and the C. elegans homolog SAX-3 and is essential for SAX-3 function in vivo. The structure reveals two levels of auto-inhibition that prevent premature activation: (1) cis blocking of the D4 dimerization interface and (2) trans interactions between opposing Robo receptors that fasten the D4-blocked conformation. Complementary experiments in mouse primary neurons and C. elegans support the auto-inhibition model. These results suggest that Slit stimulation primarily drives the release of Robo auto-inhibition required for dimerization and activation.
Wang, J. and Ding, M. (2018). Robo and Ror function in a common receptor complex to regulate Wnt-mediated neurite outgrowth in Caenorhabditis elegans. Proc Natl Acad Sci U S A 115(10): E2254-E2263. PubMed ID: 29463707
Growing axons are exposed to various guidance cues en route to their targets, but the mechanisms that govern the response of growth cones to combinations of signals remain largely elusive. This study found that the sole Robo receptor, SAX-3 (see Drosophila Robo), in Caenorhabditis elegans functions as a coreceptor for Wnt/CWN-2 (Drosophila homolog: Wnt5) molecules. SAX-3 binds to Wnt/CWN-2 and facilitates the membrane recruitment of CWN-2. SAX-3 forms a complex with the Ror/CAM-1 receptor and its downstream effector Dsh/DSH-1, promoting signal transduction from Wnt to Dsh. sax-3 functions in Wnt-responsive cells and the SAX-3 receptor is restricted to the side of the cell from which the neurite is extended. DSH-1 has a similar asymmetric distribution, which is disrupted by sax-3 mutation. Taking these results together, it is proposed that Robo receptor can function as a Wnt coreceptor to regulate Wnt-mediated biological processes in vivo.

Tuesday, October 20th - Immune Response

Jacqueline, C., Parvy, J. P., Rollin, M. L., Faugere, D., Renaud, F., Misse, D., Thomas, F. and Roche, B. (2020). The role of innate immunity in the protection conferred by a bacterial infection against cancer: study of an invertebrate model. Sci Rep 10(1): 10106. PubMed ID: 32572049
All multicellular organisms are exposed to a diversity of infectious agents and to the emergence and proliferation of malignant cells. The protection conferred by some infections against cancer has been recently linked to the production of acquired immunity effectors such as antibodies. However, the evolution of innate immunity as a mechanism to prevent cancer and how it is jeopardized by infections remain poorly investigated. This study explored this question by performing experimental infections in two genetically modified invertebrate models (Drosophila melanogaster) that develop invasive or non-invasive neoplastic brain tumors. After quantifying tumor size and antimicrobial peptide gene expression, Drosophila larvae infected with a naturally occurring bacterium had smaller tumors compared to controls and to fungus-infected larvae. This was associated with the upregulation of genes encoding two antimicrobial peptides-diptericin and drosomycin-that are known to be important mediators of tumor cell death. It was further confirmed that tumor regression upon infection was associated with an increase in tumor cell death. Thus, this study suggests that infection could have a protective role through the production of antimicrobial peptides that increase tumor cell death. Finally, this study highlights the need to understand the role of innate immune effectors in the complex interactions between infections and cancer cell communities in order to develop innovative cancer treatment strategies.
Bartolo, G., Gonzalez, L. O., Alameh, S., Valencia, C. A. and Martchenko Shilman, M. (2020). Identification of glucocorticoid receptor in Drosophila melanogaster. BMC Microbiol 20(1): 161. PubMed ID: 32539689
Vertebrate glucocorticoid receptor (GR) is an evolutionary-conserved cortisol-regulated nuclear receptor that controls key metabolic and developmental pathways. Upon binding to cortisol, GR acts as an immunosuppressive transcription factor. Drosophila melanogaster, a model organism to study innate immunity, can also be immunosuppressed by glucocorticoids. However, while the genome of fruit fly harbors 18 nuclear receptor genes, the functional homolog of vertebrate GR has not been identified. This study demonstrated that while D. melanogaster is susceptible to Saccharomyces cerevisiae oral infection, the oral exposure to cortisol analogs, cortisone acetate or estrogen, increases fly sensitivity to yeast challenge. To understand the mechanism of this steroid-induced immunosuppression, the closest genetic GR homolog was identified as D. melanogaster Estrogen Related Receptor (ERR) gene. Drosophila ERR is necessary for cortisone acetate- and estrogen-mediated increase in sensitivity to fungal infection: while ERR mutant flies are as sensitive to the fungal challenge as the wildtype flies, the yeast-sensitivity of ERR mutants is not increased by these steroids. Interestingly, the fungal cortisone analog, ergosterol, did not increase the susceptibility of Drosophila to yeast infection. The immunosuppressive effect of steroids on the sensitivity of flies to fungi is evolutionary conserved in insects, as this study showed that estrogen significantly increases the yeast-sensitivity of Culex quinquefasciatus mosquitoes, whose genome contains a close ortholog of the fly ERR gene. This study identifies a D. melanogaster gene that structurally resembles vertebrate GR and is functionally necessary for the steroid-mediated immunosuppression to fungal infections.
Li, R., Zhou, H., Jia, C., Jin, P. and Ma, F. (2020). Drosophila Myc restores immune homeostasis of Imd pathway via activating miR-277 to inhibit imd/Tab2. PLoS Genet 16(8): e1008989. PubMed ID: 32810129
Drosophila Myc (dMyc), as a broad-spectrum transcription factor, can regulate the expression of a large number of genes to control diverse cellular processes, such as cell cycle progression, cell growth, proliferation and apoptosis. However, it remains largely unknown about whether dMyc can be involved in Drosophila innate immune response. This study has identified dMyc to be a negative regulator of Drosophila Imd pathway via the loss- and gain-of-function screening. dMyc inhibits Drosophila Imd immune response via directly activating miR-277 transcription, which further inhibit the expression of imd and Tab2-Ra/b. Importantly, dMyc can improve the survival of flies upon infection, suggesting inhibiting Drosophila Imd pathway by dMyc is vital to restore immune homeostasis that is essential for survival. Taken together, this study not only reports a new dMyc-miR-277-imd/Tab2 axis involved in the negative regulation of Drosophila Imd pathway, and provides a new insight into the complex regulatory mechanism of Drosophila innate immune homeostasis maintenance.
P, P., Tomar, A., Madhwal, S. and Mukherjee, T. (2020). Immune Control of Animal Growth in Homeostasis and Nutritional Stress in Drosophila. Front Immunol 11: 1528. PubMed ID: 32849518
A large body of research implicates the brain and fat body (liver equivalent) as central players in coordinating growth and nutritional homeostasis in multicellular animals. In this regard, an underlying connection between immune cells and growth is also evident, although mechanistic understanding of this cross-talk is scarce. This study explored the importance of innate immune cells in animal growth during homeostasis and in conditions of nutrient stress. Drosophila larvae lacking blood cells eclose as small adults and show signs of insulin insensitivity. Moreover, when exposed to dietary stress of a high-sucrose diet (HSD), these animals are further growth retarded than normally seen in regular animals raised on HSD. In contrast, larvae carrying increased number of activated macrophage-like plasmatocytes show no defects in adult growth when raised on HSD and grow to sizes almost comparable with that seen with regular diet. These observations imply a central role for immune cell activity in growth control. Mechanistically, these findings reveal a surprising influence of immune cells on balancing fat body inflammation and insulin signaling under conditions of homeostasis and nutrient overload as a means to coordinate systemic metabolism and adult growth. This work integrates both the cellular and humoral arm of the innate immune system in organismal growth homeostasis, the implications of which may be broadly conserved across mammalian systems as well.
Kosakamoto, H., Yamauchi, T., Akuzawa-Tokita, Y., Nishimura, K., Soga, T., Murakami, T., Mori, H., Yamamoto, K., Miyazaki, R., Koto, A., Miura, M. and Obata, F. (2020). Local Necrotic Cells Trigger Systemic Immune Activation via Gut Microbiome Dysbiosis in Drosophila. Cell Rep 32(3): 107938. PubMed ID: 32698005
Necrotic cells elicit an inflammatory response through their endogenous factors with damage-associated molecular patterns. Blocking apoptosis in Drosophila wings leads to the necrosis-driven systemic immune response by unknown mechanisms. This study demonstrated that immune activation in response to necrotic cells is mediated by commensal gut microbiota. Removing the microbiome attenuates hyperactivation of the innate immune signaling IMD pathway in necrosis-induced flies. Necrotic cells in wings trigger Gluconobacter expansion in the gut. An isolated Gluconobacter sp. strain is sufficient for pathological IMD activation in necrosis-induced flies, while it is not inflammatory for control animals. In addition, bacterial colonization shifts the host metabolome and shortens the lifespan of necrosis-induced flies. This study shows that local necrosis triggers a pathological systemic inflammatory response through interaction between the host and the dysbiotic gut microbiome.
Vincent, C. M., Simoes da Silva, C. J., Wadhawan, A. and Dionne, M. S. (2020). Origins of Metabolic Pathology in Francisella-Infected Drosophila. Front Immunol 11: 1419. PubMed ID: 32733472
The origins and causes of infection pathologies are often not understood. Despite this, the study of infection and immunity relies heavily on the ability to discern between potential sources of pathology. Work in the fruit fly has supported the assumption that mortality resulting from bacterial invasion is largely due to direct host-pathogen interactions, as lower pathogen loads are often associated with reduced pathology, and bacterial load upon death is predictable. However, the mechanisms through which these interactions bring about host death are complex. This study shows that infection with the bacterium Francisella novicida leads to metabolic dysregulation and, using treatment with a bacteriostatic antibiotic, this pathology was shown to be the result of direct interaction between host and pathogen. Mutants of the immune deficiency immune pathway fail to exhibit similar metabolic dysregulation, supporting the idea that the reallocation of resources for immune-related activities contributes to metabolic dysregulation. Targeted investigation into the cross-talk between immune and metabolic pathways has the potential to illuminate some of this interaction.

Monday, October 19th - Gonads

Temin, R. G. (2020). Analysis of a Strong Suppressor of Segregation Distorter in Drosophila melanogaster. Genetics. PubMed ID: 32561521
Segregation Distorter (SD) is a naturally occurring male meiotic drive system in Drosophila melanogaster, characterized by almost exclusive transmission of the SD chromosome owing to dysfunction of sperm receiving the SD+ homolog. Previous studies identified at least three closely linked loci on chromosome 2 required for distortion: Sd, the primary distorting gene; E(SD) (Enhancer of SD), which increases the strength of distortion; and Rsp (Responder), the apparent target of Sd. Strength of distortion is also influenced by linked upward modifiers including M(SD) (Modifier of SD) and St(SD) (Stabilizer of SD), and by various unlinked suppressors. Although Sd is known to encode a mutant RanGAP protein, none of the modifiers have been molecularly identified. This work focuses on the genetic and cytological characterization of a strong X-linked suppressor, Su(SD), capable of restoring Mendelian transmission in SD/SD+ males. Sd and its cohort of positive modifiers appear to act semiquantitatively in opposition to Su(SD) with distortion strength depending primarily on the total number of distorting elements rather than which particular elements are present. Su(SD) can also suppress male sterility observed in certain SD genotypes. To facilitate its eventual molecular identification, Su(SD) was localized by deletion mapping to polytene region 13C7-13E4. These studies highlight the polygenic nature of distortion and its dependence on a constellation of positive and negative modifiers, provide insight into the stability of Mendelian transmission in natural populations even when a drive system arises, and pave the way for molecular characterization of Su(SD) whose identity should reveal new information about the mechanism of distortion.
Liao, S. and Nassel, D. R. (2020). Drosophila Insulin-Like Peptide 8 (DILP8) in Ovarian Follicle Cells Regulates Ovulation and Metabolism. Front Endocrinol (Lausanne) 11: 461. PubMed ID: 32849266
In Drosophila melanogaster eight insulin-like peptides (DILP1-8) are encoded on separate genes. These DILPs are characterized by unique spatial and temporal expression patterns during the lifecycle. Whereas, functions of several of the DILPs have been extensively investigated at different developmental stages, the role of DILP8 signaling is primarily known from larvae and pupae where it couples organ growth and developmental transitions. In adult female flies, a study showed that a specific set of neurons that express the DILP8 receptor, Lgr3, is involved in regulation of reproductive behavior. This study further investigated the expression of dilp8/DILP8 and Lgr3 in adult female flies and the functional role of DILP8 signaling. The only site where both dilp8 expression and DILP8 immunolabeling was in follicle cells around mature eggs. Lgr3 expression was detected in numerous neurons in the brain and ventral nerve cord, a small set of peripheral neurons innervating the abdominal heart, as well as in a set of follicle cells close to the oviduct. Ovulation was affected in dilp8 mutants as well as after dilp8-RNAi using dilp8 and follicle cell Gal4 drivers. More eggs were retained in the ovaries and fewer were laid, indicating that DILP8 is important for ovulation. The data suggest that DILP8 signals locally to Lgr3 expressing follicle cells as well as systemically to Lgr3 expressing efferent neurons in abdominal ganglia that innervate oviduct muscle. Thus, DILP8 may act at two targets to regulate ovulation: follicle cell rupture and oviduct contractions. Furthermore, it was shown that manipulations of dilp8 expression affect starvation resistance suggesting effects on metabolism. Possibly this reflects a feedback signaling between ovaries and the CNS that ensures nutrients for ovary development. In summary, it seems that DILP8 signaling in regulation of reproduction is an ancient function, conserved in relaxin signaling in mammals.
Simonet, J. C., Foster, M. J., Lynch, E. M., Kollman, J. M., Nicholas, E., O'Reilly, A. M. and Peterson, J. R. (2020). CTP synthase polymerization in germline cells of the developing Drosophila egg supports egg production. Biol Open 9(7). PubMed ID: 32580972
Polymerization of metabolic enzymes into micron-scale assemblies is an emerging mechanism for regulating their activity. CTP synthase (CTPS) is an essential enzyme in the biosynthesis of the nucleotide CTP and undergoes regulated and reversible assembly into large filamentous structures in organisms from bacteria to humans. The purpose of these assemblies is unclear. A major challenge to addressing this question has been the inability to abolish assembly without eliminating CTPS protein. This study demonstrates that a recently reported point mutant in CTPS, Histidine 355A (H355A), prevents CTPS filament assembly in vivo and dominantly inhibits the assembly of endogenous wild-type CTPS in the Drosophila ovary. Expressing this mutant in ovarian germline cells, it was shown that disruption of CTPS assembly in early stage egg chambers reduces egg production. This effect is exacerbated in flies fed the glutamine antagonist 6-diazo-5-oxo-L-norleucine, which inhibits de novo CTP synthesis. These findings introduce a general approach to blocking the assembly of polymerizing enzymes without eliminating their catalytic activity and demonstrate a role for CTPS assembly in supporting egg production, particularly under conditions of limited glutamine metabolism.
Lin, B., Luo, J. and Lehmann, R. (2020). Collectively stabilizing and orienting posterior migratory forces disperses cell clusters in vivo. Nat Commun 11(1): 4477. PubMed ID: 32901019
Individual cells detach from cohesive ensembles during development and can inappropriately separate in disease. Although much is known about how cells separate from epithelia, it remains unclear how cells disperse from clusters lacking apical-basal polarity, a hallmark of advanced epithelial cancers. Using live imaging of the developmental migration program of Drosophila primordial germ cells (PGCs), this study showed that cluster dispersal is accomplished by stabilizing and orienting migratory forces. PGCs utilize a G protein coupled receptor (GPCR), Tre1, to guide front-back migratory polarity radially from the cluster toward the endoderm. Posteriorly positioned myosin-dependent contractile forces pull on cell-cell contacts until cells release. Tre1 mutant cells migrate randomly with transient enrichment of the force machinery but fail to separate, indicating a temporal contractile force threshold for detachment. E-cadherin is retained on the cell surface during cell separation and augmenting cell-cell adhesion does not impede detachment. Notably, coordinated migration improves cluster dispersal efficiency by stabilizing cell-cell interfaces and facilitating symmetric pulling. This study demonstrates that guidance of inherent migratory forces is sufficient to disperse cell clusters under physiological settings and present a paradigm for how such events could occur across development and disease.
Wang, X., Wang, H., Liu, L., Li, S., Emery, G. and Chen, J. (2020). Temporal Coordination of Collective Migration and Lumen Formation by Antagonism between Two Nuclear Receptors. iScience 23(7): 101335. PubMed ID: 32682323
During development, cells undergo multiple, distinct morphogenetic processes to form a tissue or organ, but how their temporal order and time interval are determined remain poorly understood. This study shows that the nuclear receptors E75 and DHR3 regulate the temporal order and time interval between the collective migration and lumen formation of a coherent group of cells named border cells during Drosophila oogenesis. E75, in response to ecdysone signaling, antagonizes the activity of DHR3 during border cell migration, and DHR3 is necessary and sufficient for the subsequent lumen formation that is critical for micropyle morphogenesis. DHR3's lumen-inducing function is mainly mediated through βFtz-f1, another nuclear receptor and transcription factor. Furthermore, both DHR3 and βFtz-f1 are required for chitin secretion into the lumen, whereas DHR3 is sufficient for chitin secretion. Lastly, DHR3 and βFtz-f1 suppress JNK signaling in the border cells to downregulate cell adhesion during lumen formation.
Kumar, T., Blondel, L. and Extavour, C. G. (2020). Topology-driven protein-protein interaction network analysis detects genetic sub-networks regulating reproductive capacity. Elife 9. PubMed ID: 32901612
Understanding the genetic regulation of organ structure is a fundamental problem in developmental biology. This study used egg-producing structures of insect ovaries, called ovarioles, to deduce systems-level gene regulatory relationships from quantitative functional genetic analysis. Previous work showed that Hippo signalling, a conserved regulator of animal organ size, regulates ovariole number in Drosophila melanogaster. To comprehensively determine how Hippo signalling interacts with other pathways in this regulation, all known signalling pathway genes were screened, and Hpo-dependent and Hpo-independent signalling requirements were identified. Network analysis of known protein-protein interactions among screen results identified independent gene regulatory sub-networks regulating one or both of ovariole number and egg laying. These sub-networks predict involvement of previously uncharacterised genes with higher accuracy than the original candidate screen. This shows that network analysis combining functional genetic and large-scale interaction data can predict function of novel genes regulating development.

Friday, October 16th - RNA

Lee, W. H., Corgiat, E. B., Rounds, J. C., Shepherd, Z., Corbett, A. H. and Moberg, K. H. (2020). A Genetic Screen Links the Disease-Associated Nab2 RNA-Binding Protein to the Planar Cell Polarity Pathway in Drosophila melanogaster. G3 (Bethesda). PubMed ID: 32817074
Mutations in the gene encoding the ubiquitously expressed RNA-binding protein ZC3H14 result in a non-syndromic form of autosomal recessive intellectual disability in humans. Studies in Drosophila have defined roles for the ZC3H14 ortholog, Nab2 (aka Drosophila Nab2 or dNab2), in axon guidance and memory due in part to interaction with a second RNA-binding protein, the fly Fragile X homolog Fmr1, and coregulation of shared Nab2-Fmr1 target mRNAs. Despite these advances, neurodevelopmental mechanisms that underlie defective axonogenesis in Nab2 mutants remain undefined. Nab2 null phenotypes in the brain mushroom bodies (MBs) resemble defects caused by alleles that disrupt the planar cell polarity (PCP) pathway, which regulates planar orientation of static and motile cells via a non-canonical arm of the Wnt/Wg pathway. A kinked bristle phenotype in surviving Nab2 mutant adults additionally suggests a defect in F-actin polymerization and bundling, a PCP-regulated processes. To test for Nab2-PCP genetic interactions, a collection of PCP mutant alleles was screened for modification of a rough-eye phenotype produced by Nab2 overexpression in the eye (GMR>Nab2) and, subsequently, for modification of a viability defect among Nab2 nulls. Multiple PCP alleles dominantly modify GMR>Nab2 eye roughening and a subset rescue low survival and thoracic bristle kinking in Nab2 zygotic nulls. Collectively, these genetic interactions identify the PCP pathway as a potential target of the Nab2 RNA-binding protein in developing eye and wing tissues and suggest that PCP signaling could contribute to neurological defects that result from loss of Drosophila Nab2 or its vertebrate ortholog ZC3H14.
Vissers, L., Kalvakuri, S., ..., Bodmer, R. and de Brouwer, A. P. M. (2020). De Novo Variants in CNOT1, a Central Component of the CCR4-NOT Complex Involved in Gene Expression and RNA and Protein Stability, Cause Neurodevelopmental Delay. Am J Hum Genet 107(1): 164-172. PubMed ID: 32553196
CNOT1 is a member of the CCR4-NOT complex, which is a master regulator, orchestrating gene expression, RNA deadenylation, and protein ubiquitination. This study reports on 39 individuals with heterozygous de novo CNOT1 variants, including missense, splice site, and nonsense variants, who present with a clinical spectrum of intellectual disability, motor delay, speech delay, seizures, hypotonia, and behavioral problems. To link CNOT1 dysfunction to the neurodevelopmental phenotype observed, variant-specific Drosophila models were generated that showed learning and memory defects upon CNOT1 knockdown. Introduction of human wild-type CNOT1 was able to rescue this phenotype, whereas mutants could not or only partially, supporting the hypothesis that CNOT1 impairment results in neurodevelopmental delay. Furthermore, the genetic interaction with autism-spectrum genes, such as ASH1L, DYRK1A, MED13, and SHANK3, was impaired in the Drosophila models. Molecular characterization of CNOT1 variants revealed normal CNOT1 expression levels, with both mutant and wild-type alleles expressed at similar levels. Analysis of protein-protein interactions with other members indicated that the CCR4-NOT complex remained intact. An integrated omics approach of patient-derived genomics and transcriptomics data suggested only minimal effects on endonucleolytic nonsense-mediated mRNA decay components, suggesting that de novo CNOT1 variants are likely haploinsufficient hypomorph or neomorph, rather than dominant negative. In summary, this study provides strong evidence that de novo CNOT1 variants cause neurodevelopmental delay with a wide range of additional co-morbidities. Whereas the underlying pathophysiological mechanism warrants further analysis, the data demonstrate an essential and central role of the CCR4-NOT complex in human brain development.
Kliuchnikova, A. A., Goncharov, A. O., Levitsky, L. I., Pyatnitskiy, M. A., Novikova, S. E., Kuznetsova, K. G., Ivanov, M. V., Ilina, I. Y., Farafonova, T. E., Zgoda, V. G., Gorshkov, M. V. and Moshkovskii, S. A. (2020). Proteome-Wide Analysis of ADAR-Mediated Messenger RNA Editing during Fruit Fly Ontogeny. J Proteome Res 19(10): 4046-4060. PubMed ID: 32866021
Adenosine-to-inosine RNA editing (see Adar) is an enzymatic post-transcriptional modification which modulates immunity and neural transmission in multicellular organisms. In particular, it involves editing of mRNA codons with the resulting amino acid substitutions. This study identified such sites for developmental proteomes of Drosophila melanogaster at the protein level using available data for 15 stages of fruit fly development from egg to imago and 14 time points of embryogenesis. In total, 40 sites were obtained, each belonging to a unique protein, including four sites related to embryogenesis. The interactome analysis has revealed that the majority of the editing-recoded proteins were associated with synaptic vesicle trafficking and actomyosin organization. Quantitation data analysis suggested the existence of a phase-specific RNA editing regulation with yet unknown mechanisms. These findings supported the transcriptome analysis results, which showed that a burst in the RNA editing occurs during insect metamorphosis from pupa to imago. Finally, targeted proteomic analysis was performed to quantify editing-recoded and genomically encoded versions of five proteins in brains of larvae, pupae, and imago insects, which showed a clear tendency toward an increase in the editing rate for each of them. These results will allow a better understanding of the protein role in physiological effects of RNA editing.
Donelick, H. M., Talide, L., Bellet, M., Aruscavage, J., Lauret, E., Aguiar, E., Marques, J. T., Meignin, C. and Bass, B. L. (2020). In vitro studies provide insight into effects of Dicer-2 helicase mutations in Drosophila melanogaster. RNA. PubMed ID: 32843367
In vitro, Drosophila melanogaster Dicer-2 (Dcr-2) uses its helicase domain to initiate processing of dsRNA with blunt (BLT) termini, and its Platform.PAZ domain to initiate processing of dsRNA with 3' overhangs (ovrs). To understand the relationship of these in vitro observations to roles of Dcr-2 in vivo, in vitro effects of two helicase mutations were compared to their impact on production of endogenous and viral siRNAs in flies. Consistent with the importance of the helicase domain in processing BLT dsRNA, both point mutations eliminated processing of BLT, but not 3'ovr, dsRNA in vitro. However, the mutations had different effects in vivo. A point mutation in the Walker A motif of the Hel1 subdomain, G31R, largely eliminated production of siRNAs in vivo, while F225G, located in the Hel2 subdomain, showed reduced levels of endogenous siRNAs, but did not significantly affect virus-derived siRNAs. In vitro assays monitoring dsRNA cleavage, dsRNA binding, ATP hydrolysis, and binding of the accessory factor Loquacious-PD, provided insight into the different effects of the mutations on processing of different sources of dsRNA in flies. In vitro studies suggest effects of the mutations in vivo relate to their effects on ATPase activity, dsRNA binding, and interactions with Loquacious-PD. These studies emphasize the importance of future studies to characterize dsRNA termini as they exist in Drosophila and other animals.
Jin, H., Xu, W., Rahman, R., Na, D., Fieldsend, A., Song, W., Liu, S., Li, C. and Rosbash, M. (2020). TRIBE editing reveals specific mRNA targets of eIF4E-BP in Drosophila and in mammals. Sci Adv 6(33): eabb8771. PubMed ID: 32851185
4E-BP (eIF4E-BP) represses translation initiation by binding to the 5' cap-binding protein eIF4E and inhibiting its activity. Although 4E-BP has been shown to be important in growth control, stress response, cancer, neuronal activity, and mammalian circadian rhythms, it is not understood how it preferentially represses a subset of mRNAs. This study successfully used HyperTRIBE (targets of RNA binding proteins identified by editing) to identify in vivo 4E-BP mRNA targets in both Drosophila and mammals under conditions known to activate 4E-BP. The protein associates with specific mRNAs, and ribosome profiling data show that mTOR inhibition changes the translational efficiency of 4E-BP TRIBE targets more substantially compared to nontargets. In both systems, these targets have specific motifs and are enriched in translation-related pathways, which correlate well with the known activity of 4E-BP and suggest that it modulates the binding specificity of eIF4E and contributes to mTOR translational specificity.
Zavortink, M., Rutt, L. N., Dzitoyeva, S., Henriksen, J. C., Barrington, C., Bilodeau, D. Y., Wang, M., Chen, X. X. L. and Rissland, O. S. (2020). The E2 Marie Kondo and the CTLH E3 ligase clear deposited RNA binding proteins during the maternal-to-zygotic transition. Elife 9. PubMed ID: 32573431
The maternal-to-zygotic transition (MZT) is a conserved step in animal development, where control is passed from the maternal to the zygotic genome. Although the MZT is typically considered from its impact on the transcriptome, previous work found that three maternally deposited Drosophila RNA-binding proteins (ME31B, Trailer Hitch [TRAL], and Cup) are also cleared during the MZT by unknown mechanisms. This study showed that these proteins are degraded by the ubiquitin-proteasome system. Marie Kondo, an E2 conjugating enzyme, and the E3 CTLH ligase are required for the destruction of ME31B, TRAL, and Cup. Structure modeling of the Drosophila CTLH complex suggests that substrate recognition is different than orthologous complexes. Despite occurring hours earlier, egg activation mediates clearance of these proteins through the Pan Gu kinase, which stimulates translation of Kdo mRNA. Clearance of the maternal protein dowry thus appears to be a coordinated, but as-yet underappreciated, aspect of the MZT.

Thursday, October 15th - Adult Neural Development and Function

Kottmeier, R., Bittern, J., Schoofs, A., Scheiwe, F., Matzat, T., Pankratz, M. and Klambt, C. (2020). Wrapping glia regulates neuronal signaling speed and precision in the peripheral nervous system of Drosophila. Nat Commun 11(1): 4491. PubMed ID: 32901033
The functionality of the nervous system requires transmission of information along axons with high speed and precision. Conductance velocity depends on axonal diameter whereas signaling precision requires a block of electrical crosstalk between axons, known as ephaptic coupling. This study used the peripheral nervous system of Drosophila larvae to determine how glia regulates axonal properties. Wrapping glial differentiation depends on gap junctions and FGF-signaling. Abnormal glial differentiation affects axonal diameter and conductance velocity and causes mild behavioral phenotypes that can be rescued by a sphingosine-rich diet. Ablation of wrapping glia does not further impair axonal diameter and conductance velocity but causes a prominent locomotion phenotype that cannot be rescued by sphingosine. Moreover, optogenetically evoked locomotor patterns do not depend on conductance speed but require the presence of wrapping glial processes. In conclusion, these data indicate that wrapping glia modulates both speed and precision of neuronal signaling.
Kitatani, Y., Tezuka, A., Hasegawa, E., Yanagi, S., Togashi, K., Tsuji, M., Kondo, S., Parrish, J. Z. and Emoto, K. (2020). Drosophila miR-87 promotes dendrite regeneration by targeting the transcriptional repressor Tramtrack69. PLoS Genet 16(8): e1008942. PubMed ID: 32764744
To remodel functional neuronal connectivity, neurons often alter dendrite arbors through elimination and subsequent regeneration of dendritic branches. However, the intrinsic mechanisms underlying this developmentally programmed dendrite regeneration and whether it shares common machinery with injury-induced regeneration remain largely unknown. Drosophila class IV dendrite arborization (C4da) sensory neurons regenerate adult-specific dendrites after eliminating larval dendrites during metamorphosis. This study shows that the microRNA miR-87 is a critical regulator of dendrite regeneration in Drosophila. miR-87 knockout impairs dendrite regeneration after developmentally-programmed pruning, whereas miR-87 overexpression in C4da neurons leads to precocious initiation of dendrite regeneration. Genetic analyses indicate that the transcriptional repressor Tramtrack69 (Ttk69) is a functional target for miR-87-mediated repression as ttk69 expression is increased in miR-87 knockout neurons, and reducing ttk69 expression restores dendrite regeneration to mutants lacking miR-87 function. miR-87 was further shown to be required for dendrite regeneration after acute injury in the larval stage, providing a mechanistic link between developmentally programmed and injury-induced dendrite regeneration.
Endo, K., Tsuchimoto, Y. and Kazama, H. (2020). Synthesis of Conserved Odor Object Representations in a Random, Divergent-Convergent Network. Neuron. PubMed ID: 32814018
Animals are capable of recognizing mixtures and groups of odors as a unitary object. However, how odor object representations are generated in the brain remains elusive. This study investigated sensory transformation between the primary olfactory center and its downstream region, the mushroom body (MB), in Drosophila and show that clustered representations for mixtures and groups of odors emerge in the MB at the population and single-cell levels. Decoding analyses demonstrate that neurons selective for mixtures and groups enhance odor generalization. Responses of these neurons and those selective for individual odors all emerge in an experimentally well-constrained model implementing divergent-convergent, random connectivity between the primary center and the MB. Furthermore, this study found that relative odor representations are conserved across animals despite this random connectivity. These results show that the generation of distinct representations for individual odors and groups and mixtures of odors in the MB can be understood in a unified computational and mechanistic framework.
Damulewicz, M., Ispizua, J. I., Ceriani, M. F. and Pyza, E. M. (2020). Communication Among Photoreceptors and the Central Clock Affects Sleep Profile. Front Physiol 11: 993. PubMed ID: 32848895
Light is one of the most important factors regulating rhythmical behavior of Drosophila melanogaster. It is received by different photoreceptors and entrains the circadian clock, which controls sleep. The retina is known to be essential for light perception, as it is composed of specialized light-sensitive cells which transmit signal to deeper parts of the brain. This study examined the role of specific photoreceptor types and peripheral oscillators located in these cells in the regulation of sleep pattern. Sleep was shown to be controlled by the visual system in a very complex way. Photoreceptors expressing Rh1, Rh3 are involved in night-time sleep regulation, while cells expressing Rh5 and Rh6 affect sleep both during the day and night. Moreover, Hofbauer-Buchner (HB) eyelets which can directly contact with s-LN (v) s and l-LN (v) s play a wake-promoting function during the day. In addition, this study showed that L2 interneurons, which receive signal from R1-6, form direct synaptic contacts with l-LN (v) s, which provides new light input to the clock network.
Bidaye, S. S., Laturney, M., Chang, A. K., Liu, Y., Bockemuhl, T., Buschges, A. and Scott, K. (2020). Two Brain Pathways Initiate Distinct Forward Walking Programs in Drosophila. Neuron. PubMed ID: 32822613
An animal at rest or engaged in stationary behaviors can instantaneously initiate goal-directed walking. How descending brain inputs trigger rapid transitions from a non-walking state to an appropriate walking state is unclear. This study identify two neuronal types, P9 and BPN, in the Drosophila brain that, upon activation, initiate and maintain two distinct coordinated walking patterns. P9 drives forward walking with ipsilateral turning, receives inputs from central courtship-promoting neurons and visual projection neurons, and is necessary for a male to pursue a female during courtship. In contrast, BPN drives straight, forward walking and is not required during courtship. BPN is instead recruited during and required for fast, straight, forward walking bouts. Thus, this study reveals separate brain pathways for object-directed walking and fast, straight, forward walking, providing insight into how the brain initiates context-appropriate walking programs.
Eadaim, A., Hahm, E. T., Justice, E. D. and Tsunoda, S. (2020). Cholinergic Synaptic Homeostasis Is Tuned by an NFAT-Mediated alpha7 nAChR-K(v)4/Shal Coupled Regulatory System. Cell Rep 32(10): 108119. PubMed ID: 32905767
Homeostatic synaptic plasticity (HSP) involves compensatory mechanisms employed by neurons and circuits to preserve signaling when confronted with global changes in activity that may occur during physiological and pathological conditions. Cholinergic neurons, which are especially affected in some pathologies, have recently been shown to exhibit HSP mediated by nicotinic acetylcholine receptors (nAChRs). In Drosophila central neurons, pharmacological blockade of activity induces a homeostatic response mediated by the Drosophila α7 (Dα7) nAChR, which is tuned by a subsequent increase in expression of the voltage-dependent K(v)4/Shal channel. This study shows that an in vivo reduction of cholinergic signaling induces HSP mediated by Dα7 nAChRs, and this upregulation of Dα7 itself is sufficient to trigger transcriptional activation, mediated by nuclear factor of activated T cells (NFAT), of the K(v)4/Shal gene, revealing a receptor-ion channel system coupled for homeostatic tuning in cholinergic neurons.

Wednesday, October 14th - Signaling

Ertekin, D., Kirszenblat, L., Faville, R. and van Swinderen, B. (2020). Down-regulation of a cytokine secreted from peripheral fat bodies improves visual attention while reducing sleep in Drosophila. PLoS Biol 18(8): e3000548. PubMed ID: 32745077
Sleep is vital for survival. Yet under environmentally challenging conditions, such as starvation, animals suppress their need for sleep. Interestingly, starvation-induced sleep loss does not evoke a subsequent sleep rebound. Little is known about how starvation-induced sleep deprivation differs from other types of sleep loss, or why some sleep functions become dispensable during starvation. This study demonstrate that down-regulation of the secreted cytokine unpaired 2 (upd2) in Drosophila flies may mimic a starved-like state. A genetic knockdown strategy to investigate the consequences of upd2 on visual attention and sleep in otherwise well-fed flies, thereby sidestepping the negative side effects of undernourishment. Knockdown of upd2 in the fat body (FB) is sufficient to suppress sleep and promote feeding-related behaviors while also improving selective visual attention. Furthermore, this study shows that this peripheral signal is integrated in the fly brain via insulin-expressing cells. Together, these findings identify a role for peripheral tissue-to-brain interactions in the simultaneous regulation of sleep quality and attention, to potentially promote adaptive behaviors necessary for survival in hungry animals.
Espinoza, C. Y. and Berg, C. A. (2020). Detecting New Allies: Modifier Screen Identifies a Genetic Interaction Between Imaginal disc growth factor 3 and combover, a Rho-kinase Substrate, During Dorsal Appendage Tube Formation in Drosophila. G3 (Bethesda). PubMed ID: 32855169
Biological tube formation underlies organ development, and when disrupted, can cause severe birth defects. To investigate the genetic basis of tubulogenesis, the formation was studied of Drosophila melanogaster eggshell structures, called dorsal appendages, which are produced by epithelial tubes. Previously it was found that precise levels of Drosophila Chitinase-like proteins (CLPs), encoded by the Imaginal disc growth factor (Idgf) gene family, are needed to regulate dorsal-appendage tube closure and tube migration. To identify factors that act in the Idgf pathway, a genetic modifier screen was developed based on the finding that overexpressing idgf3 causes dorsal appendage defects with ~50% frequency. Importantly, mutant alleles identified combover (cmb), a substrate of Rho-kinase (Rok) and a component of the Planar Cell Polarity (PCP) pathway, as an idgf3-interacting gene: loss of function enhanced while gain of function suppressed the dorsal appendage defects. Since PCP drives cell intercalation in other systems, it was asked if cmb/+ affected cell intercalation in this model, but no evidence was found of its involvement in this step. Instead, it was found that loss of cmb dominantly enhanced tube defects associated with idgf3 overexpression by expanding the apical area of dorsal appendage cells. Apical surface area determines tube volume and shape; in this way, Idgf3 and cmb regulate tube morphology.
Ewen-Campen, B., Comyn, T., Vogt, E. and Perrimon, N. (2020). No Evidence that Wnt Ligands Are Required for Planar Cell Polarity in Drosophila. Cell Rep 32(10): 108121. PubMed ID: 32905771
The frizzled (fz) and dishevelled (dsh) genes are highly conserved members of both the planar cell polarity (PCP) pathway and the Wnt signaling pathway. Given these dual functions, several studies have examined whether Wnt ligands provide a tissue-scale orientation cue for PCP establishment during development, and these studies have reached differing conclusions. This issue was reexamined in the Drosophila melanogaster wing and notum using split-Gal4 co-expression analysis, multiplex somatic CRISPR, and double RNAi experiments. Pairwise loss-of-function experiments targeting wg together with other Wnt genes, via somatic CRISPR or RNAi, do not produce PCP defects in the wing or notum. In addition, somatic CRISPR against evi (aka wntless), which is required for the secretion of Wnt ligands, did not produce detectable PCP phenotypes. Altogether, these results do not support the hypothesis that Wnt ligands contribute to PCP signaling in the Drosophila wing or notum.
Jang, Y. G., Choi, Y., Jun, K. and Chung, J. (2020). Mislocalization of TORC1 to Lysosomes Caused by KIF11 Inhibition Leads to Aberrant TORC1 Activity. Mol Cells 43(8): 705-717. PubMed ID: 32759469
While the growth factors like insulin initiate a signaling cascade to induce conformational changes in the mechanistic target of rapamycin complex 1 (mTORC1), amino acids cause the complex to localize to the site of activation, the lysosome. The precise mechanism of how mTORC1 moves in and out of the lysosome is yet to be elucidated in detail in Drosophila. This study reports that microtubules and the motor protein KIF11 are required for the proper dissociation of mTORC1 from the lysosome upon amino acid scarcity. When microtubules are disrupted or KIF11 is knocked down, mTORC1 localizes to the lysosome even in the amino acid-starved situation where it should be dispersed in the cytosol, causing an elevated mTORC1 activity. Moreover, in the mechanistic perspective, this study discovered that mTORC1 interacts with KIF11 on the motor domain of KIF11, enabling the complex to move out of the lysosome along microtubules. These results suggest not only a novel way of the regulation regarding amino acid availability for mTORC1, but also a new role of KIF11 and microtubules in mTOR signaling.
Kizhedathu, A., Kunnappallil, R. S., Bagul, A. V., Verma, P. and Guha, A. (2020). Multiple Wnts act synergistically to induce Chk1/Grapes expression and mediate G2 arrest in Drosophila tracheoblasts. Elife 9. PubMed ID: 32876044
Larval tracheae of Drosophila harbour progenitors of the adult tracheal system (tracheoblasts). Thoracic tracheoblasts are arrested in the G2 phase of the cell cycle in an ATR (mei-41)-Checkpoint Kinase1 (grapes, Chk1) dependent manner prior to mitotic re-entry. This study investigated developmental regulation of Chk1 activation. This study reports Wnt signaling is high in tracheoblasts and this is necessary for high levels of activated (phosphorylated) Chk1. Canonical Wnt signaling facilitates this by transcriptional upregulation of Chk1 expression in cells that have ATR kinase activity. Wnt signaling is dependent on four Wnts (Wg, Wnt5, 6,10) that are expressed at high levels in arrested tracheoblasts and are downregulated at mitotic re-entry. Interestingly, none of the Wnts are dispensable and act synergistically to induce Chk1. Finally, this study shows that downregulation of Wnt signaling and Chk1 expression leads to mitotic re-entry and the concomitant upregulation of Dpp signaling, driving tracheoblast proliferation.
Konstantinidis, K., Bezzerides, V. J., ..., Levine, R. L. and Anderson, M. E. (2020). MICAL1 constrains cardiac stress responses and protects against disease by oxidizing CaMKII. J Clin Invest 130(9): 4663-4678. PubMed ID: 32749237
Oxidant stress can contribute to health and disease. This study shows that invertebrates and vertebrates share a common stereospecific redox pathway that protects against pathological responses to stress, at the cost of reduced physiological performance, by constraining Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity. MICAL1, a methionine monooxygenase thought to exclusively target actin, and MSRB, a methionine reductase, control the stereospecific redox status of M308, a highly conserved residue in the calmodulin-binding (CaM-binding) domain of CaMKII. Oxidized or mutant M308 (M308V) decreased CaM binding and CaMKII activity, while absence of MICAL1 in mice caused cardiac arrhythmias and premature death due to CaMKII hyperactivation. Mimicking the effects of M308 oxidation decreased fight-or-flight responses in mice, strikingly impaired heart function in Drosophila melanogaster, and caused disease protection in human induced pluripotent stem cell-derived cardiomyocytes with catecholaminergic polymorphic ventricular tachycardia, a CaMKII-sensitive genetic arrhythmia syndrome. These studies identify a stereospecific redox pathway that regulates cardiac physiological and pathological responses to stress across species.

Tuesday, October 13th - Synapse and Vesicles

Mishima, T., Fujiwara, T., Kofuji, T., Saito, A., Terao, Y. and Akagawa, K. (2020). Syntaxin 1B regulates synaptic GABA release and extracellular GABA concentration, and is associated with temperature-dependent seizures. J Neurochem. PubMed ID: 32858780
De novo heterozygous mutations in the STX1B gene (see Drosophila Syx1A), encoding syntaxin 1B, cause a familial, fever-associated epilepsy syndrome. Syntaxin 1B is an essential component of the presynaptic neurotransmitter release machinery as a SNARE protein that regulates the exocytosis of synaptic vesicles. It is also involved in regulating the functions of the SLC6 family of neurotransmitter transporters that reuptake neurotransmitters, including inhibitory neurotransmitters, such as GABA and glycine. The purpose of this study was to elucidate the molecular mechanisms underlying the development of febrile seizures by examining the effects of syntaxin 1B haploinsufficiency on inhibitory synaptic transmission during hyperthermia in a mouse model. Stx1b(+/-) mice showed increased susceptibility to febrile seizures and drug-induced seizures. In cultured hippocampal neurons, the temperature-dependent properties of neurotransmitter release and reuptake by GABA transporter-1 (GAT-1; see Drosophila Gat) at GABAergic neurons was studied using whole-cell patch-clamp recordings. The rate of spontaneous quantal GABA release was reduced in Stx1b(+/-) mice. The hyperthermic temperature increased the tonic GABAA current in wild-type (WT) synapses, but not in Stx1b(+/-) synapses. In WT neurons, recurrent bursting activities were reduced in a GABA-dependent manner at hyperthermic temperature; however, this was abolished in Stx1b(+/-) neurons. The blockade of GAT-1 increased the tonic GABAA current and suppressed recurrent bursting activities in Stx1b(+/-) neurons at the hyperthermic temperature. These data suggest that functional abnormalities associated with GABA release and reuptake in the presynaptic terminals of GABAergic neurons may increase the excitability of the neural circuit with hyperthermia.
Catalani, E., Bongiorni, S., Taddei, A. R., Mezzetti, M., Silvestri, F., Coazzoli, M., Zecchini, S., Giovarelli, M., Perrotta, C., De Palma, C., Clementi, E., Ceci, M., Prantera, G. and Cervia, D. (2020). Defects of full-length dystrophin trigger retinal neuron damage and synapse alterations by disrupting functional autophagy. Cell Mol Life Sci. PubMed ID: 32749504
Dystrophin (dys) mutations predispose Duchenne muscular disease (DMD) patients to brain and retinal complications. Although different dys variants, including long dys products, are expressed in the retina, their function is largely unknown. This study investigated the putative role of full-length dystrophin in the homeostasis of neuro-retina and its impact on synapsis stabilization and cell fate. Retinas of mdx mice, the most used DMD model which does not express the 427-KDa dys protein (Dp427), showed overlapped cell death and impaired autophagy. Apoptotic neurons in the outer plexiform/inner nuclear layer and the ganglion cell layer had an impaired autophagy with accumulated autophagosomes. The autophagy dysfunction localized at photoreceptor axonal terminals and bipolar, amacrine, and ganglion cells. The absence of Dp427 does not cause a severe phenotype but alters the neuronal architecture, compromising mainly the pre-synaptic photoreceptor terminals and their post-synaptic sites. The analysis of two dystrophic mutants of the fruit fly Drosophila melanogaster, the homozygous Dys(E17) and Dys(EP3397), lacking functional large-isoforms of dystrophin-like protein, revealed rhabdomere degeneration. Structural damages were evident in the internal network of retina/lamina where photoreceptors make the first synapse. Both accumulated autophagosomes and apoptotic features were detected and the visual system was functionally impaired. The reactivation of the autophagosome turnover by rapamycin prevented neuronal cell death and structural changes of mutant flies and, of interest, sustained autophagy ameliorated their response to light. Overall, these findings indicate that functional full-length dystrophin is required for synapsis stabilization and neuronal survival of the retina, allowing also proper autophagy as a prerequisite for physiological cell fate and visual properties.
Scott, J., Thakar, S., Mao, Y., Qin, H., Hejran, H., Lee, S. Y., Yu, T., Klezovitch, O., Cheng, H., Mu, Y., Ghosh, S., Vasioukhin, V. and Zou, Y. (2019). Apical-Basal Polarity Signaling Components, Lgl1 and aPKCs, Control Glutamatergic Synapse Number and Function. iScience 20: 25-41. PubMed ID: 31546104
Normal synapse formation is fundamental to brain function. An apical-basal polarity (A-BP) protein, Lgl1 (see Drosophila Lgl), is present in the postsynaptic density and negatively regulates glutamatergic synapse numbers by antagonizing the atypical protein kinase Cs (aPKCs). A planar cell polarity protein, Vangl2 (see Drosophila Vang), which inhibits synapse formation, was decreased in synaptosome fractions of cultured cortical neurons from Lgl1 knockout embryos. Conditional knockout of Lgl1 in pyramidal neurons led to reduction of AMPA/NMDA ratio and impaired plasticity. Lgl1 is frequently deleted in Smith-Magenis syndrome (SMS). Lgl1 conditional knockout led to increased locomotion, impaired novel object recognition and social interaction. Lgl1+/- animals also showed increased synapse numbers, defects in open field and social interaction, as well as stereotyped repetitive behavior. Social interaction in Lgl1+/- could be rescued by NMDA antagonists. These findings reveal a role of apical-basal polarity proteins in glutamatergic synapse development and function and also suggest a potential treatment for SMS patients with Lgl1 deletion.
Heinrich, L. and Ryglewski, S. (2020). Different functions of two putative Drosophila alpha(2)delta subunits in the same identified motoneurons. Sci Rep 10(1): 13670. PubMed ID: 32792569
Voltage gated calcium channels (VGCCs) regulate neuronal excitability and translate activity into calcium dependent signaling. The α(1) subunit of high voltage activated (HVA) VGCCs associates with α(2)δ accessory subunits, which may affect calcium channel biophysical properties, cell surface expression, localization and transport and are thus important players in calcium-dependent signaling. In vertebrates, the functions of the different combinations of the four α(2)δ and the seven HVA α(1) subunits are incompletely understood, in particular with respect to partially redundant or separate functions in neurons. This study capitalizes on the relatively simpler situation in the Drosophila genetic model containing two neuronal putative α(2)δ subunits, straightjacket and CG4587, and one Ca(v)1 (Ca2+-channel protein α1 subunit D) and Ca(v)2 (Cacophony) homolog each, both with well-described functions in different compartments of identified motoneurons. Straightjacket is required for normal Ca(v)1 and Ca(v)2 current amplitudes and correct Ca(v)2 channel function in all neuronal compartments. By contrast, CG4587 does not affect Ca(v)1 or Ca(v)2 current amplitudes or presynaptic function, but is required for correct Ca(v)2 channel allocation to the axonal versus the dendritic domain. It is suggested that the two different putative α(2)δ subunits are required in the same neurons to regulate different functions of VGCCs.
Motanis, H. and Buonomano, D. (2020). Decreased reproducibility and abnormal experience-dependent plasticity of network dynamics in Fragile X circuits. Sci Rep 10(1): 14535. PubMed ID: 32884028
Fragile X syndrome is a neurodevelopmental disorder associated with a broad range of neural phenotypes. Interpreting these findings has proven challenging because some phenotypes may reflect compensatory mechanisms or normal forms of plasticity differentially engaged by experiential differences. To help minimize compensatory and experiential influences, an ex vivo approach was used to study network dynamics and plasticity of cortical microcircuits. In Fmr1(-/y) circuits, the spatiotemporal structure of Up-states was less reproducible, suggesting alterations in the plasticity mechanisms governing network activity. Chronic optical stimulation revealed normal homeostatic plasticity of Up-states, however, Fmr1(-/y) circuits exhibited abnormal experience-dependent plasticity as they did not adapt to chronically presented temporal patterns in an interval-specific manner. These results, suggest that while homeostatic plasticity is normal, Fmr1(-/y) circuits exhibit deficits in the ability to orchestrate multiple forms of synaptic plasticity and to adapt to sensory patterns in an experience-dependent manner-which is likely to contribute to learning deficits.
Bernadzki, K. M., Daszczuk, P., Rojek, K. O., Pezinski, M., Gawor, M., Pradhan, B. S., de Cicco, T., Bijata, M., Bijata, K., Wlodarczyk, J., Proszynski, T. J. and Niewiadomski, P. (2020). Arhgef5 Binds alpha-Dystrobrevin 1 and Regulates Neuromuscular Junction Integrity. Front Mol Neurosci 13: 104. PubMed ID: 32587503
The neuromuscular junctions (NMJs) connect muscle fibers with motor neurons and enable the coordinated contraction of skeletal muscles. The dystrophin-associated glycoprotein complex (DGC) is an essential component of the postsynaptic machinery of the NMJ and is important for the maintenance of NMJ structural integrity. To identify novel proteins that are important for NMJ organization, a mass spectrometry-based screen was performed for interactors of alpha-dystrobrevin 1 (aDB1; see Drosophila Dyb), one of the components of the DGC. The guanidine nucleotide exchange factor (GEF) Arhgef5 (see Drosophila Ephexin) was found to be one of the aDB1 binding partners that is recruited to Tyr-713 in a phospho-dependent manner. Arhgef5 localizes to the NMJ and its genetic depletion in the muscle causes the fragmentation of the synapses in conditional knockout mice. Arhgef5 loss in vivo is associated with a reduction in the levels of active GTP-bound RhoA and Cdc42 GTPases, highlighting the importance of actin dynamics regulation for the maintenance of NMJ integrity (Bernadzki, 2020).

Monday, October 12th - Cytoskeleton and Junctions

Zhang, S., Teng, X., Toyama, Y. and Saunders, T. E. (2020). Periodic Oscillations of Myosin-II Mechanically Proofread Cell-Cell Connections to Ensure Robust Formation of the Cardiac Vessel. Curr Biol. PubMed ID: 32679105
Actomyosin networks provide the major contractile machinery for regulating cell and tissue morphogenesis during development. These networks undergo dynamic rearrangements, enabling cells to have a broad range of mechanical actions. How cells integrate different mechanical stimuli to accomplish complicated tasks in vivo remains unclear. This study explored this problem in the context of cell matching, where individual cells form precise inter-cellular connections between partner cells. To study the dynamic roles of actomyosin networks in regulating precise cell matching, focus was placed on the process of heart formation during Drosophila embryogenesis, where selective filopodia-binding adhesions ensure precise cell alignment. Non-muscle Myosin II clusters periodically oscillate within cardioblasts with ~4-min intervals. Filopodia dynamics-including protrusions, retraction, binding stabilization, and binding separation-are correlated with the periodic localization of Myosin II clusters at the cell leading edge. Perturbing the Myosin II activity and oscillatory pattern alters the filopodia properties and binding dynamics and results in mismatched cardioblasts. By simultaneously changing the activity of Myosin II and filopodia adhesion levels, it was further demonstrated that levels of Myosin II and adhesion are balanced to ensure precise connectivity between cardioblasts. Combined, a mechanical proofreading machinery of robust cell matching is proposed, whereby oscillations of Myosin II within cardioblasts periodically probe filopodia adhesion strength and ensure correct cell-cell connection formation.
Esmangart de Bournonville, T. and Le Borgne, R. (2020). Interplay between Anakonda, Gliotactin, and M6 for Tricellular Junction Assembly and Anchoring of Septate Junctions in Drosophila Epithelium. Curr Biol. PubMed ID: 32857971
In epithelia, tricellular junctions (TCJs) serve as pivotal sites for barrier function and integration of both biochemical and mechanical signals. In Drosophila, TCJs are composed of the transmembrane protein Sidekick at the adherens junction (AJ) level, which plays a role in cell-cell contact rearrangement. At the septate junction (SJ) level, TCJs are formed by Gliotactin (Gli), Anakonda (Aka), and the Myelin proteolipid protein (PLP) M6. Despite previous data on TCJ organization, TCJ assembly, composition, and links to adjacent bicellular junctions (BCJs) remain poorly understood. This study has characterized the making of TCJs within the plane of adherens junctions (tricellular adherens junction [tAJ]) and the plane of septate junctions (tricellular septate junction [tSJ]) and reports that their assembly is independent of each other. Aka and M6, whose localizations are interdependent, act upstream to localize Gli. In turn, Gli stabilizes Aka at tSJ. Moreover, tSJ components are not only essential at vertex, as it was found that loss of tSJ integrity induces micron-length bicellular SJ (bSJ) deformations. This phenotype is associated with the disappearance of SJ components at tricellular contacts, indicating that bSJs are no longer connected to tSJs. Reciprocally, SJ components are required to restrict the localization of Aka and Gli at vertex. It is proposed that tSJs function as pillars to anchor bSJs to ensure the maintenance of tissue integrity in Drosophila proliferative epithelia.
Frye, M., Stritt, S., Ortsater, H., Hernandez Vasquez, M., Kaakinen, M., Vicente, A., Wiseman, J., Eklund, L., Martinez-Torrecuadrada, J. L., Vestweber, D. and Makinen, T. (2020). EphrinB2-EphB4 signalling provides Rho-mediated homeostatic control of lymphatic endothelial cell junction integrity. Elife 9. PubMed ID: 32897857
Endothelial integrity is vital for homeostasis and adjusted to tissue demands. Although fluid uptake by lymphatic capillaries is a critical attribute of the lymphatic vasculature, the barrier function of collecting lymphatic vessels is also important by ensuring efficient fluid drainage as well as lymph node delivery of antigens and immune cells. This study identified the transmembrane ligand EphrinB2 (see Drosophila Ephrin) and its receptor EphB4 (see Drosophila Eph) as critical homeostatic regulators of collecting lymphatic vessel integrity. Conditional gene deletion in mice revealed that EphrinB2/EphB4 signalling is dispensable for blood endothelial barrier function, but required for stabilization of lymphatic endothelial cell (LEC) junctions in different organs of juvenile and adult mice. Studies in primary human LECs further showed that basal EphrinB2/EphB4 signalling controls junctional localisation of the tight junction protein CLDN5 and junction stability via Rac1/Rho-mediated regulation of cytoskeletal contractility. EphrinB2/EphB4 signalling therefore provides a potential therapeutic target to selectively modulate lymphatic vessel permeability and function.
Bloemink, M. J., Hsu, K. H., Geeves, M. A. and Bernstein, S. I. (2020). Alternative N-terminal regions of Drosophila myosin heavy chain II regulate communication of the purine binding loop with the essential light chain. J Biol Chem. PubMed ID: 32817166
This study investigated the properties of one of the four alternative exon-encoded regions within the Drosophila myosin catalytic domain. This region is encoded by alternative exons 3a and 3b and includes part of the N-terminal β-barrel. Chimeric myosin constructs (IFI-3a and EMB-3b) were generated by exchanging the exon 3-encoded areas between native slow embryonic body wall (EMB) and fast indirect flight muscle myosin isoforms (IFI). This exchange alters the kinetic properties of the myosin S1 head. The ADP release rate (k-D) in the absence of actin is completely reversed for each chimera compared to the native isoforms. Steady-state data also suggest a reciprocal shift, with basal and actin-activated ATPase activity of IFI-3a showing reduced values compared to wild-type IFI, whereas for EMB-3b these values are increased compared to wild-type EMB. ATP-induced dissociation of acto-S1 (K(1)k(+2)) is reduced for both exon 3 chimeras. Homology modeling, combined with a recently reported crystal structure for Drosophila EMB, indicate that the exon 3 encoded region in the myosin head is part of the communication pathway between the nucleotide binding pocket (purine-binding loop) and the essential light chain, emphasizing an important role for this variable N-terminal domain in regulating acto-myosin cross-bridge kinetics, in particular with respect to the force-sensing properties of myosin isoforms.
Witte, L., Linnemannstoens, K., Schmidt, K., Honemann-Capito, M., Grawe, F., Wodarz, A. and Gross, J. C. (2020). Kinesin motor Klp98A mediates apical to basal Wg transport. Development. PubMed ID: 32665246
Development and tissue homeostasis rely on the tight regulation of morphogen secretion. In the Drosophila wing imaginal disc epithelium, Wg secretion for long-range signal transduction occurs after apical Wg entry into the endosomal system, followed by secretory endosomal transport. While Wg release appears to occur from the apical and basal cell side, its exact post-endocytic fate and the functional relevance of polarized endosomal Wg trafficking is poorly understood. This study identify the kinesin-3 family member Klp98A as the master regulator of intracellular Wg transport after apical endocytosis. In the absence of Klp98A functional mature endosomes accumulate in the apical cytosol and endosome transport to the basal cytosol is perturbed. Despite the resulting Wg mislocalization, Wg signal transduction occurs normally. It is concluded that transcytosis-independent routes for Wg trafficking exist and that Wg can be recycled apically via Rab4-recycling endosomes in the absence of Klp98A.
Gerdes, J. A., Mannix, K. M., Hudson, A. M. and Cooley, L. (2020). HtsRC-Mediated Accumulation of F-actin Regulates Ring Canal Size During Drosophila melanogaster Oogenesis. Genetics. PubMed ID: 32883702
Ring canals in the female germline of Drosophila melanogaster are supported by a robust filamentous actin (F-actin) cytoskeleton, setting them apart from ring canals in other species and tissues. Previous work has identified components required for the expansion of the ring canal actin cytoskeleton but has not identified the proteins responsible for F-actin recruitment or accumulation. Using a combination of CRISPR-Cas9 mediated mutagenesis and UAS-Gal4 overexpression, it was shown that HtsRC, a component specific to female germline ring canals, is both necessary and sufficient to drive F-actin accumulation. Absence of HtsRC in the germline resulted in ring canals lacking inner rim F-actin, while overexpression of HtsRC led to larger ring canals. HtsRC functions in combination with Filamin to recruit F-actin to ectopic actin structures in somatic follicle cells. Finally, findings are presented that indicate that HtsRC expression and robust female germline ring canal expansion are important for high fecundity in fruit flies but dispensable for their fertility, a result which is consistent with an understanding of HtsRC as a newly evolved gene specific to female germline ring canals.

Friday, October 9th - Stem cells

Huyghe, A., Furlan, G., Ozmadenci, D., Galonska, C., Charlton, J., Gaume, X., Combemorel, N., Riemenschneider, C., Allegre, N., Zhang, J., Wajda, P., Rama, N., Vieugue, P., Durand, I., Brevet, M., Gadot, N., Imhof, T., Merrill, B. J., Koch, M., Mehlen, P., Chazaud, C., Meissner, A. and Lavial, F. (2020). Netrin-1 promotes naive pluripotency through Neo1 and Unc5b co-regulation of Wnt and MAPK signalling. Nat Cell Biol 22(4): 389-400. PubMed ID: 32231305
In mouse embryonic stem cells (mESCs), chemical blockade of Gsk3alpha/beta and Mek1/2 (2i) instructs a self-renewing ground state whose endogenous inducers are unknown. This study shows that the axon guidance cue Netrin-1 (see Drosophila Netrin-A and Netrin-B) promotes naive pluripotency by triggering profound signalling, transcriptomic and epigenetic changes in mESCs. Furthermore, this study demonstrates that Netrin-1 can substitute for blockade of Gsk3alpha/beta and Mek1/2 to sustain self-renewal of mESCs in combination with leukaemia inhibitory factor and regulates the formation of the mouse pluripotent blastocyst. Mechanistically, this study reveals how Netrin-1 and the balance of its receptors Neo1 (a DCC-related protein; see Drosophila Frazzled) and Unc5B (see Drosophila Unc-5) co-regulate Wnt and MAPK pathways in both mouse and human ESCs. Netrin-1 induces Fak kinase (see Drosophila Fak) to inactivate Gsk3alpha/beta (see Drosophila Shaggy) and stabilize beta-catenin while increasing the phosphatase activity of a Ppp2r2c-containing Pp2a complex to reduce Erk1/2 activity. Collectively, this work identifies Netrin-1 as a regulator of pluripotency and reveals that it mediates different effects in mESCs depending on its receptor dosage, opening perspectives for balancing self-renewal and lineage commitment.
Feng, S., Zacharioudaki, E., Millen, K. and Bray, S. J. (2020). The SLC36 transporter Pathetic is required for neural stem cell proliferation and for brain growth under nutrition restriction. Neural Dev 15(1): 10. PubMed ID: 32741363
Drosophila neuroblasts (NBs) are neural stem cells whose maintenance relies on Notch activity. NBs proliferate throughout larval stages to generate a large number of adult neurons. Their proliferation is protected under conditions of nutrition restriction. As amino acid transporters (Solute Carrier transporters, SLCs), such as SLC36, have important roles in coupling nutrition inputs to growth pathways, they may have a role in this process. For example, an SLC36 family transporter Pathetic (Path) that supports body size and neural dendrite growth in Drosophila, was identified as a putative Notch target in genome-wide studies. This study examined expression and regulation of Path in the Drosophila larval brain. Path function in NB proliferation and overall brain growth was investigated under different nutrition conditions by depleting it from specific cell types in the CNS, using mitotic recombination to generate mutant clones or by directed RNA-interference. Path is expressed in both NBs and glial cells in the Drosophila CNS. In NBs, path is directly targeted by Notch signalling via Su(H) binding at an intronic enhancer, PathNRE. This enhancer is responsive to Notch regulation both in cell lines and in vivo. Loss of path in neural stem cells delayed proliferation, consistent with it having a role in NB maintenance. Expression from pathNRE was compromised in conditions of amino acid deprivation although other Notch regulated enhancers are unaffected. However, NB-expressed Path was not required for brain sparing under amino acid deprivation. Instead, it appears that Path is important in glial cells to help protect brain growth under conditions of nutrient restriction. This study has identified a novel Notch target gene path that is required in NBs for neural stem cell proliferation, while in glia it protects brain growth under nutrition restriction.
Gadre, P., Chatterjee, S., Varshney, B. and Ray, K. (2020). Cyclin E and Cdk1 regulate the termination of germline transit-amplification process in Drosophila testis. Cell Cycle 19(14): 1786-1803. PubMed ID: 32573329
An extension of the G1 is correlated with stem cell differentiation. The role of cell cycle regulation during the subsequent transit amplification (TA) divisions is, however, unclear. This paper reports that in the Drosophila male germline lineage, the transit amplification divisions accelerate after the second TA division. The cell cycle phases, marked by Cyclin E and Cyclin B, are progressively altered during the TA. Antagonistic functions of the bag-of-marbles and the Transforming-Growth-Factor-β signaling regulate the cell division rates after the second TA division and the extent of the Cyclin E phase during the fourth TA division. Furthermore, loss of Cyclin E during the fourth TA cycle retards the cell division and induces premature meiosis in some cases. A similar reduction of Cdk1 activity during this stage arrests the penultimate division and subsequent differentiation, whereas enhancement of the Cdk1 activity prolongs the TA by one extra round. Altogether, the results suggest that modification of the cell cycle structure and the rates of cell division after the second TA division determine the extent of amplification. Also, the regulation of the Cyclin E and CDK1 functions during the penultimate TA division determines the induction of meiosis and subsequent differentiation.
Amartuvshin, O., Lin, C. H., Hsu, S. C., Kao, S. H., Chen, A., Tang, W. C., Chou, H. L., Chang, D. L., Hsu, Y. Y., Hsiao, B. S., Rastegari, E., Lin, K. Y., Wang, Y. T., Yao, C. K., Chen, G. C., Chen, B. C. and Hsu, H. J. (2020). Aging shifts mitochondrial dynamics toward fission to promote germline stem cell loss. Aging Cell: e13191. PubMed ID: 32666649
Changes in mitochondrial dynamics (fusion and fission) are known to occur during stem cell differentiation; however, the role of this phenomenon in tissue aging remains unclear. This study reports that mitochondrial dynamics are shifted toward fission during aging of Drosophila ovarian germline stem cells (GSCs), and this shift contributes to aging-related GSC loss. As GSCs age, mitochondrial fragmentation and expression of the mitochondrial fission regulator, Dynamin-related protein (Drp1), are both increased, while mitochondrial membrane potential is reduced. Moreover, preventing mitochondrial fusion in GSCs results in highly fragmented depolarized mitochondria, decreased BMP stemness signaling, impaired fatty acid metabolism, and GSC loss. Conversely, forcing mitochondrial elongation promotes GSC attachment to the niche. Importantly, maintenance of aging GSCs can be enhanced by suppressing Drp1 expression to prevent mitochondrial fission or treating with rapamycin, which is known to promote autophagy via TOR inhibition. Overall, these results show that mitochondrial dynamics are altered during physiological aging, affecting stem cell homeostasis via coordinated changes in stemness signaling, niche contact, and cellular metabolism. Such effects may also be highly relevant to other stem cell types and aging-induced tissue degeneration.
Bajpai, A., Ahmad, Q. T., Tang, H. W., Manzar, N., Singh, V., Thakur, A., Ateeq, B., Perrimon, N. and Sinha, P. (2020). A Drosophila model of oral peptide therapeutics for adult Intestinal Stem Cell tumors. Dis Model Mech 13(7). PubMed ID: 32540914
Peptide therapeutics, unlike small molecule drugs, display crucial advantages of target-specificity and the ability to block large interacting interfaces such as those of transcription factors. The transcription co-factor of the Hippo pathway, YAP/Yki, has been implicated in many cancers, and is dependent on its interaction with the DNA-binding TEAD/Sd proteins via a large Ω-loop. In addition, the mammalian Vestigial Like (VGLL) protein, specifically its TONDU domain, competitively inhibits YAP-TEAD interaction, resulting in arrest of tumor growth. This study shows that either overexpression of the TONDU peptide or its oral uptake leads to suppression of Yorkie (Yki)-driven intestinal stem cell (ISC) tumors in the adult Drosophila midgut. In addition, comparative proteomic analyses of peptide-treated and untreated tumors, together with ChIP analysis, reveal that integrin pathway members are part of the Yki-oncogenic network. Collectively, these findings establish Drosophila as a reliable in vivo platform to screen for cancer oral therapeutic peptides and reveal a tumor suppressive role for integrins in Yki-driven tumors.
Tang, R., Jiang, Z., Chen, F., Yu, W., Fan, K., Tan, J., Zhang, Z., Liu, X., Li, P. and Yuan, K. (2020). The Kinase Activity of Drosophila BubR1 Is Required for Insulin Signaling-Dependent Stem Cell Maintenance. Cell Rep 31(12): 107794. PubMed ID: 32579921
As a core component of the mitotic checkpoint complex, BubR1 has a modular organization of molecular functions, with KEN box and other motifs at the N terminus inhibiting the anaphase-promoting complex/cyclosome, and a kinase domain at the C terminus, whose function remains unsettled, especially at organismal levels. This study generated knock-in BubR1 mutations in the Drosophila genome to separately disrupt the KEN box and the kinase domain. All of the mutants are homozygously viable and fertile and show no defects in mitotic progression. The mutants without kinase activity have an increased lifespan and phenotypic changes associated with attenuated insulin signaling, including reduced InR on the cell membrane, weakened PI3K and AKT activity, and elevated expression of dFoxO targets. The BubR1 kinase-dead mutants have a reduced cap cell number in female germaria, which can be rescued by expressing a constitutively active InR. It is concluded that one major physiological role of BubR1 kinase in Drosophila is to modulate insulin signaling.

Thursday, October 8th - Signaling

Choi, H. J., Joo Cha, S., Do, H. A., Kim, H. J., Lee, J. W. and Kim, K. (2020). SCF-Slimb is critical for Glycogen synthase kinase-3beta-mediated suppression of TAF15-induced neurotoxicity in Drosophila. J Neurochem. PubMed ID: 32915460
Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disorder characterized pathologically by motor neuron degeneration and associated with aggregation of RNA-binding proteins. TATA-binding protein-associated factor 15 (TAF15) accumulates as cytoplasmic aggregates in neuronal cells, and clearance of these aggregates is considered a potential therapeutic strategy for ALS. However, the exact pathogenic mechanism of TAF15-induced neurotoxicity remains to be elucidated. Glycogen synthase kinase-3 (GSK-3) plays a critical role in the protection of ALS pathology. In this study, a transgenic fly model over-expressing human TAF15 was used to study the protective effects of Shaggy/GSK3β on TAF15-induced neuronal toxicity in Drosophila brain. Transgenic flies were examined for locomotor activity and lithium treatment. The expression level and solubility of TAF15 were assessed with western blotting, whereas immunohistochemistry was used to assess TAF15 aggregation in Drosophila brain. This study has revealed that Shaggy/GSK3β was abnormally activated in neurons of TAF15-expressing flies and its inhibition can suppress the defective phenotypes, thereby preventing retinal degeneration and locomotive activity caused by TAF15. It was also found that Shaggy/GSK3β inhibition in neuronal cells leads to a reduction in TAF15 levels. Indeed, the F-box proteins Slimb and archipelago genetically interact with TAF15 and control TAF15 protein level in Drosophila. Importantly, SCF(slimb) is a critical regulator for Shaggy/GSK3β-mediated suppression of TAF15-induced toxicity in Drosophila. The present study has provided an in vivo evidence supporting the molecular mechanism of GSK3β inhibition for protection against TAF15-linked proteinopathies.
Zhang, G. and Zhang, W. (2020). Direct protein-protein interaction network for insecticide resistance based on subcellular localization analysis in Drosophila melanogaster. J Environ Sci Health B 55(8): 732-748. PubMed ID: 32567974
This study constructed the direct protein-protein interaction network of insecticide resistance based on subcellular localization analysis. Totally 177 of 528 resistance proteins were identified and they were located in 11 subcellular localizations. Topological properties were analyzed of the network and the biological characteristics of resistance proteins, such as k-core, neighborhood connectivity, instability index and aliphatic index. They can be used to predict the key proteins and potential mechanisms from macro-perspective. The problem of resistance has not been solved fundamentally, because the development of new insecticides can't keep pace with the development speed of resistance, and the lack of understanding of molecular mechanism of resistance. As the further analysis to reduce data noise, The direct protein-protein interaction network of insecticide resistance was costructed based on subcellular localization analysis. The interaction between proteins located at the same subcellular location belongs to direct interactions, thus eliminating indirect interaction. Totally 177 of 528 resistance proteins were identified and they were located in 11 subcellular localizations. Topological properties of the network were analyzed along with the biological characteristics of resistance proteins, such as k-core, neighborhood connectivity, instability index and aliphatic index. They can be used to predict the hub proteins and potential mechanisms from macro-perspective. This is the first study to explore the insecticide resistance molecular mechanism of Drosophila melanogaster based on subcellular localization analysis. It can provide the bioinformatics foundation for further understanding the mechanisms of insecticide resistance. It also provides a reference for the study of molecular mechanism of insecticide resistance of other insects.
Berez, A., Peercy, B. E. and Starz-Gaiano, M. (2020). Development and Analysis of a Quantitative Mathematical Model of Bistability in the Cross Repression System Between APT and SLBO Within the JAK/STAT Signaling Pathway. Front Physiol 11: 803. PubMed ID: 32848815
Cell migration is a key component in development, homeostasis, immune function, and pathology. It is important to understand the molecular activity that allows some cells to migrate. Drosophila melanogaster is a useful model system because its genes are largely conserved with humans and it is straightforward to study biologically. The well-conserved transcriptional regulator Signal Transducer and Activator of Transcription (STAT) promotes cell migration, but its signaling is modulated by downstream targets Apontic (APT) and Slow Border Cells (SLBO). Inhibition of STAT activity by APT and cross-repression of APT and SLBO determines whether an epithelial cell in the Drosophila egg chamber becomes motile or remains stationary. Through mathematical modeling and analysis, this study examine how the interaction of STAT, APT, and SLBO creates bistability in the Janus Kinase (JAK)/STAT signaling pathway. This paper updates and analyzes earlier models to represent mechanistically the processes of the JAK/STAT pathway. Parameter, bifurcation, and phase portrait analyses were made, and reductions were made to the system to produce a minimal three-variable quantitative model. The manifold between migratory and stationary steady states was analyzed in this minimal model, and it was shown that when the initial conditions of the model are near this manifold, cell migration can be delayed.
Yang, D. W. and Choi, K. W. (2020). Suppression of Patronin deficiency by altered Hippo signaling in Drosophila organ development. Cell Death Differ. PubMed ID: 32737445
The microtubule network is crucial for cell structure and function. Patronin is a conserved protein involved in protecting the minus end of microtubules. Conversely, Klp10A is a kinesin-like microtubule depolymerase. This study reports the role of Drosophila Patronin and Klp10A for cell survival in developing organs. Loss of Patronin reduces the size of organs by activation of a caspase in imaginal discs. Reduced wing by Patronin RNAi is suppressed by knockdown of Spastin (Spas) but not Katanin 60, suggesting that Patronin is inhibitory to the severing function of Spas at the minus end. Patronin RNAi phenotype is also recovered by overexpressing Death-associated inhibitor of apoptosis 1 (Diap1), a Yorkie target gene. Heterozygote mutations in Hippo pathway genes, including hippo and warts (wts), suppress the Patronin RNAi wing phenotypes. Furthermore, Patronin physically interacts with Merlin and Expanded while reducing their function. Patronin and Klp10A antagonistically regulate their levels. Wing phenotypes of Patronin RNAi are rescued by knockdown of Klp10A, consistent with their antagonistic interaction. Klp10A overexpression also causes organ size reduction that is partially suppressed by Diap1 overexpression or wts heterozygote mutation. Taken together, this study suggests that the antagonistic interaction between Patronin and Klp10A is required for controlling cell survival and organ size by modulating microtubule stability and Hippo components.
Zhu, Y., Qiu, Y., Chen, W., Nie, Q. and Lander, A. D. (2020). Scaling a Dpp Morphogen Gradient through Feedback Control of Receptors and Co-receptors. Dev Cell 53(6): 724-739. PubMed ID: 32574592
Gradients of decapentaplegic (Dpp) pattern Drosophila wing imaginal discs, establishing gene expression boundaries at specific locations. As discs grow, Dpp gradients expand, keeping relative boundary positions approximately stationary. Such scaling fails in mutants for Pentagone (pent), a gene repressed by Dpp that encodes a diffusible protein that expands Dpp gradients. Although these properties fit a recent mathematical model of automatic gradient scaling, that model requires an expander that spreads with minimal loss throughout a morphogen field. This study shows that Pent's actions are confined to within just a few cell diameters of its site of synthesis and can be phenocopied by manipulating non-diffusible Pent targets strictly within the Pent expression domain. Using genetics and mathematical modeling, this study developed an alternative model of scaling driven by feedback downregulation of Dpp receptors and co-receptors. Among the model's predictions is a size beyond which scaling fails-something that was observe directly in wing discs.
Dabool, L., Hakim-Mishnaevski, K., Juravlev, L., Flint-Brodsly, N., Mandel, S. and Kurant, E. (2020). Drosophila Skp1 Homologue SkpA Plays a Neuroprotective Role in Adult Brain. iScience 23(8): 101375. PubMed ID: 32739834
Skp1, a component of the ubiquitin E3 ligases, was found to be decreased in the brains of sporadic Parkinson's disease (PD) patients, and its overexpression prevented death of murine neurons in culture. This study exposed the neuroprotective role of the Drosophila skp1 homolog, skpA, in the adult brain. Neuronal knockdown of skpA leads to accumulation of ubiquitinated protein aggregates and loss of dopaminergic neurons accompanied by motor dysfunction and reduced lifespan. Conversely, neuronal overexpression of skpA reduces aggregate load, improves age-related motor decline, and prolongs lifespan. Moreover, SkpA rescues neurodegeneration in a Drosophila model of PD. It was also shown that a Drosophila homolog of FBXO7, the F Box protein, Nutcracker (Ntc), works in the same pathway with SkpA. However, skpA overexpression rescues ntc knockdown phenotype, suggesting that SkpA interacts with additional F box proteins in the adult brain neurons. Collectively, this study discloses Skp1/SkpA as a potential therapeutic target in neurodegenerative diseases.

Wednesday October 7th - Adult Development

Behr, M. and Riedel, D. (2020). Glycosylhydrolase genes control respiratory tubes sizes and airway stability. Sci Rep 10(1): 13377. PubMed ID: 32770153
Tight barriers are crucial for animals. Insect respiratory cells establish barriers through their extracellular matrices. These chitinous-matrices must be soft and flexible to provide ventilation, but also tight enough to allow oxygen flow and protection against dehydration, infections, and environmental stresses. However, genes that control soft, flexible chitin-matrices are poorly known. This study investigated the genes of the chitinolytic glycosylhydrolase-family 18 in the tracheal system of Drosophila melanogaster. Five chitinases and three chitinase-like genes were found to organize the tracheal chitin-cuticles. Most of the chitinases degrade chitin from airway lumina to enable oxygen delivery. They further improve chitin-cuticles to enhance tube stability and integrity against stresses. Unexpectedly, some chitinases also support chitin assembly to expand the tube lumen properly. Moreover, Chitinase2 plays a decisive role in the chitin-cuticle formation that establishes taenidial folds to support tube stability. Chitinase2 is apically enriched on the surface of tracheal cells, where it controls the chitin-matrix architecture independently of other known cuticular proteins or chitinases. It is supposed that the principle mechanisms of chitin-cuticle assembly and degradation require a set of critical glycosylhydrolases for flexible and not-flexible cuticles. The same glycosylhydrolases support thick laminar cuticle formation and are evolutionarily conserved among arthropods
Zhang, S., Zhao, J., Lv, X., Fan, J., Lu, Y., Zeng, T., Wu, H., Chen, L. and Zhao, Y. (2020). Analysis on gene modular network reveals morphogen-directed development robustness in Drosophila. Cell Discov 6: 43. PubMed ID: 32637151
Genetic robustness is an important characteristic to tolerate genetic or nongenetic perturbations and ensure phenotypic stability. Morphogens, a type of evolutionarily conserved diffusible molecules, govern tissue patterns in a direction-dependent or concentration-dependent manner by differentially regulating downstream gene expression. However, whether the morphogen-directed gene regulatory network possesses genetic robustness remains elusive. In this present study, 4217 morphogen-responsive genes were collected along A-P axis of Drosophila wing discs from the RNA-seq data, and they were clustered into 12 modules. By applying mathematical model to the measured data, a gene modular network (GMN) was constructed to decipher the module regulatory interactions and robustness in morphogen-directed development. The computational analyses on asymptotical dynamics of this GMN demonstrated that this morphogen-directed GMN is robust to tolerate a majority of genetic perturbations, which has been further validated by biological experiments. Furthermore, besides the genetic alterations, it was further demonstrated that this morphogen-directed GMN can well tolerate nongenetic perturbations (Hh production changes) via computational analyses and experimental validation. Therefore, these findings clearly indicate that the morphogen-directed GMN is robust in response to perturbations and is important for Drosophila to ensure the proper tissue patterning in wing disc.
Czajkowski, E. R., Cisneros, M., Garcia, B. S., Shen, J. and Cripps, R. M. (2020). The Drosophila CG1674 gene encodes a synaptopodin 2-like related protein that localizes to the Z-disc and is required for normal flight muscle development and function. Dev Dyn. PubMed ID: 32893414
To identify novel myofibrillar components of the Drosophila flight muscles, a proteomic analysis was carried out of chemically demembranated flight muscle myofibrils, and the knockdown phenotype of a novel gene identified in the screen, CG1674, was characterized. The CG1674 protein has some similarity to vertebrate synaptopodin 2-like, and when expressed as a FLAG-tagged fusion protein, it was localized during development to the Z-disc and cytoplasm. Knockdown of CG1674 expression affected the function of multiple muscle types, and defective flight in adults was accompanied by large actin-rich structures in the flight muscles that resembled overgrown Z-discs. Localization of CG1674 to the Z-disc depended predominantly upon presence of the Z-disc component alpha-actinin, but also depended upon other Z-disc components, including Mask, Zasp52, and Sals. Re-localization of FLAG-CG1674 to the nucleus was observed in Alpha-actinin and sals knockdown animals. These studies identify and characterize a previously unreported myofibrillar component of Drosophila muscle that is necessary for proper myofibril assembly during development.
Venugopal, P., Veyssiere, H., Couderc, J. L., Richard, G., Vachias, C. and Mirouse, V. (2020). Multiple functions of the scaffold protein Discs large 5 in the control of growth, cell polarity and cell adhesion in Drosophila melanogaster. BMC Dev Biol 20(1): 10. PubMed ID: 32552730
Scaffold proteins support a variety of key processes during animal development. Mutant mouse for the MAGUK protein Discs large 5 (Dlg5) presents a general growth impairment and moderate morphogenetic defects. This study generated null mutants for Drosophila Dlg5 and shows that it owns similar functions in growth and epithelial architecture. Dlg5 is required for growth at a cell autonomous level in several tissues and at the organism level, affecting cell size and proliferation. These results are consistent with Dlg5 modulating hippo pathway in the wing disc, including the impact on cell size, a defect that is reproduced by the loss of yorkie. However, other observations indicate that Dlg5 regulates growth by at least another way that may involve Myc protein but nor PI3K neither TOR pathways. Moreover, epithelia cells mutant for Dlg5 also show a reduction of apical domain determinants, though not sufficient to induce a complete loss of cell polarity. Dlg5 is also essential, in the same cells, for the presence at Adherens junctions of N-Cadherin, but not E-Cadherin. Genetic analyses indicate that junction and polarity defects are independent. Together these data show that Dlg5 possessess several conserved functions that are independent of each other in regulating growth, cell polarity and cell adhesion. Moreover, they reveal a differential regulation of E-cadherin and N-cadherin apical localization.
Upadhyay, A., Peterson, A. J., Kim, M. J. and O'Connor, M. B. (2020). Muscle-derived Myoglianin regulates Drosophila imaginal disc growth. Muscle-derived Myoglianin regulates Drosophila imaginal disc growth. Elife 9:e51710. PubMed ID: 32633716
Organ growth and size are finely tuned by intrinsic and extrinsic signaling molecules. In Drosophila, the BMP family member Dpp is produced in a limited set of imaginal disc cells and functions as a classic morphogen to regulate pattern and growth by diffusing throughout imaginal discs. However, the role of TGFβ/Activin-like ligands in disc growth control remains ill-defined. This study demonstrates that Myoglianin (Myo), an Activin family member, and a close homolog of mammalian Myostatin (Mstn), is a muscle-derived extrinsic factor that uses canonical dSmad2-mediated signaling to regulate wing size. Ig is proposed that Myo is a myokine that helps mediate an allometric relationship between muscles and their associated appendages. Although Babo/dSmad2 signaling has been previously implicated in imaginal disc growth control, the ligand(s) responsible and their production sites(s) have not been identified. Previous in situ hybridization and RNAi knockdown experiments suggested that all three Activin-like ligands contribute to control of wing size. However, no expression of these Activin-like ligands was found in imaginal discs, with the exception of Actβ which is expressed in differentiating photoreceptors of the eye imaginal disc. It is concluded that the small wing phenotypes caused by RNAi knockdown of Actβ or daw are likely the result of off-target effects and that Myo is the only Activin-type ligand that regulates imaginal disc growth.
Zelhof, A. C., Mahato, S., Liang, X., Rylee, J., Bergh, E., Feder, L. E., Larsen, M. E., Britt, S. G. and Friedrich, M. (2020). The brachyceran de novo gene PIP82, a phosphorylation target of aPKC, is essential for proper formation and maintenance of the rhabdomeric photoreceptor apical domain in Drosophila. PLoS Genet 16(6): e1008890. PubMed ID: 32579558
The Drosophila apical photoreceptor membrane is defined by the presence of two distinct morphological regions, the microvilli-based rhabdomere and the stalk membrane. The subdivision of the apical membrane contributes to the geometrical positioning and the stereotypical morphology of the rhabdomeres in compound eyes with open rhabdoms and neural superposition. This study describes the characterization of the photoreceptor specific protein PIP82. PIP82's subcellular localization demarcates the rhabdomeric portion of the apical membrane. It was further demonstrated that PIP82 is a phosphorylation target of aPKC. PIP82 localization is modulated by phosphorylation, and in vivo, the loss of the aPKC/Crumbs complex results in an expansion of the PIP82 localization domain. The absence of PIP82 in photoreceptors leads to misshapped rhabdomeres as a result of misdirected cellular trafficking of rhabdomere proteins. Comparative analyses reveal that PIP82 originated de novo in the lineage leading to brachyceran Diptera, which is also characterized by the transition from fused to open rhabdoms. Taken together, these findings define a novel factor that delineates and maintains a specific apical membrane domain, and offers new insights into the functional organization and evolutionary history of the Drosophila retina.

Tuesday, October 6th - Adult Physiology

Armitage, E. L., Roddie, H. G. and Evans, I. R. (2020). Overexposure to apoptosis via disrupted glial specification perturbs Drosophila macrophage function and reveals roles of the CNS during injury. Cell Death Dis 11(8): 627. PubMed ID: 32796812
Apoptotic cell clearance by phagocytes is a fundamental process during development, homeostasis and the resolution of inflammation. However, the demands placed on phagocytic cells such as macrophages by this process, and the limitations these interactions impose on subsequent cellular behaviours are not yet clear. This study sought to understand how apoptotic cells affect macrophage function in the context of a genetically tractable Drosophila model in which macrophages encounter excessive amounts of apoptotic cells. Loss of the glial-specific transcription factor Repo prevents glia from contributing to apoptotic cell clearance in the developing embryo. This leads to the challenge of macrophages with large numbers of apoptotic cells in vivo. As a consequence, macrophages become highly vacuolated with cleared apoptotic cells, and their developmental dispersal and migration is perturbed. It was also shown that the requirement to deal with excess apoptosis caused by a loss of repo function leads to impaired inflammatory responses to injury. However, in contrast to migratory phenotypes, defects in wound responses cannot be rescued by preventing apoptosis from occurring within a repo mutant background. In investigating the underlying cause of these impaired inflammatory responses, it was demonstrated that wound-induced calcium waves propagate into surrounding tissues, including neurons and glia of the ventral nerve cord, which exhibit striking calcium waves on wounding, revealing a previously unanticipated contribution of these cells during responses to injury. Taken together, these results demonstrate important insights into macrophage biology and how repo mutants can be used to study macrophage-apoptotic cell interactions in the fly embryo. Furthermore, this work shows how these multipurpose cells can be 'overtasked' to the detriment of their other functions, alongside providing new insights into which cells govern macrophage responses to injury in vivo.
Zwoinska, M. K., Rodrigues, L. R., Slate, J. and Snook, R. R. (2020). Phenotypic Responses to and Genetic Architecture of Sterility Following Exposure to Sub-Lethal Temperature During Development. Front Genet 11: 573. PubMed ID: 32582294
Thermal tolerance range, based on temperatures that result in incapacitating effects, influences species' distributions and has been used to predict species' response to increasing temperature. Reproductive performance may also be negatively affected at less extreme temperatures, but such sublethal heat-induced sterility has been relatively ignored in studies addressing the potential effects of, and ability of species' to respond to, predicted climate warming. The few studies examining the link between increased temperature and reproductive performance typically focus on adults, although effects can vary between life history stages. This study assessed how sublethal heat stress during development impacted subsequent adult fertility and its plasticity, both of which can provide the raw material for evolutionary responses to increased temperature. Phenotypic and genetic variation was quantified in fertility of Drosophila melanogaster reared at standardized densities in three temperatures (25, 27, and 29°C) from a set of lines of the Drosophila Genetic Reference Panel (DGRP). Little phenotypic variation was found at the two lower temperatures with more variation at the highest temperature and for plasticity. Males were more affected than females. Despite reasonably large broad-sense heritabilities, a genome-wide association study found little evidence for additive genetic variance and no genetic variants were robustly linked with reproductive performance at specific temperatures or for phenotypic plasticity. Results on heat-induced male sterility with other DGRP results on relevant fitness traits were measured after abiotic stress, and an association was found between male susceptibility to sterility and male lifespan reduction following oxidative stress. The results suggest that sublethal stress during development has profound negative consequences on male adult reproduction, but despite phenotypic variation in a population for this response, there is limited evolutionary potential, either through adaptation to a specific developmental temperature or plasticity in response to developmental heat-induced sterility.
Weber, J. J., Kanost, M. R. and Gorman, M. J. (2020). Iron binding and release properties of transferrin-1 from Drosophila melanogaster and Manduca sexta: Implications for insect iron homeostasis. Insect Biochem Mol Biol: 103438. PubMed ID: 32735914
Transferrins belong to an ancient family of extracellular proteins. The best-characterized transferrins are mammalian proteins that function in iron sequestration or iron transport; they accomplish these functions by having a high-affinity iron-binding site in each of their two homologous lobes. Insect hemolymph transferrins (Tsf1s) also function in iron sequestration and transport; however, sequence-based predictions of their iron-binding residues have suggested that most Tsf1s have a single, lower-affinity iron-binding site. To reconcile the apparent contradiction between the known physiological functions and predicted biochemical properties of Tsf1s, this study purified and characterized the iron-binding properties of Drosophila melanogaster Tsf1 (DmTsf1), Manduca sexta Tsf1 (MsTsf1), and the amino-lobe of DmTsf1 (DmTsf1(N)). Using UV-Vis spectroscopy, this study found that these proteins bind iron, but they exhibit shifts in their spectra compared to mammalian transferrins. Through equilibrium dialysis experiments, it was determined that DmTsf1 and MsTsf1 bind only one ferric ion; their affinity for iron is high (log K'= 18), but less than that of the well-characterized mammalian transferrins (log K' ~20); and they release iron under moderately acidic conditions (pH(50) = 5.5). Iron release analysis of DmTsf1(N) suggested that iron binding in the amino-lobe is stabilized by the carboxyl-lobe. These findings will be critical for elucidating the mechanisms of Tsf1 function in iron sequestration and transport in insects.
Xu, L. and Chen, L. Y. (2020). Identification of a New Allosteric Binding Site for Cocaine in Dopamine Transporter. J Chem Inf Model. PubMed ID: 32649824
Dopamine (DA) transporter (DAT) is a major target for psychostimulant drugs of abuse such as cocaine that competitively binds to DAT, inhibits DA reuptake, and consequently increases synaptic DA levels. In addition to the central binding site inside DAT, the available experimental evidence suggests the existence of alternative binding sites on DAT, but detection and characterization of these sites are challenging by experiments alone. This study integrated multiple computational approaches to probe the potential binding sites on the wild-type Drosophila melanogaster DAT and identified a new allosteric site that displays high affinity for cocaine. This site is located on the surface of DAT, and binding of cocaine is primarily dominated by interactions with hydrophobic residues surrounding the site. Cocaine binding to this new site allosterically reduces the binding of DA/cocaine to the central binding pocket, and simultaneous binding of two cocaine molecules to a single DAT seems infeasible. Furthermore, binding of cocaine to this site was found to stabilize the conformation of DAT but alters the conformational population and thereby reduces the accessibility by DA, providing molecular insights into the inhibitory mechanism of cocaine. In addition, the results indicate that the conformations induced by cocaine binding to this site may be relevant to the oligomerization of DAT, highlighting a potential role of this new site in modulating the function of DAT.
Weigelt, C. M., Sehgal, R., Tain, L. S., Cheng, J., Esser, J., Pahl, A., Dieterich, C., Gronke, S. and Partridge, L. (2020). An Insulin-Sensitive Circular RNA that Regulates Lifespan in Drosophila. Mol Cell 79(2): 268-279. PubMed ID: 32592682
Circular RNAs (circRNAs) are abundant and accumulate with age in neurons of diverse species. However, only few circRNAs have been functionally characterized, and their role during aging has not been addressed. This study uses transcriptome profiling during aging and find that accumulation of circRNAs is slowed down in long-lived insulin mutant flies. Next, the in vivo function was tested of a circRNA generated by the sulfateless gene (circSfl), which is consistently upregulated, particularly in the brain and muscle, of diverse long-lived insulin mutants. Strikingly, lifespan extension of insulin mutants is dependent on circSfl, and overexpression of circSfl alone is sufficient to extend the lifespan. Moreover, circSfl is translated into a protein that shares the N terminus and potentially some functions with the full-length Sfl protein encoded by the host gene. This study demonstrates that insulin signaling affects global circRNA accumulation and reveals an important role of circSfl during aging in vivo.
Baccino-Calace, M., Prieto, D., Cantera, R. and Egger, B. (2020). Compartment and cell-type specific hypoxia responses in the developing Drosophila brain. Biol Open 9(8). PubMed ID: 32816692
Environmental factors such as the availability of oxygen are instructive cues that regulate stem cell maintenance and differentiation. This study used a genetically encoded biosensor to monitor the hypoxic state of neural cells in the larval brain of Drosophila. The biosensor reveals brain compartment and cell-type specific levels of hypoxia. The values correlate with differential tracheolation that is observed throughout development between the central brain and the optic lobe. Neural stem cells in both compartments show the strongest hypoxia response while intermediate progenitors, neurons and glial cells reveal weaker responses. The distance between a cell and the next closest tracheole is a good predictor of the hypoxic state of that cell. This study indicates that oxygen availability appears to be the major factor controlling the hypoxia response in the developing Drosophila brain and that cell intrinsic and cell-type specific factors contribute to modulate the response in an unexpected manner.

Monday, October 5th - Adult neural function and development

Bridi, J. C., Ludlow, Z. N., Kottler, B., Hartmann, B., Vanden Broeck, L., Dearlove, J., Goker, M., Strausfeld, N. J., Callaerts, P. and Hirth, F. (2020). Ancestral regulatory mechanisms specify conserved midbrain circuitry in arthropods and vertebrates. Proc Natl Acad Sci U S A 117(32): 19544-19555. PubMed ID: 32747566
Corresponding attributes of neural development and function suggest arthropod and vertebrate brains may have an evolutionarily conserved organization. However, the underlying mechanisms have remained elusive. This study identified a gene regulatory and character identity network defining the deutocerebral-tritocerebral boundary (DTB) in Drosophila. This network comprises genes homologous to those directing midbrain-hindbrain boundary (MHB) formation in vertebrates and their closest chordate relatives. Genetic tracing reveals that the embryonic DTB gives rise to adult midbrain circuits that in flies control auditory and vestibular information processing and motor coordination, as do MHB-derived circuits in vertebrates. DTB-specific gene expression and function are directed by cis-regulatory elements of developmental control genes that include homologs of mammalian Zinc finger of the cerebellum and Purkinje cell protein 4. Drosophila DTB-specific cis-regulatory elements correspond to regulatory sequences of human ENGRAILED-2, PAX-2, and DACHSHUND-1 that direct MHB-specific expression in the embryonic mouse brain. This study shows that cis-regulatory elements and the gene networks they regulate direct the formation and function of midbrain circuits for balance and motor coordination in insects and mammals. Regulatory mechanisms mediating the genetic specification of cephalic neural circuits in arthropods correspond to those in chordates, thereby implying their origin before the divergence of deuterostomes and ecdysozoans.
Bakshi, A., Sipani, R., Ghosh, N. and Joshi, R. (2020). Sequential activation of Notch and Grainyhead gives apoptotic competence to Abdominal-B expressing larval neuroblasts in Drosophila Central nervous system. PLoS Genet 16(8): e1008976. PubMed ID: 32866141
Neural circuitry for mating and reproduction resides within the terminal segments of central nervous system (CNS) which express Hox paralogous group 9-13 (in vertebrates) or Abdominal-B (Abd-B) in Drosophila. Terminal neuroblasts (NBs) in A8-A10 segments of Drosophila larval CNS are subdivided into two groups based on expression of transcription factor Doublesex (Dsx). While the sex specific fate of Dsx-positive NBs is well investigated, the fate of Dsx-negative NBs is not known so far. Studies with Dsx-negative NBs suggests that these cells, like their abdominal counterparts (in A3-A7 segments) use Hox, Grainyhead (Grh) and Notch to undergo cell death during larval development. This cell death also happens by transcriptionally activating RHG family of apoptotic genes through a common apoptotic enhancer in early to mid L3 stages. However, unlike abdominal NBs (in A3-A7 segments) which use increasing levels of resident Hox factor Abdominal-A (Abd-A) as an apoptosis trigger, Dsx-negative NBs (in A8-A10 segments) keep the levels of resident Hox factor Abd-B constant. These cells instead utilize increasing levels of the temporal transcription factor Grh and a rise in Notch activity to gain apoptotic competence. Biochemical and in vivo analysis suggest that Abdominal-A and Grh binding motifs in the common apoptotic enhancer also function as Abdominal-B and Grh binding motifs and maintains the enhancer activity in A8-A10 NBs. Finally, the deletion of this enhancer by the CRISPR-Cas9 method blocks the apoptosis of Dsx-negative NBs. These results highlight the fact that Hox dependent NB apoptosis in abdominal and terminal regions utilizes common molecular players (Hox, Grh and Notch), but seems to have evolved different molecular strategies to pattern CNS.
Bonnay, F., Veloso, A., Steinmann, V., Kocher, T., Abdusselamoglu, M. D., Bajaj, S., Rivelles, E., Landskron, L., Esterbauer, H., Zinzen, R. P. and Knoblich, J. A. (2020). Oxidative Metabolism Drives Immortalization of Neural Stem Cells during Tumorigenesis. Cell 182(6): 1490-1507. PubMed ID: 32916131
Metabolic reprogramming is a key feature of many cancers, but how and when it contributes to tumorigenesis remains unclear. This study demonstrates that metabolic reprogramming induced by mitochondrial fusion can be rate-limiting for immortalization of tumor-initiating cells (TICs) and trigger their irreversible dedication to tumorigenesis. Using single-cell transcriptomics, this study found that Drosophila brain tumors contain a rapidly dividing stem cell population defined by upregulation of oxidative phosphorylation (OxPhos). Targeted metabolomics and in vivo genetic screening were combined to demonstrate that OxPhos is required for tumor cell immortalization but dispensable in neural stem cells (NSCs) giving rise to tumors. Employing an in vivo NADH/NAD(+) sensor, it was shown that NSCs precisely increase OxPhos during immortalization. Blocking OxPhos or mitochondrial fusion stalls TICs in quiescence and prevents tumorigenesis through impaired NAD(+) regeneration. This work establishes a unique connection between cellular metabolism and immortalization of tumor-initiating cells.
Coelho, D. S. and Moreno, E. (2020). Neuronal Selection Based on Relative Fitness Comparison Detects and Eliminates Amyloid-beta-Induced Hyperactive Neurons in Drosophila. iScience 23(9): 101468. PubMed ID: 32866827
During adult life, damaged but viable neurons can accumulate in the organism, creating increasingly heterogeneous and dysfunctional neural circuits. One intriguing example is the aberrant increased activity of cerebral networks detected in vulnerable brain regions during preclinical stages of Alzheimer's disease. The pathophysiological contribution of these early functional alterations to the progression of Alzheimer's disease is uncertain. A unique cell selection mechanism based on relative fitness comparison between neurons is able to target and remove aberrantly active neurons generated by heterologous human amyloid-β in Drosophila. Sustained neuronal activity is sufficient to compromise neuronal fitness and upregulate the expression of the low fitness indicators Flower(LoseB) and Azot in the fly. Conversely, forced silencing of neurons restores brain fitness and reduces amyloid-β-induced cell death. The manipulation of this cell selection process, which was already proved to be conserved in humans, might be a promising new avenue to treat Alzheimer's.
Zhang, L., Yu, J., Guo, X., Wei, J., Liu, T. and Zhang, W. (2020). Parallel Mechanosensory Pathways Direct Oviposition Decision-Making in Drosophila. Curr Biol. PubMed ID: 32649914
Female Drosophila choose their sites for oviposition with deliberation. Female flies employ sensitive chemosensory systems to evaluate chemical cues for egg-laying substrates, but how they determine the physical quality of an oviposition patch remains largely unexplored. This study reports that flies evaluate the stiffness of the substrate surface using sensory structures on their appendages. The TRPV family channel Nanchung is required for the detection of all stiffness ranges tested, whereas two other proteins, Inactive and DmPiezo, interact with Nanchung to sense certain spectral ranges of substrate stiffness differences. Furthermore, Tmc is critical for sensing subtle differences in substrate stiffness. The Tmc channel is expressed in distinct patterns on the labellum and legs and the mechanosensory inputs coordinate to direct the final decision making for egg laying. This study thus reveals the machinery for deliberate egg-laying decision making in fruit flies to ensure optimal survival for their offspring.
Yalgin, C., Rovenko, B., Andjelkovic, A., Neefjes, M., Oymak, B., Dufour, E., Hietakangas, V. and Jacobs, H. T. (2020). Effects on Dopaminergic Neurons Are Secondary in COX-Deficient Locomotor Dysfunction in Drosophila. iScience 23(8): 101362. PubMed ID: 32738610
Dopaminergic (DA) neurons have been implicated as key targets in neurological disorders, notably those involving locomotor impairment, and are considered to be highly vulnerable to mitochondrial dysfunction, a common feature of such diseases. This study investigated a Drosophila model of locomotor disorders in which functional impairment is brought about by pan-neuronal RNAi knockdown of subunit COX7A of cytochrome oxidase (COX). Despite minimal neuronal loss by apoptosis, the expression and activity of tyrosine hydroxylase was decreased by half. Surprisingly, COX7A knockdown specifically targeted to DA neurons did not produce locomotor defect. Instead, using various drivers, it was found that COX7A knockdown in specific groups of cholinergic and glutamatergic neurons underlay the phenotype. Based on the main finding of this study, the vulnerability of DA neurons to mitochondrial dysfunction as a cause of impaired locomotion in other organisms, including mammals, warrants detailed investigation.

Friday, October 2nd - Disease models

Wang, Y. Y., Ma, W. W. and Peng, I. F. (2020). Screening of sleep assisting drug candidates with a Drosophila model. PLoS One 15(7): e0236318. PubMed ID: 32726319
Lately, Drosophila has been favored as a model in sleep and circadian rhythm research due to its conserved mechanism and easily manageable operation. These studies have revealed the sophisticated parameters in whole-day sleep profiles of Drosophila, drawing connections between Drosophila sleep and human sleep. This study tested several sleep deprivation protocols (mechanical shakes and light interruptions) on Drosophila and delineated their influences on Drosophila sleep. A daytime light-deprivation protocol (DD) was applied mimicking jet-lag to screen drugs that alleviate sleep deprivation. Characteristically, classical sleep-aid compounds exhibited different forms of influence: phenobarbital and pentobarbital modified total sleep time, while melatonin only shortened the latency to sleep. Such results construct the basis for further research on sleep benefits in other treatments in Drosophila. Seven herb extracts were screened, and very diverse results were found regarding their effect on sleep regulation. For instance, Panax notoginseng and Withania somnifera extracts displayed potent influence on total sleep time, while Melissa officinalis increased the number of sleep episodes. By comparing these treatments, it was possible to rank drug potency in different aspects of sleep regulation. Notably, the presence of sleep difficulties in a Drosophila Alzheimer's disease (AD) model was confirmed with an overexpression of human Aβ, and clear differences were recognized between the portfolios of drug screening effects in AD flies and in the control group. Overall, potential drug candidates and receipts for sleep problems can be identified separately for normal and AD Drosophila populations, outlining Drosophila's potential in drug screening tests in other populations if combined with the use of other genetic disease tools.
White, J. A., 2nd, Krzystek, T. J., Hoffmar-Glennon, H., Thant, C., Zimmerman, K., Iacobucci, G., Vail, J., Thurston, L., Rahman, S. and Gunawardena, S. (2020). Excess Rab4 rescues synaptic and behavioral dysfunction caused by defective HTT-Rab4 axonal transport in Huntington's disease. Acta Neuropathol Commun 8(1): 97. PubMed ID: 32611447
Huntington's disease (HD) is characterized by protein inclusions and loss of striatal neurons which result from expanded CAG repeats in the poly-glutamine (polyQ) region of the huntingtin (HTT) gene. Both polyQ expansion and loss of HTT have been shown to cause axonal transport defects. While studies show that HTT is important for vesicular transport within axons, the cargo that HTT transports to/from synapses remain elusive. This study shows that HTT is present with a class of Rab4-containing vesicles within axons in vivo. Reduction of HTT perturbs the bi-directional motility of Rab4, causing axonal and synaptic accumulations. In-vivo dual-color imaging reveal that HTT and Rab4 move together on a unique putative vesicle that may also contain synaptotagmin, synaptobrevin, and Rab11. The moving HTT-Rab4 vesicle uses kinesin-1 and dynein motors for its bi-directional movement within axons, as well as the accessory protein HIP1 (HTT-interacting protein 1). Pathogenic HTT disrupts the motility of HTT-Rab4 and results in larval locomotion defects, aberrant synaptic morphology, and decreased lifespan, which are rescued by excess Rab4. Consistent with these observations, Rab4 motility is perturbed in iNeurons derived from human Huntington's Disease (HD) patients, likely due to disrupted associations between the polyQ-HTT-Rab4 vesicle complex, accessory proteins, and molecular motors. Together, these observations suggest the existence of a putative moving HTT-Rab4 vesicle, and that the axonal motility of this vesicle is disrupted in HD causing synaptic and behavioral dysfunction. These data highlight Rab4 as a potential novel therapeutic target that could be explored for early intervention prior to neuronal loss and behavioral defects observed in HD.
Tsai, H. Y., Wu, S. C., Li, J. C., Chen, Y. M., Chan, C. C. and Chen, C. H. (2020). Loss of the Drosophila branched-chain α-keto acid dehydrogenase complex (BCKDH) results in neuronal dysfunction. Dis Model Mech. PubMed ID: 32680850
Maple syrup urine disease (MSUD) is an inherited error in the metabolism of branched-chain amino acids (BCAAs) caused by a severe deficiency of the branched chain keto-acid dehydrogenase (BCKDH) enzyme, which ultimately leads to neurological disorders. The limited therapies, including protein-restricted diets and liver transplants, are not as effective as they could be for the treatment of MSUD due to the current lack of molecular insights into the disease pathogenesis. To address this issue, a Drosophila model of MSUD was developed by knocking out the dDBT (CG5599) gene, an ortholog of the human dihydrolipoamide branched chain transacylase (DBT) subunit of BCKDH. The homozygous dDBT mutant larvae recapitulate an array of MSUD phenotypes, including aberrant BCAA accumulation, developmental defects, poor mobile behavior, and disrupted L-glutamate homeostasis. Moreover, the dDBT mutation causes neuronal apoptosis during the developmental progression of larval brains. The genetic and functional evidence generated by in vivo depletion of dDBT expression in the eye shows severe impairment of retinal rhadomeres. Further, the dDBT mutant shows elevated oxidative stress and higher lipid peroxidation accumulation in the larval brain. Therefore it is concluded from in vivo evidence that the loss of dDBT results in oxidative brain damage that may led to neuronal cell death and contribute to aspects of MSUD pathology. Importantly, when the dDBT mutants were administrated with Metformin, the aberrances in BCAA levels and motor behavior were ameliorated. This intriguing outcome strongly merits the use of the dDBT mutant as a platform for developing MSUD therapies.
Yusuff, T., Jensen, M., Yennawar, S., Pizzo, L., Karthikeyan, S., Gould, D. J., Sarker, A., Gedvilaite, E., Matsui, Y., Iyer, J., Lai, Z. C. and Girirajan, S. (2020). Drosophila models of pathogenic copy-number variant genes show global and non-neuronal defects during development. PLoS Genet 16(6): e1008792. PubMed ID: 32579612
While rare pathogenic copy-number variants (CNVs) are associated with both neuronal and non-neuronal phenotypes, functional studies evaluating these regions have focused on the molecular basis of neuronal defects. This study reports a systematic functional analysis of non-neuronal defects for homologs of 59 genes within ten pathogenic CNVs and 20 neurodevelopmental genes in Drosophila melanogaster. Using wing-specific knockdown of 136 RNA interference lines, qualitative and quantitative phenotypes were identified in 72/79 homologs, including 21 lines with severe wing defects and six lines with lethality. In fact, it was found that 10/31 homologs of CNV genes also showed complete or partial lethality at larval or pupal stages with ubiquitous knockdown. Comparisons between eye and wing-specific knockdown of 37/45 homologs showed both neuronal and non-neuronal defects, but with no correlation in the severity of defects. Disruptions were observed in cell proliferation and apoptosis in larval wing discs for 23/27 homologs, and altered Wnt, Hedgehog and Notch signaling for 9/14 homologs, including AATF/Aatf, PPP4C/Pp4-19C, and KIF11/Klp61F. These findings were further supported by tissue-specific differences in expression patterns of human CNV genes, as well as connectivity of CNV genes to signaling pathway genes in brain, heart and kidney-specific networks. These findings suggest that multiple genes within each CNV differentially affect both global and tissue-specific developmental processes within conserved pathways, and that their roles are not restricted to neuronal functions.
Tanaka, N., Okuda, M., Nishigaki, T., Tsuchiya, N., Kobayashi, Y., Uemura, T., Kumo, S., Sugimoto, H., Miyata, S. and Waku, T. (2020). Development of a brain-permeable peptide nanofiber that prevents aggregation of Alzheimer pathogenic proteins. PLoS One 15(7): e0235979. PubMed ID: 32706773
Alzheimer's disease (AD) is proposed to be induced by abnormal aggregation of amyloidβ in the brain. This study designed a brain-permeable peptide nanofiber drug from a fragment of heat shock protein to suppress aggregation of the pathogenic proteins. To facilitate delivery of the nanofiber into the brain, a protein transduction domain from Drosophila Antennapedia was incorporated into the peptide sequence. The resulting nanofiber efficiently suppressed the cytotoxicity of amyloid βby trapping amyloid β onto its hydrophobic nanofiber surface. Moreover, the intravenously or intranasally injected nanofiber was delivered into the mouse brain, and improved the cognitive function of an Alzheimer transgenic mouse model. These results demonstrate the potential therapeutic utility of nanofibers for the treatment of AD.
Zhao, X., Li, X., Shi, X. and Karpac, J. (2020). Diet-MEF2 interactions shape lipid droplet diversification in muscle to influence Drosophila lifespan. Aging Cell 19(7). PubMed ID: 32537848
Using Drosophila, a role for was uncovered for myocyte enhancer factor 2 (MEF2) in modulating diet-dependent lipid droplet diversification within adult striated muscle, impacting mortality rates. Muscle-specific attenuation of MEF2, whose chronic activation maintains glucose and mitochondrial homeostasis, leads to the accumulation of large, cholesterol ester-enriched intramuscular lipid droplets in response to high calorie, carbohydrate-sufficient diets. The diet-dependent accumulation of these lipid droplets also correlates with both enhanced stress protection in muscle and increases in organismal lifespan. Furthermore, MEF2 attenuation releases an antagonistic regulation of cell cycle gene expression programs, and up-regulation of Cyclin E is required for diet- and MEF2-dependent diversification of intramuscular lipid droplets. The integration of MEF2-regulated gene expression networks with dietary responses thus plays a critical role in shaping muscle metabolism and function, further influencing organismal lifespan. Together, these results highlight a potential protective role for intramuscular lipid droplets during dietary adaptation.

Thursday, October 1 - Adult Neural Development and Function

Nguyen, A. Q., Sutley, S., Koeppen, J., Mina, K., Woodruff, S., Hanna, S., Vengala, A., Hickmott, P. W., Obenaus, A. and Ethell, I. M. (2020). Astrocytic Ephrin-B1 Controls Excitatory-Inhibitory Balance in Developing Hippocampus. J Neurosci 40(36): 6854-6871. PubMed ID: 32801156
Astrocytes are implicated in synapse formation and elimination, which are associated with developmental refinements of neuronal circuits. Astrocyte dysfunctions are also linked to synapse pathologies associated with neurodevelopmental disorders and neurodegenerative diseases. Although several astrocyte-derived secreted factors are implicated in synaptogenesis, the role of contact-mediated glial-neuronal interactions in synapse formation and elimination during development is still unknown. This study examined whether the loss or overexpression of the membrane-bound ephrin-B1 (see Drosophila Ephrin) in astrocytes during postnatal day (P) 14-28 period would affect synapse formation and maturation in the developing hippocampus. Enhanced excitation of CA1 pyramidal neurons in astrocyte-specific ephrin-B1 KO male mice, which coincided with a greater vGlut1/PSD95 colocalization, higher dendritic spine density, and enhanced evoked AMPAR and NMDAR EPSCs. In contrast, EPSCs were reduced in CA1 neurons neighboring ephrin-B1-overexpressing astrocytes. Overexpression of ephrin-B1 in astrocytes during P14-28 developmental period also facilitated evoked IPSCs in CA1 neurons, while evoked IPSCs and miniature IPSC amplitude were reduced following astrocytic ephrin-B1 loss. Lower numbers of parvalbumin-expressing cells and a reduction in the inhibitory VGAT/gephyrin-positive synaptic sites on CA1 neurons in the stratum pyramidale and stratum oriens layers of KO hippocampus may contribute to reduced inhibition and higher excitation. Finally, dysregulation of excitatory/inhibitory balance in KO male mice is most likely responsible for impaired sociability observed in these mice. The ability of astrocytic ephrin-B1 to influence both excitatory and inhibitory synapses during development can potentially contribute to developmental refinement of neuronal circuits.
Ghimire, S. R. and Deans, M. R. (2019). Frizzled3 and Frizzled6 Cooperate with Vangl2 to Direct Cochlear Innervation by Type II Spiral Ganglion Neurons. J Neurosci 39(41): 8013-8023. PubMed ID: 31462532
Type II spiral ganglion neurons provide afferent innervation to outer hair cells of the cochlea and are proposed to have nociceptive functions important for auditory function and homeostasis. These neurons are anatomically distinct from other classes of spiral ganglion neurons because they extend a peripheral axon beyond the inner hair cells that subsequently makes a distinct 90 degree turn toward the cochlear base. As a result, patterns of outer hair cell innervation are coordinated with the tonotopic organization of the cochlea. Previously, it was shown that peripheral axon turning is directed by a nonautonomous function of the core planar cell polarity (PCP) protein VANGL2 (see Drosophila Van Gogh). Using mice of either sex it was demonstrated that Fzd3 and Fzd6 similarly regulate axon turning, are functionally redundant with each other, and that Fzd3 genetically interacts with Vangl2 to guide this process. FZD3 and FZD6 (see Drosophila Frizzled) proteins are asymmetrically distributed along the basolateral wall of cochlear-supporting cells, and are required to promote or maintain the asymmetric distribution of VANGL2 and CELSR1. These data indicate that intact PCP complexes formed between cochlear-supporting cells are required for the nonautonomous regulation of axon pathfinding. Consistent with this, in the absence of PCP signaling, peripheral axons turn randomly and often project toward the cochlear apex. Additional analyses of Porcn (see Drosophila Porcupine) mutants in which WNT secretion is reduced suggest that noncanonical WNT signaling establishes or maintains PCP signaling in this context. A deeper understanding of these mechanisms is necessary for repairing auditory circuits following acoustic trauma or promoting cochlear reinnervation during regeneration-based deafness therapies.
Jia, R., Chai, Y., Xie, C., Liu, G., Zhu, Z., Huang, K., Li, W. and Ou, G. (2020). The spectrin-based membrane skeleton is asymmetric and remodels during neural development in C. elegans. J Cell Sci 133(15). PubMed ID: 32620698
Perturbation of spectrin-based membrane mechanics (see Drosophila α Spectrin) causes hereditary elliptocytosis and spinocerebellar ataxia, but the underlying cellular basis of pathogenesis remains unclear. This study introduced conserved disease-associated spectrin mutations into the Caenorhabditis elegans genome and studied the contribution of spectrin to neuronal migration and dendrite formation in developing larvae. The loss of spectrin resulted in ectopic actin polymerization outside of the existing front and secondary membrane protrusions, leading to defective neuronal positioning and dendrite morphology in adult animals. Spectrin accumulated in the lateral region and rear of migrating neuroblasts and redistributes from the soma into the newly formed dendrites, indicating that the spectrin-based membrane skeleton is asymmetric and remodels to regulate actin assembly and cell shape during development. Spectrin was affinity-purified from C. elegans, and its binding partner ankyrin was shown to function with spectrin. Asymmetry and remodeling of the membrane skeleton might enable spatiotemporal modulation of membrane mechanics for distinct developmental events.
McEachern, E. P., Coley, A. A., Yang, S. S. and Gao, W. J. (2020). PSD-95 deficiency alters GABAergic inhibition in the prefrontal cortex. Neuropharmacology 179: 108277. PubMed ID: 32818520
Postsynaptic Density Protein-95 (see Drosophila PSD-95) is a major scaffolding protein in the excitatory synapses in the brain and a critical regulator of synaptic maturation for NMDA and AMPA receptors. PSD-95 deficiency has been linked to cognitive and learning deficits implicated in neurodevelopmental disorders such as autism and schizophrenia. Previous studies have shown that PSD-95 deficiency causes a significant reduction in the excitatory response in the hippocampus. However, little is known about whether PSD-95 deficiency will affect gamma-aminobutyric acid (GABA)ergic inhibitory synapses. Using a PSD-95 transgenic mouse model (PSD-95(+/-)), how PSD-95 deficiency affects GABAA receptor expression and function in the medial prefrontal cortex (mPFC) during adolescence was studied. The results showed a significant increase in the GABAA receptor subunit alpha1. Correspondingly, there are increases in the frequency and amplitude in spontaneous inhibitory postsynaptic currents (sIPSCs) in pyramidal neurons in the mPFC of PSD-95(+/-) mice, along with a significant increase in evoked IPSCs, leading to a dramatic shift in the excitatory-to-inhibitory balance in PSD-95 deficient mice. Furthermore, PSD-95 deficiency promotes inhibitory synapse function via upregulation and trafficking of NLGN2 and reduced GSK3beta activity through tyr-216 phosphorylation. This study provides novel insights on the effects of GABAergic transmission in the mPFC due to PSD-95 deficiency and its potential link with cognitive and learning deficits associated with neuropsychiatric disorders.
Tai, Y., Gallo, N. B., Wang, M., Yu, J. R. and Van Aelst, L. (2019). Axo-axonic Innervation of Neocortical Pyramidal Neurons by GABAergic Chandelier Cells Requires AnkyrinG-Associated L1CAM. Neuron 102(2): 358-372. PubMed ID: 30846310
Among the diverse interneuron subtypes in the neocortex, chandelier cells (ChCs) are the only population that selectively innervate pyramidal neurons (PyNs) at their axon initial segment (AIS), the site of action potential initiation, allowing them to exert powerful control over PyN output. Yet, mechanisms underlying their subcellular innervation of PyN AISs are unknown. To identify molecular determinants of ChC/PyN AIS innervation, an in vivo RNAi screen was performed of PyN-expressed axonal cell adhesion molecules (CAMs) and select Ephs/ephrins. Strikingly, it waw found the L1 family member L1CAM (see Drosophila Neuroglian) is the only molecule required for ChC/PyN AIS innervation. Further, it was shown that L1CAM is required during both the establishment and maintenance of innervation, and that selective innervation of PyN AISs by ChCs requires AIS anchoring of L1CAM by the cytoskeletal ankyrin-G/betaIV-spectrin complex. Thus, these findings identify PyN-expressed L1CAM as a critical CAM required for innervation of neocortical PyN AISs by ChCs.
Brignani, S., Raj, D. D. A., Schmidt, E. R. E., Dudukcu, O., Adolfs, Y., De Ruiter, A. A., Rybiczka-Tesulov, M., Verhagen, M. G., van der Meer, C., Broekhoven, M. H., Moreno-Bravo, J. A., Grossouw, L. M., Dumontier, E., Cloutier, J. F., Chedotal, A. and Pasterkamp, R. J. (2020). Remotely Produced and Axon-Derived Netrin-1 Instructs GABAergic Neuron Migration and Dopaminergic Substantia Nigra Development. Neuron 107(4): 684-702. PubMed ID: 32562661
The midbrain dopamine (mDA) system is composed of molecularly and functionally distinct neuron subtypes that mediate specific behaviors and show select disease vulnerability, including in Parkinson's disease. Despite progress in identifying mDA neuron subtypes, how these neuronal subsets develop and organize into functional brain structures remains poorly understood. This study generated and used an intersectional genetic platform, Pitx3-ITC, to dissect the mechanisms of substantia nigra (SN) development and implicate the guidance molecule Netrin-1 (see Drosophila Netrins) in the migration and positioning of mDA neuron subtypes in the SN. Unexpectedly, this study shows that Netrin-1, produced in the forebrain and provided to the midbrain through axon projections, instructs the migration of GABAergic neurons into the ventral SN. This migration is required to confine mDA neurons to the dorsal SN. These data demonstrate that neuron migration can be controlled by remotely produced and axon-derived secreted guidance cues, a principle that is likely to apply more generally.
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