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Tuesday, April 29th, 2024 - Disease Models

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Willot, Q., du Toit, A., de Wet, S., Huisamen, E. J., Loos, B., Terblanche, J. S. (2023). Exploring the connection between autophagy and heat-stress tolerance in Drosophila melanogaster. Proceedings Biological sciences, 290(2006):20231305 PubMed ID: 37700658
Mechanisms aimed at recovering from heat-induced damages are closely associated with the ability of ectotherms to survive exposure to stressful temperatures. Autophagy, a ubiquitous stress-responsive catabolic process, has recently gained renewed attention as one of these mechanisms. By increasing the turnover of cellular structures as well as the clearance of long-lived protein and protein aggregates, the induction of autophagy has been linked to increased tolerance to a range of abiotic stressors in diverse ectothermic organisms. However, whether a link between autophagy and heat-tolerance exists in insect models remains unclear despite broad ecophysiological implications thereof. This study explored the putative association between autophagy and heat-tolerance using Drosophila melanogaster as a model. It was hypothesized that (i) heat-stress would cause an increase of autophagy in flies' tissues, and (ii) rapamycin exposure would trigger a detectable autophagic response in adults and increase their heat-tolerance. In line with this hypothesis, flies exposed to heat-stress present signs of protein aggregation and appear to trigger an autophagy-related homoeostatic response as a result. It was further shown that rapamycin feeding causes the systemic effect associated with target of rapamycin (TOR) inhibition, induces autophagy locally in the fly gut, and increases the heat-stress tolerance of individuals. These results argue in favour of a substantial contribution of autophagy to the heat-stress tolerance mechanisms of insects.
Yoo, J., Dombrovski, M., Mirshahidi, P., Nern, A., LoCascio, S. A., Zipursky, S. L., Kurmangaliyev, Y. Z. (2023). Brain wiring determinants uncovered by integrating connectomes and transcriptomes. Curr Biol, 33(18):3998-4005. PubMed ID: 37647901
Advances in brain connectomics have demonstrated the extraordinary complexity of neural circuits. Developing neurons encounter the axons and dendrites of many different neuron types and form synapses with only a subset of them. During circuit assembly, neurons express cell-type-specific repertoires comprising many cell adhesion molecules (CAMs) that can mediate interactions between developing neurites. Many CAM families have been shown to contribute to brain wiring in different ways. It has been challenging, however, to identify receptor-ligand pairs directly matching neurons with their synaptic targets. This study integrated the synapse-level connectome of the neural circuit with the developmental expression patterns and binding specificities of CAMs on pre- and postsynaptic neurons in the Drosophila visual system. To overcome the complexity of neural circuits, focus was placed on pairs of genetically related neurons that make differential wiring choices. In the motion detection circuit, closely related subtypes of T4/T5 neurons choose between alternative synaptic targets in adjacent layers of neuropil. This choice correlates with the matching expression in synaptic partners of different receptor-ligand pairs of the Beat and Side families of CAMs. Genetic analysis demonstrated that presynaptic Side-II and postsynaptic Beat-VI restrict synaptic partners to the same layer. Removal of this receptor-ligand pair disrupts layers and leads to inappropriate targeting of presynaptic sites and postsynaptic dendrites. It is proposed that different Side/Beat receptor-ligand pairs collaborate with other recognition molecules to determine wiring specificities in the fly brain. Combining transcriptomes, connectomes, and protein interactome maps allow unbiased identification of determinants of brain wiring.
Yamada, T., Yoshinari, Y., Tobo, M., Habara, O., Nishimura, T. (2023). Nacalpha protects the larval fat body from cell death by maintaining cellular proteostasis in Drosophila. Nat Commun, 14(1):5328 PubMed ID: 37658058
Protein homeostasis (proteostasis) is crucial for the maintenance of cellular homeostasis. Impairment of proteostasis activates proteotoxic and unfolded protein response pathways to resolve cellular stress or induce apoptosis in damaged cells. However, the responses of individual tissues to proteotoxic stress and evoking cell death program have not been extensively explored in vivo. This study shows that a reduction in Nascent polypeptide-associated complex protein alpha subunit (Nacα) specifically and progressively induces cell death in Drosophila fat body cells. Nacα mutants disrupt both ER integrity and the proteasomal degradation system, resulting in caspase activation through JNK and p53. Although forced activation of the JNK and p53 pathways was insufficient to induce cell death in the fat body, the reduction of Nacα sensitized fat body cells to intrinsic and environmental stresses. Reducing overall protein synthesis by mTor inhibition or Minute mutants alleviated the cell death phenotype in Nacα mutant fat body cells. This work revealed that Nacα is crucial for protecting the fat body from cell death by maintaining cellular proteostasis, thus demonstrating the coexistence of a unique vulnerability and cell death resistance in the fat body.
Cachoux, V. M. L., Balakireva, M., Gracia, M., Bosveld, F., Lopez-Gay, J. M., Maugarny, A., Gaugue, I., di Pietro, F., Rigaud, S. U., Noiret, L., Guirao, B., Bellaiche, Y. (2023). Epithelial apoptotic pattern emerges from global and local regulation by cell apical area. Curr Biol, 33(22):4807-4826.e4806 PubMed ID: 37827152
Geometry is a fundamental attribute of biological systems, and it underlies cell and tissue dynamics. Cell geometry controls cell-cycle progression and mitosis and thus modulates tissue development and homeostasis. In sharp contrast and despite the extensive characterization of the genetic mechanisms of caspase activation, little is known about whether and how cell geometry controls apoptosis commitment in developing tissues. This study combined multiscale time-lapse microscopy of developing Drosophila epithelium, quantitative characterization of cell behaviors, and genetic and mechanical perturbations to determine how apoptosis is controlled during epithelial tissue development. Early in cell lives and well before extrusion, apoptosis commitment is linked to two distinct geometric features: a small apical area compared with other cells within the tissue and a small relative apical area with respect to the immediate neighboring cells. These global and local geometric characteristics are sufficient to recapitulate the tissue-scale apoptotic pattern. Furthermore, this study established that the coupling between these two geometric features and apoptotic cells is dependent on the Hippo/YAP and Notch pathways. Overall, by exploring the links between cell geometry and apoptosis commitment, this work provides important insights into the spatial regulation of cell death in tissues and improves understanding of the mechanisms that control cell number and tissue size.
Pai, Y. L., Lin, Y. J., Peng, W. H., Huang, L. T., Chou, H. Y., Wang, C. H., Chien, C. T., Chen, G. C. (2023). The deubiquitinase Leon/USP5 interacts with Atg1/ULK1 and antagonizes autophagy. Cell Death Dis, 14(8):540 PubMed ID: 37607937
Accumulating evidence has shown that the quality of proteins must be tightly monitored and controlled to maintain cellular proteostasis. Misfolded proteins and protein aggregates are targeted for degradation through the ubiquitin proteasome (UPS) and autophagy-lysosome systems. The ubiquitination and deubiquitinating enzymes (DUBs) have been reported to play pivotal roles in the regulation of the UPS system. However, the function of DUBs in the regulation of autophagy remain to be elucidated. This study found that knockdown of Leon/USP5 caused a marked increase in the formation of autophagosomes and autophagic flux under well-fed conditions. Genetic analysis revealed that overexpression of Leon suppressed Atg1-induced cell death in Drosophila. Immunoblotting assays further showed a strong interaction between Leon/USP5 and the autophagy initiating kinase Atg1/ULK1. Depletion of Leon/USP5 led to increased levels of Atg1/ULK1. These findings indicate that Leon/USP5 is an autophagic DUB that interacts with Atg1/ULK1, negatively regulating the autophagic process.
Li, C., Zhu, X., Sun, X., Guo, X., Li, W., Chen, P., Shidlovskii, Y. V., Zhou, Q., Xue, L. (2023). Slik maintains tissue homeostasis by preventing JNK-mediated apoptosis. Cell division. 18(1):16 PubMed ID: 37794497
The c-Jun N-terminal kinase (JNK) pathway is an evolutionarily conserved regulator of cell death, which is essential for coordinating tissue homeostasis. This study has characterized the Drosophila Ste20-like kinase Slik as a novel modulator of JNK pathway-mediated apoptotic cell death. First, ectopic JNK signaling-triggered cell death is enhanced by slik depletion whereas suppressed by Slik overexpression. Second, loss of slik activates JNK signaling, which results in enhanced apoptosis and impaired tissue homeostasis. In addition, genetic epistasis analysis suggests that Slik acts upstream of or in parallel to Hep to regulate JNK-mediated apoptotic cell death. Moreover, Slik is necessary and sufficient for preventing physiologic JNK signaling-mediated cell death in development. Furthermore, introduction of STK10, the human ortholog of Slik, into Drosophila restores slik depletion-induced cell death and compromised tissue homeostasis. Lastly, knockdown of STK10 in human cancer cells also leads to JNK activation, which is cancelled by expression of Slik. This study has uncovered an evolutionarily conserved role of Slik/STK10 in blocking JNK signaling, which is required for cell death inhibition and tissue homeostasis maintenance in development.

Monday, April 29th - Disease Models

Bennett, C. L., Dastidar, S., Arnold, F. J., McKinstry, S. U., Stockford, C., Freibaum, B. D., Sopher, B. L., Wu, M., Seidner, G., Joiner, W., Taylor, J. P., West, R. J. H., La Spada, A. R. (2023). Senataxin helicase, the causal gene defect in ALS4, is a significant modifier of C9orf72 ALS G4C2 and arginine-containing dipeptide repeat toxicity. Acta neuropathologica communications, 11(1):164 PubMed ID: 37845749
Identifying genetic modifiers of familial amyotrophic lateral sclerosis (ALS) may reveal targets for therapeutic modulation with potential application to sporadic ALS. GGGGCC (G4C2) repeat expansions in the C9orf72 gene underlie the most common form of familial ALS, and generate toxic arginine-containing dipeptide repeats (DPRs), which interfere with membraneless organelles, such as the nucleolus. This study considered senataxin (SETX), the genetic cause of ALS4, as a modifier of C9orf72 ALS, because SETX is a nuclear helicase that may regulate RNA-protein interactions involved in ALS dysfunction. After documenting that decreased SETX expression enhances arginine-containing DPR toxicity and C9orf72 repeat expansion toxicity in HEK293 cells and primary neurons, SETX fly lines were generated and the effect of SETX was evaluated in flies expressing either (G4C2)(58) repeats or glycine-arginine-50 [GR(50)] DPRs. Dramatic suppression was observed of disease phenotypes in (G4C2)(58) and GR(50) Drosophila models, and a striking relocalization of GR(50) out of the nucleolus was detected in flies co-expressing SETX. Next-generation GR(1000) fly models, that show age-related motor deficits in climbing and movement assays, were similarly rescued with SETX co-expression. It is noted that the physical interaction between SETX and arginine-containing DPRs is partially RNA-dependent. Finally, the nucleolus in cells expressing GR-DPRs was directly assessed, confirmed reduced mobility of proteins trafficking to the nucleolus upon GR-DPR expression, and found that SETX dosage modulated nucleolus liquidity in GR-DPR-expressing cells and motor neurons. These findings reveal a hitherto unknown connection between SETX function and cellular processes contributing to neuron demise in the most common form of familial ALS.
Swinter, K., Salah, D., Rathnayake, R., Gunawardena, S. (2023). PolyQ-Expansion Causes Mitochondria Fragmentation Independent of Huntingtin and Is Distinct from Traumatic Brain Injury (TBI)/Mechanical Stress-Mediated Fragmentation Which Results from Cell Death. Cells, 12(19) PubMed ID: 37830620
Mitochondrial dysfunction has been reported in many Huntington's disease (HD) models; however, it is unclear how these defects occur. This study tested the hypothesis that excess pathogenic huntingtin (HTT) impairs mitochondrial homeostasis, using Drosophila genetics and pharmacological inhibitors in HD and polyQ-expansion disease models and in a mechanical stress-induced traumatic brain injury (TBI) model. Expression of pathogenic HTT caused fragmented mitochondria compared to normal HTT, but HTT did not co-localize with mitochondria under normal or pathogenic conditions. Expression of pathogenic polyQ (127Q) alone or in the context of Machado Joseph Disease (MJD) caused fragmented mitochondria. While mitochondrial fragmentation was not dependent on the cellular location of polyQ accumulations, the expression of a chaperone protein, excess of mitofusin (MFN), or depletion of dynamin-related protein 1 (DRP1) rescued fragmentation. Intriguingly, a higher concentration of nitric oxide (NO) was observed in polyQ-expressing larval brains and inhibiting NO production rescued polyQ-mediated fragmented mitochondria, postulating that DRP1 nitrosylation could contribute to excess fission. Furthermore, while excess PI3K, which suppresses polyQ-induced cell death, did not rescue polyQ-mediated fragmentation, it did rescue fragmentation caused by mechanical stress/TBI. Together, these observations suggest that pathogenic polyQ alone is sufficient to cause DRP1-dependent mitochondrial fragmentation upstream of cell death, uncovering distinct physiological mechanisms for mitochondrial dysfunction in polyQ disease and mechanical stress.
Catterson, J. H., Minkley, L., Aspe, S., Judd-Mole, S., Moura, S., Dyson, M. C., Rajasingam, A., Woodling, N. S., Atilano, M. L., Ahmad, M., Durrant, C. S., Spires-Jones, T. L., Partridge, L. (2023). Protein retention in the endoplasmic reticulum rescues A β toxicity in Drosophila. Neurobiology of aging, 132:154-174 PubMed ID: 37837732
Amyloid β (A β) accumulation is a hallmark of Alzheimer's disease. In adult Drosophila brains, human A β overexpression harms climbing and lifespan. It's uncertain whether A β is intrinsically toxic or activates downstream neurodegeneration pathways. This study uncovers a novel protective role against A β toxicity: intra-endoplasmic reticulum (ER) protein accumulation with a focus on laminin and collagen subunits. Despite high A β, laminin B1 (LanB1) overexpression robustly counters toxicity, suggesting a potential A β resistance mechanism. Other laminin subunits and collagen IV also alleviate A β toxicity; combining them with LanB1 augments the effect. Imaging reveals ER retention of LanB1 without altering A β secretion. LanB1's rescue function operates independently of the IRE1 α/XBP1 ER stress response. ER-targeted GFP overexpression also mitigates A β toxicity, highlighting broader ER protein retention advantages. Proof-of-principle tests in murine hippocampal slices using mouse Lamb1 demonstrate ER retention in transduced cells, indicating a conserved mechanism. Though ER protein retention generally harms, it could paradoxically counter neuronal A β toxicity, offering a new therapeutic avenue for Alzheimer's disease.
Roth, J. R., de Moraes, R. C. M., Xu, B. P., Crawley, S. R., Khan, M. A., Melkani, G. C. (2023). Rapamycin reduces neuronal mutant huntingtin aggregation and ameliorates locomotor performance in Drosophila. Frontiers in aging neuroscience, 15:1223911 PubMed ID: 37823007
Huntington's disease (HD) is a neurodegenerative disease characterized by movement and cognitive dysfunction. HD is caused by a CAG expansion in exon 1 of the HTT gene that leads to a polyglutamine (PQ) repeat in the huntingtin protein, which aggregates in the brain and periphery. Drosophila models have been used to determine that Htt-PQ aggregation in the heart causes shortened lifespan and cardiac dysfunction that is ameliorated by promoting chaperonin function or reducing oxidative stress. The role of neuronal mutant huntingtin and how it affects peripheral function was further studied. Normal (Htt-PQ25) or expanded mutant (Htt-PQ72) exon 1 of huntingtin was overexpressed in Drosophila neurons and mutant huntingtin was found to cause age-dependent Htt-PQ aggregation in the brain and could cause a loss of synapsin. To determine if this neuronal dysfunction led to peripheral dysfunction, a negative geotaxis assay was performed to measure locomotor performance and it was found that neuronal mutant huntingtin caused an age-dependent decrease in locomotor performance. Next, it was found that rapamycin reduced Htt-PQ aggregation in the brain. These results demonstrate the role of neuronal Htt-PQ in dysfunction in models of HD, suggest that brain-periphery crosstalk could be important to the pathogenesis of HD, and show that rapamycin reduces mutant huntingtin aggregation in the brain.
Han, J. E., Kang, K. H., Kim, H., Hong, Y. B., Choi, B. O., Koh, H. (2023). PINK1 and Parkin rescue motor defects and mitochondria dysfunction induced by a patient-derived HSPB3 mutant in Drosophila models. Biochem Biophys Res Commun, 682:71-76 PubMed ID: 37804589
Small heat shock proteins (sHSPs) are ATP-independent molecular chaperones with the α-crystalline domain that is critical to their chaperone activity. Within the sHSP family, three (HSPB1, HSPB3, and HSPB8) proteins are linked with inherited peripheral neuropathies, including distal hereditary motor neuropathy (dHMN) and Charco-Marie-Tooth disease (CMT). This study introduced the HSPB3 Y118H (HSPB3(Y118H)) mutant gene identified from the CMT2 family in Drosophila. With a missense mutation on its α-crystalline domain, this human HSPB3 mutant gene induced a loss of motor activity accompanied by reduced mitochondrial membrane potential in fly neuronal tissues. Moreover, mitophagy, a critical mechanism of mitochondrial quality control, is downregulated in fly motor neurons expressing HSPB3(Y118H). Surprisingly, PINK1 and Parkin, the core regulators of mitophagy, successfully rescued these motor and mitochondrial abnormalities in HSPB3 mutant flies. Results from the first animal model of HSPB3 mutations suggest that mitochondrial dysfunction plays a critical role in HSPB3-associated human pathology.
Nil, Z., Deshwar, A. R., Huang, Y., ..., Mao, X., Wegner, D. J., Sisco, K., Shinawi, M., Wangler, M. F., Weksberg, R., Yamamoto, S., Costain, G., Bellen, H. J. (2023). Rare de novo gain-of-function missense variants in DOT1L are associated with developmental delay and congenital anomalies. American journal of human genetics, 110(11):1919-1937 PubMed ID: 37827158
Misregulation of histone lysine methylation is associated with several human cancers and with human developmental disorders. DOT1L is an evolutionarily conserved gene encoding a lysine methyltransferase (KMT) that methylates histone 3 lysine-79 (H3K79) and was not previously associated with a Mendelian disease in OMIM. This study has identified nine unrelated individuals with seven different de novo heterozygous missense variants in DOT1L through the Undiagnosed Disease Network (UDN), the SickKids Complex Care genomics project, and GeneMatcher. All probands had some degree of global developmental delay/intellectual disability, and most had one or more major congenital anomalies. To assess the pathogenicity of the DOT1L variants, functional studies were performed in Drosophila and human cells. The fruit fly DOT1L ortholog, grappa, is expressed in most cells including neurons in the central nervous system. The identified DOT1L variants behave as gain-of-function alleles in flies and lead to increased H3K79 methylation levels in flies and human cells. These results show that human DOT1L and fly grappa are required for proper development and that de novo heterozygous variants in DOT1L are associated with a Mendelian disease.

Friday, April 26th - Adult neural structure, development and function

Sharma, Y., Jacobs, J. S., Sivan-Loukianova, E., Lee, E., Kernan, M. J., Eberl, D. F. (2023). The retrograde IFT dynein is required for normal function of diverse mechanosensory cilia in Drosophila. Frontiers in molecular neuroscience, 16:1263411 PubMed ID: 37808471
Cilia biogenesis relies on intraflagellar transport (IFT), a conserved transport mechanism which functions bi-directionally to bring protein complexes to the growing ciliary tip and recycle signaling and transport proteins between the cilium and cell body. In Drosophila, anterograde IFT is critical for assembly of sensory cilia in the neurons of both chordotonal (ch) organs, which have relatively long ciliary axonemes, and external sensory (es) organs, which have short axonemal segments with microtubules in distal sensory segments forming non-axonemal bundles. Previous work has isolated the beethoven (btv) mutant in a mutagenesis screen for auditory mutants. Although many btv mutant flies are deaf, some retain a small residual auditory function as determined both by behavior and by auditory electrophysiology. This study molecularly characterized the btv gene and demonstrated that it encodes the IFT-associated dynein-2 heavy chain Dync2h1. Morphological changes in Johnston's organ are described as flies age to 30 days, and it was found that morphological and electrophysiological phenotypes in this ch organ of btv mutants become more severe with age. NompB protein, encoding the conserved IFT88 protein, an IFT complex B component, fails to be cleared from chordotonal cilia in btv mutants, instead accumulating in the distorted cilia. In macrochaete bristles, a class of es organ, btv mutants show a 50% reduction in mechanoreceptor potentials. Thus, the btv-encoded Dync2h1 functions as the retrograde IFT motor in the assembly of long ciliary axonemes in ch organs and is also important for normal function of the short ciliary axonemes in es organs.
Mannino, M. C., Cassidy, M. B., Florez, S., Rusan, Z., Chakraborty, S., Schoborg, T. (2023). Mutations in abnormal spindle disrupt temporal transcription factor expression and trigger immune responses in the Drosophila brain. Genetics, 225(4) PubMed ID: 37831641
The coordination of cellular behaviors during neurodevelopment is critical for determining the form, function, and size of the central nervous system (CNS). Mutations in the vertebrate Abnormal Spindle-Like, Microcephaly Associated (ASPM) gene and its Drosophila melanogaster ortholog abnormal spindle (asp) lead to microcephaly (MCPH), a reduction in overall brain size whose etiology remains poorly defined. This study provides the neurodevelopmental transcriptional landscape for a Drosophila model for autosomal recessive primary microcephaly-5 (MCPH5) and extend findings into the functional realm to identify the key cellular mechanisms responsible for Asp-dependent brain growth and development. Multiple transcriptomic signatures, including new patterns of coexpressed genes were identified in the developing CNS. Defects in optic lobe neurogenesis were detected in larval brains through downregulation of temporal transcription factors (tTFs) and Notch signaling targets, which correlated with a significant reduction in brain size and total cell numbers during the neurogenic window of development. This study also found inflammation as a hallmark of asp mutant brains, detectable throughout every stage of CNS development, which also contributes to the brain size phenotype. Finally, apoptosis was shown not to be a primary driver of the asp mutant brain phenotypes, further highlighting an intrinsic Asp-dependent neurogenesis promotion mechanism that is independent of cell death. Collectively, these results suggest that the etiology of the asp mutant brain phenotype is complex and that a comprehensive view of the cellular basis of the disorder requires an understanding of how multiple pathway inputs collectively determine tissue size and architecture.
Trombley, S., Powell, J., Guttipatti, P., Matamoros, A., Lin, X., O'Harrow, T., Steinschaden, T., Miles, L., Wang, Q., Wang, S., Qiu, J., Li, Q., Li, F., Song, Y. (2023). Glia instruct axon regeneration via a ternary modulation of neuronal calcium channels in Drosophila. Nat Commun, 14(1):6490 PubMed ID: 37838791
A neuron's regenerative capacity is governed by its intrinsic and extrinsic environment. Both peripheral and central neurons exhibit cell-type-dependent axon regeneration, but the underlying mechanism is unclear. Glia provide a milieu essential for regeneration. However, the routes of glia-neuron signaling remain underexplored. This study shows that regeneration specificity is determined by the axotomy-induced Ca(2+) transients only in the fly regenerative neurons, which is mediated by L-type calcium channels, constituting the core intrinsic machinery. Peripheral glia regulate axon regeneration via a three-layered and balanced modulation. Glia-derived tumor necrosis factor acts through its neuronal receptor to maintain calcium channel expression after injury. Glia sustain calcium channel opening by enhancing membrane hyperpolarization via the inwardly-rectifying potassium channel (Irk1). Glia also release adenosine which signals through neuronal adenosine receptor (AdoR) to activate HCN channels (Ih) and dampen Ca(2+) transients. Together, this study identified a multifaceted glia-neuron coupling which can be hijacked to promote neural repair.
Sun, Y., Li, M., Geng, J., Meng, S., Tu, R., Zhuang, Y., Sun, M., Rui, M., Ou, M., Xing, G., Johnson, T. K., Xie, W. (2023). Neuroligin 2 governs synaptic morphology and function through RACK1-cofilin signaling in Drosophila. Communications biology, 6(1):1056 PubMed ID: 37853189
Neuroligins are transmembrane cell adhesion proteins well-known for their genetic links to autism spectrum disorders. Neuroligins can function by regulating the actin cytoskeleton, however the factors and mechanisms involved are still largely unknown. Here, using the Drosophila neuromuscular junction as a model, it was revealed that F-Actin assembly at the Drosophila NMJ is controlled through Cofilin signaling mediated by an interaction between DNlg2 and RACK1, factors not previously known to work together. The deletion of DNlg2 displays disrupted RACK1-Cofilin signaling pathway with diminished actin cytoskeleton proteo-stasis at the terminal of the NMJ, aberrant NMJ structure, reduced synaptic transmission, and abnormal locomotion at the third-instar larval stage. Overexpression of wildtype and activated Cofilin in muscles are sufficient to rescue the morphological and physiological defects in dnlg2 mutants, while inactivated Cofilin is not. Since the DNlg2 paralog DNlg1 is known to regulate F-actin assembly mainly via a specific interaction with WAVE complex, the present work suggests that the orchestration of F-actin by Neuroligins is a diverse and complex process critical for neural connectivity.
Sun, M., Ma, M., Deng, B., Li, N., Peng, Q., Pan, Y. (2023). A neural pathway underlying hunger modulation of sexual receptivity in Drosophila females. Cell Rep, 42(10):113243 PubMed ID: 37819758
Accepting or rejecting a mate is one of the most crucial decisions a female will make, especially when faced with food shortage. Previous studies have identified the core neural circuity from sensing male courtship or mating status to decision-making for sexual receptivity in Drosophila females, but how hunger and satiety states modulate female receptivity is poorly understood. This study identified the neural circuit and its neuromodulation underlying the hunger modulation of female receptivity. Adipokinetic hormone receptor (AkhR)-expressing neurons inhibit sexual receptivity in a starvation-dependent manner. AkhR neurons are octopaminergic and act on a subset of Octβ1R-expressing LH421 neurons. Knocking down Octβ1R expression in LH421 neurons eliminates starvation-induced suppression of female receptivity. It was further found that LH421 neurons inhibit the sex-promoting pC1 neurons via GABA-resistant to dieldrin (Rdl) signaling. pC1 neurons also integrate courtship stimulation and mating status and thus serve as a common integrator of multiple internal and external cues for decision-making.
Nakano, H., Sakai, T. (2023). Impact of Drosophila LIM homeodomain protein Apterous on the morphology of the adult mushroom body. Biochem Biophys Res Commun, 682:77-84 PubMed ID: 37804590
A LIM homeodomain transcription factor Apterous (Ap) regulates embryonic and larval neurodevelopment in Drosophila. Although Ap is still expressed in the adult brain, it remains elusive whether Ap is involved in neurodevelopmental events in the adult brain because flies homozygous for ap mutations are usually lethal before they reach the adult stage. In this study, using adult escapers of ap knockout (KO) homozygotes, whether the complete lack of ap expression affects the morphology of the mushroom body (MB) neurons and Pigment-dispersing factor (Pdf)-positive clock neurons in the adult brain was examined. Although ap KO escapers showed severe structural defects of MB neurons, no clear morphological defects were found in Pdf-positive clock neurons. These results suggest that Ap in the adult brain is essential for the neurodevelopment of specific ap-positive neurons, but it is not necessarily involved in the development of all ap-positive neurons.

Thursday, April 25th - Synapse and Vesicles

Yamada, D., Davidson, A. M., Hige, T. (2024). Cyclic nucleotide-induced bidirectional long-term synaptic plasticity in Drosophila mushroom body. bioRxiv, PubMed ID: 37808762
Activation of the cAMP pathway is one of the common mechanisms underlying long-term potentiation (LTP). In the Drosophila mushroom body, simultaneous activation of odor-coding Kenyon cells (KCs) and reinforcement-coding dopaminergic neurons activates adenylyl cyclase in KC presynaptic terminals, which is believed to trigger synaptic plasticity underlying olfactory associative learning. However, learning induces long-term depression (LTD) at these synapses, contradicting the universal role of cAMP as a facilitator of transmission. A system was developed to electrophysiologically monitor both short-term and long-term synaptic plasticity at KC output synapses and demonstrate that they are indeed an exception where activation of the cAMP/protein kinase A pathway induces LTD. Contrary to the prevailing model, cAMP imaging finds no evidence for synergistic action of dopamine and KC activity on cAMP synthesis. Furthermore, it was found that forskolin-induced cAMP increase alone is insufficient for plasticity induction; it additionally requires simultaneous KC activation to replicate the presynaptic LTD induced by pairing with dopamine. On the other hand, activation of the cGMP pathway paired with KC activation induces slowly developing LTP, proving antagonistic actions of the two second-messenger pathways predicted by behavioral study. Finally, KC subtype-specific interrogation of synapses reveals that different KC subtypes exhibit distinct plasticity duration even among synapses on the same postsynaptic neuron. Thus, this work not only revises the role of cAMP in synaptic plasticity by uncovering the unexpected convergence point of the cAMP pathway and neuronal activity, but also establishes the methods to address physiological mechanisms of synaptic plasticity in this important model.
Wells, A., Mendes, C. C., Castellanos, F., Mountain, P., Wright, T., Wainwright, S. M., Stefana, M. I., Harris, A. L., Goberdhan, D. C. I., Wilson, C. (2023). A Rab6 to Rab11 transition is required for dense-core granule and exosome biogenesis in Drosophila secondary cells. PLoS Genet, 19(10):e1010979 PubMed ID: 37844085
Secretory cells in glands and the nervous system frequently package and store proteins destined for regulated secretion in dense-core granules (DCGs), which disperse when released from the cell surface. Despite the relevance of this dynamic process to diseases such as diabetes and human neurodegenerative disorders, mechanistic understanding is relatively limited, because of the lack of good cell models to follow the nanoscale events involved. This study employed the prostate-like secondary cells (SCs) of the Drosophila male accessory gland to dissect the cell biology and genetics of DCG biogenesis. These cells contain unusually enlarged DCGs, which are assembled in compartments that also form secreted nanovesicles called exosomes. Known conserved regulators of DCG biogenesis, including the small G-protein Arf1 and the coatomer complex AP-1, play key roles in making SC DCGs. Using real-time imaging, this study found that the aggregation events driving DCG biogenesis are accompanied by a change in the membrane-associated small Rab GTPases which are major regulators of membrane and protein trafficking in the secretory and endosomal systems. Indeed, a transition from trans-Golgi Rab6 to recycling endosomal protein Rab11, which requires conserved DCG regulators like AP-1, is essential for DCG and exosome biogenesis. These data allow development of a model for DCG biogenesis that brings together several previously disparate observations concerning this process and highlights the importance of communication between the secretory and endosomal systems in controlling regulated secretion.
Biton, T., Scher, N., Carmon, S., Elbaz-Alon, Y., Schejter, E. D., Shilo, B. Z., Avinoam, O. (2023). Fusion pore dynamics of large secretory vesicles define a distinct mechanism of exocytosis. J Cell Biol, 222(11) PubMed ID: 37707500
Exocrine cells utilize large secretory vesicles (LSVs) up to 10 μm in diameter. LSVs fuse with the apical surface, often recruiting actomyosin to extrude their content through dynamic fusion pores. The molecular mechanism regulating pore dynamics remains largely uncharacterized. This study observed that the fusion pores of LSVs in the Drosophila larval salivary glands expand, stabilize, and constrict. Arp2/3 is essential for pore expansion and stabilization, while myosin II is essential for pore constriction. Several Bin-Amphiphysin-Rvs (BAR) homology domain proteins were identified that regulate fusion pore expansion and stabilization. The I-BAR protein Missing-in-Metastasis (MIM) localizes to the fusion site and is essential for pore expansion and stabilization. The MIM I-BAR domain is essential but not sufficient for localization and function. It is concluded that MIM acts in concert with actin, myosin II, and additional BAR-domain proteins to control fusion pore dynamics, mediating a distinct mode of exocytosis, which facilitates actomyosin-dependent content release that maintains apical membrane homeostasis during secretion.
Wang, R., Fortier, T. M., Chai, F., Miao, G., Shen, J. L., Restrepo, L. J., DiGiacomo, J. J., Velentzas, P. D., Baehrecke, E. H. (2023). PINK1, Keap1, and Rtnl1 regulate selective clearance of endoplasmic reticulum during development. Cell, 186(19):4172-4188.e4118 PubMed ID: 37633267
Selective clearance of organelles, including endoplasmic reticulum (ER) and mitochondria, by autophagy plays an important role in cell health. This study describes a developmentally programmed selective ER clearance by autophagy. Parkinson's disease-associated PINK1, as well as Atl, Rtnl1, and Trp1 receptors, regulate ER clearance by autophagy. The E3 ubiquitin ligase Parkin functions downstream of PINK1 and is required for mitochondrial clearance while having the opposite function in ER clearance. By contrast, Keap1 and the E3 ubiquitin ligase Cullin3 function downstream of PINK1 to regulate ER clearance by influencing Rtnl1 and Atl. PINK1 regulates a change in Keap1 localization and Keap1-dependent ubiquitylation of the ER-phagy receptor Rtnl1 to facilitate ER clearance. Thus, PINK1 regulates the selective clearance of ER and mitochondria by influencing the balance of Keap1- and Parkin-dependent ubiquitylation of substrates that determine which organelle is removed by autophagy.
Ham, S. J., Yoo, H., Woo, D., Lee, D. H., Park, K. S., Chung, J. (2023). PINK1 and Parkin regulate IP(3)R-mediated ER calcium release. Nat Commun, 14(1):5202 PubMed ID: 37626046
Although defects in intracellular calcium homeostasis are known to play a role in the pathogenesis of Parkinson's disease (PD), the underlying molecular mechanisms remain unclear. This study shows that loss of PTEN-induced kinase 1 (PINK1) and Parkin leads to dysregulation of inositol 1,4,5-trisphosphate receptor (IP(3)R) activity, robustly increasing ER calcium release. In addition, CDGSH iron sulfur domain 1 (CISD1, also known as mitoNEET) functions downstream of Parkin to directly control IP(3)R. Both genetic and pharmacologic suppression of CISD1 and its Drosophila homolog CISD (also known as Dosmit) restore the increased ER calcium release in PINK1 and Parkin null mammalian cells and flies, respectively, demonstrating the evolutionarily conserved regulatory mechanism of intracellular calcium homeostasis by the PINK1-Parkin pathway. More importantly, suppression of CISD in PINK1 and Parkin null flies rescues PD-related phenotypes including defective locomotor activity and dopaminergic neuronal degeneration. Based on these data, it is proposed that the regulation of ER calcium release by PINK1 and Parkin through CISD1 and IP(3)R is a feasible target for treating PD pathogenesis.
Mrestani, A., Dannhauser, S., Pauli, M., Kollmannsberger, P., Hubsch, M., Morris, L., Langenhan, T., Heckmann, M., Paul, M. M. (2023). Nanoscaled RIM clustering at presynaptic active zones revealed by endogenous tagging. Life science alliance, 6(12) PubMed ID: 37696575
Chemical synaptic transmission involves neurotransmitter release from presynaptic active zones (AZs). The AZ protein Rab-3-interacting molecule (RIM) is important for normal Ca(2+)-triggered release. However, its precise localization within AZs of the glutamatergic neuromuscular junctions of Drosophila melanogaster remains elusive. CRISPR/Cas9-assisted genome engineering of the rim locus was used to incorporate small epitope tags for targeted super-resolution imaging. A V5-tag, derived from simian virus 5, and an HA-tag, derived from human influenza virus, were N-terminally fused to the RIM Zinc finger. Whereas both variants are expressed in co-localization with the core AZ scaffold Bruchpilot, electrophysiological characterization reveals that AP-evoked synaptic release is disturbed in rimV5-Znf but not in rimHA-Znf. In addition, rimHA-Znf synapses show intact presynaptic homeostatic potentiation. Combining super-resolution localization microscopy and hierarchical clustering, ∼10 RIM(HA-Znf) subclusters were identified with ∼13 nm diameter per AZ that are compacted and increased in numbers in presynaptic homeostatic potentiation.

Wednesday, April 24th - Adult Physiology

Heidarian, Y., Tourigny, J. P., Fasteen, T. D., Mahmoudzadeh, N. H., Hurlburt, A. J., Nemkov, T., Reisz, J. A., D'Alessandro, A., Tennessen, J. M. (2023). Metabolomic analysis of Drosophila melanogaster larvae lacking pyruvate kinase. G3 (Bethesda), 14(1) PubMed ID: 37792629
Pyruvate kinase (Pyk) is a rate-limiting enzyme that catalyzes the final metabolic reaction in glycolysis. The importance of this enzyme, however, extends far beyond ATP production, as Pyk is also known to regulate tissue growth, cell proliferation, and development. Studies of this enzyme in Drosophila melanogaster are complicated by the fact that the fly genome encodes 6 Pyk paralogs whose functions remain poorly defined. To address this issue, sequence distance and phylogenetic approaches were used to demonstrate that the gene Pyk encodes the enzyme most similar to the mammalian Pyk orthologs, while the other 5 Drosophila Pyk paralogs have significantly diverged from the canonical enzyme. Consistent with this observation, metabolomic studies of 2 different Pyk mutant strains revealed that larvae lacking Pyk exhibit a severe block in glycolysis, with a buildup of glycolytic intermediates upstream of pyruvate. However, this analysis also unexpectedly reveals that pyruvate levels are unchanged in Pyk mutants, indicating that larval metabolism maintains pyruvate pool size despite severe metabolic limitations. Consistent with these metabolomic findings, a complementary RNA-seq analysis revealed that genes involved in lipid metabolism and protease activity are elevated in Pyk mutants, again indicating that loss of this glycolytic enzyme induces compensatory changes in other aspects of metabolism. Overall, this study provides both insight into how Drosophila larval metabolism adapts to disruption of glycolytic metabolism as well as immediate clinical relevance, considering that Pyk deficiency is the most common congenital enzymatic defect in humans.
Sriskanthadevan-Pirahas, S., Tinwala, A. Q., Turingan, M. J., Khan, S., Grewal, S. S. (2023). Mitochondrial metabolism in Drosophila macrophage-like cells regulates body growth via modulation of cytokine and insulin signaling. Biol Open, 12(11) PubMed ID: 37850733
Macrophages play critical roles in regulating and maintaining tissue and whole-body metabolism in normal and disease states. While the cell-cell signaling pathways that underlie these functions are becoming clear, less is known about how alterations in macrophage metabolism influence their roles as regulators of systemic physiology. This was investigated by examining Drosophila macrophage-like cells called hemocytes. Knockdown of TFAM, a mitochondrial genome transcription factor, to reduce mitochondrial OxPhos activity specifically in larval hemocytes. This reduction in hemocyte OxPhos leads to a decrease in larval growth and body size. These effects are associated with a suppression of systemic insulin, the main endocrine stimulator of body growth. It was also found that TFAM knockdown leads to decreased hemocyte JNK signaling and decreased expression of the TNF alpha homolog, Eiger in hemocytes. Furthermore, genetic knockdown of hemocyte JNK signaling or Eiger expression mimics the effects of TFAM knockdown and leads to a non-autonomous suppression of body size without altering hemocyte numbers. These data suggest that modulation of hemocyte mitochondrial metabolism can determine their non-autonomous effects on organismal growth by altering cytokine and systemic insulin signaling. Given that nutrient availability can control mitochondrial metabolism, these findings may explain how macrophages function as nutrient-responsive regulators of tissue and whole-body physiology and homeostasis.
Abhilash, L., Shafer, O. T. (2023). Parametric effects of light acting via multiple photoreceptors contribute to circadian entrainment in Drosophila melanogaster. Proceedings Biological sciences, 290(2006):20230149 PubMed ID: 37700655
Circadian rhythms in physiology and behaviour have near 24 h periodicities that must adjust to the exact 24 h geophysical cycles on earth to ensure adaptive daily timing. Such adjustment is called entrainment. One major mode of entrainment is via the continuous modulation of circadian period by the prolonged presence of light. Although Drosophila melanogaster is a prominent insect model of chronobiology, there is little evidence for such continuous effects of light in the species. This study demonstrates that prolonged light exposure at specific times of the day shapes the daily timing of activity in flies. It was also established that continuous UV- and blue-blocked light lengthens the circadian period of Drosophila, and evidence is provided that this is produced by the combined action of multiple photoreceptors which, includes the cell-autonomous photoreceptor Cryptochrome. Finally, ramped light cycles were introducted as an entrainment paradigm that produces light entrainment that lacks the large light-driven startle responses typically displayed by flies and requires multiple days for entrainment to shifted cycles. These features are reminiscent of entrainment in mammalian models systems and make possible new experimental approaches to understanding the mechanisms underlying entrainment in the fly.
Dorogova, N. V., Fedorova, S. A., Bolobolova, E. U., Baricheva, E. M. (2023). The misregulation of mitochondria-associated genes caused by GAGA-factor lack promotes autophagic germ cell death in Drosophila testes. Genetica, 151(6):349-355 PubMed ID: 37819589 :
The Drosophila GAGA-factor encoded by the Trithorax-like (Trl) gene is DNA-binding protein with unusually wide range of applications in diverse cell contexts. In Drosophila spermatogenesis, reduced GAGA expression caused by Trl mutations induces mass autophagy leading to germ cell death. This work investigated the contribution of mitochondrial abnormalities to autophagic germ cell death in Trl gene mutants. Using a cytological approach, in combination with an analysis of high-throughput RNA sequencing (RNA-seq) data, it was demonstrated that the GAGA deficiency led to considerable defects in mitochondrial ultrastructure, by causing misregulation of GAGA target genes encoding essential components of mitochondrial molecular machinery. Mitochondrial anomalies induced excessive production of reactive oxygen species and their release into the cytoplasm, thereby provoking oxidative stress. Changes in transcription levels of some GAGA-independent genes in the Trl mutants indicated that testis cells experience ATP deficiency and metabolic aberrations, that may trigger extensive autophagy progressing to cell death.
Wang, L., Lin, J., Yang, K., Wang, W., Lv, Y., Zeng, X., Zhao, Y., Yu, J., Pan, L. (2023). Perilipin 1 Deficiency Prompts Lipolysis in Lipid Droplets and Aggravates the Pathogenesis of Persistent Immune Activation in Drosophila. Journal of innate immunity, 15(1):697-708 PubMed ID: 37742619
Lipid droplets (LDs) are highly dynamic intracellular organelles, which are involved in numerous of biological processes. However, the dynamic morphogenesis and functions of intracellular LDs during persistent innate immune responses remain obscure. This study induce long-term systemic immune activation in Drosophila through genetic manipulation. Then, the dynamic pattern of LDs is traced in the Drosophila fat body. Deficiency of Plin1, a key regulator of LDs' reconfiguration, blocks LDs minimization at the initial stage of immune hyperactivation but enhances LDs breakdown at the later stage of sustained immune activation via recruiting the lipase Brummer (Bmm, homologous to human ATGL). The high wasting in LDs shortens the lifespan of flies with high-energy-cost immune hyperactivation. Therefore, these results suggest a critical function of LDs during long-term immune activation and provide a potential treatment for the resolution of persistent inflammation.
Nunes, R. D., Drummond-Barbosa, D. (2023). A high-sugar diet, but not obesity, reduces female fertility in Drosophila melanogaster. Development, 150(20) PubMed ID: 37795747
Obesity is linked to reduced fertility in various species, from Drosophila to humans. Considering that obesity is often induced by changes in diet or eating behavior, it remains unclear whether obesity, diet, or both reduce fertility. This study shows that Drosophila females on a high-sugar diet become rapidly obese and less fertile as a result of increased death of early germline cysts and vitellogenic egg chambers (or follicles). They also have high glycogen, glucose and trehalose levels and develop insulin resistance in their fat bodies (but not ovaries). By contrast, females with adipocyte-specific knockdown of the anti-obesity genes brummer or adipose are obese but have normal fertility. Remarkably, females on a high-sugar diet supplemented with a separate source of water have mostly normal fertility and glucose levels, despite persistent obesity, high glycogen and trehalose levels, and fat body insulin resistance. These findings demonstrate that a high-sugar diet affects specific processes in oogenesis independently of insulin resistance, that high glucose levels correlate with reduced fertility on a high-sugar diet, and that obesity alone does not impair fertility.

Tuesday, April 16th - Disease Models

Dhankhar, J., Shrivastava, A., Agrawal, N. (2023). Amendment of Altered Immune Response by Curcumin in Drosophila Model of Huntington's Disease. Journal of Huntington's disease, 12(4):335-354 PubMed ID: 37781812
Though primarily classified as a brain disorder, surplus studies direct Huntington's disease (HD) to be a multi-system disorder affecting various tissues and organs, thus affecting overall physiology of host. Recently, it has been reported that neuronal expression of mutant huntingtin induces immune dysregulation in Drosophila and may pose chronic threat to challenged individuals. Therefore, the polyphenolic compound curcumin was tested to circumvent the impact of immune dysregulation in Drosophila model of HD. The present study examined the molecular basis underlying immune derangements and immunomodulatory potential of curcumin in HD. UAS-GAL4 system was used to imitate the HD symptoms in Drosophila, and the desired female progenies (elav > Httex1pQ25; control and elav > Httex1pQ93; diseased) were cultured on food mixed without and with 10 μM concentration of curcumin since early development. Effect of curcumin supplementation was investigated by monitoring the hemocytes' count and their functional abilities in diseased condition. Reactive oxygen species (ROS) level in cells was assessed by DHE staining and mitochondrial dysfunction was assessed by CMXros red dye. In addition, transcript levels of pro-inflammatory cytokines and anti-microbial peptides were monitored by qRT-PCR. Curcumin supplementation was found to effectively reduced higher crystal cell count and phenoloxidase activity in diseased flies. Interestingly, curcumin significantly managed altered plasmatocytes count, improved their phagocytic activity by upregulating the expression of key phagocytic receptors in HD condition. Moreover, substantial alleviation of ROS levels and mitochondria dysfunction was observed in plasmatocytes of diseased flies upon curcumin supplementation. Furthermore, curcumin administration effectively attenuated transcriptional expression of pro-inflammatory cytokines and AMPs in diseased flies. These results indicate that curcumin efficiently attenuates immune derangements in HD flies and may prove beneficial in alleviating complexities associated with HD.
Godfrey, R. K., Alsop, E., Bjork, R. T., Chauhan, B. S., Ruvalcaba, H. C., Antone, J., Gittings, L. M., Michael, A. F., Williams, C., Hala'ufia, G., Blythe, A. D., Hall, M., Sattler, R., Van Keuren-Jensen, K., Zarnescu, D. C. (2023). Modelling TDP-43 proteinopathy in Drosophila uncovers shared and neuron-specific targets across ALS and FTD relevant circuits. Acta neuropathologica communications, 11(1):168 PubMed ID: 37864255
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) comprise a spectrum of neurodegenerative diseases linked to TDP-43 (see Drosophila TDPH) proteinopathy, which at the cellular level, is characterized by loss of nuclear TDP-43 and accumulation of cytoplasmic TDP-43 inclusions that ultimately cause RNA processing defects including dysregulation of splicing, mRNA transport and translation. Complementing previous work in motor neurons, this study reports a novel model of TDP-43 proteinopathy based on overexpression of TDP-43 in a subset of Drosophila Kenyon cells of the mushroom body (MB), a circuit with structural characteristics reminiscent of vertebrate cortical networks. This model recapitulates several aspects of dementia-relevant pathological features including age-dependent neuronal loss, nuclear depletion and cytoplasmic accumulation of TDP-43, and behavioral deficits in working memory and sleep that occur prior to axonal degeneration. RNA immunoprecipitations identify several candidate mRNA targets of TDP-43 in MBs, some of which are unique to the MB circuit and others that are shared with motor neurons. Among the latter is the glypican Dally-like-protein (Dlp), which exhibits significant TDP-43 associated reduction in expression during aging. Using genetic interactions iy was shown that overexpression of Dlp in MBs mitigates TDP-43 dependent working memory deficits, conistent with Dlp acting as a mediator of TDP-43 toxicity. Substantiating these findings in the fly model, it was found that the expression of GPC6 mRNA, a human ortholog of dlp, is specifically altered in neurons exhibiting the molecular signature of TDP-43 pathology in FTD patient brains. These findings suggest that circuit-specific Drosophila models provide a platform for uncovering shared or disease-specific molecular mechanisms and vulnerabilities across the spectrum of TDP-43 proteinopathies.
Olmos, V., Thompson, E. N., Gogia, N., Luttik, K., Veeranki, V., Ni, L., Sim, S., Chen, K., Krause, D. S., Lim, J. (2024). Dysregulation of alternative splicing in spinocerebellar ataxia type 1. Hum Mol Genet, 33(2):138-149 PubMed ID: 37802886
Spinocerebellar ataxia type 1 is caused by an expansion of the polyglutamine tract in ATAXIN-1. Ataxin-1 is broadly expressed throughout the brain and is involved in regulating gene expression. However, it is not yet known if mutant ataxin-1 can impact the regulation of alternative splicing events. W RNA sequencing was performed in mouse models of spinocerebellar ataxia type 1 and mutant ataxin-1 expression abnormally leads to diverse splicing events in the mouse cerebellum of spinocerebellar ataxia type 1. The diverse splicing events occurred in a predominantly cell autonomous manner. A majority of the transcripts with misregulated alternative splicing events were previously unknown, thus allowing identification of overall new biological pathways that are distinctive to those affected by differential gene expression in spinocerebellar ataxia type 1. Evidence is provided that the splicing factor Rbfox1 mediates the effect of mutant ataxin-1 on misregulated alternative splicing and that genetic manipulation of Rbfox1 expression modifies neurodegenerative phenotypes in a Drosophila model of spinocerebellar ataxia type 1 in vivo. Together, this study provides novel molecular mechanistic insight into the pathogenesis of spinocerebellar ataxia type 1 and identifies potential therapeutic strategies for spinocerebellar ataxia type 1.
Eberwein, A. E., Kulkarni, S. S., Rushton, E., Broadie, K. (2023). Glycosphingolipids are linked to elevated neurotransmission and neurodegeneration in a Drosophila model of Niemann Pick type C. Disease models & mechanisms, 16(10) PubMed ID: 37815467
The lipid storage disease Niemann Pick type C (NPC) causes neurodegeneration owing primarily to loss of NPC1. This study employed a Drosophila model to test links between glycosphingolipids, neurotransmission and neurodegeneration. Npc1a nulls had elevated neurotransmission at the glutamatergic neuromuscular junction (NMJ), which was phenocopied in brainiac (brn) mutants, impairing mannosyl glucosylceramide (MacCer) glycosylation. Npc1a; brn double mutants had the same elevated synaptic transmission, suggesting that Npc1a and brn function within the same pathway. Glucosylceramide (GlcCer) synthase inhibition with miglustat prevented elevated neurotransmission in Npc1a and brn mutants, further suggesting epistasis. Synaptic MacCer did not accumulate in the NPC model, but GlcCer levels were increased, suggesting that GlcCer is responsible for the elevated synaptic transmission. Null Npc1a mutants had heightened neurodegeneration, but no significant motor neuron or glial cell death, indicating that dying cells are interneurons and that elevated neurotransmission precedes neurodegeneration. Glycosphingolipid synthesis mutants also had greatly heightened neurodegeneration, with similar neurodegeneration in Npc1a; brn double mutants, again suggesting that Npc1a and brn function in the same pathway. These findings indicate causal links between glycosphingolipid-dependent neurotransmission and neurodegeneration in this NPC disease model.
Blount, J. R., Patel, N. C., Libohova, K., Harris, A. L., Tsou, W. L., Sujkowski, A., Todi, S. V. (2023). Lysine 117 on ataxin-3 modulates toxicity in Drosophila models of Spinocerebellar Ataxia Type 3. Journal of the neurological sciences, 454:120828 PubMed ID: 37865002
Ataxin-3 (Atxn3) is a deubiquitinase with a polyglutamine (polyQ) repeat tract whose abnormal expansion causes the neurodegenerative disease, Spinocerebellar Ataxia Type 3 (SCA3; also known as Machado-Joseph Disease). The ubiquitin chain cleavage properties of Atxn3 are enhanced when the enzyme is itself ubiquitinated at lysine (K) at position 117: in vitro, K117-ubiqutinated Atxn3 cleaves poly-ubiquitin markedly more rapidly compared to its unmodified counterpart. How polyQ expansion causes SCA3 remains unclear. To gather insights into the biology of disease of SCA3, the following question was posited: is K117 important for toxicity caused by pathogenic Atxn3? To answer this question, transgenic Drosophila lines were generated that express full-length, human, pathogenic Atxn3 with 80 polyQ with an intact or mutated K117. It was found that mutating K117 mildly enhances the toxicity and aggregation of pathogenic Atxn3. An additional transgenic line that expresses Atxn3 without any K residues confirms increased aggregation of pathogenic Atxn3 whose ubiquitination is perturbed. These findings suggest that Atxn3 ubiquitination is a regulatory step of SCA3, in part by modulating its aggregation.
Sodders, M. J., Shen, M., Olsen, A. L. (2023). Measuring Constipation in a Drosophila Model of Parkinson's Disease. J Vis Exp, (199) PubMed ID: 37811970
Non-motor symptoms in Parkinson's disease (PD) are common, difficult to treat, and significantly impair quality of life. One prevalent non-motor symptom is constipation, which can precede the diagnosis of PD by years or even decades. Constipation has been underexplored in animal models of PD and lacks specific therapies. This assay utilizes a Drosophila model of PD in which human alpha-synuclein is expressed under a pan-neuronal driver. Flies expressing alpha-synuclein develop the hallmark features of PD: the loss of dopaminergic neurons, motor impairment, and alpha-synuclein inclusions. This protocol outlines a method for studying constipation in these flies. Flies are placed on fly food with a blue color additive overnight and then transferred to standard food the following day. They are subsequently moved to new vials with standard fly food every hour for 8 h. Before each transfer, the percentage of blue-colored fecal spots compared to the total fecal spots on the vial wall is calculated. Control flies that lack alpha-synuclein expel all the blue dye hours before flies expressing alpha-synuclein. Additionally, the passage of blue-colored food from the gut can be monitored with simple photography. The simplicity of this assay enables its use in forward genetic or chemical screens to identify modifiers of constipation in Drosophila.

Monday, April 15th - Evolution

Shpak, M., Ghanavi, H. R., Lange, J. D., Pool, J. E., Stensmyr, M. C. (2023). Genomes from historical Drosophila melanogaster specimens illuminate adaptive and demographic changes across more than 200 years of evolution. PLoS Biol, 21(10):e3002333 PubMed ID: 37824452
The ability to perform genomic sequencing on long-dead organisms is opening new frontiers in evolutionary research. These opportunities are especially notable in the case of museum collections, from which countless documented specimens may now be suitable for genomic analysis-if data of sufficient quality can be obtained. This study reports 25 newly sequenced genomes from museum specimens of the model organism Drosophila melanogaster, including the oldest extant specimens of this species. By comparing historical samples ranging from the early 1800s to 1933 against modern-day genomes, this study documents evolution across thousands of generations, including time periods that encompass the species' initial occupation of northern Europe and an era of rapidly increasing human activity. It was also found that the Lund, Sweden population underwent local genetic differentiation during the early 1800s to 1933 interval (potentially due to drift in a small population) but then became more similar to other European populations thereafter (potentially due to increased migration). Within each century-scale time period, temporal sampling allows documentation of compelling candidates for recent natural selection. In some cases, insights were gained regarding previously implicated selection candidates, such as ChKov1, for which the inferred timing of selection favors the hypothesis of antiviral resistance over insecticide resistance. Other candidates are novel, such as the circadian-related gene Ahcy, which yields a selection signal that rivals that of the DDT resistance gene Cyp6g1. These insights deepen understanding of recent evolution in a model system, and highlight the potential of future museomic studies.
Corpuz, R. L., Bellinger, M. R., Veillet, A., Magnacca, K. N., Price, D. K. (2023). The Transmission Patterns of the Endosymbiont Wolbachia within the Hawaiian Drosophilidae Adaptive Radiation. Genes, 14(8) PubMed ID: 37628597
The evolution of endosymbionts and their hosts can lead to highly dynamic interactions with varying fitness effects for both the endosymbiont and host species. Wolbachia, a ubiquitous endosymbiont of arthropods and nematodes, can have both beneficial and detrimental effects on host fitness. This study documented the occurrence and patterns of transmission of Wolbachia within the Hawaiian Drosophilidae and examined the potential contributions of Wolbachia to the rapid diversification of their hosts. Screens for Wolbachia infections across a minimum of 140 species of Hawaiian Drosophila and Scaptomyza revealed species-level infections of 20.0%, and across all 399 samples, a general infection rate of 10.3%. Among the 44 Wolbachia strains identified using a modified Wolbachia multi-locus strain typing scheme, 30 (68.18%) belonged to supergroup B, five (11.36%) belonged to supergroup A, and nine (20.45%) had alleles with conflicting supergroup assignments. Co-phylogenetic reconciliation analysis indicated that Wolbachia strain diversity within their endemic Hawaiian Drosophilidae hosts can be explained by vertical (e.g., co-speciation) and horizontal (e.g., host switch) modes of transmission. Results from stochastic character trait mapping suggest that horizontal transmission is associated with the preferred oviposition substrate of the host, but not the host's plant family or island of occurrence. For Hawaiian Drosophilid species of conservation concern, with 13 species listed as endangered and one listed as threatened, knowledge of Wolbachia strain types, infection status, and potential for superinfection could assist with conservation breeding programs designed to bolster population sizes, especially when wild populations are supplemented with laboratory-reared, translocated individuals. Future research aimed at improving the understanding of the mechanisms of Wolbachia transmission in nature, their impact on the host, and their role in host species formation may shed light on the influence of Wolbachia as an evolutionary driver, especially in Hawaiian ecosystems.
Depetris-Chauvin, A., Galagovsky, D., Keesey, I. W., Hansson, B. S., Sachse, S., Knaden, M. (2023). Evolution at multiple processing levels underlies odor-guided behavior in the genus Drosophila. Curr Biol, 33(22):4771-4785 PubMed ID: 37804828
Olfaction is a fundamental sense guiding animals to their food. How the olfactory system evolves and influences behavior is still poorly understood. This study selected five drosophilid species, including Drosophila melanogaster, inhabiting different ecological niches to compare their olfactory systems at multiple levels. First ecologically relevant natural food odorants from every species were identified and species-specific odorant preferences were identified. To compare odor coding in sensory neurons, the antennal lobe (AL) structure was analyzed, glomerular atlases were generated, and GCaMP transgenic lines were developed for all species. Although subsets of glomeruli showed distinct tuning profiles, odorants inducing species-specific preferences were coded generally similarly. Species distantly related or occupying different habitats showed more evident differences in odor coding, and further analysis revealed that changes in olfactory receptor (OR) sequences partially explain these differences. The results demonstrate that genetic distance in phylogeny and ecological niche occupancy are key determinants in the evolution of ORs, AL structures, odor coding, and behavior. Interestingly, changes in odor coding among species could not be explained by evolutionary changes at a single olfactory processing level but rather are a complex phenomenon based on changes at multiple levels.
Erkosar, B., Dupuis, C., Cavigliasso, F., Savary, L., Kremmer, L., Gallart-Ayala, H., Ivanisevic, J., Kawecki, T. J. (2023). Evolutionary adaptation to juvenile malnutrition impacts adult metabolism and impairs adult fitness in Drosophila. Elife, 12 PubMed ID: 37847744
Juvenile undernutrition has lasting effects on adult metabolism of the affected individuals, but it is unclear how adult physiology is shaped over evolutionary time by natural selection driven by juvenile undernutrition. RNAseq, targeted metabolomics, and genomics were combined to study the consequences of evolution under juvenile undernutrition for metabolism of reproductively active adult females of Drosophila melanogaster. Compared to Control populations maintained on standard diet, Selected populations maintained for over 230 generations on a nutrient-poor larval diet evolved major changes in adult gene expression and metabolite abundance, in particular affecting amino acid and purine metabolism. The evolved differences in adult gene expression and metabolite abundance between Selected and Control populations were positively correlated with the corresponding differences previously reported for Selected versus Control larvae. This implies that genetic variants affect both stages similarly. Even when well fed, the metabolic profile of Selected flies resembled that of flies subject to starvation. Finally, selected flies had lower reproductive output than controls even when both were raised under the conditions under which the selected populations evolved. These results imply that evolutionary adaptation to juvenile undernutrition has large pleiotropic consequences for adult metabolism, and that they are costly rather than adaptive for adult fitness. Thus, juvenile and adult metabolism do not appear to evolve independently from each other even in a holometabolous species where the two life stages are separated by a complete metamorphosis.
Mahdjoub, H., Khelifa, R., Roy, J., Sbilordo, S. H., Zeender, V., Perdigon Ferreira, J., Gourgoulianni, N., Lupold, S. (2023). Interplay between male quality and male-female compatibility across episodes of sexual selection. Sci Adv, 9(39):eadf5559 PubMed ID: 37774022
The processes underlying mate choice profoundly influence the dynamics of sexual selection and the evolution of male sexual traits. Consistent preference for certain phenotypes may erode genetic variation in populations through directional selection, whereas divergent preferences (e.g., genetically compatible mates) provide one mechanism to maintain such variation. However, the relative contributions of these processes across episodes of selection remain unknown. Using Drosophila melanogaster, this study followed the fate of male genotypes, previously scored for their overall reproductive value and their compatibility with different female genotypes, across pre- and postmating episodes of selection. When pairs of competitor males differed in their intrinsic quality and their compatibility with the female, both factors influenced outcomes from mating success to paternity but to a varying degree between stages. These results add further dimensions to understanding of how the interactions between genotypes and forms of selection shape reproductive outcomes and ultimately reproductive trait evolution.
Dey, M., Brown, E., Charlu, S., Keene, A., Dahanukar, A. (2023). Evolution of fatty acid taste in drosophilids. Cell Rep, 42(10):113297 PubMed ID: 37864792
Comparative studies of related but ecologically distinct species can reveal how the nervous system evolves to drive behaviors that are particularly suited to certain environments. Drosophila melanogaster is a generalist that feeds and oviposits on most overripe fruits. A sibling species, D. sechellia, is an obligate specialist of Morinda citrifolia (noni) fruit, which is rich in fatty acids (FAs). To understand evolution of noni taste preference, this study characterized behavioral and cellular responses to noni-associated FAs in three related drosophilids. Mixtures of sugar and noni FAs evoke strong aversion in the generalist species but not in D. sechellia. Surveys of taste sensory responses reveal noni FA- and species-specific differences in at least two mechanisms-bitter neuron activation and sweet neuron inhibition-that correlate with shifts in noni preference. Chemoreceptor mutant analysis in D. melanogaster predicts that multiple genetic changes account for evolution of gustatory preference in D. sechellia.

Monday, April 8th - Signaling

Zhao, A., Varady, S., O'Kelley-Bangsberg, M., Deng, V., Platenkamp, A., Wijngaard, P., Bern, M., Gormley, W., Kushkowski, E., Thompson, K., Tibbetts, L., Conner, A. T., Noeckel, D., Teran, A., Ritz, A., Applewhite, D. A. (2023). From network analysis to experimental validation: identification of regulators of non-muscle myosin II contractility using the folded-gastrulation signaling pathway. BMC molecular and cell biology, 24(1):32 PubMed ID: 37821823
The morphogenetic process of apical constriction, which relies on non-muscle myosin II (NMII; Zipper) generated constriction of apical domains of epithelial cells, is key to the development of complex cellular patterns. Apical constriction occurs in almost all multicellular organisms, but one of the most well-characterized systems is the Folded-gastrulation (Fog)-induced apical constriction that occurs in Drosophila. The binding of Fog to its cognizant receptors Mist/Smog results in a signaling cascade that leads to the activation of NMII-generated contractility. Despite knowledge of key molecular players involved in Fog signaling, this study sought to explore whether other proteins have an undiscovered role in its regulation. A computational method was developed to predict unidentified candidate NMII regulators using a network of pairwise protein-protein interactions called an interactome. First a Drosophila interactome of over 500,000 protein-protein interactions was constructed from several databases that curate high-throughput experiments. Next, several graph-based algorithms were implemented that predicted 14 proteins potentially involved in Fog signaling. To test these candidates, RNAi depletion was used in combination with a cellular contractility assay in Drosophila S2R + cells, which respond to Fog by contracting in a stereotypical manner. Of the candidates dcreened using this assay, two proteins, the serine/threonine phosphatase Flapwing and the putative guanylate kinase CG11811 (oya) were demonstrated to inhibit cellular contractility when depleted, suggestive of their roles as novel regulators of the Fog pathway.
Keyan, K. S., Salim, S., Gowda, S., Abdelrahman, D., Amir, S. S., Islam, Z., Vargas, C., Bengoechea-Alonso, M. T., Alwa, A., Dahal, S., Kolatkar, P. R., Da'as, S., Torrisani, J., Ericsson, J., Mohammad, F., Khan, O. M. (2023). Control of TGFbeta signalling by ubiquitination independent function of E3 ubiquitin ligase TRIP12. Cell Death Dis, 14(10):692 PubMed ID: 37863914
The TGFβ pathway is a master regulator of cell proliferation, differentiation, and death. Deregulation of TGFβ signalling is well established in several human diseases including autoimmune disorders and cancer. Thus, understanding molecular pathways governing TGFβ signalling may help better understand the underlying causes of some of those conditions. This study shows that a HECT domain E3 ubiquitin ligase TRIP12 controls TGFβ signalling in multiple models. Interestingly, TRIP12 control of TGFβ signalling is completely independent of its E3 ubiquitin ligase activity. Instead, TRIP12 recruits SMURF2 to SMAD4, which is most likely responsible for inhibitory monoubiquitination of SMAD4, since SMAD4 monoubiquitination and its interaction with SMURF2 were dramatically downregulated in TRIP12(-/-) cells. Additionally, genetic inhibition of TRIP12 in human and murine cells leads to robust activation of TGFβ signalling which was rescued by re-introducing wildtype TRIP12 or a catalytically inactive C1959A mutant. Importantly, TRIP12 control of TGFβ signalling is evolutionary conserved. Indeed, genetic inhibition of Drosophila TRIP12 orthologue, ctrip, in gut leads to a reduced number of intestinal stem cells which was compensated by the increase in differentiated enteroendocrine cells. These effects were completely normalised in Drosophila strain where ctrip was co-inhibited together with Drosophila SMAD4 orthologue, Medea. Similarly, in murine 3D intestinal organoids, CRISPR/Cas9 mediated genetic targeting of Trip12 enhances TGFβ mediated proliferation arrest and cell death. Finally, CRISPR/Cas9 mediated genetic targeting of TRIP12 in MDA-MB-231 breast cancer cells enhances the TGFβ induced migratory capacity of these cells which was rescued to the wildtype level by re-introducing wildtype TRIP12. This work establishes TRIP12 as an evolutionary conserved modulator of TGFβ signalling in health and disease.
Krejxova, G., Morgantini, C., Zemanova, H., Lauschke, V. M., Kovarova, J., Kubasek, J., Nedbalova, P., Kamps-Hughes, N., Moos, M., Aouadi, M., Dolezal, T., Bajgar, A. (2023). Macrophage-derived insulin antagonist ImpL2 induces lipoprotein mobilization upon bacterial infection. The EMBO journal, 42(23):e114086 PubMed ID: 37807855
The immune response is an energy-demanding process that must be coordinated with systemic metabolic changes redirecting nutrients from stores to the immune system. Although this interplay is fundamental for the function of the immune system, the underlying mechanisms remain elusive. The data of this study show that the pro-inflammatory polarization of Drosophila macrophages is coupled to the production of the insulin antagonist ImpL2 through the activity of the transcription factor HIF1α. ImpL2 production, reflecting nutritional demands of activated macrophages, subsequently impairs insulin signaling in the fat body, thereby triggering FOXO-driven mobilization of lipoproteins. This metabolic adaptation is fundamental for the function of the immune system and an individual's resistance to infection. This study demonstrated that analogically to Drosophila, mammalian immune-activated macrophages produce ImpL2 homolog IGFBP7 in a HIF1α-dependent manner and that enhanced IGFBP7 production by these cells induces mobilization of lipoproteins from hepatocytes. Hence, the production of ImpL2/IGFBP7 by macrophages represents an evolutionarily conserved mechanism by which macrophages alleviate insulin signaling in the central metabolic organ to secure nutrients necessary for their function upon bacterial infection.
Chiang, M. H., Lin, Y. C., Chen, S. F., Lee, P. S., Fu, T. F., Wu, T., Wu, C. L. (2023). Independent insulin signaling modulators govern hot avoidance under different feeding states. PLoS Biol, 21(10):e3002332 PubMed ID: 37847673
Thermosensation is critical for the survival of animals. However, mechanisms through which nutritional status modulates thermosensation remain unclear. This study shows that hungry Drosophila exhibit a strong hot avoidance behavior (HAB) compared to food-sated flies. Hot stimulus increases the activity of α'β' mushroom body neurons (MBns), with weak activity in the sated state and strong activity in the hungry state. Furthermore, it was shown that α'β' MBn receives the same level of hot input from the mALT projection neurons via cholinergic transmission in sated and hungry states. Differences in α'β' MBn activity between food-sated and hungry flies following heat stimuli are regulated by distinct Drosophila insulin-like peptides (Dilps). Dilp2 is secreted by insulin-producing cells (IPCs) and regulates HAB during satiety, whereas Dilp6 is secreted by the fat body and regulates HAB during the hungry state. Dilp2 induces PI3K/AKT signaling, whereas Dilp6 induces Ras/ERK signaling in α'β' MBn to regulate HAB in different feeding conditions. Finally, it was shown that the 2 α'β'-related MB output neurons (MBONs), MBON-α'3 and MBON-β'1, are necessary for the output of integrated hot avoidance information from α'β' MBn. These results demonstrate the presence of dual insulin modulation pathways in α'β' MBn, which are important for suitable behavioral responses in Drosophila during thermoregulation under different feeding states.
Nemoto, K., Masuko, K., Fuse, N., Kurata, S. (2023). . Dilp8 and its candidate receptor, Drl, are involved in the transdetermination of the Drosophila imaginal disc. Genes to cells 28(12):857-867 PubMed ID: 37817293
Drosophila imaginal disc cells can change their identity under stress conditions through transdetermination (TD). Research on TD can help elucidate the in vivo process of cell fate conversion. Previous work showed that the overexpression of winged eye (wge) induces eye-to-wing TD in the eye disc and that an insulin-like peptide, Dilp8, is then highly expressed in the disc. Although Dilp8 is known to mediate systemic developmental delay via the Lgr3 receptor, its role in TD remains unknown. This study showed that Dilp8 is expressed in specific cells that do not express eye or wing fate markers during Wge-mediated TD and that the loss of Dilp8 impairs the process of eye-to-wing transition. Thus, Dilp8 plays a pivotal role in the cell fate conversion under wge overexpression. Furthermore, this study found that instead of Lgr3, another candidate receptor, Drl, is involved in Wge-mediated TD and acts locally in the eye disc cells. A model is proposed in which Dilp8-Drl signaling organizes cell fate conversion in the imaginal disc during TD.
Trammell, C. E., Rowe, E. H., Char, A. B., Jones, B. J., Fawcett, S., Ahlers, L. R. H., Goodman, A. G. (2023). Insulin-mediated endothelin signaling is antiviral during West Nile virus infection. J Virol, 97(10):e0111223 PubMed ID: 37796127
Arboviruses, particularly those transmitted by mosquitoes, pose a significant threat to humans and are an increasing concern because of climate change, human activity, and expanding vector-competent populations. West Nile virus is of significant concern as the most frequent mosquito-borne disease transmitted annually within the continental United States. This study identified a previously uncharacterized signaling pathway that impacts West Nile virus infection, namely endothelin signaling (In insulin resistance the high levels of blood insulin results in increased production and activity of endothelin-1, which promotes vasoconstriction and elevates blood pressure). Additionally, it was demonstrated that results obtained from D. melanogaster can be successfully translated into the more relevant human system. These results add to the growing field of insulin-mediated antiviral immunity and identify potential biomarkers or intervention targets to better address West Nile virus infection and severe disease.

Thursday, April 4th - Behavior

Mishra, S., Sharma, N., Lone, S. R. (2023). Understanding the impact of sociosexual interactions on sleep using Drosophila melanogaster as a model organism. Frontiers in physiology, 14:1220140 PubMed ID: 37670770
Sleep is conserved across species, and it is believed that a fixed amount of sleep is needed for normal neurobiological functions. Sleep rebound follows sleep deprivation; however, continuous sleep deprivation for longer durations is believed to be detrimental to the animal's wellbeing. Under some physiologically demanding situations, such as migration in birds, the birth of new offspring in cetaceans, and sexual interactions in pectoral sandpipers, animals are known to forgo sleep. The mechanisms by which animals forgo sleep without having any obvious negative impact on the proper functioning of their neurobiological processes are yet unknown. Therefore, a simple assay is needed to study how animals forgo sleep. The assay should be ecologically relevant so it can offer insights into the physiology of the organisms. Equally important is that the organism should be genetically amenable, which helps in understanding the cellular and molecular processes that govern such behaviors. This paper presents a simple method of sociosexual interaction to understand the process by which animals forgo sleep. In the case of Drosophila melanogaster, when males and females are in proximity, they are highly active and lose a significant amount of sleep. In addition, there is no sleep rebound afterward, and instead, males engaged in sexual interactions continue to show normal sleep. Thus, sexual drive in the fruit flies is a robust assay to understand the underlying mechanism by which animals forgo sleep.
Strunov, A., Schoenherr, C., Kapun, M. (2023). Wolbachia has subtle effects on thermal preference in highly inbred Drosophila melanogaster which vary with life stage and environmental conditions. Sci Rep, 13(1):13792 PubMed ID: 37612420
Temperature fluctuations are challenging for ectotherms which are not able to regulate body temperature by physiological means and thus have to adjust their thermal environment via behavior. However, little is yet known about whether microbial symbionts influence thermal preference (T(p)) in ectotherms by modulating their physiology. Several recent studies have demonstrated substantial effects of Wolbachia infections on host T(p) in different Drosophila species. These data indicate that the direction and strength of thermal preference variation is strongly dependent on host and symbiont genotypes and highly variable among studies. By employing highly controlled experiments, this study investigated the impact of several environmental factors including humidity, food quality, light exposure, and experimental setup that may influence T(p) measurements in adult Drosophila melanogaster flies. Additionally, the effects were assessed of Wolbachia infection on T(p) of Drosophila at different developmental stages, which has not been done before. Only subtle effects were found of Wolbachia on host T(p) which are strongly affected by experimental variation in adult, but not during juvenile life stages. An in-depth analyses show that environmental variation has a substantial influence on T(p) which demonstrates the necessity of careful experimental design and cautious interpretations of T(p) measurements together with a thorough description of the methods and equipment used to conduct behavioral studies.
Roemschied, F. A., Pacheco, D. A., Aragon, M. J., Ireland, E. C., Li, X., Thieringer, K., Pang, R., Murthy, M. (2023). Flexible circuit mechanisms for context-dependent song sequencing. Nature, 622(7984):794-801 PubMed ID: 37821705
Sequenced behaviours, including locomotion, reaching and vocalization, are patterned differently in different contexts, enabling animals to adjust to their environments. How contextual information shapes neural activity to flexibly alter the patterning of actions is not fully understood. Previous work has indicated that this could be achieved via parallel motor circuits, with differing sensitivities to context. This study demonstrated that a single pathway operates in two regimes dependent on recent sensory history. The Drosophila song production system was leveraged to investigate the role of several neuron types in song patterning near versus far from the female fly. Male flies sing 'simple' trains of only one mode far from the female fly but complex song sequences comprising alternations between modes when near her. Ventral nerve cord (VNC) circuits are shaped by mutual inhibition and rebound excitability between nodes driving the two song modes. Brief sensory input to a direct brain-to-VNC excitatory pathway drives simple song far from the female, whereas prolonged input enables complex song production via simultaneous recruitment of functional disinhibition of VNC circuitry. Thus, female proximity unlocks motor circuit dynamics in the correct context. A compact circuit model was constructed to demonstrate that the identified mechanisms suffice to replicate natural song dynamics. These results highlight how canonical circuit motifs can be combined to enable circuit flexibility required for dynamic communication.
Salem, W., Cellini, B., Jaworski, E., Mongeau, J. M. (2023). Flies adaptively control flight to compensate for added inertia. Proceedings Biological sciences, 290(2008):20231115 PubMed ID: 37817597
Animal locomotion is highly adaptive, displaying a large degree of flexibility, yet how this flexibility arises from the integration of mechanics and neural control remains elusive. For instance, animals require flexible strategies to maintain performance as changes in mass or inertia impact stability. Compensatory strategies to mechanical loading are especially critical for animals that rely on flight for survival. To shed light on the capacity and flexibility of flight neuromechanics to mechanical loading, the performance of fruit flies (Drosophila) was pushed near its limit, and a control theoretic framework was implemented. Flies with added inertia were placed inside a virtual reality arena which permitted free rotation about the vertical (yaw) axis. Adding inertia increased the fly's response time yet had little influence on overall gaze stabilization performance. Flies maintained stability following the addition of inertia by adaptively modulating both visuomotor gain and damping. By contrast, mathematical modelling predicted a significant decrease in gaze stabilization performance. Adding inertia altered saccades, however, flies compensated for the added inertia by increasing saccade torque. Taken together, in response to added inertia flies increase reaction time but maintain flight performance through adaptive neural control. Overall, adding inertia decreases closed-loop flight robustness. This work highlights the flexibility and capacity of motor control in flight.
Baker, C. A., Guan, X. J., Choi, M., Murthy, M. (2024). The role of fruitless in specifying courtship behaviors across divergent Drosophila species. Sci Adv, 10(11):eadk1273 PubMed ID: 38478605
Sex-specific behaviors are critical for reproduction and species survival. The sex-specifically spliced transcription factor fruitless (fru) helps establish male courtship behaviors in invertebrates. Forcing male-specific fru (fruM) splicing in Drosophila melanogaster females produces male-typical behaviors while disrupting female-specific behaviors. However, whether the joint role of fru in specifying male and inhibiting female behaviors is conserved across species is unknown. CRISPR-Cas9 was used to force FruM expression in female Drosophila virilis, a species in which males and females produce sex-specific songs. In contrast to D. melanogaster, in which one fruM allele is sufficient to generate male behaviors in females, two alleles are needed in D. virilis females. D. virilis females expressing FruM maintain the ability to sing female-typical song as well as lay eggs, whereas D. melanogaster FruM females cannot lay eggs. These results reveal potential differences in fru function between divergent species and underscore the importance of studying diverse behaviors and species for understanding the genetic basis of sex differences.
Amin, H., Nolte, S. S., Swain, B., von Philipsborn, A. C. (2023). GABAergic signaling shapes multiple aspects of Drosophila courtship motor behavior. iScience, 26(11):108069 PubMed ID: 37860694
Inhibitory neurons are essential for orchestrating and structuring behavior. This study used one of the best studied behaviors in Drosophila, male courtship, to analyze how inhibitory, GABAergic neurons shape the different steps of this multifaceted motor sequence. RNAi-mediated knockdown of the GABA-producing enzyme GAD1 and the ionotropic receptor Rdl in sex specific, fruitless expressing neurons in the ventral nerve cord causes uncoordinated and futile copulation attempts, defects in wing extension choice and severe alterations of courtship song. Altered song of GABA depleted males fails to stimulate female receptivity, but rescue of song patterning alone is not sufficient to rescue male mating success. Knockdown of GAD1 and Rdl in male brain circuits abolishes courtship conditioning. The around 220 neurons coexpressing GAD1 and Fruitless in the Drosophila male nervous system were characterized, and inhibitory circuit motifs underlying key features of courtship behavior were proposed based on the observed phenotypes.

Monday, April 1st - Larval and Adult Neural Development, Structure and Function

Tello, J. A., Jiang, L., Zohar, Y., Restifo, L. L. (2023). Drosophila CASK regulates brain size and neuronal morphogenesis, providing a genetic model of postnatal microcephaly suitable for drug discovery. Neural Dev, 18(1):6 PubMed ID: 37805506
CASK-related neurodevelopmental disorders are untreatable. Affected children show variable severity, with microcephaly, intellectual disability (ID), and short stature as common features. X-linked human CASK shows dosage sensitivity with haploinsufficiency in females. CASK protein has multiple domains, binding partners, and proposed functions at synapses and in the nucleus. Human and Drosophila CASK show high amino-acid-sequence similarity in all functional domains. Flies homozygous for a hypomorphic CASK mutation (∆18) have motor and cognitive deficits. A Drosophila genetic model of CASK-related disorders could have great scientific and translational value. It ws demonstrated that Δ18 homozygous flies have small brains, small heads, and short bodies. When neurons from developing CASK-mutant CNS were cultured in vitro, they grew small neurite arbors with a distinctive, quantifiable 'bushy' morphology that was significantly rescued by transgenic CASK(+). As in humans, the bushy phenotype showed dosage-sensitive severity. To overcome the limitations of manual tissue trituration for neuronal culture, the design and operation of a microfluidic system was devised for standardized, automated dissociation of CNS tissue into individual viable neurons. Neurons from CASK-mutant CNS dissociated in the microfluidic system recapitulate the bushy morphology. Moreover, for any given genotype, device-dissociated neurons grew larger arbors than did manually dissociated neurons. This automated dissociation method is also effective for rodent CNS. These biological and engineering advances set the stage for drug discovery using the Drosophila model of CASK-related disorders. The bushy phenotype provides a cell-based assay for compound screening. Nearly a dozen genes encoding CASK-binding proteins or transcriptional targets also have brain-development mutant phenotypes, including ID. Hence, drugs that improve CASK phenotypes might also benefit children with disorders due to mutant CASK partners.
Maksymchuk, N., Sakurai, A., Cox, D. N., Cymbalyuk, G. S. (2023). Cold-Temperature Coding with Bursting and Spiking Based on TRP Channel Dynamics in Drosophila Larva Sensory Neurons. Int J Mol Sci, 24(19) PubMed ID: 37834085
Temperature sensation involves thermosensitive TRP (thermoTRP) and non-TRP channels. Drosophila larval Class III (CIII) neurons serve as the primary cold nociceptors and express a suite of thermoTRP channels i mplicated in noxious cold sensation. How CIII neurons code temperature remains unclear.This study combined computational and electrophysiological methods to address this question. In electrophysiological experiments, two basic cold-evoked patterns of CIII neurons were identified: bursting and spiking. In response to a fast temperature drop to noxious cold, CIII neurons distinctly mark different phases of the stimulus. Bursts frequently occurred along with the fast temperature drop, forming a peak in the spiking rate and likely coding the high rate of the temperature change. Single spikes dominated at a steady temperature and exhibited frequency adaptation following the peak. When temperature decreased slowly to the same value, mainly spiking activity was observed, with bursts occurring sporadically throughout the stimulation. The spike and the burst frequencies positively correlated with the rate of the temperature drop. Using a computational model, the distinction is explained in the occurrence of the two CIII cold-evoked patterns bursting and spiking, using the dynamics of a thermoTRP current. A two-parameter activity map (Temperature, constant TRP current conductance) marks parameters that support silent, spiking, and bursting regimes. Projecting on the map the instantaneous TRP conductance, governed by activation and inactivation processes, reflects temperature coding responses as a path across silent, spiking, or bursting domains on the map. The map sheds light on how various parameter sets for TRP kinetics represent various types of cold-evoked responses. Together, these results indicate that bursting detects the high rate of temperature change, whereas tonic spiking could reflect both the rate of change and magnitude of steady cold temperature.
Lago-Baldaia, I., Cooper, M., Seroka, A., Trivedi, C., Powell, G. T., Wilson, S. W., Ackerman, S. D., Fernandes, V. M. (2023). A Drosophila glial cell atlas reveals a mismatch between transcriptional and morphological diversity. PLoS Biol, 21(10):e3002328 PubMed ID: 37862379
glia display diverse morphologies, both across and within glial classes. This study explored the relationship between glial morphology and transcriptional signature using the Drosophila CNS, where glia are categorised into 5 main classes (outer and inner surface glia, cortex glia, ensheathing glia, and astrocytes). Single-cell RNA sequencing data of Drosophila glia were analysedin 2 well-characterised tissues from distinct developmental stages containing distinct circuit types: the embryonic ventral nerve cord (VNC) (motor) and the adult optic lobes (sensory). This analysis identified a new morphologically and transcriptionally distinct surface glial population in the VNC. However, many glial morphological categories could not be distinguished transcriptionally, and indeed, embryonic and adult astrocytes were transcriptionally analogous despite differences in developmental stage and circuit type. While extensive within-class transcriptomic diversity was detected for optic lobe glia, this could be explained entirely by glial residence in the most superficial neuropil (lamina) and an associated enrichment for immune-related gene expression. In summary, this study generated a single-cell transcriptomic atlas of glia in Drosophila, and this extensive in vivo validation revealed that glia exhibit more diversity at the morphological level than was detectable at the transcriptional level.
Nakamizo-Dojo, M., Ishii, K., Yoshino, J., Tsuji, M., Emoto, K. (2023). Descending GABAergic pathway links brain sugar-sensing to peripheral nociceptive gating in Drosophila. Nat Commun, 14(1):6515 PubMed ID: 37845214
Although painful stimuli elicit defensive responses including escape behavior for survival, starved animals often prioritize feeding over escape even in a noxious environment. This behavioral priority is typically mediated by suppression of noxious inputs through descending control in the brain, yet underlying molecular and cellular mechanisms are incompletely understood. This study identified a cluster of GABAergic neurons in Drosophila larval brain, designated as SEZ-localized Descending GABAergic neurons (SDGs), that project descending axons onto the axon terminals of the peripheral nociceptive neurons and prevent presynaptic activity through GABA(B) receptors. Remarkably, glucose feeding to starved larvae causes sustained activation of SDGs through glucose-sensing neurons and subsequent insulin signaling in SDGs, which attenuates nociception and thereby suppresses escape behavior in response to multiple noxious stimuli. These findings illustrate a neural mechanism by which sugar sensing neurons in the brain engages descending GABAergic neurons in nociceptive gating to achieve hierarchical interaction between feeding and escape behavior.
Chua, N. J., Makarova, A. A., Gunn, P., Villani, S., Cohen, B., Thasin, M., Wu, J., Shefter, D., Pang, S., Xu, C. S., Hess, H. F., Polilov, A. A., Chklovskii, D. B. (2023). A complete reconstruction of the early visual system of an adult insect. Curr Biol, 33(21):4611-4623.e4614 PubMed ID: 37774707
For most model organisms in neuroscience, research into visual processing in the brain is difficult because of a lack of high-resolution maps that capture complex neuronal circuitry. The microinsect Megaphragma viggianii, because of its small size and non-trivial behavior, provides a unique opportunity for tractable whole-organism connectomics. This study imaged the Drosophila whole head using serial electron microscopy. Its compound eye was reconstructed, and the optical properties of the ommatidia as well as the connectome of the first visual neuropil-the lamina was analyze. Compared with the fruit fly and the honeybee, Megaphragma visual system is highly simplified: it has 29 ommatidia per eye and 6 lamina neuron types. Features are reported that are both stereotypical among most ommatidia and specialized to some. By identifying the "barebones" circuits critical for flying insects, these results will facilitate constructing computational models of visual processing in insects.
Poe, A. R., Zhu, L., Tang, S. H., Valencia, E., Kayser, M. S. (2023). Energetic Demands Regulate Sleep-Wake Rhythm Circuit Development. bioRxiv, PubMed ID: 37786713
Sleep and feeding patterns lack a clear daily rhythm during early life. As diurnal animals mature, feeding is consolidated to the day and sleep to the night. Circadian sleep patterns begin with formation of a circuit connecting the central clock to arousal output neurons; emergence of circadian sleep also enables long-term memory (LTM). However, the cues that trigger the development of this clock-arousal circuit are unknown. This study identify a role for nutritional status in driving sleep-wake rhythm development in Drosophila larvae. In the 2(nd) instar (L2) period, sleep and feeding are spread across the day; these behaviors become organized into daily patterns by L3. Forcing mature (L3) animals to adopt immature (L2) feeding strategies disrupts sleep-wake rhythms and the ability to exhibit LTM. In addition, the development of the clock (DN1a)-arousal (Dh44) circuit itself is influenced by the larval nutritional environment. Finally, it was demonstrated that larval arousal Dh44 neurons act through glucose metabolic genes to drive onset of daily sleep-wake rhythms. Together, these data suggest that changes to energetic demands in developing organisms triggers the formation of sleep-circadian circuits and behaviors.
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