Altered Gut Microbiome Load and Immune Activation in a Drosophila Model of Human Tauopathy

Tau is a microtubule-associated protein that stabilizes the neuronal cytoskeleton. In the family of neurodegenerative diseases known as tauopathies, including Alzheimer’s disease (AD), frontotemporal dementia (FTD), and chronic traumatic encephalopathy (CTE), abnormal tau aggregation destabilizes microtubule structure, contributing to a cascade of cellular processes leading to neuronal cell death. The gut microbiome has increasingly become a target of neurodegenerative disease research since gut microbiome imbalances have been linked to protein aggregation and inflammation through a bidirectional axis linking the gut and brain. Accordingly, the present study examined tau-mediated changes to gut microbiome composition and immune activation in a Drosophila melanogaster model of human mutant tauopathy. Fecal deposit quantification and gastric emptying time courses suggested an abnormal food distribution and reduced gut motility in tau transgenic flies compared to controls. Tau transgenic flies also showed an increase in gut bacteria colony forming units (CFUs) from diluted fly homogenate, indicating an increased bacterial load. Finally, we showed that tau transgenic flies have a trend towards elevated systemic levels of antimicrobial peptides targeting gram-negative bacteria using qPCR, suggesting an enhanced innate immune response to bacterial insult. These data demonstrate qualifiable and quantifiable gut microbial and innate immune responses to tauopathy. Furthermore, these results provide a framework for future studies targeting the gut microbiome as a modifier of neurodegenerative disease.

In situ Nuclear Matrix preparation in Drosophila melanogaster and its use in studying the components of nuclear architecture

The study of Nuclear Matrix (NuMat) over the last 40 years has been limited to either isolated nuclei from tissues or cells grown in culture. Here, we provide a protocol for NuMat preparation in intact Drosophila melanogaster embryos and its use in dissecting the components of nuclear architecture. The protocol does not require isolation of nuclei and therefore maintains the three-dimensional milieu of an intact embryo, which is biologically more relevant compared to cells in culture. One of the advantages of this protocol is that only a small number of embryos are required. The protocol can be extended to larval tissues like salivary glands and imaginal discs with little modification. Taken together, it becomes possible to carry out such studies in parallel to genetic experiments using mutant and transgenic flies. This protocol, therefore, opens the powerful field of fly genetics to cell biology in the study of nuclear architecture.

α-Synuclein E46K Mutation and Involvement of Oxidative Stress in a Drosophila Model of Parkinson’s Disease

Parkinson’s disease (PD) is an age-associated neurodegenerative condition in which some genetic variants are known to increase disease susceptibility on interaction with environmental factors inducing oxidative stress. Different mutations in the SNCA gene are reported as the major genetic contributors to PD. E46K mutation pathogenicity has not been investigated as intensive as other SNCA gene mutations including A30P and A53T. In this study, based on the GAL4-UAS binary genetic tool, transgenic Drosophila melanogaster flies expressing wild-type and E46K-mutated copies of the human SNCA gene were constructed. Western blotting, immunohistochemical analysis, and light and confocal microscopy of flies’ brains were undertaken along with the survival rate measurement, locomotor function assay, and ethanol and paraquat (PQ) tolerance to study α-synuclein neurotoxicity. Biochemical bioassays were carried out to investigate the activity of antioxidant enzymes and alterations in levels of oxidative markers following damages induced by human α-synuclein to the neurons of the transgenic flies. Overexpression of human α-synuclein in the central nervous system of these transgenic flies led to disorganized ommatidia structures and loss of dopaminergic neurons. E46K α-synuclein caused remarkable climbing defects, reduced survivorship, higher ethanol sensitivity, and increased PQ-mediated mortality. A noticeable decline in activity of catalase and superoxide dismutase enzymes besides considerable increase in the levels of lipid peroxidation and reactive oxygen species was observed in head capsule homogenates of α-synuclein-expressing flies, which indicates obvious involvement of oxidative stress as a causal factor in SNCAE46K neurotoxicity. In all the investigations, E46K copy of the SNCA gene was found to impose more severe defects when compared to wild-type SNCA. It can be concluded that the constructed Drosophila models developed PD-like symptoms that facilitate comparative studies of molecular and cellular pathways implicated in the pathogenicity of different α-synuclein mutations.

Functional Analysis of SARS-CoV-2 Proteins in Drosophila Identifies Orf6-induced Pathogenicity Attenuated by Selinexor

Background: SARS-CoV-2 causes COVID-19 with a widely diverse disease profile that affect many different tissues. The mechanisms underlying its pathogenicity in host organisms remain unclear. Animal models for study the pathogenicity of SARS-CoV-2 proteins are lacking. Methods: Using bioinformatic analysis, we showed that the majority of the virus-host interacting proteins are conserved in Drosophila. Therefore, we generated a series of transgenic lines for individual SARS-CoV-2 genes and used the Gal4-UAS system to express them in various tissues to study their pathogenicity. Results: We found that the Nsp6, Orf6 and Orf7a transgenic flies displayed reduced trachea branching and muscle deficits resulting in “held-up” wing phenotype and poor climbing ability. Furthermore, muscle tissue in these flies showed dramatically reduced mitochondria. Since Orf6 was found to bind nucleopore proteins XPO1, we tested Selinexor, a drug that inhibits XPO1, and found that it could attenuated the Orf6-induced lethality and tissue-specific phenotypes in flies. Conclusions: Our studies here established new Drosophila models for studying the function of SARS-CoV2 genes, identified Orf6 as a highly pathogenic protein in various tissues, and demonstrated the effects of Selinexor for inhibiting Orf6 toxicity with an in vivo model system.

AMPK signaling mediates synphilin-1-induced hyperphagia and obesity in drosophila

Expression of synphilin-1 in neurons induces hyperphagia and obesity in a Drosophila model. However, the molecular pathways underlying synphilin-1-linked obesity remain unclear. Here, the Drosophila models and genetic tools were used to study the synphilin-1-linked pathways in energy balance by combining molecular biology and pharmacological approaches. We found that expression of human synphilin-1 in flies increased AMPK phosphorylation at Thr172 compared with non-transgenic flies. Knockdown of AMPK reduced AMPK phosphorylation and food intake in non-transgenic flies, and further suppressed synphilin-1-induced AMPK phosphorylation, hyperphagia, fat storage, and body weight gain in transgenic flies. Expression of constitutively activated AMPK significantly increased food intake and body weight gain in non-transgenic flies, but it did not alter food intake in the synphilin-1 transgenic flies. In contrast, expression of dominant-negative AMPK reduced food intake in both non-transgenic and synphilin-1 transgenic flies. Treatment with STO609 also suppressed synphilin-1-induced AMPK phosphorylation, hyperphagia and body weight gain. These results demonstrated that the AMPKsignaling pathway plays a critical role in synphilin-1-induced hyperphagia and obesity. These findings provide new insights into the mechanisms of synphilin-1 controlled energy homeostasis.

SNCAE46K transgenic Drosophila Model of Parkinson’s Disease Confirmed the Causative Role of Oxidative Stress

Parkinson’s disease (PD) is a class of neurodegenerative disorders in which, complex interactions of genetic and environmental agents are involved in the etiology of both sporadic and familial PD cases. α-synuclein-encoding SNCA gene is known as one of the major genetic contributors of this disease. E46K mutation in SNCA gene has not been investigated as intensive as other SNCA gene mutations including A30P and A53T. In this study, to induce PD in Drosophila flies, UAS-hSNCAWT and UAS-hSNCAE46K transgenic fly lines were constructed, where SNCA gene was over-expressed in flies brains using GAL4-UAS genetic system. Western blot analysis of head samples of SNCA-expressing flies verified SNCA expression at protein level. Light and electron microscopy analysis of ommatidial structures were performed to verify neurodegeneration as a result of α-synuclein gene overexpression in Drosophila transgenic flies. Confocal microscopy analysis of dopaminergic neuron clusters verified cell loss following SNCAE46K expression in the flies’ brain. E46K α-synuclein gene over-expression resulted in an evident decline in longevity as well as climbing ability of the flies. Biochemical studies of transgenic flies showed a remarkable decline in antioxidant enzymes activity and a significant increase in oxidative markers level as well as AchE enzyme activity. Oxidative stress has been known as a causal factor in PD pathogenesis, following expression of E46K mutant version of human SNCA gene. This Drosophila model is able to facilitate comparative studies of both molecular and cellular assays implicated in the assessment of neurotoxicity of different α-synuclein mutations.

Decision between mitophagy and apoptosis by Parkin via VDAC1 ubiquitination

VDAC1 is a critical substrate of Parkin responsible for the regulation of mitophagy and apoptosis. Here, we demonstrate that VDAC1 can be either mono- or polyubiquitinated by Parkin in a PINK1-dependent manner. VDAC1 deficient with polyubiquitination (VDAC1 Poly-KR) hampers mitophagy, but VDAC1 deficient with monoubiquitination (VDAC1 K274R) promotes apoptosis by augmenting the mitochondrial calcium uptake through the mitochondrial calcium uniporter (MCU) channel. The transgenic flies expressing Drosophila Porin K273R, corresponding to human VDAC1 K274R, show Parkinson disease (PD)-related phenotypes including locomotive dysfunction and degenerated dopaminergic neurons, which are relieved by suppressing MCU and mitochondrial calcium uptake. To further confirm the relevance of our findings in PD, we identify a missense mutation of Parkin discovered in PD patients, T415N, which lacks the ability to induce VDAC1 monoubiquitination but still maintains polyubiquitination. Interestingly, Drosophila Parkin T433N, corresponding to human Parkin T415N, fails to rescue the PD-related phenotypes of Parkin-null flies. Taken together, our results suggest that VDAC1 monoubiquitination plays important roles in the pathologies of PD by controlling apoptosis.

Oogenesis of Hematophagous Midge Forcipomyia taiwana (Diptera: Ceratopogonidae) and Nuage Localization of Vasa in Germline Cells

Forcipomyia taiwana is an irritating hematophagous midge that preferentially attacks humans and affects leisure industries in Taiwan. Understanding the female reproductive biology of such pests would facilitate the development of pest control strategies. However, knowledge about oogenesis in the genus Forcipomyia is unavailable. Accordingly, we examined the ovariole structure and features of oogenesis in terms of the oocyte and the nurse cell. After being blood-fed, we observed a high degree of gonotrophic harmony—the synchronization of developing follicles. The follicle of the F. taiwana has only one nurse cell connected to the oocyte, which is distinct among hematophagous midges. In the nurse cell, we identified the perinuclear localization of the germline marker, Vasa. The Vasa localization is reminiscent of the nuclear envelope-associated nuage observed by electron microscopy. To determine whether F. taiwana Vasa (FtVasa) is an authentic nuage component, we produced transgenic flies expressing FtVasa in the female germline and proved that FtVasa was able to be localized to Drosophila nuage. By characterizing the oogenesis and Vasa expression in the germline cells of F. taiwana, this study extends knowledge about the female reproductive biology of hematophagous midges.

PATHOLOGICAL TAU ABORTS CELL CYCLE ACTIVATION BY MIMICKING EPITHELIAL-MESENCHYMAL-TRANSITION IN NEURONS

Alzheimer’s disease (AD) is an irreversible neurodegenerative disorder which is characterized by neurofibrillary tau tangles and amyloid-ß plaques. Our laboratory uses tau transgenic Drosophila to rapidly test hypotheses along with human brain samples in order to ensure that our work is relevant to clinical endeavors. Using this approach, we identified a neurodegenerative pathway whereby pathological tau over-stabilizes filamentous actin (f-actin), leading to disrupting aberrant nuclear pleomorphisms and decondensation of heterochromatic DNA. Due to the neuronal phenotypes observed in tau-transgenic Drosophila and reentry of post-mitotic neurons into the cell cycle, we hypothesized that tau perturbs the cellular program that maintains terminal neuronal differentiation. Based on RNA-sequencing, we identified prospero as the most differentially expressed and downregulated transcript in tau transgenic flies. Prospero regulates genes that promote and maintain terminal neuronal differentiation. At the protein level, prospero is significantly reduced in tau transgenic flies at 10 days old. We find that over stabilization of f-actin or over-expression of moesin, a cytoskeletal protein known to participate in tumor progression and metastasis, depletes prospero. Using genetically manipulated prospero target genes we find a connection to neurodegeneration. These targets include a PKC, tep4, and knot-suggesting that broad cellular processes are affected by this reduction. Overall, our findings suggest that pathological tau causes cell cycle re-entry by disrupting transcription factors governing terminal neuronal differentiation as well as over-stabilization of f-actin being the driving loss of terminal neuronal differentiation and casually associated with neuronal death.