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Alzheimer's disease (AD) brains are histologically marked by the presence of intracellular and extracellular amyloid deposits, which characterize the onset of the disease pathogenesis. Increasing evidence suggests that certain nutrients exert a direct or indirect effect on amyloid ß (Aß)-peptide production and accumulation and, consequently, on AD pathogenesis. We exploited the fruit fly Drosophila melanogaster model of AD to evaluate in vivo the beneficial properties of Lisosan G, a fermented powder obtained from organic whole grains, on the intracellular Aß-42 peptide accumulation and related pathological phenotypes of AD. Our data showed that the Lisosan G-enriched diet attenuates the production of neurotoxic Aß peptides in fly brains and reduces neuronal apoptosis. Notably, Lisosan G exerted anti-oxidant effects, lowering brain levels of reactive oxygen species and enhancing mitochondrial activity. These aspects paralleled the increase in autophagy turnover and the inhibition of nucleolar stress. Our results give support to the use of the Drosophila model not only to investigate the molecular genetic bases of neurodegenerative disease but also to rapidly and reliably test the efficiency of potential therapeutic agents and diet regimens.

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Motor proteins, encoded by Kinesin superfamily (KIF) genes, are critical for brain development and plasticity. Increasing studies reported KIF's roles in neurodevelopmental disorders. Here, a 6 years and 3 months-old Chinese boy with markedly symptomatic epilepsy, intellectual disability, brain atrophy, and psychomotor retardation was investigated. His parents and younger sister were phenotypically normal and had no disease-related family history. Whole exome sequencing identified a novel heterozygous in-frame deletion (c.265_267delTCA) in exon 3 of the KIF5C in the proband, resulting in the removal of evolutionarily highly conserved p.Ser90, located in its ATP-binding domain. Sanger sequencing excluded the proband's parents and family members from harboring this variant. The activity of ATP hydrolysis in vitro was significantly reduced as predicted. Immunofluorescence studies showed wild-type KIF5C was widely distributed throughout the cytoplasm, while mutant KIF5C was colocalized with microtubules. The live-cell imaging of the cargo-trafficking assay revealed that mutant KIF5C lost the peroxisome-transporting ability. Drosophila models also confirmed p.Ser90del's essential role in nervous system development. This study emphasized the importance of the KIF5C gene in intracellular cargo-transport as well as germline variants that lead to neurodevelopmental disorders and might enable clinicians for timely and accurate diagnosis and disease management in the future.

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Obesity is associated with various metabolic disorders, such as insulin resistance and adipose tissue inflammation (ATM), characterized by macrophage infiltration into adipose cells. This study presents a new Drosophila model to investigate the mechanisms underlying these obesity-related pathologies. We employed genetic manipulation to reduce ecdysone levels to prolong the larval stage. These animals are hyperphagic and exhibit features resembling obesity in mammals, including increased lipid storage, adipocyte hypertrophy and high circulating glucose levels. Moreover, we observed significant infiltration of immune cells (hemocytes) into the fat bodies, accompanied by insulin resistance. We found that attenuation of Eiger/TNFα signaling reduced ATM and improved insulin sensitivity. Furthermore, using metformin and the antioxidants anthocyanins, we ameliorated both phenotypes. Our data highlight evolutionarily conserved mechanisms allowing the development of Drosophila models for discovering therapeutic pathways in adipose tissue immune cell infiltration and insulin resistance. Our model can also provide a platform to perform genetic screens or test the efficacy of therapeutic interventions for diseases such as obesity, type 2 diabetes and non-alcoholic fatty liver disease.

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Abnormal activation of microtubule affinity-regulating kinase 4 (MARK4) and its phosphorylation of the microtubule-associated protein tau are believed to play a role in the pathogenesis of Alzheimer's disease, and MARK4 inhibition can be a strategy to develop disease-modifying therapy. Here we report the development of a membrane-permeable peptide that inhibits MARK4 activity in an allosteric manner. The SARS-CoV-2-derived protein Orf9b inhibited MARK4-mediated tau phosphorylation in primary neurons and Drosophila. Orf9b inhibited MARK4 activity in an allosteric manner and did not inhibit the activity of MARK2, which is another MARK family member and is closely related to MARK4. Co-expression of Orf9b in the fly retina expressing human tau and MARK4 suppressed phosphorylation of tau at the microtubule-binding repeats and tau-induced neurodegeneration. We identified the minimal sequence of Orf9b required to suppress MARK4 activity and fused it to a cell-permeable sequence (TAT-Orf9b10-18_78-95). Extracellular supplementation of TAT-Orf9b10-18_78-95 inhibited MARK4 activity in primary neurons, and feeding TAT-Orf9b10-18_78-95 to a fly model of tauopathy lowered phospho-tau levels and suppressed neurodegeneration. These results suggest that TAT-Orf9b10-18_78-95 is a unique class of MARK4 inhibitor and can be used to modify tau toxicity.

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Recent research has unveiled numerous important functions of protein glycosylation in development, homeostasis, and diseases. A type of glycosylation taking the center stage is protein O-mannosylation, a posttranslational modification conserved in a wide range of organisms, from yeast to humans. In animals, protein O-mannosylation plays a crucial role in the nervous system, whereas protein O-mannosylation defects cause severe neurological abnormalities and congenital muscular dystrophies. However, the molecular and cellular mechanisms underlying protein O-mannosylation functions and biosynthesis remain not well understood. This review outlines recent studies on protein O-mannosylation while focusing on the functions in the nervous system, summarizes the current knowledge about protein O-mannosylation biosynthesis, and discusses the pathologies associated with protein O-mannosylation defects. The evolutionary perspective revealed by studies in the Drosophila model system are also highlighted. Finally, the review touches upon important knowledge gaps in the field and discusses critical questions for future research on the molecular and cellular mechanisms associated with protein O-mannosylation functions.

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Replicative DNA polymerases, such as DNA polymerase α-primase, δ and ε, are multi-subunit complexes that are responsible for the bulk of nuclear DNA replication during the S phase. Over the last decade, extensive genome-wide association studies and expression profiling studies of the replicative DNA polymerase genes in human patients have revealed a link between the replicative DNA polymerase genes and various human diseases and disorders including cancer, intellectual disability, microcephalic primordial dwarfism and immunodeficiency. These studies suggest the importance of dissecting the mechanisms involved in the functioning of replicative DNA polymerases in understanding and treating a range of human diseases. Previous studies in Drosophila have established this organism as a useful model to understand a variety of human diseases. Here, we review the studies on Drosophila that explored the link between DNA polymerases and human disease. First, we summarize the recent studies linking replicative DNA polymerases to various human diseases and disorders. We then review studies on replicative DNA polymerases in Drosophila. Finally, we suggest the possible use of Drosophila models to study human diseases and disorders associated with replicative DNA polymerases.

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Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease characterized by the progressive degeneration of specific muscles. OPMD is due to a mutation in the gene encoding poly(A) binding protein nuclear 1 (PABPN1) leading to a stretch of 11 to 18 alanines at N-terminus of the protein, instead of 10 alanines in the normal protein. This alanine tract extension induces the misfolding and aggregation of PABPN1 in muscle nuclei. Here, using Drosophila OPMD models, we show that the unfolded protein response (UPR) is activated in OPMD upon endoplasmic reticulum stress. Mutations in components of the PERK branch of the UPR reduce muscle degeneration and PABPN1 aggregation characteristic of the disease. We show that oral treatment of OPMD flies with Icerguastat (previously IFB-088), a Guanabenz acetate derivative that shows lower side effects, also decreases muscle degeneration and PABPN1 aggregation. Furthermore, the positive effect of Icerguastat depends on GADD34, a key component of the phosphatase complex in the PERK branch of the UPR. This study reveals a major contribution of the ER stress in OPMD pathogenesis and provides a proof-of-concept for Icerguastat interest in future pharmacological treatments of OPMD.

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Lycium barbarum (LB) is a famous traditional Chinese medicinal plant as well as food supplement possessing various pharmacological functions such as anti-aging and antioxidant effects. The Parkinson's disease (PD)-related kinase Pink1 plays vital role in maintaining the neuron cell homeostasis, having been recognized as a potential target for the development of anti-PD drugs. In this work, the neuroprotective effects of methanol extract of LB fruit (LBFE) were investigated using a Drosophila PD model (PINK1B9) and a human neuroblastoma SH-SY5Y cell line. We found that when LBFE was supplied to the PINK1B9 flies at 6, 12, and 18 days of age, it raised the ATP and dopamine levels at all ages, extended life span, improved motor behavior, and rescued olfactory deficits of the PINK1B9 flies. In addition, histopathological examinations indicated that muscle atrophy in thoraces of the mutant flies was significantly repaired. Finally, LBFE was able to rescue the SH-SY5Y cells against MPP+-induced neurotoxicity. This work reports for the first time the anti-PD potential of L. barbarum fruit extract in PINK1 mutant fruit flies, presenting a new viewpoint for studing the mechanism of action of LBFE.

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Neuronal intranuclear inclusion disease (NIID) is a neuromuscular/neurodegenerative disease caused by the expansion of CGG repeats in the 5' untranslated region (UTR) of the NOTCH2NLC gene. These repeats can be translated into a polyglycine-containing protein, uN2CpolyG, which forms protein inclusions and is toxic in cell models, albeit through an unknown mechanism. Here, we established a transgenic Drosophila model expressing uN2CpolyG in multiple systems, which resulted in progressive neuronal cell loss, locomotor deficiency, and shortened lifespan. Interestingly, electron microscopy revealed mitochondrial swelling both in transgenic flies and in muscle biopsies of individuals with NIID. Immunofluorescence and immunoelectron microscopy showed colocalization of uN2CpolyG with mitochondria in cell and patient samples, while biochemical analysis revealed that uN2CpolyG interacted with a mitochondrial RNA binding protein, LRPPRC (leucine-rich pentatricopeptide repeat motif-containing protein). Furthermore, RNA sequencing (RNA-seq) analysis and functional assays showed down-regulated mitochondrial oxidative phosphorylation in uN2CpolyG-expressing flies and NIID muscle biopsies. Finally, idebenone treatment restored mitochondrial function and alleviated neurodegenerative phenotypes in transgenic flies. Overall, these results indicate that transgenic flies expressing uN2CpolyG recapitulate key features of NIID and that reversing mitochondrial dysfunction might provide a potential therapeutic approach for this disorder.

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Filamins are large proteins with actin-binding properties. Mutations in FLNC, one of the three filamin genes in humans, have recently been implicated in dominant cardiomyopathies, but the underlying mechanisms are not well understood. Here, we aimed to use Drosophila melanogaster as a new in vivo model to study these diseases. First, we show that adult-specific cardiac RNAi-induced depletion of Drosophila Filamin (dFil) induced cardiac dilatation, impaired systolic function and sarcomeric alterations, highlighting its requirement for cardiac function and maintenance of sarcomere integrity in the adult stage. Next, we introduced in the cheerio gene, using CRISPR/Cas9 gene editing, three missense variants, previously identified in patients with hypertrophic cardiomyopathy. Flies carrying these variants did not exhibit cardiac defects or increased propensity to form filamin aggregates, arguing against their pathogenicity. Finally, we show that deletions of the C-term part of dFil carrying the last four Ig-like domains are dispensable for cardiac function. Collectively, these results highlight the relevance of this model to explore the cardiac function of filamins and increase our understanding of physio-pathological mechanisms involved in FLNC-related cardiomyopathies. This article has an associated First Person interview with the first author of the paper.

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Metazoans have developed strategies to protect themselves from pathogenic attack. These preserved mechanisms constitute the immune system, composed of innate and adaptive responses. Among the two kinds, the innate immune system involves the activation of a fast response. NF-κB signaling pathways are activated during infections and lead to the expression of timely-controlled immune response genes. However, activation of NF-κB pathways can be deleterious when uncontrolled. Their regulation is necessary to prevent the development of inflammatory diseases or cancers. The similarity of the NF-κB pathways mediating immune mechanisms in insects and mammals makes Drosophila melanogaster a suitable model for studying the innate immune response and learning general mechanisms that are also relevant for humans. In this review, we summarize what is known about the dynamic regulation of the central NF-κB-pathways and go into detail on the molecular level of the IMD pathway. We report on the role of the nuclear protein Akirin in the regulation of the NF-κB Relish immune response. The use of the Drosophila model allows the understanding of the fine-tuned regulation of this central NF-κB pathway.

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Silver nanoparticles (AgNPs) display unique plasmonic and antimicrobial properties, enabling them to be helpful in various industrial and consumer products. However, previous studies showed that the commercially acquired silver nanoparticles exhibit toxicity even in small doses. Hence, it was imperative to determine suitable synthesis techniques that are the most economical and least toxic to the environment and biological entities. Silver nanoparticles were synthesized using plant extracts and their physico-chemical properties were studied. A time-dependent in vitro study using HEK-293 cells and a dose-dependent in vivo study using a Drosophila model helped us to determine the correct synthesis routes. Through biological analyses, we found that silver nanoparticles' cytotoxicity and wound-healing capacity depended on size, shape, and colloidal stability. Interestingly, we observed that out of all the synthesized AgNPs, the ones derived from the turmeric extract displayed excellent wound-healing capacity in the in vitro study. Furthermore, the same NPs exhibited the least toxic effects in an in vivo study of ingestion of these NPs enriched food in Drosophila, which showed no climbing disability in flies, even at a very high dose (250 mg/L) for 10 days. We propose that stabilizing agents played a superior role in establishing the bio-interaction of nanoparticles. Our study reported here verified that turmeric-extract-derived AgNPs displayed biocompatibility while exhibiting the least cytotoxicity.

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Ten-eleven translocation (TET) proteins are crucial epigenetic regulators highly conserved in multicellular organisms. TETs' enzymatic function in demethylating 5-methyl cytosine in DNA is required for proper development and TETs are frequently mutated in cancer. Recently, Drosophila melanogaster Tet (dTet) was shown to be highly expressed in developing fly brains and discovered to play an important role in brain and muscle development as well as fly behavior. Furthermore, dTet was shown to have different substrate specificity compared with mammals. However, the exact role dTet plays in glial cells and how ectopic TET expression in glial cells contributes to tumorigenesis and glioma is still not clear. Here, we report a novel role for dTet specifically in glial cell organization and number. We show that loss of dTet affects the organization of a specific glia population in the optic lobe, the "optic chiasm" glia. Additionally, we find irregularities in axon patterns in the ventral nerve cord (VNC) both, in the midline and longitudinal axons. These morphologic glia and axonal defects were accompanied by locomotor defects in developing larvae escalating to immobility in adult flies. Furthermore, glia homeostasis was disturbed in dTet-deficient brains manifesting in gain of glial cell numbers and increased proliferation. Finally, we establish a Drosophila model to understand the impact of human TET3 in glia and find that ectopic expression of hTET3 in dTet-expressing cells causes glia expansion in larval brains and affects sleep/rest behavior and the circadian clock in adult flies.

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Hereditary spastic paraplegias (HSPs) are a group of inherited, progressive neurodegenerative conditions characterised by prominent lower-limb spasticity and weakness, caused by a length-dependent degeneration of the longest corticospinal upper motor neurons. While more than 80 spastic paraplegia genes (SPGs) have been identified, many cases arise from mutations in genes encoding proteins which generate and maintain tubular endoplasmic reticulum (ER) membrane organisation. The ER-shaping proteins are essential for the health and survival of long motor neurons, however the mechanisms by which mutations in these genes cause the axonopathy observed in HSP have not been elucidated. To further develop our understanding of the ER-shaping proteins, this study outlines the generation of novel in vivo and in vitro models, using CRISPR/Cas9-mediated gene editing to knockout the ER-shaping protein ADP-ribosylation factor-like 6 interacting protein 1 (ARL6IP1), mutations in which give rise to the HSP subtype SPG61. Loss of Arl6IP1 in Drosophila results in progressive locomotor deficits, emulating a key aspect of HSP in patients. ARL6IP1 interacts with ER-shaping proteins and is required for regulating the organisation of ER tubules, particularly within long motor neuron axons. Unexpectedly, we identified physical and functional interactions between ARL6IP1 and the phospholipid transporter oxysterol-binding protein-related protein 8 in both human and Drosophila model systems, pointing to a conserved role for ARL6IP1 in lipid homeostasis. Furthermore, loss of Arl6IP1 from Drosophila neurons results in a cell non-autonomous accumulation of lipid droplets in axonal glia. Importantly, treatment with lipid regulating liver X receptor-agonists blocked lipid droplet accumulation, restored axonal ER organisation, and improved locomotor function in Arl6IP1 knockout Drosophila. Our findings indicate that disrupted lipid homeostasis contributes to neurodegeneration in HSP, identifying a potential novel therapeutic avenue for the treatment of this disorder.

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FZR1, which encodes the Cdh1 subunit of the anaphase-promoting complex, plays an important role in neurodevelopment by regulating the cell cycle and by its multiple post-mitotic functions in neurons. In this study, evaluation of 250 unrelated patients with developmental and epileptic encephalopathies and a connection on GeneMatcher led to the identification of three de novo missense variants in FZR1. Whole-exome sequencing in 39 patient-parent trios and subsequent targeted sequencing in an additional cohort of 211 patients was performed to identify novel genes involved in developmental and epileptic encephalopathy. Functional studies in Drosophila were performed using three different mutant alleles of the Drosophila homologue of FZR1 fzr. All three individuals carrying de novo variants in FZR1 had childhood-onset generalized epilepsy, intellectual disability, mild ataxia and normal head circumference. Two individuals were diagnosed with the developmental and epileptic encephalopathy subtype myoclonic atonic epilepsy. We provide genetic-association testing using two independent statistical tests to support FZR1 association with developmental and epileptic encephalopathies. Further, we provide functional evidence that the missense variants are loss-of-function alleles using Drosophila neurodevelopment assays. Using three fly mutant alleles of the Drosophila homologue fzr and overexpression studies, we show that patient variants can affect proper neurodevelopment. With the recent report of a patient with neonatal-onset with microcephaly who also carries a de novo FZR1 missense variant, our study consolidates the relationship between FZR1 and developmental and epileptic encephalopathy and expands the associated phenotype. We conclude that heterozygous loss-of-function of FZR1 leads to developmental and epileptic encephalopathies associated with a spectrum of neonatal to childhood-onset seizure types, developmental delay and mild ataxia. Microcephaly can be present but is not an essential feature of FZR1-encephalopathy. In summary, our approach of targeted sequencing using novel gene candidates and functional testing in Drosophila will help solve undiagnosed myoclonic atonic epilepsy or developmental and epileptic encephalopathy cases.

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Sarcopenia is a syndrome characterized by progressive loss of muscle mass and function during aging. Although mitochondrial dysfunction and related metabolic defects precede age-related changes in muscle, their contributions to muscle aging are still not well known. In this study, we used a Drosophila model to investigate the role of lipophorin receptors (LpRs), a Drosophila homologue of the mammalian very low-density lipoprotein receptor (VLDLR), in mitochondrial dynamics and muscle aging. Muscle-specific knockdown of LpR1 or LpR2 resulted in mitochondrial dysfunction and reduced proteostasis, which contributed to muscle aging. Activation of AMP-activated protein kinase (AMPK) ameliorated muscle dysfunction induced by LpR1 knockdown. These results suggest that LpR1/VLDLR is a novel key target that modulates age-dependent lipid remodeling and muscle homeostasis.

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Parkinson's disease (PD) is a common neurodegenerative disorder with motor symptoms linked to the loss of dopaminergic neurons in the brain. α-Synuclein is an aggregation-prone neural protein that plays a role in the pathogenesis of PD. In our previous paper, we found that saffron; the stigma of Crocus sativus Linné (Iridaceae), and its constituents (crocin and crocetin) suppressed aggregation of α-synuclein and promoted the dissociation of α-synuclein fibrils in vitro. In this study, we investigated the effect of dietary saffron and its constituent, crocetin, in vivo on a fly PD model overexpressing several mutant α-synuclein in a tissue-specific manner. Saffron and crocetin significantly suppressed the decrease of climbing ability in the Drosophila overexpressing A30P (A30P fly PD model) or G51D (G51D fly PD model) mutated α-synuclein in neurons. Saffron and crocetin extended the life span in the G51D fly PD model. Saffron suppressed the rough-eyed phenotype and the dispersion of the size histogram of the ocular long axis in the eye of A30P fly PD model. Saffron had a cytoprotective effect on a human neuronal cell line with α-synuclein fibrils. These data showed that saffron and its constituent crocetin have protective effects on the progression of PD disease in animals in vivo and suggest that saffron and crocetin can be used to treat PD.

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Charcot-Marie-Tooth disease is the most common inherited peripheral neuropathy. Dominant mutations in the glycyl-tRNA synthetase (GARS) gene cause peripheral nerve degeneration and lead to CMT disease type 2D. The underlying mechanisms of mutations in GARS (GARSCMT2D ) in disease pathogenesis are not fully understood. In this study, we report that wild-type GARS binds the NAD+ -dependent deacetylase SIRT2 and inhibits its deacetylation activity, resulting in the acetylated α-tubulin, the major substrate of SIRT2. The catalytic domain of GARS tightly interacts with SIRT2, which is the most CMT2D mutation localization. However, CMT2D mutations in GARS cannot inhibit SIRT2 deacetylation, which leads to a decrease of acetylated α-tubulin. Genetic reduction of SIRT2 in the Drosophila model rescues the GARS-induced axonal CMT neuropathy and extends the life span. Our findings demonstrate the pathogenic role of SIRT2-dependent α-tubulin deacetylation in mutant GARS-induced neuropathies and provide new perspectives for targeting SIRT2 as a potential therapy against hereditary axonopathies.

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Cell-based screening of bioactive compounds has served as an important gateway in drug discovery. In the present report, using human neuroblastoma cells and enrolling an extensive three-step screening of 57 phytochemicals, we have identified caffeic acid phenethyl ester (CAPE) as a potent neurodifferentiating natural compound. Analyses of control and CAPE-induced neurodifferentiated cells revealed: (i) modulation of several key proteins (NF200, MAP-2, NeuN, PSD95, Tuj1, GAP43, and GFAP) involved in neurodifferentiation process; and (ii) attenuation of neuronal stemness (HOXD13, WNT3, and Msh-2) and proliferation-promoting (CDC-20, CDK-7, and BubR1) proteins. We anticipated that the neurodifferentiation potential of CAPE may be beneficial for the treatment of neurodegenerative diseases and tested it using the Drosophila model of Alzheimer's disease (AD) and mice model of amnesia/loss of memory. In both models, CAPE exhibited improved disease symptoms and activation of physiological functions. Remarkably, CAPE-treated mice showed increased levels of neurotrophin-BDNF, neural progenitor marker-Nestin, and differentiation marker-NeuN, both in the cerebral cortex and hippocampus. Taken together, we demonstrate the differentiation-inducing and therapeutic potential of CAPE for neurodegenerative diseases.

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Myosin is vital for body movement and heart contractility. Mutations in MYH7, encoding slow/ß-cardiac myosin heavy chain, are an important cause of hypertrophic and dilated cardiomyopathy, as well as skeletal muscle disease. A dominant missense mutation (R1845W) in MYH7 has been reported in several unrelated cases of myosin storage myopathy. We have developed a Drosophila model for a myosin storage myopathy in order to investigate the dose-dependent mechanisms underlying the pathological roles of the R1845W mutation. This study shows that a higher expression level of the mutated allele is concomitant with severe impairment of muscle function and progressively disrupted muscle morphology. The impaired muscle morphology associated with the mutant allele was suppressed by expression of Thin (herein referred to as Abba), an E3 ubiquitin ligase. This Drosophila model recapitulates pathological features seen in myopathy patients with the R1845W mutation and severe ultrastructural abnormalities, including extensive loss of thick filaments with selective A-band loss, and preservation of I-band and Z-disks were observed in indirect flight muscles of flies with exclusive expression of mutant myosin. Furthermore, the impaired muscle morphology associated with the mutant allele was suppressed by expression of Abba. These findings suggest that modification of the ubiquitin proteasome system may be beneficial in myosin storage myopathy by reducing the impact of MYH7 mutation in patients.

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