Reelin is a secreted glycoprotein with roles in the development of the mammalian neocortex, hippocampus, and cerebellum. This vertebrate signaling molecule also contributes to adult brain function. Mammalian Reelin increases the complexity of Drosophila Mushroom Body (MB) neurites, an effect mediated by LpR1 and LpR2, the orthologs of mammalian Reelin receptors. Paradoxically, to date, no Reelin ortholog has been described in Drosophila . Here, we report that the protein product of the uncharacterized Drosophila CG17739 gene, which we named Drospondin, shares sequence homology with vertebrate F-spondin and Reelin. We show that Drospondin is expressed in glial cells and is crucial for MB development. Our results also show that Drospondin genetically interacts with LpRs and that human Reelin rescues neuronal and brain structural defects in Drospondin-deficient flies. Furthermore, Drospondin-deficient flies exhibit altered sleep, locomotion, and social behaviors. Our results reveal that Drospondin is a Drosophila protein with similar functions to mammalian Reelin/F-spondin, that has an essential role in brain development and function, the impairment of which has profound functional consequences for the animal.

Epilepsy affects approximately 50 million people worldwide, yet more than half of individuals with a presumed genetic cause still lack a molecular diagnosis despite the identification of over 1,000 monogenic epilepsy genes. This diagnostic gap is unlikely to be resolved by improved variant detection alone, suggesting that variants affecting the same biological pathway may combine to cause disease. By studying epilepsy-associated actin regulatory genes, we identified a conserved actin/mitochondria/glutamate (AMG) pathway. We demonstrate that reduced actin polymerization promoted DRP1-mediated mitochondrial fission, increased ROS levels, and enhanced glutamatergic transmission, leading to seizures. The glial innate immune pathway, a recently recognized contributor to epilepsy, is activated when the AMG pathway is affected. Reducing mitochondrial fission with the mitochondria division inhibitor (Mdivi-1), or suppressing ROS with N-acetyl-l-cysteine amide (NACA), significantly alleviated seizures. Importantly, digenic heterozygous loss-of-function variants in AMG pathway genes combined to cause seizures, and individuals with epilepsy of unknown etiology showed an increased burden of such variants when compared with the controls. Modeling patient-specific digenic combinations in Drosophila confirmed that many combinations promote seizure susceptibility. Together, these findings establish the AMG pathway as a mechanistic framework for identifying digenic etiologies in epilepsy and highlight potential therapeutic targets.

Both neuronal and peripheral tissues become disrupted in Alzheimer's disease (AD). However, a comprehensive understanding of how AD impacts different tissues across the whole organism is lacking. Using Drosophila, we generated an AD Fly Cell Atlas (AD-FCA) based on whole-organism single-nucleus transcriptomes of 219 cell types from flies expressing AD-associated proteins, either human amyloid-β 42 peptide (Aβ42) or Tau, in neurons. We found that Aβ42 primarily affects the nervous system, including sensory neurons, while Tau induces accelerated aging in peripheral tissues. We identified a neuronal cluster enriched in Aβ42 flies, which has high lactate dehydrogenase (LDH) expression. This LDH-high cluster is conserved in 5XFAD mouse and human AD datasets. We found a conserved defect in fat metabolism from both fly and mouse tauopathy models. The AD-FCA offers new insights into how Aβ42 or Tau systemically and differentially affects a whole organism and provides a valuable resource for understanding brain-body communication in neurodegeneration.

Most Alzheimer disease (AD) susceptibility genes have poorly understood roles in the central nervous system (CNS). To address this gap, we systematically characterized 100 conserved candidate AD risk genes using a cross-species strategy in the fruit fly, Drosophila melanogaster. Genes were prioritized based primarily on human functional genomic evidence. We generated custom loss-of-function alleles for each of the conserved fly orthologs. Most of the genes are expressed in the adult brain, including 24 neuron- and 13 glia-specific expression patterns. Overall, we identify 50 candidate AD risk gene homologs with requirements for CNS structure or function, including 18 whose loss of function causes neurodegeneration (e.g., Snx6/SNX32 and ClC-a/CLCN1), 35 required for neurophysiology (e.g., Arr1/ARRB2 and stai/STMN4), and eight with diminished CNS resilience following a thermal or mechanical stress (e.g., cindr/CD2AP and Amph/BIN1). In a parallel screen, we found 28 AD risk gene homologs (e.g., Ets98B/SPI1 and Yod1/YOD1) that modify the neurotoxicity of either amyloid-β peptide or tau protein, which aggregate to form AD pathology. To translate our findings back to human AD, we used oligogenic risk scores based on gene clusters with shared nervous system phenotypes in flies, pinpointing functional pathways that differentially drive AD risk. Our results-available online via the Alzheimer's Locus Integrative Cross-species Explorer portal-reveal nervous system requirements for dozens of AD risk genes and may enable dissection of causal heterogeneity in AD.

BACKGROUND:Genome-wide association studies (GWAS) of Alzheimer's disease (AD) have identified a plethora of risk loci. However, the disease variants/genes and the underlying mechanisms have not been extensively studied. METHODS:Bulk ATAC-seq was performed in induced pluripotent stem cells (iPSCs) differentiated various brain cell types to identify allele-specific open chromatin (ASoC) SNPs. CRISPR-Cas9 editing generated isogenic pairs, which were then differentiated into glutamatergic neurons (iGlut). Transcriptomic analysis and functional studies of iGlut co-cultured with mouse astrocytes assessed neuronal excitability and lipid droplet formation. RESULTS:We identified a putative causal SNP of CLU that impacted neuronal chromatin accessibility to transcription-factor(s), with the AD protective allele upregulating neuronal CLU and promoting neuron excitability. And, neuronal CLU facilitated neuron-to-glia lipid transfer and astrocytic lipid droplet formation coupled with reactive oxygen species (ROS) accumulation. These changes caused astrocytes to uptake less glutamate thereby altering neuron excitability. CONCLUSIONS:For a strong AD-associated locus near Clusterin (CLU), we connected an AD protective allele to a role of neuronal CLU in promoting neuron excitability through lipid-mediated neuron-glia communication. Our study provides insights into how CLU confers resilience to AD through neuron-glia interactions.

The Sehgal lab presents data showing that the non-cell autonomous pathway of glial lipid droplet formation occurs during sleep and helps to resolve neuronal reactive oxygen species (ROS). This promotes neuronal function after an active day. Hence, this pathway has an important physiological function beyond its previously described role in ROS-associated diseases, including Alzheimer’s disease.

Reelin is a secreted glycoprotein with roles in the development of the mammalian hippocampus and cerebral cortex. This vertebrate signaling molecule also contributes to adult brain function. Mammalian Reelin increases the complexity of Drosophila Mushroom Body (MB) neurites, an effect mediated by LpR1 and LpR2, the orthologs of mammalian Reelin receptors. Paradoxically, to date, no Reelin ortholog has been described in Drosophila . Here, we report that the protein product of the uncharacterized Drosophila CG17739 gene, named Drospondin, shares sequence homology with vertebrate F-spondin and Reelin. We show that Drospondin is expressed in glial cells and is crucial for MB development. Our results also show that Drospondin interacts genetically with LpRs and that human Reelin rescues structural defects in Drospondin-deficient flies. Furthermore, Drospondin-deficient flies exhibit altered sleep homeostasis, locomotion, and social behaviors. Our results reveal that flies express a functional homolog of mammalian Reelin that controls the development and function of the Drosophila nervous system.

Peripheral tissues become disrupted in Alzheimer’s Disease (AD). However, a comprehensive understanding of how the expression of AD-associated toxic proteins, Aβ42 and Tau, in neurons impacts the periphery is lacking. Using Drosophila , a prime model organism for studying aging and neurodegeneration, we generated the Alzheimer’s Disease Fly Cell Atlas (AD-FCA): whole-organism single-nucleus transcriptomes of 219 cell types from adult flies neuronally expressing human Aβ42 or Tau. In-depth analyses and functional data reveal impacts on peripheral sensory neurons by Aβ42 and on various non-neuronal peripheral tissues by Tau, including the gut, fat body, and reproductive system. This novel AD atlas provides valuable insights into potential biomarkers and the intricate interplay between the nervous system and peripheral tissues in response to AD-associated proteins.

The accumulation of reactive oxygen species (ROS) is a common feature of tauopathies, defined by Tau accumulations in neurons and glia. High ROS in neurons causes lipid production and the export of toxic peroxidated lipids (LPOs). Glia uptake these LPOs and incorporate them into lipid droplets (LDs) for storage and catabolism. We found that overexpressing Tau in glia disrupts LDs in flies and rat neuron-astrocyte co-cultures, sensitizing the glia to toxic, neuronal LPOs. Using a new fly tau loss-of-function allele and RNA-mediated interference, we found that endogenous Tau is required for glial LD formation and protection against neuronal LPOs. Similarly, endogenous Tau is required in rat astrocytes and human oligodendrocyte-like cells for LD formation and the breakdown of LPOs. Behaviorally, flies lacking glial Tau have decreased lifespans and motor defects that are rescuable by administering the antioxidant N-acetylcysteine amide. Overall, this work provides insights into the important role that Tau has in glia to mitigate ROS in the brain. Goodman et al. found that Tau is critical for ROS-induced lipid droplet formation in glia from flies and mammals. Too much or too little glial Tau disrupts lipid droplets, leaving the glia susceptible to neuronal ROS-induced damage and causing phenotypes in tau-/- flies.

In this opinion article, we discuss potential connections between sleep disturbances observed in Alzheimer’s disease (AD) and Parkinson’s disease (PD) and the dysregulation of lipids in the brain. Research using Drosophila has highlighted the role of glial-mediated lipid metabolism in sleep and diurnal rhythms. Relevant to AD, the formation of lipid droplets in glia, which occurs in response to elevated neuronal reactive oxygen species (ROS), is required for sleep. In disease models, this process is disrupted, arguing a connection to sleep dysregulation. Relevant to PD, the degradation of neuronally synthesized glucosylceramides by glia requires glucocerebrosidase (GBA, a PD-associated risk factor) and this regulates sleep. Loss of GBA in glia causes an accumulation of glucosylceramides and neurodegeneration. Overall, research primarily using Drosophila has highlighted how dysregulation of glial lipid metabolism may underlie sleep disturbances in neurodegenerative diseases.

During oxidative stress neurons release lipids that are internalized by glia. Defects in this coordinated process play an important role in several neurodegenerative diseases. Yet, the mechanisms of lipid release and its consequences on neuronal health are unclear. Here, we demonstrate that lipid-protein particle release by autolysosome exocytosis protects neurons from ferroptosis, a form of cell death driven by lipid peroxidation. We show that during oxidative stress, peroxidated lipids and iron are released from neurons by autolysosomal exocytosis which requires the exocytic machinery VAMP7 and syntaxin 4. We observe membrane-bound lipid-protein particles by TEM and demonstrate that these particles are released from neurons using cryoEM. Failure to release these lipid-protein particles causes lipid hydroperoxide and iron accumulation and sensitizes neurons to ferroptosis. Our results reveal how neurons protect themselves from peroxidated lipids. Given the number of brain pathologies that involve ferroptosis, defects in this pathway likely play a key role in the pathophysiology of neurodegenerative disease.

Here, we discuss findings made using Drosophila on Alzheimer's disease (AD) risk and progression. Recent studies have investigated the mechanisms underlying glia-mediated neuroprotection in AD. First, we discuss a novel mechanism of glial lipid droplet formation that occurs in response to elevated reactive oxygen species in neurons. The data suggest that disruptions to this process contribute to AD risk. We further discuss novel mechanistic insights into glia-mediated Aβ42-clearance made using the fly. Finally, we highlight work that provides evidence that the aberrant accumulation of reactive oxygen species in AD may not just be a consequence of disease but contribute to disease progression as well. Cumulatively, the discussed studies highlight recent, relevant discoveries in AD made using Drosophila.

Treatment-resistant glioma stem cells are thought to propagate and drive growth of malignant gliomas, but their markers and our ability to target them specifically are not well understood. We demonstrate that podoplanin (PDPN) expression is an independent prognostic marker in gliomas across multiple independent patient cohorts comprising both high- and low-grade gliomas. Knockdown of PDPN radiosensitized glioma cell lines and glioma-stem-like cells (GSCs). Clonogenic assays and xenograft experiments revealed that PDPN expression was associated with radiotherapy resistance and tumor aggressiveness. We further demonstrate that knockdown of PDPN in GSCs in vivo is sufficient to improve overall survival in an intracranial xenograft mouse model. PDPN therefore identifies a subset of aggressive, treatment-resistant glioma cells responsible for radiation resistance and may serve as a novel therapeutic target.

SummaryA growing list of Alzheimer’s disease (AD) genetic risk factors is being identified, but the contribution of these genetic mutations to disease remains largely unknown. Accumulating data support a role of lipid dysregulation and excessive ROS in the etiology of AD. Here, we identified cell-specific roles for eight AD risk-associated genes in ROS-induced glial lipid droplet (LD) formation. We demonstrate that ROS-induced glial LD formation requires two ABCA transporters (ABCA1andABCA7) in neurons, the APOE receptor (LRP1), endocytic genes (PICALM,CD2AP, andAP2A2) in glia, and retromer genes (VPS26andVPS35) in both neurons and glia. Moreover, ROS strongly enhances Aβ42-toxicity in flies and Aβ42-plaque formation in mice. Finally, an ABCA1-activating peptide restores glial LD formation in the APOE4-associated loss of LD. This study places AD risk factors in a neuron-to-glia lipid transfer pathway with a critical role in protecting neurons from ROS-induced toxicity.

Transportin-2 (TNPO2) mediates multiple pathways including non-classical nucleocytoplasmic shuttling of >60 cargoes, such as developmental and neuronal proteins. We identified 15 individuals carrying de novo coding variants in TNPO2 who presented with global developmental delay (GDD), dysmorphic features, ophthalmologic abnormalities, and neurological features. To assess the nature of these variants, functional studies were performed in Drosophila. We found that fly dTnpo (orthologous to TNPO2) is expressed in a subset of neurons. dTnpo is critical for neuronal maintenance and function as downregulating dTnpo in mature neurons using RNAi disrupts neuronal activity and survival. Altering the activity and expression of dTnpo using mutant alleles or RNAi causes developmental defects, including eye and wing deformities and lethality. These effects are dosage dependent as more severe phenotypes are associated with stronger dTnpo loss. Interestingly, similar phenotypes are observed with dTnpo upregulation and ectopic expression of TNPO2, showing that loss and gain of Transportin activity causes developmental defects. Further, proband-associated variants can cause more or less severe developmental abnormalities compared to wild-type TNPO2 when ectopically expressed. The impact of the variants tested seems to correlate with their position within the protein. Specifically, those that fall within the RAN binding domain cause more severe toxicity and those in the acidic loop are less toxic. Variants within the cargo binding domain show tissue-dependent effects. In summary, dTnpo is an essential gene in flies during development and in neurons. Further, proband-associated de novo variants within TNPO2 disrupt the function of the encoded protein. Hence, TNPO2 variants are causative for neurodevelopmental abnormalities.

Tumor recurrence following radiation therapy for patients with malignant gliomas leads to nearly universally fatal outcomes. Treatment resistant glioma stem cells are thought to propagate and drive growth of these tumors, but their markers and our ability to target them specifically are not well understood. Transcriptome analyses suggest that expression of the type-I integral membrane glycoprotein podoplanin (PDPN) may be a prognostic marker in astrocytic gliomas. Here, we aim to explore whether PDPN is a glioma stem cell marker and understand its biological significance. Transcriptome and clinical outcome analyses was carried out using standard bioinformatics tools on publically available TCGA datasets. Glioma stem cell cultures were cultured in serum-free media. PDPN shRNA constructs were generated using standard cloning techniques and delivered via lentiviral transduction. Animal injection experiments were performed with a stereotactic injection apparatus and conducted with prior approval by the institutional review board. Glioma stem cell neurosphere formation assays were used to test response to irradiation, which was delivered in 2, 4 or 6 Gy doses prior to the assay. We demonstrate here that PDPN expression is an independent prognostic marker in gliomas across multiple independent cohorts comprising both high- and low-grade gliomas. PDPN expression is prominent in glioma stem cells and correlates with DNA hypermethylation-induced RT resistance. Enrichment analyses of PDPN expressing GSCs revealed that PDPN correlates with polycomb repressive complex 2 (PRC2) of which the catalytic subunit enhancer of zeste 2 (EZH2) controls DNA methylation and has been previously shown to protect GSCs from radiation-induced cell death. PDPN identifies the tumor-initiating, treatment-resistant (TITR) glioma cells responsible for radiation resistance and may serve as a novel therapeutic target.

The discovery of an expanded (GGGGCC)n repeat (termed G4C2) within the first intron of C9orf72 in familial ALS/FTD has led to a number of studies showing that the aberrant expression of G4C2 RNA can produce toxic dipeptides through repeat-associated non-AUG (RAN-) translation. To reveal canonical translation factors that impact this process, an unbiased loss-of-function screen was performed in a G4C2 fly model that maintained the upstream intronic sequence of the human gene and contained a GFP tag in the GR reading frame. 11 of 48 translation factors were identified that impact production of the GR-GFP protein. Further investigations into two of these, eIF4B and eIF4H, revealed that downregulation of these factors reduced toxicity caused by the expression of expanded G4C2 and reduced production of toxic GR dipeptides from G4C2 transcripts. In patient-derived cells and in post-mortem tissue from ALS/FTD patients, eIF4H was found to be downregulated in cases harboring the G4C2 mutation compared to patients lacking the mutation and healthy individuals. Overall, these data define eIF4B and eIF4H as disease modifiers whose activity is important for RAN-translation of the GR peptide from G4C2-transcripts.