Identification of Tau Toxicity Modifiers in the Drosophila Eye.

Drosophila is a powerful model to study human diseases thanks to its genetic tools and ease of screening. Human genes can be expressed in targeted organs and their toxicity assessed on easily scorable external phenotypes that can be used as readouts to perform genetic screens of toxicity modifiers. In this chapter, I describe how to express human Tau protein in the Drosophila eye, assess protein expression by Western blot, assess Tau toxicity by quantifying the size of the Tau-induced rough eye, and perform a genetic screen of modifiers of Tau toxicity in the Drosophila eye.

The Alzheimer susceptibility gene BIN1 induces isoform-dependent neurotoxicity through early endosome defects.

The Bridging Integrator 1 (BIN1) gene is a major susceptibility gene for Alzheimer's disease (AD). Deciphering its pathophysiological role is challenging due to its numerous isoforms. Here we observed in Drosophila that human BIN1 isoform1 (BIN1iso1) overexpression, contrary to human BIN1 isoform8 (BIN1iso8) and human BIN1 isoform9 (BIN1iso9), induced an accumulation of endosomal vesicles and neurodegeneration. Systematic search for endosome regulators able to prevent BIN1iso1-induced neurodegeneration indicated that a defect at the early endosome level is responsible for the neurodegeneration. In human induced neurons (hiNs) and cerebral organoids, BIN1 knock-out resulted in the narrowing of early endosomes. This phenotype was rescued by BIN1iso1 but not BIN1iso9 expression. Finally, BIN1iso1 overexpression also led to an increase in the size of early endosomes and neurodegeneration in hiNs. Altogether, our data demonstrate that the AD susceptibility gene BIN1, and especially BIN1iso1, contributes to early-endosome size deregulation, which is an early pathophysiological hallmark of AD pathology.

Pyk2 overexpression in postsynaptic neurons blocks amyloid ß1-42-induced synaptotoxicity in microfluidic co-cultures.

Recent meta-analyses of genome-wide association studies identified a number of genetic risk factors of Alzheimer's disease; however, little is known about the mechanisms by which they contribute to the pathological process. As synapse loss is observed at the earliest stage of Alzheimer's disease, deciphering the impact of Alzheimer's risk genes on synapse formation and maintenance is of great interest. In this article, we report a microfluidic co-culture device that physically isolates synapses from pre- and postsynaptic neurons and chronically exposes them to toxic amyloid ß peptides secreted by model cell lines overexpressing wild-type or mutated (V717I) amyloid precursor protein. Co-culture with cells overexpressing mutated amyloid precursor protein exposed the synapses of primary hippocampal neurons to amyloid ß1-42 molecules at nanomolar concentrations and induced a significant decrease in synaptic connectivity, as evidenced by distance-based assignment of postsynaptic puncta to presynaptic puncta. Treating the cells with antibodies that target different forms of amyloid ß suggested that low molecular weight oligomers are the likely culprit. As proof of concept, we demonstrate that overexpression of protein tyrosine kinase 2 beta-an Alzheimer's disease genetic risk factor involved in synaptic plasticity and shown to decrease in Alzheimer's disease brains at gene expression and protein levels-selectively in postsynaptic neurons is protective against amyloid ß1-42-induced synaptotoxicity. In summary, our lab-on-a-chip device provides a physiologically relevant model of Alzheimer's disease-related synaptotoxicity, optimal for assessing the impact of risk genes in pre- and postsynaptic compartments.

The new genetic landscape of Alzheimer's disease: from amyloid cascade to genetically driven synaptic failure hypothesis?

A strong genetic predisposition (60-80% of attributable risk) is present in Alzheimer's disease (AD). In view of this major genetic component, identification of the genetic risk factors has been a major objective in the AD field with the ultimate aim to better understand the pathological processes. In this review, we present how the genetic risk factors are involved in APP metabolism, ß-amyloid peptide production, degradation, aggregation and toxicity, innate immunity, and Tau toxicity. In addition, on the basis of the new genetic landscape, resulting from the recent high-throughput genomic approaches and emerging neurobiological information, we propose an over-arching model in which the focal adhesion pathway and the related cell signalling are key elements in AD pathogenesis. The core of the focal adhesion pathway links the physiological functions of amyloid precursor protein and Tau with the pathophysiological processes they are involved in. This model includes several entry points, fitting with the different origins for the disease, and supports the notion that dysregulation of synaptic plasticity is a central node in AD. Notably, our interpretation of the latest data from genome wide association studies complements other hypotheses already developed in the AD field, i.e., amyloid cascade, cellular phase or propagation hypotheses. Genetically driven synaptic failure hypothesis will need to be further tested experimentally within the general AD framework.

Physiological and pathological roles of FATP-mediated lipid droplets in Drosophila and mice retina.

Increasing evidence suggests that dysregulation of lipid metabolism is associated with neurodegeneration in retinal diseases such as age-related macular degeneration and in brain disorders such as Alzheimer's and Parkinson's diseases. Lipid storage organelles (lipid droplets, LDs), accumulate in many cell types in response to stress, and it is now clear that LDs function not only as lipid stores but also as dynamic regulators of the stress response. However, whether these LDs are always protective or can also be deleterious to the cell is unknown. Here, we investigated the consequences of LD accumulation on retinal cell homeostasis under physiological and stress conditions in Drosophila and in mice. In wild-type Drosophila, we show that dFatp is required and sufficient for expansion of LD size in retinal pigment cells (RPCs) and that LDs in RPCs are required for photoreceptor survival during aging. Similarly, in mice, LD accumulation induced by RPC-specific expression of human FATP1 was non-toxic and promoted mitochondrial energy metabolism in RPCs and non-autonomously in photoreceptor cells. In contrast, the inhibition of LD accumulation by dFatp knockdown suppressed neurodegeneration in Aats-metFB Drosophila mutants, which carry elevated levels of reactive oxygen species (ROS). This suggests that abnormal turnover of LD may be toxic for photoreceptors cells of the retina under oxidative stress. Collectively, these findings indicate that FATP-mediated LD formation in RPCs promotes RPC and neuronal homeostasis under physiological conditions but could be deleterious for the photoreceptors under pathological conditions.

Male-specific epistasis between WWC1 and TLN2 genes is associated with Alzheimer's disease.

Systematic epistasis analyses in multifactorial disorders are an important step to better characterize complex genetic risk structures. We conducted a hypothesis-free sex-stratified genome-wide screening for epistasis contributing to Alzheimer's disease (AD) susceptibility. We identified a statistical epistasis signal between the single nucleotide polymorphisms rs3733980 and rs7175766 that was associated with AD in males (genome-wide significant pBonferroni-corrected=0.0165). This signal pointed toward the genes WW and C2 domain containing 1, aka KIBRA; 5q34 and TLN2 (talin 2; 15q22.2). Gene-based meta-analysis in 3 independent consortium data sets confirmed the identified interaction: the most significant (pmeta-Bonferroni-corrected=9.02*10-3) was for the single nucleotide polymorphism pair rs1477307 and rs4077746. In functional studies, WW and C2 domain containing 1, aka KIBRA and TLN2 coexpressed in the temporal cortex brain tissue of AD subjects (ß=0.17, 95% CI 0.04 to 0.30, p=0.01); modulated Tau toxicity in Drosophila eye experiments; colocalized in brain tissue cells, N2a neuroblastoma, and HeLa cell lines; and coimmunoprecipitated both in brain tissue and HEK293 cells. Our finding points toward new AD-related pathways and provides clues toward novel medical targets for the cure of AD.

Using High-Throughput Animal or Cell-Based Models to Functionally Characterize GWAS Signals.

PURPOSE OF REVIEW: The advent of genome-wide association studies (GWASs) constituted a breakthrough in our understanding of the genetic architecture of multifactorial diseases. For Alzheimer's disease (AD), more than 20 risk loci have been identified. However, we are now facing three new challenges: (i) identifying the functional SNP or SNPs in each locus, (ii) identifying the causal gene(s) in each locus, and (iii) understanding these genes' contribution to pathogenesis. RECENT FINDINGS: To address these issues and thus functionally characterize GWAS signals, a number of high-throughput strategies have been implemented in cell-based and whole-animal models. Here, we review high-throughput screening, high-content screening, and the use of the Drosophila model (primarily with reference to AD). SUMMARY: We describe how these strategies have been successfully used to functionally characterize the genes in GWAS-defined risk loci. In the future, these strategies should help to translate GWAS data into knowledge and treatments.

Developmental Expression of 4-Repeat-Tau Induces Neuronal Aneuploidy in Drosophila Tauopathy Models.

Tau-mediated neurodegeneration in Alzheimer's disease and tauopathies is generally assumed to start in a normally developed brain. However, several lines of evidence suggest that impaired Tau isoform expression during development could affect mitosis and ploidy in post-mitotic differentiated tissue. Interestingly, the relative expression levels of Tau isoforms containing either 3 (3R-Tau) or 4 repeats (4R-Tau) play an important role both during brain development and neurodegeneration. Here, we used genetic and cellular tools to study the link between 3R and 4R-Tau isoform expression, mitotic progression in neuronal progenitors and post-mitotic neuronal survival. Our results illustrated that the severity of Tau-induced adult phenotypes depends on 4R-Tau isoform expression during development. As recently described, we observed a mitotic delay in 4R-Tau expressing cells of larval eye discs and brains. Live imaging revealed that the spindle undergoes a cycle of collapse and recovery before proceeding to anaphase. Furthermore, we found a high level of aneuploidy in post-mitotic differentiated tissue. Finally, we showed that overexpression of wild type and mutant 4R-Tau isoform in neuroblastoma SH-SY5Y cell lines is sufficient to induce monopolar spindles. Taken together, our results suggested that neurodegeneration could be in part linked to neuronal aneuploidy caused by 4R-Tau expression during brain development.

Genome-wide, high-content siRNA screening identifies the Alzheimer's genetic risk factor FERMT2 as a major modulator of APP metabolism.

Genome-wide association studies (GWASs) have identified 19 susceptibility loci for Alzheimer's disease (AD). However, understanding how these genes are involved in the pathophysiology of AD is one of the main challenges of the "post-GWAS" era. At least 123 genes are located within the 19 susceptibility loci; hence, a conventional approach (studying the genes one by one) would not be time- and cost-effective. We therefore developed a genome-wide, high-content siRNA screening approach and used it to assess the functional impact of gene under-expression on APP metabolism. We found that 832 genes modulated APP metabolism. Eight of these genes were located within AD susceptibility loci. Only FERMT2 (a ß3-integrin co-activator) was also significantly associated with a variation in cerebrospinal fluid Aß peptide levels in 2886 AD cases. Lastly, we showed that the under-expression of FERMT2 increases Aß peptide production by raising levels of mature APP at the cell surface and facilitating its recycling. Taken as a whole, our data suggest that FERMT2 modulates the AD risk by regulating APP metabolism and Aß peptide production.

Identification of Tau Toxicity Modifiers in the Drosophila Eye.

Drosophila is a powerful model to study human diseases thanks to its genetic tools and ease of screening. Human genes can be expressed in targeted organs and their toxicity assessed on easily scorable external phenotypes that can be used as readout to perform genetic screen of toxicity modifiers. In this chapter, I describe how to express human Tau protein in the Drosophila eye, assess protein expression by western blot, assess Tau toxicity by quantifying the size of the Tau-induced rough eye, and perform a genetic screen of modifiers of Tau toxicity in the Drosophila eye.

ADAM30 Downregulates APP-Linked Defects Through Cathepsin D Activation in Alzheimer's Disease.

Although several ADAMs (A disintegrin-like and metalloproteases) have been shown to contribute to the amyloid precursor protein (APP) metabolism, the full spectrum of metalloproteases involved in this metabolism remains to be established. Transcriptomic analyses centred on metalloprotease genes unraveled a 50% decrease in ADAM30 expression that inversely correlates with amyloid load in Alzheimer's disease brains. Accordingly, in vitro down- or up-regulation of ADAM30 expression triggered an increase/decrease in Aß peptides levels whereas expression of a biologically inactive ADAM30 (ADAM30(mut)) did not affect Aß secretion. Proteomics/cell-based experiments showed that ADAM30-dependent regulation of APP metabolism required both cathepsin D (CTSD) activation and APP sorting to lysosomes. Accordingly, in Alzheimer-like transgenic mice, neuronal ADAM30 over-expression lowered Aß42 secretion in neuron primary cultures, soluble Aß42 and amyloid plaque load levels in the brain and concomitantly enhanced CTSD activity and finally rescued long term potentiation alterations. Our data thus indicate that lowering ADAM30 expression may favor Aß production, thereby contributing to Alzheimer's disease development.

Tau phosphorylation regulates the interaction between BIN1's SH3 domain and Tau's proline-rich domain.

INTRODUCTION: The application of high-throughput genomic approaches has revealed 24 novel risk loci for Alzheimer's disease (AD). We recently reported that the bridging integrator 1 (BIN1) risk gene is linked to Tau pathology. RESULTS: We used glutathione S-transferase pull-down assays and nuclear magnetic resonance (NMR) experiments to demonstrate that BIN1 and Tau proteins interact directly and then map the interaction between BIN1's SH3 domain and Tau's proline-rich domain (PRD) . Our NMR data showed that Tau phosphorylation at Thr231 weakens the SH3-PRD interaction. Using primary neurons, we found that BIN1-Tau complexes partly co-localize with the actin cytoskeleton; however, these complexes were not observed with Thr231-phosphorylated Tau species. CONCLUSION: Our results show that (i) BIN1 and Tau bind through an SH3-PRD interaction and (ii) the interaction is downregulated by phosphorylation of Tau Thr231 (and potentially other residues). Our study sheds new light on regulation of the BIN1/Tau interaction and opens up new avenues for exploring its complex's role in the pathogenesis of AD.

Fatty acid transport proteins in disease: New insights from invertebrate models.

The dysregulation of lipid metabolism has been implicated in various diseases, including diabetes, cardiopathies, dermopathies, retinal and neurodegenerative diseases. Mouse models have provided insights into lipid metabolism. However, progress in the understanding of these pathologies is hampered by the multiplicity of essential cellular processes and genes that modulate lipid metabolism. Drosophila and Caenorhabditis elegans have emerged as simple genetic models to improve our understanding of these metabolic diseases. Recent studies have characterized fatty acid transport protein (fatp) mutants in Drosophila and C. elegans, establishing new models of cardiomyopathy, retinal degeneration, fat storage disease and dermopathies. These models have generated novel insights into the physiological role of the Fatp protein family in vivo in multicellular organisms, and are likely to contribute substantially to progress in understanding the etiology of various metabolic disorders. Here, we describe and discuss the mechanisms underlying invertebrate fatp mutant models in the light of the current knowledge relating to FATPs and lipid disorders in vertebrates.

In vivo assessment of neuronal cell death in Drosophila.

Following neuronal cell death at the cellular level and over several time points is challenging in living animal because of the difficulty of accessing and identifying individual neurons. In the eye of a living Drosophila, it is possible to visualize neurons thanks to the cornea neutralization technique. This technique can be coupled to the generation of mosaic clones by the Tomato /GFP -FLP/FRT method to identify a group of photoreceptor neurons at a single-cell resolution. This method has proved to be efficient for the study of photoreceptor development and degeneration. In this chapter, I describe this method and focus on fatp mutant photoreceptor neuron degeneration.

The Tomato/GFP-FLP/FRT method for live imaging of mosaic adult Drosophila photoreceptor cells.

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

TDP-43 loss-of-function causes neuronal loss due to defective steroid receptor-mediated gene program switching in Drosophila.

TDP-43 proteinopathy is strongly implicated in the pathogenesis of amyotrophic lateral sclerosis and related neurodegenerative disorders. Whether TDP-43 neurotoxicity is caused by a novel toxic gain-of-function mechanism of the aggregates or by a loss of its normal function is unknown. We increased and decreased expression of TDP-43 (dTDP-43) in Drosophila. Although upregulation of dTDP-43 induced neuronal ubiquitin and dTDP-43-positive inclusions, both up- and downregulated dTDP-43 resulted in selective apoptosis of bursicon neurons and highly similar transcriptome alterations at the pupal-adult transition. Gene network analysis and genetic validation showed that both up- and downregulated dTDP-43 directly and dramatically increased the expression of the neuronal microtubule-associated protein Map205, resulting in cytoplasmic accumulations of the ecdysteroid receptor (EcR) and a failure to switch EcR-dependent gene programs from a pupal to adult pattern. We propose that dTDP-43 neurotoxicity is caused by a loss of its normal function.

Tau pathology modulates Pin1 post-translational modifications and may be relevant as biomarker.

A prerequisite to dephosphorylation at Ser-Pro or Thr-Pro motifs is the isomerization of the imidic peptide bond preceding the proline. The peptidyl-prolyl cis/trans isomerase named Pin1 catalyzes this mechanism. Through isomerization, Pin1 regulates the function of a growing number of targets including the microtubule-associated tau protein and is supposed to be deregulated Alzheimer's disease (AD). Using proteomics, we showed that Pin1 is posttranslationally modified on more than 5 residues, comprising phosphorylation, N-acetylation, and oxidation. Although Pin1 expression remained constant, Pin1 posttranslational two-dimensional pattern was modified by tau overexpression in a tau-inducible neuroblastoma cell line, in our THY-Tau22 mouse model of tauopathy as well as in AD. Interestingly, in all of these systems, Pin1 modifications were very similar. In AD brain tissue when compared with control, Pin1 is hyperphosphorylated at serine 16 and found in the most insoluble hyperphosphorylated tau fraction of AD brain tissue. Furthermore, in all tau pathology conditions, acetylation of Pin1 may also contribute to the differences observed. In conclusion, Pin1 displays several posttranslational modifications, which are specific in tauopathies and may be useful as biomarker.

Drosophila fatty acid transport protein regulates rhodopsin-1 metabolism and is required for photoreceptor neuron survival.

Tight regulation of the visual response is essential for photoreceptor function and survival. Visual response dysregulation often leads to photoreceptor cell degeneration, but the causes of such cell death are not well understood. In this study, we investigated a fatty acid transport protein (fatp) null mutation that caused adult-onset and progressive photoreceptor cell death. Consistent with fatp having a role in the retina, we showed that fatp is expressed in adult photoreceptors and accessory cells and that its re-expression in photoreceptors rescued photoreceptor viability in fatp mutants. The visual response in young fatp-mutant flies was abnormal with elevated electroretinogram amplitudes associated with high levels of Rhodopsin-1 (Rh1). Reducing Rh1 levels in rh1 mutants or depriving flies of vitamin A rescued photoreceptor cell death in fatp mutant flies. Our results indicate that fatp promotes photoreceptor survival by regulating Rh1 abundance.

ER stress inhibits neuronal death by promoting autophagy.

Endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases but its relationship and role in disease progression remain unclear. Using genetic and pharmacological approaches, we showed that mild ER stress ("preconditioning") is neuroprotective in Drosophila and mouse models of Parkinson disease. In addition, we found that the combination of mild ER stress and apoptotic signals triggers an autophagic response both in vivo and in vitro. We showed that when autophagy is impaired, ER-mediated protection is lost. We further demonstrated that autophagy inhibits caspase activation and apoptosis. Based on our findings, we conclude that autophagy is required for the neuroprotection mediated by mild ER stress, and therefore ER preconditioning has potential therapeutic value for the treatment of neurodegenerative diseases.

Drosophila models of tauopathies: what have we learned?

Aggregates of the microtubule-associated protein Tau are neuropathological hallmark lesions in Alzheimer's disease (AD) and related primary tauopathies. In addition, Tau is genetically implicated in a number of human neurodegenerative disorders including frontotemporal dementia (FTD) and Parkinson's disease (PD). The exact mechanism by which Tau exerts its neurotoxicity is incompletely understood. Here, we give an overview of how studies using the genetic model organism Drosophila over the past decade have contributed to the molecular understanding of Tau neurotoxicity. We compare the different available readouts for Tau neurotoxicity in flies and review the molecular pathways in which Tau has been implicated. Finally, we emphasize that the integration of genome-wide approaches in human or mice with high-throughput genetic validation in Drosophila is a fruitful approach.