Fly Lines Research Articles

Functional Analysis of Human GBA1 Missense Mutations in Drosophila: Insights into Gaucher Disease Pathogenesis and Phenotypic Consequences.

The human GBA1 gene encodes lysosomal acid β-glucocerebrosidase, whose activity is deficient in Gaucher disease (GD). In Drosophila, there are two GBA1 orthologs, Gba1a and Gba1b, and Gba1b is the bona fide GCase encoding gene. Several fly lines with different deletions in the Gba1b were studied in the past. However, since most GD-associated GBA1 mutations are point mutations, we created missense mutations homologous to the two most common GD mutations: the mild N370S mutation (D415S in Drosophila) and the severe L444P mutation (L494P in Drosophila), using the CRISPR-Cas9 technology. Flies homozygous for the D415S mutation (dubbed D370S hereafter) presented low GCase activity and substrate accumulation, which led to lysosomal defects, activation of the Unfolded Protein Response (UPR), inflammation/neuroinflammation, and neurodegeneration along with earlier death compared to control flies. Surprisingly, the L494P (called L444P hereafter) flies presented higher GCase activity with fewer lysosomal defects and milder disease in comparison to that presented by the D370S homozygous flies. Treatment with ambroxol had a limited effect on all homozygous fly lines tested. Overall, our results underscore the differences between the fly and human GCase enzymes, as evidenced by the distinct phenotypic outcomes of mutations in flies compared to those observed in human GD patients.

Transgenic Drosophila Expressing Active Human LH Receptor in the Gonads Exhibit a Decreased Fecundity: Towards a Platform to Identify New Orally Active Modulators of Gonadotropin Receptor Activity.

The gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and their receptors are major regulators of reproduction in mammals and are absent in insects. We previously established transgenic Drosophila lines expressing a constitutively active human LH receptor variant (LHRD578Y) and the wild-type receptor (LHRwt; inactive in the absence of an agonist). That study showed that ubiquitously expression of LHRD578Y-but not of LHRwt-resulted in pupal lethality, and targeted expression in midline cells resulted in thorax/bristles defects. To further study the Drosophila model for an in vivo drug screening platform, we investigated here whether expressing LHRD578Y in the fly gonads alters reproduction, as shown in a transgenic mice model. The receptor was expressed in somatic cells of the gonads using the tissue-specific traffic jam-Gal4 driver. Western blot analysis confirmed receptor expression in the ovaries. A fecundity assay indicated that the ectopic expression of LHRD578Y resulted in a decrease in egg laying compared to control flies carrying, but not expressing the transgene (~40% decrease in two independent fly lines, p < 0.001). No significant reduction in the number of laid eggs was seen in flies expressing the LHRWT (<10% decrease compared to non-driven flies, p > 0.05). The decreased egg laying demonstrates a phenotype of the active receptor in the fly gonads, the prime target organs of the gonadotropins in mammals. We suggest that this versatile Drosophila model can be used for the pharmacological search for gonadotropin modulators. This is expected to provide: (a) new mimetic drug candidates (receptor-agonists/signaling-activators) for assisted reproduction treatment, (b) blockers for potential fertility regulation, and (c) leads relevant for the purpose of managing extra gonadotropic reported activities.

Proteomic analysis of the SMN complex reveals conserved and etiologic connections to the proteostasis network.

Molecular chaperones and co-chaperones are highly conserved cellular components that perform a variety of duties related to the proper three-dimensional folding of the proteome. The web of factors that carries out this essential task is called the proteostasis network (PN). Ribonucleoproteins (RNPs) represent an underexplored area in terms of the connections they make with the PN. The Survival Motor Neuron (SMN) complex is an assembly chaperone and serves as a paradigm for studying how specific RNAs are identified and paired with their client substrate proteins to form RNPs. SMN is the eponymous component of a large complex, required for the biogenesis of uridine-rich small nuclear ribonucleoproteins (U-snRNPs), that localizes to distinct membraneless organelles in both the nucleus and cytoplasm of animal cells. SMN protein forms the oligomeric core of this complex, and missense mutations in the human SMN1 gene are known to cause Spinal Muscular Atrophy (SMA). The basic framework for understanding how snRNAs are assembled into U-snRNPs is known. However, the pathways and mechanisms used by cells to regulate their biogenesis are poorly understood. Given the importance of these processes to normal development as well as neurodegenerative disease, we set out to identify and characterize novel SMN binding partners. We carried out affinity purification mass spectrometry (AP-MS) of Drosophila SMN complexes using fly lines exclusively expressing either wildtype or SMA-causing missense alleles. Bioinformatic analyses of the pulldown data, along with comparisons to proximity labeling studies carried out in human cells, revealed conserved connections to at least two other major chaperone systems including heat shock folding chaperones (HSPs) and histone/nucleosome assembly chaperones. Notably, we found that heat shock cognate protein Hsc70-4 and other HspA family members preferentially associated with SMA-causing alleles of SMN. Hsc70-4 is particularly interesting because its mRNA is aberrantly sequestered by a mutant form of TDP-43 in mouse and Drosophila ALS (Amyotrophic Lateral Sclerosis) disease models. Most important, a missense allele of Hsc70-4 (HspA8 in mammals) was recently identified as a bypass suppressor of the SMA phenotype in mice. Collectively, these findings suggest that chaperone-related dysfunction lies at the etiological root of both ALS and SMA.

Subtle changes in respiratory function produce big differences in life-history traits in stressed mitonuclear fly lines

Subtle changes in respiratory function produce big differences in life-history traits in stressed mitonuclear fly lines

Orange maker: a CRISPR/Cas9-mediated genome editing and screening project to generate orange-eyed DarkJedi GAL4 lines by undergraduate students

One of the greatest strengths of Drosophila genetics is its easily observable and selectable phenotypic markers. The mini-white marker has been widely used as a transgenic marker for Drosophila transgenesis. Flies carrying a mini-white construct can exhibit various eye colors ranging from pale orange to intense red, depending on the insertion site and gene dosage. Because the two copies of the mini-white marker show a stronger orange color, this is often used for selecting progenies carrying two transgenes together in a single chromosome after chromosomal recombination. However, some GAL4 lines available in the fly community originally have very strong red eyes. Without employing another marker, such as GFP, generating a recombinant chromosome with the strong red-eyed GAL4 and a desired UAS-transgene construct may be difficult. Therefore, we decided to change the red eyes of GAL4 lines to orange color. To change the eye color of the fly, we tested the CRISPR/Cas9 method with a guide RNA targeting the white gene with OK371-GAL4 and elav-GAL4. After a simple screening, we have successfully obtained multiple lines of orange-eyed OK371-GAL4 and elav-GAL4 that still maintain their original expression patterns. All of these simple experiments were performed by undergraduate students, allowing them to learn about a variety of different genetic experiments and genome editing while contributing to the fly research community by creating fruit fly lines that will be used in real-world research.

Lack of prion transmission barrier in human PrP transgenic Drosophila

While animal prion diseases are a threat to human health, their zoonotic potential is generally inefficient because of interspecies prion transmission barriers. New animal models are required to provide an understanding of these prion transmission barriers and to assess the zoonotic potential of animal prion diseases. To address this goal, we generated Drosophila transgenic for human or non-human primate PrP and determined their susceptibility to known pathogenic prion diseases, namely vCJD and classical BSE, and that with unknown pathogenic potential, namely CWD. Adult Drosophila transgenic for M129 or V129 human PrP, or non-human primate PrP developed a neurotoxic phenotype and showed an accelerated loss of survival after exposure to vCJD, classical BSE, or CWD prions at the larval stage. vCJD prion strain identity was retained after passage in both M129 and V129 human PrP Drosophila. All of the primate PrP fly lines accumulated prion seeding activity and concomitantly developed a neurotoxic phenotype, generally including accelerated loss of survival, after exposure to CWD prions derived from different cervid species, including North American white-tailed deer and muntjac, and European reindeer and moose. These novel studies show that primate PrP transgenic Drosophila lack known prion transmission barriers since, in mammalian hosts, V129 human PrP is associated with severe resistance to classical BSE prions, while both human and cynomolgus macaque PrP are associated with resistance to CWD prions. Significantly, our data suggest that interspecies differences in the amino acid sequence of PrP may not be a principal determinant of the prion transmission barrier.

Airway specific deregulation of asthma-related serpins impairs tracheal architecture and oxygenation in D. melanogaster

Serine proteases are important regulators of airway epithelial homeostasis. Altered serum or cellular levels of two serpins, Scca1 and Spink5, have been described for airway diseases but their function beyond antiproteolytic activity is insufficiently understood. To close this gap, we generated fly lines with overexpression or knockdown for each gene in the airways. Overexpression of both fly homologues of Scca1 and Spink5 induced the growth of additional airway branches, with more variable results for the respective knockdowns. Dysregulation of Scca1 resulted in a general delay in fruit fly development, with increases in larval and pupal mortality following overexpression of this gene. In addition, the morphological changes in the airways were concomitant with lower tolerance to hypoxia. In conclusion, the observed structural changes of the airways evidently had a strong impact on the airway function in our model as they manifested in a lower physical fitness of the animals. We assume that this is due to insufficient tissue oxygenation. Future work will be directed at the identification of key molecular regulators following the airway-specific dysregulation of Scca1 and Spink5 expression.

“Being Careful in Only a Perverse Way”: The Use of Aesthetic Experience in Psychoanalytic Work

ABSTRACT The author describes some of his interest in the painting of Richard Diebenkorn, the poetry of Bob Dylan, and a lifelong enjoyment of entomology and fly fishing as it comes into “play” with his analytic work. Diebenkorn’s “Notes to Myself Upon Beginning a Painting,” which were discovered after his death in 1995, are remarkably overlapping with elements of the ontological analysis of both Winnicott and Bion. The author elaborates a few elements of analytic listening from inside his personal “aesthetic matrix,” a concept so helpfully introduced in Palmer’s paper, “The Aesthetic Matrix: A Conversation Between a Painter and a Psychoanalyst.” There is special attention to a few varieties of visual and verbal imagery linked to auxiliary containing functions for the analyst.

Chaperoning the chaperones: Proteomic analysis of the SMN complex reveals conserved and etiologic connections to the proteostasis network.

Molecular chaperones and co-chaperones are highly conserved cellular components that perform variety of duties related to the proper three-dimensional folding of the proteome. The web of factors that carries out this essential task is called the proteostasis network (PN). Ribonucleoproteins (RNPs) represent an underexplored area in terms of the connections they make with the PN. The Survival Motor Neuron (SMN) complex is an RNP assembly chaperone and serves as a paradigm for studying how specific small nuclear (sn)RNAs are identified and paired with their client substrate proteins. SMN protein is the eponymous component of a large complex required for the biogenesis of uridine-rich small nuclear ribonucleoproteins (U-snRNPs) and localizes to distinct membraneless organelles in both the nucleus and cytoplasm of animal cells. SMN forms the oligomeric core of this complex, and missense mutations in its YG box self-interaction domain are known to cause Spinal Muscular Atrophy (SMA). The basic framework for understanding how snRNAs are assembled into U-snRNPs is known, the pathways and mechanisms used by cells to regulate their biogenesis are poorly understood. Given the importance of these processes to normal development as well as neurodegenerative disease, we set out to identify and characterize novel SMN binding partners. Here, we carried out affinity purification mass spectrometry (AP-MS) of SMN using stable fly lines exclusively expressing either wildtype or SMA-causing missense alleles. Bioinformatic analyses of the pulldown data, along with comparisons to proximity labeling studies carried out in human cells, revealed conserved connections to at least two other major chaperone systems including heat shock folding chaperones (HSPs) and histone/nucleosome assembly chaperones. Notably, we found that heat shock cognate protein Hsc70-4 and other HspA family members preferentially interacted with SMA-causing alleles of SMN. Hsc70-4 is particularly interesting because its mRNA is aberrantly sequestered by a mutant form of TDP-43 in mouse and Drosophila ALS (Amyotrophic Lateral Sclerosis) disease models. Most important, a missense allele of Hsc70-4 (HspA8 in mammals) was recently identified as a bypass suppressor of the SMA phenotype in mice. Collectively, these findings suggest that chaperone-related dysfunction lies at the etiological root of both ALS and SMA.

Becoming Nature: Encounters in Interspecies Contact Zones

Abstract Nature affords transformations to consumers’ social, embodied, and temporal experiences. Yet, consumer research has yet to consider how wild species contribute to and are affected by experiential consumption in nature. With data from an ethnography of fly fishing, we theorize human-fish interactions as interspecies encounters in contact zones. Our findings explain how these encounters are established, engendering processes of interspecies becoming that transform both species. We discuss how these transformations are ordered by power relationships that classify roles for entities enrolled in consumption assemblages. Often, humans exert power over other living entities by classifying them as resources for consumption. Yet we also discover more reciprocal expressions of power between humans and other species. With consumption as a major contributor to the decline of wild species populations, we discuss theoretical and practical implications of our work that are intended to stimulate further research.

LEVELS OF CARBOHYDRATES IN SHORT-LIVED STRAIN OF Drosophila melanogaster

Aim. The aim of this study was to investigate the concentrations of key energetic substrates such as glucose, fructose, and trehalose, in control and artificially selected flies shown to have shorter lifespan. Methods. For all experiments flies were maintained on standard yeast-sucrose nutrient medium consisted of 5% sucrose, 5% yeast, 1% agar and 0.18% nipagin as mold growth inhibitor. Experimental line of flies was selected on high-protein diet and previously showed short lifespan. Newly enclosed flies were kept two days for mating and used for measurement of feeding behavior and fecundity. Some flies were frozen at -80 °C for further analysis. Concentrations of glucose, fructose and trehalose were measured by Gas Chromatography/Mass Spectrometry using described protocol and expressed per wet weight. Results. Amount of glucose was significantly higher in experimental flies of both sexes. Male flies had about 70% more free glucose and females for about 45%. These differences were accompanied with higher amounts of trehalose that consist of two molecules of glucose and is a form of glucose storage in Drosophila. Trehalose content in experimental males was 2-fold higher, while in females it was higher for about 1.8-fold. Fructose content was also higher in experimental flies. This carbohydrate is metabolized through glycolysis, providing energy in the form of ATP. Beyond energy production, fructose plays a role in supporting reproductive processes such as oogenesis in females, though measure of fecundity showed no difference. Conclusions. Our results show that despite similar consumption of food and reproduction offspring of artificially selected flies have shorter lifespan. However, this effect can be explained by lower energetic status while flies store more energetic substrates such as glucose, fructose and trehalose.

The 23rd Annual Meeting of the Rocky Mountain Virology Association.

Located 50 miles west of Fort Collins, Colorado, Colorado State University's Mountain Campus in Pingree Park hosted the 23rd annual Rocky Mountain Virology Association meeting in 2023 with 116 participants. The 3-day event at the end of September consisted of 28 talks and 43 posters that covered the topics of viral evolution and surveillance, developments in prion research, arboviruses and vector biology, host-virus interactions, and viral immunity and vaccines. This year's Randall Jay Cohrs keynote presentation covered the topic of One Health and emerging coronaviruses. This timely discussion covered the importance of global disease surveillance, international collaboration, and trans-disciplinary research teams to prevent and control future pandemics. Peak fall colors flanked the campus and glowed along the multiple mountain peaks, allowing for pristine views while discussing science and networking, or engaging in mountain activities like fly fishing and hiking. On behalf of the Rocky Mountain Virology Association, this report summarizes select presentations from the 23rd annual meeting.

Expanding the Drosophila toolkit for dual control of gene expression.

The ability to independently control gene expression in two different tissues in the same animal is emerging as a major need, especially in the context of inter-organ communication studies. This type of study is made possible by technologies combining the GAL4/UAS and a second binary expression system such as the LexA system or QF system. Here, we describe a resource of reagents that facilitate combined use of the GAL4/UAS and a second binary system in various Drosophila tissues. Focusing on genes with well-characterized GAL4 expression patterns, we generated a set of more than 40 LexA-GAD and QF2 insertions by CRISPR knock-in and verified their tissue specificity in larvae. We also built constructs that encode QF2 and LexA-GAD transcription factors in a single vector. Following successful integration of this construct into the fly genome, FLP/FRT recombination is used to isolate fly lines that express only QF2 or LexA-GAD. Finally, using new compatible shRNA vectors, we evaluated both LexA and QF systems for in vivo gene knockdown and are generating a library of such RNAi fly lines as a community resource. Together, these LexA and QF system vectors and fly lines will provide a new set of tools for researchers who need to activate or repress two different genes in an orthogonal manner in the same animal.

Expanding the Drosophila toolkit for dual control of gene expression

The ability to independently control gene expression in two different tissues in the same animal is emerging as a major need, especially in the context of inter-organ communication studies. This type of study is made possible by technologies combining the GAL4/UAS and a second binary expression system such as the LexA system or QF system. Here, we describe a resource of reagents that facilitate combined use of the GAL4/UAS and a second binary system in various Drosophila tissues. Focusing on genes with well-characterized GAL4 expression patterns, we generated a set of more than 40 LexA-GAD and QF2 insertions by CRISPR knock-in and verified their tissue specificity in larvae. We also built constructs that encode QF2 and LexA-GAD transcription factors in a single vector. Following successful integration of this construct into the fly genome, FLP/FRT recombination is used to isolate fly lines that express only QF2 or LexA-GAD. Finally, using new compatible shRNA vectors, we evaluated both LexA and QF systems for in vivo gene knockdown and are generating a library of such RNAi fly lines as a community resource. Together, these LexA and QF system vectors and fly lines will provide a new set of tools for researchers who need to activate or repress two different genes in an orthogonal manner in the same animal.

NeuronBridge: an intuitive web application for neuronal morphology search across large data sets

BackgroundNeuroscience research in Drosophila is benefiting from large-scale connectomics efforts using electron microscopy (EM) to reveal all the neurons in a brain and their connections. To exploit this knowledge base, researchers relate a connectome’s structure to neuronal function, often by studying individual neuron cell types. Vast libraries of fly driver lines expressing fluorescent reporter genes in sets of neurons have been created and imaged using confocal light microscopy (LM), enabling the targeting of neurons for experimentation. However, creating a fly line for driving gene expression within a single neuron found in an EM connectome remains a challenge, as it typically requires identifying a pair of driver lines where only the neuron of interest is expressed in both. This task and other emerging scientific workflows require finding similar neurons across large data sets imaged using different modalities.ResultsHere, we present NeuronBridge, a web application for easily and rapidly finding putative morphological matches between large data sets of neurons imaged using different modalities. We describe the functionality and construction of the NeuronBridge service, including its user-friendly graphical user interface (GUI), extensible data model, serverless cloud architecture, and massively parallel image search engine.ConclusionsNeuronBridge fills a critical gap in the Drosophila research workflow and is used by hundreds of neuroscience researchers around the world. We offer our software code, open APIs, and processed data sets for integration and reuse, and provide the application as a service at http://neuronbridge.janelia.org.

Test penjanja kao mera lokomotrone sposobnosti na modelu vinske mušice za fragilni X sindrom

Introduction: Fragile X syndrome (FXS) is the most common monogenetic cause of intellectual disability (ID) and autism spectrum disorder (ASD) in humans. The Drosophila melanogaster model of FXS (dFMR1 mutants) is an excellent model for research in the field of FXS. The aim of this study was a comprehensive investigation of climbing abilities, as a measurement of locomotion, in the dFMR1B55 line as a Drosophila model of FXS. Methods: In this study, control w1118 and dFMR1B55 lines of fruit flies were used. The climbing performance of flies was examined using a climbing performance assay for groups of flies as well as for individual flies. Parameters that represent climbing ability, speed and endurance were determined. Females and males were analyzed separately. Results: This study revealed the following: (i) worse climbing performance of dFMR1B55 males in comparison to w1118 males; (ii) worse climbing success of dFMR1B55 females in comparison to w1118 females; (iii) better climbing performance of top performer males in comparison to top performer females in the group climbing test in both dFMR1B55 and w1118 groups; (iv) better, but not statistically significant, climbing performance (based on the time needed for 50% of flies to complete the task), and a higher success rate in dFMR1B55 females in comparison to dFMR1B55 males. Conclusion: According to the results of the current study, climbing impairment was proved only in dFMR1B55 males, while dFMR1B55 females had climbing abilities similar to control w1118 females.

Boosting life sciences research in Brazil: building a case for a local Drosophila stock center.

Drosophila melanogaster is undoubtedly one of the most useful model organisms in biology. Initially used in solidifying the principles of heredity, and establishing the basic concepts of population genetics and of the synthetic theory of evolution, it can currently offer scientists much more: the possibility of investigating a plethora of cellular and biological mechanisms, from development and function of the immune system to animal neurogenesis, tumorigenesis and beyond. Extensive resources are available for the community of Drosophila researchers worldwide, including an ever-growing number of mutant, transgenic and genomically-edited lines currently carried by stock centers in North America, Europe and Asia. Here, we provide evidence for the importance of stock centers in sustaining the substantial increase in the output of Drosophila research worldwide in recent decades. We also discuss the challenges that Brazilian Drosophila scientists face to keep their research projects internationally competitive, and argue that difficulties in importing fly lines from international stock centers have significantly stalled the progression of all Drosophila research areas in the country. Establishing a local stock center might be the first step towards building a strong local Drosophila community that will likely contribute to all areas of life sciences research.

Selection parameters in lines of Drosophila melanogaster Meig., obtained from populations living in territories with different levels of radiation pollution: approbation of the Crow’s method

Man-made disasters, such as the accidents at the Chornobyl NPP and the Fukushima NPP-1, have raised questions about the radiation risks associated with the use of atomic energy in a new and acute way. The purpose of the study was to investigate the features of the action of natural selection in the lines of Drosophila melanogaster Meig., obtained from natural populations living in territories with different levels of radiation pollution. One of the aims was to test Crow's method on a model object, such as Drosophila. This method makes it possible to estimate the total intensity of selection, as well as to determine the contribution of its individual components, such as differential fecundity and differential mortality. The study was carried out on three lines of Drosophila melanogaster Meig.: the Haidary line (radiation background in the territory from which the line originates: 0.12 μSv/h, β-radiation flux: 0 particles/cm2/min), the KhPTI line (radiation background: 0.12–0.20 μSv/h, β-radiation flux: 0 particles/cm2/min), the Chornobyl line (radiation background: 0.20–0.22 μSv/h, β-radiation flux: 7–8 particles/cm2/min). According to the obtained results, the lines did not differ among themselves in egg production of females. In terms of the number of adult offspring, the Chornobyl line was inferior to the Haidary and KhPTI lines by 48.9% and 57.8%, respectively. Mortality in the pre-reproductive period of development (indicator pd), which includes embryonic and pupal mortality, was the highest in the Chornobyl line and exceeded the pd value in the Haidary and KhPTI lines by 1.4 times. As a result, the Chornobyl line, obtained from the territory contaminated with radionuclides, significantly exceeded the Haidary and KhPTI lines, obtained from the territories where the radiation situation does not go beyond the norm, by both components of selection – both by the component of differential fecundity (If), and by differential mortality (lm). The total selection indices (Itot) were quite close in the Haidary and KhPTI lines, and in the Chornobyl line this index was 2,1–2,6 times higher than in the other two lines. The results of the study support the view that ionizing radiation can promote evolution by accelerating evolutionary change. They indicate an increased mortality rate, a reduced level of fitness and an increased selection pressure in the line of fruit flies, which originates from the population living in the radiation-contaminated territory in the Chornobyl exclusion zone.

APOL1-G2 accelerates nephrocyte cell death by inhibiting the autophagy pathway.

People of African ancestry who carry the APOL1 risk alleles G1 or G2 are at high risk of developing kidney diseases through not fully understood mechanisms that impair the function of podocytes. It is also not clear whether the APOL1-G1 and APOL1-G2 risk alleles affect these cells through similar mechanisms. Previously, we have developed transgenic Drosophila melanogaster lines expressing either the human APOL1 reference allele (G0) or APOL1-G1 specifically in nephrocytes, the cells homologous to mammalian podocytes. We have found that nephrocytes that expressed the APOL1-G1 risk allele display accelerated cell death, in a manner similar to that of cultured human podocytes and APOL1 transgenic mouse models. Here, to compare how the APOL1-G1 and APOL1-G2 risk alleles affect the structure and function of nephrocytes in vivo, we generated nephrocyte-specific transgenic flies that either expressed the APOL1-G2 or both G1 and G2 (G1G2) risk alleles on the same allele. We found that APOL1-G2- and APOL1-G1G2-expressing nephrocytes developed more severe changes in autophagic pathways, acidification of organelles and the structure of the slit diaphragm, compared to G1-expressing nephrocytes, leading to their premature death. We conclude that both risk alleles affect similar key cell trafficking pathways, leading to reduced autophagy and suggesting new therapeutic targets to prevent APOL1 kidney diseases.

Abeta and Tau impair retromer‐mediated cargo trafficking via distinct mechanisms

AbstractBackgroundEvidence from neuronal pathology and experimental models suggest that endolysosomal dysfunction may contribute to Alzheimer’s Disease (AD) pathogenesis, however it is unclear how Aβ and Tau directly affect endolysosomal dynamics, partially due to a lack of reporters. The retromer, including VPS35, VPS26, and VPS29, is a conserved protein complex responsible for recycling proteins within the endolysosomal pathway. Retromer regulates Tau phosphorylation and pathogenic processing of the amyloid precursor protein. Cation‐independent Mannose 6‐phosphate receptor (CIMPR) is a well‐established retromer cargo, with a WLM motif recognized by the retromer for retrieval.MethodTo track retromer‐dependent protein trafficking in vivo, I have cloned the cytoplasmic tail of CIMPR carrying the WLM motif into a pUAST‐attb vector and generated a transgenic fly line (UAS‐CIMPR‐WLM). A transgenic line carrying CIMPR, but having WLM mutated to AAA, has also been developed as a negative control (UAS‐CIMPR‐AAA). I examined the CIMPR reporter in Vps35 and Vps29 mutants, as well as established Drosophila AD models during aging.ResultThe protein level of CIMPR‐WLM was reduced by genetic ablation of Vps35, possibly due to promoted degradation. Expressed in young Vps29 mutant brains, CIMPR‐WLM showed an increased colocalization with a late endosomal/lysosomal marker Arl8, indicating a promoted delivery of retromer cargoes into the lysosome. Further, neuronal expression of CIMPR‐WLM exhibited a punctate distribution, which was abolished when WLM was mutated to AAA. Altogether, my data suggested that CIMPR‐WLM worked as a retromer‐dependent reporter. While the reporter was broadly enriched within neuropil regions in control animals (Elav&gt;CIMPR), neuronally expression of Aβ (Elav&gt; Aβ+CIMPR) led to a striking redistribution of CIMPR, shifting from neuropil to enrichment in soma, and forming large perinuclear puncta. Moreover, the retromer reporter was mislocalized in brains from 1‐day‐old Elav&gt; Aβ animals, and this result did not significantly change with aging. Interestingly, Elav&gt;Tau+CIMPR animals showed no noticeable effects on the distribution of CIMPR‐WLM on day 1, but caused a similar redistribution of CIMPR‐WLM to the perinuclei region at day 30.ConclusionThe CIMPR‐WLM reporter requires the retromer for proper cellular localization. Our findings are consistent with the notion that Aβ and Tau affect endosomal sorting, but likely via distinct mechanisms.