Aggregated Aβ1-42 Is Selectively Toxic for Neurons, Whereas Glial Cells Produce Mature Fibrils with Low Toxicity in Drosophila

Article

Aggregated Ab1-42 Is Selectively Toxic for Neurons, Whereas Glial Cells Produce Mature Fibrils with Low Toxicity in Drosophila Graphical Abstract

Authors Maria Jonson, Sofie Nystro¨m, Alexander Sandberg, ..., K. Peter R. Nilsson, Stefan Thor, Per Hammarstro¨m

Correspondence [email protected]

In Brief Jonson et al. used transgenic Drosophila to understand cell-specific response to protein aggregates in neurodegenerative disease. They demonstrate that the Alzheimer-associated peptide Ab1-42 form various amyloid structures with different toxic properties when expressed in different cell types of the brain.

Highlights d

Expressed Ab1-42 aggregates profoundly in various cell types of Drosophila

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Ab1-42 accumulates as extracellular amyloid fibrils when expressed in glial cells

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Ab1-42 is highly toxic to neurons in Drosophila Immature intracellular aggregates are more toxic than mature fibrillar Ab1-42

Jonson et al., 2018, Cell Chemical Biology 25, 1–16 June 21, 2018 ª 2018 Elsevier Ltd. https://doi.org/10.1016/j.chembiol.2018.03.006

Please cite this article in press as: Jonson et al., Aggregated Ab1-42 Is Selectively Toxic for Neurons, Whereas Glial Cells Produce Mature Fibrils with Low Toxicity in Drosophila, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.03.006

Cell Chemical Biology

Article Aggregated Ab1-42 Is Selectively Toxic for Neurons, Whereas Glial Cells Produce Mature Fibrils with Low Toxicity in Drosophila Maria Jonson,1 Sofie Nystro¨m,1 Alexander Sandberg,1 Marcus Carlback,1 Wojciech Michno,2 Jo¨rg Hanrieder,2,3 Annika Starkenberg,4 K. Peter R. Nilsson,1 Stefan Thor,4 and Per Hammarstro¨m1,5,* 1Department

of Physics, Chemistry and Biology, Linko¨ping University, Linko¨ping SE-581 83, Sweden of Psychiatry and Neurochemistry, Sahlgrenska Academy at the University of Gothenburg, 431 80 Mo¨lndal, Sweden 3Department of Molecular Neuroscience, Institute of Neurology, University College London, London W1C3BG, UK 4Department of Clinical and Experimental Medicine, Linko ¨ ping University, Linko¨ping SE-581 85, Sweden 5Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.chembiol.2018.03.006 2Department

SUMMARY

The basis for selective vulnerability of certain cell types for misfolded proteins (MPs) in neurodegenerative diseases is largely unknown. This knowledge is crucial for understanding disease progression in relation to MPs spreading in the CNS. We assessed this issue in Drosophila by cell-specific expression of human Ab1-42 associated with Alzheimer’s disease. Expression of Ab1-42 in various neurons resulted in concentration-dependent severe neurodegenerative phenotypes, and intraneuronal ringtangle-like aggregates with immature fibril properties when analyzed by aggregate-specific ligands. Unexpectedly, expression of Ab1-42 from a pan-glial driver produced a mild phenotype despite massive brain load of Ab1-42 aggregates, even higher than in the strongest neuronal driver. Glial cells formed more mature fibrous aggregates, morphologically distinct from aggregates found in neurons, and was mainly extracellular. Our findings implicate that Ab1-42 cytotoxicity is both cell and aggregate morphotype dependent.

INTRODUCTION Alzheimer’s disease (AD) is a progressive neurodegenerative disease initially affecting memory and cognition. Neuropathological hallmarks of AD include formation of extracellular amyloid plaques composed of the amyloid-b (Ab) peptide (Glenner and Wong, 1984; Masters et al., 1985), and intracellular tangles composed of the Tau protein (Lee et al., 1991). The initiating event of AD is the accumulation of Ab aggregates (Jack et al., 2013). Because AD is a disease of memory loss, it is often assumed that excitatory neurons are the sole target cells in AD patient’s brain. However, pathological aggregates of Ab are found in and around neurons, microglia, astrocytes, smooth muscle cells in the vasculature, eye epithelial cells, and choroid

plexus epithelial cells. Ab amyloid deposits provoke inflammation, resulting in an increased attention to the role of glial cells in AD pathology (Sastre et al., 2006; Heneka et al., 2015; Heppner et al., 2015). However, how the various pathological amyloidogenic species form and influence tissue tropism in AD and other neurodegenerative diseases is poorly understood (Jackson, 2014). Drosophila melanogaster is an important tool for understanding human development and disease. Although Drosophila is evolutionary divergent from humans, 75% of human disease-related genes have orthologs, or genes with similar functions, in the fly (Reiter et al., 2001). Major advantages of using Drosophila as a model system is the short generation time and the low number of chromosomes, which makes it easy to genetically modify the fly genome (Jennings, 2011). Drosophila has become a widely used model system to study neurodegenerative disorders, among them AD. Amyloid plaques in human AD consist of different variants of the Ab peptide, with the most abundant being Ab1-40 and Ab1-42. It has been shown that expression of Ab1-42 in neurons of the fly CNS is toxic and cause shortened lifespan, progressive protein accumulation, impaired locomotor behavior, and amyloid buildup (Iijima et al., 2004; Crowther et al., 2005; Bilen and Bonini, 2005; Caesar et al., 2012; Luheshi et al., 2007), mimicking the events of human disease progression. The majority of these Drosophila studies expressed different variants of Ab in the same type of neurons using the elav-Gal4 driver or in the fly eye using the GMR-Gal4 driver. The same two drivers have been utilized to express other human neurodegenerative disease proteins (Feany and Bender, 2000; Bilen and Bonini, 2005; Berg et al., 2009; Thackray et al., 2012). In addition, a few studies have examined the effect of expressing these proteins, e.g., Tau (Colodner and Feany, 2010), Huntingtin, and Ataxin-1 (Shiraishi et al., 2014) in glial cells, with strong toxicity as a result. However, there are no studies describing a systematic expression of the Ab1-42 peptide in different cell types. To approach the question whether the Ab-toxicity and aggregation pattern seen in CNS neurons of Drosophila can be observed in other cell types, or if the toxic effects are cell-type specific, we turned to five different driver lines expressing Ab1-42 in most if not all neurons, motor neurons, glial cells, and eye. Flies expressing Ab1-42 showed a reduced lifespan Cell Chemical Biology 25, 1–16, June 21, 2018 ª 2018 Elsevier Ltd. 1

Please cite this article in press as: Jonson et al., Aggregated Ab1-42 Is Selectively Toxic for Neurons, Whereas Glial Cells Produce Mature Fibrils with Low Toxicity in Drosophila, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.03.006

compared with control flies; however, in a tissue-specific manner. We used our recently reported landing-site integrated UAS-Ab1-42 transgene (Jonson et al., 2015) to minimize gene background effects. An unexpected result was that glial cellexpressing flies had the least affected lifespan and locomotor behavior even though the amount of Ab1-42 aggregates was more extensive than that observed in the strongest and most toxic neuronal models. Our study demonstrates that Ab1-42 amyloid aggregates can be experimentally induced in various cell types of Drosophila. However, the cytotoxicity appears largely selective toward neurons. Our study indicates that immature Ab1-42 aggregates and fibrils are more toxic than mature fibrils, and that the process of Ab1-42 fibrillation is influenced by cell type. This opens up for mechanistic understanding of Ab1-42 cell-specific targets of toxicity and fibril morphotype or amyloid strain propagation. This is of importance for AD, since a vast distribution of amyloid aggregate conformations in plaque cores were recently reported in various patients (Rasmussen et al., 2017). RESULTS Histological Analysis Reveals Ab Aggregates in all Ab-Transgenes To address the question whether various Drosophila cell types can form Ab aggregates, we expressed previously generated landing-site-integrated UAS-Ab1-42 transgenes (Jonson et al., 2015) with the n-syb-Gal4, C155-elav-Gal4, D42-Gal4, GMRGal4, and repo-Gal4 driver lines, to direct expression to neurons, motor neurons, the eye disc, and glia. To analyze Ab aggregates, we used the amyloid-specific luminescent-conjugated oligothiophene (LCO) p-FTAA (Aslund et al., 2009). We have previously shown that p-FTAA co-localizes with antibodies against Ab in amyloid aggregates in Drosophila and that p-FTAA is more sensitive toward intact aggregates (Berg et al., 2010). In a first analysis flies were aged for 10 days post eclosion before the brains were dissected, stained, and analyzed by confocal microscopy. Although no aggregates were found in control flies (Figure S1), we found that all genotypes expressing Ab1-42 were affected by p-FTAA-positive aggregates; however, to a very different extent (Figures 1AI–1DI and 1H). In accordance with our previous studies (Caesar et al., 2012; Jonson et al., 2015), flies expressing Ab1-42 in CNS neurons displayed aggregates in the center of the brain (Figure 1AI and 1BI). To compare expression profiles of the Gal4 driver flies, we compared UAS-nuclear-GFP (nGFP) crosses. As expected nGFP expression in the strong neuronal driver flies showed a more prominent expression pattern than for the weak neuronal driver flies (Figure S2F), and were expression declined with age (Figure S2). However, the nGFP expression and the Ab1-42 accumulation do not fully overlap. In particular Ab1-42 aggregates were not prominent in the mushroom body where nGFP expression was high. Notwithstanding most Ab1-42 aggregates in the neuronal drivers found at high resolution were intracellular (see below). The strong neuronal (n-syb-Gal4) and weak neuronal (C155elav-Gal4) driver flies showed overall similar aggregation patterns with p-FTAA staining (Figures 1AI, 1AII, 1BI, and 1BII) (Figures S4AI, S4II, S4BI, and S4BII). Flies expressing Ab1-42 in motor neurons (D42-Gal4) also reveal p-FTAA-positive 2 Cell Chemical Biology 25, 1–16, June 21, 2018

aggregates; however, to a lower extent than the two global CNS neuronal-expressing models (Figures 1CI and 1CII). The Ab1-42 accumulation was quite similar to the nGFP expression in these flies (Figure 1CIII). An unexpected observation was made when examining the pan-glial cell-expressing flies (repoGal4). Compared with the other genotypes, these flies revealed extensive amounts of fibrillar amyloid structures, despite the apparent well-being of these flies at day 10 post eclosion. The fibrillar amyloid structures were spread out throughout the whole brain, instead of the more centralized pattern observed in the neuronal drivers (Figures 1DI and 1DII). This patchy expression pattern was very similar to that of nGFP in the glial flies (Figure 1DIII). Quantification of the location and amount of p-FTAA aggregates in the 3D images showed that the aggregates were embedded within the brain for all genotypes (Figure 1E). The quantity of p-FTAA-positive aggregate foci were significantly higher for glial cells compared with the neuronal drivers (Figure 1F). Eye disc expression (GMR-GAL4/Ab1-42) flies did not show aggregates in dissected brains, arguing against extensive transfer of Ab-peptides from the retina into the brain (Figure 1G). Still, when analyzing sections of these fly heads, we found extensive loads of aggregates in the retina, confirming the presence of aggregated Ab (Figures 1H and 1JI). As anticipated, at higher magnification, nGFP expression did not co-localize with Ab aggregates (Figures 1I and 1JI). Ab aggregates are localized in the area in between the two cell nuclei layers (arrow in Figure 1JII), while nGFP expression is localized in the two layers of cell nuclei (arrowheads in Figure 1JII). To assess the toxic effect of Ab in the retina we analyzed the eye structure. The Drosophila eye is composed of more than 700 precisely arranged ommatidia (Cagan, 2009). As an effect of toxic protein expression, eye development can be affected, with the ommatidia structure disrupted, hence resulting in a so-called rough eye phenotype (Fernandez-Funez et al., 2016). We used both light microscopy and scanning electron microscopy to detect such a phenotype, but did not observe any disruption of the eye structure in these flies compared with the control flies (Figure S3). This was unexpected due to the large amount of aggregates observed by p-FTAA staining. Ab1-42 Accumulation Levels Was in Agreement with Histological Findings To analyze if the different degrees of aggregation seen in the histological analysis was due to various levels of Ab1-42 expression, we used an immunoassay from Meso Scale Discovery (MSD). Flies were aged for 10 days post eclosion, prior to homogenization. We divided our head extracts into a soluble fraction, composed of fly heads homogenized in a HEPES buffer (pH 7.3), and an insoluble fraction, obtained from heads homogenized in a HEPES buffer containing 5 M GuHCl. To capture Ab1-42 we used an anti-Ab 42 antibody (12F4), while detection was accomplished by an antibody targeting the midregion of the Ab peptide (4G8). Regardless of the cell type expressing Ab1-42, very low amounts of the peptide were detected in the soluble fractions, ranging from 0 to 9 ng mL 1 per fly (Figure 1K). The majority of Ab1-42 was found in the insoluble fraction (Figure 1K). The amounts of Ab1-42 aggregates were in agreement with nGFP expression using these driver flies (Figures 1AIII, 1BIII, and S2). As anticipated from the nGFP expression,

Please cite this article in press as: Jonson et al., Aggregated Ab1-42 Is Selectively Toxic for Neurons, Whereas Glial Cells Produce Mature Fibrils with Low Toxicity in Drosophila, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.03.006

(legend on next page)

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Please cite this article in press as: Jonson et al., Aggregated Ab1-42 Is Selectively Toxic for Neurons, Whereas Glial Cells Produce Mature Fibrils with Low Toxicity in Drosophila, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.03.006

the weaker CNS neuronal driver (C155-elav-Gal4/Ab1-42) and the motor neuron expressing flies (D42-Gal4/Ab1-42) showed moderate levels of insoluble Ab1-42 (10–20 ng mL 1 per fly), while the strong CNS neuronal driver (n-syb-Gal4/Ab1-42) displayed higher amounts of protein; around 50 ng mL 1 per fly (Figure 1K). The p-FTAA aggregate load at day 10 was, however, similar in both the strong and weak neuronal drivers (Figure 1F). In accordance with the histological analysis of p-FTAA (Figure 1F), even higher amounts of Ab1-42 were detected in the insoluble fraction of glial expressing flies (repo-Gal4/ Ab1-42) and flies expressing Ab in the retina (GMR-Gal4/ Ab1-42), 60–75 ng mL 1 per fly (Figure 1K). Aggregation of Ab1-42 Progressively Increases with Fly Age We next analyzed if the aggregate load varied at different time points. Flies were aged for 5, 10, 15, 20, or 25 days post eclosion, depending on the genotype, before the brains were dissected, stained, and analyzed by confocal microscopy. The strong neuronal driver (n-syb-Gal4) flies were only analyzed at days 5 and 10 due to their short lifespan, and displayed high amounts of protein aggregates increasing with the age of the flies (Figures 1F. S4AI, and S4AII). Weak neuronal (C155-elav-Gal4)-derived flies showed aggregates at days 5 and 10, with an increase of aggregates with age (Figures 1F, S4BI, and S4BII). When these flies were analyzed at days 15 and 20, we observed decreased amounts of aggregates compared with the earlier time points (Figures S4BIII–S4BIV). This is most likely due to the decline in expression seen for C155-elav-Gal4 flies (Figure S2) while amyloid degradation proceeded (Jonson et al., 2015). Motor neuron expressing flies showed an increase of aggregates from day 5 to 10. At later time points (days 15 and 20) the aggregate load was persistent (Figures S4CI–S4CIV). Most notably, in the glial expressing flies, we observed moderate amounts of Ab aggregates at day 0 (Figure 2A); however, already at day 5 these flies displayed a higher amount of aggregates than the strong neuronal driver (n-syb-Gal4/Ab1-42) flies at day 10, toward the very end of their lives (Figure 1F). For glial expressing flies the

aggregate load visible, by p-FTAA staining, progressively increased with age, and at day 25 the brains were filled with aggregates (Figures 2A and S4DI–S4IV). Increased Ab levels in the insoluble fraction confirmed the increase in aggregation load with age for all genotypes. The soluble levels were quite constant; 0–11 ng mL 1 per fly, over time (Figure 2C; Table S1). Glial expressing flies displayed high insoluble Ab peptide levels from 50 to 70 ng mL 1 per fly, corresponding to the levels seen for the strong neuronal driver in this study and in previous studies (Figure 2C) (Jonson et al., 2015). At days 5–20 these levels were persistent both the concentration and the aggregate load (Figures 2B and 2C). Very high levels of insoluble Ab1-42 were detected in the retina also at day 20, GMR-GAL4/Ab1-42 flies (75–100 ng mL 1 per fly) (Table S1). This was unexpected since these flies showed intact ommatidia by scanning electron microscopy (Figure S3A). Thus, Ab1-42 aggregates appeared to be rather harmless to the retina of the fly at all time points, despite very high levels of Ab1-42. The GMR-Gal4 driver was verified in the context of the toxic Arctic mutation (UAS-Ab1-42 E22G) from Crowther et al. (2005) and Caesar et al. (2012), and here, as expected, showed a strong rough eye phenotype (Figure S3B). Expression of Ab1-42 in Glial Cells has a Minor Effect on Survival To assess Ab1-42 toxicity we investigated the lifespan of each genotype. Our previous studies (Caesar et al., 2012; Jonson et al., 2015) of neuronal Ab1-42 expression in flies have indicated a good correlation between protein levels, lifespan, and activity assays, indicating that high amounts of accumulated insoluble Ab1-42 results in reduced lifespan and impaired activity. Pan-neuronal expression of the Ab1-42 peptide in the fly CNS by; n-syb-Gal4/Ab1-42 (strong) and C155-elav-Gal4/Ab142 (weak) flies, resulted in a robustly reduced lifespan compared with the control flies (Figures 3A and 3B). However, there was a clear difference between the two neuronal CNS Gal4 drivers, with a median lifespan of 9 days for the stronger (n-sybGal4/Ab1-42) driver (Figure 3A) and 15 days for the weaker

Figure 1. Histological Staining Reveals Ab Aggregates in all Genotypes at Day 10 Post Eclosion (AI–DII) Whole Drosophila brain stained with the amyloid-specific LCO, p-FTAA (green), and cell nuclei stain ToPro3 (red) at day 10 post eclosion reveal Ab aggregates in all genotypes. (AIII–DIII) Expression of nuclear-GFP in dissected adult fly brains using different Gal4 drivers at day 5 post eclosion. Scale bars, 50 mm. See also Figure S1 for p-FTAA controls and Figure S2 for Gal-4/nGFP. (E) Topographic side view of a p-FTAA- and ToPro3-stained Drosophila whole brain with indicated z stack optical sections (19 layers and depth in mm). The bottom panel shows average distribution profiles of p-FTAA aggregate location in various genotypes at day 10. Average from three brains per map. (F) Quantification of p-FTAA-positive aggregate foci (pixels) for each genotype at days 5 and 10. Note the similar quantity of aggregates in strong and weak neuronal drivers and the lower amounts in motor neurons. Glial cells are much stronger at both time points. Bars represent means ± SEM deduced from triplicate samples in independent experiments and for 19 optical sections each. (G) Whole Drosophila brain GMR-Ab1-42 stained with the amyloid-specific LCO, p-FTAA (green), and cell nuclei stain ToPro3 (red) at day 10 post eclosion shows no aggregates in brain. (H and JI) Staining of GMR-Ab1-42 eye sections with p-FTAA and ToPro3 display aggregates in the photoreceptors indicated with arrows. See Figure S3A for eye morphology. (I and JII) Expression of nGFP (white) in the fly retina at day 10. (JI and JII) Arrowheads show layers of cell nuclei that overlap with nGFP (white), and arrows show the area for Ab aggregates (green) that do not overlap with GFP expressing cells. (K) Quantification of soluble and insoluble concentrations (ng/ml per fly) of Ab1-42 in fly head extracts at day 10 post eclosion, analyzed using Meso Scale Discovery (MSD) immunoassay. Note the 10-fold higher amounts of insoluble Ab1-42, showing very strong expression in neuronal strong (nsyb), glia (repo), and retina (GMR) compared with neuronal weak (elav-C155) and motor neuron (D42). Scale bars, 50 mm. The results are representative for three independent experiments. Bars represent means ± SEM deduced from triplicate samples in three independent experiments (n = 3, with 5 flies in each set), ns, not significant; *p % 0.05, **p % 0.01, ****p % 0.0001. See also Figure S4 for p-FTAA staining over time and Table S1 for data.

4 Cell Chemical Biology 25, 1–16, June 21, 2018

Please cite this article in press as: Jonson et al., Aggregated Ab1-42 Is Selectively Toxic for Neurons, Whereas Glial Cells Produce Mature Fibrils with Low Toxicity in Drosophila, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.03.006

Figure 2. Glia Expressing Flies Show Increasing Amounts of Ab and Aggregates over Time (A) Whole Drosophila brain staining of glial expressing Ab flies with the amyloid-specific LCO, p-FTAA (green), and cell nuclei stain ToPro3 (red) from the day of eclosion until day 25 post eclosion. Scale bars, 50 mm. The results are representative for three independent experiments. (B) Quantification of p-FTAA-positive aggregate foci (pixels) at days 0, 5, 10, and 20. Bars represent means ± SEM deduced from triplicate samples in independent experiments and for 19 optical sections each. Note the exceptional difference between day 0 and 5. (C) Quantification of soluble and insoluble concentrations (ng/mL per fly) of Ab1-42 in fly head extracts of glial expressing Ab flies over time. In the soluble graph the shaded area displays the concentration of the n-syb-Ab1-42 flies. In the insoluble graph, the upper shaded area displays the concentration range observed in the highly toxic n-syb-Ab1-42 flies, while the lower shaded area shows the amounts of soluble Ab. Bars represent means ± SEM deduced from triplicate samples in three independent experiments (n = 3, with 5 flies in each set), ns, not significant; ****p % 0.0001. See also Figure S4 for p-FTAA staining over time and Table S1 for data.

(C155-elav-Gal4/Ab1-42) driver (Figure 3B). This confirmed previously described results (Crowther et al., 2005; Jonson et al., 2015), as well as correlated with the levels of Ab1-42 described above. When expressing the Ab1-42 peptide in the motor neurons of the fly, a toxicity similar to that in the weaker CNS neuronal cell flies, was seen, with a median lifespan of 15 days (Figure 3C). Unexpectedly, in spite of the substantial amount of Ab1-42 aggregates present in glial cells (repo-Gal4/Ab1-42) we observed a minor reduction of lifespan compared with the control flies, and a much longer lifespan than any of the other

genotypes expressing Ab1-42, with a median lifespan of 25 days (Figure 3D). Although different Gal4 driver control flies were similar they were not identical. Comparing the median lifespan for controls and transgenes provides a relative measure of toxicity and normalizes variations from each respective Gal4 driver line backgrounds. A high value corresponds to a high toxicity. n-syb-Gal4 control flies have a median lifespan that is more than three times as high as the n-syb-Gal4/Ab1-42 flies. C155-elav-Gal4/+ and D42-Gal4/+ have double the median lifespan of their respective Ab1-42 transgenes, while glial control Cell Chemical Biology 25, 1–16, June 21, 2018 5

Please cite this article in press as: Jonson et al., Aggregated Ab1-42 Is Selectively Toxic for Neurons, Whereas Glial Cells Produce Mature Fibrils with Low Toxicity in Drosophila, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.03.006

Figure 3. Expression of Ab1-42 in Neurons Shortens the Lifespan and Impairs the Locomotor Activity (A–D) Lifespan trajectories of Ab1-42 expressing flies (closed circles) and control (open triangles) flies using the strong CNS neuronal cell expressing n-syb-Gal4 (A), the weak CNS neuronal cell expressing C155-elav-Gal4 (B), the motor neuron expressing D42-Gal4 (C), and the glial expressing repo-Gal4 (legend continued on next page)

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Please cite this article in press as: Jonson et al., Aggregated Ab1-42 Is Selectively Toxic for Neurons, Whereas Glial Cells Produce Mature Fibrils with Low Toxicity in Drosophila, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.03.006

flies only live for about 30% longer than flies expressing Ab1-42 in their glial cells (Figure 3I; Table S2). To confirm that the Gal4 driver used in this study sufficiently targets expression of proteins to glia, we addressed lifespan by misexpressing the cell death gene reaper (rpr). To this end we crossed UAS-rpr flies with repo-Gal4 and n-sybGal4, and assessed the number of eclosing flies during a 10-day period. In both cases, we observed complete larval/ pupal lethality, with 0 flies eclosing (n = 460 UAS-rpr/TM3, Sb flies eclosing for n-syb-Gal4, and 815 for repo-Gal4). These results confirm that both drivers target glial cells and neurons with sufficient expression levels to cause complete lethality prior to eclosion. Locomotor Behavior Mimics the Lifespan Assay To complement the lifespan assay with a more sensitive functional measure of toxicity upon the nervous system, we addressed locomotor activity using the iFly activity imaging system (Jahn et al., 2011). iFly records several behavioral parameters based on the negative geotaxis of Drosophila, e.g., velocity and angle-of-movement. Velocity describes the climbing speed from the bottom of the vial to the top, and angle-of-movement describes how much the flies deviate from a perpendicular path when moving to the top of the vial. As flies age or get sick they tend to move slower and in a more disordered pattern, resulting in decreased velocity and increased angle-of-movement. By analyzing the first 20 days post eclosion we found that all genotypes expressing Ab1-42 had a decreased velocity compared with controls, but in a distinctly varied fashion (Figures 3E–3H). In agreement with the observations from the lifespan assay, the strong CNS driver (n-syb-Gal4/Ab1-42) displayed the most impaired velocity, reaching the set cutoff value of 4 mm s 1 already at day 7 (Figure 3E). The weak CNS (C155-elav-Gal4/Ab1-42) and motor neuron drivers (D42-Gal4/ Ab1-42) showed similar activity pattern throughout the assay, reaching the cutoff value at days 13 and 15, respectively (Figures 3F and 3G). In line with the lifespan results, expressing Ab1-42 in glial cells (repo-Gal4/Ab1-42) only had a minor impact on the locomotor behavior compared with the other genotypes (Figure 3H). As for the lifespan assay we can obtain a quantitative measure of toxicity as an impact on nervous system function by comparing the integrated activity (i.e., the area under the curve of velocity) normalized for controls versus each transgene. These results largely mirror the lifespan results, with strong neuronal driver flies showing a high value >6 and glial expressing flies resulting in a value close to 1 indicating low toxicity (Figure 3J). At the endpoint of the assay, day 20 post eclosion, all control flies had a velocity of 10–15 mm s 1. The results from

the iFly angle-of-movement analyses were in agreement with the velocity results (Figure S5). Taken together, the survival and the two locomotor analyses showed that expression of Ab1-42 in the neurons of the fly is highly toxic, while expression in glial cells results in mild toxicity, despite the high levels of aggregated Ab1-42 (Figures 1 and 3). Hyperspectral Analysis of Tissue Sections Reveals Differences in Ab1-42 Fibril Morphology Next, we probed molecular differences in amyloid fibril morphology, by combining two hypersensitive LCOs, q-FTAA and h-FTAA (Nystro¨m et al., 2013; Psonka-Antonczyk et al., 2016) (Figure 4A). We analyzed co-stained (q-FTAA + h-FTAA) 10-mm tissue sections using hyperspectral microscopy. From these images we calculate the emission intensity ratios at 499 and 540 nm as markers of various amyloid fibril structures. This ratio varies depending on fibrillar structure. A high ratio indicates a mature bundled fibrillar structures, whereas a low ratio indicates immature and single-filament fibril structures (Nystro¨m et al., 2013; Psonka-Antonczyk et al., 2016). Ab1-42 expressed in fly neurons, by both strong and weak drivers, showed a distinct morphology, appearing mostly in ringtangle-like structures (Figure 4A, inset), with a predominance of the h-FTAA fluorescence spectrum (low 499/540 nm ratio) (Figure 4B). This type of aggregate morphology was also observed in motor neurons, together with some extended fibrous structures (Figure 4A). In glial cells the aggregate morphology was exclusively extended with a fibrous appearance (Figure 4A). For glial cells the 499/540 nm ratio was significantly higher (Figure 4B), suggesting a higher proportion of mature amyloid fibril structure. The extended fibrous morphology was also observed from Ab1-42 expressed in the eye, and was deposited in the region of photoreceptors of the eye disc (Figure 4A). Interestingly, these structures also had a higher 499/540 nm ratio (Figure 4B). The correlation between cytotoxicity and a dominance of h-FTAA fluorescence spectra (low 499/540 nm ratio) from the deposits strongly supports the notion of nascent immature aggregates to be more toxic than mature fibrils. These observations in Drosophila mimic those found in transgenic APP/PS1 mice that show large amounts of immature h-FTAA-positive amyloid and amyloid plaque, while APP23 transgenes, which are more maturated, has higher q-FTAA fluorescence (Nystro¨m et al., 2013). Interestingly, the lifespan of APP/PS1 mice is significantly shorter than for APP23 (Ye et al., 2017). The 499/540 nm ratios in Drosophila glia showed some variations, but was higher at all time points compared with neurons (Figure 4B). Drosophila Ab1-42 amyloid did not reach the same level of maturity as Ab plaque cores in tg-mice (gray

(D) driver. Survival plots and statistical analysis were calculated using the Kaplan-Meier method (Kaplan and Meier, 1958). n = 100–300 for the lifespan assay. (E–H) Locomotor activity for each Gal4/UAS combination, analyzed by velocity using the iFly system. (I) A measure of the toxicity level comparing the median lifespan of the respective control to their Ab1-42 counterpart. A high value represents high toxicity and 1 = no toxicity. (J) A measure of the toxicity level comparing the summed activity (area under curve) for control flies and Ab1-42 expressing flies. A high value represents high toxicity and 1 = no toxicity. Significance from a paired t test between control and Ab expressing flies, over the whole curve, is marked above each column. Statistical analysis of activity data was calculated for the time point prior to reaching the cutoff value for each transgene compared with its control. Errors are marked as SD. n = 30 for the activity analyzes. *p % 0.05, **p % 0.01, ****p % 0.0001. See Table S2 for data and Figure S5 for velocity data.

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Please cite this article in press as: Jonson et al., Aggregated Ab1-42 Is Selectively Toxic for Neurons, Whereas Glial Cells Produce Mature Fibrils with Low Toxicity in Drosophila, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.03.006

Figure 4. Spectral Imaging Reveals More Mature Ab1-42 Aggregates in Glial Expressing Flies and in the Retina Cryo-sections of Drosophila heads stained with combined LCO dyes q-FTAA and h-FTAA, and imaged using hyperspectral imaging in the range 460–700 nm (excitation at 436 nm). (A) The two neuronal drivers showed intraneuronal ring-tangle-like Ab aggregates with a spectral profile from h-FTAA fluorescence (yellow/red). The tangle-like inclusions were also observed in motor neurons. In glia cells and in the retina the aggregates were exclusively extended and fibrous with a higher proportion of q-FTAA fluorescence (cyan). Chemical structures of q-FTAA and h-FTAA. (B) Spectral ratios of q-FTAA/h-FTAA (499/540 nm) from 5 to 16 regions of interest (ROI) from 3 to 9 images of each genotype, as a marker of fibril maturation. The spectral ratios of amyloid in glia cells were significantly higher than in all neuronal drivers compared at day 10. Amyloid in glia cells and retina was significantly (legend continued on next page)

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Please cite this article in press as: Jonson et al., Aggregated Ab1-42 Is Selectively Toxic for Neurons, Whereas Glial Cells Produce Mature Fibrils with Low Toxicity in Drosophila, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.03.006

area in Figure 4B). The observed variations are likely due to competing processes of amyloid growth and degradation.

cells, contribute to formation of mature bundled Ab1-42 amyloid aggregates.

Mass Spectrometry Reveals Pure Ab1-42 from all Cell Types Next, we aimed to address if Ab processing was different in different cell types. Analyzing tg-APP mice we previously showed that amyloid plaque with dominating composition of Ab1-40 is associated with more q-FTAA fluorescence than amyloid from Ab1-42 (Nystro¨m et al., 2013). Hence, in light of the LCO spectral differences obtained from the different tissue drivers, we determined the molecular composition of the Ab amyloid deposits. We employed IP using combined 6E10 and 4G8 to capture total Ab, followed by MALDI-TOF mass spectrometry. In tissues from humans or tg-mice this method extracts a pool of different Ab variants (Portelius et al., 2015). For the Drosophila head samples only one peak, centered at m/z at 4,512.5, was observed, corresponding to intact unmodified Ab1-42 (Figure 4C). Controls without the UAS were devoid of this mass peak. A very minor amount, <1% corresponds to Ab1-42 with oxidized M35. Hereby we can conclude that the LCO spectral differences and the phenotypes we observe are purely attributed to conformational differences. In addition, our quantification of Ab1-42 load by antibodies (MSD assay) is accurately determining only Ab1-42 and no other Ab alloforms are missed. These results also verify that our designed signal sequence preceding Ab1-42 (Jonson et al., 2015) is precisely processed by all the driver cell types.

Ab1-42 Amyloids are Mostly Intracellular in Neurons and Extracellular in Glia Expressing Flies To further investigate the localization of Ab1-42 amyloids in the strong neuronal (n-syb-Gal4) and glia (repo-Gal4) driver lines, we imaged brain sections using p-FTAA co-stained with ToPro3 nuclear marker. Cell-proximal aggregates were found in neuronal driver flies, and extended fibrous aggregates in between cell layers were found in glial driver flies mainly distant from cell nuclei (Figure 6A). To corroborate that the aggregates were composed of Ab1-42 we co-stained brain sections with h-FTAA, Ab antibody (4G8), and ToPro3 (Figure 6B). Both imaging experiments show localization differences identical to that found by the spectral imaging analysis (Figure 4A). To study the localization in cells expressing both a marker and Ab1-42 we generated Drosophila co-expressing Ab1-42 and membrane-bound GFP (myrGFP) in neurons or glia cells and stained with h-FTAA and ToPro3. MyrGFP mainly localizes to the cell surface (Figure 6C). For neuronal expressing flies the Ab1-42 aggregates were mainly localized within the cells proximal to the cell surface as ring-tangles (Figure 6C, left). For glial cells, on the other hand, in relation to myrGFP expressed by the same cells, these aggregates protruded from the cell and hence appeared to be extracellular (Figure 6C, right). We verified that total expression of Ab1-42 was robust by quantifying Ab1-42 and GFP in the different crosses using the MSD immunoassay (Figure 6D). As expected, due to the number of cells, the amount of myr-GFP was higher in neuronal expressing flies than glial expressing flies but the relative amount of Ab1-42 remained the same, as in previous experiments.

Elevated Concentration Drives Ab1-42 Amyloid Maturation To understand the mechanism behind the formation of mature Ab1-42 amyloid aggregates in glial cells and eyes when compared with neurons, we allowed recombinant Ab1-42 to aggregate at different concentrations in vitro. The aggregates were imaged using transmission electron microscopy (TEM) and q-FTAA + h-FTAA fluorescence. Increasing concentrations of Ab1-42 from 1 to 10–100 mM correlated with higher amounts of laterally bundled fibrils forming at higher concentrations as shown by TEM (Figure 5A). Increased concentration during fibrillation was also reflected in the LCO 499/540 nm fluorescence ratio output (Figures 5B and 5C). Hence, elevated protein concentration is one mechanism for generating mature Ab1-42 amyloid structures with a high degree of bundling as illustrated in Figure 5D. The detailed characterization of all glia cells in the adult Drosophila CNS awaits further study. Studies to date point to some 5%–10% of all cells being glia and 90%–95% being neurons in both embryo and adults (Awasaki et al., 2008; Beckervordersandforth et al., 2008). As anticipated, quantification of total nGFP expression showed slightly higher nGFP in the strong neuronal driver than in glial cells (Figure S2F). Larger differences were found for the membrane-bound myrGFP expression (Figure 6D). Hence, for Ab1-42 expressing flies, fewer cells expressing as much or more Ab1-42, will, by local crowding in glial

Mutant Ab1-42 Shows Low Cytotoxicity Also in Glia Cells To assess if the mild glia-toxic phenotype could be altered by using variably aggregation prone Ab1-42 we turned to expression of three Ab1-42 mutant variants inserted by UAS-landing site technology, that we previously showed decreased the neurotoxicity in Drosophila compared with Ab1-42 wild-type (WT) (Jonson et al., 2015). First, in vitro kinetic studies of fibril formation were performed. Ab1-42 WT, Ab1-42 A42R, and Ab1-42 A42D showed rapid rates of fibril conversion, determined using either ThT or p-FTAA (Figure S6A). These two Ab1-42 mutants and WT converted with lag times between 2 and 3 hr with p-FTAA, and WT showed a longer lag time with ThT, at 4.5 hr compared with p-FTAA, as shown previously (Aslund et al., 2009). In contrast, Ab1-42 A42W did not convert over a 24-hr time frame (Figure S6A), while being Congo red positive, showing that it had aggregated (Figures S7A–S7H). Congo red staining, and imaging under crossed polarizers of aggregates formed after 24 hr, also revealed the same pattern where all variants except for Ab1-42 A42W formed Congo red birefringent fibrils (Figures S7A–S7H). Incubation for over 72 hr did result in fibril formation of Ab1-42 A42W as deduced

higher also at other time points (day 5 and 20). As a reference, the gray regions mark maximum ratios found in amyloid plaque cores in aged APP/PS1 tg-mice overexpressing Ab1-42/Ab1-40 at approximately 5:1 ratio (Nystro¨m et al., 2013). ****p % 0.0001. Scale bars, 20 mm. (C) MALDI-TOF mass spectrometry of IP-ed Ab (6E10 + 4G8) of all tissue types at day 10 reveals exclusively Ab1-42 in all genotypes (m/z = 4,512.5). Control was devoid of Ab.

Cell Chemical Biology 25, 1–16, June 21, 2018 9

Please cite this article in press as: Jonson et al., Aggregated Ab1-42 Is Selectively Toxic for Neurons, Whereas Glial Cells Produce Mature Fibrils with Low Toxicity in Drosophila, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.03.006

Figure 5. Concentration-Dependent Fibril Maturation of Ab1-42 In Vitro (A) Transmission electron microscopy of negatively stained in vitro recombinant AbM1-42 fibrils formed at different concentrations: 1, 10, and 100 mM. Higher concentration promotes fibril stacking, as indicated by the white star (*) for very dense areas. Red squares in the top micrographs show zoomed-in areas for the bottom panel. A higher proportion of bundled fibrils appear at higher concentration (insets). (B) Hyperspectral imaging in the range 460–700 nm (excitation at 436 nm) displayed concentration-dependent aggregate morphology of AbM1-42 using the LCOs q-FTAA + h-FTAA. More solitary fibrils are found at 1 mM than at higher concentrations where fibrils are clumped together. Scale bars, 20 mm. (C) Spectral ratios of q-FTAA/h-FTAA (499/540 nm) as a marker of fibril maturation. An increased concentration results in more mature aggregates, as seen by the higher 499/540 nm ratio. ****p % 0.0001. (D) Schematic picture of confined Ab1-42 fibrils at different concentrations and showing how crowding would promote fibril stacking; 1, 10, and 100 mM.

by ThT and p-FTAA (data not shown). Congo red birefringent fibrils of Ab1-42 A42W were obtained using seeding with preformed Ab1-42 WT fibrils (Figure S7J). This indicates that the A42W residue is not inhibiting formation of the fibril structure, but appears to be a kinetic trap for spontaneous fibril forma10 Cell Chemical Biology 25, 1–16, June 21, 2018

tion. To address the effects in glia cells, UAS flies of all three Ab1-42 mutants and WT were crossed with repo-Gal4, and were assayed for lifespan (Figure 7A). All variants Ab1-42 A42R, Ab1-42 A42D, and Ab1-42 A42W were even less toxic than Ab1-42 WT (Figure 7A), as in the case for strong neuronal

Please cite this article in press as: Jonson et al., Aggregated Ab1-42 Is Selectively Toxic for Neurons, Whereas Glial Cells Produce Mature Fibrils with Low Toxicity in Drosophila, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.03.006

Figure 6. Localization of Aggregates in Neurons versus Glia Cells (A and B) Sections (10 mm) of Drosophila brains stained with the amyloid-specific LCO, p-FTAA (green), and cell nuclei stain ToPro3 (red) at day 5 post eclosion reveal different localization of Ab1-42 aggregates (A). Neurons (n-syb-Gal4) show intraneuronal ring-tangle-like Ab1-42 aggregates, while glia cells (repo-Gal4) displayed extended aggregates located in-between the two layers of cell nuclei. Arrows highlight Ab1-42 aggregates. Scale bars, 10 mm (B). Sections (10 mm) of Drosophila brains co-stained with an Ab-specific antibody (4G8) (green) and the LCO h-FTAA (red) revealed co-localization within neurons (n-sybGal4) and for extracellular amyloid from glia cells (repo-Gal4). Cell nuclei stain with ToPro3 is shown in blue. Arrows highlight Ab1-42 aggregates. Scale bars, 10 mm. (C) Sections (5 mm) of Drosophila brains co-expressing Ab1-42 and membrane bound GFP (myrGFP) in either neurons (n-syb-Gal4) or glia cells (repo-Gal4). Ab1-42 aggregates appear intracellular in neurons while extracellular in glial cells. The white mask marks a single cell and arrows indicate Ab1-42 fibrils within (neuron) or protruding from the cell (glia). Staining of Ab-aggregates was accomplished using h-FTAA (red), and cell nuclei were stained with ToPro3 (blue). Scale bars, 5 mm. (D) Quantification of total GFP and insoluble concentrations (ng/ml per fly) of Ab1-42 in fly head extracts at day 5 post eclosion, analyzed using MSD immunoassay. The concentration of insoluble Ab1-42 is not affected by the co-expression of a second protein. Bars represent means ± SEM deduced from triplicate samples.

Cell Chemical Biology 25, 1–16, June 21, 2018 11

Please cite this article in press as: Jonson et al., Aggregated Ab1-42 Is Selectively Toxic for Neurons, Whereas Glial Cells Produce Mature Fibrils with Low Toxicity in Drosophila, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.03.006

Figure 7. Glia Cells Expressing Ab1-42 Mutants Show Similar Behavior as Ab1-42 WT (A) Lifespan trajectories of flies expressing Ab1-42 WT or mutant Ab1-42 (A42D, A42R, or A42W) in glia cells (repo-Gal4). Survival plots were generated using the Kaplan-Meier method (Kaplan and Meier, 1958) (n = 100). (B) Quantification of insoluble concentrations (ng/mL per fly) of Ab1-42 in fly head extracts at day 5 post eclosion, analyzed using MSD immunoassay. ****p % 0.0001. (C) Tissue sections of Drosophila (repo-Gal4) heads stained with combined LCO dyes q-FTAA + h-FTAA and imaged using hyperspectral imaging in the range 460–700 nm after excitation at 436 nm. Scale bars, 20 mm. (D) Spectral ratios of q-FTAA/h-FTAA (499/540 nm) from 2 to 8 ROI from 9 to 16 images, as a marker of fibril maturation. The spectral ratios of amyloid in glia cells, regardless of Ab1-42 WT or mutant Ab1-42 (A42D, A42R, or A42W), were significantly higher than in all neuronal drivers compared at day 5 (cf. Figure 4B). See Figure S6 for in vitro fibril formation kinetics and eye phenotype of mutants, Figure S7 for Congo red and Table S3 for data.

expression using n-syb-Gal4 (Jonson et al., 2015). Protein concentration determinations in these flies (using antibodies 6E10 and 4G8) were consistent with previous data in neurons (Jonson et al., 2015) showing lower amounts of Ab1-42 in the mutants compared with WT (Figure 7B). This is likely due to increased degradation of the mutants possibly being more geared toward carboxy peptidase cleavage than Ab1-42 WT. We also assayed the presence of fibrils and their morphology in fly brain histological sections by LCO fluorescence (q-FTAA + h-FTAA double staining). All flies showed the presence of extended amyloid aggregates reminiscent of Ab1-42 WT but to a smaller extent as expected from the lower concentration (Figure 7C). Interestingly, when we analyzed the LCO spectral ratio all flies displayed mature fibril spectra (Figure 7D). Notably, Ab1-42 A42W did not show any aggregates in neurons (Jonson et al., 2015). This strongly suggests that 12 Cell Chemical Biology 25, 1–16, June 21, 2018

glial cells are promoting Ab1-42 fibril maturation even at a low protein concentration (all mutants) and kinetically trapped Ab1-42 (A42W mutant), rendering low cytotoxicity. Immature Ab1-42 Aggregates can Generate a Rough Eye Phenotype Previous studies of Ab1-42 expression in fly eye have demonstrated an eye phenotype ranging from severe to mild depending on expression levels of Ab1-42 (Fernandez-Funez et al., 2016; Chouhan et al., 2016). Although our UAS-Ab1-42 transgene is different we were perplexed that our UAS transgene despite the large amount of Ab1-42 did not harm the eye. We hence also here expressed the three Ab1-42 mutant variants (Jonson et al., 2015). These mutants were crossed with GMR-Gal4 and were assayed for an eye phenotype (Figures S6B–S6E). Ab1-42 WT, Ab1-42 A42R, and Ab1-42 A42D

Please cite this article in press as: Jonson et al., Aggregated Ab1-42 Is Selectively Toxic for Neurons, Whereas Glial Cells Produce Mature Fibrils with Low Toxicity in Drosophila, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.03.006

showed normal eye structure, whereas Ab1-42 A42W showed a rough eye phenotype (Figures S6B–S6C). Protein concentration determinations in these flies (using antibodies 6E10 and 4G8) were consistent with previous data in neurons (Jonson et al., 2015) and in glia cells (Figure 7B), showing a lower amount of Ab1-42 in the mutants compared with WT (Figure S6F). We also assayed the presence of fibrils and their morphology in fly eye histological sections by LCO fluorescence (p-FTAA and q-FTAA + h-FTAA double staining) (Figures S6D–S6E). Ab1-42 WT showed immense amounts of fibrils, as described above, whereas the three mutant proteins showed minute amount of aggregates. Aggregates were found in the photoreceptor regions with mainly an extended fibrillar structure, but the fluorescence was too weak to allow for hyperspectral analysis. Taken together these results show that Ab1-42 A42W being fibril formation impaired in vitro, while not toxic in glial cells, forms immature aggregates in the eye which weakly stains with amyloid ligands. This property appears to correlate with the cytotoxicity and rough eye observed in the fly eye in contrast to Ab1-42 WT. DISCUSSION Ab aggregates are found in the CNS in aging healthy humans and in AD patients. In AD patients these aggregates appear to mainly damage neurons, causing severe brain atrophy in late stages of the disease. However, Ab aggregates are widespread and are found around neurons, microglia, astrocytes, smooth muscle cells in the vasculature, epithelial cells of the eye, and the choroid plexus. The pathogenesis of these accumulations is not clearly defined. The basis for selective vulnerability of certain tissues and cell types for Ab aggregates is a largely unknown territory. Drosophila disease models allows for modeling progressive disease and reduction in overall fitness of the fly depending on expressed Ab variant. We previously conducted a systematic study of 16 different Ab variants, finding that Ab1-42 and Ab3-42 were by far the most neurotoxic variants (Jonson et al., 2015). However, the significance of studying Ab1-42 expression in different cell types of the CNS has not been thoroughly addressed. In the present study, we hence aimed to investigate whether the Ab1-42 toxicity and aggregation pattern observed in CNS neurons of Drosophila can be observed in other cell types. The transgenic models utilized for this comparative study take advantage of the GAL4-UAS system (Brand and Perrimon, 1993). We used the well-studied driver C155-elavGal4, directing expression to both neural progenitors and mature neurons of the CNS (Robinow and White, 1988, 1991; Berger et al., 2007); the improved and stronger n-sybGal4 driver, directing expression to neurons (Jonson et al., 2015); the motor neuron expressing driver D42-Gal4 (Yeh et al., 1995); the widely used Drosophila glial driver repoGal4 that is expressed in most glial cells in the CNS (Sepp et al., 2001; M.R. Freeman et al., 2003); and the well-studied GMR-Gal4 driver, directing expression to the fly eye (M. Freeman, 1996). Our results show that all cell types expressing Ab1-42 are able to form amyloid aggregates. However, the toxicity is very different: Ab1-42 in neurons is very toxic, but is less toxic when expressed in glial cells.

The roles of glial cells in modulating neurodegenerative diseases in Drosophila models have begun to be studied in recent years. Glial cells in Drosophila perform similar functions as they do in vertebrates, e.g., providing various support functions to neurons during development, acting on injury to the nervous system, being responsible for the phagocytic clearance of cellular debris and maintaining neuronal survival (Xiong and Montell, 1995; MacDonald et al., 2006; M.R. Freeman and Doherty, 2006). To our knowledge, previous studies expressing Ab1-42 in Drosophila have not targeted glial cells. Our control experiments showed that expression of the cell death gene rpr rendered no transgenic progeny to eclose carrying both Gal4 and UAS-rpr in glia (repo-Gal4). This cross provided an identical outcome as for neuronal expression (n-syb-Gal4) of this cell death protein. Hence, healthy glia cells are essential for Drosophila. This shows that the mild phenotype we observe despite the heavy Ab1-42 aggregate load must be a result of low sensitivity of glial cells toward Ab1-42. Studies expressing other proteins causing human neurodegeneration in glial cells of Drosophila reported significant toxicity mediated by protein aggregation from mutant human GFAP associated with Alexander disease (Wang et al., 2011). Expressing human Tau in glial cells resulted in the formation of fibrillary tangles and a reduced lifespan as well as a non-cell-autonomous neuronal cell death (Colodner and Feany, 2010). This study also found that the expression of Tau in both glial cells and neurons caused a striking enhancement of toxicity compared with expression in the respective tissue by itself. Studies by Kretzschmar et al. (2005) and Tamura et al. (2009) found that when expressing the poly-Q proteins Htt and Atxn-1 in neurons and glia, only glia expression resulted in degeneration of the nervous system similarly to findings from (Shiraishi et al., 2014). These studies highlight the different responses that can be seen in neurons and glia. However, and most importantly, the observation of poly-Q and Tau stands in stark contrast to the outcome of our study, where we see a much stronger response in neurons than we do when expressing Ab1-42 in glial cells. The Ab1-42 aggregates found in neurons appear intracellular, filling up the soma. The shape strongly suggests that the Ab1-42 starts to aggregate in the secretory pathway and forms ring-tangles. In contrast, glial expression and eye expression results in extended fibrous aggregates away from the cell soma. Using both the GMR-Gal4 (eye) and the repo-Gal4 (glia) drivers, the expressing cells are spatially separated from the fibrous aggregates and appear mainly extracellular. Although we lack direct evidence it is likely that these aggregates can be detrimental to neighboring cells. The consistent reduction in life span and activity of the glia expressing flies indicates that non-cell-autonomous neurotoxicity may be operative here. One particularly interesting topic in the field of neurodegeneration pertains to the identification of specific structural species associated with toxicity. We have previously showed that immature oligomeric Ab1-42 aggregates, including protofibrils formed from the Arctic mutation E22G, are severely neurotoxic and showed a pronounced cytotoxic effect in the eye with the GMR-Gal4 driver (Figure S3B). However, neurotoxicity could be mitigated by small-molecule therapy that shifted the conformational states toward mature fibrils at the expense of oligomers (Caesar et al., 2012). In the current study, we Cell Chemical Biology 25, 1–16, June 21, 2018 13

Please cite this article in press as: Jonson et al., Aggregated Ab1-42 Is Selectively Toxic for Neurons, Whereas Glial Cells Produce Mature Fibrils with Low Toxicity in Drosophila, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.03.006

turned to our novel assay for amyloid fibril maturation morphotype staging, combining two LCOs q-FTAA and h-FTAA for fluorescence microspectroscopy. Glial Ab1-42 aggregates showed a spectral profile indicating significantly more mature fibrils than that observed in aggregates triggered by each of the three neuronal drivers. These data strongly suggest that the toxic species in Drosophila are immature nascent Ab1-42 fibrils formed intracellularly. To further assess the toxicity differences as a function of aggregate structure we showed that mutant Ab1-42 A42W with impaired spontaneous fibril formation capacity was more toxic to the fly eye in vivo than mature fibrillar Ab1-42 WT despite minor accumulation of mutant protein, while less toxic than Ab1-42 E22G. In glial cells on the other hand this intrinsic property of Ab1-42 A42W was overridden by some endogenous factor that promoted fibril maturation and hence low cytotoxicity. These results underline that amyloidogenic protein sequence, local concentration, cell-specific protein homeostasis factors, and aggregate conformation are all influencing Ab1-42 cytotoxicity. Our study has, for the first time, clearly associated various structural maturation stages of Ab1-42 fibrils with cytotoxicity, and reveals rather selective vulnerability of neurons toward nascent Ab1-42 aggregates. It has been proposed that glial cells can replicate and seed Ab amyloid fibrils in mammalian brain, independent of neuronal expression (Veeraraghavalu et al., 2014). It is perplexing how neurodegenerative diseases can stay dormant for decades prior to showing symptoms. Our study suggests that glia cells, being largely resistant toward toxicity caused by Ab1-42 fibril accumulations, could function as carriers of pathogenic seeds for spreading of aggregates in the brain while staying intact. Future studies aimed at understanding the cellular and molecular basis for the vulnerability of neurons and the resilience of glia may shed some light on AD progression. SIGNIFICANCE In comparison with tg-APP mice as models for Alzheimer’s disease, which rarely show neurodegeneration, the situation is dramatically different in tg-Drosophila. Here neuronal expression of Ab1-42 leads to severe neurodegeneration. However, while being severely toxic to Drosophila neurons for three different drivers, we unexpectedly discovered strong and progressive Ab1-42 amyloidosis from glial cells while showing a mild phenotype. We showed that glia cells are indeed vital for Drosophila development, and others have demonstrated that certain aggregation-prone proteins (e.g., poly-Qs and Tau) are indeed toxic to glia. Our findings are hence surprising and imply that glia cells, being specifically insensitive for Ab1-42 aggregates, could facilitate amyloid spreading without harming the transporter cell. Furthermore from a molecular mechanistic perspective, we also show that variations in Ab1-42 fibril morphology in vivo is linked to cell expression and toxicity. Immature amyloid aggregates within neurons are more cytotoxic than mature mainly extracellular fibrils originating from glial cells. These findings from a simple model organism could have implications for how to consider treatment of Ab1-42 amyloidosis in Alzheimer’s disease. 14 Cell Chemical Biology 25, 1–16, June 21, 2018

STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d d

d

KEY RESOURCES TABLE CONTACT FOR REAGENT AND RESOURCE SHARING EXPERIMENTAL MODEL AND SUBJECT DETAILS B Drosophila Stocks METHOD DETAILS B Histological Analysis B Quantification of the Ab-Peptide Levels B Lifespan Assay B Activity Assay B Mass Spectrometry B Scanning Electron Microscopy B Recombinant Ab1-42 Mutant Fibril Formation QUANTIFICATION AND STATISTICAL ANALYSIS

SUPPLEMENTAL INFORMATION Supplemental Information includes seven figures and three tables and can be found with this article online at https://doi.org/10.1016/j.chembiol.2018. 03.006. ACKNOWLEDGMENTS This work was supported by The Swedish Brain Foundation, The Swedish Research Council, The Go¨ran Gustafsson Foundation, and Astrid and Georg Olsson to P.H., by King Gustaf V and Queen Victoria’s Freemasons’ Foundation (to S.T.), and The Swedish Alzheimer foundation (to P.H. and S.N.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. AUTHOR CONTRIBUTIONS M.J., A. Sandberg., M.C., W.M., A. Starkenberg, and P.H. performed the experiments. M.J., S.N., S.T., and P.H. conceived and designed the experiments. M.J., S.N., A. Sandberg, W.M., J.H., K.P.R.N., S.T., and P.H. analyzed the data. M.J. and P.H. wrote the paper with assistance from the other authors. DECLARATION OF INTERESTS The authors declare no competing interests. Received: July 6, 2017 Revised: January 12, 2018 Accepted: March 12, 2018 Published: April 12, 2018 REFERENCES Allan, D.W., St Pierre, S.E., Miguel-Aliaga, I., and Thor, S. (2003). Specification of neuropeptide cell identity by the integration of retrograde BMP signaling and a combinatorial transcription factor code. Cell 113, 73–86. Aslund, A., Sigurdson, C.J., Klingstedt, T., Grathwohl, S., Bolmont, T., Dickstein, D.L., Glimsdal, E., Prokop, S., Lindgren, M., Konradsson, P., et al. (2009). Novel pentameric thiophene derivatives for in vitro and in vivo optical imaging of a plethora of protein aggregates in cerebral amyloidoses. ACS Chem. Biol. 4, 673–684. Awasaki, T., Lai, S.L., Ito, K., and Lee, T. (2008). Organization and postembryonic development of glial cells in the adult central brain of Drosophila. J. Neurosci. 28, 13742–13753.

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16 Cell Chemical Biology 25, 1–16, June 21, 2018

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STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Mouse monoclonal Anti-Ab42 (clone12F4)

Nordic Biosite

Cat # 805502

SULFO-TAG-conjugated Anti-Ab (clone 4G8)

Meso Scale Discovery

Cat # D20RQ-3

Streptavidin SULFO-TAG labeled

Meso Scale Discovery

Cat # R32AD-5

Anti-Ab (clone 6E10)

Signet Laboratories

Cat # SIG 39320

Anti-Ab (clone 4G8)

Signet Laboratories

Cat # SIG 39220

Sheep anti-mouse IgG Dynabeads M-280

Thermo Fisher

Cat # 11201D

Anti-GFP antibody (biotin) ab6658

Abcam

Cat # ab6658

Anti-GFP antibody [9F9.F9] ab1218

Abcam

Cat # ab1218

h-FTAA

Laboratory of Peter Nilsson, Linko¨ping University, (Klingstedt et al., 2011)

N/A

p-FTAA

Laboratory of Peter Nilsson, Linko¨ping University, (Klingstedt et al., 2011)

N/A

q-FTAA

Laboratory of Peter Nilsson, Linko¨ping University, (Klingstedt et al., 2011)

N/A

TO-PRO-3

Life Technologies

Cat # T3605

Thioflavin T

Sigma Aldrich

Cat # T35165

Congo Red

Eastman Kodak Co

Cat # C770

Dako Fluorescent mounting medium

DAKO

Cat # S3023

Tris Wash Buffer

Meso Scale Discovery

Cat # R61TX-2

MSD Blocker A

Meso Scale Discovery

Cat # R93BA-1

Antibodies

Chemicals, Peptides, and Recombinant Proteins

Ab1-42 peptide calibrator

Meso Scale Discovery

Cat # C01LB-2

Read Buffer

Meso Scale Discovery

Cat # R92TC-2

Peptide Standard Solution (PepCalMix1)

Bruker Daltonics

Cat # 8222570

MSD 96-Well MULTI-ARRAY plate

Meso Scale Discovery

Cat # L15XA-3

Bio-Rad DC Protein Assay Kit II

BioRad

Cat # 500-0112

Bloomington Drosophila Stock Center

BDSC: 458; FlyBase: FBst0000458

Critical Commercial Assays

Experimental Models: Organisms/Strains D. melanogaster: C155-elav-Gal4

D. melanogaster: n-syb-Gal4

Laboratory of Stefan Thor, Linko¨ping University

N/A

D. melanogaster: D42-Gal4

Bloomington Drosophila Stock Center

BDSC: 8816; FlyBase: FBst0008816

D. melanogaster: repo-Gal4

Bloomington Drosophila Stock Center

BDSC: 7415; FlyBase: FBst0007415

D. melanogaster: GMR-Gal4

Bloomington Drosophila Stock Center

N/A

D. melanogaster: UAS-Ab1-42

Laboratory of Stefan Thor, Linko¨ping University

N/A

D. melanogaster: UAS-Ab1-42 A42D

Laboratory of Stefan Thor, Linko¨ping University

N/A

D. melanogaster: UAS-Ab1-42 A42R

Laboratory of Stefan Thor, Linko¨ping University

N/A

D. melanogaster: UAS-Ab1-42 A42W

Laboratory of Stefan Thor, Linko¨ping University

N/A

D. melanogaster: UAS-Ab1-42 Arctic

Obtained from D. Crowther (Crowther et al., 2005)

N/A

D. melanogaster: UAS-myr-GFP

Bloomington Drosophila Stock Center

BDSC: 32198 Flybase: FBst0032198 (Continued on next page)

Cell Chemical Biology 25, 1–16.e1–e5, June 21, 2018 e1

Please cite this article in press as: Jonson et al., Aggregated Ab1-42 Is Selectively Toxic for Neurons, Whereas Glial Cells Produce Mature Fibrils with Low Toxicity in Drosophila, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.03.006

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

D. melanogaster: UAS-nls-myc-eGFP

Obtained from D. van Meyel

FlyBase: FBtp0020527

D. melanogaster: UAS-rpr

Bloomington Drosophila Stock Center

BDSC: 5824; FlyBase: FBst0005824

D. melanogaster: Oregon-R

Bloomington Drosophila Stock Center

BDSC: 6361-6366; FlyBase: FBst0006361- FBst0006366

(Walsh et al., 2009)

N/A

Recombinant DNA DNA encoding Ab M1-42 in PetSac plasmid Software and Algorithms iFly

(Jahn et al., 2011)

N/A

Prism 6.0a

GraphPad

http://www.graphpad.com/ scientific-software/prism/

CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Per Hammarstro¨m ([email protected] or [email protected]). EXPERIMENTAL MODEL AND SUBJECT DETAILS Drosophila Stocks To direct tissue-specific protein expression of transgenic UAS Drosophila melanogaster, the Gal4/UAS system was used (Brand and Perrimon, 1993). The C155-elav-Gal4 and n-syb-Gal4 driver flies (Bloomington Stock Center #458 and our double expressor (Jonson et al., 2015), respectively) were used to direct protein expression to the nerve cells of the fly CNS, with n-syb-Gal4 being the stronger driver. D42-Gal4 directs protein expression to the fly motor neurons, repo-Gal4 directs protein expression to glial cells of the fly, and the GMR-Gal4 directs protein expression to the retina of the fly. The UAS transgenes used in this study were UAS-Ab1–42 (Jonson et al., 2015), UAS-Ab1–42 A42D (Jonson et al., 2015), UAS-Ab1–42 A42R (Jonson et al., 2015), UAS-Ab1–42 A42W (Jonson et al., 2015), UAS-nls-myc-eGFP (Allan et al., 2003), UAS-myr-GFP (Bloomington Stock Center #32198), UAS-rpr (White et al., 1994), and Oregon-R (as control). Drosophila stocks were maintained at 60% humidity at 25 C until eclosion, and 29 C post eclosion, under a 12:12 h light:dark cycle. Crosses were reared in 50 ml vials containing standard Drosophila food (corn meal, molasses, yeast and agar), while newly eclosed flies were kept in 50 ml Drosophila vials containing 7 ml agar (20 g agar, 20 g sugar dissolved in 1 l of distilled water) and yeast paste (dry bakery yeast dissolved in distilled water) in a setup of twenty female flies per vial. METHOD DETAILS Histological Analysis Whole Brain Analysis To analyze and compare whole Drosophila brains of each genotype for the presence of amyloid deposits we applied histological staining using the amyloid specific luminescent conjugated oligothiophene (LCO), p-FTAA (Aslund et al., 2009). Female flies were reared at 29 C and analyzed as previously described (Jonson et al., 2015) at day 5, 10, 15, 20 and 25 post eclosion depending on the lifespan of each genotype. A silicon rubber well was made on a poly-lysine adhesive slide (Fisher Scientific) and decapitated fly heads were dissected in PBS, using two pairs of fine forceps, and placed on the slides prior to staining. Brains were fixed in 96% ethanol for 10 minutes and rehydrated to distilled water in 2-min steps, in 70%, 50% and 0% ethanol at room temperature. Slides were washed in PBS (5 min, RT) prior to addition of 3mM p-FTAA diluted in PBS (30 min, RT). After incubation with p-FTAA, slides were washed in PBS (3x5 min, RT). To visualize cell nuclei, brains were stained with 5 mM ToPro3 (TO-PRO-3; Life technologies) diluted in PBS (15 min, RT). Slides were once again washed in PBS (5 min, RT) and rinsed in distilled water (2x5 min, RT). The silicon rubber well was removed and two cover slips were aligned on each side of the dissected Drosophila brains to produce a spacer. Nail polish was used to attach the coverslips to the slide. Slides were allowed to dry at room temperature, mounted in DAKO mounting medium (DAKO #S3023; DAKO, Glostrup, Denmark), and stored at +4 C overnight. All incubations were carried out in a dark chamber to minimize the risk of bleaching. Prior to imaging, the slides were sealed with nail polish. A minimum of three brains for each genotype and time-point were analyzed. A Zeiss LSM 780 confocal microscope was used for fluorescent images; confocal stacks were merged and processed using LSM software or Adobe Photoshop. All images were processed using the same procedure. An estimation of the aggregate load in each genotype was done by visual inspection in the fluorescence microscope together with a quantitative analysis of p-FTAA positive pixels with an intensity >50 in each optical section of the whole brain using the Zen e2 Cell Chemical Biology 25, 1–16.e1–e5, June 21, 2018

Please cite this article in press as: Jonson et al., Aggregated Ab1-42 Is Selectively Toxic for Neurons, Whereas Glial Cells Produce Mature Fibrils with Low Toxicity in Drosophila, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.03.006

software (Zeiss). Analysis of nGFP (UAS-nls-myc-eGFP) expression in the brain was performed by dissections as outlined above, and the eGFP signal was collected on a Zeiss LSM 780 system. The excitation laser 458 nm was used for p-FTAA and eGFP and 640 nm was used for ToPro3. Antibody and LCO Double Staining of Fly Brain Sections Flies corresponding to the n-syb-Gal4 and repo-Gal4 crosses with UAS-Ab1–42 were aged to day 5 post eclosion at 29 C, before they were decapitated and mounted in Tissue-Tek O.C.T. using Cryomould-specimen molds and stored in -80 C until further use. Fly heads were cut into 10mm thick sections in a cryostat and stored at -20 C. Brain sections were fixed in 70% ethanol at 4 C for 3 minutes and rehydrated in distilled water 2x2-min at room temperature. Slides were blocked and permeabilized in PBS with 5% goat serum and 0.1% triton X-100 for 30 min at RT. Anti-Ab antibody, 4G8 (Cat # SIG 39220, Signet Laboratories), was diluted 1:500 in blocking buffer and added to the sections for 1.5 h at RT. Slides were washed in PBS-T 3x10 min, RT. To visualize antibody binding to Ab sections were incubated with goat anti-mouse antibody labeled with Alexa 488 (Thermo Fisher Scientific) diluted 1:400 in blocking buffer for 1h at RT. Sections were washed 3x10 min in PBS prior to addition of 3mM h-FTAA diluted in PBS (30 min, RT). After incubation with h-FTAA, slides were washed in PBS (3x5 min, RT). To visualize cell nuclei, sections were stained with 5mM ToPro3 (TO-PRO-3; Life technologies) diluted in PBS (15 min, RT). Slides were once again washed in PBS (5 min, RT) and rinsed in distilled water (2x5 min, RT). Slides were allowed to dry at room temperature, mounted in DAKO mounting medium (DAKO #S3023; DAKO, Glostrup, Denmark), and stored at +4 C overnight. All incubations were carried out in a dark chamber to minimize the risk of bleaching. Prior to imaging, the slides were sealed with nail polish. A minimum of three sections of five brains for each genotype was analyzed. A Zeiss LSM 780 confocal microscope was used for fluorescent images and images were processed using LSM software or Adobe Photoshop. All images were processed using the same procedure. An estimation of the antibody staining and aggregate load in each genotype was done by visual inspection in the fluorescence microscope. The excitation laser 488 nm was used for anti-Ab antibody, 535 nm for h-FTAA and 640 nm was used for ToPro3. Co-expression of myr-GFP and Ab1-42 in Brain Sections Flies corresponding to the UAS-Ab1–42-myr-GFP-n-syb-Gal4 and UAS-Ab1–42-myr-GFP-repo-Gal4 crosses were aged to day 5 post eclosion at 29 C, before they were decapitated and mounted in Tissue-Tek O.C.T. using Cryomould-specimen molds and stored in -80 C until further use. Fly heads were cut into 5mm thick sections in a cryostat and stored at -20 C. The staining procedure was performed as described for whole brains above; with the exception that h-FTAA was used instead of p-FTAA. After the final wash in distilled water slides were allowed to dry at room temperature, mounted in DAKO mounting medium (DAKO #S3023; DAKO, Glostrup, Denmark), and stored at +4 C overnight. All incubations were carried out in a dark chamber to minimize the risk of bleaching. Prior to imaging, the slides were sealed with nail polish. A minimum of three sections of five brains for each genotype was analyzed. A Zeiss LSM 780 confocal microscope was used for fluorescent images and images were processed using LSM software or Adobe Photoshop. All images were processed using the same procedure. An estimation of the GFP signal and aggregate load in each genotype was done by visual inspection in the fluorescence microscope. The excitation laser 488 nm was used for GFP, 535 nm for h-FTAA and 640 nm was used for ToPro3. Spectral Analysis of Brain Sections Flies corresponding to the repo-Gal4 crosses were aged to day 5, 10 or 20 post eclosion at 29 C; the n-syb-Gal4 crosses to day 5 or 10; the D42-Gal4, C155-elav-Gal4 crosses and all the control flies to day 10 post eclosion and the GMR-GAL4 cross to day 20, before they were decapitated and mounted in Tissue-Tek O.C.T. using Cryomould-specimen molds and stored in -80 C until further use. Fly heads were cut into 10mm thick sections in a cryostat and stored at -20 C. Staining of the sections was achieved using the same procedure as for the whole brain analysis described above (Jonson et al., 2015), except for the specific LCO used. No staining of the nucleus was done. To enable spectral analysis of amyloid maturation and to distinguish between different forms of aggregates between genotypes the LCOs q-FTAA, h-FTAA, and a combination of q-FTAA+h-FTAA were used (Klingstedt et al., 2011). The concentrations of q-FTAA and h-FTAA were diluted 1:5,000 (stock conc. 1 mg/ml) and for the combination of q-FTAA+h-FTAA we used 2 parts q-FTAA and 1 part h-FTAA 1:5,000 (stock conc. 1 mg/ml). For GMR staining a stock solution diluted 1:500 was used. At least 5 fly heads of each genotype and time-point were analyzed in a Leica DM6000B epifluorescence microscope equipped with long band-pass filters and a SpectraView system (Applied Spectral Imaging). SpectraView 4.0 and a Spectra-Cube (inferometrical optical head SD 300) module with a cooled CCD-camera, through a D436/10x;E475LPv2;455DCLP emission bandpass filter set. The data was processed with the SpectraView 3.0 EXPO software. Spectra were collected in an interval of 460–750 nm with the highest spectral resolution settings (‘gas-line settings’). Micrographs were analyzed for LCO-positive aggregates. Spectral images were analyzed using GraphPad Prism 6.0a software (GraphPad Software Inc., San Diego, CA, USA) using spectral data from 5-16 regions of interest (ROIs) from 3-9 images of each genotype. Quantification of the Ab-Peptide Levels To quantify the amount of expressed Ab1-42 or GFP in each genotype the Meso Scale Discovery (MSD) immunoassay was applied. Sample Preparation Flies corresponding to n-syb-Gal4 and D42-Gal4 crosses were aged in 29 C to 5 or 10 days post eclosion due to their shorter lifespan, while flies corresponding to the repo-Gal4, GMR-Gal4 and C155-elav-Gal4 crosses were aged to 5, 10 and 20 days post eclosion, also at 29 C. Five fly heads of each genotype were homogenized in 50 ml of extraction buffer (50 mM Hepes pH 7.3, 5 mM EDTA, Protease inhibitor (CompleteTM, Roche Diagnostics)). The homogenate was incubated at room temperature for 10 min, sonicated for 4 minutes in a water bath and then centrifuged at 12 000 g for 5 minutes into a ‘‘soluble’’ and ‘‘insoluble’’ fraction. 20 ml of the Cell Chemical Biology 25, 1–16.e1–e5, June 21, 2018 e3

Please cite this article in press as: Jonson et al., Aggregated Ab1-42 Is Selectively Toxic for Neurons, Whereas Glial Cells Produce Mature Fibrils with Low Toxicity in Drosophila, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.03.006

supernatant (‘‘soluble fraction’’) was mixed with 180 ml hepes dilution buffer (25 mM Hepes pH 7.3, 1 mM EDTA, 0,1% MSD Blocker A (R93BA-4, Meso Scale Discovery, MD, USA)). The pellet (‘‘insoluble fraction’’) was homogenized in 50 ml of extraction buffer containing guanidinium HCl (5 M GnHCl, 50 mM Hepes pH 7.3, 5 mM EDTA, Protease inhibitor (CompleteTM, Roche Diagnostics)). 20 ml of the supernatant of the ‘‘insoluble fraction’’ was mixed with 980 ml hepes dilution buffer prior to analysis. All samples were stored at -80 C for at least 16 hours before use. Flies corresponding to the UAS-myr-GFP and UAS-nls-myc-eGFP crosses were aged to day 5 post eclosion and treated as above; however, both the soluble and insoluble fractions were diluted 50 times. Immunoassay The quantification of Ab1-42 in the soluble and insoluble fractions was performed using a standard binding MSD 96-Well MULTI-ARRAY plate (L15XA-3, Meso Scale Discovery, MD, USA). The plate was coated with 25 ml, 10 mg/ml of a monoclonal anti-Ab42 antibody, 12F4, (Cat # 805502, Nordic Biosite, Sweden) for 1h, RT, with gentle agitation. The plate used for analysis of Ab1-42 mutants were coated with 25 ml, 20 mg/ml of a monoclonal anti-Ab antibody, 6E10 (Cat # SIG 39320, Signet Laboratories). The plate was washed three times with 150 ml 1x Tris Wash Buffer (R61TX-2, Meso Scale Discovery, MD, USA) and blocked with 150 ml/well 1% MSD Blocker A solution (R93BA-1, Meso Scale Discovery, MD, USA) for 30 min, RT, with gentle agitation. Triplicate 25 ml aliquots of Ab1–42 peptide calibrator (C01LB-2, Meso Scale Discovery, MD, USA) (ranging from 0–10 000 pg ml-1) or fly sample were mixed with an equal amount of MSD Blocker A (2% MSD Blocker A, 0.2% Tween 20 and protease inhibitor) and added to the plate for 1h, RT, gentle agitation. The plate was washed and detection was achieved by addition of 25ml 1x SULFO-TAG—conjugated 4G8 detection antibody (D20RQ-3, Meso Scale Discovery, MD, USA) for 1h, RT, gentle agitation. The plate was once again washed and 150 ml 2X read buffer (R92TC-2, Meso Scale Discovery, MD, USA) was added to the plate. Measurements were taken in a SECTOR Imager 2400 instrument (Meso Scale Discovery, MD, USA). To adjust for variation in the protein extraction step a quantitation of the total amount of protein from each sample of fly homogenate was performed by usage of the Bio-Rad DC Protein Assay Kit II (500–0112; BioRad, CA, USA). All samples were assayed in triplicates at three independent assay occasions. To analyze flies corresponding to the GFP-crosses 10 mg/ml mouse anti-GFP antibody (ab1218, Abcam) was used as capture antibody; plates were washed in 150ml PBS-T and blocked in 150ml 1% BSA in PBS-T prior to sample or GFP calibrator (ranging from 0-1000 ng ml-1) addition mixed with an equal amount of MSD Blocker A (2% MSD Blocker A, 0.2% Tween 20 and protease inhibitor). The plate was washed with PBS-T and a secondary anti-GFP antibody (ab6658-biotin, Abcam) diluted 1:20000 was added to the plate for 1h, RT, gentle agitation. The plate was washed and detection was achieved by addition of 25ml SULFO-TAG—conjugated Streptavidin detection antibody (R32AD-5, Meso Scale Discovery, MD, USA), 1:1000 for 1h, RT, gentle agitation. The plate was once again washed and 150 ml 2X read buffer (R92TC-2, Meso Scale Discovery, MD, USA) was added to the plate. Measurements were taken in a SECTOR Imager 2400 instrument (Meso Scale Discovery, MD, USA). Lifespan Assay The lifespan assay is a standard assay for monitoring the effect of genotype or environmental conditions on Drosophila lifespan. For each assay, 100 newly eclosed female flies, corresponding to the various Gal4-crossings, were kept at 29 C at 60% humidity, in 50 ml vials (20 flies per vial) and every 2-3 days the flies were transferred to fresh vials and the number of surviving flies was recorded throughout the lifetime of all flies. The assay was run 1-3 times and a total of 100-300 flies of each genotype were assayed. The data was pooled and analyzed together. The survival times described in the study are given as median survival. GraphPad Prism 6.0a software (GrapPad Software Inc., San Diego, CA, USA) was used to generate Kaplan-Meier survival curves (Kaplan and Meier, 1958) and to run the log-rank statistical analysis. The definition of significance was p-values of less than 0.05 (*), 0.01 (**), 0.001 (***) and 0.0001 (****). Activity Assay The locomotor behavior of individual flies was recorded using a locomotor assay, iFly (Jahn et al., 2011). For each genotype, a total of 50 newly eclosed flies were collected and divided into five vials, 10 flies in each vial. Three of these vials were assayed every second/third day until the flies were to immobile to be able to record. A movie of 90 s was recorded for each vial, and every 30 s the flies were tapped to the bottom of the vial to ensure the same starting point in each movie, yielding nine movies of 30 s for each genotype and time point. The three assay vials were replenished to compensate for dead flies throughout the assay. The movies collected were processed using the iFly software and the parameters velocity and angle of movement were calculated. The data was analyzed using GraphPad Prism 6.0a software (GraphPad Software Inc., San Diego, CA, USA). Mass Spectrometry The IP was performed using a KingFisher magnetic particle processor (Thermo Scientific, Waltman, MA, USA) as described earlier with some modifications (Portelius et al., 2015). Shortly, 2,4 mg/sample of Ab specific antibodies 6E10 and 4G8 (epitope Ab 4–9 and epitope Ab 18–22 respectively, Signet Laboratories, Inc., Dedham, MA, USA), were separately cross-linked to 15ml/sample/antibody sheep anti-mouse IgG Dynabeads M-280 (Dynal ) according to the manufacturer’s description. The washed beads with bound antibody were combined and used for immunoprecipitation of the neutralized diluted insoluble (GuHCl) fraction to which Tween-20 (final concentration 0.025%; Bio-Rad Laboratories Inc.) was added and incubated. The beads/insoluble fraction was transferred to a KingFisher magnetic particle processor (polypropylene tubes; Thermo Scientific) for automated washing and elution of the Ab peptides. The supernatant was collected and dried through lyophilization (Portelius et al., 2015). e4 Cell Chemical Biology 25, 1–16.e1–e5, June 21, 2018

Please cite this article in press as: Jonson et al., Aggregated Ab1-42 Is Selectively Toxic for Neurons, Whereas Glial Cells Produce Mature Fibrils with Low Toxicity in Drosophila, Cell Chemical Biology (2018), https://doi.org/10.1016/j.chembiol.2018.03.006

For MALDI analysis, samples were reconstituted in 5 mL 50% ACN/0.1% TFA. MALDI analysis was performed using the seed layer method, including pre-spotting of a matrix seed layer (CHCA, 20 mg/mL, 90% ACN, 10% methanol (MeOH) and 0.005% TFA) and subsequent co-application of 1 mL sample mixed with 1 mL matrix (CHCA, 15 mg/mL, 50% ACN/0.1% TFA). MALDI MS was performed using an UltrafleXtreme MALDI ToF/ToF instrument (Bruker Daltonics, Bremen, Germany) operating in reflector positive mode. Data were acquired over a mass range of 200-5000 Da, with 5000 shots at 1 kHz repetition rate and the laser focus set to medium. The spectra were calibrated externally with peptide standard solution (PepCalMix1, Bruker Daltonics) spotted adjacent to the sample spots on the target. MS/MS data were acquired in ToF/ToF mode using post source decay and precursor ion selection followed by re-acceleration of isolated fragment ions (LIFT). Post LIFT mother ion suppression was applied. A number of 600 shots were collected in PARENT mode followed by laser energy increase with 35% for PSD and collection of 1000 shots for acquiring fragment ion spectra (FRAGMENT mode). Scanning Electron Microscopy Flies corresponding to the GMR-GAL4 crosses were aged to day 20 in 29 C before being collected and euthanized with ether. Flies were mounted onto 12-mm aluminum specimen stubs with an adhesive tape and air-dried for 24 hours before sputter coated with platinum. The eye morphology was analyzed using a scanning electron microscope (JEOL JSM-6320F), and images were recorded at 2503 magnification. Recombinant Ab1-42 Mutant Fibril Formation Recombinant AbM1-42 and mutants were purified essentially according to (Walsh et al., 2009) using gel filtration as the final step. Details of a refined protocol will be described elsewhere (A.S. and S.N., unpublished data). Fibril formation kinetics: Monomeric AbM1-42 and mutants were diluted with PBS to a final concentration of 10 mM containing either 2 mM ThT or 0.3 mM p-FTAA. Fibril formation assays in triplicates were run using the plate reader Infinite M1000 Pro (Tecan) with an excitation wavelength of 440 nm and a recorded emission spectra between 470 nm and 650 nm. The fibrillation kinetic measurements were performed in 37 C with 30 seconds shaking and measuring points every 30 minutes for 24 hours. All samples were also measured after 72 h of incubation. The lagtime for fibril formation was calculated using fitting to a sigmoidal function according to (Gade Malmos et al., 2017) and was analyzed using Graphpad Prism. Fits were performed using individual kinetic traces and the standard deviation was calculated from the triplicates. Unstained fibrils from wt and mutants were: Mixed with 5 mM of Congo Red. Stained fibrils were left to self-sediment over-night. 4 ml from the pelleted samples were transferred to superfrost glass slides (Thermo Fisher, Walldorf, Germany) and were allowed to dry. Dried fibrils were covered with fluorescence mounting medium (Dako, Glosrup, Denmark) and were analyzed using a Nikon light microscope equipped with polarizers for both incoming light and in front of the camera. Images were taken with open and crossed polarizers using the 20x objective. Ab1-42 fibrils formed after 96 h of incubation, formed at 1 mM, 10 mM and 100 mM were stained with 0.3 mM h-FTAA and 0.6 mM q-FTAA and transferred to superfrost glass slides (Thermo Fisher, Walldorf, Germany) and were allowed to dry. Dried fibrils were covered with fluorescence mounting medium (Dako, Glosrup, Denmark) and were analyzed using a Leica 6000B microscope equipped with a SpectralCube (ASI, Migdal Haemek, Israel). Spectral images were taken using the 40x objective. Fibrils were also put on carbon coated copper grids (Carbon-B, Ted Pella Inc.). Salt was removed with deionized water and grids were negatively stained with 2% uranyl acetate for Transmission electron microscopy (TEM) analysis using a Jeol 1230 microscope operating at 100 kV, equipped with a Gatan digital camera. QUANTIFICATION AND STATISTICAL ANALYSIS Quantitative data are presented as means ± SEM for MSD immunoassay and pixelcount analysis of p-FTAA positive aggregate foci and as means ± SD for kinetic experiments and lifespan toxicity levels. Experiments were independently repeated three times. Statistical analyses were conducted using GraphPad Prism 6.0a software (GraphPad Software Inc., San Diego, CA, USA). Statistical comparisons between groups were analyzed for significance by one-way analysis of variance (ANOVA) with Tukey’s post hoc test for pixelcount analysis, lifespan toxicity levels, spectral analysis and analysis of lag-times from the kinetic experiments. For MSD immunoassay and activity analysis statistical comparisons between groups were analyzed for significance by unpaired t-test. The survival times described in the study are given as median survival. GraphPad Prism 6.0a software (GrapPad Software Inc., San Diego, CA, USA) was used to generate Kaplan-Meier survival curves (Kaplan and Meier, 1958) and to run the log-rank statistical analysis. The definition of significance was *p%0.05, **p%0.01, ***p%0.001, ****p%0.0001.

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