# Astrocyte-to-neuron H2O2 signalling supports long-term memory formation in Drosophila and is impaired in an Alzheimer's disease model

Yasmine Rabah, Jean-Paul Berwick, Nisrine Sagar, Laure Pasquer, Pierre-Yves Plaçais, Thomas Preat

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## To cite this version:

Yasmine Rabah, Jean-Paul Berwick, Nisrine Sagar, Laure Pasquer, Pierre-Yves Plaçais, et al. Astrocyte-to-neuron H2O2 signalling supports long-term memory formation in Drosophila and is impaired in an Alzheimer's disease model. Nature Metabolism, 2025, 7 (2), pp.321-335. 10.1038/s42255-024-01189-3. hal-05389388

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Submitted on 30 Nov 2025

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# Astrocyte-to-neuron H₂O₂ signalling supports long-term memory formation in *Drosophila* and is impaired in an Alzheimer's disease model

Received: 15 June 2023

Accepted: 21 November 2024

Published online: 24 January 2025

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Yasmine Rabah<fnref n="1" />, Jean-Paul Berwick Ⓓ<fnref n="1" />,², Nisrine Sagar Ⓓ<fnref n="1" />,², Laure Pasquer<fnref n="1" />, Pierre-Yves Plaçais Ⓓ<fnref n="1" /> & Thomas Preat Ⓓ<fnref n="1" />✉

Astrocytes help protect neurons from potential damage caused by reactive oxygen species (ROS). While ROS can also exert beneficial effects, it remains unknown how neuronal ROS signalling is activated during memory formation, and whether astrocytes play a role in this process. Here we discover an astrocyte-to-neuron H₂O₂ signalling cascade in *Drosophila* that is essential for long-term memory formation. Stimulation of astrocytes by acetylcholine induces an increase in intracellular calcium ions, which triggers the generation of extracellular superoxide (O₂⁻) by astrocytic NADPH oxidase. Astrocyte-secreted superoxide dismutase 3 (Sod3) converts O₂⁻ to hydrogen peroxide (H₂O₂), which is imported into neurons of the olfactory memory centre, the mushroom body, as revealed by in vivo H₂O₂ imaging. Notably, Sod3 activity requires copper ions, which are supplied by neuronal amyloid precursor protein. We also find that human amyloid-β peptide, implicated in Alzheimer's disease, inhibits the nAChRα7 astrocytic cholinergic receptor and impairs memory formation by preventing H₂O₂ synthesis. These findings may have important implications for understanding the aetiology of Alzheimer's disease.

Reactive oxygen species (ROS) such as superoxide (O₂⁻) and hydrogen peroxide (H₂O₂) are distinctive among metabolites because of their physiological ambivalence<fnref n="1" />. On the one hand, ROS are toxic byproducts that can irreversibly oxidize macromolecules, and powerful scavenging enzymes are active to protect cells from ROS damage. The production of ROS is closely linked to the activity of the mitochondria in the production of ATP, and neurons are particularly susceptible to ROS damage due to their high energy requirements and high content of unsaturated fatty acids that are prone to peroxidation². On the other hand, ROS play a positive role by activating signalling pathways involved in physiological functions such as cell survival, growth or response to stress<fnref n="1" />,². ROS signalling occurs mainly through reversible oxidation of redox-sensitive

cysteines, which requires a sharply regulated spatiotemporal increase in ROS concentration³. In vitro studies of long-term potentiation in mice have shown that neuronal ROS signalling plays a positive role in synaptic plasticity⁴,⁵. Recent in vivo studies in *Drosophila* have shown that ROS signalling is required during development for activity-dependent plasticity in motoneurons⁶,⁷. While pioneering studies have reported that mutant mice with altered ROS metabolism display learning or memory defects²,⁸, it remains unknown how ROS signalling is recruited in vivo for memory formation. Astrocytes are known to regulate neuronal redox homeostasis and cognition in mice⁹,¹⁰. In particular, it was shown that under physiological conditions mitochondrial ROS in astrocytes stimulate nuclear factor erythroid-2-related factor 2 (NRF2) transcription

<footnote n="1">¹Energy & Memory, Brain Plasticity Unit, CNRS, ESPCI Paris, PSL Research University, Paris, France. ²These authors contributed equally: Jean-Paul Berwick, Nisrine Sagar. ✉e-mail: thomas.preat@espci.fr</footnote>
<figure><img src="image_5.png" /><figcaption>b G6PD KD in astrocytes</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>c Ca²⁺ response to nicotine in astrocytes</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>d nAChRα7 KD in astrocytes</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>e Sod3 KD in astrocytes</figcaption></figure>

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**Fig. 1 | Astrocyte-derived H₂O₂ is required for LTM formation.** *a, Nox* knockdown (KD) in adult astrocytes impaired memory after spaced conditioning (*n* = 11 (first group), *n* = 12 (second group), *n* = 15 (third group); F<sub>2.35</sub> = 17.43, *P* = 0.000006). *b, G6PD* KD in adult astrocytes impaired memory after spaced conditioning (*n* = 13 (first and second groups), *n* = 18 (third group); F<sub>2.41</sub> = 12.98, *P* = 0.00004). *c, The fluorescent calcium sensor GCaMP6f* was expressed in adult astrocytes. Nicotine stimulation (50 µM) elicited calcium increase in astrocytes, which was significantly decreased in *nAChRα7* KD (*n* = 13 (first group), *n* = 11 (second group), *t*<sub>22</sub> = 3.15, *P* = 0.005). *d, nAChRα7* KD in adult astrocytes impaired memory after spaced conditioning (*n* = 10 (first and second groups), *n* = 14 (third group); F<sub>2.31</sub> = 7.81, *P* = 0.002). *e, Sod3* KD in adult astrocytes

impaired memory after spaced conditioning (*n* = 12 (first and second groups), *n* = 15 (third group); F<sub>2.36</sub> = 16.02, *P* = 0.00001). *f, Scheme of the molecular* actors involved in astrocytes for LTM. Nox, activated by calcium entry through nAChRα7, produces superoxide O₂⁻ from O₂⁻ using the NADPH co-factor derived from the PPP. Extracellular O₂⁻ is converted to H₂O₂ by extracellular Sod3 secreted from the astrocytes. Ru5P, ribulose-5-phosphate. Data are presented as mean ± s.e.m. Genotype sample sizes are listed in the legend in order of bar appearance. Significance level of a two-sided *t*-test or the least significant Tukey pairwise comparisons following one-way analysis of variance (ANOVA). \* *P* < 0.05; \*\* *P* < 0.01; \*\*\* *P* < 0.001; \*\*\*\* *P* < 0.0001.

factor expression, which promotes glutathione biosynthesis by astrocytes and antioxidant protection of neighbouring neurons¹⁰; however, it is not known whether neurons can trigger beneficial ROS production by astrocytes, and thereby if astrocytes are involved in the initiation of neuronal ROS signalling for synaptic plasticity.

Building a mechanistic view of ROS signalling during memory formation is essential not only from a brain physiology perspective, but it could also improve our understanding of the onset of Alzheimer's disease (AD), a neurodegenerative disease characterized in particular by memory loss and defects in redox homeostasis¹¹,¹². At the neuropathological level, AD is characterized by the progressive formation in the brain of amyloid plaques that correspond to the extracellular accumulation of amyloid β (Aβ) peptide, generated by cleavage of the transmembrane amyloid precursor protein (APP), and by the formation of intracellular neurofibrillary tangles consisting of hyperphosphorylated tau protein¹³. Early synaptic dysfunction has been associated with AD¹²,¹⁴, which correlates with cognitive decline¹⁵; however, the cascade of events leading to this fatal condition remains unclear.

Here, we have addressed these key brain physiology and pathology issues in *Drosophila* by combining brain H₂O₂ imaging using recently developed roGFP2-Tsa2ΔCₘ ultrasensitive sensors¹⁶ with functional characterization of ROS-related pathways during memory formation. We report that long-term memory (LTM) formation requires the establishment of a local H₂O₂ gradient in neurons of the olfactory memory centre, which follows the activation of ROS-producing enzymes in astrocytes. This signalling cascade is impaired by AD-related Aβ42. Our results provide a new framework to understand neuroglia interactions during memory formation in normal conditions and in AD.

## Results

### Astrocytes generate extracellular ROS for long-term memory

AD has been extensively linked to defects in redox homeostasis¹¹,¹². The objective of this study was to exploit the strengths of *Drosophila* genetics and in vivo imaging capabilities to further elucidate the relationships between Aβ, ROS and cognition; however, rather than artificially increasing ROS production and assessing its impact on memory formation, our approach was to investigate the potential beneficial effects of ROS on memory formation, with the understanding that this physiological pathway might be affected by Aβ in an AD-related model. Given the important role of neuron–glia metabolic coupling in *Drosophila* memory formation¹⁷⁻¹⁹, we sought to address these questions by investigating the potential role of astrocytes in neuronal ROS signalling.

We observed that inhibiting NADPH oxidase (Nox) expression in adult astrocytes impairs olfactory LTM in *Drosophila* (Fig. 1a). The only function of the transmembrane enzyme Nox is to produce extracellular O₂⁻ from O₂⁻ and intracellular NADPH³, and therefore our observation was potentially important from a ROS signalling perspective. For this experiment, we used a well-established paradigm of associative aversive olfactory conditioning²⁰,²¹ named spaced conditioning (Methods), along with inducible and spatially controlled expression of a Nox-targeting RNAi. A single Nox enzyme exists in *Drosophila*, which shows strong sequence similarity with the human Nox5 (ref. 22). Similarly to Nox5 (ref. 3), *Drosophila* Nox is activable by Ca²⁺ thanks to intracellular EF-hands domains that bind Ca²⁺ (ref. 23).

LTM formation requiring Nox activity in astrocytes depends on de novo protein synthesis²¹ in neurons of the olfactory memory centre, the mushroom body (MB)²⁴,²⁵. Conversely, anaesthesia-resistant
<figure><img src="image_11.png" /><figcaption>**Fig. 2 | An H₂O₂ gradient forms in MB α lobes upon LTM formation.**
a, Illustration of MB structure. Axons of MB neurons follow a branching pattern that forms vertical lobes (α and α') and medial lobes (β, β' and γ). b, Images of the roGFP2-Tsa2ΔC<sub>R</sub> H₂O₂ sensor expressed in MB neurons. 3D reconstructions of image stacks (128-μm-thick stack, 1 μm Z-step) of the H₂O₂ sensor in MB neurons at the two excitation wavelengths (988 and 780 nm) (top). Cb, cell bodies; vl, vertical lobes (α and α'); ml, medial lobes (β, β' and γ). Horizontal plane with regions of interest, cell bodies and α vertical lobes delimited with dashed lines (bottom). Scale bar, 30 μm. c, The H₂O₂ level in α lobes normalized</figcaption></figure>

to the cell bodies value is increased upon spaced training, in comparison to the non-associative unpaired control (n = 13 (first group), n = 14 (second group); $t_{25} = 2.82$, *P* = 0.009). d, The H₂O₂ level in β or γ lobes normalized to the H₂O₂ level in cell bodies after spaced training was similar to the unpaired control (β, n = 9 (first group), n = 11 (second group), $t_{18} = 0.94$, *P* = 0.36; γ, n = 10 (first group), n = 9 (second group), $t_{17} = 1.30$, *P* = 0.21). Data are presented as mean ± s.e.m. Genotype sample sizes are listed in the legend in order of bar appearance. Significance level of a two-sided *t*-test. **P* < 0.01; NS, not significant, *P* > 0.05.

memory (ARM), which forms after massed conditioning (Methods) and does not depend on de novo protein synthesis²¹, was not affected by Nox knockdown in adult astrocytes (Extended Data Fig. 1a). In addition, Nox knockdown did not affect perception of the stimuli used for conditioning (Supplementary Table 1), and LTM was normal when Nox RNAi expression was not induced (Extended Data Fig. 1a). To further ensure the specificity of Nox implication in LTM, we measured the immediate memory capacity after spaced conditioning of flies expressing Nox RNAi in adult astrocytes. Our results demonstrated that Nox knockdown did not affect immediate memory, confirming that superoxide production by astrocytes is required specifically for LTM, as measured at 24 h after spaced conditioning (Extended Data Fig. 1b). Last, we confirmed the involvement of Nox in adult astrocytes for LTM with a second nonoverlapping Nox RNAi (Extended Data Fig. 1c and Supplementary Table 1). These controls were performed in a systematic manner throughout the course of this study. With regard to text simplification, knockdown flies exhibiting the aforementioned phenotypes (impaired LTM as measured at 24 h after conditioning, assessed with two RNAis; normal ARM as measured at 24 h after massed conditioning; normal immediate memory after spaced conditioning; normal stimuli perception; normal LTM in the absence of RNAi induction) will be simply reported as displaying a specific LTM defect.

The Nox substrate NADPH is mainly produced by the pentose phosphate pathway (PPP)²⁶. To study this pathway we inhibited in adult astrocytes the expression of G6PD, the enzyme catalysing the first step of the PPP²⁶, and we observed a specific LTM defect (Fig. 1b, Extended Data Fig. 1d,e and Supplementary Table 1). Given that the PPP generates many intermediates involved in various biosynthetic processes,

including fatty acids and nucleotides²⁶, an additional experiment was conducted to ensure that inhibiting the PPP in astrocytes did not affect different forms of olfactory memory in an unspecific manner. Thus, in addition to the assessment of ARM following massed conditioning and immediate memory following spaced conditioning, the middle-term memory capacity following a single conditioning cycle was evaluated after G6PD knockdown in adult astrocytes. G6PD knockdown flies displayed normal middle-term memory (Extended Data Fig. 1d). The essential role of astrocytic PPP in LTM formation was confirmed by inhibiting the expression in astrocytes of Pgd and Pgls²⁶, two other enzymes in this pathway (Extended Data Fig. 1e-g and Supplementary Table 1). Altogether, these results support the notion that astrocytic Nox generates O₂⁻ required for LTM formation.

As stated above, similar to the human Nox5, *Drosophila* Nox is activated by Ca²⁺. Acetylcholine (ACh) is the major excitatory neurotransmitter of *Drosophila* brain neurons, and in particular MB neurons are cholinergic²⁷. Among the ionotropic cholinergic receptors, the channel formed of nAChRα7 subunits is the most permeable to Ca²⁺ (ref. 28) and is expressed in human astrocytes²⁹. To examine whether nAChRα7 cholinergic stimulation induces a Ca²⁺ flux in *Drosophila* astrocytes, we expressed the GCaMP6f Ca²⁺ fluorescent reporter in astrocytes and stimulated the brains of live flies with nicotine, an nAChRα7 agonist. Nicotine stimulation induced an increased astrocytic Ca²⁺ concentration in control flies (Fig. 1c). This effect was dampened when *nAChRα7* expression was inhibited in astrocytes (Fig. 1c). We next addressed the role of astrocytic nAChRα7 in memory and showed that *nAChRα7* knockdown in adult astrocytes specifically impaired LTM (Fig. 1d, Extended Data Figs. 1b and 2a,b and Supplementary Table 1). These
results suggest that during spaced conditioning ACh release can activate Ca²⁺ signalling in astrocytes through nAChRα7 and is required for LTM. Consistent with our findings, astrocytic nAChRα7 was implicated in the persistence of fear memory in mice³⁰.

While O₂⁻ is a highly reactive and therefore toxic ROS with a short half-life, H₂O₂, which is less reactive and has a longer half-life, is more suitable for ROS signalling¹,³¹. Astrocytic Nox activity at the plasma membrane produces extracellular O₂⁻, whose conversion into H₂O₂ is catalysed by the only secreted superoxide dismutase (Sod), Sod3 (ref. 32). In the *Drosophila* brain, *Sod3* is more strongly expressed in astrocytes than in neurons, as shown by single-cell transcriptomics data³³ (Extended Data Fig. 2c). *Sod3* knockdown in adult astrocytes specifically impaired LTM (Fig. 1e and Extended Data Figs. 1b and 2d,e and Supplementary Table 1). Altogether, these results suggest that a nAChRα7–Nox–Sod3 signalling cascade takes place upon LTM formation, which is triggered by ACh stimulation of astrocytes, resulting in extracellular H₂O₂ formation (Fig. 1f).

Like the tripartite synapse in the mammalian brain³⁴, astrocytic processes overlap with synapses in the *Drosophila* brain³⁵. We therefore wondered whether astrocytes might be a source of beneficial ROS imported by neurons during LTM formation. In particular, is H₂O₂ signalling involved during LTM formation in the MB?

## An H₂O₂ gradient in MB α lobe supports LTM formation

The MB is a bilateral structure consisting of about 2,000 cholinergic neurons²⁷ in each brain hemisphere. Axons of the MB neurons form bundles and branch out, giving shape to anatomical structures called lobes that synapse with the MB output neurons. MB neurons are classified into three different subtypes: the α/β neurons, whose axons branch to form an α vertical projection and a medial β projection (Fig. 2a); the α'/β' neurons; and the γ neurons, which form a single medial γ lobe. The axonal projection of the α lobe is specifically involved in LTM formation²⁰,³⁶. We aimed to image H₂O₂ in MB neurons upon LTM formation. Recently, the ability to observe physiological H₂O₂ levels has become possible with the development of an ultrasensitive H₂O₂ fluorescent sensor, roGFP2-Tsa2ΔC<sub>R</sub>¹⁶, which we imported and characterized in *Drosophila* (Fig. 2b and Extended Data Fig. 3a).

To observe change in H₂O₂ dynamics, flies expressing the H₂O₂ sensor in the MB were imaged between 0.5 h and 2 h after spaced conditioning, a time window when early LTM-encoding events occur¹⁷,³⁷. Control flies were submitted to non-associative unpaired protocol that does not allow memory formation. Notably, in flies trained for LTM, we observed an increased H₂O₂ level in the MB α lobe (Fig. 2c), and no increase in the MB β and γ medial lobes (Fig. 2d). An additional point must be considered to interpret the H₂O₂ imaging data. As we previously demonstrated, *Drosophila* LTM formation involves activation of the PPP in MB neurons, fuelled by Glut1-mediated glucose import¹⁷. As PPP activity generates the reducing agent NADPH, its activation in the MB is expected to result in a global decrease in ROS concentration (compare with scheme in Extended Data Fig. 3b). Indeed, detailed examination of imaging data revealed a decreased H₂O₂ level in MB cell bodies in flies trained for LTM (Extended Data Fig. 3c). Notably, when glucose import was impaired in adult MB, the decreased H₂O₂ response in MB neuron cell bodies after spaced conditioning was abolished, but the H₂O₂ increase was observed in the α lobe (Extended Data Fig. 3d). Altogether, these data reveal the existence in LTM-forming flies of an H₂O₂ gradient within the MB neurons, generated by H₂O₂ influx within the α vertical lobe. These results are in agreement with the fact that the α lobe is specifically involved in LTM formation²⁰,³⁶.

To test whether astrocyte activity modulates H₂O₂ signalling in the MB LTM centre, we imaged H₂O₂ in MB neurons while interfering with the H₂O₂-producing cascade in astrocytes. The increased level of H₂O₂ in the MB α lobe was lost by inhibiting the expression of either Nox, nAChRα7, G6PD or Sod3 in adult astrocytes (Fig. 3a). These results indicate that the H₂O₂ gradient formed in MB neurons after

spaced conditioning originates from astrocytic Nox and Sod3 activity in response to ACh stimulation. To substantiate this hypothesis, we intended to deplete the extracellular H₂O₂ generated by astrocytic Sod3 during LTM formation by expressing a secreted form of the human catalase in astrocytes⁷. Catalase is responsible for catalysing the breakdown of H₂O₂ into H₂O and O₂. Notably, the typical increase in H₂O₂ observed in the α lobe following spaced conditioning was absent in the presence of the secreted catalase (Fig. 3b). In accordance with the imaging data, the LTM of flies that expressed the secreted catalase was found to be specifically impaired (Fig. 3c, Extended Data Fig. 3e and Supplementary Data Table 2). These findings substantiate the hypothesis that the H₂O₂ gradient observed in the MB during LTM formation is due to extracellular SOD3 activity.

## A redox relay cascade is required in MB for LTM formation

How does H₂O₂ produced extracellularly by astrocytic enzymes enter MB neurons upon LTM formation? Although H₂O₂ can diffuse across the plasma membrane, H₂O₂ import is strongly facilitated by channels of the aquaporin protein family³⁸. We tested the potential involvement of the main *Drosophila* aquaporin, AQP, in LTM (Fig. 4a). AQP knockdown in adult MB α/β neurons specifically impaired LTM (Fig. 4b, Extended Data Fig. 4a,b and Supplementary Table 3). The LTM defect following AQP knockdown was linked to a loss of the H₂O₂ increase in the α lobe after spaced conditioning (Fig. 4c). Three additional aquaporin orthologues have been identified in *Drosophila*: drip, bib and prip⁷. The inhibition of their expression in adult α/β MB neurons did not affect LTM (Extended Data Fig. 4c–h), indicating that AQP plays a specific role in H₂O₂ import following SOD3 activity.

Our results support the idea that H₂O₂ signalling in α/β neurons occurs for LTM formation. H₂O₂ signalling is primarily characterized by the reversible oxidation of target cysteines on signalling proteins, which subsequently modifies their activity. This is achieved either by direct action of H₂O₂ following an increase in local concentration, or through a cascade of redox-sensitive relay enzymes, starting with the Cys-based peroxidase peroxiredoxin (Prx), an H₂O₂ scavenger (Fig. 4a)³⁹,⁴⁰. We identified Prx2 as the major Prx involved in LTM formation (Fig. 4d, Extended Data Fig. 5a–c and Supplementary Table 4). Oxidized Prx are reduced by thioredoxin (Trx)⁴⁰, and we showed that *Drosophila* Trx2 is specifically involved in LTM (Fig. 4e, Extended Data Fig. 5b,d,e and Supplementary Table 4). Last, Trx is reduced by a Trx reductase (Trxr) that oxidizes NADPH into NADP⁺ in the process. We identified Trxr1 as a major actor of LTM formation in α/β neurons (Fig. 4f, Extended Data Figs. 5b and 6a,b and Supplementary Table 4). Prx H₂O₂ scavenging enzymes are involved in the general maintenance of redox homeostasis³⁹,⁴⁰. Consequently, the inhibition of Prx2 expression may potentially impact α/β MB neurons in an unspecific manner. To eliminate this possibility, we conducted an additional experiment, measuring middle-term memory after a single conditioning cycle of Prx2, Trx2 or Trxr1 knockdowns. This control is of particular significance given that middle-term memory is encoded by α/β neurons, as is LTM⁴¹. Knocking down Prx2, Trx2 or Trxr1 in α/β neurons did not affect middle-term memory, demonstrating the specificity of the effect on LTM (Extended Data Figs. 5a,d and 6a).

Altogether, these results reveal a cascade of redox-sensitive enzymes required for LTM formation in α/β MB neurons. This cascade could be involved in the regulation of gene expression required for LTM formation¹,²¹.

## Appl delivers extracellular copper for SOD3 activity

We have shown that H₂O₂ is generated extracellularly by Sod3, which requires extracellular Cu²⁺ for its catalytic activity³². Because of its potential toxicity, free Cu²⁺ is maintained at a very low concentration in cells and tissues by copper-binding proteins⁴². This raises the question of how sufficient Cu²⁺ levels can be accumulated at the MB synapses to sustain during LTM formation the activity of Sod3 secreted
<figure><img src="image_12.png" /><figcaption>**Fig. 3 | Astrocytes are a source of H₂O₂ imported into MB α lobes.**</figcaption></figure>

a, Measurements of H₂O₂ levels in MB neurons after spaced training in the context of astrocytic KDs. The increase in the α lobes/cell bodies H₂O₂ level elicited by spaced training was impaired in KD of Nox (n = 10, t₁₈ = 0.11, P = 0.91; wild-type control: n = 10, t₁₈ = 3.47, P = 0.003), G6PD (n = 10 (first and second groups), t₁₈ = 0.49, P = 0.63; wild-type control: n = 10 (first group), n = 11 (second group), t₁₉ = 3.01, P = 0.007), nAChRa7 (n = 9, t₁₆ = 0.63, P = 0.54; wild-type control: n = 10, t₁₈ = 3.57, P = 0.002) and Sod3 (n = 13 (first group), n = 9 (second group), t₂₀ = 0.33, P = 0.75; wild-type control: n = 12, t₂₂ = 3.93, P = 0.0007). Data

are presented as mean ± s.e.m. **b**, Expression of human-secreted-catalase in astrocytes abolished the increase in the α lobes/cell bodies H₂O₂ level elicited by spaced training (n = 9, t₁₆ = 1.596, P = 0.13; wild-type control: n = 9, t₁₆ = 4.136, P = 0.0008). **c**, Expression of human-secreted-catalase in astrocytes impaired 24 h memory after spaced conditioning (n = 12, F(2,33) = 4.603, P = 0.017). Data are presented as mean ± s.e.m. Genotype sample sizes are listed in the legend in order of bar appearance. Significance level of two-sided *t*-test or the least significant Tukey pairwise comparison following one-way ANOVA. \* *P* < 0.05; \*\* *P* < 0.01; \*\*\* *P* < 0.001; NS, not significant, *P* > 0.05.

by astrocytes. We hypothesized that Appl, the single fly orthologue of APP transmembrane protein, might provide Cu²⁺ for astrocyte-derived Sod3 activity based on the following observations: (1) the APP extracellular domain has several Cu²⁺ binding sites43,44 whose function remains poorly understood⁴⁵, and *Drosophila* Appl carries in its conserved E2 ectodomain the four histidines that are characteristic of the M1 high-affinity Cu²⁺ binding site⁴⁶; (2) APP enables intracellular copper to be transported out of neurons, as APP overexpression decreases Cu²⁺ content in neurons in vitro, whereas APP loss of function leads to copper accumulation⁴⁷; (3) Appl knockdown in MB neurons induces an LTM defect⁴⁸; and (4) Appl exhibits neuronal expression and is enriched in α/β MB neurons⁴⁹, which are involved in LTM²⁰,³⁶. We confirmed this strong α/β neuron expression with an HA-tagged Appl line generated by CRISPR (Fig. 5a and Extended Data Fig. 7a). Furthermore, we showed that knocking down Appl specifically in α/β neurons with RNAi expression induces an LTM defect (Fig. 5b, Extended Data Fig. 7b,c and Supplementary Table 5).

If Appl does provide copper to Sod3, the H₂O₂ gradient that forms after spaced conditioning should require Appl. Indeed, we observed

that the H₂O₂ gradient after spaced conditioning was abolished when Appl expression was inhibited in adult MB (Fig. 5c).

To further support the idea that Appl and Sod3 are involved in the same pathway, we searched for a synergistic interaction between Appl and Sod3 using mild inhibition of their expression. The objective was to demonstrate that the concomitant reduction in Appl and Sod3 expression could result in a more pronounced LTM impairment than that observed in single knockdowns. The flexibility of the GAL4/GAL80ts system, which is induced by elevated temperature, was used to express Appl RNAi for a brief period of 1 day, instead of the usual 3 days of RNAi expression. At the behavioural level, the induction of Appl or Sod3 RNAi for a single day in MB neurons and astrocytes did not result in an LTM impairment (Fig. 5d). Next, we asked whether the brief simultaneous induction of Appl and Sod3 RNAi would affect LTM, thus revealing a synergistic interaction. Strikingly, LTM was specifically abolished when both Appl and Sod3 expressions were inhibited in MB neurons and astrocytes for one day (Fig. 5d, Extended Data Fig. 7d and Supplementary Table 5). Of note, the simultaneous mild inhibition of Appl and Sod3 expressions in MB alone or astrocytes alone did not
<figure><img src="image_13.png" /><figcaption>b AQP KD in MB neurons</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>c</figcaption></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /><figcaption>d Prx2 KD in MB neurons</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>e Trx2 KD in MB neurons</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>f Trxr1 KD in MB neurons</figcaption></figure>

<figure><img src="image_19.png" /></figure>

**Fig. 4 | A redox-sensitive cascade is recruited upon H₂O₂ entry into MB neurons for LTM formation.** a, Putative redox relay cascade downstream of H₂O₂ import in MB neurons. b, AQP KD in adult α/β MB neurons impaired memory after spaced conditioning (n = 12 (first group), n = 14 (second and third groups), F₂,₃₇ = 31.24, *P* = 0.00000001). c, The increase in H₂O₂ level in α lobes/cell bodies elicited by spaced training (n = 10, t₁₈ = 2.42, *P* = 0.0261) was impaired in AQP KD in adult MB neurons (n = 9 (first group), n = 10 (second group), t₁₇ = 0.59, *P* = 0.56). d, Prx2 KD in adult α/β MB neurons impaired memory after spaced conditioning (n = 9 (first group), n = 14 (second and third groups), F₂,₃₄ = 12.08, *P* = 0.0001).

e, Prx2 KD in adult α/β MB neurons impaired memory after spaced conditioning (n = 9 (first group), n = 14 (second and third groups), F₂,₃₇ = 14.43, *P* = 0.0000007). f, Trxr1 KD in adult α/β MB neurons impaired memory after spaced conditioning (n = 10 (first group), n = 12 (second and third groups), F₂,₂₇ = 20.30, *P* = 0.0012). Data are presented as mean ± s.e.m. Genotype sample sizes are listed in the legend in order of bar appearance. Significance level of a two-sided *t*-test or the least significant Tukey pairwise comparison following one-way ANOVA. \* *P* < 0.05; \*\* \*P < 0.0001; NS, not significant, *P* > 0.05.

impact LTM (Extended Data Fig. 7e,f). Altogether, we can conclude that the inability to form LTM in double-knockdown flies results from the simultaneous mild decrease of *Appl* expression in the MB, and of *Sod3* expression in astrocytes. To further substantiate this finding, we conducted imaging of H₂O₂ dynamics in the double knockdown following brief RNAi expression. Formation of the H₂O₂ gradient after spaced conditioning was abolished in flies expressing both *Appl* and *Sod3* RNAi, whereas it remained unaltered in flies expressing a single RNAi, aligning with the behavioural data (Fig. 5e). These results outline the existence of a strong functional interaction between Appl and *Sod3*, and are in agreement with the hypothesis that Appl supplies copper for *Sod3* activity during LTM formation.

To clarify the involvement of the Appl–Sod3 interaction in the early phase of LTM formation rather than in memory recall, which occurs 24 h after conditioning in our experiments, we examined the performance of flies in which the brief *Appl* and *Sod3* RNAi expression was initiated immediately following spaced training, as opposed to before training. The memory performance of flies expressing both *Appl* and *Sod3* RNAi for 1 day after training was not affected (Extended Data Fig. 7g), whereas control flies expressing both RNAi for 1 day before training exhibited an LTM defect (Extended Data Fig. 7g). These results lend support to the hypothesis that Appl–Sod3 interaction is involved in an early step of LTM formation, rather than in LTM recall.

APP physiological function has been linked to many pathways⁵⁰,⁵¹. To further demonstrate that the LTM defect of the *Appl* knockdown is indeed due to a lack of copper, we aimed to rescue the LTM defect of the *Appl* mutant by feeding flies with copper-complemented food. Notably, the memory performance of the *Appl* knockdown was fully rescued by 1 mM copper feeding before conditioning (Fig. 6a). In agreement with this, the H₂O₂ gradient was restored after spaced conditioning

in the *Appl* knockdown supplemented with copper (Fig. 6b). Thus, copper-supplemented food can compensate for *Appl* knockdown at both the behavioural and H₂O₂ signalling levels. Altogether, these results strongly support the idea that the main function of Appl during LTM formation in MB α lobe²⁰,³⁶ is to deliver extracellular copper for *Sod3*.

This leads to the question of why the increase in H₂O₂ occurs only in the α lobe of the MB, rather than in both the α and β lobes, which are two axonal projections of the same MB neurons. We postulated that this may be due to the activity of Appl itself, given that its copper-delivering property is permissive for H₂O₂ production by *Sod3*. The expression of Appl was monitored in the α and β lobes following spaced conditioning using the *Appl-HA* line and anti-HA immunohistochemistry. The level of Appl-HA expression was normalized to that of green fluorescent protein (GFP), which was expressed homogeneously in MB α/β neurons using the GAL4/UAS system. Notably, Appl-HA expression was specifically elevated in the α lobe following spaced conditioning, whereas no increase was observed in the β lobe (Fig. 6c). Furthermore, no increase in Appl-HA was observed in the α lobe following massed conditioning, a condition that does not engage H₂O₂ signalling in α/β neurons (Extended Data Fig. 8a). These findings support the notion that an elevation in Appl-HA levels within the α lobe results in an augmentation of copper release in the surrounding area, thereby facilitating the localized activation of *Sod3* secreted by astrocytes.

To further illustrate the pivotal function of the E2 copper-binding domain of Appl in LTM, we generated via CRISPR a constitutive *Appl* mutant for histidines of the evolutionarily conserved E2 copper-binding domain. E2 histidines at positions 535 and 539 were replaced by arginines (Methods). It should be noted that these mutations were introduced into the Appl gene at its typical chromosomal location,
<figure><img src="image_20.png" /><figcaption>**Fig. 5 | Appl interacts with Sod3 during LTM formation.** a, Image (maximum intensity projection of 22 µm-thick Z-stack acquisition, Z-step 1 µm) of C-terminal HA-tagged Appl immunostaining showing the high expression of HA-tagged Appl in α/β MB neurons (green). Scale bar, 40 µm. **b, Appl KD in adult α/β neurons impaired memory after spaced conditioning (n = 12 (first group), 18 (second and third groups)). F<sub>2,45</sub> = 15.58, P = 0.000007). c, The increase in H<sub>2</sub>O<sub>2</sub> level in α lobes/cell bodies elicited by spaced training (n = 15 (first group)), n = 16 (second group), t<sub>29</sub> = 4.30, P = 0.0002) was impaired in Appl KD in adult MB neurons (n = 15, t<sub>28</sub> = 0.88, P = 0.39). d, Mild decreases in both Appl and Sod3 expression in MB neurons and astrocytes impaired memory after</figcaption></figure>

spaced conditioning (n = 12, F<sub>4,55</sub> = 13.22, P = 0.0000001). This mild decrease was obtained by activating RNAi expression for 1 day instead of 3 days. e, Mild decrease in both *Appl* and *Sod3* expression in MB neurons and astrocytes prevents increase in H<sub>2</sub>O<sub>2</sub> level elicited by spaced training (wild-type control: n = 10, t<sub>18</sub> = 3.741, P = 0.0015; *Sod3* KD: n = 10, t<sub>18</sub> = 2.651, P = 0.0163; *Appl* KD: n = 10, t<sub>18</sub> = 2.655, P = 0.0161; *Sod3* and *Appl* KD: n = 10, t<sub>18</sub> = 1.262, P = 0.2229). Data are presented as mean ± s.e.m. Genotype sample sizes are listed in the legend in order of bar appearance. Significance level of a two-sided *t*-test or the least significant pairwise Tukey comparison following one-way ANOVA. \*P < 0.05; \*\*P < 0.01; \*\*\*P < 0.001; \*\*\*\*P < 0.0001; NS, not significant, P > 0.05.

without affecting regulatory sequences. *Appl*<sup>H(S35)R,H(S39)R</sup> mutant with modified copper-binding histidines exhibited an impairment in LTM (Fig. 6d). ARM and middle-term memory remained unaltered in *Appl*<sup>H(S35)R,H(S39)R</sup> flies (Extended Data Fig. 8b), whereas middle-term memory is affected in *Appl* knockdown<sup>52</sup>. The olfactory acuity and shock reactivity of the *Appl*<sup>H(S35)R,H(S39)R</sup> mutant were normal (Supplementary Table 6). Notably, the memory defect of *Appl*<sup>H(S35)R,H(S39)R</sup> mutant was rescued by 24 h 2 mM copper feeding (Fig. 6e). These findings further substantiate the pivotal and specific role of the copper-binding property of Appl in LTM formation.

## Aβ42 inhibits astrocytic nAChRα7 and prevents H₂O₂ signalling

Following the observation that Appl is involved in the activation of H₂O₂ signalling, we wondered whether, conversely, the toxic derivative of APP, amyloid β (Aβ), can inhibit this signalling pathway. Notably,

human Aβ42 is a strong ligand for the nAChRα7 cholinergic receptor<sup>53</sup>. At very low concentrations (<100 pM), which we cannot generate in *Drosophila* using available genetic expression systems, oligomeric Aβ42 was previously shown in vitro to activate nAChRα7 (ref. 54); however, in the 10–100 nM range, Aβ42 becomes a strong inhibitor of nAChRα7, preventing activation of the receptor by its natural ligand, ACh<sup>55</sup>. As we showed here that nAChRα7 activation in astrocytes initiates an H₂O₂ signalling pathway, we postulated that secreted Aβ might impede this process. Because Appl shows no clear sequence similarity with APP at the level of the Aβ sequence<sup>56</sup>, we expressed a secreted form of the human Aβ42 peptide, as frequently performed in *Drosophila* to study the consequences of brain amyloid expression<sup>57,58</sup>. The human Aβ42 peptide was expressed with a signal sequence that ensures its secretion into the extracellular space<sup>56</sup>. After several days of constitutive human Aβ42 expression in the *Drosophila* brain, diffuse aggregates are observed, along with neurodegenerative defects<sup>57</sup>. These defects are
<figure><img src="image_21.png" /><figcaption>**Fig. 6 | Appl provides copper to sustain Sod3 activity during LTM formation.**</figcaption></figure>

a, Copper feeding (1 mM) 24 h before spaced training restored normal memory capacity in flies with an *Appl*KD in adult α/β MB neurons (two-way ANOVA, *n* = 14, copper treatment: *F*<sub>1,78</sub> = 0.94, *P* = 0.33; genotype: *F*<sub>2,78</sub> = 4.48, *P* = 0.01; interaction: *F*<sub>2,78</sub> = 6.70, *P* = 0.002). **b**, Copper feeding (1 mM) 24 h before training restored the H<sub>2</sub>O<sub>2</sub> level in α lobes/cell bodies in MB neuron presynaptic terminals of an *Appl*KD in adult MB neurons (right) (two-way ANOVA, *n* = 11, copper treatment: *F*<sub>1,40</sub> = 3.07, *P* = 0.087; training: *F*<sub>1,40</sub> = 0.92, *P* = 0.34; interaction: *F*<sub>1,40</sub> = 7.01, *P* = 0.012). In wild-type controls (left), the increase in H<sub>2</sub>O<sub>2</sub> level in the α lobes/cell bodies elicited by spaced training was not affected by copper feeding (two-way ANOVA, *n* = 11 (first, third and fourth groups), *n* = 12 (second group), copper treatment: *F*<sub>1,43</sub> = 0.71, *P* = 0.40; training: *F*<sub>1,43</sub> = 15.32, *P* = 0.0003; interaction: *F*<sub>1,43</sub> = 0.30, *P* = 0.59). c, Appl expression is increased in MB α lobes after spaced training. Control flies

received odorant and shock stimuli temporally dissociated, a condition that does not induce LTM (α lobes: *n* = 20, *t*<sub>38</sub> = 2.181, *P* = 0.0355; β lobes: *n* = 20, *t*<sub>38</sub> = 0.5444, *P* = 5894). **d**, Mutations of copper-binding histidines H(535) and H(539) affect memory after spaced training (*n* = 30, *t*<sub>58</sub> = 4.169, *P* = 0.0001). **e**, Copper feeding (2 mM) 24 h before spaced training restored normal memory capacity in *Appl*<sup>H(535),H(539)</sup> flies (two-way ANOVA, *n* = 18, copper treatment: *F*<sub>1,68</sub> = 5.603, *P* = 0.0208; genotype: *F*<sub>1,68</sub> = 17.22, *P* = 0.00009; interaction: *F*<sub>1,68</sub> = 4.932, *P* = 0.0297). Data are presented as mean ± s.e.m. Genotype sample sizes are listed in the legend in order of bar appearance. Significance level of a two-sided *t*-test or the least significant Tukey pairwise comparison following one-way ANOVA or Šidák pairwise comparison following two-way ANOVA. \* *P* < 0.05; \*\* *P* < 0.01; \*\*\* *P* < 0.001; NS, not significant, *P* > 0.05.

not observed after 3 days of Aβ42 induction<sup>57</sup>. As we aimed to evaluate the potential acute effect of Aβ42 on astrocytic nAChRα7, expression was activated in MB α/β neurons for a short 24-h period. We observed that the calcium response to nicotine was inhibited in the presence of Aβ42 (Fig. 7a), suggesting that human Aβ42 secreted by MB neurons inhibits astrocytic nAChRα7. We then showed that expressing Aβ42 in MB for 24 h affected LTM specifically (Fig. 7b, Extended Data Fig. 9a and Supplementary Table 7). Last, we showed that Aβ42 inhibits formation of the H<sub>2</sub>O<sub>2</sub> gradient in MB neurons upon spaced conditioning (Fig. 7c). These results demonstrate that an acute expression of toxic Aβ42 in young flies prevents H<sub>2</sub>O<sub>2</sub> formation by astrocytes after spaced conditioning, and therefore LTM formation.

Discussion

ROS are byproducts produced mainly by mitochondria, and they are particularly toxic for neurons². It has been suggested that ROS play a

negative role in normal cognitive aging<sup>59</sup>, and that a defect in redox homeostasis is involved in the memory impairment that is characteristic of AD<sup>59,60</sup>. Glia help neurons fighting oxidative stress in various ways, including the production of reductive glutathione<sup>10,61,62</sup> or accumulation of peroxidated lipids transferred from neurons<sup>63</sup>; however, ROS also play physiological roles¹, and modulating the level of enzymes involved in ROS metabolism affects synaptic plasticity² and learning and memory<sup>8,10</sup>. Nevertheless, the precise mechanisms controlling ROS signalling during memory formation have remained poorly known, in particular because imaging physiological ROS variations during memory formation was out of reach until the development of a genetically encoded highly sensitive sensor¹⁶. Moreover, it was not known if glia could transfer beneficial ROS to neurons for neuronal plasticity and memory formation. Our data now provide a mechanistic understanding of the role of H<sub>2</sub>O<sub>2</sub> signalling in vivo during memory formation. We have deciphered a pathway by which astrocytes deliver beneficial
$H_2O_2$ to neurons for LTM formation, a phenomenon we have named astrocyte-to-neuron $H_2O_2$ signalling (ANHOS) (Fig. 7d). The production of ROS by astrocytic Nox and Sod3 enzymes, which follows ACh neurotransmitter-induced $Ca^{2+}$ increase, ensures a local $H_2O_2$ accumulation in the MB α lobe, the specific axonal projections encoding LTM$^{20,36}$. Following $H_2O_2$ import, a signalling cascade is initiated by redox-sensitive enzymes$^{64}$ comprising Prx2, Trx2 and Trxr1 (Fig. 7d). We put forth the proposition that this cascade serves as a trigger for LTM formation through the reversible oxidation of specific cysteines in LTM-relevant signalling proteins, in conjunction with the potential direct action of $H_2O_2$ itself. Although the future investigation of these molecular changes will be important, assessing transient changes of oxidation states is technically challenging, and this will require targeting the small subpopulation of α/β neurons that respond to the odorant used during LTM conditioning$^{65}$. What is the potential connection between ANHOS and cAMP-response element binding protein (CREB), a transcription factor that has been demonstrated to play a pivotal role in LTM formation in *Drosophila* and mice$^{24,25}$? Mitogen-activated protein kinases (MAPKs) have been demonstrated to activate CREB by phosphorylation, thereby facilitating memory consolidation$^{66,67}$. Moreover, kinases of the MAPK family, such as p38α and ERK, can be activated by $H_2O_2$ signalling$^{68,69}$. It is therefore proposed that the link between $H_2O_2$ signalling in the α lobe and the regulation of transcription by CREB may imply the activation of the MAPK pathway and retrograde signalling from the axonal projection to the nucleus.

Sod3 secreted by astrocytes plays a central role in ANHOS, as it generates the $H_2O_2$ molecules that will be imported by neurons. Sod3 requires a source of free copper for its catalytic activity. Although the free copper concentration remains extremely low in the brain because of its toxicity, copper concentrations around 100 µM have been reported at the synaptic cleft in mammals$^70$. Our results in *Drosophila* suggest that an essential source of copper for the activity of Sod3 secreted by astrocytes comes from neuronal Appl, which exhibits an evolutionarily conserved copper-binding site on its extracellular domain. Indeed, *Appl* and *Sod3* knockdowns display a strong synergistic interaction (Fig. 5d,e) and feeding copper to *Appl* knockdown flies rescued the LTM defect (Fig. 6a) and reestablished the $H_2O_2$ gradient in the olfactory memory centre (Fig. 6b). In addition, mutation of two of four evolutionarily conserved histidines within the E2 copper-binding domain of Appl resulted in impaired LTM (Fig. 6d), whereas middle-term

memory remained unaltered (Extended Data Fig. 8b). Conversely, Appl knockdown affects both LTM and middle-term memory$^52$, which further delineates the specific role of the copper-binding domain in

a

<figure><img src="image_22.png" /></figure>

b

<figure><img src="image_23.png" /></figure>

c

<figure><img src="image_24.png" /></figure>

d

<figure><img src="image_25.png" /></figure>

**Fig. 7 | Secreted human Aβ42 inhibits astrocyte-neuron H₂O₂ signalling.**

a, Nicotine stimulation (50 µM) elicited increased calcium in astrocytes, which was significantly decreased when secreted human Aβ42 was expressed by MB neurons (n = 10, t₁₈ = 2.29, *P* = 0.03). b, Human Aβ42 secretion by adult α/β neurons impaired memory after spaced conditioning (n = 12, F₂,₃₃ = 18.51, *P* = 0.000004). c, Spaced training elicited an increase in the $H_2O_2$ level of the α lobes/cell bodies as compared with the unpaired control (n = 9, t₁₆ = 2.75, *P* = 0.014), which was impaired by human Aβ42 secretion by adult MB neurons (n = 9 (first group), n = 8 (second group), t₁₅ = 0.72, *P* = 0.48). d, Model of ANHOS for LTM formation. ACh release from MB neurons²⁷ upon spaced training activates astrocytic nicotinic receptor nAChRα7, which induces a calcium elevation in astrocytes, resulting in Nox activation. Nox produces extracellular $O_2^{•−}$ in the presence of NADPH co-factor derived from astrocytic PPP. In the presence of $Cu^{2+}$ delivered by MB neuronal Appl, extracellular astrocytic Sod3 converts $O_2^{•−}$ to $H_2O_2$. $H_2O_2$ enters MB neuron lobes through the AQP channel and fuels the redox-sensitive cascade composed of Prx2, Trx2 and Trxr1 enzymes. Regeneration of reduced forms of these enzymes is provided by PPP-derived NADPH produced during LTM formation¹⁷. Oxidized Prx2 and Trx2 can activate signalling cascades (to be identified), which allows LTM formation. In a parallel pathway, $H_2O_2$ can directly oxidize signalling proteins (dotted line). Aβ42 impedes ANHOS by inhibiting nAChRα7. Data are presented as mean ± s.e.m. Genotype sample sizes are listed in the legend in order of bar appearance. Significance level of a two-sided *t*-test or the least significant Tukey pairwise comparison following one-way ANOVA. \* *P* < 0.05; \*\* *P* < 0.01; NS, not significant, *P* > 0.05.
LTM. The study of the *Drosophila* histidine E2 mutant is particularly important because, to the best of our knowledge, no mouse mutants for E2 copper-binding histidines have been reported so far. Feeding copper to *Appl*E2 mutant rescued the LTM defect (Fig. 6e), outlining the essential role of the copper-binding property of *Appl* in LTM formation.

Altogether, our results suggest that the copper-binding property of Appl and its functional interaction with Sod3 are the major roles of Appl in LTM formation¹⁸. Of note, expression of extracellular Sod3 in the human brain is much stronger in astrocytes than in neurons⁷¹, as in *Drosophila*³³ (Extended Data Fig. 2c). In mammals, APP binds copper⁴³,⁴⁴; it is expressed at the presynaptic active zone of neurons, where neurotransmitters are released⁷,⁷³; and it is involved in synaptic plasticity and memory⁵¹,⁷⁴. We propose that our model developed in *Drosophila* (Fig. 7d) can be generalized to these mammalian data, leading us to conclude that APP activates astrocytic Sod3 for memory formation in mammals. In the mammalian brain, glutamate is the predominant excitatory neurotransmitter. The ANHOS cascade may be activated not only at the cholinergic synapse, but also at the glutamatergic synapse, following calcium signalling in astrocytes that results from the activity of glutamatergic neurons⁷⁵.

From a pathological standpoint, our findings demonstrate that human Aβ42 exerts an inhibitory effect on the ANHOS cascade. This is evidenced by its capacity to impede the Ca²⁺ influx observed in astrocytes following nAChRα7 stimulation, as well as the H₂O₂ increase in the MB LTM centre subsequent to spaced conditioning. It is likely that the effect of Aβ42 involves the inhibition of nAChRα7 in astrocytes, which in turn prevents the formation of ROS by astrocytic Nox and Sod3. We propose that this inhibitory process is at play during AD initiation. Indeed, several observations are in agreement with the pathophysiological hypothesis that impairment of astrocytes by diffusible Aβ42 oligomers at cholinergic synapses may occur precociously in the AD brain: (1) soluble synaptic Aβ42 correlates better with the pattern of cognitive decline in AD than amyloid plaques¹⁵; (2) nAChRα7 is expressed by human brain astrocytes, in particular in the hippocampus²⁹, a neural structure that plays a major role in episodic and spatial memory; (3) Aβ42 inhibits nAChRα7 in vitro at a nM concentration⁷⁶; and (4) in humans, pathology of the basal forebrain cholinergic neurons precedes cortical defects but is predictive of future memory deficits⁷⁷,⁷⁸. In addition, as revealed in a global data-mining survey, Sod3 has been associated with AD in several 'omics' approaches, although its implication remains to be understood⁷⁹. Our ANHOS model integrates observations of early AD and cholinergic neurons, and proposes a key role for Sod3 in AD. In normal conditions, cholinergic neurons of the basal forebrain, which send diffuse projections to many cortical areas as well as the hippocampus, are involved in memory formation⁷⁷,⁸⁰. The transition from a physiological condition to an AD condition might therefore involve a switch from an ANHOS activation by ACh to ANHOS inhibition via increased Aβ42 production.

## Methods

### Experimental model

Flies (*Drosophila melanogaster*) were raised on standard medium (inactivated yeast 6% w/v; corn flour 6.7% w/v; agar 0.9% w/v; methyl-4-hydroxybenzoate 22 mM), at 18 or 23 °C (depending on the experiments; see respective details below) and 60% humidity in a 12-h light–dark cycle. The study was performed on 1–3-day-old adult flies. For behavioural experiments, both male and female flies were used. For imaging experiments, female flies were used because of their larger size. Before imaging or behavioural experiments, we chose flies informally in a random manner from a much larger group raised together for all studies; there was no formal randomization procedure for selecting flies. Data collection and analysis were not performed blind to the conditions of the experiments. Transgenic flies were outcrossed for five generations to a reference strain carrying the *w*¹¹¹¹⁸ mutation in an otherwise Canton-Special genetic background. Because TRiP RNAi

transgenes are labelled by a γ⁺ marker, these lines were outcrossed to aγ⁺¹ *w*⁶⁷,⁶²³ strain in an otherwise Canton-Special background. All strains used in this study are described in Supplementary Table 8.

### Behavioural experiments

For behavioural experiments, flies were raised on standard medium at 18 °C and 60% humidity in a 12-h light–dark cycle. We used the GAL4/GAL80¹⁵ TARGET system⁸¹ to inducibly express RNAi constructs exclusively in the MB or astrocytes of adult flies, and not during development. To achieve the induction of RNAi expression, adult flies were kept at 30.5 °C for 3 days before conditioning, unless otherwise specified. To induce Aβ42 carrying the pre-proenkephalin signal peptide for secretion⁵⁸, adult flies were kept at 30.5 °C for 24 h before conditioning. To test the interaction of *Appl* and *Sod3*, adult flies were kept at 30.5 °C for 24 h to achieve mild expression of RNAi before conditioning (Fig. 5d and Extended Data Fig. 7g) or after conditioning (Extended Data Fig. 7g). Otherwise, for non-induced experiments, experimental flies were transferred before conditioning to fresh bottles at 18 °C.

All behavioural experiments, including the sample sizes, were conducted similarly to other studies from our laboratory¹⁷,²⁰,³⁷. Groups of 20–50 flies were subjected to one of the following olfactory conditioning protocols at 25 °C: a single training cycle (1×), five consecutive associative training cycles (5× massed training), or five associative cycles spaced by 15-min inter-trial intervals (5× spaced training). A non-associative control protocol (unpaired protocol) was also employed for imaging experiments and Appl-HA immunochemistry experiments, during which the odour and shock stimuli were delivered separately in time, with shocks occurring 3 min before the first odorant. Conditioning was performed using previously described barrel-type machines that allow the parallel training of up to six groups¹⁷,²⁰,³⁶. Throughout the conditioning protocol, each barrel was plugged into a constant air flow at 2 l min⁻¹. For a single cycle of associative training, flies were first exposed to an odorant (the CS⁺) for 1 min, while 12 pulses of 5-s long 60-V electric shocks were delivered; flies were then exposed 45 s later to a second odorant without shocks (the CS⁻) for 1 min. The odorants 3-octanol (Fluka 74878, Sigma-Aldrich) and 4-methylcyclohexanol (Fluka 66360, Sigma-Aldrich), diluted in paraffin oil to a final concentration of 2.79 × 10⁻¹ g l⁻¹, were alternately used as conditioned stimuli. During unpaired conditionings, the odour and shock stimuli were delivered separately in time, with shocks occurring 3 min before the first odorant.

Flies were kept at 18 °C on standard medium between conditioning and the memory test, except in experiments in which immediate memory was tested after spaced conditioning. The memory test was performed in a T-maze apparatus⁸², 24 h after massed or spaced training, at 25 °C. The memory test was performed at 2 h after 1× training. Each arm of the T-maze was connected to a bottle containing 3-octanol and 4-methylcyclohexanol, diluted in paraffin oil to a final concentration identical to the one used for conditioning. Flies were given 1 min to choose between either arm of the T-maze. A performance score was calculated as the number of flies avoiding the conditioned odour minus the number of flies preferring the conditioned odour, divided by the total number of flies. A single performance index value is the average of two scores obtained from two groups of genotypically identical flies conditioned in two reciprocal experiments, using either odorant (3-octanol or 4-methylcyclohexanol) as the CS⁺. The indicated 'n' is the number of independent performance index values for each genotype. To avoid giving disproportionate statistical weight to a small number of flies, rare behavioural experiments involving a group of fewer than six flies were excluded.

The shock response tests were performed at 25 °C by placing flies in two connected compartments; electric shocks were provided in only one of the compartments. Flies were given 1 min to move freely in these compartments, after which they were trapped, collected and counted. The compartment where the electric shocks were delivered
was alternated between two consecutive groups. Shock avoidance was calculated as for the memory test.

Because the delivery of electric shocks can modify olfactory acuity, our olfactory avoidance tests were performed on flies that had first been presented another odour paired with electric shocks. Innate odour avoidance was measured in a T-maze similar to those used for memory tests, in which one arm of the T-maze was connected to a bottle with odour diluted in paraffin oil and the other arm was connected to a bottle with paraffin oil only. Naive flies were given the choice between the two arms during 1 min. The odour-interlaced side was alternated for successively tested groups. Odour concentrations used in this assay were the same as for the memory assays. At these concentrations, both odorants are innately repulsive.

## In vivo calcium imaging

Calcium imaging experiments were performed on flies expressing the GCaMP6f calcium sensor in astrocytes via the alrm-GAL4 driver, in combination with UAS-GCaMP6f. Transgenes were expressed in astrocytes using the inducible tub-GAL80<sup>ts</sup>; alrm-GAL4 driver. As carried out previously in our laboratory for imaging experiments<sup>17,37</sup>, flies were raised at 23 °C to increase the expression level of genetically encoded sensors without allowing RNAi expression. Adult flies were kept at 30.5 °C for 3 days before conditioning to achieve the induction of RNAi expression.

As in all previous imaging work from our laboratory, all in vivo imaging was performed on female flies, which are preferred as their larger size facilitates surgery. A single fly was picked and prepared for imaging as previously described<sup>17,37</sup>. In brief, the head capsule was opened and the brain was exposed by gently removing the superior tra-cheae. The head capsule was bathed in artificial haemolymph solution for the duration of the preparation. The composition of this solution was: NaCl 130 mM (Sigma, S9625), KCl 5 mM (Sigma, P3911), MgCl₂ 2 mM (Sigma, M9272), CaCl₂ 2 mM (Sigma, C3881), d-trehalose 5 mM (Sigma, 9531), sucrose 30 mM (Sigma, S9378) and HEPES hemisodium salt 5 mM (Sigma, H7637). At the end of surgery, any remaining solution was absorbed and a fresh 100-µl droplet of this solution was applied on top of the brain. Two-photon imaging was performed using a Leica TCS-SP5 upright microscope equipped with a ×25, 0.95 NA water-immersion objective. Two-photon excitation was achieved using a Mai Tai Deep-See laser tuned to 910 nm. The frame rate was one image per second. Recordings were acquired in the astrocytic region in the anterior part of the brain (where MB vertical lobes are located), at approximately 30 µm depth from the top of the brain. Calcium imaging experiments with nicotine stimulation were performed as previously described<sup>17</sup>. Nicotine was freshly diluted from a commercial liquid (Sigma, N3876) into the saline used for imaging on each experimental day. A perfusion setup at a flux of 2.5 ml min⁻¹ enabled the time-restricted application of 50 µM nicotine on top of the brain<sup>17</sup>. Baseline recording was performed during 1 min, after which the saline supply was switched to drug supply. The solution reached the in vivo preparation within 30 s. The stimulation was maintained for 30 s, before switching back to the saline perfusion for an additional 5 min. The GCaMP6f intensity was measured in the anterior part of the brain with MB vertical lobes are located. GCaMP6f signal was calculated over time after background subtraction and normalized by a baseline value calculated over the 30 s preceding drug injection using MATLAB software (MathWorks).

## In vivo H₂O₂ imaging

H₂O₂ imaging experiments were performed on flies expressing the excitation ratio metric H₂O₂ sensor roGFP2-Tsa2ΔC<sub>R</sub>¹⁶ in MB neurons via the 13F02-LexA driver, in combination with LexAop-roGFP2-Tsa2ΔC<sub>R</sub> (generated in this study). Transgenes were expressed in astrocytes using the inducible tub-GAL80<sup>ts</sup>; alrm-GAL4 driver, or in MB neurons using the inducible tub-GAL80<sup>ts</sup>; VT30559-GAL4 driver. For imaging experiments, flies were raised at 23 °C, except for the imaging experiment after brief RNAi expression for which flies were raised at 18 °C (Fig. 5e). Adult

flies were kept at 30.5 °C for 3 days before conditioning to achieve the induction of RNAi expression. For experiments on conditioned flies, data were collected indiscriminately from 30 min to 2 h after training.

Surgery was performed as for calcium imaging. At the end of surgery, any remaining solution was absorbed and a fresh 100-µl droplet of saline solution was applied on top of the brain. Two-photon imaging was performed using a pulsed infra-red laser (Insight X3, Spectra Physics), coupled to a Leica SP8 Dive upright microscope equipped with a ×20, 1.0 NA water-immersion objective and non-descanned spectral hybrid detectors. Emission was collected from 500 to 570 nm.

To characterize the roGFP2-Tsa2ΔC<sub>R</sub> probe for two-photon excitation microscopy, excitation spectra were measured from 780 to 1,060 nm in steps of 8 nm. Oxidation of the probe was obtained by applying 2 mM H₂O₂ (final concentration) on top of the brain. Spectra in the basal and oxidized states were measured before and 15 min after H₂O₂ treatment, respectively. Excitation wavelengths of 780 nm and 988 nm were selected for further experiments, as they maximized the sensor emission ratio (ratio 780 nm to 988 nm) between the basal and oxidized states. Experiments after conditioning were performed as follows: on a given brain area, two z-stacks (one at each acquisition wavelength) were consecutively acquired with a step of 1 µm and a line averaging of 3. The detector gain was strictly similar between the two stacks and was set to the 988 nm excitation wavelength at the highest value to avoid any pixel saturation; however, because of differential signal strength within different cellular compartments of MB neurons, two sets of stacks were acquired for each brain: one encompassing the MB lobes, and one covering the soma and calyx area, with different detector gains. For data analysis, the average intensity of three consecutive planes of the stack was calculated for each region of interest at the two excitation wavelengths. Oxidation of the sensor by H₂O₂ was measured by the ratio of the signal collected upon 780 nm excitation over the signal collected upon 988 nm excitation (referred to as 'H₂O₂ sensor ratio'), using Leica Microsystems software LAS X Small (offline version).

## Dietary copper supplementation

Copper (II) sulfate pentahydrate (Thermo Fisher Scientific, 197730010) was added to melted standard food medium to achieve a final concentration of 1 mM (Fig. 6a,b), a concentration that does not affect survival rate under chronic exposure<sup>83</sup>. Flies were kept on regular food medium for 48 h at 30.5 °C to achieve RNAi induction and then transferred to copper-enriched medium for 24 h at 30.5 °C for both behavioural and H₂O₂ imaging experiments. No lethality was observed with this copper feeding diet. Control flies were transferred on regular medium for 24 h at 30.5 °C.

For the copper rescue of *Appl*<sup>H(S35),H(S39)</sup> flies (Fig. 6e), copper (II) sulfate pentahydrate was added to melted standard food medium to achieve a final concentration of 2 mM. Flies were kept on copper-enriched medium for 24 h at 18 °C before spaced conditioning.

## Immunohistochemistry

Before dissection, whole adult female flies (2–4 days old) were fixed in 4% paraformaldehyde in PBST (phosphate-buffered saline (PBS) containing 1% Triton X-100) at 4 °C overnight. Brains were dissected in PBS solution and rinsed three times for 20 min in PBST, blocked with 2% bovine serum albumin (Sigma-Aldrich, A9085) in PBST for 2 h at room temperature, and then incubated with primary antibodies at 1:200 dilution (rat anti-HA, Roche, ROAHAHA 11867423001, clone 3F10) in the blocking buffer (2% BSA in PBST) at 4 °C overnight. The following day, brains were rinsed three times for 20 min in PBST, and incubated with secondary antibodies at 1:400 dilution (goat anti-rat Alexa 488, Invitrogen, A11006) in blocking buffer for 3 h at room temperature. Brains were rinsed for a further three times for 20 min in PBST and were mounted in ProLong Mounting Medium (Lifetechnology) for imaging. Images were acquired with a Nikon A1R confocal microscope with a ×20 objective.
In experiments where Appl-HA expression was quantified in MB neurons following spaced conditioning, artificial GFP expression was used as an expression control. *Appl-HA/+; tub-GAL80<sup>fl/fl</sup>; VT30559-GAL4>UAS-mCD8GFP* females were raised at 18 °C and transferred for 1.5 days at 30.5 °C to induce GFP expression in MB neurons. The flies were trained using the various protocols. Two hours following conditioning, adult females were fixed in 4% paraformaldehyde in PBST (PBS containing 1% Triton X-100) at 4 °C overnight. For immunochemical analysis, brains were dissected in PBS solution and rinsed three times for 20 min in PBST. They were then blocked with 2% bovine serum albumin (Sigma-Aldrich, A9085) in PBST for 2 h at room temperature. Subsequently, the samples were incubated with primary antibodies at a dilution of 1:200 dilution for rat anti-HA (Roche, ROA-HAHA 11867423001, clone 3F10) and 1:400 dilution for mouse anti-GFP (Developmental Studies Hybridoma Bank, DSHB-GFP-12A6, clone 12A6) in blocking buffer (2% BSA in PBST) at 4 °C overnight. On the subsequent day, the brains were rinsed three times for 20 min in PBST and incubated with secondary antibodies at a dilution of 1:400 (goat anti-rat Alexa 594, Invitrogen, A11007 and goat anti-mouse Alexa 488, Invitrogen, A11029) in blocking buffer for 3 h at room temperature, and subsequently placed at 4 °C overnight. Subsequently, the brains were rinsed three times for 20 min in PBST and mounted in ProLong Mounting Medium (Life Technologies) for imaging. Images were obtained using a Nikon A1R confocal microscope with a ×20 objective.

## Western blot

Proteins were extracted from adult female heads after liquid nitrogen snap freezing and mechanical grinding in lysis buffer containing sucrose 100 mM, KH₂PO₄/HPO₄⁻ 40 mM, EDTA 30 mM, KCl 50 mM, 0.25% Triton X-100, dithiothreitol 10 mM, PMSF 0.5 mM and 1x protease inhibitors (Roche, 11836153001). Protein extracts containing loading buffer NOVEX Tricine SDS sample buffer (Thermo Fisher Scientific, LC1676) were run in NOVEX 10-20% Tricine gels (Thermo Fisher, EC66252BOX) and then transferred on nitrocellulose membranes. Membranes were saturated with 15% non-fat milk, PBS-Tween 0.2% for 1 h. Membranes were incubated with primary anti-HA antibodies diluted in PBS-Tween 0.2% blocking medium (mouse anti-HA, 1:5,000 dilution, BioLegend, 901513, clone 16B12) overnight at 4 °C under agitation. Membranes were rinsed five times for 8 min in PBS-Tween 0.2% and incubated with secondary antibodies for 2 h at room temperature under agitation (HRP anti-mouse, 1:10,000 dilution, Promega, W4021). Membranes were further rinsed five times for 8 min in PBS-Tween 0.2%. Revelation was conducted using NOVEX ECL substrate (Thermo Fisher, WP20005) and chemiluminescence was detected with ImageQuant LAS4000. The same procedure was then applied to the same membranes with anti-tubulin antibodies (mouse anti-tubulin, 1:40,000 dilution, Sigma-Aldrich, T16199, clone DM1A). The molecular weight ladder corresponds to Invitrogen SeeBlue Plus2 (LC5925).

## Quantitative PCR

The efficiency of the knockdowns (KDs) used in this study was validated by quantitative PCR with reverse transcription (RT-qPCR) to measure the messenger RNA of the target gene (Supplementary Table 9). Female flies carrying the elav-Gal4 pan-neuronal driver or the repo-Gal4 pan-glial driver were crossed either with males carrying the specified UAS-RNAi or with CS males. Fly progeny were reared at 25 °C throughout their development. Then, 0-1-day-old flies were transferred to fresh food for 1 d before RNA extraction. RNA extraction and complementary DNA synthesis were performed as described elsewhere⁶⁸,⁶⁹ using the same reagents: the RNeasy Plant Mini kit (QIAGEN), RNA MinElute Cleanup kit (QIAGEN), oligo(dT)20 primers and the SuperScript III First-Strand kit (Life Technologies). The level of complementary DNA for each gene of interest was compared against the level of the α-Tub84B (Tub, CG1913) reference cDNA. Amplification was performed using a LightCycler 480 (Roche) and the SYBR Green I Master mix

(Roche). Reactions were carried out in triplicate. The specificity and size of amplification products were assessed by melting curve analyses. Expression relative to the reference was expressed as a ratio (2 − ΔCp, where Cp is the crossing point). The primers used in this study and results of RT-qPCR data are presented in Supplementary Table 9. For KDs that did not yield memory impairments (negative results), both RNAi s were checked by qPCR. For KDs that were induced with two distinct RNAi s, at least one of the two RNAi lines was validated by qPCR. Exceptions were for those RNAi constructs that were already validated by RT-qPCR in previous studies, in which case the corresponding reference is indicated (Supplementary Table 10).

## Generation of transgenic flies

For the generation of the *LexAop-roGFP2-Tsa2ΔCR* *Drosophila* line, the p415 TEF roGFP2-Tsa2ΔCR plasmid (Addgene, #83238)¹⁶ was digested by NotI and XbaI. The resulting 1,416-bp fragment was purified by electrophoresis and cloned into a pJFRC19 plasmid (13XLexAop2-IVS-myr::GFP, Addgene, #26224)⁸⁴. The resulting construct was verified by sequencing (the molecular cloning was outsourced to RD-Biotech, France). Transgenic fly strains were obtained by site-specific embryonic injection of the resulting vector in the attP18 landing site (X chromosome), which was outsourced to Rainbow Transgenic Flies.

The *Appl-HA* line was generated using the CRISPR approach (outsourced to inDroso). The 3×HA sequence (GCCGCCGTGTACCCCTACGACGTGCCCCGACTACGCCGGCTCCTACCCCTACGACGTGCCCCGACTACGCCCCCGCCGCC) preceded by a linker (sequence: GCGTGGGGC) was inserted between the 11th exon and the 3' UTR of the *Appl* gene using a guide RNA targeting the sequence AAGTGAAA | GAGTAAGCGAGA. The genomic edition was strictly restricted to the 3×HA tag and linker, preventing any alterations due to the presence of a selection marker (scarless).

## Generation of *Appl* copper-binding mutant by CRISPR

The *Appl*<sup>H(S35)R,H(S39)R</sup> lines was generated using the CRISPR approach, which was outsourced to Rainbow Transgenic Flies. To generate *Appl*<sup>H(S35)R,H(S39)R</sup>, two guide RNAs targeting CCCACGCCTTGGCCCA CTAC|CGG and CGCGCCCTGCACAAGGAC|GGG were employed. The original wild-type genomic sequence GCCCTGCACAAGGACGGGC-CCACGCCTTGGCCCACTACCGGCACCTATTGAACTCTGG, which corresponds to the amino-acids sequence ALHKDRAH(S35)ALAH(S39) YRHLLNS, was altered to GCCCTGCACAAGGACGGGCCCCGCGC-CTTGGCCGCTACCGGCACCTATTGAACTCTGG, which corresponds to the amino-acids sequence ALHKDRAR(S35)ALAR(S39)YRHLLNS. The CRISPR strategy was designed to introduce only the specified H/R modifications, and in particular, no marker was associated with the CRISPR-induced mutation. The presence of mutations in *Appl* was confirmed by Rainbow Transgenic Flies through genomic sequencing.

*Appl* mutations were generated on a X chromosome carrying *y w* mutations in an uncontrolled genetic background. To introduce the Canton-Special background appropriate for behavioural experiments, we crossed and *y Appl*<sup>H(S35)R,H(S39)R</sup> *w* females to wild-type Canton-Special males. At the next F1 generation *y Appl*<sup>H(S35)R,H(S39)R</sup> *w/+* females were crossed to Canton-Special males. Given the close proximity of the *y* gene to *Appl* and the very low rate of recombination between these two loci (less than 0.03%, as estimated from Flybase data⁸⁵), the presence of *y* mutation was employed to recover *y Appl* mutations in a Canton-Special background. In the F2 generation, recombination between the *y Appl* and *w* loci was screened for, and *y Appl*<sup>H(S35)R,H(S39)R</sup> *w*/*Y* males in a Canton-Special background were recovered. Homozygous *y Appl*<sup>H(S35)R,H(S39)R</sup> lines was generated. Concurrently, the parental *y w* line, which was utilized to induce the CRISPR mutations, was outcrossed with the identical protocol to generate a control *y* line in a Canton-Special background.

In light of the evidence indicating that *y* mutations impact LTM (based on our own unpublished observations), to conduct behavioural
experiments we crossed *yAppl*<sup>H(S35)R,H(S39)R</sup> females to males carrying the *y+* duplication *PBac\y[+]-attP-3B\VK00033* (ref. 86) in a Canton-Special background, designated *Dp(y+)*. The offspring were evaluated in behavioural assays to determine the phenotypes of hemizygous *yAppl*<sup>H(S35)R,H(S39)R</sup>/<i>Y</i>; *Dp(y+)*/+ males compared with *y/Y; Dp(y+)*/+ males.

### Sod3 transcript levels

Relative expression of *Sod3* in astrocytes and α/β neurons was obtained from published single-cell transcriptomic data³³, according to clustering performed in the original study where astrocytes are found in cluster 10 and α/β in cluster 22.

### Quantification and statistical analysis

All data are presented as mean ± s.e.m. For behaviour experiments, two groups of about 30 flies were reciprocally conditioned, using respectively octanol or methylcyclohexanol as the CS⁺. The memory score was calculated from the performance of two groups as described above, which represents a single experimental replicate. For imaging experiments, one replicate corresponds to one fly brain. No statistical methods were used to pre-determine sample sizes but our sample sizes are similar to those reported in previous publications¹⁷,¹⁹,³⁷. Data distribution was assumed to be normal but this was not formally tested. Comparisons of the data series between two conditions were achieved by a two-tailed unpaired *t*-test. Comparisons between more than two distinct groups were made using a one-way analysis of variance (ANOVA) test, followed by Tukey pairwise comparisons between the experimental groups and their controls. ANOVA results are presented as the value of the Fisher distribution $F_{x,y}$ obtained from the data, where x is the number of degrees of freedom between groups and y is the total number of degrees of freedom for the distribution. For copper-rescue experiments (Fig. 6a,b), two-way ANOVA tests were performed, followed by Šidák's multiple comparisons test. Statistical tests were performed using the GraphPad Prism 8 software. In the figures, asterisks denote the significance level of the *t*-test, or of the least significant pairwise comparison following ANOVA, with \* *P* < 0.05; \*\* *P* < 0.01; \*\*\* *P* < 0.001; NS, not significant, *P* > 0.05. Figures were designed using Adobe Illustrator.

### Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

### Data availability

No datasets that require mandatory deposition into a public database were generated during the current study. Source data that are reported as graphs on figures and extended data figures are available as Supplementary Information alongside the paper. Additional raw data will be shared with no restriction by the corresponding authors upon request. This study made use of the SCOPE database of single-cell transcriptomics in the fly brain to compare Sod3 expression in neurons and glia (Extended Data Fig. 2c) at https://scope.aertslab.org/#/Davie_et_al_Cell_2018/Davie_et_al_Cell_2018%2FAerts_Fly_AdultBrain_Filtered_57k_loom/gene. Source data are provided with the paper.

### Code availability

This paper does not report original code. Any additional information required to reanalyse the data reported in this paper is available from the corresponding authors upon request.

### References

1. Sies, H. et al. Defining roles of specific reactive oxygen species (ROS) in cell biology and physiology. *Nat. Rev. Mol. Cell Biol.* **23**, 499–515 (2022).

2. Massaad, C. A. & Klann, E. Reactive oxygen species in the regulation of synaptic plasticity and memory. *Antioxid. Redox Signal.* **14**, 2013–2054 (2011).

3. Lambeth, J. D. & Neish, A. S. Nox enzymes and new thinking on reactive oxygen: a double-edged sword revisited. *Annu. Rev. Pathol.* **9**, 119–145 (2014).

4. Klann, E., Roberson, E. D., Knapp, L. T. & Sweatt, J. D. A role for superoxide in protein kinase C activation and induction of long-term potentiation. *J. Biol. Chem.* **273**, 4516–4522 (1998).

5. Kishida, K. T. et al. Synaptic plasticity deficits and mild memory impairments in mouse models of chronic granulomatous disease. *Mol. Cell. Biol.* **26**, 5908–5920 (2006).

6. Oswald, M. C. et al. Reactive oxygen species regulate activity-dependent neuronal plasticity in *Drosophila*. *eLife* **7**, e39393 (2018).

7. Dhawan, S. et al. Reactive oxygen species mediate activity-regulated dendritic plasticity through NADPH oxidase and aquaporin regulation. *Front. Cell. Neurosci.* **15**, 641802 (2021).

8. Levin, E. D. et al. Molecular manipulations of extracellular superoxide dismutase: functional importance for learning. *Behav. Genet.* **28**, 381–390 (1998).

9. Santello, M., Toni, N. & Volterra, A. Astrocyte function from information processing to cognition and cognitive impairment. *Nat. Neurosci.* **22**, 154–166 (2019).

10. Vicente-Gutierrez, C. et al. Astrocytic mitochondrial ROS modulate brain metabolism and mouse behaviour. *Nat. Metab.* **1**, 201–211 (2019).

11. Butterfield, D. A. & Halliwell, B. Oxidative stress, dysfunctional glucose metabolism and Alzheimer disease. *Nat. Rev. Neurosci.* **20**, 148–160 (2019).

12. Tönnies, E. & Trushina, E. Oxidative stress, synaptic dysfunction, and Alzheimer's disease. *J. Alzheimers Dis. JAD* **57**, 1105–1121 (2017).

13. Musiek, E. S. & Holtzman, D. M. Three dimensions of the amyloid hypothesis: time, space and 'wingmen'. *Nat. Neurosci.* **18**, 800–806 (2015).

14. Padmanabhan, P., Kneynsberg, A. & Götz, J. Super-resolution microscopy: a closer look at synaptic dysfunction in Alzheimer disease. *Nat. Rev. Neurosci.* **22**, 723–740 (2021).

15. Palop, J. J. & Mucke, L. Amyloid-β-induced neuronal dysfunction in Alzheimer's disease: from synapses toward neural networks. *Nat. Neurosci.* **13**, 812–818 (2010).

16. Morgan, B. et al. Real-time monitoring of basal H₂O₂ levels with peroxiredoxin-based probes. *Nat. Chem. Biol.* **12**, 437–443 (2016).

17. de Tredern, E. et al. Glial glucose fuels the neuronal pentose phosphate pathway for long-term memory. *Cell Rep.* **36**, 109620 (2021).

18. Silva, B. et al. Glia fuel neurons with locally synthesized ketone bodies to sustain memory under starvation. *Nat. Metab.* **4**, 213–224 (2022).

19. Rabah, Y. et al. Glycolysis-derived alanine from glia fuels neuronal mitochondria for memory in *Drosophila*. *Nat. Metab.* **5**, 2002–2019 (2023).

20. Pascual, A. & Préat, T. Localization of long-term memory within the *Drosophila* mushroom body. *Science* **294**, 1115–1117 (2001).

21. Tully, T., Preat, T., Boynton, S. C. & Del Vecchio, M. Genetic dissection of consolidated memory in *Drosophila*. *Cell* **79**, 35–47 (1994).

22. Kawahara, T., Quinn, M. T. & Lambeth, J. D. Molecular evolution of the reactive oxygen-generating NADPH oxidase (Nox/Duox) family of enzymes. *BMC Evol. Biol.* **7**, 109 (2007).

23. Ritsick, D. R., Edens, W. A., Finnerty, V. & Lambeth, J. D. Nox regulation of smooth muscle contraction. *Free Radic. Biol. Med.* **43**, 31–38 (2007).

24. Widmer, Y. F. et al. Multiple neurons encode CrebB dependent appetitive long-term memory in the mushroom body circuit. *eLife* **7**, e39196 (2018).

25. Hirano, Y. et al. Shifting transcriptional machinery is required for long-term memory maintenance and modification in *Drosophila* mushroom bodies. *Nat. Commun.* **7**, 13471 (2016).
26. Stincone, A. et al. The return of metabolism: biochemistry and physiology of the pentose phosphate pathway. *Biol. Rev. Camb. Philos.* **90**, 927–963 (2015).

27. Barnstedt, O. et al. Memory-relevant mushroom body output synapses are cholinergic. *Neuron* **89**, 1237–1247 (2016).

28. Séguéla, P., Wadiche, J., Dineley-Miller, K., Dani, J. A. & Patrick, J. W. Molecular cloning, functional properties, and distribution of rat brain alpha 7: a nicotinic cation channel highly permeable to calcium. *J. Neurosci.* **13**, 596–604 (1993).

29. Yu, W.-F., Guan, Z.-Z., Bogdanovic, N. & Nordberg, A. High selective expression of α7 nicotinic receptors on astrocytes in the brains of patients with sporadic Alzheimer's disease and patients carrying Swedish APP 670/671 mutation: a possible association with neuritic plaques. *Exp. Neurol.* **192**, 215–225 (2005).

30. Zhang, K. et al. Fear learning induces α7-nicotinic acetylcholine receptor-mediated astrocytic responsiveness that is required for memory persistence. *Nat. Neurosci.* **24**, 1686–1698 (2021).

31. Schieber, M. & Chandel, N. S. ROS function in redox signaling and oxidative stress. *Curr. Biol.* **24**, R453–R462 (2014).

32. Wang, Y., Branicky, R., Noë, A. & Hekimi, S. Superoxide dismutases: dual roles in controlling ROS damage and regulating ROS signaling. *J. Cell Biol.* **217**, 1915–1928 (2018).

33. Davie, K. et al. A single-cell transcriptome atlas of the aging *Drosophila* brain. *Cell* **174**, 982–998.e20 (2018).

34. Perea, G., Navarrete, M. & Araque, A. Tripartite synapses: astrocytes process and control synaptic information. *Trends Neurosci.* **32**, 421–431 (2009).

35. Kremer, M. C., Jung, C., Batelli, S., Rubin, G. M. & Gaul, U. The glia of the adult *Drosophila* nervous system. *Glia* **65**, 606–638 (2017).

36. Yu, D., Akalal, D.-B. G. & Davis, R. L. *Drosophila* α/βmushroom body neurons form a branch-specific, long-term cellular memory trace after spaced olfactory conditioning. *Neuron* **52**, 845–855 (2006).

37. Plaçais, P.-Y. et al. Upregulated energy metabolism in the *Drosophila* mushroom body is the trigger for long-term memory. *Nat. Commun.* **8**, 15510 (2017).

38. Bienert, G. P. & Chaumont, F. Aquaporin-facilitated transmembrane diffusion of hydrogen peroxide. *Biochim. Biophys. Acta* **1840**, 1596–1604 (2014).

39. Stöcker, S., Van Laer, K., Mijuskovic, A. & Dick, T. P. The conundrum of hydrogen peroxide signaling and the emerging role of peroxiredoxins as redox relay hubs. *Antioxid. Redox Signal.* **28**, 558–573 (2018).

40. Rhee, S. G. & Kil, I. S. Multiple functions and regulation of mammalian peroxiredoxins. *Annu. Rev. Biochem.* **86**, 749–775 (2017).

41. Bouzaiane, E., Trannoy, S., Scheunemann, L., Plaçais, P.-Y. & Preat, T. Two independent mushroom body output circuits retrieve the six discrete components of *Drosophila* aversive memory. *Cell Rep.* **11**, 1280–1292 (2015).

42. D'Ambrosi, N. & Rossi, L. Copper at synapse: release, binding and modulation of neurotransmission. *Neurochem. Int.* **90**, 36–45 (2015).

43. Wild, K., August, A., Pietrzik, C. U. & Kins, S. Structure and synaptic function of metal binding to the amyloid precursor protein and its proteolytic fragments. *Front. Mol. Neurosci.* **10**, 21 (2017).

44. Young, T. R., Pukala, T. L., Cappai, R., Wedd, A. G. & Xiao, Z. The human amyloid precursor protein binds copper ions dominated by a picomolar-affinity site in the helix-rich E2 domain. *Biochemistry* **57**, 4165–4176 (2018).

45. Cappai, R. et al. The amyloid precursor protein (APP) of Alzheimer disease and its paralog, APLP2, modulate the Cu/Zn-nitric oxide-catalyzed degradation of glypican-1 heparan sulfate in vivo. *J. Biol. Chem.* **280**, 13913–13920 (2005).

46. Dahms, S. O. et al. Metal binding dictates conformation and function of the amyloid precursor protein (APP) E2 domain. *J. Mol. Biol.* **416**, 438–452 (2012).

47. Eskici, G. & Axelsen, P. H. Copper and oxidative stress in the pathogenesis of Alzheimer's disease. *Biochemistry* **51**, 6289–6311 (2012).

48. Goguel, V. et al. *Drosophila* amyloid precursor protein-like is required for long-term memory. *J. Neurosci.* **31**, 1032–1037 (2011).

49. Leyssen, M. et al. Amyloid precursor protein promotes post-developmental neurite arborization in the *Drosophila* brain. *EMBO J.* **24**, 2944–2955 (2005).

50. Müller, U. C. & Zheng, H. Physiological functions of APP family proteins. *Cold Spring Harb. Perspect. Med.* **2**, a006288 (2012).

51. Ludewig, S. & Korte, M. Novel insights into the physiological function of the APP (gene) family and its proteolytic fragments in synaptic plasticity. *Front. Mol. Neurosci.* **9**, 161 (2016).

52. Bourdet, I., Preat, T. & Goguel, V. The full-length form of the *Drosophila* amyloid precursor protein is involved in memory formation. *J. Neurosci.* **35**, 1043–1051 (2015).

53. Wang, H. Y., Lee, D. H., Davis, C. B. & Shank, R. P. Amyloid peptide Aβ(1-42) binds selectively and with picomolar affinity to α7 nicotinic acetylcholine receptors. *J. Neurochem.* **75**, 1155–1161 (2000).

54. Dineley, K. T., Bell, K. A., Bui, D. & Sweatt, J. D. β-Amyloid peptide activates α7 nicotinic acetylcholine receptors expressed in Xenopus oocytes. *J. Biol. Chem.* **277**, 25056–25061 (2002).

55. Liu, Q., Kawai, H. & Berg, D. K. β-Amyloid peptide blocks the response of α7-containing nicotinic receptors on hippocampal neurons. *Proc. Natl Acad. Sci. USA* **98**, 4734–4739 (2001).

56. Guo, Q., Wang, Z., Li, H., Wiese, M. & Zheng, H. APP physiological and pathophysiological functions: insights from animal models. *Cell Res.* **22**, 78–89 (2012).

57. Iijima, K. et al. Dissecting the pathological effects of human Aβ40 and Aβ42 in *Drosophila*: a potential model for Alzheimer's disease. *Proc. Natl Acad. Sci. USA* **101**, 6623–6628 (2004).

58. Finelli, A., Kelkar, A., Song, H.-J., Yang, H. & Konsolaki, M. A model for studying Alzheimer's Aβ42-induced toxicity in *Drosophila melanogaster*. *Mol. Cell. Neurosci.* **26**, 365–375 (2004).

59. Ionescu-Tucker, A. & Cotman, C. W. Emerging roles of oxidative stress in brain aging and Alzheimer's disease. *Neurobiol. Aging* **107**, 86–95 (2021).

60. Shi, Q. & Gibson, G. E. Oxidative stress and transcriptional regulation in Alzheimer disease. *Alzheimer Dis. Assoc. Disord.* **21**, 276–291 (2007).

61. Shih, A. Y. et al. Coordinate regulation of glutathione biosynthesis and release by Nrf2-expressing glia potently protects neurons from oxidative stress. *J. Neurosci.* **23**, 3394–3406 (2003).

62. Jimenez-Blasco, D., Santofimia-Castaño, P., Gonzalez, A., Almeida, A. & Bolaños, J. P. Astrocyte NMDA receptors' activity sustains neuronal survival through a Cdk5-Nrf2 pathway. *Cell Death Differ.* **22**, 1877–1889 (2015).

63. Liu, L., MacKenzie, K. R., Putluri, N., Maletić-Savatić, M. & Bellen, H. J. The glia-neuron lactate shuttle and elevated ROS promote lipid synthesis in neurons and lipid droplet accumulation in glia via APOE/D. *Cell Metab.* **26**, 719–737.e6 (2017).

64. Mishina, N. M. et al. Which antioxidant system shapes intracellular H₂O₂ gradients? *Antioxid. Redox Signal.* **31**, 664–670 (2019).

65. Turner, G. C., Bazhenov, M. & Laurent, G. Olfactory representations by *Drosophila* mushroom body neurons. *J. Neurophysiol.* **99**, 734–746 (2008).

66. Bozon, B. et al. MAPK, CREB and zif268 are all required for the consolidation of recognition memory. *Philos. Trans. R. Soc. Lond. B Biol. Sci.* **358**, 805–814 (2003).

67. Medina, J. H. & Viola, H. ERK1/2: a key cellular component for the formation, retrieval, reconsolidation and persistence of memory. *Front. Mol. Neurosci.* **11**, 361 (2018).

68. Guyton, K. Z., Liu, Y., Gorospe, M., Xu, Q. & Holbrook, N. J. Activation of mitogen-activated protein kinase by H₂O₂. *J. Biol. Chem.* **271**, 4138–4142 (1996).
69. Barata, A. G. & Dick, T. P. A role for peroxiredoxins in H₂O₂⁻ and MEKK-dependent activation of the p38 signaling pathway. *Redox Biol.* **28**, 101340 (2020).

70. Gaier, E. D., Eipper, B. A. & Mains, R. E. Copper signaling in the mammalian nervous system: synaptic effects. *J. Neurosci. Res.* **91**, 2–19 (2013).

71. Zhang, Y. et al. Purification and characterization of progenitor and mature human astrocytes reveals transcriptional and functional differences with mouse. *Neuron* **89**, 37–53 (2016).

72. Laßek, M. et al. Amyloid precursor proteins are constituents of the presynaptic active zone. *J. Neurochem.* **127**, 48–56 (2013).

73. Guénette, S., Strecker, P. & Kins, S. APP protein family signaling at the synapse: insights from intracellular APP-binding proteins. *Front. Mol. Neurosci.* **10**, 87 (2017).

74. Lee, S. H. et al. APP family regulates neuronal excitability and synaptic plasticity but not neuronal survival. *Neuron* **108**, 676–690.e8 (2020).

75. Goenaga, J., Araque, A., Kofuji, P. & Herrera Moro Chao, D. Calcium signaling in astrocytes and gliotransmitter release. *Front. Synaptic Neurosci.* **15**, 1138577 (2023).

76. Pettit, D. L., Shao, Z. & Yakel, J. L. β-Amyloid(1-42) peptide directly modulates nicotinic receptors in the rat hippocampal slice. *J. Neurosci.* **21**, RC120 (2001).

77. Ballinger, E. C., Ananth, M., Talmage, D. A. & Role, L. W. Basal forebrain cholinergic circuits and signaling in cognition and cognitive decline. *Neuron* **91**, 1199–1218 (2016).

78. Schmitz, T. W., Nathan Spreng, R. & Alzheimer's Disease Neuroimaging Initiative. Basal forebrain degeneration precedes and predicts the cortical spread of Alzheimer's pathology. *Nat. Commun.* **7**, 13249 (2016).

79. Zhang, M. et al. Drug repositioning for Alzheimer's disease based on systematic 'omics' data mining. *PLoS ONE* **11**, e0168812 (2016).

80. Ananth, M. R., Rajebhosale, P., Kim, R., Talmage, D. A. & Role, L. W. Basal forebrain cholinergic signalling: development, connectivity and roles in cognition. *Nat. Rev. Neurosci.* **24**, 233–251 (2023).

81. McGuire, S. E., Le, P. T., Osborn, A. J., Matsumoto, K. & Davis, R. L. Spatiotemporal rescue of memory dysfunction in *Drosophila*. *Science* **302**, 1765–1768 (2003).

82. Tully, T. & Quinn, W. G. Classical conditioning and retention in normal and mutant *Drosophila melanogaster*. *J. Comp. Physiol. [A]* **157**, 263–277 (1985).

83. Bonilla-Ramirez, L., Jimenez-Del-Rio, M. & Velez-Pardo, C. Acute and chronic metal exposure impairs locomotion activity in *Drosophila melanogaster*: a model to study Parkinsonism. *Biometals* **24**, 1045–1057 (2011).

84. Pfeiffer, B. D. et al. Refinement of tools for targeted gene expression in *Drosophila*. *Genetics* **186**, 735–755 (2010).

85. Öztürk-Çolak, A. et al. FlyBase: updates to the *Drosophila* genes and genomes database. *Genetics* **227**, 211 (2024).

86. Venken, K. J. T., He, Y., Hoskins, R. A. & Bellen, H. J. P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in *D. melanogaster*. *Science* **314**, 1747–1751 (2006).

## Acknowledgements

This work was supported by the European Research Council (Advanced Grant EnergyMemo no. 741550, to T.P.), the Fondation pour la Recherche Médicale (Brixham Foundation Prize, to T.P.), the Fonds ESPCI (to T.P.), the Fondation Langlois (to T.P.), the Fondation Recherche Alzheimer (to J.-P.B.) and the Agence Nationale

pour la Recherche (MetaFlex ANR-23-CE16-0029-01 to T.P. and MetabolicBrainAging ANR-20-CE92-0047-01 to P.-Y.P.). We thank the Bloomington Drosophila Stock Center (National Institutes of Health grant P40OD018537) and the Vienna Drosophila Resource Center for providing the flies used in this study. We thank J.-M. Dura (Institute of Human Genetics, Montpellier), C. Desplan (New York University) and A. Pavlowsky (ESPCI, Paris) for fruitful discussions. We thank L. Cagninacci for help with RT-qPCR experiments.

## Author contributions

T.P. conceived and supervised the work; N.S. performed the behavioural analyses, with the support of Y.R., J.-P.B. and L.P.; P.-Y.P. developed the assay for in vivo H₂O₂ imaging using the roGFP2-Tsa2ΔC<sub>R</sub> sensor; Y.R. and J.-P.B. performed H₂O₂ imaging; Y.R. performed calcium imaging; L.P. characterized the Appl-HA line; N.S. performed the copper-rescue experiments, immunohistochemistry experiments and RT-qPCR experiments; T.P. wrote the main text of the manuscript; and Y.R., J.-P.B. and P.-Y.P. contributed to writing the manuscript.

## Competing interests

The authors declare no competing interests.

## Additional information

Extended data is available for this paper at https://doi.org/10.1038/s42255-024-01189-3.

**Supplementary information** The online version contains supplementary material available at https://doi.org/10.1038/s42255-024-01189-3.

**Correspondence and requests for materials** should be addressed to Thomas Preat.

**Peer review information** *Nature Metabolism* thanks Juan Bolaños, Efthimios Skoulakis and Sean Sweeney for their contribution to the peer review of this work. Primary Handling Editor: Yanina-Yasmin Pesch, in collaboration with the *Nature Metabolism* team.

**Reprints and permissions information** is available at www.nature.com/reprints.

**Publisher's note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

© The Author(s) 2025
a

Nox KD in astrocytes

5x massed (ARM)

<figure><img src="image_26.png" /></figure>

+/UAS-NoxRNAI HMS00429

tub-GAL80<sup>tr</sup>; alrm-GAL4+>UAS-NoxRNAI HMS00429

5x spaced (LTM)
no induction

<figure><img src="image_27.png" /></figure>

b

Nox, nAChRα7 and Sod3 KD in astrocytes

5x spaced at t₀

<figure><img src="image_28.png" /></figure>

tub-GAL80<sup>tr</sup>; alrm-GAL4/+

+/UAS-NoxRNAI HMS00429

tub-GAL80<sup>tr</sup>; alrm-GAL4+>UAS-NoxRNAI HMS00429

+/UAS-nAChRα7RNAI JF02570

tub-GAL80<sup>tr</sup>; alrm-GAL4+>UAS-nAChRα7RNAI JF02570

+/UAS-Sod3RNAI GD4801

tub-GAL80<sup>tr</sup>; alrm-GAL4+>UAS-Sod3RNAI GD4801

c

Nox KD in astrocytes

5x spaced (LTM)

5x spaced (LTM)
no induction

<figure><img src="image_29.png" /></figure>

+/UAS-NoxRNAI KK111991

tub-GAL80<sup>tr</sup>; alrm-GAL4+>UAS-NoxRNAI KK111991

5x spaced (LTM)
no induction

<figure><img src="image_30.png" /></figure>

d

G6PD KD in astrocytes

5x massed (ARM)

1x (MTM)

<figure><img src="image_31.png" /></figure>

+/UAS-G6PDRNAI KK108898

tub-GAL80<sup>tr</sup>; alrm-GAL4+>UAS-G6PDRNAI KK108898

5x spaced (LTM)
no induction

<figure><img src="image_32.png" /></figure>

e

G6PD, Pgd and Pgls KD in astrocytes

5x spaced at t₀

<figure><img src="image_33.png" /></figure>

tub-GAL80<sup>tr</sup>; alrm-GAL4/+

+/UAS-G6PDRNAI KK108898

tub-GAL80<sup>tr</sup>; alrm-GAL4+>UAS-G6PDRNAI KK108898

+/UAS-PgdRNAI HMC05959

tub-GAL80<sup>tr</sup>; alrm-GAL4+>UAS-PgdRNAI HMC05959

+/UAS-PglsRNAI HMS02626

tub-GAL80<sup>tr</sup>; alrm-GAL4+>UAS-PglsRNAI HMS02626

f

Pgd KD in astrocytes

5x spaced (LTM)

1x (MTM)

<figure><img src="image_34.png" /></figure>

+/UAS-PgdRNAI HMC05959

tub-GAL80<sup>tr</sup>; alrm-GAL4+>UAS-PgdRNAI HMC05959

<figure><img src="image_35.png" /></figure>

5x spaced (LTM)
no induction

g

Pgls KD in astrocytes

5x spaced (LTM)

5x massed (ARM)

1x (MTM)

<figure><img src="image_36.png" /></figure>

+/UAS-PglsRNAI HMS02626

tub-GAL80<sup>tr</sup>; alrm-GAL4+>UAS-PglsRNAI HMS02626

<figure><img src="image_37.png" /></figure>

5x spaced (LTM)
no induction
**Extended Data Fig. 1 |** *Controls for behavioural experiments, related to Fig. 1.*

**(a)** Memory after massed conditioning was not affected by *Nox* knockdown (KD) in adult astrocytes (n = 10 (first group), n = 11 (second and third groups), F<sub>2,29</sub> = 1.17, P = 0.32). Without *Nox* RNAi induction, memory after spaced training was normal (n = 10, F<sub>2,27</sub> = 0.67, P = 0.52). **(b)** Immediate memory after spaced conditioning was not affected by *Nox*, *nAChRα7*, or *Sod3* KD (n = 12, F<sub>6,77</sub> = 1.233, P = 0.2989). **(c)** Memories at 24 h were assessed with a second RNAi against *Nox*. *Nox* KD impaired memory after spaced training (n = 12 (first group), n = 17 (second and third groups), F<sub>2,43</sub> = 24.03, P = 0.00000009), but did not affect memory after massed training (n = 12, F<sub>2,33</sub> = 0.018, P = 0.98). Without *Nox* RNAi induction, memory after spaced training was normal (n = 10, F<sub>2,27</sub> = 0.38, P = 0.68). **(d)** Memory after massed conditioning was not affected by *G6PD* KD (n = 11, F<sub>2,30</sub> = 2.93, P = 0.07). Memory at 2 h after 1x conditioning was not affected by *G6PD* KD (n = 15, F<sub>2,42</sub> = 1.528, P = 0.2287). Without *G6PD* RNAi induction, memory after spaced training was normal (n = 10, F<sub>2,27</sub> = 0.49, P = 0.62). **(e)** Immediate

memory after spaced conditioning was not affected by *G6PD*, *Pgd* or *Pgls* KD (n = 12, F<sub>6,77</sub> = 0.4174, P = 0.8653). **(f)** *Pgd* KD impaired memory after spaced training (n = 12, F<sub>2,33</sub> = 4.92, P = 0.01), but did not affect memory after massed training (n = 10, F<sub>2,27</sub> = 0.31, P = 0.73). Memory at 2 h after 1x conditioning was not affected by *Pgd* KD (n = 15, F<sub>2,42</sub> = 3.582, P = 0.0366). Without *Pgd* RNAi induction, memory after spaced training was normal (n = 10, F<sub>2,27</sub> = 0.61, P = 0.55). **(g)** *Pgls* KD impaired memory after spaced training (n = 12, F<sub>2,33</sub> = 14.75, P = 0.00003), but did not affect memory after massed training (n = 12, F<sub>2,33</sub> = 0.24, P = 0.79). Memory at 2 h after 1x conditioning was not affected by *Pgls* KD (n = 15, F<sub>2,42</sub> = 2.078, P = 0.1379). Without *Pgls* RNAi induction, memory after spaced training was normal (n = 12, F<sub>2,33</sub> = 3.11, P = 0.06). Data are presented as mean ± SEM. Sample sizes for each genotype are listed in the legend in the order of bar appearance. Significance level of the least significant Tukey pairwise comparison following one-way analysis of variance (ANOVA): \*P < 0.05; \*\*\*P < 0.0001; ns: not significant, P > 0.05.
<figure><img src="image_38.png" /></figure>

**Extended Data Fig. 2** | **Controls for behavioural experiments, related to**

**Fig. 1.** (a) Memory after massed conditioning was not affected by *nAChRa7* KD in adult astrocytes (n = 10 (first group), n = 11 (second and third groups), F<sub>2,29</sub> = 1.07, P = 0.36). Without *nAChRa7* RNAi induction, memory after spaced training was normal (n = 12 (first and second groups), n = 11 (third group), F<sub>2,32</sub> = 0.47, P = 0.63).

(b) Memories at 24 h were assessed with a second RNAi against *nAChRa7*.

*nAChRa7* KD in adult astrocytes impaired memory after spaced training (n = 13 (first group), n = 17 (second and third groups), F<sub>2,44</sub> = 26.95, P = 0.00000002), but did not affect memory after massed training (n = 10, F<sub>2,27</sub> = 0.24, P = 0.79).

Without *nAChRa7* RNAi induction, memory after spaced training was normal (n = 10, F<sub>2,27</sub> = 0.71, P = 0.50). (c) Relative expression of *Sod3* in astrocytes and α/β MB neurons according to published single-cell transcriptomics³³. Levels of *Sod3* mRNA are higher in astrocytes as compared to α/β MB neurons (n = 1407

(first group), n = 672 (second group), t<sub>2077</sub> = 28.04, P < 0.000000000000001).

(d) Memory after massed conditioning was not affected by *Sod3* KD in adult astrocytes (n = 10 (first group), n = 11 (second and third groups), F<sub>2,29</sub> = 0.06, P = 0.94). Without *Sod3* RNAi induction, memory after spaced training was normal (n = 10, F<sub>2,27</sub> = 5.15, P = 0.01). (e) Memories at 24 h were assessed with a second RNAi against *Sod3*. *Sod3* KD in adult astrocytes impaired memory after spaced training (n = 12, F<sub>2,33</sub> = 20.88, P = 0.000001), but did not affect memory after massed training (n = 12, F<sub>2,33</sub> = 0.19, P = 0.83). Without *Sod3* RNAi induction, memory after spaced training was normal (n = 12, F<sub>2,33</sub> = 1.35, P = 0.27). Data are presented as mean ± SEM. Sample sizes for each genotype are listed in the legend in the order of bar appearance. Significance level of a two-sided t-test or the least significant Tukey pairwise comparison following one-way analysis of variance (ANOVA): ****P < 0.0001; ns: not significant, P > 0.05.
a

H₂O₂ imaging in MB neurons

13F02-LexA > LexAop-roGFP2-Tsa2ΔC<sub>R</sub>

<figure><img src="image_39.png" /></figure>

c

H₂O₂ imaging in MB neurons

13F02-LexA > LexAop-roGFP2-Tsa2ΔC<sub>R</sub>

<figure><img src="image_40.png" /></figure>

b

<figure><img src="image_41.png" /></figure>

d

H₂O₂ imaging in MB neurons

13F02-LexA > LexAop-roGFP2-Tsa2ΔC<sub>R</sub>

<figure><img src="image_42.png" /></figure>

e

Human-secreted-catalase expression in astrocytes

<figure><img src="image_43.png" /></figure>

5x spaced (LTM)
no induction

<figure><img src="image_44.png" /></figure>

**Extended Data Fig. 3 | See next page for caption.**
**Extended Data Fig. 3** | roGFP2-Tsa2ΔC~R~ imaging in MB neurons, related to Figs. 2 and 3. (a) Fluorescence excitation spectra of roGFP2-Tsa2ΔC~R~ upon 2-photon excitation in basal and oxidized states. H~2~O~2~-induced oxidation of the probe led to an increase in the excitation spectrum at 780 nm and a slight decrease at 988 nm, allowing ratiometric measurement of the oxidation level of the probe (ratio 780 nm/988 nm). (b) Intracellular pathways recruited during LTM formation in MB neurons. Upon spaced training, glucose intake in MB neuron cell bodies via the Glut1 transporter is increased to fuel the PPP, which produces reductive power in the form of NADPH²⁶. (c) Measurements of H~2~O~2~ levels in cell bodies and α lobes using the roGFP2-Tsa2ΔC~R~ H~2~O~2~ sensor expressed in MB neurons. H~2~O~2~ level (measured as 780/988 nm ratio) decreased in cell bodies upon spaced training (n = 13 (first group), n = 14 (second group), t~25~ = 2.27, P = 0.03), while it did not vary in α lobes (n = 15 (first group), n = 16 (second group), t~29~ = 0.18, P = 0.86). The H~2~O~2~ level in α lobes normalized to the cell bodies value is increased upon spaced training in adult MB neurons (values, bottom panel, same value as in Fig. 2c). (d) Glut1 KD in MB neurons abolished the

decrease in H₂O₂ level in cell bodies (n = 16 (first group), n = 15 (second group), t₂₉ = 0.25, P = 0.81, top panel), revealing an increase in H₂O₂ level in α lobes after spaced training (n = 18, t₃₄ = 2.06, P = 0.047, top panel). The H₂O₂ level in α lobes normalized to the cell bodies value is increased upon spaced training when *Glut1* was knocked down in adult MB neurons (n = 16 (first group), n = 15 (second group), t₂₉ = 2.66, P = 0.013, bottom panel). (e) Memory after massed conditioning was not affected by human-secreted-catalase expression in astrocytes (n = 12, F₂,₃₃ = 1.026, P = 0.9116). Memory at 2 h after 1x conditioning was not affected by human-secreted-catalase expression in adult astrocytes (n = 10, F₂,₂₇ = 1.171, P = 0.1304). Without human-secreted-catalase induction, memory after spaced training was normal (n = 10, F₂,₂₇ = 1.539, P = 0.8436). Data are presented as mean ± SEM. Sample sizes for each genotype are listed in the legend in the order of bar appearance. Significance level of a two-sided t-test or the least significant Tukey pairwise comparison following one-way analysis of variance (ANOVA): \*P < 0.05; \*\*P < 0.01; ns: not significant, P > 0.05.
a **AQP KD in MB neurons**

5x massed (ARM)

<figure><img src="image_45.png" /></figure>

5x spaced at t₀

<figure><img src="image_46.png" /></figure>

5x spaced (LTM)
no induction

<figure><img src="image_47.png" /></figure>

b **AQP KD in MB neurons**

5x spaced (LTM)

<figure><img src="image_48.png" /></figure>

5x massed (ARM)

<figure><img src="image_49.png" /></figure>

5x spaced (LTM)
no induction

<figure><img src="image_50.png" /></figure>

c **Bib KD in MB neurons**

5x spaced (LTM)

<figure><img src="image_51.png" /></figure>

d **Bib KD in MB neurons**

5x spaced (LTM)

<figure><img src="image_52.png" /></figure>

e **Drip KD in MB neurons**

5x spaced (LTM)

<figure><img src="image_53.png" /></figure>

f **Drip KD in MB neurons**

5x spaced (LTM)

<figure><img src="image_54.png" /></figure>

g **Prip KD in MB neurons**

5x spaced (LTM)

<figure><img src="image_55.png" /></figure>

h **Prip KD in MB neurons**

5x spaced (LTM)

<figure><img src="image_56.png" /></figure>

Extended Data Fig. 4 | See next page for caption.
**Extended Data Fig. 4** | Controls for behavioural experiments, and analysis of alternative Drosophila aquaporins, related to Fig. 4. (a) Memory after massed conditioning was not affected by *AQP* KD in adult α/β MB neurons (n = 10, F<sub>2,27</sub> = 0.40, P = 0.68). Immediate memory after spaced conditioning was not affected by *AQP* KD in adult MB neurons (n = 12, F<sub>2,33</sub> = 0.1461, P = 0.8647). Without *AQP* RNAi induction, memory after spaced training was normal (n = 12, F<sub>2,33</sub> = 1.20, P = 0.32). (b) Memories at 24 h were assessed with a second RNAi against *AQP*. *AQP* KD in adult α/β MB neurons impaired memory after spaced training (n = 12 (first group), n = 14 (second and third groups), F<sub>2,37</sub> = 21.55, P = 0.0000006), but did not affect memory after massed training (n = 10, F<sub>2,27</sub> = 0.08, P = 0.92). Without *AQP* RNAi induction, memory after spaced training was normal

(n = 12, F<sub>2,33</sub> = 1.40, P = 0.26). (c) and (d) *Bib* KD in adult α/β MB neurons did not impair memory after spaced training (c: n = 12, F<sub>2,33</sub> = 1.327, P = 0.2791; d: n = 12, F<sub>2,33</sub> = 0.0081, P = 0.99). (e) and (f) *Drip* KD in adult α/β MB neurons did not impair memory after spaced training (e: n = 12, F<sub>2,33</sub> = 0.1146, P = 0.8921; f: n = 12, F<sub>2,33</sub> = 0.2708, P = 0.7644). (g) and (h) *Prip* KD in adult α/β MB neurons did not impair memory after spaced training (g: n = 12, F<sub>2,33</sub> = 0.2466, P = 0.7829; h: n = 12, F<sub>2,33</sub> = 0.3601, P = 0.7003). Data are presented as mean ± SEM. Sample sizes for each genotype are listed in the legend in the order of bar appearance. Significance level of the least significant Tukey pairwise comparison following one-way analysis of variance (ANOVA): ****P < 0.0001; ns: not significant, P > 0.05.
a **Prx2 KD in MB neurons**

<figure><img src="image_57.png" /></figure>

□ tub-GAL80<sup>ts</sup>, c739-GAL4/+

□ tub-GAL80<sup>ts</sup>, c739-GAL4>UAS-Prx2<sup>RNAi</sup> HMS00935

1x (MTM)

<figure><img src="image_58.png" /></figure>

5x spaced (LTM)
no induction

<figure><img src="image_59.png" /></figure>

b **Prx2, Trx2 and Trxr1 KD in MB neurons**

<figure><img src="image_60.png" /></figure>

□ +/UAS-Prx2<sup>RNAi</sup> HMS00935

□ tub-GAL80<sup>ts</sup>, c739-GAL4>UAS-Prx2<sup>RNAi</sup> HMS00935

□ +/UAS-Trx2<sup>RNAi</sup> HMS00989

□ tub-GAL80<sup>ts</sup>, c739-GAL4>UAS-Trx2<sup>RNAi</sup> HMS00989

□ +/UAS-Trxr1<sup>RNAi</sup> HMS00784

□ tub-GAL80<sup>ts</sup>, c739-GAL4>UAS-Trxr1<sup>RNAi</sup> HMS00784

c **Prx2 KD in MB neurons**

<figure><img src="image_61.png" /></figure>

□ +/UAS-Prx2<sup>RNAi</sup> VSH330046

□ tub-GAL80<sup>ts</sup>, c739-GAL4>UAS-Prx2<sup>RNAi</sup> VSH330046

5x spaced (LTM)

<figure><img src="image_62.png" /></figure>

5x spaced (LTM)
no induction

<figure><img src="image_63.png" /></figure>

d **Trx2 KD in MB neurons**

<figure><img src="image_64.png" /></figure>

□ +/UAS-Trx2<sup>RNAi</sup> HMS00989

□ tub-GAL80<sup>ts</sup>, c739-GAL4>UAS-Trx2<sup>RNAi</sup> HMS00989

1x (MTM)

<figure><img src="image_65.png" /></figure>

5x spaced (LTM)
no induction

<figure><img src="image_66.png" /></figure>

e **Trx2 KD in MB neurons**

<figure><img src="image_67.png" /></figure>

□ +/UAS-Trx2<sup>RNAi</sup> HMS00603

□ tub-GAL80<sup>ts</sup>, c739-GAL4>UAS-Trx2<sup>RNAi</sup> HMS00603

5x spaced (LTM)

<figure><img src="image_68.png" /></figure>

5x spaced (LTM)
no induction

<figure><img src="image_69.png" /></figure>

**Extended Data Fig. 5** | See next page for caption.
**Extended Data Fig. 5 | Controls for behavioural experiments, related to Fig. 4.**

(a) Memory after massed conditioning was not affected by *Prx2* KD in adult α/β MB neurons (n = 8 (first group), n = 14 (second and third groups), F<sub>2,33</sub> = 0.13, P = 0.88). Memory at 2 h after 1x conditioning was not affected by *Prx2* KD in adult α/β MB (n = 12, F<sub>2,51</sub> = 2.288, P = 0.1118). Without *Prx2* RNAi induction, memory after spaced training was normal (n = 12, F<sub>2,33</sub> = 0.64, P = 0.53). (b) Immediate memory after spaced conditioning was not affected by *Prx2*, *Trx2* or *Trxr1* KD in adult α/β MB (n = 12, F<sub>6,77</sub> = 1.958, P = 0.0821). (c) Memories at 24 h were assessed with a second RNAi against *Prx2*. *Prx2* KD in adult α/β MB neurons impaired memory after spaced training (n = 12, F<sub>2,33</sub> = 16.48, P = 0.00001), but did not affect memory after massed training (n = 10, F<sub>2,27</sub> = 0.33, P = 0.73). Without *Prx2* RNAi induction, memory after spaced training was normal (n = 10, F<sub>2,27</sub> = 1.09, P = 0.35). (d) Memory after massed conditioning was not affected by *Trx2* KD in adult α/β

MB neurons (n = 8 (first group), n = 13 (second and third groups), F<sub>2,31</sub> = 0.99, P = 0.38). Memory at 2 h after 1x conditioning was not affected by *Trx2* KD in adult α/β MB (n = 18, F<sub>2,51</sub> = 0.9106, P = 0.4087). Without *Trx2* RNAi induction, memory after spaced training was normal (n = 10, F<sub>2,27</sub> = 0.23, P = 0.80). (e) Memories at 24 h were assessed with a second RNAi against *Trx2*. *Trx2* KD in adult α/β MB neurons impaired memory after spaced training (n = 12 (first group), n = 14 (second and third groups), F<sub>2,37</sub> = 14.43, P = 0.00002), but did not affect memory after massed training (n = 10, F<sub>2,27</sub> = 0.54, P = 0.59). Without *Trx2* RNAi induction, memory after spaced training was normal (n = 12, F<sub>2,33</sub> = 0.47, P = 0.63). Data are presented as mean ± SEM. Sample sizes for each genotype are listed in the legend in the order of bar appearance. Significance level of the least significant Tukey pairwise comparison following one-way analysis of variance (ANOVA): ***P < 0.001; ns: not significant, P > 0.05.
a

Trxr1 KD in MB neurons

<figure><img src="image_70.png" /></figure>

5x spaced (LTM)
no induction

<figure><img src="image_71.png" /></figure>

b

Trxr1 KD in MB neurons

<figure><img src="image_72.png" /></figure>

**Extended Data Fig. 6** | Complementary controls for behavioural experiments, related to Fig. 4. (a) Memory after massed conditioning was not affected by *Trxr1* KD in adult α/β MB neurons (n = 8 (first group), n = 13 (second and third groups), F<sub>2,31</sub> = 0.81, P = 0.45). Memory at 2 h after 1x conditioning was not affected by *Trxr1* KD in adult α/β MB (n = 18, F<sub>2,51</sub> = 2.075, P = 0.1361). Without *Trxr1* RNAi induction, memory after spaced training was normal (n = 10, F<sub>2,27</sub> = 0.05, P = 0.95). (b) Memories at 24 h were assessed with a second RNAi against *Trxr1*. *Trxr1* KD in adult α/β MB neurons impaired memory after spaced

training (n = 10, F<sub>2,27</sub> = 20.30, P = 0.000004), but did not affect memory after massed training (n = 10, F<sub>2,27</sub> = 0.09, P = 0.92). Without *Trxr1* RNAi induction, memory after spaced training was normal (n = 10, F<sub>2,27</sub> = 1.07, P = 0.36). Data are presented as mean ± SEM. Sample sizes for each genotype are listed in the legend in the order of bar appearance. Significance level of the least significant Tukey pairwise comparison following one-way analysis of variance (ANOVA): ****P < 0.0001; ns: not significant, P > 0.05.
a

Appl detection in adult head
Appl-HA/+

<figure><img src="image_73.png" /></figure>

b

Appl KD in MB neurons

5x massed (ARM)

<figure><img src="image_74.png" /></figure>

5x spaced (LTM)
no induction

<figure><img src="image_75.png" /></figure>

c

Appl KD in MB neurons

5x spaced (LTM)

<figure><img src="image_76.png" /></figure>

5x massed (ARM)

<figure><img src="image_77.png" /></figure>

5x spaced (LTM)
no induction

<figure><img src="image_78.png" /></figure>

d

Appl - Sod3 RNAi in both
MB neurons and astrocytes

5x massed (ARM)
brief induction

<figure><img src="image_79.png" /></figure>

tub-GAL80<sup>Is</sup>; alrm-GAL4, VT30559-GAL4/+

tub-GAL80<sup>Is</sup>; alrm-GAL4, VT30559-GAL4>UAS-Sod3RNAi GD4801

tub-GAL80<sup>Is</sup>; alrm-GAL4, VT30559-GAL4>UAS-AppI<sup>RNAi</sup> JF02878

tub-GAL80<sup>Is</sup>; alrm-GAL4, VT30559-GAL4>
UAS-Sod3RNAi GD4801; UAS-AppI<sup>RNAi</sup> JF02878

5x spaced (LTM)
no induction

<figure><img src="image_80.png" /></figure>

e

Appl - Sod3 RNAi in MB neurons

5x spaced (LTM)
brief induction

<figure><img src="image_81.png" /></figure>

tub-GAL80<sup>Is</sup>; VT30559-GAL4/+

+/UAS-Sod3RNAi GD4801;
UAS-AppI<sup>RNAi</sup> JF02878

tub-GAL80<sup>Is</sup>; VT30559-GAL4>
UAS-Sod3RNAi GD4801;
UAS-AppI<sup>RNAi</sup> JF02878

f

Appl - Sod3 RNAi in astrocytes

5x spaced (LTM)
brief induction

<figure><img src="image_82.png" /></figure>

tub-GAL80<sup>Is</sup>; alrm-GAL4/+

+/UAS-Sod3RNAi GD4801;
UAS-AppI<sup>RNAi</sup> JF02878

tub-GAL80<sup>Is</sup>; alrm-GAL4>
UAS-Sod3RNAi GD4801;
UAS-AppI<sup>RNAi</sup> JF02878

g

Appl - Sod3 RNAi in MB neurons and astrocytes

brief RNAi induction

before conditioning

<figure><img src="image_83.png" /></figure>

***

after conditioning

<figure><img src="image_84.png" /></figure>

tub-GAL80<sup>Is</sup>; alrm-GAL4, VT30559-GAL4/+

+/UAS-Sod3RNAi GD4801; UAS-AppI<sup>RNAi</sup> JF02878

tub-GAL80<sup>Is</sup>; alrm-GAL4, VT30559-GAL4>
UAS-Sod3RNAi GD4801; UAS-AppI<sup>RNAi</sup> JF02878

Extended Data Fig. 7 | See next page for caption.
**Extended Data Fig. 7** | APPL-HA western analysis and additional behavioural experiments, related to Fig. 5. (a) Western blot against HA-tagged Appl.

The expected size bands are observed at 99 kDa (label 1) and 15 kDa (label 2), corresponding to full-length APPL and the CTF-α fragment, respectively. CTF-α is generated by α-secretase cleavage of full-length Appl. Tubulin was used as a loading control. Twenty replicates of this Western analysis were performed. (b) Memory after massed conditioning was not affected by *Appl* KD in adult α/β MB neurons (n = 12 (first group), n = 13 (second and third groups), F<sub>2,35</sub> = 0.84, P = 0.44). Without *Appl* RNAi induction, memory after spaced training was normal (n = 10, F<sub>2,27</sub> = 1.19, P = 0.32). (c) Memories at 24 h were assessed with a second RNAi against *Appl*. *Appl* KD in adult α/β MB neurons impaired memory after spaced training (n = 12 (first group), n = 14 (second and third groups), F<sub>2,37</sub> = 11.84, P = 0.0001), but did not affect memory after massed training (n = 10, F<sub>2,27</sub> = 0.36, P = 0.70). Without *Appl* RNAi induction, memory after spaced training was normal (n = 12, F<sub>2,33</sub> = 0.52, P = 0.60). (d) Mild decreases in both *Appl* and

*Sod3* expression in adult MB neurons and astrocytes did not affect memory after massed training (n = 10, F<sub>4,45</sub> = 0.83, P = 0.515). Without *Appl* and *Sod3* RNAi induction, memory after spaced training was normal (n = 10, F<sub>2,27</sub> = 0.72, P = 0.50). (e) Mild decreases in both *Appl* and *Sod3* expression in adult MB neurons alone did not affect memory after spaced training (n = 12, F<sub>2,33</sub> = 1.18, P = 0.32). (f) Mild decreases in both *Appl* and *Sod3* expression in adult astrocytes alone did not affect memory after spaced training (n = 12, F<sub>2,33</sub> = 0.70, P = 0.50). (g) Brief 1 day induction of both *Appl* and *Sod3* RNAi in astrocytes and MB neurons before spaced conditioning impaired 24 h memory (n = 18, F<sub>2,51</sub> = 11.88, P = 0.00006), whereas brief 1 day induction of both *Appl* and *Sod3* RNAi after spaced conditioning did not impair 24 h memory (n = 18, F<sub>2,51</sub> = 0.8017, P = 0.4541). Data are presented as mean ± SEM. Sample sizes for each genotype are listed in the legend in the order of bar appearance. Significance level of the least significant Tukey pairwise comparison following one-way analysis of variance (ANOVA): **P < 0.01; ***P < 0.001; ns: not significant, P > 0.05.
a

Appl expression after conditioning
Appl-HA; tub-GAL80<sup>ts</sup>; VT30559-GAL4>UAS-GFP

<figure><img src="image_85.png" /></figure>

**Extended Data Fig. 8** | Controls for immunochemical and behavioural experiments, related to Fig. 6. (a) No change of Appl expression in MB α and β lobes after massed training (α lobes: n = 16, t<sub>30</sub> = 0.5812, P = 0.5655; β lobes: n = 16, t<sub>30</sub> = 0.3759, P = 0.7096). (b) Memory after massed conditioning was not affected in Appl<sup>H(535)R;H(539)R</sup> mutant (n = 20, t<sub>38</sub> = 1.244, P = 0.2209). Memory after

b

Appl copper-binding mutant H(535)R,H(539)R

<figure><img src="image_86.png" /></figure>

<figure><img src="image_87.png" /></figure>

<figure><img src="image_88.png" /></figure>

1x conditioning was not affected in Appl<sup>H(535)R;H(539)R</sup> mutant (n = 12 (first group), n = 14 (second group), t<sub>24</sub> = 1.191, P = 0.2452). Data are presented as mean ± SEM. Sample sizes for each genotype are listed in the legend in the order of bar appearance. Significance level of a two-sided t-test: ns: not significant, P > 0.05.
a

Aβ42 secreted by MB neurons

<figure><img src="image_89.png" /></figure>

**Extended Data Fig. 9** | **Controls for behavioural experiments, related to Fig. 7.** (a) Memory after massed conditioning was not affected by secreted human Aβ42 secretion by adult MB neurons (n = 12, F<sub>2,33</sub> = 1.31, P = 0.28). Without Aβ42 induction, memory after spaced training was normal (n = 12, F<sub>2,33</sub> = 0.77,

P = 0.47). Data are presented as mean ± SEM. Sample sizes for each genotype are listed in the legend in the order of bar appearance. Significance level of the least significant Tukey pairwise comparison following one-way analysis of variance (ANOVA): ns: not significant, P > 0.05.
<figure><img src="image_90.png" /></figure>

# Reporting Summary

Nature Portfolio wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Portfolio policies, see our [Editorial Policies](#) and the [Editorial Policy Checklist](#).

## Statistics

For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.

n/a | Confirmed

☐ ☑ The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement

☐ ☑ A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly

☐ ☑ The statistical test(s) used AND whether they are one- or two-sided
*Only common tests should be described solely by name; describe more complex techniques in the Methods section.*

☑ ☐ A description of all covariates tested

☐ ☑ A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons

☐ ☑ A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals)

☐ ☑ For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and *P* value noted
*Give *P* values as exact values whenever suitable.*

☑ ☐ For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings

☑ ☐ For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes

☑ ☐ Estimates of effect sizes (e.g. Cohen's *d*, Pearson's *r*), indicating how they were calculated

*Our web collection on statistics for biologists contains articles on many of the points above.*

## Software and code

Policy information about [availability of computer code](#)

**Data collection** In vivo imaging data were obtained on two distinct commercial Leica microscopes and acquired through the Leica proprietary softwares.

**Data analysis**
- Statistical analysis was done using GraphPad Prism 8 (GraphPad Software).
- H2O2 and calcium in vivo imaging data were analyzed manually (ROI tracing and intensity calculation) using LAS-X (3.5.7.23225) and MATLAB (9.4.0.813654) softwares.
- Figures were designed on Adobe Illustrator 2022.

For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Portfolio [guidelines for submitting code & software](#) for further information.

## Data

Policy information about [availability of data](#)

All manuscripts must include a [data availability statement](#). This statement should provide the following information, where applicable:

- Accession codes, unique identifiers, or web links for publicly available datasets

- A description of any restrictions on data availability

- For clinical datasets or third party data, please ensure that the statement adheres to our [policy](#)

No datasets that require mandatory deposition into a public database were generated during the current study. Source data that are reported as graphs on figures
and extended data figures are available as Supplementary Information alongside the paper. Additional raw data will be shared with no restriction by the corresponding authors upon request.

This study made use of the SCOPE database of single-cell transcriptomics in the fly brain to compare Sod3 expression in neurons and glia (Extended Data Fig. 2c) (https://scope.aertslab.org/#/Davie_et_al_Cell_2018/Davie_et_al_Cell_2018%2FAerts_Fly_AdultBrain Filtering_57k.loom/gene).

## Research involving human participants, their data, or biological material

Policy information about studies with human participants or human data. See also policy information about sex, gender (identity/presentation), and sexual orientation and race, ethnicity and racism.

Reporting on sex and gender not applicable

Reporting on race, ethnicity, or other socially relevant groupings not applicable

Population characteristics not applicable

Recruitment not applicable

Ethics oversight not applicable

Note that full information on the approval of the study protocol must also be provided in the manuscript.

## Field-specific reporting

Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

<figure><img src="image_91.png" /></figure>

Life sciences

<figure><img src="image_92.png" /></figure>

Behavioural & social sciences

<figure><img src="image_93.png" /></figure>

Ecological, evolutionary & environmental sciences

For a reference copy of the document with all sections, see [nature.com/documents/nr-reporting-summary-flat.pdf](https://nature.com/documents/nr-reporting-summary-flat.pdf)

## Life sciences study design

All studies must disclose on these points even when the disclosure is negative.

Sample size

Sample size choice was based on previous studies from our group such as Plaçais et al. Nat. Com. 2017, de Tredern et al. Cell Rep., 2021, and Rabah et al. Nat. Metab. 2023 for behavior and live imaging experiments.

Data exclusions

Memory assays where fewer than 6 flies were recovered after the behavioral test were excluded.

Replication

Each behavioral assay was replicated in at least 2 independent experiments. Imaging experiments were replicated in at least 4 independent experiments. All replications were successful.

All quantification were performed unblinded. Statistical parameters including the definitions and exact value of n (e.g. number of group of flies (behavior experiments), number of brains (in vivo live-imaging), deviations and p values are reported in the figures and corresponding figure legends. Statistical analysis was carried out using Prims8 (Graph Pad Software). Comparisons between two groups were performed by unpaired two-sided Student's t-test, with results given as the value tx of the t distribution, where x is the number of degrees of freedom. Comparisons among three groups were performed by one-way analysis of variance (ANOVA) with post hoc testing by the Newman-Keuls pairwise comparisons test between the experimental group and its controls (significance: p<0.05). ANOVA results are given as the value of the Fisher distribution F(x,y), where x is the number of degrees of freedom numerator and y is the total number of degrees of freedom denominator. Data are expressed as the mean ± s.e.m. with dots as individual values corresponding to a group of 40-50 flies analyzed together in a behavioral assay, to the response of a single recorded fly for calcium or H2O2 imaging.

Randomization

Flies were assigned to experimental groups based on genotypes.

Blinding

Data collection and analysis was not performed blind. Blinding is not applicable because the investigator who set up the experiment is the same person doing the analyses. However, each experiment was associated with proper controls, and sample were collected and analyzed under identical conditions.

## Reporting for specific materials, systems and methods

We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.
## Materials & experimental systems

<table><thead><tr><th>n/a</th><th>Involved in the study</th></tr></thead><tbody><tr><td>☐</td><td>Antibodies</td></tr><tr><td>☑</td><td>Eukaryotic cell lines</td></tr><tr><td>☑</td><td>Palaeontology and archaeology</td></tr><tr><td>☐</td><td>Animals and other organisms</td></tr><tr><td>☑</td><td>Clinical data</td></tr><tr><td>☑</td><td>Dual use research of concern</td></tr><tr><td>☑</td><td>Plants</td></tr></tbody></table>

## Methods

<table><thead><tr><th>n/a</th><th>Involved in the study</th></tr></thead><tbody><tr><td>☑</td><td>ChIP-seq</td></tr><tr><td>☑</td><td>Flow cytometry</td></tr><tr><td>☑</td><td>MRI-based neuroimaging</td></tr></tbody></table>

## Antibodies

### Antibodies used

Primary antibodies:

- rat anti-HA, Roche, # ROAHAHA 11867423001, clone 3F10.

- mouse anti-HA, Biolegend # 901513, clone 16B12.

- mouse anti-tubulin, Sigma Aldrich # T16199, clone DM1A.

- mouse anti-GFP Developmental Studies Hybridoma Bank, # DSHB-GFP-12A6, clone 12A6

Secondary antibodies:

- goat anti-rat Alexa 488 Invitrogen # A11029

- HRP anti-mouse Promega # W4021

- goat anti-rat Alexa 594 Invitrogen # A1107

### Validation

- rat anti-HA: doi:10.1242/bio.060335

- mouse anti-HA: doi.org/10.1038/s41467-021-20898-x

- mouse anti-alpha tubulin: doi: 10.1111/acel.13137

- mouse anti GFP: doi: 10.3390/biology10080729

## Animals and other research organisms

Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research, and Sex and Gender in Research

### Laboratory animals

Drosophila melanogaster flies were raised on standard food medium containing yeast, cornmeal and agar, on a 12h:12h light-dark cycle at 18°C with 60% humidity. The Canton-Special (CS) strain was used as the wild-type strain. All lines were out-crossed for at least three generations to flies carrying a CS wild-type background. The study was performed on 0-3-day-old adult flies. For behavior experiments, both male and female flies were used. For imaging experiments, female flies were used.

List of Drosophila strains:

tub-GAL80ts; alrm-GAL4 (de Tredern et al. 2021)

tub-GAL80ts; c739-GAL4 (Turrel et al. 2018)

tub-GAL80ts, 13F02-LexA; VT30559-GAL4 (de Tredern et al. 2021)

tub-GAL80ts, 13F02-LexA; alrm-GAL4 (This study)

tub-GAL80ts; alrm-GAL4, VT30559-GAL4 (This study)

86E01-LexA (Bloomington Drosophila Stock Center (BDSC) 54287)

tub-GAL80ts, 86E01-LexA; VT30559-GAL4 (This study)

UAS-NoxRNAi HMS00429 (BDSC 32433)

UAS-NoxRNAi KK111991 (Vienna Drosophila Resource Center (VDRC) 102559)

UAS-G6PDRNAi KK108898 (VDRC 101507)

UAS-PgdRNAi HMC05959 (BDSC 65078)

UAS-PglsRNAi HMS02626 (BDSC 42933)

UAS-nAChRa7RNAi JF02570 (BDSC 27251)

UAS-nAChRa7RNAi KK108471 (VDRC 100756)

UAS-Sod3RNAi GD4801 (VDRC 37793)

UAS-Sod3RNAi GD3774 (VDRC 8760)

UAS-Glut1RNAi KK108683 (VDRC 101365)

UAS-AQPRNAi HMC03266 (BDSC 51504)

UAS-AQPRNAi KK102737 (VDRC 109314)

UAS-Prx2RNAi HMS00935 (BDSC 34971)

UAS-Prx2RNAi VSH330046 (VDRC 330046)

UAS-Trx2RNAi HMS00989 (BDSC 34019)

UAS-Trx2RNAi HMS00603 (BDSC 33721)

UAS-Trxr1RNAi HMS00784 (BDSC 32984)

UAS-Trxr1RNAi GD9738 (VDRC 47308)

UAS-App1RNAi JF02878 (BDSC 28043)

UAS-App1RNAi KK102543 (VDRC 108312)

UAS-Aß42 (Finelli et al. 2004)

UAS-GCaMP6f (BDSC 42747)

LexAop-GCaMP6f (BDSC 44277)

LexAop-GCaMP6f; UAS-Aß42 (This study)
UAS-GCaMP6f; UAS-nAChRa7RNAi JF02570 (This study)

LexAop-roGFP-Tsa2DCR (This study)

LexAop-roGFP-Tsa2DCR; UAS-Glut1RNAi KK108683 (This study)

LexAop-roGFP-Tsa2DCR; UAS-NoxRNAi HMS00429 (This study)

LexAop-roGFP-Tsa2DCR; UAS-G6PDRNAi KK108898 (This study)

LexAop-roGFP-Tsa2DCR; UAS-nAChRa7RNAi JF02570 (This study)

LexAop-roGFP-Tsa2DCR; UAS-Sod3RNAi GD4801 (This study)

LexAop-roGFP-Tsa2DCR; UAS-AQPRNAi HMC03266 (This study)

LexAop-roGFP-Tsa2DCR; UAS-ApplRNAi JF02878 (This study)

LexAop-roGFP-Tsa2DCR; UAS-A&42 (This study)

APPL-HA (This study)

APPL-HA; tub-GAL80ts; VT30559-GAL4 (this study)

y ApplH(535)R,H(539)R (this study)

PBac{y[+]-attP-3B}VK00033 (BDSC 9750)

UAS-human-secreted-catalase (Dhawan et al. 2021)

UAS-BibRNAi GD1049 (VDRC 8893)

UAS-BibRNAi JF02771 (BDSC 27691)

UAS-DripRNAi GD1936 (VDRC 51936)

UAS-DripRNAi HMC0945 (BDSC 44661)

UAS-PripRNAi GD3499 (VDRC 8124)

UAS-PripRNAi HMC03097 (BDSC 50695)

Wild animals

The study did not involve wild animal

Reporting on sex

For behavior experiments, mixed populations of males and females were used. For imaging experiments, females were used.

Field-collected samples

The study did not involve samples collected from the field

Ethics oversight

No ethical approval or guidance was required since in this study we used Drosophila melanogaster.

Note that full information on the approval of the study protocol must also be provided in the manuscript.