Autophagy within the mushroom body protects from synapse aging in a non-cell autonomous manner

doi:10.1038/s41467-019-09262-2

. 2019 Mar 21;10(1):1318.

doi: 10.1038/s41467-019-09262-2.

Autophagy within the mushroom body protects from synapse aging in a non-cell autonomous manner

Anuradha Bhukel^{1

2}, Christine Brigitte Beuschel^{1

2}, Marta Maglione^{1

2}, Martin Lehmann³, Gabor Juhász⁴, Frank Madeo^{5

6}, Stephan J Sigrist^{7

8}

Affiliations

¹ Institute for Biology/Genetics, Freie Universität Berlin, Takustr. 6, 14195, Berlin, Germany.
² NeuroCure, Charité, Charitéplatz 1, 11007, Berlin, Germany.
³ Leibniz Forschungsinstitut Für Molecular Pharmakologie, Campus Berlin-Buch, Robert-Roessle-Str. 10, 13125, Berlin, Germany.
⁴ Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Pázmány, s. 1/C. 6.520, Budapest, H-1117, Hungary.
⁵ Institute for Molecular Biosciences, NAWI Graz, University of Graz, Humboldtstrasse 50/EG, 8010, Graz, Austria.
⁶ BioTechMed Graz, 8010, Graz, Austria.
⁷ Institute for Biology/Genetics, Freie Universität Berlin, Takustr. 6, 14195, Berlin, Germany. stephan.sigrist@fu-berlin.de.
⁸ NeuroCure, Charité, Charitéplatz 1, 11007, Berlin, Germany. stephan.sigrist@fu-berlin.de.

PMID: 30899013
PMCID: PMC6428838
DOI: 10.1038/s41467-019-09262-2

Autophagy within the mushroom body protects from synapse aging in a non-cell autonomous manner

Anuradha Bhukel et al. Nat Commun. 2019.

. 2019 Mar 21;10(1):1318.

doi: 10.1038/s41467-019-09262-2.

Authors

Anuradha Bhukel^{1

2}, Christine Brigitte Beuschel^{1

2}, Marta Maglione^{1

2}, Martin Lehmann³, Gabor Juhász⁴, Frank Madeo^{5

6}, Stephan J Sigrist^{7

8}

Affiliations

¹ Institute for Biology/Genetics, Freie Universität Berlin, Takustr. 6, 14195, Berlin, Germany.
² NeuroCure, Charité, Charitéplatz 1, 11007, Berlin, Germany.
³ Leibniz Forschungsinstitut Für Molecular Pharmakologie, Campus Berlin-Buch, Robert-Roessle-Str. 10, 13125, Berlin, Germany.
⁴ Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Pázmány, s. 1/C. 6.520, Budapest, H-1117, Hungary.
⁵ Institute for Molecular Biosciences, NAWI Graz, University of Graz, Humboldtstrasse 50/EG, 8010, Graz, Austria.
⁶ BioTechMed Graz, 8010, Graz, Austria.
⁷ Institute for Biology/Genetics, Freie Universität Berlin, Takustr. 6, 14195, Berlin, Germany. stephan.sigrist@fu-berlin.de.
⁸ NeuroCure, Charité, Charitéplatz 1, 11007, Berlin, Germany. stephan.sigrist@fu-berlin.de.

PMID: 30899013
PMCID: PMC6428838
DOI: 10.1038/s41467-019-09262-2

Abstract

Macroautophagy is an evolutionarily conserved cellular maintenance program, meant to protect the brain from premature aging and neurodegeneration. How neuronal autophagy, usually loosing efficacy with age, intersects with neuronal processes mediating brain maintenance remains to be explored. Here, we show that impairing autophagy in the Drosophila learning center (mushroom body, MB) but not in other brain regions triggered changes normally restricted to aged brains: impaired associative olfactory memory as well as a brain-wide ultrastructural increase of presynaptic active zones (metaplasticity), a state non-compatible with memory formation. Mechanistically, decreasing autophagy within the MBs reduced expression of an NPY-family neuropeptide, and interfering with autocrine NPY signaling of the MBs provoked similar brain-wide metaplastic changes. Our results in an exemplary fashion show that autophagy-regulated signaling emanating from a higher brain integration center can execute high-level control over other brain regions to steer life-strategy decisions such as whether or not to form memories.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Fig. 1**
Neuronal knockdown of atg9 or atg5 suppresses autophagy in the brain. Adult brain, 10 days (a) +/*atg9*-RNAi, (b) *elav*/*atg9*-RNAi, (d) +/*atg5*-RNAi, and (e) *elav*/*atg5*-RNAi immunostained for p62/Ref(2)p. Scale bar: 50 μm. c Quantification of p62/Ref(2)p intensity within the central brain region normalized to control (n = 15–17 independent brains; ***p < 0.001; Mann–Whitney U-test). f Quantification of p62/Ref(2)p intensity within the central brain region normalized to control (n = 23–24 independent brains; ***p < 0.001; Mann–Whitney U-test). g Selective autophagy marker p62/Ref(2)p and autophagosome-associated protein Atg8a accumulate in protein homogenate prepared from the brain of *elav*/*atg9*-RNA and *elav*/*atg5*-RNAi compared to respective controls. An amount equivalent to 1 brain was loaded onto the gel. Lipidated Atg8a (Atg8a-II) is missing upon KD of *atg5* but not when *atg9* is knocked down. In the box and whisker plots, the middle line of a box represents the median (50th percentile) and the terminal lines of a box represent the 25th and 75th percentile. The whiskers represent the lowest and the highest value

**Fig. 2**
Neuronal inhibition of autophagy impairs age-sensitive olfactory memory. a Aversive associative memory performance 3 min after training (short-term memory, STM) markedly reduced in *elav*/*atg5*-RNAi compared to +/*atg5*-RNAi (n = 17–18; ***p < 0.001; Mann–Whitney U-test). b STM significantly reduced in *elav*/*atg9*-RNAi compared to +/*atg9*-RNAi (n = 6–8; *p < 0.05; Mann–Whitney U-test). c Aversive associative memory performance at 1 h after training (mid-term memory; MTM) of *elav*/*atg5*-RNAi declined significantly compared to +/*atg5*-RNAi (n = 11–16; **p < 0.01; Mann–Whitney U-test). d MTM significantly reduced in *elav*/*atg9*-RNAi flies compared to +/*atg9*-RNAi (n = 8–12; *p < 0.05; Mann–Whitney U-test). e STM significantly reduced in *ok107*/*atg5*-RNAi flies compared to +/*atg5*-RNAi (n = 17–20; ***p < 0.001; Mann–Whitney U-test). f STM significantly reduced in *vt30559*/*atg5*-RNAi flies compared to +/*atg5*-RNAi (n = 9; ***p < 0.0001; Mann–Whitney U-test). g STM significantly reduced in *ok107*/*atg9*-RNAi flies compared to +/*atg9*-RNAi (n = 12–14; **p < 0.01; Mann–Whitney U-test). h MTM significantly reduced in *ok107*/*atg9*-RNAi flies compared to +/*atg9*-RNAi (n = 10–12; *p < 0.05; Mann–Whitney U-test). i Aversive associative memory performance at 1 h after training, anesthesia-resistant memory (ARM) and j anesthesia-sensitive memory (ASM) of *elav*/*atg9*-RNAi compared to +/*atg9*-RNAi (n = 7–10; *p < 0.05; ^nsp > 0.5; Mann–Whitney U-test). k ARM and l ASM of *ok107*/*atg9*-RNAi compared to +/*atg9*-RNAi (n = 15–17; **p < 0.01; ^nsp > 0.5; Mann–Whitney U-test). In the box and whisker plots, the middle line of a box represents the median (50th percentile) and the terminal lines of a box represent the 25th and 75th percentile. The whiskers represent the lowest and the highest value

**Fig. 3**
Mushroom body-specific suppression of autophagy induces brain-wide increase of Bruchpilot. Adult brain, 10 days (a) +/*atg5*-RNAi and (b) *elav*/*atg5*-RNAi immunostained for BRP^Nc82. Scale bar: 50 μm. c Quantification of BRP^Nc82 intensity within the central brain region normalized to control (n = 11–16 independent brains; ***p < 0.001; Mann–Whitney U-test). Adult brain, 10 days (d) +/*atg5*-RNAi and (e) *ok107*/*atg5*-RNAi immunostained for BRP^Nc82. Scale bar: 50 μm. f Quantification of BRP^Nc82 intensity within the central brain region normalized to control (n = 10–16 independent brains; ***p < 0.001; Mann–Whitney U-test). Adult brain, 10 days (g) +/*atg9*-RNAi and (h) *elav*/*atg9*-RNAi immunostained for BRP^Nc82. Scale bar: 50 μm. i Quantification of BRP^Nc82 intensity within the central brain region normalized to control (n = 14–15 independent brains; ***p < 0.001; Mann–Whitney U-test). Adult brain, 10 days (j) +/*atg9*-RNAi and (k) *ok107*/*atg9*-RNAi immunostained for BRP^Nc82. Scale bar: 50 μm. l Quantification of BRP^Nc82 intensity within the central brain region normalized to control (n = 9–12 independent brains; ***p < 0.001; Mann–Whitney U-test). m MTM of +/*atg5*-RNAi, +/*ok107*;*mb247*-Gal80 and *ok107*;*mb247*-Gal80/*atg5*-RNAi (n = 10–15; ^nsp > 0.5; One-way ANOVA; Kruskal–Walis post-test). Adult brain, 10 days (n) +/*atg5*-RNAi, (o) +/*ok107*;*mb247*-Gal80, and (p) *ok107*; *mb247*-Gal80/*atg5*-RNAi immunostained for BRP^Nc82. Scale bar: 50 μm. q Quantification of BRP^Nc82 intensity within the central brain region normalized to +/*atg5*-RNAi (n = 5–6 independent brains; ^nsp > 0.5; One-way ANOVA; Kruskal-Walis test). r STM of +/*atg9*-RNAi and *dilp2*/*atg9*-RNAi (n = 8–9; ^nsp > 0.5; Mann–Whitney U-test). Adult brain, 10 days (s) *atg9*-RNAi/+ immunostained for p62/Ref(2)p. Scale bar: 50 μm. Adult brain, 10 days (t) +/*atg9*-RNAi and (u) *dilp2*/*atg9*-RNAi immunostained for BRP^Nc82. Scale bar: 50 μm. v Quantification of BRP^Nc82 intensity within the central brain region normalized to control (n = 11–16 independent brains; ^nsp > 0.5; Mann–Whitney U-test). ^nsp > 0.01, ***p < 0.0001. In the box and whisker plots, the middle line of a box represents the median (50th percentile) and the terminal lines of a box represent the 25th and 75th percentile. The whiskers represent the lowest and the highest value

**Fig. 4**
Autophagy suppression in Mushroom body causes age-typical ultrastructural presynaptic phenotypes. Electron micrograph of Kenyon cell bodies in (a) +/*atg5*-RNAi and (b) *ok107*/*atg5*-RNAi animals. Scale bar: 500 nm. Aggregates build up in *ok107*/*atg5*-RNAi animals (highlighted in red box). Scale bar: 100 nm. Electron micrograph of Kenyon cell bodies in (c) +/*atg9*-RNAi and (d) *ok107*/*atg9*-RNAi animals. Scale bar: 500 nm. Aggregates build up in *ok107*/*atg9*-RNAi animals (highlighted in red box). Scale bar: 100 nm. Electron micrograph showing presynaptic specializations at KC-to-MBON synapses in medial MB lobes of (e) +/*atg9*-RNAi and (f) *ok107*/*atg9*-RNAi animals (outlined as yellow). Scale bar: 100 nm. g Quantification of average T-bar size in +/*atg9*-RNAi and *ok107*/*atg9*-RNAi animals (n = 240 electron micrographs across 3–5 independent animals, with at least 20 T-bars per animals; ***p < 0.001; Mann–Whitney U-test). Electron micrograph showing presynaptic specializations at PN-to-KC synapses in Calyx of (h) +/*atg9*-RNAi and (i) *ok107*/*atg9*-RNAi animals (outlines as yellow). Scale bar: 100 nm. j Quantification of average T-bar size in +/*atg9*-RNAi and *ok107*/*atg9*-RNAi animals (n = 268 electron micrographs across four independent animals, with atleast 20 t-bars per animal; ***p < 0.001; Mann–Whitney U-test). STED images of BRP spots reveal ring-shaped structures (pointed by arrowheads) within the (k, l) Calyx and (n, o) antennal lobe of +/*atg9*-RNAi and *ok107*/*atg9*-RNAi. Scale bar: 500 nm. m Comparison of BRP ring diameter between Calyx of +/*atg9*-RNAi and Calyx of *ok107*/*atg9*-RNAi (total of 2931 BRP rings across 16 independent animals, with at least five rings per animals; *p < 0.05; Mann–Whitney U-test). p Comparison of BRP ring diameter between antennal lobe of +/*atg9*-RNAi and antennal lobe of *ok107*/*atg9*-RNAi (total of 1327 BRP rings across 15 independent animals, with at least five rings per animal; **p < 0.01; Mann–Whitney U-test). In the box and whisker plots, the middle line of a box represents the median (50th percentile) and the terminal lines of a box represent the 25th and 75th percentile. The whiskers represent the lowest and the highest value

**Fig. 5**
sNPF levels decline with mushroom body-specific attenuation of autophagy and with aging. a Adult brain immunostained for sNPF-precursor. Scale bar: 50 μm. b MB lobes of +/*atg5*-RNAi and c *vt30559*/*atg5*-RNAi immunostained for sNPF precursor (Single Z-plane). Scale bar: 10 μm. d Quantification of signal intensity of sNPF peptide precursor in the MB lobes normalized to control flies (5–6 independent animals; **p < 0.01; Mann–Whitney U-test). e Subesophageal ganglion (SOG) of +/*atg5*-RNAi and f *vt30559*/atg5-RNAi immunostained for sNPF precursor (Z-projection). Scale bar: 10 μm. g Quantification of signal intensity of sNPF-peptide precursor in SOG normalized to control flies (n = 6 independent animals; *p < 0.05; Mann–Whitney U-test). MB lobes of (h) 3-day-old w1118 and (j) 30-day-old w1118 immunostained for BRP^Nc82 (single Z-plane). Scale bar: 10 μm. MB lobes of (i) 3-day-old w1118 and (k) 30-day-old w1118 immunostained for sNPF (Single Z-plane). Scale bar: 10 μm. l Quantification of signal intensity of sNPF peptide precursor in the MB lobes normalized to control flies (9–10 independent animals; ***p < 0.001; Mann–Whitney U-test). SOG of (m) 3-day-old w1118 and (o) 30-day-old w1118 immunostained for BRP^Nc82 (Single Z-plane). Scale bar: 10 μm. SOG of (n) 3-day-old w1118 and (p) 30 days w1118 immunostained for sNPF (Z-projection). Scale bar: 10 μm. q Quantification of signal intensity of sNPF-peptide precursor in SOG normalized to control flies (9–10 independent animals; ***p < 0.01; Mann–Whitney U-test). In the box and whisker plots, the middle line of a box represents the median (50th percentile) and the terminal lines of a box represent the 25th and 75th percentile. The whiskers represent the lowest and the highest value

**Fig. 6**
sNPF signaling connects autophagy suppression with presynaptic metaplasticity. Adult brain, 5 days, (a) w1118 and (b) *sNPF*^c00448 immunostained for sNPF peptide precursor. Scale bar: 50 μm. c Quantification of sNPF peptide precursor within the central brain region normalized to control flies. (n = 9–11 independent brains; ***p < 0.001; Mann–Whitney U-test). Adult brain, 5 days, (d) w1118 and (e) *sNPF*^c00448 flies, immunostained for BRP^Nc82. Scale bar: 50 μm. f Quantification of BRP^Nc82 within the central brain region normalized to control flies (n = 7–8 independent brains; *p < 0.05; Mann–Whitney U-test). Adult brain, 5 days, (g) +/*snpfr*-RNAi and (h) *ok107*/*snpfr*-RNAi flies, immunostained for BRP^Nc82. Scale bar: 50 μm. i Quantification of BRP^Nc82 within the central brain region normalized to control flies (n = 13–15 independent brains; ***p < 0.001; Mann–Whitney U-test). Electron micrograph showing presynaptic specializations at PN-to-KC synapses in Calyx of (j) +/*snpfr*-RNAi and (k) *ok107*/*snpfr*-RNAi animals. Scale bar: 100 nm. l Quantification representing the average T-bar size in +/*snpfr*-RNAi and *ok107*/*snpfr*-RNAi animals (n = 206 electron micrographs across eight independent animals, with at least 20 T-bars per animal; ***p < 0.001; Mann–Whitney U-test). In the box and whisker plots, the middle line of a box represents the median (50th percentile) and the terminal lines of a box represent the 25th and 75th percentile. The whiskers represent the lowest and the highest value

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References

1. Menzies FM, et al. Autophagy and neurodegeneration: pathogenic mechanisms and therapeutic opportunities. Neuron. 2017;93:1015–1034. doi: 10.1016/j.neuron.2017.01.022. - DOI - PubMed
1. Yin Z, Pascual C, Klionsky DJ. Autophagy: machinery and regulation. Microb. Cell. 2016;3:588–596. doi: 10.15698/mic2016.12.546. - DOI - PMC - PubMed
1. Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Cell. 2008;132:27–42. doi: 10.1016/j.cell.2007.12.018. - DOI - PMC - PubMed
1. Yang F, et al. mTOR and autophagy in normal brain aging and caloric restriction ameliorating age-related cognition deficits. Behav. Brain Res. 2014;264:82–90. doi: 10.1016/j.bbr.2014.02.005. - DOI - PubMed
1. Dong W, et al. Autophagy involving age-related cognitive behavior and hippocampus injury is modulated by different caloric intake in mice. Int. J. Clin. Exp. Med. 2015;8:11843–11853. - PMC - PubMed

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[1] Menzies FM, et al. Autophagy and neurodegeneration: pathogenic mechanisms and therapeutic opportunities. Neuron. 2017;93:1015–1034. doi: 10.1016/j.neuron.2017.01.022. - DOI - PubMed

[2] Menzies FM, et al. Autophagy and neurodegeneration: pathogenic mechanisms and therapeutic opportunities. Neuron. 2017;93:1015–1034. doi: 10.1016/j.neuron.2017.01.022. - DOI - PubMed

[3] Yin Z, Pascual C, Klionsky DJ. Autophagy: machinery and regulation. Microb. Cell. 2016;3:588–596. doi: 10.15698/mic2016.12.546. - DOI - PMC - PubMed

[4] Yin Z, Pascual C, Klionsky DJ. Autophagy: machinery and regulation. Microb. Cell. 2016;3:588–596. doi: 10.15698/mic2016.12.546. - DOI - PMC - PubMed

[5] Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Cell. 2008;132:27–42. doi: 10.1016/j.cell.2007.12.018. - DOI - PMC - PubMed

[6] Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Cell. 2008;132:27–42. doi: 10.1016/j.cell.2007.12.018. - DOI - PMC - PubMed

[7] Yang F, et al. mTOR and autophagy in normal brain aging and caloric restriction ameliorating age-related cognition deficits. Behav. Brain Res. 2014;264:82–90. doi: 10.1016/j.bbr.2014.02.005. - DOI - PubMed

[8] Yang F, et al. mTOR and autophagy in normal brain aging and caloric restriction ameliorating age-related cognition deficits. Behav. Brain Res. 2014;264:82–90. doi: 10.1016/j.bbr.2014.02.005. - DOI - PubMed

[9] Dong W, et al. Autophagy involving age-related cognitive behavior and hippocampus injury is modulated by different caloric intake in mice. Int. J. Clin. Exp. Med. 2015;8:11843–11853. - PMC - PubMed

[10] Dong W, et al. Autophagy involving age-related cognitive behavior and hippocampus injury is modulated by different caloric intake in mice. Int. J. Clin. Exp. Med. 2015;8:11843–11853. - PMC - PubMed

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Autophagy within the mushroom body protects from synapse aging in a non-cell autonomous manner

Affiliations

Autophagy within the mushroom body protects from synapse aging in a non-cell autonomous manner

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Abstract

Conflict of interest statement

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Abstract

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