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# Autophagy in synapse formation and brain wiring

Bassem A Hassan, P. Robin Hiesinger

► To cite this version:

Bassem A Hassan, P. Robin Hiesinger. Autophagy in synapse formation and brain wiring. Autophagy, 2023, 19 (10), pp.2814-2816. 10.1080/15548627.2023.2179778. hal-04530710

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https://hal.sorbonne-universite.fr/hal-04530710

Submitted on 3 Apr 2024

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Distributed under a Creative Commons Attribution 4.0 International License
# Autophagy in synapse formation and brain wiring

Bassem A Hassan, P. Robin Hiesinger

► To cite this version:

Bassem A Hassan, P. Robin Hiesinger. Autophagy in synapse formation and brain wiring. Autophagy, 2023, 19 (10), pp.2814-2816. 10.1080/15548627.2023.2179778. hal-04530710

HAL Id: hal-04530710

https://hal.sorbonne-universite.fr/hal-04530710v1

Submitted on 3 Apr 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_3.png" /></figure>

Autophagy

ISSN: (Print) (Online) Journal homepage: www.tandfonline.com/journals/kaup20

# Autophagy in synapse formation and brain wiring

Bassem A. Hassan & P. Robin Hiesinger

To cite this article: Bassem A. Hassan & P. Robin Hiesinger (2023) Autophagy in synapse formation and brain wiring, Autophagy, 19:10, 2814-2816, DOI: 10.1080/15548627.2023.2179778

To link to this article: https://doi.org/10.1080/15548627.2023.2179778

<figure><img src="image_4.png" /></figure>

© 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

<figure><img src="image_5.png" /></figure>

Published online: 02 Mar 2023.

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Submit your article to this journal

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Article views: 1036

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Autophagy

ISSN: (Print) (Online) Journal homepage: www.tandfonline.com/journals/kaup20

# Autophagy in synapse formation and brain wiring

Bassem A. Hassan & P. Robin Hiesinger

**To cite this article:** Bassem A. Hassan & P. Robin Hiesinger (2023) Autophagy in synapse formation and brain wiring, Autophagy, 19:10, 2814-2816, DOI: 10.1080/15548627.2023.2179778

**To link to this article:** https://doi.org/10.1080/15548627.2023.2179778

<figure><img src="image_3.png" /></figure>

© 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

<figure><img src="image_4.png" /></figure>

Published online: 02 Mar 2023.

<figure><img src="image_5.png" /></figure>

Submit your article to this journal

<figure><img src="image_6.png" /></figure>

Article views: 1036

<figure><img src="image_7.png" /></figure>

View related articles

<figure><img src="image_8.png" /></figure>

View Crossmark data

CrossMark
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

# Autophagy in synapse formation and brain wiring

Bassem A. Hassanª and P. Robin Hiesingerᵇ

ªInstitut du Cerveau-Paris Brain Institute (ICM), Sorbonne University, Inserm, CNRS, Hôpital Pitié Salpêtrière, Paris, France; ᵇDivision of Neurobiology, Free University of Berlin, Berlin, Germany

## ABSTRACT

A recent characterization of the role of autophagy in two different neuron types during brain development in *Drosophila* revealed two different mechanisms to regulate synapse formation. In photoreceptor neurons, autophagosome formation in synaptogenic filopodia destabilizes presump- tive synaptic contacts and thereby restricts incorrect synaptic partnerships. In dorsal cluster neurons, autophagy is actively suppressed to keep mature synapses stable during axonal branching. These findings indicate that different neuron types can require activation or suppression of synaptic autophagy during the same developmental period to ensure proper synapse formation and brain connectivity.

## ARTICLE HISTORY

Received 25 January 2023

Revised 8 February 2023

Accepted 8 February 2023

## KEYWORDS

Active zone; brain
development; filopodia;
neuron; neuronal
autophagy; synapse;
synaptic autophagy;
synaptogenesis

The role of macroautophagy/autophagy in the brain was initi- ally characterized in mice with neuron-specific loss of the core autophagy genes *Atg5* or *Atg7*. This seminal work revealed that functional neurons develop in the absence of autophagy, but are at increased risk for adult-onset degeneration. However, subsequent studies revealed that autophagy- deficient neurons, although functional, exhibit hallmarks of neurodevelopmental defects. Loss of autophagy during mouse brain development leads to increased spine density due to defective pruning after synapse formation, a hallmark of neu- rodevelopmental autism spectrum disorders. Conversely, in worms and flies, loss of neuronal autophagy can lead to reduced synapse formation. These findings suggest develop- mental roles for autophagy at the level of synapses, yet there is currently no coherent picture about a single, shared mechan- ism by which autophagy functions during brain development and synapse formation.

Our labs have recently studied the role of neuronal auto- phagy during fly brain development in two different neuronal cell types. Loss of autophagy in photoreceptor neurons leads to a stabilization of synaptogenic filopodia, which in turn leads to increased synapse formation, similar to what has been suggested for spine stabilization in mice (Figure 1A). Importantly, for fly photoreceptor neurons it could be shown that the increased number of synapses includes synaptic part- nerships not present in wild-type brains, suggesting a role for autophagic regulation as part of the genetic program for synapse-specific brain wiring. In these axon terminals, auto- phagy specifically occurs at the tips of filopodia, leading to filopodial destabilization and thereby increased filopodial dynamics that restrict synaptic partner choice (Figure 1A). In a recent study, we have now expanded the analysis of a loss of neuronal autophagy to a second, very different neuron type: dorsal cluster neurons (DCNs) [1]. These large

interneurons project contralaterally through the entire central brain to compare the visual information between the left and right hemispheres. In contrast to the unbranched photorecep- tor axons, DCN axons undergo a complicated morphogenetic program that leads to stereotypic axonal branching patterns (Figure 1B). If autophagy can destabilize axon filopodial pro- jections during development, how does a loss of autophagy affect the development of such a morphologically complicated axonal structure?

Surprisingly, loss of autophagy has no obvious effect on the morphology or synapse numbers of DCN axonal projections; instead, normal axonal branching patterns and synapses only develop under conditions of active *suppression* of autophagy. The signaling mechanism underlying this suppression requires Egfr (Epidermal growth factor receptor) function, which had previously been shown to function in DCN axon branching during early axon development. The new study now revealed an independent second, later role for Egfr sig- naling in axonal branches during synapse formation. However, loss of Egfr signaling does not affect the initial trafficking of presynaptic proteins to synaptogenic filopodia at the time synaptogenesis commences in these branches. Instead, Egfr function is specifically required to stabilize newly formed presynaptic active zones by preventing their turnover through membrane degradation (Figure 1B).

Mature presynaptic sites are marked by the active zone scaffold protein Brp. Loss of Egfr function leads to increased colocalization of Brp with markers for endomembrane degrada- tion, including the early autophagosomal marker Atg5, the late endosomal marker Rab7, and the lysosomal marker spin (spinster). Correspondingly, a reduction of endomembrane degradation through *Rab7* knockdown as well as a reduction of autophagy through *Atg6* knockdown partially rescue the synaptic connectivity defects associated with loss of Egfr

<figure><img src="image_13.png" /></figure>

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

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

<figure><img src="image_16.png" /></figure>
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

# Autophagy in synapse formation and brain wiring

Bassem A. Hassanª and P. Robin Hiesingerᵇ

ªInstitut du Cerveau-Paris Brain Institute (ICM), Sorbonne University, Inserm, CNRS, Hôpital Pitié Salpêtrière, Paris, France; ᵇDivision of Neurobiology, Free University of Berlin, Berlin, Germany

## ABSTRACT

A recent characterization of the role of autophagy in two different neuron types during brain development in *Drosophila* revealed two different mechanisms to regulate synapse formation. In photoreceptor neurons, autophagosome formation in synaptogenic filopodia destabilizes presump- tive synaptic contacts and thereby restricts incorrect synaptic partnerships. In dorsal cluster neurons, autophagy is actively suppressed to keep mature synapses stable during axonal branching. These findings indicate that different neuron types can require activation or suppression of synaptic autophagy during the same developmental period to ensure proper synapse formation and brain connectivity.

## ARTICLE HISTORY

Received 25 January 2023
Revised 8 February 2023
Accepted 8 February 2023

## KEYWORDS

Active zone; brain
development; filopodia;
neuron; neuronal
autophagy; synapse;
synaptic autophagy;
synaptogenesis

The role of macroautophagy/autophagy in the brain was initi- ally characterized in mice with neuron-specific loss of the core autophagy genes *Atg5* or *Atg7*. This seminal work revealed that functional neurons develop in the absence of autophagy, but are at increased risk for adult-onset degeneration. However, subsequent studies revealed that autophagy- deficient neurons, although functional, exhibit hallmarks of neurodevelopmental defects. Loss of autophagy during mouse brain development leads to increased spine density due to defective pruning after synapse formation, a hallmark of neu- rodevelopmental autism spectrum disorders. Conversely, in worms and flies, loss of neuronal autophagy can lead to reduced synapse formation. These findings suggest develop- mental roles for autophagy at the level of synapses, yet there is currently no coherent picture about a single, shared mechan- ism by which autophagy functions during brain development and synapse formation.

Our labs have recently studied the role of neuronal auto- phagy during fly brain development in two different neuronal cell types. Loss of autophagy in photoreceptor neurons leads to a stabilization of synaptogenic filopodia, which in turn leads to increased synapse formation, similar to what has been suggested for spine stabilization in mice (Figure 1A). Importantly, for fly photoreceptor neurons it could be shown that the increased number of synapses includes synaptic part- nerships not present in wild-type brains, suggesting a role for autophagic regulation as part of the genetic program for synapse-specific brain wiring. In these axon terminals, auto- phagy specifically occurs at the tips of filopodia, leading to filopodial destabilization and thereby increased filopodial dynamics that restrict synaptic partner choice (Figure 1A). In a recent study, we have now expanded the analysis of a loss of neuronal autophagy to a second, very different neuron type: dorsal cluster neurons (DCNs) [1]. These large

interneurons project contralaterally through the entire central brain to compare the visual information between the left and right hemispheres. In contrast to the unbranched photorecep- tor axons, DCN axons undergo a complicated morphogenetic program that leads to stereotypic axonal branching patterns (Figure 1B). If autophagy can destabilize axon filopodial pro- jections during development, how does a loss of autophagy affect the development of such a morphologically complicated axonal structure?

Surprisingly, loss of autophagy has no obvious effect on the morphology or synapse numbers of DCN axonal projections; instead, normal axonal branching patterns and synapses only develop under conditions of active *suppression* of autophagy. The signaling mechanism underlying this suppression requires Egfr (Epidermal growth factor receptor) function, which had previously been shown to function in DCN axon branching during early axon development. The new study now revealed an independent second, later role for Egfr sig- naling in axonal branches during synapse formation. However, loss of Egfr signaling does not affect the initial trafficking of presynaptic proteins to synaptogenic filopodia at the time synaptogenesis commences in these branches. Instead, Egfr function is specifically required to stabilize newly formed presynaptic active zones by preventing their turnover through membrane degradation (Figure 1B).

Mature presynaptic sites are marked by the active zone scaffold protein Brp. Loss of Egfr function leads to increased colocalization of Brp with markers for endomembrane degrada- tion, including the early autophagosomal marker Atg5, the late endosomal marker Rab7, and the lysosomal marker spin (spinster). Correspondingly, a reduction of endomembrane degradation through *Rab7* knockdown as well as a reduction of autophagy through *Atg6* knockdown partially rescue the synaptic connectivity defects associated with loss of Egfr

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

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

<figure><img src="image_15.png" /></figure>
<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /><figcaption>**Figure 1.** Regulation of synapse formation through two different mechanisms in two neuron types in Drosophila. (A) In fly photoreceptor neurons, autophagosome formation is selectively triggered at the tips of synaptogenic filopodia to regulate synapse numbers and partnerships. (B) In fly dorsal cluster neurons, autophagy is actively suppressed to stabilize synapses and branch formation.</figcaption></figure>

function. These findings suggest autophagic degradation and turnover of active zone proteins as a default state that is actively suppressed during a critical developmental period of brain wiring in DCNs.

Presynaptic active zones form concomitantly with the elabo-
rate axonal branch morphogenesis of DCN axons. A role for
synapse formation in axonal and dendritic branching is well
known as “synaptotropic growth”. During synaptotropic growth
synapse formation stabilizes branches and provides a local basis
for new probabilistic branch formation. Correspondingly, sup-
pression of autophagy during DCN synapse formation seconda-
rily leads to branch destabilization and a deterioration of the
stereotypic axonal branching pattern (Figure 1B). The conse-
quences of increased synaptic autophagy during this critical
period of DCN development thereby include altered axonal
morphology, synaptic connectivity and visual behavior.

We conclude that autophagy can restrict synapse formation through different mechanisms in two different neuron types of the fly visual system during the development of synaptic connectivity. In the non-branched axon terminals of photoreceptor neurons autophagy is actively induced at the tips of synaptogenic filopodia to restrict synapse formation and partner choice; in the highly branched axons of DCNs autophagy is actively suppressed to allow synaptotropic growth of an axonal branching pattern. Furthermore, the autophagic cargo in photoreceptor filopodia include the presynaptic seeding factors Liprin-a and RhoGAP100F/Syd-1, but not the mature active zone marker Brp (Figure 1A); by contrast, autophagic degradation induced by loss of Egfr function targets mature, brp-containing active zones (Figure 1B). These findings support a view of autophagy as a regulatory module during brain development that can be employed in a cell type-specific manner

The observation that activation versus suppression of auto-
phagy can be required in two different neuron types in the
same brain region and during the same critical window of
brain development may also affect how we view brain-wide
autophagic up- or downregulation as a therapeutic opportu-
nity. If neighboring neurons employ opposite autophagic reg-
ulation to develop proper synaptic connectivity, then levels of
autophagy must be controlled at the level of individual neu-
rons to ensure normal development.

In summary, we have identified two very different neuron
types in the fly visual system that employ the spatial and
temporal regulation of autophagy for the fine-tuning of synaptic
connectivity through selective restriction of synapse formation,
albeit through different mechanisms. In both cases cell type-
specific modulation of presynaptic autophagy during synapse
formation represents a quantitative contribution to brain wiring
that is selectable at the level of animal behavior. Based on the
diversity of autophagy-dependent synapse development in the
few neuron types analyzed thus far, we speculate that more cell-
specific roles of autophagy will emerge as more neuron types are
investigated during brain development.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

Work in the Hassan and Hiesinger lab is supported by DFG Research
Unit 5289 RobustCircuit project P2 (to B.A.H and P.R.H), DFG Research
Unit Syntophagy RP7 (Hi 1886/8), funding from the ERC Adv
101019191 (ERC) under the European Union's Horizon 2020 research
<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /><figcaption>**Figure 1.** Regulation of synapse formation through two different mechanisms in two neuron types in Drosophila. (A) In fly photoreceptor neurons, autophagosome formation is selectively triggered at the tips of synaptogenic filopodia to regulate synapse numbers and partnerships. (B) In fly dorsal cluster neurons, autophagy is actively suppressed to stabilize synapses and branch formation.</figcaption></figure>

function. These findings suggest autophagic degradation and turnover of active zone proteins as a default state that is actively suppressed during a critical developmental period of brain wiring in DCNs.

Presynaptic active zones form concomitantly with the elabo-
rate axonal branch morphogenesis of DCN axons. A role for
synapse formation in axonal and dendritic branching is well
known as “synaptotropic growth”. During synaptotropic growth
synapse formation stabilizes branches and provides a local basis
for new probabilistic branch formation. Correspondingly, sup-
pression of autophagy during DCN synapse formation seconda-
rily leads to branch destabilization and a deterioration of the
stereotypic axonal branching pattern (Figure 1B). The conse-
quences of increased synaptic autophagy during this critical
period of DCN development thereby include altered axonal
morphology, synaptic connectivity and visual behavior.

We conclude that autophagy can restrict synapse formation through different mechanisms in two different neuron types of the fly visual system during the development of synaptic connectivity. In the non-branched axon terminals of photoreceptor neurons autophagy is actively induced at the tips of synaptogenic filopodia to restrict synapse formation and partner choice; in the highly branched axons of DCNs autophagy is actively suppressed to allow synaptotropic growth of an axonal branching pattern. Furthermore, the autophagic cargo in photoreceptor filopodia include the presynaptic seeding factors Liprin-a and RhoGAP100F/Syd-1, but not the mature active zone marker Brp (Figure 1A); by contrast, autophagic degradation induced by loss of Egfr function targets mature, brp-containing active zones (Figure 1B). These findings support a view of autophagy as a regulatory module during brain development that can be employed in a cell type-specific manner

The observation that activation versus suppression of auto-
phagy can be required in two different neuron types in the
same brain region and during the same critical window of
brain development may also affect how we view brain-wide
autophagic up- or downregulation as a therapeutic opportu-
nity. If neighboring neurons employ opposite autophagic reg-
ulation to develop proper synaptic connectivity, then levels of
autophagy must be controlled at the level of individual neu-
rons to ensure normal development.

In summary, we have identified two very different neuron
types in the fly visual system that employ the spatial and
temporal regulation of autophagy for the fine-tuning of synaptic
connectivity through selective restriction of synapse formation,
albeit through different mechanisms. In both cases cell type-
specific modulation of presynaptic autophagy during synapse
formation represents a quantitative contribution to brain wiring
that is selectable at the level of animal behavior. Based on the
diversity of autophagy-dependent synapse development in the
few neuron types analyzed thus far, we speculate that more cell-
specific roles of autophagy will emerge as more neuron types are
investigated during brain development.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

Work in the Hassan and Hiesinger lab is supported by DFG Research
Unit 5289 RobustCircuit project P2 (to B.A.H and P.R.H), DFG Research
Unit Syntophagy RP7 (Hi 1886/8), funding from the ERC Adv
101019191 (ERC) under the European Union's Horizon 2020 research
<figure><img src="image_19.png" /></figure>

and innovation programme (grant agreement No. 101019191 (to P.R.H), the Investissements d'Avenir program (ANR-10-IAIHU-06), Paris Brain Institute-ICM core funding, the Roger De Spoelberch Prize and an NIH Brain Initiative RO1 grant (Office of Extramural Research, National Institutes of Health 1R01NS121874-01) (to B.A.H.; Agence Nationale de la Recherche European Research Council

Reference

[1] Dutta SB, Linneweber GA, Andriatsilavo M, et al. EGFR-dependent suppression of synaptic autophagy is required for normal neural circuit development. Curr Biol. 2023 Jan 6;33 (3):517-532.e5. S0960-9822(22)01976-5.
<figure><img src="image_18.png" /></figure>

and innovation programme (grant agreement No. 101019191 (to P.R.H), the Investissements d'Avenir program (ANR-10-IAIHU-06), Paris Brain Institute-ICM core funding, the Roger De Spoelberch Prize and an NIH Brain Initiative RO1 grant (Office of Extramural Research, National Institutes of Health 1R01NS121874-01) (to B.A.H.; Agence Nationale de la Recherche European Research Council

Reference

[1] Dutta SB, Linneweber GA, Andriatsilavo M, et al. EGFR-dependent suppression of synaptic autophagy is required for normal neural circuit development. Curr Biol. 2023 Jan 6;33 (3):517-532.e5. S0960-9822(22)01976-5.