The Role of Autophagy in Synaptic Plasticity

Informations projet

NEUROPHAGY

N° de convention de subvention: 714983

DOI

10.3030/714983

Projet clôturé

Date de signature de la CE 28 Février 2017

Date de début 1 Mars 2017

Date de fin 31 Août 2022

Financé au titre de

• EXCELLENT SCIENCE - European Research Council (ERC)

Coût total

€ 1 493 750,00

Contribution de l’UE

€ 1 493 750,00

Coordonné par

UNIVERSITE DE LAUSANNE
Switzerland

Periodic Reporting for period 4 - NEUROPHAGY (The Role of Autophagy in Synaptic Plasticity)

Période du rapport: 2021-02-01 au 2022-08-31

Résumé du contexte et des objectifs généraux du projet

Autophagy is an evolutionarily conserved process that delivers macromolecules and damaged or superfluous organelles to the lysosome for degradation. In line with these crucial functions, autophagy is indispensable for neuronal integrity and its deregulation causes severe synaptic defects that perturb behavior in mouse models and in humans alike. In line with this, autophagy impairment is genetically linked with neurodevelopmental disorders, brain aging and specific forms of neurodegeneration. However, the collective contribution of different autophagic processes to the homeostasis of synaptic proteins and to the function of neuronal subpopulations remain largely elusive.
Our work first aims at characterizing the regulation and roles of the autophagic machinery in the context of neuronal physiology and synaptic function. Moreover, we aim at developing and testing novel tools in order to assess in vivo whether controlled activation of autophagy can be beneficial in ameliorating synaptic defects and behavioral deficits in different paradigms of brain disease. To this end, we have successfully developed novel mouse models, and use them along with behavioral analyses, biochemical, cell biology and in vitro electrophysiology techniques. Moreover, we perform quantitative proteomic analyses in autophagic vesicles purified from the mouse brain.
At the end of the Neurophagy project, we have concluded that:
a) Autophagy regulates the turnover of synaptic proteins, of aggregates, of ER and mitochondrial proteins using selective autophagy receptors in the brain, under steady state, physiological conditions.
b) The autophagic content and degradome is dynamic and changes with brain maturation and aging.
c)Autophagic vesicle biogenesis is regulated by synaptic activity. Long-term synaptic depression triggers the biogenesis of these vesicles in distal dendrites in order to sequester postsynaptic cargo. This regulation is of crucial importance as it's required for synaptic plasticity.
d) Autophagy is required in the minority of parvalbumin-expressing interneurons for memory formation.
Taken together, these findings greatly enhance our understanding of how autophagy operates in the brain to safeguard synaptic function, behavior and homeostasis.

Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent

Neurophagy has contributed major achievements to the field of brain autophagy. A main objective of this project (Objective 1) was to characterize where and when autophagic vesicle biogenesis takes place in neurons. To this end, our work revealed that, while under basal conditions biogenesis is restricted to the axon tip, under conditions of synaptic plasticity biogenesis is initiated in distal dendrites. This facilitates the sequestration of postsynaptic proteins into phagophores and their retrograde transport to the soma, where they are degraded in the lysosome. Another major objective (Objective 2) was to identify autophagy substrates, focusing on synaptic proteins. We have addressed this point, taking an unbiased approach to characterize both the content and the degradome of brain autophagic vesicles. We succeeded in revealing synaptic substrates and several selective autophagy pathways that regulate the turnover of mitochondria, ER and aggregates constitutively in the brain. An additional objective (Objective 3) was to characterize the synaptic defects and ensuing behavioural deficits arising from impaired autophagy in the hippocampus. We revealed that parvalbumin-interneurons in the hippocampus serve as key cellular substrates where autophagy is required for memory formation. Moreover, we revealed that autophagy-deficiency in excitatory neurons results in spontaneous seizures that affect the hippocampus and other areas. Additionally, to the objectives described in the proposal, we also examined the requirement of autophagy in oligodendrocytes, through a fruitful collaboration with the lab of Prof. Karagogeos. Some of this work has been accepted for publication in high-tier journals or is currently under revision, as detailed in the periodic report. Moreover, the work has been disseminated to the scientific community through several invited oral presentations in international meetings, as well as by poster and short-talk presentations by my team members. Finally, this work has formed the basis for developing further our ideas and planning new projects which were successfully funded.

Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent)

In the course of the Neurophagy project, we developed tools and novel methodologies, that go beyond the state of the art and provide new opportunities for the scientific community:
a) We developed a method allowing us to immunopurify mature and intact autophagic vesicles from the mouse brain. This novel method has enabled us to perform proteomic analyses to reveal the brain content of autophagic vesicles. Moreover, it allowed us to study the heterogeneity of brain autophagic vesicles, giving rise to new projects in the lab. For example, we identified vesicles carrying the machinery to fuse with lysosomes and facilitate degradation of their content, but also others that carry a different machinery that allows them to fuse with the plasma membrane and secrete their cargo. The realization of these and possibly other subpopulations of autophagic vesicles will have a great impact in our understanding of how autophagy contributes to brain homeostasis.
b) We developed a method for measuring the autophagic flux in the brain. This method makes use of a previously engineered transgenic mouse line (pCAG::RFP-GFP-LC3), where LC3 is N-terminally tagged with two fluorophores in tandem, RFP and GFP. While GFP is pH sensitive and is quenched in the acidic environment of the lysosome, RFP is pH-insensitive and continues to fluoresce in lysosomes. This mouse was previously used to monitor autophagic flux in peripheral organs, but its use to monitor brain autophagy was hindered by technical challenges. We have developed a method that allows us to preserve fluorescence in the brain. We complemented this achievement with volumetric imaging of different brain areas and subsequent refinement of machine learning tools in IMARIS to ratiometrically analyze the data. As a result, we can now confidently analyze brain autophagic flux in vivo, in multiple brain areas. We have already used this tool to compare the flux in adolescent, adult and aged brains. However, we can envision many applications, for example in neuropathological models where autophagy is suspected to play a role, or under conditions that implicate the brain-body axis.
c) We generated two new mouse lines. The first one is a transgenic mouse allowing us to induce autophagy in a time and cell-time specific manner, and control the duration of the autophagy activation by having the option to terminate it. We are using this mouse to investigate conditions where targeted activation of autophagy in brain cells of choice may be beneficial. The second mouse is a knock-in to generate floxed alleles of Atg101. This gene is involved in the most upstream step of the initiation of biogenesis. Hence, its ablation leads to a complete loss of autophagic structures, as phagophores are not at all formed. This is not the case with existing mouse lines with floxed alleles for atg5 or atg7 which are commonly used in the field to ablate autophagy, but however, leave incomplete phagophore structures behind as they act later in the biogenesis cascade.

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