Periodic Reporting for period 4 - NEUROPHAGY (The Role of Autophagy in Synaptic Plasticity)
Berichtszeitraum: 2021-02-01 bis 2022-08-31
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.
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.