ATM phosphorylation of the actin-binding protein drebrin controls oxidation stress-resistance in mammalian neurons and C. elegans

Drebrin (DBN) regulates cytoskeletal functions during neuronal development, and is thought to contribute to structural and functional synaptic changes associated with aging and Alzheimer’s disease. Here we show that DBN coordinates stress signalling with cytoskeletal dynamics, via a mechanism involving kinase ataxia-telangiectasia mutated (ATM). An excess of reactive oxygen species (ROS) stimulates ATM-dependent phosphorylation of DBN at serine-647, which enhances protein stability and accounts for improved stress resilience in dendritic spines. We generated a humanized DBN Caenorhabditis elegans model and show that a phospho-DBN mutant disrupts the protective ATM effect on lifespan under sustained oxidative stress. Our data indicate a master regulatory function of ATM-DBN in integrating cytosolic stress-induced signalling with the dynamics of actin remodelling to provide protection from synapse dysfunction and ROS-triggered reduced lifespan. They further suggest that DBN protein abundance governs actin filament stability to contribute to the consequences of oxidative stress in physiological and pathological conditions.

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No software was used Data analysis Graph pad version 6. Fiji ImageJ 1.51n. Imaris version 8.1.2 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/reviewers upon request. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

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We analyzed high n numbers for dendritic spine analysis and whole organism experiments yielding high significance underscoring the robustness of our analysis. Standard n numbers were used for biochemical analysis.
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Neurons were imaged from 3 separate primary culture preparations. Viable neurons for each experimental group were detected by the analysis software based on the MAP2 signal. Nematode analyses were performed in triplicates at different times to take into account any possible variations between the cohorts.
Randomization Images of viable neurons and images of nematodes from all experimental groups were randomly taken. For the lifespan and fecundity assays, nematodes were randomly chosen by picking the indicated number of nematode larvae and all animals were then analyzed. No further selection of data subsets was applied.

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Reporting for specific materials, systems and methods  -/-brain or neuronal lysates (data supplied in the manuscript). PS647-DBN specific antibodies were validated using DBN-YFP phospho-mutants or phosphatase assays (Kreis et al., 2013). Other antibodies such as alpha-tubulin or pMAPK were commonly used antibodies and ran at the expected size. For immunostaining, anti-Drebrin antibodies were also validated using Dbn+/+ and Dbn-/-primary hippocampal cultures. Name any commonly misidentified cell lines used in the study and provide a rationale for their use.

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