Eps8 regulates axonal filopodia in hippocampal neurons in response to brain-derived neurotrophic factor (BDNF).
The regulation of filopodia plays a crucial role during neuronal development and synaptogenesis. Axonal filopodia, which are known to originate presynaptic specializations, are regulated in response to neurotrophic factors. The structural components of filopodia are actin filaments, whose dynamics and organization are controlled by ensembles of actin-binding proteins. How neurotrophic factors regulate these latter proteins remains, however, poorly defined. Here, using a combination of mouse genetic, biochemical, and cell biological assays, we show that genetic removal of Eps8, an actin-binding and regulatory protein enriched in the growth cones and developing processes of neurons, significantly augments the number and density of vasodilator-stimulated phosphoprotein (VASP)-dependent axonal filopodia. The reintroduction of Eps8 wild type (WT), but not an Eps8 capping-defective mutant, into primary hippocampal neurons restored axonal filopodia to WT levels. We further show that the actin barbed-end capping activity of Eps8 is inhibited by brain-derived neurotrophic factor (BDNF) treatment through MAPK-dependent phosphorylation of Eps8 residues S624 and T628. Additionally, an Eps8 mutant, impaired in the MAPK target sites (S624A/T628A), displays increased association to actin-rich structures, is resistant to BDNF-mediated release from microfilaments, and inhibits BDNF-induced filopodia. The opposite is observed for a phosphomimetic Eps8 (S624E/T628E) mutant. Thus, collectively, our data identify Eps8 as a critical capping protein in the regulation of axonal filopodia and delineate a molecular pathway by which BDNF, through MAPK-dependent phosphorylation of Eps8, stimulates axonal filopodia formation, a process with crucial impacts on neuronal development and synapse formation.
Menna
Elisabetta
E
Department of Medical Pharmacology, National Research Council Institute of Neuroscience, University of Milan, Milan, Italy.
Disanza
Andrea
A
Cagnoli
Cinzia
C
Schenk
Ursula
U
Gelsomino
Giuliana
G
Frittoli
Emanuela
E
Hertzog
Maud
M
Offenhauser
Nina
N
Sawallisch
Corinna
C
Kreienkamp
Hans-Jürgen
HJ
Gertler
Frank B
FB
Di Fiore
Pier Paolo
PP
Scita
Giorgio
G
Matteoli
Michela
M
D000199:Actins / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001369:Axons / Q000378:metabolism / Q000502:physiology; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology*; D002460:Cell Line; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D005260:Female; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011554:Pseudopodia / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D034622:RNA Interference; D051381:Rats; D014162:Transfection
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D019208:Brain-Derived Neurotrophic Factor; D003598:Cytoskeletal Proteins; C083430:Eps8 protein, mouse; D049452:Green Fluorescent Proteins; D020928:Mitogen-Activated Protein Kinases