Epsin 1 Promotes Synaptic Growth by Enhancing BMP Signal Levels in Motoneuron Nuclei

We thank Carl-Henrik Heldin (Uppsala University, Sweden) for his generous gift of the PS1 pMad antibody, Hugo Bellen, Corey Goodman, Janis Fischer, Graeme Davis, Guillermo Marques, Michael O'Connor, Kate O'Connor-Giles, and the Bloomington Drosophila Stock Center for flies strains, the Developmental Studies Hybridoma Bank at the University of Iowa for antibodies to Wit and CSP; Marie Phillips for advice on membrane fractionation; Avital Rodal, Kate O'Connor-Giles, Ela Serpe, Kristi Wharton, Mojgan Padash-Barmchi for discussions or comments on the manuscript. We also thank Jody Summers at OUHSC for her generosity in letting us to use her confocal microscope.Conceived and designed the experiments: PAV TRF LRC BZ. Performed the experiments: PAV TRF LRC SMR HB NER BZ. Analyzed the data: PAV TRF LRC SMR HB NER BZ. Wrote the paper: PAV TRF BZ.Bone morphogenetic protein (BMP) retrograde signaling is crucial for neuronal development and synaptic plasticity. However, how the BMP effector phospho-Mother against decapentaplegic (pMad) is processed following receptor activation remains poorly understood. Here we show that Drosophila Epsin1/Liquid facets (Lqf) positively regulates synaptic growth through post-endocytotic processing of pMad signaling complex. Lqf and the BMP receptor Wishful thinking (Wit) interact genetically and biochemically. lqf loss of function (LOF) reduces bouton number whereas overexpression of lqf stimulates bouton growth. Lqf-stimulated synaptic overgrowth is suppressed by genetic reduction of wit. Further, synaptic pMad fails to accumulate inside the motoneuron nuclei in lqf mutants and lqf suppresses synaptic overgrowth in spinster (spin) mutants with enhanced BMP signaling by reducing accumulation of nuclear pMad. Interestingly, lqf mutations reduce nuclear pMad levels without causing an apparent blockage of axonal transport itself. Finally, overexpression of Lqf significantly increases the number of multivesicular bodies (MVBs) in the synapse whereas lqf LOF reduces MVB formation, indicating that Lqf may function in signaling endosome recycling or maturation. Based on these observations, we propose that Lqf plays a novel endosomal role to ensure efficient retrograde transport of BMP signaling endosomes into motoneuron nuclei.Yeshttp://www.plosone.org/static/editorial#pee

Lqf is required for synaptic overgrowth and pMad retrograde transport in <i>spinster</i> mutants.

<p>(A–F) Representative images of bouton morphology and pMad levels at the NMJ (A, C, E) and motoneuron nuclei (B, D, F) in <i>Drosophila</i> 3^rd instar larval NMJs from control larvae (CS, A–B), <i>spin</i> mutants (C–D) and <i>spin;lqf</i> double mutants (E–F). Synaptic boutons are overgrown in <i>spin</i> mutants (C), and this overgrowth is suppressed in the <i>spin;lqf</i> double mutants (E). (G) Quantification of synaptic bouton number in <i>spin</i> and <i>spin;lqf</i> mutants. Taken from three different animals for each genotype, n values represent the number of NMJs quantified. (H) Quantification of pMad intensity in boutons (white bars) and motoneuron nuclei (black bars). n = 5 NMJs from three larvae and 20 nuclei from five different larvae. Error bars represent SEM. *P<0.05, **P<0.01, ***P<0.001. One-way ANOVA with Tukey's Multiple Comparison Post test. Control (CS), <i>spin</i> (<i>spin⁴/spin⁵</i>), <i>spin, lqf</i> (<i>spin⁴/spin⁵; lqf^ARI/lqf Df</i>).</p

Lqf interacts with Wishful thinking (Wit) to regulate synapse growth.

<p>(A–G) Representative images of synaptic bouton morphology in <i>Drosophila</i> 3^rd instar larval NMJs from the indicated genotypes. Compared to control boutons (A), both <i>lqf</i> (<i>lqf^ARI</i>/<i>lqf</i>^FDD9) and <i>wit</i> mutants (<i>wit</i>^B11/<i>wit</i>^A12) (B and C, respectively), have fewer boutons, as does the <i>wit, lqf</i> double mutant (<i>wit^A12</i>/<i>wit^B11</i>; <i>lqf^ARI</i>/<i>lqf</i> Df; D). Neuronal overexpression of Lqf induces synaptic growth with increased number of branches and small satellite boutons and the presence of abnormally large ‘growth cone’-like boutons (Elav^C155-Gal4/+; UAS-Lqf/+; E), which is suppressed partially by removal of a single copy of <i>wit</i> (Elav^C155-Gal4/+; UAS-Lqf/+; <i>wit^A12/+</i>; F), or suppressed completely by removal of both copies of <i>wit</i> (Elav^C155-Gal4/+; UAS-Lqf/+; <i>wit^A12/wit^B11</i>; G). H). Quantification of A–G. Error bars represent SEM, n values represent the number of animals per genotype. *P<0.05, **P<0.01, ***P<0.001. One-way ANOVA with Tukey's Multiple Comparison Post test. (I) Co-immunoprecipitation of Flag-tagged Lqf from adult brain lysates from either control flies (containing no Flag), or flies expressing Flag-tagged Lqf, shows Wit co-immunoprecipitates with Lqf. Abbreviations are as follows: Immunoprecipitation (IP); Immunoblot (IB); Input (I); Supernatant (S); Immunoprecipitate (IP).</p

pMad fails to accumulate in the nucleus of motoneurons in <i>lqf</i> mutants.

<p>(A–E) Representative images of motoneuron nuclei in <i>Drosophila</i> 3^rd instar larval ventral nerve cord (VNC). pMad signal alone is shown on the left, whereas neuronal membrane (marked by HRP, green) and pMad (red) are shown on the right. (B) In <i>lqf</i> mutants, nuclear pMad is significantly reduced relative to control (CS, A), whereas neuronal overexpression of Lqf results in an increase in nuclear localized pMad (C). Similarly, two other endocytotic mutants <i>endo</i> (D) and <i>nwk</i> (E) have increased levels of nuclear pMad. (F) Quantification of nuclear pMad levels in A–E. Error bars represent SEM. *P<0.05, **P<0.01, ***P<0.001. One-way ANOVA with Tukey's Multiple Comparison Post test. (G–H') Dissected and fixed 3^rd Instar larvae were stained with anti-CSP, and segmental nerves (which contain hundreds axons of sensory and motoneurons) were examined for general traffic defects, which would be apparent as CSP accumulations. There are no significant CSP accumulations in either the wild type or <i>lqf</i> mutants. Scale bars are 10 µm in all images. n values denote number of nuclei for each genotype, quantified from three animals per genotype. Control (CS), <i>nwk</i> (<i>nwk¹/nwk¹</i>), <i>endo</i> (<i>endo^A/endo^Δ4</i>), <i>lqf</i> (<i>lqf^ARI/lqf^FDD9</i>), Lqf^O/E (Elav^C155-Gal4/+; UAS-Lqf/+).</p