Hiw promotes Nmnat protein turnover in the injured distal axons and synapses

<p>(A) Distal axons and synapses of <i>ppk-Gal4,UAS-mCD8::RFP</i> labeled sensory neurons, located in the ventral nerve cord, either before or 24 h after injury. Transgenic <i>HA::nmnat</i> is expressed in a wild-type (WT) genetic background <i>(UAS-HA::nmnat/+; ppk-Gal4,UAS-mCD8::RFP/+)</i>, <i>hiw</i>^Δ<i>N</i> mutant <i>(hiw</i>^Δ<i>N</i><i>; UAS-HA::nmnat/+; ppk-Gal4,UAS-mCD8::RFP/+)</i> or co-expressed with <i>UBP2</i> (<i>UAS-HA::nmnat/UAS-UBP2; ppk-Gal4,UAS-mCD8::RFP/+</i>). Consistent with observations in motoneurons (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001440#pbio-1001440-g006" target="_blank">Figure 6</a>), mutation of <i>hiw</i> or co-expression of <i>UBP2</i> causes a dramatic elevation in HA-Nmnat protein, and this does not disappear after injury, in dramatic contrast to the disappearance of HA-Nmnat in wild-type (WT) animals. Of note, the nerve terminals of the <i>ppk-Gal4</i> labeled axons appear to be overgrown in <i>hiw</i> and <i>UBP2</i> expressing mutants, similar to previous descriptions in other neuron types <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001440#pbio.1001440-Wan1" target="_blank">[8]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001440#pbio.1001440-DiAntonio1" target="_blank">[41]</a>. Hence the quantification shown in (B) is normalized to the size of the nerve terminals (labeled by mCD8-RFP). Due to the expression of <i>HA-nmnat</i>, no axons are degenerating in any of the above genotypes. (B) Quantification of the average HA-Nmnat intensity in <i>ppk-Gal4</i> expressing sensory neuron axon terminals, within the most posterior four segments of the ventral nerve cord. Error bars represent standard error. ns, not significant, <i>p</i>>0.05. (C) Model: Hiw promotes multiple independent responses to injury, through independent pathways. Hiw promotes degeneration in the distal stump by down-regulating Nmnat, and concurrently regulates regeneration (and protection from degeneration <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001440#pbio.1001440-Massaro1" target="_blank">[15]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001440#pbio.1001440-Xiong2" target="_blank">[30]</a>) in the proximal stump by regulating Wnd. Since Hiw may localize and function in distal axons, injury may relieve the inhibition of Wnd by Hiw in the proximal stump. Scale bars = 12.5 µm, error bars represent standard error; ***<i>p</i><0.001; ns, not significant, <i>p</i>>0.05 in <i>t</i>-test.