Heterozygous mutation in <i>Eaat1</i>, a glial high-affinity glutamate transporter, significantly worsens behavioral deficits caused by <i>wfs1</i> deficiency.

<p>(<b>A</b>) Flies with neuronal (<i>elav</i>), neuronal and glial (<i>elav-Repo</i>) knockdown of <i>wfs1</i> or <b>(B</b>) <i>wfs1</i> mutant with PiggyBac insertion (<i>wfs1</i>^{<i>LL07290/LL07290</i>}) required longer time to complete righting reflex than control flies. n = 10–26, **<i>p</i> < 0.01 and ***<i>p</i> < 0.001 by Mann-Whitney <i>U</i>-test. (<b>C</b>) The mRNA levels of genes related to synaptic activities with focus on GABAergic as well as glutamatergic systems in fly brains with neuronal and glial knockdown of <i>wfs1</i> were determined by qRT-PCR. n = 4, *<i>p</i> < 0.05 by Student’s <i>t</i>-test. (<b>D</b>) The mRNA level of <i>Eaat1</i> in fly brains with neuronal knockdown of <i>wfs1</i> was determined by qRT-PCR. n = 4, *<i>p</i> < 0.05 by Student’s <i>t</i>-test. (<b>E</b>) Heterozygous mutation in <i>Eaat1</i> did not induce locomotor defects in aged flies (left panel). In the <i>wfs1</i> knockdown background, heterozygous mutation in <i>Eaat1</i> caused locomotor deficits (right panel) as revealed by climbing assay. Average percentages of flies that climbed to the top (white), climbed to the middle (light gray), or stayed at the bottom (dark gray) of the vials. Percentage of flies that stayed at the bottom were subjected to statistical analyses. n = 4 independent experiments. n = 5, *<i>p</i> < 0.05 by Student’s <i>t</i>-test. (<b>F</b>) A heterozygous mutation in <i>Eaat1</i> induced ER stress responses in <i>wfs1</i> knockdown background, but not in control background. The mRNA levels of <i>Xbp1-RB</i>, <i>PEK</i> and <i>Hsc70-3</i> in fly brains were determined by qRT-PCR. n = 4, *<i>p</i> < 0.05, **<i>p</i> < 0.01 and ***<i>p</i> < 0.001 by Student’s <i>t</i>-test. (<b>G</b>) mRNA levels of genes related to oxidative stress responses in fly heads carrying a heterozygous mutation in <i>Eaat1</i> were prominently elevated in <i>wfs1</i> knockdown background compared to control background, as determined by qRT-PCR. n = 4, *<i>p</i> < 0.05, **<i>p</i> < 0.01 and ***<i>p</i> < 0.001 by Student’s <i>t</i>-test. (<b>H</b>-<b>I</b>) Riluzole increased the survival time in flies with neuronal and glial knockdown of <i>wfs1</i> (<b>H</b>) and in flies carrying homozygous mutations of <i>wfs1</i> with PiggyBac insertion (<i>wfs1</i>^{<i>LL07290/LL07290</i>}) (<b>I</b>). The survival rates were determined by Kaplan-Meier survival analysis with log-rank test, and Holm-Sidak method was used for multiple comparison analysis (n = 138, <i>mcherry</i> RNAi with Riluzole 0 mM, n = 128, <i>mcherry</i> RNAi with Riluzole 5 mM, n = 102, <i>wfs1</i> RNAi with Riluzole 0 mM, n = 101, <i>wfs1</i> RNAi with Riluzole 5 mM, n = 82, <i>wfs1</i>^{LL07290/LL07290} with Riluzole 0 mM, n = 85, <i>wfs1</i>^{LL07290/LL07290} with Riluzole 5 mM). The statistical significance was indicated in the figure. Genotypes and ages of flies are described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007196#pgen.1007196.s001" target="_blank">S1 Table</a>.