Knockdown of <i>wfs1</i>, a fly homolog of Wolfram syndrome 1, in the nervous system increases susceptibility to age- and stress-induced neuronal dysfunction and degeneration in <i>Drosophila</i>

<div><p>Wolfram syndrome (WS), caused by loss-of-function mutations in the Wolfram syndrome 1 gene (<i>WFS1</i>), is characterized by juvenile-onset diabetes mellitus, bilateral optic atrophy, and a wide spectrum of neurological and psychiatric manifestations. <i>WFS1</i> encodes an endoplasmic reticulum (ER)-resident transmembrane protein, and mutations in this gene lead to pancreatic β-cell death induced by high levels of ER stress. However, the mechanisms underlying neurodegeneration caused by <i>WFS1</i> deficiency remain elusive. Here, we investigated the role of <i>WFS1</i> in the maintenance of neuronal integrity <i>in vivo</i> by knocking down the expression of <i>wfs1</i>, the <i>Drosophila</i> homolog of <i>WFS1</i>, in the central nervous system. Neuronal knockdown of <i>wfs1</i> caused age-dependent behavioral deficits and neurodegeneration in the fly brain. Knockdown of <i>wfs1</i> in neurons and glial cells resulted in premature death and significantly exacerbated behavioral deficits in flies, suggesting that <i>wfs1</i> has important functions in both cell types. Although <i>wfs1</i> knockdown alone did not promote ER stress, it increased the susceptibility to oxidative stress-, excitotoxicity- or tauopathy-induced behavioral deficits, and neurodegeneration. The glutamate release inhibitor riluzole significantly suppressed premature death phenotypes induced by neuronal and glial knockdown of <i>wfs1</i>. This study highlights the protective role of <i>wfs1</i> against age-associated neurodegeneration and furthers our understanding of potential disease-modifying factors that determine susceptibility and resilience to age-associated neurodegenerative diseases.</p></div

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>.</p

Knockdown of <i>wfs1</i> alone does not induce ER stress in the fly brains.

<p><b>(A)</b> Neuronal (<i>elav</i>), <b>(B)</b> neuronal and glial (<i>elav-Repo</i>) knockdown of <i>wfs1</i> or <b>(C</b>) <i>wfs1</i> mutant with MiMIC insertion (<i>wfs1</i>^{<i>MI14041/MI14041</i>}) did not increase mRNA levels of <i>Xbp1-RB</i>, <i>PEK</i> and <i>Hsc70-3</i> in fly brains, as determined by qRT-PCR. n = 4, *<i>p</i> < 0.05 and ***<i>p</i> < 0.001 by Student’s <i>t</i>-test. 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>.</p

Knockdown of <i>wfs1</i> in both neurons and glial cells significantly shortens the lifespan of flies and exaggerates behavioral deficits and neurodegeneration.

<p>(<b>A</b>) Knockdown of <i>wfs1</i> in both neurons and glial cells, (<b>D</b>) but not in glial cells alone, caused age-dependent locomotor deficits 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. Percentages of flies that stayed at the bottom were subjected to statistical analyses. n = 3–5 independent experiments, *<i>p</i> < 0.05 by Student’s <i>t</i>-test. (<b>B</b>) Knockdown of <i>wfs1</i> in both neurons and glial cells, (<b>E</b>) but not in glial cells alone, caused neurodegeneration in the central neuropil or optic lobes of fly brain. Representative images show the central neuropil and optic lobe in paraffin-embedded brain section with hematoxylin and eosin (HE) staining from 30-day-old flies. Scale bars: 200 μm. Percentages of vacuole areas (indicated by arrowheads in the images) in central neuropil or optic lobes are shown. n = 14–19 hemispheres, ***<i>p</i> < 0.001 by Student’s <i>t</i>-test. (<b>C</b>) Knockdown of <i>wfs1</i> in both neurons and glial cells significantly shortened lifespan of flies (n = 199, <i>wfs1</i> RNAi group or 331, control group). (<b>F</b>) Knockdown of <i>wfs1</i> in glial cells did not affect lifespan up to 30 days (n = 114, <i>wfs1</i> RNAi group or 139, control group). The lifespans of flies were determined by Kaplan-Meier survival analysis with log-rank test and 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>.</p

Oxidative stress induces <i>wfs1</i> expression, and neuronal knockdown of <i>wfs1</i> increases vulnerability to oxidative stress.

<p>(<b>A</b>) mRNA levels of <i>wfs1</i> were induced by hydrogen peroxide (H₂O₂) exposure in fly brains, as determined by qRT-PCR. n = 4, ***<i>p</i> < 0.001 by Student’s <i>t</i>-test. (<b>B</b>) Neuronal knockdown of <i>wfs1</i> significantly reduced survival time after H₂O₂ exposure compared with that of flies with <i>mcherry</i> RNAi. The survival rates of flies with H₂O₂ exposure were determined by Kaplan-Meier survival analysis with log-rank test and statistical significance was indicated in the figure (left panel) (n = 29, <i>mcherry</i> RNAi with 10% sucrose only, n = 26, <i>wfs1</i> RNAi with 10% sucrose only, n = 236, <i>mcherry</i> RNAi with H₂O₂ exposure, n = 171, <i>wfs1</i> RNAi with H₂O₂ exposure). Average survival time of <i>wfs1</i> knockdown flies with H₂O₂ exposure was significantly reduced (right panel). ***<i>p</i> <0.001 by Student’s <i>t</i>-test. (<b>C</b>) The mRNA levels of genes related to oxidative stress responses in 29-day-old fly brains with <i>wfs1</i> RNAi or <i>mcherry</i> RNAi 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. 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>.</p

Neuronal knockdown of <i>wfs1</i>, a fly homolog of <i>WFS1</i>, causes behavioral deficits and neurodegeneration.

<p>(<b>A</b>) mRNA expression levels of <i>wfs1</i> were increased upon aging in the fly brains, as determined by qRT-PCR. n = 4, ***<i>p</i> < 0.001 by Student’s <i>t</i>-test. (<b>B</b>) A schematic diagram of the <i>wfs1</i> RNAi target sites and insertion sites of MiMIC/PiggyBac for <i>wfs1</i> mutant lines. (<b>C</b>) mRNA levels of <i>wfs1</i> in heads of flies carrying the RNAi transgene targeting <i>wfs1</i> were analyzed by qRT-PCR. n = 4, ***<i>p</i> < 0.001 by Student’s <i>t</i>-test. (<b>D</b>) Knockdown of <i>wfs1</i> in neurons induced age-dependent locomotor deficits as revealed by climbing assay. Flies carrying the <i>mcherry</i> RNAi transgene did not induce the age-dependent locomotor deficits. 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. Percentages of flies that stayed at the bottom were subjected to statistical analyses. n = 7 independent experiments, *<i>p</i> < 0.05, **<i>p</i> < 0.01 and ***<i>p</i> < 0.001 by Student’s <i>t</i>-test. (<b>E</b>) Neuronal knockdown of <i>wfs1</i> caused age-dependent neurodegeneration in the central neuropil or optic lobes of fly brain. Representative images show the central neuropil and optic lobe in paraffin-embedded brain section with hematoxylin and eosin (HE) staining from 60-day-old flies. Scale bars: 200 μm. Percentages of vacuole areas (indicated by arrowheads in the images) in central neuropil or optic lobes from 20-, 30-, and 60-day-old flies were analyzed. n = 8–12 hemispheres, *<i>p</i> < 0.05 and ***<i>p</i> < 0.001 by Student’s <i>t</i>-test. (<b>F</b>) Neuronal knockdown of <i>wfs1</i> shortened the lifespan (n = 182, <i>wfs1</i> RNAi group or 358, control group). The lifespans of flies were determined by Kaplan-Meier survival analysis with log-rank test and 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>.</p

Additional file 4: of Integrated biology approach reveals molecular and pathological interactions among Alzheimer’s Aβ42, Tau, TREM2, and TYROBP in Drosophila models

Table S3. Functional enrichment of DEGs identified in Aβ42, Aβ42/TREM2WT/TYROBP, and Aβ42/TREM2R47H/TYROBP files. (XLSX 23 kb

Additional file 10: of Integrated biology approach reveals molecular and pathological interactions among Alzheimer’s Aβ42, Tau, TREM2, and TYROBP in Drosophila models

Table S9 Module membership from weighted gene co-expression network analysis for ROSMAP gene expression data. (XLSX 456 kb

Additional file 8: of Integrated biology approach reveals molecular and pathological interactions among Alzheimer’s Aβ42, Tau, TREM2, and TYROBP in Drosophila models

Table S7. Functional enrichment of DEGs identified in Tau/TREM2WT/TYROBP, Tau/TREM2R47H/TYROBP files. (XLSX 23 kb

Additional file 6: of Integrated biology approach reveals molecular and pathological interactions among Alzheimer’s Aβ42, Tau, TREM2, and TYROBP in Drosophila models

Table S5. Overlap between MSigDB gene ontology/pathway gene sets and fly-human conserved genes. (XLSX 299 kb