MOESM3 of Cognitive and emotional alterations in App knock-in mouse models of Aβ amyloidosis

Additional file 3: Fig. S3. Locomotor activity in AppNL-G-F/NL-G-F and AppNL/NL mice during the pre-shock period in the contextual fear conditioning task. The distance travelled during the pre-shock period (3-min period just prior to the first footshock) in conditioning was compared among genotypes at both 6–9 (a and b) and 15–18 (c and d) months of age. Representative images of movement tracks during the pre-shock period in each genotype at 6–9 (a) and 15–18 (c) months of age were shown. At 6–9 months of age, AppNL-G-F/NL-G-F mice exhibited a slight decrease in distance travelled during the pre-shock period in comparison with WT mice (b). At 15–18 months of age, AppNL/NL mice exhibited a significant decrease in distance travelled during the pre-shock period in comparison with WT mice (d). Locomotor activity in AppNL-G-F/NL-G-F mice was also slightly decreased in comparison with WT mice. 6–9 month-old; n = 6 WT (B6 J), n = 6 AppNL/NL, n = 9 AppNL-G-F/NL-G-F. 15–18 month-old; n = 8 WT (B6 J), n = 7 AppNL/NL, n = 7 AppNL-G-F/NL-G-F. *p < 0.05 versus WT (B6J)

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

MOESM2 of Cognitive and emotional alterations in App knock-in mouse models of Aβ amyloidosis

Additional file 2: Fig. S2. Locomotor activity of AppNL-G-F/NL-G-F and AppNL/NL mice during the first and second trials in the elevated plus maze task. The distance travelled during the 10-min test of the first and second trials in the elevated plus maze task was compared among genotypes at both 6–9 (a–d) and 15–18 (e–h) months of age. Representative images of movement tracks during the first and second trials for each genotype at 6–9 (a and c) and 15–18 (e and g) months of age were shown (closed arms are indicated by shaded areas). At 6–9 months of age, AppNL-G-F/NL-G-F mice exhibited slight increases in distance travelled during the first (b) and second (d) trials in comparison with WT mice. By contrast, locomotor activity in AppNL/NL mice was comparable with WT mice in the two trials. At 15–18 months of age, AppNL-G-F/NL-G-F mice exhibited a slight increase in movement compared to WT mice during the first (f) and second (g) trials. AppNL/NL mice moved at similar levels compared with WT mice in the two trials. 6–9 month-old; n = 8 WT (B6J), n = 8 AppNL/NL, n = 8 AppNL-G-F/NL-G-F. 15–18 month-old; n = 12 WT (B6J), n = 10 AppNL/NL, n = 11 AppNL-G-F/NL-G-F. †p < 0.05 versus AppNL/NL

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

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

Global Analysis of Phosphorylation of Tau by the Checkpoint Kinases Chk1 and Chk2 <i>in vitro</i>

Hyperphosphorylation of microtubule-associated protein tau is thought to contribute to Alzheimer’s disease (AD) pathogenesis. We previously showed that DNA damage-activated cell cycle checkpoint kinases Chk1 and Chk2 phosphorylate tau at an AD-related site and enhance tau toxicity, suggesting potential roles of these kinases in AD. The purpose of this study is to systematically identify which sites in tau are directly phosphorylated by Chk1 and Chk2. Using recombinant human tau phosphorylated by Chk1 and Chk2 <i>in vitro</i>, we first analyzed tau phosphorylation at the AD-related sites by Western blot with phospho-tau-specific antibodies. Second, to globally identify phosphorylated sites in tau, liquid chromatography–tandem mass spectrometry (LC–MS³) was employed. These systematic analyses identified a total of 27 Ser/Thr residues as Chk1- or Chk2- target sites. None of them were proline-directed kinase targets. Many of these sites are located within the microtubule-binding domain and C-terminal domain, whose phosphorylation has been shown to reduce tau binding to microtubules and/or has been implicated in tau toxicity. Among these 27 sites, 13 sites have been identified to be phosphorylated in AD brains. Since DNA damage is accumulated in diseased brains, Chk1 and Chk2 may be involved in tau phosphorylation and toxicity in AD pathogenesis

Knockdown of GSK3β/Sgg is not sufficient to suppress either mislocalization of tau to the cytosol or Aβ42-induced augmentation of tau toxicity.

<p>(A) GSK3β/Sgg negatively regulates tau binding to microtubules. RNAi-mediated knockdown of GSK3/Sgg shifts all tau species to lower apparent molecular weights, and the resultant species migrate faster than the original tau_lower species (indicated as asterisk). The levels of tau and tubulin in the lysate before sedimentation (input), in the supernatant (cytosol) and in the pellet containing microtubules (microtubule) were analyzed by western blotting by using anti-tau antibody. The same amount of proteins from each genotype was loaded. Mean ± SD, n = 4; **, <i>p</i> < 0.01, ***, <i>p</i> < 0.005 by Student's t-test. Representative blots are shown. (B) Aβ42 increased the levels of tau phosphorylated at Ser202 and those phosphorylated at Thr231. Western blots of fly heads expressing tau alone (tau) or that co-expressing tau and Aβ42 (tau+Aβ42) with anti-phospho-Ser202 antibody (pSer202), anti-phospho-Thr231 antibody (pThr231), and anti-tau antibody. Tubulin was used as a loading control. Mean ± SD, n = 5; *, <i>p</i> < 0.05 by Student's t-test. Representative blots are shown. (C) Expression of Aβ42 did not increase tau phosphorylation at either of Ser202 and Thr231 in the Sgg knockdown background. Western blots of fly heads expressing Sgg RNAi tau and (SggRNAi+tau) or that co-expressing Sgg RNAi, tau and Aβ42 (SggRNAi+tau+Aβ42) with anti-phospho-Ser202 antibody (pSer202), anti-phospho-Thr231 antibody (pThr231), and anti-tau antibody. Tubulin was used as a loading control. Mean ± SD, n = 5; no significant difference by Student's t-test (<i>p</i> > 0.05). Representative blots are shown. (D) Aβ42 causes an increase in tau levels in the cytosol fraction and reduction in tau levels in the microtubule fraction in the Sgg knockdown background. The levels of tau and tubulin in fly heads expressing Sgg RNAi and tau (SggRNAi+tau) or that co-expressing Sgg RNAi, tau and Aβ42 (SggRNAi+tau+Aβ42) before sedimentation (input), in the supernatant (cytosol) and in the pellet containing microtubules (microtubule) were analyzed by western blotting with anti-tau and anti-tubulin. Mean ± SD, n = 4; ***, <i>p</i> < 0.005 by Student's t-test. Representative blots are shown. (E) Aβ42 enhances tau-induced retinal degeneration in the Sgg knockdown background (compare SggRNAi+tau and SggRNAi+tau+Aβ42). Mean ± SE, N = 6–8, asterisks indicate significant differences in the surface area of the external eye (***, <i>p</i> < 0.005, n.s., not significant (<i>p</i> > 0.05) by one-way ANOVA with Tukey's post hoc test). Transgene expression was driven by gmr-GAL4.</p