Electrophysiological analysis of <i>atg7^flox</i> control and <i>atg7</i>-SCKO mice.

<p>Four animals at age P60~70 in each group were tested.</p><p>NS; not significant, Student’s two-tailed test</p><p>Electrophysiological analysis of <i>atg7^flox</i> control and <i>atg7</i>-SCKO mice.</p

Canonical autophagy is activated in the sciatic nerves during postnatal development.

<p>A-C. Representative IF micrographs from transverse sciatic nerve sections showing the dynamic patterns of LC3B expression and puncta formation (arrowheads) as well as LAMP1 expression during postnatal development (P14~P60 [Ad]). Note the peak LC3B immunostaining at P21 and subsequent downregulation thereafter. Scale bar = 50 μm. B. For quantitative analysis of LC3B and LAMP1 staining, the relative intensity of the staining was calculated by counting the number of pixels with an intensity unit over 50 from 0–250 units (Materials and Methods). C. Left figure; LAMP1 (red) was co-localized with S100 (green, a SC marker) (arrows) in mSCs. Right figure; LC3B staining (arrow, green) was juxtaposed with LAMP1 staining (arrowheads, red). D, E. Representative IF micrographs from a teased nerve preparation of sciatic nerves showing LC3B (D) and SC expression of LAMP1 (E) (arrow). Scale bar = 50 μm. F, G. Western blot analysis of sciatic nerve extracts from animals at P8~P60. Note the peak level of the LC3B-II form at P21. G. Quantitative analysis of the LC3B-II levels. The level of LC3B-II at P8 is an arbitrary unit (AU) of 1 (n = 3, mean±SEM). **<i>P</i><0.01.</p

Myelination profiles of sciatic nerves in <i>atg7</i>-SCKO mice.

<p>A. Representative electron micrographs of sciatic nerve cross sections from animals at P10 and in adulthood. General patterns of myelination at P10 and Remak bundle development were normal in <i>atg7</i>-SCKO mice compared with <i>atg7^flox</i> control mice. B. This scatter plot showing the g-ratio in relation to the axonal diameter at P10 displays normal myelination profiles in <i>atg7</i>-SCKO mice. C. The mean length of the internodes was measured using 130 teased nerve fibers in each group at P21 (mean±SEM). NS; <i>p</i>>0.05. D. Western blot analysis using sciatic nerve extracts from mice at P10. E. The number of myelinated fibers in the sciatic nerves. (n = 3, mean±SEM). NS; <i>p</i>>0.05. F. Frequency distribution profile of the number of axons per Remak bundle in <i>atg7^flox</i> control and <i>atg7</i>-SCKO mice. G. The mean number of axons in a Remak bundle in the sciatic nerves at P60 (n = 3, mean±SEM). NS; <i>p</i>>0.05. H. Representative electron micrographs of sciatic nerve cross sections from adult mice showing hypermyelination of small fibers (arrows). I. Scatter plot showing the g-ratio in relation to the axonal diameter in adulthood. J. Morphometric quantification of the g-ratio of adult mice showed hypermyelination of small fibers that have diameters less than 3 μm. (n = 3, mean±SEM). **<i>P</i><0.01, *<i>P</i><0.05.</p

Rapamycin suppressed the mTOR pathway and enhanced cytoplasmic changes.

<p>A. Western blot analysis showing the effect of rapamycin injection on the phosphorylation of S6 kinase (p-S6K) and LC3B modification in the sciatic nerves. B. Representative electron micrographs of sciatic nerve sections from animals at P14 after intraperitoneal injection of vehicle (Veh) or rapamycin (Rapa) for one week. C. Quantitative analysis of the abaxonal cytoplasmic area of the mSCs at P14 following injection (n = 3, mean±SEM). NS; not significant. **<i>p</i><0.01.</p

<i>atg7</i>-SCKO mice exhibited a failure in the reduction of cytoplasmic area of mSCs.

<p>A. qPCR quantification of the relative <i>atg7</i> mRNA levels (normalized to β-actin expression) in the sciatic nerves from <i>atg7^flox</i> and <i>atg7</i>-SCKO mice in adulthood (n = 3, mean±SEM). ** <i>P</i><0.01. B. Western blot analysis showing ATG7, LC3B-II and p62 levels in <i>atg7</i>-SCKO mice, compared to <i>atg7^flox</i> mice at P21. C. Representative EM images of sciatic nerves from <i>atg7^flox</i> and <i>atg7</i>-SCKO mice at P60 showing excess residual abaxonal cytoplasm in the mSCs (arrows) in <i>atg7</i>-SCKO mice. D. Quantitative analysis of the abaxonal cytoplasmic area at P60 (n = 3, mean±SEM). NS: not significant. **<i>p</i><0.01. E. Scatter plot showing the cytoplasmic area in relation to the axonal diameter at P180. Approximately 200 axons from two mice in each group were employed for the analysis. F. Representative IF micrographs of a teased nerve preparation showing the abnormal accumulation of S100 and MBP in the cytoplasm of mSCs from <i>atg7</i>-SCKO mice (arrows). Scale bar = 50 μm. G. Representative EM images of the mSCs from <i>atg7</i>-SCKO mice at P60 showing numerous ribosomes with abnormally expanded RER (arrow) in the residual abaxonal cytoplasm of the mSCs. N; nucleus, MS; myelin sheath, M; mitochondria. H. A schematic drawing showing the Cajal band and membrane apposition. I. Quantitative analysis showing the number of appositions in the mSCs from <i>atg7^flox</i> and <i>atg7</i>-SCKO mice at P60 (n = 3, mean±SEM).</p

Isocitrate protects <i>DJ-1</i> null dopaminergic cells from oxidative stress through NADP⁺-dependent isocitrate dehydrogenase (IDH)

<div><p><i>DJ-1</i> is one of the causative genes for early onset familiar Parkinson’s disease (PD) and is also considered to influence the pathogenesis of sporadic PD. DJ-1 has various physiological functions which converge on controlling intracellular reactive oxygen species (ROS) levels. In RNA-sequencing analyses searching for novel anti-oxidant genes downstream of DJ-1, a gene encoding NADP⁺-dependent isocitrate dehydrogenase (IDH), which converts isocitrate into α-ketoglutarate, was detected. Loss of <i>IDH</i> induced hyper-sensitivity to oxidative stress accompanying age-dependent mitochondrial defects and dopaminergic (DA) neuron degeneration in <i>Drosophila</i>, indicating its critical roles in maintaining mitochondrial integrity and DA neuron survival. Further genetic analysis suggested that DJ-1 controls IDH gene expression through nuclear factor-E2-related factor2 (Nrf2). Using <i>Drosophila</i> and mammalian DA models, we found that IDH suppresses intracellular and mitochondrial ROS level and subsequent DA neuron loss downstream of DJ-1. Consistently, trimethyl isocitrate (TIC), a cell permeable isocitrate, protected mammalian <i>DJ-1</i> null DA cells from oxidative stress in an IDH-dependent manner. These results suggest that isocitrate and its derivatives are novel treatments for PD associated with <i>DJ-1</i> dysfunction.</p></div

IDH inhibits oxidative stress-induced cell death in <i>DJ-1</i> null mammalian DA cells.

<p>(A) Comparison of <i>IDH1</i> mRNA expression levels in wild type (<i>WT</i>) and <i>DJ-1</i> null (<i>DJ-1</i>^<i>-/-</i>) SN4741 cells under control (CON) or H₂O₂ (H₂O₂) treatment (n = 3). (B) <i>IDH2</i> mRNA expression level change was analyzed in <i>DJ-1</i> null SN4741 cells under control (CON) or H₂O₂ (H₂O₂) treatment, with scramble (-) or Keap1 siRNA (+) (n = 3). (C) Cell viability of wild type (<i>WT</i>), <i>DJ-1</i> null (<i>DJ-1</i>^<i>-/-</i>), IDH1-overexpressing <i>DJ-1</i> null (<i>IDH1 DJ-1</i>^<i>-/-</i>) and IDH2-overexpressing <i>DJ-1</i> null (<i>IDH2 DJ-1</i>^<i>-/-</i>) SN4741 cells under 1.5 mM H₂O₂ treatment for 6 h. Cell viability was measured by MTT assay. The inset shows immunoblot of FLAG-tagged IDH1 and IDH2. β–tubulin as a loading control. (D) Propidium iodide and annexin V FITC staining of H₂O₂-treated SN4741 cells. (E) Necrotic cell death rates (n = 3). (F) Flow cytometric analysis of CM-H₂DCFDA-stained SN4741 cells. Gray-filled area: <i>WT</i> or <i>DJ-1</i> null SN4741 cells without H₂O₂ treatment; Red line: H₂O_2-treated SN4741 cells; Green line: H₂O_2-treated IDH1-overexpressing SN4741 cells; Blue line: H₂O_2-treated IDH2-overexpressing SN4741 cells. (G) Fold change in the mean of fluorescence intensity (MFI) of CM-H₂DCFDA in flow cytometric analysis (n = 3). (H) Flow cytometric analysis of MitoSOX-stained SN4741 cells. Gray-filled area: <i>WT</i> or <i>DJ-1</i> null SN4741 cells without H₂O₂ treatment; Red line: H₂O₂-treated SN4741 cells; Green line: H₂O₂-treated IDH1-overexpressing SN4741 cells; Blue line: H₂O₂-treated IDH2-overexpressing SN4741 cells. (I) Fold change in the mean of fluorescence intensity (MFI) of MitoSOX in flow cytometric analysis (n = 3). (J) Cell viability of MitoTEMPO-treated SN4741 cells under H₂O₂ treatment. Cell viability was measured by MTT assays (n = 3). Data information: Significance was determined by one-way ANOVA with Sidak correction [**, P<0.01; ***, P<0.001; NS, not significant (P>0.05)]. Error bars indicate SD.</p

IDH mRNA expression is induced by DJ-1 through Nrf2 pathway.

<p>(A-C) Comparison of <i>IDHm1</i> (A), <i>IDHm2</i> (B), or <i>IDHc</i> (C) mRNA expression levels in the whole body of wild type (<i>WT</i>), <i>DJ-1β</i> null mutants (<i>DJ-1β</i>^<i>ex54</i>) and <i>DJ-1β</i> null mutants with a heterozygous <i>Keap1</i> mutation (<i>DJ-1β</i>^<i>ex54</i> <i>Keap1</i>^<i>EY5/+</i>) under control (CON) or rotenone treatment (Rotenone) (n≥3). (D) Survival curves of wild types (<i>WT</i>), <i>DJ-1β</i> null mutants (<i>DJ-1β</i>^<i>ex54</i>), <i>Keap1</i> heterozygous mutants (<i>Keap1</i>^<i>EY5/+</i>) and <i>DJ-1β</i> null mutants with a heterozygous <i>Keap1</i> mutation (<i>DJ-1β</i>^<i>ex54</i> <i>Keap1</i>^<i>EY5/+</i>) under rotenone treatment (log-rank test: <i>DJ-1β</i>^<i>ex54</i> VS <i>WT</i> & <i>DJ-1β</i>^<i>ex54</i> VS <i>DJ-1β</i>^<i>ex54</i> <i>Keap1</i>^<i>EY5/+</i>: P<0.001, <i>Keap1</i>^<i>EY5/+</i> VS <i>DJ-1β</i>^<i>ex54</i> <i>Keap1</i>^<i>EY5/+</i>: P = 0.9896, n = 120 for <i>WT</i> and <i>DJ-1β</i>^<i>ex54</i>; n = 150 for <i>Keap1</i>^<i>EY5/+</i>; n = 140 for <i>DJ-1β</i>^<i>ex54</i> <i>Keap1</i>^<i>EY5/+</i>) (E) Survival curves of adult flies under H₂O₂ treatment (log-rank test: <i>DJ-1β</i>^<i>ex54</i> VS <i>WT</i> & <i>DJ-1β</i>^<i>ex54</i> VS <i>DJ-1β</i>^<i>ex54</i> <i>Keap1</i>^<i>EY5/+</i>: P<0.001, <i>Keap1</i>^<i>EY5/+</i> VS <i>DJ-1β</i>^<i>ex54</i> <i>Keap1</i>^<i>EY5/+</i>: P = 0.1613, n = 109 for <i>WT</i>; n = 119 for <i>DJ-1β</i>^<i>ex54</i>; n = 100 for <i>Keap1</i>^<i>EY5/+</i>; n = 120 for <i>DJ-1β</i>^<i>ex54</i> <i>Keap1</i>^<i>EY5/+</i>). All life span assays were carried out at 25°C and were repeated at least twice. (F) Comparison of <i>IDHm1</i> mRNA expression levels in the whole body of <i>hs-GAL4</i> control flies (<i>hs</i>), CncC-overexpressing files (<i>hs>CncC</i>), CncC and Keap1-overexpressing flies (<i>hs>CncC Keap1</i>) and CncC, Keap1 and DJ-1β-overexpressing flies (<i>hs>CncC Keap1 DJ-1β</i>) (n≥3). (G) Comparison of <i>IDHc</i> mRNA expression levels in the whole body of flies (n≥3). (H-I) Confocal images (H) and graphs (I) of the average number of DA neurons within DL1 and DM clusters of the adult brains from 6-day-old <i>elav-GAL4</i> control flies (<i>elav</i>), <i>DJ-1β</i> null mutants (<i>elav DJ-1β</i>^<i>ex54</i>) and CncC-overexpressing <i>DJ-1β</i> null mutants (<i>elav>CncC DJ-1β</i>^<i>ex54</i>) after rotenone treatments. DA neurons were stained with anti-TH antibody (green). (n = 30 for each genotype). Scale bars: 20 μm. (J-K) Confocal images (J) and graphs (K) of the average number of DA neurons within DL1 and DM clusters of the adult brains of the 6-day-old flies after H₂O₂ treatments. DA neurons were stained with anti-TH antibody (green). (n = 27 for <i>elav</i>; n = 30 for other genotypes). Scale bars: 20 μm. (L) Comparison of luciferase activity in control (Con) or CncC-transfected (CncC) S2 cells (n = 3). The reporter plasmid with wild type (<i>WT</i>) or ARE site-mutated (Mut) <i>IDH</i> promoter was co-transfected to quantitatively measure activation of each promoter by CncC transcription factor. The construction of <i>IDH</i> reporters were described in Materials and Methods and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006975#pgen.1006975.s002" target="_blank">S2C Fig</a>. Data information: Significance was determined by one-way ANOVA with Sidak correction [*, P<0.05; **, P<0.01; ***, P<0.001; NS, not significant (P>0.05)]. Error bars indicate SD.</p

Schematic models of the protective effects of IDH on DA neurons against oxidative stress in <i>DJ-1</i> null background.

<p>Schematic models of the protective effects of IDH on DA neurons against oxidative stress in <i>DJ-1</i> null background.</p

List of differentially expressed genes in <i>DJ-1β</i> null flies on rotenone-containing media compared to wild type controls.

<p>List of differentially expressed genes in <i>DJ-1β</i> null flies on rotenone-containing media compared to wild type controls.</p