Mitochondrial SIRT3 confers neuroprotection in Huntington's disease by regulation of oxidative challenges and mitochondrial dynamics.

SIRT3 is a major regulator of mitochondrial acetylome. Here we show that SIRT3 is neuroprotective in Huntington's disease (HD), a motor neurodegenerative disorder caused by an abnormal expansion of polyglutamines in the huntingtin protein (HTT). Protein and enzymatic analysis revealed that increased SIRT3 is a signature in several HD models, including human HD brain, which is regulated by oxidative species. While loss of SIRT3 further aggravated the oxidative phenotype, antioxidant treatment regularized SIRT3 levels. SIRT3 overexpression promoted the antioxidant effect in cells expressing mutant HTT, leading to enhanced mitochondrial function and balanced dynamics. Decreased Fis1 and Drp1 accumulation in mitochondria induced by SIRT3 expression favored mitochondrial elongation, while the SIRT3 activator ε-viniferin improved anterograde mitochondrial neurite transport, sustaining cell survival. Notably, SIRT3 fly-ortholog dSirt2 overexpression in HD flies ameliorated neurodegeneration and extended lifespan. These findings provide a link between oxidative stress and mitochondrial dysfunction hypotheses in HD and offer an opportunity for therapeutic development

SVZ cells express CB1R.

<p><b>A:</b> Detection of CB1R by Western blotting in SVZ. Lane 1 corresponds to SVZ proliferating cells, lane 2 to SVZ extract from adult C57Bl6 mice and lane 3 to the negative control (total proteins from CB1R-KO mice). <b>B–F:</b> Representative confocal digital images depicting CB1R immunoreactivity in SVZ cells after 7 days of differentiation [CB1R (in red); nestin (in green), GFAP (in green), PSA-NCAM (in green), DCX (in green), βIII tubulin (in green), MAP2 (in green) and Hoechst 33342 (used to visualize cell nuclei, in blue)]. c1, e1 and f1 are magnifications of squares in C, E and F, respectively. Scale bars = 20 µm. SVZ: subventricular zone; GFAP: Glial fibrillary acidic protein; PSA-NCAM: Polysialylated neural cell adhesion; βIII tubulin: Neuron-specific class III beta-tubulin; MAP2: Microtubule-associated protein 2; CB1R: CB1 receptor.</p

(R)-(+)-Methanandamide promotes the expression of the proneurogenic genes <i>Ngn1</i>.

<p><b>A:</b> Scheme of the protocol. <b>B:</b> Bar graph depicts the fold increase of H3K36m3 recruitment in the promoter region of <i>Ngn1</i> gene quantified by qChIP analysis. <b>C</b>: Bar graph depicts the fold increase of mRNA expression for Ngn1 protein evaluated by qRT-PCR analysis. Data are expressed as mean ± SEM. N = 4–7. *P<0.05, using Dunnett’s test for comparison with control (set to 1). H3K36m3: Histone H3 trimethylated on lysine 36; Ngn1: Neurogenin 1; qChIP: quantitative chromatin immunoprecipitation; qRT-PCR: quantitative real time polymerase chain reaction.</p

(R)-(+)-Methanandamide promotes self-renewal.

<p><b>A:</b> Experimental protocol. <b>B:</b> Bar graphs represent the number of primary and secondary neurospheres. Data are expressed as mean ± SEM. N = 6. *P<0.05, **P<0.01 and ***<i>P</i><0.001 using Dunnett’s multiple comparison test, for comparison with control; ^###P<0.001 using Dunnett’s multiple comparison test, for comparison with R-m-AEA. <b>C:</b> Protocol used for studying cell-fate. <b>D:</b> Confocal digital images of cell pairs obtained following (a) the symmetrical division of a SVZ cell into two Sox2+ cells (Sox2+/+), (b) the asymmetrical division into a Sox2+ and a Sox2- progenitor (Sox2+/−) and (c) the symmetrical terminal division into two Sox2- progenitors (Sox2−/−). Scale bars 20 µm. <b>E:</b> Bar graph illustrates the number of each type of cell divisions counted. Data are expressed as the percentage of total cell pairs and are represented as the mean ± SEM. N = 5. *P<0.05 and ***P<0.001 using Bonferroni’s multiple comparison test, for comparison with the respective controls; ^###P<0.001 using Bonferroni’s multiple comparison test, for comparison with the respective R-m-AEA. SOX2: sex determining region Y-box 2.</p

Additional file 1: Figure S1. of Histamine induces microglia activation and dopaminergic neuronal toxicity via H1 receptor activation

Histamine-induced ROS production via H1R and H4R activation in primary microglial cell cultures. Cellular ROS production induced by 100 μM histamine (H100) was blocked by an H1R antagonist (AntH1R, mepyramine maleate, 1 μM) or H4R antagonist (AntH4R, JNJ7777120, 5 μM). Data are expressed as mean ± SEM (n = 5) and as percentage of control. **P < 0.01 and ***P < 0.001, using one-way ANOVA followed by Dunnett’s test. (TIF 251 KB

(R)-(+)-Methanandamide induces the differentiation of GABAergic neurons and neuritogenesis.

<p><b>A:</b> Schematic representation of the protocol. <b>B:</b> Bar graph depicts the numbers of either VGAT- or TH/βIII tubulin-positive cells, expressed as percentage of total cells. The data are expressed as percentage ± SEM. N = 3. *<i>P</i><0.05 using unpaired Student’s t test for comparison with control. <b>C:</b> Schematic representation of the protocol used for studying neuritogenesis. <b>D:</b> Representative confocal digital images of the GFP (green), MAP2 (red), Hoechst staining (blue), in control cultures and in cultures exposed to R-m-AEA. Scale bar = 20 µm. <b>E:</b> Bar graphs depict (from left to right): total length (µm), number of primary and number of secondary ramifications of MAP2 neurites per cell. N = 3. **P<0.01 using unpaired student’s t test for comparison with control. MAP2: Microtubule-associated protein 2; TH: tyrosine hydroxylase; βIII tubulin: Neuron-specific class III beta-tubulin; VGAT: vesicular GABA transporter.</p

(R)-(+)-Methanandamide does not induce glial differentiation in SVZ cultures through CB1R activation.

<p><b>A:</b> Protocol used for studying glial differentiation. <b>B:</b> Western blot analysis of GFAP and Olig2 protein levels in SVZ. Data are expressed as mean ± SEM. N = 4. <b>C:</b> Bar graph depicts the number of GFAP and Olig2-positive cells, expressed as the percentage of total cells <i>per</i> culture. Data are expressed as mean ± SEM. N = 3. <b>D:</b> Representative fluorescent digital images of GFAP-positive cells (green), Olig2-positive cells (red) and Hoechst staining (blue nuclei). Scale bar = 50 µm.</p

Primary antibodies used for immunocytochemistry.

<p>Primary antibodies used for immunocytochemistry.</p

Additional file 3: Figure S3. of Histamine induces microglia activation and dopaminergic neuronal toxicity via H1 receptor activation

Histamine-induced Nox1 immunostaining in dopaminergic neurons in the SN in vivo. Representative confocal photomicrographs showing that histamine-induced Nox1 expression in the dopaminergic cells in the SN in vivo. Cells were stained for TH (green), Nox1 (red), and Hoechst 33342 (nuclei in blue). Scale bar 10 μm. (TIF 98 KB

Microglial phagocytic response during in vivo acute and chronic inflammatory challenge.

<p>(<b>A</b>) Experimental design and apoptosis in the DG of c57BL/6 fms-EGFP 1-mo mice injected systemically with LPS (1mg/kg; <i>n</i> = 5) or vehicle (saline; <i>n</i> = 4) 8 h prior to sacrifice. Apoptotic cells were identified by pyknosis/karryorhexis. <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.g002" target="_blank">Fig 2A</a></b> was generated from data that was originally published as part of [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.ref009" target="_blank">9</a>]. (<b>B</b>) Weighted Ph capacity of microglia (in parts per unit, ppu) in control and LPS mice. (<b>C</b>) Number of microglial cells in control and LPS mice. (<b>D</b>) Ph/A coupling in the 1-mo mouse hippocampus (in fold change) during acute inflammatory challenge. (<b>E</b>) Experimental design and representative confocal z-stacks of the DG of PND21 Swiss mice fed during gestation and lactation with a diet balanced (Ω3 bal; <i>n</i> = 7) or deficient (Ω3 def; <i>n</i> = 7) in the omega 3 polyunsaturated fatty acid, a diet that induces chronic inflammation in the hippocampus. Microglia were labeled with Iba1 (cyan) and apoptotic nuclei were detected by pyknosis/karyorrhexis (white, DAPI). Arrows point to apoptotic cells engulfed by microglia (M). Scale bars = 50 μm; z = 22.5μm. (<b>F</b>) Number of apoptotic (pyknotic/karyorrhectic) cells in mice fed with Ω3 balanced and deficient diets. (<b>G</b>) Ph index in the PND21 hippocampus (in % of apoptotic cells) in mice fed with Ω3 balanced and deficient diets. (<b>H</b>) Weighted Ph capacity of microglia (in ppu) in PND21 mice. (<b>I</b>) Histogram showing the Ph capacity distribution of microglia (in % of cells) in PND21 mice. (<b>J</b>) Total number of microglial cells (Iba1⁺) in PND21 mice. (<b>K</b>) Ph/A coupling in PND21 mice. Bars represent mean ± SEM. * indicates <i>p</i> < 0.05 and ** indicates <i>p</i> < 0.01 by one-tail Student´s <i>t</i> test. Underlying data is shown in <b><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002466#pbio.1002466.s001" target="_blank">S1 Data</a></b>.</p