Cell cycle regulation and TGF-beta signaling in Ezh2-shRNA treated cells.

<p>(<b>A</b>) Real-time quantitative PCR analysis of total RNA extracted from nc-shRNA (control; C) and Ezh2-shRNA treated NSCs and pOLs shows increase in the transcript level of cell cycle arrest genes, however the transcript level of growth differentiation factors (Gdfs) belonging to TGF-beta superfamily increased only in Ezh2-shRNA treated NSCs (consistent with ChIP-seq data)<b>.</b> (<b>B</b>) Western blot confirms the upregulation of cell cycle arrest proteins (e.g. p16, a product of Cdkn2a) and the activation of TGF-beta signaling pathway (Smads) in Ezh2-shRNA treated NSCs as compared to the control (<b>C</b>).</p

NSCs and pOLs express Ezh2 in mouse brain.

<p>(A) Hoechst (nuclear) stained section of mouse brain with rectangles indicating the subventricular zone (B) and the corpus callosum (C). (B–B”) Immunohistochemistry of mouse brain sections containing the subventricular zone (SVZ). (B’)  =  anti-nestin, (B”)  =  anti-Ezh2, (B”’) overlay of (B’) and (B”) and Hoechst nuclear staining (blue). (C–C”’) Immunohistochemistry of mouse brain sections containing the corpus callosum. (C’)  =  anti-RIP (RIP  =  oligodendrocyte marker), (C”)  =  anti Ezh2, (C”’) overlay of (C’) and (C”) and Hoechst nuclear staining. (<b>E</b>–<b>H</b>) <b><i>OPCs in corpus callosum express Ezh2 during demyelination and remyelination in cuprizone fed mice.</i></b> (<b>D</b>) Hoechst (nuclear) stained section of mouse brain with corpus callosum indicated (<b>E</b>, <b>F</b>). (<b>E</b>–<b>E”’</b>) Immunohistochemistry on corpus callosum sections of five weeks cuprizone fed mice (five weeks of demyelination  = 5WD) (<b>E’</b>)  =  anti-RIP, (<b>E”</b>)  =  anti-Ezh2, (<b>E”</b>’)  =  overlay of (<b>E’</b> and <b>E”</b>) and Hoechst nuclear staining (blue). (<b>F</b>–<b>F”’</b>) Immunohistochemistry on corpus callosum section of brain of mouse kept for five weeks on cuprizone diet followed by two weeks on normal diet (two weeks remyelination  = 2WR), (<b>F’</b>)  =  anti-RIP, (<b>F”</b>) anti-Ezh2, (<b>F”’</b>)  =  overlay of (<b>F’</b>) and (<b>F”</b>) and nuclear staining. (<b>G</b>) Western blot showing the expression of Ezh2 protein (91 kDa band) <i>in vivo</i>, in the tissue lysates prepared from SVZ and hippocampus of adult mouse brain, and in <i>in vitro</i> cultured mouse adult NSCs. (<b>H</b>) Western blot showing the loading control (beta-actin), Ezh2 and H3K27me3 protein bands in lysates from corpus callosum of control, 5WD and 2WR mouse brains. In the corpus callosum of control mouse brain, the Ezh2 band was below the detection level. (<b>I</b>) Western blot on the same lysates used in (<b>H</b>) but immunoblotted with an antibody against NG2 (an early oligodendrocyte marker). (Calibration bars in (<b>A</b>) and (<b>D</b>) represent 250 micrometers; calibration bars in (<b>B’</b>) and (<b>E’</b>) represent 100 micrometers and are valid for (<b>B’</b>–<b>C”’</b>) and (<b>E’</b>–<b>F”’</b>)).</p

Acute suppression of Ezh2 induces apoptosis in pOLs.

<p>(<b>A</b>, <b>B</b>) Phase contrast images show that pOLs retract their projections when treated with Ezh2-shRNA. (<b>C</b>, <b>D</b>) Immunostaining for apoptosis marker, cleaved caspase 3 (green) shows increase in cell death in pOLs cultures treated with Ezh2-shRNA compared to control cultures (nc-shRNA). (<b>E</b>, <b>F</b>) Co-immunostaining using anti-cleaved caspase 3 and anti-RIP (an oligodendrocyte maker) shows restriction of cell extensions and increased cell death, in cultures treated with Ezh2-shRNA. (<b>G</b>) Western blot of protein samples prepared after 24 hours of transfection shows a decrease in Ezh2 and a strong increase in cleaved caspase 3 bands in pOLs treated with Ezh2-shRNA, compared to the cultures treated with nc-shRNA (control). PCNA (a proliferation marker) expression remained about the same in both conditions<b>.</b> (<b>H</b>) Quantification of apoptotic (cleaved caspase 3-positive) and differentiating (RIP-positive) cells in Ezh2-shRNA treated and control (nc-shRNA) pOLs. Bar graphs represent mean of three independent experiments ± S.D. (Calibration bar in (<b>A</b>) represents 100 micrometers, which is valid for all the photomicrographs (<b>A</b>–<b>F</b>); ** in (<b>H</b>) represents p<0.01).</p

Target genes of Ezh2 in NSCs and pOLs.

<p>(<b>A</b>–<b>B”</b>) Association of input genomic regions with the transcription start site (TSS) of all the genes putatively regulated by the genomic regions in NSCs (<b>A</b>–<b>A”</b>) and pOLs (<b>B</b>–<b>B”</b>). (<b>A</b>, <b>B</b>) “Number of associated genes per region" graphs show how many genes in each genomic region is assigned as putatively regulating based on the association rule used. (Details of association rule see online: <a href="http://great.stanford.edu/public/html/help/index.php?title=Association_Rules" target="_blank">http://great.stanford.edu/public/html/help/index.php?title=Association_Rules</a>). (<b>A’</b>, <b>B’</b>) Graphs show the distance between input regions and their putatively regulated genes (i.e. binned by orientation and distance to TSS). The distances are divided into four separate bins: 0 to 5 kb, 5 kb to 50 kb, 50 kb to 500 kb, and >500 kb. All associations precisely at 0 (i.e. on the TSS) are split evenly between the [−5 kb, 0] and [0,5 kb] bins. (<b>A”</b>, <b>B”</b>) Binned by absolute distance to TSS (only the distance to TSS is considered, not the orientation). Functional gene ontology (GO) analysis of Ezh2 target genes (DAVID online tool) in NSCs (<b>C</b>) and pOLs (<b>D</b>). The bar graphs represent the percentage of DAVID genes (since DAVID converts user input gene IDs to corresponding DAVID gene IDs) in the list associated with particular annotation term. The corresponding line graphs represent –log10 transformation of the modified Fisher Exact P-value. Fisher Exact is adopted to measure the gene-enrichment in annotation terms. The smaller the P-value, the more enriched a GO term is for genes of the given assay. (<b>E</b>) Comparison of canonical signaling pathways enriched in Ezh2 target genes in NSCs and pOLs.</p

Acute reduction of Ezh2 induces apoptosis in NSCs.

<p>(A) Phase contrast images show that NSCs treated with Ezh2-shRNA cannot form normal neurospheres in proliferation medium. Immunostaining of dissociated neurospheres in proliferation medium shows a decrease in proliferation and increase in cell death after using Ezh2-shRNA compared to the control (nc-shRNA). (<b>B</b>) Quantification of proliferating (Ki67 positive) and apoptotic (cleaved caspase 3 positive) NSCs in control and NSC cultures treated with Ezh2-shRNA. (<b>C</b>) Western blot of protein samples prepared 48 hours after transfection shows a decrease in Ezh2 and PCNA (a proliferation marker) and an increase in cleaved caspase 3 bands in NSCs treated with Ezh2-shRNA. Bars represent mean of three independent experiments ± S.D.; Student-t-test was used to calculate significance. p<0.05 was considered significant. (Calibration bar in A represents 100 micrometers and 5 micrometers, which are valid for the phase contrast, propidium iodide and the Hoechst/Ki67, cleaved caspase 3, cleaved caspase 3/Hoechst/nestin photomicrographs, respectively (<b>A</b>); ** in (<b>B</b>) represents p<0.01).</p

Chromatin immunoprecipitation (ChIP) using IgG isotype control, anti-Ezh2, and anti-H3K27me3 antibodies.

<p>(<b>A</b>) ChIP-Ezh2-quantitative PCR (qPCR) analysis. The graph shows quantitative PCR on DNA purified from ChIP-Ezh2, using promoter primers for genes associated with selected peaks from the ChIP-Seq data. In NSCs ChIP-Ezh2, there is high enrichment (Ezh2 occupancy) of neuronal, astrocytic and oligodendrocytic lineages determining genes while in pOLs ChIP-Ezh2 only neuronal and astrocytic lineage specific genes show enrichment (occupancy by Ezh2). (<b>B</b>) ChIP-H3K27me3-qPCR analysis. A similar pattern of gene enrichment can be observed in PCR analysis on DNA purified from ChIP-H3K27me3 (trimethylation of H3K27 is a functional mark of Ezh2): in NSCs, there is high enrichment (trimethylation of H3K27) of neuronal, astrocytic and oligodendrocytic lineages determining genes while in pOLs only neuronal and astrocytic lineage specific genes show high enrichment (higher level of trimethylation of H3K27). <i>Rpl32</i> (the gene encoding the 60S ribosomal protein L32, a non-target of Ezh2) was used as a negative control. <i>Acute Ezh2 reduction in NSCs and pOLs mediated by Ezh2-shRNA.</i> (<b>C</b>) Western blot shows the reduction in Ezh2 protein in comparison to control. Lysates were prepared after <i>in vitro</i> transfection with Ezh2-shRNA and non-coding shRNA (nc-shRNA) as control, in both NSCs and pOLs. Reduction in Ezh2 protein resulted in a decrease in the global level of H3K27 tri-methylation, an Ezh2-associated gene repression mark (17 kDa band). (<b>D</b>) Derepression of tested Ezh2 target genes after knocking down Ezh2 protein expression in NSCs and pOLs. The transcript levels were quantified by real-time PCR, normalized to GAPDH (housekeeping gene) and represented as a fold change between nc-shRNA (control) and Ezh2-shRNA in both NSCs and pOLs. Bars represent mean of three independent experiments ± S.D.</p

Target genes of Ezh2 in NSCs and pOLs: genome browser views.

<p>ChIP-Seq signals (peaks) are shown for anti-Ezh2 and IgG control antibodies after loading BigWig files on UCSC genome browser. (<b>A</b>) UCSC genome browser view of the known polycomb group protein target region, the Hox locus (located on chromosome 6 in the mouse), after loading three tracks: ChIP-Seq-IgG control, ChIP-Seq-NSCs and ChIP-Seq-pOLs. (<b>B</b>) ChIP-Seq–NSCs tracks show, neuronal, oligodendrocytic and astrocytic lineage determining genes as the targets of Ezh2. Upon differentiation of NSCs towards oligodendrocytes, as shown in the ChIP-Seq-pOLs tracks, Ezh2 releases the oligodendrocytic lineage determining genes, while still suppressing neuronal and astrocytic lineage genes.</p

Additional file 1: Table S2. of Telomere shortening leads to an acceleration of synucleinopathy and impaired microglia response in a genetic mouse model

Primer sequences which were used for RT-PCR. (DOC 40 kb

Pogonia minor Makino

原著和名: ヤマトキサウ科名: ラン科 = Orchidaceae採集地: 熊本県 人吉市 田野 (肥後 人吉市 田野)採集日: 1974/8/9採集者: 萩庭丈壽整理番号: JH003082国立科学博物館整理番号: TNS-VS-95308

Additional file 3: Figure S2. of Telomere shortening leads to an acceleration of synucleinopathy and impaired microglia response in a genetic mouse model

Classification and scoring of phospho-α-synuclein and PK-PET Blot. (A) Classification of phospho-α-synuclein staining into four different scores. Representative pictures for scoring. Score 0: no p-α-synuclein staining, score 1: little staining in brainstem and DpMe, score 2: strong staining in brainstem and DpMe, score 3: strong p-α-synuclein staining in brainstem, DpMe, and cerebellum indicating severe disease progression. (B) Scoring to classify PK-PET Blot. Score 0: no PK resistant aggregates, score 1: light aggregates in brainstem and Deep Mesencepahlic nucleus (DpMe), score 2: clear PK resistant aggregates in brainstem and DpMe, score 3: dominant aggregates in brainstem and DpMe. Score 4: Prominent aggregates in brainstem, DpMe and cerebellum. (PDF 125 kb