Sirt1 regulates glial progenitor proliferation and regeneration in white matter after neonatal brain injury

Regenerative processes in brain pathologies require the production of distinct neural cell populations from endogenous progenitor cells. We have previously demonstrated that oligodendrocyte progenitor cell (OPC) proliferation is crucial for oligodendrocyte (OL) regeneration in a mouse model of neonatal hypoxia (HX) that reproduces diffuse white matter injury (DWMI) of premature infants. Here we identify the histone deacetylase Sirt1 as a Cdk2 regulator in OPC proliferation and response to HX. HX enhances Sirt1 and Sirt1/Cdk2 complex formation through HIF1α activation. Sirt1 deacetylates retinoblastoma (Rb) in the Rb/E2F1 complex, leading to dissociation of E2F1 and enhanced OPC proliferation. Sirt1 knockdown in culture and its targeted ablation in vivo suppresses basal and HX-induced OPC proliferation. Inhibition of Sirt1 also promotes OPC differentiation after HX. Our results indicate that Sirt1 is an essential regulator of OPC proliferation and OL regeneration after neonatal brain injury. Therefore, enhancing Sirt1 activity may promote OL recovery after DWMI.

Supp. Figure 4. Sirt1 expression in various glia cell types in white matter. Confocal images from NX and HX white matter of WT mice at P18. WM: white matter. Dotted lines delineate white matter. Scale bar = 100µm. Cells were stained with anti-Sirt1 and colabeled with anti-CC1 (a), anti-GFAP (d) and anti-Iba1 (g) antibodies. Inserts show an example of Sirt1 expression in a CC1 + , GFAP + , and Iba1 + at higher magnification. Graphs present the percentages of Sirt1 expressing mature OLs (b, c), astrocytes (e, f) and microglia (h, i). No changes in these cell types are observed after HX. Histograms show mean + SEM. Number in parentheses within bar indicates number of samples, n=4 brains for each condition.

Demyelination exerts an opposite effect on Sirt1 expression in progenitor cells in white matter.
Confocal images of Sirt1 distribution in control and demyelinated white matter at P60 (a, f). Scale bar = 100µm. Progenitor cells were labeled with anti-NG2 antibody, mature OLs with anti-CC1, astrocytes with anti-GFAP, and reactive microglia with anti-CD68 antibodies. Graphs present the percentage of Sirt1 + cells (b), proliferating Sirt1 + cells (c), NG2 + Sirt1 + (d), and NG2 + Sirt1 + BrdU + (e) cells in white matter and , total percentage of glial cell types (g). The percentage of mature OLs expressing Sirt1 decreases (h), whereas the percentages of Sirt1 + GFAP + (i) and Sirt1 + CD68 + (j) remain unchanged after HX. All histograms show mean + SEM.
Number in parentheses within bar indicates number of samples, n=5 brains per condition, and for each antibody.
Supp. Figure 6. Inactivation of Sirt1 in SVZ-derived OPCs does not alter the effects of neonatal HX on their proliferation. Cells isolated from NX and HX SVZ at P18 were transfected with Sirt1 siRNA and with scrambled control. OPC proliferation was assessed by double labeling with anti-Ki67 (a,b) and anti-NG2 (c,d). Scale bar = 100µm. Graphs represent the percentage of Ki67 + (b) and NG2 + (d) cells in NX and HX cultures treated with scrambled control and Sirt1 siRNA. In control and Sirt1 siRNA-transfected cultures, HX upregulates cell proliferation (b) and the percentage of NG2 + progenitor cells (d). Five NX and 4 HX brains. Histograms show mean + SEM of three cultures per condition.
Supp. Figure 7. Characterization of cell proliferation in Sirt1 F/F ;PDGFR CreER ;Rosa YFP mice under normal physiological conditions. Confocal images from white matter of NX and HX WT and Sirt1 F/F ;PDGFR CreER ;Rosa YFP mice (a,c). Cells were stained with anti-Sirt1, anti-GFP (to detect YFP) antibodies and DAPI. Dotted lines delineate white matter. WM = white matter. Scale bar = 100µm. (b) Tamoxifen injection causes a reduction of total Sirt1 + cells in Sirt1 F/F ;PDGFR CreER ;Rosa YFP mice as compared to the same genotype without TMX (b), as well as to their WT littermates (d). The percentage of Sirt1 + YFP + cells is reduced in white matter of Sirt1 F/F ;PDGFR CreER ;Rosa YFP mice (d). Number in parentheses within bar indicates number of samples. Histograms show mean + SEM, n=3 brains per condition, for each antibody. Rb/E2Fs (f) complexes in NX and HX white matter (P18) analyzed by IP and Western blot. (c,e,g) Graphs represent relative protein levels of these complexes in NX and HX white matter. HX increases Cdk2/CycE, Cdk1/CycE, Rb/Cdk2, and pRb(807/811)/Cdk2 complex formation, whereas expression of the Rb/E2F1 complex is reduced. (mean + SEM, n=3 brains for each group, actin serves as control) Supp. Figure 9. Neonatal HX induces Sirt1 translocation to cytoplasm. (a) Confocal images of white matter demonstrate predominantly nuclear intensity of Sirt1 staining in NX, which decreases after HX. (b) Intensity of Sirt1 staining is expressed in arbitrary units (a.u.) immediately (0 min), or 15, 30, 60, and 120 min after HX (Mean + SEM, n=4 brains for each condition, for each time point). (c) Graph represents percentage of Sirt1 + cells displaying cytoplasmic, nuclear, or nucleo-cytoplasmic Sirt1 in NX and HX (Mean + SEM, 2 NX, 6 HX brains). (d) Representative Western blot of subcellular fractions from white matter. Sirt1 is predominantly found in the nuclear fraction in NX, and in cytoplasm after HX. Alpha-tubulin and phospho-histone3 serves as controls for each fraction (n=3 brains for each condition). (e) Graph represents lower Sirt1 expression in nucleus and higher Sirt1 level in cytoplasm after HX. Mean + SEM (2 NX and 6 HX brains). (f) Localization of Sirt1 in cellular compartments co-stained with anti-MEK 1/2, -CytC, and -Glogin-97 antibodies demonstrates that HX increases Sirt1 expression in cytoplasm and mitochondria.
Supp. Figure 10. Mechanism of Sirt1-induced OPC proliferation after neonatal HX. Illustration of the molecular events involved in Sirt1-mediated OPC proliferation induced by HX. HX increases levels of Hif1α and NAD, which respectively enhance Sirt1 expression and activity. Activated Sirt1 deacetylates Cdk2 and Rb. The deacetylation of Cdk2 leads to Sirt1 phosphorylation at Ser47. Rb deacetylation by Sirt1 and its phosphorylation by Cdk2 causes dissociation of the Rb/E2F1 complex and release of the E2F1 transcription factor. Higher levels of unbound E2F1 promote OPC cell cycle entry and maintain their proliferative state.