TOR-mediated autophagy regulates cell death in Drosophila neurodegenerative disease

Autophagy can help cells to dispose of damaged proteins, alleviating cell death in genetic models of Huntington’s disease and retinal degradation

Enterocyte-specific inactivation of SIRT1 reduces tumor load in the APC(+/min) mouse model.

SIRT1 is a mammalian NAD(+)-dependent histone deacetylase implicated in metabolism, development, aging and tumorigenesis. Prior studies that examined the effect of enterocyte-specific overexpression and global deletion of SIRT1 on polyp formation in the intestines of APC(+/min) mice, a commonly used model for intestinal tumorigenesis, yielded conflicting results, supporting either tumor-suppressive or tumor-promoting roles for SIRT1, respectively. In order to resolve the controversy emerging from these prior in vivo studies, in the present report we examined the effect of SIRT1 deficiency confined to the intestines, avoiding the systemic perturbations such as growth retardation seen with global SIRT1 deletion. We crossed APC(+/min) mice with mice bearing enterocyte-specific inactivation of SIRT1 and examined polyp development in the progeny. We found that SIRT1-inactivation reduced total polyp surface (9.3 mm(2) vs. 23.3 mm(2), p = 0.01), average polyp size (0.24 mm(2) vs. 0.51 mm(2), p = 0.005) and the number of polyps >0.5 mm in diameter (14 vs. 23, p = 0.04), indicating that SIRT1 affects both the number and size of tumors. Additionally, tumors in SIRT1-deficient mice exhibited markedly increased numbers of cells undergoing apoptosis, suggesting that SIRT1 contributes to tumor growth by enabling survival of tumor cells. Our results indicate that SIRT1 acts as a tumor promoter in the APC(+/min) mouse model of intestinal tumorigenesis

Enterocyte-specific SIRT1 deletion increases the rate of apoptosis in the intestinal tumors of APC^+/min mice.

<p>(A) A representative western blot showing expression of the wild-type SIRT1 protein in the intestinal epithelium of APC^+/min SIRT1^+/+ mice (first lane) and a truncated version in the epithelium of APC^+/min SIRT1^−/− mice (second lane). Liver cells of the APC^+/min SIRT1^−/− mice (third lane), as well as other tissues (not shown) express the wild-type protein. Pan-actin immunostaining served as a loading control. (B) Representative photographs of unfixed small intestines (distal segments) showing similar polyp number for APC^+/min SIRT1^+/+ (right) and APC^+/min SIRT1^−/− (left) mice. Scale bar indicates 5 mm length. (C) Representative photomicrographs of typical polyps from the two groups of mice, stained with hematoxylin and eosin. Scale bar indicates 100 µm length. (D) Representative photomicrographs and a bar graph showing Ki-67 immunohistochemical staining of polyp sections from APC^+/min SIRT1^+/+ (left) and APC^+/min SIRT1^−/− (middle) mice. Scale bar indicates 100 µm length. Proliferation index for polyps from APC^+/min SIRT1^+/+ and APC^+/min SIRT1^−/− mice (right), expressed as a fraction of Ki-67 positive cells within each polyp. Bars represent means ± SEM, n = 20 polyps per group. No statistically significant difference was observed. (E) Representative photomicrographs and a bar graph showing activated (cleaved) caspase-3 immunohistochemical staining of polyp sections from APC^+/min SIRT1^+/+ (left) and APC^+/min SIRT1^−/− (middle) mice. Scale bar indicates 100 µm length. Absolute numbers of apoptotic (caspase-3 positive) cells per high power field (400 x) for polyps from SIRT1^+/+ and SIRT1^−/− mice (right). Bars represent means ± SEM, n = 25 polyps per group. ***p<0.001.</p

SIRT1 inactivation inhibits Wnt and promotes p53 a pathway.

<p><b>A)</b> Representative photomicrographs showing β-catenin immunohistochemical staining of intestinal sections from APC^+/min SIRT1^+/+ and APC^+/min SIRT1^−/− mice. Scale bar indicates 50 µm length. Polyps from both groups demonstrate intense cytoplasmic and nuclear staining for β-catenin. B) Bar graph with frequencies of crypts with indicated number of cells with nuclear β-catenin staining in normal appearing mucosa of APC^+/min SIRT1^+/+ and APC^+/min SIRT1^−/− mice. The average number of β-catenin positive cells per crypt is reduced in APC^+/min SIRT1^−/− mice compared to APC^+/min SIRT1^+/+ animals 2.1±1.2 vs 3.2±1.3 (p = 1.5×10⁻⁵, at least 100 crypts per genotype were scored). C) Representative photomicrographs of normal appearing mucosa from APC^+/min SIRT1^+/+ and APC^+/min SIRT1^−/− mice demonstrating a reduced number of basal cells with nuclear β-catenin in APC^+/min SIRT1^−/− animals. Arrows are directed toward representative basal cells with nuclear β-catenin. Scale bar indicates 25 µm length. D) Inhibition of SIRT1 with EX527 (2 µM) and cambinol (50 µM) reduces activity of the TCF/LEF driven firefly luciferase reporter (TOP FLASH) transiently transfected into SW480 cells. TOP FLASH luciferase reporter contains minimal promoter along with three TCF binding sites, which have been mutated in FOP FLASH reporters. Bars represent means ± SEM of relative firefly luciferase activity normalized to renilla luciferase activity from the thymidine kinase promoter-driven renilla reporter that was co-transfected with TOP FLASH and FOP FLASH reporters. Each transfection is carried out in quadruplicate. **p<0.01. E) SW480 cells with shRNA-mediated downregulation of SIRT1 (top: western blot for SIRT1 and actin) exhibit reduced activity of the transiently transfected TCF/LEF driven firefly luciferase reporter (TOP FLASH). Bars represent means ± SEM of relative firefly luciferase activity normalized to renilla luciferase activity from the thymidine kinase promoter-driven renilla reporter that was co-transfected with TOP FLASH and FOP FLASH reporters. Each transfection is carried out in quadruplicate. *p<0.05. F) Immunoblot for acetyl-p53, p53 and actin from cells treated with etoposide, SIRT1 inhibitor EX527 or the combination of the two drugs. Inhibition of SIRT1 leads to p53 hyperacetylation in Hct116 colon cancer cell line. Hyperacetylation of p53 is observed in cells treated with EX527 and a combination of EX527 and etoposide. Etoposide alone modestly induces p53 acetylation.</p

Sir2 suppresses transcription-mediated displacement of Mcm2-7 replicative helicases at the ribosomal DNA repeats.

Repetitive DNA sequences within eukaryotic heterochromatin are poorly transcribed and replicate late in S-phase. In Saccharomyces cerevisiae, the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA arrays (rDNA). Despite the widespread association between transcription and replication timing, it remains unclear how transcription might impinge on replication, or vice versa. Here we show that, when silencing of an RNA polymerase II (RNA Pol II)-transcribed non-coding RNA at the rDNA is disrupted by SIR2 deletion, RNA polymerase pushes and thereby relocalizes replicative Mcm2-7 helicases away from their loading sites to an adjacent region with low nucleosome occupancy, and this relocalization is associated with increased rDNA origin efficiency. Our results suggest a model in which two of the major defining features of heterochromatin, transcriptional silencing and late replication, are mechanistically linked through suppression of polymerase-mediated displacement of replication initiation complexes

Discovery of Selective SIRT2 Inhibitors as Therapeutic Agents in B-Cell Lymphoma and Other Malignancies

Genetic ablation as well as pharmacological inhibition of sirtuin 2 (SIRT2), an NAD+-dependent protein deacylase, have therapeutic effects in various cancers and neurodegenerative diseases. Previously, we described the discovery of a dual SIRT1/SIRT2 inhibitor called cambinol (IC50 56 and 59 &micro;M, respectively), which showed cytotoxic activity against cancer cells in vitro and a marked anti-proliferative effect in a Burkitt lymphoma mouse xenograft model. A number of recent studies have shown a protective effect of SIRT1 and SIRT3 in neurodegenerative and metabolic diseases as well as in certain cancers prompting us to initiate a medicinal chemistry effort to develop cambinol-based SIRT2-specific inhibitors devoid of SIRT1 or SIRT3 modulating activity. Here we describe potent cambinol-based SIRT2 inhibitors, several of which show potency of ~600 nM with &gt;300 to &gt;800-fold selectivity over SIRT1 and 3, respectively. In vitro, these inhibitors are found to be toxic to lymphoma and epithelial cancer cell lines. In particular, compounds 55 (IC50 SIRT2 0.25 &micro;M and &lt;25% inhibition at 50 &micro;M against SIRT1 and SIRT3) and 56 (IC50 SIRT2 0.78 &micro;M and &lt;25% inhibition at 50 &micro;M against SIRT1 and SIRT3) showed apoptotic as well as strong anti-proliferative properties against B-cell lymphoma cells

Perturbed maintenance of transcriptional repression on the inactive X-chromosome in the mouse brain after Xist deletion

Abstract Background The long noncoding RNA Xist is critical for initiation and establishment of X-chromosome inactivation during embryogenesis in mammals, but it is unclear whether its continued expression is required for maintaining X-inactivation in vivo. Results By using an inactive X-chromosome-linked MeCP2-GFP reporter, which allowed us to enumerate reactivation events in the mouse brain even when they occur in very few cells, we found that deletion of Xist in the brain after establishment of X-chromosome inactivation leads to reactivation in 2–5% of neurons and in a smaller fraction of astrocytes. In contrast to global loss of both H3 lysine 27 trimethylation (H3K27m3) and histone H2A lysine 119 monoubiquitylation (H2AK119ub1) we observed upon Xist deletion, alterations in CpG methylation were subtle, and this was mirrored by only minor alterations in X-chromosome-wide gene expression levels, with highly expressed genes more prone to both derepression and demethylation compared to genes with low expression level. Conclusion Our results demonstrate that Xist plays a role in the maintenance of histone repressive marks, DNA methylation and transcriptional repression on the inactive X-chromosome, but that partial loss of X-dosage compensation in the absence of Xist in the brain is well tolerated