The pro-death role of Cited2 in stroke is regulated by E2F1/4 transcription factors.

We previously reported that the cell cycle-related cyclin-dependent kinase 4-retinoblastoma (RB) transcriptional corepressor pathway is essential for stroke-induced cell death both in vitro and in vivo However, how this signaling pathway induces cell death is unclear. Previously, we found that the cyclin-dependent kinase 4 pathway activates the pro-apoptotic transcriptional co-regulator Cited2 in vitro after DNA damage. In the present study, we report that Cited2 protein expression is also dramatically increased following stroke/ischemic insult. Critically, utilizing conditional knockout mice, we show that Cited2 is required for neuronal cell death, both in culture and in mice after ischemic insult. Importantly, determining the mechanism by which Cited2 levels are regulated, we found that E2F transcription factor (E2F) family members participate in Cited2 regulation. First, E2F1 expression induced Cited2 transcription, and E2F1 deficiency reduced Cited2 expression. Moreover, determining the potential E2F-binding regions on the Cited2 gene regulatory sequence by ChIP analysis, we provide evidence that E2F1/4 proteins bind to this DNA region. A luciferase reporter assay to probe the functional outcomes of this interaction revealed that E2F1 activates and E2F4 inhibits Cited2 transcription. Moreover, we identified the functional binding motif for E2F1 in the Cited2 gene promoter by demonstrating that mutation of this site dramatically reduces E2F1-mediated Cited2 transcription. Finally, E2F1 and E2F4 regulated Cited2 expression in neurons after stroke-related insults. Taken together, these results indicate that the E2F-Cited2 regulatory pathway is critically involved in stroke injury.

Pink1 regulates FKBP5 interaction with AKT/PHLPP and protects neurons from neurotoxin stress induced by MPP.

Loss of function mutations in the PTEN-induced putative kinase 1 (Pink1) gene have been linked with an autosomal recessive familial form of early onset Parkinson's disease (PD). However, the underlying mechanism(s) responsible for degeneration remains elusive. Presently, using co-immunoprecipitation in HEK (Human embryonic kidney) 293 cells, we show that Pink1 endogenously interacts with FK506-binding protein 51 (FKBP51 or FKBP5), FKBP5 and directly phosphorylates FKBP5 at Serine in an in vitro kinase assay. Both FKBP5 and Pink1 have been previously associated with protein kinase B (AKT) regulation. We provide evidence using primary cortical cultured neurons from Pink1-deficient mice that Pink1 increases AKT phosphorylation at Serine 473 (Ser473) challenged by 1-methyl-4-phenylpyridinium (MPP+ ) and that over-expression of FKBP5 using an adeno-associated virus delivery system negatively regulates AKT phosphorylation at Ser473 in murine-cultured cortical neurons. Interestingly, FKBP5 over-expression promotes death in response to MPP+ in the absence of Pink1. Conversely, shRNA-mediated knockdown of FKBP5 in cultured cortical neurons is protective and this effect is reversed with inhibition of AKT signaling. In addition, shRNA down-regulation of PH domain leucine-rich repeat protein phosphatase (PHLPP) in Pink1 WT neurons increases neuronal survival, while down-regulation of PHLPP in Pink1 KO rescues neuronal death in response to MPP+ . Finally, using co-immunoprecipitation, we show that FKBP5 interacts with the kinase AKT and phosphatase PHLPP. This interaction is increased in the absence of Pink1, both in Mouse Embryonic Fibroblasts (MEF) and in mouse brain tissue. Expression of kinase dead Pink1 (K219M) enhances FKBP5 interaction with both AKT and PHLPP. Overall, our results suggest a testable model by which Pink1 could regulate AKT through phosphorylation of FKBP5 and interaction of AKT with PHLPP. Our results suggest a potential mechanism by which PINK1-FKBP5 pathway contributes to neuronal death in PD. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Cdc25A Is a Critical Mediator of Ischemic Neuronal Death In Vitro and In Vivo.

Dysregulation of cell cycle machinery is implicated in a number of neuronal death contexts, including stroke. Increasing evidence suggests that cyclin-dependent kinases (Cdks) are inappropriately activated in mature neurons under ischemic stress conditions. We previously demonstrated a functional role for the cyclin D1/Cdk4/pRb (retinoblastoma tumor suppressor protein) pathway in delayed neuronal death induced by ischemia. However, the molecular signals leading to cyclin D/Cdk4/pRb activation following ischemic insult are presently not clear. Here, we investigate the cell division cycle 25 (Cdc25) dual-specificity phosphatases as potential upstream regulators of ischemic neuronal death and Cdk4 activation. We show that a pharmacologic inhibitor of Cdc25 family members (A, B, and C) protects mouse primary neurons from hypoxia-induced delayed death. The major contributor to the death process appears to be Cdc25A. shRNA-mediated knockdown of Cdc25A protects neurons in a delayed model of hypoxia-induced death in vitro Similar results were observed in vivo following global ischemia in the rat. In contrast, neurons singly or doubly deficient for Cdc25B/C were not significantly protective. We show that Cdc25A activity, but not level, is upregulated in vitro following hypoxia and global ischemic insult in vivo Finally, we show that shRNA targeting Cdc25A blocks Ser795 pRb phosphorylation. Overall, our results indicate a role for Cdc25A in delayed neuronal death mediated by ischemia.SIGNIFICANCE STATEMENT A major challenge in stroke is finding an effective neuroprotective strategy to treat cerebral ischemic injury. Cdc25 family member A (Cdc25A) is a phosphatase normally activated during cell division in proliferating cells. We found that Cdc25A is activated in neurons undergoing ischemic stress mediated by hypoxia in vitro and global cerebral ischemia in rats in vivo We show that pharmacologic or genetic inhibition of Cdc25A activity protects neurons from delayed death in vitro and in vivo Downregulation of Cdc25A led to reduction in retinoblastoma tumor suppressor protein (pRb) phosphorylation. An increase in pRb phosphorylation has been previously linked to ischemic neuronal death. Our results identify Cdc25A as a potential target for neuroprotectant strategy for the treatment of delayed ischemic neuronal death.

Hepatitis B virus X (HBX) protein upregulates ß-catenin in a human hepatic cell line by sequestering SIRT1 deacetylase.

Hepatitis B virus X (HBX) protein has been reported to induce upregulation of ß-catenin, a known proto-oncogene, in p53-knockout and p53-mutant hepatic cell lines both in a GSK-3ß-dependent manner and via interaction with adenomatous polyposis coli, which results in protection from ß-catenin degradation. In this study, we describe a novel mechanism for HBX-mediated upregulation of ß-catenin. We observed that HBX interacts with SIRT1, a class III histone deacetylase. Furthermore, the presence of HBX attenuated the interaction between SIRT1 and ß-catenin, leading to protection of ß-catenin from the inhibitory action of SIRT1. Reduction of SIRT1 with siRNA or suppression of SIRT1 activity with nicotinamide upregulated ß-catenin protein levels. In contrast, enhancement of SIRT1 activity with resveratrol reduced ß-catenin protein levels. Furthermore, in Hep3B cells stably expressing HBX, overexpression of SIRT1 or treatment with resveratrol enhanced sensitivity to doxorubicin-induced apoptosis, indicating that upregulation of SIRT1 could be a therapeutic strategy for HBV-related hepatocellular carcinoma. Based on these results, we propose that HBX upregulates ß-catenin by sequestering SIRT1, which leads to anticancer drug treatment resistance.