Fang Jin

Opposing roles for p16Ink4a and p19Arf in senescence and ageing caused by BubR1 insufficiency.

Expression of p16Ink4a and p19Arf increases with age in both rodent and human tissues. However, whether these tumour suppressors are effectors of ageing remains unclear, mainly because knockout mice lacking p16Ink4a or p19Arf die early of tumours. Here, we show that skeletal muscle and fat, two tissues that develop early ageing-associated phenotypes in response to BubR1 insufficiency, have high levels of p16Ink4a and p19Arf. Inactivation of p16Ink4a in BubR1-insufficient mice attenuates both cellular senescence and premature ageing in these tissues. Conversely, p19Arf inactivation exacerbates senescence and ageing in BubR1 mutant mice. Thus, we identify BubR1 insufficiency as a trigger for activation of the Cdkn2a locus in certain mouse tissues, and demonstrate that p16Ink4a is an effector and p19Arf an attenuator of senescence and ageing in these tissues.

Whole Chromosome Instability Caused by Bub1 Insufficiency Drives Tumorigenesis through Tumor Suppressor Gene Loss of Heterozygosity

Genetic alterations that promote chromosome missegregation have been proposed to drive tumorigenesis through loss of whole chromosomes containing key tumor suppressor genes. To test this unproven idea, we bred Bub1 mutant mice that inaccurately segregate their chromosomes onto p53(+/-), Apc(Min/+), Rb(+/-), or Pten(+/-) backgrounds. Bub1 insufficiency predisposed p53(+/-) mice to thymic lymphomas and Apc(Min/+) mice to colonic tumors. These tumors consistently lacked the nonmutated tumor suppressor allele but had gained a copy of the mutant allele. In contrast, Bub1 insufficiency had no impact on tumorigenesis in Rb(+/-) mice and inhibited prostatic intraepithelial neoplasia formation in Pten(+/-) mice. Thus, Bub1 insufficiency can drive tumor formation through tumor suppressor gene loss of heterozygosity, but only in restricted genetic and cellular contexts.

Cdc20 is critical for meiosis I and fertility of female mice.

Chromosome missegregation in germ cells is an important cause of unexplained infertility, miscarriages, and congenital birth defects in humans. However, the molecular defects that lead to production of aneuploid gametes are largely unknown. Cdc20, the activating subunit of the anaphase-promoting complex/cyclosome (APC/C), initiates sister-chromatid separation by ordering the destruction of two key anaphase inhibitors, cyclin B1 and securin, at the transition from metaphase to anaphase. The physiological significance and full repertoire of functions of mammalian Cdc20 are unclear at present, mainly because of the essential nature of this protein in cell cycle progression. To bypass this problem we generated hypomorphic mice that express low amounts of Cdc20. These mice are healthy and have a normal lifespan, but females produce either no or very few offspring, despite normal folliculogenesis and fertilization rates. When mated with wild-type males, hypomorphic females yield nearly normal numbers of fertilized eggs, but as these embryos develop, they become malformed and rarely reach the blastocyst stage. In exploring the underlying mechanism, we uncover that the vast majority of these embryos have abnormal chromosome numbers, primarily due to chromosome lagging and chromosome misalignment during meiosis I in the oocyte. Furthermore, cyclin B1, cyclin A2, and securin are inefficiently degraded in metaphase I; and anaphase I onset is markedly delayed. These results demonstrate that the physiologically effective threshold level of Cdc20 is high for female meiosis I and identify Cdc20 hypomorphism as a mechanism for chromosome missegregation and formation of aneuploid gametes.

Cdc20 hypomorphic mice fail to counteract de novo synthesis of cyclin B1 in mitosis

Low expression levels of Cdc20 result in chromatin bridging and chromosome misalignment, revealing a requirement for Cdc20 in efficient sister chromosome separation and chromosome–microtubule attachment.

Identification and Functional Characterization of a Novel CACNA1C-Mediated Cardiac Disorder Characterized by Prolonged QT Intervals with Hypertrophic Cardiomyopathy, Congenital Heart Defects, and Sudden Cardiac Death

Background—A portion of sudden cardiac deaths can be attributed to structural heart diseases, such as hypertrophic cardiomyopathy (HCM) or cardiac channelopathies such as long-QT syndrome (LQTS); however, the underlying molecular mechanisms are distinct. Here, we identify a novel CACNA1C missense mutation with mixed loss-of-function/gain-of-function responsible for a complex phenotype of LQTS, HCM, sudden cardiac death, and congenital heart defects. Methods and Results—Whole exome sequencing in combination with Ingenuity variant analysis was completed on 3 affected individuals and 1 unaffected individual from a large pedigree with concomitant LQTS, HCM, and congenital heart defects and identified a novel CACNA1C mutation, p.Arg518Cys, as the most likely candidate mutation. Mutational analysis of exon 12 of CACNA1C was completed on 5 additional patients with a similar phenotype of LQTS plus a personal or family history of HCM-like phenotypes and identified 2 additional pedigrees with mutations at the same position, p.Arg518Cys/His. Whole cell patch clamp technique was used to assess the electrophysiological effects of the identified mutations in CaV1.2 and revealed a complex phenotype, including loss of current density and inactivation in combination with increased window and late current. Conclusions—Through whole exome sequencing and expanded cohort screening, we identified a novel genetic substrate p.Arg518Cys/His-CACNA1C, in patients with a complex phenotype including LQTS, HCM, and congenital heart defects annotated as cardiac-only Timothy syndrome. Our electrophysiological studies, identification of mutations at the same amino acid position in multiple pedigrees, and cosegregation with disease in these pedigrees provide evidence that p.Arg518Cys/His is the pathogenic substrate for the observed phenotype.

CD8 T cell-initiated blood brain barrier disruption is independent of neutrophil support

Blood–brain barrier (BBB) disruption is a common feature of numerous neurologic disorders. A fundamental question in these diseases is the extent inflammatory immune cells contribute to CNS vascular permeability. We have previously shown that CD8 T cells play a critical role in initiating BBB disruption in the peptide-induced fatal syndrome model developed by our laboratory. However, myelomonocytic cells such as neutrophils have also been implicated in promoting CNS vascular permeability and functional deficit in murine models of neuroinflammatory disease. For this reason, we evaluated neutrophil depletion in a murine model of CD8 T cell-initiated BBB disruption by employing traditionally used anti-granulocyte receptor-1 mAb RB6-8C5 and Ly-6G–specific mAb 1A8. We report that CNS-infiltrating antiviral CD8 T cells express high levels of granulocyte receptor-1 protein and are depleted by treatment with RB6-8C5. Mice treated with RB6-8C5, but not 1A8, display: 1) intact BBB tight junction proteins; 2) reduced CNS vascular permeability visible by gadolinium-enhanced T1-weighted magnetic resonance imaging; and 3) preservation of motor function. These studies demonstrate that traditional methods of neutrophil depletion with RB6-8C5 are broadly immune ablating. Our data also provide evidence that CD8 T cells initiate disruption of BBB tight junction proteins and CNS vascular permeability in the absence of neutrophil support.

Immunovirotherapy with measles virus strains in combination with anti–PD-1 antibody blockade enhances antitumor activity in glioblastoma treatment

BackgroundnGlioblastoma (GBM) is the most common primary malignant brain tumor and has a dismal prognosis. Measles virus (MV) therapy of GBM is a promising strategy due to preclinical efficacy, excellent clinical safety, and its ability to evoke antitumor pro-inflammatory responses. We hypothesized that combining anti- programmed cell death protein 1 (anti-PD-1) blockade and MV therapy can overcome immunosuppression and enhance immune effector cell responses against GBM, thus improving therapeutic outcome.nnnMethodsnIn vitro assays of MV infection of glioma cells and infected glioma cells with mouse microglia ± aPD-1 blockade were established to assess damage associated molecular pattern (DAMP) molecule production, migration, and pro-inflammatory effects. C57BL/6 or athymic mice bearing syngeneic orthotopic GL261 gliomas were treated with MV, aPD-1, and combination treatment. T2* weighted immune cell-specific MRI and fluorescence activated cell sorting (FACS) analysis of treated mouse brains was used to examine adaptive immune responses following therapy.nnnResultsnIn vitro, MV infection induced human GBM cell secretion of DAMP (high-mobility group protein 1, heat shock protein 90) and upregulated programmed cell death ligand 1 (PD-L1). MV infection of GL261 murine glioma cells resulted in a pro-inflammatory response and increased migration of BV2 microglia. In vivo, MV+aPD-1 therapy synergistically enhanced survival of C57BL/6 mice bearing syngeneic orthotopic GL261 gliomas. MRI showed increased inflammatory cell influx into the brains of mice treated with MV+aPD-1; FACS analysis confirmed increased T-cell influx predominantly consisting of activated CD8+ T cells.nnnConclusionsnThis report demonstrates that oncolytic measles virotherapy in combination with aPD-1 blockade significantly improves survival outcome in a syngeneic GBM model and supports the potential of clinical/translational strategies combining MV with αPD-1 therapy in GBM treatment.

The yin and yang of the Cdkn2a locus in senescence and aging

Senescence of cultured cells involves activation of the p19Arf-p53 and the p16Ink4a-Rb tumor suppressor pathways. This, together with the observation that p19Arf and p16Ink4a expression increases with age in many tissues of humans and rodents, led to the speculation that these pathways drive in vivo senescence and natural aging. However, it has been difficult to test this hypothesis using a mammalian model system because inactivation of either of these pathways results in early death from tumors. One approach to bypass this problem would be to inactivate these pathways in a murine segmental progeria model such as mice that express low amounts of the mitotic checkpoint protein BubR1 (BubR1 hypomorphic mice). These mice have a five-fold reduced lifespan and develop a variety of early-aging associated phenotypes including cachetic dwarfism, skeletal muscle degeneration, cataracts, arterial stiffening, (subcutaneous) fat loss, reduced stress tolerance and impaired wound healing. Importantly, BubR1 hypomorphism elevates both p16Ink4a and p19Arf expression in skeletal muscle and fat. Inactivation of p16Ink4a in BubR1 mutant mice delays both cellular senescence and aging specifically in these tissues. Surprisingly, however, inactivation of p19Arf has the opposite effect; it exacerbates in vivo senescence and aging in skeletal muscle and fat. These mouse studies suggest that p16Ink4a is indeed an effector of aging and in vivo senescence, but p19Arf an attenuator. Thus, the role of the p19Arf-p53 pathway in aging and in vivo senescence seems far more complex than previously anticipated.

Histone demethylase JMJD2A drives prostate tumorigenesis through transcription factor ETV1

Histone demethylase upregulation has been observed in human cancers, yet it is unknown whether this is a bystander event or a driver of tumorigenesis. We found that overexpression of lysine-specific demethylase 4A (KDM4A, also known as JMJD2A) was positively correlated with Gleason score and metastasis in human prostate tumors. Overexpression of JMJD2A resulted in the development of prostatic intraepithelial neoplasia in mice, demonstrating that JMJD2A can initiate prostate cancer development. Moreover, combined overexpression of JMJD2A and the ETS transcription factor ETV1, a JMJD2A-binding protein, resulted in prostate carcinoma formation in mice haplodeficient for the phosphatase and tensin homolog (Pten) tumor-suppressor gene. Additionally, JMJD2A cooperated with ETV1 to increase expression of yes associated protein 1 (YAP1), a Hippo pathway component that itself was associated with prostate tumor aggressiveness. ETV1 facilitated the recruitment of JMJD2A to the YAP1 promoter, leading to changes in histone lysine methylation in a human prostate cancer cell line. Further, YAP1 expression largely rescued the growth inhibitory effects of JMJD2A depletion in prostate cancer cells, indicating that YAP1 is a downstream effector of JMJD2A. Taken together, these data reveal a JMJD2A/ETV1/YAP1 axis that promotes prostate cancer initiation and that may be a suitable target for therapeutic inhibition.

Perforin competent CD8 T cells are sufficient to cause immune-mediated blood-brain barrier disruption.

Numerous neurological disorders are characterized by central nervous system (CNS) vascular permeability. However, the underlying contribution of inflammatory-derived factors leading to pathology associated with blood-brain barrier (BBB) disruption remains poorly understood. In order to address this, we developed an inducible model of BBB disruption using a variation of the Theilers murine encephalomyelitis virus (TMEV) model of multiple sclerosis. This peptide induced fatal syndrome (PIFS) model is initiated by virus-specific CD8 T cells and results in severe CNS vascular permeability and death in the C57BL/6 mouse strain. While perforin is required for BBB disruption, the cellular source of perforin has remained unidentified. In addition to CD8 T cells, various innate immune cells also express perforin and therefore could also contribute to BBB disruption. To investigate this, we isolated the CD8 T cell as the sole perforin-expressing cell type in the PIFS model through adoptive transfer techniques. We determined that C57BL/6 perforin−/− mice reconstituted with perforin competent CD8 T cells and induced to undergo PIFS exhibited: 1) heightened CNS vascular permeability, 2) increased astrocyte activation as measured by GFAP expression, and 3) loss of linear organization of BBB tight junction proteins claudin-5 and occludin in areas of CNS vascular permeability when compared to mock-treated controls. These results are consistent with the characteristics associated with PIFS in perforin competent mice. Therefore, CD8 T cells are sufficient as a sole perforin-expressing cell type to cause BBB disruption in the PIFS model.