Darren J. Baker

Clearance of p16 Ink4a -positive senescent cells delays ageing-associated disorders

Advanced age is the main risk factor for most chronic diseases and functional deficits in humans, but the fundamental mechanisms that drive ageing remain largely unknown, impeding the development of interventions that might delay or prevent age-related disorders and maximize healthy lifespan. Cellular senescence, which halts the proliferation of damaged or dysfunctional cells, is an important mechanism to constrain the malignant progression of tumour cells. Senescent cells accumulate in various tissues and organs with ageing and have been hypothesized to disrupt tissue structure and function because of the components they secrete. However, whether senescent cells are causally implicated in age-related dysfunction and whether their removal is beneficial has remained unknown. To address these fundamental questions, we made use of a biomarker for senescence, p16Ink4a, to design a novel transgene, INK-ATTAC, for inducible elimination of p16Ink4a-positive senescent cells upon administration of a drug. Here we show that in the BubR1 progeroid mouse background, INK-ATTAC removes p16Ink4a-positive senescent cells upon drug treatment. In tissues—such as adipose tissue, skeletal muscle and eye—in which p16Ink4a contributes to the acquisition of age-related pathologies, life-long removal of p16Ink4a-expressing cells delayed onset of these phenotypes. Furthermore, late-life clearance attenuated progression of already established age-related disorders. These data indicate that cellular senescence is causally implicated in generating age-related phenotypes and that removal of senescent cells can prevent or delay tissue dysfunction and extend healthspan.

BubR1 insufficiency causes early onset of aging-associated phenotypes and infertility in mice.

Faithful segregation of replicated chromosomes is essential for maintenance of genetic stability and seems to be monitored by several mitotic checkpoints. Various components of these checkpoints have been identified in mammals, but their physiological relevance is largely unknown. Here we show that mutant mice with low levels of the spindle assembly checkpoint protein BubR1 develop progressive aneuploidy along with a variety of progeroid features, including short lifespan, cachectic dwarfism, lordokyphosis, cataracts, loss of subcutaneous fat and impaired wound healing. Graded reduction of BubR1 expression in mouse embryonic fibroblasts causes increased aneuploidy and senescence. Male and female mutant mice have defects in meiotic chromosome segregation and are infertile. Natural aging of wild-type mice is marked by decreased expression of BubR1 in multiple tissues, including testis and ovary. These results suggest a role for BubR1 in regulating aging and infertility.

Naturally occurring p16 Ink4a -positive cells shorten healthy lifespan

Cellular senescence, a stress-induced irreversible growth arrest often characterized by expression of p16Ink4a (encoded by the Ink4a/Arf locus, also known as Cdkn2a) and a distinctive secretory phenotype, prevents the proliferation of preneoplastic cells and has beneficial roles in tissue remodelling during embryogenesis and wound healing. Senescent cells accumulate in various tissues and organs over time, and have been speculated to have a role in ageing. To explore the physiological relevance and consequences of naturally occurring senescent cells, here we use a previously established transgene, INK-ATTAC, to induce apoptosis in p16Ink4a-expressing cells of wild-type mice by injection of AP20187 twice a week starting at one year of age. We show that compared to vehicle alone, AP20187 treatment extended median lifespan in both male and female mice of two distinct genetic backgrounds. The clearance of p16Ink4a-positive cells delayed tumorigenesis and attenuated age-related deterioration of several organs without apparent side effects, including kidney, heart and fat, where clearance preserved the functionality of glomeruli, cardio-protective KATP channels and adipocytes, respectively. Thus, p16Ink4a-positive cells that accumulate during adulthood negatively influence lifespan and promote age-dependent changes in several organs, and their therapeutic removal may be an attractive approach to extend healthy lifespan.

Rae1 is an essential mitotic checkpoint regulator that cooperates with Bub3 to prevent chromosome missegregation

The WD-repeat proteins Rae1 and Bub3 show extensive sequence homology, indicative of functional similarity. However, previous studies have suggested that Rae1 is involved in the mRNA export pathway and Bub3 in the mitotic checkpoint. To determine the in vivo roles of Rae1 and Bub3 in mammals, we generated knockout mice that have these genes deleted individually or in combination. Here we show that haplo-insufficiency of either Rae1 or Bub3 results in a similar phenotype involving mitotic checkpoint defects and chromosome missegregation. We also show that overexpression of Rae1 can correct for Rae1 haplo-insufficiency and, surprisingly, Bub3 haplo-insufficiency. Rae1-null and Bub3-null mice are embryonic lethal, although cells from these mice did not have a detectable defect in nuclear export of mRNA. Unlike null mice, compound haplo-insufficient Rae1/Bub3 mice are viable. However, cells from these mice exhibit much greater rates of premature sister chromatid separation and chromosome missegregation than single haplo-insufficient cells. Finally, we show that mice with mitotic checkpoint defects are more susceptible to dimethylbenzanthrene-induced tumorigenesis than wild-type mice. Thus, our data demonstrate a novel function for Rae1 and characterize Rae1 and Bub3 as related proteins with essential, overlapping, and cooperating roles in the mitotic checkpoint.

Cellular senescence in aging and age-related disease: from mechanisms to therapy

Cellular senescence, a process that imposes permanent proliferative arrest on cells in response to various stressors, has emerged as a potentially important contributor to aging and age-related disease, and it is an attractive target for therapeutic exploitation. A wealth of information about senescence in cultured cells has been acquired over the past half century; however, senescence in living organisms is poorly understood, largely because of technical limitations relating to the identification and characterization of senescent cells in tissues and organs. Furthermore, newly recognized beneficial signaling functions of senescence suggest that indiscriminately targeting senescent cells or modulating their secretome for anti-aging therapy may have negative consequences. Here we discuss current progress and challenges in understanding the stressors that induce senescence in vivo, the cell types that are prone to senesce, and the autocrine and paracrine properties of senescent cells in the contexts of aging and age-related diseases as well as disease therapy.

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.

Bub1 mediates cell death in response to chromosome missegregation and acts to suppress spontaneous tumorigenesis

The physiological role of the mitotic checkpoint protein Bub1 is unknown. To study this role, we generated a series of mutant mice with a gradient of reduced Bub1 expression using wild-type, hypomorphic, and knockout alleles. Bub1 hypomorphic mice are viable, fertile, and overtly normal despite weakened mitotic checkpoint activity and high percentages of aneuploid cells. Bub1 haploinsufficient mice, which have a milder reduction in Bub1 protein than Bub1 hypomorphic mice, also exhibit reduced checkpoint activity and increased aneuploidy, but to a lesser extent. Although cells from Bub1 hypomorphic and haploinsufficient mice have similar rates of chromosome missegregation, cell death after an aberrant separation decreases dramatically with declining Bub1 levels. Importantly, Bub1 hypomorphic mice are highly susceptible to spontaneous tumors, whereas Bub1 haploinsufficient mice are not. These findings demonstrate that loss of Bub1 below a critical threshold drives spontaneous tumorigenesis and suggest that in addition to ensuring proper chromosome segregation, Bub1 is important for mediating cell death when chromosomes missegregate.

The ATM–p53 pathway suppresses aneuploidy-induced tumorigenesis

The spindle assembly checkpoint (SAC) is essential for proper sister chromatid segregation. Defects in this checkpoint can lead to chromosome missegregation and aneuploidy. An increasing body of evidence suggests that aneuploidy can play a causal role in tumorigenesis. However, mutant mice that are prone to aneuploidy have only mild tumor phenotypes, suggesting that there are limiting factors in the aneuploidy-induced tumorigenesis. Here we provide evidence that p53 is such a limiting factor. We show that aneuploidy activates p53 and that loss of p53 drastically accelerates tumor development in two independent aneuploidy models. The p53 activation depends on the ataxia-telangiectasia mutated (ATM) gene product and increased levels of reactive oxygen species. Thus, the ATM-p53 pathway safeguards not only DNA damage but also aneuploidy.

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.

Increased expression of BubR1 protects against aneuploidy and cancer and extends healthy lifespan

The BubR1 gene encodes for a mitotic regulator that ensures accurate segregation of chromosomes through its role in the mitotic checkpoint and the establishment of proper microtubule–kinetochore attachments. Germline mutations that reduce BubR1 abundance cause aneuploidy, shorten lifespan and induce premature ageing phenotypes and cancer in both humans and mice. A reduced BubR1 expression level is also a feature of chronological ageing, but whether this age-related decline has biological consequences is unknown. Using a transgenic approach in mice, we show that sustained high-level expression of BubR1 preserves genomic integrity and reduces tumorigenesis, even in the presence of genetic alterations that strongly promote aneuplodization and cancer, such as oncogenic Ras. We find that BubR1 overabundance exerts its protective effect by correcting mitotic checkpoint impairment and microtubule–kinetochore attachment defects. Furthermore, sustained high-level expression of BubR1 extends lifespan and delays age-related deterioration and aneuploidy in several tissues. Collectively, these data uncover a generalized function for BubR1 in counteracting defects that cause whole-chromosome instability and suggest that modulating BubR1 provides a unique opportunity to extend healthy lifespan.