Sci. Aging Knowl. Environ., 5 April 2006
Vol. 2006, Issue 7, p. pe9
[DOI: 10.1126/sageke.2006.7.pe9]


Aging in Check

Wei Dai, and Xiaoxing Wang

The authors are in the Division of Molecular Carcinogenesis of the Department of Medicine, New York Medical College, Valhalla, NY 10595, USA. E-mail: wei_dai{at} (W.D.)

Key Words: spindle checkpoint • aneuploidy • chromosome instability • senescence • p53 • mosaic variegated aneuploidy syndrome


Faithful chromosome segregation during mitosis is pivotal to the maintenance of genomic stability. Failure to maintain such stability during cell division inevitably leads to either cell death or malignant transformation. Therefore, eukaryotic cells have evolved sophisticated molecular mechanisms, commonly referred to as checkpoints, by which chromosome alignment and partitioning during mitosis are closely monitored. The spindle checkpoint (also referred to as the spindle assembly checkpoint or the mitotic checkpoint) ensures that cells with defective spindles or defective spindle-kinetochore interactions do not enter anaphase, the stage of mitosis when sister chromatids separate and move toward opposite poles of the spindle. Initial genetic screening has identified at least seven genes (Bub1, Bub2, Bub3, Mad1, Mad2, Mad3, and MPS1) that are required for spindle checkpoint control in the budding yeast (1-3). In the absence of such genes, mitosis progresses even when spindle function is perturbed, resulting in loss of viability rather than cell cycle arrest. The spindle checkpoint is highly conserved in mammals (4). In addition to homologs of Bub and Mad family members, several genes such as those coding for CENP-E, Rae1, and Nup98 also contribute to the spindle checkpoint function in mammalian cells (5-8). A series of mouse genetic studies during the past several years has demonstrated that major components of the spindle checkpoint are essential for early embryonic development and that haploinsufficiency of, or deficiency in, checkpoint components predispose cells to chromosomal instability and malignant transformation (6, 9, 10-12). Interestingly, an elegant study published in the 13 February issue of Journal of Cell Biology has uncovered a new function for some key components of the spindle checkpoint; it establishes a strong link between defects in Bub3 and Rae1 and the development of early aging phenotypes (13). This Perspective focuses on the unexpected function of spindle checkpoint proteins in the regulation of aging.

Bub3/Rae1 and Aging

As a cell approaches metaphase, each of the two kinetochores formed at the centromeric regions of paired sister chromatids ultimately becomes attached to spindle microtubules, such that the kinetochores are oriented toward opposite poles of the spindle (that is, the kinetochores become bi-oriented). Unattached kinetochores activate the spindle checkpoint, causing a majority of checkpoint proteins, including Bub1, Bub3, Mad1, Mad2, and CENP-E, to be associated with unattached kinetochores (14). As part of the checkpoint response, Mad2, BubR1, and Bub3 also form a complex with and sequestrate Cdc20 (15), an activator of the anaphase-promoting complex/cyclosome (APC/C), which is a ubiquitin E3 ligase that functions to promote regulated proteolysis in the ubiquitin/proteasome pathway. The attachment and bi-orientation of the kinetochores of all condensed chromosomes with the mitotic spindle leads to inactivation of the spindle checkpoint, thus allowing the full activation of APC/C. Polyubiquitination of securin, a separase inhibitor, by APC/C results in the degradation of securin by the proteasome. Activated separase then specifically cleaves cohesin, a molecular "glue" (complex) that links sister chromatids, thus allowing anaphase entry. At the molecular level, a major consequence of spindle checkpoint activation is the inhibition of APC/C, leading to accumulation of securin. In fact, several studies have clearly shown that a compromised spindle checkpoint causes enhanced degradation of securin, which is accompanied by increased chromosome instability (8, 10, 11). As a majority of tumor cells are aneuploid, it is commonly thought that deregulated control of the spindle checkpoint would result in chromosomal instability and malignant transformation. However, a new line of evidence has emerged, indicating that accelerated aging, rather than oncogenic transformation, may be the major biological manifestation of a weakened spindle checkpoint (13, 16).

Rae1, a protein participating in mRNA export (17), shares extensive amino acid sequence similarity with Bub3 (18). Rae1 also has a function in regulating the spindle checkpoint. In fact, haploinsufficiency of Rae1 causes chromosomal missegregation; ectopic expression of Rae1 is capable of rescuing the spindle checkpoint defect in cells with Bub3 haploinsufficiency (6), which suggests that these two proteins share some overlapping functions in the spindle checkpoint pathway. Although both Bub3 and Rae1 are essential for early embryonic development, haploinsufficiency of either Bub3 or Rae1 appears to be insufficient for promoting tumorigenesis (6, 19). Compared with wild-type littermates, mice with haploinsufficiency of Bub3 and/or Rae do not exhibit any enhanced susceptibility to developing spontaneous tumors, although Rae1+/– mutant mice and Bub3+/– Rae1+/– compound mutant mice develop lung adenomas at an elevated rate compared with wild type after carcinogen treatment (6).

A follow-up study by the same research group reveals that mice with haploinsufficiency of Bub3 and Rae1 have a reduced life span associated with the early onset of aging-related phenotypes (13). Between 15 and 27 months of age, mice doubly haploinsufficient for Bub3 and Rae1 display early aging characteristics, including (i) the formation of cataracts, (ii) the development of lordokyphosis (spinal curvature or humpback), (iii) a reduction in dermal thickness and in subdermal adipose, and (iv) a loss of body weight. These benign to pathological phenotypes are neither observed in wild-type mice nor found in Bub3+/– or Rae1+/– mice within 2 years, suggesting that Bub3+/– Rae1+/– compound mutations predispose mice to the early onset of aging. Consistently, these age-associated phenotypes observed in the Bub3+/– Rae1+/– mice are also present in very old (35 months of age) wild-type mice, confirming that haploinsufficiency of Rae1 and Bub3 accelerates the aging process in vivo.

Cellular and Molecular Manifestations

What are the underlying mechanisms by which haploinsufficiency of both Bub3+/– and Rae1+/– cause accelerated aging in mice? Apoptosis and senescence are two primary cellular processes that could contribute to aging (20) (see "More Than a Sum of Our Cells"). Because accelerated apoptosis, or programmed cell death, occurs in cells with defects in spindle checkpoint functions (10, 21), it is natural to propose that apoptosis might be the primary mechanism that induces premature aging in Bub3+/– Rae1+/– mice. Surprisingly, apoptosis does not seem to contribute to the accelerated aging process in these mice. Despite high levels of aneuploidy in Bub3+/– Rae1+/– mouse embryonic fibroblasts (MEFs), no major enhancements in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) and annexin V staining (indicative of apoptosis) are detectable in these cells compared with wild-type MEFs that have been passaged in culture the same number of times. Instead, Bub3+/– Rae1+/– MEFs, but not Rae1+/– or Bub3+/– MEFs, accumulate high concentrations of gene products involved in the induction of cellular senescence, including p16, p19, p21, and p53 (see Dimri Perspective and Sharpless Perspective), as well as senescence-associated beta-galactosidase (SA-beta-Gal) activity in early passage cells. Moreover, BubR1 hypomorphic MEFs (BubR1H/H) that contain about 10% of the normal concentration of BubR1 protein display significantly higher levels of SA-beta-Gal activity than do Bub3+/– Rae1+/– MEFs that have been passaged the same number of times. Consistently, BubR1H/H mice display progeroid features much earlier than Bub3+/– Rae1+/– mice. Together, these studies provide strong molecular evidence suggesting that compound mutations in Bub3 and Rae1 cause the early onset of aging because of the induction of cellular senescence.

BubR1 is also known to play a role in suppressing the early onset of aging-associated phenotypes (16). Thus, it is the impairment of the spindle checkpoint function, rather than impairment of unknown functions of Bub3 and Rae1, that contributes to the induction of senescence response. Aneuploidy is the direct consequence of a defective spindle checkpoint. It is natural to speculate that the extent of aneuploidy caused by checkpoint impairment would be tightly correlated with the timing and severity of aging. Intriguingly, existing evidence indicates that aneuploidy caused by premature sister chromatid separation due to compromised checkpoint control may not be responsible for early aging phenotypes observed in Bub3+/– Rae1+/– mice. Observations that support this conclusion include the following: (i) Although Bub3+/– Rae1+/– MEFs tend to display more aneuploidy than BubR1H/H MEFs and are frequently coupled with losses or gains of a wide spectrum of chromosomes, a lower degree of senescence is observed in Bub3+/– Rae1+/– MEFs than that in BubR1H/H MEFs. (ii) Despite progressive aneuploidy phenotypes, Bub3 or Rae1 single heterozygous mice do not show signs of early aging, thus disputing spindle checkpoint failure as the underlying cause for the progeroid features described in this study. One caveat, however, for this conclusion is that studies on aneuploidy have been performed only in a limited number of cell types (e.g., MEFs and splenocytes), which might not represent the cells/tissue types (e.g., eye, spine, and dermis) that undergo accelerated aging.

Given the observed discordance between aneuploidy and aging, what are the mechanisms by which Bub3+/– Rae1+/– mice display accelerated aging features? Because a defective spindle assembly checkpoint can impair DNA damage responses (22-24), one possibility is that DNA damage checkpoint/repair pathways are compromised in these mice, leading to the accumulation of DNA lesions that in turn activate senescence responses. Although Bub3+/– Rae1+/– MEFs exhibit growth kinetics similar to those of wild-type MEFs after treatment with various DNA-damaging agents (13), it is difficult to predict responses in other cell types based on information obtained from MEF studies.

The tumor suppressor protein p53 is central to the regulation of senescence (25-27). Evidence suggests that p53 may also play a major role in induction of premature senescence in Bub3+/– Rae1+/– MEF cells with a spindle checkpoint defect, because p53 accumulates in these cells earlier than in cells with haploinsufficiency of a single gene, correlating well with the early onset of aging in Bub3+/– Rae1+/– mice. What, then, is the signal(s) that causes the accumulation of p53 in cells haploinsufficient for both Rae1 and Bub3? There are at least two scenarios that could potentially modulate p53 expression in Bub3/Rae1 haploinsufficient cells. (i) Rae1 might directly or indirectly affect translation of p53 mRNA through its nuclear mRNA exporting function (17). However, the fact that no obvious defects in mRNA transporting have been detected in Rae1–/– blastocysts (6) does not favor the possibility. (ii) Compound deficiency of Rae1 and Bub3 might affect p53 concentrations through modulating the activity of securin, a major substrate of APC/C. In addition to its function in the spindle checkpoint signaling pathway, securin also functions as a negative regulator of p53 (28). Securin physically interacts with p53 both in vitro and in vivo; the interaction between p53 and securin results in repression of the transcriptional activation activity of p53; moreover, securin-deficient human tumor cells are more susceptible to apoptosis induced by p53 (28). Thus, it will be interesting to determine whether securin depletion augments p53 activity and further accelerates premature senescence of Bub3+/– Rae1+/– cells.

To Divide or To Senesce--A Compromise

Bub3, Rae1, and BubR1, known as spindle checkpoint components, have two distinct functions in vivo: (i) maintaining genomic stability and suppressing aneuploidy and (ii) delaying the onset of aging-associated phenotypes. At the organismal level, these two functions appear to be coordinated rather well to ensure the species' survival. This coordination is clearly manifested in individuals with mosaic variegated aneuploidy (MVA) syndrome. MVA patients, who carry biallelic mutations of BubR1 in the germline, not only are prone to cancer but also exhibit progeroid features such as the development of cataracts (29).

At the cellular level, the suppression of senescence means that cells are proliferative, which in turn could increase the possibility of having missegregated chromosomes and developing aneuploidy, eventually leading to tumor formation (see "Dangerous Liaisons" and "Led Astray" for a discussion of the relation between senescence and cancer). Multicellular organisms thus need to maintain a robust spindle checkpoint so that each cell division is closely monitored and genomic stability is maintained. Given the fact that unicellular organisms also contain Bub3 and Mad3 (the latter being a BubR1 ortholog) coupled with an intact spindle checkpoint (1, 2), maintenance of genomic stability by Bub3 and Rae1 is probably a more primitive and default function. It is conceivable that for unicellular organisms, continued cell proliferation in the presence of missegregated chromosomes is a better alternative than senescence and death. From the evolutionary point of view, this mechanism might even offer certain selective advantages. For multicellular organisms, enhanced senescence in cells with a severely impaired spindle checkpoint has probably evolved to cope with chromosomal instability. It is necessary to eliminate cells with highly unstable chromosomes because of the susceptibility of these cells to neoplastic transformation. The ability to cope with aneuploidy and to initiate senescence is likely dependent on cell/tissue types, because it has been shown in mice that haploinsufficiency of BubR1 induces a shift in tumor burden from the small intestine to the large intestine in an ApcMin/+ genetic background [the adenomatous polyposis coli (Apc) gene plays a role in maintaining genomic stability, as mutations in Apc cause aneuploidy] (30). The underlying ability for a cell- or tissue-specific response might reflect differences in the concentrations and/or activities of proteins involved in the spindle checkpoint control as well as the senescence response pathway. Alternatively, given that the number of mitotic cells varies dramatically among various organs and tissues, the mitotic index might dictate tissue specificity because rapidly dividing cells are most susceptible to loss of the spindle checkpoint function.


Deficiency in Bub3 and Rae1, as well as in BubR1, promotes the early onset of aging-associated phenotypes, indicating that spindle checkpoint failure may cause additional manifestations other than chromosomal instability in vivo. The accelerated aging phenotypes are most likely a result of activating a premature senescence program. Although the current study suggests that genomic instability or aneuploidy per se may not be a driving force for the aging process in vivo, it remains unclear whether aneuploidy is capable of influencing precocious aging in Rae1/Bub3 haploinsufficient or in BubR1-deficient mice. The tumor suppressor protein p53 appears to be an important mediator for initiating senescence and premature aging in these mice, as it is directly regulated by securin, a major downstream component of the spindle checkpoint pathway. To further establish the causal link between spindle checkpoint failure and the development of progeroid features, it is necessary to investigate whether defects in other checkpoint components such as Mad1, Mad3, and Bub1 have similar aging manifestations both in vivo and in vitro. This line of study would provide additional insights into the cause of premature senescence, which in turn might lead to the discovery of key targets for rational drug design to prevent premature aging.

April 5, 2006
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Citation: W. Dai, X. Wang, Aging in Check. Sci. Aging Knowl. Environ. 2006 (7), pe9 (2006).

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