Vicious Cycle?—Neuronal Cell Division Attempts in Control and AD Brain

The cell cycle is a bit like a roller coaster. Once it starts, there’s no going back, and should something go wrong halfway through, the consequences can be disastrous. Being terminally differentiated, neurons are particularly poor candidates for cell cycling, yet there is abundant evidence that in Alzheimer disease at least some neurons have started down that slippery slope (see related ARF live discussion and live discussion). This idea is supported by a paper in the June 27 Journal of Neuroscience. Researchers in Germany led by Thomas Arendt at the University of Leipzig have used three different methods to quantify DNA in cortical neurons. “This is the central strength of the paper, the three independent ways of getting at the same question. That is good, solid science,” said Karl Herrup, Rutgers University, Piscataway, New Jersey, who has advanced the study of cell-cycle irregularities in neurons. “I think what has gone largely unappreciated is that from an increasing number of independent labs comes a steady, almost drumbeat of data saying that something very unusual is going on with the state of the genome in the mature neuron,” he said in an interview with Alzforum.

The presence of more than the usual diploid DNA content is widely accepted as evidence that cells are cycling, but as Arendt and colleagues point out, extra DNA in neurons may also be the result of chromosome mis-segregation at the precursor cell stage, as was previously described (see Yang et al., 2003). To account for such constitutional aneuploidy, first author Birgit Mosch and colleagues correlated DNA content in normal and AD brain samples with expression of cyclin B1, a marker of early mitosis. Aneuploidy and expression of the cyclin would be evidence of true cell cycle re-entry, the authors contend.

Mosch and colleagues used slide-based cytometry, chromogenic in situ hybridization, and single cell PCR amplification to estimate DNA content. First they established that they could achieve a high degree of reliability among the three methods; then, they used the same techniques to estimate DNA content in cortical cells taken from 13 control and 13 AD postmortem brain samples (mean age of 76 and 71 years, respectively). The researchers found that about 88 percent of cells in control entorhinal cortex had the normal 2n DNA content, but this was reduced to about 77 and 70 percent in early and late AD, respectively. They also calculated that the number of cells with >2n DNA increased from about 36 neurons per square millimeter in controls to 57 and 53 neurons per square millimeter in early and advanced AD. The authors suggest that this increase is most likely attributable to disease-related reactivation of DNA replication. “This progressive shift from a diploid DNA content to a content of >2n between early to more advanced stages of the disease argues against an increase in constitutional hyperploidy to account for the increased neuronal amount of DNA in AD,” the authors write.

The researchers found that chromogenic in situ hybridization (CISH), using a probe to chromosome 17, also showed an increase in DNA content in AD neurons. Though the majority of neurons in both control and AD samples had only two hybridization spots, indicative of diploid cells, the number of cells with three spots was twofold higher, and those with four spots were four- to fivefold higher in AD samples. DNA quantification by PCR also showed that while there was a distribution peak centered at around 2.5 to 3.5 pg DNA in control cells, in neurons from AD brains there was a second peak at around 6.5-7.5 pg DNA per cell, which most likely represents tetraploid neurons. Interestingly, this distribution was not observed when Mosch and colleagues looked at neurons from the occipital cortices, which are much less vulnerable to AD pathology.

In correlating the increased DNA content with cyclin B1 expression, the authors made some interesting observations. First, they found that not all control samples looked the same. The 13 control samples could be divided into two groups. In seven of the samples, cyclin B1 was widely expressed, being found in 30 to 80 percent of all neurons (average of 42 percent). In the second group of six samples, expression of the cyclin was marginal, turning up in less than 10 percent of neurons. In AD samples the fraction of neurons expressing cyclin B1 was about 39 percent for early AD, which was similar to controls with high expression, but this increased to 61 percent in late AD. However, when the researchers looked at tetraploid cells alone, they found that none of these cells from control samples were cyclin B1 positive, suggesting they were not actively engaged in cell cycling. These cells most likely “represent a constitutional tetraploidy derived from chromosome mis-segregation during mitosis in neuronal precursor cells,” write the authors. In contrast, the majority of tetraploid neurons in AD samples were cyclin B1 positive. “This association between an elevated content of DNA and expression of cyclin B1 in AD indicates that some neurons have reactivated their cell cycle and progressed toward the S phase and beyond,” the authors write.

This is the first example of the use of slide-based cytometry and single cell PCR to measure DNA content in adult CNS, and the data support previous findings from the Arendt and other labs that cell-cycle re-entry is alive but maybe not so kicking in AD (see related ARF live discussion). There is also evidence from Herrup’s own lab that cell-cycle re-entry occurs early in the disease (see ARF related news story). In this regard, it is interesting that Mosch and colleagues found that over half of the 13 control samples had quite elevated levels of cyclin B1. Whether this is an indication of prodrome remains to be determined. In fact, exactly what role cell-cycle re-entry may play in AD pathology remains unclear. “I think this whole relationship of cell-cycle to the cell in the fully differentiated state very much needs rethinking,” suggested Herrup. “I think that in some ways developmental biologists need to step in and ask, not just in lymphocytes, but in terminally differentiated tissues such as kidney, pancreas, and multinucleated cells of muscle, Does this same sort of causal relationship with diploid status apply, or is it really unique to neurons?” He added that there may be a dimension to this biology that we have been missing all along. For more on the relationship between the cell cycle and neurons, see the recent review by Herrup and Yang (Herrup and Yang, 2007).—Tom Fagan

Comments

  1. Cycling to Nowhere
    The paper by Arendt and colleagues presents compelling evidence for DNA synthesis in terminally differentiated neurons of the AD brain, and raises a number of other questions. It is intriguing to speculate whether the increase in cyclin B1 observed in half of the aged control subjects is indicative of an increased attempt by these aging neurons to re-enter the cell cycle. Is this a precursor stage to MCI? Would these individuals have developed AD if they had lived longer? Does cyclin B1 expression remain elevated after the cells reach tetraploidy?

    Given that these polyploidy neurons are unlikely to survive (Yang et al., 2003), it seems likely that the quantitation of polyploidy and cell cycle related markers at the terminal stage of the disease is a gross underestimation of the mitogenic stimulus reaching neurons in the aging brain.

    Recently we identified an upregulation in the expression of osteopontin, a protein that is intimately involved in cell cycle progression and cell adhesion (Wung et al., 2007) in the AD brain that may be a response to neuronal remodeling induced by neuron re-entry into the cell cycle.

    It remains to be determined what signals are driving differentiated postmitotic neurons back into the cell cycle. The paper by Arendt and colleagues provides strong evidence for the “Cell Cycle Hypothesis of AD.” In this respect we have postulated that the major changes in HPG axis hormones with aging (decreased sex steroids, decreased inhibin resulting in increased activin signaling, increased GnRH, increased gonadotropins; Bowen and Atwood, 2004) alter the growth-differentiation dynamic of cells throughout the body. Put another way, the changes in reproductive hormones that occur with aging are somewhat similar to those seen during fetal life and this growth-oriented profile may induce cell proliferation. In those tissues that attempt division but are unable to undergo cytokinesis (neurons, cardiomyocytes, fibroblasts), cell loss leads to loss of function and disease, while those tissues containing cells that can divide (i.e., reproductive tissues, colonic cells, lung, liver, pancreas, etc.) continue to accumulate mutations and tend to develop neoplasia.

    References:

    Bowen RL, Atwood CS. Living and dying for sex. A theory of aging based on the modulation of cell cycle signaling by reproductive hormones. Gerontology. 2004 Sep-Oct;50(5):265-90. PubMed.

    Wung JK, Perry G, Kowalski A, Harris PL, Bishop GM, Trivedi MA, Johnson SC, Smith MA, Denhardt DT, Atwood CS. Increased expression of the remodeling- and tumorigenic-associated factor osteopontin in pyramidal neurons of the Alzheimer's disease brain. Curr Alzheimer Res. 2007 Feb;4(1):67-72. PubMed.

    Yang Y, Mufson EJ, Herrup K. Neuronal cell death is preceded by cell cycle events at all stages of Alzheimer's disease. J Neurosci. 2003 Apr 1;23(7):2557-63. PubMed.

    View all comments by Craig Atwood

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References

Webinar Citations

  1. The Cell Cycle and Alzheimer’s Disease—Let's Unite for Division!
  2. Cell Cycle Hypothesis Pedaling into Mainstream Acceptance? Results in Fly, Mouse Models Warrant a Second Look

News Citations

  1. AD Cell Cycle Reentry—Early Rather Than Late

Paper Citations

  1. Yang AH, Kaushal D, Rehen SK, Kriedt K, Kingsbury MA, McConnell MJ, Chun J. Chromosome segregation defects contribute to aneuploidy in normal neural progenitor cells. J Neurosci. 2003 Nov 12;23(32):10454-62. PubMed.
  2. Herrup K, Yang Y. Cell cycle regulation in the postmitotic neuron: oxymoron or new biology?. Nat Rev Neurosci. 2007 May;8(5):368-78. PubMed.

Further Reading

Primary Papers

  1. Mosch B, Morawski M, Mittag A, Lenz D, Tarnok A, Arendt T. Aneuploidy and DNA replication in the normal human brain and Alzheimer's disease. J Neurosci. 2007 Jun 27;27(26):6859-67. PubMed.

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