15 Apr 2021

Some neurons in the Alzheimer’s brain slip back into the cell cycle, duplicating their DNA and expressing aberrant proteins. This triggers cell death—or does it? In the March 23 Proceedings of the National Academy of Sciences USA, researchers led by Lars Ittner at Macquarie University, Sydney, and Nikolas Haass at the University of Queensland, Brisbane, Australia, offer a fresh perspective on neuronal cell-cycle re-entry. They applied a relatively new tool, the fluorescent ubiquitination-based cell-cycle indicator (FUCCI), to cultured mouse neurons to reveal those that re-engaged the cell cycle. At any given time, about 10 percent of neurons transiently expressed geminin, a protein associated with DNA synthesis, suggesting the cells were attempting to enter S phase. After adding Aβ oligomers to the culture, the number of these neurons doubled. Curiously, though, neurons expressing more geminin were more likely to survive, while those with less geminin died. “Our data would suggest that neurons in the cell-cycle-like state have adopted a resistant fate and are protected from Aβ toxicity,” Ittner and Haass wrote to Alzforum.

Others are not so sure. Rachael Neve at Massachusetts General Hospital, Boston, pointed out that S-phase cells in these cultures did not manage to replicate their DNA, and so might not be representative of cycling cells in the AD brain, which do accumulate extra genetic material. Agata Copani at the University of Catania, Italy, agreed the model has limits. “In my opinion, [the paper] does not prove convincingly that cell-cycle reactivation protects neurons from Aβ toxicity,” she wrote to Alzforum.

Nonetheless, commenters appreciated the use of the FUCCI technique, which had not been applied to neurons before. “The techniques are useful and the information is interesting,” said Elliott Mufson at the Barrow Neurological Institute in Phoenix.

Cycling cells progress from the growth phase, known as G1, to DNA synthesis (S phase), preparation for division (G2), and mitosis (M phase). It has long been known that some neurons in the AD brain aberrantly enter S phase and amplify their DNA (Apr 2003 news; Jul 2007 news; Mar 2006 webinar). This has been linked to neuroinflammation and neuron death (Aug 2002 webinar; Feb 2004 news; Nov 2009 news). 

Ittner and colleagues wanted to take a closer look at neuronal cell-cycle re-entry using FUCCI. In this assay, cells are transduced to express two cell-cycle proteins, Cdt1 and geminin, tagged with red and green fluorescent protein, respectively. These proteins are subject to the same regulatory and degradative machinery as their endogenous counterparts, allowing for a fluorescent readout of the cell-cycle stage. In G1, red Cdt1 loiters in the cell; in S or later phases, green geminin does; and during the G1/S transition, both proteins are present, producing a yellow signal.

Joint first authors Stefania Ippati and Yuanyuan Deng applied this assay to primary hippocampal neurons isolated from embryonic mice. In their post-mitotic state, neurons remain in a quiescent G1 phase often called G0, and so should express the G1 marker Cdt1. As expected, the majority of neurons fluoresced red, but at any given time, about 10 percent contained green geminin as well, suggesting these cells were in the G1/S transition. Typically, geminin expression lasted but a few hours. The data hint that neurons may have to continually repress or degrade cell-cycle proteins that nudge them toward DNA synthesis. “This suggests dynamic rather than absolute maintenance of the postmitotic state of neurons,” the authors noted.

Would the presence of toxic Aβ change this? Ippati and colleagues added synthetic Aβ42 oligomers to a concentration of 0.5 μM and examined the cultures two days later. For these experiments, they transduced cells only with geminin. The number of green cells had more than doubled to 22 percent, while cell death in the cultures skyrocketed from 10 to 55 percent. About equal numbers of green S-phase cells died as survived; alas, the authors spotted a difference between the dead and the living cells. The latter had much stronger initial geminin expression than cells that died, and this expression continued to increase over time. “We found there was a threshold of geminin expression that conferred protection,” Ittner and Haass wrote to Alzforum.

Cell Cycle Re-entry. Neurons in the cortices of APP23 mice (right) express far more geminin (green), a marker of S phase, than do those in control mice (left). [Courtesy of Ippati et al., PNAS.]

Does the same thing happen in vivo? It is not entirely clear. Using an adenoviral vector, the authors transduced APP23 and wild-type mice with the green geminin marker. Consistent with the in vitro results, APP23 mice had about twice as much green fluorescence in their cortices at 3 months of age as did controls (see image above), but it remains to be determined what, if any, role this plays in the subsequent signs of preclinical AD phenotype that develop in these mice. Direct immunostaining for geminin also labeled three times as many neurons in transgenic mice as in controls. There was no increase in DNA in these cells, in agreement with the cell culture results. The authors did not use Cdt1 in this study, because it was phototoxic during live cell imaging.

In sections from postmortem human brain as well, immunostaining detected nearly four times as many geminin-positive neurons in the six AD samples as in six samples from age-matched control tissue. The findings confirmed an increase in S-phase neurons in AD, but with these postmortem slices the authors could not examine any link to cell death.

The researchers do not believe that Aβ itself triggers cell-cycle re-entry. Rather, they think that neurons that happen to be in this state are protected from toxicity, and that these cells then maintain the early S-phase because it helps them survive. Over time, this would lead to accumulation of S-phase neurons. In future experiments, the authors will directly manipulate geminin levels and the cell-cycle phase to test this hypothesis. They also plan to do more in vivo imaging in mice, noting recent improvements to the FUCCI technique that will facilitate this.

Copani applauded the renewed focus on cell-cycle events in AD, noting that the demonstration of geminin expression in AD tissue and APP23 mice is new. That said, she does not believe the findings contradict previous work linking cell-cycle re-entry to apoptosis. Her earlier studies implicated DNA replication, not the S-phase transition, as the trigger for cell death (Copani et al., 2002; Copani et al., 2006). “In the absence of signs of DNA replication [in this study], it would seem more logical to think that checkpoint arrest at the G1/S boundary rather than an early S phase explains the patterns the authors observed,” she suggested. Perhaps this arrest keeps neurons from replicating their DNA and dying, she speculated. Thus, in her view, Ittner’s findings are compatible with earlier studies that found cell-cycle re-entry to be harmful.—Madolyn Bowman Rogers

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References

News Citations

1. AD Cell Cycle Reentry—Early Rather Than Late 16 Apr 2003
2. Vicious Cycle?—Neuronal Cell Division Attempts in Control and AD Brain 3 Jul 2007
3. ATM Links DNA Damage to Neuronal Cell Cycle Activation and Apoptosis 19 Feb 2004
4. Curbing Cell Cycle Re-entry: Window of Opportunity for NSAIDs? 24 Nov 2009

Webinar Citations

1. Cell Cycle Hypothesis Pedaling into Mainstream Acceptance? Results in Fly, Mouse Models Warrant a Second Look 12 Apr 2006
2. The Cell Cycle and Alzheimer’s Disease—Let's Unite for Division! 7 Aug 2002

Research Models Citations

1. APP23

Paper Citations

1. Copani A, Sortino MA, Caricasole A, Chiechio S, Chisari M, Battaglia G, Giuffrida-Stella AM, Vancheri C, Nicoletti F. Erratic expression of DNA polymerases by beta-amyloid causes neuronal death. FASEB J. 2002 Dec;16(14):2006-8. PubMed.
2. Copani A, Hoozemans JJ, Caraci F, Calafiore M, van Haastert ES, Veerhuis R, Rozemuller AJ, Aronica E, Sortino MA, Nicoletti F. DNA polymerase-beta is expressed early in neurons of Alzheimer's disease brain and is loaded into DNA replication forks in neurons challenged with beta-amyloid. J Neurosci. 2006 Oct 25;26(43):10949-57. PubMed.

Further Reading

News

1. Cdk5—Novel Brake on the Cell Cycle, Implications for AD? 25 Apr 2010
2. Oxidative Stress Triggers Neuronal Cell-Cycle Reentry 22 Nov 2002
3. COX-2 in Neurodegeneration: Does It Wake Up a Sleeping Cell Cycle? 22 Nov 2002
4. Linking APP with Cell Cycle Reentry and Apoptosis—One Kinase Does the Trick 9 Aug 2003