Olga Grushko and Laura Buttitta, University of Michigan
Alzheimer’s disease (AD), characterized by a progressive loss of cognitive function, is the most prevalent neurodegenerative disorder of aging. A substantial body of literature has documented evidence of cell cycle re-entry and an increase in DNA ploidy in cases of age-induced neurodegeneration and AD [1,2]. Research on human tissue samples has suggested that hyperploidy in neurons may precede clinical diagnosis of AD [3,4]. Thus, cell cycle re-entry and the resulting hyperploidy could be a proximal cause of age-related neurodegeneration, as well as a useful preclinical marker. Dissecting the precise biological significance of cell cycle re-entry in age-related neural decline will be critical to developing new approaches to combat AD.
Developing a model system to study cell cycle re-entry and aging
Extensive literature has reported that cell cycle genes become reactivated in aging brains, and that this reactivation is evolutionarily conserved among diverse species—from flies to humans [5–7]. It has been difficult, however, to observe and quantify cell cycle re-entry of postmitotic cells in the brain due to its rare and potentially transient nature [8,9]. Therefore, its frequency under normal physiological aging conditions has become a matter of debate [3,10–13]. Another barrier to progress in this field has been the difficulty in obtaining aged adult brain samples and monitoring cell cycle re-entry under normal physiological conditions. These challenges, combined with a limited ability to genetically manipulate factors influencing cell cycle re-entry in mammalian models, have hampered investigations.
Progress would be greatly facilitated by the availability of a genetically tractable system that recapitulates the features of age-associated decline in the brain under a rapid lifespan. The simple fruit fly Drosophila melanogaster is an attractive model system to study this process because it exhibits age-related neural decline , ages on the order of days instead of years, and has well-developed tools for in vivo genetic manipulations .
Our laboratory is developing genetic tools and fluorescence assays for manipulating cell cycle re-entry in the adult fly brain (Figure 1) and monitoring subsequent effects on DNA ploidy and cell death. Our long-term goal is to understand the contribution of cell cycle misregulation in the brain to age-related neural decline. We want to address several long-standing and important questions in the field, including:
- What causes cell cycle re-entry in the aging brain?
- How do cell cycle re-entry and misregulation affect cell loss and neurodegeneration?
- How does manipulation of cell cycle re-entry impact ageassociated neural decline.
|Figure 1. An aged adult Drosophila brain, showing glial-specific GFP expression and actin-selective fluorescent staining. This Drosophila brain was dissected from a 50-day-old fly (average lifespan of this strain is 45 days) that expressed nuclear-localized GFP in glial cells (green). The isolated brain was stained with rhodamine phalloidin (red) to label actin and Hoechst™ 33258 (blue) to label DNA. Image provided by Laura Buttitta and Olga Grushka, University of Michigan.|
Assaying DNA content and cell death with flow cytometry
To begin to investigate these questions, we use a sensitive, high-throughput flow cytometry–based assay to monitor changes in cellular DNA content and cell death in the fly brain (Figure 2). Our method involves dissecting the adult fly brain, dissociating the tissue to a single-cell suspension using gentle trypsinization, and then staining live cells for DNA content with the Vybrant™ DyeCycle™ Violet Stain, a cell-permeant, UV/violet laser–excitable nucleic acid dye . We also measure cell death, the eventual consequence of aging-related neural decline, using the live cell–impermeant nucleic acid dye propidium iodide (PI). The Attune™ Acoustic Focusing Cytometer allows us to measure rare events and identify small populations of cells in the fly brain, on the scale of a single brain.
Figure 2C shows data from an experiment in which adult fly brains expressing GFP in glial cells were dissected, dissociated into single cells, stained with Vybrant™ DyeCycle™ Violet Stain, and analyzed on the Attune™ Acoustic Focusing Cytometer (with blue/violet lasers) in high-sensitivity mode. After excluding doublets, cell clumps, and debris by gating (described in ), we used a dot plot of GFP fluorescence vs. DNA content to identify diploid and hyperploid cells, with PI-positive dead or dying cells backgated in pink.
In a normal aged adult fly brain, GFP-labeled glial cells exhibit limited hyperploidy but increased levels of cell death, while GFPnegative neural cells show a slight increase in hyperploidy, but less cell death. The relative levels of hyperploidy for the GFP-labeled glia and GFP-negative neurons can also be shown as a traditional DNA content histogram. Importantly, our use of individual, isolated fly brains allows us to capture any variations in DNA ploidy and cell death between isogenic animals, in order to estimate frequencies of potentially rare increases in ploidy in specific populations carrying mutations associated with neurodegeneration.
Figure 2. Experimental workflow for examining DNA ploidy and cell death in the aging adult Drosophila brain using acoustic focusing cytometry. (A) Individual adult fly brains are dissected; in this case, the strain expresses GFP in the nuclei of adult glial cells. (B) Cells from the isolated brain are dissociated in 100 μL of a PBS/trypsin solution containing Vybrant™ DyeCycle™ Violet Stain (1 μL/mL) and propidium iodide (PI, 5 μL/mL): tissues are quickly triturated by pipetting in a 1.5 mL microcentrifuge tube cap and then transferred to a microcentrifuge tube containing 400 μL of additional PBS/ trypsin/Vybrant™ DyeCycle™ Violet/PI solution and stained for 1 hr. After staining, samples are diluted with 500 μL of PBS and the microcentrifuge tube is placed in the tube holder and run on the Attune™ Acoustic Focusing Cytometer (with blue/violet lasers) in high-sensitivity mode. Doublets and cell clumps or debris are excluded by comparing DNA height and width (see  for details) and removed by gating. (C) A representative dot plot of cells from an aged adult fly brain showing GFP expression (in glial cells) and DNA content (as measured with Vybrant™ DyeCycle™ Violet Stain), with PI-positive dead and dying cells backgated in pink. Here, GFP-positive glial cells show limited hyperploidy but higher levels of cell death, while the non-glial GFP-negative cells (primarily neurons) show less cell death but slightly increased levels of hyperploidy.
Genetically manipulating cell cycle regulators
We use the binary Gal4/UAS system to activate gene expression in specific cell types in the Drosophila brain . To force cell cycle re-entry, we express a combination of cell cycle regulators—cyclin G1, cyclin D, its partner kinase Cdk4, and the cell cycle transcriptional activator E2F—which we have previously reported can reverse cell cycle exit in postmitotic tissues when overexpressed . Thus, we can simultaneously activate cell cycle genes with the Gal4/UAS system, induce GFP or another fluorescent marker in a cell type–specific manner, and monitor the ploidy and death of subpopulations of cells in the adult fly brain.
Figure 3A shows a pair of GFP fluorescence vs. DNA content dot plots comparing control cells expressing GFP under a glial-specific promoter, to GFP-positive glial cells that have been forced to re-enter the cell cycle. The cell population forced to re-enter the cell cycle exhibits increased DNA hyperploidy, including 4C and 8C cells, which is also strongly correlated with PI positivity.
We can also force cell cycle re-entry in postmitotic neurons, such as those shown in Figure 3B, and observe hallmarks of active cell cycling via incorporation of 5-ethynyl-2´-deoxyuridine (EdU) and subsequent detection using the Click-iT™ EdU Alexa Fluor™ 555 Imaging Kit. Forced cell cycle re-entry in postmitotic neurons leads to phenotypes consistent with neurodegeneration, such as mitochondrial clumping and loss  (Figure 3C).
Figure 3. Assessing hyperploidy and cell death in two different cell types of the fly brain. (A) Flies expressing GFP in glial cells were either left untreated (left panel) or induced to re-enter the cell cycle by overexpressing key cell cycle regulators (right panel). Isolated brains were assayed for DNA ploidy and cell death using the workflow described in Figure 2. GFP-expressing glia expressing cell cycle regulators show hyperploidy, including cells exhibiting 4C and 8C DNA content (red arrow). Dead and dying cells incorporate propidium iodide (PI, backgated in pink), and hyperploidy in glia is strongly correlated with PI positivity. (B) Flies expressing GFP in postmitotic neurons were either left untreated (left panels) or induced to re-enter the cell cycle by overexpressing key cell cycle regulators (right panel). After isolating the brains, cell cycle re-entry was confirmed by the incorporation of 5-ethynyl-2´-deoxyuridine (EdU) into newly synthesized DNA with the Click-iT™ EdU Alexa Fluor™ 555 Imaging Kit (yellow arrows, the yellow signal is due to overlap of GFP and Alexa Fluor™ 555 fluorescence; Cat. No. C10338). (C) Pigment-dispersing factor (PDF)–expressing postmitotic neurons (a subset of neurons influencing circadian rhythms in fly brain) were labeled with a mitochondrial-localized GFP and left untreated (left panel) or induced to re-enter the cell cycle by overexpressing key cell cycle regulators (right panel). Neurons that re-enter the cell cycle exhibit hallmarks associated with neurodegeneration, including loss of mitochondria and aberrant mitochondrial clumping (white arrows).
While hyperploidy has been observed in aged human brains and in cases of AD, it remains unclear whether hyperploidy is restricted to neurons or glia, and which cell types are responsible for the neural decline. Glia play a critical support role for maintaining neuronal survival in the brain, and disruption of their quiescence may have huge impacts on the brain. Indeed, cell cycle re-entry in glia may lead to neurodegenerative phenotypes in Drosophila [20,21].
Using the adult fly brain as our model system, we can both assay and manipulate cell cycle re-entry in multiple cell types under physiological aging conditions. Our future investigations aim to establish a new model system for examining the role of cell cycle re-entry in age-related neural decline. If successful, our work will provide information about how aging impacts critical cell cycle controls in the brain, which may suggest novel approaches to combat age-related declines in cognitive function.
Watch the JoVE video on cell cycle analysis of Drosophila tissues
Follow along with the protocol for Live cell cycle analysis of Drosophila tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain by Kerry Flegel, Dan Sun, Olga Grushko, Yiqin Ma, and Laura Buttitta (Molecular, Cellular, and Developmental Biology, University of Michigan); a subscription to JoVE is required. In this 11-minute video, the authors discuss fly dissection, tissue dissociation, DNA staining, and subsequent analysis by acoustic focusing cytometry. Their cell cycle analysis protocol provides a method for determining relative cell size, cell number, and DNA content, as well as cell type via lineage tracing or cell type–specific fluorescent protein expression. This video has been viewed thousands of times by universities and research labs worldwide.
- Herrup K, Yang Y (2007) Cell cycle regulation in the postmitotic neuron: oxymoron or new biology? Nat Rev Neurosci 8:368–378.
- Khurana V, Feany MB (2007) Connecting cell-cycle activation to neurodegeneration in Drosophila. Biochim Biophys Acta 1772:446–456.
- Arendt T, Bruckner MK, Mosch B et al. (2010) Selective cell death of hyperploid neurons in Alzheimer's disease. Am J Pathol 177:15–20.
- Yang Y, Geldmacher DS, Herrup K (2001) DNA replication precedes neuronal cell death in Alzheimer's disease. J Neurosci 21:2661–2668.
- McCarroll SA, Murphy CT, Zou S et al. (2004) Comparing genomic expression patterns across species identifies shared transcriptional profile in aging. Nat Genet 36:197–204.
- Lu T, Pan Y, Kao SY et al. (2004) Gene regulation and DNA damage in the ageing human brain. Nature 429:883–891.
- Yang Y, Mufson EJ, Herrup K (2003) Neuronal cell death is preceded by cell cycle events at all stages of Alzheimer's disease. J Neurosci 23:2557–2563.
- Mosch B, Morawski M, Mittag A et al. (2007) Aneuploidy and DNA replication in the normal human brain and Alzheimer's disease. J Neurosci 27:6859–6867.
- von Trotha JW, Egger B, Brand AH (2009) Cell proliferation in the Drosophila adult brain revealed by clonal analysis and bromodeoxyuridine labelling. Neural Dev 4:9.
- Bauer S, Patterson PH (2005) The cell cycle–apoptosis connection revisited in the adult brain. J Cell Biol 171:641–650.
- Fischer HG, Morawski M, Bruckner MK et al. (2012) Changes in neuronal DNA content variation in the human brain during aging. Aging Cell 11:628–633.
- Swartz FJ, Bhatnagar KP (1981) Are CNS neurons polyploid? A critical analysis based upon cytophotometric study of the DNA content of cerebellar and olfactory bulbar neurons of the bat. Brain Res 208:267–281.
- Pack SD, Weil RJ, Vortmeyer AO et al. (2005) Individual adult human neurons display aneuploidy: detection by fluorescence in situ hybridization and single neuron PCR. Cell Cycle 4:1758–1760.
- Martinez VG, Javadi CS, Ngo E et al. (2007) Age-related changes in climbing behavior and neural circuit physiology in Drosophila. Dev Neurobiol 67:778–791.
- Duffy JB (2002) GAL4 system in Drosophila: a fly geneticist's Swiss army knife. Genesis 34:1–15.
- Flegel K, Sun D, Grushko O et al. (2013) Live cell cycle analysis of Drosophila tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain. J Vis Exp 75:e50239.
- Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118:401–415.
- Buttitta LA, Katzaroff AJ, Perez CL et al. (2007) A double-assurance mechanism controls cell cycle exit upon terminal differentiation in Drosophila. Dev Cell 12:631–643.
- Wang X, Su B, Lee HG et al. (2009) Impaired balance of mitochondrial fission and fusion in Alzheimer's disease. J Neurosci 29:9090–9103.
- Petersen AJ, Rimkus SA, Wassarman DA (2012) ATM kinase inhibition in glial cells activates the innate immune response and causes neurodegeneration in Drosophila. Proc Natl Acad Sci U S A 109:E656–664.
- Rimkus SA, Katzenberger RJ, Trinh AT (2008) Mutations in String/CDC25 inhibit cell cycle re-entry and neurodegeneration in a Drosophila model of Ataxia telangiectasia. Genes Dev 22:1205–1220.
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