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Cell cycle regulation in the postmitotic


neuron: oxymoron or new biology?
Karl Herrup* and Yan Yang‡
Abstract | Adult CNS neurons are typically described as permanently postmitotic but there is
probably nothing permanent about the neuronal cell cycle arrest. Rather, it appears that these
highly differentiated cells must constantly keep their cell cycle in check. Relaxation of this
vigilance leads to the initiation of a cell cycle and entrance into an altered and vulnerable
state, often leading to death. There is evidence that neurons which are at risk of
neurodegeneration are also at risk of re-initiating a cell cycle process that involves the
expression of cell cycle proteins and DNA replication. Failure of cell cycle regulation might be
a root cause of several neurodegenerative disorders and a final common pathway for others.

Neuronal birthday
Most neuroscientists are comfortable with the axiom that The neuronal cell cycle
The day on which a neuron CNS neurons are ‘permanently postmitotic’. The intricate The CNS begins as a microscopic sheet of a few hun-
exits mitosis and differentiates cascades of kinases and transcription factors that regulate dred ectodermal cells (the neural plate) but rapidly
rather than re-enter a new cell the cell cycle in a HeLa or lymphocytic cell line (BOX 1) develops into a macroscopic structure with as many as a
cycle. Defined operationally as
seem removed from the day-to-day biochemistry of an hundred billion neurons in the adult human. This enor-
the last day that the adult
neuron can be labelled by adult neuron. Indeed, for decades developmental neuro- mous expansion in cell number means that the proper
exogenously applied 5-bromo- biologists have shown that the bulk of CNS neurogenesis development of the brain depends on a well-regulated
2′-deoxyuridine. takes place in a tightly packed layer of nuclei lining the pattern of cell division. This raises two questions: what
lumen of the neural tube (the ventricular zone, VZ) and molecular switches regulate the rate and number of cell
Cytokinesis
The process of physically
later in the closely apposed region known as the subven- divisions during growth of the nervous system, and
dividing the nuclear and tricular zone (SVZ). Patches of adult neural stem cells can how does the mature postmitotic neuron regulate the
cytoplasmic components be found lining the lateral ventricles — to various degrees cell cycle machinery so that it can refrain from division
of a cell in M phase into in different vertebrates — but these cells represent the for many years?
two daughter cells.
persistence of a mitotic progenitor population rather The kinetics of neurogenesis in the neural tube have
than the division of a fully differentiated adult nerve cell. been studied most carefully in the mouse neocortex.
Indeed, the concept of a neuronal birthday carries with it Here, each neuronal lineage apparently undergoes 11
the inescapable conclusion that once a neuron leaves the divisions before ceasing neuron production 1–5. The
VZ (or SVZ) it will never divide again. VZ itself seems to be highly stratified. Mitosis and
The absence of cell division is believed to be a core fea- cytokinesis occur at the ventricular surface, whereas
ture of neuronal identity. The depth of this belief is such other cell cycle activities, including DNA synthesis,
*Department of Cell Biology that, on the face of it, cell cycle regulation in the adult occur at some distance from the ventricle (FIG. 1). The
and Neuroscience, Rutgers
University, 604 Allison Road,
neuron hardly seems to be a problem at all — instead, proposed stratification of cell cycle phases is validated
Piscataway, New Jersey it seems to be more of an oxymoron. This review aims by the finding that the DNA precursors 3H-thymidine6,7
08854, USA. to stimulate the idea that cell cycle regulation is actually or 5-bromo-2′-deoxyuridine 8 (BrdU, a thymidine

Center for Translational a constant problem for mature neurons. We summarize analogue) label cells mainly in the superficial regions
Neuroscience, Department
an increasing body of evidence which illustrates that, far of the VZ when injected shortly before the animal is
of Neurosciences, Case
Western Reserve University, from being permanently postmitotic, adult neurons must killed (FIG. 1). A secondary germinative zone, the SVZ,
School of Medicine, 10900 continuously hold their cell cycle in check. This perspec- develops later just beyond the VZ. The functional dif-
Euclid Avenue, Cleveland, tive stems from the finding that cell cycle control can fail ferences between the ventricular and subventricular pro-
Ohio 44106, USA. in ‘postmitotic’ neurons. The price of failure is high and liferative regions are not entirely clear. Both are regions
Correspondence to K.H.
e-mail:
the death of neurons in many diseases may be an inexo- in which neurons undergo their final cell division (or
herrup@biology.rutgers.edu rable consequence of the re-initiation of the cell cycle in birthday). Furthermore, each region generates neurons
doi:10.1038/nrn2124 an adult neuron. as well as glia, yet there seem to be many molecular and

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Box 1 | Basics of the cell cycle cytological differences between the two regions9,10. By
the end of the developmental period, the VZ has been
Cell cycle phases depleted of all mitotic cells whereas the SVZ persists (to
G1 (Gap or Growth 1) phase. The period of growth preceding any commitment to
a greater or lesser extent in different vertebrates) at the
division. G0 is a specialized form of G1. Highly differentiated cells, which are unlikely to
divide unless provoked, are said to be in G0. During late G1 the cell commits to divide, lumen of the lateral ventricles and harbours stem cell
typically marked by the phosphorylation of the tumour suppressor gene retinoblastoma precursor populations that give rise to neurons in the
(Rb), and the activation of the transcription factor E2F1, which in association with its adult. The kinetics and function of these adult-generated
partner DP1 binds to the promoters of various cell cycle genes. cells is hotly debated11–14. However, because they are
S phase. The period of DNA synthesis. The process of replication of the entire genome is
generated by stem cells and not from the division of
completed, resulting in the doubling of the DNA content of the cell. cells that are already part of the adult neuronal circuitry,
they do not contravene the basic tenet of our review:
G2 (Gap or Growth 2) phase. A period during which the mechanical components that
a mature, differentiated nerve cell does not divide.
will organize the chromosomes and physically divide the cell are assembled.
The molecular signals that stimulate cell division
M (Mitosis) phase. An exquisitely complex cytological event that accurately divides the during neurogenesis are not entirely known, but there is
duplicated sets of chromosomes and coordinates the events of fission to produce two evidence that sonic hedgehog (SHH) signals from cer-
cells from one. Many events take place during M phase including the dephosphorylation
tain cell layers to stimulate mitotic activity in both the
of RB. Upon completion of M phase, both daughter cells re-enter G1 and a new cell cycle
VZ and in a specialized germinal region of the cerebel-
is set to begin.
lum known as the external granule cell layer. The actions
Cell cycle proteins of SHH are complex; it functions in patterning, cell fate
Proteins that drive or inhibit the cell cycle are shown in grey or pink boxes, respectively.
determination and survival as well as proliferation. SHH
Cyclins. Activator proteins that are up- or downregulated depending on the phase of seems to regulate cell division directly, via the transcrip-
the cell cycle. tion factors GLI and N-MYC15 (for a fuller discussion,
Cyclin-dependent kinases (CDKs). Serine/threonine kinases that require the binding of see REF. 16). The mechanism by which the period of
a cyclin (or related protein) for full activity. Their range of substrates is not fully defined, neuronal cell division in the VZ or SVZ is brought to an
but interfering with their activity arrests or slows the cycle. end is equally uncertain. Inherent in the above descrip-
Cyclin-dependent kinase inhibitors (CKIs). Small peptides that block cyclin/CDK activity tion is the conclusion that every neuron has a birthday
either by forming an inactive complex or by acting as a competitive CDK ligand. — a last time in embryogenesis at which 3H-thymidine
DNA replication proteins. DNA polymerases and associated proteins such as or BrdU will label its genomic DNA. This final cell divi-
proliferating cell nuclear antigen (PCNA) and mini-chromosome maintenance (MCM) sion signals the beginning of maturation, but also marks
proteins, as well as proteins that assure that each origin of replication initiates the point at which the nerve cell must put in place the
replication only once per cycle. These include origin recognition complex (ORC) mechanisms that will ensure a permanent mitotic arrest.
proteins, CDT1 and its suppressor, geminin. Despite the importance of these mechanisms, virtually
Checkpoint proteins. Members of a network of proteins that monitor DNA integrity and nothing is known about them. The tumour suppressor
arrest the cell cycle until DNA damage can be repaired136–138. retinoblastoma (RB) seems to be crucial, as null alleles
of this gene cause neurons to die after re-entering a cell
CDC6 CDT1
cycle17–20. Cyclin-dependent kinase inhibitors (CKIs)
Polymerase ORC also seem to be involved in cell cycle arrest, as the loss of
PCNA Geminin
CKI family members results in alterations in cell cycle
CDT1 kinetics21. Studies in the retina and other tissues indicate
MCM complex Origin that two subclasses of CKIs — the Ink4 class (including
p16Ink4a, p15Ink4b, p18Ink4c and p19Ink4d) and the Cip/Kip
ORC class (including p21Cip1, p27Kip1 and p57Kip2) — work
coordinately to ensure cell cycle exit22, although neither
class is necessary or sufficient to do so.
P RB
DP
E2F1 Cell cycle genes ORC Loose ends. There is surprisingly little information that
CKIs links the biochemical and molecular description of the
E2F p21Cip1
CKIs RB cell cycle with the largely cytological description of
Cyclin E p27Kip1
p19Ink4d CDK2 p57kip2 neurogenesis and migration. Cell cycle proteins are
p18Ink4c Cyclin A
Cyclin D CDK2
found in brain lysates at early developmental stages21;
p16Ink4a
p15Ink4b
CDK4/6 however, there are few immunocytochemical stud-
ies of these proteins23,24. The model shown in FIG. 1
S predicts that the nuclei of the VZ are stratified by
CDK4/6
G1 cell cycle phase, and therefore that cell cycle proteins
Cyclin D
should be expressed in layers. Indeed, the M phase
marker phosphohistone H3 is found in mitotic cells at
G2
Growth M the ventricular surface and BrdU, which labels cells
signal Cyclin B
RB in S phase, labels cells in the expected regions of the
CDK1
upper VZ20,25. Most other cell cycle proteins are not so
P RB
neatly distributed. For example, proliferating cell nuclear
antigen (PCNA), another S phase marker25, and cyclin E,

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Pia for virtually every one of the CDKs, cyclins and CKIs
(including CDK2, CDK4 and CDK6, as well as cyclins
A1, B2, D1, D2, D3, E1 and E2) survive most or all of
E2F1 the way through neurogenesis (TABLE 1). The only excep-
Cortical plate RB
CDK5
tion to this rule are embryos lacking cyclin A2 (REF. 37)
ORC2–5 or cyclin B1 (REF. 38) (the knockout of CDK1 has not
been reported). The survival of these mice may be due
Intermediate zone CDK5 to functional redundancy, as mice with mutations in
p27 two or three of these proteins die earlier. However, even
p21
Subventricular zone p57 the cyclin D triple mutant (Ccnd1–/–;Ccnd2–/–;Ccnd3–/–)
progresses to approximately embryonic day 16.5
(REF. 39), which is after most neurogenesis has occurred,
BrdU/3H-T suggesting that there is more to neuronal cell cycle
S uptake regulation than cyclins and CDKs alone. Similarly,
Cyclin E RB-deficient embryos die in the third trimester, but a
Ventricular zone G1/G2 Ki67 normally patterned brain and spinal cord develop17–19.
PCNA
Finally, the loss of the CKI p27Kip1 or p19Ink4d alters cell
Phospho- cycle kinetics during neurogenesis40,41, and the balance
M
histone H3
of different cell types that differentiate can be affected,
Ventricle but cell division eventually stops. Indeed, the lasting
Figure 1 | The developing cerebral cortex. A cross-section through the wall of the morphological changes observed in these mice are rela-
neural tube is shown. The morphological zones are noted on the left. Dividing tively subtle, given the predicted role of these proteins
neuroepithelial cells are shown in blue, radial glia in green and mature cortical neurons
in orchestrating neuronal exit from the cell cycle (for a
in yellow. Various features of the cell cycle that are important for the development of
the cerebral cortex are shown on the right. The proposed stratification of the cell cycle fuller description of such mutations in the context of the
phases (G1, S, G2 and M) in the ventricular zone (VZ) is indicated. Note that the more entire developmental process, see REF. 42). A tentative
superficial subventricular zone has continued mitotic activity but no proposed summary of how these gene products affect the three
stratification. The mature cortex is generated by each successive wave of immigrants major phases of CNS neuronal development is outlined
from the VZ resulting in an inside-out pattern of layering with the first generated in FIG. 2.
(early-born) neurons residing in the deeper layers and the last generated cells (late-
born) residing more superficially. The proteins listed on the right are examples of Failure of cell cycle arrest in development
proteins discussed in the text shown in their approximate location in the normal CNS. As described above, CNS neurons do not divide after
3
H-T, 3H-thymidine. they emigrate from the VZ or SVZ. What would happen
if a neuron lost control of its cell cycle and re-entered
Phosphohistone H3 a late G1 phase marker26, appear in cells in all strata of cell division? Although it seems paradoxical, evidence
A phosphorylated form of the the VZ. For the CKIs, the picture is just as complicated22. suggests that the neuron would probably die. We refer
DNA coating protein, histone Cyclins and cyclin-dependent kinases (CDKs) are regu- to this process as cycle-related neuronal death (CRND)
H3. Empirically, the presence lated by many factors that control their synthesis, degra- — a purposefully cautious term (see below).
of this modification is unique to
M phase.
dation and localization. Their broad distribution in the The first experimental evidence for CRND came
VZ indicates that either the predicted stratification of from two independent lines of experimentation in
Proliferating cell nuclear cell cycle phase is incorrect or that the regulation of these which RB protein was inactivated by the large transfor-
antigen proteins in dividing neuronal precursors differs from the mation (T) antigen of the SV40 virus. Inactivation of RB
(PCNA). A subunit of the DNA simple models described in most textbooks. leads to the release of the transcription factor E2F1 and
replication complex. Three
Another unexpected finding is that DNA repair the subsequent upregulation of many cell cycle genes.
PCNA proteins assemble as a
homo-trimer encircling the enzymes are active immediately after the completion of When T antigen is introduced into most cultured cells,
double helix just ahead of the neurogenesis. Mutations in genes involved in DNA dou- they are released from normal proliferation control
replication fork. ble-strand break (DSB) repair, including Xrcc427, ligase IV and become transformed. However, when T antigen is
(REF. 28) and the DNA repair polymerase, Polβ 29,30, result introduced into postmitotic neurons, they degener-
Ligase IV
in embryonic lethal phenotypes. Moreover, neuronal ate43–46. Cell division begins and BrdU is incorporated
A form of DNA ligase that
rejoins 5′ and 3′ ends of death in ligase IV knockouts can be suppressed by the into DNA, but M phase is not initiated and the neuron
apposed double-strands of introduction of a second mutation in ataxia telangiecta- dies shortly after44. As might be predicted, this process
DNA. This ligase isoform is sia mutated (Atm)31, an early component of the DSB is E2F1-dependent47.
specific for DNA repair, detection and response system. The implication of these There is also massive apoptotic neuronal death in
especially non-homologous
findings is that DSBs are normal events in early neuronal mice with homozygous null alleles of Rb17–19. In these
end joining.
differentiation. The function of these proposed breaks is animals, CRND occurs after the migrating neuroblasts
Transformed unknown32–34, but the correlation of human CNS disor- leave the ventricular region. BrdU injections a few hours
A cellular state marked by ders with defects in DSB repair genes underscores their before sacrifice have revealed that neurons in normally
escape from growth control importance in the development of the brain35,36. postmitotic regions of the maturing brain and spinal
mechanisms that normally
While we might predict that the mutation of cell cord are engaged in ectopic DNA synthesis17–19. Most
regulate the cell cycle.
Typically, transformed cells will
cycle proteins would have devastating effects on devel- of the neuronal death in Rb knockouts is dependent
form tumours in soft agar and opment and neurogenesis, a final loose end comes on both E2F1 and another transcription factor, p53
in animals. from the observation that homozygous null embryos (REF. 48,49). Detailed examination of the mutant embryos

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Table 1 | Mouse knockouts of cell cycle proteins with demonstrated CNS effects
Cell cycle Disrupted Survival Neuronal cell phenotype Other phenotypes References
phase gene(s)
G1 phase Cyclin D1 Viable Reduction of cell number in the neural Reduced body size, neurological 143,144
retina abnormalities, hypoplastic retina,
impaired proliferation of mammary
gland epithelium
Cyclin D2 Viable Loss of granule cell and stellate Females sterile, males fertile, impaired 145–147
interneurons in the cerebellum proliferation of B cells
Cyclin D1 Viable, die Severely reduced number of granule Reduced body size, problems with 148
and D2 within 3 weeks cells in the IGL and abnormal coordination movements
positioning of Purkinje cells
Cyclin D1 Most die after Reduced proliferation in retina Respiration with meconium 148
and D3 birth
pRb Embryonic Apoptotic cells in CNS and PNS Defects in erythroid, neuronal, lens 17–19,69
lethal (E12–15) and placental cell lineages. Tetraploid
complementation resulted in
completion of embryonic development
pRb and p107 Embryonic CNS findings similar to Rb–/– Apoptosis in liver 149
lethal (E11–13)
p27 kip1 Viable Cell cycle progression in the neocortical ND 41
(overexpressed) PVE, lengthening of the G1 phase, but
no apoptotic cells were found
Other Cdk5 Embryonic Cell cycle related genes expressed in ND 120,139,141
lethal (E18) cerebral cortex, defective neuronal
migration, differentiation and survival
Cell cycle Atm Death occurs Most of Atm–/– models show no signs of Immune deficiences, sterility cells are 97,150
checkpoint after 4 months neuronal death, except REF. 150, which radiosensitive
reports nigro-striatal degeneration
Atr Embryonic Apoptotic cell death in early embryo Chromosome fragmentation in cultured 151
lethal (E7) blastocysts
Chk1 Embryonic No specific report on CNS General aberrant nuclei morphology 152
lethal (E3–7) and TUNEL staining in blastocysts
Chk2 Viable No specific report on CNS Radio-resistance 153
Note that XRCC2, Ligase IV, Ku70/80, 53BP1, DNA-PKcs, RAD50, MRE11 and NBC1 are all DNA damage detection and repair proteins. Although none interferes
directly with cell cycle progression, the patterns of cell death in knockout mouse models closely approximate those found in the embryonic development of the
retinoblastoma (Rb) knockout. These mutations are often embryonic lethal. Atm, ataxia telangiectasia mutated; Atr, ataxia telangiectasia and rad3-related kinase;
Cdk5, cyclin-dependent kinase 5; Chk1, cell-cycle checkpoint kinase 1; E, embryonic day; IGL, internal granule cell layer; ND, not determined; PVE, pseudostratified
ventricular epithelium; TUNEL, terminal deoxynucleotidyl transferase labelling.

has revealed a second, less appreciated phenotype: before block cell cycle advance can prevent the death of both
their death, neurons are both morphologically and bio- PC12 cells and sympathetic neurons after either NGF
chemically immature, suggesting that RB is also required withdrawal or DNA damage52–56. Similarly, various
for neuronal differentiation20. insults including trophic factor withdrawal, excitotoxic-
This link between cell cycle suppression and final ity and toxic concentrations of amyloid-β can drive a
neuronal differentiation is found for many cell cycle lethal cell cycle in cultured CNS neurons and, critically,
genes, and seems to work both ways. Early investiga- blocking the cell cycle can prevent neuronal death57–60.
tions that explored the death response of cultured In sympathetic neurons, trophic factor withdrawal or
sympathetic neurons after withdrawal of nerve growth DNA damage leads to an E2F1-dependent upregula-
factor (NGF) revealed an unexpected increase in the tion of B-myb and C-myb (transcription factors that are
levels of cyclin D mRNA50. This indicated that loss of believed to have a role in S phase progression)61. This
trophic support during development might lead to cell results in the expression of BIM (a BCL2 homology
cycle initiation as part of the death process. Research on three domain-only molecule)62, which in turn leads to
models of target-related cell death in the mouse cerebel- cell death. In activity-deprived cerebellar granule cells,
lum51 has revealed that neuronal death is preceded by phosphorylation of the proapoptotic molecule BAD by
PC12 cells
A rat pheochromocytoma cell re-expression of cell cycle markers and DNA synthesis CDK1 appears to have a similar role63,64.
line. When treated with nerve in vivo. These correlations are strong evidence that a Thus, both in vivo and in vitro, several lines of
growth factor, PC12 cells cease neuron must regulate its cell cycle or risk death during evidence converge on the following hypothesis: ‘once
mitosis and differentiate into postmitotic maturation. a CNS neuron leaves the VZ, its cell cycle must be
cells that resemble
sympathetic neurons,
The use of in vitro models of CRND has been actively held in check. Relaxation of that vigilance
complete with processes and invaluable for establishing the causative nature of cell leads to cell cycle initiation and death, which can
functional synapses. cycle induction in the process of cell death. Drugs that follow within hours’.

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Loose ends. At first glance Rb-deficient embryos seem to Neurogenesis Migration Maturation
tell a simple story of failed cell cycle suppression leading
quickly to cell death. However, it is not clear exactly how
RB deficiency leads to neuron loss. First, the phenotype
of these mice varies among brain regions, indicating
that there is regional heterogeneity in RB dependence.
Second, the lack of RB function in a neuron is not, by
itself, sufficient to cause neuronal death. Rb–/– neurons
can survive in the mixed environment of an Rb–/–↔Rb+/+
chimeric brain 65–67, and conditional knockouts of
Rb68,69 also support this idea. This is not simply a near-
neighbour effect, as the CNS phenotype is rescued
in Rb–/–↔euploid Rb+/+;tetraploid chimeras69,70, in which
Rb–/– embryos develop in a conceptus that includes wild-
type (Rb+/+) extra-embryonic tissues. In the brains of
the resulting animals, BrdU incorporation reveals that dNTP
mutant nerve cells can begin a cell cycle but not die.
DNA
Thus, failure of cell cycle suppression is not sufficient to
lead to CRND by itself, although it might be necessary.
Dissociation of the loss of cell cycle suppression from the
process of neuronal death suggests a caveat to the final
By mutation: By mutation: By presence:
clause of the hypothesis — that death follows quickly Cyclin A2? CDK5 E2F1
after cycle initiation — a concept that is developed more Cyclin B1? RB RB
fully by considering the situation in the adult brain. Sonic hedgehog? p27, p57 CDK1
Ligase IV?
XRCC4? By mutation:
Failure of cell cycle arrest in the adult Polβ? ORC2–6
CDK5
Our hypothesis predicts that the prohibition of neuronal p21
cell division is life-long. Might examples of late-onset Figure 2 | Cell cycle proteins implicated in each of the
neurodegenerative disease be accompanied by loss of neu- three phases of neuronal development. Of the cell
ronal cell cycle control? This concept was first proposed cycle proteins, mutations in only two of the cyclins (A2
to explain the presence of a unique species of phospho- and B1) are known to interrupt the process of neurogenesis
rylated tau protein, usually found only in dividing cells, (left column); however, as a direct effect of these proteins
in the neurons of patients who had died with Alzheimer’s on the behaviour of cells within the ventricular zone (VZ)
disease (AD)71,72. A number of laboratories have reported has never been shown, it may be that these mutations
block development before the nervous system can emerge.
the re-expression of various cell cycle proteins in neurons
DNA synthesis is indicated by the deoxyribonucleotide
from patients with AD. The proteins include cyclins A73,
triphosphate (dNTP) to DNA symbol on the left of the first
B73–76, D74,76–78 and E73,78, as well as CDKs71,77,79,80, PCNA73,74, column. Migration (centre column) describes the stage in
Ki67 (REFS 73,81) and CKIs of both the Ink and Cip/Kip which the neuron ceases new mitotic activity and begins
families77,79,82,83. CDK1 may be particularly important in the early differentiation steps required to emigrate from the
this group because of the tissue culture data mentioned VZ and sub ventricular zone. Maturation (right column) is
previously63,64 , and the recent evidence of genetic link- the cytological and biochemical ‘coming of age’ of the
age to AD84,85. There are also reports of cell cycle protein neuron once it has reached its adult location. The inclusion
re-expression in amyotrophic lateral sclerosis86–88, ataxia of the transcription factor E2F1 and retinoblastoma (RB) in
telangiectasia89, Parkinson’s disease90–93, stroke94 and other this stage is by inference only, as knocking down these
proteins in the adult has not yet been shown to have an
neurodegenerative conditions95, 96. Where quantification
effect on maturation. CDK1, cyclin-dependent kinase 1;
has been carried out, it seems that around 10% of at-risk
ORC, origin recognition complex.
neurons re-express these proteins.
These findings represent correlations between ele-
vated protein expression levels and disease, not proof of the brains of patients with AD using fluorescent in situ
CRND. In fact, if the immunocytochemistry is offering hybridization (FISH) probes against unique genomic
a correct picture, it seems likely that cell cycle proteins loci75. In at-risk regions of the brain, around 4% of
from different phases are co-expressed and that their the neurons had three or four spots of hybridization
location is frequently cytoplasmic. In this context, two rather than two. Thus, the cell cycle protein expression
studies 75,76 are particularly noteworthy. One shows in AD correlates with DNA replication, suggesting that
that cells that can be immunostained with antibodies at least the first phases of a true cell cycle have begun
to cell cycle proteins, when measured as a percentage in the at-risk neurons. One clear implication of this
of neurons in a region, are as prevalent during early is that in the adult, the presence of cell cycle proteins
stages of AD as they are during end-stage illness76. The or even DNA replication on its own cannot be taken
other study investigated whether these proteins initiate as evidence of adult neurogenesis. These events could
a true cell cycle process, complete with DNA replica- easily represent the beginning of a cell death process
tion, by probing the interphase nuclei of neurons in with very much the opposite result.

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Loose ends. Cell cycle regulation in the adult neuron otherwise excellent phenocopy falls short owing to the
is an area of study in which the number of loose ends absence of neurodegeneration. The gene that is mutated
vastly exceeds the clearly-established parts of the story. in ataxia telangiectasia (Atm) signals the presence of
First, the cells never enter M phase. Condensed chro- DSBs and arrests the cell cycle to allow for DNA repair.
mosomes are not found and there is no visible evidence In all reported mouse knockouts, the sterility, immune
of spindle formation. This makes it difficult to confi- deficiency and radiosensitivity associated with the
dently characterize the process as a cell cycle as it is human condition are replicated faithfully, but their
never completed. Second, where it has been quantified, gross behaviour is normal and there is no detectable
there are too many cycling nerve cells74–76. The aver- loss of Purkinje cells108. A similar effect is found in
age course of AD is 10 years from first symptoms to mouse models of AD: amyloid-β plaques appear along
death. If, as the immunostaining suggests, 5–10% of the with behavioural abnormalities, but there is no appar-
neurons are dying at any one moment, and if a typical ent loss of neurons109. Remarkably, however, initiation
apoptotic process takes roughly 12 hours, then half of of ectopic cell cycling is preserved. In Atm-knockout
the affected population should be dead in a week or mice, cell cycle initiation can be found in Purkinje
less and 95% should be dead in less than a month. This cells and striatal neurons89. Similarly, in the R1.40 yeast
is clearly not the case. The implication is that death by artificial chromosome transgenic line, an AD model,
cell cycle in adult neurons must be a very slow process cycle anomalies begin at 6 months after birth in regions
requiring in the order of 6–12 months. Although this analogous to those in which the human disease begins,
protracted time period is unexpected, mouse models of progressively appearing in the brainstem and cortical
late onset human neurodegenerative diseases indicate regions as in AD110. Two groups examined the APP23
that, if anything, 6–12 months might be an underesti- mouse, another AD model, but disagreed on the pres-
mate of the true length of the process as discussed in ence of neuronal cell cycle events110,111. The difference
the next examples. may be in the age of the mice examined; however, the
Transgenic mouse models of human inherited dis- controversy remains unresolved. With the exception of
eases, including ataxia telangiectasia97–99 and AD100–106, this one report, a long delay between cell cycle initiation
are noteworthy because, with few exceptions 107, an and cell death is apparent in these AD mice. Although
R1.40 animals can live for two years after cell cycle events
first appear, there is no apparent decrease in neuronal
Immature Mature density and no documented loss of neurons.
The combined studies in humans and mice suggest a
modification of the hypothesis articulated above. ‘Once
a CNS neuron leaves the VZ or SVZ, its cell cycle must
be actively held in check. Relaxation of that vigilance
leads to cell cycle initiation and death, which can follow
within hours in newly generated neurons. Once neurons
are fully mature, however, neuronal death by cycle re-ini-
tiation can take from months to years, and might require
Diploid an additional stimulus to make the transition from cycle
to death’ .
This distinction between young and old neurons is
illustrated in FIG. 3. The dashed arrows in this diagram
Mitotic reflect the possible need for an extra stimulus to trigger
pressure death in a cycling adult neuron.

What is a cell cycle protein?


Proteins identified as cell cycle proteins turn up in
4n Tetraploid 4n
unlikely locations at unlikely times, to serve unlikely
functions in nerve cells that, having left the VZ and
SVZ, are supposed to have no further relationship with
the cell cycle. One example of this is the Rb gene itself.
? Neurons of the embryonic Rb-deficient nervous system
Cell death
not only fail to survive, they also fail to differentiate20.
Figure 3 | Mature and immature neurons differ in their response to cell cycle That would appear to define an embryonic time during
initiation. Healthy (yellow) immature neurons that are deprived of trophic factors or are which the protein functions, but RB — along with its
exposed to other stimuli begin a cell cycle process that rapidly leads to cell death. Mature binding partner E2F1 — are both found in the cytoplasm
neurons, however, seem to require a two step process for cell death to occur in response
and dendrites of the supposedly postmitotic neuron and
to cell cycle re-entry. Under stress, for example, a healthy adult neuron can enter a cycle
and proceed all or part of the way through S phase, but the cycle then stops just before both respond rapidly to exogenous stressors by changing
M phase. These cells are in an unusual cell biological state (green), having twice the their normally nuclear localization to a predominantly
normal DNA complement (4n), but otherwise they seem normal. The dashed arrows cytoplasmic one112–114. CKIs also have an important
leading through an intermediate stage marked by a question mark indicate the need non-cycle role in differentiation115–119, which is inde-
for a second step that moves the nerve cell from this unusual state to death (grey cell). pendent of their role in cell cycle suppression117, 119.

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Mitogen
A further example of this cycle/differentiation cells are wild type, they divide in an apparent attempt
A substance, usually a protein, duality is the ‘atypical’ cyclin-dependent kinase, to fill the gap in the tissue expected to be left by the
that induces cell division in a CDK5 (BOX 2) . CDK5 has both pro-differentiation dying cells.
receptive cell. and anti-cell cycle activities during development120. Could the cycle-positive neurons in AD and ataxia
In the adult, however, the kinase ‘diversifies’. In many telangiectasia brains be analogous to these undead
if not most CNS synapses, CDK5 forms complexes cells, triggered to die but blocked from completing the
with121 and affects the function of 122–127 various syn- process? If so, these cells might be part of the problem
aptic proteins. rather than innocent victims of the disease process. If
Perhaps the most surprising example of the in their undead state they are sending mitogenic signals
promiscuous function of cell cycle proteins is that to their neuronal neighbours, they could be pressuring
members of the origin recognition complex (ORC) otherwise healthy neurons to initiate a cell cycle (FIG. 4).
are found in synaptic fractions of adult neurons 128. If this is true, their stability and persistence could rep-
This is remarkable because the ORCs are a family resent a danger to the health of the brain. As they do
of proteins best known for binding to specific sites not die, their continued presence might weaken those
in the genome during the process of mitotic DNA around them and yet, because re-entry into a cell cycle
replication. The synaptic function of these proteins does not seem to result in cell death, not kill them. This
is unknown, although in other cell types they are would create a paradox for those who seek therapeutic
reported to form complexes with proteins other than interventions for neurodegenerative conditions such as
those of the ORC129. AD. The prediction would be that the most effective
Finally, a substantial fraction of neurons in the therapies would block new neurons from entering a
adult E2f1-knockout mouse are engaged in a strange cell cycle while simultaneously ridding the brain of the
cell cycle-like process (L. Wang and K.H., unpublished undead cells.
observations). They express high levels of cyclin A
(cytoplasmic) and PCNA (nuclear), and by FISH Conclusions and future directions
criteria they have undergone DNA replication. Taken In many ways, descriptions of adult CNS neurons as
together, the data suggest that proteins best known having ‘permanently exited the cell cycle’ or having
for their role in cell cycle progression have separate entered ‘a permanent state of G0’ are terms of conven-
functions in the fully differentiated, mitotically inactive ience, which may lack true functional meaning. In fact,
neuron. such terms might lull us into a false sense of security
— a belief that the issue of cell cycle regulation in
Are adult cycling neurons ‘undead’? neurons is an oxymoron rather than an important bio-
As described above, neurons under stress can engage logical question. We believe that the evidence reviewed
a cell-cycle-like process that leads to the constitutive here challenges us to rethink this position.
expression of proteins normally found only in cycling
cells and the acquisition of hyper-diploid DNA. Despite
these oddities, cycling cells can persist in the brain for
Box 2 | An atypical CDK
long periods. The dendrites of these neurons show
only minimal atrophy89, but the behavioural anoma- Cyclin-dependent kinase 5 (CDK5) is structurally similar to
lies found in the mouse models of both AD and ataxia other CDKs, but is said to have no role in the normal cell
telangiectasia hint at functional impairments. The cycle. It binds cyclin D, but its activity is not initiated by
this partnership. Rather, two proteins known as p35 and
question posed by this situation is: what is the nature
p39 function as cyclin equivalents and activate CDK5.
of the state in which these nerve cells find themselves?
Mice that are genetically deficient in either CDK5 or both
Data from embryonic studies indicate that cell cycle activator proteins have serious defects in neuronal
initiation in a post-VZ neuron is a first step towards migration and die before birth139–141. Intermediate filament
cell death. However, data from adult studies suggest expression in CDK5-deficient neurons in vivo and in vitro is
that the processes of cell cycle initiation and cell retarded or blocked120. Early filament types such as nestin
death are separable. Similar situations, of neurons on and βIII-tubulin remain expressed in regions of Cdk5–/–
a path to death being blocked by genetic or trophic cortex from which they are lost in the wild type, and later
means, have been reviewed recently for several species differentiation markers such as microtubule-associated
and have raised many important issues130. protein 2c are seen only at very low levels120.
In addition to its pro-differentiation functions, CDK5
Speculatively, it may be possible to draw an anal-
also appears to act as a cell cycle inhibitor. Both in vivo
ogy between the arrested cell cycle state of these
and in vitro, Cdk5–/– neurons cannot restrain their cell
neurons and a cellular state defined in the imaginal cycle and die rapidly after the re-expression of cell cycle
discs of Drosophila melanogaster. The state was first proteins and the incorporation of 5-bromo-2′-
proposed131 to describe cells that are triggered to die by deoxyuridine120. In vitro models of cycle-related neuronal
an insult such as X-rays but blocked from completing death suggest that the ‘anti-cycle’ activity of CDK5
the process by expression of an anti-apoptotic protein. requires nuclear localization (Cicero, S.A. et al.
The authors describe the resulting cells as ‘undead’ unpublished observations). Whereas in other cell death
and have shown that they have some unusual proper- models the localization of CDK5 to the nucleus is
ties131–133. For example, they send signals that serve as protective142, it would appear that CDK5 is very much
involved in cell cycle regulation in neurons.
a mitogen to neighbouring cells. If the neighbouring

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Mitotic
pressure

Figure 4 | Speculative model of how a ‘cycling’ neuron might be a threat to the health of the CNS. Analogous to
the concept of ‘undead’ neurons in the fruitfly imaginal disc, the figure illustrates a situation where cycling but living
neurons pose a problem for the remaining cells in the neighbouring brain region. If, as in the fly, the undead (green) cells
are capable of releasing a mitogenic signal (arrows), then a situation arises in which the victim may become the assailant.
The transformation of a mature neuron into this unusual state would actually enhance the local ‘mitotic pressure’, possibly
leading other neurons to enter this ‘undead’ state or to enter an apoptotic process (grey nucleus) and die (grey cell).

For billions of years, the prevailing entities (which roles of cell cycle proteins such as E2F1, RB, CDKs,
ultimately became cells) were those that divided fastest cyclins and ORC proteins in dendrites and axons.
and most efficiently. Metazoans evolved when aggre- Under normal conditions these functions exemplify
gates of these early cells were able to realize gains from efficient design. Under stress, however, they appear
cooperation and specialization that exceeded those to represent an Achilles’ heel for the neuron. While
they could eke out by living on their own. It is worth some may start off as protective (for example, p27 and
considering that by this time the ‘instinct’ to divide was CDK5), many can be dangerous to the health of the cell
buried deep in the fundamental biology of every cell. It if they end up in the wrong cellular location.
may even have preceded the emergence of a genome. If cell cycle regulation is truly a constant issue for an
Thus, the dividing instinct might be as powerful today adult neuron then an entire menu of action items seems
as it was a billion years ago. The challenge faced by a spread before us. One important initiative would be to
metazoan, therefore, is to suppress the urge to cycle drop, whenever possible, the reference to proteins as cell
among its own constituents in order to enforce a regu- cycle proteins. Each protein whose first known func-
lated body plan and allow the extreme specialization tion was to advance or retard the progression of the cell
(differentiation) of its members. Indeed, suppressing cycle appears to have alternative identities in neurons
the cell cycle is arguably the most important step in any and other differentiated cells. Restricting our focus to
differentiation programme. their role in cell cycle regulation may blind us to the full
Neurons are among the most specialized of cells, repertoire of their activities. We also need to remind
and organisms that benefit from incorporating them ourselves that the mechanics of cell cycle regulation
into their structure seem to have largely solved the can be distinct in different cell types. Textbooks tell us
problem of reining in their drive to divide; the nature of that the E2F1 protein drives the cell cycle by upregulat-
the solution(s), however, remains largely unknown. But ing the synthesis of key proteins needed for cell cycle
we may wish to rephrase questions such as what factors progression, as is indeed the case in cultured fibrob-
force a postmitotic neuron to re-enter a cell cycle in AD lasts and COS cells; however, E2f1-knockout animals
or what fail-safe mechanisms of cell cycle suppression are born and develop normally, but die in middle age
are lost in AD such that pockets of neurons can break with tumours and hyperplasias in a variety of exocrine
free of their postmitotic state and return to an earlier tissues134,135. Clearly, in some cells E2F1 functions as a
mode of existence. For neurons of the CNS it seems tumour suppressor. Finally, it seems apparent that, just
that anti-cycling kinases, cell cycle promoters turned as in real estate, the three keys to activity are location,
into cell cycle suppressors, and DSBs during or imme- location and location. We need to define where these
diately after the final cell division are all components of multifunctional proteins are in neurons at different
a strategy by which the metazoan developmental plan junctures of the developmental process and during the
imposes cell cycle arrest on its neurons. This multi- response to insult or injury. The behaviour of CDK5 in
tiered strategy involves not only changes in protein CRND suggests that its function in the nucleus is differ-
localization and altered biochemical networks, but ent from that in the cytoplasm. This is not a new insight,
perhaps rearrangements of DNA structure as well. The but it is part of a readjustment to the notion that we can
evolution of multicellular organisms appears to have avoid concerning ourselves with the regulation of the
been a stern mitotic master. cell cycle in all highly differentiated cells — especially
But evolution is also a miserly process. It tends to neurons. Indeed, to reach a fuller understanding of
adapt old tools for new uses rather than fashion new how neurons remain mitotically silent for decades, there
tools. In neurons, this is reflected by the additional is a great deal of new biology yet to learn.

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