REVIEWS

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
The day on which a neuron
exits mitosis and differentiates
rather than re-enter a new cell
cycle. Defined operationally as
the last day that the adult
neuron can be labelled by
exogenously applied 5-bromo2-deoxyuridine.

Cytokinesis
The process of physically
dividing the nuclear and
cytoplasmic components
of a cell in M phase into
two daughter cells.

*Department of Cell Biology

and Neuroscience, Rutgers
University, 604 Allison Road,
Piscataway, New Jersey
08854, USA.

Center for Translational

Neuroscience, Department
of Neurosciences, Case
Western Reserve University,
School of Medicine, 10900
Euclid Avenue, Cleveland,
Ohio 44106, USA.
Correspondence to K.H.
e-mail:
herrup@biology.rutgers.edu
doi:10.1038/nrn2124

Most neuroscientists are comfortable with the axiom that

CNS neurons are permanently postmitotic. The intricate
cascades of kinases and transcription factors that regulate
the cell cycle in a HeLa or lymphocytic cell line (BOX 1)
seem removed from the day-to-day biochemistry of an
adult neuron. Indeed, for decades developmental neurobiologists have shown that the bulk of CNS neurogenesis
takes place in a tightly packed layer of nuclei lining the
lumen of the neural tube (the ventricular zone, VZ) and
later in the closely apposed region known as the subventricular zone (SVZ). Patches of adult neural stem cells can
be found lining the lateral ventricles to various degrees
in different vertebrates but these cells represent the
persistence of a mitotic progenitor population rather
than the division of a fully differentiated adult nerve cell.
Indeed, the concept of a neuronal birthday carries with it
the inescapable conclusion that once a neuron leaves the
VZ (or SVZ) it will never divide again.
The absence of cell division is believed to be a core feature of neuronal identity. The depth of this belief is such
that, on the face of it, cell cycle regulation in the adult
neuron hardly seems to be a problem at all instead,
it seems to be more of an oxymoron. This review aims
to stimulate the idea that cell cycle regulation is actually
a constant problem for mature neurons. We summarize
an increasing body of evidence which illustrates that, far
from being permanently postmitotic, adult neurons must
continuously hold their cell cycle in check. This perspective stems from the finding that cell cycle control can fail
in postmitotic neurons. The price of failure is high and
the death of neurons in many diseases may be an inexorable consequence of the re-initiation of the cell cycle in
an adult neuron.

368 | MAY 2007 | VOLUME 8

The neuronal cell cycle

The CNS begins as a microscopic sheet of a few hundred ectodermal cells (the neural plate) but rapidly
develops into a macroscopic structure with as many as a
hundred billion neurons in the adult human. This enormous expansion in cell number means that the proper
development of the brain depends on a well-regulated
pattern of cell division. This raises two questions: what
molecular switches regulate the rate and number of cell
divisions during growth of the nervous system, and
how does the mature postmitotic neuron regulate the
cell cycle machinery so that it can refrain from division
for many years?
The kinetics of neurogenesis in the neural tube have
been studied most carefully in the mouse neocortex.
Here, each neuronal lineage apparently undergoes 11
divisions before ceasing neuron production 15. The
VZ itself seems to be highly stratified. Mitosis and
cytokinesis occur at the ventricular surface, whereas
other cell cycle activities, including DNA synthesis,
occur at some distance from the ventricle (FIG. 1). The
proposed stratification of cell cycle phases is validated
by the finding that the DNA precursors 3H-thymidine6,7
or 5-bromo-2-deoxyuridine 8 (BrdU, a thymidine
analogue) label cells mainly in the superficial regions
of the VZ when injected shortly before the animal is
killed (FIG. 1). A secondary germinative zone, the SVZ,
develops later just beyond the VZ. The functional differences between the ventricular and subventricular proliferative regions are not entirely clear. Both are regions
in which neurons undergo their final cell division (or
birthday). Furthermore, each region generates neurons
as well as glia, yet there seem to be many molecular and

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Box 1 | Basics of the cell cycle
Cell cycle phases
G1 (Gap or Growth 1) phase. The period of growth preceding any commitment to
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,
typically marked by the phosphorylation of the tumour suppressor gene retinoblastoma
(Rb), and the activation of the transcription factor E2F1, which in association with its
partner DP1 binds to the promoters of various cell cycle genes.
S phase. The period of DNA synthesis. The process of replication of the entire genome is
completed, resulting in the doubling of the DNA content of the cell.
G2 (Gap or Growth 2) phase. A period during which the mechanical components that
will organize the chromosomes and physically divide the cell are assembled.
M (Mitosis) phase. An exquisitely complex cytological event that accurately divides the
duplicated sets of chromosomes and coordinates the events of fission to produce two
cells from one. Many events take place during M phase including the dephosphorylation
of RB. Upon completion of M phase, both daughter cells re-enter G1 and a new cell cycle
is set to begin.

Cell cycle proteins

Proteins that drive or inhibit the cell cycle are shown in grey or pink boxes, respectively.
Cyclins. Activator proteins that are up- or downregulated depending on the phase of
the cell cycle.
Cyclin-dependent kinases (CDKs). Serine/threonine kinases that require the binding of
a cyclin (or related protein) for full activity. Their range of substrates is not fully defined,
but interfering with their activity arrests or slows the cycle.
Cyclin-dependent kinase inhibitors (CKIs). Small peptides that block cyclin/CDK activity
either by forming an inactive complex or by acting as a competitive CDK ligand.
DNA replication proteins. DNA polymerases and associated proteins such as
proliferating cell nuclear antigen (PCNA) and mini-chromosome maintenance (MCM)
proteins, as well as proteins that assure that each origin of replication initiates
replication only once per cycle. These include origin recognition complex (ORC)
proteins, CDT1 and its suppressor, geminin.
Checkpoint proteins. Members of a network of proteins that monitor DNA integrity and
arrest the cell cycle until DNA damage can be repaired136138.
CDC6

CDT1
ORC

Polymerase
PCNA

Geminin
CDT1

MCM complex

Origin
ORC

P RB

CKIs
p19Ink4d
p18Ink4c
p16Ink4a
p15Ink4b

DP
E2F1

Cell cycle genes

E2F
RB

ORC

Cyclin E
CDK2

Cyclin A
CDK2

Cyclin D
CDK4/6

CKIs
p21Cip1
p27Kip1
p57kip2

CDK4/6

G1

Cyclin D

Growth
signal

G2
Cyclin B
CDK1

RB
P RB

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cytological differences between the two regions9,10. By

the end of the developmental period, the VZ has been
depleted of all mitotic cells whereas the SVZ persists (to
a greater or lesser extent in different vertebrates) at the
lumen of the lateral ventricles and harbours stem cell
precursor populations that give rise to neurons in the
adult. The kinetics and function of these adult-generated
cells is hotly debated1114. However, because they are
generated by stem cells and not from the division of
cells that are already part of the adult neuronal circuitry,
they do not contravene the basic tenet of our review:
a mature, differentiated nerve cell does not divide.
The molecular signals that stimulate cell division
during neurogenesis are not entirely known, but there is
evidence that sonic hedgehog (SHH) signals from certain cell layers to stimulate mitotic activity in both the
VZ and in a specialized germinal region of the cerebellum known as the external granule cell layer. The actions
of SHH are complex; it functions in patterning, cell fate
determination and survival as well as proliferation. SHH
seems to regulate cell division directly, via the transcription factors GLI and N-MYC15 (for a fuller discussion,
see REF. 16). The mechanism by which the period of
neuronal cell division in the VZ or SVZ is brought to an
end is equally uncertain. Inherent in the above description is the conclusion that every neuron has a birthday
a last time in embryogenesis at which 3H-thymidine
or BrdU will label its genomic DNA. This final cell division signals the beginning of maturation, but also marks
the point at which the nerve cell must put in place the
mechanisms that will ensure a permanent mitotic arrest.
Despite the importance of these mechanisms, virtually
nothing is known about them. The tumour suppressor
retinoblastoma (RB) seems to be crucial, as null alleles
of this gene cause neurons to die after re-entering a cell
cycle1720. Cyclin-dependent kinase inhibitors (CKIs)
also seem to be involved in cell cycle arrest, as the loss of
CKI family members results in alterations in cell cycle
kinetics21. Studies in the retina and other tissues indicate
that two subclasses of CKIs the Ink4 class (including
p16Ink4a, p15Ink4b, p18Ink4c and p19Ink4d) and the Cip/Kip
class (including p21Cip1, p27Kip1 and p57Kip2) work
coordinately to ensure cell cycle exit22, although neither
class is necessary or sufficient to do so.
Loose ends. There is surprisingly little information that
links the biochemical and molecular description of the
cell cycle with the largely cytological description of
neurogenesis and migration. Cell cycle proteins are
found in brain lysates at early developmental stages21;
however, there are few immunocytochemical studies of these proteins23,24. The model shown in FIG. 1
predicts that the nuclei of the VZ are stratified by
cell cycle phase, and therefore that cell cycle proteins
should be expressed in layers. Indeed, the M phase
marker phosphohistone H3 is found in mitotic cells at
the ventricular surface and BrdU, which labels cells
in S phase, labels cells in the expected regions of the
upper VZ20,25. Most other cell cycle proteins are not so
neatly distributed. For example, proliferating cell nuclear
antigen (PCNA), another S phase marker25, and cyclin E,

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Pia

E2F1
RB
CDK5
ORC25

Cortical plate

Intermediate zone

CDK5
p27
p21
p57

Subventricular zone

S
Ventricular zone

BrdU/3H-T
uptake
Cyclin E
Ki67
PCNA

G1/G2

Phosphohistone H3

Ventricle

Figure 1 | The developing cerebral cortex. A cross-section through the wall of the
neural tube is shown. The morphological zones are noted on the left. Dividing
neuroepithelial cells are shown in blue, radial glia in green and mature cortical neurons
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
phases (G1, S, G2 and M) in the ventricular zone (VZ) is indicated. Note that the more
superficial subventricular zone has continued mitotic activity but no proposed
stratification. The mature cortex is generated by each successive wave of immigrants
from the VZ resulting in an inside-out pattern of layering with the first generated
(early-born) neurons residing in the deeper layers and the last generated cells (lateborn) residing more superficially. The proteins listed on the right are examples of
proteins discussed in the text shown in their approximate location in the normal CNS.
3
H-T, 3H-thymidine.

Phosphohistone H3
A phosphorylated form of the
DNA coating protein, histone
H3. Empirically, the presence
of this modification is unique to
M phase.

Proliferating cell nuclear

antigen
(PCNA). A subunit of the DNA
replication complex. Three
PCNA proteins assemble as a
homo-trimer encircling the
double helix just ahead of the
replication fork.

Ligase IV
A form of DNA ligase that
rejoins 5 and 3 ends of
apposed double-strands of
DNA. This ligase isoform is
specific for DNA repair,
especially non-homologous
end joining.

Transformed
A cellular state marked by
escape from growth control
mechanisms that normally
regulate the cell cycle.
Typically, transformed cells will
form tumours in soft agar and
in animals.

a late G1 phase marker26, appear in cells in all strata of

the VZ. For the CKIs, the picture is just as complicated22.
Cyclins and cyclin-dependent kinases (CDKs) are regulated by many factors that control their synthesis, degradation and localization. Their broad distribution in the
VZ indicates that either the predicted stratification of
cell cycle phase is incorrect or that the regulation of these
proteins in dividing neuronal precursors differs from the
simple models described in most textbooks.
Another unexpected finding is that DNA repair
enzymes are active immediately after the completion of
neurogenesis. Mutations in genes involved in DNA double-strand break (DSB) repair, including Xrcc427, ligase IV
(REF. 28) and the DNA repair polymerase, Pol 29,30, result
in embryonic lethal phenotypes. Moreover, neuronal
death in ligase IV knockouts can be suppressed by the
introduction of a second mutation in ataxia telangiectasia mutated (Atm)31, an early component of the DSB
detection and response system. The implication of these
findings is that DSBs are normal events in early neuronal
differentiation. The function of these proposed breaks is
unknown3234, but the correlation of human CNS disorders with defects in DSB repair genes underscores their
importance in the development of the brain35,36.
While we might predict that the mutation of cell
cycle proteins would have devastating effects on development and neurogenesis, a final loose end comes
from the observation that homozygous null embryos

370 | MAY 2007 | VOLUME 8

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
the way through neurogenesis (TABLE 1). The only exception to this rule are embryos lacking cyclin A2 (REF. 37)
or cyclin B1 (REF. 38) (the knockout of CDK1 has not
been reported). The survival of these mice may be due
to functional redundancy, as mice with mutations in
two or three of these proteins die earlier. However, even
the cyclin D triple mutant (Ccnd1/;Ccnd2/;Ccnd3/)
progresses to approximately embryonic day 16.5
(REF. 39), which is after most neurogenesis has occurred,
suggesting that there is more to neuronal cell cycle
regulation than cyclins and CDKs alone. Similarly,
RB-deficient embryos die in the third trimester, but a
normally patterned brain and spinal cord develop1719.
Finally, the loss of the CKI p27Kip1 or p19Ink4d alters cell
cycle kinetics during neurogenesis40,41, and the balance
of different cell types that differentiate can be affected,
but cell division eventually stops. Indeed, the lasting
morphological changes observed in these mice are relatively subtle, given the predicted role of these proteins
in orchestrating neuronal exit from the cell cycle (for a
fuller description of such mutations in the context of the
entire developmental process, see REF. 42). A tentative
summary of how these gene products affect the three
major phases of CNS neuronal development is outlined
in FIG. 2.

Failure of cell cycle arrest in development

As described above, CNS neurons do not divide after
they emigrate from the VZ or SVZ. What would happen
if a neuron lost control of its cell cycle and re-entered
cell division? Although it seems paradoxical, evidence
suggests that the neuron would probably die. We refer
to this process as cycle-related neuronal death (CRND)
a purposefully cautious term (see below).
The first experimental evidence for CRND came
from two independent lines of experimentation in
which RB protein was inactivated by the large transformation (T) antigen of the SV40 virus. Inactivation of RB
leads to the release of the transcription factor E2F1 and
the subsequent upregulation of many cell cycle genes.
When T antigen is introduced into most cultured cells,
they are released from normal proliferation control
and become transformed. However, when T antigen is
introduced into postmitotic neurons, they degenerate4346. Cell division begins and BrdU is incorporated
into DNA, but M phase is not initiated and the neuron
dies shortly after44. As might be predicted, this process
is E2F1-dependent47.
There is also massive apoptotic neuronal death in
mice with homozygous null alleles of Rb1719. In these
animals, CRND occurs after the migrating neuroblasts
leave the ventricular region. BrdU injections a few hours
before sacrifice have revealed that neurons in normally
postmitotic regions of the maturing brain and spinal
cord are engaged in ectopic DNA synthesis1719. Most
of the neuronal death in Rb knockouts is dependent
on both E2F1 and another transcription factor, p53
(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
phase

Disrupted
gene(s)

Survival

Neuronal cell phenotype

Other phenotypes

References

G1 phase

Cyclin D1

Viable

Reduction of cell number in the neural

retina

Reduced body size, neurological

abnormalities, hypoplastic retina,
impaired proliferation of mammary
gland epithelium

143,144

Cyclin D2

Viable

Loss of granule cell and stellate

interneurons in the cerebellum

Females sterile, males fertile, impaired

proliferation of B cells

145147

Cyclin D1
and D2

Viable, die
within 3 weeks

Severely reduced number of granule

cells in the IGL and abnormal
positioning of Purkinje cells

Reduced body size, problems with

coordination movements

148

Cyclin D1
and D3

Most die after

birth

Reduced proliferation in retina

Respiration with meconium

148

pRb

Embryonic
lethal (E1215)

Apoptotic cells in CNS and PNS

Defects in erythroid, neuronal, lens

and placental cell lineages. Tetraploid
complementation resulted in
completion of embryonic development

pRb and p107

Embryonic
lethal (E1113)

CNS findings similar to Rb/

Apoptosis in liver

p27 kip1
Viable
(overexpressed)

Cell cycle progression in the neocortical

PVE, lengthening of the G1 phase, but
no apoptotic cells were found

ND

41

Other

Cdk5

Embryonic
lethal (E18)

Cell cycle related genes expressed in

cerebral cortex, defective neuronal
migration, differentiation and survival

ND

120,139,141

Cell cycle
checkpoint

Atm

Death occurs
after 4 months

Most of Atm/ models show no signs of

neuronal death, except REF. 150, which
reports nigro-striatal degeneration

Immune deficiences, sterility cells are

radiosensitive

Atr

Embryonic
lethal (E7)

Apoptotic cell death in early embryo

Chromosome fragmentation in cultured

blastocysts

151

Chk1

Embryonic
lethal (E37)

No specific report on CNS

General aberrant nuclei morphology

and TUNEL staining in blastocysts

152

Chk2

Viable

No specific report on CNS

Radio-resistance

153

1719,69

149

97,150

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.

PC12 cells
A rat pheochromocytoma cell
line. When treated with nerve
growth factor, PC12 cells cease
mitosis and differentiate into
cells that resemble
sympathetic neurons,
complete with processes and
functional synapses.

has revealed a second, less appreciated phenotype: before

their death, neurons are both morphologically and biochemically immature, suggesting that RB is also required
for neuronal differentiation20.
This link between cell cycle suppression and final
neuronal differentiation is found for many cell cycle
genes, and seems to work both ways. Early investigations that explored the death response of cultured
sympathetic neurons after withdrawal of nerve growth
factor (NGF) revealed an unexpected increase in the
levels of cyclin D mRNA50. This indicated that loss of
trophic support during development might lead to cell
cycle initiation as part of the death process. Research on
models of target-related cell death in the mouse cerebellum51 has revealed that neuronal death is preceded by
re-expression of cell cycle markers and DNA synthesis
in vivo. These correlations are strong evidence that a
neuron must regulate its cell cycle or risk death during
postmitotic maturation.
The use of in vitro models of CRND has been
invaluable for establishing the causative nature of cell
cycle induction in the process of cell death. Drugs that

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block cell cycle advance can prevent the death of both

and sympathetic neurons after either NGF
withdrawal or DNA damage5256. Similarly, various
insults including trophic factor withdrawal, excitotoxicity and toxic concentrations of amyloid- can drive a
lethal cell cycle in cultured CNS neurons and, critically,
blocking the cell cycle can prevent neuronal death5760.
In sympathetic neurons, trophic factor withdrawal or
DNA damage leads to an E2F1-dependent upregulation of B-myb and C-myb (transcription factors that are
believed to have a role in S phase progression)61. This
results in the expression of BIM (a BCL2 homology
three domain-only molecule)62, which in turn leads to
cell death. In activity-deprived cerebellar granule cells,
phosphorylation of the proapoptotic molecule BAD by
CDK1 appears to have a similar role63,64.
Thus, both in vivo and in vitro, several lines of
evidence converge on the following hypothesis: once
a CNS neuron leaves the VZ, 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.
PC12 cells

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Loose ends. At first glance Rb-deficient embryos seem to
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 6567, and conditional knockouts of
Rb68,69 also support this idea. This is not simply a nearneighbour effect, as the CNS phenotype is rescued
in Rb/euploid Rb+/+;tetraploid chimeras69,70, in which
Rb/ embryos develop in a conceptus that includes wildtype (Rb+/+) extra-embryonic tissues. In the brains of
the resulting animals, BrdU incorporation reveals that
mutant nerve cells can begin a cell cycle but not die.
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
clause of the hypothesis that death follows quickly
after cycle initiation a concept that is developed more
fully by considering the situation in the adult brain.
Failure of cell cycle arrest in the adult
Our hypothesis predicts that the prohibition of neuronal
cell division is life-long. Might examples of late-onset
neurodegenerative disease be accompanied by loss of neuronal cell cycle control? This concept was first proposed
to explain the presence of a unique species of phosphorylated tau protein, usually found only in dividing cells,
in the neurons of patients who had died with Alzheimers
disease (AD)71,72. A number of laboratories have reported
the re-expression of various cell cycle proteins in neurons
from patients with AD. The proteins include cyclins A73,
B7376, D74,7678 and E73,78, as well as CDKs71,77,79,80, PCNA73,74,
Ki67 (REFS 73,81) and CKIs of both the Ink and Cip/Kip
families77,79,82,83. CDK1 may be particularly important in
this group because of the tissue culture data mentioned
previously63,64 , and the recent evidence of genetic linkage to AD84,85. There are also reports of cell cycle protein
re-expression in amyotrophic lateral sclerosis8688, ataxia
telangiectasia89, Parkinsons disease9093, stroke94 and other
neurodegenerative conditions95, 96. Where quantification
has been carried out, it seems that around 10% of at-risk
neurons re-express these proteins.
These findings represent correlations between elevated protein expression levels and disease, not proof of
CRND. In fact, if the immunocytochemistry is offering
a correct picture, it seems likely that cell cycle proteins
from different phases are co-expressed and that their
location is frequently cytoplasmic. In this context, two
studies 75,76 are particularly noteworthy. One shows
that cells that can be immunostained with antibodies
to cell cycle proteins, when measured as a percentage
of neurons in a region, are as prevalent during early
stages of AD as they are during end-stage illness76. The
other study investigated whether these proteins initiate
a true cell cycle process, complete with DNA replication, by probing the interphase nuclei of neurons in

372 | MAY 2007 | VOLUME 8

Neurogenesis

Migration

Maturation

By mutation:
CDK5
RB
p27, p57
Ligase IV?
XRCC4?
Pol?

By presence:
E2F1
RB
CDK1

dNTP
DNA

By mutation:
Cyclin A2?
Cyclin B1?
Sonic hedgehog?

By mutation:
ORC26
CDK5
p21

Figure 2 | Cell cycle proteins implicated in each of the

three phases of neuronal development. Of the cell
cycle proteins, mutations in only two of the cyclins (A2
and B1) are known to interrupt the process of neurogenesis
(left column); however, as a direct effect of these proteins
on the behaviour of cells within the ventricular zone (VZ)
has never been shown, it may be that these mutations
block development before the nervous system can emerge.
DNA synthesis is indicated by the deoxyribonucleotide
triphosphate (dNTP) to DNA symbol on the left of the first
column. Migration (centre column) describes the stage in
which the neuron ceases new mitotic activity and begins
the early differentiation steps required to emigrate from the
VZ and sub ventricular zone. Maturation (right column) is
the cytological and biochemical coming of age of the
neuron once it has reached its adult location. The inclusion
of the transcription factor E2F1 and retinoblastoma (RB) in
this stage is by inference only, as knocking down these
proteins in the adult has not yet been shown to have an
effect on maturation. CDK1, cyclin-dependent kinase 1;
ORC, origin recognition complex.

the brains of patients with AD using fluorescent in situ

hybridization (FISH) probes against unique genomic
loci75. In at-risk regions of the brain, around 4% of
the neurons had three or four spots of hybridization
rather than two. Thus, the cell cycle protein expression
in AD correlates with DNA replication, suggesting that
at least the first phases of a true cell cycle have begun
in the at-risk neurons. One clear implication of this
is that in the adult, the presence of cell cycle proteins
or even DNA replication on its own cannot be taken
as evidence of adult neurogenesis. These events could
easily represent the beginning of a cell death process
with very much the opposite result.

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Loose ends. Cell cycle regulation in the adult neuron
is an area of study in which the number of loose ends
vastly exceeds the clearly-established parts of the story.
First, the cells never enter M phase. Condensed chromosomes are not found and there is no visible evidence
of spindle formation. This makes it difficult to confidently characterize the process as a cell cycle as it is
never completed. Second, where it has been quantified,
there are too many cycling nerve cells7476. The average course of AD is 10 years from first symptoms to
death. If, as the immunostaining suggests, 510% of the
neurons are dying at any one moment, and if a typical
apoptotic process takes roughly 12 hours, then half of
the affected population should be dead in a week or
less and 95% should be dead in less than a month. This
is clearly not the case. The implication is that death by
cell cycle in adult neurons must be a very slow process
requiring in the order of 612 months. Although this
protracted time period is unexpected, mouse models of
late onset human neurodegenerative diseases indicate
that, if anything, 612 months might be an underestimate of the true length of the process as discussed in
the next examples.
Transgenic mouse models of human inherited diseases, including ataxia telangiectasia9799 and AD100106,
are noteworthy because, with few exceptions 107, an
Immature

Mature

Diploid

Mitotic
pressure

4n

Cell death

4n

Tetraploid

Figure 3 | Mature and immature neurons differ in their response to cell cycle
initiation. Healthy (yellow) immature neurons that are deprived of trophic factors or are
exposed to other stimuli begin a cell cycle process that rapidly leads to cell death. Mature
neurons, however, seem to require a two step process for cell death to occur in response
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
M phase. These cells are in an unusual cell biological state (green), having twice the
normal DNA complement (4n), but otherwise they seem normal. The dashed arrows
leading through an intermediate stage marked by a question mark indicate the need
for a second step that moves the nerve cell from this unusual state to death (grey cell).

NATURE REVIEWS | NEUROSCIENCE

otherwise excellent phenocopy falls short owing to the

absence of neurodegeneration. The gene that is mutated
in ataxia telangiectasia (Atm) signals the presence of
DSBs and arrests the cell cycle to allow for DNA repair.
In all reported mouse knockouts, the sterility, immune
deficiency and radiosensitivity associated with the
human condition are replicated faithfully, but their
gross behaviour is normal and there is no detectable
loss of Purkinje cells108. A similar effect is found in
mouse models of AD: amyloid- plaques appear along
with behavioural abnormalities, but there is no apparent loss of neurons109. Remarkably, however, initiation
of ectopic cell cycling is preserved. In Atm-knockout
mice, cell cycle initiation can be found in Purkinje
cells and striatal neurons89. Similarly, in the R1.40 yeast
artificial chromosome transgenic line, an AD model,
cycle anomalies begin at 6 months after birth in regions
analogous to those in which the human disease begins,
progressively appearing in the brainstem and cortical
regions as in AD110. Two groups examined the APP23
mouse, another AD model, but disagreed on the presence of neuronal cell cycle events110,111. The difference
may be in the age of the mice examined; however, the
controversy remains unresolved. With the exception of
this one report, a long delay between cell cycle initiation
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
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-initiation can take from months to years, and might require
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
reflect the possible need for an extra stimulus to trigger
death in a cycling adult neuron.

What is a cell cycle protein?

Proteins identified as cell cycle proteins turn up in
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
not only fail to survive, they also fail to differentiate20.
That would appear to define an embryonic time during
which the protein functions, but RB along with its
binding partner E2F1 are both found in the cytoplasm
and dendrites of the supposedly postmitotic neuron and
both respond rapidly to exogenous stressors by changing
their normally nuclear localization to a predominantly
cytoplasmic one112114. CKIs also have an important
non-cycle role in differentiation115119, which is independent of their role in cell cycle suppression117, 119.
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REVIEWS
Mitogen
A substance, usually a protein,
that induces cell division in a
receptive cell.

A further example of this cycle/differentiation

duality is the atypical cyclin-dependent kinase,
CDK5 (BOX 2) . CDK5 has both pro-differentiation
and anti-cell cycle activities during development120.
In the adult, however, the kinase diversifies. In many
if not most CNS synapses, CDK5 forms complexes
with121 and affects the function of 122127 various synaptic proteins.
Perhaps the most surprising example of the
promiscuous function of cell cycle proteins is that
members of the origin recognition complex (ORC)
are found in synaptic fractions of adult neurons 128.
This is remarkable because the ORCs are a family
of proteins best known for binding to specific sites
in the genome during the process of mitotic DNA
replication. The synaptic function of these proteins
is unknown, although in other cell types they are
reported to form complexes with proteins other than
those of the ORC129.
Finally, a substantial fraction of neurons in the
adult E2f1-knockout mouse are engaged in a strange
cell cycle-like process (L. Wang and K.H., unpublished
observations). They express high levels of cyclin A
(cytoplasmic) and PCNA (nuclear), and by FISH
criteria they have undergone DNA replication. Taken
together, the data suggest that proteins best known
for their role in cell cycle progression have separate
functions in the fully differentiated, mitotically inactive
neuron.

Are adult cycling neurons undead?

As described above, neurons under stress can engage
a cell-cycle-like process that leads to the constitutive
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
long periods. The dendrites of these neurons show
only minimal atrophy89, but the behavioural anomalies found in the mouse models of both AD and ataxia
telangiectasia hint at functional impairments. The
question posed by this situation is: what is the nature
of the state in which these nerve cells find themselves?
Data from embryonic studies indicate that cell cycle
initiation in a post-VZ neuron is a first step towards
cell death. However, data from adult studies suggest
that the processes of cell cycle initiation and cell
death are separable. Similar situations, of neurons on
a path to death being blocked by genetic or trophic
means, have been reviewed recently for several species
and have raised many important issues130.
Speculatively, it may be possible to draw an analogy between the arrested cell cycle state of these
neurons and a cellular state defined in the imaginal
discs of Drosophila melanogaster. The state was first
proposed131 to describe cells that are triggered to die by
an insult such as X-rays but blocked from completing
the process by expression of an anti-apoptotic protein.
The authors describe the resulting cells as undead
and have shown that they have some unusual properties131133. For example, they send signals that serve as
a mitogen to neighbouring cells. If the neighbouring
374 | MAY 2007 | VOLUME 8

cells are wild type, they divide in an apparent attempt

to fill the gap in the tissue expected to be left by the
dying cells.
Could the cycle-positive neurons in AD and ataxia
telangiectasia brains be analogous to these undead
cells, triggered to die but blocked from completing the
process? If so, these cells might be part of the problem
rather than innocent victims of the disease process. If
in their undead state they are sending mitogenic signals
to their neuronal neighbours, they could be pressuring
otherwise healthy neurons to initiate a cell cycle (FIG. 4).
If this is true, their stability and persistence could represent a danger to the health of the brain. As they do
not die, their continued presence might weaken those
around them and yet, because re-entry into a cell cycle
does not seem to result in cell death, not kill them. This
would create a paradox for those who seek therapeutic
interventions for neurodegenerative conditions such as
AD. The prediction would be that the most effective
therapies would block new neurons from entering a
cell cycle while simultaneously ridding the brain of the
undead cells.

Conclusions and future directions

In many ways, descriptions of adult CNS neurons as
having permanently exited the cell cycle or having
entered a permanent state of G0 are terms of convenience, which may lack true functional meaning. In fact,
such terms might lull us into a false sense of security
a belief that the issue of cell cycle regulation in
neurons is an oxymoron rather than an important biological question. We believe that the evidence reviewed
here challenges us to rethink this position.

Box 2 | An atypical CDK

Cyclin-dependent kinase 5 (CDK5) is structurally similar to
other CDKs, but is said to have no role in the normal cell
cycle. It binds cyclin D, but its activity is not initiated by
this partnership. Rather, two proteins known as p35 and
p39 function as cyclin equivalents and activate CDK5.
Mice that are genetically deficient in either CDK5 or both
activator proteins have serious defects in neuronal
migration and die before birth139141. Intermediate filament
expression in CDK5-deficient neurons in vivo and in vitro is
retarded or blocked120. Early filament types such as nestin
and III-tubulin remain expressed in regions of Cdk5/
cortex from which they are lost in the wild type, and later
differentiation markers such as microtubule-associated
protein 2c are seen only at very low levels120.
In addition to its pro-differentiation functions, CDK5
also appears to act as a cell cycle inhibitor. Both in vivo
and in vitro, Cdk5/ neurons cannot restrain their cell
cycle and die rapidly after the re-expression of cell cycle
proteins and the incorporation of 5-bromo-2deoxyuridine120. In vitro models of cycle-related neuronal
death suggest that the anti-cycle activity of CDK5
requires nuclear localization (Cicero, S.A. et al.
unpublished observations). Whereas in other cell death
models the localization of CDK5 to the nucleus is
protective142, it would appear that CDK5 is very much
involved in cell cycle regulation in neurons.

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REVIEWS

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

ultimately became cells) were those that divided fastest
and most efficiently. Metazoans evolved when aggregates of these early cells were able to realize gains from
cooperation and specialization that exceeded those
they could eke out by living on their own. It is worth
considering that by this time the instinct to divide was
buried deep in the fundamental biology of every cell. It
may even have preceded the emergence of a genome.
Thus, the dividing instinct might be as powerful today
as it was a billion years ago. The challenge faced by a
metazoan, therefore, is to suppress the urge to cycle
among its own constituents in order to enforce a regulated body plan and allow the extreme specialization
(differentiation) of its members. Indeed, suppressing
the cell cycle is arguably the most important step in any
differentiation programme.
Neurons are among the most specialized of cells,
and organisms that benefit from incorporating them
into their structure seem to have largely solved the
problem of reining in their drive to divide; the nature of
the solution(s), however, remains largely unknown. But
we may wish to rephrase questions such as what factors
force a postmitotic neuron to re-enter a cell cycle in AD
or what fail-safe mechanisms of cell cycle suppression
are lost in AD such that pockets of neurons can break
free of their postmitotic state and return to an earlier
mode of existence. For neurons of the CNS it seems
that anti-cycling kinases, cell cycle promoters turned
into cell cycle suppressors, and DSBs during or immediately after the final cell division are all components of
a strategy by which the metazoan developmental plan
imposes cell cycle arrest on its neurons. This multitiered strategy involves not only changes in protein
localization and altered biochemical networks, but
perhaps rearrangements of DNA structure as well. The
evolution of multicellular organisms appears to have
been a stern mitotic master.
But evolution is also a miserly process. It tends to
adapt old tools for new uses rather than fashion new
tools. In neurons, this is reflected by the additional

NATURE REVIEWS | NEUROSCIENCE

roles of cell cycle proteins such as E2F1, RB, CDKs,

cyclins and ORC proteins in dendrites and axons.
Under normal conditions these functions exemplify
efficient design. Under stress, however, they appear
to represent an Achilles heel for the neuron. While
some may start off as protective (for example, p27 and
CDK5), many can be dangerous to the health of the cell
if they end up in the wrong cellular location.
If cell cycle regulation is truly a constant issue for an
adult neuron then an entire menu of action items seems
spread before us. One important initiative would be to
drop, whenever possible, the reference to proteins as cell
cycle proteins. Each protein whose first known function was to advance or retard the progression of the cell
cycle appears to have alternative identities in neurons
and other differentiated cells. Restricting our focus to
their role in cell cycle regulation may blind us to the full
repertoire of their activities. We also need to remind
ourselves that the mechanics of cell cycle regulation
can be distinct in different cell types. Textbooks tell us
that the E2F1 protein drives the cell cycle by upregulating the synthesis of key proteins needed for cell cycle
progression, as is indeed the case in cultured fibroblasts and COS cells; however, E2f1-knockout animals
are born and develop normally, but die in middle age
with tumours and hyperplasias in a variety of exocrine
tissues134,135. Clearly, in some cells E2F1 functions as a
tumour suppressor. Finally, it seems apparent that, just
as in real estate, the three keys to activity are location,
location and location. We need to define where these
multifunctional proteins are in neurons at different
junctures of the developmental process and during the
response to insult or injury. The behaviour of CDK5 in
CRND suggests that its function in the nucleus is different from that in the cytoplasm. This is not a new insight,
but it is part of a readjustment to the notion that we can
avoid concerning ourselves with the regulation of the
cell cycle in all highly differentiated cells especially
neurons. Indeed, to reach a fuller understanding of
how neurons remain mitotically silent for decades, there
is a great deal of new biology yet to learn.

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Acknowledgements
This work was supported by the National Institutes of Health,
the AT Childrens Project, the Alzheimers Association and
the Coins for Alzheimers Research Trust.

Competing interests statement

The authors declare no competing financial interests.

DATABASES
The following terms in this article are linked online to:
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
BAD | B-myb | CDK1 | C-myb | cyclin D | E2F1 | NGF | p19Ink4d |
p27Kip1 | p53 | RB | SHH | T antigen
OMIM: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=OMIM
Alzheimers disease | amyotrophic lateral sclerosis | ataxia
telangiectasia | Parkinsons disease
Access to this links box is available online.

www.nature.com/reviews/neuro
2007 Nature Publishing Group