Cell cycle regulation & axonal

regeneration
Think about p75NTR?
Activation of intrinsic growth capacity by peripheral nerve injury

Peripheral nerve injury elevates intracellular cAMP levels, which activates PKA. PKA triggers gene expression through CREB, resulting in
transcriptional upregulation of regeneration-related genes such as Arginase I. Arginase I promotes the synthesis of polyamines, which
may directly regulate cytoskeleton assembly or further induce gene expression necessary for regeneration. Activation of PKA also inhibits
Rho antagonizing MAG or myelin-induced Rho activation and inhibition of neurite growth. Elevated cAMP levels also upregulate IL-6,
which, through STAT3, induces regeneration-related genes such as GAP-43. Peripheral injury additionally induces c-Jun transcription
factor–dependent regeneration-related gene expression such as integrin α7β1 CD44 and galanin. Activation of the intrinsic growth
capacity is regulated mainly at transcriptional level.
Axon regeneration in peripheral nerves

After peripheral nerve injury, myelin debris is rapidly removed by Schwann cells and macrophages. Schwann cells dedifferentiate and
downregulate all myelin proteins generating a permissive environment. ECM proteins such as laminin (LM) bind integrin receptors at
the growth cone and activate PI3K locally resulting in accumulation of active Akt at the axon/laminin contact sites. Activated Akt
phosphorylates and inactivates GSK-3β. Inactivation of GSK-3β regulates cytoskeleton-binding proteins, promoting cytoskeleton
assembly. Peripheral nerve injury also increases the neuronal intrinsic growth capacity. Locally facilitated machinery for cytoskeleton
assembly coupled with activated intrinsic growth capacity in the whole cell leads to rapid axon growth along the basal lamina tubes
(can serve as guidance cue). Neurotrophins (NT) may also participate in promoting axon regeneration through tropomyosine kinase
receptors (Trk) via a similar intracellular signaling pathway to laminin. Activated intrinsic growth capacity promotes axon regeneration
in the CNS by antagonizing the signal mediators of myelin-associated inhibitory molecules
 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.
• Marker for cell cycle phase: M phase marker phosphohistone H3; BrdU, which labels
cells in S phase ; proliferating cell nuclear antigen (PCNA), another S phase marker,
and cyclin E, a late G1 phase marker, Ki67
Basics of the cell cycle
• 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 repaired
Adapted from: Herrup K, Yang Y. 2007. Cell
cycle regulation in the postmitotic
neuron: oxymoron or new biology?
Nat Rev Neurosci 8(5):368-378
Current model for regulation of the eukaryotic cell cycle
• Passage through the cycle is
controlled by G1, S-phase,
and mitotic cyclin-dependent
kinase complexes (CdkCs)
highlighted in green. These
are composed of a regulatory
cyclin subunit and a catalytic
cyclin-dependent kinase
subunit. Protein complexes
(orange) in the Cdc34
pathway and APC pathway
polyubiquitinate specific
substrates including the S-
phase inhibitor, anaphase
inhibitor, and mitotic cyclins,
marking these substrates for
degradation by proteasomes.
These pathways thus drive the
cycle in one direction because
of the irreversibility of protein
degradation. Proteolysis of
anaphase inhibitors
inactivates the protein
complexes that connect sister
chromatids at metaphase (not
shown), thereby initiating
anaphase.
 Glossary
• D-box: (Destruction-box). A sequence element (consensus
RXXLXXXN) that was first discovered in the N terminus of mitotic
cyclins that is required for their destruction. D-boxes can be
recognized by APC/CCdc20 and by APC/CCdh1.
• APC/C: The anaphase promoting complex/cyclosome (APC/C) is a
ubiquitin ligase that has essential functions in and outside the
eukaryotic cell cycle. It is the most complex molecular machine that
is known to catalyse ubiquitylation reactions, and it contains more
than a dozen subunits that assemble into a large 1.5-MDa complex.
• CDH1: CDC20 homolog 1, one of APC/C activator protein which
regulates the ubiquitin ligase activity and substrate specificity of the
anaphase promoting complex/cyclosome (APC/C). Both Cdh1 and
Cdc20 target specific proteins for ubiquitination through the
recognition of substrates containing a destruction or D box
 HLH & bHLH proteins
• The bHLH proteins are transcription factors which are characterized by a
conserved basic helix-loop-helix structural motif and binding DNS as dimers
to modulate transcription of target genes that regulate gene expression to
promote cell differentiation and tissue-specific cellular functions.Class A
bHLH For instance, NeuroD and neurogenins (Ngn1 and Ngn2) are tissue-
specific bHLH proteins involved in neurogenesis. These tissue-specific
proteins form dimers with other ubiquitously expressed bHLH transcription
factors called E proteins, which bind to the canonical E-box sequence
CANNTG and include HEB (also called ME1a, which regulates p75NTR),
E2-2, and the E2A gene products, E12 and E47. In addition, the activity of
bHLH proteins as transcription factors is negatively regulated by the
structurally related Id proteins (inhibitors of DNA binding and/or
differentiation). Id proteins, lacks basic domain, possess the HLH domain,
through which they form dimers, and function as dominant-negative HLH
proteins to form non functional heterodimers with bHLH proteins, mainly with
E proteins. As a result, E proteins cannot form functional heterodimers with
the tissue-specific bHLH factors, leading to inhibition of differentiation., Id
proteins are involved not only in cell differentiation control but also in the
regulation of cell proliferation. This study gives us an important clue that Id
proteins perhaps maybe have a critical role in axonal regeneration.
The three-dimensional structure of APC/C
• Adapted
from :
The anaphase pr
(Peters,
2006)

Peters JM. 2006. The anaphase promoting complex/cyclosome: a machine designed to destroy. Nat Rev Mol Cell Biol 7(9):644-656
Inactivation of APC/CCdh1 at the
transition from G1 to S phase
• The inactivation of anaphase promoting
complex/cyclosomeCdh1 (APC/CCdh1) at the
end of G1 phase is important to allow the
accumulation of proteins that are required for
DNA replication and mitosis, such as cyclin A
and cyclin B. Four different mechanisms have
been proposed to contribute to this inactivation
process in vertebrate cells. 1 | During the G1
phase, the APC/C-interacting ubiquitin-
conjugating (E2) enzyme UBCH10 is itself
degraded by APC/CCdh1. This process leads to
the stabilization of those APC/CCdh1 substrates
that are ubiquitylated in a distributive manner,
such as cyclin A. 2 | Cyclin A activates cyclin-
dependent kinase-2 (Cdk2), which in turn
phosphorylates Cdh1 and thereby dissociates
Cdh1 from APC/C. 3 | Phosphorylated Cdh1 is
ubiquitylated by SCF and thereby targeted for
destruction by the 26S proteasome. 4 | The
transcription factor E2F activates the expression
of early mitotic inhibitor-1 (Emi1), and Emi1 then
inhibits the activity of APC/CCdh1. P, phosphate
 Aberrant cell cycle re-activation in postmitotic neurons leads to apoptosis

Abortive cell cycle re-entry of

postmitotic neurons. Following
growth factor and/or neuronal
activity deprivation, postmitotic
neurons leave their quiescent state
(“G0”) and re-enter the cell cycle.
However, postmitotic neurons do
not progress through the cell cycle,
but undergo apoptosis. Activation of
G1 regulators and CDC2 result in
activation of the cell-intrinsic cell
death machinery and ultimately in
apoptotic cell death of the neurons.
G1: first gap phase; S: DNA
replication; G2: second gap phase;
M: mitosis.

Becker EB, Bonni A. 2005. Beyond proliferation--cell cycle control of neuronal survival and differentiation in the developing mammalian brain. Semin Cell Dev Biol
16(3):439-448
Herrup K, Yang Y. 2007. Cell cycle regulation in the postmitotic neuron: oxymoron or new biology? Nat Rev Neurosci 8(5):368-378
A schematic diagram of protein destruction pathways mediated by the proteasome and autophagy

• Cell proteins exist in a

balance between continuous
synthesis and degradation
(i.e., turnover) which
contributes to exertion of cell
type-specific functions and
maintenance of cell
homeostasis.

• Ubiquitin-proteasome system
(UPS): an elegantly organized
multi-protease complex with
catalytic activities, plays
crucial roles in selective
degradation of short-lived
regulatory proteins as well as
proteins with aberrant
structures that should be
eliminated from the cells.
 Overview of the related references
• p75NTR E box and the interacting bHLH transcription factors are
involved in the regulation of p75LNGFR gene expression. Suggests
that class A bHLH transcription factors can repress and Id-like
negative regulators can stimulate gene expression (Chiaramello et al.,
1995).

• basic helix-loop-helix transcription factors regulate the expression of

the GAP-43 gene and that the class A ME1a and E12 proteins act as
down-regulators of GAP-43 expression (Chiaramello et al., 1996).

• Down-regulation of HLH transcription factors is required for

initiation of regenerative response to axonal injury (Kabos et al.,
2002).

• E47 and E12 basic helix-loop-helix (bHLH) proteins bind the TrkB
promoter sequences in vivo (Liu et al., 2004).

• Regeneration-associated genes which promoter region contains E-box

cis-acting element should be modulated by endogenous E-box-binding
proteins, such as class A basic helix-loop-helix proteins, E12 and
• Lasorella A, et al. 2006. Degradation of Id2 by the anaphase-
promoting complex couples cell cycle exit and axonal growth.
Nature 442(7101):471-474
Experiment with 10 hybridizations, using 5 samples of species
[Homo sapiens], using 10 arrays of array design [Affymetrix
GeneChip® Human Genome HG-U133A [HG-U133A], Affymetrix
GeneChip® Human Genome HG-U133B [HG-U133B]], producing 10
raw data files and 10 transformed and/or normalized data files
E47 is a basic Helix Loop Helix (bHLH) transcription factor that has
important roles in cell fate determination and differentiation of many
cell types. In the nervous system E47 heterodimerizes with tissue-
specific, pro-neural bHLH transcription factors and activates
downstream target genes. To identify the relevant target genes of
bHLH transcription factors in neural cells, we performed gene
expression profiling of the human neuroblastoma cell line SK-N-SH
engineered to acutely express ectopic E47 by an adenoviral vector.
The experiments were done at two time points following adenoviral
infection, 8 hours and 20 hours. Genes induced by E47 after 8 hours
are likely to be direct targets of this transcription factor.
ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/MEXP/E-MEXP-413
Protein destruction in post-
mitotic neurons.

Jackson PK. 2006. Developmental neurobiology: A

destructive switch for neurons. Nature 442(7101):365-366
A model for destruction of inhibitors
of neuronal differentiation
Cdh1-APC induces the developmental decline of
neuritic growth in cerebellar granule cells

Rossi F, Gianola S, Corvetti L. 2007. Regulation of intrinsic neuronal properties

for axon growth and regeneration. Prog Neurobiol 81(1):1-28
 Microarry experiments
• Experiment with 10 hybridizations, using 5 samples of species [Homo
sapiens], using 10 arrays of array design [Affymetrix GeneChip® Human
Genome HG-U133A [HG-U133A], Affymetrix GeneChip® Human Genome
HG-U133B [HG-U133B]], producing 10 raw data files and 10 transformed
and/or normalized data files
• E47 is a basic Helix Loop Helix (bHLH) transcription factor that has
important roles in cell fate determination and differentiation of many cell
types. In the nervous system E47 heterodimerizes with tissue-specific, pro-
neural bHLH transcription factors and activates downstream target genes.
To identify the relevant target genes of bHLH transcription factors in neural
cells, we performed gene expression profiling of the human neuroblastoma
cell line SK-N-SH engineered to acutely express ectopic E47 by an
adenoviral vector. The experiments were done at two time points following
adenoviral infection, 8 hours and 20 hours. Genes induced by E47 after 8
hours are likely to be direct targets of this transcription factor.
• ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/experiment/MEXP/E-MEXP-413/