Cell Cycle & Axonal Regeneration

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 •

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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 upor 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 minichromosome 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 Sphase 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 tissuespecific 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 ubiquitinconjugating (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 cyclindependent 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

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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.

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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).

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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).

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Down-regulation of HLH transcription factors is required for initiation of regenerative response to axonal injury (Kabos et al., 2002).

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

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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

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Lasorella A, et al. 2006. Degradation of Id2 by the anaphasepromoting 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 tissuespecific, 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 postmitotic 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 •

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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, proneural 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/