Figure 3 - uploaded by Simone Di Giovanni
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A general diagram showing the location of the lesion sites on the sciatic nerve, and the dorsal column.
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Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate to a limited extent after injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are reactivated upon injury in the PNS (Makwana et al., 2005). However the tools...
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Context 1
... representative result of a ChIP experiment in DRGs following sciatic lesion is shown (Figures 1 and 2). First, we show fragmented DNA to a length of approximately 200-1000 bp (Figure 1). Second, we demonstrate a PCR signal following ChIP from the proximal promoter of the growth associated protein-43 (GAP-43) only upon sciatic nerve injury (lane 4, Figure 2, 5'). No PCR signal is present when the animal receives a sham injury only (lane 3), and when using normal IgG serum for the IP (lane 5 and 6). To test for the specificity of the antibody, we analyzed the same DNA samples, but used a control primer set which detects a region in the 3'-UTR of our gene of interest where no occupancy is expected. PCR signals are absent from all lanes (lanes 3-6, Figure 2, 3'), except for the input signal, representing non-IP DNA samples as standard PCR controls (lane 1-2, Figure 2). This ChIP procedure can be performed following either sciatic or spinal dorsal column injury as summarized in a schematic ( Figure 3). ...
Citations
... For that, we used homozygous adult MDM4 floxed mice (MDM4 f/f ), in which MDM4 is flanked by loxP sites and can be excised by Cre recombinase. To delete MDM4 specifically in neurons giving rise to the CST, I stereotaxically injected in layer V neurons of the SMC an adeno-associated virus type I (AAV1), which is the optimal serotype to infect corticospinal circumstances, as recently reviewed by Lee and Gu [44]. In addition to modulation of substrate choice, deubiquitylation is also used to stabilize Mdm2, Mdmx and p53. ...
... Chondroitin sulphate proteoglycans (CSPGs) also contribute to the inhibitory environment of the CNS, particularly after injury 44 . CSPGs are increasingly being seen as molecules that regulate plasticity in the adult CNS (for a review, see REF. 45). ...
... Quantitative chromatin immunoprecipitation. The SimpleCHIP Enzymatic Chromatin IP Kit with magnetic beads (Cell Signalling) was used according to previously published methods 44 . Antibodies used were H3K9ac, PCAF (rabbit), H3K9me2, H3K27me3, H3K4me3 and H3K18ac. ...
Axonal regenerative failure is a major cause of neurological impairment following central nervous system (CNS) but not peripheral nervous system (PNS) injury. Notably, PNS injury triggers a coordinated regenerative gene expression programme. However, the molecular link between retrograde signalling and the regulation of this gene expression programme that leads to the differential regenerative capacity remains elusive. Here we show through systematic epigenetic studies that the histone acetyltransferase p300/CBP-associated factor (PCAF) promotes acetylation of histone 3 Lys 9 at the promoters of established key regeneration-associated genes following a peripheral but not a central axonal injury. Furthermore, we find that extracellular signal-regulated kinase (ERK)-mediated retrograde signalling is required for PCAF-dependent regenerative gene reprogramming. Finally, PCAF is necessary for conditioning-dependent axonal regeneration and also singularly promotes regeneration after spinal cord injury. Thus, we find a specific epigenetic mechanism that regulates axonal regeneration of CNS axons, suggesting novel targets for clinical application.
... Importantly, p53/CBP/GAP-43 transcriptional module is also required for facial nerve regeneration following facial nerve axotomy in vivo (Tedeschi, Nguyen, Puttagunta, et al., 2009). Accordingly, after sciatic nerve injury in DRG neurons, acetylated p53 at Lys 373 occupy 81 Gatekeeper Between Quiescence and Differentiation the promoter of GAP-43 (Floriddia, Nguyen, & Di Giovanni, 2011), indicating that p53 acetylation may be a crucial event during axonal regeneration. ...
The transcription factor and tumor suppressor gene p53 regulates a wide range of cellular processes including DNA damage/repair, cell cycle progression, apoptosis, and cell metabolism. In the past several years, a specific novel role for p53 in neuronal biology has emerged. p53 orchestrates the polarity of self-renewing divisions in neural stem cells both during embryonic development and in adulthood and coordinates the timing for cell fate specification. In postmitotic neurons, p53 regulates neurite outgrowth and postinjury axonal regeneration via neurotrophin-dependent and -independent signaling by both transcriptional and posttranslational control of growth cone remodeling. This review provides an insight into the molecular mechanisms upstream and downstream p53 both during neural development and following axonal injury. Their understanding may provide therapeutic targets to enhance neuroregeneration following nervous system injury.
... The identification of factors that play a role in both the neuronal intrinsic and extrinsic spinal environment after lesion may affect the proaxonal regenerative response and functional recovery. Recently, we have shown that the transcription factor p53 is required for axonal regeneration of the injured facial nerve and for neurite outgrowth of cultured cortical and dorsal root ganglia neurons (Di Tedeschi et al., 2009a,b;Floriddia et al., 2011). ...
... We have previously found that p53 can occupy the promoters and drive the expression of a number of genes involved in neurite and axonal outgrowth such as Coronin 1b, Rab13 (Di , and GAP-43 (Tedeschi et al., 2009a;Floriddia et al., 2011). Moreover, p53 transcriptionally enhances the expression of cGKI, thereby inhibiting semaphorin 3A-dependent growth cone collapse in primary cortical and dorsal root ganglia neurons (Tedeschi et al., 2009b). ...
Following spinal trauma, the limited physiological axonal sprouting that contributes to partial recovery of function is dependent upon the intrinsic properties of neurons as well as the inhibitory glial environment. The transcription factor p53 is involved in DNA repair, cell cycle, cell survival, and axonal outgrowth, suggesting p53 as key modifier of axonal and glial responses influencing functional recovery following spinal injury. Indeed, in a spinal cord dorsal hemisection injury model, we observed a significant impairment in locomotor recovery in p53(-/-) versus wild-type mice. p53(-/-) spinal cords showed an increased number of activated microglia/macrophages and a larger scar at the lesion site. Loss- and gain-of-function experiments suggested p53 as a direct regulator of microglia/macrophages proliferation. At the axonal level, p53(-/-) mice showed a more pronounced dieback of the corticospinal tract (CST) and a decreased sprouting capacity of both CST and spinal serotoninergic fibers. In vivo expression of p53 in the sensorimotor cortex rescued and enhanced the sprouting potential of the CST in p53(-/-) mice, while, similarly, p53 expression in p53(-/-) cultured cortical neurons rescued a defect in neurite outgrowth, suggesting a direct role for p53 in regulating the intrinsic sprouting ability of CNS neurons. In conclusion, we show that p53 plays an important regulatory role at both extrinsic and intrinsic levels affecting the recovery of motor function following spinal cord injury. Therefore, we propose p53 as a novel potential multilevel therapeutic target for spinal cord injury.
... In the last years, a number of transcription factors have been associated with axonal regeneration, such as KLF4, p53, STAT3, NFAT, RARß, c-Jun, ATF3, and Sox11 [37][38][39]. Common aspects to all these transcription factors are their association to regulation of cell death and survival in several cell types, including neurons, and their involvement in neuronal development, strongly supporting the idea that successful axonal regeneration after CNS trauma in adults can be achieved by targeting transcriptional activity and molecular mechanisms associated with development [39,40]. Further, a number of neurotrophins, including NGF, brain-derived neurotrophic factor (BDNF), and NT3/ 4, have been shown to initiate and contribute to the prosurvival and pro-growth response of axotomized peripheral or central nervous system neurons, as reviewed elsewhere [41,42]. ...
One only needs to see a salamander regrowing a lost limb to become fascinated by regeneration. However, the lack of robust axonal regeneration models for which good cellular and molecular tools exist has hampered progress in the field. Nevertheless, the nervous system has been revealed to be an excellent model to investigate regeneration. There are conspicuous differences in neuroregeneration capacity between amphibia and warm-blooded animals, as well as between the central and the peripheral nervous systems in mammals. Exploration of such discrepancies led to significant discoveries on the basic tenets of neuroregeneration in the last two decades, identifying several positive and negative regulators of axonal regeneration. Implications of these findings to the comprehension of mammalian regeneration and to the development of spinal cord injury therapies are also addressed.
... After injury, active gene transcription is necessary to synthesize new proteins needed for axon growth. Acetylated-p53, together with CBP/p300 and PCAF, selectively occupies regulatory regions upstream to the TSS of pro-neurite and axon-outgrowth genes such as Coronin 1b, Rab13, and GAP-43 during an early regenerative response (Di Tedeschi et al., 2009a;Gaub et al., 2010Gaub et al., , 2011Floriddia et al., 2011; Figure 2). Both Coronin 1b and Rab13 are part of a gene cluster involved in neuronal plasticity, whose expression increases after traumatic spinal cord injury (Di Giovanni et al., 2005). ...
Trauma in the adult mammalian central nervous system leads to irreversible structural and functional impairment due to failed regeneration attempts. In contrast, neurons in the peripheral nervous system exhibit a greater regenerative ability. It has been proposed that an orchestrated sequence of transcriptional events controlling the expression of specific sets of genes may be the underlying basis of an early cell-autonomous regenerative response. Understanding whether transcriptional fine tuning, in parallel with strategies aimed at counteracting extrinsic impediments promotes axon re-growth following central nervous system injuries represents an exciting challenge for future studies. Transcriptional pathways controlling axon regeneration are presented and discussed in this review.
Chromatin immunoprecipitation, commonly referred to as ChIP, is a powerful technique for the evaluation of in vivo interactions of proteins with specific regions of genomic DNA. Formaldehyde is used in this technique to cross-link proteins to DNA in vivo, followed by the extraction of chromatin from cross-linked cells and tissues. Harvested chromatin is sheared and subsequently used in an immunoprecipitation incorporating antibodies specific to protein(s) of interest and thus coprecipitating and enriching the cross-linked, protein-associated DNA. The cross-linking process can be reversed, and protein-bound DNA fragments of optimal length ranging from 200 to 1000 base pairs (bp) can subsequently be purified and measured or sequenced by numerous analytical methods. In this protocol, two different fixation methods are described in detail. The first involves the standard fixation of cells and tissue by formaldehyde if the target antigen is highly abundant. The dual cross-linking procedure presented at the end includes an additional preformaldehyde cross-linking step and can be especially useful when the target protein is in low abundance or if it is indirectly associated with chromatin DNA through another protein.
Restoring critical neuronal architecture after peripheral nerve injury is challenging. Although immediate regenerative responses to peripheral axon injury involve the synthesis of regeneration associated proteins in neurons and Schwann cells, an unfavourable balance between growth facilitatory and growth inhibitory signaling impairs the growth continuum of injured peripheral nerves. Molecules involved with the signaling network of tumor suppressors play crucial roles in shifting the balance between growth and restraint during axon regeneration. An understanding of the molecular framework of tumor suppressor molecules in injured neurons and its impact on stage specific regeneration events may expose therapeutic intervention points. In this review we discuss how signaling networks of the specific tumor suppressors PTEN, Rb1, p53, p27 and p21 are altered in injured peripheral nerves and how this impacts peripheral nerve regeneration. Insights into the roles and importance of these pathways may open new avenues for improving the neurological deficits associated with nerve injury. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Correlative evidence suggests that GABAergic signaling plays an important role in the regulation of activity-dependent hippocampal neurogenesis and emotional behavior in adult mice. However, whether these are causally linked at the molecular level remains elusive. Nuclear factor of activated T cell (NFAT) proteins are activity-dependent transcription factors that respond to environmental stimuli in different cell types, including hippocampal newborn neurons. Here, we identify NFATc4 as a key activity-dependent transcriptional regulator of GABA signaling in hippocampal progenitor cells via an unbiased high-throughput genome-wide study. Next, we demonstrate that GABA A receptor (GABA A R) signaling modulates hippocampal neurogenesis through NFATc4 activity, which in turn regulates GABRA2 and GABRA4 subunit expression via binding to specific promoter responsive elements, as assessed by ChIP and luciferase assays. Furthermore, we show that selective pharmacological enhancement of GABA A R activity promotes hippocampal neurogenesis via the calcineurin/NFATc4 axis. Importantly, the NFATc4-dependent increase in hippocampal neurogenesis after GABA A R stimulation is required for the suppression of the anxiety response in mice. Together, these data provide a novel molecular insight into the regulation of the anxiety response in mice, suggesting that the GABA A R/NFATc4 axis is a druggable target for the therapy of emotional disorders.
The tumor suppressor p53 is a multifunctional sensor of a number of cellular signals and pathways essential for cell biology, including DNA damage, cell cycle regulation, apoptosis, angiogenesis and cell metabolism. In the last few years, a novel role for p53 in neurobiology has emerged, which includes a role in the regulation of neurite outgrowth and axonal regeneration. p53 integrates a number of extracellular signals that involve neurotrophins and axon guidance cues to modulate the cytoskeletal response associated with neurite outgrowth at both the transcriptional and post-translational level. Here, we review our current knowledge of this topic and speculate about future research directions that involve p53 and related molecular pathways and that might advance our understanding of neurite outgrowth and axonal regeneration at the molecular level.
