cAMP is a key mediator of a number of molecules that induce growth cone chemotaxis, including netrin-1 and myelin-associated glycoprotein (MAG). Endogenous neuronal cAMP levels decline during ...development, and concomitantly axonal growth cones switch their response to cAMP-dependent guidance cues from attraction to repulsion. The mechanisms by which cAMP regulates these polarized growth cone responses are unknown. We report that embryonic growth cone attraction to gradients of cAMP, netrin-1, or MAG is mediated by Epac. Conversely, the repulsion conferred by MAG or netrin-1 on adult growth cones is mediated by protein kinase A (PKA). Furthermore, fluorescence resonance energy transfer reveals that netrin-1 distinctly activates Epac in embryonic growth cones but PKA in postnatal neurons. Our results suggest that cAMP mediates growth cone attraction or repulsion by distinctly activating Epac or PKA, respectively. Moreover, we propose that the developmental switch in growth cone response to gradients of cAMP-dependent guidance cues from attraction to repulsion is the result of a switch from Epac- to PKA-mediated signaling pathways.
A decline in developing neuronal cAMP levels appears to render mammalian axons susceptible to growth inhibitory factors in the damaged CNS. cAMP elevation enhances axon regeneration, but the cellular ...mechanisms involved have yet to be fully elucidated. Epac has been identified as a signaling protein that can be activated by cAMP independently of PKA, but little is known of its expression or role in the nervous system. We report that Epac expression is developmentally regulated in the rat nervous system, and that activation of Epac promotes DRG neurite outgrowth and is as effective as cAMP elevation in promoting neurite regeneration on spinal cord tissue. Additionally, siRNA mediated knockdown of Epac reduces DRG neurite outgrowth, prevents the increased growth promoted by cAMP elevation and also diminishes the ability of embryonic neurons to grow processes on spinal cord tissue. Furthermore, we show that asymmetric activation of Epac promotes attractive growth cone turning in a similar manner to cAMP activation. We propose that Epac plays a role in mediating cAMP-dependent axon growth and guidance, and may provide an important target for inducing axon regeneration
in vivo.
During restenosis, vascular smooth muscle cells (VSMCs) migrate from the vascular media to the developing neointima. Preventing VSMC migration is therefore a therapeutic target for restenosis. Drugs, ...such as prostacyclin analogues, that increase the intracellular concentration of cyclic adenosine monophosphate (cAMP) can inhibit VSMC migration, but the mechanisms via which this occurs are unknown. Two main downstream mediators of cAMP are protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). This study has examined the effects of the prostacyclin analogue beraprost on VSMC migration and investigated the intracellular pathways involved.
In a chemotaxis chamber, human saphenous vein VSMC migrated towards a platelet-derived growth-factor-BB (PDGF) chemogradient. Incubation with therapeutically relevant concentrations of cAMP-producing agonist beraprost significantly decreased PDGF-induced migration. Direct activation of either PKA or Epac inhibited migration whereas inhibition of PKA did not prevent the anti-migratory effect of beraprost. Direct activation of Epac also prevented hyperplasia in ex vivo serum-treated human veins. Using fluorescence resonance energy transfer, we demonstrated that beraprost activated Epac but not PKA. The mechanisms of this Epac-mediated effect involved activation of Rap1 with subsequent inhibition of RhoA. Cytoskeletal rearrangement at the leading edge of the cell was consequently inhibited. Interestingly, Epac1 was localized to the leading edge of migrating VSMC.
These results indicate that therapeutically relevant concentrations of beraprost can inhibit VSMC migration via a previously unknown mechanism involving the cAMP mediator Epac. This may provide a novel target that could blunt neointimal formation.
Abstract In the search for a cure to brain and spinal cord injury much has been learned about the inhibitory environment of the central nervous system (CNS), and yet a clinical therapy remains ...elusive. In recent years great advances have been made in understanding intracellular molecular mechanisms that transduce cell surface receptor-mediated signals that neurons receive from their environment. Many of these signalling pathways share common mechanisms, which presents the possibility that manipulating activities of key cell signalling molecules such as those regulated by 3′-5′-cyclic adenosine monophosphate (cAMP) might allow axons to simultaneously overcome the inhibitory effects of a number of extracellular ligands. The identification of Epac, a novel direct intracellular target for cAMP, has opened up a new avenue of research that is beginning to explain how cAMP can mediate a range of neuronal functions including distinct axon growth and guidance decisions. With current research tools that allow more specific activation of proteins or knock-down of their expression, as well as quantitation of protein activities in live cells, it is already becoming clear that Epac plays highly important roles in the development and function of the nervous system. Here, we focus on emerging evidence that Epac mediates cAMP-regulated axon growth and chemoattraction, and thus represents a novel target for overcoming axon growth inhibition and promoting CNS regeneration.
With increasing life expectancy, Alzheimer's disease (AD) and other dementias pose an increasing and as yet unresolved health problem. A variety of cellular models of AD has helped to decipher some ...key aspects of amyloid and tau related degeneration. The initial approach of extracellular applications of synthetic peptides has now been replaced by the introduction of amyloid precursor protein (APP) and tau genes. In the present study adenoviral transductions were exploited for gene delivery into primary rat hippocampal and dorsal root ganglion (DRG) cultures to enable comparative and mechanistic studies at the cellular level and subsequent drug testing. Time lapse experiments revealed a different pattern of cell death: apoptotic-like for APP whereas tau positive cells joined and formed clusters. Mutated human APP or tau expression caused accelerated neuronal damage and cell death (cf. EGFP: −
50% for APP at 5
days; −
40% for tau at 3
days). This reduction in viability was preceded by decreased excitability, monitored via responses to depolarising KCl-challenges in Ca
2+ imaging experiments. Additionally, both transgenes reduced neurite outgrowth in DRG neurones. Treatment studies confirmed that APP induced-damage can be ameliorated by β- and γ-secretase inhibitors (providing protection to 60–100% of control levels), clioquinol (80%) and lithium (100%); while anti-aggregation treatments were beneficial for tau-induced damage (60–90% recovery towards controls). Interestingly, caffeine was the most promising drug candidate for therapeutic intervention with high efficacy in both APP (77%) and tau-induced models (72% recovery). Overall, these cellular models offer advantages for mechanistic studies and target identification in AD and related disorders.
► AdV-delivered APP caused apoptotic-like cell death in hippocampal cultures. ► Formation of cellular clusters preceded cell death for AdV-delivered tau. ► Early changes in excitability could be restored with secretase inhibition or MB. ► Morphological scoring determined protection offered by a range of compounds. ► Axonal outgrowth and maintenance was also affected by tau and APP.
Abstract Cyclic adenosine monophosphate (cAMP) has been intensively studied in recent years in order to elucidate its contribution in intracellular signalling mechanisms that regulate axon growth and ...guidance, and also to test if its activation can promote axon regeneration after injury. Cyclic guanosine monophosphate (cGMP), however, has been given considerably less attention even though it too mediates intracellular signalling cascades activated by extracellular guidance cues. cGMP can promote neurite outgrowth in neuronal cell lines but its role in promoting growth and regeneration of primary neurons is not well established. Here, we have examined the effects of elevating cGMP activity on axon growth, guidance and regeneration in vitro. We have found that, like cAMP elevation, activation of cGMP increases rat dorsal root ganglion (DRG) neurite outgrowth on a polylysine substrate and that asymmetric cGMP elevation promotes attractive growth cone turning. When grown in an in vitro model of axon regeneration activation of cGMP alone was not sufficient to promote adult neurite outgrowth. However, when combined with cAMP elevation substantial regeneration of adult neurites is achieved, superior to that achieved with either cAMP or cGMP alone. Regeneration is enhanced still further with simultaneous application of a Nogo receptor blocking peptide, suggesting this combinatorial strategy could achieve far greater axon regeneration in vivo than targeting individual cell signalling mechanisms.
The cell signalling mechanisms underlying mammalian central nervous system axon growth and guidance change during development, such that axons that establish appropriate connectivity in the embryo ...fail to regenerate after injury to the adult nervous system. The growth cone turning assay has been used in Xenopus neurons to elucidate mechanisms of axon guidance during development. Here, we describe how we have adapted this assay for rat dorsal root ganglion neurons to study the influence of extracellular secreted factors causing growth cone attraction and repulsion. Additionally, we describe how this method can be combined with small interfering RNA and cDNA transfections to manipulate protein expression in growth cones, and fluorescence resonance energy transfer to monitor the activity of signalling pathways in live neurons. This assay provides the unique ability to manipulate and visualise the internal status of growth cone signalling whilst challenged with extracellular chemotropic signalling molecules, and can be used to develop strategies to promote axon regeneration in the mature mammalian central nervous system.
During development, the axons of retinal ganglion cell (RGC) neurons must decide whether to cross or avoid the midline at the optic chiasm to project to targets on both sides of the brain. By ...combining genetic analyses with in vitro assays, we show that neuropilin 1 (NRP1) promotes contralateral RGC projection in mammals. Unexpectedly, the NRP1 ligand involved is not an axon guidance cue of the class 3 semaphorin family, but VEGF164, the neuropilin-binding isoform of the classical vascular growth factor VEGF-A. VEGF164 is expressed at the chiasm midline and is required for normal contralateral growth in vivo. In outgrowth and growth cone turning assays, VEGF164 acts directly on NRP1-expressing contralateral RGCs to provide growth-promoting and chemoattractive signals. These findings have identified a permissive midline signal for axons at the chiasm midline and provide in vivo evidence that VEGF-A is an essential axon guidance cue.
► NRP1 is expressed by contralateral mouse retinal ganglion cell axons ► NRP1 promotes commissural axon crossing at the optic chiasm as a VEGF164 receptor ► VEGF164 acts directly on retinal growth cones as an attractive guidance signal ► VEGF164 and NRP1 are essential for normal contralateral axon growth in vivo
Millions of patients suffer from debilitating spinal cord injury (SCI) without effective treatments. Elevating cAMP promotes CNS neuron growth in the presence of growth-inhibiting molecules. cAMP's ...effects on neuron growth are partly mediated by Epac, comprising Epac1 and Epac2; the latter predominantly expresses in postnatal neural tissue. Here, we hypothesized that Epac2 activation would enhance axonal outgrowth after SCI. Using
assays, we demonstrated, for the first time, that Epac2 activation using a specific soluble agonist (S-220) significantly enhanced neurite outgrowth of postnatal rat cortical neurons and markedly overcame the inhibition by chondroitin sulfate proteoglycans and mature astrocytes on neuron growth. We further investigated the novel potential of Epac2 activation in promoting axonal outgrowth by an
rat model of SCI mimicking post-SCI environment
and by delivering S-220 via a self-assembling Fmoc-based hydrogel that has suitable properties for SCI repair. We demonstrated that S-220 significantly enhanced axonal outgrowth across the lesion gaps in the organotypic spinal cord slices, compared with controls. Furthermore, we elucidated, for the first time, that Epac2 activation profoundly modulated the lesion environment by reducing astrocyte/microglial activation and transforming astrocytes into elongated morphology that guided outgrowing axons. Finally, we showed that S-220, when delivered by the gel at 3 weeks after contusion SCI in male adult rats, resulted in significantly better locomotor performance for up to 4 weeks after treatment. Our data demonstrate a promising therapeutic potential of S-220 in SCI, via beneficial effects on neurons and glia after injury to facilitate axonal outgrowth.
During development, neuronal cAMP levels decrease significantly compared with the embryonic stage when the nervous system is established. This has important consequences following spinal cord injury, as neurons fail to regrow. Elevating cAMP levels encourages injured CNS neurons to sprout and extend neurites. We have demonstrated that activating its downstream effector, Epac2, enhances neurite outgrowth
, even in the presence of an inhibitory environment. Using a novel biomaterial-based drug delivery system in the form of a hydrogel to achieve local delivery of an Epac2 agonist, we further demonstrated that specific activation of Epac2 enhances axonal outgrowth and minimizes glial activation in an
model of spinal cord injury, suggesting a new strategy for spinal cord repair.
Millions of people worldwide are affected by traumatic spinal cord injury, which usually results in permanent sensorimotor disability. Damage to the spinal cord leads to a series of detrimental ...events including ischaemia, haemorrhage and neuroinflammation, which over time result in further neural tissue loss. Eventually, at chronic stages of traumatic spinal cord injury, the formation of a glial scar, cystic cavitation and the presence of numerous inhibitory molecules act as physical and chemical barriers to axonal regrowth. This is further hindered by a lack of intrinsic regrowth ability of adult neurons in the central nervous system. The intracellular signalling molecule, cyclic adenosine 3′,5′-monophosphate (cAMP), is known to play many important roles in the central nervous system, and elevating its levels as shown to improve axonal regeneration outcomes following traumatic spinal cord injury in animal models. However, therapies directly targeting cAMP have not found their way into the clinic, as cAMP is ubiquitously present in all cell types and its manipulation may have additional deleterious effects. A downstream effector of cAMP, exchange protein directly activated by cAMP 2 (Epac2), is mainly expressed in the adult central nervous system, and its activation has been shown to mediate the positive effects of cAMP on axonal guidance and regeneration. Recently, using ex vivo modelling of traumatic spinal cord injury, Epac2 activation was found to profoundly modulate the post-lesion environment, such as decreasing the activation of astrocytes and microglia. Pilot data with Epac2 activation also suggested functional improvement assessed by in vivo models of traumatic spinal cord injury. Therefore, targeting Epac2 in traumatic spinal cord injury could represent a novel strategy in traumatic spinal cord injury repair, and future work is needed to fully establish its therapeutic potential.
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