Table of contents
MAP kinase, neural development and neural activity
Mitogen-activated protein (MAP) kinases are ubiquitous components of many signal transduction pathways. Constitutively active variants have been isolated for every component of the extracellular-signal-regulated kinase 1 (ERK1) and ERK2 MAP kinase pathway except for the ERK itself. To create an activated ERK2 variant, ERK2 was fused to the low activity form of its upstream regulator, the MAP kinase kinase MEK1. The ERK2 in this fusion protein is active in the absence of extracellular signals. Expression of the fusion protein in mammalian cells does not activate endogenous ERK1 or ERK2. It is sufficient, however, to induce activation of the transcription factors Elk-1 and AP-1; neurite extension in PC12 cells in the absence of nerve growth factor, and foci of morphologically and growth-transformed NIH3T3 cells, if the fusion protein is localized to the nucleus. A cytoplasmic fusion protein is without effect. It is concluded that activation of ERK2 is sufficient to cause several transcriptional and phenotypic responses in mammalian cells. Nuclear localization of activated ERK2 is required to induce these events (Robinson, 1998).
Nerve growth factor (NGF) promotes differentiation of PC12 cells, which respond by conversion within hours from a chromaffin-like to a sympathetic neuron-like phenotype. NGF stimulation of PC12 cells increases the activity of two MAPKs by greater than 20-fold within minutes. They are inactivated by either protein-tyrosine phosphatases or the protein-serine/threonine phosphatase termed protein phosphatase 2A, they activate protein kinase-II, and they phosphorylate identical threonine residues on myelin basic protein to those phosphorylated by other MAPKs. These protein kinases are ERK2 and ERK1, two widely expressed MAPK isoforms. MAP kinase kinases activated by NGF are dependent on serine/threonine phosphorylation for activity and promote phosphorylation of serine/threonine and tyrosine residues on MAPKs (Gomez, 1991).
IL-6 induces differentiation of PC12 cells pretreated with nerve growth factor (NGF). The signals required for neurite outgrowth of PC12 cells were explored by using a series of mutants of a chimeric receptor consisting of the extracellular domain of the granulocyte-colony stimulating factor (G-CSF) receptor and the cytoplasmic domain of gp130, a signal-transducing subunit of the IL-6 receptor. These mutants, incapable of activating the MAP kinase cascade, also fail to induce neurite outgrowth. Consistently, a MEK inhibitor, PD98059, inhibits neurite outgrowth, showing that activation of the MAP kinase cascade is essential for the differentiation of PC12 cells. In contrast, a mutation that abolishes the ability to activate STAT3 does not inhibit neurite outgrowth; rather, neurite outgrowth is stimulated. This mutant does not require NGF pretreatment for neurite outgrowth. Dominant-negative STAT3s mimics NGF pretreatment; NGF suppresses the IL-6-induced activation of STAT3, supporting the idea that STAT3 might negatively regulate the differentiation of PC12 cells. These results suggest that neurite outgrowth of PC12 cells is regulated by the balance of MAP kinase and STAT3 signal transduction pathways, and that STAT3 activity can be regulated negatively by NGF (Ihara, 1997).
Calcium/calmodulin-dependent protein kinase II (CaMK) and p42 mitogen-activated protein kinase (MAPK) are enriched in neurons and possess the capacity to become persistently active, or autonomous, following removal of the activating stimulus. Since persistent kinase activation may be a mechanism for information storage, primary cultures of cortical neurons were used to investigate whether kinase autonomy can be triggered by bursts of spontaneous synaptic activity. Both kinases respond to synaptic stimulation, but differ markedly in their kinetics of activation and inactivation, as well as in their sensitivity to NMDA receptor blockade. While 90% of maximal CaMK activation is observed after only 10 sec of synaptic bursting, MAPK activity is unaffected at this early time and rises to only 30% of maximal after 2 min of stimulation. Following blockade of synaptic stimulation, CaMK activity decreases by 50% in 10-30 sec, while MAPK activity decays by 50% within 6-10 min. Although MAPK exhibits relatively slow activation, short periods of synaptic activity can trigger the MAPK activation process, which persisted in the absence of synaptic stimulation. Comparison of the effect of NMDA receptor blockade on synaptic activation of these kinases reveals that CaMK activity is preferentially suppressed. Since CaMK is concentrated in dendritic processes in the vicinity of synapses, synaptic calcium transients were measured in fine dendritic processes (approximately 1 microns diameter) to assess their sensitivity to NMDA receptor blockade. Calcium transients in these fine processes are reduced by up to 90% by NMDA receptor blockade, possibly accounting for the profound sensitivity of CaMK to this treatment. The sharp contrast between the regulation of CaMK and MAPK by synaptic activity indicates that they may mediate neuronal responses to different patterns of afferent stimulation. The relatively slow activation and inactivation of MAPK suggests that it may be able to integrate information from multiple, infrequent bursts of synaptic activity (Murphy, 1994).
Recombinant glia maturation factor (GMF), a 17-kDa brain protein, inhibits the activity of ERK1 and ERK2. A preliminary phosphorylation of GMF by protein kinase A (PKA) dramatically increases its inhibitory effect by over 600-fold (Ki approximately 3 nM), making it the most potent MAP kinase inhibitor ever reported. Immunoprecipitation of GMF from cell extracts coprecipitates ERK (and vice versa), suggesting the association of the two proteins in the cell. The inhibitory effect of PKA-phosphorylated GMF is specific, as it does not suppress the activity of cdc2 kinase, another proline-directed kinase. Nor does it inhibit MAP kinase kinase (MEK) and MAP kinase-activated protein (MAPKAP) kinase-2, the two enzymes immediately upstream and downstream, respectively, of ERK. Of the other three enzymes that can phosphorylate GMF, only p90 ribosomal S6 kinase (RSK; see Drosophila S6kII) enhances the inhibitory function of GMF on ERK; protein kinase C (PKC) and casein kinase II (CKII) are without effect. The inhibition of ERK by PKA-phosphorylated GMF suggests that GMF could be one of the mediators of the suppressive effect of the PKA pathway on the MAP kinase pathway. On the other hand, that RSK-phosphorylated GMF also inhibits ERK implies a negative feedback loop in the regulation of MAP kinase activity (Zaheer, 1996).
AChR-inducing activity (ARIA)/heregulin, a ligand for erbB receptor tyrosine kinases (RTKs), is likely to be one nerve-supplied signal that induces expression of acetylcholine receptor (AChR) genes at the developing neuromuscular junction. Expression of activated Ras or Raf mimicks ARIA-induction of AChR epsilon subunit genes in muscle cells; whereas dominant negative Ras or Raf blocks the effect of ARIA. ARIA rapidly activates erk1 and erk2 and inhibition of both erks also abolished the effect of ARIA. ARIA stimulates association of PI3K with erbB3, expression of an activated PI3K leads to ARIA-independent AChR epsilon subunit expression, and inhibition of PI3K abolishes the action of ARIA. Thus, synaptic induction of AChR genes requires activation of both Ras/MAPK and PI3K signal transduction pathways (Tansey, 1996).
A pathway by which calcium influx through voltage-sensitive calcium channels leads to mitogen-activated protein kinase (MAPK) activation has been characterized. In PC12 cells, membrane depolarization leading to calcium influx through L-type calcium channels activates the dual specificity MAPK kinase MEK1, which phosphorylates and activates MAPK. Calcium influx leads within 30 s to activation of the small guanine nucleotide-binding protein Ras. Moreover, activation of MAPK in response to calcium influx is inhibited by the dominant negative mutant RasAsn17, indicating that Ras activity is required for calcium signaling to MAPK. Ras is also activated by release of calcium from intracellular stores and by membrane depolarization of primary cortical neurons. The pleiotropic regulatory potential of both Ras and the MAPK pathway suggests that they may be central mediators of calcium signaling in the nervous system (Rosen, 1994).
Although Ca2+-stimulated cAMP response element binding protein- (CREB-) dependent transcription has been implicated in growth, differentiation, and neuroplasticity, mechanisms for Ca2+-activated transcription have not been defined. Extracellular signal-related protein kinase (ERK) signaling is obligatory for Ca2+-stimulated transcription in PC12 cells and hippocampal neurons. The sequential activation of ERK and Rsk2 by Ca2+ leads to the phosphorylation and transactivation of CREB. The Ca2+-induced nuclear translocation of ERK and Rsk2 to the nucleus requires protein kinase A (PKA) activation. Interestingly, Ca2+-mediated CREB phosphorylation in wild-type PC12 cells is decreased by a selective PKA inhibitor. With a high efficiency transfection protocol, expression of dominant negative PKA also attenuates Ca2+-stimulated CREB phosphorylation. In addition, treatment with the PKA inhibitors also inhibits depolarization-mediated CREB phosphorylation in primary hippocampal neurons. These results suggest that in PC12 cells and hippocampal neurons, PKA activity is required for Ca2+-induced CREB phosphorylation (Impey, 1998).
Because the nuclear translocation of ERK may be necessary for ERK-activated transcription, and PKA is required for Ca2+ stimulation of CREB phosphorylation, nuclear translocation of ERK was monitored when PKA was inhibited. To efficiently induce the nuclear translocation of ERK by Ca2+, PC12 cells were treated with KCl and a direct activator of L-type Ca2+ channels. Depolarization induces the phosphorylation of ERK and its translocation to the nucleus in both PC12 cells and hippocampal neurons. The specific PKA inhibitors inhibited the nuclear translocation of Erk in PC12 cells and hippocampal neurons. Western blotting of cytosolic fractions shows that the inhibition of Erk translocation by treatment with PKA inhibitors is not the result of an effect on Erk activation. To verify that PKA is required for the nuclear translocation of ERK, the cytosolic-to-nuclear ratio of phospho-ERK in KCl-stimulated hippocampal neurons was also quantitated. A specific PKA inhibitor significantly inhibited the translocation of ERK to the nucleus. The importance of PKA activity for ERK nuclear translocation was confirmed by transiently transfecting PC12 cells with a dominant negative PKA fused to green fluorescent protein. Only cells that express dominant negative PKA-GFP show impaired nuclear translocation of phospho-ERK. These results suggest that PKA is required for the phosphorylation and transactivation of CREB by Ca2+, because PKA is required for the nuclear translocation of ERK. However, since Rsk2 [a member of the pp90(RSK) family] is a major Ca2+-activated CREB kinase in PC12 cells, inhibition of Erk translocation should also block the activation of nuclear but not cytosolic Rsk2. Accordingly, inhibition of PKA blocks the activation of Rsk2 in the nuclear fraction but not in the cytosolic fraction. Treatment with PKA inhibitors attenuates the nuclear translocation of Rsk2. This is not surprising, because it is known that both ERK and Rsk2 are tightly associated in vivo and that they cotranslocate to the nucleus. Collectively, these data indicate that PKA may be necessary for the phosphorylation and transactivation of CREB by Ca2+, because PKA is required for the nuclear translocation of ERK and subsequent nuclear activation of the CREB kinase Rsk2. Inhibition of PKA also significantly impairs the translocation of ERK to the nucleus in response to NGF. Interestingly, coexpression of dominant negative PKA attenuates NGF-stimulated Elk1 transcriptional activation. Evidently, the modulation of ERK translocation by PKA activity plays a general role in the activation of transcription by mitogens and neurotrophic factors. NGF does not detectably elevate intracellular cAMP, suggesting that basal PKA activity is sufficient for neurotrophic factors and other strong ERK activators to induce nuclear translocation of ERK. Nevertheless, in the case of depolarization, which activates ERK to a lesser degree, the concomitant depolarization-mediated increase in cAMP levels enhances ERK translocation. These results may explain why PKA activity is required for Ca2+-stimulated CREB-dependent transcription. Furthermore, the full expression of the late phase of long-term potentiation (L-LTP) and L-LTP-associated CRE-mediated transcription requires ERK activation, suggesting that the activation of CREB by ERK plays a critical role in the formation of long lasting neuronal plasticity (Impey, 1998).
Ca2+-permeable AMPA receptors may play a key role during developmental neuroplasticity, learning and memory, and neuronal loss in a number of neuropathologies. However, the intracellular signaling pathways used by AMPA receptors during such processes are not fully understood. The mitogen-activated protein kinase (MAPK) cascade is an attractive target because it has been shown to be involved in gene expression, synaptic plasticity, and neuronal stress. Using primary cultures of mouse striatal neurons and a phosphospecific MAPK antibody, the ability of AMPA receptors to activate the MAPK cascade was addressed. In the presence of cyclothiazide, AMPA causes a robust and direct (no involvement of NMDA receptors or L-type voltage-sensitive Ca2+ channels) Ca2+-dependent activation of MAPK through MAPK kinase (MEK). This activation is blocked by GYKI 53655, a noncompetitive selective antagonist of AMPA receptors. Probing the mechanism of this activation reveals an essential role for phosphatidylinositol 3-kinase (PI 3-kinase) and the involvement of a pertussis toxin (PTX)-sensitive G-protein, a Src family protein tyrosine kinase, and Ca2+/calmodulin-dependent kinase II. Application of AMPA to rat cerebral cortical neurons has been shown to lead to a rapid increase in Ras activity and activation of MAPK. Ras-dependent activation of MAPK is usually associated with seven transmembrane receptors that couple to heterotrimeric G-proteins. AMPA activates ERK2 (p42) by causing a Ca2+-dependent association of G-protein betagamma subunits, probably Gi, with a Ras, Raf kinase, MEK complex. This novel involvement of a heterotrimeric G-protein in ionotropic AMPA receptor signaling was examined. Striatal neurons were pretreated with pertussis toxin (PTX) or PBS vehicle for 24 hr before experiments with AMPA/cyclothiazide. PTX treatment abolishes AMPA receptor activation of MAPK, indicating a role for a Gi or Go-type G-protein in the activation of MAPK by AMPA receptors in striatal neurons. Similarly, kainate activates MAPK in a PI 3-kinase-dependent manner. AMPA receptor stimulation leads to a Ca2+-dependent phosphorylation of the nuclear transcription factor CREB, which can be prevented by inhibitors of MEK or PI 3-kinase. These results indicate that Ca2+-permeable AMPA receptors transduce signals from the cell surface to the nucleus of neurons through a PI 3-kinase-dependent activation of MAPK. This novel pathway may play a pivotal role in regulating synaptic plasticity in the striatum (Perkinton, 1999).
Thus, although the specific protein-protein interactions that lead to activation of the Ras-MAPK pathway by AMPA receptors are not currently known, it seems reasonable to propose that AMPA receptor-evoked rises in cytosolic Ca2+ may trigger activation of PI 3-kinase: then, recruitment of the lipid kinase to the MAPK cascade may, as is the case with seven-transmembrane Gi/Go-type G-protein linked receptors, be orchestrated by free G betagamma subunits. The specific exchange factors regulating Ras activity after AMPA receptor stimulation also remain to be determined. An involvement of the neuron-specific guanine nucleotide exchange factor, Ras-GRF, seems plausible because it has recently been demonstrated that Ras-GRF can be activated in response to increases in intracellular Ca2+ and/or free G-protein betagamma subunits that induce phosphorylation of Ras-GRF by as yet unknown kinases. However, Ca2+/calmodulin-dependent activation of Ras-GRF does not appear to involve PTKs, thus, the results indicating that tyrosine phosphorylation may be an important step in AMPA receptor activation of MAP kinase suggests that additional Ca2+-dependent routes to Ras may be activated. It has been shown that CaM-KII can phosphorylate AMPA receptor subunits (Mammen et al., 1997), resulting in enhanced receptor currents, and this has been implicated in the strengthening of postsynaptic responses associated with synaptic plasticity. Selective inhibition of CaM-KII activity substantially reduces AMPA/cyclothiazide-evoked activation of MAPK without altering Ca2+ influx through the receptor. These data indicate that CaM-KII can be a positive modulator of AMPA receptor signaling but that in the presence of cyclothiazide the kinase probably regulates AMPA receptor-mediated MAPK activation at a point downstream of Ca2+ entry (Perkinton, 1999 and references).
Synaptic vesicle availability and mobilization are important parameters in the regulation of synaptic transmission and synaptic plasticity. Synapsins, a family of highly conserved neuronal phosphoproteins that are specifically associated with synaptic vesicles, have been implicated in the regulation of neurotransmitter release by controlling the number of vesicles available for exocytosis. Synapsins exist in all organisms with a nervous system, and are encoded by three distinct genes, synapsin I, II, and III, in most vertebrates and by a single gene, Synapsin, in Drosophila. Synapsins are the most abundant synaptic vesicle proteins, with synapsin I alone accounting for 6% of total vesicle protein. They are present in nearly all presynaptic nerve terminals, but different neurons have distinct repertoires of different synapsins (Chi, 2003 and references therein).
During action potential firing, the rate of synapsin dissociation from synaptic vesicles and dispersion into axons controls the rate of vesicle availability for exocytosis at the plasma membrane. Synapsin Ia's dispersion rate tracks the synaptic vesicle pool turnover rate linearly over the range 5-20 Hz: the molecular basis for this lies in regulation at both the calcium-calmodulin-dependent kinase (CaM kinase) and the mitogen-activated protein (MAP) kinase/calcineurin sites in the Synapsin Ia protein. These results show that CaM kinase sites control vesicle mobilization at low stimulus frequency, while MAP kinase/calcineurin sites are critical at both lower and higher stimulus frequencies. These results support a model in which the speed of dissociation of synapsin from synaptic vesicles and redistribution into the axon controls the availability of synaptic vesicles for fusion with the plasma membrane. Thus, multiple signaling pathways serve to allow synapsin's control of vesicle mobilization over different stimulus frequencies (Chi, 2003).
In the mammalian visual system, retinal ganglion cell (RGC) projections from each eye, initially intermixed within the dorsal-lateral geniculate nucleus (dLGN), become segregated during the early stages of development, occupying distinct eye-specific layers. Electrical activity has been suggested to play a role in this process; however, the cellular mechanisms underlying eye-specific segregation are not yet defined. It is known that electrical activity is among the strongest activators of the extracellular signal-regulated kinase (ERK) pathway. Moreover, the ERK pathway is involved in the plasticity of neural connections during development. The role of ERK was examined in the segregation of retinal afferents into eye-specific layers in the dLGN. The activation of this signaling cascade was selectively blocked along the retino-thalamic circuitry by specific inhibitors, and the distribution of RGC fibers in the dLGN was studied. The results demonstrate that the blockade of ERK signaling prevents eye-specific segregation in the dLGN, providing evidence that ERK pathway is required for the proper development of retino-geniculate connections. Of particular interest is the finding that ERK mediates this process both at the retinal and geniculate level (Naska, 2004).
A large body of work indicates that spontaneous retinal activity, present in the developing retina, shapes the retinogeniculate connectivity during early development. The current results show that the ERK cascade, both at the retinal and geniculate level, plays a key role in the segregation of retinal projections and that ERK activation is affected by retinal activity during this process. It should be noted that the alterations of retinogeniculate pattern observed in these experiments after pERK blockade, are very similar to the alterations due to electrical activity blockade. Indeed, pERK blockade in dLGN by ICV U0126 treatment and electrical activity blockade in dLGN by intracranial infusion of tetrodotoxin (TTX) result in a complete arrest of retinal fiber segregation. In addition, the monocular blockade of pERK, or of retinal activity, produces a retraction of projections of the treated eye and an expansion of the territory occupied by projections from untreated eye. It is unlikely that U0126 affected neuronal activity and thus altered segregation through a non-specific mechanism. Indeed, in vivo studies have demonstrated that, in animals treated with U0126, the electrophysiological properties of neurons are not affected; this is in agreement with several in vitro studies. Hence, on the basis of these results, and on the fact that retinal activity drives ERK phosphorylation in the retina and dLGN, it is suggested that retinal activity signals via ERK to regulate retinal fiber segregation in the RGCs and in the dLGN projection neurons. The proposed model is consistent with the findings that binocular blockade of all retinal activity prevents segregation, but that only simultaneous blockade of pERK in the retina and the dLGN yields the same result (Naska, 2004 and references therein).
MAPK/Erk is a protein kinase activated by neurotrophic factors involved in synapse formation and plasticity, which acts at both the nuclear and cytoplasmic level. Synapsin proteins are synaptic-vesicle-associated proteins that are well known to be MAPK/Erk substrates at phylogenetically conserved sites. However, the physiological role of MAPK/Erk-dependent synapsin phosphorylation in regulating synaptic formation and function is poorly understood. This study examined whether synapsin acts as a physiological effector of MAPK/Erk in synaptogenesis and plasticity. To this aim, an in vitro model was developed of soma-to-soma paired Helix B2 neurons, which establish bidirectional excitatory synapses. It was found that the formation and activity-dependent short-term plasticity of these synapses is dependent on the MAPK/Erk pathway. To address the role of synapsin in this pathway, non-phosphorylatable and pseudo-phosphorylated Helix synapsin mutants were generated at the MAPK/Erk sites. Overexpression experiments revealed that both mutants interfere with presynaptic differentiation, synapsin clustering, and severely impair post-tetanic potentiation, a form of short-term homosynaptic plasticity. These findings show that MAPK/Erk-dependent synapsin phosphorylation has a dual role both in the establishment of functional synaptic connections and their short-term plasticity, indicating that some of the multiple extranuclear functions of MAPK/Erk in neurons can be mediated by the same multifunctional presynaptic target (Giachello, 2010).
Effective induction of midbrain-specific dopamine (mDA) neurons from stem cells is fundamental for realizing their potential in biomedical applications relevant to Parkinson's disease. During early development, the Otx2-positive neural tissues are patterned anterior-posteriorly to form the forebrain and midbrain under the influence of extracellular signaling such as FGF and Wnt. In the mesencephalon, sonic hedgehog (Shh) specifies a ventral progenitor fate in the floor plate region that later gives rise to mDA neurons. This study systematically investigated the temporal actions of FGF signaling in mDA neuron fate specification of mouse and human pluripotent stem cells and mouse induced pluripotent stem cells. A brief blockade of FGF signaling on exit of the lineage-primed epiblast pluripotent state initiates an early induction of Lmx1a and Foxa2 in nascent neural progenitors. In addition to inducing ventral midbrain characteristics, the FGF signaling blockade during neural induction also directs a midbrain fate in the anterior-posterior axis by suppressing caudalization as well as forebrain induction, leading to the maintenance of midbrain Otx2. Following a period of endogenous FGF signaling, subsequent enhancement of FGF signaling by Fgf8, in combination with Shh, promotes mDA neurogenesis and restricts alternative fates. Thus, a stepwise control of FGF signaling during distinct stages of stem cell neural fate conversion is crucial for reliable and highly efficient production of functional, authentic midbrain-specific dopaminergic neurons. Importantly, evidence is provided that this novel, small-molecule-based strategy applies to both mouse and human pluripotent stem cells (Jaeger, 2011).
This study demonstrates a functional impact of the FGF/ERK signaling level on the course of mDA neuron differentiation of mouse and human pluripotent stem cells. Pharmacological inactivation of FGF/ERK activity upon exit of the lineage-primed epiblast pluripotent state initiates transcription activities that govern early mesencephalic patterning of both the anterior-posterior and dorsal-ventral axes, leading to the induction of mDA neural progenitor characteristics and maintenance of dopaminergic competence. The consolidation of these characteristics, however, requires a period of autocrine/paracrine FGF/ERK signaling immediately after neural induction. Either continued FGF/ERK blockade in newly derived neural progenitors, or enhancing FGF signaling activity by exogenous FGF8 in these cells, abolishes the effects of FGF receptor inhibitor PD173074. These findings demonstrate a previously unrecognized inhibitory role of FGF/ERK in the induction of ventral midbrain neural progenitors and offer a novel strategy for mDA neuron production from mouse and human pluripotent stem cells and iPSCs. Furthermore, the current method represents a simple, small-molecule-based paradigm for significantly improved efficiency and high reproducibility compared with previously reported transgene-free protocols. Importantly, this strategy directs a midbrain regional identity in the derived dopamine neurons, a property that is essential for functional integration of transplanted dopamine neurons in the Parkinsonian brain (Jaeger, 2011).
Stimulation of embryonic stem cell-derived neural progenitors with Shh and FGF8 is used by almost all dopamine differentiation protocols. However, unless combined with genetic manipulation of mDA transcription factors, such as Pitx3 or Lmx1a, the midbrain regional identity of the dopamine neurons generated has remained uncertain. Furthermore, the yield of Th+ neurons has often proved unreliable between experiments and even highly variable between different microscopic fields within a single culture. A major limiting factor is the temporal and spatial heterogeneity of embryonic stem cell-derived neural progenitors. The current findings demonstrate that the above issues can be addressed using epiblast stem cells (EpiSCs). in the absence of FGF/ERK signaling manipulation, nearly 40% of Th+ neurons generated by EpiSCs already co-expressed Pitx3. This represents a significant improvement over ESC-derived monolayer cultures, where Pitx3+ neurons are rarely observed. This improvement is likely to be due to the more synchronous conversion of EpiSCs to the neuroepithelial fate, which would allow for the effective capture of mDA-competent progenitors (Jaeger, 2011).
However, without additional FGF/ERK inhibitor treatment at the neural induction phase, the total numbers of Th+ Pitx3+ cells remained low due to the overall poor efficiency in producing Th+ cells. The early induction of both Lmx1a and Foxa2 by inhibiting FGF receptor or ERK is likely to be a key factor in the observed high efficiency in these experiments. This hypothesis is based on the following observations: (1) d5 PD-treated (EpiSC) MD cultures are highly enriched for Foxa2+ Lmx1a+ neural progenitors compared with untreated controls; (2) although Shh treatment in d5-9 MD results in comparable numbers of Foxa2+ Lmx1a+ cells in PD-primed and no-PD cultures, mDA neuron production was not enhanced in the manner observed with PD treatment; (3) replacing PD with Shh, which turned out to be a slower and less effective inducer of Lmx1a and Foxa2, also led to poor mDA production; and (4) previous reports have credited the dopaminergic-promoting activity of Lmx1a to its early transgene expression in ESC-derived neural progenitors and indicated that Lmx1a functions by cooperating with Foxa2 in specifying mDA fate during midbrain development (Jaeger, 2011).
The robust induction of Wnt1 and its targets in naïve neural progenitors is likely to be a key downstream mediator that confers the observed early induction of Lmx1a, in light of the recent finding that it can be directly regulated by Wnt1/β-catenin signaling. The same study also showed that, although Otx2 itself had no effect in promoting the expression of terminal mDA neuronal marker genes such as Th, Pitx3 and Nurr1, it significantly enhanced the regulatory effect of Lmx1a and Foxa2 on the expression of these genes. Thus, Otx2 plays a permissive role in Lmx1a/Foxa2-mediated mDA neuronal production. It is worth noting that a significant effect of FGF/ERK blockade is the maintenance of Otx2 in derived neural progenitors (Jaeger, 2011).
This study also shows that, in addition to inducing a regulatory cascade for ventralizing nascent neural progenitors, FGF/ERK inhibition suppresses forebrain specification while promoting anterior neural induction, as demonstrated by the strong and consistent repression of the forebrain regulator genes Six3 and Foxg1 and the hindbrain marker Gbx2. Thus, blocking FGF/ERK at the onset of neural induction leads to a direct and early induction of the midbrain fate at the expense of forebrain and caudal neural fates. This finding is consistent with the developmental role of FGF signaling in regionalization of the forebrain (Jaeger, 2011).
Furthermore, this study demonstrated the importance of precise temporal control of cell signaling and its cross-regulation with other signaling pathways in mDA neuronal fate specification. During development, Fgf8-mediated signaling can induce the patterned expression of many midbrain/rostral hindbrain genes and is required for normal development of the midbrain and cerebellum. Fgf8-induced Wnt1 and engrailed are key regulators of midbrain and cerebellum patterning, as well as of the differentiation and survival of dopamine neurons. In EpiSC-derived neural cultures, after an initial burst of upregulation induced by PD exposure, Wnt1 expression was subsequently reduced to a level below the no-PD control by unknown factors in the newly generated neural progenitors in d3-5 MD. This is the period when Shh, Lmx1a and Foxa2 expression levels continued to rise. Given that Shh and Wnt1 play opposing roles with regard to mDA neurogenesis, these findings suggest that the delay in FGF reactivation, which suppresses Wnt1 levels, might be crucial for achieving high numbers of Th+ neurons by consolidating Lmx1a and Foxa2 expression via Shh signaling (Jaeger, 2011).
From a technological standpoint, this study describes a novel method of mDA neuron differentiation that employs temporally controlled exposure of human and mouse pluripotent stem cells to an FGF/ERK-deficient environment. The highly reliable nature of this method was demonstrated using five independent mouse EpiSC lines, a mouse iPS cell line and two human ESC lines. This protocol offers several advantages over current methods of generating midbrain-specific DA neurons in that it is adherent culture-based and free from genetic manipulation and thus could be readily applied to other cell lines of interest. Furthermore, because it is fully chemically defined, this paradigm could be readily adapted for use in a clinical setting or scaled up for toxicity and drug screening relevant to developing new therapeutics for Parkinson's disease (Jaeger, 2011).
MAP kinase in long-term facilitation and learning
Long-term facilitation of the sensory to motor synapse in Aplysia requires gene expression. While some transcription factors involved in long-term facilitation are phosphorylated by PKA, others lack PKA sites but contain MAP Kinase (MAPK) phosphorylation sites. MAPK translocates into the nucleus of the presynaptic but not the postsynaptic cell during 5-HT-induced long-term facilitation. The presynaptic nuclear translocation of MAPK is also triggered by elevations in intracellular cAMP. Injection of anti-MAPK antibodies or of MAPK Kinase inhibitors into the presynaptic cell, blocks long-term facilitation without affecting basal synaptic transmission or short-term facilitation. Thus, MAPK appears to be specifically recruited and necessary for the long-term form of facilitation. This mechanism for long-term plasticity may be quite general: cAMP also activates MAPK in mouse hippocampal neurons, suggesting that MAPK may play a role in hippocampal long-term potentiation. One target of MAPK might be CREB2. Whereas CREB2 lacks phosphorylation sites for PKA, it has two conserved consensus sites for MAPK phophorylation (Martin, 1997).
The synaptic growth that accompanies 5-HT-induced long-term facilitation of the sensory to motor neuron connection in Aplysia is associated with the internalization of apCAM (Drosophila homolog: Fasciclin 2) at the surface membrane of the sensory neuron. Epitope tags were used to examine the fate of each of the two apCAM isoforms (membrane bound and GPI-linked). Only the transmembrane form is internalized. This internalization can be blocked by overexpression of transmembrane constructs with a single point mutation in the two MAPK consensus sites, as well as by injection of a specific MAPK antagonist into sensory neurons. These data suggest MAPK phosphorylation at the membrane is important for the internalization of apCAMs and, thus, may represent an early regulatory step in the growth of new synaptic connections that accompanies long-term facilitation (Bailey, 1997).
That MAPKs are abundantly expressed in postmitotic neurons suggests functions for this cascade in the mature nervous system. The MAPK cascade is required for hippocampal long term potentiation (LTP), a robust and widely studied form of synaptic plasticity. PD 098059, a selective inhibitor of the MAPK cascade, blocks MAPK activation in response to direct stimulation of the NMDA receptor as well as to LTP-inducing stimuli. Furthermore, inhibition of the MAPK cascade markedly attenuates the induction of LTP. PD 098059, however, has no effect on the expression of established LTP, and the MAPK cascade is not persistently activated during LTP expression. These observations provide the first demonstration of a role for the MAPK cascade in the activity-dependent modification of synaptic connections between neurons in the adult mammalian nervous system (English, 1997).
Rats were given an unfamiliar tasting solution to drink under conditions that resulted in long-term memory of that taste. The insular cortex, which contains the taste cortex, was then removed and assayed for activation of mitogen-activated protein kinase (MAPK) cascades by using antibodies to the activated forms of various MAPKs. Extracellular responsive kinase 1-2 (ERK1-2) in the cortical homogenate is significantly activated within <30 min of drinking the solution that had an unfamiliar taste, without alteration in the total level of the ERK1-2 proteins. The activity subsides to basal levels within <60 min. In contrast, ERK1-2 is not activated when the taste was made familiar. The effect of the unfamiliar taste is specific to the insular cortex. Jun N-terminal kinase 1-2 (JNK1-2) is activated by drinking the unfamiliar taste but with a delayed time course, whereas the activity of Akt kinase and p38MAPK remain unchanged. Elk-1, a member of the ternary complex factor and an ERK/JNK downstream substrate, is activated with a time course similar to that of ERK1-2. Microinjection of a reversible inhibitor of MAPK/ERK kinase into the insular cortex shortly before exposure to the novel taste in a conditioned taste aversion training paradigm attenuates long-term taste aversion memory without significantly affecting short-term memory or the sensory, motor, and motivational faculties required to express long-term taste aversion memory. It is concluded that ERK and JNK are specifically and differentially activated in the insular cortex after exposure to a novel taste, and that this activation is required for consolidation of long-term taste memory (Berman, 1998).
Mitogen-activated protein kinase (MAPK) is an integral component of cellular signaling during mitogenesis and differentiation of mitotic cells. Recently MAPK activation in post-mitotic cells has been implicated in hippocampal long-term potentiation (LTP), a potential cellular mechanism of learning and memory. The involvement of MAPK was investigated in learning and memory in rats. MAPK activation increases in the rat hippocampus after an associative learning task: contextual fear conditioning. Two other protein kinases known to be activated during hippocampal LTP, protein kinase C and -calcium/calmodulin protein kinase II, also are activated in the hippocampus after associative learning. Inhibition of the specific upstream activator of MAPK, MAPK kinase (MEK), blocks fear conditioning. Thus, classical conditioning in mammals activates MAPK, which is necessary for consolidation of the resultant learning (Atkins, 1998).
Long-term facilitation of the connections between the sensory and motor neurons of the gill-withdrawal reflex in Aplysia requires five repeated pulses of serotonin (5-HT). The repeated pulses of 5-HT initiate a cascade of gene activation that leads ultimately to the growth of new synaptic connections. Several genes in this process have been identified, including the transcriptional regulators apCREB-1, apCREB-2, apC/EBP, and the cell adhesion molecule apCAM, which is thought to be involved in the formation of new synaptic connections. The transcriptional regulators apCREB-2 and apC/EBP, as well as a peptide derived from the cytoplasmic domain of apCAM, are phosphorylated in vitro by Aplysia mitogen-activated protein kinase (apMAPK). The cDNA encoding apMAPK has been cloned; apMAPK activity is increased in sensory neurons treated with repeated pulses of 5-HT and by the cAMP pathway. These results suggest that apMAPK may participate with cAMP-dependent protein kinase during long-term facilitation in sensory cells by modifying some of the key elements involved in the consolidation of short- to long-lasting changes in synaptic strength (Michael, 1998).
Long-term facilitation (LTF) of the sensory-to-motor synapses that mediate defensive reflexes in Aplysia requires induction of the transcription factor Aplysia CCAAT/enhancer binding protein (ApC/EBP) as an early response gene. The time course of ApC/ EBP DNA binding during the induction of LTF was examined: binding activity is detected within 1 h of the sensitization treatment with serotonin, reaches a maximum at 2 h, and decreases after 6 h. How are DNA binding and the turnover of ApC/EBP regulated? Phosphorylation of ApC/EBP by mitogen-activated protein (MAP) kinase is essential for binding. MAP kinase appears to be activated through protein kinase C. ApC/EBP is degraded through the ubiquitin-proteasome pathway but phosphorylation by MAP kinase renders it resistant to proteolysis. Thus, phosphorylation by MAP kinase is required for ApC/EBP to act as a transcription activator as well as to assure its stability early in the consolidation phase, when genes essential for the development of LTF begin to be expressed (Yamamoto, 1999).
An inhibitor of MAPK activation, SL327, was used to test the role of the MAPK cascade in hippocampus-dependent learning in mice. SL327, which crosses the blood-brain barrier, was administered intraperitoneally at several concentrations to animals prior to cue and contextual fear conditioning. Administration of SL327 completely blocks contextual fear conditioning and significantly attenuates cue learning when measured 24 hr after training. To determine whether MAPK activation is required for spatial learning, SL327 was administered to mice prior to training in the Morris water maze. Animals treated with SL327 exhibit significant attenuation of water maze learning; they take significantly longer to find a hidden platform compared with vehicle-treated controls and also fail to use a selective search strategy during subsequent probe trials in which the platform is removed. These impairments cannot be attributed to nonspecific effects of the drug during the training phase; no deficit was seen in the visible platform task, and injection of SL327 following training produced no effect on the performance of these mice in the hidden platform task. These findings indicate that the MAPK cascade is required for spatial and contextual learning in mice (Selcher, 1999).
MAP kinase (ERK) translates cell surface signals into alterations in transcription. ERK also regulates hippocampal neuronal excitability during 5 Hz stimulation and thereby regulates forms of long-term potentiation (LTP) that do not require macromolecular synthesis. Moreover, ERK-mediated changes in excitability are selectively required for some forms of LTP but not others. ERK is required for the early phase of LTP elicited by brief 5 Hz stimulation, as well as for LTP elicited by more prolonged 5 Hz stimulation when paired with beta1-adrenergic receptor activation. By contrast, ERK plays no role in LTP elicited by a single 1 s 100 Hz train. Consistent with these results, ERK is activated by beta-adrenergic receptors in CA1 pyramidal cell somas and dendrites (Winder, 1999).
Long-lasting forms of synaptic plasticity like the late phase of LTP (L-LTP) typically require an elevation of cAMP, the recruitment of the cAMP-dependent protein kinase (PKA), and ultimately the activation of transcription and translation; some forms also require brain-derived neurotrophic factor (BDNF). Both cAMP and BDNF can activate mitogen-activated protein kinase (MAPK/ERK), which also plays a role in LTP. However, little is known about the mechanisms whereby cAMP, BDNF, and MAPK interact. Increases in cAMP can rapidly activate the BDNF receptor TrkB and induce BDNF-dependent long-lasting potentiation at the Schaffer collateral-CA1 synapse in hippocampus. Surprisingly, in these BDNF-dependent forms of potentiation, which are also MAPK dependent, TrkB activation is not critical for the activation of MAPK but instead appears to modulate the subcellular distribution and nuclear translocation of the activated MAPK (Patterson, 2001).
Activation of mitogen-activated protein kinase (MAPK) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) are required for numerous forms of neuronal plasticity, including long-term potentiation (LTP). LTP was induced in rat hippocampal area CA1 using theta-pulse stimulation (TPS) paired with ß-adrenergic receptor activation [isoproterenol (ISO)], a protocol that may be particularly relevant to normal patterns of hippocampal activity during learning. This stimulation results in a transient phosphorylation of p42 MAPK, and the resulting LTP is MAPK dependent. In addition, CaMKII is regulated in two, temporally distinct ways after TPS-ISO: a transient rise in the fraction of phosphorylated CaMKII and a subsequent persistent increase in CaMKII expression. The increases in MAPK and CaMKII phosphorylation are strongly colocalized in the dendrites and cell bodies of CA1 pyramidal cells, and both the transient phosphorylation and delayed expression of CaMKII are prevented by inhibiting p42/p44 MAPK. These results establish a novel bimodal regulation of CaMKII by MAPK, which may contribute to both post-translational modification and increased gene expression (Giovannini, 2001).
Extracellular signal-regulated kinases (ERK1 and 2) are synaptic signaling components necessary for several forms of learning. In mice lacking ERK1, a dramatic enhancement of striatum-dependent long-term memory, which correlates with a facilitation of long-term potentiation in the nucleus accumbens, is observed. At the cellular level, it is found that ablation of ERK1 results in a stimulus-dependent increase of ERK2 signaling, likely due to its enhanced interaction with the upstream kinase MEK. Consistently, such activity change is responsible for the hypersensitivity of ERK1 mutant mice to the rewarding properties of morphine. These results reveal an unexpected complexity of ERK-dependent signaling in the brain and a critical regulatory role for ERK1 in the long-term adaptive changes underlying striatum-dependent behavioral plasticity and drug addiction (Mazzucchelli, 2002).
The enhancement of LTP observed in the nucleus accumbens of ERK1-/- mice suggests a rather straight forward interpretation of the behavioral data. The observed phenotypes in ERK1-deficient mice are consistent with both the known effects of dopamine signaling in the striatum on locomotion and procedural forms of memory. The consequences of DA receptor activation appear to be potentiated in these mutant mice. This is most likely due to an enhanced and prolonged activation of ERK2, which results in a sustained expression of downstream transcription factors in the striatum. Increased ERK2 signaling in response to DA receptor activation in ERK1-/- mice could also result in increased locomotion, particularly during exploration of the activity boxes, which might be expected to engage dopaminergic systems. In addition, when the animals are challenged in cognitive tasks such as the two avoidance paradigms, the same underlying molecular alterations might be responsible for the observed memory enhancement (Mazzucchelli, 2002).
Tyrosine kinases have been implicated in cellular processes thought to underlie learning and memory. Tyrosine kinases play a direct role in long-term synaptic facilitation (LTF) and long-term memory (LTM) for sensitization in Aplysia. Tyrosine kinase activity is required for serotonin-induced LTF of sensorimotor (SN-MN) synapses, and enhancement of endogenous tyrosine kinase activity facilitates the induction of LTF. These effects are mediated, at least in part, through mitogen-activated protein kinase (MAPK) activation and are blocked by transcriptional and translational inhibitors. Moreover, brain-derived neurotrophic factor (BDNF) also enhances the induction of LTF in a MAPK-dependent fashion. Finally, activation of endogenous tyrosine kinases enhances the induction of long-term memory for sensitization, and this enhancement also requires MAPK activation. Thus, tyrosine kinases, acting through MAPK, play a pivotal role in LTF and LTM formation (Purcell, 2003).
Tyrosine kinase activity is required for 5HT-induced LTF at SN-MN synapses in Aplysia; the enhancement of endogenous tyrosine kinase activity facilitates the induction of LTF. While the cellular and molecular studies have focused on the tail SN-MN synapse, there are important interneuronal linkages between the tail SNs and the siphon MNs. Thus, some of the experimental manipulations used, such as bath application of BDNF, may be affecting other cells in addition to the SNs. Nonetheless, the tail SN-MN synapse has been a powerful predictor of the unique features of several forms of behavioral memory in this system (Purcell, 2003).
How might tyrosine kinase activation contribute to the induction of LTF and LTM for sensitization? In mammalian systems, the neurotrophins and their tyrosine kinase receptors have been implicated in a variety of forms of long-lasting synaptic plasticity. One possibility is that one or more neurotrophin-like growth factors may be released (either from the SNs themselves, or from other neurons in the circuit) as a result of five spaced applications of 5HT, which in turn initiates a tyrosine kinase signaling cascade in the SNs. This type of mechanism has been proposed for some forms of long-term potentiation (LTP) in the hippocampus. BDNF is thought to be released downstream of cAMP elevation in the CA1 region of hippocampus as a result of theta burst stimulation. In support of this possibility, TrkB receptor bodies, which sequester endogenous ligand, have been shown to block 5HT-induced LTF in cultured Aplysia SN-MN synapses, suggesting that a neurotrophin-like molecule or other TrkB ligand is released and is required for 5HT-induced LTF. Furthermore, application of exogenous BDNF facilitates the induction of LTF, mimicking the effect of increasing endogenous tyrosine kinase activity by application of a general tyrosine phosphatase inhibitor bpV. In addition, the enhanced induction of LTF by bpV + 5HT requires rapamycin-sensitive translation. This subset of protein synthesis is stimulated downstream of growth factor receptors and is recruited as a result of BDNF stimulation in mammalian systems. For example, BDNF application to cultured cortical neurons increases protein-synthesis in a rapamycin-sensitive manner, and rapamycin can block BDNF-induced increases in synaptic efficacy as well as late-phase LTP in the hippocampus. Therefore, in these experiments a BDNF-like molecule could be binding to a receptor tyrosine kinase in the SN synapse to initiate processes required for long-term synaptic plasticity (Purcell, 2003).
Another possibility is that binding of 5HT to its receptor might activate downstream tyrosine kinases. Consistent with this notion, there have been reports in other systems describing signaling cascades, in which G protein-coupled receptors led to activation of tyrosine kinases. A third possibility is that, under basal conditions, there is a considerable amount of tyrosine phosphatase activity that acts to oppose the induction of long-term processes. Perhaps repeated pulses of 5HT not only initiate signaling cascades that induce gene transcription, but also remove this inhibitory phosphatase constraint. This type of mechanism would explain the reduction in threshold for inducing LTF in the presence of the tyrosine phosphatase inhibitor. A similar mechanism has been postulated for the induction of hippocampal LTP. STEP, a tyrosine phosphatase associated with the NMDA receptor complex, provides a tonic inhibitory constraint on LTP induction. Protein kinase A (PKA) has been shown to phosphorylate and thus inactivate STEP. Interestingly, PKA is persistently activated downstream of repeated 5HT pulses in Aplysia tail SNs and could play a similar role in regulating tyrosine phosphatase activity (Purcell, 2003).
A second important question concerns how tyrosine kinases function in the signaling cascade that leads to LTF and LTM. Work in cultured SN-MN synapses has shown that MAPK activity is required for the induction of LTF by 5HT. Inhibiting tyrosine kinase activity blocks MAPK activation by five pulses of 5HT, suggesting that tyrosine kinase activation occurs downstream of the 5HT receptor but upstream of MAPK activation. Furthermore, application of a tyrosine phosphatase inhibitor leads to MAPK phosphorylation, suggesting that increases in endogenous tyrosine kinase function are sufficient to induce MAPK activation. This tyrosine kinase-MAPK cascade is specifically recruited by stimuli that induce long-term processes, since neither tyrosine kinase nor MAPK activity is required for the induction of STF (Purcell, 2003).
A single pulse of 5HT, paired with bpV, induces LTF, whereas neither stimulus alone is sufficient to produce long-term synaptic enhancement. However, a single pulse of 5HT does not enhance MAPK activation compared to that produced by bpV alone, indicating that additional signals deriving from 5HT signaling (independent of MAPK activation) are required to induce LTF. How might MAPK and 5HT interact to initiate long-term processes? One possibility is that PKA, activated downstream of 5HT, could regulate the subcellular distribution of MAPK. For example, in hippocampal pyramidal cells, the calcium-induced nuclear translocation of MAPK requires PKA activation. In addition, in Aplysia SNs, MAPK has been shown to translocate to the nucleus in response to five pulses of 5HT and elevation of cAMP. Therefore, an attractive model would be that a single pulse of 5HT activates PKA, which in turn facilitates nuclear translocation of MAPK (Purcell, 2003).
Another possible point of convergence between 5HT-derived signals and MAPK is at the level of gene induction in the nucleus. For example, ApCREB2, which represses CREB-mediated transcription in SNs, has consensus phosphorylation sites for MAPK but not PKA, and PKA alone is unable to relieve the repression of CREB-mediated transcription caused by ApCREB2. In contrast, the CREB activator isoform (CREB1a) can be phosphorylated by PKA. Therefore, MAPK could phosphorylate ApCREB2 and relieve its inhibition, allowing for PKA-dependent phosphorylation of CREB1a to initiate gene induction. Interestingly, injection of ApCREB2 antibodies into SN cell bodies, coupled with a single pulse of 5HT, induces LTF. This finding is similar to the current results in which application of the tyrosine phosphatase inhibitor bpV also enables the induction of LTF by a single 5HT pulse. Therefore, it is possible that tyrosine kinases, acting through MAPK, remove ApCREB2 inhibition, allowing for PKA activity (induced by the single pulse of 5HT) to initiate CREB-mediated transcription. Another important possibility is that CREB1a may be activated by MAPK via activation of p90Rsk, a pathway that has been described in mammalian systems (Purcell, 2003).
The enhanced induction of LTF by bpV + 5HT requires transcription, suggesting that tyrosine kinase activation is upstream of gene induction. This transcriptional requirement is characteristic of other forms of LTF that have been described at this synapse, many of which also require CREB activation. Thus, it is possible to predict that LTF induced by bpV + 5HT would require CREB activation as well. In addition, it has been shown that a rapamycin-sensitive subset of translation is required for the induction of LTF by tyrosine kinase activation. It has been shown that 5HT increases the rate of translation both in pleural ganglia (which contain SN cell bodies) and SN neurites, and this increase is reduced by rapamycin. The subset of translation that can be inhibited by rapamycin is thought to regulate a specific set of mRNAs that are normally translated at low levels due to complex secondary structures in their 5'-UTRs. The protein products of these unique mRNAs appear to be required for long-lasting, growth-associated forms of LTF, since rapamycin blocks the increase in synaptic efficacy and varicosity number 72 hr after LTF has been induced. Since rapamycin blocks the facilitation observed at 24 hr with bpV + 5HT treatment, these data support the idea that activation of tyrosine kinases taps into the cascade that signals for persistent, growth-associated facilitation. If this hypothesis is correct, it would be expected bpV + 5HT treatment induces the growth of new varicosities. Transforming growth factor ß, which activates a serine/threonine signaling cascade, has been shown to induce LTF of this SN-MN synapse. However, TGF is thought to be released downstream of transcriptional events. The data led to the suggestion that a tyrosine kinase cascade, perhaps activated by a neurotrophin-like molecule, is required for gene induction. Thus these data raise the possibility that several growth factors are required at different stages for the induction and maintenance of long-term synaptic plasticity and, by extension, long-term memory (Purcell, 2003).
In conclusion, it has been demonstrated that increases in tyrosine kinase activity not only enhance the induction of long-term synaptic plasticity but the induction of long-term memory as well, thus strengthening the generally accepted mechanistic relationship between long-lasting changes in synaptic efficacy and the induction and maintenance of memory. The data also add to a growing body of evidence showing that growth factors can play key roles in the formation of long-term memory (Purcell, 2003).
Learning-induced synaptic plasticity commonly involves the interaction between cAMP and p42/44MAPK. To investigate the role of Rap1 (Drosophila homolog: Roughened) as a potential signaling molecule coupling cAMP and p42/44MAPK, an interfering Rap1 mutant (iRap1) was expressed in the mouse forebrain. This expression selectively decreases basal phosphorylation of a membrane-associated pool of p42/44MAPK, impairs cAMP-dependent LTP in the hippocampal Schaffer collateral pathway induced by either forskolin or theta frequency stimulation, decreases complex spike firing, and reduces the p42/44MAPK-mediated phosphorylation of the A-type potassium channel Kv4.2. These changes correlate with impaired spatial memory and context discrimination. These results indicate that Rap1 couples cAMP signaling to a selective membrane-associated pool of p42/44MAPK to control excitability of pyramidal cells, the early and late phases of LTP, and the storage of spatial memory (Morozov, 2003).
Both cAMP and p42/44 mitogen-activated protein kinase (p42/44MAPK) are crucial for neuronal plasticity and memory storage. In Aplysia, interference with cAMP-induced activation of p42/44MAPK inhibits learning and serotonin-induced long-term facilitation of the connections between the sensory and motor neurons of the gill withdrawal reflex. In rodents, inhibitors of MAPK kinase (MEK) similarly impair cAMP-dependent LTP and spatial learning. Thus, even though it is clear that cAMP and p42/44MAPK are required for plasticity and memory storage, the specific pathways coupling cAMP and other second messengers to p42/44MAPK are not well known (Morozov, 2003 and references therein).
One reason this coupling has not yet been established is that there are several signaling pathways through which cAMP and other second messengers can regulate p42/44MAPK. For example, in cell culture, two small GTPases, Rap1 and Ras, couple cAMP and other second messengers to p42/44MAPK in different ways. (1) Ras and Rap1 are activated by different GDP/GTP-exchange factors, and these exchange factors in turn couple them to distinct upstream signaling pathways. (2) Ras and Rap1 differentially control Raf1 and B-Raf, the two kinases that phosphorylate MEK. Ras recruits both Raf1 and B-Raf to activate p42/44MAPK. In contrast, Rap1 exerts a dual regulation of p42/44MAPK by either inhibiting Raf1 or activating B-Raf. Moreover, Rap1 can also antagonize Ras. Part of this difference presumably resides in their subcellular locations. Ras is anchored to the plasma membrane, whereas Rap1 is anchored to the membrane of the endosomal compartment. These differences suggest that Rap1 and Ras may regulate distinctly different pools of p42/44MAPK, thereby phosphorylating different targets to perform different functions. Indeed, in PC12 cells, Ras-dependent activation of p42/44MAPK leads to proliferation, whereas Rap1-dependent activation leads to differentiation (Morozov, 2003 and references therein).
The existence of different upstream regulators of p42/44MAPK raises the question: which of these distinct signaling pathways are important for neuronal plasticity? One way to define their roles is to interfere with the specific components of these pathways. Interfering with Ras function in the amygdala impairs LTP and fear conditioning, while decreasing its activity in the hippocampus rescues LTP deficits and spatial learning in mice carrying a mutated allele of the neurofibromatosis gene NF1. Interfering with Rap1 function using viral transfection in cultured cells affects LTD by producing deregulation of p38 kinase. However, in contrast to the several studies on Ras, nothing is known about the role of Rap1 in the nervous system of intact animals (Morozov, 2003 and references therein).
To explore the role of Rap1 in the brain, mice were generated with a dominant interfering mutant of Rap1 (iRap1) using the forebrain-specific CamKII-alpha promoter and the tetracycline-regulated system to achieve spatial restriction and temporal reversibility in the expression of the transgene. Using this approach, it was found that interfering with Rap1 in the hippocampus results in reduced basal levels of phospho-MAPK in the membrane fraction; these changes correlate with impaired forskolin and theta frequency-induced LTP and deficient learning and memory storage. These data indicate that Rap1 regulates hippocampus memory storage by gating some forms of cAMP-dependent plasticity through the regulation of a restricted pool of membrane-associated p42/44 MAPK (Morozov, 2003).
The Trk family of receptor tyrosine kinases plays a role in synaptic plasticity and in behavioral memory in mammals. A Trk-like receptor in Aplysia, ApTrkl, is expressed in the sensory neurons, the locus for synaptic facilitation, which is a cellular model for memory formation. Serotonin, the facilitatory neurotransmitter, activates ApTrkl, which, in turn, leads to activation of ERK. Finally, inhibiting the activation of ApTrkl with the Trk inhibitor K252a or using dsRNA to inhibit ApTrkl blocks the serotonin-mediated activation of ERK in the cell body, as well as the cell-wide long-term facilitation induced by 5-HT application to the cell body. Thus, transactivation of the receptor tyrosine kinase ApTrkl by serotonin is an essential step in the biochemical events leading to long-term facilitation in Aplysia (Ormond, 2004).
ApTrkl protein is expressed in sensory neurons, which are well studied for their role in synaptic facilitation. ApTrkl is activated by a single 5 min pulse of 5-HT, raising the possibility that it may function in the induction of a synaptic tag for sensory presynaptic terminals since a 5 min pulse of 5-HT is sufficient to tag the synapse. However, one pulse of 5-HT does not activate ERK, which suggests that activation of ERK by ApTrkl may require persistent activation of the receptor. For example, Trks signal to ERK only after endocytosis, and this may not be stimulated by short bouts of receptor activation or may require activation of additional signaling pathways. Alternatively, prolonged treatments with 5-HT may recruit additional signaling components that are required in addition to ApTrkl for ERK activation (Ormond, 2004).
In Aplysia, long-term facilitation (LTF) of sensory neuron synapses requires activation of both protein kinase A (PKA) and mitogen-activated protein kinase (MAPK). 5-HT through activation of PKA regulates secretion of the sensory neuron-specific neuropeptide sensorin, which binds autoreceptors to activate MAPK. Anti-sensorin antibody blocks LTF and MAPK activation produced by 5-HT and LTF produced by medium containing sensorin that is secreted from sensory neurons after 5-HT treatment. A single application of 5-HT followed by a 2 hr incubation with sensorin produces protein synthesis-dependent LTF, growth of new presynaptic varicosities, and activation of MAPK and its translocation into sensory neuron nuclei. Inhibiting PKA during 5-HT applications and inhibiting receptor tyrosine kinase or MAPK during sensorin application blocks both LTF and MAPK activation and translocation. Thus, long-term synaptic plasticity is produced when stimuli activate kinases in a specific sequence by regulating the secretion and autocrine action of a neuropeptide (Hu, 2004).
The neuropeptide sensorin is synthesized in mechanosensory neurons in several invertebrates. Sensorin may be released from sensory neurons by electrical activity, since action potentials evoke postsynaptic responses in some follower cells as does application of exogenous sensorin. The results suggest that 5 × 5-HT increases sensorin secretion. Treatments with 5 × 5-HT produce an increase in the frequency of synaptic vesicle fusions evoking miniature excitatory potentials. Thus, an increase in the probability of fusion of vesicles containing sensorin may contribute to the increase in sensorin secretion. Vesicular release of sensorin may also contribute to activity-dependent forms of plasticity expressed at sensory neuron synapses. The released sensorin does not produce acute changes in membrane polarization or input resistance in sensory neurons or L7 or affect short-term facilitation lasting minutes produced by 1 × 5-HT. Thus, sensorin secretion and its signaling pathway are utilized to produce LTF and are not required for short-term facilitation (Hu, 2004 and references therein).
The finding that sensorin can substitute for additional applications of 5-HT supports the result that sensorin secretion regulated by 5-HT is required for LTF. LTF is produced by 1 × 5-HT + sensorin (synthetic form or native peptide released by sensory neurons into the medium following 5-HT). This LTF has properties that are similar to those produced by 5 × 5-HT. Sensorin-induced LTF depends on general protein synthesis and the more selective translation that is rapamycin-sensitive; this translation contributes to the longer-lasting forms of LTF. The block of LTF at 24 hr by rapamycin is likely to result from the actions of rapamycin on the translation of proteins in both neurites and the cell bodies. Sensorin-induced LTF is accompanied by the formation of new sensory neuron varicosities. Varicosities that form on the major processes of L7 have active zones, stain with sensorin immunoreactivity, and contain other features that are characteristic of release sites. Thus, the actions of sensorin are reminiscent of neurotrophins such as BDNF, which can be released from presynaptic terminals and contribute to some long-lasting forms of LTP in hippocampus. A TGF-ß-like molecule has been shown to contribute to LTF of sensory neuron synapses. Sensorin does not appear to act through this pathway, since TGF-ß has acute effects on sensory neurons while sensorin does not. Interestingly, TGF-ß regulates the expression of both BDNF and trkB receptors in hippocampal neurons. Perhaps a TGF-ß-like molecule released from the cells or present in hemolymph acts to regulate the expression of sensorin and its receptor (Hu, 2004 and references therein).
Because activation of ERK1/2 MAP kinase (MAPK) is critical for hippocampus-dependent memory, there is considerable interest in mechanisms for regulation of MAPK during memory formation. SCOP (suprachiasmatic nucleus [SCN] circadian oscillatory protein) is a negative regulator of K-Ras in PC12 cells. SCOP interacts directly with K-Ras through its leucine-rich repeat and inhibits K-Ras function by associating with the nucleotide-free form of K-Ras. This prevents binding of GTP to K-Ras, inhibits its activity, and prevents neurotrophin stimulation of MAPK. This study shows that MAPK and CREB-mediated transcription are negatively regulated by SCOP, and SCOP is proteolyzed by calpain when hippocampal neurons are stimulated by brain-derived neurotrophic factor (BDNF), KCl depolarization, or NMDA. Moreover, training for novel object memory decreases SCOP in the hippocampus. To determine if hippocampus-dependent memory is influenced by SCOP in vivo, a transgenic mouse strain was generated for the inducible overexpression of SCOP in the forebrain. Overexpression of SCOP completely blocked memory for novel objects. It is concluded that degradation of SCOP by calpain contributes to activation of MAPK during memory formation (Shimizu, 2007).
Cadherin-mediated interactions are integral to synapse formation and potentiation. N-cadherin is required for memory formation and regulation of a subset of underlying biochemical processes. N-cadherin antagonistic peptide containing the His-Ala-Val motif (HAV-N) transiently disrupted hippocampal N-cadherin dimerization and impaired the formation of long-term contextual fear memory while sparing short-term memory, retrieval, and extinction. HAV-N impaired the learning-induced phosphorylation of a distinctive, cytoskeletally associated fraction of hippocampal Erk-1/2 and altered the distribution of IQGAP1, a scaffold protein linking cadherin-mediated cell adhesion to the cytoskeleton. This effect was accompanied by reduction of N-cadherin/IQGAP1/Erk-2 interactions. Similarly, in primary neuronal cultures, HAV-N prevented NMDA-induced dendritic Erk-1/2 phosphorylation and caused relocation of IQGAP1 from dendritic spines into the shafts. The data suggest that the newly identified role of hippocampal N-cadherin in memory consolidation may be mediated, at least in part, by cytoskeletal IQGAP1/Erk signaling (Schrick, 2007).
Enduring forms of synaptic plasticity and memory require new protein synthesis, but little is known about the underlying regulatory mechanisms. The role of MAPK signaling in these processes has been investigated. Conditional expression of a dominant-negative form of MEK1 in the postnatal murine forebrain inhibits ERK activation and causes selective deficits in hippocampal memory retention and the translation-dependent, transcription-independent phase of hippocampal L-LTP. In hippocampal neurons, ERK inhibition blocks neuronal activity-induced translation as well as phosphorylation of the translation factors eIF4E, 4EBP1, and ribosomal protein S6. Correspondingly, protein synthesis and translation factor phosphorylation induced in control hippocampal slices by L-LTP-generating tetanization are significantly reduced in mutant slices. Translation factor phosphorylation induced in the control hippocampus by memory formation is similarly diminished in the mutant hippocampus. These results suggest a crucial role for translational control by MAPK signaling in long-lasting forms of synaptic plasticity and memory (Kelleher, 2004).
Translation of eukaryotic mRNAs is primarily regulated at the level of initiation. Studies in mitotic cells have defined 5' cap recognition and ribosomal recruitment by translation initiation factors as key events in this multistep process. Cap recognition is accomplished by eukaryotic initiation factor 4E (eIF4E), and the eIF4E-associated factor eIF4G then recruits the 40S ribosomal subunit. Cap-dependent translation accounts for the synthesis of the vast majority of cellular proteins, since all mRNAs transcribed in the nucleus bear a 5' cap. The synthesis of the translation machinery itself is additionally regulated by an inhibitory cis-acting element, termed a 5' terminal oligopyrimidine tract (5' TOP), which occurs adjacent to the cap in mRNAs encoding ribosomal proteins and translation factors (Meyuhas, 2000).
Prior work in mitotic cells has identified the ERK-dependent kinase Mnk1 as the major eIF4E kinase, indicating a dominant role for ERK signaling in eIF4E phosphorylation. The complex pattern of inducible 4E-BP1 hyperphosphorylation appears to be mediated primarily by rapamycin-sensitive mTOR-dependent pathways, while some evidence has suggested ERK-dependent modulation of Ser65 phosphorylation. Studies on the mitogen-induced hyperphosphorylation of S6 have delineated a central role for mTOR-dependent activation of S6 kinase. The current findings demonstrate a major role for the ERK pathway in the neuronal activity-induced phosphorylation of S6, eIF4E, and 4E-BP1, with consistently greater effects on eIF4E relative to S6 across all levels of analysis. Interestingly, the phosphorylation of all three factors was also found to be highly sensitive to rapamycin, with the greatest effect on S6. These observations suggest that the ERK and mTOR pathways cooperate in the coordinate regulation of cap-dependent and 5' TOP-dependent translation. Hippocampal L-LTP and serotonin-induced LTF in Aplysia have been shown to be sensitive to rapamycin, implicating mTOR-dependent translation in these processes. Translational efficiency during the establishment of long-term synaptic plasticity and memory may therefore be determined through the functional interplay of ERK- and mTOR-dependent signaling mechanisms. General translational induction of a broad range of neuronal mRNAs by such activity-dependent mechanisms may provide the protein products required for the input-specific 'capture' of long-term synaptic plasticity by 'tagged' synapses (Meyuhas, 2000).
The cAMP and ERK/MAP kinase (MAPK) signal transduction pathways are critical for hippocampus-dependent memory, a process that depends on CREB-mediated transcription. However, the extent of crosstalk between these pathways and the downstream CREB kinase activated during memory formation has not been elucidated. This study reports that PKA, MAPK, and MSK1, a CREB kinase, are coactivated in a subset of hippocampal CA1 pyramidal neurons following contextual fear conditioning. Activation of PKA, MAPK, MSK1, and CREB is absolutely dependent on Ca2+-stimulated adenylyl cyclase activity. It is concluded that adenylyl cyclase activity supports the activation of MAPK, and that MSK1 is the major CREB kinase activated during training for contextual memory (Sindreu, 2007).
One of the major objectives of this study was to identify which MAPK-activated CREB kinase is stimulated during memory formation. Furthermore, it was important to define the relationship between MAPK and cAMP signaling following training for contextual fear conditioning, and to determine why Ca2+-stimulated adenylyl cyclase activity is required for contextual memory. There are several mechanisms by which cAMP could contribute to memory, including regulation of AMPA receptor trafficking and MAPK activation. No increased PKA phosphorylation of AMPA receptors was detected following contextual fear conditioning. Consequently, focus was placed on the role of cAMP signaling in MAPK activation because of the central role played by MAPK during memory formation. Confocal imaging was used to identify individual hippocampal cells in which PKA, MAPK, and CREB kinases are activated after contextual fear conditioning. It has not been previously shown that contextual fear conditioning activates PKA, nor was it known that PKA and MAPK are activated in the same neurons in the hippocampus. Furthermore, there was no evidence for activation of specific CREB kinases following fear conditioning (Sindreu, 2007).
Training for contextual memory caused a 5- to 6-fold increase in MAPK activation in approximately 10% of CA1 pyramidal neurons in two distinct intracellular pools: a nuclear pool and a postsynaptic pool. Furthermore, PKA was activated in the same subset of neurons as MAPK, and both showed increased nuclear activities after training. MAPK activation strongly correlated with activation of MSK1, a CREB kinase. Most importantly, the training-induced increases in MAPK, PKA, and MSK1 activities were ablated in mice lacking Ca2+-stimulated adenylyl cyclase activity. It is concluded that one of the major roles of cAMP signaling in memory is to support the activation and nuclear translocation of MAPK in CA1 pyramidal neurons (Sindreu, 2007).
Signal transduction pathways are usually implicated in memory formation because they are activated in specific areas of the brain by training and inhibition of the pathway blocks memory. For example, MAPK activity is stimulated in area CA1 following training for hippocampus-dependent memory, and administration of MEK inhibitors blocks both training-induced increases in MAPK and memory formation. Ca2+-stimulated adenylyl cyclase and PKA activities are required for memory formation, suggesting that either basal PKA activity is necessary or that an increment in PKA activity contributes to memory. Using an antibody that recognizes phosphorylated PKA substrates (pPKA-s), it was discovered that PKA is not only activated in area CA1 following contextual fear conditioning, but there is also a strong correlation between neurons showing MAPK activation and those in which PKA is activated. In keeping with this, increased nuclear levels of the PKA catalytic α subunit was observed in pERK+ neurons after training. The increase in pPKA-s was readily blocked by inhibitors of PKA and lost in mice lacking Ca2+-stimulated adenylyl cyclase activity, thus validating the use of the pPKA-s antibody to monitor PKA activation (Sindreu, 2007).
The observation that fear conditioning activates MAPK selectively in area CA1 agrees with other evidence that stimulation of transcription in this area of the hippocampus is particularly important for contextual memory formation. Much less was known, however, about the identity and size of the cellular population activated during training for contextual memory, and the intracellular compartments in which MAPK is stimulated. Although this analysis focused on the role of MAPK in the nucleus because of its importance for CREB-mediated transcription, MAPK was simultaneously activated in dendrites and at distal synapses following fear conditioning. It is noteworthy that MAPK regulates a number of other proteins, including dendritic K+ channels and glutamate receptors, and it may also control dendritic protein synthesis. Thus, the parallel activation of synaptodendritic and somatonuclear pools of MAPK supports the general hypothesis that memory formation depends on several MAPK-regulated events, including synaptic activity, dendritic protein synthesis, and transcription (Sindreu, 2007).
Although CREB-mediated transcription is necessary for memory formation and depends on MAPK signaling, the CREB kinase activated by MAPK following training for contextual memory was not certain. It was particularly interesting to determine if training for contextual fear activates RSK2 or MSK1 because studies using cultured neurons have implicated both kinases in CREB-mediated transcription through the phosphorylation of transcription factors and histones. This study discovered that fear conditioning activates MSK1, but not RSK2, in CA1 neurons, and that activation of MAPK and MSK1 is tightly correlated on a cell-by-cell basis. Furthermore, the activation of MSK1 induced by training is abrogated in mice lacking Ca2+-stimulated adenylyl cyclases or by post-training inhibition of MEK1/2. This strongly implicates MSK1 in MAPK-dependent CREB phosphorylation during formation of contextual memory. The identification of MSK1, and not RSK2, as the activated CREB kinase emphasizes that signaling mechanisms inferred from cultured neuron studies do not necessarily apply in vivo. Definitive evidence as to the relative importance of both CREB kinases during memory formation may come from the use of conditional mutant mice or novel MSK1 antagonists (Sindreu, 2007).
In summary, the data indicates that stimulation of MAPK in dendrites and the nucleus following training for contextual memory depends on Ca2+-stimulated adenylyl cyclase activity and leads to the activation of the CREB kinase MSK1. Furthermore, signaling elements for CREB-mediated transcription, starting with the initial cAMP signal, and including PKA, MAPK, MSK1, and CREB, are all activated in the same subset of neurons after training. It is concluded that one of the major roles of adenylyl cyclase activity in memory is to support the activation of MAPK, MSK1, and CREB in hippocampal neurons (Sindreu, 2007).
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