An alternate cAMP pathway Epac promotes hippocampal long‐term depression
Year: 2009
Type: article
Abstract: The CA1 region of the hippocampus is a model system for the study of long-term synaptic plasticity, which is thought to play an important role in learning and memory. Specifically, studies of long-term potentiation (LTP) and long-term depression (LTD) have provided many insights into the molecular processes that govern long-term synaptic modifcations. While many signalling molecules have been implicated in the regulation of synaptic plasticity, the cyclic-adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) has proven to play a vital role (Nguyen & Woo, 2003). In particular, PKA enhances LTP through downstream phosphorylation events and gene regulation (Nguyen & Woo, 2003). Conversely, PKA activation inhibits LTD (Nguyen & Woo, 2003). Recently, two proteins were discovered that are directly activated by cAMP independent of PKA, aptly named exchange protein directly activated by cAMP (Epac) (Bos, 2006). Both Epac1 and Epac2 belong to a larger family of guanine exchange factor proteins that catalyse the activation of small G-proteins, specifically Rap1 and Rap2 (Bos, 2006). Interestingly, recent studies provide evidence that Epac and PKA can produce synergistic, and/or opposing effects within the same system (Cheng et al. 2008). What this means to researchers interested in the field of cAMP signalling pathways is that we must now re-evaluate much of the earlier seminal cAMP studies which were completed prior to the discovery of Epacs and/or the advent of specific pharmacological tools to differentiate Epac and PKA pathways. Ster and colleagues recently published an intriguing paper in The Journal of Physiology which addressed this issue by examining the involvement of this alternate cAMP pathway in synaptic plasticity (Ster et al. 2009). Several forms of LTD exist that produce long-term depression in synaptic strength including: (1) low-frequency stimulation (LFS-LTD) which functions through activation of N-methyl-d-aspartate (NMDA) receptors (Dudek & Bear, 1992) and (2) (R,S)3,5-dihydroxyphenylglycine (DHPG-LTD) which functions through activation of group I metabotropic glutamate receptors (Palmer et al. 1997). The current study revealed that Epac activation induces a new form of hippocampal LTD, unlike LFS-LTD or DHPG-LTD. The goals of this study were to: (1) establish whether Epac signalling contributes to LTD, (2) examine the underlying signalling pathway(s) responsible, to define Epac-LTD, and (3) compare Epac-LTD with the other known forms of LTD. To begin, the authors administer the Epac agonist 8-(4-chloropheynylthio)-2′-O-methyladenosine-3′-5′-cylcic monophosphate (8-pCPT) and measure the CA1 field excitatory post-synaptic potential (fEPSP). Surprisingly, and in stark contrast to the effects of PKA signalling (Nguyen & Woo, 2003), the authors demonstrate that 8-pCPT produced a significant reduction in CA1 synaptic responses compared to the pre-drug response (Ster et al. 2009). Pre-treatment with brefeldin-A (BFA), a proposed inhibitor of Epac, blocked Epac-mediated LTD (Ster et al. 2009). While the authors point out BFA is not a specific Epac antagonist, it should again be noted that this drug has multiple effects including inhibition of vesicular traffic from the Golgi apparatus, thus caution and proper controls should be used when applying this drug. Collectively these results provide evidence that Epac activation results in LTD among CA1 excitatory synapses. The next goal in this paper was to determine the signalling pathway responsible for producing Epac-LTD. Multiple mechanisms/signalling proteins have been implicated in producing LTD. A common feature in hippocampal plasticity is the trafficking of post-synaptic AMPA (α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate) receptors into or out of excitatory synapses during LTP or LTD, respectively. Using a specific peptide which blocks the internalization of post-synaptic AMPA receptors, the authors demonstrate, as a positive control, that the AMPA internalization process is required for LFS-LTD (Ster et al. 2009). Using this same peptide in conjunction with 8-pCPT treatment revealed that Epac-LTD is also dependent upon this internalization process (Ster et al. 2009). Rap, a target for Epac, was previously demonstrated to remove AMPA receptors through activation of p38-MAPK (mitogen-activated protein kinase) (Zhu et al. 2002). To determine whether the Rap–p38-MAPK signalling pathway was involved in Epac-LTD, the authors tested specific inhibitors of Rap and p38-MAPK. Pre-treatment with the Rap inhibitor in conjunction with 8-pCPT, blocked Epac-LTD (Ster et al. 2009). Likewise, blocking p38-MAPK inhibits Epac-LTD (Ster et al. 2009). Collectively, these results indicate that the Rap–p38-MAPK signalling pathway and AMPA receptor internalization are necessary for the establishment of Epac-LTD. However, the authors do not provide a definitive connection between Epac–Rap–p38-MAPK signalling and AMPA receptor internalization. An additional experiment to clearly demonstrate this would have been co-application of 8-pCPT along with inhibitors of Rap and/or p38-MAPK followed by quantification of post-synaptic AMPA receptors. Multiple forms of LTD are currently known to exist including LFS-LTD and DHPG-LTD. As the authors point out, p38-MAPK signalling was previously implicated in both of these forms of LTD. In the current study (Ster et al. 2009), the authors demonstrate that inhibiting p38-MAPK does not affect LFS-LTD or DHPG-LTD, providing evidence that Epac-LTD differs from the two known forms of LTD. To further test the difference between Epac-LTD and LFS- and DHPG-LTD, the authors use occlusion experiments. In these experiments fEPSP measurements were compared after saturation of LFS-LTD or full induction of DHPG-LTD followed by application of 8-pCPT, which resulted in no difference in the size of fEPSPs. These results indicate a common mechanism between Epac-LTD and LFS- and DHPG-LTD downstream of p38-MAPK. Though Epac-LTD purportedly shares a common downstream mechanism with LFS- and DHPG-LTD, the upstream target must differ. As previously mentioned, LFS-and DHPG-LTD are produced through the NMDA receptor and metabotropic glutamate receptor-1, respectively. Application of a cAMP analogue, which would activate both PKA and Epac results in inhibition of LTD (Kameyama et al. 1998). Thus, the receptor must only activate a pool of cAMP restricted to Epac access. The authors indicate that pituitary adenylate cyclase activating peptide receptors are putative receptors for Epac-LTD, since previous studies indicate PACAP activation produces LTD independent of PKA (Kondo et al. 1997). Initial experiments demonstrated that PACAP alone induces LTD (Ster et al. 2009), consistent with previous reports (Kondo et al. 1997). PACAP induction of LTD was inhibited by the Epac inhibitor BFA (Ster et al. 2009). Likewise, inhibition of Rap blocked PACAP-mediated LTD (Ster et al. 2009). Thus, to answer the question of whether PACAP is a potential activator of Epac, occlusion experiments were conducted. In these experiments, saturation of LTD was established using two separate treatments of PACAP followed by Epac or vice versa, followed by testing ratios of changes in the fEPSPs of CA1 excitatory synapses. Following saturation of LTD using Epac, Epac-LTD occluded PACAP-LTD (Ster et al. 2009). Similarly, saturated PACAP-LTD occluded Epac-LTD (Ster et al. 2009), both indicating a common LTD mechanism. Collectively these results provide evidence that Epac-LTD is produced through PACAP receptor activation. This paper brings up a question which is of great importance to much of the neuroscience community. As cAMP itself is a ubiquitous second messenger, how can it discern Epac vs PKA activation? This is of importance since both pathways can be stimulated by increased cAMP and depending on the target could have functionally different outcomes. The authors address this by reporting that Epac may have less affinity for cAMP than does PKA; however, this topic is still highly debated. Furthermore, they postulate that Epac activation was purportedly a result of PACAP stimulation which might result in more cAMP availability for Epac rather than PKA. Recently, one proposed mechanism has been reported which indicates that differential targeting is due to compartmentalization of cAMP by phosphodiesterases, proteins responsible for cAMP degradation (Huston et al. 2008). Ster and colleagues set out to determine the effect of Epac on synaptic plasticity and have demonstrated unequivocally, in a set of well-designed experiments, that a previously uncharacterized LTD pathway exists (Ster et al. 2009). As current scientists continue to unravel the molecular milieu of cAMP signalling, this paper and others like it, lay the foundation for proper investigation which will need to take into account the role of both PKA and Epac. We greatly appreciate support from NIH grants R01EY017594. RR016481 and P30ES014443. We would also like to thank Dr Nigel G. F. Cooper and Dr Martha E. Bickford for comments on a previous draft of this manuscript.
Source: The Journal of Physiology
Authors: Christopher M. Whitaker, Haiyang Wei
Institution: University of Louisville
Language: English
Subfield: Cellular and Molecular Neuroscience
Field: Neuroscience
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W1987699183