CaM, which is a ubiquitous protein, participates in many signalling pathways within all eukaryotic cells when bound to Ca2+. In CA1 hippocampal neurons, synaptic NMDAR activation causes a local, fast Ca2+ increase in dendritic spines allowing CaM to activate downstream effectors and resulting in LTP. In this study, we examined the function of Ng, a postysynaptic CaM-binding protein, in synaptic transmission and plasticity.
Here, we show that the ability of Ng to interact with CaM within the dendritic spines is rate limiting for synaptic potentiation and is required for LTP induction. We also show that Ng-mediated potentiation mimics LTP. The former conclusion is based on three main experimental observations. First, although Ng expression potentiates synaptic transmission, mutants of Ng that are incapable of CaM binding (Ng-IQless and Ng-SD) were not able to enhance synaptic transmission. Second, a mutant of Ng that is incapable of releasing CaM with the increase in Ca2+ concentration (Ng-SFAW) lacks the ability to potentiate synaptic transmission. Third, acute knockdown of Ng, which is essential for CaM targeting within the spine, blocks LTP induction.
The conclusion that Ng-mediated potentiation mimics LTP is supported by six main experimental observations. (1) It is activity dependent as evidenced by its blockade by TTX. (2) It is NMDAR dependent. (3) Ng expression results in CaMKII activation specifically at synaptosomes. (4) Ng expression results in GluR1 insertion into the synapses. (5) Ng-mediated insertion of GluR1 is dependent on CaM–CaMKII interaction, which results in CaMKII activation. (6) Ng-mediated potentiation occludes LTP induction.
CaM is an abundant protein that has been under intensive study, as it contributes to the activation of a diverse array of signalling cascades. Interestingly, many of these interactions reveal an apparent paradox of requiring CaM to activate opposing targets. For example, in hippocampal neurons, two Ca2+
/CaM-dependent enzymes are essential for the bidirectional balance between LTP and long-term depression (LTD). Ca2+
/CaM-dependent protein phosphatase calcineurin is required for LTD (Klee et al, 1979
; Hubbard and Klee, 1987
; Mulkey et al, 1994
; Torii et al, 1995
; Zeng et al, 2001
; Yasuda et al, 2003
). On the other hand, CaMKII is required for LTP (Miller and Kennedy, 1985
; Meyer et al, 1992
; Silva et al, 1992
; Giese et al, 1998
; Hudmon and Schulman, 2002
; Lisman et al, 2002
; Kennedy et al, 2005
; Shifman et al, 2006
). Therefore, it has been postulated that the cells may regulate CaM-mediated signalling through the regulation of the availability of its targets or CaM itself. There is experimental evidence suggesting that the cell can regulate CaM signalling through regulating local CaM pools (Toutenhoofd and Strehler, 2000
). Here, we propose that Ng spatially regulates the availability of CaM within the dendritic spine and thus favouring synaptic potentiation.
The requirement of Ng–CaM binding for Ng-mediated potentiation may suggest that Ng is enhancing transmission through a concomitant increase in the overall levels of CaM within the spine, thus increasing the sensitivity of dendritic spines to local Ca2+
changes. However, overexpression of CaM does not potentiate synaptic transmission. This is consistent with an earlier report showing that intracellular injection of 20 μM CaM did not change synaptic transmission (Wang and Kelly, 1995
). Given the high levels of CaM in neurons, it is possible that the exogenous CaM in both cases was not high enough in dendritic spines to produce synaptic potentiation. However, the same concentration of CaM (20 μM) was able to produce potentiation when co-injected with 80 μM Ca2+
(Wang and Kelly, 1995
), suggesting that the lack of effect on synaptic transmission when CaM was injected alone is unlikely to be due to the lack of enough exogenous CaM. Taken together, it is possible that Ng may be targeting CaM within the spine. Indeed, the ultrastructural localization of Ng shows that it is not randomly distributed and it is mainly localized close to the plasma membrane. This spatial localization may allow for preferential activation of targets necessary for LTP induction (e.g. CaMKII). On the other hand, an overall increase in CaM levels may not change the balance in the activities of the Ca2+
/CaM-dependent enzymes that are essential in determining the synaptic plasticity balance (e.g. CaMKII and calcineurin). Therefore, we propose that changing Ng levels within the spine may provide a tool to spatially regulate the preferential localization of CaM within the spine and thus change subsequent signalling. Interestingly, there is a close correlation between Ng levels, calcineurin, and CaMKII, that is low Ng levels are correlated with high calcineurin and low CaMKII activity (Krazem et al, 2003a
; Alzoubi et al, 2005
; Norris et al, 2005
). Thus, a decrease in Ng in the spine may decrease CaM localized close to/at the plasma membrane within the dendritic spine and shifting the balance towards easier activation of calcineurin at the expense of CaMKII. On the other hand, increasing Ng at the dendritic spine shifts CaM localization close to the plasma membrane allowing higher localized concentration of CaM and enhancing the chance of CaMKII activation, whose affinity is several folds lower than that of calcineurin towards Ca2+
/CaM (Miller and Kennedy, 1985
; Hubbard and Klee, 1987
; Meyer et al, 1992
; Hudmon and Schulman, 2002
). Further studies are warranted to explore these possibilities. Moreover, such targeting of CaM could happen in one of two ways: Ng may be recruiting more CaM into the spine, thus concentrating CaM within the dendritic spines. Alternatively, Ng may be redistributing CaM within the spine and targeting it close to or at the plasma membrane.
The findings that Ng-mediated potentiation is dependent on neuronal activity, NMDAR, and CaM binding suggest that Ng may act as a sensor to the Ca2+
signal, and increasing its levels within the spine may enhance the spine sensitivity. Thus, we have originally hypothesized that for Ng to be an effective sensor of local Ca2+
changes arising from NMDAR activation, its localization within the spine might be at or directly below the PSD. However, our immuno-EM data reveal a surprising distribution in which Ng is mainly localized extrasynaptically at the plasma membrane adjacent to, but not at, the PSD (Supplementary Figure 6B
). This may suggest a functional role of extrasynaptic NMDARs in LTP induction. Interestingly, it has been suggested that extrasynaptic NMDARs can be activated after synaptic release (Hires et al, 2008
). Further studies are needed to dissect out the function of extrasynaptic NMDARs in synaptic plasticity.
Two independent loss-of-function studies of Ng in mice argue for an important function of Ng in LTP induction (Pak et al, 2000
; Krucker et al, 2002
). However, these studies gave opposite results. One study showed an enhanced LTP by high-frequency stimuli, although the other knockout mice showed deficits in LTP induction. In both studies, the CaM-binding domain was completely deleted. The conflicting results with the Ng knockout studies may reflect different developmental problems and global changes in calcium buffering in neurons. For example, a computational study strongly suggests that the lack of Ng increases the probabilities of all Ca2+
/CaM-dependent enzymes to be activated at low Ca2+
concentration (Kubota et al, 2007
). Indeed, chronically eliminating Ng resulted in global changes in the activity of several enzymes and substrates (Wu et al, 2002
). In this study, we have combined acute knockdown of Ng using siRNA and overexpression techniques to elucidate the role of Ng in synaptic function and plasticity. Our results indicate that Ng is required for LTP induction, and sufficient to produce potentiation that mimics LTP. Our results also show that Ng–CaM interaction, although critical for Ng-mediated potentiation, is not essential in maintaining synaptic transmission.
Our data support a model in which Ng targets CaM within the dendritic spine, and acts as a sensor to local Ca2+
changes. Under normal conditions, overnight spontaneous activity is not sufficient to produce synaptic potentiation. However, there is enough targeted CaM to respond to the high increase in the local Ca2+
induced by the LTP induction protocols, resulting in potentiation (see for illustration). In the absence of Ng, however, the same induction protocols are not able to induce LTP, as there is not enough CaM targeted (spatially regulated) within the spine to allow the proper activation of subsequent targets necessary for LTP induction. In cases in which there is increased local Ng in the spine, more CaM is targeted enhancing the spine sensitivity to spontaneous overnight activity. Under these circumstances, overnight activity is sufficient to produce potentiation that mimics LTP. This model focuses on the Ng–CaM interaction, which is clearly required for Ng-mediated effects. However, this does not exclude the importance or synergism of other possible CaM-independent effects of Ng. For example, earlier studies suggest that Ng can influence the free Ca2+
concentration (Krucker et al, 2002
; Huang et al, 2004
; Kubota et al, 2008
). Further studies are warranted to test whether other mechanisms are involved in Ng-mediated potentiation in synaptic transmission.
Figure 9 Ng enhances the sensitivity of the postsynaptic terminal. (A) Under normal condition, there is enough targeted CaM that LTP induction protocols result in an increase in the synaptic strength. (B) The lack of Ng, however, results in the loss of the spatial (more ...)
This study also raises more questions regarding the details of Ng function in neurons. First, it is not clear how Ng concentrates in the dendritic spines. Earlier studies show that Ng concentrates in the dendritic spines (Watson et al, 1992
; Neuner-Jehle et al, 1996
). Moreover, an elegant computational study has also supported such a need for a higher concentration in the spines to be able to induce LTP (Zhabotinsky et al, 2006
). As mRNA for Ng does exist locally at the dendrites and several studies have supported local protein synthesis on demand, for example local CaMKII synthesis after NMDAR activation (Scheetz et al, 2000
; Aakalu et al, 2001
), the concentration of Ng at the spine could be due to local synthesis. However, GFP-tagged Ng is also concentrated in the dendritic spines relative to the dendrites (Supplementary Figure 7
). As the recombinant GFP-tagged Ng lacked the signal for the RNA to traffic to the dendrite, it is likely that Ng is anchored or retained within spines by an anchoring protein. Further studies are needed to elucidate the identity of such anchoring protein.
Once in the dendritic spine, Ng awaits for the Ca2+
increase, which is enough to release CaM to activate subsequent targets. What then is the function of Ng phosphorylation? Through its IQ motif, Ng binds to CaM (Deloulme et al, 1991
; Gerendasy et al, 1994b
). The phosphorylation of the serine residue (S36) within this IQ motif prevents Ng binding to CaM (Huang et al, 1993
). Thus, it has been hypothesized that Ng phosphorylation is the event that controls CaM availability, as phosphorylated Ng does not bind to CaM. Therefore, on Ng phosphorylation, CaM is released and the synapse can be potentiated. This hypothesis has to be revisited.
As mentioned above, the increase in the local Ca2+
is the first event. Thus, it is most likely that CaM will be released from Ng even before Ng phosphorylation. Interestingly, a recent study showed that Ng binds to phosphatidic acid (PA) in a phosphorylation-dependent manner, that is Ng binds to PA only when it is unphosphorylated (Dominguez-Gonzalez et al, 2007
). This may explain, at least partly, our finding that Ng accumulates near the plasma membrane. It is not unreasonable then to hypothesize that, although the local increase in Ca2+
triggers CaM release, the Ng phosphorylation event is important to recycle Ng to and from the plasma membrane. Further studies are needed to elucidate the function of Ng phosphorylation in synapses.
A third question that surfaces from this study is what function Ng has in synaptic plasticity as well as learning and memory deficits associated with ageing, Alzheimer's disease, schizophrenia, or hypothyroidism. Ng knockout mice exhibit spatial memory deficits. Moreover, low Ng levels are correlated with poor performance in the Morris water maze (Huang et al, 2004
). In addition, many conditions that are associated with memory and synaptic plasticity deficits are also accompanied with decreased levels of Ng in pyramidal neurons, for example ageing and hypothyroidism (Iniguez et al, 1993
; Piosik et al, 1995
; Chang et al, 1997
; Zoeller et al, 2000
; Mons et al, 2001
). Thus, there is a close correlation between decreased Ng levels and synaptic plasticity and memory deficits. Interestingly, a recent study shows that Ng is one of the major variants that correlates with schizophrenia, which may explain the cognitive deficits associated with schizophrenia (Stefansson et al, 2009
). In instances in which a condition has been corrected (e.g. hypothyroidism by levothyroxin replacement therapy), Ng level and synaptic plasticity were normalized (Alzoubi et al, 2005
). Nonetheless, agents that increase Ng levels (e.g. vitamin A) were capable of partially alleviating the ageing-induced deficits in synaptic plasticity and memory (Etchamendy et al, 2001
). Our data also show that overexpression of Ng enhances synaptic transmission in an LTP-like manner, whereas knocking it down blocks LTP. Taken together, it is thus possible that reduced Ng levels may be having a function in the induced synaptic plasticity deficits in ageing and disease. Further studies will be needed to investigate any possible function of Ng in the induced plasticity deficits.
In conclusion, our results shed light into the important function of Ng in LTP induction and provide a direct link between CaM targeting within the dendritic spines, through Ng, and the postsynaptic potentiation.