We have studied the role of Shank3 in synapse function to better understand the pathogenesis of the neurological symptoms of patients affected by Phelan-McDermid syndrome (2
). For this purpose, we used a specific shRNA to knockdown the expression of the major splice variants of Shank3 in cultured murine neurons. Our immunoblotting study using two different anti-Shank3 antibodies, one against the N terminus and one against the C terminus, indicated that, at least in neuronal cultures, Shank3 shRNA knocked down all the major Shank3-specific bands. As Wang et al.
) recently described six intragenic promoters that could potentially generate multiple splicing variants coding for several isoforms of Shank3, we cannot exclude that some minor splice variants remain at undetectable levels beyond shSank3 inhibition.
Although the proteins encoded by these three SHANK genes are structurally similar, some evidence suggests that they differ in function, in synapse-targeting properties, and in binding partners. For example, the overexpression of Shank1 induces maturation of dendritic spines without increasing their numbers, whereas the overexpression of Shank3 induces the formation of new synapses and dendritic spines (1
). Shank1 targeting to synapses is dependent on the PDZ domain, but the targeting of Shank2 and Shank3 depends on their C-terminal domain, including the sterile α motif domain (24
). Shank2 and Shank3 multimerize and form a platform or framework in the PSD that depends on Zn2+
binding to the sterile α motif domain (18
). In contrast, Shank1 does not bind Zn2+,
, but forms a large framework complex with Homer in the PSD (19
The specific function of Shank proteins in dendritic spines is probably related to the fact that all three protein variants bind directly to a number of proteins involved in actin remodeling, such as cortactin, Abp1, IRsP53, and SPIN90 (11
); they also interact indirectly with actin-remodeling proteins through binding Homer, oligophrenin, and CdC42 (45
). Available data suggest that Shank proteins functionally link glutamate receptors to the cytoskeleton, thereby regulating the size and dimensions of excitatory synapses and dendritic spines (47
Shank2 and -3 can also bind to Ab1 and LAPSER1, two proteins that translocate from the PSD to the nucleus in an activity-dependent manner and induce gene transcription and translation (48
). Finally, the fact that simple deletions of either SHANK3
, but not, up to now, SHANK1
, have been clearly implicated in the pathogenesis of mental retardation, autism and, more recently, schizophrenia, suggest that the three proteins may have different functions that cannot compensate for each other. Thus, it is important to note that we did not observe any type of compensation by the other SHANK member as neither Shank1 nor Shank2 expression increased upon Shank3 knockdown.
We found that knockdown of Shank3 specifically impaired mGluR5 signaling at synapses. In hippocampal neurons knocked down for Shank3, mGluR5 protein, but not its mRNA, is specifically reduced in the total lysate and in the synaptosomes, suggesting that Shank3 is somehow involved in mGluR5 protein stabilization. Previous work has shown that mGluR5 binds directly to Shank3 or indirectly through Homer cross-linking (12
). However, because we did not find any change in Homer expression, it is possible that the direct binding of Shank3 to mGluR5 is involved in this phenomenon. Both Shank3 and mGluR5 can be degraded by proteasomes following ubiquitination, suggesting that their interaction can reciprocally modulate their ubiquitination and stabilization (50
). However, we did not find any change in Shank3 protein expression in the mGluR5 knock-out mice.5
Thus, Shank3 might act as a stabilization platform for mGluR5. We also observed a reduction in cell surface expression of GluR1 in shShank3-treated neurons without a reduction in its protein expression. The reduction in GluR1 cell surface expression correlated with the reduced mEPSC frequency. The impaired DHPG-dependent LTD observed in shShank3-treated neurons did not result in any changes in cell surface expression of GluR1, which is down-regulated by DHPG in shCtrl-treated neurons. Our findings that CDPPB, an allosteric mGluR5 agonist, was able to rescue the mEPSC frequency in neurons knocked down for Shank3 suggest that Shank3 regulates AMPA receptor trafficking in an mGluR5-dependent manner. The reduction in cell surface GluR1 expression and in frequency of mEPSCs after knockdown of Shank3 might reflect impairment in activity-dependent synaptic recruitment of AMPA receptors at basal conditions.
Despite the observed reduction in mEPSC frequency and cell surface expression of GluR1-positive clusters in shShank3-treated neurons, our multielectrode recordings did not reveal significant changes in the spiking patterns of neurons under basal conditions. This is not surprising, because the connectivity between cultured neurons is highly redundant. Therefore, despite the differences in synaptic activity between control and Shank3-knockdown neurons in TTX-treated cultures, the composite postsynaptic potentials might well exceed the threshold for spike generation in these cells in the absence of TTX. Application of DHPG to cultured neurons led to a strong increase in the bursting rate, as previously reported for hippocampal slices (52
). Importantly, DHPG-induced up-regulation of bursting was reduced in Shank3-knockdown neurons. This result confirms the importance of Shank3 in the regulation of mGluR5-dependent signaling under physiological conditions, i.e.
in the absence of TTX. It also demonstrates the potential importance of Shank3 in mGluR5 activity-induced shaping of neural network activity.
In a recent analysis of the role of Shank3 mutations when overexpressed in hippocampal neurons, Durand et al.
) showed that all mutations analyzed modify Shank3 functions. Here, we have analyzed two of the mutations studied by Durand et al.
(their R12C corresponds to our R87C and their Shank3STOP
corresponds to our Shank3Ins). Both mutations were shown by Durand et al.
) to affect the ability of Shank3 proteins to increase the dimension of dendritic spines and modify synaptic properties. Interestingly, in our study, the expression of mutated forms of Shank3 that mimic the mutations found in autistic patients was not able to rescue DHPG-dependent ERK1/2 phosphorylation. Thus, reduction in Shank3 expression, which occurs in 22q13/Phelan-McDermid syndrome, and functional mutations in Shank3, which occur in some autistic patients, might both induce alterations in mGluR5 signaling at synapses.
Four recent studies highlight the importance of Shank3 at the molecular and behavioral levels (21
). Two of them showed that Shank3 heterozygous and homozygous male mice displayed abnormal social behavior, communication pattern, and learning and memory, as compared with wild-type littermate controls (21
). These studies revealed a strong impairment in basal synaptic transmission in CA3-CA1 connections, a reduction in GluR1 clusters and protein levels in the hippocampus, and an alteration in activity-dependent AMPAR synaptic plasticity (21
). However, the reduction in mEPSC amplitude and the “compensatory” increase in mEPSC frequency in Shank3 heterozygous mice reported by these authors were not seen in our experiments. Most importantly in this context, it should be noted that in our experiments, in contrast to the other studies, we knocked down all detectable Shank3 splice variants through shRNA treatment; this led to a suppression of Shank3 expression by 70–80% rather than by 50% as in Shank3 heterozygous mice. This could lead to such a strong reduction of mEPSC amplitudes that EPSCs would decrease below the detection limit, resulting in a reduction of mEPSC frequency, as we report here. Another difference is our observation that Shank3 plays a role in LTD induced by the mGluR type 1 agonist, DHPG, whereas Bozdagi and colleagues (54
) found that Shank3 heterozygous mice have a normal LTD induced by paired-pulse low-frequency stimulation. In view of our present study, this is not surprising considering the role of Shank3 in regulation of mGluR5 expression and considering that previous studies in mGluR5 knock-out mice have also shown normal paired-pulse low-frequency stimulation-induced LTD (55
), but impaired DHPG-induced LTD (56
Peça et al.
) have instead reported that mice genetically deleted of two major Shank3 splice variants exhibit self-injurious repetitive grooming and deficits in social interaction and these behavioral defects are caused by major alteration in the striatal synapses and cortico-striatal circuits, but not in the hippocampus. Thus, it is possible that the remaining Shank3 splice variant(s) might be sufficient to maintain normal synapse function and structure in the hippocampus. Paradoxically, the remaining Shank3 protein described by Bangash et al.
), which misses the C-terminal fragment, has a gain-of-function phenotype by reducing the NR1 subunit of NMDA receptors specifically at synapses, but not affecting synaptic AMPAR function and composition. Although this pathway should obviously also be investigated in vivo
, our data strongly suggest nevertheless that knocking down all the major Shank3 splice variants strongly affects the expression of mGluR5 receptor at the synapses.
The mGluR5 receptor plays a major role in synaptic plasticity (57
). It has been clearly demonstrated that antagonism or genetic deletion of mGluR5 impairs both acquisition and extinction of hippocampal-dependent learning tasks, such as the radial arm maze and the Morris water maze, by impairing both the late phase of hippocampal long term potentiation and mGluR-dependent LTD (58
). The occurrence of mGluR-dependent LTD in CA1 relies on activation of both ERK and PI3K-mTOR pathways (36
). A role for mGluR-LTD has been demonstrated for the formation of object recognition memory (62
The existence of a link between mGluR-LTD and cognitive disease is suggested by the finding that both hippocampal and cerebellar mGluR-LTD are altered in fragile X syndrome, a mouse model of mental retardation and autism that has led to the development of novel therapeutics for this syndrome that act on mGluR5 (64
). In contrast to our finding in Shank3-knockdown neurons, mGluR-LTD is enhanced in the fragile X syndrome mouse model (65
). This enhancement occurs because, in the absence of fragile X mental retardation protein, as in fragile X syndrome, there is a loss of steady-state translational suppression that leads to increased protein levels of fragile X mental retardation protein targeting specific mRNAs, such as those coding for activity-regulated cytoskeleton-associated protein that may enhance the magnitude of LTD (66
Notably, the use of mGluR5 antagonists or genetic reduction of mGluR5 (in mice that are heterozygous for mGluR5) can reverse multiple phenotypes in mice deficient in FMR1
, a gene encoding the fragile X mental retardation protein; these phenotypes include increased dendritic spine density and deficits in experience-dependent plasticity in the visual cortex and hippocampal-dependent learning (67
Based on this finding, we tested whether the reduced mGluR5 activity in Shank3-knockdown neurons can be rescued by an allosteric agonist of mGluR5, such as CDPPB (69
), and found that both ERK1/2 phosphorylation and mEPSC frequency were rescued by overnight treatment with CDPPB. CDPPB has been shown to be brain-penetrant and to reverse amphetamine-induced locomotor activity and amphetamine-induced deficits in prepulse inhibition in rats (70
), two models that are sensitive to antipsychotic drug treatment. These results demonstrate that positive allosteric modulation of mGluR5 produces behavioral effects and suggest that mGluR5 activity could be envisaged as a potential therapeutic target. Therefore, these findings open new possibilities for the pharmacological treatment of patients affected by Shank3 gene deletion and mutation.