Loss of synaptopodin impairs mGluR5 and protein synthesis–dependent mGluR-LTD at CA3-CA1 synapses
Department of Pharmacology and Therapeutics, McGill University, Montreal, QC H3G 1Y6, Canada
Department of Pharmacology and Therapeutics, McGill University, Montreal, QC H3G 1Y6, Canada
Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, New York, NY 10461, USA
Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, New York, NY 10461, USA
Department of Pharmacology and Therapeutics, McGill University, Montreal, QC H3G 1Y6, Canada
Department of Pharmacology and Therapeutics, McGill University, Montreal, QC H3G 1Y6, Canada
Abstract
Metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) is an important form of synaptic plasticity that occurs in many regions of the central nervous system and is the underlying mechanism for several learning paradigms. In the hippocampus, mGluR-LTD is manifested by the weakening of synaptic transmission and elimination of dendritic spines. Interestingly, not all spines respond or undergo plasticity equally in response to mGluR-LTD. A subset of dendritic spines containing synaptopodin (SP), an actin-associated protein is critical for mGluR-LTD and protects spines from elimination through mGluR1 activity. The precise cellular function of SP is still enigmatic and it is still unclear how SP contributes to the functional aspect of mGluR-LTD despite its modulation of the structural plasticity. In this study, we show that the lack of SP impairs mGluR-LTD by negatively affecting the mGluR5-dependent activity. Such impairment of mGluR5 activity is accompanied by a significant decrease of surface mGluR5 level in SP knockout (SPKO) mice. Intriguingly, the remaining mGluR-LTD becomes a protein synthesis-independent process in the SPKO and is mediated instead by endocannabinoid signaling. These data indicate that the postsynaptic protein SP can regulate the locus of expression of mGluR-LTD and provide insight into our understanding of spine/synapse-specific plasticity.
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Keywords: synaptopodin, synaptic plasticity, hippocampus, mGluR-LTD, protein synthesis
Article notes
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Received 2023 Sep 9; Accepted 2024 Feb 1; Collection date 2024 Feb.
Boxed Text
Hippocampal group I metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD), a form of learning and memory, is misregulated in many murine models of neurodevelopmental disorders. Despite extensive studies, there is a paucity of information on the molecular mechanism underlying mGluR-LTD. Previously, we reported that loss of synaptopodin (SP), an actin-associated protein found in a subset of mature dendritic spines, impairs mGluR-LTD. In the current study, we uncover the molecular and cellular deficits involved. We find that SP is required for the mGluR5–Homer interaction and uncover SP as a molecular switch for mGluR-LTD expression, as mGluR-LTD becomes protein synthesis-independent and relies on endocannabinoid signaling in SP knockout. This work provides insight into SP as a gatekeeper to regulate mGluR-LTD at hippocampal synapses.
Introduction
Synaptic plasticity, characterized by long-term potentiation (LTP) and long-term depression (LTD), is a major form of plasticity in the brain that takes place at the level of individual synapses and is thought to be an underlying mechanism for learning and memory formation (1, 2). Dendritic spines, which make up the postsynaptic component of the synapse, are the primary loci of synaptic changes during plasticity. Dendritic spines can modify their molecular composition to alter synaptic activity and undergo long-term changes in their morphology based on the pattern and the strength of synaptic transmission (3). Specifically, LTD is known to induce a decrease in synaptic transmission, leading to the weakening or the elimination of synapses that are, respectively, associated with the shrinkage and the removal of the dendritic spines (4, 5). At hippocampal Schaffer collateral-CA1 (Sc-CA1) synapses, the most well-studied form of LTD is NMDAR-LTD, which is typically induced by NMDAR activity through low-frequency stimulation (4, 6–8). Another distinct form of LTD (mGluR-LTD) that requires the activation of group 1 metabotropic glutamate receptors, mGluR1 and mGluR5 was later discovered. It was found to be an important form of synaptic plasticity as it is altered in many neurological disorders such as Fragile X syndrome (FXS) and Alzheimer's disease (9). Unlike NMDAR-LTD, hippocampal mGluR-LTD in mature animals depends on rapid de novo protein synthesis occurring in dendritic spines, astrocyte release of adenosine triphosphate (10, 11) and in some cases, presynaptic mechanisms including retrograde endocannabinoid (eCB) signaling (12–14). Interestingly, how dendritic spines respond to mGluR-LTD is controversial as some report spine loss during mGluR-LTD while others do not (15, 16). This differential expression of plasticity suggests that the spines are heterogeneous and opens up the question of how plasticity-responsive spines are different from inert spines. Recently, studies on the postsynaptic protein synaptopodin (SP), an actin-associated protein, have begun providing insight into spine-specific plasticity (17–20). SP is found specifically in a subset of mature dendritic spines and is responsible for the formation of the spine apparatus (SA), a specialized smooth endoplasmic reticulum (sER) derived organelle believed to serve as an intracellular Ca2+ reservoir in spines (19, 21). Although the exact function of SP in neurons remains largely enigmatic, it has been shown to play an indispensable role in different forms of synaptic plasticity and homeostatic plasticity (22–25). Previously, Holbro et al. have shown the spine-specific expression of mGluR-LTD being dependent on the presence of sER in spines, suggesting perhaps the involvement of SP and SA (26). Subsequently, we demonstrated that SP is indeed involved in mGluR-LTD. We have shown that during mGluR-LTD, spines containing SP are protected from elimination or shrinkage, while the spines lacking SP are lost. Moreover, in SP knockout (SPKO) mice, mGluR-LTD no longer induces spine loss, suggesting that SP is required for mGluR-LTD-induced structural plasticity (27). Although mGluR-LTD affects structural plasticity in a spine-specific and SP-dependent manner, it is unknown how mGluR-LTD-dependent functional plasticity is affected by SP in dendritic spines.
Here, to better understand the mechanisms underlying the mGluR-LTD deficit in SPKO at Sc-CA1 synapses, we assessed mGluR1 and mGluR5 activity individually in wild-type (WT) and SPKO mice and found that mGluR5 activity is impaired in the SPKO. We found that mGluR5 surface expression is reduced in SPKO mice due to decreased mGluR5 association with Homer, a major scaffolding protein for mGluR1/5. Moreover, we report that mGluR-LTD becomes protein synthesis-independent and relies instead on eCB signaling in the SPKO. These findings shed light on the function of SP in spines and during mGluR-LTD and reveal SP as a molecular switch that governs the mechanism of expression of mGluR-LTD.
Results
mGluR1 and mGluR5 differentially contribute to mGluR-LTD in absence of synaptopodin
At excitatory synapses, dendritic spines are the major locus for the expression of synaptic plasticity often manifested by alterations in both the functional and the structural properties of spines (3). Chemical activation of group 1 mGluRs with the agonist dihydroxyphenylglycine (DHPG) induces long-term synaptic depression at Schaffer collateral/Sc-CA1 synapses (mGluR-LTD) that is accompanied by the selective loss of spines that lack SP. To better understand the impact of SP on functional synaptic weakening, we induced LTD by brief application of DHPG (S-DHPG; 100 μM, 5 min) and recorded field potentials (fEPSPs) at Sc-CA1 synapses in acute slices from WT and SPKO mice (Fig. 1A). In WT, DHPG rapidly induced robust long-lasting depression of evoked fEPSPs measured 40 min after the end of DHPG application (59.1 ± 4.8% of baseline, n = 11; Fig. 1A). In contrast, in SPKO mice the DHPG-induced LTD was strongly weakened (76.5 ± 4.4% of baseline, n = 10; Figs. 1B and S1), in confirmation of our previous findings (27). Both mGluR1 and mGluR5 are known to contribute to the induction of mGluR-LTD at Sc-CA1 synapses. Both receptors are expressed in the hippocampus, both coupled to Gαq/11 to signal to the canonical phospholipase C (PLC)/IP3 pathway and have been also proposed to form heterodimers (28). However, although the receptors have been generally regarded as interchangeable and able to compensate for each other's activity, there is evidence that mGluR1 and mGluR5 can differ functionally. mGluR1 is involved in intracellular calcium release and the maintenance phase of mGluR-LTD, while mGluR5 is involved in potassium current influx and the induction phase of mGluR-LTD (29, 30).
To begin exploring the mechanisms underlying weakened mGluR-LTD in the absence of SP, we examined the individual contribution of mGluR1 and mGluR5 to plasticity. To assess mGluR1 activity, we bath-applied the selective mGluR1 antagonist LY-367385 (100 µM) during the recording of LTD-induced by DHPG. In WT, inhibition of mGluR1 significantly decreased the magnitude of the depression and blocked mGluR-LTD expression (90.5 ± 5.7% of baseline, n = 8; Fig. 1A). In SPKO mice, despite mGluR-LTD being already strongly reduced (76.5 ± 4.4% of baseline, Fig. 1B), application of LY-367385 further decreased and fully blocked LTD (93.4 ± 3.7% of baseline, n = 8; Fig. 1B). To test whether the deficit in mGluR-LTD in SPKO mice could be due to reduced expression or mislocalization of mGluR1, we examined mGluR1 protein abundance and dendritic localization in the SPKO hippocampus. We found that both the total abundance of mGluR1 determined by Western blot (WT 100.0 ± 4.4%, SPKO 110.6 ± 12.0, n = 6 mice per group; Fig. 1C) and the density of immunolabeled mGluR1 puncta in dendrites of CA1 pyramidal neuron (WT 0.39 ± 0.03/µm n = 10, SPKO 0.40 ± 0.02/µm n = 10; Fig. 1D and E) were not significantly altered in absence of SP. Moreover, in both WT and SPKO mice, ∼15% of dendritic spines in CA1 pyramidal neurons similarly displayed mGluR1 expression (Fig. 1F). Collectively, these data show that the lack of SP does not alter significantly mGluR1 expression, localization and signaling in the hippocampus and demonstrate that mGluR1 activity is essential to support the residual mGluR-LTD in SPKO mice.
mGluR5 is abundantly expressed in the hippocampus and was shown to induce mGluR-LTD (30, 31). To test the contribution of mGluR5 activity to mGluR-LTD in the absence of SP, the mGluR5-specific antagonist 2-methyl-6-(phenylethynyl) pyridine (MPEP, 10 μM) was applied to hippocampal slices during mGluR-LTD recording. As previously shown, in WT mice the DHPG-induced LTD was reduced in the presence of MPEP although not blocked (78.0 ± 5.0% of baseline, n = 10; Fig. 2A). Surprisingly, we found that inhibition of mGluR5 activity did not alter the magnitude of mGluR-LTD in SPKO mice (75.2 ± 6.7% of baseline, n = 9; Fig 2B). It is worth noting that inhibition of mGluR5 in WT (78.0 ± 5.0%) results in LTD of a magnitude similar to control untreated SPKO mice (76.5 ± 4.4%). Since these findings suggested a potential impairment in mGluR5 activity in absence of SP, we next examined total mGluR5 protein expression by Western blot and found no significant difference in WT and SPKO hippocampus (Fig. 2C). Similarly, visualization of mGluR5 by immunolabeling of WT and SPKO hippocampal slices did not reveal detectable differences in the density of total mGluR5-positive puncta (comprising intracellular and surface receptors) in CA1 dendrites (Fig. 2D and E); congruent with this, the fraction of spines containing total mGluR5 was similar between genotypes (Fig. 2F). Altogether, these data indicate that although total mGluR5 expression is not significantly affected in SPKO mice, mGluR5 function is impaired and is no longer required for mGluR-LTD in absence of SP.
mGluR5 surface level is significantly decreased in absence of synaptopodin
We reasoned that despite its normal abundance, mGluR5 expression at the neuronal surface and/or coupling to downstream signaling pathways could be altered by the lack of SP. Thus, to measure the surface level of mGluR5 and mGluR1, we immunolabeled intact (nonpermeabilized) hippocampal slices with antibodies that specifically bind the extracellular domain of either receptor. We found that the mean fluorescence intensity of mGluR5 surface puncta was significantly reduced in the stratum radiatum of area CA1 in SPKO (6.8 ± 0.7 A.U) compared to WT mice (17.5 ± 1.4 A.U) (Fig. 3A and B), whereas mGluR1 fluorescence intensity was unchanged (WT: 6.5 ± 0.8, SPKO: 5.8 ± 0.6 A.U) (Fig. 3C and D). The decrease in surface mGluR5 in SPKO mice was further confirmed in individual CA1 pyramidal cells, as indicated by the reduced density of mGluR5 surface puncta along dendrites of green fluorescent protein (GFP)-labeled pyramidal neuron in the stratum radiatum of area CA1 (Fig. 3E and F).
The clustering and mobility of mGluR5 at the neuronal surface is dependent on interaction with the scaffold protein Homer (32), which also physically connects receptors present at the plasma membrane with intracellular IP3R and RyR located in the ER (33–35). It is well established that absence of SP causes loss of the SA and, therefore, loss of stable ER within spines (21). We reasoned that lack of recruitment of Homer proteins to stable spine ER in SPKO mice might preclude local interaction with mGluR5. To explore this possibility, we immunoprecipitated mGluR5 from brain lysates of WT and SPKO mice and assessed coprecipitation of Homer (long form) by Western blot. As expected, Homer readily coprecipitated with mGluR5 in the WT; in contrast, Homer coprecipitation with the receptor was significantly reduced in SPKO mice (Fig. 3G and H) in the absence of changes in total Homer abundance. Altogether, these results suggest that the lack of SP decreases the surface abundance of mGluR5 in pyramidal neurons via mechanism(s) involving impairment of the mGluR5–Homer association that normally promotes surface clustering of the receptors. We propose that deficits in mGluR5 surface expression and coupling to Homer underlie, in whole or in part, the failure of mGluR5 to contribute to mGluR-LTD in SPKO mice.
mGluR-LTD is protein synthesis-independent in absence of synaptopodin
In juvenile and adult hippocampus, mGluR-LTD normally relies on rapid local translation of a specific group of proteins, termed “LTD proteins,” that are involved in the endocytosis and trafficking of AMPA receptors (AMPARs) (13, 36). Blocking protein synthesis or eliminating specific “LTD proteins” results in severe impairment or abolishment of mGluR-LTD (13, 37). Protein synthesis can be directly induced by activation of group 1 mGluRs (38) and polyribosomes that are present in proximity to the SA (39, 40) that was also proposed to function as a satellite secretory station (41, 42). Since the SA is absent in mice that lack SP, we tested whether de novo protein synthesis could be impaired in SPKO mice. Stimulation of protein synthesis is regulated by the extracellular signal-regulated kinases (ERK1/2) and the mechanistic target of rapamycin (mTOR) pathways that are both activated by group 1 mGluRs (36). Thus we used western blot assays with lysates of hippocampal slices treated with or without DHPG (100 μM, 5 min) to examine activation of the ERK1/2 and mTOR pathways. DHPG significantly increased ERK1/2 phosphorylation at Thr202/Tyr204 in WT, congruent with previous reports that the ERK1/2 pathway is activated and required for mGluR-LTD (Fig. 4A). In SPKO, DHPG-induced ERK1/2 phosphorylation at a level comparable to WT (Fig. 4A) indicating that activation of the ERK1/2 pathway by DHPG is not affected in absence of SP. To assess the mTOR pathway, we measured phosphorylation of the ribosomal protein S6 (Ser235/236, Ser240/244), a downstream target of mTORC1 signaling. We found that DHPG treatment did not affect S6 phosphorylation in either WT or SPKO, suggesting that group 1 mGluR signaling to mTORC1 is unchanged (Fig. 4B).
The ERK1/2 and mTOR pathways are signaling cascades that regulate protein synthesis but their activities do not directly reflect actual de novo production of proteins. For a direct assessment of how the lack of SP might affect de novo protein synthesis during mGluR-LTD, we applied DHPG to hippocampal slices while perfusing anisomycin, a protein translation inhibitor, and recorded the fEPSPs. In WT, treatment with anisomycin blocked mGluR-LTD expression (89.0 ± 7.1% of baseline; Fig. 4C). These results are in line with reports that mGluR-LTD heavily relies on de novo protein synthesis (13). Since protein synthesis is required for mGluR-LTD, we anticipated that the residual mGluR-LTD in SPKO mice would also be blocked by anisomycin. To our surprise, in SPKO mice anisomycin did not block mGluR-LTD and in fact, the treatment had no effect on the fEPSPs compared to vehicle control (81.5 ± 3.9% of baseline; Fig. 4D) indicating that the residual mGluR-LTD in SPKO mice is protein synthesis-independent.
In absence of synaptopodin mGluR-LTD is mediated via an endocannabinoid-dependent mechanism
The mechanisms of expression of mGluR-LTD at Sc-CA1 synapses undergo a developmental switch (43). Whereas mGluR-LTD in the juvenile/adult hippocampus is mediated by postsynaptic induction of protein synthesis and production of “LTD” proteins promoting the removal of synaptic AMPARs (36), in neonatal animals mGluR-LTD is protein synthesis-independent and expressed through a decrease in presynaptic function (43, 44). As the residual mGluR-LTD in SPKO mice becomes protein synthesis-independent, a critical question to address is what mechanism(s) underlie functional synaptic weakening. In many brain regions, including hippocampus, eCB signaling can decrease neurotransmitter release at excitatory synapses and induce LTD (12). Endocannabinoids are produced postsynaptically in response to group 1 mGluR activation and act as retrograde signal back to the presynaptic terminal by binding to cannabinoid receptor type 1 (CB1) thereby causing synaptic depression (12, 45). To investigate whether residual mGluR-LTD in absence of SP might be produced by a presynaptic mechanism through eCB signaling, we tested the effect of the CB1 receptor inhibitor AM-251 on mGluR-LTD in SPKO mice. Surprisingly, inhibition of the CB1 receptor in SPKO hippocampal slices blocked mGluR-LTD-induced by DHPG (91.9 ± 4.2% of baseline; Fig. 5A). In contrast, application of AM-251 to WT slices did not significantly alter the magnitude of DHPG-induced LTD (66.7 ± 3.9% of baseline; Fig. 5B). Together, these findings support the notion that mGluR-LTD is normally expressed postsynaptically but loss of SP causes a switch in the locus of plasticity to the presynaptic side.
Discussion
DHPG-induced LTD at Sc-CA1 synapses is strongly reduced in absence of SP, an actin-binding protein enriched in a subset of dendritic spines (27). In this study, we identify alterations that occur at Sc-CA1 glutamatergic synapses in absence of SP and provide evidence of its crucial role in enabling postsynaptic expression and protein synthesis dependence of mGluR-LTD. We show that activation of mGluR1 is required for both robust LTD in WT and residual LTD in SPKO mice, whereas mGluR5 no longer contributes to LTD in absence of SP. In SPKO mice, mGluR5 surface expression is selectively reduced in CA1 pyramidal neurons and its association with Homer proteins is impaired. We further find that inhibition of de novo protein synthesis which blocks mGluR-LTD in the WT has no effect on mGluR-LTD in the absence of SP. Instead, in SPKO mice mGluR-LTD is blocked by inhibition of the CB1 receptor, which is activated by endocannabinoids produced downstream of group 1 mGluR signaling. Overall, our results point to a key role of SP in enabling postsynaptic expression of mGluR-LTD.
It was previously reported that the concomitant inhibition of mGluR1 and mGluR5 is required to block induction of mGluR-LTD at Sc-CA1 synapses and that inhibition of mGluR1, but not mGluR5, can block LTD expression (30). Our results confirm previous work and further show that inhibition of mGluR1 blocks both mGluR-LTD in WT and the residual LTD observed in SPKO mice, indicating that in the absence of SP mGluR1 remains capable of promoting synaptic weakening. This conclusion is congruent with the observation that global and surface expression of mGluR1 is not altered in SPKO mice. In contrast, mGluR5 function in mGluR-LTD is lost in the absence of SP, an impairment potentially caused, in whole or in part, by the reduced surface expression of the receptor in CA1 pyramidal neurons, although a role of astrocytes, which also express mGluR5, cannot be ruled out. Deficits in mGluR5 surface expression could be linked to its reduced association with Homer scaffolds in the absence of SP. The long variants of Homer proteins form tetrameric hubs (46, 47) that crosslink postsynaptic surface mGluR1a/mGluR5 with intracellular IP3R and RyR in ER membranes facilitating Ca2+ signaling (33, 35). Association with Homer proteins also modifies the lateral mobility of mGluR5 at the neuronal surface by promoting receptor clustering and countering activity-induced lateral diffusion (32). Notably, the interaction of mGluR5 with Homer was shown to be necessary for the ability of the receptor to induce mGluR-LTD (48). In the absence of SP, the SA—a source of stable ER within spines—does not form (21), affecting the dendritic localization of ER-bound IP3R and RyR and likely the consequent recruitment of Homer scaffolds to spine ER. In addition, a putative Homer-binding motif was detected in SP isoforms suggestive of a potential direct interaction between the proteins (17). Overall, our results are consistent with a model whereby reduced mGluR5–Homer crosslinking and mGluR5 surface clusters in the absence of SP compromise the capacity of the receptor to contribute to mGluR-LTD in SPKO mice. Furthermore, Wang et al. have shown that the presence of SP in dendritic spines reduces the rate of diffusion of mGluR5 compared to spines without SP, potentially favoring its local clustering and surface stability (49). It is important to note that unlike mGluR5, mGluR1 is encoded by multiple splice variants that lack the Homer-binding motif (50, 51), with the exception of the mGluR1a variant that however is mostly expressed in interneurons in area CA1 (52). It is likely that alternative Homer-independent mechanisms, which appear to remain functional in the absence of SP, contribute to regulating mGluR1 surface expression and stability at excitatory synapses (53).
mGluR-LTD has been shown to heavily rely on de novo protein synthesis, which is regulated by signaling to the ERK and the mTOR pathways. ERK1/2 activation was shown to be required for mGluR-LTD and several studies have demonstrated that mGluR5 is responsible for ERK activation (54, 55). Here we show that ERK1/2 phosphorylation is significantly increased after DHPG stimulation in WT, in agreement with others, and that ERK1/2 is equally activated in SPKO mice. Ronesi et al. have previously shown that the disruption of mGluR5–Homer interaction in hippocampus impairs mGluR-LTD but does not affect ERK1/2 activation, similar to what we observe in SPKO mice in which mGluR5 association with Homer is impaired. Notably, in knock-in mice with a mutation in mGluR5 that abolishes its binding to Homer, mGluR-LTD becomes protein synthesis-independent (56) similar to SPKO mice.
Given the more abundant expression of mGluR5 in the hippocampus compared to mGluR1 it might be counterintuitive that mGluR1 activity is the predominant participant in mGluR-LTD at Sc-CA1 synapses. However, multiple independent reports converge to indicate that mGluR1 is critical to the regulation of synaptic weakening and synapse elimination in the central nervous system through both post- and presynaptic mechanisms (30, 57–62). At hippocampal synapses, in addition to functional synaptic weakening, mGluR1 is critically involved in the elimination of dendritic spines after mGluR-LTD supporting the idea that mGluR1, although sparse, plays an indispensable role in hippocampal mGluR-LTD (27). In the absence of SP, mGluR1 ability to promote spine loss is impaired (27) whereas here we report that its contribution to the functional synaptic weakening appears to remain intact. This divergence in mGluR1 contribution to LTD may suggest that the structural and the functional changes induced by the LTD are not coupled to each other but instead mediated via distinct pathways, such that the loss of structural plasticity would not necessarily translate to the loss of functional plasticity and vice versa. A potential scenario is that mGluR1-induced spine loss relies on postsynaptic effector mechanisms (e.g. protein synthesis) that are lost in the absence of SP whereas presynaptic expression of synaptic weakening driven by mGluR1 is preserved. Although presynaptic expression of hippocampal mGluR-LTD was mostly recorded in neonatal animal (≤2 weeks-old), there is also evidence that a presynaptic component contributes to the plasticity at later ages (7, 63). In the absence of SP, the ensuing postsynaptic deficits may unmask existing presynaptic changes during the LTD that might be further strengthened as compensatory mechanism. These results call for future studies to further investigate possible noncanonical mechanisms regulating glutamate release downstream of mGluR1 revealed by the absence of SP. Nevertheless, our results reinforce the notion that mGluR1 and mGluR5, though traditionally considered to share the same molecular pathways, are functionally different from each other.
Dendritic spines are functionally and structurally heterogeneous. The presence of SP in a subset of mushroom spines is critical for spine stabilization (64) and for the generation of stronger postsynaptic responses compared to spines lacking SP (23, 24). Spines harboring SP and SA support synaptic plasticity, as demonstrated by the findings that SPKO mice show impaired LTP (21, 25, 65) and mGluR-LTD (27). The locus of expression for mGluR-LTD can be influenced by various factors including hippocampus development, aging, subregion, and experimental protocol (14, 43, 66, 67). In this study, we show for the first time that SP, a postsynaptic protein, controls the locus of expression of mGluR-LTD. The loss of SP eliminates the de novo protein synthesis dependency of hippocampal mGluR-LTD that occurs at mature synapses and switches the system to rely on the eCB signaling that produces LTD through presynaptic mechanism as in neonatal animals (43). It is important to note that expression of SP and consequently, the formation of the SA are developmentally regulated. SP is gradually expressed in the hippocampus during the postnatal period, starting postnatally around the first week, and is strongly expressed during adult life (68, 69) parallel to the switch of mGluR-LTD from presynaptic to postsynaptic, protein synthesis-dependent mechanisms of expression.
In FXS, a neurodevelopmental disorder characterized by enhanced mGluR-LTD, basal protein synthesis is increased and SP is found more abundantly in dendritic spines (70, 71). The fragile X messenger ribonucleoprotein (FMRP)—silenced in FXS—regulates RNA translation and was found associated with the SA (72). We propose that stable SP-positive spines containing the SA provide the postsynaptic locus for mGluR-LTD and alterations in SP expression and formation/stability of the SA may contribute to abnormal synaptic plasticity in FXS and other neurodevelopmental disorders marked by altered mGluR-LTD.
Materials and methods
Animals
All procedures were according to protocols approved by the guidelines of the Canadian Council on Animal Care and the McGill University Comparative Medicine and Animal Resources animal handling protocols 5057. C57Bl6 and L15 transgenic mice expressing membrane-targeted MARCKS (myristoylated alanine-rich protein kinase C substrate)–enhanced GFP under the Thy-1 promoter in a subpopulation of CA1 cells were used as WT (73). SPKO and L15S were used as SPKO and were previously described (21, 22). Mice were fed ad libitum and housed with a 12 h light/dark cycle. WT and SPKO male mice were used for experiments.
Drugs and chemicals
S-3,5-Dihydroxyphenylglycine, 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP), (S)-(+)-α-Amino-4-carboxy-2-methylbenzeneacetic acid (LY367385), anisomycin and N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM-251) were purchased from Tocris Bioscience. DHPG and MPEP were prepared in ddH2O. LY367385 was prepared in 1.1eq NaOH solution. AM-251 and anisomycin were prepared in Dimethyl sulfoxide (DMSO). All these drugs have been shown to not have any effect on basal synaptic transmission (30, 74–76).
Electrophysiology
Hippocampal slices were obtained from P30 to P40 old WT or SPKO mice. Mice were deeply anesthetized with isoflurane and killed by decapitation. Slices (400 µm) were cut on a vibratome (Leica Microsystems, VT1200S) in a sucrose-based solution containing the following (in mM): 280 sucrose, 26 NaHCO3, 10 glucose, 1.3 KCl, 1 CaCl2, and 10 MgCl2 and were transferred at 32 °C in regular artificia cerebrospinal fluid (ACSF) containing the following (in mM): 124 NaCl, 5 KCl, 1.25 NaH2PO4, 2 MgSO4, 26 NaHCO3, 2 CaCl2, and 10 glucose saturated with 95% O2/5% CO2 (pH 7.3, 300 mOsm) for 15 min before resting at room temperature (RT) for 1 h in oxygenated (95% O2/5% CO2) ACSF. To assess mGluR-LTD, slices were placed into a heated (32 °C) recording chamber of an upright microscope (DM LFSA Microsystems) and perfused continuously with oxygenated ACSF or ACSF containing indicated drugs. S-DHPG (100 μM) was applied for 5 min. Field excitatory postsynaptic potentials (fEPSPs) were recorded in the stratum radiatum of the CA1 region by using glass microelectrodes filled with 3 M NaCl. GABAA receptor-mediated inhibition was blocked with 100 μM picrotoxin, and the area CA1 was surgically isolated from CA3 to avoid epileptiform activity. fEPSPs were elicited at 0.1 Hz by a digital stimulator that was fed by a stimulation isolator unit. All data analyses were performed with custom-written software in Igor Pro 8 (Wavemetrics). fEPSP slope was measured as an index of synaptic strength.
Immunohistochemistry
Hippocampal slices of 100 µm were obtained from P30 to P40 old L15S or L15 mice. The slices were incubated in ACSF at RT for 1 h for recovery and fixed in 0.1 M Phosphate buffer (PB) containing 4% paraformaldehyde, pH 7.4, overnight at 4 °C. After fixation, slices were washed in 0.1 M PB, permeabilized in 0.4% Triton X-100, and blocked with 1.5% heat-inactivated horse serum overnight at 4 °C. Slices were incubated with primary anti-mGluR1 antibody (1:500, BD transduction, #610965) and primary anti-mGluR5 antibody (1:250, Millipore, #3352426) in permeabilizing solution for 3 days at 4 °C, washed with PB, and incubated with antirabbit secondary antibody conjugated to DyLight 649 (1:500; Jackson ImmunoResearch Laboratories) for 3 h, washed, and mounted with DAKO Fluorescent Mounting medium (Dako Canada) onto microscope slides before imaging and subsequent blinded analysis. Spine images were taken in z stacks using a Leica Microsystems SP8 confocal microscope with oil-immersion 63× objective at 6× zoom-in. The images were deconvolved and analyzed using software Huygens (Scientific Volume Imaging) and Imaris (Oxford Instruments) respectively.
For the detection of surface level of mGluR1 and mGluR5, the protocol was the same as above except that Triton X-100 was omitted. The slices were incubated with anti-mGluR1 (1:100, Alomone labs, CAT# AGC006) and anti-mGluR5 (1:100, Alomone labs, CAT# AGC007) that target the N-terminal extracellular tail. The images were taken at the stratum radiatum of CA1 hippocampus with oil-immersion 63× objective at 0.75× zoom-in.
Immunoblotting and drug treatment
Hippocampal slices (400 µm thick) were obtained from WT and SPKO mice at p30-40. The slices were incubated in ACSF at 32 °C for 3 h before treatment with S-DHPG (100 μM) for 5 min. After treatment, the slices were rapidly homogenized in ice-cold RadioImmunoPrecipitation (RIPA) buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate, 50 mM Tris–HCl (pH 8.0) with protease and phosphatase inhibitors (1X Roche Complete Mini, 5 mM NaF, 1 mM sodium orthovandate, 1 mM phenylmethylsulfonyl fluoride). Homogenates were sonicated and centrifuged for 5 min at 13,200 rpm. The supernatant was extracted and mixed with 2× Laemmli sample buffer to produce loading samples. Samples were boiled, resolved on sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, and stained with primary antibodies overnight. The following antibodies were obtained from Cell Signaling Technology (Danvers, MA): ERK1/2 (1:2,000), p-ERK1/2-Thr202/Tyr204 (1:2,000), S6 (1:1,000), p-S6-Ser235/236-Ser240/244 (1:1,000) except anti-β-actin (Sigma-Aldrich; 1:10,000) used for loading control. mGluR1 (1:1,000, BD transduction, #610965) and mGluR5 (1:1,000, Millipore, #3352426) antibodies were blotted in control slices for WT and SPKO. Horseradish peroxidase (HRP)-conjugated secondary antibodies goat antirabbit (abcam; 1:5,000) or goat antimouse (Bio-Rad Laboratories; 1:10,000) were applied for 1 h. The blots were imaged with Amersham Imager 600 and the band intensities were analyzed using the open-source program Fiji (77).
Immunoprecipitation
Brain cortices of 1 month-old mice were manually homogenized on ice with Potter–Elvehjem tissue homogenizer and vortexing (∼60 min total, each stroke followed by 10 min incubation on ice) in a buffer of 20 mM Tris–HCl pH 7.4, 5 mM ethylenediaminetetraacetic acid (EDTA), 100 mM NaCl, 1% Triton X-100 (10 μL/mg tissue) supplemented with cocktails of protease and phosphatase inhibitors. The homogenized tissue was centrifuged at 20,500 rpm for 20 min at 4 °C and protein content in the supernatant was measured by Bradford assay. An equal amount of protein (2 mg/immunoprecipitation [IP]) was incubated overnight at 4 °C with rabbit anti-mGluR5 (7 μL/IP; Alomone Labs Cat# AGC-007, RRID:AB_2039991) bound to protein G-coupled magnetic beads (40 μL Dynabeads/sample; Invitrogen Waltham, MA). The beads were washed two times with homogenization buffer, two times with PB with 0.1% Triton X-100, and once with PB (10 min rotation at 4 °C per wash). Bound proteins were eluted in denaturing sample buffer, heated at 95 °C for 5 min and separated on 8% SDS-PAGE before transfer to nitrocellulose membrane and immunoblot with rabbit anti-pan-Homer (Santa Cruz Biotechnology Cat# sc-15321, RRID:AB_2120999; 1:250), rabbit anti-mGluR5 (Abcam Cat# ab76316, RRID:AB_1523944; 1/500), mouse anti-γ-tubulin (Sigma-Aldrich Cat# T6557, RRID:AB_477584; 1:2,500) followed by detection with HRP-conjugated secondary antibodies and chemiluminescent substrate (Immobilon Western; Millipore-Sigma, Burlington, MA, USA). Bands were visualized with Azure 600 Imaging System (Azure Biosystems, Dublin, CA, USA) and quantified with the program Fiji.
Supplementary Material
Acknowledgments
The authors thank members of the A.F. and R.A.M. laboratories for comments on the manuscript; and Francois Charron for excellent technical assistance.
Contributor Information
Pei You Wu, Department of Pharmacology and Therapeutics, McGill University, Montreal, QC H3G 1Y6, Canada.
Linjia Ji, Department of Pharmacology and Therapeutics, McGill University, Montreal, QC H3G 1Y6, Canada.
Claudia De Sanctis, Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, New York, NY 10461, USA.
Anna Francesconi, Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, New York, NY 10461, USA.
Yanis Inglebert, Department of Pharmacology and Therapeutics, McGill University, Montreal, QC H3G 1Y6, Canada.
R Anne McKinney, Department of Pharmacology and Therapeutics, McGill University, Montreal, QC H3G 1Y6, Canada.
Supplementary Material
Supplementary material is available at PNAS Nexus online.
Funding
This work was supported by National Institute of Mental Health R01MH108614 to A.F.; Canadian Institutes of Health Research MOP 86724 to R.A.M.; NSERC Discovery RGPIN-2020-06373 R.A.M. and the Norman Zavalkoff Family Foundation to R.A.M.; Richard and Edith Strauss Postdoctoral Fellowship in Medicine to Y.I.
Preprints
This manuscript was posted on a preprint: https://doi.org/10.1101/2023.08.02.551676.
Data Availability
All data generated or analyzed during this study are included in this published article.
References
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Associated Data
Supplementary Materials
Data Availability Statement
All data generated or analyzed during this study are included in this published article.