An antagonism between Spinophilin and Syd-1 operates upstream of memory-promoting presynaptic long-term plasticity
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This paper addresses the important question of presynaptic homeostasis and convincingly demonstrates antagonistic interactions between Spinophilin and Syd-1 in this process. It also provides a useful hypothesis for the downstream mechanisms.
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Abstract
We still face fundamental gaps in understanding how molecular plastic changes of synapses intersect with circuit operation to define behavioral states. Here, we show that an antagonism between two conserved regulatory proteins, Spinophilin (Spn) and Syd-1, controls presynaptic long-term plasticity and the maintenance of olfactory memories in Drosophila . While Spn mutants could not trigger nanoscopic active zone remodeling under homeostatic challenge and failed to stably potentiate neurotransmitter release, concomitant reduction of Syd-1 rescued all these deficits. The Spn/Syd-1 antagonism converged on active zone close F-actin, and genetic or acute pharmacological depolymerization of F-actin rescued the Spn deficits by allowing access to synaptic vesicle release sites. Within the intrinsic mushroom body neurons, the Spn/Syd-1 antagonism specifically controlled olfactory memory stabilization but not initial learning. Thus, this evolutionarily conserved protein complex controls behaviorally relevant presynaptic long-term plasticity, also observed in the mammalian brain but still enigmatic concerning its molecular mechanisms and behavioral relevance.
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Author Response
Reviewer #2 (Public Review):
The manuscript by Ramesh et al builds upon prior studies from the Sigrist group to examine synergistic interactions between the Spinophilin (Spn) and Syd-1 synaptic proteins and their role in regulating presynaptic homeostatic plasticity at Drosophila larval NMJs and adult olfactory memory in the Mushroom Body (MB). The authors show synergistic interactions between the two proteins in these processes, where late PHP and long-term memory are abolished in Spn mutants, but restored upon reduction of Syd-1 function in the mutants. The authors go on to show that Spn appears to act in PHP by regulating a late stage in AZ remodeling and longer-term increases in the readily releasable SV pool by controlling actin polymerization/dynamics through the Mical protein. Although key aspects of the …
Author Response
Reviewer #2 (Public Review):
The manuscript by Ramesh et al builds upon prior studies from the Sigrist group to examine synergistic interactions between the Spinophilin (Spn) and Syd-1 synaptic proteins and their role in regulating presynaptic homeostatic plasticity at Drosophila larval NMJs and adult olfactory memory in the Mushroom Body (MB). The authors show synergistic interactions between the two proteins in these processes, where late PHP and long-term memory are abolished in Spn mutants, but restored upon reduction of Syd-1 function in the mutants. The authors go on to show that Spn appears to act in PHP by regulating a late stage in AZ remodeling and longer-term increases in the readily releasable SV pool by controlling actin polymerization/dynamics through the Mical protein. Although key aspects of the overall bigger picture have been published before (Mical’s role in PHP, antagonism between Spn and Syd-1 in AZ development, AZ remodeling in MB-dependent memory), the current paper ties together many of these observations into a bigger picture of how PHP plasticity at the NMJ is established and provides support for a role for PHP-required proteins in promoting long-term memory in the adult MB through effects on AZ structure and AZ protein content/amount. The study also provides new links to the role of Spn in regulating local synaptic actin dynamics and how this alters the readily releasable pool and SV release. Some points of note are provided below.
- I’m a bit confused about the time course experiments the authors describe that seem to be contradictory in Figures 1 and 2. The authors indicate control animals transiently increase BRP AZ levels during PHP at 10 mins, but by 30 minutes this increase is gone, even though PHP remains. As such, the data in these early figures suggests increases in BRP AZ levels may support an early aspect of the PHP effect (though I note this appears controversial, as other data indicate blocking the rapid AZ remodeling by several manipulations such as Arl8 transport disruption, permits early PHP, but disrupts late PHP). In contrast, the authors show that Spn mutants do not display AZ BRP increase at 10 mins, and still show early PHP, but lack late PHP. I assume the early PHP does not require AZ remodeling or an increase in the RRP at this early time point?
We thank the reviewer for this insightful question, which to a degree is reflected also in reviewer 1´s question concerning the variability of Spn mutants when tested for PHP at 10 min PhTx treatment and thus the temporally and likely functional entanglement of induction and maintenance mechanisms.
Let us start by once again describing our findings: BRP increase is clear at 10 min PhTx treatment but is no longer measurable at 30 min PhTx treatment. Genetic elimination of BRP does not restrict PHP at 10 min PhTx (Bohme et al. 2019). However, BRP mutants are neither able to maintain PHP when PhTx treatment is extended to 30 minutes as described in Turrel et al (Turrel et al. 2022), nor in a chronic PHP paradigm of BRP, GluRIIA double mutant (Bohme et al. 2019). We suggest that the transient increase of BRP, also previously described specifically in the MB γ-neurons (Zhang et al. 2018), triggers other, longer lasting AZ changes. Indeed, we found that the increase of the critical release factor Unc13A is still present at 30 min PhTx treatment and is dependent on the “transient” BRP increase (Fig. S3B) (Turrel et al. 2022). Turrel et al also uncovered a more transient upregulation of BRP when compared to Unc13A in the MB. Here, specifically upon paired olfactory conditioning, 1 h after training, animals displayed BRP and Unc13A level increases. At 3 h post training, however, BRP levels had already plateaued, whereas Unc13A levels had increased further (Figure 1B, (Turrel et al. 2022)).
We have now added to the discussion section: “We suggest that the transient increase of BRP, also previously described specifically in the MB γ-neurons (Zhang et al. 2018), triggers other, longer lasting AZ changes. Indeed, we found that the increase of the critical release factor Unc13A is still present at 30 min PhTx treatment and is dependent on the “transient” BRP increase (Fig. S3B) (Turrel et al. 2022). Turrel et al also uncovered a more transient upregulation of BRP when compared to Unc13A in the MB. Here, specifically upon paired olfactory conditioning, 1 h after training, animals displayed BRP and Unc13A level increases. At 3 h post training, however, BRP levels had already plateaued, whereas Unc13A levels had increased further (Fig. 1B, Turrel et al).” (Line 363)
RRP increase has been shown at 10 min PhTx (Weyhersmuller et al. 2011) treatment and remains high after 30 minutes of PhTx treatment (this study).
- In relation to point 1 above, the time course seems different in MB neurons, where the AZ remodeling (noted by increases in AZ BRP) seems to take 2-3 hours. Do the authors have any ideas into why the time course of PHP AZ remodeling at larval NMJs can occur in 10 minutes, but MB neuron remodeling seems to take hours?
We thank the reviewer for this question. We specifically probed the time intervals of 10 and 30 min at the NMJ due to established protocols and technical reasons; and 1hr and 3hr in the brain due to our interest in MTM. Zhang et al (Zhang et al. 2018) previously showed that indeed BRP levels in the γ-lobe were significantly increased already after 20 min after conditioning. We in the moment can only suspect that the following differences might be relevant in this point: the differences in the peripheral and central nervous system in terms of glutamatergic motoneuron presynapses (NMJ) versus cholinergic (KC presynapses) might change temporal dynamics of AZ remodeling. Furthermore, the plasticity induction protocol, using PhTx, is potentially a somewhat more “heavy-handed” approach compared to the more subtle conditioning involving the activation of dopaminergic neurons. The more complex circuitry of the central brain might also be involved in maintaining this BRP levels increase over longer timescales than at the NMJ, which may serve some yet unknown physiological purpose in maintaining memories.
We use the NMJ PhTx assay to identify proteins involved in AZ remodeling that could also be involved in memory formation in adult flies. As of now, we have no experimental evidence of whether the AZ remodeling observed in the MB actually leads to synaptic depression or instead is a reaction to the initial short-term synaptic depression occurring. This study and Turrel et al. 2022 (Turrel et al. 2022) provide evidence for an overlap of the executory machinery involved in both mechanisms, NMJ PHP plasticity and MTM formation, as BRP, Spn, Arl8, IMAC and Aplip1 are involved specifically both in mid-term NMJ PHP (at 30 min after PhTx treatment) and in MTM.
- Could the lack of rapid BRP accumulation during early PHP in Spn mutants be secondary to the larger # of AZs in those mutants and a known rate-limiting amount of BRP available that might not be enough to go to the extra Azs?
This per se might be a relevant concern. Notably, however, acute application of Latrunculin-B in Spn mutants allowed for an increase in BRP (Figure 5g-h). Thus, a limitation in the total pool of available BRP should not be responsible for Spn mutants’ inability to accumulate BRP under PhTx treatment.
- There isn't any validation of the Spn co-IP results shown in Figure 3 through other assays, and a lot of proteins are being pulled down. I can't see some of these being real (mitochondrial translation proteins? - how could Spn gain access to the inside of the mitochondria since it's a cytosolic protein?). As such, I don't know how to value that huge group of pull-down interactions without further validation, making it difficult to sort out how relevant these really are. The genetic validation of similar phenotypes in the Mical mutant, together with rescues, supports that interaction. Not sure about the rest of that list.
We appreciate the opportunity to discuss our primary data and how we used them to generate testable hypotheses for our study. Firstly, the mitochondrial translation proteins which were identified in our Spn IPs are all nuclear encoded, means they are transcribed in the nucleus and translated in the cytoplasm. Interestingly, recent work indeed suggests that mitochondrial biogenesis in the synapse is supported by local translation (e.g. see (Kuzniewska et al. 2020)). As Spn IPs are also highly significantly enriched for cytosolic translation machinery, it is an appealing idea that Spn might be involved in coupling local translation, mitochondria and memory stabilization. As this clearly goes beyond the scope of this paper, we did not further discuss this point, and are prepared to remove these data if considered misleading.
Concerning unspecific proteins being pulled down in our IPs, we would like to emphasize that these IPs are the result of an established out protocol, which entails laborious synaptosome preparations which our lab worked out previously (Depner et al. 2014). For each condition, 4 biological replicates were performed, and mitochondrial ribosomal proteins were enriched with p<10-30 significance, and never observed in our extensive systematic work on active zone biochemistry for any other active zone protein.
In this study, we used the Spn IPs to identify putative interaction partners, with the intention to validate the physiological relevance of any positive hits through experiments, like we did in the case of Mical. We were also able to identify previously known interaction partners like Syd-1 and Nrx-1 (Muhammad et al. 2015). Obviously, we did not independently validate these findings for the large number of identified proteins, e.g. by using in vitro purified proteins (we do not consider Western probing of IPs to be independent proof of any complementary value to mass-spectrometry based quantification).
We have now added this sentence to our manuscript:
“As a validation of the list of proteins that were returned as interaction partners of Spn in this work, we were able to reconfirm previously known interactions (Muhammad et al. 2015), e.g., Syd-1 (Figure 3b) and Nrx-1 (not shown).” (Line 148)
- Are the authors worried about the fact that the Actin-GFP line they use to look at synaptic actin dynamics is driven by a GAL4, and the 2nd top hit of their Spn IP pull downs are translation regulators? Could the changes in actin-GFP they see between control and Spn mutants have anything to do with a different translation of the exogenous UAS-actin-GFP? Would have been helpful to do an endogenous stain for actin levels with an anti-actin antibody so no transcription/translation issues of a transgene would be at play. This would be easy to do for the quantification of total actin levels at the synapse.
This is per se a fully justified concern, which is hard to be fully excluded. Indeed, when preparing this manuscript, we attempted to visualize and quantify the endogenous presynaptic actin through immunostaining. However, these attempts were unsuccessful, as the very bright muscle actin staining obscures the relatively low levels of actin present close to the presynaptic AZs, even when using super-resolution light microscopy. Still, we would like to emphasize that Spn and Syd-1 antagonized each others’ function concerning apparent F-actin level (using Gal4 expression of actin-GFP). Given the known connection of Spn operating as a compartment specific F-actin breaker (Chia, Patel, and Shen 2012; Ryan et al. 2005; Nakanishi et al. 1997), we are still rather confident about our finding and its interpretation.
Concerning the FRAP analyses, we are fully confident of our findings, as the intensity of actin-GFP is internally normalized within each NMJ. Therefore, the differences in FRAP experiments should be independent of the starting amounts of actin in control and mutant animals. As we can show that the Spn/Syd-1 antagonism functions on actin dynamics as well (Figure 4j), we are sure concerning the physiological relevance of our observations.
- Are Mical levels normalized in the Spn, Syd1 double mutants, given PHP is recovered?
We thank the reviewer for the comment and agree that Mical levels should be expected to normalize upon Syd-1 heterozygosity in Spn mutants. We have now immunostained for Mical in wildtype, Spn mutants and Spn mutants with Syd-1 heterozygosity to address this question. We found that Mical levels in Spn mutants were indeed normalized upon Syd-1 heterozygosity (Figure 5 - Figure supplement 1 c-d).
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eLife assessment
This paper addresses the important question of presynaptic homeostasis and convincingly demonstrates antagonistic interactions between Spinophilin and Syd-1 in this process. It also provides a useful hypothesis for the downstream mechanisms.
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Reviewer #1 (Public Review):
The study by Ramesh et al identifies key components that support presynaptic plasticity (PHP) at Drosophila glutamatergic synapses: an accepted model for their mammalian equivalents. Specifically, they identify that PHP relies on the antagonism between Spinophilin (Spn) and Syd-1 (a Rho GTPase activating protein) to dynamically alter F-actin (de)polymerisation to facilitate increased synaptic vesicle release, thus supporting PHP. A pull-down of Spn identifies additional proteins including Mical, the over-expression of which is sufficient to rescue the excessive actin stabilisation present in an Spn loss-of-function mutant. The studies relate the mechanistic understanding of Spn to aversive mid-term olfactory memory formation formed in the mushroom bodies.
Collectively, this study represents an important …
Reviewer #1 (Public Review):
The study by Ramesh et al identifies key components that support presynaptic plasticity (PHP) at Drosophila glutamatergic synapses: an accepted model for their mammalian equivalents. Specifically, they identify that PHP relies on the antagonism between Spinophilin (Spn) and Syd-1 (a Rho GTPase activating protein) to dynamically alter F-actin (de)polymerisation to facilitate increased synaptic vesicle release, thus supporting PHP. A pull-down of Spn identifies additional proteins including Mical, the over-expression of which is sufficient to rescue the excessive actin stabilisation present in an Spn loss-of-function mutant. The studies relate the mechanistic understanding of Spn to aversive mid-term olfactory memory formation formed in the mushroom bodies.
Collectively, this study represents an important addition to the understanding of PHP and its involvement in the formation of memory. The experiments presented are carefully done and the conclusions drawn are appropriate. A potential criticism is that the study spans two big areas (PHP and memory) and that each may have been better considered as separate studies. However, this is a stylistic concern and not one that influences the insights presented by this study.
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Reviewer #2 (Public Review):
The manuscript by Ramesh et al builds upon prior studies from the Sigrist group to examine synergistic interactions between the Spinophilin (Spn) and Syd-1 synaptic proteins and their role in regulating presynaptic homeostatic plasticity at Drosophila larval NMJs and adult olfactory memory in the Mushroom Body (MB). The authors show synergistic interactions between the two proteins in these processes, where late PHP and long-term memory are abolished in Spn mutants, but restored upon reduction of Syd-1 function in the mutants. The authors go on to show that Spn appears to act in PHP by regulating a late stage in AZ remodeling and longer-term increases in the readily releasable SV pool by controlling actin polymerization/dynamics through the Mical protein. Although key aspects of the overall bigger picture …
Reviewer #2 (Public Review):
The manuscript by Ramesh et al builds upon prior studies from the Sigrist group to examine synergistic interactions between the Spinophilin (Spn) and Syd-1 synaptic proteins and their role in regulating presynaptic homeostatic plasticity at Drosophila larval NMJs and adult olfactory memory in the Mushroom Body (MB). The authors show synergistic interactions between the two proteins in these processes, where late PHP and long-term memory are abolished in Spn mutants, but restored upon reduction of Syd-1 function in the mutants. The authors go on to show that Spn appears to act in PHP by regulating a late stage in AZ remodeling and longer-term increases in the readily releasable SV pool by controlling actin polymerization/dynamics through the Mical protein. Although key aspects of the overall bigger picture have been published before (Mical's role in PHP, antagonism between Spn and Syd-1 in AZ development, AZ remodeling in MB-dependent memory), the current paper ties together many of these observations into a bigger picture of how PHP plasticity at the NMJ is established and provides support for a role for PHP-required proteins in promoting long-term memory in the adult MB through effects on AZ structure and AZ protein content/amount. The study also provides new links to the role of Spn in regulating local synaptic actin dynamics and how this alters the readily releasable pool and SV release. Some points of note are provided below.
1. I'm a bit confused about the time course experiments the authors describe that seem to be contradictory in Figures 1 and 2. The authors indicate control animals transiently increase BRP AZ levels during PHP at 10 mins, but by 30 minutes this increase is gone, even though PHP remains. As such, the data in these early figures suggests increases in BRP AZ levels may support an early aspect of the PHP effect (though I note this appears controversial, as other data indicate blocking the rapid AZ remodeling by several manipulations such as Arl8 transport disruption, permits early PHP, but disrupts late PHP). In contrast, the authors show that Spn mutants do not display AZ BRP increase at 10 mins, and still show early PHP, but lack late PHP. I assume the early PHP does not require AZ remodeling or an increase in the RRP at this early time point?
2. In relation to point 1 above, the time course seems different in MB neurons, where the AZ remodeling (noted by increases in AZ BRP) seems to take 2-3 hours. Do the authors have any ideas into why the time course of PHP AZ remodeling at larval NMJs can occur in 10 minutes, but MB neuron remodeling seems to take hours?
3. Could the lack of rapid BRP accumulation during early PHP in Spn mutants be secondary to the larger # of AZs in those mutants and a known rate-limiting amount of BRP available that might not be enough to go to the extra AZs?
4. There isn't any validation of the Spn co-IP results shown in Figure 3 through other assays, and a lot of proteins are being pulled down. I can't see some of these being real (mitochondrial translation proteins? - how could Spn gain access to the inside of the mitochondria since it's a cytosolic protein?). As such, I don't know how to value that huge group of pull-down interactions without further validation, making it difficult to sort out how relevant these really are. The genetic validation of similar phenotypes in the Mical mutant, together with rescues, supports that interaction. Not sure about the rest of that list.
5. Are the authors worried about the fact that the Actin-GFP line they use to look at synaptic actin dynamics is driven by a GAL4, and the 2nd top hit of their Spn IP pull downs are translation regulators? Could the changes in actin-GFP they see between control and Spn mutants have anything to do with a different translation of the exogenous UAS-actin-GFP? Would have been helpful to do an endogenous stain for actin levels with an anti-actin antibody so no transcription/translation issues of a transgene would be at play. This would be easy to do for the quantification of total actin levels at the synapse.
6. Are Mical levels normalized in the Spn, Syd1 double mutants, given PHP is recovered?
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