Dynamic regulation of mRNA acetylation at synapses by spatial memory in mouse hippocampus

Reviewer #1 (Public review):

Summary:

RNA modification has emerged as an important modulator of protein synthesis. Recent studies found that mRNA can be acetylated (ac4c), which can alter mRNA stability and translation efficiency. The role of ac4c mRNA in the brain has not been studied. In this paper, the authors convincingly show that ac4c occurs selectively on mRNAs localized at synapses, but not cell-wide. The ac4c "writer" NAT10 is highly expressed in hippocampal excitatory neurons. Using NAT10 conditional KO mice, decreasing levels of NAT10 resulted in decreases in ac4c of mRNAs and also showed deficits in LTP and spatial memory. These results reveal a potential role for ac4c mRNA in memory consolidation.

This is a new type of mRNA regulation that seems to act specifically at synapses, which may help elucidate the mechanisms of local protein synthesis in memory consolidation. Overall, the studies are well carried out and presented. There is some confusion over training/learning vs memory, and the precise mRNAs that require ac4c to carry out memory consolidation are not clear. The specificity of changes occurring only at the end of training, rather than after each day of training, is interesting and warrants some investigation. This timeframe is puzzling because the authors show that ac4c can dynamically increase within 1 hour after cLTP.

Strengths:

(1) The studies show that mRNA acetylation (ac4c) occurs selectively at mRNAs localized to synaptic compartments (using synaptoneurosome preps).

(2) The authors identify a few key mRNAs acetylated and involved in plasticity and memory - e.g., Arc.

(3) The authors show that Ac4c is induced by learning and neuronal activity (cLTP).

(4) The studies show that the ac4c "writer" NAT10 is expressed in hippocampal excitatory neurons and may be relocated to synapses after cLTP/learning induction.

(5) The authors used floxed NAT10 mice injected with AAV-Cre in the hippocampus (NAT10 cKO) to show that NAT10 may play a role in LTP maintenance and memory consolidation (using the Morris Water Maze).

Weaknesses:

(1) The authors use a confusing timeline for their behavioral experiments, i.e, day 1 is the first day of training in the MWM, and day 6 is the probe trial, but in reality, day 6 is the first day after the last training day. So this is really day 1 post-training, and day 20 is 14 days post-training.

(2) The authors inaccurately use memory as a term. During the training period in the MWM, the animals are learning, while memory is only probed on day 6 (after learning). Thus, day 6 reflects memory consolidation processes after learning has taken place.

(3) The NAT10 cKO mice are useful to test the causal role of NAT10 in ac4a and plasticity/memory, but all the experiments used AAV-CRE injections in the dorsal hippocampus that showed somewhat modest decreases in total NAT10 protein levels. For these experiments, it would be better to cross the NAT10 floxed animals to CRE lines where a better knockdown of NAT10 can be achieved, with less variability.

(4) Because knockdown is only modest (~50%), it is not clear if the remaining ac4c on mRNAs is due to remaining NAT10 protein or due to an alternative writer (as the authors pose).

Reviewer #2 (Public review):

This is an interesting study that shows that mRNA acetylation at synapses is dynamically regulated at synapses by spatial memory in the mouse hippocampus. The dynamic changes of ac4C-mRNAs regulated by memory were validated by methods including ac4C dot-blot and liquid 13 chromatography-tandem mass spectrometry (LC-MS/MS).

Here are some comments for consideration by readers and authors:

(1) It is known that synaptosomes are contaminated with glial tissue. In the study, the authors also show that NAT0 is expressed in glia. So the candidate mRNAs identified by acRIP-seq might also be mixed with glial mRNAs. Are the GO BP terms shown in Figure 3A specifically chosen, or unbiasedly listed for all top ones?

(2) Where does NAT10-mediated mRNA acetylation take place within cells generally? Is there evidence that NAT10 can catalyze mRNA acetylation in the cytoplasm?

(3) "The NAT10 proteins were significantly reduced in the cytoplasm (S2 fraction) but increased in the PSD fraction at day 6 after memory (Figures 5J and 5K)." The authors argue that the translocation of NAT10 from soma to synapses accounts for these changes. The increase of NAT10 protein in the PSD fraction can be understood. However, it is quite surprising that the NAT10 proteins were significantly reduced in the cytoplasm (S2 fraction), considering the amount of NAT10 in soma is much more abundant in synapses. The small increase in synaptic NAT10 might not be enough to cause a decrease in soma NAT10 protein level.

(4) It is difficult to separate the effect on mRNA acetylation and protein mRNA acetylation when doing the loss of function of NAT10.

Author response:

Reviewer #1:

Comment 1: The authors use a confusing timeline for their behavioral experiments, i.e., day 1 is the first day of training in the MWM, and day 6 is the probe trial, but in reality, day 6 is the first day after the last training day. So this is really day 1 post-training, and day 20 is 14 days post-training.

We thank this reviewer for pointing out the issue of the behavioral timeline. We will revise the behavioral timeline as suggested by this reviewer. Days 1–5 will be labeled as “Training phase day 1–5”. Day 6 will be labeled as the “Day 1 post-training” and Day 20 will be labeled as the “Day 14 post-training”.

Comment 2: The authors inaccurately use memory as a term. During the training period in the MWM, the animals are learning, while memory is only probed on day 6 (after learning). Thus, day 6 reflects memory consolidation processes after learning has taken place.

We will revise the manuscript to distinguish between "learning" and "memory." We will refer to the performance during the 5-day training period as "spatial learning" and restrict the term "memory" to the probe tests on Day 6, which reflect memory processes after learning has taken place.

Comment 3: The NAT10 cKO mice are useful... but all the experiments used AAV-CRE injections in the dorsal hippocampus that showed somewhat modest decreases... For these experiments, it would be better to cross the NAT10 floxed animals to CRE lines where a better knockdown of NAT10 can be achieved, with less variability.

We want to clarify the reason for using AAV-Cre injection rather than Cre lines. Indeed, we attempted to generate Nat10 conditional knockouts by crossing Nat10^flox/flox mice with several CNS-specific Cre lines. Crossing with Nestin-Cre and Emx1-Cre resulted in embryonic and premature lethality, respectively, consistent with the essential housekeeping function of NAT10 during neurodevelopment. We are currently using the Camk2α-Cre line which starts to express Cre after postnatal 3 weeks specifically in hippocampal pyramidal neurons (Tsien et al., 1996).

Comment 4: Because knockdown is only modest (~50%), it is not clear if the remaining ac4c on mRNAs is due to remaining NAT10 protein or due to an alternative writer (as the authors pose).

Our results suggest the existence of alternative writers. As shown in Figure 6D, we identified a population of "NAT10-independent" MISA mRNAs (present in MISA but not downregulated in NASA). Remarkably, these mRNAs possess a consensus motif (RGGGCACTAACY) that is fundamentally different from the canonical NAT10 motif (AGCAGCTG). This distinct motif usage suggests that the residual ac4C signals are not merely due to incomplete knockdown of NAT10, but reflect the activity of other, as-yet-unidentified ac4C writers. Nonetheless, we think that generation of a Nat10 knockout line with completely loss of NAT10 proteins is useful to address this reviewer’s concern.

Reviewer #2:

Comment 1: It is known that synaptosomes are contaminated with glial tissue... So the candidate mRNAs identified by acRIP-seq might also be mixed with glial mRNAs. Are the GO BP terms shown in Figure 3A specifically chosen, or unbiasedly listed for all top ones?

It is true that some ac4C-mRNAs identified by acRIP-seq from the synaptosomes are highly expressed in astrocyte, such as Aldh1l1, ApoE, Sox9 and Aqp4 (Table S3, Fig. S6H). In agreement, we found that NAT10 was also expressed in astrocyte in addition to neurons. We will show representative image for the expression of NAT10-Cre in astrocytes in the revised MS. The BP items shown in Fig. 3A were chosen from top 30 and highly related with synaptic plasticity and memory. We will show the full list of significant BP items for MISA in the revised MS.

Comment 2: Where does NAT10-mediated mRNA acetylation take place within cells generally? Is there evidence that NAT10 can catalyze mRNA acetylation in the cytoplasm?

The previous studies from non-neuronal cells showed that NAT10 can catalyze mRNA acetylation in the cytoplasm and enhance translational efficiency (Arango et al., 2018; Arango et al., 2022). In this study, we showed that mRNA acetylation occurred both in the homogenates and synapses (see ac4C-mRNA lists in Table S2 and S3). However, spatial memory upregulated mRNA acetylation mainly in the synapses rather than in the homogenates (Fig. 2 and Fig. S2).

Comment 3: "The NAT10 proteins were significantly reduced in the cytoplasm (S2 fraction) but increased in the PSD fraction..." The small increase in synaptic NAT10 might not be enough to cause a decrease in soma NAT10 protein level.

We showed that the NAT10 protein levels were increased by one-fold in the PSD fraction, but were reduced by about 50% in the cytoplasm after memory formation (Fig. 5J and K). The protein levels of NAT10 in the homogenates and nucleus were not altered after memory formation (Fig. 5F and I). Due to these facts, we hypothesized that NAT10 proteins may have a relocation from cytoplasm to synapses after memory formation, which was also supported by the immunofluorescent results from cultured neurons (Fig. S4). However, we agree with this reviewer that drawing such a conclusion may require the time-lapse imaging of NAT10 protein trafficking in living animals, which is technically challenging at this moment.

Comment 4: It is difficult to separate the effect on mRNA acetylation and protein mRNA acetylation when doing the loss of function of NAT10.

This is a good point. We agree with this reviewer that NAT10 may acetylate both mRNA and proteins. We examined the acetylation levels of -tubulin and histone H3, two substrate proteins of NAT10 in the hippocampus of Nat10 cKO mice. As shown in Fig S5C, E, and F, the acetylation levels of -tubulin and histone H3 remained unchanged in the Nat10 cKO mice, likely due to the compensation by other protein acetyltransferases. In contrast, mRNA ac4C levels were significantly decreased in the Nat10 cKO mice (Figure S5G–H). These results suggest that the memory deficits seen in Nat10 cKO mice may be largely due to the impaired mRNA acetylation. Nonetheless, we believe that developing a new technology which enables selective erasure of mRNA acetylation would be helpful to address the function of mRNA. We discussed these points in the MS (line 585-592).

References

Arango, D., Sturgill, D., Alhusaini, N., Dillman, A. A., Sweet, T. J., Hanson, G., Hosogane, M., Sinclair, W. R., Nanan, K. K., & Mandler, M. D. (2018). Acetylation of cytidine in mRNA promotes translation efficiency. Cell, 175(7), 1872-1886. e1824.

Arango, D., Sturgill, D., Yang, R., Kanai, T., Bauer, P., Roy, J., Wang, Z., Hosogane, M., Schiffers, S., & Oberdoerffer, S. (2022). Direct epitranscriptomic regulation of mammalian translation initiation through N4-acetylcytidine. Molecular cell, 82(15), 2797-2814. e2711.

Tsien, J. Z., Chen, D. F., Gerber, D., Tom, C., Mercer, E. H., Anderson, D. J., Mayford, M., Kandel, E. R., & Tonegawa, S. (1996). Subregion-and cell type–restricted gene knockout in mouse brain. Cell, 87(7), 1317-1326.