Evidence suggesting creatine as a new central neurotransmitter: presence in synaptic vesicles, release upon stimulation, effects on cortical neurons and uptake into synaptosomes and synaptic vesicles

Reviewer #1 (Public Review):

This is an interesting and somewhat unusual paper supporting the idea that creatine is a neurotransmitter in the central nervous system of vertebrates. The idea is not entirely new, and the authors carefully weigh the evidence, both past and newly acquired, to make their case. The strength of the paper lies in the importance of the potential discovery - as the authors point out, creatine ticks more boxes on criteria of neurotransmitters than some of the ones listed in textbooks - and the list of known transmitters (currently 16) certainly is textbook material. A further strength of the manuscript is the careful consideration of a list of criteria for transmitters and newly acquired evidence for four of these criteria: 1. evidence that creatine is stored in synaptic vesicles, 2. mutants for creatine synthesis and a vesicular transporter show reduced storage and release of creatine, 3. functional measurement that creatine release has an excitatory or inhibitory (here inhibitory) effect in vivo, and 4. ATP-dependence. The key weakness of the paper is that there is no single clear 'smoking gun', like a postsynaptic creatine receptor, that would really demonstrate the function as a transmitter. Instead, the evidence is of a cumulative nature, and not all bits of evidence are equally strong. On balance, I found the path to discovery and the evidence assembled in this manuscript to establish a clear possibility, positive evidence, and to provide a foundation for further work in this direction.

Reviewer #2 (Public Review):

Summary:
Bian et al studied creatine (Cr) in the context of central nervous system (CNS) function. They detected Cr in synaptic vesicles purified from mouse brains with anti-Synaptophysin using capillary electrophoresis-mass spectrometry. Cr levels in the synaptic vesicle fraction were reduced in mice lacking the Cr synthetase AGAT, or the Cr transporter SLC6A8. They provide evidence for Cr release within several minutes after treating brain slices with KCl. This KCl-induced Cr release was partially calcium-dependent and was attenuated in slices obtained from AGAT and SLC6A8 mutant mice. Cr application also decreased the excitability of cortical pyramidal cells in one third of the cells tested. Finally, they provide evidence for SLC6A8-dependent Cr uptake into synaptosomes, and ATP-dependent Cr loading into synaptic vesicles. Based on these data, the authors propose that Cr may act as a neurotransmitter in the CNS.

Strengths:
1. A major strength of the paper is the broad spectrum of tools used to investigate Cr.
2. The study provides strong evidence that Cr is present in/loaded into synaptic vesicles.

Weaknesses:
(in sequential order)
1. Are Cr levels indeed reduced in Agat-/-? The decrease in Cr IgG in Agat-/- (and Agat+/-) is similar to the corresponding decrease in Syp (Fig. 3B). What is the explanation for this? Is the decrease in Cr in Agat-/- significant when considering the drop in IgG? The data should be normalized to the respective IgG control.
2. The data supporting that depolarization-induced Cr release is SLC6A8 dependent is not convincing because the relative increase in KCl-induced Cr release is similar between SLC6A8-/Y and SLC6A8+/Y (Fig. 5D). The data should be also normalized to the respective controls.
3. The majority (almost 3/4) of depolarization-induced Cr release is Ca2+ independent (Fig. 5G). Furthermore, KCl-induced, Ca2+-independent release persists in SLC6A8-/Y (Fig. 5G). What is the model for Ca2+-independent Cr release? Why is there Ca2+-independent Cr release from SLC6A8 KO neurons?
How does this relate to the prominent decrease in Ca2+-dependent Cr release in SLC6A8-/Y (Fig. 5G)? They show a prominent decrease in Cr control levels in SLC6A8-/Y in Fig. 5D. Were the data shown in Fig. 5D obtained in the presence or absence of Ca2+? Could the decrease in Ca2+-dependent Cr release in SLC6A8-/Y (Fig. 5G) be due to decreased Cr baseline levels in the presence of Ca2+ (Fig. 5D)?
4. Cr levels are strongly reduced in Agat-/- (Fig. 6B). However, KCl-induced Cr release persists after loss of AGAT (Fig. 6B). These data do not support that Cr release is Agat dependent.
5. The authors show that Cr application decreases excitability in ~1/3 of the tested neurons (Fig. 7). How were responders and non-responders defined? What justifies this classification? The data for all Cr-treated cells should be pooled. Are there indeed two distributions (responders/non-responders)? Running statistics on pre-selected groups (Fig. 7H-J) is meaningless. Given that the effects could be seen 2-8 minutes after Cr application - at what time points were the data shown in Fig. 7E-J collected? Is the Cr group shown in Fig. 7F significantly different from the control group/wash?
6. Indirect effects: The phenotypes could be partially caused by indirect effects of perturbing the Cr/PCr/CK system, which is known to play essential roles in ATP regeneration, Ca2+ homeostasis, neurotransmission, intracellular signaling systems, axonal and dendritic transport... Similarly, high GAMT levels were reported for astrocytes (e.g., Schmidt et al. 2004; doi: 10.1093/hmg/ddh112), and changes in astrocytic Cr may underlie the phenotypes. Cr has been also reported to be an osmolyte: a hyperosmotic shock of astrocytes induced an increase in Cr uptake, suggesting that Cr can work as a compensatory osmolyte (Alfieri et al. 2006; doi: 10.1113/jphysiol.2006.115006). Potential indirect effects are also consistent with a trend towards decreased KCl-induced GABA (and Glutamate) release in SLC6A8-/Y (Fig. 5C). These indirect effects may in part explain the phenotypes seen after perturbing Agat, SLC6A8, and should be thoroughly discussed.
7. As stated by the authors, there is some evidence that Cr may act as a co-transmitter for GABAA receptors (although only at high concentrations). Would a GABAA blocker decrease the fraction of cells with decreased excitability after Cr exposure?
8. The statement "Our results have also satisfied the criteria of Purves et al. 67,68, because the presence of postsynaptic receptors can be inferred by postsynaptic responses." (l.568) is not supported by the data and should be removed.

Reviewer #3 (Public Review):

SUMMARY:
The manuscript by Bian et al. promotes the idea that creatine is a new neurotransmitter. The authors conduct an impressive combination of mass spectrometry (Fig. 1), genetics (Figs. 2, 3, 6), biochemistry (Figs. 2, 3, 8), immunostaining (Fig. 4), electrophysiology (Figs. 5, 6, 7), and EM (Fig. 8) in order to offer support for the hypothesis that creatine is a CNS neurotransmitter.

STRENGTHS:
There are many strengths to this study.
• The combinatorial approach is a strength. There is no shortage of data in this study.
• The careful consideration of specific criteria that creatine would need to meet in order to be considered a neurotransmitter is a strength.
• The comparison studies that the authors have done in parallel with classical neurotransmitters are helpful.
• Demonstration that creatine has inhibitory effects is another strength.
• The new genetic mutations for Slc6a8 and AGAT are strengths and potentially incredibly helpful for downstream work.

WEAKNESSES:
• Some data are indirect. Even though Slc6a8 and AGAT are helpful sentinels for the presence of creatine, they are not creatine themselves. Therefore, the conclusions that are drawn should be circumspect.
• Regarding Slc6a8, it seems to work only as a reuptake transporter - not as a transporter into SVs. Therefore, we do not know what the transporter is.
• Puzzlingly, Slc6a8 and AGAT are in different cells, setting up the complicated model that creatine is created in one cell type and then processed as a neurotransmitter in another.
• No candidate receptor for creatine has been identified postsynaptically.
• Because no candidate receptor has been identified, is it possible that creatine is exerting its effects indirectly through other inhibitory receptors (e.g., GABAergic Rs)?
• More broadly, what are the other possibilities for roles of creatine that would explain these observations other than it being a neurotransmitter? Could it simply be a modifier that exists in the SVs (lots of molecules exist in SVs)?
• The biochemical studies are helpful in terms of comparing relevant molecules (e.g., Figs. 8 and S1), but the images of the westerns are all so fuzzy that there are questions about processing and the accuracy of the quantification.

APPRAISAL OF WHETHER THE AUTHORS ACHIEVED THEIR AIMS AND WHETHER THE RESULTS SUPPORT THE CONCLUSIONS:
There are several criteria that define a neurotransmitter. The authors nicely delineated many criteria in their discussion, but it is worth it for readers to do the same with their own understanding of the data.

By this reviewer's understanding (and the Purves' textbook definition) a neurotransmitter: 1) must be present within the presynaptic neuron and stored in vesicles; 2) must be released by depolarization of the presynaptic terminal; 3) must require Ca2+ influx upon depolarization prior to release; 4) must bind specific receptors present on the postsynaptic cell; 5) exogenous transmitter can mimic presynaptic release; 6) there exists a mechanism of removal of the neurotransmitter from the synaptic cleft.

For a paper to claim that the work has identified a new neurotransmitter, several of these criteria would be met - and the paper would acknowledge in the discussion which ones have not been met. For this particular paper, this reviewer finds that condition 1 is clearly met.

Conditions 2 and 3 seem to be met by electrophysiology, but there are caveats here. High KCl stimulation is a blunt instrument that will depolarize absolutely everything in the prep all at once and could result in any number of non-specific biological reactions as a result of K+ rushing into all neurons in the prep. Moreover, the results in 0 Ca2+ are puzzling. For creatine (and for the other neurotransmitters), why is there such a massive uptick in release, even when the extracellular saline is devoid of calcium?

Condition 4 is not discussed in detail at all. In the discussion, the authors elide the criterion of receptors specified by Purves by inferring that the existence of postsynaptic responses implies the existence of receptors. True, but does it specifically imply the existence of creatinergic receptors? This reviewer does not think that is necessarily the case. The authors should be appropriately circumspect and consider other modes of inhibition that are induced by activation or potentiation of other receptors (e.g., GABAergic or glycinergic).

Condition 5 may be met, because the authors applied exogenous creatine and observed inhibition (Fig. 7). However, this is tough to know without understanding the effects of endogenous release of creatine. if they were to test if the absence of creatine caused excess excitation (at putative creatinergic synapses), then that would be supportive of the same.

For condition 6, the authors made a great effort with Slc6a8. This is a very tough criterion to understand for many synapses and neurotransmitters.

DISCUSSION OF THE LIKELY IMPACT OF THE WORK:
In terms of fundamental neuroscience, the story would be impactful if proven correct. There are certainly more neurotransmitters out there than currently identified.

The impact as framed by the authors in the abstract and introduction for intellectual disability is uncertain (forming a "new basis for ID pathogenesis") and it seems quite speculative beyond the data in this paper.