D-serine suppresses one-carbon metabolism by competing with mitochondrial L-serine transport

Reviewer #1 (Public review):

Summary:

The authors demonstrate the stereoselective role of D-serine in 1C metabolism, showing that D-serine competes with L-serine and inhibits mitochondrial L-serine transport. They observe expression of 1C metabolites in their metabolomics approach in primary cortical neurons treated with L-serine, D-serine, and a mixture of both. Their conclusions are based on the reduction in levels of glycine, polyamines, and their intermediates and formate. Single-cell RNA sequencing of N2a cells showed that cells treated with D-serine enhanced expression of genes associated with mitochondrial functions, such as respiratory chain complex assembly, and mitochondrial functions, with downregulation of genes related to amino acid transport, cellular growth, and neuron projection extension. Their work demonstrates that D-serine inhibits tumor cell proliferation and induces apoptosis in neural progenitor cells, highlighting the importance of D-serine in neurodevelopment.

Strengths:

D-amino acids are a marvel of nature. It is fascinating that nature decided to make two versions of the same molecule, in this case, an amino acid. While the L-stereoisomer plays well-known roles in biology, the D-stereoisomer seems to function in obscurity. Research into these novel signaling molecules is gathering momentum, with newer stereoisomers being discovered. D-serine has been the most well-studied among the different stereoisomers, and we still continue to learn about this novel neurotransmitter. The roles of these molecules in the context of metabolism is not well studied. The authors aim to elucidate the metabolic role of D-serine in the context of neuronal maturation with implications for 1C metabolism and in cell proliferation. The metabolic role of these molecules is just beginning to be uncovered, especially in the context of mammalian biology. This is the strength of the manuscript. The authors have done important work in prior publications elucidating the role of D-amino acids. The advancement of the field of D-amino acids in mammalian biology is significant, as not much is known. The presentation of RNA seq data is a valuable resource to the community, however, with caveats as mentioned below.

Weaknesses:

The following are some of the issues that come out in a critical reading of the manuscript. Addressing these would only strengthen and clarify the work.

(1) Kinetic assessment of D-serine versus L-serine: While the authors mention that D-serine is not a good substrate for SHMT2 compared to L-serine, the kinetic data are presented for only D-serine. In a substrate comparison with an enzyme, data must be presented for L-serine as well to make the conclusion about substrate specificity and affinity. Since the authors talk about one versus another substrate, there needs to be a kinetic comparison of both with Km (affinity). (Ref Figure 2 panel).

(2) Molecular Dynamics simulations, while a good first step in modeling interactions at the active site, rely on force fields. These force fields are approximations and do not represent all interactions occurring in the natural world. Setting up the initial conditions in the simulations can impact the final results in non-equilibrium scenarios. The basic question here is this: Is the simulated trajectory long enough so that the system reaches thermodynamic equilibrium and the measured properties converge? Prior studies have shown mixed results with the conclusion that properties of biological systems tend to converge in multi-second trajectories (not nanosecond scales as reported by the authors) and transition rates to low probability conformations require more time. (Ref Figure 2C).

(3) The authors use N2a cell line to demonstrate D-serine burden on primary cortical neurons. N2a is an immortalized cell line, and its properties are very different from primary neurons. The authors need to mention a rationale for the use of an immortalized cell line versus primary neurons. The transcriptomic profile of an immortalized cell line is different compared to a primary cell. Hence, the response to D-serine may vary between the two different cell types.

(4) In Figure 4D, the authors mention that D-serine activates the cleavage of caspase 3. Figure 4D shows only cleaved caspase 3 as a single band. They need to show the full blot that contains the cleaved fragments along with the major caspase 3 band.

(5) In Figure panel 4, the authors use neural progenitor cells (NPCs). They need to demonstrate that the population they are working with is NPCs and not primary neurons. There must be a figure panel staining for NPC markers like SOX2 and PAX6. Also, Figure S5 needs to be properly labeled. It is confusing from the legend what panels B-E refer to? Also, scale bars are not indicated.

(6) In Supplementary Figure panel 7F, the authors mention phosphatidyl L-serine and phosphatidyl D-serine. A chromatogram of the two species would clarify their presence as they used 2D-HPLC. On an MS platform, these 2 species are not distinguishable. Including a chromatogram of the 2 species would be helpful to the readers.

(7) The authors mention about enantiomeric shift of serine metabolism during neural development, which appears to be a discussion of prior published data from Hubbard et al, 2013, Burk et al, 2020, and Bella et a,l 2021 in Supplementary Figure panels 8 A-E. This should not be presented as a figure panel, as it gives the false impression that the authors have performed the experiment, which is clearly not the case. However, its discussion can well serve as part of the manuscript in the discussion section.

(8) The entire presentation of the section on enantiomeric shift of serine metabolism during neural development (lines 274-312) is a discussion and should be part of the discussion section and not in the results section. This is misleading.

(9) The discussion section is not well written. There is no mention of recent work related to D-serine that has a direct bearing on its metabolic properties. In the discussion section, paragraph 1, the authors mention that their work demonstrates the selective synthesis of D-serine in mature neurons as opposed to neural progenitor cells. This concept has been referred to in prior publications:

(a) Spatiotemporal relationships among D-serine, serine racemase, and D-amino acid oxidase during mouse postnatal development. PMID:14531937.

(b) D-cysteine is an endogenous regulator of neural progenitor cell dynamics in the mammalian brain. PMID:34556581.

(10) In the abstract, in lines 101 and 102, the authors mention "how D-serine contributes to cellular metabolism beyond neurotransmission remains largely unknown". In 2023, a paper in Stem Cell Reports by Roychaudhuri et al (PMID:37352848) showed that D and L-serine availability impacts lipid metabolism in the subventricular zone in mice, affecting proliferative properties of stem-cell derived neurons using a comprehensive lipidomics approach. There is no mention of this work even in the discussion section, as it bears directly on L and D-serine availability in neurons, which the authors are investigating. In the discussion section in lines 410-411, the authors mention the role of D-serine in neurogenesis, but surprisingly don't refer to the above reference. The role of D-serine in neurogenesis has been demonstrated in the Sultan et al (lines 855-857) and Roychaudhuri et al references.

(11) Both D-serine and the structurally similar stereoisomer D-cysteine (sulfur versus oxygen atom) have a bearing on 1C metabolism and the folate cycle. With reference to the folate cycle, Roychaudhuri et al in 2024 (PMID:39368613) have shown in rescue experiments in mice that supplementing a higher methionine diet provides folate cycle precursors to rescue the high insulin phenotype in SR-deficient mice. Since 1C metabolism is being discussed in this manuscript, the authors seem to overlook prior work in the field and not include it in their discussion, even when it is the same enzyme (SR) that synthesizes both serine and cysteine. Since the field of D-amino acid research is in its infancy, the authors must make it a point to include prior work related to D-serine at least, and not claim that it is not known. The known D-stereoisomers are not many, hence any progress in the area must include at least a discussion of the other structurally related stereoisomers.

(12) Racemases (serine and aspartate) in general are promiscuous enzymes and known to synthesize other stereoisomers in addition to D-serine, D-cysteine, and D-aspartate. A few controls, like D-aspartate, D-cysteine, or even D-alanine must be included in their study to demonstrate the specific actions of D-serine, especially in the N2a cell treatment experiments. Cysteine and Serine are almost identical in structure (sulfur versus oxygen atom), and both are synthesized by serine racemase (published). Cysteine has also been very recently shown to inhibit tumor growth and neural progenitor cell proliferation. (PMIDs: 40797101 and 34556581). How the authors' work relates to the existing findings must be discussed, and this would put things in perspective for the reader.

Reviewer #2 (Public review):

Summary:

This study by Suzuki et al. reports an interesting stereo-selective role of D-serine in regulating one-carbon metabolism during neurodevelopment to adapt the functional transition, probably through the competition with mitochondrial transport of L-serine. The authors provide a multi-layered set of evidence, including metabolomics, enzyme assays, mitochondrial transport competition, and functional assays in immature/neural progenitor cells, to build up a conceptual integration of D-serine as both a neurotransmitter and a metabolic regulator in the central neural system, which raises a broad potential interest to the neuroscience and metabolism communities.

Strengths:

This work provides a conceptual advance that D-serine not only serves as a traditional neurotransmitter in the central neural system but also critically contributes to metabolic regulation of neural cells. The authors performed solid metabolomic assays to validate the suppressive effect of D-serine on the one-carbon metabolic pathway, providing some evidence that D-serine competitively inhibits mitochondrial serine transport, but not directly impairs SHMT2 enzymatic activity. All these data indicate a critical role of D-serine synthesis during neural maturation and suggest a potential translational strategy for targeting serine metabolism in neural tumors.

Weaknesses:

(1) The detailed mechanism by which D-serine competes with L-serine for its mitochondrial transport is not investigated. For example, although the authors made some discussion, they did not provide direct genetic or biochemical evidence linking these effects to the specific transporters, such as SFXN1.

(2) Unlike tumor cells, where SHMT2 usually plays a predominant role in catalyzing serine/THF-derived one-carbon metabolism, normal cells may employ both SHMT1 and SHMT2 to do the work. Even under certain conditions that SHMT2-mediated one-carbon metabolism is suppressed, the activity of SHMT1 could be elevated for compensation. Thus, it is important to investigate whether D-serine affects SHMT1 activity or changes the balance between SHMT1- and SHMT2-mediated one-carbon metabolism. To this aim, the authors are strongly encouraged to perform a metabolic flux assay (MFA) by using 13C-labeled L-serine in the model cells in the presence and absence of D-serine.

(3) A defect in serine-derived one-carbon metabolism may cause multiple cellular stress responses. It is valuable to detect whether cellular NADPH/NADH, GSH, or ROS is altered before and after D-serine treatment.

(4) The physiological relevance between D-serine and neural cell maturation/death should be further tested and discussed, since the dosage of D-serine used in the in vitro assay is much higher than that in physiological conditions.

Reviewer #3 (Public review):

Summary:

This manuscript presents a comprehensive and well-executed investigation into the metabolic role of D-serine in the central nervous system. The authors provide solid evidence that D-serine competitively inhibits mitochondrial L-serine transport, thereby impairing one-carbon metabolism. This stereoselective mechanism reduces glycine and formate production, suppresses cellular proliferation, and induces apoptosis in immature neural cells and glioblastoma stem cells. Developmental analyses further reveal a physiological enantiomeric shift in serine metabolism during neurogenesis, aligning with the transition from proliferation to maturation. Overall, the study bridges developmental neurobiology, cancer metabolism, and amino acid transport, uncovering a previously unrecognized metabolic function of D-serine beyond its role in neurotransmission.

Strengths:

(1) The discovery that D-serine inhibits one-carbon metabolism by competing for mitochondrial L-serine transport-rather than through enzymatic inhibition or receptor-mediated signaling-represents a significant and previously underappreciated mechanism. This finding has broad implications for understanding metabolic regulation during neurodevelopment and offers potential relevance for targeting metabolic vulnerabilities in cancer.

(2) The authors integrate metabolomics, mitochondrial transport assays, molecular dynamics simulations, genetic and pharmacologic perturbations, transcriptomics, and both in vitro and ex vivo models. The breadth of experimental approaches, combined with the coherence of the findings across systems, provides strong support for the central conclusions and enhances the overall impact of the study.

(3) The temporal shift in D-/L-serine levels during neurodevelopment is elegantly linked to the transition from proliferative to mature neuronal states. The selective vulnerability of neural progenitors and tumor cells-contrasted with the resistance of mature neurons-highlights a biologically meaningful and potentially targetable metabolic distinction.

Weaknesses:

(1) While the authors attribute D-serine's metabolic effects to competition with mitochondrial L-serine transport, the specific identity of the transporter(s) mediating this process remains undefined. This represents a meaningful mechanistic gap, as the central conclusion depends on D-serine limiting mitochondrial L-serine availability to inhibit one-carbon metabolism.

(2) The effective concentrations of D-serine used in vitro (IC₅₀ ≈ 1-2 mM) exceed typical brain levels (~0.3 mM). While the authors acknowledge this, a more focused discussion on whether higher local D-serine concentrations could arise in specific microenvironments - such as synaptic compartments, tumor niches, or pathological states-would help contextualize the in vitro findings and strengthen their physiological relevance. For example, disruptions in D-serine clearance or altered expression of serine racemase and transporters in disease contexts could lead to localized accumulation. Moreover, differences between extracellular and intracellular D-serine pools - and the mechanisms governing their regulation - may further influence its metabolic impact in vivo.

(3) While the manuscript focuses on neural stem/progenitor cells and neural tumors, it remains unclear whether the anti-proliferative effects of D-serine are specific to neural lineages or extend to other highly proliferative non-neural cell types. A brief discussion addressing this point would help clarify the scope of D-serine's metabolic impact and whether its mechanism of action reflects a unique vulnerability in neural cells or a more general feature of proliferative metabolism. This distinction is particularly relevant for assessing the broader therapeutic potential of targeting mitochondrial L-serine transport.