Dear Editor,
The developing cerebral cortex requires precise metabolic regulation to support neurogenesis and circuit formation (
Belanger et al., 2011;
Namba et al., 2021). Glutamine synthetase (GS), which catalyzes glutamate-to-glutamine conversion, sustains neurotransmitter recycling and nitrogen homeostasis (
Tani et al., 2014). Human
GLUL mutations cause lethal neurodevelopmental disorders (
Häberle et al., 2005), and GS deletion in mice leads to postnatal death (
He et al., 2010), underscoring its essential role. While adult cortical GS deficiency triggers neurodegeneration (
Zhou et al., 2019), its function in early cortical development remains elusive, despite evidence linking astrocytic metabolism to circuit maturation (
Chung et al., 2015;
Pekny et al., 2016).
Spatiotemporal analysis revealed high GS expression in embryonic neural stem cells (NSCs) within the ventricular/subventricular zone, transitioning to S100β+ astrocytes postnatally (Figs. 1A–D and S1A–C). GS enzymatic activity followed a biphasic pattern, peaking during early postnatal stages coincident with astrocyte maturation (Fig. S1D), suggesting stage-specific roles in cortical development.
To dissect the
in vivo function of GS, we generated cortex-specific GS knockout (GS-cKO) mice by crossing
Glulflox/flox mice with
Emx1-Cre mice (Fig. 1E). This strategy induces gene ablation in radial glial progenitors from embryonic day 10.5 onward while preserving postnatal viability, thereby enabling examination of GS function across developmental stages (
Gorski et al., 2002). Efficient GS deletion was confirmed in both embryonic and postnatal forebrains (Figs. 1F and S1E–G). By postnatal day 14 (P14), GS-cKO mice exhibited reduced brain size and cortical thickness (Fig. S1H–K), accompanied by notable astrocytic morphological abnormalities, as revealed by AAV2/9-GFAP-mCherry labeling (Fig. 1G–I). In contrast, embryonic neurogenesis and neuronal migration remained largely intact (Figs. 1J, 1K, and S2A–I). Although early GS loss mainly impaired astrocyte maturation, by P28, the cortex of GS-cKO mice displayed pronounced astrocyte activation, characterized by robust GFAP upregulation, cellular hypertrophy, and thickened astrocytic processes, indicative of reactive gliosis (Fig. S3A–C). Notably, dietary glutamine supplementation partially restored astrocyte maturation, as evidenced by improved morphological complexity and enhanced process elaboration (Fig. S4A–C), highlighting the essential role of GS-derived glutamine in supporting astrocytic structural development.
GS deficiency severely impaired dendritic growth, spine density, and synaptogenesis, as shown by EGFP electroporation, Golgi staining, and synaptic marker analysis (Figs. 1M, 1N, 1Y, 1Z and S4D). Electrophysiological recordings confirmed reduced miniature excitatory and inhibitory synaptic activity (Fig. 1O–T). Remarkably, glutamine supplementation restored synaptic density, indicating metabolite dependence of GS-mediated circuit maturation (Fig. S4E–H). Consistently, GS-cKO mice displayed behavioral abnormalities in motor coordination (Figs. S5A–E, S5G, and S5H) and social interaction (Figs. 1U–X and S5F), paralleling behavioral deficits in autism and epilepsy models (
Varghese et al., 2017;
Sharma et al., 2018).
Astrocyte development was prominently disrupted in GS-cKO mice. GS deficiency reduced S100β+, Aldh1L1+, Sox2+, and Sox9+ cell numbers (Fig. 2A–H) without increasing apoptosis (Fig. S6A and S6B). Single-nucleus RNA sequencing (snRNA-seq) of P7 cortex identified major cell types but showed no change in excitatory neuron or astrocyte proportions between genotypes (Fig. 2I–K). However, analysis of astrocyte-specific gene expression revealed distinct alterations, with a downregulation of Blbp (a key regulator of astrocyte maturation) and an upregulation of ApoE (a lipid transporter implicated in neuroinjury response), respectively; in contrast, the glutamate transporter Slc1a3 (GLAST) remained unchanged in GS-cKO astrocytes (Figs. 2L, S6H, S6J, and S6M). These changes emerged by P1 (Fig. S6C–F) and were confirmed by reduced Blbp protein and increased ApoE mRNA via immunostaining and in situ hybridization (Fig. S6H–O).
Metabolically, GS loss reduced glutamine, glutamate, aspartate, and asparagine, while elevating glycine and alanine (Fig. 2M and 2N). snRNA-seq revealed selective suppression of the mTOR pathway in astrocytes (Fig. 2O and 2P). Western blotting confirmed decreased phosphorylation of S6 (mTORC1) and AKT (mTORC2), with key mTOR pathway genes (Atp6v1c1, Atp6v1g2, Eif4b) downregulated (Fig. 2P–X). Thus, GS deficiency disrupts cortical amino acid homeostasis and suppresses astrocytic mTOR signaling, likely underlying the observed astrocyte dysfunction and neuronal deficits.
Our findings establish GS as a key regulator of cortical circuit maturation through astrocyte metabolic programming. GS expression shifts from NSCs to astrocytes during cortical expansion, indicating stage-specific roles. Neurogenesis remains unaffected, likely due to maternal or placental glutamine supply (
Wu et al., 2015), but postnatal GS deficiency profoundly impairs astrocyte maturation and suppresses mTOR signaling, suggesting that mTOR inactivation is central to the observed developmental deficits.
As a metabolic sensor, mTOR critically depends on intracellular amino acid availability. Glutamine and its metabolites (e.g., α-ketoglutarate and arginine) not only directly activate mTORC1 but also fuel mTOR-dependent anabolic processes (
Jewell et al., 2015). Thus, GS loss removes both signaling input and metabolic support for mTOR, leading to selective pathway impairment. In contrast, the Hippo/YAP pathway, although sensitive to energy stress (
Sheng et al., 2024), lacks a glutamine-sensing mechanism and is therefore less affected. This specificity explains why mTOR signaling is preferentially disrupted in GS-deficient astrocytes.
mTOR suppression likely compromises astrocytic metabolic support, reducing protein synthesis and precursor provision for nucleotide and neurotransmitter production. Such deficits during critical developmental windows disrupt synaptogenesis and circuit refinement, offering a mechanistic link between GS–mTOR dysregulation and neurodevelopmental disorders, such as specific autism spectrum disorder subtypes. Meanwhile, upregulation of ApoE may initially serve as a compensatory response to metabolic stress but, when prolonged, can lead to lipid dysregulation, toxic protein accumulation, and increased susceptibility to neurodegenerative conditions, consistent with APOE ε4-associated Alzheimer’s disease (
Zheng and Wang, 2025).
Our observations align with the previously reported
Glul conditional knockout model (
Zhou et al., 2019), which also exhibited seizures, motor deficits, and reactive astrogliosis. Although our study documents cortical thinning earlier and more quantitatively, careful review of their data suggests similar trends. Differences in phenotypic presentation likely reflect variations in timing and targeting strategies; nevertheless, both models converge on severe astrogliosis by the fourth postnatal week, highlighting GS loss as a potent driver of neuroinflammatory pathology.
In conclusion, this study uncovers an essential role for GS in postnatal brain development and reveals how a metabolic enzyme can regulate cell-specific gene expression, protein translation, and morphology. These insights offer new therapeutic perspectives for treating neurological disorders involving astrocytic dysfunction and impaired cortical connectivity, such as epilepsy and autism.
Footnotes
We thank the members of the Qin laboratory for their valuable suggestions and comments. This work was supported by grants from the National Natural Science Foundation of China (Nos. 31871477, 32170971), the Natural Science Foundation of Shanghai (No. 18ZR1403800) awarded to S.Q.; as well as grants from the CAMS Innovation Fund for Medical Sciences (CIFMS, No. 2024-I2M-ZD-012), the STI2030-Major Projects (No. 2022ZD0204700), and the National Natural Science Foundation of China (No. 32170964) awarded to W.-P.G.
The authors declare that they have no competing interests, financial or non-financial, that could be perceived as influencing the work reported in this manuscript.
All animal procedures were approved by the Fudan University Institutional Animal Care and Use Committee and performed in accordance with institutional and national guidelines.
All authors have provided informed consent for participation and confirmed their agreement to publish this work.
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
S.Q., W.P., J.G., and S.D. conceived the project. S.Q., W.P., and P.G. designed experiments. P.G. performed most of the experiments. X.C. performed single-cell analysis. W.C. performed electrophysiological experiments. W.H., Y.L., G.Q., X.S., Z.W., and Z.L. assisted with the experiments and helped to analyze the data. P.G. and S.Q. wrote the article.
The Author(s) 2026. Published by Oxford University Press on behalf of Higher Education Press.
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