Clustered de novo start-loss variants in GLUL result in a developmental and epileptic encephalopathy via stabilization of glutamine synthetase

Abstract

Glutamine synthetase (GS), encoded by GLUL, catalyzes the conversion of glutamate to glutamine. GS is pivotal for the generation of the neurotransmitters glutamate and gamma-aminobutyric acid and is the primary mechanism of ammonia detoxification in the brain. GS levels are regulated post-translationally by an N-terminal degron that enables the ubiquitin-mediated degradation of GS in a glutamine-induced manner. GS deficiency in humans is known to lead to neurological defects and death in infancy, yet how dysregulation of the degron-mediated control of GS levels might affect neurodevelopment is unknown. We ascertained nine individuals with severe developmental delay, seizures, and white matter abnormalities but normal plasma and cerebrospinal fluid biochemistry with de novo variants in GLUL. Seven out of nine were start-loss variants and two out of nine disrupted 5' UTR splicing resulting in splice exclusion of the initiation codon. Using transfection-based expression systems and mass spectrometry, these variants were shown to lead to translation initiation of GS from methionine 18, downstream of the N-terminal degron motif, resulting in a protein that is stable and enzymatically competent but insensitive to negative feedback by glutamine. Analysis of human single-cell transcriptomes demonstrated that GLUL is widely expressed in neuro- and glial-progenitor cells and mature astrocytes but not in post-mitotic neurons. One individual with a start-loss GLUL variant demonstrated periventricular nodular heterotopia, a neuronal migration disorder, yet overexpression of stabilized GS in mice using in utero electroporation demonstrated no migratory deficits. These findings underline the importance of tight regulation of glutamine metabolism during neurodevelopment in humans.

Keywords: GLUL; degron motif; epileptic encephalopathies; glutamine metabolism; glutamine synthetase.

Graphical abstract

Figure 1

GLUL start-loss and splice site variant location (A) Schematic of GLUL gene structure. The location of individual start-loss and splice variants are indicated relative to the first ATG (Met1) (green); variant c.1A>G (n = 4) was recurrent in this cohort. Key methionine amino acid residues (green) and lysine amino acid residues of the degron motif (orange) are indicated. (B) An intronic variant in GLUL disrupts 5′ UTR splicing resulting in a 26 base pair deletion. Splice AI predicted a deletion of 26 base pairs resulting in the exclusion of 5′ UTR exon 2 and the first 13 base pairs of coding exon 1 (orange). Sequencing of dermal fibroblast RNA with c.−13−2A>G variant demonstrated an alternatively spliced isoform with a 26 base pair deletion, which removed 13 base pairs of 5′ UTR exon 2 and 13 base pairs of coding exon 1 and therefore excised the canonical start site. Dark boxes, coding exons; light boxes, UTR exons.

Figure 2

Start-loss variants in GLUL result in initiation of translation at methionine¹⁸ (A) Fibroblasts from individuals heterozygous for GLUL start-loss variants produce two GS isoforms. Lysates were prepared from three fibroblast cell lines from individuals heterozygous for start-loss variants (individuals 1, 6, and 8) and sex- and age-matched fibroblast controls. Western blotting was performed with GS and GAPDH antibodies (n = 3). (B) Full-length GS migrates at a higher molecular weight than start-loss GS. Full-length GS^FLAG/Strep and start-loss GS^FLAG/Strep expression constructs were transfected into HEK293_GLULKO cells. Cell extracts were probed by western blotting with FLAG and GAPDH antibodies (n = 3). (C) Full-length, start-loss, and methionine¹⁸ control GS expression constructs are stably expressed. Full-length, start-loss, and met¹⁸ GS^FLAG/Strep expression constructs were individually transfected into HEK293_GLULKO cells. Cell extracts were analyzed by western blotting with FLAG antibodies and duplicate wells were loaded. (D) N-terminal start-loss GS sequence aligns with GS_met¹⁸ sequence. GS expression constructs were transfected in HEK293_GLULKO cells. Following affinity purification and chymotrypsin enzymatic digestion, peptides were identified by MS/MS. Identified sequences are shown as dark green boxes. The most N-terminal peptide sequence and amino acid peptide positions are indicated. FL, full-length; SL, start-loss c.1A>T variant; met¹⁸, first 17 amino acids truncated.

Figure 3

Met¹⁸ GS is stable and full-length GS is unstable in high glutamine conditions (A) Schematic of cycloheximide assay protocol. Full-length and met¹⁸ GS expression constructs with C-terminal 3xFLAG- Twin-Strep-tags were transfected in HEK293_GLULKO cells. Twelve hours later, a complete media replacement was done with DMEM supplemented with high (10 mM) or low (1 mM) glutamine and treated with 100 μg/ml CHX or DMSO. Cell lysates were sampled after 6 h for western blot analysis with FLAG and GAPDH antibodies. Fold reduction was calculated as the proportion of GS in DMSO treatment relative to CHX treatment. (B) Western blots of full-length and met¹⁸ GS expression constructs in high and low glutamine. Full-length and met¹⁸ GS expression constructs were transfected into HEK293_GLULKO cells and the protocol outlined in panel A followed (n = 4). (C) Full-length GS has a significantly higher fold reduction in high glutamine than in low glutamine whereas there is no difference in fold reduction of met¹⁸ GS between high and low glutamine. Following quantification of western blot bands, fold reduction was calculated as the proportion of untreated-to-CHX-treated GS for each glutamine condition. The bar height represents average fold reduction with standard error of the mean presented. Each circle represents a biological replicate. p values were calculated using an unpaired two-tailed Student’s t test (n = 4). (D and E) Subject-derived fibroblasts and age- and sex-matched control fibroblasts were treated with or without 10 mM glutamine for 72 h, and then the protein amount of the full-length GS and the truncated GS were analyzed by quantitative immunoblot. (D) Immunoblot of GS and beta-actin (loading control). Individuals 1 and 6 cells carry a variant at the start codon. Individual 8 cells have a variant that leads to alternative splicing. Controls 1, 2, 3 are the age- and sex-matched control cells. All subject cells express both the full-length (gray arrowheads) and the truncated GS (pink arrowheads). (E) Ratio of the full-length and the truncated GS in the cells cultured with 10 mM glutamine to those cultured without glutamine. The GS band intensity was normalized by the respective beta-actin band control. Gray bars and circles indicate the full-length GS; pink bars and circles indicate the truncated GS. Each circle represents a biological replicate. Error bars indicate standard deviation. Normality was determined with the Shapiro-Wilk test, p values were calculated with a one-way ANOVA, and post-hoc testing was done using the Tukey method (n = 3). (F) Full-length, met¹⁸, and enzymatically compromised p.Arg324Cys and p.Arg341Cys GS expression constructs were transfected into HEK293_GLULKO cells for enzyme activity measurement. Following affinity purification and buffer exchange, enzymatic activity of GS was measured following manufacturer protocols. The enzymatic activity is plotted as a proportion of full-length GS activity. Error bars indicate the standard error of the mean. Each circle represents a biological replicate. p values were calculated using an unpaired two-tailed Student’s t test (n = 3). FL, full-length; met¹⁸, first 17 amino acids truncated; Gln, glutamine; DMSO, dimethyl sulfoxide; CHX, cycloheximide; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, NS, not statistically significant.

Figure 4

Human cerebral cortex GLUL expression at single-cell resolution (A) Single-nucleus RNA-seq from human prefrontal cortical cells. Data from Herring et al. Samples obtained between mid-gestation (gestational week 22) to adulthood (40 years old). (i) UMAP of snRNA-seq with 10 cell clusters colored and annotated. (ii) GLUL gene expression overlaid onto UMAP plot of cell clusters. (iii) Violin plot of GLUL expression grouped by cell cluster. (B) Single-cell RNA-seq from EGRF⁺ cells sampled from human cerebral cortices (between gestational week 21–26). Data from Fu et al. (i) UMAP of scRNA-seq with 11 cell clusters colored and annotated. (ii) GLUL gene expression overlaid onto UMAP plot of cell clusters. (iii) Violin plot of GLUL expression grouped by cell cluster.