ATM phosphorylation of CD98HC increases antiporter membrane localization and prevents chronic toxic glutamate accumulation in Ataxia telangiectasia

Abstract

Ataxia telangiectasia (A-T) is a rare genetic disorder characterized by neurological defects, immunodeficiency, cancer predisposition, radiosensitivity, decreased blood vessel integrity, and diabetes. ATM, the protein mutated in A-T, responds to DNA damage and oxidative stress, but its functional relationship to the progressive clinical manifestation of A-T is not understood. CD98HC chaperones cystine/glutamate (x_c ^-) and cationic/neutral amino acid (y⁺L) antiporters to the cell membrane, and CD98HC phosphorylation by ATM accelerates membrane localization to acutely increase amino acid transport. Loss of ATM impacts tissues reliant on SLC family antiporters relevant to A-T phenotypes, such as endothelial cells (telangiectasia) and pancreatic α-cells (fatty liver and diabetes) with toxic glutamate accumulation. Bypassing the antiporters restores intracellular metabolic balance both in ATM-deficient cells and mouse models. These findings provide new insight into the long-known benefits of N-acetyl cysteine to A-T cells beyond oxidative stress through removing excess glutamate by production of glutathione.

Figure 1. ATMi impacts mitochondrial function and glutamine oxidation in HUVECs.

(A) Correlation analysis between expression of ATM and gene sets involved in mitochondrial function from a cohort of normal human tissues (RNA-Seq counts provided by ARCHS database). (B) Representative blot showing the efficiency of ATMi in HUVECs treated with H₂O₂. (C) Flow cytometry chart showing intracellular ROS levels after KU55933 treatment (histogram shows one representative sample per condition). (D) Quantification of ROS levels in C; H₂O₂ is used as a positive control. Data represented as median ± 95%CI. (E) Representative graph showing HUVEC’s metabolic potential in response to stressors (Oligomycin and FCCP) after KU55933 and NAC treatments. (F) Representative graph showing mitochondrial function assay after KU55933 treatment, combined with NAC or Trolox. (G) Chart showing maximal respiration obtained in F. (H) Correlation analysis between expression of ATM and genes involved in glutamine deprivation (ARCHS database). (I) Representative graph showing mitochondrial function assay after specific inhibition of glucose, glutamine, or fatty acids oxidation in the presence or absence of ATMi. (J) Chart showing maximal respiration and spare respiratory capacity after inhibition of glutamine oxidation. Data represented as average ± SD. *p<0.05, **p<0.01, ***p<0.001.

Figure 2. ATMi rewires metabolism leading to glutamate accumulation and glutathione depletion.

(A) Schematic of the ¹³C₆-glucose tracing showing the major significant changes observed in glycolysis and TCA cycle after ATMi in HUVECs. (B-C) Charts showing intracellular glutamate and GSH levels in cells treated with KU55933, NAC, and in combination. (D) Chart showing mRNA levels and blots showing protein levels of GCLc and GCLm after ATMi. Data represented as average ± SD. *p<0.05, ***p<0.001, ****p<0.0001.

Figure 3. ATM modulates the xc− antiport system through phosphorylation of CD98HC.

(A) Representation of the x_c⁻ antiport involved in cystine/glutamate transport (created using SMART; www.smart.servier.com). (B) ¹⁴C-L-cystine uptake after treatment with KU55933 or KU60019; SAS and Erastin are used as specific inhibitors of x_c⁻. (C) Fluorometric analysis of extracellular levels of glutamate after ATMi. (D) Chart showing GSH levels measured at different times following ATMi, x_c⁻ inhibition (SAS) or the combination (n=2). Data represented as average ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (E) Table showing the highly conserved SQ site in SLC3A2. (F) ATM consensus motif (www.phosphosite.org). (G) Representative images of proximity ligation assay (PLA) showing the interaction between CD98HC and ATM, shRNA-ATM cells were used to show signal specificity (Magnification: 80X and 200X for insets). (H) Dot blot showing binding specificity of the newly synthesized phospho-antibody. (I) Blots to further confirm antibody specificity in cell lysates treated with alkaline phosphatase (n=2). (J) Subcellular fractionation after 2h of treatment with H₂O₂. MEK1/2 and LAMIN B1 were used as cytoplasmic and nuclear markers, respectively._-(K) Blots showing the detection of phosphorylated CD98HC, its induction by H₂O₂, and inhibition by KU60019. (L) Representative images of the photoconversion assay in HEK293 cells transfected with SLC3A2 wild type or phospho-dead (scale bar: 5μm). The line plots on the right show averaged fluorescence intensity from several cell cross-section profiles (also see Figure S4B). (M) Chart showing quantification of fluorescence intensity 24h after photoconversion (n=15, from two independent experiments).

Figure 4. ATM phosphorylation of CD98HC impacts angiogenesis.

(A) Drawing showing one of the two antiport systems involved in arginine uptake in endothelial cells (created using SMART). (B) Representative chart showing ¹⁴C-L-arginine uptake after ATMi. (C) Picture showing the parameters evaluated in the vessel formation assay. (D) Representative images of the angiogenesis assay. (E-F) Quantification of images using the Angiogenesis Analyzer from Image J. Data represented as average ± SD. *p<0.05, **p<0.01, ***p<0.001.

Figure 5. Pancreatic a and b cells are highly sensitive to ATM and x_c⁻-inhibition.

(A) Simplified illustration showing TCA cycle and ATP levels modulating insulin and glucagon release (created using SMART). (B) Ridge plots showing ATM and SLC3A2 heterogeneous expression in human pancreatic cells (data obtained from multiple scRNA-Seq studies). (C) Box plots showing quantile-normalized ATM and SLC3A2 expression in different human pancreatic cell types. (D) Representative confluency charts of a and b cells following ATMi and x_c⁻ inhibition (Erastin). (E-F) Intracellular GSH and glutamate levels after ATMi in a and b cells. (G) Representative blots showing CD98HC phosphorylation (S103) after H₂O₂ and ATMi in a and b cells. (H-I) Basal respiration of a and b cells and glycolytic function of a cells after ATMi. (J) Glucagon and insulin secretion in a and b cells after ATMi/knockout. Data represented as average ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 6. ATM deficiency impairs pancreatic islet function leading to glucose intolerance and hepatic lipid accumulation.

(A-B) Intraperitoneal and oral glucose tolerance test in Atm^+/+ and Atm^−/− mice (n=4–6). (C) Glucose measurements after intraperitoneal injection of insulin (ITT, n=4–6). Data represented as average ± SEM. (D) Representative images and quantification of lipid levels by Oil Red O staining in the liver from 6-month-old Atm^+/+ and Atm^−/− mice (n=5–6) and A-T patients. Data represented as average ± SD. (E) Representative images of glucagon staining in pancreas from Atm^+/+ and Atm^−/− mice and resultant quantification (each dot represents a single islet, n=5–8). (F-G) Representative images and quantification of glutamate and glutamine staining in pancreatic islets of Atm^+/+ and Atm^−/− mice (each dot represents a single islet, n=5–8). Data represented as median ± 95%CI. (H) Mitochondrial respiration of pancreatic islets isolated from 6-month-old Atm^+/+ and Atm^−/− mice (n=4–5). (I) Parameters evaluated in H show glucose response and mitochondrial performance in Atm^+/+ vs. Atm^−/− mice (each dot represents an animal). Data represented as average ± SEM. *p<0.05, **p<0.01, ****p<0.0001.

Figure 7. NAC supplementation rescues the metabolic defects shown in ATM-deficient mice.

(A-B) Intraperitoneal and oral glucose tolerance test in Atm^+/+ and Atm^−/− mice supplemented with NAC (n=4–8). Data represented as average ± SEM. (C) Representative images of Oil Red O staining in the liver from 6-month-old Atm^+/+ and Atm^−/− mice supplemented with NAC. (D) Representative images of glucagon staining in pancreas from Atm^+/+ and Atm^−/− mice and resultant quantification (each dot represents a single islet, n=3–8). (E) Representative images and quantification of glutamate staining in pancreatic islets of Atm^+/+ and Atm^−/− mice (each dot represents a single islet, n=3–8). Data represented as median ± 95%CI. *p<0.05, ***p<0.001, ****p<0.0001.