Warburg effect linked to cognitive-executive deficits in FMR1 premutation

Abstract

A 55-200 CGG repeat expansion in the 5'-UTR of the fragile X mental retardation 1 (FMR1) gene is known as a premutation. Some carriers are affected by the neurodegenerative disorder fragile X-associated tremor/ataxia syndrome (FXTAS), primary ovarian insufficiency, and neurobehavioral impairments. Based on the mitochondrial dysfunction observed in fibroblasts and brain samples from carriers, as well as in neurons and brains from a mouse model of the premutation, we evaluated the presence of the Warburg effect in peripheral blood mononuclear cells (PBMCs) from 30 premutation carriers with either a rebalance of the metabolism [increasing glycolysis while decreasing oxidative phosphorylation (oxphos)] or a metabolic amplification (increasing glycolysis while maintaining/increasing oxphos). Deficits in oxphos-more pronounced in FXTAS-affected subjects-were accompanied by a shift toward glycolysis, suggesting increased glycolysis despite aerobic conditions. Differential proteomics extended these findings, unveiling a decreased antioxidant response, translation, and disrupted extracellular matrix and cytoskeleton organization with activation of prosenescence pathways. Lower bioenergetics segregated with increased incidence of low executive function, tremors, below-average IQ, and FXTAS. The combination of functional and proteomic data unveiled new mechanisms related to energy production in the premutation, showing the potential of being applicable to other psychiatric disorders to identify endophenotype-specific responses relevant to neurobiology.-Napoli, E., Song, G., Schneider, A., Hagerman, R., Eldeeb, M. A. A. A., Azarang, A., Tassone, F., Giulivi, C. Warburg effect linked to cognitive-executive deficits in FMR1 premutation.

Keywords: fragile X; mitochondria; neurological disorder; proteomics; triplet nucleotide diseases.

Figure 1.

Oxygen consumption, citrate synthase activity, and RCR in PBMCs. A–E) oxphos measurements [NADH oxidase (A), succinate oxidase (B), α-glycerophosphate oxidoreductase (C), and cytochrome c oxidase activities (D)] were performed along with citrate synthase activity (E). Rates, normalized by citrate synthase, are shown in Table 2. F) The RCRu is reported as the ratio of oxygen uptake in the presence of FCCP and oligomycin in nonpermeabilized cells. Data were obtained from PBMCs from 12 controls, 21 asymptomatic premutation carriers, and 9 premutation carriers affected with FXTAS. Measurements were performed in duplicate for each sample (means ± sem). Statistical analysis was performed with ANOVA followed by Tukey’s post hoc test. P values are relative to controls.

Figure 2.

Dependence of mitochondrial outcomes on donor’s age and CGG repeats. A) The oxygen uptake of intact PBMCs (12 controls and 30 premutation carriers) was evaluated with 10 mM glucose in PBS. Oxygen uptake was recorded under these conditions (total oxygen consumption): addition of 2 µM oligomycin allowed the evaluation of the oxygen uptake resistant to this complex V inhibitor; subsequent addition of 20 nM of the uncoupler FCCP allowed assessment of the maximum oxygen uptake by the ETC when not coupled to ATP production, used for the evaluation of the SRC. Values were expressed as percentages of basal rates (means ± sem). Statistical analysis was performed with Student’s t test. B, C) Citrate synthase was plotted vs. the age (B) and CGG repeats (C) of the donor. D) The RCR plotted against the CGG repeats. The corresponding correlation parameters (Pearson’s r and P values) are also shown. Dotted lines: 95% CI (B–D).

Figure 3.

Segregation of clinical symptoms with mitochondrial function. The 95% CIs were calculated with control values for each mitochondrial outcome significantly different between controls and carriers of the premutation (citrate synthase activity and coupling). A) According to these cutoffs, all subjects were classified into 3 clusters, defined as no MD, mild MD, and severe MD. B) Means ± sem of citrate synthase activity and RCRu for each group. The P values (obtained by ANOVA followed by Tukey’s post hoc test for multiple comparisons) are as follows: 0.032 (c); 0.016 (a); and <0.0001 (b, d, e). C) The frequency of each clinical symptom (shown in Table 1) was counted within each of the 3 clusters (no MD, with mild MD, or with severe MD; A) and shown as a percentage of symptomatic individuals within any given class. Low IQ was set at <90. Executive function was considered impaired if ≤14. Statistical analyses were performed by the 1-tailed χ² test with Yates correction. Statistically significant clinical outcomes that segregated with MD were (in decreasing order of significance) low executive function (as judged by BDS-2; P = 0.0008), low IQ (P = 0.006), tremors (P = 0.045), and presence of FXTAS symptoms (P = 0.049). For these outcomes, the comparison of proportions between presence and absence of individual clinical symptoms was also computed. P values are as follows: 0.050 (d); 0.049 (g); 0.047 (e); 0.045 (h); 0.024 (c); 0.015 (f); 0.006 (a); and 0.0007 (b). D) Means ± sem of CGG repeats, IQ, and executive function scores (as judged by BDS-2) in the 3 groups. The P values (obtained by ANOVA followed by Tukey’s post hoc test for multiple comparisons) are as follows: 0.028 (a); 0.02 (e); 0.006 (d); 0.004 (c); and 0.002 (b).

Figure 4.

Dysregulation of glycolysis and bioenergetic pathways in PBMCs from asymptomatic and FXTAS-symptomatic premutation-bearing individuals. In control cells, glucose is mainly used by oxphos, followed by glycolysis (as judged by ATP production) with almost negligible values shunted to the pentose phosphate pathway. The production of ATP is mainly derived from mitochondria compared to that of glycolysis (2-fold). In cells from premutation carriers, the glycolytic flux is enhanced with glucose also shunted to the pentose phosphate pathway to generate NADPH to provide reducing equivalents to the antioxidant defenses. Some pyruvate is directed to mitochondria in cells from FXTAS-free premutation but, because of higher uncoupling and lower citrate synthase of these cells compared to controls, the ratio of mitochondrial ATP to glycolytic is 0.6. Proteomic data supported a more predominant role for the second half of the Krebs cycle (from α-ketoglutarate to oxaloacetate) probably sustained by the anaplerosis of Glu/Gln (as judged by the higher abundance of glutamate dehydrogenase). The ensuing lower production of citrate may result in lower fatty acid and cholesterol syntheses as well as arachidonate-derived products (prostaglandins). Oxaloacetate will be then used to generate Asp, Ala, pyruvate, and others. In the case of cells from carriers affected with FXTAS, a more significant flux of glucose is shunted to the pentose phosphate pathway (higher abundance of glucose-6-phosphate-dehydrogenase than controls and FXTAS-free carriers) and glycolysis (higher levels of glycolytic ATP than controls but not than FXTAS-free carriers). The production of ATP is mainly glycolytic (ratio of mitochondrial ATP to glycolytic is 2.75) supported by the higher uncoupling (vs. controls and FXTAS-free carriers) and lower citrate synthase (vs. controls) of these cells. Based on proteomic data, lower overall Krebs cycle activity is expected with (based on functional data) increased production of mitochondrial ROS and increased proton leak. The total ATP levels are visualized as a bar in which the proportion of ATP derived from glycolysis is depicted in black and that of oxphos in white. The bars representing the ATP content in cells from carriers, are characterized as a percentage of that in control cells, to visualize the lower ATP content of these cells (61 and 49% of controls, for FXTAS-free and -affected carriers, respectively). Thicker and thinner arrows symbolize increased and decreased fluxes, respectively. Abbreviations: OA, oxaloacetate; AKG, α-ketoglutarate.