ATP Synthase c-Subunit Leak Causes Aberrant Cellular Metabolism in Fragile X Syndrome

Abstract

Loss of the gene (Fmr1) encoding Fragile X mental retardation protein (FMRP) causes increased mRNA translation and aberrant synaptic development. We find neurons of the Fmr1^-/y mouse have a mitochondrial inner membrane leak contributing to a "leak metabolism." In human Fragile X syndrome (FXS) fibroblasts and in Fmr1^-/y mouse neurons, closure of the ATP synthase leak channel by mild depletion of its c-subunit or pharmacological inhibition normalizes stimulus-induced and constitutive mRNA translation rate, decreases lactate and key glycolytic and tricarboxylic acid (TCA) cycle enzyme levels, and triggers synapse maturation. FMRP regulates leak closure in wild-type (WT), but not FX synapses, by stimulus-dependent ATP synthase β subunit translation; this increases the ratio of ATP synthase enzyme to its c-subunit, enhancing ATP production efficiency and synaptic growth. In contrast, in FXS, inability to close developmental c-subunit leak prevents stimulus-dependent synaptic maturation. Therefore, ATP synthase c-subunit leak closure encourages development and attenuates autistic behaviors.

Keywords: Fragile X syndrome; autism; autism syndrome; glycolysis; mitochondria; oxidative phosphorylation; permeability transition pore; protein synthesis; repetitive mouse behavior; synaptic development; synaptic plasticity.

Figure 1.. Fmr1^−/y mitochondria have an inner mitochondrial membrane leak.

(A, B) Representative electron microscopy images of brain slices show synapses from the CA1 region of the mouse hippocampus (WT and Fmr1^−/y). (C) Group data show a decrease in mitochondrial area in Fmr1^−/y compared to WT (N=15 for Fmr1^−/y and N=17 micrographs for WT, *p=0.043). (D) The vesicle number in presynaptic boutons is reduced in Fmr1^−/y (N=11 micrographs of each condition; *p=0.0143). (E, F) Example electron micrographs of synapses from the CA1 region of the mouse hippocampus slices (WT and Fmr1^−/y). (G) Mitochondrial area/synapse area is unchanged comparing WT to Fmr1^−/y (N= 13 micrographs for WT and 23 for Fmr1^−/y). (H) Group data show electron density is increased in Fmr1^−/y compared to WT (N=23 micrographs for Fmr1^−/y and 13 for WT; *p=0.0114). (I) Representative images of mitochondrial membrane potential indicator TMRM fluorescence in isolated cortical neurons. (J) Group data show TMRM intensity is reduced in FX compared to WT (N=20–24 neurons each condition, 4 independent cultures; ****p<0.0001). (K) Cytosolic ATP levels of isolated cortical neurons are reduced in FX compared to WT (N=3 wells for each, *p<0.05). (L) Illustration of method of measurement of bath H⁺ ion concentration using the H⁺ sensitive indicator ACMA. SMVs are illustrated with mitochondrial ATP synthase F₁ facing toward the bath. ATP hydrolysis causes H⁺ ion sequestration into the lumen of the vesicles. Vesicles are impermeant to the pH indicator. Red arrows show paths of H⁺ pumping (at the side of the c-subunit) and H⁺ leak (through the center of the c-subunit) (M) Lack of sequestration of H⁺ ions into Fmr1^−/y SMVs during ATP hydrolysis by the ATP synthase (N=3 samples per condition). (N) Representative patch clamp recordings of SMVs of Fmr1^−/y and WT at the indicated holding potential. Black trace indicates open channel. Red trace shows the relatively closed channel in the same recording after Dex exposure. (O) Current voltage relationship for the group of SMV recordings shown in (P). (P) Group data of peak conductances of the independent recordings (N=6 for WT and WT+Dex; N=8 for Fmr1^−/y and Fmr1^−/y +Dex) measured from 0 pA (*p=0.0449 comparing WT to WT+Dex; **p=0.0064 comparing Fmr1^−/y to Fmr1^−/y+Dex). Linear current voltage relationship was assumed for calculation of peak conductance. In Figs. 1C, D, G, H, J, K, unpaired two-tailed Student’s t-test was used. In Fig. 1M, two-way repeated measures ANOVA followed by Sidak’s multiple comparison test was used. In Fig. 1P, paired two-way Student’s t-test was used. Data are represented as mean ± SEM. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

Figure 2.. Expression of ATP synthase subunits causing inner membrane leak is increased in Fmr1^−/y mitochondria.

(A) c- and β- subunit protein expression levels are higher in Fmr1^−/y brain mitochondria compared to those of WT (N=8 independent samples for each, **p=0.0083 and *p=0.031). (B) An example of three independent experiments of non-denaturing Native-PAGE electrophoresis of isolated brain mitochondria. Immunoblotting performed with anti-c-subunit antibody. The abundance of the free c-subunit is higher in Fmr1^−/y mitochondria compared to WT. Left set of histograms show the level of fully assembled ATP synthase (monomer plus dimer) and the level of free c-subunit in Fmr1^−/y as a percent of WT control. Right histograms: Ratio of free c-subunit to its own assembled ATP synthase within the same lane. N=3 for each condition. (C) FMRP immunoprecipitation from isolated synaptosomes pulls down ATP synthase β-subunit mRNA but not ATP synthase c-subunit mRNA (ATP5G2) as detected by RT-PCR (shown is one of N=3 independent immunoprecipitation experiments). All experiments in this figure used unpaired Student’s t-test, data are represented as mean ± SEM. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

Figure 3.. Metabolic profile of FX cortical neurons shows enhancement of glycolysis/TCA flux in FX compared to WT.

(A) Glycolysis and tricarboxylic acid (TCA) cycle schematics illustrating enzymes involved in both pathways. Enzymes increased in FX>WT in at least 2 of 3 independent cultures are labeled in green. (B) Averaged spectral counts of metabolic peptides expressed in WT and FX cortical neuron cultures. Puromycin immunoprecipitates were analyzed by LC/MS/MS after cortical neuronal cultures (DIV14) were exposed to puromycin for 15 minutes. Shown in blue are the enzymes decreased by Dex treatment in at least 2 out of 3 FX cultures (N=3 independent cultures of each condition). Metabolic peptides completely removed by Dex treatment are indicated above the graph in blue lettering. (C) Lactate levels are elevated in the culture media collected from FX primary neurons compared to those of WT. Exposure of FX neurons to Dex significantly decreases lactate levels in the media. (D) Representative immunoblots of WT and FX cortical cultures exposed to vehicle or Dex. (E) Quantification of blots shown in (D). At least 3 independent cultures were used. A set of 4 key enzymes is elevated in FX compared to WT. Dex treatment normalizes the protein levels of all enzymes in the set. (F) Representative immunoblots of WT and Fmr1^−/y mitochondria isolated from brain. (G) Quantification of blots shown in (F). At least three animals per condition. Glycolytic enzymes and pyruvate dehydrogenase protein levels are elevated in Fmr1^−/y mitochondrial fractions. In Figs. 3C and E, two-way ANOVA followed by Tukey’s multiple comparisons test was used. In Fig. 3G, unpaired two-tailed Student’s t-test was used. Data are represented as mean ± SEM. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

Figure 4.. Inhibition of the mitochondrial inner membrane leak decreases protein synthesis in FX.

(A) c-subunit depletion in human FX fibroblasts decreases the rate of protein synthesis. Representative puromycin (top), c-subunit immunoblots (middle) and protein controls (bottom) are shown at left. Quantification of the immunoblots is shown at right. (B) c-subunit overexpression in human FX fibroblasts increases the rate of protein synthesis. Representative blots of puromycin and c-subunit are shown at left as in (A). Quantification of the immunoblots is shown at right. (C) Dex decreases protein synthesis rate in FX neurons. Representative blot of puromycin incorporation in FX cortical cultures is shown in the presence of different concentrations of Dex. Cultures were treated with Dex for 2–24 hr. (D) Group data for experiments shown in (C). The group data for WT translation rates are shown in (H). N=samples from at least 3 independent cultures. (E) Puromycin incorporation into WT and Fmr1^−/y after incubation of mouse brain slices with 10 μM Dex or vehicle for 2.5 hr. (F) Group data of experiments shown in (E). N=3–6 brain slices for each condition, at least three animals per condition. (G) Representative immunoblot of puromycin incorporation showing that the rate of protein synthesis in FX cortical neurons is increased over WT neurons; exposure to 0.2 μM CsA for 7 days reduced the rate of puromycin incorporation in FX neurons. (H) Quantification of the data shown in (G) N=samples from at least 3 independent cultures. (I-M) Increase in rosettes (indicating actively translating ribosomes) in Fmr1^−/y brains is normalized to WT level by CsA exposure. Representative electron micrographs of CA1 region of hippocampal brain slices of Fmr1^−/y mouse compared to WT (R, ribosomal rosettes; M, mitochondria). (K) shows a higher magnification of the actively translating ribosomes (rosettes) shown in (J). Parallel slices from the corresponding hemisphere were incubated in 0.2 μM CsA or vehicle for 2.5 hours. Scale bars as indicated. (N) Group data of 65–76 micrographs analyzed per condition. Figs. 4A, B used unpaired two-tailed Student’s t-test. Fig. 4D, one-way ANOVA followed by Tukey’s multiple comparisons test was used. For Figs. 4F, H, N, two-way ANOVA followed by Tukey’s multiple comparisons test was used. Data are represented as mean ± SEM. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

Figure 5.. The time course of synaptic stimulation-induced changes in protein synthesis and EF2 phosphorylation is disrupted in Fmr1^−/y synaptosomes, normalized by ATP synthase leak inhibition.

(A) Representative immunoblots of synaptosomal samples harvested at the indicated time points before and after 0.2 μM D-serine stimulation. Top panels show puromycin incorporation; middle panels show p-EF2 and EF2; bottom panels show ATP synthase β subunit and protein loading control (GAPDH). CsA restores the normal pattern of response to stimulation in Fmr1^−/y synaptosomes. (B-D) Group data for experiments shown in (A): (B) for puromycin incorporation, (C) p-EF2/EF2 protein levels and (D) ATP synthase β subunit protein levels. One-way ANOVA followed by Tukey’s multiple comparisons test was used for all panels in the figure. Synaptosomes were prepared from at least three independent animals per condition. N=samples. Data are represented as mean ± SEM. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001)

Figure 6.. ATP synthase leak inhibition enhances synaptic plasticity.

(A-D) Representative micrographs of primary neurons. Insets show dendritic shafts. (E) Dendritic spines were categorized according to their morphology into mature and immature spines. Illustration depicts subtypes of spines analyzed. Histograms show FX neurons have a reduced percentage of mature spines and an increased percentage of immature spines compared to WT. The types of spines are graphed as a percent of the total number of spines counted per unit length. N=5 neurons per condition from at least 2 independent cultures. (F) 5 μM Dex treatment each day for 6 consecutive days (DIV 15–20) caused an increase in the percent of mature dendritic spines / total spines per unit length in FX neurons. Dex treatment had no effect on WT neuron spine density. N= 5 neurons per condition from at least 2 independent cultures. (G) Dex normalizes dendritic ATP levels in stimulated FX neurons. ATP levels were measured in neurons at DIV 20 using FRET-based ATP reporter ATeam YEMK. Neurons were stimulated for three min. with 10 μM D-serine and ATP values were recorded at 1 hour after stimulation. Histogram shows ATP values at 1 hour after stimulation as a percentage of WT at 1 hour after stimulation. N=3 neurons per condition; 15–30 ROIs measured per neuron. (H-K) Abnormal behavior in Fmr1^−/y mice is rescued by Dex. Two-month old mice were given 3 intraperitoneal injections of 10 mg/kg Dex or saline over 40 hours prior to behavioral testing. Repetitive behaviors (grooming and nestlet shredding) were normalized by Dex in Fmr1^−/y mice. Hyperactivity as measured by locomotor activity was normalized by Dex in Fmr1^−/y mice. In Fig. 6E, F, unpaired two-tailed Student’s t-test was used. For 6G, one-way ANOVA followed by Tukey’s multiple comparisons test was used. For Figs. 6H, I, J, K, two-way ANOVA followed by Tukey’s multiple comparisons test was used. Data are represented as mean ± SEM. (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001)