Impaired mitochondrial glutamate transport in autosomal recessive neonatal myoclonic epilepsy

Abstract

Severe neonatal epilepsies with suppression-burst pattern are epileptic syndromes with either neonatal onset or onset during the first months of life. These disorders are characterized by a typical electroencephalogram pattern--namely, suppression burst, in which higher-voltage bursts of slow waves mixed with multifocal spikes alternate with isoelectric suppression phases. Here, we report the genetic mapping of an autosomal recessive form of this condition to chromosome 11p15.5 and the identification of a missense mutation (p.Pro206Leu) in the gene encoding one of the two mitochondrial glutamate/H(+) symporters (SLC25A22, also known as "GC1"). The mutation cosegregated with the disease and altered a highly conserved amino acid. Functional analyses showed that glutamate oxidation in cultured skin fibroblasts from patients was strongly defective. Further studies in reconstituted proteoliposomes showed defective [(14)C]glutamate uniport and [(14)C]glutamate/glutamate exchange by mutant protein. Moreover, expression studies showed that, during human development, SLC25A22 is specifically expressed in the brain, within territories proposed to contribute to the genesis and control of myoclonic seizures. These findings provide the first direct molecular link between glutamate mitochondrial metabolism and myoclonic epilepsy and suggest potential insights into the pathophysiological bases of severe neonatal epilepsies with suppression-burst pattern.

Figure 1

A, Pedigree and haplotype of the family. The blackened symbols indicate affected individuals, and the haplotype segregation with EME is shown. A plus sign (+) = wild-type sequence; m = mutation. B, Physical map of the critical interval, indicating the candidate genes in this region of chromosome 11p15.

Figure 2

Respiration and mitochondrial substrate oxidation in cultured skin fibroblasts from patients and controls. A, After polarographic measurement of intact cell respiration, digitonin-permeabilized cells were loaded with adenosine diphosphate (ADP) and amino oxyacetate. This compound inhibits the aspartate-amino transferase enzyme activity (see panel C). A subsequent addition of glutamate allowed estimation of mitochondrial glutamate oxidation under phosphorylation conditions (i.e., presence of ADP). Note the lack of glutamate-triggered oxygen uptake in patient cells. A similar succinate oxidation rate was measured in control and patient cells. Numbers along the traces are nmol O₂ consumed per minute per microgram of protein. B, Various ratios showing the profound defect of glutamate oxidation (GOx) in patient cells (unblackened boxes), as compared with cell respiration (R) or succinate oxidation (SOx). Values are the means (±1 SD) of six experiments. C, Features of mitochondrial glutamate metabolism, with inner membrane–associated glutamate-H⁺ symporter (1) and aspartate/glutamate antiporter (4), and the matrix-located enzymes, glutamate dehydrogenase (2) and amino-oxyacetate–sensitive aspartate-amino transferase (3). AOA = amino oxyacetate; Asp = aspartate; Glut = glutamate; α-KG = α-ketoglutarate; and OAA = oxaloacetate. Experimental conditions were as described in the “Methods” section of appendix A (online only).

Figure 3

Kinetics of [¹⁴C]glutamate uniport and [¹⁴C]glutamate/glutamate exchange catalyzed by the wild-type and mutated GC1 proteins. Proteoliposomes were reconstituted with the recombinant wild-type (blackened symbols) or the mutated GC1 protein (unblackened symbols). At time zero, 1 μM [¹⁴C]glutamate was added to the proteoliposomes containing 10 μM glutamate (exchange [circles]) or 10 μM NaCl and no substrates (uniport [triangles]). At the indicated time intervals, the uptake of labeled substrate was stopped by addition of 30 μM pyridoxal 5′-phosphate and 10 μM bathophenantroline. Data are means (± SD) of four independent experiments. No transport activity was detected when the mutant protein was used.

Figure 4

SLC25A22 gene expression in the 15-wk-old (A–F) and 20-wk-old (G–L) fetal human brain. Sections counterstained with hematoxylin and eosin are show under bright-field (A, C, E, G, I, and K) and dark-field illumination to reveal the localization of the in situ hybridization signal (B, D, F, H, J, and L). CP = cortical plate; SN = substantia nigra; NR = nucleus ruber; SON = superior olive nucleus; SF = sylvian fissure; OC = olivary complex; and PF = pontocerebellar fibers.

Figure A1

Alignment of the primary structure of known mitochondrial glutamate carriers and aspartate/glutamate carriers. The conserved proline that is mutated in the family with EME is boxed.