Mutations in cytochrome c oxidase subunit VIa cause neurodegeneration and motor dysfunction in Drosophila

Abstract

Mitochondrial dysfunction is involved in many neurodegenerative disorders in humans. Here we report mutations in a gene (designated levy) that codes for subunit VIa of cytochrome c oxidase (COX). The mutations were identified by the phenotype of temperature-induced paralysis and showed the additional phenotypes of decreased COX activity, age-dependent bang-induced paralysis, progressive neurodegeneration, and reduced life span. Germ-line transformation using the levy(+) gene rescued the mutant flies from all phenotypes including neurodegeneration. The data from levy mutants reveal a COX-mediated pathway in Drosophila, disruption of which leads to mitochondrial encephalomyopathic effects including neurodegeneration, motor dysfunction, and premature death. The data present the first case of a mutation in a nuclear-encoded structural subunit of COX that causes mitochondrial encephalomyopathy rather than lethality, whereas several previous attempts to identify such mutations have not been successful. The levy mutants provide a genetic model to understand the mechanisms underlying COX-mediated mitochondrial encephalomyopathies and to explore possible therapeutic interventions.

Figure 1.—

Molecular identification of the levy gene. Schematic of DNA encompassing the levy gene. (A) The levy gene was first localized to genetic position 2-103 using recombination mapping of the levy¹ mutation with visible markers L² and Pin¹ on the right arm of chromosome 2. Deficiency mapping localized the levy gene cytologically between bands 59D11 and 59E. Site-specific recombination mapping in males localized the levy gene between two P-element insertion sites, chrw^k06908 and Dcp-1^k05606 (represented by triangles). (B) Results of molecular analysis of three levy deficiencies (levy², levy³, and levy⁴) generated by P-element mobilization in Dcp-1^k05606 flies suggested that the levy gene is CG17280, pita, or Dcp-1. Gaps in solid lines represent DNA deleted in these three levy deficiency strains. (C) The relative locations of genes in this region are shown as lines. The CG17280 gene was identified as levy by transforming a 2-kb DNA fragment encompassing CG17280 (represented by a line in D) into the germ line of levy¹ mutants. These levy transformants did not exhibit temperature-induced paralytic behavior. The bar at the bottom represents 1 kb of DNA in B–D.

Figure 2.—

Guanine-to-adenine transition in levy¹ DNA resulted in missplicing of CG17280 RNA. (A) levy¹ DNA contained an adenine nucleotide at the 3′-splice junction within the intron of CG17280 gene. This position was occupied by guanine in the wild-type gene as given in the BDGP database as well as in our own sequence determination of the region in CS and a sibling strain to levy¹. The 5′- and 3′-intron splicing consensus sequences are underlined. The G-to-A transition caused the levy¹ RNA to be misspliced by 1 nucleotide and resulted in a reading frame shift in exon 2, as determined by RT–PCR analysis of mutant levy RNA. (B) A schematic of missplicing of levy¹ RNA. Translated regions are shown as rectangles with the wild type shown as open rectangles. The shift in reading frame would presumably lead to replacement of 77 amino acids encoded by exon 2 with 22 missense amino acids (shaded rectangle). (C) Sequencing of levy¹ cDNA confirmed the occurrence of missplicing in levy¹ RNA. A portion of the levy¹ cDNA sequence is shown with the deduced amino acid (AA) sequence. The wild-type cDNA sequence and deduced amino acid translation are shown for comparison to those of the levy¹ mutant. The aberrant amino acid sequence resulting from a shift in the reading frame is shown in italics.

Figure 3.—

levy mutants exhibited age-dependent reduction in COX activity and ATP, but not in a marker for the mitochondrial matrix, citrate synthase. COX activity and levels of ATP were measured in adult flies kept at 29° for 1 or 7 days. COX activity is set to 100% in untreated wild-type (CS) flies for comparison purposes. COX activity was reduced in levy¹ mutants compared to that in wild-type flies (Student's t-test, P < 0.025 for 1-day old and P < 0.050 for 7-day old) and levy transformants (P < 0.005 for 1-day old and P < 0.010 for 7-day old) (A and B). Transformant flies carrying levy⁺ transgene showed enzymatic activity similar to that in the wild-type flies (P ≈ 0.2), whereas activity levels in the transformant control flies were similar to those in the mutant (P ≈ 0.2). Heterozygous levy alleles exhibited a comparable reduction of COX activity with respect to wild-type (CS) flies (C). In comparison to the wild type, a reduction in ATP appeared in 7-day-old levy¹ mutants (P < 0.025) and transformant controls (P < 0.025) after a temperature-shock regimen (E). Comparable reduction in ATP did not appear in 1-day-old (D) temperature-shocked levy¹ mutants (P ≈ 0.2) or in 7-day-old levy¹ mutants prior to being subjected to temperature shock (E) (P ≈ 0.2). There was no measurable difference between wild-type and levy mutants in regard to a classical enzymatic marker for the mitochondrial matrix, citrate synthase (F). Error bars show standard deviation from three or four measurements. T, transformant; Tc, transformant control.

Figure 4.—

Reduced life span and progressive bang-induced paralysis in levy mutants. (A) Percentage of surviving adults kept at 29° after eclosion. levy¹ flies (n = 550) died at a median age of 8 days at 29° compared to wild-type (CS) flies (n = 669), which lived to a median age of 28 days. Life span of levy transformants (n = 955) was similar to that of Canton-S flies, while transformant controls (n = 682) died about the same time as levy¹ mutants. (B) Time for recovery after testing for bang-induced paralysis for 2-, 10-, and 20-day-old (after eclosion) adults kept at 21°. At this temperature, the median life span was 100 days for wild-type flies (CS), 51 days for levy¹ mutants, 93 days for transformants, and 55 days for transformant controls. Two-day-old levy¹ mutants did not exhibit bang-induced paralytic behavior. Only a fraction of 10-day-old levy¹ mutants exhibited bang-induced paralysis, all of which recovered in <2 min. In 20-day-old levy¹ mutants, all flies paralyzed in response to bang stimulus and these took longer to recover from paralysis than their younger siblings. Numbers of 2-, 10-, and 20-day-old levy flies tested were 56, 66, and 71, respectively. Numbers of 2-, 10-, and 20-day-old transformant control flies tested were 69, 64, and 68, respectively.

Figure 5.—

levy mutants exhibited vacuolization of the optic lobes and brain, but were rescued by transformation with the wild-type gene. Epon-embedded horizontal brain sections were cut from head capsules of adults kept at 29° after eclosion. Slices through the heads of 7-day-old wild type (CS) (A) and levy transformant (C) are shown for comparison to that from the head of levy¹ mutant (B). Swiss cheese-like holes appeared in the brain and optic lobes in 7-day-old levy¹ mutants (inset). These cytological lesions also appeared in the brains and optic lobes of 7-day-old transformant control flies (data not shown). Neurodegeneration occurred in age-dependent manner (D), being absent in CS flies aged for 1 day or 6 days, and in levy¹ flies aged for 1 day. On the other hand, 19 of 20 levy¹ flies aged for 6 days showed clear neurodegeneration. Serial sections from paraffin processed flies were obtained from levy¹/levy² (E) and levy¹/levy³ (F) flies after aging them for 6 days at 29°. Neurodegeneration was observed in flies from both strains, consistent with the phenomenon seen in levy¹ mutants.

Figure 6.—

From COX deficiency to neuromuscular dysfunction. This model shows nuclear and mitochondrial genes that code for structural components of COX or its assembly factors that have been shown to be involved in mitochondrial encephalomyopathies. Until now, it has been believed that mutations in the nuclear genes coding for structural subunits of COX would be embryonic lethal. The levy¹ mutation presents the first case where mutants are viable but show mitochondrial encephalomyopathic phenotypes. In addition, a general model of some possible subsequent steps via which COX deficiency could lead to neuromuscular dysfunction is presented. The data in this article establish a causal link between a reduction in COX activity and mitochondrial encephalomyopathic effects of neurodegeneration, paralytic behavior, and premature death in Drosophila.