Human Molecular Genetics, 2003, Vol. 12, No. 4 399-413
© 2003 Oxford University Press
Constitutive knockout of Surf1 is associated with high embryonic lethality, mitochondrial disease and cytochrome c oxidase deficiency in mice
1Unit of Molecular Neurogenetics, Pierfranco and Luisa Mariani Center for the Study of Children's Mitochondrial Disorders, Istituto Nazionale Neurologico ‘C. Besta’-IRCCS, Milano, Italy, 2Laboratory of Animal Models, Istituto Regina Elena, Roma, Italy, 3Dino Ferrari Center, Department of Neuroscience, Ospedale Maggiore Policlinico-IRCCS, Milano, Italy and 4Laboratory of Molecular Neuroanatomy, Unit of Experimental Neurophysiology, Istituto Nazionale Neurologico ‘C. Besta’-IRCCS, Milano, Italy
Received October 29, 2002; Revised December 16, 2002; Accepted December 17, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We report here the creation of a constitutive knockout mouse for SURF1, a gene encoding one of the assembly proteins involved in the formation of cytochrome c oxidase (COX). Loss-of-function mutations of SURF1 cause Leigh syndrome associated with an isolated and generalized COX deficiency in humans. The murine phenotype is characterized by the following hallmarks: (1) high post-implantation embryonic lethality, affecting
90% of the Surf1-/- individuals; (2) early-onset mortality of post-natal individuals; (3) highly significant deficit in muscle strength and motor performance; (4) profound and isolated defect of COX activity in skeletal muscle and liver, and, to a lesser extent, heart and brain; (5) morphological abnormalities of skeletal muscle, characterized by reduced histochemical reaction to COX and mitochondrial proliferation; (6) no obvious abnormalities in brain morphology, reflecting the virtual absence of overt neurological symptoms. These results indicate a function for murine Surf1 protein (Surf1p) specifically related to COX and recapitulate, at least in part, the human phenotype. This is the first mammalian model for a nuclear disease gene of a human mitochondrial disorder. Our model constitutes a useful tool to investigate the function of Surf1p, help understand the pathogenesis of Surf1p deficiency in vivo, and evaluate the efficacy of treatment. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Leigh syndrome (LS, MIM 256000), or subacute necrotizing encephalomyelopathy, is an early-onset progressive neurodegenerative disorder characterized by predominant involvement of the central nervous system (CNS) (1). Affected infants show severe psychomotor delay, cerebellar and pyramidal signs, dystonia, respiratory abnormalities, incoordination of ocular movements, and recurrent vomiting. Focal symmetric lesions are found by MRI in the basal ganglia, thalamus, brainstem and posterior columns of the spinal cord (2).
LS is a genetically heterogeneous entity. However, all the biochemical defects described to date in patients with LS affect the terminal oxidative metabolism and are likely to impair energy production (1,3). An isolated, severe and generalized defect of complex IV [cytochrome c oxidase, COX (EC 1.9.3.1)], is one of the most common biochemical abnormalities associated with LS (1,4). Direct screening approaches failed to detect mutations in the COX subunit genes themselves (4). Conversely, a disease locus for LSCOX was mapped to chromosome 9q34, and analysis of a candidate gene in the region, SURF1, revealed deleterious mutations in most of the LSCOX patients investigated (5,6). Sequence analysis of SURF1 in numerous LSCOX patients, as well as in patients with other forms of COX deficiency, has provided evidence that (i) SURF1 mutations are the most common cause of LSCOX, and (ii) the association between LS and SURF1 mutations is highly specific, since no abnormalities of this gene were detected in COX defects presenting with clinical and neuropathological features different from LS (7). These conclusions are in agreement with early studies demonstrating the existence of a prevalent complementation group in LSCOX (8).
SURF1 has been previously shown to be part of a very tight, highly conserved ‘housekeeping’ gene cluster in several mammalian (9) and avian genomes (10). The Surfeit cluster contains six genes, spanning 32 kb of genomic DNA in mouse (Fig. 1). The mouse Surfeit locus maps to a region within the proximal portion of chromosome 2 (11), which is syntenic to human chromosome 9q34.
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The six SURF genes do not seem functionally or structurally related. The SURF3 gene has been identified as encoding the ribosomal protein L7a (Rpl7a) (12). The SURF4 gene encodes an integral membrane protein associated with the endoplasmic reticulum (13). The two proteins encoded by SURF5 as a result of differential splicing are cytoplasmic (14), while the protein product of the SURF6 gene is located in the nucleolus (15). The role and location of the SURF2 gene product are unknown. In both humans and mouse, a common, bi-directional promoter is shared by SURF1 and SURF2 and is located in the 70 bp intervening region between the two genes (16).
Although the precise function of SURF1 remains to be elucidated, studies on the human protein, and on the yeast homologue SHY1, indicate that the SURF1 gene product (Surf1 protein, Surf1p) is imported as a larger precursor encompassing an amino-terminal leader peptide, which is cleaved off from the mature protein (17,18). The mature protein is in turn embedded in the inner membrane of mitochondria, where it is involved in the assembly of COX (17,18). The absence of Surf1p in cultured cells from LS patients causes the accumulation of early intermediates of COX and marked reduction of the fully assembled complex (17).
To better understand the role of Surf1p and the pathogenesis of LSCOX, we have created a constitutive knockout (KO) mouse model for Surf1.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation of Surf1 knockout mice
The strategy used for the targeted disruption of the mouse Surf1 gene is shown in Figure 1A and B. We constructed a targeting vector in which exons 5–7 of the mouse Surf1 gene were replaced by the neomycin-resistance (Neor) gene. A thymidine kinase (TK) gene cassette was added adjacent to exon 8 of the Surf3 gene. Electroporation of the recombinant construct in embryonic stem (ES) cells was followed by exposure to the neomycin analogue G418 and ganciclovir. Of the 300 Neor ES clones, two clones showed evidence of homologous recombination of the targeting construct. The two positive clones were injected into blastocysts and three chimaeric mice from both cell lines were able to transmit the modified Surf1 gene to the next (F1) generation. The F1 heterozygous (Surf1+/-) mice were mated to each other, to generate homozygous knockout (Surf1-/-) animals. Confirmation of gene disruption was obtained by both Southern-blot and PCR-based analysis (Fig. 1C and D). Absence of the gene transcript and protein product in Surf1-/- KO animals was demonstrated by RT–PCR (Fig. 2A) and immunoblot analysis (Fig. 2B). As shown in Figure 2B, no Surf1p-specific cross reacting material (CRM) was detected in different tissues from Surf1-/- animals, while comparable amounts of CRM were detected in age-matched wild-type (wt) and heterozygous animals. Likewise, no Surf1 cDNA was obtained by RT–PCR from Surf1-/- post-natal mice or from several Surf1-/- early-stage embryos (E6.5–E7.5 days post-coitum, dpc; Fig. 2A). However, comparable amounts of the other Surf gene cDNAs (Surf2–6) were obtained in the same Surf1-/- mice and embryos, and in age-matched heterozygous or wt animals (Fig. 2A).
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The Surf1-/- allele is associated with high embryolethality
Homozygous Surf1-/- mice were obtained from Surf1+/- intercrosses. However, the proportion of Surf1-/- pups was 10-fold lower (2.7%) than that expected by mendelian transmission of a recessive trait (25%). This result indicates that the Surf1ko allele had a recessive phenotype, which was lethal in most of the embryos. To establish the timing of the observed embryolethality, we PCR-genotyped numerous embryos collected at different stages of development. As shown in Table 1, we found that the percentages of -/-, +/- and +/+ genotypes in blastocysts were 26, 48 and 26%, as expected by mendelian transmission of the recombinant allele. However, the percentage of Surf1-/- embryos dropped to 14% at E6.5–E7.5 dpc, to 10% at E8.5–E12, and to
2% at E13–E18 (see diagram in Fig. 3A). These results indicate that the loss of Surf1-/- embryos started at a stage as early as gastrulation (E4–E7 dpc), and continued during organogenesis (E8.5–E12 dpc), body-mass growth and organ maturation (E13–E18 dpc). Interestingly, the percentage of empty deciduae found during the different stages of embryonic development was inversely related to the percentage of Surf1-/- embryos (Table 1), indicating an increase of reabsorbed, presumably Surf1-/-, embryos during pregnancy. As shown in Figure 4A Surf1-/- embryos at different developmental stages did not show gross morphological abnormalities, but were consistently smaller in size, compared with their +/+ or -/+ littermates.
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Survival and growth rates after birth
The diagram shown in Figure 3B reports the spontaneous after-birth lethality rate of our Surf1-/- mice. A total of 34/1236 live pups had the Surf1-/- genotype. However, eight animals died within 2 days of birth. These neonate pups were smaller than their littermates (Fig. 4A), and had no milk in their stomach. Five additional animals died within 4 weeks of birth, and two within 2 months. Interestingly, all these animals were males, while spontaneous death occurred in only one female at 6 months of age. After variable periods of time, that were apparently free of clinical symptoms, these animals showed a very rapid downhill course, characterized by decreased spontaneous activity, arrested weight gain or weight loss, and polypnoea. These symptoms usually began less than 24 h from death. However, the autopsy failed to reveal gross abnormalities of viscera in the spontaneously deceased animals, although all of them were much smaller in size than the littermates (about one-half in both length and weight; Fig. 4A). No early spontaneous death was observed in either wt (+/+) or heterozygous (+/-) individuals.
Twelve KO animals (four males and eight females) are still alive and apparently free of neurological symptoms 12 months or more from birth. Figure 4B reports the growth curves of our animals. Surf1-/- males showed a significant reduction in the growth rate compared with wt animals. By contrast, the growth rate was normal in Surf1-/- females. No difference in growth was observed between wt and heterozygous animals (not shown).
Neurological examination and motor tests
Animals were periodically examined to evaluate spontaneous motor activity, reaction to stimuli, and the presence of neurological symptoms. No obvious neurological impairment was detected in the Surf1-/- population, although motor activity and reaction to stimuli seemed to be slightly reduced, as compared with the wt or heterozygous littermates. In 12 Surf1-/- animals (six males and six females, age 3–6 months) we performed several rotation-rod (Rotarod) treadmill and grip strength meter tests. The Rotarod test is used to study motor coordination and skill learning, while the grip test is used to evaluate muscle strength and endurance to fatigue. As shown in the diagram of Figure 5D and E, we found a significant, consistent reduction in the motor performance of the Surf1-/- mice in both tests, suggesting a deficit in motor activity, motor skills, exercise endurance and coordination. These results were not related to the age or sex of the animals.
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Other symptoms
Both Surf1-/- males and females showed reduced fertility. We observed very few pregnancies in Surf1-/- females, none of which was brought to term. We also observed a lower fertility rate for Surf1-/- males compared with +/- or +/+ littermates.
Ten Surf1-/- animals had a congenital deformity of the tail (Fig. 4A), which was never observed in +/- or +/+ littermates.
Biochemical findings
Figure 5A shows the average values of the activities of each respiratory complex normalized to citrate synthase (CS) in skeletal muscle of a series of 10 Surf1-/- animals (age range 1–9 months, average 3.8±3.0 months) compared with the corresponding values of a series of 10 wt age-matched littermates, taken as 100%. Figure 5B shows the average values of COX/CS activities in additional tissues, including liver, heart and brain. The data clearly demonstrate that COX was the only defective enzyme activity in the skeletal muscle of Surf1-/- mice, while the other activities were comparable to the wt cohort. Table 2 summarizes the COX/CS values obtained in muscle homogenates from our mice. No difference was detected between the wt +/+ cohort and a series of 10 age-matched +/- heterozygous littermates. Defective COX activity was clearly demonstrated in the other tissues, with liver being more severely affected than heart and brain. Defective COX (20% of the control) was also demonstrated in two fibroblast cell cultures from Surf1-/- animals (not shown). These data confirm that, as in humans, lack of Surf1p is associated with generalized COX deficiency, and that the presence of a single normal Surf1 allele is sufficient to maintain normal COX activity (and assembly).
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Table 2 also reports the COX/CS values obtained in the skeletal muscle homogenates of four Surf1-/- neonate pups that spontaneously deceased within the first day after birth, compared with six wt littermates. Although the COX/CS values from both series are moderately higher than those of the adult series, the reduction observed in Surf1-/- individuals is
23% of the controls values, similar to that observed in older animals (27% of the control values). The same was true for values obtained from two E12.5 dpc Surf1-/- embryos compared with wt control embryos of the same gestational age (not shown). Taken together, these data do not show significantly lower values of COX activity in -/- animals that underwent early spontaneous death, compared with the -/- ‘longer survivors’. Finally, a block in aerobic energy metabolism is usually associated with an increase of blood lactate, sometimes leading to severe metabolic acidosis (19). Values of plasma lactate measured in Surf1-/- animals were slightly but significantly elevated as compared with controls (Fig. 5C).
COX assembly studies by SDS–PAGE and BNE immunostaining
Normal amounts of CRM specific to COX-I subunit were detected by western blot analysis of protein extracts from several KO tissues, electrophoresed by one-dimensional denaturing SDS–PAGE (Fig. 2C). Similar results were obtained by using a specific antibody against subunit COX-Va (data not shown). These results demonstrate that the absence of Surf1p does not interfere with the steady-state levels of individual COX subunits.
To evaluate the effects of the absence of Surf1p on the assembly of COX, experiments based on two-dimensional-gel blue-native electrophoresis (2D-BNE) were performed in Surf1-/- and control fibroblast cell lines. Our specific anti-COX I monoclonal antibody was used to immunostain COX-specific subcomplexes (Fig. 2D). Four subcomplexes (S1, S2, S3 and S4) have previously been defined as COX-assembly intermediates (20). Insertion of COX-I in the inner membrane of mitochondria is the first step of COX assembly, corresponding to S1. This initial step is followed by incorporation of subunit COX-IV to form a bigger intermediate, S2. Further incorporation of the other subunits leads to the formation of an almost complete intermediate, S3, and, finally, to the fully assembled complex, corresponding to S4. As shown in Figure 2D, a prominent spot corresponding to fully assembled COX-I was present in the immunoblot containing the extract from wt mouse fibroblasts. Identical results to those obtained on wt +/+ fibroblasts were obtained on fibroblasts from a +/- heterozygous individual (not shown). However, accumulation of early intermediates (especially S1), and marked reduction in the intensity and size of the spot corresponding to fully assembled COX-I (S4) was obtained in the Surf1-/- fibroblast extract. These results indicate that the amount of fully assembled, functional COX is dramatically decreased in Surf1-/- fibroblasts. This decrease parallels the biochemical defect of COX activity measured in these cells.
Morphological findings
Skeletal muscle.
Figure 6A and B shows the results of histochemical reaction to COX carried out in muscle. In the Surf1-/- animals, COX reaction was reduced in type 1 fibres, and very low or absent in type 2 fibres. By contrast, the succinate dehydrogenase (SDH) reaction was more intense in KO muscles, indicating an increase in the number of mitochondria (Fig. 6C and D). This finding was confirmed by EM examination, which revealed marked subsarcolemmal accumulation of enlarged mitochondria (Fig. 6E and F). No difference in the distribution and relative proportion of type 1 and type 2 fibres was observed in KO muscles compared with controls by ATPase staining (data not shown).
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Liver.
COX reaction was reduced (Fig. 7A and B), while SDH reaction was more intense (Fig. 7C and D) in KO liver specimens, compared with controls. Haematoxylin and eosin staining failed to reveal lipid accumulation, fibrosis or disarray of lobules and sinusoids (not shown).
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No obvious difference was observed in COX and SDH histochemical reactions in the heart and brain specimens of KO versus wt animals (not shown).
Brain.
No evidence for neurodegeneration, abnormal vascular proliferation or necrosis was detected in thionine-stained sections from the entire rostro-caudal extent of Surf1-/- CNS specimens. In particular, the thickness and layering of the neocortex were normal, and the size and architecture of hippocampus, basal ganglia, thalamus, and subthalamic and hypothalamic regions were comparable to corresponding regions of age-matched control mice (Fig. 8A and B). At the cellular level, immunoreactivity for neurofilament proteins revealed normal somatic and dendritic morphology in mesencephalic nuclei subjected to neurodegeneration in Leigh syndrome, like neurons within the red and common oculomotor nuclei (Fig. 8C). In the spinal cord, lamina-IX motor neurons displayed normal morphology and neurofilament expression in the soma, dendrites and axons entering the ventral roots (Fig. 8D). Finally, no reactive gliosis was detected, as revealed by the normal pattern of GFAP immunostaining (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We demonstrate here that homozygous disruption of the mouse Surf1 gene leads to embryonic death in most cases. Surviving animals show a number of abnormalities including poor growth, low fertility, muscle weakness and precocious death. A skeletal abnormality in the tail was observed in several KO animals, which appears very similar to that described in the flexed tail (ft) mouse (21), but additional features of the ft trait were consistently absent in our animals. We have no explanation at the moment for this finding.
Approximately 30% of our post-natal animals died during the first month after birth, and an additional 15% died within the first 6 months of life. To verify whether precocious death was due to a more severe biochemical defect of COX, we measured COX activity on spontaneously deceased Surf1-/- neonates. However, no difference in the percentage of COX reduction was observed in these animals compared with older individuals. Phenotypic variability is not uncommon in human mitochondrial disorders. For instance, the variability in times of lethality of our model is similar to that observed in a recently reported family (22), where maternal members were homoplasmic for a mitochondrial (mt)-tRNA mutation. Several offspring of the proband died within hours of birth, whilst the mother/grandmother were apparently normal and one surviving child had LS. So, similar to the KO mice, there was substantial variability with a clearly defined mutation that affects respiratory chain enzyme production. Taken together, these results suggest that the individual genetic background and possibly epigenetic factors can markedly influence the phenotypic expression and prognosis of oxidative phosphorylation (OXPHOS) defects. Likewise, we observed a striking preponderance of early deaths in KO males, in spite of a 1:1 male-to-female ratio of live pups at birth. In addition, poor growth was significantly present in male but not in female KO animals at any age. The basis of these interesting gender skewing phenomena is presently unknown. To the best of our knowledge, no such gender differences have ever been reported in patients with LSCOX due to absence of Surf1p.
As in patients affected by LS, the absence of Surf1p in mice causes a profound, specific and diffuse decrease of COX activity. The degree of this reduction is less severe in our KO animals than it is in human patients; for instance, in skeletal muscle the reduction of COX/CS in our KO mice was 27% on average (Table 2), while in our series of SURF1null patients the average percentage was 14% (7). No data are available for the activity of COX in the brain tissue of SURF1null patients. These differences may explain in part the milder phenotype of the ‘longer survivors’ in our series of KO mice. Our 2D-BNE experiments on fibroblasts indicate that the structural basis of the biochemical defect is a substantial reduction in the amount of fully assembled enzyme and, similar to what is observed in humans, the accumulation of assembly intermediates. This result confirms the role of Surf1p as an assembly factor specific to COX. In particular, it has been proposed that the function of Surf1p may involve the incorporation of subunit II into the COX I+IV intermediate, a crucial step which is believed to produce the rapid, ‘cascade-like’ assembly of the other COX subunits (17,18). However, similar to what is observed in humans, the absence of Surf1p in our mice does not completely abolish the activity of COX and aerobic metabolism. This may offer an explanation for the small percentage of Surf1-/- animals that survive embryonic development and reach post-natal life, although the basis of the selective escape of a few concepti from embryonic death is unclear. Compensatory mechanisms due to differential expression of suppressor genes could be involved in the longer survival of our knockouts. Genetic modification of survival has been demonstrated in another model of mitochondrial disease, namely heart-specific Tfam knockouts. Tfam is an essential factor for transcription and replication of mtDNA and its absence causes loss of mtDNA and absence of OXPHOS (23). In a conditional, heart-specific Tfamnull mouse model, the onset of mitochondrial cardiomyopathy occurred during embryogenesis. Approximately 75% of the knockouts died in the neonatal period, but 25% survived for several months. Interestingly, 95% of the offspring generated by intercrosses within this population of ‘long living’ knockouts were also longer living, indicating the existence of a modifying gene affecting the life span of the knockouts (24). Unfortunately, infertility in our Surf1-/- females prevented us from exploring the same possibility by a simple genetic approach. Further investigation is necessary to verify this interesting hypothesis in our model.
In our animals, the defect of COX activity was associated with an increase of venous lactate, indicating a block of aerobic utilization of pyruvate. More importantly, distinct morphological abnormalities were present in skeletal muscle and, to a lesser degree, in liver, while no abnormalities were found in heart and brain. The muscle biopsy of our KO animals showed the presence of a mitochondrial myopathy, characterized by decrease of COX reaction in type I and, even more strikingly, in type II fibres, and an increase in number and size of mitochondria, particularly in the sub-sarcolemmal areas of the muscle fibres. Likewise, the muscle biopsy in LS shows a generalized and diffuse reduction, not a total absence, of COX, affecting equally type I and type II fibres, intrafusal fibres of the muscle spindles, and the arterial walls (25). Accumulation of mitochondria has been reported in LS, although ragged-red fibres are absent.
The morphological and biochemical abnormalities of skeletal muscle can explain the remarkable and consistent reduction of muscle strength, motor skills and endurance found in our KO mice. Failure to thrive and muscle weakness are also features of human LS (1,4). Patients are thin and weak, somatic growth is retarded and failure to thrive is a common feature of the disease. However, symptoms referable to skeletal myopathy are usually masked in LS patients by the severe neurological syndrome due to CNS involvement.
In contrast to the human phenotype, our Surf1-/- animals failed to develop progressive neurological symptoms, and no structural alterations were observed in brain and spinal cord. In particular, neuronal depletion, necrotic lesions and vascular proliferation were consistently absent in KO animals. The reason for the different neurological phenotype between humans and mice is unknown, but is probably due to different energy requirements and sensitivity to faulty OXPHOS in the brain of the two organisms. Interestingly, no neurological symptoms were observed in other mouse models of mitochondrial disease. For instance, mice carrying a large deletion of mtDNA developed a combination of mitochondrial myopathy, nephropathy and cardiopathy, but no brain lesions or neurological symptoms were observed (26), in spite of high proportion of deleted mtDNA in all tissues and organs, including the CNS. In contrast, mitochondrial late-onset neurodegeneration has been generated in mice by post-natal disruption of oxidative phosphorylation in forebrain neurons produced by conditional knockout of Tfam (27). However, also in this model, KO mice develop normally and display no overt behavioural disturbances or histological changes during the first 5 months of life. The MILON mice display reduced levels of mitochondrial DNA and mitochondrial RNA from 2 and 4 months of age, respectively, and severely respiratory chain-deficient neurons from 4 months of age. Surprisingly, these respiratory chain-deficient neurons are viable for at least one month without showing signs of neurodegeneration or major induction of defences against oxidative stress.
Taken together, these resuslts suggest that severe and prolonged neuronal respiratory chain deficiency is required for the induction of neurodegeneration in mice.
Most of our Surf1-/- concepti died during embryonic development. There are no data available on embryonic lethality in SURF1null LS patients, although we have personally observed an unusually high number of spontaneous abortions in SURF1 mutant families (V.T. and M.Z., personal observation).
The cause of massive embryonic death in our animals remains unclear. In principle, we could not exclude replacement of the mid-portion of the Surf1 gene with a Neor cassette possibly altering the expression of the other genes of the Surfeit cluster, and that this perturbation could in turn be the cause of embryonic lethality. To test this possibility, we evaluated the expression of each of the Surf genes by RT–PCR on KO and wt embryos, at a stage (E7.5 dpc) just before the occurrence of death in Surf1-/- individuals. No difference was observed in the amount of cDNA obtained for the Surf2-6 genes, compared with the cDNA specific to glyceraldehyde phosphate dehydrogenase (GAPDH), a control gene, in each KO sample versus wt samples. As expected, Surf1 cDNA was present in the wt, and absent in the KO, embryos. In addition, we notice that the intronic regions of the Surf1 gene between exons 5 and 7 are not conserved during evolution, are not known to contain any trans-regulatory element, and are well away from the bi-directional promoter common to Surf1 and Surf2 genes. Taken together, these results suggest that the expression of the Surfeit gene cluster is comparable in our KO embryos to that of wt littermates. However, since subtle differences in the quantitative expression of the Surfeit gene cluster cannot be completely ruled out by our semiquantitative RT–PCR assay, we are currently creating a transgenic murine strain expressing a recombinant Surf1 cDNA on our Surf1 knockout background. This animal strain will be used to verify whether the re-expression of Surf1p in our KO mice can rescue the COX phenotype and reduce or abolish embryonic lethality.
If embryolethality is the consequence of the absence of the Surf1 gene product itself, this could either be due to a direct role of Surf1p on ontogenesis or as a result of the reduction of COX activity and cellular respiration, or both. To date, no function has been attributed to Surf1p, other than that of being involved in the formation of the respiratory chain, and in particular in the assembly of COX. Therefore, although a direct ontogenetic function for this protein cannot be formally ruled out, it is likely that the striking reduction of the survival rate in our KO individuals is mediated by the defect of respiration caused by COX deficiency.
In our Surf1-/- model, we observed a progressive skewing from the expected mendelian ratios for a recessive trait, starting from early post-implantation stages.
MtDNA maintenance and OXPHOS are necessary for embryogenesis in mice, as shown in constitutive KO models for two factors involved in the biogenesis of mitochondria, Tfam (23) and nuclear respiratory factor 1 (NRF1) (28).
Studies on energy metabolism in the early mouse embryo have shown that the oxygen consumption remains relatively constant from zygote to morula stages, but increases in the blastocyst (E3.5 dpc) and E6.5–7.5 dpc stages (29). This is the time during which the embryo completes gastrulation, followed by initial (E8–10 dpc) and late (E11–14 dpc) organogenesis (30). All are periods of rapid cellular proliferation and differentiation in which the energy demand is high. Studies on glucose metabolism during organogenesis of the rat have shown that oxidative metabolism increases significantly in embryos and investing membranes by day 11 post coitum (31). Thus, embryos impaired in their ability to oxidatively metabolise nutrients would be expected to have difficulty during these stages of increased biosynthetic activity. This hypothesis is sustained by investigation in other mouse models characterized by impaired energy metabolism. For instance, complete embryolethality was observed in homozygous mutants for a null allele of the gene for glucose-6-phosphate isomerase, the enzyme that catalyses reversible interconversion of glucose-6-phosphate into fructose-6-phosphate at the cross-point between glycolysis and the pentose-phosphate cycle. In this model, by 8.5–9.5 dpc all null embryos were dead and partially or completely reabsorbed (32). Likewise, complete embryolethality occurred in mosaic mutants carrying a null allele for the X-linked Pdha1 gene, that encodes the alpha subunit of the E1 component of the pyruvate dehydrogenase complex (PDHC) (33). PDHC is a key enzyme for the aerobic utilization of glucose. Again, in this model all embryos were necrotic by E10.5 dpc, and were not developed significantly beyond the E8.5-dpc stage. As in our embryos, Pdha1null mosaic embryos appeared to be normally formed, suggesting that there is a uniform slowing of cellular proliferation, rather than damage to selected cell types.
Finally, metabolic adaptation in rat liver mitochondria during the first postnatal hours takes place by both rapid biochemical mechanisms, such as accumulation of adenine nucleotides in the organelles, and long-term biogenetic mechanisms requiring de novo protein synthesis (34,35). As a result, during the first postnatal hours, succinate dehydrogenase, COX and F1-ATPase activities are all markedly increased, and the proton electrochemical gradient and membrane potential reach the ‘adult’ values. These observations indicate that the first postnatal hours are a critical period during which the aerobic energy demand increases dramatically. Failure to respond to these changes, due to reduced functional reserve, may contribute to explain the fragility of our KO neonates and the high rate of lethality observed immediately after birth.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Construction of the targeting vector
A 650 bp fragment (Fig. 1A), encompassing positions 184–834 of the mouse Surfeit cluster (GenBank accession no. M14689), and containing exons 9–8 of the Surf1 gene, was PCR-amplified from mouse genomic DNA, using suitable primers, and cloned into the BamHI and SalI sites of an SK+ Bluescript vector (Clontech, Palo Alto, CA, USA). A TK cassette from human herpes virus 2 was cloned into the SalI site adjacent to the 650 bp fragment. The region encompassing position 835–1806 of the Surfeit cluster, containing exons 7–5 of the Surf1 gene, was replaced by a 1138 bp PCR fragment, encompassing nucleotide positions 451–1589 of the pMC1neo-polyA cloning vector (GenBank accession no. U43612). The 1138 bp fragment contains the coding region of the gene encoding the neomycin resistance protein under the control of the TK promoter (Neor cassette). A 5122 bp fragment (Fig. 1A), encompassing positions 1807–6929 of the Surfeit cluster, was PCR-amplified and cloned into XbaI and ClaI sites. The 5122 bp fragment included exons 4–1 of Surf1 and the entire Surf2 gene. In addition, the cytosine nucleotide at position 3204 of the Surfeit cluster was deleted by site-directed mutagenesis. This deletion, which creates a diagnostic NotI site (data not shown), leads to a frame shift of the Surf1 coding region with the formation of a TGA stop codon 40 bp downstream from the first ATG.
Before electroporation into ES cells, the entire 6910 bp recombinant construct was verified by automated nucleotide sequence analysis using the big-dye terminator kit and protocol (Applied Biosystems), on a 3100 ABI automated sequencer.
Gene targeting in ES cells and generation of chimaeras
AB1 ES cell line (a kind gift from Alan Bradley), derived from 129/SvEvBr +/Hprt-bm2 mouse substrain, was used to produce Surf1null mice. Culture of ES cells and electroporation of ClaI-linearized vector were performed as described (36).
Three hundred colonies which survived selection with G-418 (200 µg/ml) for the presence of the Neor cassette, and Gancyclovir (2 µM) for the absence of the TK cassette, were isolated and screened by PCR and Southern blot analysis for the recombinant allele (see below).
Two recombinant clones were injected in B6D2F1+C57/Bl6J blastocysts (37). Chimaeric pups were identified by the presence of agouti hair and, on maturity, mated with B6D2F1 (C57/Bl6JxDBA2) females to check for the contribution of the ES cells to the germline.
PCR and Southern blot based genotyping
To rapidly identify recombinant clones, PCR-based screening specific to the recombinant allele was carried out on post-selected ES cells (not shown). PCR amplification was carried out using a sense oligonucleotide contained within the Neor cassette (5'-GATTCGCAGCGCATCGCCTT-3') and an antisense oligonucleotide encompassing positions 86–105 in the Surfeit cluster. This region lies within exon 8 of Surf3, outside the region of recombination (5'-AAGGCTAAAGAACTCGCCAC-3'). Amplification of the recombinant allele produced an 850 bp PCR fragment, while no amplification product was obtained from the wt allele. PCR was carried out from 25 ng of genomic DNA in 50 µl containing 1xMgCl2-PCR buffer (Applied Biosystems, Branchburg, NJ, USA), 200 mM of dNTPs, 0.6 mM of each primer and 0.03 U/µl Taq polymerase (Applied Biosystems). After an initial denaturation at 94°C for 2 min, the PCR profile was 94°C for 30 s, 56°C for 30 s, 72°C for 40 s, for 40 cycles, followed by a final extension at 72°C for 5 min.
To confirm homologous recombination in PCR-positive ES clones and in recombinant animals, Southern blot analysis was performed on total genomic DNA digested with EcoRI electrophoresed through a 0.8% agarose gel. As shown in Figure 1B, two EcoRI sites, 19 kb apart from each other, are present in the Surfeit cluster, encompassing the Surf4, Surf2, Surf1, Surf3, Surf5 and part of Surf6, genes, in this order. In the recombinant allele, a third EcoRI site, which is contained within the Neor cassette at position 628 of the Genbank U43612 sequence, causes the digestion of the 19 kb EcoRI fragment into a 12.5 and a 6.5 kb fragment. The 12.5 kb fragment contains part of Surf6, the entire Surf5 and Surf3 genes, and the 3' portion of Surf1. The 6.5 kb fragment contains Surf2 and Surf4, in addition to the remaining 5' portion of Surf1. The blot was probed with a 700 bp PCR fragment, radiolabelled with [
32P]dCTP (NEN, Boston, MA, USA) using the ‘Ready-to-go’ random priming kit and protocol (Amersham, Piscataway, NJ, USA). The fragment corresponds to positions 910–1609 of the coding region of the Surf5 gene (GenBank accession no. X85169). The Surf5 gene is contained within the 19 kb EcoRI genomic fragment, but remains outside the recombinant insert of our targeting vector. A typical result of this analysis is shown in Figure 1C. The DNA from a heterozygous Surf1+/- mouse shows two hybridization bands, one of 19 kb, corresponding to the wt allele, and the second of 12.5 kb, corresponding to the recombinant KO allele. The 19 kb band is the only hybridization signal found in a sample from a homozygous Surf1+/+ wt animal, while a homozygous Surf1-/- KO animal shows only the 12.5 kb hybridization band.
The presence of the 12.5 kb recombinant allele was further confirmed by re-probing the blots with a radiolabelled fragment encompassing positions 747–1526 of the Neor cassette (Fig. 1C).
PCR-based genotyping on DNA extracted from embryos and tails of 2-week-old animals was performed by using a pair of oligonucleotide primers flanking the Neor cassette (sense primer: 5'-CATACGGAAGTCTGCATC-3'; antisense primer: 5'-ATCCTCACTGAGCCTTTC-3'). As shown in Figure 1D, the 1086 bp PCR fragment specific to the wt allele was clearly distinguishable from a 1173 bp band corresponding to the recombinant allele. PCR was carried out from 250 ng of genomic DNA in 50 µl containing 1xMgCl2-PCR buffer (Applied Biosystems), 200 mM of dNTPs, 0.6 µM of each primer and 0.03 U/µl Taq polymerase (Applied Biosystems). After an initial denaturation at 94°C for 2 min, the PCR profile was 94°C for 30 s, 57°C for 30 s, 72°C for 90 s, for 35 cycles, followed by a final extension at 72°C for 5 min.
RT–PCR analysis
Total RNA was extracted from each of eight Surf1-/- and two Surf1+/+ E7.5 dpc embryos, and from skeletal muscle of one Surf1-/- and one Surf1+/+ adult animals, using the ‘Trizol reagent’ kit (Gibco-BRL, Grand Island, NY, USA) following the manufacturer's protocol. Total RNA was used as template for reverse transcription, using the ‘cDNA cycle’ kit and protocol (Invitrogen, Carlsbad, CA, USA). Total cDNA was resuspended in a final volume of 20 µl and used for PCR amplification of individual cDNA fragments corresponding to the following genes: GAPDH, Surf1, Surf2, Surf3, Surf4, Surf5, and Surf6. Primers, PCR conditions and fragment size obtained by PCR amplification of individual cDNAs are reported in Table 3. Three microlitres of cDNA were used in each 50 µl PCR reaction containing 1xMgCl2-PCR buffer, 200 mM dNTPs, 5% DMSO, 0.6 mM of each primer and 0.03 U/µl of Taq-Gold polymerase (Invitrogen). The PCR products were separated by electrophoresis through a 1.5% ethidium-bromide stained agarose gel and visualized under a UV screen.
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Western blot analysis and 2D-BNE
Western blot analysis was performed on electroblotted one-dimensional SDS–PAGE, and 2D-BNE, as described previously (38). Approximately 100 µg non-collagenous protein were used for each sample. Chemiluminescence-based immunostaining (ECL kit, Amersham) was performed using our polyclonal antibody AS182–196, raised against an oligopeptide which is identical in Surf1p sequences from humans and mice, and two monoclonal antibodies against subunits COX I and COX Va of complex IV (Molecular Probes, Eugene, OR, USA), as described previously (17).
Morphological and biochemical analyses
For light microscopy, samples from skeletal muscle, liver, heart and brain were frozen in liquid nitrogen-cooled isopentane. Standard histologic and histochemical techniques for the detection of mitochondrial alterations (H&E, Gomori Trichrome, COX, succinate dehydrogenase) and muscle fibre distribution (ATPase pH4.3) were performed on serial cryostat cross sections as previously described (39). Electron microscopy on skeletal muscle was performed on glutaraldehyde-fixed specimens, post-embedded in epoxide resin, as described previously (40).
Biochemical assays of individual respiratory complexes, were carried out on tissue homogenates (41). Concentration of protein was measured by the Folin-Ciocalteau method (42). Specific activities of each complex were normalized to that of CS, an indicator of the number of mitochondria. Lactate was measured on blood from tail vein using the ‘Lactate reagent’ kit and protocol (Sigma, Saint Louis, MO, USA).
Neuropathology
Animal handling and cerebral tissue processing.
Three KO mice (aged 1, 3 and 10 months) and three age-matched controls were sacrificed with lethal injections of 4% chloral hydrate. The KO animals were chosen among those presenting pre-mortal symptoms (see Results), in order to maximize the chance to find significant neuropathological changes. Brains and spinal cords were rapidly dissected out, immediately fixed by immersion in 4% paraformaldehyde, and cut with a vibratome in 50 µm thick coronal sections. Serial sections were obtained from the entire rostro-caudal extension of brains and cords and collected in 0.1 M PBS at pH 7.2 with 0.01% sodium azide. One out of six sections were counterstained with 0.1% thionine and the adjacent sections reacted for immunocytochemistry (ICC) as outlined below.
Immunocytochemistry.
Free-floating sections were initially pre-treated with 1% H2O2 in PBS for 20 min to neutralize the endogenous peroxidase activity, rinsed in PBS, and incubated with 10% normal serum (NGS) and 0.2% Triton-X100 for 60 min, to mask non-specific adsorption sites and to increase the penetration of the reagents. Sections were then incubated overnight with the primary antibodies diluted 1:1000 in NGS 1%. Monoclonal anti-SMI 311 and -SMI 312 antibodies (Sternberger Monoclonals Inc., Lutherville, MD, USA), recognizing non-phosphorylated and phosphorylated neurofilaments, respectively, were selected as specific neuronal markers, whereas a monoclonal anti-GFAP antibody (Sternberger Monoclonals Inc.), recognizing the glial fibrillary acidic protein, was selected as a marker for astrocytes. After rinsing in PBS, the sections were incubated with biotinylated goat anti-mouse IgG (diluted 1:200, Jackson, West Grove, PA, USA), rinsed in PBS, and then incubated with Extravidin (1:5000, Sigma, Milano, Italy). All immunoreagents were diluted in 1% normal serum in PBS. Peroxidase staining was obtained by incubating the sections in 0.075% DAB and 0.002% H2O2. The immunoreacted sections were mounted onto gelatin-coated glass slides, air-dried, dehydrated and coverslipped with DPX. Slides were then analysed and photographed with a Nikon Microphot FXA microscope equipped with a digital Nikon camera.
Rotating rod and grip strength tests
Tests were given to 20 wt mice (10 males, 10 females) and to 12 Surf1-/- KO mice (six males and six females). All animals were 3–6 months old.
The rotating rod test was performed on a Rotarod apparatus for mice (Ugo Basile). Animals were trained for 2 days before the day of test. Six trials (three trials on two consecutive days) were performed, with a 60 min rest interval between trials. Each trial lasted for a maximum of 300 s, during which the rod accelerated linearly from 0 to 32 rpm. The amount of time for each mouse to fall from the rod was recorded for each trial. If the mouse held on the rod and rotated 360°, this time was noted and the time of the second rotation was reported as the time of falling off the rod.
A Grip Strength Meter (Ugo Basile, Varese, Italy) was used to measure the forelimb grip strength. Six trials (three trials on two consecutive days) were performed in each animal, with a 15 s rest interval between trials. Mice were held by the tail and allowed to grasp a trapeze bar with both forepaws. The mouse was then manually pulled away until the bar was released. A digital meter recorded the level of tension (in grams) exerted on the bar by the mouse.
Statistical analysis
The two-tailed, unpaired, unequal variance Student's t-test was used for statistical analysis.
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ACKNOWLEDGEMENTS |
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We thank Dr Laura Pozzi for stimulating discussion and useful advice on scientific and technical aspects of our work. This work was supported by Fondazione Telethon-Italy (grant no. 1180 to M.Z.), Fondazione Pierfranco and Luisa Mariani (Ricerca 2000 grant to M.Z.) and EU Concerted Action on Mitochondrial Biogenesis and Disease (MitEuro). We are indebted to Ms Barbara Geehan for revising the manuscript.
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FOOTNOTES |
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* To whom correspondence should be addressed at: Unit of Molecular Neurogenetics, National Neurological Institute ‘Carlo Besta’, via Temolo 4, 20126 Milan, Italy. Tel: +39 022394630; Fax: +39 022394619; Email: zeviani{at}tin.it
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