Allotopic Expression Research Articles

212. AAV-Mediated Gene Transfer Protects Against Mitochondrial Optic Neuropathy in Mice

Mutations affecting mitochondrial function lead to a variety of disorders affecting the heart, skeletal muscles, endocrine glands and central nervous system. These diseases have been hard to replicate in animals, because of the difficulty in manipulating mitochondrial DNA and the lethality of nuclear mutations affecting mitochondrial respiration. We have used an AAV-mediated RNA knockdown strategy to produce two mouse models of Leber Hereditary Optic Neuropathy (LHON). Patients with LHON typically contain mutations in one of the subunits of Complex I of the mitochondrial respiratory chain (NADH-ubiquinone oxidoreductase) and experience bilateral loss of vision beginning around age 20. In mice, blocking synthesis of subunits of this enzyme leads to the pathologic hallmarks of the disease—apoptotic death of retinal ganglion cells, demyelination and loss of axons in the optic nerve. Using these mouse models, we have tested two approaches for gene therapy of LHON. In the first strategy, we delivered the gene for mitochondrial manganese superoxide dismutase (SOD2) to limit the accumulation of reactive oxygen species that result from Complex I defects. Delivery of SOD2 caused an 18% increase (p=0.001) in the survival of retinal ganglion cells and a proportional increase in the thickness of the optic nerve. In the second therapeutic strategy, we re-coded the mitochondrial gene for the ND4 subunit of Complex I, attached a mitochondrial transport sequence to its amino terminus and expressed it as a nuclear gene (allotopic expression). Treatment of the optic nerve with AAV expressing this protein led to a 13% increase (p<0.01) in diameter of the optic nerve relative to contralateral eyes treated with a control virus. Thus both approaches prevented tissue injury resulting from defects to Complex I of the respiratory chain. These provide the first evidence in animals that gene therapy for mitochondrial disease may be possible.

Limitations of allotopic expression of mitochondrial genes in mammalian cells.

The possibility of expressing mitochondrial DNA-coded genes in the nuclear-cytoplasmic compartment provides an attractive system for genetic treatment of mitochondrial disorders associated with mitochondrial DNA mutations. In theory, by recoding mitochondrial genes to adapt them to the universal genetic code and by adding a DNA sequence coding for a mitochondrial-targeting sequence, one could achieve correct localization of the gene product. Such transfer has occurred in nature, and certain species of algae and plants express a number of polypeptides that are commonly coded by mtDNA in the nuclear-cytoplasmic compartment. In the present study, allotopic expression of three different mtDNA-coded polypeptides (ATPase8, apocytochrome b, and ND4) into COS-7 and HeLa cells was analyzed. Among these, only ATPase8 was correctly expressed and localized to mitochondria. The full-length, as well as truncated forms, of apocytochrome b and ND4 decorated the periphery of mitochondria, but also aggregated in fiber-like structures containing tubulin and in some cases also vimentin. The addition of a hydrophilic tail (EGFP) to the C terminus of these polypeptides did not change their localization. Overexpression of molecular chaperones also did not have a significant effect in preventing aggregations. Allotopic expression of apocytochrome b and ND4 induced a loss of mitochondrial membrane potential in transfected cells, which can lead to cell death. Our observations suggest that only a subset of mitochondrial genes can be replaced allotopically. Analyses of the hydrophobic patterns of different polypeptides suggest that hydrophobicity of the N-terminal segment is the main determinant for the importability of peptides into mammalian mitochondria.

Rescue of a mitochondrial deficiency causing Leber Hereditary Optic Neuropathy.

A G to A transition at nucleotide 11778 in the ND4 subunit gene of complex I was the first point mutation in the mitochondrial genome linked to a human disease. It causes Leber Hereditary Optic Neuropathy, a disorder with oxidative phosphorylation deficiency. To overcome this defect, we made a synthetic ND4 subunit compatible with the "universal" genetic code and imported it into mitochondria by adding a mitochondrial targeting sequence. For detection we added a FLAG tag. This gene was inserted in an adeno-associated viral vector. The ND4FLAG protein was imported into the mitochondria of cybrids harboring the G11778A mutation, where it increased their survival rate threefold, under restrictive conditions that forced the cells to rely predominantly on oxidative phosphorylation to produce ATP. Since assays of complex I activity were normal in G11778A cybrids we focused on changes in ATP synthesis using complex I substrates. The G11778A cybrids showed a 60% reduction in the rate of ATP synthesis. Relative to mock-transfected G11778A cybrids, complemented G11778A cybrids showed a threefold increase in ATP synthesis, to a level indistinguishable from that in cybrids containing normal mitochondrial DNA. Restoration of respiration by allotopic expression opens the door for gene therapy of Leber Hereditary Optic Neuropathy.

Rescue of a deficiency in ATP synthesis by transfer of MTATP6, a mitochondrial DNA-encoded gene, to the nucleus.

A T-->G transversion at nt 8993 in mitochondrial DNA of MTATP6 (encoding ATPase 6 of complex V of the respiratory chain) causes impaired mitochondrial ATP synthesis in two related mitochondrial disorders: neuropathy, ataxia and retinitis pigmentosa and maternally inherited Leigh syndrome. To overcome the biochemical defect, we expressed wildtype ATPase 6 protein allotopically from nucleus-transfected constructs encoding an amino-terminal mitochondrial targeting signal appended to a recoded ATPase 6 gene (made compatible with the universal genetic code) that also contained a carboxy-terminal FLAG epitope tag. After transfection of human cells, the precursor polypeptide was expressed, imported into and processed within mitochondria, and incorporated into complex V. Allotopic expression of stably transfected constructs in cytoplasmic hybrids (cybrids) homoplasmic with respect to the 8993T-->G mutation showed a significantly improved recovery after growth in selective medium as well as a significant increase in ATP synthesis. This is the first successful demonstration of allotopic expression of an mtDNA-encoded polypeptide in mammalian cells and could form the basis of a genetic approach to treat a number of human mitochondrial disorders.

Gene therapy of mitochondrial DNA mutations: a brief, biased history of allotopic expression in mammalian cells.

Successful treatment of mitochondrial DNA (mtDNA) mutations might be possible by construction of mtDNA-encoded protein genes so that they can be inserted into the nuclear genome and the protein expressed in the mitochondria (allotopic expression). This technique would require individual assembly of all 13 mtDNA-encoded protein genes with an aminoterminal leader peptide that directs the cytoplasmic translated protein to the mitochondrial membrane. The 13 allotopic genes could be inserted into the nuclear genome of a patient's stem cell that had been "cured" of its nascent mtDNA via ethidium bromide treatment (rho-zero cell). The rho-zero cell would be a uridine auxotroph, and recovery from uridine auxotrophy would indicate successful transformation. The patient's own cells could then be returned to the patient's body. With a selective advantage of recovered oxidative phosphorylation, the transformed cells could replace cells with mtDNA mutations. Results of experiments by us on allotopically expressed CHO ATPase6 and of experiments by other workers suggest that there might be competition with endogenous mtDNA-encoded proteins if the particular protein gene is not removed from the endogenous mitochondrial genomes. Thus, it is likely that all 13 mtDNA-encoded protein genes will need to be allotopically expressed, with concomitant removal of all mtDNA genomes, in order for this form of mtDNA gene therapy to be successful.

Complementation of a gl-deficient feline herpesvirus recombinant by allotopic expression of truncated gl derivatives.

The alphaherpesvirus glycoproteins gE and gI form a hetero-oligomeric complex involved in cell-to-cell transmission. The gI-deficient recombinant feline herpesvirus (FHV), FHVdeltagI-LZ, produces plaques that are only 15% the size of those of wild-type FHV. Here, we have complemented FHV(delta)gI-LZ allotopically by expressing intact gI and C-terminally truncated gI derivatives from the thymidine kinase locus. The effect on gE-gI-mediated cell-to-cell spread was assessed by plaque assay employing computer-assisted image analysis (software available at http://www.androclus.vet.uu.nl/spotter/spotter.htm+ ++). Allotopic complementation with intact gI fully restored plaque size. Deletion of the C-terminal 11 residues of gI did not affect cell-to-cell spread, whereas deletion of the complete cytoplasmic tail reduced plaque size by only 35%. Mutants expressing gI166, roughly corresponding to the N-terminal half of the ectodomain, displayed a small-plaque phenotype. Nevertheless, their plaques were reproducibly larger than those of matched gI-deficient controls, indicating that the gE-gI166 hetero-oligomer, though crippled, is still able to mediate cell-to-cell spread. Our data demonstrate that plaque analysis provides a reliable and convenient tool to measure and quantitate gE-gI function in vitro.

Bioenergetic and structural consequences of allotopic expression of subunit 8 of yeast mitochondrial ATP synthase. The hydrophobic character of residues 23 and 24 is essential for maximal activity and structural stability of the enzyme complex.

Subunit 8 (Y8), a mitochondrially encoded subunit of the F0 sector of the F1F0-ATP synthase is essential for oxidative phosphorylation. We have previously introduced the technique of allotopic expression to study the structure/function of Y8, whereby an artificial Y8 gene is expressed in the nucleus of cells lacking a functional mitochondrial Y8, thus generating assembly of a functional F1F0-ATPase complex. In this paper we show that when a gene encoding an essentially unmodified version of Y8 is allotopically expressed, ATP synthesis and hydrolysis rates, as well as efficiency of oxidative phosphorylation, were similar to those of the parental wild-type strain in which Y8 is naturally expressed in mitochondria. We then tested the requirement for the hydrophobicity of the central domain (residues 14-32), which possibly represents a transmembrane stem, by introducing adjacent negative charges at different positions of Y8. One of the variants thus generated, which carries the double substitution Leu23-->Asp, Leu24-->Asp, when expressed in a strain lacking endogenous Y8, gave rise to cells which grew very slowly by oxidative phosphorylation. Measurement of bioenergetic parameters showed two major defects in these cells relative to control cells allotopically expressing unmodified Y8. First, the activity of the F1F0-ATP synthase was significantly decreased. ATP synthesis and state 3 of respiration were reduced by approximately 30-40%. ATP hydrolysis was reduced by approximately 30% and was almost insensitive to the F0 inhibitor oligomycin. Second, the physical coupling between the two sectors of the enzyme, as well as the stability of the F1 sector itself, were affected as shown by decreased recovery of F0 sector [8, 9, b, oligomycin sensitivity-conferring protein (OSCP), d, h and f] and F1 sector (alpha, gamma, delta) subunits in immunoprecipitates of ATP synthase. This study indicates that Y8 not only performs an important role in the structure of the mitochondrial complex but also in its activity. We conclude that the hydrophobic character of amino acids 23 and 24 in the middle of the putative transmembrane stem of Y8 is essential for coupling proton transport through F0 to ATP synthesis on F1.

33] Allotopic expression of mitochondrial ATP synthase genes in nucleus of Saccharomyces cerevisiae

The technique of allotopic expression is a powerful tool in the systematic analysis of the relationship between structure and function of the mitochondrially encoded mtATPase protein, subunit 8. In particular, functional allotopic expression of subunit 8 has permitted the implementation of a range of gene manipulation and expression strategies that focus on three distinct regions of this protein: the highly conserved N-terminal functional region, the central hydrophobic region, and the positively charged C-terminal region, which plays a key role in the assembly of the subunit into mtATPase. The technique of allotopic expression provides a system to test the in vivo function of the tagged construct. The approach of allotopic expression developed in yeast provides a model for application to other biological systems. Specifically, it may be possible to redress deficiencies in mitochondrially encoded proteins of humans, associated with mitochondrial disease, by application of allotopic expression strategies. This can provide a route toward gene therapy for diseases arising from mtDNA mutations.

34] Allotopic expression of yeast mitochondrial maturase to study mitochondrial import of hydrophobic proteins

Allotopic expression of a gene implies that a new copy of a gene, localized to a new cellular compartment, for instance, from mitochondria to the nucleus, is able to confer the corresponding wild-type phenotype. Thus, in the case of the allotopic expression of a mitochondrial gene, the corresponding cytoplasmically translated product should be targeted, in an active form, to the adequate mitochondrial subcompartment. This chapter describes the methods to transfer a mitochondrial gene to the nucleus. It illustrates the example of the bI4 RNA maturase to show how a cytoplasmically translated form of this protein can complement RNA maturase mitochondrial mutations. The chapter also explains how the RNA maturase can be used as a sensitive reporter activity, to study the mitochondrial import of hydrophobic proteins, and how this can permit the development of a genetic screen to search for genes involved in this import process.

Relocation of the plastid rbcL gene to the nucleus yields functional ribulose-1,5-bisphosphate carboxylase in tobacco chloroplasts.

The conserved plastid localization of rbcL suggests that biosynthesis of the large subunit of ribulose-1,5-bisphosphate carboxylase [Rubisco; 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] in chloroplasts is required to obtain functional enzyme. To examine the validity of this hypothesis, we relocated the plastid rbcL gene to the nucleus. First, we deleted the rbcL gene from the tobacco plastid genome by targeted insertion of a selectable aadA gene encoding spectinomycin resistance. The rbcL coding region was then inserted into an expression cassette and introduced into the nuclear genome of these plants by Agrobacterium-mediated transformation. We report that the nuclear rbcL functionally complements the defective plastids when the Rubisco large subunit is targeted to chloroplasts by a transit peptide. Therefore, the evolutionary process that relocates functional plastid genes to the nucleus has not yet occurred in the case of the rbcL gene. Targeted deletion of plastid genes, combined with their allotopic expression, will provide opportunities for studying the function of plastid enzyme complexes.

The C‐terminal positively charged region of subunit 8 of yeast mitochondrial ATP synthase is required for efficient assembly of this subunit into the membrane F0sector

This paper deals with a truncated derivative of subunit 8 of yeast mitochondrial ATP synthase in which a conserved positively charged residue (Lys47) has been removed by site-directed mutagenesis together with the C-terminal residue (Leu48). This derivative has been expressed as a chimaeric precursor N9L/Y8-1(K47-STP) carrying an N-terminal cleavable leader sequence (N9L), fused by a short bridging sequence to the truncated subunit-8 passenger protein. Allotopic expression of N9L/Y8-1(K47-STP) in vivo in an aap1 mit- host yeast strain lacking endogenous subunit 8 leads to partial restoration of bioenergetic function in the transformant strain denoted T475. Import and assembly studies were carried out in vitro using target mitochondria from strain YGL-1 partially depleted in subunit 8; such controlled depletion has been previously shown to be required for the efficient assembly (monitored immunochemically) of full-length subunit 8 imported in vitro as the precursor N9L/Y8-1. It was found that N9L/Y8-1(K47-STP) synthesized in vitro was imported successfully into YGL-1 mitochondria, but no significant assembly of the truncated subunit 8 was observed in these or any other mitochondria tested. The bioenergetic defects in T475 mitochondria are ascribed to the impaired assembly of the subunit-8 variant in vivo, resulting from the truncation at Lys47. In consequence, T475 mitochondria behave as though partially depleted of subunit 8. This conclusion was supported by the ability of isolated T475 mitochondria to provide a vehicle for the efficient import and assembly of subunit 8 processed from full-length N9L/Y8-1. Two related aspects of import and assembly have been addressed as part of the analysis of truncated subunit 8. First, mitochondria from strain T2-1, an aap1 mit- mutant genetically reconstituted by allotopic expression of N9L/Y8-1, were also found to be effective in the in vitro assembly of subunit 8 derived from imported N9L/Y8-1. This suggests an intramitochondrial shortage of subunit 8 delivered by allotopic expression of N9L/Y8-1 in vivo, which may underlie the incomplete restoration of energy coupling in T2-1 mitochondria compared to those of wild-type yeast. Second, on allotopic expression of N9L/Y8-2 (containing subunit 8 directly fused to N9L) in the aap1 mit- host, a rescued transformant strain T10-1 was generated which displays bioenergetic defects superficially similar to those of T475. Processed subunit 8 clearly assembled into the ATP synthase of isolated YGL-1 mitochondria, in spite of the relatively weak import of N9L/Y8-2 in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)