Isolated deficiencies of OXPHOS complexes I and IV are identified accurately and quickly by simple enzyme activity immunocapture assays.

OXPHOS deficits are associated with most reported cases of inherited, degenerative and acquired mitochondrial disease. Traditional methods of measuring OXPHOS activities in patients provide valuable clinical information but require fifty to hundreds of milligrams of biopsy tissue samples in order to isolate mitochondria for analysis. We have worked to develop assays that require less sample and here report novel immunocapture assays (lateral flow dipstick immunoassays) to determine the activities of complexes I and IV, which are far and away the most commonly affected complexes in the class of OXPHOS diseases. These assays are extremely simple to perform, rapid (1-1.5 h) and reproducible with low intra-assay and inter-assay coefficients of variability (CVs) s (<10%). Importantly, there is no need to purify mitochondria as crude extracts of whole cells or tissues are suitable samples. Therefore, the assays allow use of samples obtained non-invasively such as cheek swabs and whole blood, which are not amenable to traditional mitochondrial purification and OXPHOS enzyme analysis. As a first step to assess clinical utility of these novel assays, they were used to screen a panel of cultured fibroblasts derived from patients with isolated deficiencies in complex I or IV caused by identified genetic defects. All patients (5/5) with isolated complex IV deficiencies were identified in this population. Similarly, almost all (22/24) patients with isolated complex I deficiencies were identified. We believe that this assay approach should find widespread utility in initial screening of patients suspected of having mitochondrial disease.

Development of the neural crest.

Mutations that affect the morphogenetic behaviour and differentiation of neural crest-derived cells in mouse embryos have been shown to alter genes that code for growth factors or growth factor receptors. Identification of these and other gene products provide opportunities to understand when and how developmentally distinct embryonic cell populations arise, and how interactions between localized developmental cues and responsive cell subpopulations can be modulated during development.

Tissue distribution of cytochrome c oxidase isoforms in mammals. Characterization with monoclonal and polyclonal antibodies.

Monoclonal and polyclonal antibodies specific to the two isoforms of subunit VIa of bovine cytochrome c oxidase were generated and used to study the tissue distribution of this subunit pair in beef, human and rat. The so-called H-(heart) form was found exclusively in heart and skeletal muscle, whereas the so-called L-(liver) form was the only isoform present in brain, kidney, liver and smooth muscle. Little or no L-form was detected in skeletal muscle. In bovine heart no subunit VIa-L was detected, while in human heart the subunit VIa-H and VIa-L isoforms were present in roughly equal proportions. These results imply that, in humans, the deficiency of a subunit VIa isoform may have a different effect on the physiology of heart then on the physiology of skeletal muscle.

Subunit specific monoclonal antibodies show different steady-state levels of various cytochrome-c oxidase subunits in chronic progressive external ophthalmoplegia.

Monoclonal antibodies recognizing the mitochondrially encoded subunits I and II, and the nuclear-encoded subunits IV, Va, Vb and VIc of human cytochrome-c oxidase were generated. These antibodies are highly specific and allow the assessment of subunit steady-state levels in crude cell extracts and tissue sections. In the experimental human cell line 143B206, which is devoid of mitochondrial DNA, immunovisualization with the antibodies revealed that the nuclear-encoded subunits IV and Va were present in amounts close to that of the parental cell line despite the absence of the mitochondrially encoded subunits. In contrast, the nuclear-encoded subunits Vb and VIc were severely reduced in cell line 143B206, suggesting that unassembled nuclear-encoded subunits are degraded at different rates. In skeletal muscle sections of a patient with chronic progressive external ophthalmoplegia known to harbor the 'common deletion' in a subpopulation of her mitochondrial DNA, most cytochrome-c oxidase activity negative fibers had greatly reduced levels of subunits I, II, Va, Vb and VIc of cytochrome-c oxidase. The steady-state level of subunit IV, however, was less affected. This was particularly evident in cytochrome-c oxidase activity negative fibers with accumulated mitochondria ('ragged-red' fibers) where immunodetection with anti-subunit IV resulted in intense staining. The data presented in this paper demonstrate that the battery of monoclonal antibodies can be employed for diagnostic purposes to analyze steady-state levels of mitochondrially and nuclear-encoded subunits of cytochrome-c oxidase.

Expression of mtDNA and nDNA encoded respiratory chain proteins in chemically and genetically-derived Rho0 human fibroblasts: a comparison of subunit proteins in normal fibroblasts treated with ethidium bromide and fibroblasts from a patient with mtDNA depletion syndrome.

Although much progress has been made in identifying genetic defects associated with mitochondrial diseases, the protein expression patterns of most disorders are poorly understood. Here we use immunochemical techniques to describe subunit expression patterns of respiratory chain enzyme complexes II (succinate dehydrogenase: SD) and IV (cytochrome c oxidase: COX) in cultured cells lacking mtDNA (Rho0 cells) derived either chemically by exposure of normal cells to ethidium bromide, or genetically in cells derived from a patient with mtDNA depletion syndrome. Both control cells and early passage patient-derived cells express a normal complement of SD and COX subunit proteins. Ethidium bromide treatment of normal cells and in vitro cell proliferation of patient-derived cells caused both populations to acquire identical Rho0 phenotypes. As expected, they lack mtDNA-encoded subunits COX-I and COX-II. In contrast, nDNA-encoded subunits are affected differentially, with some (COX-VIc) lacking and others (COX-IV, COX-Va, SD 30 and SD 70) maintained at somewhat reduced levels. We suggest that the differential stability of nDNA-encoded subunits in the absence of intact enzyme complexes is due to the ability of some, but not all, subunits to associate as partial complexes in the absence of mtDNA-encoded subunits.

Efficient hybridoma production using previously frozen splenocytes.

Splenocytes from immunized mice and rats can be frozen, thawed, and fused with myeloma cells to generate monoclonal antibody-secreting hybridoma cell lines. The hybridoma yield per splenocyte is comparable to, and often better than, the yield when fresh splenocytes are used. Practical advantages of the technique include the ability to utilize all splenocytes from a given mouse or rat, greater scheduling flexibility, and the opportunity to perform small-scale 'test fusions' to monitor the immune responses of a number of immunized animals.

A rapid method for processing very large numbers of tissue sections for immunohistochemical hybridoma screening.

A hybridoma screening format which facilitates the processing of thousands of tissue sections for immunohistochemical analysis is described. The method utilizes sterile replica transfers of monoclonal antibody-containing test supernatants from 96-well culture plates to tissue sections mounted in appropriately sized 8 X 12 arrays, and is extremely rapid and inexpensive.

Antibody-based approaches to diagnosis and characterization of oxidative phosphorylation diseases.

Mitochondrial disorders caused by defects in oxidative phosphorylation function are difficult to diagnose. Here we review the emerging use of antibody-based approaches for this diagnosis. Novel methods involving immunohistochemistry and immunocapture of defective enzymes for characterization are described that add to the arsenal of approaches available.

Differentiation of neurogenic precursors within the neural crest cell lineage.

It has been suggested that many, if not all crest-derived neurons develop from a limited subpopulation of neurogenic precursors. To develop cell-type specific markers that identify these precursors directly we have used differential screening of crest-derived cell populations known to have, or not to have, neurogenic ability. We have determined that the neuron-specific human auto-antibodies designated Anti-Hu bind to cytoplasmic and nuclear determinants not only in mature avian neurons and neuroendocrine cells but also in subpopulations of morphologically non-neuronal avian crest-derived cells. Significantly, these Anti-Hu+ non-neuronal crest-derived cells are present only in populations that have neurogenic ability and are absent from populations that lack neurogenic ability. Moreover, following additional development in vivo or in vitro, Anti-Hu+ non-neuronal crest-derived cells appear to express other neuronal traits. These results suggest that Anti-Hu-immunoreactivity is an early indicator of neurogenesis among crest-derived cells, and that Anti-Hu+ non-neuronal cells are either neurogenic precursors or immature neurons. Similarly, using the same differential screening paradigm, we have identified two monoclonal antibodies, designated 12E10 and 17F5, which also label both neurons and some apparently nonneuronal cells in neurogenic populations of neural crest cells. Anti-Hu-IR appears to precede expression of either of these two markers.

Role of neuron-target interactions in the development of a subpopulation of avian sensory neurons.

Subpopulations of sensory neurons appear in embryonic dorsal root ganglia during development. In this paper, we examine what role neuron-target interactions play in this process. Previous work has shown that alterations in the environment of developing sensory neurons regulate the proportion of neurons that express a cell surface antigen identified by the monoclonal antibody SN1 (Marusich et al.: Dev. Biol. 118: 505-510, 1986). We now report that neuron-target interactions may also act to stabilize the phenotype of sensory neurons. Thus, older SN1- neurons, which would normally remain SN1- if left undisturbed in vivo, can express SN1 immunoreactivity in vitro when they are deprived of contact with their normal peripheral targets. We also demonstrate that naive sensory neurons, i.e., those that have never made contact with peripheral targets, can be identified and maintained in culture. At least some of these naive neurons (all of which are initially SN1-) can express SN1 immunoreactivity in vitro, in the absence of contact with normal peripheral targets. We conclude that subpopulations of sensory neurons may arise from naive neurons in the absence of neuron-target interactions but that subsequent neuron-target interactions may act to stabilize or modulate subpopulation-specific phenotypes.

Identification of early neurogenic cells in the neural crest lineage.

Pluripotent neural crest cells are restricted progressively during development. The sequence of restrictions and the time(s) in early development at which such restrictions are imposed on crest-derived cells are largely unknown. We have used a human autoantibody (Anti-Hu) to characterize neurogenic populations of avian neural crest-derived cells. Anti-Hu binds specifically to neurons and neuroendocrine cells in older (greater than E4) quail embryos. Early in development, Anti-Hu also binds a subpopulation of neural crest-derived cells that lack neuronal morphology and do not express other neuronal traits. These cells may represent a putative neurogenic precursor subpopulation within the early crest cell lineage. To test this hypothesis, we have characterized Anti-Hu immunoreactivity within crest-derived populations known to have, or to lack, the ability to give rise to new neurons. We report that the presence of Anti-Hu+ nonneuronal cells is correlated with the neurogenic ability of a given cell population. Moreover, Anti-Hu+ nonneuronal cells are transient and appear to be replaced by Anti-Hu+ neuronal cells. We conclude that Anti-Hu is a very early indicator of neurogenesis among crest-derived cells and that Anti-Hu+ nonneuronal cells are either neurogenic precursors or immature neurons.

Changes in cell surface antigens during in vitro lizard myogenesis.

Indirect immunofluorescence has been used to examine surface antigens of lizard myogenic cells during in vitro differentiation. At least two developmental stage-specific surface alterations have been identified. One of these is a compositional change and involves the appearance of a cell-surface antigen(s) as the cells differentiate. This antigen(s) (Ag1422) is muscle specific and is characteristic of some rounded-up G0 myosin-positive myocytes, all stretched-back, G0 myosin-positive myocytes, and all identifiable myotubes. The antigen is not found on proliferating myoblasts, extended G1 (myosin-negative) cell-cycle-competent myoblasts or newly differentiated rounded-up, G0 myosin-positive myocytes. Pretreatment of cells with neuraminidase, trypsin, or proteinase K indicates the antigen is not present in "masked" form on normally nonreactive cells. Proteinase K is effective in the removal or destruction of the antigen, indicating it is at least partially protein in nature. The antigen is expressed in a similar developmental stage-specific fashion on early-passage myogenic cells taken from both adult lizard tail regenerates and embryonic muscle. The antibodies identifying Ag1422 can be removed by adsorption with homogenates of mature skeletal muscle. Therefore, Ag1422 is not an artifact due to in vitro conditions or the expression of a transformation antigen unique to the continuous culture line. The second alteration is an apparent restriction in the mobility of surface components (antigens and lectin receptors). Upon treatment with multivalent ligands, undifferentiated myosin-negative myoblasts exhibit rapid patching and capping of cell surface components while well-differentiated myocytes and myotubes do not. This mobility restriction is evident after the appearance of Ag1422. Treatment with cytochalasin B (15 micrograms/ml) and/or colchicine (100 microM) does not alter the restricted mobility of surface components seen on differentiated cells. Therefore, neither microfilaments nor microtubules seem to be involved in the mobility restriction. These observations are discussed in relation to current views of myogenesis.

A monoclonal antibody (SN1) identifies a subpopulation of avian sensory neurons whose distribution is correlated with axial level.

We have produced a monoclonal antibody, designated SN1, which binds to the surfaces of a subpopulation of avian sensory neurons, but not to other neurons of the peripheral or central nervous systems. The proportion of SN1(+) neurons in brachial and lumbosacral dorsal root ganglia (DRG), which innervate the wings and legs respectively, is low (30-40%), compared to the proportion (80-90%) in the lower thoracic DRG. SN1 immunoreactive fibers project to laminae I and II of the spinal cord dorsal horn, and are seen in the skin, but not the deeper tissues of older embryos. On the basis of the time of appearance, axial level-dependent distribution, and the central and peripheral projections of SN1(+) neurons, we suggest that they are cutaneous afferents that depend on interaction with peripheral targets to differentiate.

The development of an identified subpopulation of avian sensory neurons is regulated by interaction with the periphery.

The monoclonal antibody, SN1, binds to the cell surface of a subpopulation of avian sensory neurons. Dorsal root ganglia (DRG) that innervate different peripheral fields contain significantly different proportions of SN1(+) neurons. Moreover, the developmental time of appearance of these neurons suggests that they normally express SN1 immuno-reactivity only after they have made contact with their normal peripheral targets (Marusich et al., 1986). In the present paper, we test the hypothesis that the proportion of SN1(+) neurons within DRG is regulated by interactions between the developing neurons and their peripheral targets. Thus, immature DRG explanted into a defined medium show an age-dependent increase in the proportion of cells able to become SN1(+), but fail to show the large increase in number of immunoreactive neurons that occurs in vivo. Moreover, unilateral wing bud extirpations performed at E3 (prior to wing innervation) resulted in a dramatic selective decrease in the number of SN1(+) neurons within DRG that normally innervate the wing. These results support the hypothesis that peripheral targets regulate the appearance of SN1(+) neurons.

Restriction of neurogenic ability during neural crest cell differentiation.

Multipotent neural crest cells undergo developmental restrictions during embryogenesis and eventually give rise to the neurons and glia of the peripheral nervous system, melanocytes, and pheochromocytes. To understand how neuronal potential is restricted to a subpopulation of crest-derived cells, we have utilized sensitive markers of early neuronal differentiation to assess neurogenesis in crest-derived cell populations subjected to defined experimental conditions in vitro and in vivo. We describe environmental conditions that either (a) result in the irreversible loss of neurogenic potential over a characteristic time course or (b) maintain neurogenic potential among neural crest cells.

Hu neuronal proteins are expressed in proliferating neurogenic cells.

We have utilized immunochemical techniques to investigate the developmental expression of the Hu proteins, a neuron-specific family of RNA binding proteins in vertebrates. Previous work suggests that these proteins may play an important role in neuronal development and maintenance. For the present study, we developed a monoclonal antibody (MAb 16A11) that binds specifically to an epitope present in gene products of all known Hu genes, including HuD, HuC, and Hel-N1. Using brief pulses (1-2 h) of the DNA precursor analog bromodeoxyuridine (BrdU) in conjunction with MAb 16A11, we observed Hu+/BrdU+ cells in nascent sensory and sympathetic ganglia in vivo, and in populations of cultured neural crest cells. In addition, a few Hu+ cells were ambiguously BrdU+ in the neural tube. We conclude that Hu+ cells first appear in avian neurogenic populations immediately before neuronal birthdays in the peripheral nervous system, and at the time of withdrawal from the mitotic cycle in the central nervous system. Consistent with these conclusions, we have also observed neural crest-derived cells that are both Hu+ and in metaphase of the cell cycle. We suggest that Hu proteins function early in neurogenic differentiation.

Human complex I defects can be resolved by monoclonal antibody analysis into distinct subunit assembly patterns.

Complex I defects are one of the most frequent causes of mitochondrial respiratory chain disorders. Therefore, it is important to find new approaches for detecting and characterizing Complex I deficiencies. In this paper, we introduce a new set of monoclonal antibodies that react with 39-, 30-, 20-, 18-, 15-, and 8-kDa subunits of Complex I. These antibodies are shown to aid in diagnosis of Complex I deficiencies and add understanding to the genotype-phenotype relationships of different mutations. A total of 11 different patients were examined. Four patients had undefined Complex I defects, whereas the other patients had defects in NDUFV1, NDUFS2 (two patients), NDUFS4 (two patients), NDUFS7, and NDUFS8. We show here that Western blotting with these antibodies, particularly when used in conjunction with sucrose gradient studies and enzymatic activity measurements, helps distinguish catalytic versus assembly defects and further distinguishes between mutations in different subunits. Furthermore, different mutations in the same gene are shown to give very similar subunit profiles, and we show that one of the patients is a good candidate for having a defect in a Complex I assembly factor.