Sensitive and specific detection of α-synuclein in human plasma.

alpha-Synuclein (alphasyn) mutations, overexpression, misfolding, and aggregation are associated with Parkinson's disease. This protein has been intensively studied in neuronal systems. However, alphasyn is also present in extracellular fluids, such as cerebrospinal fluid and blood plasma. Recent studies have attempted to quantify its levels and compare these in various extracellular fluids of control and Parkinson's disease subjects. Data from these studies have been difficult to interpret, suggesting that more sensitive, standardized, and well-characterized assays of larger cohorts are required. Here, we describe the development of a new ELISA specifically for quantifying alphasyn in human plasma. An initial assay, using a commercial anti-alphasyn monoclonal antibody (211; Santa Cruz Biotechnology, Santa Cruz, CA) and based on a published protocol, was adapted for use in human plasma. In addition, we have developed a novel alphasyn-specific antibody for the assay that has very high sensitivity and signal:noise characteristics. Assays with either antibody showed high specificity for alphasyn, and detected it in a variety of sample types, including plasma. These assays can now be employed on large cohorts of patients and control subjects to determine whether plasma levels are altered in disease. Although measuring extracellular alphasyn levels may prove to be a useful biomarker of Parkinson's disease, it should also be a powerful tool for basic research aimed at understanding the normal and pathological physiology of alphasyn secretion. .

Differential insult-dependent recruitment of the intrinsic mitochondrial pathway during neuronal programmed cell death.

Programmed cell death contributes to neurological diseases and may involve mitochondrial dysfunction with redistribution of apoptogenic proteins. We examined neuronal death to elucidate whether the intrinsic mitochondrial pathway and the crosstalk between caspase-dependent/-independent injury was differentially recruited by stressors implicated in neurodegeneration. After exposure of cultured cerebellar granule cells to various insults, the progression of injury was correlated with mitochondrial involvement, including the redistribution of intermembrane space (IMS) proteins, and patterns of protease activation. Injury occurred across a continuum from Bax- and caspase-dependent (trophic- factor withdrawal) to Bax-independent, calpain-dependent (excitotoxicity) injury. Trophic-factor withdrawal produced classical recruitment of the intrinsic pathway with activation of caspase-3 and redistribution of cytochrome c, whereas excitotoxicity induced early redistribution of AIF and HtrA2/Omi, elevation of intracellular calcium and mitochondrial depolarization. Patterns of engagement of neuronal programmed cell death and the redistribution of mitochondrial IMS proteins were canonical, reflecting differential insult-dependencies.

Smac/DIABLO is not released from mitochondria during apoptotic signalling in cells deficient in cytochrome c.

We have characterised the apoptotic defects in cells null for cytochrome c (cyt c-/-). Such cells treated with staurosporine (STS) exhibited translocation to the mitochondria and activation of the proapoptotic signalling molecule Bax but failed to release Smac/DIABLO from these organelles, judged by both confocal microscopy and Western blotting. While reference cells expressing cytochrome c released both it and Smac/DIABLO under a variety of conditions of apoptotic induction, we have never observed release of Smac/DIABLO from cyt c-/- cells. We eliminate the possibility that proteasomal degradation of cytosolically localised Smac/DIABLO is responsible for our failure to visualise the protein outside the mitochondria. Our findings indicate an unanticipated nexus between release of cytochrome c and Smac/DIABLO from mitochondria, previously thought to be a more or less synchronised event early in apoptosis. We suggest that the failure of cyt c-/- cells to release Smac/DIABLO after recruitment of Bax to mitochondria represents an extreme manifestation of some inherent difference in the regulation of release of these two proteins from mitochondria.

Ageing and mammalian mitochondrial genetics.

Mitochondrial DNA (mtDNA) is essential for the ability of mammalian cells to generate a functional oxidative phosphorylation system. Mutations in mtDNA occur in human disease and also during ageing. Here, we address three questions concerning the occurrence and accumulation of mtDNA mutations during the lifespan of the mammalian cell. What sort of mutations accumulate with age in humans and other mammals? How is the female germ line spared from the accumulation of such mutations as occurs in many somatic tissues, so that neonates normally start life with a 'clean sheet'? Is the occurrence of mtDNA mutations associated with the functional decline of cells and tissues during ageing? We argue that mtDNA mutations in somatic cells do not just reflect a passive imprint of ageing, but they are causally associated with the loss of bioenergetic function during the ageing process.

High incidence of somatic mitochondrial DNA mutations in human ovarian carcinomas.

To investigate the potential role of somatic mitochondrial DNA (mtDNA) mutations in tumorigenesis, the occurrence of mutations in mtDNA of ovarian carcinomas was studied. We sequenced the D-loop region of mtDNA of 15 primary ovarian carcinomas and their matched normal controls. Somatic mtDNA mutations were detected in 20% (3 of 15) tumor samples carrying single or multiple changes. Complete sequence analysis of the mtDNA genomes of another 10 pairs of primary ovarian carcinomas and control tissues revealed somatic mtDNA mutations in 60% (6 of 10) of tumor samples. Most of these mutations were homoplasmic, and most were T-->C or G-->A transitions, but one represented a differential length within a run of identical C residues. A region of mtDNA sequence including the 16S and 12S rRNA genes, the D-loop and the cytochrome b gene, may represent the zone of preferred mtDNA mutation in ovarian cancer. The high incidence of mtDNA mutations found in ovarian carcinomas and other human cancers suggests that genetic instability of mtDNA might play a significant role in tumorigenesis.

Import into mitochondria of precursors containing hydrophobic passenger proteins: pretreatment of precursors with urea inhibits import.

We have studied the import into isolated yeast mitochondria of three hydrophobic passenger proteins attached to the N-terminal cleavable presequence of mitochondrial ATPase subunit 9 from Neurospora crassa. One natural precursor (pN9) contained N. crassa subunit 9; two chimaeric precursors, N9L/Y8-1 and N9L/Y9-2, respectively contained yeast mitochondrial ATPase subunits 8 and 9. In the absence of urea, pN9 and N9L/Y8-1 are imported efficiently but N9L/Y9-2 is not imported. After pretreatment of precursors in 4 M urea, binding of pN9 to mitochondria is marginally affected while its import is substantially inhibited; the binding to mitochondria of chimaeric proteins, N9L/Y8-1 and N9L/Y9-2, is greatly enhanced but no import is observed. This behaviour of import precursors containing hydrophobic passenger proteins is contrasted with that of a hydrophilic chimaeric precursor pCOXIV-DHFR, whose binding and import are enhanced by pretreatment with a high concentration of urea (8 M). The import of N9L/Y8-1 is very sensitive to the presence of low concentrations of urea in the import reaction mixture, and is abolished above 0.5 M urea although precursor binding to mitochondria is increased. By contrast, neither the import nor binding of pCOXIV-DHFR is affected directly by urea up to 0.8 M. These deleterious effects of urea on import of the chimaeric precursors N9L/Y8-1 and N9L/Y9-2 are interpreted in terms of a non-productive binding of these precursors to mitochondria, brought about by exposure of their hydrophobic domains resulting from urea unfolding. The generalization that membrane translocation of mitochondrial import precursors is enhanced by their prior unfolding in urea thus does not apply in the case of these precursors containing hydrophobic passenger proteins.

A petite mitochondrial DNA segment arising in exceptionally high frequency in a mit- mutant of Saccharomyces cerevisiae.

In cultures of the mit- mutant strain Mb12 of Saccharomyces cerevisiae (carrying a mutation in the oli2 gene), 70% of the cells are petite mutants. More than 80% of the petites from Mb12 contain a particular mtDNA segment, denoted BB5, that is 880 bp long and carries a single MboI site. Thus, in cultures of Mb12, about 56% of the cells are petites containing the defective BB5 mtDNA genome, and only 30% are mit- cells containing parental Mb12 mtDNA. The BB5 mtDNA segment is also found in petites arising from the wild-type strain J69-1B (from which Mb12 was derived), but in this case mtDNA from only five out of 24 petites produced an 880 bp band after MboI digestion. Since J69-1B cultures carry a petite frequency of about 5%, approximately 1% of cells in J69-1B cultures contain the BB5 mtDNA segment. The difference between Mb12 and J69-1B cultures is reflected in the MboI digestion patterns of the respective mtDNAs. While Mb12 mtDNA contains a grossly superstoicheiometric 880 bp MboI fragment, the corresponding fragment in J69-1B mtDNA cannot be seen on stained gels, but can be readily visualized in Southern blots hybridized to a 32P-labelled DNA probe obtained from the 880 bp MboI fragment. The BB5 mtDNA segment was shown to contain the ori1 sequence (one of several very similar sequences in wild-type mtDNA thought to act as origins of replication of mtDNA) which confers the genetic property of very high suppressiveness on petites carrying this mtDNA. The efficient replication of BB5 mtDNA may contribute to its abundance in Mb12 cultures. Nevertheless, other factors must operate to influence the abundance of the BB5 mtDNA segment in cultures of different strains, the most important of which is likely to be the rate of excision of this mtDNA segment from the parental mtDNA genome.

Insights into ATP synthase assembly and function through the molecular genetic manipulation of subunits of the yeast mitochondrial enzyme complex.

Development of an increasingly detailed understanding of the eucaryotic mitochondrial ATP synthase requires a detailed knowledge of the stoichiometry, structure and function of F(0) sector subunits in the contexts of the proton channel and the stator stalk. Still to be resolved are the precise locations and roles of other supernumerary subunits present in mitochondrial ATP synthase complexes, but not found in the bacterial or chloroplast enzymes. The highly developed system of molecular genetic manipulation available in the yeast Saccharomyces cerevisiae, a unicellular eucaryote, permits testing for gene function based on the effects of gene disruption or deletion. In addition, the genes encoding ATP synthase subunits can be manipulated to introduce specific amino acids at desired positions within a subunit, or to add epitope or affinity tags at the C-terminus, enabling questions of stoichiometry, structure and function to be addressed. Newly emerging technologies, such as fusions of subunits with GFP are being applied to probe the dynamic interactions within mitochondrial ATP synthase, between ATP synthase complexes, and between ATP synthase and other mitochondrial enzyme complexes.

Each of three positively-charged amino acids in the C-terminal region of yeast mitochondrial ATP synthase subunit 8 is required for assembly.

Each of three conserved positively-charged residues in the C-terminal region of subunit 8 of yeast (Saccharomyces cerevisiae) mitochondrial ATP synthase was replaced with isoleucine. The assembly and functional properties of the resulting variants (substituted at Arg-37, Arg-42 and Lys-47) were examined using in-vitro systems to assay import into isolated mitochondria and to monitor assembly into ATP synthase, as well as an in-vivo rescue system using host yeast cells lacking endogenous subunit 8. Each such variant was found to be impaired in assembly in vitro, after import in the form of a chimaeric protein bearing a leader sequence with mitochondrial targeting function. Import precursors bearing a duplicated-leader sequence, engendering enhanced delivery to mitochondria of the passenger variant subunit-8 proteins, enabled assembly of the (Lys-47-->Ile) variant to be detected in vitro but not that of (Arg-37-->Ile) or (Arg-42-->Ile) variants. The respiratory growth of subunit 8-deficient host cells could be rescued with the (Lys-47-->Ile) variant expressed allotopically in the nucleus. Such rescued cells were found to have an enhanced growth rate (comparable to that produced by non-mutagenized parental subunit 8) when delivered to mitochondria with the duplicated-leader sequence, as compared to the single-leader sequence. This confirms that the impediment in the (Lys-47-->Ile) variant lies in the efficiency of its assembly, rather than a functional defect, as such, arising from the loss of that positive charge. In contrast, host cells were unable to be rescued by the (Arg-37-->Ile) and (Arg-42-->Ile) variants, even when they were endowed with the duplicated leader sequence. It is concluded that the positively-charged C-terminal domain of subunit 8, common to fungal and mammalian homologues of this protein, plays a key role in its assembly into mitochondrial ATP synthase.

The assembly of yeast mitochondrial ATP synthase: subunit depletion in vivo suggests ordered assembly of the stalk subunits b, OSCP and d.

The abundance in vivo of each of three subunits b, OSCP and d, components of the stalk region of the yeast mitochondrial ATP synthase complex, was manipulated by a controlled depletion strategy. Western blots of whole cell lysates were used to study the effect of depletion of each of these subunits on the cellular levels of other subunits of the enzyme complex. A hierarchy of subunit stability was determined and interpreted to indicate the order of assembly of these three subunits of the stalk region. Thus, subunit b is assembled first, followed by OSCP and then by subunit d.

Mitochondria are the functional intracellular target for a photosensitizing boronated porphyrin.

A photosensitizing boron-containing porphyrin derivative denoted BOPP, which is selectively localised into mitochondria, has been tested on Namalwa cells, in each of two genetic configurations: rho+ cells containing normal mtDNA and mitochondrial respiratory functions, or rho0 cells lacking mtDNA and devoid of mitochondrial oxidative phosphorylation. After short-term cellular uptake for 18 h, BOPP (30 micrograms/ml) was not cytotoxic, but did show marked phototoxicity in Namalwa rho+ cells, concomitant with substantial reduction of mitochondrial respiratory activity. After long-term (3 days or more) exposure to BOPP without light, growth of Namalwa rho+ cells was inhibited at concentrations significantly above 30 micrograms/ml. At such concentrations BOPP was shown to have direct inhibitory effects on mitochondrial azide-sensitive respiration of p+ cells. By contrast, BOPP showed neither cytotoxic nor phototoxic effects in rho0 cells. These results indicate functional mitochondria to be a major cellular target in vivo after BOPP uptake and photoactivation.

The universality of bioenergetic disease and amelioration with redox therapy.

Overt mitochondrial diseases associated with mitochondrial DNA mutations are characterized by a decline in mitochondrial respiratory function. Similarly, a progressive decline in mitochondrial respiratory function associated with mitochondrial DNA mutations is clearly evidenced in aged human subjects. This communication is concerned with the development of a rat model for the study of bioenergy decline associated with the ageing process and overt mitochondrial diseases. The model involves the treatment of young rats with AZT to induce skeletal and cardiac myopathies. It has shown that there is a decline in soleus muscle function in vivo and that this decline is mirrored in the capacity of heart sub-mitochondrial particles to maintain bioenergy function. Coenzyme Q10 and several analogs were administered with AZT as potential therapeutics for the re-energization of affected tissues. Coenzyme Q10 and especially decyl Q were found to be therapeutically beneficial by both in vivo improvement in soleus muscle function and in vitro cardiac mitochondrial membrane potential capacity. Sub-mitochondrial particles were also prepared from heart mitochondria of young and aged rats. The particles prepared from the aged rats were found to have a decreased ability to maintain membrane potential as compared to those derived from the young rats.

Suppression of a nuclear aep2 mutation in Saccharomyces cerevisiae by a base substitution in the 5'-untranslated region of the mitochondrial oli1 gene encoding subunit 9 of ATP synthase.

Mutations in the nuclear AEP2 gene of Saccharomyces generate greatly reduced levels of the mature form of mitochondrial oli1 mRNA, encoding subunit 9 of mitochondrial ATP synthase. A series of mutants was isolated in which the temperature-sensitive phenotype resulting from the aep2-ts1 mutation was suppressed. Three strains were classified as containing a mitochondrial suppressor: these lost the ability to suppress aep2-ts1 when their mitochondrial genome was replaced with wild-type mitochondrial DNA (mtDNA). Many other isolates were classified as containing dominant nuclear suppressors. The three mitochondrion-encoded suppressors were localized to the oli1 region of mtDNA using rho- genetic mapping techniques coupled with PCR analysis; DNA sequencing revealed, in each case, a T-to-C nucleotide transition in mtDNA 16 nucleotides upstream of the oli1 reading frame. It is inferred that the suppressing mutation in the 5' untranslated region of oli1 mRNA restores subunit 9 biosynthesis by accommodating the modified structure of Aep2p generated by the aep2-ts1 mutation (shown here to cause the substitution of proline for leucine at residue 413 of Aep2p). This mode of mitochondrial suppression is contrasted with that mediated by heteroplasmic rearranged rho- mtDNA genomes bypassing the participation of a nuclear gene product in expression of a particular mitochondrial gene. In the present study, direct RNA-protein interactions are likely to form the basis of suppression.

Coenzyme Q10 treatment improves the tolerance of the senescent myocardium to pacing stress in the rat.

OBJECTIVE: In elderly patients the results of cardiac interventions are inferior to those in the young. A possible contributing factor is an age-related reduction in cellular energy transduction during the intervention which may induce aerobic or ischemic stress. To investigate whether coenzyme Q10 (CoQ10) improves the response to aerobic stress, functional recoveries of senescent and young rat hearts after rapid pacing were compared with or without CoQ10. METHODS: Young (4.8 +/- 0.1 months) and senescent (35.3 +/- 0.2 months) rats were given daily intraperitoneal injections of CoQ10 (4 mg/kg) or vehicle for 6 weeks. Their isolated hearts were rapidly paced at 510 beats per minute for 120 min to induce aerobic stress without ischemia. RESULTS: In senescent hearts pre-pacing cardiac work was 74% and oxygen consumption (MVO2) 66% of that in young hearts. CoQ10 treatment abolished these differences. After pacing, the untreated senescent hearts, compared to young, showed reduced recovery of pre-pacing work, (16.8 +/- 4.3 vs. 44.5 +/- 7.4%; P < 0.01). CoQ10 treatment in senescent hearts improved recovery of work, (48.1 +/- 4.1 vs. 16.8 +/- 4.3%; P < 0.0001) and MVO2 (82.1 +/- 2.8 vs. 61.3 +/- 4.0%; P < 0.01) in treated versus untreated hearts respectively. Post-pacing levels of these parameters in CoQ10 treated senescent hearts were as high as in young hearts. CONCLUSIONS: (1) Senescent rat hearts have reduced baseline function and reduced tolerance to aerobic stress compared to young hearts. (2) Pre-treatment with CoQ10 improves baseline function of the senescent myocardium and its tolerance to aerobic stress.

Mitochondrial DNA deletions parallel age-linked decline in rat sensory nerve function.

In rats, the function of sensory nerves in the hind limb declines significantly with age. Normally aging rats and rats treated neonatally with capsaicin were studied here. Quantification of vascular response and substance P in young (3 months) and old (24 months) rats showed additive effects of age and capsaicin treatment. The levels in dorsal root ganglion of a particular deletion in mitochondrial DNA (mtDNA(4834)) were about 300-fold higher in old compared to young rats. Capsaicin treatment had no significant effect on mtDNA(4834) abundance. Dorsal root ganglia of old (but not young) rats were found to contain a spectrum of multiple deletions. The abundance of mtDNA(4834) in dorsal root ganglia from individual rats correlated strongly with their decline in vascular function, even where vascular responses were systematically depressed due to prior capsaicin treatment. One possibility is that mitochondrial DNA mutations directly lead to functional decline at mitochondrial and tissue levels. Alternatively, loss of mitochondrial DNA integrity and physiological decline may be consequences of the same factor, such as oxidative stress.

Amino acid substitutions in mitochondrial ATPase subunit 6 of Saccharomyces cerevisiae leading to oligomycin resistance.

The amino acid substitutions in subunit 6 of the mitochondrial ATPase complex have been determined for 4 oligomycin resistant mutants of Saccharomyces cerevisiae. The data were obtained for each mutant by nucleotide sequence analysis of the mitochondrial oli2 gene. Amino acid substitutions conferring oligomycin resistance in subunit 6 are located in two conserved regions that are thought to form domains which span the inner mitochondrial membrane. The disposition of these amino acid substitutions is consistent with the view that these two membrane-spanning domains interact structurally and functionally with the DCCD-binding proteolipid subunit 9 in the Fo-sector.

The protonophore CCCP induces mitochondrial permeability transition without cytochrome c release in human osteosarcoma cells.

Mitochondrial permeability transition (MPT) and cytochrome c redistribution from mitochondria are two events associated with apoptosis. We investigated whether an MPT event obligatorily leads to cytochrome c release in vivo. We have previously shown that treatment of human osteosarcoma cells with the protonophore m-chlorophenylhydrazone (CCCP) for 6 h induces MPT and mitochondrial swelling without significant cell death. Here we demonstrate that release of cytochrome c does not occur and the cells remain viable even after 72 h of treatment with CCCP. Bax is not mobilized to mitochondria under these conditions. However, subsequent exposure of CCCP-treated cells to etoposide or staurosporine for 48 h results in rapid cell death and cytochrome c release that is accompanied by Bax association with mitochondria, demonstrating competency of these mitochondria to release cytochrome c with additional triggers. Our findings suggest that MPT is not a sufficient condition, in itself, to effect cytochrome c release.

Amino acid substitutions in mitochondrial ATP synthase subunit 9 of Saccharomyces cerevisiae leading to venturicidin or ossamycin resistance.

A series of mitochondrially inherited mutants of yeast has been analysed, which were previously identified as showing resistance to the antibiotics venturicidin or ossamycin and whose mutations showed tight linkage to oligomycin-resistance alleles affecting subunit 9 of the mitochondrial ATP synthase. DNA sequence analysis of the oli1 gene of these mutants has been used to define the nature of amino acid substitution in the subunit 9 protein. In the case of the two venturicidin-resistant mutants, mutations affect amino acids on the N-terminal stem of the protein, namely Gly25----Ser (venR ossS oliR) and Ala 27----Gly (venR ossS oliS). The mutations found in the two ossamycin-resistant mutants affect amino acids on the C-terminal stem of the protein; namely Leu53----Phe (vanS ossR oliR) and Leu57----Phe (venS ossR oliS). These results allow us to further develop a fine structure map of domains within the subunit 9 protein involved in antibiotic interaction.

Duplication of leader sequence for protein targeting to mitochondria leads to increased import efficiency.

We describe a novel method for enhancing protein import into mitochondria, by tandemly duplicating the N-terminal cleavable leader peptide using a gene manipulation strategy. The import into isolated yeast mitochondria of passenger proteins (yeast mitochondrial ATP synthase subunits 8 and 9 and some mutagenised derivatives) that show little or no import when endowed with one such leader (that of Neurospora crassa mitochondrial ATP synthase subunit 9) is remarkably improved when the leader is tandemly duplicated. The import of these chimaeric proteins bearing a double leader is so rapid that a series of partially processed precursor intermediates accumulates inside the mitochondria before the final proteolytic release of leader sequences from the passenger proteins. It is considered that the duplicated leader greatly accelerates delivery of the import precursors to outer membrane receptor elements and the associated translocation systems, thereby enhancing precursor uptake into mitochondria.

Amino acid substitutions in mitochondrial ATPase subunit 9 of Saccharomyces cerevisiae leading to oligomycin or venturicidin resistance.

A series of isonuclear oligomycin-resistant mutants of Saccharomyces cerevisiae carrying mutations in the mitochondrial oli1 gene has been studied. DNA sequence analysis of this gene has been used to define the amino acid substitutions in subunit 9 of the mitochondrial ATPase complex. A domain of amino acids involved in oligomycin resistance can be recognized which encompasses residues in each of the two hydrophobic portions of the subunit 9 polypeptide that are thought to span the inner mitochondrial membrane. Certain amino acid substitutions also confer cross-resistance to venturicidin: these residues define an inner domain for venturicidin resistance. The expression of venturicidin resistance resulting from one particular substitution is modulated by nuclear genetic factors.