Mitochondrial gene therapy augments mitochondrial physiology in a Parkinson's disease cell model.
ABSTRACT Neurodegeneration in Parkinson's disease (PD) affects mainly dopaminergic neurons in the substantia nigra, where age-related, increasing percentages of cells lose detectable respiratory activity associated with depletion of intact mitochondrial DNA (mtDNA). Replenishment of mtDNA might improve neuronal bioenergetic function and prevent further cell death. We developed a technology ("ProtoFection") that uses recombinant human mitochondrial transcription factor A (TFAM) engineered with an N-terminal protein transduction domain (PTD) followed by the SOD2 mitochondrial localization signal (MLS) to deliver mtDNA cargo to the mitochondria of living cells. MTD-TFAM (MTD = PTD + MLS = "mitochondrial transduction domain") binds mtDNA and rapidly transports it across plasma membranes to mitochondria. For therapeutic proof-of-principle we tested ProtoFection technology in Parkinson's disease cybrid cells, using mtDNA generated from commercially available human genomic DNA (gDNA; Roche). Nine to 11 weeks after single exposures to MTD-TFAM + mtDNA complex, PD cybrid cells with impaired respiration and reduced mtDNA genes increased their mtDNA gene copy numbers up to 24-fold, mtDNA-derived RNAs up to 35-fold, TFAM and ETC proteins, cell respiration, and mitochondrial movement velocities. Cybrid cells with no or minimal basal mitochondrial impairments showed reduced or no responses to treatment, suggesting the possibility of therapeutic selectivity. Exposure of PD but not control cybrid cells to MTD-TFAM protein alone or MTD-TFAM + mtDNA complex increased expression of PGC-1alpha, suggesting activation of mitochondrial biogenesis. ProtoFection technology for mitochondrial gene therapy holds promise for improving bioenergetic function in impaired PD neurons and needs additional development to define its pharmacodynamics and delineate its molecular mechanisms. It also is unclear whether single-donor gDNA for generating mtDNA would be a preferred therapeutic compared with the pooled gDNA used in this study.
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ABSTRACT: Mutations of mitochondrial DNA (mtDNA) are associated with a wide spectrum of disorders encompassing the myopathies, encephalopathies and cardiomyopathies, in addition to organ specific presentations such as diabetes mellitus and deafness. The pathogenesis of mtDNA mutations is not fully understood although it is assumed that their final common pathway involves impaired oxidative phosphorylation. The identification of a specific respiratory chain defect (complex I deficiency) in Parkinson's disease (PD) 10 years ago focused attention on the aetiological and pathogenetic roles that mitochondria may play in neurodegenerative diseases. There is evidence now emerging that mtDNA abnormalities may determine the complex I defect in a proportion of PD patients and it may prove possible to use biochemical analysis of platelet and cybrid complex I function to identify those that lie within this group. Respiratory chain defects of a different pattern have been identified in Huntington's disease (HD) (complex II/III deficiency) and Friedreich's ataxia (FA) complex I-III deficiency). In both these disorders, the mitochondrial abnormality is secondary to the primary nuclear mutation:CAG repeat in the huntingtin gene in HD, and GAA repeat in the frataxin gene in FA. Nevertheless, it appears that the mitochondrion may be the target of the biochemical defects that are the consequence of these mutations. There is a close and reciprocal relationship between respiratory chain dysfunction and free radical generation, and there is evidence for oxidative stress and damage in PD, HD and FA, which together with the mitochondrial defect may result in cell damage. Impaired oxidative phosphorylation and free radical generation may independently adversely affect the maintenance of mitochondrial transmembrane potential (Deltapsim). A fall in Deltapsim is an early event (preceding nuclear fragmentation) in the apoptotic pathway. It is possible therefore that mitochondrial dysfunction in the neurodegenerative disorders may result in a fall in the apoptotic threshold of neurones which, in some, may be sufficient to induce cell death whilst, in others, additional factors may be required. In any event, mitochondria present an important target for future strategies for 'neuroprotection' to prevent or retard neurodegeneration.Biochimica et Biophysica Acta 09/1998; 1366(1-2):225-33. · 4.66 Impact Factor
Article: Elimination and tolerance of a new parenteral lipid emulsion (SMOF)--a double-blind cross-over study in healthy male volunteers.[show abstract] [hide abstract]
ABSTRACT: The objective of this phase I study was to investigate plasma elimination and tolerance of a new lipid emulsion based on soybean oil, medium chain triglycerides (MCT), olive oil and fish oil (SMOF). In a double-blind, randomized, cross-over study, 12 healthy male subjects received SMOF 20% and a standard soybean oil emulsion (Lipovenoes 20%, both Fresenius Kabi). Lipid emulsions were infused at a rate of 0.125 g fat/kg body weight/h over 6 h. Before, during and up to 24 h after infusion, lipid metabolism parameters and numerous clinical, chemical and hematological parameters, vital signs and local tolerance were determined. Infusion of SMOF induced a less marked increase of serum triglyceride concentration. At the end of infusion, mean serum triglyceride concentration was significantly lower with SMOF (p < 0.05). Triglyceride half-life was significantly shorter for SMOF than for Lipovenoes (p < 0.001). Infusion of SMOF was apparently associated with higher glycerol concentration (NS). Routine laboratory parameters, vital signs and local tolerance showed no evidence of infusion-related abnormalities. SMOF was eliminated significantly faster than the standard lipid emulsion. This is of potential benefit in patients with limited triglyceride elimination capacity. The safety evaluation revealed a good systemic and local tolerance of SMOF.Annals of Nutrition and Metabolism 01/2004; 48(4):263-8. · 2.26 Impact Factor
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ABSTRACT: Mitochondrial DNA (mtDNA) deletions have been investigated in a number of neurodegenerative diseases. This study aimed to investigate the characteristics of mtDNA deletions found in single substantia nigra neurons from three patient groups: controls, Parkinson disease patients, and a patient with Parkinsonism due to multiple mtDNA deletions. We have identified 89 deletions from these neurons and examined the breakpoint characteristics of them. There was no difference in the types of mtDNA deletions detected in these neurons. These results suggest that the mechanism leading to the formation of these deletions in these three distinct groups could be the same.The American Journal of Human Genetics 02/2008; 82(1):228-35. · 10.60 Impact Factor