Differential Effects of Yeast NADH Dehydrogenase (Ndi1) Expression on Mitochondrial Function and Inclusion Formation in a Cell Culture Model of Sporadic Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder that exhibits aberrant protein aggregation and mitochondrial dysfunction. Ndi1, the yeast mitochondrial NADH dehydrogenase (complex I) enzyme, is a single subunit, internal matrix-facing protein. Previous studies have shown that Ndi1 expression leads to improved mitochondrial function in models of complex I-mediated mitochondrial dysfunction. The trans-mitochondrial cybrid cell model of PD was created by fusing mitochondrial DNA-depleted SH-SY5Y cells with platelets from a sporadic PD patient. PD cybrid cells reproduce the mitochondrial dysfunction observed in a patient's brain and periphery and form intracellular, cybrid Lewy bodies comparable to Lewy bodies in PD brain. To improve mitochondrial function and alter the formation of protein aggregates, Ndi1 was expressed in PD cybrid cells and parent SH-SY5Y cells. We observed a dramatic increase in mitochondrial respiration, increased mitochondrial gene expression, and increased PGC-1α gene expression in PD cybrid cells expressing Ndi1. Total cellular aggregated protein content was decreased but Ndi1 expression was insufficient to prevent cybrid Lewy body formation. Ndi1 expression leads to improved mitochondrial function and biogenesis signaling, both processes that could improve neuron survival during disease. However, other aspects of PD pathology such as cybrid Lewy body formation were not reduced. Consequently, resolution of mitochondrial dysfunction alone may not be sufficient to overcome other aspects of PD-related cellular pathology.

Mitochondrial quality, dynamics and functional capacity in Parkinson's disease cybrid cell lines selected for Lewy body expression.

BACKGROUND: Lewy bodies (LB) are a neuropathological hallmark of Parkinson's disease (PD) and other synucleinopathies. The role their formation plays in disease pathogenesis is not well understood, in part because studies of LB have been limited to examination of post-mortem tissue. LB formation may be detrimental to neuronal survival or merely an adaptive response to other ongoing pathological processes. In a human cytoplasmic hybrid (cybrid) neural cell model that expresses mitochondrial DNA from PD patients, we observed spontaneous formation of intracellular protein aggregates ("cybrid LB" or CLB) that replicate morphological and biochemical properties of native, cortical LB. We studied mitochondrial morphology, bioenergetics and biogenesis signaling by creating stable sub-clones of three PD cybrid cell lines derived from cells expressing CLB. RESULTS: Cloning based on CLB expression had a differential effect on mitochondrial morphology, movement and oxygen utilization in each of three sub-cloned lines, but no long-term change in CLB expression. In one line (PD63(CLB)), mitochondrial function declined compared to the original PD cybrid line (PD63(Orig)) due to low levels of mtDNA in nucleoids. In another cell line (PD61(Orig)), the reverse was true, and cellular and mitochondrial function improved after sub-cloning for CLB expression (PD61(CLB)). In the third cell line (PD67(Orig)), there was no change in function after selection for CLB expression (PD67(CLB)). CONCLUSIONS: Expression of mitochondrial DNA derived from PD patients in cybrid cell lines induced the spontaneous formation of CLB. The creation of three sub-cloned cybrid lines from cells expressing CLB resulted in differential phenotypic changes in mitochondrial and cellular function. These changes were driven by the expression of patient derived mitochondrial DNA in nucleoids, rather than by the presence of CLB. Our studies suggest that mitochondrial DNA plays an important role in cellular and mitochondrial dysfunction in PD. Additional studies will be needed to assess the direct effect of CLB expression on cellular and mitochondrial function.

Cybrid models of Parkinson's disease show variable mitochondrial biogenesis and genotype-respiration relationships.

Sporadic Parkinson's disease (sPD) is a nervous system-wide disease that presents with a bradykinetic movement disorder and frequently progresses to include depression and cognitive impairment. Cybrid models of sPD are based on expression of sPD platelet mitochondrial DNA (mtDNA) in neural cells and demonstrate some similarities to sPD brains. In sPD and CTL cybrids we characterized aspects of mitochondrial biogenesis, mtDNA genomics, composition of the respirasome and the relationships among isolated mitochondrial and intact cell respiration. Cybrid mtDNA levels varied and correlated with expression of PGC-1 alpha, a transcriptional co-activator regulator of mitochondrial biogenesis. Levels of mtDNA heteroplasmic mutations were asymmetrically distributed across the mitochondrial genome; numbers of heteroplasmies were more evenly distributed. Neither levels nor numbers of heteroplasmies distinguished sPD from CTL. sPD cybrid mitochondrial ETC subunit protein levels were not altered. Isolated mitochondrial complex I respiration rates showed limited correlation with whole cell complex I respiration rates in both sPD and CTL cybrids. Intact cell respiration during the normoxic-anoxic transition yielded K(m) values for oxygen that directly related to respiration rates in CTL but not in sPD cell lines. Both sPD and CTL cybrid cells are substantially heterogeneous in mitochondrial genomic and physiologic properties. Our results suggest that mtDNA depletion may occur in sPD neurons and could reflect impairment of mitochondrial biogenesis. Cybrids remain a valuable model for some aspects of sPD but their heterogeneity mitigates against a simple designation of sPD phenotype in this cell model.

Mitochondrial gene therapy augments mitochondrial physiology in a Parkinson's disease cell model.

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.