In the present study we have developed the argument that cybrid cells expressing platelet mtDNA from subjects with sporadic PD are meaningful models of some molecular events in sPD brains and predict reduced complex I-mediated respiration in sPD brain. This argument is based on strong correlations among relative expression levels of genes for mitochondrial ETC proteins coded for by both the nuclear and mitochondrial genomes and inter-relationships among cybrid complex I-mediated respiration, levels of an 8 kDa complex I protein that discriminates sPD from CTL brain mitochondria and expression levels for multiple mtDNA-derived complex I genes. Our findings complement those recently described in sPD cybrids created in an NT2 host cell background [25
]. In that study the authors found that sPD cybrids had reduced complex I catalytic activity, less basal ATP and greater progression into mitochondrial cell death.
In an earlier study we showed that the complex I macroassembly immunocaptured from mitochondria that had been gradient-purified from the same PD brain cases used in the present study showed increased oxidative damage and reduced rates of NADH-driven electron flow [22
]. We also showed how some of the protein oxidative damage could originate within complex I. These findings are consistent with the concept that PD brain complex I proteins become oxidatively damaged more than age-matched controls through unclear mechanisms, with the end result of reduced complex I electron passage capacity that could itself contribute to additional oxidative damage.
Because coupled mitochondrial respiration cannot be reliably assayed in frozen postmortem tissue, we examined this physiological process in intact sPD cybrid cells metabolizing glucose. The “high-resolution respirometry” approach studies respiration under regulated conditions and provides understanding of respiratory coupling and control when all metabolic feedback systems are operative in intact cells [20
]. This is a different situation compared to isolated mitochondria or permeabilized cells, where specific electron transport complex substrates are provided and maximal respiration under conditions of minimal (state 4) or maximal (state 3) ATP synthesis is measured. We have recently completed and will be reporting separately a study in which intact cybrid cell respiration, including its oxygen concentration-response [26
], was compared to that of gradient-purified mitochondria isolated from the same cybrid cells. We observed similarities and some significant differences, including in isolated mitochondria the ability to stimulate substantial respiration through complex II that is minimally present in intact cells where the vast majority of respiration is mediated through complex I.
In the present study we related sPD cybrid maximal complex I-mediated uncoupled respiration in intact cells to levels of fully assembled complex I and to levels of 8 kDa complex I subunit in the cybrids, to PD brain through the intermediaries of levels of RNA’s (cDNA’s) for mtDNA-encoded complex I genes and their relationship to levels of the same 8 kDa complex I subunit in PD brain mitochondria. While our approach is necessarily correlational and is consistent with but cannot prove that PD brain complex I-mediated respiration is reduced, we feel the results are supportive of the argument.
Although maximal complex I catalytic activity is reduced in many PD tissues including brain, it is unclear if such reductions in catalytic activity necessarily reduce respiratory capacity or instead serve as markers for inefficient electron flow that yields increased oxidative stress. Our findings suggest that this reduced complex I catalytic activity in sPD brain likely produces respiratory compromise, at least in terms of ability to engage in oxidative phosphorylation, in addition to whatever oxidative stress also results.
We also observed that sPD cybrid cells have several-fold reductions in multiple mtDNA genes. It is unclear why this is the case, but it suggests that sPD mtDNA is not propagated as well as CTL mtDNA in genetically identical host cells. There are many potential causes for this problem that can include oxidative damage to the specific mtDNA replicating DNA polymerase-gamma, defective replication due to deletions in mtDNA and stalling of replication forks, increased oxidative damage to mtDNA, or combinations of these and other processes. An important starting point is analysis of characteristics of sPD cybrid mtDNA itself. We have begun such analyses in terms of quantifying deletions present in the entire mitochondrial genome and presence of heteroplasmic mutations and will be reporting those findings separately (Quigley, et al, unpublished data).
In spite of reductions in mtDNA gene levels, sPD cybrids were able to normalize (to CTL levels) many but not all of their RNA levels for these mtDNA genes. Of the five mtDNA genes we examined, three (ND4, CO1, CO2) were normalized and two (ND2, CO3) were not normalized in four of the six sPD cybrid lines studied. Of these four sPD cybrid lines with the non-normalized ND2 and CO3 gene expression, three had the lowest respiration levels. Thus, reduced respiration in sPD cybrids relates to both overall loss of mtDNA genes and failure to normalize mitochondrial gene expression at the RNA level, at least for some mitochondrial genes. The mechanisms for how mtDNA gene expression is differentially regulated in these sPD cybrid lines are unclear.
In summary, we have conducted extensive analyses of the molecular genetic properties of sPD cybrids and compared those to sPD brains. We find substantial correlations of gene expression, particularly for both nuclear and mitochondrial genome-encoded electron transport chain genes, which indicate that sPD cybrids and sPD brains are regulating gene expression for mitochondrial respiratory function in very similar ways. Based on correlations with mitochondrial gene expression and complex I protein levels, respiration through complex I in intact sPD cybrid cells may relate to that of sPD brain. If true, sPD cybrids can serve as unique genetic models of sPD to assist both in unraveling how abnormalities in mtDNA originate and progress, and to screen therapeutics for their ability to enhance respiration through complex I. However, to consider properly the inherent heterogeneity of a sporadic disease, several different cell lines would need to be studied.
It is also important to note that most sPD postmortem brain tissues are derived from individuals who suffered from PD for many years and typically have advanced disease clinically and pathologically. In contrast, sPD cybrids are made from platelet mtDNA of patients typically at much earlier disease state. While the relationships between platelet and brain mtDNA’s are not yet known, at the minimum sPD cybrids represent an earlier disease stage compared to most postmortem sPD brain tissues. Given this situation, our findings that sPD cybrids and sPD brain tissues so closely co-regulate their mitochondrial bioenergetic systems is all the more remarkable.