ARTICLE IN BRIEF
Investigators were able to pharmacologically rescue mitochondrial deficits in induced pluripotent-derived stem cells (iPSCs) made from the fibroblasts of patients with familial Parkinson's disease.
While the most obvious signs of Parkinson's disease (PD) are the loss of vulnerable dopaminergic synapses and neurons in the nigrostriatal pathway, it has become clear in the last decade or so that other neurons in the CNS and the peripheral nervous system are also damaged. Much of the insight into other pathways has come into focus with the identification of mutations that confer disease in a small percentage of people with PD.
In a new study, published in the July 4 online edition of Science Translational Medicine, a team of investigators, led by Ole Isacson, MD, reported that that they were able to pharmacologically rescue mitochondrial deficits in induced pluripotent-derived stem cells (iPSCs) made from the fibroblasts of patients with familial PD.
Ultimately, the goal is to use the technology to make patient-specific, genetically corrected iPSCs that could be used for unraveling disease states, phenotypic screening and drug development. The model seems to work.
“My hope is that we can use iPSCs from patients to determine how responsive the cells will be to specific medicines,” said Dr. Isacson, professor of neurology and neuroscience at Harvard Medical School. “In the long-term, we should be able to use this technology to identify medicines that will correct the results of the genetic defect.”
Dr. Isacson had been asked by federal researchers to create a consortium to collect and grow iPSCs that would then be banked for research through the NINDS. As soon as the technology became available to grow pluripotent stem cells from fibroblasts in the mid-2000s, Dr. Isacson began collecting skin cells from patients with sporadic forms of PD.
For the current study, he collected fibroblasts from three patients with rare mutations in genes for leucine-rich repeat kinase 2 (LRRK2); two others (siblings) who had mutations in PTEN-induced putative kinase 1 (PINK1); and two family members who did not have a mutation or PD.
Knowing that dopaminergic cells are sensitive to chemical toxins, the investigators conducted a series of studies measuring how mitochondria respond to these chemical stressors. They looked at production of reactive oxygen species, mitochondrial respiration, proton leakage, and intraneuronal movement of mitochondria. As they expected, the iPSC-derived neural cells from people with the mutations were far more vulnerable to the chemical stressors. While the mitochondrial function was different in both the mutations studied — oxygen consumption rates were lower in patient cells with the LRRK2 mutations and higher in cells with the PINK1 mutation — they both share an increased sensitivity to oxidative stress.
They used mitochondrial toxins to see if the patient-derived cells were more sensitive to damage or death — they were. And the patient-derived neurons showed more damage then the patient-derived skin cells.
Next, the researchers attempted to rescue the toxin-exposed cells with various drug treatments that have shown promise in animal models of Parkinson's, including the antioxidant coenzyme Q10 (CoQ10) and the metabolic regulator, rapamycin. All patient-derived neurons — whether they carried LRRK2 or PINK1 mutations — had beneficial responses to CoQ10. However, the patient-derived neurons differed in their response to rapamycin; the drug helped prevent damage to neurons with LRRK2 mutations, but it did not protect the neurons with PINK1 mutations.
The collaborators will now go back and repeat these experiments using iPSC-derived from fibroblasts culled from PD patients with no family history of the disease.
Dr. Isacson and his colleagues said that the findings raise many interesting points. The genotype-specific phenotypic profiles suggest possible shared cellular disease pathways for familial forms of PD, they said. There were differential vulnerabilities to chemical stressors based on the PD gene mutation, iPSC-derived neurons showed different recovery responses to CoQ10, rapamycin and the LRRK2 inhibitor GW5074. iPSC-derived neuronal cultures from PINK1 or LRRK2 cells demonstrated increased vulnerability to valinomycin, a mitochondria depolarizing agent, compared with iPSC neurons derived from healthy control subjects. Valinomycin produces current in the mitochondrial membrane. This vulnerability can be reversed in the presence of CoQ10.
The neural cells derived from iPSC-derived lines from patients with PINK1 mutations showed more sensitivity to chemicals and this could explain why there is a greater genetic risk and an earlier age of onset than in people who inherit the LRRK2 mutation.
PD and other neurodegenerative conditions begin to show symptoms when neurons have been working for decades with no obvious problems. Even in these studies, the neurons that are generated are still young. Now, Dr. Isacson and his colleagues are transplanting the human iPSC-derived cells into mice and rat models to study how the human cells respond over much longer periods of time.
Serge Przedborski, MD, PhD, professor of neurology and pathology at Columbia University College of Physicians and Surgeons, said that it has always been a challenge studying the neurobiology of PD without having access to human neurons. But iPSC-derived neurons from people who carry mutations have “opened up a whole new area for investigation,” he said. He was one of the dozen or so investigators in the PD consortium. He concentrated on the mitochondrial tests that were done on the iPSC cells.
“We found that there are subtle changes where mitochondria seem to move in the cell in an unusual manner,” said Dr. Przedborski. “We still don't know the impact of that on the cell but even subtle alterations in sensitive cells like neurons could be sufficient to cause a chronic disease like Parkinson's.”
“Right now these findings are very descriptive but we need to understand their significance to the disease itself. Even if these are not robust findings, we all feel that this is a significant step to take to learn about the neurobiology of these diseases.”
“What we are learning is that we have tools that will allow us to have human models of disease and that we can improve cell function,” said Dr. Isacson. “The future of medicine just got a lot brighter.”
“These findings are very important,” said Margaret Sutherland, PhD, a program director at NINDS. “They suggest new opportunities for clinical trials of Parkinson's disease in which cell reprogramming technology could be used to identify the patients most likely to respond to a particular intervention.” For instance, doctors or scientists could collect skin cells from a patient before they enrolled in a study and make iPS cells and then turn those cells into a specific cell type for study. They can use the iPSC-derived cells to test responsiveness to the experimental drug.
“Basically, with the combination of the iPSC approach, plus genetics, and other PD diagnostic biomarkers as they become available, the patient cohort involved in a PD clinical trial could be better defined — leading to better patient stratification,” Dr. Sutherland continued. “This is particularly important when dealing with a complex neurological disorder, where multiple factors, environmental and genetic, contribute to disease onset and progression.”
But, said Dr. Sutherland, “although this approach to understanding potential differences in patients' responses to a particular intervention is encouraging, it is still too early to ascertain the true impact this technology will have on clinical trials and drug discovery.” The results need to be replicated and validated using additional LRRK2, PINK1 lines, and across laboratories, she said, noting this is one reason NINDS developed the iPSC repository at Coriell. It allows these and additional lines to be shared with investigators worldwide in both academia and industry.
“We need to know if this approach will be useful in sporadic PD cases, as this represents the majority of the PD patient population. Moving forward, we need to think about what stage of PD these iPSC derived neurons represent — that is, perhaps the readouts from this technology will be most applicable to pre-motor or early stage PD cases, and if so, we need to identify additional biomarkers that will assist in early detection/diagnosis of PD.”
Selina Wray, PhD, a visiting research fellow at the MRC Centre for Regenerative Medicine at The University of Edinburgh, said: “Seeing the same affected pathways in neurons from so many patients means we can be confident it is a true ‘disease’ phenotype and not just down to variability between patients. This work is really labor-intensive and represents a huge effort to generate and analyze so many cells.”
Her colleague, MRC Clinical Research Fellow Michael Devine, PhD, agreed, but noted that “clonal variation — the variation between iPSC lines derived from the same individual — can be an issue. It doesn't appear that they controlled for this.”
These iPSC and fibroblast lines are one of 200 that were developed by three iPSC consortia funded through American Recovery and Reinvestment Act (ARRA) and NINDS grants that are being made available through the NINDS Cell Repository at Coriell. Investigators can use them for research regardless of their funding source. The funding for the research was partially renewed through 2013 through a public-private partnership.