12 April 2008. For the brave patients who elected to have foreign fetal neurons implanted in their brain as an experimental treatment for Parkinson disease (PD), there must have been many agonizing thoughts. Apart from the obvious will-it-help-or-hurt questions, those volunteers must have wondered how long the grafts would hold up. The research community has wondered the same. Now it turns out that such transplants may be remarkably stable. Three separate studies, reported as brief communications in the April 6 Nature Medicine online, describe grafts surviving as long as 9-16 years in six different patients, even without immunosuppressive medication. But there is a caveat. Grafts in three patients showed signs of Lewy bodies, intracellular protein aggregates that are a hallmark of Parkinson’s, suggesting that transplanted neurons may be vulnerable to the same pathology that caused the disease in the first place. Given that the transplanted neurons were too young to develop this pathology on their own, that finding also suggests that Lewy body pathology is not cell-autonomous but induced by the microenvironment of the brain.
It is not yet clear how these findings will affect transplant research, or even whether these Lewy body-like entities have any impact on the overall efficacy of the grafts. Curt Freed, from the University of Colorado Health Science Center, and a pioneer of PD cell transplants, told Alzforum via e-mail that he finds this a positive rather than a cautionary story. Freed was not involved in these studies. “What would a kidney or liver transplant look like 14-16 years after transplant in a patient who did not receive immunosuppression? The kidney or liver would have been destroyed,” he wrote. “I find it remarkable that all three Nature Medicine reports and our experience in Colorado show that dopamine cell transplants survive and function almost indefinitely.”
The studies were led by Patrik Brundin at the Wallenberg Neuroscience Center, Lund, Sweden; Ole Isacson at McLean Hospital, Belmont, Massachusetts; and Jeffery Kordower at Rush University Medical Center, Chicago, Illinois. The European study (two patients) and the Rush study (one patient) both report signs of Lewy bodies in grafted neurons, whereas Isacson’s group found no such pattern in grafts from three different patients.
Jia-Yi Li and colleagues at the Wallenberg Neuroscience Center studied postmortem samples from two PD patients who had twice received grafts of fetal dopaminergic (DA) neurons. The first patient received a graft 16 years before death and then a second graft on the opposite side of the brain four years later. The second patient received the first graft 13 years before death and a second two years later. All grafts were densely positive for tyrosine hydroxylase (TH)-expressing neurons, suggesting good survival. The neurons had long processes and formed dense networks within the graft and the surrounding striatum (in all three studies the grafts were transplanted into the striatum to compensate for lost dopaminergic innervation resulting from damage to DA neurons in the substantia nigra). But Li and colleagues also noticed that in both cases, neurons in the graft contained α-synuclein- and ubiquitin-positive inclusions that have characteristics of Lewy bodies. α-synuclein, normally restricted to presynaptic terminals, also turned up in neuritic processes. In the patient with 12- and 16-year-old grafts, about 40 percent of TH-positive neurons contained detectable α-synuclein in the youngest graft, while about 80 percent of the cells were α-synuclein-positive in the older graft. The findings “support the notion that increased intracellular α-synuclein is time- or age-dependent, which is consistent with the fact that age could be a risk factor for Parkinson’s disease,” write the authors.
Kordower and colleagues report a similar finding from their study of a patient who received a graft 14 years before death. TH immunoreactivity showed strong survival of the DA neurons and innervation into the striatum. But again, some cells also tested positive for α-synuclein- and ubiquitin-positive bodies and also for α-synuclein in neuritic processes. For comparison, the researchers examined postmortem brain tissue from two patients who died four years after receiving grafts. Those tissue samples showed no signs of Lewy bodies. The researchers are now carrying out quantitative analysis to see just what fraction of grafted neurons is affected. “Preliminary counts suggest that more cells in the graft may display these markers than in the host,” Kordower told ARF.
Contrasting these two studies is the data from McLean. First author Ivar Mendez and colleagues carried out postmortem analysis on tissue from two patients who had received grafts nine years before death, and a third patient who had a graft for 14 years. Similar to the other studies, Mendez found that the grafts were well integrated, with TH-positive cells extending into the putamen. But unlike the other two studies, Mendez and colleagues found no signs of Lewy bodies or morphological signs of neurodegeneration. They found no α-synuclein, ubiquitin, or lipofuscin inclusions, and even though immunosuppressant medication was withdrawn six months after the transplant operations, the autopsy showed no major immune reaction to the foreign tissue.
What explains the different findings? “We tried very hard to find Lewy bodies. In fact as a byline to the other stories, Dr. Kordower sent tissues from his case so that we could apply the same techniques, and I did, indeed, find a few cells that had some kind of protein aggregate,” said Isacson in an interview with ARF. He suggested neuroinflammation as one possible explanation for the differences. Whereas Isacson’s group found no signs of inflammation, Kordower and colleagues found intense activation of microglia in the grafts, which vastly exceeded that seen in the patient’s own striatum. Brundin’s groups found that microglia surrounded the grafts, but were not strongly activated. Isacson suggested that differences in transplant technology might partly explain the different data, since solid, or tissue chunk, transplants, the kind used by Kordower’s group, are known to elicit a more extensive immune response (see Freed et al., 2001).
Kordower is not convinced that inflammation due to differences in technique explains the Lewy body pattern, even though Isacson used dispersed cell suspensions rather than the solid tissue method. “Dr. Isacson’s technique and Dr. Brundin’s are virtually identical, so I don’t think that is what’s different,” said Kordower. He also added that lots of things cause inflammation but don’t cause Lewy bodies. “This is a very specific Parkinsonian pathology, and there’s no doubt that it occurs in grafted neurons,” he said.
A separate question that needs to be answered is how representative these few cases are. Freed weighed in on the no-pathology side. “In Colorado, I believe that we have done the largest series of dopamine cell transplants in the world, a total of 61 patients since 1988, and we have seen no protein deposits in dopamine neurons up to 14 years after transplant,” he wrote to ARF. Isacson suggested that individual responses of patients may be part of the explanation for the differences. “It could simply be that we haven’t had a patient who had that kind of reaction,” he said. Kordower added that “this is one of the problems with case studies. They don’t tell you what happens; they tell you what can happen, and this data tells you that Parkinson pathology can happen in grafted neurons.”
What does all this mean for transplant treatments? “It may not be relevant, in a sense. Our patient did very well for 10 years, and that has tremendous value,” said Kordower. There is also some question as to whether the aggregates seen in the grafts are true Lewy bodies. John Trojanowski, University of Pennsylvania, Philadelphia, told ARF that ultrastructural studies would have to be carried out to be sure. Trojanowski, who collaborated with Isacson and is a coauthor on his paper, also said that there are other circumstances when synuclein turns up in the cytoplasm. During development, for example, α-synuclein is normally in the cell body. “Toward the end of term there’s a shift of expression of α-synuclein from the cell body to processes, so why couldn’t that normal developmental occurrence fail?” he suggested (see Galvin et al., 2001). Whether removing fetal tissue and transplanting it into a new environment causes such a failure remains to be tested. Freed also echoed Trojanowski’s caution about the bodies seen in the grafts. “Since the precipitation of α-synuclein and ubiquitin protein is not unique to Parkinson’s, there is no indication that the cells have developed a Parkinson condition,” he wrote.
The Future of Transplants
Though no double-blind trial has shown dopaminergic transplants to be effective in PD patients, there is evidence that patients do well in open-label follow-up, and all the researchers seemed optimistic that transplants offer good therapeutic potential. What form those transplants will take is another question. Therapeutic cloning may yield new approaches, though technical hurdles and ethical objections are holding back that technology. An alternative possibility, which avoids generating embryos, is to reprogram adult cells to assume neural cell fates. In this week’s PNAS, Isacson, collaborator Rudolf Jaenisch at the Whitehead Institute for Biomedical Research, Cambridge, and colleagues demonstrate how this strategy can work to rescue PD-like symptoms in rats.
The researchers capitalized on the recent discovery that expression of a handful of key proteins is all that is needed to turn differentiated adult cells into pluripotent stem cells. Previously, researchers at the University of Kyoto in Japan, had shown that four transcription factors, Oct4, Sox2, Kif4, and c-Myc, can reset the genome to resemble that of pluripotent stem cells (see Takahashi and Yamanaka, 2006). In this study, first author Marius Wernig and colleagues took this strategy a step further, coaxing induced pluripotent stem cells (iPS) derived from rat fibroblasts to form neural precursor cells. After persuading these precursors to form dopaminergic neurons, the researchers grafted the cells into rats missing DA neurons on one side of the brain. These animals have movement problems and tend to rotate on one side when given a stimulant such as amphetamine. However, animals that received grafts showed significantly fewer rotations per hour after eight weeks than sham-operated animals.—Tom Fagan.
Mendez I, Viñuela A, Astradsson A, Mukhida K, Hallett P, Robertson H, Tierney T, Holness R, Dagher A, Trojanowski JQ, Isacson O. Dopamine neurons implanted into people with Parkinson’s disease survive without pathology for 14 years. Nature Medicine. 2008 April 6 advanced online publication. Abstract
Kordower JH, Chu Y, Hauser RA, Freeman TB, Olanow CW. Lewy body-like pathology in long-term embryonic nigral transplants in Parkinson’s disease. Nature Medicine. 2008 April 6 advanced online publication. Abstract
Li J-Y, Englund E, Holton JL, Soulet D, Hagell P, Lees AJ, Lashley T, Quinn NP, Rehncrona S, Björklund A, Widner H, Revesz T, Lindvall O, Brundin P. Lewy bodies in grafted neurons in subjects with Parkinson’s disease suggest host-to-graft disease propagation. Nature Medicine. 2008 April 6 advanced online publication. Abstract
Wernig M, Zhao J-P, Pruszak J, Hedlund E, Fu D, Soldner F, Broccoli V, Constantine-Paton M, Isacson O, Jaenisch R. Neurons derived from reprogrammed fibroblasts functionally integrate into fetal brain and improve symptoms of rats with Parkinson’s disease. PNAS. 2008 April 7 early edition. Abstract