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I.M. and A.V. designed research, performed research, analyzed data and wrote the paper. A.A. and P.H. performed research, analyzed data and wrote the paper. K.M. analyzed data and wrote the paper. H.R., T.T., R.H. and A.D. performed research and analyzed data. J.Q.T. performed neuropathology staining, analyzed raw data, evaluated and interpreted data and wrote the paper. O.I. designed research, performed research, analyzed raw data, evaluated and interpreted data and wrote the paper.
Postmortem analysis of five subjects with Parkinson’s disease9–14 years after transplantation of fetal midbrain cell suspensions revealed surviving grafts that included dopamine and serotonin neurons without pathology. These findings are important for the understanding of the etiopathogenesis of midbrain dopamine neuron degeneration and future use of cell replacement therapies.
Despite indirect evidence of long-term survival of fetal midbrain dopamine cell suspensions in people with Parkinson’s disease1, the question remains whether grafted neurons are affected by pathogenic factors intrinsic to the parkinsonian brain.
Prominent neuropathological features of Parkinson’s disease include dopaminergic neuron loss in the substantia nigra, the presence of dystrophic neurites (Lewy neurites)2 and the presence of Lewy bodies3,4. Ultimately, the durability of transplanted fetal ventral midbrain neurons in therapeutic approaches relies on their resistance to these neurodegenerative processes. Because many aspects of these processes remain unknown, it is important to understand the effects of neurodegeneration in the parkinsonian striatum upon transplanted fetal dopamine neurons. We report histopathological findings in the brains of three subjects (referred to as subjects 4, 5 and 6) with advanced idiopathic Parkinson’s disease who had received intracerebral transplantation of fetal ventral midbrain cell suspension grafts 9–14 years previously (Supplementary Tables 1 and 2 and Supplementary Results online). Additionally, we extend the pathological analysis to two subjects who died of unrelated causes 3–4 years after transplantation (subjects 1 and 2)5. This study provides a unique and in-depth long-term postmortem analysis of the effects of neurodegeneration on grafted fetal dopamine neurons in individuals with Parkinson’s disease.
We grafted the subjects using our specific transplantation procedures (Supplementary Methods online). Postmortem analysis showed well-integrated grafts containing tyrosine hydroxylase–immunoreactive neurons with extensive neuritic outgrowth into the host putamen without displacement (Fig. 1a). The distribution of tyrosine hydroxylase–immunoreactive neurons in the grafts of subjects 4 and 5 was less homogeneous than in subjects 1, 2 and 6, in whomwe used a two-hole rotating cannula. In all grafts, the majority of tyrosine hydroxylase–immunoreactive neurons were located at the graft periphery and contained small amounts of neuromelanin; no extracellular neuromelanin was observed (Fig. 1 and Fig 2; for survival, cell preparation, injection technique and stereological analysis, see Supplementary Table 1 and Supplementary Methods).
In normal aging, α-synuclein accumulation occurs as a nonpathological phenomenon in the substantia nigra, but not in other dopamine neuronal nuclei such as the ventral tegmental area6. This explains the absence of α-synuclein accumulation in young (9–14-year-old) grafted ventral midbrain neurons (Fig. 2). In the normal brain, α-synuclein is located in presynaptic terminals, creating a fine granularity in the neuropil, and is absent in the neuronal cytoplasm6. In Parkinson’s disease, the neurodegenerative process is characterized by the accumulation of proteinaceous intraneuronal inclusions4. Initially, α-synuclein appears as a pale and diffuse cytoplasmic inclusion. During neurodegeneration, perikaryal α-synuclein coalesces and incorporates polyubiquitin chains and other proteins, forming ‘pale bodies’ that fuse to form the Lewy body7. In the five subjects, triple immunostaining for tyrosine hydroxylase, α-synuclein and ubiquitin showed a clear boundary between fetal grafts and the host striatum (Fig. 2a). As expected, α-synuclein and ubiquitin aggregates were found in cell bodies and terminals within the substantia nigra (Fig. 2b,c). Similarly, α-synuclein and ubiquitin colocalized in the Parkinson’s disease brain regions including the upper raphe nucleus, neocortex and putamen, where they were broadly distributed throughout the neuropil (Fig. 2a and data not shown). In contrast, grafted dopamine and serotoninergic neurons did not contain α-synuclein, ubiquitin or lipofuscin inclusions, and there were no other morphological signs of neurode-generation in the graft neuropil (Fig. 2). The data from the fetal ventral midbrain grafts were congruent with our previous study of synuclein during development, in which no pathological aggregates were seen at up to 16 years of age8. In addition, α-synuclein can aggregate in nonpathological conditions and therefore requires ultrastructural (electron microscopy) evidence to identify the pathology6.
Coexpression of tyrosine hydroxylase and G protein–coupled inward rectifying current potassium channel type 2 (Girk2) defines the most vulnerable population of dopamine neurons in Parkinson’s disease5. This group of dopamine neurons is located in the ventral tier of adult substantia nigra pars compacta (A9), projecting axons and terminals to motor areas of the putamen5. In contrast, most tyrosine hydroxylase and calbindin coimmunoreactive neurons in the ventral tegmental area and substantia nigra (A10) are relatively spared in Parkinson’s disease and send their axons to mesolimbic brain regions1. Grafted tyrosine hydroxylase and Girk2 coimmunoreactive neurons were located close to the graft-host interface, whereas tyrosine hydroxylase and calbindin coimmunoreactive neurons populated central areas of the graft (Fig. 1b).
We have demonstrated the presence of serotonin neurons in grafted human fetal ventral midbrain tissue (Fig. 1c), which is consistent with our previous findings and those of other groups in rodents9. Although we did not observe graft-induced dyskinesias in any subjects, a recent report in a rodent model of Parkinson’s disease describes the role of serotonin neurons in the development of l-dopa–induced dyskinesias when dopamine neurons are depleted10. Given the potential role of serotonin neurons in the development of l-dopa–induced dyskinesia, our new finding highlights the need for controlling cell composition in clinical neural transplantation11.
Immunosuppression was withdrawn 6 months after transplantation in subjects 4, 5 and 6, and the transplanted fetal cell suspensions did not elicit a major immune reaction (Supplementary Fig. 1 online). This is consistent with the postmortem study of subjects 1 and 2 that showed a minimal microglial host reaction5 but contrasts with the pronounced microglial reactions to transplanted solid pieces of fetal ventral midbrain12,13. This reduced host response may be attributed to fewer major histocompatibility complex class I–containing donor blood vessels in suspension grafts and the predominance of host-derived angiogenic processes that arise from this cell preparation method1.
In summary, we show that grafted dopamine and serotonin neurons survive without signs of neurodegeneration for up to 14 years despite ongoing degeneration of midbrain dopamine neurons and other dopamine structures in the host parkinsonian brain. The lack of degeneration in the grafts does not imply that the disease will not affect these dopamine neurons eventually. Rather, this proves that under the appropriate conditions of integration and reduced inflammatory response obtained by our methods, grafted neurons can avoid significant degeneration long term. One cannot exclude the possibility for graft involvement in the neurodegenerative process in other transplantation methods or within other subject populations. These results have major implications for the etiopathogenesis of Parkinson’s disease, as the host brain does not necessarily create conditions that cause Parkinson’s disease–related neurodegeneration in the transplanted neurons. Moreover, these findings encourage the future use of fetal- and stem cell–derived dopamine neurons for people with Parkinson’s disease14,15.
Note: Supplementary information is available on the Nature Medicine website.
This work was supported by the Canadian Institute of Health Research, Queen Elizabeth II Health Sciences Centre (I.M.), US National Institutes of Health NINDS Parkinson’s Disease Research Center of Excellence (P50 NS 39793 to O.I. and NS 053488 to J.Q.T.), The Michael Stern Foundation (O.I.), The Consolidated Anti-Aging Foundation (O.I.), The Orchard Foundation (O.I.) and the Harold and Ronna Cooper Family (O.I.). Fellowship support to K.M. was provided by the Dalhousie Medical Research Foundation and Parkinson Society Canada. We also thank O. Cooper for evaluation of tissue sections from study subjects.