NpgRJ_NM_1752 1..3

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 whom we 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 (Figs. 1 and 2; for survival, cell preparation, injection technique and stereological analysis, see Supplementary Table 1 and Supplementary Methods). In normal aging, a-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 a-synuclein accumulation in young (9–14-year-old) grafted ventral midbrain neurons (Fig. 2). In the normal brain, a-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, a-synuclein appears as a pale and diffuse cytoplasmic inclusion. During neurodegeneration, perikaryal a-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, a-synuclein and ubiquitin showed a clear boundary between fetal grafts and the host striatum (Fig. 2a). As expected, a-synuclein and ubiquitin aggregates were found in cell bodies and terminals within the substantia nigra (Fig. 2b,c). Similarly, a-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 a-synuclein, ubiquitin or lipofuscin inclusions, and there were no other morphological signs of neurodegeneration 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, a-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 © 20 08 N at u re P u b lis h in g G ro u p h tt p :/ /w w w .n at u re .c o m /n at u re m ed ic in e