Extensive graft-derived dopaminergic innervation is maintained 24 years after transplantation in the degenerating parkinsonian brain

Abstract

Clinical trials using cells derived from embryonic ventral mesencephalon have shown that transplanted dopaminergic neurons can survive and function in the long term, as demonstrated by in vivo brain imaging using (18)F-fluorodopa and (11)C-raclopride positron emission tomography. Here we report the postmortem analysis of a patient with Parkinson's disease who 24 y earlier underwent unilateral transplantation of embryonic dopaminergic neurons in the putamen and subsequently exhibited major motor improvement and recovery of striatal dopaminergic function. Histopathological analysis showed that a dense, near-normal graft-derived dopaminergic reinnervation of the putamen can be maintained for a quarter of a century despite severe host brain pathology and with no evidence of immune response. In addition, ubiquitin- and α-synuclein-positive inclusions were seen, some with the appearance of typical Lewy bodies, in 11-12% of the grafted dopaminergic neurons, reflecting the spread of pathology from the host brain to the transplants. Because the clinical benefits induced by transplantation in this patient were gradually lost after 14 y posttransplantation, our findings provide the first reported evidence, to our knowledge, that even a viable dopaminergic graft giving rise to extensive striatal reinnervation may lose its efficacy if widespread degenerative changes develop in the host brain.

Keywords: Parkinson’s disease; autopsy; reinnervation; synucleinopathy; transplantation.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Survival of transplanted dopaminergic neurons in the three graft deposits. (A, B, and F) The graft deposits were rich in TH-positive neurons, arranged in clusters at the periphery of the grafts. (C–E) Cell counting revealed a total of ∼43,000 surviving dopaminergic neurons identified by their TH-positive immunostaining and/or neuromelanin content. The surviving neurons were categorized into three different populations: cells with very dense TH staining and an elaborate healthy morphology (arrow in C), cells with weaker TH staining and poorly stained processes (arrows in D and E), and cells with neuromelanin granules but no detectable TH staining (arrowheads in E). The first and second types accounted for 76% of the total number of surviving neurons. (Scale bars: 1 mm in A, 0.5 mm in B, 20 μm in E.)

Fig. 2.

Overview of graft-derived TH-positive innervation. (A) A 3D reconstruction showing the location and size of three graft deposits in the caudal, middle, and rostral putamen. (C–K) Extent of the graft-derived TH-positive innervation throughout the postcommissural putamen (positions of the sections indicated in A, arranged in a caudal-to-rostral order). (B) In contrast, the nongrafted putamen was completely devoid of TH-positive fibers. (Scale bars: 1 mm in B, 0.5 mm in D, 20 µm in E–G, 5 mm in H–K.)

Fig. 3.

Complete reinnervation of grafted putamen. (A–E) From the core of the transplants, the graft-derived TH fibers extended throughout the postcommissural putamen, with an innervation density close to normal in the dorsal part, with a decreasing gradient dorsoventrally (A and B) and in the medial and lateral directions from the graft core (C–E). A′, B′, C′, and C′′ show quantification of TH-positive fiber density in the boxed regions (yellow bars), compared with the nongrafted side (black bars) and the innervation density recorded from an age-matched non-PD brain (dashed lines, ±SEM). (F–I) Dark-field images showing the dense varicose TH-positive innervation in the grafted putamen (H and I), similar in morphology and density to that seen in the non-PD brain (F). The nongrafted putamen was completely devoid of TH-positive terminals; only scattered neuritic profiles with the appearance of truncated preterminal axons remained (G). (Scale bars: 5 mm in C, 1 mm in D and E, 100 µm in F–I.)

Fig. S1.

Density of VMAT2-positive terminals in the grafted and nongrafted putamen. (A and B) Immunostaining for VMAT2 shows a markedly higher VMAT2-positive fiber density in the grafted putamen (B) compared with the nongrafted side (A). (C) The difference in VMAT2-positive innervation, as assessed by densitometry. (Scale bars: 200 µm in A, 20 µm in B and C.)

Fig. S2.

(A–C) Microglial reaction to the transplants. IBA1 staining showing microglia cells in the putamen of an age-matched non-PD patient (A) and in the nongrafted (B) and grafted putamen (C) of the PD patient. Quantification of IBA1-positive cells showed no difference in microglia numbers (70 in the control brain, 77 in nongrafted putamen, and 78 in the grafted one on average in each counting field). (D–G) Microglia were classified into four different morphological phenotypes, believed to represent different steps of microglial activation (14): ramified (D), intermediate (E), amoeboid (F), and round (G). No major differences between grafted and nongrafted sides and normal putamen were observed. In control brain: ramified, 89%; intermediate, 7.4%; amoeboid, 2.8%; and round, 0.8%. In nongrafted putamen: ramified, 74%; intermediate, 18%; amoeboid, 7%; and round, 0.4%. In grafted putamen: ramified, 77%; intermediate, 16%; amoeboid, 6%; and round, 0%. In addition, staining with the activated microglia marker CD68 did not reveal any major difference in the number of immunoreactive cells between nongrafted and grafted putamen (62 vs. 82). (H) CD68 staining in the rostral graft. (Scale bars: 20 µm.)

Fig. 4.

White matter atrophy and Lewy body pathology. (A) Luxol fast blue staining of a whole brain slice from the rostral part of the brain showing white matter atrophy (asterisks) and enlarged lateral ventricles (LV). (B and C) Phosphorylated (S129) α-synuclein staining showing the presence of Lewy bodies in the grafted cells (C) and in the host putamen surrounding the graft (B). (D–M) Quantification of phosphorylated α-synuclein showing that up to 12% of neuromelanin-containing cells contain Lewy bodies. Extensive Lewy pathology appears in different brain regions, including amygdala (D and I), cortex (E and J), hippocampus (F and K), substantia nigra (G and L) and the nongrafted putamen (H and M). (N–P) Confocal images illustrating a host nigral dopaminergic neuron (red, TH positive) containing a typical Lewy body (green, α-synuclein positive). (Q) Ubiquitin-positive inclusions also appeared in the grafted cells. Up to 11% of the neuromelanin-containing cells contained ubiquitin-positive aggregates. (Scale bars: 10 mm in A, 20 µm in B and C, 50 µm in D–H, 20 µm in I–Q.)