Long-term health of dopaminergic neuron transplants in Parkinson's disease patients

Abstract

To determine the long-term health and function of transplanted dopamine neurons in Parkinson's disease (PD) patients, the expression of dopamine transporters (DATs) and mitochondrial morphology were examined in human fetal midbrain cellular transplants. DAT was robustly expressed in transplanted dopamine neuron terminals in the reinnervated host putamen and caudate for at least 14 years after transplantation. The transplanted dopamine neurons showed a healthy and nonatrophied morphology at all time points. Labeling of the mitochondrial outer membrane protein Tom20 and α-synuclein showed a typical cellular pathology in the patients' own substantia nigra, which was not observed in transplanted dopamine neurons. These results show that the vast majority of transplanted neurons remain healthy for the long term in PD patients, consistent with clinical findings that fetal dopamine neuron transplants maintain function for up to 15-18 years in patients. These findings are critically important for the rational development of stem-cell-based dopamine neuronal replacement therapies for PD.

Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1. Dopamine transporter expression in the reinnervated putamen 4–14 years post-transplantation

Double immunolabeling for dopamine transporter (DAT) (green) and tyrosine hydroxylase (TH) (red) in Subjects 1 (panels A–E, graft survival of 4 years), 2 (panels F–J, graft survival of 4 years), 4 (panels K–O, graft survival of 9 years), and 5 (panels P–T, graft survival of 14 years). Panels A, F, K, and P show a low magnification composite for each subject to illustrate the grafted cell bodies and adjacent reinnervated putamen. Successively higher magnification images are illustrated in panels B–D, G–I, L–N and Q–S. Boxed areas represent the image shown in the subsequent panel. Panels E, J, O and T show a low magnification composite of the patient’s putamen and adjacent external segment of the globus pallidus. DAT immunostaining shows a robust punctate localization along dopaminergic (TH-immunoreactive) fibers in the reinnervated putamen and caudate in all grafts, even up to 14 years post-transplantation. In addition, the transplanted dopamine neurons show a healthy and non-atrophied morphology. Parallel control immunostainings, where the primary antibodies were omitted, showed no immunoreactivity of DAT or TH (data not shown). To further confirm specificity of the DAT labeling observed in the reinnervated putamen and caudate, we also examined DAT immunolabeling in adjacent anatomical regions on the same tissue sections. In the lateral and medial globus pallidi, which are regions that receive comparatively little dopaminergic innervation and normally exhibit little DAT expression in the human brain (Ciliax et al., 1999), we, as expected, observed weak DAT immunolabeling and a sharp boundary from high to low DAT and TH expression (panels E, J, O, T). We also performed immunostaining in the substantia nigra of each patient and confirmed the loss of both DAT and TH immunoreactivity (data not shown). g = graft, h = host, GPe = globus pallidus externa. Scale bars in A, E, F, J, K, O, P, T = 400μm; B, G, L, Q = 100μm; C, H, M, R = 50μm; D, I, N, S = 20μm. See also Figure S1.

Figure 2. Mitochondrial phenotype in transplanted fetal dopamine neurons 4–14 years post-transplantation

Double immunolabeling for translocase of outer mitochondrial membranes 20kDa (Tom20) (green) and tyrosine hydroxylase (TH) (red) in the host substantia nigra (left panels) and grafted dopamine neurons (middle panels) and in the host globus pallidus (right panels) in Subject 2 (panels A, A′, B, B′, C, C′, graft survival of 4 years), Subject 6 (panels D, D′, E, E′, F, F′, graft survival of 9 years) and Subject 5 (panels G, G′, H, H′, I, I′, graft survival of 14 years). Panels A′-I′ show single channel Tom20 labeling from corresponding panels A-I. In dopamine (TH-immunoreactive) neurons from the patients’ own substantia nigra, Tom20 labeling often appeared intensely labeled in the cell soma with accumulation in the perinuclear area (arrows) and little immunostaining in the dopaminergic axons and processes (panels A, A′, D, D′, G, G′). In grafted TH-immunoreactive neurons at 4 years post-transplantation Tom20 labeling was robust in the perikarya and neuronal processes (panels B, B′). At 9 and 14 years post-transplantation, Tom20 labeling was generally less intense in the grafted TH-immunoreactive neurons (panels E, E′, H, H′) compared to the Tom20 staining pattern observed in Subject 2 at 4 years post-transplantation, however, there was no abnormal accumulation of mitochondria in the cell soma as was observed in the host substantia nigra. No perinuclear accumulation or fragmentation of Tom20-labeled mitochondria was observed in the host globus pallidus (panels C, C′, F, F′, I, I′). Scale bar = 50μm

Figure 3. Mitochondria abnormalities occur independently of Lewy body pathology in host neurons and neither Lewy body pathology or mitochondrial abnormalities were found in transplanted neurons

Co-immunofluorescence labeling of a-synuclein (red) and the mitochondrial import receptor subunit, Tom20, (green) was observed in neurons within the substantia nigra of PD patient 6 (Panel A). Mitochondria in neurons containing accumulated a-synuclein in the host were either diffusely distributed throughout the cytoplasm (Cell 1) or were dense in appearance (Cell 2), (Panel A). Mitochondria were also diffusely distributed in the cytoplasm of cells without Lewy bodies (Cell 3), (Panel A). No Lewy body pathology was found in the transplanted neurons of PD patients 2 (Panel B), 5 (Panel C) and 6 (Panel D) and mitochondria were dense throughout the cytoplasm. Annotations: m = mitochondria and LB = Lewy body. Scale bar =50μm.