Long-term post-mortem studies following neurturin gene therapy in patients with advanced Parkinson's disease

Abstract

We performed post-mortem studies on two patients with advanced Parkinson's disease 8 and10 years following AAV2-neurturin (CERE120) gene therapy, the longest post-mortem trophic factor gene therapy cases reported to date. CERE120 was delivered to the putamen bilaterally in one case (10 years post-surgery), and to the putamen plus the substantia nigra bilaterally in the second (8 years post-surgery). In both patients there was persistent, albeit limited, neurturin expression in the putamen covering ∼3-12% of the putamen. In the putamen, dense staining of tyrosine hydroxylase-positive fibres was observed in areas that contained detectable neurturin expression. In the substantia nigra, neurturin expression was detected in 9.8-18.95% and 22.02-39% of remaining melanin-containing neurons in the patient with putamenal and combined putamenal and nigral gene delivery, respectively. Melanized neurons displayed intense tyrosine hydroxylase and RET proto-oncogene expression in nigral neurons in the patient where CERE120 was directly delivered to the nigra. There was no difference in the degree of Lewy pathology in comparison to untreated control patients with Parkinson's disease, and α-synuclein aggregates were detected in neurons that also stained for neurturin, RET, and tyrosine hydroxylase. These changes were not associated with antiparkinsonian benefits likely due to the limited neurturin expression. This study provides the longest term evidence of persistent transgene expression following gene delivery to the CNS and the first human results when targeting both the terminal fields in the putamen as well as the originating nigral neurons.

Keywords: Parkinson’s disease; dopaminergic neuron; gene therapy; neuroprotection; neurturin.

Figure 1

Qualitative and quantitative evaluation for NRTN expression in putamen. Photomicrographs of putamen from Parkinson’s disease subjects with combined nigral and putamenal CERE120 administration (CERE120 SN/Pt; A and B) or putamenal only (CERE120 Pt; C and D) illustrating neurturin (NRTN) immunohistochemical staining. Note NRTN was distributed within putamen in both cases. Case with combined putamenal and nigral CERE120 deliveries (A and B) displayed more intense in NRTN staining relative to putamenal deliveries only (C and D). Scale bar in D = 1 mm (applies to all). (E) Histogram depicts per cent of NRTN covering left (L) and right (R) putamen.

Figure 2

Qualitative and quantitative evaluation for NRTN expression in substantia nigra. Photomicrographs of substantia nigra from Parkinson’s disease subjects with combined nigral and putamenal CERE120 delivery (CERE120 SN/Pt, A–D) or putamenal delivery only (CERE120 Pt; E–H) illustrating neurturin (NRTN) immunohistochemical staining. Note NRTN were diffused into both sides of nigral region (A and C) in Parkinson’s disease with combined nigral and putamenal CERE120 delivery but not in Parkinson’s disease with putamenal delivery only (E and G). NRTN-positive neuros (arrows; B, D, F and H) were detected in substantia nigra of both Parkinson’s disease cases. Outlined area (C) represents substantia nigra with melanized neurons. Scale bars: H, B, D and F = 100 µm; A, C, E and G = 1.6 mm. Cavalieri analyses demonstrated per cent of NRTN covering left (L) and right (R) substantia nigra in Parkinson’s disease with combined nigral and putamenal CERE120 delivery (I). (J) Stereological analyses revealed that the density of NRTN-positive neurons was higher in Parkinson’s disease with combined nigral and putamenal CERE120 delivery (CERE120 SN/Pt) than Parkinson’s disease with putamenal delivery only (CERE120 Pt). (K) There was a higher per cent of NRTN-positive neurons in remaining melanized neurons in Parkinson’s disease with combined nigral and putamenal CERE120 delivery (CERE120 SN/Pt) than Parkinson’s disease with putamenal delivery only (CERE120 Pt).

Figure 3

TH expression was enhanced in area with NRTN distribution. Photomicrographs of putamen (A and B) and substantia nigra (C and D) from Parkinson’s disease with combined nigral and putamenal CERE120 administration illustrating neurturin (NRTN; A and C) and TH (B and D) immunohistochemical staining. Note that the TH immunostaining area constituted a relatively large proportion of the NRTN-immunohistochemical footprint. Scale bar in D = 1 mm (applies to all). Quantification of TH staining area revealed a proportion of putamen (E) and substantia nigra (F) displaying an enhanced TH expression.

Figure 4

Qualitative and quantitative evaluation for TH expression in putamen with or without CERE120 delivery. Sections through the mid-putamen show TH-immunoreactive patterns in age-matched control (A and B), Parkinson’s disease with combined nigral and putamenal CERE120 delivery (CERE120 SN/Pt; C–E), Parkinson’s disease with putamenal delivery only (CERE120 Pt; F–H), and Parkinson’s disease without gene delivery (I and J). Note that there were dense TH-positive fibres in the CERE120 delivery areas (C, D, F and G) and scattered TH-positive fibres in the areas remote from CERE120 delivery (E and H). Scale bar in D = 100 µm for B, D, E, G, H and J and 1.6 mm for A, C, F and I. (K) Densitometry revealed that putamenal areas with CERE120 (present) displayed higher TH-immunoreactive intensity similar to the levels of age-matched control whereas areas absent CERE120 (absent) exhibited much lower TH-immunoreactive intensity close to the levels of Parkinson’s disease without gene delivery. (L) Stereological analyses of TH-positive fibre density throughout putamen demonstrated that Parkinson’s disease with combined nigral and putamenal CERE120 delivery displayed higher density than the Parkinson’s disease with putamenal CERE120 delivery only. PD = Parkinson’s disease.

Figure 5

Qualitative and quantitative evaluation for TH expression in substantia nigra with or without CERE120 delivery. Sections from substantia nigra show TH-immunoreactive patterns in age-matched control (A and B), Parkinson’s disease without gene delivery (C and D), Parkinson’s disease with combined nigral and putamenal CERE120 delivery (CERE120 SN/Pt; left nigra: E and F; right nigra: G and H), and Parkinson’s disease with putamenal CERE120 delivery only (CERE120 Pt; left nigra: I and J; right nigra: K and L). Note that enhanced TH-staining neurons were observed in the nigral regions with CERE120 (E–H) but not in the nigral regions without CERE120 (I–L). Scale bar in L = 100 µm for B, D, F, H, G and J and 1.6 mm for A, C, E, G, I and K. Stereological analyses revealed that the density of TH-positive neurons was higher in Parkinson’s disease with combined nigral and putamenal CERE120 delivery(CERE120 SN/Pt), than Parkinson’s disease with putamenal CERE120 delivery only (CERE120 Pt). (N) The majority of remaining melanized neurons were TH-positive in Parkinson’s disease with combined nigral and putamenal CERE120 delivery (CERE120 SN/Pt) relative to Parkinson’s disease with putamenal CERE120 delivery. PD = Parkinson’s disease; ir = immunoreactivity.

Figure 6

Co-localization analyses of RET and TH expression in nigral neurons. Confocal microscopy images of substantia nigra from age-matched control (A–C), Parkinson’s disease with combined nigral and putamenal CERE120 delivery (CERE120 SN/Pt; D–F), Parkinson’s disease with putamenal CERE120 delivery only (CERE120 Pt; G–I), and Parkinson’s disease without gene delivery (J–L) illustrating Ret (green; A, D, G and J), TH (red; B, E, H and K), and co-localization of Ret and TH (merged; C, F, I and L). In age-matched control (A–C), strong Ret (A) and TH (B) immunoreactivities were co-localized in the nigral neurons (C). Remaining nigral neurons in Parkinson’s disease with combined nigral and putamenal CERE120 delivery displayed intense Ret (arrows; D) and TH (arrows; E) that were co-localized in melanized neurons (arrows; F). However, Ret immunoreactive intensity was markedly low in subject with putamenal CERE120 delivery only (G) similar to Parkinson’s disease (J) without gene delivery. Some remaining melanized neurons had light Ret (arrows; G) and TH (arrow; H) immunostaining and other exhibited undetectable Ret (arrowheads; G) and TH (arrowheads; H). Scale bar in L = 100 µm (applies to all). Quantitation of immunofluorescent intensities revealed that the optical density values of Ret (M) and TH (N) in Parkinson’s disease with combined nigral and putamenal CERE120 delivery were similar to age-matched control whereas subject with putamenal CERE120 delivery only were close to Parkinson’s disease without gene delivery. The optical densities of RET and TH immunofluorescent intensities were measured from nigral neurons with neuronal melanin. PD = Parkinson’s disease; ir = immunoreactivity.

Figure 7

Qualitative and quantitative evaluation for micoglial cells in putamen with or without CERE120 delivery. Sections from putamen show human leucocyte antigen-DR isotype (HLA-DR) immunoreactive patterns in age-matched control (A and B), Parkinson’s disease with combined nigral and putamenal CERE120 delivery (C and D), and Parkinson’s disease without gene delivery (E and F). Note that the intensity and morphology of HLA-DR labelling cells in brain (C and D) with CERE120 delivery were similar to the age-matched control (A and B) and Parkinson’s disease without gene delivery (E and F). Arrows in C indicate a needle track for CERE120 delivery. Scale bar in F = 100 µm for B and D and 1.6 mm for A, C, E. Stereological analyses revealed that density of HLA-DR immunoreactive (HLA-DR-ir) cells in Parkinson’s disease with CERE120 delivery was similar to age-matched control and Parkinson’s disease without gene delivery (G). PD = Parkinson’s disease; ir = immunoreactivity.