Neural transplants in patients with Huntington's disease undergo disease-like neuronal degeneration

Abstract

The clinical evaluation of neural transplantation as a potential treatment for Huntington's disease (HD) was initiated in an attempt to replace lost neurons and improve patient outcomes. Two of 3 patients with HD reported here, who underwent neural transplantation containing striatal anlagen in the striatum a decade earlier, have demonstrated marginal and transient clinical benefits. Their brains were evaluated immunohistochemically and with electron microscopy for markers of projection neurons and interneurons, inflammatory cells, abnormal huntingtin protein, and host-derived connectivity. Surviving grafts were identified bilaterally in 2 of the subjects and displayed classic striatal projection neurons and interneurons. Genetic markers of HD were not expressed within the graft. Here we report in patients with HD that (i) graft survival is attenuated long-term; (ii) grafts undergo disease-like neuronal degeneration with a preferential loss of projection neurons in comparison to interneurons; (iii) immunologically unrelated cells degenerate more rapidly than the patient's neurons, particularly the projection neuron subtype; (iv) graft survival is attenuated in the caudate in comparison to the putamen in HD; (v) glutamatergic cortical neurons project to transplanted striatal neurons; and (vi) microglial inflammatory changes in the grafts specifically target the neuronal components of the grafts. These results, when combined, raise uncertainty about this potential therapeutic approach for the treatment of HD. However, these observations provide new opportunities to investigate the underlying mechanisms involved in HD, as well as to explore additional therapeutic paradigms.

Fig. 1.

Graft morphology. (A) Low-power magnification of a coronal section stained for AChE, which identifies graft placement within the putamen of the host, at the level of the anterior commissure within the striatum of patient 1. The graft is delineated by a dotted line. P-zone volume was calculated using tissue stained for the striatal markers AChE, NADPH-d, TH, PV and CB (I). The mean percentage of P-zone volume per graft was evaluated to be 58% for patient 1 (see Results for patient 5). (B and C) Nissl staining showing the cell density discrepancy between P-zones and non-P-zones (B) as well as the notable difference in appearance between cells of the graft (black arrows in B and also C) and of the host (white arrows in B and Inset in C). (D and E) H&E staining also showing the cell-density discrepancies between P-zones and non-P-zones (D) as well as in the putamen of the host brain (E). Histograms of cell count (per square millimeter of tissue) in grafted P-zones versus host supports the observation of fewer cells within grafted P-zones than in the pathological host putamen (H). Note the morphological difference in appearance of cells in the P-zone vs. the non-P-zone (D). (F and G) Higher magnification photomicrographs of individual cells within the P-zone (F, and pointed by black arrows in D) and within the host putamen (G). In comparison to cells of the host (G), grafted cells appeared larger in diameter, ballooned, and vaculated. Abnormal chromatin deposits exclusively within transplanted cells are discernible using the H&E staining (F). These characteristics suggest ongoing grafted neuronal degeneration. The immunostaining for the apoptotic marker caspase-3 (J and K) revealed equivalent neuronal degeneration in the host and graft. (Scale bars: A, 1 mm; B, 25 μm; C and inset, 12.5 μm; D and E, 50 μm; F and G, 6.25 μm; J and K, 25 μm.) CD, caudate; GP, globus pallidus; PUT, putamen.

Fig. 2.

Comparison of graft health in short- and long-term transplants. Low- (A) and high- (B) power photomicrographs of CB immunopositive neurons in the P-zone of patient 2, 18 months posttransplantation (25) (revealed with DAB; brown color). Because of the reduced CB immunoreactivity seen in the patient 1, (C and D) 108 months posttransplantation, staining was nickel-intensified, resulting in a purple coloration. In the early stage transplants, the CB-immunoreactive neurons are easily identifiable at lower (A) and higher (B) magnification within the P-zone by staining intensity, neuronal morphology, and extensive dendritic arborization (arrows in A). In comparison, CB-immunopositive neurons in late-stage transplants were marked at lower (C) and higher (D) magnifications by their degenerative appearance, poor CB immunoreactivity, even with nickel-DAB enhancement. These neurons lacked visible cytoplasmic architecture, cell polarity, and dendritic arborization (arrows in C). Nearly all neurons observed in the grafts of patients 1 and 5 showed similar patterns of staining (data not shown), which contrasted with intense CB staining in cortical regions of the same patients (Inset C). (E) CB-medium spiny projection neuron counts performed in grafted P-zone vs. host putamen of the short-term transplant recipient demonstrated greater number of cells within the graft than the host (E). The opposite finding was observed in patient 1, analyzed a decade posttransplantation (F). (G and H) Higher magnification of NADPH-d staining also exemplifies cell-density discrepancies between grafted P-zone and non-P-zone regions (G) and the host putamen (H). Insets show high magnification of cells within the graft P-zone (G) and host putamen (H). (G) Histogram of cell counts show a similar decreased cellularity in the grafted P-zone in comparison to the host putamen. (Scale bars: A and C, 100 μm; Inset C, 25 μm; B, 25 μm; D, 12.5 μm; G and H, 50 μm; G and H Insets, 15 μm.)

Fig. 3.

Inflammatory responses to grafts. Both astrocytosis and microgliosis were evaluated and found to be consistent in all 3 hemispheres; representative photomicrographs of patient 1 are illustrated here. (A) The astrocytic response is significant in the host but minimal within the graft, as investigated by GFAP staining. The edge of the graft is demarcated by a particularly strong astrocytic response, characterized by activated astrocytes. A few nonactivated astrocytes are observable within P-zones (B). Abundant GFAP fiber staining is seen within the non-P-zone (C). These nonactivated astrocytes expressed fibers that extended throughout the non-P-zones but did not significantly invade P-zone. (D) Low-power photomicrograph of double immunostaining for the neuronal nuclear marker NeuN (brown; DAB chromogen) and the microglial marker Iba-1 (black; nickel-DAB chromogen) at the interface of a P-zone and non-P-zone and at the interface of the graft and the host. A pronounced microglial response is demonstrated cuffing the edge of the P-zone as well as within the P-zone. Microglial cuffing is seen predominantly at the interface between P-zones and non-P-zones, as opposed to the interface between the non-P-zone portion of the graft and the host. (E) Higher magnification of NeuN/Iba-1 staining at the interface graft/host. (F and G) Higher power photomicrographs depicting examples of grafted neurons intermingled with microglial cells. Of note, microglial cells were often observed engulfing neuronal elements, resembling an ongoing phagocytic event. (Scale bars: A, 25 μm; B, 20 μm; C, 100 μm; D, 300 μm; E and F, 25 μm; G, 12.5 μm.)

Fig. 4.

Graft connectivity. (A) Low-power photomicrograph of synaptophysin immunoreactivity inside the graft (dotted line). The density of synaptophysin reaction product is depicted in (B), in the form of an optical density plot taken along the line shown in (A). (C and D) High magnification of synaptophysin staining in (C) a P-zone of the graft and (D) in the host striatum. (E) Photomicrograph of TH immunostaining of the grafted P-zone (dotted line). (F) Numerous host-derived TH+ fibers are observed with seamless penetration of the graft border. (G) vGlut1 axon varicosities (black dots; arrows within the neuropile) were found closely apposed to cell bodies labeled for CB (brown, arrowheads) in both the host striatum and the transplant. (H) Example of a vGlut1-labeled axon varicosity (asterix) within the transplant P-zone exhibiting an asymmetrical synaptic contact (Between small arrows). (Scale bars: C and D, 25 μm; E, 100 μm; F, 25 μm; G, 20 μm; H, 500 nm.) av, axon varicosity; sp, spine.