Dopaminergic neural grafts after fifteen years: results and perspectives

Dyskinesia seen in the off-state, referred as graft-induced dyskinesia (GID), has emerged as a serious complication induced by dopamine (DA) cell transplantation in parkinsonian patients. Although the mechanism underlying the appearance of GID is unknown, in a recent clinical study the partial 5-HT_1A agonist buspirone was found to markedly reduce GID in three grafted patients, who showed significant serotonin (5-HT) hyperinnervation in the grafted striatum in positron emission tomography scanning (Politis et al., 2010, 2011). Prompted by these findings, this study was performed to investigate the involvement of serotonin neurons in the appearance of GID in the rat 6-hydroxydopamine model.

L-DOPA-primed rats received transplants of DA neurons only, DA plus 5-HT neurons or 5-HT neurons only into the lesioned striatum. In DA cell-grafted rats, with or without 5-HT neurons, but not in 5-HT grafts, GID was observed consistently after administration of amphetamine (1.5 mg/kg, i.p.) indicating that grafted DA neurons are required to induce GID. Strikingly, a low dose of buspirone produced a complete suppression of GID. In addition, activation of 5-HT_1A and 5-HT_1B receptors by 8-OH-DPAT and CP 94253, known to inhibit the activity of 5-HT neurons, significantly reduced GID, whereas induction of neurotransmitter release by fenfluramine administration significantly increased GID, indicating an involvement of the 5-HT system in the modulation of GID. To investigate the involvement of the host 5-HT system in GID, the endogenous 5-HT terminals were removed by intracerebral injection of 5,7-dihydroxytryptamine, but this treatment did not affect GID expression. However, 5-HT terminal destruction suppressed the anti-GID effect of 5-HT_1A and 5-HT_1B agonists, demonstrating that the 5-HT₁ agonist combination exerted its anti-GID effect through the activation of pre-synaptic host-derived receptors. By contrast, removal of the host 5-HT innervation or pre-treatment with a 5-HT_1A antagonist did not abolish the anti-GID effect of buspirone, showing that its effect is independent from activation of either pre- or post-synaptic 5-HT_1A receptors. Since buspirone is known to also act as a DA D₂ receptor antagonist, the selective D₂ receptor antagonist eticlopride was administered to test whether blockade of D₂ receptors could account for the anti-dyskinetic effect of buspirone. In fact, eticlopride produced complete suppression of GID in grafted animals already at very low dose. Together, these results point to a critical role of both 5-HT₁ and D₂ receptors in the modulation of GID, and suggest that 5-HT neurons exert a modulatory role in the development of this side effect of neuronal transplantation.

The reconstruction of midbrain dopamine (DA) circuitry through intracerebral transplantation of new DA neurons contained in embryonic ventral mesencephalon (VM) is a promising therapeutic approach for Parkinson's disease (PD). Although some of the early open-label trials have provided proof-of-principal that VM grafts can provide sustained improvement of motor function in some patients, subsequent trials showed that the functional response can be highly variable. This chapter reviews an extensive body of basic and clinical research on the survival, differentiation, and connectivity of DA neurons in VM grafts, and also looks at how these parameters are affected by certain host- and donor-specific variables. We also review how technical advances in the tools available to study the integration of grafted DA neurons, such as transgenic reporter mice, have made significant contributions to our understanding of the capacity of different DA neuronal subtypes for target-directed growth and innervation of appropriate host brain structures. Our established and on-going understanding of the capacity of grafted DA neurons to structurally and functionally integrate following transplantation forms an important basis for the refinement and optimization of VM grafting procedures, and also the development of new procedures based on the use of stem cells.

The development of neural transplantation as a treatment for Parkinson’s disease has been compromised by a lack of functional efficacy and the appearance of transplant-induced motor side-effects in some patients. Since the first reports of these graft-induced dyskinesias (GID), and the realization of their impact on the progress of the field, a great deal of experimental work has been performed to determine the underlying cause(s) of this problematic side-effect. In this review we describe the clinical phenomenon of GID, explore the different representations of GID in rodent models, and examine the various hypotheses that have been postulated to be the cause. Based on the available clinical and preclinical data we outline strategies to avoid GID in future clinical trials using fetal cell transplants or cell preparations derived from stem cells.

Following transplantation into the rat brain, porcine neuroblasts differentiate and integrate host tissue, but due to their xenogeneic nature, these cells are generally rejected within several weeks. This rejection is accompanied by infiltration of the graft by macrophages and αβT lymphocytes, but so far nothing is known about the potential role of dendritic cells (DCs) in this process. DCs are professional antigen presenting cells that have the unique ability to prime naive T cells, thereby initiating an antigen-directed immune response. Here, we provide evidence for DC recruitment following the transplantation of pig mesencephalic neural cells into the striatum of LEW.1A rats, as indicated by the high number of OX62+ cells in the rejecting graft and the absence of V65 staining. DCs were found as early as 3 and 8 days postimplantation together with ED1+ and OX42+ cells. This early recruitment, which is probably due to the surgical procedure, might be a critical step in the rejection process, enabling DCs to be loaded with xenoantigens. The number of intracerebral DCs subsequently decreased, being barely detectable in older non-infiltrated xenografts. However, DCs re-appeared as they were observed in grafts infiltrated by macrophages and T cells, a phenomenon that usually precedes graft rejection. Interestingly, we observed a tight correlation between the number of DCs and that of R7.3+ T cells infiltrating the graft. In addition, DCs were often found in close proximity to αβT cells and most expressed MHCII. Taken together, these findings give credence to a role for infiltrating DCs in the mediation of T cell responses to intracerebral xenografting.

Functional recovery following intrastriatal transplantation of fetal dopaminergic neurons in animal models of Parkinson’s disease is, at least in part, dependent on the number of surviving dopaminergic neurons and the degree of graft-derived dopaminergic reinnervation of the host striatum. In the present study, we analyzed whether continuous exposure of glial cell line-derived neurotrophic factor (GDNF) to mature dopaminergic grafts could further boost the functional outcome of widespread intrastriatal dopaminergic grafts. Rats with dopamine-denervating lesions received multiple intrastriatal transplants of fetal dopaminergic cells and graft-induced behavioral effects were analyzed in drug-induced and spontaneous motor behaviors. At three months after grafting, animals received intrastriatal injections of recombinant lentiviral vectors encoding for either human GDNF or the green fluorescent protein. Continuous exposure of GDNF to the grafts did not boost the functional recovery beyond what was observed in the control animals. Rather, in some of the spontaneous motor behaviors, animals in the GDNF-group showed deterioration as compared with control animals, and this negative effect of GDNF was associated with a down-regulation of the tyrosine hydroxylase enzyme. Based on these and our earlier results, we propose that intrastriatal administration of GDNF at the time of or shortly after grafting is highly effective in initially promoting the cell survival and fiber outgrowth from the grafts. However, once the grafts are mature, GDNF’s ability to boost dopaminergic neurotransmission follows the same dynamics as for the native nigral dopaminergic neurons, which appears to be dependent on the concentration of GDNF. Since rather low doses of glial cell line-derived neurotrophic factor at nanogram levels appear to saturate these effects, it may be critical to adjust GDNF levels using tightly regulated gene expression systems.

Both increased and decreased dyskinesias have been reported from open label clinical trials of transplantation of human embryonic dopamine rich tissue in Parkinson's disease patients. In the first double-blind clinical transplantation trial, 15% of the grafted patients developed severe postoperative dyskinesias in the “off” phase. Since then, postoperative off-medication dyskinesias have been reported from two additional series of grafted patients. However, such dyskinesias are probably not a novel phenomenon. These dyskinesias have shown a different temporal development postoperatively compared to the antiparkinsonian graft effects, and no significant relationship with the magnitude of graft-derived dopaminergic reinnervation or symptomatic relief. However, positron emission tomography studies have indicated that an unbalanced putaminal dopaminergic function may contribute to this postoperative complication. While there is little doubt that intrastriatal grafts can induce dyskinesias, these appear to differ from common drug-induced dyskinesias. The term graft-induced dyskinesias (GID) is therefore suggested to more clearly identify this complication. While GID bear some phenomenological resemblance to biphasic drug induced dyskinesias, the mechanism(s) behind this complication remains obscure. Available data are scarce but allow for hypotheses to be generated that could (and should) be addressed in experimental animals.