This article was written in Nov 2013 for my course. 

Abstract                                                                                          

Parkinson’s disease (PD) has been considered as a disease leading to debilitating motor symptoms due to the progressive focal death of dopaminergic neurons in the substantia nigra (SN). Different therapeutic strategies aiming to restore this dopaminergic network with transplantation of a variety of cells have yielded promising results both in the lab and the clinic and offered a definite proof of principle. Yet, even though clinical trials have shown that restoration of dopamine is feasible, have many times failed to produce significant and meaningful benefits and, moreover, they are often complicated by variability and the development of new abnormal motor symptoms. This has not only led to a very fertile discussion about the issues that need to be overcome but also to the realisation that new perspectives are needed towards both cell replacement therapies and PD. Understanding that cell transplantation creates novel conditions not found in the premorbid or morbid brain, while appreciating the widespread effects of PD which cannot be reduced in the focal loss of dopaminergic cells, maybe perhaps the only way to overcome the inherent obstacles posed to cell replacement therapies in PD and to further develop novel therapeutic strategies.

Introduction

Parkinson’s Disease (PD) is classically considered as a progressive, neurodegenerative disease that manifests itself clinically with the cardinal triad of tremor, bradykinesia (difficulty in initiating and executing muscle movement) and rigidity, as a result of extensive loss of dopaminergic neurons (60-80%)^{[1, 2]} located in a part of the midbrain called substantia nigra pars compacta (SNc). SNc neurons innervate the putamen of the striatum and both are part of a complex neural circuit involved in modulating movement (see figure 1 and [3,4]). However, PD’s pathophysiology is multifactorial and not well understood^{[5; for a brief review]} and even if the involvement of other parts of the brain producing motor and non-motor symptoms is recognised (see later), most current therapeutic approaches aim principally in the restoration of the dopamine levels (e.g. administration of levodopa) and amelioration of the related symptoms. Nonetheless, the presumed neuropathological specificity of PD: the focal loss of a single cell type innervating a particular structure, made it an excellent candidate for cell replacement therapy (CRT) with the ultimate view of replacing neuronal losses and restoring neuronal circuitry to the premorbid status.^[6]

The first CRT studies: defining current challenges.

In 1979, the first two preclinical studies using rat models were published.^{[8, 9]}  The dopaminergic neurons of rats were destroyed selectively by unilateral injection of 6-hydroxydopamine (6-OHDA) into the SN and thereafter rat fetal ventral mesencephalic cells (fVMCs; which include dopaminergic cells) were implanted close to the SN. The treated animals showed striatal innervation and functional improvement, although quite variable, and this sparked the interest for further research. Motor function was monitored in terms of rotational behaviour, which is induced by the resultant imbalance of dopaminergic output. The following decade, the first cell transplants were performed on patients with advanced PD (open label studies). Initially, autografts from patients’ adrenal medulla, containing dopamine-secreting cells among others, were used with mixed results and no long-term graft survival.^[10,11]  But more promising were the clinical studies with human fVMCs extracted and processed form 7-8 week embryos. Although they provided again variable results (15.24-44.52% recovery in the ‘Unified Parkinson Disease Rating Scale’ - UPDRS) they offered a ‘proof-of-principle’:^[12]  the most striking example was a patient who improved so much within 3 years that he discontinued levodopa. His improvement correlated with increased fluorodopa uptake (marker of dopaminergic activity in PET) and with recovery of motor-related cortical function as shown by H₂¹⁵O PET interrogation of regional cerebral flow.^[13,14]

However, the two double-blinded randomised controlled trials that followed by Freed et al. and Olanow et al.^[15,16] not only failed to meet their primary outcomes i.e. clinical improvement e.g. in UPDRS after 1 or 2 years compared to sham surgery, but they also revealed that 15-56% of patients receiving a graft developed severe off-medication graft induced dyskinesia (GID; stiffness and abnormal involuntary movements), which in few cases required further neurosurgical interventions.^[15] Nevertheless, fluorodopa uptake was significantly increased in the grafted group and further subgroup analysis revealed clinical improvement in young people with mild PD denoting that variance was definitely one of the limitations (figure 2.).

On the other hand, this pioneer work - though not conclusive and potentially limited methodologically (patient selection, graft processing and implantation procedure, duration of immunotherapy etc.) - delineated the main issues that need to be overcome in PD CRT. The development of GID in a significant number of patients together with the variability of the results raised many questions about issues, which are not limited just to understanding and preventing GID, but are pivotal in developing successful CRT strategies in general and will be discussed in the following sections in relation to PD.

The ‘Repair Model’ vs the ‘Composite Brains Model’

However, before considering each issue separately, it is very important to understand the overall conceptual frameworks that have been historically proposed in order to tackle them. According to a recent paper by Polgar^[17] initial and current research has been driven by what he refers as the ‘Repair Model’ which focuses on the aim of replacing neuronal losses and restoring neuronal circuitry to the premorbid status e.g. as proposed by Barker.^[6]  Yet, even if this model has been in many aspects useful, it entails several conceptual and methodological constraints to further clinical progress as it fails to take into account that - by definition - the grafted cells that are going to form functional synapses, are placed in a morbid, structurally and physiologically altered environment which does not provide the stimuli and conditions that would be found in the developing brain required for their differentiation and/or maturation. As a result this disregards the important complex interactions “between the graft, the host CNS, the patient and the environment.” Therefore, Polgar proposes the ‘Composite Brains Model’ which is based on the assumption that neural transplantation produces brains that are composed of  “two distinct but interacting neural systems: the host brain and the graft” which are “continually transforming as [they] interact with each other” and are characterised by novel properties not found in either the diseased or premorbid brain. Hence this model supports the view that CRT should not aim in anatomical/physiological restoration but in ‘enhancing neuroplasticity’ in a meaningful and beneficial way, even if this means the creation of novel circuits and conditions. With this view the abovementioned issues can be seen and discussed here under a new light.

Cell Survival

Investigations following up patients receiving fVMCs have shown that grafts can exhibit significant increase in fluorodopa uptake for several years^[13-16,18] even though these do not always correlate with clinical improvement.^[15]  Moreover, postmortem histological studies have shown in some cases abundant striatal reinnervation^[15,18,19] and cell survival between 20.000 and 120.000 cells per side, depending on graft size.^[15,16] However, despite a report^[19] suggesting that grafts do not show any signs of PD-related pathology even after 14 years (i.e. intracellular α-synuclein inclusion staining), there is strong evidence for the contrary.^[20-22] This is further supported by in vitro findings of direct cell-to-cell α-synuclein transmission and α-synuclein related neuronal apoptosis,^[23]  which may suggest that PD is actually a prion-like disease.^{[24; for a review]}

Evermore, while it is suggested that good clinical outcomes occur when the number of cells surviving is more than 100.000,^[25] the parkensonian brain seems to maintain a pro-apoptotic environment with increased levels of pro-apoptotic cytokines (e.g. TNF-α) and reduced anti-apoptotic neurotrophins (e.g. nerve growth factor, NGF, and brain-derived neurotrophic factor, BDNF).^[26,27]  Hence, it could be argued that CRT could be improved with optimisation of the host environment. Interestingly, in contrast to BDNF and NGF, Glial cell line-derived neurotrophic factor (GDNF), which is important for dopaminergic neurons is not reduced in PD, possibly due to protective mechanisms^[26] albeit not that effective; and augmentation of its expression (via Adeno-associated virus vectors) in primate models of PD has been shown to improve both motor function and restoration of the dopaminergic system.^[28] Such findings led eventually to a double-blinded clinical study by Marks et al.,^[29] but there was not any significant improvement probably due to inadequate delivery of the GDNF-analogue neurturin to the affected neurons.^[30] Nonetheless, implantation of GDNF producing cells could be an alternative strategy aiming for neuroprotection of both host and grafted dopaminergic neurons. For example, encapsulated human fibroblasts, neural stem cells and astrocytes engineered to secrete GDNF have all been found to rescue SN-neurons and significantly improve motor function in rodent models (figures 3,4).^[31-33]  

Additionally, co-implantation of polymer-encapsulated GDNF-secreting myoblasts with human fVMCs, enhances significantly fibre outgrowth compared to plain grafts. ^[34] Hence, the use of ‘cell factories’, i.e. cells producing beneficial factors, could be a good alternative or augmenting strategy in CRT.

Suitability of grafted cells

There have been many theories for the limited benefits experienced by patients receiving fVMCs and the evolution of significant GID in many of them. However, some current hypotheses focus on the actual cell composition: fVMC grafts contain two types of dopaminergic neurons (A9, A10) with different receptor complement and anatomical targets, GABA-ergic and serotonergic neurons and glia. It has been found that grafts lacking A9-neurons, which normally innervate the striatum, were rather ineffective in improving motor function of 6-OHDA-lesioned rats compared to normal grafts;^[35] hence different proportions of cells could account for the variable results. Similarly, it has been proposed that serotonergic hyperinnervetion is one of the causes of GID. Yet, there is a lot of conflicting evidence and further investigation is warranted.^[36]

However, the suitability of fVMCs grafts is limited for more reasons: their allogeneic nature (immunoreactivity, infective potential), inherent variability, limited availability (up to 8 embryos are needed for a graft) and of course ethical considerations may also hinder their use in the clinic.  Therefore, there is a shift in preclinical research towards other cell sources such as embryonic (ESCs), induced-pluripotent (iPSCs), mescencymal (MSCs) and neural stem cells (NSCs) as they can be expanded in vitro (table 1).

Implanted ESCs can differentiate in vivo but exhibit significant rates of teratoma formation.^[39] On the other hand, even if current protocols can yield more than 80% dopaminergic cells from ESCs, implantation of pre-differentiated cells results in poor cell survival.^[45] Similarly, even if iPSCs are not ethically controversial, they can also be tumorogenic. Implantation of midbrain-type cells derived from protein-based human iPSCs in rats promoted recovery but tumorogenesis too.^[43] Nonetheless, recently Kriks et al.^[46] proposed a new protocol for ESCs differentiation and that the observed tumorogenicity is not an inherent problem of ESCs but that of incomplete cell specification. Moreover, after defining the optimal stage of transplantation as that of the cell cycle exit - marked by expression of a transcription factor essential for dopaminergic phenotype called NURR1^[47]- they found that grafts not only improve motor function in murine models, but also show no hyperproliferative behaviour. In a different direction, MSCs are also very exciting due to their inherent advantages (table 1) and very promising results when grafted. It is proposed that they can act as ‘cell factories’ improving so the host environment and that they may even transdifferentiate to and/or fuse with neurons. Read [41,42] for recent reviews. The first results from a clinical open-labeled study were inconclusive but nonetheless encouraging as it seems a safe option.^[40]

Finally, implantation of embryonic NSCs has yielded poor results in rat models because of poor in vivo differentiation. However, Tan et al.,^[44] identified recently that NURR1 and another factor, Brn4, are required developmentally for their differentiation, maturation and migration, and  hence implantation of NSCs engineered to express them improves in vivo differentiation and motor function in 6-OHDA-lesioned rats.

The jury is not out yet for neither fVMCs or stem cells and further elucidation of the factors involved in development and refinement of protocols is pivotal before bringing stem cells in the clinic. In the meanwhile, the Transeuro trial led by Barker,^[7] aims to revisit fVMC grafting in PD patients by integrating current knowledge. Changes include a more circumscribed dissection of tissue and use of long-term immunosuppression of 12 months instead of 6 months or none used previously.^{[Barker,personal communication, Oct 23, 2013]} However, there are still more issues to consider.

Graft Location and integration

The actual graft location and the distribution of the transplanted cells are also very crucial. One of the first explanations for GID was the hypothesis that grafted cells are unevenly distributed: It was found that recipients suffering from GID had unbalanced increases in fluorodopa uptake.^[48] This was back up by the finding that dyskinetic behaviour was significantly more pronounced in “parkinsonian” mice receiving focal transplants than those having the same amount of cells transplanted over 6 sites in the striatum.^[49] Yet, it has been for long known that lesions of the SN lead to neuronal activity changes to other target areas involved in motor function and hence even optimal striatal innervation may not restore it.^[50] Supporters of the ‘repair model’ argue that intra-nigral transplantation would be better even though more challenging. It is

well documented that axonal growth is actively inhibited in the adult brain by axon growth-inhibitory ligands.^[52] However, 6-OHDA-lesioned mice grafted in the SN with dopaminergic cells expressing green fluorescent protein showed widespread reinnervation of target structures and especially of the striatum. (figure 5) Moreover induced expression of GDNF (via viral vector) in some of them led to further axonal outgrowth and significant functional improvement (figure 6).^[51]  This suggests that reconstruction of circuits is feasible and GDNF could be used as chemoattractant for axonal growth. A better understanding of the inhibitory signals and axonal guidance, and the development of modulators will be pivotal for successful CRTs.

In the meanwhile, a multiple target strategy involving other structures, and especially the subthalamic nucleus, is very promising for the restoration of the complex motor functions,^[53] while, according to the ‘composite brains’ models the implantation of cells in novel ways in order to restore the functional imbalance should also be an aim. Hence, even if initially counterintuitive, intrastriatal implantation of inhibitory GABAergic precursors ameliorates motor symptoms of 6-OHDA-lesioned rats^[54] probably by “rebalancing” the neuronal network and therefore could be a novel strategy.^[55]

Other host-graft interactions and patient history

Though tempting, a reduction of CRT success and failures to the abovementioned issues would not only be unjustified but would also ignore the very significant host-graft interactions and the impact of patients’ history.  One such interaction implicated in the development of GID (usually observed after immunosuppression cessation^[15]) is that of the immune system. Preclinical studies showed positive correlation of different levels of inflammation with abnormal motor behaviours in rats probably due to resultant aberrant synapses formation,^[56] suggesting that immunomodulation should be central in CRT. Similarly, the effects of long-term dopamine depletion and levodopa treatment on brain remodelling have also been considered to predict poor outcome,^[36]  like the existence of widespread pathology;^[25] potentially restricting CRT to less severe cases managed currently by pharmacotherapy. 

Nonetheless, according to Polgar^[17] there are many more aspects that need to be addressed such as the role of exercise, environmental enrichment, patient’s attitudes and neurorehabilitation: enhancing neuroplasticity is the main aim and optimising the conditions within which the patients and thence the grafts are found can be instrumental.

Just the tip of the iceberg?

The driving force behind the first trials of treating PD with CRT was its presumed neuropathological specificity and even if to date the restoration of the nigrostriatal system is the main aim, it would be awfully misleading if one would not consider it as just a starting point.

It is well established nowadays that PD is a systemic disease involving many areas of the brain and that is not at all confined to the CNS (figure 7).^[57] PD can present in multiple ways and the finding that non-motor symptoms like pain, anosmia, mood changes and sleep problems were some of the most troublesome symptoms side to the classic triad of tremor, bradykinesia and stiffness,^[58] was actually an eye-opener as non-motor symptoms are rarely addressed by therapeutic approaches. Evermore, cholinergic^[59] and noradrenergic^[60,61]  denervation and the implication of serotonergic transmission^[62]  are now recognised as important aspects of PD and future cell therapies inasmuch as animal models will need to account for them as well.

Finally, one of the most interesting effects of PD is its impact on the endogenous brain regenerative capacity. In the adult brain neurogenesis is restricted mainly in two areas called the subventricular zone and the hippocampus and tightly regulated by the local microenvironment, glia, and intracellular or secreted factors. There is clinical^[63] and preclinical^[64-66] evidence that dopaminergic denervation reduces it and it is postulated that this could be the basis for a range of PD symptoms like anosmia, anxiety, depression and cognitive difficulties.^[67] However, this realisation opens the door for the development of new CRT strategies that could either mobilise and augment endogenous replacement via neural stem cells or facilitate the differentiation and integration of transplanted cells by transforming the inhibitory environment of the affected areas.  

Even if it seems that the ‘restoration’ of the nigostriatal pathway is unquestionably not enough and that there are numerous obstacles to overcome for the achievement of only this, the increasing understanding of the control of cell fate in development and adulthood, the expansion of the possible tools that can be used and most importantly the shift in the perspective towards CRT and PD, will pave the road to a more promising future.

Conclusions

Abreviations

6-OHDA: 6-hydroxydopamine

BDNF: brain-derived neurotrophic factor

CRT: Cell replacement therapy

ESCs: embryonic stem cells

fVMC: feta ventral mesencephalic cells

GDNF: Glial cell line-derived neurotrophic factor

GID: Graft induced dyskinesia

iPSCs: induced-pluripotent stem cells

MSCs: mescencymal stem cells

NSCs: Neural stem cells

NGF: Nerve growth factor

PD: Parkinson’s disease

SN(c): Substantia nigra (pars compacta)

UPDRS: Unified Parkinson Disease Rating Scale

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