CELL REPLACEMENT THERAPY FOR PARKINSON’S DISEASE: The importance of neuronal subtype, cell source and connectivity for functional recovery

Parkinson’s disease (PD) is a neurodegenerative disorder characterised by motor deficits such as slowness in movement, difficulty in initiating movement and tremor at rest. The cause of these motor symptoms is the selective loss of mesencephalic dopaminergic (mesDA) neurons, located in the substantia nigra (SN). These neurons project axons to the striatum where they release dopamine, a neurotransmitter that controls voluntary movement. Current drug treatments restore the lost dopamine, while initially efficacious, the beneficial effects wear off resulting in severe side effects. Thus, there is a clear requirement for alternative therapeutic options. One such idea is cell replacement therapy (CRT). CRT aims to replace neurons that have degenerated in PD, with donor cells that have the potential to functionally re-integrate into the host circuitry. This involves transplantation of developing midbrain cells from aborted fetuses, (the part that form mesDA neurons), into the striatum of a PD patient. Clinical trials have demonstrated that CRT can provide long-lasting, significant clinical benefit. Although some patients do not respond as favourably. We still do not know what specific factors contribute to the success in transplantation i.e. what cells are responsible for motor recovery? Can the transplants reform damaged neuronal circuitry? Use of human fetal tissue raises several ethical issues, but are there alternative cell sources that can substitute effectively? The aim of this thesis was to understand how particular factors such as neuronal content, placement and cell source, affect functional outcome after transplantation into the rodent brain. In paper №1, I detail the neurodegenerative and behavioural outcomes in a mouse lesion model of PD, which can be used as a platform for the development of novel therapeutic strategies. I also describe the development of a novel behavioural task that is predictive of mesDA neuron cell loss in mice. Previously, it was thought that transplanted neurons could not extend axons over long distances rendering transplantation into the SN a non-viable approach. In paper №2, I describe how mesDA neurons transplanted in the adult SN of a PD mouse model, extended axons across millimetres into the striatum, functionally reforming the nigrostriatal pathway. In paper №3, I also identify the specific mesDA population (A9) that is critical for functional recovery, with transplants that lack A9 neurons failing to improve motor recovery. A potentially pre-clinical aspect of this thesis is detailed in paper №4 where I describe a robust protocol for the generation of functional mesDA neurons from human embryonic stem cells that are functional in a rat model of PD. No evidence of tumour formation was observed in the transplanted animals, a major concern when utilising a pluripotent cell source. Through understanding functional recovery in terms of neuronal subtype and connectivity, the work presented in this thesis aims to bring the prospect of CRT closer to the clinic, I also describe the generation of a very promising alternative cell source that could rival fetal tissue. Together this work contributes to making CRT a reality for the treatment of PD

The A9 dopamine neuron component in grafts of ventral mesencephalon is an important determinant for recovery of motor function in a rat model of Parkinson’s disease

Grafts of foetal ventral mesencephalon, used in cell replacement therapy for Parkinson’s disease, are known to contain a mix of dopamine neuronal subtypes including the A9 neurons of the substantia nigra and the A10 neurons of the ventral tegmental area. However, the relative importance of these subtypes for functional repair of the brain affected by Parkinson’s disease has not been studied thoroughly. Here, we report results from a series of grafting experiments where the anatomical and functional properties of grafts either selectively lacking in A9 neurons, or with a typical A9/A10 composition were compared. The results show that the A9 component of intrastriatal grafts is of critical importance for recovery in tests on motor performance, in a rodent model of Parkinson’s disease. Analysis at the histological level indicates that this is likely to be due to the unique ability of A9 neurons to innervate and functionally activate their target structure, the dorsolateral region of the host striatum. The findings highlight dopamine neuronal subtype composition as a potentially important parameter to monitor in order to understand the variable nature of functional outcome better in transplantation studies. Furthermore, the results have interesting implications for current efforts in this field to generate well-characterized and standardized preparations of transplantable dopamine neuronal progenitors from stem cells

Plug and Play Brain : Understanding Integration of Transplanted Neurons for Brain Repair

In a recent issue of Nature, Falkner et al. (2016) use chronic two-photon imaging, virus-based transsynaptic tracing, and dynamic calcium indicators to elegantly demonstrate extensive in vivo functional maturation and target-specific functional integration of transplanted embryonic mouse cortical progenitors into adult lesioned visual cortical circuits

Lineage reprogramming: A shortcut to generating functional neurons from fibroblasts.

Comment on: Pfisterer U, et al. Proc Natl Sci USA 2011; 108:10343-8

The future of stem cell therapies for Parkinson disease

Cell-replacement therapies have long been an attractive prospect for treating Parkinson disease. However, the outcomes of fetal tissue-derived cell transplants in individuals with Parkinson disease have been variable, in part owing to the limitations of fetal tissue as a cell source, relating to its availability and the lack of possibility for standardization and to variation in methods. Advances in developmental and stem cell biology have allowed the development of cell-replacement therapies that comprise dopamine neurons derived from human pluripotent stem cells, which have several advantages over fetal cell-derived therapies. In this Review, we critically assess the potential trajectory of this line of translational and clinical research and address its possibilities and current limitations and the broader range of Parkinson disease features that dopamine cell replacement based on generating neurons from human pluripotent stem cells could effectively treat in the future

Human foetal brain tissue as quality control when developing stem cells towards cell replacement therapy for neurological diseases.

Human foetal brain tissue has been used in experimental and clinical trials to develop cell replacement therapy in neurodegenerative disorders such as Parkinson's disease and Huntington's disease. These pioneering clinical studies have shown proof of principle that cell replacement therapy can be effective and is worthwhile to develop as a therapeutic strategy for repairing the damaged brain. However, because of the limited availability of foetal brain material, and difficulties in producing standardized and quality-tested cell preparations from this source, there have been extensive efforts in investigating the potential use of alternative cell sources for generating a large number of transplantable, authentic neural progenitors and neurons. In this review, we highlight the value of using human foetal tissue as a reference material for quality control of acquired cell fate of in vitro generated neurons before and after transplantation

Reconstruction of the nigrostriatal dopamine pathway in the adult mouse brain.

Transplants of fetal dopamine neurons can be used to restore dopamine neurotransmission in animal models of Parkinson's disease, as well as in patients with advanced Parkinson's disease. In these studies the cells are placed in the striatum rather than in the substantia nigra where they normally reside, which may limit their ability to achieve full restoration of motor function. Using a microtransplantation approach, which allows precise placement of small cell deposits directly into the host substantia nigra, and fetal donor cells that express green fluorescent protein under the control of the tyrosine hydroxylase promoter, we show that dopamine neuroblasts implanted into the substantia nigra of adult mice are capable of generating a new nigrostriatal pathway with an outgrowth pattern that matches the anatomy of the intrinsic system. This target-directed regrowth was closely aligned with the intrinsic striatonigral fibre projection and further enhanced by over-expression of glial cell line-derived neurotrophic factor in the striatal target. Results from testing of amphetamine-induced rotational behaviour suggest, moreover, that dopamine neurons implanted into the substantia nigra are also capable of integrating into the host circuitry at the functional level

Unilateral axonal or terminal injection of 6-hydroxydopamine causes rapid-onset nigrostriatal degeneration and contralateral motor impairments in the rat.

Unilateral injection of the catecholamine neurotoxin 6-hydroxydopamine into the axons or terminals of the nigrostriatal pathway is commonly used to model Parkinson's disease in experimental animals. Although the terminal lesion paradigm is considered to induce a more progressive lesion when compared to the axonal lesion, few studies have directly compared the early time-course for lesion development in these two models. Thus, this experiment is sought to establish the temporal pattern of nigrostriatal degeneration and emergence of contralateral motor impairment in these models. Young adult male Lister Hooded rats were used. After baseline testing on a battery of spontaneous motor tests, standard stereotaxic techniques were used to inject 6-hydroxydopamine into the nigrostriatal axons or terminals at the level of the medial forebrain bundle or striatum respectively. From the day after lesion surgery, a subset of the rats was tested for motor performance, while another subset was used for immunohistochemical analysis. Quantitative tyrosine hydroxylase immunohistochemistry revealed that although both lesions caused a similar temporal pattern of immunopositive cell loss from the substantia nigra, the terminal lesion caused a more rapid loss of immunopositive terminals from the striatum. Despite these differences in striatal dopaminergic deafferentation, both lesion types caused a profound loss of contralateral motor function from the first day after lesion surgery. These findings illustrate the rapidity of the neuropathological and behavioural consequences of either axonal or terminal injection of 6-hydroxydopamine into the nigrostriatal pathway, and further highlight the need for a more progressive model of human Parkinson's disease

Characterisation of behavioural and neurodegenerative changes induced by intranigral 6-hydroxydopamine lesions in a mouse model of Parkinson's disease.

Abstract Despite the widespread use of mice as models of Parkinson's disease there is a surprising lack of validation and characterisation of unilateral lesion models in mice and the extent of behavioural impairments induced by such lesions. The aim of the present study was to characterise the behavioural deficits observed after injection of 6-hydroxydopamine unilaterally into the substantia nigra, and correlate the behavioural impairments with the extent of damage to the mesostriatal dopaminergic pathway. We found that a recently introduced test for assessment of sensorimotor impairment, the corridor task, was particularly useful in determining lesion severity, and that this test, in combination with standard drug-induced rotation tests, can be used to select animals with profound (>/= 80%) dopaminergic lesions that are stable over time. Based on these data we propose criteria that can be used to predict the extent of lesion, classified as severe, intermediate or mild lesions of the mesostriatal pathway. The correlation of cell loss and striatal innervation with the performance in each test provides a useful tool for the assessment of functional recovery in neurorestoration and cell transplantation studies, and for the evaluation of the in vivo efficacy and performance of stem cell-derived dopamine neuron preparations