Manipulation of precursor cells for the replacement of complex circuitry lost in neurodegenerative disease

摘要

The research reported in this thesis focused on the potential of neural progenitor cells to provide a suitable source of neurones which can be used in cell replacement strategies for Parkinson's disease. Specifically, the parameters affecting the differentiation of these cells into neuronal phenotypes were addressed and increasing the survival of transplanted dopamine neurones was attempted. In addition, the in vitro capacity of adult neural progenitor cells to generate neurones was assessed and a mouse model of Parkinson's disease was established. In Chapter Three an extensive study investigating the effects of donor age and periods of in vitro proliferation on the neurogenic capacity of foetal rat neural progenitor cells revealed that these two parameters had significant effects on neuronal differentiation. While ventral mesencephalic (VM) cells isolated at embryonic day 12 generated the most neurones, increased periods of in vitro cell expansion had detrimental effects on neuronal yield. Dopamine differentiation was also severely effected by in vitro proliferation with VM cells failing to generate any dopamine neurones even after short-term expansion. In Chapter Four, in an attempt to increase the survival rate of transplanted dopamine neurones, dopamine neurones were transplanted into the striatum of rat models of Parkinson's disease in a solution containing the antioxidant, ascorbic acid. Dopamine neurones transplanted with ascorbic acid formed grafts containing more dopamine neurones compared to standard dopamine grafts, indicating a specific survival effect of ascorbic acid. The ability of adult neural progenitor cells to generate neurones has generated great excitement over the past years, however, while these cells have shown the capacity to generate neurones, the ability of these cells to differentiate into neurones of dopaminergic phenotypes has not been demonstrated. This issue was addressed in Chapter Five, where both expanded and non-expanded adult progenitors were assessed for the expression of the non-specific dopamine marker tyrosine hydroxylase (TH). Neither expanded nor non-expanded cells that differentiated into neurones were immunopositive for TH, thus indicating an inability of these cells to spontaneously differentiate into dopamine neurones. To efficiently assess the functional capacity of mouse-derived embryonic stem cells, a mouse model of Parkinson's disease was established in Chapter Six. Rotational behaviour induced following amphetamine and apomorphine challenge in mice with either unilateral 6-OHDA lesions of the medial forebrain bundle (MFB) or the striatum were compared. While both models showed rotational bias, MFB lesions were variable and unreliable, whereas more consistent dopamine loss was observed following striatal lesions. The striatal lesion therefore provided the better lesion in mice, and optimal rotational bias was elicited following 10mg/kg amphetamine challenge. To confirm rotational behaviour in this mouse model reflected dopamine loss, in Chapter Seven, mice received intrastriatal transplants of dopamine neurones. 4-6 weeks post-transplantation, significant attenuation of amphetamine-induced rotational bias was observed. This behavioural recovery not only confirmed that rotations reflect dopamine loss, but also demonstrated the suitability of this model for measuring the functional capacity of mouse-derived cell lines.