Parkinson’s disease (PD) is the second most common neurodegenerative condition to affect humans, and is characterised by the loss of dopaminergic neurons from the substantia nigra pars compacta (SNpc) in the midbrain along with the deposition of abnormal aggregates of alpha-synuclein protein in the brain which are in the form of Lewy bodies. Dopaminergic neurons from the SNpc project into a large subcortical structure known as the striatum, and positron emission tomography (PET) studies have demonstrated the dysfunction of the dopaminergic system in the striatum of patients with PD. Furthermore, immunohistochemistry studies of the striatum have identified the degeneration of dopaminergic nerve terminals and inclusions of alpha-synuclein. An aggressive and early onset form of familial PD is caused by the G51D point mutation in alpha-synuclein (G51D/+). Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has been used to generate a novel and precise rat model of PD which has the G51D mutation in rat alpha-synuclein. Wild-type (WT) and G51D/+ rats were analysed over the course of ageing (5, 10/11 and 16/17 months of age) using histological experiments and L-3,4-dihydroxy-6-18F-fluorophenylalanine (18FDOPA) PET imaging in order to determine if G51D/+ rats have abnormalities of histological staining and dopaminergic function analogous to those identified in patients with PD. Histological experiments were optimised using WT rat tissue and then used immunohistochemistry for tyrosine hydroxylase (an enzyme involved in the synthesis of dopamine) to evaluate dopamine nerve terminal integrity in the striatum of WT and G51D/+ rats. In addition, immunohistochemistry for alpha-synuclein was used to evaluate staining for alpha-synuclein in cell bodies and the neuropil within the striatum of WT and G51D/+ rats. 18F-DOPA is a well validated PET radiotracer and has been used to investigate dopaminergic function in the striatum of rats. The enzyme aromatic L-amino acid decarboxylase converts 18F-DOPA to 6-18F-fluorodopamine, which is in turn incorporated into presynaptic vesicles, and then released into the synaptic cleft following neuronal activation. PET imaging experiments were first optimised using phantoms and WT rats, then the optimised protocols were applied to studies of WT and G51D/+ rats. Results from tyrosine hydroxylase immunohistochemistry at Bregma 0.00 mm identified a trend for decreased optical density of tyrosine hydroxylase staining in the striatum of 5 month G51D/+ rats compared with age-matched WT controls (p=0.15), and in 17 month G51D/+ rats compared with age-matched WT controls (p=0.10). Semi-quantitative analysis of alpha-synuclein immunohistochemistry indicated an increased abundance of alpha-synuclein positive cell somata in the striatum, and decreased punctate terminal staining in the neuropil of G51D/+ rats compared with age-matched WT rats. 18F-DOPA PET imaging experiments indicated a trend for decreased influx rate constant (Ki) of 18F-DOPA in the striatum of 5 month old G51D/+ rats compared with age-matched WT controls (p=0.08), and a trend for decreased distribution volume ratio (DVR) of 18F-DOPA in the striatum relative to the cerebellum of 16 month old G51D/+ rats when compared with age-matched WT controls (p=0.09). 18F-DOPA PET imaging experiments also identified a trend for a decreased effective distribution volume ratio (EDVR) of 18F-DOPA in the striatum relative to the cerebellum (p=0.09) and in turn indicated increased effective dopamine turnover (EDT) (p=0.13) in the striatum of 16 month old G51D/+ rats compared with age-matched WT rats. Therefore, the results indicated abnormalities of dopaminergic function, as well as tyrosine hydroxylase and alpha-synuclein staining in G51D/+ rats compared with age-matched WT controls, and this appeared to have some features of PD in humans. Indices of EDT indicated compensatory changes in dopaminergic function in the striatum of 16 month old G51D/+ rats compared with age-matched WT rats. Additional compensatory changes in dopaminergic terminal function and tyrosine hydroxylase protein expression may be evident in 11 and 10 month old G51D/+ rats respectively compared with age-matched WT rats. The G51D/+ rat model represents an interesting model for further studies such as the underlying pathophysiology of PD. However, the phenotype observed in G51D/+ rats appeared to be less severe than that which has been observed in humans with G51D type PD.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:738909 |
| Date | January 2018 |
| Creators | Morley, Victoria Lee |
| Contributors | Kunath, Tilo ; Tavares, Adriana ; Ffrench-Constant, Charles |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/28987 |
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