Al-kushi, Abdullah. G
Pathological changes in mesostriatal neurons in a PKC-gamma mutant rat.
PhD thesis, University of Glasgow.
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The AS/AGU rat originated as a recessive mutation (agu) in a closed colony of Albino Swiss (AS) rats. The mutation is in the gene coding for the gamma isoform of protein kinase C. It is characterized by movement impairments and progressive dysfunction of the nigrostriatal dopaminergic (DA) and raphe striatal serotonergic (5-HT) systems. The movement impairments including rigidity of the hind limbs, a staggering gait, a tendency to fall over every few steps, a slight whole body tremor and difficulty in initiating movements. The dysfunction in both systems is characterised by a failure to release DA or 5-HT within the striatum and cell loss within the substantia nigra pars compacta (dopaminergic cells) and the dorsal raphe nuclei (5-HT+ve cells).
In this study, three experiments were carried out to examine the possible pathological responses of midbrain cell groups to the agu mutation in the gene coding for protein kinase C-gamma (PKC-γ).
Experiment 1 was carried out to examine levels of two groups of molecules in the midbrain cell groups using quantitative immunofluorescence microscopy of cell bodies or their surrounding neuropil (a) those molecules giving information about the capacity of midbrain aminergic cell bodies to synthesis transmitters; tyrosine hydroxylase (TH) in the dopaminergic neurons and serotonin (5-HT) in the serotonergic neurons (b) those which have been found to occur in human neurodegenerative conditions such as Parkinson’s disease: ubiquitin, parkin and α-synuclein (Lewy body proteins). Immunofluorescence levels of tyrosine hydroxylase (in dopaminergic cells of the SNC) and serotonin (in 5HT+ve cells of the dorsal raphe nuclei) were both significantly increased in AS/AGU (mutant) compared to the AS (control) rats aged 6 months and older. TH and 5-HT immunofluorescence levels were both significantly decreased in the striatum in the AS/AGU (mutant) compared to the AS (control) rat aged 12 months. Ubiquitin immunofluorescence show a gradual increase with age in AS and AS/AGU rats and the increase was much greater in the mutant in every region except the oculomotor and pontine nuclei. Parkin immunofluorescence show increases in the mutant within the SNC and the dorsal raphe nucleus and this increase was significant at older ages. Alpha-synuclein does not occur in the cell bodies of the substantia nigra or VTA but outside in the neuropil. Alpha-synuclein immunofluorescence levels progressively increased with age in both strains in the SN and VTA and were higher in the mutant. The levels of those molecules (ubiquitin, parkin and alpha-synuclein) do not differ in the striatum of mutants compared to controls.
Experiment 2 examined SNC cell bodies to look for possible strain differences in cell size or ultrastructure or any sign of cell death using light and transmission electron microscopy. The diameter (maximum and minimum) of the SNC cells and nuclei were measured in toluidine blue paraffin wax and immunoperoxidase DAB staining for TH sections. Cell diameter was reduced in the AS/AGU mutant compared to the AS control. No obvious ultrastructural differences were seen in nigrostriatal neurons of both strains. The volume fractions of mitochondria and rough endoplasmic reticulum were significantly higher in the mutant. No Lewy bodies were present.
Experiment 3 examined TH+ve nigrostriatal dopaminergic terminals in the dorsal caudate-putamen to determine whether there are (a) differences in the percentages and numbers of TH+ve terminals and (b) differences in synaptic vesicles numbers. In 12-month AS/AGU mutant, there are reduction in TH+ve terminals (40%) together with a reduction in vesicle numbers (40%) in such terminals where in 3-month AS/AGU mutant, the reduction in TH+ve terminals was more (50%) and a reduction in vesicles numbers by three quarters. TH-ve terminals are also reduced in numbers in 12 months aged AS/AGU mutant rats. In 12-month AS/AGU rats, there were significantly reduced numbers of synaptic terminals in the striatum compared to AS controls. This applied to both dopaminergic terminals (which make up 15% of the total) and to non-dopaminergic terminals. In 3-month AS/AGU rats, there is a reduction in terminal numbers, but this is restricted to the dopaminergic terminals only: non-dopaminergic ones are unaffected.
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