A role for nanor in early zebrafish development and validation of a neurotoxic zebrafish model of Parkinson’s disease
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Zebrafish are a versatile experimental model which have progressed the fields of many research areas through both genetic and pharmacological studies. In this thesis I studied a novel zygotic gene, nanor, in zebrafish and investigated expression, localisation and function of the gene during early development. I also investigated neuroprotective and neurorestorative properties of three novel Parkinson’s disease drugs, calcium channel inhibitor - isradipine, tetracycline antibiotic - minocycline and monoamine oxidase inhibitor - rasagiline using a zebrafish model of Parkinson’s disease. The transition from maternal to zygotic control of embryo development occurs with activation of the zygotic genome and degradation of maternal transcripts. This stage is known as the mid-blastula transition in zebrafish. Following this, axis patterning begins with the anteroposterior axis, the dorsoventral axis and the left/right axes. Left/right axis formation is essential for asymmetric development and correct orientation of developing organs such as the heart, brain and gut. In zebrafish, asymmetric development begins with dorsal forerunner cell migration to form a transient organ, Kupffer’s vesicle; the organ of asymmetry. Kupffer’s vesicle sends signals to the lateral plate mesoderm during somitogenesis which will result in nodal-related gene expression and correct asymmetry development in the larvae. A novel, first-wave zygotic gene in zebrafish, nanor, was discovered by our research group and has been linked to left/right asymmetry development. Nanor has 3 paralogues and 3 orthologues. The best hit following genomic sequence alignment is to a P2X7-like purinoreceptor in Austrofundulus limnaeus, a closely related genus of fish, with no known human ortholog. I investigated nanor expression by quantitative PCR and whole-mount in situ hybridisation and found highest expression at sphere and shield stage with ubiquitous expression which becomes restricted to anterior structure by 24 hours post fertilisation. A second paralogue, nanor b, then goes through a second wave of gene expression up to 120 hours post fertilisation. Transcription activator-like effector nucleases (TALEN) technology was used to knockout nanor in order to identify downstream genes. High embryonic lethality was recorded in mutant embryos with severe defects such as underdeveloped or absent eyes, brain and heads with bent bodies and severe heart oedema and blood pooling. Knockout was found to be embryonic lethal in mutants. Following this, Nanor protein-protein interactions were investigated using co-immunoprecipitation followed by mass spectrometry. Results identified over 1500 interacting proteins. However, on closer investigation the custom made anti-nanor antibody used for Nanor protein complex binding was found to be binding to an off-target protein, 14-3-3 β/α-B, with no detection of Nanor. Investigations could not be continued due to no alternative antibody available. Calcium influx into cells and Fibroblast growth factor (FGF) signalling are known to be two of the earliest signalling events in establishing left/right asymmetry. This study has found nanor to be downstream of calcium and FGF signalling following inhibition of these events using calcium channel inhibitor (isradipine) and FGF signalling inhibitor (SU5402) which resulted in increased nanor expression at 24 hours post fertilisation. Nodal-related gene southpaw, co-receptor one-eyed pinhead and downstream lefty1 showed no significant changes in relative gene expression. TRAF3IP1 (elipsa) and plakoglobin mutant lines with defective left/right asymmetry development were also investigated in this study. Nanor mRNA expression was increased in both mutant lines with no changes in southpaw or one-eyed pinhead and a slight decreased in lefty1 in elipsa mutants. Nanor was concluded to be downstream of calcium signalling, FGF signalling, elipsa and plakoglobin. Parkinson’s disease is a common, debilitating, neurodegenerative disorder for which the current most popular treatment, levodopa (L-DOPA), is symptomatic but does not slow or halt disease progression. There is an urgent, unmet need for neuroprotective or, ideally, neurorestorative drugs. Neurotoxin 6-hydroxydopamine (6-OHDA) was used to induce neuronal loss and locomotor deficit in zebrafish larvae. Three drugs (isradipine, minocycline and rasagiline) were screened for neuroprotective effects by co-treatment with the drugs and 6-OHDA from 2 to 5 days post fertilisation. Isradipine showed no neuroprotective effect during behavioural testing or immunohistochemical staining of dopaminergic cell survival. In contrast, both minocycline and rasagiline improved locomotor deficit and were neuroprotective against dopaminergic cell loss. Neurorestorative potential of each drug was also assessed by exposure to 6-OHDA from 48 hours post fertilisation (hpf) – 80 hpf followed by drug treatment up to 120 hpf. Exposure to 6-OHDA during neuroprotective (3 days) or neurorestorative (32 hours) experiments induced similar, significant locomotor deficits and neuronal loss in 5-day old larvae. Isradipine had no effect on 6-OHDA-induced locomotor deficit or neuronal loss. However, both minocycline and rasagiline improved locomotor deficit and dopaminergic cell loss following 6-OHDA exposure. This study has shown the versatility of zebrafish in the study of genes essential for early development through genetic manipulation as well as their use in pharmacological screening in neurodegenerative diseases.
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