Anatomy and Neuroscience - Theses
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ItemChanges in Retinal Ganglion Cells during Disease and Aging(2020)Healthy retinal ganglion cell (RGC) function is vital for vision, and diseases that cause RGC degeneration can be debilitating. RGC degeneration features prominently during age-related diseases such as glaucoma and diabetic retinopathy. This thesis investigates how RGCs change in models of these diseases and if similar changes can be observed in normal aging. Exploring and comparing functional, structural, and molecular changes that develop in rodent RGCs during pathological states and normal aging can expand our understanding of how they degenerate during disease. Glaucoma is characterised by gradual RGC degeneration and is usually accompanied by increased intraocular pressure (IOP). We first examined the effect of an acute, non-ischemic IOP insult on RGC activity in wildtype mice. In wildtype mice, OFF-RGCs alone showed reduced spontaneous and light-elicited activity after IOP elevation. Next, we explored the role of the P2X7-receptor (P2X7-R) following IOP elevation, as it has been suggested to contribute to RGC death in glaucoma. After IOP elevation in P2X7-R knockout mice, both ON- and OFF-RGCs exhibited reduced light-elicited activity. Staining for P2X7-R in Thy1-YFP-H mice showed greater expression on ON-RGC dendrites than in other RGC cell subtypes. This study demonstrated the dysfunction of OFF-RGCs after acute, non-ischemic IOP elevation was not prevented by P2X7-R ablation. P2X7-R knockout seemed to worsen the effects of IOP elevation as it also caused ON-RGC dysfunction. In early stages of diabetic retinopathy, there is increasing evidence for RGC degeneration, prior to perturbation of other retinal neurons. Examining individual RGC function, we found that after 4 weeks of STZ-induced diabetes OFF-RGCs showed an increase in spiking activity at a single light intensity (220 photoisomerisations/sec/rod) compared to control. No changes in RGC density, synaptic protein puncta counts or Muller glia gliosis were identified. Microglia, however, showed a reduction in volume. These changes early in diabetes, though subtle, suggest dysfunction of the retinal circuitry alongside the development of inflammation. Aging can exacerbate RGC degeneration and contribute to the development of diseases such as glaucoma and diabetes. To probe the effects of age on RGCs in greater detail we used a combination of functional, structural, and molecular techniques. By examining the transcriptomes of isolated RGCs from young and aged mice using RNA-sequencing, we found upregulated genes in the pathways for oxidative stress, protein degradation and synaptic function. The upregulation of these genes may be a defensive strategy against age-related stresses during normal, healthy aging. This appears to be supported by our finding that RGCs were not as susceptible to structural or functional loss with age in comparison to photoreceptors and other cell classes. Overall, we found RGCs were dysfunctional prior to death by using early-stage disease models. The results of this thesis provide evidence that age- and disease-related stressors may invoke divergent responses in RGCs, despite aging being a risk factor for retinopathies. Stressors like increased IOP and hyperglycaemia worsen RGC function in a subtle manner; yet aging itself does not seem to pose a threat to RGC survival or function as RGCs seem more robust when compared to other retinal neurons. Future explorations could consider whether an additive effect of aging and disease may cause RGC defence mechanisms to become compromised
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ItemRibosomal protein depletion in the Drosophila haematopoietic compartment alters cell fate determination(2020)Ribosomes are essential components of the translational machinery, required for cells to effectively double their protein content in order to undergo cell division. Despite the obvious need for ribosomes, due to the high energetic cost, ribosome biogenesis is tightly regulated by numerous pathways in response to cellular and extracellular signalling cues. Predictably, dysregulation of ribosome levels strongly correlates with disease. Increased ribosome biogenesis is observed in many cancers and decreased ribosome biogenesis underlies a class of developmental disorders collectively termed ribosomopathies. Somewhat surprisingly, given the ubiquitous need for ribosomes, loss of ribosomal proteins has also been associated with tissue overgrowth in both human disease and model organisms, including zebrafish and Drosophila. In the latter, reported mechanisms for overgrowth due to global reduction of ribosomal proteins comprise extrinsic effects, whereby a smaller hormone-secreting gland delays animals in their growth phase to result in larger wings and eyes. Many ribosomopathies are associated with lineage-specific defects, with ribosomal protein loss linked with haematopoietic compartment pathologies, particularly lineage depletion and anaemia. Although lineage depletion phenotypes are well established to arise from apoptosis induced by nucleolar stress, there is also an increased incidence of cancer associated with these ribosomopathies for which mechanisms remain unclear. This thesis has taken advantage of the powerful genetic tools available in Drosophila to achieve tissue-specific depletion, in the larval hematopoietic compartment (the lymph gland), of the two ribosomal proteins most commonly lost in the ribosomopathy Diamond Blackfan Anaemia (DBA), S19a (RpS19a) or S24 (RpS24). In contrast to the haematopoietic lineage depletion observed in human disease, we report cell-intrinsic overproliferation and tissue overgrowth following depletion of either RP. However, despite gross phenotypic similarities, depletion of RpS19a and RpS24 resulted in distinct cell death and differentiation defects. Moreover, while RpS24 resulted in the expected decrease in mature ribosomes, RpS19a depletion resulted in an inexplicable increase in ribosomes. Transcriptome and proteome analyses of RP-depleted lymph glands revealed upregulation of metabolic and signalling pathways; however, factors involved in transcription and translation were disproportionately increased at the protein level, consistent with altered translation. Although certain proteins were elevated after knockdown of either RP, some were specifically increased by RpS19a or RpS24 depletion. Of the 8 candidates tested for capacity of co-depletion to suppress overgrowth defects only 5 (STAT, TOP1, Osa, TCTP and Cdk12) suppressed the RpS24 lymph gland phenotype, suggesting the requirement for increased abundance of these factors in overgrowth. In the case of RpS19a, although depletion of dHEATR1 and TCTP supressed overgrowth, only depletion of Osa, a key component of the SWI/SNF chromatin remodelling complex, suppressed overgrowth through a restoration of progenitors. The increased Osa observed in RpS19a and RpS24 knockdown is, therefore, required for progenitor depletion and lymph gland overgrowth. Further studies are required to determine whether differential translation of Osa, and the other candidates, contributes to lymph gland overgrowth due to depletion of RpS19a or RpS24.