Anatomy and Neuroscience - Theses
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ItemThe effect of the ablation of the SEZ6 protein family on microglial structure and function in the central nervous system( 2023-09)Microglia are the resident immune cells of the brain and are responsible for mediating the central nervous system (CNS) response to infection, injury and disease. More recently, roles for microglia in the developing brain have become evident, including maintaining tissue homeostasis, vascular control, and synaptic pruning, a normal developmental process during which synaptic connections are refined. Microglia have been shown to remove weaker synaptic connections through the involvement of the complement system, particularly the complement proteins C3 and C1q which label the synapse for microglial mediated removal. Reflecting their important role in this process, microglia are the only cells of the CNS that express complement receptor (CR) 3 and lack of C1q, C3 or CR3 results in reduced synaptic pruning during development. The Seizure-related protein 6 (SEZ6) family contain similar structural domains to those of complement regulatory proteins, namely Complement Control Protein (CCP) and CUB domains and SEZ6 proteins were recently shown to protect expressing cells against complement deposition. In genetic knockout models [SEZ6 knockout (KO) or SEZ6 triple knockout (TKO) mice lacking all three SEZ6 family members], roles for SEZ6 proteins in the development and maintenance of excitatory synapses have been identified. Preliminary data from our laboratory indicate that lack of SEZ6 proteins leads to reductions in microglial biomarker proteins in synaptosome preparations from mouse brain, suggesting that SEZ6 proteins are involved in regulating microglial-mediated synapse remodelling. This thesis will explore the role of SEZ6 proteins in regulating microglial structure and function in the brain. The aims of this work were to validate the preliminary synaptosome data and determine whether loss of SEZ6 proteins results in altered microglial expression of key proteins, including the cluster of differentiation (CD) 11B protein which, together with CD18, comprises CR3. Western analysis of CD11B protein in synaptosome preparations isolated from SEZ6 TKO showed reduced CD11B levels compared to control mice, supporting our preliminary data. Interestingly, this reduction was observed in female SEZ6 TKO mice only, with no change in synaptosome CD11B levels observed in males. In order to determine whether the decrease in synaptosome CD11B was due to a decrease in total expression in microglia, CD11B protein expression was also quantified in fluorescence-activated cell sorting (FACS)-isolated microglia. No change in total CD11B protein expression was found in either male or female SEZ6 TKO mice. However, when surface expression of CD11B and F11R were quantified via flow cytometry, a reduction in the surface expression of CD11B was found in both sexes and surface F11R expression was reduced in female SEZ6 TKO mice. Work in this chapter showed that loss of SEZ6 reduced the presence of the complement receptor CR3 subunit CD11B in synaptosomes of female mice and this may be due to altered surface expression of this receptor on microglia, supporting a role for SEZ6 protein in modulating the interaction between microglia and synapses. To further investigate the interaction of microglia and synapses in the absence of SEZ6 proteins, morphology of microglia was analysed as morphological changes in microglia are indicative of an altered state and associated with the microglial response to changes in their environment. In addition, microglia density was analysed as it was hypothesized that a lower density of microglia might lead to synapses being less frequently contacted. Quantification of the microglial density and morphology revealed strong regional differences, particularly in the cerebellum compared to the rest of the brain. While the density of microglia remained unchanged in the absence of SEZ6 proteins, microglia were found to be hyper-ramified in the hippocampus and striatum of SEZ6 TKO mice. This finding further supports an altered state of microglia and changed interaction with synapses. Lastly, the effect of the lack of SEZ6 proteins on synaptic pruning was analysed to investigate whether lack of SEZ6 proteins affects microglial function. Anterograde tracers [cholera toxin subunit beta (CTB) linked to different fluorophores] were injected separately into the left and right eye of P9 mice and the overlap of retinal ganglion cell (RGC) terminal fields was analysed in the P11 dorsal lateral geniculate nucleus (dLGN). No significant difference in the RGC terminal overlap was observed between SEZ6 TKO and control mice but pruning effects could not be excluded due to the incomplete labelling of the dLGN, even after troubleshooting this challenging intravitreal injection procedure. Together, the reduction of CR3 subunits in synaptosomes and on the surface of cortical microglia as well as the hyper-ramification of microglial processes in the striatum and hippocampus indicates that microglia have an altered interaction with synapses in the absence of SEZ6 proteins. While a direct functional effect on developmental synaptic pruning in SEZ6 TKO mice could not be shown, these results indicate novel roles for SEZ6 proteins as regulators of microglial function.
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ItemThe role of microglia in regulation of vasculature and blood flow in the healthy and diabetic retina( 2022)Diabetic retinopathy is a common vascular complication of diabetes and a leading cause of blindness in those of working age. Prior to overt vascular pathology, the retina displays subtle changes to neurons, glia, and blood vessels that are likely important for disease progression. However, current treatments for diabetic retinopathy are only effective at targeting late-stage pathology. Treatments that target the early cellular changes in the diabetic retina have the potential to halt disease progression before vision is threatened. One of the earliest changes observed in the diabetic retina is a reduction in blood flow. This early vascular dysfunction has been observed in the absence of any other signs of retinopathy, suggesting it may be a key early driver of disease and a promising target for intervention. It has been suggested that the underlying cause of reduced blood flow is dysfunction of the mechanisms that regulate blood flow in the retina. However, our current understanding of these mechanisms is largely incomplete. The central aim of this thesis was, therefore, to explore how blood flow is regulated in the normal retina, and to determine how this function is altered in the diabetic retina. Recent work from our group and others have identified that microglia, the resident immune cells of the central nervous system, may play a role in regulation of blood flow. Based on this emerging evidence, our hypothesis was that microglia regulate vascular function in the retina, and that hyperglycaemia leads to changes in microglia that impair this function and result in reduced blood flow. To explore this hypothesis, we first performed RNA sequencing of retinal microglia isolated from mice lacking Cx3cr1, a chemokine receptor specific to microglia and an important regulator of many microglial functions. This revealed a role for Cx3cr1 in several possible functions related to vasculature, including vascular development, microglial-vascular adhesion, and vascular tone, which were further assessed with in vitro and in vivo imaging techniques. Imaging data revealed the Cx3cr1null retina showed increased vascular density, reduced microglial-vascular contact, and most interestingly, dilation of capillaries. This loss of vascular tone may have been due to reduced expression of angiotensin converting enzyme, a component of the renin angiotensin system (RAS), which promotes vasoconstriction. The ability of microglia to dynamically alter blood vessel diameter and hence control blood flow was then assessed by live cell imaging of the ex vivo retina. We observed frequent spontaneous calcium transients in microglia which appeared to induce vasoconstriction, which may have been mediated by purinergic signalling. Microglia also evoked vasoconstriction via a calcium-independent mechanisms, which was promoted by addition of fractalkine, the ligand for Cx3cr1. Transcriptomic data suggested FKN-Cx3cr1 signalling may promote vasoconstriction via modulation of the RAS. This was confirmed by inhibition of the RAS in the ex vivo retina, which abolished FKN-evoked vasoconstriction. As earlier work from our group has shown FKN-Cx3cr1 signalling and the microglial RAS are upregulated in the diabetic retina concurrent with reduced blood flow, we postulated that this vascular dysfunction may be caused by aberrant microglia-mediated vasoregulation. To test this, we trialled pharmacological blockade of the RAS in an animal model of type 1 diabetes. Without treatment, diabetic animals exhibited constriction of retinal capillaries, reduced blood flow, and dysfunction of inner retinal neurons. Microglia did not display classical signs of activation but did show increased accumulation on capillaries. RAS blockade successfully restored capillary diameter in the diabetic retina, but surprisingly failed to improve blood flow or neuronal function. Finally, while RAS blockade did not affect the number of microglia accumulating on capillaries, it did increase the extent to which individual microglia contacted vasculature, further alluding to the importance of the microglial RAS in regulation of retinal vascular function. In summary, our findings indicate that microglia and Cx3cr1 are important for vascular function in the retina, in particular for vascular development and maintenance of capillary tone. We also established that microglia can dynamically alter blood vessel diameter in multiple ways, suggesting these cells may be important for regulating retinal blood flow. Finally, restoration of capillary diameter by RAS blockade in the diabetic retina supports the theory that aberrant microglia-mediated vasoregulation contributes to early vascular dysfunction in diabetic retinopathy. These findings may form the basis for new treatments that can prevent vascular dysfunction in diabetic retinopathy and other CNS diseases.
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ItemThe neurovascular unit in early diabetic retinopathy: cellular changes and an emerging role for microglia( 2018)In the developed world the leading cause of blindness in working age adults is diabetic retinopathy (DR). The risk of vision loss due to diabetes increases with disease duration, and most frequently affects those who have lived with diabetes for one to two decades. For individuals with diabetes, there is a need to develop therapeutic interventions that are capable of halting or delaying degeneration of the retina as early as possible to preserve vision throughout the life of the patient. DR ultimately leads to vision loss via the death of retinal neurons, which occurs after severe dysregulation of the retinal vasculature. Retinal oedema and/or the proliferation of blood vessels both signal the advancement to a high-risk stage of vision-threatening DR, and our understanding and management of these late stages is comprehensive. However, the initial cellular changes that occur soon after the onset of diabetes and contribute to this end-stage vascular pathology are not completely understood. These include changes to the neurovascular unit: retinal blood flow, neuronal function, and glial / microglial cell reactivity. It is these early signs that are investigated in this thesis, for it is these changes at this early timepoint that hold the most promise for intervention to prevent onset of clinical vasculopathy. Using an animal model of diabetic retinopathy, the relative contribution of neurons, blood vessels, and glia/microglia to early DR pathology was assessed at two early time-points after diabetes onset. In vivo measurement of retinal blood flow, blood vessel structure, and neuronal function was undertaken; in conjunction with histological analysis of pericytes, Müller glia, astrocytes, and microglia, as well as the interactions of these cells with the vasculature. Additionally, microglia were investigated for their potential to assist in vaso-regulation using ex vivo live cell imaging and single cell population RNA-Seq. Functional measurements revealed that retinal blood flow and retinal ganglion cell function were both reduced after 4 weeks of hyperglycaemia. In depth analysis of retinal vessel structure showed capillaries to be constricted at this time-point and were also unresponsive to hyperoxic challenge. Neuronal function was further degraded by 12 weeks of diabetes, although there were no signs of cell death. Glial cells showed no difference in structure, or signs of gliosis after 4 weeks of diabetes, and did not alter contact with the vasculature. Microglia were found to be in a resting phenotype and increased their contact with retinal capillaries after 4 weeks of diabetes. The microglia specific fractalkine signalling axis was shown to initiate vasoconstriction on retinal blood vessels, a response which was absent in the diabetic retina. Vaso-active genes were identified in microglial populations, several of which were upregulated in the diabetic retinae. In summary there are two main findings to report. Firstly, this project expands our understanding of the chronopathology of cell-specific dysfunction in the early stages of DR, and indicates that capillary constriction may be a new biomarker for early stage disease. Secondly, the discovery that microglia contribute to blood flow maintenance opens new avenues for research into vascular diseases, including DR, which may unlock novel therapeutic targets.