The eye contains closely related but widely different tissues, offering a unique opportunity to investigate the phenotype and function of monocyte-derived cell populations within functionally unique microenvironments in a single complex organ. The uveal tract and retina contain rich networks of immune cells that reside and traffic through the eye, these cells having been implicated in various ocular inflammatory processes and immune-mediated diseases. One such inflammatory condition is human posterior uveitis, an autoimmune disease mainly affecting the retina. As current treatments for posterior uveitis only serve to slow down disease progression, studies using animal models, namely, experimental autoimmune uveoretinitis (EAU), have focused on determining the key cellular and molecular mediators involved in disease initiation in order to expand the potential for novel therapeutic applications. The overall purpose of experiments in this thesis was to explore monocyte-derived cell populations of the uveal tract and retina, this being achieved by utilising a novel transgenic mouse model. Cx3cr1gfp/gfp transgenic mice on both BALB/c and C57Bl/6 backgrounds contain an enhanced green fluorescent protein (eGFP) encoding cassette knocked into the Cx3cr1 gene, disrupting its expression but facilitating GFP expression under the control of the Cx3cr1 promoter. Heterozygous (Cx3cr1+/gfp) mice were generated by crossing Cx3cr1gfp/gfp mice to wild-type (WT) mice. This transgenic model allowed for the exquisite visualisation of Cx3cr1-bearing monocyte-derived dendritic cells (DC) and macrophages in ocular tissues, whilst also enabling the investigation of a potential role for Cx3cr1 in recruiting monocyte-derived cells to the eye in steady-state and inflammatory conditions.

No description available.

published_or_final_version / Electrical and Electronic Engineering / Master / Master of Philosophy

published_or_final_version / abstract / Physiology / Doctoral / Doctor of Philosophy

Amyotrophic lateral sclerosis (ALS) is an aggressive neurodegenerative disease in which motor neurons selectively degenerate, leading to paralysis and death. Rare causal mutations in FUS and TARDBP implicated RNA binding proteins and RNA metabolism in ALS disease mechanisms. The absence of faithful animal models has impeded precise understanding of the impact of ALS mutations on all functions of ALS-associated proteins. In my graduate studies, I used a novel, animal model of FUS-ALS to explore gain of function disease mechanisms and observed specific, aberrant interactions between mutant FUS and other RNA binding proteins including hnRNP U. Genetic experiments indicate loss of hnRNP U is toxic to motor neurons, suggesting mutant FUS toxicity may result from hnRNP U sequestration and loss of function. In a parallel series of experiments, I also generated novel knock-in mouse models of ALS expressing pathogenic TARDBP mutations to address the flaws of existing model systems and to study the functional consequences of disease-related mutations. We demonstrate that the ALS mutant alleles TDP-43M337V and TDP-43G298S are fully functional and are insufficient to cause age-dependent motor neuron pathology, indicating that physiological levels of mutant TDP-43 are alone insufficient to initiate disease. This model enables future exploration of the interaction between genetic and environmental factors that lead to TDP-43 toxicity in ALS and related disorders. Collectively, our findings suggest a gain of function mechanism of toxicity in which mutations and aging, with other factors, alter the local concentration of RNA binding proteins, leading motor neurons to degenerate.

Systemic lupus erythematosus (SLE) and Sj?gren?s syndrome (SS) are both heterogeneous autoimmune diseases with strong B cell aspects. A proportion of SLE and SS patients exhibit elevated serum BAFF (B cell activating factor of the TNF family); BAFF plays a key role in B cell homeostasis, survival and tolerance. BAFF transgenic (Tg) mice develop nephritis and salivary gland destruction that resemble aspects of SLE and SS respectively. Autoimmune disease development in BAFF Tg mice correlates with marginal zone (MZ) B cell expansion and the abnormal presence of MZ-like B cells outside of the spleen. The role of MZ B cells in BAFF induced autoimmune disease was analysed by crossing BAFF Tg mice with Lymphotoxin-β knockout mice (creating LTβ-BTg mice) which lack most peripheral lymph nodes, and also lack MZ B cells as a result of disrupted splenic architecture. LTβ-BTg mice were not protected against nephritis but exhibited reduced salivary gland infiltration and destruction. Indicating that the development of sialadenitis but not nephritis in BAFF Tg mice is MZ B cell dependent. Nephritis development in LTβ-BTg mice was associated with the detection of B-1 B cells in the inflamed kidneys. As B-1a B cell survival is dependent on the spleen, the contribution of B-1a B cells to nephritis development in BAFF Tg mice was assessed by crossing BAFF Tg mice to congenitally asplenic Hox11-/- mice (creating Hox11 -BTg mice). The absence of a spleen and B-1a B cells in Hox11-BTg mice delayed the nephritis development. In contrast, splenectomy of BAFF Tg mice at 12 weeks of age did not alter nephritis onset. In these mice B-1a B cells persisted in the peritoneal cavity and MZ-like B cells were detected in the periphery 8 months after surgery. In summary, nephritis development in BAFF Tg mice is unaltered by the absence of MZ B cells, but delayed in the absence of a spleen, MZ and B-1a B cells. Thus, B-1a and B-1b B cells may be potential targets for the treatment of nephritis in SLE patients with elevated BAFF.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Systemic lupus erythematosus (SLE) and Sj?gren?s syndrome (SS) are both heterogeneous autoimmune diseases with strong B cell aspects. A proportion of SLE and SS patients exhibit elevated serum BAFF (B cell activating factor of the TNF family); BAFF plays a key role in B cell homeostasis, survival and tolerance. BAFF transgenic (Tg) mice develop nephritis and salivary gland destruction that resemble aspects of SLE and SS respectively. Autoimmune disease development in BAFF Tg mice correlates with marginal zone (MZ) B cell expansion and the abnormal presence of MZ-like B cells outside of the spleen. The role of MZ B cells in BAFF induced autoimmune disease was analysed by crossing BAFF Tg mice with Lymphotoxin-β knockout mice (creating LTβ-BTg mice) which lack most peripheral lymph nodes, and also lack MZ B cells as a result of disrupted splenic architecture. LTβ-BTg mice were not protected against nephritis but exhibited reduced salivary gland infiltration and destruction. Indicating that the development of sialadenitis but not nephritis in BAFF Tg mice is MZ B cell dependent. Nephritis development in LTβ-BTg mice was associated with the detection of B-1 B cells in the inflamed kidneys. As B-1a B cell survival is dependent on the spleen, the contribution of B-1a B cells to nephritis development in BAFF Tg mice was assessed by crossing BAFF Tg mice to congenitally asplenic Hox11-/- mice (creating Hox11 -BTg mice). The absence of a spleen and B-1a B cells in Hox11-BTg mice delayed the nephritis development. In contrast, splenectomy of BAFF Tg mice at 12 weeks of age did not alter nephritis onset. In these mice B-1a B cells persisted in the peritoneal cavity and MZ-like B cells were detected in the periphery 8 months after surgery. In summary, nephritis development in BAFF Tg mice is unaltered by the absence of MZ B cells, but delayed in the absence of a spleen, MZ and B-1a B cells. Thus, B-1a and B-1b B cells may be potential targets for the treatment of nephritis in SLE patients with elevated BAFF.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease caused by the death of motor neurons in the spinal cord and brain. ALS is a genetically complex disease; diverse mutations cause motor neuron death by disrupting various interrelated pathways. To date, no therapy targeting a single factor can rescue motor neuron loss, nor is it known how or why sub-populations of motor neurons are particularly vulnerable in disease. Many studies have pointed to the Transforming Growth Factor Beta (TGF-𝝱) signaling superfamily as a modifier of disease in human patients and in animal models. Here, we have used the SOD1G93A model of ALS to investigate if and how TGF-𝝱 signaling in motor neurons changes pathology in these animals. In the first part of this study we characterize canonical TGF-𝝱 activation in motor neurons in SOD1G93A animals compared to controls. We have found that a vulnerable motor neuron subpopulation upregulates TGF-𝝱RII, a receptor necessary and unique to the classical arm of the TGF-𝝱 signaling family, in a disease dependent manner. Despite the upregulation of TGF-𝝱RII in these cells, there is not a corresponding activation of downstream canonical TGF-𝝱 effectors in diseased motor neurons. Through in vivo genetic manipulation we found that TGF-𝝱RII is dispensable in motor neurons, but that ablation of TGF-𝝱RI, a key receptor in multiple arms of the TGF-𝝱 superfamily, decreases motor neuron survival in SOD1G93A animals. To further understand how this manipulation changes TGF-𝝱 activation in motor neurons, we performed iterative indirect immunoflourescence imaging. We have identified that knocking out TGF-𝝱RI from motor neurons disrupts downstream canonical TGF-𝝱 activation in these cells. To identify how TGF-𝝱 signaling changes gene expression in these cells we have used Visium, a spatial RNAseq method, on lumbar spinal cords from these animals We have identified and are currently investigating potential downstream targets of TGF-𝝱 signaling in motor neurons in SOD1G93A animals. These data suggest that motor neurons rely on TGF-𝝱 signaling for survival in disease and that TGF-𝝱 signaling is important to the biology of a known vulnerable population of motor neurons.