Anatomy and Neuroscience - Theses
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ItemStudies on mechanisms of cell death in retinal degeneration( 2018)Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinopathies affecting approximately 1.5 million people worldwide. RP is associated with genetic mutations leading to progressive deterioration of the photoreceptors followed by degeneration of the retinal pigment epithelium (RPE). The molecular mechanisms by which these mutations drive photoreceptors to death are not well understood. Accordingly, effective therapeutic strategies to stop the progression of RP and/or restore vision have yet to be established. The current study examines mechanisms of cell death in two models of RP that affect genes in visual cycle: Pro23His (P23H) rat and rd1 mice. The P23H transgenic albino rat strains are widely used models for the most common mutation of the rhodopsin (RHO) gene associated with autosomal dominant form of RP. However, detailed phenotypic characterization of the most commonly used strain (line 3, P23H-3) is lacking. Before investigating what underlying cell death signaling pathways contribute to photoreceptors death in this animal model, P23H-related functional and structural changes were evaluated using electroretinography (ERG) and in vivo imaging. In the control Sprague-Dawley (SD, control) rats, photoreceptoral and post-photoreceptoral ERG responses enhanced from postnatal day (P)18 to 1 month, when they reached adult levels. By contrast, the function of the rod and cone pathways in the P23H-3 rat showed progressive decline from 1 month of age, which correlated with loss of photoreceptors in the outer nuclear layer (ONL) from 2 months onwards. Quantitative analysis of dying P23H-3 photoreceptors in the ONL, using TUNEL assay, revealed a biphasic pattern of the cell death, with a rapid initial phase, peaking at P18, followed by a slower progressive phase. However, activation of caspase-3 and nuclear translocation of apoptosis inducing factor (AIF) were detected rarely in the nuclei of the ONL at P18, suggesting that the classical intrinsic apoptotic pathway may not be the major contributor. Consistent with this, analysis of mitochondrial membrane potential (Δψm) using flow cytometric analysis of JC-1 fluorescent assay showed similar staining patterns for both P23H-3 and age-matched SD control cells, with no significant differences between the ratio of red/green fluorescence, that is no difference in Δψm. To further investigate other potential cell death pathways involved in the degeneration of photoreceptors in P23H-3 rats, gene expression profiling was performed using a rat cell death qPCR array on RNA isolated from P14 and P18 P23H-3 and age-matched SD rat retinae. The results revealed significant dysregulation in the expression levels of several genes associated with autophagy and necrosis. The rd1 (Pde6brd1/rd1) mouse is a well-characterised animal model of human autosomal recessive RP bearing a gene mutation in the β-subunit of phosphodiesterase 6 (Pde6b). As previous studies have debated the importance of apoptotic pathways in this model, the roles of two central molecules (BAX and BAK) was investigated in mitochondrial-mediated apoptosis during normal maturation of the retina and during rd1-associated photoreceptor degeneration. C567Bl6 mice lacking Bak (Bak-/- and Bak-/- Bax+/-) exhibited normal architecture and lamination of retina, with no significant differences in thickness of retinal layers compared to wild-type (WT) at P14. Analysis of Bak and Bax expression by droplet digital PCR (ddPCR) in rd1 mice at P14 and P18 revealed elevated expression of Bak, but not Bax, compared to the WT controls. Genetic removal of Bak in rd1 mice, with or without loss of one Bax allele, resulted in a significant and transient increase in ONL thickness compared to mice rd1 at P14, that was not maintained at P18. However, in double-knockout mice (rd1/Bak-/- Bax-/-) the ONL thickness was similar to the WT, despite the presence of the rd1 mutation. Moreover, a substantial thickening was detected in inner retinal layers including inner nuclear layer (INL), inner plexiform layer (IPL) and ganglion cell layer (GCL) when compared to age-matched rd1 and WT controls. The data indicate that in the rd1 mouse model, BAK and BAX function redundantly in intrinsic cell death pathways, which are a major contributor to photoreceptor death over and above normal developmental cell death in the maturing retina. To investigate the potential to pharmacologically target intrinsic cell death mechanisms, the efficacy of a BAK antagonist was examined in vivo. Intravitreal injection of a specific BAK inhibitor (WEHI-1250993) into rd1/Bax-/- mice at P16 significantly reduced photoreceptor death at P18 when compared to vehicle controls. While these results provide proof of principle that intravitreal administration of antiapoptotic agents may be effective for ameliorating photoreceptor death, further studies are needed to develop BAX antagonists and determine the long-term effectiveness of such therapeutic approaches. In conclusion, P23H mutation in RHO induced a progressive deterioration of retinal function and structure in transgenic rats. Multiple mechanisms of cell death possibly form a complicated network of molecular events to mediate degeneration of photoreceptors in P23H-3 animal model. However, the mitochondrial-mediated apoptosis is central to both naturally occurring developmental as well as rd1-associated cell death in mouse retina. Targeting these cell death pathways may slow the progression of photoreceptor death in individuals with RP.
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ItemImpact of lipofuscin accumulation on retinal degeneration in the mouse model of the Neuronal Ceroid Lipofuscinosis variant CLN6( 2018)Failure of the autophagy-lysosomal pathway and accumulation of lipofuscin in the retinal pigment epithelium (RPE) has been suggested to induce photoreceptor dysfunction and death in age related macular degeneration (AMD), Stargardt’s disease and neuronal ceroid lipofuscinosis (NCL). With detailed knowledge about the underlying causes and molecular mechanisms involved still elusive, mouse models of NCLs provide a useful insight into the impact of intracellular waste accumulation (liposfuscin) on cellular function. All NCL forms share the ubiquitous accumulation of lipofuscin in non-mitotic cell types such as retinal neurons and the RPE as a pathomorphological hallmark. The fundamental aim of this study was therefore to investigate the effects of lipofuscin accumulation in the naturally occurring mouse model of Battens disease, CLN6nclf, to determine the mechanisms of photoreceptor death. The time course of retinal degeneration in CLN6nclf mice was carefully characterised and correlated with changes in the intracellular waste recycling system, the autophagylysosomal pathway, in the RPE and retinal neurons. In addition, a treatment targeting the RPE called nanosecond laser therapy, which has been shown to reduce waste accumulation in the RPE in patients with AMD, was tested. Fluorescence immunohistochemistry, gross histology and twin flash electroretinography (ERG), revealed an early loss of rod photoreceptor function prior to cellular death, despite lipofuscin accumulation in all retinal cell types including the RPE. The role of the autophagy-lysosome pathway was assessed in individual retinal layers and the RPE using a novel technique for the quantification of autophagosomes and lysosomes. The results showed that rod photoreceptor death does not occur due to RPE dysfunction. Instead, rod photoreceptors showed early changes in autophagosomal-lysosomal fusion events and subsequent failure of lysosomal degradation, which resulted in specific photoreceptor dysfunction and death. Nanosecond laser treatment did not alter retinal function or retinal layer thickness in the CLN6nclf mice, suggesting that treatments targeting the photoreceptors rather than the RPE may be indicated for treatment of vision loss in NCLs. In addition to lipofuscin accumulation, changes in bio-metal levels and their intracellular distribution have also been suggested to contribute to the progression of neuronal degeneration in the CLN6 variant and retinal diseases such as AMD. However, whether changes in bio-metal distribution and concentration occur in the retina and RPE of CLN6nclf mice was unknown. To investigate this, retinal sections of CLN6nclf and control mice were assessed for bio-metal concentration using X-ray fluorescence microscopy (XFM) at the Australian Synchrotron. There were developmental changes in metal distribution and concentration in the control mouse retina with age, but no changes in metal concentration in any retinal layer or the RPE of the CLN6nclf mouse when compared to the controls. CLN6nclf mice were also treated with ZnII(atsm) and CuII(atsm) metallocomplexes that have been shown to be beneficial in different CLN6 tissues. Following oral treatment of control and CLN6nclf mice with the metallo-complexes, gross histology was used to assess individual retinal layer thickness. Treatment with zinc or copper metallo-complexes or the vehicle they were prepared in resulted in a reduction of all retinal layers compared to untreated controls, suggesting alternative treatments with improved vehicle formulations are required. Taken together these findings provide detailed evidence for the specific failure of the autophagy-lysosomal pathway, leading to an early, primary loss of rod photoreceptors in CLN6nclf mice. The study also indicates that changes in bio-metals are unlikely to contribute to retinal degeneration in CLN6nclf mice and that future treatments need to be specifically tailored to enhancing removal of intracellular waste in the photoreceptors.