Impact of lipofuscin accumulation on retinal degeneration in the mouse model of the Neuronal Ceroid Lipofuscinosis variant CLN6
AffiliationAnatomy and Neuroscience
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2020-06-04.
© 2018 Dr. Philipp Johannes von Eisenhart-Rothe
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.
Keywordslipofuscin; vision; autophagy; photoreceptor; retinal degeneration; neuronal ceroid lipofuscinosis; bio-metal
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