Date of Award

4-18-2024

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Developmental, Regenerative, & Stem Cell Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Over 250 million people globally live with visual acuity loss or blindness as an estimated 7 million people suffer from visual acuity loss in the United States. People in the United States value vision over memory, hearing, or speech and consider losing vision to be among the top four worst outcome to happen to them. The economic cost of major vision problems is estimated to increase to 373 billion dollars by the year 2050. Among the irreparable causes of vision loss, retinal degeneration stands as a chief contributor to catastrophic eye disease. The retina is a neurosensory tissue located in the back of the eye as part of the central nervous system. Made up of highly specialized neurons and support cells, the retina is the most metabolically active tissue per weight in the human body. Since these neurons are non-proliferative, the cells we have post-differentiation are the ones that we will keep through the entirety of our lifetime. It is thus critical to understand how these neurons function and are supported to best develop therapies and therapeutics to help them function properly for a lifetime. In the vast majority of retinal degeneration cases, the photoreceptor layer is the first affected layer that directly causes vision loss in patients. The photoreceptor layer in mammals is made up of both rod and cone photoreceptors, with an overwhelming majority made up of rod photoreceptors. In many cases, rod photoreceptors undergo degeneration first which leads to the subsequent loss of cone photoreceptors and visual acuity due to the lack of support from rod photoreceptors. Therefore, there is clear rationale in studying how rod photoreceptors survive and degenerate in order to understand how best to prevent a critical step in the pathogenesis of many forms of retinal degeneration. Rod photoreceptors are, as a cell type, the most energetically demanding in terms of ATP consumption. AMP-activated protein kinase (AMPK) plays a crucial role in maintaining ATP homeostasis in photoreceptor neurons. AMPK is a heterotrimeric protein consisting of alpha, beta, and gamma subunits. The independent functions of the two isoforms of the catalytic alpha subunit, PRKAA1 and PRKAA2, are uncharacterized in specialized neurons such as photoreceptors. In the following chapter, we demonstrate in mice that rod photoreceptors lacking PRKAA2, but not PRKAA1, show altered levels of cGMP, GTP, and ATP, suggesting isoform-specific regulation of photoreceptor metabolism. Furthermore, PRKAA2 deficient mice display visual functional deficits on electroretinography and photoreceptor outer segment structural abnormalities on transmission electron microscopy consistent with neuronal dysfunction, but not neurodegeneration. Phosphoproteomics identified inosine monophosphate dehydrogenase (IMPDH) as a molecular driver of PRKAA2-specific photoreceptor dysfunction, and inhibition of IMPDH improved visual function in Prkaa2 rod photoreceptor knockout mice. These findings highlight a novel, therapeutically targetable PRKAA2 isoform-specific function of AMPK in regulating photoreceptor metabolism and function through a previously uncharacterized mechanism affecting IMPDH activity. Related to ATP metabolism, NAD+ metabolism has been shown to be a critical regulator of neuronal function, particularly in rod photoreceptors. Later in this dissertation, we detail a study that investigated a molecular pathway that rescues a phenotype caused by dysregulation of NAD+ metabolism. Leber congenital amaurosis type nine is an autosomal recessive retinopathy caused by mutations of the NAD+ synthesis enzyme NMNAT1. Despite the ubiquitous expression of NMNAT1, patients do not manifest pathologies other than retinal degeneration. We demonstrate that widespread NMNAT1 depletion in adult mice mirrors the human pathology, with selective loss of photoreceptors highlighting the exquisite vulnerability of these cells to NMNAT1 loss. Conditional deletion demonstrates that NMNAT1 is required within the photoreceptor. Mechanistically, loss of NMNAT1 activates the NADase SARM1, the central executioner of axon degeneration, to trigger photoreceptor death and vision loss. Hence, the essential function of NMNAT1 in photoreceptors is to inhibit SARM1, highlighting an unexpected shared mechanism between axonal degeneration and photoreceptor neurodegeneration. These results define a novel SARM1-dependent photoreceptor cell death pathway and identifies SARM1 as a therapeutic candidate for retinopathies. Advanced age-related macular degeneration (AMD), the leading cause of blindness among people over 50 years of age, is another form of retinal degeneration characterized by atrophic neurodegeneration of photoreceptors or pathologic angiogenesis. Early AMD is characterized by extracellular cholesterol-rich deposits underneath the retinal pigment epithelium (RPE) called drusen or in the subretinal space called subretinal drusenoid deposits (SDD) that drive disease progression. However, mechanisms of drusen and SDD biogenesis remain poorly understood. Although human AMD is characterized by abnormalities in cholesterol homeostasis and shares phenotypic features with atherosclerosis, it is unclear whether systemic immunity or local tissue metabolism regulates this homeostasis. Here, we demonstrate that targeted deletion of macrophage cholesterol ABC transporters A1 (ABCA1) and -G1 (ABCG1) leads to age-associated extracellular cholesterol-rich deposits underneath the neurosensory retina similar to SDD seen in early human AMD. These mice also develop impaired dark adaptation, a cardinal feature of RPE cell dysfunction seen in human AMD patients even before central vision is affected. Subretinal deposits in these mice progressively worsen with age, with concomitant accumulation of cholesterol metabolites including several oxysterols and cholesterol esters causing lipotoxicity that manifests as photoreceptor dysfunction and neurodegeneration. These findings suggest that impaired macrophage cholesterol transport initiates several key elements of early human AMD, demonstrating the importance of systemic immunity and aging in promoting disease manifestation. Polymorphisms in genes involved with cholesterol transport and homeostasis are associated with a significantly higher risk of developing AMD, thus making these studies translationally relevant by identifying potential targets for therapy. Furthermore, we investigated the role of ABCA1 and –G1 in rod photoreceptors. Photoreceptors have high intrinsic metabolic demand and are exquisitely sensitive to metabolic perturbation. In addition, they shed a large portion of their outer segment lipid membranes in a circadian manner, increasing the metabolic burden on the outer retina associated with the resynthesis of cell membranes and disposal of the cellular cargo. Here, we demonstrate that deletion of both ABCA1 and ABCG1 in rod photoreceptors leads to age-related accumulation of cholesterol metabolites in the outer retina, photoreceptor dysfunction, degeneration of rod outer segments, and ultimately blindness. A high-fat diet significantly accelerates rod neurodegeneration and vision loss, further highlighting the role of lipid homeostasis in regulating photoreceptor neurodegeneration and vision. Lastly, we sought to investigate novel therapeutic pathways that could ameliorate the aforementioned phenotype that predisposes patients to advanced AMD. Cholesterol efflux is the first step in reverse cholesterol transport, and involves the efflux of lipid to apolipoprotein A-I, the main protein constituent of high-density lipoprotein (HDL). However, whether other functions of HDL are involved in the development of AMD is unknown. We tested the novel hypothesis that apolipoprotein M (ApoM) plays an essential role in AMD pathogenesis. ApoM binds a bioactive lipid called spinghosine-1-phosphate (S1P) to regulate diverse processes including inflammation, fibrosis, cell death, and cholesterol efflux. We have discovered that circulating ApoM protein levels are reduced in patients with AMD. Delivery of ApoM-enriched plasma attenuates the development of AMD-like features in mice, but not ApoM knockout plasma or ApoM that cannot bind to S1P does not, suggesting a critical role for the ApoM-S1P interaction in AMD pathogenesis. Knockout of S1P receptor 3 is also sufficient to recapitulate AMD-like phenotypes in mice. Transmission electron microscopy shows that S1P receptor 3 knockout mice exhibit increased lipid droplets in retinal pigment epithelial cells, just like mice with double knockout of cholesterol efflux pumps. Meanwhile, treatment with ApoM decreases the number of lipid droplets, while increasing the numbers of melanosomes. These observations led to the further hypothesis that ApoM and S1P receptor 3 may be stimulating lipophagy, the process of lipid catabolism in the melanosomes of the retinal pigment epithelium. Accordingly, knockout of the key lipophagy enzyme lysosomal acid lipase phenocopied the AMD-like phenotype. Our study revealed an exciting and new therapeutic avenue in the treatment of early AMD where there is a paucity of aggressive therapies available. Taken together, our collective and collaborative work illuminates the delicate nuances required for rod photoreceptor metabolism and even proper function from supporting cells for rod photoreceptor function and survival. Future and ongoing work warrants perspicacious consideration of cell-autonomous effects of metabolism in rod photoreceptors as well as the non-cell-autonomous effects from surrounding cells to elucidate therapeutic pathways for rod photoreceptor survival.

Language

English (en)

Chair and Committee

Rajendra Apte

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