Visual deficits stemming from retinal dysfunction are associated with Alzheimer?s Disease (AD). As a result, assessment of retinal dysfunction may be a specific and non-invasive diagnostic tool of AD at the earliest stages of pathology (Mahajan & Votruba, 2017). Two hallmarks of AD are accumulation of senile plaques, composed mainly of amyloid-? (A?), and neurofibrillary tangles (NFT), composed of hyperphosphorylated tau protein. In the retina of AD patients, the presence of senile plaques and neurofibrillary tangles is limited, but there is extensive hyperphosphorylated tau throughout the inner and outer plexiform layers (den Haan et al., 2018). We speculate that this hyperphosphorylated tau, alters glucose metabolism and oxidative stress, leading to disruption of signaling in the retina and disease progression. Indeed, metabolic diseases, diabetes and glaucoma that is not linked with intraocular pressure, are associated with AD (Mancino et al., 2018). Hence, altered metabolism may be a critical component of retinal dysfunction in AD. Still, the mechanisms underlying aging and AD related retinal problems are poorly defined, which limits development of therapies . Mitochondrial dysfunction is a critical component of aging and age-related neurodegeneration including AD. In the Parent Grant (see Specific Aims on subsequent page), we are studying the structure and dynamics of the synaptic ribbon in retinal cells, mainly bipolar cells and photoreceptors using ?flash- freeze? cryofixation electron (slam-freeze cryo-EM) microscopy, confocal microscopy with electrophysiology, immunohistochemistry, and measuring oculomotor responses. This work is mainly being done in zebrafish. In the present Supplement, we want to extend our experiments to study how glucose metabolism and oxidative stress ? elements associated with AD ? impact synaptic transmission in the retina focusing on mitochondria including mitophagy and structure and dynamics of the synaptic ribbon in retinal cells, mainly bipolar cells and photoreceptors. We will essentially overlay the question of glucose metabolism and oxidative stress on our on-going experiments. Critically, we will bring our highly refined and quantitative approach to address the role of metabolism in retinal function. In Objective 1, we will address how variations in glucose metabolism and oxidative stress impact the distribution and structure of mitochondria and ribbon synapses using slam-freeze cryo-EM microscopy (Mses. Henry-Vanisko & Akmentin & Dr. Joselevitch). This technique is central to the parent grant and will allow us to exam the impact of glucose metabolism and oxidative in quantitative detail. In Objective 2, we will address how NMDA receptors contribute to cell viability under conditions of altered glucose metabolism and oxidative stress in the retina. NMDA receptors play critical roles in cell health in the retina and are strongly linked to the neurodegeneration in AD (Wang & Reddy, 2017), and a putative treatment (neurosteroids) for glaucoma and AD act through NMDA receptors (Ishikawa et al., 2018). For these experiments, we will take advantage of NMDA receptor knock-outs we have generated to study NMDA receptor subunits in the retina and the visual system as a whole (Mr. Zoodsma & Drs. Sirotkin & Joselevitch). Finally, in Objective 3, we will obtain fish lines that express various markers for mitochondria (e.g., express TMRE which changes its fluorescence spectrum based on oxidation state)(Mandal et al., 2018)(Drs. Sirotkin & Joselevitch). In the long term, these tools will allow us to ultimately expand our studies to whole animals under chronic conditions. Our major goal with the Supplement would be to establish the feasibility of our overall approach to address glucose metabolism, oxidative stress, mitochondrial dynamics, mitophagy, and ribbon synapse structure/dynamics in the retina. If we are successful, we would develop a new research program, in collaboration with Dr. Howard Sirotkin, to continue to study glucose metabolism and oxidative stress in the retina. We would envision submitting a new RO1, possibly independent of the Parent Grant, either to NEI or NIA.
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