Browsing by keyword "Cone, David"
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Activation of mTORC1 Improves Cone Cell Metabolism and Extends Vision in Retinitis Pigmentosa Mice: A DissertationRetinitis Pigmentosa (RP) is an inherited photoreceptor degenerative disease that leads to blindness and affects about 1 in 4000 people worldwide. The disease is predominantly caused by mutations in genes expressed exclusively in the night active rod photoreceptors; however, blindness results from the secondary loss of the day active cone photoreceptors, the mechanism of which remains elusive. Here, we show that the mammalian target of rapamycin complex 1 (mTORC1) is required to delay the progression of cone death during disease and that constitutive activation of mTORC1 is sufficient to maintain cone function and promote cone survival in RP. Activation of mTORC1 increased expression of genes that promote glucose uptake, retention and utilization, leading to increased NADPH levels; a key metabolite for cones. This protective effect was conserved in two mouse models of RP, indicating that the secondary loss of cones can be delayed by an approach that is independent of the primary mutation in rods. However, since mTORC1 is a negative regulator of autophagy, its constitutive activation led to an unwarranted secondary effect of shortage of amino acids due to incomplete digestion of autophagic cargo, which reduces the efficiency of cone survival over time. Moderate activation of mTORC1, which promotes expression of glycolytic genes, as well as maintains autophagy, provided more sustained cone survival. Together, our work addresses a long-standing question of non-autonomous cone death in RP and presents a novel, mutation-independent approach to extend vision in a disease that remains incurable.
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Human gene therapy for RPE65 isomerase deficiency activates the retinoid cycle of vision but with slow rod kineticsThe RPE65 gene encodes the isomerase of the retinoid cycle, the enzymatic pathway that underlies mammalian vision. Mutations in RPE65 disrupt the retinoid cycle and cause a congenital human blindness known as Leber congenital amaurosis (LCA). We used adeno-associated virus-2-based RPE65 gene replacement therapy to treat three young adults with RPE65-LCA and measured their vision before and up to 90 days after the intervention. All three patients showed a statistically significant increase in visual sensitivity at 30 days after treatment localized to retinal areas that had received the vector. There were no changes in the effect between 30 and 90 days. Both cone- and rod-photoreceptor-based vision could be demonstrated in treated areas. For cones, there were increases of up to 1.7 log units (i.e., 50 fold); and for rods, there were gains of up to 4.8 log units (i.e., 63,000 fold). To assess what fraction of full vision potential was restored by gene therapy, we related the degree of light sensitivity to the level of remaining photoreceptors within the treatment area. We found that the intervention could overcome nearly all of the loss of light sensitivity resulting from the biochemical blockade. However, this reconstituted retinoid cycle was not completely normal. Resensitization kinetics of the newly treated rods were remarkably slow and required 8 h or more for the attainment of full sensitivity, compared with <1 h in normal eyes. Cone-sensitivity recovery time was rapid. These results demonstrate>dramatic, albeit imperfect, recovery of rod- and cone-photoreceptor-based vision after RPE65 gene therapy.
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Loss of mTOR signaling affects cone function, cone structure and expression of cone specific proteins without affecting cone survivalCones are the primary photoreceptor (PR) cells responsible for vision in humans. They are metabolically highly active requiring phosphoinositide 3-kinase (PI3K) activity for long-term survival. One of the downstream targets of PI3K is the kinase mammalian target of rapamycin (mTOR), which is a key regulator of cell metabolism and growth, integrating nutrient availability and growth factor signals. Both PI3K and mTOR are part of the insulin/mTOR signaling pathway, however if mTOR is required for long-term PR survival remains unknown. This is of particular interest since deregulation of this pathway in diabetes results in reduced PR function before the onset of any clinical signs of diabetic retinopathy. mTOR is found in two distinct complexes (mTORC1 and mTORC2) that are characterized by their unique accessory proteins RAPTOR and RICTOR respectively. mTORC1 regulates mainly cell metabolism in response to nutrient availability and growth factor signals, while mTORC2 regulates pro-survival mechanisms in response to growth factors. Here we analyze the effect on cones of loss of mTORC1, mTORC2 and simultaneous loss of mTORC1 and mTORC2. Interestingly, neither loss of mTORC1 nor mTORC2 affects cone function or survival at one year of age. However, outer and inner segment morphology is affected upon loss of either complex. In contrast, concurrent loss of mTORC1 and mTORC2 leads to a reduction in cone function without affecting cone viability. The data indicates that PI3K mediated pro-survival signals diverge upstream of both mTOR complexes in cones, suggesting that they are independent of mTOR activity. Furthermore, the data may help explain why PR function is reduced in diabetes, which can lead to deregulation of both mTOR complexes simultaneously. Finally, although mTOR is a key regulator of cell metabolism, and PRs are metabolically highly active, the data suggests that the role of mTOR in regulating the metabolic transcriptome in healthy cones is minimal.
