Activation of mTORC1 Improves Cone Cell Metabolism and Extends Vision in Retinitis Pigmentosa Mice: A Dissertation
Authors
Venkatesh, AdityaFaculty Advisor
Claudio Punzo, PhDAcademic Program
Interdisciplinary Graduate ProgramUMass Chan Affiliations
OphthalmologyDocument Type
Doctoral DissertationPublication Date
2016-04-12Keywords
Dissertations, UMMSAutophagy
Retinal Cone Photoreceptor Cells
Retinal Rod Photoreceptor Cells
Retinitis Pigmentosa
TOR Serine-Threonine Kinases
Autophagy
Retinal Cone Photoreceptor Cells
Retinal Rod Photoreceptor Cells
Retinitis Pigmentosa
TOR Serine-Threonine Kinases
Cellular and Molecular Physiology
Eye Diseases
Ophthalmology
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Show full item recordAbstract
Retinitis 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.DOI
10.13028/M2NC7GPermanent Link to this Item
http://hdl.handle.net/20.500.14038/32193Rights
Copyright is held by the author, with all rights reserved.ae974a485f413a2113503eed53cd6c53
10.13028/M2NC7G
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Activated mTORC1 promotes long-term cone survival in retinitis pigmentosa miceVenkatesh, Aditya; Ma, Shan; Le, Yun Z.; Hall, Michael N.; Ruegg, Markus A.; Punzo, Claudio (2015-04-01)Retinitis pigmentosa (RP) is an inherited photoreceptor degenerative disorder that results in blindness. The disease is often caused by mutations in genes that are specific to rod photoreceptors; however, blindness results from the secondary loss of cones by a still unknown mechanism. Here, we demonstrated that the mammalian target of rapamycin complex 1 (mTORC1) is required to slow the progression of cone death during disease and that constitutive activation of mTORC1 in cones is sufficient to maintain cone function and promote long-term cone survival. Activation of mTORC1 in cones enhanced glucose uptake, retention, and utilization, leading to increased levels of the key metabolite NADPH. Moreover, cone death was delayed in the absence of the NADPH-sensitive cell death protease caspase 2, supporting the contribution of reduced NADPH in promoting cone death. Constitutive activation of mTORC1 preserved cones in 2 mouse models of RP, suggesting that the secondary loss of cones is caused mainly by metabolic deficits and is independent of a specific rod-associated mutation. Together, the results of this study address a longstanding question in the field and suggest that activating mTORC1 in cones has therapeutic potential to prolong vision in RP.
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Loss of mTOR signaling affects cone function, cone structure and expression of cone specific proteins without affecting cone survivalMa, Shan; Venkatesh, Aditya; Langellotto, Fernanda; Le, Yun Z.; Hall, Michael N.; Ruegg, Markus A.; Punzo, Claudio (2015-06-01)Cones 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.
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Magnetic Resonance Imaging of the Rat Retina: a DissertationBhagavatheeshwaran, Govind (2008-03-04)The retina is a thin layer of tissue lining the back of the eye and is primarily responsible for sight in vertebrates. The neural retina has a distinct layered structure with three dense nuclear layers, separated by plexiform layers comprising of axons and dendrites, and a layer of photoreceptor segments. The retinal and choroidal vasculatures nourish the retina from either side, with an avascular layer comprised largely of photoreceptor cells. Diseases that directly affect the neural retina like retinal degeneration as well as those of vascular origin like diabetic retinopathy can lead to partial or total blindness. Early detection of these diseases can potentially pave the way for a timely intervention and improve patient prognosis. Current techniques of retinal imaging rely mainly on optical techniques, which have limited depth resolution and depend mainly on the clarity of visual pathway. Magnetic resonance imaging is a versatile tool that has long been used for anatomical and functional imaging in humans and animals, and can potentially be used for retinal imaging without the limitations of optical methods. The work reported in this thesis involves the development of high resolution magnetic resonance imaging techniques for anatomical and functional imaging of the retina in rats. The rats were anesthetized using isoflurane, mechanically ventilated and paralyzed using pancuronium bromide to reduce eye motion during retinal MRI. The retina was imaged using a small, single-turn surface coil placed directly over the eye. The several physiological parameters, like rectal temperature, fraction of inspired oxygen, end-tidal CO2, were continuously monitored in all rats. MRI parameters like T1, T2, and the apparent diffusion coefficient of water molecules were determined from the rat retina at high spatial resolution and found to be similar to those obtained from the brain at the same field strength. High-resolution MRI of the retina detected the three layers in wild-type rats, which were identified as the retinal vasculature, the avascular layer and the choroidal vasculature. Anatomical MRI performed 24 hours post intravitreal injection of MnCl2, an MRI contrast agent, revealed seven distinct layers within the retina. These layers were identified as the various nuclear and plexiform layers, the photoreceptor segment layer and the choroidal vasculature using Mn54Cl2emulsion autoradiography. Blood-oxygenlevel dependent (BOLD) functional MRI (fMRI) revealed layer-specific vascular responses to hyperoxic and hypercapnic challenges. Relative blood volume of the retina calculated by using microcrystalline iron oxide nano-colloid, an intravascular contrast agent, revealed a superfluous choroidal vasculature. Fractional changes to blood volume during systemic challenges revealed a higher degree of autoregulation in the retinal vasculature compared to the choroidal vasculature, corroborating the BOLD fMRI data. Finally, the retinal MRI techniques developed were applied to detect structural and vascular changes in a rat model of retinal dystrophy. We conclude that retinal MRI is a powerful investigative tool to resolve layerspecific structure and function in the retina and to probe for changes in retinal diseases. We expect the anatomical and functional retinal MRI techniques developed herein to contribute towards the early detection of diseases and longitudinal evaluation of treatment options without interference from overlying tissue or opacity of the visual pathway.