Browsing by Author "Morgan, Autumn"
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Item Assessing Metabolic Changes in the Retina & Optic Nerve During Glaucoma(2024-03-21) Sepke, Katelynn; Morgan, Autumn; Inman, DenisePurpose: Glaucoma is an optic neuropathy characterized by retinal ganglion cell (RGC) death and optic nerve degeneration. Glial cells such as astrocytes form a metabolic unit with neurons to exchange metabolic substrates and neurotransmitters. When exposed to ocular hypertension (OHT), this metabolic unit is disrupted as astrocytes undergo morphological changes in response to increased pressure. ONHAs also reduce their GLUT1 expression, further exacerbating their metabolic function. It is unknown how these changes impact RGC axon structure and function, so we aim to gain insight into the metabolic relationship between glia and neurons during glaucoma. We hypothesize that glaucoma induces metabolic strain in optic nerve head astrocytes (ONHAs), preventing the exchange of metabolites between neurons, ultimately causing a decline in RGC structure and function. Methods: We have taken a two-sided approach to studying these neural-glial interactions. First, we have induced OHT as well as glucose transport inhibition in ONHAs in vivo to examine the effect of pressure-induced stress on metabolism and the visual system. Currently, we are working in vitro to study the metabolic exchange between RGCs and ONHAs co-cultured in microfluidic chambers when the ONHAs are exposed to biaxial strain as well as GLUT1 KO. Results: Preliminary results in vivo have shown that OHT and glucose transport inhibition in ONHAs disrupt anterograde transport. However, RGCs can compensate for glucose transport inhibition in astrocytes by upregulating GLUT3 and MCT2.In vitro we expect to see RGCs respond to alterations in ONHA metabolism, similarly, upregulating their lactate transporters and relying on mitochondrial metabolism to maintain their energetic needs. Conclusion: Using this model will allow us to directly observe the metabolic changes in the neural-glial unit induced by glaucoma, ultimately providing us insight into targets for future glaucoma therapies.Item Can Nicotinamide Treatment Overcome the Effect of Monocarboxylate Transporter 2 Loss on Retinal Ganglion Cell Survival and Function? dm(2024-03-21) Murinda, Kudakwashe; Inman, Denise; Kiehlbauch, Charles; Morgan, AutumnPurpose: There is currently no cure for the vision loss in glaucoma that is characterized by retinal ganglion cell (RGC) loss and irreversible optic neuropathy. Monocarboxylate transporter 2 (MCT2), which transports pyruvate, lactate, and ketone bodies, is exclusively found in neurons such as the RGCs. We have previously shown that MCT2 is lost during glaucoma, in advance of RGC loss, and MCT2 overexpression protects RGC number and function. We sought to determine if MCT2 is necessary for RGC survival by knocking it out, and to establish whether providing oral nicotinamide (NAM) could compensate for the anticipated metabolic disruption to RGCs. Methods: To test these hypotheses, we injected tamoxifen into Thy1-ERT2-cre: MCT2fl/fl mice to conditionally knock out MCT2 from Thy1-positive RGCs. Control mice carried the MCT2 flox’d allele but were Thy1-ERT2-cre-negative. Control and experimental mice were subjected to ocular hypertension using the magnetic microbead model; separate naïve controls from each genotype were also evaluated. To test the effect of nicotinamide intervention, we repeated the same groups but added the administration of oral nicotinamide to each before inducing ocular hypertension. Intraocular pressure (IOP) was measured using the TonoLab rebound tonometer. Pattern electroretinogram (PERG) and Visual Evoked Potential (VEP) were used to analyze the RGC function. We used unbiased stereology (Stereo Investigator, Micro Brightfield) to count the number of retinal ganglion cells in the wholemount retina, and ATP levels in the retina were also measured. Axon counts were done from plastic-embedded optic nerves. Results: IOP was higher in the ocular hypertension (OHT) groups. MCT2 knockout alone did not impact IOP, nor did it exacerbate RGC function loss post-OHT. After OHT, PERG amplitude was significantly lower in the OHT and KO + OHT treatment groups (p<0.005). RGC function was preserved in the KO + NAM and OHT+NAM groups but was significantly decreased in the KO+OHT group. After OHT, MCT2 KO alone did not alter RGC density but OHT and KO + OHT groups had significantly decreased RGC density (p<0.005). There was no significant decline in RGC density in any of the nicotinamide groups. ATP production in the KO + OHT group was significantly higher (1.81 +/- 0.89 µg/µl) than in the naïve control group (0.68 +/- 0.42 µg/µl). Conclusions: MCT2 knockout alone from RGCs did not change IOP, RGC density, or PERG, suggesting that MCT2 is not necessary for RGC function and survival. Ocular hypertension decreased PERG amplitude and RGC density, and the magnitude of the decrease was not significantly worsened by MCT2 knockout. The nicotinamide groups had no significant loss in RGC density, supporting the proposed neuroprotective effect of NAM administration. These data suggest that RGCs can meet their immediate metabolic needs through means beyond MCT2, and nicotinamide can rescue RGCs in the context of glaucoma.Item Effect of Monocarboxylate Transporter 2 Loss on Retinal Ganglion Cell Survival and Function(2023) Murinda, Kudakwashe; Morgan, Autumn; Inman, Denise; Kiehlbauch, CharlesPurpose: There is currently no cure for the vision loss in glaucoma that is characterized by retinal ganglion cell (RGC) loss and irreversible optic neuropathy. Monocarboxylate transporter 2 (MCT2s) that transport pyruvate, lactate, and ketone bodies, are exclusively found in neurons such as the RGCs. We have previously shown that MCT2 is lost during glaucoma, in advance of RGC loss, and MCT2 overexpression protects RGC number and function. This study was undertaken to test whether MCT2s are necessary for RGC survival and function. Methods: To test this hypothesis, we used tamoxifen injection into Thy1-ERT2-cre: MCT2fl/fl mice to conditionally knock out MCT2 from Thy1-positive RGCs. Control mice carried the MCT2 flox’d allele but were Thy1-ERT2-cre-negative. Control and experimental mice were subjected to ocular hypertension using the magnetic microbead model; separate naïve controls from each genotype were also evaluated. Intraocular pressure (IOP) was measured using the TonoLab rebound tonometer. Pattern electroretinogram (PERG) was used to analyze RGC function. We used unbiased stereology (Stereo Investigator, Micro Brightfield) to count the number of retinal ganglion cells in wholemount retina, and ATP levels in retina were also measured. Results: IOP was higher in the ocular hypertension (OHT) groups. MCT2 knockout alone did not impact IOP, nor did it alter baseline PERG amplitude or latency. After OHT, PERG amplitude was significantly lower in the MCT2-knockout mice (p=0.0013). MCT2 knockout alone did not change RGC density. After OHT, RGC density decreased, though in this preliminary analysis, RGC density among the groups was not significantly different. ATP production in the OHT+ Tamoxifen group was significantly higher (1.81 +/- 0.89 ug/ul) than in the naïve control group (0.68 +/- 0.42 ug/ul). Conclusions: MCT2 knockout from RGCs did not change IOP or PERG, suggesting that MCT2 is not necessary for RGC survival. Ocular hypertension decreased PERG amplitude and RGC density, though the magnitude of the decrease may not have been increased by MCT2 knockout. These preliminary data suggest that RGCs are capable of meeting their immediate metabolic needs through means beyond MCT2.Item Ketogenic Diet Increases Mitophagy in a Mouse Model of Glaucoma(2023) Morgan, Autumn; Fan, Yan; Inman, DenisePurpose: We have previously shown that limiting dietary intake to high fat, low protein, and negligible carbohydrate results in mitochondrial biogenesis, and in the case of glaucoma, a reduction in neurodegeneration of retinal ganglion cells (RGCs). In this experimental follow-up study, we wanted to examine the effect of the ketogenic diet on mitophagy, or mitochondrial recycling, within the glaucomatous retina. Methods: MitoQC mice were placed on a ketogenic diet or standard rodent chow for 5 weeks and ocular hypertension (OHT) was induced via microbead injection. The MitoQC reporter mice have a pH-sensitive mCherry-GFP tag on the outer mitochondrial membrane that results in retention of red fluorescence when mitochondria bound for recycling are engulfed by lysosomes. The FIJI (ImageJ) macro MitoQC counter was used to quantify red puncta (mitolysosomes) in sectioned retina as a measure of mitophagy within the RGCs and Müller glia. Results: Mitophagy in RGCs, as measured by red puncta, was significantly decreased by ocular hypertension in the control retina (Control + OHT) in comparison to naïve control retina (Ctrl; p<0.0001). The ketogenic diet (KD) resulted in a significant increase in mitolysosomes in RGCs when compared to Ctrl (p<0.0001), Control + OHT (p<0.0001) and KD + OHT (p=0.0089). The ketogenic mice with OHT showed a significantly higher RGC-associated mitolysosome number than Control + OHT mice (p<0.0001). In contrast, mitolysosomes quantified in the Müller glia of Control + OHT mice were significantly higher than the naïve control mice (p=0.0127). Mice in the KD (p=0.0001) and KD + OHT(p=0.0005) groups had significantly greater mitolysosomes than the control Müller glia, however there was no difference in mitophagy between the Control + OHT, KD, and KD + OHT Müller glia groups. Conclusion: Our data demonstrates that mitophagy is managed differently within RGCs and Müller glia of mouse retinas. The KD promoted mitophagy within the RGCs to a degree that overcame the decline of mitophagy after OHT in the control group. Within the Müller glia, the KD was redundant because OHT alone increased mitophagy to similar levels as the KD. These findings suggest a divergence of mitochondrial homeostasis in RGCs and Müller glia that may reflect the different metabolic needs of these cell types.