Journal: Neurobiology of disease

Article Title: MCT2 Overexpression Rescues Metabolic Vulnerability and Protects Retinal Ganglion Cells in Two Models of Glaucoma

doi: 10.1016/j.nbd.2020.104944

Figure Lengend Snippet: Indices for DBA/2J Mice on the Ketogenic or Control Diets

Article Snippet: An AAV2.cre.GFP (1.0×10 13 GC/mL; Vector Biolabs, Malvern, PA) was injected into MCT2 fl/fl mouse eyes to remove the flox’d MCT2 gene through cre-mediated recombination, thereby eliminating MCT2 in the infected retinal cells.

Techniques:

Journal: Neurobiology of disease

Article Title: MCT2 Overexpression Rescues Metabolic Vulnerability and Protects Retinal Ganglion Cells in Two Models of Glaucoma

doi: 10.1016/j.nbd.2020.104944

Figure Lengend Snippet: Visual evoked potential (VEP) and optic nerve (ON) metabolism in MCT2fl/+ mice. A. MCT2fl/+ mouse eyes injected with AAV2-cre-GFP were imaged to show expression of GFP in the ganglion cell layer two weeks after infection. Successful cre recombination removes one MCT2 allele from infected cells. Scale bar= 200μm. B. VEP response amplitude was significantly reduced in MCT2fl/+ mice (n=10; p<0.01) compared to age-matched C56Bl/6 (B6) control mice (n=10). C. Representative VEP traces in B6 control (left) and MCT2fl/+ (right) mice. Response amplitude is the μV difference between the N1 trough and the P2 peak. D. The difference in baseline versus oligomycin-treatment oxygen consumption rate (OCR) calculated from the ONs of B6 control (n=3) or MCT2fl/+ mice (n=9) were significantly different for the ONs incubated in glucose (Glc; p<0.1) and 2-deoxyglucose (2-DG; p<0.001). E. Extracellular acidification rate (ECAR) was similar for B6 control (n=6) and MCT2fl/+ (n=18) within the Glc or the 2-DG groups, but the 2-DG groups for each were significantly reduced compared to Glc (p<0.0001). Error bars are SD.

Article Snippet: An AAV2.cre.GFP (1.0×10 13 GC/mL; Vector Biolabs, Malvern, PA) was injected into MCT2 fl/fl mouse eyes to remove the flox’d MCT2 gene through cre-mediated recombination, thereby eliminating MCT2 in the infected retinal cells.

Techniques: Injection, Expressing, Infection, Incubation

Journal: Neurobiology of disease

Article Title: MCT2 Overexpression Rescues Metabolic Vulnerability and Protects Retinal Ganglion Cells in Two Models of Glaucoma

doi: 10.1016/j.nbd.2020.104944

Figure Lengend Snippet: Iba1 fluorescence and microglia density in peripheral retina from D2 mice injected with AAV2:MCT2. A-D. Photomicrographs showing the expression of Iba1 (red) in microglia by immunolabeling in retinas from D2 (A), D2G (B), D2 injected with AAV2:MCT2 (C), and D2 injected with AAV2:MCT2 mice on the ketogenic diet (KD) (D). Scale bar = 35μm. DAPI nuclear stain in blue. E. D2 mice injected with AAV2:MCT2 with or without ketogenic diet showed significantly higher microglia density than untreated D2 and D2G mice (p<0.0001); n=6 retinas per group and 2–3 sections per retina. Microglia were quantified at least 200μm from the optic nerve head. F. Untreated D2 mice had significantly higher Iba1 fluorescence than untreated D2G mice (p<0.001), but significantly lower Iba1 fluorescence than either AAV2:MCT2 injected D2 mice (p<0.001) or AAV2:MCT2 injected D2 mice on the ketogenic diet (p<0.05); an average of 30 sections were analyzed per group. Error bars are SEM.

Article Snippet: An AAV2.cre.GFP (1.0×10 13 GC/mL; Vector Biolabs, Malvern, PA) was injected into MCT2 fl/fl mouse eyes to remove the flox’d MCT2 gene through cre-mediated recombination, thereby eliminating MCT2 in the infected retinal cells.

Techniques: Fluorescence, Injection, Expressing, Immunolabeling, Staining

Journal: Neurobiology of disease

Article Title: MCT2 Overexpression Rescues Metabolic Vulnerability and Protects Retinal Ganglion Cells in Two Models of Glaucoma

doi: 10.1016/j.nbd.2020.104944

Figure Lengend Snippet: MCT2 protein distribution and AAV2:MCT2 virus infection. A. Immunofluorescence of control retina to show MCT2 localization on retinal ganglion cells. The left panel is a merged image of MCT2 (red) with Vimentin (Müller glia, blue) and NeuN (RGCs, green). In the middle panel, NeuN has been removed in order to discern the red puncta along both RGC somas and Müller glia endfeet. The right panel shows MCT2 alone. Scale bar is 15μM. B. A cross-section from a 12 month-old D2 retina injected with AAV2:MCT2 two months prior. Arrowheads point to an infected RGC in the GCL that is RBPMS-positive and positive for the eGFP from the AAV2:MCT2 virus. INL=inner nuclear layer; OPL=outer plexiform layer. Scale bar=15μm. C. Fundus photograph of a 10.5 month-old DBA/2J (D2) eye injected with AAV2:MCT2, two weeks after injection (left). The arrow points to GFP-positive cells in the ganglion cell layer (GCL). D. Section of a 12 month-old D2 optic nerve from an eye injected with AAV2:MCT2. The eGFP-positive axons confirm the infection of RGCs with the virus and the widespread transport of the eGFP. Scale bar=20μm. E. Sagittal section of a 12 month-old D2 retina injected with AAV2:MCT2 two months prior. Arrows point to infected cells in the GCL that are eGFP-positive from the AAV2:MCT2 virus. Infected cells are observed in the GCL and INL. Scale bar=50μm. GCL=ganglion cell layer; INL=inner nuclear layer; OPL=outer plexiform layer.

Article Snippet: An AAV2.cre.GFP (1.0×10 13 GC/mL; Vector Biolabs, Malvern, PA) was injected into MCT2 fl/fl mouse eyes to remove the flox’d MCT2 gene through cre-mediated recombination, thereby eliminating MCT2 in the infected retinal cells.

Techniques: Infection, Immunofluorescence, Injection

Journal: Neurobiology of disease

Article Title: MCT2 Overexpression Rescues Metabolic Vulnerability and Protects Retinal Ganglion Cells in Two Models of Glaucoma

doi: 10.1016/j.nbd.2020.104944

Figure Lengend Snippet: Retinal ganglion cell soma and axon quantification and integrity. A. Retinal ganglion cell density (cells/mm2) in 12 month-old D2 retina (n=16), age-matched control strain DBA/2-Gpnmb+ (D2G) retina (n=15), 12 month-old D2 retina injected with AAV2:MCT2 (n=13), and 12 month-old D2 retina injected with AAV2:MCT2 and placed on a ketogenic diet for 8 weeks (n=8). D2G and both virus injected groups have significantly greater RGC density than 12 month-old D2 (p<0.0001). B. Wholemount retina immunolabeled for RBPMS, an RGC-specific marker, from untreated D2 and D2G retina, and D2 mouse retina injected with AAV2:MCT2, with or without the ketogenic diet (KD). Images were taken 1mm from the optic nerve head. Scale bar=25μm. C. Cross-sections of ρ-phenylenediamine labeled optic nerve from untreated D2 and D2G, then D2 mice injected with AAV2:MCT2, with or without the ketogenic diet (KD). Scale bar=10μm. D. Optic nerve (ON) axon counts from D2 (n=14), D2G (n=5), and D2 injected with AAV2:MCT2, with (n=11) or without (n=13) the ketogenic diet. D2 axon number is significantly lower than D2G and AAV2:MCT2 D2 (p<0.0001). AAV2:MCT2 D2 mice on the ketogenic diet had significantly lower ON axon number than the AAV2:MCT2 D2 mice on normal rodent chow (p<0.05). Error bars are SD.

Article Snippet: An AAV2.cre.GFP (1.0×10 13 GC/mL; Vector Biolabs, Malvern, PA) was injected into MCT2 fl/fl mouse eyes to remove the flox’d MCT2 gene through cre-mediated recombination, thereby eliminating MCT2 in the infected retinal cells.

Techniques: Injection, Immunolabeling, Marker, Labeling

Journal: Neurobiology of disease

Article Title: MCT2 Overexpression Rescues Metabolic Vulnerability and Protects Retinal Ganglion Cells in Two Models of Glaucoma

doi: 10.1016/j.nbd.2020.104944

Figure Lengend Snippet: Protein and metabolic changes in optic nerve (ON) from D2 mice injected with AAV2:MCT2. A. MCT2 protein in ON from D2 (n=11), D2G mice (n=3), and D2 mice injected with AAV2:MCT2, with (n=7) or without (n=6) the ketogenic diet. Untreated D2 MCT2 protein is significantly lower (p<0.0001) than each of the other experimental groups depicted. B. MCT1 expression is higher in both untreated D2G (n=5) and AAV2:MCT2 injected D2 (n=6) than in untreated D2 (n=5) optic nerve (p<0.05). C. pAMPK/AMPK ratio is significantly higher in untreated D2 (n=6) than D2G (n=5) and AAV2:MCT2 injected D2 (n=6) mice (p<0.001). D. PGC-1α is upregulated in D2 mice injected with AAV2:MCT2 (n=5) compared to both untreated D2 (n=5) and D2G mice (n=6) (p<0.01). E. Hexokinase activity is significantly increased in AAV2:MCT2 injected D2 mice (n=4) compared to untreated D2 (n=4). Untreated D2G (n=4) and D2 mice injected with AAV2:MCT2 on the ketogenic diet (n=6) are not different from untreated D2 (p<0.05). F. Succinate dehydrogenase activity is significantly decreased in untreated D2 (n=32 sections from 8 ON) mouse ON compared to both untreated D2G (n=36 sections from 8 ON) and AAV2:MCT2 treated D2 mice on standard chow (control diet; n=16 sections from 5 ON), p<0.0001. For the AAV2:MCT2 injected D2 mouse on the ketogenic diet, 16 sections from 5 ON were analyzed. G. Succinate dehydrogenase histochemistry of example ONs from groups as shown in panel F. Scale bar=250μm. Error bars are SD.

Article Snippet: An AAV2.cre.GFP (1.0×10 13 GC/mL; Vector Biolabs, Malvern, PA) was injected into MCT2 fl/fl mouse eyes to remove the flox’d MCT2 gene through cre-mediated recombination, thereby eliminating MCT2 in the infected retinal cells.

Techniques: Injection, Expressing, Activity Assay

Journal: Neurobiology of disease

Article Title: MCT2 Overexpression Rescues Metabolic Vulnerability and Protects Retinal Ganglion Cells in Two Models of Glaucoma

doi: 10.1016/j.nbd.2020.104944

Figure Lengend Snippet: Retinal ganglion cell (RGC) and optic nerve (ON) axon integrity, and MCT2 protein levels in mice after ocular hypertension (OHT). A. Photomicrograph of AAV2:MCT2 infection in flatmount retina without OHT; AAV2:MCT2 virus infection reporter is eGFP (green), and RGC-specific label RBPMS (red). Scale bar = 50μm. B. Intraocular pressure (IOP) elevation in the three OHT experimental groups. Each OHT group IOP was significantly increased over baseline, p<0.0001, though not statistically different from each other. Error bars are SEM. C. RGC density is significantly increased by AAV2:MCT2 injection after OHT (n=11) compared to OHT alone (n=5; p<0.01). OHT alone resulted in significant RGC loss compared to control (no OHT and no AAV2, n=6; p<0.01). D. Axon number in the ON of OHT mice (n=6) is significantly decreased compared to control (No OHT, n=6; p<0.001), then rescued by injection with AAV2:MCT2 after OHT (n=10; p<0.001). E. MCT2 protein in retina is significantly higher in the OHT group injected with AAV2:MCT2 (n=4) compared to both control (No OHT, n=4; p<0.001) and OHT alone (n=4; p<0.0001). F. MCT2 protein in ON is significantly different across groups by one-way ANOVA (p<0.05); n=5 for control (No OHT, No AAV2), n=4 for OHT, n=4 for OHT + Control AAV2, and n=10 for AAV2:MCT2 + OHT. Error bars are SD.

Article Snippet: An AAV2.cre.GFP (1.0×10 13 GC/mL; Vector Biolabs, Malvern, PA) was injected into MCT2 fl/fl mouse eyes to remove the flox’d MCT2 gene through cre-mediated recombination, thereby eliminating MCT2 in the infected retinal cells.

Techniques: Infection, Injection

Journal: Neurobiology of disease

Article Title: MCT2 Overexpression Rescues Metabolic Vulnerability and Protects Retinal Ganglion Cells in Two Models of Glaucoma

doi: 10.1016/j.nbd.2020.104944

Figure Lengend Snippet: Metabolic changes in ON from OHT mice injected with AAV2:MCT2. A. The ratio of phosphorylated AMP kinase (pAMPK) to AMPK is significantly lower in OHT mice injected with AAV2:MCT2 (n=5) compared to control mice (no OHT or virus, n=6) and OHT mice injected with AAV2:GFP (n=6; p<0.01, p<0.05 respectively). B. PGC-1α protein in ON did not differ statistically across groups. ON sample number was n=12 for the No OHT, No AAV2 group, n=3 for the Control AAV+MCT2 group, and n=4 for the AAV2:MCT2+OHT group. C. Hexokinase activity is significantly decreased in OHT mice injected with AAV2:MCT2 (n=3) compared to OHT mice alone (n=4; p<0.05). ON sample number was n=3 for the No OHT, No AAV2 group, and n=5 for the OHT Control AAV2 group. D. Mitochondria size and E. Mitochondria density in the ganglion cell layer (GCL) did not differ across the Control AAV2+OHT (n=24 retinal sections across 3 mice) and AAV2:MCT2+OHT (n=22 retinal sections across 3 mice) groups. Error bars are SD.

Article Snippet: An AAV2.cre.GFP (1.0×10 13 GC/mL; Vector Biolabs, Malvern, PA) was injected into MCT2 fl/fl mouse eyes to remove the flox’d MCT2 gene through cre-mediated recombination, thereby eliminating MCT2 in the infected retinal cells.

Techniques: Injection, Activity Assay

Journal: Neurobiology of disease

Article Title: MCT2 Overexpression Rescues Metabolic Vulnerability and Protects Retinal Ganglion Cells in Two Models of Glaucoma

doi: 10.1016/j.nbd.2020.104944

Figure Lengend Snippet: Cytochrome-c oxidase (COX) and succinate dehydrogenase (SDH) activity in OHT optic nerves. A-B. COX histochemistry in OHT mouse ON (A), and OHT with AAV2:MCT2 injection (B). Higher magnification insets of OHT control (a’) and AAV2:MCT2 + OHT (b’) show greater DAB intensity after OHT with AAV2:MCT2. Quantification of histochemistry is shown in F. Scale bar=100μm. C-D. SDH histochemistry in OHT mouse ON (C) and OHT with AAV2:MCT2 injection (D). Scale bar=1000μm. Higher magnification insets of OHT control (c’) and AAV2:MCT2 + OHT (d’) indicate greater nitroblue tetrazolium intensity after OHT with AAV2:MCT2. E. SDH activity is significantly higher in AAV2:MCT2 with OHT ON (n=9 sections across 3 mice) compared to both OHT alone (n=9 sections across 3 mice), and OHT ON injected with control AAV2 (AAV2:GFP) (n=13 sections across 3 mice; p<0.001 and p<0.05, respectively). SDH activity is significantly lower in OHT animals injected with AAV2:MCT2 than in control mice (no OHT or AAV2, n=16 sections across 3 mice). F. COX activity is significantly higher in OHT mouse ON injected with AAV2:MCT2 (n=9 sections across 3 mice) compared to OHT alone (n=9 sections across 3 mice; p<0.0001). Error bars are SD.

Article Snippet: An AAV2.cre.GFP (1.0×10 13 GC/mL; Vector Biolabs, Malvern, PA) was injected into MCT2 fl/fl mouse eyes to remove the flox’d MCT2 gene through cre-mediated recombination, thereby eliminating MCT2 in the infected retinal cells.

Techniques: Activity Assay, Injection

Journal: Neurobiology of disease

Article Title: MCT2 Overexpression Rescues Metabolic Vulnerability and Protects Retinal Ganglion Cells in Two Models of Glaucoma

doi: 10.1016/j.nbd.2020.104944

Figure Lengend Snippet: Retinal microglia and pattern electroretinogram in OHT mice. A. Microglia density did not differ between OHT eyes injected with control virus (AAV2:eGFP, n=12 sections from 3 retinas) or AAV2:MCT2 (n=16 sections from 4 retinas). B. Intensity of Iba1 immunolabeling was significantly different across groups, with virus injection having a lowering effect on fluorescence intensity of Iba1 (ANOVA, p<0.0001). Samples sizes for this analysis was n=12 retinal sections from 3 retinas per group. C. Pattern electroretinogram shows significantly higher P1 amplitude in OHT animals injected with AAV2:MCT2 (n=12 mice) than in OHT alone (n=12 mice; p<0.05). Sample size also n=12 for the No OHT, No AAV2; and the Control AAV2+OHT groups. D. N2 amplitude did not differ across experimental groups; sample number as in C. Error bars are SD. E. Example PERG traces for each of the groups represented in C and D.

Article Snippet: An AAV2.cre.GFP (1.0×10 13 GC/mL; Vector Biolabs, Malvern, PA) was injected into MCT2 fl/fl mouse eyes to remove the flox’d MCT2 gene through cre-mediated recombination, thereby eliminating MCT2 in the infected retinal cells.

Techniques: Injection, Immunolabeling, Fluorescence