Figure 1. Ldha-dependent aerobic glycolysis and outer segment maintenance in photoreceptors.

(A) Freshly explanted retinas were treated with the LDH inhibitor, sodium oxamate, for 8 hr in explant culture medium, transferred to Krebs’-Ringer's for 30 min, and lactate was measured in the supernatant. Control (n = 5), Oxamate (n = 4). (B) Freshly explanted retinas were treated with oxamate or NaCl (control) in explant culture medium for 8 hr, followed by treatment with NaN₃ or NaCl (untreated group) in Krebs’-Ringer's medium for 30 min. ATP per retina was then measured. n = 7, Control untreated; n = 8, Oxamate untreated n = 8, Control NaN₃; n = 8, Oxamate NaN₃. (C) Expression of Ldha and Ldhb as determined by IHC. Glutamine synthetase (GS), a Mueller glia-specific marker, colocalized with LDHB in the cell bodies (arrowheads), processes ensheathing the photoreceptors (arrows) and the outer limiting membrane (OLM, *). Scale bar, 50 μm. (D) ISH for Ldha and Ldhb. Ldha RNA displayed photoreceptor-enriched expression while Ldhb RNA was not observed in photoreceptors. Scale bar, 100 μm. (E, F) Freshly explanted retinas were treated with FX11 or DMSO for 8 hr and transferred to Krebs’-Ringer's for 30 min and secreted lactate was measured (E) n = 5, DMSO; n = 6, FX11, or they were transferred to Krebs’-Ringer's buffer with NaN₃ or NaCl (untreated group) for 30 min for ATP quantitation (F). ATP per retina was measured at the end of the assay. n = 8, DMSO untreated; n = 8, FX11 untreated; n = 9, DMSO NaN₃; n = 7, FX11 NaN₃. (G) Freshly explanted retinae were transferred to Krebs’-Ringer's for 30 min and secreted lactate was measured. n = 8, Bl6/J; n = 8, Ldha^fl/fl; n = 8, Rod-cre; n = 16, Rod-cre> Ldha^fl/fl; n = 8, Rod-cre> Ldha^fl/+. (H) Photoreceptor outer segment phenotype 42–45 days following in vivo electroporation of a knock-down construct (shRNA) for Ldha. CAG-mGFP was used for coelectroporation. Plasmid combinations listed on the left. Magnification of areas outlined in yellow is displayed on right with threshold-adjusted rendering to highlight inner and outer segments. Scale bar, 25 μm. (I) Quantification of inner+outer segment (IS+OS) lengths. n = 53–74 photoreceptors, 4–5 retinae. (J) Photoreceptor outer segment phenotype of dark-reared animals. Electroporated pups were transferred to dark on the day of eye opening (P11) and reared with their mothers for 3 weeks. (K) Quantification of inner+outer segment lengths of (J). n = 53–83 photoreceptors, 4–5 retinae. (L) Colored end products of redox reactions catalyzed by COX and SDH enzymes in retinal tissue. Scale bar, 200 μm. (M) IHC for SDH-A subunit in adult retina. Scale bar, 200 μm. ONL, outer nuclear layer. INL, inner nuclear layer. Data, Mean±SD. Statistics, unpaired, two-tailed t-test with Kolmogorov-Smirnov correction for panels A, E; two-way ANOVA with Tukey’s correction for panels B, F and K; one-way ANOVA with Tukey’s multiple comparison test for panels G, I.

DOI: http://dx.doi.org/10.7554/eLife.25946.003

Figure 1—figure supplement 1. Metabolic challenges of photoreceptor cell.

Schematic of the rod photoreceptor and RPE-Outer segment proximity shown on the left. photoreceptors are also ensheathed by Mueller glia that span the thickness of the retina. A meshwork of blood capillaries, the choroidal plexus, supplies nutrients and oxygen to the photoreceptors. These cells shed a fraction of their outer segment to be phagocytosed by the RPE. We estimated, based on published findings, that on diurnal basis, the shed discs account for ~70X the lipid present in the cell outside the outer segment (LaVail, 1976) and necessitate ~2X the rate of protein synthesis if shedding does not occur (Kwok et al., 2008). Thus, outer segment shedding poses a considerable biosynthetic demand on the photoreceptors. Intense metabolic activity compels judicious allocation of metabolites to competing pathways (Right). Each photoreceptor consumes ~10⁸ ATP s⁻¹ in darkness primarily via the action of Na⁺/K⁺ ATPase. Glucose oxidation can generate the ATP, though this would necessitate regulated channeling of glucose to biosynthetic vs catabolic pathways. Similarly, each photon absorption results in formation of all-trans Retinal, which needs to be reduced to complete the visual cycle using NADPH in stoichiometric amounts. NADPH also plays an important role in lipid biosynthesis and countering oxidative stress, which is a byproduct of mitochondria-based oxidative phosphorylation. The central question in understanding photoreceptor physiology thus is, how carbons are allocated toward biosynthetic vs catabolic processes? ONL, outer nuclear layer. INL, inner nuclear layer. GCL, ganglion cell layer.

DOI: http://dx.doi.org/10.7554/eLife.25946.005

Figure 1—figure supplement 2. Characterization of Ldha knockdown and mitochondrial function.

(A) Lactate dehydrogenase (LDH) catalyzes equilibrium between pyruvate and lactate. Of importance is to note the 1:1:1:1 molar stoichiometry between NAD⁺, pyruvate, lactate and NADH underscoring the concept that formation of lactate results in molar equivalent contribution to the cytosolic NAD⁺ pool which in turn serves as a cofactor to generate molar equivalents of pyruvate via the glycolytic pathway. Secreted lactate represents pyruvate-derived carbons that were unavailable to that cell for other metabolic pathways. (B) LDH is composed of four subunits with the two most common encoded by the Ldha and Ldhb genes. The five tetrameric compositions are considered to differ in the ability to produce or consume lactate, although the net reaction direction would be dictated by thermodynamics and flux considerations. (C) Retinal cross section from the 8 week old F1 progeny of Rod-cre and mT/mG parents. mtdTomato is constitutively expressed while mGFP is expressed in a cre-dependent manner. (D) Retina in (c) stained for cone arrestin, a cone photoreceptor marker. (E) Immunoblot probing for LDHA expression in 3-week-old retinal lysates of Rod-Cre> Ldha^fl/fl and age-matched Cre⁻ (Ldha^fl/fl) siblings. Six retinae from 3 mice were pooled for lysate preparation in each group. (F) IHC for Ldhb on a retinal cross section of a 6-week-old Rod-Cre> Ldha^fl/fl mouse. (G) Representative immunoblots of 293 T cells transfected with either full length (FL) or the coding region (CDS) of AU1-tagged murine LDHA driven by the CAG promoter. Cells were cotransfected with constructs encoding short hairpins targeting the murine Ldha transcripts. The short hairpin sh1 targets the 3’ untranslated region (UTR) of the mouse Ldha transcript while sh3 and sh4 target the coding region. Cox IV was used as a loading control. UT, untransfected. (H) Photoreceptor outer segment phenotype 40 days following in vivo electroporation of LDHAsh, CAG-rLDHB. CAG-mGFP was used for coelectroporation. For Ldhb staining, the gain during acquisition was adjusted to prevent oversaturation of signal intensity in overexpressing photoreceptors so as to preserve detail. Live histogram of pixel-intensity distribution was used in order to prevent clipping at the far-right end of intensities. Thus, Ldhb staining intensity in the IPL seems much lower compared to those observed in other figure panels in this study. Right panel, quantification of inner+outer segment lengths. Data, Mean±SD (n = 75 photoreceptors, 3 retinae for LDHAsh+ CAG-rLDHB group). Statistics, One-way ANOVA with Tukey’s multiple comparison test. (I) Control histochemical reactions for SDH and COX activity. SDH reaction on unfixed retinal tissue without the substrate or in presence of Malonate, a competitive inhibitor. The light blue precipitate in –Succinate reaction was observed in outer segments. It differed from the intense purple precipitate when the substrate was included and does not match SDH localization in the retina. COX was inhibited by sodium azide (+NaN₃) and failed to form the end product of the histochemical reaction. (J) Confocal image of retinal cross section stained with anti-PDHE1 antibody that recognizes a subunit of pyruvate dehydrogenase (PDH). Highest signal is seen in the photoreceptor inner segments, as well as the OPL and IPL synaptic layers. Scale bar, 25 μm.

DOI: http://dx.doi.org/10.7554/eLife.25946.006

Figure 1—figure supplement 3. Cell-autonomous effect of Ldha knockdown.

Rhodopsin staining of a 42-day-old mouse retina electroporated with a shRNA construct targeting Ldha. Magnification of a retinal cross section focusing at the edge of the electroporation boundary is shown to depict the outer segments within the electroporated patch (left side of the dotted line) and outside (right-hand-side of the dotted line). CAG-mGFP was used as a coelectroporation plasmid.

DOI: http://dx.doi.org/10.7554/eLife.25946.007

Figure 1—figure supplement 4. Mitochondrial activity after Ldha loss-of-function.

Cytochrome oxidase (COX) activity after deletion of Ldha in the rods (Rod-cre> Ldha^fl/fl, bottom right) of a 7-week animal. Age-matched heterozygous sibling (Rod-cre> Ldha^fl/+, bottom left) is included as control. For comparison, a retina from wild-type mouse (previously presented in Figure 1) is included (top panel).

DOI: http://dx.doi.org/10.7554/eLife.25946.008