NMR spectroscopy-based metabolomics of organotypic retinal explants

The retina consumes massive amounts of energy, yet its metabolism and substrate exploitation remain poorly understood. Here, we used a murine explant model to manipulate retinal energy metabolism under entirely controlled conditions and utilized 1H-NMR spectroscopy-based metabolomics, in situenzyme detection, and cell viability readouts to uncover the pathways of retinal energy production. Our experimental manipulations resulted in varying degrees of photoreceptor degeneration, while the inner retina and retinal pigment epithelium were essentially unaffected. This selective vulnerability of photoreceptors suggested very specific adaptations in their energy metabolism. Rod photoreceptors were found to rely strongly on oxidative phosphorylation, but only mildly on glycolysis. Conversely, cone photoreceptors were dependent on glycolysis but insensitive to electron transport chain decoupling. Importantly, photoreceptors appeared to uncouple glycolytic and Krebs-cycle metabolism via three different pathways: 1) the mini-Krebs-cycle, fueled by glutamine and branched-chain amino acids, generating N-acetylaspartate; 2) the alanine-generating Cahill-cycle; 3) the lactate-releasing Cori-cycle. Moreover, the metabolomic data indicated a shuttling of taurine and hypotaurine between the retinal pigment epithelium and photoreceptors, likely resulting in an additional net transfer of reducing power to photoreceptors. These findings expand our understanding of retinal physiology and pathology and shed new light on neuronal energy homeostasis and the pathogenesis of neurodegenerative diseases.

Methods
Metabolite extraction

After retinal explant culture, at P15, the tissue was quickly transferred into 80% methanol / 20% ethanol, snap-frozen in liquid nitrogen. A sample of the culture medium was taken from the same well plate as the retinal tissue, and snap-frozen in liquid nitrogen. Retinal tissue was placed in 400 µL of methanol (LC-MS grade), transferred to the 2 mL glass Covaris system-compatible tubes and 800 µL of methyl-tert-butyl ether (MTBE) was added, thoroughly mixed, and further subjected to metabolite extraction via ultra-sonication (Covaris E220 Evolution, Woburn, USA). After the extraction, 400 µL of ultrapure water were added for two-phase liquid separation. The aqueous phase was separated and evaporated to dryness. Similarly, 400 µL of the aqueous medium sample was transferred to the 2 mL glass Covaris system-compatible tube, 800 µL of MTBE was added and subjected to the ultra-sonication extraction protocol. Finally, 400 µL of methanol were added and mixed, centrifuged, and after two-phase separation the aqueous layer was separated and evaporated, to obtain a dry metabolite pellet.

Sample preparation for 1H-NMR spectroscopy measurements and data analysis

Dried metabolite pellets were resuspended in a deuterated phosphate buffer (pH corrected for 7.4) with 1 mM of 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid sodium salt (TSP) as internal standard. NMR spectra were recorded at 298 K on a 14.1 Tesla ultra-shielded NMR spectrometer at 600 MHz proton frequency (Avance III HD, Bruker BioSpin, Ettlingen, Germany) equipped with a triple resonance 1.7 mm room temperature micro probe. Short zero-go (zg), 1D nuclear Overhauser effect spectroscopy (NOESY) and Carr-Purcell-Meiboom-Gill (CPMG; 4096 scans for retinal tissue samples, 128 for medium samples) pulse programs were used for spectra acquisition. Spectra were processed with TopSpin 3.6.1 software (Bruker BioSpin).

Quantification of metabolomic data and statistical analysis

Retina tissue metabolite assignment and quantification was done on the pre-processed CPMG spectra and performed with ChenomX NMR Suite 8.5 (Chenomx Inc., Edmonton, Canada).