DOI: 10.5061/dryad.c2fqz61hr
title: NMR spectroscopy-based metabolomics of organotypic retinal explants

description: 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.
description: <strong>Metabolite extraction </strong>
 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. <strong>Sample
 preparation for <sup>1</sup>H-NMR spectroscopy measurements
 and data analysis</strong> 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-d<sub>4</sub> 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).
 <strong>Quantification </strong><strong>of
 metabolomic data </strong><strong>and statistical
 analysis</strong> 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). 
description: # NMR spectroscopy-based metabolomics of organotypic retinal explants
 [https://doi.org/10.5061/dryad.c2fqz61hr](https://doi.org/10.5061/dryad.c2fqz61hr) The dataset contains raw NMR CPMG spectra files. 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. The spectra were pre-processed using TopSpin 3.6.1 software (Bruker BioSpin). ## Description of the data and file structure Data contains Carr-Purcell-Meiboom-Gill Sequence (CPMG) experiment spectra files in the pdata 1, .1r folder. Each folder contains 1 raw NMR spectra file to be opened with Bruker TopSpin software (any current version of the free academic licenced software will be able to open these files). **The organization of the dataset, the individual data files, and the variables** The path to follow to open the spectra in each experimental replicate eg. for the control replicate 1: \Ret_Control_1\22\pdata\1\1r. The accompanying files are necesary for the function of the software so keep and download the whole folder setup. **The file types** Parameter Files The parameter files **acqu*** and **proc*** containing acquisition and processing parameters, are text files (see Section of a an acqu parameter file). All parameter files ending with ‘s’ (acqus, proc2s...) describe the status of the dataset. The other files (acqu, proc2…) contain parameter values which will be used in the next processing or acquisition (so called foreground parameters). Format of all parameter files corresponds to the JCAMP-DX standard. It allows the inclusion of vendor specific parameters, which are marked by the character sequence ##$. For this reason, all parameters TopSpin parameters in the file are preceded by this sequence. 1D Spectra Applying a 1D processing command to a 1D fid or reading a 1D slice from a 2D spectrum generates processed data. They are stored in two files **1r** and **1i**, corresponding to real and imaginary part. Like in 1D fids, the data points are stored as a sequence of data point values. Their format is given by the parameter DTYPP, the byte ordering is given by the parameter BYTORDP, both may be read from the processing status parameter file procs. Each sample folder contains a sub-folder 22 that should be kept as separated folder for correct TopSpin Software import structure. Each 22 folder contains a subfolder of **pdata**, and two acqusition files **acqu** and **acqus**. Each pdata subfolder contains one-dimensional spectra subfolder **1**. Each 1 subfolder contains **1r** spectra file, and two processing files **proc** and **procs**, necessary for function of the import. More information on data formats: [http://www.nmragenda-leiden.nl/static/Bruker_manual_4.1.4/topspin_pdf/Bruker_NMR_Data_Formats.pdf ](http://www.nmragenda-leiden.nl/static/Bruker_manual_4.1.4/topspin_pdf/Bruker_NMR_Data_Formats.pdf) NMR raw spectra of the retina tissue metabolites are **categorical** data from individual conditions follows these **condition abbreviations**: 1\) 1,9-dideoxyforskolin (1,9-DDF) in 5 biological replicates Ret_1_9_DDF_1 Ret_1_9_DDF_2 Ret_1_9_DDF_3 Ret_1_9_DDF_4 Ret_1_9_DDF_5 2\) The wildtype control retina in 5 biological replicates Ret_Control_1 Ret_Control_2 Ret_Control_3 Ret_Control_4 Ret_Control_5 3\) Shikonin treated retina in 5 biological replicates Ret_Shikonin_01 Ret_Shikonin_02 Ret_Shikonin_03 Ret_Shikonin_04 Ret_Shikonin_05 4\) electron chain uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) treated retina in 5 biological replicates Retina_FCCP_01 Retina_FCCP_02 Retina_FCCP_03 Retina_FCCP_04 Retina_FCCP_05 5\) Wildtype retina without the retinal pigment epithelium (RPE) layer in 5 biological replicates Retina_noRPE_01 Retina_noRPE_02 Retina_noRPE_03 Retina_noRPE_04 Retina_noRPE_05 We further used this dataset for annotation in commercial ChenomX software, but other oftwares such as MestReNova can be used.