Despite mounting evidence that the mammalian retina is exceptionally reliant on proper NAD+ homeostasis for health and function, the specific roles of subcellular NAD+ pools in retinal development, maintenance, and disease remain obscure. Here, we show that deletion of the nuclear-localized NAD+ synthase nicotinamide mononucleotide adenylyltransferase-1 (NMNAT1) in the developing murine retina causes early and severe degeneration of photoreceptors and select inner retinal neurons via multiple distinct cell death pathways. This severe phenotype is associated with disruptions to retinal central carbon metabolism, purine nucleotide synthesis, and amino acid pathways. Furthermore, transcriptomic and immunostaining approaches reveal dysregulation of a collection of photoreceptor and synapse-specific genes in NMNAT1 knockout retinas prior to detectable morphological or metabolic alterations. Collectively, our study reveals previously unrecognized complexity in NMNAT1-associated retinal degeneration and suggests a yet-undescribed role for NMNAT1 in gene regulation during photoreceptor terminal differentiation.

Palmitoylation is a unique and reversible posttranslational lipid modification (PTM) that plays a critical role in many cellular events, including protein stability, activity, membrane association, and protein–protein interactions. The dynamic nature of palmitoylation dictates the efficient sorting of various retinal proteins to specific subcellular compartments. However, the underlying mechanism through which palmitoylation supports efficient protein trafficking in the retina remains unclear. Recent studies show that palmitoylation can also function as a signaling PTM, underlying epigenetic regulation and homeostasis in the retina. Efficient isolation of retinal palmitoyl proteome will pave the way to a better understanding of the role(s) for palmitoylation in visual function. The standard methods for detecting palmitoylated proteins employ 3 H- or 14 C-radiolabeled palmitic acid and have many limitations, including poor sensitivity. Relatively recent studies use thiopropyl Sepharose 6B resin, which offers efficient detection of palmitoylated proteome but is now discontinued from the market. Here, we describe a modified acyl resin–assisted capture (Acyl-RAC) method using agarose S3 high-capacity resin to purify palmitoylated proteins from the retina and other tissues, which is greatly compatible with downstream processing by LC-MS/MS. Unlike other palmitoylation assays, the present protocol is easy to perform and cost-effective. Graphical abstract

Many proteins undergo post‐translational lipid modifications, which are often essential to aid in subcellular trafficking, protein‐protein interactions, and membrane association. S‐palmitoylation is a unique post‐translational lipid modification which involves reversible attachment of the 16‐ carbon fatty acid palmitate to cysteine residues on proteins. In the retina, photoreceptor neurons are responsible for capturing photons of light and converting them into electrical signals, which are subsequently sent to the brain for visual processing. Many crucial phototransduction proteins in photoreceptors, including rhodopsin, phosphodiesterase‐6, and the transducin complex, have been shown to require post‐translational lipidation for proper function. To better understand the importance of “palmitoyl” lipid modifications in retinal photoreceptor cells, our lab employed acyl‐resin assisted capture (acyl‐RAC) followed by mass‐spectrometry analysis to isolate and identify proteins in retinal tissue which undergo S‐palmitoylation. From the mass‐spectrometry data pool, our lab chose several candidate proteins to study in more detail, including the β2‐ subunit of the Na+, K+‐ATPase (ATP1B2). ATP1B2 has been shown to be the predominant β‐subunit isoform of the Na+, K+‐ATPase in photoreceptor cells and is thought to be involved in maturation and trafficking of its catalytic α‐subunit counterpart. Additionally, ATP1B2 has been shown to be required by the retinal‐specific protein retinoschisin‐1 (RS1) for its localization and association with photoreceptor and bipolar cell membranes. Although it is known that the Na+, K+‐ATPase regulates the photocurrent in photoreceptors needed to send visual information to downstream neurons, the distinct role of ATP1B2 remains unknown. Acyl‐RAC followed by immunoblot served as a complementary approach to confirm that ATP1B2 undergoes palmitoylation, whereas its homolog, the β1 isoform, does not. After palmitoylation prediction software analysis, we utilized mammalian cell culture and in vivo subretinal injection/electroporation to demonstrate that ATP1B2 is palmitoylated on the 10th amino acid. Additionally, click chemistry with the palmitate analog 17‐Octadecynoic acid, was used as an independent approach to validate ATP1B2 palmitoylation. Altogether, these data confirm that ATP1B2 is post‐translationally lipid modified by palmitoylation on the 10th amino acid. However, the functional role of palmitoylation of ATP1B2 remains unknown and warrants further investigation.Support or Funding InformationNIH 1RO1EY028959‐01, WVU bridge funding, and intramural RFDG funding award (SK)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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