Fig 2: Labelling of NAD salvage pathway machinery in human retina. A NAMPT labelling is detected across all retinal layers, localizing to both nuclear (haematoxylin + ve) and cytoplasmic (haematoxylin -ve) cellular compartments, with the most intense labeling in the INL (n = 11 eyes). B NMNAT1 labelling is strongest in nuclear layers (GCL, INL, ONL) localizing to nuclei (n = 11 eyes). C NMNAT2 labelling localized to cytoplasmic compartments and was detected across all retinal layers, with the greatest intensity in the GCL, reflecting high expression in RGCs (n = 11 eyes). D In the ONH, NAMPT followed the same trend as in the retina, with labelling localizing to both nuclear and cytoplasmic compartments. Labeling intensity did not vary along the length of the optic nerve (n = 11 eyes). E NMNAT1 again localized only to nuclei in the ONH (glial framework), and its intensity was consistent along the length of the optic nerve (n = 10 eyes). F NMNAT2 conversely, localized only to the axonal compartments of the optic nerve and was significantly greater in the first 500 µm of the ONH (n = 8 eyes), potentially reflecting the density of axons relative to glial framework here, or the need for more NMNAT2 at a critical metabolic portion of the axons. N.B. these data do not allow for direct comparison of abundances of these enzymes given the nature of antibody labelling (efficiency and concentration of the antibodies etc.). RNFL retinal nerve fiber layer, GCL ganglion cell layer, IPL inner plexiform layer, INL inner nuclear layer, OPL outer plexiform layer, ONL outer nuclear layer, PS photoreceptor segments. Scale bars = 50 µm in A–C, 500 µm in D–E (overview images) and 50 µm in D–E (inset images)

Fig 3: Retinal neurons predominantly express NAD-salvage pathway transcripts. A We examined independent datasets from single cell RNA-sequencing of normal human retina (left) and single nucleus RNA-sequencing of normal human retina (right). Expression of transcripts encoding NAD synthesizing enzyme machinery was compared across cell types of the retina. Retinal neurons demonstrate greater expression levels in a higher proportion of cells for the NAD-salvage pathway (NAMPT, NMNAT1-3) than the Preiss-Handler Pathway (NAPRT, NADSYN1). NAMPT expression is greater than NMRK1-2, suggesting a favoring of nicotinamide as an NAD-salvage pathway substrate over the alternative nicotinamide riboside. Glia and other non-neuronal support cells of the retina also favor the NAD-salvage pathway but have a greater relative expression of Preiss-Handler Pathway and NMRK1 than neurons. B NAMPT, NMNAT1, and NMNAT2 were expressed to a greater extent in RGCs over other retinal neurons. Of the non-neuronal cell types, only microglia/myeloid cells and RPE cells demonstrated strong expression of NAD synthesizing enzyme machinery. C For most cell types, when considering genes with high expression, the distribution of expression within cell types appeared normal, as demonstrated by ridge plots. In the single cell RNA-sequencing (left), distribution of NAMPT and NMNAT2 suggest the possibility of distinct populations, perhaps reflecting different RGC subtypes. This was not observed in the single nucleus sequencing, where distribution of expression appeared more normal for all cell types, with the exception of NMNAT2 in RGCs which had a greater variance, and expression of NAMPT and NMNAT3 in from RPE cells. Retinal neurons: cones, rods, horizontal cells (HCs), bipolar cells (BPs), amacrine cells (ACs), and retinal ganglion cells (RGCs). Non-neuronal retinal cells: myeloid/microglia, Müller glia, astrocytes, vascular cells, and retinal pigment epithelial cells (RPE cells)