Nicotinamide treatment robustly protects from inherited mouse glaucoma

Abstract

Nicotinamide adenine dinucleotide (NAD) is a key molecule in several cellular processes and is essential for healthy mitochondrial metabolism. We recently reported that mitochondrial dysfunction is among the very first changes to occur within retinal ganglion cells during initiation of glaucoma in DBA/2J mice. Furthermore, we demonstrated that an age-dependent decline of NAD contributes to mitochondrial dysfunction and vulnerability to glaucoma. The decrease in NAD renders retinal ganglion cells vulnerable to a metabolic crisis following periods of high intraocular pressure. Treating mice with the NAD precursor nicotinamide (the amide form of vitamin B3) inhibited many age- and high intraocular pressure- dependent changes with the highest tested dose decreasing the likelihood of developing glaucoma by ∼10-fold. In this communication, we present further evidence of the neuroprotective effects of nicotinamide against glaucoma in mice, including its prevention of optic nerve excavation and axon loss as assessed by histologic analysis and axon counting. We also show analyses of age- and intraocular pressure- dependent changes in transcripts of NAD producing enzymes within retinal ganglion cells and that nicotinamide treatment prevents these transcriptomic changes.

Keywords: Glaucoma; NAD+; axon degeneration; nicotinamide; optic nerve head cupping; retinal ganglion cell.

Figures

Figure 1.

NAM prevents optic nerve atrophy and axon loss in glaucoma. Optic nerves from control (D2-Gpnmb+; A), DBA/2J (D2; B), and treated (D2 + NAMLo, D2 + NAMHi; (C) and D) mice were sectioned and stained with PPD, which darkly stains the axoplasm of dead or dying axons. Surviving axons were counted using AxonJ (E) and optic nerve cross sectional area measured (F). There is a significant decrease in total axon number and optic nerve area in glaucomatous D2 eyes compared with controls. Nerves from NAM treated mice are indistinguishable from no glaucoma controls. Nerves were assessed at 12 months of age. NAMLo and NAMHi refer to our low and high dose of NAM. Scale bar = 100 μm (A) and 30 μm (insets). * P < 0.05, * P < 0.01, * P < 0.001.

Figure 2.

NAM prevents optic nerve cupping in glaucoma. The presence of optic nerve cupping was assessed using haematoxylin and eosin staining (H & E) and cresyl violet staining (Nissl). In control eyes (D2-Gpnmb+) there is a robust ganglion cell layer and nerve fiber layer (which contains axons from the retinal ganglion cells, denoted by the distance between the black arrows). In D2 eyes that have undergone glaucomatous neurodegeneration there is evident optic nerve cupping (asterisk), and a loss of ganglion cell layer cells and nerve fiber layer thickness. Cupping and RGC loss were absent in eyes of NAM treated mice (D2 + NAMLo), thus NAM treatment protected from all assessed signs of glaucoma. Eyes were assessed at 12 months of age. V corresponds to the central retinal vessel. GCL = ganglion cell layer, IPL = inner plexiform layer, INL = inner nuclear layer, OPL = outer plexiform layer, ONL = outer nuclear layer. Scale bar = 50 μm.

Figure 3.

NAD synthesis and NAD relevant genes in RGCs. (A) NAD synthesis. Tryptophan (Trp) is used to form NAD+de novo from diet in an 8 step pathway with nicotinic acid mononucleotide (NAMN) and nicotinic acid adenine dinucleotide (NAAD+) intermediates. Alternatively NAD+ can be produced through 2 other core pathways; the Preiss-Handler pathway from nicotinic acid (NA), or through the salvage pathway from nicotinamide (NAM). NA is used in the Preiss-Handler pathway to form NAD+ via 2 steps shared with the de novo pathway: NAMN (by nicotinic acid phosphoribosyltransferase; NAPRT1) and NAAD+ (by NAD+ synthetase; NADSYN1). In the salvage pathway, NAM is used to form NAD+ being converted by nicotinamide phosphoribosyltransferase (NAMPT) to nicotinamide mononucleotide (NMN) and subsequently to NAD+ by nicotinamide nucleotide adenylytransferase (NMNAT1, −2, and −3). NAM can also be converted to NA in the gut by bacterial PncA (nicotinamidase) and salvaged into the Preiss-Handler pathway. NAM is available in diet, but can also be produced by NAD+-consuming enzymes. Nicotinamide riboside (NR) can feed into the salvage pathway to form NAD+ by nicotinamide riboside kinases (NRK1, −2; Nmrk1, −2 as mouse genes) via NMN, or via NAM by purine nucleoside phosphorylase (NP). (B) and (C) Retinal ganglion cells exhibit age-dependent changes in NAD+ synthesis-related genes as well as further IOP-dependent declines in Nmnat2, an important NAD producing enzyme linked to axon protection. The decline in NAD is a major age-dependent risk factor for DBA/2J glaucoma. NADK (Nadk gene) is a major NAD-consuming kinase and its upregulation suggests increased NAD consumption / utilization. Differentially expressed genes (FDR < 0.05) are shown in red. Non-differentially expressed genes are shown in gray.