A bstract. Maintenance of NAD pools is critical for neuronal survival. The capacity to maintain NAD pools declines in neurodegenerative disease. We identify that low NMNAT2, the critical neuronal NAD producing enzyme, drives retinal susceptibility to neurodegenerative insults. As proof of concept, gene therapy over-expressing full length human NMNAT2 is neuroprotective. To pharmacologically target NMNAT2, we identify that epigallocatechin gallate (EGCG) can drive NAD production in neurons through an NMNAT2 and NMN dependent mechanism. We confirm this by pharmacological and genetic inhibition of the NAD-salvage pathway. EGCG is neuroprotective in rodent (mixed sex) and human models of retinal neurodegeneration. As EGCG has poor drug-like qualities, we use it as a tool compound to generate novel small molecules which drive neuronal NAD production and provide neuroprotection. This class of NMNAT2 targeted small molecules could have an important therapeutic impact for neurodegenerative disease following further drug development.
Neurodegenerative disease is a significant global health and economic burden. NAD homoeostasis is a critical factor that influences neurodegeneration and neuroprotection. Increasing levels of NAD provide neuroprotection in multiple cell and animal models of disease and in human clinical trials1. Glaucoma is one of the most prevalent neurodegenerations which affects ~80 million people worldwide2. In glaucoma, the progressive dysfunction and loss of retinal ganglion cells (RGCs; the output neuron of the retina whose axons make up the optic nerve) results in irreversible blindness. There are no clinically available neuroprotective strategies.
Recent animal and human studies have uncovered metabolic dysfunction occurring early in RGCs in glaucoma, in particular the critical dependency of RGCs on sufficient levels of NAD3,4,5. In neurons, NAD levels are maintained predominantly through the NAD-salvage pathway’s two terminal enzymes; NMNAT1 (localized to the nucleus) and NMNAT2 (localized in the cytoplasm6). Protein expression of NMNAT2 is predominantly neuronal and its NAD-producing activity is essential for survival of long axons7. We previously identified downregulation of NMNAT2 occurring in RGCs prior to neurodegeneration in the DBA/2J mouse model of glaucoma3,8. This was subsequently supported by sequencing of translating mRNAs isolated from RGC ribosomes at a degenerative timepoint (where RGC loss has occurred) in a mouse ocular hypertensive (OHT) model9. Similarly, we have demonstrated that NMNAT2 immuno-labelling is decreased in late-stage glaucoma in the human retina and optic nerve head (ONH; a critical site of injury to RGC axons), where substantial RGC death has occurred6.
Given the critical role that NMNAT2 plays in axon maintenance and degeneration, understanding how NMNAT2 levels may influence RGC degeneration is of particular importance. Whilst genetic targeting of NMNAT2 has been demonstrated to be robustly neuroprotective in other neuronal systems, there are no identified drugs or compounds that target endogenous NMNAT2 to produce high levels of NAD in neurons. In this work, we identify that epigallocatechin gallate (EGCG) drives NAD production in neurons through an NMNAT2-dependent mechanism. Using EGCG as a tool compound we develop small molecules driving neuronal NAD production through NMNAT2 which can also provide neuroprotection against RGC injury ex vivo.
