NMNAT2 is a druggable target to drive neuronal NAD production

Abstract

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.

Full text

Article https://doi.org/10.1038/s41467-024-50354-5
NMNAT2 is a druggable target to drive
neuronal NAD production
James R. Tribble
1,15
,MelissaJöe
1,15
, Carmine Varricchio
2
,AminOtmani
1
,
Alessio Canovai
1,3
, Baninia Habchi
4,5,6
, Evangelia Daskalakis
4,5
,
Romanas Chaleckis
4,5,7
, Andrea Loreto
8,9
, Jonathan Gilley
8
,
Craig E. Wheelock
4,5
, Gauti Jóhannesson
10,11
, Raymond C. B. Wong
12,13
,
Michael P. Coleman
8
, Andrea Brancale
2,14
&PeteA.Williams
1
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 pharma-
cologically target NMNAT2, we identify that epigallocatechin gallate (EGCG)
can drive NAD production in neurons through an NMNAT2 and NMN depen-
dent 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 neuro-
degeneration 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 neuro-
degenerations which affects ~80 million people worldwide2.Inglau-
coma, 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 irreversibleblindness. 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 NAD3–5.Inneurons,
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
Received: 12 May 2023
Accepted: 19 June 2024
Check for updates
1
Department of Clinical Neuroscience, Division of Eye and Vision, St. Erik Eye Hospital; Karolinska Institutet, Stockholm, Sweden.
2
School of Pharmacy and
Pharmaceutical Sciences; Cardiff University, Cardiff, Wales, UK.
3
Department of Biology, Universityof Pisa, 56127 Pisa, Italy.
4
Unit of Integrative Metabolomics,
Institute of Environmental Medicine, Karolinska Institute, Stockholm, Sweden.
5
Department of Respiratory Medicine and Allergy, Karolinska University
Hospital, Stockholm, Sweden.
6
C2VN, INRAE, INSERM, Aix Marseille University, 13007 Marseille, France.
7
Gunma Initiative for Advanced Research (GIAR),
Gunma University, Maebashi, Japan.
8
John vanGeest Centre forBrain Repair,Department of Clinical Neurosciences; Universityof Cambridge,Cambridge, UK.
9
School of Medical Sciences and Save Sight Institute, Charles Perkins Centre, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW,
Australia.
10
Department of Clinical Sciences, Ophthalmology, Umeå University, 901 85 Umeå, Sweden.
11
Wallenberg Centre of Molecular Medicine, Umeå
University, 901 85 Umeå, Sweden.
12
Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, Australia.
13
Ophthalmology,
Department of Surgery, Universityof Melbourne,East Melbourne, Victoria, Australia.
14
Vysoká škola chemicko-technologická v Praze, Prague, Czech Republic.
15
These authors contributed equally: James R. Tribble, Melissa Jöe. e-mail: pete.williams@ki.se
Nature Communications | (2024) 15:6256 1
1234567890():,;
1234567890():,;
Content courtesy of Springer Nature, terms of use apply. Rights reserved

NMNAT2 is predominantly neuronal and its NAD-producing activity is
essential for survival of long axons7. We previously identified down-
regulation 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 RGCloss 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.
Results
NMNAT2 levels modulate retinal ganglion cell susceptibility to
neurodegeneration
NMNAT2 is an essential enzyme for NAD synthesis in neurons.
NMNAT2(RNA and protein) declines in the brain in Alzheimer’s disease
and its expression is highly variable10. To explore NMNAT2 in the retina
we first queried publicly available RNA-sequencing and microarray
datasets. We demonstrate that across recombinant inbred BXD strains
(recombinant inbred lines from crosses between C57BL/6J mice (B6)
and DBA/2J mice (D2); to explore Nmnat2 variations within a non-
pathological range), Nmnat2 expression is highly variable (up to 2-fold
difference) among individual strains and is variable within indepen-
dent datasets for whole eye, retina, and midbrain (where RGC axons
terminate; Fig. 1A) suggesting variability in expression within the whole
visual system. Inthe retina, this variability is not related to the number
of RGCs (Spearman’s rank correlation r=−0.00088, P= 0.997; Fig. 1B)
which is known to vary acrossindividual mice and strains11.Supporting
this, Nmnat2 expression has a significantly greater variance than RGC
markers Pou4f1 (P< 0.001), Rbpms (P< 0.001), and Tubb3 (P= 0.006),
but not Thy1 (P= 0.124; Fig. 1B). This variability increases with age
(Fig. 1C). Likewise, data from human retina demonstrates substantial
variability of NMNAT2 gene expression across individuals (up to 45-
fold difference, with 1.8 fold difference across the interquartile range;
Fig. 1D). Single-cell and single-nucleus RNA-sequencing from human
retina confirms that retinal expression of NMNAT2 is highly variable
across individual RGCs, which have the highest average expression
among retinal neurons (Fig. 1E; NMNAT2 expression is not present in
non-neuronal cell types in the retina). We hypothesize that variable
expression of NMNAT2 may be a contributing factor to the hetero-
geneity of glaucomatous disease.
Age, genetics, and high intraocular pressure (IOP) are all con-
siderable risk factors for glaucoma. In the D2 mouse (a model of a
complex age- and IOP- dependent inherited glaucoma), Nmnat2
expression (as assessed by whole tissue microarray12,13)issignificantly
reduced prior to detectable neurodegeneration in the ONH (molecular
disease group 3, P=0.025) and continues to decline in moderate and
severe disease (molecular disease group 4, P=0.001,and5,P< 0.001;
Fig. 1F). In the retina, Nmnat2 is significantly reduced in severe disease
(molecular disease group 4, P< 0.001; Fig. 1F). However, when only
considering RGCs (FAC sorted, bulk RNA-sequencing3), Nmnat2 is
significantly decreased at a time point prior to detectable neurode-
generation in eyes with the greatest degree of transcriptional
dysfunction (molecular disease group 4, P= 0.004; Fig. 1F). Dissecting
these glaucoma risk variables, IOP alone drives Nmnat2 depletion (in
the rat bead ocular hypertensive (OHT) model of glaucoma at a time
point where IOP is high but there is no significant RGC loss; Fig. 1Gand
Supp Fig. 1A, B). Direct axotomy of RGCs (severing of the optic nerve
and followed by retina tissue culture ex vivo for 12h) also drives
Nmnat2 depletion in the absence of IOP or age (Fig. 1G). We therefore
hypothesized that low NMNAT2 leads to RGC degeneration or renders
RGCs susceptible to neurodegenerative insults. To test this, we used
mice carrying one or two, fully or semi-penetrant, gene-trapped (gt)
alleles for Nmnat2 to titrate Nmnat2 levels in the retina (Fig. 2A). In
these mice, retinal Nmnat2 was depleted to (observed vs.expected
based on allele penetrance) 80% (expected 75%; Nmnat2gtBay/+), 39%
(expected 50%; Nmnat2gtE/+), or 10% (expected 25%; Nmnat2 gtBay/gtE)of
normal levels relative to wild type controls (100%; Nmnat2+/+).
(NMNAT2 null mice and humans are perinatal lethal14). Thus, these
mice model the wide spectrum of NMNAT2 levels seen in the human
retina (Fig. 1D, E).
Low Nmnat2 (Nmnat2gtBay/gtE) results in a developmental or very
early post-natal reduction in RGC density followed by an age-related
loss of RGCs initiated between 6 and 12 months of age which is absent
in Nmnat2+/+ mice up to 22 months of age (Fig. 2B) (although further
experiments arerequired to elucidate the exacttiming of this neuronal
reduction). This represents an ocular demonstration recapitulating a
combination of the developmental loss of peripheral sensory axons
and the late onset loss of motor axons previously identified in
Nmnat2gtBay/gtE mice15. This further highlights the unique fragility of
RGCs across neuronal subtypes. Developmentally regulated RGC
death and survival is influenced by connectivity in target brain
regions16,17. Given that neurons in these brain regions will also have
reduced Nmnat2, there is likely to be a more limited capacity to
maintain RGC connectivity, which may also partially explain the
developmental and early onset degeneration of RGCs in these mice.
Even when accounting for this developmental dropout in RGC popu-
lations, RGC loss was significantly greater in Nmnat2gtBay/gtE mice fol-
lowing axotomy (but not for the single alleles; i.e.predicted75%and
50% Nmnat2)(Fig.2C). This suggests a threshold of NMNAT2 expres-
sion past which RGCs are sensitized to neurodegenerative insults
(these thresholds are only met with genetic depletion and are not met
within the normal variations within the population). This is supported
by the wide range of NMNAT2 levels in humans which are non-
pathogenic in the absence of other neurodegenerative insults (e.g.
NMNAT2 expression is highly variable in aged postmortem human
brains, and further decreased in brains with Alzheimer’s disease
(~75–100% of controls levels)10). Overexpression of full length human
NMNAT2 (hNMNAT2) in RGCs via viral gene therapy was able to over-
come RGC susceptibility induced by low genetic levels of Nmnat2 (i.e.
Nmnat2gtBay/gtE mice; Fig. 3A) and provided complete neuroprotection
in B6 mice (where normal physiological levels of Nmnat2 are present;
Fig. 3B). Nmnat2 has been demonstrated to have a neuroprotective
role in animal models of neurodegeneration9,18. Supporting this
hNMNAT2 gene therapy also provided a robust neuroprotective effect
in vivo in a rat OHT glaucoma model demonstrating efficacy against
the multifactorial insults of glaucoma (Fig. 3C).
Epigallocatechin gallate drives NAD production through an
NMN and NMNAT2-dependent mechanism
Whilst gene therapy has found success in treating monogenic eye
diseases, it is less likely to provide complete protection for complex
polygenic neurodegenerative diseases such as glaucoma, and its
licensed use is likely to face many regulatory hurdles. As such, phar-
macological targeting of NMNAT2 may be a viable alternative that will
be more amenable in a clinical trial setting. Epigallocatechin gallate
(EGCG) isa green tea polyphenol that has previously been identified as
a potential NMNAT1-3 activator (in a cell-free enzymatic assay19). We
Article https://doi.org/10.1038/s41467-024-50354-5
Nature Communications | (2024) 15:6256 2
Content courtesy of Springer Nature, terms of use apply. Rights reserved

demonstrate that EGCG, in the nM to μM range, drives NAD produc-
tion in neurons in a dose (Fig. 4A) and time (Fig. 4B) dependent
manner. This effect is not replicated in neuron-low tissues (spleen,
muscle, and liver; Fig. 4C). EGCG provides full neuroprotection against
RGC loss following axotomy (Fig. 4D) mirroring the hNMNAT2 gene
therapy and our previous work with nicotinamide (NAM; an upstream
precursor to NAD in the salvage pathway3,20). Further supporting this,
EGCG delivered orally reduced (but not fully prevented) glaucomatous
neurodegeneration in the rat OHT glaucoma model, with this neuro-
protection improving with the addition of hNMNAT2 gene therapy
(Fig. 4E). EGCG also significantly improved RGC survival in human
retina punches maintained in culture following postmortem axotomy
following enucleation (Fig. 4F). Mitochondrial impairment is a trigger
for neurite depletion of NMNAT2, resulting in neurodegeneration21.
Primary retinal neurons treated with EGCG did not degenerate to the
same degree as untreated neurons when exposed to Complex I
inhibition via rotenone (Fig. 5A). EGCG, administered orally, sig-
nificantly improved RGC survival following in vivo intravitreal rote-
none injection which results in rapid RGC loss (Fig. 5B). Cortical
neurons demonstrated a significant increase in mitochondria mem-
brane potential (via JC-1 staining) when treated with 5µMEGCGanda
significant reduction in membrane potential at 50 µM (Supp Fig. 2).
Whole green tea polyphenols (containing ~25% EGCG by weight)
recapitulated the NAD-boosting effects (Supp Fig. 3A) and neuropro-
tective effects in the retina explant model at doses with comparable
EGCG content (Supp Fig. 3B) but did not provide protection in rat OHT
(Supp Fig. 3C).
We next determined whether EGCG’s neuroprotective effects
were NMNAT2-dependent. We depleted NMN (the substrate for
NMNAT2) using FK866 (a specific NAMPT inhibitor, the upstream
enzyme to NMNAT1 and NMNAT2). When neurons are depleted of
NMN, the capacity of EGCG to increase NAD levels is significantly
Fig. 1 | NMNAT2 expression is highly variable across the visual system tissues
and within retinal ganglion cell populations, and declines following injury.
ANmnat2 expression is highly variable between individual BXD mice (note loga-
rithmic scales) across RGC relevant tissues (whole eye, n= 157; retina, n=55;
midbrain, n= 37 animals). It is important to note that these data are from different
datasets and experiments andtherefore should not be usedfor comparison across
tissues, only within tissues. BIn the retina, this variability is not related to the
number of RGCs (Spearman’srank correlation, shaded area =95% CI) and the
variancein Nmnat2 expression is significantlygreater than for RGC markersPou4f1,
Rbpms,andTubb3 (Pitman-Morgan testof variance for paired samples, two-sided).
Both the founder strains (B6 and D2) are represented in these data and neither of
these strains have the most upper or lower values (D2 = 10.69, B6 = 11.09, min for
series= 10.23, max for series = 11.29 RMA gene level). CThis variability in Nmnat2
increases with age ( <200 days, n= 157 or >350 days, n= 187). DNMNAT2expression
in human retina is also highly variable between individuals (left, n=50; right,
n=105).EWithin the cell types of the retina, RGCs demonstrate the greatest
average expression of NMNAT2 by single cell and single nucleus RNA-sequencing
(red = highest expression, peach = lowest, dot plot scaled by % of expressing cells
within types; AC amacrine cell, BP bipolarcell, HC horizontal cell, RPE retinal
pigmentepithelium) data from6and47. Even within theseindividual RGCs, NMNAT2
expression is highly variable (n=74 RGCs for scSeq, n= 2039 RGCs for NucSeq).
FIn D2 mice (a chronic, age-related mouse model of glaucoma), Nmnat2 expres-
sion declines in whole ONH at a pre/early-degenerative timepoint (stage 3) and in
retina declines in late disease (stage 4) relative to DBA/2J-GpnmbR150X (for ONH,
Group 1 (n=8),Group2 (n= 8), Group 3 (n= 6), Group 4 (n=4),Group5(n=4)
where expressionis compared to n= 5 D2-Gpnmb + ; in the retina, Group 1 (n=8),
Group 2 (n=9),Group3 (n=3), Group4(n= 10); expression is compared to n=8
D2-Gpnmb +). In sorted RGCs from D2 retina, this decline in Nmnat2 is detectable
at an early, pre-degenerative time point in RGCs with high RNA dysregulation
(Group 1 (n=9 age-matched D2-Gpnmb+ and n= 6 D2s), Group 2 (n= 6 D2s),
Group 3 (n= 10 D2s), Group 4 (n= 4 D2s). Data in F from3,8,significance as FDR).
GThis is replicated under the isolatedglaucomatousinsults of ocularhypertension
(whole optic nervesfrom rat inducible model following 7 days of high IOP (OHT),
n= 6; and normotensive controls, n= 7) and following direct RGC injury through
axotomy (whole retina in retinal explant model, 12 h culture ex vivo following
axotomy, n=5; orcontrols, n= 5; two-sided Student’st-test). For A–C:TPMtran-
scripts per million, RMA robust multiarray analysis, QUANT quantile, RPKM reads
per kilobase of exon per million. Data in A–Fwere generated through screening
publicly available datasets (see Methods). *P<0.05,**P< 0.01, ***P<0.001,NS =
non-significant (P>0.05). For box plots, the centre hinge represents the median
with upper and lower hinges representing the first and third quartiles; whiskers
represent 1.5 times the interquartile range.
Article https://doi.org/10.1038/s41467-024-50354-5
Nature Communications | (2024) 15:6256 3
Content courtesy of Springer Nature, terms of use apply. Rights reserved

reduced (30% decrease, Fig. 6A) and EGCG no longer protects from
neurodegenerative insults (Fig. 6B).ThisdemonstratesthatEGCG
requires NMN to generate NAD and to provide neuroprotection (i.e.an
NAD-salvage pathway dependent process requiring NMNAT1 or
NMNAT2 to further stimulate NAD production). The neuroprotective
effects of EGCG were also reduced in Nmnat2gtBay/gtE mice (i.e.25%
Nmnat2 levels), demonstrating an NMNAT-dependent effect (Fig. 6C).
Collectively, these data suggested that EGCG may modulate NMNAT2
by binding an allosteric site on the protein surface. However, the
crystal structure of NMNAT2 is unknown. To address this and to
identify the potential EGCG binding site on NMNAT2, we generated a
homology model using the crystal structure of NMNAT1 and NMNAT3
as templates, which was further refined by loop-modelling and
extensive molecular dynamic simulations (Fig. 7A, Supp Fig. 4A–C). We
identified three consistent potential druggable binding pockets,
separate to the catalytic pocket (NMN binding site) on the NMNAT2
surface (Fig. 7B,Supp Fig. 4D, E). In silico, EGCG was able to maintain a
stable ligand-protein complex in one of the three pockets identified
through hydrophobic contacts and hydrogen bond interactions with
the surroundingresidues (i.e. Gln71, Asp78, Lys151, and Val156) (Fig. 7B,
Supplementary Fig. 4F, H) demonstrating the potential for the direct
interaction of EGCG with NMNAT2.
EGCG is a tool compound for designing novel, NMNAT2-targeted
NAD-boosting small molecules
Although EGCG efficiently drives NAD production in in vitro and
ex vivo contexts, and is well tolerated in diet, it is poorly bioavailable
and is prone to breakdown at differentpHs (as is present throughout
the digestive system; Supp Fig. 5)22. In addition, EGCG’s structural
complexity represents a significant synthetic challenge. Together
these qualities make EGCG a poor drug for clinical translation. To
address this, we screened simplified EGCG-like analogues through a
series of step-by-step truncations of the EGCG structure and tested
the NAD-boosting capacity in cortical neurons (Group 1, Fig. 7C, D,
Supp Fig. 6, Supplementary Data 1). We assessed the effects of the
removal/replacement of the hydroxyl group, the influence of ste-
reochemistry, reducing the number of rings, and the replacement of
the catechol ring. NAD assays demonstrated that flavone core deri-
vates drastically reduce biological activity while the chromene core
partially retains biological activity. This led to the identification of
Compound 18 as the simplest structure that showed statistically
significant activity, which represented a promising drug-like hit for
lead optimization. Analogues of Compound 18 were then tested to
explore the structure−activity relationships around the chromene
core. The synthetic accessibility of this particular chemical scaffold
Fig. 2 | Reduction of NMNAT2 increases retinal ganglion cell susceptibility to
injury. A Crossing mice heterozygous for Nmnat2 gene-trap alleles gtBay (pre-
dicted 50% silencing) or gtE (predicted 100% silencing) allowed Nmnat2 titration.
In these mice, retinal Nmnat2 was depleted to (observed vs. expected based on
allele penetrance) 80% (expected 75%; Nmnat2gtBay/+,n= 10), 39% (expected 50%;
Nmnat2gtE/+,n= 12), or 10% (expected 25%; Nmnat2gtBay/gtE,n= 12) of normal levels
relative to wild type controls (100%; Nmnat2+/+,n=10).BRGC density was sig-
nificantlylower in Nmnat2gtBay/gtE retina than in Nmnat2+/+r etina at 3 months without
furtherchange at 6 months (indicating a developmental loss). By 12 months of age,
Nmnat2gtBay/gtE mice had significantly fewer RGCs than at 3 and 6 months, and this is
stable to 22 months (indicating an additional early age-related decline). For
Nmnat2+/+:3months,n=6;6months,n=8;12 months,n=8;22months,n=8;for
Nmnat2gtBay/gtE: 3 months, n=6;6months,n=8;12months,n=8;22months,n=6.
Scale bar = 20 µm. CRGC density was significantly reduced in allNmnat2 gene-trap
allele mouse strains at 3 days ex vivo following RGC axotomy (RBPMS = specific
marker of RGCs in the retina) relative to naïve controls (0 days ex vivo), and this
was greatest in Nmnat2gtBay/gtE mice supporting a threshold of Nmnat2 loss beyond
which RGC susceptibility to injury is increased (Day 0: Nmnat2+/+,n=6;
Nmnat2+/gtBay,n=6;Nmnat2+/gtE,n=6;Nmnat2gtBay/gtE,n=6;Day3: Nmnat2+/+,n=8;
Nmnat2+/gtBay,n=6;Nmnat2+/gtE,n=7; Nmnat2gtBay/gtE,n= 5; scale bar = 20 µm). For B
and C, *P<0.05,**P< 0.01, ***P< 0.001, NS = non-significant (P>0.05); One-way
ANOVA with Tukey’sHSD. For box plots, the centre hinge represents the median
with upper and lower hinges representing the first and third quartiles; whiskers
represent 1.5 times the interquartile range.
Article https://doi.org/10.1038/s41467-024-50354-5
Nature Communications | (2024) 15:6256 4
Content courtesy of Springer Nature, terms of use apply. Rights reserved

allowed for the preparation of a series of drug-like derivatives,
leading to the identification of more potent compounds very rapidly.
The removal of metabolically labile hydroxyl groups led to a decrease
in activity, but their replacement with fluorine groups on the aro-
matic ring restored the NAD-boosting activity, potentially increasing
the lipophilicity and metabolic stability of the compounds (e.g.
Compound 17, 20, 50, Fig. 7C, D, Supplementary Fig. 6). A scaffold-
hopping strategy led to the identification of three new cores: tetra-
hydroquinoline (Group 2), benzomorpholine (Group 3), and tetra-
hydroquinoxaline (Group 5), which showed a higher potency
compared to the chromene core and were selected as scaffolds for
the design of a new series of analogues. Across these iterations we
identified several small molecules with greater potency in increasing
NAD than EGCG. We selected ten top candidates (Fig. 8A) for further
testing based on their NAD-boosting capacity. All of these top ten
compounds retained NAD-salvage pathway specificity as demon-
strated by co-incubation with FK866 where the capacity to increase
NAD over control was blocked. This was greatest for compounds in
Group 4 and 5 (Fig. 8B, Supplementary Data 2). Mitochondrial
membrane potential was also assessed in these top compounds
(Supp Fig. 7). To test neuron specificity, NAD-boosting effects of the
top ten compounds were assessed in dissociated neuron-high (cor-
tex) and neuron-low (liver, muscle, spleen) tissues. Supporting the
previous findings with EGCG, the majority of the newly developed
compounds retained neuron specificity (Fig. 8C).
In order to elucidate the important structural features for the
NAD-boosting activity of these families of compounds, we built a 3D-
structure-activity relationship model using the 56 compounds tested.
This revealed critical field/steric contributions to the biological NAD-
boosting activity, such as a bulky and hydrophobic group in position 2,
and a positive field (hydrogen bond donor, positive electrostatic
potential) in position 1 (Fig. 8D). This demonstrates that these classes
of compounds could be well accommodated in the identified binding
pocket on the NMNAT2 surface, making several H-bonds and hydro-
phobic interactions with the surrounding residues, further supporting
a mechanism of action working through NMNAT2. To explore the
potential of these compounds to provide neuroprotection we tested 5
of these compounds in the retinal explant model. Two of these com-
pounds (38, 56; Groups 2 and 3) demonstrated a significant, but
incomplete, RGC survival compared to untreated controls (Supple-
mentary Fig. 8A, B) and two compounds (54, 55; Group 5) demon-
strated a significant, almost complete, protection to RGCs following
axotomy (Fig. 8E). Taken together, we demonstrate that NMNAT2 is a
druggable target to generate high levels of neuron-specificNADand
that these novel small molecules can provide neuroprotection in the
retina.
Discussion
Our study demonstrates that NMNAT2 expression is overwhelmingly
restricted to RGCs in the retina (although it is presentin the majority of
neuronal cell types in the body), is highly variable within RGCs and
across individuals, and that when expression falls below a critical
threshold, RGC degeneration is significantly compounded. This sup-
ports NMNAT2 loss (RNA and/or protein) in glaucoma as animportant
component of neurodegeneration and that individuals on the lower
spectrum of NMNAT2 expression may be more susceptible to RGC
loss. However, NMNAT2 protein levels in individual RGCs remains to
be definitively assessed which will require the development of RGC
specific tools to label NMNAT2 or highly specific antibodies to
NMNAT2 suitable for protein quantification. Similarly, NMNAT2
expression (RNA and protein) is highly variable in aged postmortem
human brains, and further decreased in brains withAlzheimer’sdisease
where lower NMNAT2 is correlated with worse cognition10.We
demonstrate that reduced Nmnat2 expression drives susceptibility to
axon injury (axotomy) and future experiments could assess this long
term in more chronic optic nerve injury models in these mice (e.g.
Fig. 3 | Genetherapy delivery of humanNMNAT2 is strongly neuroprotectiveto
retinal ganglion cells. A Overexpression of hNMNAT2 robustly protects against
RGC loss in Nmnat2gtBay/gtE (25%) retinas maintained for 3 days ex vivo following
axotomy, rescuing the RGC sensitization phenotype ofthese mice (Day 0:
Nmnat2gtBay/gtE,n=6; Day3: Nmnat2gtBay/gtE,n=6;Nmnat2gtBay/gtE +hNMNAT2,n=5;
scale bar = 20 µm). BOverexpression of hNMNAT2 in C57BL/6J mice confers com-
plete protection against RGC loss at 3 days ex vivo (n=6 retinas for all conditions).
CIn the rat ocularhypertension(OHT) model, whichrecapitulatesmany features of
human glaucoma, significant RGC loss occurs following 14 days of elevated
intraocular pressure (OHT) relative to controls (NT, normotensive). Transfection
(3 weeks prior to OHT onset) and expression of GFP alone (AAV GFP) does not
significantly alter RGC survival, but RGC survival is significantly enhanced with
hNMNAT2 expression. This demonstrates that in a complex disease (with many
neurodegenerative mechanisms) NMNAT2 gene therapy provides moderate neu-
roprotection to RGCs (NT n=12 eyes, OHT n= 9 eyes, OHT AAV GFP n=10eyes,
OHT hNMNAT2 n= 8 eyes). Scale bars = 25 µm in all images. For A,Band
C,*P<0.05,**P<0.01, ***P<0.001,NSnon-significant (P> 0.05); One-way ANOVA
with Tukey’sHSD. For boxplots, the centre hingerepresents the median with upper
and lower hinges representing the first and third quartiles; whiskers represent 1.5
times the interquartile range.
Article https://doi.org/10.1038/s41467-024-50354-5
Nature Communications | (2024) 15:6256 5
Content courtesy of Springer Nature, terms of use apply. Rights reserved

optic nerve crush at different ages). Supporting these findings,
restoring NMNAT2 expression in depleted retinas via gene therapy
removes this susceptibility to RGC degeneration and provides neuro-
protection. This neuroprotection is maintained through to a chronic
in vivo glaucoma model where the protection is not fully complete
likely due to the other OHT-related events (i.e. changes in vascular
tone, neuroinflammation) which a neuron-specific therapy cannot
overcome.
Neuroprotection through similar mechanisms has previously
been demonstrated in other neuronal systems through expression of
WldS(the protein product of which functionally recapitulates the
physiological role of NMNAT214) or through modulating the stability
and subcellular localization of NMNAT223–25. Similarly, Fang et al.9
demonstrated that gene therapy delivery of Nmnat2Δex6, a more
stable cytosolic form of NMNAT2, is also neuroprotective. In cell
culture, Nmnat2Δex6 provides stronger neuroprotection than native
Nmnat2 overexpression or WldS26. In this regard, Nmnat2Δex6
behaves functionally similar to the WldSprotein26 which has pre-
viously been demonstrated to be robustly neuroprotective in
glaucoma27–29.
A number of NAD precursors have been explored for their
potential to increase NAD across different tissues, particularly the CNS
where depletion of NAD pools drives, or contributes to, neurodegen-
eration. Nicotinamide has strong therapeutic potential given its long
clinical history, strong neuroprotection in animal models of glaucoma
and other neurodegenerative diseases30, and functional benefits
established in short-term clinical trials31,32. However, nicotinamide
requires high doses to achieve meaningful NAD increases in the CNS
and is not specific to neurons. Whilst nicotinamide riboside (NR) can
achieve greater (although more transient) NAD increases than nicoti-
namide at lower doses33, its utility in the retina is more limited due to
the low expression of NRK (required to convert NR to NMN6)which
produces only a modest 10–20% NAD increase34. Stabilized versions of
NMN, while able to raise NAD, are potentially dangerous alternatives in
NAD depleted systems (such as in neurodegenerative diseases) given
that high ratios of NMN to NAD trigger SARM1 activation and axon
degeneration35–37. Similarly, SARM1 inhibition through newly devel-
oped small molecule inhibitors are strong neuroprotective candidates
but are reliant on large pools of NAD38, which are absent during the
neurodegenerative disease cascades. While SARM1 inhibitors will limit
NAD consumption, they do not increase new NAD formation and so
their long-term use may be limited. Supporting this, Sarm1−/−does not
prevent RGC soma loss following optic nerve crush39.Intheaxon
degeneration cascade, NMNAT2 acts up-stream of SARM140,andso
offers an alternate target, particularly as restoring the capacity of
NMNAT2 activity towards normal levels is unlikely to present sig-
nificant side effect in comparison to removal of a native function (e.g.
blocking SARM1). In addition, inhibition of SARM1 could have dele-
terious effects given its protective role in preventing viral spread41,42.
Small molecules that target NMNAT2 could provide neuronaltargeted
increases in NAD to boost NAD pools and prevent the initiation of
neurodegenerative cascades. This could be relevant in many neuro-
degenerative diseases in which a depletion of NAD generating capacity
or NMNAT2 levels have been demonstrated.
Fig. 4 | EGCG increases NAD and provides a robust neuroprotection following
retinalganglion cellinjury. A EGCG increasesNAD in a dose-dependent mannerin
dissociated cortical neurons, with 50nM the lowest dose to give a significant
increase in NAD compared to untreated controls (each condition assessed in a
sample from the same biological replicate; n=4). BEGCG increases NAD in a time
dependent manner in dissociated cortical neurons. EGCG was first added to the 6-h
samples(and maintainedthroughout), 2 h later EGCG was added to the4-h sample,
etc. The 0-time sample was incubated for 6h in media without EGCG. An increased
cell viability in the samples treated with EGCG at an earlier time point may
also contribute to the 5-fold increase in NAD (each condition assessed in asample
from the same biological replicate; n=4).CEGCG significantly increased NAD in
neuron-enriched tissue (cortex; n= 4 per condition) but not in neuron-low tissues
(spleen, muscle, and liver; n= 4 per condition) suggesting a specificity towards
Nmnat2 over Nmnat1. DEGCG dissolved in the culture media robustly protects
against RGC death at 3 days ex vivo (3 DEV) following axotomy in comparison to
untreated controls (n= 6, all conditions) (interventional treatment). EIn the rat
OHT model,prophylacticoral EGCG provideda modest neuroprotectionrelative to
untreated controls at 40 mg/kg/d (n= 12), but not at 20 mg/kg/d (n= 7), although
this was improved in combination with hNMNAT2 (n= 11). FIn postmortem retinal
punches(n= 10 donor retinas)maintained ex vivofor 7 days (7 DEV) significantRGC
loss occurs which is significantly reduced by EGCG (or nicotinamide, NAM, the
precursor for NAD through the NAD-salvage pathway) relative to uncultured con-
trols (0 DEV), supporting the human utility of neuroprotection by EGCG (each
condition assessed in a sample from the same biological replicate, n=10).The
prolonged postmortem time (24–48h) results in significant RGC loss,and so in this
context EGCG is able to provide interventional neuroprotection to an already
degenerating system. Scale bars = 25 µminD,Eand50µminF.*P<0.05,**P<0.01,
***P<0.001,NSnon-significant (P> 0.05); Student’st-test to control for A,B,andC;
One-way ANOVA with Tukey’sHSD for D,E,andF. For box plots, the centre hinge
represents the median with upper and lower hinges representing the firstand third
quartiles; whiskers represent 1.5 times the interquartile range.
Article https://doi.org/10.1038/s41467-024-50354-5
Nature Communications | (2024) 15:6256 6
Content courtesy of Springer Nature, terms of use apply. Rights reserved

We identified that EGCG can increase NAD and provide neuro-
protection against RGC injury across in vitro, ex vivo, and in vivo
models (including in ex vivo human tissue). Across multiple methods
of raising cellular NAD levels in neural tissue (including via Nmnat1
gene therapy3,WldStransgene27, and high dose nicotinamide3,20)we
have consistently reached a threshold/ceiling of ~200% of control NAD
(which is sufficient for strong/complete neuroprotection in the retina
and optic nerve). EGCG at µM doses can achieve this level of NAD
increase in neuronal tissue to a therapeutic level. Critically, EGCG
increased NAD capacity in neuronal, but not non-neuronal, tissues.
Supporting that this occurs due to specificity for NMNAT2, EGCG’s
NAD boosting and neuroprotective effects are reduced or blocked in
the presence of the NAMPT inhibitor FK866 or with Nmnat2 depletion
(it is important to note that in these Nmnat2 depleted systems, ~25%
Nmnat2 activity remains. As complete Nmnat2 KO mice are not viable,
further development of conditional tools of Nmnat2 would be of
benefittofurtherconfirm these findings). Our in silico data support
the existence of a binding site for EGCG on an NMNAT2 homology
model. As a polyphenol, EGCG has a number of potential mechanisms
of action that could be providing neuroprotection including through
modulation of reactive oxygen species, cell signalling, growth factors,
autophagy, and apoptotic cascades43. Modulation of these could also
affect downstream NAD levels44. Glaucoma is a complex neurodegen-
erative disease in which these factors have previously been identified
as potential pathological mechanisms45. While EGCG did not increase
NAD in non-neuronal tissues, this does not preclude other effects
derived from these other properties if given as an oral treatment.
Considering EGCG’s poor bioavailability22, it would likely have a poor
therapeutic index.
Rather than develop a strategy fordelivering therapeutic levels of
EGCG (e.g. via encapsulation or direct targeting to the eye) we instead
used EGCG as a tool compound to rationally design novel NMNAT2
targeting small molecules. We confirmed that our top candidates
significantly increase NAD capacity in an NMN-dependent manner,
supporting specificity to the NAD-salvage pathway, whilst in silico
modelling supported the maintenance of binding capability to
NMNAT2. Compounds in the later groups of refinement (e.g.Group4
and 5) demonstrated total loss of NAD boosting activity when co-
incubated with FK866, more so than for EGCG, further supporting
development towards an NMNAT2 specific mechanism of action and
loss of other potential mechanisms (e.g. reduced effects on mito-
chondrial membrane potential compared to EGCG). These top com-
pounds were able to provide neuroprotection against RGC
degeneration ex vivo, supporting their utility and potential as neuro-
protective therapeutics in the future. Next steps could further define
the effect of these compounds at a single cell level with specificity to
RGCs. This would allow the elucidation as to whether only RGCs
require increased NAD generating capacity for neuroprotection, or
whether other neurons in the retinal circuitry contribute to this neu-
roprotection. These experiments could also assess whether subtypes
of RGCs are more or less susceptible to low or high NMNAT2 levels.
The small molecules developed here serve as a starting point for
further medicinal chemistry to develop NMNAT2-targeted drugs that
could be used in vivo, and potentially progress into human clinical
trials. The next important steps will be further drug development
focusing on ADME and toxicity in rodent and human cells lines,
toxicity in vivo, and in vivo pharmacodynamics and pharmacokinetics
to establish dose/response ranges which can then be tested in
mature, in vivo, animal models of glaucoma with multiple disease
metric assessed (i.e. visual function, optic nerve and axon
morphologies, mitochondrial dynamics). As NMNAT2 has been
implicated as a core component of axon degeneration, if properly
tested, these compounds have potential for other neurodegenerative
diseases.
In conclusion, we demonstrate that NMNAT2 is highly variable
within neuronal populations of the visual system,and that low levels of
Fig. 5 | EGCG improvesneuronal resilience to rotenone injury. A Primary retinal
neuron cultures wereestablished from P2-3 C57BL/6J mouse pups and grown for
10 days. At day 11, neurons were stressed with rotenone (1 µM) or remained vehicle
treated controls (DMSO) i n the presence of EGCG (5 µM) or controls (n= 6 wells per
condition) for 1 day. Rotenone caused a significant decrease in neuron density and
total neurite length which was prevented by EGCG treatment. BMice were either
treated with EGCG (or untreated) for 1 week prior to receiving an intravitreal
injection of 10 mM rotenone or DMSO only (vehicle). RBPMS density was assessed
1 day after intravitreal injection. Rotenone injection resulted in a significant loss of
RGCs in untreated mice ( ~40%) which was significantly mitigated by EGCG treat-
ment ( ~20% loss). DMSO only n= 6 retinas, Rotenone only n= 8 retinas, DMSO
EGCG n= 9 retinas, Rotenone EGCG n= 8 retinas. Scale bar = 25µmforB.ForAand
B,*P<0.05,**P< 0.01, ***P< 0.001, NS = non-significant (P>0.05);One-way
ANOVA with Tukey’sHSD. For box plots, the centre hinge represents the median
with upper and lower hinges representing the first and third quartiles; whiskers
represent 1.5 times the interquartile range.
Article https://doi.org/10.1038/s41467-024-50354-5
Nature Communications | (2024) 15:6256 7
Content courtesy of Springer Nature, terms of use apply. Rights reserved

NMNAT2 render RGCs susceptible to neurodegenerative insults.
Increasing NMNAT2 via gene therapy or increasing NMNAT2’sNAD
output pharmacologically robustly protects from neurodegenerative
insults. Using EGCG as a tool compound we designed, synthesized, and
tested novel small molecules that drive NAD production with specifi-
city to the NAD-salvage pathway and provide neuroprotection.
NMNAT2 targeting small molecules are ideal opportunities for clinical
translation in neurodegenerative diseases and aging.
Methods
Ethics statements
Individual study protocols were approved by Stockholm’s Committee
for Ethical Animal Research (10389-2018) or the UK Home Office (PPL
P98A03BF9). Access to human eye tissues and patient samples is fully
covered under “Studier av neuronal metabolism, biomarkörer och
neuroprotektion vid glaukom”2021-01036 (additions to base appli-
cation 2020-01525; Pete Williams).
Animal strain and husbandry
All breeding and experimental procedures were undertaken in accor-
dance with the Association for Research for Vision and Ophthalmology
Statement for the Use of Animals in Ophthalmic and Research. Animals
were housed and fed in a 12 h light/12h dark cycle with food and water
available ad libitum. Male Brown Norway rats (Rattus norvegicus)aged
16–20 weeks were purchased from SCANBUR and housed for 1 week
before beginning experiments. Male C57BL/6J mice (B6, SCANBUR)
were purchased at 10–12 weeks old and housed for 1–4weekbefore
beginning experiments. Nmnat2 gene-trap allele mouse lines
Nmnat2+/gtBay and Nmnat2+/gtE (as previously described15) were kindly
provided by Michael P. Coleman. Nmnat2+/gtBay and Nmnat2+/gtE were
subsequently bred in house (Stockholm) to produce Nmnat2 +/+,
Nmnat2+/gtBay,Nmnat2+/gtE,andNmnat2gtBay/gtE genotypes. Nmnat2 mice
(mixed sex) were used at 3, 6, and 12 months old. Tissue from mice
aged to 22-month-old were from the original Cambridge colony
housed in 12 light/dark with food and water available ad libitum. Mice
were euthanized and eyes were shipped to Stockholm for analysis.
NMNAT2 expression variability
To determine the variability of expression of NMNAT2 among geneti-
cally diverse individuals we used publicly available data from Gene-
Network (www.genenetwork.org46). For mice, BXD family populations
were assessed for Nmnat2 expression across available visual system
relevant datasets. We queried Nmnat2 mRNA expression in the eye
(n= 157; UTHSCBXD All Ages Eye RNA-Seq (Nov20) TPM Log2), in the
retina in young animals <100 days old (n= 55, DoD CDMRP Retina Affy
MoGene 2.0 ST (May15) RMA Gene and Exon Level), and in the mid-
brain (n= 37) which contains the superior colliculus, where ~80% of
RGCs project to in the mouse (VU BXD Midbrain Agilent SurePrint G3
Mouse GE (May12) Quantile). We determined whether Nmnat2
expression was related to RGC variability by performing a Spearman’s
rank correlation test comparing the average Nmnat2 expression in the
retina from GeneNetwork (DoD CDMRP Retina Affy MoGene 2.0 ST
(May15)RMA Gene and Exon Level) to the average RGC density of each
strain from Williams et al.11. From the same dataset, the variance in
expression of Nmnat2 was compared to RGC markers Pou4f1,Rbpms,
Thy1,andTubb3 by performing Paired Pitman-Morgan tests using the
var.test function in the PairedData package for R. We also queried how
Nmnat2 expression in the eye was changed by age (UTHSC Individual
BXD Adultand Aged Eye RNA-Seq(Dec20) TPM Log2) where mice were
grouped by age to either <200 days (n= 157) or >35 0 days (n= 187).For
human NMNAT2 expression in the retina, we queried retinal NMNAT2
mRNA across a sample of 50 individuals (TIGEM Human Retina RNA-
Seq (Sep16) RPKM log2) and whole retina bulk RNA-sequencing data
from The Genotype-Tissue Expression (GTEx) Project through The
Human Protein Atlas [accessed 11/22/2022]. NMNAT2 expression
Fig. 6 | EGCG increases NAD and provides neuroprotection through an NMN
and NMNAT2-dependent mechanism. A Addition of FK866 (an NAMPT inhibitor
which reduces the levels of NMN available to NMNAT2) significantly decreases the
capacity of EGCG to produce additional NAD (n= 4 cortex for all conditions). BIn
the retinal axotomy model addition of FK866 does notsignificantly alter RGC
survival compared to untreated controls. However, the neuroprotective effect of
EGCG was completely abolished in the presence of FK866, suggesting that in the
contextof a RGC injury, EGCG’s neuroprotective effect is derived through an NMN-
dependent mechanism (n= 6, all conditions). CSupporting this, in Nmnat2+/+ mice
(100% Nmnat2) EGCG provides complete neuroprotection at 3 DEV (n=4),butin
Nmnat2gtBay/gtE mice (25% Nmnat2,n=6), the neuroprotective effects of EGCG are
significantly diminished (44% RGC loss, which is comparable to untreated
Nmnat2+/+,Nmnat2+/gtBay,andNmnat2+/gtE mice). This suggests that the neuropro-
tective effects of EGCG work through an NMNAT2-dependent mechanism. Scale
bars = 25 µm in B and C. For A,B,andC,*P< 0.05, **P< 0.01, ***P<0.001,NS=non-
significant (P>0.05); One-way ANOVA with Tukey’sHSD. For box plots, the centre
hinge represents themedian with upperand lower hingesrepresentingthe first and
third quartiles; whiskers represent 1.5 times the interquartile range.
Article https://doi.org/10.1038/s41467-024-50354-5
Nature Communications | (2024) 15:6256 8
Content courtesy of Springer Nature, terms of use apply. Rights reserved

variability within individual RGCs was assessed using single-cell RNA-
sequencing data from Gautam et al.47, (GEO: GSE147979) and single-
nucleus RNA-sequencing data from Orozco et al.48, (GEO: GSE135133).
The datasets was processed using Seurat V3.2. QC was performed to
remove doublets and empty droplets by filtering cells with UMI
1000–6000 and detected genes with 500–5000. Clustering and cell
annotation ID provided by the authors were used to identify the major
cell types in the retina. To compare NMNAT2 expression across indi-
vidual RGCs, the NormalizeData() function was used to generate nor-
malized and log-transformed single-cell expression with a scale factor
of 10,000. To compare NMNAT2 expression across all retinal cell types
data were scaled using the ScaleData() function following identifica-
tion of highly variable features using the FindVariableFeatures() func-
tion (vst method; 2000 features). Nmnat2 expression across glaucoma
progression was determined from publicly available microarray gene-
expression data from whole optic nerve head (ONH) and whole retina
in 10.5-month-old DBA/2J (D2) mice12,13 (GSE26299). We explored
Nmant2 expression across the disease clusters identified by Howell
et al.12 which are based on the degree of genetic change (and corre-
spond to the degree of neurodegeneration in histological optic nerve
analysis). In the ONH, these grouping are: Group 1 = no detectable
glaucoma, limited genetic change, (n= 8); Group 2 = no detectable
glaucoma, significant genetic change, (n=8); Group 3 = no or early
glaucoma, significant genetic change, (n= 6); Group 4 = moderate
glaucomatous degeneration, significant genetic change, (n= 4); Group
5 = severe glaucomatous degeneration, significant genetic change,
(n= 4). Expression is compared to n= 5 D2-Gpnmb + . In the retina,
there are 4 groups: Group 1 = no detectable glaucoma, limited genetic
change, (n= 8); Group 2 = no detectable glaucoma, moderate genetic
change, (n= 9); Group 3 = moderate glaucomatous degeneration,
significant genetic change, (n= 3); Group 4 = severe glaucomatous
degeneration, significant genetic change (n= 10). Expression is
Fig. 7 | EGCGprovides a basisfor generatingnovel NAD-producing compounds.
AAn NMNAT2 homology model using NMNAT1 and NMNAT3 was further refined
using loop-modelling software (DaReUS-Loop)to refine a low homology domain in
the central region of the protein (green). Molecular dynamic simulations demon-
strated that the protein reached a stable conformation after 100 ns. The final
generated protein conformations demonstrated greater reliability (ERRAT, VERIFY
3D, PROVE, and Z-score). BThree potential druggable binding pockets (indepen-
dent of the NMN catalytic pocket) were identified. The docking pose of EGCG was
well accommodatedin these three different druggable pockets withcorresponding
ΔGscores<−40 kcal/mol (left). Only one ligand-protein complex (Site 1; orange)
could maintain a stable conformation over a 500 ns molecular dynamic simulation
(right), demonstrating in silico evidence that EGCG can directly bind to NMNAT2.
CGiven its poor drug-like properties, EGCG was used as a tool compound to
identify novel NAD-producing compounds. The EGCG structure was truncated in
series to identify a biologically active core which was then used in a scaffold-
hopping strategy. DAn iterative synthesis and NAD-testing (luminometry of dis-
sociated cortex neurons) pipeline was established to identify and test NAD-
producing compounds. A number of compounds with greater efficacy than EGCG
at producing NAD were identified (n= 4/condition; statistical testing in Supple-
mentaryData 1). Outliersdenoted by black circles. *P<0.05,**P< 0.01,***P<0.001,
NS non-significant (P> 0.05). Forbox plots, the centre hinge representsthe median
with upper and lower hinges representing the first and third quartiles; whiskers
represent 1.5 times the interquartile range.
Article https://doi.org/10.1038/s41467-024-50354-5
Nature Communications | (2024) 15:6256 9
Content courtesy of Springer Nature, terms of use apply. Rights reserved

compared to n= 8 D2-Gpnmb + . Nmnat2 expression in RGCs early in
glaucoma (9-month-oldD2) was explored using publicly available data
from Williams et al.3; all mice used for these experiments were con-
firmed to have no detectable neurodegeneration in the optic nerve
(n= 9 D2-Gpnmb+ and n= 26 D2). Grouping represents degree of
genetic change (1 = least, 4 = most): Group 1 (n=9age-matched D2-
Gpnmb+ and n=6 D2s), Group 2 (n= 6 D2s), Group 3 (n=10 D2s),
Group 4 (n= 4 D2s).
Human donor retina
For ex vivo human retina, de-identified eyes were acquired from the St.
Erik eye hospital corneal eye bank as waste tissue following donation
for corneal transplant. Only eyes free from known ocular or metabolic
disease were selected. Upon receipt, retinas were dissected free and
the vitreous was removed. Donor tissue details are recorded in Sup-
plementary Table 1. Retinal punches were taken (Sterile Disposable
BiopsyPunch, 2 mm diameter, MILTEX) in an arc 3 mm nasally from the
Fig. 8 | Generation of NAD-salvage pathway specificcompounds that drive NAD
production and provide neuroprotection. A We selected 10 compounds for
further testing based on their NAD-producing capacity (structures shown in com-
parison to EGCG). BFor 9 of the 10 top compounds, FK866 suppressed the NAD-
boosting effect, demonstrating that these drugs retained a mechanism of action
through the NAD-salvage pathway (n=4 cortex for all conditions; fold change in
NAD comparable to normal controls, denotedby red space; foldchange comparable
to FK866 treated normal controls, denoted by black space; statistical testing in
Supplementary Data 2). CCompounds were tested for NAD modifying capacity in
dissociated cortex, liver, muscle, and spleen (n=4 for all conditions). An NAD-
boosting effect in neuron-low tissue was only demonstrated for 2 compounds (51,
56) withboth increasingNAD in muscle by<1.2 fold relative to untreated controls. A
reduction in NAD relative to control was identified for 3 compounds in neuron-low
tissue, with compound 21 and 55 reducing NAD in spleen to ~0.8 fold, and
compound 54 reducing NAD in muscle to ~0.9 fold. Results are normalized to
untreated controls of matched tissue type. DThe compounds were used to gen-
erate a structure-activity relationship model which identified favourable (green, at
C2) and unfavourable (purple) hydrophobic regions which affect the activity, and
negative electrostatics regions (blue, at C1) which are crucial for NAD-boosting
activity. EFive of the top ten compounds were tested for neuroprotective capacity
in a retinal explant model. Compounds 54 and 55 (from group 5) demonstrated a
significant protection of RGCs,demonstratingthe potentialof these compounds to
provide neuroprotection (n=6 retina/condition). Scale bar = 20 µm. For B,C,and
E,*P< 0.05, **P<0.01,***P<0.001,NSnon-significant (P>0.05), Student’st-test to
control. For box plots, the centre hinge represents the median with upper and
lower hinges representing the first and third quartiles; whiskers represent 1.5 times
the interquartile range.
Article https://doi.org/10.1038/s41467-024-50354-5
Nature Communications | (2024) 15:6256 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved

optic nerve head. Punches were used for a retinal explant model
(described below).
Retinal explant model
Retinal explants were performed as previously described for mice20
and human retinas49. Flat mounted retinas were maintained in culture
(37 °C, 5% CO
2
) fed by Neurobasal-A media supplemented with 2 mM
L-glutamate (GlutaMAX, Gibco) 2% B27, 1% N2, and 1% penicillin/
streptomycin (all Gibco) in 6-well (mouse) and 24-well culture plates
(human) with corresponding cell culture inserts (Millicell 0.4 µmpore;
Merck). For mice, animals were euthanized by cervical dislocation,
retinas dissected free in cold HBSS and flat mounted on cell culture
inserts ganglion cell layer up. Retinas were removed from culture and
fixed in 3.7% PFA at 3 days ex vivo for cell counts or homogenized at
12 h ex vivo for qPCR (see below for details). Control eyes were pro-
cessed immediately following enucleation. For cell counts, retinas
were dissected following 1 h of fixation. For human retina, 2 punches
were immediately fixed in 3.7% PFA (control) following dissection, and
6 were explanted on to individual cell culture inserts ganglion cell layer
up (2 untreated, 2 NAM treated, 2 EGCG treated). Punches were
maintained in culture ex vivo as described above for 7 days before
fixation in 3.7% PFA. Media was changed at day 2 for the mouse, and on
alternate days for the human. For drug treatments, drugs were dis-
solved in the culture media to a concentration of: EGCG (5, 50 µMfor
mouse; 50 µM for human; Merck), NAM (500mM for human; PanReac
AppliChem), whole green tea polyphenol extract (0.1 µg, 1 µgfor
mouse; Abcam), FK866 (10 µM for mouse; Sigma). Novel compounds
were diluted to 0.5 µM, 5 µM, and 50 µMinDMSO.
Rat bead model and drug treatments
Prior to induction of ocular hypertension rats were habituated to
intraocular pressure (IOP) measurement by rebound tonometry
(Tonolab, Icare). Baseline IOP was recorded the morning before surgery
(day 0). For drug treatments, rats received drug dissolved in drinking
water 1 week prior to OHT induction, with treatment continuing for the
remainder of the experiment (14 days). Drug doses were calculated
based on an average daily intake of 15 ml of water per rat. The effective
EGCG dose was ~20 or 40 mg/kg/d. Whole green tea polyphenol extract
was given to achieve a dose of ~80 mg/kg/d. The effective whole green
tea polyphenol dose was ~80 mg/kg/d. Ocular hypertension was induced
using a paramagnetic bead model as previously described50.Ratswere
anaesthetized with an intraperitoneal injection of ketamine (37.5 mg/kg)
and medetomidine hydrochloride (1.25 mg/kg). Microbeads (Dynabead
Epoxy M-450, Thermo Fisher) were prepared in Hank’s balanced salt
solution (HBSS -CaCl
2
-MgCl
2
-phenol red, Gibco) and 6–8μlofbead
solution was injected into the anterior chamber. Beads were distributed
using a magnet to block the iridocorneal angle. Rats received either
bilateral injections (OHT) or remained bilateral unoperated (naïve),
normotensive controls (NT). IOP was measured at day 3, 7, 9, 11, and 14
post induction of OHT. IOP recordings were always performed between
9 and 10 am to avoid the effects of the circadian rhythm on IOP. Rats
were taken to either 7 days or 14 days as an end-point.
Gene therapy
Gene therapy was delivered through intravitreal injection of AAV2-
CMV-hNMNAT2-CMV-eGFP or AAV2-CMV-eGFP as a control. Mice or
rats were anaesthetized by intraperitoneal injection of ketamine and
medetomidine hydrochloride as above. Bilateral intravitrealinjections
were performed using a 33 G tri-beveled needle on a 10 µl glass syringe
(WPI). AAV diluted to 2.2 × 1011 GC/ml or AAV2-CMV-hNMNAT2-CMV-
eGFP 7.8 ×1011 GC/ml AAV2-CMV-eGFP in HBSS was injected into the
vitreous (1 µlformice,3µl for rats) and the needle maintained inplace
for 30 s for distribution. Three weeks was allowed to achieve sufficient
transduction and expression before animals were used for either the
retinalexplantmodelortheratbeadmodel.
Intravitreal rotenone model
Rotenone was delivered in vivo to the retina through intravitreal
injection20. B6 mice were pretreated with EGCG dissolved in drinking
water togive a dose of ~50 mg/kg/d or untreated. Intravitrealinjections
of either 1 μl of a 10 mM rotenone (MP Biochemicals) solution dis-
solved in DMSO (PanReac AppliChem) or DMSO only (vehicle only
control) were performed in EGCG and untreated mice. Mice were
euthanized 1 day following injection and retinas were processed for
immunofluorescent labelling of RGCs.
Primary retinal neuron culture
For primary retinal neuron cultures P2-3 B6 mice were euthanized by
decapitation and retinas were dissected. To dissociate cells, 6 retinas
were pooled in 1ml of dispase (500 U, Corning) and maintained at
37 °C for 45 min in a heating block (Thermomixer C, Eppendorf) set at
350 rpm. Cells were pelleted and resuspended in culture media for
seeding onto Poly-D-Lysine coverslips (Corning) in a 24-well plate. To
encourage the selective growth and maintenance of neurons, cells
were cultured in Neurobasal-A media supplemented with 2 mM L-
glutamate, 2% B27, 1% penicillin/streptomycin, and 50ng/ml BDNF.
Cells were cultured for 10 days with a media change every 2 days. At
day 10, neurons were stressed with rotenone (1 µMinDMSO)orDMSO
only and the media was supplemented with EGCG (5µM) or remained
untreated. At day 11, neurons were fixed with ice cold methanol for
20 min and processed for immunofluorescent labelling in the wells.
Neurons were permeabilized in 0.5% TritonX for 5 min, blocked in 1%
BSA for 30 min, incubated with anti-βIII-tubulin (NB100-1612, Novus-
Biological). Neurons were washed 3× in PBS for 5 min, incubated with
secondary antibody for 1h and washed 3× in PBS for 5 min. Coverslips
were removed and inverted on to glass slides with Fluoromount-G
mounting medium. Six images per coverslip were acquired on a Zeiss
Axioskop 2 plus epifluorescence microscope (Karl Zeiss) at 40×. Using
Imaris (Bitplane, version 9.3.1) neuron morphology was reconstructed
with the filament function to calculate the number of neuron clusters
and total neurite length. Values per sample (coverslip) were taken as
the mean of six images.
qPCR
Rat optic nerves were collected following euthanasia at 7 days post-
OHT induction (and NT controls) using pentobarbital (75 mg/kg)
followed by cervical dislocation. The brain was removed and the
optic nerves cut at the chiasm. Optic nerves were cut to 4 mm from
the end proximal to the eye before flash freezing on dry ice. Before
use, optic nerves were thawed on ice and homogenized and soni-
cated in 350 µl DNAse free water for 20 s, 30,000 min−1(VDI 12, VWR).
Half of the sample was combined with 150µl of 2× buffer RLT (Qia-
gen) with 2% β-mercaptoethanol (Fisher Scientific). Flat mounted
retina were lifted from culture inserts by gentle agitation with HBSS
and homogenized into 400 µl buffer RLT (Qiagen) with 1% β-
mercaptoethanol (Fisher Scientific) using a QIAshredder kit (Qia-
gen) according to the manufacturer’s instructions. For all tissue, RNA
was extracted using RNeasy Mini Kits (Qiagen) according to the
manufacturer’s instructions. RNA was extracted into nuclease-free
water, and RNA concentration was measured in a 1 µl sample diluted
1:200 in nuclease-free water in a spectrophotometer (BioPhot-
ometer, Eppendorf). cDNA was synthesized using 1µg of input RNA
with an iScript™cDNA Synthesis Kit and MyIQ thermocycler (both
Bio-Rad) and stored at −20°C overnight. RT-qPCR was performed
using 1 µg of input cDNA, 7.5 µl of SsoAdvanced Universal SYBR Green
Supermix and 1 µl of the following DNA templates (Prime PCR Assay,
Bio-Rad): Nmnat2 (qMmuCID0005266) and Rps18 (housekeeping;
qMmuCED0045436) in the mouse; Nmnat2 (qRnoCID0001493), and
GAPDH and TBP (housekeeping; qRnoCID0057018, qRno-
CID0057007) in the rat (all templates were species specific—Mus
musculus,Rattus norvegicus, respectively; Bio-Rad). A MyIQ
Article https://doi.org/10.1038/s41467-024-50354-5
Nature Communications | (2024) 15:6256 11
Content courtesy of Springer Nature, terms of use apply. Rights reserved

thermocycler was used with a 3 min activation and denaturation step
at 95 °C, followed by an amplification stage comprising 50 cycles of a
15 s denaturation at 95 °C and1 min annealing and plate read at 60 °C.
Analysis was performed according to the ΔΔCT method.
Immunofluorescent labelling of RGCs
Following fixation, retinal flat mounts or punches were mounted on
slides (histobond) and isolated using a hydrophobic barrier pen
(VWR). Retinas were permeabilized with 0.5% Triton X-100 (VWR) in
PBS for 30 min, blocked in 2% bovine serum albumin (BSA; Fisher
Scientific) in PBS for 30 min, and primary antibody were applied and
maintained overnight at 4 °C. Following five repeated washes of
5 min with PBS, secondary antibodies were applied and slides were
maintained at room temperature for 2 h. Tissue was washed as before
and DAPI nuclear stain (1 μg/ml in PBS) was applied for 10 min. Tissue
was washed once in PBS before being mounted using Fluoromount-G
and glass coverslips were applied. Slides were sealed with nail var-
nish. The immunofluorescent labelling of the retinas treated with
C21, C38, C54, C55 and C56 in the retinalexplant model followed this
protocol but with the following variations: retinas were permeabi-
lized with 0.1% Triton X-100 in PBS for 60 min and blocked in 2% BSA
in HBSS for 60 min. Primary antibody was applied and maintained
over three nights at 4°C, following five repeated washes for 10 min,
secondary antibody was applied and maintained for 4h at RT. Reti-
nas were washed 5 times for 10 min before staining with 5µg/mL
nuclear Hoescht 33342 nuclear stain (Thermofisher Scientific) dilu-
ted in PBS. Primary antibodies used were: anti-RBPMS (NovusBiolo-
gical, cat #NBP2-20112, lot # 42858; 1:500) with or without anti-GFP
(Abcam, cat # ab13970, lot #, GR3190550-8 and 1018753-4; 1:500) for
retinas which had received gene therapy. RGC density was assessed
by counting RBPMS+ cells in the GCL. Images were acquired on either
a Zeiss Axioskop 2 plus epifluorescence microscope or Zeiss
LSM800-Airy (both Karl Zeiss). Six images per retina were taken
equidistant at 0, 2, 4, 6, 8, and 10 o’clock abouta superior to inferior
line through the optic nerve head ( ~1000 μm eccentricity). Images
were cropped to 100 × 100 µm and RBPMS+ cells were counted using
the cell counter plugin for Fiji; counts were averaged across the 6
images. Cells were counted by a minimum of two observers blinded
to treatment.
Luminescence-based NAD assay
B6 mice were euthanized by cervical dislocation, and the whole
cortex was removed and separated by hemisphere. For experiments
where other tissues were assessed, whole spleen, the left liver lobe,
and hamstring muscles were dissected and collected. Tissue was
stored on ice in HBSS until the next step. Cortical hemispheres
(maintained separately) were submerged in 800 µl dispase (5000
Caseinolytic units, 354235, Corning). Spleen was cut into two pieces
and the pieces were put separately in 800 µl dispase. The left liver
lobe was cut into two pieces and the pieces were separately added to
800 µl dispase, and each hindlimb muscle was a separate sample
added to 800 µl dispase. Tissues were finely chopped in dispase and
were put on a heating block (ThermoMixer® C, Eppendorf) at 37 °C,
350 rpm for 30 min before dissociation by gentle trituration. Cell
concentration of the cell suspension was determined with a hemo-
cytometer (C-Chip, NanoEntek). Cortical, spleen, liver, and muscle
samples were diluted to a concentration of 2 million cells/ml with
HBSS (in which 100,000 cells were used per assay well). (Due to the
low yield ( > 50,000 RGCs/retina), NAD levels in purified RGCs were
not assessed in these studies.) Samples were incubated with com-
pounds of interest for 2 h at 37 °C and 5% CO
2
(solutions were diluted
with HBSS from 50 mM stock solution in DMSO (A3672-0100, Pan-
Reac AppliChem)). After incubation, samples were homogenized for
20 s with a handheld homogeniser (30.000 min−1, VDI 12, VWR).
Compounds which displayed activity at 5 µM but not at 50 µM were
re-tested in an updated protocol where, following treatment, sam-
ples were spun down at 7700rpm (4000 g) for 5 min, and the
supernatant was removed and exchanged with HBSS prior to
homogenization to remove drugs that may interfere with the lumi-
nometric signal. To detect NAD, detection, the NAD reagent was
prepared according to the manufacturer’s protocol (NAD/NADH-
Glo™, Promega). 50 µl of the sample was combined with 50 µlof
reagent in a 96-well plate (Nunc™F96 MicroWell™White Polystyrene
plate, ThermoFisher Scientific) and luminescence was recorded with
a plate reader (infinite® 200, Tecan) 1 h from initial mixing. Fold
changes in samples were compared to the luminescence signal in
control samples. To determine the effect of EGCG on NAD over time
the experiment followed the same procedure as above, except for
incubation time. After dissociation and diluting cell samples, EGCG
was first added to 6-h samples, and 2 h later EGCG was added to the
4-h sample, etc. The 0-h samples were incubated for 6 h in HBSS
without EGCG for 6 h. All samples were incubated at 37 °C and 5%
CO
2
throughout the time. After incubation samples were processed
in the same way as previously stated to assess NAD levels.
Measuring mitochondrial membrane potential with JC-1
Mitochondrial membrane potential change of mouse cortical cells
after treatment with EGCG and compound using JC-1 as previously
described51. Four hemispheres from whole cortexes were collected and
dissociated as described above. Samples were diluted to 1 million cells/
ml in HBSS and incubated with EGCG, C21, C37, C38, C39, C40, C51,
C52, C54, C55, C56 at 5 µMand50µMfor1.5h(37°C,5%CO
2
). JC-1
(200 µMinDMSO)wasaddedtoafinal concentration of 2 µMto
the samples and the sampleswere further incubated for 30 min (37 °C,
5% CO
2
).Cellswerespundownat7700rpm(4000g,MiniSpin®,
Eppendorf) and resuspended in 1 mL HBSS. 50 µl of each sample was
loaded on a 96-well plate (Nunc™F96 MicroWell™White Polystyrene
plate, Thermo Fisher Scientific) and the fluorescence was measured
using excitation/emission at 485/535 nm and 535/590 nm (infinite®
200, Tecan). Mitochondrial membrane potential difference (ΔΨ)
was calculated from the ratio between the two wavelength
measurements.
EGCG pH stability
EGCG (pharmaceutical secondary standard, Sigma Aldrich), was dilu-
ted in DMSO to 100 mM and diluted to 1 mM in HBSS (Gibco) adjusted
to pH 1.8, 3, 4, 5,6 and non-adjusted HBSS (pH 7.6) and kept at 37 °C.
The absorbance was measured over 190–850 nm with a micro-UV/Vis
spectrophotometer (NanoDrop™OneC, ThermoFisher Scientific) at
time point 0, 0.5, 1, 2 and 24 h.
Molecular dynamic simulation
The 3D protein structure wasmodelled using the I-TASSER52 server and
the sequence of the human NMNAT2 (UniProt code Q9BZQ4).
NMNAT1 and NMNAT3 were used as templates to guide the NMNAT2
protein design as has previously been used53. The low homology
domain inthe central regionof the protein (111–190) was refined using
DaReUS-Loop54 and successively the entire protein was subject to
molecular dynamic (MD) simulation using Desmond packaged
(Maestro, Schrödinger 2022-2, New York, NY, USA), employing OPLS4
force field in the explicit solvent and the TIP4D water model. A cubic
water box was used for the solvation of the system, ensuring a buffer
distance of approximately 12Å between each box side and the com-
plex atoms. The systems were neutralised by adding 6 sodium counter
ions. The system was then minimized and pre-equilibrated using the
default NPT relax protocol in Desmond. A 1 µsMDsimulationinNPT
ensembles at constant temperature (300K) and pressure (1atm). Data
were collected every 100 ps. Hierarchical clustering based on the
structural root-mean-squared distance (RMSD) of Cαwas used to
group the different protein conformations. The three most populated
Article https://doi.org/10.1038/s41467-024-50354-5
Nature Communications | (2024) 15:6256 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved

clusters were used to select three representative structures for sub-
sequent identification of binding sites. Molecular Operating Environ-
ment (MOE) 2022 (2022.02, Chemical Computing Group ULC,
Montreal, QC, Canada) was used to visualize the protein structures.
NMNAT2 structure validation
ProsaWeb55 and SAVES 6.0 (https://saves.mbi.ucla.edu/)wereasused
to evaluate the models generated through the modelling process. Each
test evaluates a different characteristic of the protein models based on
current information known about protein structures. These scores
were used to compare and rank the protein models.
Pocket identification, docking and validation
Each representative structure was processed using Schrödinger Site-
Map (Schrödinger Release 2022-2: SiteMap, Schrödinger) module to
identify potential protein binding pockets. Sitema p generated different
descriptors, which define the size, volume, degree of enclosure,
hydrophobicity and hydrophilicity of each pocket. All these descriptors
contribute to generating a Dscore and Sitecore, which assess the
druggability of the pockets. A consensus of the different identified
pockets among the three different representative structures was used
to select the best 3 binding pockets. For each identified pocket a 12 Å
docking grid was prepared for subsequent docking studies. The ECGC
molecule was first prepared considering the ionization states at pH
7 ± 2 and then subject to docking studies using Schrödinger Glide SP
modules precision keeping the default parameters and setting
(Schrödinger Release 2022-2: Glide, Schrödinger). Molecular mechan-
ics generalized Born surface area (MMGBSA) was used to re-score the
three output docking poses of each compound. Only the best-scored
pose for each docking was used for successively MD simulation to
assess the ligand-protein complex stability. The MD simulation was
carried out using a similar approach and was performed using the same
protocol in Desmond described above. The RMSD, pocket occupancies
and protein-ligand interactions analyses were performed using the
Simulation Interaction Diagram of Desmond. The ΔG binding values of
the protein–ligands complex was calculated using the MM/GBSA each
2.5 ns during the entire MD simulations.
Statistical analysis
All statistical analyses were performed in R. Data were tested for nor-
mality with a Shapiro–Wilk test. Normally distributed data were com-
pared by Student’st-test (one sided) or ANOVA (with Tukey’sHSD).
Non-normally distributed data analysed by a Kruskal Wallis test fol-
lowed Dunn’stests with Benjamini and Hochberg correction. Unless
otherwise stated, *P<0.05,**P<0.01,***P< 0.001, NS = non-significant
(P> 0.05). For box plots, the centre hinge represents the median with
upper and lower hinges representing the first and third quartiles;
whiskers represent 1.5 times the interquartile range.
Reporting summary
Further information on research design is available in the Nature
Portfolio Reporting Summary linked to this article.
Data availability
The data generated in this study are provided in the Supplementary
Information/Source Data file. NMNAT2 expression data are publicly
available from GeneNetwork (www.genenetwork.org) under gene
network accession codes GN1027, GN709, GN381, GN802. Whole
retina bulk RNA-sequencing data is available from The Genotype-
Tissue Expression (GTEx) Project through The Human Protein Atlas
[accessed 11/22/2022]. RGC single celland single nucleus RNA-seq data
are available through the Gene Expression Omnibus accession num-
bers GSE147979 and GSE135133. NMNAT2 expression from DBA/2 J
mice are available through the Gene Expression Omnibus accession
numbers GSE26299 and GSE90654. For protein modelling, the
sequence of human NMNAT2 used is accessible with UniProt code
Q9BZQ4. NMNAT1 and NMNAT3 structure used are available through
the RCSB Protein Data Bank, PDB code 1KQN17 for NMNAT1 and 1NUU
for NMNAT3. Source data are provided with this paper.
References
1. Verdin, E. NAD+in aging, metabolism, and neurodegeneration.
Science 350,1208–1213 (2015).
2. Tham, Y. C. et al. Global prevalence of glaucoma and projections of
glaucoma burden through 2040: a systematic review and meta-
analysis. Ophthalmology 121,2081–2090 (2014).
3. Williams, P. A. et al. Vitamin B-3 modulates mitochondrial vulner-
ability and prevents glaucoma in aged mice. Science 355,
756–760 (2017).
4. Harder, J. M. et al. Disturbed glucose and pyruvate metabolism in
glaucoma with neuroprotection by pyruvate or rapamycin. Proc.
Natl Acad. Sci. USA 117,33619–33627 (2020).
5. Tribble, J. R. et al. Midget retinal ganglion cell dendritic and mito-
chondrial degeneration is an early feature of human glaucoma.
Brain Commun. 1,fcz035(2019).
6. Tribble, J. R. et al. NAD salvage pathway machinery expression in
normal and glaucomatous retina and optic nerve. Acta Neuro-
pathol. Commun. 11, 18 (2023).
7. Lautrup,S.,Sinclair,D.A.,Mattson,M.P.&Fang,E.F.NAD
+in brain
aging and neurodegenerative disorders. Cell Metab. 30,
630–655 (2019).
8. Williams, P. A., Harder, J. M., Cardozo, B. H., Foxworth, N. E. & John,
S. W. M. Nicotinamide treatment robustly protects from
inherited mouse glaucoma. Commun. Integr. Biol. 11,e1356956
(2018).
9. Fang, F. et al. NMNAT2 is downregulated in glaucomatous RGCs,
and RGC-specific gene therapy rescues neurodegeneration and
visual function. Mol. Ther. 30,1421–1431 (2022).
10. Ali,Y.O.etal.NMNAT2:HSP90complexmediatesproteostasisin
proteinopathies. PLoS Biol. 14, e1002472 (2016).
11. Williams, R. W., Strom, R. C. & Goldowitz, D. Natural variation in
neuron number in mice is linked to a major quantitative trait locus
on Chr 11. J. Neurosci. 18,138–146 (1998).
12. Howell, G. R. et al. Molecular clustering identifies complement and
endothelin induction as early events in a mouse model of glau-
coma. J. Clin. Investig. 121,1429–1444 (2011).
13. Howell, G. R., Walton, D. O., King, B. L., Libby, R. T. & John, S. W.
Datgan, a reusable software system for facile interrogation and
visualization of complex transcription profiling data. BMC Geno-
mics 12, 429 (2011).
14. Gilley, J., Adalbert, R., Yu, G. & Coleman, M. P. Rescue of peripheral
and CNS axon defects in mice lacking NMNAT2. J. Neurosci. 33,
13410–13424 (2013).
15. Gilley,J.,Mayer,P.R.,Yu,G.&Coleman,M.P.Lowlevelsof
NMNAT2 compromise axon development and survival. Hum. Mol.
Genet 28, 448–458 (2019).
16. Carpenter,P.,Sefton,A.J.,Dreher,B.&Lim,W.L.Roleoftarget
tissue in regulating the development of retinal ganglion cells in the
albino rat: effects of kainate lesions in the superior colliculus. J.
Comp. Neurol. 251,240–259 (1986).
17. Pearson, H. E. & Stoffler, D. J. Retinal ganglion cell degeneration
following loss of postsynaptic target neurons in the dorsal lateral
geniculate nucleus of the adult cat. Exp. Neurol. 116,163–171
(1992).
18. Ljungberg, M. C. et al. CREB-activity and nmnat2 transcription are
down-regulated prior to neurodegeneration, while NMNAT2 over-
expression is neuroprotective, in a mouse model of human tauo-
pathy. Hum. Mol. Genet 21,251–267 (2012).
19. Berger, F., Lau, C., Dahlmann, M. & Ziegler, M. Subcellular com-
partmentation and differential catalytic properties of the three
Article https://doi.org/10.1038/s41467-024-50354-5
Nature Communications | (2024) 15:6256 13
Content courtesy of Springer Nature, terms of use apply. Rights reserved

human nicotinamide mononucleotide adenylyltransferase iso-
forms. J. Biol. Chem. 280, 36334–36341 (2005).
20. Tribble, J. R. et al. Nicotinamide provides neuroprotection in glau-
coma by protecting against mitochondrial and metabolic dys-
function. Redox Biol. 43,101988(2021).
21. Loreto, A. et al. Mitochondrial impairment activates the Wallerian
pathway through depletion of NMNAT2 leading to SARM1-
dependent axon degeneration. Neurobiol. Dis. 134, 104678 (2020).
22. Kim, S. et al. Plasma and tissue levels of tea catechins in rats and
mice during chronic consumption of green tea polyphenols. Nutr.
Cancer 37,41–48 (2000).
23. Milde, S., Gilley, J. & Coleman, M. P. Axonal trafficking of NMNAT2
anditsrolesinaxongrowthandsurvivalinvivo.Bioarchitecture 3,
133–140 (2013).
24. Milde,S.,Fox,A.N.,Freeman,M.R.&Coleman,M.P.Deletions
within its subcellular targeting domain enhance the axon protective
capacity of Nmnat2 in vivo. Sci. Rep. 3,2567(2013).
25. Summers, D. W., Milbrandt, J. & DiAntonio, A. Palmitoylation
enables MAPK-dependent proteostasis of axon survival factors.
Proc.NatlAcad.Sci.USA115,E8746–E8754 (2018).
26. Milde, S., Gilley, J. & Coleman, M. P. Subcellular localization
determines the stability and axon protective capacity of axon sur-
vival factor Nmnat2. PLoS Biol. 11, e1001539 (2013).
27. Williams, P. A. et al. Nicotinamide and WLDS act together to prevent
neurodegeneration in glaucoma. Front. Neurosci. 11, 232 (2017).
28. Howell, G. R. et al. Axons of retinal ganglion cells are insulted in the
optic nerve early in DBA/2J glaucoma. J. Cell Biol. 179,
1523–1537 (2007).
29. Risner, M. L. et al. Neuroprotection by WldSdepends on retinal
ganglion cell type and age in glaucoma. Mol. Neurodegener. 16,
36 (2021).
30. Cimaglia, G., Votruba, M., Morgan, J. E., André, H. & Williams, P. A.
Potential therapeutic benefitofNAD
+supplementation for Glaucoma
and Age-Related Macular Degeneration. Nutrients 12,2871(2020).
31. Hui, F. et al. Improvement in inner retinal function in glaucoma with
nicotinamide (vitamin B3) supplementation: a crossover rando-
mized clinical trial. Clin. Exp. Ophthalmol. 48,903–914 (2020).
32. De Moraes, C. G. et al. Nicotinamide and pyruvate for neu-
roenhancement in open-angle glaucoma: A phase 2 randomized
clinical trial. JAMA Ophthalmol. 140,11–18 (2022).
33. Trammell, S. A. et al. Nicotinamide riboside is uniquely and
orally bioavailable in mice and humans. Nat. Commun. 7, 12948
(2016).
34. Arizono, I. et al. Axonal protection by oral nicotinamide riboside
treatment with upregulated AMPK phosphorylation in a rat glau-
comatous degeneration model. Curr. Issues Mol. Biol. 45,
7097–7109 (2023).
35. Di Stefano, M. et al. A rise in NAD precursor nicotinamide mono-
nucleotide (NMN) after injury promotes axon degeneration. Cell
Death Differ. 22,731–742 (2015).
36. Gerdts,J.,Brace,E.J.,Sasaki,Y.,DiAntonio,A.&Milbrandt,J.SARM1
activation triggers axon degeneration locally via NAD+destruction.
Science 348,453–457 (2015).
37. Essuman, K. et al. The SARM1 toll/interleukin-1 receptor domain
possesses intrinsic NAD+cleavage activity that promotes patholo-
gicalaxonaldegeneration.Neuron 93,1334–1343.e1335 (2017).
38. Feldman, H. C. et al. Selective inhibitors of SARM1 targeting an
allosteric cysteine in the autoregulatory ARM domain. Proc. Natl
Acad.Sci.USA119, e2208457119 (2022).
39. Fernandes, K. A. et al. Role of SARM1 and DR6 in retinal ganglion cell
axonal and somal degeneration following axonal injury. Exp. Eye
Res 171,54–61 (2018).
40. Walker, L. J. et al. MAPK signaling promotes axonal degeneration by
speeding the turnover of the axonal maintenance factor NMNAT2.
Elife 6, e22540 (2017).
41. Szretter, K. J. et al. The immune adaptor molecule SARM modulates
tumor necrosis factor alpha production and microglia activation in
the brainstem and restricts West Nile Virus pathogenesis. J. Virol.
83,9329–9338 (2009).
42. Crawford, C. L. et al. SARM1 depletion slows axon degeneration in a
CNS model of neurotropic viral infection. Front Mol. Neurosci. 15,
860410 (2022).
43. Singh, B. N., Shankar, S. & Srivastava, R. K. Green tea catechin,
epigallocatechin-3-gallate (EGCG): mechanisms, perspectives and
clinical applications. Biochem Pharm. 82, 1807–1821 (2011).
44. Sun, C. et al. NAD depletion mediates cytotoxicity in human neu-
rons with autophagy deficiency. Cell Rep. 42, 112372 (2023).
45. Tribble, J. R. et al. Neuroprotection in glaucoma: mechanisms
beyond intraocular pressure lowering. Mol. Asp. Med 92,
101193 (2023).
46. Sloan, Z. et al. GeneNetwork: framework for web-based genetics. J.
Open Source Softw. 1https://doi.org/10.21105/joss.00025 (2016).
47. Gautam, P. et al. Multi-species single-cell transcriptomic analysis of
ocular compartment regulons. Nat. Commun. 12, 5675 (2021).
48. Orozco, L. D. et al. Integration of eQTL and a single-cell atlas in the
human eye identifies causal genes for age-related macular
degeneration. Cell Rep. 30,1246–1259.e1246 (2020).
49. Osborne, A., Hopes, M., Wright, P., Broadway, D. C. & Sanderson, J.
Human organotypic retinal cultures (HORCs) as a chronic experi-
mental model for investigation of retinal ganglion cell degenera-
tion. Exp. Eye Res. 143,28–38 (2016).
50. Tribble, J. R. et al. Retinal ganglion cell degeneration in a rat mag-
netic bead model of ocular hypertensive glaucoma. Transl.Vis.Sci.
Technol. 10, 21 (2021).
51. Canovai, A. et al. Pyrroloquinoline quinone drives ATP synthesis
in vitro and in vivo and provides retinal ganglion cell neuroprotec-
tion. Acta Neuropathol. Commun. 11,146(2023).
52. Yang, J. et al. The I-TASSER Suite: protein structure and function
prediction. Nat. Methods 12,7–8(2015).
53. Brunetti, L., Di Stefano, M., Ruggieri, S., Cimadamore, F. & Magni, G.
Homology modeling and deletion mutants of human nicotinamide
mononucleotide adenylyltransferase isozyme 2: new insights on
structure and function relationship. Protein Sci. 19,
2440–2450 (2010).
54. Karami, Y. et al. DaReUS-loop: a web server to model multiple loops
in homology models. Nucleic Acids Res 47,W423–W428 (2019).
55. Wiederstein, M.& Sippl, M. J. ProSA-web: interactiveweb service for
the recognition of errors in three-dimensional structures of pro-
teins. Nucleic Acids Res 35, W407–W410 (2007).
Acknowledgements
The Authors would like to thank Amelie Botling Taube for assistance
with writing and submitting ethics for donor human tissue use, as
well as Flavia Plastino, Helder André, and Virpi Luoma (and the St.
Erik Eye Hospital tissue lab/cornea transplant service) for assistance
in acquiring donor human retina for explant experiments. We thank
the staff at the Division of Eye and Vision’s animal facility for their
assistance in animal breeding and husbandry. We thank Rob Wil-
liams for his suggestions and assistance regarding GeneNetwork.
J.R.T is supported by Ögonfonden, KI Foundation Grants for Eye
Research, Loo och Hans Ostermans stiftelse, Stiftelsen Lars Hiertas
Minne, and St. Erik Eye Hospital philanthropic donations. A.L is
supported by Wellcome Trust. J.G is supported by BBSRC. G.J is
supported by the Knut and Alice Wallenberg Foundation and Region
Västerbotten. R.C.B.W is supported by the National Health and
Medical Research Council (Ideas grant) and Centre for Eye Research
Australia Foundation. M.P.C is supported by the John and Lucille van
Geest Foundation. P.A.W is supported by Karolinska Institutet in the
form of a Board of Research Faculty Funded Career Position, St. Erik
Eye Hospital philanthropic donations, Vetenskapsrådet (2018-02124
Article https://doi.org/10.1038/s41467-024-50354-5
Nature Communications | (2024) 15:6256 14
Content courtesy of Springer Nature, terms of use apply. Rights reserved

and 2022-00799), StratNeuro StartUp grant, Ögonfonden, Stiftelsen
Lars Hiertas Minne, Stiftelsen Kronprinsessan Margaretas Arbets-
nämnd för Synskadade, Karolinska Institutet Foundation Grants,
Petrus och Augusta Hedlunds Stiftelse, and The Glaucoma Foun-
dation. PAW is an Alcon Research Institute Young Investigator.
Author contributions
J.R.T. Performed experiments, performed analysis, created data visua-
lization, wrote the manuscript, M.J. Performed experiments, performed
analysis, created data visualization, wrote the manuscript, C.V. Per-
formed experiments, performed analysis, created data visualization,
wrote the manuscript, A.O. Performed experiments, A.C. Performed
experiments, performed analysis, B.H. Performed experiments, E.D.
Performedexperiments,R.C.Performedanalysis,A.L.Performed
experiments, wrote the manuscript, J.G. Wrote the manuscript, C.E.W.
Provided supervision, G.J. Provided supervision, R.C.B.W. Performed
analysis, wrote the manuscript, M.P.C. Provided resources, wrote the
manuscript, A.B. Provided resources, provided supervision, wrote the
manuscript, conceptualized experiments/methodologies, P.A.W. Per-
formed experiments, performed analysis, wrote the manuscript, con-
ceptualized ideas/experiments/methodologies.
Funding
Open access funding provided by Karolinska Institute.
Competing interests
PAWisaninventoronanawardedUSpatentheldbyTheJackson
Laboratory for nicotinamide treatment in glaucoma (“Treatment and
prevention of ocular neurodegenerative disorder”, US11389439B2).
PAW, MJ, CV, and AB are inventors on a submitted patent held
by Mim Neurosciences AB for novel NMNAT2-targeting small
molecules. All other authors declare that they have no competing
interests.
Additional information
Supplementary information The online version contains
supplementary material available at
https://doi.org/10.1038/s41467-024-50354-5.
Correspondence and requests for materials should be addressed to
Pete A. Williams.
Peer review information Nature Communications thanks Sovan Sarkar,
and the other, anonymous, reviewers for their contribution to the peer
review of this work. A peer review file is available.
Reprints and permissions information is available at
http://www.nature.com/reprints
Publisher’s note Springer Nature remains neutral with regard to jur-
isdictional claims in published maps and institutional affiliations.
Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as
long as you give appropriate credit to the original author(s) and the
source, provide a link to the Creative Commons licence, and indicate if
changes were made. The images or other third party material in this
article are included in the article’s Creative Commons licence, unless
indicated otherwise in a credit line to the material. If material is not
included in the article’s Creative Commons licence and your intended
use is not permitted by statutory regulation or exceeds the permitted
use, you will need to obtain permission directly from the copyright
holder. To view a copy of this licence, visit http://creativecommons.org/
licenses/by/4.0/.
© The Author(s) 2024, corrected publication 2024
Article https://doi.org/10.1038/s41467-024-50354-5
Nature Communications | (2024) 15:6256 15
Content courtesy of Springer Nature, terms of use apply. Rights reserved

1.
2.
3.
4.
5.
6.
Terms and Conditions
Springer Nature journal content, brought to you courtesy of Springer Nature Customer Service Center GmbH (“Springer Nature”).
Springer Nature supports a reasonable amount of sharing of research papers by authors, subscribers and authorised users (“Users”), for small-
scale personal, non-commercial use provided that all copyright, trade and service marks and other proprietary notices are maintained. By
accessing, sharing, receiving or otherwise using the Springer Nature journal content you agree to these terms of use (“Terms”). For these
purposes, Springer Nature considers academic use (by researchers and students) to be non-commercial.
These Terms are supplementary and will apply in addition to any applicable website terms and conditions, a relevant site licence or a personal
subscription. These Terms will prevail over any conflict or ambiguity with regards to the relevant terms, a site licence or a personal subscription
(to the extent of the conflict or ambiguity only). For Creative Commons-licensed articles, the terms of the Creative Commons license used will
apply.
We collect and use personal data to provide access to the Springer Nature journal content. We may also use these personal data internally within
ResearchGate and Springer Nature and as agreed share it, in an anonymised way, for purposes of tracking, analysis and reporting. We will not
otherwise disclose your personal data outside the ResearchGate or the Springer Nature group of companies unless we have your permission as
detailed in the Privacy Policy.
While Users may use the Springer Nature journal content for small scale, personal non-commercial use, it is important to note that Users may
not:
use such content for the purpose of providing other users with access on a regular or large scale basis or as a means to circumvent access
control;
use such content where to do so would be considered a criminal or statutory offence in any jurisdiction, or gives rise to civil liability, or is
otherwise unlawful;
falsely or misleadingly imply or suggest endorsement, approval , sponsorship, or association unless explicitly agreed to by Springer Nature in
writing;
use bots or other automated methods to access the content or redirect messages
override any security feature or exclusionary protocol; or
share the content in order to create substitute for Springer Nature products or services or a systematic database of Springer Nature journal
content.
In line with the restriction against commercial use, Springer Nature does not permit the creation of a product or service that creates revenue,
royalties, rent or income from our content or its inclusion as part of a paid for service or for other commercial gain. Springer Nature journal
content cannot be used for inter-library loans and librarians may not upload Springer Nature journal content on a large scale into their, or any
other, institutional repository.
These terms of use are reviewed regularly and may be amended at any time. Springer Nature is not obligated to publish any information or
content on this website and may remove it or features or functionality at our sole discretion, at any time with or without notice. Springer Nature
may revoke this licence to you at any time and remove access to any copies of the Springer Nature journal content which have been saved.
To the fullest extent permitted by law, Springer Nature makes no warranties, representations or guarantees to Users, either express or implied
with respect to the Springer nature journal content and all parties disclaim and waive any implied warranties or warranties imposed by law,
including merchantability or fitness for any particular purpose.
Please note that these rights do not automatically extend to content, data or other material published by Springer Nature that may be licensed
from third parties.
If you would like to use or distribute our Springer Nature journal content to a wider audience or on a regular basis or in any other manner not
expressly permitted by these Terms, please contact Springer Nature at
onlineservice@springernature.com