fig. S3, G and H) and also limited synapse loss
that occurs in this model (12) (fig. S3, C and D).
Lipid droplet formation was also prevented in
aged D2 retinas (fig. S3F). NAM also decreased
PARP activation and limited levels of DNA damage and transcriptional induction of HIF-1a (fig.
S3, A and B), all of which reflected less-perturbed
cellular metabolism. NAM prevented even the
earliest molecular signs of glaucoma in most
treated eyes, as assessed by RNA-seq (Fig. 2, F
and G, and fig. S7), and prevented the majority
of age-related gene expression changes within
RGCs (fig. S8). This highlights the unexpected
potency of NAM in decreasing metabolic disruption and prevention of glaucoma.
Attempting to further decrease the probability
of glaucoma, we administered a higher dose of
NAM (2000 mg/kg per day, NAMHi). NAMHi was
extremely protective, with 93% of treated eyes
having no optic nerve damage (Fig. 2B). The degree of protection afforded by administering this
single molecule is unprecedented and unanticipated. Although NAMLo demonstrates a clear
neuroprotective effect (no effect on IOP), NAMHi
lessens the degree of IOP elevation (fig. S6). This
indicates that NAM can protect against age-related
pathogenic processes in other cell types in addition
to RGCs (11, 16, 19). Therefore, NAM, a single
molecule that protects against both IOP elevation
and neural vulnerability, may have great potential
for glaucoma treatment; however, human studies
NMNAT2 (nicotinamide/nicotinic acid mononucleotide adenylyltransferase 2) is emerging as
an important NAD-producing enzyme in axons,
and protects from axon degeneration (20). Ongoing stress negatively impacts Nmnat2 expression in RGCs (q < 0.05 in D2 group 4) (fig. S7F).
This decline of NMNAT2 may induce vulnerability to axon degeneration in glaucoma. NMNAT2
expression is decreased in brains with Alzheimer's
disease and is highly variable in aged postmortem
human brains (21). Such variation in expression
may contribute to individual differences in vulnerability to various neurodegenerations.
Glaucoma is a complex disease involving mul-
tiple insults. Mechanical axon damage and local
inflammation are two important contributors in
glaucoma (22–24). To more fully assess the gen-
eral effectiveness of NAM treatment, we tested
its efficacy in two models of RGC death that are
used to model these glaucomatous insults. We
used a tissue culture model of axotomy and intra-
vitreal injections of soluble murine tumor necrosis
factor–a (TNFa), which can drive local inflam-
mation as well as mitochondrial dysfunction and
is implicated in glaucoma (25). NAM robustly
protected cultured retinas from RGC somal degen-
eration (fig. S9, A and B). NAM also protected
against a loss of PERG amplitude and cell loss in
TNFa-injected eyes (fig. S9, C to E). Given these
protections against severe acute insults, NAM
could have broad implications for treating glau-
coma and potentially other age-related neuro-
Gene therapy is an attractive approach for
overcoming compliance issues and improving
efficacy. In the eye, gene therapy has proven suc-
cessful for rare human Mendelian disorders (26).
Viral gene therapy allows a large number of cells
to be transfected potentially lifelong by delivering
a targeted gene product. To date, gene therapy
has not been successfully applied to complex human
diseases. Given that age is a common risk factor for
most glaucoma, protecting from age-related declines
in NAD may generally protect many glaucoma
patients. Thus, we sought to support NAD+-producing
cellular machinery through the overexpression
of Nmnat1, a terminal enzyme in NAD+ produc-
tion, to further test our NAD hypothesis. We
chose this approach as we reasoned that using
NAM phosphoribosyltransferase, the rate-limiting
enzyme in NAD synthesis, may pose complica-
tions due to its cytokine functions and over-
production of NAM mononucleotide, which may
participate in axon degeneration (27). D2 eyes
were injected once with AAV2.2 containing the
Nmnat1 gene and a gene for green fluorescent
protein (GFP) under a cytomegalovirus (CMV)
promoter expressed as a single transcript (Fig. 3).
Nmnat1 expression (as assessed by GFP expres-
sion) was robust in RGCs 2 weeks after injection
(expressed in >83% of RGCs) and remained robust
through to the end-stage time point (12 months)
(fig. S10). Overexpression of Nmnat1 was suffi-
cient to prevent axon and soma loss (Fig. 3, A to
D), to preserve axoplasmic transport (Fig. 3B),
and to preserve electrical activity in RGCs (PERG)
(Fig. 3E). Glaucomatous nerve damage was absent
in >70% of treated eyes. Because NMNAT1 cat-
alyzes the terminal step in NAD production, the
major protective effects of NAM treatment likely
result from driving NAD production in neurons
rather than other NAD-independent mechanisms
(but partial contributions from other mechanisms
cannot be completely excluded). We further as-
sessed the effects of combinational therapy of
Nmnat1 and NAMLo. This combination afforded
significant additional protection over Nmnat1 or
NAMLo alone, with 84% of eyes having no detect-
able glaucoma (~4-fold decreased risk of develop-
ing glaucoma). Increasing the NAM dose combined
with gene therapy may prove even more protective.
In conclusion, we show that dietary supple-
mentation with a single molecule (vitamin B3
or NAM) or Nmnat1 gene therapy significantly
reduces vulnerability to glaucoma by support-
ing mitochondrial health and metabolism. Com-
bined with established medications that lower
IOP, NAM treatment (and/or Nmnat1 gene ther-
apy) may be profoundly protective. By providing
a new molecular and metabolic link between
increased neuronal vulnerability with age and
neurodegeneration, these findings are of critical
importance for glaucoma and possibly other age-
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The data reported in this paper are available in the supplementary
materials. RNA-seq data are available through the Gene Expression
Omnibus (accession number GSE90654). The authors would like to
thank the staff of the flow cytometry, histology, gene expression
services, and computational sciences at the Jackson Laboratory;
G. Howell and R. Libby for discussion and experiment design;
K. Kizhatil for reading the manuscript; M. de Vries for assistance with
diet and organizing; B. Cardozo for colony maintenance and drug
changes; and A. Bell for intraocular pressure measurements.
The authors would also like to thank their sources of funding: the
Jackson Laboratory Fellowships (P.A. W. and J.M.H.), partial
support from EY11721 (S. W.M.J.), the Barbara and Joseph Cohen
Foundation (S. W.M.J.), and HL49277 (O.S.). S. W.M.J. is an Investigator
of The Howard Hughes Medical Institute. S. W.M.J. and P.A. W. are
inventors on a provisional patent application (no current patent
number) submitted by the Jackson Laboratory that covers
NAD-related therapies in glaucoma. S. W.M.J. holds additional
philanthropic funding from the Lano Family Foundation.
Materials and Methods
Figs. S1 to S10
Tables S1 to S4
29 September 2016; accepted 23 December 2016
760 17 FEBRUARY 2017 • VOL 355 ISSUE 6326
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