10/10/2018
1
Ageing, neuronal metabolism, and
mitochondria in glaucoma
Pete Williams
Karolinska Institutet, S:t Eriks Ögonsjukhus, Stockholm, Sweden
#2323 EVER 2018 Obergurgl Optic Nerve Meeeting SIS
petewilliamslab.com | ki.se/en/people/petewi | Twitter: @pete_the_teapot
Neurodegenerative hypothesis
No
rma
l a
gin
g Decreasing metabolic function
Increasing likelihood of mitochondrial and cellular failure
Outcome: Age-dependent decline in neuronal health
Increasing age
Increasing mitochondrial stress
Ne
uro
de
ge
ne
ratio
n
Decreasing metabolic function
Increasing vulnerability to disease-related stresses
Outcome: Increased instance of cell death and neuronal
dysfunction – neurodegenerative disease
Conspiring
factors
e.g. Genetics
Other diseases
Environment
Increasing age
Increasing mitochondrial stress
Are neurodegenerative diseases an age- and metabolism-related phenomena?
Can we target these pathways to develop broad neuroprotective strategies?
10/10/2018
2
Glaucoma:
A common neurodegeneration affecting
~80 million patients for which there are no
neuroprotective strategies
DBA/2J glaucoma
DB
A/2
JD
2-G
pn
mb
+
Iris disease
Increased IOP
RGC and
optic nerve
degeneration
GpnmbR150X
Tyrp1b
10/10/2018
3
Defining cell specific molecular processes in glaucoma
• RNA-sequencing of RGCs from DBA/2J mice (D2), plus age and sex
matched controls (D2-Gpnmb+)
• Computational analysis and pathway analysis
• Identify target pathways and molecules
• Test target molecules in DBA/2J glaucoma
Aim: identify age- and IOP- dependent molecular
changes within RGCs that precede glaucomatous
neurodegeneration
Dysregulation of mitochondrial transcripts
Mito
ch
on
dria
l :
nu
cle
ar
rea
d to
tal ra
tio
0
0.2
0.4
0.6
0.8
log
2ch
an
ge
fro
m D
2-G
pn
mb
+
-3
-2
-1
0
2
3
1
FDR < 0.05
log
2ch
an
ge
fro
m D
2-G
pn
mb
+
-2
-1
0
2
1
Oxidative phosphorylationnu derived mt transcriptsnu-RNA vs. mt-RNA
Williams et al, Science, 2017
10/10/2018
4
Early mitochondrial perturbations • RNA-seq of RGCs identifies early mitochondrial perturbations
• This is especially evident in genes encoding OXPHOS proteins
and NAD+ synthesis enzymes
• EM analysis identifies mitochondrial abnormalities in RGC
dendrites and synapses that precede detectable measures of
glaucoma (i.e. NFL thinning, axon loss, optic nerve cupping)
• Retinal NAD+ declines in an age-dependent manner
• Hypothesis: declining NAD is a critical insult that renders
aging RGCs susceptible to insults from high IOP
-lo
g p
fro
m c
on
tro
l
10
20
30
40
50
0eIF2 Mitochondrial
dysfunctionmTOR Oxidative
phosphorylation
D2
Gro
up
4
D2
Gro
up
3
D2
Gro
up
2
Ingenuity Pathway Analysis
Mito
ch
on
dria
l
crista
e a
rea
(μ
m2)
0
0.050
0.025
0.075
0.100
0.125
Dendritic Somal
** ***0.150
Co
ntr
ol
Gla
uco
ma
Williams et al, Science, 2017
NAM
NMN
NAD
NAMPT
NMNAT1
Re
tin
a p
mo
lN
AD
(t)
/ m
g p
rote
in
0
500
1000 ****
Can repleting retinal NAD levels prevent glaucoma?
NAM
NMN
NAD
NAMPT
NMNAT1
Nicotinamide (amide of vitamin B3)
Re
tin
a p
mo
lN
AD
(t)
/ m
g p
rote
in
0
500
1000 ****
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5
Repleting NAD using NAM in vivo
• Nicotinamide (NAM, the amide of vitamin B3)
• Early start (6 mo, pre-IOP elevation,
prophylactic)
• Late start (9 mo, high IOP in majority of eyes,
interventional)
Re
tin
a p
mo
lN
AD
(t)
/ m
g p
rote
in
0
500
1500
2000
1000
***
****
***
Williams et al, Science, 2017
NAM
NMN
NAD
NAMPT
NMNAT1
NAM supplementation prevents optic nerve degeneration in glaucoma
• Nicotinamide (NAM) protects from
human-relevant glaucomatous damage;
RGC loss, ON degeneration, ON
cupping
• Protects as intervention and
prophylactically
o 550 mg/kg/d NAM in water
o 2000 mg/kg/d NAM in food and water
o ~2 - 10 g/d for 60 kg human)
RGC loss
Co
ntr
ol
Gla
uco
ma
Nic
otin
am
ide
DAPI RBPMS
ON degeneration
PPD
Williams et al, Science, 2017
Williams et al, Communicative & Integrative Biology, 2017
0Pe
rce
nta
ge
ne
rve
s a
t e
ach
da
ma
ge
le
ve
l
25
50
75
100
** ***
***
NO
SE
VE
RE
Glaucoma Nicotinamide
ON cupping
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6
NAM prevents formation of abnormal mitochondria
Mito
ch
on
dria
l crista
e a
rea
(μ
m2)
0
0.050
0.025
0.075
0.100
0.125
**0.150
0.175 ***D2-Gpnmb+ D2 NAMLo
• Abnormal mitochondria accumulate in
RGC dendrites and soma following
periods of elevated IOP
• NAM prevents these changes and likely
promotes healthy mitochondrial function,
mitochondrial motility, and mitophagy
Williams et al, Science, 2017
PERG decreases in an IOP-dependent manner and is recovered by NAM treatment
PE
RG
am
plit
ud
e (
μV
)
0
2
4
6
8
10
12
14**
• Timing corresponds with early transcriptomic changes,
mitochondrial abnormalities, and synapse and dendritic loss
***
Williams et al, Science, 2017
10/10/2018
7
Further testing of declining NAD+ hypothesis
NAM
NMN
NAD
NAMPT
NMNAT1
/ WLDS
(Wallerian
Degeneration
Slow allele)
WLDS raises NAD in DBA/2J mice
Williams et al, Frontiers in Neuroscience, 2017
NAM
NMN
NAD
NAMPT
WLDS
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WLDS and nicotinamide in combination robustly protect from glaucoma
Williams et al, Frontiers in Neuroscience, 2017
Can we establish a long-term, retinal
ganglion cell specific, NAD-repleting
therapy?
10/10/2018
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Nmnat1 gene therapy in glaucoma
RB
PM
S
CT
-β
GF
P
GF
P
DA
PI
Me
rge
DA
PI
Me
rge
Retina LGN Sup. Col.
Pe
rce
nta
ge
ne
rve
s a
t e
ach
da
ma
ge
le
ve
l
25%
NO
SE
VE
RE
0%
50%
75%
100
%
*** ***
RB
PM
S+
RG
Cs /
10
0μ
m2
05
1015202530354045
0
2
4
6
8
PE
RG
am
plit
ud
e (
μV
)
*** ***
** ***
Williams et al, Science, 2017
First successful gene therapy in complex age-related disease
NAM
NMN
NAD
NAMPT
NMNAT1
Further activation of Nmnat1 and Nmnat2
• Decrease in Nampt, Nmnat2 transcript following axotomy
• Mirrors changes we see in DBA/2J glaucoma
Tribble & Williams, unpublished
P = 0.0017 P = 0.042
Dr James Tribble
10/10/2018
10
Further activation of Nmnat1 and Nmnat2
• EGCG (a major, ~30%, component of green tea polyphenols) (known
activator of Nmnat1, Nmnat2; NAD+ producing enzymes)
• EGCG and whole green tea polyphenols robustly prevent RGC loss
following axotomy in the μM range
NAM
NMN
NAD
NAMPT
NMNAT1
NMNAT2
NMNAT3EGCG
Tribble & Williams, unpublished
Hypothesis:
Metabolic decline is a more general process
for RGC vulnerability and degeneration in
glaucoma
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11
Decreasing retinal pyruvate in glaucoma
Williams et al, unpublished
in collaboration with Casson et al
• Enrichment of genes / pathways – pyruvate metabolism,
gluconeogenesis
• NAM corrects most of these changes except 2 genes (Mpc1, Gpnmb)
• MPCs are well expressed in rodent, monkey, human RGCs
• Oral pyruvate leads to increased retinal pyruvate levels
Repleting pyruvate prevents glaucoma
DBA/2J• 500 mg/kg[bw]/d
• + NAM 550 mg/kg[bw]/d groupWilliams et al, unpublished
in collaboration with Casson et al
Rat OHTAxotomy
explant culture
10/10/2018
12
• Thy1.2 CFP (blue RGCs)
• MitoY (Eno2 promoter, Cox8a gene-targeting sequence fused to N
terminus) (yellow mitochondria)
• B6J, D2, D2-Gpnmb+, and D2.WldS backgrounds, plus treatment
conditions (NAM, pyruvate (PYR))
Preventing formation of abnormal mitochondria
Williams et al, unpublished
Glaucoma as a metabolic optic neuropathy
• RGCs are a particularly vulnerable to metabolic and physical stressors
• Metabolism and mitochondrial health decline with age and is exacerbated by periods of elevated
IOP
• NAD decline renders RGC susceptible to elevated IOP
• Restoring NAD (nicotinamide, Nmnat1 gene therapy, or WLDS) improves mitochondrial health
and protects RGCs
• Targeting Nmnats prevents RGC degeneration (EGCG, GTP, NAM) following axotomy
• Further targeting metabolic decline (pyruvate) prevent RGC degeneration in multiple models of
glaucoma
• Targeting neuronal metabolic decline and mitochondria may offer safe, neuroprotective
treatments for glaucoma and other age-related neurodegeneration and ophthalmic degenerations
10/10/2018
13
Acknowledgements
• Pete Williams Lab
• James Tribble
• Simon John Lab
• Jeff Harder
• Nicole Foxworth
• Kelly Cochran
• Brynn Cardozo
• Bob Casson Lab
• Glyn Chidlow
• John Wood
• Chelsea Guymer
• Vittorio Porciatti
• Oliver Smithies
• Gareth Howell
• Rick Libby
• KI, S:t Eriks, & JAX services
• All our funders
Ageing, neuronal metabolism, and
mitochondria in glaucoma
Pete Williams
Karolinska Institutet, S:t Eriks Ögonsjukhus, Stockholm, Sweden
#2323 EVER 2018 Obergurgl Optic Nerve Meeeting SIS
petewilliamslab.com | ki.se/en/people/petewi | Twitter: @pete_the_teapot