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?

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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

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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

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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