role of sirt3 as a disease-modifying agent
TRANSCRIPT
ROLE OF SIRT3 AS A DISEASE-MODIFYING AGENT IN PARKINSON’S DISEASE
BIOD98H3Supervisor: Dr. Joanne NashMuhammad Hassan Khan
Introduction- Parkinson’s Disease• Second leading neurodegenerative disorder in the world• Motor and non-motor symptoms • Selective and irreversible loss of DA neurons and Lewy
Body formation intracytoplasmic inclusions containing abnormal aggregates of α-synuclein and ubiquitin
• Genes causing PD encoded mt proteins Mitochondrial dysfunction likely occurs early in PD pathogenesis
• No known cure
Introduction- SIRT3• Sirtuins are a seven-
membered family of NAD+-dependent deacetylases
• SIRT3 is best understood mitochondrial Deacetylase
• Mitochondria are the main source of ROS and energy production in neurons SIRT3 can play a potentially neuroprotective role by improving mitochondrial function resulting in increased survival of neurons
Introduction- α-synuclein rats• Two most widely used approaches:
1. Toxins e.g. MPTP, 6-OHDA2. Genetic e.g. α-synuclein, PINK-1
• Toxin models cause rapid decrease in DA neurons only replicate later stages of PD fail to develop LB and behavioural abnormalities
• Genetic unable to report a significant loss of DA neurons BUT
• Overexpression of human WT or A53T mutant α-synuclein progressive loss of DA neurons, behavioural deficit and α-synuclein cytoplasmic inclusions
Hypothesis• Over-expression of SIRT3 prior to injection of human
A53T α-synuclein will improve the general health of mitochondria and slow or prevent neurodegeneration in the SNc.
Methods
SIRT3/empty vector was infused into the animals on day -7 followed by α-synuclein/empty vector injection on day 0. Three weeks (21 days) after the infusion of α-synuclein behavioural assessment was performed. Brains were removed, tissue sectioned, and stereology performed. The aforementioned steps were again repeated at 6 weeks
Methods• Animal Surgery and Vector Delivery• Behavioural Analysis• Perfusion/Cryostatting/Imunohistochemistry• Antibodies• Stereology Analysis:
estimate the number of dopamine neurons in SNc. TH-postive cells were stained and counted using stereo-investigator.
• HPLC Analysis• Statistical Analysis
Results• SIRT3 was able to reverse behavioural impairment 6
weeks following administration in AAV1/2 A53T α-synuclein rats:
• A B
3 Week Behaviour
EV/EV EV/A53T SIRT3/A53T0
10
20
30
% a
sym
met
ry
6 Week Behaviour
EV/EV EV/A53T SIRT3/A53T0
10
20
30
40
50***
*
% a
sym
met
ry
Results• Effect of SIRT3 over-expression on striatal dopamine
turnover in AAV1/2 A53T α-synuclein rats:
• A B
3 week DA
EV/EV A53T/EV A53T/SIRT30
25
50
75
100
125
150
DA
as
a fu
nct
ion
of
un
lesi
on
ed s
ide
6 week DA
EV/EV A53T/EV A53T/SIRT30
25
50
75
100
125
150
DA
as
a fu
nct
ion
of
un
lesi
on
ed s
ide
Results• Effect of SIRT3 over-expression on striatal dopamine
turnover in AAV1/2 A53T α-synuclein rats:
• C D
3 Week HVA:DA
EV/EV EV/A53T SIRT3/A53T0
25
50
75
100
125
150 *
HV
A:D
A a
s a
fun
ctio
n o
fu
nle
sio
ned
sid
e
6 Week HVA:DA
EV/EV EV/A53T SIRT3/A53T0
25
50
75
100
125
150
175
200
**
HV
A:D
A a
s a
fun
ctio
n o
fu
nle
sio
ned
sid
e
Results• Effect of SIRT3 over-expression on striatal dopamine
turnover in AAV1/2 A53T α-synuclein rats:
• E F
3 Week DOPAC:DA
EV/EV EV/A53T SIRT3/A53T0
25
50
75
100
125
150 **
DO
PA
C:D
Aas
a f
un
ctio
n o
fu
nle
sio
ned
sid
e
6 Week DOPAC:DA
EV/EV EV/A53T SIRT3/A53T0
25
50
75
100
125
150
175
200
****
DO
PA
C:D
Aas
a f
un
ctio
n o
fu
nle
sio
ned
sid
e
Results• Effect of SIRT3 overexpression on dopamine cell number
in the substantia nigra pars compacta:
• A B
3 week Stereology
EV/EV EV/A53T SIRT3/A53T0
20
40
60
80
DA
cel
ls a
s %
of
nai
ve c
ells
6 week Stereology
EV/EV EV/A53T SIRT3/A53T0
20
40
60
80D
A c
ells
as
% o
f n
aive
cel
ls
Discussion• Liu et al (2015) found that SIRT3 plays a protective role
in MPTP mouse model of PD. • Wier et al (2012) found that SIRT3 mRNA levels were
upregulated with increased deposition of amyloid- β and ROS production in an AD model
• Possible protective pathways exploited by SIRT3 include:
aiding DA secretion, protective effect against ROS production, regulation of basal ATP levels, suppression of mtPTP formation, protection against fragmentation of the mitochondria and prevention against age-linked apoptosis
Discussion• Our data suggests that over-expression of SIRT3 prior to
infusion of human A53T α-synuclein is able to play a protective role in slowing or preventing neurodegeneration in the SNc.
• Additional studies investigating the effects of overexpression of SIRT3 need to be performed in other genetic and toxin models of PD.
• Further studies looking at the mechanism of action of SIRT3 in specific neurodegenerative diseases also need to be looked at.
Acknowledgements• I’d like to thank Dr. Joanne Nash, Dr. Jacqeuline Gleave,
Peter Perri, James Barber, Zainab Najarali, Abdul rehman Qureshi and Lindsay Arathoon, Dr. Stehlik and Dr. Ashok.