N0X1- MEDIATED OXIDATIVE
STRESS LEADS
TO DOPAMINE NEURON DEATH
IN PARKINSON’S DISEASE
By: Ishvinder Hadday
Rationale
■ The over-production of ROS through mitochondrial dysfunction has
been implicated in the development Parkinson’s Disease (PD)
■ Increased ROS is linked to DNA damage in dopaminergic (DA)
neurons in the Substantia Nigra (SA)
■ NADPH oxidases (NOX) play a significant part in the generation of
ROS in cells
Rationale Cont’d
■ Specific NOX isoforms localize to specific organelles causing ROS
accumulation in these organelles.
■ Nox1 is the inducible enzyme in the family and Rac1 is an
essential subunit for the activation of Nox1.
■ Expression is increased in dopaminergic neurons (DA) in the
Substantia Nigra (SN) of mice in response to oxidative stimuli and
environmental toxins like paraquat.
■ Exposure of SNDA of mice to toxins such as paraquat, induce
Nox1 activation and the over-production of ROS
Hypothesis
■ The ROS produced by the Nox1/Rac1 complex play a pivotal
role in the degeneration of nigrostriatal dopaminergic
neurons in an animal model of PD.
■ Literature supports various aspects of this hypothesis. This
study was done to bring it all together to form clear
conclusion.
■ ROS in the nucleus of SNDA is directly responsible for
nuclear DNA damage which leads to neurodegeneration
Step by Step
■ Examine NOX1 induction and activation of the NADPH oxidase system in
dopaminergic cells
■ Determine the specifics of 6-OHDA mediated NOX derived ROS production
■ Expression of NOX1 and Rac1 under different conditions and translocation to
the nucleus
■ DNA oxidative damage in SNDA cells caused by nuclear NOX1/Rac1 complex
■ Inhibition of Rac1 or Nox1 reduced SNDA cell death induced by 6-OHDA
NOX1 induction and activation of
the NADPH oxidase system in
dopaminergic cells
■ Before induction/activation, tested for the
presence of NOX components.
■ Used RT-PCR to detect the mRNAs encoding each
NOX isoform and their regulatory subunits
■ Most NOX isoforms and regulatory subunits were
detected.
Figure 1A: Dopaminergic cells contain NADPH oxidase components and 6OHDA leads to Nox1induction and Rac1 activation.
■ NOX1 was significantly induced after 6h treatment with 100 uM 6-OHDA.
■ 6-OHDA induces ROS generation and eventual cell death in a dose dependent
manner shown in 1B.
■ The activation of GTPase Rac1 has been shown to be necessary for NOX1
activation
Figure 1B and C:
Dopaminergic cells
contain NADPH
oxidase components
and 6-OHDA leads to
Nox1induction and
Rac1 activation.
■ Mitochondrial ROS was shown to
play a role in inducing NOX1 and
generating NOX1 derived ROS
(super oxide)
■ Effect of Mitochondrial ROS on
NOX1 induction tested using
respiratory complex inhibitors.
SUPPLEMENTARY FIG. S2. Nox1 induction caused by
mitochondrial respiratory complex inhibitors. N27 cells were
treated with various mitochondrial inhibitors including
rotenone (5 lM), pyridaben (5 lM), antimycin A (5 lM), or FCCP
(20 lM) overnight. Nox1 mRNA were determined by RT-PCR
(A) and protein levels were measured using Western blot (B).
(C) Mitochondrial superoxide levels, N27 cells were treated
with two doses (lM) of each inhibitor. Mitochondrial
superoxide was measured using MitoSOX. CTRL, control;
PYR, pyridaben; ANTI, antimycin A; ROT, rotenone. *p < 0.05,
**p < 0.01, + p < 0.001 vs. control.
The NADPH oxidase system plays a pivotal
role in 6-OHDA mediated ROS generation
■ Test whether the 6-ohda induced nox1 expression is responsible for superoxide
generation
■ Chemical inhibitors diphenyleneiodonium (DPI) and apocynin were used at
various concentrations
■ Superoxide was measured using the nitro blue tetrazolium (NBT) assay at 6 h
post 6-OHDA.
■ DPI significantly reduced 6OHDA-mediated ROS generation
at concentration as low as 0.1
lM
■ DPI has overall stronger effect,
possibly due to non specific
nature
■ Aponycin blocks the formation
of the NOX1/Rac1 complex
reducing ROS production
Figure 2: NADPH oxidase is responsible for 6-OHDA-mediated ROS generation in dopaminergic cells.
■ NOX1 expression was selectively inhibited using RNA interference to create a
knockdown model
■ Specifically small interfering RNA (siRNA) used to degrade NOX1 mRNA after
transcription so no proteins are produced
■ Knockdown efficiency of
siRNA was shown to be
37%, parallel to
decrease in 6-OHDA
induced ROS production
Expression of Nox1 in nigrostriatal DA
neurons of PD animal model
■ Significant increase of Nox1
expression was observed in
the (SN) after 3 days of 6OHDA striatal injection (Fig.
3A).
■ Visualized using
immunostaining indicated
coexpression of tyrosine
hydroxylase (TH) with NOX1
■ Coexpression proved
specific DA neuron NOX1
expression
FIG. 3. Striatal administration of 6-OHDA robustly increased Nox1
expression in dopaminergic neurons in the SN.
■ 3 days post 6-OHDA injection, basal Nox1 expression was significantly increased
in the nucleus and in the cytoplasm of DA neurons
■ N27 immortal SNDA cell line treated with 6-OHDA for 24h showed activated
Rac1 along with NOX1 in nuclear extracts
■ Suggests an active NOX1/Rac1 complex forms inside the nucleus after
translocation
■ NOX1 present in the nucleus of post mortem PD patients further supporting
hypothesis
■ These results demonstrated for the first time that active NOX1/Rac1 complexes
are formed in the nucleus of oxidatively stressed SNDA neurons
Nuclear Nox1/Rac1 caused DNA oxidative
damage in DA cells
■ Tested whether selective inhibition of the complex led to reduced ROS
production in the nucleus
■ Adenoassociated virus serotype 2 (AAV2) expression combined with either
Nox1shRNA or T17N dominant negative Rac1 variant were used to knockdown
Nox1 or inhibit Rac1, respectively.
■ Both methods caused reduced ROS production in N27 cells
■ Effectively proved the NOX1/Rac1 complex produces ROS in the nucleus
■ 6-OHDA treatment increased DNA oxidative damage (super oxide) indicated by
increased 8-oxo-dG immunoreactivity in the nucleus of both N27 cells and SNDA
rat neurons
■ 6-OHDA induced 8-oxo-dG immunoreactivity significantly reduced by preincubation of N27 cells with either Nox1 shRNA or T17NRac1/ AAV2 viral
particles.
■ In vivo targeting of Rac1 or Nox1 was achieved by stereotaxic delivery of AAV2
particles harboring either T17NRac1 or Nox1shRNA into the rat SN***
■ NOX1 expression and concurrent increased nuclear 8-oxo-dG immunostaining
further shows oxidative DNA damage is caused by the NOX1/Rac1 complex
*** Really cool
Inhibition of Rac1 or Nox1 reduced DA cell
death induced by 6-OHDA
■ Need to determine the mechanism of how NOX1/Rac1 complex generated
oxidative stress ROS leads to SNDA neuronal death.
■ C-Jun N-terminal kinase (JNK)-mediated signaling has been implicated as a final
common pathway of DA neuronal apoptosis
■ N27 cells expressing shNOX1 lack JNK immunostaining suggesting that the
knockdown prevented 6-OHDA induced apoptosis
■ (in vivo) 4 week pre-injection of NOX1 or Rac1 inhibitor in the SN area reduced
SNDA neuron loss after 6-OHDA treatment
The Results
■ SNDA neurons possessed the NOX1 and Rac1 subunits of the NADPH oxidase
system
■ 6-OHDA induces NOX1 and Rac1 activity
■ Mitochondrial ROS plays a role NOX1 induction
■ Induced NOX1 expression is responsible for increases ROS (super oxide)
generation
■ Active NOX1/Rac1 complexes are formed in the nucleus of oxidatively stressed
SNDA neurons
■ NOX1/Rac1 complexes are the source of oxidative stress in the nucleus of SNDA
neurons
■ NOX1/Rac1 ROS generation may lead to cell death through the JNK pathway
■ ***lead to the advancement that oxidative damage to
nuclear DNA occurs through the accumulation of
Nox1/Rac1 complex and ROS in the nucleus.
Potential Treatments
■ AAV2 mediated gene therapy effective in long term expression and modification
of targets
– persist in an extrachromosomal state without integrating into the genome of the
host cell
■ AAV2 mediated gene transfer effective in long term expression and modification
of targets
■ Drugs such DPI or apocynin function in ways that may affect various pathways
instead of just the NOX1/Rac1 complex.
The End