Activation of the Nrf2-ARE pathway by siRNA knockdown of Keap1 reduces oxidative stress and provides partial protection from MPTP-mediated neurotoxicity - PubMed (original) (raw)

Activation of the Nrf2-ARE pathway by siRNA knockdown of Keap1 reduces oxidative stress and provides partial protection from MPTP-mediated neurotoxicity

Tracy P Williamson et al. Neurotoxicology. 2012 Jun.

Abstract

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that binds to the antioxidant response element, a cis-acting regulatory element that increases expression of detoxifying enzymes and antioxidant proteins. Kelch-like ECH associating protein 1 (Keap1) protein is a negative regulator of Nrf2. Previous work has shown that genetic overexpression of Nrf2 is protective in vitro and in vivo. To modulate the Nrf2-ARE system without overexpressing Nrf2, we used short interfering RNA (siRNA) directed against Keap1. Keap1 siRNA administration in primary astrocytes increased the levels of Nrf2-ARE driven genes and protected against oxidative stress. Moreover, Keap1 siRNA resulted in a persistent upregulation of the Nrf2-ARE pathway and protection against oxidative stress in primary astrocytes. Keap1 siRNA injected into the striatum was also modestly protective against MPTP-induced dopaminergic terminal damage. These data indicate that activation of endogenous intracellular levels of Nrf2 is sufficient to protect in models of oxidative stress and Parkinson's disease.

Copyright © 2012 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. Keap1 knockdown upregulates of ARE-hPAP activity in vitro in a time-dependent and siKeap1 dependent manner

A) Confluent astrocytes were treated with siKeap1 for 24 hours followed by media change and ARE-hPAP activity levels were assayed at timepoints from 1-4 days and compared to untreated cell control. B) Four different siRNA sequences all unique to Keap1 (siKeap1 1-4) were administered for 24 hours and tested for hPAP reporter activity in confluent astrocyte culture 4 days post administration. hPAP activity of tBHQ after 24 hours administration was used as a positive control. n=6; error bars SEM; * p<0.05, siKeap1 vs. siNT (black bars) and tBHQ vs vehicle (white bars).

Figure 2

Figure 2. Keap1 knockdown protects primary astrocytes and neuronally enriched cultures incubated with siKeap1 treated astrocytes against tert-butyl hydrogen peroxide in an Nrf2-dependent manner

Confluent astrocytes were treated with siKeap1 or siNT for 24 hours four days prior to a 24 hour tBOOH challenge in A) wild-type (WT) or B) Nrf2 knockout (KO) primary astrocyte cultures; n=4, error bars SEM, * p<0.05 siKeap1 vs siNT treated cells. C) Confluent primary astrocytes were treated with siNT or siKeap1 for 24 hours, incubated in fresh medium for 24 hours and then lifted, dissociated, and coplated onto neuronally enriched cultures at a plating density of 10,000 astrocytes per 100,000 neurons for 48 hours, followed by a 24 hour tBOOH challenge. Astrocytes were either WT or KO, and plated onto WT neurons. n=3, error bars are SEM. * p< 0.05, no astrocyte addition vs siNT treated astrocytes on neuronally enriched cultures. # p <0.05 WT siNT vs WT siKeap1 astrocytes on neuronally enriched cultures; † p <0.05 KO siNT vs KO siKeap1 on neuronally enriched cultures.

Figure 3

Figure 3. Nrf2-ARE regulated proteins are increased for 6 weeks after a single treatment of siKeap1

A) Four different siRNA sequences all unique to Keap1 (siKeap1 1-4) and siNT were administered for 24 hours and tested for hPAP reporter activity in confluent astrocyte culture 3 weeks post treatment. * p<0.05, siKeap1 vs siNT. B) hPAP reporter activity and C) NQO1 activity was assayed from 4-42 days after a single 24 hour treatment with siKeap1 (black bars), siNT (light grey bars) or tBHQ (dark grey bars) in confluent astrocytes. * p<0.05, siKeap1 vs siNT; #p<0.05, tBHQ vs siNT.

Figure 4

Figure 4. Transplantation of siRNA treated astrocytes onto neuronally enriched culture protects against tBHP mediated toxicity for three weeks after siRNA administration

A) Cell viability was assayed in confluent primary astrocytes treated with siKeap1 or siNT for 24 hours and challenged with tBOOH 1,2, or 3 weeks post siRNA treatment. n=3, error bars SEM. * p<0.05, all siKeap1 vs. siNT. B) Confluent astrocytes were treated with siRNA for 24 hours, followed by medium change and subsequent lifting, dissociation, and coplating with neurons at a density of 10,000 astrocytes onto 100,000 cells in a neuronally enriched culture per well of a 96 well plate at the timepoints indicated; at all timepoints astrocytes were incubated with neurons for 48 hours followed by tBOOH challenge and assay of cell viability. n=3, error bars SEM. * p<0.05 all siKeap1 timepoints are significantly different than nontargeting siRNA treated.

Figure 5

Figure 5. hPAP activity is increased after siKeap1 injection in the striatum of ARE:hPAP animals

A) ARE-hPAP reporter mice were injected with one μL 20 μM siNT (left panel) or 20 μM siKeap1 (right panel) in the striatum and harvested 1 week postinjection and sectioned and stained for hPAP reporter activity via BCIP/NBT staining. Images are taken at 5×. B) ARE-hPAP reporter mice treated as described above and sacrificed 9 days post injection, the striatum was dissected out and snap frozen, and hPAP activity was quantitated. n=3, SEM, *p<0.05.

Figure 6

Figure 6. siKeap1 is mildly protective in the striatum after subchronic MPTP challenge in vivo

A) Mice were injected intrastriatally with 1 μL of 20 μM siKeap1 or siNT 4 days before the initiation of the subchronic MPTP protocol, and collected 7 days after MPTP injections and stained for tyrosine hydroxylase (TH) in the striatum. Images are taken at 20×. B) The striatum was also stained for GFAP (G) and DAPI (B), images taken at 40×. C) TH levels in the striatum were quantified via western blot. * p<0.05, MPTP vs vehicle. # p<0.05 siKeap1 vehicle vs siNT vehicle; # p<0.05, siKeap1 MPTP vs siNT MPTP. n=6 for quantification, with a representative n=2 shown on western blot. D) Dopamine levels were quantified via HPLC after completion of the MPTP protocol. Dopamine content shown as a ratio of ng dopamine per mg tissue wet weight. n=7-8, *p<0.05, vehicle vs MPTP treated.

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