J Mol Neurosci DOI 10.1007/s12031-013-0163-9
Silencing of Hsp90 Chaperone Expression Protects Against 6-Hydroxydopamine Toxicity in PC12 Cells Behrang Alani & Rasoul Salehi & Payam Sadeghi & Mohammad Zare & Fariba Khodagholi & Ehsan Arefian & Mazdak Ganjalikhani Hakemi & Hadi Digaleh
Received: 22 September 2013 / Accepted: 23 October 2013 # Springer Science+Business Media New York 2013
Abstract Parkinson’s disease (PD) is the second most common age-related neurodegenerative disorder that has been shown to be associated with oxidative stress. This phenomenon occurs primarily via generation of 6-hydroxydopamine (6-OHDA) in catecholaminergic neurons leading to activation of apoptosis. The 90-kDa heat shock protein (Hsp90) functions as a chaperone in maintaining the functional stability and viability of cells under a transforming pressure. Since Hsp90 binds to inactive transcription factor heat shock factor-1 (HSFB. Alani (*) : R. Salehi Department of Genetic and Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran e-mail:
[email protected] B. Alani Department of Applied Cell Science, Faculty of Medicine, Kashan University of Medical Sciences, Kashan, Iran P. Sadeghi : F. Khodagholi : H. Digaleh NeuroBiology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran P. Sadeghi : F. Khodagholi : H. Digaleh Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran M. Zare Department of Neurology, Isfahan University of Medical Sciences, Isfahan, Iran M. Zare Neuroscience Research Center, Isfahan University of Medical Sciences, Isfahan, Iran E. Arefian Department of Molecular Biology and Genetic Engineering, Stem Cell Technology Research Center, Tehran, Iran M. G. Hakemi Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
1), inhibition of Hsp90 could activate HSF-1 and transcription of heat shock element containing genes subsequently, like Hsp70 as an anti-apoptotic factor. Our trial of silencing Hsp90 expression through transfection of Hsp90 siRNAs into neuronal PC12 cells being exposed to 6-OHDA resulted in the inhibition of pro-apoptotic factors, Bax, caspase-3, and PARP and upregulation of anti-apoptotic factor, Bcl2. In this manner, our data suggest a protective role for Hsp70 as it was observed to be induced upon Hsp90 knockdown. Furthermore, our results showed that Hsp90 silencing against 6-OHDAinduced oxidative stress may associate with upregulation of nuclear factor-erythroid 2-related factor 2. In summary, we found that silencing of Hsp90 expression leads to induction of cytoprotective pathways which can protect neurons against apoptosis in a PD model. Keywords Hsp90 . siRNA . 6-OHDA . Apoptosis
Introduction Parkinson’s disease (PD) is a neurological disorder that has been shown to have a strong association with oxidative stress (Gandhi and Wood 2005; Tsang and Chung 2009). PD is mainly characterized by the presence of aggregated cytoplasmic Lewy body’s protein inclusions and degeneration of dopaminergic neurons in substantia nigra pars compacta (Dawson and Dawson 2003; Surendran and Rajasankar 2010). Oxidative stress is caused by the imbalance between the production of reactive oxygen species (ROS) and the biological system’s inability to detoxify those species (Sayre et al. 2001). Numerous studies indicate the involvement of oxidative stress in the etiology and pathogenesis of PD (Zhou et al. 2008; Schapira and Jenner 2011). One potential source of ROS in the catecholaminergic neurons includes the autooxidation of the dopamine to generate 6-hydroxydopamine
J Mol Neurosci
(6-OHDA). This subject has been demonstrated to give oxidative insults via excess production of superoxide, H2O2, and inhibition of mitochondrial complexes I and IV (Woodgate et al. 1999; Chen et al. 2005). Some studies suggested the possible role of 6-OHDA in the pathogenesis of PD, as concentration of 6-OHDA increases in the brain and urine of PD patients with L -dopa therapy (Blum et al. 2001). To elucidate the molecular pathways of neuronal death as well as symptoms of the disease and to develop neuroprotective strategy, neurotoxin 6-OHDA is widely used to produce experimental models of Parkinson’s disease both in vivo and in vitro (Jakel et al. 2005; Hanrott et al. 2006). Oxidative stress occurs as a consequence of the imbalance between the production of ROS and a decrease in the capacity of antioxidant enzymes that are involved in the oxidation, misfolding, and aggregation of vital proteins ultimately resulting in the failure of normal cell function and also a signal for the activation of the apoptotic cascade. The heat shock proteins are important class of proteins evolved to protect cells against a variety of stressful conditions, including oxidative stress and protein misfolding (Feder and Hofmann 1999). The 90-kDa heat shock protein (Hsp90) is one of the most widely studied heat shock proteins that bind to inactive transcription factor heat shock factor-1 (HSF-1) under normal or unstressed conditions (Csermely et al. 1998). In response to oxidative stress or Hsp90 inhibitors, HSF-1 dissociates from the Hsp90 complex, trimerizes, and translocates to the nucleus to activate the transcription of Hsp proteins such as Hsp70. Hsp70 is induced during cellular stress to prevent protein aggregation and facilitate protein refolding. Hsp70 also protects cells from a wide range of cellular stresses such as oxidative radicals and its direct protection against apoptosis has been discussed as well (Kalmar and Greensmith 2009; Brown 2007). Recently, molecular chaperones have emerged as a therapeutic target for the treatment of PD (Kalia et al. 2010). The KEAP1/nuclear factor-erythroid 2related factor 2 (Nrf2)/ARE axis is a well-studied mechanism by which the cell could respond to oxidative stress (Tkachev et al. 2011). Nrf2 is a bZip transcription factor and a member of the cap ‘n’ collar family that mediates cellular response to electrophiles and oxidants by upregulating antioxidant, xenobiotic-metabolizing, and other cytoprotective enzymes (Ma 2013). Nrf2 and its association with PD pathology has been discussed representing a potential locus for the development of novel therapeutics focused on induction of the Nrf2directed antioxidant pathway (Cuadrado et al. 2009). A line of studies suggested that Hsp90 inhibition by several pharmaceutical agents could increase the expression of stressinduced proteins such as Hsp70. Nowadays, short interfering RNA molecules (RNAi) have emerged as a possible method to reduce target gene expression (Mook et al. 2007). Although RNAi has not yet been well studied in models of dopaminergic neurodegeneration, there is evidence for application of
RNAi to improve the motor and neuropathological findings in a mouse model of spinocerebellar ataxia type 1. These works are in support of its possible utility for the treatment of neurodegenerative diseases such as PD (Cummings et al. 2001; Manfredsson et al. 2006). Niture and Jaiswal (2010) have discovered an interaction between Hsp90 and Keap1 during heat shock and antioxidant response that can positively regulate the expression of Nrf2. This study aimed to determine the neurotoxicity effect of heat shock proteins 90 using RNAi technology. To this end, PC12 cells were differentiated with nerve growth factor (NGF) to dopaminergic nervous cells followed by applying 6-OHDA as an oxidative stress inducer. We also interpreted the role of Hsp90 inhibitory system using small interfering RNA (siRNA) technology on the activity of Nrf2 pathway in the neural-like dopaminergic cells.
Material and Methods Materials Antibodies directed against heat shock proteins (Hsp90 and Hsp70), Bax, poly (ADP-ribose) polymerase (PARP-1), Bcl2, caspase-3, and β-actin were obtained from Cell Signaling Technology. L -glutamylcysteine synthetase (γ-GCS), Nrf2, and heme oxygenase-1 (HO-1) were obtained from Abcam Biotechnology. Lamin B2 antibody was obtained from Santa Cruz Biotechnology. Also, 6-OHDA and all other reagents were from Sigma-Aldrich (St. Louis, MO, USA). Cell culture media and sera were obtained from Gibco. Cell Culture and Differentiation Rat pheochromocytoma (PC12) cells (obtained from Pasteur Institute, Tehran, Iran) were cultured in DMEM-F12 medium, supplemented with 10 % fetal bovine serum, 5 % horse serum, and 1 % antibiotics (all from Gibco) at 37 °C under humidified atmosphere with 5 % CO2. PC12 cells were treated with 50 ng/ml NGF (Sigma, St. Louis, MO, USA) and differentiated by incubation for a week. Cell Viability PC12 cells were planted at a density of 105 cells/well in 96well plates. Then, they were treated with 6-OHDA at 75, 100, 125, 150, 175, and 200 μM for 24 h. After incubation, cell viability was determined by the conventional 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) reduction assay. The dark blue formazan crystals formed in intact cells were solubilized in dimethyl sulfoxide, and the absorbance was measured at 630 nm. Results were expressed as the percentages of reduced MTT, assuming the absorbance of control cells as 100 %.
J Mol Neurosci
Gene Silencing by siRNA For depletion of Hsp90, we used commercially available siRNAs from Ambion (Silencer® select predesigned s152162, s152163 for rat HSP90aa1 and s-153798, s235785 for rat HSP90ab1). A negative control siRNA (scrambled) was included to monitor nonspecific effects. The cells were transfected to a final concentration of 30 nM siRNAs using transfection reagent (Mir 2150 TransIT-TKO® from Mirus Bio Corporation) according to the manufacturer’s instructions. The efficiency of the transfection was examined by FACS Culibur flowcytometry (BD, USA) and fluorescent microscopy (Olympus IX71, Japan) using fluorescein-labelled siRNA (Santa Cruz Biotechnology). The knockdown efficiency was determined by real-time PCR and western blotting. For confirmation of siRNA specificity and any probable gene expression profile change (off-target effect), we conducted associated tests with different groups and concentrations (data not shown).
data analysis was done by ImageJ software. The concentrations of proteins were determined according to Bradford’s method (Bradford 1976). Glutathione Measurement as an Antioxidant Content in Level The concentration of glutathione (GSH) was determined in whole cell lysate using the dithionitrobenzoic acid method at 412 nm (Ellman, 1959). For this assessment, the absorbance of the 96-well plate containing different samples of different groups of cells was read by ELISA reader. GSH concentrations were expressed as mole per milligram protein. Statistical Analysis All data are depicted as the mean ± SEM. One-way analysis of variance followed by Tukey’s post hoc test was conducted to analyze the differences. The statistical significances were taken when p
