Effect of rifampicin pre- and post-treatment on rotenone-induced dopaminergic neuronal apoptosis and alpha-synuclein expression**☆○
Yuanlin Sun1, Guohua Zhang1, Jie Xu1, Shiwen Chen1, Enxiang Tao1, Changqing Xu2,          M. Catherine Bennett○2

1Department of Neurology, Second Affiliated Hospital of Sun Yat-sen University, Guangzhou  510120, Guangdong Province, China
2Blanchette Rockefeller Neurosciences Institute, Rockville, MD  20850, USA

Yuanlin Sun☆, Studying for doctorate, Associate professor, Department of Neurology, Second Affiliated Hospital of Sun Yat-sen University, Guangzhou 510120, Guangdong Province, China

Corresponding author: Enxiang Tao, Doctor, Professor, Doctoral supervisor, Department of Neurology, Second Affiliated Hospital of Sun Yat-sen University, Guangzhou  510120, Guangdong Province, China    
taoenxiang@yahoo.com.cn

Supported by: the Natural Science Foundation of Guangdong Province, No. 04009355*; Science and Technology Planning Project of Guandong Province, China, 05B33801003*

Abstract
BACKGROUND: Rifampicin inhibits the formation of α-synuclein multimer and protects against 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyritine (MPTP)-induced PC12 cell apoptosis.
OBJECTIVE: To compare the effect of rifampicin pre- and post-treatment on tyrosine hydroxylase and α-synuclein expression in substantia nigra pars compacta in a rat model of Parkinson’s disease.
DESIGN, TIME AND SETTING: A randomized, controlled experiment was performed at the Ex-perimental Animal Center of Sun Yat-sen University North Campus (China) from November 2006 to October 2008.
MATERIALS: Rifampicin was purchased from MD, USA; rotenone was purchased from Sigma, USA; mouse anti-rat α-synuclein monoclonal antibody was purchased from B&D, USA; and rabbit anti-rat tyrosine hydroxylase monoclonal antibody was purchased from Chemicon, USA.
METHODS: A total of 72 male, Sprague Dawley rats, aged 8 weeks, were randomly assigned to 5 groups: blank control (n = 12), rifampicin (n = 12), rotenone (n = 16), rifampicin pre-treatment (n = 16), and rifampicin post-treatment (n = 16). Parkinson’s disease model rats were established via a subcutaneous injection of rotenone (1.5 mg/kg per day) in the three treatment groups, once a day for 3 successive weeks. Rifampicin (30 mg/kg per day) was intragastrically administered in the rifam-picin pre-treatment group 3 days prior to rotenone induction and in the rifampicin post-treatment group 7 days after rotenone induction. Rats were treated with a subcutaneous injection of 1 mL/kg per day sunflower oil in the blank control group and an intragastric injection of 30 mg/kg per day ri-fampicin in the rifampicin group, once a day for 3 successive weeks in total.
MAIN OUTCOME MEASURES: Prior to treatment and in the end of the 3rd week after treatment, the rats were evaluated using the modified neurological severity score. The substantia nigra from the rats was extracted for hematoxylin-eosin staining. Western blot analysis was performed to determine tyrosine hydroxylase and α-synuclein expression.
RESULTS: Hematoxylin-eosin staining revealed a significant reduction in the number of substantia nigral neurons in the rotenone group, in addition to neurodegradation, hypopigmentation, and pyknosis. In the rifampicin pre-treatment and post-treatment groups, the number of dopaminergic neurons was significantly increased compared with the rotenone group (P < 0.01), with slight neu-ronal damage. Compared with the rotenone group, substantia nigral tyrosine hydroxylase expres-sion was significantly increased in the rifampicin pre-treatment and post-treatment groups  (P < 0.01), but α-synuclein expression and modified neurological severity scores were significantly de-creased (P < 0.01). In addition, the effect of rifampicin in the pre-treatment group was superior to the post-treatment group. There was no significant difference in tyrosine hydroxylase and α-synuclein expression, or in the modified neurological severity scores, between the blank control and rifampicin groups (P > 0.05).
CONCLUSION: Rifampicin significantly attenuated neuropathological and behavioral motor deficits induced by rotenone. Moreover, rifampicin enhanced tyrosine hydroxylase expression, but inhibited α-synuclein expression. The effect of rifampicin pre-treatment was superior to rifampicin post-treatment.
Key Words: rifampicin; rotenone; Parkinson’s disease; α-synuclein; dopaminergic neurons

INTRODUCTION
   
Parkinson’s disease (PD) is a neurodegenerative disorder that is pathologically characterized by the loss of dopamine neurons in the substantia nigra pars compacta (SNpc) and the presence of cytoplasmic inclusions (Lewy bodies) and dystrophic neuritis (Lewy neuritis) in surviving neurons[1]. Although the molecular pathogenesis of this disorder remains poorly understood, it has been suggested that α-synuclein plays a key role in the neurodegenerative process of synucleinopathies[2-3] and can be stimulated to aggregate in vitro or in vivo. The accumulation of α-synuclein in dopaminergic neurons is assumed to be a major pathogenic factor in PD. Therefore, inhibition of α-synuclein aggregation has become a plausible method for PD therapy[4].
Previous epidemiological results have shown significantly reduced morbidity of senile dementia in patients that received long-term rifampicin treatment[5]. Subsequent studies revealed that rifampicin blocked cellular β-amyloid aggregation and toxicity in cell culture[6]. Beta-amyloid and α-synuclein form amyloid-type inclusions in Alzheimer’s disease and PD, respectively, and the two diseases are often co-morbid[7], suggesting some commonality in pathologies. The cytoprotective effect of rifampicin, as well as the suppression of β-amyloid aggregation, suggests that it has therapeutic potential for PD[8]. In vitro, rifampicin inhibits α-synuclein fibrillation and disaggregates pre-formed fibrils[9]; it also attenuates 1-methyl-4-phenylpyridinium (MPP+)-mediated death of primary dopaminergic neurons in culture and protects PC12 cells against cytotoxicity and increased expression of an α-synuclein multimer in response to MPP+ exposure[10]. In vivo, rifampicin reduces MPTP-induced neurotoxicity and blocks α-synuclein aggregation in the mouse brain[11-12].
The present study established an in vivo PD model with rotenone in Sprague Dawley rats. The protective role of rifampicin was evaluated by behavioral impairment, as well as tyrosine hydroxylase (TH) and α-synuclein expressions in the substantia nigra of rats.

MATERIALS AND METHODS

Design
Randomized and controlled animal experiment.
Time and setting
The experiment was performed at the Experimental Animal Center of Sun Yat-sen University North Campus (China) between November 2006 and October 2008.
Materials
A total of 72 healthy, adult, specific pathogen-free, male, Sprague Dawley rats, aged 8 weeks and weighing 250–300 g, were obtained from the Experimental Animal Center of Sun Yat-sen University (Certificate No. SYXK (yue) 2007-0081). All treatments were in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals published by the Ministry of Science and Technology of the People’s Republic of China[13].
Main reagents and equipment are listed as follows:

Rotenone was dissolved in sunflower oil at a concentration of 1.5 mg/mL; rifampicin was diluted with 0.5% sodium carboxymethyl cellulose at a concentration of 5 mg/mL.
Methods
Animal grouping and treatment
A total of 72 rats were randomly assigned to control (n = 24) and treatment (n = 48) groups. The control group was subdivided into 2 groups: blank (n = 12) and rifampicin (n = 12). The treatment group was subdivided into 3 groups: rotenone (n = 16), rifampicin post-treatment commencing 7 days after start of rotenone treatment (n = 16), and rifampicin pre-treatment 3 days prior to start of rotenone treatment (n = 16).
According to previously described methods[14], a chronic rotenone model of PD was established in rats by subcutaneous injection of rotenone. Rotenone (1 mL/kg) was subcutaneously injected once per day for 3 weeks to the three treatment groups. Rifampicin (6 mL/kg) was administered intragastrically once per day to the rifampicin, rifampicin pre-treatment, and rifampicin post-treatment groups for 3 successive weeks. Sunflower oil (1 mL/kg) was administered intragastrically to the blank control group.
Tissue processing
Following behavioral testing, half rats from each group were sacrificed by anesthesia and the substantia nigra (end of anterior fontanelle anterior-posterior: –6.2 mm to –4.6 mm; dorsal-ventral: 7.5 mm to 8.0 mm; lateral:   0.5 mm to 2.5 mm) was rapidly dissected using previously described methods[15]. The rats were transcardially perfused with phosphate buffered saline (PBS), followed by 20 minutes of perfusion with 4% paraformaldehyde/0.1 mol/L PBS. The brains were then removed and immersed in paraformaldehyde fixative. Brain sections were dehydrated with graded ethanol, passed through xylene, and embedded in paraffin. Paraffin sections (5 μm thickness) of the substantia nigra were processed for hematoxylin-eosin (HE) staining and immunofluorescence.
The remaining rats in each group were perfused with  0.1 mol/L PBS (pH 7.4) following a lethal injection of anesthetic. A tissue block from the brain, which contained the substantia nigra, was removed and placed in PBS. The arachnoid and dura were removed and the tissue was combined with a protein extraction reagent, protease inhibitors, and 1% β-mercaptoethanol, and homogenized in the cold. Tissue homogenates were centrifuged at 10 500 r/min for 5 minutes at 4 °C. Protein concentration was detected using BCA chromatography according to previously described methods[16].
HE staining in substantia nigra
Paraffin sections were deparaffinized and rehydrated, and then stained with hematoxylin for 3 minutes, followed by eosin for 5-10 seconds. The substantia nigra was examined by light microscopy at a magnification of × 400. All cells in the substantia nigra pars compacta field were quantified from every third section for all 5 rats in each group. The number of positive neurons/mm2 was extrapolated from these quantifications.
Immunofluorescence of TH and α-synuclein in the substantia nigra
Double immunofluorescence was performed according to previously described methods[17]. Paraffin sections were deparaffinized and rehydrated, followed by blocking with 5% bovine serum albumin for 30 minutes, rabbit anti-rat TH monoclonal antibody (1: 200) and mouse anti-rat α-synuclein monoclonal antibody (1: 200) simultaneously overnight at 4 °C, and rhodamine-conjugated goat anti-rabbit IgG (1: 100) and FITC-conjugated goat anti-mouse IgG (1: 100) simultaneously for 2 hours at room temperature. The sections were examined under fluorescence microscope in the dark. All cells in the substantia nigra field were quantified in every third section from 5 rats in each group. The number of positive neurons/mm2 was extrapolated from these quantifications.
Western blot analysis of TH and α-synuclein
Western blot analysis was performed according to previously described methods[16]. Briefly, each supernatant aliquot (20 μg total protein) was loaded on a lane and the proteins were separated by electrophoresis on sodium dodecyl sulfate polyacrylamide gels. Proteins were transferred to a polyvinylidene difluoride membrane, blocked with 5% nonfat milk for 3 hours, incubated with rabbit anti-rat TH monoclonal antibody (1: 500) or mouse anti-rat α-synuclein monoclonal antibody (1: 500) at 4 °C overnight, rinsed 3 times with tris-buffered saline and 0.5% (v/v) tween 20, incubated with appropriate secondary antibodies, reacted with electrochemiluminescence-plus chemiluminescence kit, and exposed to X-ray films. Mouse anti-rat β-actin monoclonal antibody was used to confirm equal protein loading.
Images of midbrain sections were acquired with a Nikon-Eclipse TE2000-U microscope via a Nikon- T-B2.5XA color camera and were analyzed using Image-Pro Plus version 5.1 image analysis software. The protein bands were photographed using an Imager gel documentation system, and band intensities were calculated by densitometric analysis using Quantity One 1-D Analysis Software.
Grading of behavioral impairment using the modified neurological severity score
Prior to start of treatment and in the end of the 3rd week after treatment, the rats were evaluated using the modified neurological severity score, which is a behavioral scale that provides a composite analysis of motor, sensory, balance, and reflex function. The scores were assessed as previously described method[18].
Main outcome measures
Modified neurological severity scores; neuropathological changes in substantia nigra; TH and α-synuclein expression.
Statistical analysis
All results were presented as Mean ± SD. Overall differences among the groups were determined by analysis of variance. A posteriori pair-wise differences were determined using the Student-Newman-Keuls q-test. Data were analyzed using SPSS 13.0 software (SPSS, Chicago, IL, USA). A value of P < 0.05 was considered statistically significant.

RESULTS

Quantitative analysis of experimental animals
A total of 72 rats were initially included in the study. During the week 1 of treatment, 1 rat in the rotenone group, 1 in the rifampicin post-treatment group, and 2 in the rifampicin pre-treatment group died due to acute rotenone toxicity. During week 3 of treatment, 1 rat in the rotenone group and 1 in the rifampicin post-treatment group died, and both exhibited lethargy and anorexia. A total of 66 rats were included in the final analysis.  
SNpc neuropathology
HE-stained nigral sections from the different groups exhibited neuropathological differences. In the control group, the abundant and typical TH-positive dopaminergic neurons were large and darkly stained (Figure 1A). Damage was not evident in the rifampicin group, and this group was indistinguishable from the blank control group (Figure 1B). In contrast, the SNpc was severely damaged by rotenone treatment (Figure 1C). There were approximately two-thirds less neurons in this structure. In addition, many of the surviving neurons were smaller and TH expression was weaker, indicating various stages of degeneration. The rifampicin post-treatment group was statistically different from the pre-treatment group; protection against rotenone toxicity was incomplete and approximately 40% of the neurons were rescued (Figure 1D). However, many of the remaining neurons were pyknotic or exhibited irregular morphology. Approximately 78% of neurons were preserved in the rifampicin pre-treatment group (Figure 1E). The majority of remaining neurons exhibited normal morphology. Some pyknotic cells were observed, but they were less compared with the rotenone or rifampicin post-treatment groups.

TH and α-synuclein immunofluorescence in the SNpc
Rotenone treatment significantly increased the number of α-synuclein-positive cells and reduced the number of TH-positive cells, as determined by immunofluorescence. Rifampicin pre-treatment significantly attenuated, but did not abolish this change, while rifampicin post-treatment marginally reduced the number of α-synuclein-positive cells and increased the number of TH-positive cells. There was no significant difference in α-synuclein- and TH-positive cells between the blank control and rifampicin groups. In addition, there was a redistribution of α-synuclein in the cells, with more protein visible in the neurites of the substantia nigra reticulata, which were probably dendrites from inverted neurons of the ventral-most SNpc layer. The increased number of α-synuclein-positive cells was observed in all treatment groups (Table 1, Figure 2).

TH and α-synuclein expression in brain tissue
Western blot analysis of TH and α-synuclein expression demonstrated no significant differences between the blank control and rifampicin groups (Figure 3, Tables 1, 2). Compared with the blank control and rifampicin groups, TH expression was significantly reduced and α-synuclein expression was significantly increased in the rotenone, rifampicin post-treatment, and rifampicin pre-treatment groups (Figure 3, Tables 1, 2). The rotenone group exhibited significantly increased α-synuclein expression and reduced TH expression. Rifampicin pre-treatment significantly blocked these changes. However, rifampicin post-treatment was not as successful as rifampicin pre-treatment for reducing α-synuclein expression and increasing TH expression.
Rat behavioral observations
Prior to treatment, rat behavior was normal in all groups, with modified neurological severity score of 0. After 3  weeks of treatment, rats in the blank control and rifampicin groups did not exhibit impairment (modified neurological severity score = 0). The rotenone group was the most severely impaired (modified neurological severity score = 13.4 ± 2.6), as indicated by decreased activity, instability of gait, tremor, stiffness, flexion, piloerection, and muscle wasting. The rifampicin post-treatment group exhibited some of these symptoms (modified neurological severity score = 8.4 ± 2.2), but not as severe as the rotenone group. The rifampicin pre-treatment group exhibited minimal impairment (modified neurological severity score = 2.4 ± 2.3) in comparison to the rotenone and rifampicin post-treatment groups.
 

DISCUSSION

Rotenone, a potent inhibitor of mitochondrial complex I, is a pesticide used worldwide. Chronic in vivo rotenone administration induces many key features of PD[14]. The present study successfully established a rat model of PD. Chronic injections of rotenone induced specific nigrostriatal dopaminergic neurodegeneration, as well as upregulation of α-synuclein in surviving dopaminergic neurons. The neuropathological changes were accompanied by considerable deterioration of motor, sensory, balance, and reflex functions, as indicated by significantly increased modified neurological severity scores. These results indicate that the rotenone rat model is a suitable PD animal model. Rotenone is a mitochondrial toxin, which is associated with the generation of free radicals and the collapse of ATP synthesis[2]; it results in oxidative stress in dopaminergic neurons and α-synuclein aggregation[16].
The protective effects of rifampicin against rotenone-induced neurotoxicity were identified as follows. (1) Rifampicin treatment markedly reduced the modified neurological severity scores compared with rotenone treatment. The rifampicin-pretreated rats exhibited significantly less impairment than rats treated with rotenone alone. (2) Rifampicin significantly reduced neuronal loss, as measured by HE staining. Rifampicin pre-treatment was more successful than rifampicin post-treatment, which suggested that early intervention in PD could result in a better outcome. (3) Rifampicin attenuated decreased TH expression in the substantia nigra, which was induced by rotenone treatment. These results were similar to the protection conferred by rifampicin in the mouse brain against MPTP toxicity[11]. (4) Rotenone treatment significantly increased α-synuclein expression, which was consistent with previous reports[16]. Increased α-synuclein expression can be interpreted as a reactive cytoprotective mechanism or as part of the cell death cascade. There is increasing evidence of vital functional roles for α-synuclein[19-22], which suggests a cytoprotective role for α-synuclein. However, it is possible that post-translational modification could alter binding, folding, and aggregation characteristics[23-28]. These changes ultimately result in cytotoxicity. Rifampicin treatment significantly reduced α-synuclein expression compared with rotenone treatment, and rifampicin pre-treatment resulted in less α-synuclein expression than delayed treatment.
Several properties of rifampicin could be responsible for the neuroprotective mechanisms. (1) The common structure of the rifampicin derivative is a naphthohydroquinone chromophore, which is spanned by an aliphatic ansa chain, and the structural feature of a naphthohydroquinone ring may contribute to the function of a hydroxyl radical scavenger[6, 11]. (2) Rifampicin can penetrate the blood-brain barrier, resulting in a direct effect to the brain. The lipophilic ansa chain in rifampicin is primarily responsible for drug transport across the blood-brain barrier[29-30]. (3) Rifampicin reduces α-synuclein expression and prevents aggregation in vitro and in vivo[6, 31]. In a previous study, flavonoid baicalein, which contains a chromophoric naphthohydroquinone group similar to rifampicin, inhibited fibrillation of α-synuclein and disaggregated existing fibrils, in particular in the oxidized quinone form[32]. Similarly, the catecholamine dopamine inhibits α-synuclein fibrillation via covalent binding of the orthoquinone derivative of dopamine[33]. Therefore, one possible mechanism for rifampicin inhibition of α-synuclein aggregation is via nucleophilic attack by the α-synuclein lysine side chains leading to covalent modification[9]. (4) Rifampicin binds to and activates glucocorticoid receptors, and rifampicin-mediated inhibition of apoptosis and activation of caspase-3 and caspase-8 occurs in part via glucocorticoid receptor activation[34-35], although the details of this pathway remain controversial[36].
Regardless of the mechanism, or the combination of mechanisms, the present results demonstrated neuroprotective effects of rifampicin against rotenone toxicity in vivo, as evidenced by improved behavior, decreased loss of dopaminergic neurons, and reduced α-synuclein expression. Moreover, neuroprotection was conferred at doses that produced no evidence of toxicity. Rifampicin, therefore, is an extremely promising therapeutic agent for the treatment of PD. The fact that delayed treatment reduced, but did not eliminate, the deleterious effects of rotenone strongly, suggests that treatment would be most effective with early intervention in the disease process. Future efforts should focus on the identification of early markers of PD pathogenesis.

REFERENCES

[1] Shults CW. Lewy bodies. Proc Natl Acad Sci U S A. 2006;103(6): 1661-1668.
[2] Liu H, Wang XZ. Alpha-synuclein and Parkinson disease. Neural Regen Res. 2007;2(4):239-243.
[3] Xu J, Kao SY, Lee FJ, et al. Dopamine-dependent neurotoxicity of alpha-synuclein: a mechanism for selective neurodegeneration in Parkinson disease. Nat Med. 2002;8(6):600-606.
[4] Ono K, Hirohata M, Yamada M. Alpha-synuclein assembly as a therapeutic target of Parkinson's disease and related disorders. Curr Pharm Des. 2008;14(30):3247-3266.
[5] Namba Y, Kawatsu K, Izumi S, et al. Neurofibrillary tangles and senile plaques in brain of elderly leprosy patients. Lancet. 1992; 340(8825):978.
[6] Tomiyama T, Asano S, Suwa Y, et al. Rifampicin prevents the aggregation and neurotoxicity of amyloid beta protein in vitro. Biochem Biophys Res Commun. 1994;204(1):76-83.
[7] Kim S, Seo JH, Suh YH. Alpha-synuclein, Parkinson's disease, and Alzheimer's disease. Parkinsonism Relat Disord. 2004;10 Suppl 1:S9-13.
[8] Bradbury J. New hope for mechanism-based treatment of Parkinson's disease. Drug Discov Today. 2005;10(2):80-81.
[9] Li J, Zhu M, Rajamani S, et al. Rifampicin inhibits alpha-synuclein fibrillation and disaggregates fibrils. Chem Biol. 2004;11(11): 1513-1521.
[10] Xu J, Wei C, Xu C, et al. Rifampicin protects PC12 cells against MPP+-induced apoptosis and inhibits the expression of an alpha-synuclein multimer. Brain Res. 2007;1139:220-225.  
[11] Oida Y, Kitaichi K, Nakayama H, et al. Rifampicin attenuates the MPTP-induced neurotoxicity in mouse brain. Brain Res. 2006; 1082(1):196-204.
[12] Ubhi K, Rockenstein E, Mante M, et al. Rifampicin reduces alpha-synuclein in a transgenic mouse model of multiple system atrophy. Neuroreport. 2008;19(13):1271-1276.
[13] The Ministry of Science and Technology of the People’s Republic of China. Guidance Suggestions for the Care and Use of Laboratory Animals. 2006-09-30.
[14] Fleming SM, Zhu C, Fernagut PO, et al. Behavioral and immunohistochemical effects of chronic intravenous and subcutaneous infusions of varying doses of rotenone. Exp Neurol. 2004;187(2):418-429.
[15] Heffner TG, Hartman JA, Seiden LS. A rapid method for the regional dissection of the rat brain. Pharmacol Biochem Behav. 1980;13(3):453-456.
[16] Testa CM, Sherer TB, Greenamyre JT. Rotenone induces oxidative stress and dopaminergic neuron damage in organotypic substantia nigra cultures. Brain Res Mol Brain Res. 2005;134(1): 109-118.
[17] Yoo YM, Lee U, Kim YJ. Apoptosis and nestin expression in the cortex and cultured astrocytes following 6-OHDA administration. Neurosci Lett. 2005;382(1-2):88-92.
[18] Chen Y, Constantini S, Trembovler V, et al. An experimental model of closed head injury in mice: pathophysiology, histopathology, and cognitive deficits. J Neurotrauma. 1996;13(10):557-568.
[19] Jenco JM, Rawlingson A, Daniels B, et al. Regulation of phospholipase D2: selective inhibition of mammalian phospholipase D isoenzymes by alpha- and beta-synucleins. Biochemistry. 1998;37(14):4901-4909.
[20] Perez RG, Waymire JC, Lin E, et al. A role for alpha-synuclein in the regulation of dopamine biosynthesis. J Neurosci. 2002;22(8): 3090-3099.
[21] Quilty MC, King AE, Gai WP, et al. Alpha-synuclein is upregulated in neurones in response to chronic oxidative stress and is associated with neuroprotection. Exp Neurol. 2006;199(2): 249-256.
[22] Uversky VN. Neuropathology, biochemistry, and biophysics of alpha-synuclein aggregation. J Neurochem. 2007;103(1):17-37.
[23] Chandra S, Gallardo G, Fernández-Chacón R, et al. Alpha-synuclein cooperates with CSPalpha in preventing neurodegeneration. Cell. 2005;123(3):383-396.
[24] Rochet JC, Outeiro TF, Conway KA, et al. Interactions among alpha-synuclein, dopamine, and biomembranes: some clues for understanding neurodegeneration in Parkinson's disease. J Mol Neurosci. 2004;23(1-2):23-34.
[25] Andringa G, Lam KY, Chegary M, et al. Tissue transglutaminase catalyzes the formation of alpha-synuclein crosslinks in Parkinson's disease. FASEB J. 2004;18(7):932-934.
[26] Junn E, Mouradian MM. Human alpha-synuclein over-expression increases intracellular reactive oxygen species levels and susceptibility to dopamine. Neurosci Lett. 2002;320(3):146-150.
[27] Ahn M, Kim S, Kang M, et al. Chaperone-like activities of alpha-synuclein: alpha-synuclein assists enzyme activities of esterases. Biochem Biophys Res Commun. 2006;346(4): 1142-1149.
[28] Citron BA, Suo Z, SantaCruz K, et al. Protein crosslinking, tissue transglutaminase, alternative splicing and neurodegeneration. Neurochem Int. 2002;40(1):69-78.
[29] Mindermann T, Landolt H, Zimmerli W, et al. Penetration of rifampicin into the brain tissue and cerebral extracellular space of rats. J Antimicrob Chemother. 1993;31(5):731-737.
[30] Yulug B, Kilic U, Kilic E, et al. Rifampicin attenuates brain damage in focal ischemia. Brain Res. 2004;996(1):76-80.
[31] Tomiyama T, Kaneko H, Kataoka K, et al. Rifampicin inhibits the toxicity of pre-aggregated amyloid peptides by binding to peptide fibrils and preventing amyloid-cell interaction. Biochem J. 1997; 322(Pt 3):859-865.
[32] Zhu M, Rajamani S, Kaylor J, et al. The flavonoid baicalein inhibits fibrillation of alpha-synuclein and disaggregates existing fibrils. J Biol Chem. 2004;279(26):26846-26857.
[33] Conway KA, Rochet JC, Bieganski RM, et al. Kinetic stabilization of the alpha-synuclein protofibril by a dopamine-alpha-synuclein adduct. Science. 2001;294(5545):1346-1349.
[34] Calleja C, Pascussi JM, Mani JC, et al. The antibiotic rifampicin is a nonsteroidal ligand and activator of the human glucocorticoid receptor. Nat Med. 1998;4(1):92-96.
[35] Gollapudi S, Jaidka S, Gupta S. Molecular basis of rifampicin-induced inhibition of anti-CD95-induced apoptosis of peripheral blood T lymphocytes: the role of CD95 ligand and FLIPs. J Clin Immunol. 2003;23(1):11-22.
[36] Herr AS, Wochnik GM, Rosenhagen MC, et al. Rifampicin is not an activator of glucocorticoid receptor. Mol Pharmacol. 2000;57(4): 732-737.
 (Edited by Zhong GW/Ji H/Song LP)