1. Minocycline, memantine,and glycoconjugate were assessed for their ability to protect cultured primary cortical neurons against double-stranded RNA-induced neurotoxicity. 2. Minocycline but not memantine or glycoconjugate protected cultured cells and warrants further investigation.published_or_final_versio

Chang, RCC

Cheung, YT

Ho, YS

Lai, SW

So, KF

Yik, SY

Yu, MS

Hong Kong Medical Journal

HKU Scholars Hub

Title Neuroprotective effects of minocycline on double-stranded RNA-induced neurotoxicity in cultured cortical neuronsAuthor(s) Yik, SY; Yu, MS; Ho, YS; Lai, SW; Cheung, YT; So, KF; Chang,RCCCitation Hong Kong Medical Journal, 2012, v. 18 n. 1, suppl 2, p. 42-44Issued Date 2012URL http://hdl.handle.net/10722/146384Rights Creative Commons: Attribution 3.0 Hong Kong LicenseRESEARCH FUND FOR THE CONTROL OF INFECTIOUS DISEASES42      Hong Kong Med J Vol 18 No 1 Supplement 2 February 2012Neuroprotective effects of minocycline on double-stranded RNA-induced neurotoxicity in cultured cortical neuronsSY Yik 易雪怡MS Yu 余雯珊 YS Ho 何宛姍 CSW Lai 賴秀芸 YT Cheung 張婉婷 KF So 蘇國輝RCC Chang 鄭傳忠Key Messages1. Minocycline, memantine, and glycoconjugate were assessed for their ability to protect cultured primary cortical neurons against double-stranded RNA-induced neurotoxicity.2. Minocycline but not memantine or glycoconjugate protected cultured cells and warrants further investigation.Laboratory of Neurodegenerative Diseases, Department of Anatomy, Research Centre of Heart, Brain, Hormone and Healthy Aging, LKS Faculty of Medicine, The University of Hong KongSY Yik, MS Yu, YS Ho, CSW Lai, YT Cheung, KF So, RCC Chang RFCID project number: 05050032Principal applicant and corresponding author:Dr Raymond Chuen-Chung ChangDepartment of Anatomy, Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong SAR, ChinaTel: (852) 2819 9127Fax: (852) 2817 0857Email: rccchang@hkucc.hku.hkHong	Kong	Med	J	2012;18(Suppl	2):S42-4IntroductionJapanese encephalitis virus (JEV) is an RNA virus spread by infected mosquitoes. Patients may develop symptoms such as fever, chills, tiredness, headache, nausea, vomiting, confusion, and agitation. The disease can lead to serious infection in the brain (encephalitis), and in about 30% of patients severe brain damage ensues. There is no specific treatment for the disease, and the role of medication is to relieve severe symptoms.  Few studies have reported on protection of neurons subsequent to viral infection. We hypothesise that neuroprotective drugs can be used to treat Japanese encephalitis patients and safeguard neurons. We tested some neuroprotective drugs from western and Chinese medicine (minocycline, memantine, and glycoconjugate) for treating Japanese encephalitis. The double-stranded RNA analogue, pIpC, is a commonly used analogue mimicking the consequence of virus infection. Cell cultures of neurons were used to investigate whether these three drugs could attenuate any direct toxic effects of pIpC toward neurons. The underlying mechanisms of the neuroprotective effects were also investigated.  Minocycline is a semi-synthetic tetracycline derivative exhibiting biological effects of neuroprotection in different types of neurological disorders including Parkinson’s disease, amyotrophic lateral sclerosis, spinal cord injury, and cerebral ischaemia.1,2 Such neuroprotective effects are largely attributed to the inhibition of microglia and production of inflammatory factors. Minocycline can even attenuate the excitotoxicity of glutamate in the cerebrospinal fluid from amyotrophic lateral sclerosis patients. The pathology of Japanese encephalitis entails severe cerebral inflammation, which may be minimised by minocycline and so protect neurons.  Memantine is a non-competitive NMDA open channel blocker approved by the US Food and Drug Administration for neuroprotection in Alzheimer’s disease.3 Glutamate is usually released from injured or energy deficient neurons in many neurological disorders. It can further depolarise other healthy neurons and the potassium ions released from such depolarised neurons can enhance cerebral inflammation aggravating neuronal death.4 Memantine may serve as neuroprotective agent to minimise secondary brain damage exerted by glutamate.  Glycoconjugate is extracted from the anti-ageing Chinese medicine Lycium	barbarum. It is neuroprotective against the toxicity exerted from β-amyloid protein, a toxin found in the Alzheimer’s disease, as well as retinal ganglion cells in an animal model of glaucoma.5 Its cytoprotective properties may extend to other neurodegenerative diseases and even Japanese encephalitis or its dsRNA-elicited neurotoxicity. MethodsThis study was conducted from 1 June 2006 to 31 May 2008. Protection of Neuroprotective effects of minocyclineHong Kong Med J Vol 18 No 1 Supplement 2 February 2012      43neurons from the direct toxic effects of pIpC by the three drugs (minocycline, memantine, and glycoconjugate) was examined, using primary cultures of cortical neurons and human neuroblastoma SH-SY5Y cells. Primary cortical neurons were prepared from embryonic day 17 Sprague-Dawley rats (Laboratory Animal Unit, The University of Hong Kong).6 In brief, cerebral cortices were mechanically dissociated in phosphate-buffered saline with glucose (18 mM). Cells were seeded onto six-well (0.8×106 cells/well) or 12-well plates (1.0×105 cells/well) pre-coated with poly-L-lysine (25 μg/mL). The culture medium consisted of Neurobasal Medium supplemented with 2% B-27 supplement, L-glutamine (2 mM), penicillin (50 U/mL), streptomycin (50 μg/mL), and 2-mercaptoethanol. Neurons were maintained at 37ºC in a humidified atmosphere of 5% CO2 and were cultured for 7 days prior to treatment. To evaluate whether minocycline exerted neuroprotective effects, neurons were pretreated with different dosages of potential drugs for 1 h prior to exposure of pIpC. Neurons were challenged by 20 μg/mL pIpC with FuGENE 6 reagent. After 48 h, neurons were harvested for assay. General toxicity was determined by measuring lactate dehydrogenase (LDH) activity released into culture medium. Apoptosis was assessed by determining caspase-3 activity. TUNEL and DAPI nuclear staining were used to count the number of apoptotic bodies. All results obtained were expressed as mean±standard error from at least three independent experiments. Significant difference between groups was determined by one-way ANOVA, followed by Student-Newman-Keuls as a post hoc test. Fig. Effects of minocycline, memantine, and glycoconjugate on pIpC-induced neurotoxicityCultured cortical neurons were treated with (a) minocycline, (b) memantine, or (c) glycoconjugate at different dosages 1 h prior to the exposure of 20 μg/mL pIpC. Culture medium was taken for lactate dehydrogenase (LDH) assay and cells were harvested for caspase-3 assay 24 h after treatmentMin (µM)1.61.41.21.00.80.60.40.20.0Caspase-3 asseyVehiclecontrol0 1 10 20P<0.001P<0.001 P<0.001LBA (µg/mL)1.41.21.00.80.60.40.20.0Caspase-3 assey0 0.1 10 100 250VehiclecontrolP<0.05P<0.05 P<0.051.41.21.00.80.60.40.20.0LDH assayVehiclecontrol0 1 10 20Min (µM)P<0.005 P<0.001P<0.001(a)0 0.1 10 100 2501.21.00.80.60.40.20.0LDH assayVehiclecontrol LBA (µg/mL)P<0.001(c)1.61.41.21.00.80.60.40.20.0LDH assayVehiclecontrol0 0.001 0.01 0.1 1P<0.005P<0.05P<0.05(b)Mem (µM)Vehiclecontrol0 0.001 0.01 0.1 11.61.41.21.00.80.60.40.20.0Caspase-3 assey P<0.05Mem (µM)Yik et al44      Hong Kong Med J Vol 18 No 1 Supplement 2 February 2012Results and discussionpIpC exhibits direct neurotoxicity. Increased levels of dsRNA produced from JEV can be toxic to cortical neurons. Dose-dependent and time-dependent experiments were performed using primary cultures of cortical neurons and SH-SY5Y cells as two different models. Exposure of neurons to 20 μg/mL pIpC resulted in 1.23±0.02-fold increase in the release of LDH when compared to the vehicle control. At higher dosage, the release of LDH decreased to 1.15±0.03-fold. Thus, 20 μg/mL pIpC was chosen for the subsequent experiments. As pIpC could trigger its neurotoxicity via activating caspase-3, a colorimetric caspase-3-like activity assay was carried out for different dosages of pIpC. Exposure of neurons to 20 μg/mL pIpC significantly increased the activity of caspase-3 to 1.33±0.06-fold. Apart from biochemical assays, neurites were damaged by pIpC, as shown by their morphology. Under a phase contrast microscope, the cell bodies of cultured neurons in the control group appeared round and dark with a network of process. After exposure to pIpC, cultured neurons were damaged and showed fragmented neurites. Exposure of neurons to 20 μg/mL pIpC for 48 h resulted in 1.2±0.03-fold increase in the release of LDH when compared to the vehicle control. There was a slight decrease of LDH release at 72 h. The colorimetric caspase-3-like activity assay was also carried out after different durations of exposure to pIpC. Exposure of neurons to 20 μg/mL pIpC for 48 h significantly increased the activity of caspase-3 to 1.43±0.03-fold. Similar to the results of the LDH release assay, caspase-3-like activity decreased at 72 h. As the maximum increase of LDH release and caspase-3 was at 48 h, this duration was chosen for the subsequent experiments. Exposure of human dopaminergic SH-SY5Y cell neurons to 20 μg/mL pIpC resulted in 1.50±0.09-fold increase of LDH release when compared to that of the vehicle control. Exposure of neurons to 20 μg/mL pIpC significantly increased the activity of caspase-3 to 1.29±0.03-fold. These results provided further proof of the toxicity of pIpC, using SH-SY5Y cells as second cell culture model. pIpC induced neuronal apoptosis can be confirmed by recognised methods. Treatment with pIpC for 48 h increased the number of apoptotic bodies (typical nuclear condensation and fragmentation) as indicated by the increased number of TUNEL-positive neurons. TUNEL assay relied on the detection of fragmented DNA strands. In control cultures treated with vehicle, only few TUNEL-positive cells were observed. Minocycline exhibited the best neuroprotective effect on neurons based on the LDH assay; 10 or 20 μM exerted similar neuroprotection on caspase-3 activity (Fig a). We could not further increase the concentrations of minocycline as this would exceed the normal dosage for treatment and exert side-effect in patients. Memantine significantly reduced the toxicity of pIpC (both LDH release and caspase-3 activity), but its neuroprotective effect was marginal. In contrast to memantine, minocycline elicited significant neuroprotective effects on cortical neurons against direct pIpC toxicity (Fig b). Glycoconjugates from L	 barbarum elicited cytoprotective effects; L	 barbarum attenuated caspase-3 activity triggered by pIpC (Fig c). Nonetheless, only the highest dosage of L	barbarum could inhibit LDH release triggered by pIpC. Of the three different compounds, minocycline exhibited significant neuroprotective effects against direct pIpC neurotoxicity and warrants further investigation.AcknowledgementThe study was supported by the Research Fund for the Control of Infectious Diseases, Food and Health Bureau, Hong Kong SAR Government (#05050032).References 1. Tikka TM, Koistinaho JE. Minocycline provides neuroprotection against N-methyl-D-aspartate neurotoxicity by inhibiting microglia. J Immunol 2001;166:7527-33.2. Tikka TM, Vartiainen NE, Goldsteins G, et al. Minocycline prevents neurotoxicity induced by cerebrospinal fluid from patients with motor neurone disease. Brain 2002;125:722-31.3. Miguel-Hidalgo JJ, Alvarez XA, Cacabelos R, Quack G. Neuroprotection by memantine against neurodegeneration induced by beta-amyloid (1-40) Brain Res 2002;958:210-21.4. Chang RC, Hudson PM, Wilson BC, Liu B, Abel H, Hong JS. High concentrations of extracellular potassium enhance bacterial endotoxin lipopolysaccharide-induced neurotoxicity in glia-neuron mixed cultures. Neuroscience 2000;97:757-64.5. Chang RC, So KF. Use of anti-aging herbal medicine, Lycium barbarum, against aging-associated diseases. What do we know so far? Cell Mol Neurobiol 2008;28:643-52.6. Lai CS, Yu MS, Yuen WH, So KF, Zee SY, Chang RC. Antagonizing beta-amyloid peptide neurotoxicity of the anti-aging fungus Ganoderma lucidum. Brain Res 2008;1190:215-24.

Neuroprotective effects of minocycline on double-stranded RNA-induced neurotoxicity in cultured cortical neurons

http://hub.hku.hk/bitstream/10722/146384/1/content.pdf

Neuroprotective effects of minocycline on double-stranded RNA-induced neurotoxicity in cultured cortical neurons

Abstract

Similar works

Full text

Available Versions

HKU Scholars Hub