Glutaredoxin 1 downregulation in the substantia nigra leads to dopaminergic degeneration in mice
Aditi Verma, PhD1; Ajit Ray, PhD1; Deepti Bapat, MSc1; Latha Diwakar, PhD1,2; Reddy Peera Kommaddi, PhD1,2; Bernard L Schneider, PhD3; Etienne C. Hirsch, PhD4 and Vijayalakshmi Ravindranath, PhD1,2 *
1. Centre for Neuroscience, Indian Institute of Science, Bangalore-560012, India
2. Centre for Brain Research, Indian Institute of Science, Bangalore-560012, India
3. Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland
4. Institut du Cerveau – ICM Inserm U 1127, CNRS UMR 7225, Sorbonne Université, F-75013, Paris, France
Address for correspondence
Vijayalakshmi Ravindranath,
Centre for Brain Research, Indian Institute of Science, C.V. Raman Avenue, Bangalore - 560012, India, Phone: +91-80-22933433, Fax: +91-80-23603323, email: [email protected]
Keywords: Parkinson’s disease; dopaminergic neurons; glutaredoxin 1; shRNA;
tyrosine hydroxylase
Supplementary
MATERIALS AND METHODS
Animals: All experiments were performed with 3-5 months old C57BL/6 male mice (25-30 grams) obtained from Indian Institute of Science. The experiments were approved by the institutional animal ethical review board, named ‘Institutional Animal and Ethics Committee’ of Indian Institute of Science (Protocol#
CAF/Ethics/267/2012) and were carried out in accordance to institutional guidelines for the use and care of animals. All efforts were made to minimize animal suffering, reduce the number of animals used. Animals were randomly divided into either of the experimental groups, were housed in groups and had access to pelleted diet and water, ad libitum.
AAV6-sh/scRNA-Grx1 titration: Infectious particle or viral transducing unit (TU/mL) counts were determined using a quantitative PCR method by comparing the downregulation viruses against a reference AAV6-GFP viral preparation. The absolute TU/mL for the AAV6-GFP virus was estimated using FACS analysis as described before [1]. Even though our constructs carried a GFP tag, and absolute FACS based titration could not be performed due to cell death induced by Grx1 downregulation [2]. Briefly 293T cells were infected at graduated doses with purified AAV6-Grx1 shRNA/scRNA or AAV6-GFP particles. After 48 hr of transduction, cells were harvested and dsDNA containing transduced pAAV construct and host cell genomic DNA were purified. The absolute number of copies of β-globin intronic sequence (present in both the AAV and host cell genomes) were estimated using qPCR and normalized to the copies of albumin (only present in host cell genomes).
These normalized values were compared against the reference AAV6-GFP virus values to estimate TU/ml [3,4].
Stereotaxic surgery: AAV-sh/scrRNA-Grx1 or equivalent volume of PBS was stereotaxically, unilaterally injected into mouse SNpc using a Hamiltonian syringe directly connected to an automated injector. Each animal was weighed and
anesthetized by administering ketamine/xylazine mixture (i.p.). The animal was then fixed onto Dual lab Standard stereotaxic frame with the mouse/neonatal rat adaptor (Stoelting Co., Wood Dale, IL, USA). A hole was drilled into the skull with a 24G needle. A 5μL 700 Series syringe with 30G removable blunt-point needles (Hamilton Co., Reno, NV, USA) was used for injecting AAV-sh/scrRNA-Grx1 or PBS at the following coordinates: 3.2mm posterior to bregma; 1.2mm lateral to bregma; 4.5mm ventral to bregma-based on the mouse brain atlas. The injections were randomly performed to the left or right hemispheres to exclude bias. 2 L of virus carrying sh/scrRNA or vehicle (PBS) was then injected at a flow rate of 0.2 L/min using the automated injector. The health of the animal was closely monitored over the next 3 days during which it was treated with analgesic and topical antiseptic every day.
The post-surgery behavioral recordings were performed subsequently following which the animals were sacrificed for immunohistochemistry after 14 or 28 days.
Behavioral analyses: Animal behavior was recorded both pre and post-surgery to examine if Grx1 downregulation led to development of locomotor deficits. The
schedule for the behavioral paradigm is described in Fig 1.C. All equipment used for behavioral assessment was cleaned with 70% ethanol in between recording for each animal.
Rotarod analysis: The general locomotor abilities of the injected animals were assessed by evaluating their ability to balance on a rotating rod at gradually
increasing speeds. Rotarod instrument consisted of a clean rod placed at a height of 30 cm from base. Before recording data, animals were trained for 2 sessions at lowest speed. Animals were trained at lowest speed of rotation for three consecutive days before surgery and for a single day before each set of trials. Animals were tested in a group of 3 consecutive sessions, each session being a day of recording.
During each session 3 trials were performed at increasing speeds through each trial.
The initial rotation started at 12 rpm and increased to 20 rpm, 25 rpm and finally 30 rpm with each step lasting for 40 s for a total of 160 s. The time that the animal took to fall off the rod (latency) was recorded for each animal in a trial. The trials were separated by 15 min of rest. Recordings were performed during 3 such sessions separated by 7 days from the first session. Latency for falling off accelerating rotarod was measured before surgery to rule out inherent animal biases. Latency
measurements were performed for three consecutive days at three time points for upto 28 days post-surgery. Data has been represented as the average time on the rotating rod from three trials in a session calculated for every animal.
Elevated Body Swing Test (EBST): Unilateral locomotor deficits in animals post- surgery was assessed by performing elevated body swing test. Each session on a single day consisted of a single trial. Three sessions were performed post-surgery at intervals of 7 days. Each session lasted for 45s wherein the animal’s side preference was recorded after freely suspending the animal by its tail at a height of 30cm. The number of contralateral and ipsilateral turns were counted in experimentally blind manner. Percentage of contralateral turns to total turns was calculated to identify any preference of the animal.
Sacrificing of animals, brain collection and sectioning for histochemical analysis: The animals were anesthetized in an enclosed chamber. The animals were transcardially perfused with 4% w/v paraformaldehyde (PFA) as fixative to collect the brains for cryosectioning. The removed brain was dropped in 4% w/v PFA and stored at 4°C overnight for post-fixation, followed by transfer to 30% w/v sucrose in 1X PBS at 4°C till the brain sank in the solution (typically ~36-48 hr). The brains were mounted onto sectioning chucks and embedded in fresh tissue freezing medium (Leica
Microsystems Nussloch GmbH Cat# 0201 08926). Serial sections of 25 m
thickness were cut through the striatal and midbrain regions of the brain, and stored in 1X tris buffered saline (TBS) containing 0.005% w/v sodium azide at 4°C until used for staining.
Tissue processing for immunohistochemistry: Serial sections floating in TBS with sodium azide were separated in a 48-well dish for IHC. These sections were then washed twice with TBS for 5 min each. The sections were then incubated with blocking solution containing 3% bovine serum albumin (BSA) with 0.1% Triton-X 100 and 3% normal horse serum in TBS or PBS for 1 hour at room temperature followed by incubation in rabbit anti-TH (Millipore Cat# AB152, RRID: AB_390204; 1:500), rabbit anti-GFAP (Abcam Cat# ab7260, RRID:AB_305808; 1:500), rabbit anti-Iba1 (Wako Cat# 019-19741, RRID:AB_839504; 1:500) in TBST or PBS for overnight at 4°C on a rocker or Fluorescent label conjugated stain for Nissl substance,
NeuroTrace® 530/615 Red Fluorescent Nissl Stain (Thermo Fisher Scientific Cat#
N21482, RRID:AB_2620170) (1:500) for 1 hour at room temperature on rocker. This was followed by four TBST washes the sections probed for Nissl were mounted. The sections probed for TH, GFAP were incubated in secondary antibody, donkey anti- rabbit IgG H+L (Alexa Fluor® 594) (ThermoScientific Cat# A-21207, RRID:
AB_2556547) (1:1000) in TBST or PBS for 1 hour. The sections were given four washes with cold TBST or PBS for 5 min each and mounted in VECTASHIELD®
mounting medium with DAPI (Vector Laboratories, Cat# H-1200) on glass slides.
Micrographs were obtained using Zeiss Axio Imager 2 and Zeiss 780 confocal microscope.
Quantitative analysis on histological data: Stereological analysis total number of TH and Nissl positive neurons in SNpc was performed for every sixth coronal section through the midbrain using StereoInvestigator by MBF Bioscience. All data was quantified for all sections in an experimentally blind manner.
The sections stained for the dopaminergic marker, TH or neuronal marker, Nissl, were imaged on an Olympus microscope using the StereoInvestigator® (MBF
Bioscience) software to obtain tile scans of unilateral SNpc across z (vertical axis) at a magnification of 40X. Counting was performed in experimentally blind manner, offline with the optical fractionator probe [5,6] using 60 × 60 µm counting frame at x
=150 µm, y = 150 µm intervals from a random start point. A 2.5 µm guard zone, and a probe depth of 20 µm were used. The coefficient of error was below 0.1 in all animals studied. The sections probed for Nissl were counter-stained using TH antibody to enable the delineation of SNpc contour.
Intensity of TH positive terminals in striatum, was quantified by performing mean fluorescent intensity measurements using ImageJ. For each experimental condition mean fluorescent intensity was calculated as mean staining intensity in striatum - mean staining intensity in adjoining cortex of all stereological sections.
Human PD autopsy samples: Post-mortem brain material (midbrain) from PD patients (n=6) and aged matched control subjects (n=6) were used in this study.
Control brains were obtained from individuals without neurologic or psychiatric disorders. Patients with PD displayed the characteristics of the disease including akinesia, rigidity and/or resting tremor. All patients with PD were treated with dopaminergic agonists and/or L-DOPA and displayed or had displayed dyskinesia.
The clinical diagnosis was confirmed by neuropathological examination and all displayed the presence of Lewy bodies at least in the substantia nigra. Mean post- mortem delay and age at death did not differ between control subjects and patients with PD. SNpc was scraped off the surface of the slides in cold and sterile conditions following anatomical markers. Detailed case history of the samples is provided in supplementary table S1.
Carbidopa-levodopa treatment to mice: C57BL/6 male mice (3-4 months) were given a combination of carbidopa (30mg/kg body weight) and levodopa (10mg/kg body weight) dissolved in 10% (w/v) sucrose [7]. Carbidopa-levodopa (CDLD) was
administered daily per orally using gavage for 14 days. The animals were sacrificed on 15th day and SNpc was dissected out.
Assay of glutathione and protein thiols
SNpc was dissected from each of the contralateral and ipsilateral hemisphere of C57BL/6 mice after 14 days of unilateral transduction with AAV-scr/shRNA-Grx1.
Glutathione (GSH) was assayed as described previously [8]. Briefly, tissue was flash frozen and weighed and homogenized in 20 volumes potassium phosphate buffer (0.1M, pH 7.4 containing 1mM EDTA). The homogenate was then added to 2
volumes of 10% sulfosalicylic acid and centrifuged at 10,000g for 10 minutes at 4ºC.
The supernatant was then used to assay total GSH using enzymatic recycling method [9], wherein the rate of reduction of DTNB (5,5'-dithiobis-(2-nitrobenzoic
acid) or Ellman's reagent) in the presence of glutathione reductase and NADPH is measured as a function of total GSH concentration.
In order to assay total protein thiols (PrSH), the acid precipitated pellet was washed twice with 10% sulfosalicylic acid and then sonicated and resuspended in Tris-HCl (0.65M, pH 7.6 containing 3mM DTNB). Absorbance was measured after a 30 minute incubation at 412nm as described [10].
Primary cortical cultures: Primary cortical neurons were prepared at postnatal day zero (p0) to postnatal day one (p1) using cortices from C57BL/6 mice as described in Beaudoin et. al. [11]. Mouse pup was cryo-anaesthetized, alcohol-sterilized and decapitated. After the skull was carefully opened and the meninges were removed, cortices were dissected out. The neurons were then dissociated using mechanical trituration in 0.25% papain (10 Units/mg, Sigma-Aldrich Cat# P4762) solution containing 1 X Hank’s Balanced Salt Solution, 12.5 mM 2-[4-(2-hy-
droxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) buffered sterile saline, D- glucose, and 500 µg/mL penicillin-streptomycin). Cortical neurons were seeded on glass cover slips precoated with poly-D-lysine (0.1 mg/ml) in 12-well plates.
Neurobasal A (Gibco, Cat# 10888022) medium supplemented with 1.5X B-27 Supplement (Gibco, Cat# 17504044), 2 mM L-GlutaMAX (Gibco, Cat# 35050061), and 100 µg/mL penicillin-streptomycin (Gibco, Cat# 15140122) was used to grow the cells in serum-free medium conditions and maintained at 37C in 5% CO2. The cultures were transduced with ~5x108 TU of AAV-shRNA-Grx1 and AAV-scRNA- Grx1 after 18 days in vitro (DIV) and were harvested on DIV21 for RNA isolation.
RNA isolation and cDNA synthesis: All the reagents and glassware used for RNA isolation and cDNA synthesis were made RNAse free. RNA was isolated from
human autopsy brain tissue, mouse SNpc tissue and mouse primary cortical neurons using the RNeasy Plus Universal Mini Kit (Qiagen, Cat# 73404). RNA for mice SNpc was isolated using TRIzol-BCP protocol. Total RNA (200ng for human RNA and 1000ng for mouse RNA) was used for first strand cDNA synthesis using random hexamers, dNTPs and reverse transcriptase from the high capacity cDNA reverse transcription kit (Applied Biosystems, Cat# 4368814). The quality of RNA was ascertained using BioAnalyzer and the samples with RNA having RNA integrity number (RIN) less than 5 were not used for analysis.
Quantitative real time PCR: Quantitative real time PCR (qRT-PCR) was performed using SYBR green chemistry with primer pairs designed against human Grx1, - actin, GAPDH and for mouse Grx1. The nucleotide sequences for primers used for gene expression analysis and the PCR conditions are provided in supplementary table S2, S3 and S4. 18S rRNA was used as an endogenous control for
normalization when cDNA from human autopsy brain tissue was studied and for the mouse carbidopa-levodopa experiment. Samples were analyzed in duplicates or triplicates. Exclusion of outliers was performed following a widely used method of outlier removal called Median Absolute Deviation (MAD) [12].
Figure S1:
Grx1 down-regulation leads to dopaminergic degeneration in mice
Viral transduction
SNpc VTA AAV6
sh/scrRNAGrx1 mU6 CMV β-globin GFP
Contra Ipsi
SNpc
SNpc
Striatum
Control Injected
Behaviour PrSSG
GSSG
Grx1/GSH/NADPH
Glutathione Reductase
PrSH + GSSG GSH
SNpc
Figure S1: Summary of experimental design and results
Grx1 is a protein thiol/disulfide oxidoreductase that is essential for cellular protein thiol redox homeostasis. Downregulation of Grx1 through viral transduction of AAV- shRNA-Grx1 in mouse substantia nigra leads to degeneration of TH and Nissl positive neurons in SN accompanied with a loss of TH positive fibers in striatum.
Locomotor deficits are also observed in these mice.
REFERENCES
[1] C. Towne, P. Aebischer, Lentiviral and Adeno-Associated Vector-Based Therapy for Motor Neuron Disease Through RNAi, in: Methods Mol. Biol., 2009: pp. 87–108. doi:10.1007/978-1-60327-295-7_7.
[2] U. Saeed, A. Ray, R.K. Valli, A.M.R. Kumar, V. Ravindranath, DJ-1 loss by glutaredoxin but not glutathione depletion triggers Daxx translocation and cell death, Antioxid. Redox Signal. 13 (2010) 127–144. doi:10.1089/ars.2009.2832.
[3] U.-P. Rohr, F. Heyd, J. Neukirchen, M.-A. Wulf, I. Queitsch, G. Kroener-Lux, U. Steidl, R. Fenk, R. Haas, R. Kronenwett, Quantitative real-time PCR for titration of infectious recombinant AAV-2 particles, J. Virol. Methods. 127 (2005) 40–45. doi:10.1016/j.jviromet.2005.03.006.
[4] C. Towne, C. Raoul, B.L. Schneider, P. Aebischer, Systemic AAV6 Delivery Mediating RNA Interference Against SOD1: Neuromuscular Transduction Does Not Alter Disease Progression in fALS Mice, Mol. Ther. 16 (2008) 1018–
1025. doi:10.1038/mt.2008.73.
[5] H.J. Gundersen, E.B. Jensen, The efficiency of systematic sampling in stereology and its prediction, J. Microsc. 147 (1987) 229–63.
doi:10.1111/j.1365-2818.1987.tb02837.x.
[6] M.J. West, L. Slomianka, H.J.G. Gundersen, Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator, Anat. Rec. 231 (1991) 482–497.
doi:10.1002/ar.1092310411.
[7] M. Käenmäki, A. Tammimäki, J.A. Garcia-Horsman, T. Myöhänen, N.
Schendzielorz, M. Karayiorgou, J.A. Gogos, P.T. Männistö, Importance of membrane-bound catechol-O-methyltransferase in L-DOPA metabolism: A
pharmacokinetic study in two types of Comt gene modified mice, Br. J.
Pharmacol. 158 (2009) 1884–1894. doi:10.1111/j.1476-5381.2009.00494.x.
[8] B.R. Shivakumar, V. Ravindranath, Oxidative stress induced by administration of the neuroleptic drug haloperidol is attenuated by higher doses of
haloperidol, Brain Res. 595 (1992) 256–262. doi:10.1016/0006- 8993(92)91058-M.
[9] F. Tietze, Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: Applications to mammalian blood and other tissues, Anal. Biochem. 27 (1969) 502–522. doi:10.1016/0003- 2697(69)90064-5.
[10] D. Di Monte, D. Ross, G. Bellomo, L. Eklöw, S. Orrenius, Alterations in
intracellular thiol homeostasis during the metabolism of menadione by isolated rat hepatocytes, Arch. Biochem. Biophys. 235 (1984) 334–342.
doi:10.1016/0003-9861(84)90206-6.
[11] G.M.J. Beaudoin, S.H. Lee, D. Singh, Y. Yuan, Y.G. Ng, L.F. Reichardt, J.
Arikkath, Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex, Nat. Protoc. 7 (2012) 1741–1754.
doi:10.1038/nprot.2012.099.
[12] C. Leys, C. Ley, O. Klein, P. Bernard, L. Licata, Detecting outliers: Do not use standard deviation around the mean, use absolute deviation around the median, J. Exp. Soc. Psychol. 49 (2013) 764–766.
doi:10.1016/J.JESP.2013.03.013.
