LRRK2 Kinase Activity

Anonymous
timer Asked: Feb 5th, 2019
account_balance_wallet $9.99

Question Description

Paper on (LRRK2).

- the paper may not be correct ,and can suggest and explanation

Question1:

In fig 3the authors provide evidence suggesting that the role of LRRK2 is to impair neurotransmitter release(NTrelease). Is this statement True or False? Support in 300words

Question 2

Describe in a couple of sentences the role of synapsin in NT release;Based on your description, how would you explain the results in fig 3A:Is the addition of IN-1 INCREASING orDECREASING the availability of Synaptic vesicles for release?Assuming the increase/decrease is mediated via synapsin, how would LRRK2 induce the increase/decrease of NT release(does it activate Synapsin’s function or inactivatesit)?(300wordsmax)


Question 3

Describe in your own words the principle of the method that produced the results in fig 2A.What was the hypothesis the authors tried to prove?(300wordsmax)

Question 4

In fig 8 the authors propose a model where inhibition of LRRK2 results in release of SV’s from the actin cytoskeleton. Do the results in fig4c support this hypothesis?Explain brieflywhy.(300words max)

Unformatted Attachment Preview

ORIGINAL RESEARCH ARTICLE published: 27 May 2014 doi: 10.3389/fnmol.2014.00049 MOLECULAR NEUROSCIENCE LRRK2 kinase activity regulates synaptic vesicle trafficking and neurotransmitter release through modulation of LRRK2 macro-molecular complex Maria D. Cirnaru 1,2 , Antonella Marte 3 , Elisa Belluzzi 4 , Isabella Russo 4 , Martina Gabrielli 2,5 , Francesco Longo 6 , Ludovico Arcuri 6 , Luca Murru 2 , Luigi Bubacco 4 , Michela Matteoli 5,7 , Ernesto Fedele 8 , Carlo Sala 2,5 , Maria Passafaro 2 , Michele Morari 6 , Elisa Greggio 4 , Franco Onofri 3 and Giovanni Piccoli 1,2* 1 2 3 4 5 6 7 8 Division of Neuroscience, San Raffaele Scientific Institute and Vita-Salute University, Milan, Italy Department of Molecular and Cellular Pharmacology, National Research Council, Neuroscience Institute, Milan, Italy Department of Experimental Medicine, University of Genova, Genova, Italy Department of Biology, University of Padova, Padova, Italy Department of Medical Biotechnology and Translational Medicine, University of Milan, Milan, Italy Department of Medical Science and National Institute of Neuroscience, University of Ferrara, Ferrara, Italy Humanitas Clinical and Research Center, Pharmacology and Brain Pathology, Rozzano, Italy Department of Pharmacy, University of Genoa, Genoa, Italy Edited by: Kirsten Harvey, University College London, UK Reviewed by: Lu-Yang Wang, University of Toronto, Canada Nicola B. Mercuri, University of Rome, Italy R. Jeremy Nichols, The Parkinson’s Institute, USA *Correspondence: Giovanni Piccoli, San Raffaele Scientific Institute and Vita-Salute University, via Olgettina 58, 20132 Milan, Italy e-mail: piccoli.giovanni@hsr.it Mutations in Leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson’s disease (PD). LRRK2 is a complex protein that consists of multiple domains executing several functions, including GTP hydrolysis, kinase activity, and protein binding. Robust evidence suggests that LRRK2 acts at the synaptic site as a molecular hub connecting synaptic vesicles to cytoskeletal elements via a complex panel of protein-protein interactions. Here we investigated the impact of pharmacological inhibition of LRRK2 kinase activity on synaptic function. Acute treatment with LRRK2 inhibitors reduced the frequency of spontaneous currents, the rate of synaptic vesicle trafficking and the release of neurotransmitter from isolated synaptosomes. The investigation of complementary models lacking LRRK2 expression allowed us to exclude potential off-side effects of kinase inhibitors on synaptic functions. Next we studied whether kinase inhibition affects LRRK2 heterologous interactions. We found that the binding among LRRK2, presynaptic proteins and synaptic vesicles is affected by kinase inhibition. Our results suggest that LRRK2 kinase activity influences synaptic vesicle release via modulation of LRRK2 macro-molecular complex. Keywords: LRRK2, kinase, presynaptic vesicle, synaptic activity, protein interaction INTRODUCTION Parkinson’s disease (PD) is an age-related neurodegenerative disease affecting 2% of the population above 65-years and is clinically characterized by bradykinesia, rigidity, and resting tremor. The neuropathological hallmark of the disease is the progressive loss of dopaminergic neurons in the substantia nigra (Moore et al., 2005; Hardy et al., 2006). Although the majority of cases are idiopathic, mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene (PARK8; OMIM 609007) cause late-onset PD. LRRK2 mutations account for up to 13% of familial PD cases compatible with dominant inheritance (Paisan-Ruiz et al., 2004; Zimprich et al., 2004) and have been identified in 1–2% of sporadic PD patients (Aasly et al., 2005; Berg et al., 2005). LRRK2 is a large protein encompassing several functional domains including a kinase domain with feature similar to mitogen activated protein kinase kinase kinases (MAPKKK) and receptor-interacting protein kinases (RIPK) (Bosgraaf and Van Haastert, 2003; Guo et al., 2006). Several single nucleotide variants have been identified in LRRK2 (Brice, 2005). While only the common G2019S mutation, located in the kinase domain, has been consistently associated Frontiers in Molecular Neuroscience with increased kinase activity in vitro (West et al., 2005; Gloeckner et al., 2006; Greggio et al., 2006), a recent study monitoring LRRK2 autophosphorylation at Ser 1292 suggested that other pathogenic mutants possess augmented activity in the cellular context (Sheng et al., 2012). Up to now few LRRK2 substrates have been identified in in vitro studies, but none has been convincingly proved in vivo, leaving the pathophysiological relevance of the kinase activity unclear. Instead, several lines of evidence suggest that kinase activity is linked to LRRK2 dimerization (Greggio et al., 2008; Sen et al., 2009; Civiero et al., 2012) as well as subcellular distribution (Berger et al., 2010) and regulates binding to 14-3-3 proteins (Nichols et al., 2010). Accumulating data correlate LRRK2 to synaptic functions. Several studies suggested that LRRK2 is part of a protein complex that influences the trafficking of synaptic vesicles belonging to the recycling pool (Shin et al., 2008; Piccoli et al., 2011; Matta et al., 2012). The description of synaptic phenotype in LRRK2 mutant models (Tong et al., 2009; Migheli et al., 2013; Yun et al., 2013) further underlines the tight link among LRRK2, synaptic vesicle trafficking and neurotransmitter release. In this study we investigated the functional www.frontiersin.org May 2014 | Volume 7 | Article 49 | 1 Cirnaru et al. LRRK2 at the presynaptic site impact of LRRK2 kinase activity on presynaptic function and we determined functional properties of neurons upon LRRK2 pharmacological inhibition. A combination of electrophysiological, biochemical and imaging analyses suggested that LRRK2 inhibition impacts synaptic transmission acting on the organization of LRRK2 macro-molecular complex at the presynaptic site. MATERIALS AND METHODS ANIMALS, NEURON CULTURES, AND DRUGS Non-transgenic wild-type (WT) and LRRK2 knock-out (KO) mice, back-crossed on a C57BL/6J strain, were obtained from Mayo Clinic (Jacksonville, FL, USA) through a collaboration with Prof. Matthew Farrer and Dr. Heather Melrose (Hinkle et al., 2012). Animals were kept following guidelines of Ministry of Education, Universities and Research (MIUR). Neuron cultures were prepared from either mouse cortexes or hippocampi obtained from embryonic day 15.5–16.5 mice (C57BL/6J). High-density (750–1000 cells/mm2 ) and medium-density (150– 200 cells/mm2 ) neuron cultures were plated and grown as described on 12-well plastic tissue culture plates (Iwaki; Bibby Sterilin Staffordshire, UK) or on 12 mm diameter coverslips put into 24-well plastic tissue culture plates (Iwaki) (Piccoli et al., 2007). IN-1 and GSK-2578215A compounds (Tocris Bioscience, Bristol, UK) or DMSO were added to culture media at the concentrations indicated through the text. PLASMIDS AND PROTEIN PURIFICATION N-terminal 3xFLAG and myc hLRRK2 full length (hereinafter FLAG-LRRK2 and myc-LRRK2), N-terminal FLAG hLRRK2 A2106T (a kind gift of Prof. Dario Alessi, MRC, University of Dundee), LRRK2 silencing and control viral constructs vectors have been already described (Bauer et al., 2009; Nichols et al., 2009; Civiero et al., 2012). FLAG-LRRK2 was purified via affinity chromatography using FLAG-M2 agarose beads (Sigma Aldrich) as previously described (Civiero et al., 2012) from HEK293T cells transfected by lipofection using Lipofectamine 2000 (Life Technologies Carlsbad, CA, USA) according to manufacturer’s instructions. Viral particles were produced as in (Bauer et al., 2009). Neurons were infected at DIV4 and processed when indicated. IMMUNO-PRECIPITATION AND ANTIBODIES Immunoprecipitation was performed as described previously (Onofri et al., 2007) using 25 µl of settled prewashed protein G-Sepharose beads (GE-Healthcare, Freiburg, Germany) to precipitate the immunocomplexes. NaCl 150 mM, Tris 50 mM (pH 7.4), NP-40 (1% v/v), SDS (0.1% v/v) and protease and phosphatase inhibitors extracts of Percoll-purified synaptosomes obtained from rat cerebral cortex were incubated for 2 h at RT in absence or in presence of IN- 1 (1 µM) with anti-LRRK2 antibodies (10 µg/sample; MJFF C41-2 Abcam, Cambridge UK) or a control rabbit IGg (10 µg/sample; Sigma-Aldrich, St. Louis, MO, USA). The eluted proteins were separated by SDS-PAGE, transferred onto nitrocellulose membrane (GE-Healthcare) and analyzed by western-blotting. Antibodies list includes rabbit anti LRRK2 1:500 MJFF C41-2, rabbit anti LRRK2 P-Ser 935 UDD2 10(12) (Abcam), rabbit anti synapsin I 1:500 (Synaptic System, Frontiers in Molecular Neuroscience Goettingen, Germany), mouse anti actin 1:1000, mouse anti FLAG 1:1000, mouse anti myc 1:1000, mouse anti synaptophysin 1:1000 (Sigma-Aldrich St. Louis, MO, USA). The secondary antibodies (HRP-conjugated anti-mouse, anti-rabbit) (BIORAD, Hercules, CA, USA) were used in a ratio of 1:5000 coupled with the ECL chemiluminescence detection system. Immunoblots were quantified by densitometric analysis of the fluorograms (Quantity One software, Bio-Rad) obtained in the linear range of the emulsion response. IN VITRO KINASE ASSAY GST-LRRK2970−2527 (Life technologies) at the concentration of 30 nM were incubated with 500 µM LRRKtide, 100 µM 33 P-ATP (0.5 µCi) in kinase reaction buffer consisting of 25 mM TrisHCl (pH7.5), 5 mM beta-glycerophosphate, 2 mM dithiothreitol (DTT), 0.1 mM Na3 VO4 , 10 mM MgCl2 and increasing concentrations of inhibitors at 30◦ C for 1 h. Reactions were carried out in triplicate and spotted onto P81 phosphocellulose. Following different washing of phosphocellulose membranes with 75 mM phosphoric acid, 33 P incorporation into LRRKtide was quantified with Cyclone (Perkin Elmer, Alameda, CA, USA). SIZE EXCLUSION CHROMATOGRAPHY Cells transiently transfected with FLAG-LRRK2 wild-type were solubilized in lysis buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM NaVO4 , protease inhibitor cocktail (Sigma-Aldrich) and lysates were cleared for 30 min at 14,000 xg. When appropriate, proteins were further purified via FLAG immunoprecipitation as described above. Cleared lysates (0.5 ml; 5 mg total proteins) or purified proteins (0.5 ml; 1.3 µg of purified protein) were injected and separated on a Superose 6 10/300 column (GE Healthcare). The column was preequilibrated with buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl and 0.07% Triton X-100) and used at a flow rate of 0.5 ml/min. Elution volumes of standards were 7.5 ml for Blue Dextran (V0), 11.5 ml for hemocyanin from Carcinus aestuarii (900 kDa), 12 ml for thyreoglobin (669 kDa), 14 ml for ferritin (440 kDa). When appropriate, inhibitors (1 µM IN-1 and 1 µM GSK-2578215A) were applied for 90 min before lysis and kept throughout the following purification steps, including equilibration of chromatographic mobile phase. Chromatographic fractions were analyzed by dot blot. One microliter of each fraction from SEC was applied onto a nitrocellulose membrane. The membrane was blocked with 10% (w/v) milk in TBS plus 0.1% Triton (TBS-T) for 1 h and subsequently incubated with mouse monoclonal anti-Flag M2-peroxidase (Sigma-Aldrich). Immunoreactive proteins were visualized using enhanced chemiluminescence plus (ECL plus, GE Healthcare). SYNAPTIC VESICLE PURIFICATION AND LRRK2 BINDING ASSAYS Synaptic vesicles (SV) were obtained from rats by homogenization of the isolated forebrains and finally purified through the step of controlled-pore glass (CPG) chromatography (Huttner et al., 1983). After elution, purified SV were centrifuged for 2 h at 175,000 × g and resuspended at a protein concentration of 1– 2 mg/ml in 0.3 M glycine, 5 mM HEPES, 0.02% sodium azide, pH www.frontiersin.org May 2014 | Volume 7 | Article 49 | 2 Cirnaru et al. LRRK2 at the presynaptic site 7.4 (glycine buffer). Protein concentrations were determined by the Bradford or BCA assays. SDS-PAGE was performed according to Laemmli (1970). For the dissociation of endogenously bound LRRK2 purified SV (40 µg/sample) were incubated for 1 h at 30◦ C with or without IN-1 (1 µM) in glycine buffer plus 30 mM NaCl, 25 mM Tris/HCl, 2 mM DTT, 10 mM MgCl2 protease and phosphatase inhibitors. After the incubation, LRRK2 bound to SV were separated by soluble LRRK2 by high-speed centrifugation (400,000 × g for 45 min) (Messa et al., 2010). Aliquots of the resuspended pellets were subjected to SDS–PAGE and subsequent Western blotting with anti LRRK2 MJFF C41-2 (Abcam) antibody. The recovery of SV, used to correct the amounts of LRRK2 bound to SV, was determined by Western blotting with anti-synaptophysin antibody (kind gift of Prof. Paul Greengard The Rockefeller University New York USA). The binding of purified FLAG-LRRK2 to native SV was performed like below. SV (10 µg/sample) were incubated for 1 h at 0◦ C with FLAGLRRK2 (50 nM) in glycine buffer plus 30 mM NaCl, 25 mM Tris/HCl, 2 mM DTT, 10 mM MgCl2 protease and phosphatase inhibitors and 1.0 µg/ml bovine serum albumin in absence or in presence of IN-1 (1 µM). After incubation, SV-bound FLAGLRRK2 was separated by high-speed centrifugation (400,000 g for 45 min). Aliquots of the resuspended pellets were subjected to immunoblotting with anti-FLAG (Sigma-Aldrich) antibodies. The recovery of SV was determinated like above. study, the fast and continuous removal of endogenous substances released by nerve terminals rules out that endogenous glutamate is uptaken by glutamate transporters, or even activates autoreceptors. Sample collection (every 3 min) was initiated after a 20 min period of filter washout. The effect of IN-1 was evaluated on both spontaneous efflux and K+-stimulated neurotransmitter outflow. IN-1 (3 µM) was added to the perfusion medium 9 min before a 90 s pulse of 15 mM KCl, and maintained until the end of the experiment. In other experiments purified synaptosomes were prepared on Percoll gradients (Sigma-Aldrich) and incubated at 37◦ C for 15 min in presence of 0.03 µM [3 H]D-aspartate (Marte et al., 2010). A 90 s period of depolarization was applied at t = 39 min of superfusion with 15 mM KCl, substituting for an equimolar concentration of NaCl. IN-1 1 µM was added 9 min before depolarization. Fractions collected and superfused synaptosomes were counted for radioactivity by liquid scintillation counting. The efflux of radioactivity in each fraction has been expressed as a percentage of the total radioactivity present in synaptosomes at the onset of the fraction collected (fractional rate). Depolarization-evoked neurotransmitter overflow was calculated by subtracting the transmitter content of the two 3-min fractions, representing the basal release, from that in the two 3-min fractions collected during and after the depolarization pulse. SLICE ELECTROPHYSIOLOGY EXO-ENDOCYTOTIC ASSAY The endocytosis assay to monitor SV recycling was performed using rabbit polyclonal antibodies directed against the intravesicular domain of synaptotagmin1 (Synaptic System), applied for 5 min if not indicated otherwise at RT on the cultures, as described previously (Matteoli et al., 1992). Incubations with the antibody (1:400) were performed in Tyrode solution containing 124 mM NaCl, 5 mM KCl, 2 mM MgCl2 , 30 mM glucose, 25 mM HEPES, pH 7.4 and 2 mM CaCl2 . After fixation and permeabilization, a synaptophysin counter staining with mouse anti synaptophysin, 1:400 (Sigma-Aldrich) visualized the totality of SV. Acquired images were processed and quantitatively analyzed with ImageJ software as previously described (Verderio et al., 1999). Briefly, GFP positive processes were manually tracked and the number of synaptotagmin and synaptophysin positive clusters and synaptophysin positive clusters present in the region of interest were automatically counted. NEUROTRANSMITTER RELEASE Synaptosome were isolated from cerebral cortex (fronto-temporal areas) as described previously (Marti et al., 2003; Mela et al., 2004). The synaptosomal pellet was resuspended in oxygenated (95% O2, 5% CO2) Krebs solution (mM: NaCl 118.5, KCl 4.7, CaCl2 1.2, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, glucose 10). One millilitre aliquot of the suspension (∼0.35 mg protein) was slowly injected into nylon syringe filters (outer diameter 13 mm, 0.45 µM pore size, internal volume of about 100 µl; Teknokroma, Barcelona, Spain) connected to a peristaltic pump. Filters were maintained at 36.5◦ C in a thermostatic bath and superfused at a flow rate of 0.4 ml/min with a preoxygenated Krebs solution. Under the superfusion conditions adopted in the present Frontiers in Molecular Neuroscience C57Bl/6J mice were anesthetized in a chamber saturated with chloroform and then decapitated. The brain was rapidly removed and placed in an ice-cold solution containing 220 mM sucrose, 2 mM KCl, 1.3 mM NaH2 PO4 , 12 mM MgSO4 , 0.2 mM CaCl2 , 10 mM glucose, 2.6 mM NaHCO3 (pH 7.3, equilibrated with 95% O2 and 5% CO2). Coronal hippocampal slices (thickness, 250–300 µm) were prepared with a vibratome VT1000 S (Leica, Wetzlar Germany) and then incubated first for 40 min at 36◦ C and then for 30 min at room temperature in artificial CSF (aCSF), consisting of (in mM) 125 NaCl, 2.5 KCl, 1.25 NaH2 PO4 , 1 mM MgCl2 , 2 mM CaCl2 , 25 mM glucose, and 26 mM NaHCO3 (pH 7.3, equilibrated with 95% O2 and 5% CO2 ). Slices were then divided into 2 experimental groups: the first one was the control group and the second one was the group of slices incubated with the inhibitor 1 at concentration of 2 µM for at least 2 h. Slices were transferred to a recording chamber perfused with aCSF, where the concentration of CaCl2 was increased to 4 mM and MgCl2 decreased to 0.5 mM, due to the low frequency of miniature excitatory post-synaptic currents (mEPSCs) in CA1 hippocampus, at a rate of ∼2 ml/min and at 38◦ C. Whole-cell patch-clamp electrophysiological recordings were performed with an Axon Multiclamp 700 B amplifier (Molecular devices, Sunnyvale, CA USA) and using an infrared-differential interference contrast microscope. Patch microelectrodes (borosilicate capillaries with a filament and an outer diameter of 1.5 µm; Sutter Instruments, Novato, CA USA) were prepared with a four-step horizontal puller (Sutter Instruments) and had a resistance of 3–5 M. mEPSCs were recorded at a holding potential of −65 mV with an internal solution containing: 126 mM K-gluconate, 4 mM NaCl, 1 mM EGTA, 1 mM MgSO4 , 0.5 mM CaCl2 , 3 mM ATP (magnesium salt), 0.1 mM GTP mM (sodium salt), 10 mM www.frontiersin.org May 2014 | Volume 7 | Article 49 | 3 Cirnaru et al. LRRK2 at the presynaptic site glucose, 10 mM HEPES (pH adjusted to 7.3 with KOH). Access resistance was between 10 and 20 M; if it changed by >20% during the recording, the recording was discarded. All glutamatergic currents were recorded in the presence of bicuculline (20 µM) in the external solution, to block the GABAergic transmission, and lidocaine (500 µM), to block the action potentials onset. Currents through the patch-clamp amplifier were filtered at 2 kHz and digitized at 20 kHz using Clampex 10.1 Software (Molecular Devices). Analysis was performed offline with Clampfit 10.1 software (Molecular Devices). ELECTROPHYSIOLOGICAL RECORDINGS OF CULTURED NEURONS Whole-cell voltage clamp recordings were performed using a MultiClamp 700 A amplifier (Molecular devices) coupled to a pCLAMP 10 Software (Molecular Devices), and using an inverted Axiovert 200 microscope (Zeiss, Oberkochen Germany). Patch electrodes, fabricated from thick borosilicate glasses (Sutter Instruments) were pulled and fire-polished to a final resistance of 3–5 M using a two-stage puller (Narishige, Japan). Experiments were performe ...
Purchase answer to see full attachment

Tutor Answer

paula9
School: Duke University

Attached.

0

Babri Masjid Conflict
Student’s Name
Course
Instructor
Date

1

Babri Masjid-Ramjanmabhumi issue is the most entrapped and weight prompting in India,
a whole country was in the hold of aggregate strain and hatred all through the past three decades1.
It included anguish that Ramjanmabhumi such as the beginning of Ram (as demonstrated by
Hindu society), which ought to be a sacred place of affection, took the condition of cutting edge for
both, the Hindus and the Muslims. The conflict from here spread to the whole country. Ayodhya is
as of now in every one's mind, not in light of its association with Ram the God, yet on account of
the manner in which that basic power in various political social occasions made it their essential
political inspiration for clear representative increments2. This inquiry of late has transformed into
the most fundamental clarification behind a significant debilitating of between shared relationship
and communalization of Indian political process3.
Therefore, the source of the Babri Masjid conflict was a result of mixing politics and
religion. In 19th February 1991, the religious parliament, also known as the Dharam Sansad
organized by the Vishva Hindu Parishad (VHP) had the objective to rebuild Ram temple and
preserve the Hindu Culture (Dharan). It was followed up with a bike rally also organized by the
VHP which kar sevaks from across the country came and took a dip in the Sarayu River and
pledged to rebuild the temple. On the other hand, the Muslim clergy too took to the streets in Delhi
led by a sixty-two year old woman, Shah Bano, who won the right to alimony from the Supreme
Court4.

1

Bacchetta, P. (2000). Sacred space in conflict in India: The Babri Masjid affair. Growth and
Change, 31(2), 255-284.
2
Gopal, S., Thapar, R., Chandra, B., Bhattacharya, S., Jaiswal, S., Mukhia, H., ... & Verma, R. N. (1990).
The political abuse of history: Babri Masjid-Rama Janmabhumi dispute. Social scientist, 76-81.
3
Contractor, Q. (2017). “Jab Babri Masjid Shaheed Huyi”: Memories of Violence and Its Spatial Remnants
in Mumbai. In Social Dynamics of the Urban (pp. 135-151). Springer, New Delhi.
4
Ryan, Stephen. Ethnic conflict and international relations. Dartmouth Pub Co, 1995.

2

Rajiv Gandhi, the prime minister representing the government at that time, tried to calm
down the Muslim clergy by passing the law that undermines the Supreme Court ruling on Shah
Bano. The Bharatiya Janata Party (BJP) accused Rajiv Gandhi of indulging in “appeasement of
politics”5. Therefore, a percentage of the Hindus were irritated by the outrage of the Muslim
clergy. Seeing this, Rajiv Gandhi decided to pacify the Hindu lobby against accusations of
indulging in appeasement politics by facilitating the foundation-laying ceremony of a Ram
Mandir.
In February 1989, the VHP announced it would perform the Shilanayas for a Ram Mandir,
Bricks were collected from every part of the country and brought to Ayodhya for the Shilanayas
ceremony ordained by the Rajiv Gandhi government6. However, the high court had ordered there
was no construction to be carried out in the disputed land but the VHP were determined to go
against the High Court. As a result tension intensified among the citizens. On 8th November 1989,
the state was in favor of the Shilanayas ceremony after interpretation of the High Court order. The
upcoming elections would have been the reason behind the sudden change of heart. At this point,
the congress was fighting the battle on its neck despite having more than three quarter of the
members in parliament7.
After rising to power, the Prime Minister VP Singh accepted the controversial Mandal
Commission report which divided students across the country8. Upper-caste Hindu and minority

5

Engineer, Asgharali. Babri-Masjid Ramjanambhoomi Controversy. South Asia Books, 1990.
Sen, Ragini, and Wolfgang Wagner. "History, emotions and hetero-referential representations in
inter-group conflict: The example of Hindu-Muslim relations in India." Papers on Social
Representations 14 (2005): 2-1.
7
Bacchetta, Paola. "Sacred space in conflict in India: Th...

flag Report DMCA
Review

Anonymous
Excellent job

Similar Questions
Related Tags

Brown University





1271 Tutors

California Institute of Technology




2131 Tutors

Carnegie Mellon University




982 Tutors

Columbia University





1256 Tutors

Dartmouth University





2113 Tutors

Emory University





2279 Tutors

Harvard University





599 Tutors

Massachusetts Institute of Technology



2319 Tutors

New York University





1645 Tutors

Notre Dam University





1911 Tutors

Oklahoma University





2122 Tutors

Pennsylvania State University





932 Tutors

Princeton University





1211 Tutors

Stanford University





983 Tutors

University of California





1282 Tutors

Oxford University





123 Tutors

Yale University





2325 Tutors