Emphasis Type="Italic"FUS/Emphasislinked esse

Authors: Murni Tio Rujing Wen Yih Lin Lim Huashan Wang ShuoChien Ling Yi Zhao EngKing Tan

Publish Date: 2016/07/09

Volume: 135, Issue: 11, Pages: 1223-1232

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Abstract

Essential tremor ET is one of the most common adultonset neurological disorders which produce motor and nonmotor symptoms To date there are no gold standard pathological hallmarks of ET and despite a strong genetic contribution toward ET development only a few pathogenic mutations have been identified Recently a pathogenic FUSQ290X mutation has been reported in a large ETaffected family however the pathophysiologic mechanism underlying FUSlinked ET is unknown Here we generated transgenic Drosophila expressing hFUSWT and hFUSQ290X and targeted their expression in different tissues We found that the targeted expression of hFUSQ290X in the dopaminergic and the serotonergic neurons did not cause obvious neuronal degeneration but it resulted in motor dysfunction which was accompanied by impairment in the GABAergic pathway The involvement of the GABAergic pathway was supported by rescue of motor symptoms with gabapentin Interestingly we observed gender specific downregulation of GABAR and NMDAR expression and reduction in serotonin level Overexpression of hFUSQ290X also caused an increase in longevity and this was accompanied by downregulation of the IIS/TOR signalling pathway Our in vivo studies of the hFUSQ290X mutation in Drosophila link motor dysfunction to impairment in the GABAergic pathway Our findings would facilitate further efforts in unravelling the pathophysiology of ETEssential tremor ET is one of the most common adultonset ageprogressive neurological disorders affecting about 09  of world population Louis and Ferreira 2010 Despite its high prevalence the exact pathophysiology of ET remains to be elucidated ET is a heterogeneous disorder involving both motor such as tremor and nonmotor such as cognitive psychiatric dementia and sensory symptoms Chandran and Pal 2012 Unlike in neurodegenerative condition such as PD where there is a progressive loss of dopaminergic neurons Benazzouz et al 2014 no obvious neuronal loss has been reproducibly observed for ET Neuropathological evidence suggests abnormalities in the GABAergic Purkinje cells of the cerebellum and their surrounding neuronal populations which likely lead to defects in the cerebellar cortical circuits and output Louis 2016 Higher mortality rates have been reported in the cases of lateonset ET Louis et al 2007 but increased longevity has also been associated with early onset ET and observed in ET patients and their relatives Jankovic et al 1995Gene seems to play a major role on the development of ET JimenezJimenez et al 2013 although the exact cause and effect relationship is unclear This is due to limited discoveries of pathogenic ET gene mutations and lack of in vivo ET models Pathogenic risk variants have been reported for TENM4 teneurin transmembrane protein 4 and studies of this gene in zebra fish suggested its role on oligodendrocyte differentiation and myelination of CNS axons Hor et al 2015 A pathogenic mutation Q290X has also been recently identified for the FUS/TLS fused in sarcoma/translated in liposarcoma gene through exome sequencing in a large ET family from Quebec Merner et al 2012 FUS is an RNA/DNA binding protein and it is involved in the regulation of transcription RNA splicing and translational control Prasad et al 1994 Belly et al 2005 Q290X is a nonsense mutation caused by a singlebasepair substitution which results in the truncation of the FUS protein at amino acid 290 in its nuclear export signal motif Merner et al 2012 Mutations in other domains of the FUS protein are known to cause amyotrophic lateral sclerosis ALS and frontotemporal dementia FTD Vance et al 2009 Chen et al 2011Here we performed targeted expression analysis Brand and Perrimon 1993 of hFUSQ290X in Drosophila and characterised its phenotype In addition we examined the involvement of the GABAergic system the serotonergic system and the IIS/TOR signalling pathway and conducted therapeutic challengesFlies were raised on standard corn meal containing food and genetic crosses were performed at 25 °C GAL4 lines GMRGAL4 pleGAL4 DdcGAL4 and OK371GAL4 and RNAi lines to dFUS stock numbers 32990 and 34839 used were obtained from Bloomington Drosophila Stock Centre BDSC For generation of the wildtype transformation construct wildtype hFUS cDNA was excised from the pCMVFUS plasmid DNA from Origene using restriction enzymes XhoI and XbaI and subcloned into the XhoI and XbaI digested pUASTattB transformation vector The Q290X mutation was generated using the QuickChange II SiteDirected Mutagensis Kit Stratagene following the manufacturer’s instructions with the pUASTattBwildtype hFUS as the DNA template All plasmid constructs which had been subcloned into the transformation vector were verified by sequencing before sent for transgenesis at Best Gene Inc To avoid possible contribution of second site mutation which might have arisen during transgenesis the transgenic lines were backcrossed into yw from Bloomington for six generationsProteins were extracted from head homogenates of approximately 30–50 flies of the respective genotypes Adult flies were exposed to liquid nitrogen followed by shaking to separate the heads from the bodies Heads were collected and homogenised in MPER Mammalian Protein Extraction Buffer Thermo Scientific in the presence of protease inhibitor Roche Cell debris was removed by centrifugation and protein concentrations were determined by BCA protein assay Thermo Scientific 50–150 µg of total proteins of each samples were denatured by heating to 95 °C and loaded on SDS–PAGE gels After electrophoresis proteins were transferred to nitrocellulose paper blocked with 1 × PBST + 5  nonfat milk and incubated in primary antibody overnight at 4 °C This was followed by incubation in secondary antibody Protein expression was detected by chemiluminescence Thermo Scientific Primary antibodies used were rabbit antiFUS from Bethyl Labs cat A300302A 11000 dilution rabbit antiNterminal hFUS LSC unpublished result 11000 mouse antiserotonin Thermo Scientific 140 dilution and mouse antitubulin Sigma 110000 dilution Secondary antibodies used were HRPconjugated goat antimouse/rabbit Santa Cruz Biotechnology Inc 14000 dilutionBrain fixation and antibody staining were carried out according to the standard protocol Briefly adult brains were dissected in phosphate buffered saline PBS then fixed with 4  formaldehyde followed by a few washes in PBT PBS + 01  Triton X100 After blocking with 3  BSA in PBT brain samples were incubated in primary antibody rabbit antityrosine hydroxylase Sigma 11000 dilution overnight at 4 °C with rotation After removal of primary antibodies samples were washed with PBT followed by incubation in secondary antibody Cy3conjugated goat antirabbit Jackson Immunoresearch 1500 After washes samples were incubated with Vectashield and analysed by Carl Zeiss Upright Confocal MicroscopeFor adult eye microscopy heads were removed and glued to masking tapes and then photos were captured by a digital camera through eye piece of a scanning microscope Olympus Photography of wing phenotypes was carried out similarly by placing flies on CO2 gas pad on their backs

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