Patent Abstract:
to elucidate the molecular mechanisms of “ gain of toxic function ” of expanded polyglutamine stretches in cag repeat expansion diseases , the inventors established an expression system of full - length and truncated cdnas for dentatorubral - pallidoluysian atrophy and found that truncated drpla proteins containing the expanded polyglutamine stretch , but not the full - length protein , form peri - and intra - nuclear aggregates consisting of filaments and concomitant apoptosis . the apoptotic cell death was partially suppressed by transglutaminase inhibitors , cystamine and monodansyl cadaverine , raising the possibility of involvement of transglutaminase reaction . the results may provide a potential basis for the development of therapeutic measures for cag repeat expansion diseases .

Detailed Description:
aggregate formation and induction of apoptotic cell death by truncated drpla protein including expanded poluglutamine stretches . to investigate whether the full - length or truncated drpla mutant proteins exhibit structural abnormalities such as aggregate formation , or exhibit cytotoxicities , the inventors generated various deletion mutants of full - length wildtype ( coding for 19 glutamines ) and mutant ( coding for 82 glutainines ) drpla cdnas ( fig1 ). plasimids containing these cdnas were constructed as below . a full - length human drpla cdna containing a cag repeat of normal length ( 15 cag repeats ) ( pdrplan ) was constructed by ligating partial drpla cdna clones ( f1 and f15 - 20 ) 18 into a pbluescript sk (−) vector . a full - length human drpla cdna containing an expanded cag repeat ( 78 cag repeats ) ( pdrplae ) was constructed by replacing the 963 - bp ecot22i - spli segment of pdrplan with the corresponding ecot22i - spli segment of a cosmid drpla genomic clone which was isolated from a genomic cosmid library constructed from genomic dna of a patient with drpla . after the noti - bbsi fragment of pdrplan , pdrplae or pdrpla was removed , an oligonucleotide adapter containing the sequences for a noti site , methionine , the flag tag and a bbsi site ( 5 ′- gcggccgctctagagccgccaccatggactacaaagacgatgacgacaagatgaagacac - 3 ′) was ligated into a pbluescript sk (−) vector ( psk - afn and psk - afe ). the noti - sali fragment of psk - afn or psk - afe containing the segment coding for the translation initiation methionine , the flag tag and the entire drpla cdna was subcloned into a mammalian expression vector , pef - bos 38 ( pef - bos - afn and pef - bos - afe ). since there is a sequence of 5 ′- cag - caa - cag - caa upstream of the cag repeat of the drpla cdna ( this segment is not included as the number of cag repeats ), pef - bos - afn and pef - bos - afe code for 19 and 82 glutamines , respectively . deletion mutants containing an expanded cag repeat and a down - stream segment of various lengths were constructed . a segment containing 21 bp upstream of the cag repeat , the cag repeat and the 305 bp fragment downstream of the cag repeat of pdrplan were first amplified by pcr using a primer ( 5 ′- ggcggccgctctagagccgccaccatg - gactacaaagacgatgacgacaagcatcaccaccagcaacagcaa - 3 ′) containing the sequences for the flag tag and a noti linker , and a primer with the sequence 5 ′- accggtgggaaagggtagggc - 3 . the pcr products were digested with noti and nari , and then subcloned into pdrplae from which the corresponding noti - nari fragment had been removed ( pbfe ). deletions of the segment downstream of the cag repeat were generated either by exoiii / mung bean nuclease digestion of pbfe , or by pcr using pdrplae as the template . the deleted dna segments were subcloned into the pef - bos expression vector along with a multi - stop linker at the 3 ′ end . the resultant plasmids , pef - bos - fq 82 - 447 , pef - bos - fq 82 - 376 , pef - bos - fq 82 - 174 , pef - bos - fq 82 - 129 , pefbos - fq 82 - 101 , pef - bos - fq 82 - 40 and pef - bos - fq 82 - 19 , contain dna segments coding for 3 histidines , 82 glutamines and various lengths of amino acids downstream of the polyglutamine stretch ( 447 , 376 , 174 , 129 , 101 , 40 and 19 amino acids , respectively ). deletion mutants coding for 19 glutamines were also generated using similar methods . deletion mutants containing cag repeats and the upstream segment of various lengths were constructed . dna segments containing an expanded cag repeat and upstream segments of various lengths were obtained by pcr using one of the following sense primers : an anti - sense primer ( e8r : 5 - gggtcgacttatcagccctccagtgggtggggaaat - 3 ′). the pcr products were digested by noti and sali , and subcloned into the pef - bos expression vector . the resultant plasmids , pef - bos - f483 - q 82 , pef - bos - f322 - q 82 and pef - bos - f174 - q 82 contain the segments coding for the flag tag , 82 glutamines , 19 amino acids downstream of the polyglutamine stretch and upstream segments with 483 , 322 and 174 amino acids upstream of the polyglutamine stretch , respectively . the cos7 cells were transfected with plasmid thus constructed . cos7 cells were seeded in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ) containing 10 % fetal calf serum the day before transfection at 3 × 10 4 per well of an 8 - well chamber slide ( nunc inc ., naperville , ill .). the cos7 cells were transfected with 0 . 5 μg of plasmid dna using the superfect transfection reagent ( qiagen , hilden , germany ) according to the manufacturer &# 39 ; s instructions . on the cells transfected with plasmid dna , expression patterns of various drpla proteins described above were analyzed using anti - flag m5 monoclonal antibody 72 hours after transfection and the extent of apoptotic cell death was examined . cells were fixed for 30 minutes in 4 % paraformaldehyde in 0 . 1 m phosphate - buffered saline ( pbs ), permeabilized with pbs containing 0 . 02 % triton x - 100 , and incubated in 10 % normal goat serum in pbs for 30 min at room temperature ( rt ). cells were then incubated with an anti - flag m5 monoclonal antibody ( eastman kodak , new heaven , conn .) with a 1 : 500 dilution for 2 hours at rt , followed by a 1 hour - incubation with rhodamine - conjugated anti - mouse igg ( dako , glostrup , denmark ) and observed by fluorescence microscopy . immunostaining using 3 , 3 ′- diaminobenzidine tetrahydrochrolide was also carried out using the avidin - biotin peroxidase complex ( abc ) method . the cells were counter - stained with hematoxylin and examined by light microscopy . cells transfected with pef - bos - afn ecoding the full - length drpla protein containing a polyglutamine stretch of normal length ( 19 glutamines ) expressed drpla protein diffusely in the cytoplasm with a homogenous or fine granular pattern ( fig2 b ), while mock - transfected cells were not stained by the antibody ( fig2 a ). cells transfected with pef - bos - afe coding for the full - length drpla protein with an expanded polyglutamine stretch ( 82 glutamines ) ( fig2 c ) or cells transfected with pef - bos - fq 19 - 19 coding for a truncated drpla protein containing mostly the polyglutamine stretch of normal length ( 19 glutamines ) ( fig2 d ) also showed similar expression patterns to those of cells transfected with pef - bos - afn ( fig2 b ). cells transfected with pef - bosfq 82 - 19 coding for a truncated protein containing mostly the expanded polyglutamine stretch ( 82 glutamines ), however , showed distinct aggregate bodies mainly in the perinuclear cytoplasmic areas ( fig2 e , j ). these aggregate bodies were immunopositive for ubiquitin in some population , but negative for vimentin or congo red stain . with time , cells with these aggregate bodies became shrunken , and stained positively in the tunel ( terminal deoxynucleotidyl - transferase - mediated dutp - biotinnick end - labeling ) assay ( fig2 g ). these tunel - positive cells were observed frequently 72 hours after transfection ( 52 % of the cells with aggregate bodies ). on the other hand , apoptotic cells were not observed in cells transfected with pef - bos - afn , pef - bos - afe or pef - bos - fq 19 - 19 ( fig2 j ). to investigate whether full - length mutant drpla protein with the expanded polyglutamine stretch is incorporated into the aggregate bodies in the presence of aggregate bodies of truncated mutant proteins , another plasmid construct ( pegfp - q 82 - 19 ) coding for the truncated mutant protein mostly containing the expanded polyglutamine stretch fused with gfp ( green fluorescence protein ) was made . incorporation of the full - length mutant flag - tagged drpla protein into the aggregate bodies was clearly demonstrated in cells co - transfected with pef - bos - afe and pegfp - q 82 - 19 ( fig2 h , i ). such aggregation was never observed when the cells were transfected with pef - bos - afe alone ( fig2 c ). to determine whether the formation of aggregate bodies is dependent on the lengths of the mutant proteins , the inventors generated various deletion mutants of the full - length wild - type and mutant drpla cdnas ( fig1 ). aggregate formation was observed at high frequencies ( 71 - 88 %) in the cells expressing the truncated drpla proteins containing the polyglutamine stretch and the downstream region with 129 or fewer amino acids ( fq 82 - 129 , fq 82 - 101 , fq 82 - 40 or fq 82 - 19 ) ( fig3 b ). although the frequencies were low , aggregate formation was also observed in cells expressing various lengths of the upstream regions and the expanded polyglutamine stretch ( f483 - q 82 , f322 - q 82 or f174 - q 82 ) ( fig3 a ). the percentages of cells stained in the tunel reaction were high for cells transfected with pef - bos - fq 82 - 129 , pef - bos - fq 82 - 101 , pef - bos - fq 82 - 40 or pef - bos - fq 82 - 19 . as cells formed aggregate bodies exhibited apoptotic cell death detected by tunel reaction , it was indicated strongly that formation of the aggregate bodies cause apoptotic cell death . time - dependent formation of aggregate bodies was examined . imunohistochemical analysis was performed as described in example 1 . formation of the aggregate bodies was observed in 53 % of the cells expressing the flag epitope at 24 hours after transfection with pef - bos - fq 82 - 19 ( fig4 a , d ). the percentage of cells exhibiting aggregate bodies increased to 65 % ( fig4 b , d ) and to 89 % ( fig4 c , d ) at 48 and 72 hours after the transfection , respectively . the frequency of tunel - positive cells was only 1 % at 24 hours after transfection , but was increased to 9 % and 52 % at 48 and 72 hours after transfection , respectively ( fig4 d ). for the further analysis on the detailed structures of the aggregate bodies , the inventors observed the cells transfected with pef - bos - fq 82 - 19 by electron microscopy . cells were fixed with 4 % paraformaldeheide - 0 . 1 % glutaraldehyde in 0 . 1m phosphate buffer , ph 7 . 4 , for 15 min at rt . the cells were then dehydrated in a graded dimethylformamide series and embedded in lr white resin ( london resin company , berkshire , england ). ultrathin sections were cut and mounted on nickel grids . after incubation with 10 % normal goat serum in pbs for 10 minutes at rt , the sections were incubated overnight at 4 ° c . with a mouse anti - flag m5 monoclonal antibody at a dilution of 1 : 4000 . after washing with pbs , the sections were incubated with goat anti - mouse igg conjugated to 10 - nm gold ( british biocell international , cardiff , uk ; 1 : 30 dilution ) for 30 min at rt . the sections were then washed with pbs and incubated in 2 % glutaraldehyde in 0 . 1 m cacodylate buffer , ph 7 . 4 . after washing with distilled water , the sections were stained with uranyl acetate and lead citrate , and examined with a hitachi h - 7100 electron microscope . immuno - electron microscopic observation revealed that the aggregate bodies consist of fibrous aggregations mainly in perinuclear cytoplasmic areas ( fig5 a , b ). the fibrous aggregations consist of radially oriented straight or slightly curved unbranched filaments approximately 10 - 12 nm in diameter ( fig5 b , c ). no specific cell organella were found in the aggregates . these aggregate bodies were observed not only in the perinuclear areas in the cytoplasm but also occasionally in the nucleus ( fig5 b , c ). non - aggregated filaments with morphology similar to those of the aggregates were also scattered in the nuclei as well as in the cytoplasm . penetration of the nuclear membrane by filaments of the aggregates , some of which were present via the nuclear pores , was often found in these cells ( fig5 c ). these filamentous structures were observed only in cells transfected with pef - bos - fq 82 - 19 , but not in cells transfected with pef - bos - afn , pef - bos - afe or pef - bos - fq 19 - 19 . with culture time after transfection , many apoptotic bodies containing the aggregates were encountered ( fig5 d ). to investigate if similar aggregates were present in the brains of drpla patients , the inventors performed immunohistochemical analysis of the dentate nucleus in the cerebellum of drpla patients ( n = 5 ) and controls ( n = 5 ) using an anti - drpla protein polyclonal antibody . the autopsied brains were fixed with phosphate - buffered 4 % paraformaldehyde and embedded in paraffin for histological examination . immunostaining was performed using a rabbit antiubiquitin antibody ( dakopatts : 1 : 200 dilution ) or a rabbit anti - drpla protein polyclonal antibody ( 1 : 300 dilution ), which was generated against a gst - fusion protein containing amino acid residues 172 - 253 of drpla protein and affinity - purified using an affigel - 10 column ( bio - rad ) conjugated with the gst - fusion protein . presence of intranuclear inclusions , which were stained with the anti - drpla protein antibody ( fig6 a ) as well as with an anti - ubiquitin antibody ( fig6 b ), was confirmed in all the 5 drpla patients . such inclusions were never observed in the controls . the intranuclear inclusions were examined by electron microscope . cells were fixed with 3 % glutaraldehyde - 1 % paraformaldeheide in 0 . 1 m phosphate buffer , ph 7 . 4 , post - fixed in 1 % osmium tetroxide , dehydrated in a graded ethanol series and embedded in epon 812 . ultrathin sections were cut and stained with uranyl acetate and lead citrate , and examined with a hitachi h - 7100 electron microscope . electron microscopic study revealed that the intranuclear inclusions were composed of fine granular and occasionally filamentous structures ( fig6 c ). these results indicate the possibility that formation of intranuclear inclusions play a important role at onset of neurodegenerative diseases . suppression of aggregate formation and apoptotic cell death by transglutaminase inhibitors . to investigate whether the transglutaminase reaction is involved in the formation of aggregate bodies and the induction of apoptotic cell death , the inventors cultured cos7 cells in the presence of transglutaminase inhibitors ( cystamine 28 , monodansyl cadaverine ( mdc ) 29 , and putrescine 30 ), after transfection . for tilis purpose , truncated drpla proteins were expressed as fusion proteins with green fluorescence protein ( gfp ), which allowed the highly sensitive observation of viable cells . the inserts of pef - bos - fq 82 - 19 and pef - bos - fq 19 - 19 were transferred into pegfp containing the coding region for gfp . the resultant plasmid dnas ( pegfp - fq 82 - 19 and pegfp - fq 19 - 19 ) were transfected into cos7 cells . the plasmid dna was constructed as below . the segment coding for 3 histidines , the polyglutamine stretch and 19 amino acids downstream of the polyglutamine stretch of drpla cdna ( pdrplae or pdrplan ) was amplified by pcr using a primer ( 5 ′- gggaattcggatgcaccat - caccaccagcaacagcaacag - 3 ′) containing an ecori linker sequence and a primer ( 5 ′- gtggatccccgccctccagtgggtggggaaatgct - 3 ′). pcr products were digested with ecori and bamhi , and subcloned into the pegfp - n1 expression vector ( clontech , palo alto , calif .). the nucleotide sequences of all the constructs were confirmed using automated dna sequencers ( pe applied biosystems , foster city , calif .). cells transfected with pegfp - q 19 - 19 expressed the gfp fusion protein diffusely in the cytoplasm ( fig7 a ), wlile cells transfected with pegfp - q 82 - 19 exhibited formation of aggregate bodies ( fig7 c ), similarly to those transfected with pef - bos - fq 19 - 19 and pef - bos - fq 82 - 19 , respectively . cystamine did not change the expression patterns of the fusion protein in the pegfp - q 19 - 19 - transfected cells ( fig7 b ). when the cells transfected with pegfp - q 82 - 19 were cultured for 60 hours in 1 mm cystamine , however , formation of aggregate bodies was significantly suppressed from 42 % to 33 % ( p & lt ; 0 . 01 ) ( fig7 c , d , e ). nuclear fragmentation was also suppressed from 38 % to 24 % in the presence of 1 mm cystamine ( p & lt ; 0 . 05 ) ( fig7 f ). similar results were observed at 24 and 48 hours after transfection ( fig7 e , f ). on the other hand , the frequency of cells retaining a diffuse cytoplasmic expression pattern without aggregate formation was increased from 32 % ( absence of cystamine ) to 56 % ( 1 mm cystamine ) at 60 hours after transfection ( p & lt ; 0 . 01 ). the suppression of aggregate bodies and nuclear fragmentation was observed even at 100 m of cystamine and the suppression effects appeared in a dose - dependent manner ( fig8 a , c ). the effects of other transglutaminase inhibitor ( mdc ) on the aggregate formation and apoptotic cell death were also investigated . the tunel assay was performed using an in situ cell death detection kit ( boehringer mannheim , mannheim , germany ) according to the manufacturer &# 39 ; s instructions . fitc - conjugated dutp was used for the terminal deoxynucleotidyl transferase reaction . an assay for nuclear fragmentation was performed by staining cells with 5 m hoechst 33342 . quantitation was performed by analyzing 100 cells expressing the flag epitope . statistical analyses were performed using student &# 39 ; s t test . strong suppression of nuclear fragmentation by mdc was observed in a dose dependent manner ( fig8 d ). there were no significant changes in the frequencies of the cells with aggregate formation ( fig8 b ), although the sizes of the aggregate bodies were smaller when the transfected cells were cultured in the presence of mdc compared to those observed in the absence of mdc . 1 . la spada , a . r ., wilson , e . m ., lubahn , d . b ., harding , a . e . & amp ; fischbeck , k . h . androgen receptor gene mutations in x - 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