Patent Application: US-201514809279-A

Abstract:
macromolecule intracellular transduction technology based on improved cell - permeable parkin recombinant protein has been developed as a protein - based anti - neurodegenerative agent for efficient bbb - penetration to effectively deliver the recombinant protein into the brain . parkin protein , a dopaminergic neuronal cell death inhibitor , has been fused with a newly developed advanced macromolecule transduction domain and solubilization domain to increase the solubility / yield and cell -/ tissue - permeability of the recombinant protein . in addition , our newly developed amtd / sd - fused recombinant icp - parkin protein has shown bbb - permeability . both in vitro and in vivo , our icp - parkin recombinant protein improved motor skills , a typical phenotype of parkinson &# 39 ; s disease , by increasing dopamine level in the brain by suppressing apoptosis of dopaminergic neuron cells . in conclusion , icp - parkin could be applicable in clinical studies as a protein - based anti - neurodegenerative agent to treat parkinson &# 39 ; s disease by protecting dopaminergic neuron cells and regulating the secretion of dopamine .

Description:
the present invention relates to protein - based therapeutics for parkinson &# 39 ; s disease having cell - permeability applicable for the clinical studies that facilitate the transduction of biologically active macromolecules including proteins across the cell membrane . the cell - permeable parkin recombinant protein of the present invention based on amtd is artificially developed . in this invention , the aim is to develop icp - parkin by adopting novel hydrophobic cpps formatted based on the seven critical factors determined based on in - depth analysis to facilitate protein translocation across the membrane . these seven critical factors include the amino acid length ( 9 - 13 ), bending potential based on the proline position and location ( 6 ′, 7 ′, 8 ′ in the middle and 12 ′ at the end ), rigidity / flexibility ( ii : 40 - 60 ), structural formation ( ai : 180 - 220 ), amino acid composition ( a , v , i , l , and p ), and the secondary structure ( helix formation recommended ). based on these critical factors analyzed with selected published cpps , the novel hydrophobic cpps — amtds — have been designed for the development of icp - parkin proteins to enhance its ability to transduce across the cell membrane . various hydrophobic cpp have been used to enhance the delivery of protein cargoes to mammalian cells and tissues . similarly , amtd321 had been discovered to enhance the uptake of a his - tagged coding sequence of solubilization domain a ( sda ) in raw264 . 7 cells as assessed by flow cytometry . relative levels of protein uptake was 7 times that of a reference mtm12 protein , which contained 1st generation cpp ( membrane translocating motif ) and was 2 . 9 times that of a mtd85 reference protein , which contained 2nd generation cpp ( macromolecule transduction domain ). in addition , relative to 8 . 1 - fold higher protein uptake was observed with a random peptide recombinant protein ( rp38 )- fused with sda , a peptide sequence , which had an opposite property of that of amtd fig1 . similar results were obtained in nih3t3 cells using fluorescent microscopy to monitor the protein uptake . these amtd321 - mediated intracellular delivered into cells were displayed in fig2 and information of amtd321 displayed in table 1 . recombinant cargo ( parkin ) proteins fused to hydrophobic cpp could be expressed in bacteria system , purified with single - step affinity chromatography , but protein dissolved in physiological buffers ( e . g . pbs , dmem or rpmi1640 etc .) was highly insoluble and had extremely low yield as a soluble form . therefore , an additional non - functional protein domain ( solubilization domain : sd ) has been applied to fuse with the recombinant protein for improving the solubility , yield and eventually cell and tissue permeability . according to the specific aim , the selected domains are sda and sdb ( table 2 ). the amtd / sd - fused recombinant proteins have been determined for their stability and stability . the solubilization domains ( sds ) and amtds have greatly influenced in increasing solubility / yield and cell -/ tissue - permeability of the protein . therefore , we have developed highly soluble and highly stable parkin recombinant protein fused with sd ( sda and sdb ) and amtds for the clinical application . we designed 4 different types of recombinant proteins with or without the amtd and solubilization domains for parkin protein . protein structures were labeled as follows : ( i ) a cargo protein only , ( ii ) a cargo protein fused with amtd , ( iii ) a cargo protein fused with amtd and solubilization domain a ( sda ) and ( iv ) a cargo protein fused with amtd and solubilization domain b ( sdb ) ( fig3 ). each parkin recombinant protein was successfully induced by adding iptg and purified ( fig5 ). we observed a significant increase of solubility of parkin fused with either sda ( hm 321 psa ) or sdb ( hm 321 psb ), which were compared to a cargo protein only ( hp ) or cargo protein fused with only amtd ( hm 321 p ). moreover , the solubilization domains ( sda and sdb ) successfully improved relative yield of proteins compared to hp , where hm 321 psa and hm 321 psb showed 4 folds increase of solubility ( fig6 ). therefore , these results suggested that the parkin recombinant proteins fused with sds displayed a significant improvement of solubility and yields . in order to solve the problem with low solubility and yield of negative control protein ( hp ), additional set of structures for recombinant proteins were designed as shown in fig1 . recombinant proteins in set 2 were fused with sdb on c - terminal with ( hm 321 psb ) or lacking amtd ( hpsb ). these parkin recombinant proteins improve the solubility and yield by using these strategies fig1 and 14 . the amtd 321 / sd - fused parkin recombinant proteins have significantly higher cell -, tissue - permeability as compared to the parkin recombinant proteins lacking amtd321 sequence ( hp and hpsb ). collectively , even though these amtd 321 / sd - fusion parkin recombinant proteins ( hm321psa and hm321psb ) have similar solubility and yield , cellular and systemic delivery activity of amtd321 / sdb - fused parkin recombinant protein was higher than parkin recombinant protein lacking amtd321 sequence . therefore , amtd 321 / sd - fused parkin recombinant protein was determined as the most stable structure of the recombinant proteins . we investigated in the cell / tissue - permeability and biological activity of developed parkin recombinant proteins . cell permeability of parkin recombinant proteins was evaluated in raw 264 . 7 cells after 1 hour of protein treatment . fitc - labeled parkin recombinant proteins lacking amtd ( hp and hpsb ) was not detectable in raw cells . in contrast , the amtd - bearing parkin recombinant proteins , hm 321 p , hm 321 psa and hm 321 psb , showed high cell permeability ( fs . 7 and 15 ). similar results were obtained in nih3t3 cells , using fluorescence confocal laser scanning microscopy to monitor protein intracellular localization . ( fig8 and 16 ). in particular , the amtd / sd - fused parkin recombinant proteins ( hm 321 psa and hm 321 psb ) showed the highest cell permeability . these results showed that the amtd successfully abled the proteins to penetrate into the cells within short time ( 1 hour ) and improved the solubility of proteins that positively affect cell - permeability . next , we determined in vivo tissue - permeability of parkin recombinant proteins after 15 min and 30 min of intraperitoneal injection of fitc - labeled proteins ( fig1 ). the pbmc analyzed by facs showed a gain in fluorescence , indicative of the presence of fitc - labeled proteins as compared with control animals that received fitc - labeled hpsb or unconjugated fitc . one of the two parkin recombinant proteins , hm 321 psb , showed a higher intracellular signal in pbmc . the distribution of fitc - labeled proteins in different organs in cryosections analyzed by fluorescence microscopy ( fig9 and 18 ). similar results , the parkin recombinant proteins lacking amtd ( hp and hpsb ) showed limited tissue permeability in various organs ( brain , heart , lung , liver , spleen and kidney ). in contrast , amtd 321 enhanced the systemic delivery of parkin recombinant proteins in tissues ( heart , lung , liver and kidney ). to determine the blood - brain - barrier permeability by using immunohistochemical labeling ( immunohistochemistry ), tissues were immunohistochemically processed using anti - parkin ( 1 : 200 , santa cruz biotechnology ) monoclonal antibodies . parkin positive immunoreactivity was observed in brain of the hm 321 psb - treated mice , but it was not observed in brain of the hpsb - treated mice ( fig2 ). in the result of western blot , parkin antibody - positive band was only observed in group administered hm 321 psb recombinant protein ( fig2 ). the results have demonstrated that the amtd 321 / sd - fused parkin recombinant protein could be efficiently delivered to the brain by penetrating the blood - brain barrier permeability . to determine the protective effect of parkin recombinant protein on the neuronal death caused by the neurotoxin , cath . a and sh - sy5y cells were treated with 6 - hydroxydopamine ( 6 - ohda ). after treatment of 6 - ohda , these cells were pre - treated with parkin recombinant proteins and tunel assays were conducted . a large number of cell death were observed in 6 - ohda only treated group . similarly to 6 - ohda - treated group , hp lacking amtd has shown similar percentage of apoptotic cell death with the agonist only group . contrastingly , amtd 321 / sd - fused parkin recombinant proteins ( hm 321 psa and hm 321 psb ) have suppressed apoptosis to 19 . 7 and 14 . 2 % in cath . a and sh - sy5y cells , respectively (* p & lt ; 0 . 05 ). similar results have been obtained in both cath . a cells and sh - sy5y cells . these results have demonstrated that amtd 321 / sd - fused parkin recombinant proteins have neuroprotective effects in cultured neuronal cells ( fig1 and 11 ). in order to determine the effect of parkin recombinant proteins in vivo , we developed a parkinson &# 39 ; s disease —( pd -) animal model that mimics physiological and mental symptoms of parkinson &# 39 ; s disease by using a neural toxin . to induce parkinson &# 39 ; s disease - like symptoms , the neural toxin , mptp ( 1 - methyl - 4 - phenyl - 1 , 2 , 3 , 6 - tetrahydrophyridine ) was used . this mptp is converted to a toxic agent mpp + after it gets activated by monoamine oxidase ( mao - b ) in the inner mitochondrial membrane , and this selectively targets dopaminergic neuron to induce parkinson &# 39 ; s disease . to assess the motor function recovery effect of parkin recombinant proteins , swimming test was conducted . swimming activity ( 4 legged ) of each group ( diluent , mptp only , mptp + hpsb and mptp + hm 321 psb ) was measured and expressed as a percentage of the unlesioned diluent control . mptp only group showed significant decrease in the swimming activity as compared to the diluent group . similarly , hpsb - treated group showed similar result of mptp only group with 6 - ohda treated group . contrastingly , hm 321 psb - treated group showed improved motor activity . therefore , we have determined that amtd 321 / sd - fused parkin recombinant protein recovered motor function in acute mptp - induced parkinson disease mouse model ( fig2 ). to assess the motor function recovery effect of parkin recombinant proteins , gait test was performed ( fig2 ). in this experiment , the stride distance and sway distance were measured . the stride distance was significantly reduced in the mptp only and hpsb - treated group , while the sway distance was increased as compared to the diluent group . however , the hm 321 psb - treated mice showed the stride distance ( fig2 ) of similar levels as the normal group and they showed significantly reduced sway distance ( fig2 ) as compared to the mptp only and hpsb - treated group . therefore , we have determined that amtd 321 / sd - fused parkin recombinant protein improves gait function in acute mptp - induced parkinson diseased mouse model . 7 . activation of dopamine release in mptp - pd mouse model by parkin recombinant proteins to measure the dopamine level in urine , urine was collected from mice in all groups 10 h after the first treatment of parkin recombinant proteins . these urine samples have been measured by elisa . there has been statistically significant difference between mptp only and hm 321 psb - treated group in the result after 10 h . while mptp only group has shown decreased urine level , hm 321 psb - treated group have shown similar urine level as compared with the diluent group . the results have demonstrated that the amtd 321 / sd - fused parkin recombinant protein stimulates dopamine level in urine . ( fig2 ). to measure the dopamine level in the brain , dopamine level of striatal regions in all groups have been measured by elisa . striatal dopamine level in hm 321 psb - treated group was more than double compared to the mptp only and hpsb - treated group . therefore , we have determined that amtd 321 / sd - fused parkin recombinant protein causes an increase of striatal dopamine level , decreased by mptp treatment ( fig2 ). 8 . expression recovery of tyrosine hydroxylase by parkin recombinant proteins in mptp - pd model to determine the protective efficacy of dopaminergic neuron by parkin recombinant protein , immunohistochemistry was performed using an antibody for tyrosine hydroxylase , which is a marker enzyme in dopamine neurons . the number of dopaminergic neurons in the substantia nigra and the striatum region of the mice treated with amtd 321 / sd - fused parkin recombinant protein were observed and compared to the mptp only and hpsb administrated group . therefore , we have determined that amtd 321 / sd - fused parkin recombinant protein could have a neuroprotective function ( fig2 and 29 ). for this invention , cell - permeable parkin recombinant proteins have been designed and developed with the amtd . all parkin recombinant proteins fused with amtd and control recombinant proteins lacking amtd have been confirmed for their quantitative , visual cell -/ tissue - permeability and bbb - permeability . we were able to confirm that the cell - permeable amtd 321 / sd - fused parkin recombinant proteins had relatively high cell -/ tissue - permeability ( fig7 , 9 , 15 , 16 , 17 and 18 ), as well as efficient in the brain tissue delivery by penetrating through bbb ( fig1 and 20 ). to determine the biological activity of cell - permeable parkin recombinant protein , we carried out a variety of functional tests . we confirmed that the cell - permeable parkin recombinant protein has anti - apoptotic effect on the neuronal cell death caused by a neurotoxin ( 6 - ohda ) ( fig1 and 11 ), and it has a recovery effect in the pd - mice model that displayed movement dysfunction induced by neurotoxin ( mptp ) ( fig2 , 25 , 26 and 27 ). 10 . development of improved cell - permeable amtd / sd - fused parkin recombinant protein ( icp - parkin ) to suppress parkinson &# 39 ; s disease - associated phenotype many human diseases are caused by proteins with deficiency or over - expression that causes mutations such as gain - of - function or loss - of - function . we are developing protein - based therapeutics that can be efficiently delivered into the brain through the bbb penetration based on macromolecule intracellular transduction technology . it could be a new therapeutic treatment of parkinson &# 39 ; s disease in which it regulates the function of proteins changed by various genetic causes . the following examples are presented to aid practitioners of the invention , to provide experimental support for the invention , and to provide model protocols . in no way are these examples to be understood to limit the invention . our newly developed technology , amtd - based mitt , has enabled us to improve the method for developing cell - permeable recombinant proteins . the expression vectors were designed for parkin proteins fused with amtd321 and solubilization domain a ( sda ) or solubilization domain b ( sdb ). to acquire expression vectors for recombinant proteins , polymerase chain reaction ( pcr ) had been devised to amplify these recombinant proteins . the pcr reactions ( 100 ng genomic dna , 10 pmol each primer , each 0 . 2 mm dntp mixture , 1 × reaction buffer and 2 . 5 u pfu (+) dna polymerase ( doctor protein , korea )) was digested on the restriction enzyme site between bamhi ( 5 ′) and hindiii ( 3 ′) involving 35 cycles of denaturation ( 95 ° c .) for 30 seconds , annealing ( 60 ° c .) for 30 seconds , and extension ( 72 ° c .) for 2 min each . for the last extension cycle , the pcr reactions remained for 5 minutes at 72 ° c . then , they were cloned into the site of pet - 28a (+) vectors ( novagen , madison , wis ., usa ). dna ligation was performed using t4 dna ligase at 4 ° c . overnight . these plasmids were mixed with competent cells of e . coli dh5 • strain on the ice for 10 minutes . this mixture was placed on the ice for 2 minutes after it was heat shocked in the water bath at 42 ° c . for 90 seconds . then , the mixture added with lb broth media was recovered in 37 ° c . shaking incubator for 1 hour . transformant was plated on lb broth agar plate with kanamycin ( 50 • g / ml ) ( biopure , johnson , tenn .) before incubating at 37 ° c . overnight . from a single colony , plasmid dna was extracted , and after the digestion of bamhi and hindiii restriction enzymes , digested dna was confirmed by using 1 . 2 % agarose gels electrophoresis ( fig3 ). pcr primers for the his - tagged parkin recombinant proteins fused to amtd and sd are summarized in table 3 . denatured recombinant proteins were lysed using denature lysis buffer ( 8 m urea , 10 mm tris , 100 mm nah2po4 ) and purified by adding ni - nta resin . resin bound to proteins were washed 3 times with 30 ml of denature washing buffer ( 8 m urea , 10 mm tris , 20 m imidazole , 100 mm nah2po4 ). proteins were eluted 3 times with 30 ml of denature elution buffer ( 8 m urea , 10 mm tris , 250 mm imidazole ). after purification , they was dialyzed twice against a refolding buffer ( 550 mm guanidine - hcl , 440 mm l - arginine , 50 mm tris , 100 mm ndsb , 150 mm nacl , 2 mm reduced glutathione and 0 . 2 mm oxidized glutathione ). finally , they were dialyzed against a physiological buffer such as dmem at 4 ° c . until the dialysis was over 300 × 105 times . concentration of purified proteins was quantified using bradford assay according to the manufacturer &# 39 ; s instructions . after purification , they were dialyzed against dmem as indicated above . finally , sds - page analysis was conducted to confirm the presence of target protein . the amtd 321 - fused parkin proteins containing sda or sdb are cloned , expressed , purified , and prepared in a soluble form . each recombinant protein fused to amtd and / or sd was determined for their solubility and yield . solubility was scored on a 5 - point scale ranging from highly soluble proteins with little tendency to precipitate (*****) to largely insoluble proteins (*) by measuring their turbidity ( a450 ). yield ( mg / l ) in physiological buffer condition of each recombinant protein was also determined . the cell - permeable parkin recombinant proteins were observed as a single band , where the amount of the final purified protein was 13 mg / l ( fig3 ). recombinant proteins purified under the denatural condition were analyzed on 10 % sds - page gel and stained with coomassie brilliant blue . intracellular delivery of parkin recombinant proteins for quantitative cell - permeability , the amtd 321 / sd - fused parkin recombinant proteins were conjugated to fluorescein isothiocyanate ( fitc ) according to the manufacturer &# 39 ; s instructions ( sigma - aldrich , st . louis , mo .). raw 264 . 7 cells were treated with 10 • m fitc - labeled recombinant proteins for 1 hour at 37 ° c ., washed three times with cold pbs , treated with proteinase k ( 5 • g / ml ) for 10 min at 37 ° c . to remove cell - surface bound proteins . cell - permeability of these recombinant proteins were analyzed by flow cytometry ( facscalibur ; bd , franklin lakes , n . j .) using the flowjo analysis software . for a visual reference of cell - permeability , nih3t3 cells were cultured for 24 hours on a coverslip in 24 - wells chamber slides , treated with 10 μm fitc - conjugated recombinant proteins for 1 hour at 37 ° c ., and washed three times with cold pbs . treated cells were fixed in 4 % paraformaldehyde ( pfa , junsei , tokyo , japan ) for 10 minutes at room temperature , washed three times with pbs , and mounted with vectashield mounting medium ( vector laboratories , burlingame , calif .) with dapi ( 4 ′, 6 - diamidino - 2 - phenylindole ) for nuclear staining . the intracellular localization of the fluorescent signal was determined by confocal laser scanning microscopy ( lm700 , zeiss , germany ). for in vivo delivery , icr mouse ( 5 weeks old , female ) were injected intraperitoneally ( ip , 600 ug / head ) with fitc only or fitc - conjugated proteins . after 15 min and 30 min , pbmc were isolated from whole blood in mice , were analyzed by flow cytometry ( bd , guaba ). for a visual reference of tissue - permeability , 600 μg of fitc - labeled parkin recombinant proteins was administered to icr mice ( 5 weeks old , female ). two hours later , the mice are sacrificed , and liver , kidney , spleen , lung , heart and brain were isolated and embedded with an oct compound ( sakura , alphen anden rijn , neetherlands ), frozen , and then sectioned to a thickness of 20 μm . the tissue specimens are mounted on a glass and observed by fluorescence microscopy ( nikon , tokyo , japen ). for immunohistochemistry , 6 - week - old icr female mice were injected intraperitoneally with diluent ( pbs ) or with 600 • g his - tagged parkin recombinant proteins . after 2 h , mice was perfused with 0 . 9 % nacl and fixed with cold 4 % paraformaldehyde . after the brains were removed , they were post - fixed with 4 % paraformaldehyde and transferred to 30 % sucrose . the brains were cut into 30 μm coronal sections using a freezing microtome . brain cryosections ( 30 • m ) are immunostained with anti - parkin ( 1 : 100 , santa cruz biotechnology ) monoclonal antibodies , followed by biotin - conjugated goat anti - mouse secondary antibody ( vector laboratories ), and developed with avidin - biotin complex kit ( vectastain kit , vector laboratories ). for western blot analysis , mice treated with proteins were perfused with 0 . 9 % nacl . brains were isolated , and striatal region was dissected and homogenized in lysis buffer ( intron , seongnam , korea ). supernatant from the centrifugation ( 13 , 000 rpm for 10 min at 4 ° c .) is analyzed by western blot that is probed with antibodies against parkin ( 1 : 200 ) and •- actin ( 1 : 2 , 000 ). the secondary antibody is goat anti - mouse igg - hrp ( all antibodies were from santa cruz biotechnology ). terminal dutp nick - end labeling ( tunel ) assays are conducted according to the manufacturers &# 39 ; instructions ( roche ). mouse dopaminergic neuronal ( cath . a ) cells ( atcc : american type culture collection ) are plated ( 3 × 10 4 / well ) and pre - treated with 50 • m 6 - hydroxydopamine ( 6 - ohda ) for 1 h at 37 ° c . followed by the treatment with 2 . 5 • m parkin recombinant proteins for 2 . 5 h at 37 ° c ., analyzing the changes in cell survival . human brain tumor ( sh - sy5y ) cells ( korea cell line bank ) are also cultured , plated ( 3 × 10 4 / well ) and pre - treated with 100 • m 6 - hydroxydopamine ( 6 - ohda ) for 6 h followed by the treatment with 2 . 5 • m parkin recombinant proteins for 2 . 5 h at 37 ° c ., analyzing the alteration . 8 - week - old c57bl / 6 male and female mice housed in plastic cages in a temperature — and humidity — controlled room with a 12 - h light / 12h - dart cycle . mice were randomly assigned to one of four experimental groups ( diluent , mptp only , mptp + hpsb and mptp + hm 321 psb ). three groups of mice except for diluent were received intraperitoneal injections of mptp ( 15 mg / kg × 3 times / day , 2 h interval ) for three consecutive days . the neurotoxin , 1 - methyl - 4 - phenyl - 1 , 2 , 3 , 6 - tetrahydropyridine ( sigma - aldrich , st . louis , mo .) was dissolved in 0 . 9 % nacl . controls are treated with 0 . 9 % nacl for the same time period . after 3 days , mice in mptp + hpsb and mptp + hm 321 psb groups were received intraperitoneal injection of hpsb , hm 321 psb recombinant protein ( 600 μg / head , a time / day ) for five consecutive days , respectively . we confirm that animal experiments are performed in accordance with the guidelines of the institutional animal care and use committee . measurement of dopamine in urine of mptp - pd animal models treated with parkin recombinant proteins for measurement of dopamine synthesized in the urine , we collected the urine of mice in all groups on the first day of treatment of parkin recombinant protein . dopamine synthesized in the urine is measured by using a commercial elisa kit according to instructions provided by the manufacturer ( genway , san diego , calif .). in brief , rabbit anti - dopamine antibody is added to urine or tissue extract , and the immune complexes are recovered in wells coated with goat anti rabbit antibody . a second enzyme conjugated anti - dopamine antibody directed against a different epitope produces the reaction products proportional to the amount of antigen as compared against a standard curve . measurement of dopamine in brain of mptp - pd animal models treated with parkin recombinant proteins dopamine synthesized in the brain extracts is measured by using a commercial elisa kit according to instructions provided by the manufacturer ( genway , san diego , calif .). in brief , rabbit anti - dopamine antibody is added to urine or tissue extract , and the immune complexes are recovered in wells coated with goat anti rabbit antibody . a second enzyme conjugated anti - dopamine antibody directed against a different epitope produces the reaction products proportional to the amount of antigen as compared against a standard curve . assessment of motor activity with swim test of mptp - pd animal models treated with parkin recombinant proteins gross motor functions of mptp - lesioned mice are assessed by using a swim test . mice are placed in a 37 ° c . water bath and video recorded . unlesioned mice have swum using all 4 legs 98 % of the time . the percent of time of each group ( mptp only , mptp + hpsb or mptp + hm 321 psb ) spent swimming ( 4 legged ) is measured and expressed as a percentage of the unlesioned diluent control . assessment of motor activity with gait test of mptp - pd animal models treated with parkin recombinant proteins the mice were allowed to walk along a 50 cm long , 10 cm wide runway with 10 cm high walls into an enclosed box . stride length and sway length were measured as the average distance of forward movement between each stride and sway . on the last day of treatment of parkin recombinant protein , mice was perfused with 0 . 9 % nacl and fixed with cold 4 % paraformaldehyde . and then , brains were removed , post - fixed with 4 % paraformaldehyde , and transferred to 30 % sucrose . the brains were cut into 30 μm coronal sections using a freezing microtome . dopaminergic neuronal cell marker in brain - tyrosine hydroxylase ( th ) is immunostained with anti - th ( 1 : 50 , thermo scientific , rockford , usa ) monoclonal antibody , followed by biotin - conjugated goat anti - rabbit secondary antibody ( 1 : 100 , santa cruz biotechnology , santa cruz , calif .) and developed with abc kit ( vectastain kit , vector laboratories , burlingame , calif .). all experimental data using cultured cells are expressed as means s . d . for at least three independent experiments . statistical significance is evaluated using a two - tailed student &# 39 ; 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103 ( 28 ): 10793 - 8 . 13 . clark i e , dodson m w , jiang c , cao j h , huh j r , seol j h , et al . drosophila pink1 is required for mitochondrial function and interacts genetically with parkin . nature 2006 ; 441 ( 7097 ): 1162 - 6 .