alzheimer 's disease ( ad ) is a progressive brain disease affecting the elderly population , causing problems with memory , thought , and behavior .
approximately 25% of ad cases are familial ( fad ) , caused by autosomal dominant mutations in amyloid precursor protein ( app ) or the presenilin ( ps1 , ps2 ) genes [ 13 ] , while the majority of sporadic ad cases are not associated with any known mutations .
the hallmarks of alzheimer disease include intraneuronal neurofibrillary tangles , consisting of the hyperphosphorylated microtubule - associated protein tau and extracellular deposits of filaments of 42-residue amyloid  ( a ) peptide [ 4 , 5 ] .
a is the product of sequential cleavage of app by - and -secretases [ 7 , 8 ] .
nonharmful app cellular processing by - and -secretases results in a short , highly soluble , nonamylogenic p3 peptide . in alternative
amyloidogenic processing , app is hydrolyzed by - and -secretases , and three possible peptides ( a40 , a42 , a43 ) can be generated .
variability of the cleavage site of -secretase is associated with mutations in the ps1 and ps2 genes .
a peptides are secreted from the presynaptic terminal into the extracellular matrix , where fibrillary a deposits are formed outside of neurons .
some evidences suggest that the a aggregates are the critical factor which triggers a complex pathological cascade leading to neurodegeneration .
all strategies to lower brain a42 levels should be therapeutically beneficial in ad treatment . given that bace1 is the initiating enzyme in a generation , it is considered a prime target for drug in ad for reducing cerebral a levels [ 1315 ] .
rna interference ( rnai ) is an eukaryotic regulatory mechanism that uses double - stranded rna ( dsrna ) for induction of posttranscriptional gene silencing by the sequence - specific hydrolysis of homologous mrna [ 16 , 17 ] .
rnai has already been proposed for therapy of neurodegenerative diseases , including amyotrophic lateral sclerosis ( als ) , spinocerebellar ataxia ( sca ) , huntington 's disease , and alzheimer 's disease [ 2123 ] .
reduced the cleavage of app at the beta site , lowered amyloid burden , and achieved amelioration of dendritic and synaptic pathology in the hippocampus of tested animals .
other bace1 silencing studies , performed in vitro in primary neurons derived from app - transgenic mice , resulted in reduced production of app fragments , ctfs and a .
the present studies concern optimization of rnai for effective silencing of overexpressed and endogenous bace1 protein in the human / mouse / rat cell cultures .
for that purpose we used original sequences of sirnas coding synthetic sirna duplexes as well as shrna - encoding plasmids and lentiviral vector .
moreover , we analyzed the effects of the use of bace1-specific sirnas and , in consequence , bace1 silencing on the expression of selected genes involved , for example , in adult neurogenesis .
the synthesis of rna oligonucleotides was performed according to the routine phosphoramidite approach , using lca cpg glass support and commercially available nucleoside phosphoramidites ( glen research ) .
oligonucleotide synthesis was performed with an applied biosystems 394 instrument under the conditions recommended by the manufacturer .
assembly of sirna duplexes was performed by mixing the equimolar amounts of complementary sense and antisense oligoribonucleotides in pbs buffer , heating at 96c for 2 minutes , followed by slow ( 2 hours ) cooling to room temperature .
japan ) were generated by annealing two complementary 62-mer oligonucleotides containing ( i ) a 21 nucleotide ( nt ) sense strand and 21  nt antisense strand separated by loop , ( ii ) a stretch of five thymidines as the pol iii promoter termination signal , and ( iii ) both sides of the insert flanked by bspmi restriction site .
additionally , the use of the following loop sequences was explored : gtgtgctgtcc , ttcaagaga , cttcctgtca , and tagtgaagccacagatgta .
hela ( human , cervical carcinoma ) cells were cultured in rpmi 1640 medium ( gibco , brl , paisley ) supplemented with 10% heat - inactivated fetal bovine serum ( fbs ) ( gibco , brl , paisley ) , 100  u / ml penicillin , and 100  g / ml streptomycin ( polfa ) at 37c and 5%  co2 .
sh - sy5y ( human , caucasian , bone marrow neuroblastoma ) cells were cultured in 50% f12 nutrient mixture ( ham ) medium ( gibco , brl , paisley)/50% mem ( gibco , brl , paisley ) , supplemented with 15% fbs and antibiotics ( 100  mg / ml streptomycin and 100  u / ml penicillin ) .
hek293 cells ( human , embryonic kidney ) and m15 cells ( mouse , mesonephric epithelium ) were cultured in dulbecco 's mem ( sigma - aldrich co. , saint louis , mo ) , supplemented with 10% fbs and antibiotics at 37c and 5%  co2 . before transfection
transfections were performed using lipofectamine 2000 ( invitrogen ) at a 2 : 1 ratio and appropriate sirnas or plasmids encoding shrna were added . for dual fluorescence assay
, hela cells were cotransfected with plasmid dna pdsred2-n1 , 15  ng / well ( bd biosciences ) and p - bace - gfp , 70  ng / well , provided by dr .
weihong song ( the university of british columbia , vancouver , canada ) , and with sirna ( 0.15  nm final concentrations ) or plasmids ( psilencer - shrna / pcpurhu6-shrna 30  ng / well ) dissolved in opti - mem medium ( 50  l / well , gibco ) .
the cells were incubated for 5 - 6 hours and then medium with transfection mixture was replaced with the fresh , culturing medium with antibiotics .
after 48-hour incubation at 37c in atmosphere of 5%  co2 , the cells were washed three times with phosphate saline buffer ( pbs ) and lysed overnight with mixture of np-40 buffer ( 150  mm nacl , 1% igepal , 50  mm tris - hcl ( ph 7 ) , 1  mm pmsf and pbs ( ratio 1 : 3 ) at 37c ) .
cell lysates were used for fluorescence determination by a dual fluorescence assay , as described previously . shortly , fluorescence values of enhanced green fluorescent protein ( egfp ) and red fluorescent protein ( rfp ) were measured using a synergy ht reader ( bio - tek ) ; data quantification was performed using kc4 software .
excitation and emission wavelengths were as follows : gfp  
 ex = 485/20  nm and  
 em = 528/20  nm ; rfp  
 ex = 530/25  nm and  
 em = 590/30  nm .
the sirna activity was calculated as the ratio of gfp to rfp fluorescence values , averaged over eight repetitions .
the relative level of fluorescence ( gfp / rfp ) in control cells ( transfected with pbace - gfp , pdsred2-n1 and control s-0 sirna ) was taken as the reference ( 100% ) .
hek293 , m15 , sh - sy5y cells were transfected with plasmids encoding shrna expression cassettes using lipofectamine 2000 ( invitrogen ) according to the manufacturer 's protocol .
the cells were collected 36 hours after transfection , lysed with tripure isolation reagent ( roche applied science ) , and total rna was extracted and analyzed by rt - pcr . for a single reaction , 1  g of total rna was used as a template .
primers of the following sequences : for bace1 ( h / m / r ) : forward ( 633652 ) tgtggagatggtggacaacc , reverse ( 993 - 1012 ) , atctcagcataggccagccc ; for gapdh ( h ) : forward gagtcaacggatttggtcgt , reverse ttgattttggagggatctcg ; for gapdh ( m / r ) : forward gtgtgaacggatttggccgt , gapdh ( m ) reverse ttgatgttagtggggtctcg , gapdh ( r ) reverse ttgatgttagcgggatctcg were synthesized in house .
commercially available primers were used for rt - pcr amplification of genes listed in table 2 .
the reverse transcription and pcr amplification reactions were performed using onestep rt - pcr kit ( qiagen ) .
cell lysates obtained by tripure isolation reagent ( roche applied science ) were used for preparation of total protein fraction , according to manufacturer 's protocol .
protein samples ( 20  g for each lane ) were separated by 10% sds - page and semi - dry electroblotted to immobilon p pvdf membrane ( millipore corp . , mass , usa ) .
rabbit polyclonal anti - bace1 ( santa cruz biotechnology inc . ) in dilution 1 : 300 and rabbit polyclonal anti--actin ( abcam ) in dilution 1 : 5000 were used as primary antibodies .
goat antirabbit igg conjugated to alkaline phosphatase ( zymed , san francisco ) in dilution 1 : 5000 was used .
bound antibodies were visualized by reaction of alkaline phosphatase with chemiluminescence substrate ( millipore corp . ) at the visualization system ( g - box , syngene ) .
293 t cells were cultured in 10  cm dish in iscove 's modified dulbecco 's medium ( gibco / invitrogen ) , supplemented with 1% l - glutamine , 10% heat - inactivated fbs ( sigma ) and 1% antibiotics ( gibco / invitrogen ) . the tuhc pcsc - sp - pw - egfp vector   ( 20  g ) , coding for the shrna sequence , and helper vectors , pmdl ( 12  g ) , rev ( 6  g ) , and vsvg ( 8  g ) , were cotransfected into 293 t cells using a calcium - phosphate method [ 33 , 34 ] . 48 and 72 hours after transfection , culture medium was collected , pooled together , filtered , and concentrated by ultracentrifugation .
virus titers were determined by transduction of 293 t cells and enhanced gfp expression visualization using fluorescence microscopy .
hcn a94 cells ( adult rat hippocampal neural stem cells ) were cultured as described . the day before transduction , cells were passaged into 6 well plates .
then , the cells were transduced with appropriate recombinant lentiviruses : lv - si-5 , lv - si-6 , and control lv - egfp . after infection
, the hcn a94 cells were cultured in conditions specific for neuronal differentiation : in f12 media containing 1% n-2 supplement ( gibco brl , paisley ) with 1  m retinoic acid ( ra ) and 5  m forskolin ( sigma ) .
four days after infection , level of mrna of the screened proteins were determined by rt - pcr .
several target sites in human / mouse / rat ( h / m / r ) bace1 genes were selected for sirna duplexes using available tools [ 3639 ] .
the sirna duplexes used had the typical structure of 19-base pairs ( bp ) fully complementary duplex with 2-nucleotide ( nt ) overhangs at each 3 end , typically two thymidine units ( table 1 ) .
all rna strands were synthesized in house and their structures and purity were uniformly confirmed by maldi - tof ms and page electrophoresis , respectively , ( data not shown ) .
the shrna encoding sequences were designed to express rna transcripts with a hairpin structure of a 19-bp stem with a 9-nt loop : ttcaagaga ( psilencer 2.0-u6 ) , or a 21-bp stem with an 11-nt loop : gtgtgctgtcc ( pcpurhu6 ) . to investigate the silencing activity of sirna duplexes or shrna - expressing vectors we optimized a dual fluorescence reporter system ( dfa ) [ 30 , 40 ] .
the system is based on measurement of the relative fluorescence intensity of enhanced green fluorescent protein ( egfp , expressed from fusion p - bace - gfp   plasmid ) and coral ( discosoma spp . )
derived red fluorescent protein ( rfp ) , expressed in hela cells from exogenously delivered plasmids .
localization of fluorescent proteins is shown in figure 1(a ) , and interestingly bace1-egfp fusion protein localizes in the cytoplasmatic membranes , probably in the golgi apparatus and/or er compartments , while rfp protein is present in the nucleus and cytoplasm with no localization in the membrane .
all duplexes used in the experiments mediated the human target gene silencing to varying extent ( see table 1 ) , depending on the sirna concentration and target site .
variability of sirna potency is most likely the result of changed thermodynamic properties of the 5- and 3-ends of the duplexes as well as secondary structure of target mrna .
representative microscopic images of dfa control cells ( transfected with pdsred2-n1 and p - bace - gfp plasmids ) and cells additionally transfected with si-2 and si-4 duplexes are shown at figure 1(b ) .
silencing activities of sirnas or shrna - encoded plasmids directed toward endogenous bace1 mrna were screened in human ( hek293 and sh - sy5y ) and mouse ( m15 ) cell lines by semiquantitative rt - pcr and by western blot analysis . in sh - sy5y cells ,
the most active was si-4 duplex which lowered bace1 mrna level up to 34% ( 100% value consists of bace1 expression in control sample without silencing ) ( see table 1 ) .
low activity of sirna in sh - sy5y neuroblastoma cell line was probably caused by lower transfection efficiency of these cells as compared to hela cells , in which screened duplexes si-2si-4 were much more active .
fluorescence of gfp or rfp expressed from pgfp - bace or pdsred2-n1 plasmids , respectively , was ca .
1020 times higher in hela cells than in sh - sy5y cells transfected in standard conditions ( using lipofectamine at a 2 : 1 ratio ) ( see figure 2 ) .
the pcpurhu6 si-5 vector ( sequence corresponding to si-5a ) , specific for the human bace1 gene , was the most potent inhibitor in hek293 cells ( 32% of expression of the control sample ) .
additionally , pcpurhu6 si-5b , specific for mouse and rat bace1 mrna , also revealed high silencing efficiency in m15 mouse cells ( 30% of expression level of the control sample ) .
the second plasmid construct , pcpurhu6 si-6 ( sequence corresponding to si-6 ) , specific for the h / m / r bace1 gene , resulted in bace1 expression of 51% in hek293 cells and 71% in m15 cells .
the loop structure has a significant impact on export of pre - mirna or shrna from nucleus   and their silencing efficiency was proven to be the most efficient with loops ranging from 9 to 19  nt [ 27 , 28 , 36 ] .
we designed and cloned into the pcpurhu6 vector the hairpin - type rnas with si-6 sequence ( pcpurhu6 si-6 ) with the 19 - 21 base pair ( bp ) stems and with various loops : ( 1 ) pcpurhu6 si-6 ( 21  bp)-mir26 , ( 2 ) si-6 ( 19  bp ) with 9-nt uucaagaga loop , ( 3 ) si-6 ( 21  bp ) with 9-nt uucaagaga loop , ( 4 ) si-6 ( 21  bp ) with 10-nt cuuccuguca ( loop from mirna23 ) , and ( 5 ) si-6 ( 21  bp ) with 19-nt uagugaagccacagaugua ( loop from mirna30 ) ( see figure 3 ) .
all five plasmids were tested in hek293 cells and the most active was the construct 1 causing 50% lowering of bace1 mrna .
constructs 4 and 5 with mirna - origin loops mirna23 and mirna30 , respectively , demonstrated moderate silencing activity , lowering bace1 mrna by 27% and 38% , respectively . in order to validate the effects of shrnas on bace1 gene expression in rat cells model ,
the lentiviral vectors were constructed and used for the lv - si-5 and lv - si-6 lentiviruses production .
the high titer lentiviruses lv - si-5 and lv - si-6 were used for transduction of adult hippocampal neural stem cells hcn a94 .
both vectors caused significant reduction in bace1 mrna ( > 75% ) in comparison to the level of mrna expressed by control cells ( figure 4 ) . in search for additional functions of bace1 or products of bace1 proteolytic activity , we analyzed mrna levels of several genes involved in adult neurogenesis ( listed in table 2 ) .
their list consists of a transcription factor sox-2 ( essential for maintaining progenitor cells in the undifferentiated stage ) , transcription factors neurogenin 13 and neurod1 , nrsf / rest ( expressed during neuronal differentiation and responsible for activation of the expression of neuronal genes ) , synapsin i , nachii , glur2 , iii - tubulin , calbidin , bdnf , and scg10 .
additionally , we assessed expression of gfap , indicative of astrocytes ; mbp , specific for oligodendrocytes ; and genes of creb , mecp2 , app , bace2 , pkr , and gapdh .
we did not find any influence of bace1-specific sirnas on the mrna level of the above - mentioned genes .
interestingly , we found correlation between bace1 and scg10 gene expression , as bace1 inhibition by lv - si-5 and lv - si-6 resulted in simultaneous downregulation of scg10 ( figure 4 , table 2 ) .
moreover , during neurogenesis in conditions favouring the differentiation of progenitor cells the level of bace1 expression was increasing over the four days of the experiment ( data not shown ) .
many alzheimer 's disease treatments are aimed at blocking the formation of pathogenic amyloid- peptides .
both - and -secretases , due to their crucial role in the secretion of a , are supposed to be useful therapeutic targets .
successful inhibition of these enzymes may reduce the burden of amyloid--peptide in ad patients ' brains , which may slow down the neurodegeneration .
inhibition of -secretase activity can evoke severe side effects , because this protease is involved in processing of other important substrates , including notch receptor and cell surface proteins type i [ 43 , 44 ] .
therefore , bace1 , being a major -secretase participating in the toxic a generation in the brain , is a primary therapeutic target .
multiple strategies have been used to inhibit bace1 gene expression , including antisense oligonucleotides and catalytic nucleic acids [ 21 , 25 , 4548 ] . in the present studies
it was already demonstrated that silencing of bace1 decreases the amount of secreted a peptides [ 21 , 24 , 25 , 47 ] , therefore the main goal of these studies was to select sirnas that are able to reduce specifically bace1 expression in the designed experimental system .
several sirna sequences directed toward human , mouse , and rat bace1 mrna were originally designed and their functionality against overexpressed and endogenous bace1 was evaluated .
the experiments were performed in hek293 and sh - sy5y human and m15 mouse cell lines as well as in hcn a94 rat cells , where a lentiviral vector expressing active shrna was applied . as determined in the bace - gfp / rfp dual fluorescence assay , the most active were si-3 and si-4 constructs , which at 100  pm concentration reduced the level of bace - gfp mrna by ca .
these two sirnas were the most active in the same system when introduced into psilencer plasmid ( 5 and 11% of bace1 expression , resp .
interestingly , two other sirnas , si-5a and si-6 , cloned into pcpurhu6 plasmid , silenced bace1 in more than 80% , and may offer a good model system for further investigations with the use of plasmid - coded sirna .
synthetic sirnas , si-2 , si-3 , and si-4 , transfected into human sh - sy5y cells , reduced bace1 mrna and protein level to the lower extent than it was seen in dfa experiments performed in hela cells .
although sh - sy5y neuroblastoma cells endogenously express high level of -secretase and are often used in studies on neurodegeneration , their low transfection efficiency using conventional approach is the most limiting feature .
5-fold lower transfection efficiency of pegfp - c1 plasmid into sh - sy5y cells in comparison to hela cells was observed with the use of conventional polycationic transfecting agents as well as with the more sophisticated gas plasma transfection methodology . in our experiments ,
transfection of plasmid dna , assessed by the extent of expression of pgfp - bace or pdsred2-n1 plasmids in sh - sy5y cells , reached the level of only 10 20% of that in hela cells ( figure 2 ) .
si-5a specific for human bace1 mrna and si-5b , varying with one nucleotide , specific for mouse and rat homologs .
both these sirnas were rather selective toward cognate genes , as it is seen from experiments in hek293 and m15 cells with sirnas coded in pcpurhu6 plasmid ( table 1 , the last column ) .
thus , fully complementary si-5a caused selective silencing of human bace1 ( 32% of bace1 expression ) but being by one nucleotide mismatched ( uc ) with mouse bace1 mrna , it was almost inactive toward this gene ( 92% of bace1 expression ) . in contrary ,
the duplex si-5b , fully complementary to mouse bace1 mrna , was more efficient in mouse m15 cells than in hek293 human cells .
such high specificity of sirna with mutation at the position 16 of the antisense strand ( counting from the 5-end of this strand ) was previously reported by schwarz et al . for two alleles of the same gene of sod1 . to improve the knockdown efficiency of our constructs
, we designed and cloned hairpin - type rnas with the same stem sequence , but with various loops , derived from mirnas : 23 , 26b , or 30 .
all variants showed suppressive activity , but the most potent was the construct with the 21  bp stem and 11-nt loop from the microrna 26b ( 50% expression of target bace1 ) .
moreover , the efficacy of constructs with the same loop sequence did not depend on the length of stem ( 19-bp or 21-bp ) ( see the activity of constructs 2 and 3 , figure 3 ) .
finally , active sirna sequences , si-5b and si-6 , were cloned into lentiviral vector ( tuhc pcsc - sp - pw - egfp ) and used to silence the target protein in rat adult neural progenitor cells hcn a94 .
both lentivirus - coded sirnas were very efficient in bace1 silencing in hcn a94 cells , allowing to demonstrate that si-5b and si-6 duplexes , complementary to human , mouse , and rat mrna target sites , can be simultaneously used as inhibitors of bace1 gene expression in human , mouse , and rat cells , although with various efficiency in particular cells .
we asked a question , whether the activity of bace1-specific sirnas , and in consequence silencing of bace1 protein , influence the profile of expression of other genes .
such nonspecific  off - target  effects are observed when target - specific sirna molecules are used at high molar concentrations ( > 100  nm ) [ 52 , 53 ] .
subsequent analysis of mrna levels of several genes involved in neurogenesis showed that screened sirnas and bace1 silencing did not affect the expression of most of selected genes ( see table 2 ) .
unexpectedly , we observed downregulation of scg10 expression . ncbi / blast analysis has shown no significant similarity between scg10 and bace1 sequences .
therefore , our observation suggests the presence of a regulatory pathway for scg10 expression involving bace1 or products of its proteolytic activity , although this hypothesis requires additional studies .
scg10 is a neuron - specific member of the stathmin family of microtubule regulatory proteins . like stathmin
, it can bind to soluble tubulin and depolymerize microtubules in neurons . because this protein has restricted localization in neurons , expression of scg10 gene is tightly regulated , mainly by nrsf / rest transcription factor .
. it would be interesting to explain the potential metabolic pathway between -secretase and scg10 and these studies are currently in progress .
the scg10 downregulation observed during rnai - induced silencing of bace1 may constitute important limitation of therapeutic application of this antiamyloid gene therapy , especially because increased expression of scg10 is observed in the cortical and hippocampal regions of brain after the neuronal lesions . in summary , we demonstrate high silencing activity of synthetic sirnas and viral vectors - encoded shrnas against overexpressed and endogenous bace1 .
this process is fairly selective as expression of majority of screened genes involved in neurogenesis is not affected by bace1-specific sirnas nor by bace1 level reduction .
 beta - secretase ( bace1 ) is the major enzyme participating in generation of toxic amyloid - beta ( a ) peptides , identified in amyloid plaques of alzheimer 's disease ( ad ) brains . 
 its downregulation results in decreasing secretion of a. thus , bace1 silencing by rnai represents possible strategy for antiamyloid therapy in the treatment of ad . in this study , 
 a series of newly designed sequences of synthetic and vector - encoded sirnas ( psilencer , pcpurhu6 , and lentivirus ) were tested against overexpressed and endogenous bace1 in several cell lines and in adult neural progenitor cells , derived from rat hippocampus . 
 sirnas active in human , mouse , and rat cell models were shown to diminish the level of bace1 . in hcn a94 cells , two bace1-specific sirnas 
 did not alter the expression of genes of bace2 and several selected genes involved in neurogenesis ( synapsin i , iii - tubulin , calbidin , neurod1 , glur2 , creb , mecp2 , pkr ) , however , remarkable lowering of scg10 mrna , coding protein of stathmin family , important in the development of nervous system , was observed . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion