aging is a major risk factor for alzheimer 's disease ( ad ) and the number of ad patients will increase in the near future . for this reason , therapeutic treatments against this devastating disease are urgently sought for ( hardy and selkoe , 2002 ; dodel et al . , 2003 ; cummings , 2004 ; the amyloid hypothesis holds that generation and deposition of amyloid -peptide ( a ) are key events driving neurodegeneration in ad ( glenner and wong , 1984 ) . immunotherapy involving injection of synthetic a aggregates to elicit neutralizing and aggregate - breaking antibodies and passive a immunization showed promising results in delaying cognitive decline ( younkin , 2001 ; haass , 2002 ) , but also underscored the risk of side effects ( pfeifer et al . -secretases cleave several substrates and their inactivation appears to interfere with physiologically important signaling pathways ( haass , 2004 ) , but -secretase remains an obvious therapeutic target because its activity can fully be removed in mice by knocking out bace ( -site app cleaving enzyme ) without any obvious toxicity ( luo et al . , 2001 ; ohno et al . , 2004 ) . inhibitors of bace are under active study , but the development of specific , cell - permeable drugs that penetrate into the brain remains a challenging task ( kahle and de strooper , 2003 ) . here , we propose a novel approach to control a production in vivo . the approach is based on intracellular expression of single chain antibodies ( intrabodies ; biocca et al . , 1990 ; , 1988 ; marasco and dana jones , 1998 ; lobato and rabbitts , 2004 ; stocks , 2004 ) that interfere with pathologic endoproteolysis by binding close to the -secretase cleavage site of huapp ( fig . one intrabody associated within the er with newly synthesized -amyloid precursor protein ( app ) . association persisted during app transport along the secretory line , protected app from -secretase cleavage and favored the alternative cleavage by -secretase . this resulted in decreased production of the toxic a peptide and increased production of p3 . another intrabody carrying a carboxy - terminal er retention signal caused quantitative er retention and slow disposal of app , thereby virtually abolishing a production . app is a type i transmembrane protein with a single hydrophobic domain for membrane retention . the amyloidogenic processing of app produces the -amyloid peptide ( a ) through sequential cleavages by bace at the -site and by -secretase . shedding of the app ectodomain occurs through redundant proteolytic events at the cell surface ( -cut ) or in endosomes ( -cut ) by the secretases . the swedish mutation at the -site strongly favors bace cleavage of app on route to the cell surface . the 40 aa sequence of a is also depicted ( bold letters ) as well as the 3 aa exchanged in murine a ( normal fonts ) . the monoclonal antibody 1 ( paganetti et al . , 1996 ) specifically binds to the efrh tetrapeptide adjacent to the -secretase cleavage site of huapp ( fig . 1 , at position a3 - 6 ) . sfv1 consists of the light and heavy chain variable regions of 1 ( 132 and 120 residues , respectively ) covalently linked by a ggggs pentapeptide repeated three times . sfv1-kdel is a variant of the same intrabody carrying the sekdel carboxy - terminal residues of bip / grp78 to confer er retention ( munro and pelham , 1987 ) . the native signal sequence of the light chain was maintained to target the intrabodies to the er lumen . liquid chromatography mass spectrometry of secreted sfv1 expressed in human embryonic kidney 293 ( hek ) cells revealed that the signal peptide was removed at the consensus site similar to the original 1 antibody ( unpublished data ) . we first determined if sfv1 maintained the capacity of the 1 template to associate with huapp when expressed intracellularly . hek cells were transfected for expression of the swedish variant of huapp ( mullan et al . , 1992 ) with or without sfv1 . 1 d after transfection , cells were metabolically labeled with s - methionine and cysteine , chased for 10 min or 2 h , and detergent solubilized . labeled huapp was immunoprecipitated from cell lysates with specific antibodies . in mock - transfected cells , 2 a , lanes 1 and 2 ) . after a 10-min chase app is immature ( appi ; mr = 120 kd ) in the er as shown by endoh sensitivity of its single n - linked glycan ( fig . after a 2-h chase most of huapp was released from the er and the n - glycan became endoh resistant ( fig . maturation of huapp ( appm ; mr = 130 kd ) also involves tyrosine - sulfation and o - glycosylation resulting in higher mr ( weidemann et al . , 1989 ) . in cells also expressing sfv1 , the intrabody ( mr = 26 kd ) coprecipitated with appi after a 10-min chase ( fig . 2 a , lane 3 ) and association persisted through the chase ( lane 4 ) . monitoring kinetics of association revealed that the half - time for formation of the intracellular sfv1-huapp complex was 11 min ( fig . 2 c ) and that association between sfv1 and huapp did not prevent huapp maturation ( fig . the specificity of sfv1 for huapp was confirmed by the substantial reduction in the amount of sfv1 coprecipitated with a variant of app characterized by a efgh versus efrh mutation in the 1 epitope ( fig . the parental antibody 1 has strongly decreased affinity for this epitope present in mouse app . ( a ) hek cells were transfected for expression of huapp ( lanes 1 and 2 ) , sfv1 and huapp ( lanes 3 and 4 ) , or sfv1 and rgapp ( lanes 5 and 6 ) . after metabolic labeling with s - amino acids and chasing with unlabeled amino acids , huapp and rgapp were immunoprecipitated from cell extracts with a carboxy - terminal app antibody . when present , sfv1 associates and coprecipitates with huapp but not with rgapp . ( b ) analysis on 8% sds page better visualizes app maturation and endoh sensitivity . at 10 min labeled appi is still endoh sensitive ; but after 2 h app is released from the er and appm becomes endoh resistant and shows increased mr upon n - glycan modification , tyrosine - sulfation , and addition of o - glycans . ( c ) kinetics of app : sfv1 association were determined by coimmunoprecipitations and plotted as a function of the maximal amount of sfv1 coprecipitated with app . the position of mr markers of 200 , 116 , 97 , 66 , 45 , and 32 kd is shown with thin lines . next , we coexpressed huapp with sfv1-kdel to determine first if this intrabody maintained the capacity to associate with app , and second to establish if appending an er - retention sequence to an app - targeted intrabody also caused retention of huapp . cells were metabolically labeled and chased for 10 min and 3 h. sfv1-kdel had slower electrophoretic mobility than sfv1 ( fig . it rapidly associated with newly synthesized huapp as shown by coimmunoprecipitation after a 10-min chase ( fig . 3 a , lane 4 ) . unlike sfv1 , however , association of sfv1-kdel with huapp prevented export of the latter from the er . in fact , the molecular weight of app did not increase with progression of the chase ( fig . 3 a , lanes 3 and 4 ) and the protein failed to acquire endoh resistance or other posttranslational modifications even after 3 h ( fig . both intrabodies associates and coprecipitates with huapp using a carboxy - terminal antibody to app . in the presence of sfv1 , appm at the end of the 3-h chase has higher mr than newly synthesized appi ( 10 min ) . in contrast , sfv1-kdel impairs maturation of app , no mr shift is observed and appi remains endoh sensitive . ( b ) indirect immunofluorescence was performed with an antibody to app ( transfected cells in panels 1 , 3 , 5 , 7 , 9 , and 11 ) and to cnx to visualize the er ( all cells in panels 2 , 6 , and 10 ) or giantin to visualize the golgi ( panels 4 , 8 , and 12 ) . in the presence of sfv1-kdel ( panels 912 ) , app and cnx colocalizes . in sfv1 ( panels 58 ) or mock - transfected cells ( panels 14 ) , app is detected in the er and in the golgi . ( c ) immature app retained in the er by sfv1-kdel is degraded with a t1/2 of 2 h ; app disposal is partially inhibited by the proteasomal inhibitor mg132 . cytochemical analysis by indirect immunofluorescence revealed that part of huapp colocalized with the er marker calnexin ( cnx ) and part of it stained clustered regions free of cnx ( fig . 3 b , squares , panels 1 and 2 ) but stained with an antibody to the golgi marker giantin ( fig note that all cells are labeled with the markers but only transfected cells are positive for anti - app . er and golgi localization is expected for app , a secretory protein synthesized in the er and transported for maturation along the secretory pathway ( weidemann et al . , 1989 ) . expression of sfv1 did not affect the intracellular localization of huapp , which colocalized in part with cnx ( fig . 3 b , panels 5 and 6 ) but was also in perinuclear clusters stained with giantin ( fig on the other hand , expression of sfv1-kdel caused retention of huapp in the er as shown by exclusion of app from giantin - containing structures ( fig . thus , we generated one intrabody ( sfv1 ) that associated with app in the er lumen and remained associated with it during transport along the secretory line . a second intrabody ( sfv1-kdel ) was equally efficient and fast to associate with app , but prevented exit of the target protein from the er . sfv1-kdel actually acted as a killer intrabody because it triggered slow disposal of newly synthesized app in a process that was delayed by mg132 , an inhibitor of the cytosolic proteasome ( fig . we next determined if intracellular association of sfv1 and of sfv1-kdel with app affected secretase - mediated endoproteolysis resulting in the shedding of the ectodomain of this type i membrane protein . cells coexpressing huapp and sfv1 or sfv1-kdel were metabolically labeled and chased for 10 min or 2 h. to analyze protein secretion , conditioned media were harvested , boiled in sample buffer and analyzed by sds page . because of cmv - driven expression , labeled sapp ( and sfv1 ) are the major secretory products of transfected hek cells . s1 ) as the labeled proteins are still folding in the er . after a 2-h chase , ectodomain shedding of labeled huapp had occurred in mock - treated cells ( fig . s1 ) , and coexpression of sfv1-kdel virtually abolished secretase - mediated release of huapp from cells ( fig . 4 a , lane 6 ) . app with the mutated 1 epitope served again as specificity control because coexpression of sfv1 did not reduce release of the rgapp ectodomain in the extracellular media compared with controls ( fig . consequences of sfv1 and of sfv1-kdel expression on app processing and release in the extracellular medium of the app ectodomain . ( a ) 15 l of conditioned medium ( total was 1.5 ml ) were boiled in sample buffer and analyzed in sds - page for determination of radiolabeled app - ectodomain ( sapp ) release from cells at 2 h ( lane 2 ) . coexpression of sfv1 reduces sapp release from cells ( lane 4 ) and sfv1-kdel virtually abolishes release of the app ectodomain ( lane 6 ) . ( b ) the same samples analyzed in a were immunoprecipitated with an app - specific antibody to demonstrate sapp : sfv1 association . only a fraction of secreted sfv1 coprecipitates with sapp ( compare the relative ratio sapp vs. sfv1 in a and b , lanes 4 ) . ( c ) coexpressing of sfv1 does not lower secretion of the rgapp ectodomain ( lanes 2 and 4 , cond . media ; lanes 2 and 4 , anti - app ) and sfv1 does not associate with rgapp as shown by lack of coprecipitation ( lane 4 , anti - app ) . 4 , a and c , lane 4 ) , whereas virtually no labeled sfv1-kdel was detected extracellularly ( fig . 4 a , lane 6 ) , as expected for a polypeptide carrying an er retention signal . part of the secreted sfv1 was associated and coprecipitated with the secreted huapp ( fig . 4 b , lane 4 ) but not with the control protein rgapp ( fig . 4 c , lane 8) . thus , the complex between the intrabody and huapp was maintained during secretion and after shedding . from the data described above , we concluded that uncomplexed sfv1 was also released from cells . this is of interest and might have beneficial consequences in case of a therapeutic application of our approach because recognition of the efrh epitope proved essential for antibodies able to prevent formation or to disassemble preexisting a plaques ( frenkel et al . , 1998 ; moreover , passive immunization may rapidly reverse behavioral deficits in mice ( dodart et al . , exerts protective and/or therapeutic activity awaits further experimentation in an animal model for the disease . 4 d , we proved that by associating close to the -secretase cleavage site , sfv1 inhibited by > 60% the processing and release of the swedish variant of app . addition of a sekdel - retention signal led to production of a killer intrabody that retained newly synthesized app in the er preventing secretase processing and eventually leading to slow degradation of app . the endoproteolysis of appswedish consists mainly in sequential cleavages by - and -secretase releasing the soluble app ectodomain and the toxic a peptide . a minor alternative endoproteolysis initiated by -secretase releases the soluble app ectodomain and the peptide p3 ( fig . , we next determined how coexpression of sfv1 or sfv1-kdel affected production of the app metabolites a and p3 . app - derived peptides produced by hek cells were identified independently by matrix - assisted laser desorption ionization time of flight mass spectrometry ( wang et al . , 1996 ; unpublished data ) . here , we determined the peptides by combining immunoprecipitation and immunoblotting with two well - characterized antibodies to the carboxy terminus of a ( paganetti et al . , 1996 ) and electrophoretic comigration with synthetic peptides ( fig . ( a ) western blot analysis of cell lysates with a carboxy - terminal app - antibody visualizes immature ( appi ) and mature ( appm ) full - length app at steady state . maturation of huapp and rgapp is not affected by sfv1 when compared with mock conditions . on the other hand , sfv1-kdel fully retains huapp in the appi form , but shows much lower affinity for the mutated efgh epitope of rgapp . ( b ) a and p3 were identified according to their electrophoretic mobility using synthetic peptides after immunoprecipitation and western blot analysis using two carboxy - terminal a-specific antibodies . secretase - mediated endoproteolysis of appswedish mainly results in production of a ( lane 1 ) . association of sfv1 close to the -secretase cleavage site substantially reduces production of a ( lane 2 ) , whereas sfv1-kdel virtually abolishes production of a ( lane 3 ) . the intrabodies have no effect on a generation when the rg mutant of app is expressed ( lanes 46 ) . overexposition of gel ( bottom ) better visualizes the metabolite p3 , and shows that sfv1 lowers a , whereas favoring -secretase  mediated cleavage resulting in the innocuous p3 peptide ( lane 2 ) . as demonstrated above using metabolic labeling , steady - state analysis of cell lysates by western blot confirmed that sfv1 did not interfere with app maturation ( fig . 5 a , lanes 1 and 2 ) . in contrast , sfv1-kdel strongly impaired app maturation ( fig . 5 a , lane 3 ) . overexpression of appswedish produced abundant a as expected for this substrate efficiently processed by -secretase ( fig . consistent with the effects observed for the ectodomain shedding , a generation was lowered by coexpression of sfv1 ( fig . in contrast , sfv1 did not affect cleavage of the rgapp carrying the point mutation in the sfv1 epitope ( fig . 5 , a and b , lane 5 ) and sfv1-kdel slightly affected rgapp maturation ( fig . 5 a , lane 6 ) and did not affect significantly the production of a ( fig . analysis of the ratio a ( mr = 4 kd ) versus p3 ( mr = 3 kd ) produced in mock - transfected cells and in cells expressing sfv1 ( fig . 5 b , longer exposure , lanes 2 and 3 ) revealed that sfv1 augmented to a certain extent p3 , i.e. , shifted app cleavage from the - to the -site . thus , association of intrabodies to a sequence adjacent to the -secretase cleavage site interfered with the amyloidogenic processing of huapp by the -secretase as determined by reduction of release from cells of the app ectodomain and a. in contrast , the innocuous cleavage by -secretase was slightly favored as shown by increased production of p3 . in summary , we engaged the cellular protein factory , the er , to produce therapeutic agents inhibiting production of a. by rapidly associating with newly synthesized app and by escorting it during intracellular transport , sfv1 shielded app from the pathologic -secretase  mediated cleavage . by appending an er - retention sequence , we generated an intrabody that retained app in the er strongly impairing a production . the use of specific intrabodies allowed intervention in an enzymatic process leading to a human disease by targeting the substrate , rather than the modifying enzyme whose activity is at the origin of the disease . by avoiding direct intervention on the enzyme , liabilities for side effects that may be triggered when the enzyme of interest is involved in other physiologic processes are lowered . proof of principle  for an intervention with highest selectivity in pathophysiologic processes in disease . hek cells were grown in dme supplied with 10% fcs , penicillin , and streptomycin ( gibco brl ) . for transfections , cells plated at 90% confluency were transfected with expression plasmids using lipofectamine 2000 ( gibco brl ) according to the manufacturer 's instructions for adherent cells . for cotransfections , plasmids for app and intrabody expression this dna ratio makes it unlikely that cells expressing only app are present in our cultures . 17 h after transfections cells were starved for 30 min in met / cys free medium , pulsed for 10 min with 150 ci / ml s - met / cys in starvation medium and chased with dme supplemented with 5 mm of cold met / cys . cell extracts were prepared by solubilization in 800 l / dish ice - cold 2% chaps in hepes buffer saline , ph 6.8 , containing 20 mm ice - cold n - ethylmaleimide and protease inhibitors ( hbs ) . postnuclear supernatants were prepared by a 10-min centrifugation at 10,000 g and analyzed by reducing sds - page as described in molinari et al . 12% polyacrylamide gels were used , with the exception of 8% gels in the endoh analysis ( figs . 2 and 3 ) and for fig.5 a. analysis of a and p3 in fig . 5 b was done on 13% tris / bicine gels ( klafki et al . , 1996 ) . immunoprecipitations were performed using antibodies and protein a beads ( sigma - aldrich ) and 14-h incubations in a cold room . the immunoprecipitates were washed three times with hbs/0.5% chaps and resuspended in sample buffer for sds - page . gels were exposed to biomax ( kodak ) films and scanned with an agfa scanner . for endoh treatment , app was immunoprecipitated from the cell extracts with specific antibody and denatured before incubation for 1 h at 37c with 1 mu of endoh ( roche molecular biochemicals ) . for immunoblotting , protein in cell extracts or immunoprecipitates were separated by reducing sds - page and transferred to immobilon p membranes ( millipore ) before blotting with specific antibodies ( paganetti et al . , 1996 ) . for indirect immunofluorescence , hek cells were plated for 24 h on alcian blue - treated glass coverslips , washed twice with pbs , and fixed at rt for 20 min in serum - free medium hepes containing 3.7% formaldehyde . cells were washed twice in serum - free medium hepes , twice with pbs and permeabilized with 0.05% saponin ( ps ) for 15 min . cells were incubated with primary antibody diluted in ps for 45 min , washed 15 min with ps , then incubated with conjugated secondary antibody diluted in ps for 30 min . microscopic images were collected at rt using a microscope ( model e-800 ; nikon ) equipped with a 60/1.4 plan apo objective , filter cubes for cfp and yfp fluorescence , and a camera ( q - imaging ) , controlled by openlab 3.5 software . s1 shows kinetics of app maturation and disappearance from cells ( intracellular ) and of secretion of soluble app ( sapp , extracellular ) . longer persistence of the mature form of app ( appm , top gel , panel on the right ) in cells expressing sfv1 is a further indication that intrabody coexpression interferes with shedding of the app ectodomain . this is proved by the lower amount of sapp in the extracellular media ( bottom panel compare labeled sapp in lanes 4 vs. 5 ) . hek cells were grown in dme supplied with 10% fcs , penicillin , and streptomycin ( gibco brl ) . for transfections , cells plated at 90% confluency were transfected with expression plasmids using lipofectamine 2000 ( gibco brl ) according to the manufacturer 's instructions for adherent cells . for cotransfections , plasmids for app and intrabody expression this dna ratio makes it unlikely that cells expressing only app are present in our cultures . 17 h after transfections cells were starved for 30 min in met / cys free medium , pulsed for 10 min with 150 ci / ml s - met / cys in starvation medium and chased with dme supplemented with 5 mm of cold met / cys . cell extracts were prepared by solubilization in 800 l / dish ice - cold 2% chaps in hepes buffer saline , ph 6.8 , containing 20 mm ice - cold n - ethylmaleimide and protease inhibitors ( hbs ) . postnuclear supernatants were prepared by a 10-min centrifugation at 10,000 g and analyzed by reducing sds - page as described in molinari et al . 12% polyacrylamide gels were used , with the exception of 8% gels in the endoh analysis ( figs . 2 and 3 ) and for fig.5 a. analysis of a and p3 in fig . 5 b was done on 13% tris / bicine gels ( klafki et al . , 1996 ) . immunoprecipitations were performed using antibodies and protein a beads ( sigma - aldrich ) and 14-h incubations in a cold room . the immunoprecipitates were washed three times with hbs/0.5% chaps and resuspended in sample buffer for sds - page . gels were exposed to biomax ( kodak ) films and scanned with an agfa scanner . for endoh treatment , app was immunoprecipitated from the cell extracts with specific antibody and denatured before incubation for 1 h at 37c with 1 mu of endoh ( roche molecular biochemicals ) . for immunoblotting , protein in cell extracts or immunoprecipitates were separated by reducing sds - page and transferred to immobilon p membranes ( millipore ) before blotting with specific antibodies ( paganetti et al . , for indirect immunofluorescence , hek cells were plated for 24 h on alcian blue - treated glass coverslips , washed twice with pbs , and fixed at rt for 20 min in serum - free medium hepes containing 3.7% formaldehyde . cells were washed twice in serum - free medium hepes , twice with pbs and permeabilized with 0.05% saponin ( ps ) for 15 min . cells were incubated with primary antibody diluted in ps for 45 min , washed 15 min with ps , then incubated with conjugated secondary antibody diluted in ps for 30 min . microscopic images were collected at rt using a microscope ( model e-800 ; nikon ) equipped with a 60/1.4 plan apo objective , filter cubes for cfp and yfp fluorescence , and a camera ( q - imaging ) , controlled by openlab 3.5 software . s1 shows kinetics of app maturation and disappearance from cells ( intracellular ) and of secretion of soluble app ( sapp , extracellular ) . longer persistence of the mature form of app ( appm , top gel , panel on the right ) in cells expressing sfv1 is a further indication that intrabody coexpression interferes with shedding of the app ectodomain . this is proved by the lower amount of sapp in the extracellular media ( bottom panel compare labeled sapp in lanes 4 vs. 5 ) .
endoproteolysis of the -amyloid precursor protein ( app ) by - and -secretases generates the toxic amyloid -peptide ( a ) , which accumulates in the brain of alzheimer 's disease ( ad ) patients . here , we established a novel approach to regulate production of a based on intracellular expression of single chain antibodies ( intrabodies ) raised to an epitope adjacent to the -secretase cleavage site of human app . 
 the intrabodies rapidly associated , within the endoplasmic reticulum ( er ) , with newly synthesized app . 
 one intrabody remained associated during app transport along the secretory line , shielded the -secretase cleavage site and facilitated the alternative , innocuous cleavage operated by -secretase . 
 another killer intrabody with an er retention sequence triggered app disposal from the er . the first intrabody drastically inhibited and the second almost abolished generation of a. intrabodies association with specific substrates rather than with enzymes , may modulate intracellular processes linked to disease with highest specificity and may become instrumental to investigate molecular mechanisms of cellular events .