alzheimer 's disease ( ad ) is characterized by amyloid deposition in senile plaques that are mainly composed of 40- and 42-mer amyloid -proteins ( a40 and a42 )     [ 1 , 2 ] . these proteins are secreted from amyloid precursor protein ( app ) by two proteases , - and -secretases . a42 plays a more critical role in the pathogenesis of ad than a40 because of its stronger aggregative ability and neurotoxicity . oxidative stress is believed to contribute to neuronal loss in ad     [ 46 ] ; one of the proposed mechanisms of a42-induced neurotoxicity is related to the radicalization at both tyr10 and met35 accompanied by the generation of hydrogen peroxide     [ 7 , 8 ] . on the other hand , soluble oligomeric assembly of a causes cognitive impairment and synaptic dysfunction in ad     [ 9 , 10 ] . our previous investigation using solid - state nmr together with systematic proline replacement proposed a toxic conformer with a turn at positions 22 and 23 in a42 aggregates and a nontoxic conformer with a turn at positions 25 and 26 ; the former showed a potent ability to aggregate , form oligomers , and exhibit neurotoxicity . the turn formation at positions 22 and 23 along with the neighboring -sheet structure in the toxic conformer of a42 brought tyr10 and met35 close together to generate the s - oxidized radical cation at met35 , the ultimate toxic radical species , through oxidation by the phenoxy radical at tyr10 produced by redox reactions     [ 7 , 12 ] . the mutations of a are concentrated at positions 21 , 22 , and 23 ; a21 g ( flemish ) , e22 g ( arctic ) , e22q ( dutch ) , e22k ( italian ) , and d23n ( iowa ) types . these mutations may play a pathological role in cerebral amyloid angiopathy ( caa ) or familial ad ( fad ) because these mutant proteins induced neuronal death in vitro more potently than wild - type a42     . thus , glu22 and asp23 in a are considered to be key residues for neurotoxicity through the formation of radicals . recently , mori and coworkers reported that a novel mutation , in which the glu-22 residue is defective ( e22 ) , induced ad - type dementia without amyloid deposition , and that in vitro e22-a42 favorably formed low - molecular weight oligomers to inhibit long - term potentiation ( ltp ) compared with a42      and to induce synaptic alteration . therefore , the effects of the deletion at glu22 on the secondary structure , formation of radicals , and neurotoxicity are interesting from the standpoint of discussing the role of the glu-22 residue of a42 in the pathogenesis of ad . this paper describes a comprehensive study of the aggregative ability , secondary structure , radical - generating activity , neurotoxicity in primary rat cortical neuronal cell cultures , and the inhibitory activity of ltp of both e22-a40 and e22-a42 . these results were compared with those of e22p - a42 with a turn at positions 22 and 23 . their molecular weights were confirmed by matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( maldi - tof - ms ) : e22-a40 ( m / z : calcd : 4201.76 ; found : 4201.56 [ m + h ] ) , e22-a42 ( m / z : calcd : 4386.00 ; found : 4385.98 [ m + h ] ) . the aggregation kinetics of each a ( 25  m ) was estimated with the sedimentation assay using hplc . the area of absorption at 220  nm was integrated and expressed as a percentage of the control . aggregative ability of each a ( 25  m ) was evaluated by the th - t method developed by naiki and gejyo . the measurement was performed on a multidetection microplate reader powerscan ht ( dainippon sumitomo pharma ) at room temperature , as described elsewhere     . gel electrophoresis using 1020% tricine gel ( invitrogen , carlsbad , ca ) and western blots analysis were carried out according to the manufacturer 's protocol . briefly , each a was dissolved in 0.1% nh4oh at 250  m . after a 10-fold dilution by 50  mm sodium phosphate containing 100  mm nacl at ph 7.4 , the resultant peptide solution ( 25  m ) the anti - n - terminus of a antibody , 82e1 , ( immuno - biological laboratories co. , ltd . , gunma , japan ) was used at 1  g / ml as the primary antibody . each a was dissolved in 0.1% nh4oh at 250  m and diluted 10 times with 50  mm phosphate buffer ( ph 7.12 ) . the procedure was described elsewhere     . a reliable method for estimating the ability of a ( 100  m ) to produce radicals using esr esr spectrometry was performed on an emx esr spectrometer ( bruker biospin k.k . , karlsruhe , germany ) at room temperature , as described elsewhere . to evaluate the neurotoxicity of a using an mtt assay , we used undifferentiated pc12 cells , which have the potential to differentiate into neural cells , are sensitive to a , and are generally used for detecting neurotoxicity as a neurotoxicity model . near - pure neuronal cultures were obtained from the cerebral cortices of fetal rats ( 1719 days of gestation ) as described     [ 21 , 22 ] . cultures were maintained in eagle 's mem supplemented with 10% heat - inactivated fetal bovine serum or 10% heat - inactivated horse serum at 37c in a humidified 5% co2 atmosphere . to prevent the proliferation of nonneural cells , 10  m cytosine -arabinofuranoside hydrochloride was added after 5 days of plating . in all experiments mature cells    used after 1113 days in vitro . animals were treated in accordance with the guidelines of the kyoto university animal experimentation committee and the guidelines of the japanese pharmacological society . each a was dissolved in 0.02% nh4oh at 200  m and diluted on ice immediately before treatment . after 48  hr treatment , neurotoxicity was evaluated by lactate dehydrogenase ( ldh ) release assay and mtt assay . field excitatory postsynaptic potentials ( fepsps ) were recorded from the ca1 region of rat hippocampal slices ( wistar rats , male , 6 weeks old ) by electrically stimulating the schaffer collateral     . hippocampal slices were soaked in e22-a40 , e22-a42 , and e22p - a42 solution [ 20  g/200  ml phosphate - buffered saline ( pbs ) ] before high - frequency stimulation ( 5 trains consisted of four 100-hz pulses with an intertrain interval of 200  ms ) . e22-a40 and e22-a42 were examined for their aggregative ability by a sedimentation assay : hplc analysis after centrifugation of each a solution . both e22-a40 and e22-a42 aggregated at a velocity similar to a42 , while a40 hardly aggregated even after 24-hr incubation ( figure 1(a ) ) . this suggests that the ability to form aggregates of both e22-a40 and e22-a42 would be comparable to that of a42 though soluble a assemblies ( oligomers ) could not be distinguished from high - molecular weight fibrils in this assay condition ( centrifugation : 20,000  g  10  min ) . in the th - t assay , which can estimate the -sheet structure in a aggregates in contrast , the maximum fluorescence of e22-a42 did not exceed that of a42 , although the velocity of e22-a42 showing fluorescence was slightly higher than that of a42 ( figure 1(b ) ) . these data suggest that the e22 mutation accelerates the aggregation of a.         western blotting was carried out to estimate accurately the oligomerization state of a.   e22-a42 formed trimers exclusively , but e22-a40 produced dimers immediately after incubation ( figure 1(c ) ) , as did the cases in the paper by tomiyama et al .     ; however , our th - t assay results do not coincide with their results     ; under tomiyama 's conditions , both mutants showed almost no fluorescence , even after 7 days . this discrepancy of the th - t test may be due to the different conditions to make aggregates , presumably resulting in the generation of oligomers containing a -sheet structure , as ishii and coworkers suggested     [ 24 , 25 ] . to investigate the secondary structure of e22-a40 and e22-a42 , their cd spectra were measured . in the control experiment using a42 ( figure 2(c ) ) , the positive peak at 200  nm and the negative peak at 220  nm gradually increased during the 48-hr incubation , suggesting that transformation of the random organization into a -sheet structure occurred , while a40 remained mainly random ( figure 2(a ) ) . in contrast , e22-a42 formed a -sheet - rich structure immediately after dissolution ( figure 2(d ) ) . the velocity of the transformation of e22-a40 was also higher than that of a42 ( figure 2(b ) ) . these results suggest that the e22 mutation induces -sheet transformation to form a oligomers under our condition . our previous studies suggested that the radical productivity of a42 mutants at position 22 such as e22p- , e22k- , e22q- , and e22g - a42 correlated with their aggregative ability and neurotoxicity . to investigate the effect of e22 mutation in as on the radical - generating activity , esr was measured using phenyl - n - tert - butylnitrone ( pbn ) as a spin - trapping reagent ( figure 3 ) . esr signals of e22-a40 were twice more potent than those of a40 , and e22-a42 also showed slightly stronger signals than a42 . the radical productivity of the e22-as correlated basically with their ability to form oligomers and a -sheet structure ( figures 1(c ) , 2 ) . having demonstrated that e22 mutation enhanced the -sheet structure and radical productivity , we assessed the effect of this mutation on the neurotoxicity in primary rat cortical neuronal cell cultures by ldh and mtt assay ( figure 4 ) . treatment of the neurons with 120  m of wild - type a42 for 2 days induced neurotoxicity in a dose - dependent manner in the ldh test ( figure 4(a ) , left ) , in which the released ldh of the damaged cells ( mainly neurons ) was measured in the medium . e22p - a42 with a turn at positions 22 , and 23 induced stronger damage to the neurons than wild - type a42 ; cell viability was less 40% at 20  m ( figure 4(a ) , right ) . on the other hand , the difference in cell viability between the vehicle and wild - type a42 did not reach a significant level in the mtt assay even at 20  m ( figure 4(b ) , left ) . the cell viability of e22p - a42 in mtt was also about 50% at 20  m . in the mtt assay , total cells containing neurons , astrocytes , and microglia damaged by as were counted . since the neurons are more sensitive to damage than astrocytes or microglia in the primary cell cultures , the neurotoxicity  estimated by the ldh test is often stronger than that evaluated by the mtt test . it is worth noting that e22-a40 and e22-a42 as well as a40 at 20  m failed to show neurotoxicity against the primary cultures both in the ldh and mtt tests ( figure 4 ) . these results are consistent with those reported by takuma et al . ; the neurotoxicity of e22-a42 was very weak against mouse neuroblastoma neuro-2a and human neuroblastoma imr-32 . in our mtt test using rat neuroblastoma pc12 cells , the ic50 of e22-a42 and wild - type a42 was 4.6  1.1  m and 0.65  0.11  m , respectively , showing that e22-a42 was significantly less toxic than wild - type a42 . the neurotoxicity of e22-a40 ( ic50 = 10  1.0  m ) was weak as expected , but slightly stronger than that of a40 ( ic50 = 20  1.0  m ) . selkoe and coworkers suggested that a dimers are the smallest synaptotoxic species inhibiting the ltp in the pathogenesis of ad and that plaque cores are largely inactive but sequester and release dimers     . reported the more potent inhibition of ltp by e22-a42 than by wild - type a42     . figure 5 shows that e22-a40 is not such a potent inhibitor of ltp as e22-a42 , whose inhibitory potency was stronger that of than wild - type a42 , as tomiyama et al . this coincides with the previous datas that the 42-mer a showed more potent neurotoxicity than 40-mer a     . notably , e22p - a42 , which can more readily form a toxic conformer with a turn at positions 22 and 23 than wild - type a42 , inhibited the ltp more strongly than e22-a42 at an almost undetectable level after 60  min ( figure 5(b ) ) . this suggests that the conformation at positions 22 and 23 of e22p - a42 might be similar to that of e22-a42 at positions 21 and 23 . the present results suggest that e22 mutation in a accelerates the transformation of a random form into a -sheet structure ( figure 2 ) and radical productivity ( figure 3 ) but does not increase neurotoxicity in primary rat cortical neuronal cell cultures ( figure 4 ) . e22-a42 synthesized in our laboratory showed the significant formation of oligomers ( figure 1 ) and synaptotoxicity ( figure 5 ) , as reported by mori and coworkers     . in addition , e22p - a42 inhibited ltp more severely than e22-a42 ( figure 5 ) . we previously reported that e22p - a42 , with a turn at positions 22 and 23 as a pro - x corner ( x : variable amino acid residue ) , could form significant oligomers   with a -sheet - rich structure      and radicals to result in potent neurotoxicity with the formation of radicals ; therefore , e22-induced synaptotoxicity might be in part related to turn - induced radical formation . this implies that conformational change in e22-a is similar to that in e22p - a42 , but is not the same since e22-a42 exhibited no neurotoxicity , unlike e22p - a42 and wild - type a42 . it should be noted that the effects of e22 mutation on the physicochemical properties of a40 are significantly higher than those of a42 . we previously reported a comprehensive study on the aggregation , neurotoxicity , and secondary structure of a mutants at positions 2123 ( a21 g , e22 g , e22q , e22k , and d23n )     . since a40 is secreted in neurons about nine times more abundantly than a42 , in some cases a40 may play a critical role in the pathogenesis of caa or fad . in addition , the e22 mutant of a40      as well as the caa- or fad - related a40 mutants at positions 21 , 22 , and 23 have been reported to be more resistant than wild - type a40 against degradation by insulin - degrading enzyme     ; however , it remains controversial whether e22 is a familial type of ad or ad - type dementia . mori , tomiyama , and coworkers implied the intracellular accumulation of a oligomers using cultured cells      and their own developed mouse model      with e22 mutation . quite recently , we proposed the involvement of a turn at positions 22 and 23 of a in intracellular amyloidosis . thus , the increase of radical productivity by e22 mutation is in good agreement with the turn - induced oxidative stress of a42 , presumably via the interplay between tyr10 and met35 . the deletion mutation of the residue at position 22 might promote tyr10 in close proximity to the sulfur atom of met35 , inducing the effective production of radicals . in summary , e22-a42 effectively induced the transformation of a random form to a -sheet structure and the formation of radicals accompanied with oligomerization . however , the molecular mechanism of the pathology of ad of e22-a42 might be different from that of wild - type a42 since e22-a42 showed more potent synaptotoxicity but weaker neurotoxicity than wild - type a42 .
oligomers of 40- or 42-mer amyloid -protein ( a40 , a42 ) cause cognitive decline and synaptic dysfunction in alzheimer 's disease . 
 we proposed the importance of a turn at glu22 and asp23 of a42 to induce its neurotoxicity through the formation of radicals . 
 recently , a novel deletion mutant at glu22 ( e22 ) of a42 was reported to accelerate oligomerization and synaptotoxicity .   to investigate this mechanism , 
 the effects of the e22 mutation in a42 and a40 on the transformation of -sheets , radical production , and neurotoxicity were examined . 
 both mutants promoted -sheet transformation and the formation of radicals , while their neurotoxicity was negative . 
 in contrast , e22p - a42 with a turn at glu22 and asp23 exhibited potent neurotoxicity along with the ability to form radicals and potent synaptotoxicity . 
 these data suggest that conformational change in e22-a is similar to that in e22p - a42 but not the same , since e22-a42 exhibited no cytotoxicity , unlike e22p - a42 and wild - type a42 .