alzheimer 's disease ( ad ) is a progressive neurological disorder resulting from the deposition of alzheimer -amyloid peptide ( a ) as senile plaques , the appearance of neurofibrillary tangles , and selective neuronal loss .
the most abundant forms of a are 40 and 42 amino acid residues long and referred to as a40 and a42 , respectively .  
the endocytic pathway has been implicated in the extracellular secretion of a40 and a42 [ 2 , 3 ] .
these peptides are derived from the endoproteolytic cleavage of the amyloid precursor protein ( app ) with -secretase , followed by -secretase .
the endosomal contents can be either secreted from the cell [ 79 ] or transferred to the lysosome .
the endosome has been found to be quite acidic ( ph 6 ) with the recycled endosome slightly less acidic ( ph 6.5 ) [ 11 , 12 ] .
exposure of a to endosomal ph conditions has been found to induce various conformational and oligomeric states [ 1315 ] .
many oligomeric forms of a have been proposed and characterized as intermediates in the pathway to forming the amyloid fibre .
some of these structures include trimers , pentamers , high molecular weight a-derived diffusible ligands ( addls ) , protofibrils , and fibrils [ 1621 ] .  
here we present a method for generating a mixed population of a conformations using model endocytic conditions . using this method ,
we demonstrate that when a is exposed to endosomal conditions for an extended period of time , the ability of the peptide to bind and internalize into living rat adrenal pheochromocytoma ( pc12 ) cells increases with time until it reaches a maximum beyond which its ability to interact with pc12 cells diminishes drastically .
a40 and a42 were synthesized and purified as described previously . before cleavage from the resin , the fluorophore , n-(9-fluorenylmethoxycarbonyl)-n-tetramethylrhodamine-(5-carbonyl)-l - lysine ( molecular probes , eugene , or ) ( abbreviated tmr ) ,
the crude peptides were purified by hplc using a superdex tricorn 10/300  gl peptide column ( amersham biosciences , piscataway , nj ) with 30  mm nh4oh running buffer . to maintain stock peptide solutions free from fibril seeds ,
solutions were stored at ph 10 and 4c immediately after chromatographic separation of monomeric peptides .
a preparations were never lyophilized , as this process may allow for seeds to form .
these solution conditions have been previously shown to maintain the monomeric state [ 16 , 23 , 24 ] .
peptide purity and identity was confirmed using both maldi mass spectrometry and amino acid analysis .
concentrations of stock peptide solutions were determined using amino acid analysis and confirmed by either tyrosine absorbance ( 275  nm ,  = 1390  cm  m ) or tmr absorbance for labelled peptides ( 550  nm ,  = 92000  cm  m ) .
at least three separate synthesized lots of a were used in this study and each displayed identical cell association rates when compared for quality assurance .
oligomeric samples were prepared by diluting stock a samples to 30  m with 30  mm nh4oh and reducing to ph 6 with 0.2  m hcl and incubating for zero ( a ) , 3 ( a ) , 24 ( a ) , or 90 hours ( a ) in the dark at 20c .  
hydrodynamic radius ( rh ) measurements were made at 20c with a dynapro dls instrument ( protein solutions inc . ,
peptide samples ( 30  m ) were reduced to ph 6 using 0.2  mm hcl , centrifuged at 12000  g   for 3 minutes and then rapidly added to a 1  cm path length cuvette and left in the instrument .
dls data was collected at various time points over 90 hours . particle translational diffusion coefficients were calculated from decay curves of autocorrelation of light scattering data and converted to hydrodynamic radius ( rh ) with the stokes - einstein equation .
histograms of intensity versus rh were calculated using dynamics data analysis software ( protein solutions inc . , piscataway , nj ) .
a samples ( 30  m ) were either not filtered or spin - filtered for 30 minutes at 14,000  g   using 10 , 30 , 100  kda ( amicon ultra cellulose kmwo ) , or 0.1  m ( amicon pvdf ) spin filters .
absorbance at 550  nm was collected on a molecular devices spectramax m5 ( molecular devices corp . ,
sunnyvale , ca ) and graphs were created normalizing the absorbance signal from each filtered sample to the corresponding unfiltered sample .
fluorescence measurements were obtained using 200  l of 30  m a samples , within a 96-well plate , after addition of 5-fold molar excess of thioflavin - t and incubation at room temperature for 30 minutes .
emission at 485  nm was collected using 440  nm excitation on a molecular devices spectramax m5 ( molecular devices corp . ,
pc12 cells were maintained in dmem / f12 containing 10% fetal bovine serum ( hyclone , logan , ut ) with 100  units / ml penicillin and 100  g / ml of streptomycin . to induce differentiation of pc12 cells and for cell imaging , they were plated at 2.2  10  cells / cm in lab - tech chambered cover glass chambers and suspended in phenol red free dmem / f12 containing n2 supplement and 10  ng / ml ngf .
cells were differentiated for 72 hours before media was replaced and peptide treatments ( final concentration 1.5  m ) were performed .
tmr - labelled a samples ( 6  m ) were prepared in phenol red free dmem / f12 media containing n2 supplement and 10  ng / ml ngf .
either fresh media or cultured supernatants , obtained from cell culture after 72 hours , were used . to avoid interference from cell culture components , molecular weights of tmr - labelled a were obtained by selective monitoring of tmr absorbance at 550  nm .
sedimentation experiments were performed at 20c on a beckman xli analytical ultracentrifuge using an an50-ti rotor .
molecular weights were calculated using beckman xli data analysis software in which absorbance versus radial position data were fitted to the sedimentation equilibrium equation using nonlinear least - squares fitting .
three - dimensional stacks of fluorescence micrographs were taken at 20c with a confocal laser - scanning system consisting of an lsm 510 zeiss meta nlo confocal microscope with a c - apo 40x water immersion objective ( numerical aperture 1.2 ) and hene laser with a 543  nm laser line .
the displayed images were captured using zeiss lsm image version 4 and prepared using imagej version 1.37v and represent a single cross - section through the cells .
differentiated pc12 cells were treated with 50  m monodansylcadaverine ( mdc ) for 20 minutes at 37c with 5% carbon dioxide . after inhibitor treatment , a ( final concentration 1.5  m ) was added to the media containing mdc and imaged after 4 hours at 37c .
a ( final concentration 1.5  m ) was also added to differentiated pc12 cells media for 4 hours at either 37c or 4c prior to imaging .
differentiated pc12 cells were first treated with a ( final concentration 1.5  m ) for 6 hours at 37c followed by treatment with 40  nm mitotracker deep red and 50  nm lysotracker green dnd-26 for 20 minutes .
after treatment , the media was exchanged with fresh phenol red free dmem / f12 media containing n2 supplement and 10  ng / ml ngf with 1.5  m dapi for nuclear staining .
cells were imaged using argon laser with 488  nm laser line for lysotracker , hene laser with 633  nm laser line for mitotracker , hene laser with 543  nm laser for tmr - labelled a , and a tunable chameleon laser at 730  nm for two - photon excitation of dapi .
2d histograms and correlation coefficients were determined using image j version 1.42q with colocalisation threshold plugin .
differentiated pc12 cell media was replaced with media containing 0.6 to 20  m a , a , a , or 10  m melitin as a positive control for 48 hours .
cell survival was quantified using the sulforhodamine b assay , and absorbance was measured at 560  nm using molecular devices spectramax m5 ( molecular devices corp . , sunnyvale , ca ) .
lc50 values were determined as the concentration of a required to kill fifty percent of the cells from an absorbance versus a concentration plot .
to study the interaction between a and live cells , we synthesized and fluorescently labelled a40/42 .
the synthesized a was maintained in solution from purification to storage and was never lyophilized , as these solution conditions are known to significantly reduce the formation of a aggregation seeds [ 16 , 23 , 24 ] .
we covalently attached tetramethylrhodamine ( tmr ) to the n - terminus of a via a flexible glycine linker to generate tmr - a. the n - terminus of a is highly accessible even in the fibril state [ 21 , 27 , 28 ] and attaching a fluorescent label to this site has been shown to neither alter its amyloidogenic properties [ 16 , 29 , 30 ] , nor its solubility behaviour [ 31 , 32 ] .
we have previously shown that treating cultured cells with 1.5  m monomeric tmr - a42 leads to the formation of visible aggregates on the surface of pc12 cells within one hour of treatment .
we initiated our current study by investigating whether a aggregation could occur in cell culture media alone . using analytical ultracentrifugation , we measured the molecular weights of a40 and a42 present in phenol red free cell culture medium , both freshly prepared and conditioned media taken from differentiated pc12 cells 3 days postdifferentiation .
following the addition of 6  m a to each medium and subsequent 24-hour incubation at room temperature , both media preparations were centrifuged to equilibrium at room temperature in an analytical ultracentrifuge in order to determine the molecular weight of a conformations .
the molecular weights of a40 and a42 from both media preparations were measured to be approximately 4103  da and 4425  da , respectively .
these values both correspond to the expected monomeric molecular weights of a , falling within the 95% confidence intervals of 36504570  da for a40 and 36504770  da for a42 .
thus , the aggregation of a seen by confocal microscopy apparently occurs only after interaction with the differentiated pc12 cells and not with cultured supernatants .
it should be noted that the concentration previously used to treat cells ( 1.5  m ) and the concentration used for ultracentrifugation ( 6  m ) are considerably lower than the reported 20 to 50  m range required for in vitro aggregation [ 3335 ] .
since a40 and a42 are generated through endoproteolytic cleavage [ 79 ] , and the ph of the endosome and recycled cellular vesicles is equivalent to ph 6 , we characterized the a conformations formed under endosome conditions .
a ( 30  m ) was reduced to ph 6 , and hydrodynamic radius calculations were collected after zero ( a ) , 3 ( a ) , 24 ( a ) , and 90 ( a ) hours using dynamic light scattering ( figure 1 ) .
the average hydrodynamic radius of a40 was found to increase from 2.0  nm at time zero ( a40 ) to 216  nm after 90 hours at ph 6 ( a40 ) .
a more striking increase was found with a42 , beginning with 1.7  nm at time zero ( a42 ) to 451  nm after 90 hours at ph 6 ( a42 ) .
for each of these samples , the development of increasingly higher ordered aggregates was observed over time and the samples that were treated for 90 hours contained particles over 1000  nm in radii . to further investigate the relative levels of peptide aggregation at endocytic ph , we filtered the a40 and a42 samples through various molecular weight cutoff ( mwco ) spin filters ( figure 2 ) .
approximately 67% of a40 , 60% of a40 , and 55% of a40 were recovered through the 10  kda mwco spin filter and approximately 80% were recovered through the 100  kda mwco filter , except for a40 with only 60% recovered . in contrast , only 55% of a42 , a42 , and a42 were recovered through the 10  kda filter and approximately 60% were recovered through the 100  nm filter .
these results indicate that the majority of peptide conformations present under these conditions were able to pass through a 10  kda molecular weight filter , but that just over 40% could not be recovered through the 100  nm filter for the a40 , a42 , a42 , and a42 samples .
we also used thioflavin - t to assess the time - dependence of the extent of amyloid fibril formation at endocytic ph .
thioflavin - t is a dye known to shift its fluorescence from 430  nm to 490  nm upon binding specifically to the cross--structure of amyloids but not to monomeric or small oligomeric complexes [ 36 , 37 ] .
we observed enhanced thioflavin - t fluorescence at all time points ( figure 3 ) ; however , thioflavin - t bound most strongly to a40 , a42 , and a42 .
the high thioflavin - t binding to a samples suggests that these late stage a conformations are the most aggregated . using confocal microscopy
we have previously shown that monomeric a42 associates with cells more rapidly than a40 , with significant staining observable after six hours of treatment . to determine whether the aggregation state of a affects cell association , we exposed each of the a40 and a42 samples to differentiated pc12 cells and monitored the kinetics of association by confocal microscopy . upon treating cells with a40 , the cell surface association
moreover , a40 was observed to significantly internalize into these cells after only 6 hours , whereas significant internalization of a40 was only visualized around 24 hours ( figure 4 ) .
similarly , a42 internalized into differentiated pc12 cells after only one hour of treatment , whereas a42 treatment only became observable at 24 hours ( figure 4 ) .
interestingly , when differentiated pc12 cells were treated with late - stage a40 or a42 , very few cells underwent internalization or even exhibited cell surface interaction with these aggregated peptide forms ( figure 4 )
. to illustrate the contrast between these treatments , we collected the three - dimensional image slices through a cell from the base to the apex for the 6-hour treatment with a42 , a42 , and a42 ( figure 5 ) .
a42 was only observed around the periphery of the cell , whereas some a42 was located inside the cell . by contrast
, a42 treatment seemed to localize to extracellular regions and did not produce the punctate pattern as observed with the a42 sample . to quantify the frequency of cells that exhibited peptide internalization , we randomly selected five fields of view from at least three separate 6-hour treatment experiments and plotted the percentage of cells having internalized a ( figure 6 ) .
was found to have a large number of aggregates with hydrodynamic radius over 1000  nm ( figure 1 ) and were found to significantly bind thioflavin - t ( figure 3 ) , then these aggregates may favour self - association over cell association . to determine if the internalization of endocytic a is mediated through cellular processes , such as receptor - mediated endocytosis or through direct peptide - mediated processes like membrane pore formation , we monitored the effects of temperature and monodansylcadaverine ( mdc ) on internalization .
mdc is a known inhibitor of receptor - mediated clathrin - dependent endocytosis [ 38 , 39 ] . at physiological temperature ( 37c ) , after a 4-hour treatment of cells with a40 and a42 , internalization was observed ( figure 7 ) . at 4c membrane vesicle formation
when a40 or a42 association with differentiated pc12 cells was monitored at 4c , none of the cells were found to have internalized these peptides ( figure 7 ) .
similarly , when differentiated pc12 cells were treated with mdc , neither a40 nor a42 were observed within the cells .
these observations indicated that a was internalized through a cell - directed import mechanism , rather than an independent penetration route through the cell membrane .
the location of deposited intracellular a40/42 was examined using intracellular organelle markers in differentiated pc12 cells .
cells treated with a40 and a42 for 6 hours were visualized using lysotraker green dnd-26 for the lysosome , mitotracker deep red for the mitochondria , and dapi for nucleus staining ( figure 8) .
the staining pattern of each cellular organelle marker in the same image plane as the a40 and a42 treatment was determined ( figure 8) and quantified ( figure 9 ) . only the signal from the lysosome marker costained with both internalized a40 and a42 . the red signal from the peptides costained well with green signal from the lysosomes , resulting in the yellow signal in the merged image . when quantified , 71% of the lysosome signal intensity colocalised with the a40 channel and 80% with the a42 channel ( figure 9 ) .
whereas only 9% of the mitrochondria signal intensity colocalised with a40 channel and less than 1% with the a42 channel ( figure 9 ) .
the intensity of both a40 and a42 fluorescence signals was found to increase in the lysosomes over time , which may reflect the accumulation of a40/42 in the lysosome .   to determine the relative toxicity of these a conformations ,
a cytotoxicity assay was performed where differentiated pc12 cells were treated with various peptide concentrations for 48 hours ( figure 10 ) .
we calculated the lethal concentration at which 50% of cells were killed ( lc50 ) , for each of the a conformations .
we found that a40 was moderately more toxic than a40 , with a lower lc50 value ( figure 10 ) .
surprisingly , a40 was also found to have a lower lc50 value compared to a40 .
a42 was also found to be moderately more toxic than a42 , whereas a42 had a much higher lc50 value compared to a42 ( figure 10 ) .
we have developed a method to produce a collection of a conformations that differ in their extent of aggregation and investigated the interaction between these states of a40 and a42 and differentiated pc12 cells .
others have described the methods to isolate individual soluble oligomeric forms of a , using various chemical reagents and protocols [ 4144 ] .
in addition , purified oligomeric a molecules from cell culture [ 45 , 46 ] or transgenic mice   have also been monitored .
these purified sources of oligomeric a offer great potential in understanding the progression of a aggregation ; however , they can not be directly visualized over the time course of their effects on cells during maturation from earlier to later conformations .
since it is well established that a monomers can oligomerize under the physiological ph of the endosome [ 1315 ] , and a is formed through endoproteolytic cleavage , we have utilized this condition to determine which conformations are present and how these mixed conformations interact with differentiated pc12 cells .
it is known that the extent of oligomerization / fibrillation is very dependent on experimental conditions .
have indicated that a can be induced to form fibrils via dilution from 100  mm naoh to neutral ph in the presence of 10  mm hepes/100  mm nacl buffer , while dilution into phosphate buffered saline results in oligomer formation . in our experiments , the peptide was diluted from 30  mm ammonium hydroxide ( ph 10 ) to ph 6 with final condition of 1  mm ammonium chloride as the only additional chemical .
we do note that the normal human physiological concentration of ammonium chloride in blood and cerebrospinal fluid is approximately 20 to 50  m [ 49 , 50 ] , and that hyperammonemia has been linking to alzheimer type ii astrocytosis [ 51 , 52 ] .  
when extracellular monomeric a associates to the surface of cells , we speculate there are three possible outcomes : ( 1 ) it can act as a stable template to allow further a aggregation ; ( 2 ) the peptide can penetrate through the cell membrane depositing in the cytoplasm ; or ( 3 ) the peptide may be internalized into the cell within endocytic vesicles , which would result in a reduction in the surrounding ph . in the third case
, the endocytic vesicles containing a can theoretically be recycled back to the cell surface or directed to the lysosome where a further reduction in ph would occur .
we have previously visualized oligomeric a on the surface of neuronal cell lines , and here sought to determine first whether this aggregation occurred prior to cell surface deposition in the cell culture mediaor are aggregation and cell surface binding concomitant and linked processes.using ultracentrifugation analysis of a preparations either in freshly prepared cell culture media or in conditioned media removed from cultured cells after 3 days , we did not observe any conformation larger than the monomer .
these results indicate that components in our cell media or secreted factors from cultured cells are not responsible for the observed a aggregation present on the surface of neuronal cells . after adding monomeric a to cells
, we noted a maturation time for the visual appearance of a on the cell surface and in the cell interior ( figure 4 ; ) .
we postulate that the peptides on the cell surface might undergo a series of ph reductions through endocytic recycling , before becoming visible punctae on and inside the cell . since ph 6 is approximately endocytic ph , we characterized the kinetic effects of this condition on peptide conformations . exposing a40 for up to 24 hours at ph 6
, we observed an increase in the average hydrodynamic radius from 2.0  nm ( a40 ) to 183  nm ( a40 ) ( figure 1 ) .
in addition , approximately 60% of these peptides were recovered through a 10  kda mwco filter and 80% were recovered through a 100  kda mwco filter ( figure 2 ) , and only minor thioflavin - t binding was found ( figure 3 ) . following exposure of up to 24 hours at ph 6 , a42 had an increase in the average hydrodynamic radius from 1.7  nm ( a42 ) to 246  nm ( a42 ) ( figure 1 ) , approximately 55% were recovered through 10  kda mwco filter and only 60% were recovered through 100  nm filter ( figure 2 ) and more significant thioflavin - t binding was measured ( figure 3 ) .
both a40 and a42 treated for 90 hours at ph 6 were observed to have large average hydrodynamic radius particles , with 40% of these samples being withheld by a 100  nm filter and both displayed significant thioflavin - t staining . also , these early - and late - stage a samples were observed to have an ensemble of a conformations ( figure 1 ) .
the interactions of these early - and late - stage a conformations were monitored with differentiated pc12 cells . whereas late stage a40 and a42 exhibited very few interactions with cells ( figures 4 , 5 ) and internalized at a very low frequency ( figure 6 ) , early stage a40 and a42 interacted with cells rapidly ( figure 4 ) and internalized into the majority of the cells present ( figure 6 ) .
interestingly , a40 showed a more rapid cellular association and internalization compared to a40 ( figure 4 ) .
similarly , a42 was found to internalize more rapidly than a42 , indicating that a specific conformation may play a role in cell binding and internalization .
furthermore , our model of endocytic a toxicity ( figure 10 ) is in agreement with previous findings utilizing chemically - produced oligomers of a which showed that a fibrils were less toxic than soluble oligomers [ 41 , 53 ] .
our results with a are also in agreement with studies that have indicated that regions with large amyloid plaques do not correlate directly with regions of significant neuronal loss [ 54 , 55 ] .
for example , the peptide sequences corresponding to tat ( 4860 ) , penetratin , and oligoarginine are known to internalize into live cells .
cellular import of tat ( 4860 ) and penetratin was shown to be temperature dependent , indicating the possible role of endocytosis for internalization [ 56 , 57 ] .
in contrast , oligoarginine containing c - terminal tryptophan was shown to follow a nonendocytic pathway , independent of energy requirements and temperature .
another type of internalization process has been studied in the bacterium clostridium septicum , whose alpha toxin contains functional domains responsible for oligomerization and cellular pore formation . using specific domains that bind cell surface receptors ,
alpha toxin monomers oligomerize to form pores in human cells and thus impose direct entry . by exposing a to endosomal conditions
, we were able to observe internalization of a40/42 , which was inhibited at 4c or with the endocytosis inhibitor mdc ( figure 7 ) . throughout the course of our confocal experiments
, we did not observe any disruption of the cell membrane , which would have been expected if membrane channels or pores were being formed by a. thus , the structural conformation of a that was observed to internalize into cells seems to follow a cell - mediated import mechanism .
our results with the various a conformations correlate directly with previous reports that indicate the deposition of a in the lysosome [ 15 , 59 , 60 ] and with the previous study that internalized a can persist undegraded for days . from our findings taken together with previous studies , we present the following model for the interaction of a with neuronal cells ( figure 11 ) .
as monomeric a binds the cell surface , this event could lead to a conformational change allowing for the further catalytic aggregation of a. recently , it has been shown that oligomeric a binds to gm1 ganglioside and alters physical properties of the plasma membrane which stimulates the amyloidogenic processing pathway of app [ 62 , 63 ] .
a that is bound to the cell surface may become internalized and recycled , allowing for acidification and further aggregation , which in turn may stimulate additional a generation .
once the surface a reach some specific structural form , they may be internalized and directed to the lysosome for potential degradation . however , the reduced ph of the lysosome may instead cause further aggregation and persistence of a , resulting in lysosomal overload and cell death
. the recently reported high turnover rate of monomeric a [ 64 , 65 ] and the formation of large aggregate pools of a may indicate that the human body uses these mechanisms to sequester and remove the toxic a oligomers from neuronal cells . with age , the turnover rate for
monomeric a may slow down , which could then result in the initiation of our proposed mechanism and may contribute to the late onset of alzheimer 's disease .
it is also possible that endocytosis of a and lysosomal targeting are mechanisms by which the cell clears a aggregates from the cell surface .
also , certain large aggregates of a may just be too large for the cell to internalize , and they may represent the precursor for extracellular amyloid formation .
our study shows that using a simple method to generate various a conformations , the rates of cellular interaction and targeting can be followed with live cell cultures . using this model
, we found that early endocytic conformations , rather than highly aggregated late forms , serve as the major contributors to rapid cell internalization .
the mechanism of internalization likely involves a cell surface receptor - mediated process instead of peptide - mediated direct entry , resulting in accumulation in the lysosome .
this method allows for conformation - specific therapeutics and conditions to be screened with live cells , circumventing the need to purify specific a conformations .
 the amyloid peptides , a40 and a42 , are generated through endoproteolytic cleavage of the amyloid precursor protein . 
 here we have developed a model to investigate the interaction of living cells with various forms of aggregated a40/42 . 
 after incubation at endosomal ph 6 , we observed a variety of a conformations after 3 ( a3 ) , 24 ( a24 ) , and 90 hours ( a90 ) . both a4224 and a4024 
 were observed to rapidly bind and internalize into differentiated pc12 cells , leading to accumulation in the lysosome . 
 in contrast , a40/4290 were both found to only weakly associate with cells , but were observed as the most aggregated using dynamic light scattering and thioflavin - t . 
 internalization of a40/4224 was inhibited with treatment of monodansylcadaverine , an endocytosis inhibitor . 
 these studies indicate that the ability of a40/42 to bind and internalize into living cells increases with degree of aggregation until it reaches a maximum beyond which its ability to interact with cells diminishes drastically . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion