Conformational targeting of intracellular Aβ oligomers demonstrates their pathological oligomerization inside the endoplasmic reticulum

Aβ oligomers (AβOs) are crucially involved in Alzheimer’s Disease (AD). However, the lack of selective approaches for targeting these polymorphic Aβ assemblies represents a major hurdle in understanding their biosynthesis, traffic and actions in living cells. Here, we established a subcellularly localized conformational-selective interference (CSI) approach, based on the expression of a recombinant antibody fragment against AβOs in the endoplasmic reticulum (ER). By CSI, we can control extra- and intracellular pools of AβOs produced in an AD-relevant cell model, without interfering with the maturation and processing of the Aβ precursor protein. The anti-AβOs intrabody selectively intercepts critical AβO conformers in the ER, modulating their assembly and controlling their actions in pathways of cellular homeostasis and synaptic signalling. Our results demonstrate that intracellular Aβ undergoes pathological oligomerization through critical conformations formed inside the ER. This establishes intracellular AβOs as key targets for AD treatment and presents CSI as a potential targeting strategy.

T he proteolytic processing, misfolding and self-aggregation of several proteins are often associated with pathological conditions and conformational diseases. However, the identification, study and targeting of different conformations and multimeric states of a given protein in the complex context of subcellular compartments/microdomains of living cells remains a big challenge.
A most relevant example of such a challenge is represented by the amyloid-b (Ab) peptides, crucial players of Alzheimer's disease (AD) pathogenesis [1][2][3][4] . In living cells, differently than in a test tube containing only synthetic peptides, the Ab species are generated from the amyloid precursor protein (APP) by a complex process of regulated intramembrane proteolysis (RIP) 5,6 , and undergo a process of misfolding and aggregation, whose mechanism and subcellular localization(s) are still debated 7 . In particular, along the aggregation pathways, naturally occurring Ab oligomers (AbOs) are still considered mysterious entities in terms of molecular and structural composition and activity 8 , even though they are recognized as the most neurotoxic proteinaceous forms in AD 8,9 . As an additional level of complexity, APP-RIP generates different proteolytic fragments and shares the secretases (both a, b and g) with numerous different substrates 5 . Thus, the cellular biochemistry of Ab is representative of two general mechanisms operating in living cells and organisms, namely RIP and amyloid formation (proteins enriched in cross b-sheet and prone to self-aggregation) and represents not only a pathologically relevant target, but also an ideal test case for the development of new approaches for studying these processes in cells.
Conformational-sensitive antibodies are important tools for analysing amyloid assembly states and dynamics 10 . In particular, recombinant antibody fragments can be exploited as intracellular antibodies (intrabodies) for a subcellular-localized interference to block or modulate the function of target molecules 11,12 . In principle, if the intrabodies are intrinsically equipped with conformational-sensitive binding properties, they could be exploited for interference studies not currently feasible with nucleic acid targeting methods (that is, RNA interference or gene knockout), that can silence entire gene products (that is, APP or RIP machinery protein components) but not peculiar posttranslational modification products (such as AbOs). Furthermore, even though new chemical modulators and inhibitors for APP-RIP or for Ab assembly are intensively studied, their molecular selectivity and their precise subcellular delivery and actions remain not easy to control. In the current state of the art, while several conformation-and oligomeric-specific antibodies targeting the Alzheimer's AbOs have been developed 8 , they are largely not exploitable for subcellular targeting and intracellular functional studies in living cells.
We generated, by an in vivo intracellular selection in yeast cells, a panel of conformation-sensitive antibody fragments selectively recognizing AD-relevant AbO conformers 13 ideal for the expression (as genes) in mammalian cells, as intrabodies targeted to different subcellular compartments. Here we expressed the anti-AbO single-chain antibody fragment (scFv) A13 (ref. 13) as an intrabody, with the aim of intercepting AbOs at subcellular sites of their putative formation, and of attempting their functional silencing. In this way, we established a new experimental paradigm of subcellular-localized and conformational-selective interference (CSI). The intrabodybased CSI besides providing a novel approach to selectively control biologically-active AbO conformers in living cells, allows a new dissection of cellular mechanisms of AbO generation, trafficking and actions. Indeed, by exploiting CSI, we demonstrate that intracellular Ab can oligomerize into pathological forms, through critical conformations formed inside the endoplasmic reticulum (ER). The anti-AbOs intrabody selectively intercepts critical AbO conformers and controls their 'toxic' assembly in the ER, without interfering with the complex processes of maturation and processing of APP. Remarkably, the pool of targeted AbO conformers are involved in the deregulation of two independent pathways of cellular homeostasis and synaptic signalling. Our overall results firmly establish the ER as the site of formation of critical Ab conformers and validate ER-formed intracellular AbOs as a key target for AD. For these reasons, the anti-AbOs CSI can be exploitable for in vivo therapeutic applications as well as to improve our understanding of the molecular and cellular processes of AD pathogenesis, thereby uncovering new targets for drugs development. Indeed, one of the earliest events in AD pathogenesis seems to be the intraneuronal Ab oligomerization 14,15 and the synaptic targeting of AbOs 3, 16 and current studies on the intracellular Ab generation and oligomerization do not offer yet a common and conclusive opinion, also for the lack of adequate experimental tools of study.

Results
The anti-AbO scFvA13 expressed as an intrabody in mammalian cells. This study is based on scFvA13, a recombinant antibody fragment generated by a direct intracellular selection of functional intrabodies in yeast cells against the in vivo misfolded antigen Ab 13 . The scFvA13 selectively recognizes conformational Ab epitopes, as attested by the preferential binding in vitro to synthetic oligomeric assemblies, such as the amyloid-derived diffusible ligands (ADDLs 17 ), but not to the monomeric or fibrillar states 13 . Furthermore, the oligomeric-specific scFvA13purified protein was characterized for its neutralizing properties against synthetic ADDLs 13 or against endogenous Ab species 18 as well as for the recognition of pathological AbO deposits in the brain of a mouse model of familial AD (fAD) 19 and of other neurodegeneration models 20 .
The conformation sensitivity of scFvA13 is accompanied by a sequence specifity for Ab 13 , as also attested by dot blot (DB) assay (Fig. 1a) and enzyme-linked immunosorbent assay (ELISA) ( Supplementary Fig. 1), which show the lack of immunoreactivity with other proteins forming oligomers, such as a-Synuclein and lysozyme, at variance with the generic anti-oligomer A11 (ref. 21) (Supplementary Fig. 1).
Here, we tailored the scFvA13 for intrabody expression in subcellular compartments of living cells 12 , to target critical conformations of Ab oligomeric assemblies naturally occurring inside cells and to intercept AbOs where they are formed in the cell.
As a transmembrane protein, APP is cotranslationally inserted in the membrane of the ER, where it traffics through the other subcellular compartments of the secretory pathway; however, the role of the ER in the APP-RIP and Ab generation, but especially in Ab misfolding and oligomerization, remains to be deciphered (Fig. 1b). We exploited the scFvA13 intrabody, to ask the question as to where and when, during the APP maturation and trafficking, is Ab formed and oligomerized, starting from the early secretory compartment. To this aim, the scFvA13 was targeted to the ER, through a C-terminal KDEL fusion allowing its ER retention and its retrieval from the cis-Golgi, through the ER-Golgi intermediate compartment 12,22 . In detail, the cDNA of scFvA13-KDEL was stably expressed in the 7PA2 fAD cell line 23,24 (CHO cells overexpressing fAD human V717F APP mutant, Supplementary Fig. 2), which represents a widely used model of Ab oligomerization and the best characterized source of natural, pathologically relevant and bioactive AbOs 3,8,9,[24][25][26] .
Furthermore, the scFvA13-KDEL intrabody was expressed in CHO transfectants expressing wild-type human APP referred to as 7WD4 (ref. 23) ( Supplementary Fig. 2), which show much lower rates of Ab production and oligomerization in comparison with 7PA2 (Fig. 1c,d) and in human neuroblastoma SHSY5Y cells, expressing only the endogenous APP, that show effective RIP and negligible Ab oligomerization.
Measuring Ab and AbOs biochemical patterns in 7PA2 cells. The detection of the pathologically relevant Ab and AbOs biochemical patterns in 7PA2 cells is essential prerequisite to study the effects of the subcellular targeting of scFvA13 intrabody.
An Ab immunoproteomic analysis, based on the use of anti-Ab (monoclonal antibody (mAb) 6E10 and 4G8) as capturing antibodies in SELDI-TOF mass spectrometry (MS) 27 , allows directly revealing the complex profile of Ab monomers and fragments secreted by cells in the conditioned medium (CM). In this way, by comparing the 7PA2 and 7WD4 cells (respectively expressing the V717F mutant or the wild-type human APP), we measured in 7PA2 CM higher levels of secreted Ab peptides ( Fig. 1c) and, most importantly, a fAD-related higher Ab1-42:Ab1-40 ratio (Fig. 1c, Supplementary Table 1). Of note, we observed an overall unbalanced proportion of carboxy-and amino-terminally truncated Ab peptides (Supplementary Table 1) in 7PA2 CM, similar to Ab profiles in cerebrospinal fluids from human fAD 28 and representative of a fAD-linked APP misprocessing ( Supplementary Fig. 2).
The 6E10 þ 4G8 SELDI-TOF MS method reveals in 7PA2 CM, the complex profile of Ab monomeric peptides and of Ab truncated forms but does not detect immunoproteomic peaks corresponding to AbOs. Thus, we investigated the patterns of soluble Ab also by other approaches, such as western blot (WB) or DB. The WB analysis of straight (non-concentrated) 7PA2 CM shows that the Ab oligomeric bands are a small fraction (o20%) of total soluble Ab species, which mainly run in the gels as monomeric bands (Supplementary Fig. 3). Accordingly, the biologically relevant LDS/SDS-stable Ab dimers and trimers were previously recognized in 7PA2 CM as a small pool of total soluble Ab species only upon immunoprecipitation enrichment 23,24 .   Monomers and oligomers of human a-synuclein, human Ab1-42 and lysozyme were assayed by DB with scFvA13 as primary antibody (panel on the left). The scFvA13 is revealed through its C-terminal V5 tag. As a control, the same samples were respectively analysed through a mAb anti a-synuclein (a-syn), the mAb anti-Ab 6E10 and by Ponceau S (panels on the right). The recognition of scFvA13 is selective and specific for oligomers of human Ab1-42, but not for other proteins, as also shown by ELISA in Supplementary Fig. 1. (b) Cartoon illustrating APP-RIP (grey-light blue-shaded box), Ab misfolding (green-red-shaded box), Ab oligomerization (box with a red frame) and trafficking in different (sub)cellular sites (also presumptive, indicated by the question marks) as well as the localization of intrabody scFvA13-KDEL in the ER. a-, band g-secretase are represented by yellow, orange and blue arrows, respectively and Ab misfolding by a change of a green fragment into a red hairpin. Some FAD mutations, such as the hAPPV717F brought by the APP-RIP cell model 7PA2, alter the g-secretase cleavage determining pathological and unbalanced patterns of Ab peptides ( Supplementary Fig. 2). (c) Immunoproteomic (anti-Ab 6E10 þ 4G8 SELDI-TOF MS) profiles of Ab monomers and Ab truncated forms secreted by 7WD4 (CHO-hAPPwt) and 7PA2 (CHO-hAPPV717F) cells. Here, in a new way, we were able to detect LDS/SDS-stable Ab dimers and trimers by a direct WB analysis of 7PA2 CM ( Supplementary Fig. 3), with a major sensitivity if the CM is previously subjected to concentration steps. We demonstrated that the dimeric and trimeric bands are Ab-specific being selectively abolished by cell pretreatment with a g-secretase inhibitor (L-685,458) 29 and being recognized by independent anti-Ab antibodies (6E10 and 4G8) raised against different Ab epitopes ( Supplementary Figs 4,5). Finally, we established a new AbO-specific DB assay, based on the use of scFvA13, and of the other conformation-sensitive anti-AbO scFvIm3 (ref. 13), as primary antibodies ( Supplementary  Fig. 6). The scFv-based DB can selectively detect both extracellular and intracellular Ab conformers ( Supplementary  Figs 6,7). Of note, the DB approach is essential to analyse the intracellular pools of AbOs, hardly detectable by WB both for sensitivity limits and for the main cross-reactivity of the anti-Ab 6E10 and 4G8 with a large number of intracellular APP fragments ( Supplementary Fig. 7a). On the other hand, the anti-Ab WB is a good method to detect Ab monomeric bands, both in lysates and microsomes ( Supplementary Fig. 7b), which are representative of the largest pool of intracellular Ab species (both monomers and LDS/SDS unstable oligomers).
Targeting the scFvA13 intrabody to the ER. The scFvA13 intrabody was targeted to the early secretory pathway exploiting a β-Actin 94 kDa (Grp94) 78 kDa (Grp78=BiP) 55 kDa . The intrabody was detected by indirect immunofluorescence through its C-terminal V5 tag by using an anti-V5 as a primary antibody (left panels). The middle panels show, from the top the ER marker calnexin and the Golgi marker protein 58K. The panels on the right show the respective merged images (scFvA13-KDEL with calnexin or 58K) with a major co-localization of scFvA13-KDEL intrabody with calnexin, and a partial co-localization of scFvA13-KDEL intrabody with 58K, indicating the preferential KDEL-mediated ER localization of the intrabody. Scale bar, 10 mm, magnification Â 63. the ER retention/retrieval KDEL system. The subcellular localization of scFvA13-KDEL was confirmed by indirect immunofluorescence to be mainly in the ER and partially in the cis-Golgi (Fig. 2a). The effective enrichment in the ER was further demonstrated through the WB analysis of different subcellular vesicles derived by microsome fractionation of 7PA2 scFvA13-KDEL transfectants (Fig. 2b). Consistent with its retention in the ER, the scFvA13-KDEL was found not to be secreted in the extracellular medium (Fig. 2c   other cell lines (as measured by the unchanged levels of BiP) (Fig. 2d).
The effect of scFvA13-KDEL on the extracellular pools of Ab and AbOs. To better study the effects of scFvA13-KDEL on the biochemistry of Ab, AbOs and APP, we first established 7PA2 cells stably expressing the intrabody scFvA13-KDEL, referred to as 7PA2-A13K cells.
The comparative WB analysis of straight CM from 7PA2 and 7PA2-A13K cells does not reveal significant differences in the levels of Ab monomeric bands, which are representative of the largest amount of soluble Ab species (monomers and LDS/SDS unstable oligomers) ( Supplementary Fig. 3).
Instead, the amount of LDS/SDS-stable Ab dimers and trimers secreted from 7PA2 cells (detected in concentrated CM) is significantly reduced (B60% in 4G8-WB and B70% in 6E10-WB) by the expression of the scFvA13-KDEL intrabody ( Fig. 3a; Supplementary Fig. 5). Similarly, the secreted oligomeric conformers, measured by scFv-DB assay in CM, are significantly reduced (70-80%) in 7PA2-A13K ( Fig. 3b; Supplementary Fig. 6). Of note, the similar ratio of AbOs decrease, measured by WB (selective for Ab dimers and trimers with respect to the monomeric Ab bands) and by DB, further attests the oligomeric specificity of the scFv-based DB assay (in addition to the experimental control of g-secretase inhibitor treatment, Supplementary Fig. 6).
In contrast to the reduction of AbOs measured in the CM of 7PA2-A13K cells, the immunoproteomic analysis of 7PA2-A13K CM (by 6E10 þ 4G8 SELDI-TOF MS) shows a significant increase of Ab monomeric species, with respect to 7PA2 CM (Fig. 3c). Relevantly, the expression of scFvA13-KDEL does not alter the profile of g-secretase-generated Ab products (species of 37-42 amino-acid length) in 7PA2 cells, but leads, instead, to a similar increase in the absolute amounts of all Ab species (Fig. 3c, Supplementary Table 2).
Thus, the intracellular expression of scFvA13-KDEL determines a selective and significant reduction of extracellular AbOs accompanied by an opposite increase of secreted Ab monomers and truncated forms. While it is difficult to establish a precise quantitative balance between these two independent Ab pools, our results show that their variations are likely mutually complementary.
Modulation of the intracellular Ab and AbOs pools by the scFvA13-KDEL. We evaluated the effects of the scFvA13-KDEL expression on the intracellular pools of Ab and AbOs by WB and DB analyses. The anti-Ab WB analysis allows concluding that the intracellular Ab monomeric bands are not significantly changed in 7PA2-A13K lysates (Fig. 4), neither in total microsomes (Supplementary Fig. 7b), while the levels of intracellular AbO conformers (selectively detected by DB analysis in cell lysates) are significantly decreased (Fig. 4, Supplementary Fig. 7b). Of note, also the intracellular oligomers detected (in DB) by the independent A11 polyclonal antibody 21 , known to recognize the so-called 'prefibrillar oligomers' (larger than dimers and trimers) 30 , are found to be decreased in 7PA2-A13K cells (Fig. 4).
Thus, the reduction of secreted AbOs determined by the expression of scFvA13-KDEL occurs without a concomitant intracellular accumulation of Ab and with a reduction of intracellular AbO conformers, detected by scFvA13, scFvIm3 and A11 likely as distinct pools of AbOs.
Results can be explained by an interference of the scFvA13-KDEL intrabody in the formation of stable AbOs in the ER, highlighting the relevance of intracellular (as opposed to the extracellular) Ab oligomerization process in the overall balance of AbOs. We conclude that the ER-assembled AbOs are a consistent source of those secreted extracellularly and of those found intracellularly.
Of note, the expression of an Ab-unrelated intrabody (scFvR4-KDEL used as negative control) in 7PA2 cells, has no effect on the levels of extra-and intracellular Ab or AbOs ( Supplementary  Fig. 8).

ScFvA13-KDEL intrabody does not affect the APP metabolism.
To further characterize the cellular mechanisms leading to the observed changes in Ab/AbOs levels in the 7PA2-A13K cells (summarized in the Supplementary Table 3), we investigated whether the intrabody has a direct or indirect interference on the expression, maturation and processing of APP, processes which tightly influence the levels of secreted Ab (Fig. 5).
The steady-state levels of full-length APP and of APP C-terminal fragments (CTFs) in total lysates (Fig. 5b), as well as of soluble a-APP and b-APP in the extracellular medium ( Fig. 5c), are similar in 7PA2 and in 7PA2-A13K cells. Likewise, in comparative subcellular fractionation studies, the scFvA13-KDEL intrabody does not affect the intracellular maturation and processing of APP, assessed by evaluating the compartmentspecific (ER versus Golgi) N-and O-glycosylation of APP and the CTFs levels (Fig. 6a). Finally, the subcellular activities of a-band g-secretase were found not to be affected by the scFvA13-KDEL intrabody, as measured in comparative analyses of 7PA2 and 7PA2-A13K cells (Fig. 6b,c). In detail, the g-secretase activity was assayed in subcellular ER-enriched fractions after their incubation for 2 h at 37°C, by measuring the selective reduction of the APP-CTFs and the appearance of the APP intracellular domain (AICD) (Fig. 6b) (as previously described 31 ). Furthermore, we established an assay to determine both g-secretase and a-, b-secretases activities through a reversible block of intracellular trafficking from ER to Golgi with Brefeldin A (BFA). One or four hours of treatment with BFA determines: (i) a time-dependent reduction of APP-CTFs (almost complete after 4 h of treatment) indicative of g-secretase processing; (ii) a significant change in the ratio of immature/mature APP forms, with a prevalence of the immature one, indicative of the block of APP in the ER. After a 2 h washout of BFA: (i) the APP-CTFs are reestablished or de novo generated (indicative of a-, b-secretases activities likely in post-ER compartments); (ii) the maturation (glycosylation) of full-length APP, previously prevented by the BFA treatment, is restored (Fig. 6c).
All these assays show that in 7PA2 cells, the anti-AbOs scFvA13-KDEL does not affect the maturation and processing of APP, not even during its trafficking in the early secretory pathway where the intrabody resides, shuttling between ER and cis-Golgi.
Remarkably, also in other cell lines (7WD4 and SHSY5Y) with lower or negligible levels of AbOs, scFvA13-KDEL does not interfere with the physiological APP processing ( Supplementary  Fig. 9).
In conclusion, our data demonstrate that the scFvA13-KDEL intrabody acts effectively and selectively on the levels of extraand intracellular pools of AbOs, with no interference with APP expression, maturation or processing. This molecular and subcellular selectivity represents a significant experimental advance to modulate the levels of Ab peptides, in comparison with widely used approaches of g-secretase inhibition. Indeed, as expected, we found that in 7PA2 cells the g-secretase inhibitor L-685,458 (ref. 29) dramatically decreases the production of Ab and, consequently, of AbOs, but massively increases the levels of aand b-CTFs, altering also their subcellular distribution, and the levels of soluble APPs (Supplementary Fig. 10).
The lack of interference of the intrabody on the APP cellular biochemistry is noteworthy due to several functions of APP-derived fragments (that is, of soluble APPs and of AICD) 32 .
ScFvA13-KDEL selectively binds AbOs in the ER. In order to explore the selective mechanism of AbO modulation, we first investigated the interaction of the intrabody with subcellularly localized Ab/AbOs species, by microsome fractionation of 7PA2-A13K cells, followed by co-immunoprecipitation (co-IP) and WB analyses. We demonstrated the intrabody-mediated selective pulldown of LDS/SDS-stable Ab dimers, trimers and small oligomers (o20 kDa), but not of Ab monomers, neither of the full-length APP, in the ER-enriched fractions of 7PA2-A13K ARTICLE cells (Fig. 7a). The AbO pulldown was successful when freshly prepared microsome vesicles were subjected to a preliminary step of chemical cross-linking; this suggests a transient nature of the AbO-intrabody complexes (consistent with a chaperone-like interaction of the intrabody).
The binding selectivity of scFvA13-KDEL intrabody for AbOs in living cells, with respect to the monomeric Ab, is in accordance with the in vitro oligomer-selective discrimination and conformation binding properties of the scFvA13 (ref. 13), but the fact that we could prove it in the intracellular environment is noteworthy. Furthermore, the binding selectivity with respect to the APP further suggests that, in the ER lumen, the Ab moiety in the context of APP is not recognized by the scFvA13-KDEL intrabody, in line with the lack of interference with APP processing and maturation (Figs 5,6).
We suggest that the primary action of scFvA13-KDEL intrabody is to bind to early-assembled, possibly transient, AbO conformers (here detected in the co-IP/WB analysis as SDS/LDS-stable Ab dimers and trimers and small AbOs), but not to the free Ab monomers. This could inhibit a further Ab oligomerization, most likely due to the interference with critical seeding defined by the bound AbO conformers. The significant reduction of intracellular pool of AbOs measured by A11 antibody (see Fig. 4), which preferentially recognizes prefibrillar AbOs larger than dimers and trimers 30 , is also in accordance with this proposed mechanism.
The co-IP results (Fig. 7a) and the DB immunoreactivity of cell lysates with A11, scFvA13 and scFvIm3 (Fig. 4) (the latter confirmed as Ab-specific by the g-secretase inhibitor treatment ( Supplementary Fig. 7b)), demonstrate that AbOs larger than dimers and trimers exist in the intracellular environment of 7PA2 cells. We conclude that the intrabody-mediated interference in the ER with the dynamic pool of intracellular AbOs, including oligomers larger than Ab dimers and trimers, can influence also the balance of extracellular AbOs, which are mainly detected as LDS/SDS-stable dimers and trimers. The scFvA13 interferes with AbOs assembly in vitro. We studied next the effects of scFvA13 (used as purified protein) on the formation of AbOs or on preformed AbOs in vitro. Indeed, while considering obvious and well-known differences between natural and synthetic Ab samples (that is, the high concentration of synthetic Ab used in vitro and the different appearance of Ab assemblies) 33 such in vitro study could help interpreting the mechanism of conformational interference of scFvA13-KDEL intrabody. We demonstrated that the scFvA13, both at stoichiometric and substoichiometric ratios with the Ab1-42 monomeric peptide, strongly inhibits the assembly of AbOs in vitro. Indeed, in the presence of scFvA13, we observed the lack of formation of high-molecular weight (HMW) AbOs (30-80 kDa in WB analysis) from a starting solution where Ab monomers and low-molecular weight (LMW) oligomers (o20 kDa) are in rapid equilibrium (Fig. 7b). Thus, the scFvA13 blocks the assembly from LMW towards HMW AbOs (Fig. 7b). In addition, we studied whether and how the scFvA13 acts on preformed AbOs. When incubated with scFvA13, LMW and HMW AbOs undergo a change of their assembly state, as revealed by the reduction of their MWs with a concomitant significant increase of free Ab monomers (in a scFv concentration-dependent manner) ( Supplementary Fig. 11). Both these in vitro observations (dynamic interference in assembly and disassembly pathways) support our hypothesis on the mechanism of action of the scFvA13-KDEL intrabody on AbO conformers and are in accordance with the binding and pulldown into the ER and with measured overall modulation of Ab/AbOs levels in cells (reduction of AbOs and the increase of secreted Ab monomers).
Intracellular fate of scFvA13-KDEL. In order to support the mechanism by which the scFvA13-KDEL intrabody intercepts early AbO conformers in the ER and acts as a conformationalsensitive and chaperon-like assembly modulator, we better characterized the subcellular localization of scFvA13-KDEL and its possible delivery to pathways of cellular degradation.
We found that the intrabody does not localize in trans-Golgi network (TGN), neither in early nor late endosomes and in lysosomes (as demonstrated by indirect immunofluorescence, by the lack of co-localization with the subcellular markers TGN46, Rab5, Rab7 or LAMP-1, respectively) (Fig. 8a). Thus, the intrabody, as expected for a physiological and non-altered KDEL system, is mainly restricted into the ER excluding its localization in other endocytic vesicles which are crucially involved in recycling or degradation pathways. Of note, the TGN was recently identified as a crucial compartment for the synthesis of Ab40 mainly due to the APP re-routing from the plasma membrane via the retromeric pathway 34 . Also endosomal, lysosomal and the secretory pathway (ER, Golgi) were demonstrated to be sites of Ab production 7 . However, the precise localization of the scFvA13-KDEL in the ER (and, only partially, with cis-Golgi), as demonstrated, strongly support the view of a more confined action of the intrabody in these compartments. This formally establishes the ER (and cis-Golgi) as a major organelle where the control of Ab oligomerization is crucial.
We next investigated if the scFvA13-KDEL intrabody undergoes possible mechanisms of ER-associated degradation (ERAD) that acts through the ER to cytosol retrotranslocation of ER-resident proteins and their degradation by targeting the proteasome 35 . In principle, the ER-localized intrabody might escort the bound AbOs to the ERAD pathway. We observed, however, that in 7PA2-A13K cells, the levels of the intrabody do not change after 6-h treatment with the proteasome inhibitor MG132 (20 mM) (Fig. 8b). On the other hand, in 7PA2 cells stably expressing a different version of the intrabody, referred to as ARTICLE 'scFvA13-degradin' (specifically targeted to the ERAD system via C-terminal fusion with the moiety of SEL1L protein involved in the retrotranslocation process) 36 , we observed a significant increase of the intrabody levels after 6 h MG132 treatment ( Supplementary Fig. 12). Thus, the scFvA13-degradin undergoes ERAD and proteasome degradation, while the scFvA13-KDEL  Fig. 12). Mean values ± s.e.m., n ¼ 3; *Po0.01 (Student's t-test) in CHX treatment both versus control and versus MG132 treatment.
does not. As control, a 6-h treatment with cycloheximide (blocking the protein synthesis) dramatically reduces the levels of both scFvA13-KDEL (Fig. 8b) and scFvA13-degradin ( Supplementary Fig. 12) suggesting for both intrabody forms a short yet similar half-life. The short half-life of the intrabody scFvA13-KDEL is also confirmed by an in vitro assay on ER-enriched subcellular fractions, where the intrabody undergoes an almost complete degradation after 6 h of incubation at 37°C (Supplementary Fig. 13).
The overall data suggest that the scFvA13-KDEL is mainly located in the ER, does not alter the APP maturation and processing, and does not undergo ERAD process. Furthermore, the short half-life of scFvA13-KDEL, together with its moderate levels of expression, prevents its accumulation in the ER and a consequent ER stress. We conclude that observed variations of AbO levels and the overall balance of secreted Ab species are representative of a fine mechanism of conformational assembly modulation of AbO, which occurs in the ER (and in KDEL recycling compartments) where the intrabody intercepts critical conformers changing their oligomerizing fate, rather than their targeted degradation to proteasome or their subcellular re-targeting in other active endocytic pathways.

Functional consequences of the intrabody interference with
AbOs. In order to investigate the functional consequences of the selective interference of the scFvA13-KDEL intrabody with specific pools of AbO conformers, we exploited two established experimental readouts on 7PA2 cells. The AbOs secreted by 7PA2 cells induce specific AD-related synaptic dysfunctions, when administered to primary neurons, brain slices or rodent brains 3,8,9,[24][25][26] . In detail, in the primary hippocampal neurons, AbOs (contained in 7PA2 CM) can induce an impairment of the signalling mediated by extrasynaptic NR2B-containing NMDA receptors, modulating the ERK1/2 and CREB phosphorylation status 26 . Here, we demonstrate, in this experimental paradigm, that both ERK1/2 and CREB are largely dephosphorylated in the hippocampal neurons treated with 7PA2 CM but significantly less in those treated with 7PA2-A13K CM (Fig. 9a). This provides a functional demonstration that the AbO conformers targeted by the scFvA13-KDEL (and selectively immunodepleted in the 7PA2-A13K CM) are involved in the activation of a NR2B-NMDA extrasynaptic signalling, which is implicated in neurodegenerative cascades 37 .
Furthermore, we reported an independent functional demonstration of the biological relevance of the intrabody-targeted AbO conformers. Indeed, in 7PA2 cells, the activity of mTOR (mammalian target of rapamycin), a crucial mediator of cellular homeostasis and autophagy, is directly linked to the levels of endogenous Ab 38 . Here, we show that in 7PA2-A13K cells, mTOR activity is reduced back to basal levels (found in CHO cells), as measured by the phosphorylation levels of the direct downstream target p70S6K (Fig. 9b). This provides a new functional demonstration that the intracellular AbO conformers targeted by the scFvA13-KDEL are involved in the impaired homeostasis and autophagy in 7PA2 cells, through the mTOR signalling pathway.
In conclusion, the functional results, through two independent readouts, demonstrate that both extra-and intracellular pools of AbO conformers targeted by the scFvA13-KDEL intrabody, among the numerous and diverse Ab and APP-derived soluble species, constitute a crucial pool of active AbOs, directly involved both in the deregulation of specific cellular pathways linked to cellular homeostasis, and in the synaptic actions previously ascribed to the more complex CM of 7PA2 cells, and thought to be relevant in neurodegeneration and also AD pathogenesis.

Discussion
AbOs are considered the most neurotoxic Ab forms in AD brains, but they are still mysterious entities in terms of molecular and structural composition and activity 8 . Furthermore, while intracellular AbOs are emerging targets in AD studies 7,15 , their identification, study and targeting in the complex context of subcellular compartments of living cells still remains a big challenge. This is a key issue, in order to uncover and validate new molecular and cellular targets tightly linked to the Ab hypothesis, for a successful search and development of novel therapeutics for AD 8,39 .
While the processes of Ab aggregation are intensively studied at different levels of complexity (in silico, in vitro and in vivo) numberless questions remain open and to be addressed, such as the identification of the cellular mechanisms and the subcellular sites of Ab misfolding and oligomerization. Of note, in the current state of the art, this question necessarily calls for the development of new selective experimental approaches exploitable in living cells.
We face the question in a way not tackled before by establishing a new approach that we called CSI based on the subcellular targeting of a conformation-sensitive intrabody in living cells. In this way, we showed for the first time that it is possible to selectively target, inside the cell, certain oligomeric Ab conformers but not the Ab precursor protein (APP) or the monomeric Ab. This represents a significant advancement with respect to the currently used secretase inhibitors 40 and previous targeting of the Ab/APP system with intrabodies that were acting through an interference with the processing and/or maturation of APP 41 or of g-secretase components such as nicastrin 42 .
Moreover, the subcellular-localized anti-AbOs CSI, via the retention/retrieval of the KDEL intrabody in the ER, provides significant new insights into the functions of this organelle in the assembly of functionally active AbOs.
We show that the main mechanism operated by the ERresident intrabody is of a conformational assembly modulation, through a mechanism of chaperone-like folding assistance on critical AbO conformers intermediates (Fig. 10).
The CSI determines an opposite balance of the different pools of soluble Ab observed in 7PA2-A13K cells: on one hand, the extracellular LDS/SDS-stable dimers and trimers and the AbO conformers are reduced, and, on the other hand, the extracellular monomeric Ab peptides are increased, without significant changes in the ratios of Ab truncated forms.
Recent studies show the AD pathological importance of qualitative changes in the g-secretase-generated Ab products (species of 37-43 amino-acid length) 43 . Even small changes in the composition of synthetic Ab mixes can affect critically their aggregation kinetics and toxic effects; in particular, AbOs composed by fAD-like Ab42:Ab40 ratios are more stable and toxic 44 . This is in accordance with the differences observed here, between 7PA2 and 7WD4 cells. However, our results in 7PA2-A13K cells (where we observed complementary changes of the secreted AbOs and of Ab monomers/fragments) additionally show that the ER control of Ab peptides folding and assembly in vivo, even in the context of their pathological ratios, is a crucial step in the formation of pathologically relevant AbO conformers.
In 7PA2-A13K cells, the increased proportion of secreted free monomers can be interpreted as deriving from the intracellular AbO conformers intercepted by the intrabody along oligomerization pathways and representing a fingerprint of their composition (in non-assembled state). Thus, AbOs, when assembled inside the cells and then secreted, would be formed by well-defined ratios of several Ab peptides and fragments.
The Ab1-42, known to be preferentially generated in the ER 7,45,46 , could act here as a seed for oligomerization of other Ab monomers with a mechanism therefore favored by the neutral luminal pH (peculiar of the ER) and by the macromolecular crowding of this organelle. Of note, in vitro studies demonstrate that neutral pH, opposite to acid pH, favors pathways of Ab oligomerization against fibrillization 47 and that the macromolecular crowding can contribute to the amyloid aggregation 48 .
Besides its favourable physicochemical milieu, the ER has a crucial role in the protein quality control and in the physiological oligomerization of some protein complexes, making a priori this organelle a good candidate for our intrabody-targeting study. The chaperone-like mechanism proposed for the scFvA13-KDEL is consistent with the physiological role of ER-resident chaperones engaged in protein folding and/or degradation processes 35 . However, it is unknown if AbOs can be a direct target of endogenous ER chaperones; a study demonstrated that some ER chaperones indirectly regulate the Ab production through their interaction with APP impairing its maturation and processing 49 . Thus, the scFvA13-KDEL intrabody can equip the ER with new beneficial functions (otherwise lacking in mammalian cells) of selective control of critical AbO assembly. We also surmise that the KDEL system, physiologically exploited by some endogenous ER chaperones for the protein quality control, was likely essential to obtain a chaperone-like action of the intrabody (shuttling between the ER and the Golgi complex).
Several studies show that the Ab peptides can be formed in different subcellular compartments such as ER, Golgi, TGN, endosomes, lysosomes 7,34 but no study has been able to address the important question of where and how intracellular Ab peptides oligomerize. Recently, intracellular AbOs have been identified in fixed cells or brain tissues through oligomericspecific antibodies, but these antibodies do not allow functional studies of the AbOs formation and trafficking in living cells. Thus, the localization of intracellular AbOs is still an open and controversial question; as for the origin of intracellular AbOs, the prevailing hypothesis suggests the endocytosis of AbOs assembled outside of the cell. However, the extracellular space, such as the extracellular fluid of the brain, is not the ideal milieu for the Ab aggregation, first due to the very low concentration of soluble Ab (o1 nM). Accordingly, a study demonstrated that nanomolar concentrations of synthetic Ab, extracellularly administered to the cultured cells, form amyloid seeds only in the intracellular milieu upon cellular uptake, concentration and aggregation in endocytic vesicles 50 . At difference, our study is performed in living cells where a complex pattern of Ab peptides is endogenously produced through the APP-RIP occurring in several subcellular compartments. In this context, we recognized the ER as the main and crucial site to control critical events of Ab oligomerization, just exploiting the precise spatially localized subcellular intrabody probe for CSI (in the ER but not in other endocytic vesicles). However, we cannot exclude that some Ab oligomerization (or Ab super assembly onto ER-formed seeding nuclei) could also occur in other subcellular compartments (that is, along the secretory and endocytic pathways), and/or that some Ab/AbO species can reach the ER through the Golgi complex, during retrograde routes of subcellular vesicular trafficking that is, via the retromer TGN). Finally, we demonstrated that the pool of AbO conformers intercepted in the ER and modulated by the scFvA13-KDEL intrabody is functionally relevant, by exploiting two distinct functional readouts regarding the extrasynaptic activation of ERK/CREB signalling pathways in primary neurons and the phosphorylation pathway of p70S6K, a downstream target of mTOR, implicated in the impaired cellular homeostasis and autophagy. As an interesting parallel to our results, amyloidbinding compounds modulating the Ab assembly also restored the protein homeostasis during aging in Caenorhabditis elegans models 51 . Given the central role of the ER mitochondria crosstalk in maintaining cellular homeostasis, and given that 7PA2 cells show a prominent set of functional mitochondrial deficits 52 , it will be interesting to investigate whether the scFvA13-KDEL is able to restore normal mitochondrial functions in 7PA2 cells, via its interference with intracellular AbOs in the ER.
Our overall results support the idea that targeting specific conformations of Ab can be a strategy more selective and more effective than lowering its total levels. Furthermore, we show the value of the subcellular targeting for mechanistic studies on Ab oligomerization in the ER and the early secretory compartment. In addition, intrabodies can be targeted to other subcellular compartments and also to neuronal specialized districts in order to address new relevant questions on different mechanisms of AbOs trafficking and actions. Indeed, the cellular mechanisms of APP processing, Ab generation and oligomerization can present specific cell-type peculiarities, as in the case of the polarized and specialized neuronal cells. In neurons, APP undergoes fast anterograde transport to nerve terminals and is metabolized within the axonal or presynaptic vesicles into Ab peptides that are released and deposited as amyloid plaques around nerve Our study demonstrates that the ER is an organelle crucial for the assembly of biologically relevant AbOs. The ER-assembled AbOs are likely the main source of those found extracellularly and they are essential in the intra-and extracellular balance of Ab and AbO species. (c) In 7PA2-A13K cells, we propose a mechanism by which early-assembled AbO conformers are intercepted by the intrabody in the ER. Thus, the intrabody can be deputed to a selective control of the Ab misfolding (represented by a change of the green-red-shaded box with a green one) and assembly (represented by smaller red frame boxes in comparison with 7PA2 cells), equipping the ER with new functions of quality control, otherwise escaped by pathological conformations of Ab. Thus, the main mechanism operated by the ER-resident intrabody is of a conformational assembly modulation. As a consequence, the levels of intra-and extracellular pools of AbOs decrease while secreted Ab monomers increase.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4867 ARTICLE terminals 53,54 . Furthermore, synaptic activity increases Ab secretion, indicating that the presynaptic terminal is an important regulatory site for Ab generation 55,56 . Of note, intrabodies can be targeted also to neuronal specialized districts, such as pre-and post-synaptic terminals 57,58 . The demonstration of cell-specific (neuronal or not) mechanisms of subcellular AbOs trafficking and actions will be the next goal of our intrabody-based studies, having here established that CSI allows a dissection of AbOs-specific mechanisms not mediated by Ab monomers nor by APP.
In light of the controversial and open questions emerging from Ab-targeting clinical trials 39 and immunotherapy 59 in AD, we believe that our study provides a new cellular and biochemical basis for targeting AbOs inside cells, and that the CSI approach can be worthy of further preclinical and clinical applications. Of note, the conformation selectivity and the molecular specificity of the CSI approach overcome limits of methods targeting nucleic acid (genes or mRNA) or exploiting new chemical compounds that cannot achieve a comparable molecular and subcellular precision. Finally, the CSI approach can be exploited in different proteinopathies through the upstream direct selection of new functional intrabodies 60,61 against antigens that undergo in vivo misfolding, similarly to Ab 13 .

Methods
Cell lines. 7PA2 and 7WD4 cells (CHO stably transfected respectively with the human APP751 cDNA bearing the V717F fAD mutation and with the wtAPP751, kindly provided by Dr D.J. Selkoe, Harvard Medical School, Boston) were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), 2 mM glutamine and antibiotic selection (G418) as previously described 23 . SHSY5Y human neuroblastoma cells were grown in DMEM-F12 supplemented with 10% FBS and 2 mM glutamine.
Intrabody constructs. The constructs for scFv-KDEL intrabodies for the ER retention/retrieval, follow the scheme of expression box of the pscFvExpress vector 62 , with an N-terminal SecL, secretory leader sequence from an IgG1 immunoglobulin, and a C-terminal KDEL, ER retention/retrieval signal sequence. A V5 tag is inserted before the C-terminal KDEL sequence and allows the immunodetection of the scFvs, by anti-V5 antibodies. The cDNA of the anti-AbOs scFvA13 (ref. 13) (or of the Ab-unrelated scFvR4 (ref. 63)) was cloned through the restriction enzyme BssHII-NheI in frame with a C-terminal V5 tag. The secretory construct differs from the KDEL construct only for the lack of the C-terminal KDEL ER retention/retrieval signal sequence. The cDNA of scFvA13 was also cloned in the backbone vector 'scFv-degradin' (kind gift of Prof. Burrone, IGEB Trieste) 36 .
Establishment of cell lines stably expressing the intrabodies. Cells were transfected with scFv-intrabody vectors, coding for scFvA13 or scFvR4, using Lipofectamine 2000 (Invitrogen) and stable cell clones were obtained by limiting dilution in the presence of appropriate antibiotics (250 mg ml À 1 G418 and 2.5 mg ml À 1 puromycin for 7PA2 and 7WD4 cells, and only G418 for SHSY5Y cells).
We established both a polyclonal population and multiple stable single clones. In a preliminary survey, we characterized the levels of intrabody expression (by WB), its subcellular localization (by immunofluorescence) and the modulation of secreted AbOs. Then we performed the overall study on a 7PA2-A13K clone, showing an average expression level and representative of the entire polyclonal population.
Cell samples for Ab/AbOs analyses. For CM preparation, cells were grown to B90% confluence, washed twice with Dulbecco's phosphate-buffered saline (DPBS) and then conditioned in serum-free medium for 18 h. CM was collected and cleared of cell debris by centrifugation at 200g for 10 min. In comparative analysis of different cell lines, CM was prepared from cells at the same density of plating. The Ponceau S staining of nitrocellulose membrane before WB (detecting the total proteins) and the WB for the extracellular soluble protein Laminin (unpublished data) were used as normalization of WB lanes loading (see also Supplementary Information).
In straight (non-concentrated) 7PA2 CM, Ab monomers are detectable (by WB and SELDI-TOF MS), while AbOs (by WB and DB) are generally under the detection limits. In order to improve the detection of AbOs (without immunoprecipitation steps), we optimized a procedure of concentration of the CM by centrifugation steps in concentrators with a 3 kDa or a 5 kDa cut-off (VivaSpin Sartorius). We established that a 15 Â concentration allows a sensitive discrimination of AbOs (in comparative analyses of 7PA2 and 7PA2-A13K CM). The overall results demonstrate that this procedure is useful to obtain samples, whose concentrations of total Ab (determined by quantitative WB) are in the range of previous studies, and, most importantly, maintaining the effective conformational-relevant differences of starting non-concentrated samples.
Cell lysates were done in Tris-HCl pH8 50 mM, NaCl 150 mM, NP40 1% with the addition of cocktail of proteases and phosphatases inhibitors (Roche). The lysates were centrifuged at 12,000g for 10 min. The supernatant was collected and the concentration of total proteins was determined by BCA assay.
As discussed in the text, the bands at B8 kDa and at B12 kDa correspond to Ab dimers and Ab trimers, collectively named LDS/SDS-stable low MW AbOs (due to the analysis method involving LDS in the sample buffer and SDS in the polyacrylamide gel), largely characterized in 7PA2 cells 24 . The identification of the 4, 8 and 12 kDa bands as containing Ab was confirmed by the use of a g-secretase inhibitor L-685,458 (ref. 29), which almost abolishes the formation of Ab, and, as a consequence, also of its assemblies, AbOs (see also Supplementary Information).
DB analysis. Purified proteins and cellular samples were spotted onto nitrocellulose membrane filters 0.22 mm. The anti-AbOs scFvA13 (3.5 mg ml À 1 ) and scFvIm3 (4.5 mg ml À 1 ) were used as primary antibodies diluted in TBS 0.05% tween, 5% dry milk with the addition of cocktail of proteases inhibitors (Roche). The polyclonal antibody anti-oligomer A11 (ref. 21) was used 1:500 in TBS 0.05% tween, 5% dry milk. The AbO scFv-positive immunodetection was performed by anti-His tag (for cellular samples), or by anti-V5 tag (for monomers and oligomers of synthetic Ab, a-Synuclein and lysozyme), which respectively recognizes the C-terminal 6xHis tag or V5 tag of recombinant scFvs (expressed in Escherichia coli and purified, see the paragraph below). Serial dilution curves of cellular samples were preliminarily tested to obtain non-saturating condition of immunodetection. Samples were loaded as follows: 450 ng per dot of synthetic human Ab1-42; 500 ng per dot of recombinant human a-Synuclein; 500 ng per dot of lysozyme; 20 ml per dot of 15 Â concentrated 7PA2 CM and related samples (CM of g-secretase inhibitor-treated 7PA2 cells and 7PA2-A13K CM) by using Bio-Dot SF apparatus Bio-Rad; 500 ng per dot of total lysates (non-denatured).
Purified proteins assayed by DB and ELISA. Synthetic human Ab1-42 (HFIP-pretreated peptide, purchased from Anaspec) was dissolved (5 mM) in anhydrous DMSO and assembled to form AbOs as ADDLs (100 mM Ab in phenol red-free Ham's F12 medium, purchased from PanBiotech) at 4°C for 24 h 47 without shaking. Following incubation, tubes were centrifuged at 14,000g for 10 min in the cold. The supernatant (soluble ADDLs) was recovered and transferred to new tubes, and immediately frozen in dry ice and stored at À 80°C in aliquots of small volume. Several independent ADDLs preparations were assayed by DB and ELISA comprising ADDLs previously prepared and characterized for toxicity and synaptic binding 13 . As Ab monomers, we used the nonaggregated Ab1-42 diluted in ice-cold MilliQ water (to a final concentration of 100 mM Ab) and immediately frozen at À 80°C. Recombinant human a-Synuclein (Abcam), 70 mM in 100 mM NaCl, 20 mM Tris-HCl, 1 mM MgCl 2 (pH 7.5) was sonicated for 10 min in water bath sonicator, aliquoted and immediately frozen at À 80°C or oligomerized by incubating the protein solution at 4°C for 3 or 7 days without shaking (adapting conditions described 64 ). Purified lysozyme (AppliChem), from chicken egg white, was dissolved in 25 mM KH 2 PO 4 pH 2 at 45°C, consecutively filtered through 0.22 and 0.1 mm pore size syringe filters and assembled as oligomeric fibrils (0.5 mg ml À 1 in 175 mM NaCl/25 mM KH 2 PO 4 pH 2) for 3 h at 50°C (ref. 65); following a different procedure, lysozyme was also dissolved in phosphate buffer (PB) 0.1 M at pH 7.4, filtered through 0.1 mm pore size syringe filters and assembled as oligomers (7 mg ml À 1 in PB 0.1 M at pH 7.4) for 4 h at 50°C (ref. 66). The presence of oligomers in the assembled samples was confirmed by ELISA, using as primary antibodies the generic anti-oligomer A11 (ref. 21) or the AbO-specific scFvA13 (ref. 13) (see also Supplementary Information).
Ab immunoproteomic SELDI-TOF MS analysis. The anti-Ab mAbs (6E10 þ 4G8, 0.125 mg ml À 1 ) (Covance), used as Ab capturing antibodies, were incubated in a humid chamber for 2 h at room temperature (RT) to allow covalent binding to the PS20 ProteinChip Array (Bio-Rad Laboratories) 27 . Unreacted sites were blocked with Tris-HCl 0.5 M, pH 8 in a humid chamber at RT for 30 min. Each spot was washed first three times with PBS containing 0.5% (v/v) Triton X-100 and then twice with PBS. The spots were coated with 5 ml of sample (CM, straight or 10 Â concentrated in a Savant Speed Vac) and incubated in a humid chamber overnight. Each spot was washed first three times with PBS containing 0.1% (v/v) Triton X-100, twice with PBS and finally with deionized water. a-cyano-4-hydroxy cinnamic acid (Bio-Rad) was added 1 ml per spot. Mass identification was made using the ProteinChip SELDI System, Enterprise Edition (Bio-Rad).
In comparative studies (7WD4 CM versus 7PA2 CM and 7PA2 CM versus 7PA2-A13K CM), the CM was prepared from cells at the same density of plating; CM was also loaded in polyacrylamide gels, blotted and normalized by Ponceau S staining of nitrocellulose membrane (detecting the total proteins) and analysed by WB as described.
Subcellular fractionation. Subcellular fractionation on discontinuous iodixanol gradients were made as described 67 . In brief, cells grown to confluence were pelleted and resuspended in homogenization buffer (10 mM HEPES, pH 7.4, 1 mM EDTA and 0.25 M sucrose) supplemented with protease inhibitor cocktail (Roche). Cells were disrupted with a Dounce homogenizer (10 strokes) followed by five passages through a 27-gauge needle. Nuclei and unbroken cells were pelleted and the post-nuclear supernatant centrifuged for 1 h at 65,000g. The resultant vesicle pellets, nominally total microsomes, were resuspended in 0.8 ml of homogenization buffer. A discontinuous gradient of iodixanol (OptiPrep, Axis-Shield) in 0.25 M sucrose homogenization buffer, was prepared by underlayering with a syringe different density solutions ranging from 2.5 to 30%. The resuspended vesicle fractions were loaded on top of the gradient and centrifuged in a SW40Ti rotor at 40,000g for 2.5 h (4°C). The resulting gradient was collected in 12 fractions of 1 ml. Then, each fraction was denaturated and tested by WB for subcellular markers.
Intrabody-AbOs co-IP from subcellular fractions. Freshly prepared subcellular fractions (or total microsome preparations) were incubated with a cross-linker solution of dithiobis(succinimidyl propionate) (DSP, Pierce), to a final concentration of 2 mM for 30 min at RT, after that Stop Solution (1 M Tris pH 7.5) was added to a final concentration of 20 mM for 15 min. Cross-linked subcellular fractions (or total microsomes) samples were incubated with anti-V5 agarose affinity gel (Sigma) for 16 h at 4°C for the immunoprecipitation procedure. After ice-cold PBS washes (6 times 5,000 g, 5 min each), the immunoprecipitated proteins were eluted with 4 Â Tricine sample buffer (Bio-Rad) containing b-mercaptoethanol by 10 min boiling, electrophoresed on Tris-Tricine gel 16.5% (precasted gels, Bio-Rad). The semidry transfer blot was performed in nitrocellulose membrane filter 0.22 mm and the filter boiled in PBS and subjected to WB analyses (mAb 6E10 anti-Ab/APP and mAb anti-V5-HRP). Co-IP of small AbOs and intrabody was analysed in Tris-Tricine gels 16.5% for a better resolution of low MWs (o50 kDa) WB bands. The full-length APP (B110 kDa) was analysed in Tris-glycine HCl gels 10% or in Criterion Bis-Tris XT Gels 4-12% (Bio-Rad) upon elution and boiling of immunoprecipitated proteins with the appropriate sample buffers (respectively 4 Â Laemmli Sample buffer or 4 Â XT sample buffer Bio-Rad with b-mercaptoethanol).
In vitro assembly interference with synthetic Ab1-42. The human Ab1-42 peptide, HFIP-pretreated (Anaspec) was diluted (5 mM) in anhydrous DMSO and then in phenol red-free Ham's F12 medium (PanBiotech) at 100 mM as previously described 68 . Aliquots were immediately frozen and stored at À 80°C. Before aggregation, 100 mM Ab1-42 in Ham's F12 was diluted at 65 mM by adding scFvA13 or NaPB buffer (NaCl 100 mM, sodium phosphate buffer pH7, 20 Mm) as control. The scFvA13 was used at stoichiometric (65 mM) or substoichiometric (13 mM) ratio with Ab1-42. The aggregation was performed at 22°C for 24 h. For disassembly assays, stoichiometric (65 mM) or substoichiometric (13 mM) concentrations of the scFvA13 or the NaPB buffer as control were added to Ab1-42 in Ham's F12 (preassembled at the concentration of 100 mM, 22°C for 24 h) obtaining a final concentration of 65 mM Ab. The disassembly assay was performed at 22°C for 24 h.
Synthetic Ab samples, not boiled nor treated with reducing agents, were loaded (30 ng per well) in Bis-Tris XT Criterion Gels (Bio-Rad), run in MES buffer and analysed by WB, as described above by 6E10 (Covance), 4G8 (Covance) or by a rabbit mAb anti-Ab (D54D2, Cell Signalling).
ER-localized c-secretase activity. Freshly prepared ER-enriched fractions (from subcellular fractionations) were incubated at 4°C for determination of basal APP-CTFs and AICD levels, or incubated at 37°C for 2 h for APP-CTFs g-secretase cleavage and de novo AICD generation as previously described 31 . Individual fractions were subjected to WB. The detection of AICD was favored by boiling the membrane filter in PBS.
BFA treatment. Cells (at a confluence of 65-70%) were treated with BFA (10 mM) for 1 or 4 h. After treatment, cells were washed twice with PBS and lysed or were left to recover in fresh growth medium for 2 h before lysis. The lysates were subjected to WB with anti-APP C-terminal antibody (Sigma).
Other treatments. The g-secretase inhibitor treatment of 7PA2 cells were performed with 1 mM of L-685,458 (ref. 29) (purchased from Calbiochem-Millipore) for 18 h.
The proteasome inhibitor MG132 (Sigma) or the inhibitor of protein synthesis cycloheximide (Sigma) was administered at 20 and 40 mM, respectively for 6 h.
Hippocampal neuronal cultures. Primary rat hippocampal neurons were prepared from embryonic day 18 pups and maintained in vitro in Neurobasal medium with B-27 supplement and 0.5 mM L-glutamine for 14 days (14 DIV). Half the medium was exchanged every 4 days. Neurons (14 DIV) were treated for 16 h with Neurobasal medium conditioned on confluent dishes of 7PA2 cells or 7PA2-A13K cells. Cultures were lysed and subjected to WB, as described 26 .
Immunofluorescence analysis. Cells were plated on glass coverslips and cultured until reaching B70% confluence, then fixed with 1.9% (v/v) formaldehyde in DPBS (with Ca 2 þ and Mg 2 þ ) for 5 min, followed by a postfix for 10 min in 3.7% formaldehyde at RT. The coverslips were washed, permeabilized with 0.1% Triton in PBS for 5 min and blocked with 5% FBS in PBS for 1 h at RT. After blocking, cells were incubated with primary antibodies overnight at 4°C, and then with appropriate secondary antibodies (anti-rabbit or anti-mouse Alexa-Fluor 488 or Alexa-Fluor 594 (Molecular Probes)) for 1 h at RT. Coverslips were mounted with Aqua-Poly/Mount Reagent (Polysciences) and analysed by confocal microscopy.
Confocal microscopy. Microscopic images were collected at RT using a confocal laser scanning microscope (TCS SP5, Leica Microsystems) equipped with four laser lines and with HCX PL APO lambda blue 63.0 Â 1.40 OIL ultraviolet objective. To exclude cross-talk between the emission spectra of fluorophores, images were acquired using the sequential acquisition modality. The acquired images were converted into TIFF files of separated and merged channels by using Leica Application Suite 6000 (LAS AF Lite).
Statistical analysis. Student's t-test was used to evaluate differences between two study groups in normally distributed data (two samples, two tails, equal variance).
Densitometric analysis. WB bands or DB dots images were analysed by using ScionImage software (NIH Image for Windows, Beta 4.0.2).
Western Blot analysis. Full-lenght images of western blot are shown in Supplementary Figs 14,15.
The recombinant scFv proteins were prepared following established procedures for the expression and refolding of another SPLINT-derived scFv in the cytoplasm of E. coli 69 .
The purification of the refolded scFvs were achieved through a cation exchange chromatography, performed on HiTrap SP column (5 ml, GE), equilibrated with 20 mM sodium phosphate buffer, pH 7 (for scFvA13) and pH 6.5 (for scFvIm3) as A buffer. The elution of the sample was achieved with a linear gradient from 0 to 70% of B buffer (A buffer þ 1 M NaCl).
The sample was then dialysed against PBS and purified on a size exclusion chromatography column, a Superdex 75 (GE), equilibrated with PBS.