APP‐C31: An Intracellular Promoter of Both Metal‐Free and Metal‐Bound Amyloid‐β40 Aggregation and Toxicity in Alzheimer's Disease

Abstract Intracellular C‐terminal cleavage of the amyloid precursor protein (APP) is elevated in the brains of Alzheimer's disease (AD) patients and produces a peptide labeled APP‐C31 that is suspected to be involved in the pathology of AD. But details about the role of APP‐C31 in the development of the disease are not known. Here, this work reports that APP‐C31 directly interacts with the N‐terminal and self‐recognition regions of amyloid‐β40 (Aβ40) to form transient adducts, which facilitates the aggregation of both metal‐free and metal‐bound Aβ40 peptides and aggravates their toxicity. Specifically, APP‐C31 increases the perinuclear and intranuclear generation of large Aβ40 deposits and, consequently, damages the nucleus leading to apoptosis. The Aβ40‐induced degeneration of neurites and inflammation are also intensified by APP‐C31 in human neurons and murine brains. This study demonstrates a new function of APP‐C31 as an intracellular promoter of Aβ40 amyloidogenesis in both metal‐free and metal‐present environments, and may offer an interesting alternative target for developing treatments for AD that have not been considered thus far.


Introduction
[3][4][5][6] As a type-I transmembrane protein, APP is localized in cell surfaces or membranes of organelles. [1,7]Cleavage of APP by enzymes such as -, -, and -secretases releases various fragments that participate in neurotrophic activities, synaptic plasticity, and intracellular signaling. [1]Amyloid- (A) peptides with 38-43 amino acid residues are generated via the sequential cleavage of APP by and -secretases, [1][2][3][4]7,8] as illustrated in Figure 1a. A ptides self-assemble into amyloid oligomers and fibrils that trigger neurodegeneration observed in AD. [1][2][3][4]7,8] The classical amyloid cascade hypothesis suggests that extracellular A aggregates result in synaptic loss and memory impairment; [2,3] however, emerging evidence like the progression of cognitive deficits prior to forming senile plaques leads to the concept that A aggregates inside the cells may also be neurotoxic.[4,[8][9][10] Under pathological conditions, it has been identified that A species can be accumulated in perinuclear and nuclear regions rather than transported outside the cell through secretory pathways.[4,5,[9][10][11] These intracellular A aggregates induce the dysfunction of organelles and neuronal death.[4,5,[9][10][11] As depicted in Figure 1a, a cytoplasmic peptide carrying 31 amino acid residues denoted APP-C31 (APP 665-695 ) is produced upon cleavage of the C-terminal region of APP by caspases.13][14][15][16][17][18] Like A, APP-C31 contributes to neuronal toxicity leading to apoptosis and synaptic depression.[6,[12][13][14][15][16][17][18] The role of APP-C31 in facilitating the accumulation of A 42 has also been suggested, implicating its involvement in activating the amyloidogenic cleavage of APP and altering the A 42 -to-A 40 ratio.[14] The knockout of caspases or the mutation of APP from Asp664 to Ala whose role in inhibiting the generation of APP-C31 reduces the formation of A plaques, diminishes synaptic loss, and prevents neurodegeneration in AD transgenic mice.[17][18][19] Thus, these findings imply that the amount of intracellular APP-C31 is augmented and, consequently, neurotoxicity is enhanced when AD develops. Recetly, the co-localization of C-terminal APP fragments with A was observed in cellular regions, including autolysosomes, perinuclear compartments, and multi-vesicular bodies, in the brains of AD transgenic mice.[5,[20][21][22] Moreover, the studies with the C-terminal truncated APP, APP 1-664 , and mutant APP(Asp664Ala) suggested that A could activate caspases inducing the C-terminal cleavage of APP generating APP-C31.[17][18][19]22] Despite these indications, the mechanism of how APP-C31 is involved in the pathology driven by A is not known to date.
Moreover, in AD-afflicted brains, metal ion dyshomeostasis and highly concentrated metals trapped in senile plaques such as ≈400 μm and 1 mm of copper and zinc, respectively, are found. [7,23]The accumulation of A aggregates with metal ions is also detected in intracellular systems. [24,25]][25][26][27][28] In this context, a better understanding of how APP-C31 interacts with metal ions and metal-A is also of significant interest.
We questioned whether APP-C31 interacts with both metalfree A and metal-A and alters their aggregation and toxicity profiles.Thus, we evaluated the effects of APP-C31 on modifying the aggregation and toxicity of intracellular A 40 through live-cell investigations employing microinjection methods.In addition, the influence of APP-C31 on the aggregation kinetics and pathways of both metal-free and metal-bound A 40 and their detailed molecular-level interactions were determined.Moreover, the impact of A 40 incubated with APP-C31 on neuronal growth and inflammation was probed in human neurons and in vivo (murine brains).Overall, our work substantiates that an intracellular Cterminal fragment, APP-C31, can provoke the aggregation behavior and toxicity of both metal-free and metal-bound A 40 through the interactions between the peptides, which illuminates a new role of APP-C31 as an intracellular promoter in the pathology of AD.

Results and Discussion
The intracellular environment is crowded, and 20%-30% of the volume is occupied by macromolecules. [29]In these systems, effective concentrations of biomolecules are higher than their actual concentrations in the test tube due to the excluded volume effect. [29]Moreover, the spatiotemporal localizations of proteins could induce their concentrations up to a millimolar level. [30]Analyzing intracellular A and APP-C31 is hampered by heterogeneous intracellular systems, but it has been suggested that intracellular A can be accumulated exhibiting a significantly higher concentration than extracellular A. [31]Given that our investigations have the detection limit at low concentrations of peptides, we considered all these aspects to set their concentrations used for individual experiments.

Impact of APP-C31 on the Aggregation of A𝜷
To determine whether APP-C31 affects the aggregation of A at the intracellular region, microinjection experiments, which can have the high transduction efficiency of biomolecules and the precise control of injection volume to the cytosol, [32] were conducted in combination with high-resolution confocal microscopy.[34] A 40 conjugated to the HiLyte Fluor 647 (HF647) fluorophore at the N-terminus ( HF647 A 40 ) was employed to monitor intracellular A aggregates in living cells due to its similar aggregation kinetics, compared to that of fluorophore-unlabeled A 40 (Figure S1, Supporting Information), and the relatively high photostability of the HF647 fluorophore. [35,36]It should be noted that A 42 was not used for microinjection experiments because its rapid aggregation causes the clogging of injection tips.
The size of individual A 40 aggregates was determined by counting the pixels of the fluorescent area following previously reported procedures. [36,37]As described in Figure 1b, the femtoliter volume of solutions containing HF647 A 40 with and without APP-C31 was injected into the cytoplasm. [38]After 24 h incubation, HF647 A 40 in the absence of APP-C31 exhibited a cluster of spherical aggregates with an average size of ≈0.19 μm 2 , as illustrated in Figure 1c and Figure S2, Supporting Information.When HF647 A 40 with APP-C31 was introduced inside the cells, we detected enlarged A 40 aggregates with a cluster size of about 0.42 μm 2 .The intracellular distribution of HF647 A 40 aggregates was further monitored in the cells stained with the fluorescent SYTO16 dye that can visualize nuclei by binding to nucleic acids, [39] as displayed in Figure 1d and Figure S3, Supporting Information.A 40 aggregates without APP-C31 were mostly located in the cytoplasm and few clusters of them were internalized at the nucleus, as highlighted with white arrows (Figure 1d).When HF647 A 40 was injected with APP-C31 into the cells, large A 40 aggregates invaded into the nucleus.These findings support that APP-C31 can promote the aggregation of A 40 in intracellular regions and induce the deposition of A 40 aggregates particularly at the nuclear and perinuclear regions.
Our cell studies showed the striking effects of APP-C31 on intracellular A 40 aggregation and deposition; however, some experimental limitations existed.First, since the cell environment is heterogeneous and composed of many biomolecules, it is challenging to investigate the direct impact of APP-C31 on the aggregation kinetics of A.Second, due to the conjugation of a fluorophore to A 40 , which induces differences in the physiochemical property and structural conformation of A, [35] APP-C31 binding to A 40 may be affected by the presence of a fluorophore.Thus, we evaluated the effect of APP-C31 on the aggregation kinetics of unlabeled A 40 in aqueous media by the thioflavin-T (ThT) www.advancedscience.comfluorescence assay used for quantifying the formation of amyloid fibrils. [7,40,41]As summarized in Figure 2a,b, the change in fluorescence upon incubation of A 40 showed a sigmoidal curve with a lag phase followed by an elongation phase, indicating a nucleation-dependent fibrillar aggregation.The averaged lag time, t lag , and the averaged time to reach the half-maximum fluorescence intensity, t 1/2 , were analyzed to be 19.3 (±0.2) h and 21.5 (±0.1) h, respectively.When A 40 was treated with the stoichiometric amount of APP-C31, the lag phase was noticeably shortened exhibiting the decreased t lag and t 1/2 values [t lag = 6.5 (±0.2) h; t 1/2 = 7.3 (±0.2) h] that were similar to those under the suprastoichiometric conditions.Moreover, the maximum ThT fluorescence intensity where A 40 aggregation reached the plateau became lower as a function of APP-C31's concentration.Therefore, these results suggest that APP-C31 facilitates the nucleationdependent A 40 aggregation and produces different A 40 aggregates depending on the APP-C31-to-A stoichiometry.
The aggregation kinetics of A 40 with APP-C31 was also probed in the presence of seeds generated from preformed amyloid fibrils of A 40 .A monomers form nuclei via primary nucleation, which is thermodynamically unfavorable, followed by the elongation of amyloid fibrils, whereas their contact onto the surface of preformed aggregates catalyzes the generation of nuclei (i.e., secondary nucleation). [42]As shown in Figure S4, Supporting Information, A 40 seeds escalated the aggregation of A 40 with the t lag and t 1/2 values of 3.0 (±0.2) h and 3.5 (±0.2) h, respectively, which was not significantly altered with the addition of APP-C31 [t lag of 2.3 (±0.2) h; t 1/2 of 3.1 (±0.2) h].These results imply that APP-C31 could be involved in the primary nucleation of A 40 aggregation over its seed-dependent aggregation pathways.It should be noted that APP-C31 itself did not show a significant change in the secondary structure and negligibly affected the ThT fluorescence (Figures S5 and S6, Supporting Information); thus, APP-C31 itself could not generate -structured amyloid fibrils under these experimental conditions.
To verify the morphology of A 40 aggregates produced with different amounts of APP-C31, the resultant peptide samples were visualized by transmission electron microscopy (TEM).The sample of A 40 only presented small and thin fibrils after 20 h incubation, as presented in Figure 2c.In the presence of APP-C31 (1 equiv), amyloid fibrils were notably generated starting from 10 h incubation.When A 40 was incubated with 10 equiv of APP-C31, we detected A 40 protofibrils at the early aggregation stage.In addition, curvilinear fibrils appeared from A 40 treated with 10 equiv of APP-C31.Different from A 40 added with equimolar APP-C31, longer incubation of these samples showed bundles of short fibrils and a mixture of globular agglomerates and branched fibrils (marked with white arrows).Some of APP-C31 may form the globular agglomerates at its high concentration (Figure S7, Supporting Information).These observations imply that the morphological changes of A 40 aggregates may also be induced by nonfibrillar APP-C31 aggregates, in addition to monomeric APP-C31.In the case of A 42 , its aggregation kinetics [t 1/2 of 1.6 (±0.2) h] was slightly altered in the presence of APP-C31 [t 1/2 of 1.1 (±0.2) h], as shown in Figure S8, Supporting Information.Notably, the treatment of APP-C31 varied the morphology of A 42 aggregates, as presented in Figure S8c, Supporting Information.A 42 incubated with APP-C31 for 8 h was observed to generate long, thick, and straight A fibrils while A 42 without APP-C31 produced relatively shorter and thinner amyloid fibrils.These results suggest that APP-C31 could affect the polymorphic amyloid formation of A 42 .
To further probe the effect of A on APP-C31 aggregation, the molecular weight distribution of the resultant peptide species was monitored by gel electrophoresis/Western blot (gel/Western blot) employing two antibodies, including an anti-A antibody (6E10 for A) and an anti-APP C-terminus antibody (Y188 for APP-C31) (Figure S9, Supporting Information).When APP-C31 was incubated for 24 h, the molecular weight of ≈4 kDa corresponding to its monomeric form was indicated by Y188.Upon incubation of APP-C31 with A 40 , a band at ≈7 kDa with faint smearing ranging from 4 to 270 kDa additionally appeared.Given that the band at ≈7 kDa with smearing was also detected by 6E10, the complexation between APP-C31 and A 40 , instead of the aggregation of APP-C31 induced by A 40 , was suggested.Taken together, our aggregation studies corroborate that APP-C31 can accelerate the aggregation of A 40 in aqueous media as well as inside living cells.

Interaction of APP-C31 with A𝜷 40
To provide mechanistic insight into how APP-C31 modifies the aggregation profile of A, we assessed the molecular-level interaction between APP-C31 and A.Given that i) APP-C31 showed the noticeable impact on the aggregation kinetics of A 40 , compared to A 42 , and ii) the rapid aggregation of A 42 could induce the disappearance of peak signals, we used A 40 for investigating the interaction between two peptides.When A 40 incubated with APP-C31 was monitored by electrospray ionizationmass spectrometry (ESI-MS), a soft ionization method used in the characterization of protein complexes, [43] new peaks at 1342, 1610, and 2012 m z −1 were observed with +6, +5, and +4 charge states, respectively, which represent that APP-C31 binds to A 40 in a 1:1 ratio (Figure 2d).Binding events between APP-C31 and A 40 , detected by isothermal titration calorimetry (ITC), indicated the spontaneous reaction with ΔG = −5.0(±0.1) kcal mol −1 and ΔH = −1.5 (±0.3) kcal mol −1 , as presented in Figure S10, Supporting Information.The TΔS term was found to be 3.5 (±0.3) kcal mol −1 .These thermodynamic properties are curious on first sight, as a reaction in which two particles associate to form a single species is expected to be entropically disfavored.Specifically, it is the translational entropy associated with each particle amounting to roughly 10 kcal mol −1 at room temperature that disfavors associative processes reducing the number of free particles. [44]A minor but relevant feature is the loss of structural flexibility upon aggregation, which also reduces conformational entropy.The increase in entropy observed here is likely due to the release of water molecules that were tightly bound to the protein surfaces and become free water molecules as the two peptides form an association complex.The binding affinity of APP-C31 for A 40 was measured to be 149.7 (±11.4)μm, indicative of relatively weak protein-protein interactions. [45]he interaction between APP-C31 and A 40 in solution was supported by 2D 1 H- 15 N heteronuclear single quantum coherence nuclear magnetic resonance (2D 1 H-15 N HSQC NMR) spectroscopy at the amino acid residue level.As illustrated in Figure 2e and Figure S11, Supporting Information, upon The amino acid residues indicated in blue or black asterisks represent the residues that were unresolved or not significantly shifted, respectively.f) Conformations of A 40 (PDB 2LFM) [55] and APP 666-693 [PDB 3DXC; as a part of APP-C31 (APP 665-695 )] [56] used for metadynamics MD simulations.The self-recognition site of A shown in Figure 1a  treatment of APP-C31 with a solution of uniformly 15 N-labeled A 40 , we detected notable chemical shift perturbations (CSPs) of the Arg5, Glu11, His13, Gln15, Leu17, Phe20, and Ala21 residues in 15 N-labeled A 40 .This observation indicates that APP-C31 could interact with the N-terminal region and the self-recognition site of A 40 (i.e., Leu17 to Ala21; Figure 1a).Given that the selfrecognition site of A is a central hydrophobic region responsible for the oligomerization and fibrilization, [7,8] APP-C31 may alter the conformation and aggregation propensity of A (vide infra).In addition to monomeric A, we probed the interaction between APP-C31 and amyloid fibrils of A 40 by 1D 1 H NMR spectroscopy.As depicted in Figure S12, Supporting Information, the peak intensities of APP-C31 were notably diminished by ≈40% when it was incubated with A 40 fibrils, thereby supporting its potential interaction with A fibrils.Collectively, the abovementioned ESI-MS, ITC, and NMR studies verify that APP-C31 can interact with A in a relatively weak binding manner.

Potential APP-C31-A𝜷 40 Binding Modes
[48][49][50][51][52][53] As A is an intrinsically disordered peptide, multiple structural ensembles can be generated depending on experimental conditions. [54,55]In these computer simulations, we sought to use a structure of monomeric A 40 observed in aqueous solutions without organic solvents and hydrophobic micelles.Thus, the structure of A 40 obtained by solution NMR (PDB 2LFM; [55] Figure 2f), which meets our criteria, was employed as a starting conformation of monomeric A 40 in our simulations. [55]n addition, the atomistic structure of APP-C31 is not known; thus, the X-ray crystal structure of APP 666-693 excised from the intracellular domain of APP complexed with Fe65 protein (PDB 3DXC; [56] Figure 2f) was selected as the starting guess structure of APP-C31.Modelling slow processes, like the association of peptides taking place in minutes to hours of time-scale, with MD simulations is challenging.One solution to accelerating the simulation and obtaining meaningful data within a tractable computing time is to first conduct metadynamics MD simulations that allow a more rapid and efficient exploration of the wider configurational space to identify the most relevant adduct structures.Using the multi-walker metadynamics algorithm, [53] we sampled dimeric configurations with the gross metadynamics simulation time being 8.28 μs.These simulations facilitate the conformational sampling along the center-of-mass (COM) coordinate between the pair of proteins, and provide the ensemble of the dimeric interface between the two proteins. [46]As depicted in Figure S13a, Supporting Information, we found the most stable hetero-dimer conformers at the COM distance of 13 Å.The observed COM distance profile shown in Figure S13c, Supporting Information, also suggests that the association of the two proteins is reversible.
To verify and rank the relative stabilities of the dimeric structures, additional 500 ns of unbiased MD simulations without the metadynamics acceleration were conducted, where the initial structures were taken from the metadynamics MD simulations.The adduct structures from the MD simulations were evaluated based on two criteria: i) the monomers of A 40 and APP 666-693 maintain contacts throughout the course of unbiased MD simulations (Figure S14, Supporting Information); ii) seven amino acid residues displaying the pronounced CSPs are at the dimeric interface (Figure S15, Supporting Information).Figure 2g and Figure S16, Supporting Information illustrate two classes of stable APP 666-693 -A 40 dimeric adduct structures that are most consistent with our experimental results described in Figure 2e.In Model I and II, the dimeric interfaces are formed by favorable intermolecular contacts and hydrogen bonding.The amino acid residues in the N-terminal region and the self-recognition site of A 40 exhibit intermolecular ionic interactions with APP 666-693 .Both models present a salt bridge between Arg5 of A 40 and Glu683 of APP 666-693 .Other salt bridges are detected between Asp7 of A 40 and Lys688 of APP 666-693 (Model I) or Lys16 of A 40 (Model II).These salt bridges can further neutralize the electrostatic charge in the N-terminal region of A 40 , as presented in Figure S17, Supporting Information, suggesting that the protein-protein interactions reduce the electrostatic repulsion that can interfere with A aggregation. [40] Interestingly, our MD simulations indicate a conformational change in A 40 upon binding to APP-C31.As displayed in Figure 2g, the secondary structure of A 40 spanning from His13 to Asp23 becomes disordered, which is distinct from its monomeric form exhibiting an -helix in the same region (Figure 2f).In particular, the self-recognition site of A 40 highlighted in purple adopts an extended conformation.The Ramachandran plots for the amino acid residues in the selfrecognition site (Leu17 to Ala21) present their dihedral angles mostly in the region of a -sheet and an extended helix, [57] as summarized in Figure S18, Supporting Information.This observation suggests that the conformation of A 40 can be easily converted to a -strand in the presence of APP-C31.Structural changes may occur during protein association, but the extent of change we observed is notable and offers additional explanations for the dominance of the entropy term in the binding free energy.There must be a lot of intra-and intermolecular displacements of hydrogen bond networks and van der Waals and electrostatic interactions upon complexation. [3]Although we cannot explain what kinds of interactions positively and negatively contribute to the term of ∆H, a negative sign of ∆H obtained by the ITC analyses indicates the favorable non-covalent interactions with ensemble structures.As a possible scenario with our ITC and MD results, we reasoned that the favorable entropic contribution could originate from both the liberation of water molecules that were hydrogen bonded to the polar sidechain such as Gln15, [58] as well as the enhanced conformational degree of freedom (e.g., conformational entropy) of the extended backbone conformation.The transition to the extended motif increases the intrastrand conformational entropy which will help to compensate for the translational entropy penalty that must be paid during the hetero-dimer formation.Ultimately, in addition to intermolecular ionic and hydrophobic interactions and hydrogen bonds, such structural alteration in A can modify its oligomerization and fibrilization, as the self-recognition site is critically involved in A aggregation. [7,8]Overall, the results and observations by our MD simulations validate that APP-C31 possibly interacts with the N-terminal region and the selfrecognition site of A 40 , which may induce its conformational transition.

Interactions of APP-C31 with Metal Ions and Metal-A𝜷 40 and Its Impact on Metal-A𝜷 40 Aggregation
][25][26][27][28] We questioned whether APP-C31 can sequester the metal ion from metal-A 40 or form a ternary complex.The binding affinity of APP-C31 to Cu(II) and Zn(II) was probed by ITC, as shown in Figure S19, Supporting Information.Upon titration of Cu(II) or Zn(II) to APP-C31, two ITC peaks were indicated in the earlier thermograms.A sharp ITC peak was followed by a broad ITC peak, suggesting that metal binding to APP-C31 caused its aggregation based on previously reported observations. [59]Thus, although we could not determine the thermodynamic parameters of intermolecular interactions, the apparent values of the Gibbs free energy change (ΔG), the enthalpy change (ΔH), the entropy change (ΔS), and the dissociation constant (K d ) were obtained.When Cu(II) was titrated to APP-C31, an exergonic reaction was observed [ΔG = −6.5 (±0.1) kcal mol −1 ; ΔH = −0.5 (±0.1) kcal mol −1 ; TΔS = 6.0 (±0.1) kcal mol −1 ] with the K d value of 8.7 (±1.5) μm.Similarly, APP-C31 binding to Zn(II) was also exergonic [ΔG = −6.2(±0.1) kcal mol −1 ; ΔH = 0.3 (±0.1) kcal mol −1 ; TΔS = 6.5 (±0.1) kcal mol −1 ].The K d value for Zn(II)-APP-C31 was measured to be 18.0 (±4.6) μm.The increase in entropy observed here is likely due to the release of water molecules that were tightly bound to the metal ion in the first solvent shell and become free water molecules as the metal ion binds to the peptide. [60]Given that the K d values of Cu(II)-A and Zn(II)-A are 10 −10 and 10 −6 m, respectively, [7,23,26,27,61,62] APP-C31 may not be able to extract the metal ion from metal-bound A.
When Cu(II) or Zn(II) binding to APP-C31 was monitored by ESI-MS, the low peak corresponding to [Cu(II) + APP-C31] 4+ was observed, indicative of the Cu(II)-APP-C31 complexation, whereas Zn(II)-bound APP-C31 was not observed under our experimental conditions (Figure S20, Supporting Information).It is likely due to the disruption of metal-APP-C31 interactions during the transition from solution to the gas phase used for ESI-MS.Different from peptide-peptide interactions, only four to six non-covalent bonds regulate metal-peptide complexation and, thus, the bonds can be relatively easily broken in electrospray droplets. [63]Despite the experimental limitation to observe metal-peptide complexes, the interaction of APP-C31 with Cu(II)-treated A 40 was verified by ESI-MS.As illustrated in Figure 3a and  15 N HSQC NMR spectroscopy.As described in Figure S23, Supporting Information, when Cu(II) was treated to a solution of uniformly 15 N-labeled A 40 , noticeable changes in CSPs were identified at several regions of A 40 (e.g., Asp7, Ser8, Tyr10, Glu11, Gln15, Lys16, Val18, Phe19, Gly29, Ala30, and Ile31).In addition, the treatment of Cu(II) induced an overall reduction in the signal intensity of 15 N-labeled A 40 , and further decreased intensities at the N-terminal half part of A 40 due to its paramagnetic effects.These results indicate intermolecular interactions between Cu(II) and A 40 as well as complexation, consistent with previous studies. [64,65]When APP-C31 was added to Cu(II)-A 40 , significant changes in CSPs were observed at the Glu3, Arg5, Ser8, Tyr10, Glu11, Val12, Gln15, Lys16, Leu17, and Ala21 residues in 15 N-labeled A 40 (Figure 3b).All these results suggest APP-C31 binding to the N-terminal and the central self-recognition sites of Cu(II)-A 40 .Therefore, our studies manifest that APP-C31 can directly interact with metal ions and form a ternary complex with metal-A 40 .
Moving forward, the influence of APP-C31 on the aggregation of metal-A 40 was investigated by the ThT assay and TEM.When Cu(II) was incubated with A 40 , aggregates were not significantly generated up to 60 h, as depicted in Figure 3c,d.In the presence of APP-C31, the rapid amyloid aggregation of Cu(II)-A 40 was induced displaying the t lag and t 1/2 values of 18.4 (±0.8) h and 23.2 (±1.1) h, respectively.Linear amyloid fibrils with short lengths were only detected from the sample of Cu(II)-A 40 with APP-C31, as presented in Figure 3e.It is noteworthy that the noticeable fibrillary species were not obtained from Cu(II)-A 40 , contrary to our earlier work, [66] which may result from the difference in manufacturers, buffer and salt concentrations, consumables, and instruments for A aggregation. [8,40,66,67]In the case of Zn(II)-A 40 , the aggregation was initiated without the lag phase, which is a typical nucleation-independent amorphous aggregation, while APP-C31 altered Zn(II)-A 40 aggregation pathways exhibiting the biphasic growth of the aggregation (Figure 3d).TEM results indicated that the addition of APP-C31 produced a mixture of amorphous aggregates and amyloid fibrils, different from amorphous aggregates mainly obtained by incubation of Zn(II)-A 40 (Figure 3e).It should be emphasized that APP-C31 with Cu(II) and Zn(II) in the absence of A 40 did not change ThT fluorescence and, thus, amyloid fibrils did not form with metaltreated APP-C31 under our experimental conditions (Figure S24, Supporting Information).Only small globular aggregates were monitored from Cu(II)-added APP-C31 by TEM (Figure S25, Supporting Information).Overall, our investigations reveal that APP-C31 modulates the aggregation of Cu(II)-A 40 and Zn(II)-A 40 in a distinct manner.

Cytotoxicity of A𝜷 40 Associated with APP-C31
As APP-C31 accelerates the aggregation of intracellular A 40 and triggers the deposition of A 40 aggregates especially at the nuclear and perinuclear regions, as illustrated in Figure 1, its influence on the cytotoxicity associated with intracellular A 40 was monitored by microinjection experiments.A 40 species with and without APP-C31 and metal ions were injected into living cells, as described in Figure 4a, with Texas Red-labeled dextran which is an indicator for the cells treated with the samples. [34]fter 24 h incubation, cell viability was determined by counting the Texas Red-positive cells following previously reported methods. [68]After injection with either APP-C31 or A 40 , about 95% of the cells survived under our experimental conditions, as displayed in Figure 4b.When both APP-C31 and A 40 were administered inside the cells, cell death was enhanced by ≈40%.In the case of Cu(II)-and Zn(II)-added A 40 , the viability was 84% and 93%, respectively.Upon addition of metal-treated A 40 with APP-C31 inside the cells, the presence of Cu(II)-A 40 or Zn(II)-A 40 with APP-C31 induced relatively more cytotoxicity exhibiting 78% and 86% viability, respectively.Thus, our microinjection studies demonstrate that APP-C31 aggravates the cytotoxicity of intracellular A 40 with and without metal ions to different degrees.
Thus far, the role of APP-C31 in inducing apoptosis and increasing the presence of A has been suggested; [12][13][14] however, it remains elusive.We investigated whether the treatment of both APP-C31 and A 40 inside the cells induces apoptosis by applying live-cell imaging experiments using annexin V staining.Annexin V conjugated with Alexa Fluor 488 can stain externalized phosphatidylserine in apoptotic cells, thereby emitting green fluorescence. [69]The overlap between green and red fluorescence from annexin V and Texas Red-labeled dextran, respectively, indicates the cells that are injected with the samples and undergo apoptosis.The degree of the colocalization between green and red fluorescence was analyzed by Pearson's coefficient (r), a type of correlation coefficient that represents the strength of the association between the two variables. [70]When APP-C31 or A 40 was added inside the cells followed by 24 h incubation, the cells did not significantly show the green fluorescence of annexin V, as displayed in Figure 4c.The r value was ≈0.2, indicative of the poor association between apoptosis and the internalization of APP-C31 or A 40 under our experimental conditions.Notably, the injection of both APP-C31 and A 40 into the cells resulted in the intense green fluorescence of annexin V with an r value of ≈0.6.Clearly, these results support that the intracellular presence of the two peptides can cause apoptosis.
Next, the effect of A 40 species on human neuronal growth was investigated in the presence of various concentrations of APP-C31 (Figure 5a-c and Figure S26, Supporting Information).Human neurons differentiated from H9 human embryonic stem cells were prepared, and peptides were introduced into the media of human neurons, as described in Figure 5a.After 24 h and 72 h incubation, the length of neurites was measured to monitor neuronal growth.Neurons incubated with A 40 for 24 h exhibited slightly shortened neurites by ≈10%, compared to vehicleadded neurons (Figure 5b).The averaged neurite length further decreased when A 40 with stoichiometric or suprastoichiometric amounts of APP-C31 was treated to neurons.After 72 h incubation, neurons incubated with A 40 displayed the reduced length of neurites by about 35%, compared to the control.More noticeably, the addition of A 40 and various concentrations of APP-C31 to neurons led to the reduction of the neurite length by 50% to 65% and exhibited the fragmentation of neurites.These observations suggest that the A 40 -induced degeneration of neurites can be intensified by APP-C31.
On the basis of these results, the impact of APP-C31 on the toxicity of A 40 species was further probed in vivo (murine brains) through immunohistochemical investigations.As depicted in Figure 5d, A 40 was directly injected with or without APP-C31 into the hippocampus, where protein deposits and C-terminally cleaved APP are dominantly observed in the ADaffected brain. [3,6]After 10 days, the brain sections were stained with various fluorescent dyes or antibodies detecting the nucleus, A, APP-C31, and microglia.Compared to the hippocampus of the mice injected with A 40 , the 6E10-positive area was significantly enhanced when both A 40 and APP-C31 were injected (Figure 5e,f).These results imply the potential involvement of APP-C31 in the assembly of A 40 in the brain.It should be noted that the fluorescent signal of thioflavin-S in response to amyloid fibrils was not significantly detected (data not shown), which may be due to the slow fibrilization of A 40 under our experimental settings. [3]Moreover, it was found that the number of the Iba1-positive microglial cells [5] showed a 3.7-fold increase upon injection of both A 40 and APP-C31, compared to that of A 40 only.Given the association of the enhanced microglial recruitment with the activation of immune responses, [3,4] these results manifest that the A 40 -induced inflammatory response can be further amplified by addition of APP-C31.Taken together, our in vitro and in vivo studies manifest that APP-C31 can detrimentally impact the toxicity of A 40 .

Conclusion
APP-C31 is generated by cleaving the intracellular C-terminal region of APP by caspases. [1]This peptide is suggested to be a toxic APP fragment.The role of APP-C31 in synaptic damage and neurodegeneration has been identified. [1,10,12,13]][13] Moreover, it is likely that APP-C31 may alter the cellular A 42 -to-A 40 ratio that could affect amyloid aggregation and neurotoxicity. [14,71]But to date, it is not known whether APP-C31 directly interacts with A and alters its misfolding and aggregation that ultimately leads to the A-related toxicity.We report for the first time that APP-C31 directly affects the aggregation and toxicity of A.We found that metal ions such as Cu(II) and Zn(II) that are known to bind to A in AD-affiliated brains have notable influence without changing the overall impact decisively.
Our work illustrates that APP-C31 can form a hetero-dimer complex with A 40 through the contacts onto the N-terminal region and the self-recognition site of A 40 .Our computer simulations suggest that upon complexation with APP-C31, A 40 unfolds the -helical segment at the self-recognition site.This structural rearrangement facilitates the -sheet formation that is critical for the aggregation of A. [7,8,72]Notably, APP-C31 promotes the aggregation of A 40 possibly via affecting the primary nucleation step.The aggregation kinetics of A 42 is slightly influenced by APP-C31, but a noticeable change in the morphology of the resultant A 42 aggregates is observed.These results imply that APP-C31 may interact with A 42 and cause its polymorphic amyloidogenesis.We speculate that the faster fibrilization kinetics and higher aggregation propensities of A 42 than A 40 may lead to fewer intermolecular interactions between APP-C31 and A 42 . [73]In the near future, we will conduct high-resolution structural studies of A 42 aggregates with APP-C31 which can advance our better understanding of the role of APP-C31 in the aggregation of A peptides.
In the presence of metal ions, APP-C31 accelerates the aggregation of Cu(II)-A 40 and modifies the aggregation pathways of Zn(II)-A 40 .In living cells, larger A 40 aggregates are intracellularly produced in the presence of APP-C31.APP-C31 can induce the perinuclear and intranuclear deposition of A 40 aggregates.APP-C31 may assist in translocating A 40 into the perinuclear region or nuclei, consistent with previous observations that the C-terminal fragments of APP can transfer into nuclei after being complexed with other proteins. [74,75]This is further supported by the recent findings that perinuclear A aggregates are colocalized with the C-terminal region of APP. [15,16]Moreover, A 40 incubated with APP-C31 significantly suppresses the outgrowth of neurites in human neurons and notably amplifies the immune response in vivo (murine brains).Clearly, APP-C31 can exacerbate the toxicity mediated by A 40 .The toxicity of metal-bound A 40 is less than that of metal-free A 40 in the presence of APP-C31, suggesting that the altered structure and aggregation pathways of A by metal ions may affect their interactions with APP-C31 and, concomitantly, induce different toxicity pathways.Overall, our combined experimental and theoretical studies demonstrate that the interactions of APP-C31 with both metal-free A 40 and metal-A 40 transform their aggregation and toxicity profiles.

Experimental Section
Materials and Methods: All reagents were purchased from commercial suppliers and used as received unless otherwise stated.A 40 (DAE-FRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV) and A 42 (DAE-FRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA) were obtained from Peptide Institute, Inc. (Osaka, Japan) that was purified by highperformance liquid chromatography (HPLC) using YMC Pack ODS-A (YMC CO., LTD., Kyoto, Japan) and Agilent ZORBAX 300SB-C18 columns (Agilent, Santa Clara, CA, USA), respectively, or Anaspec (Fremont, CA, USA).APP-C31 (AAVTPEERHLSKMQQNGYENPTYKFFEQMQN) and HF647 A 40  Preparation of APP-C31 and A: APP-C31 and A were dissolved in ddH 2 O and ammonium hydroxide (NH 4 OH; 1% w/w, aq), respectively.After lyophilizing the resulting solutions, the peptides were stored at −80 °C.A stock solution of A was prepared by dissolving the lyophilized peptide with NH 4 OH (1% w/w, aq; 10 μL) and diluting with ddH 2 O, as previously reported. [76]A stock solution of APP-C31 was prepared by dissolving the lyophilized peptide in ddH 2 O.The concentration of A was determined by measuring the absorbance of the solution at 280 nm ( 280 = 1,450 M −1 cm −1 for A 40 ;  280 = 1,490 M −1 cm −1 for A 42 ). [76]As the extinction coefficient of APP-C31 has not been reported, the concentration of APP-C31 was determined based on the theoretical extinction coefficient ( 280 = 2,980 M -1 cm -1 ) obtained from the ExPASy ProtParam online tool. [77]hT Fluorescence Assay: A (20 μm), APP-C31 (20, 100, and 200 μm), A 40 seeds (5% v/v), and metal ions (18 μm for CuCl 2 ; 20 μm for ZnCl 2 ) were incubated with ThT (5 μm) at 37 °C with constant agitation (559 cpm) in 20 mm HEPES [4-(2-hydroxyethyl)−1-piperazineethanesulfonic acid], pH 7.4, 150 mm NaCl.A 40 seeds were prepared by incubation of A 40 (20 μm) for 40 h at 37 °C with constant agitation (250 rpm) in 20 mm HEPES, pH 7.4, 150 mm NaCl followed by ultrasonication with a Q55 sonicator (Qsonica, Newtown, CT, USA).All experiments were prepared in half-area 96-well plates that have a non-binding surface (Corning, Kennebunk, ME, USA).The fluorescence intensity was measured by a microplate reader ( ex = 445 nm;  em = 485 nm).The kinetic parameters of A aggregation were acquired by fitting the emission curves using a modified Boltzmann-sigmoidal Equation (1) in the Origin software: F 0 and A are the initial fluorescence intensity and amplitude, respectively. [40]t 1/2 indicates the half time when fluorescence reaches 50% of its maximum intensity. [40]k is the rate constant of the elongation phase.The lag time, t lag , was defined using an Equation (2). [40]Experiments were performed in triplicate.Data are presented as mean ± s.e.m. (standard error of the mean) of all experiments.Gel/Western Blot: Samples containing A (25 μm) with and without APP-C31 (25 μm) in the absence and presence of CuCl 2 or ZnCl 2 (25 μm)  were incubated in 20 mm HEPES, pH 7.4, 150 mm NaCl at 37 °C with constant agitation.After 24 h incubation, The samples (10 μL) were separated on a 10%-20% Tris-tricine gel (Invitrogen, Grand Island, NY, USA).Following separation, the proteins were transferred onto nitrocellulose membranes and blocked with bovine serum albumin (BSA, 3% w/v, Biosesang, Seongnam, Republic of Korea) in Tris-buffered saline (TBS) containing 0.1% v/v Tween-20 (TBS-T) for 3 h at room temperature.Then, the membrane was incubated with a primary antibody [an anti-A antibody (6E10; 1:2,000, Covance, Princeton, NJ, USA) or an anti-APP C-terminus antibody (Y188; 1:2,000, Abcam, Cambridge, MA, USA)] for 4 h at room temperature.After washing with TBS-T three times (7 min each), the horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:2,500 for 6E10; Cayman, Ann Arbor, MI, USA) or the goat anti-rabbit secondary antibody (1:2,000 for Y188 antibody; Promega, Madison, WI, USA) in the solution of BSA (2% w/v in TBS-T) was added to the membrane and incubated for 1 h at room temperature.The membrane was visualized by an imaging system with a homemade ECL kit. [78]To monitor both peptides in the samples, the same membrane was stripped after the visualization by treating it with hydrogen peroxide (H 2 O 2 ) for 30 min at room temperature followed by washing four times with TBS-T for 10 min each, blocking with the solution of BSA [3% w/v in TBS-T (0.01% v/v)], and incubating with the other antibody.
TEM: Samples for TEM measurements were prepared based on previously published methods. [76]Glow-discharged grids (Formvar/Carbon 300-mesh, Electron Microscopy Sciences, Hatfield, PA, USA) were treated with the resultant samples for 2 min at room temperature.The excess sample was removed using a filter paper.Each grid was washed three times with ddH 2 O and incubated with uranyl acetate (1% w/v ddH 2 O; 5 μL) for 1 min.After removing excess uranyl acetate, the grids were dried overnight at room temperature.Images of each grid were taken at 200 kV with a magnification of 29,000×.For the TEM analysis, the location of samples on the grids was randomly selected for taking more than 15 images per each grid.
CD Spectroscopy: APP-C31 (0.4 mg mL −1 ) was incubated for 0 and 40 h at 37 °C in 20 mm HEPES, pH 7.4, 150 mm NaF.The samples were prepared in a half-area 96-well plate that has a non-binding surface (Corning).The CD spectra of APP-C31 samples were collected in the range from 200 to 250 nm with a cell path length of 0.5 mm.The digital integration time, the bandwidth, and the scanning speed were 4 s, 2 nm, and 20 nm min −1 , respectively.Each spectrum was smoothed by Fourier transforms.
Cell Culture and Microinjection: The human neuroblastoma SH-SY5Y cell line was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).The cell line was maintained in media containing 50% v/v Dulbecco's Modified Eagle Medium and 50% v/v Nutrient Mixture F12 (phenol red-free DMEM/F12; GIBCO, Grand Island, NY, USA) supplemented with 10% v/v fetal bovine serum (GIBCO) and 100 U mL −1 penicillin-streptomycin (GIBCO).Cells were grown and maintained at 37 °C in a humidified atmosphere with 5% CO 2 .The cells used for the studies did not indicate mycoplasma contamination.Cells were plated on an imaging dish (35 mm; 1.0 × 10 5 cells per 1 mL) and incubated for 48 h before microinjection.The solutions of peptides were centrifuged at 5,000 rpm for 1 min to remove large aggregates that can clog a microinjection glass capillary.The solutions of either A 40 (10 μm), APP-C31 (10 μm), or both peptides in the absence and presence of CuCl 2 or ZnCl 2 (10 μm) were mixed with dextran labeled with Texas Red (0.4 mg mL −1 , 3000 MW; Invitrogen, Carlsbad, CA, USA) followed by injection into the cells using an InjectMan 4 micromanipulator and a Femtojet 4i microinjector.The injection pressure (P i ), the compensation pressure (P c ), and the injection time (t i ) were set to 50 hPa, 20 hPa, and 0.2 s, respectively.Cell viability was determined based on the previously reported procedures. [68]After microinjection, the number of Texas Red-positive cells was first counted after 3 h and it was assigned as 100% of cell viability to exclude the cells damaged by microinjection.Cells were counted after 24 h incubation and cell viability (%) was calculated.At least 100 cells from each sample were injected and analyzed.Experiments were performed at least in triplicate.Data are represented as mean ± s.e.m (standard error of the mean) of three independent experiments.Fluorescence analysis of the cells was conducted by a fluorescence microscope with an RFP light cube [excitation at 531 (±40) nm and emission at 593 (±40) nm].
Annexin V Staining: SH-SY5Y cells were plated on an imaging dish (35 mm; 0.3 × 10 5 cells per 1 mL) and incubated for 48 h.The solutions of either A 40 (10 μm), APP-C31 (10 μm), or both peptides were mixed with dextran labeled with Texas Red, and the resultant solutions were microinjected into the cells, as described above.After 24 h incubation, the cells were washed with annexin-binding buffer and stained with annexin V-Alexa Fluor 488 for detecting apoptosis following the manufacturer's instruction (FITC Annexin V/Dead Cell Apoptosis Kit; V13241, Invitrogen).Fluorescent responses of annexin V and Texas Red-labeled dextran were identified by a microscope with a GFP light cube [excitation at 470 (±22) nm and emission at 525 (±50) nm] and a Cy5 light cube [excitation at 628 (±40) nm and emission at 685 (±40) nm], respectively.At least five randomly selected fields per conditions were captured with a microscope.Pearson's correlation coefficient for the indication of co-localization was calculated with the Coloc2 plugin in Fiji software. [79]onfocal Microscopy: All experiments were conducted with A 40 conjugated to the HiLyte Fluor 647 (HF647) fluorophore at the N-terminus (Anaspec).The concentration of HF647 A 40 was determined by measuring the absorbance at 649 nm ( 649 = 250,000 M -1 cm -1 ). [36]HF647 A 40 (10  μm) with or without APP-C31 (10 μm) was microinjected into SH-SY5Y cells followed by incubation for 24 h.HF647 A 40 aggregates were monitored from three or more randomly selected fields per condition with a lateral resolution of 120 nm.A series of the Z-stack images was acquired with the step size of 2 μm.The size of HF647 A 40 aggregates was determined by the "analyze particles" program of Fiji software based on the previously reported reports. [37]All pixels above the threshold value were selected to calculate the area of HF647 A 40 aggregates.This analysis included HF647 A 40 aggregates with the size larger than 0.05 μm 2 .To stain the nuclei of cells, cells were incubated with a SYTO16 green fluorescent nucleic acid stain dye (2.5 μm; Invitrogen) for 10 min.Images were obtained by adopting a confocal microscope ( ex = 488 nm for a nucleic acid dye;  ex = 633 nm for HF647 A 40 ).
Measurement of Neurite Outgrowth: Human NPCs were differentiated into neurons for 2 d and the media was replaced by fresh media.Neurons were treated with either A (20 μm), APP-C31 (10, 20, 40, and 100 μm), or both peptides followed by incubation for 24 and 72 h.Under each condition, at least 5 randomly selected fields were captured with an Olympus IX71 microscope.The neurite of a neuron measured from the soma to the tip was analyzed using Neuron J program of Fiji software. [81]SI-MS: APP-C31 (100 μm) was incubated with A 40 (100 μm) in the absence and presence of CuCl 2 or ZnCl 2 (100 μm) for 2 h at 37 °C in 20 mm ammonium acetate, pH 7.3.The samples were prepared in microtubes (Eppendorf).Before injection into the mass spectrometer, the resultant samples were diluted by tenfold with LC-grade H 2 O.The capillary voltage, the drying gas flow, and the gas temperature were set to 5.8 kV, 12 L min −1 , and 300 °C, respectively.The measurements were conducted in triplicate.
Stereotaxic Peptide Delivery in the Mouse Brain: All experiments were performed in accordance with approved animal protocols and guidelines established by Korea Research Institute of Bioscience and Biotechnology (KRIBB).C57BL/6J (11 weeks old; 30 g) were anesthetized by the intraperitoneal injection of tribromoethanol (320 mg kg −1 , i.p.) and positioned in a stereotaxic apparatus.Mice were injected with 1 μL of APP-C31 (200 μm), 1 μL of A 40 peptide (100 μm) or 1 μL of the mixture of A 40 (100 μm) and APP-C31 (200 μm) peptides into the hippocampus (anteroposterior, −2.0 mm; mediolateral, ±1.5 mm; dorsoventral, −1.7 mm from bregma) according to the mouse brain atlas. [82]All infusions were given using a Hamilton syringe equipped with a 26-gauge beveled needle and attached to a Nanojet stereotaxic syringe pump (Chemyx, Inc., TX, USA).Infusions were delivered at a rate of 0.2 μL min −1 .After injection, the needle was left in place for an additional 5 min before being slowly retracted.After 10 days, animals were sacrificed and their brains were harvested after surgery.
ITC: All samples were dissolved in the buffered solution (20 mm HEPES, pH 7.4), and the resultant solutions were degassed for 3 min prior to the loading into the ITC instrument.To investigate the binding affinity of APP-C31 to A 40 , the final concentrations of APP-C31 (in the syringe) and A 40 (in the cell) were adjusted to 600 and 30 μm, respectively.To measure the binding affinity of metal ions to APP-C31, 2.1 mm of CuCl 2 or ZnCl 2 (in the syringe) and 100 μm of APP-C31 (in the cell) were used for experiments.Titration experiments composed of 28 injections were conducted.The injection volume was 2 μL for the first injection to minimize effects of bubbles and 10 μL for the remaining injections.To prevent A 40 aggregation, the temperature and stirring speed were set to 10 °C and 260 rpm, respectively.The initial delay was 1800 s and the reference power was 10 μcal s −1 .The heats of dilution of APP-C31, CuCl 2 , and ZnCl 2 titrated into the same buffer were also examined.The ITC thermogram and binding isotherm were displayed after subtracting the dilution heat.The binding isotherms after baseline correction were fitted to the one set of sites binding model incorporated in the MicroCal Origin 7.0 software. [84]D 1 H- 15 N HSQC NMR Spectroscopy: NMR samples were prepared as described in the previous study. [66]Uniformly 15 N-labeled A 40 (rPeptide Inc., Bogart, GA, USA) was initially dissolved in NH 4 OH (1% w/w, aq; 10 μL).After lyophilizing the resulting solution, the peptide was stored at −80 °C.A solution of the 15 N-labeled A 40 monomer was prepared by dissolving the lyophilized peptide in the chilled NaOH solution (10 mm) to make a stock concentration of ≈200 μm.For 2D 1 H-15 N HSQC measurements, the stock solution was further diluted to be 40 μm of 15 N-labeled A 40 in the buffered solution (20 mm HEPES, pH 7.4) containing 10% v/v D 2 O, and then treated with APP-C31 (200 μm).All HSQC spectra were obtained at 5 °C.Each spectrum was obtained from 256 t 1 experiments using 64 transients and 1 s recycle delay.Data were processed by NMRPipe [85] and analyzed by Sparky. [86]The assignment of backbone resonance was carried out based on the previously assigned information. [84]CSP (Δ NH ) was calculated by the Equation (3): where A standard pulse sequence with water suppression by excitation sculpting (zgesgp from Bruker Topspin using the standard parameter set) was used.For each spectrum, 128 transients were collected into 32,768 data points over a spectrum width of 20 ppm.Data were analyzed using TopSpin 3.6.1 software. [87]reparation of MD Simulations: The atomistic NMR structure of A 40 deposited in the protein data bank (PDB 2LFM) [55] was used as the initial conformation of A monomer.For APP-C31, the X-ray crystal structure of APP 666-693 excised from the intracellular domain of APP complexed with Fe65 protein (PDB 3DXC) [56] was selected as the starting conformation of the protein.MD simulations of APP 666-693 monomer, A 40 monomer, and the APP 666-693 -A 40 dimer were conducted by a GPU enabled Am-ber16 software suite. [47]Each monomer was immersed in an octahedral solvation box filled with TIP3P water molecules. [48]For the APP 666-693 -A 40 dimer, 34 distinct dimeric conformations that were sampled from metadynamics MD simulations were used as the initial conformations (vide infra).The minimal distance from the protein surface to the solvation boundary was at least 12 Å.Monovalent counterions (Na + or Cl -) were added to ensure the charge neutrality of the simulated systems.To model the peptides, the ff14SB all atom amino acid force fields parameter set was adopted. [49]MD simulations were applied to 0.2 fs time integration steps in conjunction with the Settle algorithm that fixes the bond distance between a hydrogen atom and its heavy atom partner. [50]Particle Mesh Ewald (PME) method was employed to accelerate the calculations of long-range electrostatic interactions. [88]The simulations began by 4,000 steps of energy minimizations.The temperature of each solvated model was increased from 0 to 100 K in 2 ns while restraining each carbon atom with a harmonic potential of spring constant as 2 kcal mol −1 Å −2 .The temperature was then increased from 100 to 300 K in 10 ns while restraining each C atom with a harmonic potential of spring constant as 10 kcal mol −1 Å −2 .Prior to the production periods, additional 10 ns of equilibrations was performed while keeping the temperature at 300 K and the pressure at 1 atm.The production runs lasted for 500 ns for the monomer and dimer models without any restraints to the solutes.
Metadynamics MD Simulations: Metadynamics MD simulations were employed to investigate the interfaces of the APP 666-693 -A 40 dimer.The COM distance of the two monomeric peptides, APP 666-693 and A 40 , was used as the reaction coordinate of the dimerization.Gibbs free energy profile was computed along the chosen reaction coordinates without additional processing of the simulation data. [46]Using the metadynamics MD simulations, reversible association of the two monomers was monitored followed by achieving an enriched set of the associated dimer conformations.Metadynamics MD simulations were initiated from the last step of the constant temperature-pressure MD simulations of the APP 666-693 -A 40 dimer.To enhance the convergence of the simulations, the well-tempered metadynamics method was applied with the bias temperature of 500 K. [51,52] The multi-walker method was used to diversify the dimer conformations. [53]Total 12 replicas of the dimers were simulated.Each replica was simulated for 700 ns resulting in 8.4 μs of gross simulation time.The COM distance was sampled from 5 to 40 Å and was monitored by the well-tempered metadynamics algorithm.Specifically, the 35 Å interval was discretized with equally spaced windows of 0.5 Å.As the simulation samples a certain window, the algorithm incremented the height of bias potential of the window, which promoted the simulations to sample less explored COM distance within the interval.Initially, every visitation of a certain window increased the bias potential by 0.005 kcal mol −1 , and the height of the increment gradually reduced with the help of the well-tempered metadynamics algorithm.The simulation temperature was held constant at 300 K.The convergence of the metadynamics MD simulations was identified by observing the Gibbs free energy difference (ΔG) of the bound and unbound states of the dimer.The averaged potential of mean-force (PMF) from 35 to 40 Å was determined as the energy of the unbound state, whereas that of the minimum was treated as the energy of the bound state.The energy difference began to converge after 7 μs in gross simulation time.Reversible binding and unbinding events were observed from individual metadynamics MD trajectories (Replicas A, D, H, K, and L).These observations suggested that the metadynamics MD trajectories successfully explored the underlying conformation space of the APP 666-693 -A 40 dimer interface.GPU enabled NAMD v2.12 software was used for the metadynamics MD simulations. [89]tructural Clustering of the Dimer Interfaces Sampled from Metadynamics MD Simulations: Upon completion of metadynamics MD simulations, structural cluster analysis was carried out to identify the distinct APP 666-693 -A 40 dimer interfaces.First, the dimer interfaces that show the COM distances between 10 and 15 Å were collected, where the computed Gibbs free energy profile was minimized.Those collected interfaces were subject to the structural clustering analysis using VMD software. [90]wo conformers having the backbone RMSD less than 3.0 Å were considered to be similar structures, which resulted in 34 different structural clusters.A representative conformation of each structural cluster was subject to additional 500 ns MD simulations without structural constraints.Upon completion of the constant temperature MD simulations, the COM distance between APP 666-693 and A 40 was observed.12 of the selected interfaces showed a stable COM distance profile through the course of MD simulation.The number of intermolecular contacts between APP 666-693 and the Arg5, Glu11, His13, Gln15, Leu17, Phe20, and Ala21 residues in A 40 whose CSPs were pronounced in the 2D 1 H-15 N HSQC NMR experiments was investigated.The number of intermolecular contacts was defined as the number of heavy atoms of APP 666-693 within 10.0 Å of the backbone nitrogen atoms of the seven amino acid residues in A 40 .Upon thorough inspection, Models 7 and 19 that showed the relatively stable COM distance and the intermolecular contacts congruent with the 2D 1 H- 15 N HSQC NMR studies were selected as the two representative models of the APP 666-693 -A 40 dimer interfaces and labeled as Model I and II, respectively.
Statistical Analysis: All statistical analyses were performed using Origin software. [91]The comparison between the two groups was performed with Student's t-test.Values were denoted as mean ± s.e.m. (standard error of the mean).Statistical difference was considered significant at *p < 0.05, **p < 0.01, and ***p < 0.001.All the ThT assays were performed in triplicate.For the TEM analysis, the location of samples on the grids was randomly selected for taking more than 15 images per each grid.For the cell viability assay with microinjection, at least 100 cells from each sample were injected, and the experiments were performed at least in triplicate.The size of HF647 A 40 aggregates was determined by the "analyze particles" program of Fiji software based on the previously reported studies. [37]All pixels above the threshold value were selected to calculate the area of HF647 A 40 aggregates.The analysis included HF647 A 40 aggregates with the size larger than 0.05 μm 2 .The neurite of a neuron measured from the soma to the tip was analyzed using the Neuron J program of Fiji software. [81]At least five randomly selected fields were captured with an Olympus IX71 microscope.

Figure 1 .
Figure 1.Impact of APP-C31 on the intracellular aggregation of A 40 .a) Amino acid sequences of APP-C31 (APP 665-695 ) and A 40 that are generated from the proteolytic cleavage of APP.The self-recognition site of A is highlighted in bold.b) Scheme of the microinjection experiments.c) Mean size of intracellular A 40 aggregates in the absence and presence of APP-C31 observed by confocal microscopy.Aggregates (red) with dimensions exceeding the resolution limit were analyzed from three or more randomly selected fields per condition.Conditions: [ HF647 A 40 ] = 10 μm; [APP-C31] = 10 μm; 24 h incubation.Scale bars = 5 and 10 μm.Data are represented as mean ± s.e.m. n = 52 for the group of HF647 A 40 ; n = 63 for the group of HF647 A 40 with APP-C31; *p < 0.05; Student's t-test.The size distribution of A 40 aggregates detected in both groups was summarized in Figure S2, Supporting Information.d) Z-stack images of A 40 aggregates (red) with and without APP-C31.Nuclei were stained with SYTO16 (green).HF647 A 40 aggregates observed from the nuclear region were indicated with white arrows.Individual green and red fluorescence images are presented in Figure S3, Supporting Information.Conditions: [ HF647 A 40 ] = 10 μm; [APP-C31] = 10 μm; [SYTO16] = 2.5 μm; 24 h incubation.Scale bar = 10 μm.

Figure 2 .
Figure 2. Effect of APP-C31 on the aggregation of A 40 and its interactions with A 40 .a) Scheme of the aggregation experiments.b) Degree on the aggregation of A 40 upon incubation with or without APP-C31 analyzed by the ThT assay.Experiments were carried out in triplicate.c) Morphology of the peptide aggregates produced by treatment of A 40 with or without APP-C31 detected by TEM.White arrows indicate a mixture of globular aggregates and short fibrils.Scale bar = 200 nm.Conditions: [A 40 ] = 20 μm and [APP-C31] = 20, 100, and 200 μm; 20 mm HEPES, pH 7.4, 150 mm NaCl; 37 °C; constant agitation (559 cpm).d) ESI-MS spectra of APP-C31, A 40 , and APP-C31 incubated with A 40 .Charge states are marked in the MS spectra.Conditions: [APP-C31] = 100 μm; [A 40 ] = 100 μm; 20 mm ammonium acetate, pH 7.3; 37 °C; 2 h incubation; no agitation.The samples were diluted by tenfold prior to injection to the mass spectrometer.e) Interaction of APP-C31 with monomeric A 40 analyzed by 2D 1 H-15 N HSQC NMR (900 MHz).The average of CSPs and the average + one standard deviation are indicated with solid and dashed lines, respectively.The zoomed-in images of the regions where noticeable CSPs appear are depicted in Figure S11, Supporting Information.Conditions: [ 15 N-labeled A 40 ] = 40 μm; [APP-C31] = 200 μm; 20 mm HEPES, pH 7.4; 5 °C.The amino acid residues indicated in blue or black asterisks represent the residues that were unresolved or not significantly shifted, respectively.f) Conformations of A 40 (PDB 2LFM)[55] and APP 666-693 [PDB 3DXC; as a part of APP-C31 (APP 665-695 )][56] used for metadynamics MD simulations.The self-recognition site of A shown in Figure1ais highlighted in purple.g) A representative model of the APP 666-693 -A 40 interfaces from the trajectories of MD simulations.Possible hydrogen bonds within 3.0 Å and hydrophobic interactions observed within 4.0 Å are indicated with dashed black lines.The amino acid residues involved in hydrophobic interactions are presented in the space-filling representation.The other representative model, Model II, is illustrated in Figure S16, Supporting Information.
Figure 2. Effect of APP-C31 on the aggregation of A 40 and its interactions with A 40 .a) Scheme of the aggregation experiments.b) Degree on the aggregation of A 40 upon incubation with or without APP-C31 analyzed by the ThT assay.Experiments were carried out in triplicate.c) Morphology of the peptide aggregates produced by treatment of A 40 with or without APP-C31 detected by TEM.White arrows indicate a mixture of globular aggregates and short fibrils.Scale bar = 200 nm.Conditions: [A 40 ] = 20 μm and [APP-C31] = 20, 100, and 200 μm; 20 mm HEPES, pH 7.4, 150 mm NaCl; 37 °C; constant agitation (559 cpm).d) ESI-MS spectra of APP-C31, A 40 , and APP-C31 incubated with A 40 .Charge states are marked in the MS spectra.Conditions: [APP-C31] = 100 μm; [A 40 ] = 100 μm; 20 mm ammonium acetate, pH 7.3; 37 °C; 2 h incubation; no agitation.The samples were diluted by tenfold prior to injection to the mass spectrometer.e) Interaction of APP-C31 with monomeric A 40 analyzed by 2D 1 H-15 N HSQC NMR (900 MHz).The average of CSPs and the average + one standard deviation are indicated with solid and dashed lines, respectively.The zoomed-in images of the regions where noticeable CSPs appear are depicted in Figure S11, Supporting Information.Conditions: [ 15 N-labeled A 40 ] = 40 μm; [APP-C31] = 200 μm; 20 mm HEPES, pH 7.4; 5 °C.The amino acid residues indicated in blue or black asterisks represent the residues that were unresolved or not significantly shifted, respectively.f) Conformations of A 40 (PDB 2LFM)[55] and APP 666-693 [PDB 3DXC; as a part of APP-C31 (APP 665-695 )][56] used for metadynamics MD simulations.The self-recognition site of A shown in Figure1ais highlighted in purple.g) A representative model of the APP 666-693 -A 40 interfaces from the trajectories of MD simulations.Possible hydrogen bonds within 3.0 Å and hydrophobic interactions observed within 4.0 Å are indicated with dashed black lines.The amino acid residues involved in hydrophobic interactions are presented in the space-filling representation.The other representative model, Model II, is illustrated in Figure S16, Supporting Information.
Additionally, multiple hydrogen bonds are observed in the APP 666-693 -A 40 dimeric interfaces.The Asp7 residue of A 40 constructs hydrogen bonds with Tyr687 and Asn684 of APP 666-693 in Model I and II, respectively.Furthermore, the backbone carbonyl group of Val12 (Model I) and the imidazole nitrogen atom of His13 (Model II) in A 40 produce the backbone-side chain and side chain-side chain hydrogen bonds with Asn684 of APP 666-693 , respectively.Hydrogen bonding between Lys16 adjacent to the self-recognition site of A 40 and the backbone carbonyl group of Gly681 or the hydroxyl group of Thr686 in APP 666-693 is also monitored in Model I.As indicated in Model II, Thr686 of APP 666-693 additionally forms hydrogen bonding with the backbone amide moiety of Val18 in A 40 .Moreover, the intermolecular hydrophobic contacts between the aliphatic side chain of Val18 in A 40 and the aromatic rings of Tyr682, Phe689, and Phe690 in APP 666-693 are revealed in both models.
were purchased from AnaSpec.Double-distilled water (ddH 2 O) used for all experiments was obtained from a Milli-Q Direct 16 system (Merck KGaA, Darmstadt, Germany).Trace metal contamination was removed from the solutions used for the aggregation experiments by treating Chelex (Sigma-Aldrich, St. Louis, MO, USA) overnight.The concentrations of peptides were determined by an Agilent 8453 UV−Visible spectrophotometer (Agilent).Experiments by ESI-MS were performed by an Agilent 6530 Accurate Mass Quadrupole Time-of-Flight (Q-TOF) mass spectrometer with an ESI source (Agilent).2D 1 H-15 N HSQC NMR spectroscopy was conducted by an AVANCE II-900 MHz or 700 MHz NMR spectrometer [Bruker BioSpin, Rheinstetten, Germany; Korea Basic Science Institute (KBSI), Ochang, Republic of Korea] equipped with a cryogenic probe.NMR studies of APP-C31 with A 40 fibrils were carried out on a Bruker Avance II 800 MHz NMR spectrometer (Bruker BioSpin; KBSI) equipped with a cryogenic probe.ITC was performed by a VP-ITC instrument equipped with a motor-driven syringe (Malvern Panalytical, Malvern, UK).The secondary structure of APP-C31 was analyzed by a JASCO-815 150-L CD spectropolarimeter [Jasco Inc., Tokyo, Japan; KAIST Analysis Center for Research Advancement (KARA), Daejeon, Republic of Korea].Morphological changes of peptide aggregates were monitored by a Tecnai F20 transmission electron microscope (FEI Company, Eindhoven, Netherlands; KARA).A Synergy Neo2 Hybrid Multi-Mode microplate reader (Biotek, Winooski, VT, USA) was employed to measure the fluorescence for the ThT assay.Live-cell microinjection experiments were performed by the combined system composed of an InjectMan 4 micromanipulator (Eppendorf, Hamburg, Germany) and a Femtojet 4i microinjector (Eppendorf).Fluorescence analysis of living cells was conducted by an EVOS FL fluorescence microscope (Advanced Microscopy Group, Bothell, WA, USA).Confocal microscopic images were taken by Zeiss LSM 880 (Zeiss, Zena, Germany; KARA).
Δ H and Δ N represent the change of chemical shift in the proton and nitrogen dimensions, respectively.NMR Studies with A Fibrils: The solution of fibrillar A 40 was prepared by incubation of freshly prepared A 40 (200 μm) for 40 h at 37 °C with constant agitation (250 rpm) in 20 mm HEPES, pH 7.4.A 40 fibrils were further diluted to be 100 μm in the buffered solution (20 mm HEPES, pH 7.4) containing 10% v/v D 2 O and then added with APP-C31 (20 μm).