International Journal of Molecular Sciences Article Aβ40 Aggregation under Changeable Conditions Jofre Seira Curto 1 , Maria Rosario Fernandez 1 , Josep Cladera 2 , Núria Benseny-Cases 2, * and Natalia Sanchez de Groot 1, * 1 Unitat de Bioquímica, Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain; jofre.seira@uab.cat (J.S.C.); rosario.fernandez@uab.cat (M.R.F.) 2 Unitat de Biofísica, Departament de Bioquímica i Biologia Molecular, Centre d’Estudis en Biofísica, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain; josep.cladera@uab.cat * Correspondence: nuria.benseny@uab.cat (N.B.-C.); natalia.sanchez@uab.cat (N.S.d.G.) Abstract: Homeostasis is crucial for cell function, and disturbances in homeostasis can lead to health disorders. Under normal conditions, intracellular pH is maintained between 7.35 and 7.45. Altered endosomal and lysosomal pH together with a general drop in brain pH are associated with the aggregation of amyloid-β-peptide (Aβ) and the development of Alzheimer’s disease. Under acidic conditions, close to the Aβ isoelectric point, the absence of charges favors the formation of intermolecular contacts and promotes aggregation. Here, we analyzed how pH levels affect the aggregation of Aβ40 considering the variations in brain pH and the coexistence of different aggregated conformations. Our results suggest that different macromolecular conformations can interact with each other and influence the aggregation process. In addition, we showed that neutral pH and physiological salt concentrations favor a slow aggregation, resulting in ordered, stable fibrils, with low cytotoxic effects. Overall, we highlight the complexity of the aggregation processes occurring in different physiological and pathological environments. Keywords: amyloid-β-peptide; charge repulsion; aggregation Citation: Seira Curto, J.; Fernandez, 1. Introduction M.R.; Cladera, J.; Benseny-Cases, N.; Sanchez de Groot, N. Aβ40 The intracellular space is complex and dynamic. Cellular homeostasis depends on the Aggregation under Changeable maintenance and regulation of internal conditions in time and space. Impaired homeostasis Conditions. Int. J. Mol. Sci. 2023, 24, is an age-related problem that has been observed in many neurodegenerative diseases. 8408. https://doi.org/10.3390/ In these disorders, impaired removal of misfolded and aggregated proteins can trigger a ijms24098408 cascade of events that ultimately leads to cell death [1–3]. Similarly, the maintenance of an appropriate acid–base balance is essential for the Academic Editors: Konstantin K. correct functioning of human body processes [4]. Under normal physiological conditions, Turoverov and Irina M. Kuznetsova the intracellular and interstitial pH is between 7.35 and 7.45. Changes in pH can inhibit Received: 4 April 2023 enzymatic activity, interrupt metabolic pathways, and disturb the membrane transport of Revised: 28 April 2023 proteins. Significantly lower pH has been measured in the brain and cerebrospinal fluid Accepted: 4 May 2023 (CSF) of patients with Alzheimer’s disease (AD) [5] (Figure 1A). In addition, alteration of Published: 7 May 2023 endosomal and lysosomal pH has been related with problems in amyloid-β-peptide (Aβ) clearance and the formation of extracellular Aβ plaques [2,6]. Aβ is generated through the proteolytic cleavage of α-, β-, and γ-secretases on amyloid precursor protein (APP), a single-pass transmembrane protein [7–9]. This cleavage can Copyright: © 2023 by the authors. produce different extracellular Aβ peptides with lengths ranging from 37 to 49; however, Licensee MDPI, Basel, Switzerland. the most abundant forms are Aβ40 (~90%) and Aβ42 (~10%) [10–12]. In a disease context, This article is an open access article Aβ42 is the main major constituent of the amyloid plaques in the brains of patients with distributed under the terms and conditions of the Creative Commons Alzheimer’s disease, whereas Aβ40 aggregates accumulate at the arteriolar walls in cerebral Attribution (CC BY) license (https:// amyloid angiopathy [12–14]. The two extra residues at the C-terminus Aβ42 result in creativecommons.org/licenses/by/ faster aggregation kinetics compared to Aβ40. Interestingly, under mixed conditions, 4.0/). both peptides can interact and influence each other’s aggregation [15–18]. Aβ can enter Int. J. Mol. Sci. 2023, 24, 8408. https://doi.org/10.3390/ijms24098408 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 2 of 14 Int. J. Mol. Sci. 2023, 24, 8408 2 of 13 under mixed conditions, both peptides can interact and influence each other’s aggregation [15,16,17,18]. the Aβ can enter cell via endocytosis forthe cell via endocytosis subsequent degradation forbysubsequent lysosomesdegradation by cell [2,6,19]. When lysosomes [2,6,19]. When cell homeostasis is affected, this degradation pathway can fail, homeostasis is affected, this degradation pathway can fail, causing Aβ accumulation and causing Aβ accumulation and aggregation. Additionally, the acidity of these organelles aggregation. Additionally, the acidity of these organelles can accelerate the aggregation can accelerate the aggregation process (Figure 1A) [2,6,20]. process (Figure 1A) [2,6,20]. Figure 1. pH influences the net charge of Aβ and Alzheimer’s disease development. (A) Three pieces Figure 1. pH influences the net charge of Aβ and Alzheimer’s disease development. (A) Three pieces of evidence of pH alterations in Alzheimer’s disease. (i) Poor acidification of autolysosomes results of in evidence autophagyofdysfunction pH alterationsand in Alzheimer’s precedes disease. and Aβ deposition (i) Poor acidification amyloid of autolysosomes plaque formation [2]. (ii) Inresults in autophagy dysfunction and precedes Aβ deposition and amyloid plaque formation ApoE4 astrocytes (with the E4 allele of apolipoprotein E), downregulation of the Na+/H+ exchanger [2]. (ii) In NHE6 results in over-acidification of the endosomes and pH increase in lysosomes [6]. ApoE4 astrocytes (with the E4 allele of apolipoprotein E), downregulation of the Na+/H+ exchanger (iii) Lower pH hasresults NHE6 been measured in the brains in over-acidification of and cerebrospinal the endosomes and fluid pH (CSF) of patients increase with Alzheimer´s in lysosomes [6]. (iii) Lower pH disease (AD) [5]. (B) Scheme of the Aβ N-terminal sequence, comprising histidines whose has been measured in the brains and cerebrospinal fluid (CSF) of patients with Alzheimer´s disease protonation is affected in the pH range analyzed in this study. The scheme shows the amino acid (AD) [5].and position (B)pK Scheme of the Aβand of the histidines N-terminal sequence, the net charge at eachcomprising pH. histidines whose protonation is affected in the pH range analyzed in this study. The scheme shows the amino acid position and pK of the histidines Protein and the net charge aggregation at each influenced is highly pH. by hydrophobicity and net charge [4,21,22,23,24]. When the pH approaches the isoelectric point (pI), the absence of charges Protein removes aggregation the repulsion is and forces highly influenced favors by hydrophobicity the formation of intermolecular and net charge contacts involved [4,21–24]. When in the aggregation process. The presence of salt ions can have a similar effect by shielding the the pH approaches the isoelectric point (pI), the absence of charges removes repulsion repulsive forces and favors the ionic interactions [25].formation of intermolecular Our previous studies withcontacts involvedthat Aβ40 showed in the theaggre- gation process. aggregates formedTheatpresence neutral or ofacidic salt ions pH can havehave a similar different effect and structures by shielding repulsive cell toxicities [23,24]. ionic However, the interactions [25].processes inside studies Our previous the cell are with dynamic, and pHthat Aβ40 showed can the change regularly aggregates formed at neutral or acidic pH have different structures and cell toxicities [23,24]. However, the processes inside the cell are dynamic, and pH can change regularly in organelles [4,6]. Based on this, we aim to go forward here and shed light on the different toxic protein species that can arise under moderate pH and salt concentration changes. For this purpose, we analyzed how pH levels affect the aggregation of Aβ40 considering the changeable Int. J. Mol. Sci. 2023, 24, 8408 3 of 13 conditions of the intracellular space and the pH alterations that can occur locally in the extracellular space. Based on the pH reported for the altered endosomes and lysosomes in AD (Figure 1B), we focused on a range of pH from 5.5 to 7.4 and salt from 0 to 150 mM of NaCl (the most abundant salt found in multicellular organisms [25–27] widely used in aggregation assays [25,26,28,29]). We characterized the biophysical differences between the aggregates that form under these conditions considering the possible coexistence of different aggregated conformations and their ability to interact and interconvert. We found that salt and pH modulate the structure, stability, and cellular toxicity of the aggregates. Overall, we propose that even small cellular changes in pH and salt concentration could result in a complex and variable mixture of multimeric forms, which are able to interfere with each other to different degrees. 2. Results 2.1. pH and Salt Effect on Aβ40 Aggregation Taking into consideration the different intracellular environments where the presence of Aβ40 has been detected [6], and with the aim of understanding how pH and NaCl con- centration may influence the kinetics and conformation of Aβ40 aggregates, we measured amyloid aggregation at different pH and NaCl conditions. Here, we specifically tested four different pH values (7.4, 6.8, 6.2, and 5.5) (Figure 1B), similar to those previously measured in endosomes and lysosomes (Figure 1A), and seven concentrations of NaCl ranging from 0 to physiological conditions (150 mM), to understand the extent to which the presence of this salt influences the aggregation of Aβ40 at different pH values. Aβ40 aggregation was monitored every 10 min under quiescent conditions at 37 ◦ C (Figures 2A–D and S1). In general, lag time and half-life (t1/2) decrease when the pH becomes more acidic, consistent with the charge reduction (from −3 to nearly 0) when approaching the isoelectric point (pI, 5.3) (Figures 2E,F and S2). The increase in salt has a similar effect, which can be explained by the reduction of repulsive forces exerted by the peptide’s negative charges (Figure 2E,F). However, at a pH close to the pI, the repulsion effect was lower, we did not observe any effect of NaCl on the aggregation process, and the half-lives were similar for all conditions (Figure 2D). In contrast, when the net charge was −3 (pH 7.4) and the salt concentration was low (from 0 to 25 mM), the aggregation kinetics were too slow to be measured over the time scale reported here (16 h) (Figure 2A). Kinetic analysis for longer times showed that they achieved a plateau and remained stable (Figure S3). Meisl and coworkers obtained similar results when analyzing the effect of ionic strength on Aβ42 aggregation [25]. They found that the reduction in electrostatic repulsion surrounding the peptide can accelerate the incorporation of new monomers into the fibrils, which in turn reduces the impact that the fragmentation can have on the elongation speed [25]. To study the aggregates’ conformation, we chose a 100 mM NaCl concentration, which is close to the physiological values, and two extreme pHs (7.4 vs. 5.5). We analyzed the morphology of the aggregates using transmission electron microscopy (TEM) (Figure 2F,G) and the secondary conformation using infrared spectroscopy (IR) (Figures 2H and S4). Under these experimental conditions, the aggregates at pH 7.4 are arranged into long and straight fibrillar structures that tend to group into clumps. However, at pH 5.5 the aggregates are shorter, less ordered, and more dispersed. This agrees with the IR absorbance spectra, which show the presence of oligomeric/amorphous aggregates at pH 5.5 and, in a lower amount, at pH 6.2. The IR spectra at these pH values show a characteristic band at 1690 cm−1 together with a clear beta-sheet band at 1623 cm−1 , which is associated with the presence of an antiparallel β-sheet, which is typical of oligomers and amorphous aggregates. Several studies have associated the presence of bands at 1623 cm−1 and 1690 cm−1 to antiparallel beta, whereas the sole presence of the band around 1620 cm−1 is associated with parallel (fibrillar) beta-sheet structures [30,31]. Consistently, at pH 7.4, we detected only the 1623 cm−1 absorption peak characteristic of the beta-sheet structure, suggesting the presence of fibrils (Figure 2H and Figure S4). Conversely, at a net charge Int. J. Mol. Sci. 2023, 24, 8408 4 of 13 above -5 (pH 11), no signal of β-sheet structure (1623 cm−1 ) or amorphous aggregates (1690 cm−1 ) was detected, just a wide peak at 1645 cm−1 typical of an unfolded monomeric form [32]. In agreement with this, the initial stock sample at pH 11 only showed monomeric particles Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEWunder dynamic light scattering (DLS) [33,34], and no aggregated structures 4 of 14 were observed at the TEM grid (Figure S5). Figure 2. Effect of pH on Aβ40 aggregation and conformation. We measured aggregation kinetics at Figure four 2. Effect pH different of pH on Aβ40 values: (A) 7.4,aggregation andand (B) 6.8, (C) 6.2, conformation. We pH, (D) 5.5. For each measured we also aggregation analyzed seven kinetics at four NaCldifferent pH values: concentrations ranging(A) from 7.4, 0 to(B) 1506.8, mM. (C)(E)6.2, Plotand (D) 5.5. showing For each the Aβ40 pH, we net charge at also analyzed different pH values, seven calculated with Protein NaCl concentrations rangingCalculator from 0 tov3.3 150(Chris mM.Putnam, (E) PlotScripps showing Research Institute). the Aβ40 (F) net charge at SchemepH different showing values,the repulsivewith calculated forces between Protein Aβ molecules Calculator at highPutnam, v3.3 (Chris and low Scripps pH and Research high and lowInstitute). NaCl concentrations. (G) Plot showing the relationship between the half-life (t(1/2)) of the kinetics (F) Scheme showing the repulsive forces between Aβ molecules at high and low pH and high and presented in panels (A–D) and the NaCl concentration. TEM images of aggregates formed at (H) lowpH NaCl 5.5 andconcentrations. (I) pH 7.4. (J) FTIR (G) spectra Plot showing the relationship of Aβ40 aggregates between incubated at four the half-life different (t(1/2)) pH values (5, of the kinetics 6, 7.4,presented and 10) and in for panels 24 h.(A–D) Arrows and the NaCl indicate concentration. the position TEM images of the following of aggregates wavelengths: 1690 cm formed -1, at (H)1645 pH cm and (I) 5.5-1and 1623pH cm7.4. -1. The spectrum at pH 10 demonstrates that the initial stock solution does not (J) FTIR spectra of Aβ40 aggregates incubated at four different pH values (5, contain 6, 7.4, andbeta-sheet structure. It just shows a wide band at 1645 cm−1 corresponding to random struc- 10) and for 24 h. Arrows indicate the position of the following wavelengths: 1690 cm−1 , tures, −indicating that the 1 .peptide is monomeric at the beginning of the experiments. The peaks at 16451690 1 cmcm-1and and 1623 1623 cmcm-1−indicate The spectrum the presence at pH 10 demonstrates of antiparallel that beta-sheet the initial stock solution does structures. not contain beta-sheet structure. It just shows a wide band at 1645 cm−1 corresponding to random structures,To indicating study the that the peptide aggregates’ is monomeric conformation, weatchose the beginning a 100 mM of the NaClexperiments. The peaks concentration, which at 1690 cmis−close 1 andto thecm 1623 −1 indicate the physiological values, and two presence extreme pHs of antiparallel (7.4 vs. 5.5). beta-sheet We analyzed structures. the morphology of the aggregates using transmission electron microscopy (TEM) (Figure 2.2.2F,G) pH Exchange and Cross-Seeding and the secondary conformation using infrared spectroscopy (IR) (Figures 2H and S4). ToUnder these experimental understand how pH changes conditions,can the aggregates affect at pH of the structure 7.4preformed are arrangedaggregates into long and theand straight fibrillar interaction between structures them, we thatanalyzed tend to group (i) theinto clumps. effect of pH However, changeat(Figure pH 5.5 3A) the and aggregates are shorter, less ordered, and more dispersed. (ii) the cross-seeding between aggregates formed at different pH values (Figure 3B). The This agrees with the IR absorb- TEM ance spectra,show images whichthatshow theaggregates the presence of grownoligomeric/amorphous in acidic conditions aggregates are pHat pH 5.5 and, and sensitive, in a lower amount, at pH 6.2. The IR spectra at these pH values show a characteristic band after 24 h of−1increasing the pH to neutral, the aggregates−1progressed to straight fibrils, at 1690 cm together with a clear beta-sheet band at 1623 cm , which is associated with which were shorter than those grown at pH 7.4. These fibrils were also more abundant, to a the presence of an antiparallel β-sheet, which is typical of oligomers and amorphous ag- gregates. Several studies have associated the presence of bands at 1623 cm−1 and 1690 cm−1 Int. J. Mol. Sci. 2023, 24, 8408 5 of 13 similar extent as the amorphous aggregates at acidic pH (Figure 3C). Meanwhile, when the pHREVIEW Int. J. Mol. Sci. 2023, 24, x FOR PEER of the aggregates grown at 7.4 was decreased, no significant structural changes 6 of 14 were observed (Figure 3D). Figure 3. Effect of cross-seeding and pH switch on Aβ40 aggregation and conformation. (A) Scheme Figure 3. Effect of cross-seeding and pH switch on Aβ40 aggregation and conformation. (A) Scheme showing the different steps of the pH switch assay (related to images (C,D)). (B) Scheme showing showing the different the different steps ofsteps of the pH switch the cross-seeding assay assay (related(related to images to images (E,G,H)).(C,D)). (B) Scheme The striped patternshowing indi- the different steps cates that Aβ40 of isthe cross-seeding aggregated, assay and the (related dotted patterntothat images Aβ40(E,G,H)). is soluble.The TEM striped imagespattern for the ag-indicates gregates that Aβ40 is formed: (C) with aggregated, thethe and first 24 h atpattern dotted pH 5.5 that and the Aβ40 second ones atTEM is soluble. 7.4, (D) with the images for first 24 h the aggregates at pH 7.4 and the second ones at 5.5, (G) at pH 7.4 with seeds formed at pH formed: (C) with the first 24 h at pH 5.5 and the second ones at 7.4, (D) with the first 24 h at 5.5, and (H) at pH 5.5pH 7.4 with seeds formed at pH 7.4. (E) Aggregation kinetics at pH 7.4 with and without different seed and the second ones at 5.5, (G) at pH 7.4 with seeds formed at pH 5.5, and (H) at pH 5.5 with seeds types. The fluorescence measured with seeds formed at pH 5.5 is significatively larger than with the formed at pH 7.4. seeds formed at pH (E)7.4Aggregation (p-value < 0.01).kinetics at pHplot The inside 7.4shows with noandnormalized without different fluorescenceseed types. after 16 The fluorescence measured h of aggregation. The t1/2with seedsfor in hours formed at pHsamples the different 5.5 is significatively are as follows: larger unseeded than (6.61with the seeds ± 0.10), seeds pH formed 7.4 (5.38 at pH ± 0.01), seeds 7.4 (p-value pH 6.8 < 0.01). The(5.1 ± 0.259), inside plotseeds showspH 6.2 no (4.83 ± 0.17), and normalized seeds pH 5.5 fluorescence (4.4416 h of after ± 0.19). (F) The percentage of cell viability after incubation with different concentrations of Aβ40 aggregation. The t1/2 in hours for the different samples are as follows: unseeded (6.61 ± 0.10), aggregated at different pH values (measured using the MTT assay). The plot shows triplicates of the seeds twopH 7.4 (5.38 ± independent 0.01),The assays. seeds linespH ± 0.259), 6.8 (5.1the represent seeds pH 6.2to(4.83 mean corresponding each±concentration 0.17), and seedsof thepH 5.5 (4.44 ± 0.19). added (F) The aggregates. Thepercentage significance ofagainst cell viability afterofincubation the control with 100% viability wasdifferent measured concentrations using the of Bonferroni Aβ40 test (****, aggregated <0.0001).pH at different At concentrations values (measured of 2.5using uM and the5 MTT uM ofassay). aggregates, the fluorescence The plot shows triplicates difference of the between seeds two independent formed assays. at pH The lines5.5represent and seedsthe formed meanatcorresponding pH 7.4 is also significant (p = 0.001 to each concentration of and p = 0.0003, respectively). The measurements of the different controls are shown in Figure S6. the added aggregates. The significance against the control of 100% viability was measured using the Bonferroni 2.3. AΒ40test (****, <0.0001). Aggregates ToxicityAt concentrations of 2.5 uM and 5 uM of aggregates, the fluorescence difference between seeds formed at pH 5.5 and seeds formed at pH 7.4 is also significant (p = 0.001 Less stable aggregates are associated with the formation of more endpoints and and p = 0.0003, higher respectively). cellular damage [22,35]. The measurements Along these lines, of thewe different measuredcontrols cell are shownusing viability, in Figurethe S6. MTT assay, after 24 h of incubation with Aβ40 aggregated at pH 5.5 and 7.4 (Figures 3F Int. J. Mol. Sci. 2023, 24, 8408 6 of 13 Based on the observed morphological changes and the labile character of the aggre- gates formed at acidic pH, we analyzed their ability to seed the aggregation at pH 7.4 (Figure 3E). This is important because fibril fragility is associated with the presence of shorter aggregates, the release of a larger number of growing ends, and the acceleration of the aggregation process [22,35]. Accordingly, we measured shorter half-life times (t1/2) when Aβ40 was seeded with aggregates formed at a low pH, indicating a higher seeding ability. Interestingly, the TEM images from the different seeding assays showed aggregates with similar morphology: mature fibrils that, in some cases, formed clumps (Figure 3G,H). 2.3. AB40 Aggregates Toxicity Less stable aggregates are associated with the formation of more endpoints and higher cellular damage [22,35]. Along these lines, we measured cell viability, using the MTT assay, after 24 h of incubation with Aβ40 aggregated at pH 5.5 and 7.4 (Figures 3F and S6). The colorimetric measures showed a significant decrease in viability at concentrations above 1.25 uM, but only in cells incubated with aggregates formed at pH 5.5 when the viability decreased to close to 40%. Importantly, we previously demonstrated, using µFTIR, that cell culture conditions do not affect the presence of Aβ40 amyloid fibrils formed at pH 7.4 or pH 5.5 [36]. In addition, this assay showed an increased level of oxidation in cells incubated with aggregates formed at pH 5.5 [36], in agreement with the present results. 3. Discussion 3.1. pH Effect on Aβ40 Aggregation Recent studies have recapitulated the intracellular origin of the Aβ aggrega- tes [1–3,6,19,37], which are related to autophagy impairment and pH imbalances. Lee and co-workers observed, in AD mouse models, that the poor acidification of autolyso- somes results in autophagy dysfunction and precedes Aβ deposition and amyloid plaque formation [2]. In the case of the E4 allele of apolipoprotein E (ApoE4, the strongest genetic factor in sporadic AD), downregulation of the Na+ /H+ exchanger NHE6 results in over- acidification of endosomes and inhibition of Aβ clearance [6]. In this context, in ApoE4 astrocytes, the pH of the endosome was reduced from 6.21 to 5.37, and the pH of the lysosome increased from 4.08 to 5.20. However, pH alterations were not only detected in these intracellular organel- les [11,19,29,38–42]. The postmortem brains and cerebrospinal fluid of AD patients also show a significantly lower, although moderate, pH compared to the controls. Infusion of low-pH cerebrospinal fluid, in APP-PS1 mice, increases Aβ plaque load [4]. Moreover, a pH reduction can also be found under inflammatory and apoptotic processes and at the surface of anionic phospholipid membranes [5,43]. Overall, these events could place Aβ close to pI, reducing its solubility and favoring its self-association and aggregation [4,21,22]. As the pH changes from physiological (7.4) to the pI (5.3) [44], His6, His13, and His14 are protonated successively, generating new interactions and favoring the transition from soluble to aggregated [20,26]. Acidity affects the morphology and toxicity of Aβ aggregates (Figures 2 and 3). Protein aggregation is a very sensitive process, and the interaction and rearrangement of the molecules during the aggregate assembly could be influenced by multiple factors (temperature, charges, hydrophobicity, ionic strength), resulting in different macromolecular conformations or strains [21–24,36,38,41,45]. Amorphous and less organized structures are also associated with lower stability, large reactivity, and cellular toxicity [21–24,36,39,41,45]. Thus, small changes in the Aβ uptake and degrada- tion could affect the self-assembly process [1–3,6,37,46,47]. Moreover, under changeable conditions, different conformational assemblies can coexist, interact, and influence each other’s aggregation. 3.2. Effect of pH Exchange and Cross-Seeding on Aβ40 Aggregation Here, we evaluated the effect of changes in the electrostatic repulsion on Aβ40 aggre- gation, taking into consideration the different environments that this peptide can encounter Int. J. Mol. Sci. 2023, 24, 8408 7 of 13 in the cell [1,6,22,37–39,48,49]. We also presumed that the conditions in the cell can change Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 8 of 14 and that different Aβ40 assemblies can rise and interact. Based on this assumption, we performed a series of experiments, ranging from neutral (7.4) to acidic pH (5.5) and from physiological concentrations to 0 uM of NaCl. We also analyzed the effect of pH alteration, faster (Figure decreasing the 4). pHSimilarly, from neutral the increase or increasingin NaCl reduces it from the repulsion an acidic solution.between Following the these nega- tive charges, alterations, weaccelerating analyzed the aggregation, coexistence except under acidic of assemblies grownpH, underwhen the charges different are al- conditions. ready In nearly agreementneutralized. with previousThe aggregates publications, formed at different pH our experiments valueselectrostatic support have different re- morphologies and structural content. As previously pulsion as a driving force in aggregation, modulated by the change of pH and reported, under neutral conditions, salt con- the aggregates centration are more fibrillar [23–26,38,41,50,51]. The and pH contain decrease fewerfromoligomeric 7.4 to 5.5and protofibril results in a net structures charge than under variation fromacidic -3 topH 0.2,conditions [23,24,51]. and by getting closer A to faster Aβ40 0 s pI, the aggregation aggregation process maykineticshinder be- the correct come assembly faster (Figure of4).theSimilarly, differentthe molecules, increase resulting in less ordered in NaCl reduces aggregates the repulsion betweenthanthefi- brils presenting negative charges, antiparallel acceleratingβ-sheet structures, aggregation, exceptsimilar to those under acidic pH, detected when the in charges vitro in are the already nearly early stages of neutralized. the amyloid The fibrilaggregates formation formedprocess at different [30,31]. Here,pHwe values alteredhave thedifferent aggrega- morphologies tion conditions andbystructural incubatingcontent. As previously the preformed reported, aggregates under neutral at a second different conditions, pH or by the aggregates seeding are more the process withfibrillar different and contain fewer assemblies (Figure oligomeric and protofibril 3A,B). Overall, different structures aggregates than wereunder observedacidic at pH conditions different [23,24,51]. pH values. A fastergrown Aggregates aggregation process under acidic may hinder conditions arethe pH correct sensitiveassembly and canofseedthe different aggregation molecules, stronger resulting than thosein less ordered formed aggregates at neutral pH, than fibrils resulting in presenting a larger amountantiparallel β-sheet structures, of aggregates (Figure 3E).similarThis couldto thosebe adetected consequencein vitro in the of the early different stages of the amyloid arrangements of thesefibril formation assemblies. In process general,[30,31]. Here, forms, less ordered we altered suchthe as aggregation oligomers or conditions by incubating the preformed aggregates at a second protofibrils, are associated with lower stability and higher toxicity (such as the different pH or by seeding generation the of reactive oxygen species or the disruption of membranes) [1,23,24,39,51–53]. In linewere process with different assemblies (Figure 3A,B). Overall, different aggregates with observed this, we haveat different previously pH values. reportedAggregates that, in leakagegrown under acidicthe experiments, conditions aggregates areformed pH sen- at sitive and can seed aggregation stronger than those formed pH 5.5 have a higher capacity to disrupt phosphatidylcholine model membranes than fi- at neutral pH, resulting in abrils larger amount formed of aggregates at pH 7.4 [23]. (Figure 3E). This could be a consequence of the different arrangements In relation to the stability of theIndifferent of these assemblies. general,aggregates, less ordered weforms, observed suchthatasthe oligomers aggregatesor protofibrils, are associated with lower stability and higher formed at pH 7.4 barely changed their morphology at acidic pH, while those formed at toxicity (such as the generation of pH reactive oxygen 5.5 evolved tospecies a moreor the disruption ordered form when of membranes) placed at neutral[1,23,24,39,51–53]. pH (Figure 4). In line Thiswith sug- this, we have previously reported that, in leakage experiments, gests that aggregates formed at pH 7.4 may be more stable (less pH sensitive) than those the aggregates formed atgrown pH 5.5 athave a higher pH 5.5. capacity In addition, theto disrupt smaller phosphatidylcholine size and higher abundance model membranes of aggregates than formed fibrils formed at pH 7.4 [23]. at pH 5.5 may also provide more end-points for amyloid fibril growth [22]. Figure4.4. Effect Figure Effect of pH onon Aβ40 Aβ40aggregation, aggregation,conformation, conformation,and andtoxicity. toxicity.This table This summarizes table summarizesthe the differences between the aggregates formed at pH 7.4 and pH 5.5. At these pH values, Aβ40a differences between the aggregates formed at pH 7.4 and pH 5.5. At these pH values, Aβ40 has different has net charge a different (−3 vs. net charge (−0), resulting 3 vs. in different 0), resulting repulsion in different forces forces repulsion and aggregation speeds.speeds. and aggregation Mean- while, at pH 7.4 the aggregates are ordered and pH resistant and exhibit low toxicity. At pH 5.5, the Meanwhile, at pH 7.4 the aggregates are ordered and pH resistant and exhibit low toxicity. At pH 5.5, aggregates presented abundant oligomers and protofibrils, which are pH sensitive and toxic. the aggregates presented abundant oligomers and protofibrils, which are pH sensitive and toxic. 3.3. Aβ40 Aggregation under Complex Conditions Low pH can be found in intracellular organelles as well as in the presence of oxidized or pre-apoptotic membranes. These membranes present negative charges on their sur- faces, which increase the concentration of protons and cause a local pH decrease [23]. In Int. J. Mol. Sci. 2023, 24, 8408 8 of 13 In relation to the stability of the different aggregates, we observed that the aggregates formed at pH 7.4 barely changed their morphology at acidic pH, while those formed at pH 5.5 evolved to a more ordered form when placed at neutral pH (Figure 4). This suggests that aggregates formed at pH 7.4 may be more stable (less pH sensitive) than those grown at pH 5.5. In addition, the smaller size and higher abundance of aggregates formed at pH 5.5 may also provide more end-points for amyloid fibril growth [22]. 3.3. Aβ40 Aggregation under Complex Conditions Low pH can be found in intracellular organelles as well as in the presence of oxidized or pre-apoptotic membranes. These membranes present negative charges on their surfaces, which increase the concentration of protons and cause a local pH decrease [23]. In addition, pathological vascular events, such as ischemia and microhemorrhages related to AD, can cause a lack of oxygen, leading to a local decrease in pH in the extracellular space [54]. In patients with Alzheimer’s disease, there are changes in the concentrations of several ions. For example, Vitvitsky and co-workers found an increased concentration of positive ions, Na+ and K+ , in comparison with control brains, pointing to a possible accelerated aggregation process [55]. Aβ’s pH dependence may also be associated with the formation of different conforma- tional strains and with disease heterogeneity [21,39,56]. Accordingly, seeding experiments with Aβ aggregates obtained from the brains of patients with AD affected by different phe- notypes resulted in structurally distinct fibrils [21,22,57]. In this way, oligomers have been reported to be less stable and more toxic than amyloid fibrils [1,23,24,39,51–53]. Phenotypic severity has been also associated with fibril fragility, because its fragmentation releases new growing ends that can seed new fibrils and accelerate the aggregation process [22,35]. Our results showed that the aggregates formed under acidic conditions present higher cytotoxicity and seeding. This agrees with our previous work where larger amounts of oligomers were present at lower pHs [23,24], but also suggests the presence of a larger number of endpoints. These results also point to an amplifying effect (Figure 3E); although aggregation is slow at neutral pH, the presence, at some point, of seeds formed at lower pHs could rapidly saturate the cell with highly stable aggregates and trigger the formation of extracellular plaques [2]. In fact, the study of AD mouse model APP/PS1 brains using FTIR microscopy has demonstrated the presence of non-fibrillar aggregates in situ, which is compatible with the FTIR spectra of oligomers and aggregates formed at low pH in the early stages of the disease (3–6 months). At later stages of the disease, a decrease in this type of aggregate was observed, and a higher number of fibrillary plaques were found [58]. The present work agrees with the co-existence of different macromolecular conformations and their possible cooperation triggering a harmful aggregation process [21]. In addition, it supports neutral pH and physiological salt concentrations being conditions that favor a slow aggregation process, resulting in ordered, stable, and less cytotoxic fibrils. This agrees with previous works showing that fibrils are safer aggregates than oligomers or protofibrils [22,35]. Based on these results, blocking or slowing down the aggregation process by favoring electrostatic repulsion could be an effective strategy to reduce toxic effects [2,6]. Moreover, owing to the sensitivity of aggregates to these variable conditions, it is possible to control the conformation of aggregates and their properties, a strategy already exploited by the cell to form functional amyloid structures [59]. In the case of Aβ, this could be a positive and evolutionarily selected property to activate its antimicrobial activity, since on the bacterial membranes the negative charges could favor Aβ’s aggregation and the consequent cell disruption [60]. Overall, the data presented here highlight the complexity of the aggregation processes occurring inside the cell and in the extracellular space and support the idea that the physio-pathological processes able to modify pH can play a significant role in triggering the onset of Aβ40 aggregation. Int. J. Mol. Sci. 2023, 24, 8408 9 of 13 4. Material and Methods 4.1. AB40 Peptide Preparation Synthetic Aβ40 (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV-NH2 ) was purchased from Proteogenix (Schiltigheim, France). Stock solutions were prepared by dissolving 1 mg of the peptide to a final concentration of 250 µM and adding 20 mM sodium phosphate buffer, 0.04% NH3, and NaOH to a final pH of 11. Then, the peptide was sonicated for ten minutes (Fisherbrand Pittsburgh, PA, USA, FB15051) without sweep mode and stored at −80 ◦ C until needed [23]. All samples were prepared in low protein-binding microcentrifuge tubes (ThermoFisher Scientific, Waltham, MA, USA). The pH was measured at the start and end of all the aggregation reactions to confirm that it remained stable. For the pH change assays, 100 µL of sample after 24 h of aggregation at pH 7.4 was used, and 20 mM HCl was added to reach a pH of 5.5. For aggregates formed at pH 5.5, 20 mM NaOH was added to reach a pH of 7.4. For the seeding assays, 300 µL of preformed aggregates was obtained after 24 h of aggregation and 5 min of sonication. Preformed seeds were added to the corresponding aggregation reactions to a final seed concentration of 2.5 mM. The monomeric concentration of Aβ40 used was 25 µM. The control of unseeded reaction has the buffers at pH 7.4 and 25 µM of monomeric Aβ40 (everything but the seeds). 4.2. ThT Aggregation Kinetics Thioflavin T was dissolved in Milli-Q water at 5 mM stock, filtered with a 0.2 µm filter, diluted to 0.5 mM, and stored at −20 ◦ C prior to use. ThT fluorescence was measured every 10 min using a 440 nm excitation filter and a 480 nm emission filter using bottom-optics in a plate reader (TECAN Infinite+ NANO). Samples were placed in a flat-bottom, black, non-binding 96-well plate (Greiner bio-one). A total of 100 µL of sample was added per well. Each condition was measured in triplicate. The Aβ40 peptide stock, initially at pH 11, was diluted to 25 µM in sodium phosphate buffer under the corresponding pH and salt conditions. HCl was added at a concentration of 15 mM to reach a pH of 5.5. The pH in the wells was measured both at the start and end of the experiment to ensure the pH stability throughout the aggregation process. ThT was added to 20 mM final concentration. The aggregation reaction was performed at 37 ◦ C, under quiescent conditions. The t1/2 is the time necessary, at a given condition, to reach 50% of the final fluorescence signal. The lag phase was calculated by considering its ending as the point at which it reached 10% of the final fluorescence. 4.3. Dynamic Light Scattering The monomeric state of Aβ40 at the stock solution (250 µM, pH 11) was analyzed using a NANOTRAC FLEX in situ particle size analyzer (Microtrac TM). A total of 50 µL of the sample was added to the tip of the laser of the DLS equipment, and a 90 s analysis was performed. 4.4. Transmission Electron Microscopy After 16 h of aggregation in low protein-binding microcentrifuge tubes, under the corresponding conditions, a 10 µL sample was placed onto carbon-coated copper grids, incubated for 5 min, and dried with Whatman paper. The grids were washed with dis- tilled water, negatively stained with 2% (w/v) uranyl acetate for 2 min, and then dried. Micrographs were obtained using a JEM-1400 (JEOL, Tokyo, Japan) transmission electron microscope (TEM) operated at an accelerating voltage of 80 keV. 4.5. Infrared Spectroscopy Infrared spectroscopy was performed as described by Benseny-Cases, 2007 [24]. Briefly, 100 µM peptide was incubated for 11, 15, and 24 h in sodium phosphate buffer in D2 O at the corresponding pH at 37 ◦ C under quiescent conditions. Then, 30 mL of peptide was deposited between two CaF2 windows separated by a 50 mm Teflon separator. All Int. J. Mol. Sci. 2023, 24, 8408 10 of 13 measurements were carried out on an FTIR Mattson Polaris spectrometer equipped with a liquid-nitrogen-cooled mercury–cadmium–telluride (MCT) detector at 37 ◦ C. The spec- trometer was purged continuously with dry air. For each spectrum, an average of one thousand scans were averaged at an instrumental resolution of 2 cm−1 . The spectra were recorded in the range of 400 to 4000 cm−1 . To obtain the infrared spectrum of the peptide, the spectrum of the solvent was subtracted from the sample, and all spectra were corrected for the atmospheric water vapor contribution. Each experiment was repeated three times. 4.6. Cell Cytotoxicity Assay Neuroblastoma SH-SY5Y cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 1% glutamine, and 1% (v/v) penicillin/streptomycin. The cells were maintained at 37 ◦ C and 5% CO2 in a 75 cm2 cell culture flask. Differentiation to neuronal cells started 24 h after plating by re- placement of the maintenance medium with differentiation culture medium for 7 days and refreshment every 72 h. The differentiation culture medium consisted of DMEM supple- mented with 2.5% inactivated FBS, 1% penicillin/streptomycin, 1% glutamine, and 10 µM retinoic acid. Differentiation was monitored microscopically by morphological assessment. For the cytotoxicity assay, the cells were seeded in 96-well plates and treated at a den- sity of 2 × 104 cells/well. Aβ40 at pH 5.5 and pH 7.4 was added at different concentrations, and after 24 h of incubation, cell viability was detected by MTT assay. After removing the medium, 10 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/mL) and 100µL of the medium were added to each well and incubated at 37 ◦ C for 4 h. Then, 150 µL of dimethyl sulfoxide was added to each well to dissolve the formazan after discarding the supernatant. Absorbance values were quantified using a plate reader (TECAN Spark) at a wavelength of 490 nm. For each pH, the data are expressed as a percentage of viability with respect to non-treated cells. The non-treated cells were grown in a medium containing the same amount of buffer, at the corresponding pH, but without Aβ40. Figure S6 compares these two controls (pH 7.4 and pH 5.5) and a culture of cells non-treated and without the addition of any extra buffer (neither pH 7.4 nor pH 5.5). Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/ijms24098408/s1. Author Contributions: N.B.-C. and N.S.d.G. conceived the manuscript structure. J.S.C., M.R.F., N.B.-C. and N.S.d.G. wrote the main manuscript text. J.S.C., N.B.-C. and N.S.d.G. prepared the figures and revised the manuscript. Conceptualization, N.B.-C. and N.S.d.G.; methodology, N.B.-C., N.S.d.G. and J.S.C.; validation, J.S.C.; formal analysis, J.S.C.; investigation, J.S.C., N.B.-C. and N.S.d.G.; resources, J.C., N.B.-C. and N.S.d.G.; writing—original draft preparation, J.C., J.S.C., M.R.F., N.B.-C. and N.S.d.G.; supervision, J.C., M.R.F., N.B.-C. and N.S.d.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by grants 2022-FISDUR-0072 (AGAUR), FPU21/03897, RYC2019- 026752-I, and PID2020-117454RA-I00 which is funded by MCIN/AEI/10.13039/501100011033. It was also funded by a national award from L’Oréal-UNESCO For Women in Science Programme. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: The authors would like to thank Marc Torrent Burgas (Autonomous University of Barcelona, Spain) for his critical comments on the manuscript. 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