β-amyloid

Aβ peptide is notably recognized as the etiological factor of several amyloidogenic diseases, such as Alzheimer’s disease. In this pathology, a transmembrane protein called Amyloid Precursor Protein (APP)¹⁵, whose the extracellular portion, in physiological conditions, is cleaved sequentially by α- and γ-secretases, in pathological conditions α-secretase is anticipated by β-secretase, which in cooperation with γ-secretase cuts the protein in a different position releasing the so-called Aβ40/Aβ42/Aβ43 fragments responsible of the neurotoxic properties.

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Figure 5.1.3.1: Schematic representation of non-amyloidogenic and amyloidogenic pathways in Alzheimer's disease (A) and fibril structure model (B). Figure adapted from references^16,17.

Once formed, these fragments tend to organize in highly-ordered fibrils (diameter = 7-10 nm) made of cross-β structures^16,18–20 packed together perpendicularly to the fibril axis (Figure 5.1.3.1).

Since it is not clear whether amyloid fibrils or metastable aggregates are the primary effectors of neurotoxicity and which are the factors for a neurotoxic response^21–25, it makes sense to study the aggregation process in the future optic to develop targeted therapeutics²⁶. However, a lot of research is still ongoing in this field due to the polymorphic and the transient nature of these metastable aggregates (Figure 5.1.3.2)²⁵.

Figure 5.1.3.2: Free energy schematic graph of metastable intermediates encountered in self-assembly process. Figure adapted from reference¹.

5.1.4 Peptide models: Aβ

fragment

Dealing with full-length peptides, it is obviously uncomfortable, mainly for the long synthesis and hard purification. For these reasons, many experimental studies have been carried out for

B A

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the determination of short inner peptide fragments with self-aggregating properties similar to full-length ones. For instance, Tenedis et al.^27,28 have determined that the NFGAIL hexapeptide of islet amyloid peptide (IAPP) is a good model for the full-length peptide because of the similar cytotoxic and structural properties of the parent peptides. Due to their boosted kinetics in aggregation, short peptides are very convenient tools for drawing the basilar mechanisms in self-assembly. For amyloid-β peptide aggregation study, Aβ16-22 has been denotated as a good reference model to study Alzeheimer’s pathological pathways (Figure 5.1.4.1)^1,18. In fact, it is well known that residues 17-21 represent a strong hydrophobic core essential for self-assembly of full-length peptide. So targeting this fragment with chemical modifications can be a way to alter this process by simply changing its polarity^12,18. In addition, the compresence of polar Lys16 and Glu22 give the opportunity to change morphology to nanotubes at different pH conditions, which makes this peptide an interesting tool also in material sciences^1,18,29,30.

Figure 5.1.4.1: Aβ16-22 chemical structure.

5.1.5 Amyloid-β aggregation

The mechanism of amyloid self-assembly is highly complex because of the high number of metastable aggregates involved. As evidenced in Figure 5.1.5.1, this process is characterized by a sigmoidal growth curve subdivided into three phases: lag phase, growth phase and plateau phase.

Figure 5.1.5.1: Growth curve of Amyloid-β: the sigmoidal pattern is evidence of the nucleation-dependent behaviour. The graph was obtained by Thioflavin T assay (ThT)^1,31–33. Figure adapted from reference¹.

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In the lag phase, it has been demonstrated by electron and atomic force microscopy studies that the low-molecular-aggregates formed have totally different structures to mature amyloid fibrils^31,34. In fact, in this phase dominates the primary nucleation during which monomers spontaneously aggregate in a small nucleus stable enough to prefer further growth rather than the back-dissociation^1,35. Since primary nucleation occurs without the involvement of pre-existing oligomers, the rate of this process depends only on monomeric concentration^35,36. When a critical concentration of amyloid fibrils is reached, the growth phase is triggered and the aggregate masses increase exponentially. This is possible for the secondary nucleation mechanism, which dominates this phase. Here, in fact, new aggregates are formed on the surface of pre-existing fibrils as they were templates for their formation. Therefore, differently to the previous phase, here the rate is dependent on the concentration of pre-existing fibrils via positive feedback, which explains the rapid exponential growth³². The last plateau phase shows that the process is complete and no other aggregates are forming. This is a simplistic view of the entire process, but actually secondary pathways and further oligomerisation are involved adding more complexity to kinetic analysis^31,35,36.

5.1.6 Aggregation studies: Microscopy, Fluorescence Quenching and Photo-Induced Cross-Linking

For a qualitative study on amyloid aggregates are commonly used Electron and Atomic Force Microscopy^27,37. One of the first experimental studies done on this topic was carried out by Hsieh et al.³⁸. Taking TEM images in different time points over a range of 1h-14days, they discovered that WT-Aβ16-22 self-assemblies via a ribbon-like intermediate that, at neutral pH, rearranges into fibrils, while at acidic pH rearranges into nanotubes. Through CD experiments, then, by the β-sheet content demonstrated that under both conditions the self-assembly starts with the formation of anti-parallel out-of-register β-sheets, followed by a realignment at pH=6 into in-register fibrillary strands, which corresponds to ribbons observed with TEM (Figure 5.1.6.1).

Figure 5.1.6.1: Aβ16-22 pairing modes in self-assembly: at physiological pH in-register fibrils are preferred by means of ionic interactions between K16 and E22, whereas at pH=2 out-of-register nanotubes/ribbons are favoured. Figure adapted from reference³⁸.

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These interesting results, for the first time, proved, differently from what had been demonstrated in computational studies on FF self-assembly, that at neutral pH the intermolecular pairing of negatively charged E22 carboxylate and positively charged K16 does not drive the whole process but is just responsible of the thermodynamic stabilization of already-formed species³⁸.

Another way to study amyloid aggregation is the ThT fluorescence assay (Figure 5.1.6.2). This assay relies on the fluorescence increase due to the rotational immobilization of the central bond between the benzothiazole and aniline rings upon β-sheet-rich aggregates formation concomitant with the growth phase^21,26. This rigidification and the subsequent conjugation extension makes fluorescence emission pathway the fastest process compared to the other non-radiative pathways³⁹. Unfortunately, this assay is not suitable for the detection of the small β-sheet-free aggregates, which are implicated in Alzheimer's disease neurotoxicity^21,23,40–

42.

Figure 5.1.6.2: Fluorescence intensity profiles over aggregation time (A) through ThT (B) and TAMRA (C) assays. From A to B a lag phase is observed where primary nucleation dominates, followed by B-C phase where pre-existing nuclei reorganize themselves without further accumulation. The last C-D phase is the actual growth phase where β-sheet structures are formed. Figure adapted from references²¹.

For a better understanding of β-amyloid aggregation, it is required the fluorescence self-quenching assay developed by Frieden and Garai²¹, which can be useful also for a quantitative determination of the full-time course of β-amyloid aggregation. This assay essentially relies on the fluorescence quenching due to the proximity of two fluorescent tags in the aggregate state⁴³. For Aβ1-40 and Aβ1-42 aggregation, the peptides are covalently labelled with tetramethylrhodamine (TAMRA). In Figure 5.1.6.2 fluorescence emission in both assays (ThT/TAMRA) is represented as a function of time and shows how the information collected by both experiments are totally different because of the different mechanism of action²¹. For TAMRA-Aβ1-42, an initial fast quenching is observed thanks to the first small aggregates formed.

Simultaneously, in the ThT assay no variation in fluorescence intensity is observed for the absence of rigid β-sheet structures. During the subsequent lag phase, just a slight decrease in TAMRA fluorescence is observed, while in ThT assay it is observed a slight increase in

A

B

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fluorescence. In this phase probably just a reorganization of pre-existing amorphous aggregates occurs and no monomers concur for accumulation. In the intermediate part of the graph in both experiments a fast change is observed because of the rapid Aβ1-42 self-assembly. CD studies showed that this phase is mainly responsible of most of β-sheet content. The plateau observed at the end confirms that the self-assembly process is complete and no other growth is observed^21,44. Even if ThT assays seem to be less accurate than TAMRA assays and not suitable for early-stage aggregates, they can discriminate the nature of aggregates in terms of β-sheet content^21,39.

Figure 5.1.6.3: PIC strategy. Diazirines, upon activation by irradiation as carbenes, are directly involved in cross-linking. The cross-linked structures and relative cross-linking positions are easily detected by ion-mobility bidimensional mass spectrometry. Figure adapted from reference⁴⁵.

Photo-Induced Cross-Linking (PIC)^45,46 is a more accurate technique since by irradiation triggers covalent cross-linking useful to trap reversible and/or weak supramolecular interactions into stable irreversible bonds⁴⁷. This is generally used in the lag phase where small aggregates are subjected to very fast changes in morphology so it is very important to freeze these intermediates to have a better understanding of the self-assembly process. As PIC group, nowadays, diazirines⁴⁵ are mainly employed thanks to the rapid carbene formation upon irradiation, which allows to use a very reliable temporal control in the technique. First examples in this context were made by Smith at al.⁴⁷, which incorporating 3-aryl-(trifluoromethyl)diazirine (TFMD) group in Aβ16-22 observed in mass spectrometry, upon irradiation, several aggregates obtained by indiscriminate reactivity of TFMD^45,47. A subsequent work of Preston et al.⁴⁶ demonstrated that it is possible to incorporate TFMD in Aβ16-22 with solid-phase peptide synthesis (SPPS) and exploit it in amyloid aggregation at different pH conditions: the intermolecular cross-linking gave information about the reciprocal alignment of peptides, meanwhile intramolecular cross-linking gave information about monomer conformations. All the intermediates are generally analysed with electrospray ionisation-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS)^45,46, which is able to discriminate high-mass fragments with equal molecular weight through collision-induced dissociation (CID) by means of tandem MS/MS techniques^46,47. Specifically, MS-induced cleavage occurs onto amidic nitrogen easily protonated by ESI source^48,49. After CID and second MS selection, an array of fragment