Our method include:
-Study of hereditary forms of Alzheimer's disease,
- Study of the stability and kinetics of aggregation,
- Increased stability of amyloid peptides,
-determination of key amino acid residues,
-selection of antibodies to amyloids

The figure1 shows a diagram of an amyloid peptide indicating hereditary mutations that have different distinctive features at the physiological level, and also have different characteristics in the studied physical interactions with other molecules (with amyloid peptides, oligomers, molecular complexes).

The figure1 shows those hereditary mutations that will be studied in detail for resistance, toxicity, aggregation kinetics, manifestations of Alzheimer's disease and other diseases.

Fig.1. Amino acid sequence of the ABeta(11–42) peptide, with its missense mutations being indicated. AAR numbering is given in two variants: for the peptide itself and relative to its predecessor protein APP.

Scheme of the APP region, indicating known mutations

A.1.

K16N-K16N

K16N-WT

the Aβ42 K16N peptide was less harmful to neuroblastoma cells than wild-type Aβ42
the mutant Aβ peptide formed predominantly low-n oligomers in vitro

the mutant peptide was highly toxic when mixed in equimolar amounts with wild-type Aβ mixed with wild-type Aβ it aggregated into high-n oliogmers.

A21G-A21G
(Flemish)

a) Aβ peptides carrying the Flemish mutation have relatively low aggregation propensity in vitro, including dimerization
b) Conformational changes are thought to explain the Flemish mutation's inhibitory effect on self-assembly and nucleation.

E22K- E22K
(Italian)

a) Arctic Aβ40 forms protofibrils at an increased propensity and faster rate compared with wild-type Aβ40
b) In Arctic AD brain differentially truncated abundant Aβ is deposited in plaques of variable numbers and shapes in different regions of the brain.

CAA caused by the Italian or Dutch Aβ variants is uniquely characterized by deposition of Aβ in the smooth muscle cells surrounding the cerebral vasculature

E22Q- E22Q
(Dutch)

E22Q-E22Q
(Dutch)

a) The discovery of this mutation was an early demonstration that a variant in the APP gene could cause severe amyloid deposition.
b) Extensive Aβ accumulates in the cerebral vessels, especially the meningeal arteries and the cerebro-cortical arterioles.
c) In vitro, this mutation accelerates Aβ aggregation, leading to increased fibril formation.
d) This mutation is associated with high levels of β-sheet conformation and induction of apoptosis in cerebral endothelial cells compared with wild-type Aβ

[K16N]Aβ42 was found to be less toxic than WT Aβ42 at equal concentrations. However, a mixture of the two adding up to the same concentration was equally or more toxic than WT Aβ42 alone

Fig.5. Oligomerization and toxicity of K16N substituted Aβ peptides.
(1) SH-SY5Y cells were incubated for 12 hours with either 2 μM freshly dissolved peptides (load) of Aβ40 (A) or Aβ42 (B), or oligomers (2–20x) obtained by SEC.

(2) Primary hippocampal neurons were incubated for 48 hours with 2 μM freshly dissolved peptides. Toxicity was determined by percentage of living cells compared to untreated control cells (n = 4–8). The data are presented as mean ± SEM. (*p < 0.001, **p < 0.0001). Reprinted with permission from Kaden et al [97].

(1)

(2)

[more stable]

It is characterized by high thermodynamic stability compared to other dimers, as well as the least tendency to enter into biochemical reactions to achieve equilibrium. As one of the biological consequences of finding a dimer with high thermodynamic stability, there is a low reactivity, a reduced rate of forming high-molecular complexes, including fibrils and oligomers.

[less stable]

It is characterized by reduced thermodynamic stability compared to other dimers, as well as the greatest tendency to enter into biochemical reactions to achieve equilibrium. As one of the biological consequences of the definition of a dimer with low thermodynamic stability is a high reactivity, an increased rate of formation of high-molecular complexes, including fibrils and oligomers.

Analysis of the stability of amyloid peptides taking into account hereditary mutations. The calculation was performed using the software developed by our team

E22G- E22G
(Arctic)

[less stable]

[more stable]

[ABeta]2 ______lg(cond(w))

E22K-E22K__ _____5.55431
WT-WT ___________5,5372

[less stable]

[more stable]

[ABeta]2 ______lg(cond(w))

E22K-E22K__ _____5.55431
WT-WT ___________5,5372

[less stable]

[more stable]

[ABeta]2 ______lg(cond(w))

E22K-E22K__ _____5.54094
WT-WT ___________5,5372

[less stable]

[more stable]

[ABeta]2 ________lg(cond(w))

E21G-E21G_ _____5.51966
wt-wt____________5,5372

[less stable]

[more stable]

[ABeta]2 ________lg(cond(w))

E22G-E22G_ _____5.5492
wt-wt____________5,5372

[less stable]

[more stable]

[ABeta]2 ______lg(cond(w))

E22K-E22K__ _____5.55431
WT-WT ___________5,5372

[middle value]

[less stable]

[more stable]

[ABeta]2 ______lg(cond(w))

K16N+K16N _____5,5264
wt-wt____________5,5372
K16N+wt ________5,5312

K16N+K16N ____________5,5264
wt-wt ___________________5,5372
K16N+wt _______________5,5312

E21G-E21G _____________5,51966
wt-wt ___________________5,5372

E22K-E22K_____________ 5,55431
WT-WT_________________ 5,5372

E22K-E22K _____________5,54094
WT-WT _________________5,5372

B.1.

In addition to investigating genetic mutations, we also performed a calculation for random mutations in amyloid peptides and analysed their effect on the half-life of aggregation. Estimated data of lg(cond(W)) value for such mutations are shown in Fig.6 b. The structure with the D23N substitution in the two amyloid peptides represents an antiparallel interaction in the formation of dimer, as opposed to the wild-type structure, whose formation is due to the parallel coupling of amyloid peptides.

Fig.7 Two amyloid peptide structures, with the upper structure corresponding to an interaction between wild-type amyloid peptides, and the lower structure corresponding to an interaction between two mutant forms of D23N peptides, with them forming a dimer by combining into antiparallel structures.

Figure 6: Correlation scheme for physical quantities such as lg(cond(W)) and ∆H. The value of lg(cond(W)) characterizes the stability of the biological complex, and ∆H characterizes the orderliness of the system as the system transitions from one state to another, in this case from the state of the interaction of wild-type amyloid dimers into a system of dimers given the substitutions of the selected amino acid residues.

We will pay special attention to the modified amyloid peptide pair mAβ(11−42)2 (D23N). In this dimer, two symmetric substitutions of D23N were performed. The numerical results are shown in Fig.d extreme value. As can be seen from the presented graphs, the replacement of D23N in the two polypeptide chains results in a sharp decrease in lg(cond(W)), while the ∆H values are conversely in the positive region of values (see Fig.6c). At the same time, the extreme values in the diagram d change significantly (contrast) with the rest of the values in the graph a. The necessary minimum requirement to indicate a significant disruption of molecular bounds and the transition of the system to a new position is the absence of co-directional change in the two quantities lg(cond(W)) and ∆H, which we observe in Fig.6 c and Fig. 6d. The structures of the two wild-type dimers when replacing D23N are shown in Fig.7 .

C.1.