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Review
, 24 (6)

Contributions of Mass Spectrometry to the Identification of Low Molecular Weight Molecules Able to Reduce the Toxicity of Amyloid-β Peptide to Cell Cultures and Transgenic Mouse Models of Alzheimer's Disease

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Review

Contributions of Mass Spectrometry to the Identification of Low Molecular Weight Molecules Able to Reduce the Toxicity of Amyloid-β Peptide to Cell Cultures and Transgenic Mouse Models of Alzheimer's Disease

Raluca Ştefănescu et al. Molecules.

Abstract

Alzheimer's Disease affects approximately 33 million people worldwide and is characterized by progressive loss of memory at the cognitive level. The formation of toxic amyloid oligomers, extracellular amyloid plaques and amyloid angiopathy in brain by amyloid beta peptides are considered a part of the identified mechanism involved in disease pathogenesis. The optimal treatment approach leads toward finding a chemical compound able to form a noncovalent complex with the amyloid peptide thus blocking the process of amyloid aggregation. This direction gained an increasing interest lately, many studies demonstrating that mass spectrometry is a valuable method useful for the identification and characterization of such molecules able to interact with amyloid peptides. In the present review we aim to identify in the scientific literature low molecular weight chemical compounds for which there is mass spectrometric evidence of noncovalent complex formation with amyloid peptides and also there are toxicity reduction results which verify the effects of these compounds on amyloid beta toxicity towards cell cultures and transgenic mouse models developing Alzheimer's Disease.

Keywords: Alzheimer’s Disease; amyloid-β peptides; anti-aggregating compounds; cell viability; cognitive dysfunction; in vitro models; mass spectrometry; proteolytic enzymes; spatial working memory; transgenic mouse models.

Conflict of interest statement

AD, Alzheimer’s Disease; Aβ, Amyloid-beta; APP, Amyloid Precursor Protein; FAD, Familial Alzheimer’s Disease; ABCA1 modulation, ATP binding cassette transporter A1; AD model with Npc1-deficient mice, a novel mouse model of "Juvenile Alzheimer's Disease” carrying a D1005G-Npc1 mutation; GluA1 and GluA2 subunits of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors; GluN2A, the main subunit of N-methyl-d-aspartate receptors; PSD-95, a membrane-associated guanylate kinase, is the major scaffolding protein in the excitatory postsynaptic density (PSD) and a potent regulator of synaptic strength; ESI, Electrospray ionization; MALDI, Matrix Assisted Laser Desorption and Ionization; iPSCs, Induced pluripotent stem cells; PS1, Presenilin 1; PS2, Presenilin 2; SH-SY5Y, Human neuroblastoma cell line; GAP-43, Growth-Associated Protein; NeuN, Neuronal Nuclei; SYN, Synaptophysin; SV2, Synaptic Vesicle Protein II; NSE, Neuron Specific Enolase; MAP, Microtubule Associated protein; GFAP, Glial Fibrillary Acidic Protein; SK-N-SH, Original Human Neuroblastoma Cell Line from which SH-SY5Y was derived; TH, Tyrosine Hydroxylase; GSK3 beta, Glycogen synthase kinase 3 beta; HN, Humanin peptide; Acm, Acetamidomethyl; NHS-Activated Sepharose, N-hydroxysuccinimide - Activated Sepharose; MALDI-TOF, Matrix Assisted Laser Desorption and Ionization – Time Of Flight; nano-ESI-FTICR, Nano-Electrospray Ionization - Fourier Transform Ion Cyclotron Resonance; PC12, Rat phaeochromocytoma; Bcl-2, B-cell lumphoma 2, antiapoptotic protein; Bax, Bcl-2-like protein 4, proapoptotic protein, Bax gene; icv, intracerebroventricular; ip, intraperitoneal; APPswe/PS1dE9 mice, double transgenic mouse model carrying APP Swedish mutation and deletion of presenilin 1 in exon 9; APPswe, tauP310L, PS1M146V mice, triple transgenic mouse model carrying APP Swedish mutation, Tau mutation P310L and presenilin 1 mutation M146V; 3xTg-AD mice, triple transgenic mouse model of Alzheimer’s disease; Q-TOF, Quadrupole – Time Of Flight; LC/MS, Liquid Cromatography/Mass Spectrometry; Glu-C, Endoprotease V8 from Staphylococcus aureus; N2a, mouse neuroblastoma cell line; Tg2576 mice, transgenic mice overexpressing APP695 carrying Swedish mutation K670N and M671L; TgCRND8 mice, transgenic mouse model of Alzheimer’s Disease overexpressing APP695 and carrying Swedish mutation K670N and M671L and Indiana mutation V717F; HP-β-CD, 1-hydroxypropyl-β-cyclodextrin; Tg19959 mice, transgenic mouse model of Alzheimer’s disease, which overexpress human APP with the Swedish and Indiana familial AD mutations; Fmoc, 9-Fluorenyl- methyloxycarbonyl; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; fAβ-Cu, Amyloid- beta fibrils Cu complex; ROS, Reactive Oxygen Species.

Figures

Figure 1
Figure 1
Schematic representation of the therapeutic strategy based on the finding of an Aβ aggregation inhibitor. Cleavage of the amyloid precursor protein (APP) containing 770 amino acids, by β-secretase in the extracellular domain after methionine-671 and by γ-secretase in the transmembrane domain after valine-711 and alanine-713 produces in the extracellular medium the free peptides Aβ(1–40) and Aβ(1–42). Both peptides form oligomers and fibrils. Anti-aggregating compounds which inhibit the oligomerization of the amyloid peptides are searched.
Figure 2
Figure 2
Schematic representation of the affinity mass spectrometric experiments carried out for the identification of the binding sequences of two peptides forming a complex using one immobilized binding partner. (A) Scheme of an affinity experiment using immobilized Aβ peptide. A mixture of humanin and neurotensin is incubated with the affinity medium. The mass spectrum of the supernatant should contain non-binding peptides and the mass spectrum of the elution fraction should contain the peptides presenting affinity towards amyloid-beta peptide. (B) In the excision experiment, intact humanin is incubated with immobilized Aβ followed by proteolytic cleavage of the peptide fragments that are not protected against proteolysis through the interaction. Mass spectrum of the supernatant reveals the regions from humanin which do not interact with Aβ while the mass spectrum of the elution fraction shows the region which is involved in the interaction. (C) In the extraction experiment humanin is first proteolitically cleaved, then protease inhibitors are added and the mixture is incubated with immobilized Aβ. The mass spectra of the supernatant and elution fractions obtained from both the excision and extraction experiments indicate the binding region of humanin.
Figure 3
Figure 3
Schematic representation of the affinity mass spectrometric experiments carried out for the identification of the binding region from Aβ interacting with the test substances melatonin and oleuropein. (A) In the affinity experiment, incubation of Aβ(1–40) with melatonin or oleuropein in a suitable buffer for ESI-MS analysis indicates whether a noncovalent complex is formed. (B) Complex formation followed by cleavage with specific proteolytic enzymes and analysis of the complex together with the proteolytic mixture by ESI-MS allows the identification of the binding region from Aβ. (C) Proteolysis prior to complex formation and analysis of the mixture by ESI-MS allows the identification of the peptides that are incomplete and do not maintain affinity to the tested substance.
Figure 4
Figure 4
Schematic representation of the ion mobility spectrometry-mass spectrometry experiments performed for the investigation of Aβ(1–42) oligomerization in the absence (A) or presence of the tetrapeptides Ac-VVIA (B) and VVIA-NH2 (C).
Figure 5
Figure 5
Workflow for the evaluation of the mechanism of action for potential inhibitors of Aβ oligomerization.

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