Correction: Yagolovich et al. DR5-Selective TRAIL Variant DR5-B Functionalized with Tumor-Penetrating iRGD Peptide for Enhanced Antitumor Activity against Glioblastoma. Int. J. Mol. Sci. 2022, 23, 12687.

In the original publication [...].

Dual targeting of DR5 and VEGFR2 molecular pathways by multivalent fusion protein significantly suppresses tumor growth and angiogenesis.

Destroying tumor vasculature is a relevant therapeutic strategy due to its involvement in tumor progression. However, adaptive resistance to approved antiangiogenic drugs targeting VEGF/VEGFR pathway requires the recruitment of additional targets. In this aspect, targeting TRAIL pathway is promising as it is an important component of the immune system involved in tumor immunosurveillance. For dual targeting of malignant cells and tumor vascular microenvironment, we designed a multivalent fusion protein SRH-DR5-B-iRGD with antiangiogenic VEGFR2-specific peptide SRH at the N-terminus and a tumor-targeting and -penetrating peptide iRGD at the C-terminus of receptor-selective TRAIL variant DR5-B. SRH-DR5-B-iRGD obtained high affinity for DR5, VEGFR2 and αvß3 integrin in nanomolar range. Fusion of DR5-B with effector peptides accelerated DR5 receptor internalization rate upon ligand binding. Antitumor efficacy was evaluated in vitro in human tumor cell lines and primary patient-derived glioblastoma neurospheres, and in vivo in xenograft mouse model of human glioblastoma. Multivalent binding of SRH-DR5-B-iRGD fusion efficiently stimulated DR5-mediated tumor cell death via caspase-dependent mechanism, suppressed xenograft tumor growth by >80 %, doubled the lifespan of xenograft animals, and inhibited tumor vascularization. Therefore, targeting DR5 and VEGFR2 molecular pathways with SRH-DR5-B-iRGD protein may provide a novel therapeutic approach for treatment of solid tumors.

Targeted Cytokine Delivery for Cancer Treatment: Engineering and Biological Effects.

Anti-tumor properties of several cytokines have already been investigated in multiple experiments and clinical trials. However, those studies evidenced substantial toxicities, even at low cytokine doses, and the lack of tumor specificity. These factors significantly limit clinical applications. Due to their high specificity and affinity, tumor-specific monoclonal antibodies or their antigen-binding fragments are capable of delivering fused cytokines to tumors and, therefore, of decreasing the number and severity of side effects, as well as of enhancing the therapeutic index. The present review surveys the actual antibody-cytokine fusion protein (immunocytokine) formats, their targets, mechanisms of action, and anti-tumor and other biological effects. Special attention is paid to the formats designed to prevent the off-target cytokine-receptor interactions, potentially inducing side effects. Here, we describe preclinical and clinical data and the efficacy of the antibody-mediated cytokine delivery approach, either as a single therapy or in combination with other agents.

Recent Advances in the Development of Nanodelivery Systems Targeting the TRAIL Death Receptor Pathway.

The TRAIL (TNF-related apoptosis-inducing ligand) apoptotic pathway is extensively exploited in the development of targeted antitumor therapy due to TRAIL specificity towards its cognate receptors, namely death receptors DR4 and DR5. Although therapies targeting the TRAIL pathway have encountered many obstacles in attempts at clinical implementation for cancer treatment, the unique features of the TRAIL signaling pathway continue to attract the attention of researchers. Special attention is paid to the design of novel nanoscaled delivery systems, primarily aimed at increasing the valency of the ligand for improved death receptor clustering that enhances apoptotic signaling. Optionally, complex nanoformulations can allow the encapsulation of several therapeutic molecules for a combined synergistic effect, for example, chemotherapeutic agents or photosensitizers. Scaffolds for the developed nanodelivery systems are fabricated by a wide range of conventional clinically approved materials and innovative ones, including metals, carbon, lipids, polymers, nanogels, protein nanocages, virus-based nanoparticles, dendrimers, DNA origami nanostructures, and their complex combinations. Most nanotherapeutics targeting the TRAIL pathway are aimed at tumor therapy and theranostics. However, given the wide spectrum of action of TRAIL due to its natural role in immune system homeostasis, other therapeutic areas are also involved, such as liver fibrosis, rheumatoid arthritis, Alzheimer's disease, and inflammatory diseases caused by bacterial infections. This review summarizes the recent innovative developments in the design of nanodelivery systems modified with TRAIL pathway-targeting ligands.

Application of an Autoinduction Strategy to Optimize the Heterologous Production of an Antitumor Bispecific Fusion Protein Based on the TRAIL Receptor-Selective Mutant Variant in Escherichia coli.

Autoinduction is a simple approach for heterologous protein expression that helps to achieve the high-level production of recombinant proteins in soluble form. In this work, we investigated if the application of an autoinduction strategy could help to optimize the production of bifunctional protein SRH-DR5-B, the DR5-specific TRAIL variant DR5-B fused to a VEGFR2-specific peptide SRHTKQRHTALH for dual antitumor and antiangiogenic activity. The protein was expressed in Escherichia coli SHuffle B T7, BL21(DE3), and BL21(DE3)pLysS strains. By IPTG induction, the highest expression level was in SHuffle B T7, while by autoinduction, the similar expression level was achieved in BL21(DE3)pLysS. However, in SHuffle B T7, only 45% of IPTG-induced SRH-DR5-B was expressed in soluble form, in contrast to 75% autoinduced in BL21(DE3)pLysS. The yield of purified SRH-DR5-B protein expressed by autoinduction in BL21(DE3)pLysS was 28 ± 4.5 mg per 200 ml of cell culture, which was 1.4 times higher than the yield from IPTG-induced SHuffle B T7. Regardless of the production method, SRH-DR5-B was equally cytotoxic to BxPC-3 human tumor cells expressing DR5 and VEGFR2 receptors. Thus, the production of SRH-DR5-B by autoinduction in the E. coli BL21(DE3)pLysS strain is an efficient, technologically simple, and economical technique that allows to obtain a large amount of active protein from the cytoplasmic cell fraction. Our work demonstrates that the strategy of induction of protein expression is no less important than the strain selection.

Toxicity Evaluation and Controlled-Release of Curcumin-Loaded Amphiphilic Poly-N-vinylpyrrolidone Nanoparticles: In Vitro and In Vivo Models.

Curcumin attracts huge attention because of its biological properties: it is antiproliferative, antioxidant, anti-inflammatory, immunomodulatory and so on. However, its usage has been limited by poor water solubility and low bioavailability. Herein, to solve these problems, we developed curcumin-loaded nanoparticles based on end-capped amphiphilic poly(N-vinylpyrrolidone). Nanoparticles were obtained using the solvent evaporation method and were characterized by dynamic and electrophoretic light scattering, transmission electron (TEM) and atomic force (AFM) microscopy. The average particle size was 200 nm, and the ζ-potential was -4 mV. Curcumin-release studies showed that nanoparticles are stable in aqueous solutions. An in vitro release study showed prolonged action in gastric, intestinal and colonic fluids, consistently, and in PBS. In vitro studies on epidermoid carcinoma and human embryonic kidney cells showed that the cells absorbed more curcumin in nanoparticles compared to free curcumin. Nanoparticles are safe for healthy cells and show high cytotoxicity for glioblastoma cells in cytotoxicity studies in vitro. The median lethal dose was determined in an acute toxicity assay on zebrafish and was 23 µM. Overall, the curcumin-loaded nanoparticles seem promising for cancer treatment.

DR5-Selective TRAIL Variant DR5-B Functionalized with Tumor-Penetrating iRGD Peptide for Enhanced Antitumor Activity against Glioblastoma.

TRAIL (TNF-related apoptosis-inducing ligand) and its derivatives are potentials for anticancer therapy due to the selective induction of apoptosis in tumor cells upon binding to death receptors DR4 or DR5. Previously, we generated a DR5-selective TRAIL mutant variant DR5-B overcoming receptor-dependent resistance of tumor cells to TRAIL. In the current study, we improved the antitumor activity of DR5-B by fusion with a tumor-homing iRGD peptide, which is known to enhance the drug penetration into tumor tissues. The obtained bispecific fusion protein DR5-B-iRGD exhibited dual affinity for DR5 and integrin αvß3 receptors. DR5-B-iRGD penetrated into U-87 tumor spheroids faster than DR5-B and demonstrated an enhanced antitumor effect in human glioblastoma cell lines T98G and U-87, as well as in primary patient-derived glioblastoma neurospheres in vitro. Additionally, DR5-B-iRGD was highly effective in a xenograft mouse model of the U-87 human glioblastoma cell line in vivo. We suggest that DR5-B-iRGD may become a promising candidate for targeted therapy for glioblastoma.

Optimized Heterologous Expression and Efficient Purification of a New TRAIL-Based Antitumor Fusion Protein SRH-DR5-B with Dual VEGFR2 and DR5 Receptor Specificity.

In the last two decades, bifunctional proteins have been created by genetic and protein engineering methods to increase therapeutic effects in various diseases, including cancer. Unlike conventional small molecule or monotargeted drugs, bifunctional proteins have increased biological activity while maintaining low systemic toxicity. The recombinant anti-cancer cytokine TRAIL has shown a limited therapeutic effect in clinical trials. To enhance the efficacy of TRAIL, we designed the HRH-DR5-B fusion protein based on the DR5-selective mutant variant of TRAIL fused to the anti-angiogenic synthetic peptide HRHTKQRHTALH. Initially low expression of HRH-DR5-B was enhanced by the substitution of E. coli-optimized codons with AT-rich codons in the DNA sequence encoding the first 7 amino acid residues of the HRH peptide. However, the HRH-DR5-B degraded during purification to form two adjacent protein bands on the SDS-PAGE gel. The replacement of His by Ser at position P2 immediately after the initiator Met dramatically minimized degradation, allowing more than 20 mg of protein to be obtained from 200 mL of cell culture. The resulting SRH-DR5-B fusion bound the VEGFR2 and DR5 receptors with high affinity and showed increased cytotoxic activity in 3D multicellular tumor spheroids. SRH-DR5-B can be considered as a promising candidate for therapeutic applications.

GFP-Margatoxin, a Genetically Encoded Fluorescent Ligand to Probe Affinity of Kv1.3 Channel Blockers.

Peptide pore blockers and their fluorescent derivatives are useful molecular probes to study the structure and functions of the voltage-gated potassium Kv1.3 channel, which is considered as a pharmacological target in the treatment of autoimmune and neurological disorders. We present Kv1.3 fluorescent ligand, GFP-MgTx, constructed on the basis of green fluorescent protein (GFP) and margatoxin (MgTx), the peptide, which is widely used in physiological studies of Kv1.3. Expression of the fluorescent ligand in E. coli cells resulted in correctly folded and functionally active GFP-MgTx with a yield of 30 mg per 1 L of culture. Complex of GFP-MgTx with the Kv1.3 binding site is reported to have the dissociation constant of 11 ± 2 nM. GFP-MgTx as a component of an analytical system based on the hybrid KcsA-Kv1.3 channel is shown to be applicable to recognize Kv1.3 pore blockers of peptide origin and to evaluate their affinities to Kv1.3. GFP-MgTx can be used in screening and pre-selection of Kv1.3 channel blockers as potential drug candidates.

Bispecific Antibodies for IFN-ß Delivery to ErbB2+ Tumors.

The main aim of our work was to create a full-length bispecific antibody (BsAb) as a vehicle for the targeted delivery of interferon-beta (IFN-ß) to ErbB2+ tumor cells in the form of non-covalent complex of BsAb and IFN-ß. Such a construct is a CrossMab-type BsAb, consisting of an ErbB2-recognizing trastuzumab moiety, a part of chimeric antibody to IFN-ß, and human IgG1 Fc domain carrying knob-into-hole amino acid substitutions necessary for the proper assembly of bispecific molecules. The IFN-ß- recognizing arm of BsAb not only forms a complex with the cytokine but neutralizes its activity, thus providing a mechanism to avoid the side effects of the systemic action of IFN-ß by blocking IFN-ß Interaction with cell receptors in the process of cytokine delivery to tumor sites. Enzyme sandwich immunoassay confirmed the ability of BsAb to bind to human IFN-ß comparable to that of the parental chimeric mAb. The BsAb binds to the recombinant ErbB2 receptor, as well as to lysates of ErbB2+ tumor cell lines. The inhibition of the antiproliferative effect of IFN-ß by BsAb (IC50 = 49,3 µg/mL) was demonstrated on the HT29 cell line. It can be proposed that the BsAb obtained can serve as a component of the immunocytokine complex for the delivery of IFN-ß to ErbB2-associated tumor cells.

Kinetics and Energetics of Intramolecular Electron Transfer in Single-Point Labeled TUPS-Cytochrome c Derivatives.

Electron transfer within and between proteins is a fundamental biological phenomenon, in which efficiency depends on several physical parameters. We have engineered a number of horse heart cytochrome c single-point mutants with cysteine substitutions at various positions of the protein surface. To these cysteines, as well as to several native lysine side chains, the photoinduced redox label 8-thiouredopyrene-1,3,6-trisulfonate (TUPS) was covalently attached. The long-lived, low potential triplet excited state of TUPS, generated with high quantum efficiency, serves as an electron donor to the oxidized heme c. The rates of the forward (from the label to the heme) and the reverse (from the reduced heme back to the oxidized label) electron transfer reactions were obtained from multichannel and single wavelength flash photolysis absorption kinetic experiments. The electronic coupling term and the reorganization energy for electron transfer in this system were estimated from temperature-dependent experiments and compared with calculated parameters using the crystal and the solution NMR structure of the protein. These results together with the observation of multiexponential kinetics strongly support earlier conclusions that the flexible arm connecting TUPS to the protein allows several shortcut routes for the electron involving through space jumps between the label and the protein surface.

Death Receptors DR4 and DR5 Undergo Spontaneous and Ligand-Mediated Endocytosis and Recycling Regardless of the Sensitivity of Cancer Cells to TRAIL.

Tumor necrosis factor-associated ligand inducing apoptosis (TRAIL) induces apoptosis through the death receptors (DRs) 4 and 5 expressed on the cell surface. Upon ligand stimulation, death receptors are rapidly internalized through clathrin-dependent and -independent mechanisms. However, there have been conflicting data on the role of death receptor endocytosis in apoptotic TRAIL signaling and possible cell type-specific differences in TRAIL signaling have been proposed. Here we have compared the kinetics of TRAIL-mediated internalization and subsequent recycling of DR4 and DR5 in resistant (HT-29 and A549) and sensitive (HCT116 and Jurkat) tumor cell lines of various origin. TRAIL stimulated the internalization of both receptors in a concentration-dependent manner with similar kinetics in sensitive and resistant cell lines without affecting the steady-state expression of DR4 and DR5 in cell lysates. Using the receptor-selective TRAIL variant DR5-B, we have shown that DR5 is internalized independently of DR4 receptor. After internalization and elimination of TRAIL from culture medium, the receptors slowly return to the plasma membrane. Within 4 h in resistant or 6 h in sensitive cells, the surface expression of receptors was completely restored. Recovery of receptors occurred both from newly synthesized molecules or from trans-Golgi network, as cycloheximide and brefeldin A inhibited this process. These agents also suppressed the expression of cell surface receptors in a time- and concentration-dependent manner, indicating that DRs undergo constitutive endocytosis. Inhibition of receptor endocytosis by sucrose led to sensitization of resistant cells to TRAIL and to an increase in its cytotoxic activity against sensitive cells. Our results confirm the universal nature of TRAIL-induced death receptor endocytosis, thus cell sensitivity to TRAIL can be associated with post-endocytic events.

Amphiphilic Poly(N-vinylpyrrolidone) Nanoparticles Conjugated with DR5-Specific Antitumor Cytokine DR5-B for Targeted Delivery to Cancer Cells.

Nanoparticles based on the biocompatible amphiphilic poly(N-vinylpyrrolidone) (Amph-PVP) derivatives are promising for drug delivery. Amph-PVPs self-aggregate in aqueous solutions with the formation of micellar nanoscaled structures. Amph-PVP nanoparticles are able to immobilize therapeutic molecules under mild conditions. As is well known, many efforts have been made to exploit the DR5-dependent apoptosis induction for cancer treatment. The aim of the study was to fabricate Amph-PVP-based nanoparticles covalently conjugated with antitumor DR5-specific TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) variant DR5-B and to evaluate their in vitro cytotoxicity in 3D tumor spheroids. The Amph-PVP nanoparticles were obtained from a 1:1 mixture of unmodified and maleimide-modified polymeric chains, while DR5-B protein was modified by cysteine residue at the N-end for covalent conjugation with Amph-PVP. The nanoparticles were found to enhance cytotoxicity effects compared to those of free DR5-B in both 2D (monolayer culture) and 3D (tumor spheroids) in vitro models. The cytotoxicity of the nanoparticles was investigated in human cell lines, namely breast adenocarcinoma MCF-7 and colorectal carcinomas HCT116 and HT29. Notably, DR5-B conjugation with Amph-PVP nanoparticles sensitized resistant multicellular tumor spheroids from MCF-7 and HT29 cells. Taking into account the nanoparticles loading ability with a wide range of low-molecular-weight antitumor chemotherapeutics into hydrophobic core and feasibility of conjugation with hydrophilic therapeutic molecules by click chemistry, we suggest further development to obtain a versatile system for targeted drug delivery into tumor cells.

Corrigendum: Death Receptors DR4 and DR5 Undergo Spontaneous and Ligand-Mediated Endocytosis and Recycling Regardless of the Sensitivity of Cancer Cells to TRAIL.

[This corrects the article DOI: 10.3389/fcell.2021.733688.].

Chemotherapeutic Agents Sensitize Resistant Cancer Cells to the DR5-Specific Variant DR5-B more Efficiently than to TRAIL by Modulating the Surface Expression of Death and Decoy Receptors.

TRAIL is considered a promising antitumor agent because it causes apoptosis of transformed cells without affecting normal cells. However, many types of tumors are cytokine resistant, and combination therapy with various chemotherapeutic drugs is being developed to overcome the resistance. We have demonstrated that the combination of TRAIL with doxorubicin, bortezomib, and panobinostat dramatically reduced the viability of TRAIL-resistant A549 and HT-29 cells. Chemotherapy even more efficiently sensitized cells to the DR5-specific mutant variant of TRAIL DR5-B, which does not have an affinity for decoy receptors. Bortezomib and doxorubicin greatly enhanced the surface expression of the death receptors DR5 and DR4, while panobinostat increased expression of DR5 and suppressed expression of DR4 in both cell lines. All drugs increased surface expression of the decoy receptors DcR1 and DcR2. Unlike the combined treatment, if the cells were pretreated with chemotherapy for 24 h, the cytotoxic activity of TRAIL was less pronounced, while sequential treatment of cells enhanced the effectiveness of DR5-B. The same results were obtained with agonistic anti-DR5 antibodies. Thus, the effectiveness of TRAIL was rather limited due to changes in the ratio of death and decoy receptors and DR5-specific agonists may be preferred in combination antitumor therapy regimens.

Genetically Modified DR5-Specific TRAIL Variant DR5-B Revealed Dual Antitumor and Protumoral Effect in Colon Cancer Xenografts and an Improved Pharmacokinetic Profile.

Despite the weak clinical efficacy of TRAIL death receptor agonists, a search is under way for new agents that more efficiently activate apoptotic signaling. We previously created a TRAIL DR5-selective variant DR5-B without affinity for the DR4, DcR1, DcR2, and OPG receptors and increased proapoptotic activity in tumor cells. Here we showed that DR5-B significantly inhibited tumor growth in HCT116 and Caco-2 but not in HT-29 xenografts. The antitumor activity of DR5-B was 2.5 times higher in HCT116 xenografts compared to TRAIL. DR5-B at a dose of 2 or 10 mg/kg/d for 10 days inhibited tumor growth in HCT116 xenografts by 26% or 50% respectively, and increased animal survival. Unexpectedly, DR5-B at a higher dose (25 mg/kg/d) inhibited tumor growth only during the first 8 days of drug exposure, while at the end of the monitoring, no effect or even slight stimulation of tumor growth was observed. The pharmacokinetic parameters of DR5-B were comparable to those of TRAIL, except that the half-life was 3.5 times higher. Thus, enhancing TRAIL selectivity to DR5 may increase both antitumor and proliferative activities depending on the concentration and administration regimens.

The Kinetics of Amyloid Fibril Formation by de Novo Protein Albebetin and Its Mutant Variants.

Engineering of amyloid structures is one of the new perspective areas of protein engineering. Studying the process of amyloid formation can help find ways to manage it in the interests of medicine and biotechnology. One of the promising candidates for the structural basis of artificial functional amyloid fibrils is albebetin (ABB), an artificial protein engineered under the leadership of O.B. Ptitsyn. Various aspects of the amyloid formation of this protein and some methods for controlling this process are investigated in this paper. Four stages of amyloid fibrils formation by this protein from the first non-fibrillar aggregates to mature fibrils and large micron-sized complexes have been described in detail. Dependence of albebetin amyloids formation on external conditions and some mutations also have been described. The introduction of similar point mutations in the two structurally identical α-ß-ß motifs of ABB lead to different amiloidogenesis kinetics. The inhibitory effect of a disulfide bond and high pH on amyloid fibrils formation, that can be used to control this process, was shown. The results of this work are a good basis for the further design and use of ABB-based amyloid constructs.

Crystal structure of PMGL2 esterase from the hormone-sensitive lipase family with GCSAG motif around the catalytic serine.

Lipases comprise a large class of hydrolytic enzymes which catalyze the cleavage of the ester bonds in triacylglycerols and find numerous biotechnological applications. Previously, we have cloned the gene coding for a novel esterase PMGL2 from a Siberian permafrost metagenomic DNA library. We have determined the 3D structure of PMGL2 which belongs to the hormone-sensitive lipase (HSL) family and contains a new variant of the active site motif, GCSAG. Similar to many other HSLs, PMGL2 forms dimers in solution and in the crystal. Our results demonstrated that PMGL2 and structurally characterized members of the GTSAG motif subfamily possess a common dimerization interface that significantly differs from that of members of the GDSAG subfamily of known structure. Moreover, PMGL2 had a unique organization of the active site cavity with significantly different topology compared to the other lipolytic enzymes from the HSL family with known structure including the distinct orientation of the active site entrances within the dimer and about four times larger size of the active site cavity. To study the role of the cysteine residue in GCSAG motif of PMGL2, the catalytic properties and structure of its double C173T/C202S mutant were examined and found to be very similar to the wild type protein. The presence of the bound PEG molecule in the active site of the mutant form allowed for precise mapping of the amino acid residues forming the substrate cavity.

A minimum set of stable blocks for rational design of polypeptide chains.

The aim of this work was to find a minimal set of structurally stable pentapeptides, which allows forming a polypeptide chain of a required 3D structure. To search for factors that ensure structural stability of the pentapeptide, we generated peptide sequences with no more than three functional groups, based on the alanine pentapeptide AAAAA. We analyzed 44,860 structures of peptides by the molecular dynamics method and found that 1,225 pentapeptides over 80% of the simulation time were in a stable conformation. Clustering of these conformations revealed 54 topological types of conformationally stable pentapeptides. These conformations relate to different combined elements of the protein secondary structure. So, we obtained a minimal set of amino acid structures of conformationally stable pentapeptides, creating a complete set of different topologies that ensure the formation of pre-folded conformation of protein structures.

Lynx1 and Aß1-42 bind competitively to multiple nicotinic acetylcholine receptor subtypes.

Lynx1 regulates synaptic plasticity in the brain by regulating nicotinic acetylcholine receptors (nAChRs). It is not known to which extent Lynx1 can bind to endogenous nAChR subunits in the brain or how this interaction is affected by Alzheimer's disease pathology. We apply affinity purification to demonstrate that a water-soluble variant of human Lynx1 (Ws-Lynx1) isolates α3, α4, α5, α6, α7, ß2, and ß4 nAChR subunits from human and rat cortical extracts, and rat midbrain and olfactory bulb extracts, suggesting that Lynx1 forms complexes with multiple nAChR subtypes in the human and rodent brain. Incubation with Ws-Lynx1 decreases nicotine-mediated extracellular signal-regulated kinase phosphorylation in PC12 cells and striatal neurons, indicating that binding of Ws-Lynx1 is sufficient to inhibit signaling downstream of nAChRs. The effect of nicotine in PC12 cells is independent of α7 or α4ß2 nAChRs, suggesting that Lynx1 can affect the function of native non-α7, non-α4ß2 nAChR subtypes. We further show that Lynx1 and oligomeric ß-amyloid1-42 compete for binding to several nAChR subunits, that Ws-Lynx1 prevents ß-amyloid1-42-induced cytotoxicity in cortical neurons, and that cortical Lynx1 levels are decreased in a transgenic mouse model with concomitant ß-amyloid and tau pathology. Our data suggest that Lynx1 binds to multiple nAChR subtypes in the brain and that this interaction might have functional and pathophysiological implications in relation to Alzheimer's disease.