New approaches to the immunotherapy of Alzheimer’s disease with the synthetic fragments of α7 subunit of the acetylcholine receptor

The effect of immunization with the synthetic fragments of the α7 subunit of the acetylcholine nicotine receptor on the spatial memory of mice subjected to olfactory bulbectomy, which causes the development of the neurodegenerative disease of Alzheimer’s type, was studied. NMRI mice were immunized with the KLH conjugates of two peptide fragments of the N-terminal fragment of the α7 subunit extracellular fragment, subjected to olfactory bulbectomy to cause the development of the neurodegenerative disease of Alzheimer’s type, and then the state of the spatial memory was evaluated. It was shown that 20% of bulbectomized mice immunized with N-terminal 1–23 fragment exhibited good spatial memory after training. Immunization with the peptide construct (159–167)-(179–188) consisting of two hydrophilic exposed regions of α7-subunit induced good spatial memory in 50% of bulbectomized mice, while in the control group, which received only KLH, none of animals were learned. Thus, the development of immunotherapy with peptide (159–167)-(179–188) seems to be a promising approach to prophylaxis and treatment of Alzheimer’s disease.     

Increased Level of β-Amyloid in the Brain of Bulbectomized Mice

Six weeks after bilateral olfactory bulbectomy, a peptide with molecular weight of 4 kD was revealed in extracts of the neocortex and hippocampus from mice. Using monoclonal antibodies 4G8, this peptide was identified as beta-amyloid. Its level was significantly higher in the bulbectomized animals than in sham-operated mice. The bulbectomized mice displayed sharp impairment in spatial memory when tested in the Morris water maze. The results suggest that bulbectomy initiates in the brain a pathological process similar to human Alzheimer's disease in location, biochemistry, and behavioral manifestations.     

Induction of antibodies to particular sites of the alpha7 subunit of the nicotinic acetylcholine receptor in mice of different lines

Five synthetic fragments of the N-terminal domain of the alpha7 subunit of the human nicotinic acetylcholine receptor (alpha7 nAChR) that correspond to theoretically calculated B epitopes and T helper epitopes of the protein and contain from 16 to 29 amino acid residues were tested for the ability to stimulate the formation of antibodies in mice of three lines having H-2d, H-2b, and H-2k haplotypes of the major histocompatibility complex. It was shown that, in the free (unconjugated) form, all the peptides stimulate the formation of antibodies at least in one mouse line. Most of the peptides induced the formation of antibodies in BALB/c mice (haplotype H-2d); therefore, more detailed studies were carried out on these animals. The free peptides and/or their conjugates with keyhole limpet hemocyanin were demonstrated to be capable of stimulating the formation in BALB/c mice of antibodies that bind to the recombinant extracellular N-terminal domain of (alpha7 nAChRalpha). The epitope mapping of antipeptide antibodies carried out using truncated fragments helped reveal antipeptide antibodies to four regions of the alpha7 subunit: 1-23, 98-106, 159-168, and 173-188 (or 179-188).     

Immunodepressed Status of Mice after Bulbectomy

We studied the immune response in lymphoid cells of mice subjected to bilateral olfactory bulbectomy in comparison with sham-operated animals 1.5 and 13 months after surgery. The concentration of tumor necrosis factor decreased threefold in the peripheral blood of bulbectomized mice 1.5 months after surgery. Signs of immunodepression were also observed 13 months after surgery: suppression of mitogen-stimulated proliferation of T and B lymphocytes in the spleen, inhibition of synthesis of tumor necrosis factor in peritoneal macrophages and splenocytes, and decreased macrophage NO production. According to the immune status indices and our previous data on behavioral, biochemical, and morphological changes induced in bulbectomized mice, they have common symptoms with the Alzheimer's disease. This allows us to consider such animals as a model of sporadic form of this disease rather than of a depression.     

Mitochondrial dysfunction in neocortex and hippocampus of olfactory bulbectomized mice,a model of Alzheimer’s disease

Structural and functional impairments of mitochondria in brain tissues in the pathogenesis of Alzheimer’s disease (AD) cause energy deficiency, increased generation of reactive oxygen species (ROS), and premature neuronal death. However, the causal relations between accumulation of beta-amyloid (Aβ) peptide in mitochondria and mitochondrial dysfunction, as well as molecular mechanisms underlying deleterious effects of both these factors in sporadic AD, the most common form in humans, remain unknown. Here we used olfactory bulbectomized (OBX) mice of NMRI strain as a model for sporadic AD. Five weeks after surgery, the OBX mice developed major behavioral and biochemical features of AD neurodegeneration, including spatial memory loss, increased brain levels of Aβ, and energy deficiency. Mitochondria isolated from the neocortex and hippocampus of OBX mice displayed severe functional impairments, such as low NADH oxidation rate, reduced transmembrane potential, and decreased cytochrome c oxidase (complex IV) activity that correlated with high levels of soluble Aβ1-40. Mitochondria from OBX mice showed increased contents of lipid peroxidation products, indicative of the development of oxidative stress. We found that neurodegeneration caused by olfactory bulbectomy is accompanied by energy metabolism disturbances and oxidative stress in brain mitochondria similar to those occurring in transgenic animals–familial AD models and patients with sporadic AD. Therefore, OBX mice can serve as a valid AD model for investigating the mechanisms of AD neurodegeneration, drug testing, and development of therapeutic strategies for AD treatment.     

Olfactory Bulbectomy Leads to the Development of Epilepsy in Mice

There is a clear link between epilepsy and depression. Clinical data demonstrate a 30–35% lifetime prevalence of depression in patients with epilepsy, and patients diagnosed with depression have a three to sevenfold higher risk of developing epilepsy. Traditional epilepsy models partially replicate the clinical observations, with the demonstration of depressive traits in epileptic animals. Studies assessing pro-epileptogenic changes in models of depression, however, are more limited. Here, we examined whether a traditional rodent depression model—bilateral olfactory bulbectomy—predisposes the animals towards the development of epilepsy. Past studies have demonstrated increased neuronal excitability after bulbectomy, but continuous seizure monitoring had not been conducted. For the present study, we monitored control and bulbectomized animals by video-EEG 24/7 for approximately two weeks following the surgery to determine whether they develop spontaneous seizures. All seven bulbectomized mice exhibited seizures during the monitoring period. Seizures began about one week after surgery, and occurred in clusters with severity increasing over the monitoring period. These results suggest that olfactory bulbectomy could be a useful model of TBI-induced epilepsy, with advantages of relatively rapid seizure onset and a high number of individuals developing the disease. The model may also be useful for investigating the mechanisms underlying the bidirectional relationship between epilepsy and depression.     

A synthetic fragment 60–70 of the receptor for advanced glycation end products exhibits a therapeutic effect in an animal model of Alzheimer’s disease

Six synthetic peptides overlapping a fragment 60–76 of the receptor for advanced glycation end products (RAGE) were studied on a protective effect on spatial memory of animals in the experimental model of Alzheimer’s disease. It was shown that only a peptide corresponding to the fragment 60–70 of RAGE exhibits a therapeutic activity. Intranasal administration of this peptide into bulbectomized mice, which develop neurodegenerative features of the Alzheimer type, completely protects animal memory. Thus, it was found that the N-terminal region (60–70) within the peptide sequence 60–76 of RAGE is responsible for the revealed protective effect. The synthetic peptide RAGE-(60–70) could be the basis for the development of a new drug for the treatment of Alzheimer’s disease.     

Expression and characterization of recombinant osteopontin peptides representing matrix metalloproteinase proteolytic fragments.

Osteopontin (OPN) is a secreted, arginine-glycine-aspartic acid (RGD)-containing phosphoprotein proteolytically modified by members of the matrix metalloproteinase (MMP) family. We previously defined the MMP-3 and MMP-7 cleavage sites in OPN and found increased adhesive and migratory activity of a pool of MMP-cleaved fragments compared to full-length OPN. In the present study, we performed mutational analysis of recombinant full-length OPN and generated recombinant OPN fragments corresponding to the MMP-cleaved fragments, which have apparent molecular weights of 40, 32, and 25 kD by SDS-PAGE. Single residue mutations in 167L and 211L do not abrogate MMP cleavage although processing of the putative C-terminal fragment appears to be affected by a 167L to 167A mutation. The N-terminal 40-kD fragment was a stronger adhesive substrate compared to full-length OPN despite the observation that full-length OPN displayed greater binding in soluble phase to endothelial cell surfaces. While the 32-kD fragment showed significant binding to endothelial cell surfaces, the C-terminal 25-kD fragment did not interact with cell surface. Our data indicate that the increased adhesive activity of MMP-cleaved OPN was accountable by the N-terminal 40-kD fragment. We further analyzed receptor binding, using competition with peptides representing the alpha4beta1 and alpha9beta1 binding sites in the 40-kD N-terminal fragment. Using Jurkat cells, we found that a peptide corresponding to 131ELVTDFPTDLPATE144 had no effect on cell adhesion, whereas the peptide SVVYGLR competitively inhibited cell adhesion. These results suggest that a shorter motif that is found in MMP-cleaved OPN, 162SVVYG166, is sufficient to mediate cell adhesion of Jurkat cells to receptors, including the beta1 integrins, which have been previously characterized to bind the SVVYGLR sequence.     

Intranasal administration of mitochondria improves spatial memory in olfactory bulbectomized mice

Here, we found that functionally active mitochondria isolated from the brain of NMRI donor mice and administrated intranasally to recipient mice penetrated the brain structures in a dose-dependent manner. The injected mitochondria labeled with the MitoTracker Red localized in different brain regions, including the neocortex and hippocampus, which are responsible for memory and affected by degeneration in patients with Alzheimer''s disease. In behavioral experiments, intranasal microinjections of brain mitochondria of native NMRI mice improved spatial memory in the olfactory bulbectomized (OBX) mice with Alzheimer’s type degeneration. Control OBX mice demonstrated loss of spatial memory tested in the Morris water maze. Immunocytochemical analysis revealed that allogeneic mitochondria colocalized with the markers of astrocytes and neurons in hippocampal cell culture. The results suggest that a non-invasive route intranasal administration of mitochondria may be a promising approach to the treatment of neurodegenerative diseases characterized, like Alzheimer''s disease, by mitochondrial dysfunction.     

Induction of antibodies to particular sites of the α7 subunit of the nicotinic acetylcholine receptor in mice of different lines

Five synthetic fragments of the N-terminal domain of the α7 subunit of the human nicotinic acetylcholine receptor (α7 nAChR) that correspond to theoretically calculated B epitopes and T helper epitopes of the protein and contain from 16 to 29 amino acid residues were tested for the ability to stimulate the formation of antibodies in mice of three lines having H-2^d, H-2^b, and H-2^k haplotypes of the major histocompatibility complex. It was shown that, in the free (unconjugated) form, all the peptides stimulate the formation of antibodies at least in one mouse line. Most of the peptides induced the formation of antibodies in BALB/c mice (haplotype H-2^d); therefore, more detailed studies were carried out on these animals. The free peptides and/or their conjugates with keyhole limpet hemocyanin were demonstrated to be capable of stimulating the formation in BALB/c mice of antibodies that bind to the recombinant extracellular N-terminal domain of (α7 nAChRα. The epitope mapping of antipeptide antibodies carried out using truncated fragments helped reveal antipeptide antibodies to four regions of the α7 subunit: 1–23, 98–106, 159–168, and 173–188 (or 179–188).     

Antigenic structure of the foot-and-mouth virus. VI. Functional segments of the immunodominant region of the VP1 protein of foot-and-mouth virus strains O1K and A22]

B- and T-epitopes have been localized within the protective fragments of VP1 protein, viz., 136-152 of the O1K strain and 135-159 of the A22 strain of the foot-and-mouth disease virus (FMDV). Antibodies eliciting after immunization of various animals with the 135-159 A22 peptide are directed to different sites of the peptide. Immunogenicity of fragments of the 135-159 A22 peptide on mice correlates with their activity on T-cells of the same animals and protective activity on guinea pigs. The investigations were carried out using synthetic fragments of the 136-152-O1K and 135-159-A22 peptides.     

Functional recovery after olfactory bulbectomy in rats: effect of embryonal brain grafts

Regeneration of olfactory receptor neurones after bulbectomy can lead to formation of extrabulbar synapses, the functional significance of which remains controversial. Adult hooded rats (n = 27) were bilaterally bulbectomized under pentobarbital anaesthesia. Small pieces of brain tissues (1-2 mm3; OB: olfactory bulb; St: corpus striatum) were obtained from embryos of the same strain and placed into the bulbectomy-produced cavity in contact with the exposed brain surface. Smell was tested at 2- to 3-week intervals from 7 weeks to 7 months after the operation. The latency to find hidden food gradually improved and attained the intact control level in bulbectomized rats without grafts, but remained poor in the OB and St transplanted groups. Seven to ten months after transplantation, spontaneous unit activity and unit reactions to amyl acetate vapours were examined with a carbon fibre microelectrode. Unit responses in the transplants resembled those in the normal OB, but were less pronounced. Morphological examination of the transplant and of its connections with the olfactory receptor neurones and with the host brain suggested that functional recovery was mediated by the connections of the olfactory axons with the remnants of the OB, with the anterior olfactory nucleus and/or with the frontal cortex. The adverse effect of OB and St transplants was probably due to their poor connectivity with the host brain which prevented the regenerating olfactory axons from reaching higher olfactory centres.     

Rat and Mouse Monoclonal Antibodies to Human Myelin Basic Protein

BALB/c mice and Lewis rats were immunized with human myelin basic protein and its N- and C-terminal fragments. Mouse X mouse fusions produced seven monoclonal antibodies, all of the IgG class and directed against the N-terminal fragment. Five of the antibodies seemed to be against the same epitope, between amino acid residues 92 and 118. One antibody bound between residues 45 and 91, and the remaining antibody reacted with both peptides 1-44 and 45-91. Three monoclonal antibodies, all of the IgM class, were obtained by rat X rat hybridization. Two monoclonal antibodies, raised against whole myelin basic protein and the C-terminal fragment, respectively, each bound to peptide 118-178. The remaining antibody, raised against the N-terminal fragment, bound to peptide 45-91. These monoclonal antibodies are of interest for use in clinical radioimmunoassays and for immunohistochemical investigation of the structural relationships of the myelin sheath.     

Detection of alpha7 nicotinic acetylcholine receptors with the aid of antibodies and toxins

Neuronal nicotinic acetylcholine receptors (nAChRs) containing alpha7 subunit are well represented in the brain and some non-neuronal tissues, and their malfunctioning is associated with diverse pathologies. Therefore, detection and quantification of alpha7 nAChR are important tasks. The affinity-purified antibodies were prepared against the 1-23 and 179-190 fragments of the human and rat alpha7 nAChR extracellular domain. The specificity and selectivity of these alpha7 (1-23) and alpha7 (179-190) antibodies was tested by ELISA in model systems: the E. coli-expressed alpha7 subunit extracellular domain and the pituitary cell line GH(4)C(1) stably expressing human alpha7 nAChR. On the rat brain slices two antibodies and biotinylated alpha-cobratoxin specifically stained the hippocampus region known to be rich in alpha7 nAChR. Western blot analysis revealed that in the human thalamus membranes and in rat brain membranes, antibodies alpha7 (1-23) stained a single band of 62 kDa, while the alpha7 (179-190) antibodies stained a doublet of 53-54 kDa. The results obtained show that utilization of model systems and a combination of several antibodies with appropriately labeled toxins may provide better ways for detection of alpha7 nAChR.     

Isolation and characterization of a CNBr cleavage peptide of influenza viral hemagglutinin stimulatory for mouse cytolytic T lymphocytes

A polypeptide fragment obtained by CNBr cleavage of the hemagglutinin from A/JAPAN/305/57 influenza virus has been purified by using high performance liquid chromatography. The first five N-terminal amino acids as determined by sequential Edman degradations have localized this peptide to the HA2 subunit of the hemagglutinin between residues 103 and 123. This peptide, denoted HA2(103-23), can generate both proliferative and cytolytic responses from spleen cells of BALB/c mice previously immunized with A/JAPAN/305/57. These results demonstrate that a single nonglycosylated fragment of the influenza hemagglutinin as small as 21 amino acid residues is capable of being recognized as an antigenic determinant to generate influenza CTL from primed precursors.     

Analysis of specificity of antibodies against synthetic fragments of different neuronal nicotinic acetylcholine receptor subunits

We have compared specificity of a panel of polyclonal antibodies against synthetic fragments of the alpha7 subunit of homooligomeric acetylcholine receptor (AChR) and some subunits of heteromeric AChRs. The antibody interaction with extracellular domain of alpha7 subunit of rat AChR (residues 7-208) produced by heterologous expression in E. coli and rat adrenal membranes was investigated by the ELISA method. For comparison, membranes from the Torpedo californica ray electric organ enriched in muscle-type AChR and polyclonal antibodies raised against the extracellular domain (residues 1-209) of the T. californica AChR alpha1 subunit were also used. Antibody specificity was also characterized by Western blot analysis using rat AChR extracellular domain alpha7 (7-208) and the membrane-bound T. californica AChR. Epitope localization was analyzed within the framework of AChR extracellular domain model based on the crystal structure of acetylcholine-binding protein available in the literature. According to this analysis, the 179-190 epitope is located on loop C, which is exposed and mobile. Use of antibodies against alpha7 (179-190) revealed the presence of alpha7 AChR in rat adrenal membranes.     

beta-Amyloid(1-42) binds to alpha7 nicotinic acetylcholine receptor with high affinity. Implications for Alzheimer's disease pathology

Alzheimer's disease pathology is characterized by the presence of neuritic plaques and the loss of cholinergic neurons in the brain. The underlying mechanisms leading to these events are unclear, but the 42-amino acid beta-amyloid peptide (Abeta(1-42)) is involved. Immunohistochemical studies on human sporadic Alzheimer's disease brains demonstrate that Abeta(1-42) and a neuronal pentameric cation channel, the alpha7 nicotinic acetylcholine receptor (alpha7nAChR), are both present in neuritic plaques and co-localize in individual cortical neurons. Using human brain tissues and cells that overexpress either alpha7nAChR or amyloid precursor protein as the starting material, Abeta(1-42) and alpha7nAChR can be co-immunoprecipitated by the respective specific antibodies, suggesting that they are tightly associated. The formation of the alpha7nAChR.Abeta(1-42) complex can be efficiently suppressed by Abeta(12-28), implying that this Abeta sequence region contains the binding epitope. Receptor binding experiments show that Abeta(1-42) and alpha7nAChR bind with high affinity, and this interaction can be inhibited by alpha7nAChR ligands. Human neuroblastoma cells overexpressing alpha7nAChR are readily killed by Abeta(1-42), whereas alpha7nAChR agonists such as nicotine and epibatidine offered protection. Because Abeta(1-42) inhibits alpha7nAChR-dependent calcium activation and acetylcholine release, two processes critically involved in memory and cognitive functions, and the distribution of alpha7nAChR correlates with neuritic plaques in Alzheimer's disease brains, we propose that interaction of the alpha7nAChR and Abeta(1-42) is a pivotal mechanism involved in the pathophysiology of Alzheimer's disease.     

Fluoro Jade-B Detection of Dying Cells in the SVZ and RMS of Adult Rats After Bilateral Olfactory Bulbectomy

A novel fluorochrome, Fluoro-Jade B, was used to detect dying precursor cells in the subventricular zone (SVZ) and rostral migratory stream (RMS) of adult rats after bilateral olfactory bulbectomy and in control intact rats. The animals in experimental group were left to survive 3 days and from 3 till 16 months after surgical procedure. 1. In the control animals, Fluoro-Jade B positive cells were visible in the SVZ and within the whole extent of the RMS. The number of Fluoro-Jade B positive cells increased in the elbow in comparison to the rest parts of the RMS. 2. In the experimental animals surviving either 3 days or from 3 till 16 months after bilateral olfactory bulbectomy, Fluoro-Jade B positive cells displayed the similar pattern of distribution as in the control animals. However, some quantitative differences in the labeled cells number along the rostral migratory pathway appeared. 3. The average number of degenerating cells within the control SVZ and RMS was 26.24+/- 0.686. In bulbectomized animals, regardless of survival time, an insignificant increase of Fluoro-Jade B positive cells number occurred. We can conclude that dying of precursor cells is a physiological process running within the SVZ/RMS in both control and experimental animals. Moreover, this physiological process is not influenced by survival period after bilateral olfactory bulbectomy. Our results demonstrate Fluoro-Jade B as a useful marker of dying cells.     

Reconstitution of a native-like SH2 domain from disordered peptide fragments examined by multidimensional heteronuclear NMR

The N-terminal SH2 domain from the p85alpha subunit of phosphatidylinositol 3' kinase is cleaved specifically into 9- and 5-kD fragments by limited proteolytic digestion with trypsin. The noncovalent SH2 domain complex and its constituent tryptic peptides have been investigated using high-resolution heteronuclear magnetic resonance (NMR). These studies have established the viability of the SH2 domain as a fragment complementation system. The individual peptide fragments are predominantly unstructured in solution. In contrast, the noncovalent 9-kD + 5-kD complex shows a native-like (1)H-(15)N HSQC spectrum, demonstrating that the two fragments fold into a native-like structure on binding. Chemical shift analysis of the noncovalent complex compared to the native SH2 domain reveals that the highest degree of perturbation in the structure occurs at the cleavage site within a flexible loop and along the hydrophobic interface between the two peptide fragments. Mapping of these chemical shift changes on the structure of the domain reveals changes consistent with the reduction in affinity for the target peptide ligand observed in the noncovalent complex relative to the intact protein. The 5-kD fragment of the homologous Src protein is incapable of structurally complementing the p85 9-kD fragment, either in complex formation or in the context of the full-length protein. These high-resolution structural studies of the SH2 domain fragment complementation features establish the suitability of the system for further protein-folding and design studies.