The receptor for advanced glycation end products (RAGE) plays an essential role in the development of Alzheimer's disease. Activation of RAGE causes brain inflammation, oxidative stress and secretion of beta-amyloid. It is known that the soluble isoform of receptor (sRAGE), which lacks the transmembrane and cytosolic domains, binds to ligands and prevents negative effects of the receptor activation in in vivo and in vitro experiments. We proposed that peptide fragments from sRAGE would demonstrate the same biological activity. RAGE fragments were prepared with standard Fmoc/Butprotocol of solid-phase peptide synthesis. NMRI mice were used after olfactory bulbectomy (OBX) which induced behavioral, morphological and biochemical signs of the Alzheimer's type degeneration. In 2 weeks after the surgery peptides were intranasally administrated in a dose of 20 mg per mouse during 15 days. Murine memory was tested in the Morris's water maze. We used Nissl staining to observe the morphological state of neurons in the temporal cortex and areas of CA1 and CA3 of the hippocampus. We counted the neuronal density, the percentage of normal neurons and neurons with pathologies. We selected and synthesized ten peptide fragments from unstructured surface-exposed regions of RAGE. We found that after intranasal administration of peptides only fragment (60–76) effectively prevents murine memory from impairment. Six overlapping fragments of RAGE (60–76) peptide were synthesized to find a site responding for the therapeutic effect. Tests carried out with these fragments showed that only (60–70) peptide is responsible for preventing animal memory impairment. Fragments (60–76) and (60–70) block the development of neuronal pathology in OBX mice. However, some N-terminal fragments which do not have significant activity in Morris's test also improve the morpho-functional state of neurons in OBX animals, but not so efficient in several aspects. There is a correlation between memory improvement activity and maintenance of neuronal morphology on a normal level after intranasal administration of RAGE fragment. Peptides (60–76) and (60–70) are active in both tests. Several N-terminal fragments also protect morphological state of neurons, but less noticeably, which corresponds with the absence of effect in Morris's test. Supported by RFBR Grant 15–04–01360, 17–34–80016 ”moleva.

In order to study the structural and functional specificity of different fragments of extracellular domains of the membrane receptors, their immunological blockade and analysis of changes in brain function in health and disease were carried out. Effects of the immunization against unstructured fragments of the neurotrophin receptor (p75 NTR) containing binding sites for a nerve growth factor, amyloid-beta, and proneurotrophin were studied. It was shown that immunization of NMRI mice with synthetic peptides with amino-acid residue sequences corresponding to the p75 NTR fragments 86–93 and 146–153 exerted an adverse effect both on the morpho-functional state of the cortical and hippocampal neurons and on the spatial memory of animals. In contrast, the immunization by the p75 NTR synthetic fragment 167–176 was accompanied by an improvement of these characteristics. The results of the structural-functional mapping of the extracellular domain in neuronal membrane receptors in vivo were in line with the published data of the X-ray analysis. The immunological approach developed in this work provides insight into the peculiarities of the receptor functioning in the course of the Alzheimer’s type neurodegeneration process and favors the development of effective immunological ways for selective therapy of the Alzheimer’s disease.

Прионный белок является одной из мембранных мишеней бета-амилоида, основного нейротоксического агента при болезни Альцгеймера. Нами был осуществлен выбор и синтез фрагментов прионного белка 17–33, 23–33, 95–110 и 101–115, ответственных за связывание с бета-амилоидом. Было изучено влияние иммунизации пептидами на развитие признаков болезни у животных с экспериментально индуцированной формой болезни Альцгеймера. Показано, что иммунизация свободным пептидом 17–33 и белковыми конъюгатами пептидов 23–33 и 101–115 приводит к восстановлению состояния пространственной памяти животных. Также показано, что иммунизация пептидом 17–33 приводит к снижению уровня мозгового бета-амилоида и восстановлению морфофункциональных показателей мозга.

The prion protein is considered as one of the membrane targets of the neurotoxic beta-amyloid during development of Alzheimer’s disease. We chose and synthesized peptide fragments that corresponded to the 17–33, 23–33, 95–110, and 101–115 sequences of the prion protein and are responsible for the betaamyloid binding. The effect of immunization with the peptides on the development of symptoms of Alzheimer’s disease was investigated on animals with an experimentally induced form of the disease. Immunization with either free 17–33 peptide or with protein conjugates of the 23–33 and 101–115 peptides was shown to restore spatial memory of the animals. Immunization with the 17–33 peptide was also shown to decrease the level of brain beta-amyloid and to recover morphofunctional parameters of the brain.

The Y-box binding protein 1 (YB-1) is a member of the family of DNA- and RNA binding proteins. It is involved in a wide variety of DNA/RNA-dependent events including cell proliferation and differentiation, stress response, and malignant cell transformation. Previously, YB-1 was detected in neurons of the neocortex and hippocampus, but its precise role in the brain remains undefined. Here we show that subchronic intranasal injections of recombinant YB-1, as well as its fragment YB-11−219, suppress impairment of spatial memory in olfactory bulbectomized (OBX) mice with Alzheimer’s type degeneration and improve learning in transgenic 5XFAD mice used as a model of cerebral amyloidosis. YB-1-treated OBX and 5XFAD mice showed a decreased level of brain β-amyloid. In OBX animals, an improved morphological state of neurons was revealed in the neocortex and hippocampus; in 5XFAD mice, a delay in amyloid plaque progression was observed. Intranasally administered YB-1 penetrated into the brain and could enter neurons. In vitro co-incubation of YB-1 with monomeric β-amyloid (1–42) inhibited formation of β-amyloid fibrils, as confirmed by electron microscopy. This suggests that YB-1 interaction with β-amyloid prevents formation of filaments that are responsible for neurotoxicity and neuronal death. Our data are the first evidence for a potential therapeutic benefit of YB-1 for treatment of Alzheimer’s disease.

Receptor p75 is a classical membrane receptor, which consists of an extracellular region, a transmembrane region, and an intracellular C-terminus death domain. p75 is involved in the activation of neurotrophic factors and plays an essential role in the regulation of cell functions such as proliferation, differentiation, synaptogenesis, neuronal plasticity, and survival or apoptosis. It is supposed that p75 accelerates the positive effects of neurotrophins being a part of the complex with the Trk receptor; however, in the absence of Trk receptors, the interaction between p75 and neurotrophins leads to cell apoptosis. The interest in the p75 receptor has increased dramatically in the past few years, as it has been shown that p75 may play a crucial role in the genesis of neurodegenerative disorders, in particular, Alzheimer’s disease. The interaction between p75 and β-amyloid leads to neuronal death in the brain. Identification of the p75 regions involved in the development of pathology is of great importance for understanding the mechanisms of Alzheimer’s disease and for creating targeted therapeutic agents for this disorder. In the present work, immunological approaches have been used for structure-function mapping of the extracellular domain of the p75 receptor during the development of a neurodegenerative process in olfactory bulbectomized animals. The nine extracellular regions selected on the basis of X-ray structure analysis of p75 molecule hypothetically could include β-amyloid binding sites and participate in the progression of pathology. Peptides with the amino acid sequences analogous to the selected regions were synthesized. Immunization with such fragments conjugated with hemocyanin induced the formation of antipeptide antibodies in experimental animals. However, only immunization with fragment (167–176) prevented the memory loss and neuronal death in the cortex and hippocampus of bulbectomized mice. This fragment had no effect on sham-operated mice. The results indicate that fragment (167–176) of the p75 sequence deserves further research as a potential basis of the targeted immunotherapy of Alzheimer’s disease.

Replacement cell therapy with transplantation of stem cells is a promising approach for the therapy of various neurodegenerative diseases, e.g. Alzheimer’s disease. However, the behavior of transplanted cells in the damaged tissue should be thoroughly studied before introduction of this method into clinical practice. We studied the pathways of migration of human multipotent mesenchymal stromal cells after their systemic transplantation into the brain of bulbectomized mice characterized by the development of Alzheimer-type neurodegenerative process. Immunohistochemical analysis with antibodies to human nuclear antigen (HNA) and immunofluorescent analysis of the results of transplantation of multipotent mesenchymal stromal cells carrying green fluorescent protein (GFP) gene showed that these cells can cross the blood–brain barrier and penetrate into some structures of recipient brain. Analysis of differentiation of transplanted human cells using antibodies to neurospecific enolase (NSE) or astroglial marker (GFAP) with parallel staining for human nuclear antigen revealed no neural differentiation of transplanted cells in the brains of bulbectomized animals. However, some of these cells differentiated into astrocytes, which brought us to an assumption on important role of astroglial abnormalities in the pathogenesis of Alzheimer’s disease.

Epidemiological studies demonstrated association between head injury (I-II) and the subsequent development of Alzheimer's disease (AD). Certain hallmarks of AD, e.g. amyloid-beta (A beta) containing deposits, may be found in patients following traumatic BI (TBI). Recent studies uncover the cellular prion protein, PrPC, as a receptor for soluble polymeric forms of A beta (sA beta) which are an intermediate of such deposits. We aimed to test the hypothesis that targeting of PrPC can prevent A beta related spatial memory deficits in olfactory bulbectomized (OBX) mice utilized here to resemble some clinical features of AD, such as increased level of A beta, memory loss and deficit of the CNS cholin- and serotonin-ergic systems. We demonstrated that immunization with the a.a. 95-123 fragment of cellular prion (PrP-I) recovered cortical and hippocampus neurons from OBX induced degeneration, rescued spatial memory loss in Morris water maze test and significantly decrease the A beta level in brain tissue of these animals. Affinity purified anti-PrP-I antibodies rescued pre-synaptic biomarker synaptophysin eliciting similar effect on memory of OBX mice, and protected hippocampal neurones from A beta(25-35)-induced toxicity in vitro. Immunization OBX mice with a.a. 200-213 fragment of cellular prion (PrP-II) did not reach a significance in memory protection albeit having similar to PrP-I immunization impact on A beta level in brain tissue. The observed positive effect of targeting the PrP-I by either active or passive immunization on memory of OBX mice revealed the involvement of the PrPC in AD-like pathology induced by olfactory bulbectomy. This OBX model may be a useful tool for mechanistic and preclinical therapeutic investigations into the association between PrPC and AD. (C) 2013 Elsevier Inc. All rights reserved.

Ablation of the olfactory bulbs (bulbectomy) in mice and guinea pigs evokes a neurodegenerative process which, in terms of its morphological, biochemical, and behavioral features, is similar to Alzheimer’s disease. We report here studies of the long-term sequelae of bulbectomy in rats. One year after surgery, testing of spatial memory in bulbectomized rats (BER) allowed the animals to be divided into two groups-those with good memory (BER-gm) and those with poor memory (BER-pm). Quantitative analysis of the morphofunctional state of neurons showed that BER-pm, as compared with the BER-gm group, had more marked pathological lesions in neurons of the temporal cortex and hippocampus, with significant increases in the numbers of cells showing pyknosis, karyolysis, cytolysis, and vacuolization. Both groups showed decreases in the distribution density of cells in the cortex. In terms of the level of brain β-amyloid, the study groups fell in the order: BER-pm > BER-gm > control sham-operated rats. These results provide evidence of the long-term nature of changes in the morphofunctional state of neurons in the brains of BER, correlating with their levels of spatial memory.

Olfactory bulbs removal (bulbectomy) induced neurodegeneration in the brain of mice and guinea pigs which, according to its morphological, biochemical and behavioral features was simular to manifestations of Alzheimer's disease. In the present work long-term effects of bulbectomy were examined in rats. It was shown that 1 year after the operation bulbectomized animals (BEA) could be divided into two subgroups: animals with good results of testing spatial memory(BEA-GM), and with poor memory (BEA-PM). The quantitative analysis of neurons morpho-functional state has shown more expressed pathological changes (an increase in the number of cells with pyknosis, karyolysis, cytolysis, and vacuolization) in neurons of temporal cortex and hippocampus in BEA-PM as compared to those in BEA-GM. In both animal groups the reduction of cellular density was marked in the cortex. According to the content of brain beta-amyloid the groups of experimental animals were distributed in the following order: BEA-PM>BEA-GM>control group of sham-operated rats. The results indicate the long-term changes of morpho-functional state of neurons in the brain of BEA, which correlated with the level of their spatial memory.

Our investigation is focused on further experimental verification of insufficiently studied hypothesis on the role of olfactory system abnormalities in AD genesis. Early we have shown that olfactory bulbectomized mice and rats demonstrated the increased brain level of beta-amyloid (A beta), neuronal loss in specific brain structures, deficit in the acetylcholinergic and serotoninergic systems along with impairment of memory and development of depressive-like state. Here we revealed that plaque formation was induced in the cortex and hippocampus in olfactory bulbectomized guinea-pigs, which express A beta peptides of the human amino acid sequence.

The effects of an antioxidant mixture of mineral ascorbates (MA) on the state of neurons in the temporal area of the cortex and the behavior of mice subjected to bulbectomy (BE) were studied; these mice, as demonstrated previously, are characterized by deficiency of spatial memory and the development of a neurodegenerative process in brain structures showing pathological changes in Alzheimer's disease. One month after BE, there were abnormalities in the cytoarchitectonics of the temporal area of the cortex, with loss of clarity of the boundaries between its layers because of dystrophy of pyramidal neurons and foci of loss of these cells. There were sharp increases in the numbers of neurons showing pyknosis, karyolysis, and vacuolysis on the background of decreases in neuronal density. Three weeks of treatment by addition of MA to the diet prevented the degradation of spatial memory in mice after BE and protected neurons in the temporal area of the cortex from degenerative changes. These results provide evidence for the possibility of prophylaxis of neurodestructive changes of the Alzheimer's type.

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 β-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.

The effect of antioxidant mixture of mineralascorbates (MA) on the status of neurons of brain temporal cortex and behavior of mice after olfactory bulbectomy (BE) was studied, as it was previously shown by us that these animals were characterized by a deficit of spatial memory and development of neurodegenerative process in brain structures, which are affected by Alzheimer disease. Disorganization of cytoarchitectonics of temporal cortex with the deletion of its layers as a result of dystrophy of pyramidal neurons and foci of their complete disappearance were shown 1 month after BE. The increased number of neurons with the phenomena of karyopyknosis, karyolysis and vacuolysis was observed with a concomitant reduction in neuronal density. Addition of MA to the diet for three weeks prevented the development of deterioration of spatial memory in mice after BE and protected the neurons of brain temporal cortex from the degenerative changes. The results obtained suggest the possibility of realization of prophylaxis aimed at the prevention of the development of Alzheimer-type neurodestructive processes.

This report presents results obtained from studies of the mnemotropic activity of the ergot alkaloid agroclavin. The effects of this substance were studied at concentrations of 1, 10, 25, 50, and 200 μg/kg on the learning and retention of a spatial habit in a Morris water maze. These doses of agroclavin had no effect on the process of formation of the spatial habit, but sharply disturbed retention of the habit. A long-lasting effect of memory trace disturbance was seen over a period of 48 h after dosage. On retraining, animals showed no delayed effects with agroclavin on learning a new spatial habit. The possible mechanisms for the effect of agroclavin on memory are discussed.