Effect of Transplantation of Neural Stem and Progenitor Cells on Memory in Animals with Alzheimer's Type Neurodegeneration.

The effects of systemic and intracerebral transplantation of human fetal neural stem and progenitor cells were studied on the model of olfactory bulbectomy in mice with developing signs of sporadic Alzheimer's disease. It was found that transplantation of these cells at certain stages of disease development contributed to improvement of spatial memory and preservation of hippocampal neurons in these animals.

Molecular and Cellular Mechanisms of Sporadic Alzheimer's Disease: Studies on Rodent Models in vivo.

In this review, recent data are presented on molecular and cellular mechanisms of pathogenesis of the most widespread (about 95%) sporadic forms of Alzheimer's disease obtained on in vivo rodent models. Although none of the available models can fully reproduce the human disease, several key molecular mechanisms (such as dysfunction of neurotransmitter systems, especially of the acetylcholinergic system, ß-amyloid toxicity, oxidative stress, neuroinflammation, mitochondrial dysfunction, disturbances in neurotrophic systems) are confirmed with different models. Injection models, olfactory bulbectomy, and senescence accelerated OXYS rats are reviewed in detail. These three approaches to in vivo modeling of sporadic Alzheimer's disease have demonstrated a considerable similarity in molecular and cellular mechanisms of pathology development. Studies on these models provide complementary data, and each model possesses its specific advantages. A general analysis of the data reported for the three models provides a multifaceted and the currently most complete molecular picture of sporadic Alzheimer's disease. This is highly relevant also from the practical viewpoint because it creates a basis for elaboration and preclinical studies of means for treatment of this disease.

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 (Aß) 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 Aß, 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 Aß1-40. 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.

The Y-Box Binding Protein 1 Suppresses Alzheimer's Disease Progression in Two Animal Models.

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 ß-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 ß-amyloid (1-42) inhibited formation of ß-amyloid fibrils, as confirmed by electron microscopy. This suggests that YB-1 interaction with ß-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.

[Protective Activity of Prion Protein Fragments after Immunization of Annimals with Experimentally Induced Alzheimer's Disease].

The prion protein is considered as one of the membrane targets of neurotoxic beta-amyloid during Alzheimer's disease development. We have chosen and synthesized 17-33, 23-33, 95-110 and 101-115 prion fragments involved in beta-amyloid binding. The effect of immunization with the peptides on the features of Alzheimer's disease was investigated in animals with an experimentally induced form of the disease. It was shown that immunization either with peptide 17-33 or with protein conjugates of peptides 23-33 and 101-115 increases the level of brain beta-amyloid and improves morfofunctional state of the brain.

[Fragment of Receptor for Advanced Glycation End Products Improves Memory State in a Model of Alzheimer's Disease].

A number of synthetic peptides corresponding to potentially important regions in the sequence of the four membrane proteins known as beta-amyloid cell receptors have been investigated on their ability to improve memory state in experimental model of Alzheimer's disease. Nine fragments repeating all the exposed nonstructural regions of the RAGE protein according to X-ray data, have been synthesized. The activity of these peptides and synthesized earlier immunoprotective fragments of other three proteins (acetylcholine receptor alpha7-type, prion protein and neurotrophin receptor p75) has been investigated under intranasal administration, without immune response to the peptide. Only one fragment RAGE (60-76) was shown to have a therapeutic activity improving the memory state of bulbectomized mice and leads to decreasing in the level of brain beta-amyloid.

Localization and differentiation pattern of transplanted human multipotent mesenchymal stromal cells in the brain of bulbectomized mice.

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.

Immunization with either prion protein fragment 95-123 or the fragment-specific antibodies rescue memory loss and neurodegenerative phenotype of neurons in olfactory bulbectomized mice.

Epidemiological studies demonstrated association between head injury (HI) and the subsequent development of Alzheimer's disease (AD). Certain hallmarks of AD, e.g. amyloid-ß (Aß) containing deposits, may be found in patients following traumatic BI (TBI). Recent studies uncover the cellular prion protein, PrP(C), as a receptor for soluble polymeric forms of Aß (sAß) which are an intermediate of such deposits. We aimed to test the hypothesis that targeting of PrP(C) can prevent Aß related spatial memory deficits in olfactory bulbectomized (OBX) mice utilized here to resemble some clinical features of AD, such as increased level of Aß, 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 Aß 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 Aß25-35-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 Aß 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 PrP(C) 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 PrP(C) and AD.

[Immunization witha synthetic fragment 155-164 of neurotrophin receptor p75 prevents memory loss and decreases beta-amyloid level in mice with experimentally induced Alzheimer's disease].

Neurotoxic beta-amyloid peptide plays an important role in the pathology of Alzheimer's disease. In aggregated form it binds to several proteins on the surface of the brain cells leading to their death. p75 receptor in- volved in supporting of cell balance is one of the targets for toxic beta-amyloid. We proposed that induction of antibodies against potential binding sites of p75 with beta-amyloid can be a promising approach towards new drug development for Alzheimer's disease therapy. Four potentially immunoactive fragments of p75 were chosen and chemically synthesized. Investigation of immunoprotective effect of the peptide fragments carried out in mice with experimentally induced form of Alzheimer's disease helped to reveal two fragments effectively preserving murine memory from impairment. Results obtained by ELISA biochemical analysis showed that only immunization with fragment p75 155-164 led to significant decrease in beta-amyloid level in the brain of the experimental mice. Thus, immunization with both fragments of p75 receptor is believed to be an effective tool for the development of new drugs against Alzheimer's disease.

Therapeutic effect of mesenchymal multipotent stromal cells on memory in animals with Alzheimer-type neurodegeneration.

Transplantation of human mesenchymal multipotent stromal cells improved spatial memory in bulbectomized mice with Alzheimer-type neurodegeneration. The positive effect was observed in 1 month after intracerebral transplantation and in 3 months after systemic injection of mesenchymal multipotent stromal cells. No cases of malignant transformation were noted. These findings indicate prospects of using mesenchymal multipotent stromal cells for the therapy of Alzheimer disease and the possibility of their systemic administration for attaining the therapeutic effect.

Morphofunctional state of neurons in the temporal cortex and hippocampus in relation to the level of spatial memory in rats after ablation of the olfactory bulbs.

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 beta-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.

[New approaches to the immunotherapy of Alzheimer's disease with the synthetic fragments of alpha7 subunit of the acetylcholine receptor].

The effect of immunization with the synthetic fragments of the alpha7 subunit of the acetylcholine nicotine receptor on the spatial memory of mice subjected to olfactory bulbectomy, which causes the development of the neuro-degenetrative disease of Alzheimer's type, was studied. Mice of the NMRI line were immunized with the KLH conjugates of two peptide fragments of the N-terminal fragment of the alpha7 subunit extraxcellular fragment, subjected to olfactory bulbectomy to cause the development of the neurodegenetrative disease of Alzheimer's type, and then the state of the spartial memory was evaluated. It was shown that 20% of bulbectomized mice immunized with the 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 alpha7-subunit induced good spatial memory in 50% of bulbectomized mice, while in the control group, which received only KLH, none of the animals were educated. 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.

[Morpho-functional state of neurons in temporal cortex and hippocampus in bulbectomized rats with different level 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.

[Noopept improves the spatial memory and stimulates prefibrillar beta-amyloid(25-35) antibody production in mice].

The effects of the novel proline-containing nootropic and neuroprotective dipeptide noopept (GVS-111, N-phenylacetyl-L-prolylglycine ethyl ester) were studied on NMRI mice upon olfactory bulbectomy, which had been previously shown to imitate the main morphological and biochemical signs of Alzheimer's disease (AD). The spatial memory was assessed using the Morris (water maze) test; the immunological status was characterized by ELISA with antibodies to prefibrillar beta-amyloid(25-35), S100b protein, and protofilaments of equine lysozyme, which are the molecular factors involved in the pathogenesis of AD. The control (sham-operated) animals during the Morris test preferred a sector where the safety platform was placed during the learning session. Bulbectomized animals treated with saline failed to recognize this sector, while bulbectomized animals treated with noopept (0.01 mg/kg for 21 days) restored this predominance, thus demonstrating the improvement of the spatial memory. These animals also demonstrated an increase in the level of antibodies to beta-amyloid(25-35)--the effect, which was more pronounced in the sham-operated than in bulbectomized mice. The latter demonstrated a profound decrease of immunological reactivity in a large number of tests. Noopept, stimulating the production of antibodies to beta-amyloid(25-35), can attenuate the well-known neurotoxic effects of beta-amyloid. The obtained data on the mnemotropic and immunostimulant effects noopept are indicative of good prospects for the clinical usage of this drug in the therapy of patients with neurodegenerative diseases.

Cholinopositive effect of dilept (neurotensin peptidomimetic) as the basis of its mnemotropic effect.

Dilept eliminated learning disturbances in the extrapolation avoidance test, caused by chronic injections of scopolamine alone and in combination with madopar to rats. Dilept improved the dynamics of training and parameters of spatial memory impaired by olfactory bulbectomy (operation causing hypofunction of the central cholinergic system). The detected choline-positive effect of dilept together with pronounced dopaminergic activity necessitate its further development as a drug effective in positive and negative symptoms of schizophrenia and psychotic manifestations of Alzheimer's disease.

[Immunodepressed status of mice after bulbectomy]. / Immunodepressivnoe sostoianie myshei posle bul'bectomii.

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.

Morphofunctional changes in neurons in the temporal cortex of the brain in relation to spatial memory in bulbectomized mice after treatment with mineral ascorbates.

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.

Increased level of beta-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.

[Morpho-functional changes of neurons in temporal cortex in comparison with spatial memory in bulbectomized mice after treatment with minerals and ascorbates]. / Morfofunktsional'nye izmeneniia neironov visochnoi kory mozga v sopostavlenii s prostranstvennoi pamiat'iu u bul'béktomirovannykh myshei posle primeneniia mineralaskorbatov.

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.