A. N. Samokhin

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 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.

Exogenous Hsp70 delays senescence and improves cognitive function in aging mice

Significance The compromised ability of neurons to express Heat Shock Protein 70 (Hsp70) correlates with aging-related neurodegeneration. In this study, middle-aged and old mice were treated chronically until death with human Hsp70 delivered intranasally and were investigated after 5 or 9 mo of Hsp70 treatment for their cognitive ability and synaptic density. Hsp70 treatment extended mean and maximum lifespan, improved learning and memory in old animals, increased curiosity, decreased anxiety, and helped maintain synaptic structures that degrade with age. These results provide evidence that intranasal administration of Hsp70 could have significant therapeutic potential in preserving brain tissue and memory for middle-age and old individuals and could be applied either as unique self-contained treatment or in combination with other pharmacological therapies. Molecular chaperone Heat Shock Protein 70 (Hsp70) plays an important protective role in various neurodegenerative disorders often associated with aging, but its activity and availability in neuronal tissue decrease with age. Here we explored the effects of intranasal administration of exogenous recombinant human Hsp70 (eHsp70) on lifespan and neurological parameters in middle-aged and old mice. Long-term administration of eHsp70 significantly enhanced the lifespan of animals of different age groups. Behavioral assessment after 5 and 9 mo of chronic eHsp70 administration demonstrated improved learning and memory in old mice. Likewise, the investigation of locomotor and exploratory activities after eHsp70 treatment demonstrated a significant therapeutic effect of this chaperone. Measurements of synaptophysin show that eHsp70 treatment in old mice resulted in larger synaptophysin-immunopositive areas and higher neuron density compared with control animals. Furthermore, eHsp70 treatment decreased accumulation of lipofuscin, an aging-related marker, in the brain and enhanced proteasome activity. The potential of eHsp70 intranasal treatment to protect synaptic machinery in old animals offers a unique pharmacological approach for various neurodegenerative disorders associated with human aging.

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 Alzheimers disease (AD). Certain hallmarks of AD, e.g. amyloid-β (Aβ) 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 Aβ (sAβ) which are an intermediate of such deposits. We aimed to test the hypothesis that targeting of PrP(C) can prevent Aβ related spatial memory deficits in olfactory bulbectomized (OBX) mice utilized here to resemble some clinical features of AD, such as increased level of Aβ, 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 Aβ 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 Aβ25-35-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 Aβ 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.

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.

Immunization with a 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 involved 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 development of new anti-Alzheimer’s disease treatment. 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 pro-vides a new insight into anti-Alzheimer’s disease drug design.

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, β-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.

Immunization Against Specific Fragments of Neurotrophin p75 Receptor Protects Forebrain Cholinergic Neurons in the Olfactory Bulbectomized Mice

Alzheimer’s disease (AD) is characterized by progressive cognitive impairment associated with marked cholinergic neuron loss and amyloid-β (Aβ) peptide accumulation in the brain. The cytotoxicity in AD is mediated, at least in part, by Aβ binding with the extracellular domain of the p75 neurotrophin receptor (p75NTR), localized predominantly in the membranes of acetylcholine-producing neurons in the basal forebrain. Hypothesizing that an open unstructured loop of p75NTR might be the effective site for Aβ binding, we have immunized both olfactory bulbectomized (OBX) and sham-operated (SO) mice (n = 82 and 49, respectively) with synthetic peptides, structurally similar to different parts of the loops, aiming to block them by specific antibodies. OBX-mice have been shown in previous studies, and confirmed in the present one, to be characterized by typical behavioral, morphological, and biochemical AD hallmarks, including cholinergic deficits in forebrain neurons. Immunization of OBX- or SO-mice with KLH conjugated fragments of p75NTR induced high titers of specific serum antibodies for each of nine chosen fragments. However, maximal protective effects on spatial memory, evaluated in a Morris water maze, and on activity of choline acetyltransferase in forebrain neurons, detected by immunoreactivity to specific antibodies, were revealed only for peptides with amino acid residue sequences of 155–164 and 167–176. We conclude that the approach based on immunological blockade of specific p75NTR sites, linked with the cytotoxicity, is a useful and effective tool for study of AD-associated mechanisms and for development of highly selective therapy of cholinergic malfunctioning in AD patients.

A fragment of the receptor for advanced glycation end products restores the spatial memory of animals in a model of Alzheimer’s disease

The ability of some synthetic peptides modeling the potentially important regions of four membrane proteins, known as cell targets for beta-amyloid, to restore the spatial memory of animals in an experimental model of Alzheimer’s disease has been studied. Nine fragments of the protein receptor for advanced glycation end products (RAGE), which, according to X-ray structure analysis data, repeat all its exposed nonstructural regions, have been synthesized. The effect of these peptides and of earlier synthesized immunoprotective fragments of three other proteins (alpha7-type acethylcholine receptor, the prion protein, and the neurotrophin receptor p75) has been studied on intranasal administration, which excludes the development of the immune response to the peptide. It has been shown that only one fragment, RAGE (60–76), exhibits a therapeutic activity, by restoring the spatial memory of bulbectomized mice and decreasing the level of the brain beta-amyloid.

Protective activity of fragments of the prion protein after immunization of animals with experimentally induced Alzheimer’s disease

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.

Molecular Mechanisms Underlying Neuroprotective Effect of Intranasal Administration of Human Hsp70 in Mouse Model of Alzheimer’s Disease

Heat shock protein 70, encoded by the HSPA1A gene in humans, is a key component of the machinery that protects neuronal cells from various stress conditions and whose production significantly declines during the course of aging and as a result of several neurodegenerative diseases. Herein, we investigated whether sub-chronic intranasal administration of exogenous Hsp70 (eHsp70) exerts a neuroprotective effect on the temporal cortex and areas of the hippocampus in transgenic 5XFAD mice, a model of Alzheimers disease. The quantitative analysis of neuronal pathologies in the compared groups, transgenic (Tg) versus non-transgenic (nTg), revealed high level of abnormalities in the brains of transgenic mice. Treatment with human recombinant Hsp70 had profound rejuvenation effect on both neuronal morphology and functional state in the temporal cortex and hippocampal regions in transgenic mice. Hsp70 administration had a smaller, but still significant, effect on the functional state of neurons in non-transgenic mice as well. Using deep sequencing, we identified multiple differentially expressed genes (DEGs) in the hippocampus of transgenic and non-transgenic mice. Furthermore, this analysis demonstrated that eHsp70 administration strongly modulates the spectrum of DEGs in transgenic animals, reverting to a pattern similar to that observed in non-transgenic age-matched mice, which included upregulation of genes responsible for amine transport, transmission of nerve impulses and other pathways that are impaired in 5XFAD mice. Overall, our data indicate that Hsp70 treatment may be an effective therapeutic against old age diseases of the Alzheimers type.