Natalia V. Bobkova

Bulbectomy-induced loss of raphe neurons is counteracted bt antidepressant treatment

1. Bilateral olfactory bulb ablation was performed in C57B1/6j mice (C57). Separate groups of bulbectomized mice were treated with either antidepressants (trazodone, 20 mg/kg i.p., or amitriptyline, 20 mg/kg i.p.) or saline daily for 14 consecutive days starting 14 days after surgery. 2. Celloidine-imbedded 10 microns-thick brain sections containing the nucleus raphe dorsalis (NRD) or locus coeruleus (LC) were stained for Nissl, and the number of functional and pyknotic cells was counted out of 500 total cell count for each animal in every experimental group: sham-operated, bulbectomized treated with saline or one of the two antidepressants. 3. Bulbectomy produced a significant 4 times increase in the proportion of pyknotic cells in NRD as compared to sham-operated control. Both antidepressants reversed the effect bringing the number of pyknotic cells to control level. The proportion of pyknotic cells in LC was also slightly increased (61%) in the bulbectomized mice, but only amitriptyline was able to reverse the effect. 4. Widespread degeneration of the neurons in NRD caused by bulbectomy may be involved in the serotonergic component of the bulbectomy syndrome. The ability of antidepressants to diminish bulbectomy-induced loss of NRD neurons may underlie their restorative effect on the behavior and neurochemical characteristics of bulbectomized animals.

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.

Interhemispheric EEG differences in olfactory bulbectomized rats with different cognitive abilities and brain beta-amyloid levels

Alterations in electroencephalogram (EEG) asymmetry and deficits in interhemispheric integration of information have been shown in patients with Alzheimers disease (AD). However, no direct evidence of an association between EEG asymmetry, morphological markers in the brain, and cognition was found either in AD patients or in AD models. In this study we used rats with bilateral olfactory bulbectomy (OBX) as one of the AD models and measured their learning/memory abilities, brain beta-amyloid levels and EEG spectra in symmetrical frontal and occipital cortices. One year after OBX or sham-surgery, the rats were tested with the Morris water paradigm and assigned to three groups: sham-operated rats, SO, and OBX rats with virtually normal, OBX(+), or abnormal, OBX(-), learning (memory) abilities. In OBX vs. SO, the theta EEG activity was enhanced to a higher extent in the right frontal cortex and in the left occipital cortex. This produced significant interhemispheric differences in the frontal cortex of the OBX(-) rats and in the occipital cortex of both OBX groups. The beta1 EEG asymmetry in SO was attenuated in OBX(+) and completely eliminated in OBX(-). OBX produced highly significant beta2 EEG decline in the right frontal cortex, with OBX(-)>OBX(+) rank order of strength. The beta-amyloid level, examined by post-mortem immunological DOT-analysis in the cortex-hippocampus samples, was about six-fold higher in OBX(-) than in SO, but significantly less (enhanced by 82% vs. SO) in OBX(+) than in OBX(-). The involvement of the brain mediatory systems in the observed EEG asymmetry differences is discussed.

Antidepressants suppress bulbectomy-induced augmentation of voluntary alcohol consumption in C57Bl/6j but not in DBA/2j mice

Bulbectomy has been previously shown to produce the specific antidepressant-sensitive syndrome in C57Bl/6j, but not DBA/2j mice. The present study examined the effect of the depression on voluntary alcohol consumption. Alcohol consumption and alcohol preference (% of alcohol solution in total liquid) in a free-choice, two-bottle situation was measured in C57BL/6j and DBA/2j mice after sham-operation, anosmia with 10% ZnSO4, or bulbectomy. Both anosmic and bulbectomized mice of both strains consumed more alcohol and showed stronger preference for alcohol than sham-operated mice. In DBA/2j mice both operations altered alcohol consumption of the whole population, and the effect of bulbectomy was stronger. In C57Bl/6j mice bulbectomy and, to a less degree, anosmia seemed to affect predominantly the low-drinking animals. Chronic treatment with antidepressants amitriptyline (20 mg/kg), trazodone (20 mg/kg), and imipramine (10 mg/kg), did not change alcohol consumption in sham-operated C57Bl/6j mice. In anosmic mice antidepressants decreased alcohol preference, but only amitryptyline also decreased alcohol consumption. All antidepressants decreased both alcohol consumption and preference in bulbectomized C57Bl/6j mice. In DBA/2j mice antidepressant treatment either increased, or did not alter alcohol consumption and preference in all groups, though the effects varied among individual antidepressants. The possible connection between the bulbectomy-induced behavioral syndrome and elevated ethanol consumption in C57Bl/6j mice is discussed.

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.

Interplay between recombinant Hsp70 and proteasomes: proteasome activity modulation and ubiquitin-independent cleavage of Hsp70

The heat shock protein 70 (Hsp70, human HSPA1A) plays indispensable roles in cellular stress responses and protein quality control (PQC). In the framework of PQC, it cooperates with the ubiquitin-proteasome system (UPS) to clear damaged and dysfunctional proteins in the cell. Moreover, Hsp70 itself is rapidly degraded following the recovery from stress. It was demonstrated that its fast turnover is mediated via ubiquitination and subsequent degradation by the 26S proteasome. At the same time, the effect of Hsp70 on the functional state of proteasomes has been insufficiently investigated. Here, we characterized the direct effect of recombinant Hsp70 on the activity of 20S and 26S proteasomes and studied Hsp70 degradation by the 20S proteasome in vitro. We have shown that the activity of purified 20S proteasomes is decreased following incubation with recombinant human Hsp70. On the other hand, high concentrations of Hsp70 activated 26S proteasomes. Finally, we obtained evidence that in addition to previously reported ubiquitin-dependent degradation, Hsp70 could be cleaved independent of ubiquitination by the 20S proteasome. The results obtained reveal novel aspects of the interplay between Hsp70 and proteasomes.

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.

The brain compensatory mechanisms and Alzheimer's disease progression: a new protective strategy.

Compensatory/adaptive mechanisms in the brain are hypothesized to be involved in its protection from the Alzheimers disease (AD) progression. These mechanisms are activated by malfunctioning of various brain systems: antioxidant, neurotrophic, neurotransmitter, immune, and others. Detailed analysis of compensatory/adaptive capabilities of these systems might be a start point for further discovery and development of perspective approaches for early diagnostics and treatment of AD and associated neurodegenerative disorders. AD is characterized by memory impairment, dementia, cholinergic neuron loss, amyloid-beta (Aβ) peptides deposition forming senile plaques and intracellular neurofibrillary tangles of hyperphosphorylated tau protein in the brain. This age-associated chronical neurodegenerative pathology is intensively expanding in modern human society and has been noted to start well in advance of its clinical manifestations. The “latent” period in AD progression appears to be associated with involvement of compensatory mechanisms in the brain which are able temporarily or permanently to protect it from the neurodegenerative processes even initiated by specific mutations in the amyloid precursor protein or presenilins. Understanding of principles how these mechanisms work and how manipulate them allows the developing of new approaches in early diagnostics of AD, its effective prophylactic, and therapeutic treatment. The main efforts in this field were associated with the attempts to characterize an imbalance between the neurodegenerative and regenerative processes in the brain (for review, Iqbal et al., 2014). However, most of conventional approaches are predominantly oriented on a clinical stage of the disease, when the compensatory mechanisms are at a “saturated” stage: either completely exhausted or hyperactivated, which makes them inaccessible to detailed studying. Associated with this, a lack of experimental models directly imitating the progression of AD was the main restrictive factor in the “compensatory” field. In our previous studies, the animals with surgically removed olfactory bulbs (OBX-animals) showed typical behavioral, morphological and biochemical AD hallmarks (Nesterova et al., 2008), and evident disturbances in interrelations between different brain areas (Bobkova et al., 2008). Recently, we have demonstrated in OBX-mice a biphasic time course in the AD hallmarks’ expression (in particular, the spatial memory impairment and the Aβ level rising in the hippocampus) after the bulbectomy (Bobkova et al., 2013). Protective and therapeutic capabilities of these compensatory mechanisms can be released at various levels of the brain functioning. Surprisingly, the neurofibrillary tangles and Aβ have been revealed to be able to display an antioxidant activity (Moreira et al., 2008). Furthermore, Aβ, as a chelator of some metals (cuprum, iron, and zinc), can be involved in normal neurotrophic functions in the brain. Commonly, both Aβ generation and tau-protein phosphorylation are associated with recovery of oxidative homeostasis in the cells. At early stage of AD, activated both micro- and astroglia, accompanying the disease, have been shown to be involved in englobement of both Aβ and remains of decayed neurons and, additionally, in prevention of the neurofibrillary tangle maturing. The late stage of AD is characterized by evident misbalance between Aβ accumulation and its utilization in the brain (Moreira et al., 2008). At early, but not late, stage of AD progression, the level of transthyretin, a protein inhibiting Aβ aggregation and detoxifying cell-damaging conformers, has been shown to be increased that precludes both Aβ oligomerization and tau-protein hyperphosphorilation (Buxbaum et al., 2008). A protein, neprilysin, Aβ-degrading endopeptidase, displayed similar AD progression profile in the cerebrospinal fluid (CSF) in AD patients. The compensatory brain mechanisms in these patients seem to be involved in an associative rising of both the level of clusterin, a protein associated with the clearance of cellular debris, and the extent of Aβ utilization after delivery of Aβ complexes into the neuroglial cells and/or in blood stream. In OBX-mice, we revealed a close association between memory improvement, morphobiochemical brain markers normalization and enhanced levels of an endogenous heat shock protein (HSP70) in the hippocampus (Bobkova et al., 2013). HSP70, as a chaperone, protects neurons from Aβ aggregation and toxicity whereas its capability to produce very stable complexes with the tau-protein protects them from hyperphosphorilation. Recently, we have shown that a subchronical intranasal injection of HSP70, at a small dose, effectively protected OBX-mice from the memory loss both at early and late stages of AD progression (Bobkova et al., 2014a). Depleting level of a presynaptic marker, synaptophysin, closely associated with lowering of synaptic receptor density in both AD patients, transgenic animals and OBX-mice (Bobkova et al., 2014b), has been shown to be compensated in part by enhancing level of a postsynaptic protein, PSD-95 (Leuba et al., 2008). This protein is well known to be supportive for functioning of the AMPA and NMDA receptors, which are important for memory formation. In our previous study on OBX-mice, a rising of the serotoninergic (5-HT) receptors density was revealed in the cortex and the hippocampus, that we supposed to be a compensatory reaction on the bulbectomy-produced lowering of both neuronal density and 5-HT content in the dorsal n.raphe, the main source of serotonin for the frontal brain areas (Gurevich et al., 1993). In AD patients, the levels of anti-Aβ antibodies in the serum and/or CSF may correlate with AD progression, thus, making them potentially useable as a treatment approach and/or the early diagnostic and prognostic markers for the disease (Dorothee et al., 2013). This compensatory reaction is evidently associated with Aβ elimination from the brain. However, the immunological approach has been shown to be ineffective in clinics seemingly because of its traditional use at very late stage of the disease, when the compensatory mechanisms were completely unworkable. Moreover, even at early stage of AD progression, this approach needs to be specified for the intercepting of those processes which are exactly associated with depression of compensatory mechanisms. We suggest that the selective immunological blockade of Aβ targets, mediating its neurotoxicity, might be an effective and balanced approach at early stage of AD, allowing the implementing of Aβ beneficial functions in the brain. Given close association between Aβ-produced neurotoxicity and activation of neurotransmitter receptors, we attempted to uncover those of their fragments which contained specific biding sites for Aβ. After immunization with the fragments of extracellular domains of α7-subtype either of the acetylcholine or prion receptors, evident improvement in both spatial memory and morphology of cortical and hippocampal neurons, in parallel with the Aβ level lowering, were revealed in OBX-mice (Kamynina et al., 2010; Bobkova et al., 2014b). The antibodies to these fragments had to be noticed to exert obvious protective effects on hippocampal cells culture treated with the Aβ1–42 oligomers as well (Bobkova et al., 2014b). Recently, we have performed several pilot experiments on OBX-mice with the immunological blockade of p75-neurotrophin receptors, which are well known to be involved in Aβ-induced cytotoxicity. Only immunization with two from nine selected fragments of p75 receptor, and with induction of high level of specific antibodies, showed spatial memory improvement, lowering of Aβ in cortical and hippocampal extracts, and protecting of the forebrain acetylcholine system in OBX-mice (in press). We suggest that understanding of intimate mechanisms of the neurotrophin receptors Aβ binding and use of suitable immunological tools might be one of the most perspective approaches for AD treatment. It should be mention, however, that this paper does not introduce all aspects of the brain compensatory mechanisms functioning at latent phase of AD, in particular, associated with the controlling of hormonal processes, apoptosis, astrocyte functions, and neurogenesis. Nevertheless, we hope that the main idea about these mechanisms involved in protection from AD progression might be a start point for further discovery and development of perspective approaches, based on the releasing of the endogenic brain reserves, for early diagnostics and treatment of AD and associated neurodegenerative disorders. We are grateful to all members of the laboratory staff. NB was supported by grant KOMFI 13-04-40106-H (Russia): “Structure-functional studies of π-75 receptor – molecular target for neurodegenerative diseases immunotherapy”; Grant RFBR 13-04-00633A (Russia): “Study of role of receptor for advanced glycation end products (RAGE) in mechanisms of beta-amyloid neurotoxicity in model of sporadic Alzheimers disease”.

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.

The Weak Combined Magnetic Fields Reduce the Brain β-Amyloid in an Animal Model of Sporadic Alzheimer's Disease

Subchronic effect of weak combined magnetic fields (MFs) on spatial memory and level of brain β-amyloid (βA) was studied in mice with ablation of the olfactory bulbs and control sham-operated (SO) animals. The bulbectomized (BE) mice show the main signs of Alzheimer’s type degeneration such as memory impairment, the increase of the βA level in the brain, pathology in the acetylcholinergic system, and the loss of neurons in the brain structures responsible for memory [5–7, 9]. The combined MFs consisted of the constant component 42μT and of the variable component 0.08μT The variable field was the sum of two signals of frequencies of 4.38 and 4.88 Hz. Exposure to the MFs (4 hours for 10 days) induced the reduction of the βA level in the brain of the BE mice, but did not protected their memory from impairment. However, the same MFs improved the spatial memory in SO mice. The beneficial effect of the MFs in the SO animals was prolonged and was revealed for a month after exposure to the MFs. The results suggest that the MFs can be used to prevent the Alzheimer’s disease in a group of risk as well as in other diseases involving amyloid protein deposition in different tissues.