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Neurodegeneration is associated with hypertension and disturbance in fat metabolism. The complex interaction of neurodegenerative processes with both metabolic changes and blood pressure is still not fully elucidated. Here we demonstrate that the experimentally induced tauopathy in hypertensive transgenic animals causes significant downregulation of plasma leptin (53% of control), reduction of body weight by 11%, a 1.2-fold drop of adiposity index, and decrease in HDL cholesterol level, while the fasting glucose and insulin concentration remain unchanged. Despite of these alterations we found the leptin projection circuit including the arcuate nucleus, paraventricular nucleus in hypothalamus, and nucleus tractus solitarius in the brainstem not affected by neurofibrillary pathology. Furthermore, hypertension does not alter disturbances in leptin signalling. The presented data provide further insight into neurodegeneration-induced metabolic alterations relevant for human tauopathies.

The close relationship between Alzheimer's disease (AD) and obesity was recognized many years ago. However, complete understanding of the pathological mechanisms underlying the interactions between degeneration of CNS and fat metabolism is still missing. The leptin a key adipokine of white adipose tissue has been suggested as one of the major mediators linking the obesity and AD. Here we investigated the association between peripheral levels of leptin, general metabolic status and stage of the pathogenesis in rat transgenic model of AD. We demonstrate significantly decreased levels of plasma leptin in animals with experimentally induced progressive neurofibrillary pathology, which represents only 62.3% (P = 0.0015) of those observed in normal wild type control animals. More detailed analysis showed a strong and statistically significant inverse correlation between the load of neurofibrillary pathology and peripheral levels of leptin (r = - 0.7248, P = 0.0177). We also observed a loss of body weight during development of neurodegeneration (about 14% less than control animals, P = 0.0004) and decrease in several metabolic parameters such as glucose, insulin, triglycerides and VLDL in plasma of the transgenic animals. Our data suggest that plasma leptin could serve as a convenient peripheral biomarker for tauopathies and Alzheimer's disease. Decrease in gene expression of leptin in fat tissue and its plasma level was found as one of the consequences of experimentally induced neurodegeneration. Our data may help to design rational diagnostic and therapeutic strategies for patients suffering from Alzheimer's disease or other forms of tauopathy.

Repetitive head impacts (RHI) are common in youth athletes participating in contact sports. RHI differ from concussions; they are considered hits to the head that usually do not result in acute symptoms and are therefore also referred to as "subconcussive" head impacts. RHI occur e.g., when heading the ball or during contact with another player. Evidence suggests that exposure to RHI may have cumulative effects on brain structure and function. However, little is known about brain alterations associated with RHI, or about the risk factors that may lead to clinical or behavioral sequelae. REPIMPACT is a prospective longitudinal study of competitive youth soccer players and non-contact sport controls aged 14 to 16 years. The study aims to characterize consequences of exposure to RHI with regard to behavior (i.e., cognition, and motor function), clinical sequelae (i.e., psychiatric and neurological symptoms), brain structure, function, diffusion and biochemistry, as well as blood- and saliva-derived measures of molecular processes associated with exposure to RHI (e.g., circulating microRNAs, neuroproteins and cytokines). Here we present the structure of the REPIMPACT Consortium which consists of six teams of clinicians and scientists in six countries. We further provide detailed information on the specific aims and the design of the REPIMPACT study. The manuscript also describes the progress made in the study thus far. Finally, we discuss important challenges and approaches taken to overcome these challenges. ispartof: Brain Imaging And Behavior vol:16 issue:1 ispartof: location:United States status: published

Alzheimer’s disease (AD) pathology is partly characterized by accumulation of aberrant forms of tau protein. Here we report the results of ADAMANT, a 24-month double-blinded, parallel-arm, randomized phase 2 multicenter placebo-controlled trial of AADvac1, an active peptide vaccine designed to target pathological tau in AD (EudraCT 2015-000630-30). Eleven doses of AADvac1 were administered to patients with mild AD dementia at 40 μg per dose over the course of the trial. The primary objective was to evaluate the safety and tolerability of long-term AADvac1 treatment. The secondary objectives were to evaluate immunogenicity and efficacy of AADvac1 treatment in slowing cognitive and functional decline. A total of 196 patients were randomized 3:2 between AADvac1 and placebo. AADvac1 was safe and well tolerated (AADvac1 n = 117, placebo n = 79; serious adverse events observed in 17.1% of AADvac1-treated individuals and 24.1% of placebo-treated individuals; adverse events observed in 84.6% of AADvac1-treated individuals and 81.0% of placebo-treated individuals). The vaccine induced high levels of IgG antibodies. No significant effects were found in cognitive and functional tests on the whole study sample (Clinical Dementia Rating-Sum of the Boxes scale adjusted mean point difference −0.360 (95% CI −1.306, 0.589)), custom cognitive battery adjusted mean z-score difference of 0.0008 (95% CI −0.169, 0.172). We also present results from exploratory and post hoc analyses looking at relevant biomarkers and clinical outcomes in specific subgroups. Our results show that AADvac1 is safe and immunogenic, but larger stratified studies are needed to better evaluate its potential clinical efficacy and impact on disease biomarkers. The authors present the results of a 24-month phase 2 study of AADvac1, a tau vaccine against Alzheimer’s disease. AADvac1 was safe and induced high levels of antibodies. In the whole study sample, there were no significant changes on clinical outcomes.

Immunotherapies targeting pathological tau have recently emerged as a promising approach for treatment of neurodegenerative disorders. We have previously showed that the mouse antibody DC8E8 discriminates between healthy and pathological tau, reduces tau pathology in murine tauopathy models and inhibits neuronal internalization of AD tau species in vitro.Here we show, that DC8E8 and antibodies elicited against the first-in-man tau vaccine, AADvac1, which is based on the DC8E8 epitope peptide, both promote uptake of pathological tau by mouse primary microglia. IgG1 and IgG4 isotypes of AX004, the humanized versions of DC8E8, accelerate tau uptake by human primary microglia isolated from post-mortem aged and diseased brains. This promoting activity requires the presence of the Fc-domain of the antibodies.The IgG1 isotype of AX004 showed greater ability to promote tau uptake compared to the IgG4 isotype, while none of the antibody-tau complexes provoked increased pro-inflammatory activity of microglia. Our data suggest that IgG1 has better suitability for therapeutic development.

International audience; As a result of migrations and globalization, people may face a possible increase in the incidence of central nervous system rickettsial infections (CNS R). These diseases, caused by Rickettsia species and transmitted to humans by arthropod bites, are putatively lethal. However , the diagnosis of CNS R is challenging and often delayed due to their nonspecific clinical presentation and the strict intracellular nature of rickettsiae. Furthermore, transfer of rickettsiae to the brain parenchyma is not yet understood. The aim of this review is to analyze and summarize the features and correlated findings of CNS R in order to focus attention on these intriguing but frequently neglected illnesses. We also incorporated data on CNS infections caused by Rickettsia-related microorganisms.

Pathologically altered tau protein is a common denominator of neurodegenerative disorders including Alzheimer’s disease (AD) and other tauopathies. Therefore, promising immunotherapeutic approaches target and eliminate extracellular pathogenic tau species, which are thought to be responsible for seeding and propagation of tau pathology. Tau isoforms in misfolded states can propagate disease pathology in a template-dependent manner, proposed to be mediated by the release and internalization of extracellular tau. Monoclonal antibody DC8E8, binding four highly homologous and independent epitopes in microtubule-binding domain (MTBD) of diseased tau, inhibits tau-tau interaction, discriminates between healthy and pathologically truncated tau and reduces tau pathology in animal model in vivo. Here, we show that DC8E8 antibody acts via extracellular mechanism and does not influence viability and physiological functions of neurons. Importantly, in vitro functional assays showed that DC8E8 recognises pathogenic tau proteins of different size and origin, and potently blocks their entry into neurons. Next, we examined the mechanisms by which mouse antibody DC8E8 and its humanized version AX004 effectively block the neuronal internalization of extracellular AD tau species. We determined a novel mode of action of a therapeutic candidate antibody, which potently inhibits neuronal internalization of AD tau species by masking of epitopes present in MTBD important for interaction with neuron surface Heparan Sulfate Proteoglycans (HSPGs). We show that interference of tau-heparane sulfate interaction with DC8E8 antibody via steric hindrance represents an efficient and important therapeutic approach halting tau propagation. Electronic supplementary material The online version of this article (10.1186/s40478-019-0770-y) contains supplementary material, which is available to authorized users.

Alzheimer's disease (AD) represents the most common neurodegenerative disorder. Several animal models have been developed in order to test pathophysiological mechanisms of the disease and to predict effects of pharmacological interventions. Here we examine the molecular and behavioral features of R3m/4 transgenic mice expressing human non-mutated truncated tau protein (3R tau, aa151-391) that were previously used for efficacy testing of passive tau vaccine. The mouse model reliably recapitulated crucial histopathological features of human AD, such as pre-tangles, neurofibrillary tangles, and neuropil threads. The pathology was predominantly located in the brain stem. Transgenic mice developed mature sarkosyl insoluble tau complexes consisting of mouse endogenous and human truncated and hyperphosphorylated forms of tau protein. The histopathological and biochemical features were accompanied by significant sensorimotor impairment and reduced lifespan. The sensorimotor impairment was monitored by a highly sensitive, fully-automated tool that allowed us to assess early deficit in gait and locomotion. We suggest that the novel transgenic mouse model can serve as a valuable tool for analysis of the therapeutic efficacy of tau vaccines for AD therapy.

Neuroinvasive microorganisms are suspected to play an important role in the etiopathogenesis of neurological diseases. However, direct evidence for the pathogenic function is still missing. The main aim of this study was to investigate biochemical and morphological changes that may occur as a result of an in vitro infection of rat cerebrocortical neurons by selected members of the genus Rickettsia. Our results showed that survival of the neurons is significantly reduced after the infection. Intracellular level of ATP is gradually decreased and inversely correlates with the load of rickettsiae. Immunofluorescence revealed that rickettsiae can enter the neurons and are localized in perinuclear space and also in neuronal processes. Data obtained in this study correspond to the idea of possible involvement of rickettsiae in the etiopathogenesis of various neuropathies.

Accumulation of misfolded forms of microtubule associated, neuronal protein tau causes neurofibrillary degeneration typical of Alzheimer's disease and other tauopathies. This process is accompanied by elevated cellular stress and concomitant deregulation of heat-shock proteins. We used a transgenic rat model of tauopathy to study involvement of heat shock protein 27 (Hsp27) in the process of neurofibrillary degeneration, its cell type specific expression and correlation with the amount of insoluble tau protein aggregates. The expression of Hsp27-mRNA is more than doubled and levels of Hsp27 protein tripled in aged transgenic animals with tau pathology. The data revealed a strong positive and highly significant correlation between Hsp27-mRNA and amount of sarkosyl insoluble tau. Interestingly, intracellular accumulation of insoluble misfolded tau protein in neurons was associated with overexpression of Hsp27 almost exclusively in reactive astrocytes, not in neurons. The topological dissociation of neuronally expressed pathological tau and the induction of astrocytic Hsp27, GFAP, and Vimentin along with up-regulation of microglia specific markers such as CD18, CD68 and C3 point to cooperation of astrocytes, microglia and neurons in response to intra-neuronal accumulation of insoluble tau. Our data suggest that over expression of Hsp27 represents a part of microglia-mediated astrocytic response mechanism in the process of neurofibrillary degeneration, which is not necessarily associated with neuroprotection and which in contrary may accelerate neurodegeneration in late stage of the disease. This phenomenon should be considered during development of disease modifying strategies for treatment of tauopathies and AD via regulation of activity of Hsp27.

Animal models of neurodegeneration induced by neuronal expression of truncated tau protein emerge as an important tool for understanding the pathogenesis of human tauopathies and for therapy development. Here we highlight common features of truncated tau models and make a critical assessment of possible pitfalls in their analysis. Particularly, the amount of soluble tau oligomers, which are suspected to be neurotoxic agents participating on the spreading of pathology inside the brain, may be overestimated due to a post-lysis oxidative tau oligomerization. Using a mouse brain lysate spiked with recombinant truncated and full length tau forms, we show that tau oligomers might inadvertently be produced during the isolation procedure. This finding is further corroborated by the analysis of brain lysates originated from a mouse model expressing truncated tau variant. Our results underline the necessity of thiol-protecting conditions during the analysis of tau oligomers involved in the etiopathogenesis of various tauopathies including Alzheimer's disease.

Alzheimer's disease (AD) is a multifactorial disorder; neurofibrillary pathology composed of tau protein is found side by side with amyloid-β deposits and extensive neuroinflammation. The immune system of the brain is considered as one of the factors that could influence the speed of the progression of AD neuropathology as a potential mediator of the damage induced by AD protein deposits. Alzheimer's disease pathology can be impacted by psychological stress; however, signalling pathways in background are not well known. We have explored possible avenues of how stress could influence the brain's immune system in a rat model of AD. Animals were subjected either to a single or multiple instances of immobilization stress. The analysis of a panel of immunity-related genes was used to evaluate the impact of stress on the immune response in the brain. We have identified 19 stress-responsive genes that are involved in neuroinflammation accompanying tau pathology: Nos2, Ptgs2, IL-8rb, C5, Mmp9, Cx3cr1, CD40lg, Adrb2, IL-6, IL-6r, IL-1r2, Ccl2, Ccl3, Ccl4, Ccl12, TNF-α, IL-1α, IL-1β, IL-10. Most of them are deregulated under the stress conditions also in control animals; however, the magnitude of the response to either acute or chronic stress differs. This can lead to serious influence, most probably to acceleration of neurodegenerative phenotype in diseased animals. Several of the genes (IL-1β, Casp1, Cx3cr1 and C5) are deregulated solely in tauopathic animals. The stress-induced changes in the inflammatory picture of the brain highlight the fact that the brain's immune response is highly responsive to environmental stimuli. The pattern of changes is indicative of an attempt to protect the brain in the short term, while being potentially detrimental to the response against a long-term pathological process such as neurofibrillary degeneration.

Previous evidence from post-mortem Alzheimer disease (AD) brains and drug (especially rapamycin)-oriented in vitro and in vivo models implicated an aberrant accumulation of the mammalian target of rapamycin (mTor) in tangle-bearing neurons in AD brains and its role in the formation of abnormally hyperphosphorylated tau. Compelling evidence indicated that the sequential molecular events such as the synthesis and phosphorylation of tau can be regulated through p70 S6 kinase, the well characterized immediate downstream target of mTor. In the present study, we further identified that the active form of mTor per se accumulates in tangle-bearing neurons, particularly those at early stages in AD brains. By using mass spectrometry and Western blotting, we identified three phosphoepitopes of tau directly phosphorylated by mTor. We have developed a variety of stable cell lines with genetic modification of mTor activity using SH-SY5Y neuroblastoma cells as background. In these cellular systems, we not only confirmed the tau phosphorylation sites found in vitro but also found that mTor mediates the synthesis and aggregation of tau, resulting in compromised microtubule stability. Changes of mTor activity cause fluctuation of the level of a battery of tau kinases such as protein kinase A, v-Akt murine thymoma viral oncogene homolog-1, glycogen synthase kinase 3β, cyclin-dependent kinase 5, and tau protein phosphatase 2A. These results implicate mTor in promoting an imbalance of tau homeostasis, a condition required for neurons to maintain physiological function.

Neuroendocrine and behavioral stress responses are orchestrated by corticotropin-releasing hormone (CRH) and norepinephrine (NE) synthesizing neurons. Recent findings indicate that stress may promote development of neurofibrillary pathology in Alzheimer's disease. Therefore, we investigated relationships among stress, tau protein phosphorylation, and brain NE using wild-type (WT) and CRH-knockout (CRH KO) mice. We assessed expression of phosphorylated tau (p-tau) at the PHF-1 epitope and NE concentrations in the locus coeruleus (LC), A1/C1 and A2/C2 catecholaminergic cell groups, hippocampus, amygdala, nucleus basalis magnocellularis, and frontal cortex of unstressed, singly stressed or repeatedly stressed mice. Moreover, gene expression and protein levels of tyrosine hydroxylase (TH) and CRH receptor mRNA were determined in the LC. Plasma corticosterone levels were also measured. Exposure to a single stress increases tau phosphorylation throughout the brain in WT mice when compared to singly stressed CRH KO animals. In contrast, repeatedly stressed CRH KO mice showed exaggerated tau phosphorylation relative to WT controls. We also observed differences in extent of tau phosphorylation between investigated structures, e.g. the LC and hippocampus. Moreover, CRH deficiency leads to different responses to stress in gene expression of TH, NE concentrations, CRH receptor mRNA, and plasma corticosterone levels. Our data indicate that CRH effects on tau phosphorylation are dependent on whether stress is single or repeated, and differs between brain regions. Our findings indicate that CRH attenuates mechanisms responsible for development of stress-induced tau neuropathology, particularly in conditions of chronic stress. However, the involvement of central catecholaminergic neurons in these mechanisms remains unclear and is in need of further investigation.

Misfolded, N- and C-terminally truncated tau protein is the primary constituent of neurofibrillary tangles in brains of patients afflicted with Alzheimer's disease (AD). Intracellular accumulation of misfolded and truncated tau leads to generation of cytotoxic intermediates; transgenic expression of truncated tau leads to neurological deficits, neurofibrillary degeneration, and premature death of animals. Since no cure for AD or other tauopathies is available yet, we tested the possibility for prevention of pathogenic events elicited by tau, via inhibition of its intracellular accumulation. Using a cell model conditionally expressing truncated and misfolding-prone tau protein, we showed that pathogenic forms of tau are degraded via the proteasome. We have also observed that chymotrypsin-like activity of the proteasome was significantly suppressed (a decrease of ∼29.12% in comparison to control cells; p < 0.001) as a consequence of truncated tau expression. Interestingly, the activity of the proteasome was enhanced by geldanamycin, a natural inhibitor of Hsp90. This activation resulted in accelerated degradation of misfolded tau. We suggest that non-toxic inhibitors of Hsp90, especially those which can activate the proteasome, are good candidates for the development of molecules that efficiently counteract the damaging effects of pathologically misfolded proteins.

Neurofibrillary degeneration induced by misfolded protein tau is considered to be one of the key pathological hallmarks of Alzheimer's disease (AD). In the present study, we have introduced a novel transgenic rat model expressing a human truncated tau that encompasses 3 microtubule binding domains (3R) and a proline-rich region (3R tau151-391). The transgenic rats developed progressive age-dependent neurofibrillary degeneration in the cortical brain areas. Neurofibrillary tangles (NFTs) satisfied several key histological criteria used to identify neurofibrillary degeneration in human Alzheimer's disease including argyrophilia, Congo red birefringence, and Thioflavin S reactivity. Neurofibrillary tangles were also identified with antibodies used to detect pathologic tau in the human brain, including DC11, recognizing an abnormal tau conformation and antibodies that are specific for hyperphosphorylated forms of tau protein. Moreover, neurofibrillary degeneration was characterized by extensive formation of sarkosyl insoluble tau protein complexes consisting of rat endogenous and truncated tau species. Interestingly, the transgenic rats did not show neuronal loss either in the cortex or in the hippocampus. We suggest that novel transgenic rat model for human tauopathy represents a valuable tool in preclinical drug discovery targeting neurofibrillary degeneration of Alzheimer's type.

We have previously demonstrated in a transgenic rat model of tauopathy that human misfolded truncated tau derived from Alzheimer's disease suffices to drive neurofibrillary degeneration in vivo. We employed this model to investigate the impact of truncated tau expression levels on life span, neuronal loss and the final load of neurofibrillary tangles (NFTs) in transgenic rats. Two independent transgenic lines (SHR72, SHR318), that display different expression levels of truncated tau, were utilized in this study. We found that transgene expression levels in the brain of SHR72 rats were 44% higher than in SHR318 rats and that truncated tau protein levels determined the survival rate of transgenic rats. The line with higher expression levels of truncated tau (SHR72) showed decreased median survival (222.5 days) when compared with the line with lower expression (SHR318; 294.5 days). Interestingly, NFT loads (total NFT/total neurons) were very similar in terminal stages of disease in both transgenic lines (SHR72 - 10.9%; SHR318 - 11.6%), despite significantly different expression levels of truncated tau. Moreover, mean neuron numbers in the hippocampus (CA1-3) and brain stem (gigantocellular reticular nucleus) in the two transgenic rat strains in the terminal stages of disease were similar, and did not differ significantly from those observed in age-matched non-transgenic controls. These findings suggest that the expression levels of misfolded truncated tau determine the life span in a transgenic rat model of tauopathy without causing neuronal loss or correlating with terminal NFT load.

Abnormal phosphorylation of tau protein represents one of the major candidate pathological mechanisms leading to Alzheimer’s disease (AD) and related tauopathies. Altered phosphorylation status of neuronal tau protein may result from upregulation of tau-specific kinases or from inhibition of tau-specific phosphatases. Increased expression of the protein inhibitor 1 of protein phosphatase 2A (I1PP2A) could therefore indirectly regulate the phosphorylation status of tau. As an important step towards elucidation of the role of I1PP2A in the physiology and pathology of tau phosphorylation, we developed a novel monoclonal antibody, DC63, which recognizes I1PP2A. Specificity of the antibody was examined by mass spectrometry and Western blot. This analysis supports the conclusion that the antibody does not recognize any of the other proteins of the 9-member leucine-rich acidic nuclear phosphoprotein family to which I1PP2A belongs. Immunoblot detection revealed that the inhibitor I1PP2A is expressed throughout the brain, including the hippocampus, temporal cortex, parietal cortex, subcortical nuclei and brain stem. The cerebellum displayed significantly higher levels of expression of I1PP2A than was seen elsewhere in the brain. Imunohistochemical analysis of normal human brain showed that I1PP2A is expressed in both neurons and glial cells and that the protein is preferentially localized to the nucleus. We conclude that the novel monoclonal antibody DC63 could be successfully employed as a mass spectrometry-validated molecular probe that may be used for in vitro and in vivo qualitative and quantitative studies of physiological and pathological pathways involving I1PP2A.

A novel monoclonal antibody DC39N1 specific for tau protein N-terminal insert N1 (residues 45-73) was developed. Species analysis revealed that monoclonal antibody DC39N1 did not recognize tau proteins derived from rat, mouse, bovine, swine and rabbit brain tissues and is human tau protein specific. The antibody recognized all and only those human tau isoforms that contain a tau N1 insert. DC39N1 epitope on paired helical filaments from the brain of patients with Alzheimer's disease is phosphorylation independent. Immunohistochemical analysis of Braak stages with novel antibody revealed the presence of tau aminoterminal N1 insert during evolution of neurofibrillary degeneration from early to late stages of Alzheimer's disease.

Brainstem catecholaminergic neurons significantly participate in the regulation of neuroendocrine system activity, particularly during stressful conditions. However, so far the precise quantitative characterisation of basal and stress-induced changes in gene expression and protein levels of catecholaminergic biosynthetic enzymes in these neurons has been missing. Using a quantitative reverse transcription-polymerase chain reaction method, we investigated gene expression of catecholamine biosynthetic enzymes in brainstem noradrenergic and adrenergic cell groups in rats under resting conditions as well as in acutely and repeatedly stressed animals. For the first time, we described quantitative differences in basal levels of catecholamine biosynthetic enzyme mRNA in brainstem catecholaminergic ascending and descending projecting cell groups. Moreover, we found and defined some differences among catecholaminergic cell groups in the time-course of mRNA levels of catecholaminergic enzymes following a single and especially repeated immobilisation stress. The data obtained support the assumption that brainstem catecholaminergic cell groups represent a functionally differentiated system, which is highly (but specifically) activated in rats exposed to stress. Therefore, potential interventions for the treatment of stress-related diseases need to affect the activity of brainstem catecholaminergic neurons not uniformly but with some degree of selectivity.

Stress may accelerate onset of neurodegenerative diseases in vulnerable subjects and, vice versa, neurodegeneration affects the responsiveness to stressors. We investigated the neuroendocrine response to immobilization stress in normotensive Wistar-Kyoto rats (WKY), spontaneously hypertensive rats (SHR), and transgenic rats of respective WKY and SHR strains overexpressing human truncated tau protein. Plasma levels of epinephrine, norepinephrine, and corticosterone were determined. An immobilization-induced elevation of epinephrine and norepinephrine was significantly reduced in WKY transgenic rats compared to WKY wild-type rats, while no differences were seen between SHR transgenic and SHR wild-type animals. Our data have shown that sympathoadrenal system response to stress strongly depends on both tau protein-induced neurodegeneration and genetic background of experimental animals.

We have exploited a new monoclonal antibody against the tyrosine kinase A (TrkA) nerve growth factor (NGF) receptor to block the NGF–TrkA interaction in the rat basal forebrain. The monoclonal antibody MNAC13 is a potent antagonist that prevents the binding of NGF to TrkA in a variety of systems. This antibody was used to study the maintenance of the cholinergic phenotype in the rat basal forebrainin vivo, by the implant of antibody-secreting cells. Basal forebrain cholinergic neurons (BFCNs) are greatly affected by the antibody treatment, both in terms of cell number and of cell soma size. When antibody-secreting cells are implanted at postnatal day 2 (P2), the effects observed at P8 are as severe as those obtained with anti-NGF antibodies and, interestingly, are observed also if anti-TrkA cells are implanted at P8, when anti-NGF antibodies, delivered by the same route, are no longer effective (Molnar et al., 1998). The effects induced by anti-TrkA, as those induced by anti-NGF, are reversible, but the time required for recovery and the critical period in the sensitivity of BFCNs to the functional inactivation of TrkA is twice as long than that observed when NGF is intercepted. These results demonstrate that BFCNs are more sensitive to the block of TrkA activation than they are to the block of NGF. The cloning of MNAC13 variable regions and their assembly into a functional polypeptide of reduced size (single chain Fv fragment) will allow its use in gene transfer applications.

The present study was undertaken to investigate the effects of selenite (SeIV) and selenate (SeVI) on the all-trans retinoic acid (RA)-nuclear retinoic acid receptor (RAR) complex formation in rat liver. We also present the data on the in vitro effects of SeIV on the RARalpha and the type I iodothyronine 5'-deiodinase gene expression in the GH4C1 rat pituitary tumor cells. SeIV at 1.0 micromol/L was found to reduce (p < 0.05) the RA specific binding to RAR in rat liver. Dithiothreitol (DTT), a protective agent for sulfhydryl groups, was found to be slightly effective in protecting the RAR binding properties when affected by SeIV. SeVI at 0.1 micromol/L reduced (p < 0.05) the RA specific binding to RAR in liver, as well. Seleno-L-methionine (Se-II) when compared to L-methionine did not exert any inhibitory effect on the formation of the RA-RAR complex. SeIV (up to 2.5 micromol/L) has no inhibitory effect on GH4C1 cell proliferation as well as the prolactin secretion. SeIV at 1.0 micromol/L significantly decreases the rate of mRNA synthesis and/or degradation of the alpha form of the RAR and causes the enhancement of the type I iodothyronine 5'-deiodinase gene expression in GH4C1 cells. The results based on in vitro experiments suggest that inorganic selenium may affect the RA specific binding to their cognate receptor molecules, and it may reduce expression of the gene encoding the RARalpha, with the cell vitality and the cell growth remaining unchanged.

Tau protein misfolding is a pathological mechanism, which plays a critical role in the etiopathogenesis of neurodegeneration. However, it is not entirely known what kind of stimuli can induce the misfolding. It is believed that physical and emotional stresses belong to such risk factors. Although the influence of stress on the onset and progression of Alzheimer’s disease (AD) has already been proposed, the molecular links between stresses and AD are still unknown. We have therefore focused our attention on determination of the influence of acute immobilization stress (IMO) in normal mice and mice deficient in corticotropin-releasing hormone (CRH). Specifically, we have analyzed levels of hyperphosphorylated tau proteins, bearing the AD-specific phospho-epitopes (AT-8, pT181, and PHF-1), which are implicated in the pathogenesis of AD. We found that IMO induces transient hyperphosphorylation of tau proteins regardless of continuation of the stimulus. Concerning tau modifications, detailed analysis of the mouse brain revealed that neurons in different brain regions including frontal cortex, temporal cortex, hippocampal C1 and CA3 regions, dentate gyrus as well as nucleus basalis Meynert, and several brainstem nuclei such as locus coeruleus but also raphe nucleus and substantia nigra respond similarly to IMO. The strongest tau protein phosphorylation was observed after 30 min of IMO stress. Stress lasting for 120 min led either to the disappearance of tau hyperphosphorylation or to the induction of a second wave of hyperphosphorylation. Noteworthy is the magnitude of pathological phosphorylation of tau protein in CRH and glucocorticoids deficient mice, being much lower in comparison to that observed in wild-type animals, which suggests a critical role of CRH in the pathogenesis of AD. Thus, our results indicate that hyperphosphorylation of tau protein induced by stress may represent the pathogenic event upstream of tau protein misfolding, which leads to progression or eventually initiation of neurodegeneration. The data show that CRH plays an important role in stress induced hyperphosphorylation of tau protein, which might be either a direct effect of CRH innervations in the brain or an effect mediated via the hypothalamo–pituitary–adrenal axis.

Transition of protein tau from physiologically unfolded to misfolded state represent enigmatic step in the pathogenesis of tauopathies including Alzheimer’s disease (AD). Major molecular events playing role in this process involve truncation and hyperphosphorylation of tau protein, which are accompanied by redox imbalance followed by functional deterioration of neuronal network. Recently we have developed transgenic rat model showing that expression of truncated tau causes neurofibrillary degeneration similar to that observed in brain of AD sufferers. Consequently we tested cortical and hippocampal neuronal cultures extracted from this model as a convenient tool for development of molecules able to target the mechanisms leading to and/or enhancing the process of neurodegeneration. Here we document three major pathological features typical for tauopathies and AD in cortical and hippocampal neurons from transgenic rat in vitro. First, an increased accumulation of human truncated tau in neurons; second, the hyperphosphorylation of truncated tau on the epitopes characteristic of AD (Ser202/Thr205 and Thr231); and third, increased vulnerability of the neurons to nitrative and oxidative stress. Our results show that primary neurons expressing human truncated tau could represent a cellular model for targeting tau related pathological events, namely, aberrant tau protein accumulation, tau hyperphosphorylation, and oxidative/nitrative damage. These characteristics make the model particularly suitable for detailed study of molecular mechanisms of tau induced neurodegeneration and easily applicable for drug screening.

We have previously shown that human misfolded tau proteins strongly perturb mitochondrial transport and induce accumulation of free radicals in neurons. This interference is underlying cause of increased susceptibility to oxidative stress and could be linked to excitotoxic pathways. In order to understand integral mechanisms of misfolded tau driven neurodegeneration, we have investigated the role of human truncated tau protein, derived from Alzheimer’s disease, in rat cortical neurons under the conditions of excitotoxic stress induced by glutamate. We found that primary neurons expressing truncated tau protein are highly susceptible to glutamate-induced cell death. Pre-treatment with memantine (N-methyl-d-aspartate receptor antagonist) significantly improved survival of rat neurons exposed to glutamate and its effect was associated with overall decrease of reactive oxygen species (ROS) in both transgenic and nontransgenic neurons. Interestingly, despite of this overall effect, memantine was not able to decrease misfolded tau-induced ROS level specifically in transgenic cells. Our data suggest that memantine does not interfere with specific pathological pathways induced by misfolded tau protein nevertheless is able to attenuate oxidative stress in neurons.

Intrinsically disordered proteins (IDPs) represent key mediators in many physiological as well as pathological processes. Solution-exposed cysteines of IDPs are highly reactive and therefore reducing agents are frequently included during their preparation to prevent formation of nonnative disulfides. However, reductants can potentially interfere with subsequent assays performed on the purified IDPs. Herein we report a method for purification of IDP tau in an atmosphere of inert argon, which eliminates the need for reducing agents. We have used this method for preparing several IDP tau isoforms and found it useful in the investigation of monomeric tau toxicity in rat cerebral neurons.

Truncated tau protein is the characteristic feature of human sporadic Alzheimer’s disease. We have identified truncated tau proteins conformationally different from normal healthy tau. Subpopulations of these structurally different tau species promoted abnormal microtubule assembly in vitro suggesting toxic gain of function. To validate pathological activity in vivo we expressed active form of human truncated tau protein as transgene, in the rat brain. Its neuronal expression led to the development of the neurofibrillary degeneration of Alzheimer’s type. Furthermore, biochemical analysis of neurofibrillary changes revealed that massive sarcosyl insoluble tau complexes consisted of human Alzheimer’s tau and endogenous rat tau in ratio 1:1 including characteristic Alzheimer’s disease (AD)-specific proteins (A68). This work represents first insight into the possible causative role of truncated tau in AD neurofibrillary degeneration in vivo.

The present study was undertaken in order to investigate the effects of sodium selenite on: Sodium selenite (up to 2.5 μM) has no inhibitory effect on GH4C1 cell proliferation as well as the prolactin secretion. On the other hand, 0.5 μM sodium selenite significantly decreases the rate of mRNA synthesis and/or degradation of both, the α1 form of the T3 receptor (TRα1) and the α2 isoform of the T3 receptor. At 1 μM of sodium selenine, significant changes in the electrophoretic profile of low molecular mass cytoplasmic proteins were found, moreover, sodium selenite (1 μM) also considerably affects phosphorylation of a higher molecular mass proteins. The results based on the in vitro experiments suggest that sodium selenite may affect specific processes at the pretranslational level as well as it may also take part in processes of posttranslational modification of protein(s), the cell vitality and the cell growth remaining unchanged.

The effect of 3,5,3′- l -triiodothyronine (T 3 ) on survival and morphology of primary cultured neurons of the fetal rat brain was studied. In defined conditions of serum-free culture media we found the death preventing effect of T 3 in all tested neuronal populations cultivated at high initial densities of plating (10 5 cells/cm 2 ). While the survival of cerebrocortical neurons was improved very slightly, the number of surviving hippocampal and septal neurons reached 127.2 ± 2.0% or 134.8 ± 12.3% of their respective controls. The septal neurons responded at normal physiological concentration of T 3 (1nM) in high density as well as in low density cultures (5 × 10 3 cells/cm 2 ). Moreover the treatment with 10 nM of T 3 caused significant extension of the axon elongation of septal neurons (194.5 ± 15.7%). These findings suggest the direct positive effect of T 3 on pure cell population of septal neurons derived from embryonic rat brain and support the evidence for the role of this peripheral hormone during neuritogenesis.