In Alzheimer's disease, tau protein undergoes post-translational modifications including hyperphosphorylation and truncation, which promotes two major conformational changes associated with progressive N-terminal folding. Along with the development of the disease, tau ubiquitination was previously shown to emerge in the early and intermediate stages of the disease, which is closely associated with early tau truncation at aspartic acid 421, but not with a subsequently truncated tau molecule at glutamic acid 391. In the same group of cases, using multiple immunolabeling and confocal microscopy, a possible relationship between the ubiquitin-targeting of tau and the progression of conformational changes adopted by the N-terminus of this molecule was further studied. A comparable number of neurofibrillary tangles was found displaying ubiquitin, an early conformation recognized by the Alz-50 antibody, and a phosphorylation. However, a more reduced number of neurofibrillary tangles were immunoreactive to Tau-66 antibody, a late tau conformational change marker. When double-labeling profiles of neurofibrillary tangles were assessed, ubiquitination was clearly demonstrated in tau molecules undergoing early N-terminal folding, but was barely observed in late conformational changes of the N-terminus adopted by tau. The same pattern of colocalization was visualized in neuritic pathology. Overall, these results indicate that a more intact conformation of the N-terminus of tau may facilitate tau ubiquitination, but this modification may not occur in a late truncated and more compressed folding of the N-terminus of the tau molecule.

Post-translational modifications, including phosphorylation, greatly impact the physiological function of proteins, especially those that are natively unfolded and implicated in many neurodegenerative diseases. However, structural and functional studies of such proteins require fully defined phosphorylation, including those that are not physiological. Thus, the kinases ERK2 and GSK-3β were immobilized to various superparamagnetic beads with carboxylic, aldehyde, Ni2+, or Co3+ functional groups, with a view to efficiently phosphorylate peptides and proteins in vitro. Full phosphorylation of specific synthetic peptides confirmed that beads were successfully loaded with kinases. Remarkably, enzymes covalently immobilized on carboxylated SeraMag beads remained active upon reuse, with residual activity after 10 uses 99.5 ± 0.34% for GSK-3β and 36.2 ± 2.01% for ERK2. The beads were also used to sequentially phosphorylate recombinant tau, which in vivo is a biomarker of Alzheimer's disease. Thus, a system consisting of two fully active kinases immobilized to magnetic beads is demonstrated for the first time. In comparison to soluble enzymes, the beads are easier to handle, reusable, and thus low-cost. Importantly, these beads are also convenient to remove from reactions to minimize contamination of phosphorylated products or to exchange with other kinases.

The principal aim of our study was to present norms for old and very old Czech adults on the Czech version of the Montreal Cognitive Assessment (MoCA) and investigate the influence of social and demographic factors on MoCA performance. We analyzed 540 adults aged 60 years (5-year age categories; nationally representative sample in terms of sex and educational level), who met strict inclusion criteria for the absence of neurodegenerative disorders and performed within normal range in neuropsychological assessment. Using multiple regression model, we found that MoCA performance was affected by age and education (both p<.001) but not sex. The study provides normed percentile estimates for MoCA performance stratified by age (60-74 years;75 years) and education lower versus higher. We also present percentile equivalents for the MoCA and Mini-Mental State Examination (MMSE) for use in clinical practice. We found age- and education-related effects on MoCA performance which support the use of culturally adapted normative data.

Methamphetamine (MA) is the most commonly used psychostimulant drug, the chronic abuse of which leads to neurodegenerative changes in the brain. The global use of MA is increasing, including in pregnant women. Since MA can cross both placental and haematoencephalic barriers and is also present in maternal milk, children of chronically abused mothers are exposed prenatally as well as postnatally. Women seem to be more vulnerable to some aspects of MA abuse than men. MA is thought to exert its effects among others via direct interactions with dopamine transporters (DATs) in the brain tissue. Sexual dimorphism of the DAT system could be a base of sex-dependent actions of MA observed in behavioural and neurochemical studies. Possible sex differences in the DATs of preadolescent offspring exposed to MA prenatally and/or postnatally have not yet been evaluated. We examined the striatal synaptosomal DATs (the activity and density of surface expressed DATs and total DAT expression) in preadolescent male and female Wistar rats (31–35-day old animals) exposed prenatally and/or postnatally to MA (daily 5 mg/kg, s.c. to mothers during pregnancy and lactation). To distinguish between specific and nonspecific effects of MA on DATs, we also evaluated the in vitro effects of lipophilic MA on the fluidity of striatal membranes isolated from preadolescent and young adult rats of both sexes. We observed similar changes in the DATs of preadolescent rats exposed prenatally or postnatally (MA-mediated drop in the reserve pool but no alterations in surface-expressed DATs). However, prenatal exposure evoked significant changes in males and postnatal exposure in females. A significant decrease in the activity of surface-expressed DATs was found only in postnatally exposed females sensitized to MA via prenatal exposure. MA applied in vitro increased the fluidity of striatal membranes of preadolescent female but not male rats. In summary, DATs of preadolescent males are more sensitive to prenatal MA exposure via changes in the reserve pool and those of preadolescent females to postnatal MA exposure via the same mechanism. The combination of prenatal and postnatal MA exposure increases the risk of dopaminergic deficits via alterations in the activity of surface-expressed DATs especially in preadolescent females. MA-mediated changes in DATs of preadolescent females could be still enhanced via nonspecific disordering actions of MA on striatal membranes.

Depression and anxiety disorders are highly prevalent in women. Although several studies have reported altered circulating steroids accompanying various mental disturbances, knowledge about alterations in the peripheral steroid pattern in such pathologies is incomplete. Therefore, we attempted to add to this knowledge using the simultaneous quantification of circulating steroids by gas chromatography mass spectrometry (GC-MS) in groups of premenopausal women in the follicular phase of the menstrual cycle (22 women with depression, 17 with anxiety disorders, 17 healthy controls). In addition to age-adjusted analysis of covariance (ANCOVA) followed by multiple comparisons, we developed models to successfully discriminate these groups from each other on the basis of steroid levels. Women with depression showed a reduced sulfoconjugation of steroids as well as lower levels of 7α-, 7β- and 16α-hydroxy-metabolites of C19 Δ5 steroids. Women with depression have significantly lower circulating levels of 5α/β-reduced pregnane steroids (with exception of free isopregnanolone) than women with anxiety or controls. Finally, our data indicate higher levels of estrogens in women with anxiety disorders when compared to women with depression.

Mass spectrometry coupled with bioaffinity separation techniques is considered a powerful tool for studying protein interactions. This work is focused on epitope analysis of tau protein, which contains two VQIXXK aggregation motifs regarded as crucial elements in the formation of paired helical filaments, the main pathological characteristics of Alzheimer's disease. To identify major immunogenic structures, the epitope extraction technique utilizing protein fragmentation and magnetic microparticles functionalized with specific antibodies was applied. However, the natural adhesiveness of some newly generated peptide fragments devalued the experimental results. Beside presumed peptide fragment specific to applied monoclonal anti-tau antibodies, the epitope extraction repeatedly revealed inter alia tryptic fragment 299-HVPGGGSVQIVYKPVDLSK-317 containing the fibril-forming motif 306-VQIVYK-311. The tryptic fragment pro-aggregation and hydrophobic properties that might contribute to adsorption phenomenon were examined by Thioflavin S and reversed-phase chromatography. Several conventional approaches to reduce the non-specific fragment sorption onto the magnetic particle surface were performed, however with no effect. To avoid methodological complications, we introduced an innovative approach based on altered proteolytic digestion. Simultaneous fragmentation of tau protein by two immobilized proteases differing in the cleavage specificity (TPCK-trypsin and α-chymotrypsin) led to the disruption of motif responsible for undesirable adhesiveness and enabled us to obtain undistorted structural data.

Alzheimer’s disease (AD) is one of the most serious human, medical, and socioeconomic burdens. Here we tested the hypothesis that a rat model of AD (Samaritan; Taconic Pharmaceuticals, USA) based on the application of amyloid beta42 (Abeta42) and the pro-oxidative substances ferrous sulfate heptahydrate and L-buthionine-(S, R)-sulfoximine, will exhibit cognitive deficits and disruption of the glutamatergic and cholinergic systems in the brain. Behavioral methods included the Morris water maze (MWM; long-term memory version) and the active allothetic place avoidance (AAPA) task (acquisition and reversal), testing spatial memory and different aspects of hippocampal function. Neurochemical methods included testing of the NR1/NR2A/NR2B subunits of NMDA receptors in the frontal cortex and CHT1 transporters in the hippocampus, in both cases in the right and left hemisphere separately. Our results show that Samaritan rats™ exhibit marked impairment in both the MWM and active place avoidance tasks, suggesting a deficit of spatial learning and memory. Moreover, Samaritan rats exhibited significant changes in NR2A expression and CHT1 activity compared to controls rats, mimicking the situation in patients with early stage AD. Taken together, our results corroborate the hypothesis that Samaritan rats are a promising model of AD in its early stages.

OBJECTIVE:Anxiety and mood disorders (AMD) are the most frequent mental disorders in the human population. They have recently shown increasing prevalence, and commonly disrupt personal and working lives. The aim of our study was to analyze the spectrum of circulating steroids in order to discover differences that could potentially be markers of affective depression or anxiety, and identify which steroids could be a predictive component for these diseases. METHODS:We studied the steroid metabolome including 47 analytes in 20 men with depression (group D), 20 men with anxiety (group AN) and 30 healthy controls. OPLS and multivariate regression models were used for statistical analysis. RESULTS:Discrimination of group D from controls by the OPLS method was absolute, as was group AN from controls (sensitivity=1.000 (0.839, 1.000), specificity=1.000 (0.887, 1.000)). Relatively good predictivity was also found for discrimination between group D from AN (sensitivity=0.850 (0.640, 0.948), specificity=0.900 (0.699, 0.972)). CONCLUSION:Selected circulating steroids, including those that are neuroactive and neuroprotective, can be useful tools for discriminating between these affective diseases in adult men.

BACKGROUND:Reversible acetylcholinesterase inhibitors are used in Alzheimer disease therapy. However, tacrine and its derivatives have severe side effects. Derivatives of the tacrine analogue 7-methoxytacrine (MEOTA) are less toxic.METHODS:We evaluated new derivatives of 7-MEOTA (2 homodimers linked by 2 C4-C5 chains and 5 N-alkylated C4-C8 side chain derivatives) in vitro, using the rat hippocampal choline transporter CHT1.RESULTS:Some derivatives were effective inhibitors of rat acetylcholinesterase and comparable with 7-MEOTA. All derivatives were able to inhibit CHT1, probably via quaternary ammonium, and this interaction could be involved in the enhancement of their detrimental side effects and/or in the attenuation of their promising effects. Under conditions of disrupted lipid rafts, the unfavorable effects of some derivatives were weakened. Only tacrine was probably able to stereospecifically interact with the naturally occurring amyloid-β isoform and to simultaneously stimulate CHT1. Some derivatives, when coincubated with amyloid β, did not influence CHT1. All derivatives also increased the fluidity of the cortical membranes.CONCLUSION:The N-alkylated derivative of 7-MEOTA bearing from C4 side chains appears to be the most promising compound and should be evaluated in future in vivo research.

Abnormal aggregation of Tau in glial cells has been reported in Alzheimer's disease (AD) and other tauopathies; however, the pathological significance of these aggregates remains unsolved to date. In this study, we evaluated whether full-length Tau (Tau441) and its aspartic acid421-truncated Tau variant (Tau421) produce alterations in the normal organization of the cytoskeleton and plasma membrane (PM) when transiently expressed in cultured C6-glial cells. Forty-eight hours post-transfection, abnormal microtubule bundling was observed in the majority of the cells, which expressed either Tau441 or Tau421. Moreover, both variants of Tau produced extensive PM blebbing associated with cortical redistribution of filamentous actin (F-Actin). These effects were reverted when Tau-expressing cells were incubated with drugs that depolymerize F-Actin. In addition, when glial cells showing Tau-induced PM blebbing were incubated with inhibitors of the Rho-associated protein kinase (ROCK) signaling pathway, both formation of abnormal PM blebs and F-Actin remodeling were avoided. All of these effects were initiated upstream by abnormal Tau-induced microtubule bundling, which may release the microtubule-bound guanine nucleotide exchange factor-H1 (GEF-H1) into the cytoplasm in order to activate its major effector RhoA-GTPase. These results may represent a new mechanism of Tau toxicity in which Tau-induced microtubule bundling produces activation of the Rho-GTPase-ROCK pathway that in turn mediates the remodeling of cortical Actin and PM blebbing. In AD and other tauopathies, these Tau-induced abnormalities may occur and contribute to the impairment of glial activity.

Summary There is growing evidence of the involvement of advanced glycation end products ( AGE s) in the pathogenesis of neurodegenerative processes including Alzheimer's disease ( AD ) and their function as a seed for the aggregation of Aβ, a hallmark feature of AD . AGE s are formed endogenously and exogenously during heating and irradiation of foods. We here examined the effect of a diet high in AGE s in the context of an irradiated diet on memory, insoluble Aβ 42 , AGE s levels in hippocampus, on expression of the receptor for AGE s ( RAGE ), and on oxidative stress in the vasculature. We found that AD ‐like model mice on high‐ AGE diet due to irradiation had significantly poorer memory, higher hippocampal levels of insoluble Aβ 42 and AGE s as well as higher levels of oxidative stress on vascular walls, compared to littermates fed an isocaloric diet. These differences were not due to weight gain. The data were further supported by the overexpression of RAGE , which binds to Aβ 42 and regulates its transport across the blood–brain barrier, suggesting a mediating pathway. Because exposure to AGE s can be diminished, these insights provide an important simple noninvasive potential therapeutic strategy for alleviating a major lifestyle‐linked disease epidemic.

The authors regret that one dedication to a grant support was inadvertently omitted (in the Acknowledgments). We would like to include this dedication in the form of Corrigendum. The article text was not changed. The corrected text of Acknowledgments follows: Acknowledgments This work was supported by the research project of the Czech Science Foundation, grant nr. P304/12/G069, by the project “National Institute of Mental Health (NIMH-CZ)”, grant number ED2.1.00/03.0078 (and the European Regional Development Fund), by EU project NADINE (No. 246513), by the Ministry of Education, Youth and Sports of the Czech Republic (Project CZ.1.07/2.3.00/30.0021 “Enhancement of R&D Pools of Excellence at the University of Pardubice”) and by the project of the Czech Science Foundation, grant nr. 13-26601S. The authors have declared no conflict of interest. The authors would like to apologize for any inconvenience caused. Identification and characterization of natural antibodies against tau protein in an intravenous immunoglobulin productJournal of NeuroimmunologyVol. 289PreviewThe latest therapeutic approaches to Alzheimer disease are using intravenous immunoglobulin (IVIG) products. Therefore, the detailed characterization of target-specific antibodies naturally occurring in IVIG products is beneficial. We have focused on characterization of antibodies isolated against tau protein, a biomarker of Alzheimer's disease, from Flebogamma IVIG product. The analysis of IgG subclass distribution indicated skewing toward IgG3 in anti-tau-enriched IgG fraction. The evaluation of their reactivity and avidity with several recombinant tau forms was performed by ELISA and blotting techniques. Full-Text PDF

It has been suggested that advanced glycation end (AGE) products, via cognate receptor activation, are implicated in several diseases, including Alzheimer’s disease. The NMDA receptor–nitric oxide pathway appears to be influenced by AGE products and involved in the pathogenesis of this type of dementia. In this study, C57BL/6J (WT) and transgenic (Tg2576) mice expressing human mutant amyloid precursor protein were kept on prolonged (8 months) diets containing regular or high amounts of AGE products. After the decapitation of 11-months old mice, brain tissue analyses were performed [expressions of the NR1, NR2A and NR2B subunits of NMDA receptors, activities of neuronal, endothelial and inducible nitric oxide synthase (nNOS, eNOS and iNOS)]. Moreover, levels of malondialdehyde and of human amyloid β 1–42 were estimated. We found increased activity of nNOS in WT mice maintained on a high compared to regular AGE diet; however, no similar differences were found in Tg2576 mice. In addition, we observed an increase in NR1 expression in Tg2576 compared to WT mice, both kept on a diet high in AGE products. Correlation analyses performed on mice kept on the regular AGE diet supported close links between particular subunits (NR2A–NR2B, in WT as well as in Tg2576 mice), between subunits and synthase (NR2A/NR2B–nNOS, only in WT mice) or between particular synthases (nNOS–iNOS, only in WT). Correlation analysis also revealed differences between WT mice kept on both diets (changed correlations between NR2A/NR2B–nNOS, between nNOS–eNOS and between eNOS–iNOS). Malondialdehyde levels were increased in both Tg2576 groups when compared to the corresponding WT mice, but no effects of the diets were observed. Analogously, no significant effects of diets were found in the levels of soluble or insoluble amyloid β 1–42 in Tg2576 mice. Our results demonstrate that prolonged ingestion of AGE products can influence the NMDA receptor–nitric oxide pathway in the brain and that only WT mice, not Tg2576 mice, are able to maintain homeostasis among subunits and synthases or among particular synthases. The prolonged application of AGE products enhanced differences between 11-months old Tg2576 and WT mice regarding this pathway. Observed differences in the pathway between WT mice kept on regular or high AGE diets suggest that the prolonged application of a diet low in AGE products could have beneficial effects in older or diabetic people and perhaps also in people with Alzheimer’s disease.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

BACKGROUND Overexpression of the mitochondrial enzyme 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10, which is also known as the intracellular amyloid-β peptide (Aβ) binding protein) is observed in cortical or hippocampal regions of patients with Alzheimer's disease (AD). It appears that 17β-HSD10 may play a role in the pathogenesis of AD. OBJECTIVE We investigated the possibility that levels of 17β-HSD10 in cerebrospinal fluid could be a prospective biomarker of AD. METHODS We estimated the enzyme levels in 161 people (15 non-demented controls, 52 people with mild cognitive impairment (MCI), 35 people with probable AD, or 59 people with other types of dementia) and compared them with those of Aβ(1- 42), tau, and phospho-tau. RESULTS We found significantly higher levels of 17β-HSD10 in people with MCI due to AD (to 109.9% ), with AD (to 120.0% ), or with other types of dementia (to 110.9% ) when compared to the control group. The sensitivity of the new biomarker to AD was 80.0% , and the specificity was 73.3% (compared to controls) or 52.5-59.1% (compared to other types of dementia). Results of multiple linear regression and of correlation analysis revealed AD-mediated changes in links between 17β-HSD10 and Mini Mental State Examination score. CONCLUSION It seems that changes in 17β-HSD10 start many years before symptom onset, analogous to those in Aβ1 - 42, tau, or phospho-tau and that the levels are a relatively highly sensitive but unfortunately less specific biomarker of AD. A role of 17β-HSD10 overexpression in AD is discussed.

The latest therapeutic approaches to Alzheimer disease are using intravenous immunoglobulin (IVIG) products. Therefore, the detailed characterization of target-specific antibodies naturally occurring in IVIG products is beneficial. We have focused on characterization of antibodies isolated against tau protein, a biomarker of Alzheimer's disease, from Flebogamma IVIG product. The analysis of IgG subclass distribution indicated skewing toward IgG3 in anti-tau-enriched IgG fraction. The evaluation of their reactivity and avidity with several recombinant tau forms was performed by ELISA and blotting techniques. Truncated non-phosphorylated tau protein (amino acids 155–421) demonstrated the highest reactivity and avidity index. We provide the first detailed insight into the reactivity of isolated natural antibodies against tau protein.

Patients with Alzheimer disease (AD) have simultaneous impairment in language and in several types of memory (long-term semantic, short-term episodic). Therefore we prepared a brief and easily administered screening instrument to examine these deficits in a single test in very early AD. The first task in our new test was to write down and simultaneously remember the names of 20 line drawings (long-term semantic memory, language). Then participants were immediately asked to recall and write down as many picture names as possible (immediate short-term episodic memory). We tested 50 normal elderly people (NEP) (MMSE 29±1 points) and 70 patients with very early AD fulfilling NIA-AA recommendations. The AD patients were categorized to a subgroup of 34 patients with mild cognitive impairment (MCI-AD) (MMSE 25±3 points) and a subgroup of 36 ones with mild dementia (ADD) (MMSE 23±3 points). We counted a number of pictures wrongly named or left unnamed at all (naming errors). Only a number of correctly recalled pictures (without confabulations or repetitions) in one minute was considered. The NEP wrote the picture names in significantly shorter time than MCI-AD or ADD patients (average 1,5 vs 2,6 (MCI-AD) vs 2,8 (ADD) minutes) (p < 0.01). They made significantly less naming errors than MCI-AD or ADD patients (1 vs 3 vs 4 errors) (p < 0.01). Conversely, the NEP recalled significantly more picture names than MCI-AD or ADD patients (9 vs 5 vs 3 correct names) (p < 0.001). When comparing the NEP and AD group according to ROC analysis we found an optimal cut-off score > 1 naming error or ≤ 7 correctly recalled picture names. The area under the curve was 0.76 for naming and 0.94 for recall. The novel and original test to name and recall pictures is a short, yet difficult assessment of several types of memory and language and may contribute to the diagnosis of even very early AD. Therefore it may be easily used in various clinical situations and applied in other countries due to visual nature. Supported by grant IGA NT 13183, PRVOUK 34/LF3 and NIMH ED2.1.00/03.0078.