Jozef Hanes

Identification and partial characterization of the major royal jelly protein of the honey been (Apis mellifera L.)

SUMMARYThe major royal jelly protein (MRJP) derived from the hypopharyngeal glands of the honey bee, Apis mellifera, was isolated by ionexchange chromatography. When using SDS-PAGE, the preparation exhibited only one band, which had a molecular mass of 57 kDa and consisted of at least eight components with similar isoelectric points with a range of pH 4.5—pH 5.0. Components of the MRJP were the most acidic proteins of royal jelly. The proteins from different parts of the honey bee body were analysed with antiserum raised against MRJP. Mobilities of proteins isolated directly from hypopharyngeal glands were greater than those of royal jelly proteins. The most significant difference was in the major protein. The MRJP and the major protein from hypopharyngeal glands had apparent molecular masses of 57 kDa and 55 kDa respectively. This difference reflects some modification of hypopharyngeal gland proteins.

Rat tau proteome consists of six tau isoforms: implication for animal models of human tauopathies

Human brain encompasses six tau isoforms, containing either three (3R) or four (4R) repeat domains, all of which participate in the pathogenesis of human tauopathies. To investigate the role of tau protein in the disease, transgenic rat models have been created. However, unlike humans, it has been suggested that rat brain expresses only three 4R tau isoforms. Because of the significance of the number of tau isoforms for faithful reproducibility of neurofibrillary pathology in transgenic rat models, we reopened this issue. Surprisingly, our results showed that adult rat brain contains six tau isoforms like humans. Protein expression of 4R tau isoforms was ninefold higher than 3R isoforms. Furthermore, the protein levels of tau isoforms with none, one or two N‐terminal inserts were 30%, 35%, and 35% of total tau, respectively. Moreover, amount and ratio of tau isoforms were developmentally regulated. The levels of 4R tau isoforms progressively increased from early postnatal period until adulthood, whereas the expression of 3R tau isoforms reached maximum at P10 and then gradually declined. Our results show that rat brain encompasses full tau proteome similar to humans. These findings support the use of rat as an animal model in human tauopathies research.

High-yield purification of fetal tau preserving its structure and phosphorylation pattern

The fetal type of tau phosphorylation always re-appears during pathogenesis of Alzheimers disease and related tauopathies. The major obstacle in the study of the fetal tau phosphorylation has been the lack of a simple and reproducible purification method yielding fetal tau with high purity and unmodified phosphorylation pattern. We have developed a two-step, highly efficient purification procedure of perchloric acid-extracted fetal tau by immunoaffinity chromatography and trichloroacetic acid (TCA) precipitation. The method yielded tau with more than 90% purity. Most importantly, purified fetal tau exhibited unmodified phosphorylation pattern as confirmed by phosphorylation-dependent antibodies. In summary, this purification process preserves and protects unstable phosphoresidues from dephosphorylation and allows their detailed molecular analysis especially in the pathogenesis of Alzheimers disease and related tauopathies.

Intraneuronal accumulation of misfolded tau protein induces overexpression of Hsp27 in activated astrocytes

Accumulation of misfolded forms of microtubule associated, neuronal protein tau causes neurofibrillary degeneration typical of Alzheimers 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.

Dendritic Cells as an Alternate Approach for Treatment of Neurodegenerative Disorders

Despite years of research, Alzheimer’s disease (AD) remains incurable and thus poses a major health challenge in coming years. This neurodegenerative disease belongs to a heterogeneous group of human tauopathies, characterized by the extracellular deposition of beta amyloid-Aβ and intracellular accumulation of tau protein in neuronal and glial cells, whereby tau pathology best correlates with disease progression. For decades, several disease-modifying agents were brought to clinical studies with promising efficacy in preclinical trials; however, all of the subsequent clinical trials failed. Therefore, the pursuit for therapeutic agents for the treatment of AD and other tauopathies still continue. Recent evidences show previously unidentified role of peripheral immune system in regulating the inflammatory status of the brain, mainly the dendritic cells. A decrease in functionality and count of dendritic cells has been observed in Alzheimer’s disease. Here, we discuss a potential role of dendritic cell-based vaccines as therapeutic approach in ameliorating disease pathogenesis in AD and other tauopathies.

NOVEL ULTRASENSITIVE IMMUNOASSAY DETECTING P-TAU THR217 COMPLETELY DISTINGUISHES ALZHEIMER’S DISEASE FROM FRONTOTEMPORAL LOBAR DEGENERATION

rating 6 appropriate and 8 inappropriate. Conclusions: In anticipation of the emergence of more reliable CSF analysis platforms, this presentation will provide the Alzheimer’s research community the first opportunity to review the AUC in detail. A full manuscript will also be published in the near future. The manuscript offers important guidance to healthcare practitioners, and suggestions for implementation and future research.

Proteomic Approaches for Diagnostics of Canine and Feline Dementia

Cognitive dysfunction syndrome (CDS) in pet animals constitutes a pressing problem of the modern society. Millions of senior dogs and cats undergo age-related behavioral changes that impact their social interactions. Currently, it is difficult to discriminate between normal aging and dementing processes. The situation is akin to that in humans; however, human medicine receives enormous resources that resulted in a set of current diagnostic criteria including a number of dementia assessing scales, diagnostic assays, and novel potential biomarkers. While animal well-being is not in the limelight of societal interest, the dementia diagnostics starts to catch up. Nevertheless, biochemical markers related to the animal dementia are underdeveloped, despite the fact that dogs and cats provide natural models for human dementia.

Role of the genetic background in the neurodegeneration induced by truncated tau in the transgenic rat model

allelic loss of progranulin causes very subtle growth modulatory differences in MEFs derived from progranulin knockout mice compared to those from wild-type littermate controls. Secondly, progranulin knockout MEFs showed comparable cell viability with a non-statistically significant increased susceptibility to certain apoptotic stimuli as measured by various apoptotic parameters. Thirdly, loss of progranulin did not directly lead to altered TDP-43 localization, fragmentation and/or accumulation as studied by a panel of antibodies raised against the Nor C-terminus or against recently described phosphorylation sites of TDP-43. Finally, similar to the effect observed in human cell lines, caspase activation in MEFs led to TDP43 cleavage to generate a predominantly z35 kDa C-terminal fragment. This pattern although did not resemble the pathological fragmentation observed in diseased brains. Conclusions: Our data suggests that progranulin has subtle growth modulatory activities in physiological situations and loss of progranulin alone might not be sufficient to directly cause alterations in TDP-43 expression, fragmentation and/or post-translational modification in the studied cell models. Thus, the impact of progranulin loss on the development of TDP-43 pathology in patients carrying progranulin mutations remains elusive.

Intrinsically disordered protein tau with unsheltered microtubule-binding domain become mis-disordered: Implications for neurofibrillary degeneration

Background: rTg4510 is a conditional mouse model of tauopathy that expresses mutant human tau within the mouse forebrain at 13 fold that of endogenous tau. Cognitive impairment and pre-tangle pathology within the forebrain is observed at 2.5 months. Mature tangle pathology is apparent by 4 months of age in the cortex and 5.5 months in the CA1, accompanied by significant neuronal loss. Inhibiting transgenic tau expression via doxycycline (dox) administration can reduce the cognitive deficits in rTg4510 mice. Methods: The rTg4510 inducible tau model has well-defined biochemical, pathological, and phenotypic stages, and we have now exploited this model to determine if aged neurons are predisposed to tauopathy. To explore this question, we administered dox to mothers from conception of rTg4510 litters and continued to dox weaned rTg4510 mice and controls until key pathological time points as defined in rTg4510 mice who did not receive dox (ON). In the first cohort, we aged rTg4510 mice to 2.75 months before inducing tau expression for the subsequent 2.75 months (2.75OFF/ 2.75ON mice). For the second cohort, we aged rTg4510 mice to 5.5 months before inducing tau expression for the subsequent 2.75 months (5.5OFF/ 2.75ON mice). Finally, in the third cohort, we aged rTg4510 mice to 5.5 months before inducing tau expression for an additional 5.5 months (5.5OFF/5.5ON mice). ON rTg4510 mice, rTg4510 mice continually receiving dox (OFF), and non-transgenic mice at 2.75, 5.5, 8.25, and 11 months of age were compared to the three experimental cohorts with biochemical and neuropathological measures of tauopathy. Results: rTg4510 mice in which transgenic tau expression was induced at key biochemical time points showed reduction in tau hyperphosphorylation and accumulation of the 64kD species of sarkosyl insoluble tau when compared to rTg4510 (ON) mice. Additionally, ultrastructural analysis of the sarkosyl insoluble fraction of tau from OFF/ON mice suggests the presence of amorphous, ill-defined clumps of tau protein compared to defined tau filaments in rTg4510 ON mice. Neuropathological analyses are still ongoing. Conclusions: This data clearly shows that the age of a neuron, alone, does not predispose neurons to rapid induction of tauopathy.

Molecular analysis of tau phosphorylation in a rat model of Alzheimer's disease revealed interplay between the endogenous rat tau and misfolded human Alzheimer's disease derived tau

Background: Misfolded microtubule-associated neuronal protein tau is regarded as a prime iniciator of neurodegeneration in human tauopathies. It is believed that seminal posttranslational events leading to the transition of tau protein from disordered to misordered state are associated with increased phosphorylation or truncation or a combination of the two. Recently, we have presented a rat model of Alzheimer’s disease expressing a truncated tau form derived from human sporadic AD, which exhibits drastically reduced survival caused by severe neuronal degeneration. This work revealed the toxic properties of misfolded tau protein. Methods: We have analyzed the endogenous rat tau and the misfolded (truncated) human tau expressed in the brain of the transgenic animals by Western blotting with phospho-tau specific antibodies. Results: We found that only misfolded tau protein is hyperphosphorylated on several Ser and Thr residues during the whole life of the transgenic animals. On the other hand, phosphorylated forms of the endogenous rat tau disappeared within 3 weeks after birth in both wt and transgenic animals. Furthermore, misfolded tau negatively influenced the phosphorylation level of endogenous tau, suggesting a competition between the tau forms (substrates) for kinases. Gene expression analysis of transgenic and wild type rat brains has shown that neither upregulation of kinases nor downregulation of phosphatases takes place in these transgenic animals. Conclusions: Based on our results we conclude that hyperphosphorylation of a misfolded tau is driven by its conformation, which is distinct from that of a normal tau protein, and not by an increased activity of kinases. Acknowledgement: The work was supported by a grant from Axon Neuroscience and the Slovak grant agency APVV, grants number LPP 32606, APVV VV 603-06 and VEGA 2/7129/27.