Evolving prion-like tau conformers differentially alter postsynaptic proteins in neurons inoculated with distinct isolates of Alzheimer's disease tau.

OBJECTIVES: Although accumulation of misfolded tau species has been shown to predict cognitive decline in patients with Alzheimer's disease (AD) and other tauopathies but with the remarkable diversity of clinical manifestations, neuropathology profiles, and time courses of disease progression remaining unexplained by current genetic data. We considered the diversity of misfolded tau conformers present in individual AD cases as an underlying driver of the phenotypic variations of AD and progressive loss of synapses. METHODS: To model the mechanism of tau propagation and synaptic toxicity of distinct tau conformers, we inoculated wild-type primary mouse neurons with structurally characterized Sarkosyl-insoluble tau isolates from the frontal cortex of six AD cases and monitored the impact for fourteen days. We analyzed the accumulation rate, tau isoform ratio, and conformational characteristics of de novo-induced tau aggregates with conformationally sensitive immunoassays, and the dynamics of synapse formation, maintenance, and their loss using a panel of pre-and post-synaptic markers. RESULTS: At the same concentrations of tau, the different AD tau isolates induced accumulation of misfolded predominantly 4-repeat tau aggregates at different rates in mature neurons, and demonstrated distinct conformational characteristics corresponding to the original AD brain tau. The time-course of the formation of misfolded tau aggregates and colocalization correlated with significant loss of synapses in tau-inoculated cell cultures and the reduction of synaptic connections implicated the disruption of postsynaptic compartment as an early event. CONCLUSIONS: The data obtained with mature neurons expressing physiological levels and adult isoforms of tau protein demonstrate markedly different time courses of endogenous tau misfolding and differential patterns of post-synaptic alterations. These and previous biophysical data argue for an ensemble of various misfolded tau aggregates in individual AD brains and template propagation of their homologous conformations in neurons with different rates and primarily postsynaptic interactors. Modeling tau aggregation in mature differentiated neurons provides a platform for investigating divergent molecular mechanisms of tau strain propagation and for identifying common structural features of misfolded tau and critical interactors for new therapeutic targets and approaches in AD.

Populations of Tau Conformers Drive Prion-like Strain Effects in Alzheimer's Disease and Related Dementias.

Recent findings of diverse populations of prion-like conformers of misfolded tau protein expand the prion concept to Alzheimer's disease (AD) and monogenic frontotemporal lobar degeneration (FTLD)-MAPT P301L, and suggest that distinct strains of misfolded proteins drive the phenotypes and progression rates in many neurodegenerative diseases. Notable progress in the previous decades has generated many lines of proof arguing that yeast, fungal, and mammalian prions determine heritable as well as infectious traits. The extraordinary phenotypic diversity of human prion diseases arises from structurally distinct prion strains that target, at different progression speeds, variable brain structures and cells. Although human prion research presents beneficial lessons and methods to study the mechanism of strain diversity of protein-only pathogens, the fundamental molecular mechanism by which tau conformers are formed and replicate in diverse tauopathies is still poorly understood. In this review, we summarize up to date advances in identification of diverse tau conformers through biophysical and cellular experimental paradigms, and the impact of heterogeneity of pathological tau strains on personalized structure- and strain-specific therapeutic approaches in major tauopathies.

Distinct populations of highly potent TAU seed conformers in rapidly progressing Alzheimer's disease.

Although genetic factors play a main role in determining the risk of developing Alzheimer's disease (AD), they do not explain extensive spectrum of clinicopathological phenotypes. Deposits of aggregated TAU proteins are one of the main predictors of cognitive decline in AD. We investigated the hypothesis that variabilities in AD progression could be due to diverse structural assemblies (strains) of TAU protein. Using sensitive biophysical methods in 40 patients with AD and markedly different disease durations, we identified populations of distinct TAU particles that differed in size, structural organization, and replication rate in vitro and in cell assay. The rapidly replicating, distinctly misfolded TAU conformers found in rapidly progressive AD were composed of ~80% misfolded four-repeat (4R) TAU and ~20% of misfolded 3R TAU isoform with the same conformational signatures. These biophysical observations suggest that distinctly misfolded population of 4R TAU conformers drive the rapid decline in AD and imply that effective therapeutic strategies might need to consider not a singular species but a cloud of differently misfolded TAU conformers.

Cellular Biology of Tau Diversity and Pathogenic Conformers.

Tau accumulation is a prominent feature in a variety of neurodegenerative disorders and remarkable effort has been expended working out the biochemistry and cell biology of this cytoplasmic protein. Tau's wayward properties may derive from germline mutations in the case of frontotemporal lobar degeneration (FTLD-MAPT) but may also be prompted by less understood cues-perhaps environmental or from molecular damage as a consequence of chronological aging-in the case of idiopathic tauopathies. Tau properties are undoubtedly affected by its covalent structure and in this respect tau protein is not only subject to changes in length produced by alternative splicing and endoproteolysis, but different types of posttranslational modifications that affect different amino acid residues. Another layer of complexity concerns alternate conformations-"conformers"-of the same covalent structures; in vivo conformers can encompass soluble oligomeric species, ramified fibrillar structures evident by light and electron microscopy and other forms of the protein that have undergone liquid-liquid phase separation to make demixed liquid droplets. Biological concepts based upon conformers have been charted previously for templated replication mechanisms for prion proteins built of the PrP polypeptide; these are now providing useful explanations to feature tau pathobiology, including how this protein accumulates within cells and how it can exhibit predictable patterns of spread across different neuroanatomical regions of an affected brain. In sum, the documented, intrinsic heterogeneity of tau forms and conformers now begins to speak to a fundamental basis for diversity in clinical presentation of tauopathy sub-types. In terms of interventions, emphasis upon subclinical events may be worthwhile, noting that irrevocable cell loss and ramified protein assemblies feature at end-stage tauopathy, whereas earlier events may offer better opportunities for diverting pathogenic processes. Nonetheless, the complexity of tau sub-types, which may be present even within intermediate disease stages, likely mitigates against one-size-fits-all therapeutic strategies and may require a suite of interventions. We consider the extent to which animal models of tauopathy can be reasonably enrolled in the campaign to produce such interventions and to slow the otherwise inexorable march of disease progression.

Brain-derived neurotrophic factor (BDNF) promotes molecular polarization and differentiation of immature neuroblastoma cells into definitive neurons.

Throughout development, neuronal progenitors undergo complex transformation into polarized nerve cells, warranting the directional flow of information in the neural grid. The majority of neuronal polarization studies have been carried out on rodent-derived precursor cells, programmed to develop into neurons. Unlike rodent neuronal cells, SH-SY5Y cells derived from human bone marrow present a sub-clone of neuroblastoma line, with their transformation into neuron-like cells showing a range of highly instructive neurobiological characteristics. We applied two-step retinoic acid (RA) and brain-derived neurotrophic factor (BDNF) protocol to monitor the conversion of undifferentiated SH-SY5Y into neuron-like cells with distinctly polarized axon-dendritic morphology and formation of bona fide synaptic connections. We show that BDNF is a key driver and regulator of the expression of axonal marker tau and dendritic microtubule-associated protein-2 (MAP2), with their sorting to distinct cellular compartments. Using selective kinase inhibitors downregulating BDNF-TrkB signaling, we demonstrate that constitutive activation of TrkB receptor is essential for the maintenance of established polarization morphology. Importantly, the proximity ligation assay applied in our preparation demonstrates that differentiating neuron-like cells develop elaborate synaptic connections enriched with hallmark pre- and postsynaptic proteins. Described herein findings highlight several fundamental processes related to neuronal polarization and synaptogenesis in human-derived cells, which are of major relevance to neurobiology and translational neuroscience.

Interactions of 17ß-Hydroxysteroid Dehydrogenase Type 10 and Cyclophilin D in Alzheimer's Disease.

The nucleus-encoded 17ß-hydroxysteroid dehydrogenase type 10 (17ß-HSD10) regulates cyclophilin D (cypD) in the mitochondrial matrix. CypD regulates opening of mitochondrial permeability transition pores. Both mechanisms may be affected by amyloid ß peptides accumulated in mitochondria in Alzheimer's disease (AD). In order to clarify changes occurring in brain mitochondria, we evaluated interactions of both mitochondrial proteins in vitro (by surface plasmon resonance biosensor) and detected levels of various complexes of 17ß-HSD10 formed in vivo (by sandwich ELISA) in brain mitochondria isolated from the transgenic animal model of AD (homozygous McGill-R-Thy1-APP rats) and in cerebrospinal fluid samples of AD patients. By surface plasmon resonance biosensor, we observed the interaction of 17ß-HSD10 and cypD in a direct real-time manner and determined, for the first time, the kinetic parameters of the interaction (ka 2.0 × 105 M1s-1, kd 5.8 × 104 s-1, and KD 3.5 × 10-10 M). In McGill-R-Thy1-APP rats compared to controls, levels of 17ß-HSD10-cypD complexes were decreased and those of total amyloid ß increased. Moreover, the levels of 17ß-HSD10-cypD complexes were decreased in cerebrospinal fluid of individuals with AD (in mild cognitive impairment as well as dementia stages) or with Frontotemporal lobar degeneration (FTLD) compared to cognitively normal controls (the sensitivity of the complexes to AD dementia was 92.9%, that to FTLD 73.8%, the specificity to AD dementia equaled 91.7% in a comparison with the controls but only 26.2% with FTLD). Our results demonstrate the weakened ability of 17ß-HSD10 to regulate cypD in the mitochondrial matrix probably via direct effects of amyloid ß. Levels of 17ß-HSD10-cypD complexes in cerebrospinal fluid seem to be the very sensitive indicator of mitochondrial dysfunction observed in neurodegeneration but unfortunately not specific to AD pathology. We do not recommend it as the new biomarker of AD.

Tau-Reactive Endogenous Antibodies: Origin, Functionality, and Implications for the Pathophysiology of Alzheimer's Disease.

In Alzheimer's disease (AD), tau pathology manifested by the accumulation of intraneuronal tangles and soluble toxic oligomers emerges as a promising therapeutic target. Multiple anti-tau antibodies inhibiting the formation and propagation of cytotoxic tau or promoting its clearance and degradation have been tested in clinical trials, albeit with the inconclusive outcome. Antibodies against tau protein have been documented both in the brain circulatory system and at the periphery, but their origin and role under normal conditions and in AD remain unclear. While it is tempting to assign them a protective role in regulating tau level and removal of toxic variants, the supportive evidence remains sporadic, requiring systematic analysis and critical evaluation. Herein, we review recent data showing the occurrence of tau-reactive antibodies in the brain and peripheral circulation and discuss their origin and significance in tau clearance. Based on the emerging evidence, we cautiously propose that impairments of tau clearance at the periphery by humoral immunity might aggravate the tau pathology in the central nervous system, with implication for the neurodegenerative process of AD.

Kinase-loaded magnetic beads for sequential in vitro phosphorylation of peptides and proteins.

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-3ß 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-3ß 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.

Characterization of isolated tau-reactive antibodies from the IVIG product, plasma of patients with Alzheimer's disease and cognitively normal individuals.

The presence of natural tau-reactive antibodies was reported in human blood. In this study, we isolated and characterized natural tau-reactive antibodies occurring in IVIG product Flebogamma, plasma of patients with Alzheimer's disease (AD) and older cognitively normal persons (controls). Using blotting immunoassays and ELISA, we showed reactivity of antibodies obtained from IVIG and controls against a recombinant fragment of tau (155-421aa) and aggregates present in brains of AD patients. In contrast, antibodies isolated from plasma of AD patients reacted mainly with the recombinant full-length tau form and tau monomeric forms in brain tissue.

Monoamine oxidase B is elevated in Alzheimer disease neurons, is associated with γ-secretase and regulates neuronal amyloid ß-peptide levels.

BACKGROUND: Increased levels of the pathogenic amyloid ß-peptide (Aß), released from its precursor by the transmembrane protease γ-secretase, are found in Alzheimer disease (AD) brains. Interestingly, monoamine oxidase B (MAO-B) activity is also increased in AD brain, but its role in AD pathogenesis is not known. Recent neuroimaging studies have shown that the increased MAO-B expression in AD brain starts several years before the onset of the disease. Here, we show a potential connection between MAO-B, γ-secretase and Aß in neurons. METHODS: MAO-B immunohistochemistry was performed on postmortem human brain. Affinity purification of γ-secretase followed by mass spectrometry was used for unbiased identification of γ-secretase-associated proteins. The association of MAO-B with γ-secretase was studied by coimmunoprecipitation from brain homogenate, and by in-situ proximity ligation assay (PLA) in neurons as well as mouse and human brain sections. The effect of MAO-B on Aß production and Notch processing in cell cultures was analyzed by siRNA silencing or overexpression experiments followed by ELISA, western blot or FRET analysis. Methodology for measuring relative intraneuronal MAO-B and Aß42 levels in single cells was developed by combining immunocytochemistry and confocal microscopy with quantitative image analysis. RESULTS: Immunohistochemistry revealed MAO-B staining in neurons in the frontal cortex, hippocampus CA1 and entorhinal cortex in postmortem human brain. Interestingly, the neuronal staining intensity was higher in AD brain than in control brain in these regions. Mass spectrometric data from affinity purified γ-secretase suggested that MAO-B is a γ-secretase-associated protein, which was confirmed by immunoprecipitation and PLA, and a neuronal location of the interaction was shown. Strikingly, intraneuronal Aß42 levels correlated with MAO-B levels, and siRNA silencing of MAO-B resulted in significantly reduced levels of intraneuronal Aß42. Furthermore, overexpression of MAO-B enhanced Aß production. CONCLUSIONS: This study shows that MAO-B levels are increased not only in astrocytes but also in pyramidal neurons in AD brain. The study also suggests that MAO-B regulates Aß production in neurons via γ-secretase and thereby provides a key to understanding the relationship between MAO-B and AD pathogenesis. Potentially, the γ-secretase/MAO-B association may be a target for reducing Aß levels using protein-protein interaction breakers.

Purification of Natural Antibodies Against Tau Protein by Affinity Chromatography.

Natural antibodies are now widely studied for their therapeutical potential. Therefore, their isolation and subsequent characterization is desired. Here, we describe an isolation method for natural anti-tau antibodies from human plasma by utilization of affinity chromatography with epoxy-activated copolymer resin. The evalution of isolation efficiency and avidity of isolated antibodies is decribed by modified indirect ELISA assay.

Difficulties associated with the structural analysis of proteins susceptible to form aggregates: The case of Tau protein as a biomarker of Alzheimer's disease.

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.

Identification and characterization of natural antibodies against tau protein in an intravenous immunoglobulin product.

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.