dGAE(297-391) Tau Fragment Promotes Formation of Chronic Traumatic Encephalopathy-Like Tau Filaments.

The microtubule-associated protein tau forms disease-specific filamentous aggregates in several different neurodegenerative diseases. In order to understand how tau undergoes misfolding into a specific filament type and to control this process for drug development purposes, it is crucial to study in vitro tau aggregation methods and investigate the structures of the obtained filaments at the atomic level. Here, we used the tau fragment dGAE, which aggregates spontaneously, to seed the formation of full-length tau filaments. The structures of dGAE and full-length tau filaments were investigated by magic-angle spinning (MAS) solid-state NMR, showing that dGAE allows propagation of a chronic traumatic encephalopathy (CTE)-like fold to the full-length tau. The obtained filaments efficiently seeded tau aggregation in HEK293T cells. This work demonstrates that in vitro preparation of disease-specific types of full-length tau filaments is feasible.

The engagement of microglia in tau-targeted immunotherapy in Alzheimer's disease.

Alzheimer's disease (AD) is an age-related neurodegenerative disease characterized by progressive memory decline, histopathological lesions such as amyloid ß plaques and neurofibrillary tangles, and neuroinflammation driven by glial cells. Microglia, the innate immune cells of the brain, dynamically survey their environment for signs of infection and cell damage. Although our understanding of microglia and their modes of activation has expanded in recent years, their role in AD is still not completely understood. Broad range of microglia phenotypes, from neuroinflammatory to neuroprotective, found in neurodegenerative diseases make their role difficult to discern. In this review, we summarize activities of microglia in healthy and AD brains and their possible role during immunotherapy targeted against pathological tau proteins.

Capillary electrophoresis and ultra-high-performance liquid chromatography methods in clinical monitoring of creatinine in human urine: A comparative study.

Creatinine is an important diagnostic marker and is also used as a standardization tool for the quantitative evaluation of exogenous/endogenous substances in urine. This study aimed at evaluating and comparing three analytical approaches, based on hyphenations of different separation [two-dimensional capillary isotachophoresis (CITP-CITP), capillary zone electrophoresis (CZE), ultra-high-performance liquid chromatography (UHPLC)] and detection [conductivity (CD), ultraviolet (UV), tandem mass spectrometry (MS/MS)] techniques, for their ability to provide reliable clinical data along with their suitability for the routine clinical use (cost, simplicity, sample throughput). The developed UHPLC-MS/MS, CITP-CITP-CD, and CZE-UV methods were characterized by favorable performance parameters, such as linearity (r ˃ 0.99), precision (relative standard deviation, 0.22-2.97% for the creatinine position in analytical profiles), and recovery (87.1-115.1%). Clinical data, obtained from the analysis of 24 human urine samples by a reference enzymatic method, were comparable with those obtained by the tested methods (Passing-Bablok regression and Bland-Altman analysis), approving their usefulness for the routine clinical use. In this context, the UHPLC-MS/MS method provides benefits of enhanced orthogonality/accuracy and high sample throughput (threefold shorter total analysis times than the CE methods), whereas advantages of the CE methods for routine labs are simplicity and low cost of both the instrumentation and measurements.

Profiling of Amino Acids in Urine Samples of Patients Suffering from Inflammatory Bowel Disease by Capillary Electrophoresis-Mass Spectrometry.

Urine represents a convenient biofluid for metabolomic studies due to its noninvasive collection and richness in metabolites. Here, amino acids are valuable biomarkers for their ability to reflect imbalances of different biochemical pathways. An impact of amino acids on pathology, prognosis and therapy of various diseases, including inflammatory bowel disease (IBD), is therefore the subject of current clinical research. This work is aimed to develop a capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) method for the quantification of the 20 proteinogenic amino acids in human urine samples obtained from patients suffering from IBD and treated with thiopurines. The optimized CE-MS/MS method, with minimum sample preparation (just "dilute and shoot"), exhibited excellent linearity for all the analytes (coefficients of determination were higher than 0.99), with inter-day and intra-day precision yielding relative standard deviations in the range of 0.91-15.12% and with accuracy yielding relative errors in the range of 85.47-112.46%. Total analysis time, an important parameter for the sample throughput demanded in routine practice, was shorter in ca. 17% when compared to established CE-MS methods. Favorable performance of the proposed CE-MS/MS method was also confirmed by the comparison with corresponding ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) method. Consistent data for the investigated amino acid metabolome were obtained using both methods. For the first time, the amino acid profiling by CE-MS approach was applied on the clinical IBD samples. Here, significant differences observed in the concentration levels of some amino acids between IBD patients undergoing thiopurine treatment and healthy volunteers could result from the simultaneous action of the disease and the corresponding therapy. These findings indicate that amino acids analysis could be a valuable tool for the study of mechanism of the IBD treatment by thiopurines.

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 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.

Microglia display modest phagocytic capacity for extracellular tau oligomers.

BACKGROUND: Abnormal misfolded tau protein is a driving force of neurofibrillary degeneration in Alzheimer's disease. It has been shown that tau oligomers play a crucial role in the formation of intracellular neurofibrillary tangles. They are intermediates between soluble tau monomers and insoluble tau filaments and are suspected contributors to disease pathogenesis. Oligomeric tau can be released into the extracellular space and spread throughout the brain. This finding opens the question of whether brain macrophages or blood monocytes have the potential to phagocytose extracellular oligomeric tau. METHODS: We have used stable rat primary microglial cells, rat peripheral monocytes-derived macrophages, BV2 microglial and TIB67 macrophage immortalized cell lines that were challenged by tau oligomers prepared by an in vitro aggregation reaction. The efficiency of cells to phagocytose oligomeric protein was evaluated with confocal microscopy. The ability to degrade tau protein was analyzed by immunoblotting. RESULTS: Confocal microscopy analyses showed that macrophages were significantly more efficient in phagocytosing oligomerized tau proteins than microglial cells. In contrast to macrophages, microglia are able to degrade the internalized oligomeric tau only after stimulation with lipopolysaccharide (LPS). CONCLUSIONS: Our data suggests that microglia may not be the principal phagocytic cells able to target extracellular oligomeric tau. We found that peripheral macrophages display a high potency for elimination of oligomeric tau and therefore could play an important role in the modulation of neurofibrillary pathology in Alzheimer's disease.

Efficacy assessment of an active tau immunotherapy in Alzheimer's disease patients with amyloid and tau pathology: a post hoc analysis of the "ADAMANT" randomised, placebo-controlled, double-blind, multi-centre, phase 2 clinical trial.

BACKGROUND: Tau pathology correlates with and predicts clinical decline in Alzheimer's disease. Approved tau-targeted therapies are not available. METHODS: ADAMANT, a 24-month randomised, placebo-controlled, parallel-group, double-blinded, multicenter, Phase 2 clinical trial (EudraCT2015-000630-30, NCT02579252) enrolled 196 participants with Alzheimer's disease; 119 are included in this post-hoc subgroup analysis. AADvac1, active immunotherapy against pathological tau protein. A machine learning model predicted likely Amyloid+Tau+ participants from baseline MRI. STATISTICAL METHODS: MMRM for change from baseline in cognition, function, and neurodegeneration; linear regression for associations between antibody response and endpoints. RESULTS: The prediction model achieved PPV of 97.7% for amyloid, 96.2% for tau. 119 participants in the full analysis set (70 treatment and 49 placebo) were classified as A+T+. A trend for CDR-SB 104-week change (estimated marginal means [emm] = -0.99 points, 95% CI [-2.13, 0.13], p = 0.0825]) and ADCS-MCI-ADL (emm = 3.82 points, CI [-0.29, 7.92], p = 0.0679) in favour of the treatment group was seen. Reduction was seen in plasma NF-L (emm = -0.15 log pg/mL, CI [-0.27, -0.03], p = 0.0139). Higher antibody response to AADvac1 was related to slowing of decline on CDR-SB (rho = -0.10, CI [-0.21, 0.01], p = 0.0376) and ADL (rho = 0.15, CI [0.03, 0.27], p = 0.0201), and related to slower brain atrophy (rho = 0.18-0.35, p < 0.05 for temporal volume, whole cortex, and right and left hippocampus). CONCLUSIONS: In the subgroup of ML imputed or CSF identified A+T+, AADvac1 slowed AD-related decline in an antibody-dependent manner. Larger anti-tau trials are warranted. FUNDING: AXON Neuroscience SE.

The self-perpetuating tau truncation circle.

Pathological truncations of human brain proteins represent the common feature of many neurodegenerative disorders including AD (Alzheimer's disease), Parkinson's disease and Huntington's disease. Protein truncations significantly change the structure and function of these proteins and thus can engender their pathological metamorphosis. We have shown previously that truncated forms of tau protein are contained in the core of the paired helical filaments that represent the main constituent of neurofibrillary pathology. Recently, we have identified truncated tau species of a different molecular signature. We have found that tau truncation is not produced by a random process, but rather by highly specific proteolytic cleavage and/or non-enzymatic fragmentation. In order to characterize the pathophysiology of AD-specific truncated tau species, we have used a transgenic rat model for AD expressing human truncated tau. Expression of the tau protein induces the formation of novel truncated tau species that originate from both transgenic human tau and endogenous rat tau proteins. Moreover, these truncated tau proteins are found exclusively in the misfolded fraction of tau, suggesting that they actively participate in the tau misfolding process. These findings corroborate further the idea that the appearance of truncated tau species starts a self-perpetuating cycle of further tau protein truncation leading to and accelerating tau misfolding and formation of neurofibrillary pathology.

Crystallization and preliminary X-ray diffraction analysis of tau protein microtubule-binding motifs in complex with Tau5 and DC25 antibody Fab fragments.

The Alzheimer's disease-associated protein tau is an intrinsically disordered protein with no preferred structure in solution. Under physiological conditions, tau binds to microtubules and regulates their dynamics, whereas during the development of neurodegeneration tau dissociates from microtubules, misfolds and creates highly insoluble deposits. To elucidate the determinants of tau-protein misfolding, tau peptides from microtubule-binding motifs were crystallized in complexes with Fab fragments of specific monoclonal antibodies. The crystals diffracted to 1.69 Šresolution and gave complete data sets using a synchrotron X-ray source. Molecular replacement was used to solve the phase problem.

Immunomodulation of memory-impairing protein tau in Alzheimer's disease.

BACKGROUND: Neurodegeneration induced by misfolded tau protein and neuroinflammation represent the major hallmarks of human tauopathies including Alzheimer's disease (AD). While tau driven neurodegeneration significantly correlates with disease progression, inflammation is considered to be an important factor regulating the resistance or susceptibility to AD. The emerging evidence suggests that the genes related to immunity can influence neurodegeneration. OBJECTIVE: In order to determine the role of MHC class II in the tau neurofibrillary cascade, we generated and used transgenic lines expressing human truncated tau protein in either spontaneously hypertensive rat (SHR) or Wistar-Kyoto rat (WKY) genetic background. METHODS: Brains of WKY and SHR transgenic rats and their age-matched nontransgenic littermates were examined by immunohistochemistry and RT-PCR. RESULTS: Our results clearly showed that genetic background determined the inflammatory pattern (MHC class II) induced by tau neurodegenerative cascade in the transgenic rats expressing human misfolded truncated tau. CONCLUSION: Using two transgenic rat lines with different immunogenetic backgrounds, expressing the same transgene, we conclude that genetic background is a potent modifier of the type of the immune response induced by tau neurodegeneration.

Monoclonal antibodies targeting two immunodominant epitopes on the Spike protein neutralize emerging SARS-CoV-2 variants of concern.

BACKGROUND: The emergence of new SARS-CoV-2 variants of concern B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta) that harbor mutations in the viral S protein raised concern about activity of current vaccines and therapeutic antibodies. Independent studies have shown that mutant variants are partially or completely resistant against some of the therapeutic antibodies authorized for emergency use. METHODS: We employed hybridoma technology, ELISA-based and cell-based S-ACE2 interaction assays combined with authentic virus neutralization assays to develop second-generation antibodies, which were specifically selected for their ability to neutralize the new variants of SARS-CoV-2. FINDINGS: AX290 and AX677, two monoclonal antibodies with non-overlapping epitopes, exhibit subnanomolar or nanomolar affinities to the receptor binding domain of the viral Spike protein carrying amino acid substitutions N501Y, N439K, E484K, K417N, and a combination N501Y/E484K/K417N found in the circulating virus variants. The antibodies showed excellent neutralization of an authentic SARS-CoV-2 virus representing strains circulating in Europe in spring 2020 and also the variants of concern B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta). In addition, AX677 is able to bind Omicron Spike protein just like the wild type Spike. The combination of the two antibodies prevented the appearance of escape mutations of the authentic SARS-CoV-2 virus. Prophylactic administration of AX290 and AX677, either individually or in combination, effectively reduced viral burden and inflammation in the lungs, and prevented disease in a mouse model of SARS-CoV-2 infection. INTERPRETATION: The virus-neutralizing properties were fully reproduced in chimeric mouse-human versions of the antibodies, which may represent a promising tool for COVID-19 therapy. FUNDING: The study was funded by AXON Neuroscience SE and AXON COVIDAX a.s.

ADAMANT: a placebo-controlled randomized phase 2 study of AADvac1, an active immunotherapy against pathological tau in Alzheimer's disease.

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.

Genetic background modifies neurodegeneration and neuroinflammation driven by misfolded human tau protein in rat model of tauopathy: implication for immunomodulatory approach to Alzheimer's disease.

BACKGROUND: Numerous epidemiological studies demonstrate that genetic background modifies the onset and the progression of Alzheimer's disease and related neurodegenerative disorders. The efficacious influence of genetic background on the disease pathway of amyloid beta has been meticulously described in rodent models. Since the impact of genetic modifiers on the neurodegenerative and neuroinflammatory cascade induced by misfolded tau protein is yet to be elucidated, we have addressed the issue by using transgenic lines expressing the same human truncated tau protein in either spontaneously hypertensive rat (SHR) or Wistar-Kyoto (WKY) genetic background. METHODS: Brains of WKY and SHR transgenic rats in the terminal stage of phenotype and their age-matched non-transgenic littermates were examined by means of immunohistochemistry and unbiased stereology. Basic measures of tau-induced neurodegeneration (load of neurofibrillary tangles) and neuroinflammation (number of Iba1-positive microglia, their activated morphology, and numbers of microglia immunoreactive for MHCII and astrocytes immunoreactive for GFAP) were quantified with an optical fractionator in brain areas affected by neurofibrillary pathology (pons, medulla oblongata). The stereological data were evaluated using two-way ANOVA and Student's t-test. RESULTS: Tau neurodegeneration (neurofibrillary tangles (NFTs), axonopathy) and neuroinflammation (microgliosis, astrocytosis) appeared in both WKY and SHR transgenic rats. Although identical levels of transgene expression in both lines were present, terminally-staged WKY transgenic rats displayed significantly lower final NFT loads than their SHR transgenic counterparts. Interestingly, microglial responses showed a striking difference between transgenic lines. Only 1.6% of microglia in SHR transgenic rats expressed MHCII in spite of having a robust phagocytic phenotype, whereas in WKY transgenic rats, 23.2% of microglia expressed MHCII despite displaying a considerably lower extent of transformation into phagocytic phenotype. CONCLUSIONS: These results show that the immune response represents a pivotal and genetically variable modifying factor that is able to influence vulnerability to neurodegeneration. Therefore, targeted immunomodulation could represent a prospective therapeutic approach to Alzheimer's disease.

CSF phospho-tau correlates with behavioural decline and brain insoluble phospho-tau levels in a rat model of tauopathy.

The aim of the present study was to identify the relationship between progressive neurobehavioural decline and phospho-tau levels (p-tau(181)) in the cerebrospinal fluid (CSF) and the brain in transgenic rats expressing human truncated tau protein. Behavioural analyses, as quantified using the NeuroScale scoring method, revealed that the transgenic rats fell into two main groups based on the baseline behavioural functioning: (1) mild neurobehavioural impairment (MNI, score 3.3-26) and (2) severe neurobehavioural impairment (SNI, score 36-44). SNI transgenic rats showed a significant increase in brain sarkosyl insoluble p-tau(181) when compared to their MNI counterparts. In order to determine whether CSF phospho-tau reflects the behavioural decline and increase in sarkosyl insoluble tau in the brain, p-tau(181) was measured in the CSF in a longitudinal study. The study showed a significant increase in CSF p-tau(181) during the progression of the disease from MNI to SNI. Moreover, increased levels of p-tau(181) in CSF correlated with an increase in the sarkosyl insoluble p-tau(181) levels in the brain. The increase in the CSF level of p-tau(181) during progressive behavioural decline suggests that it may represent a useful surrogate biomarker for preclinical drug development and a potential surrogate endpoint for clinical trials of disease-modifying therapy for Alzheimer's disease and related human tauopathies.

Transition of tau protein from disordered to misordered in Alzheimer's disease.

The neuronal protein tau, a member of the class of intrinsically disordered proteins, is characterized by the absence of any firm 3-D structure and high solubility when free in solution. The tau protein forms insoluble fibrils in the brain of people suffering from Alzheimer's disease (AD) and other tauopathies and plays a key role in the neurodegenerative process. Posttranslational events leading to the transition of tau from a disordered highly soluble protein to the insoluble aggregate seem to be associated with hyperphosphorylation, truncation or a combination of both. These modifications are assumed to change the native disorder of tau into a preaggregation state, which then directly initiates the fibrillization process. Conformation-specific anti-tau antibody DC11 detects pathological truncated tau forms present in AD brains but not in the normal human brain. A truncated tau protein selected from this pool of DC11-positive molecules was sufficient to initiate and drive the complete tau cascade of neurofibrillary pathology in a rat model of tauopathy. Thus, DC11 antibody recognizes the AD-specific conformation of tau not found in the normal human brain. We propose the term 'misdisordered' for this distinct conformational state of tau, which is the first step in the transition of tau from a soluble disordered protein to its insoluble, misordered aggregated form.

A simple and rapid LC-MS/MS and CE-MS/MS analytical strategy for the determination of therapeutic peptides in modern immunotherapeutics and biopharmaceutics.

Modern therapy of metabolic, neurodegenerative, inflammation, or cancer diseases is recently based on an immunotherapeutic approach. The peptide conjugates represent innovative and effective therapeutics that are better tolerated and are much more specific than small molecule-based medicines. The nature and manufacturing process of these therapeutics make their analysis very challenging. Here, two robust analytical methods based on an on-line combination of ultra-high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) and capillary electrophoresis with tandem mass spectrometry (CE-MS/MS) were developed for fast determination of immunogenic synthetic peptide (peptide sequence CADNLHKVVGQST) in a conjugate with bovine serum albumin (BSA) as a carrier protein and is a peptide, conjugate formulated with a vaccine adjuvant - Alhydrogel® 2 %. An effective non-enzymatic release step of the peptide from the final peptide conjugate based on acid hydrolysis with the use of 2% formic acid was successfully tested and implemented. The proposed methods were validated according to the ICH guideline and parameters such as linearity, precision, and accuracy, the limit of detection (LOD) or limit of quantification (LOQ) were assessed. Calibration curves were linear within the range of 1-30 µg.mL-1 and the correlation coefficients were higher than 0.99. The intraday and interday precisions were 3.2-8.1 % (UHPLC-MS/MS), 1.6-9.3 % (CE-MS/MS) and 3.6-10.3 % (UHPLC-MS/MS), 4.1-10.2 % (CE-MS/MS), respectively. The recovery ranged in the interval of 98.4-107.4 % for UHPLC-MS/MS method and 100.3-103.2 % for CE-MS/MS method. The presented approaches represent an effective tool for simple, rapid and robust quantification of immunogens in modern immunotherapeutics and other biopharmaceuticals with appropriate peptide sequences.

Environmental Enrichment Rescues Functional Deficit and Alters Neuroinflammation in a Transgenic Model of Tauopathy.

Alzheimer's disease (AD) is the most frequent neurodegenerative disorder, affecting over 44 million people worldwide. There are no effective pharmaco-therapeutic options for prevention and treatment of AD. Non-pharmacological approaches may help patients suffering from AD to significantly ameliorate disease progression. In this study, we exposed a transgenic rat model (tg) of human tauopathy to enriched environment for 3 months. Behavioral testing at 6 months of age revealed improvement in functional deficits of tg rats reared under enriched conditions, while sedentary tg rats remained severely impaired. Interestingly, enriched environment did not reduce tau pathology. Analysis of neurotrophic factors revealed an increase of nerve growth factor (NGF) levels in the hippocampus of both enriched groups (tg and non-tg rats), reflecting a known effect of enriched environment on the hippocampal formation. On the contrary, NGF levels decreased markedly in the brainstem of enriched groups. The non-pharmacological treatment also reduced levels of tissue inhibitor of metalloproteinase 1 in the brainstem of transgenic rats. Expression analysis of inflammatory pathways revealed upregulation of microglial markers, such as MHC class II and Cd74, whereas levels of pro-inflammatory cytokines remained unaffected by enriched environment. Our results demonstrate that exposure to enriched environment can rescue functional impairment in tau transgenic rats without reducing tau pathology. We speculate that non-pharmacological treatment modulates the immune response to pathological tau protein inclusions, and thus reduces the damage caused by neuroinflammation.

Humanized tau antibodies promote tau uptake by human microglia without any increase of inflammation.

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.

Evaluation of a novel immunoassay to detect p-tau Thr217 in the CSF to distinguish Alzheimer disease from other dementias.

OBJECTIVE: To investigate whether tau phosphorylated at Thr217 (p-tau T217) assay in CSF can distinguish patients with Alzheimer disease (AD) from patients with other dementias and healthy controls. METHODS: We developed and validated a novel Simoa immunoassay to detect p-tau T217 in CSF. There was a total of 190 participants from 3 cohorts with AD (n = 77) and other neurodegenerative diseases (n = 69) as well as healthy participants (n = 44). RESULTS: The p-tau T217 assay (cutoff 242 pg/mL) identified patients with AD with accuracy of 90%, with 78% positive predictive value (PPV), 97% negative predictive value (NPV), 93% sensitivity, and 88% specificity, compared favorably with p-tau T181 ELISA (52 pg/mL), showing 78% accuracy, 58% PPV, 98% NPV, 71% specificity, and 97% sensitivity. The assay distinguished patients with AD from age-matched healthy controls (cutoff 163 pg/mL, 98% sensitivity, 93% specificity), similarly to p-tau T181 ELISA (cutoff 60 pg/mL, 96% sensitivity, 86% specificity). In patients with AD, we found a strong correlation between p-tau T217 and p-tau T181, total tau and ß-amyloid 40, but not ß-amyloid 42. CONCLUSIONS: This study demonstrates that p-tau T217 displayed better diagnostic accuracy than p-tau T181. The data suggest that the new p-tau T217 assay has potential as an AD diagnostic test in clinical evaluation. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that a CSF immunoassay for p-tau T217 distinguishes patients with AD from patients with other dementias and healthy controls.

New age of neuroproteomics in Alzheimer's disease research.

Alzheimer's disease (AD) is the leading cause of dementia, a condition that gradually destroys brain cells and leads to progressive decline in mental functions. The disease is characterized by accumulation of misfolded neuronal proteins, amyloid and tau, into insoluble aggregates known as extracellular senile plaques and intracellular neurofibrillary tangles, respectively. However, only tau pathology appears to correlate with the progression of the disease and it is believed to play a central role in the progression of neurodegeneration. In AD, tau protein undergoes various types of posttranslational modifications, most notably hyperphosphorylation and truncation. Using four proteomics approaches we aimed to uncover the key steps leading to neurofibrillary degeneration and thus to identify therapeutic targets for AD. Functional neuroproteomics was employed to generate the first transgenic rat model of AD by expressing a truncated misordered form of tau, "Alzheimer's tau". The rat model showed that Alzheimer's tau toxic gain of function is responsible for the induction of abnormal tau cascade and is the driving force in the development of neurofibrillary degeneration. Structural neuroproteomics allowed us to determine partial 3D structure of the Alzheimer's filament core at a resolution of 1.6 A. Signaling neuroproteomics data lead to the identification and characterization of relevant phosphosites (the tau phosphosignalome) contributing to neurodegeneration. Interaction neuroproteomics revealed links to a new group of proteins interacting with Alzheimer's tau (tau interactome) under normal and pathological conditions, which would provide novel drug targets and novel biomarkers for treatment of AD and other tauopathies.