Changes in lipid metabolism track with the progression of neurofibrillary pathology in tauopathies.

BACKGROUND: Accumulation of tau leads to neuroinflammation and neuronal cell death in tauopathies, including Alzheimer's disease. As the disease progresses, there is a decline in brain energy metabolism. However, the role of tau protein in regulating lipid metabolism remains less characterized and poorly understood. METHODS: We used a transgenic rat model for tauopathy to reveal metabolic alterations induced by neurofibrillary pathology. Transgenic rats express a tau fragment truncated at the N- and C-terminals. For phenotypic profiling, we performed targeted metabolomic and lipidomic analysis of brain tissue, CSF, and plasma, based on the LC-MS platform. To monitor disease progression, we employed samples from transgenic and control rats aged 4, 6, 8, 10, 12, and 14 months. To study neuron-glia interplay in lipidome changes induced by pathological tau we used well well-established multicomponent cell model system. Univariate and multivariate statistical approaches were used for data evaluation. RESULTS: We showed that tau has an important role in the deregulation of lipid metabolism. In the lipidomic study, pathological tau was associated with higher production of lipids participating in protein fibrillization, membrane reorganization, and inflammation. Interestingly, significant changes have been found in the early stages of tauopathy before the formation of high-molecular-weight tau aggregates and neurofibrillary pathology. Increased secretion of pathological tau protein in vivo and in vitro induced upregulated production of phospholipids and sphingolipids and accumulation of lipid droplets in microglia. We also found that this process depended on the amount of extracellular tau. During the later stages of tauopathy, we found a connection between the transition of tau into an insoluble fraction and changes in brain metabolism. CONCLUSION: Our results revealed that lipid metabolism is significantly affected during different stages of tau pathology. Thus, our results demonstrate that the dysregulation of lipid composition by pathological tau disrupts the microenvironment, further contributing to the propagation of pathology.

Plasma Leptin Reflects Progression of Neurofibrillary Pathology in Animal Model of Tauopathy.

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

Hypertension does not alter disturbances in leptin signalling observed in experimental model of tauopathy.

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

A Novel RP-UHPLC-MS/MS Approach for the Determination of Tryptophan Metabolites Derivatized with 2-Bromo-4'-Nitroacetophenone.

Many biologically active metabolites of the essential amino acid L-tryptophan (Trp) are associated with different neurodegenerative diseases and neurological disorders. Precise and reliable methods for their determination are needed. Variability in their physicochemical properties makes the analytical process challenging. In this case, chemical modification of analyte derivatization could come into play. Here, we introduce a novel fast reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) coupled with tandem mass spectrometry (MS/MS) method for the determination of Trp and its ten metabolites in human plasma samples after derivatization with 2-bromo-4'-nitroacetophenone (BNAP). The derivatization procedure was optimized in terms of incubation time, temperature, concentration, and volume of the derivatization reagent. Method development comprises a choice of a suitable stationary phase, mobile phase composition, and gradient elution optimization. The developed method was validated according to the ICH guidelines. Results of all validation parameters were within the acceptance criteria of the guideline, i.e., intra- and inter-day precision (expressed as relative standard deviation; RSD) were in the range of 0.5-8.2% and 2.3-7.4%, accuracy was in the range of 93.3-109.7% and 94.7-110.1%, limits of detection (LODs) were in the range of 0.15-9.43 ng/mL, coefficients of determination (R2) were higher than 0.9906, and carryovers were, in all cases, less than 8.8%. The practicability of the method was evaluated using the blue applicability grade index (BAGI) with a score of 65. Finally, the developed method was used for the analysis of Alzheimer's disease and healthy control plasma to prove its applicability. Statistical analysis revealed significant changes in picolinic acid (PA), anthranilic acid (AA), 5 hydroxyindole-3-acetic acid (5-OH IAA), and quinolinic acid (QA) concentration levels. This could serve as the basis for future studies that will be conducted with a large cohort of patients.

Cost-Effective Protein Production in CHO Cells Following Polyethylenimine-Mediated Gene Delivery Showcased by the Production and Crystallization of Antibody Fabs.

Laboratory production of recombinant mammalian proteins, particularly antibodies, requires an expression pipeline assuring sufficient yield and correct folding with appropriate posttranslational modifications. Transient gene expression (TGE) in the suspension-adapted Chinese Hamster Ovary (CHO) cell lines has become the method of choice for this task. The antibodies can be secreted into the media, which facilitates subsequent purification, and can be glycosylated. However, in general, protein production in CHO cells is expensive and may provide variable outcomes, namely in laboratories without previous experience. While achievable yields may be influenced by the nucleotide sequence, there are other aspects of the process which offer space for optimization, like gene delivery method, cultivation process or expression plasmid design. Polyethylenimine (PEI)-mediated gene delivery is frequently employed as a low-cost alternative to liposome-based methods. In this work, we are proposing a TGE platform for universal medium-scale production of antibodies and other proteins in CHO cells, with a novel expression vector allowing fast and flexible cloning of new genes and secretion of translated proteins. The production cost has been further reduced using recyclable labware. Nine days after transfection, we routinely obtain milligrams of antibody Fabs or human lactoferrin in a 25 mL culture volume. Potential of the platform is established based on the production and crystallization of antibody Fabs and their complexes.

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.

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.

Therapeutic antibody targeting microtubule-binding domain prevents neuronal internalization of extracellular tau via masking neuron surface proteoglycans.

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

FUNDAMANT: an interventional 72-week phase 1 follow-up study of AADvac1, an active immunotherapy against tau protein pathology in Alzheimer's disease.

BACKGROUND: Neurofibrillary pathology composed of tau protein is closely correlated with severity and phenotype of cognitive impairment in patients with Alzheimer's disease and non-Alzheimer's tauopathies. Targeting pathological tau proteins via immunotherapy is a promising strategy for disease-modifying treatment of Alzheimer's disease. Previously, we reported a 24-week phase 1 trial on the active vaccine AADvac1 against pathological tau protein; here, we present the results of a further 72 weeks of follow-up on those patients. METHODS: We did a phase 1, 72-week, open-label study of AADvac1 in patients with mild to moderate Alzheimer's disease who had completed the preceding phase 1 study. Patients who were previously treated with six doses of AADvac1 at monthly intervals received two booster doses at 24-week intervals. Patients who were previously treated with only three doses received another three doses at monthly intervals, and subsequently two boosters at 24-week intervals. The primary objective was the assessment of long-term safety of AADvac1 treatment. Secondary objectives included assessment of antibody titres, antibody isotype profile, capacity of the antibodies to bind to AD tau and AADvac1, development of titres of AADvac1-induced antibodies over time, and effect of booster doses; cognitive assessment via 11-item Alzheimer's Disease Assessment Scale cognitive assessment (ADAS-Cog), Category Fluency Test and Controlled Oral Word Association Test; assessment of brain atrophy via magnetic resonance imaging (MRI) volumetry; and assessment of lymphocyte populations via flow cytometry. RESULTS: The study was conducted between 18 March 2014 and 10 August 2016. Twenty-six patients who completed the previous study were enrolled. Five patients withdrew because of adverse events. One patient was withdrawn owing to noncompliance. The most common adverse events were injection site reactions (reported in 13 [50%] of vaccinated patients). No cases of meningoencephalitis or vasogenic oedema were observed. New micro-haemorrhages were observed only in one ApoE4 homozygote. All responders retained an immunoglobulin G (IgG) antibody response against the tau peptide component of AADvac1 over 6 months without administration, with titres regressing to a median 15.8% of titres attained after the initial six-dose vaccination regimen. Booster doses restored previous IgG levels. Hippocampal atrophy rate was lower in patients with high IgG levels; a similar relationship was observed in cognitive assessment. CONCLUSIONS: AADvac1 displayed a benign safety profile. The evolution of IgG titres over vaccination-free periods warrants a more frequent booster dose regimen. The tendency towards slower atrophy in MRI evaluation and less of a decline in cognitive assessment in patients with high titres is encouraging. Further trials are required to expand the safety database and to establish proof of clinical efficacy of AADvac1. TRIAL REGISTRATION: The studies are registered with the EU Clinical Trials Register and ClinicalTrials.gov : the preceding first-in-human study under EudraCT 2012-003916-29 and NCT01850238 (registered on 9 May 2013) and the follow-up study under EudraCT 2013-004499-36 and NCT02031198 (registered 9 Jan 2014), respectively.

Safety and immunogenicity of the tau vaccine AADvac1 in patients with Alzheimer's disease: a randomised, double-blind, placebo-controlled, phase 1 trial.

BACKGROUND: Neurofibrillary pathology composed of tau protein is a main correlate of cognitive impairment in patients with Alzheimer's disease. Immunotherapy targeting pathological tau proteins is therefore a promising strategy for disease-modifying treatment of Alzheimer's disease. We have developed an active vaccine, AADvac1, against pathological tau proteins and assessed it in a phase 1 trial. METHODS: We did a first-in-man, phase 1, 12 week, randomised, double-blind, placebo-controlled study of AADvac1 with a 12 week open-label extension in patients aged 50-85 years with mild-to-moderate Alzheimer's disease at four centres in Austria. We randomly assigned patients with a computer-generated sequence in a 4:1 ratio overall to receive AADvac1 or placebo. They received three subcutaneous doses of AADvac1 or placebo from masked vaccine kits at monthly intervals, and then entered the open-label phase, in which all patients were allocated to AADvac1 treatment and received another three doses at monthly intervals. Patients, carers, and all involved with the trial were masked to treatment allocation. The primary endpoint was all-cause treatment-emergent adverse events, with separate analyses for injection site reactions and other adverse events. We include all patients who received at least one dose of AADvac1 in the safety assessment. Patients who had a positive IgG titre against the tau peptide component of AADvac1 at least once during the study were classified as responders. The first-in-man study is registered with EU Clinical Trials Register, number EudraCT 2012-003916-29, and ClinicalTrials.gov, number NCT01850238; the follow-up study, which is ongoing, is registered with EU Clinical Trials Register, number EudraCT 2013-004499-36, and ClinicalTrials.gov, number NCT02031198. FINDINGS: This study was done between June 9, 2013, and March 26, 2015. 30 patients were randomly assigned in the double-blind phase: 24 patients to the AADvac1 group and six to the placebo group. A total of 30 patients received AADvac1. Two patients withdrew because of serious adverse events. The most common adverse events were injection site reactions after administration (reported in 16 [53%] vaccinated patients [92 individual events]). No cases of meningoencephalitis or vasogenic oedema occurred after administration. One patient with pre-existing microhaemorrhages had newly occurring microhaemorrhages. Of 30 patients given AADvac1, 29 developed an IgG immune response. A geometric mean IgG antibody titre of 1:31415 was achieved. Baseline values of CD3+ CD4+ lymphocytes correlated with achieved antibody titres. INTERPRETATION: AADvac1 had a favourable safety profile and excellent immunogenicity in this first-in-man study. Further trials are needed to corroborate the safety assessment and to establish proof of clinical efficacy of AADvac1. FUNDING: AXON Neuroscience SE.

Truncated tau from sporadic Alzheimer's disease suffices to drive neurofibrillary degeneration in vivo.

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