Blood-brain barrier penetration of non-replicating SARS-CoV-2 and S1 variants of concern induce neuroinflammation which is accentuated in a mouse model of Alzheimer's disease.

COVID-19 and especially Long COVID are associated with severe CNS symptoms and may place persons at risk to develop long-term cognitive impairments. Here, we show that two non-infective models of SARS-CoV-2 can cross the blood-brain barrier (BBB) and induce neuroinflammation, a major mechanism underpinning CNS and cognitive impairments, even in the absence of productive infection. The viral models cross the BBB by the mechanism of adsorptive transcytosis with the sugar N-acetylglucosamine being key. The delta and omicron variants cross the BB B faster than the other variants of concern, with peripheral tissue uptake rates also differing for the variants. Neuroinflammation induced by icv injection of S1 protein was greatly enhanced in young and especially in aged SAMP8 mice, a model of Alzheimer's disease, whereas sex and obesity had little effect.

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.

[Study of local anaesthetics: part 204* determination of critical micelle concentrations of selected derivatives of pyrrolidino-m-alkoxyphenylcarbamic acid using pyrene as a probe]. / Stúdium lokálnych anestetík: cast 204* determinácia kritických micelových koncentrácií vybraných m-alkoxysubstituovaných pyrolidínoetylesterov kyseliny fenylkarbámovej vyuzitím pyrénu ako sondy.

The critical micelle concentrations of the studied derivatives of pyrrolidino-m-alkoxyphenylcarbamic acid in aqueous media at 25 °C were determined using the UV/VIS absorption spectroscopy method with pyrene as a probe. In the absorption spectra of the derivatives and pyrene, there were examined unmasked pyrene peaks, at wavelength of 336 and 320 nm for VIII Z, XIV Z and XVII Z, and in addition at 308 nm for XXIX Z. The critical micelle concentrations were defined from the plots of the sum of the unmasked pyrene peaks against the surfactant concentration that had a sigmoidal character of Boltzmann type. The cmc values of the derivatives were exponentially dependent on the number of carbons in the hydrophobic chain. From the plot of ln (cmc) against the number of carbons n with the equation ln (cmc) = -0.146-0.691n, the value of the Gibbs free energy of transfer of each methylene group of the alkoxychain from the aqueous phase into the internal hydrophobic volume of micelle was also defined: δΔG(CH(2)) = (-0.691 ± 0.023)RT. Building on the obtained results, the spherical micelle forming in the solution can be assumed.

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.

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.

Fundamental and Advanced Therapies, Vaccine Development against SARS-CoV-2.

Coronavirus disease (COVID-19) caused by the SARS-CoV-2 virus has been affecting the world since the end of 2019. The severity of the disease can range from an asymptomatic or mild course to acute respiratory distress syndrome (ARDS) with respiratory failure, which may lead to death. Since the outbreak of the pandemic, scientists around the world have been studying the genome and molecular mechanisms of SARS-CoV-2 infection to develop effective therapies and prevention. In this review, we summarize the progressive development of various treatments and vaccines as they have emerged, a year after the outbreak of the pandemic. Initially for COVID-19, patients were recommended drugs with presumed antiviral, anti-inflammatory, and antimicrobial effects that were previously used to treat other diseases. Thereafter, therapeutic interventions were supplemented with promising approaches based on antibodies, peptides, and stem cells. However, licensed COVID-19 vaccines remain the most effective weapon in combating the pandemic. While there is an enormous effort to enhance the vaccination rate to increase the entire population immunity, the production and delivery of vaccines is becoming limited in several countries. In this regard, there are new challenges needing to be addressed by combining non-pharmacological intervention with effective therapies until vaccination is accessible to all.

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.

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.

Chaperone-like antibodies in neurodegenerative tauopathies: implication for immunotherapy.

Alzheimer's disease (AD) belongs to the category of neurodegenerative tauopathies, which are characterized by intracellular and extracellular accumulation of misfolded tau. Structurally, tau belongs to the family of the intrinsically disordered proteins that are characterized by the absence of well-defined three-dimensional structure of the free protein. In the course of neurodegeneration, intrinsically disordered tau protein gains highly ordered misfolded structure. Currently it is widely accepted that misfolded tau proteins represent viable drug target for prospective therapeutic development. Until now several therapeutic approaches targeting misfolded tau were developed. Monoclonal antibodies with chaperone-like activities that would be able to neutralize the toxic gain of function of misfolded tau represent novel promising immunological concept in the treatment of AD. We suggest that antibodies as specific chaperones targeting misfolded proteins may serve as potent therapeutic drugs of AD as well as others conformational diseases.

Structure solution of misfolded conformations adopted by intrinsically disordered Alzheimer's tau protein.

Until now it was impossible to obtain atomic structure of intrinsically disordered protein (IDP) tau and/or its assembly in Alzheimer's paired helical filaments as neither of them could have been prepared in the form amenable to X-ray or NMR techniques. Using IDP tau property to attain regular tertiary structure after binding events during self-assembly or when complexed with its target we propose monoclonal antibodies as surrogate tau protein binding partners to form complexes and crystals for structure solution by X-ray technique.

A walk through tau therapeutic strategies.

Tau neuronal and glial pathologies drive the clinical presentation of Alzheimer's disease and related human tauopathies. There is a growing body of evidence indicating that pathological tau species can travel from cell to cell and spread the pathology through the brain. Throughout the last decade, physiological and pathological tau have become attractive targets for AD therapies. Several therapeutic approaches have been proposed, including the inhibition of protein kinases or protein-3-O-(N-acetyl-beta-D-glucosaminyl)-L-serine/threonine Nacetylglucosaminyl hydrolase, the inhibition of tau aggregation, active and passive immunotherapies, and tau silencing by antisense oligonucleotides. New tau therapeutics, across the board, have demonstrated the ability to prevent or reduce tau lesions and improve either cognitive or motor impairment in a variety of animal models developing neurofibrillary pathology. The most advanced strategy for the treatment of human tauopathies remains immunotherapy, which has already reached the clinical stage of drug development. Tau vaccines or humanised antibodies target a variety of tau species either in the intracellular or extracellular spaces. Some of them recognise the amino-terminus or carboxy-terminus, while others display binding abilities to the proline-rich area or microtubule binding domains. The main therapeutic foci in existing clinical trials are on Alzheimer's disease, progressive supranuclear palsy and non-fluent primary progressive aphasia. Tau therapy offers a new hope for the treatment of many fatal brain disorders. First efficacy data from clinical trials will be available by the end of this decade.

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.

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

The fetal type of tau phosphorylation always re-appears during pathogenesis of Alzheimer's 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 Alzheimer's disease and related tauopathies.

Chaperone-like antibodies targeting misfolded tau protein: new vistas in the immunotherapy of neurodegenerative foldopathies.

Neurodegenerative foldopathies are characterized by aberrant folding of proteins leading to the intracellular and extracellular accumulation of insoluble misfolded proteins. One of the most prominent protein folding disorders is Alzheimer's disease (AD). In AD, there were identified two major driving forces behind neurodegeneration, misfolded proteins tau and amyloid-beta. Tau belongs to a family of intrinsically disordered proteins that are characterized by the absence of a rigid three-dimensional structure in their natural environment. However, in disease condition, tau truncation and hyperphosphorylation could lead to tau transformation from intrinsically disordered protein into highly ordered soluble and insoluble misfolded structures. Increased understanding of the molecular mechanism underlying pathological transformation of tau protein has opened up the possibility of targeting misfolded tau for therapeutic purposes. Pharmacological research has identified several therapeutic approaches targeting directly or indirectly tau cascade. Novel promising field of AD treatment represent monoclonal antibodies with chaperon like activities that will be able to neutralize the toxic gain of function of misfolded tau and thus increase its degradation. We suggest that chaperon like antibodies targeting disease modified tau may hold promise for the successful treatment of AD and related foldopathies.

Ten Years of Tau-Targeted Immunotherapy: The Path Walked and the Roads Ahead.

Neurofibrillary pathology comprised of pathological tau protein is closely tied to a range of neurodegenerative disorders, the most common of which is Alzheimer's disease. While they are individually rarer, a range of other disorders, the tauopathies (including Pick's disease, progressive supranuclear palsy, corticobasal degeneration, primary progressive aphasia, and ∼50% of behavioral variant frontotemporal dementia cases) display pronounced underlying tau pathology. In all cases, the distribution and amount of tau pathology closely correlates with the severity and phenotype of cognitive impairment, and with the pattern and degree of brain atrophy. Successfully counteracting tau pathology is likely to halt or slow the progression of these debilitating disorders. This makes tau a target of prime importance, yet an elusive one. The diversity of the tau proteome and post-translational modifications, as well as pathophysiology of tau are reviewed. Beginning 2013, a range of tau-targeted immunotherapies have entered clinical development; these therapies, and their common themes and differences are reviewed. The manuscript provides an extensive discussion on epitope selection for immunotherapies against tau pathology, on immunological mechanisms involved in their action, and challenges such as immune senescence, vaccine design, or evolution of epitopes. Furthermore, we provide methodological recommendations for the characterization of active vaccines and antibodies, animal models, and the target itself - the diseased tau proteome.

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.

A novel monoclonal antibody DC63 reveals that inhibitor 1 of protein phosphatase 2A is preferentially nuclearly localised in human brain.

Abnormal phosphorylation of tau protein represents one of the major candidate pathological mechanisms leading to Alzheimer's disease (AD) and related tauopathies. Altered phosphorylation status of neuronal tau protein may result from upregulation of tau-specific kinases or from inhibition of tau-specific phosphatases. Increased expression of the protein inhibitor 1 of protein phosphatase 2A (I1PP2A) could therefore indirectly regulate the phosphorylation status of tau. As an important step towards elucidation of the role of I1PP2A in the physiology and pathology of tau phosphorylation, we developed a novel monoclonal antibody, DC63, which recognizes I1PP2A. Specificity of the antibody was examined by mass spectrometry and Western blot. This analysis supports the conclusion that the antibody does not recognize any of the other proteins of the 9-member leucine-rich acidic nuclear phosphoprotein family to which I1PP2A belongs. Immunoblot detection revealed that the inhibitor I1PP2A is expressed throughout the brain, including the hippocampus, temporal cortex, parietal cortex, subcortical nuclei and brain stem. The cerebellum displayed significantly higher levels of expression of I1PP2A than was seen elsewhere in the brain. Imunohistochemical analysis of normal human brain showed that I1PP2A is expressed in both neurons and glial cells and that the protein is preferentially localized to the nucleus. We conclude that the novel monoclonal antibody DC63 could be successfully employed as a mass spectrometry-validated molecular probe that may be used for in vitro and in vivo qualitative and quantitative studies of physiological and pathological pathways involving I1PP2A.

Neuronal Expression of Truncated Tau Efficiently Promotes Neurodegeneration in Animal Models: Pitfalls of Toxic Oligomer Analysis.

Animal models of neurodegeneration induced by neuronal expression of truncated tau protein emerge as an important tool for understanding the pathogenesis of human tauopathies and for therapy development. Here we highlight common features of truncated tau models and make a critical assessment of possible pitfalls in their analysis. Particularly, the amount of soluble tau oligomers, which are suspected to be neurotoxic agents participating on the spreading of pathology inside the brain, may be overestimated due to a post-lysis oxidative tau oligomerization. Using a mouse brain lysate spiked with recombinant truncated and full length tau forms, we show that tau oligomers might inadvertently be produced during the isolation procedure. This finding is further corroborated by the analysis of brain lysates originated from a mouse model expressing truncated tau variant. Our results underline the necessity of thiol-protecting conditions during the analysis of tau oligomers involved in the etiopathogenesis of various tauopathies including Alzheimer's disease.