Tau Antibodies Research Articles

Tau conformational changes in aging‐related tau astrogliopathy.

AbstractBackgroundThe presence of aging‐related tau astrogliopathy (ARTAG) is highly frequent, but poorly characterized in aging brain and neurodegeneration. Over the abnormal tau accumulation processes, tau protein undergoes pathological modifications and the sequency of these pathological events can be associated with different levels of tau aggregation.MethodTo evaluate the frequency of ARTAG in a large population‐based study, we analyzed brain sections immunostained with phosphor tau (AT8) of 1151 individuals with previous neuropathological evaluation. Further, to investigate early stages of tau protein aggregation in ARTAG, we performed additional immunohistochemistry in the amygdala of 32 cases with ARTAG pathology using the following tau antibodies: AT100 and Tau‐C3.ResultARTAG was frequently observed in cognitively normal individuals independent of concomitant neurodegenerative disease. Higher burden of Alzheimer‐type pathology was observed in ARTAG compared to non‐ARTAG. Also, presence of more than one pathology (mixed pathology) was higher in ARTAG compared to non‐ARTAG (p<0.001). Additionally, AT100 immunoreactive ARTAG was found in overall cases, while Tau‐C3 immunoreactive ARTAG was not observed in 12 cases (36%), showing different mature stage for ARTAG compared to neurofibrillary tangles.ConclusionDifferent immunological markers can be found in ARTAG and Alzheimer’s neurofibrillary pathology in early stages of tau pathological processing, suggesting different levels of abnormal tau aggregates among cases with ARTAG pathology.

In vivo neuronal deficits and microglia structural dynamics in the course of tau pathology and during antibody treatment

AbstractBackgroundWe crossed mouse models Thy‐1GCaMP6, Cx3cr1CreER:tdTomatoflox and Cx3cr1GFP with PS19 P301S tauopathy mice to visualize neuronal calcium and microglia structural dynamics in vivo during the progression of tau pathology and after tau immunotherapy.MethodBy using two‐photon live imaging in head‐restrained mice attached to a custom‐made, free‐floating treadmill, we examined calcium activity in L2/3 pyramidal neurons and microglia structural dynamics in the motor cortex during tau pathology progression and after two intravenous antibody injections targeting the truncated Asp421 epitope of tau.ResultL2/3 pyramidal neurons from young Thy‐1GCaMP6:wild‐type mice (1‐6 months old) increased their somatic activity during running periods as expected, but their littermate Thy‐1GCaMP6:PS19+/− mice did not. Importantly, acute tau immunotherapy restored neuronal function after two intravenous antibody injections (100 µg each) in Thy‐1GCaMP6:PS19+/− mice (5‐6 months old), whereas control antibody had no effect. Although tau pathology is evident in the cortex and hippocampus in 2‐3 months old Cx3cr1GFP/+:PS19+/− mice and Cx3cr1CreER/+:tdTomatoflox:PS19+/− mice, their microglia morphology did not differ from control littermate Cx3cr1GFP/+:wild‐type mice.ConclusionOur current findings indicate that functional deficits in cortical neurons associated with tau pathology start early in PS19 mice and can be alleviated by two acute tau antibody injections. Also, those neuronal functional impairments can be detected without accompanying structural changes in microglia, which suggests that neuronal deficits linked to tau accumulation precede microglial activation. Studies on the potential influence of tau antibody treatment on microglia morphology in vivo are underway.

Prophylactic effect of passive immunotherapy with tau antibody 43D in 3xTg‐AD mice

AbstractBackgroundAccumulation of abnormally hyperphosphorylated tau protein is a histopathological hallmark of Alzheimer’s disease (AD) and related tauopathies. Immunotherapy targeting tau represents a promising disease‐modifying therapeutic strategy for treating AD. We have found that immunization with monoclonal 43D antibody against tau 6‐18 weekly for 6 weeks can inhibit the seeding and spread of tau pathology templated by AD hyperphosphorylated tau, reduce both tau and Aβ pathologies and rescue cognitive impairment in 3xTg‐AD mice at mild to moderate stage of the disease. In this study, we investigated whether tau immunization with 43D prior to the onset of AD‐like pathologies can reduce AD‐like pathologies and improve cognitive function in 3xTg‐AD mice.MethodsWe immunized 3‐ to 4‐month‐old female 3×Tg‐AD mice with nine monthly doses of 15 µg of mouse monoclonal antibody 43D or, as a control, of mouse immunoglobulin G (IgG) intravenously. Age‐matched wild‐type mice treated with mouse IgG were also used as controls. The effect of immunization on cognition was analyzed by using one‐trial novel object recognition test and Morris water maze test, and the effect on tau pathology was assessed by Western blot.ResultsWe found that 3xTg‐AD mice exhibited cognitive impairment as compared to the WT mice, as determined by novel object recognition test and Morris water maze test, and that immunization with 43D antibody improved the cognitive function seen in both tests. Immunization with 43D also decreased tau pathologies in the 3xTg‐AD mice. Additionally, long‐term treatment with 43D antibody did not display side events in 3xTg‐AD mice.ConclusionsThese findings suggest that passive immunization with 43D antibody targeting proximal N‐terminal domain tau 6‐18 may be as both prophylactic and therapeutic approaches for treating AD and related tauopathies.

Brain‐Penetrable Antibody Probes for in vivo Tau Imaging

AbstractBackgroundAntibody‐based PET ligands are desirable due to their high specificity; however, their brain uptakes are limited by the blood‐brain barrier (BBB). We previously demonstrated that transport of antibody across BBB can be facilitated through interaction with the transferrin receptor (TfR)1. We report here the first in vivo PET imaging studies using BBB permeable F‐18 radiolabeled bispecific antibodies specific to tau protein in mice.MethodWe performed multiple PET scans using each of the radio‐probes synthesized via conjugation of [18F]SFB with one of the four bispecific antibody constructs: 1) 6B2G12‐ScFv8D3 (full‐size Tau antibody conjugated with TfR fragment (ScFv8D3); abbreviated as TAUb), 2) ScFv235‐ScFv8D3 (small Tau‐TfR; TAUs), 3) 3D6‐ScFv8D3 (full‐size Aβ‐TfR; Aβb) or 4) ScFv3D6‐ScFv3D6 (small Aβ‐TfR; Aβs). We used a matched control design to compare a WT with one of the two types of transgenic (tg) tauopathy mice [PS19 (P301S) or JNPL3 (P301)] in each scan. Subjects were scanned on a MOLECUBES PET/CT scanner. An initial low‐dose (244 ± 54 μCi) dynamic scan (30 min) was performed, followed by two scans at 8hr and 12hr (20 min each static scans) after injection of a high dose (1832 ± 447 μCi) of the same probe. PMOD and FireVoxel were used for imaging processing. After co‐registration of PET/CT/atlas, data were quantified as SUV and SUVR [CB as a reference], and clearance was estimated as %change/hr.ResultTAUs probe displayed highest brain uptake with more specific binding retained in the brain, as compared to TAUb and Aβs. TAUs probe showed faster brain penetration in tau‐tg mice than in WT, with differences in amygdala, basal forebrain, and hypothalamus of 66%, 62% and 85%, respectively, at 8hr post‐injection (Fig. 1). Further, higher brain uptakes (TAUs > Aβs) in the same tau‐tg mouse (Fig. 2) suggest the specific binding of TAUs probe, while Aβs probe can serve as a control due to its lack of specific binding in tau‐tg mice.ConclusionWe have successfully synthesized and evaluated four novel F‐18 bispecific antibodies via in vivo PET. TAUs probe showed most promising BBB permeability and binding specificity to tau antigen with a reasonable clearance.

Tangle and neuritic burden from neurofibrillary tangle maturity markers in the hippocampus are differentially correlated to tau‐PET, MRI, and cognitive measures

AbstractBackgroundThere is a gap in knowledge concerning which tangle maturity levels (Moloney, Alzheimers Dement, 2021) are associated with antemortem measures (tau‐PET, MRI, cognitive testing). We hypothesized antemortem measures would be more strongly correlated with markers recognizing middling to advanced tangle maturity. We additionally sought to assess the strength of the relationship with tangle and neuritic pathology recognized by tangle maturity markers.MethodPosterior hippocampi from n = 30 cases with antemortem measures ≤3 years prior to death were cut (5µm) and immunostained with tau antibodies to evaluate early (AT8), middling (PHF‐1), or advanced (2E9) tangle maturity. GENIE (pattern recognition software) separated tangle from neuritic burden (Figure). Hippocampus, parahippocampus, and fusiform were analyzed to correlate digital pathology with flortaucipir tau‐PET standard uptake value ratio (SUVr, cerebellar crus normalization), MRI volume/thickness, and cognitive measures which included Auditory Verbal Learning Test (AVLT) delayed recall, Mini‐Mental State Exam (MMSE), and Clinical Dementia Rating (CDR).ResultThe strongest relationships observed with flortaucipir were with PHF‐1 and 2E9 burden (Table). AT8 tangle and neuritic burden correlations with flortaucipir were relatively similar in the hippocampus but were stronger with neuritic pathology in parahippocampus and fusiform. PHF‐1 tangle and neuritic burden correlations were relatively similar with flortaucipir. 2E9 correlations were stronger with neuritic pathology in hippocampus. MRI measures were more strongly correlated with PHF‐1 and 2E9 burden. AT8 tangle and neuritic burden were correlated with fusiform thickness. The correlations between MRI measures and 2E9 tangle and neuritic burden were similar. Correlations between cognitive measures were similar between tangle maturity markers. AVLT delayed recall correlated with all tangle maturity markers, but neuritic burden was stronger for AT8 and 2E9 in hippocampus compared to tangle burden. MMSE correlated with neuritic burden for all tangle maturity markers. CDR remained statistically non‐significant.ConclusionInterpretation of the strength of the relationship between tau burden and antemortem measures differed across tangle maturity markers. Neuroimaging measures may be more sensitive to middle/advanced maturity markers with limited differences between tangle and neuritic pathology. These findings may be biologically relevant and imply either a limited recognition of tangles depending on antibody epitope, and/or may represent the clinicoradiologic impact of increasing neuritic burden.

Immunohistochemical detection of amyloid beta and tau proteins in the Monodelphis domestica brain

AbstractBackgroundAnimal models have been important for the understanding of Alzheimer’s disease and related dementias (ADRD). However, most of the research involving these diseases has been with rodents. Researchers have raised awareness about the need for alternative and feasible animal models in order to understand these types of diseases. To increase the knowledge of alternative animal models for the study of ADRD, the current study proposed the use of the Monodelphis domestica animal model to detect the presence of amyloid beta and tau proteins.MethodFixed brains from young adult Monodelphis were used in the present study. A standard immunohistochemistry (ABC‐DAB) staining protocol was used to detect amyloid beta and tau proteins. To detect the presence of amyloid beta, a 1:100 anti‐amyloid primary dilution was used; for tau protein, a 1:1000 anti‐tau primary dilution was utilized. Only the hippocampal region was selected for description and quantification. An Axioscan Z.1 microscope system was utilized to capture images, and image analysis was performed using ImageJ software.ResultThe area of the hippocampus of the first coronal section was 30262602.934 um2. The results from an amyloid‐beta dilution of 1:100 detected amyloid beta in a number of neurons in the hippocampus. Amyloid beta was also detected in the hippocampus of another brain section (total area: 7048008 um2). The negative control tissue did not reveal positive signal in neurons in the hippocampal area, suggesting low levels of non‐specific binding. The results for tau antibody are pending.ConclusionOur study revealed that in Monodelphis domestica animal model brain tissue, a 1:100 dilution resulted in identification of positive amyloid beta protein. However, no abnormal amyloid beta plaque formation was found with this animal model. A possible explanation was that a natural, non‐induced animal model was used in the study.

Novel Conformation-Dependent Tau Antibodies Are Modulated by Adjacent Phosphorylation Sites.

Tau proteins within the adult central nervous system (CNS) are found to be abnormally aggregated into heterogeneous filaments in neurodegenerative diseases, termed tauopathies. These tau inclusions are pathological hallmarks of Alzheimer's disease (AD), Pick's disease (PiD), corticobasal degeneration (CBD), and progressive supranuclear palsy (PSP). The neuropathological hallmarks of these diseases burden several cell types within the CNS, and have also been shown to be abundantly phosphorylated. The mechanism(s) by which tau aggregates in the CNS is not fully known, but it is hypothesized that hyperphosphorylated tau may precede and further promote filament formation, leading to the production of these pathological inclusions. In the studies herein, we generated and thoroughly characterized two novel conformation-dependent tau monoclonal antibodies that bind to residues Pro218-Glu222, but are sensitive to denaturing conditions and highly modulated by adjacent downstream phosphorylation sites. These epitopes are present in the neuropathological hallmarks of several tauopathies, including AD, PiD, CBD, and PSP. These novel antibodies will further enable investigation of tau-dependent pathological inclusion formation and enhance our understanding of the phosphorylation signatures within tauopathies with the possibility of new biomarker developments.

Analysis of clinical failure of anti-tau and anti-synuclein antibodies in neurodegeneration using a quantitative systems pharmacology model

Misfolded proteins in Alzheimer’s disease and Parkinson’s disease follow a well-defined connectomics-based spatial progression. Several anti-tau and anti-alpha synuclein (aSyn) antibodies have failed to provide clinical benefit in clinical trials despite substantial target engagement in the experimentally accessible cerebrospinal fluid (CSF). The proposed mechanism of action is reducing neuronal uptake of oligomeric protein from the synaptic cleft. We built a quantitative systems pharmacology (QSP) model to quantitatively simulate intrasynaptic secretion, diffusion and antibody capture in the synaptic cleft, postsynaptic membrane binding and internalization of monomeric and oligomeric tau and aSyn proteins. Integration with a physiologically based pharmacokinetic (PBPK) model allowed us to simulate clinical trials of anti-tau antibodies gosuranemab, tilavonemab, semorinemab, and anti-aSyn antibodies cinpanemab and prasineuzumab. Maximal target engagement for monomeric tau was simulated as 45% (semorinemab) to 99% (gosuranemab) in CSF, 30% to 99% in ISF but only 1% to 3% in the synaptic cleft, leading to a reduction of less than 1% in uptake of oligomeric tau. Simulations for prasineuzumab and cinpanemab suggest target engagement of free monomeric aSyn of only 6–8% in CSF, 4–6% and 1–2% in the ISF and synaptic cleft, while maximal target engagement of aggregated aSyn was predicted to reach 99% and 80% in the synaptic cleft with similar effects on neuronal uptake. The study generates optimal values of selectivity, sensitivity and PK profiles for antibodies. The study identifies a gradient of decreasing target engagement from CSF to the synaptic cleft as a key driver of efficacy, quantitatively identifies various improvements for drug design and emphasizes the need for QSP modelling to support the development of tau and aSyn antibodies.

Degradation of Oligomer Tau by Autophagy

AbstractBackgroundThe specific pathological findings in Alzheimer’s disease (AD) brain are senile plaques composed of amyloid‐β protein (Aβ) and neurofibrillary tangles (NFTs) composed of highly phosphorylated tau protein. It has been suggested that autophagy may play an important role in the degradation of tau proteins. In this study, we investigated whether autophagy is involved in the degradation of oligomeric tau by immunohistochemistry using autopsied AD brains.MethodFrozen brains were homogenized to produce lysates, and Western blotting (WB) was performed using anti‐LC3 antibody, a marker of autophagy. The parietal lobes of the same autopsy brains were used for immunohistochemical analysis using anti‐LC3, anti‐P62, anti‐oligomeric tau antibody (TOC‐1) and anti‐phosphorylated tau antibody (PHF‐1).ResultIn the WB of the temporal lobe, LC3‐II was generally increased in AD brains compared to controls. In addition, immunostaining showed that oligomeric tau (TOC1) and LC3 coexisted in AD brains. In AD and control brains, phosphorylated tau, which stained faintly, was found to coexist with LC3, but LC3 was not found in the cytoplasm of neurons with serious phosphorylated tau (PHF‐1) deposition. However, the coexistence of highly phosphorylated tau and p62 was remarkable. No LC3 or P62 staining was observed in the neuropil thread.ConclusionThese results suggest that oligomeric tau is degraded by autophagy and phosphorylated tau is also degraded by autophagy, but autophagy may no longer function when the degree of phosphorylated tau deposition is serious.

Learning from anti‐tau antibody clinical trials using computer simulation of spatial tau propagation

AbstractBackgroundBased on the tau spreading hypothesis, anti‐tau antibodies have been developed that can bind to and eliminate the misfolded tau protein during the short extracellular period. However, trials with tau antibodies have come up short in improving clinical outcome in Progressive Supranuclear Palsy (PSP) or Alzheimer’s Disease (AD). It is unclear whether the negative outcomes were due to insufficient target engagement and to what extent the change of tau CSF biomarkers reflected the brain pharmacodynamics.MethodA Quantitative Systems Pharmacology (QSP) computer platform of tau biology has been developed including tau species diversity and their respective concentration, neuronal activity‐dependent tau secretion, tau binding to membrane surface receptors HSPG and LRP1, tau‐antibody binding and subsequent internalization of tau at the synaptic cleft. This model is combined with a Physiology‐Based PharmacoKinetic (PBPK) model to derive brain target exposure of tau antibodies. In addition, a PBPK model is used to simulate the level of plasma and CSF biomarkers.ResultThe platform was calibrated with preclinical in‐vitro cellular assays and after injection of brain extract in Transgene mouse models and was further humanized using published data on seed‐competent tau from postmortem AD brain at different Braak stages. Combining the extracellular antibody exposure, the spatial dimensions and the dynamics of tau secretion, diffusion and binding to the membrane surface receptors, the model suggests that a very limited fraction (10‐20%) of misfolded tau protein can be captured by antibodies and cleared. Even less reduction (5‐10%) is predicted for Progressive Supranuclear Palsy due to the higher affinity of PSP‐4R tau for membrane surface receptors. The reductions in misfolded tau protein are greatest in earlier stages of the disease. With regard to epitope selection, we identify conditions where high specificity (i.e. monomeric vs misfolded tau) is superior to high sensitivity (affinity).ConclusionAdvanced Quantitative Systems Pharmacology computer modeling combining drug exposure dynamics with mechanistic modeling allows to (1) generate testable hypotheses for the outcomes of current therapeutic interventions, (2) support improved clinical trial designs in terms of dosing, patient selection, background amyloid therapy and disease stage and (3) ultimately prioritize targets for addressing tau pathology.

Integrated Mass Spectrometry‐based Pipeline for Characterization of Tau in Human Alzheimer’s Brain: Development and Comparison of Sample Preparation Methods

AbstractBackgroundTau protein aggregation into neurofibrillary tangles in the brain is a pathological hallmark of Alzheimer’s disease (AD) and related tauopathies [1]. Though the mechanism of tau destabilization is not fully understood, tau protein exhibits several allele‐specific isoforms and post‐translational modifications (PTMs) specific to tau aggregates [2]. To explore in‐depth tau species in brain tissues of AD patients, we developed novel pipelines based on different tau fractions retrieved by buffer solubility, Filter Aided Sample Preparation (FASP) or immunoprecipitation, before high‐resolution mass spectrometry (HRMS) analysis.MethodAs a proof of concept, we used frozen brain samples from two AD patients (0.6‐1.2 g per replicate, middle frontal gyrus). The samples were divided and homogenized, ultracentrifuged at 180.000xg for 30min and solubilized in different buffers successively (2 replicates): (1) SarkMethodlow‐salt buffer, 10% Triton X‐100, sarkosyl buffer, and urea buffer; (2) RIPAMethodhigh‐salt buffer (twice), RIPA buffer and urea buffer; and, (3) GnHMethodguanidine hydrochloride and urea buffer. Then, we performed an antibody‐free approach by FASP or immunoprecipitation with a panel of commercial tau antibodies and analyzed on reversed‐phase capillary liquid chromatography, coupled with a qExactive HRMS.ResultHRMS demonstrated that the RIPA method performed better than the other methods for Tau analysis: coverage of 63% (full‐length, 2N4R) and 342 tau peptide‐spectrum matches (PSMs) (Sark: 49% and 183 PSMs; GnH: 41% and 169 PSMs). PTM‐modified tau species (phosphorylated, methylated, and acetylated) were primarily seen using the RIPA method (59 PSMs; Sark: 19 PSMs, GnH: 21 PSMs). Immunoprecipitation improved tau coverage and PTM detection compared to FASP.ConclusionThese findings highlight the best performance of the RIPA method followed by immunoprecipitation for the study of AD brain tau allele‐specific isoforms and PTMs using HRMS. Better characterization of Tau molecular profile in the brain of AD patients opens the gate for a better understanding of AD pathogenesis and represents an opportunity in the biomarker field.[1] Mroczko, B. et al. Int J Mol Sci 2019, 20, E4661, https://doi.org/10.3390/ijms20194661.[2] Boyarko, B.; Hook, V. Front. Neurosci. 2021, 15, 702788, https://doi.org/10.3389/fnins.2021.702788.

Multiplex in‐vivo quantification of tau by mass spectrometry: Method optimization for comprehensive detection of tau proteoforms and biomarker identification

AbstractBackgroundNeurodegenerative diseases diagnosis is still challenging, especially at the early stages. The diagnosis of Alzheimer’s disease (AD) relies on the identification of tau proteoforms (i.e., phosphorylation) in the cerebrospinal fluid (CSF) and plasma. However, pathophysiological fluid biomarkers are still lacking for non‐AD tauopathies. Multiplex liquid chromatography (LC)‐mass spectrometry (MS/MS) offers the opportunity to quantify the vast diversity of tau proteoforms (3R and 4R isoforms, truncations, phosphorylations, acetylations). The current gold standard for tau extraction before LC‐MS/MS analysis of very low concentrations in biofluids relies on immunoprecipitation (IP) approaches. Nonetheless, IP may miss some proteoforms due to its high selectivity. Here, we tested anti‐body free and IP approaches and compared their ability to detect tau proteoforms with LC‐MS/MS.MethodWe analyzed samples of artificial CSF (recombinant tau protein in diluted serum), and human CSF (from AD and non‐AD patients). Fully 15N‐labeled tau was added as an internal standard to the samples. Regarding IP, we first assessed a panel of commercial tau antibodies (in line with the literature) coated on magnetic beads. For the antibody‐free approach, we performed perchloric acid precipitation, followed by micro‐Solid Phase Extraction (Pc‐µSPE) using Oasis hydrophilic‐lipophilic balance sorbent. Purified proteins were digested into peptides using trypsin before multiplex quantitative analysis on reversed‐phase capillary LC, coupled with a qExactive high‐resolution mass spectrometer operated in the Parallel Reaction Monitoring (PRM) acquisition mode.ResultIP optimization led to a combination of antibodies targeting the central and N‐ter domains of tau. IP and Pc‐µSPE comparison in artificial CSF showed a higher sensitivity of IP: limits of detection decreased up to a factor 5. In the human CSF, Pc‐µSPE allowed for better detection of peptides from the N‐ter to the C‐ter than IP, and a higher signal for tau proteoforms (e.g., 3R and 4R isoforms and phosphorylated tau species).ConclusionDespite better performances when dealing with canonical tau, IP was overcome by the antibody‐free sample preparation when identifying tau proteoforms with LC‐MS/MS in human CSF. The protocol is readily applicable to samples of clinical cohorts. This result opens the possibility of better detecting tau proteoforms and identifying pathophysiological biomarkers for non‐AD tauopathies.

Development and Characterization of Mouse-Specific Anti-Tau Monoclonal Antibodies: Relevance for Analysis of Murine Tau in Cerebrospinal Fluid.

Clearance of tau seeds by immunization with tau antibodies is currently evaluated as therapeutic strategy to block the spreading of tau pathology in Alzheimer's disease and other tauopathies. Preclinical evaluation of passive immunotherapy is performed in different cellular culture systems and in wild-type and human tau transgenic mouse models. Depending on the preclinical model used, tau seeds or induced aggregates can either be of mouse, human or mixed origin. We aimed to develop human and mouse tau-specific antibodies to discriminate between the endogenous tau and the introduced form in preclinical models. Using hybridoma technology, we developed human and mouse tau-specific antibodies that were then used to develop several assays to specifically detect mouse tau. Four antibodies, mTau3, mTau5, mTau8, and mTau9, with a high degree of specificity for mouse tau were identified. Additionally, their potential application in highly sensitive immunoassays to measure tau in mouse brain homogenate and cerebrospinal fluid is illustrated, as well as their application for specific endogenous mouse tau aggregation detection. The antibodies reported here can be very important tools to better interpret the results obtained from different model systems as well as to study the role of endogenous tau in tau aggregation and pathology observed in the diverse mouse models available.

Correlation of 18F-PM-PBB3 (18F-florzolotau) Tau PET Imaging with Postmortem Neuropathological Findings in A Case with Progressive Supranuclear Palsy (P9-11.015)

<h3>Objective:</h3> To examine correlations between <i>in vivo</i> positron emission tomography (PET) imaging with 18F-PM-PBB3 (18F-florzolotau) and postmortem neuropathological findings in a patient with progressive supranuclear palsy (PSP). <h3>Background:</h3> There have been no reports demonstrating tight PET-pathology associations in the same individual with non-Alzheimer-type neurodegenerative disorders, including PSP, characterized by aggregations of four-repeat tau isoforms only. 18F-florzolotau is a PET probe allowing high-contrast imaging of Alzheimer’s and non-Alzheimer tau pathologies irrespective of isoform compositions, while the utility of this tracer for quantifying the abundance of tau deposits in living cases is yet to be verified. <h3>Design/Methods:</h3> <h3>Patient:</h3> A 65-year-old male with a clinical diagnosis of PSP Richardson syndrome underwent 11C-PiB amyloid and 18F-florzolotau tau PET scans one year before autopsy. Clinical, neuroimaging, and neuropathological findings in this case were briefly summarized elsewhere (K Tagai, et al. Neuron 2021). <h3>Image analysis:</h3> The standardized uptake value ratio (SUVR) image was generated from averaged PET images at 90–110 min after injecting 18F-florzolotau. The reference region was determined using an optimized reference tissue method (K Tagai, et al. medRxiv DOI: https://doi.org/10.1101/2022.02.13.22270135). <h3>Neuropathological analysis:</h3> Quantitative analysis of tau burden was performed using an anti-phosphorylated tau antibody (AT8). A threshold-based binarization method was used for determining the mean AT-8-positive area (%area tau burden). <h3>Results:</h3> 18F-florzolotau PET showed enhanced radiosignals in the brainstem, subthalamic nucleus, and basal ganglia, and the presence of PSP-characteristic tau deposits, including the globose neurofibrillary tangle, tufted astrocytes and coiled bodies, primarily in these areas was confirmed by neuropathological assays. There was a close correlation between regional radioprobe SUVR and %area tau burden (<i>r</i><i>_s</i> = 0.63, <i>p</i> &lt;0.0001). Amyloid PET was negative by visual read. <h3>Conclusions:</h3> 18F-florzolotau PET measures reflect the abundance of tau pathologies in PSP. <b>Disclosure:</b> Dr. Endo has received personal compensation in the range of $500-$4,999 for serving as a Consultant for APRINOIA Therapeutics. Dr. Endo has received personal compensation in the range of $0-$499 for serving on a Speakers Bureau for AbbVie Inc.. The institution of Dr. Endo has received research support from JSPS KAKENHI 21K15705. Dr. Araki has nothing to disclose. Dr. Takeda has nothing to disclose. Dr. Takado has nothing to disclose. Dr. Tagai has nothing to disclose. Dr. Matsuoka has nothing to disclose. Dr. Seki has nothing to disclose. Dr. Takahata has nothing to disclose. Dr. Sahara has nothing to disclose. Hitoshi Shinotoh has nothing to disclose. Mr. Kawamura has nothing to disclose. Dr. Zhang has nothing to disclose. Dr. Honda has nothing to disclose. Prof. SHIMADA has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Guidepoint. Prof. SHIMADA has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for eli lilly. Prof. SHIMADA has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for Takeda Pharmaceutical . Prof. SHIMADA has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for Eisai. Prof. SHIMADA has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for Nihon Medi-Physics. Prof. SHIMADA has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for Biogen. Prof. SHIMADA has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for FUJIFILM Toyama Chemical. Prof. SHIMADA has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for Sunplanet . Prof. SHIMADA has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for NIPPON CHEMIPHAR. Prof. SHIMADA has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for Sumitomo Pharma. Prof. SHIMADA has received personal compensation in the range of $500-$4,999 for serving on a Speakers Bureau for GE Healthcare. Prof. SHIMADA has received personal compensation in the range of $500-$4,999 for serving as an Expert Witness for CHUGAI PHARMACEUTICAL. Prof. SHIMADA has received intellectual property interests from a discovery or technology relating to health care. Prof. SHIMADA has received publishing royalties from a publication relating to health care. Prof. SHIMADA has received publishing royalties from a publication relating to health care. Prof. SHIMADA has received publishing royalties from a publication relating to health care. The institution of Dr. Higuchi has received research support from APRINOIA Therapeutics.

Antibody Assay and Anti-Inflammatory Function Evaluation of Therapeutic Potential of Different Intravenous Immunoglobulins for Alzheimer's Disease.

Alzheimer's disease (AD) is a common neurodegenerative disease that currently has no known cure. Intravenous immunoglobulin (IVIG), which contains AD-related antibodies and has anti-inflammatory properties, has shown potential as a treatment for AD. However, the efficacy of clinical trials involving AD patients treated with IVIG has been inconsistent. Our previous study found that different IVIGs had significantly varied therapeutic effects on 3xTg-AD mice. In order to investigate the relationship between the composition and function of IVIG and its efficacy in treating AD, we selected three IVIGs that showed notable differences in therapeutic effects. Then, the concentrations of specific antibodies against β-amyloid (Aβ)42, tau, and hyperphosphorylated tau (p-tau) in three IVIGs, as well as their effects on systemic inflammation induced by lipopolysaccharide (LPS) in Balb/c mice, were analyzed and compared in this study. The results indicated that these IVIGs differed greatly in anti-Aβ42/tau antibody concentration and anti-p-tau ratio, and improved LPS-stimulated peripheral inflammation, liver and kidney injury, and neuroinflammation in Balb/c mice to varying degrees. Combined with our previous results, the efficacy of IVIG against AD may be positively correlated with its level of AD-related antibodies and anti-inflammatory ability. AD-related antibody analysis and functional evaluation of IVIG should be given sufficient attention before clinical trials, as this may greatly affect the therapeutic effect of AD treatment.

Passive immunization inhibits tau phosphorylation and improves recognition learning and memory in 3xTg-AD mice

Tau pathology is essential in the pathogenesis of Alzheimer's disease (AD) and related tauopathies. Tau immunotherapy aimed at reducing the progression of tau pathology provides a potential therapeutic strategy for treating these diseases. By screening monoclonal antibodies 43D, 63B, 39E10, and 77G7 that recognize epitopes ranging from tau's N-terminus to C-terminus, we found the 77G7, which targets the microtubule-binding domain promoted tau clearance in a dose-dependent manner by entering neuronal cells in vitro. Intra-cerebroventricular injection of 77G7 antibody reduced tau levels in the wild-type FVB mouse brain. Without influencing the levels of detergent-insoluble and aggregated tau, intravenous injection of 77G7 reduced tau hyperphosphorylation in the brain and improved novel object recognition but not spatial learning and memory in 15–18-month-old 3xTg-AD mice. These studies suggest that epitopes recognized by tau antibodies are crucial for the efficacy of immunotherapy. Immunization with antibody 77G7 provides a novel potential opportunity for tau-directed immunotherapy of AD and related tauopathies.

Selective In Vitro and Ex Vivo Staining of Brain Neurofibrillary Tangles and Amyloid Plaques by Novel Ethylene Ethynylene-Based Optical Sensors.

The identification of protein aggregates as biomarkers for neurodegeneration is an area of interest for disease diagnosis and treatment development. In this work, we present novel super luminescent conjugated polyelectrolyte molecules as ex vivo sensors for tau-paired helical filaments (PHFs) and amyloid-β (Aβ) plaques. We evaluated the use of two oligo-p-phenylene ethynylenes (OPEs), anionic OPE12- and cationic OPE24+, as stains for fibrillar protein pathology in brain sections of transgenic mouse (rTg4510) and rat (TgF344-AD) models of Alzheimer's disease (AD) tauopathy, and post-mortem brain sections from human frontotemporal dementia (FTD). OPE12- displayed selectivity for PHFs in fluorimetry assays and strong staining of neurofibrillary tangles (NFTs) in mouse and human brain tissue sections, while OPE24+ stained both NFTs and Aβ plaques. Both OPEs stained the brain sections with limited background or non-specific staining. This novel family of sensors outperformed the gold-standard dye Thioflavin T in sensing capacities and co-stained with conventional phosphorylated tau (AT180) and Aβ (4G8) antibodies. As the OPEs readily bind protein amyloids in vitro and ex vivo, they are selective and rapid tools for identifying proteopathic inclusions relevant to AD. Such OPEs can be useful in understanding pathogenesis and in creating in vivo diagnostically relevant detection tools for neurodegenerative diseases.

New unexpected role for Wolfram Syndrome protein WFS1: a novel therapeutic target for Alzheimer's disease?

New unexpected role for Wolfram Syndrome protein WFS1: a novel therapeutic target for Alzheimer's disease?

Abnormal Hyperphosphorylation of Tau in Canine Immune-mediated Meningoencephalitis.

Tau is a microtubule-associated protein involved in the assembly and stabilization of microtubules. In human medicine, hyperphosphorylation of tau is associated with microtubule instability and is considered to play a role in the progression of multiple sclerosis (MS). MS is an autoimmune neurological disease that shares many characteristics, including pathological mechanisms, with canine meningoencephalitis of unknown etiology (MUE). With this background, this study investigated the presence of hyperphosphorylated tau in dogs with MUE and experimental autoimmune encephalomyelitis (EAE). In total, eight brain samples were examined from two neurologically normal dogs, three dogs with MUE, and three canine EAE models. Anti-(phospho-S396) tau antibody was used for immunohisto-chemistry, which stained hyperphosphorylated tau. In normal brain tissues, hyperphosphorylated tau was not found. In all the dogs with EAE and one of the dogs with MUE, immunoreactivity for S396 p-tau was observed in glial cell cytoplasm and the background in the periphery of the inflammatory lesion. These results suggest for the first time that tau pathology may be involved in the progression of neuroinflammation in dogs, similar to that in human MS.

Neuronal calcium dysregulation in mice at an early stage of tauopathy and its alleviation by a tau antibody

AbstractBackgroundTau immunotherapies are promising approaches to treat Alzheimer’s disease and other tauopathies. How tau antibodies restore neuronal function remains unknown. Neuronal activity can be monitored by two‐photon calcium imaging in awake and behaving animals. Using this method, we observed altered neuronal calcium activity in the motor cortex of 12‐month‐old JNPL3 tauopathy mice during quiet wakefulness or forced running (Wu Q et al, Neurobiol. Dis. 2021). We also demonstrated that a mouse monoclonal antibody, targeting phosphorylated tau‐Ser396/Ser404, significantly restored the abnormal calcium activity and decreased pathological tau levels in these mice. Analysis of possible neuronal functional deficits and tau antibody efficacy in younger tauopathy mice warrants further investigation.MethodsSomatic calcium activity in the motor cortex of 6‐month‐old JNPL3 mice were assessed by two‐photon microscopy when animals were either resting or running on a treadmill. At this age, JNPL3 mice are in their early stage of tauopathy. The frequency, amplitude and total activity of calcium transients were analyzed and compared between wild‐type and JNPL3 mice. After the first imaging session, two doses (100 µg each) of a tau antibody were administered intravenously to JNPL3 mice four days apart. The somatic calcium activity was assessed again in these animals four days later, and compared to their neuronal calcium activity profile before tau antibody treatment.ResultsWe found abnormal cortical calcium activity in resting and running JNPL3 mice at 6 months of age. Brain levels of pathological tau correlated with the degree of calcium dysfunction. Acute treatment with a tau antibody attenuated these changes, whereas littermate JNPL3 mice treated with control IgG showed no improvements.ConclusionThese findings revealed neuronal calcium dysregulation in JNPL3 mice at an early stage of tauopathy that was partially corrected by an acute tau antibody treatment, analogous to our prior findings at a later stage of tauopathy. Together, these results indicate that neuronal dysfunction correlates with tau pathology at various stages of tauopathy, which can be consistently alleviated by a tau antibody treatment. This type of functional evaluation in vivo is a valuable way to study tau pathogenesis, and assess therapeutic efficacy at different stages of tauopathy.