Tau Antibodies Research Articles

Tau antibody 77G7 targeting microtubule binding domain suppresses oligomeric tau to seed tau aggregation

AbstractBackgroundNeurofibrillary tangle (NFT) of hyperphosphorylated tau is a hallmark of Alzheimer’s disease (AD) and related tauopathies. Tau lesion starts in the trans‐entorhinal cortex, from where it spreads to limbic regions, followed by neocortical areas. The regional distribution of NFTs associates with the progression of AD. Accumulating evidence suggests that proteopathic tau can seed tau aggregation in a prion‐like fashion in vitro and in vivo. Inhibition of the seeding activity of proteopathic tau could provide a potential therapeutic opportunity to block the propagation of tau pathology. In the present study, we investigated the role of 77G7, a monoclonal tau antibody to the microtubule‐binding repeats, in the seeding activity of proteopathic tau.MethodAffinity of 77G7 to various tau strains, including oligomeric tau (AD O‐tau), sarkosyl insoluble tau (SI‐tau), and heat stable normal tau (HS‐tau) isolated from frozen autopsied AD brains by sedimentation, was analyzed by immuno‐dot‐blots. Seeding activity of AD O‐tau was accessed by the level of aggregated tau in vitro and in cultured cells and by overlay capture assay. Tau pathology in human tau transgenic mice was induced by hippocampal injection of AD O‐tau. 77G7 was mixed with AD O‐tau or was intravenously administrated weekly for 6 weeks started one week prior to AD O‐tau injection. Tau phosphorylation and tau pathology were analyzed by immunohistochemical staining and Western blots 8‐10 weeks after AD O‐tau injection.ResultWe found that 77G7 had a higher affinity toward aggregated tau strains than un‐aggregated tau derived from AD brain. 77G7 inhibited the internalization of tau aggregates by cells, blocked AD O‐tau to capture normal tau and to seed tau aggregation in vitro and in cultured cells. AD O‐tau induced tau pathology in hippocampus of 3xTg‐AD mice was reduced by mixing 77G7 with AD O‐tau. Intravenous administration of 77G7 ameliorated site‐specific hyperphosphorylation of tau induced by AD O‐tau in the hippocampi of Tg/hTau mice.ConclusionThese findings indicate that 77G7 can effectively suppress the seeding activity of AD O‐tau and thus, could be developed as a potential immunotherapeutic drug to inhibit the propagation of tau pathology in AD and related tauopathies.

Bead‐based electrochemical gap sensors for detection of tau protein in blood to diagnosis and monitoring the tauopathy

AbstractBackgroundRecently, there is a need for a method for detecting biomarkers in blood that can be collected relatively easily for diagnosis and monitoring of dementia symptoms in Alzheimer's disease (AD) patients. Tau protein, a typical hallmark of AD, has six isoforms, and is divided into four repeating microtubule binding domains (4R) and three microtubule binding domains (3R) depending on whether exon 10 is included. According to the balance of 3R‐ and 4R‐tau, tauopathy can be distinguished. It is expected to be able to differentiate normal (NC) from AD and other dementias by measuring the tau protein in the blood.MethodIn this study, an electrochemical sensor with a novel structure was employed to detect tau in the plasma of living patients. The nanogap sensor consists of a gap electrode and a SU‐8 microwell for loading a single magnetic bead (Figure 1). A sample was prepared by reacting the surface of the magnetic beads with an antibody (total tau or anti 3R‐ or 4R‐tau antibody) and an antibody functionalized with a secondary O‐glycosylated tau (O‐g tau) antibody or phosphorylated tau (p‐tau) antibody.ResultAs a result of O‐g/p‐tau measurement, the impedance change by O‐g tau was large in NC (n=4), whereas the impedance change by p‐tau was large in AD (n=13). Patients with idiopathic Parkinson's disease (IPD) (n=3) also had more impedance changes due to p‐tau (Figure 2). As a result of measuring 3R‐ or 4R‐tau protein, the impedance change by 4R‐tau in NC was larger than that by 3R‐tau, whereas the opposite result was shown in AD. similar to the previous results, the impedance change by 3R‐tau was larger in IPD (Figure 3),ConclusionWe used a novel electrochemical gap sensor to detect tau protein in blood samples collected from living patients. As a result, it is expected to be able to differentiate NC from tauopathy and other dementias by measuring the tau protein in the blood (Figure 4).

The potentially protective role of an understudied tau isoform in Alzheimer’s disease

AbstractBackgroundAs Alzheimer’s disease (AD) progresses, tau becomes abnormally aggregated and hyperphosphorylated in selective brain regions such as the cortex and hippocampus. Surprisingly, the cerebellum remains spared from tau pathology. Given this observation, we proposed to examine possible differences in the level of various tau isoforms in the cerebellum compared to the forebrain.MethodsWe first compared the transcript and protein levels of tau isoforms between the cerebellum and other brain regions (e.g., the cortex and hippocampus) using both mouse and human brain samples. Here, we discovered that “big tau”, an understudied tau isoform previously reported to be specifically expressed in the periphery, is highly elevated in the cerebellum compared to other brain regions. Next, we performed detergent fractionation using cells expressing mutant big tau or tau441 (the longest one of six major tau isoforms) to compare the aggregation propensity between the two tau isoforms. Lastly, to test this in an animal model, we injected a newborn wild‐type mice with adeno‐associated viruses (AAV) that express either mutant big tau or tau441. After aging the mice for 6 to 9 months, we examined their brains to analyze pathological changes associated with either of these two tau isoforms.ResultsWe found that the transcript level of big tau is significantly higher in the mouse cerebellum compared to the cortex and hippocampus. Using the total tau antibody and the novel mouse big tau‐specific antibody that we generated, we confirmed that big tau protein level is also significantly elevated in the cerebellum. We obtained similar results in human tissues as both the transcript and protein levels of big tau were higher in the cerebellum than the cortex. Interestingly, our fractionation experiment using cells revealed that big tau protein is significantly less likely to aggregate compared to tau441. Consistent with these results, big tau did not become pathologically aggregated or phosphorylated compared to tau441 in the AAV‐based mouse model.ConclusionBig tau has a substantially lower tendency to become pathologically aggregated or hyperphosphorylated. The higher level of big tau in the cerebellum may potentially protect this brain region from developing tau pathology in AD.

Investigation of differentially expressed proteins through neurofibrillary tangle maturation in Alzheimer’s disease using NanoString’s GeoMx Digital Spatial Profiler

AbstractBackgroundNeurofibrillary tangles are dynamic entities with a lifespan encompassing three maturity levels: intracellular pretangles, intracellular mature tangles, and extracellular ghost tangles. Proteins are suggested to be differentially expressed throughout the tangle lifespan; however, past studies may be limited by spatial expression or number of proteins investigated. We sought to assess differences in protein expression across the tangle lifespan using GeoMxTM Digital Spatial Profiler (DSP) by NanoString, a new technology allowing for spatially derived multiplex investigation.MethodWe used DSP to measure protein expression in hippocampi from Alzheimer’s disease (n=6) and nondemented controls (n=2) using the “Human Neural Cell Profiling Core” and “Alzheimer’s Pathology” module. Regions of interest (ROIs) were classified by the major tangle maturity level recognized by a primary fluorescent conjugated tau antibody. Protein expression was normalized to the geomean of the three housekeeping genes. The tau‐positive segment was specifically analyzed for comparisons. ANOVA was used for group‐wise comparisons and t‐test using the Benjamini‐Yekutieli adjustment for multiple tests was used for pair‐wise comparisons.ResultAs expected, cytoskeletal (e.g., MAP2, Tau) and neuronal (e.g., NeuN) markers generally decreased through the neurofibrillary tangle lifespan (Figure). Markers profiled as neurodegenerative (e.g., amyloid‐β1‐42, APOE, APP) generally increased through tangle maturity levels, except for TDP‐43 which decreased. Interestingly, there was no significant difference in ubiquitin expression. Astrocytic GFAP and S100B expression was significantly increased through each maturity level. Expression of microglial‐specific markers P2RY12 and TMEM119 was significantly elevated in ghost tangles compared to mature tangles. Additional microglial/macrophage markers including Iba‐1 and CD68 were generally increased as neurofibrillary tangles matured. Interestingly, APOE expression remained low in pretangles and mature tangles before dramatically increasing in ghost tangles (Figure).ConclusionOur findings suggest that proteins are differentially expressed throughout the tangle lifespan and the majority followed a monotonically directed pattern. Non‐monotonically directed protein patterns may reflect the death of the neuron during the ghost tangle maturity level. Future studies will test the hypothesis that the microenvironment of the hippocampus is altered with increasing vulnerability to tangle pathology.

Efficacy evaluation of a novel vectorized anti‐tau antibody, targeting a C‐terminal phospho‐tau epitope, using systemic dosing of a blood brain barrier penetrant AAV capsid in mouse models of tauopathies.

AbstractBackgroundAnti‐tau immunotherapy has become a promising therapy for Alzheimer’s disease (AD) and tauopathies. With the hypothesis that tau pathology spreads via cell‐to‐cell transmission, including trans‐synaptic propagation, success of anti‐tau immunotherapy relies, in part, on the identification of efficacious antibodies and their delivery to affected or vulnerable brain regions with sufficient or enhanced exposure in the CNS. We have previously demonstrated broad distribution and expression of vectorized anti‐tau antibodies in the mouse brain using a blood brain barrier penetrant capsid, VOY101, administered intravenously (IV).MethodSeveral novel anti‐tau antibodies that met the target profile have been generated and are being evaluated in vivo (Liu et al, submitted as a separated abstract in AAIC 2022).ResultOne of the antibodies discovered, antibody 1, exhibits strong affinity for PHF‐tau, demonstrates specific binding to tau pathology on brain sections of AD and PSP patients, and potently prevents PHF seeding and propagation in vitro and in vivo. This antibody recognizes a phospho‐specific epitope in the C‐terminal region of tau and shows significant reduction of tau pathology in an AD‐PHF induced P301S hippocampal seeding and propagation model. Furthermore, we have vectorized antibody 1 into an AAV expression vector with by BBB penetrant capsid and are evaluating it in two independent mouse models of tauopathy.ConclusionThis gene therapy‐based approach has potential advantages over traditional passive immunization, including 1) continuous expression of antibody in the central nervous system (CNS) after a single gene therapy administration compared to repetitive administrations of high dose of antibody by passive immunotherapy; 2) increased CNS exposure of tau antibody relative to passive immunotherapy; and 3) the potential to target intracellular tau aggregates which are less effectively accessed by passively delivered antibody. These results add to accumulating evidence that systemic dosing of a vectorized anti‐tau antibody using a BBB‐penetrant AAV capsid results in reduced tau pathology and may represent a new single‐dose therapeutic strategy for treating various tauopathies.

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Quantifying the distribution and morphology of tau protein structures in brain tissues is key to diagnosing Alzheimer's Disease (AD) and its subtypes. Recently, deep learning (DL) models such as UNet have been successfully used for automatic segmentation of histopathological whole slide images (WSI) of biological tissues. In this study, we propose a DL-based methodology for semantic segmentation of tau lesions (i.e., neuritic plaques) in WSI of postmortem patients with AD. The state of the art in semantic segmentation of neuritic plaques in human WSI is very limited. Our study proposes a baseline able to generate a significant advantage for morphological analysis of these tauopathies for further stratification of AD patients. Essential discussions concerning biomarkers (ALZ50 versus AT8 tau antibodies), the imaging modality (different slide scanner resolutions), and the challenge of weak annotations are addressed within this seminal study. The analysis of the impact of context in plaque segmentation is important to understand the role of the micro-environment for reliable tau protein segmentation. In addition, by integrating visual interpretability, we are able to explain how the network focuses on a region of interest (ROI), giving additional insights to pathologists. Finally, the release of a new expert-annotated database and the code (\url{https://github.com/aramis-lab/miccai2022-stratifiad.git}) will be helpful for the scientific community to accelerate the development of new pipelines for human WSI processing in AD.

Site-Specific Phospho-Tau Aggregation-Based Biomarker Discovery for AD Diagnosis and Differentiation.

Tau aggregates are present in multiple neurodegenerative diseases known as "tauopathies," including Alzheimer's disease (AD), Pick's disease (PiD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). Such misfolded tau aggregates are therefore potential sources for tauopathy biomarker discovery. Using the tau antibody screening approach targeting high-molecular-weight misfolded tau aggregates, we tested several tau antibodies and a comprehensive set of site-specific phospho-tau (p-tau) antibodies targeting tau phosphorylation sites showing high frequencies in AD subjects. Our screens revealed that site-specific p-tau antibodies can not only differentiate AD from non-AD brains, but also discriminate AD from rare tauopathies PiD, PSP, and CBD brains. Differential detection of tau aggregates identified several novel p-tau sites as potential new biomarkers. As a proof-of-principle example, we showed that p-tau198 is a novel promising AD biomarker with sensitivity and specificity comparable with the existing biomarkers p-tau181 and p-tau217. Our results demonstrated that p-tau198 detection can not only differentiate AD from non-AD controls, but also diagnose AD from related 4R tauopathies PSP and CBD with AUCs of 0.96-0.99 (95% CI ranges from 0.90 to 1.00). Promisingly, p-tau198 was able to discriminate mild cognitive impairment from cognitively normal brains with an AUC of 0.75 (95% CI = 0.58-0.92). Our work provides a new avenue for developing diagnosis and differentiation tools for AD and related tauopathies.

Tau antibody 77G7 targeting microtubule binding domain suppresses proteopathic tau to seed tau aggregation.

Neurofibrillary tangle (NFT) of hyperphosphorylated tau is a hallmark of Alzheimer's disease (AD) and related tauopathies. Tau lesion starts in the trans-entorhinal cortex, from where it spreads to limbic regions, followed by neocortical areas. The regional distribution of NFTs associates with the progression of AD. Accumulating evidence suggests that proteopathic tau can seed tau aggregation in a prion-like fashion in vitro and in vivo. Inhibition of tau seeding activity could provide a potential therapeutic opportunity to block the propagation of tau pathology in AD and related tauopathies. In the present study, we investigated the role of 77G7, a monoclonal tau antibody to the microtubule-binding repeats, in repressing the seeding activity of proteopathic tau. We found that 77G7 had a higher affinity toward aggregated pathological tau fractions than un-aggregated tau derived from AD brain. 77G7 inhibited the internalization of tau aggregates by cells, blocked AD O-tau to capture normal tau, and to seed tau aggregation in vitro and in cultured cells. Tau pathology induced by hippocampal injection of AD O-tau in 3xTg-AD mice was suppressed by mixing 77G7 with AD O-tau. Intravenous administration of 77G7 ameliorated site-specific hyperphosphorylation of tau induced by AD O-tau in the hippocampi of Tg/hTau mice. These findings indicate that 77G7 can effectively suppress the seeding activity of AD O-tau and thus could be developed as a potential immunotherapeutic drug to inhibit the propagation of tau pathology in AD and related tauopathies.

Single domain antibodies targeting pathological tau protein: Influence of four IgG subclasses on efficacy and toxicity.

SummaryBackgroundEleven tau immunoglobulin G (IgG) antibodies have entered clinical trials to treat tauopathies, including Alzheimer's disease, but it is unclear which IgG subclass/subtype has the ideal efficacy and safety profile. Only two subtypes, with or without effector function, have been examined in the clinic and not for the same tau antibody. The few preclinical studies on this topic have only compared two subtypes of one antibody each and have yielded conflicting results.MethodsWe selected two single domain antibodies (sdAbs) derived from a llama immunized with tau proteins and utilized them to generate an array of Fc-(sdAb)2 subclasses containing identical tau binding domains but differing Fc region. Unmodified sdAbs and their IgG subclasses were tested for efficacy in primary cultures and in vivo microdialysis using JNPL3 tauopathy mice.FindingsUnmodified sdAbs were non-toxic, blocked tau toxicity and promoted tau clearance. However, the efficacy/safety profile of their Fc-(sdAb)2 subclasses varied greatly within and between sdAbs. For one of them, all its subtypes were non-toxic, only those with effector function cleared tau, and were more effective in vivo than unmodified sdAb. For the other sdAb, all its subtypes were toxic in tauopathy cultures but not in wild-type cells, suggesting that bivalent binding of its tau epitope stabilizes a toxic conformation of tau, with major implications for tau pathogenesis. Likewise, its subclasses were less effective than the unmodified sdAb in clearing tau in vivo.InterpretationThese findings indicate that tau antibodies with effector function are safe and better at clearing pathological tau than effectorless antibodies, Furthermore, tau antibodies can provide a valuable insight into tau pathogenesis, and some may aggravate it.FundingFunding for these studies was provided by the National Institute of Health (R01 AG032611, R01 NS077239, RF1 NS120488, R21 AG 069475, R21 AG 058282, T32AG052909), and the NYU Alzheimer's Disease Center Pilot Grant Program (via P30 AG008051).

PBPK-PD modeling for the preclinical development and clinical translation of tau antibodies for Alzheimer's disease.

Disrupted tau proteostasis and transneuronal spread is a pathological hallmark of Alzheimer’s disease. Neurodegenerative diseases remain an unmet medical need and novel disease modifying therapeutics are paramount. Our objective was to develop a mechanistic mathematical model to enhance our understanding of tau antibody pharmacokinetics and pharmacodynamics in animals and humans. A physiologically-based pharmacokinetic-pharmacodynamic (PBPK-PD) modeling approach was employed to support the preclinical development and clinical translation of therapeutic antibodies targeting tau for the treatment of Alzheimer’s disease. The pharmacokinetics of a tau antibody was evaluated in rat and non-human primate microdialysis studies. Model validation for humans was performed using publicly available clinical data for gosuranemab. In-silico analyses were performed to predict tau engagement in human brain for a range of tau antibody affinities and various dosing regimens. PBPK-PD modeling enabled a quantitative understanding for the relationship between dose, affinity, and target engagement, which supported lead candidate optimization and predictions of clinically efficacious dosing regimens.

Oral microbiome and serological analyses on association of Alzheimer's disease and periodontitis.

To investigate the association between Alzheimer's disease (AD) and periodontitis in the aspects of periodontal status, serological markers, and oral microbiome. Twenty AD and 20 healthy subjects were enrolled in this age- and gender-matched case-control study. Clinical periodontal parameters and serum biomarkers, including amyloid β42 (Aβ42 ), Tau, phosphorylated Tau (pTau), triglyceride, pro-inflammatory cytokines, and anti-Porphyromonas gingivalis lipopolysaccharide (LPS) antibody were examined. The saliva samples were analyzed for oral microbiome composition. Alzheimer's disease patients with Clinical Dementia Rating (CDR) ≥1 exhibited significantly more clinical attachment loss (CAL) than those with lower CDR. The levels of serum Tau protein, hsCRP and anti-P.gingivalis LPS antibody were markedly elevated in the AD group compared with the control group. Serum pTau protein level was positively correlated with anti-P.gingivalis LPS antibody titer. Moreover, the increased abundances of Capnocytophaga sp ora clone DZ074, Eubacterium infirmum, Prevotella buccae, and Selenomonas artemidis were detected in the AD group. Interestingly, serum levels of Aβ42, pTau, and anti-P.gingivalis LPS antibody were strongly related to the gene upregulation in human pathogen septicemia. Our study suggested the association of periodontal infection and oral microbiome with AD. Further large-scale studies with longitudinal follow-up are warranted.

A call for better understanding of target engagement in Tau antibody development

Significant efforts have been channeled into developing antibodies for the treatment of CNS indications. Disappointment with the first generation of clinical Tau antibodies in Alzheimer's disease has highlighted the challenges in understanding whether an antibody can reach or affect the target in the compartment where it is involved in pathological processes. Here, we highlight different aspects essential for improving translatability of Tau-based immunotherapy.

Highly Specific and Sensitive Target Binding by the Humanized pS396-Tau Antibody hC10.2 Across a Wide Spectrum of Alzheimer’s Disease and Primary Tauopathy Postmortem Brains

Background: Deposits of hyperphosphorylated tau fibrils are hallmarks of a broad spectrum of tauopathies, including Alzheimer’s disease (AD). Objective: To investigate heterogeneity of tau pathology across brain extracts from a broad selection of different tauopathies and examine the binding properties of the humanized pS396-tau antibody hC10.2 and six other anti-tau antibodies. Methods: 76 individual tauopathy tissue samples were analyzed in a battery of assays: immunohistochemistry, ELISA, tau aggregation assay, western blot, [3H]PI-2620 and [3H]MK-6240 tau tracer binding, and aggregated seeding activity in RD_P301S HEK293T Biosensor cells. The efficiency of seven anti-tau antibodies to engage with pathological tau species was directly compared. Results: Our data indicate that a strong correlation existed between the tau tracer binding, amount of tau aggregates, pS396-tau phosphorylation, and seeding activity. The hC10.2 antibody, which has entered clinical development, effectively engaged with its epitope across all individual cases of mid-stage and late AD, and primary tauopathies. hC10.2 was superior compared to other phospho- and total tau antibodies to prevent seeded tau aggregation in the biosensor cells. hC10.2 effectively depleted hyperphosphorylated and aggregated tau species across all tauopathy samples proportionally to the amount of tau aggregates. In AD samples, hC10.2 bound to ghost tangles which represent extracellular pathological tau species. Conclusion: S396 hyperphosphorylation is a feature of the formation of seeding-competent tau across different tauopathies and it is present both in intra- and extracellular pathological tau. hC10.2 represents an excellent candidate for a hyperphosphorylation-selective therapeutic tau antibody for the treatment of AD and primary tauopathies.

Reassessment of Neuronal Tau Distribution in Adult Human Brain and Implications for Tau Pathobiology

Tau is a predominantly neuronal, soluble and natively unfolded protein that can bind and stabilize microtubules in the central nervous system. Tau has been extensively studied over several decades, especially in the context of neurodegenerative diseases where it can aberrantly aggregate to form a spectrum of pathological inclusions. The presence of tau inclusions in the form of neurofibrillary tangles, neuropil threads and dystrophic neurites within senile plaques are essential and defining features of Alzheimer’s disease. The current dogma favors the notion that tau is predominantly an axonal protein, and that in Alzheimer’s disease there is a redistribution of tau towards the neuronal soma that is associated with the formation of pathological inclusions such as neurofibrillary tangles and neuropil threads. Using novel as well as previously established highly specific tau antibodies, we demonstrate that contrary to this overwhelmingly accepted fact, as asserted in numerous articles and reviews, in adult human brain, tau is more abundant in cortical gray matter that is enriched in neuronal soma and dendrites compared to white matter that is predominantly rich in neuronal axons. Additionally, in Alzheimer’s disease tau pathology is significantly more abundant in the brain cortical gray matter of affected brain regions compared to the adjacent white matter regions. These findings have important implications for the biological function of tau as well as the mechanisms involved in the progressive spread of tau associated with the insidious nature of Alzheimer’s disease.

Detection of astrocytic tau pathology facilitates recognition of chronic traumatic encephalopathy neuropathologic change

Traumatic brain injury (TBI) is associated with the development of a range of neurodegenerative pathologies, including chronic traumatic encephalopathy (CTE). Current consensus diagnostic criteria define the pathognomonic cortical lesion of CTE neuropathologic change (CTE-NC) as a patchy deposition of hyperphosphorylated tau in neurons, with or without glial tau in thorn-shaped astrocytes, typically towards the depths of sulci and clustered around small blood vessels. Nevertheless, although incorporated into consensus diagnostic criteria, the contribution of the individual cellular components to identification of CTE-NC has not been formally evaluated. To address this, from the Glasgow TBI Archive, cortical tissue blocks were selected from consecutive brain donations from contact sports athletes in which there was known to be either CTE-NC (n = 12) or Alzheimer’s disease neuropathologic change (n = 4). From these tissue blocks, adjacent tissue sections were stained for tau antibodies selected to reveal either solely neuronal pathology (3R tau; GT-38) or mixed neuronal and astroglial pathologies (4R tau; PHF-1). These stained sections were then randomised and independently assessed by a panel of expert neuropathologists, blind to patient clinical history and primary antibody applied to each section, who were asked to record whether CTE-NC was present. Results demonstrate that, in sections stained for either 4R tau or PHF-1, consensus recognition of CTE-NC was high. In contrast, recognition of CTE-NC in sections stained for 3R tau or GT-38 was poor; in the former no better than chance. Our observations demonstrate that the presence of both neuronal and astroglial tau pathologies facilitates detection of CTE-NC, with its detection less consistent when neuronal tau pathology alone is visible. The combination of both glial and neuronal pathologies, therefore, may be required for detection of CTE-NC.

Efficacy of a vectorized anti‐tau antibody using systemic dosing of a blood brain barrier penetrant AAV capsid in mouse models of tauopathies

AbstractBackgroundAnti‐tau immunotherapy is a promising therapy for tauopathies, including Alzheimer's disease (AD), frontotemporal dementia (FTD) and progressive supranuclear palsy (PSP). While passive immunization with anti‐tau monoclonal antibodies has been shown by several laboratories to reduce age‐dependent tau pathology and neurodegeneration in mouse tauopathy models, these studies have typically used repeated high weekly doses of antibody and reported only moderate reduction of tau pathology. To circumvent these limitations, using an adeno‐associated virus (AAV) vector to deliver a tau monoclonal antibody into hippocampus by direct injection to the location have been described. High levels of antibody and marked reduction of tau pathology is achieved by this approach. To further optimize this approach with a lesser invasive administration route, we have previously demonstrated broad distribution and expression of a vectorized anti‐tau antibody in the mouse brain using a blood‐brain barrier (BBB) penetrant AAV capsid administered intravenously (IV). This gene therapy‐based approach has potential advantages, including continuous expression of antibody in the central nervous system (CNS) after a single administration of vector, increased CNS levels of tau antibody relative to passive immunotherapy, and the potential to target intracellular tau aggregates. Here we describe studies characterizing the onset of transduction following dosing with a vectorized tau antibody and its efficacy in mouse models of tauopathy.MethodAAV vectors comprising the BBB‐penetrant AAV capsid and a transgene encoding an anti‐tau monoclonal antibody were administered by IV bolus to mouse models of tauopathy. Biodistribution and cellular tropism in the CNS were evaluated by ELISA and (or) immunostaining. Tau pathology was measured by AT100 immunohistochemistry and AT8 ELISA.ResultWe then investigated efficacy in reduction of tau pathology in mouse tauopathy models. Treatment with our vectorized antibody resulted in durable antibody expression in the CNS and a corresponding reduction in CNS insoluble pathological tau and neurofibrillary tangles.ConclusionOur results indicate that systemic dosing of a vectorized anti‐tau antibody using a BBB‐penetrant AAV capsid results in reduced tau pathology and may represent a new single‐dose therapeutic strategy for treating various tauopathies.

High‐resolution non‐invasive whole brain imaging of tauopathy in a tauopathy mouse model

AbstractBackgroundAbnormal tauopathy accumulation play an important role in tauopathy diseases including Alzheimer’s disease and Frontotemporal dementia. There is a gap in high‐resolution non‐invasive imaging tool for detecting tauopathy deposits in animal models.MethodThe binding properties of a panel of imaging probes to 4‐repeat K18 tau fibril was assessed by using Thoflavin T assay and surface plasmon resonance assay. Next we established optimal dosage for tau probes in in vivo optoacoustic imaging, and developed transcranial whole brain imaging (130 micron spatial resolution) of tauopathy in P301L model of 4‐repeat tauopathy. T2 magnetic resonance imaging at 9.4T was performed for structural information and for volume‐of‐interest analysis of optoacoustic imaging data after co‐registration.ResultIn vitro assay showed binding to recombinant K18 tau fibril using PBB5, THK5351, but not T‐807 or MK‐6240. i.v. administration of PBB5 in P301L mice led to a retention of PBB5 in tau ladened cortex and hippocampus by registering with structural magnetic resonance imaging data. The specificity of PBB5 was validated by immunohistochemistry with anti‐phosphorylated tau antibody AT‐100.ConclusionOptoacoustic imaging with PBB5 facilitates preclinical high‐resolution whole brain imaging of tauopathy, for studying the spreading and accumulation of tauopathy, and for monitoring putative treatments targeting tau.

Mass spectrometric measurement of six site‐specific tau phosphorylations in CSF and blood of Alzheimer's disease patients

AbstractBackgroundHyperphosphorylation of tau is responsible for its loss of normal physiological function, gain of toxicity and its aggregation to form NFTs. Increased phosphorylated tau is detected in Alzheimer´s disease (AD) CSF and blood, and changes in the tau phosphorylation pattern may reflect different stages of the disease progression. A better understanding on how the levels of site‐specific phosphorylations are modulated across the AD continuum in these biofluids may help to provide with a more accurate diagnosis.MethodWe developed an LC‐MS method based on parallel reaction monitoring (PRM) using Orbitrap MS to measure tau phosphorylations at positions pT181, pS199, pT205, pT217 and pT231 in CSF and plasma, and additionally p396 in CSF. Tau enrichment prior to LC‐MS differed between biofluids. For CSF, 250 µl of sample were prepared by performing partial PCA protein precipitation, followed by SPE and tryptic digestion. For plasma, 1 ml samples were subjected to immunoprecipitation using a combination of several tau antibodies, followed by tryptic digestion and SPE.ResultThe performance of the method was first evaluated by analysis of CSF from neurochemically characterized AD patients and controls (n=40). All measured phosphorylations showed significant separation between groups (p<0.001) (Fig. 1). In addition, pT181, pT217 and pT231 showed good correlations with SIMOA immunoassays. In plasma, a set of AD patients and controls (n=26) were analysed. All phosphorylations showed an increase in AD, and most significantly pT205 (p=0.003) and pT231 (p=0.008) (Fig 2). Interestingly, these two phosphorylations were the ones that showed higher correlation with p‐tau CSF (Spearman r=0.52 and r=0.63, respectively). Plasma values were also compared to the respective SIMOA immunoassays. Finally, the method was used to measure samples from the Translational biomarker In Aging and Dementia (TRIAD) cohort (n=209), consisting of young individuals (<30 years old), cognitive unimpaired, mild cognitive impairment (MCI) and AD patients.ConclusionWith our LC‐MS method to quantify site‐specific p‐tau levels, we have established the tau phosphorylation pattern in AD biofluids. Our data indicates that phosphorylation at position T231 is the one that shows a higher fold change between AD and controls both in CSF and blood.

Characterizing neurofibrillary tangle maturity of phosphorylated tau fluid biomarkers in Alzheimer's disease

Phosphorylated tau (p-tau) cerebrospinal fluid and plasma biomarkers have become more accurate in predicting Alzheimer's disease (AD) pathology. Although p-tau fluid biomarkers have been studied in vivo for nearly 20 years, tau phosphorylation sites have not been systematically studied in the context of neurofibrillary tangle maturity in the postmortem brain. Neurofibrillary tangles have a lifespan that is defined by three maturity levels: pretangles, mature tangles, and ghost tangles. Our goal was to characterize four tau phosphorylation sites (pT181, pT205, pT217, and pT231) in postmortem brain to determine which neurofibrillary tangle maturity level they preferentially recognize.The FLorida Autopsied Multi-Ethnic (FLAME) cohort was queried for cases at each Braak stage (I-VI). We excluded α-synucleinopathies, TDP-43 proteinopathies, and non-AD primary tauopathies, and those lacking available tissue. Ethnoracial diversity was prioritized for final case selection. A total of 24 cases were evaluated with each Braak stage containing 2 males and 2 females. For Braak stages IV-VI, only cases with a neuropathologic diagnosis of typical AD were selected. Posterior hippocampus was stained by immunohistochemistry for four antibodies: AT270 (pT181), pT205, pT217, and AT180 (pT231). Slides were digitized using Leica's Aperio technology to enable quantification of tau burden in the CA1 subsector and subiculum of the hippocampus.All tau phosphorylation sites recognized pretangles and mature tangles, and rarely recognized ghost tangles. Digital pathology measures of pT181, pT205, pT217, and pT231 burden showed subtle increases from Braak I-III. A greater increase was observed from Braak IV onward, except in pT217 where the burden decreased from Braak IV to Braak V before maxing out in Braak VI. pT205 and pT217 had the largest increase between Braak III and Braak VI compared to pT181 and pT231.pT181, pT205, pT217, and pT231 recognize primarily early neurofibrillary tangle maturity levels. This finding could explain why these p-tau sites are recognized in fluids before symptom onset. Future studies will compare these p-tau sites to other commonly used tau antibodies for AD research, including AT8 and PHF-1, as well as to in vivo measures in autopsied study participants who underwent antemortem biomarker evaluation.

Quantitative systems pharmacology model of the effect of tau hyperphosphorylation on synaptic plasticity.

The hyperphosphorylated tau is a principal component of neurofibrillary tangles, and has been identified as a key molecule in Alzheimer's disease. Fyn-dependent tau hyperphosphorylation plays a major role in excitotoxicity of extrasynaptic NMDAr. Long-term potentiation (LTP) of excitatory synaptic transmission is a key mechanism for learning and memory. In animal models of Alzheimer's disease (AD), tau hyperphosphorylation reduces LTP. Tau phosphorylation level in the cell is balanced through phosphorylation by protein kinases and dephosphorylation by phosphates. Tau hyperphosphorylation has been implicated in glutamate excitotoxicity and calcium released from ER (endoplasmic reticulum), which leads to synaptic dysfunction. Tau pathology is modeled often okadaic acid (OA) inhibits the ability of PP2A to dephosphorylate tau. Increases of PP2A inhibitors have been reported in AD's patients. Quantitative systems pharmacology (QSP) model of synaptic plasticity was developed to investigate influence of tau hyperphosphorylation on impairment of LTP. The model describes a calcium influx in postsynapse through acetylcholine and glutamate receptors, calcium release from EPR, depending on IP3 receptors, activation of kinase/phosphatase cascade, tau phosphorylation and AMPA receptors phosphorylation level. Phosphorylation of AMPA receptors is associated with LTP. Influence of hyperphosphorylated tau on extrasynaptic NMDAr and calcium release from ER impairs the synaptic plasticity. Various data on synaptic plasticity in presence of different effectors (amyloid beta, memantine, SSR180711 etc.), available from the literature, are used for verification. The model qualitatively describes tau hyperphosphorylation, impairment of LTP by tau hyperphosphorylation influence and effect of tau antibody, kinase and phosphatase inhibitors. It demonstrates that: 1) tau with anti-aggregant properties may lead to a robust LTP, that was even more pronounced than control tau 2) antibody to tau decreases tau hyperhyperphosphorylation and restores LTP reduced by OA influence 3) inhibition of GSK-3b kinase leads attenuates hyperphosphorylation and leads to recovery of LTP CONCLUSION: The QSP model describes an influence of tau hyperphosphorylation on receptor's excitotoxicity and released calcium from ER (high activity of PP2b, increased intracellular calcium concentration). Our model allows to explore change of LTP under the influence of tau targeting therapy.