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Phospho-Threonine-231 and MC1-tau immunoreactivity in brain. Brains from P301S and JNPL3 animals were harvested and stained with either anti tau phospho-Thr231 antibody (A) or anti-MC1-tau (B). (A): a, b, d, e Representative images of CA1 hippocampal cell layer and entorhinal cortex (EC) stained with anti-pThr231. Control mice received AAV1-CAG-eGFP injection (n = 13). Treated mice were injected with AAV1-CAG-scFvMC1 (n = 13). (A): c,f Semi-quantification of percentage of area stained by RZ3 shows a trend of reduction in the AAV1-CAG-scFvMC1 injected group, in the CA1 region of hippocampus in the JNPL3 (c, p = 0.1128, unpaired t test with Welch's correction); no trend to reduction is detected in the entorhinal cortex (EC) (f, p = 0.3458, unpaired t test with Welch's correction). (A): g, h, j, k Representative images of the CA1 region and entorhinal cortex (EC) from P301S; pThr-231 staining was performed as above. Control mice received AAV1-CAG-eGFP injection (n = 6), treated mice were injected with AAV1-CAG-scFvMC1 (n = 6); (A) i, l Semi-quantification of percentage of area stained by RZ3 in P301S mice (i, p = 0.2403; l, p = 0.2251; non parametric MannWhitney test). (B): a, b, d, e Representative images of CA1 hippocampal cell layer and entorhinal cortex (EC) stained with anti-MC1 antibody. Control mice received AAV1-CAG-eGFP injection (n = 13). Treated mice were injected with AAV1-CAG-scFvMC1 (n = 13) (c, f) Semi-quantification of percentage of area stained by MC1 shows a trend of reduction in the AAV1-CAG-scFvMC1 injected group, in the CA1 region of hippocampus (c, p = 0.0996; unpaired t test with Welch's correction); no reduction is detected in the entorhinal cortex (EC) (f, p = 0.9558; unpaired t test with Welch's correction) in JNPL3. (g, h, j, k) Representative images of the CA1 region and the entorhinal cortex (EC) from P301S; MC1 staining was performed as above. Control mice received AAV1-CAG-eGFP injection (n = 6), treated mice were injected with AAV1-CAG-scFvMC1 (n = 6). (i, l) Semi-quantification of percentage of area stained by MC1 in P301S mice (i, p = 0.4127; l, p = 0.8413; non parametric Mann-Whitney). (Olympus BH-2 bright field microscope; scale bar: 100 μm). Graphs are expressed as % Control area stained, and means +/− SEM
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Abstract With evidence supporting the prion-like spreading of extracellular tau as a mechanism for the initiation and progression of Alzheimer’s disease (AD), immunotherapy has emerged as a potential disease-modifying strategy to target tau. Many studies have proven effective to clear pathological tau species in animal models of AD, and several cli...
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... addition we performed an immunohistochemical analysis on JNPL3 (Fig. 6A a-f; Fig. 6B a-f) and P301S brain slices (Fig. 6A g-l; Fig. 6B g-l), by assessing tau pathology in the CA1 hippocampal pyramidal cell layer and in the entorhinal cortex (EC). Semi-quantification of tau phosphorylated at Thr231 and MC1-tau was performed on both CA1 (Fig. 6A c, i; Fig. 6B c, i) and EC (Fig. 6A f, l; Fig. 6B f, l). No significant ...
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... addition we performed an immunohistochemical analysis on JNPL3 (Fig. 6A a-f; Fig. 6B a-f) and P301S brain slices (Fig. 6A g-l; Fig. 6B g-l), by assessing tau pathology in the CA1 hippocampal pyramidal cell layer and in the entorhinal cortex (EC). Semi-quantification of tau phosphorylated at Thr231 and MC1-tau was performed on both CA1 (Fig. 6A c, i; Fig. 6B c, i) and EC (Fig. 6A f, l; Fig. 6B f, l). No significant changes in immunoreactivity were observed, except some ...
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... addition we performed an immunohistochemical analysis on JNPL3 (Fig. 6A a-f; Fig. 6B a-f) and P301S brain slices (Fig. 6A g-l; Fig. 6B g-l), by assessing tau pathology in the CA1 hippocampal pyramidal cell layer and in the entorhinal cortex (EC). Semi-quantification of tau phosphorylated at Thr231 and MC1-tau was performed on both CA1 (Fig. 6A c, i; Fig. 6B c, i) and EC (Fig. 6A f, l; Fig. 6B f, l). No significant changes in immunoreactivity were observed, except some trends towards decreased staining in the hippocampal region of JNPL3 mice (Fig. 6 A c, p = 0.1128; Fig. 6 B c, p = 0.0996). Tau phosphorylation at Ser396/404 and Ser202 (not shown) also failed to show significant reduction when ...
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... immunohistochemical analysis on JNPL3 (Fig. 6A a-f; Fig. 6B a-f) and P301S brain slices (Fig. 6A g-l; Fig. 6B g-l), by assessing tau pathology in the CA1 hippocampal pyramidal cell layer and in the entorhinal cortex (EC). Semi-quantification of tau phosphorylated at Thr231 and MC1-tau was performed on both CA1 (Fig. 6A c, i; Fig. 6B c, i) and EC (Fig. 6A f, l; Fig. 6B f, l). No significant changes in immunoreactivity were observed, except some trends towards decreased staining in the hippocampal region of JNPL3 mice (Fig. 6 A c, p = 0.1128; Fig. 6 B c, p = 0.0996). Tau phosphorylation at Ser396/404 and Ser202 (not shown) also failed to show significant reduction when comparing treated to non-treated ...
Citations
... Interestingly, Vitale et al. demonstrated the in vivo feasibility and efficacy of targeting pathological tau in the brain, by employing intramuscular (IM) delivery of vectorized anti-tau scFvMCI. Two different tau transgenic models received a single IM injection of AAV1-scFvMC1, showing a significant reduction of tau pathology and more interestingly even if the scFvMC1 was internalized by the microglia they could not find any inflammatory reactions in the brain [114]. However, understanding the consequence of tau knockdown at adulthood is fundamental to halt pathological tau while preserving healthy tau. ...
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline, memory loss, and behavioral impairments. Despite extensive research efforts, effective treatment options for AD remain limited. Recently, gene therapy has emerged as a promising avenue for targeted intervention in the pathogenesis of AD. This review will provide an overview of clinical and preclinical studies where gene therapy techniques have been utilized in the context of AD, highlighting their potential as novel therapeutic strategies. While challenges remain, ongoing research and technological advancement continue to enhance the potential of gene therapy as a targeted and personalized therapeutic approach for AD.
... [54] AAV-expressed scFv-MC1 Tau, Alzheimer's disease and other Tauopathies Reduces tau pathology in Tg mice. [55,56] AAV-expressed AT8-scFv Tau, Alzheimer's disease and other Tauopathies Crosses the BBB and enters neurons. [57] VHH-E4-1 and VHH-Z70 Tau, Alzheimer's disease and other Tauopathies Inhibit Tau seeding in Tg mice. ...
... An scFv antibody fragment, designated scFv-MC1 and based on the variable heavy (VH) and variable light (VL) chains of the well-characterized anti-tau monoclonal antibody MC1, was constructed, cloned into the AAV, and shown to reduce tau pathology after intracranial injection in adult JNPL3 transgenic mice, expressing human P301L-mutant tau [55]. Subsequently, the authors demonstrated that a single intramuscular injection of AAV1-scFvMC1 significantly reduces insoluble and soluble tau aggregates in the brain of two different tau transgenic mice, JNPL3 and P301S [56]. The authors detected scFvMC1 in the microglia, and, importantly, no inflammatory molecules were detected in the brain, suggesting the therapeutic potential of anti-tau scFv for AD and other tauopathies without side effects [56]. ...
... Subsequently, the authors demonstrated that a single intramuscular injection of AAV1-scFvMC1 significantly reduces insoluble and soluble tau aggregates in the brain of two different tau transgenic mice, JNPL3 and P301S [56]. The authors detected scFvMC1 in the microglia, and, importantly, no inflammatory molecules were detected in the brain, suggesting the therapeutic potential of anti-tau scFv for AD and other tauopathies without side effects [56]. AT8-scFv fragment, based on the VL and VH regions of another well-characterized anti-phospho-Tau (Ser202, Thr205) monoclonal antibody AT8, was cloned into the AAV and shown to cross the BBB, infect neurons and express AT8-scFv for at least 22 weeks [57]. ...
Recombinant antibody fragments are promising alternatives to full-length immunoglobulins, creating big opportunities for the pharmaceutical industry. Nowadays, antibody fragments such as antigen-binding fragments (Fab), single-chain fragment variable (scFv), single-domain antibodies (sdAbs), and bispecific antibodies (bsAbs) are being evaluated as diagnostics or therapeutics in pre-clinical models and in clinical trials. Immunotherapy approaches, including passive transfer of protective antibodies, have shown therapeutic efficacy in several animal models of Alzheimer ́s disease(AD), Parkinson ́s disease (PD), frontotemporal dementia (FTD), Huntington ́s disease (HD), transmissible spongiform encephalopathies (TSEs) and multiple sclerosis (MS). There are various antibodies approved by the Food and Drug Administration (FDA) for treating multiple sclerosis and two amyloid beta-specific humanized antibodies, Aducanumab and Lecanemab, for AD. Our previous review summarized data on recombinant antibodies evaluated in pre-clinical models for immunotherapy of neurodegenerative diseases. Here, we explore recent studies in this fascinating research field, give an update on new preventive and therapeutic applications of recombinant antibody fragments for neurological disorders and discuss the potential of antibody fragments for developing novel approaches for crossing the blood-brain barrier (BBB) and targeting cells and molecules of interest in the brain.
... The nanobodies reported in this study should be considered in the context of similar antibodies and related nanobodies that have previously been reported. The vast majority of reported tau conformational antibodies have been conventional IgGs (45)(46)(47)(48)(49). These antibodies have been critical to studying differences in tau fibril morphology present in different tauopathies (45), understanding the progression of tau aggregation (46, 47), and testing the effects of targeting tau aggregates using in vivo models of neurological disease (48)(49)(50). ...
... The vast majority of reported tau conformational antibodies have been conventional IgGs (45)(46)(47)(48)(49). These antibodies have been critical to studying differences in tau fibril morphology present in different tauopathies (45), understanding the progression of tau aggregation (46, 47), and testing the effects of targeting tau aggregates using in vivo models of neurological disease (48)(49)(50). Similar to our findings, these antibodies have been reported to selectively recognize aggregates in mouse and human brain tissues (45)(46)(47)(48)(49)(50). ...
... These antibodies have been critical to studying differences in tau fibril morphology present in different tauopathies (45), understanding the progression of tau aggregation (46, 47), and testing the effects of targeting tau aggregates using in vivo models of neurological disease (48)(49)(50). Similar to our findings, these antibodies have been reported to selectively recognize aggregates in mouse and human brain tissues (45)(46)(47)(48)(49)(50). Our findings that these nanobody-Fc fusion proteins demonstrate conformational specificity for recombinant fibrils (Figure 2), aggregates formed in P301S transgenic mouse tissue (Figures 3, 4), and aggregates present in Alzheimer's disease (Figures 5, 6) and progressive supranuclear palsy ( Figure 5) brain tissue samples indicate that our nanobodies have potential for further evaluation and study of tau aggregates in neurodegenerative models. ...
Single-domain antibodies, also known as nanobodies, are broadly important for studying the structure and conformational states of several classes of proteins, including membrane proteins, enzymes, and amyloidogenic proteins. Conformational nanobodies specific for aggregated conformations of amyloidogenic proteins are particularly needed to better target and study aggregates associated with a growing class of associated diseases, especially neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases. However, there are few reported nanobodies with both conformational and sequence specificity for amyloid aggregates, especially for large and complex proteins such as the tau protein associated with Alzheimer’s disease, due to difficulties in selecting nanobodies that bind to complex aggregated proteins. Here, we report the selection of conformational nanobodies that selectively recognize aggregated (fibrillar) tau relative to soluble (monomeric) tau. Notably, we demonstrate that these nanobodies can be directly isolated from immune libraries using quantitative flow cytometric sorting of yeast-displayed libraries against tau aggregates conjugated to quantum dots, and this process eliminates the need for secondary nanobody screening. The isolated nanobodies demonstrate conformational specificity for tau aggregates in brain samples from both a transgenic mouse model and human tauopathies. We expect that our facile approach will be broadly useful for isolating conformational nanobodies against diverse amyloid aggregates and other complex antigens.
... 90% JNPL3 mice exhibited motor and behavioral problems by 10 months (Lewis et al., 2000). This model is often used to evaluate the effect of different types of tau antibodies on tau pathology of AD, such as single domain antibodies (Congdon et al., 2022), phosphorylated tau-specific intrabodies (Goodwin et al., 2021), vectorized single chain variable fragment (Vitale et al., 2020), and the interaction between Aβ and tau by (Tanemura et al., 2001; a) The table is ordered by mutation site alphabetically. "-" means no data. ...
Transgenic models are useful tools for studying the pathogenesis of and drug development for Alzheimer's Disease (AD). AD models are constructed usually using overexpression or knock-in of multiple pathogenic gene mutations from familial AD. Each transgenic model has its unique behavioral and pathological features. This review summarizes the research progress of transgenic mouse models, and their progress in the unique mechanism of amyloid-β oligomers, including the first transgenic mouse model built in China based on a single gene mutation (PSEN1 V97L) found in Chinese familial AD. We further summarized the preclinical findings of drugs using the models, and their future application in exploring the upstream mechanisms and multitarget drug development in AD.
... E2 and E3 represent exon 2 and 3, respectively; M1-M4 is the microtubule-binding domain of tau. [54]; and the other investigating the feasibility of antibodies against tau in early pathological confirmation, such as MC1, a conformationdependent IgG1 antibody [55], DMR7, and SKT82 [56]. On the other hand, Pan-tau antibodies have various epitopes to recognize the tau protein's N domain or mid-domain. ...
... These therapeutic targets and strategies can be an inspiration for clinical trials in the near future. Table 1 summarizes the recent literature and its results in vivo [53][54][55][56]61,[65][66][67][68]. Table 2 provides an overview of current therapies and clinical trials under investigation and the results of these studies [27,62,63,69,70]. ...
Tubulin-associated unit (tau) has been associated with more than 25 neurological disorders–the so-called tauopathies. Hence, finding a novel therapeutic agent targeting tau to halt the progression of diseases has been of interest. Alzheimer’s disease (AD) and progressive supranuclear palsy (PSP) are the most studied tauopathies. AD is characterized by two cardinal pathological mechanisms: amyloid β (Aβ) plaques and neurofibrillary tangles (NFTs), leading to atrophy of the brain. Over the last few years, attention has been on NFTs composed of the hyperphosphorylated microtubule-associated protein tau. Tau contributes to the synaptic plasticity of axons; hyperphosphorylated and aggregated tau have been shown to increase the likelihood of cognitive impairments. PSP is also associated with tau accumulation in NFTs and neuropil threads, making this condition a candidate for tau-targeted therapies. Many tau-targeting therapies have been developed, and clinical trials are being conducted. Tau-targeting drugs are classified into four subgroups based on the pathological target: tau phosphorylation inhibitors, stabilizers of microtubules, enhancing tau clearance, and tau aggregation inhibitors. On the other hand, the desired specificity and sensitivity of tau immunotherapy agents without interrupting normal proteome are the fundamental point of tremendous attention. Starting with animal studies of these therapies to clinical trials, both are divided into passive and active immunotherapies, while passive immunotherapies are the method of desire. Targeting aggregation and phosphorylation sites of tau is the chief target of therapies. This article reviews the latest animal and clinical studies of tau-based immunotherapies and tau-targeted drugs for AD and PSP.
... TRIM21 was originally shown to mediate intracellular clearance of antibody-coated viruses by catalyzing ubiquitination of antibody bound virus and degradation via the ubiquitin proteasome system and similar data exists that TRIM21 can play a role in degrading internalized tau aggregates [116,117]. Notably, several studies show that single domain variable fragments lacking Fc domain can modulate tau pathology in vivo; such data would suggest that Fc-mediated mechanisms are not responsible for efficy of tau immunotherapies [118][119][120]. Though most therapeutic approaches have focused on using monoclonal tau antibodies, active immunization strategies have also been pursued. ...
Scientific advances over the last four decades have steadily infused the Alzheimer’s disease (AD) field with great optimism that therapies targeting Aβ, amyloid, tau, and innate immune activation states in the brain would provide disease modification. Unfortunately, this optimistic scenario has not yet played out. Though a recent approval of the anti-Aβ aggregate binding antibody, Aduhelm (aducanumab), as a “disease-modifying therapy for AD” is viewed by some as a breakthrough, many remain unconvinced by the data underlying this approval. Collectively, we have not succeeded in changing AD from a largely untreatable, inevitable, and incurable disease to a treatable, preventable, and curable one. Here, I will review the major foci of the AD “disease-modifying” therapeutic pipeline and some of the “open questions” that remain in terms of these therapeutic approaches. I will conclude the review by discussing how we, as a field, might adjust our approach, learning from our past failures to ensure future success.
... Removing either component of the epitope abolishes the binding. Reactivity to the antibody appears to be dependent based on the severity and progression of AD (Vitale et al., 2020). In vivo data coming after injections in the forebrain of transgenic P301L tau mice have shown that MC-1 could reduce tau pathology (Chai et al., 2011b;d'Abramo et al., 2013). ...
... In mouse models, both active and passive immunotherapies have been shown to be capable of reducing the formation of intracellular tau pathology as well as provided significant delays in the onset of the degenerative phenotype induced by tau Boutajangout, Ingadottir, Davies, & Sigurdsson, 2011;Chai et al., 2011a;d'Abramo, Acker, Jimenez, & Davies, 2013;Sankaranarayanan et al., 2015;Theunis et al., 2017Theunis et al., , 2013. The potential to reduce tau pathology has been also demonstrated by the use of antibody fragments like the single-chain variable fragments (scFvs) tested either in tau pathology mouse models Goodwin et al., 2021;Ising et al., 2017;Vitale et al., 2020) or Drosophila models (S. Li et al., 2021). ...
... Lately, gene therapy has gained a lot of importance, mainly using recombinant adeno-associated virus (rAAV) that are considered safer than LVs for medical applications. Thus, a deeper research of the potential use of rAAV vectors encoding tau-targeted antibodies or antibody fragments, like in the case of VHH Z70, could be used to more directly target tau within the mouse brain (Ising et al., 2017;Vitale et al., 2020), via one-time intracranial injection. However, peripheral administration provides obvious advantages compared to AAV gene therapy: a non-invasive route of injection, lack of surgery-related side effects, improved patient compliance and costs. ...
Tau is a microtubule-associated protein, best known to regulate cytoskeletal dynamics of neurons in the brain. Although the mechanisms leading to Tau aggregation in tauopathies are still ill-defined, the peptide PHF6 is described as a nucleus of Tau aggregation. Immunotherapy is proposed as a therapeutic approach in tauopathies. This strategy is indeed effective in attenuating Tau pathology in animal models, improving also cognitive and motor functions. However, it remains important to better define several parameters to design the best strategy in Tau-specific immunotherapy. For example, even-though the intracellular accumulation of Tau aggregates into paired helical filaments (PHFs) are the major events connected to neuropathological lesions, extracellular tau is lately considered as the key driver in the spread and seeding of the pathology. In view of the challenges to design the most effective Tau-specific immunotherapy, we have used antibody fragments called VHHs (Variable domain of the Heavy- chain of the Heavy-chain-only-antibodies, or nanobodies) because they are easier to generate, select and engineered compared to conventional antibodies. Of interest, VHHs can be used for intracellular applications, by engineering their ability to penetrate the cells or by direct intracellular expression. A VHH named Z70, binding the PHF6 peptide, was previously selected, characterized and optimized in the laboratory. VHH Z70 has the ability to block Tau seeding in in vitro assays. Based on these results, VHH Z70 capacity of blocking Tau seeding was investigated in THY-tau30 transgenic mouse model. For this, LVVs expressing VHH Z70, VHH Z70 fused to mCherry, VHH Z70 fused to a Fc fragment of mouse immunoglobulin (Minibody) and VHH anti-GFP for negative control, were produced and injected to one-month old mice in the hippocampus area. Exogenous human Alzheimer’s disease brain lysates were subsequently injected to induce the endogenous aggregation process. AT-8 immunostaining was chosen to define the level of the pathology in the brain. Animals injected with LVVs (lentiviral vectors) expressing VHH Z70 showed a decreases tendency of the seeded Tau pathology, but this tendency became only significant in the case of VHH Z70 fused to mCherry. For the mice treated with VHHs expressing the Minibody, the immunohistochemistry analysis did not reveal any change in the treated mice, compared to the negative control (Minibody VHH anti-GFP). Given these promising results, optimizations of VHH Z70 activity in the intracellular compartment were performed. The strategy for optimization involved random mutagenesis coupled with yeast two-hybrid for selection, in partnership with Hybrigenics company. Eight functional variants were selected from this screen with conserved epitope recognition. Determination of the affinity by surface plasmon resonance suggested that the binding of the epitope can be improved by mutations located in both the complementary determining regions and the framework regions. These variants were further tested in comparison with VHH Z70 for their ability to inhibit Tau aggregation in fluorescence-based seeding reporter cells. Three of these mutants have an in vitro inhibitory activity that matched VHH Z70 and could be used in future in vivo assays. To conclude, the results of this three-year project have established that VHH Z70 has potential in mitigating Tau accumulation, at least in a mouse model of seeding. In addition, these studies demonstrated that the Tau-specific VHHs are useful molecular tools to decipher the best target in Tau immunotherapies.
... Another study by this group on the same mice models showed that a single intramuscular (IM) injection of the previously used vectorized scFv derived from MC-1 stimulated long term production of anti-tau scFvMC1, and significantly decreased insoluble and soluble tau in different brain regions. In addition, this study highlighted the key role of microglia in clearance of scFv-tau [53]. The role of microglia in MC-1 mediated tau clearance was also assessed by Luo and colleagues, who used the P301S mice model and showed that MC-1 accelerates the uptake and clearance of pathological tau species by microglia in an Fc-dependent manner [54]. ...
Alzheimer's disease (AD) is the leading cause of dementia that has remained a major medical, sociocultural and economical challenge globally. Previously developed treatments like anticholinesterase inhibitors (AChEIs) and N-methyl-D-aspartate receptor (NMDAR) antagonists only provide short-term symptomatic improvement and do not prevent progression. Repeated setbacks and failures over the past 25 years in AD clinical trials have hindered efforts to develop effective AD treatments. Fortunately, Aducanumab, a specific anti-amyloid β antibody, has shown promising clinical results and was recently approved by the Food and Drug Administration (FDA) through an accelerated approval pathway. This has raised hopes for AD patients; however post-approval trials are necessary to estimate the true scope of its clinical benefits. We have reviewed several AD clinical studies and summarized the experience to date with Aducanumab and two other potential AD drugs including Zagotenemab (an anti-tau antibody) and Pioglitazone (nuclear Peroxisome-Proliferator Activated Receptor γ (PPARγ) agonist). These have shown mixed results so far and the next few years will be critical to elucidate and interpret their broad long-term protective effects. A concerted effort is required to understand and strengthen the translation of pre-clinical findings from these drugs to routine clinical practice.
