Single-domain antibody-based protein degrader for synucleinopathies.

Synucleinopathies are a group of neurodegenerative diseases characterized by the accumulation of α-synuclein (α-syn) in the brain, leading to motor and neuropsychiatric symptoms. Currently, there are no known cures for synucleinopathies, and treatments mainly focus on symptom management. In this study, we developed a single-domain antibody (sdAb)-based protein degrader with features designed to enhance proteasomal degradation of α-syn. This sdAb derivative targets both α-syn and Cereblon (CRBN), a substrate-receptor for the E3-ubiquitin ligase CRL4CRBN, and thereby induces α-syn ubiquitination and proteasomal degradation. Our results indicate that this therapeutic candidate enhances proteasomal degradation of α-syn, in addition to the endogenous lysosomal degradation machinery. By promoting proteasomal degradation of α-syn, we improved clearance of α-syn in primary culture and mouse models of synucleinopathy. These findings indicate that our sdAb-based protein degrader is a promising therapeutic candidate for synucleinopathies. Considering that only a small percentage of antibodies enter the brain, more potent sdAbs with greater brain entry than whole antibodies could enhance clinical benefits of antibody-based therapies.

Single-Domain Antibody-Based Protein Degrader for Synucleinopathies.

Synucleinopathies are a group of neurodegenerative diseases characterized by the accumulation of α-synuclein (α-syn) in the brain, leading to motor and neuropsychiatric symptoms. Currently, there are no known cures for synucleinopathies, and treatments mainly focus on symptom management. In this study, we developed a single-domain antibody (sdAb)-based protein degrader with features designed to enhance proteasomal degradation of α-syn. This sdAb derivative targets both α-syn and Cereblon (CRBN), a substrate-receptor for the E3-ubiquitin ligase CRL4CRBN, and thereby induces α-syn ubiquitination and proteasomal degradation. Our results indicate that this therapeutic candidate enhances proteasomal degradation of α-syn, in addition to the endogenous lysosomal degradation machinery. By promoting proteasomal degradation of α-syn, we improved clearance of α-syn in primary culture and mouse models of synucleinopathy. These findings indicate that our sdAb-based protein degrader is a promising therapeutic candidate for synucleinopathies. Considering that only a small percentage of antibodies enter the brain, more potent sdAbs with greater brain entry than whole antibodies could enhance clinical benefits of antibody-based therapies.

Tau-targeting therapies for Alzheimer disease: current status and future directions.

Alzheimer disease (AD) is the most common cause of dementia in older individuals. AD is characterized pathologically by amyloid-ß (Aß) plaques and tau neurofibrillary tangles in the brain, with associated loss of synapses and neurons, which eventually results in dementia. Many of the early attempts to develop treatments for AD focused on Aß, but a lack of efficacy of these treatments in terms of slowing disease progression led to a change of strategy towards targeting of tau pathology. Given that tau shows a stronger correlation with symptom severity than does Aß, targeting of tau is more likely to be efficacious once cognitive decline begins. Anti-tau therapies initially focused on post-translational modifications, inhibition of tau aggregation and stabilization of microtubules. However, trials of many potential drugs were discontinued because of toxicity and/or lack of efficacy. Currently, the majority of tau-targeting agents in clinical trials are immunotherapies. In this Review, we provide an update on the results from the initial immunotherapy trials and an overview of new therapeutic candidates that are in clinical development, as well as considering future directions for tau-targeting therapies.

Single-domain antibody-based noninvasive in vivo imaging of α-synuclein or tau pathology.

Intracellular deposition of α-synuclein and tau are hallmarks of synucleinopathies and tauopathies, respectively. Recently, several dye-based imaging probes with selectivity for tau aggregates have been developed, but suitable imaging biomarkers for synucleinopathies are still unavailable. Detection of both of these aggregates early in the disease process may allow for prophylactic therapies before functional impairments have manifested, highlighting the importance of developing specific imaging probes for these lesions. In contrast to the ß sheet dyes, single-domain antibodies, found in camelids and a few other species, are highly specific, and their small size allows better brain entry and distribution than whole antibodies. Here, we have developed such imaging ligands via phage display libraries derived from llamas immunized with α-synuclein and tau preparations, respectively. These probes allow noninvasive and specific in vivo imaging of α-synuclein versus tau pathology in mice, with the brain signal correlating strongly with lesion burden. These small antibody derivatives have great potential for in vivo diagnosis of these diseases.

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

BACKGROUND: Eleven 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. METHODS: We 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. FINDINGS: Unmodified 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. INTERPRETATION: These 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. FUNDING: Funding 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).

Targeting tau only extracellularly is likely to be less efficacious than targeting it both intra- and extracellularly.

Aggregation of the tau protein is thought to be responsible for the neurodegeneration and subsequent functional impairments in diseases that are collectively named tauopathies. Alzheimer's disease is the most common tauopathy, but the group consists of over 20 different diseases, many of which have tau pathology as their primary feature. The development of tau therapies has mainly focused on preventing the formation of and/or clearing these aggregates. Of these, immunotherapies that aim to either elicit endogenous tau antibodies or deliver exogenous ones are the most common approach in clinical trials. While their mechanism of action can involve several pathways, both extra- and intracellular, pharmaceutical companies have primarily focused on antibody-mediated clearance of extracellular tau. As we have pointed out over the years, this is rather surprising because it is well known that most of pathological tau protein is found intracellularly. It has been repeatedly shown by several groups over the past decades that antibodies can enter neurons and that their cellular uptake can be enhanced by various means, particularly by altering their charge. Here, we will briefly describe the potential extra- and intracellular mechanisms involved in antibody-mediated clearance of tau pathology, discuss these in the context of recent failures of some of the tau antibody trials, and finally provide a brief overview of how the intracellular efficacy of tau antibodies can potentially be further improved by certain modifications that aim to enhance tau clearance via specific intracellular degradation pathways.

Increased neuronal activity in motor cortex reveals prominent calcium dyshomeostasis in tauopathy mice.

Perturbed neuronal Ca2+ homeostasis is implicated in Alzheimer's disease, which has primarily been demonstrated in mice with amyloid-ß deposits but to a lesser and more variable extent in tauopathy models. In this study, we injected AAV to express Ca2+ indicator in layer II/III motor cortex neurons and measured neuronal Ca2+ activity by two photon imaging in awake transgenic JNPL3 tauopathy and wild-type mice. Various biochemical measurements were conducted in postmortem mouse brains for mechanistic insight and a group of animals received two intravenous injections of a tau monoclonal antibody spaced by four days to test whether the Ca2+ dyshomeostasis was related to pathological tau protein. Under running conditions, we found abnormal neuronal Ca2+ activity in tauopathy mice compared to age-matched wild-type mice with higher frequency of Ca2+ transients, lower amplitude of peak Ca2+ transients and lower total Ca2+ activity in layer II/III motor cortex neurons. While at resting conditions, only Ca2+ frequency was increased. Brain levels of soluble pathological tau correlated better than insoluble tau levels with the degree of Ca2+ dysfunction in tauopathy mice. Furthermore, tau monoclonal antibody 4E6 partially rescued Ca2+ activity abnormalities in tauopathy mice after two intravenous injections and decreased soluble pathological tau protein within the brain. This correlation and antibody effects strongly suggest that the neuronal Ca2+ dyshomeostasis is causally linked to pathological tau protein. These findings also reveal more pronounced neuronal Ca2+ dysregulation in tauopathy mice than previously reported by two-photon imaging that can be partially corrected with an acute tau antibody treatment.

Dynamics of Internalization and Intracellular Interaction of Tau Antibodies and Human Pathological Tau Protein in a Human Neuron-Like Model.

We and others have shown in various in vivo, ex vivo and cell culture models that several tau antibodies interact with pathological tau within neurons. To further clarify this interaction in a dynamic human model, we differentiated SH-SY5Y cells with retinoic acid and BDNF to create a neuron-like model. Therein, tau antibodies were primarily taken up by receptor-mediated endocytosis, and prevented toxicity of human brain-derived paired helical filament-enriched tau (PHF). Subsequently, we monitored in real-time the interaction of antibodies and PHF within endocytic cellular compartments. Cells were pre-treated with fluorescently-tagged PHF and then incubated with tau antibodies, 4E6, 6B2, or non-specific isotype control IgG1 labeled with a pH sensitive dye. The uptake and binding of the efficacious antibody, 4E6, to PHF occurred mainly within the soma, whereas the ineffective antibody, 6B2, and ineffective control IgG1, were visualized via the processes and showed limited colocalization with PHF within this period. In summary, we have developed a neuron-like model that clarifies the early intracellular dynamics of the interaction of tau antibodies with pathological tau, and identifies features associated with efficacy. Since the model is entirely human, it is suitable to verify the therapeutic potential of humanized antibodies prior to extensive clinical trials.

Tau antibody chimerization alters its charge and binding, thereby reducing its cellular uptake and efficacy.

BACKGROUND: Bringing antibodies from pre-clinical studies to human trials requires humanization, but this process may alter properties that are crucial for efficacy. Since pathological tau protein is primarily intraneuronal in Alzheimer's disease, the most efficacious antibodies should work both intra- and extracellularly. Thus, changes which impact uptake or antibody binding will affect antibody efficacy. METHODS: Initially, we examined four tau mouse monoclonal antibodies with naturally differing charges. We quantified their neuronal uptake, and efficacy in preventing toxicity and pathological seeding induced by human-derived pathological tau. Later, we generated a human chimeric 4E6 (h4E6), an antibody with well documented efficacy in multiple tauopathy models. We compared the uptake and efficacy of unmodified and chimeric antibodies in neuronal and differentiated neuroblastoma cultures. Further, we analyzed tau binding using ELISA assays. FINDINGS: Neuronal uptake of tau antibodies and their efficacy strongly depends on antibody charge. Additionally, their ability to prevent tau toxicity and seeding of tau pathology does not necessarily go together. Particularly, chimerization of 4E6 increased its charge from 6.5 to 9.6, which blocked its uptake into human and mouse cells. Furthermore, h4E6 had altered binding characteristics despite intact binding sites, compared to the mouse antibody. Importantly, these changes in uptake and binding substantially decreased its efficacy in preventing tau toxicity, although under certain conditions it did prevent pathological seeding of tau. CONCLUSIONS: These results indicate that efficacy of chimeric/humanized tau antibodies should be thoroughly characterized prior to clinical trials, which may require further engineering to maintain or improve their therapeutic potential. FUND: National Institutes of Health (NS077239, AG032611, R24OD18340, R24OD018339 and RR027990, Alzheimer's Association (2016-NIRG-397228) and Blas Frangione Foundation.

Structural characterization of monoclonal antibodies targeting C-terminal Ser404 region of phosphorylated tau protein.

Targeting tau with immunotherapies is currently the most common approach taken in clinical trials of patients with Alzheimer's disease. The most prominent pathological feature of tau is its hyperphosphorylation, which may cause the protein to aggregate into toxic assemblies that collectively lead to neurodegeneration. Of the phospho-epitopes, the region around Ser396/Ser404 has received particular attention for therapeutic targeting because of its prominence and stability in diseased tissue. Herein, we present the antigen-binding fragment (Fab)/epitope complex structures of three different monoclonal antibodies (mAbs) that target the pSer404 tau epitope region. Most notably, these structures reveal an antigen conformation similar to a previously described pathogenic tau epitope, pSer422, which was shown to have a ß-strand structure that may be linked to the seeding core in tau oligomers. In addition, we have previously reported on the similarly ordered conformation observed in a pSer396 epitope, which is in tandem with pSer404. Our data are the first Fab structures of mAbs bound to this epitope region of the tau protein and support the existence of proteopathic tau conformations stabilized by specific phosphorylation events that are viable targets for immune modulation.

Sex Differences in Autophagy Contribute to Female Vulnerability in Alzheimer's Disease.

Alzheimer's disease (AD) is the most common form of dementia, with over 5. 4 million cases in the US alone (Alzheimer's Association, 2016). Clinically, AD is defined by the presence of plaques composed of Aß and neurofibrillary pathology composed of the microtubule associated protein tau. Another key feature is the dysregulation of autophagy at key steps in the pathway. In AD, disrupted autophagy contributes to disease progression through the failure to clear pathological protein aggregates, insulin resistance, and its role in the synthesis of Aß. Like many psychiatric and neurodegenerative diseases, the risk of developing AD, and disease course are dependent on the sex of the patient. One potential mechanism through which these differences occur, is the effects of sex hormones on autophagy. In women, the loss of hormones with menopause presents both a risk factor for developing AD, and an obvious example of where sex differences in AD can stem from. However, because AD pathology can begin decades before menopause, this does not provide the full answer. We propose that sex-based differences in autophagy regulation during the lifespan contribute to the increased risk of AD, and greater severity of pathology seen in women.

Tau-targeting therapies for Alzheimer disease.

Alzheimer disease (AD) is the most common form of dementia. Pathologically, AD is characterized by amyloid plaques and neurofibrillary tangles in the brain, with associated loss of synapses and neurons, resulting in cognitive deficits and eventually dementia. Amyloid-ß (Aß) peptide and tau protein are the primary components of the plaques and tangles, respectively. In the decades since Aß and tau were identified, development of therapies for AD has primarily focused on Aß, but tau has received more attention in recent years, in part because of the failure of various Aß-targeting treatments in clinical trials. In this article, we review the current status of tau-targeting therapies for AD. Initially, potential anti-tau therapies were based mainly on inhibition of kinases or tau aggregation, or on stabilization of microtubules, but most of these approaches have been discontinued because of toxicity and/or lack of efficacy. Currently, the majority of tau-targeting therapies in clinical trials are immunotherapies, which have shown promise in numerous preclinical studies. Given that tau pathology correlates better with cognitive impairments than do Aß lesions, targeting of tau is expected to be more effective than Aß clearance once the clinical symptoms are evident. With future improvements in diagnostics, these two hallmarks of the disease might be targeted prophylactically.

Affinity of Tau antibodies for solubilized pathological Tau species but not their immunogen or insoluble Tau aggregates predicts in vivo and ex vivo efficacy.

BACKGROUND: A few tau immunotherapies are now in clinical trials with several more likely to be initiated in the near future. A priori, it can be anticipated that an antibody which broadly recognizes various pathological tau aggregates with high affinity would have the ideal therapeutic properties. Tau antibodies 4E6 and 6B2, raised against the same epitope region but of varying specificity and affinity, were tested for acutely improving cognition and reducing tau pathology in transgenic tauopathy mice and neuronal cultures. RESULTS: Surprisingly, we here show that one antibody, 4E6, which has low affinity for most forms of tau acutely improved cognition and reduced soluble phospho-tau, whereas another antibody, 6B2, which has high affinity for various tau species was ineffective. Concurrently, we confirmed and clarified these efficacy differences in an ex vivo model of tauopathy. Alzheimer's paired helical filaments (PHF) were toxic to the neurons and increased tau levels in remaining neurons. Both toxicity and tau seeding were prevented by 4E6 but not by 6B2. Furthermore, 4E6 reduced PHF spreading between neurons. Interestingly, 4E6's efficacy relates to its high affinity binding to solubilized PHF, whereas the ineffective 6B2 binds mainly to aggregated PHF. Blocking 4E6's uptake into neurons prevented its protective effects if the antibody was administered after PHF had been internalized. When 4E6 and PHF were administered at the same time, the antibody was protective extracellularly. CONCLUSIONS: Overall, these findings indicate that high antibody affinity for solubilized PHF predicts efficacy, and that acute antibody-mediated improvement in cognition relates to clearance of soluble phospho-tau. Importantly, both intra- and extracellular clearance pathways are in play. Together, these results have major implications for understanding the pathogenesis of tauopathies and for development of immunotherapies.

Harnessing the immune system for treatment and detection of tau pathology.

The tau protein is an attractive target for therapy and diagnosis. We started a tau immunotherapy program about 13 years ago and have since demonstrated that active and passive immunotherapies diminish tau pathology and improve function, including cognition, in different mouse models. These findings have been confirmed and extended by several groups. We routinely detect neuronal, and to a lesser extent microglial, antibody uptake correlating with tau pathology. Antibodies bind tau aggregates in the endosomal/lysosomal system, enhancing clearance presumably by promoting their disassembly. Extracellular clearance has recently been shown by others, using antibodies that apparently are not internalized. As most pathological tau is neuronal, intracellular targeting may be more efficacious. However, extracellular tau may be more accessible to antibodies, with tau-antibody complexes a target for microglial phagocytosis. The extent of involvement of each pathway may depend on numerous factors including antibody properties, degree of pathology, and experimental model. On the imaging front, multiple tau ligands derived from ß-sheet dyes have been developed by several groups, some with promising results in clinical PET tests. Postmortem analysis should clarify their tau specificity, as in theory and based on histological staining, those are likely to have some affinity for various amyloids. We are developing antibody-derived tau probes that should be more specific, and have in mouse models shown in vivo detection and binding to pathological tau after peripheral injection. These are exciting times for research on tau therapies and diagnostic agents that hopefully can be applied to humans in the near future.

Two novel Tau antibodies targeting the 396/404 region are primarily taken up by neurons and reduce Tau protein pathology.

Aggregated Tau proteins are hallmarks of Alzheimer disease and other tauopathies. Recent studies from our group and others have demonstrated that both active and passive immunizations reduce Tau pathology and prevent cognitive decline in transgenic mice. To determine the efficacy and safety of targeting the prominent 396/404 region, we developed two novel monoclonal antibodies (mAbs) with distinct binding profiles for phospho and non-phospho epitopes. The two mAbs significantly reduced hyperphosphorylated soluble Tau in long term brain slice cultures without apparent toxicity, suggesting the therapeutic importance of targeting the 396/404 region. In mechanistic studies, we found that neurons were the primary cell type that internalized the mAbs, whereas a small amount of mAbs was taken up by microglia cells. Within neurons, the two mAbs were highly colocalized with distinct pathological Tau markers, indicating their affinity toward different stages or forms of pathological Tau. Moreover, the mAbs were largely co-localized with endosomal/lysosomal markers, and partially co-localized with autophagy pathway markers. Additionally, the Fab fragments of the mAbs were able to enter neurons, but unlike the whole antibodies, the fragments were not specifically localized in pathological neurons. In summary, our Tau mAbs were safe and efficient to clear pathological Tau in a brain slice model. Fc-receptor-mediated endocytosis and the endosome/autophagosome/lysosome system are likely to have a critical role in antibody-mediated clearance of Tau pathology.

Antibody uptake into neurons occurs primarily via clathrin-dependent Fcγ receptor endocytosis and is a prerequisite for acute tau protein clearance.

Tau immunotherapy is effective in transgenic mice, but the mechanisms of Tau clearance are not well known. To this end, Tau antibody uptake was analyzed in brain slice cultures and primary neurons. Internalization was rapid (<1 h), saturable, and substantial compared with control mouse IgG. Furthermore, temperature reduction to 4 °C, an excess of unlabeled mouse IgG, or an excess of Tau antibodies reduced uptake in slices by 63, 41, and 62%, respectively (p = 0.002, 0.04, and 0.005). Uptake strongly correlated with total and insoluble Tau levels (r(2) = 0.77 and 0.87 and p = 0.002 and 0.0002), suggesting that Tau aggregates influence antibody internalization and/or retention within neurons. Inhibiting phagocytosis did not reduce uptake in slices or neuronal cultures, indicating limited microglial involvement. In contrast, clathrin-specific inhibitors reduced uptake in neurons (≤ 78%, p < 0.0001) and slices (≤ 35%, p = 0.03), demonstrating receptor-mediated endocytosis as the primary uptake pathway. Fluid phase endocytosis accounted for the remainder of antibody uptake in primary neurons, based on co-staining with internalized dextran. The receptor-mediated uptake is to a large extent via low affinity FcγII/III receptors and can be blocked in slices (43%, p = 0.04) and neurons (53%, p = 0.008) with an antibody against these receptors. Importantly, antibody internalization appears to be necessary for Tau reduction in primary neurons. Overall, these findings clarify that Tau antibody uptake is primarily receptor-mediated, that these antibodies are mainly found in neurons with Tau aggregates, and that their intracellular interaction leads to clearance of Tau pathology, all of which have major implications for therapeutic development of this approach.

Tau isoform composition influences rate and extent of filament formation.

The risk of developing tauopathic neurodegenerative disease depends in part on the levels and composition of six naturally occurring Tau isoforms in human brain. These proteins, which form filamentous aggregates in disease, vary only by the presence or absence of three inserts encoded by alternatively spliced exons 2, 3, and 10 of the Tau gene (MAPT). To determine the contribution of alternatively spliced segments to Tau aggregation propensity, the aggregation kinetics of six unmodified, recombinant human Tau isoforms were examined in vitro using electron microscopy assay methods. Aggregation propensity was then compared at the level of elementary rate constants for nucleation and extension phases. We found that all three alternatively spliced segments modulated Tau aggregation but through differing kinetic mechanisms that could synergize or compete depending on sequence context. Overall, segments encoded by exons 2 and 10 promoted aggregation, whereas the segment encoded by exon 3 depressed it with its efficacy dependent on the presence or absence of a fourth microtubule binding repeat. In general, aggregation propensity correlated with genetic risk reported for multiple tauopathies, implicating aggregation as one candidate mechanism rationalizing the correlation between Tau expression patterns and disease.

Methylthioninium chloride (methylene blue) induces autophagy and attenuates tauopathy in vitro and in vivo.

More than 30 neurodegenerative diseases including Alzheimer disease (AD), frontotemporal lobe dementia (FTD), and some forms of Parkinson disease (PD) are characterized by the accumulation of an aggregated form of the microtubule-binding protein tau in neurites and as intracellular lesions called neurofibrillary tangles. Diseases with abnormal tau as part of the pathology are collectively known as the tauopathies. Methylthioninium chloride, also known as methylene blue (MB), has been shown to reduce tau levels in vitro and in vivo and several different mechanisms of action have been proposed. Herein we demonstrate that autophagy is a novel mechanism by which MB can reduce tau levels. Incubation with nanomolar concentrations of MB was sufficient to significantly reduce levels of tau both in organotypic brain slice cultures from a mouse model of FTD, and in cell models. Concomitantly, MB treatment altered the levels of LC3-II, cathepsin D, BECN1, and p62 suggesting that it was a potent inducer of autophagy. Further analysis of the signaling pathways induced by MB suggested a mode of action similar to rapamycin. Results were recapitulated in a transgenic mouse model of tauopathy administered MB orally at three different doses for two weeks. These data support the use of this drug as a therapeutic agent in neurodegenerative diseases.

Phenothiazine-mediated rescue of cognition in tau transgenic mice requires neuroprotection and reduced soluble tau burden.

BACKGROUND: It has traditionally been thought that the pathological accumulation of tau in Alzheimer's disease and other tauopathies facilitates neurodegeneration, which in turn leads to cognitive impairment. However, recent evidence suggests that tau tangles are not the entity responsible for memory loss, rather it is an intermediate tau species that disrupts neuronal function. Thus, efforts to discover therapeutics for tauopathies emphasize soluble tau reductions as well as neuroprotection. RESULTS: Here, we found that neuroprotection alone caused by methylene blue (MB), the parent compound of the anti-tau phenothiaziazine drug, Rember™, was insufficient to rescue cognition in a mouse model of the human tauopathy, progressive supranuclear palsy (PSP) and fronto-temporal dementia with parkinsonism linked to chromosome 17 (FTDP17): Only when levels of soluble tau protein were concomitantly reduced by a very high concentration of MB, was cognitive improvement observed. Thus, neurodegeneration can be decoupled from tau accumulation, but phenotypic improvement is only possible when soluble tau levels are also reduced. CONCLUSIONS: Neuroprotection alone is not sufficient to rescue tau-induced memory loss in a transgenic mouse model. Development of neuroprotective agents is an area of intense investigation in the tauopathy drug discovery field. This may ultimately be an unsuccessful approach if soluble toxic tau intermediates are not also reduced. Thus, MB and related compounds, despite their pleiotropic nature, may be the proverbial "magic bullet" because they not only are neuroprotective, but are also able to facilitate soluble tau clearance. Moreover, this shows that neuroprotection is possible without reducing tau levels. This indicates that there is a definitive molecular link between tau and cell death cascades that can be disrupted.

Inhibition of tau polymerization with a cyanine dye in two distinct model systems.

In a host of neurodegenerative diseases Tau, a microtubule-associated protein, aggregates into insoluble lesions within neurons. Previous studies have utilized cyanine dyes as Tau aggregation inhibitors in vitro. Herein we utilize cyanine dye 3,3'-diethyl-9-methyl-thiacarbocyanine iodide (C11) to modulate Tau polymerization in two model systems, an organotypic slice culture model derived from Tau transgenic mice and a split green fluorescent protein complementation assay in Tau-expressing cells. In slice cultures, submicromolar concentrations (0.001 microm) of C11 produced a significant reduction of aggregated Tau and a corresponding increase in unpolymerized Tau. In contrast, treatment with a 1 microm dose promoted aggregation of Tau. These results were recapitulated in the complementation assay where administration of 1 microm C11 produced a significant increase in polymerized Tau relative to control, whereas treatment of cells with 0.01 microm C11 resulted in a marked reduction of aggregated Tau. In the organotypic slice cultures, modulation of Tau aggregation was independent of changes in phosphorylation at disease and microtubule binding relevant epitopes for both dosing regimes. Furthermore, treatment with 0.001 microm C11 resulted in a decrease in both total filament mass and number. There was no evidence of apoptosis or loss of synaptic integrity at either dose, however, whereas submicromolar concentrations of C11 did not interfere with microtubule binding, higher doses resulted in a decrease in the levels of microtubule-bound Tau. Overall, a cyanine dye can dissociate aggregated Tau in an ex vivo model of tauopathy with little toxicity and exploration of the use of these type of dyes as therapeutic agents is warranted.