Biochemical approaches to assess the impact of post-translational modifications on pathogenic tau conformations using recombinant protein.

Tau protein is associated with many neurodegenerative disorders known as tauopathies. Aggregates of tau are thought of as a main contributor to neurodegeneration in these diseases. Increasingly, evidence points to earlier, soluble conformations of abnormally modified monomers and multimeric tau as toxic forms of tau. The biological processes driving tau from physiological species to pathogenic conformations remain poorly understood, but certain avenues are currently under investigation including the functional consequences of various pathological tau changes (e.g. mutations, post-translational modifications (PTMs), and protein-protein interactions). PTMs can regulate several aspects of tau biology such as proteasomal and autophagic clearance, solubility, and aggregation. Moreover, PTMs can contribute to the transition of tau from normal to pathogenic conformations. However, our understating of how PTMs specifically regulate the transition of tau into pathogenic conformations is partly impeded by the relative lack of structured frameworks to assess and quantify these conformations. In this review, we describe a set of approaches that includes several in vitro assays to determine the contribution of PTMs to tau's transition into known pathogenic conformations. The approaches begin with different methods to create recombinant tau proteins carrying specific PTMs followed by validation of the PTMs status. Then, we describe a set of biochemical and biophysical assays that assess the contribution of a given PTM to different tau conformations, including aggregation, oligomerization, exposure of the phosphatase-activating domain, and seeding. Together, these approaches can facilitate the advancement of our understanding of the relationships between PTMs and tau conformations.

Tau here, tau there, tau almost everywhere: Clarifying the distribution of tau in the adult CNS.

The microtubule-associated protein tau has gained significant attention over the last several decades primarily due to its apparent role in the pathogenesis of several diseases, most notably Alzheimer's disease. While the field has focused largely on tau's potential contributions to disease mechanisms, comparably less work has focused on normal tau physiology. Moreover, as the field has grown, some misconceptions and dogmas regarding normal tau physiology have become engrained in the traditional narrative. Here, one of the most common misconceptions regarding tau, namely its normal cellular/subcellular distribution in the CNS, is discussed. The literature describing the presence of tau in neuronal somata, dendrites, axons and synapses, as well as in glial cells is described. The origins for the erroneous description of tau as an "axon-specific," "axon-enriched" and/or "neuron-specific" protein are discussed as well. The goal of this work is to help address these specific dogmatic misconceptions and provide a concise description of tau's normal cellular/subcellular localization in the adult CNS. This information can help refine our collective understanding of- and hypotheses about tau biology and pathobiology.

Behavioral and neuropathological characterization over the adult lifespan of the human tau knock-in mouse.

Tau is a microtubule-associated protein with a diverse functional repertoire linked to neurodegenerative disease. Recently, a human tau knock-in (MAPT KI) mouse was developed that may overcome many limitations associated with current animal models used to study tau. In MAPT KI mice, the entire murine Mapt gene was replaced with the human MAPT gene under control of the endogenous Mapt promoter. This model represents an ideal in vivo platform to study the function and dysfunction of human tau protein. Accordingly, a detailed understanding of the effects MAPT KI has on structure and function of the CNS is warranted. Here, we provide a detailed behavioral and neuropathological assessment of MAPT KI mice. We compared MAPT KI to wild-type (WT) C57BL/6j mice in behavioral assessments of anxiety, attention, working memory, spatial memory, and motor performance from 6 to 24 months (m) of age. Using immunohistological and biochemical assays, we quantified markers of glia (microglia, astrocytes and oligodendrocytes), synaptic integrity, neuronal integrity and the cytoskeleton. Finally, we quantified levels of total tau, tau isoforms, tau phosphorylation, and tau conformations. MAPT KI mice show normal cognitive and locomotor behavior at all ages, and resilience to mild age-associated locomotor deficits observed in WT mice. Markers of neuronal and synaptic integrity are unchanged in MAPT KI mice with advancing age. Glial markers are largely unchanged in MAPT KI mice, but glial fibrillary acidic protein is increased in the hippocampus of WT and MAPT KI mice at 24 m. MAPT KI mice express all 6 human tau isoforms and levels of tau remain stable throughout adulthood. Hippocampal tau in MAPT KI and WT mice is phosphorylated at serine 396/404 (PHF1) and murine tau in WT animals displays more PHF1 phosphorylation at 6 and 12 m. Lastly, we extended previous reports showing that MAPT KI mice do not display overt pathology. No evidence of other tau phosphorylation residues (AT8, pS422) or abnormal conformations (TNT2 or TOC1) associated with pathogenic tau were detected. The lack of overt pathological changes in MAPT KI mice make this an ideal platform for future investigations into the function and dysfunction of tau protein in vivo.

CRISPR screens in iPSC-derived neurons reveal principles of tau proteostasis.

A hallmark of age-associated neurodegenerative diseases is the aggregation of proteins. Aggregation of the protein tau defines tauopathies, which include Alzheimer's disease and frontotemporal dementia. Specific neuronal subtypes are selectively vulnerable to the accumulation of tau aggregates, and subsequent dysfunction and death. The mechanisms underlying cell type-selective vulnerability are unknown. To systematically uncover the cellular factors controlling the accumulation of tau aggregates in human neurons, we conducted a genome-wide CRISPRi-based modifier screen in iPSC-derived neurons. The screen uncovered expected pathways, including autophagy, but also unexpected pathways including UFMylation and GPI anchor synthesis, that control tau oligomer levels. We identify the E3 ubiquitin ligase CUL5 as a tau interactor and potent modifier of tau levels. In addition, disruption of mitochondrial function increases tau oligomer levels and promotes proteasomal misprocessing of tau. These results reveal new principles of tau proteostasis in human neurons and pinpoint potential therapeutic targets for tauopathies.

Flow cytometric isolation of drug-like conformational antibodies specific for amyloid fibrils.

Antibodies that recognize specific protein conformational states are broadly important for research, diagnostic and therapeutic applications, yet they are difficult to generate in a predictable and systematic manner using either immunization or in vitro antibody display methods. This problem is particularly severe for conformational antibodies that recognize insoluble antigens such as amyloid fibrils associated with many neurodegenerative disorders. Here we report a quantitative fluorescence-activated cell sorting (FACS) method for directly selecting high-quality conformational antibodies against different types of insoluble (amyloid fibril) antigens using a single, off-the-shelf human library. Our approach uses quantum dots functionalized with antibodies to capture insoluble antigens, and the resulting quantum dot conjugates are used in a similar manner as conventional soluble antigens for multi-parameter FACS selections. Notably, we find that this approach is robust for isolating high-quality conformational antibodies against tau and α-synuclein fibrils from the same human library with combinations of high affinity, high conformational specificity and, in some cases, low off-target binding that rival or exceed those of clinical-stage antibodies specific for tau (zagotenemab) and α-synuclein (cinpanemab). This approach is expected to enable conformational antibody selection and engineering against diverse types of protein aggregates and other insoluble antigens (e.g., membrane proteins) that are compatible with presentation on the surface of antibody-functionalized quantum dots.

Phosphomimetics at Ser199/Ser202/Thr205 in Tau Impairs Axonal Transport in Rat Hippocampal Neurons.

Our understanding of the biological functions of the tau protein now includes its role as a scaffolding protein involved in signaling regulation, which also has implications for tau-mediated dysfunction and degeneration in Alzheimer's disease and other tauopathies. Recently, we found that pseudophosphorylation at sites linked to the pathology-associated AT8 phosphoepitope of tau disrupts normal fast axonal transport through a protein phosphatase 1 (PP1)-dependent pathway in squid axoplasm. Activation of the pathway and the resulting transport deficits required tau's N-terminal phosphatase-activating domain (PAD) and PP1 but the connection between tau and PP1 was not well defined. Here, we studied functional interactions between tau and PP1 isoforms and their effects on axonal transport in mammalian neurons. First, we found that wild-type tau interacted with PP1α and PP1γ primarily through its microtubule-binding repeat domain. Pseudophosphorylation of tau at S199/S202/T205 (psTau) increased PAD exposure, enhanced interactions with PP1γ, and increased active PP1γ levels in mammalian cells. Expression of psTau also significantly impaired axonal transport in primary rat hippocampal neurons. Deletion of PAD in psTau significantly reduced the interaction with PP1γ, eliminated increases of active PP1γ levels, and rescued axonal transport impairment in neurons. These data suggest that a functional consequence of phosphorylation within S199-T205 in tau, which occurs in AD and several other tauopathies, may be aberrant interaction with and activation of PP1γ and subsequent axonal transport disruption in a PAD-dependent fashion.

Using Live-Cell Imaging to Measure the Effects of Pathological Proteins on Axonal Transport in Primary Hippocampal Neurons.

Bidirectional transport of cargos along the axon is critical for maintaining functional synapses, neural connectivity, and healthy neurons. Axonal transport is disrupted in multiple neurodegenerative diseases, and projection neurons are particularly vulnerable because of the need to transport cellular materials over long distances and sustain substantial axonal mass. Pathological modifications of several disease-related proteins negatively affect transport, including tau, amyloid-ß, α-synuclein, superoxide dismutase, and huntingtin, providing a potential common mechanism by which pathological proteins exert toxicity in disease. Methods to study these toxic mechanisms are necessary to understand neurodegenerative disorders and identify potential therapeutic interventions. Here, cultured primary rodent hippocampal neurons are co-transfected with multiple plasmids to study the effects of pathological proteins on fast axonal transport using live-cell confocal imaging of fluorescently-tagged cargo proteins. We begin with the harvest, dissociation, and culturing of primary hippocampal neurons from rodents. Then, we co-transfect the neurons with plasmid DNA constructs to express fluorescent-tagged cargo protein and wild-type or mutant tau (used as an exemplar of pathological proteins). Axons are identified in live cells using an antibody that binds an extracellular domain of neurofascin, an axon initial segment protein, and an axonal region of interest is imaged to measure fluorescent cargo transport. Using KymoAnalyzer, a freely available ImageJ macro, we extensively characterize the velocity, pause frequency, and directional cargo density of axonal transport, all of which may be affected by the presence of pathological proteins. Through this method, we identify a phenotype of increased cargo pause frequency associated with the expression of pathological tau protein. Additionally, gene-silencing shRNA constructs can be added to the transfection mix to test the role of other proteins in mediating transport disruption. This protocol is easily adaptable for use with other neurodegenerative disease-related proteins and is a reproducible method to study the mechanisms of how those proteins disrupt axonal transport.

Central role for p62/SQSTM1 in the elimination of toxic tau species in a mouse model of tauopathy.

Intracellular accumulation of filamentous tau aggregates with progressive neuronal loss is a common characteristic of tauopathies. Although the neurodegenerative mechanism of tau-associated pathology remains unclear, molecular elements capable of degrading and/or sequestering neurotoxic tau species may suppress neurodegenerative progression. Here, we provide evidence that p62/SQSTM1, a ubiquitinated cargo receptor for selective autophagy, acts protectively against neuronal death and neuroinflammation provoked by abnormal tau accumulation. P301S mutant tau transgenic mice (line PS19) exhibited accumulation of neurofibrillary tangles with localization of p62 mostly in the brainstem, but neuronal loss with few neurofibrillary tangles in the hippocampus. In the hippocampus of PS19 mice, the p62 level was lower compared to the brainstem, and punctate accumulation of phosphorylated tau unaccompanied by co-localization of p62 was observed. In PS19 mice deficient in p62 (PS19/p62-KO), increased accumulation of phosphorylated tau, acceleration of neuronal loss, and exacerbation of neuroinflammation were observed in the hippocampus as compared with PS19 mice. In addition, increase of abnormal tau and neuroinflammation were observed in the brainstem of PS19/p62-KO. Immunostaining and dot-blot analysis with an antibody selectively recognizing tau dimers and higher-order oligomers revealed that oligomeric tau species in PS19/p62-KO mice were significantly accumulated as compared to PS19 mice, suggesting the requirement of p62 to eliminate disease-related oligomeric tau species. Our findings indicated that p62 exerts neuroprotection against tau pathologies by eliminating neurotoxic tau species, suggesting that the manipulative p62 and selective autophagy may provide an intrinsic therapy for the treatment of tauopathy.

Mitochondrial oxidative stress contributes to the pathological aggregation and accumulation of tau oligomers in Alzheimer's disease.

Tau oligomers (oTau) are thought to precede neurofibrillary tangle formation and likely represent one of the toxic species in disease. This study addresses whether mitochondrial reactive oxygen species (ROS) contribute to tau oligomer accumulation. First, we determined whether elevated oxidative stress correlates with aggregation of tau oligomers in the brain and platelets of human Alzheimer's disease (AD) patient, tauopathy mice, primary cortical neurons from tau mice and human trans-mitochondrial 'cybrid' (cytoplasmic hybrid) neuronal cells, whose mitochondria are derived from platelets of patients with sporadic AD- or mild cognitive impairment (MCI)-derived mitochondria. Increased formation of tau oligomers correlates with elevated ROS levels in the hippocampi of AD patients and tauopathy mice, AD- and MCI-derived mitochondria and AD and MCI cybrid cells. Furthermore, scavenging ROS by application of mito-TEMPO/2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride, a mitochondria-targeted antioxidant, not only inhibits the generation of mitochondrial ROS and rescues mitochondrial respiratory function but also robustly suppresses tau oligomer accumulation in MCI and AD cybrids as well as cortical neurons from tau mice. These studies provide substantial evidence that mitochondria-mediated oxidative stress contributes to tau oligomer formation and accumulation.

Clioquinol Decreases Levels of Phosphorylated, Truncated, and Oligomerized Tau Protein.

The neuropathological hallmarks of Alzheimer's disease (AD) are senile plaques (SPs), which are composed of amyloid ß protein (Aß), and neurofibrillary tangles (NFTs), which consist of highly phosphorylated tau protein. As bio-metal imbalance may be involved in the formation of NFT and SPs, metal regulation may be a direction for AD treatment. Clioquinol (CQ) is a metal-protein attenuating compound with mild chelating effects for Zn2+ and Cu2+, and CQ can not only detach metals from SPs, but also decrease amyloid aggregation in the brain. Previous studies suggested that Cu2+ induces the hyperphosphorylation of tau. However, the effects of CQ on tau were not fully explored. To examine the effects of CQ on tau metabolism, we used a human neuroblastoma cell line, M1C cells, which express wild-type tau protein (4R0N) via tetracycline-off (TetOff) induction. In a morphological study and ATP assay, up to 10 µM CQ had no effect on cell viability; however, 100 µM CQ had cytotoxic effects. CQ decreased accumulation of Cu+ in the M1C cells (39.4% of the control), and both total and phosphorylated tau protein. It also decreased the activity of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) (37.3% and 60.7% levels of the control, respectively), which are tau kinases. Of note, activation of protein phosphatase 2A (PP2A), which is a tau phosphatase, was also observed after CQ treatment. Fractionation experiments demonstrated a reduction of oligomeric tau in the tris insoluble, sarkosyl soluble fraction by CQ treatment. CQ also decreased caspase-cleaved tau, which accelerated the aggregation of tau protein. CQ activated autophagy and proteasome pathways, which are considered important for the degradation of tau protein. Although further studies are needed to elucidate the mechanisms responsible for the effects of CQ on tau, CQ may shed light on possible AD therapeutics.

Frontotemporal Lobar Dementia Mutant Tau Impairs Axonal Transport through a Protein Phosphatase 1γ-Dependent Mechanism.

Pathologic tau modifications are characteristic of Alzheimer's disease and related dementias, but mechanisms of tau toxicity continue to be debated. Inherited mutations in tau cause early onset frontotemporal lobar dementias (FTLD-tau) and are commonly used to model mechanisms of tau toxicity in tauopathies. Previous work in the isolated squid axoplasm model demonstrated that several pathogenic forms of tau inhibit axonal transport through a mechanism involving activation of protein phosphatase 1 (PP1). Here, we determined that P301L and R5L FTLD mutant tau proteins elicit a toxic effect on axonal transport as monomeric proteins. We evaluated interactions of wild-type or mutant tau with specific PP1 isoforms (α, ß, and γ) to examine how the interaction contributes to this toxic effect using primary rat hippocampal neurons from both sexes. Pull-down and bioluminescence resonance energy transfer experiments revealed selective interactions of wild-type tau with PP1α and PP1γ isoforms, but not PP1ß, which were significantly increased by the P301L tau mutation. The results from proximity ligation assays confirmed the interaction in primary hippocampal neurons. Moreover, expression of FTLD-linked mutant tau in these neurons enhanced levels of active PP1, also increasing the pausing frequency of fluorescently labeled vesicles in both anterograde and retrograde directions. Knockdown of PP1γ, but not PP1α, rescued the cargo-pausing effects of P301L and R5L tau, a result replicated by deleting a phosphatase-activating domain in the amino terminus of P301L tau. These findings support a model of tau toxicity involving aberrant activation of a specific PP1γ-dependent pathway that disrupts axonal transport in neurons.SIGNIFICANCE STATEMENT Tau pathology is closely associated with neurodegeneration in Alzheimer's disease and other tauopathies, but the toxic mechanisms remain a debated topic. We previously proposed that pathologic tau forms induce dysfunction and degeneration through aberrant activation of a PP1-dependent pathway that disrupts axonal transport. Here, we show that tau directly interacts with specific PP1 isoforms, increasing levels of active PP1. Pathogenic tau mutations enhance this interaction, further increasing active PP1 levels and impairing axonal transport in isolated squid axoplasm and primary hippocampal neurons. Mutant-tau-mediated impairment of axonal transport was mediated by PP1γ and a phosphatase-activating domain located at the amino terminus of tau. This work has important implications for understanding and potentially mitigating tau-mediated neurotoxicity in tauopathies.

EFhd2 brain interactome reveals its association with different cellular and molecular processes.

EFhd2 is a conserved calcium-binding protein that is highly expressed in the central nervous system. We have shown that EFhd2 interacts with tau protein, a key pathological hallmark in Alzheimer's disease and related dementias. However, EFhd2's physiological and pathological functions in the brain are still poorly understood. To gain insights into its physiological function, we identified proteins that co-immunoprecipitated with EFhd2 from mouse forebrain and hindbrain, using tandem mass spectrometry (MS). In addition, quantitative mass spectrometry was used to detect protein abundance changes due to the deletion of the Efhd2 gene in mouse forebrain and hindbrain regions. Our data show that mouse EFhd2 is associated with cytoskeleton components, vesicle trafficking modulators, cellular stress response-regulating proteins, and metabolic proteins. Moreover, proteins associated with the cytoskeleton, vesicular transport, calcium signaling, stress response, and metabolic pathways showed differential abundance in Efhd2(-/-) mice. This study presents, for the first time, an EFhd2 brain interactome that it is associated with different cellular and molecular processes. These findings will help prioritize further studies to investigate the mechanisms by which EFhd2 modulates these processes in physiological and pathological conditions of the nervous system.

Neuronal and Glial Distribution of Tau Protein in the Adult Rat and Monkey.

Tau is a microtubule-associated protein for which the physiological functions remain a topic of vigorous investigation. Additionally, tau is a central player in the pathogenesis of several diseases such as Alzheimer's disease and several frontotemporal dementias. A critical variable to understanding tau in physiological and disease contexts is its normal localization within cells of the adult CNS. Tau is often described as an axon-specific (or enriched) and neuron-specific protein with little to no expression in glial cells, all of which are untrue. Understanding normal tau distribution also impacts interpretation of experimental results and hypotheses regarding its role in disease. Thus, we set out to help clarify the normal localization of tau in the adult CNS of middle-aged rats and rhesus macaque using the hippocampus as a representative brain structure. The physiological concentration of tau in the rat hippocampus was 6.6 µM and in white matter was 3.6 µM as determined by quantitative sandwich ELISAs. We evaluated the cellular localization of tau using multiple tau-specific antibodies with epitopes to different regions, including Tau1, Tau5, Tau7, R1, and two novel primate-specific antibodies NT9 and NT15. In the rat and monkey, tau was localized within the somatodendritic and axonal compartments, as well as a subset of neuronal nuclei. Semi-quantitative fluorescence intensity measurements revealed that depending on the specific reagent used the somatodendritic tau is relatively equal to, higher than, or lower than axonal tau, highlighting differential labeling of tau with various antibodies despite its distribution throughout the neuron. Tau was strongly expressed in mature oligodendrocytes and displayed little to no expression in oligodendrocyte precursor cells, astrocytes or microglia. Collectively, the data indicate tau is ∼3 - 7 µM under physiological conditions, is not specifically enriched in axons, and is normally found in both neurons and mature oligodendrocytes in the adult CNS. The full landscape of tau distribution is not revealed by all antibodies suggesting availability of the epitopes is different within specific neuronal compartments. These findings set the stage for better understanding normal tau distributions and interpreting data regarding the presence of tau in different compartments or cell types within disease conditions.

Tau: A Signaling Hub Protein.

Over four decades ago, in vitro experiments showed that tau protein interacts with and stabilizes microtubules in a phosphorylation-dependent manner. This observation fueled the widespread hypotheses that these properties extend to living neurons and that reduced stability of microtubules represents a major disease-driving event induced by pathological forms of tau in Alzheimer's disease and other tauopathies. Accordingly, most research efforts to date have addressed this protein as a substrate, focusing on evaluating how specific mutations, phosphorylation, and other post-translational modifications impact its microtubule-binding and stabilizing properties. In contrast, fewer efforts were made to illuminate potential mechanisms linking physiological and disease-related forms of tau to the normal and pathological regulation of kinases and phosphatases. Here, we discuss published work indicating that, through interactions with various kinases and phosphatases, tau may normally act as a scaffolding protein to regulate phosphorylation-based signaling pathways. Expanding on this concept, we also review experimental evidence linking disease-related tau species to the misregulation of these pathways. Collectively, the available evidence supports the participation of tau in multiple cellular processes sustaining neuronal and glial function through various mechanisms involving the scaffolding and regulation of selected kinases and phosphatases at discrete subcellular compartments. The notion that the repertoire of tau functions includes a role as a signaling hub should widen our interpretation of experimental results and increase our understanding of tau biology in normal and disease conditions.

TIA1 potentiates tau phase separation and promotes generation of toxic oligomeric tau.

Tau protein plays an important role in the biology of stress granules and in the stress response of neurons, but the nature of these biochemical interactions is not known. Here we show that the interaction of tau with RNA and the RNA binding protein TIA1 is sufficient to drive phase separation of tau at physiological concentrations, without the requirement for artificial crowding agents such as polyethylene glycol (PEG). We further show that phase separation of tau in the presence of RNA and TIA1 generates abundant tau oligomers. Prior studies indicate that recombinant tau readily forms oligomers and fibrils in vitro in the presence of polyanionic agents, including RNA, but the resulting tau aggregates are not particularly toxic. We discover that tau oligomers generated during copartitioning with TIA1 are significantly more toxic than tau aggregates generated by incubation with RNA alone or phase-separated tau complexes generated by incubation with artificial crowding agents. This pathway identifies a potentially important source for generation of toxic tau oligomers in tau-related neurodegenerative diseases. Our results also reveal a general principle that phase-separated RBP droplets provide a vehicle for coassortment of selected proteins. Tau selectively copartitions with TIA1 under physiological conditions, emphasizing the importance of TIA1 for tau biology. Other RBPs, such as G3BP1, are able to copartition with tau, but this happens only in the presence of crowding agents. This type of selective mixing might provide a basis through which membraneless organelles bring together functionally relevant proteins to promote particular biological activities.

Liquid-liquid phase separation induces pathogenic tau conformations in vitro.

Formation of membrane-less organelles via liquid-liquid phase separation is one way cells meet the biological requirement for spatiotemporal regulation of cellular components and reactions. Recently, tau, a protein known for its involvement in Alzheimer's disease and other tauopathies, was found to undergo liquid-liquid phase separation making it one of several proteins associated with neurodegenerative diseases to do so. Here, we demonstrate that tau forms dynamic liquid droplets in vitro at physiological protein levels upon molecular crowding in buffers that resemble physiological conditions. Tau droplet formation is significantly enhanced by disease-associated modifications, including the AT8 phospho-epitope and the P301L tau mutation linked to an inherited tauopathy. Moreover, tau droplet dynamics are significantly reduced by these modified forms of tau. Extended phase separation promoted a time-dependent adoption of toxic conformations and oligomerization, but not filamentous aggregation. P301L tau protein showed the greatest oligomer formation following extended phase separation. These findings suggest that phase separation of tau may facilitate the formation of non-filamentous pathogenic tau conformations.

Tau is not necessary for amyloid-ß-induced synaptic and memory impairments.

The amyloid hypothesis posits that the amyloid-beta (Aß) protein precedes and requires microtubule-associated protein tau in a sort of trigger-bullet mechanism leading to Alzheimer's disease (AD) pathology. This sequence of events has become dogmatic in the AD field and is used to explain clinical trial failures due to a late start of the intervention when Aß already activated tau. Here, using a multidisciplinary approach combining molecular biological, biochemical, histopathological, electrophysiological, and behavioral methods, we demonstrated that tau suppression did not protect against Aß-induced damage of long-term synaptic plasticity and memory, or from amyloid deposition. Tau suppression could even unravel a defect in basal synaptic transmission in a mouse model of amyloid deposition. Similarly, tau suppression did not protect against exogenous oligomeric tau-induced impairment of long-term synaptic plasticity and memory. The protective effect of tau suppression was, in turn, confined to short-term plasticity and memory. Taken together, our data suggest that therapies downstream of Aß and tau together are more suitable to combat AD than therapies against one or the other alone.

A human induced pluripotent stem cell-derived cortical neuron human-on-a chip system to study Aß42 and tau-induced pathophysiological effects on long-term potentiation.

INTRODUCTION: The quest to identify an effective therapeutic strategy for neurodegenerative diseases, such as mild congitive impairment (MCI) and Alzheimer's disease (AD), suffers from the lack of good human-based models. Animals represent the most common models used in basic research and drug discovery studies. However, safe and effective compounds identified in animal studies often translate poorly to humans, yielding unsuccessful clinical trials. METHODS: A functional in vitro assay based on long-term potentiation (LTP) was used to demonstrate that exposure to amyloid beta (Aß42) and tau oligomers, or brain extracts from AD transgenic mice led to prominent changes in human induced pluripotent stem cells (hiPSC)-derived cortical neurons, notably, without cell death. RESULTS: Impaired information processing was demonstrated by treatment of neuron-MEA (microelectrode array) systems with the oligomers and brain extracts by reducing the effects of LTP induction. These data confirm the neurotoxicity of molecules linked to AD pathology and indicate the utility of this human-based system to model aspects of AD in vitro and study LTP deficits without loss of viability; a phenotype that more closely models the preclinical or early stage of AD. DISCUSSION: In this study, by combining multiple relevant and important molecular and technical aspects of neuroscience research, we generated a new, fully human in vitro system to model and study AD at the preclinical stage. This system can serve as a novel drug discovery platform to identify compounds that rescue or alleviate the initial neuronal deficits caused by Aß42 and/or tau oligomers, a main focus of clinical trials.

Rho-kinase ROCK inhibitors reduce oligomeric tau protein.

Neurofibrillary tangles, one of the pathological hallmarks of Alzheimer's disease, consist of highly phosphorylated tau proteins. Tau protein binds to microtubules and is best known for its role in regulating microtubule dynamics. However, if tau protein is phosphorylated by activated major tau kinases, including glycogen synthase kinase 3ß or cyclin-dependent kinase 5, or inactivated tau phosphatase, including protein phosphatase 2A, its affinity for microtubules is reduced, and the free tau is believed to aggregate, thereby forming neurofibrillary tangles. We previously reported that pitavastatin decreases the total and phosphorylated tau protein using a cellular model of tauopathy. The reduction of tau was considered to be due to Rho-associated coiled-coil protein kinase (ROCK) inhibition by pitavastatin. ROCK plays important roles to organize the actin cytoskeleton, an expected therapeutic target of human disorders. Several ROCK inhibitors are clinically applied to prevent vasospasm postsubarachnoid hemorrhage (fasudil) and for the treatment of glaucoma (ripasudil). We have examined the effects of ROCK inhibitors (H1152, Y-27632, and fasudil [HA-1077]) on tau protein phosphorylation in detail. A human neuroblastoma cell line (M1C cells) that expresses wild-type tau protein (4R0N) by tetracycline-off (TetOff) induction, primary cultured mouse neurons, and a mouse model of tauopathy (rTG4510 line) were used. The levels of phosphorylated tau and caspase-cleaved tau were reduced by the ROCK inhibitors. Oligomeric tau levels were also reduced by ROCK inhibitors. After ROCK inhibitor treatment, glycogen synthase kinase 3ß, cyclin-dependent kinase 5, and caspase were inactivated, protein phosphatase 2A was activated, and the levels of IFN-γ were reduced. ROCK inhibitors activated autophagy and proteasome pathways, which are considered important for the degradation of tau protein. Collectively, these results suggest that ROCK inhibitors represent a viable therapeutic route to reduce the pathogenic forms of tau protein in tauopathies, including Alzheimer's disease.

Activity of the poly(A) binding protein MSUT2 determines susceptibility to pathological tau in the mammalian brain.

Brain lesions composed of pathological tau help to drive neurodegeneration in Alzheimer's disease (AD) and related tauopathies. Here, we identified the mammalian suppressor of tauopathy 2 (MSUT2) gene as a modifier of susceptibility to tau toxicity in two mouse models of tauopathy. Transgenic PS19 mice overexpressing tau, a model of AD, and lacking the Msut2 gene exhibited decreased learning and memory deficits, reduced neurodegeneration, and reduced accumulation of pathological tau compared to PS19 tau transgenic mice expressing Msut2 Conversely, Msut2 overexpression in 4RTauTg2652 tau transgenic mice increased pathological tau deposition and promoted the neuroinflammatory response to pathological tau. MSUT2 is a poly(A) RNA binding protein that antagonizes the canonical nuclear poly(A) binding protein PABPN1. In individuals with AD, MSUT2 abundance in postmortem brain tissue predicted an earlier age of disease onset. Postmortem AD brain tissue samples with normal amounts of MSUT2 showed elevated neuroinflammation associated with tau pathology. We observed co-depletion of MSUT2 and PABPN1 in postmortem brain samples from a subset of AD cases with higher tau burden and increased neuronal loss. This suggested that MSUT2 and PABPN1 may act together in a macromolecular complex bound to poly(A) RNA. Although MSUT2 and PABPN1 had opposing effects on both tau aggregation and poly(A) RNA tail length, we found that increased poly(A) tail length did not ameliorate tauopathy, implicating other functions of the MSUT2/PABPN1 complex in tau proteostasis. Our findings implicate poly(A) RNA binding proteins both as modulators of pathological tau toxicity in AD and as potential molecular targets for interventions to slow neurodegeneration in tauopathies.