Endogenous Tau Research Articles

Targeting Endogenous Tau in Seeded Tauopathy Models Inhibits Tau Spread.

The transmission of tau pathology has been proposed as one of the major mechanisms for the spatiotemporal spreading of tau pathology in neurodegenerative diseases. Over the last decade, studies have demonstrated that targeting total or pathological tau using tau antibodies can mitigate the development of tau pathology in tauopathy or Alzheimer's disease (AD) mouse models, and multiple tau immunotherapy agents have progressed to clinical trials. Tau antibodies are believed to inhibit the internalization of pathologic seeds and/or block seed elongation after seed internalization. To further address the mechanism of tau antibody inhibition of pathological spread, we conducted immunotherapy studies in mouse primary neurons and wild-type mice (females) seeded with AD patient-derived tau to induce the formation and spreading of tau pathology. Notably, we evaluated the effect of a mouse tau-specific antibody (mTau8) which does not interact with AD-tau seeds in these models. Our results show that mTau8 crosses the blood-brain barrier at levels similar to other antibodies and effectively decreases AD-tau-seeded tau pathology in vitro and in vivo. Importantly, our data suggest that mTau8 binds to endogenous intraneuronal mouse tau, thereby inhibiting the elongation of internalized tau seeds. These findings provide valuable insights into the possible mechanism underlying antibody-based therapies for treating tauopathies.Significance Statement The transmission of tau pathology plays key role in the pathoclinical progression of tauopathy. Studies have shown that tau antibody treatment can mitigate tau pathology in transgenic and spreading models of tauopathy. To explore the mechanisms involved in this procedure, we conducted immunotherapy studies on human tau seeds induced tau spreading models using a mouse tau-specific antibody (mTau8), which does not interact with human-tau seeds. Our findings in the study enhance our understanding of antibody-based therapies for tauopathies.

Taurine is essential for mouse uterine luminal fluid resorption during implantation window via the SCNN1A and AQP8 signaling.

Uterine fluid homeostasis during peri-implantation is crucial for successful embryo implantation. Taurine (Tau) plays a crucial role in regulating osmotic pressure and ion transport. However, the precise mechanisms underlying Tau-mediated regulation of uterine fluid homeostasis during peri-implantation in mice remain unclear. In this study, we generated a Tau-deficient mouse model by administering Tau-free diet to Csad knockout (Csad-/-) mice to block endogenous Tau synthesis and exogenous Tau absorption (Csad-/--Tau free). Our findings demonstrated that Csad-/-Tau free mice with diminished level of Tau exhibited decreased rates of embryo implantation and impaired fertility. Further analysis revealed that the expression of Scnn1a was down-regulated during the implantation window, while Aqp8 was upregulated in Csad-/-Tau free mice, leading to uterine luminal fluid retention and defects in luminal closure, resulting in failed embryo implantation. Additionally, it was also found that E2 inhibited uterine Csad expression and Tau synthesis, while P4 promoted them. Therefore, our findings suggest that ovarian steroid hormones regulate Csad expression and Tau synthesis, thereby affecting release and resorption of uterine luminal fluid, ultimately impacting embryo implantation success.

Tau is required for glial lipid droplet formation and resistance to neuronal oxidative stress.

The accumulation of reactive oxygen species (ROS) is a common feature of tauopathies, defined by Tau accumulations in neurons and glia. High ROS in neurons causes lipid production and the export of toxic peroxidated lipids (LPOs). Glia uptake these LPOs and incorporate them into lipid droplets (LDs) for storage and catabolism. We found that overexpressing Tau in glia disrupts LDs in flies and rat neuron-astrocyte co-cultures, sensitizing the glia to toxic, neuronal LPOs. Using a new fly tau loss-of-function allele and RNA-mediated interference, we found that endogenous Tau is required for glial LD formation and protection against neuronal LPOs. Similarly, endogenous Tau is required in rat astrocytes and human oligodendrocyte-like cells for LD formation and the breakdown of LPOs. Behaviorally, flies lacking glial Tau have decreased lifespans and motor defects that are rescuable by administering the antioxidant N-acetylcysteine amide. Overall, this work provides insights into the important role that Tau has in glia to mitigate ROS in the brain.

Depletion of TDP-43 exacerbates tauopathy-dependent brain atrophy by sensitizing vulnerable neurons to caspase 3-mediated endoproteolysis of tau in a mouse model of Multiple Etiology Dementia.

TDP-43 proteinopathy, initially disclosed in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), coexists with tauopathy in a variety of neurodegenerative disorders, termed multiple etiology dementias (MEDs), including Alzheimer's Disease (AD). While such co-pathology of TDP-43 is strongly associated with worsened neurodegeneration and steeper cognitive decline, the pathogenic mechanism underlying the exacerbated neuron loss remains elusive. The loss of TDP-43 splicing repression that occurs in presymptomatic ALS-FTD individuals suggests that such early loss could facilitate the pathological conversion of tau to accelerate neuron loss. Here, we report that the loss of TDP-43 repression of cryptic exons in forebrain neurons (CaMKII-CreER;Tardbp f/f mice) is necessary to exacerbate tauopathy-dependent brain atrophy by sensitizing vulnerable neurons to caspase 3-dependent cleavage of endogenous tau to promote tauopathy. Corroborating this finding within the human context, we demonstrate that loss of TDP-43 function in iPSC-derived cortical neurons promotes early cryptic exon inclusion and subsequent caspase 3-mediated endoproteolysis of tau. Using a genetic approach to seed tauopathy in CaMKII-CreER;Tardbp f/f mice by expressing a four-repeat microtubule binding domain of human tau, we show that the amount of tau seed positively correlates with levels of caspase 3-cleaved tau. Importantly, we found that the vulnerability of hippocampal neurons to TDP-43 depletion is dependent on the amount of caspase 3-cleaved tau: from most vulnerable neurons in the CA2/3, followed by those in the dentate gyrus, to the least in CA1. Taken together, our findings strongly support the view that TDP-43 loss-of-function exacerbates tauopathy-dependent brain atrophy by increasing the sensitivity of vulnerable neurons to caspase 3-mediated endoproteolysis of tau, resulting in a greater degree of neurodegeneration in human disorders with co-pathologies of tau and TDP-43. Our work thus discloses novel mechanistic insights and therapeutic targets for human tauopathies harboring co-pathology of TDP-43 and provides a new MED model for testing therapeutic strategies.

Tau modulation through AAV9 therapy augments Akt/Erk survival signalling in glaucoma mitigating the retinal degenerative phenotype

The microtubule-associated protein Tau is a key player in various neurodegenerative conditions, including Alzheimer's disease (AD) and Tauopathies, where its hyperphosphorylation disrupts neuronal microtubular lattice stability. Glaucoma, a neurodegenerative disorder affecting the retina, leads to irreversible vision loss by damaging retinal ganglion cells and the optic nerve, often associated with increased intraocular pressure. Prior studies have indicated Tau expression and phosphorylation alterations in the retina in both AD and glaucoma, yet the causative or downstream nature of Tau protein changes in these pathologies remains unclear. This study investigates the impact of Tau protein modulation on retinal neurons under normal and experimental glaucoma conditions. Employing AAV9-mediated gene therapy for Tau overexpression and knockdown, both manipulations were found to adversely affect retinal structural and functional measures as well as neuroprotective Akt/Erk survival signalling in healthy conditions. In the experimental glaucoma model, Tau overexpression intensified inner retinal degeneration, while Tau silencing provided significant protection against these degenerative changes. These findings underscore the critical role of endogenous Tau protein levels in preserving retinal integrity and emphasize the therapeutic potential of targeting Tau in glaucoma pathology.

Endogenous tau released from human ReNCell VM cultures by neuronal activity is phosphorylated at multiple sites.

Activity-dependent release of endogenous human tau from human ReNCell VM cultures occurs under physiological conditions.Released human tau is phosphorylated at nine sites (pSites) in the proline-rich domain and the C-terminal domain detected by AT270 (T175/T181), AT8 (S202/T205), AT100 (T212/S214), AT180 (T231), and PHF-1 (S396/S404) tau antibodies, strongly suggesting that these pSites are important for activity-dependent tau release from human ReNCell VM.In contrast, intracellular human tau proteins have different phosphorylation status among these nine pSites: AT270 and PHF-1 pSites are highly phosphorylated, but AT8 and AT180 are weakly phosphorylated, suggesting AT8 and AT180 pSites are release-sensitive phosphorylation motifs.Activity-dependent release of endogenous human tau is decreased by a tau kinase GSK-3β inhibitor SB 216763, indicating that GSK-3β-dependent phosphorylation plays an important role in activity-dependent tau release. The human ReNCell culture is an excellent model system to study mechanisms underlying physiological release of endogenous tau.

FUBP3 mediates the amyloid-β-induced neuronal NLRP3 expression.

JOURNAL/nrgr/04.03/01300535-202507000-00028/figure1/v/2024-09-09T124005Z/r/image-tiff Alzheimer's disease is characterized by deposition of amyloid-β, which forms extracellular neuritic plaques, and accumulation of hyperphosphorylated tau, which aggregates to form intraneuronal neurofibrillary tangles, in the brain. The NLRP3 inflammasome may play a role in the transition from amyloid-β deposition to tau phosphorylation and aggregation. Because NLRP3 is primarily found in brain microglia, and tau is predominantly located in neurons, it has been suggested that NLRP3 expressed by microglia indirectly triggers tau phosphorylation by upregulating the expression of pro-inflammatory cytokines. Here, we found that neurons also express NLRP3 in vitro and in vivo, and that neuronal NLRP3 regulates tau phosphorylation. Using biochemical methods, we mapped the minimal NLRP3 promoter and identified FUBP3 as a transcription factor regulating NLRP3 expression in neurons. In primary neurons and the neuroblastoma cell line Neuro2A, FUBP3 is required for endogenous NLRP3 expression and tau phosphorylation only when amyloid-β is present. In the brains of aged wild-type mice and a mouse model of Alzheimer's disease, FUBP3 expression was markedly increased in cortical neurons. Transcriptome analysis suggested that FUBP3 plays a role in neuron-mediated immune responses. We also found that FUBP3 trimmed the 5' end of DNA fragments that it bound, implying that FUBP3 functions in stress-induced responses. These findings suggest that neuronal NLRP3 may be more directly involved in the amyloid-β-to-phospho-tau transition than microglial NLRP3, and that amyloid-β fundamentally alters the regulatory mechanism of NLRP3 expression in neurons. Given that FUBP3 was only expressed at low levels in young wild-type mice and was strongly upregulated in the brains of aged mice and Alzheimer's disease mice, FUBP3 could be a safe therapeutic target for preventing Alzheimer's disease progression.

Recent insights from non-mammalian models of brain injuries: an emerging literature.

Traumatic brain injury (TBI) is a major global health concern and is increasingly recognized as a risk factor for neurodegenerative diseases including Alzheimer's disease (AD) and chronic traumatic encephalopathy (CTE). Repetitive TBIs (rTBIs), commonly observed in contact sports, military service, and intimate partner violence (IPV), pose a significant risk for long-term sequelae. To study the long-term consequences of TBI and rTBI, researchers have typically used mammalian models to recapitulate brain injury and neurodegenerative phenotypes. However, there are several limitations to these models, including: (1) lengthy observation periods, (2) high cost, (3) difficult genetic manipulations, and (4) ethical concerns regarding prolonged and repeated injury of a large number of mammals. Aquatic vertebrate model organisms, including Petromyzon marinus (sea lampreys), zebrafish (Danio rerio), and invertebrates, Caenorhabditis elegans (C. elegans), and Drosophila melanogaster (Drosophila), are emerging as valuable tools for investigating the mechanisms of rTBI and tauopathy. These non-mammalian models offer unique advantages, including genetic tractability, simpler nervous systems, cost-effectiveness, and quick discovery-based approaches and high-throughput screens for therapeutics, which facilitate the study of rTBI-induced neurodegeneration and tau-related pathology. Here, we explore the use of non-vertebrate and aquatic vertebrate models to study TBI and neurodegeneration. Drosophila, in particular, provides an opportunity to explore the longitudinal effects of mild rTBI and its impact on endogenous tau, thereby offering valuable insights into the complex interplay between rTBI, tauopathy, and neurodegeneration. These models provide a platform for mechanistic studies and therapeutic interventions, ultimately advancing our understanding of the long-term consequences associated with rTBI and potential avenues for intervention.

Lymphocyte-Activation Gene 3 Facilitates Pathological Tau Neuron-to-Neuron Transmission.

The spread of prion-like protein aggregates is a common driver of pathogenesis in various neurodegenerative diseases, including Alzheimer's disease (AD) and related Tauopathies. Tau pathologies exhibit a clear progressive spreading pattern that correlates with disease severity. Clinical observation combined with complementary experimental studies has shown that Tau preformed fibrils (PFF) are prion-like seeds that propagate pathology by entering cells and templating misfolding and aggregation of endogenous Tau. While several cell surface receptors of Tau are known, they are not specific to the fibrillar form of Tau. Moreover, the underlying cellular mechanisms of Tau PFF spreading remain poorly understood. Here, it is shown that the lymphocyte-activation gene 3 (Lag3) is a cell surface receptor that binds to PFF but not the monomer of Tau. Deletion of Lag3 or inhibition of Lag3 in primary cortical neurons significantly reduces the internalization of Tau PFF and subsequent Tau propagation and neuron-to-neuron transmission. Propagation of Tau pathology and behavioral deficits induced by injection of Tau PFF in the hippocampus and overlying cortex are attenuated in mice lacking Lag3 selectively in neurons. These results identify neuronal Lag3 as a receptor of pathologic Tau in the brain，and for AD and related Tauopathies, a therapeutic target.

Alzheimer proteopathic tau seeds are biochemically a forme fruste of mature paired helical filaments.

Aggregation prone molecules, such as tau, form both historically well characterized fibrillar deposits (neurofibrillary tangles) and recently identified phosphate-buffered saline (PBS) extract species called proteopathic seeds. Both can cause normal endogenous tau to undergo templated misfolding. The relationship of these seeds to the fibrils that define tau-related diseases is unknown. We characterized the aqueous extractable and sarkosyl insoluble fibrillar tau species derived from human Alzheimer brain using mass spectrometry and in vitro bioassays. Post-translational modifications (PTMs) including phosphorylation, acetylation and ubiquitination are identified in both preparations. PBS extract seed competent tau can be distinguished from sarkosyl insoluble tau by the presence of overlapping, but less abundant, PTMs and an absence of some PTMs unique to the latter. The presence of ubiquitin and other PTMs on the PBS-extracted tau species correlates with the amount of tau in the seed competent size exclusion fractions, with the bioactivity and with the aggressiveness of clinical disease. These results demonstrate that the PTMs present on bioactive, seed competent PBS extract tau species are closely related to, but distinct from, the PTMs of mature paired helical filaments, consistent with the idea that they are a forme fruste of tau species that ultimately form fibrils.

Tracking Tau in Neurons: How to Grow, Fix, and Stain Primary Neurons for the Investigation of Tau in All Developmental Stages.

Primary murine neurons are a well-established tool for investigating Tau in the context of neuronal development and neurodegeneration. However, culturing primary neurons is usually time-consuming and requires multiple feeding steps, media exchanges, proprietary media supplements, and/or preparation of complex media. Here, we describe (i) a relatively cheap and easy cell culture procedure for the cultivation of forebrain neurons from embryonic mice (E13.5) based on a commercially available neuronal supplement (NS21), (ii) a protocol for the cultivation of hippocampal and cortical neurons from postnatal (P0-P3) animals, and (iii) basic fixation and immunofluorescence techniques for the staining of neuronal markers and endogenous Tau. We demonstrate a staining technique, which minimizes antibody consumption and allows for fast and convenient processing of samples for immunofluorescence microscopy of endogenous Tau in primary neurons. We also provide a protocol that enables cryopreservation of fixed cells for years without measurable loss of Tau signal. In sum, we provide reliable protocols enabling microscopy-based studies of Tau in primary murine neurons.

Differentiating SH-SY5Y Cells into Polarized Human Neurons for Studying Endogenous and Exogenous Tau Trafficking: Four Protocols to Obtain Neurons with Noradrenergic, Dopaminergic, and Cholinergic Properties.

Pathological alterations of the neuronal Tau protein are characteristic for many neurodegenerative diseases, called tauopathies. To investigate the underlying mechanisms of tauopathies, human neuronal cell models are required to study Tau physiology and pathology in vitro. Primary rodent neurons are an often used model for studying Tau, but rodent Tau differs in sequence, splicing, and aggregation propensity, and rodent neuronal physiology cannot be compared to humans. Human-induced pluripotent stem cell (hiPSC)-derived neurons are expensive and time-consuming. Therefore, the human neuroblastoma SH-SY5Y cell line is a commonly used cell model in neuroscience as it combines convenient handling and low costs with the advantages of human-derived cells. Since naïve SH-SY5Y cells show little similarity to human neurons and almost no Tau expression, differentiation is necessary to obtain human-like neurons for studying Tau protein-related aspects of health and disease. As they express in principle all six Tau isoforms seen in the human brain, differentiated SH-SY5Y-derived neurons are suitable for investigating the human microtubule-associated protein Tau and, for example, its sorting and trafficking. Here, we describe and discuss a general cultivation procedure as well as four differentiation methods to obtain SH-SY5Y-derived neurons resembling noradrenergic, dopaminergic, and cholinergic properties, based on the treatment with retinoic acid (RA), brain-derived neurotrophic factor (BDNF), and 12-O-tetrade canoylphorbol-13-acetate (TPA). TPA and RA-/TPA-based protocols achieve differentiation efficiencies of 40-50% after 9 days of treatment. The highest differentiation efficiency (~75%) is accomplished by a combination of RA and BDNF; treatment only with RA is the most time-efficient method as ~50% differentiated cells can be obtained already after 7 days.

Tracking Tau in Neurons: How to Transfect and Track Exogenous Tau in Primary Neurons.

Primary murine neurons have proved to be an essential tool for the general investigation of neuronal polarity, polarized Tau distribution, and Tau-based neuronal dysfunction in disease paradigms. However, mature primary neurons are notoriously difficult to transfect with non-viral approaches and are very sensitive to cytoskeletal manipulation and imaging. Furthermore, standard non-viral transfection techniques require the use of a supportive glial monolayer or high-density cultures, both of which interfere with microscopy. Here we provide a simple non-viral liposome-based transfection method that enables transfection of Tau in low levels comparable to endogenous Tau. This allows the investigation of, for example, distribution and trafficking of Tau, without affecting other cytoskeleton-based parameters such as microtubule density or microtubule-based transport. Using this protocol, we achieve a profound transfection efficiency but avoid high overexpression rates. Importantly, this transfection method can be applied to neurons at different ages and is also suitable for very old cultures (up to 18days in vitro). In addition, the protocol can be used in cultures without glial support and at suitable cell densities for microscopy-based single cell analysis. In sum, this protocol has proven a reliable tool suitable for most microscopy-based approaches in our laboratory.

The role of microtubule-associated protein tau in netrin-1 attractive signaling.

Direct binding of netrin receptors with dynamic microtubules (MTs) in the neuronal growth cone plays an important role in netrin-mediated axon guidance. However, how netrin-1 (NTN1) regulates MT dynamics in axon turning remains a major unanswered question. Here, we show that the coupling of netrin-1 receptor DCC with tau (MAPT)-regulated MTs is involved in netrin-1-promoted axon attraction. Tau directly interacts with DCC and partially overlaps with DCC in the growth cone of primary neurons. Netrin-1 induces this interaction and the colocalization of DCC and tau in the growth cone. The netrin-1-induced interaction of tau with DCC relies on MT dynamics and TUBB3, a highly dynamic β-tubulin isotype in developing neurons. Netrin-1 increased cosedimentation of DCC with tau and TUBB3 in MTs, and knockdown of either tau or TUBB3 mutually blocked this effect. Downregulation of endogenous tau levels by tau shRNAs inhibited netrin-1-induced axon outgrowth, branching and commissural axon attraction in vitro, and led to defects in spinal commissural axon projection in vivo. These findings suggest that tau is a key MT-associated protein coupling DCC with MT dynamics in netrin-1-promoted axon attraction.

Characteristics of AD‐brain tau aggregate internalisation by human astrocytes and the responses induced

AbstractBackgroundHighly phosphorylated tau aggregates are deposited in tauopathy brain as disease progresses, driven by a prion‐like spread of tau seeds. Evidence suggests that astrocytes may influence tau spread. However, the efficiency of uptake of disease‐associated tau species is not well defined, nor are the effects of tau uptake on astrocyte reactivity and function. We investigate human astrocyte ability to internalise and clear tau aggregates derived from multiple Alzheimer’s disease (AD) cases, and their impact on astrocyte function and reactivity. We further explore alterations in endogenous tau expression, APP processing and cytoskeletal proteins following tau uptake.MethodControl human iPSC‐derived astrocytes were cultured for 70 days. Aggregated (sarkosyl‐insoluble) tau was isolated from postmortem AD brain. Post‐translational modifications (PTM) of isolated AD‐brain tau aggregates were analysed by mass spectrometry. Tau (0.1 ng/µL) and extracts from control human brain were spiked into astrocyte cultures. Internalisation of aggregated tau and the localised response of S100B and GFAP were quantified by ICC. Changes to tau, APP, functional and reactivity related genes were measured by qPCR and/or proteins by SDS‐PAGE.ResultAggregated tau from AD brain is readily internalised by human astrocytes, and sequesters GFAP and S100B. Differences in the rate of uptake, clearance and seeding of tau correlates with cytoskeletal protein accumulation and specific changes in astrocyte mRNA and protein levels. Work is in progress to relate these changes to characteristics of tau aggregates.ConclusionOur data shows that the capacity of AD brain aggregates to be internalised, cleared and induce seeding of endogenous tau varies by case. Investigations are underway to pinpoint the tau characteristics that underlie these differences. We also show that seeding ability is associated with changes in cytoskeletal elements and reactivity markers at mRNA and protein level. This work agrees with growing evidence that tau variability in AD is linked with specific cell type vulnerabilities that contribute to tau spread and disease progression.

A role of tau‐induced astrocyte dysfunction and senescence in AD

AbstractBackgroundUnder certain conditions and during aging, cellular stress and damage can trigger cellular senescence, an irreversible state of cell cycle arrest accompanied by the expression of proinflammatory mediators known collectively as the “senescent‐associated secretory phenotype” (SASP). In Alzheimer’s disease (AD), neuronal tau becomes hyperphosphorylated and misfolded, forming pathogenic soluble aggregates (tau oligomers) and destabilizing the microtubule cytoskeleton. Pathogenic soluble tau is released from neurons during neuronal activation and is transmitted in a prion‐like fashion to postsynaptic cells. In addition to neurons, tau is expressed in a variety of brain cell types including astrocytes. In the present study, we tested the central hypothesis that soluble pathogenic tau can be transmitted to astrocytes and that tau transmission to astrocytes induces cytoskeleton destabilization and cellular dysfunction, contributing to the progression of AD.MethodsTo test the molecular consequences of tau transmission, we treated primary human astrocytes with purified tau oligomers. We used advanced biochemical tools, immunocytochemistry, and live‐cell microscopy to measure cell and nuclear size, cell cycle arrest, and SASP activation. We also co cultured human astrocytes undergoing tau‐induced senescence with primary mouse or non‐human primate neurons to determine the functional impact of senescent astrocytes on neurons, then utilized immunocytochemistry and 3D image analyses to measure parameters of dendrite and spine morphology.ResultsWe found that pathogenic soluble tau is readily transmitted to primary human astrocytes, and that pathogenic tau transmission triggers phosphorylation of endogenous astrocyte tau, leading to the destabilization of the microtubule cytoskeleton. Further, we found that transmission of pathogenic tau to astrocytes potently upregulated multiple markers of cellular senescence, demonstrating for the first time tau‐induced astrocyte senescence. Density of dendritic spines and dendritic area were pronouncedly decreased in neurons co‐cultured with astrocytes undergoing tau‐induced senescence.ConclusionWe showed transmission of pathogenic tau to human astrocytes and that this event triggers astrocyte senescence in mouse models of AD tauopathy. Our data suggest that senescent astrocytes may be critical mediators of neuronal dysfunction in AD tauopathy.

Extracellular Tau Oligomers Damage the Axon Initial Segment by an Endogenous Tau‐Dependent Mechanism

AbstractBackgroundNeuronal polarity and synaptic connectivity are compromised in Alzheimer’s disease (AD) and other tauopathies. The axon initial segment (AIS) is a key structure for regulating polarity and functions of neurons. It occupies the first 20‐60 µm of the axon, comprises a diffusion barrier that segregates axon‐enriched from somatodendritic‐enriched molecules, and has a high concentration of voltage‐gated ion channels that generate action potentials. Extracellular amyloid‐β oligomers compromise AIS integrity. However, effects on the AIS of toxic tau species, including extracellular oligomers (xcTauOs) that spread tau pathology from neuron to neuron by a prion‐like process, whereas unknown. Therefore, we wanted to test the hypothesis that AIS structure is sensitive to xcTauOs.MethodPrimary cortical neurons derived from either wild type (WT), or tau knockout (KO) mice were exposed to xcTauOs or vehicle. Quantitative western blotting and immunofluorescence microscopy with an antibody against the AIS‐enriched protein TRIM46 was used to monitor effects on the AIS. The same methods were also used to compare TRIM46 and two other AIS proteins, ankyrin‐G and neurofascin‐186 in human hippocampal tissue obtained from AD and age‐matched non‐AD donors.ResultIn cultured WT, but not TKO neurons, xcTauOs cause a trend toward AIS shortening and reduce the concentration of the resident AIS protein, TRIM46, without affecting total TRIM46 levels. Lentiviral‐driven human tau expression in tau KO neurons rescues TRIM46 sensitivity to xcTauOs. In human AD hippocampus, AIS length and TRIM46 concentration within the AIS are reduced in neurons containing neurofibrillary tangles (NFTs), without affecting the overall protein levels of multiple resident AIS proteins.ConclusionThese collective findings demonstrate that in cultured neurons, xcTauOs cause partial AIS damage by a mechanism dependent on intracellular tau, thereby raising the possibility that AIS reduction in AD is caused by xcTauOs working in concert with endogenous neuronal tau.

Outbreak of tauopathy in the olfactory system of PS19 mice: an early marker for Alzheimer’s disease diagnosis?

AbstractBackgroundClinical data indicate that impairment of olfactory function is an early indicator of neurodegenerative diseases, including Alzheimer’s disease (AD)(Attems et al., 2014). Our hypothesis is that typical AD lesions could appear in olfactory neurons from the olfactory epithelium (OE). The olfactory epithelium can be easily collected in human individuals (e.g., by nasal brushing), which provides an important tool to investigate the pathology in patients. Here, we address the question of the occurrence of tauopathy in the OE of transgenic mice and its correlation with pathology in the central nervous system (CNS). We use Tau P301S mice (PS19 mice), a tauopathy mouse model. These mice express human tau protein (1N4R) carrying the P301S mutation. They develop neurofibrillary tangle‐like inclusions in the cortex and hippocampus, leading to progressive neurodegeneration at eight months (Yoshiyama et al., 2007).MethodMouse samples were collected from heterozygous PS19 and wild‐type (WT) mice. Immunohistochemical staining (DAB) was used to demonstrate the presence of hyperphosphorylated human tau protein (hTau). We investigated regions of the olfactory system to correlate with data obtained in CNS regions (e.g., hippocampal formation, entorhinal cortex). Mouse heads decalcification was also implemented to preserve the olfactory epithelium structure. Western blots were performed to monitor tau levels and tau phosphorylation pattern.ResultThe hyperphosphorylated human tau protein (Ser202, Thr205) is only detected in PS19 mice sections while the endogenous murine tau protein is expressed both in WT and PS19 mice. The localization and expression of these proteins vary according to the region (OE or CNS subregions), the age (3, 6 or 9 months), and the genotype of the mice (WT or heterozygous PS19). It also seems that tau lesions may spread between brain regions that are directly connected, notably through the olfactory pathway.ConclusionIn PS19 mice, a pathological phosphorylation pattern peaked in the OE prior to vulnerable CNS regions or olfactory bulbs. We hypothesize that tau lesions appearing early in the OE may spread in the olfactory bulb and finally reach the entorhinal cortex and piriform cortex, highlighting the significance of the olfactory system investigation as an early diagnosis tool.

BAG3 regulates RAB35 mediates the Endosomal Sorting Complexes Required for Transport/Endolysosome pathway and Tau clearance

AbstractBackgroundDeclining proteostasis with aging contributes to increased susceptibility to neurodegenerative diseases, including Alzheimer’s disease (AD). Emerging studies implicate impairment of the endosome‐lysosome pathway as a significant factor in the pathogenesis of these diseases. Our lab was the first to demonstrate that BAG3 regulates phosphorylated tau clearance. However, we did not fully define how BAG3 regulates endogenous tau proteostasis, especially in the early stages of disease progression.MethodsMass spectrometric analyses were performed to identify neuronal BAG3 interactors. Multiple biochemical assays were used to investigate the BAG3‐HSP70‐TBC1D10B‐Rab35‐Hrs regulatory networks. Live‐cell imaging was used to study the dynamic of endosomal pathway. Immunohistochemistry and immunoblotting were performed in human AD brains and BAG3 overexpressed P301S tau transgenic mice.ResultsThe primary group of neuronal BAG3 interactors identified are involved in the endocytic pathway. Among them were key regulators of small GTPases, such as the Rab35 GTPase activating protein, TBC1D10B. We demonstrated that a BAG3‐HSP70‐TBC1D10B complex attenuates the ability of TBC1D10B to inactivate Rab35. Thus, BAG3 interacts with TBC1D10B to support the activation of Rab35 and recruitment of Hrs, initiating ESCRT‐mediated endosomal tau clearance. Further,TBC1D10B shows significantly less co‐localization with BAG3 in AD brains than in age‐matched controls. Overexpression of BAG3 in P301S tau transgenic mice increased the co‐localization of phospho‐tau with the ESCRT III protein CHMP2B and reduced the levels of the mutant human tau.ConclusionWe identified a novel BAG3‐TBC1D10B‐Rab35 regulatory axis that modulates ESCRT‐dependent protein degradation machinery and tau clearance. Dysregulation of BAG3 could contribute to the pathogenesis of AD.

Hsp40 molecular chaperones are potent regulators of tau seeding and accumulation

AbstractBackgroundThe misfolding of the microtubule associated protein tau is associated with Alzheimer’s disease and other tauopathies. In tauopathies, aberrant tau spreads through synaptically connected regions of the brain by templating the misfold of endogenous tau. Work from our group and others has identified molecular chaperones as regulators of tau accumulation and toxicity, but many tau regulators have yet to be identified.MethodWe performed a screen of members from seven major chaperone families using Tau RD P301S FRET Biosensor cells. Hits were assessed by secondary knockdown studies in cultured cells and validated in tau expressing primary neurons. Additional in vitro experiments to dissect the mechanism of activity are ongoing, which will be followed by in vivo studies.ResultOur data support the use of Tau RD P301S FRET Biosensor cells as a robust screening tool to identify known and novel tau regulators. The Hsp40 family is enriched with tau regulators, including some not previously known to affect tau. Studies are ongoing to determine how these proteins alter tau levels.ConclusionMultiple members of the Hsp40 family regulate tau accumulation.