Tau Oligomers Research Articles

Alzheimer's disease as an autoimmune disorder of innate immunity endogenously modulated by tryptophan metabolites.

IntroductionAlzheimer's disease (AD) is characterized by neurotoxic immuno‐inflammation concomitant with cytotoxic oligomerization of amyloid beta (Aβ) and tau, culminating in concurrent, interdependent immunopathic and proteopathic pathogeneses.MethodsWe performed a comprehensive series of in silico, in vitro, and in vivo studies explicitly evaluating the atomistic–molecular mechanisms of cytokine‐mediated and Aβ‐mediated neurotoxicities in AD. Next, 471 new chemical entities were designed and synthesized to probe the pathways identified by these molecular mechanism studies and to provide prototypic starting points in the development of small‐molecule therapeutics for AD. ResultsIn response to various stimuli (e.g., infection, trauma, ischemia, air pollution, depression), Aβ is released as an early responder immunopeptide triggering an innate immunity cascade in which Aβ exhibits both immunomodulatory and antimicrobial properties (whether bacteria are present, or not), resulting in a misdirected attack upon “self” neurons, arising from analogous electronegative surface topologies between neurons and bacteria, and rendering them similarly susceptible to membrane‐penetrating attack by antimicrobial peptides (AMPs) such as Aβ. After this self‐attack, the resulting necrotic (but not apoptotic) neuronal breakdown products diffuse to adjacent neurons eliciting further release of Aβ, leading to a chronic self‐perpetuating autoimmune cycle. AD thus emerges as a brain‐centric autoimmune disorder of innate immunity. Based upon the hypothesis that autoimmune processes are susceptible to endogenous regulatory processes, a subsequent comprehensive screening program of 1137 small molecules normally present in human brain identified tryptophan metabolism as a regulator of brain innate immunity and a source of potential endogenous anti‐AD molecules capable of chemical modification into multi‐site therapeutic modulators targeting AD's complex immunopathic–proteopathic pathogenesis. Discussion Conceptualizing AD as an autoimmune disease, identifying endogenous regulators of this autoimmunity, and designing small molecule drug‐like analogues of these endogenous regulators represents a novel therapeutic approach for AD.

Inflammasome activation in neurodegenerative diseases.

Approximately ten million people are diagnosed with dementia annually since they experience difficulties with memory and thinking skills. Since neurodegenerative diseases are diagnosed late, most of them are difficult to treat. This is due to the increased severity of the disease during the progression when neuroinflammation plays a critical role. The activation of immune cells, especially microglia, plays a crucial role in the development of neurodegenerative diseases. Molecular sensors within these microglia, such as the NLRP3 inflammasome, are activated by signals that represent the hallmarks of neurodegenerative diseases. Here, we first summarize the two activation steps of NLRP3 inflammasome activation. Furthermore, we discuss the key factors that contribute to NLRP3 inflammasome activation in the different neuroinflammatory diseases, like Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). The prominent NLRP3 inflammasome triggers include amyloid β and tau oligomers in AD, α-synuclein in PD, and superoxide dismutase (SOD1) and TAR DNA-binding protein 43 (TDP43) in ALS. NLRP3 inhibitor treatment has shown promising results in several preclinical mouse models of AD, PD, and ALS. Finally, we postulate that current understandings underpin the potential for NLRP3 inhibitors as a therapeutic target in neurodegenerative diseases.

Oligomer-Targeting Prevention of Neurodegenerative Dementia by Intranasal Rifampicin and Resveratrol Combination - A Preclinical Study in Model Mice.

Amyloidogenic protein oligomers are thought to play an important role in the pathogenesis of neurodegenerative dementia, including Alzheimer’s disease, frontotemporal dementia, and dementia with Lewy bodies. Previously we demonstrated that oral or intranasal rifampicin improved the cognition of APP-, tau-, and α-synuclein-transgenic mice by reducing the amount of Aβ, tau, and α-synuclein oligomers in the brain. In the present study, to explore more effective and safer medications for dementia, we tested the drug combination of rifampicin and resveratrol, which is a multifunctional natural polyphenol with the potential to antagonize the adverse effects of rifampicin. The mixture was intranasally administered to APP-, tau-, and α-synuclein-transgenic mice, and their memory and oligomer-related pathologies were evaluated. Compared with rifampicin and resveratrol alone, the combinatorial medicine significantly improved mouse cognition, reduced amyloid oligomer accumulation, and recovered synaptophysin levels in the hippocampus. The plasma levels of liver enzymes, which reflect hepatic injury and normally increase by rifampicin treatment, remained normal by the combination treatment. Notably, resveratrol alone and the combinatorial medicine, but not rifampicin alone, enhanced the levels of brain-derived neurotrophic factor (BDNF) and its precursor, pro-BDNF, in the hippocampus. Furthermore, the combination showed a synergistic effect in ameliorating mouse cognition. These results show the advantages of this combinatorial medicine with regards to safety and effectiveness over single-drug rifampicin. Our findings may provide a feasible means for the prevention of neurodegenerative dementia that targets toxic oligomers.

Differential expression of tau species and the association with cognitive decline and synaptic loss in Alzheimer's disease.

Pathological tau proteins in patients with Alzheimer's disease (AD) mainly accumulate in the form of neurofibrillary tangles (NFTs) and neuritic plaques (NPs). However, the molecular properties of tau species present in NFTs and NPs are not known. We tested the hypothesis that tau species within NFT-predominant tissue (NFT_AD) are distinct and more toxic than those in NP-predominant tissue (NP_AD). We analyzed the tau species from post mortem prefrontal cortical brains of NFT_AD and NP_AD. Compared to NP_AD, NFT_AD displayed highly phosphorylated tau oligomers, possessed tau oligomers in extracellular vesicles, and the 3-repeat (3R) and 4-repeat (4R) isoforms were differentially expressed between the groups. Comparison of tau proteins isolated from NFT- versus NP-AD subjects demonstrated higher tau seeding activity in NFT subjects and a greater degree of inducing synaptic loss in cultured neurons. We propose that tau species from NFT-predominant tissues possess greater levels of degenerative properties, thereby causing synaptic loss and cognitive decline.

Single‐particle characterization of tau oligomers in solution

AbstractBackgroundProtein aggregation is the hallmark of neurodegenerative disorders such as Alzheimer’s disease (AD), and oligomeric species of microtubule‐associated protein Tau are implicated as neurotoxic species as well as promising biomarkers. The size and shape of the oligomers are critical to their toxicity. Currently existing, sensitive immunoassays for biomarker detection do, however, not provide information on the size‐ or shape‐distribution of Tau aggregates. This limitation reduces the information content from tau analyses since specific sizes and shapes of oligomeric Tau aggregates are more toxic than others. Hence, a novel approach that reveals the size‐, and the shape‐distribution of Tau oligomers rapidly in solution would be ideal.MethodHere, we use resistive‐pulse sensing with solid‐state nanopores to detect and characterize Tau oligomers on a single particle basis in solution with respect to their size and shape.ResultThe nanopore‐based approach facilitates single‐particle detection and fingerprinting of tau oligomers with regard to their size and shapes. We can determine the heterogeneity among oligomers with respect to their sizes and shapes, which is usually overlooked by averaging. We follow and compare the aggregation kinetics of wild type and AD‐associated variants of Tau.ConclusionResistive‐pulse sensing using nanopores allows rapid fingerprinting Tau oligomers in solution in a label‐free manner. The size and shape information of oligomeric species can improve the general understanding of their toxicity and facilitate the development of new prevention strategies.

Oligomeric tau disrupts nuclear envelope via binding to lamin proteins and lamin B receptor.

Accumulating evidence indicates that the nucleocytoplasmic interface is greatly disrupted in Alzheimer's disease and related disorders (ADRD). Many proteins that normally exist in the nucleus, such as RNA binding proteins, or in the nuclear membrane, such as nucleoporins, accumulate in the cytoplasm. Multiple studies also show dysmorphic nuclear membrane pathology in AD brain and in models of AD. However, the mechanism underlying changes in the nucleocytoplasmic interface are unknown. Because oligomeric tau (oTau) is the key toxic species in tauopathy, we hypothesized that interaction of oTau with nuclear membrane proteins might be responsible for the nuclear membrane pathology. We developed a light-reactive optogenetic tau::Cry2Olig system to rapidly convert tau from monomers to oligomers in primary cortical neurons. Proteins that aggregated and became insoluble in neurons with tau oligomerization were then isolated and elucidated by mass spectrometry and proteomics analysis. Tau oligomerization selectively induced nuclear membrane defects. The nuclear membrane became deformed within minutes upon light induced tau oligomerization, but not with oligomerization of just the CryOlig protein. Prolonged light exposure increased tau oligomerization, elicited tau fibrillization and led to even greater disruption of nuclear membrane structure. Analysis of the insoluble fraction revealed many proteins associated with the nuclear envelope, including Lmna, Lamb1, Lamb2, Ran, nup155, nup88, and nup98. Immunohistochemical studies displayed loss of localization of Lmna in the nuclear membrane. Studies in PS19 mice and human AD brain tissue using proximity ligation assays showed that laminB2 and lamin B Receptor (LBR) were recruited and co-localized with oTau. Immunoprecipitation of oTau complex from aged PS19 mice also demonstrated the direct binding of laminB2 and LBR to oTau in tauopathy. oTau directly binds to lamin proteins and the lamin B Receptor. These proteins also exhibit loss of solubility upon oligomerization, and show loss of nuclear membrane localization. These results suggest that oTau directly acts on proteins in the nuclear membrane to elicit disruption of the nucleocytoplasmic interface.

Amelioration of tau related pathology with a novel anti‐prion protein monoclonal antibody in an AD mouse model

AbstractBackgroundA number of studies have shown that amyloid β oligomers (Aβo) mediate their toxicity, in part, via the cellular prion protein (PrPC) on the surface of neurons. We hypothesized that tau oligomers may also mediate toxicity and spread of pathology via PrPC. We have generated a novel monoclonal anti‐PrP antibody (TW1) and assessed the efficacy of passive immunization with this antibody in a mouse model of AD with only tau related pathology.MethodHTau/PS1 transgenic AD model mice were injected at 5 months of age intraperitoneally once a week with TW1 from the age of 5 months. Control mice received only saline. Their behavior was assessed at 8 months of age and brain tissue subsequently harvested for analysis at 9 monthsResultThe treated and control mice did not show any significant differences in sensorimotor testing. Significant statistical differences were observed with the novel object recognition test in the immunized mice on percentage time spent in zones (two tailed, t‐test p = 0.0019). On the closed field symmetrical maze immunized mice performed significantly better (Day1: two tailed t‐test p=0.0001; Day2: two tailed t‐test p=0.0015; Day3: two tailed t‐test p=0.0002). Fifty percent less tau pathology was observed in the dentate gyrus with PHF‐1 immunolabeling (two tailed t‐test p= 0.02) and a 60% reduction was seen in the piriform cortex (two‐tailed, t‐test p=0.01) in treated vs control animals. CP13 immunolabeling showed a significant difference in the piriform cortex (p=0.02, two‐tailed t‐test) in treated mice versus controls. There was no significant difference in astrogliosis or microgliosis in treated versus control mice. Western blots using PHF‐1 showed that TW1 therapy reduced phosphorylated tau pathology (two‐tailed t‐test p=0.03) and improved the ratio of pathological soluble tau to tubulin (two‐tailed t‐test p=0.0006). The same effect were observed with the CP13 antibody (two‐tailed t‐test p=0.0007) and improved the ratio CP13/tubulin (two tailed, t‐test p=0.0014).ConclusionThese results indicate that passive immunization with the TW1 antibody can significantly decrease tau pathology and improve cognition in a transgenic mouse model of AD.

AD- and PSP-specific brain-derived tau oligomers engage synapses with different dynamic.

Alzheimer's Disease (AD) is characterized by gradual cognitive decline driven by the targeting of synapses by small oligomers of both Aβ (AβO) and tau (TauO), which results in synaptic dysfunction that ultimately underscores disease progression. Indeed, there is ample consensus that targeting oligomer binding to synapses would be an effective therapeutic concept for AD. However, recent failures of clinical trials of Aβ-directed therapeutics suggest reduced effectiveness of targeting Ab in clinically-manifest AD, redirecting attention onto TauO that are known to increase later in the disease timeline. In support of this vision, here we show that recombinant TauO, human brain derived tau oligomers (BDTO) and AβO target synapses with different dynamics. We used recombinant AβO and TauO as well as BDTO from AD (BDTO-AD), and from Progressive Supranuclear Palsy (BDTO-PSP). Analyses of flow-cytometry, western blot, immunofluorescence, and proteinase K digestion were performed to study the binding dynamic of the oligomers onto synaptosomes isolated from human and mouse frontal cortex and hippocampus. The synaptic dysfunction was measured by FASS-LTP assay. We found that TauO overcome AβO, becoming the prevailing species associated with the synapses. On the other hand, AβO are ineffective in competing TauO off the synapses, and at higher concentration AbO increase TauO binding to the synaptosomes. Consistent with these observations, analyses of FASS-LTP show that the suppression of long-term potentiation by TauO is not affected by the concomitant presence of AβO. Pre-digestion of synaptosomes with proteinase K abolishes the ability of TauO to overcome AβO without affecting the increased synaptic recruitment of TauO by high levels of AβO, suggesting that the former phenomenon necessitates of a protein substrate whereas the latter does not. Furthermore, we observed different synaptic engagement and internalization profiles among the analyzed BDTOs, suggesting different synaptic toxicity of TauO across the tauopathies spectrum. Our results suggest that at the advanced stages of AD, when tau levels increase, TauO become the main synaptototoxic species and likely an effective therapeutic target. Furthermore, subtle disease-specific differences in the dynamic of BDTOs engagement of synapses may underscore different clinical presentations among the tauopathy spectrum.

Characterisation of the biophysical and cellular properties of Tau35, a disease‐associated tau fragment

AbstractBackgroundTauopathies are characterised by the accumulation of intracellular tau aggregates in the brain. Tau inclusions contain various of truncated tau species, but the potential detrimental effects of these tau fragments are not well established. Tau35 is a C‐terminal fragment of wild‐type human tau, identified in tauopathy brain. We showed that minimal expression of Tau35 in transgenic mice recapitulates key features of tauopathy, including cognitive and motor dysfunction.MethodWe used small angle X‐ray scattering (SAXS), circular dichroism (CD), and transmission electron microscopy (TEM) to characterise the conformations of recombinant or aggregated Tau35 and full‐length (2N4R) tau. Tau assembly kinetics was monitored using Thioflavin T (ThT) and quantified. To assess cellular effects of tau, differentiated SH‐SY5Y cells (dSH) and mouse primary cortical neurons were exposed to Cy5‐labelled oligomeric tau and examined by immunofluorescence, western blots and live dead assay.ResultSAXS analysis showed that both Tau35 and 2N4R tau display characteristics of intrinsically disordered monomeric proteins, with Tau35 exhibiting more restricted motions. ThT kinetics indicated that Tau35 is more prone to beta‐sheet formation and this was confirmed by CD and TEM analysis. Rapid uptake of aggregated Tau35 and 2N4R tau was observed by both dSH and mouse neurons. Internalised tau accumulates with time. However no significant cell death were observed with dSH.ConclusionOur results demonstrate that Tau35, a fragment associated with human tauopathy, exhibits increased aggregation and rapid uptake into neuronal cells, which likely impacts on tau propagation.

Reduction in Aha1 chaperone preserves hippocampal neurons and prevents tau‐related pathology in rTg4510 tau transgenic mice

Several molecular chaperones have been identified as important modulators of tau protein in aging and neurodegenerative illnesses like Alzheimer's diseases. Tau-related diseases, collectively known as tauopathies, are characterized by abnormal deposition of insoluble tau protein leading to neuronal degeneration and the formation of neurofibrillary tangles in the brain. Although the exact mechanism is largely unknown, discrete molecular chaperones are known to regulate tau stability. Our laboratory demonstrated the activator of Hsp90 ATPase Activity (Aha1) potently promotes tau fibrilization and its expression positively correlates with features of tau pathology in brains from tau overexpressing mice and individuals with Alzheimer's disease. In this study, we investigate whether reduction of Aha1 levels in tau transgenic mice is sufficient to slow the progression of tau pathology in brains of rTg4510 tau transgenic mice.To test this, we designed three AAV9 plasmids targeting Aha1 and a scramble shRNA was used as control. Aha1 knockdown was confirmed in N2A cells using Western blot analysis. Following this, 4-month-old rTg4510 male and female tau transgenic mice were injected into the hippocampus with Aha1-shRNA or scramble-shRNA (control). Two months were allowed for viral expression followed by the collection of brains for protein and tissue analysis.n N2A cells, one out of three Aha1-targeting shRNAs effectively reduced Aha1 expression after 72 hours of viral transfection. In animals, Aha1 knockdown decreased pS396/404 and AT8 phosphorylated tau as well Gallyas-silver positive tau tangles in the hippocampus. Total or oligomeric tau were not affected in mice injected with AAV9-shAha1 when compared to AAV9-scramble animals. Using unbiased stereology, we found that hippocampal CA1 neurons were preserved after reducing Aha1.Our findings demonstrate that Aha1 knockdown reduces tau phosphorylated species, prevents tau tangle formation, and rescued CA1 hippocampal neurons in rTg4510 mice. Overall, these in vivo observations suggest that Aha1 knockdown could prevent development of pathological features like tangle formation in tauopathies. Although further examinations in human brain are needed, Aha1 inhibition could be a potential therapeutic target to slow or prevent tau pathologies.

Synaptic stimulation induces tau clearance by enhancing autophagosomal/lysosomal degradation.

Alterations of synaptic excitability and reduced brain metabolism are some of the earliest changes associated Alzheimer's disease (AD) pathogenesis (Reiman et al., 2004; Sperling et al., 2009). Among different approaches for therapeutics, the stimulation of synaptic activity exerts protective effects in models of AD, and deep brain stimulation (DBS) has been shown to ameliorate AD symptoms in certain patients (Sankar et al., 2015; Swaab and Bao, 2010; Tampellini, 2015). Such positive effects might be the result of changes occurring at cellular level upon activity induction, which promote cellular mechanisms correcting neuronal and synaptic dysfunctions. We have demonstrated that synaptic stimulation, via DBS or other methods, is protective in mouse models of AD and frontotemporal dementia (FTD) by enhancing autophagy and lysosomal degradation of pathologic tau, and by protecting synapses (Akwa et al., 2018; Mann et al., 2018). Our more recent investigations are revealing the involvement of Transcription factor EB (TFEB), a pivotal player in lysosomal biogenesis and autophagy (Sardiello et al., 2009), and its downstream genes in the enhancement of lysosomal activity upon stimulation. 3xTg mice received deep brain stimulation by electrode implantation in the entorhinal cortex (Mann et al., 2018). Cultured neurons were prepared from E15 PS19 mouse embryos (Akwa et al., 2018) and stimulated at 14 days in vitro (Ehlers, 2003). Confocal immunofluorescence, Western blot and statistical analyses were performed as described (Akwa et al., 2018). RT-qPCR was performed as described (Napolitano et al., 2018). DBS was able to reduce levels of hyperphosphorylated and oligomeric (but not total) tau restoring levels of synaptic proteins back to wild-type in 3xTg mice. Pathological tau clearance occurred via autophagic pathway and lysosomal activity, the latest being enhanced by synaptic stimulation. TFEB was required for tau clearance as its inhibition prevented activity-induced reduction of pathological tau. In addition, RT-qPCR data analyses revealed increase expressions of TFEB downstream genes, including ATP6-V1H, ATP6-V0D1, and mucolipin-1 in neurons during synaptic stimulation. Protective effects of DBS/synaptic stimulation against pathological tau include the involvement TFEB and the enhancement of autophagy and lysosomal degradation.

Exosomal tau with seeding activity is released from Alzheimer's disease synapses, and seeding potential is associated with amyloid beta

AbstractSynaptic transfer of tau has long been hypothesized from the human pathology pattern and has been demonstrated in vitro and in vivo, but the precise mechanisms remain unclear. Extracellular vesicles such as exosomes have been suggested as a mechanism, but not all tau is exosomal. The present experiments use a novel flow cytometry assay to quantify depolarization of synaptosomes by KCl after loading with FM2–10, which induces a fluorescence reduction associated with synaptic vesicle release; the degree of reduction in cryopreserved human samples equaled that seen in fresh mouse synaptosomes. Depolarization induced the release of vesicles in the size range of exosomes, along with tetraspanin markers of extracellular vesicles. A number of tau peptides were released, including tau oligomers; released tau was primarily unphosphorylated and C-terminal truncated, with Aβ release just above background. When exosomes were immunopurified from release supernatants, a prominent tau band showed a dark smeared appearance of SDS-stable oligomers along with the exosomal marker syntenin-1, and these exosomes induced aggregation in the HEK tau biosensor assay. However, the flow-through did not seed aggregation. Size exclusion chromatography of purified released exosomes shows faint signals from tau in the same fractions that show a CD63 band, an exosomal size signal, and seeding activity. Crude synaptosomes from control, tauopathy, and AD cases demonstrated lower seeding in tauopathy compared to AD that is correlated with the measured Aβ42 level. These results show that AD synapses release exosomal tau that is C-terminal-truncated, oligomeric, and with seeding activity that is enhanced by Aβ. Taken together with previous findings, these results are consistent with a direct prion-like heterotypic seeding of tau by Aβ within synaptic terminals, with subsequent loading of aggregated tau onto exosomes that are released and competent for tau seeding activity.

In vivo validation of therapeutic efficacy of a small molecule lead targeting tau self‐association in JNPL3 mice

AbstractBackgroundSmall molecules were screened and optimized for inhibiting tau self‐association, the initial step in the formation of soluble toxic tau oligomers and larger insoluble aggregates. This approach is differentiated from other tau aggregation inhibitor programs that have largely focused on inhibiting formation and/or dissociating large and relatively inert fibrils which could generate toxic tau oligomer seeds. Small molecules inhibitors are agnostic to specific post‐translational modifications, conformational changes or strains of tau aggregates, and have superior access to their target in the CNS relative to immunotherapeutic approaches. We have previously shown in vivo efficacy of a lead compound in preventive studies in htau (Davidowitz et al., 2020) and human tau P301L JNPL3 mouse models of tauopathy. The tau P301L mutation is the most common FTDP‐17 mutation and causes tau aggregation and motor deficits with age (Lewis et al., 2000).MethodBlinded studies of homozygous female tau 4R/0N P301L JNPL3 mice were performed at an independent laboratory where mice were treated for five months by administration of our lead compound in feed from 7 to 12 months‐of‐age. The four groups of mice in the study were baseline (7 months; n=20), vehicle, and two treatment groups (40 and 80 mg/kg doses; n=25 each). To quantify levels of Sarkosyl‐insoluble tau, self‐associated tau, and phosphorylated tau in the treated and control groups of mice ELISAs were performed (Acker et al., 2012; Forest et al., 2019). Rotarod and open field studies were performed to evaluate motor behavior.ResultEfficacy was shown in the cortex of treated mice that had reduced levels of self‐associated tau below baseline, baseline levels of insoluble tau aggregates, and reduced levels of aberrantly phosphorylated tau with statistical significance. In the hindbrain there was reduction of insoluble tau aggregates in the 40 mg/kg group compared to vehicle. Treatment groups showed improved Rotarod performance compared to baseline and vehicle groups.ConclusionTreatment with the lead compound reduced tau aggregation and improved motor behavior in aged JNPL3 mice and support preclinical development of the lead for clinical studies.

Caspase inhibition mitigates tau cleavage and neurotoxicity in iPSC‐induced neurons with the V337M MAPT mutation

Tau post-translational modifications (PTMs) are associated with progressive tau accumulation and neuronal loss in tauopathies, including forms of frontotemporal lobar degeneration (FTLD) and Alzheimer's disease (AD). Proteolytic cleavage of tau by active caspases, including caspase-6, represents an underexplored tau PTM implicated in tau pathology by generating tau species with an enhanced propensity for self-aggregation and accumulation into neuronal cytoplasmic inclusions. To elucidate the presence and temporal course of caspase activation, tau cleavage, and neuronal death, we generated two neoepitope monoclonal antibodies (mAbs) against caspase-6 tau proteolytic sites (D13 and D402) and cortical neurons (iNs) from induced pluripotent stem cells (iPSCs) with the frontotemporal dementia (FTD)-causing V337MMAPTmutation (tauV337M ). FTLD tauV337M and AD postmortem brains showed positivity for both cleaved tau mAbs as well as active caspase-6 (Fig. 1). Three month tauV337M iNs showed a three-fold increase in caspase-cleaved D402 and D13 tau protein levels and increased p-tau and oligomeric tau species compared to controls (Fig. 2). Three month tauV337M iNs also showed a significantly higher rate of neuronal death than the tauWT counterpart. Treatment with staurosporine (STS), a stress inducer, led to a 5-fold increase of cytotoxicity levels in STS-treated tauV337M iNs compared to vehicle-treated tauV337M iNs. Although STS-induced cytotoxicity was anticipated, the increase was significantly higher in STS-treated tauV337M iNs than in STS-treated control iNs. Similarly, we observed a 4-fold increase of caspase-6 activity in STS-treated tauV337M iNs compared to vehicle-treated tauV337M iNs that was reversed following co-treatment with the caspase-6 inhibitor z-VEID-fmk (Fig. 2). We propose a model in which time-dependent accumulation of caspase-cleaved tau in V337MMAPTneurons promotes neurotoxicity that is reversed by caspase-6 inhibition. Caspase-cleaved tau may be a biomarker of tauopathy, and caspases could be viable targets for therapeutic intervention against tau pathogenesis in FTLD and other tauopathies.

Tau seeds are widespread in the brain at the earliest stages of Alzheimer's disease-related changes.

Alzheimer's disease (AD) is pathologically characterized by the systematic spread of 3R/4R tau species with progressing disease. Current imaging and CSF in-vivo assessments of tau are thought to reflect more mature tangle neuropathology. However, earlier oligomeric tau species are indicated to have greater neurotoxicity than mature forms and it is largely unknown how tau seeds correlate with histological indicators of AD. Furthermore, tau co-pathology is thought to influence features and progression of other neurodegenerative diseases including Lewy body disease (LBD); yet reliable measurements of tau seeds in such diseases remains an unmet need. Here, we use tau real-time quaking-induced conversion (RT-QuIC) assays to directly measure bioactive tau seeds in brain samples across a spectrum of neurodegenerative diseases to determine tau seeding activities at different stages of AD related changes. Tau RT-QuIC exploits the seeded polymerization growth mechanism of tau filaments, with sensitivities in the low fM range, allowing quantitation of disease-associated aggregates with unprecedented sensitivity and selectivity. Tau RT-QuIC assays were used to quantitate tau seeds in mid-frontal lobe brain tissue from 64 cases including subjects with neuropathologically confirmed AD (n=15), Parkinson's disease (n=7), multiple system atrophy (n=6), LBD (n=14), frontotemporal lobar degeneration (n=4), progressive supranuclear palsy (n=6), corticobasal degeneration (n=2), early Alzheimer's changes (Braak I-III) (n=7), and controls (n=3). All cases had Braak tau staging and frontal lobes were immunohistochemically stained for tau with NFT1. 3R/4R tau seeding activity is typically 100-1000-fold higher in cases with a primary AD diagnosis compared to controls, 4R tauopathies, and synucleinopathies, consistent with our prior observations. Seeding activity positively correlated with Braak tau stage, a histological measure of tau accumulation, with increased tau seeding activities at higher Braak stages. Tau seeds are detectable in the frontal lobe even at Braak I, a stage at which frontal lobe lacks histologically visible tau deposits. Ultrasensitive RT-QuIC assays detect tau seeds at the earliest stages of disease processes, prior to histopathological evidence of more mature tau accumulations and can indicate co-occurrence of tau pathology in non-AD cases. Tau seeding activity correlates with overall Braak tau stage, raising the possibility of quantitative value of tau RT-QuIC assays.

The metabolic signaling of the nucleoredoxin-like 2 gene supports brain function

The nucleoredoxin gene NXNL2 encodes for two products through alternative splicing, rod-derived cone viability factor-2 (RdCVF2) that mediates neuronal survival and the thioredoxin-related protein (RdCVF2L), an enzyme that regulates the phosphorylation of TAU. To investigate the link between NXNL2 and tauopathies, we studied the Nxnl2 knockout mouse (Nxnl2−/−). We established the expression pattern of the Nxnl2 gene in the brain using a Nxnl2 reporter mouse line, and characterized the behavior of the Nxnl2−/− mouse at 2 months of age. Additionally, long term potentiation and metabolomic from hippocampal specimens were collected at 2 months of age. We studied TAU oligomerization, phosphorylation and aggregation in Nxnl2−/− brain at 18 months of age. Finally, newborn Nxnl2−/− mice were treated with adeno-associated viral vectors encoding for RdCVF2, RdCVF2L or both and measured the effect of this therapy on long-term potential, glucose metabolism and late-onset tauopathy. Nxnl2−/− mice at 2 months of age showed severe behavioral deficiency in fear, pain sensitivity, coordination, learning and memory. The Nxnl2−/− also showed deficits in long-term potentiation, demonstrating that the Nxnl2 gene is involved in regulating brain functions. Dual delivery of RdCVF2 and RdCVF2L in newborn Nxnl2−/− mice fully correct long-term potentiation through their synergistic action. The expression pattern of the Nxnl2 gene in the brain shows a predominant expression in circumventricular organs, such as the area postrema. Glucose metabolism of the hippocampus of Nxnl2−/− mice at 2 months of age was reduced, and was not corrected by gene therapy. At 18-month-old Nxnl2−/− mice showed brain stigmas of tauopathy, such as oligomerization, phosphorylation and aggregation of TAU. This late-onset tauopathy can be prevented, albeit with modest efficacy, by recombinant AAVs administrated to newborn mice. The Nxnl2−/− mice have memory dysfunction at 2-months that resembles mild-cognitive impairment and at 18-months exhibit tauopathy, resembling to the progression of Alzheimer's disease. We propose the Nxnl2−/− mouse is a model to study multistage aged related neurodegenerative diseases. The NXNL2 metabolic and redox signaling is a new area of therapeutic research in neurodegenerative diseases.

Contrasting Effects of Ferric and Ferrous Ions on Oligomerization and Droplet Formation of Tau: Implications in Tauopathies and Neurodegeneration.

The dysregulation of metal homeostasis is reported to enhance the aggregation of tau, a key neuronal microtubule-associated protein. Herein, we found that ferric (Fe3+) ions enhanced tau aggregation. Fe3+ and Al3+ induced tau aggregation while several trivalent metal ions such as Cr3+, La3+, and V3+ had no discernable effect on tau aggregation. Fe3+ reduced the critical concentration of tau required for the liquid-liquid phase separation (LLPS); however, Cr3+, La3+, and V3+ did not affect tau droplet formation. Dynamic light scattering, atomic force microscopic, and transmission electron microscopic analysis suggested that Fe3+ significantly increased the formation of tau oligomers and fibrils. In contrast, Fe2+ neither enhanced tau droplet formation nor increased the heparin-induced aggregation of tau. Using a tryptophan mutant (Y310W-tau) of tau, Fe3+ was found to bind to tau with four times higher affinity than Fe2+. Acrylamide quenching of the tryptophan fluorescence of Y310W-tau, 1-anilino-8-naphthalene sulfonate (ANS) fluorescence experiment, and far-UV circular dichroism analysis indicated that Fe3+ decreased the solvent exposure of the tryptophan residue, perturbed the hydrophobic surface arrangement, and disrupted the secondary structure of tau, respectively. The increase in the β-sheet content and a subsequent decrease in the disordered content of tau due to the binding of Fe3+ may favor tau aggregation. Fe3+ may enhance and stabilize the non-covalent interactions between disordered domains of tau molecules leading to tau aggregation. The data highlighted the relationship between the dysregulation of ferric ions and neurodegenerative disorders.

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 β protein (Aβ), 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.

Discovery of Hydroxybenzothiazole Urea Compounds as Multitargeted Agents Suppressing Major Cytotoxic Mechanisms in Neurodegenerative Diseases.

Multiple factors are causally responsible and/or contribute to the progression of Alzheimer's and Parkinson's diseases. The protein kinase Dyrk1A was identified as a promising target as it phosphorylates tau protein, α-synuclein, and parkin. The first goal of our study was to optimize our previously identified Dyrk1A inhibitors of the 6-hydroxy benzothiazole urea chemotype in terms of potency and selectivity. Our efforts led to the development of the 3-fluorobenzyl amide derivative 16b, which displayed the highest potency against Dyrk1A (IC50 = 9.4 nM). In general, the diversification of the benzylamide moiety led to an enhanced selectivity over the most homologous isoform, Dyrk1B, which was a meaningful indicator, as the high selectivity could be confirmed in an extended selectivity profiling of 3b and 16b. Eventually, we identified the novel phenethyl amide derivative 24b as a triple inhibitor of Dyrk1A kinase activity (IC50 = 119 nM) and the aggregation of tau and α-syn oligomers. We provide evidence that the novel combination of selective Dyrk1A inhibition and suppression of tau and α-syn aggregations of our new lead compound confers efficacy in several established cellular models of neurotoxic mechanisms relevant to neurodegenerative diseases, including α-syn- and 6-hydroxydopamine-induced cytotoxicities.

Dynamic interactions and Ca2+-binding modulate the holdase-type chaperone activity of S100B preventing tau aggregation and seeding

The microtubule-associated protein tau is implicated in the formation of oligomers and fibrillar aggregates that evade proteostasis control and spread from cell-to-cell. Tau pathology is accompanied by sustained neuroinflammation and, while the release of alarmin mediators aggravates disease at late stages, early inflammatory responses encompass protective functions. This is the case of the Ca2+-binding S100B protein, an astrocytic alarmin which is augmented in AD and which has been recently implicated as a proteostasis regulator, acting over amyloid β aggregation. Here we report the activity of S100B as a suppressor of tau aggregation and seeding, operating at sub-stoichiometric conditions. We show that S100B interacts with tau in living cells even in microtubule-destabilizing conditions. Structural analysis revealed that tau undergoes dynamic interactions with S100B, in a Ca2+-dependent manner, notably with the aggregation prone repeat segments at the microtubule binding regions. This interaction involves contacts of tau with a cleft formed at the interface of the S100B dimer. Kinetic and mechanistic analysis revealed that S100B inhibits the aggregation of both full-length tau and of the microtubule binding domain, and that this proceeds through effects over primary and secondary nucleation, as confirmed by seeding assays and direct observation of S100B binding to tau oligomers and fibrils. In agreement with a role as an extracellular chaperone and its accumulation near tau positive inclusions, we show that S100B blocks proteopathic tau seeding. Together, our findings establish tau as a client of the S100B chaperone, providing evidence for neuro-protective functions of this inflammatory mediator across different tauopathies.