Human Tauopathies Research Articles

Mice expressing P301S mutant human tau have deficits in interval timing

Interval timing is a key executive process that involves estimating the duration of an interval over several seconds or minutes. Patients with Alzheimer’s disease (AD) have deficits in interval timing. Since temporal control of action is highly conserved across mammalian species, studying interval timing tasks in animal AD models may be relevant to human disease. Amyloid plaques and tau neurofibrillary tangles are hallmark features of AD. While rodent models of amyloid pathology are known to have interval timing impairments, to our knowledge, interval timing has not been studied in models of tauopathy. Here, we evaluate interval timing performance of P301S transgenic mice, a widely studied model of tauopathy that overexpresses human tau with the P301S mutation. We employed an interval timing task and found that P301S mice consistently underestimated temporal intervals compared to wild-type controls, responding early in anticipation of the target interval. Our study indicating timing deficits in a mouse tauopathy model could have relevance to human tauopathies such as AD.

Downregulation of glob1 mitigates human tau mediated neurotoxicity by restricting heterochromatin loss and elevating the autophagic response in drosophila.

Human neuronal tauopathies are typically characterized by the accumulation of hyperphosphorylated tau in the forms of paired helical filaments and/or neurofibrillary tangles in the brain neurons. Tau-mediated heterochromatin loss and subsequent global transcriptional upsurge have been demonstrated as one of the key factors that promotes tau toxicity. We have reported earlier that expression of human tau-transgene in Drosophila induces the expression of glob1, and its restored level restricts tau etiology by regulating tau hyperphosphorylation and ROS generation via GSK-3β/p-Akt and Nrf2-keap1-ARE pathways, respectively. In view of this noted capability of glob1 in regulation of oxidative stress, and involvement of ROS in chromatin remodeling; we investigate if downregulation of glob1 restores tau-mediated heterochromatin loss in order to alleviate neurotoxicity. The tauV337M transgene was expressed in Drosophila eye by utilizing GAL4/UAS system. Expression of glob1 was depleted in tauV337M expressing tissues by co-expressing an UAS-glob1RNAi transgene by GMR-Gal4 driver. Immunostaining and wstern blot analysis suggested that tissue-specific downregulation of glob1 restores the cellular level of CBP and minimizes tau-mediated heterochromatin loss. It also assists in mounting an improved protective autophagic response to alleviate the human tau-induced neurotoxicity in Drosophila tauopathy models. Our study unfolds a novel aspect of the multitasking globin protein in restricting the pathogenesis of neuronal tauopathies. Interestingly, due to notable similarities between Drosophila glob1 and human globin gene(s), our findings may be helpful in developing novel therapeutic approaches against tauopathies.

Hebbian plasticity: the elusive missing link at the heart of Alzheimer's disease pathogenesis?

Hebbian plasticity: the elusive missing link at the heart of Alzheimer's disease pathogenesis?

Tau acetylation reduces its autophagic degradation and is a targetable pathway for human tauopathies.

Tau acetylation reduces its autophagic degradation and is a targetable pathway for human tauopathies.

Nodding syndrome: A key role for sources of nutrition?

Nodding Syndrome (NS) has occurred among severely food-stressed communities in northern Uganda and several other East African populations that, with their forced physical displacement, have resorted to nutritional support from available wild plants and fungi, some of which have neurotoxic potential. Among the latter is an agaric mushroom with an unknown content of hydrazine-generating agaritine, namely Agaricus bingensis, the unusually wide consumption of which may relate to the low serum levels of vitamin B6 in Ugandan NS subjects relative to controls. Hydrazine-related compounds induce patterns of DNA damage that promote neuropathological changes (tauopathy) reminiscent of those associated with established NS. While the cause of this childhood brain disease is unknown, we encourage increased attention to the role of malnutrition and B6 hypovitaminosis in the etiology of this devastating brain disease.

Mical modulates Tau toxicity via cysteine oxidation in vivo

Tau accumulation is clearly linked to pathogenesis in Alzheimer’s disease and other Tauopathies. However, processes leading to Tau fibrillization and reasons for its pathogenicity remain largely elusive. Mical emerged as a novel interacting protein of human Tau expressed in Drosophila brains. Mical is characterized by the presence of a flavoprotein monooxygenase domain that generates redox potential with which it can oxidize target proteins. In the well-established Drosophila Tauopathy model, we use genetic interactions to show that Mical alters Tau interactions with microtubules and the Actin cytoskeleton and greatly affects Tau aggregation propensity and Tau-associated toxicity and dysfunction. Exploration of the mechanism was pursued using a Mical inhibitor, a mutation in Mical that selectively disrupts its monooxygenase domain, Tau transgenes mutated at cysteine residues targeted by Mical and mass spectrometry analysis to quantify cysteine oxidation. The collective evidence strongly indicates that Mical’s redox activity mediates the effects on Tau via oxidation of Cys322. Importantly, we also validate results from the fly model in human Tauopathy samples by showing that MICAL1 is up-regulated in patient brains and co-localizes with Tau in Pick bodies. Our work provides mechanistic insights into the role of the Tau cysteine residues as redox-switches regulating the process of Tau self-assembly into inclusions in vivo, its function as a cytoskeletal protein and its effect on neuronal toxicity and dysfunction.

O-GlcNAcase Inhibitor ASN90 is a Multimodal Drug Candidate for Tau and α-Synuclein Proteinopathies.

Neurodegenerative proteinopathies are characterized by the intracellular formation of insoluble and toxic protein aggregates in the brain that are closely linked to disease progression. In Alzheimer’s disease and in rare tauopathies, aggregation of the microtubule-associated tau protein leads to the formation of neurofibrillary tangles (NFT). In Parkinson’s disease (PD) and other α-synucleinopathies, intracellular Lewy bodies containing aggregates of α-synuclein constitute the pathological hallmark. Inhibition of the glycoside hydrolase O-GlcNAcase (OGA) prevents the removal of O-linked N-acetyl-d-glucosamine (O-GlcNAc) moieties from intracellular proteins and has emerged as an attractive therapeutic approach to prevent the formation of tau pathology. Like tau, α-synuclein is known to be modified with O-GlcNAc moieties and in vitro these have been shown to prevent its aggregation and toxicity. Here, we report the preclinical discovery and development of a novel small molecule OGA inhibitor, ASN90. Consistent with the substantial exposure of the drug and demonstrating target engagement in the brain, the clinical OGA inhibitor ASN90 promoted the O-GlcNAcylation of tau and α-synuclein in brains of transgenic mice after daily oral dosing. Across human tauopathy mouse models, oral administration of ASN90 prevented the development of tau pathology (NFT formation), functional deficits in motor behavior and breathing, and increased survival. In addition, ASN90 slowed the progression of motor impairment and reduced astrogliosis in a frequently utilized α-synuclein-dependent preclinical rodent model of PD. These findings provide a strong rationale for the development of OGA inhibitors as disease-modifying agents in both tauopathies and α-synucleinopathies. Since tau and α-synuclein pathologies frequently co-exist in neurodegenerative diseases, OGA inhibitors represent unique, multimodal drug candidates for further clinical development.

Disrupting the Balance of Protein Quality Control Protein UBQLN2 Accelerates Tau Proteinopathy.

Tau protein accumulation drives toxicity in several neurodegenerative disorders. To better understand the pathways regulating tau homeostasis in disease, we investigated the role of ubiquilins (UBQLNs)-a class of proteins linked to ubiquitin-mediated protein quality control (PQC) and various neurodegenerative diseases-in regulating tau. Cell-based assays identified UBQLN2 as the primary brain-expressed UBQLN to regulate tau. UBQLN2 efficiently lowered wild-type tau levels regardless of aggregation, suggesting that UBQLN2 interacts with and regulates tau protein under normal conditions or early in disease. Moreover, UBQLN2 itself proved to be prone to accumulation as insoluble protein in male and female tau transgenic mice and the human tauopathy progressive supranuclear palsy. Genetic manipulation of UBQLN2 in a tauopathy mouse model demonstrated that a physiological UBQLN2 balance is required for tau homeostasis. UBQLN2 overexpression exacerbated phosphorylated tau pathology and toxicity in mice expressing P301S mutant tau, whereas P301S mice lacking UBQLN2 showed significantly reduced phosphorylated tau. Further studies support the view that an imbalance of UBQLN2 perturbs ubiquitin-dependent PQC and autophagy. We conclude that changes in UBQLN2 levels, whether because of pathogenic mutations or secondary to disease states, such as tauopathy, contribute to proteostatic imbalances that exacerbate neurodegeneration.SIGNIFICANCE STATEMENT We defined a role for the protein quality control protein Ubiquilin-2 (UBQLN2), in age-related neurodegenerative tauopathies. This group of disorders is characterized by the accumulation of tau protein aggregates, which differ when UBQLN2 levels are altered. Given the lack of effective disease-modifying therapies for tauopathies and the function of UBQLN2 in handling various disease-linked proteins, we explored the role of UBQLN2 in regulating tau. We found that UBQLN2 reduced tau levels in cell models but behaved differently in mouse brain, where it accelerated mutant tau pathology and tau-mediated toxicity. A better understanding of the diverse functions of regulatory proteins like UBQLN2 can elucidate some of the causative factors in neurodegenerative disease and outline new routes to therapeutic intervention.

Glial profiling of human tauopathy brain demonstrates enrichment of astrocytic transcripts in tau-related frontotemporal degeneration

To understand how glia may be altered in frontotemporal degeneration with tau pathology (FTD-tau), we used a NanoString glial profiling panel to measure 770 transcripts related to glial biology in human control (n = 8), Alzheimer's disease (AD) (n = 8), and FTD-tau (n = 8) dorsolateral prefrontal cortex. Compared to control, 43 genes were upregulated and 86 genes were downregulated in the FTD-tau samples. Only 3 genes were upregulated and 2 were downregulated in AD. Pathway analysis revealed many astrocyte-, microglia-, and oligodendrocyte-related pathway scores increased in FTD-tau, while neuron-related pathway scores decreased. We compared these results to a previously published proteomic dataset containing many of the same samples and found that the targeted panel approach obtained measurements for genes whose proteins were not measured in the proteomics. Our results point to the utility of multiomic approaches and marked dysregulation of glia in FTD-tau.

Molecular Investigation of the Unfolded Protein Response in Select Human Tauopathies.

Background:Tauopathies are a group of neurodegenerative diseases associated with the accumulation of misfolded tau protein. The mechanisms underpinning tau-dependent proteinopathy remain to be elucidated. A protein quality control pathway within the endoplasmic reticulum, the unfolded protein response (UPR), has been suggested as a possible pathway modulating cellular responses in a range of neurodegenerative diseases, including those associated with misfolded cytosolic tau.Objective:In this study we investigated three different clinically defined tauopathies to establish whether these diseases are accompanied by the activation of UPR.Methods:We used PCR and western blotting to probe for the modulation of several reliable UPR markers in mRNA and proteins extracted from three distinct tauopathies: 20 brain samples from Alzheimer’s disease patients, 11 from Pick’s disease, and 10 from progressive supranuclear palsy. In each disease samples from these patients were compared with equal numbers of age-matched non-demented controls.Results:Our investigation showed that different markers of UPR are not changed at the late stage of any of the human tauopathies investigated. Interestingly, UPR signatures were often observed in non-demented controls.Conclusion:These data from late-stage human cortical tissue report an activation of UPR markers within the aged brain across all cohorts investigated and further support the emerging evidence that the accumulation of misfolded cytosolic tau does not drive a disease-associated activation of UPR.

The Lonafarnib target, Rhes, is uniquely dysregulated in tauopathies: A human postmortem study

AbstractBackgroundThe farnesyltransferase inhibitor, Lonafarnib, is capable of reducing tau lesions and associated atrophy in tauopathy models through activity on the Ras GTPase, Rhes (Hernandez I et al., 2019). While hinting at a role of Rhes in tau proteinopathic etiology, it is unclear how Rhes activity changes in human tauopathies or other neurodegenerative diseases. As these models reflect rare, familial forms of tauopathy, we sought to detect evidence of Rhes changes in human sporadic tauopathy versus FTLD‐TDP and synucleinopathies.MethodAllocortical and neocortical sections from postmortem cases (Table 1) representing five different tauopathies, FTLD‐TDP, Lewy body disease, and healthy controls (HC) were immunohistochemically stained for Rhes, proteinopathic lesions (i.e. PHF‐1 positive tau inclusions, phospho‐TDP‐43, or α‐synuclein), and NeuN. Single‐nucleus RNA‐sequencing (snRNA‐seq) was also performed in a separate cohort representing HC and early AD.Result40 cases were examined in this study (Table 1). Rhes is widespread in cortical neurons, as detected by immunohistochemical staining and snRNA‐seq (Figure 1). In tauopathies, neurons either present Rhes as diffuse or punctiform, or feature an absence of Rhes (Figure 2). Accordingly, we classified neurons as Rhes type Diffuse, Punctiform, or Absent. The proportion of neurons with PHF‐1 positive tau inclusions significantly differ between the three neuronal Rhes phenotypes (p < 0.0001) with the majority of Absent type neurons harboring PHF‐1 positive tau inclusions and Diffuse type neurons mostly devoid of PHF‐1 positive tau inclusions (Figure 3, Table 2). Punctiform and Absent type neurons were not detected in neurons with phospho‐TDP‐43 inclusions or α‐synuclein inclusions (Figure 4).ConclusionGiven the distribution of PHF‐1 positive tau inclusions across different Rhes‐deliniated neuronal phenotypes, we propose a model by which Rhes takes on a punctiform phenotype then is ultimately cleared from neurons with tau lesions. This postmortem study in humans, together with in vivo and in vitro work done in familial tauopathy models, suggests the presence of a causal relationship between Rhes clearance and tau proteinopathic lesions, but not phospho‐TDP‐43 or α‐synuclein inclusions. As such, Rhes cellular phenotype represents a novel postmortem hallmark of tauopathy and supports to application of Lonafarnib to the treatment of tauopathies.

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.

Testing the neuroinflammatory role of tau‐induced transposable elements in tauopathy

AbstractBackgroundNovel mechanisms through which pathogenic tau leads to neuronal toxicity remain largely unexplored as therapeutic targets for tauopathies. Activation of retrotransposons has been identified as a mechanism by which pathogenic tau contributes to neurotoxicity (Guo et al., 2018; Sun et al., 2018). To mobilize, retrotransposons are transcribed into RNA, reverse transcribed into DNA, and reinserted into the genome in a new location. Control of retrotransposons occurs at two levels, 1) transcriptional control via the location of retrotransposons within highly condensed heterochromatin and 2) by post‐transcriptional silencing mechanisms. While activated retrotransposons can create novel mutations via the “copy‐and‐paste” mechanism, double stranded RNAs (dsRNAs) formed from transposable element transcripts trigger the innate immune response, further contributing to cell toxicity (Cuellar et al., 2017; Kassiotis & Stoye, 2016; Roulois et al., 2015). Elevation of dsRNAs is documented in models of AGS and TDP‐43 proteinopathy, where dsRNA derived from repetitive elements can stimulate an interferon response (Ahmad et al., 2018; LaRocca, et al., 2019; Rodriguez et al., 2021).We are currently testing the hypothesis that transposable element‐derived dsRNAs stimulate the immune response in tauopathy.MethodBioinformatic analysis of bidirectional transcription was completed on RNA‐sequencing data from control and tau transgenic Drosophila. dsRNAs were quantified by J2 anti‐dsRNA antibody via immunofluorescence and ELISA. Analysis of inflammatory markers in Drosophila was completed by RNA‐sequencing and with a custom NanoString Gene Expression codeset.ResultWe find that dsRNAs are elevated in the brains of tau transgenic Drosophila, in the cortex of a mouse model of tauopathy, and in post‐mortem brain tissue from human tauopathy. Based on bioinformatic analysis, we identified retrotransposons that are bidirectionally transcribed in tau transgenic Drosophila. RNA sequencing and NanoString analysis of tau transgenic flies reveals elevated levels of innate immune response gene transcripts. Loss of transposon transcriptional silencing results in similar dsRNA production and immune system activation found in tau transgenic Drosophila.ConclusionOur goal is to determine if RNA intermediates from tau‐induced transposable elements are a therapeutic target for tauopathies. Overall, our findings support a model where dsRNA production induced by loss of transcriptional transposon silencing contributes to inflammation in tauopathy

Self-assembly and cellular effect of tau35, a disease-associated tau fragment.

Tauopathies are characterised by the accumulation of intracellular tau aggregates in the brain. Tau inclusions contain phosphorylated and 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. Minimal expression of Tau35 in transgenic mice recapitulates key features of tauopathy, including cognitive and motor dysfunction. We used small angle X-ray scattering (SAXS), circular dichroism (CD), transmission electron microscopy (TEM) and Thioflavin T (ThT) assays to characterise the conformations and assembly of recombinant Tau35 and full-length (2N4R) human tau. To assess cellular effects of tau expression, differentiated SH-SY5Y (dSH) cells and mouse primary cortical neurons were exposed to Cy5-labelled aggregated recombinant tau and examined by immunofluorescence and on western blots. SAXS 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 fibril formation than 2N4R tau, which was confirmed by TEM. CD showed an increase in beta content. Notably, both Tau35 and 2N4R tau were taken up by dSH cells and mouse cortical neurons, with Tau35 internalisation occurring at a faster rate. Tau35 and 2N4R tau intracellular accumulation in both cells increased over time, with both tau species entering the nucleus. Our results demonstrate that Tau35, which is associated with human tauopathy, exhibits an increased propensity to aggregate in vitro. Our observation that Tau35 is both highly aggregating and rapidly internalised by neuronal cells, may promote its ability to propagate tau pathology in the tauopathies.

Tau pathology, neuroinflammation and adaptive immune cells in human primary tauopathies.

Primary tauopathies are neurodegenerative disorders that result from the accumulation of aggregated tau in the brain. Frontotemporal lobar degeneration with tau (FTLD-tau) is a tauopathy that underlies ∼50% of cases of frontotemporal dementia (FTD). The discovery of genetic risk variants related to innate/adaptive immunity have highlighted a potential role for neuroinflammation in FTLD. Furthermore, studies have shown microglial and astrocyte activation together with T cell infiltration in the brain of THY-Tau22 tauopathy mice. To study the relationship between tau and neuroinflammation we obtained FFPE brain tissue from 12 FTLD-MAPT, 33 Pick's Disease (PiD), 45 Progressive Supranuclear Palsy (PSP) patients and 55 controls, via Brain UK. Using immunohistochemistry, we assessed tau pathology using antibodies 8 epitopes of tau with phosphorylation are different site. The immunophenotype of microglia were assessed with phenotypic markers and their morphology (ramified, reactive, ameboid) was analysed using Iba1. Astrocytic phenotype and T cell infiltration exploration is currently ongoing. Tau epitopes AT8, AT100, PHF1, CP13 and Tau-2 were increased in PiD, PSP and FTLD-MAPT compared to controls, with expression levels significantly correlated between tau epitopes. Others are currently under analysis. Microglial markers Iba1, CD68, HLA-DR and CD64 showed no difference between groups. However, CD16 was significantly increased in FTLD-MAPT vs. control (p = 0.03) and correlations between tau and microglial markers (except with Iba1) were detected between disease groups. Morphological assessment revealed that ameboid microglia were the highest represented population in PiD and FTLD-MAPT (p<0.0001 and p<0.0001, respectively); while reactive microglia were the most abundant population in PSP (p<0.0001). Preliminary observation of T cell staining confirms their recruitment in FTLD-tau. These findings support the involvement of microglia in FTLD-tau. Additional immuno-phenotyping is necessary for further defining their role as well as T cell involvement in the disease pathogenesis.

Autophagy and lysosomal defects in cells expressing disease‐associated tau

AbstractBackgroundIntracellular accumulation of insoluble tau is an important hallmark of Alzheimer’s disease (AD) and related tauopathies. We have previously identified in human tauopathy brain a truncated tau species (Tau35), comprising the C‐terminal half with four microtubule‐binding repeats. Minimal Tau35 expression in transgenic mice results in a progressive tauopathy phenotype including tau phosphorylation and aggregation, cognitive and behavioural abnormalities and impaired protein clearance. The autophagy‐lysosomal pathway (ALP) plays a crucial role in the clearance of protein aggregates and defects in ALP are associated with the pathogenesis of AD. We sought to explore the effect of Tau35 expression on the ALP and whether autophagy is disrupted due to lysosomal dysfunction.MethodChinese hamster ovary (CHO) cells stably expressing Tau35 (CHO‐Tau35) or full‐length human 2N4R tau (CHO‐FL) were generated. Primary cortical neurons from Tau35 transgenic and wild‐type mice were cultured for 14 days in vitro(DIV) and brain homogenates were prepared from mice aged 4 and 12 months. The effect of Tau35 on the ALP was examined using immunofluorescence and western blots.ResultNuclear translocation of transcription factor EB (TFEB), a key mediator of lysosomal biogenesis, was significantly reduced in both CHO‐FL and CHO‐Tau35 cells. However, only CHO‐Tau35 cells exhibited disrupted mammalian target of rapamycin complex 1 (mTORC1) activity and autophagic flux. Expression of ALP markers, including LC3‐I/II, LAMP1, LAMP2 and cathepsin D, were also reduced in CHO‐Tau35 cells.ConclusionOur findings suggest that N‐terminally cleaved tau damages both lysosomal clearance of cellular proteins and lysosomal biogenesis. The Tau35‐expressing cultured neurons will provide a useful tool to explore molecular mechanisms underlying tau‐induced lysosomal dysfunction, which may lead to the identification of novel therapeutic targets for dementia.

Defects in the autophagy lysosomal pathway in a cell model of disease‐associated tau

Intracellular accumulation of insoluble tau is an important hallmark of Alzheimer's disease (AD) and related tauopathies. We have previously identified in human tauopathy brain a truncated tau species (Tau35), comprising the C-terminal half with four microtubule-binding repeats. Minimal Tau35 expression in transgenic mice results in a progressive tauopathy phenotype including tau phosphorylation and aggregation, cognitive and behavioural abnormalities and impaired protein clearance. The autophagy-lysosomal pathway (ALP) plays a crucial role in the clearance of protein aggregates and defects in ALP are associated with the pathogenesis of AD. We sought to explore the effect of Tau35 expression on the ALP and whether autophagy is disrupted due to lysosomal dysfunction. Chinese hamster ovary (CHO) cells stably expressing Tau35 (CHO-Tau35) or full-length human 2N4R tau (CHO-FL) were generated. Primary cortical neurons from Tau35 transgenic and wild-type mice were cultured for 14 days in vitro(DIV) and brain homogenates were prepared from mice aged 4 and 12 months. The effect of Tau35 on the ALP was examined using immunofluorescence and western blots. Nuclear translocation of transcription factor EB (TFEB), a key mediator of lysosomal biogenesis, was significantly reduced in both CHO-FL and CHO-Tau35 cells. However, only CHO-Tau35 cells exhibited disrupted mammalian target of rapamycin complex 1 (mTORC1) activity and autophagic flux. Expression of ALP markers, including LC3-I/II, LAMP1, LAMP2 and cathepsin D, were also reduced in CHO-Tau35 cells and in post-symptomatic Tau35 transgenic mice. Our findings suggest that N-terminally cleaved tau damages both lysosomal clearance of cellular proteins and lysosomal biogenesis. The Tau35-expressing cultured neurons will provide a useful tool to explore molecular mechanisms underlying tau-induced lysosomal dysfunction, which may lead to the identification of novel therapeutic targets for dementia.

Single cell transcriptomic profiling of tau pathophysiology in a novel 3D neural-glial coculture model.

Research into neurodegeneration has been hampered by a lack of systems that rapidly and accurately recapitulate neurodegenerative processes. We report development of AstAD, a model combining human iPSC derived neurons (hiNCs) and astrocytes (hiACs) in self-aggregating 3D organoids that reproducibly develop a robust range of tau pathology and neurodegeneration. AstAD is a scalable and highly manipulatable model of neurodegeneration and critical understudied glial responses. Additionally, the AstAD model induces pathology without reliance on genetic overexpression. AstAD represents an important technological advance in modeling Alzheimer's Disease (AD), and is a promising tool for identifying disease modifying treatments. The AstAD system was developed by combining advances in 3D cell modeling and tau propagation. We perform directed differentiation of wild-type iPSC to hiNCs and hiACs, and hiNCs are selectively exposed to toxic aggregations to induce pathology. Cells are combined in microwells to induce self-aggregation, and pathology develops over the course of three weeks. Tau pathogenesis in the AstAD model was correlated with human tauopathy progression by immunopathology and single cell transcriptomics, and the application of a selective inhibitor PU-H71 demonstrated the efficacy of the model to explore disease modifying treatment. The AstAD model consistently displays a robust range of tau pathology within three weeks, including progressive development of hyperphosphorylated tau at positions T181, S262, and S396/404, misfolded tau (MC1, TOMA2), and fibrillar tau (THIOS). Neurodegeneration (FlouroJadeB, LDH) is accompanied by reactive astrogliosis (GFAP), which single cell transcriptomics reveals to be driven by heat shock proteins (HSPs). The observed tau pathology and transcriptional responses were reduced by treatment with PU-H71, which eliminates dysfunctional HSP epichaperome. Together this evidence demonstrates a rapid and reversible recapitulation of tau pathogenesis. The novel AstAD model consistently recapitulates tau-induced neurodegeneration in a human system, and allows for the elucidation of neuronal and astrocytic responses. Additionally, we have demonstrated the power of AstAD to model the reduction of tau pathology by drug treatment. Continued expansion of the AstAD model will include integration of microglia. The AstAD model represents a technological advance that will allow for the rapid recapitulation of neurodegeneration in a manipulatable system that promises to accelerate drug development for AD.

Single cell transcriptomic profiling of neurodegeneration mediated by tau in a novel 3D neuron‐astrocyte coculture model

AbstractBackgroundResearch into neurodegeneration has been hampered by a lack of systems that rapidly and accurately recapitulate neurodegenerative processes. We report development of a model combining human iPSC derived neurons (hiNCs) and astrocytes (hiACs) in self‐aggregating 3D organoids that reproducibly develop a robust range of tau pathology and neurodegeneration. This system is a scalable and highly manipulatable model of neurodegeneration and critical understudied glial responses. Additionally, the model induces pathology without reliance on genetic overexpression. Our system represents an important technological advance in modeling Alzheimer’s Disease (AD), and is a promising tool for identifying disease modifying treatments.MethodsThe system was developed by combining advances in 3D cell modeling and tau propagation. We perform directed differentiation of wild‐type iPSC to hiNCs and hiACs, and hiNCs are selectively exposed to toxic aggregations to induce pathology. Cells are combined in microwells to induce self‐aggregation, and pathology develops over the course of three weeks. Tau pathogenesis in the model was correlated with human tauopathy progression by immunopathology and single cell transcriptomics, and the application of a selective inhibitor PU‐H71 demonstrated the efficacy of the model to explore disease modifying treatment.ResultThe model consistently displays a robust range of tau pathology within three weeks, including progressive development of hyperphosphorylated tau at positions T181, S262, and S396/404, misfolded tau (MC1, TOMA2), and fibrillar tau (THIOS). Neurodegeneration (FlouroJadeB, LDH) is accompanied by reactive astrogliosis (GFAP), which single cell transcriptomics reveals to be driven by heat shock proteins (HSPs). The observed tau pathology and transcriptional responses were reduced by treatment with PU‐H71, which eliminates dysfunctional HSP epichaperome. Together this evidence demonstrates a rapid and reversible recapitulation of tau pathogenesis.ConclusionOur novel model consistently recapitulates tau‐induced neurodegeneration in a human system, and allows for the elucidation of neuronal and astrocytic responses. Additionally, we have demonstrated the power to model the reduction of tau pathology by drug treatment. Continued expansion of the model will include integration of microglia. This novel model represents a technological advance that will allow for the rapid recapitulation of neurodegeneration in a manipulatable system that promises to accelerate drug development for AD.

Role of Oxygen Radicals in Alzheimer’s Disease: Focus on Tau Protein

Oxygen free radical burst is a prominent early event in the pathogenesis of Alzheimer’s disease (AD). Posttranslational modifications of Tau protein, primarily hyper-phosphorylation and truncation, are indicated as critical mediators of AD pathology. This finding is confirmed by the high levels of oxidative stress markers and by the increased susceptibility to oxygen radicals found in cultured neurons and in brains from transgenic animal models expressing toxic Tau forms, in concomitance with a dramatic reduction in their viability/survival. Here, we collect the latest progress in research focused on the reciprocal and dynamic interplay between oxygen radicals and pathological Tau, discussing how these harmful species cooperate and/or synergize in the progression of AD. In this context, a better understanding of the role of oxidative stress in determining Tau pathology, and vice versa, primarily could be able to define novel biomarkers of early stages of human tauopathies, including AD, and then to develop therapeutic strategies aimed at attenuating, halting, or reversing disease progression.