Intracellular Tau inclusions are a pathological hallmark of several neurodegenerative diseases, collectively known as the tauopathies. They include Alzheimer disease, tangle-only dementia, Pick ...disease, argyrophilic grain disease, chronic traumatic encephalopathy, progressive supranuclear palsy, and corticobasal degeneration. Tau pathology appears to spread through intercellular propagation, requiring the formation of assembled “prion-like” species. Several cell and animal models have been described that recapitulate aspects of this phenomenon. However, the molecular characteristics of seed-competent Tau remain unclear. Here, we have used a cell model to understand the relationships between Tau structure/phosphorylation and seeding by aggregated Tau species from the brains of mice transgenic for human mutant P301S Tau and full-length aggregated recombinant P301S Tau. Deletion of motifs 275VQIINK280 and 306VQIVYK311 abolished the seeding activity of recombinant full-length Tau, suggesting that its aggregation was necessary for seeding. We describe conformational differences between native and synthetic Tau aggregates that may account for the higher seeding activity of native assembled Tau. When added to aggregated Tau seeds from the brains of mice transgenic for P301S Tau, soluble recombinant Tau aggregated and acquired the molecular properties of aggregated Tau from transgenic mouse brain. We show that seeding is conferred by aggregated Tau that enters cells through macropinocytosis and seeds the assembly of endogenous Tau into filaments.
Characteristics of seed-competent Tau are unknown.
Native Tau aggregates have a higher seeding potency than recombinant Tau aggregates. Recombinant Tau acquires the conformation and potency of native Tau aggregates by seeded assembly.
Conformation determines the seeding potencies of Tau aggregates.
Understanding the properties of seed-competent Tau gives insight into disease mechanisms.
Cellular models recapitulating features of tauopathies are useful tools to investigate the causes and consequences of tau aggregation and the identification of novel treatments. We seeded rat primary ...cortical neurons with tau isolated from Alzheimer's disease brains to induce a time-dependent increase in endogenous tau inclusions. Transcriptomics of seeded and control cells identified 1075 differentially expressed genes (including 26 altered at two time points). These were enriched for lipid/steroid metabolism and neuronal/glial cell development genes. 50 genes were correlated with tau inclusion formation at both transcriptomic and proteomic levels, including several microtubule and cytoskeleton-related proteins such as Tubb2a, Tubb4a, Nefl and Snca. Several genes (such as Fyn kinase and PTBP1, a tau exon 10 repressor) interact directly with or regulate tau. We conclude that this neuronal model may be a suitable platform for high-throughput screens for target or hit compound identification and validation.
Tauopathies are a diverse class of neurodegenerative diseases characterized by the formation of insoluble tau aggregates and the loss of cellular function and neuronal death. Tau inclusions have been ...shown to contain a number of proteins, including molecular chaperones, but the consequences of these entrapments are not well established. Here, using a human cell system for seeding-dependent tau aggregation, we demonstrate that the molecular chaperones heat-shock cognate 71-kDa protein (HSC70)/heat-shock protein 70 (HSP70), HSP90, and J-domain co-chaperones are sequestered by tau aggregates. By employing single-cell analysis of protein-folding and clathrin-mediated endocytosis, we show that both chaperone-dependent cellular activities are significantly impaired by tau aggregation and can be reversed by treatment with small-molecule regulators of heat-shock transcription factor 1 (HSF1) proteostasis that induce the expression of cytosolic chaperones. These results reveal that the sequestration of cytoplasmic molecular chaperones by tau aggregates interferes with two arms of the proteostasis network, likely having profound negative consequences for cellular function.
The interneuronal propagation of aggregated tau is believed to play an important role in the pathogenesis of human tauopathies. It requires the uptake of seed-competent tau into cells, seeding of ...soluble tau in recipient neurons and release of seeded tau into the extracellular space to complete the cycle. At present, it is not known which tau species are seed-competent. Here, we have dissected the molecular characteristics of seed-competent tau species from the TgP301S tau mouse model using various biochemical techniques and assessed their seeding ability in cell and animal models. We found that sucrose gradient fractions from brain lysates seeded cellular tau aggregation only when large (>10 mer) aggregated, hyperphosphorylated (AT8- and AT100-positive) and nitrated tau was present. In contrast, there was no detectable seeding by fractions containing small, oligomeric (<6 mer) tau. Immunodepletion of the large aggregated AT8-positive tau strongly reduced seeding; moreover, fractions containing these species initiated the formation and spreading of filamentous tau pathology in vivo, whereas fractions containing tau monomers and small oligomeric assemblies did not. By electron microscopy, seed-competent sucrose gradient fractions contained aggregated tau species ranging from ring-like structures to small filaments. Together, these findings indicate that a range of filamentous tau aggregates are the major species that underlie the spreading of tau pathology in the P301S transgenic model. Significance statement: The spread of tau pathology from neuron to neuron is postulated to account for, or at least to contribute to, the overall propagation of tau pathology during the development of human tauopathies including Alzheimer's disease. It is therefore important to characterize the native tau species responsible for this process of seeding and pathology spreading. Here, we use several biochemical techniques to dissect the molecular characteristics of native tau protein conformers from TgP301S tau mice and show that seed-competent tau species comprise small fibrils capable of seeding tau pathology in cell and animal models. Characterization of seed-competent tau gives insight into disease mechanisms and therapeutic interventions.
Activation of microglia, the resident immune cells of the central nervous system, is a prominent pathological hallmark of Alzheimer's disease (AD). However, the gene expression changes underlying ...microglia activation in response to tau pathology remain elusive. Furthermore, it is not clear how murine gene expression changes relate to human gene expression networks.
Microglia cells were isolated from rTg4510 tau transgenic mice and gene expression was profiled using RNA sequencing. Four age groups of mice (2-, 4-, 6-, and 8-months) were analyzed to capture longitudinal gene expression changes that correspond to varying levels of pathology, from minimal tau accumulation to massive neuronal loss. Statistical and system biology approaches were used to analyze the genes and pathways that underlie microglia activation. Differentially expressed genes were compared to human brain co-expression networks.
Statistical analysis of RNAseq data indicated that more than 4000 genes were differentially expressed in rTg4510 microglia compared to wild type microglia, with the majority of gene expression changes occurring between 2- and 4-months of age. These genes belong to four major clusters based on their temporal expression pattern. Genes involved in innate immunity were continuously up-regulated, whereas genes involved in the glutamatergic synapse were down-regulated. Up-regulated innate inflammatory pathways included NF-κB signaling, cytokine-cytokine receptor interaction, lysosome, oxidative phosphorylation, and phagosome. NF-κB and cytokine signaling were among the earliest pathways activated, likely driven by the RELA, STAT1 and STAT6 transcription factors. The expression of many AD associated genes such as APOE and TREM2 was also altered in rTg4510 microglia cells. Differentially expressed genes in rTg4510 microglia were enriched in human neurodegenerative disease associated pathways, including Alzheimer's, Parkinson's, and Huntington's diseases, and highly overlapped with the microglia and endothelial modules of human brain transcriptional co-expression networks.
This study revealed temporal transcriptome alterations in microglia cells in response to pathological tau perturbation and provides insight into the molecular changes underlying microglia activation during tau mediated neurodegeneration.
A key histopathological hallmark of Alzheimer's disease (AD) is the presence of neurofibrillary tangles of aggregated microtubule-associated protein tau in neurons. Anle138b is a small molecule which ...has previously shown efficacy in mice in reducing tau aggregates and rescuing AD disease phenotypes.
In this work, we employed bioinformatics analysis-including pathway enrichment and causal reasoning-of an in vitro tauopathy model. The model consisted of cultured rat cortical neurons either unseeded or seeded with tau aggregates derived from human AD patients, both of which were treated with Anle138b to generate hypotheses for its mode of action. In parallel, we used a collection of human target prediction models to predict direct targets of Anle138b based on its chemical structure.
Combining the different approaches, we found evidence supporting the hypothesis that the action of Anle138b involves several processes which are key to AD progression, including cholesterol homeostasis and neuroinflammation. On the pathway level, we found significantly enriched pathways related to these two processes including those entitled "Superpathway of cholesterol biosynthesis" and "Granulocyte adhesion and diapedesis". With causal reasoning, we inferred differential activity of SREBF1/2 (involved in cholesterol regulation) and mediators of the inflammatory response such as NFKB1 and RELA. Notably, our findings were also observed in Anle138b-treated unseeded neurons, meaning that the inferred processes are independent of tau pathology and thus represent the direct action of the compound in the cellular system. Through structure-based ligand-target prediction, we predicted the intracellular cholesterol carrier NPC1 as well as NF-κB subunits as potential targets of Anle138b, with structurally similar compounds in the model training set known to target the same proteins.
This study has generated feasible hypotheses for the potential mechanism of action of Anle138b, which will enable the development of future molecular interventions aiming to reduce tau pathology in AD patients.
The levels of p-tau217 and p-tau231 in cerebrospinal fluid (CSF) are associated with early amyloid beta (Aß) changes in the brain, while the CSF levels of p-tau205 are foremost related to tau ...pathology in the later stages of the disease. To investigate if the three p-tau variants are found to the same degree in different tau structures and if their co-localization is affected by the diagnosis and presence of Aß plaques, we immunostained sections of the entorhinal cortex (EC) and inferior temporal gyrus (ITG) from non-demented controls (NC), patients with Alzheimer's disease (AD), and primary age-related tauopathy (PART) against p-tau217, p-tau231, and p-tau205 together with Methoxi-X04. An analysis using confocal microscopy showed that the co-localization variable, the Pearson correlation coefficient (PCC), was significantly higher between p-tau231 and p-tau205 in neurofibrillary tangles compared to neuropil threads and dystrophic neurites in plaques. The PCC value between all three p-tau variants in the neuropil threads was significantly lower in the ECs of patients with AD compared to the NC and in the ITGs of patients with AD, with a high Aß load compared to PART. The lowered value was associated with proportionally higher amounts of non-colocalized p-tau231 and p-tau217 compared to p-tau205, and the PCC values were negatively correlated with Aß and the tangle loads in patients with AD, but positively correlated with tangles in PART. These results suggest that the proportion of and co-localization between p-tau217, p-tau231, and p-tau205 are dependent on cellular localization and are altered in response to AD pathology in a spatial-temporal manner.
In animal cells there are several regulatory complexes which interact with 20S proteasomes and give rise to functionally distinct proteasome complexes. gamma-Interferon upregulates three immuno beta ...catalytic subunits of the 20S proteasome and the PA28 regulator, and decreases the level of 26S proteasomes. It also decreases the level of phosphorylation of two proteasome alpha subunits, C8 (alpha7) and C9 (alpha3). In the present study we have investigated the role of phosphorylation of C8 by protein kinase CK2 in the formation and stability of 26S proteasomes. An epitope-tagged C8 subunit expressed in mammalian cells was efficiently incorporated into both 20S proteasomes and 26S proteasomes. Investigation of mutants of C8 at the two known CK2 phosphorylation sites demonstrated that these are the two phosphorylation sites of C8 in animal cells. Although phosphorylation of C8 was not absolutely essential for the formation of 26S proteasomes, it did have a substantial effect on their stability. Also, when cells were treated with gamma-interferon, there was a marked decrease in phosphorylation of C8, a decrease in the level of 26S proteasomes, and an increase in immunoproteasomes and PA28 complexes. These results suggest that the down-regulation of 26S proteasomes after gamma-interferon treatment results from the destabilization that occurs after dephosphorylation of the C8 subunit.
Chaperone Function of Hsp90-Associated Proteins Bose, Suchira; Weikl, Tina; Bugl, Hans ...
Science (American Association for the Advancement of Science),
12/1996, Letnik:
274, Številka:
5293
Journal Article
Recenzirano
The Hsp90 heat shock protein of eukaryotic cells regulates the activity of proteins involved in signal transduction pathways and may direct intracellular protein folding in general. Hsp90 performs at ...least part of its function in a complex with a specific set of partner proteins that include members of the prolyl isomerase family. The properties of the major components of the Hsp90 complex were examined through the use of in vitro protein folding assays. Two of the components, FKBP52 and p23, functioned as mechanistically distinct molecular chaperones. These results suggest the existence of a super-chaperone complex in the cytosol of eukaryotic cells.
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