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Humanization of the entire murine Mapt gene provides a murine model of pathological human tau propagation.

ABSTRACT: In cortical regions of brains from individuals with preclinical or clinical Alzheimer's disease (AD), extracellular ?-amyloid (A?) deposition precedes the aggregation of pathological intracellular tau (the product of the gene microtubule-associated protein tau (MAPT)). To our knowledge, current mouse models of tauopathy reconstitute tau pathology by overexpressing mutant human tau protein. Here, through a homologous recombination approach that replaced the entire murine Mapt gene with the human ortholog, we developed knock-in mice with humanized Mapt to create an in vivo platform for studying human tauopathy. Of note, the humanized Mapt expressed all six tau isoforms present in humans. We next cross-bred the MAPT knock-in mice with single amyloid precursor protein (App) knock-in mice to investigate the A?-tau axis in AD etiology. The double-knock-in mice exhibited higher tau phosphorylation than did single MAPT knock-in mice but initially lacked apparent tauopathy and neurodegeneration, as observed in the single App knock-in mice. We further observed that tau humanization significantly accelerates cell-to-cell propagation of AD brain-derived pathological tau both in the absence and presence of A?-amyloidosis. In the presence of A?-amyloidosis, tau accumulation was intensified and closely associated with dystrophic neurites, consistently showing that A?-amyloidosis affects tau pathology. Our results also indicated that the pathological human tau interacts better with human tau than with murine tau, suggesting species-specific differences between these orthologous pathogenic proteins. We propose that the MAPT knock-in mice will make it feasible to investigate the behaviors and characteristics of human tau in an animal model.

SUBMITTER: Saito T

PROVIDER: S-EPMC6709628 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Humanization of the entire murine Mapt gene provides a murine model of pathological human tau propagation.

Saito Takashi T Mihira Naomi N Matsuba Yukio Y Sasaguri Hiroki H Hashimoto Shoko S Narasimhan Sneha S Zhang Bin B Murayama Shigeo S Higuchi Makoto M Lee Virginia M Y VMY Trojanowski John Q JQ Saido Takaomi C TC

The Journal of biological chemistry 20190704 34

In cortical regions of brains from individuals with preclinical or clinical Alzheimer's disease (AD), extracellular β-amyloid (Aβ) deposition precedes the aggregation of pathological intracellular tau (the product of the gene microtubule-associated protein tau (MAPT)). To our knowledge, current mouse models of tauopathy reconstitute tau pathology by overexpressing mutant human tau protein. Here, through a homologous recombination approach that replaced the entire murine Mapt gene w ...[more]

PMID: 31273083

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Project description:Missorting of MAPT/Tau represents one of the early signs of neurodegeneration in Alzheimer disease. The triggers for this are still a matter of debate. Here we investigated the sorting mechanisms of endogenous MAPT in mature primary neurons using microfluidic chambers (MFCs) where cell compartments can be observed separately. Blocking protein degradation pathways with proteasomal or autophagy inhibitors dramatically increased the missorting of MAPT in dendrites on the neuritic side, suggesting that degradation of MAPT in dendrites is a major determinant for the physiological axonal distribution of MAPT. Such missorted dendritic MAPT differed in its phosphorylation pattern from axonal MAPT. By contrast, enhancing autophagy or proteasomal pathways strongly reduced MAPT missorting, thereby confirming the role of protein degradation pathways in the polar distribution of MAPT. Dendritic missorting of MAPT by blocking protein degradation resulted in the loss of spines but not in overall cell toxicity. Inhibition of local protein synthesis in dendrites eliminated the missorting of MAPT, indicating that the accumulation of dendritic MAPT is locally generated. In support of this, a substantial fraction of Mapt/Tau mRNA was detected in dendrites. Taken together, our results indicate that the autophagy and proteasomal pathways play important roles in fine-tuning dendritic MAPT levels and thereby prevent synaptic toxicity caused by MAPT accumulation. Abbreviations Ani: anisomycin; Baf: bafilomycin A1; BSA: bovine serum albumin; cAMP: cyclic adenosine monophosphate; CHX: cycloheximide; DMSO: dimethyl sulfoxide; DIV: days in vitro; Epo: epoxomicin; E18: embryonic day 18; FISH: fluorescence in situ hybridization; IgG: immunoglobulin; kDa: kilodalton; Lac: lactacystin; LDH: lactate dehydrogenase; MFC: microfluidic chambers; MAPs: microtubule-associated proteins; MAPT/Tau: microtubule-associated protein tau; PVDF: polyvinylidene difluoride; PBS: phosphate-buffered saline; PRKA: protein kinase AMP-activated; RD150: round device 150; RT: room temperature; SDS: sodium dodecyl sulfate; SEM: standard error of the mean; Wor: wortmannin.

Project description:Synapses represent an important target of Alzheimer disease (AD), and alterations of their excitability are among the earliest changes associated with AD development. Synaptic activation has been shown to be protective in models of AD, and deep brain stimulation (DBS), a surgical strategy that modulates neuronal activity to treat neurological and psychiatric disorders, produced positive effects in AD patients. However, the molecular mechanisms underlying the protective role(s) of brain stimulation are still elusive. We have previously demonstrated that induction of synaptic activity exerts protection in mouse models of AD and frontotemporal dementia (FTD) by enhancing the macroautophagy/autophagy flux and lysosomal degradation of pathological MAPT/Tau. We now provide evidence that TFEB (transcription factor EB), a master regulator of lysosomal biogenesis and autophagy, is a key mediator of this cellular response. In cultured primary neurons from FTD-transgenic mice, synaptic stimulation inhibits MTORC1 signaling, thus promoting nuclear translocation of TFEB, which, in turn, induces clearance of MAPT/Tau oligomers. Conversely, synaptic activation fails to promote clearance of toxic MAPT/Tau in neurons expressing constitutively active RRAG GTPases, which sequester TFEB in the cytosol, or upon TFEB depletion. Activation of TFEB is also confirmed in vivo in DBS-stimulated AD mice. We also demonstrate that DBS reduces pathological MAPT/Tau and promotes neuroprotection in Parkinson disease patients with tauopathy. Altogether our findings indicate that stimulation of synaptic activity promotes TFEB-mediated clearance of pathological MAPT/Tau. This mechanism, underlying the protective effect of DBS, provides encouraging support for the use of synaptic stimulation as a therapeutic treatment against tauopathies.Abbreviations: 3xTg-AD: triple transgenic AD mice; AD: Alzheimer disease; CSA: cyclosporine A; DBS: deep brain stimulation; DIV: days in vitro; EC: entorhinal cortex; FTD: frontotemporal dementia; gLTP: glycine-induced long-term potentiation; GPi: internal segment of the globus pallidus; PD: Parkinson disease; STN: subthalamic nucleus; TFEB: transcription factor EB.

Project description:Defects of autophagy-lysosomal protein degradation are thought to contribute to the pathogenesis of several neurodegenerative diseases, and the accumulation of aggregation prone proteins such as MAPT/Tau in Alzheimer disease (AD). We previously showed the localization of the immunophilin FKBP4/FKBP52 in the lysosomal system of healthy human neurons suggesting its possible role in lysosome function. We also showed that decreased FKBP4 levels in AD brain neurons correlate with abnormal MAPT accumulation and aggregation. In this study, we demonstrate that FKBP4 decrease in a human neuronal cell line (SH-SY5Y) and in dorsal root ganglion (DRG) neurons from human MAPTP301S transgenic mice affected the function of the autophagy-lysosomal system under MAPT induced proteotoxic stress conditions. We show that acute MAPT accumulation in SH-SY5Y cells induced perinuclear clustering of lysosomes, triggered FKBP4 localization around the clusters and its colocalization with MAPT and MAP1LC3/LC3-positive autophagic vesicles; a similar FKBP4 localization was detected in some AD brain neurons. We demonstrate that FKBP4 decrease altered lysosomal clustering along with MAPT and MAP1LC3 secretion increase. Although ectopic FKBP4 expression could not induce autophagy under our experimental conditions, it prevented MAPT secretion after MAPT accumulation in SH-SY5Y cells implying a regulatory role of FKBP4 on MAPT secretion. Finally, we observe that FKBP4 deficiency decreased MAP1LC3-II expression and provoked MAPT accumulation during long-term stress in mouse DRG neurons. We hypothesize that the abnormal FKBP4 decrease observed in AD brain neurons might hinder autophagy efficiency and contribute to the progression of the tauopathy by modulating MAPT secretion and accumulation during MAPT pathogenesis.Abbreviations: AD: Alzheimer disease; AKT/protein kinase B: AKT serine/threonine kinase; ALP: Autophagy-lysosomal pathway; ATG: autophagy-related; BafA1: bafilomycin A1; CQ: chloroquine; CTSD: cathepsin D; DIV: days in vitro; DRG: dorsal root ganglion neurons; Dox: doxycycline; DNAJC5: DnaJ heat shock protein family (Hsp40) member C5; EL: empty lentiviral vectors; ENO2/NSE: enolase 2, gamma neuronal; FKBP4/FKBP52: FKBP prolyl isomerase 4; FTLD-Tau: frontotemporal lobar degeneration with Tau pathology; GFP: green fluorescent protein; LAMP1: lysosomal associated membrane protein 1; LDH: lactate dehydrogenase; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPT/Tau: microtubule associated protein tau; MTT: tetrazolium salt; NFTs: neurofibrillary tangles; RPE-1: retinal pigment epithelial cells; shRNA: small-hairpin ribonucleic acid; SQSTM1/p62: sequestosome 1; SD: standard deviation; SEM: standard error of the mean; SH-SY5Y: human neuroblastoma cells; Sh1 or Sh2: Lentiviral shRNA vectors inducing FKBP4 decrease; SH-52GFP: MAPT/Tau-inducible SH-SY5Y cell line constitutively expressing FKBP4-GFP; TUBB3/βIII tubulin: tubulin beta 3 class III; UPS: ubiquitin-proteasome system.

Project description:Accumulating evidence suggests that misfolded MAPT (microtubule associated protein tau), the main component of neurofibrillary tangles in tauopathies, is subject to degradation by the autophagy-lysosomal pathway. Selective autophagy is a subtype of macroautophagy that requires cargo receptors, such as OPTN (optineurin) or SQSTM1, to recognize specific targets for their sequestration within the autophagosome and their eventual degradation by the lysosome, although their roles in targeting distinct MAPT species have not been fully investigated. Using cargo receptor knockout cell lines and a seeding-based cellular assay in which neurofibrillary tangle pathology can be modeled in vitro, we reveal that while OPTN primarily targets soluble MAPT expressed in physiological conditions, SQSTM1 predominantly degrades insoluble but not soluble mutant MAPT. Endogenous SQSTM1 colocalizes with misfolded and aggregated MAPT species in vitro and in vivo, and both this colocalization and its function in MAPT clearance require both the LC3-interacting region (LIR) motif and also the PB1 self-polymerization domain of SQSTM1. Further, pathogenic MAPT accumulation reduces basal macroautophagy/autophagy in vitro and is associated with a compensatory upregulation of the lysosomal pathway in vivo. Finally, increased expression of SQSTM1 in MAPT transgenic mouse brains ameliorates MAPT pathology and prion-like spreading. Our results uncover distinct properties of selective autophagy receptors in targeting different MAPT species, implicate compromised autophagy as a potential underlying factor in mutant MAPT deposition, and demonstrate a potent and specific role of SQSTM1 in targeted clearance of pathogenic MAPT, through which it blocks neurofibrillary tangle accumulation and pathological spreading. Abbreviations: AAV: adeno-associated virus; AD: Alzheimer disease; ALP: autophagy-lysosomal pathway; ALS: amyotrophic lateral sclerosis; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; FTD: frontotemporal dementias; HD: Huntington disease; HTT: huntingtin; LIR: LC3-interacting region; NBR1: autophagy cargo receptor; NFE2L2/Nrf2: nuclear factor, erythroid derived 2, like 2; NFTs: neurofibrillary tangles; MAPT: microtubule associated protein tau; OPTN: optineurin; p-MAPT: hyperphosphorylated MAPT; PFA: paraformaldehyde; TARDBP/TDP-43: TAR DNA binding protein; TAX1BP1 Tax1: binding protein 1; ThioS: thioflavin-S; UBA: ubiquitin-associated.

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Dataset Information

Humanization of the entire murine Mapt gene provides a murine model of pathological human tau propagation.

Publications

Humanization of the entire murine <i>Mapt</i> gene provides a murine model of pathological human tau propagation.

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