Tau protein modulates an epigenetic mechanism of cellular senescence in human SH-SY5Y neuroblastoma cells.

Introduction: Progressive Tau deposition in neurofibrillary tangles and neuropil threads is the hallmark of tauopathies, a disorder group that includes Alzheimer's disease. Since Tau is a microtubule-associated protein, a prevalent concept to explain the pathogenesis of tauopathies is that abnormal Tau modification contributes to dissociation from microtubules, assembly into multimeric ß-sheets, proteotoxicity, neuronal dysfunction and cell loss. Tau also localizes in the cell nucleus and evidence supports an emerging function of Tau in DNA stability and epigenetic modulation. Methods: To better characterize the possible role of Tau in regulation of chromatin compaction and subsequent gene expression, we performed a bioinformatics analysis of transcriptome data obtained from Tau-depleted human neuroblastoma cells. Results: Among the transcripts deregulated in a Tau-dependent manner, we found an enrichment of target genes for the polycomb repressive complex 2. We further describe decreased cellular amounts of the core components of the polycomb repressive complex 2 and lower histone 3 trimethylation in Tau deficient cells. Among the de-repressed polycomb repressive complex 2 target gene products, IGFBP3 protein was found to be linked to increased senescence induction in Tau-deficient cells. Discussion: Our findings propose a mechanism for Tau-dependent epigenetic modulation of cell senescence, a key event in pathologic aging.

Tau accumulation in degradative organelles is associated to lysosomal stress.

Neurodegenerative disorders are characterized by the brain deposition of insoluble amyloidogenic proteins, such as α-synuclein or Tau, and the concomitant deterioration of cell functions such as the autophagy-lysosomal pathway (ALP). The ALP is involved in the degradation of intracellular macromolecules including protein aggregates. ALP dysfunction due to inherited defects in lysosomal or non-lysosomal proteins causes a group of diseases called lysosomal storage disorders (LSD) because of abnormal accumulation of lysosomal degradation substrates. Supporting the contribution of ALP defects in neurodegenerative diseases, deposition of amyloidogenic proteins occurs in LSD. Moreover, heterozygous mutations of several ALP genes represent risk factors for Parkinson's disease. The reciprocal contribution of α-synuclein accumulation and lysosomal dysfunction have been extensively studied. However, whether this adverse crosstalk also embraces Tau pathology needs more investigation. Here, we show in human primary fibroblasts that Tau seeds isolated from the brain of Alzheimer's disease induce Tau accumulation in acidic degradative organelles and lysosomal stress. Furthermore, inhibition of glucocerebrosidase, a lysosomal enzyme mutated in Gaucher's disease and a main risk for Parkinson's disease, causes lysosomal dysfunction in primary fibroblasts and contributes to the accumulation of Tau. Considering the presence of Tau lesions in Parkinson's disease as well as in multiple neurodegenerative disorders including Alzheimer's disease, our data call for further studies on strategies to alleviate ALP dysfunction as new therapeutic opportunity for neurodegenerative diseases and LSD.

Cancer-specific association between Tau (MAPT) and cellular pathways, clinical outcome, and drug response.

Tau (MAPT) is a microtubule-associated protein causing common neurodegenerative diseases or rare inherited frontotemporal lobar degenerations. Emerging evidence for non-canonical functions of Tau in DNA repair and P53 regulation suggests its involvement in cancer. To bring new evidence for a relevant role of Tau in cancer, we carried out an in-silico pan-cancer analysis of MAPT transcriptomic profile in over 10000 clinical samples from 32 cancer types and over 1300 pre-clinical samples from 28 cancer types provided by the TCGA and the DEPMAP datasets respectively. MAPT expression associated with key cancer hallmarks including inflammation, proliferation, and epithelial to mesenchymal transition, showing cancer-specific patterns. In some cancer types, MAPT functional networks were affected by P53 mutational status. We identified new associations of MAPT with clinical outcomes and drug response in a context-specific manner. Overall, our findings indicate that the MAPT gene is a potential major player in multiple types of cancer. Importantly, the impact of Tau on cancer seems to be heavily influenced by the specific cellular environment.

Tau protein binds to the P53 E3 ubiquitin ligase MDM2.

Tau gene mutations cause a progressive dementia and neurotoxic Tau forms deposited in neurofibrillary tangles are hallmarks of neurodegenerative tauopathies. Loss of non-canonical Tau functions may contribute to disease. In fact, Tau depletion affects the cellular response to DNA damage and tauopathies exhibit the accumulation of DNA lesions. Moreover, Tau modulates P53 activity and cell fate. Considering that MDM2 is the main antagonist of P53, we investigated, using orthogonal assays, if Tau interacts with MDM2. We report the existence in cells and brain of a Tau-MDM2 complex that, in vitro, exhibits reduced P53 ubiquitination activity in a manner sensitive to a Tau mutation. The Tau-MDM2 interaction involves the microtubule-binding domain of Tau and the acidic domain of MDM2, reminiscent of the binding of Tau to negatively charged microtubules. Notably, MDM2 accumulates aberrantly in neurofibrillary tangles. Aging-associated insults may expose a novel loss-of-function of Tau in neurodegeneration and cancer.

Tau Seeds in Extracellular Vesicles Induce Tau Accumulation in Degradative Organelles of Cells.

Clinical progression of tauopathies may result from transcellular propagation of pathogenic Tau seeds with the possible involvement of extracellular vesicles (EVs) as transport vectors. We established a cell model for investigating EV delivery of proteins, since the mechanism regulating EV cargo delivery to recipient cells is poorly understood. In our cell model, EVs are readily internalized and accumulate in degradative organelles (DOs). We then show for the first time that in this acidic compartment, profibrillogenic Tau delivered by EVs interacts with Tau expressed by the recipient cells and cause its accumulation by a process that involves the participation of autophagy. Thus, the degradative compartment of cells may represent the subcellular site initiating a cascade of events resulting in early hallmarks of tauopathies. These are characterized by seeded Tau accumulation, pathology-associated epitopes, DO stress, and cytotoxicity. The involvement of autophagy to this process and the relative accessibility of the degradative pathway for extracellular agents, support possible modes of intervention to slow down the progression of neurodegeneration.

Autoetnografía de una vivencia CODA, Children Of Deaf Adult / Autoethnography of an experience of CODA, Children Of Deaf Adult

En este trabajo autoetnográficorelato cómo la adquisición tardía de una lengua de signos per-mite un proceso de reparación y de reapropiación de una identidad CODA (Children Of Deaf Adults). Se trata de una investigación reflexiva sobre una vivencia singular como hija mayor de padres sordos, con el fin de resaltar sus complejidades y enlazarlas con conceptos sociales, cul-turales y psicológicos. En ese particular proceso de contar la propia historia, escribo en primera persona fusionando el “sujeto” y “objeto” de la investigación. Cada persona crea la realidad social y cultural al tiempo que se inspira de ella para construir su propia identidad. Así, la elección deliberada de la lengua de signos refleja una voluntad de transformación vital tanto a nivel personal como social, es una acción política. Las tiras de cómic permiten ilustrar las pro-blemáticas y representar la investigación de forma accesible con objeto de alcanzar cierto gra-do de generalización.(AU)

In this autoethnographic work I relate how the late acquisition of a sign language allows a pro-cess of repair and re-appropriation of a CODA (Children Of Deaf Adults) identity. This is a re-flective research on a singular experience as the eldest daughter of deaf parents in order to highlight its complexities and link them with social, cultural and psychological concepts. In this particular process of telling one's story, I write in the first person by fusing the “subject” and “object” of the investigation. Each personcreates the social and cultural reality while drawing inspiration from it to build his or her own identity. Thus, the deliberate choice of sign language reflects a will for vital transformation both at the personal and social level, it is a political action. The comic strips allow to illustrate the issues and represent the research in an accessible way in order to reach a certain degree of generalization.(AU)

Emerging Evidences for an Implication of the Neurodegeneration-Associated Protein TAU in Cancer.

Neurodegenerative disorders and cancer may appear unrelated illnesses. Yet, epidemiologic studies indicate an inverse correlation between their respective incidences for specific cancers. Possibly explaining these findings, increasing evidence indicates that common molecular pathways are involved, often in opposite manner, in the pathogenesis of both disease families. Genetic mutations in the MAPT gene encoding for TAU protein cause an inherited form of frontotemporal dementia, a neurodegenerative disorder, but also increase the risk of developing cancer. Assigning TAU at the interface between cancer and neurodegenerative disorders, two major aging-linked disease families, offers a possible clue for the epidemiological observation inversely correlating these human illnesses. In addition, the expression level of TAU is recognized as a prognostic marker for cancer, as well as a modifier of cancer resistance to chemotherapy. Because of its microtubule-binding properties, TAU may interfere with the mechanism of action of taxanes, a class of chemotherapeutic drugs designed to stabilize the microtubule network and impair cell division. Indeed, a low TAU expression is associated to a better response to taxanes. Although TAU main binding partners are microtubules, TAU is able to relocate to subcellular sites devoid of microtubules and is also able to bind to cancer-linked proteins, suggesting a role of TAU in modulating microtubule-independent cellular pathways associated to oncogenesis. This concept is strengthened by experimental evidence linking TAU to P53 signaling, DNA stability and protection, processes that protect against cancer. This review aims at collecting literature data supporting the association between TAU and cancer. We will first summarize the evidence linking neurodegenerative disorders and cancer, then published data supporting a role of TAU as a modifier of the efficacy of chemotherapies and of the oncogenic process. We will finish by addressing from a mechanistic point of view the role of TAU in de-regulating critical cancer pathways, including the interaction of TAU with cancer-associated proteins.

Tau affects P53 function and cell fate during the DNA damage response.

Cells are constantly exposed to DNA damaging insults. To protect the organism, cells developed a complex molecular response coordinated by P53, the master regulator of DNA repair, cell division and cell fate. DNA damage accumulation and abnormal cell fate decision may represent a pathomechanism shared by aging-associated disorders such as cancer and neurodegeneration. Here, we examined this hypothesis in the context of tauopathies, a neurodegenerative disorder group characterized by Tau protein deposition. For this, the response to an acute DNA damage was studied in neuroblastoma cells with depleted Tau, as a model of loss-of-function. Under these conditions, altered P53 stability and activity result in reduced cell death and increased cell senescence. This newly discovered function of Tau involves abnormal modification of P53 and its E3 ubiquitin ligase MDM2. Considering the medical need with vast social implications caused by neurodegeneration and cancer, our study may reform our approach to disease-modifying therapies.

Phosphorylation of nuclear Tau is modulated by distinct cellular pathways.

Post-translational protein modification controls the function of Tau as a scaffold protein linking a variety of molecular partners. This is most studied in the context of microtubules, where Tau regulates their stability as well as the distribution of cellular components to defined compartments. However, Tau is also located in the cell nucleus; and is found to protect DNA. Quantitative assessment of Tau modification in the nucleus when compared to the cytosol may elucidate how subcellular distribution and function of Tau is regulated. We undertook an unbiased approach by combing bimolecular fluorescent complementation and mass spectrometry in order to show that Tau phosphorylation at specific residues is increased in the nucleus of proliferating pluripotent neuronal C17.2 and neuroblastoma SY5Y cells. These findings were validated with the use of nuclear targeted Tau and subcellular fractionation, in particular for the phosphorylation at T181, T212 and S404. We also report that the DNA damaging drug Etoposide increases the translocation of Tau to the nucleus whilst reducing its phosphorylation. We propose that overt phosphorylation of Tau, a hallmark of neurodegenerative disorders defined as tauopathies, may negatively regulate the function of nuclear Tau in protecting against DNA damage.

Split GFP technologies to structurally characterize and quantify functional biomolecular interactions of FTD-related proteins.

Protein multimerization in physiological and pathological conditions constitutes an intrinsic trait of proteins related to neurodegeneration. Recent evidence shows that TDP-43, a RNA-binding protein associated with frontotemporal dementia and amyotrophic lateral sclerosis, exists in a physiological and functional nuclear oligomeric form, whose destabilization may represent a prerequisite for misfolding, toxicity and subsequent pathological deposition. Here we show the parallel implementation of two split GFP technologies, the GFP bimolecular and trimolecular fluorescence complementation (biFC and triFC) in the context of TDP-43 self-assembly. These techniques coupled to a variety of assays based on orthogonal readouts allowed us to define the structural determinants of TDP-43 oligomerization in a qualitative and quantitative manner. We highlight the versatility of the GFP biFC and triFC technologies for studying the localization and mechanisms of protein multimerization in the context of neurodegeneration.

Motifs in the tau protein that control binding to microtubules and aggregation determine pathological effects.

Tau pathology is associated with cognitive decline in Alzheimer's disease, and missense tau mutations cause frontotemporal dementia. Hyperphosphorylation and misfolding of tau are considered critical steps leading to tauopathies. Here, we determine how motifs controlling conformational changes in the microtubule-binding domain determine tau pathology in vivo. Human tau was overexpressed in the adult mouse forebrain to compare variants carrying residues that modulate tau propensity to acquire a ß-sheet conformation. The P301S mutation linked to frontotemporal dementia causes tau aggregation and rapidly progressing motor deficits. By comparison, wild-type tau becomes heavily hyperphosphorylated, and induces behavioral impairments that do not progress over time. However, the behavioral defects caused by wild-type tau can be suppressed when ß-sheet breaking proline residues are introduced in the microtubule-binding domain of tau. This modification facilitates tau interaction with microtubules, as shown by lower levels of phosphorylation, and by the enhanced protective effects of mutated tau against the severing of the cytoskeleton in neurons exposed to vinblastine. Altogether, motifs that are critical for tau conformation determine interaction with microtubules and subsequent pathological modifications, including phosphorylation and aggregation.

Transgenic expression of ß1 antibody in brain neurons impairs age-dependent amyloid deposition in APP23 mice.

Heterologous expression of the functional amyloid beta (Aß) antibody ß1 in the central nervous system was engineered to maximize antibody exposure in the brain and assess the effects on Aß production and accumulation in these conditions. A single open reading frame encoding the heavy and light chains of ß1 linked by the mouth and foot virus peptide 2A was expressed in brain neurons of transgenic mice. Two of the resulting BIN66 transgenic lines were crossed with APP23 mice, which develop severe central amyloidosis. Brain concentrations at steady-state 5 times greater than those found after peripheral ß1 administration were obtained. Similar brain and plasma ß1 concentrations indicated robust antibody efflux from the brain. In preplaque mice, ß1 formed a complex with Aß that caused a modest Aß increase in brain and plasma. At 11 months of age, ß1 expression reduced amyloid by 97% compared with age-matched APP23 mice. Interference of ß1 with ß-secretase cleavage of amyloid precursor protein was relatively small. Our data suggest that severely impaired amyloid formation was primarily mediated by a complex of ß1 with soluble Aß, which might have prevented Aß aggregation or favored transport out of the brain.

Carriers of the fragile X mental retardation 1 (FMR1) premutation allele present with increased levels of cytokine IL-10.

BACKGROUND: Fragile X-associated tremor/ataxia syndrome (FXTAS) is an inherited late-onset neurodegenerative disorder, characterized both by neurological and cognitive deficits. It is caused by the expansion of CGG repeats (55 to 200 repeats) in the noncoding region of the fragile X mental retardation 1 (FMR1) gene. Abnormal immunological patterns are often associated with neurodegenerative disorders and implicated in their etiology. We therefore investigated the immune status of FXTAS patients, which had not been assessed prior to this study. METHOD: Peripheral blood mononuclear cells (PBMCs) were collected from 15 asymptomatic FMR1 premutation carriers and 20 age-matched controls. Concentrations of three cytokines (IL-6, IL-8, IL-10) were measured in PBMC supernatants using ELISA assays. RESULTS: We found a significant increase in the concentration of the major anti-inflammatory cytokine IL-10 in supernatants of PBMCs derived from premutation carriers, when compared with controls (P = 0.019). This increase correlated significantly with the number of CGG repeats (P = 0.002). CONCLUSIONS: Elevated IL-10 levels were observed in all premutation carriers, before appearance of the classical neurological symptoms; therefore, IL-10 may be one of the early biomarkers of FXTAS.

Structure of the PRYSPRY-domain: implications for autoinflammatory diseases.

We determined the first structure of PRYSPRY, a domain found in over 500 different proteins, involved in innate immune signaling, cytokine signaling suppression, development, cell growth and retroviral restriction. The fold encompasses a 7-stranded and a 6-stranded antiparallel beta-sheet, arranged in a beta-sandwich. In the crystal, PRYSPRY forms a dimer where the C-terminus of an acceptor molecule binds to the concave surface of a donor molecule, which represents a putative interaction site. Mutations in the PRYSPRY domains of Pyrin, which are responsible for familial Mediterranean fever, map on the putative PRYSPRY interaction site.

Interaction of pyrin with 14.3.3 in an isoform-specific and phosphorylation-dependent manner regulates its translocation to the nucleus.

OBJECTIVE: Pyrin, the familial Mediterranean fever gene product, exists in several isoforms of unknown functions. The recombinant full-length isoform (pyrin.fl) is cytoplasmic, whereas an alternatively spliced isoform lacking exon 2 (pyrin.DeltaEx2) concentrates in the nucleus. Native pyrin, mainly consisting of pyrin.fl, is also cytoplasmic in monocytes but is predominantly nuclear in other cell types. To understand pyrin-dependent biologic pathways and to decipher the mechanisms accounting for such different patterns of subcellular compartmentalization, binding partners and posttranslational modifications of pyrin were assessed. METHODS: A yeast 2-hybrid screen was performed with pyrin.fl as the bait. The interaction identified between pyrin.fl and 14.3.3 proteins was confirmed by immunoprecipitation of (35)S-radiolabeled protein complexes; similar experiments were performed with pyrin.DeltaEx2, pyrin.fl after alkaline phosphatase treatment, and pyrin.fl mutants in which several exon 2-encoded serine residues were replaced by nonphosphorylatable alanines. The subcellular localization of the different wild-type and mutated pyrin proteins was assessed by immunofluorescence. RESULTS: Two members of the 14.3.3 protein family were identified as pyrin partners. Whereas pyrin.fl interacted with 14.3.3tau and 14.3.3epsilon, these interactions did not occur with pyrin.DeltaEx2. Pyrin.fl was phosphorylated, and this modification mediated 14.3.3 binding. Serines 208, 209, and 242, within exon 2, acted as critical residues in the interaction between pyrin.fl and 14.3.3. When an S208-S209-S242A pyrin.fl triple mutant or wild-type pyrin.fl in the presence of an inhibitor of 14.3.3-ligand interactions was used, promotion of nuclear translocation of pyrin was observed. CONCLUSION: These results disclose the role played by 14.3.3 in the regulation of the subcellular compartmentalization of pyrin in a phosphorylation- and isoform-dependent manner. They also reconcile the observations made in vitro with those made in vivo, while providing a direct link between 14.3.3-dependent pathways and pyrin.

The tumor necrosis factor alpha-dependent activation of the human mediterranean fever (MEFV) promoter is mediated by a synergistic interaction between C/EBP beta and NF kappaB p65.

MEFV is a gene expressed specifically in myeloid cells and whose mutations underlie an autosomal recessive auto-inflammatory disease, called familial Mediterranean fever (FMF), characterized by recurrent episodes of serosal inflammation. This gene, which encodes a protein with unclear physiological functions, has been shown to be up-regulated by the pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha). However, the mechanism of this regulation is unknown, and the MEFV promoter is still to be characterized. Here, we show that 243 bp of the 5'-flanking region of the human MEFV gene are sufficient to direct high level expression of MEFV in TNFalpha-treated cells. The TNFalpha-induced expression of MEFV is dependent on both NFkappaB p65 and C/EBPbeta that bind to evolutionarily conserved sites located, in the human promoter, at positions -163 and -55, respectively. As shown by a series of transcription and gel shift assays performed with wild-type and mutated promoter sequences, these two transcription factors act differently on the TNFalpha-dependent transcription of MEFV: C/EBPbeta is the key regulatory factor required to confer cell responsiveness to TNFalpha, whereas NFkappaB p65 increases this response by means of a synergistic interaction with C/EBPbeta that is dependent on the integrity of the identified -55 C/EBP binding site. Given the phenotype of patients with FMF, this C/EBP-NFkappaB interaction may represent a key step in the control of an inflammatory response that is abnormally high in this disease. These data, which shed novel light on the pathophysiology of FMF, represent an unusual example of cross-talk between C/EBP and NFkappaB pathways in TNFalpha signaling.

Familial Mediterranean fever among patients from Karabakh and the diagnostic value of MEFV gene analysis in all classically affected populations.

OBJECTIVE: Familial Mediterranean fever (FMF) is an autosomal-recessive disorder that is common in Armenian, Turkish, Arab, and Sephardic Jewish populations. Its clinical diagnosis is one of exclusion, with the patients displaying nonspecific symptoms related to serosal inflammation. MEFV gene analysis has provided the first objective diagnostic criterion for FMF. However, in the absence of an identified mutation (NI/NI genotype), both the sensitivity of the molecular analyses and the involvement of the MEFV gene in FMF are called into question. The present study was designed to further evaluate the diagnostic value of MEFV analysis in another population of Mediterranean extraction. METHODS: The MEFV gene was screened for mutations in 50 patients living in Karabakh (near Armenia) who fulfilled the established criteria for FMF. In addition, we analyzed published series of patients from the above-mentioned at-risk populations. RESULTS: The mutation spectrum in Karabakhian patients, which consisted of only 6 mutations (with 26% of NI alleles), differed from that reported in Armenian patients. Strikingly, among patients from Karabakh and among all classically affected populations, the distribution of genotypes differed dramatically from Hardy-Weinberg equilibrium (P = 0.0016 and P < 0.00001, respectively). These results, combined with other population genetics-based data, revealed the existence of an FMF-like condition that, depending on the patients' ancestry, was shown to affect 85-99% of those with the NI/NI genotype. CONCLUSION: These data illuminate the meaning of negative results of MEFV analyses and show that in all populations evaluated, most patients with the NI/NI genotype had disease that mimicked FMF and was unrelated to the MEFV gene. Our findings also demonstrate the high sensitivity of a search for very few mutations in order to perform a molecular diagnosis of MEFV-related FMF.