Multilevel investigation of Tau pathology: from the cytoplasm to the nucleus

Tau protein has been discovered in 1975 from brain tissues and its main function in neurons is to bind and stabilize microtubules. This observation was followed by the identification of Tau as one of the main actors able to induce neuronal toxicity in a group of neurodegenerative disorders named tauopathies. From these discoveries, the scientific community has invested great efforts to elucidate the mechanisms involving Tau and to find a way to prevent its pathological effects. Tau toxicity is due to its displacement from microtubules, progressive aggregation of the protein and spreading in several brain areas causing neuronal dysfunction and death. These are considered the central events leading to neurodegeneration. However, recently it has been demonstrated that Tau is located not only on microtubules or in the cytoplasmic aggregates but also in other subcellular regions, in particular in dendrites and in the nuclear compartment where it exerts functions related to synaptic transmission and genome protection, respectively. In order to investigate the dynamics of Tau from physiological conditions to destabilization and aggregation, we developed a FRET-based biosensor, the Conformational Sensitive Tau (CST), able to determine the conformational changes of Tau during the progression of the pathology. We showed that in physiological conditions, in living cells, Tau binds microtubules with a paperclip conformation. After drug treatments or tauopathy-related mutations, the conformation of Tau opens indicating an impairment of Tau binding to tubulin. Finally, by treating cells with different kinds of Alzheimer\u2019s aggregates, the CST displaced from microtubules and formed FRET-positive intracellular inclusions demonstrating that it is a powerful tool to study also aggregation. The CST employment allowed the characterization of a particular mutation associated to Pick\u2019s disease, Q336H. Remarkably, we found that this mutation induces a closer conformation of Tau and a higher affinity to tubulin, an effect that is opposite to previously discovered mutations. We applied the CST to develop a cell-based aggregation assay to screen compounds impairing Tau pathology. A first screening identified the ERK kinase inhibitor PD-901 as a compound reducing Tau aggregation. Moreover, to test the efficacy of therapeutic compounds in vivo, a transgenic zebrafish expressing the CST is under development to establish a zebrafish-based aggregation platform. The signal of the CST is detectable not only in the cytoplasm but also in the nucleus, however, the FRET analysis in this compartment revealed that nuclear Tau conformation is probably more open and relaxed. We investigated the nuclear function of Tau and we found that the increase in the soluble pool of Tau enhances its translocation into the nucleus and, concomitantly, the nuclear Tau alters the expression of VGluT1, a diseaserelevant gene, indicating that Tau has a role in gene expression modulation. We observed that the increase in VGluT1 expression mediated by Tau causes neuronal hyperexcitability in hippocampal primary neurons, an event typical of the first stages of AD. We found that this Tau function is impaired by the P301L mutation and by pathological aggregation. To identify other possible genes modulated by Tau we performed an RNAseq experiment and we found a global gene expression alteration that strongly resembles the late mild cognitive impairment. The investigation of molecular mechanisms involving nuclear Tau indicates that it competes with HDAC1 for the binding with TRIM28, a nuclear protein involved in heterochromatin formation. This competition causes the delocalization of HDAC1 from the nucleus modifying the chromatin structure and leading to VGluT1 increased levels, suggesting that Tau modulates the gene expression by altering the chromatin condensation. In conclusion, the CST developed in this study allows to follow in real time the pathological process depicting the early and the late stages of aggregation; thus, it is now at the bases of two screening platforms for the drug discovery and validation in reporter cells and in a transgenic model. In addition, this study demonstrated for the first time that Tau in the nuclear compartment modulates the expression of genes probably by altering the chromatin structure and this role seems to be strongly related to mild cognitive impairment stages when Tau is destabilized and partially aggregated

Il Fondo Atlante per la gestione dei Npl's. Il caso Veneto Banca

Il mio lavoro analizza quelli che sono definiti come il male dei bilanci bancari, i cosiddetti Npl's, ossia crediti deteriorati. Nella prima parte dell'elaborato analizzeremo le varie tipologie di tali crediti, e le diverse modalità attraverso le quali è possibile gestirle. Passeremo poi ad analizzare la situazione bancaria italiana in merito a questo aspetto,soffermandoci sulle banche di maggiori dimensioni, approfondiremo il Fondo che tanto ha fatto e fa parlare di se, il Fondo Atlante, e chi in realtà gestisce tale fondo, ed infine passeremo ad un caso pratico, ossia il caso Veneto Banca, e la sua acquisizione da parte del Fondo stesso

The Q336H MAPT mutation linked to Pick's disease leads to increased binding of tau to the microtubule network via altered conformational and phosphorylation effects

Tauopathies are neurodegenerative disorders characterized by Tau aggregation. Genetic studies on familial cases allowed for the discovery of mutations in the MAPT gene that increase Tau propensity to detach from microtubules and to form insoluble cytoplasmic Tau aggregates. Recently, the rare mutation Q336H has been identified to be associated with Pick's disease (PiD) and biochemical analyses demonstrated its ability to increase the microtubules (MTs) polymerization, thus revealing an opposite character compared to other Tau mutations studied so far. Here we investigated the biophysical and molecular properties of Tau(Q336H) in living cells by the employment of the conformational Tau biosensor CST. We found that this mutation alters Tau conformation on microtubules, stabilizes its binding to tubulin, and is associated with a paradoxical lower level of Tau phosphorylation. Moreover, we found that this mutation impacts the cytoskeletal complexity by increasing the tubulin filament length and the number of branches. However, despite these apparently non-pathological traits, we observed the formation of intracellular inclusions confirming that Q336H leads to aggregation. Our results suggest that the Tau aggregation process might be triggered by molecular mechanisms other than Tau destabilization or post-translational modifications which are likely to be detrimental to neuronal function in vivo

Data Rate vs. Maximum Reach in a Data Center Interconnect Scenario Exploiting Wideband InP Mach-Zehnder Modulators

A new Mach-Zehnder DP-IQ ultra-wideband indium phosphide modulator with integrated optical semiconductor amplifiers has been characterized for time domain simulations to investigate data rate versus maximum range in a DCI scenario

Machine Learning Aided Control of Ultra-Wideband Indium Phosphide IQ Mach-Zehnder Modulators

A digital model of a dual-polarization IQ ultra-wideband indium phosphide Mach-Zehnder modulator is obtained through machine learning techniques. The model is used to test optimization algorithms that automatically set the modulator control voltages under different operative conditions finding the optimum bias point

Tau Modulates VGluT1 Expression

Abstract Tau displacement from microtubules is the first step in the onset of tauopathies and is followed by toxic protein aggregation. However, other non-canonical functions of Tau might have a role in these pathologies. Here, we demonstrate that a small amount of Tau localizes in the nuclear compartment and accumulates in both the soluble and chromatin-bound fractions. We show that favoring Tau nuclear translocation and accumulation, by Tau overexpression or detachment from MTs, increases the expression of VGluT1, a disease-relevant gene directly involved in glutamatergic synaptic transmission. Remarkably, the P301L mutation, related to frontotemporal dementia FTDP-17, impairs this mechanism leading to a loss of function. Altogether, our results provide the demonstration of a direct physiological role of Tau on gene expression. Alterations of this mechanism may be at the basis of the onset of neurodegeneration

Spot‐on SERS Detection of Biomolecules with Laser‐Patterned Dot Arrays of Assembled Silver Nanowires

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Severe acute respiratory syndrome coronavirus 2 infection leads to Tau pathological signature in neurons

COVID-19 has represented an issue for global health since its outbreak in March 2020. It is now evident that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection results in a wide range of long-term neurological symptoms and is worryingly associated with the aggravation of Alzheimer’s disease. Little is known about the molecular basis of these manifestations. Here, several strain variants were used to infect SH-SY5Y neuroblastoma cells and K18-hACE C57BL/6J mice. The Tau phosphorylation profile and aggregation propensity upon infection were investigated on cellular extracts, subcellular fractions, and brain tissue. The viral proteins spike, nucleocapsid, and membrane were overexpressed in SH-SY5Y cells, and the direct interaction and effect on Tau phosphorylation were checked using immunoblot experiments. Upon infection, Tau is phosphorylated at several pathological epitopes associated with Alzheimer’s disease and other tauopathies. Moreover, this event increases Tau’s propensity to form insoluble aggregates and alters its subcellular localization. Our data support the hypothesis that SARS-CoV-2 infection in the central nervous system triggers downstream effects altering Tau function, eventually leading to the impairment of neuronal function

Identification of an ERK inhibitor as a therapeutic drug against tau aggregation in a new cell-based assay

Formation of Tau aggregates is a common pathological feature of tauopathies and their accumulation directly correlates with cytotoxicity and neuronal degeneration. Great efforts have been made to understand Tau aggregation and to find therapeutics halting or reversing the process, however, progress has been slowed due to the lack of a suitable method for monitoring Tau aggregation. We developed a cell-based assay allowing to detect and quantify Tau aggregation in living cells. The system is based on the FRET biosensor CST able to monitor the molecular dynamic of Tau aggregation in different cellular conditions. We probed candidate compounds that could block Tau hyperphosphorylation. In particular, to foster the drug discovery process, we tested kinase inhibitors approved for the treatment of other diseases. We identified the ERK inhibitor PD-901 as a promising therapeutic molecule since it reduces and prevents Tau aggregation. This evidence establishes the CST cell-based aggregation assay as a reliable tool for drug discovery and suggests that PD-901 might be a promising compound to be tested for further preclinical studies on AD

The polymorphism L412F in TLR3 inhibits autophagy and is a marker of severe COVID-19 in males

The polymorphism L412F in TLR3 has been associated with several infectious diseases. However, the mechanism underlying this association is still unexplored. Here, we show that the L412F polymorphism in TLR3 is a marker of severity in COVID-19. This association increases in the sub-cohort of males. Impaired macroautophagy/autophagy and reduced TNF/TNFα production was demonstrated in HEK293 cells transfected with TLR3L412F-encoding plasmid and stimulated with specific agonist poly(I:C). A statistically significant reduced survival at 28 days was shown in L412F COVID-19 patients treated with the autophagy-inhibitor hydroxychloroquine (p = 0.038). An increased frequency of autoimmune disorders such as co-morbidity was found in L412F COVID-19 males with specific class II HLA haplotypes prone to autoantigen presentation. Our analyses indicate that L412F polymorphism makes males at risk of severe COVID-19 and provides a rationale for reinterpreting clinical trials considering autophagy pathways. Abbreviations: AP: autophagosome; AUC: area under the curve; BafA1: bafilomycin A1; COVID-19: coronavirus disease-2019; HCQ: hydroxychloroquine; RAP: rapamycin; ROC: receiver operating characteristic; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TLR: toll like receptor; TNF/TNF-α: tumor necrosis factor