modulation-tau-subcellular-localization-as-tool-to-investigate

Modulation of Tau Subcellular Localization as a Tool to Investigate the Expression of Disease-related Genes

Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, ...

Scuola Normale Superiore

Recent advances in fluorescence microscopy provided tools for the investigation and the analysis of the viral replication steps in the cellular context. In the HIV field, the current visualization systems successfully achieve the fluorescent labeling of the viral envelope and proteins, but not the genome. Here, we developed a system able to visualize the proviral DNA of HIV-1 through immunofluorescence detection of repair foci for DNA double-strand breaks specifically induced in the viral genome by the heterologous expression of the I-SceI endonuclease. The system for Single-Cell Imaging of HIV-1 Provirus, named SCIP, provides the possibility to individually track integrated-viral DNA within the nuclei of infected cells. In particular, SCIP allowed us to perform a topological analysis of integrated viral DNA revealing that HIV-1 preferentially integrates in the chromatin localized at the periphery of the nuclei.

Single-cell imaging of HIV-1 provirus (SCIP).

The ability to visualize fluorescent HIV-1 particles within the nuclei of infected cells represents an attractive tool to study the nuclear biology of the virus. To this aim we recently developed a microscopy-based fluorescent system (HIV-IN-EGFP) that has proven valid to efficiently visualize HIV-1 complexes in the nuclear compartment and to examine the nuclear import efficiency of the virus. The power of this method to investigate viral events occurring between the cytoplasmic and the nuclear compartment is further shown in this study through the analysis of HIV-IN-EGFP in cells expressing the TRIMCyp restriction factor. In these cells the HIV-IN-EGFP complexes are not detected in the nuclear compartment, while treatment with MG132 reveals an accumulation of HIV-1 complexes in the cytoplasm. However, the Vpr-mediated transincorporation strategy used to incorporate IN fused to EGFP (IN-EGFP) impaired viral infectivity. To optimize the infectivity of the HIV-IN-EGFP, we used mutated forms of IN (E11K and K186E) known to stabilize the IN complexes and to partially restore viral infectivity in transcomplementation experiments. The fluorescent particles produced with the modified IN [HIV-IN(K)EGFP_IN(E)] show almost 30% infectivity as compared to wild-type NL4.3. Detailed confocal microscopy analysis revealed that the newly generated viral particles resulted in HIV-1 complexes significantly smaller in size, thus requiring the use of brighter fluorophores for nuclear visualization [HIV-IN(K)sfGFP_IN(E)]. The second-generation visualization system HIV-IN(K)sfGFP_IN(E), in addition to allowing direct visualization of HIV-1 nuclear entry and other viral events related to nuclear import, preserves intact viral properties in terms of nuclear entry and improved infectivity.

Second generation imaging of nuclear/cytoplasmic HIV-1 complexes.

Recent advances in fluorescence microscopy allow three-dimensional analysis of HIV-1 preintegration complexes in the nuclei of infected cells. To extend this investigation to gammaretroviruses, we engineered a fluorescent Moloney murine leukemia virus (MLV) system consisting of MLV-integrase fused to enhanced green fluorescent protein (MLV-IN-EGFP). A comparative analysis of lentiviral (HIV-1) and gammaretroviral (MLV) fluorescent complexes in the nuclei of infected cells revealed their different spatial distributions. This research tool has the potential to achieve new insight into the nuclear biology of these retroviruses. 

Comparative Analysis of HIV-1 and Murine Leukemia Virus Three-Dimensional Nuclear Distributions.

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.

Tau Modulates VGluT1 Expression.

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.

Article	Year
Single-cell imaging of HIV-1 provirus (SCIP). Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 23513220	2013
Second generation imaging of nuclear/cytoplasmic HIV-1 complexes. AIDS research and human retroviruses\| PubMed ID: 24798748	2014
Comparative Analysis of HIV-1 and Murine Leukemia Virus Three-Dimensional Nuclear Distributions. Journal of virology\| PubMed ID: 26962222	2016
Tau Modulates VGluT1 Expression. Journal of molecular biology\| PubMed ID: 30664870	2019
Identification of an ERK Inhibitor as a Therapeutic Drug Against Tau Aggregation in a New Cell-Based Assay. Frontiers in cellular neuroscience\| PubMed ID: 31496937	2019

Valentina Quercioli

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