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

Auditory cortex hypoperfusion: a metabolic hallmark in Beta Thalassemia

Abstract Background Sensorineural hearing loss in beta-thalassemia is common and it is generally associated with iron chelation therapy. However, data are scarce, especially on adult populations, and a possible involvement of the central auditory areas has not been investigated yet. We performed a multicenter cross-sectional audiological and single-center 3Tesla brain perfusion MRI study enrolling 77 transfusion-dependent/non transfusion-dependent adult patients and 56 healthy controls. Pure tone audiometry, demographics, clinical/laboratory and cognitive functioning data were recorded. Results Half of patients (52%) presented with high-frequency hearing deficit, with overt hypoacusia (Pure Tone Average (PTA) > 25 dB) in 35%, irrespective of iron chelation or clinical phenotype. Bilateral voxel clusters of significant relative hypoperfusion were found in the auditory cortex of beta-thalassemia patients, regardless of clinical phenotype. In controls and transfusion-dependent (but not in non-transfusion-dependent) patients, the relative auditory cortex perfusion values increased linearly with age (p < 0.04). Relative auditory cortex perfusion values showed a significant U-shaped correlation with PTA values among hearing loss patients, and a linear correlation with the full scale intelligence quotient (right side p = 0.01, left side p = 0.02) with its domain related to communication skills (right side p = 0.04, left side p = 0.07) in controls but not in beta-thalassemia patients. Audiometric test results did not correlate to cognitive test scores in any subgroup. Conclusions In conclusion, primary auditory cortex perfusion changes are a metabolic hallmark of adult beta-thalassemia, thus suggesting complex remodeling of the hearing function, that occurs regardless of chelation therapy and before clinically manifest hearing loss. The cognitive impact of perfusion changes is intriguing but requires further investigations

Mitochondrial dysfunction and mitophagy defects in LRRK2-R1441C Parkinson's disease models

Mutations in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene have been identified as one of the most common genetic causes of Parkinson’s disease (PD). The LRRK2 PD-associated mutations LRRK2G2019S and LRRK2R1441C, located in the kinase domain and in the ROC-COR domain, respectively, have been demonstrated to impair mitochondrial function. Here, we sought to further our understanding of mitochondrial health and mitophagy by integrating data from LRRK2R1441C rat primary cortical and human induced pluripotent stem cell-derived dopamine (iPSC-DA) neuronal cultures as models of PD. We found that LRRK2R1441C neurons exhibit decreased mitochondrial membrane potential, impaired mitochondrial function and decreased basal mitophagy levels. Mitochondrial morphology was altered in LRRK2R1441C iPSC-DA but not in cortical neuronal cultures or aged striatal tissue, indicating a cell-type-specific phenotype. Additionally, LRRK2R1441C but not LRRK2G2019S neurons demonstrated decreased levels of the mitophagy marker pS65Ub in response to mitochondrial damage, which could disrupt degradation of damaged mitochondria. This impaired mitophagy activation and mitochondrial function were not corrected by the LRRK2 inhibitor MLi-2 in LRRK2R1441C iPSC-DA neuronal cultures. Furthermore, we demonstrate LRRK2 interaction with MIRO1, a protein necessary to stabilize and to anchor mitochondria for transport, occurs at mitochondria, in a genotype-independent manner. Despite this, we found that degradation of MIRO1 was impaired in LRRK2R1441C cultures upon induced mitochondrial damage, suggesting a divergent mechanism from the LRRK2G2019S mutation

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

Multilevel investigation of Tau pathology: molecular characterization of nuclear Tau function and drug screening against aggregation

Tau protein is a neuronal microtubule (MT) associated protein whose aggregation is one of the hallmarks of several neurodegenerative diseases known as tauopathies, such as Alzheimer’s disease (AD) and frontotemporal dementia (FTDP-17). At the onset of the pathology, Tau undergoes hyperphosphorylation which leads to destabilisation and detachment from MTs and to an increase in the soluble pool of Tau. Once it is detached from the MTs, the protein is more prone to aggregation so intracellular toxic aggregates deposition starts. Although aggregation has a crucial role in Tau pathology, an increasing body of evidence suggests that some non-canonical functions of Tau can account for some fundamental aspects of the pathology as well. As a matter of fact, our lab demonstrated that nuclear Tau is able to modulate the expression of the vesicular glutamate transporter VGluT1, which happens to be upregulated in the early phases of tauopathies. Moreover, we demonstrated that the increase in the soluble pool of Tau enhances Tau translocation into the nucleus thus increasing VGluT1 expression. This finding has great relevance since it might describe at the cellular level what happens in the early phases of the disease. In this thesis, I am investigating the mechanisms underlying this new function of Tau protein. In particular, I am focusing on the hypothesis that Tau might be involved in chromatin remodelling. In fact, it has been found out that Tau interacts with TRIM28 that is a member of a protein complex that includes the histone deacetylase HDAC1. Moreover, I am investigating the effects of the pathological mutation P301L, associated with FTDP-17, and the effects of Tau aggregation on VGluT1 expression. Nowadays, no disease modifying drugs against tauopathies are available and all the attempts to target other lesions linked to these diseases failed. For this reason, there is an increasing interest in targeting the pathological forms of Tau in order to develop a Tau-centric treatment against tauopathies. To fill this gap, I set-up an in vitro screening assay to test potential therapeutic molecules against Tau aggregation. The lab recently developed the FRET-based Conformational Sensitive Tau sensor (CST) that allows studying the conformation of Tau protein both in physiological and pathological conditions, thus providing a clear imaging read-out of different features of the aggregates inside the cell. I exploited the peculiar biophysical properties of CST to screen drugs against Tau pathology. The in vitro assay exploits a CST reporter SH-SY5Y cell line, where Tau aggregation is induced with synthetic Tau seeds. This system tests the ability of candidate compounds to interfere with the process of aggregation or even to revert it. In conclusion, this thesis focuses on two aspects relevant for Tau pathology: the molecular characterization of a new function for Tau that might be relevant in the early phases of pathology to make it a potential therapeutic target, and the development of an in vitro drug screening to evaluate the therapeutic potential of two candidate compounds

Tau-dependent HDAC1 nuclear reduction is associated with altered VGluT1 expression

During AD pathology, Tau protein levels progressively increase from early pathological stages. Tau altered expression causes an unbalance of Tau subcellular localization in the cytosol and in the nuclear compartment leading to synaptic dysfunction, neuronal cell death and neurodegeneration as a consequence. Due to the relevant role of epigenetic remodellers in synaptic activity in physiology and in neurodegeneration, in particular of TRIM28 and HDAC1, we investigated the relationship between Tau and these epigenetic factors. By molecular, imaging and biochemical approaches, here we demonstrate that Tau altered expression in the neuronal cell line SH-SY5y does not alter TRIM28 and HDAC1 expression but it induces a subcellular reduction of HDAC1 in the nuclear compartment. Remarkably, HDAC1 reduced activity modulates the expression of synaptic genes in a way comparable to that observed by Tau increased levels. These results support a competitive relationship between Tau levels and HDAC1 subcellular localization and nuclear activity, indicating a possible mechanism mediating the alternative role of Tau in the pathological alteration of synaptic genes expression

Metabolomic change due to combined treatment with myo-inositol, D-chiro-inositol and glucomannan in polycystic ovarian syndrome patients: a pilot study

Polycystic ovarian syndrome (PCOS) is a highly variable syndrome and one of the most common female endocrine disorders. Although the association inositols-glucomannan may represent a good therapeutic strategy in the treatment of PCOS women with insulin resistance, the effect of inositols on the metabolomic profile of these women has not been described yet

Single-cell spatial transcriptomic and translatomic profiling of dopaminergic neurons in health, aging, and disease

Summary: The brain is spatially organized and contains unique cell types, each performing diverse functions and exhibiting differential susceptibility to neurodegeneration. This is exemplified in Parkinson’s disease with the preferential loss of dopaminergic neurons of the substantia nigra pars compacta. Using a Parkinson’s transgenic model, we conducted a single-cell spatial transcriptomic and dopaminergic neuron translatomic analysis of young and old mouse brains. Through the high resolving capacity of single-cell spatial transcriptomics, we provide a deep characterization of the expression features of dopaminergic neurons and 27 other cell types within their spatial context, identifying markers of healthy and aging cells, spanning Parkinson’s relevant pathways. We integrate gene enrichment and genome-wide association study data to prioritize putative causative genes for disease investigation, identifying CASR as a regulator of dopaminergic calcium handling. These datasets represent the largest public resource for the investigation of spatial gene expression in brain cells in health, aging, and disease